Sialated diazeniumdiolate: A new sialidase-activated nitric oxide donor

Article abstract of DOI:10.1021/ol048397p

(Chemical Equation Presented) A new sialated diazeniumdiolate has been synthesized, and the glycosylation product was exclusively an α anomer. This new nitric oxide donor exhibited significantly improved stability as compared to its parent diazeniumdiolate salts, and it could be efficiently hydrolyzed by neuraminidase to release nitric oxide with a K_m of 0.14 mM. The sialic acid-NO conjugate would be a valuable prodrug that targets NO to influenza viruses.

Full text of DOI:10.1021/ol048397p

ORGANIC
LETTERS
2004
Vol. 6, No. 23
4203-4205
Sialated Diazeniumdiolate: A New
Sialidase-Activated Nitric Oxide Donor
T. Bill Cai, Dongning Lu, Megan Landerholm, and Peng George Wang*
Departments of Biochemistry and Chemistry, The Ohio State UniVersity,
Columbus, Ohio 43210
pgwang@chemistry.ohio-state.edu
Received August 12, 2004
ABSTRACT
A new sialated diazeniumdiolate has been synthesized, and the glycosylation product was exclusively an
r
anomer. This new nitric oxide
donor exhibited significantly improved stability as compared to its parent diazeniumdiolate salts, and it could be efficiently hydrolyzed by
neuraminidase to release nitric oxide with a Km of 0.14 mM. The sialic acid−NO conjugate would be a valuable prodrug that targets NO to
influenza viruses.
Nitric oxide (NO) is a gaseous molecule that serves as a
mediator of many physiological events.1 To utilize this active
small molecule in therapy and research, NO is usually
released from a stable prodrug form, which is also known
as an NO donor.2 Design and synthesis of site-specific NO
donors are current tasks in our laboratory. We have attached
NO releasing moieties to carriers, which can be recognized
by certain targets. We reported several classes of enzyme-
activated NO donors previously. For example, the peptide-
diazeniumdiolate conjugates were synthesized by the attach-
ment of a diazeniumdiolate to the substrates of prostate
specific antigen (PSA), and they were demonstrated to release
NO upon the activation by PSA.3 In addition, the conjugates
of cephalosporin with 3-morpholinosydnonimine (SIN-1)
release NO in the presence of â-lactamase.4 It was also found
that the diazeniumdiolate functional group could be attached
to the anomeric position of monosaccharide, and those
glycosylated diazeniumdialates could be hydrolyzed by
glycosidases.5 Here, we report the development of a new
sialated diazeniumdiolate which promises to be utilized as
an NO donor that targets influenza viruses.
Diazeniumdiolates (formerly known as NONOates) are
important NO donors in biomedical research.6 The major
feature of this class of NO donors is that they can spontane-
ously release NO under physiological conditions with a range
of half-lives from a few seconds to several days. Since O2-
alkylated N-diazeniumdiolates are stable compounds, many
O2-substituted diazeniumdiolates have been prepared which
would decompose under a variety of conditions to regenerate
the unsubstituted NO releasing moieties.7 In our ongoing
research, we focused on the development of O2-protected
diazeniumliolates.
The cytotoxicity of NO to viruses is an exciting area which
can potentially provide better understanding of human
immunity to viral infections as well as provide more
information on antiviral drug designs.8 The influenza virus
is especially attractive to us, since the replication of influenza
A and B viruses could be severely impaired by NO donors.9
Hemagglutinin (HA) and neuraminidase are embedded in the
lipid bilayer of influenza virus to bind and cut the terminal
sialic acid, respectively. Neuraminidase (a type of sialidase)
(1) Ignarro, L. J. Nitric oxide: biology and pathobiology; Academic
Press: San Diego, 2000.
(2) Wang, P. G.; Xian, M.; Tang, X.; Wu, X.; Wen, Z.; Cai, T.; Janczuk,
A. J. Chem. ReV. 2002, 102, 1091.
(3) Tang, X.; Xian, M.; Trikha, M.; Honn, K. V.; Wang, P. G.
Tetrahedron Lett. 2001, 42, 2625.
(6) Keefer, L. K. Chemtech 1998, 28, 30.
(7) Hrabie, J. A.; Keefer, L. K. Chem. ReV. 2002, 102, 1135.
(8) Guidotti, L. G.; McClary, H.; Loudis, J. M.; Chisari, F. V. J. Exp.
Med. 2000, 191, 1247.
(4) Tang, X.; Cai, T.; Wang, P. G. Bioorg. Med. Chem. Lett. 2003, 13,
1687.
(5) Wu, X.; Tang, X.; Xian, M.; Wang, P. G. Tetrahedron Lett. 2001,
42, 3779.
(9) Rimmelzwaan, G. F.; Baars, M. M. J. W.; DeLijster, P.; Fouchier,
R. A. M.; Osterhaus, A. D. M. E. J. Virol. 1999, 73, 8880.
10.1021/ol048397p CCC: $27.50
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Published on Web 10/21/2004

