Antibodies mimic natural oxidosqualene-cyclase action in steroid ring a formation

Article abstract of DOI:10.1021/ja993054l

Cationic cyclizations are among the most demanding reactions that have been catalyzed by antibodies. These studies provided valuable mechanistic insights while opening up the possibility of formation of steroidal carbon frameworks. However, they have involved substrates that contained an aryl sulfonate group adjacent to a primary carbon center not observed in natural cationic cyclization processes. This paper presents an extension of our earlier work, now focusing on substrates analogous to those seen in triterpene biosynthesis. Three antibodies, 15D6, 20C7, and 25A10, have been generated by immunization with an 4-aza-steroid aminoxide hapten (termed HA8) that initiate the cationic cyclization of an oxidosqualene derivative and catalyze the formation of ring A of the lanosterol nucleus at neutral pH. 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 a functional group at the head (epoxide trigger) and the tail (amide functionality) of the otherwise hydrophobic poly-ene chain. The assay involved solubilization of the substrates with 0.2% surfactant. 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 in the field of enzymic poly-ene cyclizations suggest that subtle changes in the substrate structure may well lead to multi-ring formation under the influence of the current catalyst.

Full text of DOI:10.1021/ja993054l

40
J. Am. Chem. Soc. 2000, 122, 40-45
Antibodies Mimic Natural Oxidosqualene-Cyclase Action in Steroid
Ring A Formation
Jens Hasserodt,* Kim D. Janda,* and Richard A. Lerner*
Contribution from the Department of Chemistry and The Skaggs Institute for Chemical Biology,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
ReceiVed August 23, 1999
Abstract: Cationic cyclizations are among the most demanding reactions that have been catalyzed by antibodies.
These studies provided valuable mechanistic insights while opening up the possibility of formation of steroidal
carbon frameworks. However, they have involved substrates that contained an aryl sulfonate group adjacent
to a primary carbon center not observed in natural cationic cyclization processes. This paper presents an extension
of our earlier work, now focusing on substrates analogous to those seen in triterpene biosynthesis. Three
antibodies, 15D6, 20C7, and 25A10, have been generated by immunization with an 4-aza-steroid aminoxide
hapten (termed HA8) that initiate the cationic cyclization of an oxidosqualene derivative and catalyze the
formation of ring A of the lanosterol nucleus at neutral pH. 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 a functional group at the head (epoxide trigger) and the tail (amide functionality) of the otherwise
hydrophobic poly-ene chain. The assay involved solubilization of the substrates with 0.2% surfactant. 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 in the field of enzymic poly-ene cyclizations suggest
that subtle changes in the substrate structure may well lead to multi-ring formation under the influence of the
current catalyst.
Introduction
frameworks. However, biomimetic mono- or sesquiterpenoid-
like substrates containing nonnatural leaving groups were used
in these preceding investigations. Consequently, two remaining
questions are whether antibodies are capable of cyclizing
triterpene substrates and whether they can trigger cyclization
starting from the natural leaving group, i.e., an epoxide oxygen.
Enzyme or catalytic-antibody poly-ene cyclizations can be
divided into three stages: initiation, propagation, and termina-
tion. Crystallographic data from an antibody-catalyzed tandem
cyclization have provided evidence that during propagation a
strategy similar to nature’s8 was adopted within the combining
site. This antibody mainly utilized aromatic amino acid residues
for the stabilization of high-energy cationic intermediates.9
During such an electrophilic addition event, precise orientation
of a π-quadrupole moment and close proximity of the neighbor-
ing double bond toward their respective carbocation leads to
an efficient decrease in activation energy that culminates in
barrierless collapse.10 The initiation event as observed with
oxidosqualene or squalene is believed to be triggered by an
acidic catalytic motif (his-asp for squalene-hopene cyclase,
SHC) which is buried at the bottom of the active cavity.
N-oxides can strongly bind to this motif as has been demon-
(6) (a) Hasserodt, J.; Janda, K. D.; Lerner, R. A. J. Am. Chem. Soc. 1997,
119, 5993-5998. (b) Hasserodt, J.; Janda, K. D. Tetrahedron 1997, 53,
11237-11256.
(7) Paschall, C.; Hasserodt, J.; Jones, T.; Lerner, R. A.; Janda, K. D.;
Christianson, D. W. Angew. Chem., Int. Ed. Engl. 1999, 38, 1743-1747.
(8) (a) Wendt, K. U.; Poralla, K.; Schulz, G. E. Science 1997 277, 1811-
1815. (b) Wendt, K. U.; Lenhardt, A.; Schulz, G. E. J. Mol. Biol. 1999
286, 175-187. (c) Starks, C. M.; Back, K.; Chappell, J.; Noel, J. P. Science
1997, 277, 1815-1820. (d) Lesburg, C. A.; Zhai, G.; Cane, D. E.;
Christianson, D. W. Science 1997, 277, 1820-1824.
(9) (a) Dougherty, D. Science 1996, 275, 1800-1804. (b) Lesburg, C.
