Development of GlcNAc-inspired iminocyclitiols as potent and selective N -acetyl-β-hexosaminidase inhibitors

Article abstract of DOI:10.1021/cb100011u

Human N-acetyl-β-hexosaminidase (Hex) isozymes are considered to be important targets for drug discovery. They are directly linked to osteoarthritis because Hex is the predominant glycosidase released by chondrocytes to degrade glycosaminoglycan. Hex is also associated with lysosomal storage disorders. We report the discovery of GlcNAc-type iminocyclitiols as potent and selective Hex inhibitors, likely contributed by the gain of extra electrostatic and hydrophobic interactions. The most potent inhibitor had a K_i of 0.69 nM against human Hex B and was 2.5 × 10⁵ times more selective for Hex B than for a similar human enzyme O-GlcNAcase. These glycosidase inhibitors were shown to modulate intracellular levels of glycolipids, including ganglioside-GM2 and asialoganglioside-GM2.

Full text of DOI:10.1021/cb100011u

ARTICLE
Development of GlcNAc-Inspired
Iminocyclitiols as Potent and Selective
N-Acetyl-␤-Hexosaminidase Inhibitors
Ching-Wen Ho†,‡,§, Shinde D. Popat†, Ta-Wei Liu†, Keng-Chang Tsaiʈ, Meng-Jung Ho†,Ќ
,
Wei-Hung Chen†, An-Suei Yangʈ,‡, and Chun-Hung Lin†,ʈ,‡,Ќ,
*
†Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, ‡Chemical Biology and Molecular Biophysics, Taiwan
International Graduate Program, Academia Sinica, Taipei, Taiwan, §Department of Chemistry, National Tsing-Hua
University, Hsin-Chu, Taiwan, ʈThe Genomics Research Center, Academia Sinica, Taipei, Taiwan, ЌInstitute of Biochemical
Sciences, National Taiwan University, Taipei, Taiwan
-Acetyl-␤-hexosaminidase (Hex) is a member
of the lysosomal hydrolase family that cata-
lyzes the hydrolysis of terminal, non-reducing
ABSTRACT Human N-acetyl-␤-hexosaminidase (Hex) isozymes are considered
to be important targets for drug discovery. They are directly linked to osteoarthri-
tis because Hex is the predominant glycosidase released by chondrocytes to de-
grade glycosaminoglycan. Hex is also associated with lysosomal storage disorders.
We report the discovery of GlcNAc-type iminocyclitiols as potent and selective
Hex inhibitors, likely contributed by the gain of extra electrostatic and hydropho-
bic interactions. The most potent inhibitor had a Ki of 0.69 nM against human Hex
B and was 2.5 ϫ 105 times more selective for Hex B than for a similar human en-
zyme O-GlcNAcase. These glycosidase inhibitors were shown to modulate intracel-
lular levels of glycolipids, including ganglioside-GM2 and asialoganglioside-GM2.
N
N-acetyl-␤-D-glucosamine (GlcNAc) and N-acetyl-␤-D-
galactosamine (GalNAc) residues in glycoproteins, gan-
gliosides, and glycosaminoglycans (1, 2). There are two
major Hex isozymes in humans, Hex A and Hex B. Hex A
is a heterodimer composed of ␣ and ␤ subunits that
are encoded by two related genes, HEXA and HEXB, re-
spectively. Hex B is a homodimer made up of two iden-
tical ␤-subunits (3, 4). Hex S exists as a minor isozyme
consisting of two ␣-subunits. Human Hex isozymes are
important because of the role they play in osteoarthritis
and lysosomal storage disorders. Hex is the dominant
glycosaminoglycan-degrading glycosidase released by
chondrocytes into the extracellular milieu. The Hex en-
zymes are the predominant glycosidases in the synovial
fluid from patients with osteoarthritis (5). Inhibition of
this enzyme can prevent or even reverse cartilage ma-
trix degradation, representing a new avenue in the de-
velopment of therapeutic treatments for osteoarthritis
(5−7).
The Hex enzymes are also linked to lysosomal stor-
age disorders that are characterized by the intralysoso-
mal accumulation of glycolipids, including ganglioside-
GM2 and asialoganglioside-GM2 (GA2) (8). Deficiency of
either Hex ␣- or ␤-subunit results in clinically devastat-
ing neurological diseases. Tay-Sachs disease and Sand-
hoff disease arise from mutations in HEXA and HEXB, re-
spectively (9). Although enzyme replacement therapy
(ERT) has been primarily utilized to treat patients suffer-
ing from lysosomal storage disorders, currently there is
*Corresponding author,
chunhung@gate.sinica.edu.tw.
