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Catalytic antibodies and biomimetics
Catalytic antibodies and biomimetics Bernard S. Green The Hebrew University, Jerusalem, Israel Of the various approaches being studied to mimic the catalytic properties of enzymes, catalytic antibody research is advancing most rapidly and successfully; the discovery of new reactions and new catalytic antibody-producing haptenic structures continues unabated. One of the highlights of the past year was the design and synthesis of a catalytically active peptide. The overall area of catalytic antibodies and biomimetics will be prominent in future biotechnotogical applications, as further advances are made and the nature of the catalyzed reactions becomes better understood. Current Opinion in Biotechnology 1991, 2:395-400
Introduction Catalysis of chemical reactions which display reactionselectivity as well as substrate-selectivity was once the purview of enzymes alone. In recent years, however, nonenzymatic contenders for inclusion in this class of selective catalysts have been discovered. Catalytic antibodies (or 'abzylnes') have attracted the most attention and will be the main focus of this review. Protein- or peptide-based catalysts can now be designed and synthesized de n o v a T h e first example of a peptide from this class was reported last year [1"]. Although chemists have devoted considerable ingenuity and extraordinary skill to the design and synthesis of many nonprotein organic molecules, which may exhibit selective binding and sometimes highly enhanced reaction rates, few of these biomimetic systems are true catalysts, exhibiting turnover. A comprehensive review of these fascinating systems will not be attempted in the limited space here. The research reported in this review is still decidedly 'preapplication' with regard to biotechnology, but the time is clearly approaching for at least some of these potential applications, such as catalytic antibody-based biosensors [2"], to be used successfully.
Catalytic antibodies Antibodies have a functional region which, like enzymes, bind other molecules with high affinity and specificity. As both molecules are composed of the same amino acid building blocks, an antibody binding site may satisfy the
requirements of an enzyme active site. That is, it may utilize the difference in binding energies of the transition state (TS) and of the substrate to catalyze chemical reactions, as well as provide appropriate amino acid functional groups for chemical catalysis. By virtue of their essentially unlimited range of rec_ggniti0n sites, antibodies may provide an enorm-o-u-s repertoire of new, tailormade, enzyme-like catalysts. Furthermore, extraordinary advances have been made in genetic engineering of antibody molecules, including novel ways of producing fragments with combining sites that have greater diversity than those produced by an immunized animal [3"]. Additionally, the incorporation of metal ion [ 4 " , 5 " ] or other cofactors within antibody-combining sites is possible. Thus, there is virtually no limit to the kinds of reactions that can be considered using antibodies. The techniques for generating catalytic antibodies are basically the same as those for conventional binding monoclonal antibodies (mAbs). However, two aspects are crucial for success: the design of the hapten and the process of selecting those few mAbs that are catalytic out of the larger number of mAbs that merely bind the hapten. The first aspect requires a thorough understanding of the reaction mechanism. Haptens can be TS analogues or other structures designed to induce complementary binding sites which function as catalysts. The haptenic structures used so far have been largely lira: ited to relatively easily synthesized compounds, whose TS analogue natures have been based on enzyme inhibition. This has resulted in many successful examples of antibody-catalyzed reactions. Haptens which fail to elicit the expected catalytic antibody, even though they are based on powerful enzyme inhibitors, have also been reported, however [6.]. The future will undoubtedly see far more ambitious syntheses of haptens which mimic
Abbreviations catmAb~catalytic rnonoclonal antibody; CDR---complementarity-determining region; mAb~monoclonal antibody; TS--transition state; VIP--vasoactiveintestinal peptide. ~) Current Biology Ud ISSN 0958-1669
395
396
Biochemicalengineering TSs of reactions more effectively and meet the demands of more difficult (i.e. higher activation energy) and complex chemical reactions.
atively charged structures which closely mimic the TS for ester or amide hydrolysis, and their powerful inhibition of esterases and peptidases.
It is also expected that the development of improved catalytic antibody selection processes will receive greater attention. The large numbers of individual antibodysecreting clones produced by hybridoma technology demands at least a screening for hapten binding. However, because the hapten is only a crude mimic of the true TS, this is cert,ainty inadequate. A screen based on a 'short TS analogue' has been found to be useful [7"] in selecting catalytic monoclonal antibodies (catmAbs) which exhibit tumover and are not product-inhibited. Catalytic antibody research awaits the development of techniques to directly screen hybridoma supematants for catalytic activity rather than the lengthy isolation, purification and individual assay steps used today [8]. Alternatively, genetic screens may be used [3"]. Such methods will ease the problem of product inhibition which invariably plagues many of the catmAbs that have been elicited to date.
The positively charged N-methylpyridinium hapten (Fig. lb) includes tetrahedral geometry at the hydroxy group. It was hoped that a complementary negatively charged group on the antibody combining site would act as a general acid/base or nucleophilic catalyst for a related substrate (Fig. lc) im'oMng a TS such as Fig. ld. Indeed, seven of the 23 mAbs specific for N-methylpyridinium catalyzed the hydrolysis of ester, whereas none of the 21 mAbs raised against the uncharged pyridine control hapten were catalytic [9"].
Hydrolytic reactions The majority of the hydrolytic antibodies reported so far have been raised against phosphonate haptens (Fig la). The rationale for their use includes the tetrahedral, neg-
(a) .
Even more surprising is the induction of a catalytic antibody raised against a neutral hapten (Fig. le), which solely incorporates the tetrahedral geometry of the TS [6.]. This antibody hydrolyzed both ester (Fig. lf) and carbonate (Fig. lg). Experimental and theoretical studies have all emphasized the importance of electrostatic interactions in stabilizing hydrolytic transition states. Thus, the hydrolytic activity of this antibody is unexpected. Perhaps this result best emphasizes the opportunities in the catmAb field that are based partly on the vast numbers of different possible binding sites that are available via the immune system. The first mutagenesis study of a catalytic antibody based on structural data has been reported [10.]. (An earlier mutagenesis study relied on affinity labelling of the
(b) 0I1., oOH R~ P~O-R'
~
R Positively charged N-rnethylpyridiniumhapten
Phosphonate hapten
(c)
(d) OH"
~ R Uncharged control hapten
%°zzR
(e)
C02H
Intermediate or transition state
Ester
Neutral hapten
(f)
NO2
NO2
o/'~o
Ester
/
Carbonate
Fig. 1 Examplesof haptensagainstwhich hydrolyticantibodieshavebeen raisedrecently(a-e),and someof the substratesused(f,g).
