Design of highly efficient and selective transfer reaction of nitrosyl group to dC and dMC resulting in specific deamination

Article abstract of DOI:10.1081/NCN-200060312

Nitric oxide (NO) is an important endogenous regulatory molecule, and S-nitrosothiols are believed to play a significant role in NO storage, transport, and delivery. Based on the ability to generate NO in vivo, S-nitrosothiols can be used as therapeutic d

Full text of DOI:10.1081/NCN-200060312

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DESIGN OF HIGHLY EFFICIENT AND SELECTIVE
TRANSFER REACTION OF NITROSYL GROUP TO dC AND
dMC RESULTING IN SPECIFIC DEAMINATION
Md. Monsur Ali a , Fumi Nagatsugi a , Shizuka Nakayama a , Md. Rowshon Alam b , Takeshi
Kawasaki b & Shigeki Sasaki a
a CREST, Japan Science and Technology Agency , Japan
b Graduate School of Pharmaceutical Sciences, Kyushu University , Fukuoka, Japan
Published online: 15 Nov 2011.
To cite this article: Md. Monsur Ali , Fumi Nagatsugi , Shizuka Nakayama , Md. Rowshon Alam , Takeshi Kawasaki &
Shigeki Sasaki (2005) DESIGN OF HIGHLY EFFICIENT AND SELECTIVE TRANSFER REACTION OF NITROSYL GROUP TO dC AND
dMC RESULTING IN SPECIFIC DEAMINATION, Nucleosides, Nucleotides and Nucleic Acids, 24:5-7, 721-724, DOI: 10.1081/
NCN-200060312
To link to this article: http://dx.doi.org/10.1081/NCN-200060312
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5--7):721–724, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200060312
DESIGN OF HIGHLY EFFICIENT AND SELECTIVE TRANSFER
REACTION OF NITROSYL GROUP TO dC AND dMC RESULTING
IN SPECIFIC DEAMINATION
5
Md. Monsur Ali, Fumi Nagatsugi, and Shizuka Nakayama
CREST, Japan
Science and Technology Agency, Japan
5
Md. Rowshon Alam and Takeshi Kawasaki
Graduate School of Pharmaceutical
Sciences, Kyushu University, Fukuoka, Japan
5
Shigeki Sasaki
CREST, Japan Science and Technology Agency, Japan
5
Nitric oxide (NO) is an important endogenous regulatory molecule, and S-nitrosothiols are believed to
play a significant role in NO storage, transport, and delivery. Based on the ability to generate NO in vivo,
S-nitrosothiols can be used as therapeutic drugs. In this study, we have developed an innovative method for
sequence- and base-specific delivery of NO to a specific site of DNA followed by specific deamination.
INTRODUCTION
Chemical reactions to DNA may cause the point mutation to lead diseases. For
example, the reaction of nitrous acid was deaminated of primary amino groups to
convert dA into dI, dC into dU, and dG into dX, resulting in mutation.[1] Since
chemical reactions to DNA occur nonselectively, and mutations are caused in a
random manner. If we can control chemical reactions to DNA in a specific manner,
they can be used as chemical tools to cause a selective point mutation on the target
DNA. We have previously reported that 2-amino-6-vinylpurine skeleton (1) ex-
hibited efficient cross-linking with selectivity toward cytidine.[2] The high selectivity
and efficiency of this cross-linking reaction is attributable to the proximity effect. As
an expansion of this design concept, we have designed a NO-transfer reaction from
S-nitroso thioguanine to an imino tautomer of cytosine (Figure 1). In this article, we
wish to report the development of an innovative method for sequence- and base-
specific delivery of NO to a specific site of DNA followed by specific deamination.
Address correspondence to Fumi Nagatsugi, Graduate School of Pharmaceutical Sciences, Kyushu
University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
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722
Md. M. Ali et al.
FIGURE 1 Design of the novel NO transfer reaction.
RESULTS AND DISCUSSION
Reaction in a complex between S-nitroso thioguanine and an imino tautomer
of cytosine was simulated by ab initio calculation, and it was suggested that NO
transfer from S to N would take place together with hydrogen transfer from N3 of
cytosine to N1 of thioguanine with relatively low activation energy.
The synthesis of ODNs incorporating S-nitroso-thioguanosine (2) is summa-
rized in Scheme 1. The ODN bearing thioguanosine (3) was prepared according to
the methods reported previously.[3] Nitrosation of 3 was carried out by a NO
transfer reaction with S-nitroso-N-acetylpenicillamine (SNAP) under alkaline
conditions. This reaction proceeded in good yields to give the ODN bearing S-
nitroso guanosine.
An interstrand NO transfer reaction was investigated with ODN (4) and its
complementary ODN (5; X = dC or dmC) in 50 mM MES buffer containing 100
mM NaCl at pH 7 at 25°C, and followed by HPLC (Figure 2A). The transfer
reaction yields were obtained by the conversion ratio of 4 to 3, as shown in Figure
2B. A rapid transfer reaction of 4 was observed with dC or dmC bearing ODN (5).
On the other hand, the NO transfer reaction did not take place with the ODNs (5)
having dT, dA, or dG at the target site or with ODN (7--9) having dC at a non-
target site. These results have suggested that the transfer reaction proceeds with high
SCHEME 1 Synthesis of the ODN incorporating S-nitroso guanosine.