Received for review November 6, 2009
and accepted February 24, 2010.
Published online February 27, 2010
10.1021/cb100011u
© 2010 American Chemical Society
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Figure 1. Structures of inhibitors 1؊6.
no such treatment for patients with GM2-gangliosidosis fers high affinity with glycosidases by providing a good
(10). It is known that ERT is associated with a number of
disadvantages, such as extreme high cost and failure
to cross the bloodϪbrain barrier, making it necessary
to develop other alternatives (11). One promising alter-
native is to use inhibitors of Hex enzymes as pharmaco-
logical chaperones (also called chemical chaperones)
to increase the stability of defective lysosomal enzymes
and thus maintain their metabolic functions at physi-
ologically sustainable levels (12, 13). A high-throughput
screening was applied to search for drug-like inhibitors,
leading to identification of three effective pharmacologi-
cal chaperones in patient fibroblasts (13).
Even though Hex isozymes are suitable targets for
drug discovery, currently there are two major challenges
to limit their use. The first issue is achieving sufficient in-
hibitor potency. Second, oftentimes the Hex inhibitors
also inhibit a similar enzyme, O-GlcNAcase (an enzyme
hydrolyzing terminal GlcNAc residue from O-linked gly-
coproteins), which uses the same catalytic mechanism
(14, 15). O-GlcNAcase is categorized in the Glycoside
Hydrolase Family 84 based on the CAZy database (16,
17), whereas the Hex enzymes are classified in Family
20. In eukaryotic cells, many proteins are modified by
O-linked GlcNAc on serine and threonine residues (18,
19). The dynamic addition and removal of O-GlcNAc resi-
dues requires the action of two essential enzymes,
namely, O-GlcNAc transferase (20, 21) and
transition-state mimicry at physiological pH. We previ-
ously developed 1,5-dideoxy-1,5-imino-L-fucitol deriva-
tives with success by incorporating additional aglycons
(groups attached to the iminocyclitol ring) to generate
potent and selective inhibitors against ␣-fucosidase
(29−31). We sought to determine if iminocyclitol deriva-
tives designed in this study such as 2-acetamido-1,2,5-
trideoxy-1,5-imino-D-glucitols (GlcNAc-type iminocycliti-
ols including compound 1 and related analogues, see
Figure 1) are potent inhibitors of Hex, as well as what
chemical modifications are able to afford selectivity to
distinguish between Hex and O-GlcNAcase. The struc-
ture information of human Hex B (32) reveals that the vi-
cinity of the enzyme active site contains multiple nega-
tive charges. This feature is, nevertheless, not found in
the structure of O-GlcNAcase (15). Our strategy is thus to
incorporate various groups to the ring nitrogen of com-
pound 1 to acquire additional electrostatic and other in-
teractions. The most potent inhibitor was uncovered to
show a Ki of 0.69 nM against human Hex B and a Ki of
2.1 nM against Hex A. The inhibition was 2.5 ϫ 105
times more selective for Hex B than for a similar hu-
man enzyme O-GlcNAcase. The inhibitors were shown
to cause the accumulation of gangliosides GM2 and
GA2 by inhibiting lysosomal Hex isozymes, whereas a
similar dosage did not affect the level of O-GlcNAc-
modified proteins.
O-GlcNAcase, respectively (22, 23). A number of inhibi-
tors have been developed to specifically recognize
O-GlcNAcase (14, 24, 25). Recently the development of
potent and selective Hex inhibitors has started to draw
attention, such as Gal-PUGNAc by Vocadlo et al., GlcNAc-
statins by van Aalten et al., and pyrrolidines by Fleet
and co-workers (26−28).
Iminocyclitiols have demonstrated great potential in
the therapeutic development, as evidenced by N-(2-
hydroxyethyl)-deoxynojirimycin (Miglitol) for the treat-
RESULTS AND DISCUSSION
We initially synthesized compound 1 (Figure 1) by modi-
fication of Vasella’s procedure (scheme S1A) (33−39).
