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J. Am. Chem. Soc. 1997, 119, 11554-11555
Synthesis of Water-Soluble Paclitaxel Derivatives by
Enzymatic Acylation
Yuri L. Khmelnitsky,† Cheryl Budde,† J. Michael Arnold,†
Alexander Usyatinsky,† Douglas S. Clark,*,‡ and
Jonathan S. Dordick*,§
EnzyMed, Inc., 2501 Crosspark Road
Oakdale Research Park, Iowa City, Iowa 52242
Department of Chemical Engineering
UniVersity of California
Berkeley, California 94720
Department of Chemical and Biochemical Engineering
UniVersity of Iowa, Iowa City, Iowa 52242
ReceiVed September 3, 1997
Figure 1. Two-step enzymatic modification of paclitaxel resulting in
paclitaxel 2′-adipic acid (29) and paclitaxel 2′-adipoylglucose (30).
Reaction conditions are described in the text.
Paclitaxel,1 a diterpenoid originally isolated from the bark
of the Pacific yew, Taxus breVifolia, is a powerful antimitotic
agent2 that acts by promoting tubulin assembly into stable
aggregated structures. Although paclitaxel has shown tremen-
dous potential as an anticancer compound,3 its use as an
anticancer drug is compromised by its poor aqueous solubility.
For this reason, a number of water-soluble paclitaxel prodrugs
have been synthesized that contain hydrophilic or charged
functionalities attached to specific sites on the paclitaxel
molecule.4
Acylation at the 2′ position (for the structure of paclitaxel,
see Figure 1) can be a very effective strategy for improving the
water solubility of paclitaxel.4 Interestingly, acylation of the
C-2′ hydroxyl eliminates microtubule stabilization but not
cytotoxicity, which is consistent with the hydrolytic regeneration
of paclitaxel from pro-paclitaxel within the cell.5 Water soluble
pro-paclitaxels modified at the 2′ position include arylsulfonyl
ethoxycarbonates and thiodiglycolic esters synthesized by
Nicolaou et al.,4a the most soluble of which were 100-1000
times more soluble than paclitaxel.
of two-step enzymatic acylation depicted in Figure 1. In the
first step, the starting compound is reacted with a bifunctional
acylating agent to give an activated acyl derivative, which is
then used as a complex acyl donor in the second step of the
derivatization procedure. In accordance with this strategy, the
starting point of the present work was to identify an appropriate
enzyme catalyst suitable for acylation of paclitaxel in the first
step. After a wide range of enzymes and solvents8 were tested,
thermolysin (an extracellular protease from Bacillus thermo-
proteolyticus rokko) suspended in anhydrous tert-amyl alcohol
was identified to be the most effective catalyst for paclitaxel
acylation.9 In particular, this enzyme-catalyzed acylation of
paclitaxel with a bifunctional acyl donor, divinyl adipate, as
determined by TLC and HPLC. The reactivity of thermolysin
toward paclitaxel was enhanced ca. 20-fold by lyophilizing the
enzyme in the presence of KCl prior to use.10 Using the salt-
activated enzyme preparation (5.7 mg/mL protein), ca. 90%
conversion of paclitaxel (14 mM solution) was obtained in 96
h in the presence of 45 mM divinyl adipate. Following
termination of the reaction,11 two products were isolated from
the reaction mixture via preparative HPLC. The identities of
In the present work, we establish for the first time that
paclitaxel can be enzymatically derivatized6 in an organic
solvent7 to generate new potential prodrugs possessing high
solubility in water. Our approach is based on a unique strategy
† EnzyMed, Inc.
1
‡ University of California.
these products were determined by mass and H NMR spec-
§ University of Iowa.
troscopies to be paclitaxel 2′-vinyl adipate (7, major) and
7-epipaclitaxel 2′-vinyl adipate (14, minor) (Table 1). Isolated
yields of the products (based on the starting amount of
paclitaxel) were 60 and 18%, respectively. Thus, thermolysin
is an extremely regioselective enzyme toward the 2′-hydroxyl
moiety of paclitaxel, as no other hydroxyl groups on the
paclitaxel molecule were esterified in the enzymatic reaction.
In addition to divinyl adipate, several other straight-chain vinyl
esters were suitable for the thermolysin-catalyzed acylation of
paclitaxel under conditions described above for divinyl adipate.
(1) For reviews on paclitaxel see: (a) Kingston, D. G. I. Trends
Biotechnol. 1994, 12, 222. (b) Rowinsky, E. K.; Onerto, N.; Canetta, R.
