RAPID SOLID PHASE SYNTHESIS OF OLIGONUCLEOTIDES USING PHOSPHORUS OXYCHLORIDE ACTIVATION

Abstract: A method for the synthesis of oligonucleotides which comprises the steps of packing a flow reactor with supported nucleoside comprised of a first nucleoside attached at its 5'-position of an insoluble solid support, activating said support-nucleoside by treatment with phosphorus oxychloride in the presence of a basic catalyst to provide a 3'-dichlorophosphoryl group on said first nucleoside, coupling a second similar or dissimilar nucleoside, unprotected at the 3'-and 5'-position, to said first nucleoside by condensation with said dichlorophosphoryl group and repeating said activation and coupling step until the desired oligonucleotide sequence is obtained. The oligonucleotide may then be recovered by cleavage from the support and purified, if required.

Description: RAPID SOLID PHASE SYNTHESIS OF OLIGONUCLEOT DΞS USING PHOSPHORUS OXYCHLORIDE ACTIVATION Technical Field This invention relates to the synthesis of oligo- nucleotides. More particularly, the invention is directed to the solid-phase synthesis of oligonucleo- tides using phosphorous oxychloride activation. Background Art Ribo- and deoxyribooligonucleotides are of extreme interest and importance currently because of the explosive development of the recombinant DNA field, for which "libraries" of codon and other oligonucleotide sequences are required. The synthesis of these mole¬ cules is also of interest simply because of the great challenge they present to the synthetic organic chemist. However, despite the fact that the synthesis of several small nucleic acids has now been achieved, implying that the chemical methodology has been developed fully, procedures are still extremely tedious and rarely pro¬ duce good yields of oligonucleotides. In fact, the recent synthesis of the yeast alanine t-RNA gene by Khorana and co-workers required 20 man-years! Even the application of the solid-phase approach has failed to result in " generally applicable methods which result in the rapid synthesis of high yields of oligonucleotides. This can, for the most part, be traced to the complex- ity of the chemistry of nucleosides and nucleotides. Internucleotide bond formation involves the phosphory- lation of sugar hydroxyl groups which have low reacti¬ vities, especially the secondary alcohol at the 3'- position. Coupling yields are, therefore, invariably relatively low (rarely more than 80-90%) , resulting in very low recoveries of oligonucleotides. For example, the maximum theoretical yield of a pentanucleotide is only 32% if the coupling yield is only 80% at each coupling , . However, because of the possibility of num¬ erous side reactions which can occur on deprotection or because of the sensitivity of glycosidic linkages or the heterocyclic base in the molecule, yields are often reduced even further. Syntheses, therefore, result in complicated mixtures which contain "deletion" and truncated sequences which are invariably difficult and tedious to separate. Because of these problems, a plethora of protect¬ ing groups has been developed for temporarily blocking functional groups during the formation of the inter- nucleotide phosphodiester bond. A number of synthetic approaches have also emerged. The first of these, the so-called phosphodiester method, originally developed by horana and co-workers, utilizes a 3'-protected nucieoside 5'-phosphate (II) which is activated (for example with dicyσlohexylcarbodiimide) and coupled with a 5'-protected nucieoside (I) (I) (ID (III) DCC=dicyclohexylcarbodiimide R and R' = protecting groups The protecting groups (R and R' ) must be stable to the synthetic conditions but must be selectively removable in order to extend the oligonucleotide chain. In the ribooligonucleotide series, the added problems of 2'-3' phosphate interchange and consequent need for protection exist. This scheme is also further compli¬ cated by the possibility of side reactions associated with the reactivity of the phosphate oxygen atom [asterisked in (III) ] which remains unprotected throughout the synthesis. For this reason, the so- called "phosphotriester approach" was developed by Letsinger in which this phosphate oxygen atom is also blocked by a suitable protecting group throughout the synthesis. In an even more recent approach, the so-called "phosphite method", also developed by Letsinger, nucleosides are activated as their phosphites (IV) (i.e. phosphorus in oxidation state +3 rather than phosphorus in oxidation state +5 as in phosphates) and the phosphite oxidized to the phosphate after the coupling reaction, i.e.: OR' CH 3 -0-P-Cl (IV) OR* R and R' a=ρrotecting groups Although the above description refers to prior