A novel solid phase approach to Aia‐containing peptides

A strategy was developed to directly assemble 4‐amino‐1,2,4,5‐tetrahydro‐indolo[2,3‐c]‐azepin‐3‐ones on solid‐phase‐supported peptide sequences. Fmoc‐ and Boc‐based strategies were investigated. The Fmoc‐strategy approach strongly depends on the peptide sequence being synthesized while the Boc‐based synthesis leads to excellent results for all the selected peptide analogs. The method was applied to prepare Aia‐analogs of several bioactive peptides containing one or more Trp‐residues which were shown to be important for biological recognition. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Facile synthesis of orthogonally protected amino acid building blocks for combinatorial N-backbone cyclic peptide chemistry.

Protected Nalpha-(aminoallyloxycarbonyl) and Nalpha-(carboxyallyl) derivatives of all natural amino acids (except proline), and their chiral inverters, were synthesized using facile and efficient methods and were then used in the synthesis of Nalpha-backbone cyclic peptides. Synthetic pathways for the preparation of the amino acid building units included alkylation, reductive amination and Michael addition using alkylhalides, aldehydes and alpha,beta-unsaturated carbonyl compounds, and the corresponding amino acids. The resulting amino acid prounits were then subjected to Fmoc protection affording optically pure amino acid building units. The appropriate synthetic pathway for each amino acid was chosen according to the nature of the side-chain, resulting in fully orthogonal trifunctional building units for the solid-phase peptide synthesis of small cyclic analogs of peptide loops (SCAPELs). Nalpha-amino groups of building units were protected by Fmoc, functional side-chains were protected by t-Bu/Boc/Trt and N-alkylamino or N-alkylcarboxyl were protected by Alloc or Allyl, respectively. This facile method allows easy production of a large variety of amino acid building units in a short time, and is successfully employed in combinatorial chemistry as well as in large-scale solid-phase peptide synthesis. These building units have significant advantage in the synthesis of peptido-related drugs.     

2-(N-Fmoc)-3-(N-Boc-N-methoxy)-diaminopropanoic acid, an amino acid for the synthesis of mimics of O-linked glycopeptides

Amino acids with N-alkylaminooxy side chains have proven effective for the rapid synthesis of neoglycopeptides. Chemoselective reaction of reducing sugars with peptides containing these amino acids provides glycoconjugates that are structurally similar to their natural counterparts. 2-(N-Fmoc)-3-(N-Boc-N-methoxy)-diaminopropanoic acid (Fmoc: 9-fluorenylmethoxycarbonyl; Boc: t-butyloxycarbonyl) was synthesized from Boc-Ser-OH in >40% overall yield and incorporated into peptides by standard Fmoc chemistry based solid phase peptide synthesis. The resulting peptides are efficiently glycosylated and serve as mimics of O-linked glycopeptides. The synthesis of this derivative greatly expands the availability of the N-alkylaminooxy strategy for neoglycopeptides.     

Fmoc-Protein Synthesis: Preparation of Peptide Thioesters Using a Side-Chain Anchoring Strategy

A side-chain anchoring approach for preparation of peptide thioesters by Fmoc SPPS is reported. This strategy involves the side-chain anchoring of trifunctional amino acids, such as Lys, Glu, Gln, Asp and Asn, for peptide elongation and the post-chain assembly introduction of thioester functionality. This approach allows for the use of standard nucleophilic Fmoc peptide synthesis cycles, which are generally incompatible with thioester-based resin-linkages. The strategy was successfully demonstrated by the straightforward Fmoc syntheses of a model RANTES(1--33) thioester peptide. The Fmoc prepared RANTES(1--33) thioester peptide was then ligated to RANTES(34--68), folded and purified to give the RANTES protein.     

Solid phase synthesis of hydrophobic difficult sequence peptides on BDDMA-PS support.

