New Class of Glycoside Hydrolase Mechanism-Based Covalent Inhibitors: Glycosylation Transition State Conformations

Article abstract of DOI:10.1021/jacs.7b05065

The design of covalent inhibitors in glycoscience research is important for the development of chemical biology probes. Here we report the synthesis of a new carbocyclic mechanism-based covalent inhibitor of an α-glucosidase. The enzyme efficiently catalyzes its alkylation via either an allylic cation or a cationic transition state. We show this allylic covalent inhibitor has different catalytic proficiencies for pseudoglycosylation and deglycosylation. Such inhibitors have the potential to be useful chemical biology tools.

Full text of DOI:10.1021/jacs.7b05065

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Communication
A New Class of Glycoside Hydrolase Mechanism-Based Covalent
Inhibitors: Glycosylation Transition State Conformations
Saeideh Shamsi Kazem Abadi, Michael Tran, Anuj Yadav, Pal
John Pal Adabala, Saswati Chakladar, and Andrew J. Bennet
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b05065 • Publication Date (Web): 19 Jul 2017
Downloaded from http://pubs.acs.org on July 19, 2017
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A New Class of Glycoside Hydrolase Mechanism-Based Covalent In-
hibitors: Glycosylation Transition State Conformations
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Saeideh Shamsi Kazem Abadi, Michael Tran, Anuj Yadav, Pal John Pal Adabala, Saswati Chak-
2
2,
ladar, and Andrew J. Bennet *
1
2
Departments of Biochemistry and Molecular Biology, and Chemistry Simon Fraser University, 8888 University
Drive, Burnaby, British Columbia, V5A 1S6, Canada
RECEIVED DATE (automatically inserted by publisher); E-mail:bennet@sfu.ca
Supporting Information Placeholder
ABSTRACT: The design of covalent inhibitors in glycosci-
ence research is important for the development of chemical
biology probes. Here we report the synthesis of a new carbo-
cyclic mechanism-based covalent inhibitor of an α-
glucosidase. The enzyme efficiently catalyzes its alkylation
via either an allylic cation or a cationic transition state. We
show that this allylic covalent inhibitor has very different
catalytic proficiencies for pseudo-glycosylation and deglyco-
sylation. Such inhibitors have the potential to be useful
chemical biology tools.
Figure 1. Proposed mechanism of a GH13 retaining α-
glucosidase; for clarity, some hydroxyl groups are not shown.
1
The catalytic transfer of carbohydrate moieties frequently
involves anomeric positive charge delocalization onto the
endocyclic oxygen atom; we show for the first time that an
alkene can perform the same task. Of note, enzymes that
either add or remove carbohydrates are often critical to cel-
Conformations are shown for the Michaelis complex ( S
3
), the
4
glucopyranosylium ion-like transition state ( H ) and the
3
4
enzyme-bound intermediate ( C ).
1
1-3
lular regulation. The enzymes that remove sugar residues
by hydrolysis of glycosidic bonds are called glycoside hydro-
4
lases (GHs). Nature has evolved several strategies for cataly-
sis by GHs, with most enzymes using a pair of active site as-
partic and/or glutamic acid (Asp/Glu) residues.
5
-7
Figure 2. Structures of the mechanism-based covalent inhib-
itor 1 (GH36 enzyme), and the GH13 inhibitors (2 and 3)
evaluated as covalent inhibitors in the current study.
