a tandem hydrolysis–1,6-elimination reaction.9 We have dem-
onstrated that it is possible to obtain an abzyme that can activate
a tripartate system without incorporating the ‘drug’ into the
TSA by attaching the carrier protein to a position on the TSA
that corresponded to the point of attachment of the ‘drug’ to the
tripartate prodrug. Although ST51 can catalyze the hydrolysis
of N-methylcarbamates at physiological pH, the rate of the
reaction is too slow for an accurate kinetic analysis and effective
prodrug activation. It has been estimated that for ADEPT
systems, a kcat value of about 1.0 s21 is required.2c Nevertheless,
the approach outlined here should be readily applicable to
tripartate prodrugs of type 1 bearing moieties that are more
amenable to antibody–catalyzed hydrolysis, under physio-
logical conditions, than N-methylcarbamates, which are highly
challenging substrates for antibody catalysis.8 An N-H carba-
mate linkage between the specifier and linker, of the type
recently exploited by Blackburn and coworkers for abzyme
catalysis,2c should be very suitable to the approach reported
here.11 However, tripartate prodrugs of type 1 bearing such a
moiety cannot be used as substrates for ST51 since ST51 will
not hydrolyze N-H carbamate 8, amide 9, or even ester 10.
Studies to elucidate the mechanism of ST51 are in progress and
we are continuing our work to develop abzymes that can trigger
tripartate prodrugs of type 1 under physiological conditions.
We thank the Canadian Institutes of Health Research (CHIR)
for financial support of this work. We would also like to thank
Katherine Majewska and Parham Daneshvar for assistance in
synthesizing compounds 8–10.
Scheme 2
Notes and references
1 For recent reviews see: K. N. Syrigos and A. A. Epenetos, Anticancer
Res., 1999, 19, 605; W. A. Denny and W. R. Wilson, J. Pharm.
Pharmacol., 1998, 50, 387.
2 (a) H. Miyashita, Y. Karaki, M. Kikuchi and I. Fujii, Proc. Natl. Acad.
Sci, USA, 1993, 90, 5337; (b) D. A. Campbell, B. Gong, L. M.
Kochersperger, S. Yonkovich, M. A. Gallop and P. G. Schultz, J. Am.
Chem. Soc., 1994, 116, 2165; (c) P. Wentworth, A. Datta, D. Blakey, T.
Boyle, L. J. Partridge and G. M. Blackburn, Proc. Natl. Acad. Sci USA,
1996, 93, 799; (d) D. Shabat, C. Rader, B. List, R. A. Lerner and C. F.
Barbas III, Proc. Natl. Acad. Sci. USA, 1999, 96, 6925.
3 G. Winter and C. Milstein, Nature, 1991, 349, 293.
4 P. L. Carl, P. K. Chakavarty and J. A. Katzenellenbogen, J. Med. Chem.,
1981, 24, 479.
5 S. D. Taylor, M. J. Chen, A. N. Dinaut and R. A. Batey, Tetrahedron,
1998, 54, 4223.
6 L. J. Liotta, P. A. Benkovic, G. P. Miller and S. J. Benkovic, J. Am.
Chem. Soc., 1993, 115, 350; F. Tanaka, K. Kinoshita, R. Tanimura and
I. Fujii, J. Am. Chem. Soc., 1996, 118, 2332.
7 Since the primary objective of these studies was to determine if ST51
could trigger the tripartate system, it was not necessary to use an actual
drug to make the model ‘prodrug’. Indeed, for these studies any amine-
bearing molecule would have sufficed for the ‘drug’ so long as it (1) had
no resemblance to the specifier (PNP) portion of the prodrug, (2) was
relatively easy to detect and quantitate, (3) had a charged moiety that
would enhance the solubility of the prodrug and, (4) was readily
available and allowed for economic and facile synthesis of the model
prodrug. Tryptophan was chosen since it satisfied all of these
requirements.
kinetics, and approximately equimolar quantities of PNP and
Trp were detected for the duration of the time the reactions were
monitored. When following the production of PNP, ST51
exhibited a kcat = 0.075 h21 and a Km = 137 mM. Similar
values were obtained from data following the production of Trp.
We also found that ST51 was unable to catalyze the hydrolysis
of Z-Trp. Taken together, these results indicate that the
production of Trp is not a result of hydrolysis of the N-H
carbamate moiety, but rather initial hydrolysis of the N-
methylcarbamate followed by the spontaneous fragmentation
reaction and that the abzyme-catalyzed step is slow compared to
the fragmentation reaction. We also found that ST51 was
capable of catalyzing the activation of 3 with multiple turnover.
This indicates that 5, which is produced as an intermediate
during the reaction, does not inactivate the abzyme after a single
turnover by reacting with crucial residues in the active site.
Assuming that the spontaneous rate of hydrolysis of the N-
8 A. N. Dinaut, M.-J. Chen, A. Marks, R. A. Batey and S. D. Taylor,
Chem. Commun., 2000, 385.
9 While our work was in progress, Shabat et al. reported abzyme-
catalyzed activation of tripartate prodrugs by a tandem retro-aldol–
retro-Michael reaction (see ref. 2d). Although the abzyme was not
originally designed to act as a prodrug activator (see also ref. 10),
generic tripartate prodrug activation was achievable because of the
broad substrate specificity of the abzyme.
10 J. Wagner, R. A. Lerner and C. F. Barbas, Science, 1995, 270, 1797.
11 For tripartate prodrugs of type 1 based upon N-H carbamates of the kind
examined by Blackburn and coworkers (see ref. 2c), the specifier would
be an amino acid and atom X in Scheme 1 would be oxygen and so the
self-immolative intermediate 2, would be a phenol derivative. Phenols
also undergo the spontaneous 1,6-elimination reaction. For a recent
example of this see: I. Niculescu-Duvaz, D. Niculescu-Duvaz, F.
Friedlos, R. Spooner, J. Martin, R. Marais and C. J. Springer, J. Med.
Chem., 1999, 42, 2485.
1
methylcarbamate moiety in 3 is similar to that of 7 (t = 5.7
2
years in 100 mM bicine, 100 mM NaCl, 5% DMSO, pH 9.0)
then the rate enhancement (kcat/kuncat) with 3 is about 5000-fold.
This is only slightly less than the rate enhancement found for
ST51 using substrate 7 (6500-fold).8 It is also important to note
that 3 and 7 exhibit similar Km values (266 mM for 7 and 137 mM
for 3). These results indicate that the ‘drug’ portion of the
prodrug is not an important recognition site for the antibody-
catalyzed reaction and this is consistent with our hapten
design.
In summary, we have reported the first example of antibody-
catalyzed activation of a model tripartate prodrug of type 1 by
Chem. Commun., 2001, 1386–1387
1387