Solid-phase synthesis of chemotactic peptides using alpha-azido acids.

Four chemotactic peptides, For-Met-Xxx-Phe-OMe, with an alpha,alpha-disubstituted amino acid at position 2 have been synthesized by the azido acid method [Meldal M, Juliano MA, Jansson AM. 1997. Azido acids in a novel method of solid-phase peptide synthesis. Tetrahedron Lett. 38: 2531-2534] on solid-phase, and were tested for biological activity. Dipropylglycine in the central position (Xxx) was found to be as active as the natural chemotactic peptide for chemotactic activity toward human neutrophils. Higher yields were obtained than previously reported solution-phase syntheses of chemotactic peptides, and EEDQ was used successfully for the difficult solid-phase formylation of amino groups.     

Conformation studies on oligoalanines, substance P, and the myoglobin sequence (66-73). Circular dichroism of polyethyleneglycol-bound peptides]

The conformation of polyethylene glycol-bound peptides, synthesized by the liquid-phase method, was investigated. This marcromolecular C-terminal protecting group is transparent in the visible and the ultraviolet range to 190 nm and solubilizes peptides in many different solvents. The CD spectra of the polymer-bound myoglobin sequence 66–73 and of the biologically active undecapeptide “substance P” were measured in each step of the synthesis. In both examples the formation of a secondary structure during the growth of the peptide chain was found. In the hydrophobic octapeptide containing the myoglobin sequence 66–73, the influence of either the blocked or the free N-terminal amino group on the conformation was observed. The blocked octapeptide in trifluoroethanol showed a higher degree of α-helix contribution than in its free state. The conformation of the polyethylene glycol-bound nona- and decaalanine in trifluoroethanol and water was determined. The peptide with a free amino end group has β-conformation in trifluoroethanol as well as in water. The corresponding N-Boc-protected derivatives show helical structure. The amino end group has a decisive influence on the formation of β-structure. The method of CD investigation of polymer-bound peptide sequences during the peptide synthesis in solution enables one to determine the influence of protecting groups and the chain end of a peptide on its conformation. It is also possible to study the relationship between the secondary structure, the chain length, and the kinetic of the coupling reaction in different solvents. Since the crystallization method for the liquid-phase peptide synthesis allows one to synthesize peptides in very short time, a new method of studying peptide conformations is opened.     

Development of the efficient preparation method for thermoresponsive elastin-like peptides using liquid-phase synthesis combined with fragment condensation strategy

Elastin-like peptides (ELPs) are synthetic peptides that mimic the characteristic hydrophobic amino acid repeat sequences of elastin and exhibit temperature-dependent reversible self-assembly properties. ELPs are expected to be used as temperature-responsive biomolecular materials across diverse industrial and research fields, and there is a requirement for a straightforward method to mass-produce them. Previously, we demonstrated that phenylalanine-containing ELP analogs, namely, (FPGVG)_n, can undergo coacervation with short chains (n = 5). The Fmoc solid-phase peptide synthesis method is one strategy used to synthesize these short ELPs. However, owing to its low reaction efficiency, an efficient method for preparing ELPs is required. In this study, efficient preparation of ELPs was investigated using a liquid-phase synthesis method with a hydrophobic benzyl alcohol support (HBA-tag). Because HBA-tags are highly hydrophobic, they can be easily precipitated by the addition of poor solvents and recovered by filtration. This property allows the method to combine the advantages of the simplicity of solid-phase methods and the high reaction efficiency of liquid-phase methods. By utilizing liquid-phase fragment condensation with HBA-tags, short ELPs were successfully obtained in high yield and purity. Finally, the temperature-dependent response of the ELPs generated through fragment condensation was assessed using turbidity measurements, which revealed a reversible phase transition. Consequently, the ELPs exhibited a reversible phase transition, indicating successful synthesis of ELPs via fragment preparation with tags. These findings provide evidence of the potential for mass production of ELPs using this approach.     

Synthesis of the C-terminal decapeptide of bovine insulin B-chain.

The liquid-phase synthesis of a decapeptide corresponding to the last 10 amino acid residues of bovine insulin B-chain is described. Modified monofunctional polyethylene glycol containing benzyl bromide functional group was used as the soluble polymeric support. Cleavage of the fully-protected peptide from the polymer was achieved with 1N NaOH in dioxane. The protected peptide was purified by chromatography on Sephadex LH-20. The protecting groups of a sample were removed with anhydrous HF, and the unprotected crude decapeptide was purified by ion-exchange chromatography on carboxymethyl-cellulose. Both peptides were tested for the racemization of individual amino acids by the gas chromatographic method. The results showed that no residue had been significantly racemized.     

