RA Lerner
Antibody-catalyzed phosphate triester hydrolysis. Rosenblum, Jonathan S.; Lo, Lee-Chiang; Li, Tingyu; Janda, Kim D.; Lerner, Richard A. Dep. Chem., Scripps Res. Inst., La Jolla, CA, 92037, USA. Angew. Chem., Int. Ed. Engl. (1995), 34(20), 2275-7.
CODEN: ACIEAY; ISSN: 0570-0833. Journal written in English. AN 1995:930418
From molecular diversity to catalysis: lessons from the immune system. Schultz, Peter G.; Lerner, Richard A. Howard Hughes Med. Inst., Univ. California, Berkeley, CA, 94720, USA. Science (Washington, D. C.) (1995), 269(5232), 1835-42. CODEN: SCIEAS; ISSN: 0036-8075. Journal; General Review written in English. CAN 123:249709
A review, with 75 refs. By combining the enormous mol. diversity of the immune system with basic mechanistic principles of chem., one can produce catalytic antibodies that allow control of reactions in ways heretofore not possible. Mechanistic and structural studies of these antibodies are also providing insights into important aspects of enzymic catalysis and the evolution of catalytic function. Moreover the ability to harness and rationally direct the immune response to generate selective catalysts for reactions ranging from pericyclic and redox reactions to cationic rearrangement reactions underscores the chem. potential of this and other large combinatorial libraries.
Antibody Catalysis of Multistep Reactions: An Aldol Addition Followed by a Disfavored Elimination. Koch, Thomas; Reymond, Jean-Louis; Lerner, Richard A. Department of Molecular Biology, Scripps Research Institute, La Jolla, CA, 92037, USA. J. Am. Chem. Soc. (1995), 117(37), 9383-7. CODEN: JACSAT; ISSN: 0002-7863. Journal written in English. AN 1995:797736
The intramol. aldol condensation of keto-aldehyde 1 yields a substituted 2-benzyl-3-hydroxy-cyclohexanone 2 and subsequently 2-benzyl-2-cyclohexenone (3). The sequence involves four individual reaction steps. Three of these steps can be accelerated using general acid-base catalysis to effect proton transfer at or near the a-carbon of the ketone involved in the condensation, which is at the homobenzylic position relative to the arom. group of the substrate (Ar). An antibody to the corresponding N-benzyl-N-methylpiperidinium hapten 5 was found to catalyze the entire reaction sequence. This antibody seems to act purely as a general base and does not catalyze the carbon-carbon bond forming step. Catalysis of the aldol elimination is selective for the disfavored trans-elimination with a single enantiomer of stereoisomer 2a. Catalysis is suppressed by incubating the atnibody with a carboxyl-specific reagent, suggesting that a carboxyl group acts as a general base to catalyze the sequence. The antibody is approx. 2.0 « 105 times more reactive than acetate for catalysis of the sequence. These expts. demonstrate that catalysis of reactions with several consecutive transition states is possible using catalytic antibodies.
Random mutagenesis of staphylococcal nuclease and phage display selection. Light, James; Lerner, Richard A. Department Molecular Biology, Scripps Research Institute, La Jolla, CA, 92037, USA. Bioorg. Med. Chem. (1995), 3(7), 955-67. CODEN: BMECEP; ISSN: 0968-0896. Journal written in English. CAN 123:277142
Multiple cycles of mutagenesis and phage display selection have been investigated as a method for obtaining enzymes with altered catalytic properties. A library of staphylococcal nuclease mutants displayed on phage was created by error-prone PCR mutagenesis and selected for binding to thymidine- or guanosine-contg. substrate analogs. After discarding non-binders, the binding mutants were then subjected to further mutagenesis and selection rounds. After four mutagenesis and selection cycles, the catalytic properties of some of the resulting nucleases were studied and one nuclease with nine accumulated mutations was found to have a two-fold redn. in kca5t for DNA hydrolysis, but a two-fold increase in kcat/Km for hydrolysis of a thymidine contg. small mol. substrate. The possibility of this technique for in vitro evolution of enzyme properties is discussed.
Chemical characterization of a family of brain lipids that induce sleep. Cravatt, Benjamin F.; Prospero-Garcia, Oscar; Siuzdak, Gary; Gilula, Norton B.; Henriksen, Steven J.; Boger, Dale L.; Lerner, Richard A. Dep. Chemistry, Scripps Res. Inst., La Jolla, CA, 92307, USA. Science (Washington, D. C.) (1995), 268(5216), 1506-9. CODEN: SCIEAS; ISSN: 0036-8075. Journal written in English. CAN 123:30094
A mol. isolated from the cerebrospinal fluid of sleep-deprived cats has been chem. characterized and identified as cis-9,10-octadecenoamide. Other fatty acid primary amides in addn. to cis-9,10-octadecenoamide were identified as natural constituents of the cerebrospinal fluid of cat, rat, and human, indicating that these compds. compose a distinct family of brain lipids. Synthetic cis-9,10-octadecenoamide induced physiol. sleep when injected into rats. Together, these results suggest that fatty acid primary amides may represent a previously unrecognized class of biol. signaling mols.