Our modified synthesis has two advantages. The start-
ing material (GlcNAc) to synthesize compound 1 is avail-
able at a large scale and low cost. The procedure al-
lows the flexibility of either incorporating any
substituent to the ring nitrogen (Supplementary Scheme
ment of type II diabetes. This class of molecules often of- S1A) or modifying the N-acyl group at C2 position (i.e.,
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TABLE 1. Inhibition constants and selectivity ratios of inhibitors 1؊6 for human Hex B and human
O-GlcNAcasea
Ki of O-GlcNAcase (␮M)
Ki of Hex B (␮M)
Selectivity ratio (Ki O-GlcNAcase/Ki Hex B)
1
2
3
4
5
6
23.6 Ϯ 0.97
107.7 Ϯ 9.17
101.5 Ϯ 6.47
175.6 Ϯ 2.15
155.1 Ϯ 9.87
145.0 Ϯ 1.77
0.54 Ϯ 0.25
43
51,000
85,000
250,000
91
0.0021 Ϯ 0.00038
0.0012 Ϯ 0.00023
0.00069 Ϯ 0.000077
1.7 Ϯ 0.56
0.0267 Ϯ 0.00111
5,400
aValues are the means of three determinations Ϯ SD.
the N-acetyl group can be hydrolyzed before the final
deprotection, followed by acylation).
The enzyme activity was assayed by using
4-methylumbelliferyl N-acetylglucosamine (MUG) as the
substrate. Hex A was separated from other Hex
isozymes by the known procedure (Supplementary Fig-
ure S1), and so was Hex B (40). In consistence with the
previous result (33), compound 1 is an inhibitor of Hex B
(Ki ϭ 0.54 ␮M).
(please see pages 8 and 9 of Supporting Information
for the detailed information). The derivatives from five
carboxylic acids (C9, C10, D11, E4 and F4 shown in
Supplementary Figure S2) and three aldehydes (A-4a,
B-2a and B-3a in Supplementary Figure S2) were found
to give more potent inhibition than others (Supplemen-
tary Figure S3). These eight molecules were individually
prepared, purified, and examined for the inhibition. The
most selective and potent inhibitor of Hex B was ob-
tained when compound 2 was coupled with
To acquire additional electrostatic interactions as
aforementioned, the ring nitrogen of compound 1 was
attached with several aminoalkyl groups of different
chain length. The derivative containing N-aminoheptyl
group (compound 2 in Figure 1) was found to be the
most potent. Kinetic analysis indicated that compound
2 is a competitive inhibitor of Hex B (Ki ϭ 2.1 nM).
To compare the selective inhibition between Hex B
and O-GlcNAcase, human O-GlcNAcase was prepared
for examination (see Supporting Information) (23, 41).
Compounds 1 and 2 had Ki values of 23.6 and
107.7 ␮M for human O-GlcNAcase and 0.54 and
0.0021 ␮M for Hex B, respectively (Table 1). The ratio
Ki O-GlcNAcase/Ki Hex B revealed that compound 2 is 51,000
times more selective for Hex B than for O-GlcNAcase,
whereas compound 1 is only 43 times more selective.
The inhibitory potential of compound 2 was further
enhanced by derivatization with a variety of groups in-
cluding 180 carboxylic acids (for amide bond formation)
(30, 31) and 40 aldehydes (for reductive amination)
that were purchased from commercial sources. Their
molecular structures were shown in Supplementary Fig-
ure S2. After the reactions were complete, the resulting
mixtures were diluted without purification and assayed
for the Hex B activity by using MUG as the substrate
p-methoxybenzaldehyde (B-3a) by reductive amination.
As a matter of fact, the reaction produced two products
including the monosubstituted product 3 and disubsti-
tuted product 4. Their yields were dependent on the
amount of NaBH3CN and the aldehyde in the reaction
(Supplementary Scheme S1B). Both compounds 3 and
Figure 2. Lineweaver؊Burk analysis of Hex B steady-state kinetics in
the presence of compound 4. The concentrations of compound 4 used
were 3.12 nM (⌬), 1.56 nM (9), 0.78 nM (▫), 0.39 nM (ꢀ), and 0.00 (Œ).
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Figure 3. Computational molecular models of inhibitor 4 in the active site of human Hex B (PDB code, 1NOW) as shown in ribbon (Hex B) and stick (in-
hibitor 4) form (A) and with respect to the Hex B surface (B). The carbon atoms of inhibitor 4 are colored in magenta. The green, orange, and yellow
dotted lines represent tentative hydrogen bonding donor؊acceptor pairs, hydrophobic, and electrostatic interactions of inhibitor 4 with Hex B, re-
spectively. The molecular models were created using the software PyMOL. A) Side chains of Hex B residues within 7 Å radius centered on inhibitor 4
are also shown in stick explicitly, including carbon (gray), nitrogen (blue), and oxygen (red) atoms. Trace of the Hex B backbone is shown as blue
tube. The secondary structure elements are shown in ribbon form, and the important residues involved in inhibitor 4 binding are labeled. B) The col-
ors red, blue, green, and gray on the Hex B surface indicate oxygen, nitrogen, carbon, and hydrogen atoms, respectively.
