A new procedure for the phosphorylation of nucleosides: Application to the discovery of inhibitors of HIV integrase

Article abstract of DOI:10.1081/NCN-100002419

A new phosphorylating agent for nucleosides, 2-O-(4,4′-dimethoxytrityl) ethylsulfonylethan-2′-yl-phosphate (1), has been developed by us. In the many examples studied by us, phosphorylation yields were found to be very high (about 90%). The procedure appe

Full text of DOI:10.1081/NCN-100002419

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 739–742 (2001)
A NEW PROCEDURE FOR THE
PHOSPHORYLATION OF NUCLEOSIDES:
APPLICATION TO THE DISCOVERY OF
INHIBITORS OF HIV INTEGRASE
Vasu Nair∗ and Michael Taktakishvili
Department of Chemistry, The University of Iowa,
Iowa City, Iowa 52242
ABSTRACT
A new phosphorylating agent for nucleosides, 2-O-(4,4ꢀ-dimethoxytrityl)
ethylsulfonylethan-2ꢀ-yl-phosphate (1), has been developed by us. In the many
examples studied by us, phosphorylation yields were found to be very high
(about 90%). The procedure appears to be remarkably general and can be uti-
lized for the phosphorylation of many biomolecules. Successful application of
this phosphorylation method has contributed to the discovery of inhibitors of
HIV integrase in our laboratory.
INTRODUCTION
Phosphorylation reactions are ubiquitous in nature. Non-natural phosphory-
lated nucleosides are of interest for biological studies involving various nucleotide
metabolizing enzymes and as potential therapeutic agents (1–4). Chemical method-
ologies for solution-phase phosphorylations are of critical importance in the prepa-
ration of these compounds. Several requirements need to be fulfilled for efficient
phosphorylation of nucleosides. The hydroxyl functions of the phosphate group
being introduced need to be protected (4). These phosphate protecting groups must
possess stability with respect to reaction conditions of subsequent synthetic steps
but must also have the property of selective lability at the termination of synthesis.
∗Corresponding author.
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Copyright 2001 by Marcel Dekker, Inc.
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Scheme 1.
Current methods of chemical phosphorylation have disadvantages that range from
lengthy procedures, instability of intermediates, difficulty of purification, problems
associated with removal of phosphate protecting groups, issues of monitoring of
reaction, and, in many cases low yields in the phosphorylation and deprotection
steps (5–18).
We describe herein a new method for phosphorylation of nucleosides and
nucleotides which utilizes more effective phosphodiester instead of phosphotriester
chemistry and surmounts the problems associated with stability, lability, purifica-
tion and yields in phosphorylation reactions discussed above. The new phosphory-
lating reagent is 2ꢀ-O-(4,4ꢀ-dimethoxytrityl)ethylsulfonylethan-2-yl-phosphate (1).
It can be synthesized easily from sulfonyldiethanol by selective monotritylation
followed by phosphorylation with POCl3/1,2,4-triazole/triethylamine and work up
with NaHCO3 (Scheme 1) (19). When 1 is activated by coupling reagents such as
TPS-TAZ or TPS-NT, it phosphorylates primary and secondary alcohol groups in
nucleosides with remarkable efficiency (of the order of 90%). Deprotection of the
phosphodiester intermediate formed after the phosphorylation step is carried out
in only one step, but that step constitutes the normal work-up of the reaction. The
precursor nucleoside does require protection prior to the phosphorylation step. An
example is the 3ꢀ-phosphorylation of protected deoxyadenosine which proceeds in
90% overall yield under the conditions shown in Scheme 2.
We have examined many examples of 3ꢀ- as well as 5ꢀ-phosphorylation of
nucleosides to establish the generality of this procedure (20). The results are com-
pletely reproducible. The phosphorylation methodology can be utilized in the syn-
thesis of more complex and sensitive molecules, including those that are potent
inhibitors of HIV integrase. An example of the utilization of this phosphorylation
Scheme 2.

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Scheme 3.
method in the synthesis of one HIV integrase inhibitor is illustrated in Scheme 3.
The yield of phosphorylated dinucleotide (HPLC purified) was 79%.
ACKNOWLEDGMENT
We thank the NIH (NIAID) for support of our research.
REFERENCES
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4.20–4.10 (m, 2H, DMTrOCH2), 3.65–3.45 (m, 2H, CH2OP), 3.20–3.15 (m, 2H,
DMTrOCH2CH2), 3.10–3.05 (m, 2H, CH2CH2OP). 31P (CDCl3): δ − 4.90 (br,s).
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