M.; Arbuck, S. G.; Sem. Oncol. 1992, 19, 646. (c) Nicolaou, K. C.; Dai,
W. M.; Guy, R. K. Angew. Chem., Int. Ed. Eng. 1994, 33, 15. (d) Kingston,
D. G. I. Pharmacol. Ther. 1991, 52, 1.
(2) Schiff, P. B.; Fant, J.; Horwitz, S. B. Nature 1979, 277, 665.
(3) (a) Rowinsky, E. K.; Cazenave, L. A.; Donehower, R. C. J. Nat.
Cancer Inst. 1990, 82, 1247. (b) McGuire, W. P.; Rowinsky, E. K.;
Rosenshein, N. B.; Grumbine, F. C.; Ettinger, D. S.; Armstrong, D. K.;
Donehower, R. C. Ann. Int. Med. 1989, 111, 273.
(4) (a) Nicolaou, K. C.; Riemer, C.; Kerr, M. A.; Rideout, D.; Wrasidlo,
W. Nature 1993, 364, 464. (b) Deutsch, H. M.; Glinski, J. A.; Hernandez,
M.; Haugwitz, R. D.; Narayanan, V. L.; Suffness, M.; Zalkow, L. H. J.
Med. Chem. 1989, 32, 788. (c) Mathew, A. E.; Mejillano, M. R.; Nath, J.
P.; Himes, R. H.; Stella, V. J. J. Med. Chem. 1992, 35, 145. (d) Ueda, Y.;
Wong, H.; Matiskella, J. D.; Mikkilineni, A. B.; Farina, V.; Fairchild, C.;
Rose, W. C.; Mamber, S. W.; Long, B. H.; Kerns, E. H.; Casazza, A. M.;
Vyas, D. M. Bioorg. Med. Chem. Lett. 1994, 4, 1861. (e) Zhou, Z.; Kingston,
D. G. I.; Crosswell, A. R. J. Nat. Prod. 1991, 54, 1607.
(8) Over 50 different commercially available lipases and proteases were
screened for paclitaxel acylation using vinyl butyrate as the acyl donor.
The following enzymes were found to possess paclitaxel acylation activ-
ity: R-chymotrypsin, subtilisin Carlsberg, and thermolysin. Among these
enzymes thermolysin showed the highest activity (ca. 3- and 40-fold higher
than R-chymotrypsin and subtilisin, respectively) and, therefore, was used
as a catalyst in all subsequent reactions.
(5) (a) Kingston, D. G. I.; Samaranayake, G.; Ivey, C. A. J. Nat. Prod.
1990, 53, 1. (b) Potier, P. Chem. Soc. ReV. 1992, 113.
(6) Recently, two enzymes from Nocardioides strains (isolated from soil)
were shown to catalyze the regioselective hydrolysis of the 10-acetyl and
13-side chain esters. Since these enzymatic reactions were carried out in
aqueous solutions, they resulted in low productivities (due to the insolubility
of paclitaxel) and hydrolytic (and hence degradative) reactions. Hanson,
R. L.; Wasylyk, J. M.; Nanduri, V. B.; Cazzulino, D. L.; Patel, R. N.; Szarka,
L. J. J. Biol. Chem. 1994, 269, 22145.
(7) For reviews on enzymatic catalysis in organic solvents, see: (a)
Dordick, J. S. In Applied Biocatalysis; Blanch, H. W. Clark, D. S., Eds.;
Marcel Dekker: New York, 1991; Vol. 1, pp 1-51. (b) Klibanov, A. M.
Acc. Chem. Res. 1990, 23, 114. (c) Khmelnitsky, Yu. L.; Levashov, A. V.;
Klyachko, N. L.; Martinek, K. Enzyme Microb. Technol. 1988, 10, 710.
(9) The ability of thermolysin to catalyze a transesterification reaction,
such as that used in paclitaxel acylation, has never been observed before.
This unusual finding reveals an interesting new feature of the zinc-containing
protease which has been used as a catalyst for synthesis of peptides:
Miyanaga, M.; Tanaka, T.; Sakiyama, T.; Nakanishi, K. Biotechnol. Bioeng.
1995, 46, 631.
(10) Salt-activated thermolysin was prepared following the published
procedure (Khmelnitsky, Yu. L.; Welch, S. H.; Clark, D. S.; Dordick, J. S.
J. Am. Chem. Soc. 1994, 116, 2647).
(11) The workup of the reaction mixture included removal of the
suspended enzyme by centrifugation and evaporation of the solvent under
vacuum.
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