Design of an in vitro biocatalytic cascade for the manufacture of islatravir

Science ◽  
2019 ◽  
Vol 366 (6470) ◽  
pp. 1255-1259 ◽  
Author(s):  
Mark A. Huffman ◽  
Anna Fryszkowska ◽  
Oscar Alvizo ◽  
Margie Borra-Garske ◽  
Kevin R. Campos ◽  
...  
Keyword(s):  
Chemical Synthesis ◽  
Directed Evolution ◽  
Building Blocks ◽  
Hiv Treatment ◽  
Pharmaceutical Manufacturing ◽  
Enzymatic Reactions ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions

Enzyme-catalyzed reactions have begun to transform pharmaceutical manufacturing, offering levels of selectivity and tunability that can dramatically improve chemical synthesis. Combining enzymatic reactions into multistep biocatalytic cascades brings additional benefits. Cascades avoid the waste generated by purification of intermediates. They also allow reactions to be linked together to overcome an unfavorable equilibrium or avoid the accumulation of unstable or inhibitory intermediates. We report an in vitro biocatalytic cascade synthesis of the investigational HIV treatment islatravir. Five enzymes were engineered through directed evolution to act on non-natural substrates. These were combined with four auxiliary enzymes to construct islatravir from simple building blocks in a three-step biocatalytic cascade. The overall synthesis requires fewer than half the number of steps of the previously reported routes.

scholarly journals One-step catalytic asymmetric synthesis of all-syn deoxypropionate motif from propylene: Total synthesis of (2R,4R,6R,8R)-2,4,6,8-tetramethyldecanoic acid

2016 ◽  
Vol 113 (11) ◽  
pp. 2857-2861 ◽  
Author(s):  
Yusuke Ota ◽  
Toshiki Murayama ◽  
Kyoko Nozaki
Keyword(s):  
Building Blocks ◽  
Single Step ◽  
Enzymatic Reactions ◽  
Stereogenic Centers ◽  
Complex Building ◽  
One Step ◽  
Simple Molecules ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Major Acid

In nature, many complex structures are assembled from simple molecules by a series of tailored enzyme-catalyzed reactions. One representative example is the deoxypropionate motif, an alternately methylated alkyl chain containing multiple stereogenic centers, which is biosynthesized by a series of enzymatic reactions from simple building blocks. In organic synthesis, however, the majority of the reported routes require the syntheses of complex building blocks. Furthermore, multistep reactions with individual purifications are required at each elongation. Here we show the construction of the deoxypropionate structure from propylene in a single step to achieve a three-step synthesis of (2R,4R,6R,8R)-2,4,6,8-tetramethyldecanoic acid, a major acid component of a preen-gland wax of the graylag goose. To realize this strategy, we focused on the coordinative chain transfer polymerization and optimized the reaction condition to afford a stereo-controlled oligomer, which is contrastive to the other synthetic strategies developed to date that require 3–6 steps per unit, with unavoidable byproduct generation. Furthermore, multiple oligomers with different number of deoxypropionate units were isolated from one batch, showing application to the construction of library. Our strategy opens the door for facile synthetic routes toward other natural products that share the deoxypropionate motif.

Computationally Augmented Retrosynthesis: Total Synthesis of Paspaline A and Emindole PB

2018 ◽  
Author(s):  
Timothy Newhouse ◽  
Daria E. Kim ◽  
Joshua E. Zweig
Keyword(s):  
Chemical Synthesis ◽  
Total Synthesis ◽  
Small Molecules ◽  
First Principles ◽  
Quantum Chemical ◽  
Computational Analysis ◽  
Empirical Evaluation ◽  
Synthetic Strategy ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions

The diverse molecular architectures of terpene natural products are assembled by exquisite enzyme-catalyzed reactions. Successful recapitulation of these transformations using chemical synthesis is hard to predict from first principles and therefore challenging to execute. A means of evaluating the feasibility of such chemical reactions would greatly enable the development of concise syntheses of complex small molecules. Herein, we report the computational analysis of the energetic favorability of a key bio-inspired transformation, which we use to inform our synthetic strategy. This approach was applied to synthesize two constituents of the historically challenging indole diterpenoid class, resulting in a concise route to (–)-paspaline A in 9 steps from commercially available materials and the first pathway to and structural confirmation of emindole PB in 13 steps. This work highlights how traditional retrosynthetic design can be augmented with quantum chemical calculations to reveal energetically feasible synthetic disconnections, minimizing time-consuming and expensive empirical evaluation.

scholarly journals Use of isotopically labeled substrates reveals kinetic differences between human and bacterial serine palmitoyltransferase

2019 ◽  
Vol 60 (5) ◽  
pp. 953-962 ◽  
Author(s):  
Peter J. Harrison ◽  
Kenneth Gable ◽  
Niranjanakumari Somashekarappa ◽  
Van Kelly ◽  
David J. Clarke ◽  
...  
Keyword(s):  
In Vivo Studies ◽  
Serine Palmitoyltransferase ◽  
Biochemical Pathways ◽  
Catalytic Mechanisms ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
The Impact ◽  
Sphingolipid Biosynthesis

Isotope labels are frequently used tools to track metabolites through complex biochemical pathways and to discern the mechanisms of enzyme-catalyzed reactions. Isotopically labeled l-serine is often used to monitor the activity of the first enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT), as well as labeling downstream cellular metabolites. Intrigued by the effect that isotope labels may be having on SPT catalysis, we characterized the impact of different l-serine isotopologues on the catalytic activity of recombinant SPT isozymes from humans and the bacterium Sphingomonas paucimobilis. Our data show that S. paucimobilis SPT activity displays a clear isotope effect with [2,3,3-D]l-serine, whereas the human SPT isoform does not. This suggests that although both human and S. paucimobilis SPT catalyze the same chemical reaction, there may well be underlying subtle differences in their catalytic mechanisms. Our results suggest that it is the activating small subunits of human SPT that play a key role in these mechanistic variations. This study also highlights that it is important to consider the type and location of isotope labels on a substrate when they are to be used in in vitro and in vivo studies.

scholarly journals Ionic liquids as (co)solvents for enzymatic reactions

2006 ◽  
Vol 12 (3) ◽  
pp. 181-186 ◽  
Author(s):  
Muzafera Paljevac ◽  
Maja Habulin ◽  
Zeljko Knez
Keyword(s):  
Ionic Liquid ◽  
Ionic Liquids ◽  
Immobilized Lipase ◽  
Rhizomucor Miehei ◽  
Reaction Rates ◽  
Synthetic Activity ◽  
Enzymatic Reactions ◽  
Lipozyme Rm Im ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions

Ionic liquids are low melting point salts that represent an exciting new class of reaction solvents. Many reactions show advantages when carried out in ionic liquids, either with regard to enhanced reaction rates, improved selectivity, or easier reuse of catalysts. To ascertain the influence of ionic liquids on the enzyme activity, three different ionic liquids 1-butyl-3-methylimidazolium chloride ([bmim] [CI]) 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim] [PF6]) and 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF4]) were synthesized and investigated as potential media for the hydrolysis of carboxymethyl cellulose, catalyzed by non-immobilized cellulase from Humicola insolens (Celluzyme 0,7T) and for ester synthesis, catalyzed by immobilized lipase from Rhizomucor miehei (Lipozyme RM IM). Enzyme-catalyzed reactions were performed in a batch stirred reactor at atmospheric pressure. Celluzyme 0,7T showed better activity in hydrophobic ionic liquid ([bmim] [PF6]), as compared to hydrophilic ionic liquid ([bmim] [BF4]). In the case of Lipozyme RM IM, the synthetic activity of the enzyme was strongly reduced by incubating the enzyme in ionic liquids.

scholarly journals Catalytic iron-carbene intermediate revealed in a cytochromeccarbene transferase

