NBS-DMSO as a nonaqueous nonbasic oxidation reagent for the synthesis of oligonucleotides

Article abstract of DOI:10.1016/S0960-894X(03)00754-6

A new method for the oxidation of nucleoside phosphite triester into phosphate triester under nonbasic and nonaqueous conditions using NBS-DMSO in CH₃CN has been developed. The utility of this method for solution- and solid-phase synthesis of oligonucleotide is demonstrated.

Full text of DOI:10.1016/S0960-894X(03)00754-6

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3537–3540
NBS–DMSO as a Nonaqueous Nonbasic Oxidation Reagent for
the Synthesis of Oligonucleotides
Matthew C. Uzagare,a Kamlesh J. Padiya,a Manikrao M. Salunkhea
and Yogesh S. Sanghvib,*
aThe Institute of Science, 15 Madam Cama Road, Mumbai 400 032, India
bIsis Pharmaceuticals, 2292 Faraday Avenue, Carlsbad, CA 92008, USA
Received 17 October 2002; accepted 8 July 2003
Abstract—A new method for the oxidation of nucleoside phosphite triester into phosphate triester under nonbasic and nonaqueous
conditions using NBS–DMSO in CH3CN has been developed. The utility of this method for solution- and solid-phase synthesis of
oligonucleotide is demonstrated.
# 2003 Elsevier Ltd. All rights reserved.
Synthetic oligonucleotides are an emerging class of drug
molecules that are assembled via phosphoramidite
chemistry. The quality and yield of oligonucleotides
made by this protocol depends on four key steps namely
detritylation, coupling, oxidation and capping. Among
these oxidation is a crucial step that converts an inter-
nucleosidic phosphite triester linkage P(III) to the corre-
sponding phosphate triester linkage P(V).1 The
development of efficient methods for such oxidation is an
important area of research particularly when therapeutic
oligonucleotides will be required in large quantities.2
exposure of aqueous base may not be appropriate for
the base labile protecting groups, particularly with
modified bases containing labile protecting groups.4
Due to these problems, we have focused our attention
towards the development of reagents useful for oxida-
tion under nonbasic and nonaqueous conditions. Sev-
eral strategies including nitrogen dioxide (N2O) in
dichloromethane,5 m-chloroperbenzoic acid (MCPBA)
in dichloromethane,6 tert-butyl hydroperoxide (TBHP)/
toluene solution,7 bis(trimethylsilyl)peroxide in di-
chloromethane or an acetonitrile dichloromethane mix-
ture,8 dimethyldioxirane in dichloromethane,9 (1S)-
(+)(10-camphorsulphonyl)oxaziridine (CSO)10 in ace-
tonitrile and ethyl(methyl)dioxirane in dichloro-
methane11 have been reported in the literature. These
methods have some limitations for large-scale applica-
tions such as toxicity of N2O and MCPBA, and the
undesired cleavage of 50-O-dimethoxytrity group that
results when m-chlorobenzoic acid is liberated in the
MCPBA oxidation step. Reagents like TBHP in decane,
bis(trimethylsilyl)peroxide and CSO are very expensive.
Additionally, silylated peroxides and dimethyldioxirane
are explosive reagents. Dimethyldioxirane is not avail-
able commercially on large-scale and undesirable oxi-
dative modifications of thymidine,12 uracil12 and
adenine bases have been reported with this reagent.13
Currently, iodine in THF/water/pyridine (or lutidine) is
used for oxidation in conventional oligonucleotide
synthesis.3 Although this method of oxidation is widely
accepted for small-scale synthesis, it is not suitable for
the large-scale synthesis of oligonucleotides required for
antisense therapeutics. On large-scale, the presence of
iodine, water and base compromises the quality and
yield of the product and increases the overall cost. For
example, controlled pore glass is a frequently used sup-
port that holds water tightly and complete removal of
water before the coupling step requires excessive wash-
ings with acetonitrile. This increases the volume and
cost of the solvent used. Presence of iodine in the reagent
imparts a pale coloration to the oligonucleotide product
that is not desirable for therapeutic applications. Lastly,
These limitations with nonbasic and nonaqueous
reagents reported in the literature encouraged us to
investigate the applications of CCl4-dimethyl sulfoxide
*Corresponding author at current address: Rasayan Inc., 2802 Crystal
Ridge Road, Encinitas, CA 92024, USA. Tel.: +1-760-944-1541; fax:
+1-760-944-1543; e-mail: rasayan@sbcglobal.net
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00754-6

