An expeditious and efficient synthesis of β-d-galactopyranosyl-(1→3)-d-N-acetylglucosamine (lacto-N-biose) using a glycosynthase from Thermus thermophilus as a catalyst

Article abstract of DOI:10.1016/j.tetasy.2009.05.007

Mutant glycosynthases or transglycosidases obtained from a Thermus thermophilus β-d-glycosidase (TtbGly) efficiently catalyzed the synthesis of β-(1→3)-disaccharides. Unfortunately, this regioselectivity was changed to the β-(1→4) one when N-acetylglucosa

Full text of DOI:10.1016/j.tetasy.2009.05.007

Tetrahedron: Asymmetry 20 (2009) 1243–1246
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Tetrahedron: Asymmetry
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An expeditious and efficient synthesis of b-D-galactopyranosyl-
(1?3)-D-N-acetylglucosamine (lacto-N-biose) using a glycosynthase
from Thermus thermophilus as a catalyst
*
Ayité D’Almeida, Marina Ionata, Vinh Tran, Charles Tellier, Michel Dion, Claude Rabiller
Biotechnologie, Biocatalyse, Biorégulation (UMR CNRS 6204), Nantes University, 2, rue de la Houssinière, BP 92208, F-44322 Nantes, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Mutant glycosynthases or transglycosidases obtained from a Thermus thermophilus b-D-glycosidase (Ttb-
Gly) efficiently catalyzed the synthesis of b-(1?3)-disaccharides. Unfortunately, this regioselectivity was
changed to the b-(1?4) one when N-acetylglucosamine derivatives were used as acceptors, thus preclud-
Received 16 March 2009
Accepted 8 May 2009
Available online 6 June 2009
ing the possibility of synthesizing
GlcpNAc, which are useful synthons for the synthesis of antigen determinants. In contrast, we show in
this work that, in the presence of phenyl 2-amino-1-thio-b- -glucopyranoside, the ‘normal’ b-(1?3) reg-
ioselectivity of E338G TtbGly glycosynthase takes place. Thus, transglycosylations using -galactosyl or
-glucosyl fluorides gave the corresponding phenyl b- -glycopyranosyl-(1?3)-2-amino-2-deoxy-1-
thio-b- -glucopyranosides in high yields (88–97%). Subsequent selective N-acylation followed by NBS/
water deprotection of the thiophenyl group afforded lacto-N-biose in high overall yields.
Ó 2009 Elsevier Ltd. All rights reserved.
D-Galp-b-(1?3)-D-GlcpNAc (lacto-N-biose) or D-Glcp-b-(1?3)-D-
D
a
a
D
D
1. Introduction
tions.13,14 We have validated the Withers and Planas strategy with
this b- -glycosidase by producing very efficient mutant glyco-
D
Carbohydrate ligands play an important role in a number of bio-
logical mechanisms and there remains a need for green and low
cost methods for their preparation despite the considerable devel-
opment of efficient synthetic methods in this field. Over the last
decades, enzyme-catalyzed reactions have proved to be very effi-
cient in the construction of the glycosidic bond due to their stere-
oselectivity. For this purpose, retaining glycoside hydrolases is
particularly useful, but their use was limited by the competition
between hydrolysis and transglycosylation, both reactions cata-
lyzed by these enzymes. However, site-directed mutagenesis, a
strategy developed at the same time by Withers1–7 and Planas8–12
on retaining b-glycosidases, afforded new enzymes called glyco-
synthases. This approach was based on the mutation of the active
nucleophile site, an aspartate or a glutamate, by a neutral amino-
synthases able to catalyze the one-step synthesis of b-(1?3) disac-
charides.15,16 In addition, we have developed a new strategy based
on directed evolution techniques which provided clones com-
pletely devoid of their hydrolytic activity while keeping a high
transfer capability.17,18 These enzymes were called transglycosid-
ases. Combining the two strategies, we have described the
two-step synthesis of 4-nitrophenyl
(1?3)- -Glcp 2, an analogue of antigen H1, as shown in Scheme
1 (overall yield 35%).19 Unfortunately, when aryl 2-(acylamino)-
2-deoxy-b- -glucopyranosides [compounds necessary for the syn-
thesis of antigen HI: -Fucp- -(1?2)- -Galp b-(1?3)- -GlcpNAc]
L-Fucp-a-(1?2)-D-Galp-b-
D
D
L
a
D
D
were used as acceptors, the T. thermophilus glycosynthases, or the
evolved T. thermophilus transglycosidases obtained by directed
evolution, both catalyzed the synthesis of
D-Galp b-(1?4)-D-
acid (glycine, serine or alanine) and the use of donors with
a
GlcpNAc disaccharides and not that of the b-(1?3) regioisomer.20
Thus, an alternative would be to replace the bulky acetamido
group of the acceptor by the amino group, assuming that the latter
would not induce any significant conformational changes in the
active site of the enzyme.
