Transition State-Based Sialyltransferase Inhibitors: Mimicking Oxocarbenium Ion by Simple Amide

Article abstract of DOI:10.1021/acs.jmedchem.6b01644

In the new transition-state based sialyltransferase inhibitors, an amide group was placed at the corresponding C-2 position of CMP-sialic acid to mimic the geometry and charge distribution in the transition state, and simple aromatic or aliphatic rings were used instead of the sialic acid moiety. All synthetic compounds exhibited excellent α(2-6)-sialyltransferase inhibition, resulting in up to a 2600-fold higher affinity for the enzyme than CMP-Neu5Ac, suggesting that amide is a key element for simulating transition-state features.

Full text of DOI:10.1021/acs.jmedchem.6b01644

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Brief Article
Transition State-Based Sialyltransferase Inhibitors:
Mimicking Oxocarbenium Ion by Simple Amide
Jian Guo, Wenming Li, Weiwei Xue, and Xin-Shan Ye
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01644 • Publication Date (Web): 06 Feb 2017
Downloaded from http://pubs.acs.org on February 7, 2017
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Transition State-Based Sialyltransferase Inhibitors: Mimicking
Oxocarbenium Ion by Simple Amide
Jian Guo,† Wenming Li,† Weiwei Xue,‡ and Xin-Shan Ye*,†
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†State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Xue Yuan
Road No. 38, Beijing 100191, China
‡School of Pharmaceutical Sciences and Innovative Drug Research Centre, Chongqing University, Daxuecheng South Road
No. 55, Chongqing 401331, China
Supporting Information
ABSTRACT: In the new transition-state-based sialyltransferase inhibitors, an amide group was placed at the corresponding C-2
position of CMP-sialic acid to mimic the geometry and charge distribution in the transition-state, and simple aromatic or aliphatic
rings were used instead of sialic acid moiety. All synthetic compounds exhibited excellent α(2-6)-sialyltransferase inhibition, result-
ing in up to a 2600-fold higher affinity for the enzyme than CMP-Neu5Ac, suggesting that amide is a key element for simulating
transition-state features.
 INTRODUCTION
Glycosidases and glycosyltransferases are involved in the bio-
synthesis of glycoconjugates associated with intercellular
recognition, metastasis, immune response, etc., and represent
viable therapeutic targets.1-6 Of particular interest is the devel-
opment of transition-state analogues as potent inhibitors for
the discovery of new therapeutic agents,7-14 as Pauling pro-
posed in 1946 that inhibitors closely imitating the structure of
the transition-state in an enzyme-catalyzed process could bind
more tightly to the active site of the enzyme than ground-state
substrates.15 Many different kinds of glycosidase inhibitors
based on the transition-state analogues have been reported and
some of them have been developed into clinic drugs success-
fully. However, only very limited glycosyltransferase inhibi-
tors based on this strategy were reported, and only a few could
exhibit inhibition in the nanomolar range. Obviously, how to
mimic the transition-state in the glycosyltransferase-catalyzed
process more efficiently needs to be further explored.
Of all glycosyltransferases, sialyltransferase (ST) is un-
Figure 1. Mechanism of the sialylation reactions.
doubtedly one of the most important enzymes due to their
biological significance.16-19 Potent ST inhibitors could have
potential applications in both glycobiology as probes and clin-
ic treatment as anticancer, immunoregulation drugs, etc. Alt-
hough current transition-state-based ST inhibitors are thought
to possess poor cellular permeability because of charged phos-
phate groups, it is possible to solve this problem through pro-
drug or other drug delivery strategies. Encouraged by these
facts, and as a continuation of our interest in this field,20-22 we
deemed it an important goal to search for new transition-state
analogues as potent ST inhibitors. According to the proposed
SN1-like mechanism of the ST-catalyzed sialylation reac-
tions,23-28 the glycosyl donor in the transition state may involve
the partial dissociation of the CMP moiety
with increased distance from the anomeric carbon, and the
formation of a trigonal planar oxocarbenium ion (Figure 1).
Recognition of these salient features of the transition-state
could guide the design of potential inhibitors. Compounds 1−4
stand for four types of successful ST transition-state analogue
inhibitors (Figure 2).12,13,21,29 In these inhibitors, functional
groups with planar sp2 carbons such as olefins and aryls, or
functional groups with a planar conformation such as cyclo-
pentane, were adopted to imitate the planar configuration of
the oxocarbenium ion. It is generally believed that the separat-
ed charges of the donor in transition-state will be stabilized by
the ST, though what factors are responsible for this stabiliza-
tion remains an open question. There is a higher chance that
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the charged sialyl oxocarbenium mutually interacts with polar
high affinity based on Schmidt’s SAR study were also intro-
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amino-acid residues, the dipole of α-helix, or other factors at
the active site of ST to realize the transient stability of transi-
tion-state. However, both the partially positive charge distribu-
tion between the anomeric carbon and the ring-oxygen, as well
as the resulting interaction with ST have never been taken into
account in the previous inhibitor design, and usual nonpolar
planar functional groups could not mimic the situation here
effectively.
duced into the inhibitors, such as keeping CMP with another
adjacent negative charge center and an increased distance be-
tween the presumptive anomeric carbon and the CMP.13, 29, 34
In addition, the reported inhibitors 5g and 5h also need to be
synthesized for comparison and control research. Compound
5g, just lacking the amide bond, possesses the very similar
structure to 5c.21 Compound 5h is the most potent inhibitor
among the simplified transition-state analogue inhibitors in
which aromatic ring is used to mimic sialyl oxocarbenium.35
After the synthetic work is completed, the inhibitory activities
of these target molecules against α(2−6)-sialyltransferase will
be evaluated.
