A chemical synthesis of GlcNAcβ(1→4)GlcUA-UDP to elucidate the catalytic mechanism of hyaluronic acid synthases (HAS)

Article abstract of DOI:10.1055/s-2007-970766

A first chemical synthesis of GlcNAcβ(1→4)GlcUA-UDP is described here. This compound can be an essential tool to elucidate the catalytic mechanism of hyaluronic acid synthases (HAS) and this synthetic strategy contributes to the synthesis of various UDP-s

Full text of DOI:10.1055/s-2007-970766

818
LETTER
A Chemical Synthesis of GlcNAcb(1→4)GlcUA–UDP to Elucidate the
Catalytic Mechanism of Hyaluronic Acid Synthases (HAS)
C
a
hemical Synthesi
i
s
of
G
l
r
cN
A
c
b
(
1
o
→
4)GlcU
A
n
-UD
P
ao Takaku,*a,b Hide-ki Ishida,c,1 Masaya Fujita,c Toshiyuki Inazu,d Hideharu Ishida,a,b Makoto Kiso*a,b
Faculty of Applied Biological Sciences, United Graduate School of Agricultural Science, Gifu University,
Yanagido, Gifu-shi, Gifu 501-1193, Japan
b
c
d
CREST, Japan Science and Technology Corporation (JST), Yanagido, Gifu-shi, Gifu 501-1193, Japan
The Noguchi Institute, Kaga, Itabashi-ku, Tokyo 173-0003, Japan
Department of Applied Chemistry, School of Engineering, and Institute of Glycotechnology, Tokai University,
Kitakaname, Hiratsuka-shi, Kanagawa 2591-292, Japan
Fax +81(58)2932918; E-mail: hironao55@yahoo.co.jp; E-mail: kiso@cc.gifu-u.ac.jp
Received 20 November 2006
group was added for protection of the sterically hindered
3-OH position of GlcUA, which could be removed under
neutral conditions, since GlcUA is prone to undergo b-
elimination under basic conditions.
Abstract: A first chemical synthesis of GlcNAcb(1→4)GlcUA–
UDP is described here. This compound can be an essential tool to
elucidate the catalytic mechanism of hyaluronic acid synthases
(HAS) and this synthetic strategy contributes to the synthesis of
various UDP-sugars which include modified GlcUA moieties.
HNAc
COOMe
O
Key words: carbohydrates, enzymes, nucleotides, oligosaccha-
rides, synthesis
AcO
AcO
O
BnO
O
P
O
OAc
BnO
O
OBn
OBn
key compound 10
Hyaluronic acid (HA), an important component of the
extracellular matrix, is a linear polysaccharide composed
of a repeating disaccharide unit of GlcNAcb(1→4) and
GlcUAb(1→3), and plays an important role in a wide va-
riety of biological processes.2 Hyaluronic acid synthases
(HAS), responsible for the synthesis of HA, have been
cloned from bacteria and mammalian cells.3 They have
been even known as unique glycosyltransferases, which
can catalyze two kinds of UDP-sugars (GlcNAc–UDP
and GlcUA–UDP). They are classified into two families
based on their primary structure as well as their enzyme
characteristics.4 However, the three-dimensional structure
of HASs responsible for catalytic mechanism remains
unclear.
COO–
O
O
N
HNAc
HO
HO
O
NH
HO
O
OH
O
O
OH
O
O
O P O P O
OH OH
OH OH
GlcNAcβ(1-4)GlcUA-UDP (12)
Figure 1 Structure of disaccharide-1-phosphate and UDP-
disaccharide
Coupling of the donor 17 and the suitably protected accep-
tor 3 prepared from 28 in the presence of N-iodosuccin-
imide (NIS)–trifluoromethanesulfonic acid (TfOH) and
4 Å molecular sieves gave the disaccharide 4 in 86%
yield. Deprotection of the Troc group and the subsequent
selective acetylation of the amine gave 5 in 85% yield.
Dess–Martin oxidation of 6-OH at the reducing end sugar,
the further oxidation of the intermediate aldehyde to a car-
boxylic acid by the use of NaClO2 in phosphate buffer,9
and the following methyl esterification gave 6 with 88%
overall yield in three steps. Selective removal of the 2-(tri-
methylsilyl)ethyl (SE) group in 6 with trifluoroacetic acid
(TFA) afforded 7 in 92% yield (Scheme 1).
We were interested in to obtaining the information about
the catalytic mechanism, i.e. whether the HA elongating
reaction occurs at the non-reducing end or at the reducing
end of sugar chain, and also which substrate among
GlcNAc–UDP and GlcUA–UDP serves as a primer of the
elongation reaction. We believed that our target com-
pound [GlcNAcb(1→4)GlcUA–UDP] could be an essen-
tial tool for performing the above-mentioned tasks.
Moreover, only a few reports about the enzymatic synthe-
sis of GlcUA–UDP are available.5,6 We describe herein
the first chemical synthesis of GlcNAcb(1→4)GlcUA–
UDP which has the GlcUA–UDP moiety. Our synthetic
strategy could be extended for the synthesis of various
UDP-sugars including modified GlcUA structures.
In order to obtain the a-monophosphate derivative of
the disaccharide, coupling of the trichloroacetimidate
derivative with dibenzylphosphate was carried out
(Scheme 2).10 Compound 7 was converted into the imi-
date 8 in a moderate yield,11 which was directly coupled
with dibenzylphosphate in CH2Cl2 for one day to give
the unexpected b-oriented monophosphate derivative 9 as
the major product. Treatment of 9 with TfOH in CH2Cl2
at 0 °C did not give the a compound. Alternatively, forma-
tion of lithium alkoxide of 7 by the action of lithium
hexamethyldisilazide (LHMDS) followed by the coupling
For the synthesis of GlcNAcb(1→4)GlcUA–UDP, the
protected GlcNAcb(1→4)GlcUA-1-phosphate derivative
10 (Figure 1) was prepared as a key compound. Benzyl
SYNLETT 2007, No. 5, pp 0818–0820
1
6.
0
3.
2
0
0
7
Advanced online publication: 08.03.2007
DOI: 10.1055/s-2007-970766; Art ID: U13906ST
© Georg Thieme Verlag Stuttgart · New York

