Selective antibody-catalyzed solvolysis of endo-2-norbornyl mesylate [9]

Full text of DOI:10.1021/ja990896b

J. Am. Chem. Soc. 1999, 121, 10227-10228
Scheme 1
10227
Selective Antibody-Catalyzed Solvolysis of
endo-2-Norbornyl Mesylate
Lifu Ma, Elizabeth H. Sweet, and Peter G. Schultz*
Department of Chemistry, The Scripps Research Institute,
0550 North Torrey Pines Road, La Jolla, California 92037
1
ReceiVed March 19, 1999
1
2
Extensive experimental and computational data have now
firmly established that endo-2-norbornyl mesylate 1 undergoes
solvolysis through an ionization process to initially form an
unsymmetrical localized (classical) cation 3, which is converted
by the participation of the C1-C6 sigma bond to the more stable
nonclassical cation 4. The nonclassical cation is calculated to be
3
more stable than the classical cation by about 6 kcal/mol. The
bridged intermediate 4, involving delocalization of σ electrons,
has a plane of symmetry and is achiral. Attack by water at C-1
or C-2 results in equal amounts of the enantiomeric exo-norbornyl
Scheme 2
4
alcohols 5 (Scheme 1). The norborneol product is exclusively
5
exo since reaction occurs from the direction opposite of that of
the bridging interaction. The rate-determining step for the
6
solvolysis reaction involves the formation of transition state 2.
7
Antibodies elicited to an analogue of this transition state might
be expected to sufficiently stabilize the initially formed localized
cation 3 relative to the nonclassical ion 4 or slow the conversion
of the former to the latter, such that reaction predominantly occurs
from the classical cation 3. Alternatively, the asymmetrical
antibody combining site could differentiate the C-1 and C-2
positions of the nonclassical ion 4 so that they are attacked by
nucleophile with unequal facility, resulting in enantiomerically
enriched exo-2-norborneol. In either case, the antibody-catalyzed
reaction would give products characteristic of the “classical”
norbornyl cation.
albumin via a five carbon linker. A total of sixteen monoclonal
antibodies specific for 6 were obtained by standard methods.
9
Twelve of the antibodies were found by ELISA assays to bind
racemic hapten 6 and were subsequently purified by protein G
affinity chromatography and analyzed for catalytic activity. The
solvolysis of racemic substrate 1 was carried out with or without
antibodies at 22 °C in aqueous 10 mM phosphate, 100 mM NaCl,
pH 7.4 buffer (PBS). The reaction mixture was quenched by rapid
extraction with ethyl acetate and quantitatively assayed by gas
chromatography (GC)10 with toluene as the internal standard. One
antibody (15M3) showed a significant rate enhancement over the
uncatalyzed reaction in an initial screen for 2-norborneol product.
Only the exo-product was isolated, and no endo-2-norborneol was
detected.10 This result is consistent with a C-O versus S-O bond
To test this notion, we generated antibodies to an analogue (6)
of transition state 2 that mimics both the developing positive
charge on the C-2 position and the developing negative charge
of the sulfonate leaving group.7 The synthesis of racemic hapten
,8
6
was carried out as shown in Scheme 2; hapten 6 was linked to
the carrier proteins keyhole limpet hemocyanin and bovine serum
(1) (a) Olah, G. A.; Schleyer, P. v. R. Carbonium Ions; Wiley-Inter-
science: New York, 1992. (b) Brown, H. C.; Schleyer, P. v. R. The
Nonclassical Ion problem; Plenum Press: New York, 1977. (c) Grob, C. A.
Acc. Chem. Res. 1983, 16, 426-431. (d) Brown, H. C. Acc. Chem. Res. 1983,
1
6, 432-440. (e) Olah, G. A.; Prakash, G. K. S.; Saunders, M. Acc. Chem.
Res. 1983, 16, 440-448. (f) Walling, C. Acc. Chem. Res. 1983, 16, 448-
11
4
54.
cleavage mechanism. Antibody 15M3 was further purified by
ion-exchange (Mono Q) chromatography and characterized in
detail.
(
2) (a) Schleyer, P. v. R.; Sieber, S. Angew. Chem., Int. Ed. Engl. 1993,
3
2, 1606-1608. (b) Sieber, S.; Schleyer, P. v. R.; Vancik, H.; Mesic, M.;
Sunko, D. E. Angew. Chem., Int. Ed. Engl. 1993, 32, 1604-1606. (c) Kirmse,
W.; Herpers, E. Angew. Chem., Int. Ed. Engl. 1991, 30, 1018-1020. (d) Koch,
W.; Liu, B.; DeFrees, D. J.; Sunko, D. E.; Vancik, H. Angew. Chem., Int. Ed.
