WO1996003417A1

WO1996003417A1 - Improved methods of detritylation for oligonucleotide synthesis

Info

Publication number: WO1996003417A1
Application number: PCT/US1995/009322
Authority: WO
Inventors: Ivan Habus; Sudhir Agrawal
Original assignee: Hybridon, Inc.
Priority date: 1994-07-25
Filing date: 1995-07-24
Publication date: 1996-02-08
Also published as: AU3143695A

Abstract

New methods of synthesizing oligonucleotides are disclosed. The methods result in improved yields by reducing or eliminating depurination that often occurs during detritylation. The method comprises detritylating DMT-blocked oligonucleotides with dichloroacetic acid in combination with a lower alcohol (e.g., methanol or ethanol) or 1H-pyrrole. The method is advantageously used to synthesize oligonucleotides up to about 150 monomers long.

Description

IMPROVED METHODS OF DETRITYLATION FOR OLIGONUCLEOTIDE SYNTHESIS

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to improved methods of oligonucleotide synthesis. In particular, it relates to improved methods of cleaving the 4',4"-dimethoxytrityl sugar-protecting group from an oligonucleotide chain during oligonucleotide synthesis. Description of the Related Art

Since Zamecnik and Stephenson, Proc. Natl Acad. Set USA 75, 280-284 (1978) first demonstrated virus replication inhibition by synthetic oligonucleotides, great interest has been generated in oligonucleotides as therapeutic agents. In recent years the development of oligonucleotides as therapeutic agents and as agents of gene expression modulation has gained great momentum. The greatest development has been in the use of so-called antisense oligonucleotides, which form Watson-Crick duplexes with target mRNAs. Agrawal, Trends in Biotechnology 10, 152-158 (1992) extensively reviews the development of antisense oligonucleotides as antiviral agents. See also Uhlmann and Peyman, Chem. Rev. 90, 543 (1990).

Also important, but somewhat less developed is the so-called antigene oligonucleotide approach, in which oligonucleotides form triplexes with target DNA duplexes through Hoogsteen base pairing. Chang and Pettitt, Prog. Biophys.

Molec. Biol 58, 225-257 (1992), have recently reviewed developments in this latter approach. Triplex formation has been observed between DNA and various types of oligonucleotides. Further developments in the triplex approach have been described by Cooney et al., Science 241, 456-459 (1988), Latimer et al.,

Nucleic Acids Res. 22, 1549-1561 (1989), Kibler-Herzog et al., Nucleic Acids Res.

18, 3545-3555 (1990), Xodo et al., /. Molec Biol 19, 5625-5631 (1991), Young et al., Proc Natl Acad. Set USA 88, 10023_^10100 (1991), Praseuth et al., Proc. Natl Acad. Set USA 85, 1349-1353 (1988), and Giovannangeli et al., /. Am. Chem.

Soc 113, 7775-7777 (1991).

Both the antisense and antigene oligonucleotide approaches have as their goal gene expression modulation, which is beneficial in understanding gene expression, both in vitro and in vivo, and in therapeutic treatment of diseases or conditions involving gene expression.

Various methods have been developed for the synthesis of oligonucleotides for such purposes. See generally, Methods in Molecular Biology, Vol 20: Protocols for Oligonucleotides and Analogs (S. Agrawal, Ed., Humana Press, 1993); Oligonucleotides and Analogues: A Practical Approach (F. Eckstein, Ed., 1991); Uhlmann and Peyman, supra. Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al., /. Molec. Biol 72, 209 (1972), discloses phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 34, 3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and Caruthers, Tetrahedron Lett. 22, 1859- 1862 (1981), discloses the use of deoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik, U.S. Patent No. 5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.

Both of these modern approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild, Tetrahedron Lett. 28, 3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochemistry 23, 3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager et al., Biochemistry

27, 7237 (1988), discloses synthesis of oligonucleotide phosphoramidates using phoshoramidite chemistry. Agrawal et al., Proc. Natl Acad. Set USA 85, 7079- 7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.

