A chemoenzymatic approach for the synthesis of unnatural disaccharides containing D-galacto- or D-fucofuranosides

Article abstract of DOI:10.1002/ejoc.200500525

Unusual diglycosides composed of D-hexofuranosyl entities were prepared by a chemoenzymatic route using the α-L-arabinofuranosidase, AbfD3. The required, unprotected monosaccharidic donors were first prepared according to multi-step syntheses. Since one g

Full text of DOI:10.1002/ejoc.200500525

FULL PAPER
A Chemoenzymatic Approach for the Synthesis of Unnatural Disaccharides
Containing
D
-Galacto- or -Fucofuranosides
D
Ronan Euzen,[a] Gérald Lopez,[a] Caroline Nugier-Chauvin,*[a] Vincent Ferrières,*[a]
Daniel Plusquellec,[a] Caroline Rémond,[b] and Michael O’Donohue[b]
Keywords: Glycofuranosides / α-l-Arabinofuranosidase / Oligosaccharides / Carbohydrates / Enzymes / Enzymatic
glycosylation
Unusual diglycosides composed of
were prepared by a chemoenzymatic route using the α-
binofuranosidase, AbfD3. The required, unprotected mono-
saccharidic donors were first prepared according to multi-
step syntheses. Since one goal of this study was the investi-
gation of donor –1 subsite in the active site of the enzyme,
D
-hexofuranosyl entities
tions on the side arm. These substrates were then used in
AbfD3-catalysed hydrolyses to determine the parameters Km
and kcat and in AbfD3-catalysed transglycosylation to evalu-
ate their ability to serve as donor/acceptor. Four disaccha-
rides were thus isolated and characterised, two resulting
from β-(1,2) connection along with two β-(1,3)-regioisomers.
L-ara-
we focused on
D-fucofuranosyl and 6-deoxy-6-fluoro-D-
galactofuranosyl derivatives which present stereochemical
similarities with L-arabinose series, but also structural varia-
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
ies have explored the use of glycoside hydrolases for the
synthesis of oligosaccharides characterised by the presence
of furanosyl residues, and more particularly hexofuranosyl
entities. As part of our ongoing interest in oligosaccharides
and glycoconjugates containing alternate furanoid and py-
ranoid residues,[6,7] we are also involved in the development
of new chemoenzymatic approaches for the preparation of
such rare bioactive carbohydrates. Indeed, galactofurano-
syl-containing oligosaccharides are crucial constituents of
polysaccharides in the cell walls in bacteria, fungi and para-
sitic microorganisms including some clinically significant
pathogens.[8] Since these glycosides are not present in mam-
mals, structurally well-defined, synthetic analogues are po-
tentially antigenic and, as such, are of great interest for the
development of new pharmacophores and new therapies.[9]
However, those oligofuranosides are generally difficult to
prepare according to classical methods of glycosidic synthe-
sis. It is therefore highly desirable to explore new ap-
proaches and notably methodologies that combine the ver-
satility of chemical methods and the simplicity and pre-
cision of enzymes. In particular, enzymes are likely to facili-
tate both the regio- and diastereoselective control of glyco-
Introduction
Retaining glycoside hydrolases exhibiting transglycosyla-
tion activity are increasingly involved in the synthesis of
oligosaccharides and glycoconjugates.[1] It is well-estab-
lished that these enzymes perform hydrolysis using a
double-displacement mechanism: (i) The formation of a co-
valently-linked enzyme-substrate intermediate, (ii) the reac-
tion of the enzyme-substrate intermediate with nucleophilic
molecules. In the case of transglycosylation, these glycosyl
acceptors, generally O-nucleophiles, compete with water to
generate oligosaccharides or hydrolysed sugars.[2] Conse-
quently, glycosides may be synthesised with glycoside hy-
drolases as catalysts under either thermodynamically or
kinetically controlled conditions. However, as the former
approach generally requires sophisticated procedures and
often provides coupling products in poor yields, kinetic
control of synthesis is largely preferred. In order to generate
a reactive intermediate, suitable activated glycosyl donors
including di- and oligosaccharides,[3]aryl glycosides[4] and
glycosyl fluorides,[1a,5] are required. To date, very few stud-
[a] Ecole Nationale Supérieure de Chimie de Rennes, UMR CNRS
6052 Synthèses et Activations de Biomolécules, Institut de Chi- sidic coupling without having recourse to the use of protect-
mie de Rennes,
ing groups. Up to now, few galactofuranosidases have been
Avenue du Général Leclerc, 35700 Rennes, France
identified[10] and, to the best of our knowledge, none of
Fax: +33-223238058
E-mail: vincent.ferrieres@ensc-rennes.fr
these biocatalysts have been used as synthetic tools for the
preparation of galactofuranosyl-containing oligosaccha-
rides. Nevertheless, on the basis of structural similarities,
we expected that d-galactofuranosyl residues and stereo-
chemically related analogues could be recognized as either
donors or acceptors by more commonly available furanosyl
[b] Institut National de la Recherche Agronomique, UMR INRA
614 Fractionnement des Agroressources et Emballage,
8 rue Gabriel Voisin, BP 316, 51688 Reims, France
Fax: +33-326355369
E-mail: michael.odonohue@univ-reims.fr
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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DOI: 10.1002/ejoc.200500525
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Chemoenzymatic Synthesis of Unnatural Disaccharides
FULL PAPER
hydrolases such as α-l-arabinofuranosidases (E.C. diated synthesis of novel furanose-containing disaccharides
3.2.1.55). These enzymes are glycoside hydrolases which are that have never been obtained through classical multi-step
naturally involved in the degradation of plant biomass synthesis.
through the catalysis of hydrolysis of α-(1Ǟ2)-, α-(1Ǟ3)-,
and/or α-(1Ǟ5)-l-arabinofuranosidic linkages in arabino-
xylans, arabinans and other l-arabinose-containing com-
pounds.[11] In an original report, Rémond et al.[12] reported
the use of the thermostable arabinofuranosidase produced
by Thermobacillus xylanilyticus (AbfD3) as an efficient tool
to catalyze transglycosylation in the presence of various
alcohols as well as p-nitrophenyl glycosides. More recently,
an exo-arabinanase isolated from Penicillium chysogenum
has been found to possess trans-arabinobiosylation activity
on various acceptors such as aliphatic alcohols and com-
mon monosaccharides.[13] Based on the characterisation of
Figure 1. Chemical structures of targeted 6-modified galactofur-
anosides 1 and 2.
the isolated α-l-arabinofuranosidase encoding gene,
AbfD3, the arabinofuranosidase from T. xylanilyticus be-
longs to the vast family 51 within the glycoside hydrolase
classification, which groups enzymes into families on the
basis of amino acid sequence similarity.[14] This family is
exclusively composed of retaining arabinofuranosidases
which catalyze hydrolysis leading to net conservation of the
anomeric configuration. Previous studies based on site-di-
Results and Discussion
Synthesis of Activated Donors
The p-nitrophenyl hexofuranosides 1 and 2 can be re-
rected mutagenesis, have led to the localisation of both acid- garded as variants of d-galactofuranose because all three
base and nucleophilic catalytic sites, Glu176 and Glu298, and possess a common molecular backbone structure. Since
a third glutamate residue (Glu28) that is not directly in- AbfD3 performs both hydrolysis and transglycosylation
volved in the catalytic mechanism, but which is probably a with p-nitrophenyl β-d-galactofuranoside, we decided to
key determinant of substrate recognition in the –1 subs- prepare the desired activated donors. In order to limit the
ite.[15] In two recent studies,[16] the glycosynthetic ability of number of synthetic steps, n-octyl β-d-galactofuranoside
AbfD3 was further investigated using a variety of candi- (3), that was specifically obtained by galactosylation of n-
dates as acceptors and/or donors. Likewise, it was shown octanol promoted by ferric chloride,[17] was used as a com-
that AbfD3 can catalyze the self-condensation of aromatic mon building block. Accordingly, d-fucofuranose derivative
glycosides derived from α-l-arabinofuranose (α-l-Araf), β- 4[6a] was obtained as described previously and used as a
d-xylopyranose (β-d-Xylp) and β-d-galactofuranose (β-d- donor for the glycosylation of p-nitrophenol (Scheme 1).
