Peptidic catalysts developed by combinatorial screening methods

In recent years, oligopeptides have been developed as efficient catalysts for a range of important organic reactions, including acylation, silylation, oxidation, ester hydrolysis and aldol reactions. With many peptidic catalysts, high yields and chiral induction can be achieved under mild reaction conditions. Discovery and optimization of these catalysts typically involves the testing of compound collections and is therefore strongly linked to advances in combinatorial screening methods. This review summarizes recent developments in the field of catalytically active short-chain peptides, highlighting the combinatorial techniques that have facilitated their discovery.     

Localization properties of fluorescence cytochemical enzyme procedures

Fluorescence enzyme cytochemical procedures will contribute significantly to biomedical problems where knowledge of the enzymic composition of individual cells is important. Compared with the number of absorbance enzyme cytochemical methods, relatively few fluorescence procedures have been reported. In this paper, the merits of the described methods are discussed. A distinction is made between methods with and without a capture reaction. Only a few methods satisfy the requirement of accurate localization of the final product and high signal to noise ratios. Thus, there still is a need for valid fluorescence cytochemical enzyme methods. It is concluded that the bottle neck for valid fluorescence cytochemical enzyme methods is the development of efficient fluorogenic capture reactions for the primary enzyme products.     

Convenient methods for the synthesis of P1-farnesyl-P2-indicator diphosphates

To access P1-farnesyl-P2-indicator diphosphates, more efficient methods for the synthesis of farnesyl-phosphate and diphosphates were developed. The procedures reported here provide more flexible conditions than the conventional imidazolide and morpholidate coupling methods. Milder conditions for the synthesis of sensitive allylic diphosphates and greatly improved reaction efficiencies provide access to novel reagents for analysis of diphosphate-based enzymatic reactions.     

Chemical modifications of bile acids under high-intensity ultrasound or microwave irradiation

High-intensity ultrasound (HIU) and microwave (MW) irradiation, having emerged as effective promoters of organic reactions, were exploited for the synthesis of bile acids derivatives. Esterification, amidation, hydrolysis, oxidation, and reduction were investigated. Compared to conventional methods, both techniques proved much more efficient, increasing product yields and dramatically cutting down reaction times. Scaled-up studies are now under way.     

The efficiency of reactant site sampling in network-free simulation of rule-based models for biochemical systems

Rule-based models, which are typically formulated to represent cell signaling systems, can now be simulated via various network-free simulation methods. In a network-free method, reaction rates are calculated for rules that characterize molecular interactions, and these rule rates, which each correspond to the cumulative rate of all reactions implied by a rule, are used to perform a stochastic simulation of reaction kinetics. Network-free methods, which can be viewed as generalizations of Gillespie's method, are so named because these methods do not require that a list of individual reactions implied by a set of rules be explicitly generated, which is a requirement of other methods for simulating rule-based models. This requirement is impractical for rule sets that imply large reaction networks (i.e. long lists of individual reactions), as reaction network generation is expensive. Here, we compare the network-free simulation methods implemented in RuleMonkey and NFsim, general-purpose software tools for simulating rule-based models encoded in the BioNetGen language. The method implemented in NFsim uses rejection sampling to correct overestimates of rule rates, which introduces null events (i.e. time steps that do not change the state of the system being simulated). The method implemented in RuleMonkey uses iterative updates to track rule rates exactly, which avoids null events. To ensure a fair comparison of the two methods, we developed implementations of the rejection and rejection-free methods specific to a particular class of kinetic models for multivalent ligand-receptor interactions. These implementations were written with the intention of making them as much alike as possible, minimizing the contribution of irrelevant coding differences to efficiency differences. Simulation results show that performance of the rejection method is equal to or better than that of the rejection-free method over wide parameter ranges. However, when parameter values are such that ligand-induced aggregation of receptors yields a large connected receptor cluster, the rejection-free method is more efficient.     

