Synthesis and yeast - mediated bioreduction of α -keto- β -lactams bearing a functionalized and rigid side chain

Bakers’ yeast ( Saccharomyces cerevisiae ) mediated reductions were investigated for the potential of a fermentation approach towards novel paclitaxel C-13 side-chain analogs bearing a polar methoxy-functionality in combination with a rigid structural core unit. Synthesis of correspondingly substituted α -keto- β -lactam compounds as biotransformation precursors was carried out following a straightforward strategy involving Staudinger [2+2] cyclization. Bioreductions were carried out using growing whole-cells of S. cerevisiae and the dia-and enantioselectivity of the process was studied.


Introduction
The introduction of enzymes as catalysts into synthetic organic chemistry opened up a variety of new transformations.The employment of microorganisms provides access to enzymes that are difficult to use in an isolated form.In addition, such organisms are self-propagating sources of the desired biocatalyst and all cofactors necessary for the reaction.Therefore, the use of such "biocatalytic reagents" has spread among synthetic organic chemists over the past decade.Since fermentations are generally carried out in aqueous buffers the use of organic solvents and heavy metals in the course of a synthesis is minimized, hence this methodology representing a "green chemistry" approach is of increasing interest for industrial applications.
Bakers' yeast (Saccharomyces cerevisiae) has been the first and most popular whole-cell biocatalyst, particularly for asymmetric reductions of carbonyl compounds.Reductions catalyzed by this organism tolerate a large variety of ketones and side-reactions are rarely observed.This broad substrate acceptance is due to the presence of a number of reductase enzymes, some of which have been isolated and characterized.
While α-keto acids and derivatives thereof have received relatively less attention than β-keto esters as substrates for yeast reductions, the resulting α-hydroxy carbonyl compounds are equally valuable chiral building blocks and several routes to these compounds have been devised.Among a number of biologically interesting targets microbial reduction of α-keto-β-lactams allows access to the paclitaxel side chain.In this contribution we are disclosing initial studies of the bioreduction of functionalized precursors of this type, aimed at the subsequent synthesis of modified taxoids.
Taxol (paclitaxel; R' = phenyl, R'' = benzoyl, R''' = acetyl) and Taxotere (docetaxel; R' = phenyl, R'' = tert.-butoxycarbonyl,R''' = H) are block-buster chemotherapeutic agents (Scheme 1).The compounds are used in clinical therapy against ovarian, breast, and non-small cell lung cancer and preclinical screens have revealed significant activity against resistant murine melanoma.Paclitaxel was originally extracted in small quantities from the bark of the Pacific yew tree, Taxus brevifolia, and was quickly regarded as a most promising anti cancer agent.The compound class promotes microtubule assembly in vitro and, consequently, upsets the delicate dynamic equilibrium between soluble dimeric tubules and the microtubule polymers.The stabilization of the latter inhibits the late part of phase G 2 and M of cell mitosis in cancer cells.Due to this particular mode of action paclitaxel represents an important addition to the cancer armamentarium.
Presently, the drug is prepared via a semisynthetic process utilizing baccatin III, which is isolated from needles of the Atlantic yew tree.The precursor represents the polycyclic core structure of taxol and the biologically highly relevant C-13 side chain incorporated by the "βlactam approach" (Scheme 1).
A number of structure-activity studies suggests that significant changes in the pharmacological activity of the compound can be achieved by modifications of the C-13 side chain.This led to the development of a number of "second generation" taxoids with improved administration profiles and/or cytotoxic potential.
In this context we are presenting our synthetic efforts to incorporate rigid cyclic systems as key structural elements of novel C-13 side chain analogs bearing additional functional groups.The key step in our sequence is the stereoselective bioreduction of appropriate α-keto-β-lactams using whole yeast cells in a fermentation process.Previously, we could demonstrate, that yeast cells carry the required biocatalytic arsenal to generate the natural side chain precursor for  Subsequent cyclization to the β-lactam core 7a-c was carried out via a Staudinger-type methodology: The intermediate ketene was either generated in a one pot procedure from acetoxy acetic acid 6a using POCl 3 or from acid chloride 6b in the presence of excess NEt 3 .Due to sterical hindrance by the quaternary carbon adjacent to the cyclization site, the cycloaddition reaction was rather sluggish and required several days to reach complete conversion.For such prolonged reaction times, stability of the imine under the conditions applied became a limiting factor, as in all cases aldehyde 4a-c was recovered.This decomposition of the imines 5a-c gave rise to significant amounts of p-anisidine, which led to the formation of amid 8 as by-product of the cyclization reaction.The observed problems with sterically demanding reactions partners in the Staudinger reaction are consistent with a significantly decreased yield for the conversion to the corresponding 4-tert.-butyl-azetidinoneas found in a previous study.
While lactams 7a and 7b were formed as cisand trans-isomers in approximately equal amounts, tetrahydropyran derivative 7c was isolated in cis-form, exclusively.Assignment of diastereomers is based on previous studies of coupling constants for azetidin protons H-3 and H-4.Hydrolysis of the acetoxy protecting group under basic conditions yielded alcohols 9a-c in a similar cis/trans ratio.Subsequent oxidation using P 2 O 5 /DMSO finally gave α-keto-β-lactams 10a-c in good overall yields after chromatographic purification.
Bioreductions with commercial bakers' yeast were carried out in 10% glucose solution supplemented by 1 equivalent of β-cyclodextrin to improve solubility and membrane penetration of the substrates and to decrease toxicity vis-à-vis the living cells.Biotransformations at 30°C in an orbital shaker required 2-5 days to reach completion (Scheme 3, Table 2).Alcohols 9a-c were obtained in good chemical yields as cis-isomers, exclusively, when using ordinary bakers' yeast obtained at a local supermarket.However, enantioselectivity of the microbial transformation was low for all ketone precursors.This result was rather unexpected, as previous yeast mediated reductions of α-keto-β-lactams displayed a distinguished diastereo-and enantioselectivity.In the case of substrates 10a-c the responsible enzyme seems unable to differentiate the existing chiral center of the racemic substrate.Consequently, the reduction is mainly governed by thermodynamic effects, leading to the energetically most stable product.In the case of the azetidine core this is the cis-substitution pattern of groups in position 3 and 4, consistent with the chemical reduction of ketones 10a-c using sodium borohydride.Previous bioreduction studies with phenyl (10d) and tert.-butyl(10e) ketolactams utilizing whole yeast cells showed a distinguished trend (Scheme 3): Using commercial bakers' yeast, racemic substrate 10d gave complete conversion to two diastereomeric alcohols cisand trans-9d.Stereochemistry at the secondary alcohol was R in both cases.While trans-9d was obtained almost exclusively as (3R,4R) enantiomer, cis-9d was only obtained in moderate optical purity, however predominantly in (3R,4S) configuration as suitable precursor for paclitaxel.A control experiment with the mutant yeast strain ATCC 26403 gave a classical kinetic resolution, yielding only cis-9d, however, again in moderate enantiomeric excess, as in the previous transformation.This strain is deficient in fatty acid synthase (FAS), which suggests that FAS is responsible for the formation of trans-9d and this enzyme is stereoselective.
The low selectivity in the bioreduction to cis-9d was attributed to the similarity of the 4phenyl substituent and the PMP protecting group.Consequently, switching to a 4-tert.-butylprecursor, a key structural element of second generation butitaxels, was expected to improve the selectivity of the yeast mediated reductions.This effect was successfully demonstrated for ketone 10e, which was converted diastereoselectively to cis-(3R,4S)-9e and trans-(3R,4R)-9e in high optical purity.
Based on the structural similarity of the sterically strained compounds, we had expected a similar trend for the biotransformation of ketones 10a-c.However, these compounds seem to be unsuitable substrates for FAS, as no trans-alcohols were formed in the biocatalytic conversion.A control experiment with 10b, using the mutant strain ATCC 26403 in a suitable growth media containing fatty acid supplements, did not show any conversion to alcohols 9b.As a result, ketones 10a-c were only accepted by a still not identified enzyme, which is responsible for the formation of cis-products but with rather limited stereoselectivity for the secondary alcohol functionality generated in the biocatalytic process.The biotransformation of substrates 10a-c is most likely the result of a single enzyme, while for the bioreduction of 10d/e a two enzyme process seems most likely.
Nevertheless, such conclusions should be handled with caution, as yeast harbors a large variety of different reductases with partially overlapping substrate specificity.Unambiguous assignment of enzyme activity and stereoselectivity can only be made after biotransformations using isolated enzymes or recombinant expression systems with a suitable genetically clean background.Such experiments are currently under way in our laboratory.

