Synthesis of the bradykinin B1 antagonist [desArg10]HOE 140 on 2-chlorotrityl resin

Summary This paper describes the synthesis of the bradykinin B₁ antagonist [desArg¹⁰]HOE 140 (d-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-d-Tic-Oic-OH) by the solid-phase method. This synthesis is predicted to be a difficult one because the C-terminal sequence d-Tic-Oic, when linked to the resin, could easily undergo an intramolecular aminolysis, releasing the corresponding diketopiperazine. This reaction is greatly favored by the imino acidic structure of these two residues and by the d-configuration of the second one. When using the Fmoc strategy, the reaction takes place during the Fmoc removal with piperidine. In this case, it has been suggested previously that it is possible to prevent the side reaction by reducing the time of the base treatment. In our hands, this expedient worked correctly for the synthesis of a test tetrapeptide (H-Gly-Pro₃-OH), but under the same conditions the synthesis of the target peptide failed completely. In contrast, the use of the very hindered 2-chlorotrityl resin reduced diketopiperazine formation to undetectable levels.     

Synthesis of cyclic penta- and hexapeptides: A general synthetic strategy on DAS resin

The active part or receptor-binding sequence of peptide hormones can usually be defined by a span of 4–8 amino acids. Cyclic penta- and hexapeptides are excellent model systems for performing conformational and structure-function studies on this class of bioactive molecules. A synthetic scheme has been devised comprising solid-phase Fmoc chemistry followed by resin cleavage, cyclization in solution, and, finally, side-chain deprotection. A new resin, DAS, cleaved under weak acid conditions, is an excellent solid-phase synthesis support, and HBTU or PyBOP are the activation reagents of choice, not only during synthesis, but also for the cyclization reaction. Three cyclic peptides were synthesized using this method, one requiring extensive side-chain protection, and this method has general applicability for any cyclic pentapeptide or hexapeptide, giving good yields and high purity.     

Silyl linker-based approach to the solid-phase synthesis of Fmoc glycopeptide thioesters

An efficient solid-phase synthesis of Fmoc (glyco)peptide thioesters is described. Fmoc x Ser x OAll and Fmoc x Thr x OAll bound to resin with a silyl ether linker were deallylated by Pd(0) catalysis and condensed with thiophenol, benzyl mercaptane, and ethyl 3-mercaptopropionate by activation with DCC/HOBt. The thioesters were released from the resin either by treatment with CsF-AcOH or by acidic hydrolysis. The effectiveness of this silyl linker strategy is further demonstrated by the synthesis of more complex (glyco)peptide thioesters 25, 26 and 27 involving N-->C and C-->N peptide elongation.     

Chemical synthesis of NS-1 region of hepatitis C-viral polyprotein fragments: a comparison of PS-BDODMA resin and merrifield resin

Three peptide fragments selected from the NS-1 region of hepatitis C-viral polyprotein (Leu-Ile-Asn-Thr-Asn-Ala-Ser-Trp-His-Ala-Asn-Arg-Thr-Ala-Leu-Ser Asn-Asp Ser-Lys Leu Asn Thr-Gly Ala NH(2), Leu-lle Asn Thr Asn Ala Ser-Trp-His-Ala-Asn-Arg-Thr Ala NH(2) and Leu-Asn-Cys(Acm)-Asn-Asp-Ser-Leu-Asn-Thr-Ala-NH(2)) have been synthesized on PS-BDODMA resin. The synthetic capability of the resin PS-BDODMA resin was compared with Merrifield resin. The peptides were synthesized by the stepwise fluoren-9-yl methoxycarbonyl (Fmoc) solid-phase method. The synthesized peptides were purified by HPLC and the identity of the peptides was established by mass spectrum and amino acid analysis. The synthesis of these peptides illustrates the application of the PS-BDODMA resin for the synthesis of long chain peptides in high yield and homogeneity compared to the Merrifield resin.     

A simple oxazolidine linker for solid-phase synthesis of peptide aldehydes

A very simple and cheap linker has been used for solid-phase synthesis of peptide aldehydes. Protected amino acid aldehydes are immobilized on 2-Cl(trt) resin as oxazolidine formation via diethanolamine. After classical Fmoc SPPS, treatment of the resin with AcOH/DCM/H(2)O (8:1:1) affords peptide aldehydes in high yield and purity.     

