Composition of matter comprising an imprinted matrix exhibiting selective binding interactions through chelated metals

The invention provides an imprinted matrix which exhibits selective binding interactions through metal chelates with a predetermined molecule or biological particle. Also provided is a preformed, fluid-imprinted matrix having sufficient rigidity to maintain selective binding interaction with a predetermined molecule or biological particle through interactive moieties. Methods for producing such matrices are additionally provided.

BACKGROUND OF THE INVENTION 
This invention relates to affinity ligands in general and, more 
particularly, to methods of producing highly selective matrices with 
reversible binding characteristics. 
Separation and purification accounts for a very large percentage of the 
production cost of proteins and many specialty chemicals used for human 
therapeutics. Considerable effort is being expended on developing and 
optimizing techniques for the large-scale separation and purification of 
proteins. The difficulties lie in the high degrees of purity required for 
human therapeutics and therefore the extreme selectivity that is required 
for the separation process. These requirements result in complex, 
multi-step processes with concomitant high cost and low yield. Similar 
considerations hold true for specialty chemicals. 
High selectivity in purification appears to be incompatible with low cost. 
For example, precipitation processes, or even ion-exchange chromatography, 
are relatively inexpensive operations, but they are also relatively 
nonselective and often must be accompanied by additional purification 
steps. Affinity separations often give a very high degree of purification 
in a single step, but biologically derived ligands such as monoclonal 
antibodies which are used in affinity chromatography are very costly, are 
unstable, and are not particularly easy to recycle or sterilize. 
Affinity separations have been developed which exploit the affinity 
exhibited by proteins for metal ions. This property has been utilized in 
immobilized metal-affinity chromatography (IMAC) of proteins from natural 
sources and from recombinant organisms. A related technique, known as 
ligand-exchange chromatography, has been used in the purification of 
specialty chemicals such as amino acid derivatives and chiral precursors. 
In these techniques, a chelated metal ion with at least one available 
coordination site is covalently attached to a solid support and used as an 
affinity ligand to retain molecules which exhibit metal-coordinating 
moieties on their surfaces. Examples of such metal coordinating moieties 
are the amino acid side chains of histidine and cysteine on the surfaces 
of proteins. IMAC holds a number of important advantages over the use of 
biologically derived affinity ligands as recognition agents in protein 
separations. The small metal chelates generally used in metal-affinity 
separations are stable under a wide range of solvent conditions and 
temperatures. As a result, they can be recycled numerous times with 
negligible loss in performance. Other advantages of metal-affinity 
separations include the high metal loadings and therefore high protein 
capacities that can be attained and the relative ease of product elution 
and ligand regeneration. Proteins bound to chelated metals are easily 
removed by lowering the pH or by introducing a metal-binding ligand such 
as imidazole, and metal-affinity columns are regenerated simply by 
replenishing the supply of chelated metal. Metal chelate ligands have the 
additional advantage of being inexpensive. 
Although metal-affinity separations are attractive from a number of 
economic and practical viewpoints, the metal-affinity ligands used are, 
unfortunately, not nearly as selective as biologically derived molecules 
such as antibodies. For example, chromatography on iminodiacetic 
acid-bound Cu(II), the most commonly used metal chelate, distinguishes 
proteins primarily by their surface histidine contents. While such current 
metal-affinity separations distinguish among proteins that contain widely 
different numbers of exposed histidines, it becomes more difficult to 
separate those with similar numbers of histidines. 
One method to create polymeric matrices which exhibit selective binding 
interactions is to prepare polymers by a technique known as molecular 
imprinting or template polymerization. The technique is reviewed in Ekberg 
and Mosbach, TIBTECH 7:92-96 (1989), and in Wulff, Am. Chem. Soc. Symp. 
Ser. 308:186-230 (1986), and which describe molecular imprinting of small 
organic molecules and amino acids. Imprinting utilizes a template molecule 
with which to orchestrate the synthesis of individual monomers into a 
polymer matrix. The resulting matrix exhibits a large number of 
complementary interactions between the monomers and template molecule and 
can be viewed as a molecular "mold-like" structure. Such polymers are 
capable of specific recognition of template molecules and have been 
exclusively limited to small molecules. 
A major disadvantage of this molecular imprinting technique is that the 
chemistry involved in synthesizing such polymer matrices is largely 
limited to organic solvents. While organic solvents can be used with small 
organic molecules and amino acids, they cannot be used with biological 
macromolecules or particles since they result in denaturation and 
inactivation of such molecules. Another disadvantage is that a large 
number of interactions are needed to selectively recognize a molecule as 
large as a protein. It is extremely difficult to synthesize materials with 
such a large number of complementary interactions. 
There thus exists a need for inexpensive compositions which exhibit the 
high selectivity of biologically derived affinity ligands toward 
molecules, including macromolecules and biological particles, and yet 
retain reversible interactive and stability properties of metal-chelate 
ligands and imprinted polymers. The present invention satisfies these 
needs and provides related advantages as well. 
SUMMARY OF THE INVENTION 
The invention provides an imprinted matrix which exhibits selective binding 
interactions through metal chelates with a predetermined molecule or 
biological particle. Also provided is a preformed, fluid imprinted matrix 
having sufficient rigidity to maintain selective binding interaction with 
a predetermined molecule or biological particle through interactive 
moieties. Methods for producing such matrices are additionally provided.

DETAILED DESCRIPTION OF THE INVENTION 
References are cited throughout the specification. These references in 
their entirety are incorporated by reference into the specification to 
more fully describe the state of the art to which it pertains. 
The invention is directed to a novel class of compositions and methods of 
making such compositions for imprinting predetermined molecules and 
biological particles. The compositions are tailored to selectively 
recognize essentially any desired molecule or aggregate of molecules. 
Imprinting methods are efficient, inexpensive, and yield matrices which 
are stable under a variety of conditions. Thus, the imprinted matrices are 
desirable alternatives to expensive and unstable biologically derived 
recognition molecules. The imprinted matrices are applicable in a variety 
of research, therapeutic, diagnostic and manufacturing methods. 
In one embodiment, the imprinted matrices are composed of polymers 
containing chelated metals which bind functional groups on protein 
surfaces. Chelated metals provide optimal energetic requirements for 
imprinted matrices since a relatively small number of interactions are 
needed for selective recognition. The selective recognition is due to the 
spatial matching of metal ions, for example copper ions, in a rigid 
polymer to that of metal-coordinating amino acid residues, for example 
histidines, on the protein surface. Spatial matching is performed by 
polymerization of the monomers and cross-linking agents in the presence of 
the protein. Metal-chelating monomers are preorganized prior to 
polymerization to complement the distribution of coordinating amino acids 
on the protein surface. Polymerization locks the preorganized monomers 
into a rigid matrix. Thus, the protein acts as a template to geometrically 
arrange metal-chelates in the correct three-dimensional structure for 
selective recognition. The protein template is removed following 
polymerization, and the resulting imprinted matrix can exhibit selective 
and reversible affinity for the template protein. Much lower affinities 
are also observed for proteins whose spatial distribution of coordinating 
residues does not complement the distance between metal ions. 