Encoded reaction cassette for the highly sensitive detection of the making and breaking of chemicals bonds. Fenniri, Hicham; Janda, Kim D.; Lerner, Richard A. Dep. Mol. Biol. and Chem., Scripps Res. Inst., La Jolla, CA, 92037, USA. Proc. Natl. Acad. Sci. U. S. A. (1995), 92(6), 2278-82. CODEN: PNASA6; ISSN: 0027-8424. Journal written in English. CAN 123:103752
A reaction cassette has been designed for the highly sensitive detection of the making and breaking of chem. bonds. The system is envisioned as a companion device to be used in the search for antibody and other novel catalysts. The cassette also may have important clin. applications in the design of diagnostic reagents. In its fully encoded format, this methodol. is capable of both detecting and decoding chem. events.
Oxepane Synthesis Along a Disfavored Pathway: The Rerouting of a Chemical Reaction Using a Catalytic Antibody. Janda, Kim D.; Shevlin, Charles G.; Lerner, Richard A. Department of Molecular Biology, The Scripps Research Institute, La Jolla, LA, 92037, USA. J. Am. Chem. Soc. (1995), 117(9), 2659-60. CODEN: JACSAT; ISSN: 0002-7863. Journal written in English. CAN 122:290083
An efficient synthesis of an oxepane is described using antibody catalysis to reroute a chem. reaction along a disfavored pathway. The reaction proceeds efficiently (kcat = 0.89 min-1) with a high degree of regio- and enantioselectivity. This result again illustrates how catalysts induced by the experimenter can be paired with appropriate substrates to uniquely control complex reaction coordinates.
Cerebrodiene: a brain lipid isolated from sleep-deprived cats. Lerner, Richard A.; Siuzdak, Gary; Prospero-Garcia, Oscar; Henriksen, Steven J.; Boger, Dale L.; Cravatt, Benjamin F. Departments of Chemistry and Neuropharmacology, The Scripps Research Inst., La Jolla, CA, 92037, USA. Proc. Natl. Acad. Sci. U. S. A. (1994), 91(20), 9505-8. CODEN: PNASA6; ISSN: 0027-8424. Journal written in English. CAN 121:252049
The authors report the isolation of a heretofore unrecognized brain lipid that is present in cerebrospinal fluid of sleep-deprived cats. The mol. appears to be a long-chain base structurally related to sphingosine and sphinganine in which a second unsatd. bond has been introduced. An increase in the degree of unsatn. of a key membrane component is expected to have important physiol. consequences.
Antibody catalyzed cationic cyclization. Li, Tingyu; Janda, Kim D.; Ashley, Jon A.; Lerner, Richard A. Dep. Mol. Biol. Chem., Scripps Res. Inst., La Jolla, CA, 92037, USA. Science (Washington, D. C.) (1994), 264(5163), 1289-93. CODEN: SCIEAS; ISSN: 0036-8075. Journal written in English. CAN 121:204864
Two major goals for the design of new catalysts are the facilitation of chem. transformations and control of product outcome. An antibody to hapten I has been induced that efficiently catalyzes a cationic cyclization in which the acyclic olefinic sulfonate ester substrate II is converted almost exclusively (98 percent) to the cyclic alc. III. The key to the catalysis of the reaction and the restriction of the product complexity is the use of antibody binding energy to rigidly enforce a concerted mechanism in accord with the design of the hapten. Thus, the ability to direct binding energy allows the experimenter to dictate a reaction mechanism which is an otherwise difficult task in chem. New catalysts for cationic cyclization may be of general use in the formation of carbon-carbon and carbon-heteroatom bonds leading to multiring mols. including steroids and heterocyclic compds.
Crossing Extreme Mechanistic Barriers by Antibody Catalysis: Syn Elimination to a Cis Olefin. Cravatt, Benjamin F.; Ashley, Jon A.; Janda, Kim D.; Boger, Dale L.; Lerner, Richard A. Department of Molecular Biology, Scripps Research Institute, La Jolla, CA, 92307, USA. J. Am. Chem. Soc. (1994), 116(13), 6013-14. CODEN: JACSAT; ISSN: 0002-7863. Journal written in English. CAN 121:82220
An antibody induced to hapten I catalyzed exclusively the disfavored syn elimination of an acyclic substrate II to a cis (Z) olefin III. In the absence of antibody, only anti elimination of acyclic substrate II to provide the trans olefin IV was obsd. The exclusive catalysis of the syn elimination of II in the absence of an observable background rate underscores the power of catalytic antibodies to accelerate disfavored chem. reactions with high efficiency and selectivity. This reversal of exclusive anti to syn elimination likely represents the most energetically disfavored transition yet catalyzed by an antibody.
