Lewis acid catalysed methylation of N‐(9H‐fluoren‐9‐yl)methanesulfonyl (Fms) protected lipophilic α‐amino acid methyl esters

This work reports an efficient Lewis acid catalysed N‐methylation procedure of lipophilic α‐amino acid methyl esters in solution phase. The developed methodology involves the use of the reagent system AlCl₃/diazomethane as methylating agent and α‐amino acid methyl esters protected on the amino function with the (9H‐fluoren‐9‐yl)methanesulfonyl (Fms) group. The removal of Fms protecting group is achieved under the same conditions to those used for Fmoc removal. Thus the Fms group can be interchangeable with the Fmoc group in the synthesis of N‐methylated peptides using standard Fmoc‐based strategies. Finally, the absence of racemization during the methylation reaction and the removal of Fms group were demonstrated by synthesising a pair of diastereomeric dipeptides. Copyright © 2015 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.     

Design,synthesis and biological activity of cell‐penetrating peptide‐modified octreotide analogs

Four novel octreotide analogs with cell‐penetrating peptides (CPPs) at the N‐terminus or C‐terminus were synthesized by a stepwise Fmoc solid‐phase synthesis strategy. The synthesized peptides were analyzed and characterized using reverse phase HPLC and MALDI‐TOF mass spectrometry. The antiproliferative activity of the analogs was tested in vitro on human gastric (SGC‐7901) and hepatocellular cancer (BEL7402) cell lines using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Interestingly, these analogs showed a higher anticancer activities than the parent octreotide except CMTPT03 analog. The results demonstrate that the designed octreotide analogs enhance their anticancer activity after linking together the CPPs to octreotide at the N‐terminus, and are potential molecules for future use in cancer therapy and drug targeting. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and conformation of an analog of the helix‐loop‐helix domain of the Id1 protein containing the O‐acyl iso‐prolyl‐seryl switch motif

Synthetic peptides reproducing the helix‐loop‐helix (HLH) domains of the Id proteins fold into highly stable helix bundles upon self‐association. Recently, we have shown that the replacement of the dipeptide Val‐Ser at the loop–helix‐2 junction with the corresponding O‐acyl iso‐dipeptide leads to a completely unfolded state that only refolds after intramolecular O → N acyl migration. Herein, we report on an Id HLH analog based on the substitution of the Pro‐Ser motif at the helix‐1–loop junction with the corresponding O‐acyl iso‐dipeptide. This analog has been successfully synthesized by solid‐phase Fmoc chemistry upon suppression of DKP formation. No secondary structure could be detected for the O‐acyl iso‐peptide before its conversion into the native form by O → N acyl shift. These results show that the loop–helix junctions are determinant for the folded/unfolded state of the Id HLH domain. Further, despite the high risk of DKP formation, peptides containing O‐acyl iso‐Pro‐Ser/Thr units are synthetically accessible by Fmoc chemistry. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Design and Synthesis of Fmoc‐Thr[PO(OH)(OPOM)] for the Preparation of Peptide Prodrugs Containing Phosphothreonine in Fully Protected Form

The design and efficient synthesis of N‐Fmoc‐phosphothreonine protected by a mono‐(pivaloyloxy)methyl (POM) moiety at its phosphoryl group (Fmoc‐Thr[PO(OH)(OPOM)]‐OH, 1 , is reported. This reagent is suitable for solid‐phase syntheses employing acid‐labile resins and Fmoc‐based protocols. It allows the preparation of phosphothreonine (pThr)‐containing peptides bearing bis‐POM‐phosphoryl protection. The methodology allows the first reported synthesis of pThr‐containing polypeptides having bioreversible prodrug protection, and as such it should be useful in a variety of biological applications.     

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 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.     

Synthesis and application of Nα‐Fmoc‐Nπ‐4‐methoxybenzyloxymethylhistidine in solid phase peptide synthesis

The 4‐methoxybenzyloxymethyl (MBom) group was introduced at the N^π‐position of the histidine (His) residue by using a regioselective procedure, and its utility was examined under standard conditions used for the conventional and the microwave (MW)‐assisted solid phase peptide synthesis (SPPS) with 9‐fluorenylmethyoxycarbonyl (Fmoc) chemistry. The N^π‐MBom group fulfilling the requirements for the Fmoc strategy was found to prevent side‐chain‐induced racemization during incorporation of the His residue even in the case of MW‐assisted SPPS performed at a high temperature. In particular, the MBom group proved to be a suitable protecting group for the convergent synthesis because it remains attached to the imidazole ring during detachment of the protected His‐containing peptide segments from acid‐sensitive linkers by treatment with a weak acid such as 1% trifluoroacetic acid in dichloromethane. We also demonstrated the facile synthesis of Fmoc‐His(π‐MBom)‐OH with the aid of purification procedure by crystallization to effectively remove the undesired τ‐isomer without resorting to silica gel column chromatography. This means that the present synthetic procedure can be used for large‐scale production without any obstacles. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Preventing aspartimide formation in Fmoc SPPS of Asp‐Gly containing peptides — practical aspects of new trialkylcarbinol based protecting groups

