Exploring the synthetic potency of the first furanothioglycoligase through original remote activation

Article abstract of DOI:10.1039/c1ob06227a

Thioglycosidic bonds are of utmost importance in biomolecules as their incorporation led to more stable glycomimetics with potential drug activities. Until now only chemical methods were available for their incorporation into glycofuranosyl conjugates. Herein, we wish to describe the use of the first furanothioglycoligase for the preparation of a great variety of thioaryl derivatives with moderate to excellent yields. Of great interest, a stable 1-thioimidoyl arabinofuranose, classically used in chemical glycosylation, was able to efficiently act as a donor through an original enzymatic remote activation mechanism. Study of the chemical structure as well as the nucleophilicity of the thiol allowed us to optimize this biocatalyzed process. As a consequence, this mutated enzyme constitutes an original, mild and eco-friendly method of thioligation. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c1ob06227a

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PAPER
Exploring the synthetic potency of the first furanothioglycoligase through
original remote activation†
Me´lanie Almendros,a,b Dantcho Danalev,a,b Marc Franc¸ois-Heude,a,b Pascal Loyer,c Laurent Legentil,a,b
Caroline Nugier-Chauvin,a,b Richard Daniellou*a,b and Vincent Ferrie`res*a,b
Received 22nd July 2011, Accepted 20th September 2011
DOI: 10.1039/c1ob06227a
Thioglycosidic bonds are of utmost importance in biomolecules as their incorporation led to more
stable glycomimetics with potential drug activities. Until now only chemical methods were available for
their incorporation into glycofuranosyl conjugates. Herein, we wish to describe the use of the first
furanothioglycoligase for the preparation of a great variety of thioaryl derivatives with moderate to
excellent yields. Of great interest, a stable 1-thioimidoyl arabinofuranose, classically used in chemical
glycosylation, was able to efficiently act as a donor through an original enzymatic remote activation
mechanism. Study of the chemical structure as well as the nucleophilicity of the thiol allowed us to
optimize this biocatalyzed process. As a consequence, this mutated enzyme constitutes an original, mild
and eco-friendly method of thioligation.
Introduction
Carbohydrates play an important part in a vast array of biological
processes and glycomimetics are currently becoming a powerful
class of novel therapeutics.1 Amongst them, thioglycosides, in
which a sulfur atom has replaced the glycosidic oxygen atom,
are tolerated by most biological systems and are less sensitive
to acid/base or enzyme-mediated hydrolysis. Such compounds
have already been demonstrated to be valuable tools as good
chemical donors for synthetic purpose,2,3 as stable intermediates
Fig. 1 General mechanism of the furanothioglycoligase.
in X-ray crystallographic analysis of proteins4 and, of particular
the literature, although the corresponding mutated furanosidases
interest, as competitive inhibitors of a wide range of glycosidases
have been easily obtained.13,14 In that case the scientific key seems
involved in numerous diseases.5 Their methods of preparation
to rely on the chemical synthesis of both furanosyl substrates. Still,
have recently been reviewed.6 One promising strategy relies on
glycofuranosyl-conjugates are widespread in nature and, notably,
the use of eco-friendly and versatile enzymes. Thioglycoligases,7
are present in numerous pathogens responsible for global threats
mainly developed by the team of Withers, represent mutant
such as tuberculosis, leishmaniosis, leprae, etc.15,16 In addition,
retaining glycosidases with low hydrolytic activities that are able
they are completely absent from mammals, except in the case of
to efficiently perform the synthesis of S-linked oligosaccharides
the D-ribose and 2-deoxy-D-ribose in nucleic acids, and thus can
and thioglycoproteins.8–12 Since they are lacking the catalytic
constitute original molecular structures for the development of
acid/base amino acid residue (Fig. 1), such enzymes generally
drugs or immunostimulating additives in vaccines.17–20
required particular glycosyl donors owning good leaving groups
As a consequence, we were interested in demonstrating the
like dinitrophenyl derivatives, for example, and deoxythio sugars
thioglycoligase potency of the E173A mutant of the a-L-
as better nucleophilic acceptors. However, up to now, only
arabinofuranosidase Araf 51 from Clostridium thermocellum14 for
examples related to pyranosyl hydrolases have been described in
the preparation of thiodisaccharides (Fig. 1). First, the prereq-
uisite was the chemical synthesis of both a stable furanosyl
aEcole Nationale Supe´rieure de Chimie de Rennes, CNRS, UMR 6226, Av-
donor owning a better leaving group and various thiol acceptors.
Secondly, enzymatic reactions were performed and helped us in
gaining insights into the substrate specificity and the mechanism
of the biocatalyzed process. Finally, application of this enzymatic
procedure allowed us to synthesize various S-arabinofuranosides,
thus revealing an efficient and general method of ligation of sugars
onto thiophenol derivatives.
enue du Ge´ne´ral Leclerc, CS 50837, 35708, Rennes Cedex 7, France. E-mail:
richard.daniellou@ensc-rennes.fr; Fax: +33-2-23-23-80-46; Tel: +33-2-23-
23-80-96
bUniversite´ Europe´enne de Bretagne, France
cInserm UMR S-991, Liver Metabolisms and Cancer, Universite´ de Rennes
1, 35033, Rennes, France
† Electronic supplementary information (ESI) available: Spectra of com-
pounds 7, 10, 11, 12, 13, 14 and 15. See DOI: 10.1039/c1ob06227a
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Interestingly, 3 displays a similar conformation as the arabinofura-
nosyl moiety of the arabinotrioside present in the -1 subsite in the
crystal structure previously reported (Fig. 3).14 As expected, the
aglycone undergoes hydrophobic stacking with Trp-178 in the +1
subsite. More interestingly, this thioimidoyl leaving group is axial
to the nucleophile Glu-292 that is poised for in-line nucleophilic
Results and discussion
The arabinofuranosidase Araf 51 is a thermostable glycosidase
able to catalyze the release of L-arabinose from a range of plant
polysaccharides. Its substrate specificity as well as its structural
determination by X-ray analysis have already been published.14
Recently, this native enzyme has also been demonstrated to be very
efficient in promoting auto-condensation reactions, thus leading
to a great variety of oligofuranosides.18 The E173A mutant of this
biocatalyst was overexpressed in our laboratory using common
procedures previously described14 and easily purified by heating
the mixture for one minute at 70 ◦C to precipitate out all other
proteins. This procedure afforded us tens of milligrams of protein.
Then, we focused our attention on the chemical synthesis of a
convenient glycosyl donor as well as numerous acceptors.
