From 1,4-Disaccharide to 1,3-Glycosyl Carbasugar: Synthesis of a Bespoke Inhibitor of Family GH99 Endo-α-mannosidase

Article abstract of DOI:10.1021/acs.orglett.8b03260

Understanding the enzyme reaction mechanism can lead to the design of enzyme inhibitors. A Claisen rearrangement was used to allow conversion of an α-1,4-disaccharide into an α-1,3-linked glycosyl carbasugar to target the endo-α-mannosidase from the GH99 glycosidase family, which, unusually, is believed to act through a 1,2-anhydrosugar "epoxide" intermediate. Using NMR and X-ray crystallography, it is shown that glucosyl carbasugar α-aziridines can act as reasonably potent endo-α-mannosidase inhibitors, likely by virtue of their shape mimicry and the interactions of the aziridine nitrogen with the conserved catalytic acid/base of the enzyme active site.

Full text of DOI:10.1021/acs.orglett.8b03260

Letter
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
From 1,4-Disaccharide to 1,3-Glycosyl Carbasugar: Synthesis of a
Bespoke Inhibitor of Family GH99 Endo-α-mannosidase
Dan Lu, Sha Zhu, Lukasz F. Sobala, Ganeko Bernardo-Seisdedos, Oscar Millet,
†
,¶
†,¶
‡
§
§
†
∥,⊥
‡
,†
Yongmin Zhang, Jesus Jimen
†
́
ez-Barbero,
Gideon J. Davies, and Matthieu Sollogoub*
Sorbonne Universite,
́
CNRS, Institut Parisien de Chimie Molec
́
ulaire, UMR 8232, 4 place Jussieu, 75005 Paris, France
‡
York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, U.K.
§
∥
Protein Stability and Inherited Diseases Laboratory and Molecular Recognition and Host−Pathogen Interactions, CIC bioGUNE,
Bizkaia Technology Park, Building 800, 48160 Derio, Spain
⊥
Ikerbasque, Basque Foundation for Science, Maria Diaz de Haro 3, 48013 Bilbao, Spain
S Supporting Information
*
ABSTRACT: Understanding the enzyme reaction mechanism can lead to the design of enzyme inhibitors. A Claisen
rearrangement was used to allow conversion of an α-1,4-disaccharide into an α-1,3-linked glycosyl carbasugar to target the
endo-α-mannosidase from the GH99 glycosidase family, which, unusually, is believed to act through a 1,2-anhydrosugar
“
epoxide” intermediate. Using NMR and X-ray crystallography, it is shown that glucosyl carbasugar α-aziridines can act as
reasonably potent endo-α-mannosidase inhibitors, likely by virtue of their shape mimicry and the interactions of the aziridine
nitrogen with the conserved catalytic acid/base of the enzyme active site.
6
nderstanding enzyme reaction mechanisms is essential for
the rational design of inhibitors. Glycoside hydrolases
GH), also called glycosidases, are a vast group of enzymes that
important class of enzymes are α-1,3-disaccharide analogues,
and these fall into two categories: those which mimic the charge
and those which mimic its shape. In the first class, we find the
usual deoxymannojirimycin (1), isofagomine (2), or noeur-
U
(
are classified, based upon their amino acid sequence similarity,
4
,7
into over 150 distinct families in the carbohydrate active
omycin derivatives. A clear influence of the amine position has
been demonstrated. It has also been shown that the glucal
derivative 3 is an inhibitor of the enzyme due to its shape, which
corresponds to a H conformation. Here, we decided to
combine both properties in a new inhibitor and create an amine
with a likely E/ H conformation. To achieve both the half-
chair conformation and introduce an amino group, we decided
to synthesize an aziridine on a carbasugar for obvious stability
reason. Furthermore, according to the mechanism, the catalytic
acid E336 of Bacteroides xylanisolvens GH99 (BxGH99) should
be in its acidic form and therefore prone to release a proton to
the aziridine; hence we decided to investigate the synthesis and
inhibitory ability of glucosyl carbasugar α-aziridine 4 (Scheme
).
