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Description  |
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This invention relates to the preparation of immobilized enzymes with a
high degree of resistance to thermal inactivation through covalent
attachment to phospholipid layers, which are already covalently linked to
solid supports through long chain spacer molecules. Also included in the
invention are processes for the synthesis of appropriately functionalized
phospholipids suitable for the dual role of binding to bioactive
substances as well as to modified solid supports.
BACKGROUND OF THE INVENTION
Enzymes find extensive applicability in diverse areas such as food
processing, enantioselective organic synthesis, production of
pharmaceuticals, clinical diagnosis/treatment, extracorporeal affinity
chromatography, waste management, environmental analysis/pollution control
and biosensors. As industrial catalysts, they offer a number of advantages
over conventional chemical catalysts due to their high catalytic activity,
substrate specificity, the mild conditions involved in their use, minimal
by-product formation and no environmental pollution risk. However the two
main disadvantages relating to their utility are their instability and the
economic factor. The practical use of enzymes often requires elevated
temperatures to increase productivity, prevent microbial contamination,
improve the solubility of substrates and reduce the viscosity of the
reaction medium. On the other hand, the stability of enzymes is affected
by conditions such as heat, contact with chemicals and organic solvents,
all of which cause denaturation. Amongst these, heat is by far the most
important factor for the loss of the biological activity of enzymes and
some correlation exists between thermal stability and other kinds of
stabilization such as resistance to proteolysis. Thermal inactivation of
enzymes is initiated by the partial reversible unfolding of their native
structure which is followed by irreversible configurational/conformational
changes. Processes such as aggregation, formation of "scrambled
structures", cleavage of disulfide bridges, peptide bond hydrolysis,
racemization of amino acid residues, deamidation, dissociation of
prosthetic groups, isopeptide bond formation and oxidation of thiol/indole
groups have been implicated during heat mediated denaturing of enzymes.
Enhancement of the thermal stability could alleviate most, if not all, of
the problems associated with the use of native enzymes for various
applications. Thermostabilization strategies followed during the past
three decades consist of (i) addition of substances, (ii) chemical
modification, (iii) cross-linking, (iv) use of anhydrous solvents
(non-aqueous media), (v) protein engineering and (vi) immobilization. Of
these, the immobilization technique is the most extensively used one for
imparting thermal stability to enzymes. Enzymes immobilized on suitable
substrates possess considerable advantages over those used in the soluble
phase. They often show marked increase in stability and may be used in
bioreactors for continuous processing, thereby cutting down on the costs
in comparison with reactors utilizing these biocatalysts in solution. For
example, using immobilized aminoacylase, the cost of amino acid production
is reduced by 40% as against the soluble enzyme. In addition, immobilized
biocatalysts are easily removable from reaction mixtures and have enhanced
shelf life.
By definition, an immobilized enzyme is a protein physically localized in a
certain region of space or converted from a water-soluble mobile state to
a water-insoluble immobile condition. Protocols used for immobilizing
enzymes can be categorized according to whether the protein becomes
immobile by chemical binding or by physical retention. These consist of
(i) binding of enzyme molecules to carriers through covalent bonds, (ii)
by adsorptive interactions (physisorption), (iii) entrapment into gels,
beads or fibres, (iv) cross-linking or co-crosslinking with bifunctional
reagents and (v) encapsulation in microcapsules or membranes. Of these,
the adsorptive procedures have become more or less obsolete due to the
fact that the surfaces produced are too unstable to withstand mechanical
stresses and chemical treatments involved in industrial processes.
Immobilization through cross-linking has met with limited success because
of the large amounts of enzyme required, the uncontrollable nature of the
reaction which may lead to inactivation and the unsuitable mechanical
properties of the resulting surfaces. The main disadvantages of the
microencapsulation technique are that the molecular weight of the
substrate has to be very low to allow diffusion across the membranous
barrier and the capsules are very prone to enzyme leakage as they are
relatively fragile. Furthermore, the polysaccharide-based polymeric
materials used for entrapping enzymes into gels or beads suffer from the
fact that strict sterile operating conditions must be maintained to
prevent the growth of bacteria and fungii. With acrylamide monomers used
for entrapment purposes, the conditions of photopolymerization may
generate localized temperatures up to 60.degree. C. causing denaturing of
the enzyme. With other polymeric systems, problems of enzyme loading,
viability and stability have to be overcome for industrial applications.