anomeric configurations (such as -glycosyl fluorides). These
a
glycosidases provided very high transglycosidation yields because
they were unable to hydrolyze the transglycosylation product. In
our laboratory, we have cloned and characterized a thermophilic
b-glycosidase from Thermus thermophilus. This enzyme has been
proven to accept b-
b-D-galactosides as donors while the b-1,3 regioselectivity was
D-fucosides (main activity), b-D-glucosides and
2. Results and discussion
observed in the hydrolytic and in the transglycosylation reac-
Herein we report the successful synthesis of phenyl b-
pyranosyl-(1?3)-2-amino-2-deoxy-1-thio-b- -glucopyranoside 4
using phenyl 2-amino-1-thio-b- -glucopyranoside 3 as an acceptor
substrate. Disaccharide 4 easily affords the 2-acetamido analogue 5
D-galacto-
D
D
* Corresponding author. Tel.: +33 (0)2 51 12 57 32; fax: +33 51 12 56 37.
E-mail address: claude.rabiller@univ-nantes.fr (C. Rabiller).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.05.007

1244
A. D’Almeida et al. / Tetrahedron: Asymmetry 20 (2009) 1243–1246
Glycosynthase from
Thermus thermophilus
OH
OH
OH
OH
OH
OH
O
O
O
HO
O
HO
O
O
O
+
HO
HO
HO
1
Ph
Ar
OH
OH
OH
OH
OH
OH
F
OH
Ar
O
α-L-transfucosidase from
O
O
HO
O
Thermotoga maritima
O
O
HO
2
1
Ar
+
OH
O
OH
HO
OH
O
OH
HO
OH
Scheme 1. Two-step enzymatic synthesis of
L-Fucp-
a-(1?2)-
D
-Galp-b-(1?3)-
D
-Glcp 2.17
OH
OH
OH
OH
OH
Glycosynthase E338G from
Thermus thermophilus
OH
O
O
O
HO
O
HO
O
S
S
Ph
+
HO
HO
Ph
HO
4
OH
NH3+ Cl-
NH3+ Cl-
Ac2O, pyridine
OH
OH
3
F
OH
OH
OH
OH
NBS / H2O
OH
O
O
HO
O
O
O
HO
O
HO
OH
S
HO
Ph
NHAc
OH
6
NHAc
OH
5
Scheme 2. Synthesis of
D-Galp b-(1?3)-D-GlcpNAc (lacto-N-biose) 6.
via selective acylation of nitrogen. Next, NBS/H2O deprotection of
the thiophenyl group gives b- -galactopyranosyl-(1?3)-2-acetam-
ido-2-deoxy-b- -glucopyranose 6 (Scheme 2).
The choice of the S-Ph group at the anomeric position for the
acceptor was made since we have already shown that the presence
of a b-aryl anomeric group is necessary for either the catalytic
activity of wild type glycosidase from T. thermophilus or for the de-
rived glycosynthases to ensure a correct positioning of the acceptor
in the active site.15 Thus, the synthesis of 3 was achieved according
to Scheme 3.21
reactions were observed in the presence of GalF: transglycosyla-
tion and the spontaneous hydrolysis of the fluoride. When operat-
ing at 37 °C and with a sufficient amount of enzymatic activity, the
latter reaction slowly resulted in mixtures containing low amounts
of galactose. In previous work, we had shown that the glycosynth-
ases from T. thermophilus did not catalyze the hydrolysis of GalF or
its self-condensation, which led to only one transglycosylation
product.15 Our experimental results confirmed our predictions:
acceptor 3 was a very good substrate for the enzyme and disaccha-
ride 4 (structure established by means of NMR spectroscopy, see
Section 4) could be selectively obtained in high yields. Moreover,
we have already reported that our glycosynthase also accepted
D
D
Alternatively, 3 can be prepared via a slightly shorter strategy
starting from 2-(acetylamino)-2-deoxy-2,3,4,6-tetra-O-acetyl-b-D-
glucopyranoside.22
a
-glucopyranosyl fluoride (GlcF) as a donor.15 Thus, in order to
