U.S. patent application number 10/181435 was filed with the patent office on 2003-07-17 for molecularly imprinted polymer.
Invention is credited to Chen, Beining, Day, Richard M, Pileiska, Olena V, Pileisky, Sergiy A, Subrahmanyam, Sreenath, Turner, Anthony P F.
Application Number | 20030134296 10/181435 |
Document ID | / |
Family ID | 9884186 |
Filed Date | 2003-07-17 |
United States Patent
Application |
20030134296 |
Kind Code |
A1 |
Chen, Beining ; et
al. |
July 17, 2003 |
Molecularly imprinted polymer
Abstract
To facilitate preparation of a molecularly imprinted polymer
(MIP) which specifically binds a particular template substance, a
virtual library of functional monomers is screened to find those
that interact strongly with a molecular model of the substance. A
selected few are then "annealed" with the substance in a computer
simulation. This shows the optimum ratios of template substance to
individual monomers. An appropriate mixture of monomers is then
polymerised in the presence of the template to produce a MIP.
Inventors: |
Chen, Beining; (Cranfield,
GB) ; Day, Richard M; (Navestock, GB) ;
Subrahmanyam, Sreenath; (Chennai, IN) ; Pileisky,
Sergiy A; (Cranfield, GB) ; Pileiska, Olena V;
(Cranfield, GB) ; Turner, Anthony P F; (North
Crawley, GB) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Family ID: |
9884186 |
Appl. No.: |
10/181435 |
Filed: |
November 15, 2002 |
PCT Filed: |
January 25, 2001 |
PCT NO: |
PCT/GB01/00324 |
Current U.S.
Class: |
435/6.16 ;
435/7.1; 436/518; 703/11 |
Current CPC
Class: |
C08F 2/44 20130101; C08F
291/00 20130101; C08F 251/00 20130101; G01N 2600/00 20130101; C08F
289/00 20130101 |
Class at
Publication: |
435/6 ; 435/7.1;
703/11; 436/518 |
International
Class: |
C12Q 001/68; G01N
033/53; G06G 007/48; G06G 007/58; G01N 033/543 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 25, 2000 |
GB |
0001513.1 |
Claims
1. A method of preparing a molecularly imprinted polymer which is
adapted to interact with a template substance, the method
comprising the steps of: (a) producing a virtual library of
molecular models of functional monomers having polymerisable
portions and binding portions capable of reversibly binding to a
template substance; (b) providing a molecular model of the template
substance; (c) screening the virtual library with the molecular
model of the template substance and determining a plurality of the
monomers found to bind strongly to it; (d) providing a chemical
reaction system containing said plurality of monomers and said
template substance and effecting polymerisation of said monomers to
produce a molecularly imprinted polymer.
2. A method according to claim 1 wherein said screening step (c)
involves (c-i) calculating interaction energies between individual
monomers and the template substance to identify a plurality of said
monomers able to bind relatively strongly to the template
substance; and (c-ii) using molecular mechanical calculations to
estimate how said plurality of monomers would bind to said template
substance, thereby determining optimum ratios of template substance
to monomers; and wherein said step (d) employs a reaction system
containing amounts of template substance and monomers in accordance
with said ratios.
3. A process according to claim 1 or claim 2 wherein said
functional monomers in said virtual library comprise one or more of
vinyl monomers, allyl monomers, acetylenes, acrylates,
methacrylates, amino acids, nucleosides, nucleotides,
carbohydrates, phenols, heterocycles, aniline, and their
derivatives.
4. A process according to claim 1, 2 or 3 wherein said template
substance is selected from biological receptors, nucleic acids,
immunosuppressants, hormones, heparin, antibiotics, vitamins, drugs
or synthetic molecules possessing biological activity, cell
components and components of viruses such as carbohydrates, lipids,
saccharides, nucleoproteins, mucoproteins, lipoproteins, peptides
and proteins, glycoproteins, glucosaminoglycanes and steroids.
5. A polymer as produced by the process of any preceding claim.
6. Use of a polymer as produced by the process of any of claims 1-4
for selectively binding the template substance or a related
compound.