4 showed greater inhibition of Hex B as compared with
compound 2. In particular, compound 4 had a Ki of 0.69
nM for Hex B and was 250,000 times more selective
for Hex B than for O-GlcNAcase (Table 1). Similarly, the
same compound exhibited a Ki of 2.1 nM
for Hex A. The double reciprocal plot clearly
showed compound 4 to be a competitive in-
hibitor (Figure 2). The chemical modifica-
tions from compound 1 to 4 resulted in a sig-
nificant enhancement in both the potency
and selectivity.
Meanwhile we also carried out the inhibi-
tion study of Bacteroides fragilis
O-GlcNAcase (Bf O-GlcNAcase, Supplemen-
tary Table S1), because the enzyme shares
ϳ40% sequence identity with human homo-
logue. In fact, O-GlcNAcase from a similar
strain, Bacteroides thetaiotaomicrometer (Bt
O-GlcNAcase, showing 74% sequence iden-
tity as compared to Bf O-GlcNAcase), was
proven highly similar to the human homo-
logue (15). The similarity was further cor-
roborated with our inhibition result (Supple-
mentary Table S1) because the inhibition
Figure 4. Immunocytochemical analysis of intracellular GA2 ganglioside level. Mouse microglia
cells (BV2) cells were cultured in the presence or absence of 100 nM biotin-conjugated inhibitor
(6) and then triple stained with polyclonal antibodies against GA2 ganglioside (green, panels B
and E), polyclonal antibodies against biotin (red, panels C and F), and 4=,6=-diamidino-2-
phenylindole to stain nuclear DNA (blue, panels A and D). GA2 ganglioside accumulated after BV2
was incubated with the inhibitor for 16 h, as shown by significant green fluorescence. The two
right-column panels G؊L represent higher magnification from selected single cells in panels
A؊F, respectively. Bars ؍ 30 ␮m
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pattern of Bf O-GlcNAcase is consistent with that of hu-
man O-GlcNAcase.
Although the Hex and O-GlcNAcase active sites are
structurally similar, the surrounding residues are ex-
tremely different. Thus, in principle it would be pos-
sible to develop highly selective inhibitors for both Hex
and O-GlcNAcase if suitable aglycones could be found.
Figure 3 presents a computational modeling analysis of
human Hex B (PDB code, 1NOW) in complex with inhibi-
tor 4. A hydrophobic cleft, enclosed by residues W424,
D426, Y450, and L453, is located on the protein surface
near the active site; this cleft was found to accommo-
date the aminoheptyl moiety of inhibitor 4. The termi-
nal amino group is protonated in solution and likely
forms electrostatic interactions with D426, consistent
with previous data (7). The conversion of the amino
group of compound 2 to an azide (compound 5, shown
in Figure 1) not only led to the elimination of the positive
charge but also greatly diminished the inhibition up to
810 fold (from Ki ϭ 0.0021 ␮M of compound 2 to Ki ϭ
1.7 ␮M of 5). Additionally, the p-methoxybenzyl substit-
uents of inhibitor 4 were surrounded by a hydrophobic
pocket composed of residues P448, L450, K462, Y463,
and V466. On the other hand, the aglycon of compound
4 [i.e., bis(4-methoxybenzyl)aminoheptyl] could not
find a matched pocket in O-GlcNAcase, leading to lower
binding affinity with O-GlcNAcase than with Hex A/B.
Because the structure of human O-GlcNAcase is cur-
rently unavailable, the X-ray structure of Bt O-GlcNAcase
(PDB code, 2J4G) was utilized for the purpose of compu-
tational modeling. E355 of human Hex B and D243 of
Bt O-GlcNAcase were found to be close to the ring nitro-
gen of compound 1 at a distance of 3.9 and 4.6 Å, re-
spectively (Supplementary Figure S4-1 and S4-2). The
shorter distance in human Hex B may provide com-
pound 1 with a better starting point during optimiza-
tion of the inhibition selectivity, because the inhibitors
1Ϫ6 are all more selective for Hex B than for
Figure 5. Inhibitors 1, 4, and PUGNAc were shown to increase levels of
GM2 in microglia (BV2) cells to a different degree. The cells were
treated with 200 nM of each inhibitor for 10 days and extracted with
chloroform/methanol for further HP-TLC analysis (A). In each experi-
ment, the control cells (designated as “؊”) were grown without addi-
tion of inhibitor, in parallel to the inhibitor-treated cells (designated as
“؉”). The spots corresponding to GM2 were further quantified by
densitometric analyses (B).
nM) for 16 h, as shown in Figure 4, panels AϪL. The
amount of GA2 was proportional to the concentration
of inhibitor 6 and was reached a plateau at 20 nM of in-
hibitor 6, as demonstrated by the dot-blot analysis
(Supplementary Figure S5). Furthermore, after BV2 cells
were treated with compound 1, 4, or PUGNAc (200 nM of
each) for 10 days, glycosphingolipids were extracted
from the cells for analysis of ganglioside level by using
high-performance thin-layer chromatography (HP-TLC).