In another embodiment, ordered fluids such as bilayer membranes are used as 
imprint matrices instead of solid polymers. Although metals can provide 
the binding interactions, the preorganized structure of bilayer membranes 
enables imprinting of large molecules, such as proteins, viruses, or cells 
without necessitating the use of metal-chelates as affinity ligands. 
Instead, interactive moieties which bind to functional groups on the 
template molecule through hydrogen binding, electrostatic interactions, 
hydrophobic interactions, and van der Waals forces can be used. Metal 
coordination can also be used. The interactive moieties are attached to 
the head groups of lipids that are used to form a membrane which is then 
exposed to the template molecule. The lipids are free to diffuse within 
the membrane, which allows alignment of interactive moieties with 
functional groups on the template molecule. Once the geometrical spacing 
is organized, the lipids are locked in place by chemical cross-linking, 
forming a two-dimensional imprinted matrix of the template protein. 
Removal of the template protein yields a membrane with sufficient rigidity 
to retain spatial organization of interactive moieties for selective and 
reversible recognition of the template molecule. 
As used herein, the term "imprinted matrix" refers to a molecular mold-like 
structure which has preorganized interactive moieties complementing the 
spacing of binding sites on a template. The interactive moieties can be, 
for example, chemical groups or affinity ligands. A specific example of an 
affinity ligand is iminodiaoetic acid-bound copper(II). The geometrical 
organization of interactive moieties imparts selective binding 
characteristics for the template substance onto the imprinted matrix. 
"Imprinting," as used herein, is the act of spatially organizing the 
interactive moieties to complement binding sites on a template. Examples 
of imprinted matrices are, for example, styrene-iminodiacetate 
metal-chelating monomer cross-linked with ethylene glycol dimethacrylate 
and stabilized lipid vesicles that contain iminodiacetate-metal head 
groups and that have been polymerized via diene groups in their lipid 
tails. 
As used herein, the term "selective binding interactions" refers to 
preferential and reversible binding exhibited by an imprinted matrix for 
its template molecule compared to other non-template molecules. Selective 
binding includes both affinity and specificity of the imprinted matrix for 
its template molecule. 
As used herein, the term "preformed fluid imprint matrix" refers to a 
substance composed of many monomers which are free to diffuse within a 
two-dimensional space and are organized into a definable structure. A 
fluid imprint matrix must be able to retain its overall structural 
integrity when bound to a template molecule. Such monomers of a fluid 
imprint matrix can be, for example, lipids such as 
phosphotidylethanolamine and phosphotidylcholine which have been modified 
in their hydrophilic head groups with interactive moieties Two-dimensional 
diffusion of the monomers allows complementary spatial organization with 
the template substance. A specific example of a preformed fluid imprint 
matrix is a lipid bilayer. 
As used herein, the term "predetermined molecule" refers to a substance 
which has been selected to be used as a template for imprinting. The 
template molecule can be, for example, small organic molecules such as 
amino acids, 1,4-naphthyl methyl imidazole and 1,4-benzyl imidazole; 
larger molecules such as peptides, oligonucleotides, proteins, DNA, and 
polysaccharides. Such larger molecules are referred to herein as 
"macromolecules". A template protein is a specific example of a 
predetermined molecule, (i.e., a protein), which has been selected as a 
template for imprinting. A "predetermined biological particle," as used 
herein, refers to template molecules which are composed of aggregates of 
macromolecules such as cells and viruses. 
The invention provides an imprinted matrix exhibiting selective binding 
interactions through chelated metals with a predetermined molecule or 
biological particle. 
The invention also provides a method of imprinting a predetermined molecule 
or biological particle. The method consists of: (a) combining a 
predetermined molecule or biological particle and polymerizable imprint 
matrix monomers containing chelated metals under conditions where the 
imprint matrix monomer binds the predetermined molecule or biological 
particle through interactions with the chelated metals; (b) forming an 
imprinted matrix from the imprint matrix monomers, the imprint matrix 
having sufficient rigidity to maintain selective binding interactions 
between the matrix and the predetermined molecule or biological particle; 
and (c) removing the predetermined molecule or biological particle from 
the imprinted matrix. 
Small molecules, macromolecules and biological particles are each 
characterized by an unique spatial distribution of binding sites. Such 
binding sites can be, for example, nitrogen atoms, amino acid side chains, 
groups of amino acid side chains, or whole macromolecules. Unique 
arrangements of binding sites can be used in a general scheme for 
synthesizing polymers capable of recognizing predetermined molecules or 
biological particles. For example, specific proteins are each 
characterized by an unique spatial distribution of surface amino acids. 
Each surface amino acid constitutes a potential binding site for a variety 
of different functional groups. Correct spatial arrangement of 
complementary functional groups and amino acid binding sites allows 
selective recognition of the protein by the synthesized polymer. 
Attempts to orchestrate the synthesis of a large number of weak 
interactions have yielded matrices which do not exhibit strong binding and 
high selectivity toward the template molecule. The problem can be 
attributed to the difficulty of synthesizing compounds that accurately 
place and retain functional groups in the desired configuration. 
Additionally, the problem is compounded by the fact that a large number of 
complementary interactions are needed when each individual interaction, 
such as hydrogen bonding or van der Waals forces, is relatively weak. As 
described herein, however, such problems can be overcome for synthesizing 
imprinted matrix polymers by correct placement of just a few (about 2 to 
4) complementary functional groups which exhibit relatively strong and 
reversible interactions. Functional groups which demonstrate such binding 
properties are certain chelated metals. 
Molecular imprinting utilizes the template molecule to create selective 
binding sites, thereby obviating the tedious synthesis of prearranged 
complementary groups. The basic steps for imprinting into a polymer matrix 
using metal-chelates are shown schematically in FIG. 1 for an imprinting 
process using protein as template. The binding sites illustrated on the 
template protein of FIG. 1 are histidines and are denoted as a "H". Free 
polymerizable monomers containing chelated metals are also shown. 
