Glycosynthase-catalysed syntheses at pH below neutrality

Article abstract of DOI:10.1016/j.bmcl.2003.08.037

The use of the new glycosynthase Ta-β-GlyE386G and of the already known Ss-β-GlyE387G for the synthesis of interesting 4-methylumbelliferyl disaccharides and for the galactosylation of α- and β-xylosides of 4-penten-1-ol is reported. The results show sati

Full text of DOI:10.1016/j.bmcl.2003.08.037

Bioorganic & MedicinalChemistry Letters 13 (2003) 4039–4042
Glycosynthase-Catalysed Syntheses at pH below Neutrality
Antonio Trincone,a,* Assunta Giordano,a Giuseppe Perugino,b Mose Rossib
and Marco Moraccib
aIstituto di Chimica Biomolecolare C.N.R., Via Campi Flegrei 34, 80072 Pozzuoli, Napoli, Italy
bInstitute of Protein Biochemistry C.N.R., Via Pietro Castellino 111, 80131 Napoli, Italy
Received 22 May 2003; revised 1 July 2003; accepted 20 August 2003
Abstract—The use of the new glycosynthase Ta-b-GlyE386G and of the already known Ss-b-GlyE387G for the synthesis of inter-
esting 4-methylumbelliferyl disaccharides and for the galactosylation of a- and b-xylosides of 4-penten-1-ol is reported. The results
show satisfactory yields of reaction in presence of low excesses of acceptors and demonstrated that the high activity of these
enzymes at pH below neutrality is applicable in the transfer of glucose as well as of galactose from the preferred 2-NP-based
donors.
# 2003 Elsevier Ltd. All rights reserved.
Chromophoric oligosaccharides such as nitrophenyl and
4-methylumbelliferyl glycosides are of widespread
interest for the kinetic analysis of hydrolytic activities
and to define the mode of action of particular enzymes
(i.e., exo- or endo-glycosidases). These compounds have
proved to be a valuable tools in different field of appli-
cative interest such as clinical, biological and food
chemistry.1ꢀ3 n-Pentenylgyl cosides are stabel to a wide
range of reagents and are readily activated by halonium
ion, thus they are excellent substrates for a wide variety
of reactions occurring at anomeric center in oligo-
saccharide synthesis in the so called Fraser–Reid meth-
odology.4 The enzymatic synthesis of these sugar
building blocks could then shorten the synthetic routes
by coupling enzymatic and chemical methodologies thus
improving overall yield in a synthetic plan.4 One of the
synthetic strategies for these molecules generally rely
upon the availability of the appropriate carbohydrate
sequence as found in naturalsugar poylmers and
applying in turn the aglycones by chemical methods.5
the enzyme and can be hydrolysed back thus reducing
yields of transfer products.
Recently the synthesis catalysed by glycosynthases
emerged as significant development in in this field.6,7 By
using these enginereed enzymes, which lack the nucleo-
phile residue in their active site, the products can accu-
mulate in the reaction mixture due to the loss of the
hydrolytic activity of the biocatalyst for the products.
