CA2088255A1 - Hydroxyl-protecting groups orthogonally removable by reduction and their use in the chemical synthesis of oligonucleotides - Google Patents
Hydroxyl-protecting groups orthogonally removable by reduction and their use in the chemical synthesis of oligonucleotidesInfo
- Publication number
- CA2088255A1 CA2088255A1 CA002088255A CA2088255A CA2088255A1 CA 2088255 A1 CA2088255 A1 CA 2088255A1 CA 002088255 A CA002088255 A CA 002088255A CA 2088255 A CA2088255 A CA 2088255A CA 2088255 A1 CA2088255 A1 CA 2088255A1
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- Canada
- Prior art keywords
- hydroxyl
- group
- different
- same
- halogeno
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
- C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
Hydroxyl-protecting groups orthogonally removable by reduction with a liquid reducing agent are disclosed. The novel hydroxyl-protecting groups are particularly useful in the chemical synthesis of linear and branched oligonucleotide structures, as they are readily removed from the protected molecule with mild reagents such as dithionite. Examples of such hydroxyl-protecting groups include the 2-methylene-9,10-anthraquinone (Maq) carbonate ester and the p-nitrobenzyl carbonate ester.
Description
~0~)2/0~53~ PCT/US~1/05~-~0882~
HYDROXYL-PROTECTING GROUPS
ORTHOGONALLY REMOVABLE BY REDUCTION AND
THEIR USE IN THE CHEMICAL SYNTHESIS OF OLIGONUCLEOTIDES
Description Technical Field This invention relates generally to hydroxyl-protecting groups and more particularly relates t~
hydroxyl-protecting groups which are orthogonally removable by reduction with liquid reducing agents and which are especially useful in the chemical synthesis of oligonucleoti~es.
~ack~round With the advent of hybrid DNA technology and the e~plosion in the ability to isolate, purify and assay a wide variety of natural productsl, there is an increasing need for rapid and efficient methods of preparing and purifying oligomers o~ nucleic acids and amino acids.
With nucleic acids, it is typically necessary to synthesize sequences for use as lin~er~, adapters, synthetic genes, and synthetic rPgulatory sequences, as well as for use as probes, primers, and the like. Many procedures have been developed for producing oligomers of nucleotides, or "oligonucleotidesl'. These procedures for the most part rely on initial attachment of a f irst nuc}eotide to a solid support, followed by the se~uential :addition of subse~uent nucleotide units, with each :~30 addition involving a number of chemical reactions.
The two primary methods of oligonucleotide synthesis, which are well-established in the art, are the so-called "phosphotxiester" and 'Iphosphoramidite'' methods (descri~ed at some length in the references oited below) .
-.
- : ~ .: ...~:-. .
.;. . . ..
wo sun2s3~ Pcr/ussl/os~-~ 2-In the most prevalen~ schemes for both methods, the oligonucleotide chain grows by nucleophilic attack of the 5'-OH of the immobilized oligomer on an activated 3'-phosphate or phosphoramidite function o~ a soluble 5'-protected nucleotide building block. Other key stepsinclude the acid deprotection of the 5'-0-(4,4'-dimethoxytrityl) group (DMT) in the phosphotriester method, and, in the phosphoramidite process, the oxidation of the phosphite triester to the phosphate triester. _ _ Other methods of oligonucleotide synthesis are also known, including 5'-to-3' syntheses which use a ~-cyanoethyl phosphate protectin~ group (De Napoli et al., Gazz~ Chim. It:al. 114:65 (1984); Rosenthal et al., lS Tetrahedron Lett. 24:1691 ~1983); Belagaje and ~rush, Nucleic Acids Res. 10:6295 (1977); ~ramer and Koster, i~n~QW~ C~em. Int. Ed. Enql. 7:473 (1968); and Blackburn et al., ~. Chem. Soc. C, 243~ 67)).
All of these methods of sy~nthesizing oligonucleotides involve the use of 3'- and 5'-hydroxyl-protecting groups. Many of the hydroxyl-protecting groups used in oligonucleotide synthesis present some problems. For example, it is obviously desirable that a hydroxyl-protecting group be "orthogonal," i.e., removable with reagents that do not affect the remainder of the molecule, including other blocking or protecting groups which may be present. Some of the known hydroxyl-protecting groups are not completely "orthogonal". Also, many of the currently used hydroxyl-protecting groups, e.g., the levulinyl group, require removal with harsh re~gents (e.g., acid in the case of dimethoxytrityl).
~ The need for harsh reagents can damage a growing ; oligonucleotide chain and, furthermore, severely limits the number and type of protecting groups which may be employed elsewhere in the molecule during synthesis.
.- . : : . . . : . ., W092/0253~ PCT/US91/0;~-_3_ 20~8255 Finally, it is desirable that the hydroxyl-protecting group be chemically stable in relation to whatever reagents are to be used in the chemical reactions involving the remainder of the molecule. It has proved difficult to find hydroxyl-protectiny groups which are chemically stable as ~bound" during use yet which are readily removable with relatively mild reagents. The invention is directed to orthogonal hydroxyl-protecting groups which are in fact quite stable while bound to the protected molecule, but which are nevertheless easily removable post-reaction with mild reagents. The presen, invention makes use of protecting groups which, when bound to the protected molecule, are in an oxidized, stable state, but which upon reduction become labile and are thus readily removable. The novel hydroxyl-protecting groups may also be used when there is more than one hydroxy~ group present in the molecule to be protected. These protecting groups have been found by the inventors herein to be extremely versatile and invaluable as hydroxyl-protecting groups in general and more particularly in the chemical ~,ynthesis of oligonucleotides.
In addition to the refere~nces cited and discussed in the preceding section, the following references also relate to one or more aspects of the present invention.
D.S. Kemp et al., Tetrahedron Letters, No. 12, pp. 1031-1034 (1977), describe the use of Maq esters as carboxyl protecting groups, specifically for use in the chemical synthesis of peptides.
N. Balgobin et al., Chemica ScriPta 20:198-200 (~982), describe the use of 2-oxymethyleneanthraquinone as a terminal phosphate protecting group in the chemical synthesis of DNA and RNA.
: . . ' :
.
:
-: ; . . :. . : :
- ~'. ' ~ ; ': ' ' ' , WO ~/02533 PCI/~'S91/05~-2 0~-8 2~ ~ _4_ R.L. Blankespoor et al., ~. Orq. Chem. 49:4441-46 (1984), describe the use of the 2-methylene-9,10-anthraquinone (Maq) ester to bind a y-aminobutyric acid.
The focus is on the development of an improved delivery system for neurotransmitters (i.e., such as ~-amino-butyric acid (GABA)). The authors note that the Maq ester is cleavable upon electroreduction to give the corresponding hydroquinone.
Disclosure of the Invention Accordingly, it is a primary object of the invention to provide methods and reagents for protecting hydroxyl groups, particularly during the chemical synthesis of oligonucleotides.
It is another object of the invention to provide orthogonally removable hydroxyl-protecting groups which are rendered labile and removable upon reduction with a liquid reducing agent.
It is still another object: of the invention to provide a multifunctional nucleic acid derivatized at the N4-position with an oxyalkylene moiety -(CH2)X-OR where R
is a hydroxyl-protecti~g group as will be described in detail herein.
It is yet another object of the invention to provide oligonucleotide chains containing such multifunctional nucleic acids.
It is a further object of the invention to provide a method of protecting a hydroxyl group of a hydroxyl-containing compound during chemical reaction of other functional groups contained within the compound which involves, prior to such chemical reaction, reacting the hydroxyl group to be protected with a chloroformate derivative of the desired protecting species.
It is still a further object of the invention to provide an improved method for chemically synthesizing - : . . :: : ,:
. . : .: : . : : : : . ' W~2/n253~ PCT/US91/~57~
-5- 20882~
oligonucleotides from nucleotide monomers. The improvement is directed to the use of certain orthogonally removable hydroxyl-protecting groups as will be described herein.
It is yet a further object oî the invention to provide a method of making a branched oligonucleotide s~ructure, which involves derivatizing a linear oligo-nucleotide at the N4-position of cytosine residues with secondary oligonucleotide chains, using the orthogonally removable hydroxyl-protecting groups of the invention at the N4 "branch points" during synthesis.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will ~ecome apparent to those skilled in the art on examination of the following, or may be learned by practice of the invention.
In one aspect of the invention, a method for prot~cting a hydroxyl group o~ a hyclroxyl-containing compound during chemical reaction oi other functional qroups contained within the compouncl is provided. The method involves reaction with a protecting species to give rise to a protected or "blocked`' hydroxyl group -OR, wherein R is in a stable oxidized form as bound, but which is readily removable upon reduction with a liquid reduc~ng agent.
In other aspects of the invention, methods for synthesizing linear and branchPd oligonucleotides are provided which make use of orthogonally removable hydroxyl-protecting groups that are -endered labile and thus readily removable upon reduction.
In still other aspects of the invention, multifunctional nucleic acids containing orthogonally removable hydroxyl-protecting groups bound to the N4-position of cytosine through an oxyalkylene linka~e are . :
:~ . . : : :
W092/0253~ PCT/~1S91/OS~'-2 o~2~5 6 provided. Such multifunctional nucleic acids are useful in the synthesis of branched oligonucleotide structures by virtue of the orthogonally removable group at the N4-position. Oligonucleotide chains containing such S multifunctional nucleic acids are provided as well.