Identified by comparison with the authentic standards,
GM2 was significantly more increased in the treatment
with compound 4 than that with 1 or PUCNAc (Figure 5).
To study the effects of O-GlcNAcase inhibition in hu-
O-GlcNAcase.
To determine if the inhibitors were functional in vivo,
compound 2 was conjugated with biotin (to give com-
pound 6, shown in Figure 1) for the purpose of fluores-
cence imaging and affinity chromatography. Even
though the Ki value of compound 6 increased from 2.1
nM (compound 2) to 26.7 nM, compound 6 was 5400
times more selective for Hex B as compared with
O-GlcNAcase. GA2 in mouse microglia cells (BV2) accu- man cells, activity of compounds 1, 2,and 4 in the hu-
mulated if the cells were treated with compound 6 (100 man 293T cell line were evaluated by Western blot
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There are several strategies to enhance the potency
and selectivity of enzyme inhibitors, such as acquisition
of additional interaction and better mimicry of the tran-
sition state. Our previous development of ␣-fucosidase
inhibitors, for instance, incorporated an extra aglycon to
the iminocyclitol, resulting in Ͼ1000-fold enhancement
in the binding affinity (29). The observed enhancement
in the potency and selectivity was also documented in
the successful development of several potent and selec-
tive O-GlcNAcase inhibitors, including PUGNAc pentana-
mide (45), GlcNAcstain (24), and NAG-thiazoline deriva-
tives (14, 25), where a hydrophobic aglycone and/or
N-acyl group play a critical role (45). A review was re-
cently published that covers the O-GlcNAcase inhibi-
tors in depth (46). With respect to Hex inhibitors, the
single change in the configuration from PUGNAc to Gal-
PUGNAc successfully distinguished between Hex A/B
and O-GlcNAcase (28). Fleet’s, Nitoda’s, and Wong’s
groups also prepared pyrrolidines to give potent inhibi-
tion against Hex (6, 27, 47). GlcNAcstain was also modi-
fied to give nanomolar inhibitors against Hex A/B and
O-GlcNAcase (26). Recently the 2-acetamino-1,2-
dideoxynojirimycin derivatives not only were reported
as Hex inhibitors but also utilized to enhance the im-
paired Hex activity in adult Tay-Sachs and infantile
Sandhoff cell lines (48). Our synthesized GlcNAc-type
iminocyclitiols can significantly improve the potency
and selectivity by gaining extra electrostatic and hydro-
phobic interactions.
Summary and Significance. GlcNAc-type iminocycli-
tiols were developed and optimized for the Hex inhibi-
tion by incorporating various groups to the ring nitrogen
of compound 1. Among the derived products, the best
inhibitor (compound 4) is highly selective for Hex A (Ki ϭ
2.1 nM) and Hex B (Ki ϭ 0.69 nM), while it inhibits
O-GlcNAcase only at much higher level (Ki ϭ 175 ␮M).
The inhibitor was shown to increase GM2- and GA2-
ganglioside levels in cultured cells but not affect the
level of O-GlcNAc-modified proteins. The result is prom-
ising for further development of therapeutic agents for
various Hex-related diseases.
Figure 6. Immunoblot analysis indicated that inhibitors 1,
2, and 4 do not increase the level of O-GlcNAc-modified
proteins, in contrast to the two known O-GlcNAcase inhibi-
tors PUGNAc and STZ (positive controls). 293T cells were
treated with 10 ␮M of each inhibitor for 16 h and analyzed
for the level of O-GlcNAc-modified proteins by Western
blot (upper panel). In contrast, the level of GAPDH was
about equal in each lane (lower panel).
analysis with an anti-O-GlcNAc antibody (14). Two
known O-GlcNAcase inhibitors, PUGNAc (Ki ϭ 35 nM)
and STZ (Ki ϭ 14 ␮M), were employed as the positive
control (26, 42−44). No significant accumulation in
O-GlcNAc levels was observed when the human cells
was treated with 10 ␮M of compounds 1, 2, and 4
(Figure 6). The results echo those of the previous in
vitro study that compound 4 is much more selective for
Hex A/B than for O-GlcNAcase. Furthermore, the concen-
tration of compound 4 can be increased up to 50 ␮M
without altering the level of O-GlcNAc-modified proteins,
in contrast to the enhanced level at 200 ␮M (Supple-
mentary Figure S6). In accordance with Supplementary
Figures S5 and S6, we conclude that compound 4 can
display the desirable potency and selectivity for Hex B at
the range of 20 nMϪ50 ␮M without affecting the
O-GlcNAcase activity.
for the synthetic schemes. Unless otherwise mentioned, all the
reactions were conducted using anhydrous solvents under an ar-
gon atmosphere. Analytical TLC was performed on precoated
plates (Merck, Kieselel 60 F254) with a 250-␮m layer thickness.
