WO1991002040A1 - Cyclodextrin labels for nucleic acid and biochemical analysis - Google Patents

Cyclodextrin labels for nucleic acid and biochemical analysis Download PDF

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Publication number
WO1991002040A1
WO1991002040A1 PCT/US1990/004375 US9004375W WO9102040A1 WO 1991002040 A1 WO1991002040 A1 WO 1991002040A1 US 9004375 W US9004375 W US 9004375W WO 9102040 A1 WO9102040 A1 WO 9102040A1
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Prior art keywords
cyclodextrin
label
labels
nucleic acid
labeled
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PCT/US1990/004375
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French (fr)
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Kenneth M. Kosak
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Kosak Kenneth M
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Publication of WO1991002040A1 publication Critical patent/WO1991002040A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G01N21/763—Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals

Definitions

  • This invention relates to nucleic acid sequence analysis using nonradioactive, luminescent cyclodextrin labels.
  • cyclodextrins that also include a fluorescent or chemi luminescent substance, and are used as .labels coupled to nucleotides. It has been discovered that cyclodextrin labels provide new properties and advantages, described below. As far as can be determined, such labels are novel compositions and their use is unknown in the prior art.
  • Chemilumi escent substances have been used as labels by chem ⁇ ically coupling them directly to certain ligands, antibodies and other substances for use in immunoassays.
  • An acridinium ester label is used by Bounaud, J-Y., et al , Clin. Chem. 34/12, 2556- 2560 (1988); luminol is used by Tokinaga, D. , et al , in U.S. Pat. No. 4,628,035 (1986); and luciferin is used by Schaeffer, J.M., et al , in U.S. Pat. No. 4,665,022 (1987).
  • colloidal, lipophilic particles such as liposomes
  • ligand and a fluorescent or chemi luminescent laDel adjacently bound to the surface of the particle.
  • They are used in immunoassays where ste ⁇ c interaction between a binding receptor and the label modulates the signal.
  • Chemiluminescent substances have been used as labels by chem ⁇ ically coupling them directly to certain ligands, antibodies and other substances for use in assays.
  • An acridinium ester label is used by Bounaud, J-Y., et al , Clin. Chem. 34/12, 2556-2560 (1988); luminol- is used by Tokinaga, D. , et al , in U.S. Pat. No. 4,628,035 (1986); and luciferin is used by Schaeffer, J.M., et al , in U.S. Pat. No. 4,665,022 (1987).
  • the im unoassay inventions of the prior art are fundamentally different compositions and methods from the instant invention where a cyclodextrin label is coupled to a nucleotide or nucleic acid primer, for use in nucleic acid sequencing.
  • Previously cited prior art immunoassay inventions provide no teaching or suggestion on how to perform nucleic acid sequence analysis. Recently, fluorescence and chemi luminescence has been used in nucleic acid hybridization analysis.
  • a homogeneous feature is provided through the hybri ⁇ dization of two labeled DNA probes with the nucleic acid analyte wherein a "transformation radical” (eg. OH ⁇ ) , is generated by a catalyst on one probe which converts an apoluminescer on the other probe to a luminescer.
  • a transformation radical eg. OH ⁇
  • the Miller invention makes* no disclosure of cyclodextrins and suffers the limitation of requiring two labeled probes, wherein the number of label molecules per probe is limited to the number of coupling sites available on said probe.
  • Gen-Probe Inc. San Diego, CA, has disclosed a procedure called PACETM, wherein acridinium ester label is coupled to a nucleic acid probe for use in a nucleic acid hybridization assay.
  • PACETM a procedure called PACETM, wherein acridinium ester label is coupled to a nucleic acid probe for use in a nucleic acid hybridization assay.
  • the procedure does not teach or suggest any of the cyc ⁇ lodextrin labels and methods of the instant invention.
  • ECL enhanced chemiluminescence
  • fluorescent labels for sequencing DNA and RNA. Examples are Prober, J.M., et al , Science 238, 336-341 (1987), and Smith, L.M., et al , Nature (London) 321, 674-679 (1986).
  • fluorescent methods require an incident light source for generation of detectable signal. This produces the inherent background problems of reflected incident light, Raman scattering, and nonspecific fluorescence of other substances in the sample.
  • cyclodextrins have been used primarily for chromatography,*- catalysts, and fluorescent or chemiluminescent enhancement.
  • cyclodextrin labels provide new properties and with unexpected advantages. For instance, they provide potentially higher signal efficiency, versatility in label colors, while maintaining uniform chemical and physical properties.
  • This invention provides a nucleic acid test method that for the first time has all of the following useful properties:
  • luminescent labels detected in a "black” background avoid problems of fluorescence and allow for more sensitivity
  • cyclodextrin complexes provide potentially higher signal efficiency in aqueous solutions
  • this invention is intended for use in assays empl ⁇ oying photomultipliers or charge-coupled device (CCD) cameras, with computerized data collection and reduction. It is also suitable for photodiode detection (eg. Aizawa, M. , et al , Anal. Lett. 17(B7), 555-564, 1984), or photographic detection and recording. Still another suitable use of the instant invention is in continuous flow and/or solid phase detection systems, (eg. Van Zoonen, P., et al , Anal. Chim. Acta. 167, 249-256, 1985; and Anal. Chim. Acta. 174, 151-161, 1985), including chromatographic methods. Also, an object of the instant invention is to provide reagents, containers and other components in the form of a mer ⁇ cantile kit, to carry out the methods of the instant invention.
  • the instant invention can be used with a variety of test formats and detection systems.
  • luminometers such as portable or automated, "tube lumin ⁇ meters” and other automated instruments that read 96 well, microtiter plates could be used.
  • the instant inven ⁇ tion provides advantages of speed, sensitivity, simplicity and versatility previously unknown or suggested in the art of nuc ⁇ leic acid sequence analysis.
  • Ligand A ligand is defined as a selectively bindable mater ⁇ ial, that has a selective affinity for or is bound by a usually, but not necessarily, larger specific binding body or "partner", in a ligand binding reaction.
  • ligand binding reactions are well known and include selective affinity binding between anti ⁇ gen and antibody, biotins and avidins, lectins and glycopro- teins, receptors and hormones, substrates or cofactors and enz ⁇ ymes, and between restriction enzymes and nucleic acids, among others.
  • Ligands are also capable of being bound to non- biological types of binding substances such as chelators, cavitands, resins and surfactants.
  • the ligand In immunoassays and nucleic acid tests, the ligand would be the antigen, hapten or comple ⁇ ment that would be bound by or to its corresponding antibody. In the case of an enzyme, a ligand would be the substrate or the coenzyme.
  • Other substances that are capable of being bound as ligands by organic or biological substances are proteins, histones, enzymes, enzyme fractions and derivatives, hormones, vitamins, steroids, polypeptides, carbohydrates, lipids, bio- tins, biotin derivatives, fc receptors, antibiotics, drugs, digoxins, pesticides, nucleic acid polymers, oligonucleotides, and substances used or modified such that they function as lig ⁇ ands.
  • Ligands also include various substances with selective affinity for ligators that are produced through recombinant DNA, genetic and olecu'lar engineering.
  • Ligator A ⁇ gator is defined as a specific binding body or "partner" that is usually, but not necessarily, larger than the ligand it can bind to. For the purposes of this invention, it is a specific substance or material or chemical or “reactant” that is capable of selective affinity binding with a specific ligand in a ligand binding reaction.
  • a ligator can be a protein such as an antibody, a nonprotein binding body or a "specific reactor.”
  • ligators When binding ligands, ligators would include anti ⁇ bodies including monoclonal antibodies, chimeric antibodies and fractions thereof, enzymes, plasma proteins, avidins, strep- tavidins, chalones, cavitands, thyroglobulin, intrinsic factor, globulins, biological receptors, viruses, cell membrane derivat ⁇ ives, chelators, surfactants, organometallic substances, staphy- lococcal protein A, protein G, ribosomes, bacteriophages, cyto- chromes, lectin ⁇ , certain resins, organic polymers, cyclodex ⁇ trins, and catalysts.
  • Ligators also include various substances with selective affinity for ligands that are produced through recombinant DNA, genetic and molecular engineering. Under suit ⁇ able conditionsj ligators can be used as labels wherein they are coupled to an appropriate
  • nucleic Acid A nucleic acid is defined as any nucleic acid sequence from any source that is suitable for use in the instant invention. Said nucleic acid includes all types of RNA, all types of DNA, oligonucleotides and other genetic materials including synthetjc nucleic acid polymers. Also included are DNA and/or RNA fragments, and derivatives from any tissue, cells, nuclei, chromosomes, cytoplasm, mitochondria, ribosomes, and other cellular sources. Also included are modified and der- ivatized nucleic acid sequences including those that are coupled to or associated with other substances such as proteins, lec- tins, histones, pol peptides, carbohydrates, lipid ⁇ , resins, steroids, hormones and enzymes.
  • nucleic acid may be pretreated by well known methods before use in the instant invention.
  • nucleic acid may be extracted, purified, amplified, denatured by various means, immobilized, and/or suitably deriv- atized as needed.
  • Luminescence Luminescence is defined as the product of a luminescent reaction.
  • a luminescent reaction for purposes of this invention, is defined as nonradioactive, electromagnetic radiation or light produced by some means of electronic excita ⁇ tion or ionization of molecules or atoms, in the absence of an incident light source. Specifically, this includes photons emitted through a chemical or biochemical reaction such as oxid ⁇ ation, peroxide cleavage or ionization. However, it would exclude certain physical energy sources for electronic excita ⁇ tion that require an incident light source to cause photon emis ⁇ sion. Although certain methods that do use an incident light source (eg.
  • Chemi 1uminescence For the purposes of this invention, che - i luminescent (CL) substances are inorganic and organic sub ⁇ stances that generate light during their chemical change or decomposition. Chemiluminescent substances can be readily activated by various inorganic oxidizers and do not require enz ⁇ ymes or other proteins to produce luminescence. However, certain chemi luminescent substances can also be activated through enzymatic reactions that generate peroxide as a by ⁇ product, which subsequently reacts with the chemiluminescent substance. A chemiluminescent substance, or luminescer, can be coupled to and/or complexed with a cyclodextrin label that is detected through activation of the CL substance and measurement of the light produced.
  • chemiluminescent substances that would be useful in this invention include a number of compounds such as luminols, isoluminols, aminobutylethyl-isoluminols (ABEI), aminobutylethyl-'naphthalene-isoluminols (ABEN) and any other cyclic or acyclic hydrazides.
  • dioxe ⁇ tanes include various dioxe ⁇ tanes, includedini 3-phosphate-9H-xanthene-9-ylidineadamantanes, tert-butyldimethylsi loxyl-substituted and/or adamantyl- substituted dioxetanes, and other dioxetane and dioxetanone der ⁇ ivatives and precursors, 2,4,5-triphenylimidazones (lophines), acridines, acridine and acridinium esters and salts, including derivatives and precursors, indole-3-pyruvic acid, aryl Grignard reagents, riboflavin, lucigenin ⁇ , 9, 10-bis(phenylethynyl )- anthracenes (BPEA), 9, 10-bis(phenylethynyl )naphthacenes (BPEN), luciferins, phthalazin
  • Bioluminescence Bioluminescence is light produced by a bio- luminescent (BL) reaction using certain light generating proteins or protein-containing substances that can be extracted from various biolu inescent organisms.
  • BL sub ⁇ stances are luciferases and photoproteins.
  • the preparations and components of the instant invention are synthesized by coupling labels, ligands, ligators, nucleic acids, support materials and other substances in various combinations as described below. Said coupling can be through noncovalent, "attractive” binding or through covalent, electron- pair bonds.
  • Functional Group A functional group is defined here as a potentially reactive site on a substance where one or more atoms are ava lable for covalent coupling to some other substance. Some substances have functional groups as part of their struc ⁇ ture such as those provided by amino acid residues on certain proteins. Other substances may require chemical "activation" of their functional groups to produce aldehydes, ketones or other useful groups. Also, functional groups can be added to various substances through derivatization or substitution reactions.
  • Examples of functional groups are aldehydes, amines, amides, azides, carboxyls, carbonyls, epoxys, hydroxyls, ketones, metals, phosphates, sulfhydryls, sulfonyls, thioethers, phenolic hydroxyls, indoTes, bromines, chlorines, iodines, and others.
  • the prior art has shown that most, if not all of these func ⁇ tional groups can be incorporated into or added to cyclodex ⁇ trins, ligands, ligators, nucleic acids and support materials if not already present.
  • a coupling agent (or cross!inking agent), is defined as a chemical substance or energy that produces and/or reacts with functional groups on a target substance so that cov ⁇ alent coupling or conjugation can occur with the target subs ⁇ tance. Because of the stability of covalent coupling, this is often the preferred method.
  • nucleotide, nucleic acid, ligand, or ligator the appropriate coupling agent is used to produce the necessary active functional group or react with it.
  • energy type coupling agents are ultraviolet, visible and radioactive radiation that can promote coupling or crosslinking of certain substances.
  • photochemical coupling agents disclosed in U.S. Pat. No. 4,737,454, among others.
  • enzymes that produce covalent coupling such as nucleic acid polymerases and ligases, among others.
  • Covalent coupling or conjugation can be done through functional groups using coupling agents such as glutaraldehyde, formaldehyde, cyanogen bromide, azides, p- benzoquinone, succinic anhydrides, carbodiimides, maleimides, epichlorohydrin, periodic acid, ethyl chlorofor ate, dipyridyl disulphide and polyaldehydes.
  • coupling agents such as glutaraldehyde, formaldehyde, cyanogen bromide, azides, p- benzoquinone, succinic anhydrides, carbodiimides, maleimides, epichlorohydrin, periodic acid, ethyl chlorofor ate, dipyridyl disulphide and polyaldehydes.
  • bifunctional imidoesters such as dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl 3,3'-dithiobis-propionimidate (DTBP), and 2- iminothiolane (Traut's reagent); bifunctional NHS esters such as disuccinimidyl suberate (DSS) , bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES) , disuccinimidyl (N,N'-diacetylhomocystein) (DSAH), disuccinimidyl tartarate (DST), dithiobis(succinimidyl propionate) (DSP), and ethylene glycol bis(succinimidyl succinate) (EGS), including various derivatives such as their sulfo- forms; heterobifunctional reagents such as N
  • FNPA N-hydroxysuccinimidyl-4- azidobenzoate
  • NHSAB N-hydroxysuccinimidyl-4-azidosal icylic acid
  • MABI methyl-4-azidobenzoimidate
  • PNP-DTP p- nitrophenyl-2-diazo-3,3,3-trifluoropropionate
  • 2- diazo-3,3,3-trifluoropropionyl chloride N-succinimidyl-6(4'- az do-2'-nitropheny!amino)hexanoate
  • SADP N-succinimidyl (4- azidophenyl )1 ,3'-dithiopropionate
  • SADP sulfosuccinimidyl-2- (m-azido-o-nitobenzamido)-ethyl-l ,3'-dithiopropionate
  • SASD 1 ,3'-dithiopropionate
  • Certain coupling agents may be less suitable than others due to adverse modification of the cyclodextrins, nucleotides, nuc ⁇ leic acids, ligands, ligators, and components of the instant invention being coupled. In these cases, routine precautions by one skilled in the art of covalent coupling can be taken to pre ⁇ vent such difficulties.
  • Intermediate Coupling Substance In addition to covalently coupling through functional groups, it i ⁇ also useful to include intermediate substances which function as "spacer" molecules between the materials being covalently coupled. These interme ⁇ diate substances may be desirable to provide additional coupling sites and thereby increase the amount of label coupled to a nuc ⁇ leotide, ligand or ligator, and/or overcome steric hindrance and/or introduce certain other desirable properties, such as energy transfer.
  • intermediate substances they are coupled to components of the instant invention by using the appropriate functional groups and coupling agents. Then, the desired nucleic acid, nucleotide, ligand or ligator is coupled to the available sites on the intermediate substance and thereby be coupled indirectly to said components of the instant inven ⁇ tion.
  • intermediate coupling substances examples include proteins, polypeptides, polyamino acids, glycoprotein ⁇ , lipopro- teins, nucleic acid polymers, oligonucleotides, DNA, RNA, carbo ⁇ hydrates, polysaccharides, polyglutamic acid, poly(acrylamides) , poly(allylamines) , lipids, glycolipids and certain synthetic polymers, nylons and surfactants.
  • suitable intermediate coupling substances are the polymers disclosed in U.S. Pat. No. 4,645,646.
  • Various materials may be incorporated into the components of the instant invention to impart additional properties and thereby improve their usefulness in certain applications.
  • the addition of ferrous or magnetic particles may be used to give cyclodextrin labels magnetic properties (Ithakis- sios, D.S., Clin. Chim. Acta 84(1-2), 69-84, 1978). This may be useful for various manipulations such as dispensing, transfer ⁇ ring, washing and separating.
  • sub ⁇ stances and components that are described as being in close proximity or close association are close enough so that they share the same molecular or chemical environment. Said close proximity can be due to their being coupled to a common subs ⁇ tance or through their being coupled to each other. In any case, said substances or components may not necessarily interact with each other but one can effect said environment of the other.
  • a suitable enzyme in close proximity to a cyclo ⁇ dextrin label would be able to produce a product that could activate a fluorescent or luminescent substance inside the cyc ⁇ lodextrin label.
  • a fluorophore or CL compound, coupled in close proximity to a cyclodextrin molecule, is able to form an inclusion complex with said cyclodextrin, and/or participate in an energy transfer reaction.
  • Cyclodextrins A cyclodextrin (CD), is an oligosaccharide composed of glucose monomers coupled together to form a conical, hollow molecule with a hydrophobic interior or cavity.
  • Said cyclodextrins (CD's), of the instant invention can be any suit ⁇ able cyclodextrin, including alpha-, beta-, and gamma- cyclodextrins, and their analogs, isomers, and derivatives. Also included are altered forms such as crown ether-like com ⁇ pounds prepared by Kandra, L. , et al , J. Indus. Phenom.
  • a cyclodextrin label is defined herein as any cyclodextrin of suitable size that is capable of complex- ing or combining with one or more molecules to form an "inclu ⁇ sion complex", and wherein said cyclodextrin also has one or more suitable functional groups available for coupling to a sub ⁇ stance to be labeled.
  • Said inclusion complex is defined herein as said cyclodextrin, functioning as a "host” molecule, combined with one or more "guest” molecules that are contained or bound, wholly or partially, within the hydrophobic cavity of said cyc ⁇ lodextrin.
  • guest molecules that are contained or bound, wholly or partially, within the hydrophobic cavity of said cyc ⁇ lodextrin.
  • Cyclodextrin Fltiorophore Labels A cyclodextrin fluorophore label is defined herein as a cyclodextrin label wherein said guest molecule is any suitable fluorophore described below, including any suitable fluorescent, phosphorescent, or scintil- lator substance, or organic dye.
  • a cyclodextrin chemi- luminescent (CL), label is defined herein as a cyclodextrin label wherein said guest molecule is any suitable CL substance previously described, combined with said CD to form a light emitting inclusion complex.
  • a new form of cyclodextrin CL label comprises said inclusion complex wherein said CL substance is any polyaromatic hydrazide CL compound, disclosed in the copend- ing patent application SN 363,081, filed June 8, 1989, previ ⁇ ously described.
  • a cyclodextrin catalyst label is defined herein as a cyclodextrin label wherein said CD host functions as an "artificial enzyme", and said guest molecule is a substance that functions as a chemical substrate.
  • said chemical substrate comes in contact with said cyclodextrin cata ⁇ lyst label under appropriate conditions, it is modified to pro ⁇ prise a detectable signal directly or indirectly (eg. Ikeda, VanEtten, Hirai, below).
  • said signal can be due to the production of a colorimetric or fluorimetric or chemiluminescent substance.
  • said CD catalyst labels can be synthesized to bind specific substrates and catalyze specific reactions.
  • CD catalyst labels eg. size, aff ⁇ inity, reactivity, etc.
  • several labels can be used to label different substances so that their presence is detectable in the same solution.
  • each CD catalyst label could be distinguished from the others by the production of a different product which produced a correspondingl different signal, such as a different color. .
  • Said cyclodextrin catalyst labels can be couple to a variety of substances, such as ligands, antigens, antibodies, nucleo- tides, nucleic acids, primers and chain terminators, as well as to a variety of magnetic particles for use and retrieval in chemical processes, including organic synthesis and detoxifica ⁇ tion.
  • a cyclodextrin energy transfer label is a cyclodextrin label wherein said guest molec ⁇ ule is any suitable fluorophore, or fluorescent, phosphorescent, or scintillator substance, laser dye, or organic dye, and wherein one or more suitable energy producing substances are coupled to the cyclodextrin surface.
  • Said energy producing sub ⁇ stances can include suitable CL substances, BL substances, per- oxyoxalates, dioxetanes, dioxetanones, oxalate-type esters, cat ⁇ alysts, enzymes (eg. oxidases), and other substances, that under suitable conditions, can chemically generate ionization, perox ⁇ ide decomposition and/or electronic excitation energy which can be transferred to guest molecules inside said complex.
  • Yet another new label provides a plurality of said cyclodex ⁇ trin energy transfer label, that can be readily coupled to any suitable substance, in numbers that are greater than the number of available sites on said substance as described previously for multiple CD labels.
  • a cyclodextrin tracer is a component of this invention composed of a nucleoside, nucleotide, ligand, ligator, nucleic acid, or other specific binding substance that has been coupled to or associated with a cyclodextrin label.
  • a cyclodextrin tracer can be a fluorescent or luminescence tracer that has the specific binding properties of the nucleotide, lig ⁇ and or ligator 1n combination with the potential for emitting light. Said fluorescent or luminescence cyclodextrin tracer is detected by activating the label and measuring the light pro ⁇ quizd.
  • a cyclodextrin tracer can also be said specific binding subs ⁇ tance coupled to said CD catalyst label.
  • Cyclodextrin labels coupled to avidins and streptavidins would be useful for subsequent coupling to a biotinylated nuc ⁇ leic acid.
  • cyclodextrin labeled antibodies can be coupled to nucleic acids with the appropriate antigen coupled to it.
  • a cyclodextrin tracer can also be any nucleotide, or RNA, or DNA, or nucleic acid probe that has been covalently or non- covalently coupled to a cyclodextrin label.
  • Peroxyoxalates are defined in the instant invention as organic substances that contain as part of their structure, an oxalate derivative that can decompose in the presence of H2O2 or other peroxides and thereby activate or electronically excite a fluorophore or fluorescer to emit light.
  • peroxyoxalates useful in the instant invention are; oxalyl chloride, bis(3-trifluoromethyl-4-nitropheny!
  • TBMNPO bis(2 » 4-dinitrophenyl )oxalate
  • DNPO bis(2,4,6- trichlorophenyl )oxalate
  • TCPO bis(2,4,5-trichloro-6- carbopentoxyphenyl )oxalate
  • CPPO bis(2,4,5-trichloro-6- carbopentoxyphenyl )oxalate
  • ⁇ oxalyl is[(trifluoromethylsulfonyl )imino]ethylene ⁇ bis[4-methyl- morpholinium trifluoromethane sulfonate] (METQ), among others, including analogs, precursors and derivatives such as mixed anhydrides, amides, sulfonamides, and N-trifyloxamides of oxa- 1ates.
  • METQ morpholinium trifluoromethane sulfonate
  • fluoro ⁇ phores are defined as any suitable dye, and/or fluorescent, phosphorescent, scintillator substance that can form a light emitting complex with a cyclodextrin of the instant invention.
  • fluorophores are defined here as not chemiluminescent by the definitions of the instant invention. Therefore, fluorophores of this invention do not undergo a chem ⁇ ical decomposition during light emission.
  • fluorophores useful in the instant invention are aminofluoranthrenes, anthracenes, diphenyl anthracenes, 8- anilo-1-naphthalenes, europium chelates including 4,7- bis(chlorosulfonyl )-1 , 10-phenanthroline-2,9-dicarboxy1ic acids, ruthenium chelates, fluoresceins, fluorescamines, luciferins, N- methylacridones, perylenes, rhodamine B, rubrenes, ruthenium dipyridines, blue fluorescent proteins, green fluorescent proteins, photoproteins, and their analogs and derivatives, among others.
  • scinti1lators are fluorophores that include light emitting com ⁇ pounds generally used in scintillation counting systems and as laser dyes. Under suitable conditions, they are useful in the instant invention as fluorescent acceptors, and in energy trans ⁇ fer systems such as with cyclodextrins and other preparations described below.
  • Examples are: acriflavins, aminoacridines, 7- amino-4-methycoumarins, carbostyrri Is, coumarins, 2,5- diphenyloxazoles, (PPO), 1 ,4-bis[5-phenyl-2-oxazoyl]benzenes (POPOP), and other oxazoles, P-terphenyls, rhodamines, and analogs and derivatives of these.
  • PPO 2,5- diphenyloxazoles
  • POPOP 1 ,4-bis[5-phenyl-2-oxazoyl]benzenes
  • POPOP 1,4-bis[5-phenyl-2-oxazoyl]benzenes
  • Other useful scinti1lators incorporated herein by reference, can be found under "Laser Dyes", 877-880, Sigma Catalogue, 1989, Sigma Chem. Co., St. Louis, MO.
  • Fluorophore Activation The fluorophores of the instant invention emit light after electronic excitation or "activa ⁇ tion", from some outside energy source.
  • Said outside energy source can be physical, such as an incident light or radiation source (fluorescence), an electrical current (electrolumines ⁇ cence), sonication (triboluminescence) , or heat (thermolumines ⁇ cence), or said energy source can be a chemical reaction (energy transfer 1uminescence) .
  • Said chemical reaction can be any suitable reaction known to activate fluorophores, including those involving acridinium esters, various hydrazides, enzymes, dioxetanes, dioxetanones, oxalate ⁇ , and peroxyoxalates.
  • suitable activation reactions that are applicable to the instant invention are reviewed by Cilento, G. , et a1 , Photochem. Photobiol. 48, 361-368 (1988).
  • fluorophores are activated through energy transfer from a chemiluminescent reaction, such as peroxide decomposition of peroxyoxalate.
  • chemi luminescent activation of peroxyoxalates, acridinium esters, various hydrazides, and other CL substances involve the use of a peroxide such as H2O2.
  • H2O2 can be added directly to a reaction mixture of CD fluorophore label and peroxyoxalate, in suitable buffer solution.
  • H2O2 can be generated by enzymes such as glucose oxidase and suitable substrate.
  • peroxides are suitable, such as t-butyl hydroperoxide, benzoyl peroxide, lino- lenic hydroperoxide, peroxylauric acid, cholesterol hydroper ⁇ oxide, cumene hydroperoxide, with additional examples disclosed by Cathcart, R., et al , Anal. Biochem. 134, 111-116 (1983), among others.
  • Said energy transfer is generally from a chemical source.
  • activation can include energy transfer from any suit ⁇ able substance wherein said energy is first generated by said physical source ⁇ in said substance, which is then exposed to said fluorophore.
  • fluorophores of the instant invention can convert some of this energy to visible light. Examples of this approach are disclosed by Tseng et al , U.S. Pat. Nos. 4,338,213 and 4,407,743; Arthen et al , U.S. Pat. No. 4,401,585; Kamhi et al , U.S. Pat No. 4,404,513; Frenzel , U.S. Pat. No. 4,433,060 and Berthold et al , U.S. Pat No. 4,435,509.
  • compositions of the instant invention it will be understood by those skilled in the art of organic synthesis methods, that there are certain limitations and conditions as to what compositions will comprise a suitable label. Said limitations include types and numbers of derivatives used, steric properties, fluorescent properties of fluorophores or scintillators, excitation energy transfer requirements, oxidation requirements in aqueous solution, stability at room temperature and solubility, among others.
  • cyclodextrins there are limitations as to which fluorophores or CL compounds can be used to form inclusion complexes with certain cyclodex ⁇ trins.
  • nucleic acid for the purposes of the instant invention, the appropriate nucleic acid, probe, nucleoside, nucleotide or "artificial dinucleotide" (eg. Sessler, supra), is labeled through a suit ⁇ able functional group. Said labeling is done while retaining its ability to function in ol igonucleotide binding and/or hybri ⁇ dization and/or synthesis including use as a primer or as a chain terminator in nucleic acid sequence analysis.
  • an artificial dinucleotide eg. Sessler, supra
  • Said functional group can be any previously described func ⁇ tional group already present on the labeled molecule, or can be introduced through known chemical or photochemical means.
  • said functional group is a free amino group such as an aliphatic amine coupled to the purine or pyrimidine base, or coupled to the sugar moiety.
  • a suitable aliphatic amine can be coupled to the desired nuc ⁇ leotide sugar for use in an ol igonucleotide primer by the method of Smith, L.M., et al , Nucleic Acids Res. 13, 2399-2412 (1985), Smith, L.M., et al , Nature (London) 321, 674-679 (1986), and Atkinson, T. , et al , IN "01 igonucleotide Synthesis: A Practical Approach", Gait, M.J., ed. , IRL, Oxford, (1984).
  • Chain terminators are defined herein as any suitable chain- terminating nucleotide such as dideoxyribonucleotide triphos- phates, useful in nucleic acid sequence analysis. Methods suit ⁇ able for synthesizing amino derivatized, chain-term nating dideoxyribonucleotide triphosphates, are described by Prober, J.M., et al , Science 238, 336-341 (1987) and Sanger, F., et al , Proc. Nat! . Acad, Sci . USA 74, 5463-5467 (1977), including nuc ⁇ leoside derivatizing methods of Robins, M.J., et al , J. Org. Chem.
  • Th-ese methods can be suitably modified as needed to make available an amino group on the appropriate nucleotide for coupling to the labels of this invention.
  • This procedure can be applied to the synthesis of the appro ⁇ priate nucleotides, primers or chain-terminating dideoxyribo- nucleoside triphosphates, including those of dideoxycytidine (ddCTP), dideoxythymidine (ddTTP), dideoxyadenosine (ddATP), dideoxyguanosine (ddGTP), and dideoxyuridine (ddUTP), triphos- phates.