Retaining glycoside hydrolases with two catalytic Asp/Glu
residues operate via two sequential inversions of configura-
tion. The first results in the formation of a covalent glycosyl-
enzyme intermediate (Figure 1) and the second, not shown,
involves intermediate hydrolysis. The transition states (TSs)
for glycosylation and deglycosylation possess pyranosylium
ion-like character, and the six-membered ring adopts one of
Previously, we utilized the requirement for transition state
charge delocalization, a fundamental factor in catalysis by
most GHs, in our design of a cyclopropyl-containing mecha-
nism-based covalent inhibitor (1) of
galactosidase (GH36).
a
retaining α-
Here we describe the synthesis of
two carbocyclic analogues of -glucose (2 and 3) that are
1
4,15
6,8
D
several allowed conformations. In the current example,
9
covalent inhibitors of a GH13 retaining α-glucosidase. We
show that these compounds lead to a single covalent labeling
of the enzyme, and importantly that the rate constants for
retaining GH13 α-glucosidases react via pyranosylium ion-
like TSs that are traversed during the catalytic cycle. Moreo-
ver, the pyranoside conformation at the TSs is postulated to
4
10
'
pseudo'-glycosylation and deglycosylation for these inhibi-
be a H half-chair. Also, for GH13 enzymes the structure of
3
4
11
tors are distinct and provide insight into the conformational
itinerary for this GH family.
the enzyme bound intermediate is a C chair (Figure 1).
1
Kinetic isotope effect data suggests that the productive
Michaelis complex is not a ground state chair conformation,
We synthesized 2 and 3 (Scheme 1) from 4, which we made
1
12,13
but is likely a S skew boat (Figure 1).
in four steps (42% yield) from 2,3,4,6-tetra-O-benzyl-D-
3
16
glucopyranose. First, we acetylated the tertiary alcohol in 4
to give 5, which underwent a palladium-catalyzed [3,3]-
sigmatropic rearrangement to give the pseudo anomeric ace-
17
tate 6. Deacetylation gave allylic alcohol 7, which was sub-
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jected to a Furukawa modified Simmons-Smith cyclopropa-
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1
2
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2
3
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5
5
5
6
nation to give 8. Both 7 and 8 underwent facile S Ar reac-
N
tions followed by global debenzylation with BCl to give 2
3
and 3, respectively.
Scheme 1. Synthesis of carbocyclic inhibitors 2 and 3.
BnO
HO
BnO
BnO
AcO
BnO
BnO
BnO
1
) LiHMDS
) AcCl
2%
Pd(II)
OAc
OBn
2
EtOAc
5
BnO
OBn
BnO
OBn
BnO
4
5
6
8
BnO
BnO
BnO
BnO
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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0
1
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0
1
2
3
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0
1
2
3
4
5
6
7
8
9
0
NaOMe
Zn(Et)
2 2 2
, CH I
OH
OH
MeOH
PhMe
7
7% (2 steps)
81%
BnO
F
OBn
BnO
1) NaH,
OBn
NO
7
1
) NaH,
NO
2
F
2
2) BCl
3
,
CH
9% (2 steps)
NO
2
Cl
2
2) BCl
3
,
CH
42% (2 steps)
NO
2 2
Cl
HO
HO
HO
4
Figure 4. Reactivation kinetics for covalently inhibited α-
2
HO
2
glucosidase. Shown in red circles are the data for the dealkyl-
ation of enzyme labeled by inhibitor 2, while the blue circles
represent reactivation of enzyme treated with 3. The solid
lines represent the best non-linear fits to a standard first-
order rate equation. Conditions were T=25 °C, sodium phos-
phate buffer (50 mM, pH 6.84, [BSA] = 1 mg/ml).
HO
O
HO
O
OH
OH
2
3
We tested carbasugar analogues 2 and 3 for their activity
against a yeast α-glucosidase. Shown in Figure 3 are the
measured pseudo-first-order rate constants for the loss of
enzyme activity, each data point is calculated from remain-
ing enzyme activity as a function of preincubation time, as a
function of the concentration of the carbasugar analogue.
Notably, the allylic inhibitor 2 (red circles) is less active than
the bicyclo[4.1.0]heptyl inactivator 3 (blue circles).
