Sialyltransferase inhibitors: Consideration of molecular shape and charge/hydrophobic interactions

Article abstract of DOI:10.1016/j.carres.2012.12.017

In order to evaluate the importance of molecular shape of inhibitor molecules and the charge/H-bond and hydrophobic interactions, we synthesized three types of molecules and tested them against a sialyltransferase. The first type of compounds were designe

Full text of DOI:10.1016/j.carres.2012.12.017

Carbohydrate Research xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Sialyltransferase inhibitors: consideration of molecular shape
and charge/hydrophobic interactions
Rishi Kumar, Ravindranath Nasi, Milan Bhasin, Nam Huan Khieu, Margaret Hsieh, Michel Gilbert,
⇑
Harold Jarrell, Wei Zou , Harold J. Jennings
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to evaluate the importance of molecular shape of inhibitor molecules and the charge/H-bond and
hydrophobic interactions, we synthesized three types of molecules and tested them against a sialyltrans-
ferase. The first type of compounds were designed as substrate mimics in which the phosphate in
CMP-Neu5NAc was replaced by a non-hydrolysable, uncharged 1,2,3-triazole moiety. The second type
of compound contained a 2-deoxy-2,3-dehydro-acetylneuraminic moiety which was linked to cytidine
through its carboxylic acid and amide linkers. In the third type of compound the sialyl phosphate was
substituted by an aryl sulfonamide which was then linked to cytidine. Inhibition study of these cytidine
conjugates against Campylobacter jejuni sialyltransferase Cst 06 showed that the first type of molecules
are competitive inhibitors, whereas the other two could only inhibit the enzyme non-competitively.
The results indicate that although the binding specificity may be guided by molecular shape and H-bond
interaction, the charge and hydrophobic interactions contributed most to the binding affinity.
Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
Received 31 October 2012
Received in revised form 10 December 2012
Accepted 13 December 2012
Available online xxxx
Keywords:
Sialyltransferase inhibitors
Sialic acid
2-Deoxy-2,3-dehydro-acetylneuraminic
acid
Click chemistry
1,2,3-Triazole
Amino acid
1. Introduction
NH2
NH2
N
Sialylated glycoproteins and glycolipids are ubiquitous in mam-
malian cells and play important roles in various fundamental phys-
iological and pathological processes, such as cell–cell adhesion,
immune defense, tumor cell metastasis, and inflammation.1–5 Bio-
synthesis of sialylated glycoconjugates can be achieved by using
sialyltransferases (ST), a family of glycosyltransferases, that cata-
lyze the transfer of sialic acid residues to non-reducing terminal
galactose or another terminal sialic acid attached to oligosaccharide
chains of glycoproteins and glycolipids, using cytidine monophos-
phate N-acetylneuraminic acid (CMP-Neu5Ac) as the donor. Studies
have shown elevated sialyltransferase activity leads to over expres-
sion of sialylated glycoconjugates in human colorectal cancer,
breast carcinoma, leukemia cell lines, and metastatic tissues.6–10
Characteristic differences in the occurrence of sialic acids in healthy
and in diseased human tissues have also been reported.11,12 Thus,
sialyltransferase inhibitors may provide an alternative chemother-
apeutic treatment of cancer and also an invaluable tool to study
ST-dependent biological processes.13
N
N
O
O
O
N
O
O
P
P
O
O
O
O
O
P
P
OH
O
O
O
OH
OH OH
O
OH
O
O
OH OH
HO
O
OH
AcHN
B
A
OH
Figure 1. Transition-state sialyltransferase inhibitors: molecules with charge.
state28–31 involved in the glycosyl transfer within the active site
of the enzyme. Schmidt15,32–34 has designed and synthesized inhib-
itors based on transition state analogs. They reported compounds A
and B (Fig. 1), transition-state mimics, based on the proposed
mechanism of sialyl transfer involving partial dissociation of the
CMP moiety and the formation of a planar oxocarbenium structure
in the transition state.28–31 In compound A,15 they used a 2-deoxy-
2,3-dehydroneuraminic acid residue and introduced another nega-
tively charged phosphate group in order to achieve trigonal planar
geometry similar to the transition-state and to increase charge
interactions. Similarly, in compound B32 they replaced the neuram-
inyl residue with an aryl ring keeping intact the cytidine residue.
Both molecules exhibited good inhibition, but from a therapeutic
A common approach toward the development of effective
sialyltransferase inhibitors is to create mimic analogs based on
the donor14–22 (CMP-Neu5Ac), acceptor23–27 or the transition
⇑
Corresponding author. Tel.: +1 613 991 0855; fax: +1 613 952 9092.
E-mail address: wei.zou@nrc-cnrc.gc.ca (W. Zou).
0008-6215/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.12.017
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2
R. Kumar et al. / Carbohydrate Research xxx (2013) xxx–xxx
NH2
N
cytidine (1) was treated with a catalytic amount of 2,2,6,6-tetra-
methyl-1-piperidinyloxy (TEMPO) and stoichiometric amount of
the oxidant, [bis(acetoxy)iodo]benzene (BAIB) in acetonitrile–water
(1:1) that led to an acid (2),42 which was then treated with propar-
gylamine along with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-
imide (EDC) in DMF to give the desired propargyl derivative (3).
The other propargyl derivative (7) was also synthesized starting
from 1 in four steps, including intermediate, N-acetyl-50-azido-
20,30-di-O-isopropylidene cytidine (4), obtained by treatment of 1
with methanesulfonyl chloride and pyridine followed by substitu-
tion by sodium azide in DMF. N-Deacetylation with base followed
by N-protection with di-tert-butyloxycarbonate gave N-(t-butyl-
oxycarbonyl)-50-azido-20,30-di-O-isopropylidene cytidine (5), which
was then reduced to N-(t-butyloxycarbonyl)-50-amino-20,30-di-O-
isopropylidene cytidine (6). Interchange of N-acetyl protection to
N-Boc protection was necessary as catalytic hydrogenation of 4
failed to produce the desired amine (6). Condensation of amine (6)
with propiolic acid along with coupling reagents EDC–HOBt in
DMF yielded the desired propargyl derivative (7).
OH
OH
HO
COOH
non-charged linker
O
N
O
AcHN
O
HO
C
OH OH
NH2
OH
O
N
O
HO
HO
AcHN
non-charged linker
N
O
O
OH
OH OH
D
NH2
N
O
non-charged linker
N
O
R
Synthesis of 2-azido-2-deoxy-N-acetyl-a-neuraminic acid deriv-
O
ative 9 started from fully acetylated 2-chloro-2-deoxy-b-neurami-
nic acid methyl ester, which under a phase transfer catalysis
process reacted with sodium azide to yield acetylated neuraminyl
azide (8).43 Since the [3+2] cycloaddition between alkynes (3 and
7) and 8 failed under click-chemistry conditions, as reported by
Linhardt,44 the O-acetyl groups of 8 were removed under basic con-
ditions to yield neuraminyl azide 9. [3+2] Cycloaddition of propargyl
derivatives 3 and 9 in the presence of CuSO4 and sodium ascorbate
was then performed at 60 °C in a mixture of the t-BuOH and water
(1:1 v/v), which successfully gave the desired triazole 11 in 78%
yield. Similarly, triazole 10 was obtained from the propargyl deriva-
tives 7 and 9 in 81% yield. The addition products, 10 and 11, were
treated with aqueous TFA to yield the deprotected desired C-type
compounds, 12 and 13, respectively, which were fully characterized
by spectroscopic analyses. The proton and carbon signals in D2O
were assigned unambiguously with 1D- and 2D-NMR techniques.
Using similar [3+2] cycloaddition a triazole linkerwas introduced
in type-D compound 19 (Scheme 3) which involves the coupling of
alkyne derivative 17 and azide 4. O-Deacetylation of 2,3-dehydro-
neuraminic methyl ester 14 under basic conditions followed by
treatment with 2,2-dimethoxypropane and p-toluenesulfonic acid
(PTSA) in DMF afforded the 8,9-di-O-isopropylidene-2,3-dehydro-
neuraminic methyl ester 15 in 58% overall yield. When treated with
0.1 N NaOH/MeOH (1:1) the methyl ester was hydrolyzed to afford
the free carboxylic acid derivative 16. Condensation of 16 and
propargyl amine using coupling reagents EDC–HOBt in DMF yielded
the desired propargyl derivative 17. Click chemistry was performed
on 17 and the azide 4 using CuI in MeCN which successfully gave
triazole derivative 18. Sequential removal of N-acetyl and isopropyl-
idene protection in 18 by using NaOMe–MeOH solution and aqueous
TFA–water solution afforded the desired triazole linker D-type
molecule 19.
In addition, other linkers such as sulfonamide (Scheme 3) and
peptides (Scheme 4) were also introduced in the construction of
other D-type molecules (21 and 24). Intermediate 20 for sulfon-
amide linker D-type molecule 21 was synthesized by reacting
sulfamoyl chloride with 1 in DMA. This sulfonamide (20) was
coupled with carboxylic acid 16 and the subsequent deprotection
afforded 21 in good yield (Scheme 3). In order to prepare peptide
linked type-D molecules (24a–e), Fmoc chemistry was employed
as outlined in Scheme 4. The synthetic sequence involved con-
densation of amine derivative 6 with protected amino acids,
Fmoc-Gly-OH, Fmoc-Gln(Tr)-OH, Fmoc-Lys(Boc)-OH, Fmoc-As-
p(OtBt)-OH, and Fmoc-Trp(Boc)-OH. The coupling reaction was
carried out using EDC–HOBt coupling reagents to furnish Fmoc-
protected coupled products in 65–70% yields. Removal of the
E
OH OH
Figure 2. Three types of molecules as potential sialyltransferase inhibitors.
perspective they have serious drawbacks because of their poor bio-
availability. The charged phosphate linkage imparts poor cellular
permeability and is prone to phosphatase activity resulting in a
significant or total loss of activity.
