New glycosidase activated nitric oxide donors: Glycose and 3-morphorlinosydnonimine conjugates

Article abstract of DOI:10.1021/jo050010o

(Chemical Equation Presented) To achieve site specific delivery of nitric oxide (NO), a new class of glycosidase activated NO donors has been developed. Glucose, galactose, and N-acetylneuraminic acid were covalently coupled to 3-morphorlinosydnonimine (SIN-1), a mesoionic heterocyclic NO donor, via a carbamate linkage at the anomeric position. The β-glycosides were successfully prepared for these conjugates, while the α-glycosidic compounds were very unstable. The new stable sugar-NO conjugates could release NO in the presence of glycosidases. Such NO prodrugs may be used as enzyme activated NO donors in biomedical research.

Full text of DOI:10.1021/jo050010o

New Glycosidase Activated Nitric Oxide Donors: Glycose and
3-Morphorlinosydnonimine Conjugates
T. Bill Cai, Dongning Lu, Xiaoping Tang, Yalong Zhang, Megan Landerholm, and
Peng George Wang*
Departments of Biochemistry and Chemistry, The Ohio State University, Columbus, Ohio 43210
pgwang@chemistry.ohio-state.edu
Received January 3, 2005
To achieve site specific delivery of nitric oxide (NO), a new class of glycosidase activated NO donors
has been developed. Glucose, galactose, and N-acetylneuraminic acid were covalently coupled to
3-morphorlinosydnonimine (SIN-1), a mesoionic heterocyclic NO donor, via a carbamate linkage
at the anomeric position. The â-glycosides were successfully prepared for these conjugates, while
the R-glycosidic compounds were very unstable. The new stable sugar-NO conjugates could release
NO in the presence of glycosidases. Such NO prodrugs may be used as enzyme activated NO donors
in biomedical research.
Introduction
role of carbohydrates in cell recognition and internaliza-
tion processes, since sugar derivatives can be delivered
by hexose transporters on the cell membrane.4 Two types
of carbohydrate-linked NO donors have been developed
in our group. The first type is the sugar-S-nitrosothiol.
For example, Glyco-SNAP-1 (1) is a water soluble and
stable NO donor, which showed superior cytotoxicity in
vitro and is commercially available for biological re-
search.5 The second type is the sugar-NONOate (di-
azeniumdiolates are used instead of NONOates in recent
nomenclature). For example, glucose-NONOate (2) could
release NO by the action of glycosidases.6 Our previous
studies also showed that carbohydrates could stabilize
the NO releasing moieties. The sugar-linked approach
could be a good solution for the instability that most NO
donors suffer from. Here we report new glycosyl carbam-
ate linked NO donors (3). The carbohydrate moiety
ensures that the NO donor compound is inactive until it
encounters an appropriate activating enzyme, such as a
glycosidase, and also increases the water solubility. We
are interested in the development of carbamate-linked
sugars, because this linkage is stable in aqueous solution
and could be hydrolyzed by glycosidases7 and no other
toxic groups are released from the linkage portion of the
molecule.
Nitric oxide (NO) is a small gaseous molecule that
serves as a mediator of many physiological events.1 NO
can be easily converted into a variety of reactive nitrogen
species (RNS), such as dinitrogen trioxide (N2O3), nitro-
gen dioxide (NO2), and the peroxynitrite anion (ONOO-).2
While these RNS are highly reactive, their reactivity can
be utilized to achieve beneficial results. It has been
reported that sufficient generation of RNS can lead to
cellular dysfunction and cytotoxicity.3 By controlling the
generation of a RNS to a localized area in which it is
desirable to kill cells, such as in a solid tumor, the
reactivity of the RNS can be used to achieve a desired
result.
To achieve site-specific delivery of NO, spontaneous NO
releasing agents can be converted into stable prodrugs
by attaching them to carriers, which can be specially
recognized by certain targets. We have designed and
synthesized novel NO donors by taking advantage of the
* Corresponding author. Phone: ++1-614-292-9884. Fax: ++1-614-
688-3106.
(1) (a) Ignarro, L. J.; Murad, F. Nitric Oxide: Biochemistry, Molecular
Biology, and Therapeutic Implications; Academic: San Diego, CA, 1995.
(b) Lancaster, J. Nitric Oxide: Principles and Actions; Academic: San
Diego, CA 1996.
(2) Fukuto, J. M.; Cho, J. Y.; Switzer, C. H. In Nitric Oxide:
Biochemistry, Molecular Biology, and Therapeutic Implications; Ig-
narro, L. J., Ed.; Academic: San Diego, CA, 2000; pp 23-40.
(3) (a) Koppenol, W. H. Free Radical Biol. Med. 1998, 25, 385. (b)
Murphy, M. P.; Packer, M. A.; Scarlett, J. L.; Martin, S. W. Gen.
Pharmacol. 1998, 31, 179. (c) Douki, T.; Cadet, J.; Ames, B. N. Chem.
Res. Toxicol. 1996, 9, 3.
(4) Wong, A.; Toth, I. Curr. Med. Chem. 2001, 8, 1123.
(5) Hou, Y.; Wang, J.-Q.; Ramirez, J.; Wang, P. G. Methods Enzymol.
1999, 301, 242.
(6) Wu, X.; Tang, X.; Xian, M.; Wang, P. G. Tetrahedron Lett. 2001,
42, 3779.
(7) De Graaf, M.; Pinedo, H. M.; Quadir, R.; Haisma, H. J.; Boven,
E. Biochem. Pharmacol. 2003, 65, 1875.
10.1021/jo050010o CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/23/2005
3518
J. Org. Chem. 2005, 70, 3518-3524

New Glycosidase Activated Nitric Oxide Donors
producing 4-nitrophenyl (peracetyl-D-glycosyl)carbonate
(7a and 7b) as R/â mixtures (molar ratio 1: 3). The R/â
mixtures could be separated in the following step. The
coupling reaction of SIN-1 with protected glycosyl car-
bonate was carried out at room temperature in anhy-
drous pyridine.14 The crude product was purified by silica
gel chromatography and the anomeric mixtures of the
products were separated. For this step, since SIN-1 itself
was unstable in the solution, the best yield obtained was
40%. Then the acetyl protecting groups were removed in
anhydrous methyl alcohol with sodium methoxide as a
catalyst. This deprotection step required several hours
at room temperature before the reaction mixture was
neutralized with a strong acidic resin (Amberlyst 15 ion-
exchange resin). Complete assignment of the chemical
shifts of 9a and 9b was based on the 2D COSY and
HMQC NMR spectra. The â configuration at the ano-
meric postion was also confirmed by the coupling con-
stant (7.5 and 8.0 Hz) between H1 and H2. The depro-
tection of the R anomers of 8a and 8b failed. NMR
showed that the heterocyclic ring decomposed. It seems
that R conjugates were unstable after removal of acetyl
protecting groups.
