CA2253969A1 - Non-nucleotide linking reagents - Google Patents

Abstract

Non-nucleotide reagents capable of forming oligomers with nucleotide units are disclosed, together with intermediates for synthesizing such non-nucleotide reagents, oligomers incorporating such reagents, kits containing such reagents and methods for their use in forming oligomers with nucleotide units.

Description

Wo 97/43451 pcTluss7lo9os4 Description Non-Nucleotide ~ inkin~ R~pen Technical Field The present invention relates generally to the use of non-nucleotide reagents as monomeric units in oligonucleotides.

Ba~k.ground of the Invention In both rcsearch applir~tio~ and clinical ~ gn~sis, a known technique for d~L~ inillg the presence of a particular nucleotide sequence (the "target nucleotide sequenr.e") in either RNA or DNA is to pclroll-- a nucleic acid hybridization assay.
In such an assay, a nucleotide probe, typically an oligonucleotide, is selectr~l having a nucleotide sequence complPm~nt~ry to at least a portion of the target nucleotide sequence. Typically, the probe is labeled to provide a means whereby the plesellce of the probe can be readily detected.
When the labeled probe is exposed to a sarnple suspected of co,.l~i..i..g the target nucleotide sequence, under hybridizing conditions, the target sequence will hybridize with such a labeled probe. The presence of the target sequence in the sample can then be deLe~ ed qualitatively or qu~ ively, usually after sepd~alillg hybridized and non-hybridized probes and deLc~ ing the presence or amount of the labeled probe which hybridized to the test sample.
Prior m~tho~ for linking a label to a nucleotide probe have generally utilized a single label att~rhP l to a nucleotide ml~m~meric unit, and then incol~o~aling one or more of the nucleotide l"onoll,eric units into the probe. For example, analogs of dUTP and UTP col.~ g a biotin moiety have been chrmir~lly syn~hrci7~ and inco~ora~ed into polynucleotides (P.R. Langer et al., Proc. Nat. Acad. Sci. USA 78:6633 (1981)). Such biotin-labeled nllrleotitles maythen be inco~ dled into nucleic acid probes of biological or synthetic origin.
Other methods for labeling nucleotide probes have been proposed which allow labels to be randomly linked to nucleotides in a nucleotide mlll~im~r.
Numerous proposals have been made for inco~o~dLhlg multiple mo~ifilo~

CA 022F,3969 1998-11-09 WO 97t434Sl PCT/US97/09094 nucleotides or non-nucleotide monomeric units into oligonucleotides with a view towards enhancing the ~lçtect~bility of the labeled probe and the target nucleotide sequence.
However, it has been dell~o~sll~led that use of such labeled nucleotides in a S probe can reduce the stability of the hybrid formed with a target nucleotide sequence, particularly when multiple labels are present. Such reduced hybrid stability has been d~rn~-n~trated for nucleic acid probes of biological origin possessing multiple biotin moieties, for synthetic oligonucleotides possçssi~-e multiple fluorescein labels, as well as for synthetic oligonucleotides possessing biotin and fluorescein labels.
In addition, derivatives of nucleotide linking phosphate groups have been disclosed, the nucleophilic moiety of which can be labeled following their incorporation into an oligonucleotide. However, such compounds, being based on nucleotide derivatives, would be expected to exhibit some of the disadvantages discussed above for nucleotide based derivatives.
More recently, 2-amino-1,3-propanediol structures have been used to label oligonucleotides with reporter groups (Nelsen, P.S. et al., Nuc. Acids Res. 20:6253 (1992)). However, these structures appear to demonstrate low coupling efficiency, and thus low yield of labeled oligonucleotides which furthermore must be carefully purified before they can find use as probes for target sequçn~-es.
Thus it is considered desirable to provide a non-nucleotide reagent which demo~ a~es high coupling efficiency and thus provides higher yield of labeled oligomer.
Furthermore, it is also considered desirable to provide such a reagent which will allow the rçsll1t~nt oligomers to anneal and hybridize with efficiencies approaching those of oligomers which contain only native nucleotide monompric units.

.

- CA 022~3969 1998-11-09 PCr/US 97/09094 ~P~US 2 3 J~ 1998 Disclosure of the ~nvention The present invention provides non-nucleotide reagents capable of forming an oligomer with nucleotide units, said reagents comprising compounds of the formula:
Rl - X - CH2 -/&- CH2 - R3 X' xl2 wherein R' is selected from the group con~i~ting of hydrogen, acid-sensitive, base-stable blocking groups and acyl capping groups;
X is selected from the group consisting of O, S, NH and N=N;
Xl is a substituted or unsubstituted C5 to C7 cyclic moiety incorporating the carbon atom of the formula;
X2 is selected from the group consisting of O, S, CH2, NH and N=N; and X3 is hydrogen or a linking functional group which is capable of linking with a functional moiety; and R3 is a linking group of the formula X4 x6 (a)-OP or (b)-OP=o wherein X4is halogen or subsli~uled amino, X5is alkyl, alkoxy or phenoxy, or a cyano derivative thereof, X6 is halogen, amino or O, and X7is alkyl, alkoxy or aryloxy, or may be H only if x6 is O, or R3 is a bond, either directly or through an i~lel ",~ te group, to a solid support.

~tl~Srirl~

CA 022~3969 1998-11-09 Such reagents can be used to label or otherwise incorporate desirable funr,tion~lhi~s into oligomers, utili7.ing conventional automated nucleotide synthetic protocols.
The present reagents preserve the natural three carbon internucleotide phosphatedi~t~nre, so as to preserve the hybri-1i7.~tion and ~nn~ling properties of the nucleotide duplex.
Also provided in the present invention are interm-o,tli~t~s useful for producingsuch non-nucleotide reagents, oligomers incorporating such reagents, kits cont:~ining such reagents and methods for use of the reagents in forming oligomers with nucleotide units.
Brief Description of the Draw;ng~
Figure 1 is a schematic depiction of the synthetic protocol of Example l(a), steps I and II;
Figure 2 is a sçh~m~tic depiction of the synthetic protocol of Example l(a), steps III, IV and V;
Figure 3 is a sr,h~m~tic depiction of the synthetic protocol of Example l(b);
Figure 4 is a sc-hem~tic depiction of the synthetic protocol of Example l(c);
and Figure 5 is a sçhem~tic depiction of the synthetic protocol of Example l(f).

CA 022~3969 1998-11-09 Detailed Description of the Invention The present invention provides non-nucleotide reagents capable of forrning an oligomer with nucleotide units, said reagents comprising compounds of the formula:
Rl - X - CH2 - C - CH2 - R3 xl x2 wherein Rl is selected from the group consisting of hydrogen, acid-sensitive, base-stable blocking groups and acyl capping groups;
X is selected from the group consisting of O, S, NH and N= N;
X' is a substituted or unsub~liluled C5 to C7 cyclic moiety incorporating the carbon atom of the formula;
X2 is selected from the group consisting of O, S, CH2, NH and N=N; and X3 is hydrogen, or a linking functional group which is capable of linking with a functional moiety; and R3 is a linking group of the formula X4 x6 (a)-OP or (b)-OP=o wherem X4is halogen or subsliluled amino, X5is alkyl, alkoxy or phenoxy, or a cyano derivative thereof, X6 is halogen, amino or O, and X7is alkyl, alkoxy or aryloxy, or may be H only if x6 is O, or R3 is a bond, either directly or through an interm~li~te group, to a solid support.