Inhibition studies have shown that the cytidine portion of the
donor is important for binding with the active or regulatory site of
the enzyme, though sialic acid may not be essential.35,36 The phos-
phodiester of CMP-Neu5Ac may also contribute to the binding, but
it is unclear to what extent, as derivatives void of the phosphodiester
linker also exhibitedsignificant ST inhibitingactivity.37,38 Since non-
substrate mimic inhibitors of sialyltransferases (e.g., flavonoids,39
Soyasaponin,40 and, Lithocholic acid analogs41) do not share the
structural characteristics of CMP-Neu5Ac, interaction through their
hydrophobic moieties with sialyltransferases becomes critical in the
inhibition either competitively or non-competitively.
To address whether non-charged molecules devised from
compounds A and B can be effective competitive inhibitors to sialyl-
transferase, we designed a library of three distinct classes of mole-
cules containing two distinct features, a non-charged linker and an
increase, in type D–E, in hydrophobicity and planarity of the
replaced sialic acid unit (Fig. 2). In C-type molecules the phosphodi-
ester linkage of the substrate CMP-Neu5Ac was replaced with a non-
ionic triazole moiety. Such mimics still contain both the sialic acid
and cytidine residues responsible for binding to the sialyltransferase
active site. In the D-type molecules, instead of sialic acid, a 2-deoxy-
2,3-dehydroacetylneuraminyl moiety was connected to cytidine by
a triazole or a sulfamide linkage. This might help to achieve trigonal
planarity at the anomeric center of the transition state involved dur-
ing glycosylation, while also by slightly increasing hydrophobicity.
Type E compounds lack both sialic acid and a phosphodiester linkage
that were replaced, respectively, by an aryl group and an isosteric
sulfamide linker. The present work describes the synthesis and
preliminary inhibition studies of above three types of molecules.
2. Results and discussion
2.1. Synthesis
Using click chemistry, two C-type compounds were obtained
from 2-azidosialic acid (9) and propargyl derivatives of cytidine (3
and 7) (Schemes 1 and 2). N-Acetyl-20,30-di-O-isopropylidene
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R. Kumar et al. / Carbohydrate Research xxx (2013) xxx–xxx
3
NHAc
N
NHAc
N
NHBoc
N
NHBoc
N
N
O
N
O
N
O
HO
N3
N3
N
O
H2N
O
O
O
O
c
e
d
O
O
O
O
O
O
O
O
1
4
5
6
a
f
NHAc
NHAc
N
NHBoc
N
N
H
H
N
O
N
O
HO
O
O
N
O
N
O
N
O
O
b
O
O
O
O
O
O
O
3
2
7
Scheme 1. Reagents and conditions: (a) TEMPO, BAIB, MeCN–H2O (1:1 v/v), 3 h, 0 °C–rt, 84%; (b) propargylamine, EDC–HOBt, DMF, 24 h, rt, 76%; (c) (i) MsCl/Py, DCM; (ii)
NaN3, DMF, 80 °C, 72%; (d) (i) NaOMe/MeOH, 1 h, 0 °C; (ii) (Boc)2O, Et3N, THF, 24 h, rt, 69%; (e) Pd/C, MeOH, 92%; (f) propiolic acid, EDC–HOBt, DMF, 24 h, rt, 72%.
OAc
OAc
AcO
COOMe
N3
O
AcHN
AcO
8
OH
HO
OH
COOMe
N3
NHBoc
N
NHAc
N
O
AcHN
HO
H
N
9
H
N
N
O
O
O
N
O
O
O
a
a
O
O
O
O
O
7
3
OH
OH
OH
OH
HO
HO
NHBoc
N
NHAc
N
COOMe
COOMe
N
N
N
N
O
H
N
N
H
N
AcHN
N
AcHN
N
O
HO
HO
O
O
N
O
O
O
O
O
O
O
10
11
b
b
OH
OH
OH
OH
HO
9
HO
9
1
1
NH2
NH2
COOH
COOH
8
8
6
6
5'''
6'''
N3'''
N3'''
5'''
N
N
AcHN 7
N
3'
AcHN 7
N
O
O
2
2
N
1'
N
1'
5
H
N
5
HO
4
H
4
3
HO
3
N
O 6'''
O
N1'''
1''
O
N1''' O
5''
2'
2'
5''
4''
3'
O
1''
2''
O
4''
2''
3'O' H OH
3O'' H OH
13
12
t
Scheme 2. Reagents and conditions: (a) CuSO4ꢀ5H2O, sodium ascorbate, BuOH/H2O/DCM (2:1:1 v/v), 12 h, 60 °C, 82% for 10, 78% for 11; (b) TFA–H2O, DCM, 2 h, 0 °C, 1 M,
NaOH–H2O.
Fmoc group using piperidine produced intermediates (22a–e),
followed by coupling with acid 16 yielded compounds 23a–e.
Global deprotection using aqueous TFA gave another five type-
D compounds 24a–e, which were fully characterized by spectro-
scopic analyses.
Next, type-E molecules were constructed by coupling various
substituted benzoic acids with the sulfamide 20. The intermediates
were subsequently deprotected to afford (25a–r). The couplings
were achieved indirectly using N-succinimide esters of benzoic
acids. Direct coupling between benzoic acids and sulfamide, using
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4
R. Kumar et al. / Carbohydrate Research xxx (2013) xxx–xxx
OH
O
OAc
O
OH
O
O
O
O
a
b
O
OMe
AcO
AcO
AcHN
OMe
O
OH
O
O
AcHN
AcHN
OH
OAc
OH
14
16
15
NHBoc
N
N
O
N3
O
NHBoc
N
5
OH
O
O
O
OH
O
O
c
O
N
H
O
N
O
N
O
N
d
N
O
AcHN
O
N
H
O
AcHN
OH
18
17
OH
O
O
NH2
N3'''
OH
O
5'''
9
1'
3'
O
6
2'
8
1
7
6'''
e
HO
N
2
3
N1'''
O
N
5''
H
5
N
HO
O
N
1''
2''
4
AcHN
4''
3''
OH
19
OH OH
OH
O
NHAc
N
NHAc
N
O
O
OH
O
AcHN
N
O
H2N
O
S
O
N
O
HO
OH
16
f
O
O
O
g
O
O
O
O
20
1
NHAc
N
NH2
N
3''
5''
6''
OH
O
OH
O
1
9
O
O
8
6
7
N
O
N1''
1'
O
HO
NH
O
HO
NH
O
O
2
5'
3'
h
S
S
O
5
O
HO
AcHN
HO
AcHN
O
O
3
O
4'
4
2'
OH
OH
O
O
OH OH
21
Scheme 3. Reagents and conditions: (a) (i) NaOMe, MeOH; (ii) DMP, PTSA, DMF, 3 h, rt, 58% for two steps; (b) NaOH, MeOH (quantitative); (c) propargylamine, EDC–HOBt,
DMF, 24 h, rt, 56%; (d) CuI, DIPEA, MeCN, 1 h, rt, 68%; (e) TFA, H2O, 1 h, 0 °C, 67%; (f) sulfamoyl chloride, DMA, 0 °C, 4 h, rt, 68%; (g) EDC, HOBt, DMF, 24 h, 0 °C; (h) TFA, H2O,
1 h, 0 °C, 59% for two steps.
dicyclohexylcarbodiimide (DCC) and DMAP as described by Wid-
lanski45 also gave 25a. The synthesis of 25d was also achieved by
activating the salicylic acid with 1,10-carboxydiimidazole and then
using DCC to couple with the sulfamide.
docking study with the enzyme Cst-I suggests 13 could have two
binding sites with two different binding energies, which is corrob-
orated by the two observed inhibition constants. Modeling shows
that 13 is able to bind to the enzyme’s active site as well as to an-
other site just off the actual active site. The calculated free energy
of binding at the active site is ꢁ6.75 kcal/mol and ꢁ5.19 kcal/mol
at the other site. (More detailed discussion can be found in Supple-
mentary data.)
2.2. Inhibition studies
Three different types (C, D, and E) of molecules synthesized
above were tested against
a
-(2,3)-sialyltransferase Cst-06. Cst-06
D-Type molecules, in general, were found to be poorer inhibi-
tors than C-type molecules. The highest inhibition observed among
the D-type molecules was around 30% for 21 and 19. These two
molecules differ in the linkage structure with the triazole in 19
being replaced with sulfamide in 21. The poor inhibition was likely
due to the lack of charge interaction, as indicated by the better
inhibition of 24c which carries a negative charged amino acid
(aspartic acid). However, the kinetics showed that the inhibition
was not competitive. We did not further investigate the unknown
binding site of the sialyltransferase. Although the compounds are
analogs of a known transition state inhibitors (see Fig. 1A) the lack
of phosphate group and charge interaction seems to be detrimental
to the binding affinity at the active site. The results also suggest
is a fusion protein with Cst-I of Campylobacter jejuni bound with
maltose and belongs to GT-42 superfamily.46 The results of inhibi-
tion are presented in Table 1.
