Fig. 1
with lactoside 1 (1.7 g) and sialic acid (1 equiv.) at the beginning of
the fed-batch phase. TLC analysis of the fermentation time course
showed that 1 accumulated transiently into the cell and was
converted into the more polar sialyllactoside 3, which accumulated
first in the intracellular fraction, before being partly released in the
supernatant. Both fractions were purified as previously described,
by adsorption on activated charcoal followed by ion exchange
chromatography, affording 840 mg of the expected trisaccharide 3.
The structure of this sialyllactoside was confirmed by mass
Scheme 2 Preparation of GM
then Me S; ii) cysteamine hydrochloride (10 equiv.), H
RT; iii) NaBH CN, BSA, H O, 37 uC; iv) pentafluorophenyl 4-(pyrrol-1-
yl)butanoate, Et N, DMF, RT, 94% (2 steps).
3
conjugates. i) O
3
, MeOH, 250 uC, 1 h
2
2
O, UV (254 nm),
3
2
3
+
+
1
spectrometry (m/z: 696 [M + H] ; 718 [M + Na] ) as well as by H
and C NMR spectroscopy, which also demonstrated the good
13
trivial and acceptors have to be screened to extend our
methodology.
purity of the product. As allyl b-lactoside proved to be a good
To demonstrate the field of application offered by this
2
acceptor for the synthesis of 3, the corresponding GM saccharidic
approach, two GM glycoconjugates were prepared. The protein
3
portion GalNAcb1,4(NeuAca2,3)Galb1,4Glc was prepared.
5
Cultivation of strain TA05 on glycerol supplemented with 1
conjugate 11 (Scheme 2) was obtained by ozonolysis of 3, followed
by reductive amination of aldehyde 9 with BSA as previously
7
described by Hall. After purification by filtration on a P6 biogel
(
1.5 g) and NeuAc, afforded the allyl tetrasaccharide 4 (1.25 g),
13
1
which was characterized by H, C NMR and mass spectrometry
after purification as described earlier.
3
cartridge, the GM content (26 mol of trisaccharide per mol of
10
protein) was determined by a phenol–sulfuric acid assay.
Propargyl b-lactoside 2, which was prepared on 50 g scale in the
laboratory, was also efficiently internalized and glycosylated within
the living factory process. Saccharidic portions of GM and GM
The pyrrole derivative 12 was further synthesized to develop a
microarray for the study of carbohydrate–protein interactions.
Pyrrole conjugates can be electropolymerized into thin electro-
3
2
bearing the terminal alkyne function at the reducing end were
efficiently prepared following the previous procedure. The tri- and
tetrasaccharides 5 (670 mg) and 6 (900 mg) were isolated following
the same procedure from 1-litre culture (1.7 g and 3 g of 2
respectively) and were characterized by MS and FT-IR spectro-
11
conducting films that have proved to be remarkable biosensors.
Compound 12 was prepared through radical addition of a
cysteamine linker onto the allyl group of 3. The reaction was
carried out in water by UV irradiation (254 nm) at room
temperature for 48 hours with an excess of cysteamine hydro-
2
1
scopy. The characteristic CMC vibrational band at 2114 cm
indicated the presence of the alkynyl group. NMR spectra of 5 and
confirmed the oligosaccharidic structures, although the proton
12
chloride (10 equiv.). The resulting amine 10 was freeze dried and
directly used in the next reaction without prior treatment, although
it can be purified by Dowex1 50W64 cation exchange resin. The
pyrrole motif was finally introduced by coupling 10 with an excess
6
and carbon signals of the triple bond were sometimes missing on
1
3
spectra recorded in D O. This phenomenon, which was dependent
2
of a pentafluorophenyl activated carboxylic pyrrole (see ESI{) in
anhydrous dimethylformamide in the presence of triethylamine.
After purification by flash chromatography on silica gel, 12 was
isolated in 94% yield after the two steps.
on the NMR sample preparation, could be suppressed in DMSO-
d6. However, the resolutions of the spectra were dramatically
decreased. For unambiguous characterization, NMR experiments
2
were finally recorded in D O after reduction of the alkyne group
In conclusion we have demonstrated that b-lactosides bearing
latent chemical functions such as an allyl and a propargyl group,
are efficiently internalized by the lactose permease into metabo-
lically engineered E. coli cells. These lactosyl acceptors can be
glycosylated by appropriate glycosyltransferases giving access, on
the gram scale, to conjugatable forms of GM2 and GM3
saccharidic portions. Synthesis of GM2 glycoclusters based on
click chemistry, is currently underway and will be reported
by hydrogenolysis over Pd(OH)2.
Other b-lactosides with nitrogen-containing aglycons were also
tested as potential acceptors in the fermentation process. N-Allyl
acetamide b-lactoside failed to be internalized into the cell whereas
an azido ethyl lactoside gave poor results. The polarity of the
aglycon seems to have a significant influence on the ability to be
internalized, showing that the choice of suitable acceptors is not
This journal is ß The Royal Society of Chemistry 2005
Chem. Commun., 2005, 2558–2560 | 2559
Fort, Sebastien