Galf), a hexose that is configurationally related to α-l-Araf. Initially, tin(iv) chloride[18] was chosen as the promoter for
These self-condensation reactions were characterised by this reaction and subsequent deacylation afforded the target
variable degrees of regioselectivity. In the case of α-l-Araf, fucofuranoside 1 in 46% yield. This modest result was at-
the major product of transglycosylation was an α-l-(1,2)- tributed to the strength of the Lewis acid which probably
arabinobioside, although other non-characterised species gave rise to degradation of both donor and product. There-
were generated. In the case of β-d-Galf, regioselectivity was fore, we decided to susbtitute tin(iv) chloride by a milder
very tight and the dominant species was a β-d-(1,2)-galacto- promoter. After experimentation, best results were obtained
furanobioside. Therefore, in order to pursue our investiga- with the boron trifluoride–etherate complex in the presence
tion into the selectivity of AbfD3, it appears useful to ex- of triethylamine.[19] Under such conditions and after
tend the rationale of the previous study. Likewise, in this Zemplen deacylation, the required fucofuranoside 1 was
study we have synthesised other novel donors that consti- isolated in a significantly higher (66%) yield for two steps.
tute structural variations of the galactofuranosyl skeleton.
The preparation of the fluorinated target 2 required a
This strategy has allowed us to examine the effects of sub- free primary hydroxy which was differentiated from the sec-
stitution of the primary hydroxy group on the 4-C- ondary ones according to a three-step procedure (trityl-
branched exocyclic arm of galactofuranosides on (i) sub- ation, benzylation, selective hydrolysis) in 62% overall
strate binding in the –1 subsite of AbfD3 and on (ii) the yield.[20] Substitution of 6-hydroxy in 5 was further per-
reactivity of secondary hydroxy functions owing to varia- formed using the well-known fluorinating agent diethylami-
tion of inductive effects. In this paper, we report the original nosulfur trifluoride (DAST) (Scheme 1, path A). Unfortu-
synthesis of required hexofuranosyl donors 1 and 2 that are nately, initial attempts under standard conditions[21] re-
galactofuranosyl derivatives modified at the 6 position, i.e. sulted in the degradation of 5 and/or the target compound
p-nitrophenyl β-d-fucofuranoside (p-NP-β-d-Fucf, 6-deoxy- 6. The lability of these molecules was undoubtedly pro-
galactofuranosyl derivative) 1 and p-nitrophenyl 6-deoxy-6- voked by the high acidity of the medium. Indeed, the ad-
fluoro-β-d-galactofuranoside (p-NP-6F-β-d-Galf) 2 (Fig- dition of triethylamine to the reaction medium attenuated
ure 1). In a second part, we have examined the AbfD3-me- this effect and allowed the synthesis of the fluoro derivative
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C. Nugier-Chauvin, V. Ferrières et al.
FULL PAPER
Scheme 1. (a) i. p-NPOH, BF3·OEt2, Et3N, CH2Cl2; ii. MeONa, MeOH (66%); (b) DAST, Et3N, CH2Cl2 (47%); (c) NaBrO3, Na2HSO3,
H2O, AcOEt (80%); (d) Ac2O, Pyr (97%); (e) i. SOCl2, Pyr, CH2Cl2; ii. RuCl3, NaIO4, CH3CN, H2O, CH2Cl2 (89%); (f) i. TBAF, THF,
Me2CO; ii. H2SO4, H2O (86%); (g) H2, Pd(OAc)2, AcOH, AcOEt (92%) (h) Ac2O, H2SO4, CH2Cl2 (98%); (i) i. p-NPOH, BF3·OEt2,
Et3N, CH2Cl2; ii. MeONa, MeOH (61%).
6, which was obtained in moderate but classical 47% yield.
Having obtained 7, the precursor 12 was prepared. First
Subsequently, debenzylation was performed under standard 7 was acetylated (Ǟ 11) and then smooth acetolysis[26]
hydrogenolysis conditions. However, pallado-catalysed de- yielded 12, which was subsequently used to glycosylate p-
protection could not be completed, even in the presence of NPOH. Once again, it is interesting to note that the pres-
excess catalyst. This phenomenon was attributed to the ence of a fluorine atom, albeit distant from the anomeric
presence of a fluorine atom at the primary position since centre, was detrimental for the coupling reaction promoted
the fucofuranosyl counterpart was readily deprotected un- by tin(iv) chloride. This is possibly due to the strong inter-
der similar conditions. To surmount this difficulty, we actions between the fluoroglycosyl donor 12 and the Lewis
turned our attention towards alternative methods and, after acid. Fortunately, the softer promoter boron trifluoride–tri-
experimentation, best results were obtained using an oxidat- ethylamine prepared in situ was an interesting alternative
ive procedure involving sodium bromate.[22] In this way, 6- to tin(iv) chloride. Finally, transesterification afforded the
fluorofuranoside 7 was obtained in 80% yield and in 20% target activated donor 2 in 61% yield for the last two steps.
overall yield over seven steps starting from 3. In order to
improve this result, we also investigated a second fluorina-
tion approach based on sulfate ring opening by fluoride
Hydrolytic Activity of Abfd3 on p-Nitrophenyl Furanosides
1 and 2
anion[23] (Scheme 1, path B). For this purpose, dihydrox-
ylated compound 8 was prepared in 79% yield from 3 as
described by Reynolds (acetonation, benzylation, selective
The specific activities of AbfD3 on the galactofuranoside
hydrolysis).[24] Further treatment by thionyl chloride gave a analogues 1 and 2 were evaluated and compared to those
mixture of two inseparable cyclic diastereoisomeric sulfites obtained in the presence of p-NP-α-l-Araf or p-NP-β-d-
with a 1.3:1 ratio. Oxidation by sodium periodate was cata- Galf (Table 1). The specific activities of AbfD3 on 1 and 2
lysed by ruthenium(iii) chloride[25] and led to the desired were lower than the activity observed on p-NP-α-l-Araf,
5,6-sulfate 9. Subsequent nucleophilic substitution with tet- but were significantly higher than that estimated for p-NP-
rabutylammonium fluoride (TBAF) followed by in situ β-d-Galf.[16] Investigation of the kinetic parameters re-
acidic desulfation gave compound 10 in 86% yield for the vealed that the use of 1 and 2 as substrates caused only
last two steps. The 5-hydroxy now being unprotected, re- minor increases in Km value when compared to the Km
moval of other benzyl groups in 2 and 3 positions was cata- value of the hydrolysis of p-NP-α-l-Araf. Importantly, these
lytically performed under hydrogen to efficiently afford the increases were insignificant when compared to the increase
desired 6-fluorofuranoside 7. In conclusion, both pathways in Km value that was observed when p-NP-β-d-Galf was
required seven steps, but the overall yield calculated from 3 used as the substrate.[16b] These results suggest that 1 and 2
was higher for path B (55%) than for path A (20%).
are well recognized by AbfD3 and highlight the drastic ef-
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Chemoenzymatic Synthesis of Unnatural Disaccharides
FULL PAPER
Table 1. Hydrolytic parameters[a] of AbfD3 towards furanoside substrates.