C-Type molecules 12 and 13 with a triazole linkage as mimic of
the phophodiester linkage47–49 and a
a
-anomeric configuration of
sialic acid showed more than 50% inhibition at 500 M. The Ki for
13 is 160 M which is twice lower than the Km of CMP-Neu5Ac
(400
M).46 The Lineweaver–Burk double reciprocal plot of mole-
cule 13 as shown in Figure 3 suggested a mixed inhibition toward
Cst-06. The two inhibition constants calculated were Kic = 222.7
and Kiu = 984.2 M. Since the Kic is 4–5 times less than the Kiu, the
inhibition can be considered ‘predominantly competitive’.50 The
l
l
l
lM
l
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R. Kumar et al. / Carbohydrate Research xxx (2013) xxx–xxx
5
NHBoc
NHBoc
N
N
R
R
22a R = H
OH
N
O
N
O
FmocHN
HN
H2N
22b R = (CH2)4NHBoc
22c R = (CH2)2COOtBu
22d R = (CH2)2CONHTr
H2N
O
O
O
O
a
O
O
O
O
22e R =
6
N
OH
O
Boc
O
O
OH
R
b
O
AcHN
16
OH
NHBoc
N
OH
O
23a R = H
O
N
O
HN
O
N
H
23b R = (CH2)4NHBoc
23c R = (CH2)2COOtBu
23d R = (CH2)2CONHTr
O
O
O
AcHN
OH
O
O
23e R =
c
N
Boc
NH2
24a R = H
N3'''
5'''
24b R = (CH2)4NH2
24c R = (CH2)2COOH
24d R = (CH2)2CONH2
OH
O
1
R
1'
9
6'''
O
O
N1'''
1''
O
8
2'
6
HN
7
5''
HO
N
2
3
H
5
HO
O
4''
3''
24e R =
2''
4
OH
AcHN
OH OH
N
H
Scheme 4. Reagents and conditions: (a) (i) EDC, HOBt, DMF, 24 h, rt, 52–63%; (ii) piperdine, CH2Cl2, 30 min, rt; (b) EDC, HOBt, DMF, 24 h, 0 °C; (c) TFA, H2O, 2 h, rt, 60–70%.
that a combination of cytidine and a neuraminyl group without
charge interaction is not enough to have an effective binding at
Table 1
the active site, which may consequently lead to another binding
site likely stabilized by hydrophobic and hydrogen bonding inter-
actions. This is supported by the fact that although cytidine is
not an inhibitor of sialyltransferases,51 yet cytidine monophos-
phate (CMP) does competitively inhibit sialyltransferase35 and
polysialyltransferase activities.52 The cytidine may play a key role
in binding specificity, but the affinity could be largely contributed
through charge interaction from additional phosphate. Another
example illustrating the importance of charge interaction is
Tamiflu which as a prodrug becomes a powerful neuraminidase
inhibitor only after the ester is hydrolyzed to allow charge interac-
tion and effective binding.53
Non-sugar analogs, such as an unsaturated bicyclic system with
a conjugated carboxylate group are used as a replacement of neu-
raminyl group,54 and sugar moiety attached to a bulky thiazole like
compounds has also been tested as potent inhibitors.55 These
experiments encouraged us to explore E-type compounds as po-
tential sialyltransferase inhibitors. E-type molecules contain a sul-
fonamide group as a surrogate of phosphate group (see Scheme 5).
Meanwhile, the neuraminyl group was replaced by aryl/heteroaryl
groups to further explore if the potency of the inhibition could be
improved as observed by others,32 where 50-triazole nucleoside
Inhibition of a
-(2,3)-sialyltransferase Cst-06a
Compound % Inhibition at 500
C-Type
12
13
l
M
Compound % Inhibition at 500
E-Type
lM
51.7 (58.5)b
58.5 (55.0)
25a
25b
27.2 (22.5)
31.7 (33.5)
(Ki = 160 l
M)b
25c
25d
25e
25f
25g
25h
25i
25j
25k
25l
28.0 (29.0)
38.1
39.5 (44.1)
43.1 (44.6)
22.3
28.7
12.7
15.9
D-Type
19
21
24a
24b
24c
24d
24e
29.2
33.5
10.5
10.7
30.5
19.1
25.1
40.5 (43.1)
29.5
25m
25n
78.4 (75.6)b
79.4 (73.0)
(Ki = 87 l
M)b
25o
25p
25q
25r
9.0
50.1 (48.6)
27.4
33.8
a
Data were average from a duplicated enzyme inhibition assay performed at
37 °C. Data in bracket were repeated results.
analogs were found to be competitive inhibitors of
transferase. Compounds 25m and 25n gave the best inhibition,
70–80% at 500 M. But, the kinetics suggest the inhibition is not
a-2,3-sialyl-
b
Inhibition was confirmed using FCHASE lactoside as substrate by monitoring
the formation of sialyllactoside (data not shown). The assays were performed at
37 °C for 20 min using 50 mM HEPES, pH 7.0, 10 mM MgCl2, 250
l
M CMPNeuAc,
l
500 Lac-FCHASE 6-(5-fluoresceincarboxamido)-hexanoic acid succinimidyl
lM
competitive, which is inconsistent with the results of similar com-
pounds reported.32 Distinctively, the substitution of m-NO2 of the
aryl group is critical, but not the carboxyl/carboxylate group as
indicated by comparing the inhibition of 25i, 25j–25m, and 25n,
ester. All reactions were stopped with 0.35 M Na2CO3. The samples were analyzed
by CE with a P/ACE MDQ CE system equipped with a laser module 488 (Beckmann
coulter). The percentage conversion of the FCHASE-Lac to product was calculated by
the integration of the trace peaks using the MDQ 32 Karat software.
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6
R. Kumar et al. / Carbohydrate Research xxx (2013) xxx–xxx
1/v (µM/min)
4.2. General methods
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
NMR spectra were recorded on a spectrometer with tetrameth-
ylsilane or the residual signal of the solvent as the internal stan-
dard. Chemical shifts are quoted in ppm and J values in Hz. Mass
spectra were recorded on a Quadrupole time-of-flight mass spec-
trometer using electrospray ionization (ESI) or time-of-flight mass
spectrometer using matrix-assisted laser desorption/ionization
(MALDI). Analytical thin-layer chromatography was performed
on precoated plates of silica gel and visualized with H2SO4–H2O
(1:20 v/v) followed by heating. Flash column chromatography
was performed using silica gel (230–400 mesh). All solvents and
reagents were purified and dried according to standard procedures.
-3
-2
-1
0
1
2
3
1/S (mM)
Figure 3. Lineweaver–Burk plot for inhibition of compound 13 against
sialyltransferase (C. jejuni).
a-2,3-
4.3. General procedure for synthesis of NHS ester
To a solution of carboxylic acid (1.0 equiv) in THF (10 mL) at
0 °C were added N-hydroxysuccinimide (1.0 equiv) and DCC
(1.0 equiv). The resulting mixture was stirred for 30 min at 0 °C
and 2 h at room temperature. The reaction mixture was filtered
to remove the precipitates, and the filtrate was concentrated under
reduced pressure. Purification by flash chromatography (hexane/
EtOAc) afforded the desired N-hydroxysuccinimidyl esters.
respectively, suggesting a strong interaction occurred only be-
tween –NO2 group and the enzyme. Since all other analogs are poor
inhibitors, it is plausible that a specific binding between m-NO2
aryl group and the enzyme contributes to the inhibition.
Time based inhibition studies were also carried out to explore
whether increasing the pre-incubation of enzyme with inhibitor
would affect the inhibition. Although pre-incubation of com-
pounds with the enzyme for 10 min led to slightly better inhibi-
tion, prolonged pre-incubation did not further improve inhibition.
4.4. General procedure for acylation from NHS esters
To a solution of N-hydroxysuccinimidyl ester (1.0 equiv) in DMF
(10 mL) at 0 °C were added protected sulfamoyl cytidine (20)
(1.5 equiv) and Cs2CO3 (2.0 equiv). The reaction mixture was
warmed to room temperature and stirred for 16–20 h. The reaction
solution was concentrated under reduced pressure to a residue
which was extracted with EtOAc (100 mL), and the filtrate was
concentrated. Purification by flash chromatography (EtOAc/MeOH
10:1 v/v) afforded the desired compounds.
3. Conclusions
Through molecular structure variations and its correlation to
inhibition against a sialyltransferase it was observed that mole-
cules based on substrate mimics are more likely to be a competi-
tive inhibitor. The results also show that the charge interaction
plays a pivotal role in enhancing binding affinity. Increasing hydro-
phobicity of a molecule may improve its binding but risk sacrific-
ing its specificity. Perhaps, the better way to improve the
bioavailability of a sialyltransferase inhibitor is not to eliminate
the charge as we tried, but rather to temporary mask the charge
and make a pro-inhibitor. It should be noted that inhibitors against
a bacterial sialyltransferase often do not inhibit human enzymes
due to their limited structural homology.
4.5. General procedure for direct acylation
To a solution of carboxylic acid (1.0 equiv) and protected sulfa-
moyl cytidine (20) (1.0 equiv) in DMF (10 mL) at ambient temper-
ature were added DCC (1.0 equiv) and DMAP (1.0 equiv). The
resulting mixture was stirred for 3 days at ambient temperature.
The reaction mixture was concentrated under reduced pressure.
Purification by flash chromatography (EtOAc/MeOH 10:1 v/v)
afforded the desired compounds.
4. Experimental section
4.1. Inhibition assay
4.6. General procedure for TFA deprotection
A solution of N-acetyl-50-O-[N-acyl(sulfamoyl)]-20,30-O-isopro-
pylidenecytidine in 30% TFA (MeOH–H2O 1:1 v/v) was stirred for
2 h at 0 °C to give the desired product. The reaction mixture was
concentrated under reduced pressure. Purification by size-exclu-
sion gel chromatography (Biogel-P2, water) afforded the pure com-
pound (25).
