Organic Process Research & Development 2000, 4, 199−204
Synthesis of Antisense Oligonucleotides: Replacement of
3H-1,2-Benzodithiol-3-one 1,1-Dioxide (Beaucage Reagent) with Phenylacetyl
Disulfide (PADS) As Efficient Sulfurization Reagent: From Bench to Bulk
Manufacture of Active Pharmaceutical Ingredient
Zacharia S. Cheruvallath, Recaldo L. Carty, Max N. Moore, Daniel C. Capaldi, Achim H. Krotz, Patrick D. Wheeler,
Brett J. Turney, Stephen R. Craig, Hans J. Gaus, Anthony N. Scozzari, Douglas L. Cole, and Vasulinga T. Ravikumar*
Isis Pharmaceuticals, 2290 Faraday AVenue, Carlsbad California 92008, U.S.A.
Abstract:
ates differ from natural DNA by the replacement of one of
two nonbridging oxygen atoms by a sulfur atom at each
internucleotide linkage.
It is demonstrated that phosphorothioate oligodeoxyribonucle-
otides can be synthesized on scales from 1 µmol to 150 mmol
using phenylacetyl disulfide (PADS) as an efficient and eco-
nomical replacement for Beaucage reagent. A 0.2 M solution
of PADS in a mixture of 3-picoline and acetonitrile (1:1 v/v) as
solvent with 60-120 s contact time efficiently (>99.6%) sulfu-
rizes phosphite triesters to phosphorothioate triester linkages.
Phenylacetyl disulfide reagent is inexpensive and scaleable and
is currently being used by us for the manufacture of antisense
phosphorothioate oligodeoxyribonucleotide active pharmaceuti-
cal ingredients (API).
With the first antisense drug (Vitravene) being approved
by the U.S. Food and Drug Administration and recommended
by the European panel for marketing as a treatment for CMV
retinitis in AIDS patients and with several systemic drugs
potentially reaching the market in the next several years, the
development of economical and environmentally safe meth-
ods for the synthesis of high quality oligonucleotides has
become a major focus of our research.12-25 Typically,
synthesis of oligonucleotides on scales up to 150 mmol is
performed in a cyclic manner on automated solid-phase
synthesizers using phosphoramidite derivatives of protected
nucleosides with 4,4′-dimethoxytrityl (DMT) protection of
the 5′-hydroxyl group, benzoyl protection for adenine (dAbz)
and cytosine (dCbz), and isobutyryl protection for guanine
(dGibu).
Introduction:
Antisense oligonucleotides as modulators of gene expres-
sion represent an exciting new drug technology.1-9 Phos-
phorothioate oligodeoxyribonucleotides are now among the
most intensively investigated nuclease-resistant antisense
analogues, as evidenced by a number of ongoing clinical
trials and one FDA approved drug.10,11 A major advantage
of the antisense strategy is its potential specificity of action.
In principle, an antisense oligonucleotide can be designed
to target a single gene within the human genome, creating a
specific therapeutic for any disease for which a causative or
contributory gene is known. Structurally, the phosphorothio-
Phosphoramidite chemistry26-28 requires sulfurization after
each coupling. It is crucial that the sulfur transfer step be
(12) It is roughly estimated that for a systemic dosing for treatment of a chronic
disease about 700-900 kg per year of drug is needed to meet the present
demand.
(13) Scozzari, A. N. In Large Scale Oligonucleotide Synthesis: Production,
Purification and Analysis; IBC Conference, October 28-29, 1997; San
Diego, CA, 1997
(14) Krotz, A. H.; Cole, D. L.; Ravikumar, V. T. Bioorg. Med. Chem. 1999, 7,
435.
(15) Krotz, A. H.; Carty, R. L.; Moore, M. N.; Scozzari, A. N.; Cole, D. L.;
Ravikumar, V. T. Green Chem. 1999, manuscript submitted for publication.
(16) Ravikumar, V. T.; Krotz, A. H.; Cole, D. L. Tetrahedron Lett. 1995, 36,
6587.