Solid phase synthesis of oligonucleotides by the foregoing methods involves the same generalized protocol. Briefly, this approach comprises anchoring the 3 '-most nucleoside to a solid support functionalized with amino and/or hydroxyl moieties and subsequently adding the additional nucleosides in stepwise fashion. Desired internucleoside linkages are formed between the 3 ' functional group of the incoming nucleoside and the 5 ' hydroxyl group of the 5 ' - most nucleoside of the growing, support-bound oligonucleotide. To prevent coupling of the incoming oligonucleotides at unwanted sites (such as at the amino moiety of the nucleoside base), these sites are protected with various blocking groups (such as N-acyl derivatives). See Oligonucleotides and Analogues, supra. In particular, to prevent dimerization (or higher multimerization) between incoming nucleosides, the 5 ' hydroxyl group of the nucleosides is also protected, most frequently with 4 ',4"-dimethoxytrityl (DMT). When appropriate, such as in the phosphoramidate approach to oligonucleotides synthesis, capping is followed by oxidation of the internucleoside linkage.

Following capping and oxidation, the 5 ' hydroxyl group is detritylated to allow the next nucleoside to link to that site. The next nucleoside is added, the unreacted sites capped, and the oligonucleotide detritylated. This procedure is repeated in stepwise fashion until the desired, full length oligonucleotide is synthesized. The oligonucleotide is then cleaved from the solid support and the various protecting groups removed.

Variations on this generalized protocol exist - the most evident reason being to provide for modifications of, for example, interaucleotide linkages. All protocols, however, will include the foregoing procedures.

Of particular interest here is the detritylation reaction. Strong protic acids have been utilized as effective detritylating agents. Stawinski et al., Nucleic Acids Res. 4, 353 (1977), reported the use of 2% benzene sulfonic acid in CH₃CI/CH₃OH (7:3, v/v) at 0^°C. Stawinski et al. theorized that an aromatic acid would have an affinity for the aromatic rings of dimethoxytrityl group, thereby localizing acid catalysis to this part of the molecule. Stawinski et al. hoped to overcome the problem of depurination that frequently occurs during the detritylation reaction. While improving on the prior art method of using 80% acetic acid, Stawinski et al. still observed 4% depurination. Roelen et al., Nucleic

Acids Res. 16, 7633 (1988), also used 2% benzenesulphonic acid, but in CH₂CyCH₃OH (7:3, v/v) at 20°C. Gaffney et al., Tetrahedron 40, 3 (1984), employed three 1 minute treatments of cold 2% benzenesulphonic acid in

CH₂a₂/CH₃OH (7:3, v/v). Matteucci and Caruthers, Tetrahedron Lett. 21, 3243 (1980), tested 2% toluene sulfonic acid in CHCl₃/CH₃OH (7:3) and found rapid depurination at 18°C. 0.5% toluene sulfonic acid in CHCl₃/CH₃OH gave better results, but the authors found the protic reagent less desirable than aprotic reagents (discussed infra).

Reese et al., Nucleic Acids Res. 13, 5215 (1985), used phenyl dihydrogen phosphate in chloroform/propanol (95:5, v/v) at room temperature, but they observed just a 54% yield, due, they theorized, to loss of the 2^,-0- protecting group. A number of other publications disclose the use of non-aromatic protic acids to cleave DMT. Reese et al., Nucleosides & Nucleotides 10, 81 (1991), used trifluoroacetic acid in methylene chloride, and Sproat and Gait, in Oligonucleotide Synthesis: A Practical Approach, p. 83 (Gait, M J., Ed., IRL Press, 1984), used 3% dichloroacetic acid in 1,2-dichloroethane. Sinha et al., Nucleic Acids Res. 12, 4539 (1984) used 3% trichloroacetic acid in 1% CH₃OH/CH₃N0₂. Wolter et al.,

Nucleosides & Nucleotides 5, 65-77 (1986), employed 3% trichloroacetic acid in CH₂C1₂/CH₃N0₂/CH₃0H (80/19/1).