Catalytic antibodies and biomimetics Green
complementarity-determining region (CDR) to identify target residues for modification [11].) Two residues in the heavy chain variable region of the phosphorylcholinebinding mAb S107 were substituted with amino acids that might participate in catalysis. When a Tyr residue in the CDR, which had been s h o w n not to be involved in binding or catalysis, was substituted by His, hydrolysis was accelerated eightfold. By comparison, all modifications carried out on an Arg present in the CDR, whose positively charged side chain is believed to stabilize the oxyanion intermediate (TS), lowered the activity of S107. This mutagenesis study was performed on an antibody which is highly homologous to one whose crystal structure has been determined. As crystal structures of catalytic antibodies become available (none have been reported so far), many additional mutagenesis studies can be anticipated.
is a serious problem in such reactions, and the product is bound more strongly than either of the reactants.
In an earlier mAb-catalyzed Diels-Alder reaction, the problem of product inhibition was overcome by using a reaction in which the product is intrinsically unstable and, once formed, decomposes spontaneously to give a new substance which is released by the antibody [13]. Another important potential application of catmAbs in organic chemistry is the development of specific reagents for the selective addition or removal of a group or function. An example is the selective removal of protecting groups. (CatmAbs may also obviate the need for protecting groups in many reactions.) The first reported example in this direction involves the removal of methoxytrityl groups [14°]. Organic reactions may be further enhanced by using organic solvents rather than aqueous solutions. Hydrolysis [15"] and oxidation [16o] reactions have been studied in organic media and such approaches should greatly increase the scope of reactions possible using water-soluble reactants.
O t h e r reactions
The potential of catalytic antibodies is particularly evident when the reaction being catalyzed is one for which enzymes are not available. Such is the case with the Diels-Alder reaction (one of the most import~ant synthetic organic reactions) which has been catalyzed by an antibody raised against a tricyclic hapten (Fig. 2) [12,]. Three new chiral centres are produced in this reaction which represents the synthesis of a relatively complex molecule, in comparison to the antibody-catalyzed reactions reported so far. Although the question of asymmetric synthesis was not addressed in this particular example, experience has shown that catmAbs generally display a very high degree of chiral discrimination (as do binding mAbs) and should afford enantiomer-specific asymmetric synthesis [8]. As might be expected, product inhibition
An important future development will certainly be the extended incorporation of cofactors such as metal ions or complexes into specific sites within the combining site. The antibody would then primarily exert its traditional and reliable role as a selective, tight binder, and a properly positioned metal ion "complex would function as the chemical catalyst. A peptide hydrolysis using this approach has been reported [17] and other recent advances have im,olved the elegant genetic engineering of a metal-binding site into an antibody (although this has not yet been exploited for chemical catalysis) [4°o,5o.]. In order to mimic cytochrome P450-1ike activity, antibodies against metalloporphyrin were used to catalyze an epoxidation reaction [16"]. An antibody that catalyzes the met-
O + HN x , . , ~ 0 V
-R ,
CO2"
>
O N',,~OvCOf
H
o
o
Reactants
Product m
o
~
N
-
R HN y O v C O
o Tricyclic hapten (transition state
analogue)
0 Transition state
2
Fig. 2 Catalysis of the Diels-Alder reaction by an antibody raised against a tricyclic hapten. The tricyclic hapten is a stable synthesized analogue of the transition state depicted. It was used to raise a rnonoclonal antibody which catalyzes the Diels-Alder reaction shown.
397
398 Biochemicalengineering alation of porphyrin has also been developed [18-]; the antibody apparently bends the porphyrin into a conformarion that allows metal ions to be incorporated more easily. One of these metalloporphyrin-binding antibodies was also used to catalyze peroxidation reactions [19"]. The detailed mechanisms of all the observed mAb-catalyzed reactions remain to be elucidated, although initial mechanistic studies are beginning to be reported [20o,21o]. One result of the large number of possible antibody catalysts that can be obtained from each hapten is that the mechanisms may vary among catmAbs raised against the same TS analogue. The mechanism may even differ from that anticipated from the structure of the TS analogue hapten [20-]. A study of equilibrium constants in the presence and absence of antibody has highlighted the ability of catmAbs to shift molecular equilibria [22°]. The appearance of naturally occurring highly active, selecrive peptide-hydrolyzing catalytic antibodies [23] was a great surprise and has raised many questions. Further work on this human IgG autoantibody which hydrolyzes the Glnl6-Metl7 bond of vasoactive intestinal peptide (VIP), a 28-amino-acid neuropeptide, corroborate the earlier results, as well as providing additional information [24",25.]. However, the intriguing aslSects of how this, the most powerfully active catalytic antibody yet described, is elicited and exerts its effect remain to be elucidated.
clonal catalytic antibodies'). Continuing investigations in this largely unexplored area include pyridoxal-catalyzed amino acid mproton exchange [27] and hydrolysis [28] reactions. A different kind of 'imprinting' has also been reported in which an enzyme was lyophilized with an unnatural D-inhibitor. The resulting enzyme exhibited a surprising 'memory' for the D-configurarion substrate [29"].
Conclusion On the basis of the growing number and complexity of reported catmAb-mediated reactions, an impressive army of potential applications to chemistry, biotechnology and medicine are being made available. Although they cannot compete with enzymes for catalytic efficiency, the powerful aspect of rational design makes the catmAb approach especially attractive. Much further research will be required to gain a better understanding of the mechanisms of these new reactions and to elaborate ways of improving desired catmAb properties. The application of new techniques for obtaining antibodies and antibodylike molecules with catalytic properties and the d e n o v o design of catalytic peptides promises to provide many exciting new developments in biomimetic catalysis.
References and recommended reading Catalytic peptides After many attempts and a variety of preliminary advances towards the goal of d e n o v o design of a peptide-based catalyst, a peptide has been designed and synthesized which has approximately 1% of the activity of natural cbymottypsin for an unactivated, alkyl ester substrate (this represents a rate enhancement of around 105 over the uncatalyzed reaction) [1..]. This peptide displays substrate specificity, multiple ( > 100) tumovers, is inhibited by chymotrypsin inhibitors, and is inactivated by hearing (but reactivated by cooling and lyophilization). This is an impressive result and encourages future work in this exciting area.
Templated polymers The polymerization of functional monomers about a template or 'print' molecule, and the subsequent removal of this substance from the rigid polymer, produces sites which display marked preference for molecules which share the structure and enantiomeric selectivity of the template molecule [26]. If the print molecule is a TS analogue, the polymers may function as catalysts (these may be considered as 'synthetic instmcrional poly-
Papers of special interest, published v,ithin the annual period o f re~iew, have been highlighted as: • of interest •, of outstanding interest 1. •.
