Solid-phase synthesis of functionalized bis-peptides

We demonstrate the first solid-phase synthesis of highly functionalized bis-peptides. Bis-peptides are ladder oligomers composed of stereochemically pure, cyclic bis-amino acids joined by substituted diketopiperazine linkages. They have a shape-programmable backbone that is controlled by controlling the stereochemistry and sequence of the monomers within each oligomer. Functionalized bis-peptides are assembled using a new amide bond forming reaction (acyl-transfer coupling) that we have previously developed and a novel activation strategy that allows the sequential formation of penta- and hexa-substituted diketopiperazines from extremely hindered N-alkyl-alpha,alpha-disubstituted amino acids. We present mechanistic evidence that acyl-transfer coupling is competitive with direct acylation in the formation of hindered amide bonds. We also detail the synthesis of four functionalized bis-peptides, and that by combining bis-peptides with amino acids through diketopiperazine linkages, bis-peptides can mimic the display of residues i, i+4, i+7 of an alpha-helical peptide.     

Mutual Amino Acid Catalysis in Salt-Induced Peptide Formation Supports this Mechanism's Role in Prebiotic Peptide Evolution

The presence of some amino acids and dipeptides under the conditions of the salt-induced peptide formation reaction (aqueous solution at 85 °C, Cu(II) and NaCl) has been found to catalyze the formation of homopeptides of other amino acids, which are otherwise produced only in traces or not at all by this reaction. The condensation of Val, Leu and Lys to form their homodipeptides can occur to a considerable extent due to catalytic effects of other amino acids and related compounds, among which glycine, histidine, diglycine and diketopiperazine exhibit the most remarkable activity. These findings also lead to a modification of the table of amino acid sequences preferentially formed by the salt-induced peptide formation (SIPF) reaction, previously used for a comparison with the sequence preferences in membrane proteins of primitive organisms     

Formation of Dendrolasin,Sesquirosefuran, Perillene and Rosefuran by Biomimetic Autooxidation

To estimate the steric distance between the bitter taste determinant sites in peptides, some cyclic dipeptides, amino acid anilides, amino acid cyclohexylamides, and benzoyl amino acids were synthesized and their tastes were evaluated. The diketopiperazine ring of cyclic dipeptides acted as a bitter taste determinant site due to its hydrophobicity. The steric distance between 2 sites was estimated as 4.1 Å from the molecule models of cyclic dipeptides composed of typical amino acids in the bitter peptides. Due to the hypothesis of two bitter taste determinant sites, which bind with the bitter taste receptor via a “binding unit” and a “stimulating unit,” a mechanism for the bitterness in peptides was postulated.     

The joint evolution of defence and inducibility against natural enemies

The aminoacyl-tRNA synthetases (aaRSs) ensure the fidelity of the translation of the genetic code, covalently attaching appropriate amino acids to the corresponding nucleic acid adaptor molecules-tRNA. The fundamental role of aminoacylation reaction catalysed by aaRSs implies that representatives of the family are thought to be among the earliest proteins to appear. Based on sequence analysis and catalytic domain structure, aaRSs have been partitioned into two classes of 10 enzymes each. However, based on the structural and sequence data only, it will not be easily understood that the present partitioning is not governed by chance. Our findings suggest that organization of amino acid biosynthetic pathways and clustering of aaRSs into different classes are intimately related to one another. A plausible explanation for such a relationship is dictated by early link between aaRSs and amino acids biosynthetic proteins. The aaRSs catalytic cores are highly relevant to the ancient metabolic reactions, namely, amino acids and cofactors biosynthesis. In particular we show that class II aaRSs mostly associated with the primordial amino acids, while class I aaRSs are usually related to amino acids evolved lately. Reasoning from this we propose a possible chronology of genetic code evolution.     

Solid phase synthesis of a functionalized bis-peptide using "safety catch" methodology

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides(1). Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides(2), which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield(1), but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation. Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an α-amine)(3,4). Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.     

Synthesis of a reported calpain inhibitor isolated from Streptomyces griseus

The reported diketopiperazine calpain inhibitor, cis-L-L-3,6-bis-(4-hydroxybenzyl)-1,4-dimethylpiperazine-2,5-dione 1, and its analogues 3 and 4 were synthesized from the corresponding amino acids. The previously assigned structure of 1 is confirmed but neither synthetic 1 nor its N-methylphenylalanine analogues 3 and 4 inhibit porcine erythrocyte calpain I.     