Several reviews have appeared in the scientific and patent literature on
the available choices of substrates and the protocols for covalently
binding enzymes on them. The substrates in vogue range from inorganic
materials such as porous glass, ceramics, silica and metal/metal oxides to
organic materials such as the natural polymers cellulose, chitin and
agarose and synthetic products like acrylates, polyamides, derivatized
polystyrene and redox systems like polypyrrole. Biomolecules like the
avidin-biotin system or bovine serum albumin are also being utilized.
However, because of the problems of microbial growth on organic supports,
and the consequent loss of activity, collapse of the structure and product
contamination, there has been an increasing interest in the use of
inorganic support materials, especially silica, controlled pore glass and
ceramics.
The two factors to be considered in the selection of a method for the
covalent linkage of an enzyme to a support are: the type of functional
groups on the protein through which binding to the support is to be
accomplished (and consequently the type of chemical reactions to the
employed) and the physical/chemical characteristics of the support
material with appropriate reactive functionalities grafted onto their
surface. The functional groups on the enzymes which are available for
covalent bonding are (1) amino (eta-amino groups of lysine and arginine
and the N-terminal amino moieties of the polypeptide chains), (2) carboxyl
groups of aspartic and glutamic acid and the C-terminal moieties, (3)
phenol rings of tyrosine, (4) sulfhydryl groups of cysteine, (5) hydroxyls
of serine, threonine and tyrosine, (6) the imidazole groups of histidine
and (7) the indole groups of tryptophan. In practice, most of the covalent
coupling reactions involve the amino, carboxy and mercapto moieties on the
amino acids in the protein structure. The solid supports, in turn, must
carry functional groups such as carboxyl, amino, formyl, epoxy, halo
(chloro or bromo) and hydroxyl. A majority of solid supports either carry
hydroxyls on their surfaces or can be easily modified by chemical or
electrochemical means to introduce such hydroxylic groups.
Chemical reactions most commonly used for the interaction of the
functionalities in the enzyme with those on the support materials consist
of (1) the nucleophilic displacement of the surface hydroxyls on the
supports activated with a sulphonyl chloride, 2-fluoro pyridinium tosylate
or cyanuric chloride by the amino group on the protein, (2) nucleophilic
addition of the protein amino group to a surface hydroxyl on the support
which is activated with cyanogen bromide or carbonyldiimidazole or a
chloroformate; or an analogous nucleophilic addition of the protein amino
group to a carboxyl on the support surface which is activated as its
N-hydroxysuccinimide ester, azide or with a diimide, (3) electrophilic
addition of a diazonium functionality formed from an aromatic amino moiety
on the support to the tyrosine residues on the enzyme, (4) electrophilic
addition of the mercapto group on the cysteine moiety of the enzyme to a
maleimide function introduced onto the surface of the support, and (5)
cross-linking a surface amino group on the support to an amino group on
the enzyme with a bifunctional reagent such as glutaraldehyde.
The thermal stability of enzymes covalently attached to support materials
is significantly enhanced in comparison with the native enzyme. For
example, Hayashi et al. (J. Appl. Polym. Sci. 1992, 44, 143) have observed
that papain immobilized on polymethyl L-glutamate exhibited an activity up
to three times higher than the native enzyme when maintained at 70.degree.
C. in buffer solution for one hour. The free papain loses 90% of its
initial activity at 75.degree. C. within 45 minutes. Raghunath and
coworkers (Biotechnol. Bioeng. 1984, 26, 104) have demonstrated that
urease immobilized on collagen-poly(glycidyl methacrylate) graft copolymer
support was thermally stable up to 70.degree. C. and 40 days when stored
at 4.degree. C. in a buffer solution. Davidenko et al. (Chem. Abstr. 1985,
102, 127894) have reported that urease adsorbed on carbon fibres is stable
up to 65.degree. C. and retained 90% of its activity when stored for a
month at 4.degree.-5.degree. C. Thermal stabilization up to 70.degree. C.
in buffer solutions was also reported for chymotripsin by multi-point
covalent attachment to aldehyde-agarose gels (Guisen et al. Biotechnol.
Bioeng. 1991, 38, 1144) and for glucoamylase on periodate oxidized dextran
(Lenders and Chricton, Biotechnol. Bioeng. 1988, 31,267). Asakura et al.
