Conformational Behaviour of Azasugars Based on Mannuronic Acid

Article abstract of DOI:10.1002/cbic.201700080

A set of mannuronic-acid-based iminosugars, consisting of the C-5-carboxylic acid, methyl ester and amide analogues of 1deoxymannorjirimicin (DMJ), was synthesised and their pH-dependent conformational behaviour was studied. Under acidic conditions the methyl ester and the carboxylic acid adopted an “inverted” ¹C₄ chair conformation as opposed to the “normal” ⁴C₁ chair at basic pH. This conformational change is explained in terms of the stereoelectronic effects of the ring substituents and it parallels the behaviour of the mannuronic acid ester oxocarbenium ion. Because of this solution-phase behaviour, the mannuronic acid ester azasugar was examined as an inhibitor for a Caulobacter GH47 mannosidase that hydrolyses its substrates by way of a reaction itinerary that proceeds through a ³H₄ transition state. No binding was observed for the mannuronic acid ester azasugar, but sub-atomic resolution data were obtained for the DMJ?CkGH47 complex, showing two conformations—³S₁ and ¹C₄—for the DMJ inhibitor.

Full text of DOI:10.1002/cbic.201700080

Accepted Article
Title: Conformational behaviour of mannuronic acid based azasugars
Authors: Erwin van Rijssel, Antonius Janssen, Alexandra Males,
Gideon Davies, Gijsbert van der Marel, Hermen S. Overkleeft,
and Jeroen Codée
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10.1002/cbic.201700080
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Conformational behaviour of mannuronic acid based azasugars
Erwin R. van Rijssel,[a] Antonius P. A. Janssen,[a] Alexandra Males,[b] Gideon J. Davies,[b] Gijsbert A.
van der Marel,[a] Herman S. Overkleeft,*[a] and Jeroen D. C. Codée*[a]
Dedicated to the memory of Professor Dr Werner Reutter
Abstract: A set of mannuronic acid based iminosugars, comprising
the C-5-carboxylic acid, -methyl ester and -amide analogues of 1-
deoxymannorjirimicin (DMJ), was synthesized and their pH
dependent conformational behavior studied. Under acidic conditions
the methyl ester and the carboxylic acid took up an “inverted” 1C4
chair conformation as opposed to the “normal” 4C1 chair at basic pH.
This conformational change is explained by the stereoelectronic
effects of the ring substituents and it parallels the behavior of the
mannuronic acid ester oxocarbenium ion. Because of this solution
phase behavior, the mannuronic acid ester azasugar was probed as
an inhibitor for a Caulobacter GH47 mannosidase that hydrolyzes its
equatorially positioned substituents.[4] These effects can be
explained by the more favorable interaction of the axially
positioned electronegative oxygen substituents with the positive
charge present on the azasugar ring in a protonated state and
the (partial) positive charge of oxocarbenium ion (-like)
intermediates in glycosylation reactions.[4-7]
.
In mannuronic acids, mannosides of which the C6-OH is
oxidized to a carboxylic acid functionality, the carboxylic acid
has a profound effect on the conformation and reactivity of the
pyranoside.[8] In the context of the construction of bacterial
oligosaccharides we have studied the glycosylation behaviour of
a variety of mannuronic acid donors in detail and we have found
these to be unexpectedly reactive.[9] In addition, glycosylations
involving these donors proceed with an extraordinary selectivity
to provide 1,2-cis glycosidic linkages. These findings have been
rationalized through the conformational preferences of (partially)
positively charged mannuronic acid oxocarbenium ion (-like)
intermediates that are governed by the ring substituent effects.
These species prefer to adopt a “flipped” ring structure and in
the 3H4 (-like) oxocarbenium ion all substituents take up the most
stabilizing (or least destabilizing) orientation: the C2-OR pseudo-
equatorial and the C3-, C4-OR and C5-COOR groups pseudo-
axial. Indeed DFT calculations indicate that the 3H4
oxocarbenium ion is significantly more stable than the alternative
(“non ring-flipped”) 4H3 ion (See Scheme 1A).[10]
3
substrates following a reaction itinerary that proceeds through a H4
transition state. No binding was observed for the mannuronic acid
ester azasugar, but sub-atomic resolution data were obtained for the
DMJ-CkGH47 complex, showing two conformations, 3S1 and 1C4, for
the DMJ inhibitor.
Introduction
Stereoelectronic substituent effects have a profound effect on
the three-dimensional structure of molecules. Where
substituents on a cyclic compound generally have a preference
for an (pseudo)-equatorial position for steric reasons, the
electronic spatial preferences depend on different forces such as
interactions.[1]
The
A)
RO
RO
charge-charge
and
dipole-dipole
RO
RO
CO2R
conformation and reactivity of carbohydrates is determined to a
large extent by the nature and orientation of the substituents.
