CA2132987A1 - Molecular imaging method - Google Patents

Abstract

Disclosed are chemically-produced specific binding, "molecular imaged" sorbents which reversibly bind a preselected macromolecule by spacially matched multipoint interactions between functional groups synthesized on the surface of the sorbent and functional groups on the surface of the macromolecule. Also disclosed are methods of producing such sorbents. The sorbents typically are high surface area solids comprising surface binding regions which have charged groups, metal coordinating groups, hydrophobic moieties, or various combinations thereof anchored thereto and spaced in the mirror image of complementary interactive groups on a surface of the macromolecule.

Description

.'0~3/19&~ PCT/US93/02~23 , 2 1 ~ 7 MOLEWLAR IMAGING METHOD
-BACKGROUND OF T~E INVENTION

This invention relates to solid sorbents having surface defining sites capable of selectively binding a preselected macromolecule, useful in the separation of a target solute from a complex mixture and in various types of analyses. The invention also relates to a la~ily of synthetic techniques useful in fabricating such surfaces.

Adsorption of macromolecules such as proteins to surfaces involves attraction at multiple sites through hydrophobic, electros~atic, and hydrogen bonding~
Surfaces used in chromatographic packing materials therefore have a high density of ionic, hydrophobic or hydroxyl containing groups available for this adsorption process. The interface between the surface and adsorbed proteins may cover between about l0-l00 surface sroups on the sorbent~ depending on the surface density of the charged or other groups and on the size of the protein.
Adsorption typically occurs through 5 to l0 groups on the surface of the protein~ so there is a large excess of surface functional groups. As the surface density of - - functional groups on a sorbent decreases, the strength of protein adsorption typically decreases rapidly. Although the number of groups on the sorbent surface is more than--~~ 25 adeguate for binding, the groups are not distributed properly in space.

The effect is illustrated schematically in Figures lA
and lB. In Figure lA, the accessible surface area of a protein, depicted at l0, h~s five dispersed anion groups, - all of which lie close to one or more cation groups wos3/ls&~ PCT/US93/02~ ~
~3'~8~ - 2 -disposed at high density in a field on the surface 12 of the adsorbent. As shown in Figure lB, at lower surface density, the protein will be less avidly bound, as the spatial distribution of the anions on the protein surface do not match up well with the positioning of the cations on the sorbent. `~
. ..~
Of course, real behavior differs in several respects from the oversimplified situation depicted, as, for - 10 example, 1) charged groups are randomly positioned on the sorbent, 2) adsorption occurs in three dimensions, e.g., the charge pair in the square shown in Figure lB may be spaced apart in a direction normal to the plane of the paper, 3) the protein may have cation groups on its surface which will be repelled by the cation surface and 4) there are other physical interactions at work in addition to electrostatic attraction.
i This complimentary adsorption phenomenon is used most widely in chromatographic processes involving purification and analysis of analytes exploiting differential sorption properties~ of~solutes in a mixed solution. Those who manufacture chromatographic systems generally seek to make the;surface of the sorbent as homogeneous as possible, and 25 to have~ a~high density of functional groups. - -~- ~ Complementarity is based on the presence of a single set -~ - of functional groups on the sorbent surface being complement~ary with a subset of the functional groups on the analyte. In adsorption chromatography, for example, silanol groups at the surface of silica are used to associate with solutes through hydrogen bonding. This - generally is achieved in an organic solvent where hydrogen -~-~ - `
bonding is strong. In ion exchange chromatography, as noted above, a charged surface interacts with a molecular~
35 species of opposite charge through electrostatic -~-~-~ ~ -l093"9 ~ 2 1 3 2 9 8 7 PCT~US93/02623 interaction. The dri~ing force for interaction is based in part on enthalpic changes upon bindin~ and in part upon entropic effects from the displacement of water at the surface of both the sorbent and the sorbate. In reversed-S phase and hydrophobic interaction chroma~ography, the - entropic effect is exploited to its fullest as hydrophobic molecules are forced against the sorbent surface to minimize their hydrophobic contact area with the relati~ely polar solvent. Immobilized metal affinity chromatography is yet another example of the participation of complementary functional groups in the adsorption process. In this system, immobilized metal coordination compounds interact in the presence of metaI such as zinc or copper with histidine on an accessible exterior surface of a polypeptide. This association causes the differential adsorption of polypeptides based on number and spatial arrangement of histidines. All of these systems exploit a surface having a random high ligand density. No attempt is made to match specific structural features of the molecule with structural features of the sorbent surface.

Affinity chromatography is based on exploitation of -- - biological systems to achieve intermolecular docking and ~ 25 adsorption. In this system, the surface of the sorbent is caused to mimic a biological substance which naturally _ assaciates with a polypeptide. Affinity interactions ~ -- - generally are based on multiple phenomenon including electro~tatic attraction, hydrophobic interaction, jhydrogen bonding, and stereochemical interfit.
, :

, , _ WO93/19 ~ PCT/US93/02~
t~l3'~9~ .

Reversible binding interactions between pairs of biological macromolecules such as ligands and receptors or antibodies and antigens have been exploited widely to construct systems taking advantage of the exquisite 5 specificity and affinity of these interactions. Affinity chromatography often involves the immobilization of specific binding protein, previously typically polyclonal antisera, but now commonly monocl~nal antibody, to a high surface area solid matrix such as a porous particulate 10 material packed in a column. The feed mixture is passed through the column where the target solute binds to the immobilized binding protein. The column then is washed and the target substance~subsequently eluted to produce a fraction of higher purity. Solid material comprising such 15 specific binding surfaces also are used in immunoassay where immobilized binding protein is used to capture - selectively and thereby separate an analyte in a sample.

~There has been steady ! sometimes dramatic improvement 20 in methods for producing specific binding protein useful in such contexts and for immobilizing them on surfaces.
Thus, monoclonal antibodies largely replaced polyclonal antisera obviating the need to purify the antibodies from bleedings, enabling_epitope-specific binding, and ~- 25 established a technology capable theoretically of -- t praducing~ industrial quantities of these valuable compounds. More recently, advances in protein engineering and~ recombinant expression have~permitted the design and ~-~
manufacture of totally synthetic binding sites mimicking 30 the antigen binding domains of the natural antibodies.
, -~ While this technology is very useful it is not without - its drawbacks. The binding proteins are high molecular ~ weight biological macromolecules whose function depend on :

~ . .
..

'093/19&~ 2 .9 8 7 PCT/US93/~2623 maintenance of a tertiary structure easily altered upon exposure to relatively mild condition in use or storage.
Furthermore, while it is now within the skill of the art to prepare antibodies or their biosynthetic analogs having spe ificity for a predetermined target molecule, the preparative technique are time-consuming and costly, purification is difficult, and the techniques for immobilizing them onto surfaces at high density while maintaining activity is imperfect. Furthermore, when such specific binding surfaces are used for the purification of substances intended for therapeutic or prophylactic use in vivo, they introduce a risk of contamination of the product by foreign biological material. This complicates quality control, increases the complexity of the design of a purification system, and increases the expense and time reguired to obtain regulatory approval of the drug.

Molecular recognition is an important phenomenon in biological systems. The area involved in the interface between the surface and the analyte can be as small as lO
to lO0 square A in the case of amino acids and monosacharides and range to as large as thousands of A in the interface between polypeptides forming quaternary - - structure. At the level between about lO-lO0 square A
surface area in the interface, man has been successful in mimicking natu~e. This is the basis for msdern affinity _ chromatography discussed above. However, the ability to ~ -- - discriminate could be increased by using a-broader surface - ~rea at the interface. I
It is an object of this inventlon to provide rationally designed, stable, inexpensive to manufacture _ surfaces on solid materials comprising a mul~iplicity of ~=~~ ~ ~ site which reversibly, nonco~alently bind with hiqh _ wo93/ls&~ PCT/US93/026 213 ~9~7 6 -~
specificity and affinity a preselected target molecule.
Another object is to provide such materials adapted for use in various types of analyses involving specific binding which heretofore have been limited to the use of immobilized macromolecules of biological origin. Still another object is to provide solids having surfaces containing specific binding sites useful for both preparative and analytical chromatographic separations, which, as compared with conventional affinity chromatography surfaces, are more durable, useful over a greater range of conditions, and less expensive to manufacture. Still another object is to provide a family of synthetic techniques which permit synthesis of rationally designed surfaces containing a multiplicity of regions which, through a combination of spatially matched electrostatic attraction, hydrophobic interaction, chelation, hydrogen bonding, and/or stereochemical interfit, are capable of binding to any given macromolecular surface.
These and other objects and features of ~he invention will be apparent from the drawing, description, and claims which follow.

-VO93/19&~ 2 1 3 2 9 8 7 PCT/US93/02623 Summary of the Invention The invention relates to novel sorbents as compositions of matter and methods of making a sorbent 5 useful for binding a preselected molecule at its surface by complementary functional group interaction. Due to this complementarity, there is a selective, reversible association between the molecule and the surface. This association may be used in the purification of the molecule, in its detection or quantitation, and in its removal from a complex system. The methods for making such specific binding surfaces are termed herein ~'molecular imaging" methods. The surface is said to be an ~imaged surface." Practice of the invention provides high 1~ surface area chromatography matrix material, molecular-specific sorbents, and catalytically active surfaces. These materials are synthesized as disclosed hereln by covalently adhering, in a way that is geometrically controlled at least in the direction parallel and preferably also in a direction normal to an underlying surface plane, a plurality of charged groups, hydrophobic groups, metal coordination groups, and various combinations thereof, to form a mirror image of groups complementary to them on a molecular surface of a target -- 2-S --macromolecule. These groups preferably are spaced about a hydrophilic undersurface rich in hydrogen containing groups and electronegative atoms such as oxygen, nitrogen, phospharus~, or-sulfur which take part in formation of ' hydrogen bonds.
- More specifically, in a first aspec~, the invention pro~ides a solid material defining a binding surface which comprises a multiplicity of regions capable of selective ----~'~-'~-''binding of a preselected macromolecule having a plurality ~ 35 of ionizable groups spaced about its molecular surface.

.

W093/l9~ g ~ PCT/US93/026 Each of the regions comprise a plurality of charged moieties bonded, preferably covalently bonded, to the surface or a coating adhered to the surface~ and disposed in spaced-apart relation within the region in a mirror 5 image and charged inverse of at least a subset of the ionizable groups on the surface of ~he macromolecule.
These regions bind the preselected molecule preferentially to other molecules by ~irtue of the spatially matched electrostatic attraction between the surface of the molecule and the binding surface.