Direct selection for a catalytic mechanism from combinatorial antibody libraries. Janda, Kim D.; Lo, Chih Hung L.; Li, Tingyu; Barbas, Carlos F., III; Wirsching, Peter; Lerner, Richard A. Dep. Mol. Biol. Chem., Scripps Res. Inst., La Jolla, CA, 92037, USA. Proc. Natl. Acad. Sci. U. S. A. (1994), 91(7), 2532-6. CODEN: PNASA6; ISSN: 0027-8424. Journal written in English. CAN 120:265164
Semisynthetic combinatorial antibody library methodol. in the phage-display format was used to select for a cysteine residue in complementarity-detg. regions. Libraries were panned with an a-phenethyl pyridyl disulfide that undergoes disulfide interchange. Out of 10 randomly picked clones, two contained an unpaired cysteine, one of which was studied. The antibody catalyzed the hydrolysis of the corresponding thioester where the electrophilic carbonyl occupies the three-dimensional space that was defined by the reactive sulfur atom during selection. The reaction operates by covalent catalysis. Although the steady-state rate enhancement relative to the activated thiol ester substrate is modest, hydrolysis of the acylated cysteine intermediate is remarkably efficient with a catalytic advantage of about four orders of magnitude. The results suggest that iterative mechanism-based selection procedures can recapitulate the enzymic mechanisms refined through evolution.
Antibody-Catalyzed Activation of Unfunctionalized Olefins for Highly Enantioselective Asymmetric Epoxidation. Koch, Andreas; Reymond, Jean Louis; Lerner, Richard A. Departments of Molecular Biology and Chemistry, Scripps Research Institute, La Jolla, CA, 92037, USA. J. Am. Chem. Soc. (1994), 116(2), 803-4. CODEN: JACSAT; ISSN: 0002-7863. Journal written in English. CAN 120:163836
Antibodies raised against transition state intermediates have been shown to catalyze a no. of chem. transformations. In this study the antibody-catalyzed oxidn. at carbon, i.e. enantioselective epoxidn. of unfunctionalized alkenes, is reported. Epoxidn. of the cyclopentene I with antibody 20B11 gave the epoxide II in 98% enantiomeric excess.
Control of the exo and endo pathways of the Diels-Alder reaction by antibody catalysis. Gouverneur, Veronique E.; Houk, K. N.; de Pascual-Teresa, Beatriz; Beno, Brett; Janda, Kim D.; Lerner, Richard A. Dep. Mol. Biol., Scripps Res. Inst., La Jolla, CA, 92037, USA. Science (Washington, D. C., 1883-) (1993), 262(5131), 204-8. CODEN: SCIEAS; ISSN: 0036-8075. Journal written in English. CAN 120:106152
Catalytic antibodies that control the reaction pathways of the Diels-Alder cycloaddn. have been generated. One antibody catalyzes the favored endo and the other the disfavored exo pathway to yield the resp. cis and trans adducts in the enantiomerically pure form. A comparison of the x-ray structure of the hapten with the calcd. geometry of the transition structure showed that [2.2.2] bicyclic compds. are excellent mimics of the transition state of the Diels-Alder reaction. To achieve catalysis and the high degree of stereoselectivity shown here, the antibody must simultaneously control the conformation of the individual reactants and their relation to each other. In the case of the disfavored process, binding energy must be used to reroute the reaction along a higher energy pathway. The rerouting of reaction pathways has become a major focus of antibody catalysis and other disfavored reactions can be expected to be catalyzed so long as the energy barrier is not extreme. The energy requirements needed for abs. control of all of the stereoisomers of many Diels-Alder reactions fall in the energy range (.apprx.20 kcal per mol) deliverable by antibody binding.
A highly specific metal-activated catalytic antibody. Wade, Warren S.; Ashley, Jon A.; Jahangiri, Guiti K.; McElhaney, Gregory; Janda, Kim D.; Lerner, Richard A. Dep. Mol. Biol., Scripps Res. Inst., La Jolla, CA, 92037, USA. J. Am. Chem. Soc. (1993), 115(11), 4906-7. CODEN: JACSAT; ISSN: 0002-7863. Journal written in English. CAN 119:154730
Catalytic antibodies share many mechanistic features found in enzymes, including catalysis by transition-state stabilization, entropic effects, catalytic functional groups and the use of cofactors. A method by which catalytic antibody efficiency may be improved would be to introduce multiple combinations of these enzymic traits in an antibody combining site and have them function in a harmonious manner. Here, results are provided which provide a foundation for achieving such goals. The hapten I was employed as the immunogen; the structural features of I are such that the Ig is forced to bind to distinct metal entities rather than multidentate metal complexes. Of 23 antibodies screened, one, IgG 84A3, showed significant substrate hydrolysis in the presence of zinc. Catalysis was dependent upon the presence of zinc and was specific for the pyridine ester II as substrate. A min. mechanism consistent with obsd. kinetic results is presented.
Direct selection of antibodies that coordinate metals from semisynthetic combinatorial libraries. Barbas, Carlos F., III; Rosenblum, Jonathan S.; Lerner, Richard A. Dep. Chem., Scripps Res. Inst., La Jolla, CA, 92037, USA. Proc. Natl. Acad. Sci. U. S. A. (1993), 90(14), 6385-9. CODEN: PNASA6; ISSN: 0027-8424. Journal written in English. CAN 119:154683
An iterative strategy for the selection of catalytic metalloantibodies is described. The first stage of this strategy is validated by the selection of semisynthetic antibodies that coordinate a variety of different metal ions and the metal oxide magnetite. These results have implications not only for the development of catalytic metalloantibodies but also for the development of reagents for magnetic resonance imaging, delivery of radioisotopes, and purifn. of recombinant proteins.