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a
Figure 2. Typical transition-state analogue inhibitors. h/l stands
for higher or lower moving spots on the TLC as described in the
original references.
Figure 4. Designed transition-state analogues with amide bonds.
 RESULTS AND DISCUSSION
Considering the configuration and charge distribution of the
oxocarbenium, an inhibitor equipped with a both planar and
highly polarized functional group to mimic the characteristics
of the anomeric position of the sialyl oxocarbenium will look
more like the real transition-state.15 The amide bond is one of
the most important functional groups in chemistry and biology.
It is characterized as a both trigonal planar and high-polarity
group due to the strong resonance interaction between the nN
and π*c=o orbitals (Figure 3), resulting in easy formation of
dipole-dipole interaction and hydrogen bond connections
through both nitrogen and oxygen sites.30 These characteristics
have been utilized in the design of glycosidase inhibitors,31-33
but no related work was reported in the design of glycosyl-
transferase inhibitors. If an amide bond is placed at the corre-
sponding anomeric position of transition-state analogues of
CMP-Neu5Ac, will it make the inhibitors and the interaction
between the inhibitors and STs look more like the real transi-
tion-state? This similarity may lead to the discovery of potent
ST inhibitors.
The synthetic route of target compounds 5a−5h was shown in
Scheme 1. Since some final target compounds such as 5a, 5e
and 5f, were sensitive to strong bases and acids, the selection
of proper protective groups in 7 and 8 was crucial. Through
initial screening, Cbz and benzyl were prioritized as protective
groups of cytidine and phosphate. They could be easily re-
moved under neutral transfer hydrogenation, while not affect-
ing the cytosine group.36 Another issue that needs to be noted
is the formation of a new chiral center in 7. Since it has been
confirmed that the absolute configuration of this chiral center
did not show any regular influence on the final inhibitory ac-
tivity,35 and as it was not the focus of our research, no special
attention was paid to the asymmetric synthesis.
With above key points in mind, the synthesis of 5 started
from the known materials 6 containing the simple amides, the
focus of our attention, and which are easily accessible through
one step following the reported procedure. Alcohols 6 were
oxidized to the corresponding aldehydes, which were reacted
with dibenzyl phosphonate to afford the corresponding α-
hydroxy-phosphonates 7. The condensation of 7 with cytidine-
phosphitamide 8 in the presence of H-tetrazole, which was
followed by oxidation with tert-butyl hydroperoxide, afforded
9 as a mixture of four diastereoisomers. The global deprotec-
tion of 9 using 10% Pd/C and 1,4-cyclohexadiene in DMF
provided the final target molecules 5. After filtration and puri-
fication by C-18 reverse-phase column chromatography (H2O
→ H2O/MeOH 5:1), the products 5 were collected in the form
of two diastereoisomers, which were further separated by pre-
parative RP-HPLC. Finally, after converting to their corre-
sponding sodium salts by ion-exchange (IR 120 Na+) and ly-
ophilization from water, the pure products 5-s and 5-l were
obtained as white amorphous solids (s stands for the compo-
Figure 3. Resonance description of the amide bond.
To verify this hypothesis, compounds 5a−5f with an amide
bond were designed (Figure 4). According to the previous
work by Schmidt et al., the Neu5Ac moiety is dispensable.13
Considering the synthetic accessibility, simple aromatic or
aliphatic rings were used to mimic the Neu5Ac residue, and an
amide bond was placed at the corresponding C-2 position of
the sialyl oxocarbenium. Moreover, other positive factors for
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nent with a shorter retention time while l stands for the com-
5a-s showed the highest inhibitory activity with Ki =
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ponent with a longer retention time in preparative RP-HPLC).
0.016±0.005 μM, and its affinity to α(2-6)-sialyltransferase is
2600-fold higher than that of the natural substrate CMP-
Neu5Ac, even stronger than 5h. The 2600-fold enhancement
of affinity indicates compound 5a-s is the most potent sialyl-
transferase inhibitors up to now. Compound 5d-s also showed
comparable activity as 5h. By comparing 5a and 5b with 2,
and 5c with 5g, it is obvious that the insertion of an amide
group here presents a positive effect on increasing the affinity,
with up to an almost 100-fold increase. This clearly emphasiz-
es that the polar amide may be better mimic functional groups
than simple nonpolar planar groups, which is consistent with
our expectation. As an additional proof, in terms of 5e and 5f,
succinimide and phthalimide are different from other amides,
in which the extra carbonyl makes the charge density of nitro-
gen-atom in amides decrease largely, thus the charge-
separated structure functions less in the resonance structure,
and the dipole moment is obviously reduced,38 therefore mak-
ing 5e and 5f just as a configuration mimic, and exhibiting the
similar inhibitory activity to compound 2.