Scheme 2. Kinetic scheme for the covalent inhibition
of GH13 yeast α-glucosidase by carbasugar analogues
2
and 3.
kinact
kreact
E + I
E:I
E-I
E + P
Ki
We tried to identify the sites of labeling by incubating the
enzyme with excess inhibitor, followed by ESI tandem mass
spectrometry (MS/MS) of the tryptic (and peptic) peptides
obtained by digestion of both the inactivated and the un-
treated enzymes. Unfortunately, we were unable to obtain
satisfactory peptide fragmentation that remained covalently
modified after tryptic digestion. However, we showed that
yeast α-glucosidase is singly labeled by the expected mass
addition of the carbon skeleton portions of 2 and 3 to the
molecular weight of the enzyme (Figure S1 Supporting In-
formation). That is, the mass spectrum of enzyme shows a
single peak for the native enzyme at 67275.7, while that after
reaction with 2 shows the intact enzyme and a mono-
alkylated species (C H O = 158.1) at 67433.9, and the mass
7
10
4
Figure 3. Kinetics for covalent inhibition of yeast α-
glucosidase. Shown in red circles are the data for allylic in-
hibitor 2, while the blue circles represent labeling by bicyclic
inhibitor 3. Error bars that are not visible are encompassed
within the symbol. The dashed lines are the best non-linear
fits to a standard Michaelis–Menten equation. Conditions
were T=25 °C, sodium phosphate buffer (50 mM, pH 6.84,
[BSA] = 1 mg/ml).
spectrum for the enzyme modified by 3 displays a single peak
at 67448.8, which corresponds to addition of the carbocyclic
skeleton of 3 (C H O = 172.1 + H).
8
12
4
We then measured the reactivation rate constants for the
covalently-modified enzyme (Figure 4). Remarkably, the
recovery of enzyme activity following inhibition by allylic
inhibitor 2 (red circles) is more rapid than that for bicy-
clo[4.1.0]heptyl inactivated enzyme (blue circles). The kinetic
data were fit to the standard kinetic scheme for covalent
inhibition (Scheme 2) and the derived rate and equilibrium
constants are tabulated in Table 1.
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Table 1. Kinetic parameters for the covalent inhibition and reactivation of yeast α-glucosidase by the inhibitors
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
and 3. Conditions were T=25 °C, sodium phosphate buffer (50 mM, pH 6.84, [BSA] = 1 mg/ml).
–
1
–1 –1
–1
Inactivator
Ki (
ꢀ
M)
kinact (s )
t½ (mins)
12.8
kinact/K (M s )
kreact (s )
t½ (hrs)
16.3
I
–
4
–5
2
3
570 ± 90
285 ± 45
(9.05
(1.82
±
0.63)
×
10
1.59
6.4
±
0.27
(1.19
(2.17
±
±
0.07)
0.14)
×
×
10
–
3
–6
±
0.14)
×
10
6.3
±
1.1
10
88.7
1,4
The data in Table 1 shows two remarkable features: (i)
B boat). That is, the evolved reaction coordinate for α-
rate-
determining non-chemical step, exhibits a second-order
both the first- (kinact) and second-order (kinact/K ) rate con-
glucopyranoside hydrolysis, which involves
a
i
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
12
stants for inactivation of yeast α-glucosidase are larger for
the bicyclic inhibitor; and (ii) the reactivation of labeled en-
zyme (kreact) is faster for the allylic covalent adduct.
4
–1 –1
rate constant (kcat/Km = 6.7 × 10 M s ) for 4-nitrophenyl α-
glucopyranoside. However, in order to assess the efficiency
12
18
19-21
of covalent labeling it is important to calculate the relative
enzymatic proficiencies for formation of the covalent glyco-
Both allylic and cyclopropylcarbinyl compounds
un-
dergo S 1 reactions at accelerated rates via allylic and non-
classical bicyclobutonium cationic intermediates,
N
22,23
syl-enzyme intermediates (kcat/K × 1/kuncat or kinact/K ×
respec-
m
i
1
4,27,28
1/kuncat).
tively. Of note, distinct conformations are required for for-
mation of delocalized carbocations from inhibitors 2 and 3.