The use of the foregoing acids and protocols, however, causes modifications of the protected purine base 4'-benzoyl-2^/-deoxyadenosine and, to a lesser extent, 6'-isobutyryl-2'-deoxyguanosine during detritylation. The result is depurination and chain cleavage during the final amide deprotection step with ammonium hydroxide. This problem becomes particularly acute in the synthesis of long oligonucleotides, where it is known that high yields require prolonged detritylation periods. Wolter et al., supra; Seliger et al., /. Chromatogr. 476, 49-57 (1989); and Seliger et al., /. Chromatogr. 397, 141-151 (1987).

Many Lewis acids, such as A1C1₈, ZnCl₂, ZnBr„ SnCl₄, and TiCl₄ have been examined under a variety of conditions as alternative detritylating agents. Matteucci and Caruthers, /. Am. Chem. Soc. 103, 3185 (1981), and Kohli et al., Tetrahedron Lett. 21, 2683 (1980). The use of a stronger, rather aggressive Lewis acid (BF₃/methanol) for detritylation has been reported. Engels, Angew. Chem. Int. Ed. Engl 19, 148 (1979), and Mitchell et al., Nucleic Acids Res. 18, 5321 (1990). Among the alternative detritylating agents, zinc bromide has been the subject of widest interest in the scientific community. Gaffney et al., supra; Matteucci et al., supra; Matteucci and Caruthers, supra; Kohli et al., supra;

Kierzek et al., Tetrahedron Lett. 22, 3761 (1981); Koster et al., Tetrahedron Lett. 24, 747 (1983); Ohtsuka et al., Tetrahedron 40, 47 (1984); Kumar and Poonian, /. Org. Chem. 49, 4905 (1984); Josephson et al., A Chem. Scand. B38, 539 (1984); Teese and Skone, Nucleic Acids Res. 13, 5215 (1985); and Ito et al., Nucleic Acids Res. 10, 1755 (1982). Zinc bromide does not cause significant depurination.

Both the selective removal of the nucleoside N-acyl protecting group by zinc bromide in the presence of alcohols (Kierzek et al., supra) and the rate of trityl removal decrease rapidly as the growing oligonucleotide chain becomes longer (Ito et al., supra, and Adams et al.,/. Am. Chem. Soc 105, 661 (1983)), however, effectively limiting the length of the sequence. As an alternative, Adams et al., supra, used dichloroacetic acid (pK, = 1.5). Dichloroacetic acid is now widely used for deprotecting the 5 ' hydroxyl group prior to coupling the next nucleoside in automated DNA synthesis on a solid support. Dichloroacetic acid (as well as trichloroacetic acid) suffers from the disadvantage of causing depurination, however.

Thus, depurination of oligonucleotide chains arising from the detritylation step during solid phase oligonucleotide synthesis remains a problem. Methods of circumventing this problem have not been entirely satisfactory as they tend to limit the length of the oligonucleotide that can be synthesized. Consequently, new methods of detritylation are desirable.

SUMMARY OF THE INVENTION

The present invention provides improved methods of oligonucleotide synthesis. In particular, we have discovered a method of detritylation that substantially improves the overall yield of desired oligonucleotide, both reducing the level of depurination that generally occurs in the prior art methods and allowing for synthesis of long oligonucleotides. The improved methods comprise detritylating a 5 ' DMT protected solid support-bound mononucleoside or oligonucleotide by contacting it with a detritylating agent comprising dichloroacetic acid in combination with a lower alcohol and/or IH-pyrrole. We have found that dichloroacetic acid (preferably at a concentration of about 2%) in the presence of about 0.1% lower alcohol (such as, but not limited to, methanol or ethanol) and/or 0.1-1.0% IH-pyrrole is a highly effective non- depurinating detritylating agent. Using these methods, increases in yield of from about 50% to 125% are observed. Moreover, because the present methods result in substantially less depurination, very good yields are obtained in the synthesis of extremely long oligomers, having length of up to at least about 150 monomers.