Engl. 1990, 29, 183-185. (e) Kirmse, W.; Minkner, D. Angew. Chem., Int.
Ed. Engl. 1993, 32, 385-387.
The first-order rate constants and kinetic parameters for both
the uncatalyzed and antibody-catalyzed reactions were derived
from the initial velocities by using the computer program
(
3) (a) Solomon, J. J.; Field, F. H. J. Am. Chem. Soc. 1976, 98, 1567-
1
569. (b) Schleyer, P. v. R.; Chandrasekhar, J. J. Org. Chem. 1981, 46, 225-
27. (c) Farcasiu, D. J. Org. Chem. 1981, 46, 223-225.
(9) Kohler, G.; Milstein, C. Nature 1975, 256, 495-497.
2
(10) Gas chromatographic separation of the product mixture of solvolysis
reactions was carried out on a methyl silicone capillary column (HP-1, 25 m
× 0.2 mm) with a Hewlett-Packard 5890 Series II gas chromatograph. GC
conditions: injector temperature ) 245 °C, detector temperature ) 250 °C,
oven temperature, initially at 50 °C for 6 min and then raised to 200 °C at 20
°C/min. The retention times for exo-2-nornorneol 5, endo-norbornyl mesylate
1, and the internal standard toluene are 8.9, 13.1, and 2.9 min, respectively.
Endo-2-norborneol (Aldrich) has a retention time of 9.3 min under these
conditions. All compounds are stable under these conditions, and no
decomposition of materials was observed.
(
4) (a) Winstein, S.; Trifan, D. S. J. Am. Chem. Soc. 1949, 71, 2953-
2
1
1
954. (b) Winstein, S.; Trifan, D. S. J. Am. Chem. Soc. 1952, 74, 1147-
154. (c) Winstein, S.; Trifan, D. S. J. Am. Chem. Soc. 1952, 74, 1154-
160.
(
5) Nevell, T. P.; Salas, E.; Wilson, C. L. J. Chem. Soc. 1939, 1188-
199.
6) Brown, H. C.; Ravindranathan, M.; Rao, C. G.; Chloupek, F. J.; Rei,
1
(
M.-H. J. Org. Chem. 1978, 43, 3667-3678.
(
(
7) Schultz, P. G.; Lerner, R. A. Science 1995, 269, 1835-1842.
8) (a) Li, T.; Lerner, R. A.; Janda, K. D. Acc. Chem. Res. 1997, 30, 115-
(11) (a) Veeravagu, P.; Arnold, R. T.; Eigenmann, E. W. J. Am. Chem.
Soc. 1964, 86, 3072-3075. (b) Tsuji, Y.; Kim, S. H.; Saeki, Y.; Yatsugi, K.;
Fujio, M.; Tsuno, Y. Tetrahedron Lett. 1995, 36, 1465-1468. (c) Schreiner,
P. R.; Schleyer, P. v. R.; Schaefer, H. F. J. Org. Chem. 1997, 62, 4216-
4228. (d) Lee, W. H.; Maskill, H.; Menneer, I. D. J. Chem. Soc., Chem.
Commun. 1993, 503-504. (e) Bentley, T. W.; Bowen, C. T.; Brown, H. C.;
Chloupek, F. J. J. Org. Chem. 1981, 46, 38-42.
1
1
21. (b) Shokat, K. M.; Leumann, C. J.; Sugasawara R.; Schultz, P. G. Nature
989, 338, 269-271. (c) Reymond, J.-L.; Janda, K. D.; Lerner, R. A. Angew.
Chem., Int. Ed. Engl. 1991, 30, 1711-1713. (d) Yu, J.; Hsieh, L. C.;
Kochersperger, L.; Yonkovich, S.; Stephans, J. C.; Gallop, M. A.; Schultz, P.
G. Angew. Chem., Int. Ed. Engl. 1994, 33, 339-341. (e) Li, T.; Janda, K. D.;
Lerner, R. A. Nature 1996, 379, 326-328.