Among glycosynthases, the thermophilic representatives
can be used efficiently in inverting (with a-glycosyl
fluoride as donors) or retaining (with aryl b-glycosides
as donors) reactions. In the latter case, while mesophilic
members can react only with highly activated 2,4-dini-
trophenyl glycosides, stable 2-nitrophenyl glycosides
can be used by the thermophilic biocatalysts with
sodium formate (2–4 M) acting as externalbiomimick-
ing nucleophile. In addition different biodiversity in
terms of regioselectivity of the reaction conducted with
thermophilic enzymes is observed. These biocatalysts
are also characterized by a high degree of reactivation
compared to the mesophilic counterparts.6 Moreover, it
has been recently observed that at pH’s below neutrality
in diluted (50 mM) sodium formate buffers, the kcat
values of three thermophilic glycosynthases (Ss-b-
GlyE387G, Ta-b-GlyE386G, CelBE372A) can be res-
cued at levels comparable to the wild type enzymes or
17-fold higher than the kcat values observed at high
concentration of sodium formate, thus improving the
efficiency of the hydrolytic reaction in terms of reaction
time and amounts of enzyme used.8 In these new reaction
The enzymatic approach using glycoside hydrolases is
based on reverse hydrolysis or transglycosylation. The
latter is performed by the action of two active-site car-
boxylic acids, one acting as nucleophile, the other as an
acid-base catalyst. The major drawback of this
approach is that the reaction products are substrates for
*Corresponding author: Tel.: +39-1818-675-095; fax: +39-0818-041-
770; e-mail: atrincone@icmib.na.cnr.it
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.037

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A. Trincone et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4039–4042
conditions, the three mutants (Ss-b-GlyE387G, Ta-b-
GlyE386G, CelBE372A) produced 2-nitrophenyl
oligoglucosides with different selectivity and degree of
polymerization when 2-nitrophenyl-b-glucoside was
used as substrate (donor and acceptor).8 The thermo-
philic nature of these biocatalysts coupled with their
high activity in diluted buffers are interesting issues
considering the improved solubility of donors and
acceptors and some practicalbenefits, in the monitoring
and purification procedures, due to the absence of
molar concentration of nucleophiles. Practical applica-
tion for long reaction times is secured by the good sta-
ratio of activities of wild types versus mutant enzymes
for 2-NP-Glc and cellobiose respectively, shifts from ca.
2 to 332 for T. aggregans and from 4 to 52 for S. solfa-
taricus. These findings lead to the benefit of using dif-
ferent aryl-based structures as acceptors thus obtaining
usefulcompounds in moderate-high yiedl. The sub-
strates we chose for the reactions10,11 are 4-methy-
lumbelliferyl b-d-glucoside and a- and b-xylosides of 4-
penten-1-ol; the use of acceptors with b-glycosidic lin-
kages is a unique characteristic of mutant enzymes in
that these molecules are completely hydrolyzed by the
wild type. Blank experiments (GOD analysis) secured
that no significant (<1% in 3 h and ca. 11% in 24 h)
chemical hydrolysis of aryl-donor occurred in the reac-
tion time intervals. The reaction10 conducted using the
mutant of Ta-b-Gly and 2-NP-glucoside as donor and
4-methylumbelliferyl b-d-glucoside as acceptor, furn-
ished two disaccharides: 4-methylumbelliferyl laminar-
ibioside, 1 (4-MU-Lam, 40%) and 4-methylumbelliferyl
cellobioside, 2 (4-MU-Cell, 60%) (Table 2).
ꢁ
bility expressed by these thermophilic proteins at 65 C
in 50 mM sodium formate buffer, pH 4.8
In this communication we report on the preparation of
chromophoric oligosaccharides and on the galactosyla-
tion of xylosides of 4-penten-1-ol exploiting the new
glycosynthase from Thermosphaera aggregans and the
already known Ss-b-GlyE387G6 in these new reaction
conditions.9
The synthesis was conducted at three different pH in 50
