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A Novel Approach to the Synthesis of the Taxol Side Chain: Adapting S. Cerevisiae as Enantioselective Reagent for the Reduction of a-Keto-b-lactams

Marko D. Mihovilovic*a, Margaret M. Kayserb, Sonia Rodriguezc
and Jon D. Stewartc

a Vienna University of Technology, Institute of Organic Chemistry, Getreidemarkt 9, A-1060 Vienna, Austria (EU), b University of New Brunswick, Department of Physical Science, P.O.Box 5050, Saint John, N.B., Canada E2L 4L5, c University of Florida, Department of Chemistry, P.O.Box 117200, Gainesville FL 32611-7200, USA

Taxol as a Cancer Drug

Since its discovery Taxol® had experienced many ups and downs as a highly potent cancer drug. The compound was finally approved for treatment of a great variety of different types of cancer, especially breast, ovarian, and lung cancer.

As the production of Taxol is still far from being optimized, there is still a lot of research going on in this field (1). Up to now a large scale total synthesis is lacking and the access by isolation of the bark of the Pacific Yew tree is very limited. The precursor Baccatin III can be isolated from the needles of the Atlantic Yew tree as a quickly regrowable resource. An industrial process takes advantage of the b-lactam method to introduce the side chain into this polycyclic core (2).

Our approach to synthesize this key intermediate is based on the enantioselective reduction of an a-keto-b-lactam precursor. We studied several natural, mutant, and engineered yeast strains for their selectivity in the chiral resolution step involved.


Yeast as a Biocatalyst

BakerÔs yeast is well established as a bioorganic reducing agent for ketones and ketoesters (3). Its cheap and easy accessibility and the ease of cultivating this microorganism made it popular among organic chemists. Moreover, the use of yeast represents an environmentally compatible technique ÷ a feature becoming increasingly important ÷ and the organism is pathogenically benign. Apart from technical problems such as solubility problems of the starting material in aqueous cultures and/or possible toxicity of the substrate to yeast, one of the major downsides of using yeast for reductions is the fact that these reactions are frequently but not always highly enantioselective. In substrates with a preexisting chiral center, enantioselectivity as well as diastereoselectivity may be low. This can be attributed to the presence of several reductases with overlapping substrate acceptabilities (4). In an effort to find a selective reducing agent for a-keto-b-lactams we have studied some natural, mutated, and bioengineered yeast strains.


Synthesis of the Keto-Precursor

The synthesis of the carbonyl precursor 4 is straightforward:

Cyclization of ester 1 with imine 2 in a modified Staudinger reaction using LDA gives the azetidine ring system in excellent yield. Deprotection of the ketal 3 requires rather forcing conditions (75% H2SO4), however yields of the ketone 4 are high.

Four isomers are possible after reaction: the enantiomeric cis products 5 and the trans compounds 6. The required stereoconfiguration for the Taxol side chain is (3R,4S)-5.


Results and Discussion of the Biotransformations

All biotransformations were carried out in a 2:1 ratio of biocatalyst to substrate (10mM in reaction media). In the case of commercial BakerÔs yeast, a large excess (200x) of the microorganism was also performed. The reductions were followed by chiral HPLC.

Assuming that the si/re-face attack model is valid, the predominantly active enzyme for a-keto-b-lactams seems to be a D-enzyme giving predominantly the R-enantiomer.


The R-alcohol with the correct stereo configuration for Taxol is the predominantly formed cis-isomer (3R,4S)-5 in all reactions. This indicates that the major enzyme system responsible for the biotransformation belongs to the D-family. Plots of the reaction progress indicate that all strains exhibit resolution of the racemic starting material 4. Formation of the diastereomeric trans-compound (3R,4R)-6 is observed after consumption of substantial amounts of starting material. The yields of the reductions on complete conversion were quantitative in most cases.


In the laboratory strain INVSC1 the D-reductase seems to be expressed in excess compared to ordinary Baker╬s yeast. Hence much lower amounts of biocatalyst were necessary to give complete conversion in comparable times combined with better resultion capability.

Much slower conversion rates and yields of alcohols were detected with the mutant strain ATCC 26403 with a deficiency in the fatty acid synthetase expression system that carries D-reaductase activity. However, the fact that still reduction to an R-alcohol could be observed indicate the potential presence of another D-enzyme.

The genetically engineered strain INVSC1(pSRG14) overexpressing the aldo-keto reductase L-1 exhibits a slower conversion that can be attributed to the still present activity of the D-enzyme.

The fact that all yeast strains studied produced similar amounts of the cis-isomer (3S,4R)-5 after comparable times indicate that a-keto-b- lactams might be accepted by another reductase giving S-alcohols. Only minor traces of the corresponding diastereomeric compound (3S,4S)-5 were detected.


Final Product Distribution:


Structural assignment of (3R,4R)-6 was carried out by X-ray diffraction of derivative 7 utilizing the heavy atom effect of bromine to determine the absolute configuration.


X-ray of (3R,4R)-7



The above results suggest that a member of the D-reductase family ÷ tentatively fatty acid synthetase ÷ accepts a-keto-b- lactams as substrats for the reduction to a key intermediate in the Taxol synthesis. Further studies to prove the identity of the major enzyme for the conversion to the desired lactam (3R,4S)-5 are presently in progress aiming towards the development of an engineered strain with optimized selectivity for the presented transformation.



This work was in part funded by the Austrian Science Fund (FWF) project no. CHEM-J01471.

We would like to thank Dr. Fernande D. Ronchon, Département de chimie, Université du Québec à Montréal, for performing X-ray diffraction and structure elucidation of compound 7.



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