HAH.'~ KW, Kt.tS WA, STEWARTJM: Design and Synthesis o f a Peptide ttaving Chymotrypsin-Like Esterase Activity. Science 1990, 248:1544-1546. The first de notv9 design and synthesis of a peptide with catabxic actMty. Using acet)t tyrosine ethst ester, a ch}rnotolasin substrate, as a 'template', a 73-residue peptide was designed by computer modelling to mimic the active site of chymotwpsin. The chyrnottTpsin His57-Asp102-Ser195 ('catab'tic triad') residues were maintained in the positions and orientations o f those found from ct)~tal structure analysis by using a bundle of four parallel amphipathic or-helices which were linked at their carboxTl ends. 2. •
BLACKBUKNGF, TALLEYDB, BOOTH PM, DUaFOR CN, MARaXN Mr, NAPPERAn, REESAR: Potentiometric Biosensor Employhag Catalytic Antibodies as the Molecular Recognition Element. A n a l Cbem 1990, 62:2211-2216. A protot~l~e potentiometric biosensor has been constructed to demonsteam the feasibility o f the concept o f catabxic antibody-based biosensots. This ire'tires a micro-pti electrode that is in close contact ~ith a catmAb which catabxes the hydrolysis of phen)t acetate. The hydrogen ions produced are detected by the electrode. (Another electrode modified vdth irrelevant antibodies serves as a control.) The biosensor displa)~ selective response for phenyt acetate, is reversible after multiple ( > 80) cTcles, and shows no deterioration of catmAb activity on standing. 3. WI.WI'ERG, MItS'rEIN C: Man-Made Antibodies. Na tu r e 1991, •. 349:293-299. A comprehensive review of new approaches for genetically engineering antibodies ~xith exciting sisions of future possibilities, including in vitro mimicking and even bypassing animals in the production of antibodies with new properties.
Catalytic antibodies and biomlmetics Green 4. **
IVERSONBL, IVERSON SA, ROBERTS VA, GETZOFF ED, TAL\'ER JA, BEI',q,7.OVICSJ, LER,\XRRA: Metalloantibodies. Science 1990, 249:659-662. This paper and [5**] describe the impressive design, construction and properties of an antibody containing a metal-ion-binding site. The lightchain region of a single chain, fluorescein-binding, variable domain antibody was modified with three ttis residues which v;ere selected on the basis of known zinc-binding enz~ne (carbonic anhydrase B) cr~tal structures and known conserved antibody structures. In addition, a Tyr residue which it wxs thought might interfere ~ t h metal binding ~as changed to Leu. The mutant antibody binds copper ion (binding constant, 106M - 1 ) and also zinc and cadmium: Cu2+ >Zn z+ > C d 2+. The authors pros'ide e~idence that the combining site simultaneously binds the metal ions with fluorescein. This achievement may lead to the engineering of particularly effec~-e metalloantibody-catabxed chemical reactions. 5. ••
ROBERTSVA, IVERSONBI~ IVERSON SA, BENKOVICSJ, LEKNER R&, GE'rZOFF ED~ TA~'ERJA: Antibody Remodeling: A General Solution to the Design of a Metal-Coordination Site in an Antibody Binding Pocket. Proc Natl Acad Sci USA 1990, 87:6654-6658.
See [4.*1. 6. •
StlOKAT KM, KO MK, SC&'%AN TS, KOCHERSPERGER I~ YO,\'KO',aCHS, TH.MSR/VONGS S, 8CHULTZ PG: Catalytic Antibodies: a New Class of Transition-State Analogues used to Elicit Hydrolytic Antibodies. Angew C13em Int Ed Engl 1990, 29:1296-1303. A review ~ c h includes the details on the first use of an uncharged hapten to elicit esterolytic catab'tic antibodies. The authors do not speculate on the reasons for the success of this study, but are to be commended for also repotting interesting unsuccessful experiments. None of the eight elicited coformycin-specific mAbs accelerated the deamination of adenosine to isosine, even though coformycin is one of the most powerful enz~ane inhibitors knob-n, inhibiting adenosine deaminase ~Sth K~ =
2.5 pM.
7. .
TAX~TIKDS, ZEMEL RIL ARAD-YEmN R, GREEN BS, ESmt,,R Z: Simple Method of Selecting Catalytic Monoclonal Antibodies that Exhibit Turnover and Specificity. Biochemistry 1990, 29:9916--9921. Provides a 'recipe' for raising catmAbs successfully. A truncated hapten, which maximizes the relative contribution of those structural elements that are unique to the "IS, is used to select for the eatmAbs. This paper also presents an approach to rule out enzyme or other catabxic agents as being responsible for antibody catab~is. 8.
POLIACKSJ, HSIUN P, SCHUL'rZ PG: Stereospecific Hydrolysis of Alkyl Esters by Antibodies. J Am Chem Soc 1989, 111:5961-5962.
9.
JANDA KD, WELNHOUSE MI, SCIILOEDER D3|, LEIL\'ER RA, • BENKOVICSJ: Bait and Switch Strategy for Obtaining Catalytic Antibodies with Acyl-Transfer Capabilities. J Am C/~m Soc 1990, 112:1274-1275. A new hapten structure affords esterotytic catmAbs. 10. ,
JACKSONDY, PRUDENTJR, BALD',XY',IEP, SCtlULTZ PG: A Mutagenesis Study of a Catalytic Antibody. Proc Nail Acad Sci USA 1991, 88:58--62. Residues Arg52H and Tyr33H of the phosphoryl choline-binding antibody S107 were each ,,-ailed in order to elucidate the effects that these amino acids have upon the hydrob'sis ofpnitrophenyl choline carbonate. The importance of electrostatic interactions for S107-catatyzed hydrolysis is emphasized and the potential to increase appreciably the catab'tic activity by judiciously altering amino acid residues is demonsttated. 11. 12. ,
BALDX~X'~ E, SCHULTZPG: Generation of a Catalytic Antibody by Site-Directed Mutagenesis. Science 1989, 245:1104-1107.
BRAISTEDAC, SCHULTZPG: An Antibody-Catalyzed Bimolecular Diels--Alder Reaction. J Am Chem Soc 1990, 112:74307431. Kinetic parameters are presented for a mAb-catab~ed Diels-Alder reaction. This paper and [13] highlight the importance of product-release design in antibody-catalyzed reactions.
13.
HILVERTD, ttILL KW, NAREDD , AUDITOR /~LM: Antibody Catal}'sis of a Diels-Alder Reaction. J Am Cbem Soc 1989, 111:9261-9262.
