News and Publications

Catalytic Antibodies: Organic Synthesis, Catalysis, and New Hapten Strategies

The emphasis of our research is the symbiosis of chemistry and biology from a synthetic organic perspective. Specifically, we are exploring the scope and application of antibodies as catalysts in organic synthesis and as in vitro analytical tools. In addition, we are developing new techniques for hapten design. Last, using antibodies, we produced a novel assay for the detection of nerve gases.

Antibody Catalysis and Organic Synthesis

Recent work in catalysis expanded the boundary of antibody-catalyzed reactions and offers further insight into the facility with which proteins can manipulate reactive intermediates and evolve catalytic ability for reactions previously thought to be within and beyond the scope of biological catalysis.

Cationic cyclizations are among the most demanding reactions that have been catalyzed by antibodies. Studies on such reactions provided valuable mechanistic insights and opened up the possibility of formation of steroidal carbon frameworks. However, the reactions involved substrates that contained an aryl sulfonate group adjacent to a primary carbon center not observed in natural cationic cyclization. Recent advances are an extension of our earlier work; we are now focusing on substrates analogous to those used in triterpene biosynthesis.

Three antibodies, 15D6, 20C7, and 25A10, generated by immunization with an 4-aza-steroid aminoxide hapten, initiate the cationic cyclization of an oxidosqualene derivative and catalyze the formation of ring A of the lanosterol nucleus at neutral pH (Fig. 1). Antibody HA8-25A10 kinetically resolved its racemic substrates. Design of the substrate was based on a dual-anchor model for specific binding that consists of displaying functional groups at the head (epoxide trigger) and the tail (amide functionality) of the otherwise hydrophobic polyene chain. No ring formation was detected in the absence of antibody catalyst. The uncatalyzed epoxide hydrolysis was slow and did not deprive the antibody of substrate. Observations of enzymic polyene cyclizations suggest that subtle changes in the substrate structure may lead to multiring formation under the influence of the current catalyst.

The stereoselective formation of monocyclic ring systems reflects the ability of the antibody catalysts to exert control over the initiation process. Thus, the step that requires particular enzymic assistance in biosynthetic oxidosqualene cyclization has been catalyzed by an antibody. However, propagation of cyclization for this particular class of substrates did not occur. Experiments are ongoing to accomplish such propagation.

When we used the catalytic antibody 6G6 to optimize conditions for a kinetic resolution to furnish S-(+)-naproxen, we made a number of further discoveries. First, the antibody can hydrolyze enantioselectively its chosen substrate, the p-(methylsulfonyl)phenyl ester of naproxen, in media with a high organic content (>50%), thus surmounting initial solubility problems associated with the scale-up of this system. Furthermore, under these stringent conditions, the proficiency of 6G6 actually increases because of a decrease in the observed K_m of the substrate and alleviates inhibition by the phenolic product.

Second, the generality of the hydrolysis reaction that 6G6 can catalyze was revealed in experiments with several panels of substrate analogs. Modifications in the phenolic leaving group are not tolerated well, although small changes can be accommodated; for example, replacement of the p-methylsulfonyl substituent with the corresponding sulfoxide provides a substrate that can be turned over more efficiently by 6G6. However, 6G6 can chirally discriminate at the sulfoxide center. This characteristic limits the use of racemic p-(methylsulfoxyl)phenol as a derivitizing agent for resolution of chiral acids but offers the prospect of antibody-mediated access to homochiral arylsulfoxides. Finally, the naphthyl moiety of naproxen is not an essential recognition element for 6G6. The antibody can process the p-(methylsulfonyl)phenyl ester of a range of aryl and nonaryl propionic acids.

