A molecular mechanism for the enzymatic methylation of nitrogen atoms within peptide bonds

This work was financially supported by ETH Zürich, University of Minnesota, the Swiss National Science Foundation (grant nos. 31003A_149512 and 200021–159713), Wellcome Trust (094476/Z/10/Z), ERC (NCB-TNT 339367), and BBSRC (BB/R018189/1).The peptide bond, the defining feature of proteins, governs peptide chemistry by abolishing nucleophilicity of the nitrogen. This and the planarity of the peptide bond arise from the delocalization of the lone pair of electrons on the nitrogen atom into the adjacent carbonyl. While chemical methylation of an amide bond uses a strong base to generate the imidate, OphA, the precursor protein of the fungal peptide macrocycle omphalotin A, self-hypermethylates amides at pH 7 using S-adenosyl methionine (SAM) as cofactor. The structure of OphA reveals a complex catenane-like arrangement in which the peptide substrate is clamped with its amide nitrogen aligned for nucleophilic attack on the methyl group of SAM. Biochemical data and computational modeling suggest a base-catalyzed reaction with the protein stabilizing the reaction intermediate. Backbone N-methylation of peptides enhances their protease resistance and membrane permeability, a property that holds promise for applications to medicinal chemistry.Publisher PDFPeer reviewe

Engineering an omega-Transaminase for the Efficient Production of a Chiral Sacubitril Precursor

An omega-transaminase was engineered for the efficient production of a chiral precursor to sacubitril, (2R,4S)-5-([1,1'-biphenyl]-4-yl)-4-amino-2-methylpentanoic acid, a key component in the blockbuster heart failure drug Entresto®. Starting from an enzyme with trace activity and preference for the undesired diastereoisomer, eleven rounds of enzyme evolution were performed. The resultant variant, CDX-043, showed high productivity giving 90% conversion at 75 g/L substrate concentration with 1% enzyme loading with respect to the substrate in 24 h and without the use of an organic co-solvent. The product diastereomeric purity towards the desired (2R,4S)-stereoisomer was > 99.9:0.1 d.r. This variant also exhibited high process robustness and could tolerate reaction temperatures up to 65 °C and isopropylamine concentrations of at least 2 M. A structural analysis of the enzyme variants gave insight into how the mutations affected activity and selectivity. This new enzyme variant allows for the efficient and cost-effective production of sacubitril at large scale

Toolbox study for application of hydrogen peroxide as a versatile, safe and industrially-relevant green oxidant in continuous flow mode

Hydrogen peroxide embodies an ideal oxidant in terms of atom-economy, availability and green metrics. However, its use has been limited because of risks associated with disproportionation and the exothermic potential of oxidizing reaction mixtures. This study aims to showcase the versatility of hydrogen peroxide in a range of oxidations, while ensuring that such processes operate practically and with a defined and minimized risk. To offset the hazards of using peroxides, continuous-flow equipment was utilized to limit the volume of unquenched peroxides and to maintain process control. A methodological approach was established relying on kinetic and calorimetric understanding. Finally, scalability was highlighted in an organocatalysed heterocyclic N-oxidation using a cascade of stirred tank reactors with nitrogen flushing

WHO 2004 criteria and CK19 are reliable prognostic markers in pancreatic endocrine tumors

BACKGROUND: It is difficult to predict the biologic behavior of pancreatic endocrine tumors in absence of metastases or invasion into adjacent organs. The World Health Organization (WHO) has proposed in 2004 size, angioinvasion, mitotic activity, and MIB1 proliferation index as prognostic criteria. Our aim was to test retrospectively the predictive value of these 2004 WHO criteria and of CK19, CD99, COX2, and p27 immunohistochemistry in a large series of patients with long-term follow-up. DESIGN: The histology of 216 pancreatic endocrine tumor specimens was reviewed and the tumors were reclassified according to the 2004 WHO classification. The prognostic value of the WHO classification and the histopathologic criteria necrosis and nodular fibrosis was tested in 113 patients. A tissue microarray was constructed for immunohistochemical staining. The staining results were scored quantitatively for MIB1 and semiquantitatively for CK19, COX2, p27, and CD99. The prognostic value of these markers was tested in 93 patients. RESULTS: The stratification of the patients into 4 risk groups according to the 2004 WHO classification was reliable with regard to both time span to relapse and tumor-specific death. In a multivariate analysis, the CK19 status was shown to be independent of the WHO criteria. By contrast, the prognostic significance of COX2, p27, and CD99 could not be confirmed. CONCLUSIONS: The 2004 WHO classification with 4 risk groups is very reliable for predicting both disease-free survival and the time span until tumor-specific death. CK19 staining is a potential additional prognostic marker independent from the WHO criteria for pancreatic endocrine tumors

Toward a scalable synthesis and process for EMA401. Part III: Using an engineered phenylalanine ammonia lyase enzyme to synthesize a non-natural phenylalanine derivative

A process using engineered phenylalanine ammonia lyase (PAL) enzymes was developed as part of an alternative route to a key intermediate of olodanrigan (EMA401). In the first part of the manuscript, the detailed results from a screening for the optimal reaction conditions are presented, followed by the discussion of several work-up strategies investigated. In the PAL catalyzed reaction, 70–80% conversion of a cinnamic acid derivative to the corresponding phenylalanine derivative could be achieved. The phenylalanine derivative was subsequently telescoped to a Pictet-Spengler reaction with formaldehyde and the corresponding tetrahydroisoquinoline derivative was isolated in 60–70% yield with >99.9:0.1 er. Based on our screenings, carbonate/carbamate buffered ammonia at 9–10 M NH3 concentration and pH 9.5–10.5 were found as the optimal conditions. Enzyme loadings down to 2.5wt% (E:S 1:40 w/w) could be achieved and substrate concentrations between 3–9 v/w (1.17–0.39 M) were found to be compatible with the reaction conditions. A temperature gradient was applied in the final process: a pre-equilibrium was established at 45 °C, before making use of the temperature-dependence of the entropy term with subsequent cooling to 20 °C and achieving maximum conversion. This temperature gradient also allowed balancing enzyme stability (low at 45 °C, high at 20 °C) with activity (high at 45 °C, low at 20 °C) in order to achieve optimal conversion (low at 45 °C, high at 20 °C). From the various work-up operations investigated, a sequence consisting of denaturation of the enzyme, followed by NH3/CO2 removal by distillation, acidification and telescoping to the subsequent Pictet-Spengler cyclization was our preferred approach. The process presented in this study is a more sustainable, shorter and more cost effective alternative to the previous process

High-affinity inhibitors of Zymomonas mobilis tRNA-guanine transglycosylase through convergent optimization.

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Application of Transition-Metal Catalysis, Biocatalysis and Flow Chemistry as State-of-the-Art Technologies in the Synthesis of LCZ696

LCZ696 is a novel treatment for patients suffering from heart failure that combines the two active pharmaceutical ingredients sacubitril and valsartan in a single chemical compound. While valsartan is an established drug substance, a new manufacturing process suitable for large-scale commercial production had to be developed for sacubitril. The use of chemocatalysis, biocatalysis, and flow chemistry as state-of-the-art technologies allowed to efficiently build up the structure of sacubitril and achieve the defined performance targets