  • ddCTP dideoxycytidine
  • ddTTP dideoxythymidine
  • ddATP dideoxyadenosine
  • ddGTP dideoxyguanosine
  • ddUTP dideoxyuridine
  • the starting compound is any appropriate nucleoside that has the necessary groups protected such as the hydroxyl groups pro ⁇ tected as p-toluyl esters and wherein the heterocyclic base has been mono-halogenated (eg. iodinated after Robbins, supra).
  • Said halogenated nucleoside is coupled to an appropriate, tetra- hydropyranyl acetal (THP) protected hydroxyalkyne, such as 2-(3- butynloxy)tetrahydro-2H-pyran.
  • THP protected hydroxyal- kynes can be suitably synthesized by known methods from 5-hexyn- 1-ol, among others.
  • Said coupling is done by combining said halogenated nucleo ⁇ side (2-3 gm), with said THP protected hydroxyalkyne (500-700 mg), in 160 ml of deoxygenated triethylamine, with addition of appropriate amounts (50-70 mg) of bis(triphenylphosphine)palladium chloride, herein called (Ph3P)2PdCl2 , and Cul.
  • the mixture is stirred at 55 °C under N2 for 4 Hr or until coupling is completed.
  • a glass lined pressure bomb in a heated oil bath is used.
  • the product a THP-hydroxyalkyne nucleoside
  • a THP-hydroxyalkyne nucleoside is collected as a yel ⁇ low oil by evaporation, dissolved in chloroform, and washed with 5% disodium EDTA/H2O.
  • the product is recrystal1ized by redis- solving in chloroform, precipitating with methanol, collected by filtration and drying.
  • THP-hydroxyalkyne nucleoside is converted to a triphos- phate u ⁇ ing known method ⁇ such as those described by Tener, G.M., J. Am. Chem. Soc. 83, 159-168 (1961), and Hoad, D.E., et al , J. Am. Chem. Soc. 87, 1785-1788 (1965), to produce the THP- hydroxyalkyne nucleoside triphosphate.
  • the THP protective group is removed to expose (deprotect), the alkyne hydroxyl for coupling to the labels of the instant invention.
  • Said THP on said THP-hydroxyalkyne nuc ⁇ leoside triphosphate (2-3 gm) is removed by acid catalysis in a solution of 30 ml of CH2CI2/methanol/CF3CO2H (15:10:5), at 25 °C for 0.5-1 Hr, and recrystal1 ized as above.
  • Said nucleoside with added alkyne hydroxyl is suitably coupled directly to a label of the instant invention through a suitable amino group provided on the label as described below.
  • the hydroxyl can be converted to a hemisuccinate using succinic anhydride as described by Steiner, A.L., et al , Proc. Nat. Acad. Sci . U.S.A. 64, 367-373 (1969), among others.
  • sebacoyl dichlorides can be used as disclosed by Bailey, J.M., et al , IN: "The Reticuloendothelial System and Atheros ⁇ clerosis", Diluzio, N.R., et al , eds., Plenum Press, NY, (1967).
  • a variety of coupling agents that will crosslink said hydroxyl with an amino group, such as epoxys eg.
  • deprotected alkyne hydroxyl is suitably sulfonylated by reacting with an appropriate sulfonylating reagent ⁇ uch as tosyl chlor ⁇ ide.
  • an appropriate sulfonylating reagent ⁇ uch as tosyl chlor ⁇ ide.
  • 200-300 mg of said nucleoside triphosphate with deprotected alkyne hydroxyl is combined with 200-300 mg of p-toluenesulfonyl chloride in 20 ml of pyridine and stirred at 25 °C for 18 Hr, evaporated and recrystal!ized.
  • the product, tosylated-hy roxyalkyne nucleoside triphosphate can then be coupled to a label of the instant invention.
  • Said tosylated-hydroxyalkyne nucleoside can be further deriv ⁇ atized through well known methods to replace the tosyl ted hydroxyl with an amino group or a sulfhydryl group, among others, and subsequently coupled to a suitably derivatized label of this invention.
  • said amino-de ⁇ vatized nucleo ⁇ side is readily coupled to the N-hydroxysuccinimide derivatized labels described below.
  • said sulfhydryl-derivatized nucleo ⁇ side is readily coupled to the maleimido-derivatized or sulfhydryl-derivatized labels described below.
  • Said coupling can also be done through many well known coupling agents previ ⁇ ously described.
  • the p-toluyl esters are removed to deprotect the corresponding hydroxyls by treatment with .1 N sodium methoxide in anhydrous methanol at 25 °C for 5-6 hrs.
  • Any suitable nucleotide, with appropriately protected hydroxyls and other groups as needed, is treated with mercuric acetate by the method of Dale, R.M.K., et al , Proc. Nat. Acad. Sci . USA 70, 2238-2242 (1973), to produce the corresponding 5- mercuriacetate or 7-mercuriacetate derivative.
  • Said mercuriacetate derivative is suitably treated with thiocyanogen (prepared by combining bromine with Pb(SCN)2 in appropriate solvent), to convert said mercuriacetate derivat ⁇ ive to a thiocyanate derivative.
  • thiocyanogen prepared by combining bromine with Pb(SCN)2 in appropriate solvent
  • Said thiocyanate derivative is then converted to an S- alkyl thiocarbamate (amino) derivative by hydrolysis, or to an S-alkyl-N-alkylamino derivative by treatment with a suitable diamino substance, described below.
  • said thiocyanate derivative is treated with chlorine to give the sulfonyl chloride derivative, which can be further derivatized as previously described.
  • the purpose is to provide a cyclodextrin label that can be readily coupled to a variety of nucleotides, ligands, ligators, antibodie ⁇ , nucleic acid ⁇ , primers and chain terminators. Improvements provide for coupling a plurality of cyclodextrin labels in numbers that are greater than the number of available sites on the labeled molecule*..
  • the general approach is; (1) to produce or modify, if needed, one or more functional groups on the outside of said cyclodextrin molecule, (2) combine under appropriate conditions, said cyclodextrin and a CL substance, scintillator or fluorophore to synthesize said inclusion complex, or a suit ⁇ able chemical substrate to produce a detectable signal.
  • cyclodextrins are carbohydrates, they can be suitably derivatized and coupled through well known procedures used for other carbohydrates, especially through available hydroxyl groups. For instance, vicinal hydroxyl groups on the cyclodex ⁇ trin can be appropriately oxidized to produce aldehydes. In addition, any functional group can be suitably added through well known methods while preserving the cyclodextrin structure and complexing properties.
  • Examples are: esterification, acyla- tion, oxidation, halogenation, hydrolysis, reactions with hydrazines and other amines, including the formation of osazone ⁇ , acetals, aldehydes, amides, imides, carbonyls, esters, isopropyl idenes, sulfonates, sulfonyls, sulfonamides, nitrates, carbonates, metal salts, hydrazones, glycosones, mercaptals, and suitable combinations of these.
  • Said functional groups are then available for the coupling of one or more cyclodextrin molecules to a bifunctiona! reagent and/or to an appropriate ligand, lig ⁇ ator or nucleic acid. Said coupling can be done before forming said complex or afterward.
  • the purpose is to provide new CD derivatives and labels that are; (1) water soluble, (2) form complexes with CL substances, scinti1lators, fluorophores or substrates, (3) can be readily coupled to the desired substance, and (4) have the needed "spacer arm” (eg. O'Carra, supra), to overcome steric inter ⁇ ference after coupling to the labeled substance.
  • the spacer arm is of 4 or more carbon atoms in length and can include aliphatic, aromatic and heterocyclic structures.
  • Suitable coupling agents for preparing CD labels of the instant invention can be a variety of reagents previously descr ⁇ ibed, including well known crosslinkers such as epichlorohydrin, isocyanates, and formaldehyde, used to polymerize CD's.
  • crosslinkers such as epichlorohydrin, isocyanates, and formaldehyde, used to polymerize CD's.
  • Other suitable crosslinkers are various epoxy compounds including propylene oxide, 1 ,2-diethoxyethane, 1 ,2,7,8-diepoxyoctane, 2,3- epoxy-1-propanol (glycidol), 2,3-epoxy-1 ,4-butanediol diglycidy! ether, (eg. Gramera, or Case, or Johnson, or Parmerter, supra).
  • Derivatizing is defined as the chemical modification of a CD through addition of any functional group and/or other substance.
  • Capping is defined herein as coupling any suitable chemical sub ⁇ stance to two or more sites on the CD molecule so that said sub ⁇ stance spans the area between the coupled sites.
  • the CD's used herein can be suitably complexed with one or more guest molecules and/or derivatized and/or capped before or after their incorporation into the labels of the instant inven ⁇ tion.
  • said derivatizing and/or capping can be a done to produce CD's with the desired substances coupled to specific locations on the CD molecule.
  • scint 1lators or fluorophores modifications that increase affinity between the host CD and guest(s) are preferred.
  • the host CD's of this invention are preferably derivatized (eg. methylated), and/or capped by various means to increase host-guest affinity, provided that suitable functional groups are left for coupling to the substances to be labeled.
  • said capping substance is coupled between ⁇ ite ⁇ at the primary or secondary "end" of the CD molecule, forming a bridge across either (or both) opening(s), that includes ⁇ uit- able hydrophobic groups in said capping substance.
  • Said capping substances can be coupled directly to available hydroxyls on the CD, or they can be coupled to suitable functional groups such as; diamino compounds to iodinated CD, or azido compounds to sulfonylated hydroxyls, and/or through "spacer arms" added to the CD.
  • Suitable capping substance ⁇ are 6-methylamino-deoxy and 6-methylamino-6-deoxy derivatives transformed to the correspond ⁇ ing N-formyl compounds, imidazoles, m,m'-benzophenone-disulfonyl chloride, p,p'-stilbene-disulfonyl chloride, diphenylmethane- p,p'-di ⁇ ulfonyl chloride, terephthaloyl chloride, dianhydrides such as 3,3' ,4,4'-benzophenonetetracarboxylic dianhydride and 3,4,9, 10-perylenetetracarboxylic anhydride, amino compounds such as bismark brown, N,N'-bis(3-aminophenyl-3,4,9, 10-perylenetetra- carboxylic diimide, 1 ,4-bis(3-aminopropyl )piperizine, direct yellow, azido compounds such as 2,6-bis(4-azid
  • Primary and/or secondary hydroxyl groups can be selectively protected and deprotected using known methods during derivatizing and/or capping procedures, to provide selec ⁇ tive coupling at the primary or secondary end of the CD molec ⁇ ule, as desired. For instance, formation of protective esters (eg. benzoates using benzoyl chloride), and selective cleavage (deprotection) , of primary ' esters using anhydrous alcoholysis (eg. Boyer, supra), provides mostly primary hydroxyls for deriv- atization. After derivatization and/or coupling the primary hydroxyls, the secondary hydroxyls can be deprotected for addi ⁇ tional derivatization, coupling and/or capping.
  • protective esters eg. benzoates using benzoyl chloride
  • selective cleavage deprotection
  • secondary hydroxyls can be deprotected for addi ⁇ tional derivatization, coupling and/or capping.
  • the substance to be labeled can be a suitable protein, including antibodies and avidins or streptavidin, or ligands, or nucleic acids.
  • the substance to be labeled is an aminc-derivatized primer or chain terminator previously descr ⁇ ibed.
  • CD sulfonylation of CD (or CD polymer) before or after protection of specific hydroxyl groups
  • a suitable sulfonylating reagent such as p-toluenesulfonyl (tosyl) chloride, mesitylenesul- fonyl chloride or naphthalenesulfonyl chloride, among others, in anhydrous pyridine, for 3-5 Hrs at room temperature (RT).
  • a suitable sulfonylating reagent such as p-toluenesulfonyl (tosyl) chloride, mesitylenesul- fonyl chloride or naphthalenesulfonyl chloride, among others, in anhydrous pyridine, for 3-5 Hrs at room temperature (RT).
  • Dialdehyde CD is prepared by oxidation of CD using known methods (eg. Royer or Kobayashii, supra), with sodium metaperiodate.
  • a more selective'procedure is to oxidize the CD with an oxidizing enzyme (eg. glucose oxidase), in ⁇ uitable buffer solution (eg. 0.2 M phosphate saline, pH 5-7).
  • Said ⁇ ulfonylated CD i ⁇ ⁇ uitably iodinated so that it will couple to primary amino groups, using known methods (eg. Ikeda or Iwakura, supra).
  • 10 gm of ⁇ ulfonylated CD is com ⁇ bined with 12 gm of Nal on 200 ml of methanol, and mix at 70 °C for 48-60 Hrs.
  • the iodinated CD product is collected by precip ⁇ itation with acetone and purified by column chromatography.
  • Said iodinated CD or said dial-CD is coupled through the amino group to a suitable amino-carboxylic acid to provide the desired length of spacer arm.
  • suitable amino-carboxylic acids are; 4-aminobutyric acid, 6-aminohexanoic acid, 7-aminoheptanoic acid, 8-aminocaprylic acid, 12-aminododecanoic acid, and other aliphatic, or aromatic, or heterocyclic carboxylic acids with an available amino group for coupling.
  • Coupling to said iodinated CD is done in a suitable solvent such as dimethylformamide (DMF), mixing 10 gm of iodinated CD with a molar excess of amino-carboxylic acid (eg. 10 gm of 6- aminohexanoic acid), at 100 °C for 24 Hrs.
  • a suitable solvent such as dimethylformamide (DMF)
  • amino-carboxylic acid eg. 10 gm of 6- aminohexanoic acid
  • the product, CD- carboxylic acid is concentrated and purified by column chroma ⁇ tography.
  • Coupling to said dial-CD is done by reductive alkylation.
  • a suitable buffer eg. 0.1 M borate, pH 7.5-8.5, 0.1-0.5 M triethanolamine
  • 10 gm of dial CD is mixed with a molar excess of amino-carboxylic acid (eg. 10 gm of 12-aminodecanoic acid), at RT for 1-2 Hrs.
  • the Schiff's base coupling is stabilized by- suitable reduction with NaBH4 (eg. 0.1-1 mg/ml ) , for 1-12 Hrs.
  • the product, CD-carboxylic acid is concentrated and purified by column chromatography and dried for subsequent reactions a ⁇ needed.
  • D Preparation of NHS-CD Derivatives.
  • said CD- carboxyl ic acid is combined with N-hydroxysuccinimide and an aromatic carbodiimide such as N,N-dicyclohexylcarbodiimide, at approximately equimolar ratios and reacted at RT for 1-3 Hrs.
  • N-hydroxysuccinimide cyclodextrin (NHS-CD) is separated in the filtrate from precipitated dicyclohexylurea, collected by evaporation and purified by chromatography.
  • a CD (or multiple CD, below), is suitably protected and/or deprotected as needed and a sulfonylated CD is prepared as des ⁇ cribed previously.
  • Amino groups can be introduced into CD by reaction of said sulfonylated CD with azide compounds including hydrazine, and 2,6-bis(4-azidobenzylidene)-4-methyleyelohexanone (eg. Ikeda, supra), or coupling to diamines as described by Kawaguchi , or Matsui , supra.
  • a "monoamino" CD wherein one amino group has been coupled, can be prepared through known methods, includ ⁇ ing limited or sterically determined monosulfonylation, and/or by specific protection and deprotection schemes.
  • Said sulfonylated CD is suitably iodinated as described prev ⁇ iously.
  • Said iodinated CD is coupled through an amino group to a suitable diamino substance.
  • suitable diamino substances are; 1 ,4-diaminobutane, 1 ,6-diaminohexane, 1 ,7-diaminoheptane, 1,8- diaminooctane, 1 , 10-diaminodecane, 1 , 12-diaminododecane, and other aliphatic, or aromatic, or heterocyclic carboxylic acids with two available amino groups for coupling.
  • Coupling is done in a suitable solvent such as dimethylformamide (DMF), mixing 10 gm of iodinated CD with a molar excess of the diamino substance (eg. 10-20 gm of 1 ,6-diaminohexane), at 100 °C for 24 Hrs.
  • a suitable solvent such as dimethylformamide (DMF)
  • mixing 10 gm of iodinated CD with a molar excess of the diamino substance eg. 10-20 gm of 1 ,6-diaminohexane
  • the product, amino-CD is concentrated and purified by column chro ⁇ matography.
  • Said amino groups introduced by various methods can be suit ⁇ ably protected by reaction with a halogenated alkylphthalimide such as N-(4-bromobutyl )phthal imide.
  • a halogenated alkylphthalimide such as N-(4-bromobutyl )phthal imide.
  • an amino group is deprotected by reaction with hydrazine in suitable solvent.
  • Al ⁇ o said diamino sub ⁇ tances of various chain lengths car. be suitably derivatized before coupling. For instance, they can be "half protected*' * as trif1uoroacetamidoalkanes at one of the amino ends, as described by Guilford, H., et a! , Biochem. Soc. Trans.
  • NHS-CD derivative ⁇ can be prepared by coupling NHS esters directly to said amino-CD's.
  • said NHS ester i ⁇ a bifunctional NHS coupling agent with a ⁇ uitable spacer arm.
  • NHS esters of iodoacids can be coupled to said amino-CD's.
  • Suitable iodoacids for use in this invention are iodopropionic acid, iodobutyric acid, iodohexanoic acid, iodohippuric acid, 3-iodotyrosine, among others.
  • the appropriate NHS ester is prepared by known methods (eg. Rector, supra).
  • iodopropionic acid and N- hydroxy ⁇ uccinimide are mixed in anhydrou ⁇ dioxane at RT for 1-2 Hrs, the precipitate removed by filtration, and the NHS iodopropionic acid ester is collected in the filtrate. Said NHS iodopropionic acid ester is then coupled to the amino-CD.
  • NHS coupling agents for use in this invention have been previously described, including DSS, bis(sulfosuccinimidyl )suberate (BS 3 ), DSP, DTSSP, SPDP, BSOCOES, DSAH, DST, and EGS, among others.
  • a sulfhydryl group is added to said amino-CD, suitably prepared as described previously, by coupling the appropriate thiolating agent to the available amino group.
  • thiolation of available amino groups can be done by known meth ⁇ ods using S-acetylmercaptosuccinic anhydride (SAMSA), (eg. Klotz, Rector, or Lui , supra), SIAB, or 2-iminothiolane (eg. Traut, supra).
  • SAMSA S-acetylmercaptosuccinic anhydride
  • SIAB 2-iminothiolane
  • Sulfhydryl ⁇ can al ⁇ o be introduced through reaction of avail ⁇ able hydroxyls with a suitable epoxy compound.
  • a suitable epoxy compound For instance, epichlorohydrin or another suitable epoxy cross! inker previously- described, is coupled to CD (preferably immobilized by a cleav- able coupling agent), wherein free epoxy groups are produced. Said free epoxy groups are then reacted with sodium thiosulfate to produce thiosulphate e ⁇ ters (eg. Carlsson, supra). Said thiosulphate esters are subsequently reduced to sulfhydryls with dithiothreitol .
  • Sulfhydryls can be used for disulfide coupling to other available sulfhydryls on the desired substance to be labeled such as antibodies, or avidins, or streptavidin, or ligands, or nucleic acids, or primers, or chain terminators.
  • Said available ⁇ ulfhydryls may be native, or introduced by thiolation of said substance before coupling.
  • said sulfhydryl cont ⁇ aining CD label is coupled to any maleimide derivative of pro ⁇ tein, nucleic acid or biotin, (eg. biotin-maleimide) or iodoacetyl derivatives such as N-iodoacetyl-N'- biotinylhexylenediamine.
  • maleimido-cyclodextrin (mal-CD), derivative of this invention is suitable for coupling to native or introduced sulf ⁇ hydryls on the desired substance to be labeled in a single step.
  • a maleimido group is added to the amino-CD, ⁇ uitably prepared as described previously, by coupling a suitable hetero- bifunc onal coupling agent to the available amino group.
  • Said hetero-Difunctional coupling agent consists of a suitable spacer arm with a maleimide group at one end and an NHS ester at the other end. Examples are previously described and include MBS, SMCC, SMPB, SPDP, among others.
  • the reaction is carried out so that the NHS ester couples to the available amino group on the CD, leaving the maleimide group free for subsequent coupling to an available sulfhydryl on the substance to be labeled.
  • Said -substance to be labeled can be a protein such as an antibody, or avidin, or streptavidin, or lig ⁇ ands, or nucleic acids, or primers, or chain terminators suit ⁇ ably derivatized with a sulfhydryl group.
  • Biotinylated CD i ⁇ produced by combining said amino-CD deriv ⁇ ative with a known N-hydroxysuccinimide derivative of biotin in appropriate buffer such as 0.1 M phosphate, pH 8.0, reacting for up to 1 hour at room temperature.
  • suitable buffer such as 0.1 M phosphate, pH 8.0
  • biotin derivat- ive ⁇ that can be u ⁇ ed are, biotin-N-hydroxysuccinimid ⁇ , biotin- amidocaproate N-hydroxysuccinimide ester or sulfosuccinimidyl 2- (biotinamino)ethyl-l ,3'-dithiopropionate, among others.
  • Said biotinylated CD can be used to couple a plurality of CD labels to any biotinylated compound. For instance, by combining dilute solutions of said biotinylated CD with avidin or strep ⁇ tavidin in the appropriate molar ratio, 1, 2 or 3 biotinylated CD molecules will couple to the avidin or streptavidin and pro ⁇ quiz a complex with one or more biotin-binding sites still available.
  • biotinylated com ⁇ pound to be labeled, such as antibody, or avidins, or streptav ⁇ idin, or ligands, or nucleic acids, or primers, or chain terminators, and allowed to couple through the remaining biotin- binding site.
  • a photochemical CD label is a CD label (including multiple CD labels, below), that pos ⁇ esses a photoreactive coupling group in its composition, for coupling said label to any suitable subs ⁇ tance.
  • the photoreactive group is an aryl azide which upon exposure to light, generates a highly reactive nitrene coupling group.
  • Photo-CD labels can be synthesized by several methods.
  • said amino-CD derivatives previously described can be derivatized with a suitable bifunctional coupling agent that will couple to amino groups at one end and provide a photo ⁇ reactive group at the other end.
  • a suitable bifunctional coupling agent that will couple to amino groups at one end and provide a photo ⁇ reactive group at the other end.
  • Some examples are NHS-ASA, ANB-NOS, APG, EADB, HSAB, MABI, SANPAH, SADP, SAND, Sulfo-SADP, Sulfo-SANPAH, and SASD, (all available from Pierce Chemical Co., IL.), including carbenes and nitrenes disclosed by Knowles, J.R., Ace. Chem. Res. 5, 155-160, (1972), among others.
  • a suitable procedure is to combine, in a dark environment, said amino-CD (0.5-1.0 gm) in phosphate buffered saline (PB), pH 8.5, or a suitable anhydrous solvent such as N,N dimethyl- formamide (DMF), with NHS-ASA (1-2 gm), and let react 1 to 2 hours at 0 °C.
  • the photo-CD label product is purified by column chromatography, dried, and stored in the dark until used for coupling. Coupling is initiated by mixing the photo-CD label with the substance to be labeled, in suitable solvent, and irradiating with u.v. light for 5-20 minutes at RT to induce cross!inking.
  • said sulfhydryl derivatized CD labels can be derivatized with a suitable bifunctional coupling agent that will couple to sulfhydryl groups at one end and pro ⁇ vide a photoreactive group at the other end.
  • a suitable bifunctional coupling agent that will couple to sulfhydryl groups at one end and pro ⁇ vide a photoreactive group at the other end.
  • a multiple CD label is defined herein as a CD label wherein a plurality of CD's are coupled together.
  • Said multiple CD ( [CDjn , wherein n 2 or more), labels of this invention over ⁇ come the problem of labeling a substance with a plurality of CD molecules that are greater than the number of coupling sites available.
  • These labels allow the covalent coupling of a plura ⁇ lity of CD molecules to proteins such as antibody, avidin or streptavidin, or ligands, antigens, or nucleic acids, or primers, or chain terminators, and other substances, such as magnetic particles, through the appropriate functional groups.
  • One composition comprises two or more cyclodextrin labels coupled to an appropriate intermediate compound so that at least one functional group is left available for subsequent coupling to the substance to be labeled.
  • Said functional group is coupled directly to the substance to be labeled or, derivatized with any suitable coupling agent such as succinimidyl, maleim- idyl, imidoester, aldehyde, or photoactive agents including nitrenes, among others previously described.
  • Said intermediate compound can be any of the intermediate coupling substances previously described including said hydroxy- lated compounds, carbohydrates, sulfur containing and amino con ⁇ taining compounds.
  • compositions comprises a grouping of cyclodextrin mol ⁇ ecules coupled to each other through various known means to form a dimer, trimer or polymer of cyclodextrin molecules.
  • Said grouping of CD's al ⁇ o includes suitable functional groups for derivatizing and/or coupling to (labeling), the desired subs ⁇ tance.
  • said CD grouping can be coupled to an intermediate substance that includes a suitable functional group or coupling agent to facilitate said labeling.
  • said functional group or agent i ⁇ appropriately protected, if nece ⁇ ary, during coupling of ⁇ aid CD grouping to ⁇ aid intermediate compound. Said func ⁇ tional group i ⁇ then de-protected for coupling and/or derivat ⁇ ization.
  • a label produced by the following method employs an esterified hydroxylated compound as an intermediate coupling compound.
  • Hydroxylated compounds such as oligosacharrides, celluloses, dextrans, polysaccharides, polysorbates, hyaluromc acids, heparins, polyene antibiotics, polyvinyl alcohols (eg. Szejtli, supra), oligonucleotides, protein ⁇ , polypeptide ⁇ , and polyaminoacids, among others, are suitable wherein a carboxylic acid group is available. If necessary, said carboxylic acid group can be added by known methods. Hydroxylated carboxylic acids such as cholic acid, gallic acid, digallic acid, citrazinic acid, fluorescin, or polytyrosine, including their variou ⁇ derivative ⁇ , can also be used.
  • the desired carboxylic acid group of said hydroxylated compound is suitably protected by known methods.
  • one or more carboxy ⁇ lic acid groups can be reacted with a suitable alcohol to form a protective ester.
  • a suitable alcohol for example, a benzyl alcohol is used to pro ⁇ prise a hydroxylated benzyl ester.
  • Cyclodextrin which can be suitably complexed, derivatized and/or capped before coupling, is combined with said hydroxy ⁇ lated protected ester and a suitable coupling agent.
  • coupling is done with epichlorohydrin, or an epoxy compound pre ⁇ viously described, in suitable solvent, and mixed under suitable conditions until coupling is completed between a plurality of said CD and the available hydroxyls on the protected ester.
  • the product a plurality of CD molecules coupled to said hydroxylated ester (ester-[CD]n ) , is appropriately treated if necessary to protect any remaining uncoupled hydroxyl groups on the CD molecules.
  • Said ester-[CD] n is suitably hydrolyzed (eg. hydrogen bromide in acetic acid), to cleave the benzyl ester and produce multiple CD's coupled to a carboxylic acid (acid-[CD]n ) .
  • Said acid-[CD] n is combined with N-hydroxysuccinimide in anhydrous dimethyl ⁇ formamide (DMF), and mixed with N,N'-dicylohexylcarbodiimide for 2 hours to overnight at RT. Dicylohexyl urea is removed by pre ⁇ cipitation after adding a few drops of glacial acetic acid.
  • hydroxylated compounds may be used.
  • flavone derivatives and analogs including dihydroxyflav- ones (chrysins), trihydroxyflavones (apigenins), pentahydroxy- flavone ⁇ (morins), hexahydroxyflavones ( yricetins) , flavyliums, quercetins, fisetins; various antibiotics including teramycins, tetracyclines, chlorotetracyclines, clomocyclines, guamecy- clines, amphotericins, filipins, fungichromins; various cevine derivatives and analogs including verdines, desatrines, vera- tridine; various sulfur and mercapto derivatives and analogs including dihydroxy-2-mercaptopyrimidines, ethylmercapto hydrox- ybenzoate ⁇ , 6-hydroxy-1 ,3-benzox
  • CD labels can be synthesized with a more predictable number, of CD molecules per label, giving new advantages of uniform structure and chemical proper ⁇ ties.
  • These CD labels also are suitable for coupling a plural ⁇ ity of CD molecules to a sub ⁇ tance in a single step.
  • the synthesis approach is to immobilize the initial CD molecule to a solid support, and then attach additional CD molecules to the first in a controlled, step-wise manner. After sufficient CD's have been linked together, the entire group is cleaved from the solid support fcr use as a [CD] n label.
  • each CD mol ⁇ ecule (or dimer, or trimer), that is coupled has a well defined structure to facilitate the production of CD labels with uniform and consistent properties.
  • Said solid support can be in the form of particles, beads, fibers, plates, and tubing walls, and composed of styrenes, acrylamides, silica gels, solid or porous glass, metals, dextrans, nylons, and celluloses, among others that are suitably derivatized as needed and compatible with the reactions used.
  • the coupling agent used to couple the initial CD to the sup ⁇ port is preferably one that is readily cleaved when desired, and the coupling agent used to couple subsequent CD's is preferably noncleavable.
  • the initial coupling agent is also a bifunctional reagent such as those with a cleavable disulfide group, including DTBP, DSP, DTSSP, EADB, SPDP, and photoactive couplers like DTBPA, SADP, SAND, and SASD.
  • Other suitable agents are periodate cleavable, such as DST and sulfo-DST, and hydroxylamine cleavable at the ethyl ester linkage, such as EGS and sulfo-EGS.
  • the label is cleaved from the sup ⁇ port after synthesis, leaving a suitable functional group for subsequent coupling.
  • the remaining functional group i ⁇ converted to an NHS ester by various known means for coupling to an amino group on the substance to be labeled.
  • the noncleavable coupling agent used can be from a variety of reagents previously described, including well known crosslinkers used to polymerize CD's.
  • a highly porous support eg. porous glass beads or predried silica gel, 5-6 gm
  • a highly porous support is amino-derivatized with a .0% (v/v), solution of (3-aminopropyl )trimethoxysi lane in toluene (150 ml) at 150 °C for 6 Hrs and suitably washed with toluene, then acetone, then methanol, and dried.