Assays were performed based on procedures described by Goss-
elin et al. (15) on microtiter plate. The hydrolysis of CMP-NeuAc in
the absence of enzyme was excluded from enzyme-catalyzed
activities. Pre-incubation for 5 min at 37 °C in a temperature con-
trolled microplate reader (Bio-Rad Benchmark Microplate Reader)
was followed prior to the addition of CMP-NeuAc. Each of the
microwell contained 10 mM of magnesium chloride, 10 mM of
manganese chloride, 55 mM HEPES (Fluka) at pH 7.0, 50 mM of
potassium chloride, 0.22 mg/ml Fraction V Bovine Serum Albumin
(Sigma–Aldrich), 2.8 mM phospho(enol)pyruvic acid tri(cyclohexyl
ammonium) salt (PEP) 4 mM ATP (Sigma), 0.45 mM NADH (Sigma).
3.6 U of pyruvate kinase (Sigma), 5.4 U of Lactic dehydrogenase
(Sigma), 5 mU of nucleotide monophosphate kinase, 25 mM of lac-
4.7. 50-O-[N-(Benzoyl)sulfamoyl]cytidine (25a)
White solid, ½a 2D0
ꢂ
+36.9 (c 0.27, H2O); 1H NMR (400 MHz, D2O) d
7.75 (d, J = 7.8 Hz, 2H, H-200, H-600), 7.61 (d, J = 7.6 Hz, 1H, H-6), 7.43
(dd, J = 7.8 and 6.7 Hz, 1H, H-400), 7.32 (dd, J = 7.6 and 7.6 Hz, 2H,
H-300, H-500), 5.76 (d, J = 3.4 Hz, 1H, H-10), 5.69 (d, J = 7.8 Hz, 1H,
H-5), 4.36 (d, J = 11.4 Hz, 1H, H-5a0), 4.26 (dd, J = 11.6 and 3.6 Hz,
1H, H-5b0), 4.17–4.13 (m, 3H, H-20, H-30, H-40); 13C NMR
(100 MHz, D2O) d 166.2 (2 ꢃ CO), 144.2 (C-4), 141.3 (C-6), 136.3
(C-100), 132.3 (C-400), 128.5 (C-200, C-300, C-500, C-600), 96.4 (C-5), 89.9
(C-10), 81.5 (C-40), 74.1 (C-20), 69.5 (C-30), 68.1 (C-50), MALDI-HRMS:
Calcd for C16H19N4O8S [M+H]+ 427.0918, Found: 427.0912.
tose, 250
(CST06) in a final volume of 200
l
M of CMP-NeuAc, and 0.25 mU of sialyltransferase
L. All assays were designed to
l
limit the CMP-Neu5Ac consumption to 10–15%, in order to get reli-
able initial rates. All results were duplicated. Kinetic data were
analyzed using LEONARA program.56
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R. Kumar et al. / Carbohydrate Research xxx (2013) xxx–xxx
7
NH2
N3
NHAc
N
NHAc
N
5
6
H
N
R1''
O
H
N1
O
N
O
N
O
H2N
O
R
N
O
5'
S
S
S
O
O
O
RCOOH
b
O
O
4'
1'
2'
O
O
O
O
O
O
a
3O' H OH
O
O
O
O
25
20
25 (i, k, m, o)
R =
c
AcHN
MeO
NO2
a
b
c
OMe
25 (j, l, n, p)
OH
OMe
OMe
e
f
d
NO2
COOMe
COONa
COOMe
g
i
h
NaOOC
MeOOC
O2N
j
COONa
k
l
O2N
COOMe
COONa
o
COOMe
n
q
m
N
N
COONa
N
O
O
H
r
p
Scheme 5. Reagents and conditions: (a) DCC, DMAP, DMF, 48 h, rt; or (i) NHS, DCC, THF, 4 h, 0 °C; (ii) CsCO3, DMF, 12–15 h, 0 °C–rt; (b) TFA, H2O, MeOH, 2–4 h, rt; (c) NaOH
(pH 11.5), 10 h, rt.
4.8. 50-O-{N-[(400-Acetylamino-phenyl)acetyl]sulfamoyl}cytidine
(25b)
7.32 (ddd, J = 8.2, 7.2 and 1.8 Hz, 1H, H-400), 6.81 (ddd, J = 7.9, 7.3
and 1.1 Hz, 1H, H-500), 6.77 (dd, J = 8.2 and 1.1 Hz, 1H, H-300), 5.85
(d, J = 8.0 Hz, 1H, H-5), 5.64 (d, J = 3.5 Hz, 1H, H-10), 4.41 (dd,
J = 11.9 and 1.9 Hz, 1H, H-5a0), 4.32 (dd, J = 11.7 and 3.9 Hz, 1H,
H-5b0), 4.22 (dd, J = 5.1 and 3.7 Hz, 1H, H-20), 4.18–4.14 (m, 2H,
H-30, H-40); 13C NMR (100 MHz, D2O) d 174.2, 159.1 (CO), 148.3
(C-4), 144.4 (C-6), 134.9 (C-100), 130.1 (C-200), 119.9 (C-300), 118.2
(2C, C-600, C-500), 117.1 (C-300), 94.9 (C-5), 91.1 (C-10), 81.9 (C-40),
73.7 (C-20), 69.3 (C-30), 68.8 (C-50); MALDI-HRMS: Calcd for
White solid, ½a 2D0
ꢂ
+36.0 (c 0.3, H2O); 1H NMR (400 MHz, D2O) d
7.64 (d, J = 7.6 Hz, 1H, H-6), 7.39 (d, J = 8.1 Hz, 2H, Ar-H), 7.28 (d,
J = 8.1 Hz, 2H, Ar-H), 5.95 (d, J = 7.6 Hz, 1H, H-5), 5.83 (d,
J = 2.1 Hz, 1H, H-10), 4.51 (m, 2H, H-5ab0), 4.31 (m, 1H, H-40), 4.19
(m, 2H, H-20, H-30), 3.65 (s, 2H, CH2), 2.16 (s, 3H, Ac); 13C NMR
(100 MHz, D2O) d 172.8, 159.1, 148.4, 143.5, 136.4, 130.2, 130.0,
122.3, 121.5, 95.1, 89.7, 81.3, 73.8, 69.5, 68.8, 43.0, 23.0.
C
16H18N4NaO9S [M+Na]+ 465.0686, Found: 465.0637.
4.9. 50-O-[N-(2-Nitrobenzoyl)sulfamoyl]cytidine (25c)
4.11. 50-O-[N-(3,5-Dimethoxybenzoyl)sulfamoyl]cytidine (25e)
White solid, ½a 2D0
ꢂ
+15.0 (c 0.04, H2O); 1H NMR (400 MHz, D2O) d
Amorphous solid, ½a D20
ꢂ
+6.0 (c 0.4, H2O); 1H NMR (400 MHz,
7.91 (dd, J = 8.2 and 1.0 Hz, 1H, H-600), 7.74 (d, J = 7.6 Hz, 1H, H-6),
7.62 (ddd, J = 7.6, 7.4 and 1.2 Hz, H-400), 7.49 (ddd, J = 8.2, 7.4 and
1.6 Hz, 1H, H-500), 7.46 (dd, J = 7.6 and 1.2 Hz, 1H, H-300), 5.86 (d,
J = 7.6 Hz, 1H, H-5), 5.84 (d, J = 4.3 Hz, 1H, H-10), 4.39 (dd, J = 11.5
and 2.1 Hz, 1H, H-5a0), 4.31 (dd, J = 11.5 and 2.7 Hz, 1H, H-5b0),
4.25–4.23 (m, 1H, H-40), 4.21 (dd, J = 4.9 and 4.7 Hz, 1H, H-30),
4.19 (dd, J = 4.9 and 4.5 Hz, 1H, H-20); 13C NMR (100 MHz, D2O) d
175.1 (CO), 165.4 (CO), 156.7 (C-4), 146.0 (C-6), 141.8 (C-200),
134.5 (C-100), 134.4 (C-300), 130.6 (C-600), 128.6 (C-500), 24.4 (C-400),
96.4 (C-5), 89.4 (C-10), 81.8 (C-40), 74.1 (C-20), 69.6 (C-30), 68.3 (C-
D2O) d 7.90 (d, J = 7.6 Hz, 1H, H-60), 7.08 (br s, 2H, Ar-H), 7.73 (br
s, 1H, Ar-H), 6.00 (d, J = 7.6 Hz, 1H, H-5), 5.89 (d, J = 3.9 Hz, 1H,
H-10), 4.56 (dd, J = 2.0 and 12.1 Hz, 1H, H-5a0), 4.49 (dd, J = 4.2
and 12.1 Hz, 1H, H-5b0), 4.30 (m, 3H, H-20, H-30, H-40), 3.87 (s, 6H,
OMe); 13C NMR (100 MHz, D2O) d 173.6, 160.1, 159.1, 148.6,
144.2, 138.0, 109.9, 109.3, 106.6, 104.4, 94.9, 90.7, 81.8, 73.7,
69.1, 68.7, 55.7, 55.6; MALDI-HRMS: Calcd for C18H23N4O10
S
[M+H]+ 487.1129, Found: 487.1152.
4.12. 50-O-[N-(2,6-Dimethoxy-3-nitrobenzoyl)sulfamoyl]
cytidine (25f)
50); MALDI-HRMS: Calcd for C16H18N5O10
Found: 472.0746.