The foregoing merely summarizes certain aspects of the present invention and is not intended, nor should it be construed, to limit the invention in any way. All patents and publications cited herein are hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and IB display the results of capillary gel electrophoresis of oligonucleotides synthesized by the present methods.

Figure 2 displays a polyacrylamide gel electrophoresis of crude oligonucleotide SEQ. ID. NOs.: 1 and 2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention comprises an improved method of oligonucleotide synthesis. The standard method of synthesis involves initially loading a 4 ',4*- dimethoxytrityl (DMT) 5 ' hydroxyl protected nucleoside on a functionalized solid support, capping the unreacted functionalized sites with a blocking group, oxidizing the internucleoside linkage if appropriate, and detritylating the 5 ' hydroxyl, thereby providing a functional group to which the next nucleoside can couple. The process of adding DMT protected nucleoside, capping, oxidizing, and detritylating is repeated until the full length oligonucleotide is synthesized. The oligonucleotide is then cleaved from the solid support, the remaining base and phosphate protecting groups removed, and the resulting oligonucleotide purified. In one embodiment, the present method comprises a method of synthesizing an oligonucleotide on a solid support using DMT-protected nucleosides, the improvement comprising detritylating a support-bound, DMT- protected mononucleoside or oligonucleotide by contacting it with a detritylating agent comprising dichloroacetic acid in combination with a lower alcohol, IH- pyrrole, or a combination thereof for a time sufficient to detritylate substantially all the DMT-protected mononucleosides or oligonucleotides.

In a second embodiment, the present invention comprises a method of synthesizing an oligonucleotide by incorporating a new manner of detritylation.

The method comprises: a. providing a support-bound, 5 ' DMT-protected mononucleoside or oligonucleotide; b. detritylating the support-bound, 5 ' DMT-protected mononucleoside or oligonucleotide by contacting it with a detritylating agent for a time sufficient to detritylate substantially all of the support-bound, 5 ' DMT-protected mononucleoside- or oligonucleotide, wherein the detritylating agent comprises dichloroacetic acid in combination with a lower alcohol, 1H- pyrrole, or a mixture thereof; c. removing unreacted detritylation reactants, products, and by¬ products from the detritylated mononucleoside or oligonucleotide by washing with one or more solvents for a time sufficient to remove substantially all unreacted detritylation reactants, products and by-products;

In a third embodiment of the present invention, we provide a method of synthesizing an oligonucleotide having a desired sequence comprising: a. coupling the 5 ' DMT-protected nucleoside to a solid support by contacting the support with a 5 ' DMT-protected nucleoside to foπn a support-bound DMT-protected mononucleoside and a DMT-protected mononucleoside-bound support or, if the 5 ' DMT- protected nucleoside is not the first nucleoside of the desired sequence, coupling the 5 ' DMT-protected nucleoside to a 5' unprotected support-bound oligonucleotide to form a 5 ' DMT- protected support-bound oligonucleotide; b. capping unreacted functional groups by contacting the DMT- protected mononucleoside- or oligonucleoside-bound support with a capping agent; c. oxidizing the internucleoside linkage if required; d. detritylating the 5 ' position of the DMT-protected support-bound mononucleoside or oligonucleotide by contacting it with a detritylating agent for a time sufficient to detritylate substantially all of the support-bound, 5 'DMT-protected mononucleoside- or oligonucleotide, wherein the detritylating agent comprises dichloroacetic acid in combination with a lower alcohol, IH- pyrrole, or a mixture thereof; e. removing unreacted detritylation reactants, products, and by¬ products from the detritylated mononucleoside or oligonucleotide by washing with one or more solvents for a time sufficient to remove substantially all unreacted detritylation reactants, products and by-products; f. repeating a) through e) until an oligonucleotide having the desired sequence is synthesized; g. cleaving the oligonucleotide from the support; h. removing protecting groups from the oligonucleotide. As used herein, the term "functional group," means any moiety susceptible to forming a covalent bond with a nucleoside. Such functional groups are generally -OH and -NH₂. A "functionalized support" is a solid support having functional groups. Whether oxidation of the internucleoside linkage is required will depend on the mode of synthesis. Oxidation of the internucleoside linkage is required, for instance, when the phosphoramidate and H-phosphate approaches of oligonucleotide synthesis are employed. Those skilled in the art will recognize when oxidation is required. The skilled artisan will appreciate that any method of oligonucleotide synthesis consistent with the present invention can be used. Many such methods (including protocols for coupling a nucleoside to a solid support and support-bound nascent oligonucleotide, capping unreacted functional sites, oxidizing the internucleoside linkage, cleaving the oligonucleotide from the support, and deprotecting the oligonucleotide) are known in the art. E.g.,

