Practical Synthesis of the Fluorogenic Enzyme Substrate 4-Methylumbelliferyl α- L -Idopyranosiduronic Acid

Article abstract of DOI:10.1055/s-0040-1708021

A practical and concise synthesis of 4-methylumbelliferyl α- l -idopyranosiduronic acid, a fluorogenic enzyme substrate diagnostic for α- l -iduronidase, was accomplished. It features successive radical bromination and radical reduction of easily accessible methyl 4-methyl umbelliferyl-2,3,4-tri- O -acetyl-β- d -glucouronate in four steps with 28percent overall yield.

Full text of DOI:10.1055/s-0040-1708021

SYNLETT
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© Georg Thieme Verlag Stuttgart · New York
2020, 31, 1083–1086
letter
1083
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J. Tian et al.
Letter
Synlett
Practical Synthesis of the Fluorogenic Enzyme Substrate
4-Methylumbelliferyl -L-Idopyranosiduronic Acid
Jiameng Tiana
Wenliang Ouyanga
Yanling Heb
MeO2C
AcO
AcO
MeO2C
AcO
AcO
MeO2C
AcO
AcO
O
O
O
NBS, CCl4
hn, reflux
2 h, 83%
O
O
O
O
O
O
O
O
O
OAc
OAc
OAc
Br
Br
Qianqian Ninga
Jiang Baia
Br
Haixin Ding*a
Qiang Xiao*a
0
0
0
0-0
0
0
3-2
8
1
2-
7
7
7
8
Bu3SnH, Et3B
CH2Cl2, –78 °C to r.t
O
O
O
O
O
O
OAc
O
OH
a Key Laboratory of Organic Chemistry in Jiangxi Province,
Institute of Organic Chemistry, Jiangxi Science &
Technology Normal University, Nanchang 330013,
P. R. of China
MeO2C
HO2C
O
OA
OH
OAc
OH
dinghaixin_2010@163.com
xiaoqiang@tsinghua.org.cn
b Beijing Landbridge Technology Co., Ltd., No. 3, Gaobeidian
North Road, Chaoyang District, Beijing, P. R. of China
Received: 03.03.2020
Accepted after revision: 27.03.2020
Published online: 17.04.2020
used fluorogenic enzyme substrate was 4-methylumbel-
liferyl -L-idopyranosiduronic acid (1), which could be hy-
drolyzed by -L-iduronidase, releasing the fluorogenic 7-
hydroxy-4-methylcoumarin (3, excitation at 365 nm, emis-
sion at 450 nm; Scheme 1).6 During development and man-
ufacture of the diagnostic kits for IDUA, a large amount of
enzyme substrate 1 was required. Although enzyme sub-
strate 1 is commercially available, it is not financially feasi-
ble to use on any scale (25 mg, 1150 US dollars, www.bio-
synth.com). Thus, a practical synthetic method that can be
easily scaled at low-cost is needed.
DOI: 10.1055/s-0040-1708021; Art ID: st-2020-d0124-l
Abstract A practical and concise synthesis of 4-methylumbelliferyl -
L-idopyranosiduronic acid, a fluorogenic enzyme substrate diagnostic
for -L-iduronidase, was accomplished. It features successive radical
bromination and radical reduction of easily accessible methyl 4-methyl-
umbelliferyl-2,3,4-tri-O-acetyl--D-glucouronate in four steps with 28%
overall yield.
Key words -L-Iduronidase, 4-methylumbelliferyl -L-idopyrano-
siduronic acid, radical reduction, radical bromination, fluorogenic en-
zyme substrate, synthesis
-L-Iduronidase (IDUA; EC 3.2.1.76) belonging to the
glycoside hydrolase family 39 in the CAZy (Carbohydrate-
Active enZYmes database), hydrolyses the corresponding L-
iduronic acid 2 (IdoA) from glycosaminoglycans (GAGs).1 A
lack of IDUA could cause a dangerous build-up of GAGs
within cells, specifically inside the lysosomes, and could
further lead to mucopolysaccharidosis type I (MPS I).2 The
symptoms of MPS I include progressive mental retardation,
an enlarged and deformed skull, valvular heart defects, and
joint contractures.3 MPS I is inherited in an autosomal re-
cessive pattern and it occurs approximately 1 in 100,000
live births.