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(DMSO) mixture as an oxidative reagent for oligonu-
cleotide synthesis.14 Volatility, toxicity and carcinogeni-
city issues have increasingly limited use of CCl4 in the
chemical industry. To avoid the use of halogenated
compounds, herein we report the use of N-bromosucci-
nimide (NBS)–DMSO as a safer and green reagent15 for
the oxidation of oligonucleotide phosphite P(III) to
their corresponding phosphate P(V).
–COR, –OCH2CH2CN) remain unaffected under the
oxidation conditions. The key to success was based on
the use of the right amount of reagents (NBS–DMSO–
CH3CN; w/v/v 0.02/0.07/1), freshly crystallized NBS and
anhydrous solvent. Reducing the amount of NBS–
DMSO compromised the purity of the dimers (<90%
HPLC).
The proposed mechanism for NBS–DMSO mediated
oxidation of P(III) to P(V) is shown in Scheme 2. The
first step appears to be halogenation of the P(III) species
with NBS to furnish an intermediate 5 which undergoes
addition of DMSO to provide an activated sulfonium
ion 6. This eliminates the dimethyl sulfide resulting in
the formation of stable P(V) product 4.
In order to develop appropriate conditions for the oxi-
dation cycle required during solid-phase oligonucleotide
synthesis, we first studied the NBS–DMSO mediated
oxidation in solution phase using a dinucleoside phos-
phite triester 3 (Scheme 1). The synthesis of dimer 3 was
accomplished by coupling of nucleoside phosphor-
amidite 1 with nucleoside 2 in the presence of 1H-tetra-
zole at room temperature for 5 min in anhydrous
CH3CN. Product 3 was isolated in good yields (89%)
after chromatographic purification. Interestingly, the
low cost of DMSO has allowed oxidation of various
compounds on large-scale.16 Similarly, NBS has been
reported to be an effective reagent for the oxidation of a
variety of functional groups.17 In this study we utilised
for the first time a combination of these two reagents for
the oxidation of P(III) to P(V) species. After significant
efforts, oxidation of 3 was successfully accomplished
with NBS–DMSO in CH3CN to furnish phosphate 4 in
90% isolated yield. The progress of the reaction was
monitored by 31P NMRspectroscopy indicating that 3
(d 144.2) was oxidized completely to furnish 4 (d 1.88).
All of the possible sixteen dimers having different com-
binations of ABz, T, Gibu, CBz were prepared following
the NBS–DMSO protocol in >90% isolated yield. The
Next, the solution-phase conditions were applied to the
solid-phase synthesis on an automated DNA synthesi-
zer. Synthesis of d(CATGG) was undertaken because it
represents all four natural bases. Initial experiments
with similar concentrations of NBS–DMSO (w/v 0.02/
0.07 in CH3CN) furnished ꢀ50% full-length product by
HPLC in the crude mixture. The 2-fold increase in the
concentrations and contact time of NBS–DMSO affor-
ded better results with ꢀ70% full-length product. The
oligonucleotide synthesis was performed in triplicate on
controlled pore glass (CPG) bound dG via a succinyl
linker.18 The modified synthesis cycle is shown in Table
1. Each monomer coupling was affected in the average
yield of 95% as determined by the colourimetric method
of released DMTr cation. The final oligonucleotide was
cleaved from solid support by ammonium hydroxide
treatment.3 The 31P NMRspectrum of the crude oligo-
nucleotide product had no signal due to the trivalent
phosphorus indicating quantitative oxidation. The
1
UV, H NMRand HPLC analysis indicated that the
heterocyclic moiety and all protecting groups (DMTr,
Scheme 1. Solution-phase synthesis of 16-dimers. Reagents and con-
ditions: (i) 0.1 mmol 1, 0.1 mmol 2, CH3CN, 1-H tetrazole, rt, 2 min,
85–90% 3; (ii) 0.1 mmol 3, 6 mL NBS/DMSO/CH3CN (w/v/v 0.02/
0.07/1), rt, 5 min, 90–95% 4.
Scheme 2. Proposed mechanism for oxidation.
Table 1. Synthesis cycle
Step no.
Operation
Reagent
Time (min)
1
2
3
4
5
6
Washing
Detritylation
Coupling
Washing
Capping
Oxidation
CH3CN
3% CHCl2COOH–CH2Cl2
0.1 M Phosphoramidite–CH3CN + 0.5 M 1H-tetrazole–CH3CN
CH3CN
0.5
1.8
1.1
0.2
0.4
2
Ac2O/2,6-lutidine/ THF (1:1:2) + 6.5% DMAP–THF
NBS–DMSO–CH3CN (W/V/V 0.04/0.14/1)

