The UDP-Galp mutase catalyzed isomerization: Synthesis and evaluation of 1,4-anhydro-β-d-galactopyranose and its [2.2.2] methylene homologue

Article abstract of DOI:10.1039/b917409e

The synthesis of 1,4-anhydro-β-d-galactopyranose (1,5-anhydro-α- d-galactofuranose), a proposed intermediate in the ring contraction isomerisation catalyzed by UDP-galactopyranose mutase, together with its [2.2.2] bicyclic methylene homologue, synthesised as a possible competitive inhibitor or alternative substrate, are reported. Neither compound was found to be an inhibitor or substrate for UDP-galactopyranose mutase from Klebsiella pneumoniae.

Full text of DOI:10.1039/b917409e

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
The UDP-Galp mutase catalyzed isomerization: synthesis and evaluation of
1,4-anhydro-b-D-galactopyranose and its [2.2.2] methylene homologue†
Ali Sadeghi-Khomami,a Tatiana J. Forcada,a Claire Wilson,a David A. R. Sandersb and Neil R. Thomas*a
Received 24th August 2009, Accepted 23rd December 2009
First published as an Advance Article on the web 8th February 2010
DOI: 10.1039/b917409e
The synthesis of 1,4-anhydro-b-D-galactopyranose (1,5-anhydro-a-D-galactofuranose), a proposed
intermediate in the ring contraction isomerisation catalyzed by UDP-galactopyranose mutase, together
with its [2.2.2] bicyclic methylene homologue, synthesised as a possible competitive inhibitor or
alternative substrate, are reported. Neither compound was found to be an inhibitor or substrate for
UDP-galactopyranose mutase from Klebsiella pneumoniae.
for the essential coenzyme (reduced FAD) in catalysis32 includ-
ing hydride transfer,33,34 single electron transfer,35,37 nucleophilic
Introduction
attack,36,43,44 charge stabilization and a structural role.34,37,45 Fur-
thermore, during catalysis the galactose moiety of the substrate
has been proposed to take a variety of different forms including
an acyclic adduct,34,36,38 monocyclic ring (anomeric radical or
oxonium ring)35,37,41 and bicyclic39,40 intermediates.
D-Galactose is extensively distributed in nature as a constituent of
oligosaccharides, polysaccharides and glycoconjugates. However
the furanoid configuration (galactofuranose, Galf ) has yet to
be found in mammals, its presence is apparently restricted to
bacteria, protozoa, fungi and plants.1–8 Therefore the biosynthesis
of Galf , given that it plays critical roles in cellular viability and
virulance, would seem to be an appealing target for potential
antimicrobial and antifungal drug development.9–13 One major
family of pathogenic bacteria that utilise Galf are the Mycobac-
teria that include the bacteria responsible for the tuberculosis and
leprosy. Every year, tuberculosis (TB) kills around 3 million people
worldwide, more than AIDS and malaria combined, causing the
World Health Organisation (WHO) to declare TB a global health
emergency in 1993.14 Mycobacterium tuberculosis (M. tuberculosis)
the causative agent of TB, has a unique cell envelope responsible
for pathogen resistance to antibiotics and harmful conditions. The
cell wall component is composed of a mycolyl arabinogalactan
polymer attached to the peptidoglycan. The galactan component
is an oligomer of 15–30 Galf residues that are alternately linked
b(1→5) and b(1→6).15–24 It has been demonstrated that the
precursor of Galf residue is UDP-a-D-galactofuranose (UDP-
Galf , 3), which in turn is formed from UDP-a-D-galactopyranose
(UDP-Galp, 1) by the flavoenzyme UDP-galactopyranose mutase
(UDP-Galp mutase, E.C.5.4.99.9).25–30 It has been shown that
Galf , and therefore the mutase action, is essential for the viability
of mycobacteria.31 Understanding the catalytic mechanism of
the mutase will hopefully lead to new directions in anti-TB
therapy.32 Although a number of studies on the mutase has been
conducted,33–42 the mechanism of substrate turnover is still not
fully understood. Reviewing currently available biochemical and
kinetic data indicates several different roles have been suggested
Through position isotope exchange (PIX) and 13C-NMR
spectroscopy monitoring, it has been demonstrated that the
cleavage of the glycosidic C–O bond takes place during the
mutase catalytic cycle.34,39 Since the axial 4-OH of Galp has
a special stereoelectronic role that facilitates the hydrolysis of
galactopyranosides,46 it has been suggested that it functions as
an internal nucleophile in the above reaction.4,39 Thus, bicyclic
structure 2 has been proposed as an intermediate in the mutase
catalysis (Scheme 1). Furthermore, selective production of Galf
under acidic conditions from derivatives of 2,47–52 chemically
supports this proposal. Additionally, the weak inhibitory effect
of a UMP-(1C)-phosphonate-2 has been reported as indirect
evidence for validity of bicyclic intermediate in Scheme 1.40
Scheme 1 The UDP-Galp mutase catalysed reaction with putative
bicyclic intermediate.
