Investigation of the specificity of an α-l-arabinofuranosidase using C-2 and C-5 modified α-l-arabinofuranosides

Article abstract of DOI:10.1016/j.carres.2007.06.001

The synthesis of three novel glycosyl donors presenting the same scaffold as α-l-arabinofuranose but modified at the C-2 or C-5 positions has been achieved. Furthermore, chemoenzymatic syntheses using the α-l-arabinofuranosidase AbfD3 and these unnatural

Full text of DOI:10.1016/j.carres.2007.06.001

Carbohydrate Research 342 (2007) 2202–2211
Investigation of the specificity of an a-L-arabinofuranosidase
using C-2 and C-5 modified a-L-arabinofuranosides
a
a,
b
b
*
´
´
Gerald Lopez, Caroline Nugier-Chauvin, Caroline Remond and Michael O’Donohue
aEcole Nationale Supe´rieure de Chimie de Rennes, UMR CNRS 6226 Sciences Chimiques de Rennes,
`
`
´
´ ´
Equipe Synthese Organique et Systemes Organises, Avenue du General Leclerc, F-35700 Rennes, France
bLaboratoire de Technologie Enzymatique et Physico-Chimie des Agroressources, UMR URCA/INRA FARE,
8 Rue Gabriel Voisin, BP 316, F-51688 Reims, France
Received 17 April 2007; received in revised form 25 May 2007; accepted 2 June 2007
Available online 9 June 2007
Abstract—The synthesis of three novel glycosyl donors presenting the same scaffold as a-L-arabinofuranose but modified at the C-2
or C-5 positions has been achieved. Furthermore, chemoenzymatic syntheses using the a-L-arabinofuranosidase AbfD3 and these
unnatural furanosides were investigated. The use of the novel p-nitrophenyl furanoside donors revealed that AbfD3 can perform
transglycosylation with the C-5 deoxygenated donor but not with the C-2 modified one. These results emphasize the vital role
for OH-2 in AbfD3 substrate recognition.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Glycofuranosides; a-L-Arabinofuranosidase; Deoxygenated furanosides; Modified substrates; Enzymatic glycosylation
1. Introduction
catalyzed syntheses of furanose-containing oligosaccha-
rides have been published.9–12
Biocatalytic methods are useful additions to the classical
sugar chemist’s toolbox.1 Among these tools, retaining
O-glycosidases are carbohydrate-acting enzymes that
perform catalysis through a two-step mechanism that in-
volves enzyme glycosylation, followed by nucleophile-
promoted deglycosylation. When used for synthetic
applications, O-glycosidases often display only moder-
ate regioselectivity and conversion yields. Nevertheless,
they are generally considered to be useful because they
are readily available and catalytically versatile.2–5 The
efficiency of O-glycosidases is emphasized by the fact
that some can accept unnatural substrates, displaying
modifications of the sugar moiety and/or a variety of
aglycone groups.6–8 However, to the best of our knowl-
edge, apart from the synthesis of fructooligosaccharides
by fructosidases, very few examples of O-glycosidase-
In previous studies, an a-L-arabinofuranosidase of
bacterial origin (AbfD3) has been successfully employed
for the synthesis of novel alkyl-glycosides13 and fura-
nose-containing disaccharides.14,15 In the case of disac-
charide synthesis, it was shown that AbfD3 can
catalyze the self-condensation of both p-nitrophenyl a-
L-arabinofuranoside and p-nitrophenyl b-D-galactofur-
anoside. Similarly, using these p-nitrophenyl furanosides
as donors and b-D-xylosides as acceptors mixed disac-
charides could be obtained. Depending on the donor/
acceptor ratio, the reactions occurred with variable
degrees of regioselectivity towards both (1!2)- and
(1!3)-linkages, which reflects the previously observed
bond specificity of AbfD3 operating in hydrolytic
mode.16 Other authors have previously suggested that
there is a causal relationship between the bond specific-
ity of an O-glycosidase working in the hydrolytic mode
and regiospecificity during transglycosylation.17 More
recently, we demonstrated that AbfD3 can also cata-
lyze syntheses involving galactofuranosyl analogues
*
Corresponding author. Tel.: +33 (0)2 23 23 80 66; fax: +33 (0)2 23 23
80 46; e-mail: Caroline.Nugier@ensc-rennes.fr
0008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.06.001

transesterification. Gratifyingly, methyl a-L
-arabino-
furanoside 2 was clearly identified on the basis of NMR
spectroscopy in accordance with the literature data.21
The singlet signal for H-1 was found at d 4.8 ppm in
G. Lopez et al. / Carbohydrate Research 342 (2007) 2202–2211
2203
O
NO2
OH
O
R
1
R'
the H NMR spectrum while the C-1 resonated at d
108.6 ppm, typical values for a-L-arabinofuranosides.22
The preparation of the fluorinated target 6 required a
free primary hydroxyl group, which was first differenti-
ated from the secondary ones according to a conven-
tional tritylation–benzoylation protocol.23 However,
the fluorination step was inefficient and led to a poor
overall yield (14%). Therefore, we obtained the 5-mono-
fluorinated compound 3 via an alternative route through
direct substitution of the 5-hydroxyl group using the
well-known fluorinating agent diethylaminosulfur triflu-
oride (DAST) on the unprotected sugar 2. Using previ-
ously reported procedures,24,25 the treatment of 2 with
an excess of DAST at 0 ꢁC afforded 3 in a moderate,
but largely improved (48%) yield as the only a-anomer.
Unfortunately, attempts to use less than a sixfold excess
of DAST afforded an inextricable mixture. Difficulties
associated with the introduction of fluorine at a primary
position in pentofuranoses have already been encoun-
tered by others.26 Indeed, upon DAST treatment a
methoxy shift from C-1 to C-5 of methyl b-D-ribofur-
anosides has been observed. Moreover, diverse side
reactions often occur when fluorine is introduced into
sugar derivatives via nucleophilic substitution using
DAST reagent. In particular, anhydro compounds and
cyclic sulfites (during work-up) are generated when
unprotected sugars are used.27 In our case, the forma-
tion of the fluorinated compound 3 was accompanied
by the generation of a degradation compound that
was observed in the final fractions after chromato-
graphic purification. Incorporation of fluorine in 3 was
confirmed by NMR data that were coherent with
R
R'
p
p
p
p
NP α-L-Ara
f
OH OH
NP 5-F-α-L-Ara
f
6
F
OH
OH
H
NP 5-deoxy-α-L-Ara
f
f
10
H
NP 2-deoxy-α-L-Ara
15 OH
Figure 1. p-Nitrophenyl activated C-2- and C-5-modified a-L-arabino-
furanosides.
modified at the C-6 position, that is, p-nitrophenyl b-D-
fucofuranoside and p-nitrophenyl-6-deoxy-6-fluoro-b-D-
galactofuranoside. These reactions were also character-
ized by diverse regioselectivity in favor of the b-(1!2)
linkage.18 Encouraged by these results, it appears useful
to pursue this approach and to aim at a better control
of regioselectivity through the use of tailor-made
substrates.
In this work we have attempted to manipulate the
regioselectivity of AbfD3-catalyzed transglycosylation
reactions by synthesizing a series of unnatural donors
that display structural modifications at their C-2 or C-
5 positions. We have thus synthesized the three donors,
p-nitrophenyl 5-deoxy-5-fluoro-a-L-arabinofuranoside
6, p-nitrophenyl 5-deoxy-a-L-arabinofuranoside 10 and
p-nitrophenyl
2-deoxy-a-L-arabinofuranoside
15
(Fig. 1). Following synthesis, these glycosides were
tested to determine if these deoxygenated analogues
could be recognized as substrates in AbfD3-catalyzed
reactions.