This influence becomes apparent in glycosylation reactions,
where the amount, nature and orientation of the hydroxyl groups,
protected with electron withdrawing esters or more electron
neutral ether groups, determine the overall reactivity.[2] It has
long been known that in glycosylations, axial substituents are
less deactivating or “disarming” than their equatorially positioned
equivalents.[3] Similarly, the basicity of iminosugars (or
“azasugars”), carbohydrates of which the endocyclic oxygen is
replaced by an amine, is influenced by the orientation of the ring
substituents and azasugars bearing more axially positioned
hydroxyl groups are more basic than their stereoisomers bearing
OR
CO2R
O
O
OR
B)
HO
HO
O
OH
O
H
OH
N
NH
OH
N
OH
HO
HO
DMJ, 2
kifunensine, 1
C)
O
R
O
OH
OH
NH
R
H
HO
HO
HO
H
N
HO
H
[a]
Dr E.R van Rijssel, A.P.A. Janssen, Prof. dr H.S. Overkleeft, Prof. dr
G.A. van der Marel, Dr J.D.C. Codée
Leiden Institute of Chemistry
O
O
O
OH
NH
OH
NH
OH
NH
MeO
HO
HO
HO
HO
HO
H2N
HO
HO
Leiden University
Einsteinweg 55, 2333 CC Leiden, The Netherlands
E-mail: jcodee@chem.leidenuniv.nl
A. Males, Prof. dr G.J. Davies
3
4
5
[b]
York Structural Biology Laboratory, Department of Chemistry
The University of York
YO10 5DD, United Kingdom
Figure 1. A) Conformational equilibrium for the mannuronic acid
oxocarbenium ion. B) Structures of the mannosidase inhibitors kifunensine (1)
and 1-deoxymannojirimycin (DMJ, 2) in their 1C4 conformation. C) Mannuronic
acid azasugars studied here.
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Carbohydrate processing enzymes, such as glycoside
hydrolases, may induce a chemical transformation by forcing the
carbohydrate substrate into an unusual conformation.[11] a-
Mannosidases that belong to the CAZY family GH47, are
inverting glycoside hydrolases that cleave a-1,2-mannosidic
linkages. The mammalian GH47 mannosidases can be found in
the Golgi and endoplasmatic reticulum (ER) where they cleave
mannose residues from N-glycans, thereby playing an important
role in protein biosynthesis and quality control. The mechanism
by which these hydrolases cleave the 1,2-mannosidic bonds is
notable as they employ an unusual catalytic itinerary. The
intermediate azide and subsequent crystallization from ethanol
gave 2-amino-6-bromo-lactone (8) as its hydrochloric acid salt in
55%. Treatment of this salt with triethylamine in methanol led to
ring opening and intramolecular bromide displacement by the C2
amine to give crude azasugar methyl ester 3. Purification of this
compound from the triethylammonium and sodium salts formed
in the reaction proved difficult, because of the high polarity of the
compound as well as the lability of the methyl ester towards
hydrolysis. Attempts to crystallize the compound were to no avail.
Therefore, all hydroxyl groups in
3 were capped with
trimethylsilyl groups[16] to allow for the purification of the
compound by chromatography. After desilylation, the pure
methyl ester 3 was obtained as its hydrochloric acid salt. DMJ
(2) was synthesized from 3 by a sodium borohydride mediated
reduction and was obtained in 29% yield after column
chromatography. D-Mannuronic acid azasugar 4 and amide 5
3
substrate that is to be cleaved binds in a 3,OB / S1 conformation
and is hydrolyzed in a reaction that proceeds through a
transition state in which the mannose ring adopts a 3H4
conformation.[12] Kifunesine (1, Scheme 1B), a potent inhibitor of
the mannosidase I enzyme, has been shown to adopt a ring
flipped 1C4 conformation and a similar conformation was found
for 1-deoxymannojirimycin (DMJ, 2, Scheme 1B) bound in the
active site of the yeast homologue Saccharomyces cerevisiae.[12]
We were inspired by the conformation of the inhibitors of the
GH47 enzymes to explore the behaviour of mannuronic acid
based azasugars. We here report on the synthesis of
mannuronic acid based azasugars 3, 4, and 5 (Scheme 1C) and
a study on their conformational behaviour. We show that the
stereoelectronic effects that determine the structure of the
mannuronic acid oxocarbenium ions also impact the three-
dimensional structure of these azasugars and that protonation of
the ring nitrogen can induce a ring flip leading to an axial rich 1C4
conformation in solution. We build on this to show how
were obtained from
3 through saponification with sodium
hydroxide or aminolysis with methanolic ammonia, respectively.
Br
OH OH
O
O
a
HO
Ca2+
HO
O
O
2
HO
OH OH
Br
6
7
b
Br
d
e
f
O
2
4
5
OH
NH
O
MeO
HO
HO
HO
c
deoxymannojirimycin, the "parent" compound, binds to
a
O
bacterial GH47 enzyme from Caulobacter sp. K31[12] but also
that, unfortunately despite improved solution behaviour, the
mannuronic acid derivatives do not bind to the GH47 enzyme,
likely by virtue of their altered C5-substitutent.
