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# c12) United States Patent 

# Leuck et al. 

# (54) PROCESS FOR THE SYNTHESIS OF OLIGONUCLEOTIDES 

(75) Inventors: Michael Leuck, Hamburg (DE); Andreas Wolter, Hamburg (DE); Alfred Stumpe, Hamburg (DE) 

(73) Assignee: Sigma-Aldrich Co. LLC, St. Louis, MO (US) 

( *) Notice: Subject to any disclaimer, the term ofthis patent is extended or adjusted under 35 U.S.C. 154(b) by 736 days. 

(21) Appl. No.: 11/853,662 

(22) Filed: Sep. 11, 2007 

## (65) Prior Publication Data 

US 2008/0064867 Al Mar. 13, 2008 

(60) Provisional application No. 60/825,175, filed on Sep. 11, 2006. 

(51) Int. Cl. C07H 21102 (2006.01) C07H 21104 (2006.01) 

(52) U.S. Cl. .................................................... 536/25.31 

(58) Field of Classification Search ........................ None See application file for complete search history. 

## (56) References Cited 

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Griffin et al., "The Synthesis of Oligonucleotides-IV Preparation of Dinucleoside Phosphates from 2,5’-Protected Ribonucleoside Derivatives".* 

Greene et al., "Protective groups in Organic Synthesis", published 1999 by John Wiley and Sons, chapter 2, Protection for the Hydroxylgroup, Including 1,2-and l,3~Diols, pp. 17-244.* 

Chattopadhyaya et al., "Chemical Synthesis of a Tridecanucleoside dodecaphosphate sequence of SV 40 DNA" Nucleic Acids Research (1980) vol. 8 No. 8, pp. 2039-2053.* 

Zegelaar-Jaarsveld, "Iodonium Ion-Assisted Synthesis ofTetrameric Fragments Corresponding to the Cell Wall Phenolic Glycolipids of Mycobacterium kansasii serovars II and IV" Tetrahedron (1996) vol. 52 No. 10, pp. 3575-3592.* 

Kamimura et al., "Protection of Imide Group of Uracil Moiety by Means of2,2,2-trichloro-tert-butyloxycarbonyl chloride: A selective synthesis of2’-O-methyluridine" Chemistry Letters (1982) pp. 965- 968.* 

## Related U.S. Application Data 

## U.S. PATENT DOCUMENTS 

## OTHER PUBLICATIONS 

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# (IO) Patent No.: US 8,138,330 B2 

# Mar.20,2012 (45) Date of Patent: 

Cook et al., "Use of Chloroacetic Anhydride for the Protection of Nucleoside Hydroxyl Groups" Journal of Organic Chemistry ( 1970) vol. 35 No. 6, pp. 1940-1943.* 

Protective Groups in Organic Synthesis, Third Edition, by Theodora Greene and Peter Wuts, published 1999 by John Wiley and Sons, Inc, pp. 160-165.* 

Griffin et al., "The Synthesis of Oligonucleotides-IV Preparation of Dinucleoside Phosphates from 2,5’-Protected Ribonucleoside Derivatives" Tetrahedron (1968) vol. 24 pp. 639-662.* 

Beaucage et al., "Advances in the Synthesisof Oligonucleotides by the Phosphoramidite Approach"; Tetrahedron; 1992; pp. 2223-2311; vol. 48; No. 12. 

Beaucage et al., "The Synthesis of Modified Oligonucleotides by the Phosphoramidite Approach and Their Applications"; Tetrahedron; 1993; pp. 6123-6194; vol. 49; No. 28. 

Bergmann et al., "The 2-Dansylethoxycarbonyl ( ~2 { [ 5(Dimethylamino )naphthalen-1-y ]sulfonyl }ethoxycarbonyl; Dnseoc) Group for Protection of the 5’-Hydroxy Function in Oligodeoxyribonucleotide Synthesis"; Helvetica Chimica Acta; 1994; pp. 203-215; vol. 77. 

Bergmann et al., "Ally! as Internucleotide Protecting Group in DNA Synthesis to be Cleaved off by Ammonia"; Tetrahedron; 1995; pp. 6971-6976; vol. 51, No. 25. 

Fearon eta!.; "Investigation of the ’n-1’ impurity in phosphorothioate oligodeoxynucleotides synthesized by the solid-phase beta› cyanoethyl phosphoramidite method using stepwise sulfurization"; Nucleic Acids Research; 1995; pp. 2754-2761; vol. 23; No. 14. 

Gryaznov et al., "Selective O-phosphitilation with nucleoside phoshoramidite reagents"; 1992; Nucleic Acids Research; pp. 1879- 1882; vol. 20; No. 8. 

Honda et al., "Synthesis of Oligoribonucleotides by Use of S,S› Diphenyl N-Monomethoxytrityl Ribonucleoside 3’-Phosphorodithioates"; Tetrahedon; 1984; pp. 153-163; vol. 40; No. 1. 

Iwai et al., "5-Levulinyl and 2’tetrahydrofuranyl protection for the synthesis of oligoribonucleotides by the phosphoramidite approach"; Nucleic Acids Research; 1988; pp. 9443-9456; vol. 18; No. 20. 

Primary Examiner - Eric S Olson 

(74) Attorney, Agent, or Firm - Polsinelli Shughart PC 

## (57) ABSTRACT 

The present invention discloses novel methods for the syn› thesis of oligonucleotides with nucleoside phosphoramidites on solid supports. The methods comprise the stepwise chain assembly of oligonucleotides on supports with 5’-acyl phos› phoramidites. The synthesis cycles consist of a front end deprotection step which is conducted with a solution of a primary amine or a phenolate, a phosphoramidite coupling step with a 5’-acyl nucleoside phosphoramidite in the pres› ence of an activator, a phosphite oxidation step and an optional capping step. The novel methods improve the quality of synthetic oligonucleotides due to the irreversibility of the front end deprotection step, which prevents the formation of deletion sequences, and due to the avoidance of acidic reagents in the synthesis cycles, which prevent the formation of depurination side products. The invention further discloses novel nucleoside phosphoramidite compositions wherein the phosphoramidites carry acyl front end protective groups which are cleavable with primary amines or phenolates. The invention is applicable to the synthesis of oligodeoxyribo› nucleotides, oligoribonucleotides and oligonucleotides with modifications in their sugar or phosphate groups. 

US008138330B2 

## 23 Claims, 6 Drawing Sheets 

(Continued) 