can catalyze the hydrolysis of the R-O-glycosidic linkage
of N-acetylneuraminic acid (NeuAc, 1) connected to a variety
of aglycons on the cellular surface.10 We have tried to
synthesize N-acetylneuraminic acid-NO donor conjugates
as novel NO prodrugs targeting influenza viruses. If these
NO donors could bind and be hydrolyzed by influenza
neuraminidases, the NO will be released around viruses and
obtain the targeting effect. Our proposed synthetic NeuAc-
NO conjugates can also be used as probes to selectively
deliver NO to viruses. Such conjugates may also be used to
test a neuraminidase-activated prodrug design for antiviral
therapy.
different pH. Compound 5 was very stable in both neutral
and basic (pH 10, NaOH) solution and did not decompose
within several days, while in acidic (pH 1, HCl) solution it
showed a half-life about 19.6 h. Additionally, this compound
decomposed readily after addition of neuraminidase (EC
3.2.1.18, from Clostridium perfringens, Sigma). The decom-
position went steadily, as determined by the decay of the
absorbance at 255 nm (Figure 1). The enzymatic kinetics
PYRRO/NO (3), which has a half-life about 3 s, was
chosen as a representative of diazeniumdiolates.11 N-Acetyl-
neuraminyl chloride (2), which was prepared from N-
acetylneuraminic acid (1), was utilized as the sialadation
donor. Acetonitrile was used as the solvent for the reaction,
since it is an excellent solvent to obtain the R-anomer.
Compound 2 was stirred with 3 in anhydrous acetonitrile at
room temperature. The reaction was monitored by TLC. After
2 days, the starting material totally disappeared and there
was one product shown on TLC. After column chromatog-
raphy on silica gel, the R-anomer of sialyl diazeniumdiolate
(4) was obtained in 70% yield (Scheme 1). Since the
Scheme 1
Figure 1. UV absorbance changing of 5 in the presence (O) and
in the absence (b) of neuraminidase. Neuraminidase (0.05 mg/mL)
and 5 (0.15 mM) were incubated in 150 mM phosphate buffer, pH
7.4, 37 °C. UV was measured at 255 nm.
were measured on a UV-vis spectrophotometer fitted with
an electrically thermostated cell block. The Michaelis
constant (Km) was determined from the Lineweaver-Burk
plot. The Km (0.14 mM) was smaller than Km values (0.6-
1.6 mM) for oligosaccharides as substrates,14 which indicated
the binding between 5 and neuraminidase was stronger than
the natural sialosides. The kcat was calculated as 1.92 s-1,
revealing that the enzymatic hydrolysis was efficient.
The enzymatic decomposition of 5 was also examined by
NO measurement. The amount of NO generated could be
measured with an electrochemical ISO-NO Mark-II isolated
nitric oxide meter (World Precision Instruments, Inc. Sara-
sota, Florida). As shown in Figure 2, 5 was stable in the
buffer solution (pH 5.0), whereas a substantial amount of
NO was generated after the addition of neuraminidase. The
NO released from PYRRO/NO could not be shown in Figure
2, since its half-life is only 2.8 s, and the NO signal
disappeared within 30 s. In aerobic solution, NO is released
but it is consumed by the NO/O2 reaction and dissipates by
diffusion from the solutions. This is the reason that only a
small amount of NO was detected. In addition, the Griess
method15 also showed the formation of nitrite ion from the
enzymatic decomposition of 5, because the NO was oxidized
R-anomer is thermodynamically disfavored and there is no
suitable neighboring participation at C-3, it would be difficult
to control the anomeric selectivity. However, the result came
out successfully. The reason might be the formation of
â-acetonitrilium ion as an intermediate which provides
predominantly R-sialosides.12 Then deprotection of the acetyl
and ester groups afforded the first sialated diazeniumdiolate
(5). The anomeric configuration was determined as R by the
chemical shift of H3e (2.70 ppm).13
This novel NO donor (5) was water-soluble and very stable
in the solid state. It could be stored at room temperature
without any decomposition for months. The stability of this
novel NO donor was tested in aqueous solutions with
(10) Air, G. M.; Laver, W. G. Proteins 1989, 6, 341.
(11) Saavedra, J. E.; Billiar, T. R.; Williams, D. L.; Kim, Y.-M.; Watkins,
S. C.; Keefer, L. K. J. Med. Chem. 1997, 40, 1947.
(12) Nicolaou, K. C.; Hummel, C. W.; Bockovich, N. J.; Wong, C.-H.
J. Chem. Soc., Chem. Commun. 1991, 870.
(14) Bouwstra, J. B.; Deyl, C. M.; Vliegenthart, J. F. G. Biol. Chem.
Hoppe-Seyler 1987, 368, 269.
(13) Dabrowski, U.; Friebolin, R.; Brossmer, R.; Supp, M. Tetahedron
Lett. 1979, 4637.
(15) Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper, P. L.; Wishnok,
J. S.; Tannenbaum, S. R. Anal. Biochem. 1982, 126, 131.
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Org. Lett., Vol. 6, No. 23, 2004

