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Biomedical subjects

K D Janda

Publications and source records attributed to K D Janda.

At least 55 records · Page 3Linked to original sources
PubMed1999

A comparison of flexible and constrained haptens in eliciting antibody catalysts for paraoxon hydrolysis.

A new amine-oxide hapten was employed as an antigen, producing seven monoclonal antibodies (mAbs) from a panel of 20 that catalyzed paraoxon hydrolysis. The current hapten design differs from that previously described in that the molecule is inherently more flexible than its constrained predecessor. One of the seven antibody catalysts, mAb 1H9, showed the highest activity and was selected for detailed study. At pH = 8.77, the catalytic hydrolysis of paraoxon by mAb 1H9 followed Michaelis Menten kinetics affording a k(cat) = 3.73 x 10(-4) min(-1) and a Km = 1.12 mM with a rate acceleration k(cat)/k(uncat) = 56. The hapten was found to be a competitive inhibitor of antibody-catalyzed paraoxon hydrolysis with a Ki = 0.54 mM. A comparison of both the number and proficiency of antibody catalysts obtained when utilizing a flexible versus constrained hapten indicates that, for paraoxon hydrolysis, constrained haptens elicit superior catalysts, suggesting that further development should begin with the use of constrained haptens in producing more proficient antibody catalysts for paraoxon hydrolysis.

Aminocaproates↗
PubMed1999

A combined parallel synthesis and screening of macrocyclic lanthanide complexes for the cleavage of phospho di- and triesters and double-stranded DNA.

A parallel synthesis of macrocyclic lanthanide-ligand complexes 4Ln has been developed in conjunction with a parallel screening of these ligands for catalysis of phosphate ester hydrolysis. Complexes 4Ln were screened on a 96-well plate reader for their ability to catalyze the hydrolysis of a variety of phosphate esters efficiently. The hydrolysis of bis(4-nitrophenyl) phosphate (BNPP) 5 and p-nitrophenylethyl phosphate 6 was accelerated by up to 150-fold in the presence of the complex 4cGd. The cleavage of a double-stranded DNA plasmid with this same complex obeyed saturation kinetics following a Michaelian model (K(m) = 7.4 microM, kcat = 4.5 x 10(-3) min-1). Our findings demonstrate how a combination of parallel synthesis and screening can expedite compound access, accelerate catalyst identification, and thereby dramatically increase the speed of finding good ligand-metal combinations.

Catalysis↗
PubMed1998

Soluble polymer traceless linker investigations: solvent effects on the desulfonylation of polyethylene glycol (PEG) substituted aryl alkyl sulfones with sodium amalgam.

The soluble polymer polyethylene glycol containing a traceless aryl alkyl sulfone linker was found to be resistant to reductive cleavage with sodium amalgam (Na/Hg). Detailed investigations revealed that the solvent plays an important role in the desulfonylation reaction; subsequently it was determined that MeOH/DMF (1/8) is an ideal solvent system for the cleavage of this traceless linker.

Alloys↗
PubMed1998

Investigating highly crosslinked macroporous resins for solid-phase synthesis.

The washing efficiencies of a chromophore and the reaction rates of a classical esterification reaction are improved with macroporous resins (MRs) relative to a classical Merrifield resin. Furthermore, Wacker-oxidation of a MR bound alkene yielded the expected methylketone product whereas an alkene bound to a low-crosslinked Merrifield resin gave no product, a function of the relative permeability of each of these resins to the aqueous solvent conditions employed.

Cross-Linking Reagents↗
PubMed1998

A bait and switch hapten strategy generates catalytic antibodies for phosphodiester hydrolysis.