The synthesis and study of side-chain lactam-bridged peptides

Side-chain lactam bridges linking amino acid residues that are spaced several residues apart in the linear sequence offer a convenient and flexible method for introducing conformational constraints into a peptide structure. The availability of a variety of selectively cleavable protecting groups for amines and carboxylic acids allows for several approaches to the synthesis of monocyclic, dicyclic, and bicyclic lactam-bridged peptides by solid-phase methods. Multicyclic structures are also accessible, but segment-condensation syntheses with solution-phase cyclizations are most likely to provide the best synthetic approach to these more complex constrained peptides. Lactam bridges linking (i, i + 3)-, (i, i + 4), and (i, i + 7)-spaced residue pairs have all proven useful for stabilization of alpha helices, and (i, i + 3)-linked residues have also been demonstrated to stabilize beta-turns. These structures are finding an increasing number of applications in protein biology, including studies of protein folding, protein aggregation, peptide ligand-receptor recognition, and the development of more potent peptide therapeutics. Defining the functional roles of the amphiphilic alpha-helices in medium-sized peptide hormones, and studying helix propagation from rigid, alpha-helix initiating bicyclic peptides are among the most exciting developments currently underway in this field.     

Solid-phase synthesis of neurokinin A antagonists. Comparison of the Boc and Fmoc methods.

During the preparation of the NK-2 selective tachykinin antagonist MEN 10208 (Thr-Asp-Tyr-D-Trp-Val-D-Trp-D-Trp-Arg-NH2) and its analogs by the solid-phase method employing the Boc strategy routinely used in our laboratory, we encountered difficulties in the coupling of hydrophobic amino acids D-Trp and Val. To study the coupling problems several syntheses of MEN 10208 and analogs were carried out with different activation strategies. These syntheses yielded considerable amounts of deletion sequences even though a negative Kaiser test was obtained after each coupling. Inaccessibility of the free amino group of the growing peptide due to steric hindrance of the hydrophobic residues during coupling, and for the ninhydrin complex during the Kaiser test, may account, at least in part, for the unsatisfactory synthetics results and for the false-negative ninhydrin tests. Repetition of each synthesis with the Fmoc strategy on a newly developed DOD resin for peptide amides using the DCC/HOBt chemistry gave superior results in terms of the yield and purity of the crude peptides. Therefore, the Fmoc strategy appears to offer advantages over the Boc method for the preparation of these peptides containing hydrophobic amino acids.     

The primary structure identification of a corn peptide facilitating alcohol metabolism by HPLC-MS/MS

The aim of this study is to identify the primary structure of corn peptides (CPs) with a facilitating alcohol metabolism effect. Corn protein was hydrolyzed by Alcalase first. The hydrolysate, crude corn peptides (CPs), was then fractionated through ultrafiltration technology. The primary structure of a peptide from the fraction (Mm<5kDa) was identified by HPLC-MS/MS, coupled with the peptide sequence retrieval using the MS-MS online database. The amino acid sequence of the peptide was determined as Q-L-L-P-F, and the pentapeptide was synthesized by Fmoc solid-phase peptide synthesis (SPPS) method. Its ability to facilitate alcohol metabolism was evaluated in vivo. Results showed that the synthetic peptide (10mg/kg) had a higher ability to eliminate alcohol in vivo compared to the mixed peptides (Mm<5kDa, 200mg/kg). In conclusion, the pentapeptide Q-L-L-P-F has a potent ability in facilitating alcohol metabolism, and this pentapeptide is the main bioactive component in the mixed peptides obtained from corn.     

DEPBT as an efficient coupling reagent for amide bond formation with remarkable resistance to racemization

3-(Diethoxyphosphoryloxy)-1, 2, 3-benzotriazin-4(3H)-one (DEPBT) is an effective coupling reagent for synthesis of linear and cyclic peptides by both solution and solid-phase peptide synthesis. DEPBT mediates amide bond formation with remarkable resistance to racemization. When DEPBT is used as a coupling reagent, it is not necessary to protect the hydroxy group of the amino component (such as tyrosine, serine, and threonine) and the imidazole group in the case of histidine. The high efficiency of DEPBT and its utility have been demonstrated in the syntheses of complex natural products such as ramoplanin A2, ramoplanose aglycon, ustiloxin, and teicoplanin aglycon.     

Solid-phase precipitation and extraction, a new separation process applied to the isolation of synthetic peptides.