(Polym.-Plast. Technol. Eng. 1989, 28, 453) immobilized alkaline
phosphatase on Bombyx mori silk fibroin by cyanogen bromide and diazo
coupling methods and have shown that while the free enzyme was totally
deactivated at 65.degree. C., the enzyme coupled by the diazonium
procedure retained 30% of its activity, in comparison with 10% for the
cyanogen bromide-modified product. Yabushita and coworkers (Chem. Pharm.
Bull. 1988, 36, 954) have shown that urokinase immobilized on an
ethylene-vinyl acetate copolymer matrix retained more than 50% of its
initial activity when kept for 8 hours at 45.degree. C., while the soluble
enzyme lost almost all of its activity in 3 hours.
Margolin and coworkers (Eur. J. Biochem. 1985, 146, 625) effected a
comparative evaluation of the stability and activity of enzymes
immobilized on water-soluble and water-insoluble supports. Employing poly
(N-ethyl-4-vinyl pyridinium bromide) (a polycationic support) and poly
(methylacrylic acid) (a polyanionic support) for immobilizing a series of
enzymes, these authors showed that pronounced thermal stabilization of
penicillin amidase and urease could be achieved only if these enzymes are
on the precipitated supports (in the insoluble form) and covalently
attached to the polyelectrolyte nucleus. Thus, the thermal stability of
polyelectrolyte complex-bound penicillin amidase increased seven-fold at
pH 5.7, 60.degree. C. and three hundred-fold at pH 3.1, 25.degree. C.,
compared to the native enzyme. For urease, the thermal stabilization
increases twenty-fold at pH 5, 70.degree. C.
The role of phospholipids as protective agents for maintaining the activity
of antibodies, enzymes and receptors is well-documented. There is
considerable evidence concerning the requirement of a lipid environment
for sustaining the activity of enzymes. For example, it has been shown
that a lipid-modified glucose oxidase enzyme electrode offers greater
selectivity and stability for the analysis of glucose. Phospholipids may
act as modulators of enzymatic reactions in addition to their role as
obligatory cofactors for some membrane enzymes. Thus, it was shown
(Niedzwiecka et al., Acta Biochim. Biophys. Hung., 1990, 25,47), that the
purified lymphocyte 5'-nucleotidase reconstituted into lipid bilayer
demonstrates remarkable stability on storage at 4.degree. C. The liposome
incorporated enzyme from chicken gizzard is five times more stable at
56.degree. C. than the enzyme in the detergent solution, indicating that
the phospholipids play a role in preventing the denaturing process.
Rosenberg, Jones and Vadgama (Biochim. Biophys. Acta 1992, 1115, 157)
encapsulated glucose oxidase in liposomes and found that electrodes coated
with a nitro-cellulose membrane carrying these liposome-enzyme
formulations exhibited extended linear range of response. The enzyme
activity was found to be partially dictated by the liposomal bilayer
permeability, and therefore, the enzyme affinity for its substrate could
be regulated by using liposomes prepared from different lipids such as
dimyristoyl, dipalmitoyl and distearoyl-phosphatidylcholine. It has also
been shown by Kotowski and Tien (Bioelectrochem. Bioenerg. 1988, 19, 277)
that glucose oxidase could be covalently immobilized on a
polypyrrole-supported bilayer lipid membrane surface and the
enzyme-substrate reaction could be followed by cyclic voltammetry. The
phospholipid functions as an electric switch during this analysis, besides
supplying the natural biomembrane-type environment to the enzyme.
Besides thermal inactivation, the extent of activity exhibited by an
immobilized enzyme is also dependent upon aspects such as the chemical
procedure used to effect immobilization, the spacer chain length and the
pH of the buffering medium in which the enzyme-substrate reactions are
carried out. For example, Comfort et al. (Biotechnol. Bioeng. 1988, 32,
554) evaluated the immobilization yields of heparinase and bilurubin
oxidase on agarose and acrylic beads activated by four different reagents,
viz. cyanogen bromide, carbonyldiimidazole, oxirane and tresyl chloride,
respectively. They found that while heparinase was bound in 90% yield
(with 50% active enzyme) by the cyanogen bromide method, bilurubin oxidase
was preferentially linked. (50-55% maximum yield, with 25-30% active
enzyme) by the tresyl chloride and oxirane displacement. However, in both
cases, nearly 40-50% of the immobilized enzymes were leached out when
allowed to stand in buffer for a short time. Przybyt and Sugier (Anal.