1
reported values.28 In the H NMR spectrum of 3, the
2. Results and discussion
resonances for each of the diastereotopic hydrogens on
C-5 appeared as a doublet of a doublet of a doublet,
3
2.1. Synthesis of a-L-arabinofuranoside analogues
with JH,F magnitudes of 47.2 and 47.5 Hz. Further-
more, in the 13C NMR spectrum a doublet signal,
Our first attempts to synthesize the desired activated
donors 6 and 10 starting from an anomeric mixture of
methyl L-arabinofuranoside failed in the first step.
Indeed, as previously observed on a galactofuranose
derivative,19 differentiation between the primary and
secondary hydroxyl groups of methyl L-arabinofuran-
oside by tritylation or tosylation was extremely tricky
and led to very poor product yields. Consequently,
methyl a-L-arabinofuranoside 2 was used as a common
building block for the syntheses of both targets (Scheme
1). First, a multi-gram scale synthesis of per-O-benzoyl-
ated methyl L-arabinofuranoside 1 was performed using
a conventional method.15 Next, using a previously re-
ported protocol,20 the a-anomer of 1 was quantitatively
characterized by a high coupling constant (1JC,F
=
186.7 Hz), was found for C-5.
Subsequently, compound 5 was synthesized by benzo-
ylation of 3 leading to the precursor 4, which was sub-
jected to acetolysis. The overall yield for the two steps
was 79%. Finally, 5 was used as a donor for the glyco-
sylation of p-nitrophenol. This reaction was promoted
by tin(IV) chloride,29,30 leading to a moderate (50%)
´
yield of the desired arabinofuranoside 6 after Zemplen
deacylation. The formation of the reducing arabino-
furanosyl derivative during the course of the reaction
led us to suspect that the acidic conditions were respon-
sible for partial degradation of the donor 5 and/or the
product 6. Therefore, the less aggressive promoter,
boron trifluoride–triethylamine (prepared in situ),31
´
crystallized from ethanol leading to 2 after Zemplen

2204
G. Lopez et al. / Carbohydrate Research 342 (2007) 2202–2211
OBz
O
i
OMe
L-Arabinose
BzO
OBz
1
ii
OH
O
OH
O
OBz
O
OMe
OMe
OMe
vii
iii
HO
F
F
TsO
3
2
7
OH
OH
OBz
viii
iv
OBz
O
OMe
OBz
O
OMe
4
OBz
8
OBz
ix
v
OBz
O
OBz
O
OAc
OAc
F
9
5
OBz
vi
OBz
x
O
NO2
O
NO2
OH
O
OH
O
F
10
6
OH
OH
Scheme 1. Reagents and conditions: (i) (a) AcCl–MeOH, 3 h, rt, (b) BzCl, pyridine, 2 h, rt (100%); (ii) (a) cryst. EtOH, (b) NaOMe–MeOH, 3 h, rt
(50%); (iii) (a) TsCl, DMAP, pyridine, overnight, rt, (b) BzCl, pyridine, overnight, rt (79%); (iv) NaBH4, DMF, 6 h, 65 ꢁC (55%); (v) Ac2O, H2SO4,
CH2Cl2, overnight, rt (95%); (vi) (a) p-nitrophenol, BF3ÆOEt2, Et3N, CH2Cl2, 3 h, rt, (b) NaOMe–MeOH, 3 h, rt (55%); (vii) DAST, CH2Cl2, 1 h,
0 ꢁC to rt (48%); (viii) BzCl, pyridine, overnight, rt (80%); (ix) Ac2O, H2SO4, CH2Cl2, overnight, rt (99%); (x) (a) p-nitrophenol, BF3ÆOEt2, Et3N,
CH2Cl2, 3 h, rt, (b) NaOMe–MeOH, 3 h, rt (59%).
was used to obtain the p-nitrophenyl 5-deoxy-5-fluoro-
a-L-arabinofuranoside donor 6, after deprotection, with
a significantly improved yield (59%) and excellent
a-stereoselectivity.
deacylation. This sequence afforded pure p-nitrophenyl
5-deoxy-a-L-arabinofuranoside 10 in 52% yield for the
last three steps. The coupling constants observed
between H-1 and H-2 (3J1,2 = 1.8 Hz for 6 and 1.3 Hz
for 10) and the C-1 signals in the 13C NMR spectra of
6 and 10 (at d 107.7 ppm and d 105.4 ppm, respectively)
allowed us to ascertain the a configuration of these
novel glycosyl donors.
For many years, chemical 2-deoxygenation of pento-
furanoses has been used with variable success to study
ribonucleosides. Considerable improvements to the
deoxygenation of carbohydrate-type compounds have
been achieved thanks to the Barton and McCombie
radical deoxygenation.35 Reductive deoxygenation of
phenyl thionocarbonate from unhindered secondary
alcohols usually proceeds smoothly using tri-n-butyltin
hydride and a free radical initiator. As illustrated in
The deoxygenated donor 10 was prepared through
selective tosylation of the primary hydroxyl group in
2, followed by benzoylation to afford the known furan-
oside 732 in good yield (79% for the two steps). Sodium
borohydride reduction of 7 afforded the deoxygenated
arabinofuranoside 8 with a moderate 55% yield. In this
case, sodium borohydride reduction was preferred to the
usually more convenient lithium aluminum hydride
reduction of partially benzylated furanosides,33,34 due
to the greater tolerance of sodium borohydride towards
benzoyl participating groups. Acetolysis of 8 led to the
production of the donor 9, followed by a one-pot p-
´
nitrophenyl glycosidation reaction and final Zemplen

G. Lopez et al. / Carbohydrate Research 342 (2007) 2202–2211
2205
Si
O
NO2
O pNP
Si
O
O pNP
OH
O
O
O
Si
O
O
Si
i
ii
O
HO
O
O
11
12
13
OH
OH
OPTC
Si
O pNP
O pNP
O
OH
O
O
Si
iv
iii
O
O
HO
14
15
Scheme 2. Reagents and conditions: (i) TIPSCl2, pyridine, 1 h, rt (85%); (ii) PTCCl, overnight, rt (100%); (iii) n-Bu3SnH, AIBN, toluene, overnight,
80 ꢁC (63%); (iv) TBAF–THF, overnight, rt (95%).
Scheme 2, the initial synthesis of p-nitrophenyl 2-deoxy-
a-L-erthyro-pentofuranoside, 15, involved simultaneous
protection of the 3- and 5-positions of the known p-
nitrophenyl a-L-arabinofuranoside 1136,37 by selective
release, two novel species were formed, although a large
amount of non-reacted substrate remained. These prod-
ucts were isolated as previously described18 and identi-
fied by NMR spectroscopy.
silylation
with
1,3-dichloro-1,1,3,3-tetra-
Analysis of 13C NMR data for the major product, 16,
revealed a downfield shift of the C-2I of the reducing 5-
deoxy-L-arabinofuranoside from 81.9 (for 10) to
88.06 ppm (for 16), as well as weak upfield shifts
(Dd = ꢀ1.4 ppm and ꢀ1.9 ppm) for the signals of the
neighboring carbons C-1I and C-3I. Moreover, an in-
tense three-bond coupling between C-2I (88.06 ppm)
isopropyldisiloxane in pyridine to give 12 with the
expected reported 85% yield.38 Phenoxythiocarbonyl
chloride (PTC-Cl) was shown to quantitatively convert
12 into the phenyl thionocarbonate derivative 13.
Standard reduction using 1.5 equiv of tri-n-butyltin
hydride and 0.2 equiv of AIBN,39,40 followed by TBAF
desilylation afforded the novel compound 15 in good
yield. The very important upfield shift of the C-2 of
the 2-deoxygenated arabinofuranoside from 92.2 (for
13) to 42.5 ppm (for 15) was consistent with such a
chemical modification.21
1
and H-1II (5.06 ppm) in the H–13C (HMBC) spectrum
allowed the unambiguous identification of 16 as a
(1!2)-linked disaccharide. Finally, the small coupling
constant observed between H-1II and H-2II
ðJ1II ¼ 1:2 HzÞ clearly indicated that the glycosidic
2II
;
bond in 16 exhibited a-stereochemistry (Scheme 3). On
the basis of similar data analysis, the minor product
17 was identified as a a-(1!3) disaccharide.