This article illustrates the successful and efficient solid phase assembly of hydrophobic difficult sequence peptides following both t-Boc and Fmoc chemistry. The peptides were synthesized on an optimized 1,4-butanediol dimethacrylate-crosslinked polystyrene support (BDDMA-PS). Four difficult sequence test peptides, VAVAG, VIVIG, QVGQVELG and VQAAIDYING, were synthesized in relatively good yield and purity without any aggregation problems. The peptides were assembled on chloromethylated and 4-hydroxymethylphenoxymethyl (HMP) BDDMA-PS resins. The peptides were fabricated using Boc amino acid 1-hydroxybenzotriazolyl and Fmoc amino acid pentafluorophenyl active esters in coupling reactions. The peptides after synthesis were cleaved from the polymeric support by exposing the peptidyl resin to 90% trifluroacetic acid/5% thioanisole/5% EDT mixture. The HPLC and MALDI TOF MS studies of the peptides revealed the high homogeneity of the synthesized peptides. Chloromethylated resin having a functional group loading of 1.14 mmol Cl/g was used for the synthesis. The yield and homogeneity of these peptides synthesized using the new support were high when compared with the conventional DVB-PS resin.     

Synthesis of differentially protected N-acylated reduced pseudodipeptides as building units for backbone cyclic peptides.

Backbone cyclization has become an important method for generating or stabilizing the bioactive conformation of peptides without affecting the amino acid side-chains. Up to now, backbone cyclic peptides were mostly synthesized with bridges between N-amino- and N-carboxy-functionalized peptide bonds. To study the influence of a more flexible backbone on the biological activity, we have developed a new type of backbone cyclization which is achieved via the N-functionalized moieties of acylated reduced peptide bonds. As described in our previous publications, the formation of N-functionalized dipeptide units facilitates the peptide assembly compared with the incorporation of N-alkyl amino acids. Besides the racemization-free synthesis of Fmoc-protected pseudodipeptide esters with reduced peptide bonds, the new type of backbone modification allows the use of a great variety of omega-amino- and alpha,omega-dicarboxylic acids differing in chain length and chemical properties. Best results for the coupling of the omega-amino- and alpha,omega-dicarboxylic acids to the reduced peptide bond were obtained by the formation of mixed anhydrides with alkyl chloroformates. Whereas the protecting group combination of Z/OBzl in the dipeptide unit and Boc/OtBu for the N-functionalized moiety leads to the formation of 2-ketopiperazine during hydrogenation, the combination of Fmoc/OtBu and Alloc/OAll is very suitable for the synthesis of backbone cyclic peptides on solid support.     

Solid-phase synthesis of phosphopeptides

We report the solid-phase synthesis of peptides containing O-phosphoserine. Coupling was with commercially available Fmoc-amino acid pentafluorophenyl esters, with base used at each cycle to cleave Fmoc. Phosphorylation of those serine residues left unprotected on the peptide-resin was achieved with dibenzylphosphochloridate, and finally trifluoroacetic acid was used to remove side-chain protecting groups (including the benzyl groups used for the phosphate), and to cleave the peptide from the resin in the same step. This synthetic strategy enables the preparation of peptides with individual, selectively phosphorylated residues. Alternative approaches to introduce protected phosphate and continue with coupling of further amino acids were less advantageous due to the lability of the phosphate group to base and to steric hindrance.     

Self‐assembly of short peptides composed of only aliphatic amino acids and a combination of aromatic and aliphatic amino acids

The morphology of structures formed by the self‐assembly of short N‐terminal t‐butyloxycarbonyl (Boc) and C‐terminal methyl ester (OMe) protected and Boc‐deprotected hydrophobic peptide esters was investigated. We have observed that Boc‐protected peptide esters composed of either only aliphatic hydrophobic amino acids or aliphatic hydrophobic amino acids in combination with aromatic amino acids, formed highly organized structures, when dried from methanol solutions. Transmission and scanning electron microscopic images of the peptides Boc‐Ile‐Ile‐OMe, Boc‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe and Boc‐Trp‐Ile‐Ile‐OMe showed nanotubular structures. Removal of the Boc group resulted in disruption of the ability to form tubular structures though spherical aggregates were formed. Both Boc‐Leu‐Ile‐Ile‐OMe and H‐Leu‐Ile‐Ile‐OMe formed only spherical nanostructures. Dynamic light scattering studies showed that aggregates of varying dimensions were present in solution suggesting that self‐assembly into ordered structures is facilitated by aggregation in solution. Fourier transform infrared spectroscopy and circular dichroism spectroscopy data show that although all four of the protected peptides adopt well‐defined tertiary structures, upon removal of the Boc group, only H‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe had the ability to adopt β‐structure. Our results indicate that hydrophobic interaction is a very important determinant for self‐assembly and presence of charged and aromatic amino acids in a peptide is not necessary for self‐assembly. Copyright © 2012 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.     

