News and Publications

Catalytic Antibodies, Synthetic Proteins, and Annonaceous Acetogenins

Catalytic Antibodies

Our ongoing research projects in antibody catalysis encompass new reactions, new catalysts, and new concepts in both basic and applied aspects of the field. Our main efforts can be summarized as follows:

Although the solution photochemical reaction of the ketone 1 (in Fig. 1) yields mainly the cleavage products 2 and 3, in the presence of antibodies to compound 5, this Norrish type II reaction results in the selective formation of cis cyclobutanol (compound 4 in Fig. 1). Furthermore, the fact that compound 4, which consists of 2 asymmetric centers, is obtained as a single diastereomer, makes this photoproduct a valuable building block for the synthesis of natural products.

An aldolase antibody, 24H6, obtained from immunization with haptens 6a and 6b (Fig. 1), has an active-site lysine residue with a perturbed pK_a of 7.0. This antibody catalyzes both the aldol addition and the retrograde aldol fragmentation with a broad range of substrates that differ structurally from the hapten. We observed similar behavior with the aldolase antibody 38C2, which was elicited against a much smaller hapten. This observation suggests that in reactive immunization with 1,3-diketones, the hapten structure governs the chemistry but not the overall organization of the active site. Hammett correlation studies of the 38C2- and 24H6-catalyzed aldol and retro-aldol reactions revealed that although the 2 antibodies have broad substrate specificities, they use slightly different mechanisms.

We also developed an antibody-metalloporphyrin assembly that catalyzes the enantioselective oxidation of aromatic sulfides to sulfoxides. Antibody SN37.4 was elicited against a water-soluble tin(IV) porphyrin containing an axial α-naphthoxy ligand. The catalytic assembly consisting of antibody SN37.4 and a ruthenium(II) porphyrin cofactor has the characteristics of an enzyme, such as predetermined substrate selectivity, enantioselective delivery of oxygen to the substrate, and Michaelis-Menten saturation kinetics.

Antibody 38C2 catalyzes the deuterium exchange reaction with a variety of ketones and aldehydes; all reactions were carried out in deuterium under neutral conditions. In addition to the regioselectivity and chemoselectivity of these reactions, the catalytic rates (k_cat) and rate-enhancement values (k_cat/k_un) are the highest values ever observed with catalytic antibodies.

We also studied an antibody-retinal assembly that mimics the opsin shift of the naturally occurring visual pigments. Both experiments and calculations indicate that the aldolase antibody 33F12 covalently binds all-trans retinal via a protonated Schiff base with a lysine residue. This chromophore, which has a remarkable red shift (140 nm) in the presence of 33F12, is a useful model system for studying the factors that contribute to the opsin shift.

During our ongoing efforts to develop catalytic antibodies for organometallic reactions, we discovered a new chemistry of platinum. A novel, air- and moisture-stable dihydrido(methyl)platinum(IV) complex, TpPtH₂Me (compound 8 in Fig. 1), was formed as the sole product by the reaction of TpPtMeCO (compound 7 in Fig. 1) with water.

Novel Catalytic Activities With Synthetic Proteins

We developed a new strategy for the assembly of large polypeptides on a solid support that uses a highly stable safety-catch acid-labile linker. This amide-generating linker is compatible with a wide range of N-terminal protecting groups and ligation chemistries. The usefulness of the method was indicated by a 3-segment synthesis of vMIP I, a chemokine that contains all 20 natural amino acids and has 2 disulfide bonds. This strategy facilitates the synthesis of large polypeptides and can be applied in the assembly of protein libraries.

A profound change in the catalytic activity and mechanism of 4-oxalocrotonate tautomerase (4-OT) was accomplished by creating a rationally designed single amino acid mutation. Although the wild-type enzyme catalyzes only the tautomerization of oxalocrotonate, its bifunctional mutant, P1A, catalyzes 2 mechanistically distinct reactions: the original tautomerization reaction and the decarboxylation of oxaloacetate. These findings support the theory that new enzymatic activity can evolve in a continuous manner.