2018 ◽  
Vol 115 (28) ◽  
pp. 7308-7313 ◽  
Author(s):  
Russell D. Lewis ◽  
Marc Garcia-Borràs ◽  
Matthew J. Chalkley ◽  
Andrew R. Buller ◽  
K. N. Houk ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Computational Methods ◽  
Active Site ◽  
Noncovalent Interactions ◽  
Chemical Transformations ◽  
Rhodothermus Marinus ◽  
Geometrical Constraints ◽  
Carbene Transfer ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions

Recently, heme proteins have been discovered and engineered by directed evolution to catalyze chemical transformations that are biochemically unprecedented. Many of these nonnatural enzyme-catalyzed reactions are assumed to proceed through a catalytic iron porphyrin carbene (IPC) intermediate, although this intermediate has never been observed in a protein. Using crystallographic, spectroscopic, and computational methods, we have captured and studied a catalytic IPC intermediate in the active site of an enzyme derived from thermostableRhodothermus marinus(Rma) cytochromec. High-resolution crystal structures and computational methods reveal how directed evolution created an active site for carbene transfer in an electron transfer protein and how the laboratory-evolved enzyme achieves perfect carbene transfer stereoselectivity by holding the catalytic IPC in a single orientation. We also discovered that the IPC inRmacytochromechas a singlet ground electronic state and that the protein environment uses geometrical constraints and noncovalent interactions to influence different IPC electronic states. This information helps us to understand the impressive reactivity and selectivity of carbene transfer enzymes and offers insights that will guide and inspire future engineering efforts.

Recent Advance in Organocatalyzed Asymmetric Reduction of Prochiral Ketones: An Update

Synthesis ◽  
2021 ◽  
Author(s):  
Xu-Long Qin ◽  
Li-Jun Xu ◽  
Fu-She Han
Keyword(s):  
Asymmetric Reduction ◽  
Building Blocks ◽  
Short Review ◽  
Chiral Alcohols ◽  
Comprehensive Overview ◽  
Synthetic Intermediates ◽  
Prochiral Ketones ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions ◽  
Easy Operation

Chiral alcohols are important synthetic intermediates or building blocks for the diverse synthesis of drugs, agrochemicals, and natural products. Asymmetric reduction of prochiral ketones has been the most popularly investigated method for accessing chiral alcohols. In this regard, the organocatalyzed asymmetric reduction as a complementary of transition-metal- and enzyme-catalyzed reactions have attracted tremendous interest in the past decades due to the nature of metal-free and easy operation, as well as, principly, the ease of recovery and reuse of catalysts. Following up a comprehensive overview on organocatalyzed asymmetric reduction of prochiral ketones in early 2018, this short review is intended to summarize the recent progress in this area from the beginning of the year 2018 to the end of Aug. 2021.

scholarly journals Star-shaped Triazine-derivatives: would they crossbind SARS-CoV-2 spike helices?

2021 ◽  
Author(s):  
julio coll
Keyword(s):  
Neural Network ◽  
Severe Acute Respiratory Syndrome ◽  
Chemical Synthesis ◽  
Building Blocks ◽  
Water Soluble ◽  
Fusion Inhibition ◽  
Host Membrane ◽  
Bottom Cavity ◽  
Triazine Derivatives

This work describes synthesizable water-soluble Triazine-derivatives computationally crossbinding the S spike helices of Severe Acute Respiratory Syndrome coronavirus (SARS)-CoV-2. The "spring-loaded switch-folding” (S-SLSF) α-helices included in the S homotrimer top-to-bottom cavity and implicated in viral-host membrane fusion were targeted by star-shaped Trihydroxyl-Triphenyl-Triazines (TTT) leads at subnanomolar binding-scores. Exploration of in silico leads among millions of molecular candidates, included several similar searches, core-replacement, fragment extensions, or convolutional neural network deep-screening combined with hundreds of water-soluble lead-derivatives identified by manual iterations and commercially available building-blocks for chemical synthesis. The lead-derivatives are briefly discussed for in vitro validation and possibilities of fusion inhibition substituting mutations.