Studies of oligoadenylate formation on a poly(U) template

We have studied a variety of condensation reactions involving poly (U) as template and isomeric adenosine dinucleotides as substrates. We find that [3'-5']-linked dinucleotides such as A3pA and pA3pA are better acceptors than the corresponding [2'-5']-linked compounds, while ImpA2pA is a better donor than ImpA3pA. The reaction between A2pA and ImpA3pA, for example, yields only 4% of product while the reaction of A3pA with ImpA2pA yields 86% of product. The more efficient condensation reactions of dimers are about as efficient as the self-condensation of ImpA. They yield a few percent of material in which five or more substrate molecules are linked together. The percentage of the natural [3'-5']-linkage in the product varies greatly, from as little as 1% to as much as 45%.     

Application of dimeric orthopalladated complex in Suzuki-Miyaura cross coupling reaction under microwave irradiation and conventional heating

The Suzuki-Miyaura reaction of various aryl halides using [Pd{C₆H₂(CH₂CH₂NH₂)-(OMe)₂,3,4} (μ-Br)]₂ have been investigated. This orthopalladated complex is an efficient, stable and non-sensitive to air and moisture catalyst for the hetrocoupling reaction in DMF as the reaction solvent at 130 °C. The combination of dimeric complex as homogenous catalyst and microwave irradiation can be very useful and efficient methods in organic synthesis, so the application of microwave irradiation have been investigated using homogenous dimeric complex [Pd{C₆H₂(CH₂CH₂NH₂)-(OMe)₂,3,4} (μ-Br)]₂. Application of dimeric complex as catalyst caused to produce the desired coupling products and the using of microwave irradiation improving the yields of the reactions and shortening the reaction times.     

Localization properties of fluorescence cytochemical enzyme procedures

Summary Fluorescence enzyme cytochemical procedures will contribute significantly to biomedical problems where knowledge of the enzymic composition of individual cells is important. Compared with the number of absorbance enzyme cytochemical methods, relatively few fluorescence procedures have been reported. In this paper, the merits of the described methods are discussed. A distinction is made between methods with and without a capture reaction. Only a few methods satisfy the requirement of accurate localization of the final product and high signal to noise ratios. Thus, there still is a need for valid fluorescence cytochemical enzyme methods. It is concluded that the bottle neck for valid fluorescence cytochemical enzyme methods is the development of efficient fluorogenic capture reactions for the primary enzyme products.In honour of Prof. P. van DuijnSupported (in part) by the Foundation for Medical Research (FUNGO), which is subsidized by the Netherlands Organization for the Advancement of Pure Research     

Improved strategies for electrochemical 1,4-NAD(P)H2 regeneration: A new era of bioreactors for industrial biocatalysis

Industrial enzymatic reactions requiring 1,4-NAD(P)H₂ to perform redox transformations often require convoluted coupled enzyme regeneration systems to regenerate 1,4-NAD(P)H₂ from NAD(P) and recycle the cofactor for as many turnovers as possible. Renewed interest in recycling the cofactor via electrochemical means is motivated by the low cost of performing electrochemical reactions, easy monitoring of the reaction progress, and straightforward product recovery. However, electrochemical cofactor regeneration methods invariably produce adventitious reduced cofactor side products which result in unproductive loss of input NAD(P). We review various literature strategies for mitigating adventitious product formation by electrochemical cofactor regeneration systems, and offer insight as to how a successful electrochemical bioreactor system could be constructed to engineer efficient 1,4-NAD(P)H₂-dependent enzyme reactions of interest to the industrial biocatalysis community.     