A.; Caruthers, J. M.; Paschall, C. M.; Christianson, D. W. Curr. Op. Struct.
Biol. 1998, 8, 695-703.
(10) Jenson, C.; Jorgensen, W. L. J. Am. Chem. Soc. 1997, 119, 10846-
10854.
Poly-ene cyclizations are a fascinating class of biosynthetic
unimolecular rearrangement reactions.1 A large number of
cyclase enzymes accept their respective substrate out of a pool
of five natural candidates2 to generate the astonishing diversity
observed in the natural product class of the terpenes. A subclass
among those enzymes transforms the triterpene squalene or its
2,3-epoxy derivative into pentacyclic (bacteria and protozoae)
or, in the case of eukaryotes, either tetracyclic (via chair-boat-
chair folding of oxidosqualene) or tetra- or pentacyclic products
(via chair-chair-chair folding), respectively. Bacterial cyclases
are considered primitive due to their lack of complete substrate
and/or product specificity and the simple, thermodynamically
favored cyclization mechanism involved; in contrast, the eucary-
otic lanosterol synthase (OSC) has apparently reached maximum
possible fidelity for the disfavored processes it carries out.3,4
Cationic cyclizations are among the most demanding reactions
that have been catalyzed by antibodies.5 These reactions have
been studied mechanistically and recently the crystal structure
of an antibody, which catalyzes a tandem cationic cyclization,6
has been elucidated.7 The outgrowth of this work opened up
the possibility of catalytic formation of steroidal carbon
(1) Sacchettini, J. C.; Poulter, C. D. Science 1997, 277, 1788-1789.
(2) Geranyl-, farnesyl-, and geranylgeranyl pyrophosphate, squalene, and
2, 3-(S)-oxidosqualene.
(3) (a) Abe, I.; Rohmer M.; Prestwich, G. D. Chem. ReV. 1993, 93, 2189-
2206. (b) Ourrisson, G.; Rohmer, M.; Poralla, K. Ann. ReV. Microbiol. 1987,
41, 301-333.
(4) For current developments in nonenzymic poly-ene cyclizations, see:
(a) Sen, S. E.; Zhang, Y. z.; Smith, S. M. J. Org. Chem. 1998, 63, 4459-
4465. (b) Xing, X.; Demuth, M. Synlett 1999, S1, 987-989.
(5) (a) Li T.; Janda K. D.; Ashley J. A.; Lerner R. A. Science 1994,
264, 1289-1293. (b) Hasserodt, J.; Janda, K. D.; Lerner, R. A. J. Am. Chem.
Soc. 1996, 118, 11654-11655. (c) Li. T.; Janda, K. D.; Lerner, R. A. Nature
1996, 379, 326-327. (d) Li, T.; Lerner, R. A.; Janda, K. D. Acc. Chem.
Res. 1997, 30, 115-121.
10.1021/ja993054l CCC: $19.00 © 2000 American Chemical Society
Published on Web 12/18/1999

Catalytic Antibody Oxidosqualene-Cyclase Action
J. Am. Chem. Soc., Vol. 122, No. 1, 2000 41
Figure 1. Antibody HA8-25A10 was induced by lithocholic acid-
derived hapten 5 and stereoselectively catalyzed monocyclization of
oxidosqualene derivatives 1 and 2 at neutral pH in an aqueous
environment.
strated with various potent oxidosqualene cyclase inhibitors11
and ultimately seen through a crystal structure.8a
These combined observations coupled with results from
antibody-catalyzed intramolecular hydroxyl attack on epoxides12
led to the design and synthesis of optically pure hapten 5, which
is composed of a 4-aza-steroid skeleton derived from lithocholic
acid (Figure 1).13 Lithocholic acid was chosen as a convenient
starting material because it displays complete trans fusion
between its rings B, C, and D. The configuration in its derivative
5 mimics a chair-chair-chair folding of the poly-ene substrate
and thus represents a simpler cyclization mechanism compared
to the one that OSC controls. Hapten 5 was linked through its
“tail” (representing the latter part of the cyclization cascade) to
keyhole limpet hemocyanin (KLH) so that the N-oxide moiety
representing the initiation site would be most deeply buried in
the antibody combining site.14 The complete antigen was then
used to obtain a set of 25 monoclonal antibodies by standard
hybridoma protocol.
