Solid phase oligonucleotide synthesis by using β-CE H-phosphonate approach

Article abstract of DOI:10.1081/SCC-120002704

A new approach to the solid phase synthesis of oligonucleotide is described, which is based on oxidative coupling of nucleoside 3′-β-CE H-phosphonates in presence of N-Bromosuccinimide (NBS) as a coupling agent.

Full text of DOI:10.1081/SCC-120002704

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: http://www.tandfonline.com/loi/lsyc20
SOLID PHASE OLIGONUCLEOTIDE SYNTHESIS BY
USING β-CE H-PHOSPHONATE APPROACH
Kamlesh J. Padiya , Nikhil U. Mohe & Manikrao M. Salunkhe
To cite this article: Kamlesh J. Padiya , Nikhil U. Mohe & Manikrao M. Salunkhe (2002) SOLID
PHASE OLIGONUCLEOTIDE SYNTHESIS BY USING β-CE H-PHOSPHONATE APPROACH,
Synthetic Communications, 32:6, 917-922, DOI: 10.1081/SCC-120002704
To link to this article: http://dx.doi.org/10.1081/SCC-120002704
Published online: 16 Aug 2006.
Submit your article to this journal
Article views: 24
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=lsyc20
Download by: [FU Berlin]
Date: 04 May 2017, At: 00:04

SYNTHETIC COMMUNICATIONS, 32(6), 917–922 (2002)
SOLID PHASE OLIGONUCLEOTIDE
SYNTHESIS BY USING b-CE
H-PHOSPHONATE APPROACH
Kamlesh J. Padiya, Nikhil U. Mohe, and
*
Manikrao M. Salunkhe
Department of Chemistry, The Institute of Science,
15, Madam Cama Road, Mumbai-400 032, India
ABSTRACT
A new approach to the solid phase synthesis of oligonucleo-
tide is described, which is based on oxidative coupling
of nucleoside 30-b-CE H-phosphonates in presence of
N-Bromosuccinimide (NBS) as a coupling agent.
In the recent years the antisense oligonucleotides have been proposed
as a major class of new pharmaceuticals. The need for the production of
oligonucleotides has thus led to the investigation of alternative synthetic
strategies aimed at increased purity and better yields. Today, most of the
antisense oligonucleotides are synthesised either by the phosphoramidite
approach1 or H-phosphonate,2 the condensation and oxidation being the
key steps involved in the synthesis.
Despite of the efficiency of the DNA synthesis, impurities inevitably
accumulate, the synthesis of a 20 mer oligonucleotide involves more than
100 separate chemical steps, each with a finite possibility of producing
*Corresponding author.
917
Copyright & 2002 by Marcel Dekker, Inc.
www.dekker.com

918
PADIYA, MOHE, AND SALUNKHE
an undesirable side reaction.3 Thus the development of any alternative
strategy which would decrease the number of synthetic steps, without ham-
pering the yields and purity of the synthesis would itself justify the effort.
In our previous reports we have described the solution phase oligo-
nucleotide synthesis by using Alkyl H-phosphonates.4 During the synthesis
a general drawback was observed that there was about 1% weight reduction
of the total amount due to the repeated precipitation and filtration steps,
resulted in the low yield of the oligonucleotides. Therefore we have exploited
the application of the nucleoside 30-b-CE H-phosphonate 1, as versatile
starting material for the solid phase oligonucleotide synthesis via oxidative
coupling method. The nucleoside-b-CE H-phosphonates were prepared con-
veniently from corresponding phosphoramidites (Scheme 1).
Scheme 1.
Although the synthon 1 has been reported to be completely resistant to
activation in presence of strong condensing agents,5 it forms sufficiently
reactive intermediate in presence of activating agents such as iodine or
NBS and undergoes oxidative coupling with the free hydroxy group of
the support bound nucleoside (Scheme 2).
To set up a standard synthetic protocol for solid phase synthesis of
oligonucleotides using b-CE-H-phosphonates, the effect of different cou-
pling agents, solvent and bases on coupling efficiency were studied by
preparing a dimer d(TT), a coupling time of 2 min being given in each
case. The results are summarized in Table 1. From all these studies a stan-
dard synthetic protocol for solid phase synthesis of oligonucleotide, as
shown in Table 2, was set up. To study the effect of type of nucleoside
base on coupling efficiency all the four dimers d(AT), d(GT), d(CT) and
d(TT) were prepared using 0.4 M NBS in CH3CN/TEA (4 : 1, v/v) as cou-
pling agent, contact time with the support being 2 min. The results are
compiled in Table 3.