art syntheses of 2'-deoxyribooligonucleotides, through appropriate protection of the 2'-hydroxyl group, the same approaches have been applied to the synthesis of ribooligonucleotides. The approaches described above are, for the most part, effective in producing low to moderate yields of oligonucleotides but all require the synthesis of pro¬ tected nucieoside derivatives which can be both time- consuming and expensive. There is, therefore, a need for a reproducible method which rapidly leads to the highest possible yields of oligonucleotides. Further¬ more, the method should, if possible, use relatively inexpensive, unprotected nucleosides as starting materials. Disclosure of the Invention Accordingly, it is an object of the invention to provide a reproducible method which rapidly provides high yields of oligonucleotides. Another object of the invention is a method for the preparation of oligonucleotides that enables the use of unprotected nucleosides in the synthesis. Yet another object of the invention is to provide an inexpensive method for synthesizing oligonucleotides which avoids the need for deprotection steps and the possibility of side reactions so that yields are sub¬ stantially improved. A further object of the invention is to provide a solid-phase method for the facile, large scale produc¬ tion of oligonucleotides. These requirements are satisfied by a novel, solid- phase synthesis that is based on the use of phosphorus oxychloride for activation and unprotected nucleosides as the "building blocks" for oligonucleotide synthesis. In accordance with the present invention oligo¬ nucleotides are synthesized by a method comprising the steps of activating a support-nucleoside comprised of a first nucieoside attached at its 5 '-position to an insoluble solid support by treatment with phosphorus oxychloride in the presence of a basic catalyst to provide a 3'-dichlorophosphoryl group on said first nucieoside, coupling a second similar or dissimilar nucieoside, unprotected at the 3'- and 5'-position, to said first nucieoside by condensation of said dichloro¬ phosphoryl group with said second nucieoside at the 5'- position of said second nucieoside, and repeating said activation and coupling steps until the desired oligo¬ nucleotide sequence is obtained. The oligonucleotide sequence or chain may then be recovered by cleaving the linkage between the support and oligonucleotide formed. The oligonucleotide is then purified, if required. The synthesis of the invention is advantageously carried out in a flow reactor. In a preferred aspect of the invention, a pressurized flow reactor is used and all phases of the synthesis are preferably carried out under a pressure of at least atmospheric plus 25 2 psi or 0.0003625 dyne/cm (i.e. at least 40 psi or 2 0.00058 dyne/cm ). Generally the pressures fall in 2 the range of at least 100 psi or 0.00145 dyne/cm up 2 to 1000 psi or 0.0145 dyne/cm , although pressures up 2 to 10,000 psi or 0.145 dyne/cm or more may be used. This reactor maximizes the efficiency of all of the operations in the synthetic cycle through the applica¬ tion of the mass action effect, which forces reactions to completion, thereby maximizing both rates and yields in coupling reactions. The flow reactor also minimizes side reactions through efficient removal of excess reagents and also by-products of reactions. Scale-up of syntheses is also more facile than in conventional, shaken reactors. In an embodiment of the invention, the support- nucieoside is formed in the flow reactor as the initial step of the synthesis. According to this embodiment of the invention, the flow reactor is packed with an insoluble solid support containing substituent groups reactive with a nucieoside to form a stable linkage with said nucieoside, passing in continuous flow a first nucieoside through said packed reactor to couple sai.d first nucieoside to said support by said linkage at the 5'-position of said nucieoside and thereby form a- support-nucleoside, activating said support-nucleoside by passing phosphorus oxychloride in the presence of a basic catalyst through said reactor to provide a 3'- dichlorophosphoryl group on said first nucieoside, coupling a second similar or dissimilar nucieoside, unprotected at the 3' or 5' position, to said nucieoside by condensation of said dichlorophosphoryl group with said second nucieoside at the 5'-position of said second nucieoside, and repeating said activation and coupling steps until the desired oligonucleotide sequence is obtained. Modes for Carrying Out the Invention In the synthesis of the present invention, an insoluble solid support or matrix, advantageously in bead form, such as any of the conventional solid-phase polymeric substrates