˚
attack (2.9 A). Similarly, the hydrogen of the OH-2 of the
arabinofuranosyl moiety interacts with the nucleophile O2 atom of
˚
Glu-292 (1.8 A). In addition, the NH group of the benzimidazoyl
aglycone also interacts with the other nucleophile O1 atom of
˚
the same glutamate residue (2.2 A). Consequently, enzymatic
remote activation of the leaving group seems to occur directly
with Glu-292 present in the active site, i.e. the nucleophile residue
commonly responsible for the formation of the glycosyl–enzyme
intermediate. To our knowledge, such activation constitutes the
first report of an enzymatic remote activation of a stable donor
in glycosidases. More importantly, as all the members of this
class of enzymes working with retention of configuration share
a common mechanism,‡ such results pave the way for the further
development of thioimidoyl furanoses and pyranoses, classically
used in chemical glycosylation, in biocatalyzed reactions.
Chemical synthesis of donors and acceptors
p-Nitrophenyl glycosides like 1 are well known as synthetic
substrates of glycosidases and, as a consequence, most of them
are commercially available (Fig. 2). On the contrary, thiogly-
coligases require glycosyl donors with good leaving groups that
do not need acid catalysis, for example, dinitrophenyl groups,
to allow formation of the glycosyl-enzyme intermediate (Fig.
1). Unfortunately, in the case of furanosides, the preparation
of dinitrophenyl- or chloronitrophenyl L-arabinofuranosides 2
involves tedious reactions and leads to unstable water-sensitive
compounds.13,21 Moreover, such chemical procedures were hardly
reproducible and allowed us to produce these compounds only in
very low yields. In order to overcome this problem, unprotected
1-thioimidoyl arabinofuranoses like 3 were thought to represent
good donors since i) they proved to be very stable in water (no
degradation was observed by NMR in 2 days), and ii) upon remote
activation, their aglycone constitutes good leaving groups which
enhanced their reactivity in the glycosylation process.22–24
Fig. 3 Docking of 3 in the active site of the acid/base mutant of Araf 51.
With this original potential enzymatic donor in hand, we turned
our attention to the chemical synthesis of the 5-deoxy-5-thio-
a-L-arabinofuranosides 7 (Scheme 1). Two kinds of derivatives
were considered: the methyl 7a for its ease of preparation and
the benzyl 7b or the p-methoxyphenyl 7c since such groups
are known to be good mimics of sugar entities and they can
be easily visualized using standard analytical methods. Starting
from readily accessible protected methyl arabinofuranoside 4,25
a simple Zemplen deprotection gave us 5a in quantitative yield.
Alternatively, the sugar 4 was also activated by acetolysis and
the desired aromatic groups were introduced using common
glycosylation reactions promoted by a borontrifluoride–etherate
complex and the corresponding alcohols. Zemplen deprotection
afforded us 5b–c with 63% and 71% overall yield, respectively.
Then, Mitsunobu conditions were used in order to substitute the
Fig. 2 Substrates of the wild-type arabinofuranosidase 1 and the
thioglycoligase mutant 2–3.
On this basis, we thus envisioned that 3 should be well-
recognized by Araf 51 and might act as an efficient glycosyl donor
for enzymatic reactions, eventually through remote activation of
the aglycone thanks to an acidic residue close to the active site.
The biocatalyzed hydrolysis of 3 in the presence of the wild-
type Araf 51 was followed by 1H NMR spectroscopy in D2O
(data not shown). The spectra rapidly exhibited the appearance
of signals corresponding to those of the anomeric protons of
L-arabinose, thus confirming the substrate potency of the 1-
thioimidoyl arabinofuranose. This was unambiguously confirmed
using a competitive assay in the presence of 1 (1 mM) at pH =
7.0, a concentration equal to 0.15 mM of 3 being necessary to
prevent 50% of the hydrolysis of the former. Docking experiments
were further performed in order to assess the role of the aglycone.
‡ See the Carbohydrate Active Enzyme Database at www.cazy.org
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Therefore, a set of commercially available thiols§ (50 eq.), both
alkyl and aryl, were chosen based on the pKa of the thiol moiety
(Table 1). As envisioned, octane thiol, owning the highest pKa,
was totally unreactive in our conditions (entry 2). Interestingly, all
aryl thiols were able to act as substrates and led to compounds
11–18 with moderate to excellent yields (50–98%, entries 4–12).
The only exception was the benzyl mercaptan (entry 3), which,
unsurprisingly with respect to its pKa of 9.43, led to only 5% yield
of the thioglycoside 10. However, the ligation reaction seems to be
general for aryl thiols of pKa between 8.75 to 5.83. In our case,
the best result was obtained with the thiophenol as emphasized by
the 98% yield for derivative 15 (entry 8). Substituents like chlorine
or a methyl group in the ortho, meta and para position did not
prevent the function of the biocatalyst (entries 5–6 and 10–11 for
examples). In addition, the enzyme was able to tolerate many other
chemical functions (OH, NH2, CO2H) and was shown to be fully
selective for the creation of the thioglycosidic linkage (entries 4, 6
and 12). It should also be noted that DTT (pKa of 9.2 and 10.1)
doesn’t seem to participate in the reaction since we never observed
its incorporation in the product. Consequently the E173A mutant
of the Araf 51 constitutes an original and powerful method to
create a new thioglycosidic bond using mild and eco-compatible
conditions.
Scheme 1 Chemical synthesis of 5-deoxy-5-thio-a-L-arabinofuranosides:
i) MeoNa (0.01 M), MeOH, rt: 5a (100%), 7a (100%), 7b (94%), 7c (98%);
ii) a) Ac2O, cat. H2SO4, CH2Cl2, rt, b) BF3–OEt2 (3.1 eq.), Et3N (0.08 eq.),
alcohol (1.2 eq.), CH2Cl2, rt, 24 h: 5b (71%), 5c (63%); iii) a) thioacetic
acid (1.8 eq.), PPh3 (1.7 eq.), DIAD (1.7 eq.), THF, rt, overnight, b)
Ac2O/pyridine (1/1), rt, 3 h: 6a (69%), 6b (74%), 6c (74%).
primary alcohol with a thioacetyl function. After concentration
and filtration on a silica pad, the resulting crude compounds were
directly peracetylated to facilitate their purification. Compounds
6a–c, obtained with 69 to 74% yields, were stored at -20 ◦C
and finally deprotected using sodium methoxide to give 7 just
before enzymatic reaction in order to prevent the formation of
their corresponding disulfide. The absence of the latter was easily
verified by checking the chemical shift of the 13C NMR signal
corresponding to C-5, around 27 ppm for the thiol and 43.5 ppm
for its disulfide counterpart.