In preferred embodiments, the binding surface is substantially free of bound charged moieties in exress of those which bind to the ionizable groups on the preselected molecule. $he binding surface preferably comprises a coating adhered to the surface of a solid particulate material useful, for example, in chromatography, and comprising, fôr example, particulate styrene divinylbenzene. The charged moieties may comprise negatively charged groups such as carboxylate, sulfonate, phosphate, or phosphonate. Carboxyl groups currently are preferred. The charged moieties also may comprise positively charged groups such as primary, secondary, tertiary or quarternary amines. These charged moieties preferably are bonded to the solid matrix or ~o an adherent coating constituting the binding surface through flexible oligomeric chains anchored to the underlying surface so that the spaced apart charged moieties define a conformationally compliant charged surface, and the charged moieties are disposed at varying distances from the surface of the underlying substrate so as to match, at least to some extent, surface topography of the preselected macromolecular species. Preferably, each binding region on the surface presents an interfacing --3~ surface area of at least 50 square A, preferably at least VO 93/lg&~ ~ 1 3 2 9 ~ 7 PCT/US93/02623 _ ~00 square A, and most preferably over 1000 square A or more. An important advantage of the invention is that the interfacing area of binding can be much larger than that of an antigen-antibody interaction. The binding surface underlying the spaced apart charged residues preferably is an oxygen rich hydrophilic polymer surface. Imaged - surfaces may be synthesized to selectively adsorb various biological macromolecules and are weil suited for selectiveIy sorbing proteins such as na~ural or synthetic lymphokines, cytokines, hormones, growth factors, peptides, morphogens, enzymes, cofactors, ligands, receptors, antibodies and other valuable proteins and polypeptides. They may also be designed to sorb analogs of intermediates in organic reactions thereby to produce catalytic surfaces mimicking the behavior of enzymes.
' ~ , The spatially dispersed charged moieties bonded to the bind-ing æurface may be present in combination with one or more hydrophobic patches disposed at a location within the binding~region~which interface~with one or more patches on the~ surface of the macromolecule. The surfaces also may inclu~de one or more~metal coordinating moiety disposed at locations in each bindinq region to form, in the presence of a~coordinating metal ion, metal coordinàting-bonds

2~5~-~etween~the~coordinating moiety and an imidàzole residue such~as~histidine esposed on the~surface of a macromolecule~.

In~a second~aspect, the invention provides a solid , sorbent material defining a binding surface having regions which selectivély bind a preselected organic macromolecule through one or more me~al coordinating bonds between the 1~ ~- .
I - - - _ , -, :
, -~ ~ .
:
:

WOg3/1~&~ PCT/US93/02~

~ ,32981 sorbent surface and imidazole residues spaced about amolecular surface of the macromolecul . Each region on the binding surface comprises one or more metal coordinating moieties, again disposed in spaced-apart relation within the region ;in~a mirror image of at least a subset of the imidazole residues. In the presence of coordinating metal ions, the surface regions bind the preselected molecule preferentially to other molecules by multipoint spatially matched metal coordination bonds between the coordinatin~ moieties on the sorbent surface and imidizole residues, e.g., histidine residues, on the surface of the preselected macromolecule. This type of surface can bind selectively with high affinity particularly well to proteins having multiple exposed histidine residues.

In still another aspect, the invention provides such a solid material which defines a binding surface comprising regions which selectively bind through multiple hydrophobic patches. Each region on the binding surface has plural hydrophobic moieties, surrounded by hydrnphilic surface, bonded to the binding surface and again disposed in spaced-apart relation within the region in a mirror image of at least a subset_of the hydrophobic patches on the surface of the preselected molecule. Such imaged regions bind by spatially matched hydrophobic interaction to the molecular surface of the preselected compound ; preferentially to others.

Imaged surfaces containing multiple regions which exploit various combinations of these effects, and especially those which extend over surface area of 1000 -square A or more, provide powerful, stable binding systems ~093/19&~ 2 1 3 2 9 8 7 PCT/US93/02623 = approachin~, equalling, or even exceeding the discriminatory capabilities of the binding molecules of the immune system.

The preferred method of fabricating these molecular imaged surfaces also comprise an important aspect of the invention. Broadly, after selecting the target macromolecule, the synthesis of the molecular image on the surface of a solid is conducted by contacting a solution of the preselected macromolecule with a specially derivatized activated surface produced, for example, as disclosed herein, permitting or inducing reaction between certain groups on the surface of the preselected molecule and the derivatized surface, and then converting remaining 1~ reactive moieties on the derivatized surface to inactive `^
form. Next, the covalent bonds between the imagîng molecule and the surface are cleaved, or the preselected imaging molecule is digested while leaving residues of the macromolecule covalently bound to the surface~ Then, the surface is ~developed~ to convert the remaining residues ; into matching, covalently attached, charged, hydrophobic, or metal coordinating groups, or by producing charge at each cleava~e point . The optimal strategy for imaging a particular macromolecule may be tiscerned using _ ~5--computerized~protein and other macromolecule modeling techniques as~ disclosed herein.

~ = -More specifically, appropriately spaced ionizable groups may be produced on a surface by providing as a starting material a solid having a surface layer of moieties co~alently reactive with ionizable groups, ~~contacting the surface layer with a preselected - polyaminoacid macromolecule under conditions in which the : _ ~--- ion-izable groups of the molecule react with the surface by iS multipoint formaeion of covalent bonds between at least .. , ., .. .. .. .,- . , .. , .. , . .. . . ~ . ..

W093/19~ 3`~9 ~1 PCT/US93/O~C~-some of the ionizable groups and the molecular surface, and then digesting the amino acid polymer by hydrolysing peptide bonds, using strong base or enzymatic hydrolysis, leaving an amino acid residue, covalently bonded to the S surface, at each position where an ionizable group had reacted. Next, the amino~groups or the carboxylic acid groups of each of the bound amino acids is derivatized to leave a charge opposite in sign and in spa~e to the charge of the ionizable groups on the surface of the preselected peptide bonded amino acid polymer. This results in the production on the surface of spatially distributed charged groups in a mirror image and charge inverse of the reacted subset of the ionizable groups on the molecular surface.

The approach to producing spatially specific metal coordinating compounds is similar but distinct. In this case, a solid substrate material having a surface layer of moieties covalently reactive with an organic, nitrogen containing, polycarboxylic acid metal coordinating compound is required~ This surface layer may be the same type~of~surface layer as is used in making electrostatic molecular imaged surfaces. This material is used in a heterogeneous reaction togethe~ with the preselected molecule containing an imidazole residue and a metal ion, under conditions to produce multipoint formation of coordinated metal ion links between at least some of the imidazole residues in the preselect macromolecule and molecules of the coordination compound, and to produce ~ovalent bonds between at least a subset of the covalently reactive moieties on the surface of the solid material and the coordination compound. Next, the metal ions are I removed from the reaction mixture, e.g., by chelation, to --produce on the surface of the material a multiplicity of V093/19844 2 1 3 2 9 ~ ~ Pcr/us93/o2623 regions comprising plural, covalently bonded metal coordinating compound molecules spaced apart within the regions in the mirror image of the imidazole residues on the macromolecule.

The approach to producing hydrophobic surface in the binding regions, appropriately located to interact with the macromolecule by hydrophobic attraction, involves prereacting the preselected molecule with an amphipathic molecule. The term amphipathic molecule, as used herein, refers to a molecule comprising a hydrophobic moiety, such as a hydrocarbon, halocarbon, or aromatic residue, which associates with a hydrophobic patch on the target molecule, and a covalently reactive group, such as an amine, carboxylic, aldehyde, or epoxy residue, adapted to react with the activated surface~ One then provides a starting material having, e.g., the same co~alently reactive hydrophilic surface as is used to derivatize with charged or metal coordinatin~ groups, and reacts this solid starting material with the amphipathic molecule/preselected molecule complex, held together by hydrophobic-hydrophobic interaction. This reaction pr~duces a sorbent comprising hydrophobic bonding interaction sites, which associate with at least a subset --25 o~ the hydrophobic patches on the surface of the preselected macromolecule, and which are co~alently bonded to the surface through the covalently reactive moieties on the solid binding surface and the covalently reactive - groups of the amphipathic molecules. The preselected molecule then is desorbed from the surface by breaking the - hydrophobic-hydrophobic attraction to yield a surface ~~ comprising regions wherein plural, covalently anchored _ hydrophobic moieties are spaced within a hydrophilic field -= -~~~~in the mirror image of the hydrophobic patches on the molecolar surface of the preselected macromolecule.

W093/19&~ PCT/US93/Ot~

~3`19~ - 14 -In preferred aspects of the method, high surface area solid material is used, e.g., a perfusive matrix material such as is disclosed in U.S. Patent No. 5,019,270, and the first step in the manufacturing process is to produce a uniform, adherent, hydro~ilic, derivatizable coating about the entirety of the surface of the solid matrix, e.g., in accordance with the method disclosed in U.S.
Patent No~ 5,030,352. Next, the coating is derivatized with oligomer chains of reactive monomers, e.g., comprising aldehyde or epoxy groups to praduce a field of active filaments. A soluti~n of the imaging macromolecule 'i next is placed in contact with the derivatized, reac~ive surface of the matrix. As the molecule comes in contact with the derivatized surface, nucleophilic amine groups exposed on the molecule surface covalently react with a subset of the epoxy or aldehyde groups on the sur ace of the matrix. A schiff base is formed in the case of aldehyde coupling which is reduced to a secondary amine.
The support-protein complex next is hydrolysed, breaking peptide bonds linking amino acids in the protein, and all remaining epoxy groups in the case of base catalyzed hydrolysis of the epoxy support matrix. This leaves a single amino acid covalently bonded to the surface through its amino side chain leaving a free amino group and a free carboxylic acid group at each point where amines on the polypeptide reacted with the activated surface~ To make an imaged anion surface, one derivatizes the amino terminal of the bound amino acids, e.g., by converting them to amidates usin~ an anhydride, thereby leaving carboxylic acid groups and their characteristic negative charge at each point on the surface corresponding to amino groups on the surface of the polypeptide~ ;

~093/19&~ 2 1 3 2 ~ 8 ~ PCT/US93/OZ623 Sorbents comprising cations covalently bonded and spaced in the mirror image of plural exposed anions on the molecular surface of a macromolecule can be produced with an analo~ous strategy using different chemistry. In this case, one starts with a sorbent surface derivatized with, for example, a terminal amine group having an adjacent vicinal hydroxyl group (-CHOH-CH2-NH2). Vpon exposure of the imaging molecule to the surface of in the presence of a water soluble carbodiimide such as 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide, amide bonds are formed between carboxylate ions on the surface of the macromolecule and the amine groups on the surface of the sorbent. Oxidation with periodate cleaves the remaining -CHOH-CH2-NH2 to produce a bonded aldehyde (-CHO) group.
This group will react with any lysine, arginine, or N-terminal amino groups on the macromolecule that are - - located at the sorbent-macromolecule interface, forming a schiff base. Sodium borohydride is then used to convert the residual surface aldehyde groups to primary alcohols and schiff bases to secondary amines. Next hydrolysis in, for example, potassium hydroxide, leaves an imaged sorbent surface having cationic amine groups bonded to the scrbent surface in locations opposite the anionic carboxylic acid - -groups on the imaged macromolecuIe. Electrostatic imaging ~5--of-anionic species is carried out with molecules that have ~- an excess of anionic functional groups. For this reason, only a ~smaller number of cationic amino acids are ~onded -~t~ the surface in this process and will have little effect - on the adsorption of anionic species.
To produce mirror imaging points of hydrophobicity to ~~ -induce hydrophobic-hydrophobic interaction, i.e., a _pecific binding reverse phase sorbent~ one mixes together he- imaging molecule having one or more hydrophobic .

