RESUMO
Although optical pure amino alcohols are in high demand due to their widespread applicability, they still remain challenging to synthesize, since commonly elaborated protection strategies are required. Here, a multi-enzymatic methodology is presented that circumvents this obstacle furnishing enantioenriched 1,3-amino alcohols out of commodity chemicals. A Type I aldolase forged the carbon backbone with an enantioenriched aldol motif, which was subsequently subjected to enzymatic transamination. A panel of 194 TAs was tested on diverse nine aldol products prepared through different nucleophiles and electrophiles. Due to the availability of (R)- and (S)-selective TAs, both diastereomers of the 1,3-amino alcohol motif were accessible. A two-step process enabled the synthesis of the desired amino alcohols with up to three chiral centers with de up to >97 in the final products.
RESUMO
Thiamine diphosphate (ThDP) dependent enzymes are useful catalysts for asymmetric C-C bond formation through benzoin-type condensation reactions that result in α-hydroxy ketones. A wide range of aldehydes and ketones can be used as acceptor substrates; however, the donor substrate range is mostly limited to achiral α-keto acids and simple aldehydes. By using a unifying retro-biosynthetic approach towards acyl-branched sugars, we identified a subclass of (myco)bacterial ThDP-dependent enzymes with a greatly extended donor substrate range, namely functionalized chiral α-keto acids with a chain length from C4 to C8 . Highly enantioenriched acyloin products were obtained in good to high yields and several reactions were performed on a preparative scale. The newly introduced functionalized α-keto acids, accessible by known aldolase-catalyzed transformations, substantially broaden the donor substrate range of ThDP-dependent enzymes, thus enabling a more general use of these already valuable catalysts.
Assuntos
Açúcares , Tiamina , Aldeídos , Biocatálise , Cetoácidos , Cetonas/química , Especificidade por Substrato , Tiamina Pirofosfato/metabolismoRESUMO
A cell-free enantioselective transformation of the carbon atom of CO2 has never been reported. In the urgent context of transforming CO2 into products of high value, the enantiocontrolled synthesis of chiral compounds from CO2 would be highly desirable. Using an original hybrid chemoenzymatic catalytic process, we report herein the reductive oligomerization of CO2 into C3 (dihydroxyacetone, DHA) and C4 (l-erythrulose) carbohydrates, with perfect enantioselectivity of the latter chiral product. This was achieved with the key intermediacy of formaldehyde. CO2 is first reduced selectively by 4e- by an iron-catalyzed hydroboration reaction, leading to the isolation and complete characterization of a new bis(boryl)acetal compound derived from dimesitylborane. In an aqueous buffer solution at 30 °C, this compound readily releases formaldehyde, which is then involved in selective enzymatic transformations, giving rise either (i) to DHA using a formolase (FLS) catalysis or (ii) to l-erythrulose with a cascade reaction combining FLS and d-fructose-6-phosphate aldolase (FSA) A129S variant. Finally, the nature of the synthesized products is noteworthy, since carbohydrates are of high interest for the chemical and pharmaceutical industries. The present results prove that the cell-free de novo synthesis of carbohydrates from CO2 as a sustainable carbon source is a possible alternative pathway in addition to the intensely studied biomass extraction and de novo syntheses from fossil resources.
RESUMO
A two-enzyme cascade reaction plus in situ oxidative decarboxylation for the transformation of readily available canonical and non-canonical L-α-amino acids into 2-substituted 3-hydroxy-carboxylic acid derivatives is described. The biocatalytic cascade consisted of an oxidative deamination of L-α-amino acids by an L-α-amino acid deaminase from Cosenzaea myxofaciens, rendering 2-oxoacid intermediates, with an ensuing aldol addition reaction to formaldehyde, catalyzed by metal-dependent (R)- or (S)-selective carboligases namely 2-oxo-3-deoxy-l-rhamnonate aldolase (YfaU) and ketopantoate hydroxymethyltransferase (KPHMT), respectively, furnishing 3-substituted 4-hydroxy-2-oxoacids. The overall substrate conversion was optimized by balancing biocatalyst loading and amino acid and formaldehyde concentrations, yielding 36-98% aldol adduct formation and 91- 98% ee for each enantiomer. Subsequent in situ follow-up chemistry via hydrogen peroxide-driven oxidative decarboxylation afforded the corresponding 2-substituted 3-hydroxycarboxylic acid derivatives.