Antibody catalysis of difficult chemical transformations. Schultz, Peter G.; Lerner, Richard A. Dep. Chem., Univ. California, Berkeley, CA, 94720, USA. Acc. Chem. Res. (1993), 26(8), 391-5. CODEN: ACHRE4; ISSN: 0001-4842. Journal; General Review written in English. CAN 119:111763
A review, with 28 refs., on antibody catalysis. Topics include disfavored chem. reactions, control of regio- and enantioselectivity of reactions, control of ortho transfers, and transesterification reactions.
Antibody catalyzed hydrolysis of enol ethers. Reymond, Jean Louis; Jahangiri, Guiti K.; Stoudt, Catherine; Lerner, Richard A. Dep. Mol. Biol., Scripps Res. Inst., La Jolla, CA, 92037, USA. J. Am. Chem. Soc. (1993), 115(10), 3909-17. CODEN: JACSAT; ISSN: 0002-7863. Journal written in English. CAN 119:72007
The pH profile of the antibody 14D9 catalyzed, enantioselective hydrolysis of enol ether Z-MeOCH:CMeCH2C6H4CONH(CH2)2OH-p (I) between pH = 3.1 and pH = 7.2 has been measured in both H2O and D2O at 20ĄC. The kinetic solvent isotope effect is (kH/kD)cat = 1.75 for the antibody catalyzed reaction and (kH/kD)uncat = 1.92 to 190 mM at high pH. Satn. of the catalytic activity is obsd. at low pH. These observations are consistent with general acid catalysis by an ionizable side chain with pK = 5.2, presumably a carboxyl group, in the active site. A max. rate acceleration kcat/kuncat = 8200 is obtained at the high pH end of the profile, and a max. turnover no. of 9.75 « 10-5 s-1 is obtained at the low pH end. Enol ethers, e.g. Z-MeOCH:CHCH2C6H4CONH(CH2)2OH-p (II), III, IV, are also catalytically hydrolyzed by 14D9. The max. turnover numbered measured is 0.39 s-1 with III at pH = 6.0 at 20ĄC. The catalytic effect kcat/kuncat is influenced by the structure of the enol ether. Catalysis increases by a factor of 12 between II and its b-Me analog I and by a factor of 34 between the six-membered ring enol ether IV and its five-membered ring analog III. These rate effects may reflect the principle of strain in catalysis. They suggest that hydrophobic interactions directly participate in transition-state stabilization, which is unexpected for an acid-base reaction usually discussed in terms of proton relay mechanisms. The implication of these findings for the design and improvement of antibody catalysts is discussed.
Antibody catalysis of a disfavored chemical transformation. Janda, Kim D.; Shevlin, Charles G.; Lerner, Richard A. Dep. Mol. Biol., Scripps Res. Inst., La Jolla, CA, 92037, USA. Science (Washington, D. C., 1883-) (1993), 259(5094), 490-3. CODEN: SCIEAS; ISSN: 0036-8075. Journal written in English. CAN 118:191474
Org. reactions are often limited by stereoelectronic constrains that appear along the reaction coordinate. An antibody has been generated that overcomes these constraints and catalyzes a highly disfavored chem. transformation. The antibody facilitates the difficult 6-endo-tet ring closure of an epoxy-alc. I (X = H, OMe, NHAc) to form a tetrahydropyran II. The catalyzed process is in formal violation of what has become known as Baldwin's rules for ring-closure reactions. In addn. to controlling the regiochem. of the disfavored cyclization reaction, these catalytic antibodies resolve enantiomeric substrates to afford a stereochemically pure product. The principles demonstrated in this study may be applicable to other disfavored chem. processes.
Catalytic antibodies from combinatorial libraries. Chen, Y. C. Jack; Danon, Tami; Sastry, Lakshmi; Mubaraki, Monica; Janda, Kim D.; Lerner, Richard A. Dep. Mol. Biol., Scripps Res. Inst., La Jolla, CA, 92037, USA. J. Am. Chem. Soc. (1993), 115(1), 357-8. CODEN: JACSAT; ISSN: 0002-7863. Journal written in English. CAN 118:54819
The first isolation of a catalytic antibody from a combinatorial library expressed in Escherichia coli is reported and catalytic properties of the antibody are characterized. Combinatorial libraries should allow more facile access to catalytic antibodies.
Antibodies without immunization. Lerner, Richard A.; Kang, Angray S.; Bain, Jim D.; Burton, Dennis R.; Barbas, Carlos F., III. Dep. Chem., Scripps Res. Inst., La Jolla, CA, 92037, USA. Science (Washington, D. C., 1883-) (1992), 258(5086), 1313-14. CODEN: SCIEAS; ISSN: 0036-8075. Journal written in English. CAN 118:37189
A discussion is presented of the synthesis of combinatorial libraries for antibodies in which the complementarity detg. regions are encoded by synthetic nucleic acids randomized at most positions.