-WO 93/19844 PCr/US93/02 patches on an exterior surface and an amphipathic molecule comprising a hydrophobic moiety, e.g., a hydrocarbon or halocarbon, and an opposing moiety comprising a group -covalently reactive with an activated surface of the type----described above. For example, the covalently reactive group may be an amine or carbox~late group. When the amphipathic molecule and imaging molecule are brought together in relatively hydrophilic media, the hydrophobic regions associate to exclude water molecules between their hydrophobic contact surfaces. This complex is then reacted as disclosed above such that, at the end of the , synthetic scheme, the hydrophobic end of each amphipathic ¦ molecule extends through a covalent linkage from the - - ~~ -~
surface of the sorbent and is located in space such that lS it interfits with a hydrophobic patch on the surface of ; the imaging moleule. This technique is particularly - powerful when the molecule is electrostatically attracted by charge on the surface.
:
The preferred approach to produce sorbent material having an image surface which binds selectively to macromolecuIes ha~ing imidazole residues on its surface, :i e.g., proteins having exposed histidine residues, involves reacting the imidazole containing macromolecule in the presence of copper or other metal ion and a metal coordinating compound such as iminodiacetic acid (IDA).
This results in formation of a copper coordination complex between imidazole moieties cn the surface of the ~arget protein and the IDA moieties. The nucleophilic nitrogen in the Lminodiacetic acid moiety then can be reacted with aldehyde or epoxy groups in the reaction schemes noted above so that, at the conclusion of the synthesis, an IDA
- moiety is covalently bonded to the surface of the sorbent at the precise location in space matching the imidazole residues on the imaging macromolecule. This technique .
.
,. s ., . .. . . . ,.. , ,~ ... . ,, , , , ~ , .

YO93/19&~ ~13 ~ 9 8 7 PCT/US93/02623 also most preferably is used with charge group matching, but may be used separately. ~-~

It will be seen that a key to synthesis of molecular imaged surface is to orient an appropriate surface of the - target molecule in face-to-face relation with the sorbent surface. In accordance with the invention, the ~
relationship of the imaging molecule to the sorbent surface may be permitted to occur relatively randomly, in - lO which case a ~polyclonal~ sorbent will be produced, i.e., ~ ~~-one in which the multiplicity of binding regions on the ~ ~~ ` ;
- sorbent surface contain the mirror images of different presented surfaces of the macromolecule. However, - , sorbents having a higher frequency of regions imaged to a given face of the imaging molecule can be produced by using several strategies,- e.g., taking steps to assure i~ more consistent orientation of the molecule during early stages of the imaging process, or using peptide analogs of a surfac~e region of the macromolecule sou~ht to be imaged.
- 20 Thus, for example, the presence of anti-chaotropic salts ~¦ such as- sodium sulfate in solution with the macromolecule at the imaging stage will induce the more hydrophobic face - ~ of the target macromolecule to contact the actiYated sorbent~surface. Alternatively, one may include anionic -25 -or cationic groups on the acti~ated surface to "dock" a ~ surface of the macromolecule rich in moieties of the - ~ opposite-charge by electrostatic attraction.

- ~
.
, ;, -. - . - . -----~ ~3 ~ 9 8 PCT/US93/026 Brief Description of the Drawinq Figures lA and lB are drawings schematically illustratin~ the nature of adsorption of macromoleculë~~- ~
onto a high density and low density cationic surface, (anion exchange) respectively. -Figure 2A, 2B, and 2C are illustrations which depictthe relationship of a protein or other }arge macromolecule and a surface imaged as disclosed herein~ Figure ~A:-a~d~-2B are "plan view" illustrations looking through an adssrbed protein onto the molecular imaged s~rface.
Figure 2C is an illustration taken in cross-sPction~
showing the nature of the molecular imaged sorbent and the l~ protein adsorbed thereon, and illustrating how the use of oligomeric filaments extending from the sorbent surface - - can accommodate varying molecular topology on the surface of proteins.

Figures 3A, 3B, and 3C depict exemplary activated surfaces of the type useful as a starting point in the synthesis of molecular imaged surfaces of the invention.

Figures 4A-4E are molecular diagrams useful in explaining how to ma~e a molecular imaged surface having anionic groups spaced thereabout in the mirror image of cationic grGups on a preselected protein, starting with the activated surface illustrated in Figure 3A. -Figures 5A-5D are molecular diagrams similar to those in Figure 4 but using the activated surface of Figure 3B
i~ place of 3A, and ending with an imaged surface comprising appropriately spaced ~both horizontally and vertically with respect to the substrate) a~ionic charges :

3~ in the molecular image of the preselected molecule. ---YO93/198~ ~13 ~ 9 8 7 PCT/US93/02623 Figures 6A-6E are molecular diagrams illustrating how to make a molecular imaged surface beginning with an activated substrate comprising a high density of anioni~, carboxylic acid groups where~y the imaging molecule is oriented with respect to the surface by electrostatic forces, i.e., presents its most positively charged surface to the sorbent.

Figures 7A-7E are molecular diagrams illustrating how to make a molecular imaged sorbent having plural, spaced-apart cationic groups, and beginning with an imaging macromolecule having~plural anionic groups which are attracted electrostatically to the surface in the first stage of the synthesis.
Figure 8A illustrates a macromolecule having a hydrophobic patch, e.g., a protein having a region high in ~j amino acids with aliphatic or aromatic side chains, in !~
association with two amphipathic molecules comprising a hydrophobic region linked to a primary amine, and disposed in contact with an activated surface of the t~pe il illustrated in Figures 3B and 5A. Figure 8~ shows the imaged surface resulting from the starting point of 8A
having both anionic groups and a~hydrophobic patch -- Z5 dis~oséd in the mirror image of the protein depicted in Figure-~8A.

-~~- ~ ~ -Figure-~A-~depicts a macromolecule having a pair of amine side~groups and an imidazole side group complexed through a metal ion to an iminodiacetic acid moiety, with the macromolecule and complex disposed adjacent an activated surface of the type illustrated in Figure 3A.
~- ~ Fig~re~9B illustrates a molecular imaged sorbent made by i follDw~ng a preferred synthesis disclosed herein from the ~ ~ ~~~ 35 star~ng point of Figure 9A, and Figure 9C shows the maged surface of Figure 9A comprising a pair of anionic WO 93tl9~ PCT/US93/02~

~13298 1 groups disposed in the mirror image of the amine groups on the surface of the macromolecule and an iminodia~etic acid moiety properly disposed with respect to the surface of the imaging protein to form a metal coordinatinq bond with its imidazole side group and its multipoint-electrostatic attraction.

Figures lOA-lOE are elution profiles illustrating the properties of an imaged column. In each case, the shaded peak represents the imaging molecule, here-Lysozyme.
=: :
Figure 11 depicts superposed elution profiles (pHz6.2, ~.0-1.0 M NaCl gradient, 5 m}/min in 5 min.) of the same Lysozyme/Cytochrome C mixtures on a Lysozyme imaged column and on a strong cation exchange column.
.
Figure 12 is a flow chart useful in describing how to optimize imaged surfaces using computer aided design principles.
Like reference characters in the respective drawn figures indicate corresponding parts.
- . -:

,; ~-,~ .
~- :

.

~ 1 3 ~ 9 8 7 PCT/US93/0~623 The Nature of the Molecular Imaqed Surface The highest specificity of bindin~ between a biomolecule and a surface currently is achieved using affinity interaction between, for example, antibodies and antigen, receptors and ligands, lectins and their receptors, avidin and biotin, etc. Both strength and specificity are important in such specific binding reactions. Affinity based systems often involve binding constants in the range of 106 to lO9M 1, and can be as high as lOlsM 1. Surfaces capable of specific, tight-binding with a preselected molecule currently are produced by exploiting naturally occurring bîological binding systems. These systems in turn exploit a combination of 1~ electrostatic interaction, hydrophobic-hydrophobic interaction, hydrogen bonding, and stereospecific interfit - to achieve high affinity selective binding.

This application discloses how specific binding sites can be~produced on surfaces without resort to the productionr colle tion, and~attachment of biological binding mo}ecules such as antibodies or receptors.
Binding~to sorbent surfaces of the invention is selective, i.e.~, shows a preference for the imaged molecule versus other molecules, and reversible, i.e., involves no covalent~ bonding. Selective binding, as used herein, means that the surface binds the imaging macromolecule in preference to others. Reversible binding, as used herein, means that binding is achieved without formation of covalent bonds. The chemically defined binding sites of the invention tend to be more stable, resist leaching from ~he surface, can be reproducibly synthesized, need not - expose product to biological materials, and obviate the risk of contamination of product by biomolecules incident - 3~ to product pùrification. This process, a type of rational 1~
i' ~, ,. . . .

WO93/19&~ PCT/US93/026 ~3~931 - 22 -surface design, involves the creation of a specific binding sorbent surface, herein called an "imaged"
surface, which is complementary to a surface of a molecule of interest, hereinafter referred to as the ~imaging"
molecule.
.
Adsorption of molecules at a surface is based on the existence of complementarity between at least some functional groups on the mo}ecule and those at the surface. Figures lA and lB, discussed above, exemplify adsorption of a protein onto the surface 12 of a strong cation resin (Figure lA) and a weak cation resin (Figure lB). As should be apparent, the high density cation sorbent of Figure lA will result in a more tightly adhered lS protein as there is a high frequency of multipoint electrostatic attraction between negative groups on the surface of the protein and positive groups on the sorbent surface 12. Neither the weak nor strong cation exchange sorbent exhibits specificity for any given macromolecule.
In contrast, Figure 2A depicts a region of a sorbent surface l2 containing only five cationic groups. However, as illustrated, these groups are arranged on sorbent surface 12 such that they are opposite in space to the 2~ five negative charges on the surface of the protei~ lO~
This distribution of positive charges in this region of the surface, because it represents the mirror image of the negative charges on the surface of the protein lO, ~ ~- -~~
specifically bind protein lO in preference to other proteins where the charge distribution does not match.
Thus, although the charge density of the cationic moiet-ies in the sorbent surface 12 of Figure 2A is less tha~ the charge density in Figures lA or lB, protein lO will adhere to the imaged surface of Flgure 2A with greater~aff:inity, ~093/19&~ ~ 1 3 2 9 ~ 7 ~CT/US93/~2623 and far greater specificity, than it will to the surfaces in Figure l.

