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1.
Chimia (Aarau) ; 78(3): 108-117, 2024 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-38547011

RESUMO

Excelzyme, an enzyme engineering platform located at the Zurich University of Applied Sciences, is dedicated to accelerating the development of tailored biocatalysts for large-scale industrial applications. Leveraging automation and advanced computational techniques, including machine learning, efficient biocatalysts can be generated in short timeframes. Toward this goal, Excelzyme systematically selects suitable protein scaffolds as the foundation for constructing complex enzyme libraries, thereby enhancing sequence and structural biocatalyst diversity. Here, we describe applied workflows and technologies as well as an industrial case study that exemplifies the successful application of the workflow.


Assuntos
Engenharia de Proteínas , Proteínas , Humanos , Suíça , Universidades , Biocatálise , Proteínas/química , Engenharia de Proteínas/métodos
2.
Angew Chem Int Ed Engl ; 63(30): e202405152, 2024 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-38739413

RESUMO

Biocatalysis provides an attractive approach to facilitate synthetic reactions in aqueous media. Motivated by the discovery of promiscuous aminolysis activity of esterases, we exploited the esterase from Pyrobaculum calidifontis VA1 (PestE) for the synthesis of carbamates from different aliphatic, aromatic, and arylaliphatic amines and a set of carbonates such as dimethyl-, dibenzyl-, or diallyl carbonate. Thus, aniline and benzylamine derivatives, aliphatic and even secondary amines could be efficiently converted into the corresponding benzyloxycarbonyl (Cbz)- or allyloxycarbonyl (Alloc)-protected products in bulk water, with (isolated) yields of up to 99 %.


Assuntos
Aciltransferases , Carbamatos , Esterases , Água , Esterases/metabolismo , Esterases/química , Carbamatos/química , Carbamatos/metabolismo , Carbamatos/síntese química , Água/química , Aciltransferases/metabolismo , Aciltransferases/química , Biocatálise , Estrutura Molecular , Aminas/química , Aminas/metabolismo
3.
J Org Chem ; 87(7): 4955-4960, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-35317556

RESUMO

A highly efficient asymmetric synthesis of the IDO inhibitor navoximod, featuring the stereoselective installation of two relative and two absolute stereocenters from an advanced racemic intermediate, is described. The stereocenters were set via a crystallization-induced dynamic resolution along with two selective ketone reductions: one via a biocatalytic ketoreductase transformation and one via substrate-controlled hydride delivery from LiAlH(Ot-Bu)3. Following this strategy, navoximod was synthesized in 10 steps from 2-fluorobenzaldehyde and isolated in 23% overall yield with 99.7% ee and high purity.


Assuntos
Inibidores Enzimáticos , Indóis , Inibidores Enzimáticos/farmacologia , Imidazóis , Estereoisomerismo
4.
Bioorg Med Chem Lett ; 48: 128242, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34217829

RESUMO

Therapeutic oligonucleotides require the addition of multiple chemical modifications to the nucleosidic scaffold in order to improve their drug delivery efficiency, cell penetration capacity, biological stability, and pharmacokinetic properties. This chemical modification pattern is often accompanied by a synthetic burden and by limitations in sequence length. Here, we have synthesized a nucleoside triphosphate analog bearing two simultaneous modifications at the level of the sugar (LNA) and the backbone (thiophosphate) and have tested its compatibility with enzymatic DNA synthesis which could abrogate some of these synthetic limitations. While this novel analog is not as well tolerated by polymerases compared to the corresponding α-thio-dTTP or LNA-TTP, α -thio-LNA-TTP can readily be used for enzymatic synthesis on universal templates for the introduction of phosphorothioated LNA nucleotides.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Oligonucleotídeos Fosforotioatos/biossíntese , Conformação de Ácido Nucleico , Oligonucleotídeos Fosforotioatos/química
5.
Chimia (Aarau) ; 75(7): 605-613, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34523401

RESUMO

Ipatasertib is a potent small molecule Akt kinase inhibitor currently being tested in Phase III clinical trials for the treatment of metastatic castration-resistant prostate cancer and triple negative metastatic breast cancer. In this paper an overview of the development achievements towards the commercial manufacturing process is given. The convergent synthesis consists of ten steps with eight isolated intermediates and utilizes a wide range of chemical techniques and technologies to build-up this complex drug. All three stereocenters are introduced using enzyme or metal catalysis.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias , Humanos , Masculino , Neoplasias/tratamento farmacológico , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico
6.
Chemistry ; 26(69): 16281-16285, 2020 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-33017078