Modes for Carryin~ Out the Invention l. Definitions:
As used herein the terms "oligonucleotide" and "polynucleotide" shall be generic to polydeoxyribo~
nucleotides (containing 2'-deoxy-D-ribose or modified forms thereof), to polyribonucleotides (containing D-ribose or modified forms thereof), and to any other type of polynucleotide which is an N-glycoside of a purine or pyrimidine base, or of a modified purine or pyrimidine base. The term "nucleoside" will similarly be generic to ri~onucleosides, deoxyribonucleosides, or to any other nucleoside which is an N-glycoside of a purine or pyrimidine base, or of a modified purine or pyrimidine ba~e. There is no intended distinction in length between the term "oligonucleotide" and "polynucleotide" and these terms will be used interchangeably. These oligonucleotides and polynucleotides may be single-stranded or double-stranded, typically single-stranded.
~lso, the oligonucleotides of the present invention are normally of from about 2 to about 2000 monomer units, and more typically, for most probe-based applications, fxom about 2 to about lOO monomer units.
"Derivatizable" nucleotides as used herein are nucleotides mqdified so as to include at the 4- position of a pyrimidine, e.g., cytosine, a functional group w~ich can react with the protecting species described hereln in which, furthermore, can be used to initiat~ synthesis of secondary oligonucleotide chains in the preparation of branched oligonucleotide structures. An example of a . ~
, WO ~2/02~3~ PCl /US~1/05''- ~`
.
~7~ 2 0882~ a derivatizable nucleotide is one which has been modified at the 4-position with an oxyalkylene moiety so that a free hydroxyl group is present at that position of the molecule.
A hydroxyl group that is "protected" is one that has been reacted with a protecting moiety such that the resulting protected group will not be susceptible to any undesired chemical reaction during the synthetic step or steps during which the protecting group is present.
By "stability" of the hydroxyl-protected compound or of the~hydroxyl-protecting group when covalently bound to -the hydroxyl-containing compound, is meant substantial absence of steric interference as well as inherent chemical stability, i.e., resistance to attack and/or degradation.
By "lower alkyl" and "lower alkoxy" are meant alkyl and alkoxy substituents, respectively, having from about 1 to 8, more typically from about 1 to 6, carbon atoms.
, Where aromatic substituents, are indicated, it is to be understood that each indiviclual aromatic ring may be substituted at one or more carbon atoms with moieties which do not adversely affect function or reactivity.
2. Hydroxyl Group Protection: i The method o~ the invention is thus useful for protecting a free hydroxyl group of a hydroxyl-containing compound so as to preserve the hydroxyl functionality during chemical reaction or chemical conversion of other functionalities present on the molecule. In general terms, the hydroxyl group to be protected is reacted with a protecting species to give rise to a moiety -OR. In a preferred embodiment, R is Maq or a derivative thereof.
In such a case, R may be represented by the structural ~ormula : : --, ' ' ' . " ~ ' .
W0~2/0253~ PCr/~lS9l/052'.
20882~ -8-~' () - ~- O ~ R
R O
in which R' is hydrogen, aryl, or aralkyl, the Ri may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower ~ alkyl and lower alkoxy, the Rj may be the same or differ- -~
ent and are selected rrom the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy;
i is zero, 1, 2 or 3; and j is zero, 1, 2, 3 or 4.
In this structure, R' is preferably hydrogen or phenyl. The Ri and Rj, as indicate~, may represent any one of a number of different substil:uents. The substituents can be selected to render the protecting ; moiety more easily reduced and thus more readily removed ~r~m the protected hydroxyl functio~ality. Alterna-tivoly, substituents may be selected which render the group more difficult to remove and thus more stable when bound. Substitution at the 1, 4, 5 and/or 8 positions will give rise to the greatest effect. ,:~
In an alternative em~odiment, R is I :
- 1-O-CH~ O~
Rk in which k is zero, 1, ~, 3 or 4; and the Rk may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy. A preferred example of such a species is p-nitrobenzyl.
~' ' ' ` ` :' . ';' ' , ' " " : ' ' : ' . : ' W O 92/02533 PC~r/US91tOS2~, ~9~ io882~5 The free hydroxyl group to be protected is derivatized with a protecting species as just described preferably by reaction with the chloroformate derivativP.
That is, to provide a protecting group which is Maq or a S Maq derivative, reaction would be carried out between the hydroxyl-containing compound and the chlororormate ~' O
O ' !l --~, ,, O
Where R is p-nitrobenzyl or a derivative thereof, again, reaction would preferably be carried out with the chloroformate C-O-CH, ~`"10 Rk Reaction is preferably carried out in an anhydrous solvent at a relatively low temperature, i.e., lower than about 20C, more preferably at or below about O~C. The chloroformate derivatives themselves may be readily synthesized from the hydroxymethyl analog with triphosgen.
Although these reducible hydroxyl-protecting groups may be used in conjunction with a wide variety of molecular structures and synthetic steps, they have been found to be particularly useful in the chemical synthesis of oligonucleotides, including short oligonucleotide chains useful for probe-type applications as well as lonyer and/or more complex, e.g., branched oligonucleotide structures. The inventors herein have 3~ found these reducible hydroxyl-pro~ecting groups to De an : : .. . .
. . , WO ~2/0253~ PCT/US91/05'~-2088~S
excellent alternative to the dimethoxytrityl (DMT) and pixyl groups at the S'-hydroxyl position. Use at the 3'-hydroxyl of either a nucleotide or an oligonucleotide chain is also possible.
S A further application of the hydroxyl-protecting groups of the invention, as will be described in more detail below, is in the bloc~ing of an exocyclic hydroxyl group present at the N~-position of a 5-methyl cytosine residue. A protecting group is necessary in such a structure when the N4-position of cytosine ~~~ ~~~ residues, i.e., contained within an oligonucleotide - chain, are used as branch points for the synthesis of secondary oli~onucleotide chains orthogonal to the backbone of the starting material.
While several of the references cited and discussed hereinabove disclose the ~Ise of reducible protecting groups ~or phosphate or c:arboxyl moieties, the use of such structures for the protection of hydroxyl species is new and provides a number of important and 2~ distinct advantagQs. First o~ all, relatively mild reagents can be used to reduce the bound protecting group and thus render it labile and removable. In the case of Maq, for example, dithionite may ~e used. This is in contrast to the need for a reagent such as acid, which is required when the dimethoxytrityl group is used for hydroxyl protection. The ability to use mild reagents minimizes the likelihood of damage to the oligonucleotide structure being synthesi~ed. These reducible protecting groups are also orthogonal in that they are quite specific and not chemically vulnerable to most chemical reagents unless reduced. Again, these reducible protecting groups work by binding to the hydroxyl gro~p to be protected in an oxidized, chemically very stable state, but are rendered labile when reduced. With the Maq ester for example, cleavage at the ester site is . .
:: : . : , , : . :. , , . ;.,. :, : . ,, ., ~ . , , W~2/0Z53~ PCT/US91/05~'-2~882~5 --11-- ` ... ~`
expected to be rapid for the hydroquinone form (see, e.g., D.S. Kemp et al., cited above), while in the quinone form, resistance to cleavage would be expected.
Finally, one additional and critically important advantage should ~e noted. This is that use of these hydroxyl pro~ecting groups in DNA synthesis substantially reduces the likelihood of depurination when acid-l~bile protection is employed, and thus eliminates the corresponding large loss in yield. This is an important advantage for all of the DNA synthesis applications - discussed herein as well as others which might be envisioned by those skilled in the art upon reading the present disclosure.
3. Chemical Synthesis of Oligonucleotides Using Orthogonally Removable Hydroxyl-Protecting Groups:
As noted above, an importiant application o~ the present hydroxyl-protecting groups iand methods is in the chemical synthesis of both linear and branched oligo-nucleotides. As is now well-known in the art, methods for syn~hesizin~ oli~onucleotides typically involve sequential addition of 3'-blocked and 5'-blocked nucleo-tide monomers to the terminal 5'-hydroxyl group of a growing oligonucleotide chain, where each addition is effected by nucleophilic attack of the terminal 5'-~5 hydroxyl group of the growing chain on the 3'-position of the added monomer, which is typically a phosphorus derivative such as a phosphotriester, phosphoramidite, or the like. Such procedures are described in detail in the references cited and discussed in the "Background"
section herein.
Another aspect of the invention thus involves use of orthogonally removable, reducible hydroxyl-protecting groups as either said 3'- or 5'-blocking groups or both. The use of the reducible protecting ~roups is preferred at the 5'-position as an alternative : , . . : . ~.
,:,, : ,-: . :: , :. :
- . . ~ , - : :: :: : . : . : .
~, :: - :. ,:::: :: : - :: . :: ::
. - . : .: ::: : ,: : .:: : ~ :. . : ,., : .: .
WQ92/~25~ PCT~IS91/0~
2V8825~ -12-to the well-known protecting moieties dimethoxytrityl and pixyl.
The hydroxyl-protecting groups of the invention are additionally useful in the fo~mation of branched oligonucleotide structures, e.g., nucleic acid multimers useful in "amplified" nucleic acid hybridization assays, as described in applicants' European Patent Application Serial No. 88.309697.6. As described in that application, the N4-position of cytosine residues within an oligonucleotide chain is modified so as to contain an oxyalkyiene moiety which may then be derivatized to give rise to secondary oligonucleotide chains in a branched structure. The referenced application describes the use of the levulinyl group as the hydroxyl-protecting moiety lS at the N4-position (the levulinyl group requires removal with hydra2ine or a similar reagent, which can give rise to destabilization).