conventionally employed for the synthesis of polynucleotides or polypeptides can be utilized. Typical of such polymeric resins are cross- linked polystyrene resins, polyaσrylamide derivatives, silica gel, porous glass, clays, celite, crosslinked dextran, and similar insoluble solid supports which either naturally contain reactive sites for coupling with nucleosides by condensation with the 5'-positioned hydroxy groups of the nucleosides or which can be pro¬ vided with such rea ' ctive sites. In practise, rigid, non-swollen resins are preferred such as those of the macroporous type or silica-based. Typical of the reactive sites either naturally present or provided the solid support are groups con- densible with nucleosides such as carboxyl groups, alkyl, aralkyl or aryl halide groups, alcohol groups, amine groups and the like. The preferred reactive sites are carboxyl groups. A variety of methods for deriving these carboxyl group-containing insoluble solid supports (generally identified below as / n ) are known in the art and frequently involve the inclusion of spacer groups to extend the distance of the carboxyl group from the support or polymer ~ backbone for the purpose of facilitating the coupling reaction to the nucieoside. For example, polystyrene (VI) may be deri- vatized readily to first form an aminometh 1 ted deriva¬ tive (VII), i.e. ~ ~ ~ This resin (VTI) may then be further derivatized with spacer groups terminated in carboxyl groups, i.e. (VIII) Alternatively, polystyrene may be derivatized directly to a carboxyl-containing support (XI) by Friedel-Crafts acylation using activated dicarboxylic acids such as succinic anhydride: A1C1 ®o + / n — - cχ ∞ -*- - ∞2 H (IX) Spacer groups may be added to this resin through use of ω-amino acids , i . e. 1. DCC (IX) + H-,N- (CH- H CO^Me » 2 2 n 2 2 . OH " C0 2 H In the case of macroporous polyacrylic acid, the resin may be further derivatized to the polyacrylamide derivative in order to remove the carboxyl groups from proximity to the polymer backbone, through use of ω- amino acids, i.e. <τv SOC1- 1. H 2 N — CH 2'n C0 2 Me L; C ~ 0 2- H (? coci 2. OH Supports of the silica-type (silica gel, porous gel, porous glass, etc.) are first derivatized by treat¬ ment with triethoxyaminopropyl silane to provide amine- functionalized supports (XI) , which may be transformed to carboxyl-containing support (XII) by similar methods to those described above for polystyrene, i.e. (Si)- OH + (EtO*y Si- -CH 2 y NH 2 (si)- O-Si - H^ NH, (XI) (XI) C — 6CH 2 - C0 2 Me/DCC (si)- O-Si — e CH 2 -Y-- NH-CO - CH 2 -J ** ^ C0 2 H (XII) Using these or similar supports containing carboxyl groups, the first nucieoside in the desired sequence is attached to the support via an ester linkage to the 5'- hydroxyl group, i.e. It is probably not necessary to protect the 3'-hydroxyl function in the nucieoside because of the much greater reactivity of the 5'-hydroxyl group. However, if the 3'-hydroxyl is temporarily protected as the monomethoxy- trityl derivative, cleavage of this group under mild, acidic conditions (e.g. 1% to 5% trifluoroac tic acid in benzene) permits rapid quantitation of the extent of incorporation of the nucieoside on the support by spectro- photometric techniques. In the case of adenine, quanine and cytosine-containing nucleosides, "minimal protection" of amine functions on the bases improves yields through elimination of the possibility of amine phosphorylation. The resin-nucleoside (XIII) (R = H) is packed into the column reactor and activated by treatment with phosphorus oxychloride in the presence of a basic cata¬ lyst, i.e. ] Cl - PP - Cl II TMP = trimethyl phosphate O (xiv) The phosphorus oxychloride and tertiary base are advan- tageously made up in the form of an activating solution employing a suitable solvent for both the base and POCl^ • The preferred solvent for this reaction is trimethyl phosphate and the preferred basic catalyst is imidazole and its derivatives, such as 1-methylimidazole, a tertiary base such as diethylaniline, and the like, which have been found to promote the most rapid phosphory¬ lation. Coupling with a second similar or dissimilar nucieoside is effected by passing a solution of the nucieoside, a tertiary base and catalytic amounts of water in trimethyl phosphate through the column, i.e. OH Once again, unprotected nucleosides may be used because of the vast differences in reactivity between the 5 ' - and 3' hydroxyl groups. (Additional specificity may be pro¬ vided by the addition of trace amounts of water to the reaction mixture) . Couplings are rapid (typically 5-30 minutes for the phosphorylation step and 10-60 minutes for the phosphodiester formation step) and yields are also high (up to 