In this regard, we set out to further investigate the involvement
of the bicyclic intermediate 2 in UDP-Galp catalyzed reaction.
Previously, we have shown that a carbasugar analogue of 2
is not an inhibitor for M. tuberculosis mutase, this finding
indirectly indicated that during turnover the presence of a
bicyclic intermediate is unlikely.53 Here we report the synthesis
of the proposed bicyclic intermediate 2 in 5 steps and its effect
on the turnover of the natural substrate 3 in K. pneumoniae
mutase system. Also the synthesis and inhibitory effect of a
methylene homologue 4 of the proposed bicyclic intermediate
aSchool of Chemistry, Centre for Biomolecular Sciences, University of
Nottingham, University Park, Nottingham, United Kingdom NG7 2RD.
E-mail: neil.thomas@nottingham.ac.uk
bDepartment of Chemistry, University of Saskatchewan, Saskatoon, S7N
5C9, Canada
† Electronic supplementary information (ESI) available: Experimental
details for 5–11, 13–18, enzyme assay conditions and spectra for products;
table comparing oxanorbornane NMR data. CCDC reference number
287123. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/b917409e
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2, designed as a potential competitive inhibitor with reduced
ring strain and better stability, is reported in 8 steps. During
the preparation of this manuscript, Caravano et al. reported
the synthesis and evaluation of 1,4-anhydrogalactopyranose with
UDP-Galp mutase.54 We noticed that the 1H and 13C- NMR data
they provide on 1,4-anhydrogalactopyranose does not agree with
our data and is less consistent with the data on protected forms of
1,4-anhydrogalactopyranose that have been reported previously
in the literature (see also supporting information for a tabulated
comparison†).51,55,56 Several 2,7-dioxabicyclo[2.2.1]heptane57 and
isoquinuclidine58 compounds that bear a substantial structural
similarity to 2 and 4 have been evaluated as inhibitors of
glycosidases although only the latter displayed any significant
inhibitory activity.
Since the instability of the desired oxanorbornane systems
under acidic, basic, oxidative and reducing conditions have been
documented,64 we therefore selected benzyl ethers (Bn) as suitable
protecting groups for the 2-, 3- and 6-hydroxyls in our synthetic
strategy because of their mild deprotection conditions. Addition-
ally, Bz and TBS protecting groups were recently described as
being unsuitable for the cyclization step.52 To generate compound
9 with a free 4-hydroxyl group, regioselective ring opening of 4,6-
benzylidene was required, and this has been well documented.
Although 4,6-benzylidene (1,3-dioxane) formation occurs cleanly
from D-glucose via Evans’ procedure,65,66 in our hands with D-
galactose we always obtained low to moderate yield (30–50%) of
5 together with unreacted starting materials from the reaction
mixture. Excess dimethyl benzyl acetal and higher temperature
caused formation of the alternative regioisomeric acetals, 3,4-
benzylidenes (1,3-dioxolane) 6 and 7 (Fig. 1). Finally changing
solvent and acid catalyst to match Clausen’s procedure66 provided
5 in quantitative yield.
Results and discussion
Formation of derivatives of 2 at high temperature or under py-
rolysis conditions have previously been reported,56,59,60 suggesting
that 2 is sufficiently stable to allow its preparation. Target 2 could
possibly be achieved under basic conditions via 1,4-trans cycliza-
tion from either appropriately protected D-glucose with a leaving
group at C-4 or a protected D-galactose with a leaving group at the
a-anomeric carbon in analogy with preparation of 1,4-anhydro-
a-D-glucose.49,51,56,59,61–63 The main drawback of these methods was
that the introduction of good leaving groups requires additional
steps and the resulting compounds are usually unstable and prone
to polymerization. Also cyclization via anomeric oxocarbeniums
(requiring acidic conditions), increases the possibility of by-
product formation. Therefore an alternative method introduced
Fig. 1 Regioisomeric acetals formed between methyl-a-D-galactoside and
benzaldehyde.
Protection of the remaining hydroxyls of 5 was achieved using
a standard benzylation procedure67 under basic conditions to give
8. It should be noted that decomposition of the acid-labile 1,3-
dioxane ring of 8 after 24 h storage in chloroform solution was
clearly observed by IR and NMR spectroscopy. The product 8
was therefore purified by crystallization and X-ray crystallography
data confirmed that the phenyl ring takes the expected lower
energy equatorial position in the 1,3-dioxane ring system (CCDC
287123†). Reductive ring opening of 8 using the most common
regioselective protocol (NaCNBH3 and HCl/THF)68,69 provided 9
as well as minor by-products 10 and 11. Use of CF3CO2H/DMF
or Et3SiH/CF3SO3H systems70–72 did not improve the yield or
selectivity in this step. In accordance with Larsson and co-
workers,73 our spectroscopic data supported the existence of
internal hydrogen bonding between the proton of 4-OH and the
adjacent 3-O. Interestingly we observed on TLC an unusually high
Rf for 9 compared with the more hydrophobic starting material
8, which could be explained by internal hydrogen bonding. The
optical rotation of 10 did not agree with that published by
Lucas and Schuerch,74 but it was in good agreement with that
reported by Ek and co-workers.75 Cyclization of 9 was achieved in
moderate yield using anhydrous FeCl3 catalyst in a dilute solution
of acetonitrile after 1 h gentle reflux at 60 ◦C, running the reaction
under vigorous reflux increased formation of by-products and the
risk of decomposition. Although FeCl3 can be used to deprotect
benzyl groups, the application of a coordinating solvent (CH3CN)
reduced the possibility of deprotection occurring. We found that
increasing the reaction time (e.g. 12 h) could decrease the yield
of cyclization step and it produced highly polar by-products.
