Semi-automated high throughput combinatorial solid-phase organic synthesis.

A semi-automated technique for massive parallel solid-phase organic synthesis based on a "split only" strategy is described. Two different types of purpose-oriented reaction vessels are used. The initial steps are performed in domino blocks, and the resin-bound intermediates then split into wells of a micro plate for the last combinatorial step. The domino block is a reaction block for manual and semi-automatic parallel solid-phase organic synthesis that simplifies liquid exchange and integrates common synthetic steps. The synthesis in micro plates does not use any filter for separation of resin beads from the supernatant liquid, and allows high throughput parallel synthesis on solid phase to be performed. This technique, documented on examples of diverse disubstituted benzenes, includes the use of gaseous cleavage in the last synthetic step and allows the synthesis of thousands of compounds per day in mg quantities.     

The use of optical spectroscopy in combinatorial chemistry

Infrared and Raman spectroscopy allow direct spectral analysis of the solid‐phase, thus avoiding the tedious cleavage of compounds from the solid support. With diagnostic bands in starting materials or products, infrared and Raman spectroscopy are efficient in monitoring each reaction step directly on the solid phase. Consequently, infrared and Raman spectroscopy have evolved as the premier analytical methodology for direct analysis on the solid support. While infrared transmission spectroscopy is a general analytical method for resin samples, internal reflection spectroscopy is especially suited for solid polymer substrates known as “pins” or “crowns.” Single bead analysis is done best by infrared microspectroscopy, whereas photoacoustic spectroscopy allows totally nondestructive analysis of resin samples. With an automated accessory, diffuse reflection spectroscopy provides a method for high throughput on‐bead monitoring of solid‐phase reactions. Providing identification based on molecular structure, HPLC‐FTIR is, therefore, complementary to LC‐MS. Additionally, Raman spectroscopy as a complement to infrared spectroscopy can be applied to resin samples and—using a Raman microscope—to single beads. Fluorometry as an extremely sensitive spectroscopic detection method allows rapid quantification of organic reactions directly on the resin. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng (Comb Chem) 61:179–187, 1998/1999.     

Fmoc‐based peptide thioester synthesis with self‐purifying effect: heading to native chemical ligation in parallel formats

The chemical synthesis of proteins has facilitated functional studies of proteins due to the site‐specific incorporation of post‐translational modifications, labels, and non‐proteinogenic amino acids. Moreover, native chemical ligation provides facile access to proteins by chemical means. However, the application of the native chemical ligation reaction in the synthesis of parallel formats such as protein arrays has been complicated because of the often cumbersome and time‐consuming synthesis of the required peptide thioesters. An Fmoc‐based peptide thioester synthesis with self‐purification on the sulfonamide ‘safety‐catch’ linker widens this bottleneck because HPLC purification can be avoided. The method is based on an on‐resin cyclization–thiolysis reaction sequence. A macrocyclization via the N‐terminus of the full‐length peptide followed by a thiolytic C‐terminal ring opening allows selective detachment of the truncation products and the full‐length peptide. A brief overview of the chemical aspects of this method is provided including the optimization steps and the automation process. Furthermore, the application of the cyclization–thiolysis approach combined with the native chemical ligation reaction in the parallel synthesis of a library of 16 SH3‐domain variants of SHO1 in yeast is described, demonstrating the value of this new technique for the chemical synthesis of protein arrays. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

An HPLC‐ESMS study on the solid‐phase assembly of C‐terminal proline peptides

DKP formation is a serious side reaction during the solid‐phase synthesis of peptide acids containing either Pro or Gly at the C‐terminus. This side reaction not only leads to a lower overall yield, but also to the presence in the reaction crude of several deletion peptides lacking the first amino acids. For the preparation of protected peptides using the Fmoc/tBu strategy, the use of a ClTrt‐Cl‐resin with a limited incorporation of the C‐terminal amino acid is the method of choice. The use of resins with higher loading levels leads to more impure peptide crudes. The use of HPLC‐ESMS is a useful method for analysing complex samples, such as those formed when C‐terminal Pro peptides are prepared by non‐optimized solid‐phase strategies. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Facile preparation of the N‐acetyl‐glucosaminylated asparagine derivative with TFA‐sensitive protecting groups useful for solid‐phase glycopeptide synthesis