RESUMO
The congested nature of quaternary carbons hinders their preparation, most notably when stereocontrol is required. Here we report a biocatalytic method for the creation of quaternary carbon centers with broad substrate scope, leading to different compound classes bearing this structural feature. The key step comprises the aldol addition of 3,3-disubstituted 2-oxoacids to aldehydes catalyzed by metal dependent 3-methyl-2-oxobutanoate hydroxymethyltransferase from E. coli (KPHMT) and variants thereof. The 3,3,3-trisubstituted 2-oxoacids thus produced were converted into 2-oxolactones and 3-hydroxy acids and directly to ulosonic acid derivatives, all bearing gem-dialkyl, gem-cycloalkyl, and spirocyclic quaternary centers. In addition, some of these reactions use a single enantiomer from racemic nucleophiles to afford stereopure quaternary carbons. The notable substrate tolerance and stereocontrol of these enzymes are indicative of their potential for the synthesis of structurally intricate molecules.
Assuntos
Aldeídos/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidroximetil e Formil Transferases/metabolismo , Cetoácidos/metabolismo , Aldeídos/química , Sítios de Ligação , Biocatálise , Domínio Catalítico , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Hidroximetil e Formil Transferases/química , Hidroximetil e Formil Transferases/genética , Cetoácidos/química , Mutagênese Sítio-Dirigida , Estereoisomerismo , Especificidade por SubstratoRESUMO
Nitrogen heterocycles are structural motifs found in many bioactive natural products and of utmost importance in pharmaceutical drug development. In this work, a stereoselective synthesis of functionalized N-heterocycles was accomplished in two steps, comprising the biocatalytic aldol addition of ethanal and simple aliphatic ketones such as propanone, butanone, 3-pentanone, cyclobutanone, and cyclopentanone to N-Cbz-protected aminoaldehydes using engineered variants of d-fructose-6-phosphate aldolase from Escherichia coli (FSA) or 2-deoxy-d-ribose-5-phosphate aldolase from Thermotoga maritima (DERA Tma ) as catalysts. FSA catalyzed most of the additions of ketones while DERA Tma was restricted to ethanal and propanone. Subsequent treatment with hydrogen in the presence of palladium over charcoal, yielded low-level oxygenated N-heterocyclic derivatives of piperidine, pyrrolidine and N-bicyclic structures bearing fused cyclobutane and cyclopentane rings, with stereoselectivities of 96-98 ee and 97:3 dr in isolated yields ranging from 35 to 79%.
RESUMO
The asymmetric aldol addition reaction mediated by aldolases is recognized as a green and sustainable method for carbon-carbon bond formation. Research in this area has unveiled their unprecedented synthetic potential toward diverse, new chemical structures; novel product families; and even as a technology for industrial manufacturing processes. Despite these advances, aldolases have long been regarded as strictly selective catalysts, particularly for nucleophilic substrates, which limits their broad applicability. In recent years, advances in screening technologies and metagenomics have uncovered novel C-C biocatalysts from superfamilies of widely known lyases. Moreover, protein engineering has revealed the extraordinary malleability of different carboligases to offer a toolbox of biocatalysts active towards a large structural diversity of nucleophile substrates. Herein, the nucleophile ambiguity of native and engineered aldolases is discussed with recent examples to prove this novel concept.
Assuntos
Aldeído Liases/química , Aldeído Liases/genética , Aminoácidos/química , Aminoácidos/genética , Biocatálise , Domínio Catalítico/genética , Bactérias Gram-Negativas/enzimologia , Mutagênese Sítio-Dirigida/métodos , Mutação , EstereoisomerismoRESUMO
The uptake of a large variety of contaminants of emerging concern (CECs) by crops has already been reported, and the occurrence of phase II metabolites or conjugates has only been detected in plant cell cultures. However, the extent of their formation under cropping conditions is largely unknown. In this study, an analytical strategy to assess the conjugation of 11 CECs in lettuce (Lactuca sativa L.) irrigated with different concentrations (0, 0.05, 0.5, 5, and 50 µg L-1) of CECs was developed. The methodology involved enzymatic digestion with ß-glucosidase to obtain the total fraction (free form + conjugates) of CECs. The conjugation fraction was then obtained based on the difference. The highest extent of conjugation (i.e., 27 to 83%) was found with the most hydrophobic compounds, such as bisphenol A, carbamazepine, methyl paraben, and triclosan. So, the CEC conjugate fraction cannot be neglected in the estimate of human daily intake.