1
0.1021/ja990896b CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/16/1999

10228 J. Am. Chem. Soc., Vol. 121, No. 43, 1999
Communications to the Editor
enantiomerically pure. The enantiomeric enrichment of exo-2-
norborneol from the antibody-catalyzed reaction was further
confirmed by the determination of optical rotations. While the
product from the uncatalyzed solvolysis reaction displays no
20
optical activity, the specific rotation [R] for the exo-2-norborneol
from the antibody-catalyzed reaction was determined to be +
2
3
.97° (CHCl , c 0.5). In comparison to the reported specific
15
25
rotations for (+)-(1R,2R,4S)-5 {[R] ) +3.06° (CHCl
and (-)-(1S,2S,4R)-5 {[R] ) -3.14° (CHCl
3
, c 3)}
25
3
, c 3.1)}, respec-
tively, the antibody-catalyzed solvolysis reaction of racemic 1
afforded (+)-(1R,2R,4S)- and (-)-(1S,2S,4R)-5 (98:2 ratio).
To determine whether the antibody-catalyzed reaction occurs
through an S
N N
L or S 2 substitution mechanism, we synthesized
R-D
a trideuterated substrate (7). The kinetic isotope effect
(V/
R-H
K)/ (V/K) was measured by direct determination of the ratios
of labeled to unlabeled substrate and product in a mixture by mass
spectroscopy.16 The observed isotope effect for the uncatalyzed
solvolysis reaction is 1.15 ( 0.03, while the antibody-catalyzed
(PBS, 22 °C) reaction affords an isotope effect of 1.33 ( 0.04,
based on four determinations. A secondary isotope effect of 1.15
for the uncatalyzed reaction is consistent with formation of a
nonclassical ion (Scheme 1).17 The kinetic isotope effect of 1.33
for the antibody-catalyzed solvolysis reaction is also consistent
Figure 1. Comparison of the product distribution from the antibody-
catalyzed and uncatalyzed solvolysis reactions: (A) 500 MHz H NMR
1
3
spectrum (in CDCl ) of the (R)-Mosher’s ester from exo-2-norborneol
isolated from the antibody-catalyzed solvolysis of racemic 1 and (B) 500
1
3
MHz H NMR (in CDCl ) of the (R)-Mosher’s ester from exo-2-
norborneol isolated from the uncatalyzed solvolysis of 1. Insets are
expanded signals of the nonequivalent 1-hydrogen and 4-hydrogen
N
with an S 1-type mechanism, but one in which greater rehybrid-
1
9
groups in the norbornyl moiety for diastereomeric (R)-Mosher’s esters.
ization occurs at the 2 position of substrate 1 relative to the
uncatalyzed reaction, possibly due to enhanced stabilization of
the classical cation.
DYNAFIT.12 The background rate of solvolysis of mesylate 1
-
4
-1
Antibody 15M3 not only shows a high degree of substrate
specificity; it also yields products characteristic of the classical
2-norbornyl cation. This might arise from selective stabilization
of the cation relative to the nonclassical ion in an asymmetric
antibody combining site. Alternatively, the antibody might slow
the rate of conversion of the initially formed C-2 cation to the
nonclassical ion relative to the rate of trapping by solvent. If the
antibody-catalyzed solvolysis reaction is occurring through to the
nonclassical cation intermediate, then the trajectory of solvent
attack is controlled such that reaction is favored at the C-2 position
of the achiral nonclassical cation (4).18 In any case this study
demonstrates that antibodies can control the energetics of complex
reaction coordinates to a remarkable degree.
was determined to be 9.0 × 10 min at 22 °C in PBS buffer.
Antibody-catalyzed solvolysis of 1 was found to follow Michae-
-
1
m
lis-Menten kinetics with a kcat of 1.7 min , a K of 1.5 mM,
and a resulting kcat/kuncat of 1900. Furthermore, the antibody-
catalyzed reaction was inhibited by hapten 6: least-squares Dixon
analysis afforded a K
significantly lower than that of the K
i
value of 9.2 ( 0.5 µM. The K
i
value is
, indicating that the antibody
m
preferentially binds to the charged transition-state analogue 6
relative to substrate 1. The conversion rate of enantiomerically
pure (1S,2R,4R)-113 to exo-2-norborneol at 22 °C in the presence
of 14 µM antibody 15M3 was identical to the rate of the
background reaction, demonstrating that only the (1R,2S,4S)-1
enantiomer is a substrate for the antibody-catalyzed reaction. The
antibody-catalyzed reaction was found to be insensitive to pH
variations (pH 6.5-8.5).
Acknowledgment. This work was supported by the National Institutes
of Health and a postdoctoral fellowship from the Cancer Research Fund
of the Damon Runyon-Walter Winchell Foundation (L.M., DRG-1416).
