α-l-Arabinofuranosylated pyrrolidines as arabinanase inhibitors

Article abstract of DOI:10.1039/c1cc13675e

As part of continued efforts to understand the mechanisms of 1,5-α-l-arabinanases better, some arabinan-like iminosugar oligosaccharides were synthesized. An iminosugar analogue of arabinobiose was found to be a good inhibitor of the arabinanase Arb93A fr

Full text of DOI:10.1039/c1cc13675e

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COMMUNICATION
a-L-Arabinofuranosylated pyrrolidines as arabinanase inhibitorswz
Ethan D. Goddard-Borger,ya Raphael Carapito,zb Jean-Marc Jeltsch,b Vincent Phalip,b
Robert V. Sticka and Annabelle Varrot*c
Received 20th June 2011, Accepted 5th July 2011
DOI: 10.1039/c1cc13675e
As part of continued efforts to understand the mechanisms
of 1,5-a-L-arabinanases better, some arabinan-like iminosugar
oligosaccharides were synthesized. An iminosugar analogue of
arabinobiose was found to be a good inhibitor of the arabinanase
Arb93A from Fusarium graminearum. Structures were determined
for complexes of this inhibitor with wild-type Arb93A and a
catalytically inactive mutant.
inverting hydrolases.7 GH93 arabinanases are thought to be
retaining hydrolases—an assignment that was initially made
on the basis of transglycosylation experiments because rapid
mutarotation of the a-arabinobiose product precluded assignment
1
by H NMR experiments.4,8 Retention results from a double
displacement mechanism where a covalent glycosyl-enzyme
intermediate is formed and subsequently hydrolyzed via
oxacarbenium-ion-like transition states. Two catalytic residues
are usually involved, with one acting as an acid/base residue and
the other as a nucleophile. Structures have been determined for
the GH93 arabinanases Arb93A from Fusarium graminearum
and Abnx from Penicillium chrysogenum 31B in complex with
arabinobiose. They reveal two carboxylate residues that are
ideally positioned to function as a catalytic acid/base and a
nucleophile (Glu170 and Glu242 in Arb93A, respectively),
thereby supporting the assignment of these enzymes as retaining
hydrolases.9 On the basis of this information, subsequent
mutagenesis studies confirmed the essential role that these
residues play in catalysis.4,10
Plant arabinan, a component of pectin, is a polysaccharide
possessing a backbone of repeating (1-5)-a-L-arabinofuranosyl
units, known as linear arabinan, adorned with branching
a-(1-2)- and a-(1-3)-linked L-arabinofuranosides. The
saccharification of arabinan requires the co-operative action
of many a-L-arabinofuranosidases, hailing from glycoside
hydrolase (GH) families 3, 43, 51, 54, 62 and 93,1,2 as defined
by the Carbohydrate Active enZyme (CAZy) database (available
at http://www.cazy.org/).3 Glycoside hydrolases from families
43 and 93 are arabinanases that hydrolyze linear arabinan into
smaller oligosaccharides. GH43 enzymes present an endo
mode of action,2 whilst GH93 enzymes are exo-hydrolases
that liberate arabinobiose from the non-reducing end of linear
arabinan.4,5 These enzymes, like the many others recruited by
various organisms to degrade plant cell-wall polysaccharides,
are of great interest to those seeking to create renewable
chemical commodities from plant biomass.
Further studies of the mechanisms employed by arabinanases,
particularly those from GH family 93, with regard to conforma-
tional itinerary and transition state stabilisation, would be
aided by appropriate small molecule inhibitors, just as other
inhibitors have helped to delineate the details of other GH
mechanisms. Iminosugars have proven to be invaluable tools
for the study of the glycoside hydrolase mechanisms, irrespective
of debate as to whether or not they are true transition state
mimics.11–14 Simple iminosugars are relatively poor inhibitors
of glycanases, as they occupy very little of these enzymes’
large, catalytically active, substrate-binding cleft.15 However,
glycosylated iminosugars occupy much more of the substrate-
binding site and have a greater affinity for the enzyme of
interest.15–17 To make such tools available for continuing
mechanistic studies of arabinanases, compounds 1 and 2, the
deoxyiminosugar equivalents of arabinobiose and arabinotriose,
respectively, were synthesised and evaluated as arabinanase
inhibitors (Fig. 1).
Enzyme-mediated hydrolysis of glycosidic bonds proceeds
with either a net inversion or retention of the substrate’s anomeric
configuration.6 GH43 arabinanases are well established as
a School of Biomedical, Biomolecular and Chemical Sciences,
The University of Western Australia, 35 Stirling Highway, Crawley,
WA 6009, Australia. E-mail: rvs@cyllene.uwa.edu.au
b Institut de recherche de l’Ecole de biotechnologie de Strasbourg,
´
Boulevard Sebastien Brandt-BP 10413, 67412 ILLKIRCH Cedex,
France. E-mail: phalip@unistra.fr; Fax: +33 368855330;
Tel: +33 368854820
c
´
CERMAV-CNRS, affiliated with Universite Joseph Fourier and
ICMG, BP 53, 38041 Grenoble cedex 9, France.
