The first chemical synthesis of novel MeO-3-GlcUA derivative of hyaluronan-based disaccharide to elucidate the catalytic mechanism of hyaluronic acid synthases (HASs)

Article abstract of DOI:10.1016/j.tetlet.2009.09.042

The first chemical synthesis of MeO-3-GlcUAp(1 →3)GlcNAc-UDP to elucidate the catalytic mechanism of hyaluronic acid synthases (HASs) is described. Construction of the desired β(1 →3)-linked disaccharide 10 was achieved very efficiently by coupling MeO-3-GlcUA donor 3 with the suitable protected GlcNTroc acceptor 4 using BF₃Et₂O as Lewis acid. Chemoselective removal of anomeric NAP, phosphorylation, hydrogenation, coupling with UMP-morpholidate, and finally complete deprotection gave the target compound 1 in good yield.

Full text of DOI:10.1016/j.tetlet.2009.09.042

Tetrahedron Letters 50 (2009) 6543–6545
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The first chemical synthesis of novel MeO-3-GlcUA derivative
of hyaluronan-based disaccharide to elucidate the catalytic
mechanism of hyaluronic acid synthases (HASs)
Guohua Wei a,b, Vipin Kumar a, Jun Xue a, Robert D. Locke a, Khushi L. Matta a,
*
a Cancer Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
b State Key laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The first chemical synthesis of MeO-3-GlcUAb(1?3)GlcNAc-UDP to elucidate the catalytic mechanism of
hyaluronic acid synthases (HASs) is described. Construction of the desired b(1?3)-linked disaccharide 10
was achieved very efficiently by coupling MeO-3-GlcUA donor 3 with the suitable protected GlcNTroc
acceptor 4 using BF3ꢀEt2O as Lewis acid. Chemoselective removal of anomeric NAP, phosphorylation,
hydrogenation, coupling with UMP-morpholidate, and finally complete deprotection gave the target
compound 1 in good yield.
Received 2 September 2009
Accepted 8 September 2009
Available online 12 September 2009
Keywords:
MeO-3-GlcUA
Hyaluronic acid synthases (HASs)
Oligosaccharides
Phosphorylation
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
properties of HA, are preferably executed using low-molecular-
weight HA fragments rather than polymeric HA.5 Thus, syntheses
Hyaluronic acid (HA), a vital extracellular matrix component of
vertebrate tissues, has been implicated in numerous physiological
and biological phenomena.1 HA is a linear biopolymer composed of
thousands of alternating repeating disaccharide units of glucuronic
acid [GlcUAb(1?3)] and N-acetylglucosamine [GlcNAcb(1?4)].2
The enzymes that catalyze the polymerization of HA, the hyaluro-
nan synthases (HASs), are unique glycosyltransferases that have
both selective GlcNAcb(1?4) transferase and GlcUAb(1?3) trans-
ferase activities.3 There is an ambiguity regarding the directionality
of chain elongation of HA biosynthesis, that is, whether the addi-
tion of monosaccharide units occurs at the reducing or non-reduc-
ing terminus of growing polysaccharide chains. Although, many
groups have investigated this problem extensively, till date there
is no accurate knowledge regarding the mechanism of HA biosyn-
thesis and hence only minimal information is available on the
molecular basis for HASs catalytic action. Consequently, our under-
standing of the detailed mechanisms whereby hyaluronan influ-
ences cell behavior is still incomplete.3
of well-defined HA fragments is required to provide adequate
material for carrying out such investigations. Consequently, several
strategies have been reported for the construction of HA fragments
containing either a GlcUA or a GlcNAc at the reducing end.6
Moreover, it was observed that the deoxy analogs obtained by the
substitution of one of the –OH groups of basic enzyme acceptor with
–F, –OMe, –N3, –NH2, etc. sometimes result in highly specific accep-