Initially, the protein preorganizes the metal ions; that is, it binds to 
the monomers which contain the chelated metal ions. The chelated metal can 
be, for example, kinetically labile ions such as Cu.sup.2+, or they can be 
inert such as Pt.sup.2+ or Ru.sup.2+. Next, the protein-monomer complex 
is allowed to polymerize in the presence of a cross-linking agent as shown 
in second step set forth in FIG. 1. The cross-linking agent produces 
covalent bonds between diene groups on adjacent monomers. Chemical groups 
other than dienes can also be used. One skilled in the art knows what 
groups will work and can select the appropriate monomer and cross-linking 
agent. Polymerization should be sufficient to produce a rigid matrix which 
maintains the preorganized spatial arrangement of chelated metals. This 
requirement can be obtained by either polymerization in a large amount of 
cross-linking agent or, alternatively, adding other monomers which do not 
contain chelated metals in addition to a moderate amount of cross-linking 
agent. Metal ions polymerized into the resulting polymer therefore match 
the spacing of histidines on the protein surface and are immobilized 
within a solid matrix. Finally the protein is extracted, leaving behind 
the imprinted polymer matrix as shown in the third step set forth in FIG. 
1. The metal used for imprinting can be removed and replaced with another 
metal if that metal is more suitable for the particular application. Such 
metals can be, for example, those described above or Ni.sup.2+, Zn.sup.2+, 
Co.sup.2+, Cd.sup.2+, or Ca.sup.2+. The resultant matrix will bind the 
protein with which it was imprinted with higher affinity than other, 
similar, though not identical, species. 
Metal coordination as the primary reversible interaction in conjunction 
with proper geometric arrangement of such functional groups allows for 
high affinity in the presence of about 2 to 4 specific interactions. Only 
a few interactions are required because the strength of a single 
interaction is relatively high (3-10 kcal/mol for metal coordination as 
compared to 1 kcal/mol for hydrogen bonds). 
The energetics of metal coordination and its relevance to template 
recognition is illustrated for a protein in FIGS. 2a and 2b. For example, 
if the spacing between histidines in a protein is matched by the spacing 
between metal ions on the imprinted matrix, so that both histidines can 
bind at once (FIG. 2a), the overall binding constant will be much higher 
than if only a single histidine can bind (FIG. 2b). Typical values of the 
association constant for interactions between single histidines and a 
copper-bound metal chelate such as iminodiacetic acid-bound Cu(II) are 
K=10.sup.3.6 M.sup.-1 in water. Thus the apparent overall binding constant 
for the case shown in FIG. 2b is about 2.times.10.sup.3.6, while that for 
the case shown in FIG. 2a can be much larger. The magnitude of the binding 
constant for the case shown in FIG. 2a can be quite large, as high as 
about (10.sup.3.6).sup.2 =10.sup.7.2 M.sup.-1, depending on the 
flexibility of the protein and the imprinted matrix, strain induced upon 
binding, and other factors. With three properly positioned metal atoms, 
binding constants on the order of about 10.sup.9 -10.sup.10 M.sup.-1 are 
conceivable. In contrast, if three hydrogen bonds are used, the maximum 
stability constant that can be expected is only about 10.sup.4 -10.sup.5 
M.sup.-1, five orders of magnitude weaker. 
Certain copper chelates are ideally suited for imprinting macromolecules 
such as proteins. As described above, copper has a high, reversible 
binding affinity for electron-rich histidines. Histidine is a relatively 
rare amino acid, comprising about 2% of the amino acids in proteins, of 
which only about half are surface exposed. Therefore, a protein of about 
200 kD will contain an average of about two histidines on its surface. 
This natural histidine distribution is ideal for imprinting proteins with 
copper as the chelated metal. However, different molecules, macromolecules 
and biological particles will have different distributions of functional 
groups which have affinity for metals other than copper. Ruthenium and 
nickel are examples of such other metals which can be bound by a chelating 
agent and used to imprint a substrate. The metal ion used for forming the 
imprinted matrix must be able to coordinate functional groups on the 
template molecule. The chelate used to bind the metal must leave at least 
one metal coordination site available for binding to the template 
molecule. One skilled in the art knows what functional groups on different 
print substrates have affinity for which metal-chelates and knows how to 
substitute the appropriate metal-chelate for a particular print substrate. 
The invention provides a preformed fluid imprinted matrix having sufficient 
rigidity to maintain selective binding interactions through interactive 
moieties with a predetermined molecule or biological particle. 
The invention also provides a method of imprinting a predetermined molecule 
or biological particle which consists of: (a) combining said predetermined 
molecule or biological particle and a preformed fluid imprint matrix 
containing polymerizable monomers and interactive moieties under 
conditions where the fluid imprint matrix binds the predetermined molecule 
or biological particle through the interactive moieties; (b) providing 
sufficient rigidity to the fluid imprint matrix so as to maintain 
selective interactions between the matrix and the predetermined molecule 
or biological particle to form an imprinted matrix; and (c) removing the 
predetermined molecule or biological particle from the imprinted matrix. 
Spatial arrangement of complementary functional groups can also be 
performed in a preformed fluid imprint matrix. A polymerizable lipid 
monolayer, membrane or vesicle are specific examples of preformed fluid 
imprint matrices. Polymerizable membranes are formed from amphiphilic 
lipid monomers that contain a lipid "tail" and a hydrophilic "head group." 
Some fraction of these polymerizable monomers contain interactive moieties 
attached to the head group. These monomers are free to diffuse laterally 
within the membrane prior to polymerization. Such diffusion of the 
interactive monomers within an overall stable membrane structure allows 
imprinted matrices to be made without the use of chelated metals for 
specific interactions. Such interactions can be hydrogen bonds, 
hydrophobic interactions, van der Waals forces, and electrostatic 
interactions between an interactive moiety on the membrane surface and 
binding sites on the print substrate. Therefore, "interactive moiety" as 
used herein, refers to a chemical group capable of binding a template 
molecule through hydrogen bonds, hydrophobic interactions, van der Waals 
forces, electrostatic interactions or through metal coordination. The 
interactive moieties can be the head groups of the lipids or moieties 
specifically attached to the head groups which will have affinity for 
binding sites on a print substrate. Functional groups possessing positive 
or negative charges are examples of such attached moieties. Additionally, 
chelated metals can be employed as the interactive moiety. FIG. 3a shows 
chelated copper attached to lipid monomers which are assembled into a 
membrane structure. 
The interactive moiety can also be placed on compounds which are soluble in 
lipid membranes. The compounds can then be added to lipid membranes to 
incorporate the moieties into the membrane. Porphyrin is an example of 
such a compound. Porphyrin can embed itself into lipid membranes and once 
embedded it is free to diffuse laterally within the membrane. As with the 
monomers previously described, such compounds can be separately prepared 
and stored. Different derivatized monomers can then be selected for 
different applications. 
The interactive moieties arrange themselves by lateral diffusion to 
complement the print substrate's binding sites (FIG. 3b). The spatial 
organization can be locked into place to form a rigid structure incapable 
of lateral diffusion by a variety of means. For example, fluidity can be 
decreased by polymerization of vinyl, methacrylate, diacetylene, diene, 
isocyano, and styrene substituents in either the hydrocarbon chains or 
polar head groups of the lipid monomers. Diene groups on the lipid tails 
can be polymerized to form rigid structures using radical initiators, UV 
radiation or qamma radiation (FIG. 3c). 