AMENDED SH~

CA 022~3969 1998-11-09 PCT/US 97/~9~94 IP~WS 2 3 JUN 1998 In the disclosure which follows, the following terms will have the in~lic~ted me~nings unless a contrary mP~ning is otherwise apparen~ from the context in which the term is used.
As used herein, the term "nucleotide" is taken to mean a subunit of a nucleic acid consisting of a phosphate group, a five carbon sugar and a nitrogen-cont~ining base. The term is also taken to include analogs of such subunits.
As used herein, the term "nucleotide oligomer" or "oligomer" is taken to mean a chain of nucleotides linked by phosphodiester bonds or analogs thereof.
As used herein, the term "nucleotide oligomer co..~ ing non-nucleotide monomers" is taken to mean an oligomer complised of nucleotide units together with non-nucleotide monomeric units linked by phosphodiester bonds or analogs thereof.
The present invention provides a non-nucleotide reagent which can be coupled synthetically with nucleotide monomeric units to produce a defined sequence oligomer with a backbone comprised of nucleotide and non-nucleotide monomeric units.
In the formula first provided above, n1 ~ r~ ~ ~H - R3 r~ -- A -- ~--~2 -- ~ ' 2 Xl Rl is a substit~lent group which is int~n-le l to be removed to farili~ate linkage with other units in the backbone structure of a nucleotide oligomer cont~ining non-nucleotide monoll~els. As such, Rl is generally selected from thegroup con~i~ting of hydrogen, acid-sensitive, base-stable blocking groups and acyl capping groups. Such groups are well known in the art, and include, for example,triphenylmethyl compounds, and alkoxy derivatives thereof, such as ~im~thoxytriphenyl (DMT) groups.
The group identified as X functions in part to Ill~il,l;.il~ proper intramolecular (li~t~nre in the non-nucleotide reagent when functioning as a monomeric unit.

AMENE~~D SHI~

llJ~ 97/O90~ 1~
II~IS 2 3 JUN 1998 Typically, X is selected from the group consisting of O, S, NH and N=N, althoughother atoms, or groups of atoms, could also serve in this capacity. Most commonly, X will be O.
The groups identified as Xl, X2, and X3 are substituent groups which are s intentled to facilitate linkage with other functional moieties, and other functional groups, which may be desired to be included in a nucleotide oligomer cont~ining non-nucleotide monomers.
Due to the chemical nature of the present non-nucleotide reagent, it may be positioned at any desired point within the nucleotide oligomer sequence. Thus it is possible to design a wide variety of pro~,e,lies into oligomers which contain both nucleotide and non-nucleotide monomeric units. Such prop~ ies include the ~tt~rhment of specific moieties herein termed "functional moieties" at any desired location within the oligomer. Such moieties can include (but are not limited to)detect~ble labels (including enzymatic, fluorogenic, radioactive, çhemihlminescent, and the like), interc~l~ting agents, metal chelators, drugs, hormones, ploteills, peptides, radical generators, nucleolytic agents, proteolytic agents, catalysts,specific binding agents (including biotin, antigens, haptens, antibodies, receptors, and the like), and other subst~nres of biological hlLle~.~, together with agentswhich modify DNA transport across a biological barrier, (such as a membrane), and substances which alter the solubility of a nucleotide mllltimrr. Thus it is possible to position such labels and agents adjacent to any desired nucleotide.
The groups X', X2, and X3 will col"~,ise a substituent to the carbon backbone of the formula in which: Xl is a sub~ ed or unsubstituted C5 to C, cyclic moiety incorporating the carbon atom of the formula; x2 is selected from the group con~i.cting of O, S, CH2, NH and N=N; and X3 iS a linking functional groupwhich is capable of linking with a functional moiety.
In the present reagent, the rigidity of the chrmir-~l structure of X ' provides that desirable feature of extending the linkage group and functional moiety away fromthe oligomeric backbone structure, thereby substantially enh~nring the coupling ~ 7~t ~

CA 022~3969 1998-11-09 W O 97/43451 PCTrUS97/09094 efficiency of the reagents of the present invention. Commonly, X I will be suks~ i or u~lsub~liLuled cyclohexane.
The group identi~led as x2 functions as a linking and modifiable reactive group. Typically, x2 is selected from the group consisting of O, S, NH, CH2, andN=N, although other atoms, or groups of atoms, could also serve in this capacity.
Most commonly, x2 will be NH.
In the formula, X3 is hydrogen, or a linking functional group which can be of any length a~ro~,idle to the particular functional moiety selected Typically, X3 is a group of the formula - CO - (CH2)" - NH - functional moiety.
wherein n is an integer from 0 to 20. It is of course within the invention to add the functional moiety to the reagent prior to, or after, the inclusion of the reagent as a monomeric unit in an oligomer. In addition, the functional moiety can also serve as a bond to a solid support.
In the formula, R3 is a substituent group which is int~n(1e-1 to facilitate linkage with other units in the backbone structure of a nucleotide oligomer cont~ining non-nucleotide monomers or to solid supports and the like. Typically,such linkage will be acco~ lished by automated methodologies, such as ?l~lo~ t~dDNA/RNA synthetic protocols. As such, R3 is generally selected from the group CollSis~ g of phosphodiesters, phosphotriesters, phosphites, phosphor~mi-1ites, H-phosphonates, alkyl-phosphonates, and phosphorothioates. Such groups are well known in the art, and include, for example, phosphorus linking group of the formula X4 x6 (a)-Op or (b)-Op = O

wherein X4is halogen or substit~te~l amino, Xs is alkyl, alkoxy or phenoxy, or a cyano derivative thereof, CA 022~3969 1998-11-09 W O 97/43451 PCT~US97/09Og4 Xfi is halogen, amino or O, and X7is alkyl, alkoxy or aryloxy, or may be H only if x6 is O, or R3 is a bond, either directly or through an intermediate group, to a solid support.
As ~ cussed above, the present non-nucleotide reagents will possess a linker functionality to which desired chemi~l moieties have been or can be att~hed, either prior to or after i.~ i..g the synthesis of the nucleotide oligomer.
In general, the techniques for linking moieties to the linker arm will be similar to the techniques known for linking labels to groups on proteins. Examples of useful chemistries include a reaction of alkyl amines with active esters, active imines, aryl fluorides or isothiocyanates, and the reaction of thiols with maleimides, haloacetyls, etc. (see generally Means, G.M. and R.E. Feeney, "Chemical Modification of Proteins" Holden-Day Inc. (1971); R.E. Feeney, Int. J. Pep~ide ProteinRes. 29:145-161 (1987)).
As di.~cu~sed above, due to the chemical nature of the present non-nucleotide reagent, it may be positioned at any desired point within the nucleotide oligomer sequence. Thus it is possible to design a wide variety of propc.lies into oligomers which contain both nucleotide and non-nucleotide monomeric units. Such prol)elLies include the att~chm~nt of specific functional moieties at any desired location within the oligomer.
Other benefits provided by the practice of the present invention include the ability to immobilize the defined sequence to a solid support by employing the linker arm filnction~lity conjoined to a rh~mir~l moiety of the support in order to construct, for example, nucleotide affinity ~u~oll~. Multiple ch~mic~l moieties can also be incorporated into the oligomer through multiple non-nucleotide monomeric units in a particular nucleotide oligomeric sequence.
One can also provide oligomers which differ from naturally OC~;ul~ g polynucleotides in that they include altered activities by utili7.ing protei~s and enzymes which act on polynucleotides. For example, the pl~r~mPnt of the non-nucleotide monomeric unit on the 3' te~ of an otherwise pure polynucleotide will impart resistance to degradation by snake venom phosphodiesterases, or providing specific cleavage sites for selected nucleases.