2.2. Chemoenzymatic synthesis of furanose-containing
disaccharides
This result was rather expected because the analo-
gously modified D-galactofuranoside derivative, p-nitro-
phenyl b-D-fucofuranoside, also led to the synthesis of
a-(1!2)- and a-(1!3)-linked disaccharides.18 More-
over, according to HPTLC analysis of the AbfD3-
catalyzed condensation of p-nitrophenyl-5-deoxy-a-L-
arabinofuranoside 10, the regioselectivity of the trans-
glycosylation appeared to be low (16/17, 1.5:1) as previ-
ously observed using the galactofuranosyl derivative
To test the suitability of modified arabinofuranosyl
donors for AbfD3-catalyzed synthesis, compounds 6,
10, and 15 were incubated with AbfD3 (12 IU) at
60 ꢁC for short periods. After the incubation, high
performance thin-layer chromatography (HPTLC) was
used to detect transglycosylation products. Using mod-
ified arabinofuranoside 10, the HPTLC chromatogram
revealed that after 30 min, concomitant to p-nitrophenol
O pNP
OH
OH
O
O pNP
OH
O
O
α-L-arabinofuranosidase (AbfD3)
O
O
O pNP
+
OH
O
OH
O
OH
OH
OH
10
16
17
Scheme 3. Structures of the disaccharides, 16 and 17, which were obtained from the AbfD3-catalyzed condensation of 10.

2206
G. Lopez et al. / Carbohydrate Research 342 (2007) 2202–2211
reduced at the C-6 position, that is p-nitrophenyl-b-D-
fucofuranoside.18
4. Experimental
4.1. General methods
In a similar way, the furanosyl donor 6 was incu-
bated with AbfD3 and the course of the reaction was
monitored by HPTLC. However, while the major prod-
uct appeared to be p-nitrophenol, traces of two insepa-
rable other products (suspected to be disaccharides due
to the HPTLC profile) could be detected attesting that
hydrolysis of compound 6 largely overwhelmed the
autocondensation pathway. Overall, these results sug-
gest that both C-5 deoxygenated a-L-arabinofuran-
osides are good substrates for hydrolysis but rather
poor ones for the transglycosylation process. Indeed,
incubation of the furanoside 10 in the described condi-
tions led to very poor yield (<5%) of the isolated
disaccharides.
Finally, when the 2-deoxy analogue 15 was incubated
with AbfD3 in the same conditions, HPTLC analysis
failed to reveal any reaction products. Moreover, the
free sugar could not be detected by charring the plate
with a general acidic reagent, confirming that AbfD3
is not active towards this analogue. As suspected, this
result indicates that the presence of the OH-2 group in
the donor sugar is crucial for substrate recognition by
AbfD3.
Optical rotations were measured on a Perkin–Elmer 341
polarimeter. Melting points were determined on a Reic-
hert microscope and are uncorrected. Thin-layer chro-
matography (TLC) analyses were conducted on E.
Merck 60 F254 Silica Gel non-activated plates and com-
pounds were revealed using a 5% solution of H2SO4 in
EtOH followed by heating. High performance TLC
(HPTLC) experiments were performed on a CAMAG
TLC SCANNER 3, designed for densitometric measure-
ment of thin-layer chromatograms up to 200 · 200 mm
in size and controlled by the newly designed software
WINCATS. TLC (HPTLC) analyses were carried out with
detection by UV absorption (300 nm) and when neces-
sary charring with 5% solution of H2SO4 in EtOH.
For column chromatography, Geduran Si 60 (40–
63 lm) Silica Gel was used. All new compounds were
determined to be >95% pure by 1H NMR spectroscopy.
Preparative TLC was performed using 0.25 mm Silica
1
Gel 60 plates (E. Merck). H, 13C, HMQC, and COSY
NMR spectra were recorded on a Bruker ARX 400
¨
spectrometer at 400 MHz for 1H, 100 MHz for 13C,
and 376 MHz for 19F analyses. 1D and 2D spectra of
the disaccharides were all recorded on a Bruker ARX
¨
1
3. Conclusion
500 spectrometer at 500 MHz for H and 126 MHz for
13C. CDCl3 was employed as an NMR solvent for pro-
tected sugars and D2O or CD3OD for the free sugars.
Chemical shifts are given in d-units (ppm) measured
downfield from Me4Si. Disaccharide-associated carbons
and protons are designated with the superscript ‘I’ for
the reducing residue and ‘II’ for the non-reducing
residue. The HRMS were measured at the CRMPO
(Rennes, France) with an MS/MS ZabSpec TOF Micro-
mass using m-nitrobenzylic alcohol as a matrix and
accelerated cesium ions for ionization.
This study clearly demonstrates that besides cleavage,
the AbfD3 furanosidase is able to catalyze transglycosyl-
ation reactions with C-5 modified p-nitrophenyl arabi-
nofuranosides and confirmed that AbfD3 possesses the
ability to synthesize both a-(1!2)- and a-(1!3)-linked
regioisomeric disaccharides from the C-5 deoxygenated
substrate 10. The preparation of three original arabino-
furanosyl donors, p-nitrophenyl 5-deoxy-a-L-arabino-
furanoside, p-nitrophenyl 5-deoxy-5-fluoro a-L-arabino-
furanoside, and p-nitrophenyl 2-deoxy-a-L-erythro-
pentofuranoside, has allowed us to demonstrate that
the presence of OH-5 is not required for AbfD3 sub-
strate recognition. On the other hand, the present data
also underline the importance of OH-2 for substrate rec-
ognition. This is coherent with structural data available
for other GH-51 arabinofuranosidases10,41 that indicate
that OH-2 is involved in at least two interactions, one
with the catalytic nucleophile and the other with the
adjacent Asn residue. Consequently, this present state
of affairs precludes the use of a C-2 deoxygenation ap-
4.2. Methyl a-L-arabinofuranoside (2)21
Compound 142 (10 g, 67 mmol) was re-crystallized from
ethanol to give methyl 2,3,5-tri-O-benzoyl-a-L-arabino-
furanoside 1a with 50% maximal yield as a white solid.
Compound 1a (30 g, 80 mmol) was then added to a 1 N
MeONa solution in MeOH (200 mL). After stirring for
3 h at room temperature, the reaction mixture was neu-
tralized by adding glacial HOAc and concentrated under
reduced pressure. The resulting crude product was puri-
fied by column chromatography (CH2Cl2–MeOH, 4:1)
to yield 2 (13 g, 100%) as a white solid (CH2Cl2–MeOH,
proach
oligosaccharides.
for
the
synthesis
of
(1!5)-linked
Finally, like many other transglycosylating O-glycosi-
dases, hydrolysis by AbfD3 is favored with respect
to transglycosylation. Further development of this strat-
egy aiming at reducing water activity is now under-
way.
20
1
4:1, Rf = 0.28); mp 143 ꢁC; ½aꢁD ꢀ7.2 (c 1, MeOH); H
NMR (D2O, 400 MHz): 4.81 (d, 1H, H-1,
d
J1,2 = 1.5 Hz), 4.21–4.13 (m, 2H, H-2, H-4, J2,3
=
0
3,3 Hz, J3,4 = 5.9 Hz, J4,5 = 3.6 Hz, J4;5 ¼ 5:6 Hz),

G. Lopez et al. / Carbohydrate Research 342 (2007) 2202–2211
2207
0
4.05 (dd, 1H, H-3), 3.92 (dd, 1H, H-5, J5;5 ¼ 12:2 Hz),
3.25 (s, 1H, OCH3); 13C NMR (D2O, 100 MHz): d
108.6 (C-1), 84.2 (C-4), 80.9 (C-2), 76.7 (C-3), 61.5 (C-
5), 55.2 (OCH3). ESIMS m/z calcd for [C6H12O5]Na+:
187.0582, found: 187.0587.
