Synthesis of α-P-modified nucleoside diphosphates with ethylenediamine

Article abstract of DOI:10.1021/ja055179y

This report describes a one-pot synthesis of α-P-borano-, α-P-thio-, and α-P-seleno-modified nucleoside diphosphate analogues that are otherwise difficult to obtain. The key step involves the intramolecular nucleophilic attack by an amino group in 5 to re

Full text of DOI:10.1021/ja055179y

Published on Web 11/11/2005
Synthesis of r-P-Modified Nucleoside Diphosphates with Ethylenediamine
Ping Li,† Zhihong Xu,† Hongyan Liu,† Charlotta K. Wennefors,† Mikhail I. Dobrikov,†
Ja´nos Ludwig,‡ and Barbara Ramsay Shaw*,†
Department of Chemistry, Duke UniVersity, Box 90346, Durham, North Carolina 27708-0346, and DeVeloGen AG,
Rudolf Wissel Str. 28, 37079 Go¨ttingen, Germany
Received July 29, 2005; E-mail: brshaw@chem.duke.edu
Scheme 1. One-Pot Synthesis of NDPRB, NDPRS, and NDPRSe
At the forefront of antiviral therapeutics has been the design of
new classes of nucleosides and nucleotides.1 Most nucleoside
analogues become active after a series of phosphorylation steps
(through mono- and diphosphates) to their triphosphate forms.1
Thus, nucleoside di- (NDP) and triphosphates (NTP) are ubiquitous
biological molecules, and their analogues could have important
diagnostic and therapeutic applications. For example, phospho-
rothioate analogues have been well studied and employed to
determine enzymatic mechanisms.2 Recently, boranophosphate
analogues3 have shown promising therapeutic applications in
antiviral drug research.4 The Rp-R-boranotriphosphate is the pre-
ferred isomer as a chain terminator with viral reverse transcriptase,4a,b,5
while the viral DNA repair by pyrophosphorolysis is significantly
reduced compared with its parent nucleotide.5c-f In addition, an
increased phosphorylation activity of the human NDP kinase
(NDPK) toward nucleoside Rp-R-boranodiphosphate (Rp-NDPRB)
is observed.5a-c Although these R-P-modified nucleotide analogues
have wide applications, their preparation, especially NDPRB,6
remains a challenge.7
with Ethylenediaminea
The successful preparation of various R-P-modified NTP ana-
logues 9 by the Ludwig-Eckstein approach8 prompted us to explore
its use for the synthesis of R-P-modified NDP analogues in
conjunction with ethylenediamine, which has been shown to act as
a dephosphorylating reagent.9 On the basis of these findings, we
have developed a novel method for the synthesis of R-P-modified
NDP analogues 7, as outlined in Scheme 1. Since our main interest
was the borane analogues, the applicability of this procedure for
the synthesis of NDPRB was examined using all eight common
deoxyribo- and ribonucleosides.
The opening of the cyclic structure 4 by nucleophilic attack
usually occurs at the phosphate moiety rather than at the phosphorus
atom bearing the borane group.10 Therefore, treatment of intermedi-
ates 4a-h with ethylenediamine resulted in NDPRB 6a-h and
cyclic phosphorodiamidate 8.11 The formation of these two products
could be explained by ring opening of 4 first to 5, followed by a
second, intramolecular nucleophilic displacement. It is worth
mentioning that such a two-step mechanism has suggested a new
concept for designing drugs targeting Ras, a major protein
responsible for the formation of human tumors.12a Furthermore, the
use of diamine derivatives in the ring opening reaction12b may also
suggest novel solutions for NTP derivatives with controlled stability.
We demonstrate that the same method can also be employed to
synthesize NDPRS and NDPRSe13 analogues. The final products