Peptidomimetics Through Click Chemistry: Synthesis of Novel β-Keto Triazole Acids from <Emphasis Type="Italic">N</Emphasis>-Protected Amino Acids

An efficient procedure for the preparation of azidomethylketones from N-urethane protected amino acids and their application in Cu(I) catalyzed azide-alkyne cycloaddition reaction are described. The synthesis has been carried out under mild conditions with all the commonly used urethane protected (Fmoc, Boc and Z) amino acids and the desired azides/triazoles were obtained in good yields. Incorporation of these triazole amino acids into small peptides generating dipeptidomimetics containing β-keto triazole units has also been demonstrated.     

Evaluation of new base-labile 2-(4-nitrophenylsulfonyl) ethoxycarbonyl (Nsc)-amino acids for solid-phase peptide synthesis.

The 2-(4-nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group is a new base-labile protecting group for solid-phase peptide synthesis, completely interchangeable with the fluorenylmethoxycarbonyl (Fmoc) protecting group, but with certain advantages. In this paper, we report a methodology with Nalpha-Nsc-protected amino acids for the synthesis of some melanotropins important to our research, namely, gamma-melanocyte-stimulating hormone (gamma-MSH), its [Nle3]-analogue, and a cyclic alpha-MSH/beta-MSH hybrid. We developed an efficient protocol for the synthesis of the cyclic MSH analogue that yielded this peptide in >98% purity. The gamma-MSH synthesis, which gave problems with both the Boc and Fmoc strategies, yielded the desired peptide by Nsc-chemistry but was accompanied by side products. Finally, the Nle3-gamma-MSH analogue was synthesized more efficiently using the Fmoc strategy, suggesting that Nsc-chemistry might not be the best methodology for certain sequences.     

The synthesis and spectroscopic analysis of the neurotoxic prion peptide 106-126: Comparative use of manual Boc and Fmoc chemistry

Summary A peptide corresponding to residues 106–126 of the human prion protein (PrP) possesses the neurotoxic and amyloidogenic properties of the infectious form of the parental protein. This peptide is now identified as a ‘difficult sequence’ and synthesis using conventional manual Fmoc chemistry was unsuccessful with acylation terminating at a central core of hydrophobic amino acids. The use of tetramethylfluoroformamidinium hexafluorophosphate and 1-methyl-2-pyrrolidone as anti-aggregatory agents in the coupling steps improved the synthesis but still resulted in an incomplete peptide. The incorporation ofN-(2-hydroxy-4-methoxybenzyl)protection at glycine residues 119 and 124 enabled synthesis of the full length peptide in low yield. Synthesis using Boc chemistry within situ neutralisation gave the full length peptide in high yield.     

Solid-phase route to Fmoc-protected cationic amino acid building blocks

Diamino acids are commonly found in bioactive compounds, yet only few are commercially available as building blocks for solid-phase peptide synthesis. In the present work a convenient, inexpensive route to multiple-charged amino acid building blocks with varying degree of hydrophobicity was developed. A versatile solid-phase protocol leading to selectively protected amino alcohol intermediates was followed by oxidation to yield the desired di- or polycationic amino acid building blocks in gram-scale amounts. The synthetic sequence comprises loading of (S)-1-(p-nosyl)aziridine-2-methanol onto a freshly prepared trityl bromide resin, followed by ring opening with an appropriate primary amine, on-resin N(β)-Boc protection of the resulting secondary amine, exchange of the N(α)-protecting group, cleavage from the resin, and finally oxidation in solution to yield the target γ-aza substituted building blocks having an Fmoc/Boc protection scheme. This strategy facilitates incorporation of multiple positive charges into the building blocks provided that the corresponding partially protected di- or polyamines are available. An array of compounds covering a wide variety of γ-aza substituted analogs of simple neutral amino acids as well as analogs displaying high bulkiness or polycationic side chains was prepared. Two building blocks were incorporated into peptide sequences using microwave-assisted solid-phase peptide synthesis confirming their general utility.     