We also found that enzymes of the 4-OT superfamily, including 4-OT itself, its P1A mutant, and macrophage migration inhibitory factor, also efficiently catalyze the asymmetric aldol addition reaction. These enzymes have remarkable rate accelerations, which are comparable to those reported for natural aldolase enzymes. These results suggest that the 4-OT superfamily may represent a general scaffold for the catalysis of reactions involving imine and enamine intermediates.

Annonaceous Acetogenins

The single-step, tandem oxidative polycyclization reaction with rhenium(VII) reagents was developed as a powerful method by which polyene alcohols can be converted into polytetrahydrofuran products in a single step. A set of rules has been deduced to predict the product's configuration in this reaction.

Annonaceous acetogenins, particularly those with adjacent bis-tetrahydrofuran rings, have remarkable cytotoxic, antitumor, antimalarial, immunosuppressive, pesticidal, and antifeedant activities. More than 350 different acetogenins have been isolated from only 35 of 2300 plants of the family Annonaceae. We developed synthetic approaches that can be used to generate chemical libraries of stereoisomeric acetogenins. These efforts have resulted in the total synthesis of several naturally occurring acetogenins, including asimicin, bullatacin, trilobacin, rolliniastatin, solamin, reticulatacin, goniocin, cyclogoniodenin T, and mucocin (Fig. 2), and many nonnatural stereoisomers. This work is being done in collaboration with S.C. Sinha, The Scripps Research Institute. Avedissian, H., Sinha, S.C., Yazbak, Y., Sinha, A., Neogi, P., Sinha, S.C., Keinan, E. Total synthesis of asimicin and bullatacin. J. Org. Chem. 65:6035, 2000.

Brik, A., D'Souza, L.J., Keinan, E., Grynszpan, F., Dawson, P.E. Lessons from the enamine mechanism of aldolase antibodies exercised in the tautomerase enzymes. J. Am. Chem. Soc., in press.

Brik, A., Keinan, E., Dawson, P.E. Protein synthesis by solid phase chemical ligation using a safety catch linker. J. Org. Chem. 65:3829, 2000.

Grynszpan, F., Keinan, E. Opsin shift in an aldolase antibody. Bioorg. Med. Chem. Lett. 9:2419, 1999.

Haskel, A., Keinan, E. Formation of a stable dihydridoalkyl Pt(IV) complex by water activation. Organometallics 18:4677, 1999.

Haskel, A., Keinan, E. Tris-pyrazolyl borate platinum methyl complex: A useful protecting group of electron-deficient acetylenes. Tetrahedron Lett. 40:7861, 1999.

Nimri, S., Keinan, E. Antibody-metalloporphyrin catalytic assembly mimics natural oxidation enzymes. J. Am. Chem. Soc. 121:8978, 1999.

Shulman, A., Shabat, D., Barbas, C.F. III, Keinan, E. Teaching catalytic antibodies to undergraduate students: An organic chemistry lab experiment. J. Chem. Educ. 76:977, 1999.

Shulman, A., Sitry, D., Shulman, H., Keinan, E. Highly efficient antibody-catalyzed deuteration of carbonyl compounds. J. Org. Chem., in press.

Shulman, H., Eberhard, A., Eberhard, C., Ulitzur S., Keinan, E. Highly sensitive and rapid detection of antibody catalysis by luminescent bacteria. Bioorg. Med. Chem. Lett. 10:2353, 2000.

Shulman, H., Keinan, E. Catalysis of the Kemp elimination by natural coals. Org. Lett. 23:3747, 2000.

Shulman, H., Keinan, E. Substrate-selective mechanisms in biocatalysis demonstrated with a versatile and efficient aldolase antibody. Bioorg. Med. Chem. Lett. 9:1745, 1999.

Shulman, H., Makarov, C., Ogawa, A.K., Romesberg, F., Keinan, E. Chemically reactive immunogens lead to functional convergence of the immune response. J. Am. Chem. Soc. 122:10743, 2000.

Sinha, A., Sinha, S.C., Sinha, S.C., Keinan, E. Total synthesis of trilobin. J. Org. Chem. 64:2381, 1999,

Sinha, S.C., Sinha, S.C., Keinan, E. Total synthesis of squamotacin. J. Org. Chem. 64:7067, 1999.