Nerve Gas Detection

Issues related to chemical warfare agents are currently of great importance in national security and world affairs. The lethal compounds sarin (6 in Fig. 2), soman, and VX have been feared as the "nuclear weapons" of the poorer nations because the manufacture of these compounds is relatively simple and the starting materials are inexpensive and readily available. Because these and other nerve gas agents are formed from, and naturally degrade to, methylphosphonic acid (MPA), a convenient method is needed to detect both manufacture and use of nerve gas agents. Therefore, the development of a simple, portable, and inexpensive immunoassay kit would be valuable for monitoring treaty compliance and for detecting MPA during military operations.

Because of immunogenicity problems, monoclonal antibodies that bind MPA could not be successfully obtained. Consequently, we reasoned that if MPA were readily derivatized with recognition elements, as found in the MPA derivative 7 (Fig. 2), monoclonal antibodies that bound the derivative could be elicited by using a structurally congruent, immunogenic hapten (8 in Fig. 2). In this way, the presence of MPA itself could be assessed indirectly through formation and detection of the derivative 7. After immunization, a panel of 11 monoclonal antibodies that bound compound 7 (Fig. 2) were isolated. Of these, the antibody CDC27B4 had the highest affinity (K_d ~1 µM) as detected by a competition enzyme-linked immunosorbent assay.

Unlike existing methods, our procedure is inexpensive, sensitive, convenient, and powerful and requires a minimum of sample preparation. Although refinements are necessary to establish a field kit, we think that our method can complement other methods for detection of MPA. Efforts to develop a field kit and adaptation of the antibody method for detection of other compounds related to chemical warfare agents are in progress.

New Hapten Strategies

Since the inception of catalytic antibodies, numerous hapten strategies have evolved for the generation of antibody hydrolases. Specifically, these strategies can be grouped as approaches that use transition-state analogs, bait and switch, or, more recently, reactive immunization. To extract more powerful catalysts with designer substrate specificity from the murine immune system, we are constantly developing new hapten strategies. Our latest approach, which we dub the "combigen" (for combination immunogen) strategy, combines transition-state stabilization and bait-and-switch strategies in the same hapten molecule. We think that this rationale will lead to the most powerful antibody hydrolases yet isolated.

Hapten 9 (Fig. 3), which incorporates a ß-ammonium phosphate motif locked in a syn geometry by the anhydroribitol ring system, is the first practical strategy for rational incorporation of a general base residue in an antibody combining site close to the developing transition state loci during acyl transfer processes. The scope is such that these reactions may involve either general base catalysis of intramolecular nucleophilic attack (of a proximal hydroxyl group) or general base catalysis via activation of a water molecule. This approach offers broad application across a plethora of different substrates (e.g., compounds 10a-10m in Fig. 3) for acyl transfer reactions.

Initial studies revealed several antibodies that catalyze the hydrolysis of various functional groups observed to proceed via acyl transfer, all elicited from the same hapten (9 in Fig. 3). The key to the success of our strategy is the additional catalytic power imparted by transition-state stabilization and a catalytic mechanism, a direct result of the stereoelectronics of hapten 9 and thus the complementary binding site of the immunoglobulins generated.

Publications

Brümmer, O., La Clair, J.J., Janda, K.D. A colorimetric ligand for mercuric ion. Org. Lett. 1:415, 1999.

Brümmer, O., Wentworth, P., Jr., Weiner, D.P., Janda, K.D. Phosphorodithioates: Synthesis and evaluation of new haptens for the generation of antibody acyl transferases. Tetrahedron Lett. 40:7307, 1999.

Cordova, A., Reed, N.N., Ashley, J.A., Janda, K.D. Convenient synthesis of l-proline benzyl ester. Bioorg. Med. Chem. Lett. 9:3119, 1999.

Datta, A., Wentworth, P., Jr., Shaw, J.P., Simeonov, A., Janda, K.D. Catalytically distinct antibodies prepared by the reactive immunization versus transition state analogue hapten manifolds. J. Am. Chem. Soc. 121:10461, 1999.

Gao, C., Brümmer, O., Mao, S., Janda, K.D. Selection of human metalloantibodies from a combinatorial phage single-chain antibody library. J. Am. Chem. Soc. 121:6517, 1999.