Figure 2B illustrates still another principle of the molecular imaging technology disclosed herein.
Specifically, in Figure 2B, a different protein 14 is depicted as having a pair of hydrophobic patches 16, 16', three negatively charged groups, and a surface histidine residue. The histidine residue has an imidazole side chain which, as is known, can be attached to a metal coordinating co~pound by complexation with metal such as copper or zinc~ In Figure 2B, the molecular imaged surface 12 comprises a corresponding pair of hydr-ophobic patches 17, 17', three appropriately spaced positive ions, and a covalently linked iminodiacetic acid (IDA) metal coordinating molecule disposed opposite the position of ~ the histidine residue on the protein 14. As illustra~ed, the protein 14 will associate with imaged surface 12 of Figure 2B with high specificity and affinity. ~hen the protein attains its proper orientation, three positive charges properly spaced on the surface of the sorbent 12, a pair of hydrophobic patches, and, in the presence of copper ions, a metal coordinating bond, all acts simultaneously to hold protein 14 in position. It will immediately be appare~t that, for example, surface 12 of Figure 2B will readily discriminate between macromolecule --- 14 and maoromolecule lO.
, Figure 2C illustrates still another aspect of the molecular imaging technology disclosed herein. This drawing schematically illustrates a l'cross-sec~ional" view through an imaged surface l2 and yet another different protein, here depicted as 18. The bottom surface 20 of ,, WO93/19 ~ PCT/US93/026 2 ~3 2~ 8~ - 24 -the protein 18 comprises peaks and valleys, or a mDlecular topology, defined by the three dimensional structure of the macromolecule. Viewed from left to riqht, the surface of the protein comprises first a pair of cationic groups, e.g., amine side chains in a protein such as those on the lysine or ar~inine amino acid residues, a histidine residue disposed `in a "valley" on the protein surface, a hydrophobic patch 16, and an anionic group, e.g., a carboxylic acid side chain such as is present on amino acid residues like aspartic acid or glutamic acid. In the sorbent of Figure 2C, random length oligomer chain "filaments" covalently bonded directly to the matrix or to an adherent coating on the matrix comprising the sorbent surface 12, extend upwardly and have appended charged groups opposite in sign to those on the face 20 of the protein 18, a metal chelating group disposed~opposite the histidine residue, and a hydrophobic moiety 17 disposed opposite the hydrophobic patch 16 on protein 18.

From the foregoing it should be apparent that proper disposition of charges, hydrophobic patches, and metal - coordination groups, both within the plane of a sorbent surface 12 and in a direction more or less perpendicular to the plane~, if embodied in a real structure, could~-Z5 produce chemical, as opposed to biological, bindin-g-sites-~
of high specificity and affinity. In this case the imaged surface is a copy, counterpart, or likeness of the target _ molecule displaying matching opposite charge, matching--~-hydrophobic patches, and/or matching metal chelating --points which together interact chemospecifically with themaged molecule and bind selectively and reversibly-to the molecule, or at least display significant preferential adsorption of the imaged molecule from a complex mixture.

~093/19&~ 3 ~ 9 8 7 PCT/US93/02~23 Several approaches have been envisioned to achieve these goals. The presently preferred approach involves reacting the target macromolecule with an activated surface and leaving behind complement~ry functional groups. The remainder of the specification will disclose how to make and use such molecular imaged surfaces, and will discuss certain properties of such materials.

The Nature of the Solid Matrix Sorbents having molecular imaged surfaces produced in accordance with the invention have many uses. Chief among these is affinity chromatography purification procedures, activated sorbents for the removal of a target molecule from a mixture, e.g., a toxin from food, and specific binding assays such as are used widely to detect the presence or concentration of biological molecules, toxins, contaminants, drugs and the }ike in samples such as water, body fluids, and~various plant~and animal matter extracts.
In many~of these uses the solid~substrate, or matrix, - ideally should have as high a ratio of surface area to volume;as is practical. Since it often wil~l be desirable to transpart aqueous solutions containing biological lecules such~as proteins, carbohydrates, lipids, 2~ steroids~and the like into contact with the surface to selectively~bind or to ind~ce a chemical change in a component~in thè liquid phase,~it~is often ad~antageous to use a riqid~so}id having-;a uniform hydrophilic surface and a geometry which pèrmits convecti~e transport of solutes ,-30 to the imaged surface. A rigid, high mechanical strength material~ permits high pressure flow without crushing.
Perfusive matrices are preferred. Methods for making perfusive matrix materials, the nature and unique geometry of these terials, and vario=s of their ~dvantages are WO93/19&~ PcT/uss3/o26 2 ~3 29 8~ - 2~ -disclosed in detail in U.S. Patent No. 5,019,270 issued May 28, 1991 and assigned to the owner of this application. The preferred material for fabricating perfusive matrices is polymeric material such as polystyrene divinylbenzene ! ;Qreferably synthesized as disclosed in the above-referenced ~.S. Paten~ in particulate form. There are various ways of providing on the surface of the inert and hydrophobic styrene based matrix material a hydrophilic coating well suited for interaction with aqueous solutions of biological macromolecules. The currently preferred methods for providing such coatings are disclosed in U.S. Patent No.
5,030,352 issued July 9, 1991, and assigned to the Purdue Research Foundation of West Lafayette, Indiana. The ~352 patent discloses how to provide an adherent, crosslinked hydrophilic, easily derivatized coating onto the surface of particulate and other types of matrix material. The coatings are compatible with protein solutions and are extraordinarily versatile, permitting various types of activated sroups, oligomers, polymer chains and the like -~ ~ to be fixed to the surface as desired. The disclosures of both the foreqoing patents are incorporated herein by ~- reference.
.
- 25 Three exemplary activate~ surfaces suitable as ~~ -~
start~ing points for the production of the imaged surfaces - of the invention are disclosed, respectively, in Figures _ -- 3A, 3B~, and 3C. The first of these represents a portion ~- -of a solid matrix 12, shown in cross-section, having a high density of epoxy groups covalently bound to a hydrophilic surface coating adhered to matrix ma~eriaL 1~
- for example, in accordance with the procedure disclosed in the above-referenced Purdue patent. Matrix material of_ -this type may be produced from POROS2 brand chromatography-matrix material, e.g., POROS~ OH, and are available ~093/19&~ 2 13 ~ 9 8 7 PCT/US93/02623 commercially as POROSR EP from PerSeptive Biosystems, Inc., of Cambridge, Massachuset~s. Epoxy groups react with amine groups to produce an alcohol group and a secondary amine covalent linkage. The alcohol group contributes to the hydrophilieity of the surface. The secondary amine linkage forms a strong covalent bond which is exploited as disclosed below to make various types of molecular imaged surfacesO

Figure 3B discloses another type of activated surface, preferred in many instances, comprising an oligomer of - acrolein (acryloylaldehyde), which is characterized by a hydrocarbon backbone having aldehyde groups (CHO~
branching from alternate carbon atoms. This type of activated surface, having oligomers ranging from l to 20 monomer units, i.e., a, b, c, d, and e are between about 1 and about 20, can be produced from POROSR-OH, commericially available from PerSeptive Biosystems, Inc., as disclosed herein, by reaction with acrolein in the presence of cerium. Aldehyde groups also react readily with primary nitrogen atoms to produce secondary nitrogen ~ linkages and water in the presence of sodium cyanogen - borohydride. Other types of a1dehyde activated surfaces are available commercially, e.g., POROSR AL depicted in Figure 3C. Both the epoxy groups and the aldehyde groups can be further derivatized to form, for example, hydroxyls, carboxylic acid groups, or amine groups using -- - conventional chemistry. These may be used as starting materiaIs in various synthetic schemes as disclosed herein 30 ! to produce molecular imaged surfaces.

An important aspect of the activated surface 12 of Figure 3B is that the surface presents a very high density ; of active aldehyde groups present not only over the

4 PCI`/US93/026, 2l3;~98~

entirety of the surface of substrate 12 but also extending away from the surface. ~ach filament comprises a series of aldehyde side groups appended from a flexible hydrocarbon chain. Reactive moieties on the surface of S the imaging molecule can react with the aldehydes buried within or adjacent the surface of the field of filaments, as dictated by the surface shape of the imaging molecule.
Furthermore, the filaments can flex and bend laterally to conform to a shape as required by making minor spatial adjustments. This type of activated surface, i.e., a surface having chemically-active groups disposed on oligomer units extending upwardly from the surface, permits synthesis of molecular imaged surfaces which can approximate or match the peaks and valleys on the surface of the imaging macromolecule as illustrated in Figure 2C.
It also assures multipoint formation of charged or other groups.

~It should be noted that the epoxy and aldehyde groups shown in Figure 3 are illustra~ive and preferred, but are by no means the only such groups that can be used. As will be apparent from the disclosure below, the nature of the activated surface groups can vary widely, depending on the particul~r imaging chemistry used in the manufacture 2~ of the imaged surface. ~- --One important chemical feature of these startingmaterials~is the surface density of the surface anchored ~-~-active groups. If, for example, a pair of charges 3~ disposed on a surface of a macromolecu~e to be imaged are five angstroms apart, then the active ~roups on the sorbent surface must be at least this close together to be useful in a molecular imaging process. On the other hand, a starting material having 9 or 10 active groups per~~0~ -VO93/19844 2 1 3 ~ 9 8 7 PCr/USg3/02623 -- square angstroms would be operative, although perhaps not optimal, in imaging a molecular surface of, for example, 2000 square angstroms, involving spaced apart charge or other surface features at least 10-20 A apart. It thus can be seen that the surface spacing of active groups on an imageable surface is directly analogous to grain size in photographic surfaces, and that different surface densities may be used, depending on the resolution required.
The Imaqing Molecule Essentially any macromolecule may be imaged in accordance with the procedures disclos2d herein. The term "macromolecule," as used herein, refers to molecules ha~ing an imageable surface area af at least SO square A i Proteins are currently preferred. Smaller peptides may also be used, and certain of the procedures disclosed herein may be used to form molecular imaqes of gIy~oproteins, polysaccharides, polynucleic acids and o~her large molecules. Generally, the interfacing area of the imaged surface and the imaging molecule (i.e., the area of interface between sorbent and sorbate) should be ~ at least about S0 square A, more preferably 100 A, and 2S often will exceed 1,000 A.

- - _ It generally is preferable to limit the number of ~~ ~-- -distinct surfaces on a given macromolecule-imaged in a give~ synthesis. This is because it would be possible to create 10-20 different images of the surface of a - - ~ macromolecule and that each could have a different binding ~ constant. It is also important, particularly in the case of proteins, to avoid during the imaging stage high , _ _ , . .
~ ~oncentrations of org~nic sol~ent, extremes in p~, or wo93~1s&~ PCT/US93/026 ~i3 ~9 8~ - 30 -elevated temperature. All of these tend to alter thethree dimensional structure of the protein or other imaging biological molecule and to create a false image of the molecule, not reflecti~e of its native character.
An important aspect of molecular imaging therefore involves the orientation of the imaging molecule with respect to the surface to be imaged. When using the covalent immobilization synthetic route disclosed herein, molecular orientation can be achieved by using an anti-chaotropic salt such as sodium sulfate, to drive the protein to the surface and promote hydrophobic interaction. Alternatively, charge groups can be included -at the surface to orient the imaging molecule in a 15 naturally most favored binding conformation, i.e., one l:
presenting a molecular face rich in the opposite charge.
, Another approach to promoting homogeneous imaged : : bi~ding regions on the sorbent surface is to use pep~ide analogs of a surface region of the target protein as the imaging molecule. Thus, digested samples of the imaging protein, or randomly generated peptides, may be screened, for example, by affinity chromatography using monoclonal antibody, or using an imaged surface produced as disclosed -:25 herein, to obtain a short, e.g., 5-20 amino acid, peptide --which mimics the charge or other surface fea~ure distribution of the imaging:protein. Nethods for -producing such peptides are known in the art. -~
Alternatively, rapidly growing data bases storing X-ray diffraction and NMR data from various macromolecules of importance, and programs which display images of proteins and the like based on such data and on amino acid se~uence ---information, may be used to determine sequence o~ a ~0 g3/19&~ 2 1 3 2 .~ 8 7 PCT/U~93/02623 --- peptide which mimicks the surface structure of a given macromolecule. Use of such peptides as the im~ging molecule may be preferable for cost purposes when synthesizing large quantities of imaged sorbent. They also can provide a source of analogs of short-lived intermediates useful in the preparation of catalytic surfaces, and in any event provide a means of promoting image homogeneity by very significantly reducing the number of surfaces available for imaging during manufacture of the sorbents of this invention.