In the present method, the branched oligonucleotide structure is made by ~irst providing an oligonucleotide chain the cytosine residues of which have been N4-derivatized to give -(CH2)X-OR moieties wherein R
is as defined above, capping the 3'- and 5'-terminal hydroxyl groups of the chain, removing the hydroxyl-protecting groups R by treatment with a liquid reducing agent, thereby giving rise to free hydroxyl groups bound through an alkylene linking group to the N4-position, and finally synthesizing secondary oligonucleotide chains at the free hydrox~l groups which then serve as the branch points.
HYDROXYL-PROTECTING GROUPS
ORTHOGONALLY REMOVABLE BY REDUCTION AND
THEIR USE IN THE CHEMICAL SYNTHESIS OF OLIGONUCLEOTIDES
Description Technical Field This invention relates generally to hydroxyl-protecting groups and more particularly relates t~
hydroxyl-protecting groups which are orthogonally removable by reduction with liquid reducing agents and which are especially useful in the chemical synthesis of oligonucleoti~es.
~ack~round With the advent of hybrid DNA technology and the e~plosion in the ability to isolate, purify and assay a wide variety of natural productsl, there is an increasing need for rapid and efficient methods of preparing and purifying oligomers o~ nucleic acids and amino acids.
With nucleic acids, it is typically necessary to synthesize sequences for use as lin~er~, adapters, synthetic genes, and synthetic rPgulatory sequences, as well as for use as probes, primers, and the like. Many procedures have been developed for producing oligomers of nucleotides, or "oligonucleotidesl'. These procedures for the most part rely on initial attachment of a f irst nuc}eotide to a solid support, followed by the se~uential :addition of subse~uent nucleotide units, with each :~30 addition involving a number of chemical reactions.
The two primary methods of oligonucleotide synthesis, which are well-established in the art, are the so-called "phosphotxiester" and 'Iphosphoramidite'' methods (descri~ed at some length in the references oited below) .
-.
- : ~ .: ...~:-. .
.;. . . ..
wo sun2s3~ Pcr/ussl/os~-~ 2-In the most prevalen~ schemes for both methods, the oligonucleotide chain grows by nucleophilic attack of the 5'-OH of the immobilized oligomer on an activated 3'-phosphate or phosphoramidite function o~ a soluble 5'-protected nucleotide building block. Other key stepsinclude the acid deprotection of the 5'-0-(4,4'-dimethoxytrityl) group (DMT) in the phosphotriester method, and, in the phosphoramidite process, the oxidation of the phosphite triester to the phosphate triester. _ _ Other methods of oligonucleotide synthesis are also known, including 5'-to-3' syntheses which use a ~-cyanoethyl phosphate protectin~ group (De Napoli et al., Gazz~ Chim. It:al. 114:65 (1984); Rosenthal et al., lS Tetrahedron Lett. 24:1691 ~1983); Belagaje and ~rush, Nucleic Acids Res. 10:6295 (1977); ~ramer and Koster, i~n~QW~ C~em. Int. Ed. Enql. 7:473 (1968); and Blackburn et al., ~. Chem. Soc. C, 243~ 67)).
All of these methods of sy~nthesizing oligonucleotides involve the use of 3'- and 5'-hydroxyl-protecting groups. Many of the hydroxyl-protecting groups used in oligonucleotide synthesis present some problems. For example, it is obviously desirable that a hydroxyl-protecting group be "orthogonal," i.e., removable with reagents that do not affect the remainder of the molecule, including other blocking or protecting groups which may be present. Some of the known hydroxyl-protecting groups are not completely "orthogonal". Also, many of the currently used hydroxyl-protecting groups, e.g., the levulinyl group, require removal with harsh re~gents (e.g., acid in the case of dimethoxytrityl).
~ The need for harsh reagents can damage a growing ; oligonucleotide chain and, furthermore, severely limits the number and type of protecting groups which may be employed elsewhere in the molecule during synthesis.
.- . : : . . . : . ., W092/0253~ PCT/US91/0;~-_3_ 20~8255 Finally, it is desirable that the hydroxyl-protecting group be chemically stable in relation to whatever reagents are to be used in the chemical reactions involving the remainder of the molecule. It has proved difficult to find hydroxyl-protectiny groups which are chemically stable as ~bound" during use yet which are readily removable with relatively mild reagents. The invention is directed to orthogonal hydroxyl-protecting groups which are in fact quite stable while bound to the protected molecule, but which are nevertheless easily removable post-reaction with mild reagents. The presen, invention makes use of protecting groups which, when bound to the protected molecule, are in an oxidized, stable state, but which upon reduction become labile and are thus readily removable. The novel hydroxyl-protecting groups may also be used when there is more than one hydroxy~ group present in the molecule to be protected. These protecting groups have been found by the inventors herein to be extremely versatile and invaluable as hydroxyl-protecting groups in general and more particularly in the chemical ~,ynthesis of oligonucleotides.
In addition to the refere~nces cited and discussed in the preceding section, the following references also relate to one or more aspects of the present invention.
D.S. Kemp et al., Tetrahedron Letters, No. 12, pp. 1031-1034 (1977), describe the use of Maq esters as carboxyl protecting groups, specifically for use in the chemical synthesis of peptides.
N. Balgobin et al., Chemica ScriPta 20:198-200 (~982), describe the use of 2-oxymethyleneanthraquinone as a terminal phosphate protecting group in the chemical synthesis of DNA and RNA.
: . . ' :
.
:
-: ; . . :. . : :
- ~'. ' ~ ; ': ' ' ' , WO ~/02533 PCI/~'S91/05~-2 0~-8 2~ ~ _4_ R.L. Blankespoor et al., ~. Orq. Chem. 49:4441-46 (1984), describe the use of the 2-methylene-9,10-anthraquinone (Maq) ester to bind a y-aminobutyric acid.
The focus is on the development of an improved delivery system for neurotransmitters (i.e., such as ~-amino-butyric acid (GABA)). The authors note that the Maq ester is cleavable upon electroreduction to give the corresponding hydroquinone.
Disclosure of the Invention Accordingly, it is a primary object of the invention to provide methods and reagents for protecting hydroxyl groups, particularly during the chemical synthesis of oligonucleotides.
It is another object of the invention to provide orthogonally removable hydroxyl-protecting groups which are rendered labile and removable upon reduction with a liquid reducing agent.
It is still another object: of the invention to provide a multifunctional nucleic acid derivatized at the N4-position with an oxyalkylene moiety -(CH2)X-OR where R
is a hydroxyl-protecti~g group as will be described in detail herein.
It is yet another object of the invention to provide oligonucleotide chains containing such multifunctional nucleic acids.
It is a further object of the invention to provide a method of protecting a hydroxyl group of a hydroxyl-containing compound during chemical reaction of other functional groups contained within the compound which involves, prior to such chemical reaction, reacting the hydroxyl group to be protected with a chloroformate derivative of the desired protecting species.
It is still a further object of the invention to provide an improved method for chemically synthesizing - : . . :: : ,:
. . : .: : . : : : : . ' W~2/n253~ PCT/US91/~57~
-5- 20882~
oligonucleotides from nucleotide monomers. The improvement is directed to the use of certain orthogonally removable hydroxyl-protecting groups as will be described herein.
It is yet a further object oî the invention to provide a method of making a branched oligonucleotide s~ructure, which involves derivatizing a linear oligo-nucleotide at the N4-position of cytosine residues with secondary oligonucleotide chains, using the orthogonally removable hydroxyl-protecting groups of the invention at the N4 "branch points" during synthesis.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will ~ecome apparent to those skilled in the art on examination of the following, or may be learned by practice of the invention.
In one aspect of the invention, a method for prot~cting a hydroxyl group o~ a hyclroxyl-containing compound during chemical reaction oi other functional qroups contained within the compouncl is provided. The method involves reaction with a protecting species to give rise to a protected or "blocked`' hydroxyl group -OR, wherein R is in a stable oxidized form as bound, but which is readily removable upon reduction with a liquid reduc~ng agent.
In other aspects of the invention, methods for synthesizing linear and branchPd oligonucleotides are provided which make use of orthogonally removable hydroxyl-protecting groups that are -endered labile and thus readily removable upon reduction.
In still other aspects of the invention, multifunctional nucleic acids containing orthogonally removable hydroxyl-protecting groups bound to the N4-position of cytosine through an oxyalkylene linka~e are . :
:~ . . : : :
W092/0253~ PCT/~1S91/OS~'-2 o~2~5 6 provided. Such multifunctional nucleic acids are useful in the synthesis of branched oligonucleotide structures by virtue of the orthogonally removable group at the N4-position. Oligonucleotide chains containing such S multifunctional nucleic acids are provided as well.
Modes for Carryin~ Out the Invention l. Definitions:
As used herein the terms "oligonucleotide" and "polynucleotide" shall be generic to polydeoxyribo~
nucleotides (containing 2'-deoxy-D-ribose or modified forms thereof), to polyribonucleotides (containing D-ribose or modified forms thereof), and to any other type of polynucleotide which is an N-glycoside of a purine or pyrimidine base, or of a modified purine or pyrimidine base. The term "nucleoside" will similarly be generic to ri~onucleosides, deoxyribonucleosides, or to any other nucleoside which is an N-glycoside of a purine or pyrimidine base, or of a modified purine or pyrimidine ba~e. There is no intended distinction in length between the term "oligonucleotide" and "polynucleotide" and these terms will be used interchangeably. These oligonucleotides and polynucleotides may be single-stranded or double-stranded, typically single-stranded.
~lso, the oligonucleotides of the present invention are normally of from about 2 to about 2000 monomer units, and more typically, for most probe-based applications, fxom about 2 to about lOO monomer units.