Applying our strategy to other heterocyclic NO donors,
more carbohydrate and NO donor conjugates could be
developed. Mesoionic oxatriazole-5-imines (GEAs) are
structurally isosteric to sydnonimines. They are potent
anti-platelet, fibrinolytic, thrombolytic, and bronchiec-
tatic agents.15 We have synthesized one member of this
type of NO donors, GEA 3162 (10), according to a
reported procedure.16 Coupling of the GEA 3162 with
4-nitrophenyl (2,3,4,6-tetra-O-acetyl-R/â-D-galactopyra-
nosyl)carbonate (7b) following a previously established
method gave the desired Gal-GEA3162 conjugates (11)
in fair yields (Scheme 2). However, deprotection of acetyl
groups caused the decomposition of the oxatriazole ring.
The synthesis of sialic acid/SIN-1 conjugate was slightly
different from that of other monosaccharides due to the
one additioanl carboxyl at the anomeric carbon (Scheme
3). After the protection of carboxyl and hydroxyl groups
with methyl ester and acetyl groups, the free anomeric
OH form of sialic acid (13)17 was coupled with 4-nitro-
phenyl chloroformate in the presence of triethylamine to
give 14. The following reaction with SIN-1 failed in the
previously described condition. Optimization of the reac-
tion conditions showed that this reaction worked better
in aqueous THF solution, which could give the protected
sialic acid/SIN-1 conjugate (15) in 40% yield. The ano-
meric configurations of 14 and 15 could not be deter-
mined by NMR until the deprotected compound was
obtained. The following two steps of deprotection afforded
the sialic acid/SIN-1 conjugate (16) successfully. The
anomeric configuration of the final product was deter-
mined as a â-sialoside, since the chemical shift of H3e
FIGURE 1. Structures of sugar-NO conjugates and SIN-1
The cytotoxicity of RNS against viruses is an exciting
area in which we can potentially gain better understand-
ing of the human immunity to viral infection and discover
more information on antiviral drug design.8 The influenza
virus is especially attracting 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 the influenza virus to
bind and cut the terminal sialic acid. Therefore we also
synthesized an N-acetylneuraminic acid-NO donor con-
jugate as an NO prodrug targeting the influenza virus.
If this kind of prodrug is able to bind and be hydrolyzed
by influenza neuraminidases, the NO will be released
around the viruses and obtain the targeting effect. Our
proposed synthetic sialic acid-NO conjugates can also be
used as probes to selectively deliver RNS to the virus.
Such conjugates may also be used to test an idea of
neuraminidase activated prodrug design for antiviral
therapy.
3-Morphorlinosydnonimine (SIN-1) (4) was first chosen
as a model NO donor moiety.10 SIN-1 is a vasodilator and
is believed to release NO nonenzymatically. At physi-
ological or alkaline pH, SIN-1 undergoes rapid hydrolysis
to the ring-open form from which the NO radical is
released. The activation of SIN-1 also produces the
superoxide anion, which combines with NO to produce
the highly reactive peroxynitrite anion. This reactive
species can damage biomolecules and induce cytotoxic-
ity.11
Results and Discussion
SIN-1 (4) as one of the starting materials was synthe-
sized from 4-aminomorpholine by a two-step transforma-
tion following literature procedures.12 Glucose and ga-
lactose derivatives (6a and 6b) with unprotected anomeric
hydroxyl groups were obtained by treating peracetylated
glycoses with benzylamine in THF at an ambient tem-
perature.13 After purification by silica gel column chro-
matography, the glycose was coupled with 1 equiv of
4-nitrophenyl chloroformate in the presence of 3 equiv
of triethylamine in methylene chloride at 0 °C, thereby
(8) Guidotti, L. G.; McClary, H.; Loudis, J. M.; Chisari, F. V. J. Exp.
Med. 2000, 191, 1247.
(14) Azoulay, M.; Escriou, V.; Florent, J.-C.; Monneret, C. J.
Carbodydr. Chem. 2001, 20, 841.
(9) Rimmelzwaan, G. F.; Baars, M. M. J. W.; De Lijster, P.; Fouchier,
R. A. M.; Osterhaus, A. D. M. E. J. Virol. 1999, 73, 8880.
(10) (a) Schonafinger, K. IL Farmaco 1999, 54, 316. (b) Newton, C.
G.; Ramsden, C. A. Tetrahedron 1982, 38, 2965.
(11) Virag, L.; Szabo, E.; Gergely, P.; Szabo, C. Toxicol. Lett. 2003,
140-141, 113.
(15) (a) Karup, G.; Preikschat, H.; Wilhelmsen, E. S.; Pedersen, S.
B.; Marcinkiewicz, E.; Cieslik, K.; Gryglewski, R. J. Pol. J. Pharmacol.
1994, 46, 541. (b) Corell, T.; Pedersen, S. B.; Lissau, B.; Moilanen, E.;
Metsae-Ketelae, T.; Kankaanranta, H.; Vuorinen, P.; Vapaatalo, H.;
Rydell, E.; Andersson, R.; Marcinkiewicz, E.; Korbut, R.; Gryglewski,
R. J. Pol. J. Pharmacol. 1994, 46, 553.
(12) Masuda, K.; Imashiro, Y.; Kaneko, T. Chem. Pharm. Bull. 1970,
18, 128.
(13) Sim, M. M.; Kondo, H.; Wong, C.-H. J. Am. Chem. Soc. 1993,
115, 2260.