HO
Cl
NH3
3
8
Scheme 2. Synthesis of DMJ and its C5 analogs for this study. Reaction
conditions: a) (i) HBr, AcOH; (ii) MeOH, 26% over 2 steps; b) (i) NaN3,
acetone; (ii) H2, Pd/C, HCl (aq.), MeOH, 55% over 2 steps; c) (i) Et3N, MeOH,
quant.; (ii) HMDS, CuSO4.H2O (cat.), CAN; (iii) MeOH, AcCl (cat), 70% over
three steps. d) NaBH4, EtOH, 29%; e) NaOH, H2O, quant.; f) NH3, MeOH,
quant..
Results and Discussion
The synthesis of DMJ 2 and its C5 analogs was achieved
according to the route devised by Wrodnigg and co-workers.[13]
As depicted in Scheme 2, methyl mannuronic acid ester
azasugar 3 was obtained in four steps from the commercially
available calcium D-gluconate monohydrate (6).[14] The
gluconate 6 was treated with HBr in acetic acid to form 3,5-di-O-
With the set of azasugars in hand we established their pKa
values by titration and investigated their conformational
behaviour at different pH* (the pH measured in D2O) values by
NMR spectroscopy. Table 1 summarizes the results of these
studies. For DMJ a pKa value of 7.4 was measured, which is in
line with the pKa previously established for this compound
(7.5).[4c] The pKa values of methyl ester 3, amino acid 4 and
amide 5 were determined to be 5.3, 7.5 and 5.8, respectively.
The drop in pKa value for the ester and the amide is a clear
manifestation of the electron withdrawing effect of the carboxylic
acid ester and amide functionalities. At higher pH*, where acid 4
is deprotonated, the electron withdrawing effect of the
carboxylate is lowered because of its negative charge.
acetyl-2,6-dideoxy-2,6-dibromo-D-manno-1,4-lactone after
a
series of acid catalyzed transformations (i.e. substitution of the
C2 and C6 hydroxyl groups, intramolecular ring closure and
acetylation of the remaining hydroxyl groups). Next the acetyl
groups at O3 and O5 were removed in an acid catalyzed
transesterification with methanol to provide the pure
dibromolactone 7 after crystallization from chloroform/water in
26% yield over the two steps. Regioselective displacement of
the C2-bromide with an azide occurred with retention of
configuration, explained by Bock et al.[15] with epimerization of
the C2-bromide to the more reactive glucose configured
dibromide and subsequent regioselective substitution by the
azide. Thereafter palladium catalyzed reduction of the
Table 1. pKa values for compounds 2-5 and observed and calculated coupling
constants and determined 4C1 : 1C4 conformer ratio
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Mannuronic acid 4 can occur in three different charged states:
J3-4 (calc.)
4C1
NMR Ratio
Compound
pH*
J3-4 (obs.)
pKa
4C1:1C4
the fully protonated state, the neutral zwitterionic state and the
1
1C4
negatively charged state. In Figure 4, the H NMR spectra of 4
are shown from pH* 1 to pH* 12. Again large chemical shift
changes are observed upon changing pH* (especially for H5
shifting from d = 3.9 to d = 2.9 ppm). Also a small change in
coupling constants is apparent. The J3,4 changes from 9.8 Hz at
high pH* to 8.8 Hz at neutral pH* to 8.3 Hz at acidic pH*. Thus,
in line with the conformational behaviour of methyl ester 3,
mannonic acid 4 can change its conformation in a pH-dependent
manner.
9.0
3.3
2
2
3
3
4
4
4
5
5
3
3
9
9.5
9.5
9.4
7.5
9.8
8.8
8.3
9.7
9.3
9.2
4.8
100:0
100:0
99:1
7.4
9.4
4.3
2
9.4
3.9
8
5.3
7.5
Figure 5 displays the collection of 1H NMR spectra for amide 5 at
different pH* values. Smaller changes are observed for the
chemical shift change of H5 and there is no significant change of
the coupling constants, indicating minimal conformation changes
going from high to low pH* for this azasugar.
9.5
4.9
2
56:44
100:0
94:6
9.3
3.1
11
9.1
3.8
5
9.5
4.7
1
75:25
100:0
100:0
99:1
9.0
2.8
9
5.8
9.1
4.7
2
9.3
4.2
MeOD
Figure 2. 400 MHz 1H NMR spectra for DMJ (2) at different pH*; spectra are
referenced to residual methanol.
MeOD
(+TFA)
9.3
4.7
1:99
Figures 2-5 show the 1H NMR spectra of azasugars 2-5
recorded at varying pH*. In Figure 2, the 1H NMR spectra of
DMJ (2) in D2O at pH* 1-12 are collected. From pH* 1 to pH* 6.5
no changes are observed in either chemical shifts or coupling
constants. The coupling constants are indicative of a “normal”
4C1 chair conformation for the azasugar ring. Going from pH* 6.5
to pH* 12 a significant shift in chemical shift is observed for all
ring protons, with the direct neighbours of the amino group
experiencing the largest shift. No changes occur in the coupling
constants of the ring protons, indicating that no major
conformation change takes place.
Figure 3. 1H NMR spectra for 2,6-dideoxy-2,6-imino-mannuronic acid methyl
ester (3) at different pH*; spectra are referenced to residual methanol.