Km [mm]
kcat [s–1]
kcat/Km [mM–1·s–1]
Specific activity [IU·mg–1]
p-NP-α-l-Araf
p-NP-β-d-Galf
p-NP-β-d-Fucf 1
p-NP-6F-β-d-Galf 2
3.47 0.45
Ͼ 50[b]
5.72 0.44
7.59 1.81
425.79 29.02
15.02 0.46
193.13 19.97
39.26 5.21
122.58 24.35
0.13 0.02
33.70 6.05
5.28 1.96
465
0.64
119
28
[16b]
[a] Each value represents the mean of 3 measurements. [b] The Km values were estimated to be extremely high and thus incompatible with
the solubility and availability of the substrate.[16b]
fect that is caused by the presence of an extra hy- Abfd3-Catalysed Synthesis of Homo-Disaccharides
droxymethyl group at the 5-C of the arabinofuranosyl skel-
eton. Moreover, AbfD3-mediated hydrolysis of 1 and 2 was
When compounds 1 or 2 (5 mm) were incubated with 12
characterised by a 2.2 to 10.8-fold lower activity (kcat) and IU AbfD3 at 60 °C for short periods (not exceeding
3.6 to 23.2-fold lower catalytic efficiency (kcat/Km) com- 30 min), transglycosylation products were detected by high
pared with p-NP-α-l-Araf. These results can be considered performance thin layer chromatography (HPTLC) analysis.
in the light of the findings of a recently published study in In both cases, in addition to the starting furanosides and
which the three-dimensional structure of another family 51 the products of hydrolysis (reducing fucose or 6-deoxy-6-
α-l-arabinofuranosidase was reported.[27] This work em- fluorogalactose and p-nitrophenol), two novel species could
phasises that the arabinofuranosyl moiety at the –1 subsite be detected at 300 nm (Figure 2).
of the active site is tightly bound by a large number of hy-
These products were isolated after one pass by prepara-
drogen bonds, including some with the 5-OH. The data pre- tive TLC using a normal stationary phase and identified
sented here using the synthetic derivatives 1 and 2 indicate by careful examination of the corresponding NMR spectra.
that the addition of different hydrophobic groups, CH3 and Since the 13C NMR spectra revealed the presence of only
CH2F, respectively, to the 5-C of the arabinofuranosyl skel- two peaks between δ = 104 and 110 ppm, it was concluded
eton does not necessarily have a major effect on substrate that all four products correspond to disaccharides 13–16
binding at the donor subsite. Indeed the addition of a (Figure 3). A more precise analysis of the 13C NMR spec-
methyl group has a relatively minor effect on both substrate troscopic data for 13, the major disaccharide, obtained from
binding and catalysis, whereas the presence of fluorine is p-NP-β-d-Fucf 1, showed a downfield shift of the 2a-C of
mainly detrimental for catalysis. In contrast, it is interesting the reducing d-fucofuranosyl moiety from 81.5 (for, 1) to
to note that the most detrimental effect on hydrolysis and 87.6 ppm, as well as weak upfield shifts (Δδ = –1.3 ppm
substrate recognition is induced by the presence of a pri- and –2.3 ppm) for the signals of the neighbouring carbons,
mary hydroxy on 6-C (β-d-Galf). Presumably, this addition 1a-C and 3a-C, respectively. Moreover, an intense three-
constitutes a steric hindrance for binding and, by possibly bond coupling between 2a-C (δ = 87.6 ppm) and 1b-H (δ =
participating in new hydrogen bonds, might also cause the 5.11 ppm) on 1H-13C (HMBC) spectrum allowed the unam-
collapse of the normal hydrogen bonding network. Never- biguous identification of 13 as a (1,2)-disaccharide. Finally,
theless, in previous work it was shown that, despite being a the small coupling constant observed between 1b-H and 2b-
poor substrate for hydrolysis, p-NP-β-d-Galf can act as a H (J1b,2b = 1.4 Hz) clearly indicated that the glycosidic
donor for transglycosylation. Therefore, in this work we as- bond in 13 exhibited β-stereochemistry. The minor disac-
sumed that 1 and 2 might also fulfil this role.
charide 14 produced in the same reaction as 13 was also
Figure 2. HPTLC chromatograms at 300 nm for the AbfD3-catalysed transformations of 1 (a) and 2 (b).
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C. Nugier-Chauvin, V. Ferrières et al.
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Figure 3. Structures of disaccharides 13, 14 and 15, 16 obtained from the AbfD3-catalysed condensation of 1 and 2, respectively.
isolated and analysed. For this compound, the 13C chemical
Overall, the analysis of the products 13–16 revealed that
signal for 3a-C was significantly downfield shifted (Δδ = AbfD3-mediated transglycoylation of 1 and 2 was charac-
+4.8 ppm), whereas 2a-C and 4a-C exhibited upfield- terised by moderately relaxed regioselectivity. This is in
shifted signals (Δδ = –2.3 ppm and –1.5 ppm, respectively). sharp contrast to the AbfD3-mediated transglycosylation
1
These data corroborated the results obtained from the H- reactions involving p-NP-β-d-Galf, but is similar to the sit-
13C (HMBC) spectrum which showed a three-bond coup- uation observed in reactions involving p-NP-α-l-Araf. This
ling between 3a-C (δ = 82.2 ppm) and 1b-H (δ = 5.14 ppm), suggests that although p-NP-β-d-Galf is an extremely poor
the latter being characterised by a low coupling constant substrate for hydrolysis, mainly because it is poorly recog-
with 2b-H (J1b,2b = 1.1 Hz). This result lead us to conclude nized by AbfD3, the radically altered binding mode for this
that the two monosaccharide moieties in 14 are linked substrate in the –1 and/or +1 subsites of AbfD3 might actu-
through a β-(1,3) glycosidic bond.
ally increase discrimination of reactivity towards the 2-OH
As expected, the structural analysis of the products 15 vs. 3-OH of the furanosyl acceptor molecule.
and 16 arising from the action of AbfD3 on p-NP-6F-β-d-
Galf (2) revealed similar results. The major compound 15
was identified as a β-(1,2) disaccharide (Figure 3) on the
Conclusions
basis of 1D and 2D NMR experiments. Indeed, O-2 glycos-
Here we provide a further demonstration of the catalytic
ylation involved a rather large, downfield shift of 2a-C (Δδ versatility of the α-l-arabinofuranosidase AbfD3 and its
= +4.7 ppm) and the HMBC spectrum showed a three- usefulness for the synthesis of unnatural disaccharides con-
bond coupling between this carbon atom (δ = 87.6 ppm) taining hexofuranosyl entities. This study was based on the
and a signal at δ = 5.12 ppm assigned to 1b-H. According use of two hexofuranosides whose structures are related to
to HPTLC analysis of AbfD3-catalysed condensation of 2 α-l-Araf and whose electronic and steric properties were
(Figure 2, b), the regioselectivity of the reaction appeared chosen to be intermediate between those of α-l-Araf and
to be higher than the one involving p-NP-β-d-Fucf 1 and β-d-Galf, i.e. d-fucofuranosyl and 6-deoxy-6-fluoro-d-gal-
largely orientated towards the formation of the β-(1,2) di- actofuranosyl. In particular, the kinetic parameters deter-
saccharide 15 (15/16 = 6.6:1). However, the minor disaccha- mined for the AbfD3-catalysed hydrolysis of 1 reveal that
ride 16 produced in this reaction was characterised by 1D p-NP-β-d-Fucf is a very good substrate. Moreover, the
and 2D NMR (COSY) experiments. As expected, the data analysis of transglycosylation products has confirmed that
showed a downfield chemical shift of +4.7 ppm for 3a-C using hexofuranosides 1 and 2, AbfD3 can synthesise both
and upfield chemical shifts of –1.0 and –2.3 ppm for 2a-C β-(1,2) and β-(1,3) regioisomeric disaccharides. Such β-(1,2)
and 1a-C, respectively. Moreover, low coupling constants and β-(1,3)-linked galactofuranosides were previously iden-
between 1b-H and 2b-H appeared to be relevant for two β- tified in polysaccharides of Talaromyces species and were
glycosidic linkages for 15 and 16 (J1b,2b = 1.6 Hz and used as chemotaxonomic markers.[28] Consequently, these