Identification of alternative products and optimization of 2-nitro-5-thiocyanatobenzoic acid cyanylation and cleavage at cysteine residues

The reagent 2-nitro-5-thiocyanatobenzoic acid (NTCB) is commonly used to cyanylate and cleave proteins at cysteine residues, but this two-step reaction requires lengthy incubations and produces highly incomplete cleavages. In previous reports, incomplete cleavage was attributed to a competing beta-elimination reaction that converts cyanylated cysteine to dehydroalanine. In this study, previously unidentified side reactions of the NTCB cleavage were discovered and beta-elimination was not the major reaction competing with peptide bond cleavage. A major side reaction was identified as carbamylation of lysine residues. Carbamylation could be minimized by desalting the cyanylation reaction before cleavage or by reducing the reactant concentrations, but both methods suffered from further reductions in cleavage efficiency. Based on model peptide studies, poor cleavage was primarily caused by a mass neutral rearrangement of the cyanylated cysteine which produced a cleavage-resistant, nonreducible product. The formation of this product could be minimized by using stronger nucleophiles for the cleavage reaction. We discovered that base-catalyzed nucleophilic cleavage could be achieved with many amino-containing compounds. Most notably, glycine is capable of promoting efficient cleavage. In addition, efficient NTCB cleavage can be performed in a simple one-step method without a prior cyanylation step, rather than the previously described two-step reaction.     

Isocyanide-based consecutive Bargellini/Ugi reactions: an efficient method for the synthesis of pseudo-peptides containing three amide bonds

Isocyanide-based consecutive Bargellini/Ugi multicomponent reactions as a combinatorial strategy have been developed for the synthesis of new class of pseudo-peptides. Via Bargellini reaction 3-carboxamido-isobutyric acids are prepared using acetone, chloroform, sodium hydroxide, and isocyanides. Then, using Ugi multicomponent reaction strategy, pseudo-peptides containing three amide bonds are synthesized using the Bargellini reaction product, aldehydes, amines, and isocyanides. This is an efficient and eco-friendly approach for easy access to wide variety of structurally diverse, drug-like pseudo-peptides from cheap and readily available precursors in high yields.

     

Stochastic modelling of biochemical systems of multi-step reactions using a simplified two-variable model

Background

A fundamental issue in systems biology is how to design simplified mathematical models for describing the dynamics of complex biochemical reaction systems. Among them, a key question is how to use simplified reactions to describe the chemical events of multi-step reactions that are ubiquitous in biochemistry and biophysics. To address this issue, a widely used approach in literature is to use one-step reaction to represent the multi-step chemical events. In recent years, a number of modelling methods have been designed to improve the accuracy of the one-step reaction method, including the use of reactions with time delay. However, our recent research results suggested that there are still deviations between the dynamics of delayed reactions and that of the multi-step reactions. Therefore, more sophisticated modelling methods are needed to accurately describe the complex biological systems in an efficient way.

Results

This work designs a two-variable model to simplify chemical events of multi-step reactions. In addition to the total molecule number of a species, we first introduce a new concept regarding the location of molecules in the multi-step reactions, which is the second variable to represent the system dynamics. Then we propose a simulation algorithm to compute the probability for the firing of the last step reaction in the multi-step events. This probability function is evaluated using a deterministic model of ordinary differential equations and a stochastic model in the framework of the stochastic simulation algorithm. The efficiency of the proposed two-variable model is demonstrated by the realization of mRNA degradation process based on the experimentally measured data.

Conclusions

Numerical results suggest that the proposed new two-variable model produces predictions that match the multi-step chemical reactions very well. The successful realization of the mRNA degradation dynamics indicates that the proposed method is a promising approach to reduce the complexity of biological systems.

     

The effect of electrochemical regeneration upon the enzymatic catalysis of a thermodynamically unfavorable reaction