Figure 2. Synthesis of oxidosqualene-derived substrates.
by Ceruti et al.16c Subsequently, 6c was transformed into
substrate 1 with silver oxide. The various amide derivatives of
1 have been prepared by standard coupling procedures. With
the present substrate design, the risk of product inhibition is
likely to be low since formation of ring A of the steroid nucleus
bearing a 3-hydroxyl group will diminish resemblance to the
4-aminoxide hapten design. The assay conditions for initial
screening included solubilization of the hydrophobic substrates
with a surfactant and running the reactions in phosphate buffer
at neutral pH. The reactions were monitored simultaneously by
thin-layer chromatography (TLC) (staining with anisaldehyde)
and HPLC (210 nm). This dual assay system was necessary
since simple UV monitoring by HPLC only showed products
still containing two or three double bonds due to a low extinction
coefficient. For example, a tetracyclized product like lanosterol
derivative 9 or 10 could easily be TLC-detected in low
concentrations through staining while remaining invisible in
HPLC analyses coupled with UV detection.17
Results and Discussion
Initial testing for catalysis was carried out with two oxi-
dosqualene derivatives, 1 and 2, bearing two functional groups
at their head and tail. This dual-anchor design, which is also
displayed in hapten 5, is expected to significantly increase
substrate specificity while hydrophobic interactions should be
the main contributor to binding energy.15 The synthesis of 1
and 2 was accomplished as shown in Figure 2. Compound 6a
was obtained via the Curphey/van Tamelen procedure.16a
Protection as a dioxolane, another bromohydrine formation to
form 6b, deprotection, and hydrogen bromide elimination to
6c16b were carried out by a sequence similar to that published
(11) Cerutti, M.; Delprino, L.; Cattel, L.; Bouvier-Nave, P.; Duriatti, A.;
Schuber, F.; Benveniste, P. J. Chem. Soc., Chem. Commun. 1985, 1054-
1055.
(12) Janda, K. D.; Shevlin, C. G.; Lerner, R. A. Science 1993, 259, 490-
493.
(13) Hasserodt, J.; Janda, K. D.; Lerner, R. A. Bioorg. Med. Chem. In
press.
(14) Please note the reversal of substrate orientation when comparing
this strategy (and the strategy by SHC) with an earlier concept. See ref 3b.
Three antibodies (15D6, 20C7, and 25A10), all obtained from
immunization with hapten 5, were selected for their ability to
(15) Mader, M. M.; Bartlett, P. A. Chem. ReV. 1997, 97, 1281-1301.
(16) (a) van Tamelen, E. E.; Curphey, T. J. Tetrahedron Lett. 1962, 121-
124. (b) Xiao, Y.; Prestwich, G. D. J. Labelled Compd. Radiopharm. 1991,
29, 883-890. (c) Ceruti, M.; Rocco, F.; Viola, F.; Balliano, G.; Milla, P.;
Arpicco, S.; Cattel, L. J. Med. Chem. 1998, 41, 540-554.

42 J. Am. Chem. Soc., Vol. 122, No. 1, 2000
Hasserodt et al.
or chloroform. There, cyclization is observed, presumably via
concerted oxirane cleavage. Under the aqueous conditions
maintained in the antibody-catalyzed process, the presence of
an abundant nucleophile evidently favors a reaction path
involving water attack on the oxirane over anchimeric assistance
by the neighboring double bond (despite the fact that cyclization
would involve a thermodynamically favorable chair conforma-
tion). Also, the model reactions yielded complex mixtures of
products in addition to the desired cyclized product, ranging
from acyclic ketones to fluorohydrines.
Cyclohexenols 3a and 4a bearing an endocyclic 4,5-double
bond were the principal products (93%) formed by all three
catalysts from substrates 1 and 2, respectively (Figure 1).20 This
ring system represents ring A of the lanosterol skeleton.
Regioisomers 3b and 4b carrying an exocyclic double bond at
C4 were found as minor products (7%) (in Figure 3, the traces
for 3a and 3b are superimposed due to limitations of the column
used).21,22 Interestingly, compounds 3b and 4b feature the same
ring system as Achilleol A, biosynthesized by the plant Achillea
odorata (Figure 6, A).23
Any enantioselectivity by HA8-25A10 could not be deter-
mined by chiral HPLC. The optical antipodes of 3a were not
separated likely due to the incompatibility of the hydrophobic
analytes with the relatively hydrophilic commercial chiral phases
(e.g. amylose-based Chiralpak AD). Chiral GC on a â-cyclo-
dextrin column also failed because of the low volatility of the
methylated derivatives of 1 and 3. Thus, 3a,b was transformed
into its methyl ester with diazomethane and then into the (S)-
R-methoxy-R-trifluoromethyl-R-phenylacetic acid (MTPA) es-
ter.24 Proton NMR revealed only the presence of one major
enantiomer as judged by the dd signals for H1 (93:7 for esters
of 3a,b). This result was supported by fluorine NMR that
showed only one major signal and three insignificant ones.25
However, an unambiguous determination of the enantiomeric
excess was difficult due to the presence of 3b (the signals of
which cannot be distinguished reliably from those of 3a). Hence,
signals of the MTPA ester of 3b could be mistaken for a minor
amount of the MTPA diastereomer derived from the minor
enantiomer of 3a.