90-95% per coupling) . The synth¬ esis is continued by repeating the phosphorylation and coupling steps until the desired oligonucleotide sequence is obtained. The product is finally cleaved from the polymeric support by any of the standard methods used in the art. The method selected in any given case will be dependant principally on the particular linkage being cleaved. For instance, in the case of an ester linkage, cleavage is obtained by hydrolysis. Hydrolysis can be accomplished by one of several techniques, includ¬ ing saponification with alkali, treatment with amonia or hydrazine, etc. Purification of the final product, if required, may be carried out by standard, ion exchange techniques using, e.g. DEAE-Sephadex, QAE-Sephadex, etc. Although the above description refers to the synthesis of 2'-deoxyribooligonucleotides,. through appropriate choice of protecting groups for the 2'-hydroxyl- group well known to one skilled in the art, the same method can be employed for the synthesis of ribooligonucleotides. The nucleosides useful in the production of oligo¬ nucleotides include any of the 3 1 - and 5'-hydroxyl group containing nucleosides. Illustrative of these complex compounds are thymidine, uridine, deoxyuridine, cytidine, deoxycytidine, adenosine, deoxyadenosine, guanosine, deoxyguanosine and the like. As aforementioned, in the preferred embodiment of the invention, rapid, large scale production of oligonucleotides can be obtained by conducting the entire series of coupling reactions in a pressurized flow reac¬ tor. Elevated reactor pressures can be generated by use of commercial pressurizing equipment and methods. For example, any of the commercially available recipro¬ cating pumps capable of generating the required pressures can be used and the reactants and reagents pumped directly into and through the reactor. Alternatively, the reactants and reagents may be pumped through the reactor by means of pressurization with an inert gas such as nitrogen and the pressure in the reactor regulated by controlling the volume of inert gas released to transfer the reactants and reagents into and through the reactor. A simple method of delivering reactants and reagents under the high pressure required for this method involves the use of a conventional high pressure liquid chroma- tography apparatus. The columns of such a system can serve as reactors for the synthesis and the high per- formance pump generally found in such an apparatus may be easily adapted to pump reagents and reactants through the column reactor. However, such an apparatus is limited to small scale synthesis since the maximum possible flow rates in such a system are too low (generally less than ιo ml per minute) for larger scale synthesis. ' The following examples are included to further illustrate the present invention. In all of the examples, th.e----nitrogen-pressurized flow system de¬ scribed in Example 2 of United States Patent No. 4,192,798, incorporated herein by reference, was employed. ----Example- l Preparation of Adipoyl-aminomethy1 Polystyrene To a stirred suspension of macroporous polystyrene (Amberlite XAD-2) in 200 ml of 1:1 trifluoroacetic acid: methylene choloride is added 0.45 ml (5 mmol) hydroxy- methylphthalamid . After 5 hrs the resin is filtered and washed with 1:1 trifluoroacetic acid: methylene chloride, methylene chloride and ethanol. The resin is suspended in 250 ml of ethanol containing 12 ml hydrazine and refluxed for 24 hr. The resin is filtered and washed with hot ethanol. The resin is dried in vacuum, then resuspended in 100 ml of dimethylacetamide. Adipic acid monomethyl ester, 10 mol (1.6 g) is added, and the suspension cooled to 0°C. A solution of dicyclohexylcarbodiimide, 10 mmol (2.1 g) , in 10 ml dimethylacetamide is added and after 1 hr at 0-5°C, the suspension is shaken for 24 hr at room temperature. Benzoyl chloride, 1 ml, and pyridine " , 4 ml, are then added to block any unreacted amino groups. After 4 hr, the resin is filtered and washed with dimethylacetamide, methanol and methylene chloride. The resin is dried, then saponified by shaking with 100 ml of 1:1 ' N sodium hydroxide-dioxane for 24 hr at room temperature. The resin is filtered and washed with N hydrochloric acid, water, and methanol. After drying a sample of the resin is titrated and shown to " have 0.3 mmol/g of free carboxyl groups. Example 2: Preparation of Poly (acrylamidoundeca-πcic acid) To a mechanically stirred suspension of 10 g dried macroporous polyacrylic acid (Bio-Rex 70-H ) (10 mmol/g. of carboxyl groups) in 100 ml methylene chloride is added 150 mmol (31 g) of phosphorous penthachloride. After shaking 6 hrs at room temperature, the resin is filtered and washed with methylene chloride. After