STD-NMR
Biocatalyzed-thioligation
In addition, no inhibition was observed when performing a com-
petitive assay with both benzyl and phenyl mercaptan. Compound
10, even with a ratio up to 1 : 10 (donor 3 : 10), was also not able to
inhibit the enzymatic reaction thus ruling out a possible product
inhibition. Therefore, one question was still present in this study:
is the benzyl mercaptan simply less reactive than the thiophenol
or does the Araf 51 E173A recognize it less? Saturation Transfer
Difference (STD) NMR experiments were performed in order
to solve this problem.27 This technique allowed us to determine
rapidly the binding epitopes of the thioglycosides 10 and 15 (Fig.
4). Normally, an STD value of 100% is arbitrarily attributed to
the highest peak. Here, as it corresponded to the peak of the 5
protons of the aromatic, a value of 500% was given. As depicted
in Fig. 4, the S-phenyl arabinofuranoside 15 revealed to be tightly
bound to the enzyme. Moreover, the aglycon seemed to be as
well recognized by the protein as the sugar moiety, emphasizing
once again the importance of the hydrophobic stacking with Trp-
178 in the +1 subsite. Similar values were obtained for the S-
benzyl arabinofuranoside 10. This simple observation led us to
the conclusion that the recognition does not represent a major
problem but more surely that the benzyl mercaptan is less reactive
than the thiophenol during this biocatalyzed procedure.
The thioimidoyl donor 3 was then allowed to react with the
various 5-deoxy-5-thio-arabinofuranosides 7a–c (2 eq.) in the
presence of the thioglycoligase (Scheme 2) and dithiothreitol (1.6
eq., DTT). Experiments were performed in 50 mM phosphate
buffer at pH = 8.0 and 37 ◦C. Reactions were monitored by
thin layer chromatography until complete consumption of the
donor (24 h). The resulting compounds were purified by flash
column chromatography and subsequently analyzed by NMR
spectroscopy.
Scheme 2 Biocatalyzed synthesis of thiofuranosides 8–19.
Unexpectedly, in all three cases, we were not able to detect the
formation of the desired thioglycoside 8. In fact, 3 was totally
converted into its hydrolysis product (L-arabinopyranose) and
the thio-sugars 7 were partially oxidized as disulfides. These
observations led us to worry about i) the recognition of the
carbohydrates 7 by the active-site of the mutated enzyme and
ii) more importantly, their reactivity as nucleophiles towards the
ligation reaction. Since 7 represent slightly modified substrates
compared to the natural ones, we envisioned the second point to
be the major problem. Taking into account results obtained in the
native chemical ligation reaction of peptides,26 our hypothesis was
that aryl thiols constitute better nucleophiles than carbohydrates
7 and might act as good acceptors in this biocatalyzed ligation
reaction.
Fig. 4 S-Bn and S-ph arabinofuranosides 10 and 15. STD effects
observed by NMR are indicated in italics for each proton.
§ First reactions were performed with 2 eq. but yields were limited to 30%.
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Table 1 Thiols tested in the thioligase reaction
potential drug activities. In addition to well-known chemical
procedures, we have developed an original method using the
first furanothioglycoligase. As a prerequisite, both donors and
acceptors were prepared using standard chemical reactions with
good overall yields. Interestingly, thioimidoyl glycosides like 3
were shown to represent a promising new class of stable substrate
that can undergo remote activation inside glycosidases. As they
are easily available, their further use even in the pyranose form
will with undoubtedly help us in increasing the synthetic potency
of new thioglycoligases. Moreover, even if unreactive in our
case, three different 5-deoxy-5-thio furanosides are now available
for such studies. Even more importantly, insights were also
gained on the detailed mechanism of this particular mutated
enzyme. In total accordance with what was previously described,
Araf 51 constitutes a tremendous synthetic tool due to its great
versatility toward the acceptor recognition subsite i.e. furanosides,
pyranosides, aryl, thioaryl, etc. In addition, the nucleophilicity of
the thiols seems to represent the major parameter that influences
the yield of the biocatalyzed reaction. Experiments are currently in
progress to extend this enzymatic thioligation to more challenging
biological objects.
Entry
1
R2-SH
pKa
Product (yield%)
10.60a
8(—)
2
3
10.00
9.43
9(—)
10(5)
4
5
8.75
6.81
11(60)
12(50)
6
7
6.79
6.75
13(52)
14(81)
Experimental
General procedures
Thin layer chromatography (TLC) analyses were conducted on E.
Merck 60 F254 Silica Gel non activated plates and compounds
were visualized by UV-absorption at 254 nm, by dipping in
a 5% solution of orcinol in 5% solution of H2SO4 in EtOH
followed by heating. Preparative chromatography purifications
were performed on Geduran Si 60 (40–63 mm) Silica Gel. Optical
rotations were measured on a Perkin–Elmer 341 polarimeter.
NMR spectra were recorded with a Bru¨ker ARX 400 spectrometer
8
9
6.61
6.36
15(98)
16(67)
1
at 400 MHz for H and 100 MHz for 13C. Chemical shifts are
given in d-units (ppm) measured from the solvent signal. Coupling
constants J were calculated in hertz (Hz). Abbreviations were used
to present signal multiplicity: s (singlet), d (doublet), t (triplet), m
(multiplet), dd (doublet doublet). Mass spectra were measured
with a MS/MS ZabSpec TOF Micromass using m-nitrobenzylic
alcohol as a matrix and accelerated caesium ions for ionization.
10
6.11
17(77)
11
12
5.83
5.83
18(73)
19(84)
Docking experiment
Rotatable bonds in the ligand were assigned with Autodock
Tools – an accessory program that allows the user to interact with
Autodock. Docking simulations were performed with AutoDock
4.2 using a Lamarckian genetic algorithm. The standard docking
procedure was used for a rigid protein and a flexible ligand
whose torsion angles were identified (for 100 independent runs per
ligand). A grid of 60, 60 and 60 points in x, y, and z directions was
built, centered on the nucleophile residue Glu-292. A grid spacing
a Estimated value by comparison with methyl and ethyl mercaptan.