Specifically, enzyme-catalyzed labeling within the active site
requires a conformation in which a π-type molecular orbital
can assist glycosidic C–O bond cleavage, a process that oc-
curs from an oxygen n-type lone pair for natural substrates.
In the current study, the cyclopropyl-containing inhibitor 3
requires a pseudo-equatorial aglycone for effective σ-bond
NO
2
NO
2
NO
2
O
O
O
O
9
10
11
Figure 6. Structures of the model compounds (9–11) used to
estimate relative catalytic proficiencies.
24-26
Due to the extremely slow spontaneous hydrolysis rates of
participation (Fig. 5, Panel A),
in carbocyclic inhibitor 2 entails a pseudo-axial aglycone
Fig. 5, Panel B).
while allylic participation
29
glycosides, we used the kinetic data for the unsubstituted
14
model compounds 9 and 11 and we made 10 (Figure 6), by
(
standard procedures (Supporting Information). To evaluate
the spontaneous rate constant for the pH-independent hy-
drolysis of 10 at 25 °C we extrapolated kinetic data acquired
at higher temperatures (Supporting Information, Table S1).
We then calculated the relative rate constants for covalent
labeling of the enzyme (pseudo-glycosylation) and for cleav-
age of the glycosidic bond in the enzyme intermediate
(pseudo-deglycosylation). Listed in Table 2 are the second-
order rate constants (kcat/Km and kinact/K ) for yeast α-
i
glucosidase
reacting
with
4-nitrophenyl
α-D-
1
2
glucopyranoside and our two covalent inhibitors 2 and 3,
the uncatalyzed first-order rate constants for model com-
pound hydrolysis, and the relative catalytic proficiencies
(
(
CP), with a value of 1.0 for the 4-nitrophenyl glucoside
kcat/K × 1/k ) .
m
uncat rel
Table 2. Relative enzymatic proficiencies for glyco-
side hydrolysis and covalent-labeling by 2 and 3.
Figure 5. Conformations for π-type orbital participation
blue atoms at back) into the σ* of the glycosidic C–O bond;
(
the C6 hydroxyl group is omitted for clarity. Panel A) α-
cyclopropyl inhibitor 3; B) α-allylic compound 2; C) Merca-
tor projection of six-membered ring conformations. The cur-
rently accepted reaction coordinate for a GH13 enzyme is
indicated by the orange box. Possible conformations for a
pyranosylium ion-like TS are shown in red. Bisected confor-
mations for cyclopropyl assisted ionization are shown in teal
with the closest to the enzymatic reaction coordinate being
circled. The two lowest energy conformations for an allylic
cation (between C5–C6–C1) are labeled in rose (bold font)
with the proposed reaction coordinate for 2 circle by the rose
box.
–
1 a
core structure kcat/K or kinact/K
kuncat (s )
CPrel
m
i
–
1 –1
(M s )
b
–5
10
pyranosyl
67,000
4.61
4.20
1.49
×
1.0
–
8
cyclohexenyl
1.59
6.4
×
10
0.028
0.0033
–6
× 10
bicyclo[4.1.0]
a
Rate constant extrapolated to 25 °C, Supporting Infor-
mation (Table S2). Data taken from reference .
b
12
Notably, the catalytic proficiency for covalent labeling by 2
is higher than the corresponding value for reaction with 3,
despite the cyclopropyl inhibitor exhibiting a larger second-
5
,6
Based on current theories that GH13 enzymes stabilize
4
1
pyranosylium ion-like H TSs from a bound S Michaelis
order rate constant (kinact/K ) for enzyme labeling.
i
3
3
5
,6
complex, we propose that our bicyclo[4.1.0]carbasugar 3
reacts from the bisected geometry, required for bicyclobuto-
nium ion formation, that is closest to that for the catalyzed-
hydrolysis reactions of GH13 enzymes (Figure 5, panel A, a
Even though the ground state conformations that permit
2
π-bond participation in 2 ( H half-chair or a B boat) are
3
1,4
removed from the GH13 α-glucosidase reaction coordinate
3
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(
orange box, Figure 5, panel C) it is clear that the enzyme
available free of charge on the ACS Publications website at
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
stabilizes formation of an allylic cation-like TS, which should
http://pubs.acs.org.
