Scheme 1. Synthesis of Phosphoramidite 11
pyridine,9 affording methyl S-(4,4-dimethoxytrityl)-2-
mercaptoacetate. The crude ester was then treated with
LiAlH4 in THF to give 10 in 90% yield.10 Condensation of
10 with i-Pr2NPCl2 and i-Pr2NEt in anhydrous MeCN
proceeded smoothly, as indicated by 31P NMR analysis of
the reaction mixture. Complete conversion of i-Pr2NPCl2 (δP
170 ppm) to the phosphoramidite 11 (δP 148 ppm) occurred
within 2 h at 25 °C. Purification of the reaction product was
accomplished by silica gel chromatography, affording 11 in
an isolated yield of 82%. The parameters for optimal
coupling efficiency of 11 were first investigated by perform-
ing manual syntheses of 5′-phosphate/thiophosphate mo-
noester derivatives of commercial deoxyribonucleosides
covalently attached to controlled-pore glass (CPG) through
a 3′-O-succinyl linker (12a-d, Scheme 2). Typically, a 0.1
M solution of activated 11 in MeCN was mixed with 5′-O-
detritylated 12a-d for 3 min. A treatment with 0.1 M ethyl-
(methyl)dioxirane11,12 in CH2Cl2 for 1 min or 0.05 M 3H-
1,2-benzodithiol-3-one-1,1-dioxide13 in MeCN for 2 min was
performed and resulted in the formation of 13a-d or 14a-d
in yields exceeding 95%. Exposure of 13a-d or 14a-d to
3% TCA in CH2Cl2 for 9 min and then to a solution of 1.2%
(w/v) DTT and 5% (v/v) i-Pr2NEt in H2O for 1 h at ambient
temperature produced 15a-d or 16a-d. Subsequent reaction
with MeNH2 gas (∼2.5 bar) for 30 min or concd NH4OH
for 10 h at 55 °C cleaved the nucleobase protecting groups
and released 17a-d or 18a-d from the solid support. When
12a-d is replaced with 19a-d under identical conditions,
the corresponding deoxyribonucleoside 3′-phosphate/thio-
phosphate monoesters 20a-d or 21a-d are produced in
yields comparable ((3%) to those obtained when employing
6 or 8 as the phosphorylating reagent. The phosphate
monoesters 17a-d and 20a-d were analyzed by RP-HPLC
(data shown in the Supporting Information) and exhibited
chromatographic profiles identical to those of authentic
deoxyribonucleoside 5′-monophosphates or 3′-monophos-
phates obtained from commercial sources. To further assess
the scope and limitations of 11 as a phosphorylating reagent,
the preparation of oligonucleotide 5′-phosphate/thiophosphate
monoesters was undertaken. Specifically, the automated
Figure 1. Reagents for the phosphorylation of nucleosides and
oligonucleotides.
namely, 4,3 6,4 and 7,5 require elevated temperature condi-
tions (concd NH4OH, 55-60 °C) that are incompatible with
the preparation of oligonucleotides functionalized with
thermosensitive phosphotriester groups. Furthermore, the
coupling efficiency of 4, 5,6 and 7 cannot be easily monitored
because each reagent is devoid of any reporter group.
Whereas reagent 87 also requires an elevated temperature
to produce phosphorothioate monoester 26 within a reason-
able period of time, the H-phosphonate reagent 98 is
incompatible with automated phosphoramidite chemistry for
solid-phase oligonucleotide synthesis. Given the limitations
of reagents 4-9 in the context of our studies, we decided to
develop a phosphorylating reagent that would be: (i)
compatible with automated phosphoramidite chemistry; (ii)
functionalized with a reporter group to permit accurate
evaluation of its coupling efficiency; and (iii) capable of
generating thiophosphate monoester derivatives of oligo-
nucleotides, such as in 3, under mild temperature conditions
(∼23 °C) to prevent premature thermolytic cleavage of these
thiophosphate protecting groups.
The phosphorylating agent 11 was designed to fulfill all
of the above requirements and was prepared in three steps
from methyl 2-mercaptoacetate (Scheme 1). Specifically,
methyl 2-mercaptoacetate was first functionalized with the
DMTr reporter group upon reaction with DMTrCl in
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