  • a suitable diamino substance such as 1 ,12-diaminododecane is coupled to said amino-derivatized support through a suitable cleavable disulfide coupling agent such as DSP.
  • the solvents used can be anhydrous such as methanol, methylene chloride, or pyridine or they can be suitable aqueous buffer solutions, as conditions require.
  • Excess DSP in pyr dine is added to said amino-derivatized support and allowed to couple, after which uncoupled DSP is removed.
  • Excess 1 ,12-diaminododecane is added to the support and allowed to couple to the previously coupled DSP, after which excess 1 ,12-diaminododecane is removed, giving 1,12- diaminododecane-coupled support.
  • Said 1 ,12-diaminododecane-coupled support is then com ⁇ bined with excess DSS to allow coupling of the DSS to the coupled 1 ,12-diaminododecane.
  • the excess DSS is removed, giving DSS-activated 1 , 12-diaminododecane coupled to the support.
  • CD amino derivatized
  • said amino derivatized CD is a mono-amino preparation with a suitable spacer arm, wherein all of the CD molecules have the amino group on the same (eg. primary), side. Said CD molecules are allowed to couple and the excess is removed, leaving CD's coupled to the 1 ,2-diaminododecane through the DSS coupling agent.
  • Said coupled CD's are then suitably oxidized (eg. sodium periodate or oxidase enzyme), to produce dialdehydes on the coupled CD's (dial-CD).
  • dial-CD is then added excess, amino-derivatized CD so that coupling occurs between the dialdehyde of the previously coupled CD and the amino group of said amino-derivatized CD's added, and excess CD is removed.
  • the dialdehyde coupling procedure of this step is repeated until the desired number of CD's have been coupled.
  • the [CD]n label composition is recovered from the support by cleaving the initial coupling agent used.
  • the cleavable disulfide is treated with a suitable reducing agent such as dithiothreitol, among others.
  • a suitable reducing agent such as dithiothreitol, among others.
  • the Schiff bases are suitably reduced (eg. NaBH.t ) .
  • the released CD label will have a sulfhydryl group available for subsequent coupling to any suitable substance.
  • said sulfhydryl is converted to an NHS ester by coupling it to a heterobifunctional reagent with maleimide-NHS groups at opposite ends.
  • n a multiple of 1 or more
  • the gen ⁇ eral structure- is: i CD ! n -SPACER-NHS D. Multiple CD Label with Intermediate Substance.
  • the CD molecules are coupled to the same intermediate compound.
  • suitable derivatiza ⁇ tion a wide variety of substances are suitable as intermedi ⁇ ates.
  • CHAPS is reduced with dithiothreitol to convert the sulfonate group to a sulfhydryl (eg. Carlsson, supra), to produce 3-[(3-cholamidopropyl )-dimethylammonio]-1- mercaptopropane (hereinafter CHAMP).
  • a suitable amino-derivatized support is prepared as des ⁇ cribed above. Said amino groups are then suitably thiolated to provide sulfhydryls (or 2-pyridyl disulphides using SPDP). Said CHAMP is coupled through its sulfhydryl to the available thiol or 2-pyridyl disulphide to form a cleavable disulfide linkage to the support, and produce a CHAMP coupled support.
  • Said CHAMP coupled support is treated with a suitable noncleavable, hydroxyl crosslinking agent such as epichlorohy ⁇ drin, among others, to give an epoxy-activated CHAMP, and exposed to an excess of CD molecules, that have been suitably derivatized as needed for coupling.
  • a suitable noncleavable, hydroxyl crosslinking agent such as epichlorohy ⁇ drin, among others.
  • the product is 1, 2, or 3 CD molecules coupled to said CHAMP through the hydroxyl groups.
  • Said product is suitably cleaved and recovered from the support by treating with a suitable reducing agent such as dithiothreitol, among others.
  • a suitable reducing agent such as dithiothreitol, among others.
  • the released CD label will have a sulfhydryl group available for subsequent coupling to any suit ⁇ able substance.
  • said sulfhydryl is converted to an NHS ester by coupling it to a heterobifunctional reagent with maleimide-NHS groups at opposite ends.
  • the CD label can be further modified wherein specific der ⁇ ivatization and/or capping reactions are performed while the disulfide group is protected.
  • the desired guest molecules can be included before or after coupling of the CD's.
  • cleavable groups eg. Ji , supra
  • other useful functional groups can be incorporated into the label that remain protected until cleaved. For instance, initial coupling through a cleavable ester will produce a carboxylic acid or hydroxyl group on the CD label after cleavage.
  • a variety of protecting and deprotecting schemes can be adapted to serve as temporary coupling sites on a solid support for synthesis of said CD labels. The major requirement is that subsequent reactions for coupling CD's do not cleave the label before synthesis is completed.
  • a suitable amino-derivatized support is prepared as described above. Said amino groups are then suit ⁇ ably coupled to a dianhyride such as 3,4,9,10- perylenetetracarboxylic dianhydride to form an imide.
  • a suitably protected amino-CD is coupled to the other end of the immobilized dianhydride.
  • the label is cleaved by treatment with hydrazine, leaving an amino group on the label.
  • this procedure can also be done on a hydroxylated support material.
  • n a multiple of 1 or more
  • the gen ⁇ eral structure is: lCDln !CD!n-INTERMEDIATE-NHS lCDln
  • label compositions allow labeling a substance with a plurality of CD's i ⁇ hat are greater than the number of coupling sites available.
  • the use of a carbohydrate provides new proper ⁇ ties for coupling a plurality of CD's to a variety of other sub ⁇ stances such as lectins, cell receptors, nucleic acids, proteins such as antibody, avidin or streptavidin, and other substances.
  • a useful label is produced by using one or more carbohydrates or saccharides as part of the label .
  • Said carbohydrate is any carbohydrate including oligosaccharides, mono-, di- and polysac- charides, amino sugars, sulfo-sugars, deoxysugars, glycosides, lectin-binding carbohydrates, aldoses, ketoses, pentoses, arab- inoses, riboses, xyloses, hexoses, glucoses, fructoses, galac- toses, mannoses, sorboses, glucosamines, sucroses, lactoses, maltoses, raffinoses, soluble starches, amylopectins, pectins, agars, agaroses, dextrans, celluloses, hyaluronic acids, hepa- rins, and any suitable polymers, derivatives and analogs of car ⁇ bohydrates.
  • One approach is to couple two or more CD molecules to said carbohydrate either directly or through an intermediate coupling agent.
  • the resulting label can then be bound noncovalently to the appropriate lectin or receptor that binds the carbohydrate on the label .
  • the approach is to couple two or more CD molecules to said carbohydrate either directly or through an intermediate so that at least one functional group is left available on the label.
  • Said functional group is then coupled to the substance to be labeled or, derivatized with any suitable coupling agent such as succinimidyl, maleimidyl, imidoester, aldehyde, or photoactive agents including nitrenes, among others previously described.
  • any suitable coupling agent such as succinimidyl, maleimidyl, imidoester, aldehyde, or photoactive agents including nitrenes, among others previously described.
  • said functional group is appropriately protected, if necessary, dur ⁇ ing coupling.
  • Said functional group is then de-protected for coupling and/or derivatization.
  • Another method for introducing said functional groups into said carbohydrate is to derivatized a small number of the exist ⁇ ing hydroxyl groups with a bifunctional coupling agent that will not react significantly during coupling of one or more CD's. A suitable number of hydroxyl groups are left for coupling said carbohydrate to said CD.
  • various amino groups that may be present on said carbohydrate can be derivatized using known methods to produce an appropriate pro ⁇ tecting group such as a benzyl ester.
  • said carbohydrate can have one or more functional groups preferentially derivatized.
  • pairs of hydroxyls are reacted with appropriate aldehydes or ketones to produce protective derivat ⁇ ives such as acetals (eg. isopropylidenes) , which can be subse ⁇ quently hydrolyzed.
  • protective derivat ⁇ ives such as acetals (eg. isopropylidenes) , which can be subse ⁇ quently hydrolyzed.
  • pairs of vicinal hydroxyls are oxidized to produce aldehyde groups. Said oxidation suitably is done with sodium periodate or with oxidiz ⁇ ing enzymes using known methods, so that sufficient hydroxyls are left un-oxidized for coupling with CD molecules.
  • PREPARATION X CD Labels with Different Colored Guests
  • a major advantage of the CD labels of the instant invention is the ease with which different guest molecules can be com- plexed with said CD labels to produce easily distinguishable labels.
  • the distinguishing feature is the different light emis ⁇ sion wavelengths (or peak emission, or "color”), obtained from different guest molecules when they are activated or electroni ⁇ cally excited. >_,
  • Said emission can be generated by electronically exciting the guest molecule through various means such as by chemical reac ⁇ tion (eg. energy transfer), by an incident light source (eg. fluorescence and phosphorescence), electrically (eg. electrolum ⁇ inescence), or heat (eg. thermoluminescence) . Also, through the choice of appropriate CL substance as a guest, various colors of chemiluminescent emission are possible.
  • chemical reac ⁇ tion eg. energy transfer
  • an incident light source eg. fluorescence and phosphorescence
  • electrically eg. electrolum ⁇ inescence
  • heat eg. thermoluminescence
  • said guest molecules are efficient emitters that form high affinity inclusion complexes with said CD label host, with a suitable shape and size compatible with the CD molecules used. It is possible to produce CD labels with guests that have a diversity of size and shape and color, yet are contained in the same sized CD host molecule. In applications where it is desirable to have several different colored guests that also have similar chemical and physical characteristics, the choice of guests can be derived from the same chemically related group.
  • any scint 11ator or fluorophore described prev ⁇ iously, or any aromatic nucleus including acridine, anthracene, benzene, biphenyl, biphenylene, fluorene, fluorescein, naphthacene, naphthalene, pentacene, pentalene, perylene, phenanthrene, among others, can have its emission wavelength altered or "shifted" by coupling or derivatizing with one or more specific groups.
  • Said wavelength-altering group can be any suitable substance including hydrogens, oxygens, nitrogens, sul- fur ⁇ , halogens, metals, methyls, ethyls, toluyls, and any suit ⁇ able functional group, among others.
  • Suitable guest molecules are; fluorescein dyes such as 9-(carboxyethyl )-3-hydroxy-6-oxo-6H-xanthenes . (eg. SF-505, SF-512, SF-519, and SF-526, of Prober, supra), acridines anthracenes, naphthalenes, and the fluorophores previously des ⁇ cribed, among others.
  • fluorescein dyes such as 9-(carboxyethyl )-3-hydroxy-6-oxo-6H-xanthenes . (eg. SF-505, SF-512, SF-519, and SF-526, of Prober, supra), acridines anthracenes, naphthalenes, and the fluorophores previously des ⁇ cribed, among others.
  • fluorescein dyes such as 9-(carboxyethyl )-3-hydroxy-6-oxo-6H-xanthenes . (eg.
  • a CD label with a "captured guest” stabilizes the label complex and overcomes the problem of the CD host and guest molecule separating under various conditions.
  • a captured guest is any (one or more), guest molecules that are captured by, or coupled in close proximity to, the CD host so that they cannot separate by the normal pro ⁇ Des of diffusion. Said capturing is accomplished through physical entrapment by the CD host, or by covalent coupling of the guest in the immediate vicinity of the CD host.
  • physical entrapment can be done by capping both ends of the host CD, thereby entrapping the guest molecule.
  • Another useful method is to couple two host CD's together so that said guest molecule is entrapped between them (eg. "duplex cyclodextrin" of Tabushi, supra), and wherein the outside ends are too small and/or are capped to prevent escape of the guest.
  • Said guest is captured by covalent coupling when the guest is coupled by various suitable means to the CD host or to an inter ⁇ mediate substance in close proximity. Said coupling is done after the guest enters the host CD. Or, said guest can be coupled through a suitable spacer arm of sufficient length (eg. 6 or more carbons), to allow the guest to enter the host CD after coupling (eg. Ueno, supra).
  • guest molecule to use will depend on the color desired for said label, fluorescent and/or excitation efficiency, energy transfer efficiency, and compatibility with its intended use.
  • the desired guest molecule eg. scintillator or fluorophore or CL compound
  • suitable guest molecules are; carboxylic fluorescein dyes such as 9- (carboxyethyl )-3-hydroxy-6-oxo-6H-xanthenes (eg.
  • the desired guest molecule can be coupled through an available amino group, such as with acridine yellow, acrifla- vines, aminoanthracenes, 9-(methylaminomethyl )anthracenes, aminofluoranthenes, aminofluorenes, aminofluorenones, aminopyrenes, and others, using appropriate bifunctional coupl ⁇ ing agents, or a carbodiimide method.
  • the desired guest molec ⁇ ule can also be * coupled through an available sulfhydryl (eg.
  • sulfonates 8-anili-1- naphthalene sulfonates (ANS), 2-p-toluidinylnaphthalene sulfo ⁇ nates (TNS), among others.
  • ANS 8-anili-1- naphthalene sulfonates
  • TMS 2-p-toluidinylnaphthalene sulfo ⁇ nates
  • a variety of previously described coupling agents can be used for coupling through an available carboxyl , amino or sulfhydryl on the host CD.
  • said carboxylic acid or amino group can be introduced onto the guest through suitable derivatization, as with europium chelates and ruthenium chelates (eg. dipyridines of Ege, supra) .
  • said host CD can be suitably capped if desired, to increase the affinity between the coupled guest and CD host.
  • a suitable scheme for synthesizing guest-CD labels of the instant invention is as follows;
  • amino-CD's are prepared wherein a suitable amino group is preferably coupled to one end.
  • a suitable amino group is preferably coupled to one end.
  • CD hydroxyls are protected with benzoate esters, the primary end is selectively deprotected and amino derivatized (eg. Szejtli or Boyer, supra).
  • a suitable amino-deri atized solid support is prepared as previously described for the preparation of immobilized [CD]n labels, above.
  • To said support is coupled an intermediate com ⁇ pound through a cleavable disulfide as previously described, and activated, such as DSS-activated 1 ,12-diami ododecane or epoxy- activated CHAMP.
  • one or more of said amino-CD's is coupled to said activated intermediate on the support through the available CD amino group.
  • Remaining hydroxyl groups on the resulting immobi ⁇ lized CD's are deprotected as needed and amino-derivatized with a suitable spacer arm.
  • said hydroxyls can be oxidized to dialdehydes, or treated with epichlorohydrin, and coupled to diaminohexane.
  • treated with acetic or succinic anhydride to give carboxy- late ⁇ that are converted to NHS esters through reaction with carbodiimides and N-hydroxysuccinimide, and then coupled to diaminohexane.
  • said immobilized CD's are prepared with one or more amino groups available, attached through suitable spacer arms, to the CD's. Said immobilized CD's can now be coupled to a variety of fluorophores (or CL compounds), using any suitable coupling agent for coupling to amino groups.
  • Coupling to fluorophores with available carboxylic acid groups such as carboxylic fluorescein dyes such as SF-505, SF- 512, SF-519, and SF-526, (see Prober, supra), 9- acridinecarboxylic acid, 1-fluorenecarboxylic acid, indoleacetic acid, 10-methylanthracene-9-carboxaldehydes, mordant oranges, mordant reds, mordant yellows, naphthylacetic acids, N-(4- nitrobenzoyl )-6-aminocaproic acid, phenolphthalein carbinol dis- ulfate, protoporphyrins, 1-pyrenebutyric acid, quinolinecar- boxylic acid, retinoic acid, rhodamine B's, among others, is done by converting an available carboxylate on said fluorophore to an NHS ester through reaction with a carbodiimide and N- hydroxysuccin
  • Coupling to fluorophores with sulfhydryls may require that potential sulfhydryls are made available by reduction of sulfo ⁇ nates on the fluorophore using suitable reducing agents (eg. Carlsson, supra).
  • suitable reducing agents eg. Carlsson, supra.
  • fluorophores with reducable sulf ⁇ onates are 8-an lo-1-naphthalene sulfonate, 2-p- toluidinylnaphthalene-6-sulfonate, acid blacks, acid blues, acid greens, acid reds, acid violets, acid yellows, alizarins, direct blues, direct reds, texas reds, and others.
  • coupling to said immobilized CD is done by combining said immobilized CD with any suitable hetero- bifunctional , ,amino-sulfhydryl-coupling agent previously descr ⁇ ibed, such as MBS, SIAB, among others, and said fluorophore, under suitable conditions for coupling.
  • any suitable hetero- bifunctional , ,amino-sulfhydryl-coupling agent previously descr ⁇ ibed such as MBS, SIAB, among others, and said fluorophore, under suitable conditions for coupling.
  • Photoreactive Coupling is done by adding said staining fluorophore to said
  • Coupling of CD's to fluorophores can also be done by combin ⁇ ing in the dark, said immobilized CD with any suitable hetero- bifunctional , photoreactive-a ino-coupling agent previously des ⁇ cribed, such as HSAB, NHS-ASA, among others, under suitable con ⁇ ditions for coupling. Then, said fluorophore is added and the photoreactive group is activated with a suitable light source to initiate coupling.
  • any suitable hetero- bifunctional , photoreactive-a ino-coupling agent previously des ⁇ cribed, such as HSAB, NHS-ASA, among others, under suitable con ⁇ ditions for coupling.
  • n a multiple of 1 or more
  • the gen ⁇ eral structure is:
  • a new CD label composition with potentially greater luminesc ⁇ ent efficiency can be synthesized by coupling certain light and/or energy collecting substances (herein called “antenna” substances), to said CD labels.
  • Said antenna substances can be coupled to said label in various ways to promote the most effi ⁇ cient activity.
  • said antennas can be suitably coupled to the CD molecule, to the fluorophore guest, or to an intermediate substance that is part of the CD label.
  • suitable antenna substances are aromatic compounds, folic acids, carotenoids, retinols, retinals, rhodopsins, chlorophylls, blue fluorescent proteins (eg. from bacteria), green fluorescent proteins (eg. from renilla, etc.), tryptophan and/or tyrosine-containing substances (eg. polypeptides) , and various derivatives, analogs and precursors of said antenna sub ⁇ stances.
  • the entire group is cleaved from the solid support as described previously, providing the free label with a functional group for coupling to any suitable substance.
  • n a multiple of 1 or more
  • the gen ⁇ eral structure is:
  • the N-succinimidyl reactive group on NHS- [CD]n reacts with primary and secondary aliphatic amines on the protein.
  • a suitable protein such as antibody, or avidin (or streptavidin) is labeled by mixing 50 micrograms of protein in 0.2 ml of 0.1 M phosphate buffer (PB), pH 8, with approximately 10 micro!iters of acetonitrile contain ⁇ ing 2.5 micrograms of dissolved NHS-[CD]n , in a glass vial for 15 minutes..
  • PB phosphate buffer
  • the mixture is applied to a column of G25 equilibrated with PBS composed of PB, pH 6.3, containing 0.15 M NaCl and 0.02 sodium azide.
  • the column is eluted with PBS and labeled fractions in the void volume are pooled and concentrated if necessary, by vacuum dialysis.
  • the reaction conditions may be varied appropriately, such as the inclusion of an intermediate coupling substance, to obtain a labeled protein or nucleic acid that is suitably coupled to a plurality of NHS-[CD]n and sufficiently retains the binding pro ⁇ perties needed for use as a tracer.
  • Such conditions that may be appropriately varied are, amounts of reagents, times, tempera ⁇ ture and the use of other compatible buffers, solvents and addi ⁇ tives.
  • said NHS-[CD]n is substituted for one of said labels from previous preparations wherein said label contains an N-hydroxysuccinimidyl coupling group.
  • said NHS-[CD]n can be substituted for one of said labels from previous preparations wherein said label contains a coupling group or functional group that is not an N-hydroxysuccinimidyl coupling group.
  • conditions can be appropriately modified by one skilled in the art. PREPARATION XIII.
  • CD Labeled Nucleic Acid Probes Any known method for preparing DNA, oligonucleotide, or RNA probes may be used for synthesizing probes for coupling to the CD labels of the instant invention, provided the method is suit ⁇ ably modified when necessary, to use biotinylated nucleotides or allylamine derivatized nucleotides or some other nonradioactive label or ligand in place of the radioactive label.
  • a suitable DNA probe is prepared by cloning the desired DNA into M13mp8 bacteriophage DNA, using standard methods. Dur ⁇ ing synthesis of the complementary strand of the single stranded M13 vector, biotinylated nucleotides or allylamine derivatized nucleotides or some other nonradioactive label or ligand is incorporated into it.
  • the vector DNA can be left double stranded and suitably labeled with biotin, allylamine or appropriate ligands using well known methods including DNA intercalators, psoralens and crosslinkers, among others.
  • a suitable oligonucleotide probe is synthesized that is complementary to the nucleic acid sequence to be detected using standard methods.
  • the probe includes appropriate biotinylated, allylamine derivatized, or ligand coupled nucleotides.
  • a suitable RNA probe is prepared using standard methods such as transcription from the appropriate DNA template contain ⁇ ing appropriate vectors such as pSP and/or pT7 and/or pT3, among others.
  • suitable nucleotides for incorpora ⁇ tion are bio-11-UTP and/or allylamine UTP.
  • the probe is suitably biotinylated during or after synthesis using known methods and the necessary reagents for fragmentation, purifica ⁇ tion and biotinylation, which can be purchased as pre-packaged kits from several sources. Examples are, Enzo Biochemicals, NY, and Bethesda Research Laboratories, MD.
  • a photoactive coupler may also be desirable to use a photoactive coupler and introduce a "spacer arm", between the biotin or allylamine and th ⁇ nucleic acid sequence.
  • a spacer arm is desirable because it helps prevent binding interference due to steric hindrance.
  • the nick-translation procedure may be used.
  • Several commercial kits with reagents and instructions are also available for this procedure.
  • the nucleic acid sequence to be hybridized in the double stranded probe would have to be denatured to produce the single stranded form before use.
  • any desired CD label can be noncovalently coupled to it.
  • any CD that is coupled to avidin or streptavidin can be noncovalently coupled to the probe by allowing the coupled avidin or streptavidin to bind to the probe.
  • noncovalent coupl ⁇ ing is to incorporate or produce an antigen or hapten into the probe composition. Then, the appropriate, specific antibody will noncovalently couple to that antigen.
  • the probe can be sulf nated or have digoxin, digoxigenin, or another suitable antigen incorporated into the composition, and then allowed to couple to the appropriate labeled antibody. This way, any CD label that is coupled to an antibody, can be non ⁇ covalently cou ⁇ ,ed to a probe.
  • any CD label that can be coupled through said groups can be coupled to the probe using well known methods.
  • different colored probes can be prepared by labeling with different colored CD fluorophore labels, previously described.
  • Said test kit can contain one or more of the said preparations as needed, and other appropriate reagents and/or solutions for performing the intended assays, with suitable containers and instructions for storage and use.
  • Said preparations and reagents can be packaged in various forms including frozen and/or lyophilized.
  • the principle of this method is based on the use of labeled, dideoxyribonucleoside triphosphates (chain terminators), which cause termination of oligonucleotide synthesis when incorporated into the chain in place of normal nucleotides, (eg. Sanger, or Prober, supra).
  • chain terminators which cause termination of oligonucleotide synthesis when incorporated into the chain in place of normal nucleotides, (eg. Sanger, or Prober, supra).
  • the terminators compete with their normal nuc ⁇ leotide analogs and terminate the primer-initiated, template- dependent synthesis at specific positions corresponding to the substituted nucleotide. This produces labeled oligonucleotide fragments of various lengths which are subsequently separated by size and detected through the incorporated label.
  • CD labels are used wherein each chain terminator is coupled to a CD label, or [CD]n label, complexed with a different colored guest fluorophore or a different guest CL compound.
  • CD label or [CD]n label, complexed with a different colored guest fluorophore or a different guest CL compound.
  • This has the advantage of allowing the use of a wide range of guest molecules that are inside the CD host so that chemical and physical differences between CD labels are minimized.
  • inclusion of the fluorophore or CL compound by the CD label enhances the signal generating efficiency and detec-abi1ity of the label.
  • amino derivatives of the chain terminator triphos- phates are each coupled to different colored NHS-CD labels (suitably, CD labeled dideoxyuridine (ddUTP), can be used in RNA analysis).
  • Said NHS- CD labels can also be multiple CD labels.
  • said labeled chain terminators are prepared so that they meet the requirements of, (1) the fluorophore guests are suitably efficient emitters, (2) the emission spectra or colors, are easily distinguishable for each terminator, and (3) the label-s do not significantly impair sequencing reactions of the chain terminators.
  • nucleic acid template is suitably prepared by restriction enzyme frag ⁇ mentation and purification.
  • said nucleic acid templates and suitable primer are combined to form annealed template-primer.
  • Said annealed template-primer is then combined, in a reaction mixture in appropriate buffer, with suf ⁇ ficient amounts of the appropriate nucleotides and suitable synthesizing enzymes such as polymerases, transcriptases, and others, to promote oligonucleotide synthesis (primer extension).
  • the corresponding CD-labeled chain terminators are added and appropriately incubated to allow termination of synthesis with the formation of CD-labeled oligonucleotide fragments.
  • Said CD-labeled fragments are removed from unincorporated label and separated by size. Said labeled fragments are detected and discriminated by the color of light generated through activation of the CD label.
  • a section of M13mp18 DNA (3 micrograms), is annealed with 60 ng of 17 bp primer.
  • the annealed template- primer is combined with 250 picomoles each of deoxyadenosine triphosphate (dATP), deoxycytosine triphosphate (dCTP), deoxy- thymidine triphosphate (dTTP), and deoxyguanosine triphosphate (dGTP), or, 2'-deoxy-7-deazoguanosine triphosphate (c 7 dGTP), in place of dGTP (see Prober, supra).
  • dATP deoxyadenosine triphosphate
  • dCTP deoxycytosine triphosphate
  • dTTP deoxy- thymidine triphosphate
  • dGTP deoxyguanosine triphosphate
  • c 7 dGTP 2'-deoxy-7-deazoguanosine triphosphate
  • the buffer used is 60 mM tris HC1 , pH 8.3, 7.5 mM MgCl ⁇ , 75 mM NaCl , 0.5 M dithiothreitol and 17 units of AMV reverse transcriptase.
  • excess of each CD-labeled chain terminator 100-1000 picomoles
  • the resulting CD-labeled fragments are removed from unincorporated label by gel column chromatography and separated by size to determine the number of bases per fragment. Separa ⁇ tion is done using any suitable method such as polyacrylamide gel electrophoresis or high pressure liquid chromatography, (HPLC).
  • Said CD fluorophore labeled fragments are activated by any suitable energy transfer reaction described previously, such as combining them with a suitable peroxyoxalate and peroxide.
  • TCPO is added (eg. 600 mg/1 in 10 -3 M phosphate buffer, pH 6), and in a dark environment, the activation is started with the addition of H2O2 (eg. 10 _5 -10- 6 M).
  • H2O2 eg. 10 _5 -10- 6 M
  • Other sub ⁇ stances are suitably included in the buffer such as ethyl acetate, stabilizers and surfactants, as needed.
  • Said reagents can be combined with said CD labeled fragments to activate in said gel. Alternatively, fragments removed from the gel or HPLC column, are detected in suitable containers or a continuous flow detection system.
  • Detection is done in the dark with a photometer or other light detector that can discriminate between the emission wavelengths (eg. four colors), of the CD fluorophore labels used.
  • said light detector employs photomultipliers, or charge coupled devices (CCD), (eg. video camera), or photodi- ode ⁇ with appropriate filters and/or grids as needed.
  • CCD charge coupled devices
  • a scan ⁇ ning type of system eg. using fiber optics
  • the light emission data is collected in a com ⁇ puterized, automated system. Using known sequencing and statistical methods, it can then be determined which labeled nucleotide (by color), terminated synthesis at each base incre ⁇ ment (by fragment size), of the nucleic acid chain being ana ⁇ lyzed. METHOD II. Nucleic Acid Sequencing with CD Labeled Primers The principle of this method is similar to Method I, above, except that labeled oligonucleotide primers are used instead of labeled chain terminators (eg. Sanger or Smith, supra). In the method of -the instant invention, CD labels are used wherein each primer is coupled to a CD label, or [CD]n label, complexed with a different colored guest fluorophore or a different guest CL compound. ,
  • oligonucleotide primers are prepared, each labeled with a CD fluorophore- label complexed with a different colored guest molecule, as described previously.
  • Said labels can also include captured guests, and various derivatives and/or capping, as des ⁇ cribed previously.
  • an amino derivative of a primer eg. an oligonucleotide with a single aliphatic amino group at the 5' terminus
  • a primer eg. an oligonucleotide with a single aliphatic amino group at the 5' terminus
  • a NHS-CD label e.g. an oligonucleotide with a single aliphatic amino group at the 5' terminus
  • multiple NHS-CD label ed with a different colored guest fluorophore.
  • said labeled primers are prepared so that they meet the requirements of (1 ) the fluorophore guests are suitably efficient emitters, (2) the emission spectra or colors, are easily distinguishable, and (3) the labels do not significantly impair sequencing reactions.
  • nucleic acid template is suitably prepared by restriction enzyme frag ⁇ mentation and purification. Under appropriate conditions, sepa ⁇ rate base specific reactions are performed for each base to be detected, wherein said nucleic acid template and suitably CD- labeled primer are combined to form annealed template-primer-CD.
  • Said annealed template-primer-CD is then combined, in a reac ⁇ tion mixture in appropriate buffer, with sufficient amounts of the appropriate nucleotides and suitable synthesizing enzymes such as Klenow, T7 or Taq polymerases, reverse transcriptases (eg. from avian myeloblastosis virus (AMV)), and others, to pro ⁇ mote oligonucleotide synthesis (primer extension).
  • suitable synthesizing enzyme will depend on conditions of the sequenc ⁇ ing reaction.
  • the corresponding chain terminators are added and appro ⁇ priately incubated to allow termination of synthesis with the formation of CD-labeled oligonucleotide fragments.
  • the resulting CD-labeled fragments are suitably separated by size to determine the number of bases per fragment. Separation is done using any suitable method such as polyacrylamide gel electrophoresis or high pressure liquid chromatography, (HPLC).
  • Said CD fluorophore labeled fragments are activated and detected as described previously in Method I, above.