mM sodium formate/formic solutions. The donor con-
version at pH 5 is not significant even after 96 h; at pH
3, 75% conversion of donor was reached in 3 h and the
overall yield of the disaccharides 1 and 2 is 65% after
purification and acetylation for NMR spectroscopy
study.12 At pH 4 the conversion reached 93% after 9 h
and the isolated yield of 1 and 2 is 60%. Unreacted 4-
MUG is recovered during product purification. These
data are in agreement with the reported increase of
enzymatic activity observed at acidic pH.8 The yields
are remarkably high considering the low, 2-fold molar
excess of the acceptor used. Trace amounts of glucose
and of the disaccharides of the donor are observed. No
trisaccharidic product formation was observed in TLC
analysis of the reaction mixtures. Elongation to tri-
saccharides was investigate using as acceptors: p-nitro-
phenyl-b-d-lactoside, the disaccharide 1 and phenyl
thiocellobioside13 (Table 2). No reaction was observed
in the first case, while poor yields of trisaccharides were
obtained for the phenyl thiocellobioside. Using 4-MU-
Lam (1) as acceptor, 16% yield of two trisaccharides (3a
and in lesser amount 3b) was obtained in a reaction
conducted at equimolar donor/acceptor ratio. The ana-
lysis of 1H NMR spectra, COSY and 1H–13C NMR
correlations permit to establish the trisaccharidic nature
of products and the interglycosidic linkages for 3a as
b-Glc-(1-4)-b-Glc-(1-3)-b-Glc-MU.12
In Table 1 the specificity of mutant and wild type
enzymes is reported for different substrates. 2-Nitro-
phenyl glucoside is efficiently cleaved by the glyco-
synthase of T. aggregans as well as by the glycosynthase
of Sulfolobus solfataricus. The loss of activity is 80–90%
by changing the position of nitro group in the aglycon
(2-NP-Glc vs 4-NP-Glc). Moreover on 4-methy-
lumbelliferyl b-d-glucoside, Ta-b-GlyE386G retains
only 3.6% of the activity expressed on 2-NP-Glc (Table
1). It is interesting to note, as a basic foundation of
glycosynthase activity of these biocatalysts, that the
Table 1. Substrate specificity (U mgꢀ1) of Thermosphaera aggregans
glycosynthase and Ss-b-GlyE387G in 50 mM sodium formate buffer,
pH 3 as compared to the wild types
Substrates
Ta-b-Gly
Ss-b-Gly
wt
m
wt
m
2-NP-Glc
72.2
129.8
252.6
63.3
2-NP-Gal140.9
2-NP-Xyl10.4
4-NP-Glc
4-MUG
Cellobiose
6.3
6.4
218.6
19.1
2.7
2.5
149.8
61.7
33.5
33.8
12.0
4.7
14.9
9.7
1.2
88.2
61.9
0.39
Aryl-glycosides and cellobiose substrates were assayed at 5 and 200
mM finalconcentration, respectively, in 50 mM sodium formate buffer
pH 3.0 (see ref 8 for details); wt, wild type; m, mutant.
Table 2. Syntheses conducted with Ta-b-GlyE386G and Ss-b-GlyE387G
Donor
Acceptor
Enzyme
Products
Yield (%)
2-NP-Glc
2-NP-Glc
2-NP-Glc
2-NP-Glc
2-NP-Gal
2-NP-Gal
2-NP-Gal
4-MUG
PNP-Lac
Ph-S-Cell
1
b-Xyl-4P
a-Xyl-4P
b-Xyl-4P
Ta-b-GlyE386G
Ta-b-GlyE386G
Ta-b-GlyE386G
Ta-b-GlyE386G
Ta-b-GlyE386G
Ss-b-GlyE387G
Ss-b-GlyE387G
1, 2
—
—
3a, 3b
4
60–65
—
Poor
16
34
5a, 5b
4
33
40
2-NP, 2-nitrophenyl; Glc, glucose; Gal, galactose; 4-MUG, 4-methylumbelliferyl b-d-glucoside; PNP-Lac, p-nitrophenyl-b-d-lactoside; Ph-S-Cell,
phenyl thio-cellobioside; 1, 4-methyl umbelliferyl-b-d-laminaribioside; 3a, b-Glc-(1-4)-b-Glc-(1-3)-b-Glc-MU; 3b, b-Glc-b-Glc-b-Glc-MU; b-Xyl-4P,
4-penten-1-yl b-d-xylopyranoside; a-Xyl-4P, 4-penten-1-yl a-d-xylopyranoside; 4, b-Gal-(1-3)-b-Xyl-4P; 5a, b-Gal-(1-3)-a-Xyl-4P; 5b, b-Gal-(1-4)-a-
Xyl-4P.

A. Trincone et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4039–4042
4041
The glycosynthases from Ta-b-Gly and Ss-b-Gly remain
active also on 2-NP-Gal (Table 1) and we used this poten-
tialto investigate the transfer of gaal ctose to the xyol sides
of 4-penten-1-ol13 for the synthesis of the disaccharidic
Zambardino of the NMR service of ICB-Naples, for
running NMR spectra. The present research was par-
tially supported by the Italian PNPRA research project.
unit (or regioisomers) present in xyloglucan oligosacchar-
14
ides possessing severalbioolgicalactivities.