drying in vacuum, the resin is resuspended in tetrahydrofuran, (100 ml). Triethylamine, 2.8 ml (20 mmol) is added followed by aminoundecanoic acid methyl ester hydro- chloride, 2.5 g (10 mmol) . After 24 hrs at room temperature, 20 ml (200 mmol) n-butylamine is added to block unreacted acid chloride functions. After 6 hrs, the resin is filtered and washed with tetrahydro¬ furan, methanol and water. The resin is saponified for 24 hrs with N sodium hydroxide-dioxane as in Example 1. Titration of the resultant poly (aery- lamidoundecanoic acid) resin shows i-2 mmol/g of carboxyl groups. Example 3: Attachment of thymidine to Adipoyl-ami-riαmet-hyl Resin To a suspension of 1 g adipoyl-aminomethyl poly¬ styrene (carboxyl substitution 0.3 mmol/g) in 10 ml dimethylacetamide, 0.51 g (1 mmol) of 3'-methoxytrityl- thymidine and 0.12 g (1 mmol) of dimethylaminopyridine is added. The suspension is maintained at 0-5°C for 1 hr, then shaken at room temperature for 24 hrs. The suspension is recooled to 0°C and an additional 0.2 g of dicyclohexylcarbodiimide plus 0.1 ml ( 1 mπr.1) n- butylamine are added. After 24 hrs of shaking, the resin is filtered and washed with dimethylaceta-ride, methanol ' and methylene chloride. The dry 3'-m hoxyl- trityl-5'-thymidyl-adipoylaminomethyl resin is weighed, then treated with 50 ml of 5% trifluoroacetic acid in benzene for 30 min at room temperature. The resin suspension is filtered and washed with 5% trifluoroacetic acid in benzene until the wash is nearly colorless. The filtrate is taken up to a known volume and an aliquot diluted for visible spectrum. The substitution is calculated based on the known extinction of the peak at 480 nm for methoxytritylcarbonium ion. The 5'- thymidyl-adipolyaminomethyl polystyrene resin is washed with benzene, methanol and methylene chloride, then dried in vacuum. Example 4: Preparation of Thymidylyl (3'-5') thymidine on Adipoyl-aminomethyl Polystyrene 5'-Thymidyl-adipoylaminomethyl polystyrene (0.5 g) (substitution 0.1 mmol/g) is packed in a stainless steel column with dioxane wash at 1000 psi or 0.0145 2 dyne/cm . The resin is washed with 100 ml trimethyl- phosphate, then a freshly prepared solution of 100 ml 0.5M phosphorous oxychloride - 0.5M imidazole in trimethyl- phosphate passed through the column for 20 min at room temperature. The column is washed with trimethylphosphate until a negative chloride test with silver nitrate is obtained for the effluent IOO ml required) . A solution of 100 ml 0.1 M thymidine - 0.1 M imidazole in trimethyl- phosphate is then passed through the column over 60 in at room temperature. The column is finally washed with triraethylphosphate until the effluent is UV-transparent. The column is unpacked and the resin washed on a funnel with water, then resuspended in 20 ml of N sodium hydroxide. The suspension is shaken for 24 hrs at room temperature. The resin is filtered and washed with several portions of water. The total filtrate is neutralized with Dowex 50 pyridinium, 20 cc. The suspension is filtered and the filtrate evaporated to dryness. The residue is redissolved in water and applied to a column of QAE-Sephadex bicarbonate. The column is eluted with a gradient of ammonium bicarbonate. Effluent is monitored at 26.7 nm. The peak for dinucleotide was collected and lyophilized to give 50-80% yield of product. Purity is confirmed by HPLC on a hatmann 10/25 SAX anion exchange column or a Unimetrics Lichrosorb RP-18 column. Example 5: Preparation of thymidylyl (3'-5' )—hymidylyl- thymidine on Adipoylaminomethyl Polystyrene Resin The 5'-thymidylyl-(3'-5' )-thymidine-adipoyl- aminomethyl resin of Example 4 is submitted to one more cycle of 3'-phosphorylation followed by coupling to free thymidine. The oligonucleotide is then cleaved with sodium hydroxide and purified as in Example 4. Example 6: Attachement of N-benzoyldeoxycytidine to Poly (acryloylaminoundecanoic acid) Resin 3'-Methoxytrityl-N-benzoyldeoxycytidine is attached to poly (acryloylaminoundecanoic acid) resin and the resultant resin detritylated by the procedure given in Example 3. Example 7: Preparation of deoxycytidylyl -(3'-5' )- thymidylyl-(3 τ -5' )-deoxycytidine on Poly (acryloylaminoundecanoic acid) 5'-deoxycytidylyl-poly (acryloylaminoundecanoic acid) resin from Example 6 is converted to 3'-dichloro- phosphoryl resin and subsequently coupled to free thymidine as in Example 4. The dinucleotide resin is submitted to a further cycle of phosphorylation followed by coupling to free N-benzoyldeoxycytidine. The oligonucleotide is cleaved from the resin and simultaneously deprotected of N-benzoyl groups by treatment with N NaOH for 24 hrs. Purification is accomplished on QAE-Spenhadex bicarbonate resin as for Example 4.

