Synthesis of 4-methylumbelliferyl α-D-mannopyranosyl-(1→6)- β-D-mannopyranoside and development of a coupled fluorescent assay for GH125 exo-α-1,6-mannosidases

Article abstract of DOI:10.1016/j.bmc.2013.05.062

Certain bacterial pathogens possess a repertoire of carbohydrate processing enzymes that process host N-linked glycans and many of these enzymes are required for full virulence of harmful human pathogens such as Clostridium perfringens and Streptococcus pneumoniae. One bacterial carbohydrate processing enzyme that has been studied is the pneumococcal virulence factor SpGH125 from S. pneumoniae and its homologue, CpGH125, from C. perfringens. These exo-α-1,6-mannosidases from glycoside hydrolase family 125 show poor activity toward aryl α-mannopyranosides. To circumvent this problem, we describe a convenient synthesis of the fluorogenic disaccharide substrate 4-methylumbelliferone α-d-mannopyranosyl-(1→6)-β-d- mannopyranoside. We show this substrate can be used in a coupled fluorescent assay by using β-mannosidases from either Cellulomonas fimi or Helix pomatia as the coupling enzyme. We find that this disaccharide substrate is processed much more efficiently than aryl α-mannopyranosides by CpGH125, most likely because inclusion of the second mannose residue makes this substrate more like the natural host glycan substrates of this enzyme, which enables it to bind better. Using this sensitive coupled assay, the detailed characterization of these metal-independent exo-α-mannosidases GH125 enzymes should be possible, as should screening chemical libraries for inhibitors of these virulence factors.

Full text of DOI:10.1016/j.bmc.2013.05.062

Bioorganic & Medicinal Chemistry 21 (2013) 4839–4845
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 4-methylumbelliferyl
a
-D-mannopyranosyl-(1?6)-b-D-
mannopyranoside and development of a coupled fluorescent assay
for GH125 exo-a-1,6-mannosidases
Lehua Deng a, Polina Tsybina b, Katie J. Gregg c, Renee Mosi a, Wesley F. Zandberg a, Alisdair B. Boraston c,
David J. Vocadlo a,b,
⇑
a Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia V5A 1S6, Canada
b Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia V5A 1S6, Canada
c Department of Biochemistry and Microbiology, University of Victoria, PO Box 3055, STN CSC, Victoria, British Columbia V8W 3P6, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 11 June 2013
Certain bacterial pathogens possess a repertoire of carbohydrate processing enzymes that process host
N-linked glycans and many of these enzymes are required for full virulence of harmful human pathogens
such as Clostridium perfringens and Streptococcus pneumoniae. One bacterial carbohydrate processing
enzyme that has been studied is the pneumococcal virulence factor SpGH125 from S. pneumoniae and
Keywords:
Mannosidase
Glycoside hydrolase
Coupled enzyme assay
Disaccharide
b-Mannose
N-glycan
Glycan catabolism
its homologue, CpGH125, from C. perfringens. These exo-
family 125 show poor activity toward aryl -mannopyranosides. To circumvent this problem, we
describe a convenient synthesis of the fluorogenic disaccharide substrate 4-methylumbelliferone
mannopyranosyl-(1?6)-b- -mannopyranoside. We show this substrate can be used in a coupled fluores-
cent assay by using b-mannosidases from either Cellulomonas fimi or Helix pomatia as the coupling
enzyme. We find that this disaccharide substrate is processed much more efficiently than aryl -manno-
a-1,6-mannosidases from glycoside hydrolase
a
a-D-
D
a
pyranosides by CpGH125, most likely because inclusion of the second mannose residue makes this sub-
strate more like the natural host glycan substrates of this enzyme, which enables it to bind better. Using
this sensitive coupled assay, the detailed characterization of these metal-independent exo-a-man-
nosidases GH125 enzymes should be possible, as should screening chemical libraries for inhibitors of
these virulence factors.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
S. pneumoniae and its homologue, CpGH125, from Clostridium per-
fringens.3,4 These enzymes have been recently characterized as
Certain bacterial pathogens possess a repertoire of carbohy-
drate processing enzymes that process host N-linked glycans and
many of these enzymes are required for full virulence of harmful
human pathogens such as Clostridium perfringens and Streptococcus
pneumoniae.1,2 Several proposals regarding the functional role of
these enzymes in virulence have been advanced including pro-
cessed monosaccharides serving as a carbon source for growth
and biofilm formation, the exposure of cell surface receptors for
adherence, and modulation of the host immune response.2
Although the ability to process glycans is emerging as being criti-
cally important in pathogenesis, knowledge of the biochemical
properties of these bacterial carbohydrate processing enzymes that
cleave human glycans is rather limited.
highly specific exo-a
-1,6-mannosidases3 that comprise GH125
(for a discussion of the sequence based classification system of gly-
coside hydrolases see the carbohydrate active enzymes (CAZy)
database5 at http://www.cazy.org/). This new family of glycoside
hydrolases, unlike divalent metal-dependent
a-mannosidases
from GH families 38, 47, and 92, use a metal-independent inverting
catalytic mechanism.3
These exo-
highly activated monosaccharide substrates such as 2,4-dinitro-
phenyl -mannopyranoside (1) and natural substrates contain-
ing
a-1,6-mannosidases show poor activity against even
a-D
a-D-mannopyranosyl-(1?6)-mannopyranose units (Fig. 1)
have no convenient fluorogenic reporter group.3 Structural studies,
however, have revealed the mannose unit binding to the +1 subsite
of a well defined active site, forming multiple carbohydrate protein
interactions. The specific and extensive interactions of these
One such bacterial carbohydrate processing enzyme that has
been studied is the pneumococcal virulence factor SpGH125 from
enzymes, their high specificity for the
a1,6-mannobiose linkage,
⇑
coupled with mannopyranosides bearing even a very good 2,4-
dinitrophenol leaving group being poorly processed, suggests that
Corresponding author. Tel.: +1 778 782 3530.