1.4 Hz, respectively).
results open new opportunities for the use of AbfD3 as an
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Chemoenzymatic Synthesis of Unnatural Disaccharides
FULL PAPER
was performed by using 0.25 mm silica gel Si 60 plates
(200×200 mm, E. Merck). Optical rotations were measured with a
efficient biocatalyst for the synthesis of a variety of hexofur-
anosyl-containing oligosaccharides. Taken together with the
three-dimensional structure of AbfD3, which will be pub-
lished in the near future, these data provide insight into the
way in which different donor compounds are bound in the
–1 subsite. Likewise, it is expected that such knowledge will
help us to optimise the use of AbfD3 for synthetic purposes.
1
Perkin–Elmer 341 polarimeter. H, 13C, HMBC and COSY NMR
spectra were recorded with a Bruker ARX 400 spectrometer at
1
400 MHz for H, 100 MHz for 13C and 376 MHz for 19F analyses.
One-dimensional and two-dimensional spectra of the disaccharides
13–16 were all recorded with a Bruker Avance 500 spectrometer at
1
500 MHz for H and 125 MHz for 13C. Chemical shifts are given
in δ units (ppm) measured downfield from Me4Si. Assignments of
1H and 13C for carbohydrate residues of compounds 1, 2, 6, 7, 9–
12 are given in Table 2 and Table 3. Disaccharides carbon atoms
and protons are designated with the prefix “a” for the reducing
residue and “b” for the other one. Microanalyses were performed
by the Service de Microanalyse de l’ICSN (Gif sur Yvette, France)
and by the Service de Microanalyse du Centre Régional de Mesures
Physiques de l’Ouest (CRMPO, Rennes, France). The HRMS were
measured at the CRMPO with a MS/MS ZabSpec TOF Micro-
mass.
Experimental Section
General Remarks: Melting points were determined with a Reichert
microscope and are uncorrected. Thin layer chromatography (TLC)
analyses were conducted with E. Merck 60 F254 Silica Gel nonacti-
vated plates, and compounds were revealed using a 5% solution
of H2SO4 in EtOH followed by heating. High performance TLC
(HPTLC) experiments were performed with a CAMAG TLC
SCANNER 3, designed for densitometric measurement of thin-
layer chromatograms of 100×100 mm in size and controlled by the
newly designed software WinCATS. For column chromatography,
Geduran Si 60 (40–63 μm) Silica Gel was used. Preparative TLC
p-Nitrophenyl β-D
-Fucofuranoside (1): To a solution of 4[6a] (196 mg,
0.59 mmol) in CH2Cl2 (4 mL), were successively added p-nitrophe-
nol (123 mg, 0.88 mmol), Et3N (41 μL, 0.29 mmol) and BF3·OEt2
1
Table 2. H NMR (400 MHz) chemical shifts and coupling constants (H,H and H,F) for compounds 6–7, 9–12 and 2.
Compound
δ (ppm), J (Hz)
4-H 5-H
(J4,5
1-H
2-H
3-H
6a-H
(J6a,6b/J6a,F
6b-H
(J6b,5/J6b,F)
(J1.2
)
(J2,3
)
(J3,4
)
)
(J5,6a/J5,F
)
)
6[b]
5.04
(Ͻ 1.0)
4.85
(1.8)
5.06
(1.3)
5.04
(Ͻ 1.0)
5.01
(Ͻ 1.0)
6.33
(4.6)
6.19
(Ͻ 1.0)
5.66
(2.0)
3.98
(3.3)
3.93
(4.0)
4.03
(3.3)
4.01
(2.6)
5.04
(1.8)
5.32
(7.1)
5.18
(1.8)
4.28
(4.3)
4.01
(6.9)
4.02
(6.6)
3.96
(6.6)
4.06
(6.2)
5.00
(5.6)
5.58
(6.4)
5.11
(5.1)
4.20
(6.4)
4.08
(3.3)
3.88
(3.8)
4.21
(4.8)
4.12
(2.8)
4.26
(4.1)
4.25
(7.1)
4.40
(4.3)
4.05
(3.3)
3.83
4.72–4.31
(nd/nd)[c]
4.48
(9.4/46.8)
4.65–4.46
(nd/nd)
4.43
4.72–4.31
(4.3/nd)
4.42
(6.8/47.8)
4.65–4.46
(6.8/–)
4.43
(5.8/47.1)
4.55
(5.1/46.5)
4.66–4.44
(4.1/ nd)
4.66–4.44
(5.1/nd)
4.42
(6.3/17.3)
3.97–3.91
(4.8/–)
4.96
(7.4/–)
3.97–3.89
(5.8/–)
5.39
(5.6/16.3)
5.25
(4.1/20.9)
5.36
7[a]
9[b]
10[b]
11[b]
12α[b]
12β[b]
2[a]
(nd/47.1)
4.58
(9.7/46.8)
4.66–4.44
(nd/nd)
4.66–4.44
(nd/nd)
4.46
(5.1/16.5)
3.98
(5.1/14.5)
(9.6/46.8)
(6.8/47.8)
[a] Recorded in CD3OD. [b] Recorded in CDCl3. [c] nd: not determined.
Table 3. 13C NMR (400 MHz) chemical shifts and coupling constants (C,F) for compounds 6–7, 9–12 and 2.
Compound
1-C
2-C
3-C
4-C
(J4–C,F
5-C
(J5–C,F
6-C
(J6–C,F)
)
)
6[b]
7[a]
106.1
109.5
88.3
83.5
82.6
78.4
80.0
(7.0)
83.2
(6.0)
78.4
80.5
(6.0)
79.3
(5.0)
78.6
(6.0)
81.6
(6.0)
85.0
(6.0)
76.0
(19.0)
70.2
(20.0)
80.0
69.7
(21.0)
69.9
(22.0)
71.3
(21.0)
69.8
(21.0)
70.0
(20.0)
84.3
(171.0)
85.3
(169.0)
68.9
84.1
(171.0)
81.1
(173.0)
81.0
(173.0)
80.8
(172.0)
85.1
9[b]
106.1
106.4
87.4
87.3
82.5
83.2
10[b]
11[b]
12α[b]
12β[b]
2[a]
105.5
93.2
81.4
75.5
80.7
83.3
76.6
73.3
76.3
77.9
99.1
107.7
(169.0)
[a] Recorded in CD3OD. [b] Recorded in CDCl3.