4.5. 1-O-Acetyl-2,3-di-O-benzoyl-5-deoxy-5-fluoro-a,b-L-
arabinofuranose (5)
A solution of 4 (150 mg, 0.4 mmol) in dry CH2Cl2
(4 mL) was prepared and acetic anhydride (151 lL,
1.6 mmol) was added, followed by a catalytic amount
of H2SO4. After stirring overnight at room temperature,
the reaction mixture was neutralized by Et3N (3 mL) at
0 ꢁC and concentrated under reduced pressure. The
crude product was dissolved in CH2Cl2 (20 mL), washed
(3 · 5 mL of water), dried on MgSO4, and concentrated
under reduced pressure. The resulting crude product was
purified by column chromatography (petroleum ether–
EtOAc, 6:4) to yield 5 (160 mg, 99%) as a colorless oil
(petroleum ether–EtOAc, 8:2, Rf = 0.43); 1H NMR
(CDCl3, 400 MHz): d 8.10–7.30 (m, 10H, Harom). a-
Anomer: 6.39 (s, 1H, H-1), 5.59 (s, 1H, H-2), 5.45 (d,
1H, J3,4 = 4.1 Hz, H-3), 4.72 (dddd, 2H, J4,5 = 3.8 Hz,
4.3. Methyl 5-deoxy-5-fluoro-a-L-arabinofuranoside (3)
A solution of 2 (700 mg, 4.8 mmol) in dry CH2Cl2
(20 mL) was prepared, then DAST (3.6 mL, 29 mmol)
was added dropwise at 0 ꢁC. After warming to room
temperature and stirring for 1 h, MeOH (10 mL) was
added at 0 ꢁC. The mixture was concentrated and puri-
fied by flash chromatography (petroleum ether–EtOAc,
2:3) to give 3 (340 mg, 48%) as a white solid (petroleum
20
ether–EtOAc, 3:2, Rf = 0.42); mp 136 ꢁC; ½aꢁD +27.4 (c
1
1, MeOH); H NMR (D2O, 400 MHz): d 4.82 (s, 1H,
0
H-1), 4.63 (ddd, 1H, J4,5 = 4.1 Hz, J5;5 ¼ 10:7 Hz,
0
0
J4;5 ¼ 2:8 Hz, J5,F = 47.2 Hz, J5 ;F ¼ 46:5 Hz, H-5, H-
50), 4.48 (dtd, 1H, J4,F = 25.4 Hz, H-4), 2.20 (s, 3H,
OAc). b-Anomer: 6.53 (d, 1H, H-1, J1,2 = 4.6 Hz), 5.81
(dd, 1H, J2,3 = 7.1 Hz, J3,4 = 6.1 Hz, H-3), 5.60 (dd,
0
J5,F = 47.5 Hz, H-5), 4.43 (ddd, 1H, J4;5 ¼ 5:1 Hz,
J5 ;F ¼ 47:2 Hz, H-50), 4.05 (dtd, 1H, J3,4 = 5.9 Hz,
0
J4,F = 24.6 Hz, H-4), 3.93 (dd, 1H, J1,2 = 1.5 Hz, H-2),
3.87 (dd, 1H, H-3), 3.25 (s, 3H, OCH3); 13C NMR
0
1H, H-2), 4.71 (m, 2H, J5,F = 47.2 Hz, J5 ;F ¼ 47:2 Hz,
(D2O, 100 MHz): d 108.8 (C-1), 82.6 (C-5, J5,F
=
H-5, H-50), 4.30 (m, 1H, J4,F = 22.9 Hz, H-4), 2.10 (s,
3H, OAc); 13C NMR (CDCl3, 100 MHz): d 169.6–
155.5 (CO), 135.0–128.3 (Carom). a-Anomer: 99.8 (C-
186.7 Hz), 82.6 (C-4, J4,F = 18.5 Hz), 80.8 (C-2), 75.7
(C-3, J3,F = 7.2 Hz), 55.3 (OCH3); 19F NMR (D2O,
376 MHz): d ꢀ239 (F-5). ESIMS m/z calcd for
[C6H11O4F]Na+: 189.0539, found: 189.0540.
1), 84.7 (C-4, J4,F = 19.2 Hz), 82.0 (C-5, J5,F
175.1 Hz), 80.9 (C-2), 76.9 (C-3, J3,F = 6.9 Hz), 21.5
(OAc). b-Anomer: 93.6 (C-1), 82.9 (C-5, J5,F
=
=
4.4. Methyl 2,3-di-O-benzoyl-5-deoxy-5-fluoro-a-L-
arabinofuranoside (4)
175 Hz), 81.0 (C-4, J4,F = 19.2 Hz), 76.1 (C-2), 74.5
(C-3, J3,F = 6.9 Hz), 21.45 (OAc); 19F NMR (D2O,
376 MHz): d ꢀ229 (F-5 b); ꢀ231 (F-5 a). ESIMS m/z
calcd for [C21H19O7F]Na+: 425.1013, found: 425.1009.
Following the preparation of a solution of 3 (125 mg,
0.75 mmol) in dry pyridine (8 mL), benzoyl chloride
(350 lL, 3 mmol) was added dropwise at 0 ꢁC. After
stirring overnight at room temperature the reaction mix-
ture was partitioned between CH2Cl2 (30 mL) and water
(5 mL) and then decanted. The organic layer was
washed with 1 N aqueous HCl (5 · 5 mL), satd aq NaH-
CO3 (5 · 5 mL), dried on MgSO4, and concentrated
under reduced pressure. The resulting crude product
was purified by column chromatography (petroleum
ether–EtOAc, 9:1) to yield 4 (225 mg, 80%) as a colorless
4.6. p-Nitrophenyl 5-deoxy-5-fluoro-a-L-arabinofuran-
oside (6)
To a solution of 5 (100 mg, 0.25 mmol) in CH2Cl2
(3 mL), BF3ÆOEt2 (79 lL, 0.62 mmol) then Et3N
(18 lL, 0.13 mmol) were added at 0 ꢁC. After stirring
10 min at 0 ꢁC, para-nitrophenol (42 mg, 0.3 mmol)
was added and the reaction mixture was stirred for 3 h
at room temperature. Water (15 mL) was added and
the mixture was diluted with CH2Cl2 (20 mL). After
decantation, the organic layer was washed with 1 N
aqueous HCl (5 · 10 mL), satd aq NaHCO3
(5 · 10 mL), dried on MgSO4, and concentrated under
reduced pressure. The crude residue was dissolved in
1 N MeONa–MeOH solution and then stirred during
3 h at room temperature. After neutralization by glacial
HOAc and concentration under reduced pressure, the
resulting crude product was purified by column chroma-
tography (CH2Cl2–MeOH, 9:1) to yield 6 (40 mg, 59%
20
oil (petroleum ether–EtOAc, 3:2, Rf = 0.7); ½aꢁD +11.2
(c 1, CDCl3); 1H NMR (CDCl3, 400 MHz): d 8.10–
7.30 (m, 10H, Harom), 5.45 (s, 1H, H-2), 5.36 (d, 1H,
J3,4 = 5.1 Hz, H-3), 5.11 (s, 1H, H-1), 4.81 (d, 1H,
J4,5
=
3.3 Hz, J5,F = 47.2 Hz, H-5), 4.70 (d, 1H,
J4;5 ¼ 3:3 Hz, J5 ;F ¼ 46:7 Hz, H-50), 4.36 (dtd, 1H,
J4,F = 25.3 Hz, H-4), 3.41 (s, 3H, OCH3); 13C NMR
(CDCl3, 100 MHz): d 166.3–165.8 (CO), 134.0–128.9
(Carom), 107.4 (C-1), 82.6 (C-4, J4,F = 18.5 Hz), 82.5
(C-5, J5,F = 174.3 Hz), 81.8 (C-2), 77.3 (C-3), 55.5
(OCH3); 19F NMR (D2O, 376 MHz): d ꢀ230 (F-5).