Methods for early diagnosis of MPS I include measure-
ment of glycosaminoglycans in urine, IDUA enzyme activity
in blood, and sequencing of the IDUA gene.4 Among those
methods, using a fluorogenic enzyme substrate to detect
enzymatic activity of IDUA stands as one of the most conve-
nient approaches, with merits of being highly sensitive,
easy-to-use, and economical.5 The first reported and widely
O
O
O
OH
HOOC
O
1
OH
OH
OH
OH
HOOC
O
+
HO
O
O
OH
OH
2
3
Scheme 1 -L-Iduronidase hydrolysis of 4-methylumbelliferyl -L-
idopyranosiduronic acid (1)
While enzyme substrate 1 has been widely used, only a
few synthetic routes have been reported. Early in 1978, it
was firstly prepared by oxidation of 4-methylumbelliferyl
-L-idopyranoside (4) with oxygen in aqueous alkaline me-
© 2020. Thieme. All rights reserved. Synlett 2020, 31, 1083–1086
Georg Thieme Verlag KG, Rüdigerstraße 14, 70469 Stuttgart, Germany

1084
J. Tian et al.
Letter
Synlett
O
O
O
O
O
O
MeO2C
AcO
AcO
OH
OH
O
HO
Path d
O
HOOC
O
Path a
O
O
O
OAc
Br
C-6 selective oxidation
Ref. 7
this paper
OH
OH
OH
OH
7
1
4
Mitsunobu-type
glycosylation
Ref. 9, 10
C-5 selective
bromination
Path c
HgBr2-promoted
glycosylation
Ref. 8
Path b
MeO2C
AcO
AcO
OH
OAc
O
OAc
O
O
MeO2C
F
MeO2C
O
O
O
OAc
OAc
OAc
OAc
OAc
8
6
5
Scheme 2 Retrosynthetic analysis of fluorogenic enzyme substrate 4-methylumbelliferyl -L-idopyranosiduronic acid (1)
dium using platinum black as catalyst (Scheme 2, Path a).7
The efficiency of this critical oxidation step was low (below
22% yield), and the precursor 4 was also not readily accessible.
In 1989, glycosylation of methyl (2,3,4-tri-O-acetyl--L-
idopyranosyl fluoride)uronate (5) with trimethylsilylated
7-hydroxy-4-methylcoumarin (3) to generate 1 was report-
ed (Scheme 2, Path b).8 The key intermediate 5 was synthe-
sized by photochemical bromination of methyl (2,3,4-tri-O-
acetyl--D-glucopyranosyl fluoride)uronate followed by re-
duction of its 5-bromo derivative by Bu3SnH.
Recently, this substrate 1 was also prepared using
Mitsunobu-type glycosylation of 7-hydroxy-4-methylcouma-
rin (3) and methyl 2,3,4-tri-O-acetyl-L-idopyranuronate (6)
by Hung’s group9 and our group10 (Scheme 2, Path c). The
glycosylation donor 6 was synthesized either using diace-
tone--D-glucose as starting material in eight steps9 or by
using methyl 1,2,3,4-tetra-O-acetyl--D-glucoronate as
starting material in four steps,10 respectively.
Although three methods have been successfully devel-
oped to prepare substrate 1, three challenges still remained
unsolved: (1) the glycosylation always gave a mixture of 
and  isomers, which requires tedious silica chromatogra-
phy to separate the desired  isomer; (2) the yields of the
desired  isomer were low; (3) the corresponding glyco-
sylation donor, either methyl 2,3,4-tri-O-acetyl-L-idopyra-
nuronate (6) or 1,2,3,4,6-penta-O-acetyl--L-idopyranose,
was difficult to prepare because the L-idose moiety is not
naturally available.11
To tackle those challenges, we envisaged that the de-
sired 4-methylumbelliferyl -L-idopyranosiduronic acid (1)
could be achieved simply by reversing the C-5 configuration
by radical reduction of bromide 7, which could be obtained
through the photobromination of readily accessed methyl
4-methylumbelliferyl -D-glucouronate (8, Scheme 2, Path
d). If this hypothesis were to work, it could resolve all the
challenges mentioned above and provide a new and effi-
cient synthetic approach in stereoselective manner to 4-
methylumbelliferyl -L-idopyranosiduronic acid (1).