E-mail: varrot@cermav.cnrs.fr; Fax: +33 476547203;
Tel: +33 476037634
w Figures were drawn with the PyMOL Molecular Graphics System,
Schrodinger, LLC.
¨
z Electronic supplementary information (ESI) available: Experimental
data and a table of data collection and refinement statistics are
provided. See DOI: 10.1039/c1cc13675e
y Current address: Department of Chemistry, 2036 Main Mall,
University of British Columbia, Vancouver, Canada V6T 1Z1.
´
z Current address: Physiopathologie et Medecine Translationnelle.4
Rue Kirschleger, 67085 Strasbourg cedex, France.
Fig. 1 Arabinan-like oligosaccharides and their corresponding
iminosugar analogues.
c
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carbamoyl protecting group with neat trifluoroacetic acid
provided the desired compound as its trifluoroacetic acid salt
2ꢀCF3CO2H. The rapid assembly of compounds 1 and 2 is a
fine demonstration of the utility of this selective glycosylation
strategy for the assembly of linear arabinan-like oligosaccharides.
The pseudo-disaccharide 1ꢀCF3CO2H was evaluated as
an inhibitor of the GH93 exo-1,5-arabinanase Arb93A; it
inhibited this enzyme competitively with a Ki of 3.0 ꢁ 0.3 mM.
A comparison of this dissociation constant to KM values
previously determined for the natural substrates arabinotriose
(3.6 mM), arabinotetraose (9.3 mM), arabinopentaose (4.0 mM),
arabinohexaose (2.0 mM) and linear arabinan (0.75 mM)
indicates that the inhibitor binds some 200–2500 times better
than known substrates, making it a relatively good inhibitor.4
To gain some insight into the nature of the interactions
between Arb93A and 1, the 3-D structures of pseudo-
disaccharide 1 in complex with the wild-type enzyme and its
acid/base mutant E242A were determined at 1.60 A and 1.85 A
resolution, respectively. The small variations observed among
the three molecules of the mutant structure or between the
mutant and wild-type structures result from differences in
crystal contacts. Examination of the first electron density
difference map revealed without ambiguity that compound 1
occupied the ꢂ1 and ꢂ2 subsites of the enzyme (Fig. 2). As is
typical of GHs, complexation of Arb93A with a ligand did not
result in any major local or global conformational changes.
Very small readjustments of several amino acid side-chains
were observed between the complex of arabinobiose and
compound 1, presumably to optimise the hydrogen bonding
network of each complex.
Scheme 1 Synthesis of the iminosugar oligosaccharides 1 and 2.
The syntheses of compounds 1 and 2 were realized
by extending a selective glycosylation strategy reported by
Du et al.,18 where unprotected arabinoside acceptors were
glycosylated preferentially at the C5 primary hydroxyl group.
Iminosugar 3 was prepared from L-arabinose in seven steps, as
previously described,19 then converted into the corresponding
tert-butyl carbamate 4 (Scheme 1). The donor required for the
assembly of pseudo-disaccharide 1, the trichloroacetimidate 5,
was prepared according to literature procedures.18,20 This
imidate was used to synthesise the disaccharide donor 6,
necessary for the assembly of pseudo-trisaccharide 2, using
conventional approaches.
The L-arabinosyl moiety of 1 is found within the ꢂ2 subsite
and makes similar interactions to those previously described
for the Arb93A–arabinobiose complex.4 Within this subsite,
C5 is surrounded by several residues, including Tyr62, Tyr100
and Glu60, which permits recognition of the non-reducing end
of arabinan and appears to be a determinant of the enzyme’s
exo-hydrolase activity. The iminosugar moiety of 1 occupies
the ꢂ1 subsite, as expected, and exists in a 4TN conformation.
The side-chain carboxylate of the catalytic nucleophile
Glycosylation of carbamate 4 using the monosaccharide
donor 5 proceeded well, providing the pseudo-disaccharide 7
in good yield (Scheme 1). Structural assignment of 7, a
tert-butyl carbamate, was complicated by severe broadening
1
of both the H and 13C NMR spectra; acetylation of the free
hydroxyl groups and removal of the carbamoyl group were
necessary to confirm the stereo- and regio-chemical outcome
of the glycosylation reaction (see ESIz).21,22 Similarly, glyco-
sylation of carbamate 4 with the disaccharide donor 6 provided
the pseudo-trisaccharide 8, the structure of which was confirmed
by chemical modification (see ESIz).