www.eurjoc.org
Eur. J. Org. Chem. 2005, 4860–4869
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4865

C. Nugier-Chauvin, V. Ferrières et al.
FULL PAPER
(187 μL, 1.48 mmol). After stirring for 1 h at room temperature,
the mixture was diluted with CH2Cl2 (20 mL) and washed with a
saturated solution of aqueous NaHCO3 until complete discolour-
ation. The aqueous layers thus obtained were extracted with
CH2Cl2 (2×10 mL), and the combined organic layers were dried
(MgSO4), filtered and finally concentrated. The residue was puri-
fied by flash chromatography on silica gel eluting with light petro-
leum/AcOEt, 3:2. The fractions containing p-nitrophenyl 2,3,5-tri-
O-acetyl-β-d-fucofuranoside Rf = 0.39 (light petroleum/AcOEt,
3:2) were collected and concentrated under reduced pressure. To
the isolated mixture, a 0.1 m solution of sodium methylate (590 μL,
0.06 mmol) in MeOH (10 mL) was added. After stirring at room
temperature overnight, the mixture was neutralised by glacial
AcOH and concentrated under diminished pressure. A flash
chromatography on silica gel eluting with CH2Cl2/MeOH (9:1) af-
forded 1 as a pale yellow foam with a two-step yield of 66%
a chromatographic purification (CH2Cl2/MeOH, 49:1) gave 7
(1.00 g, 92%) as a white solid. M.p. (light petroleum) 48 °C. [α]2D0
= –83.5 (c = 0.3, MeOH). TLC (1:1 light petroleum/AcOEt): Rf =
1
0.17. 19F NMR (CD3OD): δ = –230 ppm. H NMR (CD3OD): δ
2
3
(carbohydrate ring protons, see Table 2) = 3.68 (dt, J = 9.7, J =
3
6.6 Hz, 1 H, OCH2CH2), 3.42 (dt, J = 6,6 Hz, 1 H, OCH2CH2),
1,61–1,55 (m, 2 H, OCH2CH2), 1.38–1.30 [m, 10 H, (CH2)5], 0.90
(t, 3J = 6.9 Hz, 3 H, CH3) ppm. 13C NMR (CD3OD): δ (carbo-
hydrate ring carbon atoms, see Table 3) = 69.0 (OCH2CH2), 33.0,
30.7, 30.5, 30.4, 27.2, 23.7 [(CH2)6], 14.4 (CH3) ppm. HRMS
(ESI+): [C14H27FO5 + Na]+: calcd. 317.1740; found 317.1748.
C14H27FO5 (294.4): calcd. C 57.12, H 9.25; found C 56.85, H 9.11.
n-Octyl 2,3-Di-O-benzyl-5,6-O-sulfonyl-β-D-galactofuranoside (9):
Pyridine (3.5 mL, 43.3 mmol) and thionyl chloride (1.5 mL,
20.6 mmol) diluted in CH2Cl2 (5 mL) were added dropewise to a
cooled (0 °C) solution of 8 (4.92 g, 10.4 mmol) in CH2Cl2 (25 mL).
After 10 min at room temperature, the reaction mixture was parti-
tioned between CH2Cl2 (200 mL) and 5% aqueous HCl (25 mL)
and decanted. The organic layer was further washed with 5% aque-
ous HCl (2×25 mL), saturated aqueous Na2CO3 (2×25 mL) and
brine (2×25 mL). Yield could be improved by treating the resulting
aqueous layer with CH2Cl2 (3×20 mL). Finally, the combined or-
ganic layers were dried (MgSO4), and the residue could be used
without further purification. Nevertheless, main physical data were
determined directly from the mixture of both diastereoisomeric
thionyl intermediates. TLC (light petroleum/AcOEt, 5:1): Rf = 0.34.
(111 mg, 0.39 mmol). [α]2D0 = –144.2 (c = 0.3, MeOH), [α]2D0[18]
=
–145.0 (c = 1.4, MeOH). TLC (CH2Cl2/MeOH, 9:1): Rf = 0.26.
NMR spectroscopic data were compatible with those previously
published.[18a]
n-Octyl 2,3,5-Tri-O-benzyl-6-deoxy-6-fluoro-β-D-galactofuranoside
(6): To a solution of 5 (2.04 g, 3.63 mmol) in CH2Cl2 (41 mL) were
successively added Et3N (1 mL, 7.12 mmol) and DAST (567 μL,
4.33 mmol). After stirring at room temperature for 15 min, the re-
action was quenched by a 2:1 solution of Et3N and MeOH (6 mL)
and concentrated. The resulting residue was then purified by flash
chromatography eluting with light petroleum/AcOEt (19:1 Ǟ 9:1)
to give the fluoro-compound 6 (962 mg) in 47% yield. [α]2D0 = –53.8
HRMS (ESI+): [C28H38O7S
+
Na]+: calcd. 541.2236; found
541.2247. C28H38O7S (518.7): calcd. C 64.84, H 7.38, S 6.18; found
(c = 1.0, CH2Cl2). TLC (light petroleum/AcOEt, 9:1): Rf = 0.44. C 64.81, H 7.43, S 6.17.
19F NMR (CDCl3): δ = –227 ppm. 1H NMR (CDCl3): δ (carbo-
Diastereoisomer a: 1H NMR (CDCl3): δ = 7.31–7.17 (m, 10 H,
hydrate ring protons, see Table 2) = 7.38–7.22 (m, 15 H, C6H5),
C6H5), 4.99 (d, J1,2 = 1.5 Hz, 1 H, 1-H), 4.52–4.37 (m, 6 H, 5-H,
6a-H, OCH2Ph), 4.25–4.19 (m, 2 H, 4-H, 6b-H), 3.95 (dd, J2,3
2
3
4.73–4.31 (m, 8 H, H-6a, H-6b, OCH2Ph), 3.65 (dt, J = 9.9, J =
=
3
6.6 Hz, 1 H, OCH2CH2), 3.38 (dt, J = 6.9 Hz, 1 H, OCH2CH2),
2
3.0 Hz, 1 H, 2-H), 3.87 (dd, J3,4 = 6.9 Hz, 1 H, 3-H), 3.63 (dt, J
= 9.6, 3J = 6.8 Hz, 1 H, OCH2CH2), 3.33 (dt, 1 H, 3J = 6.9 Hz,
OCH2CH2), 1.53–1.46 (m, 2 H, OCH2CH2), 1.27–1.12 [m, 10 H,
1.59–1.53 (m, 2 H, OCH2CH2), 1.34–1.27 [m, 10 H, (CH2)5], 0.88
(t, 3J = 7.1 Hz, 3 H, CH3) ppm. 13C NMR (CDCl3): δ (carbo-
hydrate ring carbon atoms, see Table 3) = 138.0, 137.8, 137.7 (Cipso
C6H5), 128.5, 128.4, 128.4, 128.3, 128.1, 128.0, 127.9, 127.9 (C6H5),
3
(CH2)5], 0.80 (t, J = 6.8 Hz, 3 H, CH3) ppm. 13C NMR (CDCl3):
δ = 137.3 (Cipso C6H5), 128.6, 128.2, 128.1 (C6H5), 106.6 (1-C), 87.8
(2-C), 83.8 (3-C), 82.8 (5-C), 79.9 (4-C), 72.3, 72.2 (OCH2Ph), 68.2
(OCH2CH2), 67.3 (6-C), 31.9, 29.5, 29.4, 26.2, 24.1, 22.7 [(CH2)6],
4
73.6 (d, JC,F = 2 Hz, OCH2Ph), 72.1, 72.0 (OCH2Ph), 67.8
(OCH2CH2), 31.9, 29.6, 29.4, 29.4, 26.2, 22.8 [(CH2)6], 14.2
(CH3) ppm. HRMS (ESI+): [C35H45FO5 + Na]+: calcd. 587.3149;
found 587.3154. C35H45FO5 (564.7): calcd. C 74.44, H 8.03; found
C 74.41, H 7.97.