E-mail address: dvocadlo@sfu.ca (D.J. Vocadlo).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.062

4840
L. Deng et al. / Bioorg. Med. Chem. 21 (2013) 4839–4845
A
B
OH
O
HO
HO
HO
OH
O
OH
HO
O
OH
O
HO
O
HO
HO
HO
HO
HO
NO2
HO
OH
O
O
OH
O
O
HO
O
HO
O
HO
O
HO
O
O
HO
OR
HO
HO
OR
NO2
HO
NHAc
NHAc
α-Man-(1-6)-β-Man-(1-4)-β-GlcNAc-R
α-Man-dNP (1)
α-Man-(1-6)-α-Man-(1-6)-β-Man-(1-4)-β-GlcNAc-R
OH
O
HO
HO
GH2
HO
GH125
OH
HO
HO
β-mannosidase
α-mannosidase
OH
O
O
O
HO
O
O
O
O
O
HO
HO
O
O
O
HO
Man
Man
α-Man-β-Man-4MU (2)
β-Man-4MU (3)
Figure 1. Structures of natural substrates of GH125 enzymes and the coupled assay system. (A) Natural oligosaccharide substrates and a poor previously identified
chromogenic substrate (1) for GH125 enzymes. (B) The proposed coupled assay system using 4-methylumbelliferone -mannopyranosyl-(1?6)-b- -mannopyranoside (
-mannopyranoside (b-Man-4MU, 3), which is a substrate that
a-
D
D
a-
Man-b-Man-4MU, 2). This substrate is hydrolyzed by a GH125 enzyme to generate 4-methylumbelliferone b-
can be cleaved by a GH2 b-mannosidase to liberate the 4-methylumbelliferone fluorophore.
D
the mannose residue binding to the +1 subsite of these enzymes
facilitates catalysis.3 Given that GH125 C. perfringens and S. pneu-
moniae enzymes seem not to have a defined +2 subsite, we felt that
we could install a fluorogenic leaving group in the b-configuration
pendent to the mannose residue binding in the +1 subsite. To ad-
dress this question and develop a convenient activity assay that
could be used to screen for inhibitors of GH125 enzymes we
describe the synthesis of a fluorogenic disaccharide substrate. 4-
tions (Scheme 1). We elected to separate the anomers by O-
acetylation (7a and b) followed by silica gel chromatography and
subsequent de-O-acetylation to yield the pure anomers (8a and
b). With the pure
a-hemiacetal in hand we used the Mitsunobu
approach developed by Garegg for the formation of aryl
b-mannosides8 and generalized by Cocinero et al.9 Coupling of
2,3:4,6-di-O-isopropylidene-D-mannopyranose (8a) with 4-meth-
ylumbelliferone in the presence of diisopropyl azodicarboxylate
(DIAD) and triphenylphosphine furnished an anomeric mixture
(5:1 in favour of the desired b-anomer) of 4-methylumbelliferyl
Methylumbelliferone
ranoside ( -Man-b-Man-4MU, 2) should be a better mimic of the
natural host glycan substrates as compared to -Man-dNP (1)
and we therefore expect that GH125 enzymes should efficiently
cleave the 1,6-glycosidic linkage to liberate 4-methylumbellifer-
one b- -mannopyranoside (Fig. 1). A coupled assay could therefore
be developed by including a b-mannosidase in the reaction to
hydrolyze the liberated 4-methylumbelliferone b- -mannopyrano-
side and release 4-methylumbelliferone, which can be detected
conveniently (Fig. 1). Here we detail the synthesis of -man-b-
man-4MU and the implementation of this sensitive coupled assay
for GH125 exo- -1,6-mannosidases.
a-D-mannopyranosyl-(1?6)-b-D-mannopy-
a
a
2,3:4,6-di-O-isopropylidene-D-mannopyranoside (9a and b), from
which the desired b-anomer (9b) was conveniently obtained
by crystallization in high yield (Scheme 2). Selective removal of
the 4,6-O-isopropylidene afforded 4-methylumbelliferyl 2,3-O-
a
D
isopropylidene-b-D-mannopyranoside (10), which was selectively
D
glycosylated using HgCN2 as the catalyst and per-O-acetyl-manno-
pyranosyl bromide as the donor following the general approach
reported by Khan et al.6 Zemplen de-O-acetylation followed by
hydrolytic cleavage of the 2,3-isopropylidene protecting group
a
a
afforded the target fluorogenic substrate
(Scheme 2).
a-Man-b-Man-4MU (2)
2. Results and discussion
2.2. Evaluation of 4-methylumbelliferyl a-D-mannopyranosyl-
2.1. Chemical synthesis of 4-methylumbelliferyl
a-D
-
(1?6)-b-
D-mannopyranoside (a-Man-b-Man-4MU, 2 as a
mannopyranosyl-(1?6)-b-
D
-mannopyranoside (a-Man-b-Man-
substrate for CpGH125 exo-a
-mannosidase
4MU, 2)
The coupled assay we envisioned depended on a GH125 exo-
-mannosidase acting to liberate methylumbelliferyl b- -manno-
As a first step we set out to synthesize
which we speculated would be a better substrate than simple aryl
-mannopyranosides for GH125 -mannosidases. The synthesis
of 4-nitrophenyl -mannopyranosyl-(1?6)-b- -mannopyrano-
a
-Man-b-Man-4MU (2),
a
D
pyranoside (b-Man-4MU), which could then be processed by a
b-mannosidase. We therefore considered various b-mannosidases
and felt that the Cellulomonas fimi b-mannosidase known as CfMa-
n2A10, which was shown by Zechel et al. as being highly active
against Man-4MU,11 would be a good candidate coupling enzyme.
CfMan2A is a retaining glycoside hydrolase that uses nucleophilic
and acid–base catalysis, having a pH optimum of 7.511 which is
compatible with the activity of CpGH125. We first evaluated the
activity of CfMan2A toward b-Man-4MU (Fig. 2) and found the
kinetic constants (Table 1) to be similar to those reported in the lit-
erature.11 We next evaluated CfMan2A as a coupling enzyme with
CpGH125. We kept CpGH125 at a constant concentration of 1 nM
and set out to establish an adequate concentration of CfMan2A that
a-
D
a
a
-D
D
side has been reported6 but we felt that a fluorogenic substrate
would be more desirable to have for a high sensitivity assay. Nev-
ertheless, the route developed by Khan and coworkers involving
Koenigs–Knorr glycosylation to form the intersaccharide linkage
appeared to be efficient and so we used a related approach differ-
ing primarily in the preparation of the b-mannoside acceptor.