CA 022~3969 1998-11-09 WO 97/43451 PCTIUS97tO9094 Hybridization probes may also be constructed by interspersing hybridizable nucleotide monomeric units and non-nucleotide monomeric units. For example, a mixed synthesis of nucleotide and non-nucleotide monomers can be pe~ro~ ed whereby a defined sequence of nucleotide monomers are synthesized followed by a sequence of one or more non-nucleotide monomeric units, optionally followed by asecond block of a defined sequence of nucleotide monomers.
The present invention also provides the ability to construct synthetic probes which ~imlllt~n~ously detect nucleotide mllltim~rs which differ by one or more base pairs. This can be accomplished by using the non-nucleotide reagents desc-il,ed herein to replace the nucleotides in a probe with non-nucleotide monolllel ic units at selected sites where differences occur in the nucleotide sequence of the varioustarget nucleotide sequences.
In selected embodiments of the invention, labeled hybridization probes are constructed as oligomers with a defined sequence comprised of nucleotide and non-nucleotide monomers. Such non-nucleotide monomeric units can be grouped in a selected region or inle.~ sed throughout the sequence of the nucleotide oligomer.
The non-nucleotide monomeric units can be chlomir~lly labeled for use in hybridization reactions.
In the present invention, the non-nucleotide reagent is provided in a lllamle which permits it to be added in a stepwise fashion to produce a mixed nucleotide, non-nucleotide oligomer employing current DNA/RNA synthesis methods. Such reagents would normally be added in a stepwise ~ l.,r to attach the collesl,onding monomeric unit to an increasing oligonucleotide chain which is covalently immobilized to a solid support. Typically, the first nucleotide is ~tt~( h~d to the support through a cleavable ester linkage prior to the initiation of synthesis. In the present invention, the non-nucleotide reagent can be provided conveniently linked to such solid ~U~Ol LS, for example, to controlled pore glass (CPG), to resins, polymers such as poly~Lylelle, and the like. Stepwise extension of the oligonucleotide chain is normally carried out in the 3 ' to 5 ' direction. Such nucleic acid synthesis methods are provided, for example, in S.A. Narang, "Synthesis and , CA 022~3969 1998-11-09 Applications of DNA and RNA," Ar~lPmic Press (1987) and in M.J. Gait "Oligonucleotide Synthesis," IRL Press, Washington, D.C. (1984).
When synthesis is complete, the oligomer is cleaved from the support by hydrolyzing the ester linkage and the nucleotide originally ~tt~rllPd to the support becomes the 3' terminus of the resulting oligomer. Accordingly, the present invention provides both a reagent for plep~ g oligomers which contain a mixture of nucleotide and non-nucleotide monomeric units, together with methods for tili7.ing such reagents in the construction of such oligomers.
Typically, the present reagents will possess two coupling groups so as to permit the stepwise inclusion into a oligomer of nucleotide and non-nucleotide monomeric units. The first of said coupling groups will have the pr~.,.Ly that it can couple efflciently to the terminus of a growing chain of monomeric units. The second of said coupling groups is capable of further e~le~ -g, in a stepwise fashion, the growing chain of mixed nucleotide and non-nucleotide ml-nomers.
This typically requires that the second coupling group be inactivated while the first coupling group is coupled, so as not to substantially couple at that time, the second coupling group can thereafter be activated so as to then couple the non-nucleotide monomeric unit. The inactivation is preferably accomplished with a prole~ g group on the second coupling group, which can then be removed to activate the second coupling group. It is also considered to be within the scope of the invention that such "inactivation" and "activation" might be accomplished simply by çh~nging reaction conditions (e.g. pH, te~ er~ture, concenllaLion of reagents, and t_e like) with second coupling groups of suitable ch~mic~l structure which also lend themselves to inactivation and activation by such techniques. Such coupling groups permit the adjacent ~tt~çhmPnt of either nucleotide or non-nucleotide monomeric units. It is considered desirable that such coupling groups operate through coupling and deprotection steps which are compatible with standard ~uLollla~ed DNA
synthesis methods.
Such methods typically require that synthesis occur unidirection~lly and that all coupling cleavage and deprotection steps occur under "nonadverse conditions"

CA 022~3969 1998-11-09 that is they do not substantially adversely effect the oligomer backbone and itsvarious components.
Thus, the present invention provides oligomers cont~ining the present non-nucleotide reagents, as well as methods for using such reagents in the synthesis of oligomers cont~ining both nucleotide and non-nucleotide units.
The invention further provides interrnediates which are useful to synthesize the present non-nucleotide reagents. One embodiment of such an interrnP~i~te is provided by the formula:
Rl - X - CH2 - C - CH2 - R3 Xl ' X2 wherein R' is hydrogen; X is oxygen;
Xl taken together with the carbon atom of the formula is cyclohexane, x2 is NH, and X3 is H; and R3 is OH.
In this embodiment, the interme~i~te is of a structure similar to that of the present reagents, without having the functional groups included at R', X3 and R3.
J 20 In order to facilitate the use of the present reagents, kits for use in constructing oligomer can be provided to simplify practice of the method described above. The kit will typically contain a receptacle adapted to hold one or more individual reagent containers and at least a first container cont~ining (1) a reagent in accordance with the formula Rl-X-CH2-C-CH2-R3 Xl x2 wherein R', X, X', X2, X3, and R3 are as previously defined. The reagent can be CA 022~3969 1998-11-09 ~FAtl~ 2 3 JUN 1998 provided as a solution comprising a solvent and the reagent or (2) the reagent in an amount ~propliate to make up the desired concentration when solvent from anothercontainer is used to fill the reagent container to a predetermined level.
In many cases, the kit will also contain at least a second container cont~ining (1) a reagent used in the synthesis of oligomers, or (2) a reagent used in the detection of the functional moiety included in the subject reagent, or containers with both such materials. Such reagents are well known in the art and require no further description here. Specific examples are given in the general examples of the invention set out below. Approl~liate instructions for carrying out the method of the invention will also be included in the kit.
The following examples serve to illustrate certain plefelled embodhl,en~
and aspects of the present invention and are not to be construed as limiting thescope thereof.

Experimental In the experimental disclosure which follows, all weights are given in grams (g), milligrams (mg), micrograms (~g), nanograms (ng), or picograms (pg), all amounts are given in moles (mol), millimoles (mmol), micromoles (~mol), nanomoles (nmol), picomoles (pmol), or ~llltollloles (fmol), all concentrations are given as percent by volume (%), proportion by volume (v:v), molar (M), millim-)lar (mM), micromolar (,uM), nanomolar (nM), picomolar (pM), or normal (N), all volumes are given in liters (L), milliliters (mL), or microliters (~L), and linear measurements are given in millimeters (mm), or nanometers (nm) unlessotherwise in~ie~ted The following examples serve to demonstrate the synthesis of reagents of the present invention, as well as their use in forming oligomers with nucleotide units in accordance with the invention.
In the examples, the following abbreviations are used: "CX" is intended to refer to cyclohexane, "Bz" is intended to refer to benzoyl, "CED" is intended torefer to cyanoethyl N,N-diisopropyl phosphoramidite and "LC" is inten~ed to refer to long chain.

