Polymer supported carbodiimide strategy for the synthesis of N-acylated derivatives of deoxy- and ribo purinenucleosides using active esters

Article abstract of DOI:10.1016/j.bmcl.2005.07.078

A cost-effective synthetic strategy has been used for the selective protection of the exocyclic amino function of purine nucleosides. Instead of using the common protecting groups in their chloride or anhydride forms, the less expensive and nontoxic acidic form was chosen. The acids were first activated to an active ester form using DCC and further successfully used for N-acylation of purine nucleosides. The contamination of the N-acylated product with DCU was inconvenient and was avoided by use of polymer supported- carbodiimide that has the additional advantage of reusability.

Full text of DOI:10.1016/j.bmcl.2005.07.078

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5045–5048
Polymer supported carbodiimide strategy for the synthesis of
N-acylated derivatives of deoxy- and ribo purinenucleosides
using active esters
a,
a
Snehlata Tripathi, Krishna Misra and Yogesh S. Sanghvi
b
*
a
Nucleic Acids Research Laboratory, Department of Chemistry, University of Allahabad, Allahabad 211002, India
b
Rasayan Incorporation, 2802 Crystal Ridge Road, Encinitas, CA 92024-6615, USA
Received 1 July 2005; revised 29 July 2005; accepted 29 July 2005
Available online 8 September 2005
Abstract—A cost-effective synthetic strategy has been used for the selective protection of the exocyclic amino function of purine
nucleosides. Instead of using the common protecting groups in their chloride or anhydride forms, the less expensive and nontoxic
acidic form was chosen. The acids were first activated to an active ester form using DCC and further successfully used for N-acyl-
ation of purine nucleosides. The contamination of the N-acylated product with DCU was inconvenient and was avoided by use of
polymer supported-carbodiimide that has the additional advantage of reusability.
ꢀ
2005 Elsevier Ltd. All rights reserved.
8
During the past few years, oligodeoxyribonucleotides
ODNs) have become the focus of active research due
to recognition of antisense compounds as potential ther-
the N-acylated nucleosides. Jones procedure involves
(
prior silylation of hydroxyl functions for transient pro-
tection, followed by selective acylation of the amino
function. Strategies developed to selectively protect the
amino function have led to the discovery of several com-
1
–4
apeutic agents.
One such compound, VitraveneTM,
has been approved by the FDA for the treatment of
CMV-retinitis. Several other ODNs have demonstrated
9
–17
petent groups
from our laboratory.
with a number of groups reported
18–23
5
pre-clinical efficacy and a number of antisense oligonu-
cleotide drugs are undergoing human clinical trials for
6
the treatment of viral infections, cancer, AIDS, and a
range of inflammatory disorders.
Conventionally, all reagents that have been reported so
far for the protection of exocyclic function of nucleo-
sides are either in chloride or anhydride form. In the
present communication, installation of the protecting
group has been carried out by an entirely different meth-
od. Instead of using acyl halides, the acids have been
activated to their corresponding esters using p-nitrophe-
nol. The in situ activated esters were used as protecting
reagents and coupled with the exocyclic amino function
to generate the –CO–NH– bond in the presence of DCC,
a well-established strategy in peptide synthesis. While
using DCC as a coupling agent, the contamination of
the product with dicyclohexylurea (DCU) is very com-
mon. Water-soluble EDCI was reported as an alterna-
tive reagent, which can be subsequently eliminated by
an aqueous extraction. Also, more soluble forms of
One of the most important steps in the chemical synthe-
sis of ODNs is selective protection and deprotection of
different nucleophilic sites within the monomeric nucle-
oside building blocks, including protection of exocyclic
amino groups of the nucleoside bases. The classical pro-
7
cedure developed by Khorana and co-workers for both
deoxy- and ribonucleosides involves peracylation of the
nucleosides via acylation of both the hydroxyl groups of
sugar and the exocyclic amino group simultaneously,
followed by selective hydrolysis of the esters, leaving
Keywords: Oligodeoxyribonucleotides; Acylation; DCC; PS-cyclo-
hexylcarbodiimide.
24
DCC have been reported, removable with DCM. To
overcome the contamination problem, polymer support-
ed-cyclohexylcarbodiimide (PSCC) is successfully used
in the present work, which can be easily separated from
the reaction mixture through filtration. The advantage
*
Corresponding author at present address: Center for Emerging and
Re-emerging Pathogens, Department of Biochemistry and Molecular
Biology, A-920H, MSB, NJMS-UMDNJ, 185 South Orange Ave-
nue, Newark-07103, NJ, USA. Tel.: +1 973 97 25 32 2; fax: +1 973 97
2
5
28 657; e-mail: snehlatatripathi@yahoo.com
0
960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.078