Selective protection of the 2–4 and 6 positions was achieved by
formation of 2,3:4,6-di-O-isopropylidene-D-mannopyranose (6) as
previously described by Gelas and Horton using 2-methoxypro-
pene with catalytic p-toluenesulfonic acid7 with minor modifica-

L. Deng et al. / Bioorg. Med. Chem. 21 (2013) 4839–4845
4841
OAc
O
OH
O
OAc
O
AcO
AcO
HO
HO
AcO
b
a
AcO
AcO
HO
OH
AcO
OAc
D-Mannose
Br
4
5
c
O
O
O
O
O
O
O
O
O
O
O
d
O
O
O
+
OR
O
OH
OR
7a: R = Ac
8a: R = H
7b: R = Ac
8b: R = H
6
e
e
Scheme 1. Synthesis of the mannose donor (5) and acceptor precursor (8a). Reagents and conditions: (a) Ac2O, pyridine, rt; (b) 33% HBr–HOAc, rt, 84% over two steps; (c) 2-
methoxypropene, p-TsOHꢀH2O, DMF, 0 °C to rt, overnight, 70%; (d) Ac2O, pyridine, rt, 100% (44% for 7a, 56% for 7b); (e) NaOMe, MeOH, rt, 97% for 8a, 95% for 8b.
O
O
O
O
O
O
O
O
a
O
O
O
O
O
+
O
O
O
O
O
OH
O
O
O
9b
8a
9a
OAc
O
AcO
AcO
AcO
O
b
c
HO
HO
9b
O
O
O
O
O
O
O
O
HO
O
O
O
O
10
11
OH
O
OH
O
HO
HO
HO
HO
HO
HO
d
e
OH
O
O
O
O
O
O
HO
HO
O
O
O
HO
O
O
O
12
2
Scheme 2. Synthesis of
a-Man-b-Man-4MU. Reagents and conditions: (a) 4-methylumbelliferone, PPh3, DIAD, toluene, rt, 85% (14% for 9a, 71% for 9b); (b) 80% aq. HOAc, rt;
(c) donor 5, HgCN2, MeCN, 4 Å MS, rt, 64% (for two steps); (d) NaOMe, MeOH, rt, 60%; (g) TFA, CHCl3/H2O (100:1), rt, 88%; (e) 80% aq HOAc, 60–70 °C, 3 h, 80%.
Figure 2. Validation of CfMan2A activity toward 4-methylumbelliferyl b-
coupled assay. (A) Michaelis–Menten kinetics for the activity of CfMan2A (1 nM) toward the monosaccharide substrate b-Man-4MU (3). (B) CfMan2A concentrations of
greater than 100 nM are sufficient to ensure no dependence on CfMan2A concentration for the assay of CpGH125 (1 nM) activity toward the disaccharide substrate -Man-b-
M (triangles). Error bars represent the
D-mannopyranoside (b-Man-4MU) and determining the required CfMan2A concentration for the
a
Man-MU (2). Symbols represent the assay performed using
a-Man-b-Man-MU concentrations of either 180 lM (circles) or 60 l
standard deviation of triplicate measurements.
could be used to ensure that CpGH125 is rate limiting when mon-
itoring the production of 4-methylumbelliferone.
We found that the reaction velocities did not change signifi-
cantly when concentrations of b-mannosidase of 100 nM or greater
were used (Fig. 2). We therefore decided to use a concentration of
140 nM for all subsequent coupled assays. Surprisingly, during
control experiments we observed that CfMan2A showed low
activity toward
a-Man-b-Man-4MU (2). We therefore evaluated

4842
L. Deng et al. / Bioorg. Med. Chem. 21 (2013) 4839–4845
Table 1
having a much more activated leaving group. Notably, when using
-Man-dNP (1) as a substrate for CpGH125, saturation kinetics
Kinetic parameters of enzymes employed in the assay
a
were not observed (Fig. 3), enabling us to determine only the
second-order rate constant for this substrate (Table 1). These data
Enzyme
Substrate
kcat
(min-1)
Km
M)
kcat/Km
-1
(l
(min-1
l
M
)
CfMan2Aa b-Man-4MU (3)
1880
-Man-b-Man-4MU (2) 294
-Man-dNP (1) ND
5
68
6
17
2
suggest that binding of
In contrast, when using
saturation kinetics and a Km value of 380
a
-Man-dNP (1) to the enzyme is quite poor.
-Man-b-Man-4MU (1), we observed
M which, though Km
CpGH125b
CpGH125c
a
a
6 380 20 0.77 0.05
ND
a
1.80 0.006 ꢂ 10ꢃ3
l
a
b
c
is a kinetic parameter, suggests this substrate is bound better by
CpGH125. This finding is consistent with structural studies of
CpGH125, which shows there is a +1 subsite that forms extensive
contacts with a bound mannose residue.3
Conditions: 1 nM CfMan2A.
Conditions: 1 nM CpGH125, 140 nM CfMan2A.
Conditions: 9050 nM CpGH125.
CfMan2A activity against 2,4-ditrophenyl
a
-
D
-mannopyranoside
3. Conclusions
(aMan-dNP, 1) to check for potential a-mannosidase contamina-
tion but observed no activity against this substrate (data not
shown), which suggests that CfMan2A has low endo-b-mannosi-
dase activity. Fortunately, however, the level of this endo-mannos-
idase activity was very low; when using the same concentration of
CfMan2A as in the coupled enzyme assays (140 nM), the rates of
For GH125 enzymes, as exemplified using CpGH125, we find
that
to
a
-Man-b-Man-4MU (2) is a far superior substrate as compared
a
-Man-dNP (1). The coupled assay using CfMan2A as the cou-
pling b-mannosidase shows high sensitivity and enables us to ob-
serve traditional Michaelis-Menten kinetics for CpGH125. The
CfMan2A catalyzed hydrolysis of
than ꢁ1% the rate observed in the presence of CpGH125 (1 nM)