7. A procedure of computer aided rational design techniques for the
rapid development and optimization of molecularly imprinted
polymers (MIPs) which includes screening of a virtual library of
functional monomers for their interaction with a template molecule
and selection of those monomers giving a strong complex with the
template for polymer preparation.
8. The procedure of claim 7 wherein the template is selected from
biological receptors, nucleic acids, immunosuppressants, hormones,
heparin, antibiotics, vitamins, drugs or synthetic molecules
possessing biological activity, cell components and components of
viruses such as carbohydrates, lipids, saccharides, nucleoproteins,
mucoproteins, lipoproteins, peptides and proteins, glycoproteins,
glucosaminoglycanes and steroids.
9. A computer-aided rational design technique for the rapid
development and optimization of MIPs which comprises placing
functional monomers around a template and using molecular mechanics
to simulate, pre-arrangement of the functional monomers with
template in the monomer mixture.
10. Computer-aided rational design technique for the rapid
development and optimization of molecularly imprinted polymers
which includes two steps: first, screening of a virtual library of
functional monomers for their interaction with a template molecule;
second, selection of those monomers which form a strong complex
with the template for use in a refining step, when two or more
functional monomers are placed around the template and molecular
mechanics is used to simulate pre-arrangement of the functional
monomers with template in the monomer mixture.
11. A computer-aided rational design technique for the rapid
development and optimization of molecularly imprinted polymers as
described in claim 7, claim 9 or claim 10 where parameters of the
modeling and screening suitably comprising one or more of
dielectric constants, temperature chosen for "annealing" procedure,
and type of interactions, are adjusted according to the real
polymerization or re-binding conditions.
12. A virtual library of functional monomers for use in the
procedure of any of claims 7-11 containing at least two monomers
which possess polymerizable residues and residues able to interact
with a template through one or more of electrostatic, hydrophobic,
van-der-Waals forces, dipole-dipole interactions and reversible
covalent bonds, the monomers being selected from vinyl monomers,
allyl monomers, acetylenes, acrylates, methacrylates, amino acids,
nucleosides, nucleotides, carbohydrates, phenols, heterocycles,
aniline, and their derivatives.
Description
TECHNICAL FIELD
[0001] The present invention relates to methods and means for use
in the preparation of molecularly imprinted polymers ("MIPs"), to
the MIPs that can be created using them, and to applications of the
MIPs.
[0002] The present invention particularly relates to an application
of computer-aided rational design techniques for the rapid
development and optimization of polymers that act as synthetic
receptors. Specifically the imprinted polymers are synthesized by a
polymerization of individual monomers with or without cross-linker
in the presence of template molecule(s), which can be small
molecules such as drugs, pesticides, peptides as well as large
molecules such as carbohydrates, nucleic acids and proteins.
BACKGROUND ART
[0003] Molecularly imprinted polymers (MIPs), materials with
artificially created receptor-like recognition properties, have
recently attracted significant attention as potential substitutes
for unstable receptors and antibodies in affinity chromatography,
membranes, capillary electrophoresis and sensor technology (U.S.
Pat. Nos. 5,110,833, 5,587,273, 5,756,717, 5,728,296, 5,786,428 and
5,849,215). Among the factors limiting their practical application
is the absence of a general procedure for polymer synthesis.
Several attempts have been made to develop a general procedure for
rational design of the imprinted polymers (Nicholls I. A. (1995):
Thermodynamic consideration for the design of and ligand
recognition by molecularly imprinted polymers, Chem. Lett.,
1035-1036; Whitcombe M. J., Martin L., Vulfson E. N.
[0004] (1998): Predicting the selectivity of imprinted polymers.
Chromatography, 47, 457-464; Takeuchi T., Fukuma D., Matsui J.
(1999): Combinatorial molecular imprinting: an approach to
synthetic polymer receptors. Anal. Chem., 71, 285-290). In the best
cases they give rules or hints, indicating how the MIP should be
made in order to possess a certain level of specificity. Thus it is
recommended that the polymerization should be performed in a
hydrophobic solvent in order to produce a material able to interact
with the template through electrostatic interactions. At the same
time the choice of monomers, solvent and polymerization conditions
in most cases depends on general principles, personal experience or
information about similar systems. In some extreme cases it has
been necessary to produce and investigate hundreds of polymers in
order to optimize MIP monomer composition (Takeuchi et al. op.
cit).