(16) Karup, G. L.; Preikschat, H. F.; Corell, T. N.; Lissau, B. G.;
Clausen, F. P.; Petersen, S. B.; Alhede, B. I. F. WO Patent 9213847,
1992; CA 118:38925.
(17) Marra, A.; Sinay, P. Carbohydr. Res. 1989, 190, 317.
J. Org. Chem, Vol. 70, No. 9, 2005 3519

Cai et al.
SCHEME 1. Synthesis of Glucose, Galactose, and SIN-1 Conjugatesa
a Reagents and conditions: (a) BnNH2, THF, rt, 30 h; (b) p-NO2C6H4OCOCl, NEt3, CH2Cl2, rt, 4.5 h; (c) 4, pyridine, rt, 12 h; (d) NaOMe,
MeOH, rt, 3 h.
SCHEME 2. Synthesis of Galactose and GEA 3126
Conjugatea
a Reagents and conditions: (a) 7b, pyridine, rt, 12 h (31%).
SCHEME 3. Synthesis of Sialic Acid and SIN-1
Conjugatea
FIGURE 2. The UV absorbance changing of 9b. 9b (0.04 mM)
was incubated in 150 mM phosphate buffer at pH 7.4 and 37
°C in the presence (b) and in the absence (O) of â-galactosidase
(0.05 mg/mL). UV was measured at 307 nm.
FIGURE 3. Lineweaver-Burk plot of 9a as the substrate of
a Reagents and conditions: (a) Ac2O, HClO4, 2 h, 75%; (b)
â-glucosidase. â-Glucosidase (0.5 mg/mL) and substrate ([S])
p-NO2C6H4OCOCl, NEt3, CH2Cl2, 4.5 h, 64%; (c) 4, NaHCO3,
9a (0.1, 0.2, 0.4, 0.8, 1.25 mg/mL) were incubated in 150 mM
Tris-CF3COOH buffer for 10 s at pH 7.30.
THF-H2O, 10 h, 40%; (d) NaOCH3, CH3OH, 2 h then NaOH, H2O.
10 h, 68%.
the decomposition of the SIN-1. To study the details of
(2.28 ppm) filled in the range of 2.32 ( 0.08 ppm
enzymatic kinetics, both 9a and 9b were incubated with
â-glucosidase and â-galactosidase, respectively, and then
the solutions were analyzed by LC-MS (Shimadzu, 2010A).
The kinetic data were obtained by the Lineweaver-Burk
plots for 9a (Figure 3) and 9b (Figure 4). Km, Vmax, and
kcat are listed in Table 1. The Km for both substrates is
within the ranges of known substrates (from about 0.05
to 85 mM). The turnover numbers (kcat) are relatively
large in the range of known substrates. It indicates that
both 9a and 9b are good substrates of the correspondent
glycosidases when enzymes are fully saturated with
substrates. At low substrate concentrations, the hydroly-
sis is still efficient, since kcat/KM is also large. From the
kinetic data, we can see that glycosidases showed good
catalytic efficiency for this type of carbohydrate conju-
gates with carbamate linkages.
according to the general rule.18 There was no R anomers
obtained after column chromatography. The reason may
be the anomeric effect, which made the thermodynami-
cally favored â anomer dominant in the products. It was
shown that this â-sialoside was not the substrate of the
neuraminidase, which hydrolyzes the R-sialosides.
To test our design, the decomposition of 9b by â-ga-
lactosidase was detected by the decay of the absorbance
at 307 nm (Figure 2), which is the characteristic of the
NO donating heterocycle. The enzymatic hydrolysis was
measured on a UV-vis spectrophotometer fitted with an
electrically thermostated cell block. The half-life of 9b
with â-galactosidase was estimated to be 0.8 h, while 9b
was fairly stable in the buffer without the enzyme. It was
noticed that 9b decomposed very slowly in the UV
spectrophotometer. The reason may be that UV causes
3520 J. Org. Chem., Vol. 70, No. 9, 2005

New Glycosidase Activated Nitric Oxide Donors
FIGURE 4. Lineweaver-Burk plot of 9b as the substrate of
â-galactosidase. â-Galactosidase (0.5 mg/mL) and substrate
([S]) 9b (0.1, 0.2, 0.4, 0.8, 1.25 mg/mL) were incubated in 150
mM Tris-CF3COOH buffer for 10 s at pH 7.30.
FIGURE 5. Measurement of NO profiles of 9a, 9b, and SIN-
1: ([) SIN-1 (100 µM) and SOD (0.3 mg/mL) were incubated,
(9) 9a (100 µM) and SOD (0.3 mg/mL) were incubated with â-
glucosidase (0.005 mg/mL), and (O) 9b (100 µM) and SOD (0.3
mg/mL) with â-galacosidase (0.005 mg/mL) were incubated,
all in 150 mM phosphate buffer at pH 7.4 and 37 °C.
TABLE 1. The Kinetic Parameters of 9a/9b as
Substrates of Glycosidases
KM
(mM)
Vmax
(mM/s)
kcat
kcat/KM
substrate
(s-1
)
(mM- 1s-1
)
9a
9b
0.311
1.47
0.181
0.327
2.75 × 105
8.87 × 105
6.54 × 105
4.45 × 105
SIN-1. A possible decomposition pathway of 9b is pro-
posed in Scheme 4. â-Galactosidase first hydrolyzes the
glycosidic linkage of 9b to generate SIN-1. At the
physiological condition, SIN-1 undergoes rapid hydrolysis
to form the ring-opened product, SIN-1C. NO radical is
released in this process.21 Stoichiometric amounts of
superoxide anions (•O2-) are formed as a result of oxygen
reduction. Since NO is known to react with O2 at an
almost diffusion-controlled rate,22 peroxynitrite (OONO-)
production is inevitable. Since most of the NO released
from both glycosides was converted to peroxynitrite,
which could isomerize to nitrate (NO3-), only a small
amount of nitrite was detected by Griess assay. Peroxy-
nitrite is a well-known active species that can cause
damage to biomolecules.11
TABLE 2. Nitrite Concentration in the Incubation of
9a/9b with or without the Glycosidasesa
substrate (23 µM)
[NO2-]/µM
SIN-1
3.1
negligible
2.4
0.3
2.8
9a
9a + â-glucosidase
9b
9b + â-galactosidase
•
-
a The amount of nitrite released was measured by using the
Griess method. Glycosidases (0.05 mg/mL) and 9a or 9b (23 µM)
were incubated for 17 h in 150 mM phosphate buffer at pH 7.4
and 37 °C. UV absorbance was measured at 548 nm.