The reac-
References and Notes
tions11 were conducted at 1–5 molar excess of acceptor by
adding aliquots of the substrate to the reaction mixtures.
Conversions are still satisfactory being ca. 90 and 60% for
S. solfataricus and Thermosphaera enzyme, respectively in
24 h reaction time. The yields, calculated on the substrate
converted, are similar for both enzymes, in the range 35–
40%. Much more interesting is the change in regioselec-
tivity for the two anomers of the xyloside used. Essentially
1. Weder, J. K. P.; Kaiser, P. K. J. Chromatogr. A 1995, 698,
181.
2. Planas, A.; Abel, M.; Oscar, M.; Palası, J.; Pallares, C.;
Viladot, J. L. Carbohydr. Res. 1998, 310, 53.
3. Planas, A. Biochim. Biophys. Acta 2000, 1543, 361.
4. Fraser-Reid, B.; Udodong, U. E.; Wu, Z.; Ottoson, H.;
Merritt, J. R.; Rao, C. S.; Roberts, C.; Madsen, R. SynLett
1992, 927.
5. Viladot, J.L.; Stone, B.; Driguez, H.; Planas, A. Carbohydr.
Res. 1998, 311, 95.
6. Moracci, M.; Trincone, A.; Rossi, M. J. Mol. Cat. B: Enz.
2001, 4-6, 155.
7. Zechel, D. L.; Withers, S. G. Acc. Chem. Res. 2000, 33, 11.
8. Perugino, G.; Trincone, A.; Giordano, A.; van der Oost, J.;
Kaper, T.; Rossi, M.; Moracci, M. Biochemistry 2003, 42,
8484.
9. The Thermosphaera aggregans glycosynthase (E386G) 0.43
mg/mL and Ss-b-glyE387G (0.38 mg/mL) were used as bio-
catalysts. They were obtained by mutation as previously
described in refs 6 and 8.
12
a singl e (4, b-Gal-(1-3)-b-Xyl-4P>95%) regioisomer
was obtained when using b-Xyl-4P: while a 33% yield of
two disaccharides 5a and 5b was obtained using a-Xyl-
4P by the use of the glycosynthase Ss-b-glyE387G in
19:81 ratio, respectively (Table 2). Using Thermosphaera
mutant enzyme a similar yield of compound 4 is
obtained when using b-Xyl-4P. The difference of
regioselectivity observed for the Ss-b-glyE387G with
respect to the anomeric configuration of the acceptor (b-
Xyl-4P/a-Xyl-4P) is for the first time observed for a
glycosynthase and confirms the well known phenom-
enon revealed for the wild type glycoside hydrolases.15
10. 2-nitrophenyl b-d-glucoside (0.083 mmol) was dissolved in
three different 50 mM formic acid/sodium formate buffer
solutions at pH 3.02, 4.02 and 5.00 (2 mL). The 4-methyl-
umbelliferyl b-d-glucoside, 0.165 mmol, was added and the
reactions started in sealed vials at 65 ꢁC by adding 350 mL of
the enzyme. No significant (less than 5%) chemical hydrolysis
of donor and acceptor was observed in these conditions by
blank experiment. After 5 h, the reaction was neutralized with
sodium carbonate; reverse phase RP-18 short column and
preparative silica-gel TLC (EtOAc/MeOH/H2O, 70:20:10 by
vol) furnished disaccharides 1 and 2 which were in turn sub-
jected to standard acetylation procedures (Ac2O/pyr over-
night) for NMR spectroscopy studies.
11. Donor: 2-nitrophenyl-b-d-galactoside (74 mmol). Accep-
tor: b-(pent-4-en-1-yl)-d-xylopyranoside (0.36 mmol). Buffer
system: sodium formate 50 mM pH 4. Enzyme: Ta-b-
glyE386G 96 mg. Temperature: 65 ꢁC. The donor was con-
verted at ca. 60% in 24 h. Yield of the reaction 34%. b-(pent-
4-en-1-yl)-galactosyl-(b-1-3)-d-xylopyranoside (>95%).
