A short, novel, and cheaper procedure for oligonucleotide synthesis using automated solid phase synthesizer

Article abstract of DOI:10.1081/NCN-120021968

Dimethylthiuram disulfide (DTD) has been developed as an efficient thiolation reagent during automated synthesis of oligonucleotides using phosphoramidite chemistry. Simultaneous thiolation and capping was accomplished by mixing DTD with capping solution B, which saved 20% of solvent consumption and compressed the four-step synthesis cycle to three. Large-scale (1 mmol) synthesis of phosphorothioate oligonucleotides has been demonstrated with excellent yield and purity.

Full text of DOI:10.1081/NCN-120021968

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A Short, Novel, and Cheaper Procedure for
Oligonucleotide Synthesis Using Automated Solid Phase
Synthesizer
Quanlai Song a , Zhiwei Wang a & Yogesh S. Sanghvi a b
a Isis Pharmaceuticals, Inc., Carlsbad, California, USA
b Isis Pharmaceuticals, Inc., 2292 Faraday Avenue, Carlsbad, CA, 92008, USA
Version of record first published: 31 Aug 2006.
To cite this article: Quanlai Song, Zhiwei Wang & Yogesh S. Sanghvi (2003): A Short, Novel, and Cheaper Procedure for
Oligonucleotide Synthesis Using Automated Solid Phase Synthesizer, Nucleosides, Nucleotides and Nucleic Acids, 22:5-8,
629-633
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 629–633, 2003
A Short, Novel, and Cheaper Procedure for Oligonucleotide
Synthesis Using Automated Solid Phase Synthesizer
*
Quanlai Song, Zhiwei Wang, and Yogesh S. Sanghvi
Isis Pharmaceuticals, Inc., Carlsbad, California, USA
ABSTRACT
Dimethylthiuram disulfide (DTD) has been developed as an efficient thiolation
reagent during automated synthesis of oligonucleotides using phosphoramidite
chemistry. Simultaneous thiolation and capping was accomplished by mixing
DTD with capping solution B, which saved 20% of solvent consumption and
compressed the four-step synthesis cycle to three. Large-scale (1 mmol) synthesis
of phosphorothioate oligonucleotides has been demonstrated with excellent yield
and purity.
Key Words: Antisense; Sulfur-transfer reagent; Dimethylthiuram disulfide;
Oligonucleotide synthesis.
INTRODUCTION
The use of antisense oligonucleotides represents a powerful new strategy in the
development of therapeutic agents that act by the selective inhibition of gene expres-
sion. With increasing number of antisense products in clinic and successful outcome
of these clinical trials, large-scale production of oligonucleotides at low-cost would
become mandatory. Therefore, developing low-cost approaches for oligonucleotide
*Correspondence: Yogesh S. Sanghvi, Isis Pharmaceuticals, Inc., 2292 Faraday Avenue,
Carlsbad, CA 92008, USA; Fax: (760) 603-4655; E-mail: ysanghvi@isisph.com.
629
DOI: 10.1081/NCN-120021968
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

630
Song, Wang, and Sanghvi
synthesis are of obvious importance and widespread interest. Automated synthesis
on solid-supports using phosphoramidite chemistry has been the most successful
method for the large-scale synthesis of oligonucleotide-based drugs.[1] Detritylation,
coupling, oxidation and capping are the four key steps during automated synthesis
of oligonucleotides. During synthesis of phosphorothioate oligonucleotides oxida-
tion of phosphite P(III) into phosphorothioate triester P(V) is accomplished via a
sulfur-transfer step. Elemental sulfur was first used for this step, which is poorly
soluble in organic solvents commonly used for automated synthesis. Also the rate
of conversion of P(III) into P(V) product is not fast and facile with elemental sulfur.
For these reasons, many sulfur-transfer reagents have been developed during past
decade.[2] Among a dozen reagents described in the literature, 3H-1,2-benzo-
dithiol-3-one 1,1-dioxide (Beaucage reagent)[3] and phenylacetyl disulfide (PADS)[4]
are the two most notable and widely used reagents at the present time on large-scale.
Both are soluble in organic solvents and produce efficient and rapid sulfurization
kinetics. Although these two reagents are currently used on industrial scale, they
have certain limitations. For example, Beaucage reagent is expensive, long-term stabi-
lity in solution is not optimal and the cyclic sulfoxide by-product formed during
sulfurization is an efficient oxidizing agent which increases the levels of phospho-
diesters during packed-bed automated synthesis. Sulfurization with PADS is accom-
plished in combination with 3-picoline. Unfortunately the smell of this mixture is
obnoxious and upon standing it changes color to a dark solution. Thus, our inves-
tigation for an alternative sulfur-transfer reagent that is cheap and user friendly
led us to dimethylthiuram disulfide (DTD) as a reagent of choice.
CHEMISTRY
In 1991, Vu and Hirschbein first reported on the application of tetraethylthiuram
disulfide (1) as a sulfur-transfer reagent.[5] We found 1 attractive as a sulfur-transfer
reagent for three reasons. First, ease of synthesis and low cost. Second, proven utility
for small-scale oligonucleotide synthesis and third its atom efficiency.[6] Based on
these criterions, we embarked on an efficient synthesis of 1 and its analogs using a
common approach. Herein, we report one-pot synthesis of disulfides 1–4 in excellent
yield (> 90%; Sch. 1) starting from alkylamine and carbon disulfide which are both
cheap and commercially available on industrial scale. The products are isolated as
stable pale yellow crystalline solid that is highly soluble in organic solvents.a
aPreparation of dimethylthiuram disulfide (4): NaOH (80 g, 2 mol) was dissolved in water
(500 mL) and the solution was cooled to 0ꢀC. THF (200 mL), methylamine (40% in water,
170 mL, 2 mol) and carbon disulfide (120 mL, 2 mol) were added over a period of 30 min to
aq. NaOH solution while stirring at 0ꢀC. Crushed ice (1.5 Kg) was added to the reaction mix-
ture, followed by acetic acid (300 mL). Hydrogen peroxide (30%, 100 mL, 1 mol) was added
gradually while stirring over 15 min while maintaining the reaction temperature below 5ꢀC.
Heptane (800 mL) was added to the reaction mixture and stirred for additional 30 min. The
product was filtered off and washed with aq. acetic acid (2%, 5ꢁ200 mL) and heptanes
(2ꢁ200 mL). The product was air dried until constant weight (ꢂ1 day) to furnish off-white
solid (205 g, 97%). M.p. 98–100ꢀC (dec.)