RESUMO

Stereoselective catalysts for the Pictet-Spengler reaction of tryptamines and aldehydes may allow a simple and fast approach to chiral 1-substituted tetrahydro-ß-carbolines. Although biocatalysts have previously been employed for the Pictet-Spengler reaction, not a single one accepts benzaldehyde and its substituted derivatives. To address this challenge, a combination of substrate walking and transfer of beneficial mutations between different wild-type backbones was used to develop a strictosidine synthase from Rauvolfia serpentina (RsSTR) into a suitable enzyme for the asymmetric Pictet-Spengler condensation of tryptamine and benzaldehyde derivatives. The double variant RsSTR V176L/V208A accepted various ortho-, meta- and para-substituted benzaldehydes and produced the corresponding chiral 1-aryl-tetrahydro-ß-carbolines with up to 99 % enantiomeric excess.


Assuntos
Carbolinas , Caminhada , Biocatálise , Catálise , Estereoisomerismo
7.
Chembiochem ; 18(11): 1022-1026, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28334484

RESUMO

Amine transaminase (ATA) catalyzing stereoselective amination of prochiral ketones is an attractive alternative to transition metal catalysis. As wild-type ATAs do not accept sterically hindered ketones, efforts to widen the substrate scope to more challenging targets are of general interest. We recently designed ATAs to accept aromatic and thus planar bulky amines, with a sequence-based motif that supports the identification of novel enzymes. However, these variants were not active against 2,2-dimethyl-1-phenyl-propan-1-one, which carries a bulky tert-butyl substituent adjacent to the carbonyl function. Here, we report a solution for this type of substrate. The evolved ATAs perform asymmetric synthesis of the respective R amine with high conversions by using either alanine or isopropylamine as amine donor.


Assuntos
Aminas , Evolução Molecular Direcionada , Engenharia de Proteínas/métodos , Transaminases/genética , Aminação , Substituição de Aminoácidos , Biocatálise , Simulação por Computador , Especificidade por Substrato
8.
Chembiochem ; 18(17): 1703-1706, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28722796

RESUMO

NADP(H)-dependent imine reductases (IREDs) are of interest in biocatalytic research due to their ability to generate chiral amines from imine/iminium substrates. In reaction protocols involving IREDs, glucose dehydrogenase (GDH) is generally used to regenerate the expensive cofactor NADPH by oxidation of d-glucose to gluconolactone. We have characterized different IREDs with regard to reduction of a set of bicyclic iminium compounds and have utilized 1 H NMR and GC analyses to determine degree of substrate conversion and product enantiomeric excess (ee). All IREDs reduced the tested iminium compounds to the corresponding chiral amines. Blank experiments without IREDs also showed substrate conversion, however, thus suggesting an iminium reductase activity of GDH. This unexpected observation was confirmed by additional experiments with GDHs of different origin. The reduction of C=N bonds with good levels of conversion (>50 %) and excellent enantioselectivity (up to >99 % ee) by GDH represents a promiscuous catalytic activity of this enzyme.


Assuntos
Glucose 1-Desidrogenase/metabolismo , Iminas/metabolismo , Bacillus subtilis/enzimologia , Biocatálise , Cromatografia Gasosa , Glucose/metabolismo , Iminas/química , Espectroscopia de Ressonância Magnética , NADP/metabolismo , Oxirredução , Estereoisomerismo , Especificidade por Substrato
9.
Org Biomol Chem ; 14(43): 10249-10254, 2016 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-27739550

RESUMO

Application of amine transaminases (ATAs) for stereoselective amination of prochiral ketones represents an environmentally benign and economically attractive alternative to transition metal catalyzed asymmetric synthesis. However, the restrictive substrate scope has limited the conversion typically to non-sterically demanding scaffolds. Recently, we reported on the identification and design of fold class I ATAs that effect a highly selective asymmetric synthesis of a set of chiral aromatic bulky amines from the corresponding ketone precursors in high yield. However, for the specific amine synthetic approach extension targeted here, the selective formation of an exo- vs. endo-isomer, these biocatalysts required additional refinement. The chosen substrate (exo-3-amino-8-aza-bicyclo[3.2.1]oct-8-yl-phenyl-methanone), apart from its pharmacological relevance, is a demanding target for ATAs as the bridged bicyclic ring provides substantial steric challenges. Protein engineering combining rational design and directed evolution enabled the identification of an ATA variant which catalyzes the specific synthesis of the target exo-amine with >99.5% selectivity.