Donor: 2-nitrophenyl-b-d-galactoside (0.24 mmol). Acceptor:
a-(pent-4-en-1-yl)-d-xylopyranoside (0.40 mmol). Buffer sys-
tem: sodium formate 50 mM pH 4. Enzyme Ss-b-glyE387G,
95 mg. The donor was added in three aliquots of 0.08 mmol at
time intervals during 4 h of reaction time to the reaction mix-
ture to keep the molar excess of the acceptor in the range of
1–5. Temperature: 65 ꢁC. The donor was converted at ca. 90%
in 24 h. Yield of the reaction 33%. a-(Pent-4-en-1-yl)-galacto-
syl-(b-1-3)-d-xylopyranoside (19%), a-(pent-4-en-1-yl)-galac-
tosyl-(b-1-4)-d-xylopyranoside (81%).
The high rescue of hydrolytic activities of thermophilic
glycosynthases,8 obtained at low pH, coupled with high
resistance to criticalreaction conditions to which these
biocatalysts have to face (high temperature, low pH,
high concentrations of organics), prompted us to
explore new strategies for the exploitation of these
enzymes on different substrates. The overall results
reported in this communication demonstrate that gly-
cosynthases from T. aggregans and S. solfataricus at pH
below neutrality have the ability to produce glycosy-
lated products in presence of various acceptors. Carbo-
hydrate elongation to tri- and higher compounds is
possible for T. aggregans glycosynthase but the yield fall
seriously down indicating steric/electronic limitations in
the active site as also previously observed for this
enzyme with 2-nitrophenyl b-d-glucoside.8 However it is
worth noting that despite the low molar excess of
acceptors (1.9–1.0) for the synthesis of 4-methyl-
umbelliferyl di- and trisaccharides, a ca. 65 and 16%
yields, respectively, were obtained. The residual activity
on 2-NP-Gal of both glycosynthases was also exploited
to achieve satisfactory yields of regioisomers of Gal-Xyl
present in the oligosaccharins.14,16 It is of interest that
b-configuration of the acceptor permits to obtain a sin-
gle disaccharide out of three possible products. How-
ever the most abundant b-Gal-(1-4)-a-Xyl-4P could be
utilized in a conceivable chemical coupling for the pro-
duction of saccharides present in the region between
glycosaminoglycan (GAG) chains and protein parts in
serine-linked connective tissue proteoglycan.19
Donor: 2-nitrophenyl b-d-galactoside (0.10 mmol). Acceptor:
b-(pent-4-en-1-yl)-d-xylopyranoside (0.51 mmol). Buffer sys-
tem: sodium formate 50 mM pH 4. Enzyme Ss-b-glyE387G 76
mg. Temperature: 65 ꢁC. The donor was converted at ca. 90%
in 24 h. Yield of the reaction 40%. b-(Pent-4-en-1-yl)-galacto-
syl-(b-1-3)-d-xylopyranoside (>95%).
1
12. 1H NMR spectra, COSY and H–13C NMR correlations
generally permit the assignments of chemical shifts as below
indicated for peracetylated derivatives of compounds
obtained. In the COSY spectrum of the product, following the
correlations through pyranosidic protons starting from the
aglycon-linked sugar, it is easy to detect the position of glyco-
Acknowledgements
The authors wish to thank E. Pagnotta for its skillful
technical assistance and A. Maiello, V. Mirra and S.

4042
A. Trincone et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4039–4042
sylation for the upfield shift of the signal due to the absence of
acetylgroup. Diagnostic 13C correlation signals are indicated.
1 (CDCl3): 5.03 (H1), 5.30 (H2), 4.00 (H3 b-O-linked to the
1.68 (28.4); a-d-xylopyranosyl moiety H1-H5: 4.90 (95.6),
4.78, 4.08 (75.8), 4.92, 3.75–3.67; b-d-galactopyranosyl moiety
H1-H6: 4.66 (101.1), 5.13, 5.01, 5.38, 3.91, 4.20-4.09. 13C dis-
accharidic moiety: 101.1, 95.6, 75.8, 72.9, 71.1, 70.6, 69.2, 69.0,
66.9, 61.0, 58.6. 5b b-Gal-(1-4)-a-Xyl-4P (CDCl3) pent-4-en-1-yl
moiety: 5.79 (137.8), 4.95 (115.1), 3.69–3.39 (67.7), 2.10 (30.0),
1.68 (28.4); a-d-xylopyranosyl moiety H1–H5: 4.92 (95.8), 4.72,
5.39, 3.78 (76.6), 3.60; b-d-galactopyranosyl moiety H1-H6: 4.48
(101.1), 5.09, 5.01, 5.37, 3.89, 4.10. 13C disaccharidic moiety:
101.1, 95.8, 76.6, 71.3, 70.9, 70.8, 70.2, 69.1, 66.8, 61.1, 59.1.