˚
of 0.375 A and a distance-dependent function of the dielectric
constant were used for the calculation of the energetic map. The
default settings were used for all parameters. At the end of docking,
the ligand with the most favorable free energy of binding was
selected as the resultant complex structure. All calculations were
carried out on Apple based machines using Mac Os X.6. The
Conclusions
Thioglycosidic bonds are of utmost importance in biomolecules
as their incorporation leads to more stable glycomimetics with
8374 | Org. Biomol. Chem., 2011, 9, 8371–8378
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resultant structure file was analyzed using Pymol 1.3 visualization
programs.
allowed to warm up to room temperature and stirred overnight
under nitrogen. After concentration in vacuo, the residue was
suspended in EtOAc (50 mL) before filtration through a silica
pad. The resulting filtrate was concentrated and dissolved in
dry pyridine (5 mL). Acetic anhydride (5 mL) was added and
the mixture was stirred at room temperature for 3 h. After
concentration in vacuo and co-distillation with toluene (3 ¥ 15 mL),
the resulting oil was diluted with EtOAc (100 mL) and successively
washed with 5% aqueous HCl (3 ¥ 20 mL), saturated solution of
aqueous NaHCO3 (3 ¥ 20 mL) and water (3 ¥ 20 mL). After drying
over MgSO4 and concentration in vacuo, the residue was purified
by column chromatography on silica gel (95 : 5 toluene/EtOac) to
afford 6.
General procedure for the Zemplen deprotection
The peracetylated carbohydrate (0.15 mmol) was dissolved in
MeOH (3 mL) and 0.1 M MeONa in MeOH (2.7 eq) was added.
The mixture was stirred at room temperature for 15 min. After
neutralization with amberlite IR-120 (H+ form), the resin was
filtered off and the MeOH removed under pressure. The residue
was finally purified by column chromatography on silica gel (9 : 1
CH2Cl2/MeOH) to afford 5a–c or 7a–c.
Benzyl a-L-arabinofuranoside (5b)
Methyl 2,3-di-O-acetyl-5-deoxy-5-thioacetyl-a-L-
arabinofuranoside (6a)
To a solution of 1-O-acetyl-2,3,5-tri-O-benzoyl-a-L-arabino-
furanoside (4.0 g, 7.82 mmol) in anhydrous dichloromethane
(40 mL) were successively added BF3·OEt2 (2.9 mL, 24.5 mmol)
and Et3N (0.54 mL, 0.62 mmol) at 0 ◦C. After stirring for 10 min,
benzyl alcohol (9.28 mmol) was added. The reaction mixture was
stirred for 24 h at room temperature, then diluted with water
(80 mL) and extracted with dichloromethane (150 mL). The
organic layer was washed with 1 N aqueous HCl (3 ¥ 40 mL)
followed by a saturated solution of aqueous NaHCO3 (3 ¥ 40 mL).
After drying over MgSO4 and concentration, the residue was
deprotected using the general Zemplen procedure to afford 5b
as a colourless oil (1.3 g, 71%): [a]2D0 -12.3 (c 1.00, MeOH); Rf 0.33
(9 : 1 CH2Cl2/MeOH);1H NMR (CD3OD): d 7.38–7.25 (m, 5H, H
arom), 4.94 (d, 1H, J1,2 = 1.5 Hz, H-1), 4.75 (d, 1H, J = 11.9 Hz,
CH2 benzyl), 4.53 (d, 1H, CH2 benzyl), 4.03 (dd, 1H, J2,3 = 3.8 Hz,
H-2), 3.99 (ddd, 1H, J3,4 = 6.4 Hz, J4,5a = 3.1 Hz, J4,5b = 5.5 Hz,
H-4), 3.86 (dd, 1H, H-3), 3.78 (dd, 1H, J5a,5b = 12.0 Hz, H-5a), 3.66
(dd, 1H, H-5b); 13C NMR (CD3OD): d 139.3 (C-1¢), 129.3 (C-2¢,
C-6¢), 129.0 (C-4¢), 128.6 (C-3¢, C-5¢), 108.5 (C-1), 85.5 (C-4), 83.7
(C-2), 78.9 (C-3), 70.0 (CH2 benzyl), 63.0 (C-5); HRESIMS: Calcd
for C12H16NaO5 [M + Na]+: m/z 263.0667; found: m/z 263.0666.
Colourless oil (0.78 g, 69%): [a]2D0 -65.5 (c 0.95, CHCl3); Rf 0.33
(9 : 1 toluene/EtOac);1H NMR (CDCl3): 5.01 (dd, 1H, J2,3
=
2.0 Hz, H-2), 4.89 (dd, 1H, J3,4 = 5.7 Hz, H-3), 4.87 (d, 1H, H-1),
4.17 (ddd, 1H, J4,5a = 4.9 Hz, J4,5b = 6.9 Hz, H-4), 3.77 (s, 3H,
OCH3), 3.38 (dd, 1H, J5a,5b = 13.9 Hz, J4,5a = 2.6 Hz, H-5a), 3.18
(dd, 1H, H-5b), 2.36 (s, 3H, SCOCH3), 2.11 (s, 3H, OCOCH3),
2.10 (s, 3H, OCOCH3); 13C NMR (CDCl3): d 194.8 (SCOCH3),
170.2 (OCOCH3), 169.9 (OCOCH3), 106.7 (C-1), 81.8 (C-2), 80.5
(C-4), 79.3 (C-3), 55.1 (OCH3), 31.3 (C-5), 30.6 (SCOCH3), 20.9
(2x OCOCH3); HRESIMS: Calcd for C12H18NaO7S [M + Na]+:
m/z 329.0670; found: m/z 329.0668.
Benzyl 2,3-di-O-acetyl-5-deoxy-5-thioacetyl-a-L-
arabinofuranoside (6b)
Colourless oil (1.1 g, 74%): [a]2D0 -55.3 (c 0.88, CHCl3); Rf 0.45
(95 : 5 toluene/EtOac);1H NMR (CDCl3): 7.28–7.18 (m, 5H, H
arom), 5.04 (dd, 1H, J2,3 = 2.0 Hz, H-2), 4.98 (d, 1H, H-1), 4.84
(dd, 1H, J3,4 = 5.6 Hz, H-3), 4.67 (d, 1H, J = 12.1 Hz, CH2 benzyl),
4.46 (d, 1H, CH2 benzyl), 4.14 (ddd, 1H, J4,5a = 4.9 Hz, J4,5b
=
4¢-Methoxyphenyl a-L-arabinofuranoside (5c)
6.9 Hz, H-4), 3.31 (dd, 1H, J5a,5b = 13.8 Hz, H-5a), 3.10 (dd, 1H,
H-5b), 2.28 (s, 3H, SCOCH3), 2.02 (s, 3H, OCOCH3), 2.01 (s,
3H, OCOCH3); 13C NMR (CDCl3): d 195.9 (SCOCH3), 170.2
(OCOCH3), 169.8 (OCOCH3), 137.3 (C-1¢), 128.5 (C-2¢, C-6¢),
128.4 (C-4¢), 127.9 (C-3¢, C-5¢), 104.8 (C-1), 81.9 (C-2), 80.7 (C-4),
79.3 (C-3), 69.0 (CH2 benzyl), 31.3 (C-5), 30.6 (SCOCH3), 20.9
(2x OCOCH3); HRESIMS: Calcd for C18H22NaO7S [M + Na]+:
m/z 405.0983; found: m/z 405.0983.
