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.     

Orthogonal protecting groups for N(alpha)-amino and C-terminal carboxyl functions in solid-phase peptide synthesis

For the controlled synthesis of even the simplest dipeptide, the N(alpha)-amino group of one of the amino acids and the C-terminal carboxyl group of the other should both be blocked with suitable protecting groups. Formation of the desired amide bond can now occur upon activation of the free carboxyl group. After coupling, peptide synthesis can be continued by removal of either of the two protecting groups and coupling with the free C-terminus or N(alpha)-amino group of another protected amino acid. When three functional amino acids are present in the sequence, the side chain of these residues also has to be protected. It is important that there is a high degree of compatibility between the different types of protecting groups such that one type may be removed selectively in the presence of the others. At the end of the synthesis, the protecting groups must be removed to give the desired peptide. Thus, it is clear that the protection scheme adopted is of the utmost importance and makes the difference between success and failure in a given synthesis. Since R. B. Merrifield introduced the solid-phase strategy for the synthesis of peptides, this prerequisite has been readily accepted. This strategy is usually carried out using two main protection schemes: the tert-butoxycarbonyl/benzyl and the 9-flourenylmethoxycarbonyl/tert-butyl methods. However, for the solid-phase preparation of complex or fragile peptides, as well as for the construction of libraries of peptides or small molecules using a combinatorial approach, a range of other protecting groups is also needed. This review summarizes other protecting groups for both the N(alpha)-amino and C-terminal carboxyl functions.     

The effect of thyroxine and related compounds on the aerobic myeloperoxidase--catalysed oxidation of NADH

Thyroxine concentrations as low as 1 microM significantly stimulate compound III formation during aerobic oxidation of NADH by highly purified myeloperoxidase. This increased compound III formation is paralleled by an increased oxidation of NADH. Stimulation of various thyronine and tyrosine analogues was in the order T4 greater than T3 greater than 3,5-T2 (or triiodothyropropionic acid). Thyronine and diiodotyrosine had no significant effect. From the potencies of the various thyronines to stimulate compound III formation, the following structural features seem necessary: (1) Substitution of thyronine with four iodine atoms. (2) An amino group on the alanine side chain. (3) Both aromatic rings of thyronine.     

Simultaneous Post-cysteine(S-Acm) Group Removal Quenching of Iodine and Isolation of Peptide by One Step Ether Precipitation

The S-acetamidomethyl (Acm) protecting group is widely used in the chemical synthesis of peptides that contain one or more disulfide bonds. Treatment of peptides containing S-Acm protecting group with iodine results in simultaneous removal of the sulfhydryl protecting group and disulfide formation. However, the excess iodine needs to be quenched or adsorbed as quickly as possible after completion of the disulfide bond formation in order to minimize side reactions that are often associated with the iodination step. We report a simple method for simultaneous post-cysteine (Acm) group removal quenching of iodination and isolation. Use of large volumes of diethyl ether for direct precipitation action of the oxidized peptide from the 90 or 95% aqueous acetic acid solution affords nearly quantitative recovery of largely iodine-free peptide ready for direct purification. It was successfully applied to the synthesis of various peptides including human insulin-like peptide 3 analogues. Although recovery yields were comparable to the traditionally used ascorbic acid quenching method, this new approach offers significant advantages such as more simple utility, minimal side reactions, and greater cost effectiveness.     

Site-specific incorporation of (aminooxy)acetic acid into proteins

By employing a general biosynthetic method for the elaboration of proteins containing unnatural amino acid analogues, we incorporated (aminooxy)acetic acid into positions 10 and 27 of Escherichia coli dihydrofolate reductase. Introduction of the modified amino acid into DHFR was accomplished in an in vitro protein biosynthesizing system by readthrough of a nonsense (UAG) codon with a suppressor tRNA that had been activated with (aminooxy)acetic acid. Incorporation of the amino acid proceeded with reasonable efficiency at codon position 10 but less well at position 27. (Aminooxy)acetic acid was also incorporated into position 72 of DNA polymerase beta. Peptides containing (aminooxy)acetic acid have been shown to adopt a preferred conformation involving an eight-membered ring that resembles a gamma-turn. Accordingly, the present study may facilitate the elaboration of proteins containing conformationally biased peptidomimetic motifs at predetermined sites. The present results further extend the examples of ribosomally mediated formation of peptide bond analogues of altered connectivity and provide a conformationally biased linkage at a predetermined site. It has also been shown that the elaborated protein can be cleaved chemically at the site containing the modified amino acid.     