Regarding NMR interpretation of oxanorbornane systems, it
55
˚
by Aberg and Ernst for the synthesis of b-glycosans was applied.
In this approach a relatively stable potential leaving group has
to be activated in the cyclization step using catalytic anhydrous
FeCl3 in acetonitrile as a coordinating solvent. Thus synthesis of
putative bicyclic intermediate 2 requires access to protected O-
methyl 4-hydroxy-a-D-galactoside 9 (Scheme 2).
Scheme 2 Reagents and conditions: i) Benzaldehyde dimethyl acetal, CSA,
CHCl3, reflux, 100%; ii) NaH, DMF, 0 ◦C; iii) BnBr, Bu4NI, 85%; iv) 3 A
˚
MS, NaCNBH3, THF, HCl/diethyl ether, 9 70%, 10 10%, 11 8%; v) 9,
FeCl3, CH3CN, reflux, 40%; vi) H2, Pd/C (10%), EtOH, EtOAc, 100%.
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should be noted that the coupling constants between the bridge
head proton 4-H and the endo vicinal protons 3-H and 5-H
are ~ 0 Hz due to the dihedral angle ~ 90◦. Moreover, the fixed
W shape arrangement of 2-H and 4-H causes a measurable long-
range coupling in the 1D spectrum of oxanorbornane systems
(4J ~ 1 Hz). Finally, hydrogenolysis of 12 gave 2 in an overall 24%
yield after 5 steps. Optical rotation and NMR data confirmed
that 2 adopted the rigid 1,4B conformation. We couldn’t detect
any change in NMR spectra of 2 after two weeks storage in D2O
at 22 ◦C, however ring opening and oligomerisation reactions
to give dimers, trimers and tetramers rapidly occurred in the
presence of acid catalysts e.g. HCl (see supporting information†).
Therefore, although 2 was not as reactive as many enzyme-
generated intermediates, it could potentially be easily activated by
protonation of the bridging oxygen inside the enzyme active site.
A ring expanded homologue of 2 was identified as a potential
inhibitor or alternative substrate for UDP-Galp mutase. The
synthesis of the [2.2.2] bicyclic methylene homologue 4 utilised
a similar synthetic strategy to that described for 2. The synthesis
started from the more accessible O-methyl a-D-glucoside rather
than O-methyl a-D-galactoside to avoid potential complications
in the benzylidene formation step (Fig. 1). Access to the required
free 4-OH protected glucoside 15 was achieved via benzylidena-
tion, benzyl protection and regioselective ring opening reduction
(Scheme 3). Installation of the extra methylene group was achieved
via Swern oxidation of 15 followed by Wittig olefination and
hydroboration-oxidation to afford 20. Whilst the oxidation and
methylenation reactions proceeded efficiently, the hydroboration-
oxidation was not highly regioselective, leading to a product
distribution of 20 (35%), 21 (26%), and 22 (4%) suggesting that
internal delivery of the borane on the a-face through coordination
to the adjacent oxygens is occurring to preferentially form 20 over
its stereoisomer 22. According to Takahashi and co-workers, the
diastereoselectivity of hydroboration-oxidation can depend on the
ratio of reagents.76 This could be examined further to improve on
the low yield observed in preparation of 20. Our data were not
1H-NMR analysis (J2,3 ~ 10 Hz), the predominant pyranoside
conformation of 20–22 in D2O was assigned to be 4C1. Thus
our chemical modifications at C-4 of the hexoses did not change
the conformational distribution of these compounds. Internal
cyclisation under the same mild conditions, as described for the
synthesis of 12, gave 23 in considerable higher yield (70% vs. 40%).
This is probably due to the increased strain which exists in the
[2.2.1] oxanorbornane systems compared with the [2.2.2] bicyclic
systems. Long-range coupling constants were also observed in the
1D-NMR spectra of the [2.2.2] bicyclic systems (see experimental
data). Finally hydrogenolysis of 23 resulted target 4 in ca. 1%
overall yield in 8 steps from O-methyl a-D-glucoside.