LETTER
Chemical Synthesis of GlcNAcb(1→4)GlcUA-UDP
819
OAc
O
AcO
AcO
SPh
TrocHN
1
NHR2
OR1
O
TMS
b
d
AcO
AcO
O
OR
O
O
BnO
TMS
O
HO
OBn
OAc
O
BnO
4: R1 = R2 = Troc
OBn
c
2: R = H
5: R1 = H, R2 = Ac
a
3: R = Troc
COOMe
O
HNAc
O
HNAc
COOMe
O
TMS
e
AcO
AcO
AcO
AcO
O
O
BnO
O
BnO
O
OAc
OH
OBn
OBn
OAc
6
7
Scheme 1 Preparation of GlcNAcb(1→4)GlcUA derivative. Reagents and conditions: (a) TrocCl, pyridine–CH2Cl2 (1:1), 0 °C, 2 h, 95%; (b)
NIS, TfOH, CH2Cl2, MS 4A, –30 °C, 3.5 h, 86%; (c) Zn, Ac2O, AcOH, THF, r.t., 2.5 h, 85%; (d) (i) Dess–Martin periodinane, NaHCO3,
CH2Cl2, r.t., 3 h; (ii) NaClO2, 2-methyl-2-butene, NaH2PO4, t-BuOH, H2O, r.t., 12 h; (iii) MeOH, WSC {water-soluble carbodiimide; 1-[3-(di-
methylamino)propyl]-3-ethylcarbodiimide hydrochloride}, pyridine, CH2Cl2, r.t., 4 h, 88% (3 steps); (e) TFA, CH2Cl2, r.t., 2 h, 92% (a/b =
10:3).
with tetrabenzylpyrophosphate gave the suitably pro- UMP-morpholidate13 with 1H-tetrazole12 in moderately
tected GlcNAcb(1→4)GlcUA-a-1-phosphate derivative basic condition (DMF–pyridine, 3:1) reduced side reac-
1
1012 in 67% yield. Significant signals in the H NMR tions. Subsequent deprotection of the acetyl groups and
spectrum of 10 at d = 5.88 (dd, 1 H, J1,2 = 3.4 Hz, 3J1,P
=
the methyl ester in MeOH–H2O–Et3N (7:3:1)14 at 20 °C
7.4 Hz, H-1 of GlcUA), and the signal in the 31P NMR of gave the desired GlcNAcb(1→4)GlcUA–UDP (12)15 in
10 at d = –1.83 (s, 1 P) were observed, confirming that the 52% yield in two steps after isolation and purification by
newly formed linkage between the disaccharide moiety reverse-phase column HPLC and gel-filtration column
and phosphoric acid moiety was a.
HPLC (Scheme 3).
Removal of the benzyl groups in 10 by hydrogenation In conclusion, we have succeeded in the first synthesis of
over 20% palladium hydroxide on carbon gave 11 in 94% GlcNAcb(1→4)GlcUA–UDP by the chemical approach.
yield. Several conditions to construct the UDP structure This procedure could be extended to the synthesis of
by the use of phosphoamidate did not give any desirable various UDP-sugars which contain the modified GlcUA-
result. Condensation of 11 with UMP-imidazolate5 in an- 1-phosphate moiety. We also believe that the compound
hydrous DMF at room temperature gave many byproducts 12 will be an essential tool to gain information on the
with b-elimination. This may be due to the basic condi- catalytic mechanism of hyaluronic acid syntheses.
tions which were generated during the preparation of
UMP-imidazolate. On the other hand, coupling of 11 with
O
HNAc
NH
N
O
COOMe
O
O
UMP-imidazolate: R =
UMP-morpholidate: R =
N
N
AcO
AcO
c
O
R1
N
no reaction
BnO
O
O
OAc
P
O–
R
O
O
BnO
R2
a
8: R1 = H, R2 = OC(=NH)CCl3
9: R1 = OP(=O)(OBn)2, R2 = H
OHOH
HNAc
b
7
COOMe
O
AcO
AcO
O
HO
a
O
trace
O
d
HNAc
COOMe
O
OH
AcO
OAc
O
O
P
OH
NEt3
R1O
AcO
O
OAc
R1O
OH
OR2
11
10: R1 = Bn, R2 = P(=O)(OBn)2
11: R1 = H, R2 = P(=O)(OH)2·NEt3
b
e
12
Scheme 2 Preparation of disaccharide monophosphate derivative.
Reagents and conditions: (a) trichloroacetonitrile, DBU, CH2Cl2, 0 °C,
2 h, 79%; (b) dibenzylphosphate, CH2Cl2, r.t., 24 h, (a form: 8%, b
form: 77%); (c) dibenzylphosphate, TfOH, CH2Cl2, 0 °C, 12 h; (d)
LHMDS, tetrabenzylpyrophosphate, THF, –78 °C → 0 °C, (a form:
67%, b form: 24%); (e) H2 gas, Pd(OH)2/C, MeOH, r.t., 18 h, 94%.
Scheme 3 Synthesis of GlcNAcb(1→4)GlcUA–UDP. Reagents
and conditions: (a) UMP-imidazolate, DMF, r.t., 1d; (b) (i) UMP-
morpholidate, 1H-tetrazole, DMF–pyridine (3:1), r.t., 2 d; (ii)
MeOH–H2O–Et3N (7:3:1), 20 °C, 2 d; (iii) RP column HPLC; (iv)
gel-filtration column HPLC, 52% (from 11).
Synlett 2007, No. 5, 818–820 © Thieme Stuttgart · New York