S
[M+H]+ 472.0768,
Amorphous solid, ½a D20
ꢂ
+20.0 (c 0.4, H2O); 1H NMR (400 MHz,
4.10. 50-O-[N-(2-Hydroxybenzoyl)sulfamoyl]cytidine (25d)
D2O) d 8.32 (d, J = 9.4 Hz, 1H, Ar-H), 7.89 (d, J = 8.2 Hz, 1H, H-6),
7.11 (d, J = 9.4 Hz, 1H, Ar-H), 5.81 (br s, 1H, H-10), 5.75 (d,
J = 8.2 Hz, 1H, H-5), 4.88 (dd, J = 12.1 and 2.0 Hz, 1H, H-5a0), 4.67
(dd, J = 12.1 and 4.2 Hz, 1H, H-5b0), 4.46 (m, 1H, H-40), 4.40 (m, 1H,
White solid, ½a 2D0
ꢂ
+25.8 (c 0.15, H2O). 1H NMR (400 MHz, D2O) d
7.75 (d, J = 8.0 Hz, 1H, H-6), 7.69 (dd, J = 7.9 and 1.8 Hz, 1H, H-600),
Please cite this article in press as: Kumar, R.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2012.12.017

8
R. Kumar et al. / Carbohydrate Research xxx (2013) xxx–xxx
H-30), 4.29 (m, 1H, H-20), 3.94 (s, 3H, OMe), 3.89 (s, 3H, OMe); 13C
NMR (100 MHz, D2O) d 161.2, 157.0, 155.4, 148.1, 144.8, 139.4,
131.0, 126.2, 113.8, 103.7, 89.6, 87.0, 77.0, 69.4, 64.5, 59.6, 59.3, 52.5.
7.56 (d, J = 7.5 Hz, 1H, H-6), 5.74 (d, J = 3.4 Hz, 1H, H-10), 5.64 (d,
J = 7.4 Hz, H-5), 4.36 (dd, J = 11.6 and 1.9 Hz, 1H, H-5a0), 4.28 (dd,
J = 11.6 and 3.8 Hz, 1H, H-5b0), 4.19–4.11 (m, 3H, H-20, 30, 40); 13C
NMR (100 MHz, D2O) d 175.3 (2 ꢃ CO), 165.5 (CO), 156.8 (C-4),
141.5 (C-6, C-400), 138.6 (C-100), 128.8, 128.4, 96.3 (C-5), 90.1 (C-
10), 81.5 (C-40), 74.1 (C-20), 69.5 (C-30), 68.3 (C-50); MALDI-HRMS:
Calcd for C17H17N4Na2O10S [M+Na]+ 515.0455, Found: 515.0435.
4.13. 50-O-[N-(2-Carboxylatobenzoyl)sulfamoyl]cytidine sodium
salt (25h)
Amorphous solid, ½a D20
ꢂ
+14.2 (c 0.18, H2O); 1H NMR (400 MHz,
4.18. 50-O-[N-(3-Methoxycarbonyl-5-nitrobenzoyl)sulfamoyl]
cytidine (25m)
D2O) d 7.58 (d, J = 7.5 Hz, 1H, H-6), 7.48 (m, 2H, Ar-H), 7.35 (m,
2H, Ar-H), 5.92 (d, J = 7.6 Hz, 1H, H-5), 5.76 (d, J = 3.7 Hz, 1H, H-
10), 4.40 (dd, J = 11.5 and 2.2 Hz, 1H, H-5a0), 4.30 (dd, J = 11.5 and
3.7 Hz, 1H, H-5b0), 4.19–4.13 (m, 3H, H-20, 30, 40); 13C NMR
(100 MHz, D2O) d 175.7 (2 ꢃ CO), 165.6 (CO), 156.7 (C-4), 141.7
(C-6), 135.6, 130.0, 128.2, 96.4 (C-5), 90.7 (C-10), 80.9 (C-40), 73.7
(C-20), 69.2 (C-30), 68.8 (C-50); MALDI-HRMS: Calcd for
Amorphous solid, ½a D20
ꢂ
+12.0 (c 0.01, H2O); 1H NMR (400 MHz,
D2O) d 8.75–8.73 (m, 2H), 8.64 (dd, J = 1.6 and 1.4 Hz, 1H), 7.87
(d, J = 8.0 Hz, 1H, H-6), 5.93 (d, J = 7.8 Hz, 1H, H-5), 5.71 (d,
J = 3.3 Hz, 1H, H-10), 4.41 (dd, J = 11.7 and 1.8 Hz, 1H, H-5a0), 4.31
(dd, J = 11.7 and 3.1 Hz, 1H, H-5b0), 4.21–4.18 (m, 3H, H-20, 30, 40),
3.85 (s, 3H, OCH3); 13C NMR (100 MHz, D2O) d 171.8 (2 ꢃ CO),
165.3 (CO), 162.4 (C-4), 148.2 (C-6), 143.9 (C-10), 138.9 (C-500),
135.0 (C-300), 131.7 (C-200), 127.6 (C-600), 127.1 (C-400), 95.1 (C-5),
90.4 (C-10), 81.9 (C-40), 74.0 (C-20), 69.2 (C-30), 68.2 (C-50), 53.4
(OCH3); MALDI-HRMS: Calcd for C18H20N5O12S [M+H]+ 530.0824,
Found: 530.0793.
C
17H18N4NaO10S [M+H]+ 493.0636, Found: 493.0635.
4.14. 50-O-[N-(3-Methoxycarbonylbenzoyl)sulfamoyl]cytidine
(25i)
Amorphous solid, ½a D20
ꢂ
+53.8 (c 0.07, H2O); 1H NMR (400 MHz,
D2O) d 8.29 (dd, J = 1.8 and 1.6 Hz, 1H, H-200), 8.02 (ddd, J = 7.8,
1.6 and 1.4 Hz, 1H, H-400), 7.97 (ddd, J = 7.8, 1.8 and 1.2 Hz, 1H,
H-600), 7.76 (d, J = 8.0 Hz, 1H, H-6), 7.45 (dd, J = 7.8 and 7.8 Hz,
1H, H-500), 5.83 (d, J = 8.0 Hz, 1H, H-5), 5.65 (d, J = 3.6 Hz, 1H, H-
10), 4.45 (dd, J = 11.7 and 1.8 Hz, 1H, H-5a0), 4.33 (dd, J = 11.9 and
3.9 Hz, 1H, H-5b0), 4.21–4.13 (m, 3H, H-20, 30, 40), 3.80 (s, 3H,
OCH3); 13C NMR (100 MHz, D2O) d 173.0, 168.6, 159.1, 148.3 (C-
4), 144.3 (C-6), 135.9 (C-100), 133.5 (C-300), 133.2 (C-60), 133.1 (C-
400), 129.4 (C-200), 129.2 (C-500), 94.9 (C-5), 90.7 (C-10), 81.8 (C-40),
73.8 (C-30), 69.2 (C-20), 68.8 (C-50), 53.0 (OCH3); MALDI-HRMS:
Calcd for C18H21N4O10S [M+H]+ 485.0973, Found: 485.0935.
4.19. 50-O-[N-(3-Carboxylato-5-nitrobenzoyl)sulfamoyl]cytidine
sodium salt (25n)
Amorphous solid; 1H NMR (400 MHz, D2O) d 8.66–8.65 (m, 2H),
8.56–8.54 (m, 1H), 7.77 (d, J = 8.0 Hz, 1H, H-6), 5.82 (d, J = 8.0 Hz,
1H, H-5), 5.69 (d, J = 3.4 Hz, 1H, H-10), 4.41 (dd, J = 11.9 and
1.9 Hz, 1H, H-5a0), 4.32 (dd, J = 11.9 and 3.6 Hz, 1H, H-5b0), 4.20–
4.15 (m, 3H, H-20, 30, 40); 13C NMR (100 MHz, D2O) d 172.5, 172.0,
161.8 (CO), 148.0 (C-6), 143.4 (C-4), 138.2, 135.1, 134.7, 126.7,
126.2, 125.7, 95.4 (C-5), 90.5 (C-10), 81.8 (C-40), 73.9 (C-20), 69.3
4.15. 50-O-[N-(3-Carboxylatobenzoyl)sulfamoyl]cytidine sodium
salt (25j)
(C-30), 68.3 (C-50); MALDI-HRMS: Calcd for C17H17N5NaO12
[M+H]+ 538.0487, Found: 538.0458.
S
Amorphous solid, 1H NMR (400 MHz, D2O) d 8.20 (br s, 1H,
H-200), 7.88–7.85 (dd, J = 7.6 and 1.0 Hz, 2H, H-400, H-600), 7.60 (d,
J = 7.6 Hz, 1H, H-6), 7.36 (dd, J = 7.8 and 7.8 Hz, 1H, H-500), 5.75
(d, J = 3.7 Hz, 1H, H-10), 5.68 (d, J = 6.1 Hz, 1H, H-5), 4.37 (dd,
J = 11.7 and 2.0 Hz, 1H, H-5a0), 4.27 (dd, J = 11.7 and 3.7 Hz, 1H,
H-5b0), 4.20–4.12 (m, 3H, H-20, 30, 40); 13C NMR (100 MHz, D2O) d
175.4 (2 ꢃ CO), 174.8 (CO), 165.7 (C-4), 157.1 (C-6), 141.4 (C-100),
136.4 (C-300), 132.4 (C-400), 131.1 (C-600), 128.8 (C-200), 128.5 (C-500),
96.4 (C-5), 90.0 (C-10), 81.6 (C-40), 74.1 (C-20), 69.4 (C-30), 68.1 (C-
50); MALDI-HRMS: Calcd for C17H18N4NaO10S [M+H]+ 493.0636,
Found: 493.0691.