tors or inhibitors for the individual enzymes.7 For example, Scott and
Viola found that 3-fluoro and 4-fluoro analogs of
higher affinity substrates than -glucose for aldolase reductase but
2-fluoro and 4-fluoro analogs of -glucitol were inactive acceptors
D-glucose were
D
D
for sorbitol dehydrogenase.7a It was noticed by us that the activities
of only Gal 3-O-sulfotransferases and not sialyltransferases (Sia-T)
were adversely affected by Galb(1?4)GlcNAcb(1?6)[F-3-Galb-
(1?3)]GalNAc
[Galb(1?3)]GalNAc
2,6(N)Sia-T activity but not of
a
-OBn. Amazingly, F-4-Galb(1?4)GlcNAcb(1?6)-
-OBn was found to be an inhibitor of
2,3(N)Sia-T activity.7b The deoxy
a
a
a
analogs of disaccharide-peracetylated GlcNAcb1–3Galb-O-naph-
thalenemethanol containing –H, –F, –N3, –NH2, or –OCH3 at C-30
and C-40 positions of the terminal N-acetylglucosamine residue pre-
sumably inhibit one or more galactosyltransferases in vivo, thereby
blocking sLeX formation and experimental tumor cell metastasis.7c
Thus, functionalization of hydroxyl group of an HA fragment struc-
ture by substituting with –F, –OMe, –N3, –NH2, etc. may result in
highly specific acceptors or inhibitors for the individual HAS. In this
context, we have reported very recently the first chemical synthesis
It was identified recently that the small HA fragments behave as
potent activators of immunocompetent cells which play a decisive
role in the development of T cell-mediated immune responses.4
Also, studies such as NMR spectroscopic investigations and the
mechanism of degradation, performed to understand the chemical
* Corresponding author. Fax: +1 716 845 8768.
E-mail address: khushi.matta@roswellpark.org (K.L. Matta).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.09.042

6544
G. Wei et al. / Tetrahedron Letters 50 (2009) 6543–6545
of F-4-GlcUAb(1?3)GlcNAc-UDP for investigating the catalytic
mechanism of HASs.8 In general, OMe derivatives have similar po-
tency and their synthesis requires cheap and less hazardous re-
agents than their respective fluoro derivatives. Therefore, we
became interested in developing a facile synthesis of these OMe
derivatives and report herein the first chemical synthesis of MeO-
3-GlcUAb(1?3)GlcNAc-UDP (1) which may have the potential to
be a specific donor for the HASs.
MeOOC
MeOOC
a
O
O
AcO
AcO
ONAP
AcO
AcO
AcO
AcO
OC(NH)CCl3
5
6
b
MeOOC
HOOC
c
O
O
BzO
HO
ONAP
ONAP
ONAP
HO
HO
BzO
HO
8
7
2. Results and discussion
d
It was envisaged that MeO-3-GlcUAb(1?3)GlcNAc-1-phos-
phate derivative 2 would be an ideal intermediate to generate tar-
get molecule 1 (Fig. 1). The phosphate derivative 2 could in turn be
obtained by the glycosylation of known GlcNTroc acceptor 48 by
MeO-3-GlcUA imidate donor 3. To avoid the possible formation
of side product due to transacetylation during O-glycosylation,
benzoyl group was preferred over acetyl group for protection of
the 2-OH position of GlcUA in donor 3.9 We preferred to utilize
2-naphthylmethyl (NAP) as an anomeric-protecting group in 3
and 4, because of its ease in removal by DDQ oxidation.10 Due to
the straightforward transformation of N-Troc into N-Ac, Troc group
was chosen as a temporary protecting group for nitrogen in the
glycosyl acceptor 4.11
MeOOC
e
O
BzO
3
MeO
BzO
9
Scheme 1. Preparation of donor 3. Reagents and conditions: (a) NAPOH, TMSOTf,
CH2Cl2, 4 Å MS, 0 °C, 2 h, 80%; (b) (i) NaOMe, MeOH, rt, 6 h, 90%; (ii) LiOH, THF, H2O,
0 °C, 4 h, 95%; (c) (i) Bz2O, DMF, 85 °C., 4 h, 86%; (ii) Bz2O, Py, DMAP, DMF, rt, 40 h,
96%; (iii) NaOAc, MeOH, DMF, rt, 10 h, 92%; (d) MeI, Ag2O, CH2Cl2, 4 Å molecular
sieves, rt, 20 h, 92%; (e) (i) DDQ, CH2Cl2–MeOH (4:1), rt, 12 h, 89%; (ii) CCl3CN, DBU,
CH2Cl2, 0 °C, 2 h, 78%.