Specificity of antibody-catalyzed transesterifications using enol esters: a comparison with lipase reactions. Fernholz, Erhard; Schloeder, Diane; Liu, Kevin K. C.; Bradshaw, Curt W.; Huang, Hongmei; Janda, Kim; Lerner, Richard A.; Wong, Chi Huey. Dep. Chem., Scripps Res. Inst., La Jolla, CA, 92037, USA. J. Org. Chem. (1992), 57(17), 4756-61. CODEN: JOCEAH; ISSN: 0022-3263. Journal written in English. CAN 117:89596
A catalytic antibody elicited against phosphonate PhCHMeOP(O)(O-)CH2C6H4NHCO(CH2)3CO2H-p is studied. The antibody catalyzes not only the hydrolysis of ester (S)-PhCHMeO2CCH2C6H4NHCO(CH2)3CO2H-p, but also the transfer of acyl group from enol ester p-AcNHC6H4CH2CO2CH:CH2 in aq. soln. to a no. of alcs. structurally and sterically related to (S)-1-phenylethanol. Comparison of the antibody reactions to lipase-catalyzed transesterifications indicates that the lipase reactions can only be carried out in org. solvents, and in a representative case the antibody-catalyzed reaction is as effective as the enzymic reaction, both with a kcat.apprx.6 min-1. Some esters with high enantiomeric purity were prepd. on mg scales based on the antibody-catalyzed transesterifications in aq. soln. Although a no. of alcs. and esters can be used as substrates for the lipase and antibody reactions, the antibody catalysis is more predictable with regard to the substrate specificity and stereoselectivity.
Encoded combinatorial chemistry. Brenner, Sydney; Lerner, Richard A. Dep. Chem., Scripps Res. Inst., La Jolla, CA, 92037, USA. Proc. Natl. Acad. Sci. U. S. A. (1992), 89(12), 5381-3. CODEN: PNASA6; ISSN: 0027-8424. Journal written in English. CAN 117:43905
The diversity of chem. synthesis and the power of genetics are linked to provide a powerful, versatile method for drug screening. A process of alternating parallel combinatorial synthesis is used to encode individual members of a large library of chems. with unique nucleotide sequences. After the chem. entity is bound to a target, the genetic tag can be amplified by replication and utilized for enrichment of the bound mols. by serial hybridization to a subset of the library. The library of the tagged chems. is termed an encoded combinatorial chem. library. The nature of the chem. structure bound to the receptor is decoded by sequencing the nucleotide tag. For example, with a library of peptides, each peptide was linked to a specifically designed oligodeoxynucleotide (genetic tag). Each of these oligodeoxynucleotides had a signature (unique) sequence corresponding to the linked peptide and was flanked by sequences recognizable by designed DNA primers of the polymerase chain reaction (PCR). After a desired member of this library of peptide-oligonucleotides was selected by screening (e.g., selected by binding to a target), this desired member was then identified by PCR amplification of the signature nucleotide sequence. Detn. of the signature sequence from PCR amplified DNA gave the identity of the desired peptide. Thus, this method can be used to identify a few members in a large chem. library.
Semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem. Barbas, Carlos F., III; Bain, J. D.; Hoekstra, Denise M.; Lerner, Richard A. Dep. Mol. Biol., Scripps Res. Inst., La Jolla, CA, 92037, USA. Proc. Natl. Acad. Sci. U. S. A. (1992), 89(10), 4457-61. CODEN: PNASA6; ISSN: 0027-8424. Journal written in English. CAN 117:5755
The properties of naivete and large diversity are considered to be essential starting features for combinatorial antibody libraries that eschew immunization by evolution in vitro. Large libraries with such properties were prepd. by using random oligonucleotide synthesis, which has the potential to create .apprx.1020 complementarity-detg. regions for antibody heavy chains. When combined with light chains and expressed on phage surfaces, high-affinity antibodies could be selected from 5.0 « 107 Escherichia coli transformants. Remarkably, antibodies selected only for binding displayed both general structural features known to be important in nature's own antibodies and specific consensus sequences thought to be crit. for interaction with the hapten against which the library was selected. Semisynthetic and ultimately totally synthetic combinatorial libraries when coupled with mutation and selection procedures should replace immunization for generation of reagent, therapeutic, and catalytic antibodies.
Substituent effects of an antibody-catalyzed hydrolysis of phenyl esters: further evidence for an acyl-antibody intermediate. Gibbs, Richard A.; Benkovic, Patricia A.; Janda, Kim D.; Lerner, Richard A.; Benkovic, Stephen J. Dep. Chem., Pennsylvania State Univ., University Park, PA, 16802, USA. J. Am. Chem. Soc. (1992), 114(9), 3528-34. CODEN: JACSAT; ISSN: 0002-7863. Journal written in English. CAN 116:193413
The hydrolysis of a series of para-substituted Ph esters p-(p-RC6H4O2CCH2)C6H4NHCO(CH2)3CO2H (R = O2N, Ac, CHO, Cl, Me) by the monoclonal catalytic antibody NPN43C9 was investigated. The apparent pKa obsd. in the kcat/Km-pH profiles shifts from 8.9 (p-nitrophenyl ester) up to a max. of 9.5 (p-methylphenyl ester). A correlation of the antibody-catalyzed rates of hydrolysis of the esters vs. the s parameter affords a r value of 2.3, indicative of a hydrolytic mechanism proceeding via the attack of a neutral nitrogen nucleophile, and contrasting with the low r value expected for general base catalyzed hydrolysis. There was an inverse solvent deuterium isotope effect on the antibody-catalyzed hydrolysis of the p-chlorophenyl ester at pH > 10. These observations are consistent with a previous proposal (Benkovic, et al., 1990) that this antibody employs a multistep kinetic pathway which involves the formation of a covalent acyl-antibody intermediate.