General base catalysis supplied by the histidine-12 (H-12) residue of ribonuclease (RNase) A has long been appreciated as a major component of the catalytic power of the enzyme. In an attempt to harness the catalytic power of a general base into antibody catalysis of phosphodiester bond hydrolysis, the quaternary ammonium phosphate 1 was used as a bait and switch hapten. Based on precedence, it was rationalized that this positively charged hapten could induce a counter-charged residue in the antibody binding site at a locus suitable for it to deprotonate the 2'-hydroxyl group of the anhydroribitol phosphodiester substrate 2. After murine immunization with hapten 1, mAb production yielded a library of 35 antibodies that bound to a BSA-1 conjugate. From this panel, two were found to catalyze the cyclization-cleavage of phosphodiester 2. Kinetic studies at pH 7.49 (Hepes, 20 mM) and 25 degreesC showed that the most active antibody, MATT.F-1, obeyed classical Michaelis-Menten kinetics with a Km = 104 microM, a kcat = 0.44 min-1, and a kcat/kuncat = 1.7 x 10(3). Hapten 1 stoichiometrically inhibits the catalytic activity of the antibody. MATT.F-1 is the most proficient antibody-catalyst (1.6 x 10(7) M-1) yet generated for the function of phosphodiester hydrolysis and emphasizes the utility of the bait and switch hapten paradigm when generating antibody catalysts for processes for which general-base catalysis can be exploited.

Antibodies, Catalytic↗
PubMed1998

An antibody exo Diels-Alderase inhibitor complex at 1.95 angstrom resolution.

A highly specific Diels-Alder protein catalyst was made by manipulating the antibody repertoire of the immune system. The catalytic antibody 13G5 catalyzes a disfavored exo Diels-Alder transformation in a reaction for which there is no natural enzyme counterpart and that yields a single regioisomer in high enantiomeric excess. The crystal structure of the antibody Fab in complex with a ferrocenyl inhibitor containing the essential haptenic core that elicited 13G5 was determined at 1.95 angstrom resolution. Three key antibody residues appear to be responsible for the observed catalysis and product control. Tyrosine-L36 acts as a Lewis acid activating the dienophile for nucleophilic attack, and asparagine-L91 and aspartic acid-H50 form hydrogen bonds to the carboxylate side chain that substitutes for the carbamate diene substrate. This hydrogen-bonding scheme leads to rate acceleration and also pronounced stereoselectivity. Docking experiments with the four possible ortho transition states of the reaction explain the specific exo effect and suggest that the (3R,4R)-exo stereoisomer is the preferred product.

Antibodies, Catalytic↗
PubMed1998

Investigations of azapeptides as mimetics of Leu-enkephalin.

Solution syntheses of azapeptide pentamers 2, 3, and 4 were accomplished. The binding affinity of these azapeptides and azatide 1 were examined in the context of monoclonal antibody 3-E7 known to strongly bind the [Leu5]enkephalin sequence.

Enkephalin, Leucine↗
PubMed1998

Catalytic antibodies.

Following the end of the first decade in catalytic antibody research, recent efforts are reflecting a more introspective view of the field. Notably, X-ray crystal structure analyses of antibody catalysts are permitting an increased understanding of the evolution and modus operandi of these remarkable biocatalysts. Additionally, the breadth and scope of new antibody-catalyzed reactions and novel hapten design strategies have continued to flourish with support from an increasing number of contributors to this ever expanding area.

Antibodies↗
PubMed1997

On roads not taken in the evolution of protein catalysts: antibody steroid isomerases that use an enamine mechanism.

Reactive immunization has emerged as a new tool for the study of biological catalysis. A powerful application resulted in catalytic antibodies that use an enamine mechanism akin to that used by the class I aldolases. With regard to the evolution of enzyme mechanisms, we investigated the utility of an enamine pathway for the allylic rearrangement exemplified by Delta5-3-ketosteroid isomerase (KSI; EC 5.3.3.1). Our aldolase antibodies were found to catalyze the isomerization of both steroid model compounds and steroids. The kinetic and chemical studies showed that the antibodies afforded rate accelerations up to a factor of 10(4) by means of an enamine mechanism in which imine formation was the rate-determining step. In light of our observations and the enzyme studies by other workers, we suggest that an enamine pathway could have been an early, viable KSI mechanism. Although this pathway is amenable to optimization for increased catalytic power, it appears that certain factors precluded its evolution in known KSI enzymes.