  • DNA fragments can also be labeled by the incorporation of a single CD-labeled dideoxy nucleotide at the 3' end with terminal deoxy- nucleotidyl transferase (eg. Trainor, G.L., et al , Nucl . Acid Res. 16, 11846, 1988).
  • Another suitable application for this invention is the so called "DNA fingerprinting" method disclosed by Livak, K. , et al , Amer. J. Human Gen. 43, A150 (1988), wherein the appropriate CD-labeled nucleotides are substituted for Livak's fluorescent nucleotides.
  • any of the CD labels of the instant invention can also be used to label genetic probes in nucleic acid hybridization assays such as those disclosed in copending patent application SN 363,081.
  • the different colored CD labels of the instant inven ⁇ tion can be substituted in place of the chemiluminescent labels of said previous disclosures and suitably activated by energy transfer or other means as described herein.
  • nucleic acid probes each suitably coupled with a different colored CD fluorophore label, are suit ⁇ ably hybridized to sample nucleic acid that has been suitably immobilized, or, "sandwich” hybridized to sample previously hybridized to other nucleic acid.
  • the hybridized probes are activated by any suitable energy transfer reaction (eg peroxyoxalates and peroxides), as described above.
  • the colors of emitted light are suitably detected and/or recorded as described above, to indicate which probes hybridized.
  • a homogeneous assay can be used wherein hybridization of the probes triggers activation of the CD labels.
  • One homo ⁇ geneous method would have a peroxide generating enzyme, such as glucose oxidase, coupled in close proximity to the sample nuc ⁇ leic acid. Then, after CD labeled probe is hybridized to the sample, H2O2 is generated by the enzyme in close proximity to the bound probe, in the presence of substrate such as glucose. With available peroxyoxalate, an energy transfer reaction occurs, which activates said CD fluorophore labels to emit light.
  • the different colored CD fluorophore labels of the instant invention are also usable as labels for probes in the test devices previously described in copending application SN 363,081, wherein said glucose oxidase, energy transfer reac ⁇ tion can be used for activation.
  • nucleic acid hybridization schemes are possible such as the use of CD catalyst labels, and various hybridization assays that employ magnetic particles, among others.
  • any of the CD labels of the instant invention can also be used in immunoassays.
  • CD label compositions and methods for their use in immunoassays are disclosed in U.S. patent applica ⁇ tion SN 418,843, filed Oct. 10, 1989, the contents of which are hereby incorporated by reference.
  • said liposomes can be suitably replaced with the different colored CD labels of the instant invention and provide new and versatile methods for immunoassay.
  • antibodies are suit ⁇ ably incubated and bound to sample antigen that has been suit ⁇ ably immobilized.
  • said antibodies are suitably bound in a "sandwich" immunoassay to sample antigen previously bound by captor antibody; After removal of unbound antibody, the labeled, bound antibodies are activated by any suitable energy transfer reaction (eg. peroxyoxalates and peroxides), as descr ⁇ ibed above.
  • suitable energy transfer reaction eg. peroxyoxalates and peroxides
  • the colors of emitted light are suitably detected and/or recorded as described above, to indicate which antibodies bound and therefore which antigens were present.
  • a homogeneous immunoassay can be used wherein binding of the CD labeled antibodies triggers activation of the CD labels.
  • One homogeneous method would have a peroxide generating enzyme, such as glucose oxidase, coupled in close proximity to the sample antigen. Then, after CD labeled antibody is bound to the sample., H2O2 is generated by the enzyme in close proximity to the bound antibody, in the presence of substrate such as glu ⁇ cose. With ava lable peroxyoxalate, an energy transfer reaction occurs, whfich activates said CD fluorophore labels to emit light.
  • a peroxide generating enzyme such as glucose oxidase
  • said CD catalyst labels can be coupled to suit- able genetic probes for use in hybridization assays.
  • a nucleic acid probe suitably coupled with a suitable CD catalyst label eg. histamine derivatized
  • a suitable CD catalyst label eg. histamine derivatized
  • sample nucleic acid that has been suitably immobi ⁇ lized, or, "sandwich” hybridized to sample previously hybridized to other nucleic acid.
  • the hybridized probe is exposed to a suitable substrate.
  • said histamine derivatized CD catalyst label will hydrolyze p-nitrophenyl acetate in suitable solvent, to p-nitrophenol , detectable by an increase in spectrophotometric absorbance at 400 nm.
  • a major advantage for using CD catalyst labels is their resi ⁇ tance to harsh conditions (eg. in nucleic acid hybridiza ⁇ tion), compared to enzyme labels in the prior art.
  • Said CD catalyst labels can also be used to great advantage in immunoassays.
  • a specific antibody suitably coupled with a suitable CD catalyst label (eg. histamine deriv ⁇ atized)
  • a suitable CD catalyst label eg. histamine deriv ⁇ atized
  • said antibody is suitably bound in a "sandwich" immunoassay to sample antigen previously bound by captor antibody.
  • the labeled, bound antibody is exposed to a suitable substrate.
  • said histamine derivatized CD catalyst label will hydrolyze p-nitrophenyl acetate in suitable solvent, to p- nitrophenol, detectable by an increase in spectrophotometric absorbance at 400 nm.
  • Said CD catalyst labels can also be used in chemical synthesis or detoxification, wherein said CD catalyst labels are coupled to a suitable solid support or to magnetic particles. For instance, when coupled to said magnetic particles, they are exposed to a suitable solution containing the chemical substrate to be catalytically reacted upon (eg. oxidation, hydrolysis, transformation, etc.), such as in a slurry in a continuous flow system. After sufficient reaction, said magnetic particles are suitably collected by magnetic force and recycled.
  • a suitable solution containing the chemical substrate to be catalytically reacted upon eg. oxidation, hydrolysis, transformation, etc.
  • any suitable hollow molecule or substance with a hydrophobic interior can be substituted for said cyclo ⁇ dextrins.
  • Said substitute substances can be composed of any other carbohydrates such as soluble or colloidal polymers and helical segments of amyloses.
  • celluloses agars or agaroses
  • other substances such as proteins, lipids, lip- oproteins, nucleic acids, surfactants, virus coat proteins, organic molecules, as well as porous particles of silicas, acrylamides, nylons, polystyrenes, resins, metals and cellu ⁇ loses, and combinations of these.
  • calixarenes examples include cyclophanes, and certain crown ethers, including their analog ⁇ , derivatives, and precursors. With appropriate modification, these other substances can be used in place of cyclodextrins in the preparations and methods of the instant invention.
  • examples of calixarenes are disclosed by Gutsche, CD., et al , J. Indus. Phenom. Molec. Recog. Chem. 7, 61-72 (1989).
  • cyclophanes examples include Bukownik, R.R., et al , J. Organ.
  • New and useful composition ⁇ can also be synthesized wherein previously described cyclodextrin label preparations, above, can be used as labels inside any suitable liposomes that have lig ⁇ ands, ligators or nucleic acids coupled to their surface.
  • Said liposomes can include the polymerized liposomes and "macro" lip ⁇ osomes disclosed in copending patent application SN 101,392.
  • said polyaromatic CL labels described in the copending patent application described above can be further improved wherein any ⁇ uitable cyclodextrin l ⁇ included in the label composition to form a complex with said label and enhance light emi ⁇ sion.

Abstract

This invention provides a method of qualitative and quantitative nucleic acid sequence analysis using luminescent cyclodextrin (CD), labels for dideoxy-nucleotide chain terminators and oligonucleotide primers. The CD labels are nonradioactive and detected in a ''black'' background, avoiding problems of fluorescence and allowing more sensitivity. Through formation of inclusion complexes with various fluorophores, the CD labels provide the versatility of different colored labels, with potentially higher signal efficiency in aqueous solutions. The CD labels have similar structures and thereby provide simplicity of synthesis and use, with more uniform chemical/physical properties, even with different colored labels. The invention is intended for high volume nucleic sequencing employing photomultipliers or charge-coupled device cameras for detection. Through the combination of these features, this invention provides advantages of speed, sensitivity, simplicity and versatility previously unknown or suggested in the art of nucleic acid sequence analysis.

Description

CYCLODEXTRIN LABELS FOR NUCLEIC ACID AND BIOCHEMICAL ANALYSIS
FIELD OF THE INVENTION This invention relates to nucleic acid sequence analysis using nonradioactive, luminescent cyclodextrin labels.
RELATED PATENT APPLICATIONS
This is a PCT application that hereby claims priority through US patent application entitled, "Nucleic Acid Sequence Analysis Using Luminescent Cyclodextrin Labels", SN 389,796, filed August 4, 1989, and US patent application entitled "I munoassay Using Cyclodextrin Labels", SN 418,843, filed Oct. 10, 1989, the con¬ tents of both are hereby incorporated herein by reference. The inventor in the copending applications is the same as in the instant application.
DESCRIPTION OF THE PRIOR ART
The demand is increasing for more rapid, sensitive and non- radioactive methods for nucleic acids sequencing. The instant invention addresses this problem through cyclodextrins that also include a fluorescent or chemi luminescent substance, and are used as .labels coupled to nucleotides. It has been discovered that cyclodextrin labels provide new properties and advantages, described below. As far as can be determined, such labels are novel compositions and their use is unknown in the prior art.
Chemilumi escent substances have been used as labels by chem¬ ically coupling them directly to certain ligands, antibodies and other substances for use in immunoassays. An acridinium ester label is used by Bounaud, J-Y., et al , Clin. Chem. 34/12, 2556- 2560 (1988); luminol is used by Tokinaga, D. , et al , in U.S. Pat. No. 4,628,035 (1986); and luciferin is used by Schaeffer, J.M., et al , in U.S. Pat. No. 4,665,022 (1987).
An interesting method is disclosed by Litman, et al , U.S. Pat No. 4,318,707. This is a homogeneous assay method employing a light absorbing particle with a ligand or ligand receptor coupled to it. When a corresponding analyte with a fluorescent or chemi luminescent label is subsequently bound to the particle, light emission is quenched.
Another method is employed by E.F. Ullman, et al , U.S. Pat. No. 4,193,983 whereby colloidal, lipophilic particles, such as liposomes, have a ligand and a fluorescent or chemi luminescent laDel adjacently bound to the surface of the particle. They are used in immunoassays where steπc interaction between a binding receptor and the label modulates the signal.
Of interest is U.S. Pat No. 4,372,745 by Mandle, et al , 1983, that describes several useful assay methods employing an "encap¬ sulated fluoregcer." Said "encapsulation" is accomplished by coupling the fluorescer to glass beads or by loading it into colloidal-sized liposomes which have biological substances con¬ jugated to their surface.
Also of interest is U.S. Patent No. 4,483,929, by F.C. Szoka, 1984, which discloses liposomes loaded with a "reporter composi¬ tion" that may include fluorescent, spin-label or chemi lumines¬ cent substances, and have antibodies coupled to them. Szoka does not mention cyclodextrins or disclose nucleic acids or nuc- leotides coupled to his liposomes.
Kricka, L.J., et al , U.S. Pat. No. 4,598,044 (1986), T.P. Whitehead, et al , Nature(London) 305, 158-159 (1983), G.H.G. Thorpe, et al , Clin. Chem. , 30, 806-807 (1984), and I. Sampson, et al , Analyt. Lett., 18(B11), 1307-1320 (1985), have demonstrated "enhanced" luminescent immunoassays wherein a pei— oxidase label is used to activate luminol.
Chemiluminescent substances have been used as labels by chem¬ ically coupling them directly to certain ligands, antibodies and other substances for use in assays. An acridinium ester label is used by Bounaud, J-Y., et al , Clin. Chem. 34/12, 2556-2560 (1988); luminol- is used by Tokinaga, D. , et al , in U.S. Pat. No. 4,628,035 (1986); and luciferin is used by Schaeffer, J.M., et al , in U.S. Pat. No. 4,665,022 (1987).
The im unoassay inventions of the prior art are fundamentally different compositions and methods from the instant invention where a cyclodextrin label is coupled to a nucleotide or nucleic acid primer, for use in nucleic acid sequencing. Previously cited prior art immunoassay inventions provide no teaching or suggestion on how to perform nucleic acid sequence analysis. Recently, fluorescence and chemi luminescence has been used in nucleic acid hybridization analysis.
Of interest is U.S. Pat. No. 4,670,379, by J.A. Miller, 1987, and foreign patents by Kourilsky, et al , U.K. 7913031, Pub. #2019408A; and Heller, et al , Europ. 82303701.5, Pub. #0070687 (1983) and Europ. 82303699.1, Pub. #0070687 (1982). The Miller invention is based on the use of an "apoluminescer" , wherein a nonfluorescent label is converted to a fluorescent form through catalysis. A homogeneous feature is provided through the hybri¬ dization of two labeled DNA probes with the nucleic acid analyte wherein a "transformation radical" (eg. OH~ ) , is generated by a catalyst on one probe which converts an apoluminescer on the other probe to a luminescer.
Of particular interest is the" disclosure by Miller of luminol as an apoluminescer coupled directly to DNA (Example III). This probe composition has been disclosed by Kosak, U.S. Pat. No. 4,604,364, (see col.4 for DNA and luminol, col.6 for periodate coupling agent) .
The Miller invention makes* no disclosure of cyclodextrins and suffers the limitation of requiring two labeled probes, wherein the number of label molecules per probe is limited to the number of coupling sites available on said probe.
Gen-Probe Inc., San Diego, CA, has disclosed a procedure called PACE™, wherein acridinium ester label is coupled to a nucleic acid probe for use in a nucleic acid hybridization assay. The procedure does not teach or suggest any of the cyc¬ lodextrin labels and methods of the instant invention.
Also of interest is an "enhanced chemiluminescence" (ECL), procedure used by Amersham Corp., Arlington Heights, IL, 60005, that employs luminol to detect nucleic acids.
Previously cited prior art provides no disclosure on cyclo¬ dextrin labels or teaching on using said labels in nucleic acid sequence analysis. As will be seen in the disclosures to fol¬ low, the instant invention overcomes these limitations through the use of cyclodextrin labeled nucleotides detected by chemi- luminescence.
Recently, some workers have employed fluorescent labels for sequencing DNA and RNA. Examples are Prober, J.M., et al , Science 238, 336-341 (1987), and Smith, L.M., et al , Nature (London) 321, 674-679 (1986). However, fluorescent methods require an incident light source for generation of detectable signal. This produces the inherent background problems of reflected incident light, Raman scattering, and nonspecific fluorescence of other substances in the sample.
In the prior art of cyclodextrins, there is no apparent dis¬ closures for using cyclodextrins as labels, particularly for labeling nucleotides or oligonucleotides as in the instant invention. Rather, cyclodextrins have been used primarily for chromatography,*- catalysts, and fluorescent or chemiluminescent enhancement.
Of particular interest is the disclosures by Woolf, E.J., et al , J. Luminesc. 39, 19-27 (1987), and Grayeski , M.L., et al , J. Luminesc. 33,115 (1985). These workers show that cyclodextrins can significantly enhance chemiluminescent emissions. However, there is no teaching of cyclodextrin labels or suggestion for there use in nucleic acid sequence analysis.
Some recent reviews on cyclodextrins are: Atwood J.E.D., et al , Eds., "Inclusion Compounds", vols. 2 & 3, Academic Press, NY (1984); Bender, M.L., et al , "Cyclodextrin Chemistry", Springer- Verlag, Berlin, (1978) and Szejtli, J., "Cyclodextrins and Their Inclusion Complexes", Akademiai Kiado, Budapest, Hungary (1982).
SUMMARY OF THE INVENTION
It is the object of this invention to provide a means of qualitative and quantitative nucleic acid sequence analysis which avoids the disadvantages of previous methods, and results in a new and more versatile method.
It has been discovered that cyclodextrin labels provide new properties and with unexpected advantages. For instance, they provide potentially higher signal efficiency, versatility in label colors, while maintaining uniform chemical and physical properties.
This invention provides a nucleic acid test method that for the first time has all of the following useful properties:
1. safer, nonradioactive materials;
2. luminescent labels detected in a "black" background, avoid problems of fluorescence and allow for more sensitivity;
3. cyclodextrin complexes provide potentially higher signal efficiency in aqueous solutions;
4. versatility and ease in providing different colored labels;
5. simplicity of synthesis and use through more uniform chemical/physical properties, even with different colored labels;
6. more potential host-guest stability through derivatives and "captured" guest compositions;
7. versatility and ease in one-step coupling of single or multiple labels;
8. system is readily automated for high volume nucleic acid sequencing.
The combination of these features provides new label composi¬ tions and methods unanticipated or suggested in the prior art. In addition, this invention is intended for use in assays empl¬ oying photomultipliers or charge-coupled device (CCD) cameras, with computerized data collection and reduction. It is also suitable for photodiode detection (eg. Aizawa, M. , et al , Anal. Lett. 17(B7), 555-564, 1984), or photographic detection and recording. Still another suitable use of the instant invention is in continuous flow and/or solid phase detection systems, (eg. Van Zoonen, P., et al , Anal. Chim. Acta. 167, 249-256, 1985; and Anal. Chim. Acta. 174, 151-161, 1985), including chromatographic methods. Also, an object of the instant invention is to provide reagents, containers and other components in the form of a mer¬ cantile kit, to carry out the methods of the instant invention.
With suitable modifications, the instant invention can be used with a variety of test formats and detection systems. For instance, luminometers such as portable or automated, "tube luminσmeters" and other automated instruments that read 96 well, microtiter plates could be used.
Through the combination of these features, the instant inven¬ tion provides advantages of speed, sensitivity, simplicity and versatility previously unknown or suggested in the art of nuc¬ leic acid sequence analysis.
DETAILED DESCRIPTION OF THE INVENTION
For the purposes of disclosing this invention, certain words, phrases and terms used herein are defined as follows:
Ligand A ligand is defined as a selectively bindable mater¬ ial, that has a selective affinity for or is bound by a usually, but not necessarily, larger specific binding body or "partner", in a ligand binding reaction. Such ligand binding reactions are well known and include selective affinity binding between anti¬ gen and antibody, biotins and avidins, lectins and glycopro- teins, receptors and hormones, substrates or cofactors and enz¬ ymes, and between restriction enzymes and nucleic acids, among others. Ligands are also capable of being bound to non- biological types of binding substances such as chelators, cavitands, resins and surfactants. In immunoassays and nucleic acid tests, the ligand would be the antigen, hapten or comple¬ ment that would be bound by or to its corresponding antibody. In the case of an enzyme, a ligand would be the substrate or the coenzyme. Other substances that are capable of being bound as ligands by organic or biological substances are proteins, histones, enzymes, enzyme fractions and derivatives, hormones, vitamins, steroids, polypeptides, carbohydrates, lipids, bio- tins, biotin derivatives, fc receptors, antibiotics, drugs, digoxins, pesticides, nucleic acid polymers, oligonucleotides, and substances used or modified such that they function as lig¬ ands. Ligands also include various substances with selective affinity for ligators that are produced through recombinant DNA, genetic and olecu'lar engineering.
Ligator A ϋgator is defined as a specific binding body or "partner" that is usually, but not necessarily, larger than the ligand it can bind to. For the purposes of this invention, it is a specific substance or material or chemical or "reactant" that is capable of selective affinity binding with a specific ligand in a ligand binding reaction. A ligator can be a protein such as an antibody, a nonprotein binding body or a "specific reactor." When binding ligands, ligators would include anti¬ bodies including monoclonal antibodies, chimeric antibodies and fractions thereof, enzymes, plasma proteins, avidins, strep- tavidins, chalones, cavitands, thyroglobulin, intrinsic factor, globulins, biological receptors, viruses, cell membrane derivat¬ ives, chelators, surfactants, organometallic substances, staphy- lococcal protein A, protein G, ribosomes, bacteriophages, cyto- chromes, lectinβ, certain resins, organic polymers, cyclodex¬ trins, and catalysts. Ligators also include various substances with selective affinity for ligands that are produced through recombinant DNA, genetic and molecular engineering. Under suit¬ able conditionsj ligators can be used as labels wherein they are coupled to an appropriate substance.
Nucleic Acid A nucleic acid is defined as any nucleic acid sequence from any source that is suitable for use in the instant invention. Said nucleic acid includes all types of RNA, all types of DNA, oligonucleotides and other genetic materials including synthetjc nucleic acid polymers. Also included are DNA and/or RNA fragments, and derivatives from any tissue, cells, nuclei, chromosomes, cytoplasm, mitochondria, ribosomes, and other cellular sources. Also included are modified and der- ivatized nucleic acid sequences including those that are coupled to or associated with other substances such as proteins, lec- tins, histones, pol peptides, carbohydrates, lipidε, resins, steroids, hormones and enzymes.
When appropriate, said nucleic acid may be pretreated by well known methods before use in the instant invention. For inst¬ ance, the nucleic acid may be extracted, purified, amplified, denatured by various means, immobilized, and/or suitably deriv- atized as needed.
Luminescence Luminescence is defined as the product of a luminescent reaction. A luminescent reaction, for purposes of this invention, is defined as nonradioactive, electromagnetic radiation or light produced by some means of electronic excita¬ tion or ionization of molecules or atoms, in the absence of an incident light source. Specifically, this includes photons emitted through a chemical or biochemical reaction such as oxid¬ ation, peroxide cleavage or ionization. However, it would exclude certain physical energy sources for electronic excita¬ tion that require an incident light source to cause photon emis¬ sion. Although certain methods that do use an incident light source (eg. delayed fluorescence), have been called "luminesc¬ ent" in the prior art, by definition in this invention, those are considered fluorescent methods. Therefore, fluorescence, phosphorescence, apoluminescence and radioactive photon emission are excluded from the luminescent definition.
The prior art has distinguished between two types of lumines¬ cence (Seitz, et al , Anal. Chem. Vol 46, No. 2, p.188A, 1974). One type is chemiluminescence and the other is bioluminescence. For the purposes of this invention, they are defined as follows:
Chemi 1uminescence For the purposes of this invention, che - i luminescent (CL) substances are inorganic and organic sub¬ stances that generate light during their chemical change or decomposition. Chemiluminescent substances can be readily activated by various inorganic oxidizers and do not require enz¬ ymes or other proteins to produce luminescence. However, certain chemi luminescent substances can also be activated through enzymatic reactions that generate peroxide as a by¬ product, which subsequently reacts with the chemiluminescent substance. A chemiluminescent substance, or luminescer, can be coupled to and/or complexed with a cyclodextrin label that is detected through activation of the CL substance and measurement of the light produced.
Examples of chemiluminescent substances that would be useful in this invention include a number of compounds such as luminols, isoluminols, aminobutylethyl-isoluminols (ABEI), aminobutylethyl-'naphthalene-isoluminols (ABEN) and any other cyclic or acyclic hydrazides. Also included are various dioxe¬ tanes, includini 3-phosphate-9H-xanthene-9-ylidineadamantanes, tert-butyldimethylsi loxyl-substituted and/or adamantyl- substituted dioxetanes, and other dioxetane and dioxetanone der¬ ivatives and precursors, 2,4,5-triphenylimidazones (lophines), acridines, acridine and acridinium esters and salts, including derivatives and precursors, indole-3-pyruvic acid, aryl Grignard reagents, riboflavin, lucigeninε, 9, 10-bis(phenylethynyl )- anthracenes (BPEA), 9, 10-bis(phenylethynyl )naphthacenes (BPEN), luciferins, phthalazine diones, including their trimethoxy and dimethylamino[c,a]benz analogs, isothiocyanate derivatives and other derivatives. Also included are conjugates of these sub¬ stances to proteins, carbohydrates, lipids, nucleic acids or suitable polymers. Also included are reversibly inactivated forms of said chemiluminescent substances.
Other examples of chemiluminescent substances that would be useful in this invention can be found in the following refer¬ ences which are hereby incorporated into the instant invention, including references therein, by reference:
Adam, W., et al , Chem. Ber. 116, 839-846, (1983);
Boguslaski, et al , U.S. Pat Nos. 4,355,165 and 4,363,759;
Braun, J., et al , Clin. Chem. 32/5, 743-747 (1986),
Buckler, et al , U.S. Pat. Nos. 4,331,808 and 4,334,069;
Burdo, T.G., et al , Anal. Chem. 47, 1639-1644 (1975),
Campbell, A.K., et al , IN: "Methods of Biochemical Analysis", vol. 31, 317-416, D. Glick, Ed., (1985);
DeLuca, M.A.,ed. "Methods in Enzymology", LVII, Academic Press, NY (1978).,
Hertl , et al t U.S. Pat No. 4,410,633;
Kim, J.B., et al , Clin. Chem 28, 1120 (1982),
Kohen, F., et al , Steroids 36, 421 (1980);
Konishi, E. et al , J. Clin. Microbiol. 12, 140-143 (1980);
Kricka, L.J., et al , Analyst 108, 1274-1296 (1983); Mai er , J r . , U . S . Pat Nos . 4 , 1 04 , 029 and 4 , 1 81 , 650 ;
McCapra, F., Ace. Chem. Res. 9, 201-208 (1976),
McCapra, F., et al , Photochem. Photobiol . 4, 1111-1121 (1965),
Schaap, A., et al , Tetrahed. Lett. 28, No. 9, 935-938 (1987),
Schaap, A., et al , Tetrahed. Lett. 28, No. 11, 1155-1158 (1987),
Schaap, A., et al , Tetrahed. Lett. 28, No. 11, 1159-1162 (1987),
Schuster, G.B., et al , "Bioluminescence and Chemi lumines¬ cence", 23-29, Academic Press, NY (1981),
Simpson, J.S.A., et al , "Bioluminescence and Chemi lumines¬ cence", 673-679, Academic Press, NY (1981),
Simpson, J.S.A., et al , Nature (London) 279, 646 (1979);
Thorpe, G.H.C., et al , IN: "Clinical and Biochemical Lumines¬ cence", 21-42, Kricka, L.J., et al , Eds., Marcel Dekker, NY (1982),
Weeks, I., 'et al , Clin. Chem. 29/8, 1474-1479 (1983),
Weeks, I. et al , Clin. Chem. 29, 1480 (1983),
Wehry, E.L., Anal. Chem. 58, 13R-33R (1986), (review)
Wynberg H., et al , "Bioluminescence and Chemiluminescence" , 687-689, Academic Press, NY (1981), among others.
Bioluminescence Bioluminescence is light produced by a bio- luminescent (BL) reaction using certain light generating proteins or protein-containing substances that can be extracted from various biolu inescent organisms. Examples of BL sub¬ stances are luciferases and photoproteins.
Coup! ing The preparations and components of the instant invention are synthesized by coupling labels, ligands, ligators, nucleic acids, support materials and other substances in various combinations as described below. Said coupling can be through noncovalent, "attractive" binding or through covalent, electron- pair bonds.
Many methods for covalently coupling (or crosslinking) anti¬ bodies, antigens, haptens, proteins, carbohydrates and lipids to ligands and ligators are known and, with appropriate modifica¬ tion, could be used to couple the desired substances through their "functional groups" for use in this invention.
Functional Group A functional group is defined here as a potentially reactive site on a substance where one or more atoms are ava lable for covalent coupling to some other substance. Some substances have functional groups as part of their struc¬ ture such as those provided by amino acid residues on certain proteins. Other substances may require chemical "activation" of their functional groups to produce aldehydes, ketones or other useful groups. Also, functional groups can be added to various substances through derivatization or substitution reactions.
Examples of functional groups are aldehydes, amines, amides, azides, carboxyls, carbonyls, epoxys, hydroxyls, ketones, metals, phosphates, sulfhydryls, sulfonyls, thioethers, phenolic hydroxyls, indoTes, bromines, chlorines, iodines, and others. The prior art has shown that most, if not all of these func¬ tional groups can be incorporated into or added to cyclodex¬ trins, ligands, ligators, nucleic acids and support materials if not already present.
Coupling Agent A coupling agent (or cross!inking agent), is defined as a chemical substance or energy that produces and/or reacts with functional groups on a target substance so that cov¬ alent coupling or conjugation can occur with the target subs¬ tance. Because of the stability of covalent coupling, this is often the preferred method. Depending on the chemical makeup or functional group on the cyclodextrins, nucleotide, nucleic acid, ligand, or ligator, the appropriate coupling agent is used to produce the necessary active functional group or react with it.
With appropriate modifications by one skilled in the art, the coupling methods referenced below, including references con¬ tained therein, are applicable to the synthesis of the prepara¬ tions and components of the instant invention and are hereby incorporated by reference, herein:
Blair, A.H., et al , J. Immunol. Methods 59, 129-143 (1983),
Erlanger, B.F., Pharmacol. Rev. 25, 271-280 (1973),
Kenyon, G.L., et al , "Novel Sulfhydryl Reagents", Methods in Enzymology 47, 407-430 (1977),
Mather, N.K., et al , Eds., "Polymers as Aids in Organic Chemistry", Chapter 2, Academic Press, N.Y. (1980).
Examples of energy type coupling agents are ultraviolet, visible and radioactive radiation that can promote coupling or crosslinking of certain substances. Examples are photochemical coupling agents disclosed in U.S. Pat. No. 4,737,454, among others. Also useful in synthesizing components of the instant invention are enzymes that produce covalent coupling such as nucleic acid polymerases and ligases, among others.
When the coupling agent is a chemical substance, it can pro¬ vide the linkage for synthesizing the preparations and compon¬ ents of the instant invention. Covalent coupling or conjugation can be done through functional groups using coupling agents such as glutaraldehyde, formaldehyde, cyanogen bromide, azides, p- benzoquinone, succinic anhydrides, carbodiimides, maleimides, epichlorohydrin, periodic acid, ethyl chlorofor ate, dipyridyl disulphide and polyaldehydes.