To investigate the stereoselectivity of the solvolysis reaction,
we followed the spontaneous solvolysis of racemic substrate 1
to completion and isolated the resulting product and subsequently
converted it to Mosher’s ester14 by reacting the formed exo-2-
norborneol product with (R)-(+)-R-methoxy-R-trifluorometh-
ylphenylacetyl chloride. The exo-2-norborneol derived from the
uncatalyzed solvolysis reaction was found to be an equimolar
JA990896B
(15) Irwin, A. J.; Jones, J. B. J. Am. Chem. Soc. 1976, 98, 8476-8482.
(16) To obtain sufficient signal-to-noise, we purified the 2-exo-norborneol
products from an equimolar mixture of substrate 1 and substrate 7 and
subsequently converted them to the corresponding higher molecular weight
4-nitrobenzoate esters.
1
mixture of two diastereomers as shown by its H NMR spectrum
(17) This value is in accordance with the previously determined value (1.20
(Figure 1B), indicating a racemic mixture of exo-2-norborneol 5
at 50 °C) for solvolysis of 2-deuterium-endo-norbornyl brosylate (Lee, C. C.;
Wong, E. W. C. J. Am. Chem. Soc. 1964, 86, 2752-2753 and Can. J. Chem.
1965, 43, 2254-2264). The introduction of deuterium in the C-5 and C-6
positions has a negligible isotope effect for the solvolysis of endo-2-norbornyl
derivatives (Sunko, D. E.; Borcic, S. Isotope Effects in Chemical Reactions;
Van Nostrand-Reinhold: New York, 1970).
formed by solvolysis of 1. The antibody-catalyzed solvolysis
reaction of racemic 1 was carried out at 22 °C in the presence of
3
0 µM antibody 15M3 and terminated (<40% conversion) by
extraction of exo-2-norborneol product with ethyl acetate. After
purification by flash chromatography on silica gel (elution with
(18) The exclusive formation of (+)-(1R,2R,4S)-5 from the antibody-
catalyzed solvolysis reaction of (+)-(1R,2S,4S)-1 also indicates that no hydride
1
0% ethyl acetate in hexane), this product was converted to its
2
shifts from the C-3 to the C-2 position or 1,2 shifts of a CH group from the
Mosher’s esters and determined by NMR analysis to be 96%
C-1 to the C-2 position occur in the antibody combining site. These
intramolecular rearrangements of 2-norbornyl cations have been observed in
acidic media or with alkyl substitutions. The 3,2 hydride shifts usually take
place from the exo side. See (a) Kleinfelter, D. C.; Schleyer, P. v. R. J. Am.
Chem. Soc. 1961, 83, 2329-2335. (b) Collins, C. J.; Cheema, Z. K.; Werth,
R. G.; Benjamin, B. M. J. Am. Chem. Soc. 1964, 86, 4913-4917. (c) Berson,
J. A.; Hammons, J. H.; WcRowe, A. W.; Bergman, R. G.; Remanick, A.;
Houston, D. J. Am. Chem. Soc. 1967, 89, 2561-2562. (d) Berson, J. A.;
Hammons, J. H.; WcRowe, A. W.; Bergman, R. G.; Houston, D. J. Am. Chem.
Soc. 1967, 89, 2563-2569.
(
12) Kuzmic, P. Anal. Biochem. 1996, 237, 260-273. For more information
about DYNAFIT, see http://www.biokin.com.
13) The (1S,2R,4R)-endo-2-norborneol was prepared by fractional recrys-
(
3
b,c
20
tallization as described and displayed specific optical rotation of [R]
)
3
b
24
+
2.07° (CHCl
3
, c 10.1) [lit. [R] ) + 1.89° (CHCl
3
, c 10)]. The alcohol
was converted to the corresponding mesylate derivative with an optical rotation
20
of [R] ) + 12.31° (CHCl , c 10).
3
(
14) (a) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512-519.
(
b) Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969, 34, 2543-
(19) The assignment of the proton chemical shifts for the (R)-Mosher’s
ester of 2-exo-norborneol is based on a comparison of the experimental data
with the reported values for 2-exo-norborneol: Abraham, R. J.; Rowan, A.
E. Magn. Reson. Chem. 1988, 26, 1027-1035.
2
3
1
549. (c) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1969, 90, 3732-
738. (d) Yamaguchi, S. Asymmetric Synthesis; Academic Press: 1983; Vol.
(Analytical Methods), pp 125-152.