14. •
IVERSONBL~ CAMERONKE, JAFL~NGIRI GK, PASTERNAKDS: Selective Cleavage of TriD'l Protecting Groups Catalyzed by an Antibody. J A m C/.rem Soc 1990, 112:5320-5323. A posit-ely charged tris(4-methoxTphen)Ophosphonium hapten elicited catmAbs ~ l i c h catabxed the cleavage of the corresponding trimethox3"trit~t ether. Although the rate enhancement was too modest to be practical, the simple expedient of charge stabilization for the positively charged "IS demonstrates the feasibility of this concept for other reactions involving carbocations and it may be useful for facile selective protecting group cleavage. 15.
J.~NDAKD, ASHLEYJA, JoNxs "I"51,McLEoo DA, SCHLOEDERDM, WELNHOUSE MI: Immobilized Catalytic Antibodies in Aqueous and Organic Solvents. J A m O~m Soc 1990, 112:8886-8888. Esterobxic eatmAbs were co~xIendy immobilized on glass beads and found to be catab'tically active. Rcac~.ity in organic solvents was significantly reduced for the systems studied. •
16.
KEINANE, SLNHASC, SL\~L,k-BAGCtllA, BENORYE, GHOZI MC, GREEN BS: Towards Antibody-Mediated MetaUoPorphyrin Chemistry. Pure Appl Ogem 1990, 62:2013-2019. Antibodies were raised against a Sn4+ porphyrin. Several mAb--Mn3+ complexes were able to catalyze epoxidation of St}Tene in reactions which proceeded in dry organic solvents using b'ophilized complexes and iodosobenzene. •
ESHHAR Z,
17.
I~XRSONBL, LEm\'ERRA: Sequence-Specific Peptide Cleavage Catalyzed by an Antibody. Science 1989, 243:1184-1188.
18.
COCHRANAG, SCHULTZ PG: Antibody-Catalyzed Porphyrin Metallation. J A m Chem Soc 1990, 112:9414-9415. Using an N-methylporph?xin (a structure ~ahich is a powerful inhibitor of enzyme-catabzed insertion of Fe2+ into porph~Tin and is believed to mimic the distorted porph?xin TS for this reaction), catmAbs mtaich catable metallation (Znz +, Cu2 +, Co2 +, and MnZ+) of porph)xins were elioted. •
19.
COCHR&N AG, SCHUtTZ PG: Peroxidase Activity of an Antibody-tleme Complex. Science 1990, 249:781-783. The anti-N-me!hylporph)xin-metallation catmAb studied ~as inhibited b.y Fe3 + mesoporphyfin. The latter complex catal~xes oxidation (hydrogen peroxide ks source of oxygen) of several t}pical peroxidase substrates. The authors consider the intriguing extension of this approach to mAbs raised against N-substituted porphyrins, in order to provide binding sites for both a metalloporph)Tin and substrate (analogous to the N-substiment) and highly selectixx,-ox3gen transfer reactions. •
20. .
BEr,XOVICSJ, ADAMSJA, BORDERSCL JR, J&\'DAKD, LER,\XRRA: The Enz)artic Nature of Antibody Catalysis: Development of Multistep Kinetic Processing. Science 1991, 250:1135-1139. Kinetic studies upon a mAb that catabxes hydrob~is of a/>nitrophenyl ester and amide. Although raised against a simple TS analogue for hydrolysis and thus expected to stabilize a.tetmhedral "IS, the authors suggest a cox'alent intermediate mechanism. 21.
JANDAKD, ASHLEYJA, JONT_~TM, McLEODDA, SCHLOEDERDM, WEINHOUSE.MI, LERNERRA, GraBS RA, BENKOVICPA, HILtlORST R, BENKO',aCSJ: Catalytic Antibodies with Acyl-Transfer Capabifities: Mechanistic and Kinetic Im-estigations. J Am Cbem Soc 1991, 113:291-297. Use of H2180 along with kinetic studies to elucidate mechanisms for mAb-catalyzed ester and pnitroanilide hydrob~es. •
22. ,
JANJICN, TRAMO:'¢rANoA: Chemical Equilibrium at an Antibody Binding Site: Catal)aic Efficiency Defined by a Haldane Relationship. Biochemistry 1990, 29:8867-8872. Further study of the anti-fluorescein mAb which catabxes the reversible addition of sulfite ion to xanthenone. A kinetic anab~sis highlights the antibody's ability to shift molecular equilibria and suggests the possibilit},of catmAb induction without detailed TS or mechanistic information. 23.
PAULS, VOILE DJ, BEACHCM, JOHNSONDR, POWELLMJ,/~LZSSEY RJ: Catalytic ttydrolysis of Vasoactive intestinal Peptide by Human Autoantibody. Science 1989, 244:1158--1162.
399
400
Biochemical engineering 24. j
PAULS, VOU_EDJ, Mzl S: AtFmity Chromatography of a Catalytic Autoantibody to Vasoactive Intestinal Peptide. J Immunol 1990, 145:1196--1199. After 2076-fold purification of catab'tic antibody on a V1P al]irtity colunto, the reaction specificity and Km values were the same. 25. •
PAULS, VOILE DJ, PO~ILL MJ, /~kSSEY RJ: Site Specificity of a Catalytic Vasoactive Intestinal Peptide Antibody. J Biol (~.~em 1990, 265:11910-11913. A study of VIP fragments for activity with the catabxic antibody. The binding epitope includes residues 22-28 at the carbox3-1 end which, most surprisingly, are distant from site of bond-cleavage at residues 16-17. 26.
Wtrl~ G: Polymeric Reagents and Catalysts. Amer Cbem Soc S3~np Series 1986, 308:186-230.
27.
ANDERSON El, MOSBACH K: Molecular Imprinting of the Coenzyme-Substrate Analogue N-P~widoxyl-L-Phenylaninanilide. Maleromol Chem Rapid Commun 1989, 10:491--495.
28.
ROBINSONDK, ~dOSBACIIK: Molecular Imprinting of a Transition State Analogue Leads to a Polymer Exhibiting Esterolytic Actis'ity. J C.bem Soc (3~em Comm 1989, 969-970.
29. •
STAHLM, MANSSON ]~lO, ~,]OSBACH K: The Synthesis of a D-Amino Acid Ester in an Organic Media with AIpha-Chymotrypsin Modified by a Bio-lmprinting Procedure. BioteclJ Lett 1990, 12:161-166. Quite unexpected biocatab~ts that are hard to classify even though they are based on enzymes, have been discovered. When ct-ch~Tnot[3~sin is bx~philized ~ t h a D-configurated inhibitor and then placed in an organic solvent, the inhibitor is removed and the enzyme now has D-amino acid specificity.
