Javier González-Sabín

CAL-B-catalyzed resolution of some pharmacologically interesting β-substituted isopropylamines

Abstract Some pharmacologically active amines such as amphetamine, the isomeric o-, m- and p-methoxyamphetamines, 4-phenylbutan-2-amine and mexiletine, as well as their corresponding acetamides, have been prepared in high yields and with very high enantiomeric excesses. The method consists of the Candida antarctica lipase B (CAL-B)-mediated enantioselective acetylation of racemic amines using ethyl acetate as solvent and acyl donor. The enzyme follows Kazlauskas’ rule with all amines, (R)-amides being obtained as the major enantiomer in all cases. From the conversion values measured for both enantiomers, it can be deduced that the size of the substituents attached to the stereocenter is responsible for the enantioselectivity and rate of some of these reactions.

Regioselective enzymatic acylation of complex natural products: expanding molecular diversity

Enzymatic catalysis has become a common tool in both academia and industrial chemistry. The efforts of chemists over recent decades have led to the rationalization of the mechanism of action of biocatalysts, which have been routinely incorporated into many synthetic sequences. Nowadays, a further step consists in expanding the application of enzymes to the modification of complex molecular scaffolds common to many pharmaceutical leads isolated from nature. Regioselective enzymatic acylation is a process which has been profitably applied for this purpose in recent times, leading to new drugs with improved activity, stability and pharmacokinetic properties. This tutorial review provides an overview of this subject employing two classes of enzymes, hydrolases and acyltransferases, in the recently concluded decade although some representative older studies are commented upon, if required. We shall place special emphasis on those examples in which the novel acylated derivatives have improved the activity or properties of the parental molecules.

A Novel Mithramycin Analogue with High Antitumor Activity and Less Toxicity Generated by Combinatorial Biosynthesis

Mithramycin is an antitumor compound produced by Streptomyces argillaceus that has been used for the treatment of several types of tumors and hypercalcaemia processes. However, its use in humans has been limited because of its side effects. Using combinatorial biosynthesis approaches, we have generated seven new mithramycin derivatives, which differ from the parental compound in the sugar profile or in both the sugar profile and the 3-side chain. From these studies three novel derivatives were identified, demycarosyl-3D-β-d-digitoxosylmithramycin SK, demycarosylmithramycin SDK, and demycarosyl-3D-β-d-digitoxosylmithramycin SDK, which show high antitumor activity. The first one, which combines two structural features previously found to improve pharmacological behavior, was generated following two different strategies, and it showed less toxicity than mithramycin. Preliminary in vivo evaluation of its antitumor activity through hollow fiber assays, and in subcutaneous colon and melanoma cancers xenografts models, suggests that demycarosyl-3D-β-d-digitoxosylmithramycin SK could be a promising antitumor agent worthy of further investigation.

Elucidating the Biosynthetic Pathway for the Polyketide-Nonribosomal Peptide Collismycin A: Mechanism for Formation of the 2,2′-bipyridyl Ring

The gene cluster for the bipyridyl compound collismycin was characterized from Streptomyces sp. CS40. Sequence analysis of a 46.7 kb DNA region revealed 27 open reading frames, 23 of which are involved in collismycin biosynthesis. Eight insertional inactivation mutants were generated in the sequenced region to prove its involvement in collismycin biosynthesis, define the boundaries of the cluster, functionally characterize some genes, and isolate two biosynthetic intermediates. A model for collismycin biosynthesis--which includes the conversion of lysine into picolinic acid, participation of a polyketide synthase-non-ribosomal peptide synthetase system, and some further modifications--is proposed. The biosynthetic pathway would include an unusual NRPS-mediated incorporation of a cysteine residue, possibly through a Michael addition and followed by the extension of the peptide chain by leucine incorporation and later removal by amidohydrolase.

From a Sequential to a Concurrent Reaction in Aqueous Medium: Ruthenium‐Catalyzed Allylic Alcohol Isomerization and Asymmetric Bioreduction

The ruthenium-catalyzed redox isomerization of allylic alcohols was successfully coupled with the enantioselective enzymatic ketone reduction (mediated by KREDs) in a concurrent process in aqueous medium. The overall transformation, formally the asymmetric reduction of allylic alcohols, took place with excellent conversions and enantioselectivities, under mild reaction conditions, employing commercially and readily available catalytic systems, and without external coenzymes or cofactors. Optimization resulted in a multistep approach and a genuine cascade reaction where the metal catalyst and biocatalyst coexist from the beginning.

The chromomycin CmmA acetyltransferase: a membrane-bound enzyme as a tool for increasing structural diversity of the antitumour mithramycin

Mithramycin and chromomycin A3 are two structurally related antitumour compounds, which differ in the glycosylation profiles and functional group substitutions of the sugars. Chromomycin contains two acetyl groups, which are incorporated during the biosynthesis by the acetyltransferase CmmA in Streptomyces griseus ssp. griseus. A bioconversion strategy using an engineered S. griseus strain generated seven novel acetylated mithramycins. The newly formed compounds were purified and characterized by MS and NMR. These new compounds differ from their parental compounds in the presence of one, two or three acetyl groups, attached at 3E, 4E and/or 4D positions. All new mithramycin analogues showed antitumour activity at micromolar of lower concentrations. Some of the compounds showed improved activities against glioblastoma or pancreas tumour cells. The CmmA acetyltransferase was located in the cell membrane and was shown to accept several acyl‐CoA substrates. All these results highlight the potential of CmmA as a tool to create structural diversity in these antitumour compounds.