Computationally Augmented Retrosynthesis: Total Synthesis of Paspaline A and Emindole PB

2018 ◽  
Author(s):  
Timothy Newhouse ◽  
Daria E. Kim ◽  
Joshua E. Zweig
Keyword(s):  
Chemical Synthesis ◽  
Total Synthesis ◽  
Small Molecules ◽  
First Principles ◽  
Quantum Chemical ◽  
Computational Analysis ◽  
Empirical Evaluation ◽  
Synthetic Strategy ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions

The diverse molecular architectures of terpene natural products are assembled by exquisite enzyme-catalyzed reactions. Successful recapitulation of these transformations using chemical synthesis is hard to predict from first principles and therefore challenging to execute. A means of evaluating the feasibility of such chemical reactions would greatly enable the development of concise syntheses of complex small molecules. Herein, we report the computational analysis of the energetic favorability of a key bio-inspired transformation, which we use to inform our synthetic strategy. This approach was applied to synthesize two constituents of the historically challenging indole diterpenoid class, resulting in a concise route to (–)-paspaline A in 9 steps from commercially available materials and the first pathway to and structural confirmation of emindole PB in 13 steps. This work highlights how traditional retrosynthetic design can be augmented with quantum chemical calculations to reveal energetically feasible synthetic disconnections, minimizing time-consuming and expensive empirical evaluation.

scholarly journals Factors like Dilution and Mixing Influence Enzymatic Reactions

2020 ◽  
Vol 19 (4) ◽  
Author(s):  
Mahin Basha Syed ◽  
Venkatanagraraju Erumalla
Keyword(s):  
Enzyme Activity ◽  
Substrate Concentration ◽  
Enzymatic Reaction ◽  
Enzyme Reaction ◽  
Enzyme Concentration ◽  
Enzymatic Reactions ◽  
First Report ◽  
Enzyme Substrate ◽  
Catalyzed Reactions ◽  
Enzyme Catalyzed Reactions

Enzyme-catalyzed reactions were influenced by many factors. The enzyme reacts with the substrate and converts it into products. Enzymes are influenced by temperature, pH, enzyme concentration, and substrate concentration. This paper evaluates the hypothesis of factors that may influence enzyme activity. Two more factors that affects enzyme activity are dilution and mixing. In enzyme-substrate reactions, the small amount of dilution and mixing will not affect the enzyme activity. Dilution and mixing do not slowdowns the enzyme reaction but it enhances the enzymatic reaction up to a certain limit. Increase in dilution results in less interaction of enzyme substrate, which causes a decrease in the rate of reactions. To the best of our knowledge, this is the first report to shows that, factors like mixing and dilution also affect enzyme and substrate reactions.

scholarly journals In vitro biosynthesis of ATP from adenosine and polyphosphate

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Chuanqi Sun ◽  
Zonglin Li ◽  
Xiao Ning ◽  
Wentian Xu ◽  
Zhimin Li
Keyword(s):  
Adenosine Kinase ◽  
Inorganic Polyphosphate ◽  
One Pot ◽  
Atp Production ◽  
Wide Ph Range ◽  
Catalyzed Reactions ◽  
In Vitro Biosynthesis ◽  
Enzyme Catalyzed Reactions

AbstractAdenosine triphosphate (ATP) acts as a crucial energy currency in vivo, and it is a widely used energy and/or phosphate donor for enzyme-catalyzed reactions in vitro. In this study, we established an in vitro multi-enzyme cascade system for ATP production. Using adenosine and inorganic polyphosphate (polyP) as key substrates, we combined adenosine kinase and two functionally distinct polyphosphate kinases (PPKs) in a one-pot reaction to achieve chain-like ATP regeneration and production. Several sources of PPK were screened and characterized, and two suitable PPKs were selected to achieve high rates of ATP production. Among these, Sulfurovum lithotrophicum PPK (SlPPK) exhibited excellent activity over a wide pH range (pH 4.0–9.0) and synthesized ATP from ADP using short-chain polyP. Furthermore, it had a half-life > 155.6 h at 45 °C. After optimizing the reaction conditions, we finally carried out the coupling-catalyzed reaction with different initial adenosine concentrations of 10, 20, and 30 mM. The highest yields of ATP were 76.0, 70.5, and 61.3%, respectively. Graphical Abstract

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