The NO releasing ability of 9a and 9b in the presence
and absence of glycosidases was evaluated by measuring
the formation of nitrite ion, since nitrite ion is generated
from the oxidation of NO radical by oxygen. The nitrite
(NO2-) anion in solution was quantified by the Griess
method.19 The results are summarized in Table 2. Both
9a and 9b were relatively stable under physiological
conditions without enzymes. The trace amount of nitrite
formed might come from the photodecomposition of the
heterocycle.20 In contrast, SIN-1 decomposed quite easily
in the aqueous solution. Compounds 9a and 9b generated
comparable amounts of NO2- compared to SIN-1 only in
the presence of glycosidases.
The hydrolysis of the glycosides was also studied by
monitoring the NO release with an Electrochemical ISO-
NO marker II isolated Nitric Oxide Meter manufactured
by World Precision Instruments, Inc. NO was not de-
tected when 9a or 9b was incubated in aqueous solution
at neutral pH, even in the presence of glycosidases. This
is presumed to be because NO/O2- reacted so rapidly that
NO is consumed. Yet, when 0.3 mg/mL of superoxide
dismutase (SOD) was present, NO formation was ob-
served (Figure 5). This property is the same as that for
Conclusions
In summary, we have prepared a new class of glycosi-
dase dependent NO donors, glycosyl carbamate of nitric
oxide donors. SIN-1 as an NO releasing moiety was
coupled with glucose, glactose, and sialic acid via well-
defined chemical steps. The carbohydrate-SIN-1 conju-
gates are much more stable than SIN-1 itself. We also
found that glycosyl carbamate of NO donors could be
hydrolyzed by glycosidases and form NO. Such prodrugs
may find applications in biological research and Antibody-
Directed Prodrug Therapy (ADEPT).
Experimental Section
2,3,4,6-Tetra-O-D-glucopyranose (6a). A solution of pen-
taacetate-D-glucose (3.9 g, 10.0 mmol) and BnNH2 (1.2 mL,
11.0 mmol) in THF (45 mL) was stirred at ambient temper-
ature for 30 h. The solvent was removed and the residue was
dissolved in CH2Cl2 (100 mL), then washed with 1 N HCl (100
mL × 2) and water (100 mL) successively. The organic layer
was concentrated and chromatographed on silica gel with
EtOAc/hexane (2:1-1:1), giving 6a (3.2 g, 9.2 mmol, 92%) as
(18) Dabrowski, U.; Friebolin, R.; Brossmer, R.; Supp, M. Tetahedron
Lett. 1979, 48, 4637.
(19) Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper, P. L.;
Wishnok, J. S.; Tannenbaum, S. R. Anal. Biochem. 1982, 126, 131.
(20) Ullrich, T.; Oberle, S.; Abate, A.; Schroder, H. FEBS Lett. 1997,
406, 66.
(21) Bohn, H.; Schonafinger, K. J. Cardiovasc. Pharmacol. 1989, 14
(Suppl. 11), S6.
(22) Huie, R. E.; Padmaja, S. Free Radical Res. Commun. 1993, 18,
195.
J. Org. Chem, Vol. 70, No. 9, 2005 3521

Cai et al.
SCHEME 4. Proposed NO Releasing from 9b
1
a 3:1 (R/â) mixture of anomers as judged by NMR. H NMR
anhydrous methanol (5 mL) was added NaOCH3 to adjust the
solution pH value to 8-9. The reaction mixture was stirred
at room temperature for several hours and then strong acid
ion-exchange resin was added to neutralize the reaction
mixture. After removing the solvent in vacuo, the residue was
purified by silica gel chromatography with CH2Cl2/CH3OH (4:
1) as an eluent. 9a (10 mg, 26.6 mmol) was obtained as a
colorless syrup in quantitative yield. UV 307 nm; 1H NMR (500
MHz, CD3OD) δ 8.14 (s, 1H, H-4′), 5.39 (d, 1H, J ) 7.5 Hz,
H-1), 3.95 (t, 4H, J ) 5.0 Hz, H-6′′ and H-2′′), 3.83 (dd, 1H, J
) 11.5, 2.0 Hz, H-6), 3.66 (dd, 1H, J ) 12.0, 5.0 Hz, H-6), 3.62
(t, 4H, H-3′′ and H-5′′), 3.40 (t, 1H, J ) 8.5 Hz, H-3), 3.38-
3.32 (m, 2H, H-5 and H-4), 3.32∼3.29 (m, 1H, H-2); 13C NMR
(125 MHz, CD3OD) δ 169.0 (carbamate), 101.4 (C-4′), 97.4 (C-
1), 78.6, 78.0, 74.0, 71.2 (C-2,3,4,5), 65.6 (C-2′′ and C-6′′), 62.5
(C-6), 55.5 (C-3′′ and C-5′′); ESI-MS m/z 377 [M + H]+, 399
[M + Na]+, 753 [2M + H]+, 775 [2M + Na]+; ESI-HRMS calcd
for C13H20N4O9Na (M + Na+) m/z 399.1122, found m/z 399.1120.
2,3,4,6-Tetra-O-D-galactopyranose (6b). 6b was prepared
from 5b and obtained as foams (92%). 1H NMR (400 MHz,
CDCl3) δ 5.50-5.30 (m, 3H), 5.20-5.00 (m, 2H), 4.69 (d, 0.25H,
J ) 6.6 Hz), 4.43 (m, 1H), 4.10-4.00 (m, 3H), 4.00-3.90 (m,
0.25H), 3.00-2.70 (m, 1H), 2.13, 2.11, 2.06, 2.02, 2.01, 1.96 (s,
15H); 13C NMR (100 MHz, CDCl3) δ 95.8, 90.4 (C-1).
