Inhibition of microbial β-N-acetylhexosaminidases by 4-deoxy- and galacto-analogues of NAG-thiazoline

Article abstract of DOI:10.1016/j.bmcl.2014.09.066

NAG-thiazoline is a well-established competitive inhibitor of two physiologically relevant glycosidase families - β-N-acetylhexosaminidases (GH20) and β-N-acetylglucosaminidases (GH84). Based on the different substrate flexibilities of these enzyme groups, we designed and synthesized the 4-deoxy derivative of NAG-thiazoline aiming at the selective inhibition of GH20 β-N-acetylhexosaminidases. One GH84 and two GH20 microbial glycosidases were employed as model enzymes for the inhibition assays. Surprisingly, the new compound 4-deoxy-thiazoline exhibited no activity inhibition with either of the enzyme families of interest. Unlike with the substrates, the 4-hydroxyl group of the inhibitor's sugar ring seems to be crucial for binding the inhibitor to the active sites of these enzymes.

Full text of DOI:10.1016/j.bmcl.2014.09.066

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5321–5323
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of microbial b-N-acetylhexosaminidases
by 4-deoxy- and galacto-analogues of NAG-thiazoline
a
a,
a
Jana Krejzová a,b, Lubica Kalachova a, Petr Šimon a, , Helena Pelantová , Kristyna Slámová , Vladimír Kren
⇑
´
ˇ
a
ˇ
Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, CZ 14220 Praha 4, Czech Republic
b Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Technická 5, CZ 16628 Praha 6, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
NAG-thiazoline is a well-established competitive inhibitor of two physiologically relevant glycosidase
families—b-N-acetylhexosaminidases (GH20) and b-N-acetylglucosaminidases (GH84). Based on the dif-
ferent substrate flexibilities of these enzyme groups, we designed and synthesized the 4-deoxy derivative
of NAG-thiazoline aiming at the selective inhibition of GH20 b-N-acetylhexosaminidases. One GH84 and
two GH20 microbial glycosidases were employed as model enzymes for the inhibition assays. Surpris-
ingly, the new compound 4-deoxy-thiazoline exhibited no activity inhibition with either of the enzyme
families of interest. Unlike with the substrates, the 4-hydroxyl group of the inhibitor’s sugar ring seems to
be crucial for binding the inhibitor to the active sites of these enzymes.
Received 11 August 2014
Revised 11 September 2014
Accepted 12 September 2014
Available online 2 October 2014
Keywords:
NAG-thiazoline
Enzyme inhibition
b-N-Acetylhexosaminidase
O-GlcNAcase
Ó 2014 Elsevier Ltd. All rights reserved.
Lysosomal b-N-acetylhexosaminidases (b-Hex; GH20) and cyto-
plasmatic b-N-acetylglucosaminidases (O-GlcNAcases; GH84) were
identified as structurally and functionally related glycosidases with
different abundances, substrate specificities and therefore biologi-
cal functions.1 In humans, a lack of these enzymes causes severe
neurodegenerative disorders such as Tay-Sachs’ and Sandhoff’s
lysosomal storage disorders (GH20) and Alzheimer’s disease
(GH84). For the study of the individual enzymes in vivo, inhibitors
specific for just one of these enzyme families are required in order
to avoid the generation of complex phenotypes. With respect to
the substrate-assisted catalytic mechanism and active site archi-
tecture shared by both of these glycosidase families,2 the design
and synthesis of group-specific inhibitors has been developing rap-
idly in last few decades.
of the parent compound, moreover, the selectivity of the new
derivatives was too low for their practical application.
In this work we focused on the inhibition of GH20 and GH84
glycosidases based on their fundamental difference in substrate
specificity: while the GH84 O-GlcNAcases are strictly specific for
the hydrolysis of GlcNAc units, the typical feature of GH20
b-Hex is that they also accept the respective galacto-epimer
(GalNAc)8 and, as a special case, the 4-deoxy-derivative phenyl
2-acetamido-2,4-dideoxy-b-D-xylo-hexopyranoside can be utilized
by some fungal b-N-acetylhexosaminidases with no significant loss
of activity.9 Based on this finding, the galacto- (2) and 4-deoxy (3)
derivatives of NAG-thiazoline (1) (Fig. 1) were synthesized and
tested as inhibitors of the bacterial and fungal b-Hex and bacterial
O-GlcNAcase employed as model enzymes.
One of the most studied inhibitors of the GH20 and GH84 gly-
Whereas the known NAG-thiazoline (1)3 as well as Gal-thiazo-
line (2)10 were prepared according to Knapp‘s procedure, the newly
designed 4-deoxy-thiazoline (3) was prepared from 2-acetamido-
cosidases is NAG-thiazoline (20-methyl-2-acetamido-2-deoxy-
a-
D
-glucopyranosyl-[2,1-d]-D
20-thiazoline, 1),3 which acts as a stable
mimic of the reaction intermediate NAG-oxazoline. Since its first
synthesis by Knapp and co-workers, it has been used as a lead
structure for various derivatizations.2,4–6 However, with the single
exception of a highly selective derivative named thiamet G,7 no
other synthetic analogue of the inhibitor 1 reached the effectivity
2-deoxy-
D
-glucose (GlcNAc; 4) by the multistep synthesis shown
⇑
Corresponding author. Tel.: +420 296442766; fax: +420 296442509.
E-mail addresses: slamova@biomed.cas.cz, slamova.kristyna@gmail.com
(K. Slámová).
Present address: Institute of Organic Chemistry and Biochemistry, Academy of
Sciences of the Czech Republic, Flemingovo nám. 2, CZ 16610 Praha 6, Czech Republic.
Figure 1. Structures of inhibitors used in this study.
http://dx.doi.org/10.1016/j.bmcl.2014.09.066
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