SOLID PHASE OLIGONUCLEOTIDE SYNTHESIS
919
Scheme 2.
Table 1. Effect of Type of Coupling Agent, Solvent, and Base Used
for the Solid Phase Synthesis of d(TT)
Coupling
System
% Coupling
Efficiency
Sl. No.
1
CCl4/Py (4 : 1 v/v)
CCl4/CH3CN/Py (1 : 4 : 1 v/v)
4/CH3CN/TEA (1 : 4 : 1 v/v)
I2, 0.4 M in CH2Cl2/Py (4 : 1 v/v)
I2, 0.4 M in CH3CN/Py (4 : 1 v/v)
I2, 0.4 M in CH3CN/Py (4 : 1 v/v)
NBS, 0.4 M in CH2Cl2/Py (4 : 1 v/v)
NBS, 0.4 M in CH3CN/Py (4 : 1 v/v)
NBS, 0.4 M in CH3CN/TEA (4 : 1 v/v)
68
70
70
78
82
89
91
96
98
2
3CCl
4
5
6
7
8
9
The UV, 1H-NMR and HPLC analysis of these product indicated that
the heterocyclic moiety and all protecting group (DMTr, –COR and
–CH2CH2CN) remains unaffected during the synthetic cycle. Additionally,
the RP HPLC profile of the product matches with that of the respective
authentic sample prepared by the solid phase phosphoramidite method.
The overall procedure for oligonucleotide assembly is represented by
the synthesis of d(AATTTTCAGAATTG), starting from dG-lcaa-CPG-
500A (0.5 mmol scale, loading 40 mmol/gm). The trityl assay confirmed an
average coupling efficiency of 98%. Finally conventional deprotection,
(conc. aq NH3, 55ꢀC, 16 h) cleavage and purification by using a NAP-10

920
PADIYA, MOHE, AND SALUNKHE
Table 2. Oligonucleotide Synthetic Cycle
Time in
Min
Sl. No.
Operation
Washing
Detritylation 3% Cl2CHCOOH-CH2Cl2
0.1 M b CE-H-phosphonate-
Reagent
1
2
CH3CN
0.5
1.8
2.0
3Coupling
CH3CN þ 0.4 M NBS-
CH3CN/TEA (4 : 1, v/v)
CH3CN
Ac2O/2,6-lutidine/THF
(1 : 1 : 2) þ 6.5% DMAP-THF
CH3CN
4
5
Washing
Capping
0.2
0.4
6
Washing
0.5
Table 3. Effect of the Nucleoside Bases on Coupling Efficiency
% Coupling
Efficiency
Sl. No.
Dimer
1
2
d(AT)
d(CT)
98.2
98.0
3d(GT)
4
98.5
d(TT)
98.6
column, afforded the oligo 92% homogeneous by HPLC. The overall yield
of the synthesis was 77%. The above synthesised oligonucleotide was char-
acterised by comparing the PAGE and RP HPLC retention time with that of
authentic sample having the same sequence synthesised by the phosphor-
amidite approach. The enzyme artifact (snake venom phosphodiesterase
and calf intestine alkaline phosphatase) showed no base modification or
wrong linkages, as the oligomer afforded dA, dC, dG and dT in the pre-
dicted amounts.
The results outline a method whereby nucleoside b-CE H-Phos-
phonate can be used as synthons for the solid phase synthesis of oligonucleo-
tides. The synthons are easily prepared via a readily scaleable procedure5 and
are stable towards normal laboratory conditions, a particularly useful feature
being the activation with NBS or I2 to directly yields the dinucleoside
phosphate as the only detectable product (31P NMR d À0.157 ppm).
Although the oxidative coupling of nucleoside synthons has been car-
ried out previously,6 the described approach is unique and adds to the