Synthesis, characterization, and evaluation of PS-PPDC resin: a novel flexible cross-linked polymeric support for solid-phase organic synthesis

The present study describes the synthesis of different mole densities of poly(propylene glycol)dimethacrylate cross-linked resins using monomer units such as styrene and 4-chloromethyl styrene and its evaluation as an ideal support toward different stages of solid-phase peptide synthesis. Free radical generated aqueous suspension polymerization has been followed for polymerization and the formation of resin was characterized using infrared and carbon-13 spectroscopic techniques. Surface morphology of resin was examined by scanning electron microscopy. The polymerization reaction was investigated with respect to the effect of amount of cross-linking agent to verify the swelling, loading, and the mechanical stability of resin. Solvent imbibition abilities in commonly used solvents were measured and compared to commercially available Merrifield as well as reported styrene-acryloyloxyhydroxypropyl methacrylate-tripropyleneglycol diacrylate (SAT resins. The chemical inertness of the support was also checked with different reagents used for solid-phase peptide synthesis. The suitability of support was demonstrated by synthesizing biologically potent Endothelin class of linear peptides by Fmoc strategy and compared to SAT resin. The purities of synthetic peptides were analyzed by high-performance liquid chromatography and corresponding masses by matrix-assisted laser desorption/ionisation-time of flight analysis.     

Synthesis of Aβ[1‐42] and its derivatives with improved efficiency

It has been proved that the principal component of senile plaques is aggregates of β‐amyloid peptide (Aβ) in cases of one of the most common forms of age‐related neurodegenerative disorders, Alzheimer's disease (AD). Although the synthetic methods for the synthesis of Aβ peptides have been developed since their first syntheses, Aβ[1‐42] is still problematic to prepare. The highly hydrophobic composition of Aβ[1‐42] results in aggregation between resin‐bound peptide chains or intrachain aggregation which leads to a decrease in the rates of deprotection and repetitive incomplete coupling reactions during 9‐flurenylmethoxycarbonyl (Fmoc) synthesis. In order to avoid aggregation and/or disrupt internal aggregation during stepwise Fmoc solid phase synthesis and to improve the quality of crude products, several attempts have been made. Since highly pure Aβ peptides in large quantities are used in biological experiments, we wanted to develop a method for a rational synthesis of human Aβ[1‐42] with high purity and adequate yield. This paper reports a convenient methodology with a novel solvent system for the synthesis of Aβ[1‐42], its N‐terminally truncated derivatives Aβ[4‐42] and Aβ[5‐42], and Aβ[1‐42] labeled with 7‐amino‐4‐methyl‐3‐coumarinylacetic acid (AMCA) at the N‐terminus using Fmoc strategy. The use of 10% anisole in Dimethylformamide/Dichloromethane (DMF/DCM) can substantially improve the purity and yield of crude Aβ[1‐42] and has been shown to be an optimal coupling condition for the synthesis of Aβ[1‐42]. Anisole is a cheap and simple aid in the synthesis of ‘difficult sequences’ where other solvents are less successful in the prevention of aggregation during the synthesis. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd.     

A general method for preparation of peptides biotinylated at the carboxy terminus.

A method for the preparation of a biotinylated resin that can be elongated by standard methods of solid-phase peptide synthesis to give peptides biotinylated at the carboxy terminus is described. This methodology is particularly important for the preparation of biotinylated peptides in which a free amino terminus is required. Coupling of N epsilon-9-fluorenylmethoxycarbonyl-(Fmoc)-N alpha-tert-butyloxycarbonyl(Boc)-L- lysine to p-methylbenzhydrylamine resin, followed by removal of the Fmoc protecting group and reaction with (+)-biotin-4-nitrophenyl ester yielded N alpha-Boc-biocytin-p-methyl-benzhydrylamine resin. The utility of this resin was tested by the synthesis of a biotinylated peptide, Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg-biocytin-NH2, for use as an in vitro substrate for myristoyl-CoA:protein N-myristoyltransferase (NMT), the enzyme that catalyzes protein N-myristoylation. Analysis of the peptide derivative by HPLC and mass spectrometry revealed a single major product of the expected mass, indicating that the biotin group survived cleavage and deprotection with HF. The biotinylated peptide served as a substrate for NMT, and the resulting myristoylated peptide could be quantitatively recovered by adsorption to immobilized avidin.     