Experimental Section
General Procedures.Unless otherwise noted, chemicals and microbial growth media were purchased from commercial suppliers and used without further purification.All solvents were distilled prior to use.Flash column chromatography was performed on silica gel 60 from Merck (40-63µm).Kugelrohr distillation was carried out using a Büchi GKR-51 apparatus.Melting points were determined using a Kofler-type Leica Galen III micro hot stage microscope and are

General procedure for hydrolysis to alcohols 9
A solution (5%) of compound 7 was treated with the half volume of a 1:1 mixture of 2N KOH and THF at 0°C and stirred at this temperature until conversion was completed.The reaction mixture was poured into water and extracted with EtOAc.The combined organic layers were washed with brine, dried over Na 2 SO 4 , filtered, and concentrated.

General procedure for the yeast reduction
Commercial bakers' yeast (4g, obtained from a local supermarket) was added to a 10% solution of glucose in water (100mL) and incubated for 10 minutes at 30°C.Ketone 10 (100mg in 1mL dioxane) and β-cyclodextrin (1 equiv.)were added to this fermentation culture.The mixture was incubated for 2-5 days at 30°C.The biomass was separated by centrifugation (5min at 3000 rpm).After extraction with EtOAc the combined organic layers were dried over Na 2 SO 4 , filtered, and concentrated.

Table 2 .
Yeast mediated reductions of α-keto-β-lactams to cisand trans-alcohols 9 a data obtained in this study; b data from ref. Error!Bookmark not defined.;c data from ref. Error!Bookmark not defined.;d not applicable Issue in Honor of Prof. Lubor Fišera ARKIVOC 2005 (v) 33-44 ISSN 1424-6376 Page 37 © ARKAT USA, Inc

ARKIVOC 2005 (v) 33-44 ISSN 1424-6376 Page 38
NMR-spectra were recorded from CDCl 3 or DMSO-d 6 solutions on a Bruker AC 200 (200 MHz) or Bruker Avance UltraShield 400 (400 MHz) spectrometer and chemical shifts are reported in ppm using TMS as internal standard.Combustion analysis was carried out in the Microanalytic Laboratory, University of Vienna.Enantiomeric excess was determined by HPLC Na 2 SO 4 (4 equiv.)and molecular sieves (3Å) were added to aldehyde 4 (5% solution in dry CH 2 Cl 2 ).After stirring for 24 hours at RT the mixture was passed through a bed of Celite ® and concentrated.2N HCl and extracted with CH 2 Cl 2 .The combined organic layers were washed with satd.NaHCO 3 solution, dried over Na 2 SO 4 , filtered and concentrated.