during the coupled assay. CfMan2A therefore processes -Man-b-
a-Man-b-Man-4MU (2) were less
results show
processed with a kcat/Km value that is 400-fold higher than that ob-
served for dNP- Man. We find that Man2A from C. fimi shows low
a-Man-b-Man-4MU (2) saturates CpGH125 and is
a
a
Man-4MU (2) over 11,000-fold more slowly than CpGH125. This
contribution to substrate turnover can be considered minor for
the purposes of these coupled assays and indicates CfMan2A is
useful for routine coupled assays of CpGH125 using this disaccha-
ride substrate. Given the known structure of CfMan2A,12 any CfMa-
endo-b-mannosidase activity, which may limit its utility in this
coupling system for certain applications. In those cases, the
H. pomatia b-mannosidase is likely to prove useful because this
enzyme shows no detectable endo-b-mannosidase activity toward
this disaccharide substrate. With this sensitive coupling system
available, a more detailed characterization of these interesting
n2A-catalyzed hydrolysis of
a-Man-b-Man-4MU (2) is somewhat
surprising given that the 6-hydroxyl group in the Michaelis com-
plex of CfMan2A shows this group is constrained within the en-
zyme active site. This 6-hydroxyl group, however, points toward
the solvent and it is possible some rearrangement in the enzyme
active site might enable it to tolerate a second mannose residue
appended to this group. We verified CfMan2A activity against
metal-independent GH125 exo-a-mannosidases should be possible
and screening chemical libraries for inhibitors of these virulence
factors should be feasible. Such inhibitors could prove to eventu-
ally be valuable research tools13 for studying the roles of these
enzymes in the host-pathogen interaction and may open a route
to the generation of new antibiotics to combat pathogens such as
S. pneumoniae.
a-Man-b-Man-4MU (2) using capillary electrophoresis (see the
Supplementary data). Given that CfMan2A appears to show some
low endo-b-mannosidase activity, we realized that this assay may
not be useful in all circumstances. For this reason we also
evaluated a commercial b-mannosidase that is prepared from Helix
pomatia (Sigma). This b-mannosidase showed no detectable activ-
4. Materials and methods
4.1. General
ity against a-Man-b-Man-4MU (2) and was also successfully used
in the coupled assay system with the details provided in the
methods section (data not shown).
Given the assay using CfMan2A was sufficiently robust to eval-
uate CpGH125 enzyme activity, we carried out a kinetic evaluation
Unless otherwise indicated, reagents and starting materials
were purchased from Sigma–Aldrich, TCI America, or Alfa Aesar
and were used without purification. 1H and 13C NMR spectra were
obtained on a BrukerAV600 (600 MHz for 1H and 150.8 MHz for
13C) spectrometer. Except where indicated, deuterated chloroform
(CDCl3) was used as the solvent with CHCl3 (dH 7.26) or CDCl3 (dC
77.0) being used as internal standards. NMR spectra run in D2O
used internal CH3OH (dH 3.34, dC 49.0) as the standard. High
of CpGH125 against
system with -Man-b-Man-4MU (2) (Fig. 3). We find that
CpGH125 processes -Man-b-Man-4MU (2) 400-fold more
efficiently than -Man-dNP (1) (Table 1) despite -Man-dNP (1)
a-Man-dNP (1) as well as using this coupled
a
a
a
a
Figure 3. CpGH125 activity toward disaccharide and monosaccharide substrates. (A) Michaelis-Menten kinetics for the activity of CpGH125 (1 nM) toward the disaccharide
substrate -Man-b-Man-4MU (2) using the coupled assay in the presence of CfMan2A (140 nM). (B) Activity of CpGH125 (9050 nM) toward the monosaccharide substrate
Man-dNP. Error bars represent the standard deviation of triplicate measurements.
a
a-

L. Deng et al. / Bioorg. Med. Chem. 21 (2013) 4839–4845
4843
resolution mass spectra were recorded using a Bruker micrOTOF-II
LC–mass spectrometer. Flash chromatography was performed on
BDH silica gel with the specified solvents. Thin-layer chromatogra-
phy (TLC) was effected on Merck silica gel 60 F254 aluminum-
backed plates that were stained by heating (6200 °C) with 5%
sulfuric acid in EtOH. Percentage yields for chemical reactions as
described are quoted only for those compounds that were purified
by recrystallization or by column chromatography, and the purity
was assessed by 1H NMR spectroscopy. All solvents were
purchased from Sigma–Aldrich, Caledon, or Anachemia and all
solvents except DMF and MeCN were distilled before use and dried
according to the methods of Burfield and Smithers.14 CpGH125 and
SpGH125 were expressed as previously described.3 C. fimi Man2A
was a kind gift from Professor S. G. Withers (University of British
Columbia).
(s, 3H), 1.47 (s, 3H), 1.38 (s, 3H), 1.31 (s, 3H). 13C NMR (CDCl3,
100 MHz): d 168.50, 109.77, 99.63, 91.31, 75.11, 74.50, 72.07,
63.63, 61.68, 28.85, 27.95, 26.01, 20.81, 18.63.
4.2.3.2. Compound 7b.
1H NMR (CDCl3, 400 MHz): d 6.07 (s,
1H), 4.81 (dd, J = 6.0, 3.6 Hz, 1H), 4.65 (d, J = 6.0, 1H), 4.35 (dq,
J = 10.0, 4.0 Hz, 1H), 4.05 (dd, J = 8.8, 6.0 Hz, 1H), 4.01-3.96 (m,
2H), 2.02 (s, 3H), 1.43 (s, 3H), 1.41 (s, 3H), 1.33 (s, 3H), 1.29 (s,
3H). 13C NMR (CDCl3, 100 MHz):
d 169.21, 113.09, 109.17,
100.65, 84.93, 82.10, 79.18, 72.76, 66.66, 26.85, 25.81, 25.01,
24.53, 20.92.
4.2.4. 2,3:4,6-di-O-Isopropylidene-
a-D-mannopyranose (8a), and
2,3:4,6-di-O-isopropylidene-b-
D
-mannopyranose (8b)7
Compound 7a (950 mg, 3.14 mmol) was dissolved in MeOH
(15 mL) and NaOMe was added to adjust the pH of the solution
to above 10, after which the reaction was stirred at rt for 2 h.
The reaction was judged complete by TLC and then neutralized
using Amberlite RH-120 (H+) resin, filtered and concentrated under
vacuum. The residue was dried under high vacuum to afford the ti-
tle compound 8a (796 mg, 97%).
4.2. Chemical synthesis
4.2.1. 2,3,4,6-tetra-O-Acetyl-
-Mannose (10 g, 55.5 mmol) was dissolved in dry pyridine
a-D
-mannopyranosyl bromide (5)6
D
(50 mL), and Ac2O (50 mL) was added at 0 °C, the resulting reaction
mixture was stirred at rt overnight. The solvent was concentrated
in vacuo, the residue was co-evaporated with toluene (4 ꢂ 20 mL),
and then dried under vacuum to afford the title compound 4
(21.7 g crude), which was used directly in the next step without
purification. Per-O-acetate 4 (1.84 g, 4.71 mmol) was dissolved in
33% HBr–HOAc (15 mL), the resulting solution was stirred at rt
for 5 h, after which EtOAc (100 mL) was added and the reaction
mixture, and poured into ice-cooled water, washed with cold
water, cold sat. NaHCO3, cold brine, and then dried (MgSO4). After
filtration the filtrate was dried in vacuo to afford the title com-
pound 5 (1.62 g, 84% over two steps). The spectral data was in
agreement with the literature.6
Compound 7b was subjected to the same procedure (1.40 g,
4.63 mmol) to afford the title compound 8b (1.14 g, 97%).