On‐resin conversion of Cys(Acm)‐containing peptides to their corresponding Cys(Scm) congeners

The Acm protecting group for the thiol functionality of cysteine is removed under conditions (Hg²⁺) that are orthogonal to the acidic milieu used for global deprotection in Fmoc‐based solid‐phase peptide synthesis. This use of a toxic heavy metal for deprotection has limited the usefulness of Acm in peptide synthesis. The Acm group may be converted to the Scm derivative that can then be used as a reactive intermediate for unsymmetrical disulfide formation. It may also be removed by mild reductive conditions to generate unprotected cysteine. Conversion of Cys(Acm)‐containing peptides to their corresponding Cys(Scm) derivatives in solution is often problematic because the sulfenyl chloride reagent used for this conversion may react with the sensitive amino acids tyrosine and tryptophan. In this protocol, we report a method for on‐resin Acm to Scm conversion that allows the preparation of Cys(Scm)‐containing peptides under conditions that do not modify other amino acids. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Comparison of the solid-phase fragment condensation and phase-change approaches in the synthesis of salmon I calcitonin.

Salmon I calcitonin was synthesized using both phase-change and conventional solid-phase fragment condensation (SPFC) approaches, utilizing the Rink amide linker (Fmoc-amido-2,4-dimethoxybenzyl-4-phenoxyacetic acid) combined with 2-chlorotrityl resin and the Fmoc/tBu(Trt)-based protection scheme. Phase-change synthesis, performed by the selective detachment of the fully protected C-terminal 22-mer peptide-linker from the resin and subsequent condensation in solution with the N-terminal 1-10 fragment, gave a product of slightly less purity (85 vs. 92%) than the corresponding synthesis on the solid-phase. In both cases salmon I calcitonin was easily obtained in high purity.     

Solid-Phase synthesis and stability of triple-helical peptides incorporating native collagen sequences

A generally applicable solid-phase methodology has been developed for the synthesis of triple-helical polypeptides incorporating native collagen sequences. Three nascent peptide chains are C-terminal linked through one N^α-amino and two N^ε-amino groups of Lys, while repeating Gly-Pro-Hyp triplets induce triple helicity. Different protecting group strategies, including several three-dimensionally orthogonal schemes, have been utilized for the synthesis of four homotrimeric triple-helical polypeptides (THPs) of 79–124 residues, three of which incorporate native type IV collagen sequences. Highly efficient assemblies were achieved by 9-fluorenylmethoxycarbonyl (Fmoc) N^α-amino group protection, in situ 2-(1H-benzotriazole-l-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate mediated couplings, and 1,8-diazabicyclo [5.4.0] undec-7-ene mediated Fmoc group removal. THPs were characterized by Edman degradation sequencing, size-exclusion chromatography, mass spectrometry, reversed-phase high performance liquid chromatography, and CD spectroscopy. THP thermal stabilities ranged from 35 to 59°C, with chain length and Hyp content being the influential factors. Melting temperatures and van't Hoff enthalpies for peptide triple-helical denaturation could be correlated well to Hyp content. The THP synthetic protocol developed here will allow for the study of both structure and biological activity of specific collagen sequences in homotrimeric and heterotrimeric forms. © 1993 John Wiley & Sons, Inc.     

An efficient, asymmetric solid-phase synthesis of benzothiadiazine-substituted tetramic acids: potent inhibitors of the hepatitis C virus RNA-dependent RNA polymerase

An efficient, asymmetric solid-phase synthesis of benzothiadiazine-substituted tetramic acids is reported. Starting from commercially available chiral Fmoc-protected alpha-amino acids loaded onto Wang resin, Fmoc removal, reductive amination followed by amide bond formation, and base-catalyzed cyclization with simultaneous cleavage from the resin provided the desired products. Compounds described are potent inhibitors of the hepatitis C virus RNA-dependent RNA polymerase.     