Scheme 1. Synthesis of target compounds 5.
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Table 1. Affinity of CMP-Neu5Ac (Km) to recombinant
human ST6Gal-I and inhibition data of the desired inhibi-
tors.a
compound
inhibition
Km or Ki (μM)
Km/Ki
at 1 μM (%)
CMP-Neu5Ac
5a-s
5a-l
41.630± 6.660d
0.016 ± 0.005
0.174 ± 0.039
0.106 ± 0.029
0.122 ± 0.027
0.060 ± 0.015
0.165 ± 0.023
0.020 ± 0.005
0.045 ± 0.022
0.224 ± 0.045
88.88
61.68
69.73
67.70
76.98
65.70
86.34
80.30
58.83
15.06
34.46
14.35
43.25b,d
12.82b,d
87.58
2601.9
239.3
392.7
341.2
693.8
252.3
2081.5
925.1
185.8
5b-s
5b-l
5c-s
5c-l
5d-s
5d-l
5e-s
5e-l
5f-s
5f-l
Reagents and conditions: a. i) DMSO, (COCl)2, then Et3N; ii)
5.852 ± 0.908d
7.1
5g-s
5g-l
dibenzyl phosphonate, Et3N; b. i) H-tetrazole; ii) tBuOOH; c. 1,4-
a
cyclohexadiene, Pd/C. Yield of crude 5 (2 steps from 7) after
0.019 ± 0.005d
0.20 ± 0.05c
1.0 ± 0.2c
2191.0
230.0
46.0
column chromatography on RP-18 silica gel.
5h
2-h
The inhibition rates and kinetic constants of Ki against re-
combinant human ST6Gal-I (aa 44-406) were carried out
based on the UV/RP-HPLC method developed by Schmidt
group with some modifications.37 The potent inhibitors 5h and
5g with known Ki values were selected as the control. In our
assay, the Km for CMP-Neu5Ac was determined to be 41.63 ±
6.66 μM (see Supporting Information, Figure S1), comparable
to the reported Km value 46 ± 7 μM.13 The inhibition rates of
all the target compounds were obtained at 1 μM level, and
subsequently the Ki values were determined for the com-
pounds whose inhibitory rates were superior to 50% (Table 1).
To our delight, all designed analogues showed potent inhibito-
ry activity, among which 5a−5d exhibited excellent α(2-6)-
sialyltransferase inhibition in the nanomolar range. Compound
2-l
aFor details of the procedures, see the Supporting Information.
13
c
bInhibition at 10 μM (%). Reported data in the reference.
dReported data in the reference.21
In order to explore the putative binding mode, molecular
docking simulations were performed using 5a with human
ST6Gal-I crystal structure (Figure 5). The optimized docking
results showed that the CMP moiety in the inhibitors (S)-5a
and (R)-5a binds the similar site with CMP in the complex of
ST6Gal-I and CMP, and the other moiety in the two inhibitors
binds different sites (Figures 5A–5C). The phenyl in (S)-5a is
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oriented to a hydrophobic region formed by Pro259, Tyr 275
General Information. All chemicals purchased were rea-
and Trp257, which is similar to the recent simulation results.39
It was suggested that the basic histidine residue at catalytic site
(His370 in hST6Gal I) may help to stabilize development of
the cationic transition-state.40 Recent crystal structure research
on hST6Gal-I implied the developing sialyl oxocarbenium in
transition-state could mutually interact with the imidazole side
chain of His370 and/or 6’-hydroxyl group of galactose accep-
tor through concerted mechanism.41 In our docking simulation,
the amide bond in (S)-5a was located in the proximity of resi-
dues such as His370, Asn233 and Ser323, where there is an
analogous position to anomeric carbon of donor. Considering
that the distance between them is within or near hydrogen-
binding distance, it is possible that a slight movement of resi-
due could result in either a strong hydrogen bond or an elec-
trostatic interaction. We speculated one or more of above fac-
tors contributed to the increased affinity.
gent grade and used without further purification unless other-
wise stated. All reactions were carried out under anhydrous
conditions using flame-dried glassware with freshly distilled
solvents, unless otherwise noted. Reactions were monitored by
TLC on silica gel 60 F254 precoated on aluminum plates (E.