Preparation of a Molec~lar Ima~ed Anionic Surface In one embodiment, a molecular imaged surface is lS produced by contacting a protein comprising plural exposed lysine or arginine residues, with their characteristic primary amine side chains, with an epoxide surface such as is illustrated in Figure 3A, or with the aldehyde group-derivatized surface of Figure 3B or 3C. After reaction between the aldehydes or epoxides and the amine groups, the protein is digested with strong base, or enzymatically using a mixture of proteolytic enzymes, pronase, or the - like, to leave only the corresponding lysine or arginine ~ ~ amino acids at the precise relative locations of these ~~~ 25 residues-in the protein. The positively charged amines are neutralized through acylation leaving a negatively - _charged carboxyl group at the exact location of a ~- ~- positively charged amine on the imaging protein. 5urface hydroxyl groups, which may be esterified during the acylation step, may be converted back to hydroxyl form by -- - - hydrolysis. Such an imaged surface will bind the imaging - molecule selectively and reversibly through multipoint _ electrostatic attraction while all other proteins will - __ ~ ~ interact only very weakly as if they were encountering a very weak anion surface.

W093/l9~ PCT/US93/026 ~l32987 Details of how the foregoing synthetic technique is conducted are shown in Figures 4A through 4E. Referring to the drawing, Figure 4A illustrates an activated surface 12 derivatized with plural epoxy groups and, disposed in S solution and oriented close to surface 12, a protein, here depicted as amide-bonded amino acids including, for - purposes of illustrating the technology of the invention, a central arginine residue flanked by intervening amino acid sequences and a pair of lysine residues. As shown, the lysine residues comprise a side group consisting of C4H8-NH2; the arginine residue also has a side chain terminating in an NH2 group. The purpose of the synthetic procedure is to provide on the surface 12 negatively - charged moieties located in space about surface 12 such lS that they match the location of the NH2 groups pendant from the side chains of the lysine and arginine residues - constituting a portion of the surface of the imaging protein. For ease of explanation, in Figures 4B and following, the protein backbone is represented simply by a line extending horizontally, and only the side groups are identified.

As~shown in Figure 4B, amine groups react with~
adjacent epoxy groups forming covalent bonds through 25 secondary amines linking surface~ 12 and the protein. In --the presence of weak base, such as sodium phosphate, pH:9, - unreacted~epoxy groups are opened to form hydrophilic dihydroxy~l compounds-. Next, the reaction mixture- is -- ~
treatéd with strong base such as KOH so as to thoroughly ~
hydrolyze the protein. One to three normal potassium hydroxide is suitable for this step. Proteolytic enzymes - may also be used. ~he result is shown is Figure 4C, --wherein only the two lysine and single arginine residues ~¦
; remain. Note the molecular structure on the termini of ---the covalently-linked chains estending from surface 12 WO93/l9&W 2 1 3 2 9 8 7 PCT/US93/02623 -- comprise the amino and carboxyl groups characteristic of amino acids. Next, the intermediate imaged surface illustrated in Fi~ure 4C is treated with, for example, acetic anhydride ~C~3CO)2O in appropriate solvent such as pyridine, to acylate the amine groups. This results in derivatization and removal of the positive charge region of the amino acid as shown in Figure 4D~ leaving behind the negatively-charged carboxylic acid groups located precisely opposite the amine groups on the protein originally used to initiate the imaging procedure.

Figure 4E illustrates the function of the imaged surface. As shown, the imaging protein presented together with other solutes in solution, when encountering the imaged surface, binds preferentially as the amine group in the lysine and arginine side chains ~dock" with the - - carboxylic acid groups by electrostatic attraction. Thus, the relationship of the molecular imaged surface and the imaging protein is as illustrated in Figure 2A, i.e., selective sorption occurs by virtue of spatially-matched anionic and cationic groups attached respectively to the imaged surface 12 and the surface of the imaging molecule.

- Note also in Figure 4E that the surface 12 is covered --- 25--~with plural OH groups. These can take part in hydrogen bonding and can increase the affinity constant of binding between the ~maging macromolecule and the imaged surface.

Referring to Figure 5A through SD, another series of molecular diagrams similar to t~ose set forth in Figure 4 - are shown which differ from Figure 4 in that substrate 12 - is an aldehyde derivatized startiAg material of the type .
_ :

WO93/l~&~ PCT/US93/026 illustrated in Figure 3B. As shown in Figure 5A, as the amine group of, for example, a lysine side chain, comes into contact with an aldehyde group pendent from a filament some distance from the substrate 12, it reacts to S form a secondary amine linking the protein to the surface as shown in Figure 5B. Residual aldehyde groups are reduced to primary alcohols by sodium borohydride. In the presence of strong base, the peptide bonds linking the amino acids of the protein together are hydrolyzed. This results in a structure such as illustrated in Figure 5C in which, at each location where the protein had an amino side chain, an amino acid residue remains with its characteristic amine and carboxylic acid groups. The structure next is treated to acylate the amine groups using an acylating reagent such as acetic anhydride to produce the structure of Figure 5D having a negatively charged carboxylic acid located on surface 12 in position to ma*e with the various amine groups on the exposed side chains of the imaging protein.
- As noted above, orientation of an imaging molecule prior to or during the reactive imaging step can be ~-~ accomplished by either hydrophobic or electrostatic interaction. An advantage of the electrostatic interaction is that it will provide an orientation which will maximize the number of interfacing charged sites involved in the synthesis. Figure 6 illustrates one example of this type of reaction scheme which, in this case, allows electrostatically oriented reactive imaging in production of the imaged surface comprising plural, spaced apart anions.
, Figure 6A depicts a protein, here shown simply as lines terminating in amino side groups, interfacing with a 3~ reactive surface 12, here comprising alpha-carboxyl beta hydroxylic filaments extending upwardly from surface 12.

~093/19844 ~ 1 ~ 2 9 ~ 7 PCT/US93/02623 - The negative charged polarity of the carboxyl groups and the positive charged polarity of the amine groups on the protein interact to settle the molecular surface of the protein into the activated substrate surface during the imaging procedure. Unlike the previous examples where essentially instantaneous reaction occurs upon contact between an amine group and an epoxy or aldehyde group, in this instance no reaction occurs spontaneously, and equilibrium can be established between the ima~ing molecule and the surface to be imaged. This promotes production of relatively few separate molecular surface images, i.e., tends to make all of the binding regions more nearly alike in their distribution of charge, and tends to orient the imaging molecule with its most lS positively charged surface interfacing the sorbent.

In the presen~e of EDAC, peptide bonds are formed between the amine groups and the carboxylic acid groups on the sorbent surface as shown in Figure 6B. Next, in the : 20 presence of periodate, free carboxylic acid groups are converted to aldehyde groups (Figure 6C~ and then in the presence of sodium borohydride to alcohol groups 5Fi~ure 6D). Next, strong base such as XOH is used to hydrolyze ~ ~ all peptide bonds leaving carboxylic acid groups ~ 2~ covalently bonded to the now imaged surface, spaced thereabout in the mirror image of the amine side groups on - the imaging protein.
.
Preparation of a Molecular Ima~ed Cationic Surface 3~
- - ~ Figure 7A through 7E illustrates how to make still -: another embodiment of the imaged surface of the invention.
- In this case, again, the Lmaging molecule is steered to -~ _ ~the surface by electrostatic forces such that the imaged surface of the protein is the area which is most anionic.

WQ93/19 ~ PCT/US93/026:

~32 9 8~ - 36 -Formation of the surface begins when a protein, here shown as comprising, from left to right in Figure 7A, an amino acid such as aspartic acid having an anionic carboxylic acid side chain, and an amino acid such as glutamic acid with another carboxylic acid side chain, ~nd a lysine having a cationic amine group on its side chain. Again, the imaging protein is permitted to reach equilibrium with the surface such that carboxylate groups on its side chains are electrostaticaIly attracted by primary amine groups covalently attached directly to the matrix 12 or to a coating adhering to the matrix. An activated surface comprising a field of amine groups can be synthesized using a variety of techniques. Al~ernatively, conventional, commercially available, polyimine or polyamine cation exchange resins may be used.

'~'' Negative charge on the surface of the protein is attracted to positive char~e on the surface of the activated surface. As indicated by a double-headed arrow on the right of Figure 7A, interfacing amine groups would repel one another. Treatment of the reacting system with EDAC prod,u~es amide bonds between carboxylic acid side groups and the amine groups on the sorbent as shown in Figure 7B. Strong oxidation_in periodate (IO4) liberates methylamine from the surface and coverts the terminal alcohols into aldehyde groups as~shown in Figure 7C.
Schiff base formation can occur between the resulting aldehyde group and the primary amine group on the lysine side chain provided the density of the aldehyde groups is ,, 30 high enough so that an amine group is in close prox~mity.
Upon treatment with sodium borohydride, the aldehyde groups are converted to alcohol groups, and the schiff base is converted to secondary amine as shown in Figùre 7D. Next, strong based hydrolyzes all peptide bonds WO93~19&~ 2 1 3 ~ 9 8 7 PCT~US93/026?3 - leaving, as shown in Figure 7E, amine group oovalently linked to the surface 12 and disposed opposite the carboxylate groups of the side chains of the imaging protein. Where imaging protein originally had an arginine or lysine residue, it becomes linked with the imaged surface.

Figures 8A and 8B illustrate the method of synthesis of an imaged sorbent having anions and hydrophobic groups covalently linked to the sorbent surface and disposed so as to cooperatively attract a protein having on its molecular surface a plurality of cations and a hydrophobic patch. Figure 8A shows an activated surface 12 of the type illustrated in Figure 3B comprising filaments extending from the surface, eaeh of which have plural pendent aldehyde groups. The imaging protein here is - -- depicted as having a hydrophobic patch 16 disposed between a lysine and an argenine residue.

Prior to mixing the aldehyde activated surface and the pro~ein together to begin the imaging step, amphipathic molecules, here illustrated as amine soaps having a hydrophobic e.g., hydrocarbon, tail 17 and a covalently linked amine group, are mixed together under conditions in 25--which-~he hydrophobic portion of the soap molecules associate and become embedded in the hydrophobic patch 16 on .the_surface of the pratein. ~hereafter, imaging and subsequent synthesis of the image surface is conducted essentially as disclosed above with respect to Figures 5A
through 5D. The rasult is shown in Figure 8B~

.
.