Populations of hydrophobic amino acids within protein globular domains: Identification of conserved “topohydrophobic” positions

The 3D structural comparison of families of divergent homologous domains revealed two main populations of hydrophobic amino acids, one with a low and the other with a significantly higher mean solvent accessibility, allowing two regions of the core of protein globular domains to be distinguished. The side chains of hydrophobic amino acids in topologically conserved positions (positions in the structural alignment where only hydrophobic amino acids are found), which we call topohydrophobic positions, are considerably less dispersed than those of the other amino acids (hydrophobic or not). Mean distances between gravity centers of amino acids in topohydrophobic positions are significantly shorter than those for non-topohydrophobic positions and show that the corresponding amino acids are almost all in direct contact in the inner core of globular domains. This study also showed that the small number of topohydrophobic positions is a characteristic of the structural differences between proteins of a family. This criterion is independent of the sequence identity between the sequences and of the root-mean-square distance between their corresponding structures. Using sensitive sequence alignment processes it will be possible, for many protein families, to identify topohydrophobic positions from sequences only. Proteins 33:329–342, 1998. © 1998 Wiley-Liss, Inc.     

A subset of Actinobacillus pleuropneumoniae in vivo induced promoters respond to branched-chain amino acid limitation

Actinobacillus pleuropneumoniae is the causative agent of a necrotizing hemorrhagic pleuropneumonia in swine. In this study, we investigate the possibility that the limitation of branched-chain amino acids is a stimulus that A. pleuropneumoniae will encounter during infection and will respond to by up-regulation of genes involved in branched-chain amino acid biosynthesis and virulence. Actinobacillus pleuropneumoniae genetic loci that are specifically induced during infection were screened in vitro for expression in response to limitation of branched-chain amino acids. Of 32 in vivo induced promoter clones screened in vitro, eight were induced on chemically defined medium without isoleucine, leucine and valine as compared to complete chemically defined medium. We identify the genomic context of each clone and discuss its relevance to branched-chain amino acid limitation and virulence. We conclude that limitation of branched-chain amino acids is a cue for expression of a subset in vivo induced genes, including not only genes involved in the biosynthesis of branched-chain amino acids, but also other genes that are induced during infection of the natural host. These results suggest that limitation of branched-chain amino acids may be one of an array of environmental cues responsible for the induction of virulence-associated genes in A. pleuropneumoniae.     

A new theoretical model for the origin of amino acid homochirality

Amino acid homochirality, as a unique behavior of life, could have originated synchronously with the genetic code. In this paper, phosphoryl amino-acid-5′-nucleosides with P-N bond are postulated to be a chiral origin model in prebiotic molecular evolution. The enthalpy change in the intramolecular interaction between the nucleotide base and the amino-acid side-chain determines the stability of the particular complex, resulting in a preferred conformation associated with the chirality of amino acids. Based on the theoretical model, our experiments and calculations show that the chiral selection of the earliest amino acids for L-enantiomers seems to be a strict stereochemical/physicochemical determinism. As other amino acids developed biosynthetically from the earliest amino acids, we infer that the chirality of the later amino acids was inherited from the precursor amino acids. This idea probably goes far back in history, but it is hoped that it will be a guide for further experiments in this area.     

Main-chain conformational tendencies of amino acids

A Ramachandran plot is a visual representation of the main-chain conformational tendencies of an amino acid. Despite forty years of research, the shape of Ramachandran plots is still a matter of debate. The issue in making a Ramachandran plot based on experimental data is deciding whether sparse data represent genuine conformations. We present here a simple solution to settle the ambiguities of the sparse data, and explain how we verified the accuracies of our plots using an independent dataset. To obtain our results, we then measured the pair-wise distances of main-chain conformational tendencies among amino acids, and showed that the conformational relationships of amino acids are well preserved in a two-dimensional map, leading to the conclusion that the conformational diversity space of amino acids is largely two dimensional. We further noticed that amino acids in early and late evolutionary stages are located in different zones in the two-dimensional map. In addition to these conclusions, we here present an amino acid substitution table derived from experimental data.     

Fullerene-based amino acids and peptides.

Recent advances in the chemistry of fullerene have allowed the synthesis of many classes of novel fullerene derivatives. Among these classes, fullerene-based amino acids and peptides are particularly interesting, both for structural studies and biological applications. In this review, we will discuss our own achievements in this rapidly growing field. In particular, the application of fulleroproline (Fpr) amino acids and peptides to medicinal chemistry and material science will be highlighted.     

Arbitrariness is not enough: towards a functional approach to the genetic code

Arbitrariness in the genetic code is one of the main reasons for a linguistic approach to molecular biology: the genetic code is usually understood as an arbitrary relation between amino acids and nucleobases. However, from a semiotic point of view, arbitrariness should not be the only condition for definition of a code, consequently it is not completely correct to talk about “code” in this case. Yet we suppose that there exist a code in the process of protein synthesis, but on a higher level than the nucleic bases chains. Semiotically, a code should be always associated with a function and we propose to define the genetic code not only relationally (in basis of relation between nucleobases and amino acids) but also in terms of function (function of a protein as meaning of the code). Even if the functional definition of meaning in the genetic code has been discussed in the field of biosemiotics, its further implications have not been considered. In fact, if the function of a protein represents the meaning of the genetic code (the sign’s object), then it is crucial to reconsider the notion of its expression (the sign) as well. In our contribution, we will show that the actual model of the genetic code is not the only possible and we will propose a more appropriate model from a semiotic point of view.     