To garner information about possible enantiospecific substrate
consumption, two consecutive reactions were allowed to proceed
to approximately 15% and 50% conversion of racemic 1,
respectively (Figure 4). The remainder of 1 was recovered,
methylated as above, and hydrolyzed to the glycol by 3%
perchloric acid. The obtained glycol was then transformed
selectively into the (R)-MTPA ester via the secondary hydroxyl
group. Singlets a (1.181 ppm) and b (1.140 ppm) correspond
to the diastereotopic methyl groups and signal c (5.105 ppm)
to the chiral center of a single diastereomer. Multiplets d+d′
(4.995 ppm) are the superimposed signals of both diastereomers
corresponding to the neighboring olefinic position (determined
Figure 3. LC-MS chromatograms after 20 and 49 h of incubation.
Conditions: IgG HA8-25A10 (24.6 µM), substrate 1 (400 µM), room
temperature, 0.2% TWEEN 80, 50 mM PB, pH 7.0.
catalyze the initiation event. Comparatively large reactions (40
mL total volume, 17 µM IgG, 9 mg substrate) yielded enough
product (2.3 mg for 3a,b) to allow characterization by NMR
and LC-MS. High performance liquid chromatography (HPLC)
coupled with an electrospray mass spectrometer (ESI-MS) is
an ideal analytical system to monitor the antibody-catalyzed
reaction. Since an isomerization reaction is analyzed, m/z values
for substrates and products are the same, or, in the case of
quenching of the terminal carbocations, increase by 18. The
most prominant ions of interest were observed by Selected Ion
Monitoring (SIM), which greatly enhances the sensitivity of the
mass spectrometer for the detection of possible minor products.
Figure 3 shows two chromatograms that provide a window into
the 25A10-catalyzed transformation of 1 at different time
intervals. Comparison of the retention time of 3a,b with that of
substrate and background product (glycol) revealed that the
products have resulted from elimination as a termination
pathway. Antibody 25A10 appears to effectively exclude water
from the corresponding microenvironment in the active site as
programmed by the hapten. Despite the strictly neutral condi-
tions, substrate depletion due to hydrolysis to the corresponding
glycol is clearly observed but negligible.
(20) The proton NMR spectrum displayed signals at 0.84 (s, 3H, C2-
methyl), 0.97 (s, 3H, C2-methyl), and 3.47 ppm (dd, 1H, CHOH) consistent
with the spectrum of Lanosterol-derived acid 9 and amide 10. It also showed
a signal at 5.16 ppm (m, 3H) for the three unchanged isoprenoidal double
bonds and at 5.25 ppm (m, 1H) for the endocyclic double bond. The latter
chemical shift is in accord with spectra from products with similar double
bond substitution patterns in the literature.6
(21) Two characteristic proton NMR shifts for 3b were detected at 4.60
(1H) and 4.90 (1H) ppm, which is in accord with literature data.6,23,28
(22) The presence of a third, minor isomer with a double bond between
C3 and C4 was not observed.
No ring formation was detected in the absence of antibody.
This contrasts results of BF3-18 and CCl3COOH-catalyzed19
transformations of a model compound (2,3-oxido-2,6-dimeth-
ylhept-6-ene) in organic solvents such as benzene, diethyl ether,
(28) Barrero, A. F.; Manzaneda, E. A.; Manzaneda, R. A. Tetrahedron
1990, 46, 8161-8168.
(17) See Supporting Information for synthesis scheme and NMR
characterization.
(18) Goldsmith, D. J. J. Am. Chem. Soc. 1962, 84, 3913-3918.
(19) Corey, E. J.; Cheng, H.; Baker, C. H.; Matsuda, S. P. T.; Li, D.;
Song, X. J. Am. Chem. Soc. 1997, 119, 1277-1288.
(29) (a) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512-
519. (b) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092-4096.
(30) See Supporting Information.

Catalytic Antibody Oxidosqualene-Cyclase Action
J. Am. Chem. Soc., Vol. 122, No. 1, 2000 43
Figure 4. Determination of substrate enantiospecificity. Analysis was performed on remaining 1 after ca. 15% (spectrum 2: 9.0 mg) and ca. 50%
(spectrum 3: 1.5 mg) consumption of the racemic substrate by the antibody. Spectrum 1: Authentic material. The remaining substrate was prepared
1
for H-NMR analysis by methylation (CH2N2), hydrolysis (HClO4), and transformation into its (R)-MTPA ester. Enlarged regions are from 1.12-
1.25 and 4.90-5.25 ppm.
Figure 5. Lineweaver-Burk plot and Michaelis-Menten diagram
(inset) of the reaction of IgG HA8-25A10 with substrate 1. Condi-
tions: [IgG] ) 5 µM; 37 °C; 0.2% TWEEN 80; 50 mM phosphate
buffer, pH 7.0; [1] ) 100, 200, 400, 600, 800 µM. Complete saturation
of the antibody was not possible due to solubility limits of 1. Inset
shows curves at hapten/inhibitor concentrations: [5] ) 0.0, 5.0, 7.5,
10.0 µM.
