Microwave assisted high yielding preparation of N-protected 2′-deoxyribonucleosides useful for oligonucleotide synthesis

Article abstract of DOI:10.1081/NCN-120006833

A rapid and high yielding method for the synthesis of precursors of synthons for DNA synthesis, N-protected 2′-deoxyribonucleosides is described, which occur under mild conditions using microwave irradiation. The desired material, N-protected nucleosides, was obtained in 93-96% yield in few minutes. The final products were then characterized by H-NMR and MALDI-TOF and compared with the standard samples. The method is amenable to small to moderate scale of synthesis.

Full text of DOI:10.1081/NCN-120006833

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MICROWAVE ASSISTED HIGH YIELDING PREPARATION OF
N-PROTECTED 2′-DEOXYRIBONUCLEOSIDES USEFUL FOR
OLIGONUCLEOTIDE SYNTHESIS
N. S. Rao a , P. Kumar a , V. K. Chauhan a , B. S. Garg b & K. C. Gupta c
a Nucleic Acids Research Laboratory, Mall Road , Centre for Biochemical Technology ,
University Campus, Delhi, 110 007, India
b Department of Chemistry , University of Delhi , Delhi, 110 007, India
c Nucleic Acids Research Laboratory, Mall Road , Centre for Biochemical Technology ,
University Campus, Delhi, 110 007, India
Published online: 31 Aug 2006.
To cite this article: N. S. Rao , P. Kumar , V. K. Chauhan , B. S. Garg & K. C. Gupta (2002) MICROWAVE ASSISTED HIGH
YIELDING PREPARATION OF N-PROTECTED 2′-DEOXYRIBONUCLEOSIDES USEFUL FOR OLIGONUCLEOTIDE SYNTHESIS, Nucleosides,
Nucleotides and Nucleic Acids, 21:4-5, 393-400, DOI: 10.1081/NCN-120006833
To link to this article: http://dx.doi.org/10.1081/NCN-120006833
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 2164&5), 393±400 62002)
MICROWAVE ASSISTED HIGH YIELDING
PREPARATION OF N-PROTECTED
20-DEOXYRIBONUCLEOSIDES USEFUL FOR
OLIGONUCLEOTIDE SYNTHESIS
N. S. Rao,1 P. Kumar,1 V. K. Chauhan,1 B. S. Garg,2
and K. C. Gupta1,*
1Nucleic Acids Research Laboratory, Centre for Biochemical
Technology, Mall Road, University Campus, Delhi±110 007, India
2Department of Chemistry, University of Delhi,
Delhi±110 007, India
ABSTRACT
A rapid and high yielding method for the synthesis of precursors of
synthons for DNA synthesis, N-protected 20-deoxyribonucleosides is
described, which occur under mild conditions using microwave irradia-
tion. The desired material, N-protected nucleosides, was obtained in 93±
96% yield in few minutes. The ®nal products were then characterized by
1H-NMR and MALDI-TOF and compared with the standard samples.
The method is amenable to small to moderate scale of synthesis.
INTRODUCTION
Synthetic oligonucleotides are ®nding numerous applications in the ®eld
of molecular biology, genetics and other related areas. These are required in
minute 6micrograms) to large quantities depending upon the type of appli-
cations. In particular, the potential of synthetic oligonucleotides as future
*Corresponding author. Fax: ‡91 11 7667471; E-mail: kcgupta@cbt.res.in
393
Copyright # 2002 by Marcel Dekker, Inc.
www.dekker.com