To test the hypothesis, we prepared crystalline methyl
1,2,3,4-tetra-O-acetyl--D-glucoronate (10) on a 100 g scale
from commercially available D-glucurono-6,3-lactone in a
one-pot-two-step reaction in 45% overall yield (Scheme
3).12 Then, glycosyl bromide 11 was obtained using 33% HBr
in acetic acid in 95% yield.13 Without further purification,
glycosylation of glycosyl bromide 11 and 7-hydroxy-4-
methylcoumarin (3) with freshly prepared Ag2O in dry ace-
tonitrile smoothly delivered the corresponding glycoside 8
on 50 g scale in 87% yield.14
Although the initial trial of the following photobromi-
nation of glycoside 8 with N-bromosuccinimide (NBS) in
CCl4 under a tungsten lamp gave a complex mixture,15 to
our satisfaction, the glycosylic bond was intact. Detailed
NMR analysis of the crude mixture showed that an exces-
sive bromination at the 4′-methyl position occurred, which
gave glycoside 12 besides the desired stereoselective C-5 -
bromination product 7. Additional optimization of the pho-
tobromination revealed that the formation of 12 was inevi-
table, and the reaction temperature was critical. Higher
temperatures resulted in significant amounts of colored by-
products. Eventually, using 1.5 equivalents NBS in CCl4 at
gentle reflux afforded a mixture of 7 and 12 in 83% overall
yield. Because bromide 12 is unstable and the mixture was
basically inseparable by silica gel chromatography, the
product mixture of the bromination product was not fur-
ther purified and directly used for the next stage in the syn-
thesis.
The initial exploration of the radical reduction of the
mixed bromide glycoside proved problematic. The classic
reduction method using Bu3SnH with 2,2′-azobis(2-methyl-
propionitrile) (AIBN) as initiator gave complex mixtures.16
However, employing freshly crystallized AIBN gave an im-
proved yield. As the C-5 radical intermediate is involved in
the reaction mechanism, the formation of both the desired
© 2020. Thieme. All rights reserved. Synlett 2020, 31, 1083–1086

1085
J. Tian et al.
Letter
Synlett
-L-idopyranuronate 13 and the byproduct -D-glucoronate
8 was unavoidable. After systematic screening of radical
initiators (AIBN, Et3B) and reducing agents (Bu3SnH,
Ph3SnH; see the Supporting Information for detailed opti-
mization), the combination of Et3B and Bu3SnH in THF at –
78 °C was found to give the best reduction results and dis-
tribution of 13 (yield 77%, 8/13 = 1:1). Although careful sili-
ca chromatography could give pure desired L-idopyranuro-
nate 13,17 we found that the partially deprotected products
14 and 15 could be much more readily separated. So, the
mixture of 8 and 13 was subjected to removal of all acetyl
groups to afford the corresponding glycoside 14 (29% over-
all in three steps from glycoside 8) and 15, respectively, af-
ter silica gel purification.
Finally, saponification of methyl ester 14 with 2 M
aqueous NaOH at 0 °C, followed by neutralization with
Dowex-50(H+) provided 4-methylumbelliferyl -L-idopyra-
nosiduronic acid (1) in 95% yield.17 Its structure was con-
firmed by spectroscopic analyses, and its purity was above
97% according to HPLC analysis. An -L-iduronidase hydro-
lysis assay further confirmed its structure and bioactivity.
In summary, we have developed a concise and practical
method for the synthesis of 4-methylumbelliferyl -L-
idopyranosiduronic acid (1) from readily prepared methyl
2,3,4-tri-O-acetyl-methylumbelliferyl--D-glucoronate (8)
in four steps in 28% overall yield.18,19 This newly established
approach involves successive radical photochemical bromi-
nation and radical reduction as the key steps. This method
uses naturally available D-glucurono-6,3-lactone as the
starting material, with much lower time- and cost-consum-
ing purification of the intermediates. Compared with previ-
ous reported methods, it is far less tedious and easier to
perform. Additionally, this strategy may be adapted to the
synthesis of a wide range of fluorogenic IDUA substrates,
especially for fluorescent coumarin-derived IDUA sub-
strates, and work is ongoing in our laboratory.