(1) Crooke, S. T. In Antisense Therapeutics in Biotechnology & Genetic
Engineering ReViews; Intercept Ltd.: Hampshire, UK, 1998; Vol. 15, pp
121-157.
(2) Crooke, S. T. In Basic Principles of Antisense Therapeutics in Handbook
of Experimental Pharmacology: Antisense Research & Application; Crooke,
S. T., Ed.; Springer-Verlag: Berlin, Germany, 1998.
(3) Crooke, S. T. Antisense Nucleic Acid Drug DeV. 1998, 8, 115.
(4) Crooke, S. T. In Burger’s Medicinal Chemistry and Drug DiscoVery; John
Wiley: New York, 1995 and references therein.
(17) Turney, B. J.; Cheruvallath, Z. S.; Andrade, M.; Cole, D. L.; Ravikumar,
V. T. Nucleosides Nucleotides 1999, 18, 89.
(18) Cheruvallath, Z. S.; Wheeler, P. D.; Cole, D. L.; Ravikumar, V. T.
Nucleosides Nucleotides 1999, 18, 485.
(19) Eleuteri, A.; Capaldi, D. C.; Cole, D. L.; Ravikumar, V. T. Nucleosides
Nucleotides 1999, 18, 475.
(5) Monia, B. P.; Johnston, J. F.; Muller, M.; Geiger, T. H.; Fabbro, D. Nat.
Med. 1996, 2, 668.
(6) Knorre, G. K.; Vlassov, V. V.; Zarytova, V. F.; Lebedev, A. V.; Federova,
O. S. Design and Targeted Reactions of Oligonucleotide DeriVatiVes;
Knorre, G. K., Vlassov, V. V., Zarytova, V. F., Lebedev, A. V., Federova,
O. S., Eds.; CRC Press: Boca Raton, FL, 1994.
(20) Ravikumar, V. T.; Andrade, M.; Wyrzykiewicz, T. K.; Scozzari, A. N.;
Cole, D. L. Nucleosides Nucleotides 1995, 14, 1219.
(21) Krotz, A. H.; Klopchin, P.; Cole, D. L.; Ravikumar, V. T. Bioorg. Med.
Chem. Lett. 1997, 7, 73.
(22) Krotz, A. H.; Klopchin, P.; Cole, D. L.; Ravikumar, V. T. Nucleosides
Nucleotides 1997, 16, 1637.
(7) Crooke, S. T.; Lebleu, B. Antisense Res. Appl. 1993.
(8) Cook, P. D. Anti-Cancer Drug Des. 1991, 6, 585.
(23) Capaldi, D. C.; Scozzari, A. N.; Cole, D. L.; Ravikumar, V. T. Org. Process.
Res. DeV. 1999, 3, 485.
(9) Sanghvi, Y. S. In ComprehensiVe Natural Products Chemistry; Barton, D.
H. R., Nakanishi, K., Eds.; DNA and Aspects of Molecular Biology, Vol.
7; Kool, E. T., Ed.; Pergamon Press: Elmsford, NY, 1999; p 285.
(10) Kisner, D. L. 12th International Roundtable on Nucleosides and Nucleotides,
September 19, 1996; La Jolla, CA, 1996.
(24) Eleuteri, A.; Capaldi, D. C.; Krotz, A. H.; Cole, D. L.; Ravikumar, V. T.
Org. Process Res. DeV. 2000, manuscript submitted for publication.
(25) Eleuteri, A.; Cheruvallath, Z. S.; Capaldi, D. C.; Cole, D. L.; Ravikumar,
V. T. Nucleosides Nucleotides 1999, 18, 1803.
(26) McBridge, L. C.; Caruthers, M. H. Tetrahedron Lett. 1983, 24, 245.
(27) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1992, 48, 2223.
(28) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 6123.
(11) More than 18 oligodeoxyribonucleotide phosphorothioates are currently
under clinical investigation for the treatment of various diseases.
10.1021/op990077b CCC: $19.00 © 2000 American Chemical Society and The Royal Society of Chemistry
Published on Web 12/10/1999
Vol. 4, No. 3, 2000 / Organic Process Research & Development
•
199