Assuntos
Aminas/química , Aminas/síntese química , Engenharia de Proteínas , Transaminases/genética , Transaminases/metabolismo , Biocatálise , Domínio Catalítico , Técnicas de Química Sintética , Cetonas/química , Modelos Moleculares , Rhodobacteraceae/enzimologia , Estereoisomerismo , Especificidade por Substrato , Transaminases/química
10.
J Labelled Comp Radiopharm ; 59(14): 635-639, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27196363

RESUMO

Various agonists of the trace amine-associate receptor 1, under consideration as potential clinical development candidates, were labelled with carbon-14 for use in preclinical in vitro and in vivo drug metabolism studies. Herein, the [14 C]-radiosynthesis of 2-phenyl-substituted morpholines 1 is described. After evaluating and optimizing different synthetic routes, 4-iodonitrobenzene 3 was selected as starting material for the 14-step synthesis. Incorporation of carbon-14 into the acetyl moiety allowed a safe and efficient synthesis of [14 C]-labelled 4-nitroacetophenone 2 in five steps and 38% yield. Further transformation of 2 to the target compounds 1 was achieved in a 9-step synthesis. In a representative example, [14 C]-labelled 1 was obtained in an overall yield of 11% and was isolated in >99% radiochemical purity and a specific activity of 47 mCi/mmol.


Assuntos
Radioisótopos de Carbono/química , Morfolinas/química , Morfolinas/síntese química , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Técnicas de Química Sintética , Marcação por Isótopo , Morfolinas/isolamento & purificação , Morfolinas/farmacologia , Estereoisomerismo
11.
Chembiochem ; 16(12): 1749-56, 2015 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-26044455

RESUMO

Recent investigations on imine reductases (IREDs) have enriched the toolbox of potential catalysts for accessing chiral amines, which are important building blocks for the pharmaceutical industry. Herein, we describe the characterization of 20 new IREDs. A C-terminal domain clustering of the bacterial protein-sequence space was performed to identify the novel IRED candidates. Each of the identified enzymes was characterized against a set of nine cyclic imine model substrates. A refined clustering towards putative active-site residues was performed and was consistent both with our screening and previously reported results. Finally, preparative scale experiments on a 100 mg scale with two purified IREDs, IR_20 from Streptomyces tsukubaensis and IR_23 from Streptomyces vidiochromogenes, were carried out to provide (R)-2-methylpiperidine in 98% ee (71% yield) and (R)-1-methyl-1,2,3,4-tetrahydroisoquinoline in >98% ee (82% yield).


Assuntos
Proteínas de Bactérias/genética , Iminas/química , Modelos Moleculares , Oxirredutases/genética , Proteínas de Bactérias/química , Domínio Catalítico , Estrutura Molecular , Oxirredutases/química , Oxirredutases/metabolismo , Bibliotecas de Moléculas Pequenas/química
12.
Commun Chem ; 7(1): 46, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38418529

RESUMO

Semi-rational enzyme engineering is a powerful method to develop industrial biocatalysts. Profiting from advances in molecular biology and bioinformatics, semi-rational approaches can effectively accelerate enzyme engineering campaigns. Here, we present the optimization of a ketoreductase from Sporidiobolus salmonicolor for the chemo-enzymatic synthesis of ipatasertib, a potent protein kinase B inhibitor. Harnessing the power of mutational scanning and structure-guided rational design, we created a 10-amino acid substituted variant exhibiting a 64-fold higher apparent kcat and improved robustness under process conditions compared to the wild-type enzyme. In addition, the benefit of algorithm-aided enzyme engineering was studied to derive correlations in protein sequence-function data, and it was found that the applied Gaussian processes allowed us to reduce enzyme library size. The final scalable and high performing biocatalytic process yielded the alcohol intermediate with ≥ 98% conversion and a diastereomeric excess of 99.7% (R,R-trans) from 100 g L-1 ketone after 30 h. Modelling and kinetic studies shed light on the mechanistic factors governing the improved reaction outcome, with mutations T134V, A238K, M242W and Q245S exerting the most beneficial effect on reduction activity towards the target ketone.

13.
Org Lett ; 25(49): 8927-8931, 2023 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-38051775

RESUMO

The first examples of a practical procedure for a lipase-catalyzed dynamic kinetic resolution of PEGylated N-alkyl amino esters is reported. This method allows for the preparation of a broad range of aromatic and aliphatic enantiomerically enriched N-alkyl unnatural amino acids in up to 98% yield and 99% ee. We have found that PEGylated esters have a significant solubility advantage and improved reactivity over traditional hydrophobic lipase substrates, thereby allowing for efficient and scalable dynamic kinetic resolution (DKR) under aqueous conditions.