.... . . . .. .. ......

wos3/l9&~ PCT/US93/026' ~3;~93~ 38 -As illustrated, the result of the synthesis is that anions covalently linked to the surface 12 are disposed in position to interact with cations on the surface of the imaging protein, and hydrophobic groups are positioned to interact by hydrophobic-hydrophobic attraction with the hydrophobic patch on the protèin. The hydrophobic amphipathic molecules become bonded to the surface through secondary amine linkages in precisely the same way as the - lysine or arginine residues. The amphipathic molecule may comprise, for example, a compound of the formula R-X, where R is a hydrophobic group such as a saturated or unsaturated hydrocarbon, halocarbon, either cyclic, branched, or straight chained, or an aryl group or heterocyclic nucleus, and X is a reactive group which will associate and can form covalent bonds with an appropriate activated surface. X may be, for example, an amine group as exemplified above, or a carboxylate group. When the activated surface comprises a field of amine groups, x may be an epoxy or aldehyde group. ~his procedure can be used with or without parallel formation of charged groups. It can also be used to produce imaged reverse phase matrix materials that selectively bind predetermined macromolecular species by multipoint matching hydrophobic interaction.
- Figure 9A-9C disclose the synthesis scheme for --- locating on the surface of the sorbent a metal -- -- coordinating compound, here iminodiacetic acid, at a point designed to form a metal coordinating bond in the presence 30 ! of a metal with an imidizole side chain. Figure 9A
illustrates an activated surface, here exemplified as an epoxy surface of the type in ~igure 3A, in~erfacing with a protein which, prior to the imaging step, has been mixed with i~inodiacetic acid and metal ion such as copper to W093/19&~ ~ 1 3 ~ 9 8 7 PCT/US93/02623 form -a metal coordination complex between the imidizole and IDA groups, shown in the center of Figure 9A.
Flanking the central histidine residue having the imidizole side group is a pair of lysine residues.
Formation of the imaged surface occurs using a reaction scheme as illustrated in Figures 4A through 4E with the exception that a chelating agent such as EDTA is added to the reaction mixture after hydrolysis of the protein to remove the coordinating metal, thereby leaving a iminodiacetic acid residue opposite in space from the location of the histidine residue on the protein as illust~ated in Figure 9B. Figure 9C schematically illustrates how the image surface of 9B would interact with the imaging molecule. As shown, in the presence of a l~ metal such as copper (indicated in Figure 9C as M~) a metal coordinating bond forms between the LmidizoIe ~- ~ residue and the iminodiacetic acid residue at the same time as cations on the surface of the protein and anions on the sorbent surface interact by electrostatic attraction. This procedure also can be used above, to make imaged metal coordinating~matrix materials having multipoint metal coordinating bond links, or together with the~ s~imultaneous production of hydrophobic interactive fu~ctionaIities. ~ _ ~~~
The invention will be understood further from the following, nbn}imiting examples.

From the foregoing it will be appreciated that there , 30 are many alternative strategies for producing a particular imaged surface, and that, to optimize selecti~ity and affinity-for a particular imaging macromolecule, multiple syntheses_may be desireable, with each synthesis followed .
by anaiys~is~to guide the next iteration, thereby to more ., WO93~19 ~ PCT/US93/026.
c~3~gQOrl closely attain the desired sorbent properties. An illustration of this general approach sppears in the examples below.

It is contemplated in accordance with the invention that computer aided design will play an important role in facilitating development of particular imaged surfaces.
An example of how software for modeling protein and other macromolecular structure may be exploited to advantage is shown in Figure 12. As illustratedl a given macromolecule, here "protein X" can simply be subjected to reactive imaging as disclosed herein to produce a "prototype" Lmaged surface. If the surface is to be used for low volume procedures, such as analysis, the prototype 1~ may suffice. However, if structural data for protein X is known, its three-dimensional configuration and relevant surface properties may be discerned using commercially available molecular modeling softwesr in a general purpose computer. Thus, f O example, depending on available data on the macromolecule of interest, it may be possible to discover at least the presence, approximate spacing, and relative positions of one or more hydrophobic patches, histidine residues, or charged amino acids on particular ~-surfaces on the macromolecule. This information may be used to~aid the chemist in deciding which approach might be successful and which would not, greatly decreasing the --- work in~oIved. For example, whether metal chelating ~~~ -~ ~hould be used, whether hydrophobic patch imaging alone may be successful, whether anti-chaotropic salts should be used in the Lmaging step, and if so, what face of the mole~ule likely wil} be imaged, and what features are on that face, all can be determined by modeling. Thus, as in ~ many engineering challenges, computer aided design - techniques can give insights which streamline and shorten the design process.

WO93/19&~ 21 3 ~ 9 8 7 PCT/US93/02623 In situations where a large volume of sorbent will be reguired, or where it is desired to make a highly "monoclonal" imaged sorbent, in accordance with the invention, one can find, analyze, and then synthesize a peptide having a structure which mimicks a surface of a desired macromole~ule, and then can use the peptide in a reactive imaging process. Alternati~e ways to implement this approach also are disclosed in Figure 12. Thus, peptides generated from partially hydrolyæed protein X, some other protein source, or a synthetic peptide mixture, may be screened for binding to a prototype imaged surface, e.g., using affinity chromatography with differential elution using gradient eluents, to identi~y a peptide which binds to a prototype surface. This peptide then itself may be used to make a second surface, which in turn is tested for binding to protein X. If the new surface has the desired binding properties, large amounts of the -~ imaging peptide may be synthesized and used to Image production quantities of the imaged sorbent. If not, anothe~ peptide can be imaged, and the process repeated.

Again CAD can benefit this process. Thus, analysis of a surface peptide sequence may reveal a surface mimicking 2~ peptide. It- t~en can be sequenced, synthesized, optionally screened on a prototype imaged surface, and used in reactL~ imaging to produce a new surface. This new sur~ace~ ca~ again be tested using protein X, as well as th-e identified peptide or other peptides. Again, once a surface having the desired properties emerges, it can be duplicate~-in large volumes using the peptide as the Imaging-macromolecule, and will bind selectively and with desired-affinity to protein X.

.

WO93/19&~ PCT/US93/02~;
2~3 ~ 42 -.

Example 1. Imaging lysozyme POROSR-OH (PerSeptive Biosystems, Cambridge, MA) was prepared according ~o USP 5,030,352 using an epichlorohydrin-glycidol copolymer that was subsequently crosslinked to produce a matrix rich in surface hydroxyl groups. This material was brominated with PBr3 and subsequently derivatized with sorbitol in the presence of strong base. The resultin~ surface, rich in diols, was oxidized with NaIO4, then imaged with lysozyme (30 mg/gram of beads) in the presence of 1.6M Na2SO4, 0.1 M phospha~e buffer (pH 9.0) for 20 hours in the presence of NaCNB~
At the end of the reaction, excess aldehyde groups were reduced by NaBH4. The bound protein was hydrolyzed with 4M KOH for 16 hours and subsequently acylated with acetic anhydride for 2 hours. Excess ester groups were hydrolyzed for 2 hours with 0~5 M KOH. The result was an imaged sorbent of ~he type illustrated in Figure 4E with - 20 anions on the surface spaced in the mirror Lmage of amine con~aining side groups on the surface of lysozyme.

Fourier Transform Infrared Spectrospy (FTIR) was used to analyze the progress of the reaction scheme. Thus, the generated spectra of lysozyme alone, lysozyme immobilized onto the sorbitol activated surface, immobilized lysozyme with a spec~rum of the base matrix subtracted ou~, the surface of the base matrix after hydrolysis, and surface matrix after acylation, all were compared. The spectra of lysozyme alone and the lysozyme on the base matrix were, ~ ~ as expected, very similar. The spectrum after hydrolysis clearly illustrated the absence of lysozyme with loss of -- the characteristic maximum bands at 3300, 1650, and 1550 ---- cm~1, ~lso, the spectrum of the support after acylation ~093/19&~ 2 1 3 ~ 9 ~ 7 PCT/US93/02623 showed a pair of bands at 1750 and 1200 cm~l corresponding to the presence of acetate esters. Lastly~ the imaged support was titrated with 0.1 M KOH in 5 microliter increments. No measurable ionic capacity on the surface of the sorbent could be detected, indicating that anionic charge density was extremely low, as expected~

The sorbent then was packed in a 4.6 x 100 mm column, and cytochrome C and lysozyme were applied to the surface with a gradient of increasing sodium chloride from 0 to 1 - M at pH 6. A cytochrome C peak was eluted from the column a~ very low ionic strength, followed by a separate peak at only a slightly higher concentration indicating elution of lysozyme.
The conclusion from this experiment, showing rather weak lysozyme binding to the Lmaged surface, is that while a cation exchange surface was su cessfully manufactured as : indicated by the behavior of the column and by the FTIR
: ~ 2~ spectra, based on the rather low salt concentration (approximately 0.3 M) needed to elute the lysozyme in the gradient mode, the density of active groups on the surface of the starting material was probably too low, resulting : in the creation of too few points of ionic interaction _ between the surface of the lysozyme and ~he binding regions on the sorbent. Accordingly, in an attempt to impro~e aff~ni~y, the experiment was repeated with a higher de-nsity-of activated groups on the surface of the -starting material.

.:

.. .. . . ... , . . . . .

WO93/19&~ P~T/US93/026 ,gy~l Example 2.
-Polystyrene divinylbenezene POROSh sorbent was againtreated with epichlorohydrin-glycidol copolymer to produce a field of hydroxyl groups about the surface of the perfusive particulate material. This starting material was then suspended in 750 milliliters of water, degassed by vacuum and nitrogen, and added to 25 ml acrolein and 12.5 ~ cerium sulfate. The mixture was stirred for 8 hours at room temperature under nitrogen, then the beads were washed with water, sulfuric acid, water, and acetone, and dried in a vacuum oven at 60C. This procedure resl~lted in the production of a aldehyde activated filamented surface of the type illustrated in Figure 3B.
The surface density of groups available is m~ch higher than the epoxy activated surface of Figure 3A and Example - l as the aldehydes not only cover the surface of the POROS~ support but also extend from polymer filaments.

Thirty mg of lysozyme were dissolved in 2.5 ml of 0.1 M phosphate buffer, pH 9Ø Next, 12.6 mL 2.0 M sodium sulfate in 0.1 M phosphate buffer, pH 9, was mixed with the lysozyme, and the solution was added to 2 grams of the ~ acrolein activated bead preparation._ This mixture was - ~ 25 shaken at room temper~ture for 3 hours, then 100 mg sodium borohydride added with shaking for another hour. The beads then were washed with water, suspended in 50 ml 4 M
KOH, and stirred with reflux for 16 hours. The beads were then washed with water and acetone and dried in a vacuum oven of 60C. Next, they were suspended in 25 ml pyridine followed by addition of 25 ml acetic anhydride. The mixture was stirred under reflux for 2 more hours, and the beads were then washed with water and acetone and dried.
~- . I

~093/19&~ 2 1 3 2 9 8 7 PCT/US93/026~3 The progress of the synthesis was again traced with FTIR. The same series of infrared spectra were produced as discussed above with respect ~o Example l. However, titration with 0.1 KOH revealed an ionic capacity of about 1 ~M per ml.