Other coupling agents useful in the instant invention are: bifunctional imidoesters such as dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl 3,3'-dithiobis-propionimidate (DTBP), and 2- iminothiolane (Traut's reagent); bifunctional NHS esters such as disuccinimidyl suberate (DSS) , bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES) , disuccinimidyl (N,N'-diacetylhomocystein) (DSAH), disuccinimidyl tartarate (DST), dithiobis(succinimidyl propionate) (DSP), and ethylene glycol bis(succinimidyl succinate) (EGS), including various derivatives such as their sulfo- forms; heterobifunctional reagents such as N-5-azido-2-nitrobenzoyl- oxysuccinimide (ANB-NOS), p-azidophenacyl bromide, p- a∑idophenylglyoxal , 4-fluoro-3-nitrophenyl azide (FNPA), N- hydroxysuccinimidyl-4-azidobenzoate (HSAB), m-maleimidobenzoyl- N-hydroxysuccinimide ester (MBS), methyl-4-azidobenzoimidate (MABI), p-nitrophenyl 2-diazo-3,3,3-trifluoropropionate, N- succinimidyl-6(4'-azido-2'-nitropheny1amino) hexanoate (Lomant's reagent II), succinimidyl 4-(N-maleimidomethyl )cyclohexane-1- carboxylate (SMCC), succinimidyl 4-(p-maleimidophenyl )butyrate (SMPB), N-succinimicyl (4-azidophenyldithio)propionate (SADP), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and N-(4- azidophenylthio)phthalimide (APTP), including various derivat¬ ives such as their sulfo- forms; homobifunctional reagents such as 1 ,5-difluoro-2,4- dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenylsulfone, 4,4'- diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), p- phenylenediisothiocyanate (DITC), carbonylbis(L-methionine p- nitrophenyl ester), 4,4'-dithiobisphenylazide and erythritolbis- carbonate, including various derivatives such as their sulfo- forms ; photoreactive coupling agents such as N-5-azido-2- nitrobenzoylsuccinimide (ANB-NOS), p-azidophenacyl bromide (APB), p-azidophenyl glyoxal (APG), N-(4- azidophenylthio)phthalimide (APTP), 4,4'-dithio-bis-phenylazide (DTBPA), ethyl 4-azidophenyl-1 ,4-dithiobutyrimidate (EADB), 4- fluoro-3-nitropheny! azide (FNPA), N-hydroxysuccinimidyl-4- azidobenzoate (HSAB) r N-hydroxysuccinimidyl-4-azidosal icylic acid (NHS-ASA), methyl-4-azidobenzoimidate (MABI), p- nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 2- diazo-3,3,3-trifluoropropionyl chloride, N-succinimidyl-6(4'- az do-2'-nitropheny!amino)hexanoate (SANPAH) , N-succinimidyl (4- azidophenyl )1 ,3'-dithiopropionate (SADP), sulfosuccinimidyl-2- (m-azido-o-nitobenzamido)-ethyl-l ,3'-dithiopropionate (SAND) , sulfosuccinimidyl (4-azidophenyldithio)propionate (Sulfo-SADP) , su1fosuccin -midy1-6-(4'-azido-2'-nitropheny1amino)hexanoate (Sulfo-SANPAH) , sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl- . 1 ,3'-dithiopropionate (SASD), and derivatives and analogs of these reagents, among others. The structures and references for use are given for many of these reagents in, "Pierce Handbook and General Catalog", Pierce Chemical Co., Rockford, IL, 61105.
Certain coupling agents may be less suitable than others due to adverse modification of the cyclodextrins, nucleotides, nuc¬ leic acids, ligands, ligators, and components of the instant invention being coupled. In these cases, routine precautions by one skilled in the art of covalent coupling can be taken to pre¬ vent such difficulties.
Intermediate Coupling Substance In addition to covalently coupling through functional groups, it iε also useful to include intermediate substances which function as "spacer" molecules between the materials being covalently coupled. These interme¬ diate substances may be desirable to provide additional coupling sites and thereby increase the amount of label coupled to a nuc¬ leotide, ligand or ligator, and/or overcome steric hindrance and/or introduce certain other desirable properties, such as energy transfer. When using intermediate substances, they are coupled to components of the instant invention by using the appropriate functional groups and coupling agents. Then, the desired nucleic acid, nucleotide, ligand or ligator is coupled to the available sites on the intermediate substance and thereby be coupled indirectly to said components of the instant inven¬ tion.
Examples of such intermediate coupling substances are proteins, polypeptides, polyamino acids, glycoproteinε, lipopro- teins, nucleic acid polymers, oligonucleotides, DNA, RNA, carbo¬ hydrates, polysaccharides, polyglutamic acid, poly(acrylamides) , poly(allylamines) , lipids, glycolipids and certain synthetic polymers, nylons and surfactants. Also included as suitable intermediate coupling substances are the polymers disclosed in U.S. Pat. No. 4,645,646.
Various materials may be incorporated into the components of the instant invention to impart additional properties and thereby improve their usefulness in certain applications. For instance, the addition of ferrous or magnetic particles may be used to give cyclodextrin labels magnetic properties (Ithakis- sios, D.S., Clin. Chim. Acta 84(1-2), 69-84, 1978). This may be useful for various manipulations such as dispensing, transfer¬ ring, washing and separating.
All of the synthesis methods incorporated herein by reference can be modified as needed, by coupling components of the instant invention under conditions that do not irreversibly inactivate them.
Close Proximity For the purposes of this invention, sub¬ stances and components that are described as being in close proximity or close association, are close enough so that they share the same molecular or chemical environment. Said close proximity can be due to their being coupled to a common subs¬ tance or through their being coupled to each other. In any case, said substances or components may not necessarily interact with each other but one can effect said environment of the other.
For example, a suitable enzyme in close proximity to a cyclo¬ dextrin label would be able to produce a product that could activate a fluorescent or luminescent substance inside the cyc¬ lodextrin label. Also, a fluorophore or CL compound, coupled in close proximity to a cyclodextrin molecule, is able to form an inclusion complex with said cyclodextrin, and/or participate in an energy transfer reaction.
Cyclodextrins A cyclodextrin (CD), is an oligosaccharide composed of glucose monomers coupled together to form a conical, hollow molecule with a hydrophobic interior or cavity. Said cyclodextrins (CD's), of the instant invention can be any suit¬ able cyclodextrin, including alpha-, beta-, and gamma- cyclodextrins, and their analogs, isomers, and derivatives. Also included are altered forms such as crown ether-like com¬ pounds prepared by Kandra, L. , et al , J. Indus. Phenom. 2, 869- 875 (1984), and higher ho ologues of cyclodextrins, such as those prepared by Pulley, et al , Biochem. Biophys. Res. Comm. 5, 11 (1961), and soluble dimers, trimers and polymers. These references, including references contained therein, are applicable to the synthesis of the preparations and components of the instant invention and are hereby incorporated by refer¬ ence, herein.
Cyclodextrin Labels A cyclodextrin label is defined herein as any cyclodextrin of suitable size that is capable of complex- ing or combining with one or more molecules to form an "inclu¬ sion complex", and wherein said cyclodextrin also has one or more suitable functional groups available for coupling to a sub¬ stance to be labeled. Said inclusion complex is defined herein as said cyclodextrin, functioning as a "host" molecule, combined with one or more "guest" molecules that are contained or bound, wholly or partially, within the hydrophobic cavity of said cyc¬ lodextrin. Depending on the type of guest molecule used, and the method of activation, a variety of labels with unique prop¬ erties are possible.
Cyclodextrin Fltiorophore Labels A cyclodextrin fluorophore label is defined herein as a cyclodextrin label wherein said guest molecule is any suitable fluorophore described below, including any suitable fluorescent, phosphorescent, or scintil- lator substance, or organic dye.
Cyclodextrin Chemiluminescent Labels A cyclodextrin chemi- luminescent (CL), label is defined herein as a cyclodextrin label wherein said guest molecule is any suitable CL substance previously described, combined with said CD to form a light emitting inclusion complex. A new form of cyclodextrin CL label comprises said inclusion complex wherein said CL substance is any polyaromatic hydrazide CL compound, disclosed in the copend- ing patent application SN 363,081, filed June 8, 1989, previ¬ ously described.
Cyclodextrin Catalyst Labels A cyclodextrin catalyst label is defined herein as a cyclodextrin label wherein said CD host functions as an "artificial enzyme", and said guest molecule is a substance that functions as a chemical substrate. When said chemical substrate comes in contact with said cyclodextrin cata¬ lyst label under appropriate conditions, it is modified to pro¬ duce a detectable signal directly or indirectly (eg. Ikeda, VanEtten, Hirai, below). Depending on the chemical substrate used, said signal can be due to the production of a colorimetric or fluorimetric or chemiluminescent substance. With suitable derivatization, said CD catalyst labels can be synthesized to bind specific substrates and catalyze specific reactions. Through appropriate choice of CD catalyst labels (eg. size, aff¬ inity, reactivity, etc.), several labels can be used to label different substances so that their presence is detectable in the same solution. For instance, each CD catalyst label could be distinguished from the others by the production of a different product which produced a correspondingl different signal, such as a different color. .
Said cyclodextrin catalyst labels can be couple to a variety of substances, such as ligands, antigens, antibodies, nucleo- tides, nucleic acids, primers and chain terminators, as well as to a variety of magnetic particles for use and retrieval in chemical processes, including organic synthesis and detoxifica¬ tion.
Cyclodextrin Energy Transfer Labels A cyclodextrin energy transfer label is a cyclodextrin label wherein said guest molec¬ ule is any suitable fluorophore, or fluorescent, phosphorescent, or scintillator substance, laser dye, or organic dye, and wherein one or more suitable energy producing substances are coupled to the cyclodextrin surface. Said energy producing sub¬ stances can include suitable CL substances, BL substances, per- oxyoxalates, dioxetanes, dioxetanones, oxalate-type esters, cat¬ alysts, enzymes (eg. oxidases), and other substances, that under suitable conditions, can chemically generate ionization, perox¬ ide decomposition and/or electronic excitation energy which can be transferred to guest molecules inside said complex.
Yet another new label provides a plurality of said cyclodex¬ trin energy transfer label, that can be readily coupled to any suitable substance, in numbers that are greater than the number of available sites on said substance as described previously for multiple CD labels.
Cyclodextrin Tracer A cyclodextrin tracer is a component of this invention composed of a nucleoside, nucleotide, ligand, ligator, nucleic acid, or other specific binding substance that has been coupled to or associated with a cyclodextrin label. A cyclodextrin tracer can be a fluorescent or luminescence tracer that has the specific binding properties of the nucleotide, lig¬ and or ligator 1n combination with the potential for emitting light. Said fluorescent or luminescence cyclodextrin tracer is detected by activating the label and measuring the light pro¬ duced.
A cyclodextrin tracer can also be said specific binding subs¬ tance coupled to said CD catalyst label.
Cyclodextrin labels coupled to avidins and streptavidins would be useful for subsequent coupling to a biotinylated nuc¬ leic acid. Similarly, cyclodextrin labeled antibodies can be coupled to nucleic acids with the appropriate antigen coupled to it. A cyclodextrin tracer can also be any nucleotide, or RNA, or DNA, or nucleic acid probe that has been covalently or non- covalently coupled to a cyclodextrin label.
Peroxyoxalates Peroxyoxalates are defined in the instant invention as organic substances that contain as part of their structure, an oxalate derivative that can decompose in the presence of H2O2 or other peroxides and thereby activate or electronically excite a fluorophore or fluorescer to emit light. Examples of peroxyoxalates useful in the instant invention are; oxalyl chloride, bis(3-trifluoromethyl-4-nitropheny! )oxalate (TFMNPO), bis(2»4-dinitrophenyl )oxalate (DNPO), bis(2,4,6- trichlorophenyl )oxalate (TCPO), bis(2,4,5-trichloro-6- carbopentoxyphenyl )oxalate (CPPO), 4,4'-
{oxalyl is[(trifluoromethylsulfonyl )imino]ethylene}bis[4-methyl- morpholinium trifluoromethane sulfonate] (METQ), among others, including analogs, precursors and derivatives such as mixed anhydrides, amides, sulfonamides, and N-trifyloxamides of oxa- 1ates.
Fluorophores For the purposes of this invention, fluoro¬ phores (also called fluorescers) , are defined as any suitable dye, and/or fluorescent, phosphorescent, scintillator substance that can form a light emitting complex with a cyclodextrin of the instant invention. Although sometimes called "luminescent" by some in the prior art, fluorophores are defined here as not chemiluminescent by the definitions of the instant invention. Therefore, fluorophores of this invention do not undergo a chem¬ ical decomposition during light emission.
Some examples of fluorophores useful in the instant invention are aminofluoranthrenes, anthracenes, diphenyl anthracenes, 8- anilo-1-naphthalenes, europium chelates including 4,7- bis(chlorosulfonyl )-1 , 10-phenanthroline-2,9-dicarboxy1ic acids, ruthenium chelates, fluoresceins, fluorescamines, luciferins, N- methylacridones, perylenes, rhodamine B, rubrenes, ruthenium dipyridines, blue fluorescent proteins, green fluorescent proteins, photoproteins, and their analogs and derivatives, among others.
Scinti1lators For the purposes of the instant invention, scinti1lators are fluorophores that include light emitting com¬ pounds generally used in scintillation counting systems and as laser dyes. Under suitable conditions, they are useful in the instant invention as fluorescent acceptors, and in energy trans¬ fer systems such as with cyclodextrins and other preparations described below. Examples are: acriflavins, aminoacridines, 7- amino-4-methycoumarins, carbostyrri Is, coumarins, 2,5- diphenyloxazoles, (PPO), 1 ,4-bis[5-phenyl-2-oxazoyl]benzenes (POPOP), and other oxazoles, P-terphenyls, rhodamines, and analogs and derivatives of these. Other useful scinti1lators, incorporated herein by reference, can be found under "Laser Dyes", 877-880, Sigma Catalogue, 1989, Sigma Chem. Co., St. Louis, MO.
Useful examples of peroxyoxalates, their synthesis and activation, and of fluorophores or fluorescers useful in the instant invention, can be found in the following references which are hereby incorporated into the instant invention by >' * reference:
Brandt, R. , et al , Anal. Biochem. 11, 6-9 (1965)
Bollyky, L.T., et al , J. Org. Chem. 33, 250 (1968);
Bollyky, L.T., et al , J. Org. Chem. 33, 4266 (1968);
Diamandis, E.P., Clin. Biochem. 21, 139-150 (1988);
Goto, T., et al , Tetrah. Lett. No. 49, 4299-4302 (1969)
Grayeski , M.L., et al , Anal. Chim. Acta. 183, 207-215 (1986)
Grayeski , M.L., et al , Anal. Biochem. 136, 277-284 (1984);
Gubitz, G., et al , Anal. Chem. 57, 2071-2074 (1985); Guilbault, G.G. , et al , Anal. Chem. 40, 1256-1263 (1968)
Ikariyama, Y., et al , Biochem. Biophyε. Res. Comm. 128, 987- 992 (1985)
Kricka, L.J., et al , U.S. Pat. No. 4,598,044 (1986);
Mohan, A.G., et al , J. Chem. Ed. 51, 528-529 (1974);
Rauhut, M.M., Ace. Chem. Res. 2, 80-87 (1969),
Rauhut, M.M., et al , Photochem. Photobiol . 4, 1097-1110 (1965)
Rauhut, M.M., et al , J. Org. Chem. 30, 3587-3592 (1965),
Rauhut, M.M., et al , J. Am. Chem. Soc. 88, 3604 (1966);
Rauhut, M.M., et al , J. Am. Chem. Soc. 89, 6515 (1967);
Sigvardson, K.W. , et al , Anal. Chem. 56, 1096-1102 (1984);
Tseng, S-S.,et al , J. Org. Chem. 44, 4113-4116 (1979)
Van Zoonen, P., et al , Anal. Chim. Acta. 167, 249-256 (1985)
Williams III, D.C., et al , Anal. Chem. 48, 1003-1006 (1976), among others.
Fluorophore Activation The fluorophores of the instant invention emit light after electronic excitation or "activa¬ tion", from some outside energy source. Said outside energy source can be physical, such as an incident light or radiation source (fluorescence), an electrical current (electrolumines¬ cence), sonication (triboluminescence) , or heat (thermolumines¬ cence), or said energy source can be a chemical reaction (energy transfer 1uminescence) .
Said chemical reaction can be any suitable reaction known to activate fluorophores, including those involving acridinium esters, various hydrazides, enzymes, dioxetanes, dioxetanones, oxalateε, and peroxyoxalates. In addition to those disclosed in references previously cited, other suitable activation reactions that are applicable to the instant invention are reviewed by Cilento, G. , et a1 , Photochem. Photobiol. 48, 361-368 (1988).
Suitably, fluorophores are activated through energy transfer from a chemiluminescent reaction, such as peroxide decomposition of peroxyoxalate. Generally, chemi luminescent activation of peroxyoxalates, acridinium esters, various hydrazides, and other CL substances involve the use of a peroxide such as H2O2. For instance, said H2O2 can be added directly to a reaction mixture of CD fluorophore label and peroxyoxalate, in suitable buffer solution. Or, H2O2 can be generated by enzymes such as glucose oxidase and suitable substrate. However, under appropriate conditions, other peroxides are suitable, such as t-butyl hydroperoxide, benzoyl peroxide, lino- lenic hydroperoxide, peroxylauric acid, cholesterol hydroper¬ oxide, cumene hydroperoxide, with additional examples disclosed by Cathcart, R., et al , Anal. Biochem. 134, 111-116 (1983), among others.
Said energy transfer is generally from a chemical source. However, activation can include energy transfer from any suit¬ able substance wherein said energy is first generated by said physical sourceε in said substance, which is then exposed to said fluorophore.
By exposing them to certain high energy chemical reactions such as those involving oxami-des or hypochlorites, fluorophores of the instant invention can convert some of this energy to visible light. Examples of this approach are disclosed by Tseng et al , U.S. Pat. Nos. 4,338,213 and 4,407,743; Arthen et al , U.S. Pat. No. 4,401,585; Kamhi et al , U.S. Pat No. 4,404,513; Frenzel , U.S. Pat. No. 4,433,060 and Berthold et al , U.S. Pat No. 4,435,509.
Others in the prior art have used chemically coupled chemi- luminescent substances that are capable of energy transfer. Examples of these are: Patel , A., et al . Anal. Biochem. 129, 162 (1983); Kohen, F., et al , FEBS Letters 104, 201 (1979); Campbell, A.K., et al , Biochem. J. 216, 185-194 (1983); and Patel, A., Clin. Chem. 29, 1604 (1983). With suitable modifica¬ tion, the activation methods of the foregoing references can be used in the instant invention and are incorporated herein by reference.
EXAMPLES
In the examples to follow, percentages are by weight unless indicated otherwise. During the synthesis of the compositions of the instant invention, it will be understood by those skilled in the art of organic synthesis methods, that there are certain limitations and conditions as to what compositions will comprise a suitable label. Said limitations include types and numbers of derivatives used, steric properties, fluorescent properties of fluorophores or scintillators, excitation energy transfer requirements, oxidation requirements in aqueous solution, stability at room temperature and solubility, among others. In addition, it will be understood in the art of cyclodextrins that there are limitations as to which fluorophores or CL compounds can be used to form inclusion complexes with certain cyclodex¬ trins.
Specifically, it is known that smaller fluorophore molecules are preferably used to complex with the smaller, alpha cyclodex¬ trins. Whereas larger cyclodextrins are less limited, except that a "close fit" is generally preferred for stronger complex- ing affinity and higher fluorescent and/or luminescent enhance¬ ment.
The terms "suitable" and "appropriate" refer to methods known to those skilled in the art that are needed to perform the des¬ cribed reaction or procedure, within said limitations and condi¬ tions. Generally, said limitations can be determined empiri¬ cally, and through the references to follow, the methods of which are hereby incorporated herein by reference. For example, organic synthesis reactions, including cited references therein, that can be useful in the instant invention are described in "The Merck Index", 9, pages ONR-1 to ONR-98, Merck & Co., Rah- way, NJ (1976), and suitable protective methods are described by T.W. Greene, "Protective Groups in Organic Synthesis", Wiley- Interscience, NY (1981), among others. For synthesis of nucleic acid probes, sequencing and hybridization methods, see "Molec¬ ular Cloning", 2nd edition, T. Maniatis, et al , Eds., Cold Spring Harbor Lab., Cold Spring Harbor, NY (1989).
All reagents and substances listed, unless noted otherwise, are commercially available from Aldrich Chemical Co., Wis. 53233; Sigma Chemical Co., Mo. 63178; Pierce Chemical Co., II. 61105; Molecular Probes, Junction City, Oregon; or Research Organics, Cleveland, Ohio. Or, said subεtanceε are available or can be synthesized through referenced methods, including "The Merck Index", 9, Merck & Co. , Rahway, NJ (1976).
Additional references cited are hereby incorporated herein by reference.
Buckler, et al , U.S. Pat. No. 4,331,808 (1982),
Langer, P.R. , et al , Proc. Nat! . Acad. Sci . USA 78, 6633-6637 (1981)
Patel, A., et al , Clin. Chem. 29/9, 1604-1608 (1983),
Roswell , D.F., et al , J. Amer. Chem. Soc. 92, 4855-4860 (1970),
Roberts, D.R. , et al , J. Amer. Chem. Soc. 92, 4861-4867 ( 1 970 ) ,
Robins, M.J., et al , Can. J. Chem. 60, 554-557 (1981)
Sessler, J.L., et al , J. Indus. Phenom. Mol . Recog. Chem. 7, 19-26 (1989)
Theodoropulos, S. U.S. Pat. No. 4,600,775 (1986),
Wei, C.C., et al , Tetrahedron Lett., No. 39, 3559-3562 (1971),
Accounts Chem. Res. 3, 54 (1970) J. Organ. Chem. 32, 1198 (1967),
Figure imgf000023_0001
J. Amer. Chem. Soc. 89, 3944 (1967). PREPARATION I. Nucleic Acid Derivatives
For the purposes of the instant invention, the appropriate nucleic acid, probe, nucleoside, nucleotide or "artificial dinucleotide" (eg. Sessler, supra), is labeled through a suit¬ able functional group. Said labeling is done while retaining its ability to function in ol igonucleotide binding and/or hybri¬ dization and/or synthesis including use as a primer or as a chain terminator in nucleic acid sequence analysis.
Said functional group can be any previously described func¬ tional group already present on the labeled molecule, or can be introduced through known chemical or photochemical means. Suitably, said functional group is a free amino group such as an aliphatic amine coupled to the purine or pyrimidine base, or coupled to the sugar moiety.
A. Preparation of Amino-Derivatized Primers.
A suitable aliphatic amine can be coupled to the desired nuc¬ leotide sugar for use in an ol igonucleotide primer by the method of Smith, L.M., et al , Nucleic Acids Res. 13, 2399-2412 (1985), Smith, L.M., et al , Nature (London) 321, 674-679 (1986), and Atkinson, T. , et al , IN "01 igonucleotide Synthesis: A Practical Approach", Gait, M.J., ed. , IRL, Oxford, (1984).
Another suitable method, using putrescinyl groups, is descr¬ ibed by Takeda, T. , et al , Nucleic Acids Res. Symposium Series 12, 75-78 (1983), and a method reacting diamines with a 5'- terminal phosphoroimidazol ide is described by Chu, B.C.F., et al , Nucleic Acids Res. 11, 6513 (1983). Another well known method is the use of allylamine derivatized nucleotideε (eg. Langer, supra), some of which are available commercially. Said derivatized primers can then be coupled through an available amino or other group, to a label of this invention, as described below.
B. Preparation of Amino-Derivatized Chain Terminators.
Chain terminators are defined herein as any suitable chain- terminating nucleotide such as dideoxyribonucleotide triphos- phates, useful in nucleic acid sequence analysis. Methods suit¬ able for synthesizing amino derivatized, chain-term nating dideoxyribonucleotide triphosphates, are described by Prober, J.M., et al , Science 238, 336-341 (1987) and Sanger, F., et al , Proc. Nat! . Acad, Sci . USA 74, 5463-5467 (1977), including nuc¬ leoside derivatizing methods of Robins, M.J., et al , J. Org. Chem. 48, 1854-1862 (1983). The foregoing methods are incorpor¬ ated herein by reference. Th-ese methods, among others, can be suitably modified as needed to make available an amino group on the appropriate nucleotide for coupling to the labels of this invention.
C. Preparation of Hydroxyl-Derivatized Nucleosides.
It has been discovered that labeling can be accomplished through other functional groups, providing new qualities and advantages.
1. Introducing aJHydroxyl Group for Labeling.
This procedure can be applied to the synthesis of the appro¬ priate nucleotides, primers or chain-terminating dideoxyribo- nucleoside triphosphates, including those of dideoxycytidine (ddCTP), dideoxythymidine (ddTTP), dideoxyadenosine (ddATP), dideoxyguanosine (ddGTP), and dideoxyuridine (ddUTP), triphos- phates.
The starting compound is any appropriate nucleoside that has the necessary groups protected such as the hydroxyl groups pro¬ tected as p-toluyl esters and wherein the heterocyclic base has been mono-halogenated (eg. iodinated after Robbins, supra). Said halogenated nucleoside is coupled to an appropriate, tetra- hydropyranyl acetal (THP) protected hydroxyalkyne, such as 2-(3- butynloxy)tetrahydro-2H-pyran. Other THP protected hydroxyal- kynes can be suitably synthesized by known methods from 5-hexyn- 1-ol, among others.
Said coupling is done by combining said halogenated nucleo¬ side (2-3 gm), with said THP protected hydroxyalkyne (500-700 mg), in 160 ml of deoxygenated triethylamine, with addition of appropriate amounts (50-70 mg) of bis(triphenylphosphine)palladium chloride, herein called (Ph3P)2PdCl2 , and Cul. The mixture is stirred at 55 °C under N2 for 4 Hr or until coupling is completed. With volatile alkynes, a glass lined pressure bomb in a heated oil bath is used. The product, a THP-hydroxyalkyne nucleoside, is collected as a yel¬ low oil by evaporation, dissolved in chloroform, and washed with 5% disodium EDTA/H2O. The product is recrystal1ized by redis- solving in chloroform, precipitating with methanol, collected by filtration and drying.
Said THP-hydroxyalkyne nucleoside is converted to a triphos- phate uεing known methodε such as those described by Tener, G.M., J. Am. Chem. Soc. 83, 159-168 (1961), and Hoad, D.E., et al , J. Am. Chem. Soc. 87, 1785-1788 (1965), to produce the THP- hydroxyalkyne nucleoside triphosphate.
With other appropriate hydroxyls protected as needed (eg. by p-toluyl esters), the THP protective group is removed to expose (deprotect), the alkyne hydroxyl for coupling to the labels of the instant invention. Said THP on said THP-hydroxyalkyne nuc¬ leoside triphosphate (2-3 gm) is removed by acid catalysis in a solution of 30 ml of CH2CI2/methanol/CF3CO2H (15:10:5), at 25 °C for 0.5-1 Hr, and recrystal1 ized as above.
2. Coupling Through the Added Hydroxyl.
Said nucleoside with added alkyne hydroxyl is suitably coupled directly to a label of the instant invention through a suitable amino group provided on the label as described below. For instance, the hydroxyl can be converted to a hemisuccinate using succinic anhydride as described by Steiner, A.L., et al , Proc. Nat. Acad. Sci . U.S.A. 64, 367-373 (1969), among others. Also, sebacoyl dichlorides can be used as disclosed by Bailey, J.M., et al , IN: "The Reticuloendothelial System and Atheros¬ clerosis", Diluzio, N.R., et al , eds., Plenum Press, NY, (1967). A variety of coupling agents that will crosslink said hydroxyl with an amino group, such as epoxys (eg. 1 ,4-butanediol diglycidyl ether (BDE), Vretblab, below), epichlorohydrin, sul¬ fonyls, carbonyls, chlorocarbonates, anhydrides, or cyanogen bromide, among others, can be used with appropriate modification to ensure that the labeled nucleotides (or nucleosides) , are sti11 functional .
3. Sulfonylation of the Added Hydroxyl for Additional Deπv- atizing. With other appropriate hydroxyls protected as needed, said deprotected alkyne hydroxyl is suitably sulfonylated by reacting with an appropriate sulfonylating reagent εuch as tosyl chlor¬ ide. For instance, 200-300 mg of said nucleoside triphosphate with deprotected alkyne hydroxyl is combined with 200-300 mg of p-toluenesulfonyl chloride in 20 ml of pyridine and stirred at 25 °C for 18 Hr, evaporated and recrystal!ized. The product, tosylated-hy roxyalkyne nucleoside triphosphate, can then be coupled to a label of the instant invention.
4. Other Coupling Schemes Using the Added Hydroxyl.
Said tosylated-hydroxyalkyne nucleoside can be further deriv¬ atized through well known methods to replace the tosyl ted hydroxyl with an amino group or a sulfhydryl group, among others, and subsequently coupled to a suitably derivatized label of this invention. For instance, said amino-deπvatized nucleo¬ side is readily coupled to the N-hydroxysuccinimide derivatized labels described below. Or, said sulfhydryl-derivatized nucleo¬ side is readily coupled to the maleimido-derivatized or sulfhydryl-derivatized labels described below. Said coupling can also be done through many well known coupling agents previ¬ ously described.
5. Deprotection of Other Hydroxyls.
Suitably, the p-toluyl esters are removed to deprotect the corresponding hydroxyls by treatment with .1 N sodium methoxide in anhydrous methanol at 25 °C for 5-6 hrs.
D. Nucleotides Derivatized Through Mercury Substitution.
This is another method suitable for the preparation of certain amino derivatized nucleosides and nucleotides useful in the instant invention.
1. Any suitable nucleotide, with appropriately protected hydroxyls and other groups as needed, is treated with mercuric acetate by the method of Dale, R.M.K., et al , Proc. Nat. Acad. Sci . USA 70, 2238-2242 (1973), to produce the corresponding 5- mercuriacetate or 7-mercuriacetate derivative.
2. Said mercuriacetate derivative, with appropriately pro¬ tected hydroxyls and other groups as needed, is suitably treated with thiocyanogen (prepared by combining bromine with Pb(SCN)2 in appropriate solvent), to convert said mercuriacetate derivat¬ ive to a thiocyanate derivative.
3. Said thiocyanate derivative is then converted to an S- alkyl thiocarbamate (amino) derivative by hydrolysis, or to an S-alkyl-N-alkylamino derivative by treatment with a suitable diamino substance, described below. Alternatively, under appro¬ priate conditions, said thiocyanate derivative is treated with chlorine to give the sulfonyl chloride derivative, which can be further derivatized as previously described.