BS Green, Department of Pharmaceutical Chemistry, Faculty of Medicine, School of Pharmacy, The Hebrew University, PO Box 12065, Jerusalem 91120, Israel.
Introduction Catalysis of chemical reactions which display reactionselectivity as well as substrate-selectivity was once the purview of enzymes alone. In recent years, however, nonenzymatic contenders for inclusion in this class of selective catalysts have been discovered. Catalytic antibodies (or 'abzylnes') have attracted the most attention and will be the main focus of this review. Protein- or peptide-based catalysts can now be designed and synthesized de n o v a T h e first example of a peptide from this class was reported last year [1"]. Although chemists have devoted considerable ingenuity and extraordinary skill to the design and synthesis of many nonprotein organic molecules, which may exhibit selective binding and sometimes highly enhanced reaction rates, few of these biomimetic systems are true catalysts, exhibiting turnover. A comprehensive review of these fascinating systems will not be attempted in the limited space here. The research reported in this review is still decidedly 'preapplication' with regard to biotechnology, but the time is clearly approaching for at least some of these potential applications, such as catalytic antibody-based biosensors [2"], to be used successfully.
Catalytic antibodies Antibodies have a functional region which, like enzymes, bind other molecules with high affinity and specificity. As both molecules are composed of the same amino acid building blocks, an antibody binding site may satisfy the
requirements of an enzyme active site. That is, it may utilize the difference in binding energies of the transition state (TS) and of the substrate to catalyze chemical reactions, as well as provide appropriate amino acid functional groups for chemical catalysis. By virtue of their essentially unlimited range of rec_ggniti0n sites, antibodies may provide an enorm-o-u-s repertoire of new, tailormade, enzyme-like catalysts. Furthermore, extraordinary advances have been made in genetic engineering of antibody molecules, including novel ways of producing fragments with combining sites that have greater diversity than those produced by an immunized animal [3"]. Additionally, the incorporation of metal ion [ 4 " , 5 " ] or other cofactors within antibody-combining sites is possible. Thus, there is virtually no limit to the kinds of reactions that can be considered using antibodies. The techniques for generating catalytic antibodies are basically the same as those for conventional binding monoclonal antibodies (mAbs). However, two aspects are crucial for success: the design of the hapten and the process of selecting those few mAbs that are catalytic out of the larger number of mAbs that merely bind the hapten. The first aspect requires a thorough understanding of the reaction mechanism. Haptens can be TS analogues or other structures designed to induce complementary binding sites which function as catalysts. The haptenic structures used so far have been largely lira: ited to relatively easily synthesized compounds, whose TS analogue natures have been based on enzyme inhibition. This has resulted in many successful examples of antibody-catalyzed reactions. Haptens which fail to elicit the expected catalytic antibody, even though they are based on powerful enzyme inhibitors, have also been reported, however [6.]. The future will undoubtedly see far more ambitious syntheses of haptens which mimic
Abbreviations catmAb~catalytic rnonoclonal antibody; CDR---complementarity-determining region; mAb~monoclonal antibody; TS--transition state; VIP--vasoactiveintestinal peptide. ~) Current Biology Ud ISSN 0958-1669
395
396
Biochemicalengineering TSs of reactions more effectively and meet the demands of more difficult (i.e. higher activation energy) and complex chemical reactions.
atively charged structures which closely mimic the TS for ester or amide hydrolysis, and their powerful inhibition of esterases and peptidases.
It is also expected that the development of improved catalytic antibody selection processes will receive greater attention. The large numbers of individual antibodysecreting clones produced by hybridoma technology demands at least a screening for hapten binding. However, because the hapten is only a crude mimic of the true TS, this is cert,ainty inadequate. A screen based on a 'short TS analogue' has been found to be useful [7"] in selecting catalytic monoclonal antibodies (catmAbs) which exhibit tumover and are not product-inhibited. Catalytic antibody research awaits the development of techniques to directly screen hybridoma supematants for catalytic activity rather than the lengthy isolation, purification and individual assay steps used today [8]. Alternatively, genetic screens may be used [3"]. Such methods will ease the problem of product inhibition which invariably plagues many of the catmAbs that have been elicited to date.
The positively charged N-methylpyridinium hapten (Fig. lb) includes tetrahedral geometry at the hydroxy group. It was hoped that a complementary negatively charged group on the antibody combining site would act as a general acid/base or nucleophilic catalyst for a related substrate (Fig. lc) im'oMng a TS such as Fig. ld. Indeed, seven of the 23 mAbs specific for N-methylpyridinium catalyzed the hydrolysis of ester, whereas none of the 21 mAbs raised against the uncharged pyridine control hapten were catalytic [9"].
Hydrolytic reactions The majority of the hydrolytic antibodies reported so far have been raised against phosphonate haptens (Fig la). The rationale for their use includes the tetrahedral, neg-
(a) .
Even more surprising is the induction of a catalytic antibody raised against a neutral hapten (Fig. le), which solely incorporates the tetrahedral geometry of the TS [6.]. This antibody hydrolyzed both ester (Fig. lf) and carbonate (Fig. lg). Experimental and theoretical studies have all emphasized the importance of electrostatic interactions in stabilizing hydrolytic transition states. Thus, the hydrolytic activity of this antibody is unexpected. Perhaps this result best emphasizes the opportunities in the catmAb field that are based partly on the vast numbers of different possible binding sites that are available via the immune system. The first mutagenesis study of a catalytic antibody based on structural data has been reported [10.]. (An earlier mutagenesis study relied on affinity labelling of the
(b) 0I1., oOH R~ P~O-R'
~
R Positively charged N-rnethylpyridiniumhapten
Phosphonate hapten
(c)
(d) OH"
~ R Uncharged control hapten
%°zzR
(e)
C02H
Intermediate or transition state
Ester
Neutral hapten
(f)
NO2
NO2
o/'~o
Ester
/
Carbonate
Fig. 1 Examplesof haptensagainstwhich hydrolyticantibodieshavebeen raisedrecently(a-e),and someof the substratesused(f,g).