New Procedure for Oligonucleotide Synthesis
631
Scheme 1. Synthesis and structure of thiuram disulfide analogs.
Disulfides 1–4 were tested for their ability to convert triethyl phosphite 5 into
triethyl phosphorothioate 6 in CD3CN solution at room temperature. The conver-
sion was monitored by 31P NMR spectroscopy. Reaction of disulfide 1 with 5
furnished the expected phosphorothioate (68 ppm) as a major product and 18% minor
products (16–18 ppm). Similarly reaction of disulfides 2 and 3 with 5 furnished sig-
nificant amount of undesirable phosphate products (16–18 ppm). Interestingly, the
efficacy of DTD (4) as a sulfur-transfer agent was excellent with >99% product
formation. Both PADS and Beaucage reagent gave identical results, confirming
that conversion of 5 into 6 was a reliable method for quick screening of various
sulfur-transfer reagents.
Next, stability and solubility of DTD was tested in various solvents for extended
period of time. DTD is highly soluble in THF (1 M) and relatively less in CH3CN
(0.3 M). Addition of base to a solution of DTD rapidly degraded the product.
Whereas, addition of an acid such as 4-chlorophenol stabilized a 0.3 M DTD in
THF for 3-days. Similarly addition of acetic acid to a solution of DTD significantly
increased the stability of the reagent (>30-days) without compromising the sulfur-
transfer ability. The exceptional stability of DTD in presence of acetic acid encou-
raged us to mix it with capping solution B, which contains acetic anhydride in
CH3CN hoping that oxidation and capping steps could be performed simul-
taneously. The shorter protocol (3-steps vs. 4-steps) may result in reducing solvent
wash, time savings in overall synthesis cycle and make DTD solution stable.
Therefore, synthesis of ISIS-14803, a 20-mer phosphorothioate oligonucleotide
was undertaken using the new 3-step protocol on OligoPilot II synthesizer at
173–193 mmol scale. The first two reaction cycles, detritylation (3% DCA in toluene)
and coupling (0.2 M amidite with 1-H tetrazole) were unchanged. In order to accom-
plish oxidation and capping in one step, 0.3 M DTD was added to 20% acetic an-
hydride in CH3CN (capping B solution), and capping solution A was left unaltered
(N-methylimidazole=pyridine=CH3CN, 2:3:5, v=v). The contact time during syn-
thesis for combined step was 4 min and 2 column volume of solution was utilized
for the reaction. Rest of the synthesis cycle and procedures are similar to literature

632
Song, Wang, and Sanghvi
Table 1. Data on crude oligonucleotides.
S-transfer
reagent
Scale
(m mol)
OD=
m mol
Trityl-
on %
CH3CN
use (L)
Synthesis
Time (min)
Oligo
P ¼ 0%
PADS
DTD
PADS
DTD
14803
14803
AffinitakTM
AffinitakTM
173
169
193
193
148
149
151
152
74.7
76.2
72.0
73.0
7.0
5.1
8.7
4.9
2.5
2.0
5.4
4.5
470
350
540
450
ISIS 14803 Sequence: CTGCTCATGGTGCACGGTCT.
AffinitakTM Sequence: GTTCTCGCTGGTGAGTTTCA.
method.[1] Identical sequence was made as a control using PADS for sulfurization.
The data on crude oligonucleotide made by DTD and PADS as sulfur-transfer
reagent is summarized in Table 1. Clearly, the data indicates that there was
no obvious difference between the two syntheses. Subsequent purification of two
oligonucleotides further confirmed that both were equally pure and furnished
the expected yield.
The synthesis was repeated with DTD on larger scale (1 mmol) using another
20-mer phoshorothioate oligonucleotide (AffinitakTM). Same oligonucleotide using
PADS was also synthesized as a control. The data in Table 1 indicates that DTD
works as good as PADS on larger scale. More importantly, the overall solvent
(CH3CN) consumption for synthesis with DTD is reduced by 20% compared to
the synthesis done with PADS due to elimination of a synthesis=wash cycle. The
results indicate that quality and yield of the product is not compromised when
DTD is employed in a three-step mode as described above. The products were
analyzed by HPLC, CGE, 31P NMR and LC MS method.
CONCLUSIONS
In summary, use of DTD as a sulfur-transfer reagent may provide following
advantages over PADS and other conventional reagents.
–
–
–
–
–
–
DTD is inexpensive compared to other sulfur-transfer reagents ($10=Kg).
Overall usage of CH3CN is reduced by 20% which is a significant cost savings.
Due to reduced solvent consumption, there is less waste solvent to dispose.
The total synthesis time was reduced by 25% which increases the throughput.
The atom efficiency with DTD is excellent due to its small size.
Use of DTD is safe and convenient on large-scale.
ACKNOWLEDGMENT
We are thankful to Dr. Douglas L. Cole for his support and encouragement
throughout this project.

New Procedure for Oligonucleotide Synthesis
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633
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