4.20. 50-O-{[N-(2-Methyl ethanoate)benzoyl]sulfamoyl}cytidine
(25o)
Amorphous solid, ½a D20
ꢂ
+37.3 (c 0.08, H2O); 1H NMR (400 MHz,
D2O) d 7.74 (d, J = 8.0 Hz, 1H, H-6), 7.51 (dd, J = 7.6 and 1.2 Hz,
1H, H-600), 7.42 (ddd, J = 7.5, 7.4 and 1.4 Hz, 1H, H-400), 7.30 (ddd,
J = 7.6, 7.4 and 1.2 Hz, 1H, H-500), 7.21 (dd, J = 7.1 and 0.4 Hz, 1H,
H-300), 5.94 (d, J = 8.0 Hz, 1H, H-5), 5.65 (d, J = 3.7 Hz, 1H, H-10),
4.54 (dd, J = 11.8 and 2.0 Hz, 1H, H-5a0), 4.45 (dd, J = 11.8 and
3.1 Hz, H-5b0), 4.22–4.15 (m, 3H, H-20, 30, 40), 3.78 (d, J = 2.4 Hz,
2H, CH2), 3.53 (s, 3H, OCH3); 13C NMR (100 MHz, D2O) d 174.7,
171.5, 159.1 (CO), 148.3 (C-4), 144.6 (C-6), 133.4 (C-100), 133.2 (C-
200), 132.5 (C-600), 132.4 (C-300), 128.8 (C-400), 128.0 (C-5’’), 95.0 (C-
5), 90.4 (C-10), 81.2 (C-40), 73.1 (C-30), 70.1 (C-20), 68.8 (C-50), 52.7
4.16. 50-O-[N-(4-Methoxycarbonylbenzoyl)sulfamoyl]cytidine
(25k)
White amorphous solid, ½a D20
ꢂ
+60.0 (c 0.4, H2O); 1H NMR
(OCH3), 39.0 (CH2); MALDI-HRMS: Calcd for C19H23N4O10
S
[M+H]+ 499.1129, Found: 499.1099.
(400 MHz, D2O) d 7.90 (d, J = 8.5 Hz, 2H, Ar-H), 7.81 (d, J = 8.8 Hz,
2H, Ar-H), 7.72 (d, J = 7.7 Hz, 1H, H-6), 5.80 (d, J = 7.7 Hz, 1H, H-
5), 5.69 (d, J = 3.7 Hz, 1H, H-10), 4.38 (dd, J = 11.8 and 1.8 Hz, 1H,
H-5a0), 4.29 (dd, J = 11.8 and 3.7 Hz, 1H, H-5b0), 4.20–4.12 (m, 3H,
H-20, 30, 40), 3.80 (s, 3H, OCH3); 13C NMR (100 MHz, D2O) d 174.6,
168.8, 161.0, 150.9 (C-4), 143.5 (C-6), 140.8 (C-100), 132.4 (C-400),
129.5 (C-600, C-200), 128.6 (C-300, C-500), 95.3 (C-5), 90.5 (C-10), 81.9
(C-40), 74.0 (C-20), 69.3 (C-30), 68.3 (C-50), 52.9 (OCH3); MALDI-
HRMS: Calcd for C18H21N4O10S [M+H]+ 485.0973, Found: 485.0939.
4.21. 50-O-{[N-(2-Ethanoxylato)benzoyl]sulfamoyl}cytidine
sodium salt (25p)
Amorphous solid, ½a D20
ꢂ
+51.4 (c 0.04, H2O); 1H NMR (400 MHz,
D2O) d 7.69 (d, J = 7.6 Hz, 1H, H-6), 7.47 (dd, J = 7.6 and 1.2 Hz,
1H, H-600), 7.30 (ddd, J = 7.6, 7.4 and 1.4 Hz, 1H, H-400), 7.19 (ddd,
J = 7.6, 7.4 and 1.2 Hz, 1H, H-500), 7.13 (d, J = 7.6 Hz, 1H, H-300),
5.82–5.80 (m, 2H, H-10, 5), 4.35 (dd, J = 11.7 and 2.1 Hz, 1H, H-
5a0), 4.28 (dd, J = 11.7 and 3.3 Hz, 1H, H-5b0), 4.22–4.14 (m, 3H,
H-20, 30, 40), 3.68 (d, J = 8.8 Hz, 2H, CH2); 13C NMR (100 MHz, D2O)
d 173.8, 170.7, 165.6, 156.9 (C-4), 142.1 (C-6), 141.7 (C-100), 131.7
(C-200), 131.2 (C-500), 130.9 (C-300), 130.5 (C-400), 128.9 (C-600), 96.3
(C-5), 90.6 (C-10), 80.9 (C-40), 73.7 (C-20), 69.2 (C-30), 68.8 (C-50),
4.17. 50-O-[N-(4-Carboxylatobenzoyl)sulfamoyl]cytidine sodium
salt (25l)
Amorphous solid, ½a D20
ꢂ
+20.0 (c 0.08, H2O); 1H NMR (400 MHz,
D2O) d 7.76 (d, J = 8.4 Hz, 2H, Ar-H), 7.71 (d, J = 8.4 Hz, 2H, Ar-H),
Please cite this article in press as: Kumar, R.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2012.12.017

R. Kumar et al. / Carbohydrate Research xxx (2013) xxx–xxx
9
41.7 (CH2); MALDI-HRMS: Calcd for C18H20N4NaO10
507.0792, Found: 507.0818.
S
[M+H]+
(C-100), 82.3 (C-300), 76.5 (C-6), 73.5 (C-200), 70.3 (C-400), 70.0 (C-8),
67.8 (C-7), 67.1 (C-4), 63.2 (C-9), 50.6 (C-10), 49.9 (C-5), 42.5 (C-
500), 36.6 (C-20), 22.2 (–NAc).
4.22. 50-O-[N-(200-Oxo-2H-chromene-300-carbonyl)sulfamoyl]
cytidine (25q)
Compound 24d: Amorphous solid, ½a D20
ꢂ
+9 (c 0.3, H2O); 1H NMR
000,5000
(400 MHz, D2O) d 7.60 (d, J6
= 7.6 Hz, 1H, H-6000), 6.02 (d, 1H, H-
5000), 5.85 (d, J3,4 = 2.4, 1H, H-3), 5.81 (d, J1 ,2 = 4.4 Hz, 1H, H-100),
4.52 (dd, J4,5 = 8.8 Hz, 1H, H-4), 4.44 (dd, 1H, H-10), 4.34 (m, 2H,
H-200, 6), 4.13 (m, 3H, H-300, 400, 5), 3.97 (m, 1H, H-8), 3.89 (dd,
J9,8 = 2.8, J9a,9b = 16.0 Hz, 1H, H-9a), 3.69 (m, 2H, H-9b, 7), 3.60
(m, 2H, H-500a, 500b), 2.38 (m, 2H, 2 ꢃ H-30), 2.09 (s, 3H, NAc), 2.10
(m, 2H, H-20), 13C NMR (100 MHz, D2O) d 178.0, 174.9, 173.4,
166.3, 163.5, 157.6, 145.3, 142.0 (C-6000), 109.0 (C-3), 96.3 (C-5000),
91.0 (C-100), 81.7 (C-300), 76.5 (C-6), 73.5 (C-200), 70.7 (C-400), 69.9
(C-8), 67.9 (C-7), 67.1 (C-4), 63.1 (C-9), 53.6 (C-10), 49.9 (C-5),
40.7 (C-500), 31.1 (C-3000), 26.8 (C-2000), 22.2 (–NAc); MALDI-HRMS:
Calcd for C25H38N7O13 [M+H]+ 644.2522, Found: 644.2491.
00 00
Amorphous solid, ½a D20
ꢂ
+17.0 (c 0.4, H2O); 1H NMR (400 MHz,
D2O) d 8.45 (s, 1H, Ar), 7.96 (d, J = 7.6 Hz, 1H, H-6), 7.76 (m, 2H,
Ar), 7.45 (m, 2H, Ar), 6.10 (d, J = 7.6 Hz, 1H, H-5), 5.89 (d,
J = 2.1 Hz, 1H, H-10), 4.49 (m, 2H, H-5ab0), 4.36 (br s, 1H, H-40),
4.31 (m, 2H, H-20, 30); 13C NMR (100 MHz, D2O) d 172.8, 159.1,
151.9, 147.0, 143.8, 134.3, 129.7, 125.6, 123.2, 118.3, 116.5, 95.2,
90.3, 81.8, 73.9, 69.2, 68.6; MALDI-HRMS: Calcd for C19H19N4O10
S
[M+H]+ 495.0816, Found: 495.0836.