Catalytic antibodies and biomimetics Green
complementarity-determining region (CDR) to identify target residues for modification [11].) Two residues in the heavy chain variable region of the phosphorylcholinebinding mAb S107 were substituted with amino acids that might participate in catalysis. When a Tyr residue in the CDR, which had been s h o w n not to be involved in binding or catalysis, was substituted by His, hydrolysis was accelerated eightfold. By comparison, all modifications carried out on an Arg present in the CDR, whose positively charged side chain is believed to stabilize the oxyanion intermediate (TS), lowered the activity of S107. This mutagenesis study was performed on an antibody which is highly homologous to one whose crystal structure has been determined. As crystal structures of catalytic antibodies become available (none have been reported so far), many additional mutagenesis studies can be anticipated.
is a serious problem in such reactions, and the product is bound more strongly than either of the reactants.
In an earlier mAb-catalyzed Diels-Alder reaction, the problem of product inhibition was overcome by using a reaction in which the product is intrinsically unstable and, once formed, decomposes spontaneously to give a new substance which is released by the antibody [13]. Another important potential application of catmAbs in organic chemistry is the development of specific reagents for the selective addition or removal of a group or function. An example is the selective removal of protecting groups. (CatmAbs may also obviate the need for protecting groups in many reactions.) The first reported example in this direction involves the removal of methoxytrityl groups [14°]. Organic reactions may be further enhanced by using organic solvents rather than aqueous solutions. Hydrolysis [15"] and oxidation [16o] reactions have been studied in organic media and such approaches should greatly increase the scope of reactions possible using water-soluble reactants.
O t h e r reactions
The potential of catalytic antibodies is particularly evident when the reaction being catalyzed is one for which enzymes are not available. Such is the case with the Diels-Alder reaction (one of the most import~ant synthetic organic reactions) which has been catalyzed by an antibody raised against a tricyclic hapten (Fig. 2) [12,]. Three new chiral centres are produced in this reaction which represents the synthesis of a relatively complex molecule, in comparison to the antibody-catalyzed reactions reported so far. Although the question of asymmetric synthesis was not addressed in this particular example, experience has shown that catmAbs generally display a very high degree of chiral discrimination (as do binding mAbs) and should afford enantiomer-specific asymmetric synthesis [8]. As might be expected, product inhibition
An important future development will certainly be the extended incorporation of cofactors such as metal ions or complexes into specific sites within the combining site. The antibody would then primarily exert its traditional and reliable role as a selective, tight binder, and a properly positioned metal ion "complex would function as the chemical catalyst. A peptide hydrolysis using this approach has been reported [17] and other recent advances have im,olved the elegant genetic engineering of a metal-binding site into an antibody (although this has not yet been exploited for chemical catalysis) [4°o,5o.]. In order to mimic cytochrome P450-1ike activity, antibodies against metalloporphyrin were used to catalyze an epoxidation reaction [16"]. An antibody that catalyzes the met-
O + HN x , . , ~ 0 V
-R ,
CO2"
>
O N',,~OvCOf
H
o
o
Reactants
Product m
o
~
N
-
R HN y O v C O
o Tricyclic hapten (transition state
analogue)
0 Transition state
2
Fig. 2 Catalysis of the Diels-Alder reaction by an antibody raised against a tricyclic hapten. The tricyclic hapten is a stable synthesized analogue of the transition state depicted. It was used to raise a rnonoclonal antibody which catalyzes the Diels-Alder reaction shown.
397
398 Biochemicalengineering alation of porphyrin has also been developed [18-]; the antibody apparently bends the porphyrin into a conformarion that allows metal ions to be incorporated more easily. One of these metalloporphyrin-binding antibodies was also used to catalyze peroxidation reactions [19"]. The detailed mechanisms of all the observed mAb-catalyzed reactions remain to be elucidated, although initial mechanistic studies are beginning to be reported [20o,21o]. One result of the large number of possible antibody catalysts that can be obtained from each hapten is that the mechanisms may vary among catmAbs raised against the same TS analogue. The mechanism may even differ from that anticipated from the structure of the TS analogue hapten [20-]. A study of equilibrium constants in the presence and absence of antibody has highlighted the ability of catmAbs to shift molecular equilibria [22°]. The appearance of naturally occurring highly active, selecrive peptide-hydrolyzing catalytic antibodies [23] was a great surprise and has raised many questions. Further work on this human IgG autoantibody which hydrolyzes the Glnl6-Metl7 bond of vasoactive intestinal peptide (VIP), a 28-amino-acid neuropeptide, corroborate the earlier results, as well as providing additional information [24",25.]. However, the intriguing aslSects of how this, the most powerfully active catalytic antibody yet described, is elicited and exerts its effect remain to be elucidated.
clonal catalytic antibodies'). Continuing investigations in this largely unexplored area include pyridoxal-catalyzed amino acid mproton exchange [27] and hydrolysis [28] reactions. A different kind of 'imprinting' has also been reported in which an enzyme was lyophilized with an unnatural D-inhibitor. The resulting enzyme exhibited a surprising 'memory' for the D-configurarion substrate [29"].
Conclusion On the basis of the growing number and complexity of reported catmAb-mediated reactions, an impressive army of potential applications to chemistry, biotechnology and medicine are being made available. Although they cannot compete with enzymes for catalytic efficiency, the powerful aspect of rational design makes the catmAb approach especially attractive. Much further research will be required to gain a better understanding of the mechanisms of these new reactions and to elaborate ways of improving desired catmAb properties. The application of new techniques for obtaining antibodies and antibodylike molecules with catalytic properties and the d e n o v o design of catalytic peptides promises to provide many exciting new developments in biomimetic catalysis.
References and recommended reading Catalytic peptides After many attempts and a variety of preliminary advances towards the goal of d e n o v o design of a peptide-based catalyst, a peptide has been designed and synthesized which has approximately 1% of the activity of natural cbymottypsin for an unactivated, alkyl ester substrate (this represents a rate enhancement of around 105 over the uncatalyzed reaction) [1..]. This peptide displays substrate specificity, multiple ( > 100) tumovers, is inhibited by chymotrypsin inhibitors, and is inactivated by hearing (but reactivated by cooling and lyophilization). This is an impressive result and encourages future work in this exciting area.
Templated polymers The polymerization of functional monomers about a template or 'print' molecule, and the subsequent removal of this substance from the rigid polymer, produces sites which display marked preference for molecules which share the structure and enantiomeric selectivity of the template molecule [26]. If the print molecule is a TS analogue, the polymers may function as catalysts (these may be considered as 'synthetic instmcrional poly-
Papers of special interest, published v,ithin the annual period o f re~iew, have been highlighted as: • of interest •, of outstanding interest 1. •.