There are many ways to synthesize carbasugars, including
those containing a disaccharide motif. The most direct way
1
,2
enzymes database (CAZy). The vast majority of these
enzymes can be divided into two classes according to their
reaction mechanism and the relative configuration of substrate
and product: those that act with retention or inversion of
anomeric configuration. The retaining glycosidases utilize a
double nucleophilic displacement which results in net retention
of configuration at the anomeric center. The retaining reaction
classically involves nucleophilic amino acid side chains and
proceeds via a covalent glycosyl−enzyme intermediate. Some
4
7
5
4
4
5
enzymes also use neighboring group participation reactions
3
(
notably those active on β-linked GlcNAc). Recently, a new
neighboring-group-retaining mechanism involving an inter-
mediate epoxide and hence no covalent bond to the enzyme
was proposed for endo-α-mannosidases and endomannases (α-
Glc-1,3-α-Man-OR and α-Man-1,3-α-Man-OR substrates) of
1
8
9
4
,5
the GH99 family. In this putative mechanism, nucleophilic
attack by the manno-O2 atom allows catalysis via a 1,2-anhydro
sugar “epoxide” intermediate (Scheme 1). In terms of shape and
10
would be to start directly from the corresponding disaccharide,
but the necessary 1,3-disaccharides (like Glc-α-1,3-Glc) are rare
and expensive. The synthesis of the two monosaccharides and
charge changes, a positive charge is very likely to be developed,
4
and the conformation of the pyranose changes from C of the
1
4
4
substrate to the E/ H of the epoxide. Based on these
Received: October 12, 2018
5
considerations, inhibitors that have been designed for this
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b03260
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
a
Scheme 1. Postulated Enzymatic Hydrolytic Mechanism for Bacteroides xylanisolvens GH99 (BxGH99)
a
Catalysis occurs through a 1,2-anhydrosugar intermediate (top) and known and designed (this work) substrate analogues (below); residue
numbering as in BxGH99.
their subsequent assembly seemed tedious. We therefore
decided to use a trick based on the use of Claisen rearrangement
to make carbasugars nicely developed by Nagarajan on
monosaccharides. In this reaction, a glucal functionalized
with an alkene in position 6 affords a carbaglucal with an
aldehyde in position 6. This is due to the symmetry of the
molecule, and in the operation, the protecting group in position
maltal 5, which in turn could undergo Claisen rearrangement to
give keto-alkene 6. This operation is key to the total synthesis as
we obtain not only the desired 1,3-pseudodisaccharide but also
the alkene which will be converted into the desired epoxide 7
and aziridine 4 (Scheme 3).
Maltal was prepared according to Haworth, with a slight
modification in the deprotection step (see the Supporting
Information). Next, the differentiation of the two primary
hydroxyl groups was required. For this, we decided to protect
the glucoside with a benzylidene group. This reaction had to be
performed with great care (Scheme 4) by controlling the
amount of camphor sulfonic acid used (0.1 equiv). Then, the
other primary alcohol could be selectively silylated, and
subsequent benzylation and desilylation afforded the alcohol
1
1
12
4
becomes the one in position 3 in the new numbering of the
carbasugar (Scheme 2).
Scheme 2. Claisen Rearrangement Leading to Interchange of
Positions 3 and 4 from the Glucal to the Carbaglucal
8
. Oxidation using Dess-Martin periodinane followed by
olefination produced dialkene 9. Claisen rearrangement on 9
was performed under microwave irradiation at 240 °C and was
immediately followed by aldehyde reduction to afford alcohol
1
0 in 76% yield (Scheme 4).
With alkene 10 in hand, we investigated its aziridination. We
opted for a three-step route: epoxidation, azide ring opening,
13
This approach was used to synthesize a 1,3-pseudodisacchar-
ide from a 1,4-disaccharide. The synthesis would start from
maltose, which would be converted into the doubly unsaturated
and Staudinger azide reduction aziridination; this strategy also
operates with overall inversion of configuration of the three-
membered ring. We first benzylated the remaining hydroxyl in
Scheme 3. Retrosynthetic Analysis for the Synthesis of Aziridine 4
B
DOI: 10.1021/acs.orglett.8b03260
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Scheme 4. Key Claisen Rearrangement and Preparation of Substrate 10
Scheme 5. Synthesis of Aziridine Carbasugars 4
1
6
1
0 to give 11, which was epoxidized using methyl-
Final deprotection, using Birch conditions, afforded the
unprotected aziridinated pseudosugar 4 (Scheme 5).