METHODS
Synthesis of Compounds 2؊4. Due to space limitation, the
synthesis and characterization of compounds 1, 5 and 6 are
available in Supporting Information. See Schemes S1A and S1B
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Flash column chromatography was carried out with silica gel
0.05Ϫ1.5 mM substrate in water. The reactions of O-GlcNAcase
Mallinckrodt Type 60 (230Ϫ400 mesh). Reagents of the high-
(200 ␮L) contained 2.5 nM enzyme in 50 mM NaH2PO4, 100 mM
NaCl, 0.1% BSA, pH 6.5, and 0.05Ϫ1.5 mM substrates in wa-
ter. All assays were performed in triplicate at 30 °C for 25 min.
The O-GlcNAcase activity was measured in a time-dependent
manner, in contrast to the measurement of the Hex A (or Hex
B) activity where the reaction was stopped by addition of a
5-fold excess quenching buffer (200 mM sodium glycine buffer,
pH 10.8). Both enzymes were stable over the time course in their
respective buffers (data not shown). Enzyme activity was mea-
sured by fluorescence spectroscopy of the release of
est purity were purchased from Sigma and Acros. 1H and 13
C
NMR spectra were recorded on 400-MHz instruments with CDCl3
[␦H 7.26, ␦C 77.0 (central line of a triplet)] or CD3OD [␦H 3.30 (cen-
tral line of a quintet), ␦C 49.0 (central line of a quintet)] as the in-
ternal standard. Optical rotations were measured on a digital po-
larimeter with a cuvette of 10 cm length at ambient temperature.
N-[1(7-Amino-heptyl)]-2-acetamido-1,2,5-trideoxy-1,5-imino-D-
glucitol (2). Compound 15 (see Supplementary Scheme S1A;
50.0 mg, 0.08 mmol) was deprotected by hydrogenolysis to gen-
erate 18.4 mg of compound 2 in 71% yield. [␣]20D ϭ ϩ1.50 (c
0.12, MeOH); 1H NMR (400 MHz, CD3OD) ␦ 3.89Ϫ3.99 (m, 3H,
H-6, H-2), 3.52 (dd, J ϭ 9.3, 9.1 Hz, 1H, H-4), 3.43 (dd, J ϭ 9.9,
9.2 Hz, 1H, H-3), 3.22 (dd, J ϭ 11.6, 4.4 Hz, 1H, H-1equa.),
3.05Ϫ3.09 (m, 1H, H-7), 2.96 (t, J ϭ 7.5 Hz, 2H, H-13),
4-methylumbelliferone with an excitation wavelength of 360
nm and an emission wavelength of 460 nm. The data were fit
to the MichaelisϪMenten equation using the software Kaleida-
Graph to determine the Km values. (Please see Supporting Infor-
mation regarding to the preparation and biochemical character-
ization of Hex A and Hex B)
The Ki values of compounds 1Ϫ6 were determined using
steady-state kinetics where the MUG concentrations were used
at 3- to 5-fold Km values. To give an ideal progress curve, an ap-
propriate enzyme concentration (0.06 nM of Hex and 2.5 nM of
O-GlcNAcaes) and inhibitor concentration (0.3 nMϪ200 ␮M)
were used. The mode of inhibition was verified by the
2.78Ϫ2.84 (m, 1H, H-7), 2.53Ϫ2.60 (m, 1H, H-5), 2.51 (dd, J
ϭ 11.6, 11.1 Hz, 1H, H-1axial), 2.01 (s, 3H, Ac), 1.60Ϫ1.70 (m,
4H), 1.39Ϫ1.47 (m, 6H); 13C NMR (135 MHz, CD3OD) ␦ 173.88,
76.53, 71.79, 67.78, 58.48, 54.52, 53.49, 51.15, 40.85, 30.69,
28.46, 27.77, 27.15, 25.14, 22.93; HRMS (FAB) m/z calcd for
C
15H32N3O4 (M ϩ Hϩ) 318.2393, found 318.2363.