(400 MHz, CDCl3) δ 4.41 (d, 1H, J ) 5.6 Hz, H-1 of â-anomer),
3.89 (d, 1H, J ) 3.2 Hz, H-1 of R-anomer); 13C NMR (100 MHz,
CDCl3) δ 96.0, 90.3 (C-1).
4-Nitrophenyl (2,3,4,6-Tetraacetyl-D-glucopyranosyl)-
carbonate (7a).14 To a cooled solution (0 °C) of 6a (2.48 g,
7.13 mmol) in anhydrous CH2Cl2 (50 mL) were successively
added 4-nitrophenyl chloroformate (1.72 g, 8.53 mmol) and
triethylamine (2.96 mL, 21.3 mmol). After being stirred for
4.5 h at room temperature, the crude mixture was diluted with
another portion of CH2Cl2 (50 mL) and then washed with brine
and water. The CH2Cl2 layer was dried over anhydrous Na2-
SO4 then concentrated. After purification by silica gel chro-
matography with EtOAc/hexane (1:2) as an eluent, 7a (3.28
g, 6.39 mmol, 90%) was obtained as a 1:3 (R/â) mixture of
anomers as judged by 1H NMR. 1H NMR (500 MHz, CDCl3) δ
6.28 (d, 1H, J ) 3.0 Hz, H-1 of R-anomer), 5.66 (d, 1H, J ) 8.0
Hz, H-1 of â-anomer); 13C NMR (125 MHz, CDCl3) δ 95.8, 94.1
(C-1); ESI-MS m/z 536 [M + Na]+, 1049 [2M + Na]+.
N-(2,3,4,6-Tetra-O-acetyl-â-D-glucopyranosyl)carbonyl-
3- morpholinosydnonimine (8a). To a solution of 7a (1.53
g, 2.98 mmol) in anhydrous pyridine (20 mL) was added 4 (678
mg, 3.28 mmol). The reaction mixture was stirred overnight
and then the solvent was removed in vacuo to give a sticky
oil. The residue was purified by silica gel chromatography with
EtOAc/CH2Cl2 (1:1) as an eluent. 8a (568 mg, yield 35%) and
the R-anomer (164 mg, 0.301 mmol, yield 10%) were obtained
as foams. 8a: 1H NMR (500 MHz, CDCl3) δ 7.70 (s, 1H, H-4′),
5.72 (d, 1H, J ) 8.5 Hz, H-1), 5.26 (t, 1H, J ) 9.0 Hz, H-3),
5.20∼5.10 (m, 2H, H-4 and H-2), 4.25 (dd, 1H, J ) 12.5, 4.0
Hz, H-6), 4.12 (dd, 1H, J ) 12.0, 2.0 Hz, H-6), 3.97 (t, 4H, J )
5.0 Hz, H-2′′ and H-6′′), 3.85 (dd, 1H, J ) 4.0, 2.0 Hz, H-5),
3.54 (t, 4H, J ) 5.0 Hz, H-3′′ and H-5′′), 2.06-1.98 (m, 12H,
COCH3 × 4); 13C NMR (125 MHz, CDCl3) δ 174.3 (C-
carbamate), 170.7 (CdO of Ac), 170.2 (CdO of Ac), 169.4 (Cd
O of Ac × 2), 158.3 (C-5′), 99.8 (C-4′), 93.3 (C-1), 73.1 (C-3),
72.2 (C-5), 70.4 (C-2), 67.8 (C-4), 65.4 (C-2′′ and C-6′′), 61.5
(C-6), 54.5 (C-3′′ and C-5′′), 20.7 (CH3 of Ac × 2), 20.6 (CH3 of
Ac × 2); ESI-MS m/z 545 [M + H]+, 567 [M + Na]+, 1089 [2M
+ H]+, 1111 [2M + Na]+; HRMS calcd for C21H28N4O13Na (M
+ Na+) m/z 567.1545, found m/z 567.1564. R-Anomer: 1H NMR
(500 MHz, CDCl3) δ 7.77 (s, 1H, H-4′), 6.31 (d, 1H, J ) 3.5
Hz, H-1), 5.63 (t, 1H, J ) 10.0 Hz, H-3), 5.15 (t, 1H, J ) 10.0
Hz, H-4), 5.06 (dd, 1H, J ) 10.0, 4.0 Hz, H-2), 4.28-4.26 (m,
2H, H-5 and H-6), 4.10-4.00 (m, 1H, H-6), 3.97 (t, 4H, J )
5.0 Hz, H-2′′ and H-6′′), 3.54 (t, 4H, J ) 5.0 Hz, H-3′′ and H-5′′),
2.10-1.97 (m, 12H, COCH3 × 4); 13C NMR (125 MHz, CDCl3)
δ 174.7 (C-carbamate), 170.7 (CdO of Ac), 170.0 (CdO of Ac),
169.8 (CdO of Ac), 169.6 (CdO of Ac), 159.2 (C-5′), 99.7 (C-
4′), 90.1 (C-1), 70.1 (C-3), 69.6 (C-2), 69.0 (C-5), 68.0 (C-4), 65.4
(C-2′′ and C-6′′), 61.5 (C-6), 54.6 (C-3′′ and C-5′′), 20.7 (CH3 of
Ac), 20.6 (CH3 of Ac × 2), 20.6 (CH3 of Ac); ESI-MS m/z 545
[M + H]+, 567 [M + Na]+, 1089 [2M + H]+, 1111 [2M + Na]+.
N-(â-D-Glucopyranosyl)carbonyl-3-morpholinosydnon-
imine (9a). To a solution of 8a (15 mg, 27.5 mmol) in
4-Nitrophenyl (2,3,4,6-Tetraacetyl-D-galactopyranosyl-
)carbonate (7b).14 7b was prepared from 6b and obtained as
1
foams (88%). H NMR (500 MHz, CDCl3) δ 6.33 (d, 1H, J )
3.0 Hz, H-1 of R-anomer), 5.63 (d, 1H, J ) 8.0 Hz, H-1 of
â-anomer); 13C NMR (125 MHz, CDCl3) δ 96.3, 94.8 (C-1).