Chim. Acta 1990, 239, 269) investigated the activity of urease immobilized
on oxidized tungsten electrodes by electrochemistry. The covalent binding
protocol followed by these authors consisted of initially silanizing the
metal oxide surface with gamma-aminopropyltriethoxysilane and then
cross-linking the enzyme with either cyanuric chloride or hexamethylene
diisocyanate or glutaraldehyde. They found that the lifetime of the enzyme
electrodes with the cyanuric chloride linker was only one day. In
comparison, the lifetimes of electrodes prepared by employing
glutaraldehyde and the diisocyanate cross-linkers were 29 and 22 days,
respectively. The life-time of the enzyme electrode, obtained by the
direct cross-linking of the metal oxide surface with the enzyme through
hexamethylene diisocyanate (without prior silanization) was 19 days.
Furthermore, these authors noted profound effects on the electrode
response due to factors such a the nature of the buffer, its concentration
and ionic strength.
The importance of the spacer chain length towards the retention of the
activity of an immobilized enzyme on a given surface has been demonstrated
by several groups of workers. For instance, Kennedy and Cabral (in Methods
in Enzymology, Vol. 135, pp. 117-130, Academic Press, San Diego, 1987)
examined the linking of glucoamylase to control pore glass activated with
titanium tetrachloride. The substrates were initially treated with ammonia
(no carbon spacer), 1,2-diaminoethane (a two-carbon spacer) and
hexamethylene diamine (a 6-carbon spacer) and then cross-linked with the
enzyme through glutaraldehyde. The six carbon spacer-carrying substrate
exhibited an activity retention of 12% relative to the activity of the
soluble enzyme, while the figures were 1.5% and 3.2% for the no carbon and
two carbon spacer, respectively. Jayakumari and Pillai (J. Appl. Polym.
Sci. 1991, 42, 583) observed that the direct coupling of papain to
carboxylated polystyrene yielded only 5% active enzyme, while binding of
the same enzyme to the same support through glutaric anhydride
cross-linker produced 30% of active enzyme. However, the maximum activity
retention (54%) was obtained when papain was linked to hydroxymethyl
polystyrene through polyethylene glycol (PEG 600) cross-linker. These
authors also demonstrated that increasing cross-link densities decreased
the total immobilization yields as well as the amount of active enzyme.
Furthermore, rigid supports lowered total/active enzyme yields in
comparison with flexible supports. Schuhmann et al. (J. Amer. Chem. Soc.
1991, 113, 1394) showed that the electrical communication between the
redox centres of glucose oxidase and vitreous carbon electrodes is more
effective when a long chain diamine was used to cross-link the aldehyde
functionalities of ferrocene and those of glucose oxidase obtained by the
oxidation with periodate. Reduction of electron-transfer distances between
the redox centre of the enzyme and the peripherally bound ferrocene relay
and between the relay and the electrode due to penetration of the relay to
a sufficient depth by the enzyme was postulated to be responsible for
their observations. Kobayashi et al. (J. Colloid Interface Sci. 1991, 141,
505) have reacted microfine magnetic particles of magnetite with APTES and
then cross-linked the surface with a protease; thermolysin, with
glutaraldehyde. They also utilized
omega-aminohexylaminopropyltrimethoxysilane,
4-aminobutylaminopropyltrimethoxysilane and
2-aminoethyl-aminopropyltrimethoxysilane and showed that maximum enzymatic
activity was exhibited by the hexyl-silane (50% higher than with APTES).
The report of Williamson et al. (Anal. Letters 1989, 22, 803), however,
contradicts the above findings on the spacer length, when an antibody,
rather than an enzyme, is immobilized to a support. These authors
covalently attached anti-T.sub.2 mycotoxin monoclonal antibodies on quartz
fibres by three techniques. The first two consist of the activation of the
surface hydroxyls of quartz with p-toluene sulphonyl chloride or
p-nitrophenylchloroformate, followed by the direct attachment of the
antibody. The third method involves initial silanization of quartz with
APTES followed by cross-linking of the antibody with glutaraldehyde.
Almost the same amount of activity was found to be exhibited by the
antibody on all of the above three surfaces. However, the thermal
stability of the antibody on the APTES-modified surface at 50.degree. C.
was considerably better than the antibody surfaces prepared with the other
two reagents. Significantly, treatment of the sulphonyl chloride or
chloroformate activated quartz with hexamethylene diamine, prior to the
immobilization of the antibody with glutaraldehyde, did not improve the
activity of the bound antibody, in spite of the six-carbon spacer.