HAH.'~ KW, Kt.tS WA, STEWARTJM: Design and Synthesis o f a Peptide ttaving Chymotrypsin-Like Esterase Activity. Science 1990, 248:1544-1546. The first de notv9 design and synthesis of a peptide with catabxic actMty. Using acet)t tyrosine ethst ester, a ch}rnotolasin substrate, as a 'template', a 73-residue peptide was designed by computer modelling to mimic the active site of chymotwpsin. The chyrnottTpsin His57-Asp102-Ser195 ('catab'tic triad') residues were maintained in the positions and orientations o f those found from ct)~tal structure analysis by using a bundle of four parallel amphipathic or-helices which were linked at their carboxTl ends. 2. •
BLACKBUKNGF, TALLEYDB, BOOTH PM, DUaFOR CN, MARaXN Mr, NAPPERAn, REESAR: Potentiometric Biosensor Employhag Catalytic Antibodies as the Molecular Recognition Element. A n a l Cbem 1990, 62:2211-2216. A protot~l~e potentiometric biosensor has been constructed to demonsteam the feasibility o f the concept o f catabxic antibody-based biosensots. This ire'tires a micro-pti electrode that is in close contact ~ith a catmAb which catabxes the hydrolysis of phen)t acetate. The hydrogen ions produced are detected by the electrode. (Another electrode modified vdth irrelevant antibodies serves as a control.) The biosensor displa)~ selective response for phenyt acetate, is reversible after multiple ( > 80) cTcles, and shows no deterioration of catmAb activity on standing. 3. WI.WI'ERG, MItS'rEIN C: Man-Made Antibodies. Na tu r e 1991, •. 349:293-299. A comprehensive review of new approaches for genetically engineering antibodies ~xith exciting sisions of future possibilities, including in vitro mimicking and even bypassing animals in the production of antibodies with new properties.
Catalytic antibodies and biomlmetics Green 4. **
IVERSONBL, IVERSON SA, ROBERTS VA, GETZOFF ED, TAL\'ER JA, BEI',q,7.OVICSJ, LER,\XRRA: Metalloantibodies. Science 1990, 249:659-662. This paper and [5**] describe the impressive design, construction and properties of an antibody containing a metal-ion-binding site. The lightchain region of a single chain, fluorescein-binding, variable domain antibody was modified with three ttis residues which v;ere selected on the basis of known zinc-binding enz~ne (carbonic anhydrase B) cr~tal structures and known conserved antibody structures. In addition, a Tyr residue which it wxs thought might interfere ~ t h metal binding ~as changed to Leu. The mutant antibody binds copper ion (binding constant, 106M - 1 ) and also zinc and cadmium: Cu2+ >Zn z+ > C d 2+. The authors pros'ide e~idence that the combining site simultaneously binds the metal ions with fluorescein. This achievement may lead to the engineering of particularly effec~-e metalloantibody-catabxed chemical reactions. 5. ••
ROBERTSVA, IVERSONBI~ IVERSON SA, BENKOVICSJ, LEKNER R&, GE'rZOFF ED~ TA~'ERJA: Antibody Remodeling: A General Solution to the Design of a Metal-Coordination Site in an Antibody Binding Pocket. Proc Natl Acad Sci USA 1990, 87:6654-6658.
See [4.*1. 6. •
StlOKAT KM, KO MK, SC&'%AN TS, KOCHERSPERGER I~ YO,\'KO',aCHS, TH.MSR/VONGS S, 8CHULTZ PG: Catalytic Antibodies: a New Class of Transition-State Analogues used to Elicit Hydrolytic Antibodies. Angew C13em Int Ed Engl 1990, 29:1296-1303. A review ~ c h includes the details on the first use of an uncharged hapten to elicit esterolytic catab'tic antibodies. The authors do not speculate on the reasons for the success of this study, but are to be commended for also repotting interesting unsuccessful experiments. None of the eight elicited coformycin-specific mAbs accelerated the deamination of adenosine to isosine, even though coformycin is one of the most powerful enz~ane inhibitors knob-n, inhibiting adenosine deaminase ~Sth K~ =
2.5 pM.
7. .
TAX~TIKDS, ZEMEL RIL ARAD-YEmN R, GREEN BS, ESmt,,R Z: Simple Method of Selecting Catalytic Monoclonal Antibodies that Exhibit Turnover and Specificity. Biochemistry 1990, 29:9916--9921. Provides a 'recipe' for raising catmAbs successfully. A truncated hapten, which maximizes the relative contribution of those structural elements that are unique to the "IS, is used to select for the eatmAbs. This paper also presents an approach to rule out enzyme or other catabxic agents as being responsible for antibody catab~is. 8.
POLIACKSJ, HSIUN P, SCHUL'rZ PG: Stereospecific Hydrolysis of Alkyl Esters by Antibodies. J Am Chem Soc 1989, 111:5961-5962.
9.
JANDA KD, WELNHOUSE MI, SCIILOEDER D3|, LEIL\'ER RA, • BENKOVICSJ: Bait and Switch Strategy for Obtaining Catalytic Antibodies with Acyl-Transfer Capabilities. J Am C/~m Soc 1990, 112:1274-1275. A new hapten structure affords esterotytic catmAbs. 10. ,
JACKSONDY, PRUDENTJR, BALD',XY',IEP, SCtlULTZ PG: A Mutagenesis Study of a Catalytic Antibody. Proc Nail Acad Sci USA 1991, 88:58--62. Residues Arg52H and Tyr33H of the phosphoryl choline-binding antibody S107 were each ,,-ailed in order to elucidate the effects that these amino acids have upon the hydrob'sis ofpnitrophenyl choline carbonate. The importance of electrostatic interactions for S107-catatyzed hydrolysis is emphasized and the potential to increase appreciably the catab'tic activity by judiciously altering amino acid residues is demonsttated. 11. 12. ,
BALDX~X'~ E, SCHULTZPG: Generation of a Catalytic Antibody by Site-Directed Mutagenesis. Science 1989, 245:1104-1107.
BRAISTEDAC, SCHULTZPG: An Antibody-Catalyzed Bimolecular Diels--Alder Reaction. J Am Chem Soc 1990, 112:74307431. Kinetic parameters are presented for a mAb-catab~ed Diels-Alder reaction. This paper and [13] highlight the importance of product-release design in antibody-catalyzed reactions.
13.
HILVERTD, ttILL KW, NAREDD , AUDITOR /~LM: Antibody Catal}'sis of a Diels-Alder Reaction. J Am Cbem Soc 1989, 111:9261-9262.