Molecular Biology, v. 20 supra; Oligonucleotides and Analogues, supra; and Uhlmann and Peyman, supra.

It is known that in order to obtain increased yields, extended detritylation and washing periods are required for synthesis of long nucleotides. Detritylation for too long, however, results in depurination, while too short a detritylation period will result in incomplete detritylation. Detritylation should be conducted for a period of time sufficient to remove substantially all DMT groups, but should not be so long as to result in substantial depurination. Washing should be conducted for a length of time sufficient to remove substantially all unreacted reagents, products, and byproducts from the nascent, support-bound oligonucleotide. In particular, as demonstrated below, long washing periods are necessary in the present invention to eliminate extensive accumulation of failure sequences due to the alcohol, especially N-1 and N-2 (i.e., sequences have one and two fewer monomers, respectively, as compared to the desired oligonucleotide sequence).

The optimum detritylation and washing times for any particular synthesis can be determined in a routine manner by following the methods disclosed herein. For example, by repeatedly (concurrentK or serially) synthesizing the desired oligonucleotide on an analytical scale using different detritylation and wash periods and then measuring the percent yield, one may determine the optimum times that meet the requirements of the particular application. Such issues as overall time for oligonucleotide synthesis and acceptable yield may be important considerations in determining the acceptable times. The DMT-on mononucleoside or oligonucleotide (i.e., the mononucleoside or oligonucleotide bearing a 5' DMT group) may be subjected to detritylating conditions for about 60 to 400 seconds, preferably for about 100 to about 360 seconds, and most preferably for about 310 seconds, such periods of time being sufficient to detritylate substantially all of the 5 ' DMT-protected, support-bound mononucleoside or oligonucleotide. Even with such an extended detritylation period, the presence of the lower alcohols and/or IH-pyrrole results in acceptable yields.

Washing is generally conducted for about 10 to 200 seconds, preferably for about 100 to about 150 seconds, and most preferably for about 130 seconds. Such periods of time are sufficient to remove substantially all unreacted detritylating reagents, products, and by-products from the support-bound mononucleoside or oligonucleotide.

In the most preferred embodiment, dichloroacetic acid is used in a concentration of about 2%.

In one embodiment of the invention, a lower alcohol is used with dichloroacetic acid for detritylation. Any lower alcohol may be used in the present invention. By lower alcohol it is meant a -C₆ alcohol such as, but not limited to, methanol, ethanol, or propanol. In the most preferred embodiments the alcohol is methanol or ethanol and is used in a concentration of about 0.1%.

In another embodiment, IH-pyrrole is used with dichloroacetic acid. IH-pyrrole is preferably used in a concentration of about 0.1-1.0%.

Any suitable solvent may be used for detritylation. Most preferably the solvent is CH₂C1₂. Any suitable solvent or solvent system may be used to wash after detritylation. The solvent or solvent system must, however, dissolve dichloroacetic acid, the lower alcohol or IH-pyrrole (whichever is used) and DMT⁺ as well as any other byproducts. The most preferred washing agent is acetonitrile.