1
In Figure 3, the H NMR spectra of methyl ester 3 at different
pH* values are displayed. Because hydrolysis of the methyl
ester was observed above pH* 8, no spectra were recorded
above this pH*. Large chemical shift changes are seen with
increasing pH*. Especially H5 undergoes a large chemical shift
change and shifts from d 4.04 at pH* 2 to 3.22 at pH* 8. Also a
change in coupling constants is observed for the ring protons.
For example, the J3,4 changes from 9.4 Hz at basic pH* to 7.5 Hz
at acidic pH*, indicative of a change in conformation of the
azasugar ring. At high pH* the azasugar adopts a single
conformation, while both the 1C4 and 4C1 conformers are present
at low pH* (vide infra).
Figure 4. 1H NMR spectra for 2,6-dideoxy-2,6-imino-mannonic acid (4) under
different pH*; spectra are referenced to water.
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difference between the ester and amide is notable, because
both functional groups, the C5 carboxylic acid ester and C5
carboxamide respectively, have a similar effect on the basicity of
the azasugars. The electron withdrawing effect of both groups
leads to a significant drop in the pKa values for 3 and 5, with the
strongest electron withdrawing functionality -the ester- having
the strongest inductive effect. The conformational flip of ester 3
and acid 4 can be accounted for by taking into consideration that
electron withdrawing groups prefer to occupy an axial position
on
a positively charged pyranose ring to minimize their
destabilizing effect.[4-7] The fact that amide 5 does not change its
conformation to accommodate this intrinsic preference may be
due to internal hydrogen bonds that can be formed between the
amide -NH2 and the C4-OH which provides an extra stabilizing
factor in the 4C1 amide.[18]
Figure 5. 1H NMR spectra for 2,6-dideoxy-2,6-imino-mannuronic amide (5) at
different pH*; spectra are referenced to water.
1
4
To establish the ratio of C4 and C1 conformers for the different
azasugars we used DFT calculations to determine the coupling
constants of the two conformers of both the protonated and
deprotonated azasugars (See SI for details).[17] Table 1 shows
the measured coupling constants (J3,4) for the four azasugars at
4
low and high pH*, the calculated J3,4 values for the C1 and 1C4
conformers and the ratio of the two conformers, established from
the measured average coupling constants. As can be seen from
Table 1, there is good agreement between the calculated and
measured coupling constants at high pH*. With the two values
1
4
for J3,4 the ratio of the C4 and C1 conformers was established
and it is clear that DMJ 2 takes up a single C1 conformation at
4
Figure 6. DMJ Methyl ester (3) in MeOD (±0.7 mL). The non-protonated
azasugar is shown on top (no TFA added), the protonated azasugar on the
bottom (25 μl TFA added). Spectra are referenced to CD2HOD.
both low and high pH values. For the methyl ester 3 the situation
is different. With the calculated values for the coupling constants
of both conformers (J3,4 = 9.5 Hz and 4.9 Hz, for the 1C4 and 4C1
azasugars, respectively) and the measured average coupling
constant (J3,4 = 7.5 Hz) the ratio of the two conformers was
established to be 56:44, indicating that the two chair conformers
are equally stable. In a similar vein the ratio of the two chair
conformers of the acid (4) was determined at three different pH
values. As can be seen in Table 1, at high pH, the anionic
azasugar 4 is present as a single conformer while the measured
average coupling constant at pH = 5 indicates a 94:6 mixture of
conformers. At low pH two conformers are observed in a 75:25
The NMR results show that DMJ analogues having a methyl
ester or carboxylic acid at C5 (as in 3 and 4, respectively) can
change their conformation from the 4C1 chair to the opposite 1C4
chair upon protonation. This conformational change is seen
even in a highly polar medium such as water and is significantly
enhanced in a more apolar solvent (MeOD). The nature of the
substituent at the C5 of the DMJ analogues is of major
importance, as DMJ (2) and the C5 amide DMJ (5) do not
display a conformational change upon changing pH. The
difference between the ester and amide is notable, because
both functional groups, the C5 carboxylic acid ester and C5
carboxamide respectively, have a similar effect on the basicity of
the azasugars. The electron withdrawing effect of both groups
leads to a significant drop in the pKa values for 3 and 5, with the
strongest electron withdrawing functionality -the ester- having
the strongest inductive effect. The conformational flip of ester 3
and acid 4 can be accounted for by taking into consideration that
electron withdrawing groups prefer to occupy an axial position
1
4
4C1 : C4 ratio. For the amide 5, at both high and low pH the C1
chair is almost exclusively present.
To investigate the conformational behaviour in a less polar
environment the azasugar showing the largest conformational
change, methyl ester 3, was investigated MeOD. In Figure 6 the
spectra of the non-protonated and protonated azasugar are
depicted. In this medium the J3,4 coupling constant changes from
9.2 Hz to 4.8 Hz upon protonation, indicating that the non-
4
protonated azasugar resides in the C1 conformation where the
protonated species is found in the opposite 1C4 conformation.