ESIMS m/z calcd for [C20H19O6F]Na+: 397.1063, found:
397.1059.
0
0
(two steps)) as a white solid (CH2Cl2–MeOH, 9:1,
20
Rf = 0.43); mp 132 ꢁC; ½aꢁ ꢀ9.5 (c 1, MeOH); 1H
D
NMR (CD3OD, 400 MHz): d 8.21 (d, 2H, J = 9.2 Hz,

2208
G. Lopez et al. / Carbohydrate Research 342 (2007) 2202–2211
H-30, H-50), 7.20 (d, 2H, J = 9.4 Hz, H-20, H-40), 5.67 (d,
1H, J1,2 = 1.8 Hz, H-1), 4.55 (dddd, 2H, J4,5 = 2.8 Hz,
diluted with CH2Cl2 (50 mL), washed with satd aq
NaHCO3 (5 · 20 mL), dried on MgSO4, and concen-
trated under reduced pressure. The resulting crude prod-
uct was purified by column chromatography (petroleum
ether–EtOAc, 9:1) to yield 8 (1.2 g, 55%) as a white solid
(petroleum ether–EtOAc, 9:1, Rf = 0.32); mp 165 ꢁC;
0
0
0
J4;5 ¼ 4:8 Hz, J5;5 ¼ 10:4 Hz, J5,F = 51.4 Hz, J5 ;F
¼
51:4 Hz, H-5, H-50), 4.32 (m, 1H, J2,3 = 4.3 Hz, H-2),
4.16 (dddd, 1H, J3,4 = 6.9 Hz, J4,F = 24.3 Hz, H-4),
4.12 (dd, 1H, H-3); 13C NMR (CD3OD, 100 MHz): d
164.9 (C-10), 143.2 (C-40), 126.7 (C-30, C-50), 117.7 (C-
20, C-60), 107.7 (C-1), 84.6 (C-4, J4,F = 19.1 Hz), 83.6
(C-2), 83.3 (C-5, J5,F = 170.2 Hz), 77.3 (C-3,
J3,F = 6.9 Hz); 19F NMR (D2O, 376 MHz): d ꢀ232 (F-
5). ESIMS m/z calcd for [C11H12NO6F]Na+: 296.0546,
found: 296.0543. Anal. Calcd for C11H12NO6F: C,
48.36; H, 4.43. Found: C, 48.39; H, 4.52.
20
1
½aꢁD ꢀ12.7 (c 1, CHCl3); H NMR (CDCl3, 400 MHz):
d 8.10–7.40 (m, 10H, Harom), 5.48 (d, 1H, J1,2
1.5 Hz, H-2), 5.22 (ddd, 1H, J2,3 = 2.0 Hz, J3,4
=
=
5.3 Hz, H-3), 5.10 (s, 1H, H-1), 4.39 (q, 1H,
J4,5 = 6.4 Hz, H-4), 3.50 (s, 3H, OMe), 1.58 (d, 3H, H-
5); 13C NMR (CDCl3, 100 MHz): d 169.6–165.3 (CO),
134.2–125.2 (Carom), 113.2 (C-1), 87.3 (C-2), 86.2 (C-
3), 79.5 (C-4), 55.1 (OMe), 19.9 (C-5). ESIMS m/z calcd
for [C20H20O6]Na+: 379.1158, found: 379.1154.
4.7. Methyl 2,3-di-O-benzoyl-5-O-tosyl-a-L-arabino-
furanoside (7)
4.9. 1-O-Acetyl-2,3-di-O-benzoyl-5-deoxy-a,b-L-arabino-
furanose (9)
Tosyl chloride (2.6 g, 13.7 mmol) and DMAP were
added successively (560 mg, 4.6 mmol) to a solution of
2 (1.5 g, 9.1 mmol) in pyridine (50 mL). The reaction
mixture was stirred overnight at room temperature.
Water was added (30 mL) and the mixture was diluted
with CH2Cl2 (50 mL). After decantation, the organic
layer was washed with 1 N aqueous HCl (5 · 20 mL),
satd aq NaHCO3 (5 · 20 mL), dried on MgSO4, and
concentrated under reduced pressure. The crude product
(2.55 g) was dissolved in pyridine (80 mL) and then
benzoyl chloride (4 mL, 32 mmol) was added dropwise.
The reaction mixture was stirred overnight at room
temperature. Water was added (30 mL) and the mixture
was diluted with CH2Cl2 (50 mL). After decantation, the
organic layer was washed with 1 N aqueous HCl
(5 · 20 mL), satd aq NaHCO3 (5 · 20 mL), dried on
MgSO4, and concentrated under reduced pressure. The
resulting crude product was purified by column chroma-
tography (petroleum ether–EtOAc, 3:2) to yield 7 (3.8 g,
79% (two steps)) as a white solid (petroleum ether–
EtOAc, 3:2, Rf = 0.61); mp 141 ꢁC, lit.32 mp 150 ꢁC;
To a solution of 8 (620 mg, 1.74 mmol) in dry CH2Cl2
(20 mL), acetic anhydride (660 lL, 7 mmol) was added
followed by a catalytic amount of H2SO4. After stirring
overnight at room temperature, the reaction mixture
was neutralized by Et3N (20 mL) at 0 ꢁC and concen-
trated under reduced pressure. The crude product was
diluted by CH2Cl2 (40 mL), washed (3 · 20 mL of
water) and dried on MgSO4, and concentrated under
reduced pressure. The resulting crude product was
purified by column chromatography (petroleum ether–
EtOAc, 9:1) to yield 9 (635 mg, 95%) as a colorless oil
(petroleum ether–EtOAc, 9:1, Rf = 0.25); 1H NMR
(CDCl3, 400 MHz): d 8.30–7.50 (m, 10H, Harom); a-
Anomer: 6.51 (s, 1H, H-1), 5.74 (d, 1H, J1,2 = 1.3 Hz,
H-2), 5.38 (ddd, 1H, J2,3 = 2.5 Hz, J3,4 = 4.6 Hz, H-3),
4.64 (ddd, 1H, J4,5 = 6.4 Hz, H-4), 2.25 (s, 3H, OAc),
1.68 (d, 3H, H-5). b-Anomer: 6.69 (d, 1H, J1,2
4.6 Hz, H-1), 5.84 (dd, 1H, J2,3 = 6.4 Hz, H-2), 5.75
(dd, 1H, J3,4 = 5.1 Hz, H-3), 4.46 (ddd, 1H, J4,5
=
=
20
20
½aꢁD ꢀ32.4 (c 1, CHCl3), lit.32 ½aꢁD ꢀ30.1 (c 1, CHCl3);
6.6 Hz, H-4), 2.28 (s, 3H, OAc), 1.73 (d, 3H, H-5); 13C
NMR (CDCl3, 100 MHz): d 169.6–155.5 (COBz),
135.0–125.4 (Carom). a-Anomer: 99.8 (C-1), 81.8 (C-2),
81.7 (C-4), 81.6 (C-3), 21.5 (OAc), 19.5 (C-5). b-Ano-
mer: 95.0 (C-1), 81.0 (C-2), 80.6 (C-4), 80.4 (C-3), 21.6
(OAc), 21.5 (C-5). ESIMS m/z calcd for [C21H20O7]Na+:
407.1107, found: 407.1107.