CYCLODEXTRIN COMPOSITIONS
The purpose is to provide a cyclodextrin label that can be readily coupled to a variety of nucleotides, ligands, ligators, antibodieε, nucleic acidε, primers and chain terminators. Improvements provide for coupling a plurality of cyclodextrin labels in numbers that are greater than the number of available sites on the labeled molecule*..
For synthesis, the general approach is; (1) to produce or modify, if needed, one or more functional groups on the outside of said cyclodextrin molecule, (2) combine under appropriate conditions, said cyclodextrin and a CL substance, scintillator or fluorophore to synthesize said inclusion complex, or a suit¬ able chemical substrate to produce a detectable signal.
Since cyclodextrins are carbohydrates, they can be suitably derivatized and coupled through well known procedures used for other carbohydrates, especially through available hydroxyl groups. For instance, vicinal hydroxyl groups on the cyclodex¬ trin can be appropriately oxidized to produce aldehydes. In addition, any functional group can be suitably added through well known methods while preserving the cyclodextrin structure and complexing properties. Examples are: esterification, acyla- tion, oxidation, halogenation, hydrolysis, reactions with hydrazines and other amines, including the formation of osazoneε, acetals, aldehydes, amides, imides, carbonyls, esters, isopropyl idenes, sulfonates, sulfonyls, sulfonamides, nitrates, carbonates, metal salts, hydrazones, glycosones, mercaptals, and suitable combinations of these. Said functional groups are then available for the coupling of one or more cyclodextrin molecules to a bifunctiona! reagent and/or to an appropriate ligand, lig¬ ator or nucleic acid. Said coupling can be done before forming said complex or afterward.
Additional examples of cyclodextrins, CL substances, scinti1- lators and fluorophores, including chemical methods for modify¬ ing and/or derivatizing cyclodextrins that are useful in the instant invention are described in the following references which are hereby incorporated herein by reference.
Atwood J.E.D., et al , Eds., "Inclusion Compounds", vols. 2 &
3, Academic Press, NY (1984)
Bender, M.L., et al , "Cyclodextrin Chemistry", Springer-
Verlag, Berlin, (1978)
Aldrichimica Acta 19(3), 74 (1986)
Anal. Biochem. 66, 104 (1975)
Bergeron, R.J., et al , Bioσrgan. Chem. 5, 121-126 (1976)
Boger, J., et al , Helvet. Chim. Acta 61, 2190-2218 (1978)
Biochem. Biophys. Res. Commun. 51, 666 (1973)
Biochem. 19, 2287 (1980)
Breslow, R., et al , J. Amer. Chem. Soc. 92/4, 1075-1077
(1970)
Buckler, S.A., et al , U.S. Pat. No. 3,472,835 (1969) Carlsson, .J. r et al , Eur. J. Biochem. 59, 567-572 (1975) Case, L.C., U.S. Pat. No. 3,502,601 (1970) and 3,510,471
(1970)
Casu, B., et al , Carbohydrate Res. 76, 59 (1979) Cramer, F., et al , Chem: Ber. 103, 2138 (1970) Ege, D., et al , Anal. Chem. 56, 2413-2417 (1984) Emert, J., et al , J. Amer. Chem. Soc. 97, 670 (1975) Erlanger, B. ., Pharmacol. Rev. 25, 271-280 (1973) FEBS Lett. 37, 269 (1973)
Folkman, J., et al , Science 243, 1490-1493 (1989) Fujimura, K. , et al , Anal. Chem. 55, 446-450 (1983) Furue, M.A. , et al , Polymer. Lett. 13, 357 (1975) Gramera, R.E., et al , Fr. Demande 1, 584, 917 (1968) Grayeski, M.L., et al , Anal. Biochem. 136, 277-284 (1984) Grayeski, M.L., et al , J. Luminesc. 33, 115 (1985) Harada, A., et al , Macromolecules 9, 701 and 705 (1976) Hatano, M., et al , Japan Kokai 77,71,583 (1977) Hirai, H., J. Indus. Phenom. 2, 455-466 (1984) Ikeda, T., et al , J. Indus. Phenom. 2, 669-674 (1984) Ikeda, T., et al , J. Indus. Phenom. 5, 93-98 (1987) Iwakura, Y., et al , J. Amer. Chem. Soc. 97/15, 4432-4434
(1975)
Ji, T.H., Biochim. et Biophys. Acta. 559, 39-69 (1979) Johnson, C.K. U.S. Pat. No. 3,654,261 (1972) Kawaguchi , Y., et al , Anal. Chem. 55, 1852-1857 (1983) Klotz, I.M., et al , Arch. Biochem. Biophys. 96, 605-612
et al , Biochem. 18, 690-697 (1979) et al , Chem. Lett., Oct., 1037-1040 (1976) , , et al , FEBS Lett. 43, 169-175 (1974)
Figure imgf000029_0001
Japan Kokai 77,121,096 (1977) Parmerter, S.M. , U.S. Pat. No. 3,426,011 (1969) and 3,453,257 (1969)
Patonay, G. , et al , Anal. Chem. 57, 569-571 (1985) Royer, G.P., et al , Biochem. Biophys. Res. Comm. 64, 478-484 (1975)
Siege! , B. , et a!, J. Amer. Chem. Soc. 99/7, 2309-2312 (1977) Smolkova-Keulemansova, E. ,. J. Chromatog. 251, 17-34 (1982) Szejtli, J., "Cyclodextrinε and Their Inclusion Complexes", Akademiai Kiado, Budapest, Hungary (1982)
Szejtli, J., et al , Hung. Patent 19,626 (1978) et al , J. Amer. Chem. Soc. 98/24, 7855-7856
et al , Tetrahed. Lett. No. 29, 2503-2506 (1977) Ace. Chem. Res. 15, 66-72 (1982) et al , Biochem. 12, 3266-3273 (1973)
Figure imgf000029_0002
t al , J. Indus. Phenom. 2, 555-563 (1984) VanEtten, R.L., et al , J. Amer. Chem. Soc. 89/13 3242-3253 and 3253-3262 (1967)
Vretblad, P., FEBS Lett. 47, No. 1, 86-89, Oct., (1974) Woolf, E.J., et al , J. Luminesc. 39, 19-27 (1987) Zhang, L., et al , J. Luminesc. 40 & 41 , 266-267 (1988) The purpose is to provide new CD derivatives and labels that are; (1) water soluble, (2) form complexes with CL substances, scinti1lators, fluorophores or substrates, (3) can be readily coupled to the desired substance, and (4) have the needed "spacer arm" (eg. O'Carra, supra), to overcome steric inter¬ ference after coupling to the labeled substance. Preferably, the spacer arm is of 4 or more carbon atoms in length and can include aliphatic, aromatic and heterocyclic structures.
Suitable coupling agents for preparing CD labels of the instant invention can be a variety of reagents previously descr¬ ibed, including well known crosslinkers such as epichlorohydrin, isocyanates, and formaldehyde, used to polymerize CD's. Other suitable crosslinkers are various epoxy compounds including propylene oxide, 1 ,2-diethoxyethane, 1 ,2,7,8-diepoxyoctane, 2,3- epoxy-1-propanol (glycidol), 2,3-epoxy-1 ,4-butanediol diglycidy! ether, (eg. Gramera, or Case, or Johnson, or Parmerter, supra). Also useful are methods employing acrylic esters such as m- nitrophenyl acrylates, and hexamethylenediamine and p- xylylenediamine complexes (eg. Furue, or Harada, or Hatano, or Ogata, supra), and aldehydes, ketones, alkyl ha!ides, alcyl ha!ides, silicon halides, isothiocyanates, and epoxides (eg. Buckler, supra). These procedures must be suitably modified to meet the solubility and other requirements to produce the CD labels of this invention.
A. Derivatizing and Capping Cyclodextrins. Derivatizing is defined as the chemical modification of a CD through addition of any functional group and/or other substance. Capping is defined herein as coupling any suitable chemical sub¬ stance to two or more sites on the CD molecule so that said sub¬ stance spans the area between the coupled sites.
The CD's used herein can be suitably complexed with one or more guest molecules and/or derivatized and/or capped before or after their incorporation into the labels of the instant inven¬ tion. In addit-fon, said derivatizing and/or capping can be a done to produce CD's with the desired substances coupled to specific locations on the CD molecule. In the preparation of CD labels for use as hosts for CL substances, scint 1lators or fluorophores, modifications that increase affinity between the host CD and guest(s) are preferred. For instance, the host CD's of this invention are preferably derivatized (eg. methylated), and/or capped by various means to increase host-guest affinity, provided that suitable functional groups are left for coupling to the substances to be labeled.
B. Capping and Derivatizing Substances.
Preferably, said capping substance is coupled between εiteε at the primary or secondary "end" of the CD molecule, forming a bridge across either (or both) opening(s), that includes εuit- able hydrophobic groups in said capping substance. Said capping substances can be coupled directly to available hydroxyls on the CD, or they can be coupled to suitable functional groups such as; diamino compounds to iodinated CD, or azido compounds to sulfonylated hydroxyls, and/or through "spacer arms" added to the CD. Suitable capping substanceε are 6-methylamino-deoxy and 6-methylamino-6-deoxy derivatives transformed to the correspond¬ ing N-formyl compounds, imidazoles, m,m'-benzophenone-disulfonyl chloride, p,p'-stilbene-disulfonyl chloride, diphenylmethane- p,p'-diεulfonyl chloride, terephthaloyl chloride, dianhydrides such as 3,3' ,4,4'-benzophenonetetracarboxylic dianhydride and 3,4,9, 10-perylenetetracarboxylic anhydride, amino compounds such as bismark brown, N,N'-bis(3-aminophenyl-3,4,9, 10-perylenetetra- carboxylic diimide, 1 ,4-bis(3-aminopropyl )piperizine, direct yellow, azido compounds such as 2,6-bis(4-azidobenzylidene)-4- methylcyclohexanone, and thionyl chloride derivative of aurin- tricarboxylic acid, among otherε (eg. Szejtli, Emert, Tabuεhi , or Cramer, εupra) .
C. Protected Cyclodextrins.
Primary and/or secondary hydroxyl groups (or derivatives), can be selectively protected and deprotected using known methods during derivatizing and/or capping procedures, to provide selec¬ tive coupling at the primary or secondary end of the CD molec¬ ule, as desired. For instance, formation of protective esters (eg. benzoates using benzoyl chloride), and selective cleavage (deprotection) , of primary' esters using anhydrous alcoholysis (eg. Boyer, supra), provides mostly primary hydroxyls for deriv- atization. After derivatization and/or coupling the primary hydroxyls, the secondary hydroxyls can be deprotected for addi¬ tional derivatization, coupling and/or capping.
PREPARATION II. N-hydroxysuccinimidyl Cyclodextrin (NHS-CD) Labels
This is a method for synthesizing new cyclodextrin labels (including multiple CD labels, below), wherein an N- hydroxyεuccinimidyl (NHS), coupling group is included in the label composition to provide for labeling any εuitable substance with an available amino group, in a single step. The substance to be labeled can be a suitable protein, including antibodies and avidins or streptavidin, or ligands, or nucleic acids. Suitably for this invention, the substance to be labeled is an aminc-derivatized primer or chain terminator previously descr¬ ibed.
A. Preparation of Sulfonylated Cyclodextrin.
A variety of suitable methods are known for sulfonylation of CD (or CD polymer) before or after protection of specific hydroxyl groups (eg. Bergeron, Boger or Ueno, supra), and/or capping of the CD (eg. Emert or Tabushi, supra). Suitably, CD (10 gm), is combined with a suitable sulfonylating reagent (20 gm), such as p-toluenesulfonyl (tosyl) chloride, mesitylenesul- fonyl chloride or naphthalenesulfonyl chloride, among others, in anhydrous pyridine, for 3-5 Hrs at room temperature (RT).
B. Preparation of Dialdehyde Cyclodextrin (Dial-CD). Dialdehyde CD is prepared by oxidation of CD using known methods (eg. Royer or Kobayashii, supra), with sodium metaperiodate. A more selective'procedure is to oxidize the CD with an oxidizing enzyme (eg. glucose oxidase), in εuitable buffer solution (eg. 0.2 M phosphate saline, pH 5-7).
C. Preparation of a Carboxylic Acid CD Derivatives.
Said εulfonylated CD iε εuitably iodinated so that it will couple to primary amino groups, using known methods (eg. Ikeda or Iwakura, supra). Suitably, 10 gm of εulfonylated CD is com¬ bined with 12 gm of Nal on 200 ml of methanol, and mix at 70 °C for 48-60 Hrs. The iodinated CD product is collected by precip¬ itation with acetone and purified by column chromatography.
Said iodinated CD or said dial-CD is coupled through the amino group to a suitable amino-carboxylic acid to provide the desired length of spacer arm. Suitable amino-carboxylic acids are; 4-aminobutyric acid, 6-aminohexanoic acid, 7-aminoheptanoic acid, 8-aminocaprylic acid, 12-aminododecanoic acid, and other aliphatic, or aromatic, or heterocyclic carboxylic acids with an available amino group for coupling.
Coupling to said iodinated CD is done in a suitable solvent such as dimethylformamide (DMF), mixing 10 gm of iodinated CD with a molar excess of amino-carboxylic acid (eg. 10 gm of 6- aminohexanoic acid), at 100 °C for 24 Hrs. The product, CD- carboxylic acid, is concentrated and purified by column chroma¬ tography.
Coupling to said dial-CD is done by reductive alkylation. In a suitable buffer (eg. 0.1 M borate, pH 7.5-8.5, 0.1-0.5 M triethanolamine) , 10 gm of dial CD is mixed with a molar excess of amino-carboxylic acid (eg. 10 gm of 12-aminodecanoic acid), at RT for 1-2 Hrs. The Schiff's base coupling is stabilized by- suitable reduction with NaBH4 (eg. 0.1-1 mg/ml ) , for 1-12 Hrs. The product, CD-carboxylic acid, is concentrated and purified by column chromatography and dried for subsequent reactions aε needed. D. Preparation of NHS-CD Derivatives.
In a suitable anhydrous solvent such as DMF, said CD- carboxyl ic acid is combined with N-hydroxysuccinimide and an aromatic carbodiimide such as N,N-dicyclohexylcarbodiimide, at approximately equimolar ratios and reacted at RT for 1-3 Hrs. The product, N-hydroxysuccinimide cyclodextrin (NHS-CD), is separated in the filtrate from precipitated dicyclohexylurea, collected by evaporation and purified by chromatography.
PREPARATION III. Amino-Cyclodextrin (Amino-CD) Derivativeε
A CD (or multiple CD, below), is suitably protected and/or deprotected as needed and a sulfonylated CD is prepared as des¬ cribed previously. Amino groups can be introduced into CD by reaction of said sulfonylated CD with azide compounds including hydrazine, and 2,6-bis(4-azidobenzylidene)-4-methyleyelohexanone (eg. Ikeda, supra), or coupling to diamines as described by Kawaguchi , or Matsui , supra.
Also, when desired, a "monoamino" CD, wherein one amino group has been coupled, can be prepared through known methods, includ¬ ing limited or sterically determined monosulfonylation, and/or by specific protection and deprotection schemes.
A. Diamino Derivatives.
Said sulfonylated CD is suitably iodinated as described prev¬ iously. Said iodinated CD is coupled through an amino group to a suitable diamino substance. Suitable diamino substances are; 1 ,4-diaminobutane, 1 ,6-diaminohexane, 1 ,7-diaminoheptane, 1,8- diaminooctane, 1 , 10-diaminodecane, 1 , 12-diaminododecane, and other aliphatic, or aromatic, or heterocyclic carboxylic acids with two available amino groups for coupling. Coupling is done in a suitable solvent such as dimethylformamide (DMF), mixing 10 gm of iodinated CD with a molar excess of the diamino substance (eg. 10-20 gm of 1 ,6-diaminohexane), at 100 °C for 24 Hrs. The product, amino-CD, is concentrated and purified by column chro¬ matography.
B. Protected Amino Groups.
Said amino groups introduced by various methods can be suit¬ ably protected by reaction with a halogenated alkylphthalimide such as N-(4-bromobutyl )phthal imide. After other suitable der¬ ivatizing, coupling and/or capping has been done, an amino group is deprotected by reaction with hydrazine in suitable solvent. Alεo, said diamino subεtances of various chain lengths car. be suitably derivatized before coupling. For instance, they can be "half protected*'* as trif1uoroacetamidoalkanes at one of the amino ends, as described by Guilford, H., et a! , Biochem. Soc. Trans. 3, 438 (1975), before coupling, and then suitably depro¬ tected such as by hydrolysis or alcoholysis. Yet another suit¬ able method involves the coupling of THP-protected amino- alkynes, previously described, to said iodinated CD, and sub¬ sequent deprotection as needed.
Under appropriate conditions, NHS-CD derivativeε can be prepared by coupling NHS esters directly to said amino-CD's. Preferably, said NHS ester iε a bifunctional NHS coupling agent with a εuitable spacer arm.
For instance, NHS esters of iodoacids can be coupled to said amino-CD's. Suitable iodoacids for use in this invention are iodopropionic acid, iodobutyric acid, iodohexanoic acid, iodohippuric acid, 3-iodotyrosine, among others. Before coupl¬ ing to said amino-CD, the appropriate NHS ester is prepared by known methods (eg. Rector, supra).
For instance, equimolar' amounts of iodopropionic acid and N- hydroxyεuccinimide are mixed in anhydrouε dioxane at RT for 1-2 Hrs, the precipitate removed by filtration, and the NHS iodopropionic acid ester is collected in the filtrate. Said NHS iodopropionic acid ester is then coupled to the amino-CD.
Also, other suitable NHS coupling agents for use in this invention have been previously described, including DSS, bis(sulfosuccinimidyl )suberate (BS3), DSP, DTSSP, SPDP, BSOCOES, DSAH, DST, and EGS, among others.
PREPARATION IV. Sulfhydrl-Cyclodextrin (SH-CD) Derivatives
A sulfhydryl group is added to said amino-CD, suitably prepared as described previously, by coupling the appropriate thiolating agent to the available amino group. For instance, thiolation of available amino groups can be done by known meth¬ ods using S-acetylmercaptosuccinic anhydride (SAMSA), (eg. Klotz, Rector, or Lui , supra), SIAB, or 2-iminothiolane (eg. Traut, supra). The sulfhydryl can be exposed through disulfide cleavage.
Sulfhydrylε can alεo be introduced through reaction of avail¬ able hydroxyls with a suitable epoxy compound. For instance, epichlorohydrin or another suitable epoxy cross! inker previously- described, is coupled to CD (preferably immobilized by a cleav- able coupling agent), wherein free epoxy groups are produced. Said free epoxy groups are then reacted with sodium thiosulfate to produce thiosulphate eεters (eg. Carlsson, supra). Said thiosulphate esters are subsequently reduced to sulfhydryls with dithiothreitol .
Sulfhydryls can be used for disulfide coupling to other available sulfhydryls on the desired substance to be labeled such as antibodies, or avidins, or streptavidin, or ligands, or nucleic acids, or primers, or chain terminators. Said available εulfhydryls may be native, or introduced by thiolation of said substance before coupling. Alternatively, said sulfhydryl cont¬ aining CD label is coupled to any maleimide derivative of pro¬ tein, nucleic acid or biotin, (eg. biotin-maleimide) or iodoacetyl derivatives such as N-iodoacetyl-N'- biotinylhexylenediamine.
PREPARATION V. Maleimido-Cyclodextrin Labels
The maleimido-cyclodextrin (mal-CD), derivative of this invention is suitable for coupling to native or introduced sulf¬ hydryls on the desired substance to be labeled in a single step.
A maleimido group is added to the amino-CD, εuitably prepared as described previously, by coupling a suitable hetero- bifunc onal coupling agent to the available amino group. Said hetero-Difunctional coupling agent consists of a suitable spacer arm with a maleimide group at one end and an NHS ester at the other end. Examples are previously described and include MBS, SMCC, SMPB, SPDP, among others.
The reaction is carried out so that the NHS ester couples to the available amino group on the CD, leaving the maleimide group free for subsequent coupling to an available sulfhydryl on the substance to be labeled. Said -substance to be labeled can be a protein such as an antibody, or avidin, or streptavidin, or lig¬ ands, or nucleic acids, or primers, or chain terminators suit¬ ably derivatized with a sulfhydryl group. PREPARATION VI. Biotinylated Cyclodextrin
Biotinylated CD iε produced by combining said amino-CD deriv¬ ative with a known N-hydroxysuccinimide derivative of biotin in appropriate buffer such as 0.1 M phosphate, pH 8.0, reacting for up to 1 hour at room temperature. Exampleε of biotin derivat- iveε that can be uεed are, biotin-N-hydroxysuccinimidε, biotin- amidocaproate N-hydroxysuccinimide ester or sulfosuccinimidyl 2- (biotinamino)ethyl-l ,3'-dithiopropionate, among others.
Said biotinylated CD can be used to couple a plurality of CD labels to any biotinylated compound. For instance, by combining dilute solutions of said biotinylated CD with avidin or strep¬ tavidin in the appropriate molar ratio, 1, 2 or 3 biotinylated CD molecules will couple to the avidin or streptavidin and pro¬ duce a complex with one or more biotin-binding sites still available. Then, said complex is added to the biotinylated com¬ pound to be labeled, such as antibody, or avidins, or streptav¬ idin, or ligands, or nucleic acids, or primers, or chain terminators, and allowed to couple through the remaining biotin- binding site.
PREPARATION VII. Photochemical Cyclodextrin (Photo-CD) Labels
A photochemical CD label is a CD label (including multiple CD labels, below), that posεesses a photoreactive coupling group in its composition, for coupling said label to any suitable subs¬ tance. Typically, the photoreactive group is an aryl azide which upon exposure to light, generates a highly reactive nitrene coupling group. Photo-CD labels can be synthesized by several methods.
For instance, said amino-CD derivatives previously described can be derivatized with a suitable bifunctional coupling agent that will couple to amino groups at one end and provide a photo¬ reactive group at the other end. Some examples are NHS-ASA, ANB-NOS, APG, EADB, HSAB, MABI, SANPAH, SADP, SAND, Sulfo-SADP, Sulfo-SANPAH, and SASD, (all available from Pierce Chemical Co., IL.), including carbenes and nitrenes disclosed by Knowles, J.R., Ace. Chem. Res. 5, 155-160, (1972), among others.
A suitable procedure is to combine, in a dark environment, said amino-CD (0.5-1.0 gm) in phosphate buffered saline (PB), pH 8.5, or a suitable anhydrous solvent such as N,N dimethyl- formamide (DMF), with NHS-ASA (1-2 gm), and let react 1 to 2 hours at 0 °C. The photo-CD label product is purified by column chromatography, dried, and stored in the dark until used for coupling. Coupling is initiated by mixing the photo-CD label with the substance to be labeled, in suitable solvent, and irradiating with u.v. light for 5-20 minutes at RT to induce cross!inking.
Also, said sulfhydryl derivatized CD labels, previously des¬ cribed, can be derivatized with a suitable bifunctional coupling agent that will couple to sulfhydryl groups at one end and pro¬ vide a photoreactive group at the other end. Some examples are APB, and APTP, among others.
PREPARATION VIII. Multiple CD (TCDln) Labels
A multiple CD label is defined herein as a CD label wherein a plurality of CD's are coupled together. Said multiple CD ( [CDjn , wherein n = 2 or more), labels of this invention over¬ come the problem of labeling a substance with a plurality of CD molecules that are greater than the number of coupling sites available. These labels allow the covalent coupling of a plura¬ lity of CD molecules to proteins such as antibody, avidin or streptavidin, or ligands, antigens, or nucleic acids, or primers, or chain terminators, and other substances, such as magnetic particles, through the appropriate functional groups.
One composition comprises two or more cyclodextrin labels coupled to an appropriate intermediate compound so that at least one functional group is left available for subsequent coupling to the substance to be labeled. Said functional group is coupled directly to the substance to be labeled or, derivatized with any suitable coupling agent such as succinimidyl, maleim- idyl, imidoester, aldehyde, or photoactive agents including nitrenes, among others previously described.
Said intermediate compound can be any of the intermediate coupling substances previously described including said hydroxy- lated compounds, carbohydrates, sulfur containing and amino con¬ taining compounds.
Another composition comprises a grouping of cyclodextrin mol¬ ecules coupled to each other through various known means to form a dimer, trimer or polymer of cyclodextrin molecules. Said grouping of CD's alεo includes suitable functional groups for derivatizing and/or coupling to (labeling), the desired subs¬ tance. Also, said CD grouping can be coupled to an intermediate substance that includes a suitable functional group or coupling agent to facilitate said labeling.
During the synthesis reactions, said functional group or agent iε appropriately protected, if neceεεary, during coupling of εaid CD grouping to εaid intermediate compound. Said func¬ tional group iε then de-protected for coupling and/or derivat¬ ization.
A. A Multiple CD Label Coupled Through Hydroxylated Com¬ pounds.
A label produced by the following method, employs an esterified hydroxylated compound as an intermediate coupling compound. Hydroxylated compounds such as oligosacharrides, celluloses, dextrans, polysaccharides, polysorbates, hyaluromc acids, heparins, polyene antibiotics, polyvinyl alcohols (eg. Szejtli, supra), oligonucleotides, proteinε, polypeptideε, and polyaminoacids, among others, are suitable wherein a carboxylic acid group is available. If necessary, said carboxylic acid group can be added by known methods. Hydroxylated carboxylic acids such as cholic acid, gallic acid, digallic acid, citrazinic acid, fluorescin, or polytyrosine, including their variouε derivativeε, can also be used.
In any case, the desired carboxylic acid group of said hydroxylated compound is suitably protected by known methods. For instance, under appropriate conditions, one or more carboxy¬ lic acid groups can be reacted with a suitable alcohol to form a protective ester. For example, a benzyl alcohol is used to pro¬ duce a hydroxylated benzyl ester.
Cyclodextrin, which can be suitably complexed, derivatized and/or capped before coupling, is combined with said hydroxy¬ lated protected ester and a suitable coupling agent. Suitably, coupling is done with epichlorohydrin, or an epoxy compound pre¬ viously described, in suitable solvent, and mixed under suitable conditions until coupling is completed between a plurality of said CD and the available hydroxyls on the protected ester..
The product, a plurality of CD molecules coupled to said hydroxylated ester (ester-[CD]n ) , is appropriately treated if necessary to protect any remaining uncoupled hydroxyl groups on the CD molecules.
Said ester-[CD]n is suitably hydrolyzed (eg. hydrogen bromide in acetic acid), to cleave the benzyl ester and produce multiple CD's coupled to a carboxylic acid (acid-[CD]n ) . Said acid-[CD]n is combined with N-hydroxysuccinimide in anhydrous dimethyl¬ formamide (DMF), and mixed with N,N'-dicylohexylcarbodiimide for 2 hours to overnight at RT. Dicylohexyl urea is removed by pre¬ cipitation after adding a few drops of glacial acetic acid. The product, a plurality of CD molecules coupled to an N-hydroxy- εuccinimidyl derivative (NHS-[CD]n), iε collected by evapora¬ tion.
Under suitable conditions and appropriate derivatization if needed, other hydroxylated compounds may be used. Examples are; various flavone derivatives and analogs including dihydroxyflav- ones (chrysins), trihydroxyflavones (apigenins), pentahydroxy- flavoneε (morins), hexahydroxyflavones ( yricetins) , flavyliums, quercetins, fisetins; various antibiotics including teramycins, tetracyclines, chlorotetracyclines, clomocyclines, guamecy- clines, amphotericins, filipins, fungichromins; various cevine derivatives and analogs including cevadines, desatrines, vera- tridine; various sulfur and mercapto derivatives and analogs including dihydroxy-2-mercaptopyrimidines, ethylmercapto hydrox- ybenzoateε, 6-hydroxy-1 ,3-benzoxathiol-2-oneε, chromotropic acids, 2-mercapto-5-aminobenzimidazoles, and the reduced sulfhy¬ dryl form of 3-[(3-cholamidopropyl )-dimethylammonio]-1- propaneεulfonate (CHAPS); various sulfhydryl coupling derivat¬ ives and analogs including 5-iodoacetamidosalicylic acids, difluorescein isothiocarbamidocystamines, hydroxylated N- dansylaziridines, hydroxylated S-mercuric N-dansylcysteineε, hydroxylated N-(1-pyrene)maleimideε, hydroxylated N-(3- pyrene)maleimides, hydroxylated N-(1-anilonaphthy1-4)maleimides; various dye derivatives and analogs including alizarins, acid alizarin violets, acid greens, acid reds, hematoxylins, rosolic acids (aurins), phenol reds, phenyl-*-trihydroxy-6-fluorones; var¬ ious amino dye derivatives and analogs including basic fuchsins, pararosani1 ines, cresyl violet-isomalei iides, cresyl violet- isophthalimides; various fluorescein deri'vatives and analogε including hydroxyfluoresceins, fluoresceiin isothiocyanates, fluorescein acids, fluorescein-isomaleimideε, fluorescein- isophthalimides; various steroid derivatives and analogs includ- ing cnolesterols, digoxigeninε; various coumarin derivatives and analogs including dihydroxycoumarinε (esculeti s) , dicumarols; anthracene derivatives and analogs including dithranols, methyl anthracene triols, dihydroxyquinoneε, chrysarobinε, chrysophanic acids, emodins, secalonic acids; various amino derivatives and analogs including dopas, dopamineε, epinephrineε, norepineph- rines (arterenσls) , gallamides, trihydroxybenzamides, 4- hydroxybenzamide, 4-hydroxy-3-methoxybenzylamine HC1 , 3,4- dihydroxybenzyl mine, 4-hydroxybenzoic hydrazide, 4-hydroxy-2- naphthylamine and 4-hydroxy-2-naphthoic hydrazide, hydroxy biphenyl amines, lacmoids, melamines; various carboxylic acid compounds including aurintricarboxylic acids, chelidonic acids, chrome azurol S, diethylenetriaminepentaacetic acids, hematopor- phyrinε, methythymol blueε, thymolphthalei ε, thiophenemalonic acidε; variouε phenolphthalein derivatives and analogs including phenolphthaleins, cresolphthaleins; ellagic acids; various per- ylene derivatives and analogs including hypericins; ethyl pentahydroxy-naphthalene diones; trihydroxyacetophenones; dihydroxypyrimidine-5-carboxylic acidε; flavaspidic acids; any appropriate polyamino acids and their derivatives and analogs with available hydroxyls, including polytyrosineε, polytyrosine- lyεines, polytyrosine-lysine-cysteins, polytryptophans; and var¬ ious hydroxylated polymers and their derivativeε and analogε, including polymerε of hydroxymethylphenols, hydroxylated poly¬ styrenes, hydroxylated polyethylene glycols, hydroxylated poly- acrylamides, polyhydroxybutyric acids, hydroxyated biphenyl car¬ boxylic acidε, lucigenins, among others.