Compound 5c was synthesized as previously described for 5b
using 4-methoxyphenol (9.28 mmol). White solid (1.3 g, 63%):
[a]2D0 -131.4 (c 1.00, MeOH); Rf 0.33 (9 : 1 CH2Cl2/MeOH);1H
NMR (CD3OD): d 6.97 (d, 2H, J = 9.2 Hz, H-2¢, H-6¢), 6.80 (d,
2H, H-3¢, H-5¢), 5.38 (d, 1H, J1,2 = 2.0 Hz, H-1), 4.19 (dd, 1H,
J2,3 = 4.2 Hz, H-2), 4.04 (ddd, 1H, J3,4 = 6.5 Hz, J4,5a = 3.1 Hz,
J4,5b = 5.1 Hz, H-4), 3.94 (dd, 1H, H-3), 3.76 (dd, 1H, J5a,5b
=
12.1 Hz, H-5a), 3.73 (s, 3H, OCH3), 3.64 (dd, 1H, H-5b); 13C
NMR (CD3OD): d 156.4 (C-4¢), 152.4 (C-1¢), 119.2 (C-2¢, C-6¢),
115.4 (C-3¢, C-5¢), 108.7 (C-1), 85.9 (C-4), 83.7 (C-2), 78.2 (C-3),
62.8 (C-5), 56.0 (OCH3); HRESIMS: Calcd for C12H16NaO6 [M +
Na]+: m/z 279.0844; found: m/z 279.0848.
4¢-Methoxyphenyl 2,3-di-O-acetyl-5-deoxy-5-thioacetyl-a-L-
arabinofuranoside (6c)
Colourless oil (1.1 g, 74%): [a]2D0 -94.1 (c 1.00, CHCl3); Rf 0.44
(95 : 5 toluene/EtOac);1H NMR (CDCl3): 6.97 (d, 2H, J = 9.2 Hz,
H-2¢, H-6¢), 6.82 (d, 2H, H-3¢, H-5¢), 5.51 (d, 1H, H-1), 5.30
(dd, 1H, J2,3 = 2.2 Hz, H-2), 5.00 (dd, 1H, J3,4 = 5.5 Hz, H-3),
4.37 (ddd, 1H, J4,5a = 5.2 Hz, J4,5b = 6.5 Hz, H-4), 3.77 (s, 3H,
OCH3), 3.38 (dd, 1H, J5a,5b = 14.1 Hz, H-5a), 3.22 (dd, 1H, H-
5b), 2.36 (s, 3H, SCOCH3), 2.14 (s, 6H, 2xOCOCH3); 13C NMR
(CDCl3): d 194.8 (SCOCH3), 170.2 (OCOCH3), 169.8 (OCOCH3),
155.4 (C-4¢), 150.2 (C-1¢), 118.5 (C-2¢, C-6¢), 114.7 (C-3¢, C-5¢),
105.1 (C-1), 81.9 (C-2), 81.3 (C-4), 78.8 (C-3), 55.8 (OCH3), 31.1
General procedure for the Mitsunobu reaction and the following
peracetylation
Thioacetic acid was purified prior to its use by low-temperature
distillation under nitrogen. To a cooled solution of thioacetic
acid (1.8 eq.) and 5 (1 eq.) in dry THF (20 mL) was added a
cooled solution of triphenylphosphine (1.7 eq.) and diisopropyl
azodicarboxylate (1.7 eq.) in dry THF (20 mL). The reaction was
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Org. Biomol. Chem., 2011, 9, 8371–8378 | 8375
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(C-5), 30.6 (SCOCH3), 20.9 (2x OCOCH3); HRESIMS: Calcd for
C18H22NaO8S [M + Na]+: m/z 421.0933; found: m/z 421.0929.
pressure and the residue was purified by column chromatography
(9 : 1 CH2Cl2/MeOH) on silica gel to afford the desired alkyl/aryl
1-thio-a-L-arabinofuranoside.
Methyl 5-deoxy-5-thio-a-L-arabinofuranoside (7a)
Colourless oil (27 mg, 100%): [a]2D0 -3.1 (c 0.24, MeOH); Rf 0.70
Benzyl 1-thio-a-L-arabinofuranoside (10)
(9 : 1 CH2Cl2/MeOH);1H NMR (CD3OD): 4.75 (d, 1H, J1,2
=
Colourless oil (0.5 mg, 5%): [a]2D0 -35.0 (c 0.06, MeOH); Rf 0.3 (9 : 1
CH2Cl2/MeOH);1H NMR (CD3OD): d 7.37–7.20 (m, 5H, C6H5),
4.93 (d, 1H, J1,2 = 3.8 Hz, H-1), 4.00 (ddd, 1H, J3,4 = 7.2 Hz, J4,5b
5.4 Hz, J4,5a = 3.1 Hz, H-4), 3.91 (d, 1H, J = 12.8 Hz, CH2 benzyl),
3.90 (dd, 1H, H-2), 3.86 (dd, 1H, J2,3 = 7.2 Hz, J3,4 = 4.4 Hz, H-3),
3.81 (dd, 1H, J5a,5b = 12.1 Hz, H-5a), 3.80 (d, 1H, CH2 benzyl),
3.68 (dd, 1H, H-5b); 13C NMR (CD3OD): d 136.4 (C-1¢), 132.6
(C-2¢, C-6¢), 129.9 (C-3¢, C-5¢), 128.2 (C-4¢), 88.0 (C-1), 84.2 (C-4),
84.0 (C-2), 77.1 (C-3), 61.1 (C-5), 34.3 (CH2 benzyl); HRESIMS:
Calcd for C12H16NaO4S [M + Na]+: m/z 279.0667; found: m/z
279.0666.