Structure of Gialpha1.GppNHp, autoinhibition in a galpha protein-substrate complex.

The structure of the G protein Gialpha1 complexed with the nonhydrolyzable GTP analog guanosine-5'-(betagamma-imino)triphosphate (GppNHp) has been determined at a resolution of 1.5 A. In the active site of Gialpha1. GppNHp, a water molecule is hydrogen bonded to the side chain of Glu43 and to an oxygen atom of the gamma-phosphate group. The side chain of the essential catalytic residue Gln204 assumes a conformation which is distinctly different from that observed in complexes with either guanosine 5'-O-3-thiotriphosphate or the transition state analog GDP.AlF4-. Hydrogen bonding and steric interactions position Gln204 such that it interacts with a presumptive nucleophilic water molecule, but cannot interact with the pentacoordinate transition state. Gln204 must be released from this auto-inhibited state to participate in catalysis. RGS proteins may accelerate the rate of GTP hydrolysis by G protein alpha subunits, in part, by inserting an amino acid side chain into the site occupied by Gln204, thereby destabilizing the auto-inhibited state of Galpha.     

Carbohydrate is linked through ethanolamine to the C-terminal amino acid of Trypanosoma brucei variant surface glycoprotein.

The C-terminal amino acid of the variant surface glycoprotein from Trypanosoma brucei is glycosylated. For two variant proteins that terminate in an aspartic acid and a serine residue respectively, it was shown that the sugar side chain is linked through ethanolamine to the alpha-carboxy group of the amino acid.     

Block copolymerization of α-amino acid N-carboxyanhydride initiated by vinyl polymers having a terminal amino group and the characterization of the block copolymers

Poly(methyl methacrylate) and polystyrene having terminal amino groups were synthesized by the radical polymerization of those monomers in the presence of 2-mercaptoethylammonium chloride as a chain-transfer agent. By the terminal group analysis and the molecular weight determination of the polymers, 0.5–1.3 amino groups were found in a chain of poly(methyl methacrylate) and 0.5–2.5 amino groups in a chain of polystyrene. Using these polymers having a terminal amino group as an initiator, the block polymerization of α-amino acid N-carboxyanhydride (NCA) was carried out. In the polymerizations of Glu(OBzl) NCA and Lys(Z) NCA by the poly(methyl methacrylate) initiator, the terminal amino group underwent a nucleophilic addition reaction to NCA and initiated the polymerization, yielding A-B-type block copolymers in a high yield. The same was observed in the polymerizations of Gly(OBzl) NCA and Lys(Z) NCA by the polystyrene initiator. By eliminating the protecting groups of the side chains of the polypeptide segment, the block copolymers poly(methyl methacrylate)-poly(Glu), poly(methyl methacrylate)-poly(Lys), polystyrene-poly(Glu) and polystyrene-poly(Lys) were synthesized with little side reactions. The side chain amino groups of poly(Lys) segment in the poly(methyl methacrylate)-poly(Lys) block copolymers were sulphonated or stearoylated successfully.     

Genetic introduction of a diketone-containing amino acid into proteins

An orthogonal tRNA/aminoacyl-tRNA synthetase pair was evolved that makes possible the site-specific incorporation of an unnatural amino acid bearing a beta-diketone side chain into proteins in Escherichia coli with high translational efficiency and fidelity. Proteins containing this unnatural amino acid can be efficiently and selectively modified with hydroxylamine derivatives of fluorophores and other biophysical probes.     

Pyridoxamine protects protein backbone from oxidative fragmentation

Oxidative damage to proteins is one of the major pathogenic mechanisms in many chronic diseases. Therefore, inhibition of this oxidative damage can be an important part of therapeutic strategies. Pyridoxamine (PM), a prospective drug for treatment of diabetic nephropathy, has been previously shown to inhibit several oxidative and glycoxidative pathways, thus protecting amino acid side chains of the proteins from oxidative damage. Here, we demonstrated that PM can also protect protein backbone from fragmentation induced via different oxidative mechanisms including autoxidation of glucose. This protection was due to hydroxyl radical scavenging by PM and may contribute to PM therapeutic effects shown in clinical trials.     