N~n ~!~E~

- -~IIJS 97 / 09 0 9 I~AJlls ~3 ;~U~ 7 Example 1 Reagents in accordance with the present invention can be synthesized in accordance with chemical synthetic techniques well known in the art. The following synthetic protocols demonstrate the synthesis of selected compound within the scope of the present invention.

(a) Synthesis of Reagent compound 1 wherein R' is dimethoxytriphenyl (DMT), X is O, X'-X2-X3 is cyclohexane - NH - CO - biotin, and R3 is phosphorarnidite.

0~ H

[COMPOUND 1]
The synthetic protocol for Compound 1 is outlined below and depicted in Figures 1 and 2:
Step I: Synthesis of BzO-CX
To an ice-cold solution of 500g 4,4-bis(hydroxymethyl)-1-cyclohexene in 3.0L of pyridine, was added dropwise 1.03L of benzoyl chloride. The reaction mixture was stirred at room te~ eldt~e overnight, when TLC analysis (ethyl acetate/hexane 1:4 v/v) intlic~tçd the reaction to be complete. The reaction wasquenrhe~ by the addition of lOOmL water, followed by stirring at room temperature for 1 hour.
The reaction mixture was evaporated in vacuo to afford a syrupy residue.
This was dissolved in methylene chloride and washed with 5% aqueous NaHCO3 AMI~NDED SHEB, CA 022~3969 1998-11-09 W O 97/43451 PCT~US97/09094 solution. The organic solution was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford 1,500g of crude product This product was purified by column chlullla~ography over silica gel, using ethyl acetate/hexane (1:4, v/v) to elute the product (yield 1,050g). The desiredproduct was dried under high vacuum for 2 days.
Step II: Sy~tl~D;s of NH2-CX
To a stirred solution of 120g of BzO-CX in 300mL diglyme, under argon, was added dropwise a solution of 5.5g NaBH4 in 150mL of diglyme. The reaction mixture was slowly heated to 70~C. A solution of 23.4mL of BF3-Et2O in 30mL
diglyme was then slowly added to the reaction mixture and the resulting Il~i~Lul~, stirred at 70~C for 1 hour. The reaction was qllenrlled by the addition of 2.5mLwater. This was followed by the addition of 50g of hydroxylamine-O-sulfonic acidand the reaction mixture was heated at 100~C for 3 hours. The reaction llli~lul~, was cooled to room l~ ralule and then extracted into 1.2L of methylene chloride.The organic extract was washed with 500mL water, followed by 5% NaHCO3 solution (2 X 300mL). The organic layer was dried over anhydrous sodium sulfate,filtered, and solvents removed by rotary evaporation to give 270g of crude product.
Purification of the product by silica gel column chlo..lalography, using a solvent system colllplisillg of 2.5-8.0% m~th~nol in methylene chloride to elute the product, afforded 50g of pure 4-amino isomer of NH2-CX. Small qu~ntities of the undesired 3-amino isomer of NH2-CX were also formed.
Step III: Sy~ .;s of BzO-CX-Biotin To 13.5g of the amino compound NH2-CX obtained above, was added 200mL of methylene chloride. To the reslllting solution was added 18.8g of Biotinyl N-hydroxysuccinimide ester (Biotin-NHSu) dissolved in 200mL of DMF.
The reaction mixture was stirred for 15 min at room tell,~lalule, followed by the addition of 10.3mL triethylamine. The reaction was allowed to proceed for 1.5 hours, when TLC analysis revealed the reaction to be complete.
Methylene chloride was removed by rotary evaporation, and the resulting residue treated with 30mL methanol and with 25mL of 10% aqueous sodium carbonate solution. The solution was stirred at room lell,p~,laLule for 1 hour and CA 022~3969 1998-11-09 WO 97/43451 PCTI(IS97/09094 then extracted with 1.2L of ethyl acetate. The organic layer was washed with brine (2 X 400mL), dried over anhydrous sodium sulfate, filtered, and solvents removedin vacuo to finally give 22g of crude product.
This product was purified by column chrolllatography over silica gel, using gradient elution with 2.5-8.0% methanol in methylene chloride to yield lSg.
Step IV: Synthesis of DMT-CX-Biotin To a stirred solution of 14.3g of BzO-CX-Biotin in 200mL DM~, was added 20mL of 25 % sodium methoxide in methanol, and the reslllting ~ ule stirred at 0-5~C for 1 hour. The pH of the solution was then adjusted to 7.0 by the addition of 60g Dowex 50X8-100 resin to the reaction nli~luie followed by stirring for lS min.
The resin was filtered off and the filtrate evaporated to remove DMF. The reslllting residue was dissolved in 10mL methylene chloride and the product precipil~led by the addition of 100mL hexane. The product was then dried under high vacuum.
lS The crude product obtained in this manner was azeotroped twice withpyridine and then dissolved in 300mL pyridine. To this was added 8.13g of DMT-Cl and the reaction IllixLule stirred at room lelll~ldLule, under argon, for l.Shours. The reaction was ql~enrhPcl by the addition of SmL mPth~nol. The reactionllli~lule was taken up in lL methylene chloride, the organic extract washed withS % NaHCO3 solution, and then dried over anhydrous sodium sulfate. Evaporation of the solvents in vacuo afforded 26g of crude product, which was purified by column chlolllal~graphy over silica gel, eluting with methylene chloride/mPth~n-)1 (100:4 v/v) to yield 5.7g.
Step V: Sy~lL. -;c of Biotin-CX-CED P~ s~horamidite The intermP~ tp obtained in step IV above was converted to the corresponding phosphoramidite using standard methods. Thus, 4.0g of DMT-CX-Biotin was dissolved in 40mL methylene chloride and the res~lting solution treated with 2.3mL of 2-cyanoethyl-N,N,N',N'-tetraisopro~ylphosphoro~ mitlitP~ and 600mg of DIPA-tetrazole salt. After 15 hours at room l~ alul~" the reaction was quenched by addition of 0.5mL methanol. The reaction llli~lule was poured into 400mL methylene chloride and the organic layer washed with 5~ sodium Wo 97/4345l PCT/USg7/09094 bicarbonate solution (2 X 100mL), and then dried over anhydrous sodium sulfate.
Removal of solvents by rotary evaporation gave 5.8g of crude product, which was purified by column chromatography over silica gel, eluted with CH2CI2/m~th~nr~l/
TEA (100:2:1, vlvlv) to yield 3.6g of pure Compound 1.

.. .. .

PCT~JS 97 / 09 0 9 A
-18- IPSWS %3 JlJN 199 (b) Synthesis of Reagent compound 2 wh~rein R' is dimethoxytriphenyl (DMT), X is O, X'-X2-X3 is cyclohexane - NH -CO(CH2)5NHCOCF3, and R3 is phosphoramidite.