In our efforts to develop a universal solution to the problem of aspartimide formation in Fmoc SPPS, we investigated the application of our new β‐trialkylmethyl protected aspartic acid building blocks to the synthesis of peptides containing the Asp‐Gly motif. The N^α‐Fmoc aspartic acid β‐tri‐(ethyl/propyl/butyl)methyl esters were used in the synthesis of the classic model peptide scorpion toxin II (VKDGYI), and their effectiveness in minimising aspartimide formation during extended piperidine treatments was evaluated. Furthermore, we compared their efficacy against that of the commonly used approach of adding acids to the Fmoc deprotection solution. Finally, we applied our aspartic acid building blocks to the stepwise Fmoc SPPS of teduglutide, a human GLP‐2 analogue, whose synthesis is made challenging by extensive aspartimide formation. In all experiments, our approach led to almost complete reduction of aspartimide formation with accompanied suppression of aspartic acid epimerisation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Site‐specific labeling of synthetic peptide using the chemoselective reaction between N‐methoxyamino acid and isothiocyanate

Site‐specific labeling of synthetic peptides carrying N‐methoxyglycine (MeOGly) by isothiocyanate is demonstrated. A nonapeptide having MeOGly at its N‐terminus was synthesized by the solid‐phase method and reacted with phenylisothiocyanate under various conditions. In acidic solution, the reaction specifically gave a peptide having phenylthiourea structure at its N‐terminus, leaving side chain amino group intact. The synthetic human β‐defensin‐2 carrying MeOGly at its N‐terminus or the side chain amino group of Lys¹⁰ reacted with phenylisothiocyanate or fluorescein isothiocyanate also at the N‐methoxyamino group under the same conditions, demonstrating that this method is generally useful for the site‐specific labeling of linear synthetic peptides as well as disulfide‐containing peptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

O‐Acyl isopeptide method: development of an O‐acyl isodipeptide unit for Boc SPPS and its application to the synthesis of Aβ1‐42 isopeptide

The O‐acyl isopeptide method was developed for the efficient preparation of difficult sequence‐containing peptide. Furthermore, development of the O‐acyl isodipeptide unit for Fmoc chemistry simplified its synthetic procedure by solid‐phase peptide synthesis. Here, we report a novel isodipeptide unit for Boc chemistry, and the unit was successfully applied to the synthesis of amyloid β peptide. Combination of Boc chemistry and the isodipeptide unit would be an effective method for the synthesis of many difficult peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

General method for selective labelling of double‐chain cysteine‐rich peptides with a lanthanide chelate via solid‐phase synthesis

The use of lanthanides in preference to radioisotopes as probes for various biological assays has gained enormous popularity. The introduction of lanthanide chelates to peptides/proteins can be carried out either in solution using a commercially available labelling kit or by solid‐phase peptide synthesis using an appropriate lanthanide chelate. Herein, a detailed protocol for the latter is provided for the labelling of peptides or small proteins with diethylenetriamine‐N, N, N″, N″‐tetra‐tert‐butyl acetate‐N′‐acetic acid (DTPA) chelate or other similar chelates on a solid support using a chimeric insulin‐like peptide composed of human insulin‐like peptide 5 (INSL5) A‐chain and relaxin‐3 B‐chain as a model peptide. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Heterotrimeric collagen peptides containing functional epitopes. Synthesis of single‐stranded collagen type I peptides related to the collagenase cleavage site

Synthetic collagen peptides containing larger numbers of Gly‐Pro‐Hyp repeats are difficult to purify by standard chromatographic procedures. Therefore, efficient strategies are required for the synthesis of higher molecular weight collagen‐type peptides. Applying the Fmoc/tBu chemistry, a comparative analysis of the standard stepwise chain elongation procedure on solid support with the procedure based on the use of the synthons Fmoc‐Gly‐Pro‐Hyp(tBu)‐OH and Fmoc‐Pro‐Hyp‐Gly‐OH was performed. The crude products resulting from the stepwise elongation procedure and from the use of Fmoc‐Gly‐Pro‐Hyp(tBu)‐OH clearly revealed large amounts of microheterogeneities that result from incomplete imino acid acylation as well as from diketopiperazine formation with cleavage of Gly‐Pro units from the growing peptide chain. Conversely, by the use of the Fmoc‐Pro‐Hyp‐Gly‐OH synthon, the quality of the crude products was significantly improved; moreover, protection of the Hyp side chain hydroxyl function is not required using the Fmoc/tBu strategy. With this optimized synthetic procedure, relatively large collagen‐type peptides were obtained in satisfactory yields as highly homogeneous compounds. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Side‐chain thioamides as fluorescence quenching probes

Thioamides, single atom oxygen‐to‐sulfur substitutions of canonical amide bonds, can be valuable probes for protein folding and protease studies. Here, we investigate the fluorescence quenching properties of thioamides incorporated into the side‐chains of amino acids. We synthesize and incorporate Fmoc‐protected, solid‐phase peptide synthesis building blocks for introducing N^ε‐thioacetyl‐lysine and γ‐thioasparagine. Using rigid model peptides, we demonstrate the distance‐dependent fluorescence quenching of these thioamides. Furthermore, we describe attempts to incorporate of N^ε‐thioacetyl‐lysine into proteins expressed in Escherichia coli using amber codon suppression.     