The resulting fluid imprinted matrices have a large surface area accessible 
for protein binding and can be used, for example, in chromatographic 
separations, in drug delivery, and for biosensors. Because the 
polymerization occurs in aqueous solution under mild conditions, fluid 
imprinted matrices are preferentially suited to maintaining the integrity 
of biological macromolecules such as proteins and viruses. 
The invention provides a polymerizable imprint matrix monomer capable of 
chelating metals. The imprint matrix monomer can have the structure of the 
monomer shown in Example III. Polymerizable fluid imprint matrix monomers 
capable of chelating metals are also provided. Such fluid imprint matrix 
monomers can be, for example, the monomer of Example IX or X. 
It is understood that using any of the imprinting methods described above, 
the order of attachment of metal chelates or interactive moieties to a 
monomer and template molecule does not matter. For example, monomers can 
first be covalently derivatized with metal chelates and then combined with 
the template molecule. Alternatively, the binding between a template 
molecule and metal chelates can take place first followed by attachment of 
the chelating agent to an activated monomer. One skilled in the art will 
know how to perform such reactions regardless of the order to achieve 
complementary functional groups for imprinting. 
The invention provides a method of using an imprinted matrix for separating 
a predetermined molecule or biological particle from a material containing 
the predetermined molecule or biological particle. The method includes the 
steps of: (a) contacting the material with an imprinted matrix having 
selective binding interactions for the predetermined molecule or 
biological particle to form an imprinted matrix-bound complex; (b) 
separating the imprinted matrix-bound complex from the material; and (c) 
recovering the predetermined molecule or biological particle from the 
imprinted matrix-bound complex. 
Once produced, the imprinted matrices described herein can be used in 
essentially any procedure involving affinity ligands. The matrices are 
more stable than biologically produced affinity ligands and advantageously 
provide easy recovery of the separated molecules due to the reversible 
nature of their interaction. For example, an imprinted matrix can be used 
instead of immobilized antibodies in affinity chromatography for isolating 
a particular molecule. Imprinted matrices can also be used for targeting 
therapeutics to cells, separation of viruses from a sample and for 
separation of cells or organelles from a sample. Material containing the 
molecule or biological particle is passed over the matrix and eluted under 
mild conditions after unbound material is first washed away. 
The invention provides a method of using an imprinted matrix for detecting 
the presence of a predetermined molecule or biological particle in a 
material containing said predetermined molecule or biological particle. 
The method consists of: (a) contacting the material with an imprinted 
matrix having selective binding interactions for the predetermined 
molecule or biological particle to form an imprinted matrix-bound complex; 
and (b) detecting said imprinted matrix-bound complex. 
The imprinted matrixes described herein can also be used in diagnostic 
procedures. The presence of an analyte in a sample can be determined using 
an imprinted matrix which selectively recognizes the analyte. The sample 
containing the analyte is treated with the matrix so as to allow selective 
binding. The imprinted matrix-bound complex can then be detected using 
methods known to one skilled in the art. For example, any one of a variety 
of detectable probes can be attached to the matrix either before or after 
imprinting. Such detectable probes can include, for example, agents whose 
fluorescence changes upon formation of the imprinted matrix-bound complex. 
Complexation can also be detected by measuring changes in certain physical 
properties of the imprint matrix, such as electrical conductivity. 
The following examples are intended to illustrate but not limit the 
invention. 
EXAMPLE I 
Synthesis of Metal Chelating Monomers 
This example shows the synthesis of metal-chelating monomer 
N-(4-vinyl)-benzyl iminodiacetic acid. The structure is given below: 
##STR1## 
Iminodiacetic acid (8 g; 60 mmol) was dissolved in 120 ml of 50% aqueous 
methanol containing 4 g (100 mmol) of NaOH and the resulting solution was 
warmed up to 60.degree. C. While maintained at this temperature, 10.8 g 
(70 mmol) of 4-vinyl benzylchloride dissolved in 30 ml of methanol was 
added slowly. After half of the 10.8 g was added, 4 g of NaOH dissolved in 
15 ml of methanol was added followed by addition of the remaining benzyl 
chloride. The reaction mixture was kept at this temperature for 45 
minutes. Methanol was distilled off under vacuum to half of its volume. 
The reaction mixture was allowed to cool and was extracted with diethyl 
ether (3.times.100 ml) and the combined organic phase was discarded. The 
aqueous phase was acidified with 6N HCl to pH 2.5 at which point a white 
solid mass precipitated. The crude product thus obtained was purified by 
recrystallization from water (twice) to give a 40% yield. 
EXAMPLE II 
Synthesis of Copper-Chelating Monomers 
This example shows the synthesis of copper-chelating monomer copper(II) 
N-(4-vinyl)-benzyl iminodiacetic acid.2.sub.2 O. The structure of the 
final product is shown below: 
##STR2## 
The starting material for synthesis was that produced in Example I. 
Initially, 5 g (20 mmol) of N-(4-vinyl)benzyl iminodiacetic acid was 
suspended in 25 ml of water and was neutralized with 1N NaOH to pH 7.0. To 
this solution, 3.4 g (20 mmol) of Cu(II)Cl.sub.2 .multidot.2H.sub.2 O 
dissolved in 15 ml water was added slowly. The resulting deep blue 
solution was allowed to stir for 5 hours. Subsequently the solvent was 
removed under vacuum. The residue was treated with 40 ml methanol and 
filtered off to remove the insoluble inorganic salts. The filtrate was 
concentrated to half of its volume and left in the refrigerator to give 
bright blue crystals at a 65% final yield. 
EXAMPLE III 
Synthesis of Water-Soluble Metal-Chelating Monomers 
This example shows the synthesis of novel water-soluble metal-chelating 
monomer N-[2-dicarboxymethyl)-aminoethyl methacrylamide]. The structure of 
the final product is shown below: 
##STR3## 
Starting with 1,2-ethylene diamine, this compound was synthesized in four 
steps as described below: 
(i) N-tBoc 1,2-Ethylene Diamine 
To 6 g (100 mmol) 1,2--ethylene diamine dissolved in 25 ml of dioxane, 4.4 
g (20 mmol) of di-tert-butyl dicarbonate in 40 ml of dioxane was added 
very slowly with stirring over a period of 4 hours. The reaction mixture 
was allowed to stir for 24 hours and the solvent was removed under vacuum. 