"Derivatizable" nucleotides as used herein are nucleotides mqdified so as to include at the 4- position of a pyrimidine, e.g., cytosine, a functional group w~ich can react with the protecting species described hereln in which, furthermore, can be used to initiat~ synthesis of secondary oligonucleotide chains in the preparation of branched oligonucleotide structures. An example of a . ~
, WO ~2/02~3~ PCl /US~1/05''- ~`
.
~7~ 2 0882~ a derivatizable nucleotide is one which has been modified at the 4-position with an oxyalkylene moiety so that a free hydroxyl group is present at that position of the molecule.
A hydroxyl group that is "protected" is one that has been reacted with a protecting moiety such that the resulting protected group will not be susceptible to any undesired chemical reaction during the synthetic step or steps during which the protecting group is present.
By "stability" of the hydroxyl-protected compound or of the~hydroxyl-protecting group when covalently bound to -the hydroxyl-containing compound, is meant substantial absence of steric interference as well as inherent chemical stability, i.e., resistance to attack and/or degradation.
By "lower alkyl" and "lower alkoxy" are meant alkyl and alkoxy substituents, respectively, having from about 1 to 8, more typically from about 1 to 6, carbon atoms.
, Where aromatic substituents, are indicated, it is to be understood that each indiviclual aromatic ring may be substituted at one or more carbon atoms with moieties which do not adversely affect function or reactivity.
2. Hydroxyl Group Protection: i The method o~ the invention is thus useful for protecting a free hydroxyl group of a hydroxyl-containing compound so as to preserve the hydroxyl functionality during chemical reaction or chemical conversion of other functionalities present on the molecule. In general terms, the hydroxyl group to be protected is reacted with a protecting species to give rise to a moiety -OR. In a preferred embodiment, R is Maq or a derivative thereof.
In such a case, R may be represented by the structural ~ormula : : --, ' ' ' . " ~ ' .
W0~2/0253~ PCr/~lS9l/052'.
20882~ -8-~' () - ~- O ~ R
R O
in which R' is hydrogen, aryl, or aralkyl, the Ri may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower ~ alkyl and lower alkoxy, the Rj may be the same or differ- -~
ent and are selected rrom the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy;
i is zero, 1, 2 or 3; and j is zero, 1, 2, 3 or 4.
In this structure, R' is preferably hydrogen or phenyl. The Ri and Rj, as indicate~, may represent any one of a number of different substil:uents. The substituents can be selected to render the protecting ; moiety more easily reduced and thus more readily removed ~r~m the protected hydroxyl functio~ality. Alterna-tivoly, substituents may be selected which render the group more difficult to remove and thus more stable when bound. Substitution at the 1, 4, 5 and/or 8 positions will give rise to the greatest effect. ,:~
In an alternative em~odiment, R is I :
- 1-O-CH~ O~
Rk in which k is zero, 1, ~, 3 or 4; and the Rk may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy. A preferred example of such a species is p-nitrobenzyl.
~' ' ' ` ` :' . ';' ' , ' " " : ' ' : ' . : ' W O 92/02533 PC~r/US91tOS2~, ~9~ io882~5 The free hydroxyl group to be protected is derivatized with a protecting species as just described preferably by reaction with the chloroformate derivativP.
That is, to provide a protecting group which is Maq or a S Maq derivative, reaction would be carried out between the hydroxyl-containing compound and the chlororormate ~' O
O ' !l --~, ,, O
Where R is p-nitrobenzyl or a derivative thereof, again, reaction would preferably be carried out with the chloroformate C-O-CH, ~`"10 Rk Reaction is preferably carried out in an anhydrous solvent at a relatively low temperature, i.e., lower than about 20C, more preferably at or below about O~C. The chloroformate derivatives themselves may be readily synthesized from the hydroxymethyl analog with triphosgen.
Although these reducible hydroxyl-protecting groups may be used in conjunction with a wide variety of molecular structures and synthetic steps, they have been found to be particularly useful in the chemical synthesis of oligonucleotides, including short oligonucleotide chains useful for probe-type applications as well as lonyer and/or more complex, e.g., branched oligonucleotide structures. The inventors herein have 3~ found these reducible hydroxyl-pro~ecting groups to De an : : .. . .
. . , WO ~2/0253~ PCT/US91/05'~-2088~S
excellent alternative to the dimethoxytrityl (DMT) and pixyl groups at the S'-hydroxyl position. Use at the 3'-hydroxyl of either a nucleotide or an oligonucleotide chain is also possible.
S A further application of the hydroxyl-protecting groups of the invention, as will be described in more detail below, is in the bloc~ing of an exocyclic hydroxyl group present at the N~-position of a 5-methyl cytosine residue. A protecting group is necessary in such a structure when the N4-position of cytosine ~~~ ~~~ residues, i.e., contained within an oligonucleotide - chain, are used as branch points for the synthesis of secondary oli~onucleotide chains orthogonal to the backbone of the starting material.
While several of the references cited and discussed hereinabove disclose the ~Ise of reducible protecting groups ~or phosphate or c:arboxyl moieties, the use of such structures for the protection of hydroxyl species is new and provides a number of important and 2~ distinct advantagQs. First o~ all, relatively mild reagents can be used to reduce the bound protecting group and thus render it labile and removable. In the case of Maq, for example, dithionite may ~e used. This is in contrast to the need for a reagent such as acid, which is required when the dimethoxytrityl group is used for hydroxyl protection. The ability to use mild reagents minimizes the likelihood of damage to the oligonucleotide structure being synthesi~ed. These reducible protecting groups are also orthogonal in that they are quite specific and not chemically vulnerable to most chemical reagents unless reduced. Again, these reducible protecting groups work by binding to the hydroxyl gro~p to be protected in an oxidized, chemically very stable state, but are rendered labile when reduced. With the Maq ester for example, cleavage at the ester site is . .
:: : . : , , : . :. , , . ;.,. :, : . ,, ., ~ . , , W~2/0Z53~ PCT/US91/05~'-2~882~5 --11-- ` ... ~`
expected to be rapid for the hydroquinone form (see, e.g., D.S. Kemp et al., cited above), while in the quinone form, resistance to cleavage would be expected.
Finally, one additional and critically important advantage should ~e noted. This is that use of these hydroxyl pro~ecting groups in DNA synthesis substantially reduces the likelihood of depurination when acid-l~bile protection is employed, and thus eliminates the corresponding large loss in yield. This is an important advantage for all of the DNA synthesis applications - discussed herein as well as others which might be envisioned by those skilled in the art upon reading the present disclosure.
3. Chemical Synthesis of Oligonucleotides Using Orthogonally Removable Hydroxyl-Protecting Groups:
As noted above, an importiant application o~ the present hydroxyl-protecting groups iand methods is in the chemical synthesis of both linear and branched oligo-nucleotides. As is now well-known in the art, methods for syn~hesizin~ oli~onucleotides typically involve sequential addition of 3'-blocked and 5'-blocked nucleo-tide monomers to the terminal 5'-hydroxyl group of a growing oligonucleotide chain, where each addition is effected by nucleophilic attack of the terminal 5'-~5 hydroxyl group of the growing chain on the 3'-position of the added monomer, which is typically a phosphorus derivative such as a phosphotriester, phosphoramidite, or the like. Such procedures are described in detail in the references cited and discussed in the "Background"
section herein.
Another aspect of the invention thus involves use of orthogonally removable, reducible hydroxyl-protecting groups as either said 3'- or 5'-blocking groups or both. The use of the reducible protecting ~roups is preferred at the 5'-position as an alternative : , . . : . ~.
,:,, : ,-: . :: , :. :
- . . ~ , - : :: :: : . : . : .
~, :: - :. ,:::: :: : - :: . :: ::
. - . : .: ::: : ,: : .:: : ~ :. . : ,., : .: .
WQ92/~25~ PCT~IS91/0~
2V8825~ -12-to the well-known protecting moieties dimethoxytrityl and pixyl.
The hydroxyl-protecting groups of the invention are additionally useful in the fo~mation of branched oligonucleotide structures, e.g., nucleic acid multimers useful in "amplified" nucleic acid hybridization assays, as described in applicants' European Patent Application Serial No. 88.309697.6. As described in that application, the N4-position of cytosine residues within an oligonucleotide chain is modified so as to contain an oxyalkyiene moiety which may then be derivatized to give rise to secondary oligonucleotide chains in a branched structure. The referenced application describes the use of the levulinyl group as the hydroxyl-protecting moiety lS at the N4-position (the levulinyl group requires removal with hydra2ine or a similar reagent, which can give rise to destabilization).
In the present method, the branched oligonucleotide structure is made by ~irst providing an oligonucleotide chain the cytosine residues of which have been N4-derivatized to give -(CH2)X-OR moieties wherein R
is as defined above, capping the 3'- and 5'-terminal hydroxyl groups of the chain, removing the hydroxyl-protecting groups R by treatment with a liquid reducing agent, thereby giving rise to free hydroxyl groups bound through an alkylene linking group to the N4-position, and finally synthesizing secondary oligonucleotide chains at the free hydrox~l groups which then serve as the branch points.
4. Multifunctional Nucleic Acids and Oligo-nucleotides Containing the Same:
In another embodiment, the present invention encompasses multifunctional nucleic acids derivatized so as to contain the moiety -(CH2)X-OR at the N4-position, wherein R is as defined above, as well as oligonucleo-W~2/0253~ PCT/US91/0521~
2 0 8 8 ~ ~ ~
tides containing such derivatized mul~ifunctional nucleic acids. The multifunctional nucleic acids have the structure Z
I (~) R~- ~
~
. _ .. _ ..