1
14.2 (CH3) ppm. Diastereoisomer b: H NMR (CDCl3): δ = 7.31–
7.17 (m, 10 H, C6H5), 4.99–4.94 (m, 1 H, 5-H), 4.93 (d, J1,2
1.3 Hz, 1 H,1-H), 4.53–4.37 (m, 5 H, 6a-H, OCH2Ph), 4.27 (dd,
J6a,6b = 8.4, J5,6b = 5.3 Hz, 1 H, 6b-H), 4.00 (dd, J3,4 = 6.9, J4,5
=
n-Octyl 6-Deoxy-6-fluoro-β-D-galactofuranoside (7). Procedure A:
=
An aqueous solution of NaBrO3 (33 mL, 4.71 g, 31.21 mmol) was
added to a solution of 6 (1.96 g, 3.47 mmol) in AcOEt (40 mL).
This mixture was incubated at room temperature for 10 min, and
then an aqueous solution of NaHSO3 (83 mL, 6.40 g, 31.24 mmol)
was added. In order to complete the debenzylation, the mixture
was vigorously stirred for 30 min before more NaBrO3 (2.00 g,
13.25 mmol) was added. After 30 min, the reaction mixture was
diluted with AcOEt (60 mL) and a 20% aqueous solution of
Na2S2O3 (40 mL) and stirred for more 20 min. The resulting sus-
pension was filtered and the organic layer successively washed with
a 20% aqueous solution of Na2S2O3 (4×30 mL) and a saturated
solution of NaHCO3 (2×30 mL). The aqueous layer was then ex-
tracted with AcOEt (4×30 mL), and the combined organic layers
were dried (MgSO4), concentrated under diminished pressure and
chromatographed (light petroleum/AcOEt, 1:1) to afford 7 (820 mg,
80%) as a white solid. Procedure B: A solution of dibenzylated
4.3 Hz, 1 H, 4-H), 3.93 (dd, J2,3 = 3.0 Hz, 1 H, 2-H), 3.83 (dd, 1
3
H, 3-H), 3.59 (dt, 2J = 9.7, J = 6.6 Hz, 1 H, OCH2CH2), 3.32 (dt,
3J = 6.6 Hz, 1 H, OCH2CH2), 1.53–1.46 (m, 2 H, OCH2CH2),
3
1.27–1.13 [m, 10 H, (CH2)5], 0.80 (t, J = 7.1 Hz, 3 H, CH3) ppm.
13C NMR (CDCl3): δ = 137.2 (Cipso C6H5), 128.6, 128.2, 128.1
(C6H5), 106.4 (1-C), 87.7 (2-C), 82.9 (3-C), 79.7 (4-C), 78.9 (5-C),
72.4, 72.2 (OCH2Ph), 68.3 (6-C), 68.1 (OCH2CH2), 31.9, 29.5, 29.4,
29.3, 26.2, 22.7 [(CH2)6], 14.2 (CH3) ppm. The crude oil previously
obtained was diluted in a CH2Cl2/CH3CN/H2O mixture (2:2:3,
77 mL) before adding NaIO4 (4.46 g, 20.9 mmol) and rutheni-
um(iii) chloride (10 mg). This solution was vigorously stirred at
room temperature for 30 min and then diluted with CH2Cl2
(200 mL). After separation, the organic layer was washed twice
with water (50 mL) and the combined aqueous layers extracted
with CH2Cl2 (3×25 mL). Finally, all organic phases were dried
with MgSO4, concentrated under diminished pressure before chro-
intermediate 10 (1.77 g, 3.73 mmol) in AcOH/AcOEt (1:1, 18 mL) matographic purification (light petroleum/AcOEt, 9:1) to give 9
added of palladium(ii) acetate (177 mg) was stirred for 72 h under
1 atm of hydrogen. After completion of the deprotection, the sol-
vents were removed by co-evaporation with MeOH (5×50 mL) and
(4.95 g) in 89% yield for two steps. [α]2D0 = –58.2 (c = 1.1, CH2Cl2).
TLC (light petroleum/AcOEt, 5:1): Rf = 0.30. H NMR (CDCl3):
δ (carbohydrate ring protons, see Table 2) = 7.40–7.26 (m, 10 H,
1
4866
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2005, 4860–4869

Chemoenzymatic Synthesis of Unnatural Disaccharides
FULL PAPER
C6H5), 4.65–4.46 (m, 6 H, 6-aH, 6-bH, OCH2Ph), 3.69 (dt, 2J = 1,2,3,5-Tetra-O-acetyl-6-deoxy-6-fluoro-β-
D-galactofuranose (12):
9.6, 3J = 6.6 Hz, 1 H, OCH2CH2), 3.41 (dt, 3J = 6.9 Hz, 1 H,
Ac2O (50 μL, 0.53 mmol) and H2SO4 (1.4 μL, 0.03 mmol) were suc-
OCH2CH2), 1.62–1.55 (m, 2 H, OCH2CH2), 1.35–1.21 [m, 10 H, cessively added to a solution of 11 (56 mg, 0.13 mmol) in dry
(CH2)5], 0.89 (t, 3J = 7.1 Hz, 3 H, CH3) ppm. 13C NMR (CDCl3): δ
CH2Cl2 (560 μL). After stirring at room temperature for 24 h, the
(carbohydrate ring carbon atoms, see Table 3) = 137.1, 137.0 (Cipso
reaction was quenched by adding Et3N, the solvent removed under
C6H5), 128.7, 128.3, 129.2, 128.2 (C6H5), 72.5, 72.3 (OCH2Ph), 68.4 diminished pressure and the crude residue purified by chromatog-
(OCH2CH2), 31.9, 29.5, 29.4, 29.3, 26.2, 22.7 (OCH2CH2), 14.2
(CH3) ppm. HRMS (ESI+): [C28H38O8S + K]+: calcd. 573.1925;
found 573.1923. C28H38O8S (534.7): calcd. C 62.90, H 7.16, S 6.00;
found C 62.81, H 7.11, S 5.98.
raphy (light petroleum/AcOEt, 4:1 Ǟ 3:2). This acetolysis pro-
cedure yielded an inseparable anomeric mixture of 12 (α/β = 1:4.2,
46 mg, 98%) as a colourless oil. TLC (light petroleum/AcOEt, 3:2):
Rf = 0.40. HRMS (ESI+): [C14H19FO8 + Na]+: calcd. 373.0911;
found 373.0916. C14H19FO9 (350.3): calcd. C 48.00, H 5.47; found
1
C 48.05, H 5.57. α-Anomer: 19F NMR (CDCl3): δ = –233 ppm. H
n-Octyl
2,3-Di-O-benzyl-6-deoxy-6-fluoro-β-D-galactofuranoside
(10): To a solution of 9 (4.30 g, 8.04 mmol) in acetone (43 mL) was
added a 1 m solution of TBAF in THF (12.1 mL, 12.1 mmol) and
stirred at room temperature for 1 h. After concentration, the crude
oil was diluted with THF (43 mL) before adding a solution of con-
centrated H2SO4 (1.3 mL, 24.4 mmol) and H2O (434 μL,
24.1 mmol). The reaction media was stirred at room temperature
for 15 min and then neutralised with Et3N, concentrated under re-
duced pressure and partitioned between AcOEt (200 mL) and brine
(50 mL). The organic phase was washed with aqueous saturated
Na2CO3 and the aqueous layers extracted with AcOEt (3×25 mL).