Claims: IT IS CLAIMED: 1. A method for the synthesis of oligonucleotides which comprises the steps of activating a support-nucleo- side comprised of a first nucieoside attached at its 5'- position to an insoluble solid support by treatment with phosphorus oxycholoride in the presence of a basic catalyst to provide a 3'-dichlorophosphoryl group on said first nucieoside, coupling a second similar or dissimilar nucieoside, unprotected at the 3'-and 5'-position, to said first nucieoside by condensation of said dichloro¬ phosphoryl group with said second nucieoside at the 5'- position of said second nucieoside, and repeating said activation and coupling steps until the desired oligo- nucleotide sequence is obtained. 2. A method according to Claim 1 wherein the oligonucleotide sequence is recovered by cleaving it from said support. 3. A method according to Claim 1 wherein the synthesis is conducted in a flow reactor. . A method according to Claim 3 wherein the flow reactor is pressurized. 5. A method according to Claim 4 wherein the pressure in the reactor is at least 40 psi or 0.00058 dyne/ 6. A method according to Claim 1 wherein the first nucieoside is attached at the 5'-position at the insoluble support via an ester linkage. 7. A method according to Claim 1 wherein the support is crosslinked polystyrene containing reactive groups condensible with a nucieoside. 8. A method according to Claim 1 wherein the support is a crosslinked polyacrylamide containing reactive condensible with a nucieoside. 9. A method according to Claim 1 wherein the support is a silica based support containing reactive groups condensible with a nucieoside. 10. A method for the synthesis of oligonucleo- tides which comprises the steps of packing a flow reactor with an insoluble solid support containing sub- stituent groups reactive with a nucieoside to form a stable linkage with said nucieoside, passing in continuous flow a first nucieoside through said packed reactor to couple said first nucieoside to said support by said linkage at the 5'-position of said nucieoside and thereby form a support-nucleoside, activating said support- nucleoside by passing phosphorus oxychloride in the presence of a basic catalyst through said reactor to provide a 3'-dichlorophosphoryl group on said first nucieoside, coupling a second similar or dissimilar nucieoside, unprotected at the 3'- ' and 5'-position, to said first nucieoside by condensation of said dichloro¬ phosphoryl group with said second nucieoside at the 5'- position of said nucieoside, and repeating said activation and coupling step until the desired, oligonucleotide sequence is obtained. 11. A method according to Claim 10 wherein the oligonucleotide sequence is recovered by cleaving it from said support. 12. A method according to Claim 10 wherein the flow reactor is pressurized. 13. ' A method according to Claim 12 wherein the pressure in the reactor is at least 40 psi or 0.00058 2 - dyne/cm . 14. A method according to Claim 10 wherein the first nucieoside is attached at the 5'-position to the insoluble support via an ester linkage. 15. A method according to Claim 10 wherein the support is crosslinked polystyrene containing reactive groups condensible with a nucieoside. 16. A method according to Claim 10 wherein the support is a crosslinked polyacrylamide containing reactive groups condensible with a nucieoside. 17. A method according to Claim 10 wherein the support is a silica based support containing reactive groups condensible with a nucieoside. 18. A method according to Claim 14 wherein the oligonucleotide is recovered by hydrolyzing the ester linkage between the support and oligonucleotide sequence to cleave said sequence from the support.

Inventors: FULLER WILLIAM DEAN, GOODMAN MURRAY, VERLANDER MICHAEL S
Applicants: BIORESEARCH INC
Application year: 1982
Main classification: C07H21
IPC codes: C07H21