Antibody-catalyzed hydrolysis of glycosidic compounds. Reymond, Jean Louis; Janda, Kim D.; Lerner, Richard A. Dep. Mol. Biol., Scripps Res. Inst., La Jolla, CA, 92037, USA. Angew. Chem. (1991), 103(12), 1690-2 (See also Angew. Chem., Int. Ed. Engl., 1991, 30(12) 1711-13). CODEN: ANCEAD; ISSN: 0044-8249. Journal written in German. CAN 116:106605
Glycosidic compds., e.g. I, were hydrolyzed in the presence of antibodies prepd. from piperidines II [R1 = CH2C6H4(CONHCH2CH2OH)-p,R2 = H, Me; R1 = Me, R2 = CH2C6H4(CONHCH2CH2CH)-p] and proteins. Glycosides III were also prepd. and were good competitive inhibitors of antibody-catalyzed hydrolysis of I.
Enantiofacial protonation by catalytic antibodies. Fujii, Ikuo; Lerner, Richard A.; Janda, Kim D. Dep. Chem., Scripps Res. Inst., La Jolla, CA, 92037, USA. J. Am. Chem. Soc. (1991), 113(22), 8528-9. CODEN: JACSAT; ISSN: 0002-7863. Journal written in English. CAN 116:17714
Catalytic antibodies (abzymes) induced to achiral hapten I catalyzed the hydrolysis of an enol ester. In addn., 1 of these abzymes was able to influence the enantiofacial protonation of the enolate intermediate to afford the optically enriched a-substituted cyclohexanone II. This is the 1st demonstration of an abyzme which induces asymmetry by enantiotopic face protonation.
An unexpectedly efficient catalytic antibody operating by ping-pong and induced fit mechanisms. Wirsching, Peter; Ashley, Jon A.; Benkovic, Stephen J.; Janda, Kim D.; Lerner, Richard A. Dep. Chem., Scripps Res. Inst., La Jolla, CA, 92037, USA. Science (Washington, D. C., 1883-) (1991), 252(5006), 680-5. CODEN: SCIEAS; ISSN: 0036-8075. Journal written in English. CAN 116:2751
A transition state analog was used to produce a mouse antibody that catalyzes transesterification in water. The antibody behaved as a highly efficient catalyst with a covalent intermediate and the characteristic of induced fit. Whereas some features of the catalytic pathway were programmed when the hapten was designed and reflect favorable substrate-antibody interactions, other features were a manifestation of the chem. potential of antibody diversity. The fact that antibodies recapitulate mechanisms and pathways previously thought to be a characteristic of highly evolved enzymes suggests that once an appropriate binding cavity is achieved, reaction pathways commensurate with the intrinsic chem. potential of proteins ensue.
Asymmetric induction via a catalytic antibody. Ikeda, Shoji; Weinhouse, Michael I.; Janda, Kim D.; Lerner, Richard A.; Danishefsky, Samuel J. Dep. Mol. Biol. Chem., Res. Inst. Scripps Clin., La Jolla, CA, 92032, USA. J. Am. Chem. Soc. (1991), 113(20), 7763-4. CODEN: JACSAT; ISSN: 0002-7863. Journal written in English. CAN 115:158765
An enantiospecific synthesis was developed to provide transition state mimic/hapten I. I induced the first catalytic antibody capable of enantioselective hydrolysis of meso diester II. A comparison is made between this abzyme and its enzymic counterpart, cholinesterase.
At the crossroads of chemistry and immunology: catalytic antibodies. Lerner, Richard A.; Benkovic, Stephen J.; Schultz, Peter G. Dep. Chem., Scripps Res. Inst., La Jolla, CA, 92037, USA. Science (Washington, D. C., 1883-) (1991), 252(5006), 659-67. CODEN: SCIEAS; ISSN: 0036-8075. Journal; General Review written in English. CAN 115:108973
A review with 90 refs., of the generation and use of catalytic antibodies and of the wide array of chem. reactions that they catalyze. In some cases, rates approaching those of enzymes have been achieved, but typically the antibody-catalyzed reactions proceed with rates 103-106 faster than the uncatalyzed reaction. The generation and use of antibodies (1) to stabilize neg. and pos. charged transition states, (2) as entropic traps, and (3) with catalytic groups and cofactors in their combining sites are discussed.