Antibodies, Catalytic↗
PubMed1997

Making chemistry selectable by linking it to infectivity.

The link between recognition and replication is fundamental to the operation of the immune system. In recent years, modeling this process in a format of phage-display combinatorial libraries has afforded a powerful tool for obtaining valuable antibodies. However, the ability to readily select and isolate rare catalysts would expand the scope of library technology. A technique in which phage infection controlled the link between recognition and replication was applied to show that chemistry is a selectable process. An antibody that operated by covalent catalysis to form an acyl intermediate restored phage infectivity and allowed selection from a library in which the catalyst constituted 1 in 10(5) members. Three different selection approaches were examined for their convenience and generality. Incorporating these protocols together with well known affinity labels and mechanism-based inactivators should allow the procurement of a wide range of novel catalytic antibodies.

Antibodies, Catalytic↗
PubMed1997

Chemical selection for catalysis in combinatorial antibody libraries.

For the past decade the immune system has been exploited as a rich source of de novo catalysts. Catalytic antibodies have been shown to have chemoselectivity, enantioselectivity, large rate accelerations, and even an ability to reroute chemical reactions. In many instances catalysts have been made for reactions for which there are no known natural or man-made enzymes. Yet, the full power of this combinatorial system can only be exploited if there was a system that allows for the direct selection of a particular function. A method that allows for the direct chemical selection for catalysis from antibody libraries was so devised, whereby the positive aspects of hybridoma technology were preserved and re-formatted in the filamentous phage system to allow direct selection of catalysis. This methodology is based on a purely chemical selection process, making it more general than biologically based selection systems because it is not limited to reaction products that perturb cellular machinery.

Animals↗
PubMed1997

Synthesis on soluble polymers: new reactions and the construction of small molecules.

To avoid the heterogeneous reaction conditions of solid-phase chemistry, liquid-phase synthesis provides homogeneity through the use of soluble polymer supports that can be selectively precipitated and filtered to achieve product purification. Peptides, oligonucleotides, and oligosaccharides have been synthesized by this method. Furthermore, combinatorial libraries of small molecules have been produced by liquid-phase synthesis to aid in the search for new pharmaceutical agents including compounds active against HIV. Soluble polymeric reagents have also been developed for greater ease and efficiency in organic synthesis.

Indicators and Reagents↗
PubMed1996

Cationic cyclopropanation by antibody catalysis.

Reactions involving highly reactive carbocations play a central role in many important chemical processes, such as cyclization reactions. However, the potential for controlling the pathways of such reactions to obtain energetically disfavoured (but desirable) products has been hard to realize because of the difficulties inherent in controlling the conformation and chemical environment of the carbocation intermediates. Antibody catalysts, with their high specificity and binding energies, can provide the degree of conformational and chemical control necessary for directing such reactions. Here we show how antibody catalysis can guide cationic cyclization reactions selectively to form products (in high yield) that would otherwise be highly disfavoured. Most notable is the formation of a strained bicyclic compound containing a rare cyclopropane group. To explain our results, we propose a common reaction scheme in which the key step is the formation of a highly reactive protonated cyclopropane intermediate; subtle structural modifications to the substrate (the compound on which the catalytic antibody acts) lead to dramatic differences in the structure of the final product.

Antibodies, Catalytic↗
PubMed1996

Liquid-phase combinatorial synthesis: in search of small-molecule enzyme mimics.

The applications, advantages and recent advances in liquid-phase combinatorial chemistry using poly-(ethyleneglycol) as a soluble polymer support are reviewed. Our recent efforts towards the synthesis of peptide-based catalysts on polyethyleneglycol are reported. The screening of libraries of peptides for catalysis is discussed.

Biphenyl Compounds↗