News and Publications
The Skaggs Institute For Chemical Biology
Scientific Report 1999-2000
Catalytic Antibodies: Organic Synthesis, Catalysis, and New Hapten Strategies
K.D. Janda, Y. Abe,* J.A. Ashley, O. Brümmer, R.A. Carrera, B. Clapham,
A. Cordova, C. Gao, H. Han, J. Hasserodt, T. Hoffman, S. Isomura, L. Jones, G.
Kaufmann, G.-T. Kim, R. Kubiak, D.M. Kubitz, M. Kubitz, K.-J. Lee, H. Liu, J.A.
López-Pelegrín, U.F. Mansoor, R. Manzotti, S. Mao, M. Matsushita,
G.P. McElhaney, K. Nicholas,** N.N. Reed, T. Reger, A. Simeonov, C. Spanka,***
J.D. Toker, P. Toy, M. Tremblay, A. Vaino, A.D. Wentworth, P. Wentworth, Jr.,
M. Wenz, P. Wirsching, B. Zhou, R.A. Lerner
* Fujisawa Pharmaceutical Co., Tsukuba, Japan
** University of Oklahoma, Norman, OK
*** Norvartis Pharma AG, Basel, Switzerland
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
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 Km 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 (Kd ~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.
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,
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 Lewisx and Lewisx. 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.
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.
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,