Practical tethering of vitamin B on a silica surface via its phosphate
1
group and evaluation of its activity
a
a
b
a
Ch. Vartzouma, M. Louloudi,* I. S. Butler and N. Hadjiliadis*
a
Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece. E-mail: nhadjil@cc.uoi.gr
Department of Chemistry, McGill University, 801 Sherbrooke St. West, Montreal, Quebec H3A 2K6,
Canada
b
Received (in Cambridge, UK) 26th November 2001, Accepted 19th December 2001
First published as an Advance Article on the web 13th February 2002
1
A convenient immobilization of thiamine pyrophosphate
molecules on a silica surface through the phosphate group is
developed, leading to a very active heterogenised biocatalyst
for pyruvate decarboxylation.
and 1538 cm2 which are attributed to coupling of the
pyrimidine ring (8a) with the d(NH
pyrimidine ring vibration (8b) respectively.
2
) group and to a pure
11 13
C CP MAS
NMR spectrum of the tethered TPP displayed signals which
characterize the immobilized biomolecule.† The 31P CP MAS
NMR spectrum presents the isotropic chemical shift at 211
Integration of bio- and chemo- catalysis together with materials
science provides the opportunity to design and develop new
materials for innovative applications. Hybrid organic–inorganic
composite materials are one of the most attractive targets
achievable by this co-operative process. Many of them have
been developed by chemically modified silica gels with organic
3 4
ppm (relative to 85% H PO ) accompanied by two sidebands.
This feature confirms the presence of two slightly non-
12
equivalent 31P nuclei, as expected in the present case. A
13
Herzfeld–Berger analysis gave chemical shift anisotropy
tensor components of 75.5, 8.5 and 2117.1 ppm (average =
1
29
functionalities. The fixation of active biomolecules via cova-
211.0 ppm). The CP MAS Si NMR spectrum of untreated
lent attachment to a silica surface for biotechnological processes
silica gave a typical spectrum for a silica sample showing two
2
3
is a remarkable aspect of this research area. Functionalised
major resonance peaks at 2101 and 2111 ppm assigned to Q
3
4
alkoxysilanes such as 3-aminopropyltriethoxysilane, 3-sulfa-
(Si(OSi)
3
OH) and Q (Si(OSi)
4
) groups and a third, quite weak
4
nylpropyltrimethoxysilane,
monoethoxydimethylsilylbuta-
signal, at 292 ppm attributed to Si with two OH groups
nal, cyanoethyltriethoxysilane,6 3-(triethoxysilyl)propyl iso-
5
(designated Q ). In the CP MAS Si NMR spectrum of
2 29
cyanate,7
(3-glycidyloxypropyl)trimethoxysilane
8
or
modified silica an enhancement of the Q peak and absence of
4
9
2
iodopropyltrimethoxysilane have been used as connecting
links. To overcome the limited range of available silanes, post-
modification of the grafted functions on the silica surface is
often a necessary synthetic step.10 However, the usually
adopted multi-stage post-modification process for linking the
silica surface with the active group of the biomolecule, in
general, is problematic because it occludes the preparation of
these materials. Here we report a one-step synthesis for the
the Q signal were observed indicating a decrease in the number
of Si–OH groups; this is consistent with the loss of protons on
the OH groups of the silica upon phosphorylation. There was,
however, no new signal arising from the Si–O–P moiety of the
solid. Either this peak is too weak to be distinguished from the
3
4
14
noise, or it is obscured by the strong Q and Q signals. To
prepare a complex of modified silica, [Th-OP ·x-
SiO , with Zn(II), an excess of ZnCl is added in methanol. The
resulting material, [(ZnCl ·Th-OP ·ySiO , had a
2 6 n
O -SiO3/2]
2
2
tethering of vitamin B
1
on a silica surface via the phosphate
2
)
2
2
O
6
-SiO3/2
]
m
2
group of the biomolecule. Furthermore, in the novel process, no
additional functional spacer is required. Evaluation of the
catalytic activity of the novel material for pyruvate decarbox-
ylation showed that it is a more efficient catalyst than the
homogeneous system.
ratio of zinc to thiamine molecule equal to 2+1. The amount of
Zn(II) was determined by back-titration of the remaining
amount of Zn(II) into the solution. The 13C CP MAS NMR data
for the metallated material clearly indicated metal coordination
to the pyrimidine N1A atom of the thiamine molecule.† This is
also supported by DRIFTS data where the coupled n (8a) +
A solution of thiamin pyrophosphate chloride hydrochloride,
TPP (0.5 g in 10 ml of ethanol with 2 equiv. of Et
3
N) containing
d(NH
2
) vibration as well as the n (8b) band of the pyrimidine
2
1
11
suspended silica gel (1.25 g, average pore diameter 60 Å) was
refluxed for 2 h, and then the recovered solid was washed with
methanol and dried at 60 °C under vacuum (Scheme 1). The
achieved loading is ca. 0.3 mmol TPP per gram of modified
silica, determined by elemental analysis.
ring were shifted at ca. 1667 and 1544 cm respectively. The
31
P CP MAS isotropic chemical shift was at 29.7 ppm, while a
Herzfeld–Berger analysis13 gave d11, d22 and d33 at 73.1, 210.3
and 292.7 ppm respectively, indicating some change in the
chemical structure of the phosphate moiety due to the Zn(II
)
Diffuse reflectance FTIR (‘DRIFTS’) data of the material,
showed absorption bands of the thiamine molecule at ca. 1682
approach. Thus, the metal-binding properties of the function-
alised surface were dominated by the chemistry of the TPP
molecule.
Thiamin enzymes catalyze the decarboxylation of a-keto-
1
5
acids and the transfer of aldehyde or acyl groups in vivo. The
holoenzymes depend on the cofactors thiamin pyrophosphate
2+
2+ 15
(
TPP) and bivalent metal ions such as Mg , or Ca . There
was some evidence that thiamine itself, in protein-free model
systems, catalyzes pyruvate decarboxylation.16 Here, based on
GC-MS data, we confirm this ability in a protein-free system.
Furthermore, we show that a significant improvement of the
catalytic process can be achieved in the case of a catalytic
reaction instead of a stoichiometric one. Pyruvate decarboxyla-
tion catalyzed by TPP occurs via two different procedures, in
presence (A) or not (B) of acetaldehyde (Scheme 2).
To examine the effectiveness of TPP, both procedures have
been followed. Interestingly, the catalytic activity of the
immobilized TPP remained intact after its mild anchoring
procedure. The pyruvate+TPP molar ratio used was 10+1 and
Scheme 1 Preparation of the immobilized TPP biocatalyst.
5
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CHEM. COMMUN., 2002, 522–523
This journal is © The Royal Society of Chemistry 2002