DISCLOSURE OF INVENTION
[0005] The present invention describes a new method for rational
choice of the functional monomers for the preparation of MIPs.
[0006] The present invention describes a computer-aided rational
design techniques for the rapid development and optimization of
molecularly imprinted polymers, which includes screening of a
virtual library of functional monomers for their interaction with
the template molecule and selection of those monomers forming a
strong complex with the template for polymer preparation. The
procedure of monomer selection includes several stages (it is
important to note that each individual step as well as their
combination can be used separately for the design of MIPs and are
covered by the present invention). First, a virtual library of
molecular models of functional monomers is produced containing
molecules that possess polymerizable residues and residues able to
interact with a template, e.g. through electrostatic, hydrophobic
van-der-Waals forces, dipole-dipole interactions and/or reversible
covalent bonds. Secondly, a molecular model of the template
molecule is prepared. Charges for each atom are calculated, and the
structure of the template and monomers refined using molecular
mechanical methods. Thirdly, each of the entries in the virtual
library is probed for their possible interaction with the template
molecule. The monomers giving the highest binding score represent
the best candidates for polymer preparation. Fourthly, copies of
the most ideal hits are placed around the target. Simulated
annealing is then used to simulate pre-arrangement of the
functional monomers with template in the monomer mixture prior to
polymerization. At the end of the program, the number and the
position of the functional monomers are examined The type and
quantity of the monomers participating in the complex with template
indicate the type and ratio of the template and monomers in an
optimized MIP composition. Finally a mixture of monomers
corresponding to this composition is polymerised in the presence of
the template to produce a MIP.
[0007] Main embodiments include:
[0008] 1. A computer-aided rational design technique for the rapid
development and optimization of molecularly imprinted polymers
(MIPs), which includes design of a virtual library of functional
monomers and screening of their interaction with a template
molecule. Functional monomers should possess polymerizable residues
and residues able to interact with the template, e.g. through
electrostatic, hydrophobic van-der-Waals forces, dipole-dipole
interactions and/or reversible covalent bonds. The library of
functional monomers may include one or more of vinyl monomers,
allyl monomers, acetylenes, acrylates, methacrylates, amino acids,
nucleosides, nucleotides, carbohydrates, phenols, heterocycles,
aniline, and their derivatives.
[0009] 2. Design of a molecular model of the template using
molecular mechanical methods. The template molecule may be selected
from biological receptors, nucleic acids, hormones, heparin,
antibiotics, vitamins, drugs, cell components and components of
viruses such as carbohydrates, saccharides, nucleoproteins,
mucoproteins, lipoproteins, peptides and proteins, glycoproteins,
glucosaminoglycanes and steroids.
[0010] 3.Screening of the virtual library of the functional
monomers for their ability to form molecular complex with the
template. The monomers giving the highest binding score represent
the best candidates for polymer preparation.
[0011] 4. Placing of the functional monomers (or monomers giving
the highest binding score) around the template and using molecular
mechanics in order to simulate pre-arrangement of the functional
monomers with template in the monomer mixture prior to
polymerization. At the end of the program, the number and the
position of the functional monomers are examined The type and
quantity of the monomers participating in the complex with template
indicate the type and ratio of the template and monomers in an
optimized MIP composition.
[0012] 5. The parameters of the modeling and screening (dielectric
constants, temperature chosen for "annealing" procedure, type of
interactions) could be modified in order to simulate the real
polymerization or re-binding conditions.
BRIEF DESCRIPTION OF THE DRAWINGS:
[0013] FIG. 1. Depicts a virtual library of the functional monomers
used for the development of MIP, specific for atrazine.
[0014] FIG. 2. Shows the functional monomers from the virtual
library giving the highest binding score when interacting with
atrazine.
[0015] FIG. 3. Depicts interactions between functional monomers and
atrazine in an optimized monomer-template complex.
[0016] FIG. 4. Recovery of the atrazine by polymers, based on
itaconic acid, acrylamide and AMPSA. 100 ml of herbicide solution
in water (10.sup.-9 M) were filtered through 100 mg of polymer and
eluted with 90% methanol containing 10 mM HCl.