1H NMR (CDCl3, 400 MHz): d 8.20–7.30 (m, 14H,
H
aromBz + Ts), 5.50 (d, 1H, J1,2 = 1.0 Hz, H-2), 5.32
(d, 1H, J3,4 = 4.6 Hz, H-3), 4.45 (d, 2H, J4;5
¼
J4;5 ¼ 8:4 Hz, H-5, H-50), 4.40 (dd, 1H, H-4), 3.50 (s,
3H, OMe), 2.40 (s, 3H, CH3Ts); 13C NMR (CDCl3,
100 MHz): d 169.6–165.3 (COBz), 134.2–125.2 (Carom),
107.9 (C-1), 82.1 (C-2), 81.8 (C-4), 78.5 (C-3), 69.7
(C-5), 55.8 (OMe), 22.2 (CH3Ts). ESIMS m/z calcd for
[C27H26O9S]Na+: 549.1195, found: 549.1195.
0
4.10. p-Nitrophenyl 5-deoxy-a-L-arabinofuranoside (10)
To a solution of 9 (455 mg, 1.18 mmol) in CH2Cl2
(6 mL), BF3ÆOEt2 (374 lL, 2.95 mmol) then Et3N
(84 lL, 0.6 mmol) were added at 0 ꢁC. After stirring
10 min at 0 ꢁC, p-nitrophenol (200 mg, 1.42 mmol) was
added and the reaction mixture was stirred during 3 h
at room temperature. Water (30 mL) was added and
the mixture was diluted with CH2Cl2 (50 mL). After
decantation, the organic layer was washed with 1 N
4.8. Methyl 2,3-di-O-benzoyl-5-deoxy-a-L-arabinofuran-
oside (8)
A solution of 7 was prepared (3.2 g, 6.1 mmol) in DMF
(60 mL), then NaBH4 (1.2 g, 30.4 mmol) was added pro-
gressively. The reaction mixture was stirred 6 h at 65 ꢁC.
After neutralization with glacial HOAc, the mixture was

G. Lopez et al. / Carbohydrate Research 342 (2007) 2202–2211
2209
aqueous HCl (5 · 20 mL), satd aq NaHCO3
(5 · 20 mL), dried on MgSO4, and concentrated under
reduced pressure. The crude residue was dissolved in
1 N MeONa–MeOH solution and stirred during 3 h at
room temperature. After neutralization by glacial
HOAc and concentration under reduced pressure, the
resulting crude product was purified by column chroma-
tography (CH2Cl2–MeOH, 9:1) to yield 10 (165 mg, 55%
solution of 12 (480 mg, 0.9 mmol) in pyridine (10 mL).
The reaction mixture was stirred overnight at room
temperature. Water was added (10 mL) and then the
mixture was diluted with CH2Cl2 (25 mL). After decan-
tation, the organic layer was washed with 1 N aqueous
HCl (5 · 10 mL), satd aq NaHCO3 (5 · 10 mL), dried
on MgSO4, and concentrated under reduced pressure.
The resulting crude product was purified by column
chromatography (petroleum ether–EtOAc, 9:1) to yield
(two steps)) as a beige solid (CH2Cl2–MeOH, 9:1,
20
Rf = 0.43); mp 139 ꢁC; ½aꢁ ꢀ29.2 (c 1, MeOH); 1H
13 (584 mg, 100%) as a yellow oil (petroleum ether–
D
20
NMR (CD3OD, 400 MHz): d 8.30 (d, 2H, J = 7.4 Hz,
H-30, H-50), 7.20 (d, 2H, J = 7.4 Hz, H-20, H-40), 5.90
(s, 1H, H-1), 4.43 (dd, 1H, J1,2 = 1.3 Hz, J2,3 = 3.6 Hz,
H-2), 4.23 (q, 1H, J3,4 = 6.1 Hz, J4,5 = 6.4 Hz, H-4),
3.91 (dd, 1H, H-3), 1.45 (d, 3H, H-5); 13C NMR
(CD3OD, 100 MHz): d 164.5 (C-10), 143.3 (C-40), 126.5
(C-30, C-50), 116.9 (C-20, C-60), 105.4 (C-1), 81.9 (C-2),
81.7 (C-3), 81.3 (C-4), 26.4 (C-5). ESIMS m/z calcd for
[C11H13NO6]Na+: 278.0641, found: 278.0639. Anal.
Calcd for C11H13NO6: C, 51.77; H, 5.13. Found: C,
51.91; H, 5.22.
EtOAc, 9:1, Rf = 0.68); ½aꢁ +25.4 (c 1, CHCl3); 1H
D
NMR (CDCl3, 400 MHz): d 8.15 (d, 2H, J = 7.3 Hz,
H-30, H-50), 7.35 (m, 5H, Harom PTC), 7.26 (d, 2H,
J = 7.3 Hz, H-20, H-40), 6.25 (s, 1H, H-1), 5.93 (dd,
1H, J1,2 = 1.1 Hz, J2,3 = 5.21 Hz, H-2), 4.61 (dd, 1H,
J3,4 = 4.5 Hz, H-3), 3.99–3.94 (m, 3H, H-5, H-4), 1.38–
1.32 (m, 4H, CHi-Pr), 1.06–1.01 (m, 24H, CH3i-Pr);
13C NMR (CDCl3, 100 MHz): d 192.3 (C@S), 164.9
(C-10), 152.8 (Cipso PTC), 149.4 (C-40), 126.5 (C-30,
C-50), 122.5 (Carom PTC), 116.9 (C-20, C-60), 99.5
(C-1), 92.2 (C-2), 74.5 (C-4), 73.8 (C-3), 64.3 (C-5),
18.1 (CHi-Pr), 15.9 (CH3i-Pr). ESIMS m/z calcd for
[C30H43NO9SSi2]Na+: 672.2095, found: 672.2090.
4.11. p-Nitrophenyl 3,5-O-(1,1,3,3-tetraisopropyldisilox-
ane)-a-L-arabinofuranoside (12)
4.13. p-Nitrophenyl 2-deoxy-3,5-O-(1,1,3,3-tetra-
isopropyldisiloxane)-a-L-arabinofuranoside (14)
A solution of 1137,38 (170 mg, 0.62 mmol) in pyridine
(6 mL) was prepared and TIPSCl2 (395 lL, 1.24 mmol)
was added. The reaction mixture was stirred overnight
at room temperature. Water was added (20 mL) and
then the mixture was diluted with CH2Cl2 (20 mL).
After decantation, the organic layer was washed with
1 N aqueous HCl (5 · 10 mL), satd aq NaHCO3
(5 · 10 mL), dried on MgSO4, and concentrated under
reduced pressure. The resulting crude product was puri-
fied by column chromatography (petroleum ether–
EtOAc, 9:1) to yield 12 (270 mg, 85%) as a brown oil
A solution of 13 was prepared (590 mg, 0.91 mmol) in
toluene (30 mL), then n-Bu3SnH (282 lL, 1.37 mmol)
and AIBN (32 mg, 0.19 mmol) were added successively.