Although we do not intend to limit the invention by any theory, we theorize that the lower alcohol or IH-pyrrole acts bifunctionally. First, they act as proton scavengers, decreasing the activity of the acid. Second, they decrease the reaction of cleaved DMT⁺ with the free 5 ' 0^".

The methods of the present invention are suitable for synthesizing any type of oligonucleotide. Examples of oligonucleotides for which the present - 14 - methods are suitable are RNA, DNA or RNA/DNA hybrids, each of which can be unmodified or modified in any number of positions, including the sugar phosphate backbound and/or the nucleoside base. Examples of the types of oligonucleotides that may be synthesized and isolated with the methods of the present invention include, but are not limited to, those having the following modified internucleoside linkages: phosphodiesters, phosphorothioates, phosphorodithioates, phosphoroamidates, methyl- or other alkyl-phosphonates, carbonates, and carbamates. Any unmodified base (e.g., A, G, C, T, and U) may be used as can any modified base suitable for incorporation by solid phase synthesis. Modifications may occur in any number of nucleotides and may occur alone or in any combination. Oligonucleotide synthesis may be accomplished in any manner consistent with application of the presently disclosed methods. E.g., Methods in Molecular Biology, Vol 20, supra; Uhlmann and Peyman, Chem Rev. 90, 543 (1990); Oligonucleotides and Analogues: A Practical Approach, supra,. Any solid support that can be or is derivatized for solid phase oligonucleotide synthesis can be used in the present invention. As used herein, the terms "derivatized" and "functionalized" are used interchangeably and, when used in relation to a solid support, mean that the support has reactive moieties, preferably hydroxyl and/or amino moieties, suitable for oligonucleotide synthesis. A number of such supports are known in the art. Among these are low cross- linking polystyrene, polyamide, polyamide bonded silica gel, cellulose, silica gel, controlled pore glass, polystyrene/PEG "tentacle" copolymer, and high cross- linking polystyrene. See Methods of Molecular Biology, Vol 20, supra, and references cited therein. In the most preferred embodiment, the solid support is controlled pore glass (CPG).

The following Examples are offered for illustrative purposes only and are not intended, nor should they be construed, as limiting the invention in any way.

EXAMPLES Example 1

Effect of DCA/Alcohol Detritylation on Depurination The following two oligonucleotides were synthesized as described below:

5^/_ TCTCTCGCAC CCATCTCTCT CCTTCTCTCT CGCACCCATC TCTCTCCTTC TCTCTCGCAC CCATCTCTCT CCTTCTCTCT CGCACCCATC TCTCTCCTTC

T-3'

SEOIDNO∑πSl-mer):

5^/_ TCTCTCGCAC CCATCTCTCT CCTTCTCTCT CGCACCCATCTCTCTCCTTC TCTCTCGCAC CCATCTCTCT CCTTCTCTCT CGCACCCATC TCTCTCCTTC TCTCTCGCAC CCATCTCTCT CCTTCTCTCT CGCACCCATC TCTCTCCTTC

T-3'

Oligonucleotides SEQ. ID. NO. 1 and SEQ. ID. NO. 2 were assembled using a Milligen/Bioresearch (Burlington, MA) 8700 Series DNA synthesizer and commercially available phosphoramidite monomers (Millipore, Burlington, MA). The 101-mer SEQ. ID. NO. 1 was synthesized on a controlled pore glass (CPG)

CPG-1000 solid support (43.8 μmol/g). (CPG, Inc., Fairfield, NJ) Seliger et al., /. Chromatogr. 476, 49 (1989). The 151-mer SEQ. ID. NO. 2 was synthesized on CPG-2000 (18.4 μmol/g). (CPG, Inc. Fairfield, NJ) Id. Standard protocols as detailed for the 8700 synthesizer were used throughout, except where otherwise indicated. Crude products, after cleavage from the solid support, were subjected to capillary gel electrophoresis (Andrus in Methods in Molecular Biology:, Vol 26

Protocols for Oligonucleotide Conjugates, Synthesis and Analytical Techniques, pp.