B. Multiple CD Labels Immobilized on a Solid Support.
It has been discovered that a [CD]n label can be synthesized with a more predictable number, of CD molecules per label, giving new advantages of uniform structure and chemical proper¬ ties. These CD labels also are suitable for coupling a plural¬ ity of CD molecules to a subεtance in a single step. The synthesis approach is to immobilize the initial CD molecule to a solid support, and then attach additional CD molecules to the first in a controlled, step-wise manner. After sufficient CD's have been linked together, the entire group is cleaved from the solid support fcr use as a [CD]n label.
The CD molecules used in this procedure can be suitably der¬ ivatized and/or capped before coupling to incorporate other desirable features. However, it is preferred that each CD mol¬ ecule (or dimer, or trimer), that is coupled, has a well defined structure to facilitate the production of CD labels with uniform and consistent properties.
A variety of suitable materials, such as those used in chro¬ matography (eg. Smokova-Keulemansova, supra), can be used for a solid support. Said solid support can be in the form of particles, beads, fibers, plates, and tubing walls, and composed of styrenes, acrylamides, silica gels, solid or porous glass, metals, dextrans, nylons, and celluloses, among others that are suitably derivatized as needed and compatible with the reactions used.
The coupling agent used to couple the initial CD to the sup¬ port is preferably one that is readily cleaved when desired, and the coupling agent used to couple subsequent CD's is preferably noncleavable. Suitably, the initial coupling agent is also a bifunctional reagent such as those with a cleavable disulfide group, including DTBP, DSP, DTSSP, EADB, SPDP, and photoactive couplers like DTBPA, SADP, SAND, and SASD. Other suitable agents are periodate cleavable, such as DST and sulfo-DST, and hydroxylamine cleavable at the ethyl ester linkage, such as EGS and sulfo-EGS. In any case, the label is cleaved from the sup¬ port after synthesis, leaving a suitable functional group for subsequent coupling. Suitably, the remaining functional group iε converted to an NHS ester by various known means for coupling to an amino group on the substance to be labeled.
The noncleavable coupling agent used can be from a variety of reagents previously described, including well known crosslinkers used to polymerize CD's.
C. Multiple CD Label Containing a CD Grouping.
In this example, a highly porous support, (eg. porous glass beads or predried silica gel, 5-6 gm), is amino-derivatized with a .0% (v/v), solution of (3-aminopropyl )trimethoxysi lane in toluene (150 ml) at 150 °C for 6 Hrs and suitably washed with toluene, then acetone, then methanol, and dried.
A suitable diamino substance such as 1 ,12-diaminododecane is coupled to said amino-derivatized support through a suitable cleavable disulfide coupling agent such as DSP. The solvents used can be anhydrous such as methanol, methylene chloride, or pyridine or they can be suitable aqueous buffer solutions, as conditions require.
1. Excess DSP in pyr dine is added to said amino-derivatized support and allowed to couple, after which uncoupled DSP is removed. Excess 1 ,12-diaminododecane is added to the support and allowed to couple to the previously coupled DSP, after which excess 1 ,12-diaminododecane is removed, giving 1,12- diaminododecane-coupled support.
2. Said 1 ,12-diaminododecane-coupled support is then com¬ bined with excess DSS to allow coupling of the DSS to the coupled 1 ,12-diaminododecane. The excess DSS is removed, giving DSS-activated 1 , 12-diaminododecane coupled to the support.
3. To said DSS-activated 1 ,12-diaminododecane is added excess, suitably amino derivatized, CD. Preferably said amino derivatized CD is a mono-amino preparation with a suitable spacer arm, wherein all of the CD molecules have the amino group on the same (eg. primary), side. Said CD molecules are allowed to couple and the excess is removed, leaving CD's coupled to the 1 ,2-diaminododecane through the DSS coupling agent.
4. Said coupled CD's are then suitably oxidized (eg. sodium periodate or oxidase enzyme), to produce dialdehydes on the coupled CD's (dial-CD). To said coupled dial-CD is then added excess, amino-derivatized CD so that coupling occurs between the dialdehyde of the previously coupled CD and the amino group of said amino-derivatized CD's added, and excess CD is removed. The dialdehyde coupling procedure of this step is repeated until the desired number of CD's have been coupled.
5. The [CD]n label composition is recovered from the support by cleaving the initial coupling agent used. In this case, the cleavable disulfide is treated with a suitable reducing agent such as dithiothreitol, among others. Under appropriate condi¬ tions, the Schiff bases are suitably reduced (eg. NaBH.t ) . The released CD label will have a sulfhydryl group available for subsequent coupling to any suitable substance.
Suitably, said sulfhydryl is converted to an NHS ester by coupling it to a heterobifunctional reagent with maleimide-NHS groups at opposite ends.
Schematically, wherein n = a multiple of 1 or more, the gen¬ eral structure- is: i CD ! n -SPACER-NHS D. Multiple CD Label with Intermediate Substance.
Using an intermediate substance, the CD molecules are coupled to the same intermediate compound. With suitable derivatiza¬ tion, a wide variety of substances are suitable as intermedi¬ ates. In this example, CHAPS is reduced with dithiothreitol to convert the sulfonate group to a sulfhydryl (eg. Carlsson, supra), to produce 3-[(3-cholamidopropyl )-dimethylammonio]-1- mercaptopropane (hereinafter CHAMP).
1. A suitable amino-derivatized support is prepared as des¬ cribed above. Said amino groups are then suitably thiolated to provide sulfhydryls (or 2-pyridyl disulphides using SPDP). Said CHAMP is coupled through its sulfhydryl to the available thiol or 2-pyridyl disulphide to form a cleavable disulfide linkage to the support, and produce a CHAMP coupled support.
2. Said CHAMP coupled support is treated with a suitable noncleavable, hydroxyl crosslinking agent such as epichlorohy¬ drin, among others, to give an epoxy-activated CHAMP, and exposed to an excess of CD molecules, that have been suitably derivatized as needed for coupling. The product is 1, 2, or 3 CD molecules coupled to said CHAMP through the hydroxyl groups.
3. Said product is suitably cleaved and recovered from the support by treating with a suitable reducing agent such as dithiothreitol, among others. The released CD label will have a sulfhydryl group available for subsequent coupling to any suit¬ able substance. Suitably, said sulfhydryl is converted to an NHS ester by coupling it to a heterobifunctional reagent with maleimide-NHS groups at opposite ends.
These procedures can be suitably modified wherein additional chemical and steric advantages are realized during synthesis. For instance, before the final label is cleaved from the sup¬ port, the CD label can be further modified wherein specific der¬ ivatization and/or capping reactions are performed while the disulfide group is protected. Also, the desired guest molecules can be included before or after coupling of the CD's.
Through the use of other cleavable groups (eg. Ji , supra), or coupling agents in place of DSP, other useful functional groups can be incorporated into the label that remain protected until cleaved. For instance, initial coupling through a cleavable ester will produce a carboxylic acid or hydroxyl group on the CD label after cleavage. A variety of protecting and deprotecting schemes can be adapted to serve as temporary coupling sites on a solid support for synthesis of said CD labels. The major requirement is that subsequent reactions for coupling CD's do not cleave the label before synthesis is completed.
For instance, a suitable amino-derivatized support is prepared as described above. Said amino groups are then suit¬ ably coupled to a dianhyride such as 3,4,9,10- perylenetetracarboxylic dianhydride to form an imide. The, a suitably protected amino-CD is coupled to the other end of the immobilized dianhydride. After appropriate synthesis of a mul¬ tiple CD as before, the label is cleaved by treatment with hydrazine, leaving an amino group on the label. Suitably, this procedure can also be done on a hydroxylated support material.
Schematically, wherein n = a multiple of 1 or more, the gen¬ eral structure is: lCDln !CD!n-INTERMEDIATE-NHS lCDln
PREPARATION IX. Multiple CD Molecules Coupled to Carbohydrates
These label compositions allow labeling a substance with a plurality of CD's i^hat are greater than the number of coupling sites available. The use of a carbohydrate provides new proper¬ ties for coupling a plurality of CD's to a variety of other sub¬ stances such as lectins, cell receptors, nucleic acids, proteins such as antibody, avidin or streptavidin, and other substances.
A useful label is produced by using one or more carbohydrates or saccharides as part of the label . Said carbohydrate is any carbohydrate including oligosaccharides, mono-, di- and polysac- charides, amino sugars, sulfo-sugars, deoxysugars, glycosides, lectin-binding carbohydrates, aldoses, ketoses, pentoses, arab- inoses, riboses, xyloses, hexoses, glucoses, fructoses, galac- toses, mannoses, sorboses, glucosamines, sucroses, lactoses, maltoses, raffinoses, soluble starches, amylopectins, pectins, agars, agaroses, dextrans, celluloses, hyaluronic acids, hepa- rins, and any suitable polymers, derivatives and analogs of car¬ bohydrates.
One approach is to couple two or more CD molecules to said carbohydrate either directly or through an intermediate coupling agent. The resulting label can then be bound noncovalently to the appropriate lectin or receptor that binds the carbohydrate on the label .
For covalent labeling, the approach is to couple two or more CD molecules to said carbohydrate either directly or through an intermediate so that at least one functional group is left available on the label. Said functional group is then coupled to the substance to be labeled or, derivatized with any suitable coupling agent such as succinimidyl, maleimidyl, imidoester, aldehyde, or photoactive agents including nitrenes, among others previously described. During the synthesis reactions, said functional group is appropriately protected, if necessary, dur¬ ing coupling. Said functional group is then de-protected for coupling and/or derivatization.
Another method for introducing said functional groups into said carbohydrate is to derivatized a small number of the exist¬ ing hydroxyl groups with a bifunctional coupling agent that will not react significantly during coupling of one or more CD's. A suitable number of hydroxyl groups are left for coupling said carbohydrate to said CD.
For example, before coupling to said CD molecules, various amino groups that may be present on said carbohydrate can be derivatized using known methods to produce an appropriate pro¬ tecting group such as a benzyl ester.
Alternatively, before coupling, said carbohydrate can have one or more functional groups preferentially derivatized. On certain carbohydrates, pairs of hydroxyls are reacted with appropriate aldehydes or ketones to produce protective derivat¬ ives such as acetals (eg. isopropylidenes) , which can be subse¬ quently hydrolyzed. Also, on certain carbohydrates, pairs of vicinal hydroxyls are oxidized to produce aldehyde groups. Said oxidation suitably is done with sodium periodate or with oxidiz¬ ing enzymes using known methods, so that sufficient hydroxyls are left un-oxidized for coupling with CD molecules. PREPARATION X. CD Labels with Different Colored Guests
A major advantage of the CD labels of the instant invention is the ease with which different guest molecules can be com- plexed with said CD labels to produce easily distinguishable labels. The distinguishing feature is the different light emis¬ sion wavelengths (or peak emission, or "color"), obtained from different guest molecules when they are activated or electroni¬ cally excited. >_,
Said emission can be generated by electronically exciting the guest molecule through various means such as by chemical reac¬ tion (eg. energy transfer), by an incident light source (eg. fluorescence and phosphorescence), electrically (eg. electrolum¬ inescence), or heat (eg. thermoluminescence) . Also, through the choice of appropriate CL substance as a guest, various colors of chemiluminescent emission are possible.
Preferably, said guest molecules are efficient emitters that form high affinity inclusion complexes with said CD label host, with a suitable shape and size compatible with the CD molecules used. It is possible to produce CD labels with guests that have a diversity of size and shape and color, yet are contained in the same sized CD host molecule. In applications where it is desirable to have several different colored guests that also have similar chemical and physical characteristics, the choice of guests can be derived from the same chemically related group.
For instance, any scint 11ator or fluorophore described prev¬ iously, or any aromatic nucleus including acridine, anthracene, benzene, biphenyl, biphenylene, fluorene, fluorescein, naphthacene, naphthalene, pentacene, pentalene, perylene, phenanthrene, among others, can have its emission wavelength altered or "shifted" by coupling or derivatizing with one or more specific groups. Said wavelength-altering group can be any suitable substance including hydrogens, oxygens, nitrogens, sul- furε, halogens, metals, methyls, ethyls, toluyls, and any suit¬ able functional group, among others.
Some examples of suitable guest molecules are; fluorescein dyes such as 9-(carboxyethyl )-3-hydroxy-6-oxo-6H-xanthenes .(eg. SF-505, SF-512, SF-519, and SF-526, of Prober, supra), acridines anthracenes, naphthalenes, and the fluorophores previously des¬ cribed, among others. PREPARATION XI. CD Labels with Captured Guests (Guest-CD)
A CD label with a "captured guest" (guest-CD), stabilizes the label complex and overcomes the problem of the CD host and guest molecule separating under various conditions. For the purposes of this invention, a captured guest is any (one or more), guest molecules that are captured by, or coupled in close proximity to, the CD host so that they cannot separate by the normal pro¬ cesses of diffusion. Said capturing is accomplished through physical entrapment by the CD host, or by covalent coupling of the guest in the immediate vicinity of the CD host.
For instance, physical entrapment can be done by capping both ends of the host CD, thereby entrapping the guest molecule. Another useful method is to couple two host CD's together so that said guest molecule is entrapped between them (eg. "duplex cyclodextrin" of Tabushi, supra), and wherein the outside ends are too small and/or are capped to prevent escape of the guest.
Said guest is captured by covalent coupling when the guest is coupled by various suitable means to the CD host or to an inter¬ mediate substance in close proximity. Said coupling is done after the guest enters the host CD. Or, said guest can be coupled through a suitable spacer arm of sufficient length (eg. 6 or more carbons), to allow the guest to enter the host CD after coupling (eg. Ueno, supra).
The choice of guest molecule to use will depend on the color desired for said label, fluorescent and/or excitation efficiency, energy transfer efficiency, and compatibility with its intended use.
A. Coupling guests to CD Hosts or Intermediates.
The desired guest molecule (eg. scintillator or fluorophore or CL compound), can be converted to a carbonyl chloride deriva¬ tive by treatment of an available carboxylic acid with thionyl chloride, which can subsequently be coupled through the appro¬ priate spacer arm to the host CD. Some examples of suitable guest molecules are; carboxylic fluorescein dyes such as 9- (carboxyethyl )-3-hydroxy-6-oxo-6H-xanthenes (eg. SF-505, SF-512, SF-519, and SF-526, of Prober, supra), amino-chloronaphthalenes, anilo-naphthalene sulfonic acids, naphthylacetic acids, methyl- naphtha!eneacetates, dansyl chlorides, dinitro-naphthalenes, to!uidinylnaphtha!ene-sulfonates, 10-methy1anthracene-9-carbox- aldehydes, 10-chloro-9-anthraceπemethanols, chloro-diphenyl- anthracenes, 9-f-luσrenylmethyl chloroformate, fluorenone-4- carbonyl chloride, 1-fluorenecarboxylic acid, dinitrofluorenes, pyrenebutyric acids, rhodamines, folic acids, among others.
Or, the desired guest molecule can be coupled through an available amino group, such as with acridine yellow, acrifla- vines, aminoanthracenes, 9-(methylaminomethyl )anthracenes, aminofluoranthenes, aminofluorenes, aminofluorenones, aminopyrenes, and others, using appropriate bifunctional coupl¬ ing agents, or a carbodiimide method. The desired guest molec¬ ule can also be*coupled through an available sulfhydryl (eg. produced by reduction of sulfonates), as in 8-anili-1- naphthalene sulfonates (ANS), 2-p-toluidinylnaphthalene sulfo¬ nates (TNS), among others. A variety of previously described coupling agents can be used for coupling through an available carboxyl , amino or sulfhydryl on the host CD. If necessary, said carboxylic acid or amino group can be introduced onto the guest through suitable derivatization, as with europium chelates and ruthenium chelates (eg. dipyridines of Ege, supra) . Also, said host CD can be suitably capped if desired, to increase the affinity between the coupled guest and CD host.
B. Guest-CD Label Synthesis.
A suitable scheme for synthesizing guest-CD labels of the instant invention is as follows;
1. Through known procedures for selective derivatization, amino-CD's are prepared wherein a suitable amino group is preferably coupled to one end. For instance, CD hydroxyls are protected with benzoate esters, the primary end is selectively deprotected and amino derivatized (eg. Szejtli or Boyer, supra).
2. A suitable amino-deri atized solid support is prepared as previously described for the preparation of immobilized [CD]n labels, above. To said support is coupled an intermediate com¬ pound through a cleavable disulfide as previously described, and activated, such as DSS-activated 1 ,12-diami ododecane or epoxy- activated CHAMP.
3. Then, one or more of said amino-CD's is coupled to said activated intermediate on the support through the available CD amino group. Remaining hydroxyl groups on the resulting immobi¬ lized CD's are deprotected as needed and amino-derivatized with a suitable spacer arm. For instance, said hydroxyls can be oxidized to dialdehydes, or treated with epichlorohydrin, and coupled to diaminohexane. Or, treated with acetic or succinic anhydride to give carboxy- lateε that are converted to NHS esters through reaction with carbodiimides and N-hydroxysuccinimide, and then coupled to diaminohexane.
4. In any case, said immobilized CD's are prepared with one or more amino groups available, attached through suitable spacer arms, to the CD's. Said immobilized CD's can now be coupled to a variety of fluorophores (or CL compounds), using any suitable coupling agent for coupling to amino groups.
The following examples are illustrative. a. Amino Fluorophores.
Coupling to fluorophores with available amino groups, such as acid blacks, acridine yellow, acriflavine, 2-aminoanthracene, 6- aminochrysene, 1-amino-4-chloro-naphthalene, 3- aminofluoranthene, 2-aminofluorene, 2-amino-9-fluorenone, 2- amino-3-chloro-7-nitro-9-fluorenone, 1-aminopyrene, bismark browns, N,N"-bis(3-aminophenyl )-3,4,9,10-peryleneteracarboxyl c diimide, ethidium bromide, fluoresceinamine, reactive blues, among others, is done by combining said immobilized CD with any suitable bifunctional, amino-coupling agent previously descr¬ ibed, such as DMA, DMS, DSS, among others, and said fluorophore, under suitable conditions for coupling. b. Carboxylic Acid Fluorophores.
Coupling to fluorophores with available carboxylic acid groups, such as carboxylic fluorescein dyes such as SF-505, SF- 512, SF-519, and SF-526, (see Prober, supra), 9- acridinecarboxylic acid, 1-fluorenecarboxylic acid, indoleacetic acid, 10-methylanthracene-9-carboxaldehydes, mordant oranges, mordant reds, mordant yellows, naphthylacetic acids, N-(4- nitrobenzoyl )-6-aminocaproic acid, phenolphthalein carbinol dis- ulfate, protoporphyrins, 1-pyrenebutyric acid, quinolinecar- boxylic acid, retinoic acid, rhodamine B's, among others, is done by converting an available carboxylate on said fluorophore to an NHS ester through reaction with a carbodiimide and N- hydroxysuccinimide. The product, an NHS-fluorophore ester, is then coupled to an available amino group on said immobilized CD. c. Sulfhydryl Fluorophores.
Coupling to fluorophores with sulfhydryls may require that potential sulfhydryls are made available by reduction of sulfo¬ nates on the fluorophore using suitable reducing agents (eg. Carlsson, supra). Examples of fluorophores with reducable sulf¬ onates are 8-an lo-1-naphthalene sulfonate, 2-p- toluidinylnaphthalene-6-sulfonate, acid blacks, acid blues, acid greens, acid reds, acid violets, acid yellows, alizarins, direct blues, direct reds, texas reds, and others. With an available sulfhydryl available, coupling to said immobilized CD is done by combining said immobilized CD with any suitable hetero- bifunctional , ,amino-sulfhydryl-coupling agent previously descr¬ ibed, such as MBS, SIAB, among others, and said fluorophore, under suitable conditions for coupling. d. Staining Fluorophores.
Coupling to "staining" fluorophores which have active coupl¬ ing groups or agents already attached, such as alcian blues, 4- chloro-7-nitrobenzo-2-oxa-1 ,3-diazole (NBD chloride), dansyl chlorides, europium chelates including 4,7-bis(chlorosulfonyl )- 1 ,10-phenanthroline-2,9-dicarboxylic acids, 9-fluorenyl ethyl chloroformate, fluorenone-4-carbonyl chloride, fluoreεcamine, and anhydride or isothiocyanate derivatives of various dyes such as fluoresceins, perylenes, rhodamines, and tetramethylrhodamines, among others, is done by adding said staining fluorophore to said immobilized under appropriate con¬ ditions for coupling. e. Photoreactive Coupling.
Coupling of CD's to fluorophores can also be done by combin¬ ing in the dark, said immobilized CD with any suitable hetero- bifunctional , photoreactive-a ino-coupling agent previously des¬ cribed, such as HSAB, NHS-ASA, among others, under suitable con¬ ditions for coupling. Then, said fluorophore is added and the photoreactive group is activated with a suitable light source to initiate coupling.
Schematically, wherein n = a multiple of 1 or more, the gen¬ eral structure is:
Figure imgf000050_0001
f. Antenna Substances.
A new CD label composition with potentially greater luminesc¬ ent efficiency can be synthesized by coupling certain light and/or energy collecting substances (herein called "antenna" substances), to said CD labels. Said antenna substances can be coupled to said label in various ways to promote the most effi¬ cient activity. For instance, said antennas can be suitably coupled to the CD molecule, to the fluorophore guest, or to an intermediate substance that is part of the CD label. Examples of suitable antenna substances are aromatic compounds, folic acids, carotenoids, retinols, retinals, rhodopsins, chlorophylls, blue fluorescent proteins (eg. from bacteria), green fluorescent proteins (eg. from renilla, etc.), tryptophan and/or tyrosine-containing substances (eg. polypeptides) , and various derivatives, analogs and precursors of said antenna sub¬ stances.
After a sufficient number of captured guest fluorophores have been coupled to said CD's, the entire group is cleaved from the solid support as described previously, providing the free label with a functional group for coupling to any suitable substance.
Schematically, wherein n = a multiple of 1 or more, the gen¬ eral structure is:
Figure imgf000051_0001
C. Capped Guest CD Labels.
Another scheme has been discovered for synthesizing said guest-CD labels that include the use of capped CD's wherein said capping also provides a means for coupling. When CD's are capped with reagents that produce ketones, the available ketone is subsequently reacted with an amino-carboxyl ic acid or diamino compound to produce the corresponding oxime coupling. For inst¬ ance, capping with terephthaloyl chloride produces ketones that can be reacted with carboxymethoxylamine hemihydrochloride, or hydrazinobenzoic acid, among others, to introduce a carboxylic acid group that can be coupled through a mixed anhydride reac¬ tion (eg. Erlanger, supra), or derivatized to give an NHS coupl¬ ing group.
PREPARATION XII. NHS-rCDln Labeled Protein
In this example, the N-succinimidyl reactive group on NHS- [CD]n , previously described, reacts with primary and secondary aliphatic amines on the protein. A suitable protein such as antibody, or avidin (or streptavidin) is labeled by mixing 50 micrograms of protein in 0.2 ml of 0.1 M phosphate buffer (PB), pH 8, with approximately 10 micro!iters of acetonitrile contain¬ ing 2.5 micrograms of dissolved NHS-[CD]n , in a glass vial for 15 minutes..
Then, 0.1 ml of a 10 mg/ml solution of lysine monohydro- chloride in PB, pH 8, is added, mixed and let stand 15 minutes. The mixture is applied to a column of G25 equilibrated with PBS composed of PB, pH 6.3, containing 0.15 M NaCl and 0.02 sodium azide. The column is eluted with PBS and labeled fractions in the void volume are pooled and concentrated if necessary, by vacuum dialysis.
The reaction conditions may be varied appropriately, such as the inclusion of an intermediate coupling substance, to obtain a labeled protein or nucleic acid that is suitably coupled to a plurality of NHS-[CD]n and sufficiently retains the binding pro¬ perties needed for use as a tracer. Such conditions that may be appropriately varied are, amounts of reagents, times, tempera¬ ture and the use of other compatible buffers, solvents and addi¬ tives.
Alternativel , said NHS-[CD]n is substituted for one of said labels from previous preparations wherein said label contains an N-hydroxysuccinimidyl coupling group. Also, said NHS-[CD]n can be substituted for one of said labels from previous preparations wherein said label contains a coupling group or functional group that is not an N-hydroxysuccinimidyl coupling group. In this case, conditions can be appropriately modified by one skilled in the art. PREPARATION XIII. CD Labeled Nucleic Acid Probes Any known method for preparing DNA, oligonucleotide, or RNA probes may be used for synthesizing probes for coupling to the CD labels of the instant invention, provided the method is suit¬ ably modified when necessary, to use biotinylated nucleotides or allylamine derivatized nucleotides or some other nonradioactive label or ligand in place of the radioactive label.
A. A suitable DNA probe is prepared by cloning the desired DNA into M13mp8 bacteriophage DNA, using standard methods. Dur¬ ing synthesis of the complementary strand of the single stranded M13 vector, biotinylated nucleotides or allylamine derivatized nucleotides or some other nonradioactive label or ligand is incorporated into it. Alternatively, the vector DNA can be left double stranded and suitably labeled with biotin, allylamine or appropriate ligands using well known methods including DNA intercalators, psoralens and crosslinkers, among others.
B. A suitable oligonucleotide probe is synthesized that is complementary to the nucleic acid sequence to be detected using standard methods. The probe includes appropriate biotinylated, allylamine derivatized, or ligand coupled nucleotides.
C. A suitable RNA probe is prepared using standard methods such as transcription from the appropriate DNA template contain¬ ing appropriate vectors such as pSP and/or pT7 and/or pT3, among others.
During probe synthesis, suitable nucleotides for incorpora¬ tion are bio-11-UTP and/or allylamine UTP. Also, the probe is suitably biotinylated during or after synthesis using known methods and the necessary reagents for fragmentation, purifica¬ tion and biotinylation, which can be purchased as pre-packaged kits from several sources. Examples are, Enzo Biochemicals, NY, and Bethesda Research Laboratories, MD.
It may also be desirable to use a photoactive coupler and introduce a "spacer arm", between the biotin or allylamine and thδ nucleic acid sequence. A spacer arm is desirable because it helps prevent binding interference due to steric hindrance.
When preparing a biotinylated or allylamine derivatized probe using double stranded DNA, the nick-translation procedure may be used. Several commercial kits with reagents and instructions are also available for this procedure. The nucleic acid sequence to be hybridized in the double stranded probe would have to be denatured to produce the single stranded form before use.
When a probe has been biotinylated, then virtually any desired CD label can be noncovalently coupled to it. For inst¬ ance, any CD that is coupled to avidin or streptavidin can be noncovalently coupled to the probe by allowing the coupled avidin or streptavidin to bind to the probe.
Alternatively, another suitable scheme for noncovalent coupl¬ ing is to incorporate or produce an antigen or hapten into the probe composition. Then, the appropriate, specific antibody will noncovalently couple to that antigen. For instance, the probe can be sulf nated or have digoxin, digoxigenin, or another suitable antigen incorporated into the composition, and then allowed to couple to the appropriate labeled antibody. This way, any CD label that is coupled to an antibody, can be non¬ covalently couρ,ed to a probe.
Alternatively, when a probe is synthesized to provide primary amino groups such as through ally!amines, polyethyleneimines, and/or any other functional groups, and/or any appropriate lig¬ and, then any CD label that can be coupled through said groups can be coupled to the probe using well known methods. For exam¬ ple, different colored probes can be prepared by labeling with different colored CD fluorophore labels, previously described.
Suitable probe synthesis methods, which are hereby incorpor¬ ated into the instant invention by reference, are described by:
Luehrsen, K.R., et al , Biotechniques 5, 660 (1987);
Langer-Safer, P.R., et al , Proc. Nat! . Acad. Sci . USA 78, 6633 (1981);
Leary, J.J.,et al , Proc, Nat! . Acad. Sci. USA 80, 4045-4049 (1983);
Melton, D.A. , et al , Nucl . Acids Res. 12, 7035-7056 (1984);
Rigby, P.W.J., et al , J. Mol . Biol . 113, 237-251 (1977);
Ruth, J.L., et al , Nucleosides and Nucleotides 6(1&2), 541 (1987);
Ruth, J.L., et al , DNA 4, 93 (1985);
Ruth, J.L., et al, Fed. Proc. 43, 2048 (1984);
Seyfert, H-M. BRL Focus 7, 3-4 (1985); among others. PREPARATION XIV. Mercantile Kits
Any of the preparations disclosed are readily incorporated into a mercantile test kit. Said test kit can contain one or more of the said preparations as needed, and other appropriate reagents and/or solutions for performing the intended assays, with suitable containers and instructions for storage and use. Said preparations and reagents can be packaged in various forms including frozen and/or lyophilized.
The methods and applications disclosed in the foregoing references are hereby incorporated into the instant application by reference.
METHOD I.
Nucleic Acid Sequencing with CD Labeled Chain Terminators
The principle of this method is based on the use of labeled, dideoxyribonucleoside triphosphates (chain terminators), which cause termination of oligonucleotide synthesis when incorporated into the chain in place of normal nucleotides, (eg. Sanger, or Prober, supra). The terminators compete with their normal nuc¬ leotide analogs and terminate the primer-initiated, template- dependent synthesis at specific positions corresponding to the substituted nucleotide. This produces labeled oligonucleotide fragments of various lengths which are subsequently separated by size and detected through the incorporated label.