1.8 Hz, H-1), 3.95 (dd, 1H, J2,3 = 4.0 Hz, H-2), 3.94 (ddd, 1H,
J3,4 = J4,5b = 6.4 Hz, J4,5a = 4.6 Hz, H-4), 3.85 (dd, 1H, H-3), 3.37
(s, 3H, OCH3), 2.80 (dd, 1H, J5a,5b = 13.9 Hz, H-5a), 2.70 (dd, 1H,
H-5b); 13C NMR (CD3OD): d 106.7 (C-1), 85.2 (C-4), 83.7 (C-2),
80.7 (C-3), 55.3 (OCH3), 27.4 (C-5).
=
Benzyl 5-deoxy-5-thio-a-L-arabinofuranoside (7b)
Colourless oil (36 mg, 94%): [a]2D0 -7.2 (c 0.07, MeOH); Rf 0.76
(9 : 1 CH2Cl2/MeOH);1H NMR (CD3OD): 7.39–7.32 (m, 5H, H
arom), 4.95 (d, 1H, J1,2 = 1.8 Hz, H-1), 4.75 (d, 1H, J = 11.9 Hz,
CH2 benzyl), 4.53 (d, 1H, CH2 benzyl), 4.06 (dd, 1H, J2,3 = 4.0 Hz,
H-2), 4.01 (ddd, 1H, J3,4 = 6.6 Hz, J4,5b = 6.4 Hz, J4,5a = 4.6 Hz,
H-4), 3.89 (dd, 1H, H-3), 2.84 (dd, 1H, J5a,5b = 13.9 Hz, H-5a),
2.73 (dd, 1H, H-5b); 13C NMR (CD3OD): d 139.2 (C-1¢), 129.3,
129.0, 128.6 (C-2¢, C-3¢, C-4¢, C-5¢, C-6¢), 108.4 (C-1), 85.1 (C-4),
83.9 (C-2), 81.0 (C-3), 70.2 (CH2 benzyl), 27.4 (C-5).
4¢-Aminophenyl 1-thio-a-L-arabinofuranoside (11)
Colourless oil (5.5 mg, 60%): [a]2D0 -113.7 (c 0.21, MeOH); Rf
0.3 (9 : 1 CH2Cl2/MeOH);1H NMR (CD3OD): d 7.29 (d, 2H, J =
8.5 Hz, H-2¢, H-6¢), 6.65 (d, 2H, H-3¢, H-5¢), 4.99 (d, 1H, J1,2
=
5.0 Hz, H-1), 3.92 (dd, 1H, J2,3 = 5.1 Hz, H-2), 3.92–3.85 (m, 2H,
H-3, H-4), 3.75 (dd, 1H, J5a,5b = 12.0 Hz, J4,5a = 2.5 Hz, H-5a), 3.61
(dd, 1H, J4,5b = 4.5 Hz, H-5b); 13C NMR (CD3OD): d 149.7 (C-4¢),
136.7 (C-2¢, C-6¢), 121.1 (C-1¢), 116.5 (C-3¢, C-5¢), 93.8 (C-1), 84.0
(C-4), 82.6 (C-2), 77.5 (C-3), 62.5 (C-5); HRESIMS: Calcd for
C11H15NaNO4S [M + Na]+: m/z 280.06195; found: m/z 280.0619.
4¢-Methoxyphenyl 5-deoxy-5-thio-a-L-arabinofuranoside (7c)
Colourless oil (40 mg, 98%): [a]2D0 -71.7 (c 0.06, MeOH); Rf 0.77
(9 : 1 CH2Cl2/MeOH);1H NMR (CD3OD): 6.88 (d, 2H, J = 9.2 Hz,
H-2¢, H-6¢), 6.73 (d, 2H, H-3¢, H-5¢), 5.27 (d, 1H, J1,2 = 2.0 Hz,
H-1), 4.12 (dd, 1H, J2,3 = 4.4 Hz, H-2), 3.98 (ddd, 1H, J3,4 = 6.6 Hz,
J4,5b = 6.2 Hz, J4,5a = 4.8 Hz, H-4), 3.87 (dd, 1H, H-3), 3.64 (s, 3H,
OCH3), 2.74 (dd, 1H, J5a,5b = 13.9 Hz, H-5a), 2.63 (dd, 1H, H-
5b); 13C NMR (CD3OD): d 156.5 (C-1¢), 152.3 (C-4¢), 119.4 (C-2¢,
C-6¢), 115.5 (C-3¢, C-5¢), 108.7 (C-1), 85.5 (C-4), 83.9 (C-2), 80.3
(C-3), 56.0 (OCH3), 27.4 (C-5).
4¢-Methylphenyl 1-thio-a-L-arabinofuranoside (12)
Colourless oil (4.5 mg, 50%): [a]2D0 -115.8 (c 0.24, MeOH); Rf 0.5
(9 : 1 CH2Cl2/MeOH);1H NMR (CD3OD): d 7.41 (d, 2H, J 8.0 Hz,
H-2¢, H-6¢), 7.14 (d, 2H, H-3¢, H-5¢), 5.20 (d, 1H, J1,2 = 4.6 Hz,
H-1), 3.97 (dd, 1H, J2,3 = 5.3 Hz, H-2), 3.96–3.92 (m, 1H, H-4),
3.93 (dd, 1H, J3,4 = 7,5 Hz, H-3), 3.77 (dd, 1H, J5a,5b = 12.1 Hz,
J4,5a = 2.6 Hz, H-5a), 3.64 (dd, 1H, J4,5b = 4.6 Hz, H-5b), 2.32
(s, 3H, CH3); 13C NMR (CD3OD): d 140.7 (C-4¢), 138.7 (C-1¢),
133.4 (C-2¢, C-6¢), 130.6 (C-3¢, C-5¢), 93.3 (C-1), 84.2 (C-4), 83.2
(C-2), 77.6 (C-3), 62.4 (C-5), 21.1 (CH3); HRESIMS: Calcd for
C12H16NaO4S [M + Na]+: m/z 279.0667; found: m/z 279.0669.
Expression and purification of Araf 51 E173A
Araf 51 E173A was produced in Escherichia coli strain BL21
DE3 cells harbouring the appropriate recombinant plasmid,14
cultured in LB (Luria–Bertani) broth containing 50 mg L-1
kanamycin at 37 ◦C. Cells were grown to mid-exponential
phase [A600 (absorbance) of 0.7], at which point isopropyl b-D-
thiogalactopyranoside (IPTG) was added to a final concentration
of 1 mM. The cultures were then incubated for a further 16 h at
37 ◦C. The cells were harvested by centrifugation at 4 ◦C for 30 min
and recombinant proteins were purified from cell-free extracts by
sonication followed by 15 min heat denaturation step at 70 ◦C. A
final centrifugation at 4 ◦C for 30 min was performed.