Enzyme activity assays
UDP-galactopyranose mutase (UDP-galactopyranose mutase,
E.C. 5.4.99.9) from Klebsiella pneumonia was purified as de-
scribed previously.78 Compounds 2 and 4 were tested against the
purified enzyme examining the reaction in the reverse direction
in which UDP-Galf is converted to UDP-Galp using a HPLC
assay according to Sanders et al.79 Neither 2 nor 4 display any
significant inhibition activity against the enzyme, with 15% and
17% inhibition respectively being observed with the compounds
at 4 mM. Both compounds therefore had IC50 values > 1 mM. No
evidence for 2 or 4 being utilised as alternative substrates in the
presence of UDP was observed.
Conclusion
The failure of 2 and 4 to either competitively inhibit or be used
as substrates for UDP-Galp mutase provides further evidence that
bicyclic 1,4-anhydro-b-D-galactopyranose is not an intermediate
in the UDP-Galp mutase catalyzed reaction,53,54 in accord with
the observations of Caravano et al.54,80 and is consistent with our
previous observations with the carbasugar analogue of 1,4-b-D-
anhydrogalactopyranose.53 It is well established that much of the
binding affinity of enzymes for sugar-nucleotide substrates comes
from the nucleoside-diphosphate portion (UDP, IC50 = 200 mM
1
in agreement with the literature in H-NMR assignments of 2-
H and 6b-H in 18 and the optical rotation of 21.77 Based on
◦
˚
Scheme 3 Reagents and conditions: i) Benzaldehyde dimethyl acetal, CSA, CHCl3, reflux, 92%; ii) NaH, DMF, 0 C; iii) BnBr, Bu4NI, 84%; iv) 3 A
MS, NaCNBH3, THF, HCl/diethyl ether, 15 83%, 16 5%, 17 3%; v) DMSO, TFAA, DCM, Et3N, -78 → -40 ◦C, 80%; vi) n-BuLi, Ph3PCH3Br, THF,
-60 ◦C → rt, 79%; vii) BH3·THF, THF, then H2O2/NaOH, 20 35%, 21 26%, 22 4%; viii) 20, FeCl3, CH3CN, reflux, 70%; ix) H2, Pd/C (10%), EtOH,
EtOAc, 70%.
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for UDP-Galp mutase).81 A comparison of the binding affinities
of UDP-Galf (Km = 22 mM and 194 mM under reducing
conditions),34,45 UDP-Galp (Km = 600 mM)38 and UDP (Ki =
37 mM)36 for the UDP-Galp mutase from Klebsiella pneumoniae
indicates that most of the substrate binding affinity of the enzyme
is directed towards the UDP portion of these molecules. Sinay¨ et al.
reported that UDP-C-a-D-1,4-anhydrogalactopyranose in which
the galactose moiety is locked in a bicyclic 1,4B boat conformation
that is attached to uridine monophosphate via a phosphonate was
a weak competitive inhibitor (42–53% inhibition at 1 mM; 32–34%
with the reduced enzyme) whilst UDP-C-a-D-galactofuranose,
a non-isomerable analogue of the substrate exhibits moderately
higher inhibition with the native enzyme (81–91% at 1 mM), but
lower inhibition with the reduced enzyme (2–14%)40 suggesting
that the 1,4-anhydro-b-D-galactopyranose structure contributes
little to the binding of the former compound. These observations
strengthen the argument that UDP-Galp mutase is operating
by one of the proposed alternative radical/nucleophilic flavin
mechanisms.35–37,44
aluminium–backed silica gel plates supplied by E. Merck, A.
G. Darmstad, Germany (silica gel 60 F254, thickness 0.2 mm,
Art. 5554). Chromatograms were initially examined under UV
light and then visualised with aqueous potassium permanganate
(dip), followed by warming of the TLC plate with a heat gun.
All anhydrous reactions were carried out in oven-dried glassware
◦
(>180 C), which was cooled in desiccator and was subjected to
vacuum-N2 flashing before use. Molecular sieves were activated
before applications by storing in an oven (>180 ◦C) for 12 h.
Where necessary ether and THF were refluxed and distilled from
sodium-benzophenone ketyl, and DCM from calcium hydride,
immediately before use. Anhydrous DMF was prepared through
overnight stirring with calcium hydride, followed by distillation
under reduced pressure. It was then collected over pre-activated
˚
3 A molecular sieves. Chloroform were distilled from P2O5
˚
(3% w/v) and distillates were collected over pre-activated 4 A
molecular sieves. Dimethyl benzylacetal was dried overnight over
˚
pre-activated 4 A molecular sieves, filtered, distilled in vacuo and
˚
collected over 4 A molecular sieves. Light petroleum (pet. ether)
refers to the fraction boiling in the range 40–60 ◦C that was
redistilled before use. Methyl hexopyranosides were dried over
P2O5. Organic extracts were dried over MgSO4 before evaporation,
unless otherwise specified. Evaporations were achieved using a
Bu¨chi rotary evaporator followed by drying at <1 mmHg using
an Edwards rotary vacuum pump. An Edwards Freeze-dryer
was used for lyophilization of samples. Except where specified
all reagents were purchased from commercial sources and were
used without further purification. Yields are reported for isolated,
chromatographically homogenous and analytically pure products.