In this study, a novel N‐acetyl‐glucosaminylated asparagine derivative was developed. This derivative carried TFA‐sensitive protecting groups and was derived from commercially available compounds only in three steps. It was applicable to the ordinary 9‐fluorenylmethoxycarbonyl (Fmoc)‐based solid‐phase peptide synthesis (SPPS) method, and the protecting groups on the carbohydrate moiety could be removed by a single step of TFA cocktail treatment generally used for the final deprotection step in Fmoc‐SPPS. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

p‐Methoxyphenol: A potent and effective scavenger for solid‐phase peptide synthesis

During the final step of t‐Boc/Bzl, solid‐phase peptide synthesis (SPPS)‐protecting groups from amino acids (aa) side chains must be removed from the target peptides during cleavage from the solid support . These reaction steps involve hydrolysis with hydrogen fluoride (HF) in the presence of a nucleophile (scavenger), whose function is to trap the carbocations produced during S_N1‐type reactions. Five peptide sequences were synthesised for evaluating p‐methoxyphenol effectiveness as a potent scavenger. After the synthesis, the resin–peptide was then separated into two equal parts to be cleaved using two scavengers: conventional reactive p‐cresol (reported in the literature as an effective acyl ion eliminator) and p‐methoxyphenol (hypothesised as fulfilling the same functions as the routinely used scavenger). Detailed analysis of the electrostatic potential map (EPM) revealed similarities between these two nucleophiles, regarding net atomic charge, electron density distribution, and similar pKa values. Good scavenger efficacy was observed by chromatography and mass spectrometry results for the synthesised molecules, which revealed that p‐methoxyphenol can be used as a potent scavenger during SPPS by t‐Boc/Bzl strategy, as similar results were obtained using the conventional scavenger.     

Strategies for the highly efficient synthesis of erythropoietin N‐glycopeptide hydrazides

A convergent synthesis for erythropoietin (EPO) 1‐28 N‐glycopeptide hydrazides was developed. In this approach, EPO 1‐28 peptides were synthesized on the solid phase and converted to C‐terminal hydrazides after cleavage from the resin. After selective deprotection of the Asp24 side chain, the desired glycosylamine was coupled by pseudoproline‐assisted Lansbury aspartylation. Although the initial yields of the EPO 1‐28 glycopeptides were satisfactory, they could be markedly improved by increasing the purity of the peptide using a reversed‐phase high‐performance liquid chromatography (RP‐HPLC) purification of the protected peptide.     

Microwave‐assisted TFA cleavage of peptides from Merrifield resin

Microwave‐assisted (MW) reactions are of special interest to the chemical community due to faster reaction times, cleaner reactions and higher product yields. The adaptation of MW to solid phase peptide synthesis resulted in spectacular syntheses of difficult peptides. In the case of Merrifield support, used frequently in synthesis of special peptides, the conditions used in product cleavage are not compatible with off‐resin monitoring of the reaction progress. The application of MW irradiation in product removal from Merrifield resin using trifluoroacetic acid (TFA) was investigated using model tetrapeptides and the effects were compared with standard trifluoromethanesulphonic acid (TFMSA) cleavage using elemental analysis as well as chromatographic (HPLC) and spectroscopic (IR) methods. The deprotection of benzyloxycarbonyl and benzyl groups in synthetic bioactive peptides was analyzed using LC‐MS and MS/MS experiments. In a 5 min microwave‐assisted TFA reaction at low temperature, the majority of product is released from the resin, making the analytical scale MW‐assisted procedure a method of choice in monitoring the reactions carried out on Merrifield resin due to the short reaction time and compatibility with HPLC and ESI‐MS conditions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Assessing the correlation of microscopy‐based and volumetry‐based measurements for resin swelling in a range of potential greener solvents for SPPS

The degree of resin swelling in a particular solvent system is one of the critical parameters for solid‐phase peptide synthesis (SPPS) and for solid‐phase synthesis in general. Methods used for measuring the degree of resin swelling include microscopy‐based and volumetry‐based methods. This study describes and compares the use of both methods for a number of commercially available resins commonly used in SPPS, with a range of solvents, which have been identified in the literature as ‘greener’ than DCM, DMF and NMP. The results were analysed by statistical methods, and a significant correlation between the two distinct methods has been demonstrated for the first time. The results will likely be used, in conjunction with other literature methods, to help in choosing both the resin and solvent system for greener SPPS, as well as for continuous flow SPPS, which is of growing importance.     