have five coplanar carbon atoms. Thus, we reason that the
2
AUTHOR INFORMATION
Corresponding Author
*bennet@sfu.ca
enzyme binds 2 in a H half-chair with a resulting catalyzed
3
formation of an E allylic cation or allylic cation-like TS (rose
3
box, Figure 5), a species that is conformationally similar to
4
the glycosylation TS ( H ). In the case of covalent inhibitor 3,
3
σ-bond participation requires a bisected geometry; however,
the resultant cation likely remains in the original bisected
geometry due to the high rotational barrier in bicyclobutoni-
Notes
The authors declare no competing financial interests.
3
0
um ions. We conclude that covalent labeling by 2, relative
to 3, involves a reaction coordinate that more closely match-
es that of the natural substrates.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACKNOWLEDGMENT
We thank Ms. Kyung-Mee Moon, Mr. Jason Rogalski, and Dr.
Leonard Foster for mass spectral data acquisition. This work
was supported by a Natural Sciences and Engineering Re-
search Council of Canada Discovery Grant (AJB: #121348-
Table 3. Relative proficiencies for dealkylation of the
yeast α-glucosidase covalent intermediates.
–
1 a
2012).
core
kdeglyc or kreact kuncat (s )
CPrel
–
1
structure
(s )
REFERENCES
–
1 b
–5
–8
pyranosyl
>29 s
4.61
4.20
1.49
×
10
1.0
(
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–
5
–4
cyclohexenyl 1.19
×
×
10
×
10
<4.6
<2.4
×
×
10
(
–
6
–6
10
–6
bicyclo[4.1.0] 2.17
10
×
10
a
b
(3)
Schnaar, R. L.; Gerardy-Schahn, R.; Hildebrandt, H. Phys-
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(4)
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deglycosylation are markedly different (Table 3) than those
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glucopyranosyl enzyme intermediate is hydrolyzed much
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1
(7)
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Sinnott, M. Carbohydrate Chemistry and Biochemistry:
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1
Structure and Mechanism; 2nd ed.; RSC Publishing: Cambridge,
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ate so that it undergoes hydrolysis to form α-glucopyranose
1
in a S skew boat are much less effective at promoting cleav-
3
(10)
Speciale, G.; Thompson, A. J.; Davies, G. J.; Williams, S. J.
age of the covalent enzyme-intermediates by formation of
allylic and bicyclobutonium ion-like TSs, which based on the
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Finally, we envision that these two families of covalent in-
hibitors will be useful research tools for biological studies.
(
Soc. 1997, 119, 11147-11154.
Our covalent inhibitors, unlike other inactivators such as
(14)
Chakladar, S.; Wang, Y.; Clark, T.; Cheng, L.; Ko, S.; Vo-
31-33
cyclophellitol and analogues
that irreversibly label glyco-
cadlo, D. J.; Bennet, A. J. Nat. Commun. 2014, 5, 5590.
side hydrolases, show a time dependent loss and return of
enzymatic activity. Moreover, we should be able to custom-
ize the rates of covalent-labeling (by changing the leaving
group) and reactivation (by choosing either the cyclohexenyl
or the bicyclo[4.1.0]heptyl skeleton). That is, our two classes
of reversible covalent inhibitors could be used to monitor
cellular responses to time-dependent changes in GH activity.
Also, if the rates for each process (pseudo-glycosylation and
deglycosylation) both depend on the pyranosylium ion-like
(15)
Adamson, C.; Pengelly, R.; Shamsi Kazem Abadi, S.; Chak-
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4
3
2,5 5,6,10
2509-2520.
TS ( H , H , B , or B)
and a conformation for orbital
3
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2,5
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logue for their particular GH.
4
(
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