Acanthoscurrin fragment 101-132: total synthesis at 60 degrees C of a novel difficult sequence

Glycine-rich proteins (GRPs) serve a variety of biological functions. Acanthoscurrin is an antimicrobial GRP isolated from hemocytes of the Brazilian spider Acanthoscurria gomesiana. Aiming to contribute to the knowledge of the secondary structure and stepwise solid-phase synthesis of GRPs' glycine-rich domains, we attempted to prepare G(101)GGLGGGRGGGYG(113)GGGGYGGGYG(123) GGY(126)GGGKYK(132)-NH(2), acanthoscurrin C-terminal amidated fragment. Although a theoretical prediction did not indicate high aggregation potential for this peptide, repetitive incomplete aminoacylations were observed after incorporating Tyr(126) to the growing peptide-MBHA resin (Boc chemistry) at 60 degrees C. The problem was not solved by varying the coupling reagents or solvents, adding chaotropic salts to the reaction media or changing the resin/chemistry (Rink amide resin/Fmoc chemistry). Some improvement was made when CLEAR amide resin (Fmoc chemistry) was used, as it allowed for obtaining fragment G(113)-K(132). NIR-FT-Raman spectra collected for samples of the growing peptide-MBHA, -Rink amide resin and -CLEAR amide resin revealed the presence of beta-sheet structures. Only the combination of CLEAR-amide resin, 60 degrees C, Fmoc-(Fmoc-Hmb)Gly-OH and LiCl (the last two used alternately) was able to inhibit the phenomenon, as proven by NIR-FT-Raman analysis of the growing peptide-resin, allowing the total synthesis of desired fragment Gly(101)-K(132). In summary, this work describes a new difficult sequence, contributes to understanding stepwise solid-phase synthesis of this type of peptide and shows that, at least while protected and linked to a resin, this GRP's glycine-rich motif presents an early tendency to assume beta-sheet structures.     

High-yield, solid-phase synthesis of humanin, an Alzheimer's disease associated, novel 24-mer peptide which contains a difficult sequence.

Humanin is a novel, 24-mer residue bioactive peptide, which antagonizes Alzheimer's disease (AD) related neurotoxicity and offers a hope for developing new therapeutics against AD. Access to adequate amounts of pure humanin is a prerequisite for further, thorough, investigation of the pharmacological properties and therapeutic potency of the peptide. Until now, humanin has been obtained mainly by molecular biology techniques. In this work the Fmoc solid-phase synthesis of humanin on an in-house prepared 2-Cl-tritylamidomethyl polystyrene resin is described fully. Special precautions, i.e. prolonged deprotection steps, should be taken to achieve a high overall yield, since humanin seems to contain a 'difficult sequence' (R4G5F6S7C8L9) near its highly lipophilic, biologically important region L9L10L11L12.     

Problem of aspartimide formation in Fmoc-based solid-phase peptide synthesis using Dmab group to protect side chain of aspartic acid.

The sequence-dependent, acid- or base-catalysed aspartimide formation is one of the most serious side reactions in solid-phase synthesis of peptides containing aspartic acid. In the present work, we investigated the susceptibility of 4-(N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino)benzyl (Dmab), an aspartic acid beta-carboxy side-chain protecting group, for aspartimide formation. As a model, 15-amino acid-residue galanin fragment analogue containing the Asp-Ala motif was used during Fmoc-based solid-phase synthesis. Our study showed a strong tendency of Dmab-protected peptide to form aspartimide with unusual high efficiency. Furthermore, to investigate the susceptibility of Asp-Ala motif for aspartimide formation during the synthesis using Asp(ODmab), a 5-amino acid-residue galanin fragment LGPDA, different types of resin linkers, variety of Fmoc-deprotection conditions and coupling methods were applied.     