Micro‐Solid Oxide Fuel Cell Membranes Prepared by Aerosol‐Assisted Chemical Vapor Deposition

Free‐standing electrolyte membranes for low‐temperature micro‐solid oxide fuel cells (micro‐SOFCs) are prepared by aerosol‐assisted chemical vapor deposition (AA‐CVD), a cost‐effective, non‐vacuum thin‐film deposition technique. Thin, yttria‐stabilized zirconia (YSZ) membranes (50–400 nm) as well as bilayer membranes of YSZ and gadolinia‐doped ceria are prepared at temperatures of 600 °C and below. AA‐CVD, which is a gas‐phase deposition method, allows for the synthesis of precursor‐free crystalline layers, thereby limiting the development of tensile stress. High membrane survival rates of around 90% are thus obtained. The columnar structure of the electrolyte ensures high oxygen‐ion conductivity and results in negligible ohmic losses. Using sputtered platinum electrodes, the demonstration of a micro‐SOFC based on AA‐CVD electrolyte is achieved and first power density data of 166 mW cm^‐2 at 410 °C is obtained.     

New preparation techniques for molecular and in‐situ analysis of ancient organic micro‐ and nanostructures

Organic microfossils preserved in three dimensions in transparent mineral matrices such as cherts/quartzites, phosphates, or carbonates are best studied in petrographic thin sections. Moreover, microscale mass spectrometry techniques commonly require flat, polished surfaces to minimize analytical bias. However, contamination by epoxy resin in traditional petrographic sections is problematic for the geochemical study of the kerogen in these microfossils and more generally for the in situ analysis of fossil organic matter. Here, we show that epoxy contamination has a molecular signature that is difficult to distinguish from kerogen with time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS). This contamination appears pervasive in organic microstructures embedded in micro‐ to nano‐crystalline carbonate. To solve this problem, a new semi‐thin section preparation protocol without resin medium was developed for micro‐ to nanoscale in situ investigation of insoluble organic matter. We show that these sections are suited for microscopic observation of Proterozoic microfossils in cherts. ToF‐SIMS reveals that these sections are free of pollution after final removal of a <10 nm layer of contamination using low‐dose ion sputtering. ToF‐SIMS maps of fragments from aliphatic and aromatic molecules and organic sulfur are correlated with the spatial distribution of organic microlaminae in a Jurassic stromatolite. Hydrocarbon‐derived ions also appeared correlated with kerogenous microstructures in Archean cherts. These developments in analytical procedures should help future investigations of organic matter and in particular, microfossils, by allowing the spatial correlation of microscopy, spectroscopy, precise isotopic microanalyses, and novel molecular microanalyses such as ToF‐SIMS.     

Hydrophobic benzyl amines as supports for liquid‐phase C‐terminal amidated peptide synthesis: application to the preparation of ABT‐510

C‐terminal amidation is one of the most common modification of peptides and frequently found in bioactive peptides. However, the C‐terminal modification must be creative, because current chemical synthetic techniques of peptides are dominated by the use of C‐terminal protecting supports. Therefore, it must be carried out after the removal of such supports, complicating reaction work‐up and product isolation. In this context, hydrophobic benzyl amines were successfully added to the growing toolbox of soluble tag‐assisted liquid‐phase peptide synthesis as supports, leading to the total synthesis of ABT‐510 ( 2 ). Although an ethyl amide‐forming type was used in the present work, different types of hydrophobic benzyl amines could also be simply designed and prepared through versatile reductive aminations in one step. The standard acidic treatment used in the final deprotection step for peptide synthesis gave the desired C‐terminal secondary amidated peptide with no epimerization. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Preparation of tri(ethylene glycol) grafted core‐shell type polymer support for solid‐phase peptide synthesis

A core‐shell type polymer support for solid‐phase peptide synthesis has been developed for high coupling efficiency of peptides and versatile applications such as on‐bead bioassays. Although various kinds of polymer supports have been developed, they have their own drawbacks including poor accessibility of reagents and incompatibility in aqueous solution. In this paper, we prepared hydrophilic tri(ethylene glycol) (TEG) grafted core‐shell type polymer supports (TEG SURE) for efficient solid‐phase peptide synthesis and on‐bead bioassays. TEG SURE was prepared by grafting TEG derivative on the surface of AM PS resin via biphasic diffusion control method and subsequent acetylation of amine groups which are located at the core region of AM PS resin. The performance of TEG SURE was evaluated by synthesizing several peptides. Three points can be highlighted: (1) easy control of loading level of TEG, (2) improved efficiency of peptide synthesis compared with the conventional resins, and (3) applicability of on‐bead bioassays.     