[0017] FIG. 5. Depicts interactions between functional monomers and
methylated creatinine in an optimized monomer-template complex.
[0018] FIG. 6. Interaction between phthalic dialdehyde, mercaptan
group (OPA reagents) and primary amine (I); thioacetal formation
(II); formation of fluorescent complex between thioacetal and
primary amine (III).
[0019] FIG. 7. Depicts imprinting factors of the polymers MIP A,
MIP B and MIP C calculated for creatinine and creatine. Imprinted
factors were calculated using equation
I=.DELTA.F(MIP)/.DELTA.F(Blank) where .DELTA.F is change in
fluorescence for MIP and blank polymers, correspondingly.
Measurements were made in 100 mM sodium phosphate buffer, pH 7.0
(Ex=320 nm; Em=410 nm).
MODES FOR CARRYING OUT THE INVENTION
[0020] The first embodiment describes the design of a virtual
library of functional monomers which possess polymerizable residues
and residues, able to interact with a template through
electrostatic, hydrophobic van-der-Waals forces, dipole-dipole
interactions or reversible covalent bonds. The library of
functional monomers should contain at least two, and preferably
more monomers which can be vinyl monomers, allyl monomers,
acetylenes, acrylates, methacrylates, amino acids, nucleosides,
nucleotides, sugars and saccharides, carbohydrates, phenols,
heterocycles, aniline, and their derivatives. Preferable monomers
are these able to interact with the template through non-covalent
interactions and be polymerized through a radical mechanism.
[0021] The second embodiment describes the design of a molecular
model of the template using molecular mechanical methods. The
template is selected from a group including biological receptors,
nucleic acids, immunosuppressants, hormones, heparin, antibiotics,
vitamins, drugs, cell components and components of viruses such as
carbohydrates, lipids, saccharides, nucleoproteins, mucoproteins,
lipoproteins, peptides and proteins, glycoproteins,
glucosaminoglycanes and steroids.
[0022] The third embodiment describes screening the virtual library
of functional monomers for their ability to form a molecular
complex with the template. The monomers giving the highest binding
score represent the best candidates for polymer preparation and can
be used directly for the polymer synthesis as part of a monomer
mixture.
[0023] The fourth embodiment describes a refining step for
optimizing the monomer composition. It includes placing of one or
more functional monomers, preferably the monomers giving the
highest binding score around the template, and using molecular
mechanics to simulate pre-arrangement of the functional monomers
with template in the monomer mixture prior to polymerization. At
the end of the program, the number and the position of the
functional monomers are examined. The refining step has three
goals: first, to evaluate the quantity of the monomer units which
should be used in complexation with template; second, to determine
if cocktails of monomers form a stronger complex with the template
than individual monomers; third, to check possible interactions
between the functional monomers in a monomer mixture, both positive
(stabilizing complex) and negative (competing). The type and
quantity of the monomers participating in the complex with template
(first shell layer), or their combination with the monomers which
interact with the first shell layer of monomers, stabilizing
complex (second shell layer) indicate the type and ratio of the
template and monomers in an optimized MIP composition. All
components of the refining process can be used and accounted for
individually or in combination in the optimization of the monomer
composition.
[0024] The sixth embodiment describes "fitting" of the modeling and
screening parameters (dielectric constants, temperature chosen for
"annealing" procedure, type of interactions) to real polymerization
or re-binding conditions. If, for example, the template is not
soluble in organic solvents then the dielectric constant could be
changed from vacuum to water or the constant of the solvent used
for polymerization. The temperature in the "annealing" procedure
can be also adjusted to the one applied during polymerization or
the re-binding step.
[0025] The present invention will now be further described in
detail by reference to the following, examples, which are intended
to illustrate some of the possibilities, but are in no way intended
to limit the scope of the invention.