4.23. 50-O-[N-(1H-Benzotriazole-500-carbonyl)sulfamoyl]cytidine
(25r)
Compound 24e: Amorphous solid, ½a D20
ꢂ
+9 (c 0.4, H2O); 1H NMR
(400 MHz, D2O) d 7.61, 7.48, 7.32, 7.24, 7.17 (tryptophan), 7.30 (d,
Amorphous solid, ½a D20
ꢂ
+26.0 (c 0.3, H2O); 1H NMR (400 MHz,
J6
= 7.6 Hz, 1H, H-6000), 5.86 (d, 1H, H-5000), 5.86 (d, J3,4 = 2.4, 1H,
000,5000
00 00
D2O) d 8.47 (s, 1H, Ar), 8.03 (d, J = 8.2 Hz, 1H, H-6), 7.89 (m, 2H,
Ar), 5.89 (d, J = 7.6 Hz, 1H, H-5), 5.81 (d, J = 3.9 Hz, 1H, H-10), 4.55
(dd, J = 2.0, and 12.1 Hz, 1H, H-5a0), 4.47 (dd, J = 4.2 and 12.1 Hz,
1H, H-5b0), 4.32 (m, 3H, H-20, 30, 40); 13C NMR (100 MHz, D2O) d
174.3, 159.5, 149.1, 143.9, 134.4, 126.8, 125.7, 116.8, 116.3,
113.9, 94.8, 90.6, 81.9, 73.9, 69.2, 68.4; MALDI-HRMS: Calcd for
H-3), 5.59 (d, J1 ,2 = 4.4 Hz, 1H, H-100), 4.76 (t, 1H, H-10), 4.50 (dd,
J4,5 = 8.8 Hz, 1H, H-4), 4.30 (d, 1H, H-6), 4.10 (m, 1H, H-5), 3.95
(m, 3H, H-200, 300, 400), 3.87 (m, 1H, H-9a), 3.68 (m, 3H, H-7, 8, 9b),
3.52 (dd, 1H, H-500a), 3.40 (dd, 1H, H-500b), 3.34 (d, 2H, 2 ꢃ H-20),
2.10 (s, 3H, NAc); 13C NMR (100 MHz, D2O) d 175.8, 174.9, 173.1,
166.3, 160.8, 150.4, 145.2, 144.1 (C-6000), 109.0 (C-3), 95.4 (C-5000),
91.1 (C-100), 82.3 (C-300), 76.5 (C-6), 73.5 (C-200), 70.3 (C-400), 70.0
(C-8), 67.8 (C-7), 67.1 (C-4), 63.2 (C-9), 50.6 (C-10), 49.9 (C-5),
42.5 (C-500), 36.6 (C-20), 22.2 (NAc).
C
16H17N7NaO8S [M+Na]+ 490.0757, Found: 490.0779.
4.24. Synthesis of compounds (24a–e)
The coupled products (23a–e) were dissolved in CH2CH2 (5 ml)
and treated with aqueous TFA (90%, 2 mL). The mixture was stirred
for 2 h after which solvents were removed and the residue was
purified by silica gel column chromatography to give the target
compounds 24a–e.
4.25. Synthesis of compound (21)
To a solution of 16 (212 mg, 0.64 mmol) and 20 (303 mg,
0.83 mmol) in anhydrous DMF were added WSC (1 mmol) and
HOBt (1 mmol) at 0 °C. The reaction mixture stirred under nitrogen
for overnight. The solvent was removed and residue was purified
using silica gel column chromatography. The resulted product
was dissolved in CH2CH2 (5 ml) and treated with aqueous TFA
(90%, 2 ml). The mixture was stirred for 2 h after which solvents
were removed and the residue was purified by silica gel column
Compound 24a: Amorphous solid, ½a D20
ꢂ
ꢁ17 (c 0.5, H2O): 1H
NMR (400 MHz, D2O) d 7.64 (d, J6
= 7.6 Hz, 1H, H-6000), 6.15 (d,
000,5000
1H, H-5000), 5.89 (d, J3,4 = 2.4, 1H, H-3), 5.81 (d, J1 ,2 = 4.4 Hz, 1H,
00 00
H-100), 4.52 (dd, J4,5 = 8.8 Hz, 1H, H-4), 4.36 (d, J6,5 = 10.8 Hz,
1H, H-6), 4.32 (dd, J2 ,3 = 4.4 Hz, 1H, H-200), 4.13 (m, 3H, H-300, 400,
00 00
500), 4.05 (d, J = 6.4 Hz, 2H, 10-CH2), 3.95 (m, 1H, H-8), 3.88 (dd,
J9,8 = 2.8, J9a,9b = 16.0 Hz, 1H, H-9a), 3.67 (m, 2H, H-9b, 7), 3.61
(m, 2H, H-50a, 50b), 2.12 (s, 3H, NAc); 13C NMR (100 MHz, D2O) d
178.0, 174.9, 173.4, 166.3, 163.5, 157.7, 145.3, 142.0 (C-6000),
109.0 (C-3), 96.3 (C-5000), 91.0 (C-100), 81.7 (C-300), 76.5 (C-6), 73.5
(C-200), 70.6 (C-400), 69.9 (C-8), 67.9 (C-7), 67.1 (C-4), 63.1 (C-9),
49.9 (C-5), 42.7 (C-10), 40.7 (C-500), 22.2 (NAc); MALDI-HRMS: Calcd
for C22H32N6O12Na [M+Na]+ 595.1970, Found: 595.1960.
chromatography to give 21 (227 mg, 59%). ½a D20
ꢂ
+32 (c 0.5, H2O);
1H NMR (400 MHz, D2O) d 7.85 (d, J6 ,5 = 7.6 Hz, 1H, H-600), 6.08
00 00
(d, J6 ,5 = 7.6 Hz, 1H, H-500), 5.95 (d, J3,4 = 3.6 Hz, 1H, H-3), 5.84 (d,
00 00
J1 ,2 = 2.4 Hz, 1H, H-10), 4.49 (m, 1H, H-40), 4.46 (m, 1H, H-20),
0
0
4.39 (m, 1H, H-30), 4.29 (m, 4H, H-4, 5, 6, 5a0), 4.07 (dd,
J5b ,5a = 11.2, J5b ,4 = 8.8 Hz, 1H, H-5b0), 3.94 (ddd, J8,7 = 3.9,
J8,9a = 3.0, J8,9b = 13.6 Hz, 1H, H-8), 3.89 (dd, J9a,9b = 12 Hz, 1H, H-
9a), 3.49 (m, 2H, H-7, 9b) 2.08 (s, 3H, NAc); 13C NMR (100 MHz,
D2O) d 174.8 (NAc), 169.8 (C-1), 166.1 (C-4000), 157.3 (C-2), 141.5
(C-6000), 108.7 (C-3), 96.4 (C-5000), 89.8 (C-10), 81.4 (C-40), 75.7 (C-
6), 74.1, 69.9 (C-8), 69.3 (C-20), 68.2 (C-4), 67.8 (C-7), 63.1 (C-9),
0
0
0
0
Compound 24b: Amorphous solid, [
a
]
ꢁ31 (c 0.5, H2O); 1H
D
= 7.6 Hz, 1H, H-6000), 6.21 (d,
000,5000
NMR (400 MHz, D2O) d 7.87 (d, J6
1H, H-5000), 5.84 (d, J3,4 = 2.4, 1H, H-3), 5.78 (d, J1 ,2 = 4.4 Hz, 1H,
H-100), 4.54 (dd, J4,5 = 8.8 Hz, 1H, H-4), 4.36 (m, 3H, H-200, H-6, H-
10), 4.16 (m, 3H, H-300, 400, 500), 3.91 (m, 2H, H-8, H-9a), 3.68 (m,
4H, H-7, 9b, 50a, 50b), 2.98 (t, 2H, 2 ꢃ H-50), 2.08 (s, 3H, NAc), 1.85
(m, 2H, 2 ꢃ H-20), 1.69 (m, 2H, 2 ꢃ H-40), 1.45 (m, 2H, 2 ꢃ H-30);
13C NMR (100 MHz, D2O) d 175.0, 174.0, 166.3, 164.2, 145.4,
144.6 (C-6000), 109.0 (C-3), 95.1, 91.5, 82.3, 76.5, 73.5, 70.6, 70.1,
67.7, 67.1, 63.1, 61.8, 53.8, 49.9, 40.8, 39.3, 30.7, 26.4, 22.2, 22.1,
13.3.
00 00
54.5 (C-5), 22.2 (NAc); MALDI-HRMS: Calcd for C20H30N5O14
S
[M+H]+ 596.1505, Found: 596.1514.
4.26. Synthesis of N-acetyl-20,30-isopropylidine-50-O-
sulfamoylcytidine (20)
To a solution of N-acetyl-20,30-isopropylidinecytidine (5.0 g,
1.0 equiv) in DME (10 ml) at 0 °C was added sulfamoyl chloride
(4.4 g, 2.5 equiv) over 15 min under nitrogen atmosphere. The
reaction mixture was warmed to room temperature and stirred
for 16 h, and was then diluted with ice-cold brine solution and ex-
tracted with EtOAc. The organic layer was dried with anhydrous
Na2SO4 and concentrated. Purification by silica gel chromatogra-
phy (EtOAc/methanol, 15:1) gave desired product as a white solid
(4.7 g, 76% yield). 1H NMR (400 MHz, CD3OD) d 8.04 (d, J = 7.4 Hz,
1H, H-6), 7.37 (d, J = 7.4 Hz, 1H, H-5), 5.83 (d, J = 1.8 Hz, 1H,
Compound 24c: Amorphous solid; 1H NMR (400 MHz, D2O) d
= 7.6 Hz, 1H, H-6000), 6.20 (d, 1H, H-5000), 5.88 (d,
000,5000
7.82 (d, J6
J3,4 = 2.4, 1H, H-3), 5.79 (d, J1 ,2 = 4.4 Hz, 1H, H-100), 4.76 (m, 1H,
H-10), 4.54 (dd, J4,5 = 8.8 Hz, 1H, H-4), 4.35 (m, 2H, H-4, 6), 4.13
(m, 3H, H-300, 400, 5), 3.91 (m, 2H, H-8, 9a), 3.67 (m, 2H, H-7, 9b),
3.62 (m, 2H, H-500a, 500b), 2.88 (d, 2H, H-20a, 20b), 2.08 (s, 3H,
NAc). 13C NMR (100 MHz, D2O) d 175.8, 174.9, 173.1, 166.3,
160.8, 150.4, 145.2, 144.1 (C-6000), 109.0 (C-3), 95.4 (C-5000), 91.1
00 00
Please cite this article in press as: Kumar, R.; et al. Carbohydr. Res. (2013), http://dx.doi.org/10.1016/j.carres.2012.12.017

10
R. Kumar et al. / Carbohydrate Research xxx (2013) xxx–xxx
H-10), 5.01 (dd, J = 6.4 and 1.8 Hz, 1H, H-20), 4.87 (dd, J = 6.3 and
3.5 Hz, 1H, H-30), 4.79 (br s, 2H, NH2), 4.46–4.43 (m, 1H, H-40),
4.36 (dd, J = 10.7 and 3.9 Hz, 1H, H-50a), 4.29 (dd, J = 10.7 and
5.5 Hz, 1H, H-50b), 2.15 (s, 3H, NHCOCH3), 1.52 (s, 3H, CH3), 1.32
(s, 3H, CH3); 13C NMR (100 MHz, CD3OD) d 165.5 (CO), 157.9 (C-
4), 150.6 (C-6), 114.8 (CMe2), 98.3 (C-5), 97.4 (C-10), 87.3 (C-40),
86.6 (C-20), 82.8 (C-30), 70.2 (C-50), 27.5 (CH3), 25.5 (CH3), 24.7
(NHCOCH3); MALDI-HRMS: Calcd for C14H20N4NaO8S [M+Na]+
427.0900, Found: 427.0912.