A simple protocol for combinatorial cyclic depsipeptide libraries sequencing by matrix‐assisted laser desorption/ionisation mass spectrometry

Short cyclic peptides have a great interest in therapeutic, diagnostic and affinity chromatography applications. The screening of ‘one‐bead‐one‐peptide’ combinatorial libraries combined with mass spectrometry (MS) is an excellent tool to find peptides with affinity for any target protein. The fragmentation patterns of cyclic peptides are quite more complex than those of their linear counterparts, and the elucidation of the resulting tandem mass spectra is rather more difficult. Here, we propose a simple protocol for combinatorial cyclic libraries synthesis and ring opening before MS analysis. In this strategy, 4‐hydroxymethylbenzoic acid, which forms a benzyl ester with the first amino acid, was used as the linker. A glycolamidic ester group was incorporated after the combinatorial positions by adding glycolic acid. The library synthesis protocol consisted in the following: (i) incorporation of Fmoc‐Asp[2‐phenylisopropyl (OPp)]‐OH to Ala‐Gly‐oxymethylbenzamide‐ChemMatrix, (ii) synthesis of the combinatorial library, (iii) assembly of a glycolic acid, (iv) couple of an Ala residue in the N‐terminal, (v) removal of OPp, (vi) peptide cyclisation through side chain Asp and N‐Ala amino terminus and (vii) removal of side chain protecting groups. In order to simultaneously open the ring and release each peptide, benzyl and glycolamidic esters were cleaved with ammonia. Peptide sequences could be deduced from the tandem mass spectra of each single bead evaluated. The strategy herein proposed is suitable for the preparation of one‐bead‐one‐cyclic depsipeptide libraries that can be easily open for its sequencing by matrix‐assisted laser desorption/ionisation MS. It employs techniques and reagents frequently used in a broad range of laboratories without special expertise in organic synthesis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Chloramine-T in radiolabeling techniques. IV. Penta-O-acetyl-N-chloro-N-methylglucamine as an oxidizing agent in radiolabelling techniques

Chloramine-T (CAT) is commonly used in radiolabeling of bioactive molecules by halogenation. CAT is used to release radioactive elemental iodine by oxidation of its salts. Unfortunately, CAT is a strong oxidizing agent and can cause significant damage to peptides and proteins. This may lower the yield of the iodination reaction and may produce undesirable side products. Recently, it was found that the in situ formation of N-chlorosecondary amines, by the addition of secondary amines to CAT prior to exposure to the substrate, can reduce the oxidative damage caused by CAT. To simplify the method, we prepared penta-O-acetyl-N-chloro-N-methylglucamine (NCMGE) as a solid N-chlorosecondary amine. The chemical reactivity of NCMGE toward a model amino acid, 1-aminocyclohexane carboxylic acid, was compared with that of chloramine-T. In the presence of the model amino acid, CAT lost all its chlorine titer within 60 min while NCMGE retained 99% of its chlorine titer. NCMGE was compared to CAT for the iodination of l-tyrosine and leucine enkephalin. For both substrates, the NCMGE method produced larger or equal yields of the monoiodo and diiodo products and less decomposition. It is proposed that the method employing NCMGE to release diatomic iodine is more convenient and efficient for radiolabeling peptides and proteins than currently used methods.     

Changes in the structure of thyroglobulin following the administration of thyroid-stimulating hormone.

In each of three separate experiments, female guinea pigs in groups of 20 were given 4 units of thyroid-stimulating hormone (TSH) each day for 3 days, while controls were given saline. Na125I was injected on the 3rd day, and the animals were killed 22 hours later. The pooled throids of each group were homogenized, and thyroglobulin was purified by one of the following methods: gel filtration on Sephadex G-200 followed by density gradient ultracentrifugation, two sequential filtrations on 4 percent agarose, or filtration on 4 percent agarose followed by Sephadex G-200. TSH administration was associated with the folling changes in thyroglobulin: (1) an increase in the ratio of tri-iodothyronine to thyroxine; (2) a decrease in dissociation of the 19 S to the 12 S form; (3) an alteration in its pattern on gel electrophoresis in sodium dodecyl sulfate-urea; and (4) changes in its amino acid composition, with significant increases in the content of lysine (by 15 percent), isoleucine (by 15 percent), and methionine (by 7 percent) relative to leucine. Over-all, there were no significant changes in the content of iodine, fucose, hexosamine, or sialic acid. These data show that TSH can alter the composition of thyroglobulin independently of its effects on iodine content. We suggest that these changes may stem from alterations in the subunit composition of thyroglobulin. There were also small but significant variations in amino acid composition among the three preparations of thyroglobulin from saline-treated animals and among the three from the TSH-treated. This finding shows that thyroglobulin can be heterogeneous in its protein portion as well as in its iodine content.     