Assuntos
Aminoácidos , Ésteres , Ésteres/química , Aminoácidos/química , Catálise , Lipase/metabolismo , Cinética , Polietilenoglicóis , Estereoisomerismo
14.
Sci Rep ; 12(1): 3019, 2022 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-35194101

RESUMO

Alcohol oxidation for the generation of carbonyl groups, is an essential reaction for the preparation of fine chemicals. Although a number of chemical procedures have been reported, biocatalysis is a promising alternative for more sustainable and selective processes. To speed up the discovery of novel (bio)catalysts for industrial applications, efficient screening approaches need to be established. Here, we report on an enzyme-mediated alcohol oxidation screening platform to rapidly detect the activities and selectivities of three classes of biocatalysts; ketoreductases (KREDs), alcohol oxidases (AlcOXs) and laccase-mediator systems (LMSs) with diverse substrates.

15.
J Med Chem ; 63(18): 10287-10306, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32787079

RESUMO

Despite the broad implications of the cannabinoid type 2 receptor (CB2) in neuroinflammatory processes, a suitable CB2-targeted probe is currently lacking in clinical routine. In this work, we synthesized 15 fluorinated pyridine derivatives and tested their binding affinities toward CB2 and CB1. With a sub-nanomolar affinity (Ki for CB2) of 0.8 nM and a remarkable selectivity factor of >12,000 over CB1, RoSMA-18-d6 exhibited outstanding in vitro performance characteristics and was radiofluorinated with an average radiochemical yield of 10.6 ± 3.8% (n = 16) and molar activities ranging from 52 to 65 GBq/µmol (radiochemical purity > 99%). [18F]RoSMA-18-d6 showed exceptional CB2 attributes as demonstrated by in vitro autoradiography, ex vivo biodistribution, and positron emission tomography (PET). Further, [18F]RoSMA-18-d6 was used to detect CB2 upregulation on postmortem human ALS spinal cord tissues. Overall, these results suggest that [18F]RoSMA-18-d6 is a promising CB2 PET radioligand for clinical translation.


Assuntos
Piridinas/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Receptor CB2 de Canabinoide/metabolismo , Animais , Encéfalo/diagnóstico por imagem , Radioisótopos de Flúor/química , Humanos , Ligantes , Masculino , Simulação de Acoplamento Molecular , Estrutura Molecular , Tomografia por Emissão de Pósitrons , Piridinas/síntese química , Piridinas/farmacocinética , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Ratos Wistar , Medula Espinal/diagnóstico por imagem , Baço/diagnóstico por imagem , Relação Estrutura-Atividade , Trítio/química
16.
J Org Chem ; 73(13): 4895-902, 2008 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-18517254

RESUMO

A new, enantioselective synthesis of the influenza neuraminidase inhibitor prodrug oseltamivir phosphate 1 (Tamiflu) and its enantiomer ent-1 starting from cheap, commercially available 2,6-dimethoxyphenol 10 is described. The main features of this approach comprise the cis-hydrogenation of 5-(1-ethyl-propoxy)-4,6-dimethoxy-isophthalic acid diethyl ester (6a) and the desymmetrization of the resultant all-cis meso-diesters 7a and 7b, respectively. Enzymatic hydrolysis of the meso-diester 7b with pig liver esterase afforded the (S)-monoacid 8b, which was converted into cyclohexenol 17 via a Curtius degradation and a base-catalyzed decarboxylative elimination of the Boc-protected oxazolidinone 14. Introduction of the second amino function via S(N)2 substitution of the corresponding triflate 18 with NaN3 followed by azide reduction, N-acetylation, and Boc-deprotection gave oseltamivir phosphate 1 in a total of 10 steps and an overall yield of approximately 30%. The enantiomer ent-1 was similarly obtained via an enzymatic desymmetrization of meso-diester 7a with Aspergillus oryzae lipase, providing the (R)-monoacid ent-8a.


Assuntos
Antivirais/síntese química , Ácidos Dicarboxílicos/química , Oseltamivir/síntese química , Estrutura Molecular
17.
Org Lett ; 19(18): 4806-4809, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28858516

RESUMO

A highly efficient asymmetric synthesis of the Akt kinase inhibitor ipatasertib (1) is reported. The bicyclic pyrimidine 2 starting material was prepared via a nitrilase biocatalytic resolution, halogen-metal exchange/anionic cyclization, and a highly diastereoselective biocatalytic ketone reduction as key steps. The route also features a halide activated, Ru-catalyzed asymmetric hydrogenation of a vinylogous carbamic acid to produce α-aryl-ß-amino acid 3 in high yield and enantioselectivity. The API was assembled in a convergent manner through a late-stage amidation/deprotection/monohydrochloride salt formation sequence.

