News and Publications
The Skaggs Institute For Chemical Biology
Scientific Report 1999-2000
Catalytic Antibodies, Synthetic Proteins, and Annonaceous Acetogenins
E. Keinan, F. Grynszpan,* H. Itzhaky, A. Haskel, S. Nimri, O. Levy, S. Saphier,
H. Shulman, A. Shulman, A. Yazbak, S.C. Sinha, P. Neogi, H. Avedissian, L.J.
D'Souza, S.F. Lu, C. Lo, A. Brik, P.E. Dawson**
* Department of Molecular Biology, The Scripps Research Institute
** Departments of Cell Biology and Chemistry, The Scripps Research Institute
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:
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 pKa 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
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 (kcat) and rate-enhancement
values (kcat/kun) 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.
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.
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.