A new method for separation and purification is described. The process, referred to as solid-phase precipitation and extraction (SPPE), was developed and applied to postcleavage isolation of synthetic peptides. The technique uses normal approaches of chromatography and solid-phase extraction sorbents with a precipitation or drying procedure so that the sorbent becomes a support matrix for thin-film deposition of the compounds of interest. This procedure causes precipitated compounds of interest to be trapped on the large surface area or in the pores of the matrix so that by-products and impurities can be removed by strong wash solvents. In application to solid-phase peptide synthesis chemistry, by-products from the cleavage and deprotection are selectively extracted from the crude sample mixture under mild conditions. In comparison to the ether precipitation method used in peptide chemistry, the SPPE process provides isolated products that are 14-17% (w/w) higher purity.     

Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry

The adaptation of sequences of chemical reactions to a solid-phase format has been essential to the automation, reproducibility, and efficiency of a number of biotechnological processes including peptide and oligonucleotide synthesis and sequencing. Here we describe a method for the site-specific, stable isotopic labeling of cysteinyl peptides in complex peptide mixtures through a solid-phase capture and release process, and the concomitant isolation of the labeled peptides. The recovered peptides were analyzed by microcapillary liquid chromatography and tandem mass spectrometry (microLC-MS/MS) to determine their sequences and relative quantities. The method was used to detect galactose-induced changes in protein abundance in the yeast Saccharomyces cerevisiae. A side-by-side comparison with the isotope-coded affinity tag (ICAT) method demonstrated that the solid-phase method for stable isotope tagging of peptides is comparatively simpler, more efficient, and more sensitive.     

Synthetic peptides in biochemical research

Synthetic peptides play an important role in many areas of biological research. Advances in synthetic chemistry and automation over the past few years have resulted in increasingly reliable and rapid syntheses. As a result, peptides are now frequently employed in immunological studies, structural studies, as enzyme substrates, in ligand/receptor studies, and as probes for a range of molecular interactions. This review describes solid-phase peptide synthesis and the applications of synthetic peptides in molecular biology and biochemistry.     

Solid-phase peptide synthesis (SPPS), C-terminal vs. side-chain anchoring: a reality or a myth

Here we review the strategies for the solid-phase synthesis of peptides starting from the side chain of the C-terminal amino acid. Furthermore, we provide experimental data to support that C-terminal and side-chain syntheses give similar results in terms of purity. However, the stability of the two bonds that anchor the peptide to the polymer may determine the overall yield and this should be considered for the large-scale production of peptides. In addition, resins/linkers which do not subject to side reactions can be preferred for some peptides.     

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.     

Solid-phase synthesis of peptides with C-terminal asparagine or glutamine. An effective, mild procedure based on N alpha-fluorenylmethyloxycarbonyl (Fmoc) protection and side-chain anchoring to a tris(alkoxy)benzylamide (PAL) handle

Attempts to anchor Fmoc-asparagine or glutamine as p-alkoxybenzyl esters for solid-phase peptide synthesis are fraught with difficulties. A convenient and effective method to prepare peptides with C-terminal asparagine or glutamine involves quantitative attachment of N alpha-Fmoc-C alpha-tert.-butyl aspartate or glutamate via the free omega-carboxyl groups to a tris(alkoxy)benzylamino (PAL) support. Chain elongation proceeds normally by standard Fmoc chemistry, and treatment with acid, e.g., CF3COOH--CH2Cl2, 90 min at 25 degrees, releases the desired peptides in greater than 95% yields without side reactions at the C-terminus. Feasibility of the approach has been demonstrated by the syntheses of the C-terminal octapeptide from human proinsulin, H-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-OH, and the serum thymic factor pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-OH.     

A Dde resin based strategy for inverse solid-phase synthesis of amino terminated peptides, peptide mimetics and protected peptide intermediates.

This report describes a Dde resin based attachment strategy for inverse solid-phase peptide synthesis (ISPPS). This attachment strategy can be used for the synthesis of amino terminated peptides with side chains and the carboxyl terminus either protected or deprotected. Amino acid t-butyl esters were attached through their free amino group to the Dde resin. The t-butyl carboxyl protecting group was removed by 50% TFA, and inverse peptide synthesis cycles performed using an HATU/TMP based coupling method. Protected peptides were cleaved from the resin with dilute hydrazine. Side chain protecting groups could then be removed by treatment with TFMSA/TFA. The potential of this approach was demonstrated by the synthesis of several short protected and unprotected peptides in good yield and with low epimerization. Its potential for peptide mimetic synthesis was demonstrated by the synthesis of two peptide trifluoromethylketones.     

Synthesis of of beta-amyloid precursor peptide and presenilin segments.

