Taxol Side Chain Research Articles

Concise synthesis of the Taxol side chain and demethoxy-4-epi-cytoxazone via oxazoline formation through intramolecular benzylic substitution of a bis-trichloroacetimidate

A concise and efficient method for synthesizing the Taxol side chain via the corresponding oxazoline intermediate was developed. The oxazoline ring is formed via an SN1 mechanism to ensure that the trans-oxazoline stereochemistry is retained. This process was induced by intramolecular benzylic substitution of a 1,2-bis-trichloroacetimidate, which was obtained from a known, enantiomerically pure diol. Demethoxy-4-epi-cytoxazone was also obtained from the intermediary trans-oxazoline 3b.

Biochemical characterization of acyl activating enzymes for side chain moieties of Taxol and its analogs

Taxol (paclitaxel) is a very widely used anticancer drug, but its commercial sources mainly consist of stripped bark or suspension cultures of members of the plant genus Taxus. Taxol accumulates as part of a complex mixture of chemical analogs, termed taxoids, which complicates its production in pure form, highlighting the need for metabolic engineering approaches for high-level Taxol production in cell cultures or microbial hosts. Here, we report on the characterization of acyl-activating enzymes (AAEs) that catalyze the formation of CoA esters of different organic acids relevant for the N-substitution of the 3-phenylisoserine side chain of taxoids. On the basis of similarities to AAE genes of known function from other organisms, we identified candidate genes in publicly available transcriptome data sets obtained with Taxus × media. We cloned 17 AAE genes, expressed them heterologously in Escherichia coli, purified the corresponding recombinant enzymes, and performed in vitro assays with 27 organic acids as potential substrates. We identified TmAAE1 and TmAAE5 as the most efficient enzymes for the activation of butyric acid (Taxol D side chain), TmAAE13 as the best candidate for generating a CoA ester of tiglic acid (Taxol B side chain), TmAAE3 and TmAAE13 as suitable for the activation of 4-methylbutyric acid (N-debenzoyl-N-(2-methylbutyryl)taxol side chain), TmAAE15 as a highly efficient candidate for hexanoic acid activation (Taxol C side chain), and TmAAE4 as suitable candidate for esterification of benzoic acid with CoA (Taxol side chain). This study lays important groundwork for metabolic engineering efforts aimed at improving Taxol production in cell cultures.

Catalytic Asymmetric Epoxidation of Aldehydes with Two VANOL-Derived Chiral Borate Catalysts.

A highly diastereo- and enantioselective method for the epoxidation of aldehydes with α-diazoacetamides has been developed with two different borate ester catalysts of VANOL. Both catalytic systems are general for aromatic, aliphatic, and acetylenic aldehydes, giving high yields and inductions for nearly all cases. One borate ester catalyst has two molecules of VANOL and the other only one VANOL. Catalysts generated from BINOL and VAPOL are ineffective catalysts. An application is shown for access to the side-chain of taxol.

Improved Dose-Response Relationship of (+)-Discodermolide-Taxol Hybrid Congeners

(+)-Discodermolide is a microtubule-stabilizing agent with potential for the treatment of taxol-refractory malignancies. (+)-Discodermolide congeners containing the C-3'-phenyl side chain of taxol (paclitaxel) were synthesized based on computational docking models predicting this moiety would fill an aromatic pocket of β-tubulin insufficiently occupied by (+)-discodermolide, thereby conferring improved ligand-target interaction. It was recently demonstrated, however, that the C-3'-phenyl side chain occupied a different space, instead extending toward the M-loop of β-tubulin, where it induced a helical conformation, hypothesized to improve lateral contacts between adjacent microtubule protofilaments. This insight led us to evaluate the biological activity of hybrid congeners using a panel of genetically diverse cancer cell lines. Hybrid molecules retained the same tubulin-polymerizing profile as (+)-discodermolide. Since (+)-discodermolide is a potent inducer of accelerated senescence, a fate that contributes to drug resistance, congeners were also screened for senescence induction. Flow cytometric and transcriptional analysis revealed that the hybrids largely retained the senescence-inducing properties of (+)-discodermolide. In taxol-sensitive cell models, the congeners had improved dose-response parameters relative to (+)-discodermolide and, in some cases, were superior to taxol. However, in cells susceptible to senescence, EMax increased without concomitant improvements in EC50 such that overall dose-response profiles resembled that of (+)-discodermolide.

ChemInform Abstract: Rhodium‐Catalyzed Enantioselective Hydrogenation of Tetrasubstituted α‐Acetoxy β‐Enamido Esters: A New Approach to Chiral α‐Hydroxyl‐β‐amino Acid Derivatives.