277-300 (S. Agrawal, Ed., Humana Press, 1994)), and PAGE (Figure 2). The percent yield (averaged over three separate column syntheses) of the content of N, N-1, and N-2 content were determined based on capillary-gel electrophoresis analysis of crude product. The results are presented in Table 1.

The results demonstrate that when 0.1% methanol (run nos. 6, 8 & 14),

0.1% ethanol (run no. 10), 0.1% IH-pyrrole (run no. 12) and 1.0% IH-pyrrole

(run no. 11) are added to 2.0% DCA in methylene chloride during detritylation, a significant (50-125%) increase in yield of desired oligonucleotide is observed.

By contrast, no such increase was observed when only 2.0% DCA in methylene chloride was used with an extended detritylation cycle (run no. 2) or when 2.0% DCA in methylene chloride containing 0.1% methanol were used in combination with standard detritylation and extended washing cycles (run no. 5). Capillary gel electrophoresis (CGE) data is presented in Figure 1. The run nos. refer to those presented in Table 1. CGE analysis was carried out using a Beckman (Fullerton, CA) System Gold "PERSONAL" Chromatograph, P/ACE System 220 supplied with P/ACE UV Absorbance Detector, and "ECAP" ssDNAlOO Gel Column, 47 cm in length. The crude oligonucleotides were electrokinetically loaded onto the column by applying 5 kV for 15 seconds, followed by analysis applying 14.1 kV for 80 minutes, and tris-borate 7 M urea buffer. Detection was at 254 nm.

Figure 2 presents a polyacrylamide gel electrophoresis analysis of crude oligonucleotides SEQ ID NO 1 and SEQ ID NO 2. The run nos. refer to those in Table 1. The crude oligonucleotides were labeled with γ-³²P-ATP and analyzed by electrophoresis on 6% polyacrylamide gel containing 8 M urea.

SEQUENCE LISTING

(1) GENERAL INFORMAΗON:

(i) APPLICANT: Habus, Ivan Agrawal, Suhdir

(ii) TITLE OF INVENTION: Improved Methods of Detritylation for

Oligonucleotides Synthesis

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Allegretti & Witcoff, Ltd.

(B) STREET: 10 S. Wacker Drive Suite 3000

(C) CITY: Chicago

(D) STATE: Illinois

(E) COUNTRY: U.S.A.

(F) ZIP: 60606

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: WordPerfect 5.1 for DOS

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICAΗON:

(vϋi) ATTORNEY/ AGENT INFORMAΗON:

(A) NAME: Greenfield, Michael S.

(B) REGISTRATION NUMBER: 37,142

(C) REFERENCE/DOCKET NUMBER: 94,414

(ix) TELECOMMUNICAΗON INFORMAΗON:

(A) TELEPHONE: (312)715-1000

(B) TELEFAX: (312)715-1234

(2) INFORMAΗON FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 101 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ϋi) HYPOTHETICAL: NO (iv) ANTI-SENSE: Yl (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

TCTCTCGCAC CCATCTCTCT CCTTCTCTCT CGCACCCATC TCTCTCCTTC TCTCTCGCAC 60 CCATCTCTCT CCTTCTCTCT CGCACCCATC TCTCTCCTTC T 101

(2) INFORMAΗON FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 151 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

TCTCTCGCACCCATCTCTCTCCTTCTCTCTCGCACCCATCTCTCTCCTTCTCTCTCGCAC 60 CCATCTCTCTCCTTCTCTCTCGCACCCATCTCTCTCCTTCTCTCTCGCACCCATCTCTCT 120 CCTTCTCTCTCGCACCCATCTCTCTCCTTCT 151

Claims

What is claimed is:

1. An improved method of synthesizing an oligonucleotide on a solid support using DMT-protected nucleosides, the improvement comprising detritylating a support-bound, DMT-protected mononucleoside or oligonucleotide by contacting it with a detritylation agent comprising dichloroacetic acid in combination with a lower alcohol, IH-pyrrole, or a combination thereof for a time sufficient to detritylate substantially all the DMT-protected mononucleosides or oligonucleotides.