OH
OH
MeO2C
O
O
a
HO
O
AcO
OAc
AcO
OAc
O
9
10
b
MeO2C
AcO
AcO
O
OAc
Br
11
HO
O
O
O
c
3
MeO2C
AcO
AcO
O
O
O
OAc
8
d
MeO2C
MeO2C
AcO
AcO
O
O
AcO
AcO
O
O
O
O
O
O
+
OAc
OAc
Br
Br
7
12
Br
e
MeO2C
AcO
AcO
O
O
O
O
O
O
O
OAc
OAc
+
MeO2C
O
13
8
OAc
OAc
f
MeO2C
HO
HO
O
O
O
O
O
O
O
OH
OH
+
MeO2C
O
Funding Information
15
14
OH
OH
g
This project was supported by the National Natural Science Founda-
tion of China (Grant No. 21676131, 21462019), the Bureau of Science
& Technology of Jiangxi Province (Grant No. 20143ACB20012), the Ed-
ucation Department of Jiangxi Province (Grant No.170673), and the
Jiangxi Science & Technology Normal University (Doctor Startup Fund
2018BSQD022).
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Supporting Information
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OH
Supporting information for this article is available online at
Scheme 3 Synthesis of fluorogenic enzyme substrate 4-methylumbel-
liferyl -L-idopyranosiduronic acid (1). Reagents and conditions: (a) i) cat.
NaOH, MeOH, 0 °C, ii) Ac2O, NaOAc, 90 °C, 2 h, 45% for two steps; (b)
33% HBr in HOAc, 0 °C to r.t., 3 h, 95%; (c) Ag2O, MeCN, r.t., 4 h, 87%;
(d) NBS, CCl4, h, reflux, 2 h, 83%; (e) Bu3SnH, Et3B, CH2Cl2, –78 °C to
r.t., 5 h, 77%; (f) cat. NaOMe, MeOH, r.t. 4 h, 92%; (g) 2 M NaOH,
THF/H2O (1:1), 0 °C, 3 h; Dowex-50 (H+), 95%.
https://doi.org/10.1055/s-0040-1708021.
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References and Notes
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(17) Characterization Data of Compound 13
Mp 89–91 °C; []D25 –08.1 (c 0.068, CH2Cl2). 1H NMR (400 MHz,
DMSO-d6):  = 7.75 (d, J = 8.8 Hz, 1 H, H-5), 7.16 (s, 1 H, H-8),
7.11 (d, J = 8.9 Hz, 1 H, H-6), 6.28 (s, 1 H, H-3), 6.02 (s, 1 H, H-1′),
5.16 (s, 1 H, H-3′), 5.04 (s, 2 H, H-2′, H-4′), 4.99 (s, 1 H, H-5′),
3.66 (s, 3 H, OCH3), 2.41 (s, 3 H, 4-CH3), 2.16 (s, 3 H, OAc), 2.10
(s, 3 H, OAc), 2.05 (s, 3 H, OAc). 13C NMR (101 MHz, DMSO-d6): 
= 169.1 (OAc), 169.0 (C × 2, OAc), 167.3 (COOMe), 159.8 (C-2),
158.1 (C-7), 154.2 (C-9), 153.1 (C-4), 126.7 (C-5), 114.8 (C-10),
113.4 (C-6), 112.2 (C-3), 103.8 (C-8), 95.2 (C-1′), 67.5 (C-5′), 66.8
(C-3′), 66.4(C-4′), 66.0 (C-2′), 52.3 (OCH3), 20.5 (C × 2, OAc), 20.3
(OAc), 18.0 (4-CH3). HRMS (ESI+): m/z calcd for C23H25O12 [M +
H]+: 493.1341; found: 493.1346.