14. •
IVERSONBL~ CAMERONKE, JAFL~NGIRI GK, PASTERNAKDS: Selective Cleavage of TriD'l Protecting Groups Catalyzed by an Antibody. J A m C/.rem Soc 1990, 112:5320-5323. A posit-ely charged tris(4-methoxTphen)Ophosphonium hapten elicited catmAbs ~ l i c h catabxed the cleavage of the corresponding trimethox3"trit~t ether. Although the rate enhancement was too modest to be practical, the simple expedient of charge stabilization for the positively charged "IS demonstrates the feasibility of this concept for other reactions involving carbocations and it may be useful for facile selective protecting group cleavage. 15.
J.~NDAKD, ASHLEYJA, JoNxs "I"51,McLEoo DA, SCHLOEDERDM, WELNHOUSE MI: Immobilized Catalytic Antibodies in Aqueous and Organic Solvents. J A m O~m Soc 1990, 112:8886-8888. Esterobxic eatmAbs were co~xIendy immobilized on glass beads and found to be catab'tically active. Rcac~.ity in organic solvents was significantly reduced for the systems studied. •
16.
KEINANE, SLNHASC, SL\~L,k-BAGCtllA, BENORYE, GHOZI MC, GREEN BS: Towards Antibody-Mediated MetaUoPorphyrin Chemistry. Pure Appl Ogem 1990, 62:2013-2019. Antibodies were raised against a Sn4+ porphyrin. Several mAb--Mn3+ complexes were able to catalyze epoxidation of St}Tene in reactions which proceeded in dry organic solvents using b'ophilized complexes and iodosobenzene. •
ESHHAR Z,
17.
I~XRSONBL, LEm\'ERRA: Sequence-Specific Peptide Cleavage Catalyzed by an Antibody. Science 1989, 243:1184-1188.
18.
COCHRANAG, SCHULTZ PG: Antibody-Catalyzed Porphyrin Metallation. J A m Chem Soc 1990, 112:9414-9415. Using an N-methylporph?xin (a structure ~ahich is a powerful inhibitor of enzyme-catabzed insertion of Fe2+ into porph~Tin and is believed to mimic the distorted porph?xin TS for this reaction), catmAbs mtaich catable metallation (Znz +, Cu2 +, Co2 +, and MnZ+) of porph)xins were elioted. •
19.
COCHR&N AG, SCHUtTZ PG: Peroxidase Activity of an Antibody-tleme Complex. Science 1990, 249:781-783. The anti-N-me!hylporph)xin-metallation catmAb studied ~as inhibited b.y Fe3 + mesoporphyfin. The latter complex catal~xes oxidation (hydrogen peroxide ks source of oxygen) of several t}pical peroxidase substrates. The authors consider the intriguing extension of this approach to mAbs raised against N-substituted porphyrins, in order to provide binding sites for both a metalloporph)Tin and substrate (analogous to the N-substiment) and highly selectixx,-ox3gen transfer reactions. •
20. .
BEr,XOVICSJ, ADAMSJA, BORDERSCL JR, J&\'DAKD, LER,\XRRA: The Enz)artic Nature of Antibody Catalysis: Development of Multistep Kinetic Processing. Science 1991, 250:1135-1139. Kinetic studies upon a mAb that catabxes hydrob~is of a/>nitrophenyl ester and amide. Although raised against a simple TS analogue for hydrolysis and thus expected to stabilize a.tetmhedral "IS, the authors suggest a cox'alent intermediate mechanism. 21.
JANDAKD, ASHLEYJA, JONT_~TM, McLEODDA, SCHLOEDERDM, WEINHOUSE.MI, LERNERRA, GraBS RA, BENKOVICPA, HILtlORST R, BENKO',aCSJ: Catalytic Antibodies with Acyl-Transfer Capabifities: Mechanistic and Kinetic Im-estigations. J Am Cbem Soc 1991, 113:291-297. Use of H2180 along with kinetic studies to elucidate mechanisms for mAb-catalyzed ester and pnitroanilide hydrob~es. •
22. ,
JANJICN, TRAMO:'¢rANoA: Chemical Equilibrium at an Antibody Binding Site: Catal)aic Efficiency Defined by a Haldane Relationship. Biochemistry 1990, 29:8867-8872. Further study of the anti-fluorescein mAb which catabxes the reversible addition of sulfite ion to xanthenone. A kinetic anab~sis highlights the antibody's ability to shift molecular equilibria and suggests the possibilit},of catmAb induction without detailed TS or mechanistic information. 23.
PAULS, VOILE DJ, BEACHCM, JOHNSONDR, POWELLMJ,/~LZSSEY RJ: Catalytic ttydrolysis of Vasoactive intestinal Peptide by Human Autoantibody. Science 1989, 244:1158--1162.
399
400
Biochemical engineering 24. j
PAULS, VOU_EDJ, Mzl S: AtFmity Chromatography of a Catalytic Autoantibody to Vasoactive Intestinal Peptide. J Immunol 1990, 145:1196--1199. After 2076-fold purification of catab'tic antibody on a V1P al]irtity colunto, the reaction specificity and Km values were the same. 25. •
PAULS, VOILE DJ, PO~ILL MJ, /~kSSEY RJ: Site Specificity of a Catalytic Vasoactive Intestinal Peptide Antibody. J Biol (~.~em 1990, 265:11910-11913. A study of VIP fragments for activity with the catabxic antibody. The binding epitope includes residues 22-28 at the carbox3-1 end which, most surprisingly, are distant from site of bond-cleavage at residues 16-17. 26.
Wtrl~ G: Polymeric Reagents and Catalysts. Amer Cbem Soc S3~np Series 1986, 308:186-230.
27.
ANDERSON El, MOSBACH K: Molecular Imprinting of the Coenzyme-Substrate Analogue N-P~widoxyl-L-Phenylaninanilide. Maleromol Chem Rapid Commun 1989, 10:491--495.
28.
ROBINSONDK, ~dOSBACIIK: Molecular Imprinting of a Transition State Analogue Leads to a Polymer Exhibiting Esterolytic Actis'ity. J C.bem Soc (3~em Comm 1989, 969-970.
29. •
STAHLM, MANSSON ]~lO, ~,]OSBACH K: The Synthesis of a D-Amino Acid Ester in an Organic Media with AIpha-Chymotrypsin Modified by a Bio-lmprinting Procedure. BioteclJ Lett 1990, 12:161-166. Quite unexpected biocatab~ts that are hard to classify even though they are based on enzymes, have been discovered. When ct-ch~Tnot[3~sin is bx~philized ~ t h a D-configurated inhibitor and then placed in an organic solvent, the inhibitor is removed and the enzyme now has D-amino acid specificity.
BS Green, Department of Pharmaceutical Chemistry, Faculty of Medicine, School of Pharmacy, The Hebrew University, PO Box 12065, Jerusalem 91120, Israel.