Finally, the combined organic layers were dried (MgSO4), the sol-
vent removed and the residue chromatographically purified (light
petroleum/AcOEt, 9:1 Ǟ 5:1). This procedure afforded the target
compound 10 as colourless oil (3.28 g, 86%). [α]2D0 = –69.9 (c = 1.1,
CH2Cl2). TLC (5:1 light petroleum/AcOEt): Rf = 0.20. 19F NMR
NMR (CDCl3): δ (carbohydrate ring protons, see Table 2) = 2.13,
2.12, 2.11, 2.08 (4 s, 12 H, CH3CO) ppm. 13C NMR (CDCl3): δ =
carbohydrate ring carbon atoms (see Table 3), 170.1, 169.9, 169.5,
169.3 (CO), 21.1, 20.9, 20.7, 20.5 (CH3CO). β-Anomer: 19F NMR
1
(CDCl3): δ = –231 ppm. H NMR (CDCl3): δ (carbohydrate ring
protons, see Table 2) = 2.14, 2.12, (4 s, 12 H, CH3CO) ppm. 13C
NMR (CDCl3): δ (carbohydrate ring carbon atoms, see Table 3) =
170.1, 169.9, 169.5, 169.2 (CO), 21.1, 20.9, 20.7 (CH3CO) ppm.
p-Nitrophenyl 6-Deoxy-6-fluoro-β-D-galactofuranoside (2): To a
solution of 12 (218 mg, 0.62 mmol) in CH2Cl2 (4 mL), were suc-
cessively added p-nitrophenol (130 mg, 0.93 mmol), Et3N (44 μL,
0.31 mmol) and BF3·OEt2 (197 μL, 1.56 mmol). After stirring at
room temperature for 1 h, the mixture was diluted with CH2Cl2
(20 mL), and washed with a saturated solution of aqueous
NaHCO3 until complete discolouration. The aqueous layers thus
obtained were extracted with CH2Cl2 (2×10 mL) and the com-
bined organic layers were dried (MgSO4), filtered and finally con-
centrated. The residue was purified by flash chromatography on
silica gel eluting with light petroleum/AcOEt (7:3). The fractions
containing p-nitrophenyl 2,3,5-tri-O-acetyl-6-deoxy-6-fluoro-β-d-
galactofuranoside [TLC (light petroleum/AcOEt, 3:2): Rf = 0.42]
were collected and concentrated under reduced pressure. To the
isolated mixture dissolved in anhydrous MeOH (12 mL), a decimo-
lar solution of sodium methylate (620 μL, 0.06 mmol) in MeOH
was added. After stirring overnight at room temperature, the mix-
ture was neutralised by glacial AcOH and concentrated under re-
duced pressure. A flash chromatography on silica gel eluting with
CH2Cl2/MeOH (9:1) afforded 2, as a colourless solid with a two-
step yield of 61% (115 mg, 0.38 mmol). Mp 144 °C. [α]2D0 = –197.5
(c = 0.4, MeOH). TLC (CH2Cl2/MeOH, 9:1): Rf = 0.53. 19F NMR
(CD3OD): δ = –230 ppm. 1H NMR (CD3OD): δ (carbohydrate ring
1
(CDCl3): δ = –229 ppm. H NMR (CDCl3): δ (carbohydrate ring
protons, see Table 2) = 7.38–7.28 (m, 10 H, C6H5), 4.62–4.49 (m, 4
H, OCH2Ph), 3.67 (dt, 2J = 9.7, 3J = 6.9 Hz, 1 H, OCH2CH2), 3.40
3
3
(dt, J = 6.6 Hz, 1 H, OCH2CH2), 2.37 (d, J = 7.6 Hz, 1 H, OH),
1.61–1.54 (m, 2 H, OCH2CH2), 1.35–1.21 [m, 10 H, (CH2)5], 0.88
(t, 3J = 7.1 Hz, 3 H, CH3) ppm. 13C NMR (CDCl3): δ (carbo-
hydrate ring carbon atoms, see Table 3) = 137.7, 137.3 (Cipso C6H5),
128.6, 128.5, 128.1, 128.1, 128.0, 127.9 (C6H5), 72.4, 72.1
(OCH2Ph), 67.9 (OCH2CH2), 31.9, 29.6, 29.4, 29.4, 26.2, 22.8
[(CH2)6], 14.2 (CH3) ppm. HRMS (ESI+): [C28H39FO5 + K]+:
calcd. 513.2419; found 513.2421. C28H39FO5 (474.6): calcd. C
70.86, H 8.28; found C 70.85, H 8.31.
n-Octyl 2,3,5-Tri-O-acetyl-6-deoxy-6-fluoro-β-D-galactofuranoside
(11): Acetylation of 7 (780 mg, 2.65 mmol) was performed at room
temperature in dry pyridine (6.5 mL, 79.71 mmol) using Ac2O
(7.5 mL, 79.86 mmol) as acylating agent. After 48 h stirring, con-
centration under reduced pressure and co-distillation with MeOH
(3×10 mL), the crude oil was diluted with AcOEt (100 mL) and
successively washed with 5% aqueous HCl (2×20 mL), saturated
aqueous Na2CO3 (2×20 mL) and brine (20 mL). The resulting
aqueous layers were then extracted with AcOEt (3×10 mL) and
the combined organic layers were dried (MgSO4) and concentrated.
Finally, purification of the crude oil by flash chromatography (light
petroleum/AcOEt, 4:1) yielded the desired product 11 (1.08 g,
97%). [α]2D0 = –47.4 (c = 0.6, CH2Cl2). TLC (light petroleum/Ac-
3
protons, see Table 2) = 8.22 (d, J = 9.4 Hz, 2 H, Hm C6H4), 7.21
(d, 2 H, Ho C6H4) ppm. 13C NMR (CD3OD): δ (carbohydrate ring
carbon atoms, see Table 3) = 163.4 (Cipso C6H4), 143.6 (Cp C6H4),
126.6 (Cm C6H4), 117.6 (Co C6H4) ppm. HRMS (ESI+):
[C12H14FNO7 + Na]+: calcd. 342.0391; found 342.0388.