N‐(ureidoethyl)amides of cyclic enkephalin analogs

Novel N‐(ureidoethyl)amides of cyclic enkephalin analogs have been synthesized. The p‐nitrophenyl carbamate of 1‐Boc‐1,2‐diaminoethane was coupled with 4‐methylbenzhydrylamine (MBHA) resin. The Boc group was removed by treatment with HCl/dioxane, and the peptide chain was assembled using Boc strategy. For deprotection of amino function, HCl/dioxane was used. D ‐Lys or D ‐Orn were incorporated in position 2, and the side chains of Lys, Orn, Dab, or Dap in position 5 were protected with Fmoc group. Side chain protection was removed by treatment with 55% piperidine in DMF, and cyclization was achieved by treatment with bis‐(4‐nitrophenyl)carbonate to form a urea bridge. The peptide was cleaved from the resin by treatment with 45% TFA in DCM. The peptides were tested in the guinea‐pig ileum (GPI) and mouse vas deferens (MVD) assays. Divers opioid activities were observed, depending on the size of the ring. In comparison with [Leu⁵]enkephalin, all peptides were more active in the GPI assay (between 125 and 12 times), and some of them were also more potent in the MVD assay. The conformational propensities of each peptide were determined using the EDMC method in conjunction with NMR experiments. This approach allows treating the dynamical behavior of small peptides properly. The results were compared with those obtained previously for corresponding nonsubstituted amides and are in agreement with the biologically active conformation proposed by us earlier. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Solid-phase synthesis of MEN 11270, a new cyclic peptide kinin B2 receptor antagonist.

An efficient synthesis of the cyclic decapeptide MEN 11270 [H-DArg1-Arg2 Pro3-Hyp4-Gly5-Thi6-Dab7-DTic8-Oic9-Arg10 c(7gamma - 10alpha)] was developed. Two three-dimensional orthogonal strategies were applied and compared: Fmoc/Tos/Boc (procedure A) and Fmoc/Pmc/Dde (procedure B). Both resulted in a 23-step strategy comprising the stepwise solid-phase chain assembly of the linear protected peptide, partial deprotection, solution-phase cyclization and final full deprotection. The stepwise assembly of the linear peptide was optimized by double coupling and acylation time prolongation for critical residues (Tic, Dab, Thi, Pro). O-(7-azabenzotriazol-1-yl)-N,N,N',N' tetramethyluronium (HATU) was preferred as coupling reagent for Dab. In the cyclization step, the partial racemization of Arg10 (31% using 1-ethyl-3-(3'-dimethyl-aminopropyl) carbodiimide/1-hydroxybenzotriazole (EDC/HOBt) as activation system) was reduced to 3% with HATU. The final deprotection was performed in the presence of dimethylsulfide (procedure A) and thiocresol (procedure B) as scavengers, to avoid the sulfation of Hyp side chain. The final compound and the main by-products were characterized by mass spectroscopy (MS), nuclear magnetic resonance (NMR) and racemization test. Procedure B produced operationally simpler and more efficient results than A (28% overall yield versus 4%).     

Practical approach to solid-phase synthesis of C-terminal peptide amides under mild conditions based on a photolysable anchoring linkage

Several 3-nitro-4-(N-protected aminomethyl)benzoic acids; with protection provided by tert.-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), trifluoroacetyl (Tfa), dithiasuccinoyl (Dts), or phthaloyl (Phth), have been prepared by reproducible routes. Synthesis of Dts-handle 6 illustrates some particularly novel and efficient chemistry, and is preferred over more intricate routes to Boc-handle 3 and Fmoc-handle 4. The five handles were each evaluated for their application to the synthesis of peptide amides. Coupling onto amino-functionalized supports provided a general starting point for peptide chain assembly. The handle amino function was deblocked (Boc, Fmoc, Dts), the C-terminal residue was coupled as its N alpha-protected free acid, and ultimately the ortho-nitrobenzylamide anchorage linkage was cleaved photolytically to give the corresponding amide. Starting with handles 3, 4, and 6, several free and protected peptide amides were synthesized.     