Compound 3. 4-Methoxybenzaldehyde (5.15 mg, 0.038
mmol) was added to a solution of compound 2 (10.0 mg, 0.032
mmol) in methanol (2 mL), as shown in Supplementary Scheme
S1B. NaBH3CN (6.0 mg, 0.096 mmol) was then added slowly to
the solution. The resulting mixture was stirred for 36 h at RT. Af-
ter evaporation to remove the reaction solvent, the residue was
purified by silica gel column chromatography with CHCl3/MeOH/
NH4OH (6:3.5:0.5) to give 8.2 mg of product 3 as a colorless
syrup at 60% yield. [␣]20D ϭ ϩ3.00 (c 0.001, MeOH); 1H NMR
(400 MHz, CD3OD) ␦ 7.43 (d, J ϭ 8.6 Hz, 2H), 7.01 (d, J ϭ 8.6
Hz, 2H), 4.14 (s, 2H), 3.88Ϫ4.01 (m,3H, H-6, H-2), 3.83 (s, 3H,-
OCH3), 3.52 (t, J ϭ 9.4 Hz, 1H, H-3), 3.28Ϫ3.40 (m, 2H, H-4,
H-1equa.), 3.10Ϫ3.17(m, 1H, H-7), 3.06 (t, J ϭ 7.9 Hz, 2H,
H-13), 2.77Ϫ2.84(m, 1H, H 7), 2.41Ϫ2.60 (m, 2H, H-5,
H-1axial), 1.99 (s, 3H, Ac), 1.61Ϫ1.74 (m, 4H), 1.32Ϫ1.45 (m,
6H); 13C NMR (135 MHz, CD3OD) ␦ 173.91, 162.35, 132.65 (2C),
124.51, 115.74 (2C), 76.42, 71.53, 67.92, 56.01, 54.36, 53.63,
52.05 (2C), 50.88, 46.78, 29.89, 27.84, 27.52, 27.14, 25.31,
22.86; HRMS (FAB) m/z calcd for C23H40N3O5 (M ϩ Hϩ)
438.2968, found 438.2958.
LineweaverϪBurk plot. The Ki values were determined by the
double reciprocal plot (1/V vs 1/[S]) to give an apparent Km (the
Km value obtained in the presence of inhibitors). The secondary
plot was generated by plotting the apparent Km values as a func-
tion of inhibitor concentration. Ki was determined by calculat-
ing the negative value of the resulting x intercept.
Immunofluorescence Image Analysis. Commercial antibodies
used in this study were purchased from Abcam (rabbit antibi-
otin, rabbit anti-LIMP ll, and rabbit anti-GA2), Abnova (mouse
anti-GAPDH), and Invitrogen (4=,6=-diamidino-2-phenylindole
[DAPI], goat antimouse Alexa Fluor 594, and goat antirabbit Al-
exa Fluor 488). For immunofluorescence staining (Figure 4), BV2
cells were grown on glass coverslips and fixed in 4% paraform-
aldehyde for 15 min. Fixed cells were washed briefly with PBS
containing 0.5% (v/v) Triton-X100 and stained with antibodies
as indicated. Coverslips were mounted with Prolong antifade re-
agent (Molecular Probes), and each fluorescent dye was im-
aged using a Leica TCS SP2 Confocal Microscope and Incuba-
tion System (Leica, Germany).
Compound 4. 4-Methoxybenzaldehyde (54.0 mg, 0.38 mmol)
was added to a solution of compound 2 (30.0 mg, 0.095 mmol)
in methanol. NaBH3CN (27.0 mg, 0.39 mmol) was then slowly
added to the solution, as shown in Supplementary Scheme S1B.
After stirring at RT for 36 h, the reaction mixture was evapo-
rated and purified by silica gel column chromatography with
CHCl3/MeOH (3:1) to give 34.2 mg of compound 4 as a color-
less syrup at 65% yield. [␣]20D ϭ ϩ3.75 (c 0.0016, MeOH); 1H
NMR (400 MHz, CD3OD) ␦ 7.27 (d, J ϭ 8.5 Hz, 4H), 6.91 (d, J ϭ
8.5 Hz, 4H), 3.83Ϫ3.88 (m, 3H, H-6, H-2), 3.80 (s, 6H, -OCH3),
3.61 (bs, 4H, NϪCH2), 3.42 (t, J ϭ 9.1, 1H, H-3), 3.23 (dd, J ϭ
10.0, 9.0 Hz, 1H, H-4), 3.04 (dd, J ϭ 11.3, 4.4 Hz, 1H, H-1equa.),
2.77Ϫ2.84 (m, 1H, H-7), 2.47Ϫ2.57 (m, 3H, H-7, H-13),
2.10Ϫ2.19 (m, 2H, H-5, H-1axial), 1.98 (s, 3H, Ac), 1.39Ϫ1.58
(m, 4H), 1.22Ϫ1.32 (m, 6H); 13C NMR (135 MHz, CD3OD) ␦
173.75, 161.31 (2C), 132.43 (4C), 115.22 (4C), 114.91 (2C),
77.57, 72.66, 67.67, 59.55, 58.42 (2C), 55.95 (2C), 55.50,
53.63, 53.48, 51.85, 30.16, 28.41, 28.05, 26.44, 25.65, 22.90;
HRMS (FAB) m/z calcd for C31H48N3O6 (M ϩ Hϩ) 558.3543, found
558.3530.