EXAMPLE 1
Computer Aided Design of an MIP Specific for Atrazine
[0026] a) The workstation used to generate molecular models and
perform simulation was a Silicon Graphics Octane running the IRIX
6.4 operating system. The workstation was configured with two 195
MHz reduced instruction set processors, 712 MB memory and a 12 Gb
fixed drive. The system was used to execute the software packages
SYBYL (Tripos). The virtual library contained the most frequently
used functional monomers able to form non-covalent interactions
with the template atrazine. These are methacrylic acid, acrylic
acid, itaconic acid, acrolein, acrylamido-(2-methyl)-propane
sulfonic acid (AMPSA), 2-vinylpyridine, 4-vinylpyridine,
1-vinylimidazole allylamine, N,N'-diethyl aminoethyl methacrylamide
(DEAEM), acrylonitrile, 2-hydroxyethyl methacrylate,
p-divinylbenzene, m-divinylbenzene, urocanic acid, urocanic ethyl
ester, ethylene glycol dimethacrylate,
N,N'-methylene-bis-acrylamide, styrene, acrylamide and, where
appropriate, their protonated or deprotonated forms (see FIG.
1).
[0027] The LEAPFROG algorithm was used to analyze binding between
monomers and template. 100,000 iterations were completed in
[0028] approximately 8 hours, results from each run were examined
and the empirical binding score was rank to the binding
interactions between the functional monomers and atrazine. The best
five are shown in FIG. 2. Their calculated binding scores were:
1 Itaconic Acid -37.05 kcal.mol.sup.-1 Acrylamide -35.78
kcal.mol.sup.-1 2-Acryl-2-methyl-1-propanesulph- onic -32.64
kcal.mol.sup.-1 acid N',N-Methylbisacrylate -31.63 kcal.mol.sup.-1
Acrylic acid -29.61 kcal.mol.sup.-1
[0029] b) The top three monomers (itaconic acid, acrylamide and
2-acryl-2-methyl-1-propanesulfonic acid) were next "packed" around
the template using a solvation experiment. Simulated annealing was
used to optimize the organization of the monomers around the
template molecule. Annealing conditions were fixed as 1000K-10K.
Equilibrium length was determined as 10000 ps. Energy minimization
was performed to a 0.01 kcal mol.sup.-1 convergence for each
iteration. At the end of the program, the number and the position
of the functional monomers were examined (FIG. 3). The type and
quantity of the monomers participating in the complex with template
indicate the type and ratio of the template and monomers in an
optimized MIP composition. Accordingly to simulation atrazine forms
in the model composition complex with 2 molecules of itaconic acid
(first shell layer). The rest of the monomers are displaced from
the complex as result of a competition process. At the same time,
attention can also be paid to three molecules of acrylamide, which
form a second shell layer interacting with itaconic acid from the
first shell layer. The optimization of the MIP composition gives a
template-monomers ratio 1 (atrazine):2 (itaconic acid):3
(acrylamide).
[0030] The calculated energy of the complex is based primarily on
two figures: -29.0 kCal Mol.sup.-1 for steric factors and -35.1
kCal Mol.sup.-1 for electrostatic interactions.
[0031] c) The synthesis of polymers was made with solutions of
corresponding monomers, as identified by computer simulation, using
dimethylformamide as solvent and 80% cross-linker (ethyleneglycol
dimethacrylate). Compositions are shown in Table 1.
2TABLE 1 Composition of the computational polymers specific for
atrazine. Functional Polymer Atrazine monomer EGDM.sup.1 DMF.sup.2
Initiator.sup.3 AMPSA- 215 mg 1044 mg AMPSA 4 ml 4 ml 50 mg MIP
AMPSA- -- 1044 mg AMPSA 4 ml 4 ml 50 mg Blank AA-MIP 215 mg 284.32
mg AA 2 ml 2 ml 50 mg AA-Blank -- 284.32 mg AA 2 ml 2 ml 50 mg
IA-MIP 215 mg 520 mg IA 3 ml 3 ml 50 mg IA-Blank -- 520 mg IA 3 ml
3 ml 50 mg .sup.1EGDM- ethylene glycol dimethacrylate .sup.2DMF-
dimethylformamide .sup.31,1'-Azobis(cyclohexanecarbonitrile)
[0032] The polymerisation was initiated by adding 1% azobis
(cyclohexane carbonitrile) and heating of the monomer mixture
during 12 hours. The resulting polymer was ground, sieved and
sedimented in acetone, giving a suspension with an average particle
size of 45-106 .mu.m. 100 ml of herbicide solution in water
(10.sup.-9 M) were filtered through 100 mg of polymer and eluted
with 90% methanol containing 10 mM HCl. The herbicide concentration
was measured using test-system based on the thylakoid membranes and
Hill reaction (Piletskaya E. V. et al. (1999). Anal. Chem. Acta,
391, 1-7). A good correlation was found between the polymer
affinity and the monomer binding score (FIG. 5). It was found that
the MIPs designed using the computational approach had superior
binding and recognition properties in comparison with a MIP
designed using the conventional approach (based on methacrylic acid
as a functional monomer). Additional polymer was prepared as
described before using a template-monomers ratio 1 (atrazine):2
(itaconic acid):3 (acrylamide). This polymer has shown an atrazine
binding similar to IA-based MIP with 100% efficiency of atrazine
recovering (data not shown).