The enzymic nature of antibody catalysis: development of multistep kinetic processing. Benkovic, Stephen J.; Adams, Joseph A.; Borders, C. L., Jr.; Janda, Kim D.; Lerner, Richard A. Dep. Chem., Pennsylvania State Univ., University Park, PA, 16802, USA. Science (Washington, D. C., 1883-) (1990), 250(4984), 1135-9. CODEN: SCIEAS; ISSN: 0036-8075. Journal written in English. CAN 114:97318
Detailed kinetic investigations of a catalytic antibody that promotes the hydrolyses of an anilide and Ph ester showed that this catalyst uses a multistep kinetic sequence resembling that found in serine proteases to hydrolyze its substrates, although antibody was elicited to a single transition-state analog. Like the serine proteases, the antibody catalyzed the hydrolysis reactions through a putative covalent intermediate, but unlike the enzymes it may use OH- to cleave the intermediates. Nevertheless, the antibody was a potent catalyst with turnover at higher pH values rivaling that of chymotrypsin. This anal. also revealed that turnover by the antibody is ultimately limited by product desorption, suggesting that improvements in catalytic efficiency may be achieved by judicious changes in the structure of the substrate, so that it is not superimposable on that of the eliciting hapten.
Catalytic antibodies with acyl-transfer capabilities: mechanistic and kinetic investigations. Janda, Kim D.; Ashley, Jon A.; Jones, Teresa M.; McLeod, Donald A.; Schloeder, Diane M.; Weinhouse, Michael I.; Lerner, Richard A.; Gibbs, Richard A.; Benkovic, Patricia A.; et al. Dep. Mol. Biol., Scripps Clin. Res. Found., La Jolla, CA, 92037, USA. J. Am. Chem. Soc. (1991), 113(1), 291-7. CODEN: JACSAT; ISSN: 0002-7863. Journal written in English. CAN 114:20044
Antibodies have been shown to catalyze acyl-transfer reactions. The processes by which they perform such tasks have often been postulated but largely remain unknown. An extended study is presented on 3 different monoclonal antibodies that catalyzed the hydrolysis of an alkyl ester and an aryl amide bond. Antibodies 2H6 and 21H3 catalyzed the hydrolysis of an unactivated benzyl ester and show exquisite specificity for substrates with either the R or S configuration, resp., whereas 43CF9 catalyzes the cleavage of a p-nitroanilide amide bond. New substrates were synthesized and buffer-assisted reactions were employed to det. antibody-substrate fidelity. Oxygen-18 incorporation expts. were performed providing evidence that these antibody-mediated reactions proceed through attack at the acyl carbonyl, and excluding the possibility of an SN2 displacement mechanism for the ester hydrolysis reaction. A pH-rate profile study in H2O and D2O was performed on antibody 43C9. This revealed an apparent pKa of .apprx.9 involved in catalysis, but both the presence and absence of a solvent isotope effect in the pH-dependent and -independent regions suggested a multistep reaction pathway may be operative.
Crystal structures of an antibody to a peptide and its complex with peptide antigen at 2.8 .ANG. Stanfield, Robyn L.; Fieser, Terry M.; Lerner, Richard A.; Wilson, Ian A. Dep. Mol. Biol., Res. Inst. Scripps Clin., La Jolla, CA, 92037, USA. Science (Washington, D. C., 1883-) (1990), 248(4956), 712-19. CODEN: SCIEAS; ISSN: 0036-8075. Journal written in English. CAN 113:150304
The 3-dimensional structures of an antibody to a peptide and its complex with the peptide antigen were detd. at 2.8 .ANG. resoln. The antigen is a synthetic 19-amino acid peptide homolog of the C helix of myohemerythrin (Mhr). The unliganded Fab' crystals are orthorhombic with 2 mols./asym. unit, whereas the complex crystals are hexagonal with 1 mol./asym. unit. The Fab' and the Fab'-peptide complex structures were solved independently by mol. replacement methods and have crystallog. R factors of 0.197 and 0.215, resp., with no water mols. included. The N-terminal portion of the peptide sequence (N-Glu-Val-Val-Pro-His-Lys-Lys) is clearly interpretable in the electron d. map of the Fab'-peptide complex and adopts a well-defined type II b-turn in the concave antigen binding pocket. This same peptide amino acid sequence in native Mhr is a-helical. The peptide conformation when bound to the Fab' is inconsistent with binding of the Fab' to native Mhr, and suggests that binding can only occur to conformationally altered forms of the native Mhr or to apo-Mhr. Immunol. mapping previously identified this sequence as the peptide epitope, and its fine specificity correlates well with the structural anal. The binding pocket includes a large percentage of hydrophobic residues. The buried surfaces of the peptide and the antibody are complementary in shape and cover 460 .ANG.2 and 540 .ANG.2, resp. These 2 structures now enable a comparison of a specific monoclonal Fab' both in its free and antigen complexed state. While no major changes in the antibody were obsd. when peptide was bound, there were some small but significant side chain and main chain rearrangements.