(18) Characterization Data of Compound 14
25
Mp 119–121 °C; []D –111.7 (c 0.081, CH3OH). 1H NMR (400
MHz, DMSO-d6):  = 7.73 (d, J = 8.5 Hz, 1 H, H-5), 7.07–7.04 (m,
2 H, H-8, H-6), 6.26 (s, 1 H, H-3), 5.70 (d, J = 4.9 Hz, 1 H, H-1′),
5.57 (d, J = 5.8 Hz, 1 H, 2′-OH), 5.38–5.36 (m, 2 H, 3′-OH, 4′-OH),
4.65 (d, J = 4.3 Hz, 1 H, H-5′), 3.82–3.78 (m, 1 H, H-4′), 3.67–3.63
(m, 4 H, H-3′, COOMe), 3.58–3.53 (m, 1 H, H-2′), 2.40 (s, 3 H, 4-
CH3). 13C NMR (101 MHz, DMSO-d6):  = 170.0 (COOMe), 160.0
(C-2), 159.6 (C-7), 154.3 (C-9), 153.2 (C-4), 126.6 (C-5), 114.2
(C-10), 113.3 (C-6), 111.8 (C-3), 103.3 (C-8), 99.0 (C-1′), 71.8 (C-
5′), 71.4 (C-3′), 70.7 (C-2′), 70.0 (C-4′), 51.7 (OCH3), 18.1 (4-CH3).
HRMS (ESI+): m/z calcd for C17H19O9 [M + H]+: 367.1024; found:
367.1029.
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Bioorg. Khim. 1989, 15, 1411. (b) Voznyi, Y. V.; Kalicheva, I. S.;
Galoyan, A. A. Bioorg. Khim. 1987, 13, 1655.
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J.-C.; Chang, S. W.; Liao, C. C.; Chi, F. C.; Chen, C. S.; Wen, Y. S.;
Wang, C. C.; Kulkarni, S. S.; Puranik, R.; Liu, Y. H.; Hung, S. C.
Chemistry 2004, 10, 399.
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Chem. 2020, 40, 215.
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2013, 49, 3275. (b) Lee, J. C.; Chang, S. W.; Liao, C. C.; Chi, F. C.;
Chen, C. S.; Wen, Y. S.; Wang, C. C.; Kulkarni, S. S.; Puranik, R.;
Liu, Y. H.; Hung, S. C. Chem. Eur. J. 2004, 10, 399. (c) Pellissier, H.
Org. Prep. Proced. Int. 2002, 34, 441.
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Synth. 2016, 93, 200.
(13) (a) Mikula, H.; Matscheko, D.; Schwarz, M.; Hametner, C.;
Froehlich, J. Carbohydr. Res. 2013, 370, 19. (b) Chen, D. Z.; Wu,
T.; Zhao, Z.; Lin, X. Q.; Yang, T.; Yang, J. J. Chem. Res. 2013, 37,
671.
(19) Characterization Data of 4-Methylumbelliferyl -L-Idopyra-
nosiduronic Acid (1)
25
Mp 205–207 °C; []D –38.3 (c 0.021, CH3OH). 1H NMR (400
MHz, DMSO-d6):  = 7.87(s, 1 H, H-8), 7.65 (d, J = 8.8 Hz, 1 H, H-
5), 7.34 (dd, J = 8.8, 2.4 Hz, 1 H, H-6), 6.21 (s, 1 H, H-3), 5.51 (d,
J = 7.0 Hz, 1 H, 2′-OH), 5.19 (d, J = 4.9 Hz, 1 H, H-1′), 4.96 (d, J =
3.8 Hz, 1 H, 3′-OH), 3.99 (d, J = 6.2 Hz, 1 H, H-3′), 3.25–3.17 (m, 3
H, H-2′, H-4′, H-5′), 2.40 (s, 3 H, 4-CH3). 13C NMR (101 MHz,
DMSO-d6):  = 172.1 (COOH), 160.8 (C-2), 160.4 (C-7), 154.6 (C-
9), 153.4 (C-4), 126.0 (C-5), 113.6 (C × 2, C-10, C-6), 111.3 (C-3),
103.6 (C-8), 97.3 (C-1′), 74.4 (C-5′), 73.1 (C-3′), 72.5 (C-4′), 71.1
(C-2′), 18.1 (4-CH3). HRMS (ESI+): m/z calcd for C16H17O9 [M +
H]+: 353.0867; found: 353.0868.
(14) (a) Nasseri, S. A.; Betschart, L.; Opaleva, D.; Rahfeld, P.; Withers,
S. G. Angew. Chem. Int. Ed. 2018, 57, 11359. (b) Anderson, F. B.
Clin. Chim. Acta 1965, 12, 669.
© 2020. Thieme. All rights reserved. Synlett 2020, 31, 1083–1086