R!O I
~,0~
y ORI
wherein R is a hydroxyl protecting group that can be removed and replaced, without affect~ing Rl or R2, by reduction with a liquid reducing agent; Rl is a phosphorus derivative that enables addition of nucleotides to the 5'-position of an oligonucleotide chain duri~g chemical synthesis; R2 is a protecting group that is generally base-stable and acid-sensitive; R3 is selected from the gsoup consisting of hydrogen, methyl, I, Br and F; R4 is hydrogen or methyl; and Z is selected from the group consisting of : ~
W092/0253~ PCTJ~'S91/052', 208825~ -14-'; ,1 (I) ~ CH, J ,~ C--O ' o ~') 11 ~ 1) ' ~CH.! ~--~1--C ~CH,) ~-- O - :
1 0 , , (~ .
~CH.I~ ~ - C - ~CH-\~ ICH~
1') ~ ll;
(CH,)~-- ~H ~ ICH,jV O
1", (1) - (CH. - CH~ - O)~ - ; ~d ~) (1) ! CH,!,--O
wherein x and y may be the same or different and are intege~s in the range of 1 to 8 inclusive. (The designations "tl)" and "(2)" at the Z linkage indicate the orientation of the Z linker moiety.) In this structure, it is preferred that Z be -(CH2)X-, R1 be a phosphoramidite, a phosphodiester ~r a phosphotriester, while it is similarly preferred that R2 be dimethoxytrityl or pixyl. R, as described throughout the present application, is an orthogonally removable : - : . , ,: ., . "
W0~2/02~3~ PCT/US91/052~- ~
20882~ `
hydroxyl-protecting group reducible with a liquid reducing agent to give rise to a labile, easily removable species.
oligonucleotide chains containing these modified cytosine residues, i.e., derivatized modified nucleotides as just described, thus have the s~ructure R
z 1 !'`
. . . _ . _ ,~, Rj O~ ~
b~ckbone - j bac~bone in which R, R3, R4, Z, x and y are as definad above.
It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, that the foregoing description as well as the examples which follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the s~ope of the invention will be apparent to those skilled in the art to which the invention pertains.
,.. .
., . .. .: . , . , :
.: ,. :: ,, . : :. -.
W0~2/02533 PCT/US91/052~-208~25~
Exam~le 1 Use of Carbonate Esters of 2-(hvdroxYmethYl)-anthra~uinone (MAC Derived from Methoxyanthra~uinone Oxycarbonyll to Protect Exocyclic Alkyl Hydroxyl Group _in Branchinq Monomers The MAQ moiety (methoxyanthraquinone) has been utilized to protect: a) carboxylic acids as MAQ esters (MAC; D.S. Kemp and J. Reczek, Tetrahedron letters 12, p 1031-1034 ~1977)), b) amines as a MAQ urethane (R.L.
Blankespoor, A.N.K. Law and L.L. Miller, Journal of organic Chemistry 49, p 4441-~446 (1984)), and c) phosphate diesters as the MAQ phosphotriester ~N.
Balgobin, M. Kwaitkowski and J. Chattopadhyaya, Chemica Scripta 20, p 198-200 ~1982)).
Deprotection of the MAC c:arbonate esters is effected by treatment with sodium clithionite under neutral conditions. The MAC group is orthogonal to other protecting groups (PG) used in DNA chemical synthesis, i.e., it can be removed under condi.tions that do not destabilize other protecting groupc; used (see table), nor does the sodium dithionite solution damage the native DNA
bases. The use of MAC for hydroxyl group protection has not been reported. The mild conditions for its removal may make it useful in protection of S'-hydroxy groups in DNA and RNA synthesis.
2-(Hydroxymethyl)anthraquinone is converted to the corresponding chloroformate (MAC-Cl) with triphosgen.
The MAC-Cl reacts specifically with the primary hydroxy group of N-4-(6-hydroxyhexyl)-5'-DMT-5-methyl-2'-deoxycytidine.
The synthesis conditions for making large quantities (25 millimole scale) have been worked out, and the synthesis of branched DNA molecules has been conducted.
~ : :: .: :. : : ;
W0~2/02533 PCT/US91/05~
.
-17- 20882~3 Table 1.
Stabilit~ of Selected Protectinq Groups Yields are reported in %, where 100% indicates no deprotection and/or modification as judged by TLC
analysis after 1 and 18 hours. The sodium dithionite solution was prepared by dissolving 1 gram of solid sodium dithionite in 20 ml of 1 M triethylammoniu~
bicarbonate, followed by addition of 20 ml dioxane.
Functionalitv in DNA 1 hour 18 hours succinate 100 100 A (benzyl) 100 >95 C (benzyl) 100 >go G (isobutyl) 100 100 BM2 (deprotected MAC) 100 100 BM2 (levulinyl) 100 100 p_O! cyanoethyl >85 < 5 P-O-methyl 100 >95 PreParation of 2-anthra~uinonemethoxv chloro~ormate ~MAC-Cl~: A 0.1 molar solution of 2-(hydroxymethyl)-anthraquinone (MAQ-OH) was prepared by dissolving 25 mmole (5.g5 g) in 250 ml dioxane. The yellow solution was filtered and the solvent removed by evaporation to remove watar. The residue was redissolved in 200 ml dioxane and pyridine (2 ml; 25 mmole) was added. This solution was added dropwise to a stirred solution of triphosgen (2.5 q; 25 Meq) in 50 ml CH2C12 at 0C. After ended addition the mixture was stirred at 20C for 18 hours. The mixture was diluted with 800 ml ethyl acetate and the organic phase washed with 3 x 600 .- .. , . -.. , ,: ,.. ::. .,: .: ; ,, :.: ~ .
WO 92t0253~ PCl/US91/05~
20~8~S~
':
ml 80% saturated aqueous NaCl solution. After drying of the organic phase over NaSO4 the solvent was removed in vacuo to give a yellow solid, which was dissolved in CH2CL2 (250 ml; 0.1 M). This solution was used without further purification.
Preparation of 5`-DMT-N-4-tO-2-anthraquinone-methoxycarbonyl-6-ox~hexyl)-5-methvl-2'-deoxycytidine 3'-P-~ N-diisoDro~ylmethylphosphoramidite (`'E Base" or "~
To a solution of N-4-(6-hydroxyhexyl)-5`-DMT-5-methyl-2'deoxycytidine (17 mmole), prepared as previo~- y described (Horn and Urdea, NAR vol; 17:17, p. 695g-696, (1989)), in 200 ml methylene chloride was added pyridine (40 mmole) and the mixture was cooled to 0C. A
solution of ~AC-Cl (20 mmole) in 200 ml of CH212 was added dropwise and le~t stirring for 10 minutes. TLC
analysis (silica plates developed with 10~
methanollCH2CL2) showed that the slarting material had been completely consumed. The reaction mixture was dilut~d with 400 ml ethyl acetat~ and the organic phase ~xtra~tQd with 2 x 300 ml 5~ NaHCO3 and 80% saturated aqueous NaCl. After drying of the organic phase over Na2S04 for 30 minutes followed by filtration the solvent was removed in vacuo. The product was purified by silica gel chromatography using a gradient of methanol (0-6%) in ~ CH2Cl2 to give 13 g of pure product (~5% yield).
The nucleoside N-4-tO-anthraquinone-methoxycarbonyl-6-oxyhexyl)-5'-DMT-5-methyl-2'-deoxycytidine (14.4 mmole) was dissolved in CH2Cl2 (50 ml) containinq 70 mmole DIPEA. After cooling to 0C
N,N-diisopropylaminomethoxychlorophosphine was added (2.72 ml; 14 mmole). The phosphitylating agent was added in small portions until 95% of the starting material had been consumed. The reaction mixture was then diluted with ethyl acetate t300 ml), extracted with 2 x 300 ml 5% Na~C03 then 2 ~ 300 ml 80% saturated aqueous NaCl and .. , .. - . ,. ~ . . .
.: , :, .. .
, , ., ' ; ~ :::' - . . ~ ::
; . . .
;: ~
W0~2/0253~ PCT/US91/05~
-19- 2~882~
finally dried over solid Na SO~. The solvent was removed in vacuo.
The crude phosphoramidite was purified by silica gel chromatography using the solvent system methylene chloride/ethyl acetate/triethylamine (49:49:2 v/v), and the fractions containing the product were pooled and concentrated. After coevaporation with toluene, the purified phosphoramidite was dissolved in toluane and added with rapid stirring to 800 ml of cold hexanes (-50C). the resulting precipitate was rapidly collected by filtration and dried in high vacuum for la hours to give 12.4 g of a slightly yellow solid (81%
yield). NMR 31p 8 145 ppm.
A DNA oligomer was synthesized on a 3000 A CPG
support ~40 mg) with the sequence 3'-TCC-GTA-TCC-TGG-GCA-CAG-TTE (MAC)15 using the standard 1 micromole scale program using methyl phosphoramidite on the ABI 380B.
The support was next treated with a solution of lg Na2S204 in 20 ml lM TEA~/10 ml dioxane for 30 minutes to remov~ the MAC group. After filtration and washing with water and CH3CN the solid support was dried. The secondary synthesis to introduce the secondary sequence "X" ~as performed on ABI 380B using a special double condensatian cycle to incorporate 15 identical copies of the sequence 3'-GT~-AGT-5' ("X"). During synthesis DMT
removal was achieved with 3% DCA in toluene/3~ TCA in CH2C12 ~l:lv/v) using high flow rate. Complete deprotection of the is secondary site branched DNA was achieved with 3% DCA in toluene to remove DMT groups and thiopheno~`/TEA/dioxane to remove methyl groups from phosphotriesters on the solid-supported fragment. The fragment was rèleased with NH40H at 20C for 1 hour and exocyclic N-protecting groups were removed with hot NH~OH
at 60C for 18 hours. After removal of the volatile solvent, the product was analyzed by PAGE.