7a-j were isolated in good yields (Table 1) along with the
H-phosphonate as the major byproduct in about 15% yield, which
might derive from intermediate 2.8c
a See Supporting Information for R1, R2, and the base protection.
as described in Table 1. The absolute configurations of NDPRB
were recently determined by cocrystallization with NDPK,5c in
which the first and second eluting diastereomers from HPLC are
Rp and Sp isomers, respectively.4d,6 Since the group priorities around
R-P for NDPRS and NDPRSe are opposite to those of NDPRB,
and assuming the same order of elution in HPLC, the assignment
of configuration for ADPRS and ADPRSe are Sp and Rp for the
first and second eluting diastereomers, respectively.
The assignment of the absolute configuration for both P-
diastereomers was further confirmed by 1H NMR. A difference in
the chemical shift (∆δ) for H8 of ADP analogues is observed
between the two diastereomers, as shown in Table 2. The H8 signal
Modification at the R-phosphate group produces a pair of
P-diastereomers, which were successfully resolved by RP-HPLC
† Duke University.
‡ DeveloGen AG.
9
16782
J. AM. CHEM. SOC. 2005, 127, 16782-16783
10.1021/ja055179y CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Synthesis and HPLC Profile of R-Modified NDP
Moreover, opposite stereospecificity toward ADPRB was observed
for CK (Sp preferred) and PK (Rp preferred), and the preferred
isomer bound more tightly than ADP.
Analogues
HPLC conditionb tR (min) [area %]
In summary, we have developed an efficient and convenient
protocol to synthesize R-P-modified NDP analogues that are
otherwise difficult to obtain. The absolute configurations of
P-diastereomers were confirmed by analysis of their 1H NMR.
Affinity studies revealed that the NDPRB is potentially useful in
antiviral research. This protocol guarantees the availability of these
biologically important compounds for further study.
compound
yielda
MeOH %
Rp isomer
Sp isomer
dADPRB
dGDPRB
dCDPRB
dTDPRB
ADPRB
GDPRB
CDPRB
UDPRB
ADPRS
ADPRSe
7a
7b
7c
7d
7e
7f
7g
7h
7i
22
32
30
31
28
26
29
30
30
19
9
9
6
6
8
7
5
8
9
8
5.58 [45.2]
10.69 [49.3]
10.41 [48.0]
6.72 [48.9]
8.53 [50.5]
10.73 [51.5]
10.10 [50.4]
6.57 [59.9]
11.11 [48.8]
10.34 [51.1]
11.97 [54.8]
13.29 [50.7]
17.48 [52.0]
9.02 [51.1]
17.24 [49.5]
18.38 [48.5]
13.62 [49.6]
10.19 [40.1]
7.09 [51.2]
5.47 [48.9]
Acknowledgment. Acknowledgment is made to the NIH grants
(R01-AI-52061 and 2R01-GM-57693) to B.R.S.
7j
Supporting Information Available: Experimental procedures and
spectroscopic data. This material is available free of charge via the
Internet at http://pubs.acs.org.
a Yield calculated in percentage by UV. b Waters Delta-Pak C18, 15 µm,
100 Å, 3.9 × 300 mm eluted at 1 mL/min with isocratic condition of 100
mM TEAB (pH ) 8.0) and MeOH.
Table 2. Chemical Shifts of R-Modified ADP Analogues in D2Oa
References
compound
H8
H2
H1
′
H2
′
H3
′
H4
′
H5
′
H5′′
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I-Rp
II-Sp
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I-Sp
8.45 8.08 5.99 4.60 4.47 4.23 4.11 4.00
8.43 8.08 5.99 4.62 4.39 4.24 4.11 4.00
8.51 8.03 5.94 4.61 4.47 4.22 4.15 4.05
8.44 8.03 5.94 4.61 4.47 4.22 4.15 4.05
8.57 8.10 5.99 4.66 4.49 4.26 4.19 4.09
8.48 8.09 5.99 4.64 4.47 4.27 4.16 4.11
II-Rp
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Figure 1. Conformation of P-diastereomers of ADP analogues.
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be responsible for the last phosphorylation of some antiviral NDP
analogues.16 By using a fluorescence quenching technique,17 the
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