Analysis of Ganglioside Levels and O-GlcNAc-Modified
Proteins. BV2 cells were cultured for 24 h in 10-cm tissue cul-
ture dishes containing DMEM. Experiments were initiated by
plating cells onto a 10-cm plate at approximately 10% conflu-
ence and adding 0.2Ϫ1.0 ␮M compound 1, 4, or PUGNAc. The
cells were allowed to approach confluence after 4Ϫ5 days and
split 1:3 to a 15-cm plate. After another 4Ϫ5 days of growth in
media containing the tested inhibitors, the cells were grown to
approximately 95% confluence. The cells were then washed
once with cold PBS and harvested in 15 mL of PBS. The cells
were collected by centrifugation at 250 ϫ g for 10 min, lysed,
and extracted for obtaining gangliosides according to previous
procedure (49). The resulting samples were subjected to a Folch
partition in CHCl3/MeOH/H2O of 4:2:1 (v:v:v) (50−52). The up-
per layer was collected, dried, and redissolved in CHCl3/MeOH
of 2:1 (v:v). Later, 1 mL of water was added, followed by centrifu-
gation to remove the upper layer. The lower layer was further ex-
tracted by CHCl3/MeOH/0.1%NaCl of 1:10:10 (v/v/v). The result-
ing mixture (50Ϫ60 ␮g) was then applied on a HP-TLC plate
(Merck) along with a total of 1 ␮g of a mixture of GM1, GM2,
GM3, and GD1d (Sigma) as the authentic standards. Analysis
and staining for gangliosides was carried out according to previ-
ous protocol (53). The plate was run in a solvent system consist-
ing of CHCl3/MeOH/H2O (containing 0.2% CaCl2) of 55:45:10 (v/
v/v), followed by staining with CeSO4/H2SO4.
Kinetic Analysis of O-GlcNAcase, Hex A, and Hex B. All kinetics
experiments of O-GlcNAcase, Hex A, and Hex B were determined
using the fluorogenic substrate MUG (Sigma). Standard reac-
tions of Hex A or Hex B (200 ␮L) contained 0.06 nM enzyme in
50 mM citric acid, 100 mM NaCl, 0.1% BSA, pH 4.25, and
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14. Macauley, M. S., Whitworth, G. E., Debowski, A. W., Chin, D., and
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15. Dennis, R. J., Taylor, E. J., Macauley, M. S., Stubbs, K. A., Turken-
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(2006) Structure and mechanism of a bacterial ␤-glucosaminidase
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␤-galactosidase and some ␤-glucosidases, Protein Sequences Data
Anal. 4, 61–62.
Regarding the analysis of O-GlcNAc levels, 293T cells were
grown on 6-cm plates and allowed to reach approximately 80%
confluence. Cells were incubated with inhibitors 1, 2, 4, PUG-
NAc, or STZ (10 ␮M of each) for 16 h, harvested, and lysed as
mentioned previously. Levels of O-GlcNAc-modified proteins and
GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (as a
loading control) were analyzed by Western blots. Briefly, cell pel-
lets were lysed in 200 ␮L of lysis buffer, sonicated, and centri-
fuged to remove cell debris. The cleared cell lysates were then
separated by SDSϪPAGE, blotted, and probed for levels of
O-GlcNAc-modified proteins and GAPDH.
Acknowledgment: This work was supported by National Sci-
ence Council (NSC 97-2628-M-001-016-MY3 and 95-2113-M-
001-027-MY3) and Academia Sinica, Taiwan. The data of mass
spectrometric analysis were acquired at the NRPGM Core Facili-
ties for Proteomics and Glycomics, Academia Sinica. We thank
Mr. Sue-Ming Chang for his assistance in NMR experiments.
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Supporting Information Available: This material is available
free of charge via the Internet at http://pubs.acs.org.
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