820
H. Takaku et al.
LETTER
H-5¢), 3.57 (td, J1,2 = 3.4 Hz, J2,3 = 9.2 Hz, 3J2,P = 2.8 Hz, 1
Acknowledgment
H, H-2), 3.75 (s, 3 H, COOMe), 3.85 (t, J2,3 = 9.2 Hz, J3,4
=
This work was financially supported in part by CREST of JST
(Japan Science and Technology Corporation), and a Grant-in-Aid
for Scientific Research to M.K. (No. 1701007).
9.2 Hz, 1 H, H-3), 3.86 (dd, J5¢,6¢a = 2.3 Hz, J6¢a,6¢b = 12.6 Hz,
1 H, H-6¢a), 3.97 (t, J3,4 = 9.2 Hz, J4,5 = 9.7 Hz, 1 H, H-4),
3.99 (q, J1¢,2¢ = 8.6 Hz, J2¢,NH = 9.2 Hz, 1 H, H-2¢), 4.14 (dd,
J5¢,6¢b = 4.0 Hz, J6¢a,6¢b = 12.6 Hz, 1 H, H-6¢b), 4.32 (d, J4,5
=
9.7 Hz, 1 H, H-5), 4.61 (d, 1 H, CH2Ph), 4.64 (d, J1¢,2¢ = 8.6
Hz, 1 H, H-1¢), 4.67, 4.75, 4.98 (3 × d, CH2Ph, 3 H), 5.02–
5.11 (m, 6 H, H-3¢, H-4¢, CH2Ph), 5.66 (d, J2,NH = 9.2 Hz, 1
H, NH), 5.88 (dd, J1,2 = 3.4 Hz, 3J1,P = 7.4 Hz, 1 H, H-1),
7.20–7.33 (m, 20 H, CH2Ph). 13C NMR (126 MHz, CDCl3):
d = 20.5, 20.6, 20.6 (Me of OAc), 23.1 (Me of NAc), 53.0
(COOMe), 54.4 (C-2¢), 61.6 (C-6¢), 68.0 (C-4¢), 69.2
(JCH2Ph,P = 4.8 Hz, POCH2Ph), 69.5 (JCH2Ph,P = 4.8 Hz,
POCH2Ph), 71.4 (C-5), 71.9 (C-5¢), 73.1 (C-3¢), 73.3
(CH2Ph), 75.3 (CH2Ph), 77.7 (JC-2,P = 7.2 Hz, C-2), 78.4 (C-
4), 78.7 (C-3), 94.9 (JC-1,P = 6.0 Hz, C-1), 100.0 (C-1¢),
127.0, 127.0, 127.0, 127.0, 127.0, 128.0, 128.0, 128.0,
128.0, 128.0, 128.0, 135.0, 136.0, 136.0, 137.0, 139.0, 169.0
(C=O of OAc-4¢), 170.0 (C=O of COOMe), 170.0 (C=O of
References and Notes
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(b) Hoch, M.; Heinz, E.; Schmidt, R. R. Carbohydr. Res.
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NAc), 171.0 (C=O of OAc-6¢), 171.0 (C=O of OAc-3¢). 31
P
NMR (202 MHz, CDCl3): d = –1.83 (s, 1 P). MS (MALDI–
TOF, +ve): m/z [M + Na]+ calcd for C49H56NNaO18P:
1000.31; found: 1000.21. Compound 12: [a]D +8.75° (c =