2¢-Hydroxyphenyl 1-thio-a-L-arabinofuranoside (13)
Colourless oil (5.0 mg, 52%): [a]2D0 +122.9 (c 0.07, MeOH); Rf 0.4
(9 : 1 CH2Cl2/MeOH);1H NMR (CD3OD): d 7.49 (dd, 1H, J5¢,6¢
7.7 Hz, J4¢,6¢ = 1.8 Hz, H-6¢), 7.18 (ddd, 1H, J3¢,4¢ = 8.2 Hz, J4¢,5¢
=
=
General procedure for the enzymatic ligation
7.2 Hz, H-4¢), 6.83 (dd, 1H, J3¢,5¢ = 1.1 Hz, H-3¢), 6.83 (ddd, 1H,
H-5¢), 5.42 (d, 1H, J1,2 = 4.6 Hz, H-1), 4.19 (dd, 1H, J2,3 = 4.2 Hz,
2¢-Benzimidazolyl 1-thio-a-L-arabinofuranoside
3 (10.0 mg,
35.4 mmol) and dithiothreitol (8.9 mg, 1.6 eq.) were dissolved
in tris(hydroxymethyl)aminomethane/HCl buffer (50 mM, pH 8,
10 mL). To the mixture were added successively at 37 ◦C the
enzyme Araf 51 E173A (0.22 mg mL-1, 45 nmol, 5 mL) freshly
warmed at 37 ◦C and the thiol/thiophenol derivative (50 eq.). The
reaction was monitored by TLC until disappearance of the donor
(generally 3 h). Then the mixture was concentrated under reduced
H-2), 4.06 (dd, 1H, H-3), 3.84 (ddd, 1H, J3,4 = 8.4 Hz, J4,5b =
4.8 Hz, J4,5a = 3.8 Hz, H-4), 3.75 (dd, 1H, J5a,5b = 11.7 Hz, H-5a),
3.70 (dd, 1H, H-5b); 13C NMR (CD3OD): d 158.8 (C-2¢), 136.3
(C-6¢), 131.0 (C-4¢), 121.3 (C-5¢), 120.6 (C-1¢), 116.6 (C-3¢), 93.0
(C-1), 87.1 (C-4), 79.5 (C-2), 78.0 (C-3), 63.2 (C-5); HRESIMS:
Calcd for C11H14NaO5S [M + Na]+: m/z 281.04597; found: m/z
281.0457.
8376 | Org. Biomol. Chem., 2011, 9, 8371–8378
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©

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4¢-Methoxyphenyl 1-thio-a-L-arabinofuranoside (14)
3.80 (dd, 1H, J5a,5b = 12.1 Hz, J4,5a = 2.6 Hz, H-5a), 3.68 (dd, 1H,
J5a,5b = 12.1 Hz, J4,5b = 4.6 Hz, H-5b); 13C NMR (CD3OD): d 139.1
(C-3¢), 135.5 (C-1¢), 131.3 (C-5¢), 131.2 (C-2¢), 130.2 (C-6¢), 128.0
(C-4¢), 92.9 (C-1), 84.6 (C-4), 83.5 (C-2), 77.6 (C-3), 62.3 (C-5);
HRESIMS: Calcd for C11H13ClNaO4S [M + Na]+: m/z 299.0121;
found: m/z 299.0123.
Colourless oil (7.8 mg, 81%): [a]2D0 -137.5 (c 0.24, MeOH); Rf
0.5 (9 : 1 CH2Cl2/MeOH);1H NMR (CD3OD): d 7.44 (d, 2H, J =
8.8 Hz, H-2¢, H-6¢), 6.85 (d, 2H, H-3¢, H-5¢), 5.03 (d, 1H, J1,2
4.6 Hz, H-1), 3.90 (t, 1H, J2,3 = 4.6 Hz, H-2), 3.90–3.86 (m, 2H,
H-3, H-4), 3.75 (s, 3H, OCH3), 3.73 (dd, 1H, J5a,5b = 12.1 Hz,
J4,5a = 2.2 Hz, H-5a), 3.59 (dd, 1H, J4,5b = 4.6 Hz, H-5b); 13C NMR
(CD3OD): d 161.3 (C-4¢), 136.2 (C-2¢, C-6¢), 125.8 (C-1¢), 115.4
(C-3¢, C-5¢), 93.6 (C-1), 84.1 (C-4), 82.9 (C-2), 77.5 (C-3), 62.4 (C-
5), 55.8 (OCH3); HRESIMS: Calcd for C12H16NaO5S [M + Na]+:
m/z 295.0616; found: m/z 295.0612.
=
4¢-Carboxyphenyl 1-thio-a-L-arabinofuranoside (19)
Colourless oil (8.5 mg, 84%): [a]2D0 -142.1 (c 0.61, MeOH); Rf 0.35
(15 : 1 AcOEt/AcOH);1H NMR (CD3OD): d 7.81 (d, 2H, J =
8.4 Hz, H-2¢, H-6¢), 7.42 (d, 2H, H-3¢, H-5¢), 5.30 (d, 1H, J1,2
=
4.6 Hz, H-1), 3.93 (dd, 1H, J2,3 = 4.6 Hz, H-2), 3.91–3.84 (m, 2H,
H-3, H-4), 3.69 (dd, 1H, J5a,5b = 12.1 Hz, J4,5a = 2.6 Hz, H-5a), 3.61
(dd, 1H, J4,5b = 4.6 Hz, H-5b); 13C NMR (CD3OD): d 131.0 (C-2¢,
C-6¢), 130.4 (C-3¢, C-5¢), 92.4 (C-1), 84.6 (C-4), 83.6 (C-2), 77.7
(C-3), 62.4 (C-5); HRESIMS: Calcd for C12H14NaO6S [M + Na]+:
m/z 286.0511; found: m/z 285.0514.
Phenyl 1-thio-a-L-arabinofuranoside (15)
Colourless oil (8.6 mg, 98%): [a]2D0 -145.2 (c 0.62, MeOH); Rf 0.5
(9 : 1 CH2Cl2/MeOH);1H NMR (CD3OD): d 7.52–7.49 (m, 2H,
H-2¢, H-6¢), 7.31–7.21 (m, 3H, H-3¢, H-4¢, H-5¢), 5.26 (d, 1H, J1,2
=
4.6 Hz, H-1), 3.98 (dd, 1H, J2,3 = 4.8 Hz, H-2), 3.97–3.93 (m,
1H, H-4), 3.91 (dd, 1H, J3,4 = 7.2 Hz, H-3), 3.77 (dd, 1H, J5a,5b
12.1 Hz, J4,5a = 2.6 Hz, H-5a), 3.64 (dd, 1H, J5a,5b = 12.1 Hz, J4,5b
=
=
STD-NMR
4.6 Hz, H-5b); 13C NMR (CD3OD): d 136.4 (C-1¢), 132.6 (C-2¢,
C-6¢), 129.9 (C-3¢, C-5¢), 128.2 (C-4¢), 93.1 (C-1), 84.3 (C-4), 83.4
(C-2), 77.6 (C-3), 62.4 (C-5); HRESIMS: Calcd for C11H14NaO4S
[M + Na]+: m/z 265.0510; found: m/z 265.0503.