Merck). Spots were detected under UV (254 nm) light and/or
by staining with acidic ceric ammonium molybdate. 1H NMR,
and 13C NMR spectra were recorded on an Avance III Bruker-
600 or a Varian INOVA-500 or an Advance DRX Bruker-400
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o
spectrometer at 25 C. 31P NMR spectra were recorded on an
Advance DRX Bruker-400 or a JEOL AL-300 spectrometer at
o
25 C. Chemical shifts (in ppm) were calibrated with the sol-
vent residual peak. Mass spectra were recorded by using a
Waters Xevo G2 Q-TOF or a Bruker APEX IV 70e FT-MS
spectrometer. Optical rotation was determined by a Rudolph
Research Analytical Autopol IV polarimeter. Preparative
HPLC was performed on Agela Venusil XBP preparative C-18
reversed-phase column (10 μm, 22 ×250 mm) with Gilson
HPLC by the buffer system: MeCN/TEAB (0.05 M, pH
7.2−7.5). The purity of all the final compounds was deter-
mined to be ≥95% (see Table S2) by HPLC analysis, which
was performed on an Agilent 1260 Infinity system equipped
with VWD detector and the data were collected at 254 nm.
General Procedure for the Synthesis of 7. To a solution of
DMSO (3.0 mmol, 3.0 equiv) in dried DCM (12 mL) at -78 °C
was added oxalyl chloride (2.5 mmol, 2.5 equiv) dropwise and
stirred for 15 min. Then a solution of alcohol 6 (1.0 mmol, 1.0
equiv) in DCM (2.0 mL) was added into above reaction
mixture. After stirring for another 50 min at -78 °C, TEA (6.0
mmol, 6.0 equiv) was added over 5 min. Then the cooling bath
was removed, and the reaction mixture was allowed to warm
to r.t. and stirred for 0.5 h. The reaction mixture was diluted
with DCM and washed with 1 M HCl and sat. aq. NaHCO3
successively. The combined organic phases were dried with
Na2SO4 and concentrated under reduced pressure to afford the
crude intermediate aldehyde. To a solution of the intermediate
aldehyde in dried DCM (10 mL) was added dibenzyl phos-
phonate (1.4 mmol, 1.4 equiv) and TEA (2.0 mmol, 2.0 equiv)
successively. After stirring at r.t. for 2 h, the reaction mixture
was concentrated under reduced pressure and purified by col-
umn chromatograph to afford 7 as colorless oil.
General Procedure for the Synthesis of 5. To a solution of
7 (0.2 mmol, 1.0 equiv) in DCM (6.0 mL) were added 8 (0.28
mmol, 1.4 equiv) and H-tetrazole (0.4 mmol, 2.0 equiv) under
argon atmosphere. After stirring at r.t. for 1 h, TBHP (0.56
mmol, 5.5 M in decane, 2.8 equiv) was added and the reaction
mixture was stirred for another 1 h. Then the reaction mixture
was diluted with DCM, and washed with sat. aq. NaHCO3.
The resulting organic layer was dried over Na2SO4, filtered,
concentrated to dryness to give crude product 9 as colorless oil.
The crude product 9 could be used for deprotection direcly, or
used after preliminary purification by column chromatography
on silica gel. To the solution of crude 9 in DMF (2.0 mL) were
added Pd/C (equal mass of 9, 10%) in portions under argon
atmosphere and then 1,4-cyclohexadiene (7.2 mmol, 60 equiv)
was added. The reaction mixture was stirred at r.t. for 24 h.
Then the Pd/C was removed by filtration and filtrate was con-
centrated. The residue was purified by column chromatog-
raphy on RP-18 silica gel (H2O → H2O/MeOH 5:1) to give
crude product 5. The diastereoisomers were further separated
by preparative RP-HPLC (the buffer system: MeCN/TEAB,
0.05 M, pH 7.2−7.5) and converted to the form of sodium salt
Figure 5. Molecular docking simulations for inhibitors (S)-5a and
(R)-5a with hST6Gal I crystal structure (PDB code 4js2). (A)
Putative binding mode of (S)-5a. (B) Putative binding mode of
(R)-5a. (C) Superimposition of (S)-5a (green), (R)-5a (purple)
and CMP (yellow) in the crystal structure (PDB code 4js2).
 CONCLUSION
In summary, in an effort to explore new transition-state ana-
logues for the development of potent ST inhibitors, an amide
bond was introduced to mimic the anomeric position in the
transition-state of CMP-Neu5Ac, and simple aromatic or ali-
phatic rings were used to replace Neu5Ac moiety, in coopera-
tion with CMP and phosphonate, which lead to a series of
potent inhibitors, especially 5a-s as the most potent ST inhibi-
tor up to now. Considering amide’s planar configuration and
strong dipolar structure, it is an excellent group for mimicking
characteristics of anomeric position in sialyl oxocarbenium
transition-state, and plays a key role in high affinity of ST
transition-state based inhibitors. Further applicability of this
strategy to other glycosyltransferases is underway.