4.2.4.1. Compound 8a.
1H NMR (CDCl3, 400 MHz): d 5.40 (s,
1H), 4.19–4.15 (m, 2H), 3.85 (dd, J = 9.2, 4.0 Hz, 1H), 3.80–3.68
(m, 3H), 3.61 (br s, 1H), 2.07 (s, 3H), 1.53 (s, 3H), 1.50 (s, 3H),
1.41 (s, 3H), 1.34 (s, 3H). 13C NMR (CDCl3, 100 M Hz): d 109.44,
99.76, 92.64, 76.19, 74.58, 72.67, 62.02, 61.43, 28.90, 28.09,
26.09, 18.75.
4.2.4.2. Compound 8a.
1H NMR (CDCl3, 400 MHz): d 5.35 (s,
1H), 4.78 (dd, J = 6.0, 3.6 Hz, 1H), 4.58 (d, J = 6.0, 1H), 4.40–4.34
(m, 1H), 4.15 (dd, J = 6.8, 4.8 Hz, 1H), 4.08–4.00 (m, 2H), 1.44 (s,
3H), 1.43 (s, 3H), 1.36 (s, 3H), 1.30 (s, 3H). 13C NMR (CDCl3,
100 MHz): d 112.56, 109.05, 101.12, 85.44, 80.05, 79.56, 73.23,
66.42, 26.71, 25.76, 25.08, 24.38.
4.2.2. 2,3:4,6-di-O-Isopropylidene-
-Mannose (3.6 g, 20 mmol) was dissolved in anhydrous DMF
D
-mannopyranose (6)7
D
(12 mL) and cooled to 0 °C. p-TsOHꢀH2O (19 mg, 1.0 mmol) and
2-methoxypropene (3.2 mL, 60 mL) were added to the resulting
solution. The reaction mixture was stirred at 0 °C for 3 h, after
which 2-methoxypropene (2.0 mL) was added by syringe. The mix-
ture was allowed to warm to rt gradually over 14 h. The reaction
mixture was neutralized by addition of Et3N (0.20 mL) and the
product partitioned between ether (40 mL) and water (40 mL).
The aqueous phase was collected and extracted with ether
(3 ꢂ 10 mL). The combined organic layers were washed with brine
(20 mL) and dried using MgSO4. After filtration and concentration
in vacuo, the product was isolated from the resulting gum by crys-
tallization using ethyl acetate/petrol to afford the desired product
6 (3.64 g, 70%) as a white solid containing a mixture of anomers.
4.2.5. 4-Methylumbelliferone 2,3:4,6-di-O-isopropylidene-
mannopyranoside (9a), and 4-methylumbelliferone 2,3:4,6-di-
O-isopropylidene-b- -mannopyranoside (9b)
a-D-
D
Compound 8a (796 mg, 3.06 mmol), 4-methylumbelliferone
(809 mg, 4.59 mmol), and PPh3 (1.20 g, 4.59 mmol) were dissolved
in anhydrous toluene (40 mL). To the reaction mixture was added
diisopropyl azodicarboxylate (DIAD, 0.90 mL, 4.59 mmol) and the
resulting solution was stirred at rt overnight. The mixture was di-
luted with EtOAc (200 mL), washed with 1 N NaOH (200 mL), sat-
urated NaHCO3 (200 mL), and brine (200 mL), after which the
organic layer was dried (MgSO4). After filtration and removal of
the solvent in vacuo, the residue was purified by gradient silica
gel column chromatography (Hex:EtOAc, 3:1–2:1) to afford 9a
(180 mg crude) and 9b (905 mg, 71%).
4.2.3. 1-O-Acetyl-2,3:4,6-di-O-isopropylidene-
mannopyranose (7a) and 1-O-acetyl-2,3:4,6-di-O-
isopropylidene-b-
-mannopyranose (7b)7
a-D-
D
Hemiacetals 6 (4.64 g, 17.8 mmol) were dissolved in dry pyri-
dine (40 mL). To the resulting mixture was added Ac2O (4.0 mL,
40 mmol) and the reaction mixture was stirred overnight at rt
The solution was concentrated under high-vacuum and co-evapo-
rated with toluene (4 ꢂ 10 mL) to afford a syrupy residue, which
4.2.5.1. Compound 9a.
1H NMR (CDCl3, 400 MHz): d 7.51 (d,
J = 8.8 Hz, 1H), 7.00 (J = 2.4 Hz, 1H), 6.94 (dd, J = 8.8, 2.8 Hz, 1H),
6.16 (d, J = 0.8 Hz, 1H), 5.81 (s, 1H), 4.40 (d, J = 6.0 Hz, 1H), 4.30
(dd, J = 8.0, 5.6 Hz, 1H), 3.81 (dd, J = 10.0, 8.0 Hz, 1H), 3.80-3.70
(m, 2H), 3.66–3.58 (m, 1H), 2.38 (d, J = 0.8 Hz, 3H), 1.58 (s, 3H),
1.51 (s, 3H), 1.40 (s, 3H), 1.38 (s, 3H). 13C NMR (CDCl3, 100 MHz):
d 160.96, 158.46, 154.82, 152.20, 125.65, 114.94, 113.23, 112.82,
109.94, 103.97, 99.80, 96.04, 75.66, 74.71, 72.27, 62.61, 61.67,
28.87, 28.05, 26.11, 18.69, 18.58.
was dissolved in EtOAc. The desired the
a-anomer, 7a (2.40 g,
44%), was crystallised from EtOAc/petrol and the mother liquid
afforded the b-anomer product 7b (3.00 g, 56%).