Metalloantibodies. Iverson, Brent L.; Iverson, Sheila A.; Roberts, Victoria A.; Getzoff, Elizabeth D.; Tainer, John A.; Benkovic, Stephen J.; Lerner, Richard A. Dep. Mol. Biol., Res. Inst. Scripps Clin., La Jolla, CA, 92037, USA. Science (Washington, D. C., 1883-) (1990), 249(4969), 659-62. CODEN: SCIEAS; ISSN: 0036-8075. Journal written in English. CAN 113:128608
A metalloantibody has been constructed with a coordination site for metals in the antigen binding pocket. The Zn(II) binding site from carbonic anhydrase B was used as a model. Three histidine residues have been placed in the light chain complementarity detg. regions of a single chain antibody mol. In contrast to the native protein, the mutant displayed metal-dependent fluorescence-quenching behavior. This response was interpreted as evidence for metal binding in the three-histidine site with relative affinities in the order Cu(II) > Zn(II) > Cd(II). The presence of metal cofactors in Igs should facilitate antibody catalysis of redox and hydrolytic reactions.
Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Huse, William D.; Sastry, Lakshmi; Iverson, Sheila A.; Kang, Angray S.; Alting-Mees, Michelle; Burton, Dennis R.; Benkovic, Stephen J.; Lerner, Richard A. Dep. Mol. Biol., Res. Inst. Scripps Clin., La Jolla, CA, 92037, USA. Science (Washington, D. C., 1883-) (1989), 246(4935), 1275-81. CODEN: SCIEAS; ISSN: 0036-8075. Journal written in English. CAN 112:74970
A novel bacteriophage lambda vector system was used to express in Escherichia coli a combinatorial library of Fab fragments of the mouse antibody repertoire. The system allows rapid and easy identification of monoclonal Fab fragments in a form suitable for genetic manipulation. It was possible to generate, in 2 wk, large nos. of monoclonal Fab fragments against a transition state analog hapten. These methods may supersede present-day hybridoma technol. and facilitate the prodn. of catalytic and other antibodies.
Sequence-specific peptide cleavage catalyzed by an antibody. Iverson, Brent L.; Lerner, Richard A. Dep. Mol. Biol., Res. Inst. Scripps Clin., La Jolla, CA, 92037, USA. Science (Washington, D. C., 1883-) (1989), 243(4895), 1184-8. CODEN: SCIEAS; ISSN: 0036-8075. Journal written in English. CAN 110:208493
Monoclonal antibodies have been induced that are capable of catalyzing specific hydrolysis of the Gly-Phe bond of peptide substrates an neutral pH with a metal complex cofactor. The antibodies were produced by immunizing with a Co(III) triethylenetetramine (trien)-peptide hapten. These antibodies as a group are capable of binding trien complexes of not only Co(III) but also of numerous other metals. Six peptides were examd. as possible substrates with the antibodies and various metal complexes. Two of these peptides were cleaved by several of the antibodies. One antibody was studied in detail, and cleavage was obsd. for the substrates with the trien complexes of Zn(II), Ga(III), Fe(III), In(III), Cu(II), Ni(II), Lu(III), Mg(II), or Mn(II) as cofactors. A turnover no. of 6 « 10-4 per s was obsd. for these substrates. These results demonstrate the feasibility of the use of cofactor-assisted catalysis in an antibody binding site to accomplish difficult chem. transformations.
Metalloselective anti-porphyrin monoclonal antibodies. Schwabacher, Alan W.; Weinhouse, Michael I.; Auditor, Maria Teresa M.; Lerner, Richard A. Dep. Mol. Biol., Res. Inst. Scripps Clin., La Jolla, CA, 92037, USA. J. Am. Chem. Soc. (1989), 111(6), 2344-6. CODEN: JACSAT; ISSN: 0002-7863. Journal written in English. CAN 110:133286
A new class of metalloproteins is reported. Monoclonal antibodies have been prepd., using Co(III) and Fe(III) complexes of meso-tetra-(4-carboxyphenyl)porphine (TCPP) as antigen. The binding affinities of three of the antibodies for a series of metal complexes of TCPP have been measured. In all cases studied, the metal complex used as antigen is bound 101-104 fold more tightly than other metal complexes of the same ligand.
Catalytic antibodies. Lerner, Richard A.; Tramontano, Alfonso. USA. Sci. Am. (1988), 258(3), 58-60, 65-70. CODEN: SCAMAC; ISSN: 0036-8733. Journal; General Review written in English. CAN 108:184718
A review and discussion with no refs. of catalytic antibodies (antibodies produced to catalyze reactions). These antibodies may be useful in biotechnol.
Catalytic antibodies. Tramontano, Alfonso; Janda, Kim D.; Lerner, Richard A. Dep. Mol. Biol., Res. Inst. Scripps Clin., La Jolla, CA, 92037, USA. Science (Washington, D. C., 1883-) (1986), 234(4783), 1566-70. CODEN: SCIEAS; ISSN: 0036-8075. Journal written in English. CAN 106:80805
Monoclonal antibodies elicited to haptens that are analogs of the transition state for hydrolysis of carboxylic esters behaved as enzymic catalysts with the appropriate substrates. These substrates were distinguished by the structural congruence of both hydrolysis products with haptenic fragments. The haptens were potent inhibitors of this esterolytic activity, in agreement with their classification as transition state analogs. Mechanisms were proposed to account for the different chem. behavior of these antibodies with 2 types of ester substrates. The generation of an artificial enzyme through transition state stabilization by antibodies was thus demonstrated. These studies indicate a potentially general approach to catalyst design.