The association between enzymatic and electrochemical reactions, enzymatic electrocatalysis, had proven to be a very powerful tooth in both analytical and synthetic fields. However, most of the combinations studied have involved enzymatic catalysis of irreversible or quasi-irreversible reaction. In the present work, we have investigated the possibility of applying enzymatic electrocatalysis to a case where the electrochemical reaction drives a thermodynamically unfavorable reversible reaction. Such thermodynamically unfavorable reactions include most of the oxidations catalyzed by dehydrogenases. Yeast alcohol dehydrogenase (E.C. 1.1.1.1) was chosen as a model enzyme because the oxidation of ethanol is thermodynamically very unfavorable and because its kinetics are well known. The electrochemical reaction was the oxidation of NADH which is particularly attractive as a method of cofactor regeneration. Both the electrochemical and enzymatic reactions occur in the same batch reactor in such a way that electrical energy is the only external driving force. Two cases were experimentally and theoretically developed with the enzyme either in solution or immobilized onto the electrode's surface. In both cases, the electrochemical reaction could drive the enzymatic reaction by NADH consumption in solution or directly in the enzyme's microenvironment. However even for a high efficiency of NADH consumption, the rate of enzymatic catalysis was limited by product (acetaldedehyde) inhibition. Extending this observation to the subject of organic synthesis catalyzed by dehydrogenases, we concluded that thermodynamically unfavorable reaction and can only be used in a process if efficient NAD regeneration and product elimination are simultaneously carried out within the reactor.     

Novel chalcogenides of thymidine and uridine: synthesis, properties and applications

A facile and efficient methodology has been developed for the synthesis of dithymidine and di-uridine derived disulfides using benzyltriethylammonium tetrathiomolybdate as a sulfur transfer reagent. However, a similar reaction of thymidine derivative with tetraethylammonium tetraselenotungstate as a selenium transfer reagent resulted in the formation of an unexpected cyclic diselenide. The disulfide derivatives of nucleosides have been used as precursors in a tandem disulfide cleavage-Michael addition/ring opening reactions to construct aminoacid and carbocyclic derivatives of nucleosides.     

A theoretical treatment of damped oscillations in the transient state kinetics of single-enzyme reactions

An extension of the available kinetic theory for reactions in the transient state is presented which establishes that single-enzyme reactions may exhibit damped oscillations under the conditions of standard kinetic experiments performed by stopped-flow techniques. Such oscillations may occur for reasonable magnitudes of rate constants in the enzymic reaction mechanism and at physiological concentrations of enzyme and substrate. In the simplest reaction systems, the oscillations will be strongly damped and lead to progress curves resembling those of a reaction governed by standard exponential transients; statistical regression methods may then have to be applied for their detection and characterization. The observation that single-enzyme reactions may exhibit oscillatory behaviour points to a previously unrecognized possible source of the damped oscillations observed in metabolic systems such as the pathways of glycolysis or photosynthesis.     

Exploring De Novo metabolic pathways from pyruvate to propionic acid

Industrial biotechnology provides an efficient, sustainable solution for chemical production. However, designing biochemical pathways based solely on known reactions does not exploit its full potential. Enzymes are known to accept non‐native substrates, which may allow novel, advantageous reactions. We have previously developed a computational program named Biological Network Integrated Computational Explorer (BNICE) to predict promiscuous enzyme activities and design synthetic pathways, using generalized reaction rules curated from biochemical reaction databases. Here, we use BNICE to design pathways synthesizing propionic acid from pyruvate. The currently known natural pathways produce undesirable by‐products lactic acid and succinic acid, reducing their economic viability. BNICE predicted seven pathways containing four reaction steps or less, five of which avoid these by‐products. Among the 16 biochemical reactions comprising these pathways, 44% were validated by literature references. More than 28% of these known reactions were not in the BNICE training dataset, showing that BNICE was able to predict novel enzyme substrates. Most of the pathways included the intermediate acrylic acid. As acrylic acid bioproduction has been well advanced, we focused on the critical step of reducing acrylic acid to propionic acid. We experimentally validated that Oye2p from Saccharomyces cerevisiae can catalyze this reaction at a slow turnover rate (10^?3 s^?1), which was unknown to occur with this enzyme, and is an important finding for further propionic acid metabolic engineering. These results validate BNICE as a pathway‐searching tool that can predict previously unknown promiscuous enzyme activities and show that computational methods can elucidate novel biochemical pathways for industrial applications. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:303–311, 2016     