Gao, C., Mao, S., Lo, C.-H.L., Wirsching, P., Lerner, R.A., Janda, K.D. Making artificial antibodies: A format for phage display of combinatorial heterodimeric arrays. Proc. Natl. Acad. Sci. U. S. A. 96:6025, 1999.

Garibay, P., Toy, P.H., Hoeg-Jensen, T., Janda, K.D. Application of a new solid-phase resin: Benzamide ortho-lithiation and the synthesis of a phthalide library. Synlett 9:1438, 1999.

Han, H., Yoon, J., Janda, K.D. Azatides as peptidomimetics: Solution and liquid phase synthesis. Methods Mol. Med. 23:87, 1999.

Hasserodt, J., Janda, K.D., Lerner, R.A. Antibodies mimic natural oxidosqualene-cyclase action in steroid ring A formation. J. Am. Chem. Soc. 122:40, 2000.

Hasserodt, J., Janda, K.D., Lerner, R.A. A class of 4-aza-lithocholic acid-derived haptens for the generation of catalytic antibodies with steroid synthase capabilities, Bioorg. Med. Chem. 8:995, 2000.

Lee, K.-J., Angulo, A., Ghazal, P., Janda, K.D. Soluble-polymer supported synthesis of a prostanoid library: Identification of antiviral activity. Org. Lett. 1:1859, 1999.

Mao, S., Gao, C., Lo, C.-H.L., Wirsching, P., Wong, C-H., Janda, K.D. Phage-display library selection of high-affinity human single-chain antibodies to tumor-associated carbohydrate antigens sialyl Lewis^x and Lewis^x. Proc. Natl. Acad. Sci. U. S. A. 96:6953, 1999.

Pelegrín, J.A.L., Janda, K.D. Solution and soluble polymer supported asymmetric synthesis of six-membered ring prostanoids. Chem. Eur. J. 6:1917, 2000.

Sieber, F., Wentworth, P., Jr., Janda, K.D. Exploring the scope of poly(ethylene glycol) (PEG) as a soluble polymer matrix for the Stille cross-coupling reaction. J. Comb. Chem. 1:540, 1999.

Taylor, M.J., Yli-Kauhaluoma, J.T., Ashley, J.A., Wirsching, P., Lerner, R.A., Janda, K.D. The α-keto amide group: A new motif for the elicitation of catalytic antibodies for acyl-transfer reactions. J. Chem. Soc. Perkin Trans. 19:1133, 1999.

Toker, J.D., Wentworth, P., Jr., Hu, Y., Houk, K.N., Janda, K.D. Antibody-catalysis of a bimolecular asymmetric 1,3-dipolar cycloaddition reaction. J. Am. Chem. Soc. 122:3244, 2000.

Toy, P.H., Janda, K.D. Liquid-phase organic chemistry: The use of soluble polymers as supports for organic synthesis. Acc. Chem. Res. 33:546, 2000.

Toy, P.H., Janda, K.D. New supports for solid-phase organic synthesis: Development of polystyrene resins containing tetrahydrofuran derived cross-linkers. Tetrahedron Lett. 40:6329, 1999.

Vaino, A.R., Goodin, D.B., Janda, K.D. Investigating resins for solid-phase organic synthesis: The relationship between swelling and microenvironment as probed by EPR and fluorescence spectroscopy. J. Comb. Chem. 2:3309, 2000.

Wentworth, A.D., Wentworth, P., Jr., Mansoor, U.F., Janda, K.D. A soluble polymer-supported triflating reagent: A high-throughput synthetic approach to aryl and enol triflates. Org. Lett. 2:477, 2000.

Wentworth, P., Jr., Janda, K.D. Catalytic antibodies. Compr. Asymmetric Catal. I-III. 3:1403, 1999.

Yoon, J., Han, H., Janda, K.D. Solution and soluble polymer syntheses of azatides and aza peptides. Adv. Amino Acid Mimetics Peptidomimetics 2:247, 1999.