. . - . , , : . : ;: :: . : .,. . . . .- , . . ..
- . . . : : . . : ~ .: . . .
W092/02533 PCT/~IS91/0522~
20882~ ~o :
By analogy, when N-4-(O-levulinyl-6-oxyhexyl)-5-methyl-2'-deoxycytidine is used the lev group was removed with a solution of 0.5 M hydrazine hydrate in pyridine/ace~ic acid (4:1v/v) for 90 minutes prior to secondary synthesis.
:
.- ~ : ::: : ,., ~
`"' . ' - ' . -; . ' . ' .
' : : ' , ' ' . . .' ' . : . : ' ' ' ' ':' . ; ' ' ~ ' . ' .
. ' . . . ` . ' ' ' ' ', '.' "', .' ' ''' , ' . ~' , ' ' " ' ' ' 1 ' '~ ' , ' : . '. ' '.'. ' " ' ~ ' ':: ' ', " ,' , .' ' ..".~ ' ' ~ :, ' ' , ' ' ' ':' ' ' ' ' . ' , .' ' .' ': ' ,' ' ~ ' . " ~ . ' . ' . . ' . , .
., " ,'', ' ', ' , . ' ' ,'
In another embodiment, the present invention encompasses multifunctional nucleic acids derivatized so as to contain the moiety -(CH2)X-OR at the N4-position, wherein R is as defined above, as well as oligonucleo-W~2/0253~ PCT/US91/0521~
2 0 8 8 ~ ~ ~
tides containing such derivatized mul~ifunctional nucleic acids. The multifunctional nucleic acids have the structure Z
I (~) R~- ~
~
. _ .. _ ..
R!O I
~,0~
y ORI
wherein R is a hydroxyl protecting group that can be removed and replaced, without affect~ing Rl or R2, by reduction with a liquid reducing agent; Rl is a phosphorus derivative that enables addition of nucleotides to the 5'-position of an oligonucleotide chain duri~g chemical synthesis; R2 is a protecting group that is generally base-stable and acid-sensitive; R3 is selected from the gsoup consisting of hydrogen, methyl, I, Br and F; R4 is hydrogen or methyl; and Z is selected from the group consisting of : ~
W092/0253~ PCTJ~'S91/052', 208825~ -14-'; ,1 (I) ~ CH, J ,~ C--O ' o ~') 11 ~ 1) ' ~CH.! ~--~1--C ~CH,) ~-- O - :
1 0 , , (~ .
~CH.I~ ~ - C - ~CH-\~ ICH~
1') ~ ll;
(CH,)~-- ~H ~ ICH,jV O
1", (1) - (CH. - CH~ - O)~ - ; ~d ~) (1) ! CH,!,--O
wherein x and y may be the same or different and are intege~s in the range of 1 to 8 inclusive. (The designations "tl)" and "(2)" at the Z linkage indicate the orientation of the Z linker moiety.) In this structure, it is preferred that Z be -(CH2)X-, R1 be a phosphoramidite, a phosphodiester ~r a phosphotriester, while it is similarly preferred that R2 be dimethoxytrityl or pixyl. R, as described throughout the present application, is an orthogonally removable : - : . , ,: ., . "
W0~2/02~3~ PCT/US91/052~- ~
20882~ `
hydroxyl-protecting group reducible with a liquid reducing agent to give rise to a labile, easily removable species.
oligonucleotide chains containing these modified cytosine residues, i.e., derivatized modified nucleotides as just described, thus have the s~ructure R
z 1 !'`
. . . _ . _ ,~, Rj O~ ~
b~ckbone - j bac~bone in which R, R3, R4, Z, x and y are as definad above.
It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, that the foregoing description as well as the examples which follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the s~ope of the invention will be apparent to those skilled in the art to which the invention pertains.
,.. .
., . .. .: . , . , :
.: ,. :: ,, . : :. -.
W0~2/02533 PCT/US91/052~-208~25~
Exam~le 1 Use of Carbonate Esters of 2-(hvdroxYmethYl)-anthra~uinone (MAC Derived from Methoxyanthra~uinone Oxycarbonyll to Protect Exocyclic Alkyl Hydroxyl Group _in Branchinq Monomers The MAQ moiety (methoxyanthraquinone) has been utilized to protect: a) carboxylic acids as MAQ esters (MAC; D.S. Kemp and J. Reczek, Tetrahedron letters 12, p 1031-1034 ~1977)), b) amines as a MAQ urethane (R.L.
Blankespoor, A.N.K. Law and L.L. Miller, Journal of organic Chemistry 49, p 4441-~446 (1984)), and c) phosphate diesters as the MAQ phosphotriester ~N.
Balgobin, M. Kwaitkowski and J. Chattopadhyaya, Chemica Scripta 20, p 198-200 ~1982)).
Deprotection of the MAC c:arbonate esters is effected by treatment with sodium clithionite under neutral conditions. The MAC group is orthogonal to other protecting groups (PG) used in DNA chemical synthesis, i.e., it can be removed under condi.tions that do not destabilize other protecting groupc; used (see table), nor does the sodium dithionite solution damage the native DNA
bases. The use of MAC for hydroxyl group protection has not been reported. The mild conditions for its removal may make it useful in protection of S'-hydroxy groups in DNA and RNA synthesis.
2-(Hydroxymethyl)anthraquinone is converted to the corresponding chloroformate (MAC-Cl) with triphosgen.
The MAC-Cl reacts specifically with the primary hydroxy group of N-4-(6-hydroxyhexyl)-5'-DMT-5-methyl-2'-deoxycytidine.
The synthesis conditions for making large quantities (25 millimole scale) have been worked out, and the synthesis of branched DNA molecules has been conducted.
~ : :: .: :. : : ;
W0~2/02533 PCT/US91/05~
.
-17- 20882~3 Table 1.
Stabilit~ of Selected Protectinq Groups Yields are reported in %, where 100% indicates no deprotection and/or modification as judged by TLC
analysis after 1 and 18 hours. The sodium dithionite solution was prepared by dissolving 1 gram of solid sodium dithionite in 20 ml of 1 M triethylammoniu~
bicarbonate, followed by addition of 20 ml dioxane.
Functionalitv in DNA 1 hour 18 hours succinate 100 100 A (benzyl) 100 >95 C (benzyl) 100 >go G (isobutyl) 100 100 BM2 (deprotected MAC) 100 100 BM2 (levulinyl) 100 100 p_O! cyanoethyl >85 < 5 P-O-methyl 100 >95 PreParation of 2-anthra~uinonemethoxv chloro~ormate ~MAC-Cl~: A 0.1 molar solution of 2-(hydroxymethyl)-anthraquinone (MAQ-OH) was prepared by dissolving 25 mmole (5.g5 g) in 250 ml dioxane. The yellow solution was filtered and the solvent removed by evaporation to remove watar. The residue was redissolved in 200 ml dioxane and pyridine (2 ml; 25 mmole) was added. This solution was added dropwise to a stirred solution of triphosgen (2.5 q; 25 Meq) in 50 ml CH2C12 at 0C. After ended addition the mixture was stirred at 20C for 18 hours. The mixture was diluted with 800 ml ethyl acetate and the organic phase washed with 3 x 600 .- .. , . -.. , ,: ,.. ::. .,: .: ; ,, :.: ~ .
WO 92t0253~ PCl/US91/05~
20~8~S~
':
ml 80% saturated aqueous NaCl solution. After drying of the organic phase over NaSO4 the solvent was removed in vacuo to give a yellow solid, which was dissolved in CH2CL2 (250 ml; 0.1 M). This solution was used without further purification.
Preparation of 5`-DMT-N-4-tO-2-anthraquinone-methoxycarbonyl-6-ox~hexyl)-5-methvl-2'-deoxycytidine 3'-P-~ N-diisoDro~ylmethylphosphoramidite (`'E Base" or "~
To a solution of N-4-(6-hydroxyhexyl)-5`-DMT-5-methyl-2'deoxycytidine (17 mmole), prepared as previo~- y described (Horn and Urdea, NAR vol; 17:17, p. 695g-696, (1989)), in 200 ml methylene chloride was added pyridine (40 mmole) and the mixture was cooled to 0C. A
solution of ~AC-Cl (20 mmole) in 200 ml of CH212 was added dropwise and le~t stirring for 10 minutes. TLC
analysis (silica plates developed with 10~
methanollCH2CL2) showed that the slarting material had been completely consumed. The reaction mixture was dilut~d with 400 ml ethyl acetat~ and the organic phase ~xtra~tQd with 2 x 300 ml 5~ NaHCO3 and 80% saturated aqueous NaCl. After drying of the organic phase over Na2S04 for 30 minutes followed by filtration the solvent was removed in vacuo. The product was purified by silica gel chromatography using a gradient of methanol (0-6%) in ~ CH2Cl2 to give 13 g of pure product (~5% yield).
The nucleoside N-4-tO-anthraquinone-methoxycarbonyl-6-oxyhexyl)-5'-DMT-5-methyl-2'-deoxycytidine (14.4 mmole) was dissolved in CH2Cl2 (50 ml) containinq 70 mmole DIPEA. After cooling to 0C
N,N-diisopropylaminomethoxychlorophosphine was added (2.72 ml; 14 mmole). The phosphitylating agent was added in small portions until 95% of the starting material had been consumed. The reaction mixture was then diluted with ethyl acetate t300 ml), extracted with 2 x 300 ml 5% Na~C03 then 2 ~ 300 ml 80% saturated aqueous NaCl and .. , .. - . ,. ~ . . .