5046
S. Tripathi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5045–5048
2
9
of using PSCC is that the urea byproduct can be readily
2
converted back to the active carbodiimide and reused.
cancers, the anticipated demand for modified ODNs
may be on the metric ton scale in the near future and
we envisaged the present method to effectively facilitate
cost reduction in the synthesis of ODNs.
6
6
0
6
N -Bz-2 -dA, N -phenoxyacetyl-2 -dA, N -i-Bu-2 -dG,
0
2
0
6
N -Bz-A, N -phenoxyacetyl-A, and N -i-Bz-G have
6
2
been prepared using this approach.
The PSCC was purchased from Argonaut Technologies,
San Carlos, CA. H NMR spectra were recorded on
1
The strategy presented here eliminates the need for using
conventional two-step procedures. The N-acylation
achieved is found to be absolutely selective and most
importantly, acylation of the sugar hydroxyl group is
not observed. The advantages envisaged in this process
are significant. One of these happens to be the crystalline
nature of the product, that is, N-acylated nucleosides,
which is a deciding factor while streamlining the purifi-
cation process, as well as improving yields of the
N-acylated products. However, contamination of the
Bruker DRX 300. Small amounts of all the prepared
N-acylated derivatives were hydrolyzed to get back the
starting nucleosides for confirmation of their structures.
Percentage purification of the products was checked on
HPLC (Pharmacia LKB-DBF) using reverse-phase col-
umns. N-Acylated nucleosides were purified using a
Whatman RP-C18 column using 20 mM ammonium ace-
tate buffer (pH 3.5) as buffer A and 20 mM ammonium
acetate buffer (pH 3.5)/methanol (90:10 v/v) as buffer B
at a flow rate of 0.75 mL/min, with detection at 260 nm
and column temperature set at 30 ꢁC. Buffer B (30%)
was passed for 5 min and was increased as a linear gra-
dient for 10 min. Buffer B (100%) was run for another
40 min. The mass characterization was done on a Jeol-
SX 102/DA-6000 spectrometer using argon as the FAB
gas (6 kV, 10 mA).
2
7
product with DCU is the outcome undesired of this
process. To decrease the DCU content, many molar ra-
tios have been attempted both at the active ester forma-
tion step as well as the nucleoside protection step
(
Table 1). While attempting benzoylation of dA/A, five
molar ratios have been tried, 1:1:1, 1:1:1.25, 1:1:1.5,
:1:1.75, and 1:1:2 (acid:nucleoside:DCC), respectively.
1
While using a 1:1:1 ratio, contamination with DCU
was found to be minimal with the yield also being low
General method for N-acylation of nucleosides using DCC
(Scheme 1). Recrystallized and dried benzoic acid/phe-
noxyacetic acid (1 mmol) was dissolved in anhyd 1,4-di-
oxane (5 mL) under an atmosphere of nitrogen. A
solution of p-nitrophenol (1.2 mmol) in anhyd 1,4-diox-
ane (5 mL) was added to it. Protection from moisture
was essential. The reaction mixture was made basic by
addition of pyridine (0.5 mL) and TEA (0.5 mL). After
10 min of stirring, DCC (1.25 mmol) was added to it
[DCC was melted in a sealed tube after weighing (mp
(
50%). A ratio of 1:1.25 was found to be optimal, result-
ing in 80% yield with a low level of contamination
Table 1). This strategy has been successfully applied
(
to other protecting groups, such as phenoxyacetyl and
isobutyryl (data not shown), and the products were
found to be in the same ratio. Using this strategy, N-
0
0
protected derivatives of 2 -dG and 2 -dA using three
new protecting groups have been synthesized and are
reported elsewhere.
2
8
3
5 ꢁC) and then mixed to the r.m.]. The reacting mixture
In the quest for an alternative to DCC, polymer-bound
was allowed to stir at room temperature for 2 h. Com-
pletion of the reaction was assessed by the absence of
the starting material on TLC. The reaction mixture
was cooled to 0 ꢁC and anhyd 2 -dA/A (1 mmol) sus-
pended in 5 mL of pyridine was added to the activated
ester (1) with the help of a syringe gradually over a
2
5
carbodiimide was used successfully, yielding a very
promising result with an enhanced yield, as well as being
a simplified purification process. Since PS-carbodiimide
is easy to separate from the reaction mixture, a facile
method has been successfully developed. The used poly-
mer-supported reagent can further be recycled (PSCC–
PSCU–PSCC), as previously mentioned. PSCC makes
this strategy further versatile, as well as cost-effective.
The regenerated PSCC was found to be stable at room
temperature and useful for another round of N-acyla-
tion. We have used regenerated PSCC four consecutive
times and the yield of the protected derivatives was
found to be comparable, further confirming the reusabil-
ity of PSCC. With the potential for antisense technology
in the treatment of inflammation, viral diseases, and
0
DCC/PSCC
RCOOH+HO
NO2
R+ COO
NO2
Dioxane,Py.,TEA
1
NH2
N
NHCOR
N
N
N
N
N
N
N
HO
O
HO
1
+Py.,0˚C,2h
O
OH R’
HO R’
R = -Bz,-phenoxyacetyl
2
R’ = H/OH
O
O
N
N
N
NH
N NHCOR
NH
NH2
6
Table 1. Synthesis of N -Benzoyl-dA using different molar ratios of
N
N
O
HO
HO
acid, nucleoside, and DCC
O
1+Py.,0˚C,2h
Molar ratio acid: Yield FAB-MS of benzoic FAB-MS of
(%)
OH R’
OH R’
3
6
N -benzoyl-dA
nucleoside:DCC
p-nitrophenyl ester
R=-iBu
R’=H/OH
1
1
1
1
1
:1:1
50
80
82
86
94
242
242
351
350
350
351
348
:1:1.25
:1:1.5
:1:1.75
:1:2
242
241.8
242
PSCC = PS
Scheme 1.
O
N=C=N