Assuntos
Celulases/metabolismo , Produtos Agrícolas/metabolismo , Lactuca/metabolismo , Poluentes Químicos da Água/metabolismo , Irrigação Agrícola , Compostos Benzidrílicos/análise , Compostos Benzidrílicos/metabolismo , Carbamazepina/análise , Carbamazepina/metabolismo , Glicosilação , Parabenos/análise , Parabenos/metabolismo , Fenóis/análise , Fenóis/metabolismo , Metabolismo Secundário , Triclosan/análise , Triclosan/metabolismo , Poluentes Químicos da Água/análiseRESUMO
Pyruvate-dependent aldolases exhibit a stringent selectivity for pyruvate, limiting application of their synthetic potential, which is a drawback shared with other existing aldolases. Structure-guided rational protein engineering rendered a 2-keto-3-deoxy-l-rhamnonate aldolase variant, fused with a maltose-binding protein (MBP-YfaU W23V/L216A), capable of efficiently converting larger pyruvate analogues, for example, those with linear and branched aliphatic chains, in aldol addition reactions. Combination of these nucleophiles with N-Cbz-alaninal (Cbz=benzyloxycarbonyl) and N-Cbz-prolinal electrophiles gave access to chiral building blocks, for example, derivatives of (2S,3S,4R)-4-amino-3-hydroxy-2-methylpentanoic acid (68 %, d.r. 90:10) and the enantiomer of dolaproine (33 %, d.r. 94:6) as well as a collection of unprecedented α-amino acid derivatives of the proline and pyrrolizidine type. Conversions varied between 6-93 % and diastereomeric ratios from 50:50 to 95:5 depending on the nucleophilic and electrophilic components.
Assuntos
Aldeído Liases/química , Escherichia coli/enzimologia , Ácido Pirúvico/química , Aldeídos/química , Aminoácidos/química , Compostos Heterocíclicos com 2 Anéis/química , Modelos Moleculares , Estrutura Molecular , Prolina/análogos & derivados , Prolina/química , Ligação Proteica , Pirrolidinas/química , Estereoisomerismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
A structure-guided engineering of fructose-6-phosphate aldolase was performed to expand its substrate promiscuity toward aliphatic nucleophiles, that is, unsubstituted alkanones and alkanals. A "smart" combinatorial library was created targeting residues D6, T26, and N28, which form a binding pocket around the nucleophilic carbon atom. Double-selectivity screening was executed by high-performance TLC that allowed simultaneous determination of total activity as well as a preference for acetone versus propanal as competing nucleophiles. D6 turned out to be the key residue that enabled activity with non-hydroxylated nucleophiles. Altogether 25 single- and double-site variants (D6X and D6X/T26X) were discovered that show useful synthetic activity and a varying preference for ketone or aldehyde as the aldol nucleophiles. Remarkably, all of the novel variants had completely lost their native activity for cleavage of fructose 6-phosphate.
Assuntos
Frutose-Bifosfato Aldolase/metabolismo , Cetonas/metabolismo , Cristalografia por Raios X , Frutose-Bifosfato Aldolase/química , Cetonas/química , Modelos Moleculares , Estrutura MolecularRESUMO
d-Fructose-6-phosphate aldolase (FSA) was probed for extended nucleophile promiscuity by using a series of fluorogenic substrates to reveal retro-aldol activity. Four nucleophiles ethanal, propanone, butanone, and cyclopentanone were subsequently confirmed to be non-natural substrates in the synthesis direction using the wild-type enzyme and its D6H variant. This exceptional widening of the nucleophile substrate scope offers a rapid entry, in good yields and high stereoselectivity, to less oxygenated alkyl ketones and aldehydes, which was hitherto impossible.
