Real time phase detection based online monitoring of batch fermentation processes

Industrial fermentations conducted in a batch or semi-batch mode demonstrate significant batch-to-batch variability. Current batch process monitoring strategies involve manual interpretation of highly informative but low frequency offline measurements such as concentrations of products, biomass and substrates. Fermentors are also fitted with computer interfaced instrumentation, enabling high frequency online measurements of several variables and automated techniques which can utilize this data would be desirable. Evolution of a batch fermentation, which typically uses complex medium, can be conceptualized as a sequence of several distinct metabolic phases. Monitoring of batch processes can then be achieved by detecting the phase change events, also termed as singular points (SP). In this work, we propose a novel moving window based real-time monitoring strategy for SP detection based only on online measurements. The key hypothesis of the strategy is that the statistical properties of the online data undergo a significant change around an SP. The strategy is easily implementable and does not require past data or prior knowledge of the number or time of occurrence of SPs. The efficacy of the proposed approach has been demonstrated to be superior compared to that of reported techniques for industrially relevant model organisms. The proposed approach can be used to decide offline sampling timings in real time.     

The polyamide method of solid phase peptide and oligonucleotide synthesis

A versatile system of solid-phase peptide synthesis based on polar polyamide resins, a range of reversible peptide-resin linkage agents, and Nα-t-butoxycarbonyl or fluorenylmethoxycarbonyl-amino acids has been developed. Principles used in the design of the method are discussed and illustrated by synthesis of a number of natural peptides. Application of these principles to oligodeoxyribonucleotide synthesis has provided for the first time a practical solid phase method in the nucleotide field.     

An amalgamation of solid phase peptide synthesis and ribosomal peptide synthesis

Expressed protein ligation (EPL) is a protein semisynthesis technique that allows the site-specific introduction of unnatural amino acids and biophysical probes into proteins. In the present study, we illustrate the utility of the approach through the generation of two semisynthetic proteins bearing spectroscopic probes. Dihydrofolate reductase containing a single (13)C probe in an active site loop was generated through the ligation of a synthetic peptide-alpha-thioester to a recombinantly generated fragment containing an N-terminal Cys. Similarly, c-Crk-II was assembled by the sequential ligation of three recombinant polypeptide building blocks, allowing the incorporation of (15)N isotopes in the central domain of the protein. These examples showcase the scope of the protein ligation strategy for selective introduction of isotopic labels into proteins, and the protocols described will be of value to those interested in using EPL on other systems.     

A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis

The success of solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl (Fmoc) amino acids is often limited by deleterious side reactions which occur during TFA peptide-resin cleavage and side-chain deprotection. The majority of these side reactions modify susceptible residues, such as Trp, Tyr, Met, and Cys, with TFA-liberated side-chain protecting groups and linkers. The purpose of this study was to assess the relative effectiveness of various scavengers in suppressing these side reactions. We found that the cleavage mixture 82.5% TFA : 5% phenol : 5% H2O : 5% thioanisole : 2.5% EDT (Reagent K) was maximally efficient in inhibiting a great variety of side reactions. Synthesis and cleavage of 10 peptides, each containing 20-50 residues, demonstrated the complementarity of Fmoc chemistry with Reagent K for efficient synthesis of complex peptides.     

Methods and protocols of modern solid phase peptide synthesis

The purpose of this article is to delineate strategic considerations and provide practical procedures to enable non-experts to synthesize peptides with a reasonable chance of success. This article is not encyclopedic but rather devoted to the Fmoc/tBu approach of solid phase peptide synthesis (SPPS), which is now the most commonly used methodology for the production of peptides. The principles of SPPS with a review of linkers and supports currently employed are presented. Basic concepts for the different steps of SPPS such as anchoring, deprotection, coupling reaction and cleavage are all discussed along with the possible problem of aggregation and side-reactions. Essential protocols for the synthesis of fully deprotected peptides are presented including resin handling, coupling, capping, Fmoc-deprotection, final cleavage and disulfide bridge formation.     

Monitoring of solid phase peptide synthesis by FT-IR spectroscopy

Aggregation phenomena of growing peptides on the resin have seldom been investigated. We report here how conformations are determined by FT-IR spectroscopy. Therefore the sequence 80–99 of HIV 1-protease was synthesized. After every coupling a resin sample was taken out of the reaction column and a FT-IR spectrum recorded. The results were compared with the UV monitoring obtained from another synthesis of the same peptide. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Neurotrophic peptide aldehydes: solid phase synthesis of fellutamide B and a simplified analog

A combination of solid phase and solution phase synthetic methods have been used to complete the total synthesis of the neurotrophic lipopeptide aldehyde fellutamide B (2). The beta-hydroxy aliphatic tail was prepared by regioselective reductive opening of a cyclic sulfate, and later coupled to a solid phase resin. The synthetic compound was then examined in cytotoxicity and nerve growth factor (NGF) induction assays. A simplified analog of fellutamide B also showed activity.     

Solid phase peptide synthesis in water VI: evaluation of water-soluble coupling reagents for solid phase peptide synthesis in aqueous media

Solid phase peptide synthesis requires large amounts of organic solvents, the safe disposal of which is an important environmental issue. Peptide synthesis, if performed in water and using less or nontoxic reagents, circumvents the disposal problem. Our ultimate aim is to develop an "environment-friendly" solid phase peptide synthesis (SPPS) methodology. Previously, we showed that SPPS in water is feasible. To perform SPPS in water, the coupling reagent must be water-soluble and maintain its reactivity in water. For this report, we tested the efficacy of the water-soluble coupling reagents, 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), towards SPPS in water. We successfully synthesized Leu-enkephalin amide on a solid support suspended in aqueous 50% EtOH using DMT-MM and 2-(4-sulfophenylsulfonyl)ethoxycarbonylamino acids.     