,~"1 ~c~
~?~Hco [COMPOUND 21 The synthetic protocol for Compound 2 is outlirled below and depicted in Figure 3:
Step I: Synthesis of BzO-CX-Linker To 15.0g of the NH2-CX interm~li~te (prepa~ in step 2 of Example la) dissolved in 150mL methylene chloride, was added dropwise a solution of 21.2g 6-trifluoro~cet~mido-caproic acid N-hydroxysuccinimide ester (Linker-NHSu) in - ~ 150mL methylene chloride. The resulting mixture was stirred at room tempela~re for 15 min and then treated with 12. lmL triethylamine. After 90 min stirring atroom tt~ elature, TLC analysis (CH2C12/meth~n- l, 9:1) in~ ted that the reactionhad gone to completion.
Methylene chloride was removed by rotary evaporation, the resultin~ residue treated with 150mL methanol, followed by 30mL of 10% aqueous Na2CO3 solution, and the mixture stirred for 1 hour at room Len~elature. After this tirne, the reaction rnixture was poured into 1.0L CH2C12 and the organic extract washed with 5 % sodium bicarbonate solution. After drying over anhydrous sodium sulfate, thesolvents were evaporated in vacuo to afford 22g of crude product. Flash . ~

CA 022~3969 1998-11-09 chlolllalographic purification using CH2Cl2/mPth~nol (100:3, v/v) as the eluent,afforded 10.6g of pure product.
Step II: Synthesis of DMT-CX-Linker To a stirred solution of 10.3g BzO-CX-Linker in 100mL DMF, was added 15mL of 25% sodium methoxide in methanol, and the resl~lting ~ Lule stirred at 0-5 ~C for 1 hour. The pH of the solution was then adjusted to 7.0 by the ~ ition of 45g Dowex 50X8-100 resin to the reaction mixture followed by stirring for 15 min.
The resin was filtered off and the filtrate evapolal~d to remove DMF. The reslllting residue was dissolved in 5mL methylene chloride and the product precipitated by the addition of 50mL hexane. The product was then dried under high vacuum.
The crude product obtained in this manner was azeotl-)~ed twice with pyridine and then dissolved in 100mL pyridine. To this was added 5.9g of DMT-CI and the reaction Illi~lUI~ stirred at room te~ cl~lule, under argon, for 1.5 hours. The reaction was quçn~h~d by the addition of 3mL m~th~nol and stirred for30 min. The reaction ~ lule was taken up in 500mL methylene chloride, the organic extract washed with 5 % NaHCO3 solution (300mL X 2), and then dried over anhydrous sodium sulfate. Evaporation of the solvents in vacuo afforded 11.9g of crude product, which was purified by column chromatography over silica gel, eluting with methylene chloride/m~th~nol (100:2 v/v) to yield 5.7g.
Step III: Synthesis of N-Linker-CX-CED PLosl,~G.~lllidite The intermediate obtained in step II above was converted to the corresponding phosphoramidite using standard methods. Thus, 3.0g of DMT-CX-Linker was dissolved in 50mL methylene chloride and the resl~lting solution treated with 2.2mL of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodi~mirlite and 560mg of DIPA-tetrazole salt. After 15 hours at room tellly~raluie~ the reactionwas quenched by addition of 1.0mL meth~nol. The reaction llli~lule was poured into 300mL methylene chloride, the organic layer washed with 5 % sodium bicarbonate solution (2 X 80mL), and then dried over anhydrous sodium sulfate.
Removal of solvents by rotary evaporation gave 4.3g of crude product, which was W O 97143451 PCT~US97/09094 purified by column chromatography over silica gel, eluted with CH2Cl2/m~th~nol/
TEA (100:1:1, vlvlv) - yield 3. lg of pure Compound 2.

97 / 09 C9 ~
liPEA/llS 2 3 JUN 1998 (c) Synthesis of Reagent compound 3 wherein R' is dimethoxytriphenyl (DMT), X is O, X'-X2-X3 is cyclohexane - NH - CO - fluorescein, and R3 is phosphoramidite.
>~1 ~ ~~
D~ ro .~ .

[COMPOUND 3]
The synthetic protocol for Compound 3 is outlined below and depicted in Figure 4:
Step I: Synthesis of ~amino-1,1-bis(hydroxymethyl) cyclohexane 2S To 30.6g of NH2-CX dissolved in 400mL of mPth~nol was added 39.3rnL of a solution of sodium methoxide (25 % w/v) in meth~nol. The reaction ~ ure was stirred at arnbient temperature for 1 hour under anhydrous conditions. The reaction . was monitored by TLC using a mixture of methylene chloride:methanol (9: 1) as solvent. Solvents were removed by rotary evaporation and the residue treated with 60mL water, cooled in an ice bath, and then neutralized by the slow addition of hydrochloric acid. The reaction mixture was extracted with methylene chloride (4X 100mL), and the aqueous portion concc.lLlated in vacuo to give 21.5g of product cont~ining sodium chloride. The residue was treated with 200mL methanol, filtered, and solvents removed in vacuo to give 13.2g of the desired product.
Step II: 5-(& 6-) Carboxy Fluorescein Dipivaloate To a solution of 25g 5-(&- 6)-carboxy fluorescein in 200mL pyridine, was added 25.8g of diisopropylethyl arnine and the resulting mixture cooled to -10~C.
To the cooled solution was added dropwise 32.8mL of pivaloyl chloride, and the AMi~NDE9 ~

CA 022~3969 1998-11-09 uli~ule stirred for 2 hours under argon. The reaction lui~lule was allowed to warm up to room temperature over 2 hours. The reaction uli~ule was evaporated to dryness, and the residue extracted with 1.0L methylene chloride. The organic extract was washed with water (2 X 500mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford 43.4g of crude product. Flash cllrollla~ographic purification of this crude product (silica gel, CH2Cl2/MeOH
gradient elution 2-8 % MeOH) afforded 22.0g of pure product.
Step III: 5-(& 6-)Carboxyfluorescein Dipivaloyl Succinin~dyl Ester To a solution of 28.3g 5-(and 6-) carboxyfluorescein dipivaloate in 250mL
methylene chloride, under argon, was added 7.1g N-hydroxysuccinimide followed by 13g of DCC. The reaction mixture was stirred at ambient temperature overnightunder anhydrous conditions. The reaction lni~lule was filtered and the filtrate evaporated to dryness to give 39g of crude product. This product was purified bycolumn chromatography over silica gel, using ethyl acetate/hexane (1: 1, v/v) as the eluent to yield 27.4g.
Step IV: Fluoresc~; CX-aII~ide 13.2g of 4-amino-1,1-bis(hydroxymethyl)cyclohexane was azeotroped with SOmL of anhydrous DMF using rotary evaporation at 55~C. To this was added 75mL anhydrous DMF followed by 19.5g of 5-(& 6-)carboxyfluoresce~n dipivaloyl succinimidyl ester under argon. 3.3g of triethylamine was added and the reactionmixture stirred at room temperature overnight under anhydrous conditiorls. The solution was evaporated to dryness using rotary evaporation at 55~tS~C. The residue was extracted into 500mL methylene chloride and the organic extract washed with water (2 X 100mL). After drying over anhydrous sodium sulfate, solvents were removed in vacuo to afford 32g of crude product. Flash chromatographic purification of this crude product (silica gel, CH2Cl2/MeOH, gradient elution 3-6% MeOH) afforded 7.4g of pure product.
Step V: Sylllh~ ~;c of DM T-CX-FIU01~3~
To 7.4g of product obtained from step IV above, was added 50mL of anhydrous pyridine and the mixture azeotroped. The residue was dissolved in 50mL anhydrous pyridine. To this was added 4.6g of DMT-Cl and the reaction CA 022~3969 1998-11-09 WO 97/434Sl PCT/US97/09094 mixture stirred at room te~ dl~ overnight, under argon. The reaction was ql1~n~h~cl by the addition of 10mL mPth~nnl and stirred for 30 min.
The reaction llli~lule was taken up in 250mL methylene chloride, the organic extract washed with 75mL of a 5 % NaHCO3 solution, and then dried over anhydrous sodium sulfate. Evaporation of the solvents in vacuo afforded 13.5g ofcrude product, which was purified by column chronlalography over silica gel, eluting first with 1.0 L hexane:ethyl acetate (6.5:3.5, v/v) & then with 2.0 L
hexane:ethyl acetate (1:1, v/v) to yield 5.8g.
Step VI: Synthesis of FIUG~'~SC~ CX CED Phosphoramidite The interm~di~t~ obtained in step V above was converted to the collespollding phosphoramidite using standard methods. Thus, 5.8g of DMT-CX-Fluorescein was dissolved in lOOmL anhydrous methylene chloride and the res--lting solution treated with 3.0g of diisopropylethylamine, followed by 1.9g of 2-cyanoethyl N,N-diisopropylchlorophosphoro~mi-lit~. After 2 hours at room te~ c,dluie, the reaction was quen~h~d by addition of l.OmL meth~n-)l. The reaction llli~ le was poured into 200mL methylene chloride, the organic layer washed with 5 % sodium bicarbonate solution (2 X 75mL), and then dried over anhydrous sodium sulfate. Removal of solvents by rotary evaporation gave 8.0g ofcrude product, which was purified by column chromatography over silica gel, eluted with hexane/ethyl acetate/ TEA (gradient elution - 30.0 to 35.0 % ethyl acetate in hexane conl;~;ni~.g 0.5 % TEA) to yield 4.2g of pure Compound 3.