The NMR results show that DMJ analogues having a methyl
ester or carboxylic acid at C5 (as in 3 and 4, respectively) can
change their conformation from the 4C1 chair to the opposite 1C4
chair upon protonation. This conformational change is seen
even in a highly polar medium such as water and is significantly
enhanced in a more apolar solvent (MeOD). The nature of the
substituent at the C5 of the DMJ analogues is of major
importance, as DMJ (2) and the C5 amide DMJ (5) do not
display a conformational change upon changing pH. The
on
a positively charged pyranose ring to minimize their
destabilizing effect.[4-7] The fact that amide 5 does not change its
conformation to accommodate this intrinsic preference may be
due to internal hydrogen bonds that can be formed between the
amide -NH2 and the C4-OH which provides an extra stabilizing
factor in the 4C1 amide.[18]
Having established that the mannuronic acid azasugars readily
undergo a ring flip upon protonation we probed the binding of
the azasugars in the binding pocket of an α-1,2-mannosidase,
GH47, from Caulobacter K31 strain. All four compounds were
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tested for binding using X-ray crystallography and isothermal
titration calorimetry. Initially we analysed the binding of the
parent compound DMJ 2.
suggested that the likely reason would be steric clashes with
residues in the active site, in particular E427. To test this
hypothesis, a mutant was produced containing an E427A
mutation to increase the size of the active site; however
attempts at complexes with this variant still did not allow
observation of 3-5 in the -1 subsite of the enzyme (data not
shown) suggesting that further steric clashes might also be
contributing to lack of inhibition.
DMJ 2 binds to CkGH47 with a KD of 481 nM (determined by
isothermal titration calorimetry, Figure 7A). Although DMJ
binding is essentially as observed previously for the mammalian
GH47 structures,[19] the sub-atomic resolution data (See SI), in
this case allows us to observe DMJ bound in the active site of
CkGH47 in two different ring conformations (Figure 7B). In the -1
subsite, the conformation of DMJ is in both 3S1 and 1C4, each
with a modelled occupancy of 0.5. Both conformations are
consistent with the conformational itinerary of GH47 in which the
substrate is moulded in a 3S1 conformation in the Michaelis
Conclusions
Mannuronic acid based azasugars can change their
conformation upon protonation of the endocyclic amine from a
“normal” 4C1 chair to the inverted 1C4 chair conformation. The
molecules thereby position their substituents such that they are
optimally positioned to accommodate the positive charge.
Although the conformational behavior of any other glycuronic
3
complex to react via a H4 transtion state forming the product in
a
1C4 conformation.[12,19,20] This dual-conformation observation
could be perhaps explained by the proximity of the pH of the
crystallisation conditions, 6.5, to the pKa of DMJ, 7.5 and the
protonation of the species; although one cannot deconvolute
which conformer relates to which protonation state).
acid
based
azasugars,
having
different
substituent
configurations has not yet been studied in detail, it is likely that
the spatial preferences of the substituents in the mannuronic
acid azasugar work in concert to affect the ring flip. This
behavior is in line with the conformational effects observed for
fully protected mannuronic acid glycosyl donors and therefore
the results described here provide an extra indication that the
positive charge at the anomeric center of a mannuronic acid
oxocarbenium ion is responsible for the observed unusual ring
flip. This intriguing ring-flipping behavior pointed to the potential
use of the mannuronic acid azasugars as inhibitors for
mannosidases that hydrolyze their substrates through a ring
flipped conformational itinerary. Unfortunately, the mannuronic
acid azasugars did not bind to the studied GH47 mannosidase.
Although the concept of chemically-flipped inhibitors worked in
solution, especially in low polarity buffers, they sadly highlight
the challenges of conformationally-specific enzyme inhibition.
For whilst the introduction of favourable chemistry including, in
some cases, locking groups frequently introduces substituents
that prevent binding for steric reasons - in enzyme active centres
that have evolved to harness the interactions of and thus distort
unsubstituted sugars. For example, the elegant locking of a
mannoside mimic into B2,5 conformation with a three-carbon
bridge[21] - in order to target B2,5 transition-state mannosidases
specifically, simply resulted in steric clashes with the target
b-mannosidase and no inhibition of the wild type enzyme.[22]
Indeed, whilst the concept of conformation-specific targeted
inhibition is one of the most exciting in glycochemistry, it is only
rarely achieved - the use of ring-flipped kifunensine 1 to inhibit
"Southern Hemisphere" mannosidases is one of the very few
where a conformationally-restrained inhibitor works and has
indeed found considerable application in cell biology.[23] The
challenge therefore remains to provide the specific tools and
therapeutic compounds required for cellular or patient use, whilst
also maintaining binding to the target enzyme.