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J. Krejzová et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5321–5323
OH
OH
OBz
O
OBz
O
Cl
AcCl, MeOH,
reflux, 1.5h
BzCl, pyridine
reflux, 1.5h
SO2Cl2, pyridine
4 °C, 18h
Bu3SnH, VAZO,
HO
HO
O
HO
HO
O
HO
BzO
toluene,reflux, 2h
BzO
OH
NHAc
AcHN
AcHN
AcHN
OCH3
OCH3
OCH3
4
5
6
7
OBz
O
OH
OBz
O
OBz
MeONa, MeOH
RT, 3h
Lawesson reagent
toluene,80 °C, 1.5h
1. Ac2O, H2SO4
RT, 4h
2. Ac2O,ZnCl2
50 °C, 1h
O
O
OAc
BzO
HO
BzO
BzO
N
N
AcHN
AcHN
S
S
OCH3
9
8
10
Scheme 1. Synthesis of 4-deoxy-thiazoline 3.
3
Table 1
Inhibition of microbial glycosidases by NAG-thiazoline-based inhibitors (substrate pNP-GlcNAc, pH 7, 25 °C)
Enzyme
KI (l
M)
KI (
lM)
KI (lM)
NAG-thiazoline (1)6
Gal-thiazoline (2)
4-Deoxy-thiazoline (3)
b-Hex
T. flavus
S. plicatus
B. thetaiotaomicron
42.7 1.9
24.0 5.0
0.029 0.003
393 33
90.8 6.3
n.d.
1888 474
9900 7000
1831 97
O-GlcNAcase
n. d. = not determined.
in Scheme 1. The synthesis of 4-deoxy-thiazoline (3) is based on
two key steps: 4-deoxygenation and thiazoline ring formation,
both requiring the synthesis of suitable protected precursors,
formed in a series of protection and deprotection reactions.
In the first step, the anomeric hydroxyl group of N-acetylgluco-
samine (4) was selectively methylated with methanol under catal-
ysis with acetyl chloride to yield 5,11 which was then selectively
benzoylated using benzoyl chloride in pyridine under reflux to
afford the 3,6-di-O-benzoyl derivative 6.12 Deoxygenation of the
hydroxyl group at C-4 in compound 6 was accomplished in two
steps employing reductive dechlorination.13 Compound 6 was trea-
ted with sulfuryl chloride in pyridine at 0 °C to yield the 4-chloro
derivative (7, 62%) in the galacto-configuration. Compound 7 was
then dechlorinated with tri-n-butyltin hydride in the presence of
a catalytic amount of 1,10-azobis(cyclohexanecarbonitrile) in anhy-
drous toluene, yielding the key intermediate methyl 2-acetamido-
deoxy-b-D-glucosaminide (pNP-GlcNAc) as a chromogenic sub-
strate are summarized in Table 1, the Lineweaver–Burk plots of
the experiments with inhibitors 2 and 3 are shown in the Supple-
mentary material (Figs. S1–S5).
In this study, we aimed at designing an inhibitor based on the
key difference in substrate specificities between the two otherwise
closely related glycosidase families 20 and 84. We employed the
fact that only the GH20 b-N-acetylhexosaminidases utilize the
galacto-configured substrates and some of these enzymes, mainly
those of fungal origin, were shown to also readily accept the 4-
deoxy substrate.9 As it has already been shown previously,14 Gal-
thiazoline (2) is a specific inhibitor of GH20 glycosidases, even
though its inhibition potency is somehow lower than that of
NAG-thiazoline, which we also observed in our experiments. What
we found interesting was the lack of inhibition of both tested
enzyme groups by the newly designed compound 4-deoxy-thiazo-
line (3). With respect to our previous results with the 4-deoxy sub-
strates, we expected that the GH20 b-N-acetylhexosaminidases
would be inhibited by this modified thiazoline, however, it seems