Amyloid-beta as a "difficult sequence" in solid phase peptide synthesis

The phenomenon of "difficult sequence" has long frustrated chemists in their efforts to assemble peptides that contain such sequences by solid phase synthesis methods. A variety of remedial measures are available to minimize or even abolish the negative impact of these sequences during synthesis. These include the use of elevated temperatures and stronger acylating reagents. Amyloid-beta, a fragment of the amyloid precursor protein, contains 40-43 residues and possesses a C-terminal sequence that is particularly resistant to ready solid phase synthesis making it a "difficult sequence" peptide. This review focuses on approaches to successfully assemble the peptide by both Boc- and Fmoc solid phase synthesis.     

Suppressing the epimerization of endothioamide peptides during Fmoc/t‐Bu‐based solid phase peptide synthesis

Despite a number of intriguing utilities associated with thioamide‐containing peptides and proteins in the context of biophysics, pharmacology and chemical biology, it has hitherto remained as one of the underexplored territories of peptidomimetics. The synthesis of long mono to multiply substituted endothioamide peptides is invariably accompanied with severe epimerization, oxoamide formation and various other undesired side reactions, resulting in messy product profiles. This has completely restrained their use as novel chemical tools for biological studies. During the chain elongation of an N‐terminally located thioamide peptide using the Fmoc/t‐Bu chemistry, it becomes vulnerable to the repetitive basic treatments as required for such chemistry. The incompatibility of thioamide moiety with bases as well as strong coupling reagents leads to epimerization as well as other side reactions due to its nucleophilicity, resulting in the loss of the stereochemical identity of the thioamidated amino acid residue. An easy‐to‐implement and efficient protocol to synthesize long (>10‐mer) endothioamide peptides, significantly suppressing epimerization and other side reactions using 10% piperidine/dimethylformamide for 1 min, is reported herein. The novelty of the protocol is shown through the efficient synthesis of a number of 10–12‐mer mono to multiply thioamide‐substituted peptides with broad substrate scopes. The utility of the protocol in the context of protein engineering and chemical protein synthesis is also shown through the synthesis of a thioamide version of the 16‐mer peptide from the B1 domain of protein G. Such a protocol to synthesize long endothioamide peptides would open up avenues toward engineering and accessing novel thiopeptide and thioprotein‐based chemical tools, the synthesis of which had been a serious hurdle thus far. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Determining the availability of activated amino acids for solid phase peptide synthesis

Data concerning the reaction characteristics of each amino acid are of fundamental importance for providing optimum reaction conditions and high yields during polypeptide synthesis. Loss of activated amino acid as a function of time would have a pronounced effect on the efficiency of synthesis. An assay procedure is described which quantitatively determines the availability of DCC activated amino acids as a function of preincubation time. The assay was employed to follow the inactivation of t-BOC.Leu(DCC) and indicated a significant loss of activated amino acid.     

Facile production of mono-substituted urea side chains in solid phase peptide synthesis

A method is reported for the straightforward generation of urea-containing peptides during Boc solid phase peptide synthesis. Primary amine side chains are converted to mono-alkyl ureas in two steps via an intermediate p-nitrophenyl carbamate. Use of p-methoxybenzyl amine as an ammonia equivalent affords mono-alkyl final products from standard resin cleavage methods, without the need for additional steps. The reaction is highly efficient and applicable to variable length side chains and peptides.     

Mass spectrometric monitoring of solid phase phosphate triester synthesis of DNA fragments

A new and convenient pyrolysis mass spectrometric approach for monitoring solid phase phosphate triester synthesis of oligodeoxyribonucleotides in 5'-3' direction has been developed. The base-typical fragment ions produced by pyrolysis at 280 degrees C of the polymer-bound oligonucleotide triesters in the mass spectrometer permit the analytical monitoring of DNA chain growth, using simple mathematical operations. The base composition can be determined directly from the polymer. In addition, minor nucleosides can easily be detected.     

Solid phase synthesis of C-terminal peptide amides: development of a new aminoethyl-polystyrene linker on the Multipin solid support.

A new aminoethyl-polystyrene linker, stable at low concentrations of TFA, has been developed for the solid phase synthesis of peptide amides. The described linker is stable under conditions which remove Bu(t) protecting groups (30-50% TFA in DCM) and the desired product can be finally cleaved off the solid support in 95% TFA (5% H2O). Model peptide amides and other N-alkylated peptide amides have been successfully synthesized in good yield and purity.     

Large-scale production of peptides using the solid phase continuous flow method. Part 2: preparative synthesis of a 26-mer peptide thrombin inhibitor

A preparative method for the preparation of large peptides is described. An advantageous theoretical weight of peptide/weight of starting resin ratio (tP_w/R_w) of about 0.3 was successfully experimented. The esterification of the first amino acid was realized with a racemization of less than 1%. The study of the coupling conditions led to the use of a diluted acylating mixture that allowed a 56% consumption of the amino acid derivatives (percentage use of amino acids) introduced in the synthesis. The cost analysis of the synthesis showed that the recovery of the amino acid derivatives was not worthwhile. © 1998 European Peptide Society and John Wiley & Sons, Ltd.