Details for the synthesis and analytical data of compounds 5–11
and 13–18 are given in the supporting information.†
Experimental
General information
NMR spectra were recorded on a Bruker AV 400 (1H at 400
and 13C at 100 MHz) instrument. Chemical shifts (d in ppm)
are given relative to internal standard (Tetramethylsilane (TMS)
in CDCl3) or residual solvent peak (chloroform at 7.26 ppm
in CDCl3 and water at 4.70 ppm in D2O). Coupling constants
(J) values are in Hz and are reported for couplings over three
bonds unless otherwise specified. Gaussian resolution enhance-
ment were used to determine coupling constants of complicated
peaks. Assignments were made by comparison of chemical shifts,
peak multiplicities, J values and 1H-1H COSY spectra. The
1,4-Anhydro-2,3,6-tri-O-benzyl-b-D-galactopyranose
Methyl 2,3,6-tri-O-benzyl-a-D-galactopyranoside
12.
(0.8 g,
9
1
abbreviation “app.” (apparent) in H-NMR assignments refers
1.7 mmol), and anhydrous FeCl3 (94.4 mg, 0.59 mmol) were
refluxed in anhydrous acetonitrile (200 ml) for 1 h. The reaction
mixture was concentrated in vacuo and the residue was purified by
flash chromatography on a pre-neutralized (triethylamine) silica
gel column (hexanes-EtOAc, 3 : 1) to give the bicyclic acetal 12
(290 mg, 40%) as a colourless oil. Rf 0.6 (hexanes-EtOAc, 3 : 1);
[a]D25 +57.4 (c 1.12 in CHCl3), (lit.,51,55 +57, c 1 in CHCl3); [Found:
C, 74.38; H, 6.52%; (M+Na)+ 455.1843. C27H28O5 requires C,
74.98; H, 6.53%; (M+Na)+ 455.1834]; vmax (CHCl3)/cm-1 2926 s
(CH), 2867 s (OC–H), 1952w, 1874w, 1812w, 1738w (overtone,
comb, Ph), 1603w, 1496w (C–C, Ar), 1454 s sharp (CH2), 1360 s,
1084vs, 968s; dH (CDCl3) 3.34 (1H, dd, J6a,6b 9.4 and J6a,5 8.5,
6a-H), 3.43 (1H, dd, J6b,6a 9.4 and J6b,5 5.0, 6b-H), 3.54 (1H,
app. br. d, J ~ 1.2, 3-H or 4-H), 3.82 (1H, obscured dd, J5,6a
8.5, J5,6b 5.0 and J5,4 ~ 0, 5-H), 3.83 (1H, obscured ddd, J2,1
to the appearance of the multiplet observed in the spectrum where
this differs from the expected peak shape. Hydrogen and carbons
were numbered for NMR assignment use standard carbohydrate
numbering. The sub-assignment of “a” and “b” in 1H-NMR was
based on the appearance of corresponding signals of higher and
lower field, respectively. Carbon spectra were recorded with proton
broadband decoupling. Assignments were verified using DEPT
and 1H-13C HMQC experiments. The central peak of chloroform
at 77.1 ppm was used as the internal reference for spectra run in
CDCl3. Acetone in D2O (30.63 ppm, Me) was used as external
reference for spectra run in D2O. Mass spectra were determined
using EI (VG 70E, 70 eV, ref. PFK) and ESI (Micromass
LCT, ref. Erythromycin). Elemental analyses were conducted on
an Exeter analytical, Inc. CE-440 Elemental Analyser. Melting
points were recorded on a Stuart Scientific SMP3 digital melting
point apparatus and are uncorrected. Infrared (IR) spectra were
recorded in the range of 4000–500 cm-1 using a Perkin Elmer
1600 series FT-IR spectrometer. Optical rotation was measured
with a digital Jasco polarimeter DIP-370 in a 0.5 dm cell at
ambient temperature (23 1 ◦C) and [a]D values are given in units
of 10-1 deg cm2 g-1. Chromatography is medium pressure flash
chromatography and was performed according to the method of
Still et al.82 using Fluorochem silica gel (35–70 mm) with the eluent
specified. Thin layer chromatography was performed on precoated
4
2.3 and J2,3 ~ J2,4 ~ 1.4, 2-H), 4.46 and 4.58 (AB, each 1H, d,
JA,B 11.7, CHAHBPh), 4.49 and 4.53 (AB, each 1H, d, JA,B 12.0,
CHAHBPh), 4.50 (2H, coincident d, JA,B 9.1, CHAHBPh), 4.585
(1H, app. br. d, J ~ 1.4, 4-H or 3-H), 5.48 (1H, br. d, J1,2 2.3, 1-H),
7.28–7.34 (15H, m, 3xPh); dC (CDCl3) 69.9 (CH2, C-6), 71.2,
72.4, 73.6 (each CH2, 3xCH2Ph), 74.3 (CH, C-5), 81.4 (CH, C-4
or C-3), 82.9 (CH, C-3 or C-4), 87.3 (CH, C-2), 98.7 (CH, C-1),
127.94, 128.0, 128.1, 128.5, 128.8 (all CH, 3xPh), 137.4, 137.5,
137.9 (each C, 3xPh); HRMS (ESI): found (M+Na)+ 455.1843.