Herein, the reaction shown in Scheme 2 was catalyzed by the
compare the reactivities of GalF and GlcF in the presence of 3,
the results obtained with both fluorides under different experi-
mental conditions are shown in Table 1. As expected, phenyl
glycosynthase E358G from T. thermophilus, using the readily avail-
able
a
-galactopyranosyl fluoride (GalF)12 as a donor. Only two
OH
OAc
OH
O
O
O
HO
HO
AcO
AcO
HO
HO
b
a
OH
OAc
OH
N
N
NH2
C6H4-pOCH3
C6H4-p OCH3
c
OH
O
OAc
OAc
O
e
f
OAc
d
O
HO
HO
AcO
AcO
AcO
AcO
O
AcO
AcO
S-Ph
S-Ph
OAc
NH3+Cl-
NHTFA
OAc
NHTFA
NH3+Cl-
3
Scheme 3. Synthesis of phenyl 1-amino-deoxy-1-thio-b-
D-glucopyranoside 3. Reagents and conditions: (a) p-CH3O–C6H4–CHO, NaOH 1 mol/L (95%); (b) (Ac)2O, pyridine,
DMAP (97%); (c) HCl 5 mol/L, acetone (98%); (d) TFA, pyridine, CH2Cl2 95%) ; (e) PhSH, BF3O(Et)2 (75%); (f) (i) NaOH/MeOH 5 mol/L, (ii) HCl 2 mol/L, MeOH (95%).

A. D’Almeida et al. / Tetrahedron: Asymmetry 20 (2009) 1243–1246
1245
Table 1
4.1.1. Phenyl b-
thio-b- -glucopyranosides 4 and 7
GalF (182 g/mol, 255 mg, 1.4 mmol) or GlcF (182 g/mol, 218 mg,
1.2 mol) and phenyl 2-amino-1-thio-b- -glucopyranoside (HCl
D-glycopyranosyl-(1?3)-2-amino-2-deoxy-1-
Yields (*calculated from the acceptor) obtained for the synthesis of phenyl b-D-
glycopyranosyl-(1?3)-2-amino-2-deoxy-1-thio-b-D-glucopyranosides 4 and 7
D
Donor
Fluoride (mmol/L)
Acceptor 3 (mmol/L)
Yield* for 4 or 7
D
salt, 307.8 g/mol, 308 mg, 1 mmol), were dissolved in a phosphate
buffer (150 mmol/L, pH 7, 10 mL). Then, 2 mL of glycosynthase
E338G sol. prepared as previously described24 was added and the
reaction was allowed to proceed at 37 °C until complete consump-
tion of the fluoride (about 20 h). After removal of the solvent under
reduced pressure, purification by silica gel chromatography (8:4:1
CHCl3–MeOH–NH4OH, Rf of 4, 0.24 and Rf of 7, 0.25) afforded
381 mg of pure 4 (free amine, white solid, yield 88%, mp 159–
GalF
GalF
GlcF
GlcF
100
140
100
120
100
100
100
100
82
88
92
97
b-D-glucopyranosyl-(1?3)-2-amino-2-deoxy-1-thio-b-D-glucopy-
ranoside 7 (structure established by means of NMR spectroscopy,
see Section 4) was obtained in slightly higher yields than 4.
Moreover, as expected, the chemoselective N-acylation (vs O-
acylation) and the deprotection of the thiophenyl aglycon group
afforded nearly quantitatively lacto-N-biose 5 (80% overall yield
from 3, see Section 4). A drawback of our strategy, inherent in
the T. thermophilus enzyme specificity, is the required presence of
an S- or O-phenyl anomeric group on the acceptor. Thus, these re-
sults should be compared to other approaches using glycosidases
able to accept free N-acetylglucosamine as a substrate. For exam-
ple, Xanthomonas manihotis b-
tion of lacto-N-biose from p-nitrophenyl b-
and 2-acetamido-2-deoxy-
-glucopyranose in 50% yield;23 how-
ever this result was obtained with more than a 10-fold excess of
the acceptor. Scaling-up this reaction under such conditions would
probably lead to difficulties in the high yielding separation of lacto-
N-biose. Furthermore, the free amino disaccharides 4 and 7 ob-
tained by our method offer the opportunity to synthesize different
useful amido derivatives which could be incorporated into
oligosaccharides.