As a control, the acrolein derivatized surface was exposed to the reaction scheme described above but in the absence of any imaging agent. Elution experiments on this type of material showed essentially no lysozyme retention on the control surface. Lysozyme could be eluted at less than 100 nM NaCl.

The chromatographic performance of the imaged surface was evaluated by packing a 4.6 x 100 mm column with the material produced as disclosPd above. The plot of a gradient elution of lysozyme at p~ 8 is shown in Figure 10A. The column was loaded with a 100 ~l injection of 1 mg/ml lysozyme equilibrated with tris buffer at pH 8.
three minute gradient to 1 M NaCl (27 mS conducti~ityt elutes the bound lysozyme at about 17 mS.

The~performance of this lysozyme imaged column can best be~illustrated by comparison to a high capacity strong cation exchange column. Figure 10B shows the gradient separation of lysozyme and cytochrome C on a comm~rcially--available cation exchange column (POROSR
~S/M~. Lysozyme elutes at 0.55 M NaCl (15 mS) while , 30 cytochrome C elutes at 0.42 M NaCl, ~11 mS, where 27 mS is approximately equal to 1 M NaCl). In contrast, Figure 10C
shows the same test with the lysozyme imaged column. In this caser lysozyme elutes at 0.6 M NaCl tl6 mS) while .
cytochrQme C elutes at 0.15 M NaCl (4 mS). The difference 21329~ - 46 -between these two surfaces is that lysozyme is strongly bound to both but cytochrome C binds weakly to the lysozyme imaged column. The same separation profile on the imaged column is illustrated when lysozyme is mixed with other proteins. Figure lOD shows that chymotrypsinogen binds weakly while lysozyme is bound tightly. Figure lOE shows that lysozyme is bound tightly while ribonuclease is bound weakly.

Additional work suggests that these results are essentially duplicated at pH 6.2. ~owever, since the functional groups on the surface of the imaged column are weak anionic groups, i.e., carboxyl, one would expect the column to lose its capacity with decreasing pH. As predicted, at pH 4.5, the imaged column does not bind lysozyme well, eluting at the bsginning of the gradient, while cytochrome C is completely unretained. This contras*s with the strong cation exchanger used in Figure lO~ which binds both proteins well at pH 4.5.
If one seeks to use the lysozyme imaged column in an on/off affinity mode, a small amount of sodium chloride can be included in the sample and wash buffers. In this case, the imaged column selectively binds lysozyme ~ 25 essentially exclusively from a mixture of lysozyme and cytochrome C provided the feed contains lOO mM sodium chloride. Elution is then conducted by increasing sodium --- -- chloride content to l.O M.

Figure ll shows a direct comparison of gradient - - elution between lysozyme and cytochrome-C conducted under ~-- ~ precisely the same conditions, on a lysozyme imaged column -- (top plot) and on a strong cation exchange (SCX) column - _ (POROSR ~S/M). While lysozyme binds strongly to both surfaces, cytochrome C, with a pl of 9, binds weakly to the Lmaged surface.

W093/19~ 2 ~ 3 ~ 9 8 7 PCT/US93/02623 A model of protein adsorption to ion exchange columns has been developed by Regnier et al. (see: The Role of Protein Structure in Chromatographic Behavior Science, Volume 238, page 319, 16 October 1987). This model correlates solute retention to the concentration of eluent by the following equation:
k'=I
DZ

-l0 where k~ is a volumetric chromatographic retention factor, I is a constant related to the binding constant, D is the concentration of salt used as eluent, and Z is a constant reflec~ive of the number of interaction sites between the protein and the surface.
Isocratic experiments were perfonmed to map the retention beha~ior of lysozyme and cytochrome C on strong cation exchange and on the imaged column. Plots of log k~
versus D for lysozyme and cytochrome C, followed by linear regression analysis allowed an estima~e of the constants I
and Z as shown in the table below.