AbstractThe methodology provides valuable building blocks for the synthesis of biologically active compounds which is exemplified by the transformation of one product into the corresponding α‐hydroxy‐β‐amino acid, an intermediate of the taxol side chain.

Novel chemo-enzymatic route to a key intermediate for the taxol side-chain through enantioselective O-acylation. Unexpected acyl migration

A new enzymatic strategy has been devised for the preparation of a key intermediate for the taxol side-chain from N-hydroxymethylated cis-3-acetoxy-4-phenylazetidin-2-one. Burkholderia cepacia (PS-IM)-catalyzed acylation of the primary OH group with an excess of vinyl butyrate (10equiv.) furnished the corresponding diester and unreacted monoester with excellent enantiomeric excess values (ee≥98%). Traces of undesirable enantiomeric diacetylated product and diol due to intramolecular acyl migration (∼6% mole fractions) were also detected. Finally, (2R,3S)-3-phenylisoserine hydrochloride (ee=99%), a key intermediate for the taxol side-chain, was prepared from the corresponding diester enantiomer through acidic hydrolysis.

ChemInform Abstract: Highly Diastereoselective Reaction of α‐Diazoesters with Aryl Alcohols and Isatin Imines: Rapid Access to Oxindole‐Derived α‐Alkoxy‐β‐Amino Acid Derivatives with Two Adjacent Quaternary Carbon Centers.

A stereoselective synthesis of oxindole-derived α-alkoxy-β-amino acid derivatives was achieved in high yield with excellent diastereoselectivity through the three-component reaction of methyl 2-diazoarylacetate, aryl alcohols, and isatin-derived ketimines by using Rh2(OAc)4 (1 mol-%) as the catalyst. The products are very useful for the synthesis of analogues of the gastrin receptor antagonist AG-041R, spirooxindolyl β-lactams, the oxindole-derived side chain of Taxol, and natural product cytoxazone and aminopyrrolo[2,3-b]indoline derivatives.

An investigation of nitrile transforming enzymes in the chemo-enzymatic synthesis of the taxol sidechain.

Paclitaxel (taxol) is an antimicrotubule agent widely used in the treatment of cancer. Taxol is prepared in a semisynthetic route by coupling the N-benzoyl-(2R,3S)-3-phenylisoserine sidechain to the baccatin III core structure. Precursors of the taxol sidechain have previously been prepared in chemoenzymatic approaches using acylases, lipases, and reductases, mostly featuring the enantioselective, enzymatic step early in the reaction pathway. Here, nitrile hydrolysing enzymes, namely nitrile hydratases and nitrilases, are investigated for the enzymatic hydrolysis of two different sidechain precursors. Both sidechain precursors, an openchain α-hydroxy-β-amino nitrile and a cyanodihydrooxazole, are suitable for coupling to baccatin III directly after the enzymatic step. An extensive set of nitrilases and nitrile hydratases was screened towards their activity and selectivity in the hydrolysis of two taxol sidechain precursors and their epimers. A number of nitrilases and nitrile hydratases converted both sidechain precursors and their epimers.

Highly Diastereoselective Reaction of α‐Diazoesters with Aryl Alcohols and Isatin Imines: Rapid Access to Oxindole‐Derived α‐Alkoxy‐β‐amino Acid Derivatives with Two Adjacent Quaternary Carbon Centers

AbstractA stereoselective synthesis of oxindole‐derived α‐alkoxy‐β‐amino acid derivatives was achieved in high yield with excellent diastereoselectivity through the three‐component reaction of methyl 2‐diazoarylacetate, aryl alcohols, and isatin‐derived ketimines by using Rh2(OAc)4 (1 mol‐%) as the catalyst. The products are very useful for the synthesis of analogues of the gastrin receptor antagonist AG‐041R, spirooxindolyl β‐lactams, the oxindole‐derived side chain of Taxol, and natural product cytoxazone and aminopyrrolo[2,3‐b]indoline derivatives.

ChemInform Abstract: Organocatalytic Asymmetric Mannich Reactions of 5H‐Oxazol‐4‐ones: Highly Enantio‐ and Diastereoselective Synthesis of Chiral α‐Alkylisoserine Derivatives.

The first organocatalytic Mannich reaction of 5H-oxazol-4-ones with various readily prepared aryl- and alkylsulfonimides has been developed. Two commercially available pseudoenantiomeric Cinchona alkaloids-derived tertiary amine/ureas have been demonstrated as the most efficient catalysts to access the opposite enantiomers of the Mannich products with equally excellent enantio- and diastereoselectivities. From the Mannich adducts, important α-methyl-α-hydroxy-β-amino acid derivatives, such as the α-methylated C-13 side chain of taxol and taxotere, can be conveniently prepared.