Figure 6. Monocyclization of oxidosqualene as part of a regular
metabolic pathway (A) and as a result of an imperfect fit of a nonnatural
substrate of oxidosqualene-lanosterol cyclase (B).
approximately 50% conversion. Further analysis of the (R)- and
(S)-MTPA esters24b derived from the minor enantiomer in the
recovered substrate confirmed that (S)-1, in accordance with
the absolute configuration of 5, is the true substrate of HA8-
25A10. If the transition state adopts a chair conformation, then
it can be assumed that products 3a,b show (1S,3R)-configuration
as seen in the monocyclic products shown in Figure 6.
by H,H-COSY). The decreasing intensities for one signal set
unequivocally prove the preferred consumption of one enanti-
omer as seen with natural OSC.19 In contrast, certain SHCs have
been observed to accept both enantiomers of oxidosqualene,
their nonnatural substrate.3 Spectrum 3 allowed the estimation
of an enantiomeric excess of 80% for the residual substrate after
Kinetic analysis was carried out with the most proficient
catalyst (HA8-25A10) and its best substrate (1). This catalyst

44 J. Am. Chem. Soc., Vol. 122, No. 1, 2000
Hasserodt et al.
was initially tested for optimal performance with a variety of
commercial surfactants.25 HA8-25A10 showed maximum rates
with TWEEN 80 and decanoyl-N-methylglucamide. The latter
was dropped due to its low solubilizing power and high cost.
All others impaired catalysis by various degrees with lauryl sul-
fobetaine as the only ionic detergent almost destroying catalytic
activity. Cosolvents such as DMF and DMSO were ruled out
for their significant reduction of catalytic rate as well as their
strong absorption at 210 nm during HPLC assays where these
solvents tended to bury the signals in question.
interactions and thus adding additional force to the folding of
the poly-ene chain.
Conclusion
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 needs particular
enzymic assistance in biosynthetic oxidosqualene cyclization19
has been catalyzed by an antibody. However, propagation of
cyclization for this particular class of substrates was not
observed. Can multicyclization be accomplished by use of these
catalytic antibodies? A recent finding showed that OSC produces
a monocyclic product exclusively when charged with an 2,3-
oxidosqualene analogue carrying two ethyl groups in place of
the original methyls at C10 and C15 (Figure 6, B).28 Evidently,
minute changes in the substrate substitution pattern can dictate
a productive versus a nonproductive conformation for the poly-
ene chain in the active site. Consequently, the lack of consti-
tutional congruency between the design of hapten 5 and
substrates 1 and 2 may well be the cause for antibody HA8-
25A10 to only produce monocyclic products. Theoretically, the
best match to the hapten would include a hydrogen substituent
at C10 in the substrate.29 The single substitution at this location
with a methyl group likely prevented productive binding for
propagation. Fifty-three years ago, the equivalent for this
conclusion was pointedly expressed by Linus Pauling for
antibody-antigen recognition: "..., but that interference is
caused [between a haptenic group and its antibody] by replacing
a hydrogen atom (radius 1.2 Å) by a methyl group."30
IgG HA8-25A10 showed saturation kinetics with Vmax
)
0.172 µM min-1, kcat ) 0.017 min-1, KM ) 387 µM and a
catalytic efficiency kcat/KM ) 44 M-1 min-1 (taking into account
half the concentration of racemic substrate employed) (Figure
5). For comparison, only a few studies reporting kinetic
parameters of triterpene cyclases are available to date. These
tend to vary significantly because eukaryotic oxidosqualene
cyclases are notoriously unstable during purification. While kcat
) 3.38 min-1 and Km ) 18 µM were found for the cyclase of
a yeast strain,19 another report quotes a kcat ) 0.003 min-1 and
a similar Km ) 15 µM.26 Also, a recent study characterized
squalene-hopene cyclase (SHC) from the thermoacidophilic
prokaryote Alicyclobacillus acidocaldarius that catalyzes a
pentacyclization. The reaction proceeds by a simpler mechanism
without any group migrations. This enzyme exhibits a kcat of
300 min-1 and a Km of 16.7 µM for squalene at its optimal
temperature of 60 °C.27 Naturally, caution has to be taken when
comparing the values exhibited by HA8-25A10 to the ones
above, since a monocyclization is weighed against a multicy-
clization. At this point, the performance of the antibody and
the triterpene cyclases may only be comparable in the substrate
binding mode (including diffusion and the folding process) and
possibly the mechanism of initiation. Future investigations will
be used to unravel potentially important residues and the
mechanism involved.
Experimental Section
1
General. H NMR (500 MHz) and (13C NMR (125 MHz) spectra
were recorded on a Bruker AMX-500 instrument. Chemical shifts (δ)
1
are given in ppm relative to CHCl3 in CDCl3 (7.27 ppm, H; 77.00
ppm, 13C). High-resolution mass spectra (HRMS) were recorded at The
Scripps Research Institute on a VG ZAB-ZSE mass spectrometer under
fast atom bombardment (FAB) conditions. LC-MS analyses were
performed on a Hewlett-Packard LC/MSD 1100 instrument.