Assuntos
Aldeído Liases/metabolismo , Aldeídos/química , Frutose-Bifosfato Aldolase/metabolismo , Frutosefosfatos/química , Cetonas/química , Aldeído Liases/química , Catálise , Frutose-Bifosfato Aldolase/química , Estrutura Molecular , EstereoisomerismoRESUMO
Intramolecular benzoin reactions catalyzed by benzaldehyde lyase from Pseudomonas fluorescens biovarâ I (BAL) are reported. The structure of the substrates envisaged for this reaction consists of two benzaldehyde derivatives linked by an alkyl chain. The structural requirements needed to achieve the intramolecular carbon-carbon bond reaction catalyzed by BAL were established. Thus, a linker consisting of a linear alkyl chain of three carbon atoms connected through ether-type bonds to the 2 and 2' positions of two benzaldehyde moieties, which could be substituted with either Cl, Br, or OCH3 at either the 3 and 3' or 5 and 5' positions, were suitable substrates for BAL. Reactions with 61-84 % yields of the intramolecular product and eeâ values between 64 and 98 %, were achieved.
Assuntos
Aldeído Liases/metabolismo , Benzoína/metabolismo , Pseudomonas fluorescens/enzimologia , Benzoína/química , Cristalografia por Raios X , Modelos Moleculares , Estrutura MolecularRESUMO
Glycogen synthase (GS) and glycogen phosphorylase (GP) are the key enzymes that control, respectively, the synthesis and degradation of glycogen, a multi-branched glucose polymer that serves as a form of energy storage in bacteria, fungi and animals. An abnormal glycogen metabolism is associated with several human diseases. Thus, GS and GP constitute adequate pharmacological targets to modulate cellular glycogen levels by means of their selective inhibition. The compound 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) is a known potent inhibitor of GP. We studied the inhibitory effect of DAB, its enantiomer LAB, and 29 DAB derivatives on the activity of rat muscle glycogen phosphorylase (RMGP) and E. coli glycogen synthase (EcGS). The isoform 4 of sucrose synthase (SuSy4) from Solanum tuberosum L. was also included in the study for comparative purposes. Although these three enzymes possess highly conserved catalytic site architectures, the DAB derivatives analysed showed extremely diverse inhibitory potential. Subtle changes in the positions of crucial residues in their active sites are sufficient to discriminate among the structural differences of the tested inhibitors. For the two Leloir-type enzymes, EcGS and SuSy4, which use sugar nucleotides as donors, the inhibitory potency of the compounds analysed was synergistically enhanced by more than three orders of magnitude in the presence of ADP and UDP, respectively. Our results are consistent with a model in which these compounds bind to the subsite in the active centre of the enzymes that is normally occupied by the glucosyl residue which is transferred between donor and acceptor substrates. The ability to selectively inhibit the catalytic activity of the key enzymes of the glycogen metabolism may represent a new approach for the treatment of disorders of the glycogen metabolism.
Assuntos
Arabinose/química , Arabinose/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glicogênio/metabolismo , Imino Furanoses/química , Imino Furanoses/farmacologia , Álcoois Açúcares/química , Álcoois Açúcares/farmacologia , Animais , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/metabolismo , Glicogênio Fosforilase/antagonistas & inibidores , Glicogênio Fosforilase/metabolismo , Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase/metabolismo , Simulação de Acoplamento Molecular , Ratos , Solanum tuberosum/efeitos dos fármacos , Solanum tuberosum/enzimologia , Solanum tuberosum/metabolismoRESUMO
The thorough analysis of highly complex NMR spectra using pure shift NMR experiments is described. The enhanced spectral resolution obtained from modern 2D HOBS experiments incorporating spectral aliasing in the (13) C indirect dimension enables the distinction of similar compounds exhibiting near-identical (1) H and (13) Câ NMR spectra. It is shown that a complete set of extremely small Δδ((1) H) and Δδ((13) C) values, even below the natural line width (1 and 5 ppb, respectively), can be simultaneously determined and assigned.
Assuntos
Misturas Complexas/química , Espectroscopia de Ressonância Magnética/métodos , Isótopos de Carbono/análise , Hidrogênio/análise , EstereoisomerismoRESUMO
The introduction of aromatic residues connected by a C-C bond into the non-reducing end of carbohydrates is highly significant for the development of innovative structures with improved binding affinity and selectivity (e.g., C-aril-sLex). In this work, an expedient asymmetric "de novo" synthetic route to new aryl carbohydrate derivatives based on two sequential stereoselectively biocatalytic carboligation reactions is presented. First, the benzoin reaction of aromatic aldehydes to dimethoxyacetaldehyde is conducted, catalyzed by benzaldehyde lyase from Pseudomonas fluorescens biovar I. Then, the α-hydroxyketones formed are reduced by using NaBH4 yielding the anti diol. After acetal hydrolysis, the aldol addition of dihydroxyacetone, hydroxyacetone, or glycolaldehyde catalyzed by the stereocomplementary D-fructose-6-phosphate aldolase and L-rhamnulose-1-phosphate aldolase is performed. Both aldolases accept unphosphorylated donor substrates, avoiding the need of handling the phosphate group that the dihydroxyacetone phosphate-dependent aldolases require. In this way, 6-C-aryl-L-sorbose, 6-C-aryl-L-fructose, 6-C-aryl-L-tagatose, and 5-C-aryl-L-xylose derivatives are prepared by using this methodology.