To the residue 40 ml of water was added and stirred for 1/2 hour. The 
insoluble N,N'-bis t-Boc diamine was removed by filtration. The aqueous 
solution was extracted with dichloromethane (4.times.40 ml) and the 
combined organic phase was dried over Na.sub.2 SO.sub.4. The solvent was 
evaporated under vacuum to give an oily residue which was recrystallized 
from isopropanol giving a white solid. The yield was 60%. 
(ii) N-(2-amino)-ethyl Methacrylamide HCl 
3.2 g (20 mmol) N-tBoc ethylene diamine and 2.53 g (25 mmol) were dissolved 
in 30 ml chloroform. This reaction mixture was cooled in an ice bath and 
to it 2.5 g (24 mmol) methacryloyl chloride dissolved in 10 ml chloroform 
was added slowly. After complete addition the reaction mixture was allowed 
to warm up to room temperature and stirred for 4 additional hours. The 
chloroform solution was successively washed with 5% aq Na.sub.2 CO.sub.3, 
water and brine and dried over Na.sub.2 SO.sub.4. The chloroform was 
removed under vacuum and the product was recrystallized from ether:hexane. 
The yield was 72%. 
(iii) Conversion of N-(2-amino)-ethyl 
Methacrylamide.HCl to an Amine 
The above t-Boc protected methacrylamimide was converted to the amine by 
dissolving 3 g of this compound in 20 ml ethyl acetate and to it 5 ml of 
6N HCl was added with rapid stirring. The reaction mixture was stirred for 
2 hours and then the solvent was removed under vacuum. The oily residue 
was washed with ether which crystallized upon standing giving a 
hygroscopic solid. The yield was 90%. 
(iv) Synthesis of 3 
1.65 g (10 mmol) of the above amino methacrylamide was dissolved in 15 ml 
of water and was neutralized with 1M NaOH to pH 8.0. 3.5 g (25 mmol) of 
bromoacetic acid dissolved in 10 ml of water was added slowly to the 
acrylamide solution. The temperature of the reaction mixture was kept 
below 4.degree. C. and the pH of the medium was maintained around 9-10 
with the intermittent addition of 1M NaOH. After completion of addition, 
the reaction mixture was allowed to warm up to room temperature and 
stirred for 24 hours. The reaction mixture was subsequently acidified with 
2N HCl to pH 5 and was extracted with ethyl acetate (3.times.40 ml) to 
remove unreacted bromoacetic acid. The volume of the water was reduced to 
half under vacuum and was acidified to pH approximately 2.5. The white 
precipitate thus obtained was recrystallized from water. The yield was 
60%. 
EXAMPLE IV 
Preparation of Water-Soluble Copper(II)-Chelating Monomer 
This example shows the preparation of water-soluble metal-chelating monomer 
copper(II) N-[2-dicarboxymethyl)-aminoethyl methacrylamide]. The starting 
material was that synthesized in Example III. 
N-[2-dicarboxymethyl)-aminoethyl methacrylamide] (1.22 g) was suspended in 
15 ml water and slowly neutralized with 1N NaOH to pH 7.0. 0.85 g of 
Cu(II)Cl.sub.2 .multidot.2H.sub.2 O dissolved in 5 ml water was slowly 
added to this monomer solution. The resulting blue solution was stirred at 
room temperature for 4 hours. Subsequently, the water was removed under 
vacuum and 10 ml methanol was added. The insoluble residue was filtered 
off and the methanolic solution was maintained at -20.degree. C. to obtain 
the desired copper salt in the form of blue crystals. The crystals were 
filtered, dried under vacuum, and stored under argon. The final yield was 
60%. 
EXAMPLE V 
Preparation of Imprinted Polymers: Small Molecule Templates 
This example demonstrates the preparation of metal-chelating polymers and 
the reversible removal and replacement of copper ions in the polymer. 
Further, the selectivity of the polymer for its specific bisimidazole 
template is clearly demonstrated. 
Selective polymers were synthesized on small template molecules that can be 
considered as protein analogs in that they contain functional groups that 
are similar to histidine. Specifically, polymers described in this example 
are able to distinguish between the below two, very similar bisimidazole 
compounds (1 and 2). These compounds are so similar that they cannot be 
separated using high-resolution techniques such as reverse phase HPLC. 
They also are indistinguishable by ligand-exchange (metal-affinity) 
chromatography on commercially available Cu(II)IDA supports. 
##STR4## 
Synthesis of the metal-chelating monomer used for template polymerization 
is described in Examples I and II. For preparation of the imprinted 
polymer, two molar equivalents of copper(II) N-(4-vinyl)-benzyl 
iminodiacetic acid.multidot.2H.sub.2 O were dissolved in methanol with one 
equivalent of bisimidazole template (1 or 2) and allowed to equilibrate. 
The solution color became deeper blue, indicating complexation between the 
imidazole moieties and vacant Cu(II) coordination sites. Polymerizations 
were carried out using a 5:95 molar ratio of monomer to crosslinker 
(ethylene glycol dimethacrylate). After adding the crosslinking agent, the 
mixture was flushed with argon and polymerization was initiated at 
65.degree. C. by free radical initiation with AIBN. The reaction was 
allowed to proceed for 24 hours at 65.degree. C., after which time the 
blue, solid polymer was cooled, ground into small particles (150-250 
.mu.), and washed with methanol to remove unbound template and other 
soluble material. 
The bisimidazole templates were removed by washing with 3N HCL (in 
methanol). Under these conditions, the templates are removed without 
affecting the metal ion chelated in the polymer. The copper ions were 
removed by treating the template-free polymers with excess 0.1MEDTA 
solution. The copper could be reloaded by treating the matrix with excess 
CuCl.sub.2. 
The resulting two polymers, P1 and P2, were prepared using 1 and 2 as the 
template, respectively. The polymerization conditions and the amounts of 
template and Cu(II) eluted from the polymers are listed in Table I. 
TABLE I 
______________________________________ 
POLYMERIZATION CONDITIONS AND WORKUP 
OF TEMPLATED POLYMERS 
[CU(II) template 
Polymer (mmol/g [template] recovered 
Cu(II) 
recovered 
mixture) (mmol/g) (mmol/g) 
(mmol/g) 
______________________________________ 
P1 0.52 (1) 0.26 0.26 0.47 
P2 0.53 (2) 0.27 0.26 0.49 
______________________________________ 
The binding selectivities for the bisimidazole substrates were evaluated by 
equilibrating the Cu(II)-loaded polymers with bisimidazole molecules (both 
template and non-template structures). For these experiments, the polymers 
were treated with an excess of the substrate for 30 hours at room 
temperature. Then the polymers were filtered and washed thoroughly with 
equilibration solvent (methanol:water). The substrate contents of the 
combined washings were used to determine the uptake by the polymers. The 
results of binding studies on polymers P1 and P2 are summarized in Table 
II. Each polymer prefers its template substrate. For example, a gram of P1 
, synthesized using structure 1 as the template, binds 50% more 1 (0.33 
mmol) than 2 (0.22 mmol). 
