(IO)  Patent No.:  US 8,138,330 B2  Mar.20,2012  (45)  Date of Patent:  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  (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.  (57)  ABSTRACT  23 Claims, 6 Drawing Sheets  OTHER PUBLICATIONS  U.S. PATENT DOCUMENTS  (56)  References Cited  (65)  Prior Publication Data  Related U.S. Application Data  4,415,732 A  11/1983 Caruthers et al.  4,458,066  A  7/1984 Caruthers et al.  4,500,707  A  2/1985 Caruthers et al.  4,668,777  A  5/1987 Caruthers et al.  4,973,679  A  11/1990 Caruthers et al.  5,132,418 A  7/1992 Caruthers et al.  RE34,069 E  9/1992 Koster et al.  5,519,126 A *  5/1996 Hecht  536/24.3  6,222,030 Bl  4/2001 Dellinger et al.  6,609,195 B2  8/2003 Dover  6,887,990 Bl*  5/2005 Bhan et al.  536/25.31  6,989,442 B2 *  1/2006 Vargeese .  536/25.31  US008138330B2  US 2008/0064867 Al  Mar. 13, 2008  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.*  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.  (Continued)  Primary Examiner  - Eric S Olson  (74)  Attorney, Agent, or Firm  - Polsinelli Shughart PC  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. 