Scheme 2. NO-Releasing Mechanism of 5
with that of 3 after 0.5 h at 37 °C, while no nitrite was formed
from 5 without the enzyme. In addition, 5 could also slowly
release NO in acidic solution. The two assays confirmed the
decomposing pathway of 5 (Scheme 2). Neuraminidase first
hydrolyzes the glycosidic bond, and then the diazenium
diolate automatically decomposes to release NO.
Since sialic acids and enzymes that metabolize them have
been implicated in the pathogenicity of a number of
infectious microorganisms,17 this class of NO donors would
have great potential in biomedical applications. Further
development of this class of novel NO donors is currently
underway.
Figure 2. Time course of NO release from 5 in the presence (O)
and in the absence (b) of neuraminidase. Neuraminidase (0.05 mg/
mL) and 5 (0.2 mM) were incubated in 100 mM acetate buffer,
pH 5.0, 25 °C.
to nitrite by oxygen from air through a cascade of reactions16
(Table 1). At pH 7, nitrite formation from 5 was comparable
Acknowledgment. This project is supported by a research
grant from the NIH (GM54074).
Table 1. Nitrite Formation from 5 and 3a
Supporting Information Available: Synthetic procedures
and characterization data for compounds 2-5 and enzyme
assay methodology. This material is available free of charge
via the Internet at http://pubs.acs.org.
substrate (23 µM)
[NO2-] (µM)
3
5
5.6
negligible
4.1
5 + neuraminidase
5 in HCl (pH 1)
0.2
OL048397P
a The amount of nitrite released was measured using the Griess method.
Neuraminidase (0.05 mg/mL) and 5 (23 µM) were incubated for 0.5 h in
150 mM phosphate buffer, pH 7.4, 37 °C. UV absorbance was measured at
548 nm.
(16) Wishnok, J. S.; Glogowski, J. A.; Tannenbaum, S. R. Methods
Enzymol. 1996, 268, 130.
(17) (a)Traving, C.; Schauer, R. Cell. Mol. Life Sci. 1998, 54, 1330. (b)
Corfield, T. Glycobiology 1992, 2, 509.
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