161 °C, ½a 2D2
¼ ꢁ23:7 (c 0.35 MeOH), Calcd for C18H27NO9S: C,
ꢀ
49.88; H, 6.24; N, 3.23; S, 7.39. Found: C, 49.52; H, 6.32; N, 3.52;
S, 7.21) and 407 mg of pure 7 (white solid, yield 94%, mp 165–
167 °C, ½a 2D2
¼ ꢁ29:6 (c 0.45 MeOH), Calcd for C18H27NO9S: C,
ꢀ
49.88; H, 6.24; N, 3.23; S, 7.39. Found: C, 49.59; H, 6.22; N, 3.41;
S, 7.26).
Compound 4: 1H NMR [500 MHz, D2O, reference: (CH3)3Si–CH2–
CH2–CH2–SO3Na]: d 7.65 (2H, Ph), 7.49 (3H, Ph), 4.98 (1H, d, J
10.5 Hz, H-1), 4.60 (1H, d, J 7.5 Hz, H-10), 3.95 (1H, dd, J 10.3,
8.7 Hz, H-3), 3.93 (1H, dd, J ꢁ12.4, 2.3 Hz, H-6a), 3.93 (1H, dd, J
3.3 Hz, H-40), 3.78 (1H, dd, J ꢁ12.4, 5.5 Hz, H-6b), 3.73 (1H, ddd, J
not determined, H-50), 3.69 (1H, dd, J 9.9, 3.3 Hz, H-30), 3.68 (1H,
dd, J 9.9, 8.7 Hz, H-4), 3.63 (1H, dd, J 9.9, 7.5 Hz, H-20), 3.57 (1H,
ddd, J 9.9, 5.5, 2.3 Hz, H-5), 3.34 (1H, dd, J 10.5, 10.3 Hz, H-2). 13C
NMR (125 MHz, D2O, reference: (CH3)3Si–CH2–CH2–CH2–SO3Na]:
d 135.1 (2 ꢂ CH Ph), 133.0 (C, Ph), 132.2 (2 ꢂ CH Ph), 131.7
(1 ꢂ CH Ph), 105.9 (C-10), 86.7 (C-1), 85.9 (C-3), 82.6 (C-5), 78.2
(C-50), 75.2 (C-4), 73.6 (C-20), 71.1 (C-40), 70.4 (C-30), 63.5 (C-60),
63.2 (C-6), 56.3 (C-2).
D
-galactosidase allowed the prepara-
D
-galactopyranoside
D
Compound 7: 1H NMR [500 MHz, D2O, reference: (CH3)3Si–CH2–
CH2–CH2–SO3Na]: d 7.65 (2H, Ph), 7.49 (3H, Ph), 4.82 (1H, d, J
10.4 Hz, H-1), 4.61 (1H, d, J 7.9 Hz, H-10), 3.79 (1H, dd, J -12.3,
2.3 Hz, H-60a), 3.78 (1H, dd, J 10.3, 9.5 Hz, H-3), 3.75 (1H, dd, J
ꢁ12.2, 2.2 Hz, H-6a), 3.64 (1H, dd, J ꢁ12.3, 5.9 Hz, H-60b), 3.63
(1H, dd, J ꢁ12.2, 5.5 H-6b), 3.51 (1H, dd, J 9.2, 9.5 Hz, H-4), 3.45
(1H, dd, J 8.1, 7.9 Hz, H-40), 3.40 (1H, ddd, J 9.2, 5.5, 2.2 Hz, H-5),
3.40 (1H, ddd, J 7.9, 5.9, 2.3 Hz, H-50), 3.34 (1H, dd, J 9.4, 7.9 Hz,
H-20), 3.33 (1H, dd, J 9.4, 8.1 Hz, H-30), 3.18 (1H, dd, J 10.4,
10.3 Hz, H-2). 13C NMR (125 MHz, D2O, reference: (CH3)3Si–CH2–
3. Conclusion
In conclusion, we have shown the possibility of circumventing
the lack of regioselectivity of T. thermophilus glycosynthase towards
N-acetylglucosamine derivatives. Thus, phenyl 2-amino-1-thio-b-
glucopyranoside, an acceptor well recognized by these enzymes,
allowed the synthesis of phenyl b- -glycopyranosyl-(1?3)-2-
amino-2-deoxy-1-thio-b- -glucopyranosides in high yields, the
D-
D
D
CH2–CH2–SO3Na]:
d
135.1 (2 ꢂ CH Ph), 133.0 (C, Ph), 132.2
latter being easily converted to the corresponding N-acetyldisac-
charides.