Substitution determination of Fmoc‐substituted resins at different wavelengths

In solid‐phase peptide synthesis, the nominal batch size is calculated using the starting resin substitution and the mass of the starting resin. The starting resin substitution constitutes the basis for the calculation of a whole set of important process parameters, such as the number of amino acid derivative equivalents. For Fmoc‐substituted resins, substitution determination is often performed by suspending the Fmoc‐protected starting resin in 20% (v/v) piperidine in DMF to generate the dibenzofulvene–piperidine adduct that is quantified by ultraviolet–visible spectroscopy. The spectrometric measurement is performed at the maximum absorption wavelength of the dibenzofulvene–piperidine adduct, that is, at 301.0 nm. The recorded absorption value, the resin weight and the volume are entered into an equation derived from Lambert–Beer's law, together with the substance‐specific molar absorption coefficient at 301.0 nm, in order to calculate the nominal substitution. To our knowledge, molar absorption coefficients between 7100 l mol^?1 cm^?1 and 8100 l mol^?1 cm^?1 have been reported for the dibenzofulvene–piperidine adduct at 301.0 nm. Depending on the applied value, the nominal batch size may differ up to 14%. In this publication, a determination of the molar absorption coefficients at 301.0 and 289.8 nm is reported. Furthermore, proof is given that by measuring the absorption at 289.8 nm the impact of wavelength accuracy is reduced. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.     

Rapid semi-on-line monitoring of Fmoc solid-phase peptide synthesis by matrix-assisted laser desorption/ionization mass spectrometry

A simple yet highly effective application of matrix-assisted laser desorption/ionization massspectrometry (MALDI-MS) for the rapid monitoring of Fmoc solid-phase peptide synthesisis described. A few beads of the resin are removed at any desired step during synthesis, thefully protected peptide is cleaved from the resin and an MS spectrum of the analytes presentis produced. Some standard side-chain protecting groups may be cleaved off during samplepreparation for MS analysis; however, these cleavages are readily identified. Using thisapproach, incomplete amino acid acylations are readily detected in approximately the sametime as by traditional tests such as ninhydrin. The semi-on-line method also lends itself toready optimization of synthesis protocols and to the examination of resin-bound peptide sidereactions which may not be detectable by chemical means.     

Rapid semi-on-line monitoring of Fmoc solid-phase peptide synthesis by matrix-assisted laser desorption/ionization mass spectrometry

Summary A simple yet highly effective application of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the rapid monitoring of Fmoc solid-phase peptide synthesis is described. A few beads of the resin are removed at any desired step during synthesis, the fully protected peptide is cleaved from the resin and an MS spectrum of the analytes present is produced. Some standard side-chain protecting groups may be cleaved off during sample preparation for MS analysis; however, these cleavages are readily identified. Using this approach, incomplete amino acid acylations are readily detected in approximately the same time as by traditional tests such as ninhydrin. The semi-on-line method also lends itself to ready optimization of synthesis protocols and to the examination of resin-bound peptide side reactions which may not be detectable by chemical means.     

Solid-phase synthesis of the glycopeptide of human glycophorin AM bearing the consecutive sialyl-T antigen

Fmoc solid-phase synthesis of the N-terminal glycopentapeptide of human glycophorin AM, bearing the consecutive sialyl-T antigen, was accomplished using glycosylated amino acid building blocks on a weakly acid-labile resin with high efficiency. The benzylated glycopeptide was treated with TMSOTf-thioanisole in TFA and then with aq NaHCO3 to afford the deprotected glycopeptide in good yield.     

Application of solid-phase extraction to agar-supported fermentation

Agar-supported fermentation (Ag-SF), a variant of solid-state fermentation, has recently been improved by the development of a dedicated 2 m² scale pilot facility, Platotex. We investigated the application of solid-phase extraction (SPE) to Ag-SF in order to increase yields and minimize the contamination of the extracts with agar constituents. The selection of the appropriate resin was conducted on liquid-state fermentation and Diaion HP-20 exhibited the highest recovery yield and selectivity for the metabolites of the model fungal strains Phomopsis sp. and Fusarium sp. SPE applied to Ag-SF resulted in a particular compartmentalization of the culture. The mycelium that requires oxygen to grow migrates to the top layer and formed a thick biofilm. The resin beads intercalate between the agar surface and the mycelium layer, and trap directly the compounds secreted by the mycelium through a “solid–solid extraction” (SSE) process. The resin/mycelium layer is easily recovered by scraping the surface and the target metabolites extracted by methanol. Ag-SF associated to SSE represents an ideal compromise for the production of bioactive secondary metabolites with limited economic and environmental impact.