Hapten 5 competitively inhibited the catalytic antibody with
a Ki of 9 µM, demonstrating that the combining site carries the
catalytic activity (Figure 5). Since the overall scaffolding in
hapten 5 mimics propagation there is a certain risk of product
inhibition in the case of multicyclization. The presence of
lanosterol derivatives 9 and 10, bearing the same tail function-
alities as seen in substrates 1 and 2, in the assay mixture did
not result in any inhibition. Thus, it is reasonable to assume
that 9 and 10 do not have a significant affinity toward HA8-
25A10. However, only an experiment in the presence of a
multicyclized product derived from lithocholic acid (but display-
ing the same substituents and configuration as ring A in
lanosterol) will ultimately yield whether hapten 5 is sufficiently
distinct from the desired products.
Two other derivatives of 2 were also tested with antibody
HA8-25A10. While anilide 7 was not transformed, first experi-
ments with nitroanilide 8, featuring an aminohexanoate spacer,
indicated it to be a substrate (Figure 2). This supports the
conclusion that hapten 5 is buried entirely in the combining
site, as was targeted by using a particularly long linker in this
study. Additionally, the natural triterpene substrate 2,3-oxi-
dosqualene in its racemic form was found not to be transformed
by the antibody underlining the importance of the amide group
in 1 and 2. These results are in accord with the true aim of
hapten design 5, namely to induce a combining site that would
specifically recognize the “head” and “tail” of the substrate
thereby assisting the presumably less-specific hydrophobic
Reactions were monitored by thin-layer chromatography (TLC),
using 0.25 mm Merck Silicagel Glass Plates (60F-254), fractions being
visualized by staining with p-anisaldehyde with subsequent heat
application. Column chromatography was carried out with Mallinckrodt
SilicAR 60 silicagel (40-63 µm). Reagent grade solvents for chro-
matography were obtained from Fisher Scientific. Reagents and
anhydrous solvents were obtained from Aldrich Chemical Co. and used
as is. Reported yields were determined after purification to homoge-
neous material.
22,23-Epoxy-1,1′,2-trisnorsqualene-3-carboxylic Acid (1). A solu-
tion of AgNO3 (20 mg in 0.38 mL H2O) was added to a solution of
NaOH (21 mg in 0.38 mL H2O) with stirring. After 5 min, 22,23-
epoxy-1,1′,2-trisnorsqualene-3-aldehyde 6c16b (47 mg, 0.117 mmol) in
1.6 mL of THF was added to the suspension of black Ag2O in one
portion. After 40 min, 0.19 mL of a fresh suspension of Ag2O was
prepared according to the method above and added to the reaction
mixture. Eighty minutes later, TLC indicated the reaction to be
complete. Citric acid (5%) was added, and the mixture was extracted
several times with ether. The combined organic phases were washed
with brine and dried with MgSO4, and the residue was purified by
silicagel chromatography (hexanes/ethyl acetate 8:1 f 5:1, Rf ) 0.27
(27) (a) Feil, C.; Poralla, K. Eur. J. Biochem. 1996, 242, 51-55. (b)
Sato, T.; Kanai, Y.; Hoshino, T. Biosci. Biotechnol. Biochem. 1998, 62,
407-411.
(28) Hoshino, T.; Ishibashi, E.; Kaneko, K. J. Chem. Soc., Chem.
Commun. 1995, 2401-2402.
(29) This hypothesis will now be tested by synthesizing substrates
displaying various methylation patterns to further probe antibody HA8-
25A10.
(26) Moore, W. R.; Schatzmann, G. L. J. Biol. Chem. 1992, 267, 22003-
22006.
(30) Pauling, L. Chem. Eng. News 1946, 24, 1375-1377.

Catalytic Antibody Oxidosqualene-Cyclase Action
J. Am. Chem. Soc., Vol. 122, No. 1, 2000 45
Hybridoma Production. See ref 5b.
1
[2:1], stains gray-blue). Yield: 28 mg (0.067 mmol, 57%). H NMR
(CDCl3): 1.27 (3H, s), 1.31 (3H, s), 1.60 (3H, s), 1.61 (3H, s), 1.62
(6H, s), 1.63 (2H, m), 1.96-2.03 (8H, m), 2.06-2.11 (4H, m), 2.15
(2H, m), 2.31 (2H, m), 2.45 (2H, m), 2.74 (1H, t), 5.14-5.19 (4H, m).
13C NMR (CDCl3): 16.0, 18.7, 24.8, 26.5, 26.6, 27.4, 28.2, 32.9, 34.3,
36.3, 39.5, 39.7, 58.6, 64.3, 124.3, 124.4, 124.9, 125.3, 132.9, 133.9,
134.8, 135.0, 178.8. HRMS (FAB, NBA/NaI) calcd for C27H44O3 (M
+ Na+) 439.3188; found 439.3173.