Table I

~~~ - Ly~ozyme Cytochrome-C
Imaged SCX Imaged SCX

. . :
I 0-.25- - 0.03 0.006 0.003 Z - 2.5 5.0 2.5 4.5 3~
As shown,-lysozyme interacts with a strong cation exchange surface (SCX) through fi~e sites and with the imaged surface through only 2.5 sites. This indicates either bl~nding through a different contact region on the , WO93/19 ~ PCT/US93/0262 2 i3 2 9 8~ - 48 -respective surfaces (consistent with use of the anti-chaotropic salt in the imaging process to salt out the lysozyme) or in low charge density with only 2 to 3 sites per molecule cross sectional area. The latter implies that other solutes of similar size should interact with this surface through at~;most 2.5 sites. This prediction is verified, at least in the case of cytochrome C, which has a Z number of 2.5 on the anti-lysozyme imaged column.

Although lysozyme`binds through only half as many sites on the imaqed surface as compared with the conventional strong cation exchange column, the binding constant with the ima:ged surface is about ten times higher than on the strong cation exchanger. This provides strong evidence for cooperative binding with the imaged surface.
The observation is further strengthened by the large ~ difference in ionic capacity between the imaged surface (1 M/ml) and the strong cation exchange surface (50 ~M/ml).
Finally, one~can compare the relative binding strengths on ~- 20 the-~two~surfaces. On the strong cation exchanger, lysozyme~binds ten times stronger than does cytochrome C, ; through~an~eguivalsnt number of~sites. Lysozyme binds 40 - times~ as~strongly to the imaged surface as does cytochrome ~ ;C.~ Fron~tal loading experiments;suggest the binding ~ ~ - ~;25~ constant~between~lysozymè and the Lmaged surface, assuming a Lang uir~isotherm~to describe;the binding process, is abou~ 2~X~106~N~ Compared to antibody antigen reactions~, this~is on~the low end. However, given that only 2.5~ sites are involved, it represents a rather strong ~I; 30 interaction.

,.,,, ~,, , , , .- :
:~ ~ .
; :, :

WO93/19&~ 2 1 3 ~ 9 ~ 7 PCT/US93/02623 Example -3 Bovine serum albumin (BSA) has a pI of 5~7. At pH 6 or 8, its surface therefore will be negative in charge.
S While BSA has surface amines that can be used in the reactive imaging process described in ~xample 2, electrostatic repulsion would be expected to interfere with the adsorption of BSA at such an imaged surface.
With this premise, BSA was Lmaged following the procedure of Example 2. FTI~ spectra of the reaction scheme indicated that the imaging process has proceeded as expected. The ionic capacity of the materiaL was measured to be about 1.7 ~M/ml. I
.:
Chromatographic evaluation of this BSA imaged surface was performed in analogous fashion ~o that described in ~ Example 3. Elution profiles of lysozyme and cytochrome C
from~this surfacP showed that lysozyme is weakly retained, ~ needing~only about 0.2 M NaCl to elute, while cytochrome C
-~ 20 is~unretained. As predicted, BSA binds to this BSA imaged ~surface only weakly. It is proposed that electrostatic repul~sion is the reason for this behavior. Thus, a cation exchange~surface is formed which binds lysozyme through weak cation exchange, does not bind cytochrome C, and binds its target poorly with the mechanistic explanation off~ered~above. ~
" ~
Retention maps wére-generated to characterize the binding of lysozyme and cytochrome C to this BSA imaged j 30 surface in a way similar to that set forth above in - Example 2. Bind-ing~of cytochrome-C to this surface was ~ too weak to allow a reliable estimate of Z or I. The Z
; number for lyso-yme was 4.2 while the I number was 0.004. !-~-and 0.003, respeçtively. The higher charge density, though not specific for lysozyme, does seem to increase WO93~19~ PCT/US93/026' ~3 ~93~ 50 -the interaction sites to 4.2. The binding constant for lysozyme onto the BSA imaged surface is understandably low, comparable to that of cytochrome C on the lysozyme image surface. -S
Binding strength derives in part from the number ofinteraction sites. As discussed above, an overall binding strength number ( I ) is related to the binding constant (K) between a solute and a surface ligand. In example 2, K
was measured as 2 X lO6M-l for lysozyme and the lysozyme imaged surface. Based on this measured K and the ratio of I values for various surfaces, one can determine K values for other related surfaces. Assuming that the overall binding constant is the product of individual interaction constants, one can further calculate an average single site binding constant (K~/Z) as shown in the table below.
- - !
Table II

Estimated Average Single Site Binding Constant for Lysozyme on:

SCX 115 ' ``
.
- ~ys-Imaged 330 - 25 ~S~-Imaged 12 = , .
- -- -- The foregoing analysis clearly shows the cooperative binding of lysozyme to the lysozyme imaged surface. While the BSA imaged and SCX surface behave similarly, the ~ ~ Lysozyme imaged surface binds 30 times stronger per -- ~- interation site. I
--_ --213~87 WO 93/19844 PCr/US93/02623 The nvention may be embodied in other specif ic f orm .
P.ccordingly, other embodiments are within the f ollowing claims .

Claims

What is claimed is:

1. A solid material defining a binding surface comprising a multiplicity of regions which selectively kind a preselected organic molecule having a plurality of ionizable groups spaced about a molecular surface thereof, each said region comprising a plurality of charged moieties bonded to said binding surface and dispose in spaced-apart relation within said region in a mirror image and charge inverse of at least a subset of said ionizable groups whereby said regions bind by spatially matched electrostatic attraction to the molecular surface of said preselected molecule preferentially to other molecules.

2. A composition of matter comprising:
the solid material of claim 1; and a multiplicity of organic molecules, each said organic molecule defining a plurality of ionizable groups spaced about a-molecular surface thereof, bound by spatially matched multipoint electrostatic attractions to said binding regions of said solid material.

3. The invention of claim 1 or 2 wherein said binding surf ace comprises a coating adhered to the surface of said solid material.

4. The invention of claim 1 or 2 wherein said solid material comprises organic polymeric particulate material.

5. The invention of claim 1 or 2 wherein said solid material comprises a perfusive matrix.

6. The invention of claim 1 or 2 wherein said binding surface is substantially free of bound charged moieties in excess of those which bind to the ionizable groups of said organic molecule.

7. The invention of claim 1 or 2 wherein said charged moieties comprise negatively charged moieties selected from the group consisting of carboxylate, sulfonate, phosphate and phosphonate moieties.

8. The invention of claim 1 or 2 wherein said charged moieties comprise carboxyl groups.

9. The invention of claim 1 or 2 wherein said charged moieties comprise positively charged moieties selected from the group consisting of primary amines, secondary amines, tertiary amines, and quaternary ammonium.

10. The invention of claim 1 or 2 wherein said charged moieties are bonded to said binding surface through flexible moieties and said spaced-apart charged moieties define a conformationally compliant charged surface.

11. The invention of claim 10 wherein said flexible moieties comprise oligomers varying in length whereby individual said charged moieties are disposed varying distances apart from said-binding surface.

12. The material of claim 1 wherein said preselected organic molecule defines a hydrophobic patch on said molecular surface, at least a subset of said regions further comprising:

a hydrophobic moiety within said region at a location disposed to interface with said patch when said preselected molecule is bound to said region by multipoint electrostatic attraction.

13. The composition of claim 2 wherein said organic molecule further comprises a hydrophobic patch within said molecular surface and said binding regions comprise a hydrophobic moiety which interfaces with said patch.

14. The material of claim 12 wherein said preselected organic molecule comprises an imidazole residue on said molecular surface and wherein at least a subset of said regions further comprise a metal coordinating moiety bonded to said binding surface at a location in said region disposed to form, in the presence of coordinating metal ions, a metal coordinating bond between said imidazole residue and said coordinating moiety.

15. The material of claim 14 wherein said coordinating moiety comprises an iminodiacetic acid moiety.

16. The composition of claim 2 or 13 wherein said organic molecule comprises an imidazole residue on said molecular surface complexed through a metal ion to a metal coordinating compound bonded to the surface of said regions.

17. The invention of claim 1 or 2 wherein a subset of said binding regions comprise spatial patterns of charged moieties in a mirror image and charge inverse of at least subset of ionizable groups spaced about a second molecular surface of said organic molecule.

18. The invention of claim 1 or 2 wherein said organic molecule comprises a peptide bonded amino acid polymer.

19. The invention of claim 1 or 2 wherein said molecular surface and said regions have an interfacing area of at least about 50 square angstroms.

20. The invention of claim 1 or 2 wherein said molecular surface and said regions have an interfacing area of at least about 500 square angstroms.

21. The invention of claim 1 or 2 wherein said organic molecule comprises an analog of an intermediate in an organic reaction or a polypeptide analog of a surface region of a macromolecule.

22. The invention of claim 1 or 2 wherein said binding surface is an oxygen rich polymeric hydrophilic surface.

23. A solid material defining a binding surface comprising a multiplicity of regions which selectively bind a preselected organic molecule having an imidazole residue on a molecular surface thereof, each said region comprising a metal coordinating moiety bonded to said binding surface and disposed in said region in a mirror image position of said residue, whereby in the presence of coordinating metal ions, said region binds by multipoint spatially matched attractions including at least one metal coordination bond between said imidazole residues and said coordinating moieties, to the molecular surface of said preselected molecule preferentially to other compounds.

24. The material of claim 23 wherein said binding surface is substantially free of bound metal coordinating moieties in excess of those which bind to the residues of said molecules.

25. The material of claim 23 wherein said metal coordinating moiety is an iminodiacetic acid moiety.

26. The material of claim 23 wherein said organic molecule defines a hydrophobic patch on said molecular surface, said region further comprising:
a hydrophobic moiety within said region at a location disposed to interface with said patch when said preselected polymer is bound to said region of said solid material.

27. The material of claim 23 or 26 wherein said preselected organic molecule comprises an ionizable group on said molecular surface and said regions comprise a charged moiety bound to said binding surface at a location opposite said ionizable group when said preselected molecule is bound to regions of said solid material.

28. The material of claim 23 or 26 wherein said regions bind an analog of an intermediate in an organic reaction or a polypeptide analog of a surface region of a macromolecule.

29. The material of claim 23 wherein said organic molecule has plural imidazole residues and each said region comprises a plurality of said metal coordinating moieties.

30. A solid material defining a binding surface comprising a multiplicity of regions which selectively bind a preselected organic molecule having a hydrophobic patch on a molecular surface thereof, each said region comprising at least one hydrophobic moiety, surrounded by hydrophilic surface, bonded to said binding surface, and disposed within said region in a mirror image position to said hydrophobic patch, whereby said regions bind by spatially matched attractions including at least one hydrophobic-hydrophobic interaction, to the molecular surface of said preselected compound preferentially to other compounds.

31. The material of claim 30 wherein said preselected organic molecule comprises an imidazole residue on said molecular surface and wherein at least a subset of said regions further comprise a metal coordinating moiety bonded to said binding surface at a location in said region disposed to form, in- the presence of coordinating metal ions and when said preselected molecule is bound to said regions, a metal coordinating bond between said imidazole residue and said coordinating moiety.

32. The material of claim 30 or 31 wherein said preselected organic molecule comprises an ionizable group on said molecular surface and said regions comprise a charged moiety bound to said binding surface at a location opposite said ionizable group when said preselected molecule is bound to regions of said solid material.

33. The material of claim 30 wherein said organic molecule has plural hydrophobic patches spaced about the molecular surface thereof and at least a subset of said regions comprise plural said hydrophobic moieties in mirror image position to bind with said patches.

34. The material of claim 30 wherein said region binds an analog of an intermediate in an organic reaction or a polypeptide analog of a surface region of a macromolecule.

36. In the method of separating solutes in a mixture comprising passing the mixture through a matrix which differentially binds individual solutes in said mixture and then desorbing solutes bound to said matrix, the improvement comprising:
providing a matrix comprising the material of claim 1, 12, 14, 23, 26, 30 or 56 wherein said preselected organic molecule is a target solute in said mixture; and passing said mixture through said matrix into contact with said surface to preferentially bind said target solute to said binding regions.

37. The method of claim 36 wherein said matrix comprises a perfusive matrix.

38. In a specific binding assay wherein the presence or concentration of an analyte is determined by binding analyte in a sample to the surface of an insoluble support, the improvement wherein said solid support comprises the material of claim 1, 12, 14, 23, 26, 30 or 56 and said analyte comprises said preselected molecule.

39. A method of producing the solid material of claim 1, the method comprising the steps of:

A. providing a solid material having a surface layer of moieties covalently reactive with said ionizable groups;
B. contacting said surface layer with said preselected macromolecule under conditions sufficient to react the ionizable groups of said macromolecule to said surface by multipoint formation of covalent bonds between at least a subset of said ionizable groups on said molecular surface and said reactive moieties;
C. converting remaining reactive moieties in said surface material to an uncharged state not spontaneously reactive with said ionizable groups, D. cleaving each of the covalent bonds produced in step B to release said preselected macromolecule from said surface; and E. producing a charge opposite in sign and in space to the charge of said ionizable groups at each point of cleavage thereby to produce on said surface spatially distributed charged groups in a mirror image and charge inverse of the subset of said ionizable groups on said molecular surface.

40. A method of producing the solid material of claim 1, the method comprising the steps of:
A. providing a solid material having a surface layer of moieties covalently reactive with said ionizable groups;
B. contacting said surface layer with said preselected macromolecule under conditions sufficient to react the ionizable groups of said macromolecule to said surface by multipoint formation of covalent bonds between at least a subset of said ionizable groups on said molecular surface and said reactive moieties;

C. digesting the amino acid polymer by hydrolyzing peptide bonds to leave an amino acid residue at each covalent bond produced in step B; and D. removing or derivatizing the amino group or the carboxylic acid group of said amino acid residues to leave a charge opposite in sign and space to the charge of said ionizable groups thereby to produce on said surface spatially distributed charged groups in a mirror image and charge inverse of the subset of said ionizable groups on said molecular surfaces.

41. The method of claim 39 or 40 wherein said surface layer comprises charged groups opposite in sign to said subset of ionizable groups and said contacting step B
occurs after a molecular surface of said preselected polymer is attracted electrostatically to said surface layer.

42. The method of claim 39 or 40 wherein step B is conducted in the presence of an anti-chaotropic salt.

43. The method of claim 40 wherein said surface layer of covalently reactive moieties comprise-epoxy or aldehyde groups and said ionizable groups comprise amino side chains.

44. The method of claim 39 wherein said surface layer of covalently reactive moieties comprise carboxylic or amine residues.

45. The method of claim 40 wherein said preselected macromolecule has an imidazole residue on said molecular surface, the method comprising the additional step of:

reacting said preselected macromolecule with a metal ion and a metal coordinating compound comprising a covalently reactive group prior to step C, and removing the metal ion to leave a metal coordinating moiety covalently bonded to said surface layer.

46. The method of claim 40 wherein said preselected macromolecule has a hydrophobic patch on said molecular surface, the method comprising the additional step of:
reacting said preselected macromolecule with an amphipathic molecule, comprising a hydrophobic molecular moiety and a covalently reactive group, prior to step C, whereby a hydrophobic moiety is covalently bonded to said surface layer.

47. A method of producing the solid material of claim 23, the method comprising the steps of:
A. providing a solid material having a surface layer of moieties covalently reactive with an organic, nitrogen-containing, polycarboxylic acid metal coordinating compound;
B. reacting a said coordinating compound with said preselected macromolecule, a metal ion, and said surface under conditions to form a coordinated metal ion link between the imidazole residue on said preselected macromolecule and said coordination compound and a covalent bond between and said coordination compound and one of said covalently reactive moieties; and C. removing the metal ion from the product of step B
to produce on said surface a covalently bonded metal coordination compound molecule within said regions located to form a metal coordination complex with said imidazole residues on a said molecular surface of said preselected macromolecule when the molecular surface thereof reversibly adsorbs to the surface of said solid material.

48. The method of claim 47 or 45 wherein said coordinating compound is iminodiacetic acid.

49. The method of claim 47 wherein said preselected macromolecule has a plurality of said imidazole residues on its molecular surface and, during steps B and C, a plurality of said covalently bonded metal coordinating molecules is produced.

50. A method of producing the solid material of claim 30, the method comprising the steps of:
A. providing a solid material having a hydrophilic surface layer of covalently reactive moieties;
B. reacting said surface, said macromolecule, and an amphipathic molecule, comprising a linked hydrophobic molecular moiety and a covalently reactive group, under conditions to produce formation of hydrophobic-hydrophobic bonding interactions between the hydrophobic patch on the surface of said preselected macromolecule and the hydrophobic moiety of said amphipathic molecule and a covalent bond between the covalently reactive group of said amphipathic molecule and said covalently reactive moiety on said surface; and C. desorbing the macromolecule from the surface by breaking the hydrophobic-hydrophobic attractions produced in step B to produce on said surface a multiplicity of regions comprising hydrophobic moieties within a hydrophilic field located to interface with the hydrophobic patch on said molecular surface of said polymer when said molecular surface reversibly adsorbs to the surface of said solid material.

51. The method of claim 50 wherein said preselected macromolecule has a plurality of said hydrophobic patches on its molecular surface and, during steps B and C, a plurality of said hydrophobic moieties located to interfere is produced.

52. The method of claim 39, 40, 47, or 50 wherein said regions binds an analog of an intermediate in an organic reaction or a polypeptide analog of a surface region of a said macromolecule.

53. A method of producing a specific binding surface on a solid material which noncovalently binds a preselected molecule by spacially matched electrostatic attraction, metal coordinating bond formation, hydrophobic-hydrophobic interaction, or a combination thereof, the method comprising the steps of:
identifying a polypeptide mimicking a surface region of said preselected molecule; and using said polypeptide in the method of claim 39, 40, 47, or 50.
54. The method of claim 53 wherein said identifying step is conducted by screening a mixture of peptides synthesized randomly or produced by partial digestion of a protein.

55. The method of claim 53 wherein said identifying step is conducted by analyzing surface features of said molecule through computer modeling based on structural data measured by physiochemical examination of said molecule.

56. The material of claim 1 wherein said preselected organic molecule comprises an imidazole residue on said molecular surface and wherein at least a subset of said regions further comprise a metal coordinating moiety bonded to said binding surface at a location in said region disposed to form, in the presence of coordinating metal ions, a metal coordinating bond between said imidazole residue and said coordinating moiety.

57. The material of claim 56 wherein said coordinating moiety comprises an iminodiacetic acid moiety.