The above brief summary of the thermal and a thermal factors responsible
for the deactivation of enzymes indicates that even immobilized enzymes
are not stable above 60.degree.-70.degree. C. In a number of instances,
nearly 50% of the immobilized enzyme is leached out by washing with a
buffer or detergent. Use of cross-linkers during the immobilization of the
enzymes also has a detrimental effect on the retention of the activity by
the immobilized biomolecules. Recent advances in the isolation of
thermostable enzymes utilize thermophilic bacteria and considerable
thermal stability has been claimed for the enzymes made by this route.
However, a recent report by Brosnan and coworkers (Eur. J. Biochem. 1992,
203, 225) demonstrates that alpha-amylase isolated from Bacillus
stearothermophilus is irreversibly deactivated at 90.degree. C. in 1.9
minutes at pH 5.0.
Although a large number of publications in documented literature have
clearly indicated that phospholipids exert a stabilizing effect on the
activity of enzymes, enzyme preparations so far known have only utilized
encapsulations in phospholipid liposomes. In two earlier patents (U.S.
Pat. No. 4,824,529 {1989] and U.S. Pat. No. 4,637,861 [1987]), as well as
in a recent publication (Anal. Chim. Acta 1989, 225, 369), we have
demonstrated that phospholipids can be covalently attached to different
kinds of supports. As analogues of natural biomembranes, these
phospholipids are expected to impart greater stability than hitherto known
to enzymes, if the two bio-entities could be covalently linked. It is
envisaged that the combination of a suitable spacer chain and
immobilization to a support through a phospholipid would enable the
formation of thermally very stable enzyme systems with extended
operational and storage stabilities in the solid state (without any
buffers), for a variety of applications.
It is therefore an object of the present invention to provide new compounds
suitable as spacers as well as linkers for the covalent immobilization of
enzymes and other biologically active substances either directly or
through an intermediate compound, onto a substrate.
It is a further object of the present invention to provide new
phospholipids suitable for covalent binding to the substrate through a
spacer compound and to the bioactive molecule.
It is another object of the present invention to provide preparations
comprising immobilized biologically active substances, e.g. enzymes, bound
to the substrate through the spacer compounds and optionally also through
the phospholipids.
It is still another object of the present invention to provide methods for
the preparation of the spacers and phospholipids utilized in the present
invention.
SUMMARY OF THE INVENTION
According to the invention, it is proposed to link enzymes (or other
biologically active molecules) to selected substrates through certain
spacer compounds, for example alkoxysilanes and preferably also through
phospholipid intermediates which are bound to the silanized substrate and
to the biologically active substance.
Preferably, the substrate is a solid material having sufficient functional
groups selected from hydroxyl, carboxylic, amino, mercapto and aldehyde
groups to enable the spacer compound (alkoxysilane or a diamine or a
dicarboxylic acid) to be attached to the substrate.
The substrate may be an inorganic material such as a metal, semiconductor
(silica or quartz) or ceramic (e.g. alumina); an organic polymer (either a
naturally occurring material such as cellulose or chitin or agarose, or a
synthetic product, like modified teflon) and a biomolecule, e.g. protein
or whole cell, provided that the above-defined functional groups are
present or can be incorporated onto the surface of this substrate. On
metallic substrates, hydroxyl groups can be incorporated by oxidation and
subsequent hydration.
The biologically active substances, referred to herein also as bioactive
substances, suitable for the purpose of the present invention, are
enzymes, antibodies, antigens and other proteins, i.e. compounds with
polypeptide structure. Certain other molecules such as DNA or hormones
(with polypeptide structure) are also suitable.
The enzyme or another bioactive substance is covalently linked to the
phospholipid as opposed to encapsulation in liposomes proposed in the
prior art.
Accordingly, this invention relates, in one aspect, to new preparations
comprising, in general terms, a solid substrate and a biologically active
substance linked covalently to the substrate through a spacer compound
having 7-18 carbon atoms in its alkyl chain.
The spacer compound may be an alkoxysilane, a dicarboxylic acid or a
diamine.
In another aspect, the invention relates to new preparations comprising a
substrate, a phospholipid covalently linked to the substrate through a
spacer compound having 7-18 carbon atoms in its alkyl chain, and a
bioactive substance covalently linked to the phospholipid.