14
(
trifluoromethyl)dioxirane generated in situ to give the α-
epoxide 12 in 46% yield and the β-epoxide 13 in 26% yield,
which were separated by silica gel flash chromatography. The
stereochemistry of these epoxides was determined by a NOESY
experiment. α-Epoxide 12 displayed cross-correlations between
H-5 and H-3, whereas in β-epoxide 13, H-5 showed cross-
correlations with H-3 but also with H-1 and H-2 (Scheme 5 and
Supporting Information). This reaction therefore produced the
undesired α-epoxide as the major product, but all our attempts
to improve the situation with various epoxidation reactions gave
lower yields. For instance, mCPBA, which was previously often
The properties of aziridine 4 were investigated with GH99
family members Bt (Bacteorides thetaiotaomicron) and BxGH99.
A crystal structure of the BxGH99 enzyme in complex with the
aziridine 4 at a resolution of 1.27 Å (PDB accession: 6HMG)
was obtained by soaking of BxGH99 crystals with an aqueous
solution of the compound. The nitrogen atom of the aziridine
makes a 2.6 Å hydrogen bond with one of the side chain oxygen
atoms of BxGH99 residue E336, which acts as an acid/base in
the proposed GH99 mechanism (Scheme 1). In a ternary
complex with the aziridine and α-1,2-mannobiose (obtained at a
resolution of 1.03 Å, PDB 6HMH), this distance changes to 2.7
15
used to synthesize epoxides from cyclohexenes polyols, did not
improve the selectivity or the yield. Further treatment of the
isolated epoxide 13 with sodium azide furnished a mixture of
Å. The interaction is shown in Figure 1A. In both cases, the
4
pseudopyranose ring is in a H conformation, as predicted for
5
1
,2-trans azido alcohols 14 and 15, which under the action of
the azirdine and likely resembles the intermediate/transition
state of the reaction. Indeed, it binds similarly to ManGlucal and
PPh afforded α-aziridine 16 in 52% yield after these two steps.
3
C
DOI: 10.1021/acs.orglett.8b03260
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
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Figure 1. (A) Divergent stereoview (“wall-eyed”) of 4 in complex with BxGH99. Catalytic residues surrounding the −1 subsite, and the interaction
−
3
4
between the aziridine N and E336 Oε is shown. The 2mF − DF synthesis contoured at 1 e /Å . (B) Comparison of the ligands observed in the H
0
c
5
7
conformation in complex with BxGH99. Green, aziridine 4; gold, ManGlucal (PDB 5M5D); gray, GlcChex (PDB 5MEL). The sugar moiety in the
2 subsite is not shown. (C) Result of NMR titration of BtGH99 with 4. The protein concentration in the solution was 58 μM.
−
1
7
4
GlcChex (Figure 1B), which was also observed in a H
ORCID
5
conformation in complex with BxGH99 (PDB 5M5D and
Jesus Jimenez-Barbero: 0000-0001-5421-8513
́
7
5
MC8).
Gideon J. Davies: 0000-0002-7343-776X
Matthieu Sollogoub: 0000-0003-0500-5946
Using NMR, we have determined the dissociation constant
D
15
(K ) of the aziridine with N-labeled BtGH99 to be 11.5 ± 0.9
Author Contributions
¶D.L. and S.Z. contributed equally.
μM (Figure 1C). The compound, therefore, binds to BtGH99
with an affinity 2-fold higher than that of GlcDMJ (24 μM) and
slightly higher than that of ManGlucal (15 μM). However, it is
not a potent inhibitor in comparison with GlcIFG, ManIFG, or
Notes
The authors declare no competing financial interest.
ManNOE, all of which bind to BtGH99 with a K in the
D
7
nanomolar range.
ACKNOWLEDGMENTS
In conclusion, we have devised a strategy to synthesize 1,3-
glycosyl carbasugars designed to interact with the family GH99
endo-α-mannosidases. The obtained inhibitor has improved
activity in comparison with the shape mimics but does not reach
the affinity of isofagomine or noeuromycin analogues. This
probably indicates that the aziridine is not charged owing to its
■
We thank Chinese Scholarship Council (CSC) and Diamond
Light Source for access to beamlines I02 and I04 (proposal
mx9948) that contributed to the results presented here.
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E
DOI: 10.1021/acs.orglett.8b03260
Org. Lett. XXXX, XXX, XXX−XXX