 EXPERIMENTAL SECTION
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by ion-exchange resin (IR 120 Na+) and lyophilized from wa-
0.31, H2O). H NMR (400 MHz, D2O) δ 7.81 (d, J = 7.6 Hz,
1H), 5.99 (d, J = 7.6 Hz, 1H), 5.83 (d, J = 3.6 Hz, 1H), 4.80
(dd, J = 15.3, 9.9 Hz, 1H), 4.17 – 4.09 (m, 2H), 4.08 – 4.01 (m,
2H), 4.00 – 3.94 (m, 1H), 3.61 – 3.51 (m, 1H), 3.46 – 3.36 (m,
1H), 3.33 – 3.17 (m, 2H), 1.84 – 1.53 (m, 4H); 13C NMR (100
MHz, D2O) δ 168.1, 166.1, 157.6, 141.5, 96.5, 89.4, 82.4 (d, J
= 9.0 Hz), 74.2, 72.3 (dd, J = 143.0, 7.0 Hz), 68.9, 64.0 (d, J =
5.0 Hz), 47.4, 46.8, 25.5, 23.5; 31P (121.5 MHz, D2O) δ 8.52
(d, JP,P = 31.7 Hz), -0.46 (d, JP,P = 29.3 Hz); HRMS (ESI)
calcd. for C15H23N4O12P2 [M-H]- 513.0793, found 513.0804.
4
5
6
7
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ter to give 5-s and 5-l as amorphous solids (s stands for the
component with a shorter retention time while l stands for the
component with a longer retention time).
Trisodium cytidin-5’-yl-(2-benzamido-1-phosphonato-
ethyl)-phosphate (5a). 5a-s: [α]3D0 = +9.09 (c = 0.22, H2O); 1H
NMR (400 MHz, D2O) δ 7.64 (d, J = 7.6 Hz, 1H), 7.59 (d, J =
7.4 Hz, 2H), 7.43 (t, J = 7.4 Hz, 1H), 7.33 (t, J = 7.6 Hz, 2H),
5.83 (d, J = 7.6 Hz, 1H), 5.59 (d, J = 3.5 Hz, 1H), 4.36 (qd, J =
10.0, 3.0 Hz, 1H), 4.10 (dd, J = 10.7, 4.5 Hz, 1H), 4.04 – 3.84
(m, 5H), 3.55 – 3.41 (m, 1H); 13C NMR (100 MHz, D2O) δ
170.2, 165.4, 156.6, 141.3, 133.1, 132.4, 128.8, 127.0, 96.3,
89.6, 82.4 (d, J = 8.2 Hz), 74.4, 71.9 (dd, J = 155.0, 8.0 Hz),
68.9, 64.5 (d, J = 5.2 Hz), 41.9 (d, J = 3.8 Hz); 31P NMR
(121.5 MHz, D2O) δ 14.6 (d, JP,P = 22.0 Hz), 1.1 (d, JP,P = 24.4
Hz); HRMS (ESI) calcd. for C18H23N4O12P2 [M-H]- 549.0793,
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Trisodium
cytidin-5’-yl-((2-oxo-2-(piperidin-1-yl)-1-
phosphonato-ethyl)-phosphate (5d). 5d-s: [α]3D0 = +6.40 (c =
1
0.25, H2O); H NMR (400 MHz, D2O) δ 7.81 (d, J = 7.6 Hz,
1H), 5.99 (d, J = 7.5 Hz, 1H), 5.82 (d, J = 3.9 Hz, 1H), 5.05
(dd, J = 15.9, 10.1 Hz, 1H), 4.20 – 4.01 (m, 4H), 3.95 (dd, J =
9.8, 4.4 Hz, 1H), 3.50 – 3.55 (m, 2H), 3.43 – 3.29 (m, 1H),
3.24 – 3.07 (m, 1H), 1.55 – 1.22 (m, 6H); 13C NMR (125 MHz,
D2O) δ 168.5, 166.7, 158.1, 142.3, 97.2, 90.2, 83.4 (d, J =
8.63 Hz), 74.9, 72.3 (d, J = 152 Hz), 70.0, 65.3, 48.6, 44.9,
1
found 549.0797. 5a-l: [α]3D0 = -2.14 (c = 0.28, H2O); H NMR
(400 MHz, D2O) δ 7.68 (d, J = 7.7 Hz, 1H), 7.62 (d, J = 7.6
Hz, 2H), 7.43 (t, J = 7.4 Hz, 1H), 7.34 (t, J = 7.6 Hz, 2H), 5.79
(d, J = 7.6 Hz, 1H), 5.61 (d, J = 3.1 Hz, 1H), 4.36 (qd, J = 10.0,
2.6 Hz, 1H), 4.14 (dd, J = 11.7, 4.0 Hz, 1H), 4.07 – 3.96 (m,
3H), 3.92 – 3.83 (dd, J = 13.6, 9.8 Hz, 2H), 3.58 – 3.44 (m,
1H); 13C NMR (100 MHz, D2O) δ 170.1, 165.1, 156.2, 141.2,
133.0, 132.4, 128.8, 127.0, 96.1, 89.6, 82.3 (d, J = 9.0 Hz),
74.5, 72.1 (dd, J = 155.0, 6.0 Hz), 68.6, 63.8 (d, J = 5.0 Hz),
41.9 (d, J = 3.0 Hz); 31P NMR (121.5 MHz, D2O) δ 14.6 (d,
JP,P = 22.0 Hz), 1.2 (d, JP,P = 24.3 Hz); HRMS (ESI) calcd. for
C18H23N4O12P2 [M-H]- 549.0793, found 549.0812.