Deprotection of the pseudo-disaccharide 7 began with a
Zemplen transesterification, which presumably gave the corres-
´
ponding pentol. This product was treated with dilute hydro-
chloric acid and lyophilized, with the intention that this would
remove the carbamoyl protecting group. Unfortunately, this
protocol produced a mixture of products, which, by 13C NMR
spectroscopy, appeared to contain 1ꢀHCl, L-arabinose and
pyrrolidinium chloride 3ꢀHCl. More dilute hydrochloric acid
and/or lower temperatures failed to prevent the partial hydrolysis
of the pseudo-disaccharide’s glycosidic bond. To avoid this
problem, anhydrous trifluoroacetic acid was used to remove
the carbamoyl group; the pseudo-disaccharide 1 was obtained
as the trifluoroacetic acid salt 1ꢀCF3CO2H in high yield. The
pseudo-trisaccharide 8 was deprotected in the same manner as
the pseudo-disaccharide 7; debenzolyation and removal of the
Fig. 2 Electron density for compound 1 bound to wild-type Arb93A.
2Fobs–Fcalc electron density at 0.95 eAꢂ3. Interactions are represented
by dashed lines.
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(Glu170) bridges the pyrrolidine ring, making interactions
with both the pyrrolidine nitrogen (N4–OE2, 2.80 A) and
the hydroxyl group at C2 (O2–OE1, 2.64 A). The pyrrolidine
nitrogen also interacts indirectly with the hydroxyl group of
Tyr337 by way of a water molecule. The catalytic acid/base
residue Glu242 does not interact directly with the inhibitor
and its mutation to alanine does not appear to perturb the
binding of compound 1 to Arb93A, since the inhibitor’s
position, orientation and conformation are equivalent to those
in the wild-type structure. It is interesting to note that in the
mutant complex two water molecules are found in proximity
to the positions previously occupied by OE1 and OE2 of
the carboxylate side-chain of Glu242. The pyrrolidine likely
exists in a protonated state on enzyme, in line with previous
structural and kinetic studies of GH iminosugar inhibitors,
but since the pKa of 1,4-dideoxy-1,4-imino-L-arabinitol is 7.6,
and crystallisation occurred at pH 7.5, this cannot be stated
with certainty.17,23,24
close to E3, since it should have significant oxacarbenium ion
character, which requires C4–O4–C1–C2 to be close to coplanar.
The iminosugar oligosaccharides presented here represent
the first small molecule arabinanase inhibitors and are an
important step towards the development of further tools to
delineate the mechanisms by which these enzymes effect
catalysis. Other competitive inhibitors comprising at least
two L-arabinose units and possessing an amine or sp2 hybri-
disation at the anomeric position, as well as mechanism-based
inhibitors that lead to an accumulation of the glycosyl-enzyme
intermediate, will be important tools in future mechanistic
studies of these enzymes.
Notes and references
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The distortion that a pyranoside ring undergoes during
electrophilic migration, which proceeds in accordance with
Stoddart’s pseudo-rotational itinerary, has been demonstrated
for a number of GHs.24,25 Some structural studies of enzyme–
iminosugar complexes have shown that these inhibitors can adopt
ring conformations similar to those expected at the transition
state.24 Nevertheless, owing to the lack of good mechanistic
probes, little is known about the reaction coordinates utilised
by O-furanosidases, which can be described by the furanose
pseudorotational wheel of Altona and Sundaralingam.26 Thus,
it is worth considering the relevance of the present enzyme–
inhibitor complex to the conformational itinerary of Arb93A.
The 4TN twist conformation of the pyrrolidine (Fig. 3)
within the ꢂ1 subsite is a low energy conformer on the
pseudorotational energy map calculated for a-L-arabinofurano-
sides that present a gt orientation of the exo-cyclic hydroxy-
methyl group.27 This is similar to the conformation that might
be expected for the Michaelis complex, as seen for several
retaining GH51 a-L-arabinofuranosidases, such as AbfA from
Geobacillus stearothermophilus T-6.28 This conformation
places the glycosidic bond in an axial orientation and therefore
allows in-line nucleophilic attack at the anomeric centre. In
structures of the product complex of both Arb93A and Abnx
with arabinobiose the ring conformation of the L-arabinose
residue in the ꢂ1 subsite is close to an E3 conformation with
the anomeric carbon slightly above the plane.4,10 The transition
state would then be expected to have a southern conformation
14 S. G. Withers, M. Namchuk and R. Mosi, in Iminosugars as
glycosidase inhibitors: nojirimycin and beyond, ed. A. E. Stutz,
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Fig. 3 Overlay of the active site of Arb93A in wall-eye stereo. The
structures shown are wild-type Arb93A in complex with arabinobiose
(magenta) or compound 1 (green).
28 K. Hovel, D. Shallom, K. Niefind, V. Belakhov, G. Shoham,
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9686 Chem. Commun., 2011, 47, 9684–9686
This journal is The Royal Society of Chemistry 2011