394
RAO ET AL.
drugs has thrown new challenges before the organic chemists to develop
rapid and economical processes for the synthesis of these molecules.
N-Protected nucleosides are one of the key intermediates in the preparation
of synthons required for oligonucleotide synthesis. Basically, two approaches
are being followed for the synthesis of N-protected nucleosides. The ®rst one,
developed by Khorana et al.,[1,2] is based on peracylation of nucleosides with
an appropriate acylating reagent followed by selective hydrolysis of ester
functions to obtain the desired N-acylated nucleosides in 660±70%) yields. In
the other protocol, Jones et al.[3] have proposed a new approach based on
`transient protection' for the synthesis of these molecules. The method
involves the use of trimethylsilyl chloride for transient protection of hydroxyl
groups of nucleosides followed by in situ protection of exocyclic amino
functionalities with an acylating reagent. The reaction proceeds in one pot
and yields are generally high. However, large-scale synthesis following this
protocol is not economical, as it involves the use of large excess of the
reagents and yields are not as high as one expects at small scale. In another
modi®ed method, McGee et al.[4] have reported one pot, high yielding pro-
cess for the synthesis of N-isobutyryl-20-deoxyguanosine based on the tran-
sient protection approach. All of these methods[1±4] are either quite time
consuming or requiring a large excess of the reagents, making them eco-
nomically unviable at large scale synthesis.
Recently, a number of researchers have reported organic reactions
under microwaves.[5±14] The yields obtained are generally high in relatively
shorter times. The technique has been used both in the presence of solvent as
well as in dry conditions using di€erent types of supporting materials, viz.,
silica gel, alumina, montmorillonite etc. These reactions are carried out in
polar solvents having high dielectric constant as well as boiling points, such
as, dimethylformamide 6DMF), dimethylacetamide 6DMA), acetic acid
6AcOH), N-methylpyrrolidone 6NMP).
In this communication, we describe a rapid and economical protocol,
which utilizes the fast reaction kinetics under microwave irradiation to obtain
N-protected nucleosides in shorter times as well as in high yields.[15] Both the
steps, i.e. peracylation and saponi®cation, were carried out under micro-
waves. Selective protection of 20-deoxycytidine was also achieved in higher
yield.
RESULTS AND DISCUSSION
The demand of synthetic oligonucleotides is growing at exponential rate
for various biological applications where these are required in large numbers
as well as in large quantities. In order to meet this growing demand of
synthetic oligonucleotides, it is necessary that suitable methods be developed
to synthesize N-protected-20-deoxynucleosides, precursors of building blocks

MICROWAVE ASSISTED SYNTHESIS
395
Scheme 1. Scheme for the preparation of N-protected-20-deoxyribonucleosides.
used for oligonucleotide synthesis, rapidly and economically. In the proposed
study, the following points were kept in mind, 6a) the process should be
straightforward and high yielding, 6b) it should not involve specialized che-
micals and reagents as well as equipments, and 6c) the synthesized products
must match the corresponding standard N-protected-20-deoxynucleosides.
Keeping these factors into consideration, a rapid and high yielding
process has been developed for the preparation of N-protected-20-deoxy-
ribonucleosides under microwaves. The method involves a two stepprocess.
The ®rst one involves N- and O-acylation of 20-deoxyribonucleosides
6Scheme 1) followed by the second one, i.e. saponi®cation, to yield the
desired N-protected-20-deoxyribonucleosides.
20-Deoxycytidine was N- and O-acylated with benzoic anhydride 65 eq.)
in dimethylformamide in the presence of an acylating hypernucleophilic
catalyst, DMAP, under microwaves. The progress of the reaction was
monitored on tlc and no exposure was given longer than 8s 6525 W). After
every exposure, the contents were allowed to cool to room temperature to
avoid overheating and evaporation of the solvent. Reaction proceeded
almost quantitatively. After usual work-up, the desired N- and O-protected-
20-deoxycytidine was isolated and compared with the standard sample.
The other two deoxyribonucleosides were also peracylated under iden-
tical conditions except that benzoic anhydride 67.5 eq.) in case of deo-
xyadenosine and isobutyric anhydride 610 eq.) in case of deoxyguanosine
were employed. The fully protected compounds were compared with respect
to their Rfs on tlc with standard samples.
The next step involves the selective saponi®cation of peracylated
nucleosides. The optimum concentration of the alkali for saponi®cation was
determined following kinetic studies under microwaves. The above men-
tioned stepwas found to be complete in few minutes in a homogeneous
mixture of pyridine:methanol:water 61:1:1, v/v). The proposed method was
studied at three di€erent scales, viz., 0.5, 10 and 25 mmol. The irradiation
time required under microwaves in each case was found to be di€erent for
selective saponi®cation. After complete saponi®cation, as monitored on tlc,
the reaction mixture was neutralized followed by evaporation of the solvent.