TABLE II 
______________________________________ 
UPTAKE OF BISIMIDAZOLE SUBSTRATES BY 
TEMPLATED POLYMERS 
substrate contacted with 
substrate bound 
Polymer polymer (mmol/g polymer) 
(mmol/g polymer) 
______________________________________ 
P1 1.31 (1) 0.33 
P1 1.52 (2) 0.22 
P2 1.45 (2) 0.24 
P2 1.6 (1) 0.17 
______________________________________ 
Binding experiments were also carried out using mixtures of compounds 1 and 
2 in order to determine the binding selectivities of the templated 
polymers. In addition, competitive binding was measured to the copper-free 
polymers. The binding preference for the template structure was also 
observed in competitive binding experiments when the polymer P1 contains 
copper (Table III). The separation factor for this (unoptimized) material 
is 1.17. A separation factor of 1.15 is observed for substrate 2 versus 1 
on polymer P2. With the copper removed, the polymers exhibited very little 
selectivity for a particular substrate compound. Furthermore, the binding 
capacities of the copper-free polymers were much less than those of the 
metallated materials (&lt;20%). 
TABLE III 
______________________________________ 
COMPETITIVE BINDING OF BISIMIDAZOLE 
COMPOUNDS 1 AND 2 TO TEMPLATED POLYMERS 
P1 AND P2. POLYMERS ARE CONTACTED WITH 
EXCESS BISIMIDAZOLE 
mole ratio of 1:2 
mole ratio of 1:2 
Polymer contacted with polymer 
bound to polymer 
______________________________________ 
P1 1.0 1.17 (.alpha..sub.1,2 = 1.17) 
P1 (copper free) 
1.0 1.04 
P2 1.0 0.87 (.beta..sub.2,1 = 1.15 
P2 (copper free) 
1.0 0.96 
______________________________________ 
These results demonstrate that two very similar metal-coordinating 
compounds can be separated on the templated polymers and that the chelated 
metal is critical to the separation. As mentioned previously, compounds 1 
and 2 are inseparable using high-resolution techniques such as reverse 
phase HPLC. In order to determine the individual concentrations in a 
mixture of the two, it was necessary to resort to proton NMR spectroscopy 
at high field (300 MHz). The fact that the template polymerized materials 
that were synthesized, without optimization, can distinguish these two 
compounds with a respectable separation factor is highly significant. 
These results established: 1) the successful preparation of macroporous 
metal-chelating polymers, 2) the polymers will reversibly bind Cu(II), 3) 
the Cu(II)-loaded polymers form coordination complexes with 
imidazole-containing compounds, and 4) these polymers exhibit selectivity 
for the template with which they were synthesized. 
EXAMPLE VI 
Metal Replacement Within an Imprinted Matrix 
This example demonstrates the replacement of copper by another metal ion, 
nickel, in the polymer P1 prepared in Example V. 
The copper ions of polymer P1 were removed by treating the template-free 
polymers with excess 0.1 MEDTA solution. The polymer was then reloaded 
with nickel by treating the material with excess NiSO.sub.4, followed by 
washing with aqueous buffer. The Ni(II) binding capacity of P1 is 0.45 
mmol/g polymer. 
EXAMPLE VII 
Preparation of Imprinted Polymers: Protein Templates 
This example demonstrates the preparation of methacrylamide polymer beads 
with a protein template. It also demonstrates the removal of the copper 
ion used during polymerization and reloading of the polymer with copper or 
another metal ion. 
One mg copper(II) N-[2-dicarboxymethyl)aminoethyl methacrylamide], prepared 
as described in Example IV, is initially dissolved in 1 ml MOPS buffer pH 
7.5 (prepared from 1 M solution of 3-N (morpholino) propane sulfonic acid 
and 1M KOH) and is added to 2 ml of a solution of 10 mg of horse heart 
myoglobin dissolved in the same buffer. The protein-copper-monomer complex 
is allowed to equilibrate at room temperature with gentle shaking under a 
nitrogen atmosphere. 3.5 mg of N-methyl methacrylamide comonomer and 40 mg 
of 1,4 bis(methacryloyl)piperazine crosslinker dissolved in buffer are 
added to the protein solution, and 0.1 mg ammonium persulfate and 10 .mu.l 
tetramethylethylenediamine (TEMED) are added to initiate polymerization. 
The reaction mixture is stirred gently under nitrogen for 48 hours at room 
temperature to polymerize. After polymerization, the resulting material is 
lyophilized and ground to achieve appropriate particle sizes. The 
particles are washed extensively with buffer and 0.1M NaCl to remove 
nonspecifically-bound template protein and other soluble components. 
Removal of template protein and copper bound by metal-affinity interaction 
to the polymer is achieved by washing the polymer beads with 0.1M EDTA 
solution. Alternatively, the protein can be removed by treating the 
polymer with a reducing agent such as cysteine. 
For reloading of metal ions, the protein- and copper-free polymer above is 
treated with an excess of 0.1M CuSC.sub.4 (or other copper salt) solution 
to reload the polymer with copper ion. To load the polymer with a metal 
ion other than copper, the polymer is treated with an excess of a salt of 
that metal ion in solution. For example, NiSO.sub.4, ZnCl.sub.2 are 
appropriate for loading Ni and Zn metal ions, respectively. 
The choice of the comonomer, crosslinker, and the relative ratios of these 
used for template polymerization dictate many of the physical properties 
of the resulting polymer, including nonspecific adsorption, porosity, and 
matrix rigidity. While porosity will influence loading capacity and mass 
transfer resistances, the microscopic rigidity will strongly influence the 
capability of these materials to recognize the template protein. If the 
spatial arrangement of metal ions exactly matches the histidine spacing on 
the protein, then protein binding is enhanced by increasing rigidity. This 
phenomenon is a manifestation of the entropic source of the chelate 
effect; fewer degrees of freedom mean a smaller entropy loss upon binding 
at the second and later sites. On the other hand, too much rigidity can be 
detrimental if binding induces small changes in metal or histidine 
orientation and this causes strain in the system. 
EXAMPLE VIII 
Preparation of Imprinted Polymers: Protein Templates Using Thin-layer 
Template Polymerization on Porous Silica 
The limited accessibility of the specific binding sites in a rigid 
macroporous polymer can pose difficulties for binding large proteins and 
for the recovery of functional template protein. For certain applications, 
polymer beads may not exhibit the macroscopic physical properties that are 
optimal for large-scale application, such as high mechanical stability or 
open pore structure. In order to prepare selective binding polymers in 
conjunction with a well-defined macroporous structure, an alternative 
procedure is to polymerize in a thin film over the surface of porous 
beads, or surfaces. This procedure involves the polymerization of the 
metal-chelating monomer-protein assembly in the presence of macroporous 
silica beads which bear polymerizable groups on their surfaces. A similar 
process has been applied to the preparation of silica packings for HPLC 
separation of textile dyes, Norrlow, O., Glad, M., Mosbach, K., J. 