C27H28O5 requires (M+Na)+, 455.1834.
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1,4-Anhydro-b-D-galactopyranose
(1,5-Anhydro-a-D-galacto-
dry THF (20 ml) under N2 at 0 ◦C was added dropwise BH3·THF
1 M (1.12 ml; 1.12 mmol). The solution was stirred at rt for 14 h.
After cooling to 0 ◦C, NaOH 2M (2.24 ml; 4.48 mmol) and 30%
v/v H2O2 solution (0.64 ml; 5.61 mmol) were successively added
dropwise. After stirring for 1 h at 0 ◦C, stirring was continued
at rt for additional 4 h. The solution was poured into brine,
dried with MgSO4 and concentrated in vacuo. Silica gel column
chromatography (pet. ether-EtOAc from 99 : 1 to 2 : 3) yielded 20
(189 mg, 35%) as a colourless oil. Rf 0.22 (EtOAc-hexanes, 2 : 3);
[a]D25 +45.2 (c 1.01 in CHCl3), (lit.,77 +45.5◦, c 1.01 in CHCl3);
vmax (CHCl3)/cm-1 3488 m br (hydrogen bonded OH), 2904 s
(CH), ~2850s (OC–H), 1951w, 1882w, 1810w, ~1720w (overtone,
comb, Ph), 1603w, 1496w (C–C, Ar), 1454 m sharp (CH2), 1093vs,
1048vs; dH (CDCl3) 2.36 (1H, dddd, J3,4 ~ J4,4a¢ ~ J4,4b¢ 5.8 and J4,5
2.3, 4-H), 3.22 (1H, br. s, 4¢-OH), 3.39 (3H, s, -OCH3), 3.60 (1H,
dd, J6a,6b 10.4 and J6a,5 5.0, 6a-H), 3.63 (1H, dd, J6b,6a 10.4 and J6b,5
5.8, 6b-H), 3.71 (1H, dd, J2,3 10.3 and J2,1 4.0, 2-H), 3.80 (1H, br.
dd, J4¢a,4¢b 11.4 and J4¢a,4 5.8, 4¢a-H), 3.97 (1H, dd, J4b¢,4a¢ 11.4 and
furanose) 2. A suspension of 10% Pd/C (193 mg) in absolute
ethanol (15 ml) was added to a stirred mixture of per-benzylated
bicyclic acetal 12 (0.185 mmol, 80 mg) in glacial acetic acid (4 ml)
and absolute ethanol (10 ml). The resulting mixture was stirred
at room temperature under an atmosphere of hydrogen. After
one hour the reaction mixture was filtered through Celite and
the filtrate co-evaporated with toluene (3 ¥ 5 ml) to give 2 in
quantitative yield. Rf 0.2 (EtOAc–MeOH, 8.5 : 1.5); [a]2D5 +87 (c
0.85 in water), (lit.,60 +118, c 0.2 in water); dH (D2O) 3.40 (1H,
dd, J6a,6b 11.9, J6a,5 6.0, 6a-H), 3.46 (1H, dd, J6b,6a 11.9 and J6b,5
5.3, 6b-H), 3.64 (1H, app. br. d, J 1.3, 4-H or 3-H), 3.70 (1H, app.
4
br. t, J 5.6, 5-H), 3.82 (1H, ddd, J2,1 2.5, J2,3 ~ 1.8 and J2,4 1.5,
2-H), 4.45 (1H, app. br. d, J ~ 1.8, 3-H or 4-H), 5.46 (1H, d, J1,2
2.5, 1-H); dC (D2O) 61.75 (CH2, C-6), 75.4 (CH, C-5), 76.5 (CH,
C-4 or C-3), 81.2 (CH, C-2), 83.9 (CH, C-3 or C-4), 99.6 (CH,
C-1); HRMS (ESI): found (M+Na)+ 185.0435. C6H10O5 requires
(M+Na)+, 185.0426.
J
4b¢,4 5.8, 4b’-H), 4.07 (1H, obscured ddd, J5,6b 5.8, J5,6a 5.0 and J5,4
Methyl 2,3,6-tri-O-benzyl-4-deoxy-4-C-(methylene)-a-D-
xylohexopyranoside 19
2.3, 5-H), 4.07 (1H, obscured dd, J3,2 10.3 and J3,4 5.8, 3-H), 4.58
(2H, s, CHAHBPh), 4.66 (1H, d, J1,2 4.0, 1-H), 4.67 and 4.83 (AB,
each 1H, d, JA,B 12.1, CHAHBPh), 4.72 and 4.78 (AB, each 1H,
d, JA,B 11.6, CHAHBPh), 7.28–7.39 (15H, m, 3xPh); dC (CDCl3)
43.6 (CH, C-4), 55.2 (CH3, -OCH3), 57.9 (CH2, C-4¢), 67.9 (CH,
C-5), 70.5 (CH2, C-6), 73.1, 73.4, 73.7 (each CH2, 3xCH2Ph), 76.6
(CH, C-2), 78.6 (CH, C-3), 98.7 (CH, C-1), 127.67,127.73, 127.8,
127.9, 128.0, 128.36, 128.41, 128.5 (all CH, 3xPh), 137.4, 138.2,
138.3 (each C, 3xPh); HRMS (ESI): found (M+Na)+ 501.2227.
