Application of Dmb-Dipeptides in the Fmoc SPPS of Difficult and Aspartimide-Prone Sequences

Mutter’s pseudoproline dipeptides and Sheppard’s Hmb derivatives are powerful tools for enhancing synthetic efficiency in Fmoc SPPS. They work by exploiting the natural propensity of N-alkyl amino acids to disrupt the formation of the secondary structures during peptide assembly. Their use results in better and more predictable acylation and deprotection kinetics, enhanced reaction rates, and improved yields of crude products. However, these approaches have certain limitations: pseudoproline dipeptides can only be used for sequences containing serine or threonine, and the coupling of the amino acid following the Hmb residue can be extremely difficult. To alleviate some of these shortcomings, we have prepared a range of Fmoc-Aaa-(Dmb)Gly-OH dipeptides and tested their efficacy in the synthesis of a number of challenging hydrophobic peptides. We also compared the efficiency of N-Dmb against N-Hmb backbone protection in preventing aspartimide formation in the Fmoc SPPS of peptides containing the Asp-Gly sequence.     

Investigation of the automated solid-phase synthesis of a 38mer peptide with difficult sequence pattern under different synthesis strategies

Difficult peptides are a constant challenge in solid-phase peptide synthesis. In particular, hydroxyl amino acids such as serine can cause severe breakdowns in coupling yields even several amino acids after the insertion of the critical amino acid. This paper investigates several methods of improving synthesis yields of difficult peptides including the use of different resins, activators and the incorporation of a structure-breaking pseudoproline dipeptide building block both alone and in combination with each other.     

Synthesis of “Difficult” Fluorescence Quenched Substrates of Granzyme C

The synthesis of fluorescence quenched peptide substrates of granzyme C is presented. These peptides which incorporate some unusual amino acids and have “difficult sequence” elements, in some cases could not be prepared by standard Fmoc-based SPPS. Application of three different contemporary strategies, namely the use of pseudoproline dipeptides, PEG-based solid supports and the application of microwave heating were able to provide for successful synthesis of our desired substrate peptides.     

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.     

The synergy of ChemMatrix resin and pseudoproline building blocks renders RANTES, a complex aggregated chemokine

Traditionally, solid-phase synthesis has relied on polystyrene-based resins for the synthesis of all kinds of peptides. However, due to their high hydrophobicity, these resins have certain limitations, particularly in the synthesis of complex peptides, and in such cases, poly(ethylene glycol) (PEG)-based resins are often found to give superior results. Another powerful strategy for expediting the assembly of complex peptides is to employ pseudoproline dipeptides. These derivatives disrupt the interactions among chains that are usually the cause of poor coupling yields in aggregated sequences. Here we report on an efficient stepwise solid-phase synthesis of RANTES (1-68) by combining the advantages of the totally PEG-based ChemMatrix resin and pseudoproline dipeptides.     

A Novel Route to Pseudoproline (Ψ<Superscript>H,H</Superscript>Pro)-Containing Dipeptides Building Blocks

In view of the synthetic and biological interest of pseudoproline (Ψ^R,RPro)-containing peptides, we have investigated a new strategy to these target compounds, taking into account the existence of the well-known ring–chain tautomerism occurring in the NH-free pseudoproline unit. Indeed, the NH-free oxaprolines derived from β-hyroxyamino acid (Ser, Thr) are rarely isolable in contrast to the cysteine-derived thiaproline. The strategy developed herein is based on the use of 2:3 adducts (amino acid–formaldehyde) resulting from the condensation of amino acids (Ser, Thr, Cys) with paraformaldehyde. The latter may exist as an equilibrium mixture of 1,4-diaza-3,9-dioxabicyclo[4.4.1]undecane (2) and isomeric N,N-methylenebis (oxazolidine) or -(thiazolidine) (3). Coupling these 2:3 adducts with a C-activated amino acid using conventional procedure afforded C2-unsubstituted pseudoproline (Ψ^H,HPro)-containing dipeptides. This strategy was applied to both oxa- and thiaprolines. Such result clearly established the usefulness of these 2:3 adducts in peptide synthesis as they allow to trap the non isolable NH-free oxaprolines. The isomerization process 2 3 appeared to play a major role in this procedure, as illustrated by the peculiar case of the serine-derived 2:3 adduct 2a where no coupling occurred.     