Chromatogr. 299:29 (1984), and to prepare packings characterized by very 
high selectivities for particular dialdehydes, Wulff, G., Heide, B., 
Helfmeir, G. S., J. Am. Chem. Soc. 108:1089 (1986). 
Silica beads possess predetermined pore sizes and mechanical properties, 
and these can be chosen for optimum large-scale operation. The polymer 
forms as a thin layer on the pore surfaces whose areas can be very large. 
The resulting materials are "pellicular" packings and should give maximum 
accessibility of the binding sites without seriously compromising binding 
capacity. 
Preparation of methacrylate-modified silica were prepared using wide-pore 
silica particles reacted with a large excess of 3-methacryloxypropyl 
trimethoxysilane under inert atmosphere in toluene, Norrlow, O., Glad, M., 
Mosbach, K., J. Chromatogr. 299:29 (1984). 
One g methacrylated silica in 10 ml 50 mM sodium phosphate, 0.5M NaCl pH 
7.0 was deareated under vacuum for 30 minutes with stirring. A solution of 
10 mg horse heart myoglobin in 10 ml of the same buffer was added to the 
silica, along with 1 mg of 
copper(II)-N-[2-(dicarboxymethyl)-aminoethyl]methacrylamide. 40 mg of 1,4 
bis-methacryloyl)piperazine was added. The mixture was allowed to stand 
for approximately 1 hour at room temperature. The initiator solution was 
prepared by combining 10 mg of ammonium peroxydisulfate with 100 .mu.l 
tetramethylethylenediamine in 10 ml distilled water. This mixture was 
slowly added over 4 hours to the protein/monomer/silica mixture with 
gentle stirring. The reaction vessel was left for an additional 48 hours 
at room temperature. 
After the reaction was complete, the supernatant was removed by filtration. 
The silica beads were washed extensively with 1M NaCl, 0.1 MEDTA. 6.2 mg 
of the original protein was recovered during the filtration and wash 
steps. 
EXAMPLE IX 
Synthesis of Polymerizable Monomers for Fluid Imprinted Matrices 
This example demonstrates the synthesis of the polymerizable 
metal-chelating amphiphilic lipid bis 
1,2-(2,4-octadecadienoyl)-sn-glycerol-3-(propyl)iminodiacetic acid (I). 
This monomer contains an iminodiacetic acid metal-chelating group in the 
polar, solvent accessible portion of the molecule. The structure of the 
amphiphile is shown below. 
##STR5## 
3-aminopropyl solketal was synthesized as described by Misiura et al., 
Nucleic Acids Res. 18:4345-4354 (1990) with the following modifications. 
Briefly, 13 g of 2-cyanopropyl solketal in 100 .mu.l of anhydrous ether 
was added to 3.5 g LiAlH.sub.4 in 250 .mu.l of anhydrous ether over 30 
minutes, and the mixture was refluxed for 6 hours. After cooling 50 ml of 
10% (w/w) aqueous NaOH was added to the mixture, while stirring. The 
reaction mixture was filtered and the solids refluxed with 100 ml ether 
for an additional 2 hours. The combined ether phases were evaporated in 
vacuo, and the residue was distilled under reduced pressure. The pure 
3-aminopropyl solketal was dissolved in 40 ml of methanol. A solution of 
3.05 g chloroacetic acid in 60 ml of water adjusted to pH 11 was slowly 
added to the methanol solution. The pH of the reaction medium was 
maintained at pH 11 by intermittent addition of NaOH, and the reaction 
mixture was stirred for 24 hours at 35.degree. C. The mixture was 
subsequently extracted with ether and the methanol evaporated. The pH of 
the resulting solution was brought to 1.8-2.0, at which point the 
3-(iminodiacetic acid)ethyl glycerol crystallized. 
An alternative procedure to synthesize this compound is by alkylation of 
the 3-aminoethyl solketal with ethylchloroacetate and Na.sub.2 CO.sub.3 in 
acetonitrile. Briefly, the mixture was refluxed for 24 hours and the 
carbonate was removed by filtration. The 3-(diethyl iminodiacetate) 
solketal was purified by flash chromatography using chloroform:methanol 
[9:1] as the eluant. The solketal protecting group and the ester were 
cleaved in dry MeOH/DMF with 6N HCl. 
2,4-octadecadienal was synthesized according to Ringsdorf, H., J 
Macromolec. Sci. Chem. 15:1013-1026 (1981). Briefly, 2,4- octadecadienoic 
acid was synthesized from 2,4-octadecadienal following the silver dioxide 
oxidation method of Corey et al., J. Am. Chem. Soc. 90:5616-5617 (1968). 
The coupling of the acyl chains to the 3-(iminodiacetic acid)propyl 
glycerol was carried out using dicyclohexyl carbodiimide 
(DCC)/4-N,N-dimethyl amino pyridine (DMAP) in DMF or CHCl.sub.3, as 
outlined by Hupfer et al., Chem. Phys. Lipids 33:355-374 (1983). 
An alternative approach to synthesizing the above monomer involves treating 
2,4-octadecadienoic acid chloride with 3-(iminodiacetic acid)ethyl 
glycerol by an interfacial condensation approach. The 2,4-octadecadienoic 
acid chloride was synthesized by oxalyl chloride treatment of the acid 
following the procedure for unsaturated acids of Serrano et al., 
Macromolecules 18:1999-2005 (1985). 
EXAMPLE X 
Synthesis of Metal-chelating Monomers for Polymerizable Fluid Imprinted 
Matrices 
This example demonstrates the synthesis of the polymerizable 
metal-chelating amphiphilic lipid 
rac-1,2-bis(2,4-octadecadienoyl)-sn-glycerol-3-phosphoryl 
(ethyl)iminodiacetic acid. The structure is shown below and contains an 
iminodiacetic acid metal-chelating group in the polar, solvent accessible 
portion of the molecule as does the monomer of Example IX. 
(II) 
Briefly, 2,4- octadecadienoyl chloride is synthesized as described in 
Example IX. The solketal (1,2-isopropylidene-sn-glycerol) is reacted with 
.beta.,.beta.,.beta.-trichlorethylcarbonate solketal to produce 
3-.beta.,.beta.,.beta.-trichloroethylcarbonate solketal, as described by 
Baer, E., Biochem. Prep. 2:31 (1952). The isopropylidene group is cleaved 
by HCl hydrolysis to yield (50%) 
3-.beta.,.beta.,.beta.-trichloroethylcarbonate-sn-glycerol, Pfieffer, F. 