The reaction mixture was stirred overnight at 80 ꢁC and
concentrated under reduced pressure. The resulting
crude product was purified by column chromatography
(petroleum ether–EtOAc, 9:1) to yield 14 (584 mg, 63%)
as a brown oil (petroleum ether–EtOAc, 9:1, Rf = 0.39);
20
½aꢁD +48.4 (c 1, CHCl3); 1H NMR (CDCl3, 400 MHz): d
20
(petroleum ether–EtOAc, 9:1, Rf = 0.32); ½aꢁD ꢀ65.9 (c
8.25 (d, 2H, J = 7.3 Hz, H-30, H-50), 7.18 (d, 2H,
J = 7.4 Hz, H-20, H-40), 6.30 (s, 1H, H-1), 4.48 (ddd,
1H, J2a,3 = 6.7 Hz, J2b,3 = 2.1 Hz, J3,4 = 3.2 Hz, H-3),
4.06–4.01 (m, 2H, H-5), 3.56–3.51 (m, 1H, H-4), 2.45
(dddd, 2H, J1,2a = 1.3 Hz, J1,2b = 4.5 Hz, H-2), 1.29–
1.23 (m, 4H, CHi-Pr), 1.16–1.12 (m, 24H, CH3i-Pr);
13C NMR (CDCl3, 100 MHz): d 162.6 (C-10), 142.9
(C-40), 126.5 (C-30, C-50), 116.9 (C-20, C-60), 101.6 (C-
1), 75.2 (C-4), 64.7 (C-3), 60.3 (C-5), 45.1 (C-2), 18.2
(CHi-Pr), 13.9 (CH3i-Pr). ESIMS m/z calcd for
[C23H39NO7Si2]Na+: 520.2163, found: 520.2166.
1, CHCl3); 1H NMR (CDCl3, 400 MHz): d 8.20 (d,
2H, J = 7.4 Hz, H-30, H-50), 7.10 (d, 2H, J = 7.4 Hz,
H-20, H-40), 5.70 (s, 1H, H-1), 4.61 (dd, 1H, J1,2
=
1.1 Hz, J2,3 = 3.3 Hz, H-2), 4.23 (dd, 1H, J3,4 = 4.4 Hz,
H-3), 3.99–3.91 (m, 2H, H-5), 3.88–3.93 (m, 1H, H-4),
3.11 (s, 1H, –OH), 1.31–1.26 (m, 4H, CHi-Pr), 1.07–
1.02 (m, 24H, CH3i-Pr); 13C NMR (CDCl3, 100 MHz):
d 164.7 (C-10), 143.8 (C-40), 126.5 (C-30, C-50), 116.9
(C-20, C-60), 104.1 (C-1), 82.2 (C-2), 74.3 (C-4), 72.5
(C-3), 62.3 (C-5), 16.1 (CHi-Pr), 12.9 (CH3i-Pr). ESIMS
m/z calcd for [C23H39NO8Si2]Na+: 536.2112, found:
536.2117.
4.14. p-Nitrophenyl 2-deoxy-a-L-erythro-pentofuranoside
(15)
4.12. p-Nitrophenyl 2-O-phenylthionocarbonate-3,5-O-
(1,1,3,3-tetraisopropyldisiloxane)-a-L-arabinofuranoside
(13)
Compound 14 (100 mg, 0.20 mmol) was added to a solu-
tion of TBAF 1 M in THF (10 mL). The reaction mix-
ture was stirred overnight at room temperature and
concentrated under reduced pressure. The resulting
crude product was purified by column chromatography
Phenylthionocarbonate chloride (355 lL, 2.1 mmol) and
DMAP (250 mg, 2.1 mmol) were added successively to a

2210
G. Lopez et al. / Carbohydrate Research 342 (2007) 2202–2211
(CH2Cl2–MeOH, 20:1) to yield 15 (23 mg, 88%) as a yel-
low solid (CH2Cl2–MeOH, 20:1, Rf = 0.35); mp 123 ꢁC;
(D2O, 500.13 MHz): d 8.17 (d, 2H, J = 9.2 Hz, H-30,
H-50), 7.12 (d, 2H, J = 9.3 Hz, H-20, H-60), 5.78 (s,
1H, H-1a), 5.08 (s, 1H, H-1b), 4.44 (dd, 1H, J1a,2a
<1 Hz, J2a,3a = 2.1 Hz, H-2a), 4.20 (t, 1H,
J4a,5a = 6.5 Hz, H-4a), 4.02 (dd, 1H, J1b,2b <1 Hz,
J2b,3b = 4.4 Hz, H-2b), 3.98 (t, 1H, J4b,5b = 6.3 Hz, H-
4b), 3.81 (dd, 1H, J3a,4a = 5.2 Hz, H-3a), 3.65 (dd, 1H,
J3b,4b = 6.7 Hz, H-3b), 1.29 (d, 3H, H-5a), 1.25 (d, 3H,
20
1
½aꢁD +79.4 (c 1, acetone); H NMR (D2O, 400 MHz): d
8.15 (d, 2H, J = 7.3 Hz, H-30, H-50), 7.11 (d, 2H,
J = 7.4 Hz, H-20, H-40), 6.16 (s, 1H, H-1), 4.21 (ddd,
1H, J2a,3 = 6.3 Hz, J2b,3 = 2.9 Hz, J3,4 = 3.7 Hz, H-3),
3.99–3.95 (m, 1H, H-4), 3.77–3.71 (m, 2H, H-5), 2.24
(dddd, 2H, J1,2a = 1.1 Hz, J1,2b = 4.7 Hz, H-2); 13C
NMR (D2O, 100 MHz): d 161.4 (C-10), 142.9 (C-40),
126.4 (C-30, C-50), 117.6 (C-20, C-60), 102.6 (C-1), 86.2
(C-4), 71.4 (C-3), 63.1 (C-5), 42.5 (C-2). Anal. Calcd
for C11H13NO6: C, 51.77; H, 5.13. Found: C, 51.84;
H, 5.23.
13
H-5b); C NMR (D2O, 125.76): d 161.30 (C-10),
142.20 (C-40), 126.05 (C-30, C-50), 116.68 (C-20, C-60),
106.81 (C-1b), 105.15 (C-1a), 86.93 (C-3a), 81.60 (C-
2b, C-3b), 79.98 (C-2a), 79.27 (C-4a, C-4b), 17.59 (C-
5a, C-5b); HRMS (ESI+) for C16H21NO9Na: m/z
[M+Na]+ calcd 394.1114, found: 394.1117.
4.15. Autocondensation reactions
A 5 mM aqueous solution of 6 or 10 (1.4 mL) was incu-
bated with 12 IU AbfD3 with magnetic stirring for 1 h
at 60 ꢁC. Reactions were quenched by enzyme denatur-
ation at 100 ꢁC for 10 min. After lyophilization, the
residue was dissolved in water (0.1 mL) and the auto-
condensation products were purified by preparative
TLC using 4:1 CH2Cl2–MeOH as the mobile phase.
The desired products were detected by UV absorption
(254 nm), collected from plates and extracted with 1:1
CH2Cl2–MeOH (4 mL). After filtration and freeze-
drying, the isolated disaccharides 16 and 17 were
characterized by NMR and high resolution mass
spectrometry.
Acknowledgements
Funding for G.L. was provided by the French Ministry
of Education, Research and Technology. We wish to
thank J. P. Guegan (ENSCR), S. Sinbandhit (Centre
Regional de Mesures Physiques de l’Ouest, CRMPO,
University of Rennes 1, France), and P. Guenot and
´
´
´
P. Jehan (CRMPO) for recording HRMS spectra.
References
1. Krˇen, V.; Thiem, J. J. Chem. Soc. Rev. 1997, 26, 463–473.
2. Monsan, P.; Paul, F. FEMS Microbiol. Rev. 1995, 16,
187–192.
3. Scigelova, M.; Singh, S.; Crout, D. H. G. J. Mol. Catal. B:
Enzym. 1999, 6, 483–494.