Organocatalytic Asymmetric Mannich Reactions of 5H‐Oxazol‐4‐ones: Highly Enantio‐ and Diastereoselective Synthesis of Chiral α‐Alkylisoserine Derivatives

AbstractThe first organocatalytic Mannich reaction of 5H‐oxazol‐4‐ones with various readily prepared aryl‐ and alkylsulfonimides has been developed. Two commercially available pseudoenantiomeric Cinchona alkaloids‐derived tertiary amine/ureas have been demonstrated as the most efficient catalysts to access the opposite enantiomers of the Mannich products with equally excellent enantio‐ and diastereoselectivities. From the Mannich adducts, important α‐methyl‐α‐hydroxy‐β‐amino acid derivatives, such as the α‐methylated C‐13 side chain of taxol and taxotere, can be conveniently prepared.

Microwave-Assisted Synthesis of Taxol Side-Chain Precursor from Malonic Acid

Microwave-Assisted Synthesis of Taxol Side-Chain Precursor from Malonic Acid

Abstract 2789: Interaction of (+)-discodermolide-Taxol hybrids with microtubules

Abstract Taxol and discodermolide are both microtubule stabilizing agents that bind to the β-tubulin subunit of microtubules. Based on our results that the hydrophobic binding pocket of β-tubulin is occupied by the Taxol side chain but not by (+)-discodermolide, and that the two drugs act synergistically in cancer cell lines, a small library of (+)-discodermolide-Taxol hybrids was synthesized (J. Med. Chem. 54:6319, 2011). Cytotoxicity assays demonstrated a 2-9-fold increase in antiproliferative activity by the hybrids compared to discodermolide in the human cancer cell lines, A549 and MCF-7. Hybrids containing a tether of 3 carbons that connect discodermolide with the Taxol side chain, exhibited the best activity. Tubulin assembly assays were performed with the two most potent hybrid molecules. Like discodermolide, these two hybrids increased tubulin assembly rapidly without a lag period. Tubulin polymerization studies using purified bovine brain tubulin demonstrated that they also had the greatest effect on the formation of polymerized microtubules. These results also were observed in intact A549 cells and in 100,000 x g supernatants prepared from these cells. We have previously shown that [3H]2-(m-azidobenzoyl)Taxol photolabels a peptide containing amino acid residues 217-231 of β-tubulin. A 5-fold molar excess of unlabeled compound inhibited the photolabeling of purified bovine brain tubulin by 94%, demonstrating the specificity of this photolabeling. Microtubule stabilizing agents (MSAs), such as Taxol, epothilone B, discodermolide and ixabepilone, each at a 5-fold molar excess, inhibited the photolabeling by 24%, 92%, 100% and 41%, respectively, indicating that discodermolide is the most potent inhibitor of the photolabeling. In contrast, two other MSAs, laulimalide and peloruside that are known to bind to a different site in β-tubulin, exhibited stimulatory effects on the photolabeling. Both drugs, at a 5-fold molar excess, increased the labeling by 30-40%. [3H]2-(m-azidobenzoyl)Taxol (0.5 - 20 µM) was used to study the kinetics of the inhibitory effects of the hybrid molecules on photoaffinity labeling of tubulin. Discodermolide-Taxol hybrids inhibited the photolabeling of bovine brain tubulin in a dose dependent manner. The concentrations that inhibited by 50% were lowest for the two most potent hybrid molecules. Therefore, the tubulin polymerization activity and the binding affinity of the hybrids to β-tubulin correlated with their antiproliferative activity. Other biological properties of the discodermolide-Taxol hybrids including senescence and antitumor activity are being evaluated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2789. doi:1538-7445.AM2012-2789

Using biocatalysis to integrate organic chemistry into a molecular biology laboratory course

Current cutting-edge biomedical investigation requires that the researcher have an operational understanding of several diverse disciplines. Biocatalysis is a field of science that operates at the crossroads of organic chemistry, biochemistry, microbiology, and molecular biology, and provides an excellent model for interdisciplinary research. We have developed an inquiry-based module that uses the mutagenesis of the yeast reductase, YDL124w, to study the bioorganic synthesis of the taxol side-chain, a pharmacologically important molecule. Using related structures, students identify regions they think will affect enzyme stereoselective, design and generate site-specific mutants, and then characterize the effect of these changes on enzyme activity. This laboratory activity gives our students experience, working in a scientific discipline outside of biology and exposes them to techniques and equipment they do not normally work with in a molecular biology course. These inter-disciplinary experiences not only show the relevance of other sciences to biology, but also give our students the ability to communicate more effectively with scientists outside their discipline.