2. The method of claim 1 wherein the dichloroacetic acid is used in a concentration of about 2.0%.

3. The method of claim 2 wherein the lower alcohol is methanol or ethanol.

4. The method of claim 2 wherein IH-pyrrole is used.

5. The method of claim 3 wherein the methanol or ethanol is used in a concentration of about 0.1%.

6. The method of claim 4 wherein the IH-pyrrole is used in a concentration of about 0.1 to 1.0%.

7. A method of synthesizing an oligonucleotide having a desired sequence comprising: a. providing a support-bound, 5 ' DMT-protected mononucleoside or oligonucleotide; b. detritylating the support-bound, 5 ' DMT-protected mononucleoside or oligonucleotide by contacting it with a detritylating agent for a time sufficient to detritylate substantially all of the support-bound, 5 ' DMT-protected mononucleoside- or oligonucleotide, wherein the detritylating agent comprises dichloroacetic acid in combination with a lower alcohol, IH- pyrrole, or a mixture thereof; c. removing unreacted detritylation reactants, products, and by¬ products from the detritylated mononucleoside or oligonucleotide by washing with one or more solvents for a time sufficient to remove substantially all unreacted detritylation reactants, products and by-products;

8. The method of claim 8 wherein the dichloroacetic acid is used in a concentration of about 2.0%.

9. The method of claim 8 wherein the lower alcohol is methanol or ethanol.

10. The method of claim 9 wherein IH-pyrrole is used.

11. The method of claim 9 wherein the methanol or ethanol is used in a concentration of about 0.1%.

12. The method of claim 10 wherein the IH-pyrrole is used in a concentration of about 0.1 to 1.0%.

13. A method of synthesizing an oligonucleotide having a desired sequence comprising: a. coupling the first nucleoside of the desired sequence to a solid support by contacting the support with a 5 ' DMT-protected nucleoside to form a support-bound DMT-protected mononucleoside and a DMT-protected mononucleoside-bound support or, if the 5 ' DMT-protected nucleoside is not the first nucleoside of the desired sequence, coupling the 5 ' DMT- protected nucleoside to a 5 ' unprotected support-bound oligonucleotide to form a DMT-protected support-bound oligonucleotide; b. capping unreacted functional groups by contacting the DMT- protected mononucleoside- or oligonucleoside-bound support with a capping agent; c. oxidizing the internucleoside linkage if required; d. detritylating the 5 ' position of the DMT-protected support-bound mononucleoside or oligonucleotide by contacting it with a detritylating agent for a time sufficient to detritylate substantially all of the support-bound, DMT-protected mononucleoside- or oligonucleotide, wherein the detritylating agent comprises dichloroacetic acid in combination with a lower alcohol, IH- pyrrole, or a mixture thereof; e. removing unreacted detritylation reactants, products, and by¬ products from the detritylated mononucleoside or oligonucleotide by washing with one or more solvents for a time sufficient to remove substantially all unreacted detritylation reactants, products and by-products; f. repeating a) through e) until an oligonucleotide having the desired sequence is synthesized; g. cleaving the oligonucleotide from the support; h. removing protecting groups from the oligonucleotide.

14. The method of claim 13 wherein the protic acid is dichloroacetic acid.

15. The method of claim 14 wherein the dichloroacetic acid is used in a concentration of about 2.0%.

16. The method of claim 15 wherein the lower alcohol is methanol or ethanol.

17. The method of claim 15 wherein the IH-pyrrole is used.

18. The method of claim 16 wherein the methanol or ethanol is used in a concentration of about 0.1%.

19. The method of claim 17 wherein the IH-pyrrole is used in a concentration of about 0.1 to 1.0%.