.: , :, .. .
, , ., ' ; ~ :::' - . . ~ ::
; . . .
;: ~
W0~2/0253~ PCT/US91/05~
-19- 2~882~
finally dried over solid Na SO~. The solvent was removed in vacuo.
The crude phosphoramidite was purified by silica gel chromatography using the solvent system methylene chloride/ethyl acetate/triethylamine (49:49:2 v/v), and the fractions containing the product were pooled and concentrated. After coevaporation with toluene, the purified phosphoramidite was dissolved in toluane and added with rapid stirring to 800 ml of cold hexanes (-50C). the resulting precipitate was rapidly collected by filtration and dried in high vacuum for la hours to give 12.4 g of a slightly yellow solid (81%
yield). NMR 31p 8 145 ppm.
A DNA oligomer was synthesized on a 3000 A CPG
support ~40 mg) with the sequence 3'-TCC-GTA-TCC-TGG-GCA-CAG-TTE (MAC)15 using the standard 1 micromole scale program using methyl phosphoramidite on the ABI 380B.
The support was next treated with a solution of lg Na2S204 in 20 ml lM TEA~/10 ml dioxane for 30 minutes to remov~ the MAC group. After filtration and washing with water and CH3CN the solid support was dried. The secondary synthesis to introduce the secondary sequence "X" ~as performed on ABI 380B using a special double condensatian cycle to incorporate 15 identical copies of the sequence 3'-GT~-AGT-5' ("X"). During synthesis DMT
removal was achieved with 3% DCA in toluene/3~ TCA in CH2C12 ~l:lv/v) using high flow rate. Complete deprotection of the is secondary site branched DNA was achieved with 3% DCA in toluene to remove DMT groups and thiopheno~`/TEA/dioxane to remove methyl groups from phosphotriesters on the solid-supported fragment. The fragment was rèleased with NH40H at 20C for 1 hour and exocyclic N-protecting groups were removed with hot NH~OH
at 60C for 18 hours. After removal of the volatile solvent, the product was analyzed by PAGE.
. . - . , , : . : ;: :: . : .,. . . . .- , . . ..
- . . . : : . . : ~ .: . . .
W092/02533 PCT/~IS91/0522~
20882~ ~o :
By analogy, when N-4-(O-levulinyl-6-oxyhexyl)-5-methyl-2'-deoxycytidine is used the lev group was removed with a solution of 0.5 M hydrazine hydrate in pyridine/ace~ic acid (4:1v/v) for 90 minutes prior to secondary synthesis.
:
.- ~ : ::: : ,., ~
`"' . ' - ' . -; . ' . ' .
' : : ' , ' ' . . .' ' . : . : ' ' ' ' ':' . ; ' ' ~ ' . ' .
. ' . . . ` . ' ' ' ' ', '.' "', .' ' ''' , ' . ~' , ' ' " ' ' ' 1 ' '~ ' , ' : . '. ' '.'. ' " ' ~ ' ':: ' ', " ,' , .' ' ..".~ ' ' ~ :, ' ' , ' ' ' ':' ' ' ' ' . ' , .' ' .' ': ' ,' ' ~ ' . " ~ . ' . ' . . ' . , .
., " ,'', ' ', ' , . ' ' ,'
Claims (15)
1. A multifunctional nucleic acid having the structure:
wherein:
R is a hydroxyl protecting group that can be removed and replaced, without affecting R1 or R2, by reduction with a liquid reducing agent;
R1 is a phosphorus derivative that enables addition of nucleotides to the 5'position of an oligonucleotide chain during chemical synthesis;
R2 is a protecting group that is generally base-stable and acid-sensitive;
R3 is selected from the group consisting of hydrogen, methyl, I, Br and F;
R4 is hydrogen or methyl; and Z is selected from the group consisting of (2) (1) (2) (1) (2) (1):
(2) (1) (2) (1): and (2) (1) wherein x and y may be the same or different and are integers in the range of 1 to 8 inclusive.
wherein:
R is a hydroxyl protecting group that can be removed and replaced, without affecting R1 or R2, by reduction with a liquid reducing agent;
R1 is a phosphorus derivative that enables addition of nucleotides to the 5'position of an oligonucleotide chain during chemical synthesis;
R2 is a protecting group that is generally base-stable and acid-sensitive;
R3 is selected from the group consisting of hydrogen, methyl, I, Br and F;
R4 is hydrogen or methyl; and Z is selected from the group consisting of (2) (1) (2) (1) (2) (1):
(2) (1) (2) (1): and (2) (1) wherein x and y may be the same or different and are integers in the range of 1 to 8 inclusive.
2. The multifunctional nucleic acid of claim 1 wherein R1 is a phosphoramidite, a phosphodiester or a phosphotriester.
3. The multifunctional nucleic acid of claims 1 or 2 wherein R2 is dimethoxytrityl or pixyl.
4. The multifunctional nucleic acid of claim 1 wherein R has the structure:
in which:
R' is hydrogen, aryl or aralkyl;
the Ri may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy;
the Rj may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy;
i is zero, 1, 2 or 3; and j is zero, 1, 2, 3 or 4.
in which:
R' is hydrogen, aryl or aralkyl;
the Ri may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy;
the Rj may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy;
i is zero, 1, 2 or 3; and j is zero, 1, 2, 3 or 4.
5. The multifunctional nucleic acid of claim wherein R' is hydrogen or phenyl and i and j are both zero.
6. The multifunctional nucleic acid of claim 3 wherein R is 2-oxymethyleneanthraquinone (Maq).
7. The multifunctional nucleic acid of claim 1 wherein R is in which:
k is 0, 1, 2, 3 or 4; and the Rk may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy.
k is 0, 1, 2, 3 or 4; and the Rk may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy.
8. The multifunctional nucleic acid of claim 7 wherein the Rk are hydrogen.
9. A oligonucleotide chain containing at least one modified cytosine residue each having the structure:
wherein:
R is a hydroxyl protecting group that can be removed and replaced, without affecting R1 or R2, by reduction with a liquid reducing agent;
R3 is selected from the group consisting of hydrogen, methyl, I, Br and F;
R4 is hydrogen or methyl; and Z is selected from the group consisting of (2) (1) (2) (1) (2) (1) (2) (1) (2) (1); and (2) (1) wherein x and y may be the same or different and are integers in the range of 1 to 8 inclusive.
wherein:
R is a hydroxyl protecting group that can be removed and replaced, without affecting R1 or R2, by reduction with a liquid reducing agent;
R3 is selected from the group consisting of hydrogen, methyl, I, Br and F;
R4 is hydrogen or methyl; and Z is selected from the group consisting of (2) (1) (2) (1) (2) (1) (2) (1) (2) (1); and (2) (1) wherein x and y may be the same or different and are integers in the range of 1 to 8 inclusive.
10. The oligonucleotide chain of claim 9 wherein R has the structure:
;
in which:
R' is hydrogen, aryl or aralkyl;
the Ri may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy;
the Rj may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy;
i is zero, 1, 2 or 3; and j is zero, 1, 2, 3 or 4.
;
in which:
R' is hydrogen, aryl or aralkyl;
the Ri may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy;
the Rj may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy;
i is zero, 1, 2 or 3; and j is zero, 1, 2, 3 or 4.
11. A method of protecting a hydroxyl group of a hydroxyl-containing compound during chemical conversion of other moieties therein, comprising, prior to said chemical conversion, reacting the compound with a protecting species having the structure:
in which:
R' is hydrogen, aryl or aralkyl;
the Ri may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy;
the Rj may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy;
i is zero, 1, 2 or 3; and j is zero, 1, 2, 3 or 4.
in which:
R' is hydrogen, aryl or aralkyl;
the Ri may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy;
the Rj may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy;
i is zero, 1, 2 or 3; and j is zero, 1, 2, 3 or 4.
12. A method of protecting a hydroxyl group of a hydroxyl-containing compound during chemical conversion of other functional groups contained therein, comprising, prior to said chemical conversion, reacting the compound with a protecting species having the structure in which:
k is 0, 1, 2, 3 or 4; and the Rk may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy.
k is 0, 1, 2, 3 or 4; and the Rk may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy.
13. In a method for preparing oligonucleotides from nucleotide monomers, the method comprising sequential addition of 3'-blocked and 5'-blocked nucleotide monomers to the terminal 5'-hydroxyl group of the growing oligonucleotide chain, wherein the improvement comprises:
employing as said 3' blocking group or as said 5' blocking group a moiety which is orthogonally removable by reduction with a liquid reducing agent.
employing as said 3' blocking group or as said 5' blocking group a moiety which is orthogonally removable by reduction with a liquid reducing agent.
14. The method of claim 13 wherein the orthogonally removable moiety is in which:
R' is hydrogen, aryl or aralkyl;
the Ri may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy;
the Rj may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy;
i is zero, 1, 2 or 3; and j is zero, 1, 2, 3 or 4.
R' is hydrogen, aryl or aralkyl;
the Ri may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy;
the Rj may be the same or different and are selected from the group consisting of amino, nitro, halogeno, hydroxyl, lower alkyl and lower alkoxy;
i is zero, 1, 2 or 3; and j is zero, 1, 2, 3 or 4.