4.16. p-Nitrophenyl 5-deoxy-a-L-arabinofuranosyl-
(1!2)-5-deoxy-a-L-arabinofuranoside (16)
(ꢂ1 mg accumulated from a few preparative plates)
HPTLC (CH2Cl2–MeOH, 4:1) Rf = 0.46; 1H NMR
(D2O, 500.13 MHz): d 8.17 (d, 2H, J = 9.3 Hz, H-30,
H-50), 7.11 (d, 2H, J = 9.3 Hz, H-20, H-60), 5.81 (d,
1H, J1a,2a = 1.7 Hz, H-1a), 5.06 (d, 1H, J1b,2b = 1.2 Hz,
H-1b), 4.24 (dd, 1H, J2a,3a = 4.9 Hz, H-2a), 4.09 (dd,
1H, J4a,5a = 6.4 Hz, J3a,4a = 7.5 Hz, H-4a), 4.03 (dd,
1H, J2b,3b = 3.9 Hz, H-2b), 3.92 (t, 1H, J4b,5b = 6.5 Hz,
H-4b), 3.86 (dd, 1H, H-3a), 3.60 (dd, 1H,
J3b,4b = 6.7 Hz, H-3b), 1.25 (d, 3H, H-5a), 1,07 (d, 3H,
4. Lee, R.; Mansey, D.; Watson, A.; Duncan, K.; Rithner,
C.; McNeil, M. Anal. Biochem. 1996, 242, 1–7.
5. Cote, G. L.; Tao, B. Y. Glycoconjugate J. 1990, 7, 145–
162.
6. Williams, S. J.; Withers, S. G. Carbohydr. Res. 2000, 327,
27–46.
´
´
´
ˇ
´
7. Fialova, P.; Weignerova, L.; Rauvolfova, J.; Prikrylova,
´
V.; Pisˇvejcova, A.; Ettrich, R.; Kuzma, M.; Sedmera, P.;
Krˇen, V. Tetrahedron 2004, 60, 693–701.
8. Tauss, A.; Greimel, P.; Rupitz, K.; Steiner, A. J.; Stutz, A.
¨
E.; Withers, S. G.; Wrodnigg, T. M. Tetrahedron: Asym-
metry 2005, 16, 159–165.
9. Saha, B. C. Biotechnol. Adv. 2000, 18, 403–423.
10. Ho¨vel, K.; Shallom, D.; Niefind, K.; Belakhov, V.;
Shoham, G.; Baasov, T.; Shoham, Y.; Schomburg, D.
EMBO J. 2003, 22, 4922–4932.
11. Miletti, L. C.; Marino, K.; Marino, C.; Colli, W.; Alves,
M. J. M.; de Lederkremer, R. M. Mol. Biochem. Parasitol.
2003, 127, 85–88.
12. Wallis, G. L. F.; Hemming, F. W.; Peberdy, J. F. Biochim.
Biophys. Acta 2001, 1525, 19–28.
13
H-5b); C NMR (D2O, 125.76): d 161.20 (C-10),
142.10 (C-40), 126.10 (C-30, C-50), 116.43 (C-20, C-60),
107.63 (C-1b), 103.99 (C-1a), 88.06 (C-2a), 81.80 (C-
2b), 81.58 (C-3b), 79.78 (C-3a), 79.37 (C-4b), 79.08 (C-
4a), 17.23 (C-5b), 16.93 (C-5a); HRMS (ESI+) for
C16H21NO9Na: m/z [M + Na]+ calcd 394.1114, found:
394.1116.
´
13. Remond, C.; Ferchichi, M.; Aubry, N.; Plantier-Royon,
4.17. p-Nitrophenyl 5-deoxy-a-L-arabinofuranosyl-
(1!3)-5-deoxy-a-L-arabinofuranoside (17)
R.; Portella, C.; O’Donohue, M. J. Tetrahedron Lett. 2002,
43, 9653–9655.
´
14. Remond, C.; Plantier-Royon, R.; Aubry, N.; Maes, E.;
(ꢂ1 mg accumulated from a few preparative plates)
HPTLC (CH2Cl2–MeOH, 4:1) Rf = 0.49; 1H NMR
Bliard, C.; O’Donohue, M. J. Carbohydr. Res. 2004, 330,
2019–2025.

G. Lopez et al. / Carbohydrate Research 342 (2007) 2202–2211
2211
´
15. Remond, C.; Plantier-Royon, R.; Aubry, N.; O’Donohue,
29. Chiocconi, A.; Marino, C.; de Lederkremer, R. M.
Carbohydr. Res. 2000, 323, 7–13.
30. Marino, C.; Chiocconi, A.; Varela, O.; de Lederkremer, R.
M. Carbohydr. Res. 1998, 311, 183–189.
31. Lee, Y. S.; Rho, E. S.; Min, Y. K.; Kim, B. T.; Kim, K. H.
J. Carbohydr. Chem. 2001, 20, 503–506.
32. Ferrier, R. J.; Hurford, J. R. Carbohydr. Res. 1974, 38,
125–131.
M. J. Carbohydr. Res. 2005, 340, 637–644.
16. Debeche, T.; Cummings, N.; Connerton, I.; Debeire, P.;
O’Donohue, M. J. Appl. Environ. Microbiol. 2000, 66,
1734–1736.
17. Mala, S.; Dvorakova, H.; Hrabal, B.; Kralova, B. Carbo-
hydr. Res. 1999, 322, 209–218.
`
18. Euzen, R.; Lopez, G.; Nugier-Chauvin, C.; Ferrieres, V.;
´
`
Plusquellec, D.; Remond, C.; O’Donohue, M. J. Eur. J.
33. Euzen, R.; Ferrieres, V.; Plusquellec, D. J. Org. Chem.
Org. Chem. 2005, 4860–4869.
2005, 70, 847–855.
`
`
19. Euzen, R.; Ferrieres, V.; Plusquellec, D. Carbohydr. Res.
34. Gelin, M.; Ferrieres, V.; Lefeuvre, M.; Plusquellec, D.
2006, 341, 2759–2768.
20. Callam, C. S.; Lowary, T. L. J. Chem. Educ. 2001, 78, 73–
74.
21. Mizutani, K.; Kasai, R.; Nakamura, M.; Tanaka, O.;
Matsuura, H. Carbohydr. Res. 1989, 185, 27–38.
22. Bock, K.; Pedersen, C. Adv. Carbohydr. Chem. Biochem.
1983, 41, 27–66.
Eur. J. Org. Chem. 2003, 1285–1293.
35. Barton, D. H. R.; McCombie, S. W.; Stuart, W. J. Chem.
Soc., Perkin Trans. 1 1975, 1574–1585.
36. Fielding, A. H.; Hough, L. Carbohydr. Res. 1965, 1, 327–
329.
37. Kelly, M. A.; Sinnott, M. L.; Widdows, D. Carbohydr.
Res. 1988, 181, 262–266.
23. Cociorva, O. M.; Lowary, T. L. Carbohydr. Res. 2004,
339, 853–865.
38. Mastihubova, M.; Szemesova, J.; Biely, P. Tetrahedron
Lett. 2003, 44, 1671–1673.
24. Cottam, H. B.; Wasson, D. B.; Shih, H. C.; Raychaudh-
ury, A.; Di Pasquale, G.; Carson, D. A. J. Med. Chem.
1993, 36, 3424–3430.
25. Card, P. J. J. Org. Chem. 1983, 48, 393–395.
26. Lloyd, A. E.; Coe, P. L.; Walker, R. T.; Howarth, O. W. J.
Fluorine Chem. 1993, 60, 239–250.
27. Dax, K.; Albert, M.; Ortner, J.; Paul, B. J. Carbohydr.
Res. 2000, 327, 47–86, and references therein.
28. Michalik, M.; Hein, M.; Frank, M. Carbohydr. Res. 2000,
327, 185–218.
39. Robins, M. J.; Wilson, J. S.; Hansske, F. J. Am. Chem.
Soc. 1983, 105, 4059–4065.
40. Nawrot, B.; Pankiewicz, K. W.; Zepf, R. A.; Watanabe,
K. A. J. Carbohydr. Chem. 1988, 7, 95–114.
41. Taylor, E. J.; Smith, N. L.; Turkenburg, J. P.; D’Souza, S.;
Glilbert, H. J.; Davies, G. J. Biochem. J. 2006, 395, 31–
37.
42. Timpa, J. D.; Griffin, G. W. Carbohydr. Res. 1984, 131,
185–196.