396
RAO ET AL.
1
Table 1. H-NMR and Mass Spectra of N-Protected-20-deoxyribonucleosides
MALDI-TOF
6m=z); matrix: 2,5-
dihydroxybenzoic acid
N-Protected-20-
Deoxyribonucleosides
1H-NMR 6DMSO-d6) d
6ppm)
N-Benzoyl-20-deoxycytidine
3.7 6m, 3H), 3.9 6m, 1H), 4.3
6m, 1H), 6.1 6m, 1H), 7.3±
8.0 6m, 5H), 8.1 6s, 1H), 8.4
6d, 1H)
332 6M‡H)
6Expected 331)
N-Benzoyl-20-
deoxyadenosine
1.25 6s, 1H), 2.5 6s, 1H), 3.6
6m, 2H), 4.1 6s, 1H), 4.3 6d,
1H), 6.5 6m, 2H), 7.3±7.9
6m, 5H), 8.1 6d, 1H)
356.6 6M‡H)
6Expected 355)
N-Isobutyryl-20-
deoxyguanosine
1.2 6d, 6H), 2.3 6m, 1H), 2.6
6m, 1H), 2.8 6m, 1H), 3.7
6m, 2H), 6.3 6t, 1H), 8.1
6s, 1H)
338.5 6M‡2H)
6Expected 336)
The residual mass was triturated with diethylether to get rid of acidic
impurities 6benzoic acid released from peracylated deoxyadenosine and
deoxycytidine and isobutyric acid from deoxyguanosine). Inorganic impu-
rities including the remaining sodium benzoate, isobutyrate or acetate pre-
sent in the product was taken care of by a silica gel column
chromatography. The eluted fractions containing desired products were
pooled together and concentrated on a rotary evaporator to obtain the
desired N-protected-20-deoxyribonucleosides in high yields. These com-
1
pounds were characterized by H-NMR and MALDI-TOF 6Table 1) and
compared with the corresponding standard sample of N-protected-20-deox-
yribonucleosides. Figure 16a,b,c) shows mass spectra of N-protected-20-
deoxyribonucleosides.
Taking advantage of the better nucleophilicity of exocyclic amino group
of cytidine, a simple and rapid method has also been developed for its
selective protection under microwaves. An overall yield of 84% of N-ben-
zoyl-20-deoxycytidine was obtained in just 125s.
EXPERIMENTAL PART
General: All solvents and reagents used in the present investigation were
puri®ed prior to their use. Benzoic anhydride, isobutyric anhydride, diiso-
proylethylamine and 4-dimethylaminopyridine were obtained from Sigma
Chemical Company, USA. 20-Deoxyribonucleosides 6dA, dC, dG) were
purchased from Yuki Gosei Kogyo Co., Japan. Other solvents and reagents
were procured from local suppliers.

MICROWAVE ASSISTED SYNTHESIS
397
Figure 1. MALDI-TOF spectra of 6a) N-benzoyl-dA, 6b) N-benzoyl-dC and 6c) N-isobutyryl-
dG. Matrix used: 2,5-dihydroxybenzoic acid; Laser power : 104; Mode : Linear; positive ion.
The domestic microwave oven 6BPL, India, 800 W) was used in the
present study. The oven was set at 525 W 62450 MHz for each reaction) and
the reaction mixture was irradiated for a particular duration, followed by
cooling down the reaction mixture to room temperature and re-irradiated
until the reaction was found to be complete on tlc. The frequency and
duration of exposures are mentioned in the parenthesis against each reaction.
N-Protected-20-deoxyribonucleosides prepared in the present investiga-
1
tion were characterized by H-NMR 6300 MHz, Bruker-Avance DPX 300)
and MALDI-TOF 6SEQ IV, Kratos, UK, Nitrogen laser operating at
337 nm). Standard samples of N-protected 20-deoxyribonucleosides were
prepared following either of the reported procedures [1±4].
Synthesis of N-Benzoyl-30, 50-Di-O-benzoyl-20-deoxycytidine
20-Deoxycytidine 62.27 g, 10 mmol) was taken in a conical ¯ask
and suspended in 25 mL of anhydrous N,N-dimethylformamide 6DMF).
Diisopropylethylamine 68.71 mL, 50 mmol) and 4-dimethylaminopyridine
6DMAP) 6610 mg, 5 mmol) were added to the reaction ¯ask followed by the
addition of an acylating reagent, benzoic anhydride 611.3 g, 50 mmol). The
reaction mixture was irradiated for 64s 6868s). The reaction was monitored
on tlc after each exposure till completion. The solvent was removed on a
rotary evaporator under vacuum to obtain a syrupy material, which was
taken upin chloroform 6100 mL) and subjected to washings with cold 5% aq.
sodium bicarbonate 64650 mL) and water 62650 mL), respectively. The
organic phase was separated, dried over anhydrous sodium sulfate and
concentrated to get the desired N-benzoyl-30,50-di-O-benzoyl-20-deoxy-