Methylamide 2. A solution of 1 (134 mg, 322 µmol) in dry DMF
(2.7 mL) was treated with methylamine hydrochloride (21.7 mg, 322
µmol), HATU (135 mg, 354 µmol), and diisopropylethylamine (DIEA)
(167 mg, 1288 µmol, 4 equiv). After the mixture was stirred for 3 h at
room temperature, 1 N citric acid was added, the mixture was extracted
with ether, and the combined organic phases were extracted with water
and brine and then dried over MgSO4. Column chromatography
(hexanes/ethyl acetate 6:1 f pure ethyl acetate, Rf ) 0.20 [1:1], stains
light brown) yielded a colorless oil (112 mg, 81%). 1H NMR (CDCl3):
1.27 (3H, s), 1.31 (3H, s), 1.61 (3H, s), 1.64 (3H, s), 1.60-1.70 (6H,
m), 1.96-2.03 (8H, m), 2.06-2.11 (4H, m), 2.15 (2H, m), 2.28-2.32
(4H, m), 2.71 (1H, t), 2.80 (3H, d), 5.10-5.21 (4H, m), 5.54 (1H, s,
broad). HRMS (FAB, NBA/NaI) calcd for C28H47NO2 (M + H+)
430.3685; found 430.3704.
Anilide 7. Same procedure as for 2, involving 1 (40 mg, 96 µmol),
aniline (9 mg, 96 µmol), HATU (40 mg, 106 µmol), and DIEA (50
mg, 384 µmol) in dry DMF (800µL). Column chromatography
(hexanes/ethyl acetate 20:1 f 3:1, Rf ) 0.42 [1:1], stains gray-brown)
yielded a colorless oil (29 mg, 61%). 1H NMR (CDCl3): 1.39 (3H, s),
1.59 (3H, s), 1.60 (3H, s), 1.62 (3H, s), 1.64 (3H, m), 1.96-2.03 (8H,
m), 2.06-2.11 (4H, m), 2.15 (2H, m), 2.39-2.46 (4H, m), 2.72 (1H,
t), 5.13-5.16 (3H, m), 5.24 (1H, t), 7.09 (1H, t), 7.30 (2H, t), 7.50
(2H, d), 7.63 (1H, s, broad). HRMS (FAB, NBA/NaI) calcd for C33H49-
NO2 (M + Na+) 514.3661; found 514.3673.
Nitroanilide 8. Same procedure as for 2, involving 1 (147 mg, 353
µmol), 6-aminocaproic p-nitroanilide25 (93 mg, 394 µmol), HATU (161
mg, 106 µmol), and DIEA (102 mg, 788 µmol) in dry DMF (3.2 mL).
Column chromatography (hexanes/ethyl acetate 10:1 f pure ethyl
acetate, Rf ) 0.38 [ethyl acetate]) yielded a colorless oil (53 mg, 23%).
1H NMR (CDCl3): 1.26 (3H, s), 1.30 (3H, s), 1.38 (2H, m), 1.54 (2H,
m), 1.59 (9H,m), 1.61 (3H, s), 1.62 (1H, m), 1.76 (2H, m), 1.94-1.20
(8H, m), 2.04-2.08 (5H, m), 2.15 (1H, m), 2.29 (4H, s), 2.42 (3H, t),
2.72 (1H, t), 3.25 (2H, q), 5.12-5.16 (4H, m), 5.94 (1H, t, broad),
7.80 (2H, d), 8.17 (2H, d), 9.07 (1H, s, broad). 13C NMR (CDCl3):
16.0, 18.7, 24.6, 24.9, 26.6, 27.4, 28.2, 29.1, 35.4, 36.3, 37.0, 39.0,
39.5, 39.6, 58.5, 64.3, 119.0, 124.3, 124.4, 124.9, 125.5, 133.3, 133.9,
134.8, 135.0, 143.0, 144.6, 172.2, 173.4.
Screening of Anti-5 Antibodies. All 25 clones were first tested in
reactions with a total volume of 200 µL in microvials. These consisted
of a 50 µL stock solution of substrate (800 µM in 50 mM phosphate
buffer PB, pH 7.0, 0.2% Triton-X 100), varying amount of IgG stock
solution depending on individual clone (in PB), and PB (remaining
volume to complete total). Final concentrations: [IgG] ) 16.5 µM;
[S] ) 200 µM. After incubation (48 h, 37 °C), all reactions were directly
analyzed by HPLC (Phenomenex Luna 3µ C8(2) 150 × 4.6 mm;
isocratic at CH3CN/H2O 85/15 (0.1% TFA); 210 nm). IgGs that showed
substrate depletion were reintroduced into 2 mL reactions with the same
concentrations as above. The samples obtained by extraction with 2
mL of CHCl3, centrifugation at 3000 rpm for 10 min to separate phases,
and evaporation of the solvent from the organic phases were spotted
on TLC plates and analyzed in ethyl acetate/ethanol 9:1. For substrate
1, anisaldehyde stained background product (glycol) brown, products
blue, and substrate brown again (in the order of increasing Rf values).
LC-MS. Reactions of HA8-25A10 with substrate 1 were analyzed
with Phenomenex Luna 3µ C18(2) 30 × 2.00 mm, 0.8 mL/min flow,
32 °C, gradient [“A” ) H2O, 0.05% TFA]/[“B” ) CH3CN, 0.05% TFA]
50% B-1 min-60% B-6 min-80% B, then 100% B. MS detector
was set to 399.5 (M - H2O + H+), 417.5 (M + H+) and 435.5 (M +
H2O + H+) (SIM).