4.2.3.1. Compound 7a.
1H NMR (CDCl3, 400 MHz): d 6.26 (s,
1H), 4.15 (dd, J = 7.6, 5.6 Hz, 1H), 4.10 (dd, J = 5.6, 0.8 Hz, 1H),
3.85 (dd, J = 10.8, 5.6 Hz, 1H), 3.74 (dd, J = 10.0, 8.0 Hz, 1H), 3.69
(t, J = 10.4 Hz, 1H), 3.55 (td, J = 10.4, 5.6 Hz, 1H), 2.07 (s, 3H), 1.51
4.2.5.2. Compound 9b.
1H NMR (CDCl3, 400 MHz): d 7.51 (d,
J = 8.8 Hz, 1H), 7.00 (J = 2.4 Hz, 1H), 6.98 (dd, J = 8.8, 2.4 Hz, 1H),
6.17 (s, 1H), 5.55 (d, J = 3.2 Hz, 1H), 4.47 (dd, J = 6.8, 3.2 Hz, 1H),

4844
L. Deng et al. / Bioorg. Med. Chem. 21 (2013) 4839–4845
4.40-4.30 (m, 2H), 3.93 (dd, J = 10.4, 4.8 Hz, 1H), 3.61 (t, J = 10.4 Hz,
1H), 3.51 (td, J = 10.4, 4.8 Hz, 1H), 2.40 (d, J = 1.2 Hz, 3H), 1.61 (s,
3H), 1.49 (s, 3H), 1.42 (s, 6H). 13C NMR (CDCl3, 100 MHz): d
161.00, 159.54, 154.86, 152.27, 125.59, 114.94, 113.61, 112.76,
111.87, 104.22, 99.72, 96.02, 75.94, 73.25, 71.21, 66.12, 63.03,
28.91, 26.82, 25.62, 18.95, 18.63.
at rt for 3.5 h, then the solvent was removed under vacuo, the res-
idue was co-evaporated with toluene (4 ꢂ 5.0 mL), and then was
applied on a silica gel column and purified by flash chromatogra-
phy (CH2Cl2:MeOH, 2:1) to afford the title product 10 (23.0 mg,
88%). 1H NMR (CD3OD, 400 MHz): d 7.71 (d, J = 6.8 Hz, 1H), 7.14
(d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.8, 2.4 Hz, 1H), 6.20 (d, J = 1.2 Hz,
1H), 5.28 (d, J = 0.8 Hz, 1H), 4.84 (hidden in water peak, 1H), 4.12
(d, J = 1.2 Hz, 1H), 3.96–3.83 (m, 4H), 3.70 (dd, J = 12.0, 2.4 Hz,
1H), 3.66–3.55 (m, 5H), 3.51 (ddd, J = 9.6, 5.2, 2.4 Hz, 1H), 2.45
(d, J = 1.2 Hz, 3H). 13C NMR (CD3OD, 100 MHz): d 164.03, 161.75,
156.10, 155.85, 127.31, 116.12, 115.34, 112.82, 104.85, 101.64,
99.57, 77.38, 75.13, 74.47, 72.35, 72.34, 72.14, 68.56, 68.42,
67.79, 62.82, 18.74. HR-MS: calcd for C22H29O13 (M+H)+, 501.1603;
Found: 501.1601; Calcd for C22H28NaO13 (M+Na)+, 523.1422; Found:
4.2.6. 4-Methylumbelliferone 2,3-O-isopropylidene-b-D-
mannopyranoside (10)
Compound 9b (130 mg, 0.311 mmol) was dissolved in an 80%
aqueous HOAc solution (4.0 mL). The resulting solution was stirred
at rt for 5 h and the mixure was then concentrated under high vac-
uum, co-evaporated with toluene (6 ꢂ 10 mL), after which the res-
idue was dissolved in MeOH and co-evaporated with toluene
(2 ꢂ 10 mL). The residue was dried under high vacuum to yield
the glycoside 10 (115 mg, 98%), which was used directly in the
next step.1H NMR (CDCl3, 400 MHz): d 7.47 (dd, J = 7.2, 2.4 Hz,
1H), 6.98-6.95 (m, 2H), 6.11 (d, J = 1.2 Hz, 1H), 5.54 (d, J = 2.8 Hz,
1H), 4.43 (dd, J = 7.2, 2.8 Hz, 1H), 4.35 (t, J = 2.8 Hz, 1H), 4.20 (dd,
J = 6.0, 2.8 Hz, 1H), 3.85-3.74 (m, 2H), 3.61 (dt, J = 10.0, 4.0 Hz,
1H), 2.35 (d, J = 1.2 Hz, 3H), 1.56 (s, 3H), 1.41 (s, 3H). 13C NMR
523.1419; Calcd for
539.1143.
C
22H28KO13 (M+Na)+, 539.1161; Found:
4.2.9. HPLC purification of 4-methylumbelliferone a-D-
mannopyranosyl-(1?6)-b-D-mannopyranoside (a-Man-b-Man-
4MU, 2)
a-Man-b-Man-4MU was purified using an Agilent Eclipse XDB-
(CDCl3, 100 MHz):
114.77, 113.48, 112.50, 111.30, 103.73, 95.66, 79.09, 75.46, 73.29,
68.41, 62.28, 26.90, 25.66, 18.56.
d
161.21, 159.59, 154.68, 152.59, 125.68,
C18 reversed-phase semi-preparative (9.4 ꢂ 250 mm) column. The
disaccharide was injected and eluted using a linear gradient of
MeCN (4–20% over 30 min) at a flow rate of 2 mL/min. The reten-
tion time for the disaccharide under these conditions was typically
4.2.7. 4-Methylumbelliferyl 2,3,4,6-tetra-O-acetyl-
pyranosyl-(1?6)-2,3-O-isopropylidene-b- -mannopyranoside
(11) and 4-methylumbelliferyl -mannopyranosyl-(1?6)-2,3-
O-isopropylidene-b- -mannopyranoside (12)
a
-
D
-manno-
about 19.5 min. For all assays, the a-Man-b-Man-4MU used was
found to be P95% pure by HPLC.
D
a-D
D
4.3. Enzyme assays
To the MeCN (2.5 mL) solution of donor 5 (256 mg, 0.622 mmol)
0
was added 4 ÅA molecular sieves and HgCN2 (78.6 mg, 0.311 mmol)
and the mixture was stirred at rt for 10 min. A MeCN (2.5 mL) solu-
tion of compound 10b (115 mg, 0.304 mmol) was then added to
the reaction mixture and the solution was stirred at rt overnight.