Md. M. Ali et al.
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FIGURE 2 NO transfer reaction using 4 and 5. (A) HPLC analysis of NO-transfer reaction between 4 and 5
(X = C), HPLC conditions: column: X Terra MS C18 (2.1 Â 150 mm), solvent: A: 0.1 M TEAA Buffer B: CH3CN,
B: 10% to 20%/10 min, 20% to 100%/20 min linear gradient, flow rate: 0.3 mL/min, monitored at 254 nm.
(B) Conversion yield from 4 to 3; Closed circles and curve correspond to the reaction with 5 (X = dC or dmC); the
thick line corresponds to the reaction with 5 (X = dT, dA, or dG) or 7--9.
selectivity due to the efficient proximity effect between the S-NO thioguanosine and
the target dC or dmC in the duplex.
As 5 and 6 could not be separated by HPLC, the peak containing 5 and 6 was
isolated. Their nitrosyl species were analyzed by a method established by the
Nagano group,[4] in which the diaminofluorescein derivative (DAF2) reacts with the
NO+ species to generate fluorescent DAF2-triazole. The increase of DAF2-triazole
obtained from the isolated peak including 5 and 6 correlates well with the
conversion of 4 to 3.
Since nitrosylation of an amino group of a nucleoside is reported to cause
deamination,[1] we next analyzed deaminated products. After the NO transfer
reaction with ODN (5; X = dC or dmC), the mixture was kept under acidic
conditions, and the peak of ODN (6; X = dC or dmC) was isolated by HPLC. A
portion of the isolated ODN (6) was subjected to enzymatic hydrolysis with VPDE
and BAP and was analyzed by HPLC. In addition to the major peaks
corresponding to dA, dG and dC, dC-diazoate and dU (for ODN 6; X = dC) or
dmC, dmC-diazoate, and dT (for ODN 6; X = dmC) were observed.
Table 1 summarizes the fates of the nitrosylated dC or dmC, showing specific
deamination, especially of dmC. Another portion of the ODN (6) isolated after
acidic treatment was analyzed with DAF2 to show that the NO level decreased to
80% for X = dC and to 50% for dmC compared to those analyzed immediately after
NO-transfer reaction. These results suggest that NO-species in ODN (6) remain
after acidic treatment and spontaneously return to dC or dmC when the ODN is
hydrolyzed to nucleosides.

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Md. M. Ali et al.
TABLE 1 The Fates of the Nitrosylated dC or dmC after Enzyme Digestion
R=Ha
R=Meb
78%
45%
0%
0%
14%c
13%c
8% (dU)
42% (dU)
aThe NO-transfer reaction mixture was kept at pH 5 for 4 d.
bThe NO-transfer reaction mixture was kept at pH 5 for 1 d in the presence of CaCl2.
cThe peak of HPLC was identified by comparison with the authentic sample.[5,6]
CONCLUSION
We have developed the innovative method for nitrosyl group transfer to dC
and dmC with sequence and base specificity under mild conditions.[7] The
selectivity and efficiency of NO transfer followed by deamination exhibited in this
study are extremely high compared to the conventional methods of using NO gas
or other nitrosating agents. Investigation on effects of the nitrosylated dC in DNA
polymerization reactions is now ongoing in our group.
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