On the use of N‐dicyclopropylmethyl aspartyl‐glycine synthone for backbone amide protection

To prevent aspartimide formation and related side products in Asp‐Xaa, particularly Asp‐Gly‐containing peptides, usually the 2‐hydroxy‐4‐methoxybenzyl (Hmb) backbone amide protection is applied for peptide synthesis according to the Fmoc‐protocols. In the present study, the usefulness of the recently proposed acid‐labile dicyclopropylmethyl (Dcpm) protectant was analyzed. Despite the significant steric hindrance of this bulky group, N‐terminal H‐(Dcpm)Gly‐peptides are quantitatively acylated by potent acylating agents, and alternatively the dipeptide Fmoc‐Asp(OtBu)‐(Dcpm)Gly‐OH derivative can be used as a building block. In contrast to the Hmb group, Dcpm is inert toward acylations, but is readily removed in the acid deprotection and resin‐cleavage step. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Solution‐phase submonomer diversification of aza‐dipeptide building blocks and their application in aza‐peptide and aza‐DKP synthesis

Aza‐peptides have been used as tools for studying SARs in programs aimed at drug discovery and chemical biology. Protected aza‐dipeptides were synthesized by a solution‐phase submonomer approach featuring alkylation of N‐terminal benzophenone semicarbazone aza‐Gly‐Xaa dipeptides using different alkyl halides in the presence of potassium tert‐butoxide as base. Benzophenone protected aza‐dipeptide tert‐butyl ester 31c was selectively deprotected at the C‐terminal ester or N‐terminal hydrazone to afford, respectively, aza‐dipeptide acid and amine building blocks 36c and 40c, which were introduced into longer aza‐peptides. Alternatively, removal of the benzophenone semicarbazone protection from aza‐dipeptide methyl esters 29a–c led to intramolecular cyclization to produce aza‐DKPs 39a–c. In light of the importance of aza‐peptides and DKPs as therapeutic agents and probes of biological processes, this diversity‐oriented solution‐phase approach may provide useful tools for studying peptide science. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Fullerene‐based inhibitors of HIV‐1 protease

A series of Fmoc‐Phe(4‐aza‐C₆₀)‐OH of fullerene amino acid derived peptides have been prepared by solid phase peptide synthesis, in which the terminal amino acid, Phe(4‐aza‐C₆₀)‐OH, is derived from the dipolar addition to C₆₀ of the Fmoc‐Nα‐protected azido amino acids derived from phenylalanine: Fmoc‐Phe(4‐aza‐C₆₀)‐Lys₃‐OH ( 1 ), Fmoc‐Phe(4‐aza‐C₆₀)‐Pro‐Hyp‐Lys‐OH ( 2 ), and Fmoc‐Phe(4‐aza‐C₆₀)‐Hyp‐Hyp‐Lys‐OH ( 3 ). The inhibition constant of our fullerene aspartic protease PRIs utilized FRET‐based assay to evaluate the enzyme kinetics of HIV‐1 PR at various concentrations of inhibitors. Simulation of the docking of the peptide Fmoc‐Phe‐Pro‐Hyp‐Lys‐OH overestimated the inhibition, while the amino acid PRIs were well estimated. The experimental results show that C₆₀‐based amino acids are a good base structure in the design of protease inhibitors and that their inhibition can be improved upon by the addition of designer peptide sequences. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and mass spectrometry analysis of quaternary cryptando‐peptidic conjugates

The bicyclic amines in the form of cryptands, the crown ether analogs, were used in the synthesis of cryptando‐peptidic conjugates with simultaneous formation of quaternary ammonium nitrogen moiety. A series of model cryptando‐peptidic conjugates at the peptide N‐terminus was efficiently prepared by the standard Fmoc solid phase synthesis. Tandem mass spectrometric analysis of the obtained conjugates has shown the specific fragmentation pattern during MS/MS experiment. The obtained cryptandic quaternary ammonium group undergoes the Hofmann elimination during collision‐induced dissociation fragmentation followed by the ethoxyl group elimination. The presented quaternization of cryptands by iodoacetylated peptides is relatively easy and compatible with standard solid‐phase peptide synthesis. Additionally, the applicability of such peptide derivatives and their isotopologues selectively deuterated at the α‐carbon in the quantitative LC‐MS analysis was analyzed. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.