(2 ꢂ CH Ph), 131.7 (1 ꢂ CH Ph), 106.1 (C-10), 89.3 (C-1), 89.3 (C-
3), 83.2 (C-50), 82.9 (C-5), 78.7 (C-30), 72.5 (C-4), 71.2 (C-40), 63.9
(C-6), 63.7 (C-60), 57.3 (C-2).
4. Experimental
4.1.2. Phenyl b-
D
-galactopyranosyl-(1?3)-2-acetamido-2-
4.1. General procedures
deoxy-1-thio-b-
D-glucopyranoside 5
Disaccharide 4 (free amino group, 433 g/mol, 43 mg, 0.1 mmol)
Glycosynthase E338G solution was prepared according to a pro-
cedure already described by us.24 Chemicals were supplied by Al-
drich and used without further purification. The course of the
reactions was followed by means of TLC (precoated Silica Gel 60
sheets Merck F254) and proton NMR spectroscopy. The compo-
nents of the reaction mixtures were separated by silica gel chroma-
tography. Analysis of the 1H and 13C NMR resonances and
subsequent structure assignments were carried out using standard
2D sequences (COSY, HMBC, HMQC, TOCSY correlations). The
structures of the b-(1,3)-regioisomers were established on the ba-
sis of the identification of the C-3 carbon (shifted at lower fields).
The spectra were recorded with a Bruker DRX500 spectrometer
operating at 500 MHz for 1H and 126 MHz for 13C. Due to their sol-
ubility in water, the spectra of the saccharides were recorded in
D2O and the chemical shifts (in ppm) are quoted from the reso-
nance of methyl protons of sodium 3-(trimethylsilyl)-propanesulf-
onate (DSS) used as an internal reference.
was dissolved in 3 mL of MeOH. Then, sodium bicarbonate (84 mg,
1 mmol) and acetic anhydride (102 g/mol, 100 lL, 1 mmol) were
added. The mixture was maintained at rt for 45 min while stirring.
After this time, TLC plates indicated the total consumption of 5.
Next, MeOH was distilled under reduced pressure, purification by
silica gel chromatography (8:4:1 CHCl3–MeOH–NH4OH, Rf of 5,
0.36) afforded 45 mg of pure 5 (white solid, yield 94%, mp 162–
164 °C, Calcd for C20H29NO10S: C, 50.53; H, 6.10; N, 2.95; S, 6.74.
Found: C, 50.27; H, 6.22; N, 3.12; S, 6.59). 1H NMR [500 MHz,
D2O, reference: (CH3)3Si–CH2–CH2–CH2–SO3Na]: d 7.65 (2H, Ph),
7.49 (3H, Ph), 4.93 (1H, d, J 10.6 Hz, H-1), 4.42 (1H, d, J 7.7 Hz, H-
10), 3.92 (1H, dd, J 10.6, 10.3 Hz, H-2), 3.91 (1H, dd, J ꢁ12.5,
2.1 Hz, H-6b), 3.90 (1H, dd, J 3.3, nd Hz, H-40), 3.82 (1H, dd, J
10.3, 8.2 Hz, H-3), 3.76 (1H, dd, J –12.5, 5.2 Hz, H-6a), 3.69 (1H,
ddd, J nd, H-50), 3.63 (1H, dd, J 10.0, 3.3 Hz, H-30), 3.57 (1H, dd, J
10.0, 8.2 Hz, H-4), 3.52 (1H, ddd, J 10.0, 5.2, 2.1 Hz, H-5), 3.50
(1H, dd, J 10.0, 7.7 Hz, H-20). 13C NMR (125 MHz, D2O, reference:

1246
A. D’Almeida et al. / Tetrahedron: Asymmetry 20 (2009) 1243–1246
2. Jahn, M.; Stoll, D.; Warren, R. A. J.; Szabo, L.; Singh, P.; Gilbert, H. J.; Ducros, V.
M. A.; Davies, G. J.; Withers, S. G. Chem. Commun. 2003, 1327–1329.
3. Jahn, M.; Chen, H.; Muellegger, J.; Marles, J.; Warren, R. A. J.; Withers, S. G.
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4. Mayer, C.; Zechel, D. L.; Reid, S. P.; Warren, R. A.; Withers, S. G. FEBS Lett. 2000,
466, 40–44.
(CH3)3Si–CH2–CH2–CH2–SO3Na]: d 183.5 (CO), 135.5 (2 ꢂ CH Ph),
134.5 (C Ph), 132.2 (2 ꢂ CH Ph), 130.9 (1 ꢂ CH Ph), 106.2 (C-10),
88.8 (C-1), 86.3 (C-3), 82.3 (C-5), 78.0 (C-50), 75.2 (C-30), 73.4 (C-
20), 71.2 (C-40), 71.2 (C-4), 63.7 (C-6), 63.5 (C-60), 25.6 (CH3).