Hypochlorous acid-mediated protein oxidation: how important are chloramine transfer reactions and protein tertiary structure?

Hypochlorous acid (HOCl) is a powerful oxidant generated from H2O2 and Cl- by the heme enzyme myeloperoxidase, which is released from activated leukocytes. HOCl possesses potent antibacterial properties, but excessive production can lead to host tissue damage that occurs in numerous human pathologies. As proteins and amino acids are highly abundant in vivo and react rapidly with HOCl, they are likely to be major targets for HOCl. In this study, two small globular proteins, lysozyme and insulin, have been oxidized with increasing excesses of HOCl to determine whether the pattern of HOCl-mediated amino acid consumption is consistent with reported kinetic data for isolated amino acids and model compounds. Identical experiments have been carried out with mixtures of N-acetyl amino acids (to prevent reaction at the alpha-amino groups) that mimic the protein composition to examine the role of protein structure on reactivity. The results indicate that tertiary structure facilitates secondary chlorine transfer reactions of chloramines formed on His and Lys side chains. In light of these data, second-order rate constants for reactions of Lys side chain and Gly chloramines with Trp side chains and disulfide bonds have been determined, together with those for further oxidation of Met sulfoxide by HOCl and His side chain chloramines. Computational kinetic models incorporating these additional rate constants closely predict the experimentally observed amino acid consumption. These studies provide insight into the roles of chloramine formation and three-dimensional structure on the reactions of HOCl with isolated proteins and demonstrate that kinetic models can predict the outcome of HOCl-mediated protein oxidation.     

Structure-activity study at positions 3 and 4 of human neuropeptide S

Neuropeptide S (NPS) has been identified as the endogenous ligand of a previously orphan receptor now named NPSR. Previous studies demonstrated that the N-terminal sequence Phe(2)-Arg(3)-Asn(4) of the peptide is crucial for biological activity. Here, we report on a focused structure-activity study of Arg(3) and Asn(4) that have been replaced with a series of coded and non-coded amino acids. Thirty-eight human NPS analogues were synthesized and pharmacologically tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPSR. The results of this study demonstrated the following NPS position 3 structure-activity features: (i) the guanidine moiety and its basic character are not crucial requirements, (ii) an aliphatic amino acid with a linear three carbon atom long side chain is sufficient to bind and fully activate NPSR, (iii) the receptor pocket allocating the position 3 side chain can accommodate slightly larger side chains at least to a certain degree [hArg, Arg(NO2) or Arg(Me)2 but not Arg(Tos)]. Position 4 seems to be more sensitive to amino acids replacement compared to position 3; in fact, all the amino acid replacements investigated produced either an important decrease of biological activity or generated inactive derivatives suggesting a pivotal role of the Asn(4) side chain for NPS bioactivity.     

Histidyl-tRNA synthetase.

Histidyl-tRNA synthetase (HisRS) is responsible for the synthesis of histidyl-transfer RNA, which is essential for the incorporation of histidine into proteins. This amino acid has uniquely moderate basic properties and is an important group in many catalytic functions of enzymes. A compilation of currently known primary structures of HisRS shows that the subunits of these homo-dimeric enzymes consist of 420-550 amino acid residues. This represents a relatively short chain length among aminoacyl-tRNA synthetases (aaRS), whose peptide chain sizes range from about 300 to 1100 amino acid residues. The crystal structures of HisRS from two organisms and their complexes with histidine, histidyl-adenylate and histidinol with ATP have been solved. HisRS from Escherichia coli and Thermus thermophilus are very similar dimeric enzymes consisting of three domains: the N-terminal catalytic domain containing the six-stranded antiparallel beta-sheet and the three motifs characteristic of class II aaRS, a HisRS-specific helical domain inserted between motifs 2 and 3 that may contact the acceptor stem of the tRNA, and a C-terminal alpha/beta domain that may be involved in the recognition of the anticodon stem and loop of tRNA(His). The aminoacylation reaction follows the standard two-step mechanism. HisRS also belongs to the group of aaRS that can rapidly synthesize diadenosine tetraphosphate, a compound that is suspected to be involved in several regulatory mechanisms of cell metabolism. Many analogs of histidine have been tested for their properties as substrates or inhibitors of HisRS, leading to the elucidation of structure-activity relationships concerning configuration, importance of the carboxy and amino group, and the nature of the side chain. HisRS has been found to act as a particularly important antigen in autoimmune diseases such as rheumatic arthritis or myositis. Successful attempts have been made to identify epitopes responsible for the complexation with such auto-antibodies.     