26.6, 26.0, 24.5; 31P NMR (121.5 MHz, D2O) δ 9.90 (d, JP,P
=
31.7 Hz), 0.70 (d, JP,P = 29.3 Hz); HRMS (ESI) calcd. for
C16H25N4O12P2 [M-H]- 527.0950, found 527.0962. 5d-l: [α]3D0 =
1
+7.14 (c = 0.28, H2O); H NMR (400 MHz, D2O) δ 7.81 (d, J
= 7.6 Hz, 1H), 5.99 (d, J = 7.4 Hz, 1H), 5.83 (d, J = 2.9 Hz,
1H), 5.01 (dd, J = 16.0, 10.1 Hz, 1H), 4.20 – 4.12 (m, 2H),
4.06 – 3.90 (m , 3H), 3.55-3.49 (m, 2H), 3.38 – 3.26 (m, 1H),
3.21 – 3.09 (m, 1H), 1.55 – 1.29 (m, 6H); 13C NMR (100 MHz,
D2O) δ 167.7, 164.6, 155.6, 142.2, 96.4, 89.5, 82.7 (d, J = 9.0
Hz), 74.2, 71.6 (dd, J = 144, 7.0 Hz), 69.0, 64.2 (d, J = 5.0 Hz),
47.9, 44.2, 25.8, 25.2, 23.8; 31P NMR (121.5 MHz, D2O) δ
9.82 (d, JP,P = 26.9 Hz), 0.80 (d, JP,P = 29.3 Hz); HRMS (ESI)
calcd. for C16H25N4O12P2 [M-H]- 527.0950, found 527.0972.
Trisodium cytidin-5’-yl-(2-(2,5-dioxopyrrolidin-1-yl)-1-
phosphonato-ethyl)-phosphate (5e). 5e-s: [α]3D0 = +18.93 (c =
Trisodium
cytidin-5’-yl-(2-oxo-2-(phenylamino)-1-
phosphonato-ethyl)-phosphate (5b). 5b-s: [α]3D0 = -8.00 (c =
1
0.35, H2O); H NMR (400 MHz, D2O) δ 7.65 (d, J = 8.0 Hz,
1H), 7.30 – 7.27 (m, 2H), 7.23 – 7.18 (m, 2H), 7.06 – 7.02 (m,
1H), 5.83 (d, J = 7.6 Hz, 1H), 5.66 (d, J = 3.6 Hz, 1H), 4.63
(dd, J = 10.4, 15.6 Hz, 1H), 4.13 (ddd, J = 2 .0, 4.4, 12 Hz,
1H), 4.11 – 4.00 (m, 3H), 3.92 (t, J = 4.0 Hz, 1H); 13C NMR
(100 MHz, D2O) δ 168.8, 165.1, 156.2 (d, J = 3.0 Hz), 141.3,
136.5, 129.1, 125.6, 121.6, 96.4, 89.5, 82.5 (d, J = 8.7 Hz),
75.1 (d, J = 141, 7.8 Hz), 74.3, 69.0, 64.9 (d, J = 5.3 Hz); 31P
NMR (162 MHz, D2O) δ 9.96 (d, JP,P = 29.2 Hz), -0.12 (d, JP,P
= 27.5 Hz); HRMS (ESI) calcd. for C17H21N4O12P2 [M-H]-
535.0637, found 535.0657. 5b-l: [α]3D0 = +24.21 (c = 0.19,
1
0.34, H2O); H NMR (400 MHz, D2O) δ 7.82 (d, J = 8.0
Hz ,1H), 6.02 (d, J = 7.6 Hz, 1H), 5.80 (d, J = 3.1 Hz, 1H),
4.36 (qd, J = 10.9, 2.6 Hz, 1H), 4.18 – 4.11 (m, 2H), 4.07 (td,
J = 12.3, 7.3, 2.7 Hz, 2H), 3.93 (ddd, J = 12.3, 5.7, 3.7 Hz,
1H), 3.82 (ddd, J = 14.3, 11.2, 6.5 Hz, 1H), 3.59 (d, J = 14.2
Hz, 1H), 2.62 (s, 4H); 13C NMR (100 MHz, D2O) δ 181.5,
164.9, 156.0, 142.1, 96.3, 89.5, 83.0 (d, J = 9.0 Hz), 74.2, 70.7
(dd, J = 155.0, 8.0 Hz), 69.3, 64.5 (d, J = 5.0 Hz), 40.3 (d, J =
1
H2O); H NMR (400 MHz, D2O) δ 7.63 (d, J = 7.6 Hz, 1H),
7.25 – 7.23 (m, 2H), 7.17 – 7.12 (m, 2H), 7.00 – 6.96 (m, 1H),
5.78 (d, J = 7.6 Hz, 1H), 5.56 (d, J = 3.2 Hz, 1H), 4.59 (dd, J =
10.4, 15.6 Hz, 1H), 4.19 (dd, J = 3.2, 11.2 Hz, 1H), 4.07 –
3.96 (m, 3H), 3.84 (t, J = 3.2 Hz, 1H); 13C NMR (100 MHz,
D2O) δ 169.7, 166.4, 157.7, 141.5, 137.3, 129.7, 126.1, 121.6,
97.1, 90.3, 82.8 (d, J = 9.4 Hz), 76.0 (dd, J = 139, 8.1 Hz),
75.1, 69.1, 64.7 (d, J = 4.4 Hz); 31P NMR (162 MHz, D2O) δ
8.75 (d, JP,P = 27.5 Hz), 0.31 (d, JP,P = 27.5 Hz); HRMS (ESI)
calcd. for C17H21N4O12P2 [M-H]- 535.0637, found 535.0649.