Samples were prepared in 0.5 mL of D2O and contained ~30 mmol
of 10 or 15. STD-NMR spectra were recorded on a Bruker
Avance III 400 MHz spectrometer. Processing of all data was
performed on a PC with Bruker Topspin v2.0 software. After the
determination of the optimal conditions, i.e. temperature, delay
between pulse (d20) and molecular ratio (protein/ligand), STD-
NMR experiments were performed at 283 K as followed. The
protein (1 : 100 ratio) was saturated on resonance at 0.7 ppm and
off resonance at 40 ppm with a cascade of 40 selective gaussian-
shaped pulses of 50 ms duration with a 100 ms delay between each
pulse. The total duration of the saturation time was set to 2s. A
total of 256 scans/STD-NMR experiment was acquired. A WA-
TERGATE sequence was used to suppress residual HOD signal.
A spin lock filter with strength of 5 KHz and duration of 10 ms
was also applied to suppress the protein background. A similar
experiment with no enzyme was used as reference in order to
verify the absence of STD effect in these experimental conditions.
Intensities of all STD effects were calculated though integrals over
3¢-Methoxyphenyl 1-thio-a-L-arabinofuranoside (16)
Colourless oil (6.5 mg, 67%): [a]2D0 -185.2 (c 0.25, MeOH); Rf 0.5
(9 : 1 CH2Cl2–MeOH);1H NMR (CD3OD): d 7.21 (t, 1H, J4¢,5¢
8.4 Hz, H-5¢), 7.10–7.08 (m, 2H, H-2¢, H-6¢), 6.83 (dd, 1H, J4¢,6¢
1.6 Hz, H-4¢), 5.30 (d, 1H, J1,2 = 4.6 Hz, H-1), 4.00 (dd, 1H, J2,3
=
=
=
4.8 Hz, H-2), 4.00–3.95 (m, 1H, H-4), 3.93 (dd, 1H, J3,4 = 7.3 Hz,
H-3), 3.79 (dd, 1H, J5a,5b = 12.2 Hz, J4,5a = 2.8 Hz, H-5a), 3.79 (s,
3H, OCH3), 3.67 (dd, 1H, J4,5b 4.6 Hz, H-5b); 13C NMR (CD3OD):
d 161.3 (C-3¢), 137.6 (C-1¢), 130.7 (C-5¢), 124.6 (C-6¢), 117.7 (C-2¢),
113.9 (C-4¢), 93.0 (C-1), 84.4 (C-4), 83.4 (C-2), 77.7 (C-3), 62.4 (C-
5), 55.7 (OCH3); HRESIMS: Calcd for C12H16NaO5S [M + Na]+:
m/z 295.0616; found: m/z 295.0619.
1
the respective signals in H NMR reference spectra. The largest
4¢-Chlorophenyl 1-thio-a-L-arabinofuranoside (17)
STD effect in each spectrum was set to 100% and relative intensities
were determined, as common for non-refined STD effects. Hence,
sufficient comparisons of relative STD effects between sugars were
possible, but absolute binding intensities could not be determined.
Colourless oil (7.5 mg, 77%): [a]2D0 -107.6 (c 0.46, MeOH); Rf
0.4 (9 : 1 CH2Cl2/MeOH);1H NMR (CD3OD): d 7.51 (d, 2H, J =
8.7 Hz, H-2¢, H-6¢), 7.32 (d, 2H, H-3¢, H-5¢), 5.27 (d, 1H, J1,2
4.6 Hz, H-1), 3.99 (dd, 1H, J2,3 = 4.8 Hz, H-2), 3.99–3.95 (m,
1H, H-4), 3.93 (dd, 1H, J3,4 = 7.3 Hz, H-3), 3.78 (dd, 1H, J5a,5b
=
Acknowledgements
=
12.1 Hz, J4,5a = 2.6 Hz, H-5a), 3.66 (dd, 1H, J4,5b = 4.8 Hz, H-5b);
13C NMR (CD3OD): d 135.3 (C-4¢), 134.3 (C-1¢), 134.0 (C-2¢, C-
6¢), 130.0 (C-3¢, C-5¢), 93.1 (C-1), 84.5 (C-4), 83.4 (C-2), 77.7 (C-3),
62.4 (C-5); HRESIMS: Calcd for C11H13ClNaO4S [M + Na]+: m/z
299.0121; found: m/z 299.0112.
We are grateful to the Agence Nationale de la Recherche (ANR
JCJC06_140075) for financial supports and for a grant to M. A.,
and to the Agence des Universite´s Francophones (AUF) for a grant
to D. D. We also thank Prof. G. J. Davies for providing the wild type
and the E173A mutant of Araf 51, Prof. S. J. Withers for helpful
discussions, and Jean-Paul Gue´gan (ENSCR) for recording NMR
spectra.
3¢-Chlorophenyl 1-thio-a-L-arabinofuranoside (18)
Colourless oil (7.1 mg, 73%): [a]2D0 -206.5 (c 0.40, MeOH); Rf 0.4
(9 : 1 CH2Cl2/MeOH);1H NMR (CD3OD): d 7.54 (dd, 1H, J2¢,6¢
1.8 Hz, J2¢,4¢ = 1.3 Hz, H-2¢), 7.44 (ddd, 1H, J5¢,6¢ = 7.2 Hz, J4¢,6¢
=
=
Notes and references
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A. van der Marel, Chem. Soc. Rev., 2005, 34, 769.
1.5 Hz, H-6¢), 7.29 (dd, 1H, J4¢,5¢ = 7.9 Hz, H-5¢), 7.26 (ddd, 1H,
H-4¢), 5.34 (d, 1H, J1,2 4.4 Hz, H-1), 4.01 (dd, 1H, J2,3 = 4.6 Hz,
H-2), 4.00–3.97 (m, 1H, H-4), 3.95 (dd, 1H, J3,4 = 7.2 Hz, H-3),
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 8371–8378 | 8377
©

View Online
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