398
RAO ET AL.
cytidine in almost quantitative yield 65.28 g, 98%). The sample was com-
pared with the authentic sample with respect to Rf value on tlc.
20-Deoxyguanosine 610 mmol) and 20-deoxyadenosine 610 mmol) were
also derivatized in similar manner except the following changes. In case of 20-
deoxyguanosine, the acylating reagent was isobutyric anhydride 616 mL,
100 mmol) with irradiation time of 120s 61568s), while 20-deoxyadenosine
required benzoic anhydride 616 g, 75 mmol) and irradiation time of 96s
61268s). The fully protected nucleosides were obtained in 97% and 98%,
respectively. The triacylated deoxyribonucleosides were compared with
standard samples.
General Procedure for Synthesis of N-Protected-20-deoxynucleosides
The peracylated material, N-benzoyl-30,50-di-O-benzoyl-20-deoxy-
cytidine, 65.39 g, 10 mmol), was dissolved in a mixture of pyridine 6100 mL)
and methanol 6100 mL) and 0.3 N sodium hydroxide 6100 mL, 30 mmol) in
a conical ¯ask and subjected to irradiation under microwaves. The progress
of the reaction was monitored on tlc. The reaction was found to be complete
in 36s 6666s). The reaction mixture was then neutralized by adding glacial
acetic acid 61.8 mL) and the resulting solution concentrated to a syrupy mass,
which was triturated with diethyl ether 64650 mL) to get rid of benzoic acid.
The residual mass, containing the desired material and the inorganic salts,
such as sodium acetate and sodium benzoate, was dissolved in methanol
6100 mL) and adsorbed slowly on to a silica gel 6100 g) column packed in
methanol. The column was eluted slowly with methanol and the fractions
containing the desired product were pooled together. The title compound was
obtained by concentrating the methanol solution in 96% yield 63.17 g). The
1
title compound was characterized by H-NMR and MALDI-TOF 6matrix
assisted laser desorption ionization-time of ¯ight).
Similar procedure was used for 20-deoxyadenosine and 20-deoxy-
guanosine derivatives 610 mmol of each) except the following changes. The
irradiation time required for the completion of hydrolysis of 30,50-diester
functions of 20-deoxyadenosine and 20-deoxyguanosine were 24s 6466s) and
30s 6566s), and the yields were found to be 95.8% and 93.4%, respectively
after passing through silica gel column. These were also characterized by 1H-
NMR and MALDI-TOF.
Large Scale Preparation of N-Protected-20-deoxynucleosides 825 mmol)
The selective hydrolysis of 30,50-diester functions in N-acyl-30,50-di-O-
acyl-20-deoxynucleosides at 25 mmol was achieved using the following
conditions. In case of N-isobutyryl-30,50-di-O-isobutryl-20-deoxyguanosine,

MICROWAVE ASSISTED SYNTHESIS
399
0.05M sodium hydroxide was used with irradiation time of 54s 6669s),
N-benzoyl-30,50-di-O-benzoyl-20-deoxycytidine required 0.1 M sodium
hydroxide concentration with irradiation time of 36s 6469s) and 0.05 M
sodium hydroxide was employed for N-benzoyl-30,50-di-O-benzoyl-20-deoxy-
adenosine with irradiation time of 27s 6369s). The yields of the N-protected
nucleosides were, however, the same as obtained at 10 mmol scale.
Small Scale Preparation of N-Protected-20-deoxynucleosides 80.5 mmol)
Selective hydrolysis of 30,50-ester functions in N-acyl-30,50-di-O-acyl-20-
deoxynucleosides at 0.5 mmol was achieved using the following conditions.
In case of N-isobutyryl-30,50-di-O-isobutryl-20-deoxyguanosine, 0.3M sodium
hydroxide was used with irradiation time of 30s 6566s), N-benzoyl-30,50-di-
O-benzoyl-20-deoxycytidine required 0.2 M sodium hydroxide concentration
with irradiation time of 12s 6266s) and 0.3 M sodium hydroxide was
employed for 36s 6666s) for N-benzoyl-30,50-di-O-benzoyl-20-deoxyadeno-
sine. The yields of the N-benzoyl-2-deoxyadenosine 6dAbz), N-isobutyryl-20-
deoxyguanosine 6dGibu) and N-benzoyl-20-deoxycytidine 6dCbz) nucleosides
were obtained in 98%, 98.5% and 98%, respectively.
Selective N-Benzoylation of 20-Deoxycytidine
20-Deoxycytidine 62.27 g, 10 mmol) taken in pyridine 640 mL) in a
conical ¯ask was charged with diisopropylethylamine 63.48 mL, 20 mmol)
and benzoic anhydride 65.65 g, 25 mmol). The reaction mixture was sub-
jected to microwave irradiation 6800 W). The progress of the reaction was
monitored on tlc plate after each exposure. The reaction was found to be
complete in 125s 65625s). The mixture was then concentrated on a rotary
evaporator to obtain a syrupy mass, which was taken up in water 6100 mL)
and extracted with dichloromethane 63650 mL). The organic phase was
separated and discarded. The aqueous phase was concentrated to obtain the
title compound in 84% yield.
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