M. C. Uzagare et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3537–3540
3539
ratio of the integration of PO (d 0.91) and PS (d 57.32)
signals clearly indicated that there was no desulphur-
isation of phosphorothioate linkage during the NBS–
DMSO based oxidative step. The LC–MS data (not
shown) for 8 further confirmed the structure and purity
of the product.
In summary, NBS–DMSO in CH3CN is a useful
reagent for the oxidation of nucleoside phosphite into
phosphate under nonbasic and nonaqueous conditions.
We demonstrated that this reagent could be used for
synthesis of mixed backbone oligonucleotides in good
yields and high purity both by solid as well as solution-
phase synthesis. The easy accessibility, low-cost, safe
handling and disposal of this new oxidation system is
very attractive for the synthesis of oligonucleotides on a
large scale.
Scheme 3. Solid-phase synthesis of tetramer (n=2). Reagents and
conditions: (i) NBS/DMSO/CH3CN (w/v/v 0.04/0.14/1), rt, 2 min,
CH3CN wash; (ii) detritylation 3% CHCl2COOH–CH2Cl2, 1.8 min;
(iii) NH4OH 55 ꢁC.
Acknowledgements
One of the authors, M.C.U., is grateful to Isis Pharma-
ceuticals for financial assistance and gift of various che-
micals and reagents. We are also thankful to Dr.
Douglas Cole for his encouragement and support
throughout this project.
References and Notes
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integrity of the oligonucleotide was further confirmed
by enzymatic hydrolysis of purified d(CATGG) which
indicated the expected ratio of the natural bases.
The NBS–DMSO oxidation system was further eval-
uated for use in the synthesis of mixed backbone oligo-
nucleotides due to their potential therapeutic
applications.19 A T-tetramer was chosen as a test
sequence to demonstrate the capabilities of NBS–
DMSO. Tetramer 7 containing a terminal P(III) linkage
was synthesized following traditional phosphoramidite
chemistry using Beaucage reagent for thiolation. Oxi-
dation of 7 with the NBS–DMSO system on solid sup-
port using the conditions described in Table 1 provided
8 in excellent yield (Scheme 3). The purity of tetramer 8
was assessed by 31P NMRspectrum ( Fig. 1). The 1:2

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and Reducing Agents; Burke, S. D.; Danheiser, R. L. Eds. John
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18. Oligonucleotides were synthesized on 5 mmol scale using
an ABI-394 DNA synthesizer following a protocol descri-
bed in Table 1. Doubling the ratio of NBS–DMSO (0.04/
0.14) relative to the amount used for solution phase synth-
esis, improved the yield (95%) of tetramer 8 during solid-
phase synthesis. The NBS–DMSO–CH3CN solution was
prepared freshly prior to the use. General experimental
conditions: Commercial nucleoside loaded CPG (500 A, 50–
60 umol/g) was used for all syntheses. When the synthesis
was complete, the cleavage and deprotection were carried
out by treatment with concentrated ammonium hydroxide
solution (5 mL) at 55 ꢁC for 12 h. CPG was removed by
filtration, and ammonium hydroxide solution was dried by
lyophilization. The crude product was analyzed by reverse-
phase HPLC, 31P NMRand MS (see ref 2 for more
details).
19. Zhou, W.; Agrawal, S. Bioorg. Med. Chem. Lett. 1998, 8,
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