Identification of Mithramycin Analogues with Improved Targeting of the EWS-FLI1 Transcription Factor

Purpose: The goal of this study was to identify second-generation mithramycin analogues that better target the EWS-FLI1 transcription factor for Ewing sarcoma. We previously established mithramycin as an EWS-FLI1 inhibitor, but the compounds toxicity prevented its use at effective concentrations in patients. Experimental Design: We screened a panel of mithralogs to establish their ability to inhibit EWS-FLI1 in Ewing sarcoma. We compared the IC50 with the MTD established in mice to determine the relationship between efficacy and toxicity. We confirmed the suppression of EWS-FLI1 at the promoter, mRNA, gene signature, and protein levels. We established an improved therapeutic window by using time-lapse microscopy to model the effects on cellular proliferation in Ewing sarcoma cells relative to HepG2 control cells. Finally, we established an improved therapeutic window using a xenograft model of Ewing sarcoma. Results: EC-8105 was found to be the most potent analogue and was able to suppress EWS-FLI1 activity at concentrations nontoxic to other cell types. EC-8042 was substantially less toxic than mithramycin in multiple species but maintained suppression of EWS-FLI1 at similar concentrations. Both compounds markedly suppressed Ewing sarcoma xenograft growth and inhibited EWS-FLI1 in vivo. Conclusions: These results provide a basis for the continued development of EC-8042 and EC-8105 as EWS-FLI1 inhibitors for the clinic. Clin Cancer Res; 22(16); 4105–18. ©2016 AACR.

Asymmetric reduction of prochiral ketones by using self‐sufficient heterogeneous biocatalysts based on NADPH‐dependent ketoreductases

The development of cell-free and self-sufficient biocatalytic systems represents an emerging approach to address more complex synthetic schemes under nonphysiological conditions. Herein, we report the development of a self-sufficient heterogeneous biocatalyst for the synthesis of chiral alcohols without the need to add an exogenous cofactor. In this work, an NADPH-dependent ketoreductase was primarily stabilized and further co-immobilized with NADPH to catalyze asymmetric reductions without the addition of an exogenous cofactor. As a result, the immobilized cofactor is accessible, and thus, it is recycled inside the porous structure without diffusing out into the bulk, as demonstrated by single-particle in operando studies. This self-sufficient heterogeneous biocatalyst was used and recycled for the asymmetric reduction of eleven carbonyl compounds in a batch reactor without the addition of exogenous NADPH to achieve the corresponding alcohols in 100 % yield and >99 % ee; this high performance was maintained over five consecutive reaction cycles. Likewise, the self-sufficient heterogeneous biocatalyst was integrated into a plug flow reactor for the continuous synthesis of one model secondary alcohol, which gave rise to a space-time yield of 97-112 g L-1  day-1 ; additionally, the immobilized cofactor accumulated a total turnover number of 1076 for 120 h. This is one of the few examples of the successful implementation of continuous reactions in aqueous media catalyzed by cell-free and immobilized systems that integrate both enzymes and cofactors into the solid phase.

Developing a Biocascade Process: Concurrent Ketone Reduction-Nitrile Hydrolysis of 2-Oxocycloalkanecarbonitriles

A stereoselective bioreduction of 2-oxocycloalkanecarbonitriles was concurrently coupled to a whole cell-catalyzed nitrile hydrolysis in one-pot. The first step, mediated by ketoreductases, involved a dynamic reductive kinetic resolution, which led to 2-hydroxycycloalkanenitriles in very high enantio- and diastereomeric ratios. Then, the simultaneous exposure to nitrile hydratase and amidase from whole cells of Rhodococcus rhodochrous provided the corresponding 2-hydroxycycloalkanecarboxylic acids with excellent overall yield and optical purity for the all-enzymatic cascade.

Programming cascade reactions interfacing biocatalysis with transition-metal catalysis in Deep Eutectic Solvents as biorenewable reaction media

The first application of Deep Eutectic Solvents (DESs) in the asymmetric bioreduction of ketones has been accomplished for purified ketoreductases (KREDs). The performance of the biocatalysts was enhanced by increasing the percentage of neoteric solvent in DES-buffer mixtures. At a buffer content of 50% (w/w) and even 20% (w/w), the combination of either choline chloride (ChCl)/glycerol (Gly) (1 : 2) or ChCl/sorbitol (1 : 1) proved to be most effective for achieving up to >99% conversion and up to >99% enantiomeric excess of the corresponding secondary alcohols. Moreover, this reaction medium was used to perform the first example of a chemoenzymatic cascade process in DES-buffer mixtures, namely the ruthenium-catalysed isomerisation of racemic allylic alcohols coupled with a further enantioselective bioreduction, in both sequential and concurrent modes.