11.0 Hz), 28.1; 31P NMR (121.5 MHz, D2O) δ 13.94 (d, JP,P
=
17.0 Hz), 1.27 (d, JP,P = 17.1 Hz); HRMS (ESI) calcd. for
C15H21N4O13P2 [M-H]- 527.0586, found 527.0588. 5e-l: [α]3D0 =
-2.05 (c = 0.29, H2O); 1H NMR (400 MHz, D2O) δ 7.84 (d, J =
7.7 Hz, 1H), 6.06 (d, 1H), 5.81 (d, J = 3.6 Hz, 1H), 4.33 (qd, J
= 10.9, 2.5 Hz, 1H), 4.20 – 4.12 (m, 2H), 4.07 (td, J = 4.7, 2.3
Hz, 1H), 4.05 – 3.95 (m, 2H), 3.81 (ddd, J = 14.3, 11.1, 6.2 Hz,
1H), 3.59 (d, J = 14.1 Hz, 1H), 2.61 (s, 4H); 13C NMR (100
MHz, D2O) δ 181.5, 163.8, 154.6, 142.4, 96.2, 89.4, 83.0 (d, J
= 9.0 Hz), 74.3, 70.2 (dd, J = 154.0, 7.0 Hz ), 69.1, 64.2 (d, J
= 5.0 Hz), 40.4 (d, J = 10.0 Hz), 28.1; 31P NMR (121.5 MHz,
D2O) δ 13.87 (d, JP,P = 17.0 Hz), 1.18 (d, JP,P = 19.6 Hz);
HRMS (ESI) calcd. for C15H21N4O13P2 [M-H]- 527.0586,
found 527.0598.
Trisodium
cytidin-5’-yl-(2-oxo-2-(pyrrolidin-1-yl)-1-
phosphonato-ethyl)-phosphate (5c). 5c-s: [α]3D0 = -3.39 (c =
1
0.35, H2O); H NMR (400 MHz, D2O) δ 7.80 (d, J = 7.6 Hz,
1H), 5.99 (d, J = 7.6 Hz, 1H), 5.82 (d, J = 4.3 Hz, 1H), 4.84
(dd, J = 15.5, 9.7 Hz, 1H), 4.12 – 4.03 (m, 4H), 3.93 – 3.85 (m,
1H), 3.60 – 3.52 (m, 1H), 3.51 – 3.43 (m, 1H), 3.33 – 3.18 (m,
2H), 1.86 – 1.60 (m, 4H); 13C NMR (100 MHz, D2O) δ 167.9,
165.2, 156.4, 141.9, 96.4, 89.5, 82.9 (d, J = 8.8 Hz), 74.3, 72.2
(dd, J = 145, 8.0 Hz), 69.5, 64.7 (d, J = 5.1 Hz), 47.5, 46.8,
25.5, 23.6; 31P (121.5 MHz, D2O) δ 8.81 (d, JP,P = 31.7 Hz), -
0.61 (d, JP,P = 31.7 Hz); HRMS (ESI) calcd. for C15H23N4O12P2
[M-H]- 513.0793, found 513.0793. 5c-l: [α]3D0 = +20.65 (c =
Trisodium cytidin-5’-yl-(2-(1,3-dioxoisoindolin-2-yl)-1-
phosphonato-ethyl)-phosphate (5f). 5f-s: [α]3D0 = -3.09 (c =
1
0.30, H2O); H NMR (400 MHz, D2O) δ 7.69 – 7.60 (m, 5H),
5.77 (d, J = 7.6 Hz, 1H), 5.59 (d, J = 3.0 Hz, 1H), 4.51 (dd, J =
19.5, 8.7 Hz, 1H), 4.06 – 3.72 (m, 7H); 13C NMR (100 MHz,
D2O) δ 171.1, 165.8, 156.9, 142.2, 135.3, 132.2, 124.1, 96.8,
90.3, 83.1 (d, J = 7.6 Hz), 75.1, 72.2 (dd, J = 152.1, 7.1 Hz),
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o
69.4, 65.1 (d, J = 4.8 Hz), 40.2; 31P NMR (162 MHz, D2O) δ
13.36 (d, JP,P = 14.5 Hz), 0.21 (d, JP,P = 14.9 Hz); HRMS (ESI)
calcd. for C19H21N4O13P2 [M-H]- 575.0586, found 575.0599.
and being heated at 95 C for 2 min. Then the samples were
o
centrifuged at 4 C with 14000 rpm for 20 min and analyzed
with RP-HPLC. The Ki was obtained with non-linear regres-
sion analysis computed by using GraphPad Prism 6.