13. Phenyl thio-cellobioside was obtained by standard routes
starting from a-d-cellobiose octacetate as described in ref 17.
a- and b-xylosides of 4-penten-1-ol were obtained starting
from acetylated monosaccharide (a-xylose) and using BF3-
etherate as catalyst (total yield 23%, a/b anomers 1:1) as
described in ref 18.
0
externalsugar), 5.10 (H4), 3.87 (H5), 4.20 (H6), 4.63 (H1 ),
4.92 (H20), 5.15 (H30), 5.13 (H4), 3.70 (H5), 4.40–4.05 (H6).
13C signals: 100.9, 98.4, 78.6, 72.8, 72.5, 72.2, 71.8, 71.1, 68.0
(ꢂ2), 62.0, 61.6. 2 (C6D6): 4.80 (H1), 5.50 (H2), 5.39 (H3), 3.49
(H4 b-O-linked to the external sugar), 3.20 (H5), 4.50–4.00
(H6), 4.20 (H10), 5.18 (H20), 5.37 (H30), 5.19 (H4), 3.30 (H5),
4.40–3.90 (H6). 13C signals: 100.7, 98.2, 76.2, 73.2, 72.8, 72.3,
72.0, 71.5, 71.0, 67.7, 61.7, 61.5. 3a (CDCl3) carbohydrate
moiety (CDCl3): 5.03/98.4 (H1/C1 aryl linked glucose), 3.98/
78.4 (H3/C3 aryl linked glucose), 4.60/100.8 (H1/C1 internal
glucose), 3.72/75.2 (H4/C4 internal glucose), 4.50/100.7 (H1/
C1 externalgulcose). The trisaccharidic nature of
established by the presence of three anomeric signals in the 13
3b was
C
NMR and by the TLC Rf comparable to that of 3a. 4 b-Gal-
(1-3)-b-Xyl-4P (CDCl3) pent-4-en-1-ylmoiety: 5.77 (137.9),
5.04 (114.9), 3.72–3.41 (68.2), 2.10 (29.9), 1.68 (28.5); b-d-
xylopyranosyl moiety H1-H5: 4.39 (100.1), 4.86, 3.84 (76.7),
4.87, 4.05–3.39; b-d-galactopyranosyl moiety H1–H6: 4.58
(101.3), 5.13, 4.99, 5.35, 3.89, 4.20-4.09. 13C disaccharidic
moiety: 101.3, 100.1, 76.7, 71.5, 71.0, 70.5, 69.1, 68.6, 66.9,
61.4, 61.0. 5a b-Gal-(1-3)-a-Xyl-4P (CDCl3) pent-4-en-1-yl
moiety: 5.79 (137.6), 4.95 (115.1), 3.69–3.39 (67.6), 2.10 (30.1),
14. Fry, S. C. Trends in Plant Science 1996, 1, 326.
15. Nilsson, K. G. Carbohydr. Res. 1987, 15, 95.
16. Watt, D. K.; Brash, D. J.; Larsen, D. S.; Melton, D. L.;
Simpson, J. Carbohydr. Res. 2000, 325, 300.
17. Buskas, T.; Garegg, J. P.; Konradsson, P.; Maloisel, J. L.
Tetrahedron: Asymmetry 1994, 5, 2187.
18. Lahmann, M.; Thiem, J. Carbohydr. Res. 1997, 299, 23.
19. Fukase, K.; Yasukochi, T.; Suda, Y.; Yoshida, M.;
Kusumoto, S. Tetrahedron Lett. 1996, 37, 6763.