Nsc and Fmoc Nalpha-amino protection for solid-phase peptide synthesis: a parallel study.

The 2-(4-nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group is an alternative to Fmoc for Nalpha-protection in solid-phase peptide synthesis. Nsc-amino acids may be particularly suitable for automatic synthesizers, in which the amino acids are stored in solution, and the incorporation of residues prone to racemization such as Cys and His. Owing to the hydrophilicity of the Nsc group, these derivatives are useful for the preparation of protected peptides in convergent solid-phase peptide synthesis strategies.     

Optimized syntheses of Fmoc azido amino acids for the preparation of azidopeptides

The rise of CuI‐catalyzed click chemistry has initiated an increased demand for azido and alkyne derivatives of amino acid as precursors for the synthesis of clicked peptides. However, the use of azido and alkyne amino acids in peptide chemistry is complicated by their high cost. For this reason, we investigated the possibility of the in‐house preparation of a set of five Fmoc azido amino acids: β‐azido l ‐alanine and d ‐alanine, γ‐azido l ‐homoalanine, δ‐azido l ‐ornithine and ω‐azido l ‐lysine. We investigated several reaction pathways described in the literature, suggested several improvements and proposed several alternative routes for the synthesis of these compounds in high purity. Here, we demonstrate that multigram quantities of these Fmoc azido amino acids can be prepared within a week or two and at user‐friendly costs. We also incorporated these azido amino acids into several model tripeptides, and we observed the formation of a new elimination product of the azido moiety upon conditions of prolonged couplings with 2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate/DIPEA. We hope that our detailed synthetic protocols will inspire some peptide chemists to prepare these Fmoc azido acids in their laboratories and will assist them in avoiding the too extensive costs of azidopeptide syntheses. Experimental procedures and/or analytical data for compounds 3 – 5 , 20 , 25 , 26 , 30 and 43 – 47 are provided in the supporting information. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.     

Solid-phase synthesis of peptides containing phosphoserine using phosphate tert.-butyl protecting group

In this paper, we report the solid-phase synthesis of peptides containing O-phosphonoserine using BOP as coupling reagent. Commercially available Fmoc amino-acids linked to p-alkoxybenzyl resin were used in the first step and Alloc amino acids in the following. Alloc group was removed by catalytic hydrostannolytic cleavage. Acid-labile side-chain protecting groups (including phosphate residue) were used. Thus, both removal of side-chain protecting groups and cleavage of the phosphopeptide from the resin were achieved in one step by treatment with TFA. Alloc serine was phosphorylated by the phosphoramidite method. This strategy enables the preparation of peptides with selectively phosphorylated residue and overcomes problems due to repetitive treatments with TFA and final cleavage with HF.     

Peptide synthesis containing a B-cell and a T-cell epitope on dextran beads and evaluation of humoral response against bead-peptide construct

Summary Cross-linked dextran beads were chemically modified with Fmoc-βAla to give an amino functionalized support suitable for solid-phase peptide synthesis. On this support, a peptide compriising B-cell and T-cell epitopes in tandem was synthesized manually by Fmoc procedure monitoring the coupling reaction by bromophenol blue procedure and standard Kaiser test. The quality of peptide synthesis was verified by RP-HPLC and mass spectrometry. The size and morphologic characteristics of dextran beads were conserved after peptide synthesis. Furthermore, we analyzed immunogenicity in mice of the T-B peptide on dextran beads compared with T-B peptide immunogenicity when administered in CFA/IFA. In both cases, titers were high and there was not a significant difference in antibody titers between groups (p<0.05). But in contrast CFA they are biocompatible and did not induce any adverse reaction at the site of injection. This experiment suggests that the peptides synthesis on dextran beads can be used to raise the immunogenicity of synthetic peptide in vaccines or therapeutics.