(d) Synthesis of Reagent compound 4 wherein R' is dimethoxytriphenyl (DMT), X is O, Xl-X2-X3 is cyclohexane - NH -CO(CH2)5NHCOCF3, and R3 is -OCOCH2CH2CONH - CPG.
s ~) ' ~COC~C~CONH
~H2 Dh~l'--O C~2~
/ ~ / NHCO W ~ H COC~3 .. .

[CO~POUND 4]
The synthetic protocol for Compound 4 is outlined below:
Step 1: Preparation of N-Linker ~-~crin~te:
To a stirred solution of l.95g of DMT-N-Linker-CX in 20mL anhydrous methylene chloride, was added lOOmg of 4-dimethylaminopyridine followed by 1.2g of succinic anhydride. The resulting solution was stirred at room temperature for 15 hours. The reaction mixture was qllçnrh.od by the addition of lOmL of a 5 %
~solution of sodium bicarbonate in water and the mixture stirred for 30 min. Thecrude reaction mixture was then evaporated to dryness in vacuo and the resultingresidue extracted twice with 50mL of methylene chloride.
The organic extract was washed with 5 % aqueous citric acid solution (2 X
20mL) and then dried over anhydrous sodiurn sulfate. Evaporation of the solventsin vacuo afforded l.9g of the crude product. This product was purified by flash chromatography over silica gel using CH2Cl2/CH3OH (100:5, v/v) as the eluant.
Step 2: Preparation of N-Linker-CPG:
To a suspension of 4.0g of LCAA-CPG in 14mL of methylene chloride in a 50mL round bottomed flask, was added 105mg of N-Linker succinate and 0.7mL of triethylamine. This was followed by the addition of 20mg of anhydrous ~MEND~D SHEET' hydroxybenzotriazole and 70mg of benzotrizolyl-N-oxy-tris(dimethylamino) phosphonium hex~flllorophosphate (BOP reagent).
The resulting mixture was gently shaken for 2 hours, then filtered, washed with methylene chloride (lOmL X 2), and air dried. The solid was l~ r~lled to a lOOmL round bottom flask, treated with 36mL pyridine, 4mL of acetic anhydride and 0.4mL of N-methylimidazole, and the resulting suspension shaken overnight.
The llli~lule was then suction filtered, and the solid washed with n~eth~n- I (lOmL X

3). The solid was washed further with methylene chloride (lOmL X 3), followed by anhydrous ether (lOmL X 3). The solid was air dried and then finally dried overnight under high vacuum.

CA 022=,3969 1998-11-09 PCTlIJS 97~09~9 ~ 2 ~ J~ 9 (e) Synthesis of Reagent compound 5 wherein R' is dimethoxytriphenyl (DMT), X is O, X'-X2-X3 is cyclohexane - NH - CO - biotin, and R3 is -OCOCH2CH2CONH - CPG.
~' COC}~C~CO~H o o/ ,~
~CH2 HN 'NH
DMI ~--CH~
~_NHCO

[COMPOUND ~1 The synthetic protocol for Compound 5 is illustrated in Figure 5, and outlined below:
Step 1: Preparation of Biotin sl~crin~te:
To a stirred solution of 2.0g of DMT-CX-Biotin in 20mL anhydrous methylene chloride, was added 100mg of 4-dimethylaminopyridine followed by 1.2g of succinic ar~ydride. The resulting solution was stirred at room temperature for 15 hours at which point TLC analysis of the reaction mixture (9:1 _ CH2CL2/CH30H) in-lir~t~d that the reaction was complete.
The reaction rnixture was quenrl ed by the addition of 10mL of a 5 %
solution of sodium bicarbonate in water and the rnixture stirred for 30 min. Thecrude reaction mixture was then evaporated to dryness in vacuo and the resultingresidue extracted twice with 50mL of methylene chloride. The orgar~ic extract was washed with 5 % aqueous citric acid solution (20mL X 2) and then dried over anhydrous sodium sulfate. Evaporation of the solvents in vacuo afforded 2.4g of the crude product. This product was purified by flash chromatography over silicagel using CH2Cl2/CH3OH (100:4, v/v) as the eluant.

4~1 PCT/US97tO9094 Step 2: Preparation of Biotin-CPG:
To a suspension of 4.0g of LCAA-CPG in 14mL of methylene chloride in a 50mL round bottomed flask, was added 110mg of Biotin succinate and 0.7rnL of triethylamine. This was followed by the addition of 20mg of anhydrous S hydroxyben_otriazole and 70mg of BOP reagent. The resulting llli~Ul~, was gently shaken for 2 hours, then filtered, washed with methylene chloride (20rnL X 2), and air dried. The solid was transferred to a 100mL round bottom flask, treated with36mL pyridine, 4mL of acetic anhydride and 0.4mL of N-methylimi~7ole, and the resulting suspension shaken overnight.
The llli~tUl e was then suction filtered, and the solid washed with m.oth~nnl (lOmL X 3). The solid was washed further with methylene chloride (lOmL X 3), followed by anhydrous ether (lOmL X 3). The solid was air dried and then finallydried overnight under high vacuum. The loading of the biotin derivatized CPG wasd~lell-lined to be 31.3~mole/gram using standard methods.

~P~JS 2 3 J UN 1998 fl Synthesis of Reagent compound 6 wherein R' is dimethoxytriphenyl (DMT), X is O, X'-X2-X3 is cyclohexane - NH - CO(CH2)5NHCO -biotin, and R3 is phosphoramidite.