Solvent was then removed in vacuo, the residue was taken up in
EtOAc (40 mL) and filtered. The solution was washed with water,
10% KI, saturated NaHCO3, and brine. After drying (MgSO4) and fil-
tration the solvent was removed in vacuo to yield a gummy resi-
due. Purification by gradient flash column silica chromatography
(Hex:EtOAc, 1:1–1:1.5 to 100% EtOAc) afforded intermediate disac-
charide 11 (141 mg of crude material). Disaccharide 11 (141 mg,
0.199 mmol) was dissolved in MeOH (6.0 mL), and NaOMe was
added to adjust the pH of the reaction mixture to be 11 as esti-
mated by litmus paper. The solution was stirred at rt for 3 h after
which time Amberlite RH (H+) 120 resin was added to neutralize
the base. The mixture was filtered and concentrated in vacuo to af-
ford a pale yellow gum. The desired product was purified from the
residue by silica gel flash column chromatography (CH2Cl2:MeOH,
10:1) to afford disaccharide 12 (65.1 mg, 60%).1H NMR (CD3OD,
400 MHz): d 7.71 (d, J = 8.8 Hz, 1H), 7.09 (d, J = 2.4 Hz, 1H), 7.06
(dd, J = 8.8, 2.4 Hz, 1H), 6.19 (d, J = 2.4 Hz, 1H), 5.64 (d, J = 2.8 Hz,
1H), 4.67 (d, J = 1.6 Hz, 1H), 4.49 (dd, J = 6.8, 2.4 Hz, 1H), 4.29 (t,
J = 6.8 Hz, 1H), 3.98 (dd, J = 10.4, 7.2 Hz, 1H), 3.85 (dd, J = 10.4,
4.8 Hz, 1H), 3.74-3.65 (m, 4H), 3.63-3.53 (m, 2H), 3.45 (ddd,
J = 9.6, 5.2, 2.4 Hz, 1H), 3.41 (dd, J = 3.2, 1.6 Hz, 1H), 2.46 (d,
J = 1.2 Hz, 3H), 1.57 (s, 3H), 1.42 (s, 3H). 13C NMR (CD3OD,
4.3.1. Assay of 4-methylumbelliferone b-D-mannopyranoside (b-
Man-4MU, 3) as a substrate for C. fimi Man2A
Fluorescence assays were performed in 96-well black plates
(Nunc). A stock of substrate was prepared in DMSO and diluted
to obtain the desired substrate concentrations and to ensure a final
DMSO concentration of 2% in all reactions. At this DMSO concen-
tration, the enzyme activity was within 5% of the activity as com-
pared to control assays conducted in the absence of DMSO. The
total assay volume was 100 lL. The buffer used in all reactions
was PBS (pH = 7.4) and the reaction was preincubated at 37 °C
for 5 min prior to initiation of the reaction by the addition of CfMa-
n2A to a final concentration of 1 nM. The production of 4-methyl-
umbelliferone was monitored for 10 min by fluorescence
spectroscopy using
a fMax plate reader (Molecular Devices)
equipped with a 355/485 nm filter (excitation/emission) pair. The
reaction velocity was determined by a linear fit of the data using
SoftMAX Pro software (Version 1.3.1). Relative fluorescence values
were converted to 4-methylumbelliferone concentration using a
standard curve generated in the same buffer and DMSO conditions
with a correlation coefficient of 0.9999.
4.3.2. Assay of 2,4-dinitrophenyl
a-D-mannopyranoside (a-Man-
dNP, 1) as a substrate for C. fimi Man2A
Assays were performed as described above with the following
variations. 96-well transparent polystyrene plates (Nunc) were
used and absorbance values were read continuously at 415 nm
on a fMax plate reader (Molecular Devices) over 2 h at 37 °C. Sub-
strate concentrations between 0.25 and 2.45 mM were assayed
100 MHz):
d 163.81, 161.64, 156.11, 155.79, 127.49, 116.00,
115.16, 112.79, 112.02, 104.46, 101.70, 97.06, 81.29, 76.19, 75.10,
74.40, 72.39, 71.98, 69.34, 68.43, 67.53, 62.72, 27.69, 26.20, 18.76.
HR-MS: calcd for C25H33O13 (M+H)+, 541.1916; Found: 541.1931;
Calcd for C25H32NaO13 (M+Na)+, 563.1735; Found: 563.1745.
using a concentration of 7 lM CfMan2A.
4.3.3. Assay of 2,4-dinitrophenyl
a-D-mannopyranoside (a-Man-
dNP, 1) as a substrate for CpGH125
4.2.8. 4-Methylumbelliferyl
mannopyranoside ( -Man-b-Man-4MU, 2)
Compound 12 (28.2 mg, 52.2 mol) was dissolved in a mixed
solution of TFA: H2O:CHCl3 (2.5:1:100, 2.0 mL in total), and stirred
a
-
D
-mannopyranosyl-(1?6)-b-
D
-
Assays were performed in 96-well transparent polystyrene
plates (Nunc). Absorbance values at 415 nm were monitored using
a fMax plate reader over three minutes. All reactions were per-
formed at 37 °C and were carried out in PBS buffer (pH 7.4) with
a
l

L. Deng et al. / Bioorg. Med. Chem. 21 (2013) 4839–4845
4845
a total assay solution volume of 100
converted to dNP concentrations using a path length of 0.326 cm
and an extinction coefficient of 12,890 Mꢃ1 cmꢃ1,10
l
L. Absorbance values were
every 2–3 h. The CfMan2A enzyme concentration was 7
the beginning of the assay and 24
The control sample contained 1 mM
l
M at
l
M at the end of the reaction.
a-Man-b-Man-MU and buffer
.
with no enzyme present. The control and reaction samples were
frozen and later analyzed using micellar electrokinetic chromatog-
raphy on a ProteomeLab PA800 (Beckman–Coulter) instrument.
Electrophoresis was performed under normal polarity in a 60 cm
4.3.4. Coupled assay of 4-methylumbelliferone
mannopyranosyl-(1?6)-b- -mannopyranoside (
4MU, 2) using CpGH125 and CfMan2A
Fluorescence assays were performed in 96-well black plates
(Nunc). A stock of -Man-b-Man-4MU (2) was prepared in DMSO
a-D-
a-Man-b-Man-
D
(to detector) ꢂ50
using a fixed applied voltage of 30 kV (resulting in a current of
approximately 40 A) in background electrolyte consisting of boric
acid (50 mM), sodium dodecyl sulphate (50 mM) and -cyclodex-
lm (internal diameter) fused silica capillary
a
and diluted to obtain the desired substrate concentrations to ensure
a final DMSO concentration of 2% in all reactions as described above.
l
c
The total assay volume was 100
l
L. The buffer used in all reactions
trin (11 mM), pH 9.0. All buffer components were of the highest
purity commercially available. The capillary was thermostated at
25 °C and samples were kept at 10 °C unless otherwise stated.