15. A method of making a branched oligonucleotide structure, comprising capping the 3'- and 5'-terminal hydroxyl groups of the oligonucleotide chain of claim 12, removing the R moieties by treatment with a liquid reducing agent to give free hydroxyl groups bound through an alkylene linking group to the N-4 positions, and synthesizing secondary oliqonucleotide chains at said free hydroxyl groups.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US558,881 | 1990-07-27 | ||
| US07/558,881 US5430138A (en) | 1990-07-27 | 1990-07-27 | Hydroxyl-protecting groups attached to cytidine nucleotide compounds which are orthogonally removable |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2088255A1 true CA2088255A1 (en) | 1992-01-28 |
Family
ID=24231370
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002088255A Abandoned CA2088255A1 (en) | 1990-07-27 | 1991-07-24 | Hydroxyl-protecting groups orthogonally removable by reduction and their use in the chemical synthesis of oligonucleotides |
Country Status (7)
| Country | Link |
|---|---|
| US (2) | US5430138A (en) |
| EP (1) | EP0543906B1 (en) |
| JP (1) | JP3081882B2 (en) |
| AT (1) | ATE150760T1 (en) |
| CA (1) | CA2088255A1 (en) |
| DE (1) | DE69125380T2 (en) |
| WO (1) | WO1992002533A1 (en) |
Families Citing this family (32)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5430138A (en) * | 1990-07-27 | 1995-07-04 | Chiron Corporation | Hydroxyl-protecting groups attached to cytidine nucleotide compounds which are orthogonally removable |
| TW323284B (en) * | 1992-03-23 | 1997-12-21 | Novartis Ag | |
| US5770725A (en) * | 1992-05-25 | 1998-06-23 | Gosselin; Gilles | Phosphotriester type biologically active compounds |
| US5849905A (en) * | 1994-11-23 | 1998-12-15 | Centre National De La Recherche Scientifique | Biologically active phosphotriester-type nucleosides and methods for preparing same |
| FR2692265B1 (en) | 1992-05-25 | 1996-11-08 | Centre Nat Rech Scient | BIOLOGICALLY ACTIVE COMPOUNDS OF THE PHOSPHOTRIESTER TYPE. |
| US6020482A (en) * | 1992-05-25 | 2000-02-01 | Gosselin; Gilles | Phosphotriester type biologically active compounds |
| US5955591A (en) * | 1993-05-12 | 1999-09-21 | Imbach; Jean-Louis | Phosphotriester oligonucleotides, amidites and method of preparation |
| US5597909A (en) * | 1994-08-25 | 1997-01-28 | Chiron Corporation | Polynucleotide reagents containing modified deoxyribose moieties, and associated methods of synthesis and use |
| US5639875A (en) * | 1995-02-01 | 1997-06-17 | Hybridon, Inc. | Methods for H-phosphonate syntheis of oligonucleotides using triphosgene |
| WO1997014812A2 (en) * | 1995-10-16 | 1997-04-24 | Chiron Corporation | Method of screening for factors that modulate gene expression |
| AU2462497A (en) | 1996-04-17 | 1997-11-19 | Hubert Koster | A combinatorial protecting group strategy for multifunctional molecules |
| US6187536B1 (en) | 1997-02-18 | 2001-02-13 | Thomas Jefferson University | Methods of identifying and detecting pancreatic cancer |
| US7135333B1 (en) | 1997-08-07 | 2006-11-14 | Thomas Jefferson University | Compositions that specifically bind to colorectal cancer cells and methods of using the same |
| US6120995A (en) * | 1997-08-07 | 2000-09-19 | Thomas Jefferson University | Compositions that specifically bind to colorectal cancer cells and methods of using the same |
| AU6270499A (en) * | 1998-09-29 | 2000-04-17 | Phylos, Inc. | Synthesis of codon randomized nucleic acids |
| WO2000056749A1 (en) | 1999-03-24 | 2000-09-28 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | N-acylphosphoramidites and their use in oligonucleotide synthesis |
| WO2001073132A1 (en) | 2000-03-27 | 2001-10-04 | Thomas Jefferson University | Compositions and methods for identifying and targeting cancer cells of alimentary canal origin |
| JP2004526411A (en) * | 2000-09-18 | 2004-09-02 | トマス・ジエフアーソン・ユニバーシテイ | Compositions and methods for identifying and targeting gastric and esophageal cancer cells |
| US6932943B1 (en) | 2001-01-26 | 2005-08-23 | Third Wave Technologies | Nucleic acid synthesizers |
| US7435390B2 (en) * | 2001-01-26 | 2008-10-14 | Third Wave Technologies, Inc. | Nucleic acid synthesizers |
| DE10133779A1 (en) * | 2001-07-16 | 2003-02-06 | Chemogenix Gmbh | Preparation of polynucleotides useful in gene technology involves reacting free 5'-hydroxy group of a oligonucleotide with a hydroxy group |
| US7355037B2 (en) * | 2001-12-03 | 2008-04-08 | The United States Of America As Represented By The Department Of Health And Human Services | Thermolabile hydroxyl protecting groups and methods of use |
| AUPR975201A0 (en) * | 2001-12-24 | 2002-01-24 | Unisearch Limited | Enzymatic redox labelling of nucleic acids |
| DE10247790A1 (en) * | 2002-10-14 | 2004-04-22 | Chemogenix Gmbh | Solid phase synthesis of labelled oligonucleotide conjugate, by nucleophilic substitution of labile protecting group on terminal hydroxy group with labeling reagent |
| AU2003302743B2 (en) * | 2002-12-23 | 2008-09-04 | Dynavax Technologies Corporation | Branched immunomodulatory compounds and methods of using the same |
| AU2004230927B9 (en) | 2003-04-13 | 2011-12-01 | Enzon Pharmaceuticals, Inc. | Polymeric oligonucleotide prodrugs |
| US9809824B2 (en) * | 2004-12-13 | 2017-11-07 | The United States Of America, Represented By The Secretary, Department Of Health And Human Services | CpG oligonucleotide prodrugs, compositions thereof and associated therapeutic methods |
| CA2609622C (en) | 2005-05-26 | 2013-01-15 | Eragen Biosciences, Inc. | Reagents for the improved synthesis of isoguanosine-containing oligonucleotides |
| KR100834812B1 (en) | 2006-07-04 | 2008-06-05 | 삼성전자주식회사 | Handoff appaturus and method for reduced session movement in mobile communication system |
| AU2007281709B2 (en) | 2006-08-11 | 2013-04-18 | Rutgers, The State University Of New Jersey | Dual-sensitizer-containing luminescent compounds, conjugates, and uses thereof |
| US9221759B2 (en) | 2010-01-13 | 2015-12-29 | Rutgers, The State University Of New Jersey | Fluorophore chelated lanthanide luminescent probes with improved quantum efficiency |
| WO2020236618A1 (en) * | 2019-05-17 | 2020-11-26 | Ionis Pharmaceuticals, Inc. | Synthesis of oligomeric compounds comprising phosphorothioate diester and phosphate diester linkages |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5118802A (en) * | 1983-12-20 | 1992-06-02 | California Institute Of Technology | DNA-reporter conjugates linked via the 2' or 5'-primary amino group of the 5'-terminal nucleoside |
| US4843122A (en) * | 1984-01-30 | 1989-06-27 | Enzo Biochem, Inc. | Detectable molecules, method of preparation and use |
| EP0305643B1 (en) * | 1984-04-18 | 1994-01-05 | Whitby Research Incorporated | N-6 thienyl substituted adenosine derivatives as cardiac vasodilators |
| US5367066A (en) * | 1984-10-16 | 1994-11-22 | Chiron Corporation | Oligonucleotides with selectably cleavable and/or abasic sites |
| US5093232A (en) * | 1985-12-11 | 1992-03-03 | Chiron Corporation | Nucleic acid probes |
| US4910300A (en) * | 1985-12-11 | 1990-03-20 | Chiron Corporation | Method for making nucleic acid probes |
| US4837311A (en) * | 1987-06-22 | 1989-06-06 | Hoffman-La Roche Inc. | Anti-retroviral compounds |
| CA1339351C (en) * | 1987-10-15 | 1997-08-26 | Michael S. Urdea | Nucleic acid multimers and amplified nucleic acid hybridization assays using same |
| US5430138A (en) * | 1990-07-27 | 1995-07-04 | Chiron Corporation | Hydroxyl-protecting groups attached to cytidine nucleotide compounds which are orthogonally removable |
-
1990
- 1990-07-27 US US07/558,881 patent/US5430138A/en not_active Expired - Lifetime
-
1991
- 1991-07-24 EP EP91915182A patent/EP0543906B1/en not_active Expired - Lifetime
- 1991-07-24 CA CA002088255A patent/CA2088255A1/en not_active Abandoned
- 1991-07-24 JP JP03513745A patent/JP3081882B2/en not_active Expired - Fee Related
- 1991-07-24 AT AT91915182T patent/ATE150760T1/en not_active IP Right Cessation
- 1991-07-24 WO PCT/US1991/005227 patent/WO1992002533A1/en active IP Right Grant
- 1991-07-24 DE DE69125380T patent/DE69125380T2/en not_active Expired - Lifetime
-
1995
- 1995-04-13 US US08/421,798 patent/US5703218A/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| DE69125380D1 (en) | 1997-04-30 |
| DE69125380T2 (en) | 1997-08-28 |
| EP0543906A4 (en) | 1993-07-14 |
| JP3081882B2 (en) | 2000-08-28 |
| US5703218A (en) | 1997-12-30 |
| EP0543906A1 (en) | 1993-06-02 |
| ATE150760T1 (en) | 1997-04-15 |
| US5430138A (en) | 1995-07-04 |
| JPH05509317A (en) | 1993-12-22 |
| EP0543906B1 (en) | 1997-03-26 |
| WO1992002533A1 (en) | 1992-02-20 |
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