Scale-Up for Identification of Product 3a,b and Its Transforma-
tion into the MTPA Ester. Reaction setup: 40 mL total volume; 10
mL HA8-25A10 (10.3 mg/mL) in PB (50 mM, pH 7.0); 0.858 mL
substrate 1 (25 mM, 8.92 mg) in PB (15% Triton-X 100); 29.1 mL PB
(50 mM, pH 7.0); 37 °C. After 4 days, no further progress of the
reaction could be detected. The slightly turbid solution was then
extracted with CHCl3, phase separation was achieved by fitration
through Celite, the organic phase was dried (MgSO4), treated with an
ether solution of CH2N2 for 20 min, and evaporated. The residue was
passed through a short silicagel column with the aid of ethyl acetate to
remove the detergent. A second column then provided methylated
products 3a,b (hexanes/ethyl acetate 10:1 f 3:1, Rf ) 0.33 [3:1], stains
blue). Yield: 2.3 mg (5.35 µmol, 50%, calculated for consumption of
(S)-1) and ca. 5 mgof methylated 1 (82% recovered material, remainder
1
consisted of the glycol that is formed in the background reaction). H
NMR (3a,b, CDCl3): 0.84 (3H, s), 0.98 (3H, s), 1.57-1.73 (12H, m),
1.97-2.08 (14H, m), 2.12-2.24 (1H, m), 2.30 (2H, m), 2.40 (2H, m),
3.47 (1H, dd), 3.67 (3H, s), 4.61 (1H, m, 3b), 4.88 (1H, m, 3b), 5.15
(3H, m), 5.25 (1H, m). This material was subsequently transformed
into its (S)-MTPA ester with (R)-(-)-R-methoxy-R-(trifluoromethyl)-
phenyl acid chloride according to a literature procedure.24a HRMS for
the MTPA ester of 3a,b (FAB, NBA/NaI) calcd for C38H53F3O5 (M +
Na+) 669.3743, found 669.3773.
Tris-nor-lanosterol-24-carboxylic Acid (9).31 This already pub-
lished compound has been synthesized from commercial lanosterol
(TCIAmerica No. C0427) by a novel procedure outlined in the
Acknowledgment. Financial support was provided by The
Scripps Research Institute (J.H.), The National Institutes of
Health (GM-43858), The Skaggs Institute for Chemical Biology
(K.D.J.). Help with the hybridoma work by Ping Fan and Alisa
Moore, as well as expert assistance with the LC-MS analyses
by Geoffrey Barker and Dr. Gary Siudzak, is gratefully
acknowledged.
1
Supporting Information. H NMR (CD3OD): 0.73 (3H, s), 0.80 (3H,
s), 0.90 (3H, s), 0.93 (3H, d), 0.97 (3H, s), 1.00 (3H, s), 2.23 (1H, m),
3.14 (1H, dd). The proton NMR spectrum is in accord with that in the
literature.31 HRMS (FAB, NBA/NaI) calcd for C27H44O3 (M + Na+)
439.3188; found 439.3177.
N-Methyl-tris-nor-lanosterol-24-carboxamide (10). This com-
pound has been synthesized according to the procedure for methylamide
2: 3-Acetyl-tris-nor-lanosterol-24-carboxylic acid25 (77 mg, 168 µmol),
methylamine hydrochloride (11.8 mg, 174 µmol), HATU (73 mg, 192
µmol), DIEA (90.5 mg, 698 µmol, 4 equiv), and 1.5 mL of anhydrous
DMF. Column chromatography (hexanes/ethyl acetate 5:1 f ethyl
acetate, Rf ) 0.40 [ethyl acetate], stains gray-blue) yielded 68 mg (86%)
of product. This material was then deacylated by refluxing it for 10
min in 9 mL of MeOH (containing 230 mg NaOH). Rf(10) ) 0.37,
Supporting Information Available: A listing of proton/
carbon NMR spectra of 1 and 2 and their synthetic intermediates,
of 7-10, proton NMRs of antibody products 3a,b, its (S)-MTPA
ester, 4a,b, determination of absolute configuration of true
substrate (1), detergent evaluation, typical LC-MS and conven-
tional HPLC chromatograms of the antibody-catalyzed reaction
as well as product distribution (PDF). This material is available
free of charge via the Internet at http://pubs.acs.org.
1
with tailing [ethyl acetate], stains gray-blue also. H NMR (CDCl3):
2.24 (1H, m), 2.80 (3H, d), 3.23 (1H, dd), 5.51 (1H, s, broad). LRMS
(FAB, NBA/NaI) calcd for C28H47NO2 (M + Na+) 453; found 453.
(31) Bernassau, J. M.; Fetizon, M. Synthesis 1975, 795-796 and
references therein.
JA993054L