Sample was injected at the anode by applying a pressure of
0.5 psi for 10 s and detected by measuring the absorbance at
280 nm at a rate of 4 Hz. A time course assay was performed in
which the same sample was monitored by directly injecting reac-
tion aliquots (ꢁ15 nL) into the capillary after 0.18, 1.5, 2.5, 3.5,
4.5, and 7 h. The initial CfMan2A concentration in these assays
was PBS (pH = 7.4) and CfMan2A was preincubated at 37 °C for
5 min prior to the initiation of the reaction by the addition of
CpGH125. The final CfMan2A concentration in all assays was
140 nM and the final concentration of CpGH125 used in all assays
was 1 nM. The production of 4-methylumbelliferone was monitored
over 10 min by fluorescence spectroscopy using a fMax plate reader.
4.3.5. Coupled assay of 4-methylumbelliferone
a-D-
mannopyranosyl-(1?6)-b- -mannopyranoside (
4MU, 2) using CpGH125 and H. pomatia b-mannosidase
D
a
-Man-b-Man-
was
500
(6.57
13.57
tration was 20
7
l
M
while the
M. Additional CfMan2A was added to the mixture after 2 h
enzyme added; final enzyme concentration was
M) and 4 h (6.44 M enzyme added; final enzyme concen-
a-Man-b-Man-MU concentration was
l
As an alternative approach, a b-mannosidase from H. pomatia
(Sigma) was investigated as the coupling enzyme instead of CfMa-
n2A. H. pomatia b-mannosidase, obtained as a suspension in 3 M
ammonium sulphate buffer (pH = 4), was buffer exchanged into
PBS (pH 7.4) prior to use by use of a 5 kDa MWCO filter apparatus
from Millipore. First, fluorescence assays to test the activity of
H. pomatia b-mannosidase were performed in 96-well black plates
lM
l
l
lM). Although the final calculated enzyme concen-
tration was 20
l
M at the end of the assay (7 h), it is likely that the
active enzyme was present at a lower concentration because of en-
zyme instability leading to loss of activity over the course of the
experiment.
(Nunc) using a total assay volume of 100
reactions was PBS (pH = 5.95) containing 1% DMSO and varying
concentrations of the substrate 4-methylumbelliferone b- -man-
lL. The buffer used in all
Acknowledgements
D
nopyranoside (b-Man-4MU, 3). The mixture was preincubated at
37 °C for 5 min prior to initiation of the reaction by the addition
of H. pomatia b-mannosidase to yield a final enzyme concentration
of 100 nM. The production of 4-methylumbelliferone was moni-
tored continuously for 10 minutes by fluorescence spectroscopy
using a fMax plate reader (Molecular Devices) fitted with the 355
/ 485 nm filter pair. The reaction velocity was determined by a linear
fit of the data using SoftMAX Pro software (Molecular Devices). Rel-
ative fluorescence values were converted to 4-methylumbelliferone
concentration using a standard curve (correlation coefficient of
0.9999) generated using the same buffer. Using these conditions
we obtained the following kinetic parameters for the action of the
commercial H. pomatia b-mannosidase preparation assuming all
We thank Professor S. G. Withers (University of British Colum-
bia) for C. fimi Man2A and Professor A. Bennet (Simon Fraser Uni-
versity) for access to a fluorimeter. This work was supported by a
Canadian Institutes of Health Research Operating Grant (FRN
86610). D.J.V. is a Canada Research Chair in Chemical Glycobiology
and A.B.B. is a Michael Smith Foundation for Health Research Ca-
reer Scholar. D.J.V. and A.B.B. thank the Natural Sciences and Engi-
neering Research Council of Canada (NSERC) for support through
E.W.R Steacie Memorial Fellowships.
Supplementary data
protein is active enzyme; Km = 170
9 l
M, kcat = 16.4 0.3 min-1,
l
M -1. The kcat/Km value is some
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.05.062.
and kcat/Km = 9.6 0.5 ꢂ 10-2 min-1
200-fold less than that measured for CfMan2A. On the basis of this
observation we determined the final protein concentration of the H.
pomatia b-mannosidase preparation used in the coupled assay
References and notes
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2. King, S. J. Mol. Oral Microbiol. 2010, 25, 15.
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7. Gelas, J.; Horton, D. Carbohydr. Res. 1978, 67, 371.
should be in the range of 0.5 to 1 lM when employing the same
coupled assay conditions as described for CfMan2A and CpGH125.
4.3.6. Control reactions
In order to evaluate any background enzyme activities or spon-
taneous hydrolysis of substrates, fluorescence measurements were
taken of the following control mixtures over a period of 10 min
using a fMax plate reader equipped with a 355/485 nm filter pair.
8. Garegg, P. J. Carbohydr. Res. 1979, 73, 313.
a
-Man-b-Man-MU was incubated in PBS with CpGH125 alone,
Man-b-Man-MU was incubated in PBS with CfMan2A alone, and
-Man-b-Man-MU was incubated in PBS with no enzyme present.
a-
9. Cocinero, E. J.; Stanca-Kaposta, E. C.; Scanlan, E. M.; Gamblin, D. P.; Davis, B. G.;
Simons, J. P. Chemistry 2008, 14, 8947.
10. Stoll, D.; He, S.; Withers, S. G.; Warren, R. A. Biochem. J. 2000, 351, 833.
11. Zechel, D. L.; Reid, S. P.; Stoll, D.; Nashiru, O.; Warren, R. A.; Withers, S. G.
Biochemistry 2003, 42, 7195.
12. Offen, W. A.; Zechel, D. L.; Withers, S. G.; Gilbert, H. J.; Davies, G. J. Chem.
Commun. 2009, 2484.
13. Gloster, T. M.; Vocadlo, D. J. Nat. Chem. Biol. 2012, 8, 683.
14. Burfield, D. R.; Smithers, R. H. J. Org. Chem. 1983, 48, 2420.
a
Control reactions were performed for each experiment.
4.3.7. Capillary electrophoresis reactions
a-Man-b-Man-MU (1 mM) was incubated with CfMan2A for 7 h
at 37 °C in PBS containing 1% DMSO, with fresh enzyme added