N-(2,3,4,6-Tetra-O-acetyl-â-D-galactopyranosyl)carbo-
nyl-3-morpholinosydnonimine (8b). 8b was prepared from
7b and obtained as foams (40%). 8b: 1H NMR (500 MHz,
CDCl3) δ 7.73 (s, 1H, H-4′), 5.69 (d, 1H, J ) 8.5 Hz, H-1), 5.41
(d, 1H, J ) 3.5 Hz, H-4), 5.36 (dd, 1H, J ) 10.0, 8.5 Hz, H-2),
5.09 (dd, 1H, J ) 10.0, 3.5 Hz, H-3), 4.14∼4.09 (m, 2H, H-6
and H-6), 4.09-4.04 (m, 1H, H-5), 3.97 (t, 4H, J ) 5.0 Hz, H-2′′
and H-6′′), 3.55 (m, 4H, H-3′′ and H-5′′), 2.15-1.96 (m, 12H,
COCH3 × 4); 13C NMR (125 MHz, CDCl3) δ 174.2 (C-
carbamate), 170.3 (CdO of Ac), 170.2 (CdO of Ac), 170.1 (Cd
O of Ac), 169.4 (CdO of Ac), 158.2 (C-5′), 100.0 (C-4′), 93.8
(C-1), 71.1 (C-3 and C-5), 67.9 (C-2), 66.9 (C-4), 65.9 (C-2′′ and
C-6′′), 60.9 (C-6), 54.5 (C-5′′ and C-4′′), 20.8 (CH3 of Ac), 20.6
(CH3 of Ac × 2), 20.5 (CH3 of Ac); ESI-MS m/z 545 [M + H]+,
567 [M + Na]+, 1089 [2M + H]+, 1111 [2M + Na]+; HRMS
calcd for C21H28N4O13Na (M + Na+) m/z 567.1545, found m/z
567.1518. R-Anomer (12%): 1H NMR (500 MHz, CDCl3) δ 7.76
(s, 1H, H-4′), 6.35 (d, 1H, J ) 3.5 Hz, H-1), 5.52-5.50 (m, 2H,
H-3 and H-4), 5.29 (dd, 1H, J ) 11.0, 3.5 Hz, H-2), 4.44 (t, 1H,
J ) 7.0 Hz, H-5), 4.15-4.00 (m, 2H, H-6 and H-6), 4.10-4.00
(m, 1H, H-6), 3.97 (t, 4H, J ) 5.0 Hz, H-2′′ and H-6′′), 3.53 (t,
4H, J ) 5.0 Hz, H-3′′ and H-5′′), 2.14-1.94 (m, 12H, Ac × 4);
13C NMR (125 MHz, CDCl3) δ 174.7 (C-carbamate), 170.3 (Cd
O of Ac), 170.3 (CdO of Ac), 170.1 (CdO of Ac), 169.7 (CdO of
Ac), 159.4 (C-5′), 99.6 (C-4′), 90.7 (C-1), 68.1 (C-5), 67.7 (C-3),
67.5 (C-4), 66.9 (C-2), 65.3 (C-2′′ and C-6′′), 61.2 (C-6), 54.6
3522 J. Org. Chem., Vol. 70, No. 9, 2005

New Glycosidase Activated Nitric Oxide Donors
(C-3′′ and C-5′′), 20.7 (CH3 of Ac), 20.6 (CH3 of Ac), 20.6 (CH3
of Ac × 2); ESI-MS m/z 545 [M + H]+, 567 [M + Na]+, 1089
[2M + H]+, 1111 [2M + Na]+.
13 (400 mg, 0.815 mmol) in anhydrous CH2Cl2 (8 mL) was
successively added 4-nitrophenyl chloroformate (197 mg, 0.978
mmol) and triethylamine (247 mg, 2.45 mmol). After being
stirred for 4.5 h at room temperature, the crude mixture was
diluted with CH2Cl2 (50 mL). Column chromatography with
N-(â-D-Galactopyranosyl)carbonyl-3-morpholinosyd-
nonimine (9b). 9b was prepared from 8b and obtained as a
syrup (100%). UV 307 nm; 1H NMR (500 MHz, CD3OD) δ 8.16
(s, 1H, H-4′), 5.35 (d, 1H, J ) 8.0 Hz, H-1), 3.96 (t, 4H, J ) 5.0
Hz, H-6′′ and H-2′′), 3.88 (d, 1H, J ) 3.0 Hz, H-4), 3.74-3.66
(m, 3H, H-6, H-6 and H-2), 3.65-3.60 (m, 5H, H-5, H-3′′ and
H-5′′), 3.56-3.52 (m, 1H, H-3); 13C NMR (125 MHz, CD3OD)
δ 175.3 (C-carbamate), 160.6 (C-5′), 101.5 (C-4′), 98.1 (C-1),
77.4 (C-5), 74.9 (C-3), 71.3 (C-2), 71.1 (C-4), 66.6 (C-2′′ and
C-6′′), 62.3 (C-6), 55.4 (C-3′′ and C-5′′); ESI-MS m/z 377 [M +
H]+, 399 [M + Na]+, 775 [2M + Na]+; ESI-HRMS calcd for
C13H20N4O9Na (M + Na+) m/z 399.1122, found m/z 399.1129.
N-(2,3,4,6-Tetra-O-acetyl-â-D-galactopyranosyl)carbo-
nyl-3-(3,4-dichlorophenyl)-1,2,3,4-oxatriazole-5-imine (11).