In yet another aspect, the present invention proposes a new method of
making the above-defined structures, the method comprising:
(a) providing a selected solid substrate having sufficient functional
groups selected from hydroxyl, carboxyl, amino, mercapto and aldehyde on
its surface,
(b) binding an alkoxysilane (or a long chain dicarboxylic acid or a long
chain diamine) to the functional groups of the substrate, and,
(c) binding the biologically active substance to said alkoxysilane (or
other spacer).
Alternatively, the method comprises the following steps:
(a) providing a selected solid substrate having the required functional
groups selected from carboxyl, hydroxyl, amino, mercapto or aldehyde on
its surface,
(b) binding a spacer compound to the functional groups of the substrate,
(c) binding a phospholipid to the spacer molecule, and
(d) binding a biologically active substance to the phospholipid.
In a preferred embodiment of the invention, the bioactive substance is an
enzyme. Urease was selected for laboratory tests, but the invention is not
limited thereto.
Where a phospholipid is a part of the immobilized structure of the
invention, a number of spacer compounds may be utilized for bonding the
phospholipid to the support. Alkoxysilanes with an open chain having from
7 to 18 carbons atoms, aliphatic dicarboxylic acids and diamines with
similar alkyl chains can be used as the spacer compounds.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings which illustrate the invention in more detail,
FIG. 1 is a representation of chemical reactions leading to the generation
of silanized surfaces,
FIG. 2 illustrates the immobilization of an enzyme, urease, on silanized
surfaces of FIG. 1, by: a) direct coupling, b) activation by
N-hydroxysuccinimide, and c) activation by carbonyldiimidazole,
FIG. 3 illustrates the synthesis of bifunctional phospholipids and their
coupling to support and to the enzyme,
FIG. 4 illustrates the preparation and use of bifunctional
phosphatidylethanolamines
DETAILED DESCRIPTION OF THE INVENTION
Experimental
Silicon wafers were used as substrates. The wafers (10 cm in diameter,
p-doped, natural oxide grown) were purchased from Avrel Colo., Santa
Clara, Calif. Silica gel Davisil (trademark), Grade 645, 60-100 mesh, 150
.ANG. (angstrom), 99+% purity) was obtained from Aldrich.
All solvents were reagent grade samples further purified by drying with an
appropriate drying agent and distilled prior to use. The following
products: glutaric anhydride, 10-undecylenic acid, 11-aminoundecanoic
acid, chloroplatinic acid, t-butyldimethylsilylchloride, triethoxysilane,
dimethylaminopyridine, N-hydroxysuccinimide, carbonyldiimidazole,
dicyclohexylcarbodiimide, di-t-butylcarbonate, sebacoyl chloride,
aminopropyltriethoxysilane and trifluoroacetic anhydride were purchased
from Aldrich.
Glycerophosphoryl choline cadmium chloride complex,
ethyldimethylaminopropyl carbodiimide, Urease (U2000) were purchased from
Sigma.
Lyso 1-palmitoyl phosphatidylethanolamine was supplied by Avanti, Urease
(URE3) was supplied by Biozyme, Urease substrate solution by J. D.
Biologicals and aminopropyldimethylethoxysilane by Petrarch.
Synthesis of Omega-Functional Alkyl Triethoxysilanes
1. Preparation of 11-triethoxysilylundecanoic acid methyl ester:
a) 10-Undecylenic acid (20 mmoles) was dissolved in methanol (25 ml) and
concentrated hydrochloric acid (0.5 ml) added. The mixture was reflexed
for five hours, the excess methanol distilled off and the residue treated
with cold sodium bicarbonate solution (5% aq., 200 ml). The crude methyl
ester was extracted with ether, the ether layer washed with water, dried
with magnesium sulphate and concentrated on a rotary evaporator to yield a
colorless liquid (yield almost quantitative). Distillation of this product
under vacuum gave the pure ester, b.p. 104.degree.-5.degree. C./0.1 mm.
b) Hydrosilylation--The pure methyl ester (2 g) was treated with
triethoxysilane (3 ml) under nitrogen with the addition of chloroplatinic
acid (10 mg). After stirring at room temperature for 12 hours, the mixture
was refluxed for 2 hours. The excess silane reagent was removed under
vacuum and the residue extracted with pentane under nitrogen. The extract
was filtered and the filtrate concentrated under vacuum to yield the
desired product as a colorless liquid Yield 3.5 g). FABMS: MH.sup.+, m/z
363, [MH-EtOH].sup.+, m/z 318 (100%); FTIR: .sup..nu. C=O 1731 cm.sup.-1,
.sup..nu. Si-O 1102, 1081 cm.sup.-1.