It seems likely that the beta-amyloid precursor protein (APP) and the presenilins (PS-1/2) play important roles in the development of Alzheimer's disease (AD). Attempts to mimic the biochemical actions of these proteins are often made by the application of fragments of these proteins. However, the synthesis of these segments by conventional methods of peptide synthesis is problematic. We have synthesized several C-terminal fragments of APP and PS-1/2 by solid-phase synthesis through combination of automatic and manual methods of synthesis. This permits solution of the 'difficult sequences' in the solid-phase synthesis of these peptides. Some details of the syntheses of nine segments are presented in this paper.     

Application of gel-phase 19F NMR spectroscopy for optimization of solid-phase synthesis of a hydrophobic peptide from the signal sequence of the mucin MUC1.

This paper describes the manual Fmoc/t-Bu solid-phase synthesis of a difficult nine-residue hydrophobic peptide LLLLTVLTV from one of the signal sequences that flank the tandem repeat of the mucin MUC1. Gel-phase 19F NMR spectroscopy was used as a straightforward method for optimization of the solid-phase synthesis. Different approaches were applied for comparative studies. The strategy based on modified solid-phase conditions using DIC/HOAt for coupling, DBU for Fmoc deprotection, and the incorporation of the pseudo proline dipeptide Fmoc-Leu-Thr(psiMe, Me pro)-OH as a backbone-protecting group was found to be superior according to gel-phase 19F NMR spectroscopy. Implementation of the optimized Fmoc protocol enabled an effective synthesis of signal peptide LLLLTVLTV.     

On choosing the right ether for peptide precipitation after acid cleavage.

Methyl tert-butyl ether (MTBE) and diethyl ether (DEE) tend to be regarded as interchangeable for the 'cold ether' workup concluding the final acidolytic cleavage and deprotection step of solid-phase peptide syntheses. However, the use of MTBE to precipitate peptides from strong acid solutions is shown to give rise to t-butyl alkylation byproducts, readily detectable by MALDI-TOF MS. The problem can attain undesirable dimensions in the cleavage of peptide resins containing high proportions of aromatic residues, particularly in peptide nucleic acid (PNA) syntheses. In those cases, DEE workup is advisable, as it consistently leads to cleaner products.     

Tools for investigating peptide-protein interactions: peptide incorporation of environment-sensitive fluorophores via on-resin derivatization

This protocol presents the peptide incorporation of environment-sensitive fluorophores derived from the dimethylaminophthalimide family. The procedure utilizes anhydride precursors of 4-dimethylaminophthalimide (4-DMAP) or 6-dimethylaminonaphthalimide (6-DMN), whose syntheses are described in a related protocol from these authors. In this protocol, the fluorophores are directly incorporated after solid-phase peptide synthesis (SPPS) via on-resin derivatization of peptides prepared using commercially available diamino acids, which are Alloc-protected on the side-chain amino group. The time required to complete the procedure depends on the size and number of peptides targeted. As an alternative to this approach, the corresponding fluorescent amino acids can be obtained in an Fmoc-protected form for convenient use as building blocks in SPPS. This option is described in a related protocol by these authors.     

Synthetic Approaches to Disulfide-free Circular Bovine Pancreatic Trypsin Inhibitor (c-BPTI) Analogues

Several approaches were investigated with the goal to obtain disulfide-free circularized analogues of the 58-residue small protein bovine pancreatic trypsin inhibitor (BPTI). These approaches include (1) a semisynthesis that uses as a starting point naturally occurring BPTI and takes advantage of the native proximity of the C- and N-termini; (2) a synthesis in which a peptide thioester prepared by stepwise Fmoc solid-phase chemistry is cyclized by a solution native chemical ligation step; (3) a stepwise Fmoc solid-phase synthesis of a protected circularly permuted linear sequence, followed by an attempted selective activation and head-to-tail cyclization; and (4) a stepwise Fmoc solid-phase synthesis of the same analogue, but using a different disconnection point, that features backbone amide linker (BAL) anchoring and attempted on-resin cyclization. The first two of these approaches were indeed successful in providing the desired target molecules in excellent purities and respectable yields, and could well be amenable to generalization. It is not yet clear whether or not the latter two approaches could be salvaged by modifications in the details of the chemical procedures applied.Taken in part from the February 2004 Ph.D. thesis of Judit Tulla-Puche. A preliminary report of portions of this work has appeared (Tulla-Puche et al., 2004).Dedicated to the memory of Bruce Merrifield (July 15, 1921--May 14, 2006), mentor and friend, whose conceptualization and development of solid-phase peptide synthesis opened new chapters of the chemical and biological sciences