Assuntos
Aldeído Liases/química , Aldeídos/química , Benzoína/química , Carboidratos/síntese química , Di-Hidroxiacetona/química , Escherichia coli/química , Frutosefosfatos/síntese química , Aldeído Liases/metabolismo , Biocatálise , Carboidratos/química , Escherichia coli/metabolismo , Frutosefosfatos/química , Estrutura MolecularRESUMO
Two novel arginine-based cationic surfactants were synthesized using as biocatalyst papain, an endopeptidase from Carica papaya latex, adsorbed onto polyamide. The classical substrate N (α)-benzoyl-arginine ethyl ester hydrochloride for the determination of cysteine and serine proteases activity was used as the arginine donor, whereas decyl- and dodecylamine were used as nucleophiles for the condensation reaction. Yields higher than 90 and 80 % were achieved for the synthesis of N (α)-benzoyl-arginine decyl amide (Bz-Arg-NHC10) and N (α)-benzoyl-arginine dodecyl amide (Bz-Arg-NHC12), respectively. The purification process was developed in order to make it more sustainable, by using water and ethanol as the main separation solvents in a single cationic exchange chromatographic separation step. Bz-Arg-NHC10 and Bz-Arg-NHC12 proved antimicrobial activity against both Gram-positive and Gram-negative bacteria, revealing their potential use as effective disinfectants as they reduced 99 % the initial bacterial population after only 1 h of contact. The cytotoxic effect towards different cell types of both arginine derivatives was also measured. Bz-Arg-NHCn demonstrated lower haemolytic activity and were less eye-irritating than the commercial cationic surfactant cetrimide. A similar trend could also be observed when cytotoxicity was tested on hepatocytes and fibroblast cell lines: both arginine derivatives were less toxic than cetrimide. All these properties would make the two novel arginine compounds a promising alternative to commercial cationic surfactants, especially for their use as additives in topical formulations.
Assuntos
Antibacterianos/farmacologia , Arginina/análogos & derivados , Arginina/farmacologia , Tensoativos/farmacologia , Antibacterianos/síntese química , Antibacterianos/isolamento & purificação , Arginina/síntese química , Arginina/isolamento & purificação , Biocatálise , Sobrevivência Celular/efeitos dos fármacos , Cromatografia por Troca Iônica , Eritrócitos/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hemólise , Células Hep G2 , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Papaína/química , Tensoativos/síntese química , Tensoativos/isolamento & purificaçãoRESUMO
α,α-Disubstituted α-amino acids are central to biotechnological and biomedical chemical processes for their own sake and as substructures of biologically active molecules for diverse biomedical applications. Structurally, these compounds contain a quaternary stereocenter, which is particularly challenging for stereoselective synthesis. The pyridoxal-5'-phosphate (PLP)-dependent L-serine hydroxymethyltransferase from Streptococcus thermophilus (SHMT(Sth); EC 2.1.2.1) was engineered to achieve the stereoselective synthesis of a broad structural variety of α,α-dialkyl-α-amino acids. This was accomplished by the formation of quaternary stereocenters through aldol addition of the amino acids D-Ala and D-Ser to a wide acceptor scope catalyzed by the minimalist SHMT(Sth) Y55T variant overcoming the limitation of the native enzyme for Gly. The SHMT(Sth) Y55T variant tolerates aromatic and aliphatic aldehydes as well as hydroxy- and nitrogen-containing aldehydes as acceptors.