Modeling of uncertainties in biochemical reactions

Mathematical modeling is an indispensable tool for research and development in biotechnology and bioengineering. The formulation of kinetic models of biochemical networks depends on knowledge of the kinetic properties of the enzymes of the individual reactions. However, kinetic data acquired from experimental observations bring along uncertainties due to various experimental conditions and measurement methods. In this contribution, we propose a novel way to model the uncertainty in the enzyme kinetics and to predict quantitatively the responses of metabolic reactions to the changes in enzyme activities under uncertainty. The proposed methodology accounts explicitly for mechanistic properties of enzymes and physico‐chemical and thermodynamic constraints, and is based on formalism from systems theory and metabolic control analysis. We achieve this by observing that kinetic responses of metabolic reactions depend: (i) on the distribution of the enzymes among their free form and all reactive states; (ii) on the equilibrium displacements of the overall reaction and that of the individual enzymatic steps; and (iii) on the net fluxes through the enzyme. Relying on this observation, we develop a novel, efficient Monte Carlo sampling procedure to generate all states within a metabolic reaction that satisfy imposed constrains. Thus, we derive the statistics of the expected responses of the metabolic reactions to changes in enzyme levels and activities, in the levels of metabolites, and in the values of the kinetic parameters. We present aspects of the proposed framework through an example of the fundamental three‐step reversible enzymatic reaction mechanism. We demonstrate that the equilibrium displacements of the individual enzymatic steps have an important influence on kinetic responses of the enzyme. Furthermore, we derive the conditions that must be satisfied by a reversible three‐step enzymatic reaction operating far away from the equilibrium in order to respond to changes in metabolite levels according to the irreversible Michelis–Menten kinetics. The efficient sampling procedure allows easy, scalable, implementation of this methodology to modeling of large‐scale biochemical networks. Biotechnol. Bioeng. 2011;108: 413–423. © 2010 Wiley Periodicals, Inc.     

An approach for catalyst design in artificial photosynthetic systems: focus on nanosized inorganic cores within proteins

Some enzymes can be considered as a catalyst having a nanosized inorganic core in a protein matrix. In some cases, the metal oxide or sulfide clusters, which can be considered as cofactors in enzymes, may be recruited for use in other related reactions in artificial photosynthetic systems. In other words, one approach to design efficient and environmentally friendly catalysts in artificial photosynthetic systems for the purpose of utilizing sunlight to generate high energy intermediates or useful material is to select and utilize inorganic cores of enzymes. For example, one of the most important goals in developing artificial photosynthesis is hydrogen production. However, first, it is necessary to find a “super catalyst” for water oxidation, which is the most challenging half reaction of water splitting. There is an efficient system for water oxidation in cyanobacteria, algae, and plants. Published data on the Mn–Ca cluster have provided details on the mechanism and structure of the water oxidizing complex as a Mn–Ca nanosized inorganic core in photosystem II. Progress has been made in introducing Mn–Ca oxides as efficient catalysts for water oxidation in artificial photosynthetic systems. Here, in the interest of designing efficient catalysts for other important reactions in artificial photosynthesis, a few examples of our knowledge of inorganic cores of proteins, and how Nature used them for important reactions, are discussed.     

Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis.

Microwave energy represents an efficient manner to accelerate both the deprotection and coupling reactions in 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS). Typical SPPS side reactions including racemization and aspartimide formation can occur with microwave energy but can easily be controlled by routine use of optimized methods. Cysteine, histidine, and aspartic acid were susceptible to racemization during microwave SPPS of a model 20mer peptide containing all 20 natural amino acids. Lowering the microwave coupling temperature from 80 degrees C to 50 degrees C limited racemization of histidine and cysteine. Additionally, coupling of both histidine and cysteine can be performed conventionally while the rest of the peptide is synthesized using microwave without any deleterious effect, as racemization during the coupling reaction was limited to the activated ester state of the amino acids up to 80 degrees C. Use of the hindered amine, collidine, in the coupling reaction also minimized formation of D-cysteine. Aspartimide formation and subsequent racemization of aspartic acid was reduced by the addition of HOBt to the deprotection solution and/or use of piperazine in place of piperidine.