S. Tripathi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5045–5048
5047
period of 15 min. After 10 min, a second batch of DCC
1.25 mmol) was added again and the r.m. was allowed
to stir for a further 2 h. The reaction mixture was filtered
under vacuum to remove the precipitated DCU and the
filtrate was evaporated to a gum under vacuum. It was
by reverse filtration and replaced by 10% aqueous
NaOH (5 mL) and fresh dichloromethane (20 mL).
The suspension was shaken further for 1 h and the aque-
ous supernatant was removed. The polymer was washed
with THF (2· 25 mL) to remove excess water and dried
under vacuum to furnish PSCC.
(
then dissolved in 5% aqueous NaHCO solution and
3
extracted with DCM (4· 5 mL). The organic layer was
washed with water to remove released p-nitrophenol un-
til the water became colorless, dried over Na SO , and
Acknowledgments
2
4
filtered and evaporated to dryness. The residue was
extracted in diethyl ether and crystallized further to fur-
nish the product (2). The above-described method was
repeated by substituting isobutyric acid in place of ben-
Thanks are due to RSIC, CDRI, Lucknow, India, for
IR, mass, and NMR spectra. Authors are thankful to
Dylan Harris for critical reading of the manuscript.
Financial support from ISIS Pharmaceuticals, CA, is
gratefully acknowledged.
0
zoic/phenoxyacetic acid with 2 -dG/G to get the N-acyl-
ated derivatives (3). The H NMR data were found to be
1
8
comparable to the earlier reported literature. The FAB-
MS for N -phenoxyacetyl-dA/A and N -isobutyryl-dG/
6
2
G were found to be comparable to the authentic samples
(
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