AbfD3 Production and Activity Measurement: Recombinant arabin-
ofuranosidase AbfD3 expressed from the plasmid-borne AbfD3
gene was produced and purified from Escherichia coli cells as de-
scribed previously.[15,29] The hydrolytic activity of AbfD3 was quan-
tified after incubation of the enzyme with p-NP furanosides (5 mm
in 900 μL 50 mm sodium acetate, pH 5.8) at 60 °C. In each case
1
OEt, 4:1): Rf = 0.31. 19F NMR (CDCl3): δ = –231 ppm. H NMR
2
(CDCl3): δ (carbohydrate ring protons, see Table 2) = 3.64 (dt, J
= 9.7, 3J = 6.6 Hz, 1 H, OCH2CH2), 3.43 (dt, 3J = 6.4 Hz, 1 H, 100 μL of enzyme solution was employed, but the amounts of en-
OCH2CH2), 2.14, 2.09, 2.08 (3 s, 9 H, CH3CO), 1.61–1.52 (m, 2 zyme were adapted for the different substrates: p-NP-α-l-Araf, 0.10
3
H, OCH2CH2), 1.37–1.21 [m, 10 H, (CH2)5], 0.87 (t, J = 6.9 Hz,
3 H, CH3) ppm. 13C NMR (CDCl3): δ (carbohydrate ring carbon
atoms, see Table 3) = 170.1, 169.7 (CO), 67.9 (OCH2CH2), 31.9,
IU, p-NP-β-d-Galf 1.60 IU, p-NP-6F-β-d-Galf, 0.32 IU and p-NP-
β-d-Fucf, 0.11 IU. Continuous release of p-nitrophenol was mea-
sured at 401 nm. One unit of activity corresponds to the amount
29.4, 29.3, 26.0, 22.7 [(CH2)6], 20.9, 20.7 (CH3CO), 14.2 of enzyme releasing 1 μmol of p-nitrophenol per minute. Initial rate
(CH3) ppm. HRMS (ESI+): [C20H33FO8 + Na]+: calcd. 443.2057;
found 443.2060. C20H33FO8 (420.5): calcd. C 57.13, H 7.91; found
C 57.39, H, 7.91.
conditions and a suitable substrate concentration (0.5 to 40 mm)
were used in order to determine the kinetic parameters Km and kcat
from Lineweaver–Burk plots.
Eur. J. Org. Chem. 2005, 4860–4869
www.eurjoc.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4867

C. Nugier-Chauvin, V. Ferrières et al.
FULL PAPER
Transglycosylation Reactions: A 5 mm aqueous solution of 1 or 2
(1.4 mL) was incubated with 12 IU AbfD3 under magnetic stirring
for 30 min at 60 °C. Reactions were quenched by enzyme denatur-
ation at 100 °C for 10 min. After lyophilizing, the residue was dis-
solved in water (100 μL) and the products of transglycosylation
reactions were purified by preparative TLC using 4:1 CH2Cl2/
H, 2b-H), 4.01–3.98 (m, 1 H, 5a-H), 3.90–3.88 (m, 1 H, 4b-H),
3.86–3.83 (m, 1 H, 5b-H), 3.84 (dd, J3b,4b = 6.5 Hz, 1 H, 3b-H)
ppm. 13C NMR (D2O): δ = 160.9 (Cipso C6H4), 142.3 (Cp C6H4),
125.8 (Cm C6H4), 117.3 (Co C6H4), 107.2 (1b-C), 105.4 (1a-C), 86.1
(6a-C, J6a,F = 160.3 Hz), 84.2 (6b-C, J6b,F = 161.2 Hz), 82.9 (4a-
C), 82.6 (3a-C), 82.3 (2a-C), 81.6 (4b-C), 81.5 (2b-C), 76.4 (3b-C),
MeOH as the mobile phase. The desired products were detected by 71.3 (5b-C), 69.9 (5a-C) ppm.
UV absorption (254 nm), collected from the plates and extracted
Supporting Information: (see also the footnote on the first page of
this article) One-dimensional and two-dimensional correlation
NMR spectra for new monosaccharides and disaccharides.
with 1:1 CH2Cl2/MeOH (4 mL). After filtration and freeze-drying,
the isolated disaccharides 13, 14 and 15, 16, respectively obtained
form 1 and 2, were characterised by NMR and high resolution
mass spectrometry.
Acknowledgments
p-Nitrophenyl β-D-Fucofuranosyl-(1Ǟ2)-β-D-fucofuranoside (13):
HPTLC (CH2Cl2/MeOH, 4:1): Rf = 0.64. 1H NMR (D2O): δ = 8.16
3
Bursaries for R. E. and G. L were provided by the French Ministry
of Education, Research and Technology. Part of this work was fin-
ancially supported by the lycoffal initiative. We wish to thank J. P.
Guégan (ENSCR), S. Sinbandhit (Centre Régional de Mesures
Physiques de l’Ouest, CRMPO, University of Rennes 1, France)
and A. Bondon (University of Rennes 1) for recording NMR spec-
tra and P. Guenot and P. Jéhan (CRMPO) for recording HRMS
spectra. Likewise, N. Aubry is thanked for her assistance with the
acquisition of kinetic data and G. Paës is thanked for his useful
comments.
(d, J = 9.3 Hz, 2 H, Hm C6H4), 7.16 (d, 2 H, Ho C6H4), 5.84 (s, 1
H, 1a-H), 5.11 (d, J1b,2b = 1.4 Hz, 1 H, 1b-H), 4.32 (dd, J1a,2a
Ͻ
1, J2a,3a = 4.0 Hz, 1 H, 2a-H), 4.12 (dd, J3a,4a = 6.0 Hz, 1 H, 3a-
H), 4.02 (dd, J2b,3b = 3.8 Hz, 1 H, 2b-H), 3.87–3.82 (m, 2 H, 4a-
H, 3b-H), 3.77–3.72 (m, 1 H, 5b-H), 3.70–3.64 (m, 1 H, 4b-H),
3.55–3.48 (m, 1 H, 5a-H), 1.13 (d, J5a,6a = 6.11 Hz, 3 H, 6a-H),
0.99 (d, 1 H, 6b-H, J5b,6b = 6.5 Hz) ppm. 13C NMR (D2O): δ =
161.2 (Cipso C6H4), 142.1 (Cp C6H4), 126.1 (Cm C6H4), 116.7 (Co
C6H4), 107.3 (1b-C), 104.3 (1a-C), 87.6 (2a-C), 87.5 (4b-C), 87.2
(4a-C), 81.6 (2b-C), 77.3 (3b-C), 75.3 (3a-C), 66.8 (5b-C), 62.5 (5a-
C), 18.2 (6a-C, 6b-C) ppm. HRMS (ESI+): [C18H25NO11 + Na]+:
calcd. 454.1325; found 454.1331.
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HPTLC (CH2Cl2/MeOH, 4:1): Rf = 0.68. 1H NMR (D2O): δ = 8.19
(d, 3J = 9.3 Hz, 2 H, Hm C6H4), 7.17 (d, 3J = 9.3 Hz, 2 H, Ho
C6H4), 5.79 (d, J1a,2a = 1.1 Hz, 1 H, 1a-H), 5.14 (d, J1b,2b = 1.4 Hz,
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= 4.8 Hz, 1 H, 3a-H), 4.07 (dd, J2b,3b = 3.1 Hz, 1 H, 2b-H), 3.99–
3.93 (m, 2 H, 4a-H, 5a-H), 3.87 (dd, J3b,4b = 6.1 Hz, 1 H, 3b-H),
3.83 (m, 1 H, 5b-H), 3.69 (t, J4b,5b = 6.1 Hz, 1 H, 4b-H), 1.22 (d,
J5a,6a = 6.9 Hz, 3 H, 6a-H), 1.19–1.16 (m, 3 H, 6b-H) ppm. 13C
NMR (D2O): δ = 161.2 (Cipso C6H4), 141.9 (Cp C6H4), 126.1 (Cm
C6H4), 116.8 (Co C6H4), 106.6 (1b-C), 105.7 (1a-C), 87.6 (4b-C),
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D
1
4:1): Rf = 0.62. H NMR (D2O): δ = 8.16 (d, J = 9.1 Hz, 2 H, Hm
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3.87 (m, J6b,F = 47.1 Hz, 2 H, 6b-H), 4.06 (dd, J2b,3b = 3.1 Hz, 1
4868
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Org. Chem. 2005, 4860–4869

Chemoenzymatic Synthesis of Unnatural Disaccharides
FULL PAPER
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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