Synthesis of disulfated peptides corresponding to the N‐terminus of chemokines receptors CXCR6 (CXCR61–20) and DARC (DARC8–42) using a sulfate‐protecting group strategy

Tyrosine sulfation is a post translational modification that occurs on integral membrane and secreted proteins, and is required for mediating crucial biological processes. Until recently the synthesis of sTyr peptides, especially those containing multiple sTyr residues, were among the most challenging peptides to prepare. We recently described an efficient strategy for Fmoc‐based solid phase synthesis of sTyr peptides in which the sulfate group in the sTyr residue(s) is protected with a DCV group (FmocTyr(SO₃DCV)OH, 1 ). After cleavage of the peptide from the support the DCV group is removed by hydrogenolysis. Here we demonstrate that sTyr peptides containing Met or Trp residues can be prepared using our sulfate‐protecting group strategy by preparing peptides corresponding to residues 1–20 of chemokine receptor CXCR6 and 8–42 of chemokine receptor DARC. Removing the DCV groups at the end of the syntheses was readily achieved, without any reduction of the indole ring in Trp, by performing the hydrogenolysis in the presence of triethylamine. These conditions were found to be particularly efficient for removing the DCV group and superiour to our original conditions using H₂, ammonium formate, Pd/C. The presence of Met was found not to interfere with the removal of the DCV group. The use of pseudoproline dipeptides and N‐backbone protection with the 2,4‐dimethoxybenzyl group were found to be very effective tactics for preventing aggregation and aspartimide formation during the synthesis of these peptides. We also report an alternative and more cost effective synthesis of amino acid 1 . Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Fast Fmoc synthesis of hAmylin1-37 with pseudoproline assisted on-resin disulfide formation.

Human amylin (1-37) and the (1-13) fragment were synthesized with and without pseudoproline dipeptides. Thallium (III) trifluoroacetate, a mild oxidant, was used to cyclize the peptides by forming a disulfide bridge from C(2) to C(7). On the basis of our model studies, incorporation of a pseudoproline dipeptide decreases the amount of time necessary for the crude linear amylin (1-13) to cyclize on the resin. Without pseudoproline dipeptides, the 1-37 crude linear amylin was not pure enough to undergo the cyclization reaction. Following the cyclization studies, the synthesis time of the linear human amylin (1-37) was systematically reduced from 58 h to 8.5 h by shortening the reaction times. Cyclization and cleavage times were also reduced to 1.5 h.     

The spin label amino acid TOAC and its uses in studies of peptides: chemical, physicochemical, spectroscopic, and conformational aspects

We review work on the paramagnetic amino acid 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid, TOAC, and its applications in studies of peptides and peptide synthesis. TOAC was the first spin label probe incorporated in peptides by means of a peptide bond. In view of the rigid character of this cyclic molecule and its attachment to the peptide backbone via a peptide bond, TOAC incorporation has been very useful to analyze backbone dynamics and peptide secondary structure. Many of these studies were performed making use of EPR spectroscopy, but other physical techniques, such as X-ray crystallography, CD, fluorescence, NMR, and FT-IR, have been employed. The use of double-labeled synthetic peptides has allowed the investigation of their secondary structure. A large number of studies have focused on the interaction of peptides, both synthetic and biologically active, with membranes. In the latter case, work has been reported on ligands and fragments of GPCR, host defense peptides, phospholamban, and β-amyloid. EPR studies of macroscopically aligned samples have provided information on the orientation of peptides in membranes. More recent studies have focused on peptide-protein and peptide-nucleic acid interactions. Moreover, TOAC has been shown to be a valuable probe for paramagnetic relaxation enhancement NMR studies of the interaction of labeled peptides with proteins. The growth of the number of TOAC-related publications suggests that this unnatural amino acid will find increasing applications in the future.     

Multiple peptide synthesis using a single support (MPS3)

An automated multiple peptide synthesis method to synthesize, cleave, and purify several peptides simultaneously in a single batch has been developed. The technique is based on the synthesis of multiple peptides on a single solid phase support and is easily adapted to manual or to automated methods. The approach relies on coupling of amino acid mixtures to the resin and it has been found that DCC/HOBt gives the best coupling performance. Fast Atom Bombardment Mass Spectrometry (FAB-MS) was used to rapidly and efficiently identify the peptides in each synthetic mixture which significantly assisted the purification process by HPLC. The method has been successfully applied to the synthesis of magainin 2 and angiotensinogen peptides.     

Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences

This protocol for solid-phase peptide synthesis (SPPS) is based on the widely used Fmoc/tBu strategy, activation of the carboxyl groups by aminium-derived coupling reagents and use of PEG-modified polystyrene resins. A standard protocol is described, which was successfully applied in our lab for the synthesis of the corticotropin-releasing factor (CRF), >400 CRF analogs and a countless number of other peptides. The 41-mer peptide CRF is obtained within approximately 80 working hours. To achieve the so-called difficult sequences, special techniques have to be applied in order to reduce aggregation of the growing peptide chain, which is the main cause of failure for peptide chemosynthesis. Exemplary application of depsipeptide and pseudoproline units is shown for synthesizing an extremely difficult sequence, the Asn(15) analog of the WW domain FBP28, which is impossible to obtain using the standard protocol.     