0.4, H2O). 1H NMR (500 MHz, D2O): d = 1.95 (s, 3 H, NAc),
3.36–3.37 (m, 1 H, H-5¢), 3.38–3.43 (m, 2 H, H-3¢, H-4¢),
3.50 (td, J1,2 = 4.0 Hz, J2,3 = 9.2 Hz, 3J2,P = 2.9 Hz, 1 H, H-
2), 3.59 (t, J1¢,2¢ = 8.6 Hz, 1 H, H-2¢), 3.64 (t, J2,3 = 9.2 Hz, 1
H, H-3), 3.64–3.68 (m, 2 H, H-4, H-6¢a), 3.81 (d, 1 H, H-
6¢b), 4.01 (d, 1 H, H-5), 4.08 (ddd, JRib5a,Rib5b = 12.0 Hz,
3JRib5a,P = 2.3 Hz, 1 H, H-5a of Rib), 4.13 (ddd, JRib5a,Rib5b
=
3
12.0 Hz, JRib5b,P = 2.3 Hz, 1 H, H-5b of Rib), 4.16–4.18 (m,
1 H, H-4 of Rib), 4.24–4.28 (m, 2 H, H-2 of Rib, H-3 of Rib),
4.42 (d, J1¢,2¢ = 8.6 Hz, 1 H, H-1¢), 5.49 (dd, J1,2 = 4.0 Hz,
3J1,P = 7.4 Hz, 1 H, H-1), 5.86 (d, JUra5,Ura6 = 8.6 Hz, 1 H,
H-5 of Ura), 5.87 (d, 1 H, H-1 of Rib), 7.86 (d, JUra5,Ura6 = 8.6
Hz, 1 H, H-6 of Ura). 13C NMR (126 MHz, D2O): d = 23.4
(C of Me), 56.4 (C-2¢), 61.6 (C-6¢), 65.8 (JC-5,P = 3.9 Hz,
C-5 of Rib), 70.4 (C-3 of Rib), 70.7 (C-5¢), 72.1 (C-3), 72.3
(JC-2,P = 9.6 Hz, C-2), 74.1 (C-5), 74.9 (C-4¢), 75.2 (C-2 of
Rib), 77.0 (C-3¢), 81.0 (C-4), 84.3 (JC-4,P = 9.6 Hz, C-4 of
Rib), 89.4 (C-1 of Rib), 96.3 (JC-1,P = 5.8 Hz, C-1), 101.0
(C-1¢), 104.0 (C-5 of Ura), 142.0 (C-6 of Ura), 153.0 (C-4 of
Ura), 167.0 (C-2 of Ura), 176.0 (COOH), 176.0 (C=O of
NAc). 31P NMR (202 MHz, D2O): d = –10.3 (d, J = 21.8 Hz,
1 P, P of Rib), –12.1 (d, J = 21.8 Hz, 1 P, P of GlcUA). MS
(MALDI–TOF, –ve): m/z [M – H]– calcd for C23H34N3O23P2:
782.10; found: 781.96.
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(15) The selected physical data of key compounds is listed.
Compound 10: [a]D +17.6° (c = 1.7, CHCl3). 1H NMR (500
MHz, CDCl3): d = 1.91 (s, 3 H, NAc), 1.94, 1.98, 2.01 (3 ×
s, 9 H, OAc), 3.49 (ddd, J5¢,6¢a = 2.3 Hz, J5¢,6¢b = 4.0 Hz, 1 H,
Synlett 2007, No. 5, 818–820 © Thieme Stuttgart · New York

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