18.
Nat Chem ; 8(11): 1076-1082, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27768108

RESUMO

The use of transaminases to access pharmaceutically relevant chiral amines is an attractive alternative to transition-metal-catalysed asymmetric chemical synthesis. However, one major challenge is their limited substrate scope. Here we report the creation of highly active and stereoselective transaminases starting from fold class I. The transaminases were developed by extensive protein engineering followed by optimization of the identified motif. The resulting enzymes exhibited up to 8,900-fold higher activity than the starting scaffold and are highly stereoselective (up to >99.9% enantiomeric excess) in the asymmetric synthesis of a set of chiral amines bearing bulky substituents. These enzymes should therefore be suitable for use in the synthesis of a wide array of potential intermediates for pharmaceuticals. We also show that the motif can be engineered into other protein scaffolds with sequence identities as low as 70%, and as such should have a broad impact in the field of biocatalytic synthesis and enzyme engineering.


Assuntos
Aminas/metabolismo , Transaminases/metabolismo , Aminas/química , Sítios de Ligação , Biocatálise , Domínio Catalítico , Cinética , Mutagênese Sítio-Dirigida , Engenharia de Proteínas , Quinonas/química , Quinonas/metabolismo , Estereoisomerismo , Especificidade por Substrato , Transaminases/química , Transaminases/genética
19.
Protein Eng Des Sel ; 18(7): 345-57, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15930043

RESUMO

Pyruvate decarboxylase from Zymomonas mobilis (PDC) and benzoylformate decarboxylase from Pseudomonas putida (BFD) are thiamine diphosphate-dependent enzymes that decarboxylate 2-keto acids. Although they share a common homotetrameric structure they have relatively low sequence similarity and different substrate spectra. PDC prefers short aliphatic substrates whereas BFD favours aromatic 2-keto acids. These preferences are also reflected in their carboligation reactions. PDC catalyses the conversion of benzaldehyde and acetaldehyde to (R)-phenylacetylcarbinol and predominantly (S)-acetoin, whereas (R)-benzoin and mainly (S)-2-hydroxypropiophenone are the products of BFD catalysis. Comparison of the X-ray structures of both enzymes identified two residues in each that were likely to be involved in determining substrate specificity. Site-directed mutagenesis was used to interchange these residues in both BFD and PDC. The substrate range and kinetic parameters for the decarboxylation reaction were studied for each variant. The most successful variants, PDCI472A and BFDA460I, catalysed the decarboxylation of benzoylformate and pyruvate, respectively, although both variants now preferred the long-chain aliphatic substrates, 2-ketopentanoic and 2-ketohexanoic acid. With respect to the carboligase activity, PDCI472A proved to be a real chimera between PDC and BFD whereas BFDA460I/F464I provided the most interesting result with an almost complete reversal of the stereochemistry of its 2-hydroxypropiophenone product.


Assuntos
Carboxiliases/genética , Carboxiliases/metabolismo , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Piruvato Descarboxilase/genética , Piruvato Descarboxilase/metabolismo , Especificidade por Substrato/genética , Zymomonas/enzimologia , Zymomonas/genética , Sequência de Aminoácidos , Mutagênese Sítio-Dirigida , Alinhamento de Sequência , Tiamina Pirofosfato/metabolismo
20.
J Med Chem ; 56(23): 9789-801, 2013 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-24224654

RESUMO

Starting from the weakly active dual CatS/K inhibitor 5, structure-based design supported by X-ray analysis led to the discovery of the potent and selective (>50,000-fold vs CatK) cyclopentane derivative 22 by exploiting specific ligand-receptor interactions in the S2 pocket of CatS. Changing the central cyclopentane scaffold to the analogous pyrrolidine derivative 57 decreased the enzyme as well as the cell-based activity significantly by 24- and 69-fold, respectively. The most promising scaffold identified was the readily accessible proline derivative (e.g., 79). This compound, with an appealing ligand efficiency (LE) of 0.47, included additional structural modifications binding in the S1 and S3 pockets of CatS, leading to favorable in vitro and in vivo properties. Compound 79 reduced IL-2 production in a transgenic DO10.11 mouse model of antigen presentation in a dose-dependent manner with an ED50 of 5 mg/kg.


Assuntos
Catepsinas/antagonistas & inibidores , Inibidores de Cisteína Proteinase/síntese química , Animais , Ciclopentanos/química , Inibidores de Cisteína Proteinase/farmacocinética , Humanos , Camundongos , Prolina/análogos & derivados , Relação Estrutura-Atividade
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