Synthesis and secondary structure of loop 4 of myelin proteolipid protein: effect of a point mutation found in Pelizaeus-Merzbacher disease.

To study the effects of a point mutation found in Pelizaeus-Merzbacher disease (PMD) on the physicochemical and structural properties of the extracellular loop 4 of the myelin proteolipid protein (PLP), we synthesized the peptide PLP(181-230)Pro215 and one mutant PLP(181-230)Ser215 with regioselective formation of the two disulphide bridges Cys200-Cys219 and Cys183-Cys227. As conventional amino acid building blocks failed to give crude peptides of good quality we had to optimize the synthesis by introducing pseudoproline dipeptide building blocks during the peptide elongation. In peptide Pro215 the first bridge Cys200-Cys219 was obtained after air oxidation, but in peptide Ser215 because of aggregation, dimethyl sulfoxide (DMSO) oxidation had to be used. The second bridge Cys183-Cys227 was obtained by iodine oxidation of both Cys (acetamidomethyl, Acm)-protected peptides. The secondary structures of the parent and mutant loops were analysed by circular dichroism (CD) in the presence of trifluoroethanol (TFE) and sodium dodecyl sulphate (SDS) as a membrane mimetic. Analysis of the spectra showed that the content of alpha-helix and beta-sheet varied differently for both peptides in TFE and SDS solutions, demonstrating the sensitivity of their conformation to the environment and the differences in their secondary structure. The ability of both peptides to insert into the SDS micelles was assayed by intrinsic tryptophan fluorescence.     

Chiral Symmetry Breaking in Peptide Systems During Formation of Life on Earth

Chiral symmetry breaking in complex chemical systems with a large number of amino acids and a large number of similar reactions was considered. It was shown that effective averaging over similar reaction channels may result in very weak effective enantioselectivity of forward reactions, which does not allow most of the known models to result in chiral symmetry breaking during formation of life on Earth. Models with simple and catalytic synthesis of a single amino acid, formation of peptides up to length five, and sedimentation of insoluble pair of substances were considered. It was shown that depending on the model and the values of the parameters, chiral symmetry breaking may occur in up to about 10% out of all possible unique insoluble pair combinations even in the absence of any catalytic synthesis and that minimum total number of amino acids in the pair is 5. If weak enantioselective forward catalytic synthesis of amino acids is present, then the number of possible variants, in which chiral symmetry breaking may occur, increases substantially. It was shown that that the most interesting catalysts have zero or one amino acid of “incorrect” chirality. If the parameters of the model are adjusted in such a way to result in an increase of concentration of longer peptides, then catalysts with two amino acids of incorrect chirality start to appear at peptides of length five. Models of chiral symmetry breaking in the presence of epimerization were considered for peptides up to length three. It was shown that the range of parameters in which chiral symmetry breaking could occur significantly shrinks in comparison to previously considered models with peptides up to length two. An experiment of chiral symmetry breaking was proposed. The experiment consists of a three-step cycle: reversible catalytic synthesis of amino acids, reversible synthesis of peptides, and irreversible sedimentation of insoluble substances.     

Optimization of solid-phase synthesis of difficult peptide sequences via comparison between different improved approaches

Summary. Different approaches are applied to avoid the strong aggregation of the difficult peptide sequences, which is considered as the main reason for incomplete acylation and deprotection reactions hindering the synthesis of these sequences. Temporary protection of amide nitrogen of peptide bond using 2-hydroxy-4-methoxybenzyl (Hmb) and 2,4,6-timethoxybenzyl (Tmob) amino acid derivatives, introduction of D-Ala or Pro residues in the peptide chain sequences and utilization of microwave energy are proved to be useful methods in the enhancement of solubility and in the hindrance of the aggregation during the solid-phase synthesis of oligoalanine. Oligoalanine is chosen to demonstrate the difficult sequences and to compare the efficiencies of these methods.     

Dipeptide synthesis catalyzed by aminoacyl-tRNA synthetases from Bacillus stearothermophilus

A new approach to enzymatic peptide synthesis by using aminoacyl-tRNA synthetase (ARS) as a catalyst has been investigated. Four ARSs (AspRS, HisRS, LeuRS and TyrRS) have been purified from a thermophilic bacterium, Bacillus stearothermophilus. By using TyrRS as a catalyst, tyrosine and leucinamide were shown to be condensed in the presence of ATP to give tyrosylleucinamide. In this manner, all of the ARSs investigated catalyzed the peptide synthesis reactions. TyrRS did not have strict specificity for the amino acid derivatives used as substrates and even D-amino acids were incorporated into peptides fairly easily in this enzymatic reaction. Preparative scale synthesis of L-Tyr-L-LeuNH2 was carried out and from this the scope and limitation of this new enzymatic reaction as a tool to the peptide synthesis has been described.     