Figure 7. Binding of DMJ 2 to CkGH47. (a) ITC-derived thermodynamics of
binding. The stoichiometry, n, was 0.96 ± 0.01 sites. The association constant,
KA, was 2.1x106 ± 3.3x105 M-1. The enthalpy change, ΔH, was 1140 ± 150 kcal
mol-1. (b) Divergent wall-eyed stereo electron density for the structure of
CkGH47 in complex with DMJ 2 (with the two conformations shown with
grey/purple bonds). The map shown is a maximum-likelihood / sA weighted
2Fobs-Fcalc, contoured at 0.87 electrons / Å3. The active centre calcium is
shown as a green sphere and a water - likely equating to the nucleophilic
water in catalysis, is shown as a red sphere. Key active centre residues
discussed in the text are labelled.
The structure confirms proposals made by others, and us,
concerning the catalytic apparatus.[12,19,20] Briefly, catalytic base,
E365, is hydrogen bonded to the O6 of DMJ, 2.6 Å. It is held in
place by the nucleophilic water, which in turn is coordinated by
calcium. The indirect route to protonic assistance is by the Oε2
of E121, facilitated by a water molecule, which is hydrogen
bonded to O4 of DMJ. The riding hydrogens of this bond are
visible, matching the level of detail of structure by Thompson et
al.[12a] With the assistance of a metal ion, the O2-C2-C3-O3
4
torsion angle of a C1 conformation is tightened from ~60° to 0–
15°, consistent with the known conformational pathway of GH47
via a 3H4 transition state.[12,19,20]
Unfortunately, despite promising solution characteristics, Table 1,
we were not able to detect binding of mannuronic acid
derivatives 3, 4 or 5. Simple modelling of these compounds in
the active centre, using the DMJ complex as a template,
Experimental Section
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General methods for organic synthesis: All reagents were of
commercial grade and used as received unless stated otherwise.
Reactions were performed at room temperature unless stated otherwise.
Molecular sieves (4Å) were flame dried before use. Flash column
chromatography was performed on silica gel (40-63 µm). 1H and 13C
NMR spectra were recorded on a Bruker AV 600, Bruker AV 400 or a
Bruker DPX 400 spectrometer in D2O or CD3OD. Chemical shifts (δ) are
given in ppm relative to the solvent residual signals. Coupling constants
(J) are given in Hz. All given 13C spectra are proton decoupled.
Compound names are given using the standard iminosugar
nomenclature numbering.
evaporated with dry toluene, put under argon and suspended in dry
MeOH (10 ml). The suspension was cooled to 0 °C before addition of
distilled triethylamine (1.0 ml, 7.2 mmol) and the resulting clear solution
was stirred overnight. The reaction mixture was concentrated under
reduced pressure yielding the methyl ester as a white semi-crystalline
solid. The residue was put under argon, dissolved in dry EtOH
(molsieves, 10 ml) and cooled to 0°C. Sodium borohydride (709 mg, 19
mmol) was added and the suspension was stirred overnight. Dry MeOH
(20 ml) was added before the mixture was filtered, concentrated under
reduced pressure and co-evaporated with 1M HCl in MeOH (3x, 10 ml).
The residue was purified by column chromatography (1:1 EtOAc/EtOH "
100% EtOH) yielding a pure sample of DMJ (2) in 29% yield (105 mg,
0.50 mmol). 1H NMR (399 MHz, D2O): δ 3.99 (1H, dt, J = 2.9, 1.6 Hz, C-
3), 3.76 (1H, dd, J = 12.5, 3.9 Hz, C-7), 3.71 (1H, dd, J = 12.5, 5.5 Hz, C-
7a), 3.60 (1H, t, J = 9.7 Hz, C-5), 3.53 (1H, dd, J = 9.6, 3.1 Hz, C-4), 3.03
(1H, dd, J = 14.2, 2.8 Hz, C-2a), 2.80 (1H, dd, J = 14.2, 1.5 Hz, C-2b),
2.57 (1H, ddd, J = 9.7, 4.9, 3.4 Hz, C-6). 13C NMR (101 MHz, MeOD): δ
74.4 (C-4), 67.6 (C-5), 67.5 (C-3), 62.4 (C-6), 59.6 (C-7), 48.9 (C-2).
[α]20D: -14.0° (c = 0.5, MeOH).
2,6-dibromo-2,6-dideoxy-D-mannono-1,4-lactone (7): Calcium D-
gluconate monohydrate 5 (126 g, 280 mmol) was put under an argon
atmosphere before being dissolved in 33% HBr in acetic acid (500 ml,
3.0 mol). The reaction mixture was stirred for 18 hours to form acetylated
6. MeOH (1 l) was added and the mixture was refluxed for 2 hours. After
refluxing the mixture was concentrated to half the volume under reduced
pressure before adding another 500 ml of MeOH. The reaction was left to
stir overnight after which the mixture was concentrated resulting in a
slightly oily residue. This was co-evaporated with 100 ml of MeOH and
three times with H2O. The residue was extracted with diethyl ether (4 x
100 ml), the organic layers were combined, dried with MgSO4, filtered
and concentrated under vacuum yielding a yellow oily residue. This was
crystallized from CHCl3 / H2O to yield a white crystalline solid (44 g, 146
mmol, 26% yield). 1H NMR (400 MHz, D2O): δ 5.20 (1H, d, J = 4.5 Hz,
C-2), 4.64 (2H, m, C-4, C-3), 4.19 (1H, m, C-5), 3.77 (1H, dd, J = 11.4,
2,4 Hz, C-6a), 3.65 (1H, dd, J = 11.4, 4.9 Hz, C-6b). 13C NMR (101 MHz,
D2O): δ 174.0 (C-1), 81.6 (C-4), 69.1 (C-3), 66.2 (C-5), 47.6 (C-2), 36.6
(C-6). Melting point: 130 °C. [α]20D: +58,6° (c = 1, MeOH).