that the 4-hydroxyl of the glycosyl moiety is instrumental for the
proper binding of the inhibitor in the active sites of the respective
enzymes.
In summary, we have designed and synthesized the 4-deoxy
derivative of NAG-thiazoline, a generally recognized strong com-
petitive inhibitor of GH20 b-N-acetylhexosaminidases and GH84
b-N-acetylglucosaminidases, aiming at the selective inhibition of
GH20 enzymes. Unlike the known 4-deoxy substrates, the analo-
gous transition state mimicking thiazoline 3 was found to be a poor
inhibitor of all model enzymes from both glycosidase families
employing the substrate-assisted catalytic mechanism. We sup-
pose that the 4-hydroxyl moiety in the equatorial configuration
of the inhibitor molecule is crucial for its strong binding to the
active sites of these glycosidases.
3,6-di-O-benzoyl-2,4-dideoxy-a-D-glucopyranoside (8, 93%).
Two-step conversion of compound 8 into acetate followed by ano-
merization resulted into the corresponding b-acetate (9, 64%)
required for the thiazoline ring formation.3 Compound 9 was then
converted with Lawesson’s reagent in anhydrous toluene3 at 80 °C
into the protected 4-deoxy-thiazoline (10, 82%). In the final step,
Zemplén deacylation of the benzoyl groups gave the desired
product 4-deoxy-thiazoline (3, 76%). To avoid any inconsistent
results of the enzymatic assays, we performed the 1H NMR stability
experiment in D2O at ambient temperature confirming that
compound 3 is stable for at least 24 h, which is sufficient for the
kinetic experiments. Experimental details and spectral data of the
new compounds can be found in the Supplementary material.
Three microbial model enzymes of the studied glycosidase fam-
ilies were used to test the inhibition potency of Gal-thiazoline (2)
and 4-deoxy-thiazoline (3): b-N-acetylhexosaminidase (GH20)
from the filamentous fungus Talaromyces flavus and the one from
the bacterium Streptomyces plicatus; and the b-N-acetylglucosa-
minidase (GH84) from Bacteroides thetaiotaomicron. The enzymes
were expressed and purified as described in our previous work.6
NAG-thiazoline (1) was used as the benchmark inhibitor here,
the inhibition constants for the studied enzymes with NAG-thiaz-
oline were reported recently.6 The results of the inhibition assays
performed at pH 7, 25 °C using p-nitrophenyl 2-acetamido-2-
Acknowledgements
This work was supported by the Czech Science Foundation
grant P207/11/0629, by EU project NOVOSIDES FP7-KBBE-2010-
4-265854 (co-funded by MSMT 7E11011) and by the research con-
cept RVO61388971 (Institute of Microbiology, Prague). Authors

J. Krejzová et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5321–5323
5323
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would like to thank to Prof. D. Vocadlo (SFU, Burnaby, Canada) for
very kind providing the gene of O-GlcNAcase from Bacteroides
thetaiotaomicron.
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Supplementary data
ˇ
8. Weignerová, L.; Vavrušková, P.; Pišvejcová, A.; Thiem, J.; Kren, V. Carbohydr. Res.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.09.
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