Assuntos
Aminoácidos/biossíntese , Glicina Hidroximetiltransferase/metabolismo , Engenharia de Proteínas , Streptococcus thermophilus/enzimologia , Glicina Hidroximetiltransferase/química , Modelos Moleculares , Difração de Raios XRESUMO
D-Fructose-6-phosphate aldolase (FSA) is a unique catalyst for asymmetric cross-aldol additions of glycolaldehyde. A combination of a structure-guided approach of saturation mutagenesis, site-directed mutagenesis, and computational modeling was applied to construct a set of FSA variants that improved the catalytic efficiency towards glycolaldehyde dimerization up to 1800-fold. A combination of mutations in positions L107, A129, and A165 provided a toolbox of FSA variants that expand the synthetic possibilities towards the preparation of aldose-like carbohydrate compounds. The new FSA variants were applied as highly efficient catalysts for cross-aldol additions of glycolaldehyde to N-carbobenzyloxyaminoaldehydes to furnish between 80-98 % aldol adduct under optimized reaction conditions. Donor competition experiments showed high selectivity for glycolaldehyde relative to dihydroxyacetone or hydroxyacetone. These results demonstrate the exceptional malleability of the active site in FSA, which can be remodeled to accept a wide spectrum of donor and acceptor substrates with high efficiency and selectivity.
Assuntos
Acetaldeído/análogos & derivados , Aldeído Liases/genética , Aldeídos/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Engenharia de Proteínas , Acetaldeído/metabolismo , Aldeído Liases/metabolismo , Dimerização , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Frutosefosfatos/metabolismo , Modelos Moleculares , Engenharia de Proteínas/métodosRESUMO
A straightforward chemoenzymatic synthesis of four uncovered casuarine stereoisomers is described. The strategy consists of L-fuculose-1-phosphate aldolase F131A-variant-catalyzed aldol addition of dihydroxyacetone phosphate to aldehyde derivatives of 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) and its enantiomer (LAB) and subsequent one-pot catalytic deprotection-reductive amination. DAB and LAB were obtained from dihydroxyacetone and aminoethanol using D-fructose-6-phosphate aldolase and L-rhamnulose-1-phosphate aldolase catalysts, respectively. The new ent-3-epi-casuarine is a strong inhibitor of α-d-glucosidase from rice and of rat intestinal sucrase.
Assuntos
Aldeído Liases/antagonistas & inibidores , Aldeído Liases/química , Alcaloides/síntese química , Alcaloides/farmacologia , Arabinose/química , Fosfato de Di-Hidroxiacetona/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Etanolamina/química , Frutose-Bifosfato Aldolase/antagonistas & inibidores , Inibidores de Glicosídeo Hidrolases/síntese química , Inibidores de Glicosídeo Hidrolases/farmacologia , Imino Furanoses/química , Oryza/química , Pirróis/síntese química , Pirróis/farmacologia , Sacarase/antagonistas & inibidores , Sacarase/química , Álcoois Açúcares/química , Alcaloides/química , Animais , Inibidores Enzimáticos/química , Frutose-Bifosfato Aldolase/química , Inibidores de Glicosídeo Hidrolases/química , Estrutura Molecular , Pirróis/química , Ratos , EstereoisomerismoRESUMO
Statins are an important class of drugs used to lower blood cholesterol levels and are often used to combat cardiovascular disease. In view of the importance of safe and reliable supply and production of statins in modern medicine and the global need for sustainable processes, various biocatalytic strategies for their synthesis have been investigated. In this work, a novel biocatalytic route to a statin side chain precursor was investigated in a one-pot cascade reaction starting from the protected alcohol N-(3-hydroxypropyl)-2-phenylacetamide, which is oxidized to the corresponding aldehyde in the first reaction step, and then reacts with two equivalents of acetaldehyde to form the final product N-(2-((2S,4S,6S)-4,6-dihydroxytetrahydro-2H-pyran-2-yl)ethyl)-2-phenylacetamide (phenylacetamide-lactol). To study this complex reaction, an enzyme reaction engineering approach was used, i.e. the kinetics of all reactions occurring in the cascade (including side reactions) were determined. The obtained kinetic model together with the simulations gave an insight into the system and indicated the best reactor mode for the studied reaction, which was fed-batch with acetaldehyde feed to minimize its negative effect on the enzyme activity during the reaction. The mathematical model of the process was developed and used to simulate different scenarios and to find the reaction conditions (enzyme and coenzyme concentration, substrate feed concentration and flow rate) at which the highest yield of phenylacetamide-lactol (75%) can be obtained. In the end, our goal was to show that this novel cascade route is an interesting alternative for the synthesis of the statin side chain precursor and that is why we also calculated an initial estimate of the potential value addition.