R., Miao, C. K. Weisbach, J. A., J. Org. Chem. 35:221-224 (1970). Crude 
product is reacted directly with 2,4-octadecadienoyl chloride in the 
presence of triethylamine. Selective hydrolysis using zinc-glacial acetic 
acid reagent affords the 1,2--bis(2,4-octadecadienoyl)-sn-glycerol, which 
is purified by recrystallization from petroleum ether. 
2-bromoethyl phosphoric dichloride is synthesized from POCl.sub.3 and 
2-bromoethanol according to the method of Eibl et al., Chem. Phys. Lipids 
22:1-8 (1978). The 1,2-bis(2,4-octadecadienoyl)-sn-glycerol is reacted 
with 2-bromoethyl phosphoric dichloride in the presence of triethylamine 
at 0.degree. C. in dichloroethane with stirring. Precipitated 
triethylammonium hydrochloride is removed by filtration, and the filtrate 
is evaporated. The subsequent workup is adopted from Hupfer et al., supra. 
Briefly, the resulting 
1,2-bis(2,4-octadecadienoyl)-sn-glycerol-3-phosphoryl bromoethyl ester is 
reacted directly with iminodiacetic acid at pH 10-11 in methanolwater. 
Alternatively, this synthesis can be performed by reaction of 
1,2-bis(2,4-octadecadienoyl)-sn-glycerol-3-phosphoryl bromoethyl ester 
with diethyl iminodiacetate in DMF. After coupling, the ester 
functionality is removed by selective hydrolysis. 
The resulting product is purified by Florisil chromatography as described 
by Hupfer et al., Chem. Phys. Lipids 33:355-374 (1983). Alternatively, 
silical gel chromatography can be employed using CHCl.sub.3 /CH.sub.3 
OH/NH.sub.3 as eluent. 
EXAMPLE XI 
Synthesis of Comonomers for Fluid Imprinted Matrices 
This example demonstrates the synthesis of the polymerizable lipid 
rac-1,2-bis(2,4-octadecadienoyl)-sn-glycerol-3-phosphoric acid (III). This 
polymerizable lipid is shown below and does not contain a metal-chelating 
group. It serves as a comonomer for liposome formation. 
##STR6## 
Briefly, 2,4--octadecadienoyl chloride is synthesized as described in 
Example IX. rac-.alpha.-glyceroliodohydrin is synthesized from solketal 
(1,2-isopropylidene-sn-glycerol) tosyl chloride and sodium iodide 
according to Baer and Fischer, J. Am. Chem. Soc. 70:609 (1948). 
rac-.alpha.-glycerol-iodohydrin is reacted with 2,4-octadecadienoyl 
chloride in the presence of triethylamine in dry DMF. This product can 
also be made by reacting 2,4-octadecadienoic acid with 
rac-.alpha.-glycerol-iodohydrin using dicyclohexylcarbodiimide 
(DCC)/4-N,N-dimethylaminopyridine (DMAP) reagent method as described in 
Example X. 
The polymerizable phosphatidic acid lipid is synthesized as described by 
Rosenthal, Methods Enzymol. 35:429 (1975). Silver di-tert-butyl phosphate 
is added to bis(2,4--octadecadienoyl) glycerol-3-iodohydrin in anhydrous 
chloroform. The mixture is reacted at room temperature for one hour and 
the precipitated silver iodide is removed by centrifugation. Tert-butyl 
ester groups are removed by gaseous HCl bubbled through chloroform. The 
lipid is isolated in the form of its barium salt and is purified according 
to Rosenthal, Supra. 
EXAMPLE XII 
Preparation of Fluid Imprinted Matrices: Protein Templates 
This example describes the preparation of protein-imprinted liposomes by 
template polymerization. In this process, the ability of amphiphilic 
molecules to self-assemble into ordered bilayer structures is exploited. A 
lipid containing an interactive moiety in the solvent-accessible portion 
of the molecule can diffuse laterally in the bilayer membrane to match the 
distribution of complementary functional groups on the protein surface. 
The liposomes of this example are composed of a small amount of 
metal-chelating lipid monomer (synthesized in Examples IX or X) and a 
large amount of lipid comonomers (synthesized in Example XI). Furthermore, 
all the lipid components of the bilayer are polymerizable in that they 
contain reactive moieties which allow stabilization of the templated 
liposome by covalent cross-linking. To "fix" the imprinted spatial 
distribution of metal ions on the membrane surface, the lipids are 
polymerized in the presence of the template molecule using UV light and/or 
a radical initiator. Alternatively, polymerization can be induced using 
gamma-radiation. When the template protein has been removed, the resulting 
liposome has a high affinity for that molecule via the particular 
distribution of metal ions fixed on the liposome surface. 
Small amounts of the Cu(II)IDA-lipids of Example IX or Example X are 
combined with the non-metal-containing comonomer amphiphile of Example XI 
to prepare small unilamellar vesicles. Standard techniques known to one 
skilled in the art are used and are described in New, R.R.C., ed., 
Liposomes: A Practical Approach, Oxford University Press, New York (1990). 
The optimal relative ratio of metal-chelating monomer to comonomer is 
dictated by the choice of protein and its surface histidine content, as 
well as by the desired selectivity and protein-binding capacity. For 
example, preparation of liposomes that selectively bind myoglobin (4-5 
surface histidines), a relative molar ratio of 0.01 I:III is used. 
After the liposomes are formed, they are treated with 0.1M CuSC.sub.4 
solution to load the metal-chelating lipids with Cu(II). The liposomes are 
extensively washed to remove all unbound Cu(II). Alternatively, the 
liposome can be formed using the metallated form of monomers which 
obviates the need for the Cu(II)-loading step. 
The vesicles are equilibrated with a solution of the template protein (at 
neutral pH or above) to allow for the diffusion of the metal-containing 
amphiphiles to match the surface histidyl distribution of the template 
protein. The liposome-protein complexes are gradually cooled below the 
phase transition temperature of the mixed membrane to prevent dissociation 
of the complex and liposome coalescence. The polymerization is carried out 
in a well-stirred quartz reaction vessel exposed to a powerful UV source. 
The extent of polymerization is monitored spectrophotometrically by the 
disappearance of the dienoyl absorbance at 254 nm. 
Subsequent to polymerization, the removal of the template protein and the 
copper ions from the liposome is accomplished by treating the liposomes 
with EDTA or other reagents which bind the metal ion, as described in the 
previous examples. 
The references referred to above are hereby incorporated by reference. 
Although the invention has been described with reference to the 
presently-preferred embodiment, it should be understood that various 
modifications can be made by those skilled in the art without departing 
from the invention. Accordingly, the invention is limited only by the 
following claims.