C29H34O6 requires (M+Na)+ 501.2253.
A solution of n-butyl lithium 1.6 M in hexanes (2.30 ml;
3.68 mmol) was added dropwise to a suspension of methylt-
riphenylphosphonium bromide (1.32 g, 3.61 mmol) in dry THF
(15 ml) maintained under atmosphere of N2 at -60 ◦C. After
stirring for 1 h, a solution of methyl 2,3,6-tri-O-benzyl-a-D-xylo-
4-hexulopyranoside 18 (1.13 g, 2.45 mmol) in dry THF (5 ml)
was added dropwise. After stirring for 30 min at -50 ◦C, stirring
was continued for 15 h at rt. The solution was poured into
aqueous saturated solution of NH4Cl (20 ml) and the resulting
mixture extracted with Et2O (3 ¥ 20 ml). The organic layer
was dried (MgSO4) and concentrated in vacuo. Silica gel column
chromatography, pet. ether-EtOAc (from 99 : 1 to 1 : 1), yielded 19
as colourless syrup (891 mg, 79%). Rf 0.38 (hexanes-EtOAc, 4 : 1);
[a]2D5 +58.5 (c 1.1 in CHCl3), (lit.,77 +59.2◦, c 1 in CHCl3); vmax
(CHCl3)/cm-1 2907 s (CH), 2873 s (OC–H), 1951w, 1880w, 1812w,
Methyl 2,3,6-tri-O-benzyl-4-C-(methyl)-a-D-glucopyranoside
21. Compound 21 was a by-product in the synthesis of 20, It
was separated by silica column chromatography (EtOAc-hexanes,
2 : 3) as colourless oil (139 mg, 26%). Rf 0.38 (hexanes-EtOAc,
3 : 2); [a]2D5 +12.6 (c 1.08 in CHCl3), (lit.,77 +103.2, c 1 in
CHCl3); vmax (CHCl3)/cm-1 3518 m br (hydrogen bonded OH in
6-membered ring), 2911 s (CH), 2875 s (OC–H), 1951w, 1877w,
1810w, 1723w (overtone, comb, Ph), 1604w, 1496w (C–C, Ar),
1454 m sharp (CH2), 1367 m, 1122 s, 1074vs; dH (CDCl3) 1.18
(3H, s, 4-CH3), 2.52 (1H, br. s, 4-OH), 3.41 (3H, br. s, -OCH3),
3.42 (1H, obscured dd, J2,3 10.1 and J2,1 4.0, 2-H), 3.57 (1H, dd,
J6a,6b 9.8 and J6a,5 7.0, 6a-H), 3.73 (1H, dd, J6b,6a 9.8, J6b,5 5.0,
6b-H), 3.81 (1H, d, J2,3 10.1, 3-H), 3.90 (1H, dd, J5,6a 7.0 and J5,6b
5.0, 5-H), 4.53 and 4.59 (AB, each 1H, d, JA,B 11.9, CHAHBPh),
4.60 (1H, d, J1,2 4.0, 1-H), 4.63 and 4.79 (AB, each 1H, d, JA,B
12.1, CHAHBPh), 4.80 and 4.96 (AB, 1H each, d, JA,B 11.6,
CHAHBPh), 7.27–7.39 (15H, m, 3xPh); dC (CDCl3) 15.8 (CH3,
C-4¢), 55.1 (CH3, -OCH3), 68.9 (CH2, C-6), 70.8 (CH, C-5), 73.3,
73.5, 75.7 (all CH2, 3xCH2Ph), 74.2 (C, C-4), 78.5 (CH, C-2), 83.4
(CH, C-3), 98.0 (CH, C-1), 127.6, 127.7, 127.75, 127.81, 128.0,
128.39, 128.42 (all CH, Ph), 137.8, 138.2, 139.1 (all C, Ph); HRMS
(ESI): found (M·+Na)+ 501.2247. C29H34O6 requires (M·+Na)+
501.2253.