2. Preparation of 11-triethoxysilyl undecylenic acid t-butyldimethysilyl
ester:
a) 10-Undecylenic acid (20 mmoles) was dissolved in DMF (20 ml) to which
t-butyldimethylsilyl chloride (4.5 g) and imidazole (100 mg) were added.
The mixture was stirred at room temperature under nitrogen for 24 hours.
Excess solvent and silane reagent were removed under vacuum and the
residue extracted with pentane. The pentane extract was dried and
concentrated on a rotary evaporator to yield the crude ester, which was
purified by distillation under vacuum, b.p. 110.degree.-112.degree. C./0.1
mm, yield quantitative. FTIR: .sup..nu. C=O 1716 cm.sup.-1.
b) Hydrosilylation--This reaction was carried out under the same conditions
as described under 1b. The product was characterized by FABMS: MH.sup.+,
m/z 449 (10%), [MH-EtOH].sup.+, m/z 403 (100%); FTIR: .sup..nu. C=O 1716
cm.sup.-1, .sup..nu. Si-O 1102 and 1088 cm.sup.-1.
Generation of the Omega-Carboxylic Silylated Surfaces 1 and 2
1. Formation of the carboxylic surface 1 (see FIG. 1)
a) Silanization of silica surfaces by 3-dimethylethoxysilyl 1 propanamine
was carried out by treating the cleaned surfaces with a solution of
gamma-aminopropyldimethylethoxysilane 3 (2% in toluene, 20 ml) and
refluxing for six hours under nitrogen. The substrates were then removed
and washed with chloroform, methanol and acetone in that order. Surface
characterization was effected by XPS and ellipsometry.
b) Treatment of the silanized surface with glutaric anhydride--the above
substrate 3a was suspended in THF (25 ml) and glutaric anhydride 4 (500
mg) was added. The mixture was refluxed under nitrogen for 12 hours, the
substrate removed from the solution and washed extensively with alcohol.
The resulting surface was characterized by XPS and ellipsometry.
2. Formation of the carboxydecyl dimethylsilylated surface 2
A. From 11-triethoxysilyl undecanoic acid methyl ester 8 (FIG. 1)
a) Silanization of silica substrates with the methyl ester 8
The substrate was suspended in toluene containing the methyl ester 8 (2%
solution) at room temperature under nitrogen overnight. It was then
removed and washed thoroughly with dichloromethane and dried under vacuum
for several hours.
b) Hydrolysis of the methyl ester function--after sodium hydroxide, sodium
carbonate, potassium t-butoxide were found to cleave the surface-to-silane
siloxane bond, it was attempted to use a non-basic approach. The methyl
ester-containing substrates were refluxed for 24 hours with lithium iodide
(200 mg) in DMF (20 ml). The substrates were recovered and washed
thoroughly with distilled water and vacuum dried before XPS and
ellipsometric analysis. Alternatively, the methyl ester moiety can be
removed by refluxing with trimethylchlorosilane (5 ml) and sodium iodide
(500 mg) for six hours.
B. From 11-triethoxysilyl undecanoic acid t-butyldimethylsilyl ester (9a):
a) silanization of silica substrates with the ester 9a--this reaction was
carried out at room temperature under nitrogen in toluene solution as
described under the methyl ester 8. Surface analysis was done by
ellipsometry and XPS.
b) hydrolysis of the t-butyldimethylsilyl ester moiety--the hydrolysis of
the silyl ester was accomplished by suspending the substrate from reaction
a) in aqueous methanolic hydrochloric acid (1:1, 10%, 10 ml) for three
hours. The substrate was washed copiously with water and dried. Surface
analysis was done by the usual techniques.
Immobilization of Urease on the Carboxyl-Functionalized silanized surfaces
1 & 2
A. Direct Immobilization
The carboxylic surface 1 or 2 (100 mg) was suspended in distilled water and
treated with EDC (5 mg) for 12 hours, the supernatant liquid decanted off
and the substrate treated with urease (1 mg) in distilled water (1 ml) for
a period of 48 hours at 5.degree. C. The supernatant liquid was carefully
drawn off and its enzymatic activity determined by spectrophotometry after
dilution to 10 ml. The substrate was thoroughly washed with distilled
water and kept u | | |