Microwave‐assisted cleavage of Alloc and Allyl Ester protecting groups in solid phase peptide synthesis

Orthogonal protection of amino acid side chains in solid phase peptide synthesis allows for selective deprotection of side chains and the formation of cyclic peptides on resin. Cyclizations are useful as they may improve the activity of the peptide or improve the metabolic stability of peptides in vivo. One cyclization method often used is the formation of a lactam bridge between an amine and a carboxylic acid. It is desirable to perform the cyclization on resin as opposed to in solution to avoid unwanted side reactions; therefore, a common strategy is to use –Alloc and –OAllyl protecting groups as they are compatible with Fmoc solid phase peptide synthesis conditions. Alloc and –OAllyl may be removed using Pd(PPh₃)₄ and phenylsilane in DMF. This method can be problematic as the reaction is most often performed at room temperature under argon gas. It is not usually done at higher temperatures because of the fear of poisoning the palladium catalyst. As a result, the reaction is long and reagent–intensive. Herein, we report the development of a method in which the –Alloc/–OAllyl groups are removed using a microwave synthesizer under atmospheric conditions. The reaction is much faster, allowing for the removal of the protecting groups before the catalyst is oxidized, as well as being less reagent–intensive. This method of deprotection was tested using a variety of amino acid sequences and side chain protecting groups, and it was found that after two 5‐min deprotections at 38°C, all –Alloc and –OAllyl groups were removed with >98% purity. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

The synthesis of oligonucleotides containing a primary amino group at the 5''-terminus.

Oligonucleotides containing a primary amino group at their 5'-termini have been prepared and further derivatised with amino specific probes. The sequence required is prepared using standard solid phase phosphoramidite techniques and an extra round of synthesis is then performed with N-monomethoxytrityl-0-methoxydiisopropylaminophosphinyl 3-aminopropan(1)ol. After cleavage from the resin, removal of the phosphate and base protecting groups and purification gives a monomethoxytrityl-NH(CH2)3PO4-oligomer. The monomethoxytrityl group can be removed with acetic acid to give the desired amino containing oligomer. The amino group can be further derivatised with amino specific probes yielding fluorescent or biotinylated oligonucleotide products.     

Preparation of 'side-chain-to-side-chain' cyclic peptides by Allyl and Alloc strategy: potential for library synthesis.

Automated and manual deprotection methods for allyl/allyloxycarbonyl (Allyl/Alloc) were evaluated for the preparation of side-chain-to-side-chain cyclic peptides. Using a standard Allyl/Alloc deprotection method, a small library of cyclic peptides with lactam bridges (with seven amino acids) was prepared on an automatic peptide synthesizer. We demonstrate that the Guibe method for removing Allyl/Alloc protecting groups under specific neutral conditions [Pd(PPh3)4/PhSiH3)/DCM] can be a useful, efficient and reliable method for preparing long cyclic peptides on a resin. We have also manually synthesized a cyclic glucagon analogue containing 24 amino acid residues. These results demonstrated that properly controlled palladium-mediated deprotection of Allyl/Alloc protecting groups can be used to prepare cyclic peptides on the resin using an automated peptide synthesizer and cyclic peptides with a long chain.     

What Amino Acid Properties Affect Protein Evolution?

We studied 10 protein-coding mitochondrial genes from 19 mammalian species to evaluate the effects of 10 amino acid properties on the evolution of the genetic code, the amino acid composition of proteins, and the pattern of nonsynonymous substitutions. The 10 amino acid properties studied are the chemical composition of the side chain, two polarity measures, hydropathy, isoelectric point, volume, aromaticity, aliphaticity, hydrogenation, and hydroxythiolation. The genetic code appears to have evolved toward minimizing polarity and hydropathy but not the other seven properties. This can be explained by our finding that the presumably primitive amino acids differed much only in polarity and hydropathy, but little in the other properties. Only the chemical composition (C) and isoelectric point (IE) appear to have affected the amino acid composition of the proteins studied, that is, these proteins tend to have more amino acids with typical C and IE values, so that nonsynonymous mutations tend to result in small differences in C and IE. All properties, except for hydroxythiolation, affect the rate of nonsynonymous substitution, with the observed amino acid changes having only small differences in these properties, relative to the spectrum of all possible nonsynonymous mutations. Received: 2 January 1998 / Accepted: 25 April 1998