10 1,1 V~ OP--O~ ~

DMI--o~H2~7 C~
- CO,~?~H

[COMPOUND 61 The synthetic protocol for Compound 6 is outlined below and depicted in Figure 5:
Step I: Synthesis of BzO-CX-Biotin-LC
To 32.0g of the NH2-CX intermediate (prepared as discussed in Example la) dissolved in 500mL anhydrous methylene chloride, was added dropwise a solution of 39.5g LC Biotin-NHSu (prepared by the reaction of 6-aminocaproic acid with the ~', NHSu-ester of biotin) in 500mL anhydrous DMF. The resulting mixture was stirred at room temperature for 15 min and then treated with 12. lmL triethylamine.
After 2 hours stirring at room temperature, TLC analysis (CH2Cl2/methanol, 9: 1)indicated that the reaction had gone to completion. Methylene chloride was removed by rotary evaporation, the resulting solution treated with m~th~nol (20mL) followed by 10% aqueous Na2CO3 (20mL), and the mixnlre stirred for 1 h at room temperature. After this time, the reaction mixture was poured into 2.0 L ethyl acetate and the organic extract washed with brine (500mL X 2). After drying over~nhydrous sodium sulfate, the solvents were evaporated in vacuo to afford 64g ofcrude product. Flash chromatographic purification of this crude product (silica gel, CH2Cl2/MeOH, gradient elution 5-8 % MeOH) afforded 30g of pure product.

CA 022~3969 1998-11-09 W O 97/43451 PCT~US97/09094 Step II: Synthesis of DMT-CX-Biotin-LC
To a stirred solution of 29.8g of BzO-CX-Biotin-LC in 300mL DMF, was added 34.5mL of 25% sodium methoxide in meth~nc)l, and the resulting mixture stirred at 0-5~C for 1 hour. The pH of the solution was then adjusted to 7.0 by the S addition of 100g Dowex 50X8-100 resin to the reaction llli~LUle followed by stirring for 15 min. The resin was filtered off and the filkate evaporated to remove DMF.The resulting residue was dissolved in 60mL methylene chloride and the product precipitated by the addition of 200mL hexane. The product was then dried under high vacuum.
The crude product obtained in this manner was azeotroped twice with pyridine and then dissolved in 500mL pyridine. To this was added 14.0g of DMT-Cl and the reaction mixture stirred at room temperature, under argon, for 1.5 hours. The reaction was quenrhed by the addition of 5mL ll-e~ ol and stirred for30 min. The reaction llli~Lule was taken up in 1.5L methylene chloride, the organic extract washed with 5% NaHCO3 solution (500rnL X 2), and then dried over anhydrous sodium sulfate. Evaporation of the solvents in vacuo afforded 26.0g ofcrude product, which was purified by column chromatography over silica gel, eluting with methylene chloride/meth~n- l (100:8 v/v) to yield 12.0g.
Step III: Synthesis of 'BuBz-DMT-CX-Biotin-LC
To a stirred solution of 12.0g of DMT-CX-Biotin in 300mL al~,ydlous pyridine, was added 13.0mL TMSCI and the mixture stirred at room lel.,p~ lu for 2 hours, under argon. This was followed by the addition of 4.4mL of 4-tert-butyl benzoyl chloride to the reaction llli~tu~e and the reaction was allowed toproceed at room te~l,pel~tu~e for 3 hours. The reaction was worked up by the addition of 80mL water followed by stirring for 1 hour at room telllpel~lu,e. The reaction mixture was evaporated to remove most of the pyridine and the resultin~residue dissolved in 1.0L methylene chloride. The organic extract was washed with 5 ~ NaHCO3 solution (300mL X 2), and then dried over anhydrous sodium sulfate.
Removal of solvents by rotary evaporation gave 18.3g of crude product.

CA 022~3969 1998-11-09 Flash chrolnatographic purification of this crude product (silica gel, EtOAc/CH2Cl2/MeOH, gradient elution with solutions cont~ining 5-10 parts MeOH
in EtOAc/ CH2Cl2, 50:50 parts, vlvlv~ afforded 8.3g of pure product.
Step IV: Synthesis of LC-Biotin-CX-CED Phosphoran~idite The interm~ t~ obtained in step III above was converted to the corresponding phosphoramidite using standard methods. Thus, 8.2g of 'BuBz-DMT-CX-Biotin-LC was dissolved in 100mL methylene chloride and the rçsul~ing solution treated with 4.0mL of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphoro~ ite and 600mg of DIPA-tetrazole salt. After 15 hours at room tel"?eralule, the reaction was quçnrh~ by addition of 0.5mL
m~th~nol. The reaction ~ni~lule was poured into 1.0L methylene chloride, the organic layer washed with 5% sodium bic~llbul.ate solution (2 X 300rnL), and then dried over anhydrous sodium sulfate. Removal of solvents by rotary evaporation gave 10.5g of crude product, which was purified by column chrolllalography over silica gel, and eluted with CH2CI2/m~th~nol/TEA (100:1:1, vlvlv) to yield 5.0g of pure Compound 6.

CA 022~3969 1998-ll-09 W O 97/43451 PCTrUS97/09094 Example 2 Reagents in accordance with the present invention can be incol~or~led into oligomers comprising nucleotide and non-nucleotide units, by sub~ ;,.g the present non-nucleotide reagents in place of selected nucleotide units in ~L~dardnucleotide synthesis protocols, such as automated DNA/RNA synthesis protocols.

(a) Use of Biotin-CX by direct binding to poly~ly,~.le plates:
Synthetic oligomers, cont~ining one or more biotin residues as part of the sequence, were labelled with a 5'-phosphate group. This 5'-phosphate moiety is then used to covalently bind the oligomer onto polystyrene microtiter plates.
The unbound probes are washed off and the bound probes are det~cte~ by the reaction of biotin with streptavidin conjugated to ~Ik~lin~ phosph,.l;.$e. The ~lk~lin~ phosphatase catalyses the hydrolysis of a chromogenic substrate.
Thus, 30mers were synthesized which included a 5' phosphate moiety and biotin residue labels at positions 13, 19 and 25 (5' _ 3'). Two dirr.,le~l oligomers were synth~ci7.ed one with Biotin-CX phosphoramidite plepaled in acco~dance withExample 1 and the second oligomer with Biotin-dC (a commercially available nucleosidic biotin phosphoramidite).
Each of the oligomers was diluted to a conce~ lion of 10fmol/~L in dictilled water. The oligomers were della~ui~d by heating at 95~C for 10 min, followed by cooling over ice for 10 min. The applo~,iate amount of dtn~Luled oligomers were added to the wells of cold Covalink NH modules, followed by the ~1~1itinn of EDC buffer co..~ MeIm and then overnight in~ub~tion at 50~C.
Unbound probes were washed off and the bound oligomers were detected by binding of streptavidin conjugated to alk~lin~ phosphatase. pNPP was used as thesubstrate for the enzyme and the development of color was molliLoled at 405nm.
The above experiment was repeated using both oligomers at a concentration of Sfmol/,uL. The signal i~e~ilies obtained using the two dirre.ellL biotin structures were colll~ ed. At Sfmol/~L, biotin-CX gave 95 % of the reading (OD 405nm) of biotin-dC. At 10fmol//1L, biotin-CX gave 106% of the reading of biotin-dC.