=
1720w (overtone, comb, Ph), 1655w (C C–H), 1496w (C–C, Ar),
1454 m sharp (CH2), 1354 m, 1083vs, 1049vs; dH (CDCl3) 3.45
(3H, s, -OCH3), 3.50 (1 H, dd, J2,3 9.6 and J2,1 3.8, 2-H), 3.70 (1H,
dd, J6a,6b 10.1 and J6a,5 6.1, 6a-H), 3.82 (1H, dd, J6b,6a 10.1 and
J6b,5 4.8, 6b-H), 4.34 (1H, obscured app. br. dd, J6a,5 6.1, J6b,5 4.8
4
and 4J5,4¢a ~ J5,4¢b 1, 5-H), 4.36 (1H, obscured app. br. dt, J2,3 9.6,
4
4J3,4¢a ~ J3,4¢b 1, 3-H), 4.59 and 4.64 (AB, each 1H, d, JA,B 11.9,
CHAHBPh), 4.69 and 4.87 (AB, each 1H, d, JA,B 12.1, CHAHBPh),
4.71 (1H, d, J1,2 3.8, 1-H), 4.74 and 4.82 (AB, each 1H, d, JA,B 11.4,
4
CHAHBPh), 4.99 (1H, app. br. s, J4¢a,4¢b ~ 2 and 4J4¢a,5 ~ J4¢a,3 1, 4¢a-
4
H), 5.38 (1H, ddd, J4¢b,4¢a 2 and 4J4¢b,5 ~ J4¢b,3 1, 4¢b-H), 7.26–7.42
(15H, m, 3xPh); dC (CDCl3) 55.3 (-OCH3), 67.7 (CH, C-5), 69.6
(CH2, C-6), 73.5, 73.6, 73.9 (each CH2, 3xCH2Ph), 79.2 (CH, C-
3), 81.5 (CH, C-2), 98.8 (CH, C-1), 107.7 (C=CH2), 127.6, 127.72,
127.74, 128.0, 128.35, 128.38 (all CH, 3xPh), 137.9, 138.3, 138.5
(each C, 3xPh), 142.6 (C=CH2); HRMS (ESI): found (M+Na)+
483.2126. C29H32O5 requires (M+Na)+ 483.2147.
Methyl 2,3,6-tri-O-benzyl-4-deoxy-4-C-(hydroxymehtyl)-a-D-
glucopyranoside 22. Compound 22 was a by-product in the
synthesis of 20, It was separated by silica column chromatography
(EtOAc-hexanes, 2 : 3) as colourless oil (22.8 mg, 4%). Rf 0.14
(hexanes-EtOAc, 3 : 2); dH (CDCl3) 1.70 (1H, dd, J4¢-OH,4¢a ~ J4¢-OH,4¢b
6.3, 4¢-OH), 1.87 (1H, dddd, J4,3 ~ J4,5 10.6 and J4,4¢a ~ J4,4¢b 3.5,
Methyl 2,3,6-tri-O-benzyl-4-deoxy-4-C-(hydroxymehtyl)-a-D-
galactopyranoside 20
To
a
solution of methyl 2,3,6-tri-O-benzyl-4-deoxy-4-C-
(methylene)-a-D-xylohexo-pyranoside 19 (516 mg; 1.12 mmol) in
1600 | Org. Biomol. Chem., 2010, 8, 1596–1602
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4-H), 3.38 (3H, s, -OCH3), 3.52-3.61 (1H, obscured m, 4¢a-H),
3.61 (1H, obscured dd, J2,3 9.3 and J2,1 3.5, 2-H), 3.64 (2H,
coincident d, J 3.5, 6a-H and 6b-H), 3.70 (1H, ddd, J4¢b,4¢a 11.9,
1-H); dC (D2O) 34.0 (CH, C-4), 57.3 (CH2, C-4¢), 62.1 (CH2, C-6),
74.07 (CH, C-5 or C-3), 74.13 (CH, C-3 or C-5), 75.1 (CH, C-2),
92.4 (CH, C-1); HRMS (ESI): found (M+Na)+ 199.0592, C7H12O5
requires (M+Na)+ 199.0582.
J
4¢b,4¢-OH 6.3 and J4¢b,4 3.5, 4¢b-H), 3.84 (1H, ddd, J5,4 10.6, J5,6a ~ J5,6b
3.5, 5-H), 3.91 (1H, dd, J3,4 10.6 and J3,2 9.3, 3-H), 4.49 and 4.63
(AB, each 1H, d, JA,B 11.9, CHAHBPh), 4.68 and 4.79 (AB, 1H
each, d, JA,B 11.9, CHAHBPh), 4.68 (1H, obscured d, J1,2 3.5, 1-H),
4.69 and 5.01 (AB, each 1H, d, JA,B 11.4, CHAHBPh), 7.30–7.38
(15H, m, 3xPh); dC (CDCl3) 46.0 (CH, C-4), 55.2 (-OCH3), 59.5
(CH2, C-4¢), 68.2 (CH, C-5), 70.5 (CH2, C-6), 72.9, 73.5, 75.2 (all
CH2, 3xCH2Ph), 75.4 (CH, C-3), 81.5 (CH, C-2), 98.4 (CH, C-1),
127.8, 127.9, 128.1, 128.39, 128.43, 128.46, 128.6 (all CH, 3xPh),
137.7, 138.2, 138.5 (each C, 3xPh); HRMS (ESI): found (M+Na)+
501.2235. C29H34O6Na requires (M+Na)+ 501.2253.
Acknowledgements
We would like to thank Michael R. McNeil at Colorado State
University for initial inhibition experiments. A.S.-K. would like to
thank the Iranian Government for funding.
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