Methyl 2,6-dideoxy-2,6-imino-D-mannonate hydrochloride (3): 2-
amino-6-bromo-2,6-dideoxy-D-mannono-1,4-lactone hydrochloride (7,
0.60 g, 2.2 mmol), was co-evaporated thrice with dry toluene, put under
argon and suspended in dry MeOH (12 ml). The suspension was cooled
to 0 °C before addition of distilled triethylamine (1.2 ml, 8.7 mmol) and
the resulting clear solution was stirred overnight. The reaction mixture
was concentrated under reduced pressure before being taken up in
acetonitrile (15 ml) charged with 1,1,1,3,3,3-hexamethyldisilazane (2.5 ml,
12 mmol) and copper sulphate pentahydrate (cat.). After 1 hour, the
reaction mixture was concentrated and a fraction of 234 mg (0.57 mmol)
was purified by column chromatography (1-2.5% 1,4-dioxane/DCM) to
give 162 mg (0.40 mmol) of the per-TMSylated compound. The protected
product was put under argon, dissolved in MeOH (8 ml) and acetyl
chloride (1 eq) added to generate HCl in situ. The mixture was stirred for
0.5 hour, after which the compound was concentrated and coevaporated
with MeOH yielding the title compound (98 mg, 0.40 mmol, 70% over 2
steps). 1H NMR (400 MHz, MeOD) δ 4.40 (dd, J = 5.3, 4.8 Hz, 1H, C-5),
4.17 (ddd, J = 9.5, 4.1, 2.8 Hz, 1H, C-3), 4.09 (d, J = 4.4 Hz, 1H, C-6),
3.86 (dd, J = 5.6, 2.7 Hz, 1H, C-4), 3.82 (s, 3H, OCH3), 3.44 (dd, J =
12.2, 9.6 Hz, 1H, C-2a), 3.13 (dd, J = 12.2, 4.2 Hz, 1H, C-2b). 13C NMR
(101 MHz, MeOD) δ 168.1 (C-7), 71.1 (C-4), 69.9 (C-5), 64.2 (C-3), 58.8
(C-6), 53.4 (OCH3), 43.5 (C-2). [α]20D: +31.8 (c = 1, MeOH).
2-amino-6-bromo-2,6-dideoxy-D-mannono-1,4-lactone hydrochloride
(8): 2,6-Dibromo-2,6-dideoxy-D-mannono-1,4-lactone (6, 5.0 g, 16.5
mmol) was put under argon and dissolved in dry acetone (MgSO4, 100
ml). Sodium azide (15.0 g, 231 mmol) was added and the suspension
was refluxed for 20 hours. The mixture was filtrated and the filtrate
concentrated under reduced pressure. The residue was dissolved in H2O
(50 ml) and extracted with diethyl ether (5 x 100 ml), the organic layers
were combined, dried over MgSO4, filtered and concentrated under
reduced pressure to give a brown oil which was identified as the 2-azido
compound but included some of its diastereoisomer. 1H NMR (400 MHz,
D2O): δ 4.68 (1H, dd, J = 4.5, 3.3 Hz, C-3), 4.56 (1H, d, J = 4.6 Hz, C-2),
4.46 (1H, dd, J = 9.2, 2.7 Hz, C-4), 4.09 (1H, m, C-5), 3.69 (1H, dd, J =
11.4, 2.7 Hz, C-6a), 3.56 (1H, dd, J = 11.5, 4.9 Hz, C-6b). 13C NMR (101
MHz, D2O): δ 174.1 (C-1), 81.0 (C-4), 69.6, 65.7 (C-3, C-5), 62.3 (C-2),
36.6 (C-6). The crude compound (16.5 mmol) was put under argon and
dissolved in MeOH (100 ml). Palladium on activated carbon (10%, 300
mg, 0.3 mmol) and HCl (37% in H2O, 10 ml, 121 mmol) were added and
the suspension charged with hydrogen atmosphere. The reaction mixture
was stirred for 22 hours after which the catalyst was filtered off over a
Whatman microfilter. The filtrate was concentrated under reduced
pressure and co-evaporated once with HCl (37% in H2O, 60 ml), thrice
with toluene (60 ml) and once with CHCl3 (50 ml). Crystallization from
Sodium 2,6-dideoxy-2,6-imino-D-mannonate (4): Methyl 2,6-dideoxy-
2,6-imino-D-mannonate hydrochloride (3, 24 mg, 0.10 mmol), was
dissolved in H2O (0.5 ml). A sodium hydroxide solution (1M aq., 170 μl,
0.17 mmol) was added and the mixture stirred for 2 hours. The mixture
was concentrated under reduced pressure yielding the title compound,
pure but with added sodium hydroxide. 1H NMR (400 MHz, D2O): δ 4.01
(1H, m, C-3), 3.71 (1H, t, J = 9.7 Hz, C-5), 3.60 (1H, dd, J = 9.6, 3.2 Hz,
C-4), 3.01 (1H, dd, J = 14.6, 2.7 Hz, C-2a), 2.95 (1H, d, J = 9.8 Hz, C-6),
2.75 (1H, dd, J = 14.6, 1.6 Hz, C-2b). 13C NMR (101 MHz, D2O): δ 178.4
(C-7), 74.1 (C-4), 70.6 (C-5), 69.1 (C-3), 65.2 (C-6), 47.9 (C-2). [α]20D: -
7.2 (c = 1, MeOH).