We next investigated multistep synthesis of the crucial phos-
phate intermediate 2 (Scheme 2). Coupling of the MeO-3-GlcUA
donor 3 and the known acceptor 48 was carried out with BF3ꢀEt2O
in the presence of 4 Å molecular sieves in toluene to afford the de-
sired disaccharide 10 with complete b1?3 stereoselectivity in 83%
yield.6e,8,12b,15 Detrimental effect on the yield of the above-men-
tioned glycosylation reaction was observed upon substitution of
dichloromethane as a solvent. Also, TMSOTf was found ineffective
for the same glycosylation as a promoter in comparison with
BF3ꢀEt2O. It is worth mentioning that the glycosylation reaction re-
ported here is one among the few procedures available for efficient
synthesis of b1?3 linkage with high b-stereoselectivity and high
yield.8,15d Most of the literature available on the synthesis of
b1?3 linkage suffers from certain drawbacks such as poor b-selec-
tivity and low yield.6e,k,n,12b,15b,c Following above glycosylation
reaction, MeO-3-GlcUAb1?3-linked disaccharide 10 could be ac-
cessed with complete b1?3 stereoselectivity and in high yield.
Next, replacement of the N-Troc group in 10 by an N-acetyl group
was effected with Zn-Ac2O and the subsequent acetylation of the
free amine offered 11 in 88% yield.11 Chemoselective removal of
NAP in 11 with DDQ in CH2Cl2–MeOH (4:1)10 provided anomeric-
free hydroxyl disaccharide 12 in 89% yield. Phosphorylation of 12
with tetrabenzyl pyrophosphate16 gave the benzyl-protected ano-
Scheme 1 outlines the synthesis of donor 3. Synthesis of imidate
3 was initiated with known GlcUA donor 5.12 Induction of anomer-
ic NAP protection in 5 was executed by the glycosylation of NAP-
OH with donor 5 in the presence of TMSOTf at 0 °C to afford 6 in
80% yield. Deacetylation of 6 with NaOMe–MeOH followed by
saponification of the methyl ester using LiOH gave 7 in good yield.
2,4-di-O-acylated derivative 8 was prepared from 7 following a 3-
step procedure. Lactonization of 7 with benzoic anhydride in DMF,
followed by complete benzoylation using DMAP-pyridine, and fi-
nally methanolysis of the lactone ring in the presence of anhydrous
NaOAc afforded the desired 2,4-di-O-acylated derivative 8 in 76%
yield for three steps.13 O-Methylation of the 3-hydroxyl group of
8 with CH3I and freshly prepared Ag2O provided 9 in 92% yield. Re-
moval of anomeric NAP protection of 9 was carried out using
DDQ10 followed by imidation14 to afford the MeO-3-GlcUA donor
3 in high yield (Scheme 1).
O
HO
HO
NH
HOOC
BzO
O
O
O
O
O
N
O
O
MeO
AcHN
OH
O P O P O
meric phosphate 2 as the desired
a anomer in high yield. Promi-
OH OH
nent signals in the 1H NMR spectrum of 2 at d = 5.68 (dd,
J1,2 = 3.4 Hz, J1,P = 6.4 Hz, 1 H, H-1 of GlcNAc), and the signal in
the 13P NMR of 2 at d = ꢁ1.90 (s, 1P) were observed, confirming
OH
HO
1
the
a linkage between the disaccharide and phosphoric acid moie-
BzO
ties. Deprotection of the benzyl group in 2 by hydrogenation over
palladium catalyst gave 13 in 90% yield Scheme 2. Coupling of 13
with UMP-morpholidate in the presence of 1H-tetrazole in DMF-
pyridine (3:1)16 followed by deprotection of the benzoyl groups
and the methyl ester using 3 M NaOH afforded the target com-
pound 117 in 45% yield over two steps after isolation and purifica-
tion by reverse-phase column HPLC and gel-filtration column
HPLC.