EXAMPLE 2
Computer Aided Design of the MIP Specific for Creatinine
[0033] a) The virtual library used for the design of an MIP
specific for creatinine contained 20 monomers as used in Example 1
and one additional monomer--polymerizable thioacetal formed by
allyl mercaptan and o-phthalic dialdehyde, which was necessary for
creatinine sensing. Creatinine analogue--N,N'-dimethylcreatinine
was used as a template instead of creatinine in order to prevent
covalent interactions between the template and the functional
monomers. The computer simulation was performed in the same way as
described in Example 1. The optimized composition contains
template-monomers ratio 1 (N,N'-dimethylcreatinine):- 3
(thioacetal):1 (urocanic ethyl ester) (FIG. 5).
[0034] b) Three polymers were synthesized using a conventional
selection of monomers (with cross-linker only and with methacrylic
as functional monomer) and computational selection. Three
additional blank polymers were prepared in the absence of template.
The composition of the monomer mixtures is shown in Table 2.
[0035] O-phthalic dialdehyde (563 mg) and allyl mercaptan (330 mg)
were dissolved in 2 ml of DMSO and mixed with methylated creatinine
(131 mg). Functional monomers, methacrylic acid (258 mg) (MIP B) or
urocanic acid ethyl ester (138 mg) (MIP C) and azobis (cyclohexane
carbonitrile) (ACC) (50 mg) were added to the monomers, the
solution was purged with nitrogen and left for 1 hour at room
temperature. The polymerization was initiated by heating overnight
at 80.degree. C. The resulting polymer was ground, sieved and
sedimented in acetone, giving a suspension with an average particle
size of 5 .mu.m. Three additional blank polymers were prepared in
the absence of template.
3TABLE 2 Composition of polymers designed using conventional and
computational approaches Urocanic acid ethyl Polymer AM OPA MA
ester Creatinine MIP A 330 ul 563 mg -- -- 113 mg (.mu.mol)
(.mu.mol) (.mu.mol) Blank A 330 ul 563 mg -- -- -- (.mu.mol)
(.mu.mol) MIP B 330 ul 563 mg 258 ul -- 113 mg (.mu.mol) (.mu.mol)
(.mu.mol) (.mu.mol) Blank B 330 ul 563 mg 258 ul -- -- (.mu.mol)
(.mu.mol) (.mu.mol) MIP C 330 ul 563 mg 138.13 113 mg (.mu.mol)
(.mu.mol) mg (.mu.mol) (.mu.mol) Blank C 330 ul 563 mg 138.13 --
(.mu.mol) (.mu.mol) mg (.mu.mol) AM - allyl mercaptan OPA -
o-phtalic dialdehyde; MA - methacrylic acid;
[0036] All the monomer mixtures contained 2 ml of EDMA--ethylene
glycol dimethacrylate and 2 ml of DMSO--dimethyl sulfoxide with 50
mg of AIBN--azobis (isobutyronitrile).
[0037] c) Polymer recognition properties were analyzed by measuring
fluorescence changes generated by a polymer suspension following
the reaction between primary amines and polymer thioacetal leading
to formation of an isoindole complex (FIG. 6). The imprinting
factor, calculated for MIPs, clearly indicates the superior
selectivity of the polymer designed using the computational
approach in comparison with polymers designed using a traditional
approach (FIG. 7).
* * * * *