3H, NAc); 13C NMR (100 MHz, D2O) d 175.1 (C-1), 171.8 (NAc),
170.4 (C-500), 162.9 (C-2000), 152.3 (C-4000), 144.2 (C-20), 124.8 (C-10),
95.1 (C-5000), 91.7 (C-100), 91.2 (C-2), 82.2 (C-400), 74.3 (C-6), 73.5
(C-300), 71.4 (C-8), 70.1 (C-200), 68.2 (C-4), 68.0 (C-7), 62.8 (C-9),
51.6 (C-5), 39.6 (C-3), 34.1 (C-30), 22.2 (NAc); MALDI-HRMS: Calcd
for C23H32N8O13Na [M+Na]+ 651.1987, Found: 651.1969.
4.29. Molecular docking experiment of compound 13 into the
sialyltransferase Cst-I using Autodock4
4.27. Synthesis of compound (19)
Coordinates of the
a-2,3-sialyltransferase Cst-I were extracted
from the X-ray crystal structure of the CMP-3FNeuAc (CSF) com-
plex obtained from the Protein Data Bank (2P2V). All ‘hetero’ atoms
including those belonging to CSF ligand, waters, chloride ions, and
ethylene glycol were removed. Hydrogen atoms were added auto-
matically by the Biopolymer module of InsightII (Accelrys, San Die-
go, 1995).
Using the same InsightII package, the inhibitor structure was
built with the Biopolymer module. It was then subjected to a
300-step energy minimization with a quasi-Newton BFGS method
using the DISCOVER-3 program (CFF91 forcefield) to alleviate bad
atomic contacts.
A solution of aqueous trifluoroacetic acid (90%, 2 mL) was added
drop wise to a solution of the compound 18 (190 mg, 0.264 mmol)
in CH2Cl2 at 0 °C, and the mixture was stirred for 1 h at rt. Then,
solvents were removed under reduced pressure and co-evaporated
with toluene. The residue was purified by column chromatography
to give 19 (86 mg, 58%). ½a D20
ꢂ
+11 (c 0.5, H2O); 1H NMR (400 MHz,
= 7.6 Hz, 1H, H-6000), 5.94 (d, J
D2O) d 7.96 (s, 1H, H-10), 7.10 (d, J6
6000,5000
000, 5000
= 7.6 Hz, 1H, H-5000), 5.84 (d, J3,4 = 3.1 Hz, 1H, H-3), 5.79 (d,
J1 ,2 = 2.5 Hz, 1H, H-100), 4.88 (d, J5a ,5b =15.2, J5a ,4 = 3.2 Hz, 1H,
00 00
00
00
00 00
H-5a00), 4.78 (m, 1H, H-5b00), 4.58 (dd, 1H, H-10), 4.51 (dd, J 2 ,
00
= 8.9 Hz, 1H, H-200), 4.38 (m, 1H, H-40), 4.33 (d, J6,5 = 10.5 Hz,
300
An AUTODOCK Tool (ADT) program—a graphical interface of Auto-
dock, was used to process the ligands and the receptor for the
docking. Atomic charges using the Gasteiger PEOE and Kollman
methods were added to the ligand and the receptor, respectively,
then non-polar hydrogens were merged. Atom types of the ligand
molecule and atomic solvation parameters for the protein were as-
signed automatically using ADT. The ligand was initially put at the
binding location of the CSF ligand by spatially overlaying the corre-
sponding cytidine ring. For each atom type, a grid map centered on
the ligand of 61 ꢃ 61 ꢃ 61 points with a spacing of 0.375 Å was
pre-calculated using AUTOGRID4 program as well as an electrostatic
potential map and a desolvation map with the same spatial set-
tings were also pre-calculated.
For data production, a set of 100 docking experiments was per-
formed with a modified AUTODOCK4 program on a Linux computer
using a Lamarckian Genetic Algorithm (LGA) method. The original
AUTODOCK 4 program was modified to remove the overall molecular
rotation of the ligand during docking of the ligand to the receptor
(H. Jarrell, personal communication). A population of 250 random
individuals and a maximum of 10,000,000 energy evaluations or
27,000 generations were used in each LGA docking run where a
pseudo-Solis and Wets local search method having a maximum
of 300 iterations was also used.
1H, H-6), 4.22 (dd, J4,5 = 5.6 Hz, 1H, H-4), 4.12 (m, 2H, H-300, 5),
3.91 (ddd, J8,7 = 4.0, J8,9a = 3.2, J8,9b= 13.6 Hz, 1H, H-8), 3.87 (d, 1H,
H-9a), 3.67 (m, 2H, H-9b, 7) 2.10 (s, 3H, NAc); 13C NMR
(100 MHz, D2O) d 174.9 (CO), 166.2 (C-1), 164.9 (CO), 157.4
(C-2), 145.9 (C4000), 144.6 (C-20), 141.6 (C-6000), 125.3 (C-10), 108.6
(C-3), 96.2 (C-5000), 91.4 (C-100), 80.5 (C-400), 76.4 (C-6), 73.1, 70.0
(C-8), 69.8 (C-200), 67.8 (C-4), 67.1 (C-7), 63.1 (C-9), 50.7 (C-500),
49.9 (C-5), 34.5 (C-30), 22.2 (NAc); MALDI-HRMS: Calcd for
C
23H32N8O11Na [M+Na]+ 619.2083, Found: 619.2043.
4.28. Synthesis of compounds 12 and 13
A solution of aqueous trifluoroacetic acid (90%, 2 mL) was added
dropwise to a solution of the compound 10 or 11 in CH2Cl2 at 0 °C,
and the mixture was stirred for 2 h at rt. Then, solvents were re-
moved under reduced pressure and co-evaporated with toluene.
The residue was re-dissolved in methanol (2 ml) and treated with
1 M NaOH solution at 0 °C for 12 h. The reaction solution was ad-
justed to pH 2 with Amberlite 120 (H+), the solution was filtered
to remove the resin, and the filtrate was concentrated in vacuo.
The residue was loaded onto
a column of Bio-gel P-2
(2 ꢃ 100 cm) and eluted with water. The fractions containing the
product were pooled and lyophilized to afford 12 and 13.
The docking experiment allowed the ligand’s tortions to be var-
ied as well molecular translation of the ligand. The docked struc-
tures were clustered with a tolerance of 2 Å and ranked based on
their estimated free energy of binding.
Compound 12: Yield 53%,½a D20
ꢂ
+8 (c 0.2, H2O); 1H NMR
(400 MHz, D2O) d 8.68 (s, 1H, H-10), 7.84 (d, J6
= 7.6 Hz, 1H, H-
000,5000
6000), 6.03 (d, J5
= 7.6 Hz, 1H, H-5000), 5.81 (d, J1 ,2 = 3.2 Hz, 1H,
00 00
000,6000
00 00
H-100), 4.41 (d, J2 ,3 = 5.6 Hz, 1H, H-200), 4.27 (m, 1H, H-400), 4.24
(dd, J3 ,4 = 4.0 Hz, 1H, H-300), 3.96 (m, 2H, H-4, 5), 3.92 (m, 4H, H-
00 00
Acknowledgements
500a, 500b, 8, 9a), 3.66 (m, 2H, H-7, 9b), 3.37 (dd, J3a,3b = 11.5,
J3a,4 = 4.0 Hz, 1H, H-3a), 2.27 (dd, J3b,4 = 10.5, 1H, H-3b), 2.14 (s,
3H, NAc); 13C NMR (100 MHz, D2O) d 175.2 (C-1), 170.5 (NAc),
162.5 (C-40), 160.5 (C-2000), 144.3 (C-4000), 140.5 (C-20), 124.8 (C-10),
95.1 (C-5000), 91.8 (C-100), 91.3 (C-2), 82.3 (C-400), 74.4 (C-6), 73.5
(C-300), 71.4 (C-8), 70.2 (C-200), 68.2 (C-4), 68.0 (C-7), 62.9 (C-9),
51.6 (C-5), 39.6 (C-3), 39.7 (C-30), 22.2 (NAc); MALDI-HRMS: Calcd
for C23H32N8O13Na [M+Na]+ 651.1987, Found: 651.1970.
This is NRC-CNRC Publication No. 50022. We thank Mr. Ken
Chan and Mr. Jacek Stupak (IBS/NRC) for MS analysis and Mrs. Mar-
ie-France Goneau (IBS/NRC) for the assistance in FCHASE-based
inhibition assay.
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Compound 13: Yield 48%, ½a D20
ꢂ
+23 (c 0.3, H2O); 1H NMR
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