11 was prepared from 10 and 7b and obtained as a red powder
(31%). 11: 1H NMR (500 MHz, CDCl3) δ 8.35 (d, 1H, J ) 2.5
Hz), 8.10 (dd, J ) 8.5, 2.5 Hz), 7.80 (d, 1H, J ) 8.5 Hz), 5.73
(d, 1H, J ) 8.5 Hz, H-1), 5.41-5.38 (m, 2H, H-4 and H-2), 5.09
(dd, 1H, J ) 10.0, 3.5 Hz, H-3), 4.14∼4.06 (m, 2H, H-6, H-5),
2.13, 2.01, 1.99, 1.97 (4 OAc); 13C NMR (125 MHz, CDCl3) δ
209.8, 170.6, 170.4, 170.3, 169.5 (CdO of Ac), 157.0, 140.2,
135.6, 132.4, 123.7, 120.8, 94.3 (C-1), 71.7 (C-3), 71.4 (C-5),
68.0 (C-2), 67.1 (C-4), 61.2 (C-6), 21.0, 20.9, 20.8 (CH3 of Ac);
MS (ESI) m/z 627 [M + Na]+, 1231 [2M + Na]+; HRMS calcd
for C22H22Cl2N4O12Na (M + Na+) m/z 627.0503, found m/z
627.0488. R-Anomer (26%): 1H NMR (500 MHz, CDCl3) δ 8.35
(d, 1H, J ) 2.5 Hz), 8.10 (dd, J ) 9.0, 2.5 Hz), 7.80 (d, 1H, J
) 9.0 Hz), 6.41 (d, 1H, J ) 3.5 Hz, H-1), 5.52-5.48 (m, 2H,
H-3 and H-4), 5.37 (dd, 1H, J ) 11.0, 3.5 Hz, H-2), 4.47 (t, 1H,
J ) 6.5 Hz, H-5), 4.11 (m, 2H, H-6), 2.16, 2.04, 2.01, 1.99 (4Ac);
13C NMR (125 MHz, CDCl3) δ 209.6, 170.4, 170.2, 170.1, 170.0
(CdO of Ac), 156.7, 140.1, 135.4, 132.3, 132.1, 123.4, 120.5,
91.4 (C-1), 68.6 (C-5), 67.6 (C-3), 67.6 (C-4), 66.7 (C-2), 61.3
(C-6), 20.7, 20.7, 20.6 (CH3 of Ac); MS (ESI) m/z 627 [M + Na]+,
1231 [2M + Na]+.
1
ethyl acetate afforded 14 (342 mg, 64%) as a white syrup. H
NMR (500 MHz, CD3OD) δ 8.34 (d, 2H, J ) 9 Hz), 7.52 (d,
2H, J ) 9 Hz), 5.41 (dd, 1H, J ) 6.5, 2 Hz), 5.24 (td, 1H, J )
11, 5 Hz), 5.17 (td, 1H, J ) 6, 2.5 Hz), 4.44 (dd, 1H, J ) 13,
2.5 Hz), 4.34 (dd, 1H, J ) 10.5, 2 Hz), 4.11 (t, 1H, J ) 10.5
Hz), 4.09 (dd, 1H, J ) 12, 7 Hz), 3.83 (s, 3H), 2.68 (dd, 1H, J
) 13.5, 5 Hz), 2.12 (s, 3H), 2.03-1.99 (m, 1H), 2.01 (s, 3H),
2.00 (s, 3H), 1.97 (s, 3H), 1.88 (s, 3H); 13C NMR (125 MHz,
CD3OD) δ 172.3, 171.1, 170.6, 170.4, 170.3, 165.8, 155.2, 149.8,
146.1, 125.3, 122.0, 100.8, 73.1, 70.4, 68.4, 67.7, 62.0, 53.0, 48.5,
35.6, 21.6, 19.7, 19.6, 19.5, 19.4; ESI-MS 679 (M + Na+), 695
(M + K+); HRMS calcd for C27H32N2O17Na (M + Na+) m/z
679.1593, found m/z 679.1589.
N-[Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-di-
deoxy-â-D-glycero-D-galacto-2-nonulopyranosyl)onate]-
carbonyl-3-morpholinosydnonimine (15). To a suspension
of 4 (1.68 mmol, 350 mg) in water (3 mL) was added NaHCO3
(1.85 mmol, 155 mg) with ice-cooling and stirring. After 10
min a solution of 14 (1.4 mmol, 900 mg) in THF (5 mL) was
added dropwise with stirring. The mixture was further stirred
overnight. Then THF was removed in vacuo with subsequent
extraction with ethyl acetate (10 mL) 3 times. The extract was
applied on column chromatography (CHCl3:CH3OH, 20:1). The
product 4 was collected as white foam (400 mg) (40%). 1H NMR
(300 MHz, CD3OD) δ 8.12 (s, 1H), 5.38 (dd, 1H, J ) 6.5, 2
Hz), 5.22 (td, 1H, J ) 11, 5 Hz), 5.10 (td, 1H, J ) 6, 2.5 Hz),
4.43 (dd, 1H, J ) 13, 2.5 Hz), 4.14 (dd, 1H, J ) 10.5, 2 Hz),
4.11-4.02 (m, 2H), 3.95 (t, 4H, J ) 5 Hz), 3.74 (s, 3H), 3.63 (t,
4H, J ) 5 Hz), 2.56 (dd, 1H, J ) 13.5, 5 Hz), 2.09 (s, 3H),
2.06-2.01 (m, 1H), 1.98 (s, 6H), 1.90 (s, 3H), 1.88 (s, 3H); 13
C
NMR (125 MHz, CD3OD) δ 174.4, 172.2, 171.2, 170.6, 170.3,
168.2, 157.7, 100.8, 96.9, 71.6, 70.1, 69.4, 67.4, 65.3, 61.6, 54.2,
52.,1, 48.7, 36.4, 21.5, 19.6 (2×), 19.5 (2×); ESI-MS 688 (M +
H+), 710 (M + Na+); HRMS calcd for C27H37N5O16Na (M + Na+)
m/z 710.2128, found m/z 710.2160.
Methyl 5-Acetamido-3,5-dideoxy-â-D-galacto-2-nonu-
lopyranosonate (12).23 N-Actylneuraminic acid (800 mg, 2.6
mmol) was dissolved in dry methanol (100 mL) and CF3COOH
(0.2 mL) was added. The reaction mixture was stirred at room
temperature for 3 days until the solution became clear. Then
the solvent was removed under reduced pressure and the
residue coevaporated twice with dry methanol (5 mL). The
product was used without purification.