Native chemical ligation in dimethylformamide can be performed chemoselectively without racemization

Native chemical ligation of unprotected peptides in organic solvents has been previously reported as a fast, efficient, and suitable method for coupling of hydrophobic peptides. However, it has not been determined whether the reaction can be carried out without possible side reactions or racemization. Here, we present a study on the chemoselectivity of this method by model reactions designed to test the reactivity of Arg and Lys side chains as well as that of α‐amino groups. A possible racemization of the C‐terminal amino acid of the N‐terminal peptide was also investigated. The results show that ligation in organic solvents can be conducted chemoselectively without side reactions with other nucleophilic groups. Furthermore, no racemization of the C‐terminal amino acid was observed if both educts were added simultaneously. Thus, native chemical ligation can be performed either in aqueous buffer systems or in organic solvents paving the way for the synthesis of larger hydrophobic peptides and/or membrane proteins. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Rhesus monkey gastroenteropancreatic hormones: relationship to human sequences

The amino acid sequences of the gastroenteropancreatic peptides of Old World mammals are generally well-conserved. However, only the glucagons and vasoactive intestinal polypeptides (VIP) have been shown to be identical among the species studied to date. Rhesus monkey (Macaca mulatta) insulin has been shown to be identical with human insulin. The question addressed in this study is whether other gastroenteropancreatic peptides are identical to the human peptides. Purification and sequencing of glucagon, pancreatic polypeptide, VIP and insulin confirmed their identity with the corresponding human peptides. However, the 17 amino acid monkey gastrin is identical to dog gastrin and differs from human gastrin by substitution of methionine for leucine at position 5 from the N-terminus and alanine for glutamic acid in position 10. If additional rhesus monkey tissues become available, it would be of interest to determine whether other gastrointestinal peptides also differ from the corresponding human peptides.     

Chemical mechanism to account for artifactual formation of shortened peptides with free alpha-amino groups in solid phase peptide synthesis

The formation of terminated peptides with free alpha-amino groups has often been observed in stepwise solid phase peptide synthesis. This has been attributed to variable accessibility in regions of the swollen crosslinked resin supports. It is now shown that impurities in the amino acid reagents are responsible for these by-products. Thus, sec.-butyloxycarbonylamino acids were isolated from tert.-butyloxycarbonylamino acids after treatment with trifluoroacetic acid under standard deprotection conditions for the removal of the tert.-butyloxycarbonyl (Boc) group. Direct reverse phase HPLC analysis of Boc-amino acids from commercial sources also showed the sec.-Boc-amino acids as impurities present at varying levels. The sec.-Boc group was stable to treatment at room temperature with trifluoroacetic acid in dichloromethane (1:1, v/v) (half-life 7 years), but was removed by HF-anisole under the standard conditions of cleavage and deprotection of assembled peptides. In model syntheses, the level of terminated free peptides corresponded to the level of preexisting sec.-Boc-amino acid impurities present in the Boc-amino acid reagents. Use of Boc-amino acids with no detectable sec.-Boc resulted in negligible levels (less than 0.05%) of terminated peptides. The problem is thus readily overcome by the use of pure Boc-amino acid starting materials and is not a reflection of a shortcoming inherent to the polymer supported nature of solid phase syntheses as has been previously suggested.     

Application of arylsulphonyl side-chain protected arginines in solid-phase peptide synthesis based on 9-fluorenylmethoxycarbonyl amino protecting strategy.

One of the main problems still hampering solid-phase peptide synthesis using orthogonal protection strategies based on the 9-fluorenylmethoxycarbonyl amino protecting group is the difficult removal of currently used arginine arylsulphonyl guanidino protecting groups. Poor acid liability of 4-methoxy-2,3,6-trimethylbenzenesulphonyl-protected arginine has led to the popularity of the newer 2,2,5,7,8- pentamethylchroman-6-sulphonyl guanidino protecting group. This group was initially believed to have liability to trifluoroacetic acid, the reagent commonly used to simultaneously deprotect peptides and detach them from the synthesis resin, comparable to tert.-butyl and trityl type protecting groups used for the protection of other peptide side-chain functionalities. In a comparison of three established cleavage/deprotection mixtures we have shown that this is not always the case, particularly in multiple arginine peptides. We have found that only hard-acid deprotection with trimethylsilyl bromide reliably removed both arylsulphonyl guanidino protecting groups from a variety of arginine-containing peptides.