In the method of the instant invention, CD labels are used wherein each chain terminator is coupled to a CD label, or [CD]n label, complexed with a different colored guest fluorophore or a different guest CL compound. This has the advantage of allowing the use of a wide range of guest molecules that are inside the CD host so that chemical and physical differences between CD labels are minimized. In addition, inclusion of the fluorophore or CL compound by the CD label enhances the signal generating efficiency and detec-abi1ity of the label.
A. DNA Sequencing Method.
Four chain terminator preparations are prepared, each labeled with a CD fluorophore label complexed with a different colored guest molecule, as described previously. Said labels can also include captured guests, and various derivatives and/or capping, as described previously.
Suitably, amino derivatives of the chain terminator triphos- phates, dideoxycytidine (ddCTP), dideoxythymidine (ddTTP), dide- oxyadenosine (ddATP), and dideoxyguanosine (ddGTP), are each coupled to different colored NHS-CD labels (suitably, CD labeled dideoxyuridine (ddUTP), can be used in RNA analysis). Said NHS- CD labels can also be multiple CD labels.
In any case, said labeled chain terminators are prepared so that they meet the requirements of, (1) the fluorophore guests are suitably efficient emitters, (2) the emission spectra or colors, are easily distinguishable for each terminator, and (3) the label-s do not significantly impair sequencing reactions of the chain terminators.
From the nucleic acid to be sequenced, such as DNA, nucleic acid template is suitably prepared by restriction enzyme frag¬ mentation and purification. Under appropriate conditions, said nucleic acid templates and suitable primer are combined to form annealed template-primer. Said annealed template-primer is then combined, in a reaction mixture in appropriate buffer, with suf¬ ficient amounts of the appropriate nucleotides and suitable synthesizing enzymes such as polymerases, transcriptases, and others, to promote oligonucleotide synthesis (primer extension). After a suitable incubation period, the corresponding CD-labeled chain terminators are added and appropriately incubated to allow termination of synthesis with the formation of CD-labeled oligonucleotide fragments.
Said CD-labeled fragments are removed from unincorporated label and separated by size. Said labeled fragments are detected and discriminated by the color of light generated through activation of the CD label.
For example, a section of M13mp18 DNA (3 micrograms), is annealed with 60 ng of 17 bp primer. The annealed template- primer is combined with 250 picomoles each of deoxyadenosine triphosphate (dATP), deoxycytosine triphosphate (dCTP), deoxy- thymidine triphosphate (dTTP), and deoxyguanosine triphosphate (dGTP), or, 2'-deoxy-7-deazoguanosine triphosphate (c7dGTP), in place of dGTP (see Prober, supra). The buffer used is 60 mM tris HC1 , pH 8.3, 7.5 mM MgCl∑ , 75 mM NaCl , 0.5 M dithiothreitol and 17 units of AMV reverse transcriptase. After incubating at 42 °C for 12 minutes, excess of each CD-labeled chain terminator (100-1000 picomoles), is added and incubated an addition 30 minutes or until terminal labeling is completed. The resulting CD-labeled fragments are removed from unincorporated label by gel column chromatography and separated by size to determine the number of bases per fragment. Separa¬ tion is done using any suitable method such as polyacrylamide gel electrophoresis or high pressure liquid chromatography, (HPLC).
B. Activation and Detection.
Said CD fluorophore labeled fragments are activated by any suitable energy transfer reaction described previously, such as combining them with a suitable peroxyoxalate and peroxide. Suitably, TCPO is added (eg. 600 mg/1 in 10-3 M phosphate buffer, pH 6), and in a dark environment, the activation is started with the addition of H2O2 (eg. 10_5-10-6 M). Other sub¬ stances are suitably included in the buffer such as ethyl acetate, stabilizers and surfactants, as needed. Said reagents can be combined with said CD labeled fragments to activate in said gel. Alternatively, fragments removed from the gel or HPLC column, are detected in suitable containers or a continuous flow detection system.
Detection is done in the dark with a photometer or other light detector that can discriminate between the emission wavelengths (eg. four colors), of the CD fluorophore labels used. Suitably, said light detector employs photomultipliers, or charge coupled devices (CCD), (eg. video camera), or photodi- odeε with appropriate filters and/or grids as needed. When reading light emission directly from fragments in a gel, a scan¬ ning type of system (eg. using fiber optics), can be used to detect the variously sized fragments and record light intensity, color and location in the gel.
Suitably, the light emission data is collected in a com¬ puterized, automated system. Using known sequencing and statistical methods, it can then be determined which labeled nucleotide (by color), terminated synthesis at each base incre¬ ment (by fragment size), of the nucleic acid chain being ana¬ lyzed. METHOD II. Nucleic Acid Sequencing with CD Labeled Primers The principle of this method is similar to Method I, above, except that labeled oligonucleotide primers are used instead of labeled chain terminators (eg. Sanger or Smith, supra). In the method of -the instant invention, CD labels are used wherein each primer is coupled to a CD label, or [CD]n label, complexed with a different colored guest fluorophore or a different guest CL compound. ,
A. DNA Sequencing Method.
Four oligonucleotide primers are prepared, each labeled with a CD fluorophore- label complexed with a different colored guest molecule, as described previously. Said labels can also include captured guests, and various derivatives and/or capping, as des¬ cribed previously.
Suitably, an amino derivative of a primer (eg. an oligonucleotide with a single aliphatic amino group at the 5' terminus), is coupled to a NHS-CD label, or multiple NHS-CD label, complexed with a different colored guest fluorophore.
In any case, said labeled primers, are prepared so that they meet the requirements of (1 ) the fluorophore guests are suitably efficient emitters, (2) the emission spectra or colors, are easily distinguishable, and (3) the labels do not significantly impair sequencing reactions.
From the nucleic acid to be sequenced, such as DNA, nucleic acid template is suitably prepared by restriction enzyme frag¬ mentation and purification. Under appropriate conditions, sepa¬ rate base specific reactions are performed for each base to be detected, wherein said nucleic acid template and suitably CD- labeled primer are combined to form annealed template-primer-CD.
Said annealed template-primer-CD is then combined, in a reac¬ tion mixture in appropriate buffer, with sufficient amounts of the appropriate nucleotides and suitable synthesizing enzymes such as Klenow, T7 or Taq polymerases, reverse transcriptases (eg. from avian myeloblastosis virus (AMV)), and others, to pro¬ mote oligonucleotide synthesis (primer extension). The choice of synthesizing enzyme will depend on conditions of the sequenc¬ ing reaction. Immediately, or after a suitable incubation period, the corresponding chain terminators are added and appro¬ priately incubated to allow termination of synthesis with the formation of CD-labeled oligonucleotide fragments.
The resulting CD-labeled fragments are suitably separated by size to determine the number of bases per fragment. Separation is done using any suitable method such as polyacrylamide gel electrophoresis or high pressure liquid chromatography, (HPLC).
B. Activation and Detection.
Said CD fluorophore labeled fragments are activated and detected as described previously in Method I, above.
Also, under suitable conditions for improving fluorescent methods, it may be desirable to activate said CD-labeled frag¬ ments of Methods I and II by an incident light source. This will provide the advantages of different colors with the advantage of using CD labels which provide similar chemical and physical properties with potentially higher fluorescent emission over fluorophore labels alone.
The versatility of the instant invention is seen in the application of the appropriate CD-labeled nucleotides in RNA sequencing using reverse transcriptases. In this approach, DNA fragments can also be labeled by the incorporation of a single CD-labeled dideoxy nucleotide at the 3' end with terminal deoxy- nucleotidyl transferase (eg. Trainor, G.L., et al , Nucl . Acid Res. 16, 11846, 1988).
Another suitable application for this invention is the so called "DNA fingerprinting" method disclosed by Livak, K. , et al , Amer. J. Human Gen. 43, A150 (1988), wherein the appropriate CD-labeled nucleotides are substituted for Livak's fluorescent nucleotides.
Other new methods have been discovered for replacing and improving upon the fluorescent nucleic acid sequencing methods of the prior art. For example, using suitable fluorophore labels in nucleic acid sequencing (eg. Prober or Smith, supra), the problems of fluorescent labels can be eliminated by activat¬ ing said labels through a suitable energy transfer reaction as described previously. Suitably, said labels can be activated with peroxyoxalate and peroxide, and detected as described in method I, supra, and/or by suitable modification of the method of Mahant, V.K., et al , Anal. Chim. Acta 145, 203-206 (1983), among others. An additional improvement is possible by adding free CD molecules to said labels to allow complexing of said free CD's with said fluorophores before said activation. OTHER APPLICATIONS It has been discovered that, with appropriate modification, any of the CD labels of the instant invention can also be used to label genetic probes in nucleic acid hybridization assays such as those disclosed in copending patent application SN 363,081. The different colored CD labels of the instant inven¬ tion can be substituted in place of the chemiluminescent labels of said previous disclosures and suitably activated by energy transfer or other means as described herein.
A. Use in Hybridization Assays.
For instance, several nucleic acid probes, each suitably coupled with a different colored CD fluorophore label, are suit¬ ably hybridized to sample nucleic acid that has been suitably immobilized, or, "sandwich" hybridized to sample previously hybridized to other nucleic acid. After removal of unhybridized probe, the hybridized probes are activated by any suitable energy transfer reaction (eg peroxyoxalates and peroxides), as described above. The colors of emitted light are suitably detected and/or recorded as described above, to indicate which probes hybridized.
Also, a homogeneous assay can be used wherein hybridization of the probes triggers activation of the CD labels. One homo¬ geneous method would have a peroxide generating enzyme, such as glucose oxidase, coupled in close proximity to the sample nuc¬ leic acid. Then, after CD labeled probe is hybridized to the sample, H2O2 is generated by the enzyme in close proximity to the bound probe, in the presence of substrate such as glucose. With available peroxyoxalate, an energy transfer reaction occurs, which activates said CD fluorophore labels to emit light.
For instance, the different colored CD fluorophore labels of the instant invention are also usable as labels for probes in the test devices previously described in copending application SN 363,081, wherein said glucose oxidase, energy transfer reac¬ tion can be used for activation.
Other nucleic acid hybridization schemes are possible such as the use of CD catalyst labels, and various hybridization assays that employ magnetic particles, among others.
Tests for specific nucleic acid sequences using single layer hybridization and, sandwich hybridization are well known in the art. A variety of methods and their applications are known that could be suitably modified to be used with the labels of instant invention and thereby produce new advantages and applications not previously disclosed or realized. Examples which are hereby incorporated into the instant invention by reference, are descr¬ ibed by:
Bers, G., et al , Biotechniques 3, 276-288 (1985) Brigati, D.J.,et al , Virol. 126, 32-50 (1983) Chan, V.T.W., et al , Nucl . Acids Res. 13, 8083-8091 (1985) Ehrat, M. , et al , Clin. Chem. 32/9, 1622-1630 (1986) Ellwood, M.S., et al , Clin. Chem. 32, 1631-1636 (1986) Feinberg, A.P., et al , Anal. Biochem. 132, 6-13 (1983) Fitts, R., et al , Applied Environ. Microbiol. 46, 1146-1151 (1983)
Gomes, S.A., et al , J. Virologic. Meth. 12, 105-110 (1985) Hames, B.D., et al , eds., IN: "Nucleic Acid Hybridization : A Practical Approach", IRL Press, Washington, DC, (1985);
Harper, M.E., et al , Proc. Nat! . Acad. Sci. USA 83, 772-776 (1986)
Isfort, R.J., et al , Biotech. 6, 138-141 (1988) Ish-Horowic∑, D.,et al , Nucl. Acids Res. 9, 2989-2998 (1981) Kazazian, Jr., H.H., Clin. Chem. 31/9, 1509-1513 (1985) Langer, P.R., et al , Proc. Nat! . Acad. Sci. USA 78, 6633-6637 (1981 )
Leary, J.J., et al , Proc. Natl . Acad. Sci. USA 80, 4045-4049 (1983)
Love, J., et al , Amer. Clin. Prod. Rev. 4, 12-16 (Aug 1985) Messing, J., in "Methods in Enzymology", Vol. 101, 20-79 (1983)
Palva, A.M., J. Clin. Microbiol. 18, 92-100 (1983) Rashtchian, A., et al , Clin. Chem. 33/9, 1526-1530 (1987) Reed, K.C., et al , Nucl. Acids Res. 13, 7207-7221 (1985). Rigby, P.W.J., et al , J. Mol . Biol. 113, 237-252 (1977) Sauls, CD., et al , Clin. Chem. 31/6, 804-811 (1985) Sheldon, E. , et al , Clin. Chem. 33/8, 1368-1371 (1987) Sigma Chemical Co., Technical Bull. No. PROBE-2 (1988) Spector, S.A., et al , Clin. Chem. 31/9, 1514-1520 (1985) Vary, C.P.H., et al , Clin. Chem. 32/9, 1696-1701 (1986). Virtanen, M., et al , Lancet, Feb.19, 381-383 (1983) Yokota, H., et al , Biochim. Biophys. Acta 868, 45-50 (1986) B. Use in Immunoassays.
It has also been discovered that, with appropriate modifica¬ tion, any of the CD labels of the instant invention can also be used in immunoassays. CD label compositions and methods for their use in immunoassays are disclosed in U.S. patent applica¬ tion SN 418,843, filed Oct. 10, 1989, the contents of which are hereby incorporated by reference. Also, in the heterogeneous immunoassay methods disclosed in copending U.S. patent applica¬ tion SN 101,392* said liposomes can be suitably replaced with the different colored CD labels of the instant invention and provide new and versatile methods for immunoassay.
For instance,, several specific antibodies, each suitably coupled with a different colored CD fluorophore label, are suit¬ ably incubated and bound to sample antigen that has been suit¬ ably immobilized. Or, said antibodies are suitably bound in a "sandwich" immunoassay to sample antigen previously bound by captor antibody; After removal of unbound antibody, the labeled, bound antibodies are activated by any suitable energy transfer reaction (eg. peroxyoxalates and peroxides), as descr¬ ibed above. The colors of emitted light are suitably detected and/or recorded as described above, to indicate which antibodies bound and therefore which antigens were present.
Also, a homogeneous immunoassay can be used wherein binding of the CD labeled antibodies triggers activation of the CD labels. One homogeneous method would have a peroxide generating enzyme, such as glucose oxidase, coupled in close proximity to the sample antigen. Then, after CD labeled antibody is bound to the sample., H2O2 is generated by the enzyme in close proximity to the bound antibody, in the presence of substrate such as glu¬ cose. With ava lable peroxyoxalate, an energy transfer reaction occurs, whfich activates said CD fluorophore labels to emit light.
Other immunoassay schemes are possible such as the use of CD labeled antigen in a competitive assay, and various immunoassays that employ magnetic particles, among others. c- Use of CD Catalyst Labels.
The surprising versatility of the CD labels of the instant invention is also evident in the application of CD catalyst labels, described above.
For instance, said CD catalyst labels can be coupled to suit- able genetic probes for use in hybridization assays. In one method, a nucleic acid probe suitably coupled with a suitable CD catalyst label (eg. histamine derivatized), is suitably hybridized to sample nucleic acid that has been suitably immobi¬ lized, or, "sandwich" hybridized to sample previously hybridized to other nucleic acid. After removal of unhybridized probe, the hybridized probe is exposed to a suitable substrate. For exam¬ ple, said histamine derivatized CD catalyst label will hydrolyze p-nitrophenyl acetate in suitable solvent, to p-nitrophenol , detectable by an increase in spectrophotometric absorbance at 400 nm. A major advantage for using CD catalyst labels is their resiεtance to harsh conditions (eg. in nucleic acid hybridiza¬ tion), compared to enzyme labels in the prior art.
Said CD catalyst labels can also be used to great advantage in immunoassays. For instance, a specific antibody, suitably coupled with a suitable CD catalyst label (eg. histamine deriv¬ atized), is suitably incubated and bound to sample antigen that has been suitabiy immobilized. Or, said antibody is suitably bound in a "sandwich" immunoassay to sample antigen previously bound by captor antibody. After removal of unbound antibody, the labeled, bound antibody is exposed to a suitable substrate. For example, said histamine derivatized CD catalyst label will hydrolyze p-nitrophenyl acetate in suitable solvent, to p- nitrophenol, detectable by an increase in spectrophotometric absorbance at 400 nm.
Said CD catalyst labels can also be used in chemical synthesis or detoxification, wherein said CD catalyst labels are coupled to a suitable solid support or to magnetic particles. For instance, when coupled to said magnetic particles, they are exposed to a suitable solution containing the chemical substrate to be catalytically reacted upon (eg. oxidation, hydrolysis, transformation, etc.), such as in a slurry in a continuous flow system. After sufficient reaction, said magnetic particles are suitably collected by magnetic force and recycled.
D. Other Substanceε as Labels.
Suitably, other substances that have the necessary properties to form an inclusion complex with said CL substance, BL subs¬ tance or fluorophore previously described, or any fluorescent, phosphorescent, or scintillator substances, laser dyes, or organic dyes, can be used to produce new and useful composi- tions. For instance any suitable hollow molecule or substance with a hydrophobic interior, can be substituted for said cyclo¬ dextrins. Said substitute substances can be composed of any other carbohydrates such as soluble or colloidal polymers and helical segments of amyloses. Also usable are celluloses, agars or agaroses, and other substances such as proteins, lipids, lip- oproteins, nucleic acids, surfactants, virus coat proteins, organic molecules, as well as porous particles of silicas, acrylamides, nylons, polystyrenes, resins, metals and cellu¬ loses, and combinations of these.
E. Other Host Moleculeε as Labels.
It has been discovered that certain other subεtances are suitable as hosts in forming inclusion complexes and/or as cata¬ lysts, which can provide labels with new and unexpected advantages and properties. Examples are calixarenes, cyclophanes, and certain crown ethers, including their analogε, derivatives, and precursors. With appropriate modification, these other substances can be used in place of cyclodextrins in the preparations and methods of the instant invention. Examples of calixarenes are disclosed by Gutsche, CD., et al , J. Indus. Phenom. Molec. Recog. Chem. 7, 61-72 (1989). Examples of cyclophanes are disclosed by Bukownik, R.R., et al , J. Organ. Chem. 53, (1988), Murakami, Y., et al , J. Chem. Soc. Perkin Trans. I., 1289-1299 (1988), Murakami, Y., et al , J. Indus. Phenom. 2, 35-47 (1984), and Reid, W., et al , Tetrahedron 44, No. 11, 3399-3404 (1988). Examples of crown ethers are dis¬ closed by Benetollo, F. , et al , J. Indus. Phenom. 5, 165-168 (1987), and Shin ai , S. , et al , J. Indus. Phenom. 2, 111-118 (1984). These references, including references contained therein, are applicable to the synthesis of the preparations and components of the instant invention and are hereby incorporated by reference, herein.
New and useful compositionε can also be synthesized wherein previously described cyclodextrin label preparations, above, can be used as labels inside any suitable liposomes that have lig¬ ands, ligators or nucleic acids coupled to their surface. Said liposomes can include the polymerized liposomes and "macro" lip¬ osomes disclosed in copending patent application SN 101,392.
Under appropriate conditions, said polyaromatic CL labels described in the copending patent application described above, can be further improved wherein any εuitable cyclodextrin lε included in the label composition to form a complex with said label and enhance light emiεsion.
While the invention has been described with reference to certain specific embodimentε, it is understood that changes may be made by one skilled in the art and it would not thereby depart from the spirit and scope of the invention which is limited only by the claims appended hereto.

Claims

1. A method for preparing a cyclodextrin tracer, comprising the coup!irig of;
(1) A cyclodextrin molecule, selected from the group con¬ sisting of cyclodextrins, cyclodextrin derivatives, and cyclo¬ dextrin labels, and wherein said cyclodextrin molecule includes an inclusion compound selected from the group consisting of fluorophores, scintillators, chemiluminescent substances, dyes, and substrates, to;
(2) a specific binding substance to be labeled, selected from the group consisting of ligands, ligators and nucleic acids.
2. The method of claim 1 wherein said cyclodextrin molecule includes a substance selected from the group consisting of cap¬ tured guests, antenna substances, capping substances, and acti¬ vators.
3. The method of claim 1 wherein said cyclodextrin molecule includes a substance for coupling to said specific binding subs¬ tance, that is selected from the group consisting of spacers and intermediate substances.
4. The method of claim 1 wherein said cyclodextrin molecule is a plurality of cyclodextrin molecules, coupled to said speci¬ fic binding substance.
5. The method of claim 1 wherein said specific binding subs¬ tance is selected from the group consisting of antigens, anti¬ bodies, biotins, avidins, streptavidins, lectins, and receptors.
6. A method for sequencing a nucleic acid sample using cyc¬ lodextrin labeled, chain terminators, comprising;
(1) bringing together under conditions conducive to nuc¬ leic acid synthesis;
(a) fragments of said nucleic acid sample annealed to suitable oligonucleotide primer, with;
(b) synthesizing enzymes and nucleotides for nucleic acid synthesis with; (c) a plurality of chain terminator nucleotides each previously coupled to a cyclodextrin label, wherein said nucleic acid synthesis is specifically terminated by incorporation of said chain terminatorε to produce a plurality of corresponding cyclodextrin labeled fragments, and;
(2) separation of said cyclodextrin labeled fragments by size, and;
(3) detection of said cyclodextrin labeled fragments.
7. The method of claim 6 wherein said cyclodextrin label is a cyclodextrin fluorophore label and said detection comprises detection of emitted light from said cyclodextrin fluorophore label by activating said label through an energy transfer reac¬ tion.
8. The method of claim 6 wherein said cyclodextrin label is a cyclodextrin chemiluminescent label and said detection com¬ prises detection of emitted light from said cyclodextrin fluoro¬ phore label by activating said label through a chemiluminescent reaction.
9. The method of claim 6 wherein said cyclodextrin label is a cyclodextrin catalyst label and said detection comprises detection of a reaction product produced from a substrate.
10. The method of claim 6 wherein said cyclodextrin label is a cyclodextrin energy transfer label.
11. The method of claim 6 wherein said cyclodextrin label is a cyclodextrin captured guest label.
12. The method of claim 6 wherein said cyclodextrin label includes an antenna substance.
13. A method for sequencing a nucleic acid sample using cyc¬ lodextrin labeled primer, comprising;
(1) bringing together under conditions conducive to nuc¬ leic acid syntheεis;
(a) fragments of said nucleic acid sample annealed to ol igonucleotide primer that has been coupled to a cyclodextrin l abel , wi th ;
(b) synthesizing enzymes and nucleotides for nucleic acid synthesis with;
(c) a plurality of chain terminator nucleotides wherein said nucleic acid synthesis is specifically terminated by incorporation of said chain terminators to produce a plurality of corresponding cyclodextrin labeled primer fragments, and;
(2) separation of said cyclodextrin labeled primer frag¬ ments by size, and;
(3) detection of said cyclodextrin labeled primer frag¬ ments.
14. The method of claim 13 wherein said cyclodextrin label is a cyclodextrin fluorophore label and said detection comprises detection of emitted light from said cyclodextrin fluorophore label by activating said label through an energy transfer reac¬ tion.
15. The method of claim 13 wherein said cyclodextrin label is a cyclodextrin chemiluminescent label and said detection com¬ prises detection of emitted light from said cyclodextrin fluoro¬ phore label by activating said label through a chemiluminescent reaction.
16. The method of claim 13 wherein said cyclodextrin label is a cyclodextrin catalyst label and said detection comprises detection of a reaction product produced from a substrate.
17. The method of claim 13 wherein said cyclodextrin label is a cyclodextrin energy transfer label.
18. The method of claim 13 wherein said cyclodextrin label is a cyclodextrin captured guest label.
19. The method of claim 13 wherein said cyclodextrin label includes an antenna substance.
20. A method for sequencing a nucleic acid sample using fluorophore labeled chain terminators, comprising;
(1) bringing together under conditions conducive to nuc- leic acid synthesis;
(a) fragments of said nucleic acid sample annealed to suitable oligonucleotide primer, with;
(b) synthesizing enzymes and nucleotides for nucleic acid synthesis with;
(c) a plurality of fluorophore labeled, chain terminator nucleotides wherein said nucleic acid synthesis is specifically terminated by incorporation of said chain terminators to produce a plurality of corresponding fluorophore labeled fragments, and;
(2) separation of said fluorophore labeled fragments by size, and;
(3) detection of emitted light from said fluorophore labeled fragments by activating said inclusion complexes through an energy transfer reaction.
21. The method of claim 20 wherein said detection of emitted light includes combining said fluorophore labeled fragments with free cyclodextrin molecules to form inclusion complexes and activating said inclusion complexes through an energy transfer reaction.
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WO1995002700A1 (en) * 1993-07-13 1995-01-26 Abbott Laboratories Fluorescent polymer labeled conjugates and intermediates
WO1995009247A1 (en) * 1993-09-29 1995-04-06 Institut National De Recherche Et De Securite Pourla Prevention Des Accidents Du Travail Et Des Maladies Professionnelles Method for performing a bioassay for identifying and assaying genes, proteins and other molecules
WO1995032739A1 (en) * 1994-06-01 1995-12-07 Hybridon, Inc. Cyclodextrin cellular delivery system for oligonucleotides
US5661040A (en) * 1993-07-13 1997-08-26 Abbott Laboratories Fluorescent polymer labeled conjugates and intermediates
WO1998026287A1 (en) * 1996-12-12 1998-06-18 Cis Bio International Non-aggregated fluorescent conjugates and method for obtaining them
US6120987A (en) * 1997-12-12 2000-09-19 Cis Bio International Non-aggregated fluorescent conjugates and the process for their preparation
FR2878853A1 (en) * 2004-12-07 2006-06-09 Biocydex Soc Par Actions Simpl Process for obtaining fluorescent complex, comprises establishment of contact between modified cyclodextrins and fluorochromes, agitation of the complex and extraction of soluble fraction
WO2010066908A1 (en) * 2008-12-12 2010-06-17 Eurogentec S.A. Use of cyclodextrins to improve the specificity, sensitivity and yield of nucleic acid amplification reactions
WO2014171946A1 (en) * 2013-04-18 2014-10-23 Empire Technology Development, Llc Methods and systems for labeling and detecting defects in a graphene layer
US9260711B2 (en) 2012-09-13 2016-02-16 Ge Healthcare Uk Limited Solid matrix for one step nucleic acid amplification
US10890848B2 (en) 2017-12-20 2021-01-12 Samsung Electronics Co., Ltd. Material for detecting photoresist and method of fabricating semiconductor device using the same
CN112229991A (en) * 2020-10-16 2021-01-15 厦门圣科环保科技股份有限公司 Nano-capture device and preparation method and application thereof

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Cited By (21)

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Publication number Priority date Publication date Assignee Title
WO1995002700A1 (en) * 1993-07-13 1995-01-26 Abbott Laboratories Fluorescent polymer labeled conjugates and intermediates
US5661040A (en) * 1993-07-13 1997-08-26 Abbott Laboratories Fluorescent polymer labeled conjugates and intermediates
WO1995009247A1 (en) * 1993-09-29 1995-04-06 Institut National De Recherche Et De Securite Pourla Prevention Des Accidents Du Travail Et Des Maladies Professionnelles Method for performing a bioassay for identifying and assaying genes, proteins and other molecules
FR2710655A1 (en) * 1993-09-29 1995-04-07 Inst Nat Rech Securite Method for performing a biological test for identifying and assaying genes, proteins and other molecules
WO1995032739A1 (en) * 1994-06-01 1995-12-07 Hybridon, Inc. Cyclodextrin cellular delivery system for oligonucleotides
US5605890A (en) * 1994-06-01 1997-02-25 Hybridon, Inc. Cyclodextrin cellular delivery system for oligonucleotides
US5616565A (en) * 1994-06-01 1997-04-01 Hybridon, Inc. Cyclodextrin cellular delivery system for oligonucleotides
US5691316A (en) * 1994-06-01 1997-11-25 Hybridon, Inc. Cyclodextrin cellular delivery system for oligonucleotides
WO1998026287A1 (en) * 1996-12-12 1998-06-18 Cis Bio International Non-aggregated fluorescent conjugates and method for obtaining them
FR2757162A1 (en) * 1996-12-12 1998-06-19 Cis Bio Int NON-AGREGATED FLUORESCENT CONJUGATES
US6120987A (en) * 1997-12-12 2000-09-19 Cis Bio International Non-aggregated fluorescent conjugates and the process for their preparation
FR2878853A1 (en) * 2004-12-07 2006-06-09 Biocydex Soc Par Actions Simpl Process for obtaining fluorescent complex, comprises establishment of contact between modified cyclodextrins and fluorochromes, agitation of the complex and extraction of soluble fraction
WO2010066908A1 (en) * 2008-12-12 2010-06-17 Eurogentec S.A. Use of cyclodextrins to improve the specificity, sensitivity and yield of nucleic acid amplification reactions
JP2012511317A (en) * 2008-12-12 2012-05-24 ウロジャンテック、ソシエテ、アノニム Use of cyclodextrins to improve the specificity, sensitivity and yield of nucleic acid amplification reactions
US9260711B2 (en) 2012-09-13 2016-02-16 Ge Healthcare Uk Limited Solid matrix for one step nucleic acid amplification
WO2014171946A1 (en) * 2013-04-18 2014-10-23 Empire Technology Development, Llc Methods and systems for labeling and detecting defects in a graphene layer
US9297768B2 (en) 2013-04-18 2016-03-29 Empire Technology Development Llc Methods and systems for labeling and detecting defects in a graphene layer
CN105122044B (en) * 2013-04-18 2018-01-02 英派尔科技开发有限公司 Mark and the method and system for detecting the defects of graphene layer
US10890848B2 (en) 2017-12-20 2021-01-12 Samsung Electronics Co., Ltd. Material for detecting photoresist and method of fabricating semiconductor device using the same
CN112229991A (en) * 2020-10-16 2021-01-15 厦门圣科环保科技股份有限公司 Nano-capture device and preparation method and application thereof
CN112229991B (en) * 2020-10-16 2022-08-16 厦门圣科环保科技股份有限公司 Nano-capture device and preparation method and application thereof

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