MeOOC
O
BzO
O
BzO
O
O
MeO
AcHN
OBz
O P OBn
OBn
2
MeOOC
BzO
O
O
+
BzO
BzO
ONAP
MeO
HO
3. Conclusions
BzO
TrocHN
OC(NH)CCl3
3
4
In summary, we have successfully accomplished the first chem-
Figure 1. Retrosynthesis of target molecule MeO-3-GlcUAb(1?3)GlcNAc-UDP (1).
ical synthesis of MeO-3-GlcUAb(1?3)GlcNAc-UDP following a

G. Wei et al. / Tetrahedron Letters 50 (2009) 6543–6545
6545
BzO
BzO
MeOOC
MeOOC
a
O
b
O
O BzO
O BzO
BzO
ONAP
BzO
ONAP
3 + 4
O
O
MeO
MeO
TrocHN
AcHN
BzO
BzO
10
11
c
BzO
BzO
MeOOC
d
e
MeOOC
d
O
O
O
BzO
BzO
O
2
BzO
BzO
1
O
O
O
OH
MeO
MeO
AcHN
AcHN
BzO
OBz
O P OH
O
Et3NH
13
12
Scheme 2. Synthesis of MeO-3-GlcUAb(1?3)GlcNAc-UDP 1. Reagents and conditions: (a) BF3ꢀEt2O, toluene, 4 Å MS, 0 °C, 2 h, 83%; (b) Zn, Ac2O, AcOH, THF, rt, 4 h, 88%; (c)
DDQ, CH2Cl2–MeOH (4:1), rt, 12 h, 89%; (d) LHMDS, [(BnO)2P]2O, THF, ꢁ78 to 0 °C, 3 h, 81%; (e) H2, Pd/C, EtOAc–MeOH (1:1), Et3N, rt, 9 h, 90%; (f) (i) UMP-morpholidate, 1H-
tetrazole, DMF-py (3:1), rt, 2 d; (ii) 3 M NaOH, MeOH, rt, 10 h; (iii) RP column HPLC; gel-filtration column HPLC, 45% from 13.
8. Wei, G.; Kumar, V.; Xue, J.; Locke, R. D.; Matta, K. L. Tetrahedron Lett. 2009, 50,
5920–5922.
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9. Brown, R. T.; Carter, N. E.; Mayalrap, S. P.; Scheinmann, F. Tetrahedron 2000, 56,
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pound would serve as a novel substrate to investigate the catalytic
mechanism of HASs.
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We acknowledge grant support from DOD (W81XWH-06-1-
0013) and support, in part, by the NCI Cancer Center Support Grant
to the Roswell Park Cancer Institute (P30-CA016056).
References and notes
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ꢁ12.5 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d (ppm) = 3.75 (s, 3H), 3.82 (s,
3H), 4.22 (t, 1 H, J = 9.6 Hz), 4.41 (d, 1H, J = 9.6 Hz), 5.10 (dd, 1H, J = 3.6, 9.6 Hz),
6.07 (t, 1H, J = 10.2 Hz), 6.36 (d, 1H, J = 3.6 Hz), 7.35–8.01 (m, 10 H, Ph), 8.74 (s,
1H); 13C NMR (100 MHz, CDCl3): d (ppm) = 52.5, 57.6, 69.4, 70.2, 72.8, 79.8,
90.1, 92.3, 159.7, 167.5, 168.8, 169.1. ESI-HRMS: calcd for C24H22Cl3NO9Na
[M+Na]+ 596.0253, found: 596.0250. Compound 10: 1H NMR (400 MHz, CDCl3):