EtOH
yielded
2-amino-6-bromo-2,6-dideoxy-D-mannono-1,4-lactone
hydrochloride (8) as white crystals (2.6 g, 9.2 mmol, 55% over two steps).
1H NMR (400 MHz, D2O): δ 4.83 (1H, dd, J = 4.8, 2.8 Hz, C-3), 4.63 (1H,
dd, J = 9.2, 2.7 Hz, C-4), 4.59 (1H, d, J = 4.9 Hz, C-2), 4.20 (1H, m, C-5),
3.77 (1H, dd, J = 11.5, 2.6 Hz, C-6a), 3.65 (1H, dd, J = 11.4, 5.0 Hz, C-
6b). 13C NMR (101 MHz, D2O): δ 172.1 (C-1), 78.9 (C-4), 64.1 (C-3), 62.9
2,6-dideoxy-2,6-imino-D-mannonic amide (5): 2-amino-6-bromo-2,6-
dideoxy-D-mannono-1,4-lactone hydrochloride (7, 500 mg, 1.8 mmol),
was co-evaporated thrice with dry toluene, put under argon and
suspended in dry MeOH (10 ml). The suspension was cooled to 0 °C
before addition of distilled triethylamine (1.0 ml, 7.2 mmol) and the
resulting clear solution was stirred overnight. The reaction mixture was
concentrated under reduced pressure yielding the methyl ester as a
white semi-crystalline solid. The residue was dissolved in 6M ammonia in
MeOH (10 ml, 60 mmol) and was stirred overnight. The reaction mixture
(C-5), 50.2 (C-2), 33.7 (C-6). Melting point: 207 °C (decomposed). [α]20
+41.6° (c = 1, MeOH).
:
D
1-Deoxymannojirimycin (2): 2-amino-6-bromo-2,6-dideoxy-D-mannono-
1,4-lactone hydrochloride (7, 501 mg, 1.8 mmol), was three times co-
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was concentrated under reduced pressure yielding 2,6-dideoxy-2,6-
imino-D-mannonic amide (5) in quantitative yield. An analytical sample
was made by crystallisation from pure MeOH (133 mg, 0.76 mmol, 42%).
1H NMR (399 MHz, D2O): δ 3.97 (1H, m, C-3), 3.70 (1H, t, J = 9.7 Hz, C-
5), 3.57 (1H, dd, J = 9.6, 3.1 Hz, C-4), 3.07 (1H, d, J = 9.8 Hz, C-6), 2.99
(1H, dd, J = 14.6, 2.7 Hz, C-2a), 2.75 (1H, dd, J = 14.6, 1.6 Hz, C-2b).
13C NMR (101 MHz, DMSO): δ 173.5 (C-7), 74.9 (C-4), 70.0 (C-5), 68.7
(C-3), 63.3 (C-6), 49.1 (C-2). [α]20D: -31,6° (c = 0.5, H2O). HR-MS: [M+H+]
calculated for C6H12O4N2: 177.08698; found: 177.08683
[9]
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Acknowledgements
We thank the European Research Council (ERC-2011-AdG-290836
“Chembiosphing”, to H.S.O).
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Keywords: Azasugar • Conformation • Stereoelectronic effects •
Inhibitor design • Mannosidase
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[18] It has previously been observed that mannuronic acid ester thiodonors
take up a 1C4 conformation when fully protected, while they reside in a
4C1 conformation when the C4-OH is free. This contrasting
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hydrogen bond between the C5-carboxylic acid ester and the C4-OH in
the 4C1 chair conformation.
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Entry for the Table of Contents (Please choose one layout)
Layout 1:
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Mannuronic acid azasugars can
Erwin R. van Rijssel, Antonius P. A.
Janssen, Alexandra Males] Gideon J.
Davies, Gijsbert A. van der Marel,
Herman S. Overkleeft,* and Jeroen D.
C. Codée*[
undergo
a
conformational change
from the conventional 4C1 to the 1C4
chair upon protonation. This behaviour
was shown to be dependent on the
functionality at C5. Binding is studied
Page No. – Page No.
with
a
GH47 mannosidase that
employs
a
hydrolysis
itinerary
Conformational behaviour of
mannuronic acid based azasugars
proceeding through a 3H4 transition
state.
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