1
5f-l: [α]3D0 = +8.00 (c = 0.35, H2O); H NMR (400 MHz, D2O)
6
δ 7.74 – 7.53 (m, 4H), 7.48 (d, J = 7.6 Hz, 1H), 5.64 (d, J =
7.5 Hz, 1H), 5.42 (d, J = 2.3 Hz, 1H), 4.53 (dd, J = 19.6, 9.6
Hz, 1H), 4.08 – 3.74 (m, 7H); 13C NMR (100 MHz, D2O) δ
171.2, 166.5, 157.6, 141.4, 135.2, 132.1, 124.1, 96.9, 90.1,
82.2 (d, J = 9.1 Hz), 75.2, 73.0 (d, J = 159.8 Hz), 68.6, 63.9,
40.7; 31P NMR (162 MHz, D2O) δ 13.40 (d, JP,P = 12.0 Hz), -
0.61 (d, JP,P = 12.5 Hz); HRMS (ESI) calcd. for C19H21N4O13P2
[M-H]- 575.0586, found 575.0603.
7
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
Procedures for preparing compounds 6, 7, and 8, HPLC purity of
the target compounds, details of biological assay, and NMR spec-
tral data (PDF)
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Molecular formula strings (CSV)
RP-HPLC Based Sialyltransferase Inhibition Assay. The
assay was performed according to the UV/RP-HPLC method
developed by Schmidt group.35 Recombinant hST6Gal-I was
purchased from R&D (aa 44-406, 7620-GT), CMP-Neu5Ac
was available from Sigma and p-nitrophenyl LacNAc was
purchased from Toronto Research Chemicals Inc. Each vial of
enzyme was diluted with the storage buffer (25 mM Tris, 150
mM NaCl and 50% (v/v) Glycerol, pH 7.5) into 20 ng/μL,
AUTHOR INFORMATION
Corresponding Author
*Phone: +86-10-82805736; e-mail: xinshan@bjmu.edu.cn.
Notes
The authors declare no competing financial interest.
o
split into small portions and stored at -80 C before use. The
ACKNOWLEDGMENTS
enzyme-catalyzed reaction was performed in the assay buffer
(0.5% (w/v) Triton X-100, 2 mM MnCl2, 25 mM Mes buffer,
pH 6.5).42 RP-HPLC analysis was performed with Agilent
1260 Infinity system and data were collected at 300 nm. Col-
umn: Agilent ZORBAX SB-C18 Stable Bond Analytical 4.6 ×
250 mm, 5-Micron. Eluent: 0.1% FA (pH = 2.66): CH3CN =
85/15 at 1.0 mL/min. Acetonitrile was HPLC grade, water was
Milli Q quality, solutions were filtered through 0.45 μm mem-
branes. In all assays, the consumption of CMP-Neu5Ac was
limited less than 15% in order to get reliable initial rates.
Determination of the CMP-Neu5Ac Km. The donor CMP-
Neu5Ac was diluted to 1000, 500, 250, 125, 50 μM. The en-
zyme was diluted to 13.6 ng/12 μL with the assay buffer. To a
0.6 mL microtube were added 6.0 μL of donor, 6.0 μL of 5
mM acceptor, 12.0 μL of enzyme and 6.0 μL of assay buffer
(total volume: 30 μL). Then the mixtures were mixed and in-
This work was financially supported by the grants
(2012CB822100, 2013CB910700) from the Ministry of Science
and Technology of China, and the National Natural Science
Foundation of China (21232002, 81473084).
ABBREVIATIONS USED
CMP, cytidine monophosphate; hST6Gal-I, human β-galactoside
α-2,6-sialyltransferase I; Neu5Ac, N-acetylneuraminic acid; RP,
reversed phase; ST, sialyltransferase; TEAB, triethylammonium
bicarbonate.
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cubated in water bath at 37 C for 30 min. The reactions were
quenched by adding 200 μL of deionized water and being
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heated at 95 C for 2 min. Then the samples were centrifuged
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at 4 C with 14000 rpm for 20 min and analyzed with RP-
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Determination of the inhibitor Ki. The donor CMP-
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× Ki to 0.4 × Ki). The enzyme was diluted to 20 ng/12 μL with
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inhibitor, 6.0 μL of donor, 6.0 μL of 5 mM acceptor and 12.0
μL of enzyme (total volume: 30 μL). Then the mixtures were
o
mixed and incubated in water bath at 37 C for 30 min. The
reactions were quenched by adding 200 μL of deionized water
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