5. Tolborg, J. F.; Petersen, L.; Jensen, K. J.; Mayer, C.; Jakeman, D. L.; Warren, R. A.
J.; Withers, S. G. J. Org. Chem. 2002, 67, 4143–4149.
4.1.3. b-
glucopyranoside 6
Phenyl b- -glycopyranosyl-(1?3)-2-acetamido-2-deoxy-1-thio-
-glucopyranoside 5 (475 g/mol, 48 mg, 0.1 mmol) was dissolved
D-Galactopyranosyl-(1?3)-2-acetamido-2-deoxy-D-
6. Viladot, J.-L.; Canals, F.; Batllori, X.; Planas, A. Biochem. J. 2001, 355, 79–86.
7. Nashiru, O.; Zechel, D. L.; Stoll, D.; Mohammadzadeh, T.; Warren, R. A. J.;
Withers, S. G. Angew. Chem., Int. Ed. 2001, 40, 417–420.
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9. Faijes, M.; Perez, X.; Perez, O.; Planas, A. Biochemistry 2003, 42, 13304–13318.
10. Faijes, M.; Imai, T.; Bulone, V.; Planas, A. Biochem. J. 2004, 380, 635–641.
11. Fairweather, J. K.; Faijes, M.; Driguez, H.; Planas, A. ChemBioChem 2002, 3, 866–
873.
12. Malet, C.; Planas, A. FEBS Lett. 1998, 440, 208–212.
13. Fourage, L.; Dion, M.; Colas, B. Glycoconjugate J. 2001, 17, 377–383.
14. Chiffoleau-Giraud, V.; Spangenberg, P.; Dion, M.; Rabiller, C. Eur. J. Org. Chem.
1999, 757–763.
15. Drone, J.; Feng, H.-y.; Tellier, C.; Hoffmann, L.; Tran, V.; Rabiller, C.; Dion, M.
Eur. J. Org. Chem. 2005, 1977–1983.
D
b-
D
in 2 mL of distilled water. Then, N-bromosuccinimide (178 g/mol,
35.6 mg, 0.2 mmol) was added and the mixture was stirred for
2 h at rt. At this time, the disaccharide was completely consumed
as revealed by TLC. Purification of 6 by silica gel chromatography
(2:1:1 nBuOH–AcOH–water, Rf of 6, 0.28), afforded 37 mg of 6
(white solid, yield 97%, mp 175–177 °C, ½a D22
¼ þ3:1 (equilibrium
ꢀ
value
a, b anomers) (c 0.65 MeOH), Calcd for C14H25NO11: C,
43.86; H, 6.53; N, 3.66. Found: C, 43.62; H, 6.38; N, 3.52). 1H and
13C NMR spectra were identical to those already described.23
16. Marton, Z.; Tran, V.; Tellier, C.; Dion, M.; Drone, J.; Rabiller, C. Carbohydr Res.
2008, 343, 2939–2946.
17. Osanjo, G.; Dion, M.; Drone, J.; Solleux, C.; Tran, V.; Rabiller, C.; Tellier, C.
Biochemistry 2007, 46, 1022–1033.
Acknowledgements
18. Feng, H.-Y.; Drone, J.; Hoffmann, L.; Tran, V.; Tellier, C.; Rabiller, C.; Dion, M.
J. Biol. Chem. 2005, 280, 37088–37097.
19. Osanjo, G., Thesis Nantes, France, 2007.
20. Hoffmann, L.; Tran, V.; Rabiller, C.; Tellier, C.; Dion, M. Protein Eng.Des. Sel.,
submitted for publication.
21. Silva, D. J.; Wang, H.; Allanson, N. M.; Jain, R. K.; Sofia, M. J. J. Org. Chem. 1999,
64, 5926–5929.
Thanks are due to the French Ministry of Education and Re-
search, to the Centre National de la Recherche Scientifique and to
the Region des Pays de la Loire (VANAM Program) for financially
supporting this work.
22. Benakli, K.; Zha, C.; Kerns, R. J. J. Am. Chem. Soc. 2001, 123, 9461–9462.
23. Vetere, A.; Miletich, M.; Bosco, M.; Paoletti, S. Eur. J. Biochem. 2000, 267, 942–
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