CA 022~3969 1998-ll-09 (b) Use of Biotin-CX in a hylJ.;.l;~;o-~ assay:
The oligomer Alu-011 is a 56mer with one internal biotin residue at position 25 (5' ~ 3'), designed to be comple.. l~.y to the template Alu-OllA. Two different oligomers were synthesi7~d: One with Biotin-CX and the other with Biotin-dC. Oligomers were synthesized in the Trityl-ON mode and then cartridge purified using PolyPak (Glen Research Inc.) reverse phase cartridges. These two oligomers were used in hybridization assays to detect the template Alu-01 lA.
The template Alu-01 lA was bound to CovaLink polystyrene microtiter plates using the 5'-phosphate group as described above. The probes were diluted to 25fmol/~L in hybridization buffer and 100~4L of the diluted probes was added to the wells and inr~b~tç-l at 42~C for from S hours to overnight. Excess probes were washed off with buffer and the bound biotin-labelled probes were cletected as described above.
The signal intçn~iti~S obtained using the two dirr.,~ biotin structures were colllpal~. Under identical conditions, Biotin-CX labelled oligomers generated approximately a 5-fold stronger signal than oligomers labelled with Biotin-dC.

All publications and patent applications cited in this specification are hereby incorporated by rere,ellce as if they had been specifically and individually inrliç~ted to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and underst~n-ling, it will be apparent to those of ordinary skill in the art in light of the ~ olosllre that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended clai~ns.

Claims (21)

Claims:

1. A non-nucleotide reagent which is capable of forming a oligomer with nucleotide units, said reagent comprising a compound of the formula:

wherein R1 is selected from the group consisting of hydrogen, acid-sensitive, base-stable blocking groups and acyl capping groups;
X is selected from the group consisting of O, S, NH and N=N;
X1 is a substituted or unsubstituted C5 to C7 cyclic moiety incorporating the carbon atom of the formula;
X2 is selected from the group consisting of O, S, CH2, NH and N=N; and X3 is hydrogen, or a linking functional group which is capable of linking with a functional moiety; and R3 is a phosphorus linking group of the formula (a) or (b) wherein X4 is halogen or substituted amino, X5 is alkyl, alkoxy or phenoxy, or a cyano derivative thereof, X6 is halogen, amino or O, and X7 is alkyl, alkoxy or aryloxy, or may be H only if X6 is O, or R3 is a bond, either directly or through an intermediate group, to a solid support.

2. A reagent as recited in claim 1, wherein R1 is selected from the group consisting of triphenylmethyl compounds and alkoxy derivatives thereof.

3. A reagent as recited in claim 2, wherein R1 is dimethoxytriphenyl.

4. A reagent as recited in claim 1, wherein X is O.

5. A reagent as recited in claim 1, wherein X1 is cyclohexane.

6. A reagent as recited in claim 1, wherein X2 is NH.

7. A reagent as recited in claim 1, wherein X3 is a linking group of the formula - CO - (CH2)n - NH -wherein n is an integer from 0 to 20.

8. A reagent as recited in claim 1, wherein X3 further comprises a functional moiety.

9. A reagent as recited in claim 8, wherein the functional moiety is selected from the group consisting of labels, metal chelators and specific binding agents.

10. A reagent as recited in claim 9, wherein the functional moiety is a fluorescent label.

11. A reagent as recited in claim 10, wherein the fluorescent label is fluorescein.

12. A reagent as recited in claim 9, wherein the functional moiety is a specific binding agent.

13. A reagent as recited in claim 12, wherein the specific binding agent is biotin.

14. A reagent as recited in claim 1, wherein R3 is selected from the group consisting of phosphodiesters, phosphotriesters, phosphites, phosphoramidites, H-phosphonates, alkyl-phosphonates, and phosphorothioates.

15. A reagent as recited in claim 1, wherein R3 comprises a bond, either directly or through an intermediate group, to a solid support.

16. A reagent as recited in claim 15, wherein the intermediate group comprises - O - CO(CH2)2 - CO -.

17. An oligomer having both nucleotide and non-nucleotide units, at least one of said non-nucleotide units comprising a compound of the formula:

wherein R1 is selected from the group consisting of hydrogen, acid-sensitive, base-stable blocking groups and acyl capping groups, or R1 is a bond to an adjacent monomeric unit;
X is selected from the group consisting of O, S, NH and N=N;
X1 is a substituted or unsubstituted C5 to C7 cyclic moiety incorporating the carbon atom of the formula;
X2 is selected from the group consisting of O, S, CH2, NH and N=N; and X3 is hydrogen, or a linking functional group which is capable of linking with a functional moiety; and R3 is a phosphorus linking group of the formula or wherein X4 is halogen or substituted amino, X5 is alkyl, alkoxy or phenoxy, or a cyano derivative thereof, X6 is halogen, amino or O, and X7 is alkyl, alkoxy or aryloxy, or may be H only if X6 is O, or R3 is a bond to an adjacent monomeric unit or directly or through an intermediate group a solid support, with the proviso that at least one of R1 and R3 is a bond.

18. A method for preparing an oligomer having both nucleotide and non-nucleotide units, comprising coupling at least one non-nucleotide unit comprising a compound of the formula:

wherein R1 is selected from the group consisting of hydrogen, acid-sensitive, base-stable blocking groups and acyl capping groups, or R1 is a bond to an adjacent monomeric unit;
X is selected from the group consisting of O, S, NH and N=N;
X1 is a substituted or unsubstituted C5 to C7 cyclic moiety incorporating the carbon atom of the formula;
X2 is selected from the group consisting of O, S, CH2, NH and N=N; and X3 is hydrogen, or a linking functional group which is capable of linking with a functional moiety; and R3 is a phosphorus linking group of the formula (a) or (b) wherein X4 is halogen or substituted amino, X5 is alkyl, alkoxy or phenoxy, or a cyano derivative thereof, X6 is halogen, amino or O, and X7 is alkyl, alkoxy or aryloxy, or may be H only if X6 is O, or R3 is a bond, either directly or through an intermediate group, to an adjacent monomeric unit or a solid support, to at least one nucleotide monomeric unit by a bond at either R1 or R3.

19. A kit for preparing an oligomer having both nucleotide and non-nucleotide units, comprising a receptacle adapted to hold one or more individual reagent containers; and a first container containing a reagent in accordance with the formula:

wherein R1 is selected from the group consisting of hydrogen, acid-sensitive, base-stable blocking groups and acyl capping groups;
X is selected from the group consisting of O, S, NH and N=N;
X1 is a substituted or unsubstituted C5 to C7 cyclic moiety incorporating the carbon atom of the formula;
X2 is selected from the group consisting of O, S, CH2, NH and N=N; and X3 is hydrogen, or a linking functional group which is capable of linking with a functional moiety; and R3 is a phosphorus linking group of the formula (a) or (b) wherein X4 is halogen or substituted amino, X5 is alkyl, alkoxy or phenoxy, or a cyano derivative thereof, X6 is halogen, amino or O, and X7 is alkyl, alkoxy or aryloxy, or may be H only if X6 is O, or R3 is a bond, either directly or through an intermediate group, to a solid support.

20. A kit as recited in claim 19, further comprising a second container containing (1) a reagent used in the synthesis of oligomers, or (2) a reagent used in the detection of a functional moiety associated with said reagent.

21. A chemical intermediate for synthesizing a non-nucleotide reagent which is capable of forming a oligomer with nucleotide units, said intermediate comprising a compound of the formula:

wherein R1 is hydrogen;
X is oxygen;
X1 is cyclohexane, X2 is NH, and X3 is H; and R3 is OH.