SOLID PHASE OLIGONUCLEOTIDE SYNTHESIS
921
arsenal for the preparation of oligonucleotides and oligonucleotide analogs.
In conclusion, the described oxidative coupling reaction in the presence of
NBS as the activating agent is fast, with good yield of coupling as well as
purity in the solid phase synthesis of oligonucleotide. Moreover, the method
does not require a separate oxidation step, hence less time consuming. The
use of an inexpensive activating agent like NBS as compared to the sublimed
1H-tetrazole used in phosphoramidite method is an additional advantage.
EXPERIMENTAL
All the 31P nuclear magnetic resonance spectra were recorded on
Bruker AMX 500 instrument. 31P NMR chemical shifts quoted are down-
field from 85% H3PO4. The HPLC analysis was carried out on RP C-18
column using Perkin Elmer, LC pump 250 system in combination with
Perkin Elmer UV/Vis LC290 spectrophotometric detector, the solvent
system being triethyl ammonium acetate (TEAA) (pH ¼ 7.0) and aceto-
nitrile gradient of 1% minÀ1, starting from 0%; flow rate 1 ml minÀ1. The
UV analysis was carried out on a Shimadzu 260 A spectrophotometer using
water as blank. Analytical TLC were conducted on precoated plates of
E-Merck kieselgel 60-F254 (0.2 mm).
Acetonitrile was distilled from CaH2 and was stored over activated 3ꢀA
molecular sieves, 4-(dimethylamino)pyridine (DMAP) and 1-H Tetrazole
were purchased from Aldrich. N-bromosuccinimide was purchased from
Spectrochem, India and was used after recrystallisation. All the nucleoside
phosphoramidites were purchased from Glen Research, U.S.A.
General Procedure for the Synthesis of 50-O-(p,p0-Dimethoxytrityl)-
20-deoxynucleoside 30-(2 Cyanoethyl) H-Phosphonate (1)
To a solution of 50-O-(p,p0-dimethoxytrityl)20-deoxynucleoside 30-(2
cyanoethyl) N,N 0-diisopropylphosphoramidite (1 mmol) in anhydrous
acetonitrile (10 ml) was added 0.30 ml of H2O and 0.10 gm of tetrazole.
The mixture was stirred at ambient temperature and the reaction was
followed by TLC [solvent system:-ethylacetate : petroleum ether (60/40 b.p)
(8 : 2, v/v)]. After completion of the reaction, the solution was concentrated
and the residue was partitioned between chloroform and water. The organic
phase was dried over anhydrous Na2SO4 and evaporated to yield 1. The
product obtained were dried with co-evaporation with anhydrous CH3CN
1
and were characterised by H and 31P-NMR spectra.

922
PADIYA, MOHE, AND SALUNKHE
ACKNOWLEDGMENTS
We would like to thank The Department of Atomic Energy, Mumbai
for financial assistance. Nikhil U. Mohe would like to thank The
Department of Science and Technology, New Delhi, for the financial assis-
tance. National facility of high field NMR, TIFR, Mumbai is gratefully
acknowledged.
REFERENCES
1. (a) Beaucage, S.L.; Iyer, R.P.; Caruthers, M.H. Tetrahedron 1992,
48, 2223; (b) Ramon, E.; Valeriy, S.; Caruthers, M.H. Tetrahedron
1990, 46, 721; (c) Dorman, M.A.; McBride, L.J.; Caruthers,
M.H. Tetrahedron 1984, 40, 95; (d) McBride, L.J.; Caruthers, M.H.
Tetrahedron Lett. 1984, 24, 245; (e) Beaucage, S.L.; Caruthers, M.H.
Tetrahedron Lett. 1981, 22, 1859.
2. (a) Andrus, A.; Efcavitch, J.W.; McBride, L.J.; Giusti. Tetrahedron
Lett. 1988, 29, 861; (b) Sakatsume, O.; Yamane, H.; Takaku, H.;
Yamamoto, N. Nucleic Acid Res. 1990, 18, 3327.
3. Eckstein, F. Oligonucleotide and Analogues: A Practical Approach.
Oxford University Press, 1991; pp. 19.
4. Padiya, K.J.; Salunkhe, M.M. Biorg. Med. Chem. 2000, 8, 3 3 7.
5. (a) Garegg, P.J.; Regberg, T.; Stawinski, J.; Stomberg, R. Chemica Scr.
1985, 25, 280; (b) Garegg, P.J.; Regberg, T.; Stawinski, J.; Stomberg, R.
Chemica Scr. 1986, 26, 59.
6. Michelson, A.M.; Todd, A.R. J. Chem. Soc. 1955, 2632.
Received in the Netherlands March 30, 2001