d (ppm) = 3.36 (s, 3H), 3.40 (s, 3H), 3.91–3.93 (m, 1H), 4.32–4.34 (m, 2H), 4.38–
4.42 (m, 2H), 4.48–4.52 (m, 2H), 4.55–4.57 (m, 1H), 4.82 (d, 1H, J = 8.2 Hz, H-
10), 5.02 (d, 1H, J = 8.0 Hz, H-1), 5.06–5.13 (m, 3H), 5.17 (t, 1H, J = 9.6 Hz), 5.24
(t, 1H, J = 9.6 Hz), 5.34 (bs, 1H), 5.50 (t, 1 H, J = 9.6 Hz), 7.20–8.20 (m, 27H); 13C
NMR (100 MHz, CDCl3): d (ppm) = 53.5, 57.2, 58.7, 63.5, 70.5, 70.9, 71.3, 71.8,
72.1 (2C), 72.7, 74.6, 80.8, 95.6, 99.8, 100.3, 153.2, 165.2, 166.8, 167.1, 169.0,
169.7; ESI-HRMS: calcd for C56H50Cl3NO17Na [M+Na]+ 1136.2037, found:
1136.2035. Compound 2: 1H NMR (400 MHz, CDCl3): d (ppm) = 1.83 (s, 3H),
3.31 (s, 3H), 3.51 (s, 3H), 3.82 (d, J = 10.0 Hz, 1H), 4.17 (t, J = 9.8 Hz, 1 H), 4.21–
4.26 (m, 2H), 4.39–4.49 (m, 2H), 4.67–4.74 (m, 2H), 4.97–5.10 (m, 6H), 5.40 (t,
J = 8.8 Hz, 1H), 5.68 (dd, J1,2 = 3.4 Hz, J1,P = 6.4 Hz, 1H, H-1), 5.76 (d, J = 9.2 Hz,
NHAc), 7.26–8.20 (m, 30H). 13C NMR (100 MHz, CDCl3): d (ppm) = 20.1, 51.7,
52.3, 57.8 (JC-2,P = 8.1 Hz, C-2), 62.1, 68.2, 68.7, 69.4 (JCH2Ph,P = 4.9 Hz, POCH2Ph),
69.7 (JCH2Ph,P = 4.9 Hz, POCH2Ph), 71.6, 72.4, 73.8, 74.2, 79.8, 96.4 (JC-
1,P = 6.9 Hz, C-1), 99.4 (C-10), 164.4, 165.5, 166.4, 168.6, 168.9, 169.4. 31P
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NMR (202 MHz, CDCl3):
d
(ppm) = ꢁ1.90 (s, 1P). ESI-HRMS: calcd for
C58H56NO19PNa [M+Na]+ 1124.3077, found: 1124.3080. Compound 1: 1H NMR
(400 MHz, D2O): d (ppm) = 1.93 (s, 3H), 3.53 (s, 3H), 3.56 (m, 1H), 3.60 (t,
J = 9.6 Hz, 1H), 3.63–3.88 (m, 6H), 3.93 (d, J = 9.2 Hz, 1H), 4.07 (ddd,
J2,1 = 3.4 Hz, J2,3 = 10.2 Hz, J2,P = 3.2 Hz, 1H, H-2), 4.10–4.13 (m, 2H), 4.15–4.18
0
0
(m, 1H), 4.20–4.24 (m, 2H), 4.31 (t, J = 9.6 Hz, 1H), 4.43 (d, J1 ,2 = 8.6 Hz, 1H, H-
10), 5.40 (dd, J1,2 = 3.2 Hz, J1,P = 7.8 Hz, 1H, H-1), 5.79 (d, 1H), 5.83 (d, 1H), 7.83
(d, 1H). 13C NMR (100 MHz, D2O): d (ppm) = 23.4, 54.5, 57.6 (JC-2,P = 8.6 Hz, C-
2), 62.8, 66.0 (d, J = 3.8 Hz), 70.0, 71.1, 71.6, 72.5, 74.2, 74.8, 75.5, 77.2, 81.2,
84.8 (d, J = 9.8 Hz), 89.5, 96.5 (JC-1,P = 6.0 Hz, C-1), 101.3, 104.1, 141.8, 153.4,
167.4, 175.5, 176.1. 31P NMR (202 MHz, D2O): d (ppm) = ꢁ11.4 (d, J = 22.4 Hz),
ꢁ13.1 (d, J = 22.6 Hz). ESI-HRMS: calcd for C24H36N3O23P2 [MꢁH]ꢁ 796.1210,
found: 796.1214.
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