N-(5-Acetamido-3,5-dideoxy-â-D-glycero-D-galacto-2-
nonulopyranosyl)carbonyl-3-morpholinosydnonimine
(16). Compound 15 (0.29 mmol, 200 mg) was dissolved in
methanol (30 mL) and 0.1 M NaOCH3 in methanol (0.68 mL)
was added. The mixture was stirred for 2 h at room temper-
ature. Strong acid ion-exchange resin was added to neutralize
the base. The resin was filtered off and twice washed with
methanol. The solvent was concentrated in vacuo and the
residue was dissolved in 0.1 M NaOH (70 mL). The solution
was stirred overnight. After neutralization with acidic resin,
the solution was concentrated and loaded on C-18 silica gel.
Washing with water gave product as an off-white powder (100
Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
â-D-glycero-D-galacto-2-nonulopyranosonate (13).17 To a
stirred, warmed (40 °C) solution of acetic anhydride (2.88 g,
2.7 mL) and aqueous 60% perchlorice acid (20 µL) was added
12 (2.6 mmol) during 30 min in small portions. The mixture
was stirred for 2 h at 40 °C, then cooled to room temperature,
diluted with cold water (40 mL), saturated with ammonium
chloride, and extracted with dichloromethane (3 × 100 mL).
The combined extracts were washed with saturated aqueous
sodium hydrogencarbonate (40 mL) and water (40 mL), dried
(MgSO4), and concentrated to give a white solid. Crystalliza-
tion from ethyl acetate-hexane gave 2 (1.00 g, 75%) as white
1
mg, 68%). H NMR (500 MHz, D2O) δ 7.91 (s, 1H), 3.95 (td,
1H, J ) 10, 5 Hz), 3.83 (t, 4H, J ) 5 Hz), 3.79-3.76 (m, 1H),
3.69 (d, 1H, J ) 10.5 Hz), 3.63 (m, 1H), 3.55 (dd, 1H, J ) 12.5,
3 Hz), 3.47 (t, 4H, J ) 5 Hz), 3.44 (d, 1H, J ) 8 Hz), 3.36 (dd,
1H, J ) 12, 6 Hz), 2.28 (dd, 1H, J ) 13.5, 5 Hz), 1.87 (s, 3H),
1.60 (t, 1H, J ) 13 Hz); 13C NMR (125 MHz, D2O), δ 174.9,
174.6, 173.8, 158.7, 101.2, 98.9, 72.2, 71.3, 68.0, 66.9, 65.4, 63.0,
53.9, 51.7, 39.3, 22.2; ESI-MS 506 (M + H+), 528 (M + Na+),
544 (M + K+); HR-MS calcd for C18H27N5O12Na (M + Na+) m/z
528.1548, found m/z 528.1551.
1
needles, mp 147-148 °C; H NMR (400 MHz, CDCl3), δ 5.79
(m, 1H, NH), 5.36 (m, 1H), 5.26-5.16 (m, 2H), 4.65 (br, 1H),
4.52 (dd, 1H, J ) 12 Hz), 4.21-4.12 (m, 2H), 4.02 (dd, J ) 12
Hz, 8 Hz), 3.85 (s, 3H), 2.28-2.16 (m, 2H), 2.14, 2.10, 2.02,
2.01, and 1.89 (5 s, 15H); 13C NMR (100 MHz, CDCl3), 171.2
(2×), 170.5 (2×), 170.4, 169.3, 95.1, 71.6, 71.3, 69.5, 68.3, 62.8,
53.7, 49.6, 36.3, 23.4, 21.3, 21.1 (2×), 21.0 (2×); ESI-MS 514.2
(M + Na+).
Measurement of Enzymatic Kinetics. Six 9a or 9b
solutions of concentrations from 0.2 to 5 µM (0.5 mL) were
incubated for 10 s with 10 µL of â-glucosidase or â-galactosi-
dase (50 mg/mL), respectively. Then the solutions were injected
into HPLC for analysis. The concentrations of substrates were
quantified by the areas of the corresponding peaks. The
reaction rates and concentrations of substrates were treated
Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-2-(4-nitro-
phenoxycarbonyloxy)-3,5-dideoxy-â-D-glycero-D-galacto-
2-nonulopyranosonate (14). To a cooled solution (0 °C) of
with the classical Lineweaver-Burk plot to obtain Km, Vmax
,
and kcat. The final data were the average of three repetitive
(23) Bandgar, B. P.; Hartmann, M.; Schmid, W.; Zbiral, E. Justus
Liebigs Ann. Chem. 1990, 1185.
experiments.
J. Org. Chem, Vol. 70, No. 9, 2005 3523

Cai et al.
Measurement of Nitrite Formation. The stock solution
of 9b (10 mM) was diluted to 0.023 mM with phosphate buffer
(pH 7.4). Next the solution of â-galactosidase (E.C. 3.2.1.23,
from Aspergillus Oryzae) in phosphate buffer (pH 7.4) was
added, and the mixture was incubated at 37 °C for about 17 h
before the addition of Griess reagent [150 µL of 1% sulfani-
amide in 1 M HCl and 150 µL of 0.1% N-(1-naphthyl)-
ethylenediamine in 1 M HCl]. The absorbance of the solution
was then measured at 548 nm. Calibration curves were made
from known concentrations of aqueous NaNO2 in the same
buffer solution so as to determine the amount of NO2- formed
after the reactions.
NO solutions with current changes. For the assay, 9a, super-
oxide dismutase (SOD) (0.3 mg/mL), and â-glucosidase (0.005
mg/mL) (E.C. 3.2.1.21) were incubated in pH 7.4 phosphate
buffer. The current changes were recorded every minute.
Acknowledgment. Financial support from the Na-
tional Institutes of Health (GM54074) is greatly ap-
preciated.
Supporting Information Available: Copies of NMR
spectra of synthesized compound 4, 6 through 11, and 13
through 16. This material is available free of charge via the
Internet at http://pubs.acs.org.
Measurement of Nitric Oxide. NO measurement was
carried out with an Electro-chemical ISO-NO Mark II Isolated
Nitric Oxide Meter (World Precision Instruments, Inc.). The
calibration plot was obtained by measurements of standard
JO050010O
3524 J. Org. Chem., Vol. 70, No. 9, 2005