Enzymatic ligation for synthesis of single‐chain analogue of monellin by transglutaminase*

Monellin, a sweet protein, consists of two noncovalently associated polypeptide chains: an A chain of 44 amino acid residues and a B chain of 50 residues. Microbial transglutaminase (MTGase) was used for ligation of the monellin subunits without any protecting groups, and without activation of the C^α‐carboxyl group at the C‐terminus. Since a peptide fragment LLQG is a good substrate for MTGase to form an amide bond between the γ‐amide group of the Gln residue and the ε‐amino group of Lys, a monellin B chain analogue in which LLQG was elongated at the C‐terminus (B‐LLQG) was synthesized by solid‐phase synthesis. The monellin A chain analogue in which KGK was elongated at the N‐terminus (KGK‐A) was synthesized by the same method as that of the B chain analogue. The KGK‐A chain and the B‐LLQG chain were coupled by MTGase to give single‐chain analogue of monellin. The single‐chain analogue of monellin was characterized by analytical reverse phase high performance liquid chromatography, electrospray ionization, and amino acid analyses. All analyses gave satisfactory results. The single‐chain analogue of monellin was more heat stable than natural monellin. © 1999 John Wiley & Sons, Inc. Biopoly 50: 193–200, 1999     

Microwave-assisted Solid Phase Peptide Synthesis on High Loaded Resins

Kinetic of diffusion of reactants from the solution into the resin beads is a key factor of the efficiency of reaction in solid phase synthesis. We demonstrated in this paper that stirring is required to obtain homogeneous solution during the short coupling steps in microwave-assisted SPPS. Different types of resin, loading rate and coupling reactants were investigated to highlight this observation.     

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.     

Peptide synthesis ‘in water’ by a solution‐phase method using water‐dispersible nanoparticle Boc‐amino acid

Regulatory pressure has compelled the chemical manufacturing industry to reduce the use of organic solvents in synthetic chemistry, and there is currently a strong focus on replacing these solvents with water. Here, we describe an efficient in‐water solution‐phase peptide synthesis method using Boc‐amino acids. It is based on a coupling reaction utilizing suspended water‐dispersible nanoparticle reactants. Using this method, peptides were obtained in good yield and with high purity. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of peptide thioesters via an N–S acyl shift reaction under mild acidic conditions on an N‐4,5‐dimethoxy‐2‐mercaptobenzyl auxiliary group

An efficient method of peptide thioester synthesis is described. The reaction is based on an N‐4,5‐dimethoxy‐2‐mercaptobenzyl (Dmmb) auxiliary‐assisted N‐S acyl shift reaction after assembling a peptide chain by Fmoc‐solid phase peptide synthesis. The Dmmb‐assisted N‐S acyl shift reaction proceeded efficiently under mildly acidic conditions, and the peptide thioester was obtained by treating the resulting S‐peptide with sodium 2‐mercaptoethanesulfonate. No detectable epimerization of the amino acid residue adjacent to the thioester moiety in the case of Leu was found. The reactions were also amenable to the on‐resin preparation of peptide thioesters. The utility was demonstrated by the synthesis of a 41‐mer peptide thioester, a phosphorylated peptide thioester and a 33‐mer peptide thioester containing a trimethylated lysine residue. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of Taxol and Docetaxel by Using 10‐Deacetyl‐7‐xylosyltaxanes

A mixture of taxols was prepared from 10‐deacetyl‐7‐xylosyltaxanes by three‐step reactions: redox, acetylation, and deacetylation. The mixture was separated by column chromatography on silica gel to afford Taxol, Taxol B (Cephalomannine) and Taxol C. The mixture of Taxol B and Taxol C was converted to Docetaxel by Schwartz's reagent. The structures of Taxol and Docetaxel were characterized by HPLC, ¹H‐NMR, ¹³C‐NMR and MS. This synthetic process has expanded the source of biomass for the chemical semi‐synthesis of Taxol and Docetaxel, reduced the production costs, and increased the biomass resource of taxanes.     

A convenient microwave‐enhanced solid‐phase synthesis of short chain N‐methyl‐rich peptides

Structural modification of the peptide backbone via N‐methylation is a powerful tool to modulate the pharmacokinetic profile and biological activity of peptides. Here we describe a rapid and highly efficient microwave(MW)‐assisted Fmoc/tBu solid‐phase method to prepare short chain N‐methyl‐rich peptides, using Rink amide p‐methylbenzhydrylamine (MBHA) resin as solid‐phase support. This method produces peptides in high yield and purity, and reduces the time required for Fmoc‐N‐methyl amino acid coupling. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.