Chemo- and biocatalytic strategies to obtain phenylisoserine, a lateral chain of Taxol by asymmetric reduction

Enriched ethyl 3-benzamido-2-hydroxy-3-phenylpropanoate (protected phenylisoserine), the chiral side chain of Taxol, was obtained via asymmetric reduction with transition metal–diphoshine complexes or with whole cells of non-conventional yeasts. Asymmetric hydrogenation was carried out using different approaches: hydrogenation of the tetra-substituted double bond of (E)-1-benzamido-3-ethoxy-3-oxo-1-phenylprop-1-en-2-yl ethyl oxalate 1 with Ir(I)–diphosphine complexes in the presence of TEA, hydrogenation of the carbonyl group of racemic ethyl 3-benzamido-2-oxo-3-phenylpropanoate 2 with Ru(II)–diphoshine complexes in the presence of a Lewis acid and finally a two-step enzymatic transformation of (E)-1-benzamido-3-ethoxy-3-oxo-1-phenylprop-1-en-2-yl ethyl oxalate 1 catalyzed by whole cells of yeasts bearing cell-bound esterases and dehydrogenases.

Flow chemistry approach for partial deuteration of alkynes: synthesis of deuterated taxol side chain

The partial deuteration of alkynes proceeds via a flow chemistry approach under Lindlar’s heterogeneous catalysis to provide cis-dideuterated olefins in good yields. Further, dideuterated olefin has been successfully utilized for Sharpless asymmetric dihydroxylation followed by Mitsunobu reaction for the synthesis of deuterated taxol side chain.

Total synthesis and biological evaluation of a series of macrocyclic hybrids and analogues of the antimitotic natural products dictyostatin, discodermolide, and taxol.

The design, synthesis, and biological evaluation of a series of hybrids and analogues of the microtubule-stabilizing anticancer agents dictyostatin, discodermolide, and taxol is described. A 22-membered macrolide scaffold was prepared by adapting earlier synthetic routes directed towards dictyostatin and discodermolide, taking advantage of the distinctive structural and stereochemical similarities between these two polyketide-derived marine natural products. Initial endeavors towards accessing novel discodermolide/dictyostatin hybrids led to the adoption of a late-stage diversification strategy and the construction of a small library of methyl-ether derivatives, along with the first triple hybrids bearing the side-chain of taxol or taxotere attached through an ester linkage. Biological assays of the anti-proliferative activity of these compounds in a series of human cancer cell lines, including the taxol-resistant NCI/ADR-Res cell line, allowed the proposal of various structure-activity relationships. This led to the identification of a potent macrocyclic discodermolide/dictyostatin hybrid 12 and its C9 methoxy derivative 38, accessible by an efficient total synthesis and with a similar biological profile to dictyostatin.

A Strategy to Synthesize Taxol Side Chain and (−)-epi Cytoxazone via Chiral Brønsted Acid-Rh2(OAc)4 Co-catalyzed Enantioselective Three-Component Reactions

A new approach to synthesize optically active β-amino-α-hydroxyl acid derivatives via chiral Brønsted acid-Rh(2)(OAc)(4) cocatalyzed three-component reactions of diazo acetates with alcohols and imines is reported. A matched reaction system was identified to give the products in moderate diastereoselectivity and good enantioselectivity. Application of this methodology is demonstrated in the efficient synthesis of a taxol side chain and (-)-epi-cytoxazone.

A new enzymatic strategy for the preparation of (2 R,3 S)-3-phenylisoserine: a key intermediate for the Taxol side chain

Burkholderia cepacia lipase PS-IM catalysed the hydrolysis of racemic ethyl 3-amino-3-phenyl-2-hydroxypropionate with excellent enantioselectivity ( E >200), when the reaction was performed with added H 2O as a nucleophile, in iPr 2O, at 50 °C. The hydrolysis of the less reactive enantiomeric ethyl 3-amino-3-phenyl-2-hydroxypropionate with 18% HCl afforded the corresponding enantiomerically pure (2 R,3 S)-3-amino-3-phenyl-2-hydroxypropionic acid hydrochloride, a key intermediate for the Taxol side chain.

Chemoenzymatic preparation of intermediates for the taxol side chain and analogs

In the present study, optically active cis and trans α-hydroxy-β-lactams were prepared by Baker's yeast reduction of a racemic α-keto-β-lactam and by the hydrolysis of a racemic α-acetoxy β-lactam with the resting cells of Bacillus Subtilis.