Isolation and partial characterization of cyclosporin synthetase from a cyclosporin non-producing mutant of Beauveria nivea

Cyclosporin synthetase was isolated from a cyclosporin non-producing mutant of Beauveria nivea, strain YP 582. The enzyme has a molecular mass in the range of active cyclosporin synthetase and also contains 4'-phosphopantetheine as a prosthetic group. It is able to activate all constituent amino acids of cyclosporin A as thioesters and to carry out specific N-methylation reactions. Overall synthesis of the undecapeptide cyclosporin A in the presence of all necessary substrates was not observed, but the formation of the diketopiperazine cyclo-(D-alanyl-N-methyl-leucyl). This diketopiperazine represents a partial sequence of the cyclosporin molecule. It could be detected in the mycelium of the non-producing strain, whereas mycelium of the producing strain 7939/45 did not contain this compound. The results suggest that the inability of this mutant to produce cyclosporin A is caused by a mutation of the polypeptide chain of cyclosporin synthetase.     

A novel nuclear localization signal in human DNA topoisomerase I

DNA topoisomerase (topo) I is a nuclear enzyme that plays an important role in DNA metabolism. Based on conserved nuclear targeting sequences, four classic nuclear localization signals (NLSs) have been proposed at the N terminus of human topo I, but studies with yeast have suggested that only one of them (amino acids (aa) 150-156) is sufficient to direct the enzyme to the nucleus. In this study, we expressed human topo I fused to enhanced green fluorescent protein (EGFP) in mammalian cells and demonstrated that whereas aa 150-156 are sufficient for nuclear localization, the nucleolar localization requires aa 157-199. More importantly, we identified a novel NLS within aa 117-146. In contrast to the classic NLSs that are rich in basic amino acids, the novel NLS identified in this study is rich in acidic amino acids. Furthermore, this novel NLS alone is sufficient to direct not only EGFP into the nucleus but also topo I; and the EGFP.topo I fusion driven by the novel NLS is as active in vivo as the wild-type topo I in response to the topo I inhibitor topotecan. Together, our results suggest that human topo I carries two independent NLSs that have opposite amino acid compositions.     

Investigation of the Interaction between the Large and Small Subunits of Potato ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase), a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Current evidence indicates that the two subunit types play distinct roles in enzyme function. Recently the heterotetrameric structure of potato AGPase has been modeled. In the current study, we have applied the molecular mechanics generalized born surface area (MM-GBSA) method and identified critical amino acids of the potato AGPase LS and SS subunits that interact with each other during the native heterotetrameric structure formation. We have further shown the role of the LS amino acids in subunit-subunit interaction by yeast two-hybrid, bacterial complementation assay and native gel. Comparison of the computational results with the experiments has indicated that the backbone energy contribution (rather than the side chain energies) of the interface residues is more important in identifying critical residues. We have found that lateral interaction of the LS-SS is much stronger than the longitudinal one, and it is mainly mediated by hydrophobic interactions. This study will not only enhance our understanding of the interaction between the SS and the LS of AGPase, but will also enable us to engineer proteins to obtain better assembled variants of AGPase which can be used for the improvement of plant yield.     

Lipophilicity of amino acids

Summary The lipophilicity (or hydrophobicity) of amino acids is an important property relevant for protein folding and therefore of great interest in protein engineering. For peptides or peptidomimetics of potential therapeutic interest, lipophilicity is related to absorption and distribution, and thus indirectly relates to their bioactivity. A rationalization of peptide lipophilicity requires basic knowledge of the lipophilicity of the constituting amino acids. In the present contribution we will review methods to measure or calculate the lipophilicities of amino acids, including unusual amino acids, and we will make a comparison between various lipophilicity scales.     

The optical properties of alanine and proline diketopiperazines

The optical properties of the diketopiperazine chromophore of the cyclic dipeptides have been investigated as a function of molecular conformation. The rotatory strengths of L -alanyl–L -alanine diketopiperazine and L -prolyl–L -proline diketopiperazine have been calculated as a function of the angle of fold of the diketopiperazine ring. The results of these theoretical calculations have been compared with experimental circular dichroism and optical rotatory dispersion data. It is shown that the observed optical properties of these molecules can be explained only if their diketopiperazine rings are folded in opposite directions. The direction of fold is established for each molecule. In solution, the diketopiperazine ring of L -alanyl-L -alanine diketopiperazine is folded in the direction opposite to that found by X-ray diffraction analysis of crystals. It has been observed that the degree of conservatism of the π → π* couplet of L -propyl–L -proline diketopiperazine depends markedly upon the nature of the solvent that is used. In addition, a shoulder has been discovered in the CD spectrum of L -alanyl–L -alanine diketopiperazine, which may not be directly attributable to the n → π* and π → π* transitions of the peptide chromophores.     

The effect of dietary amino acid composition on egg production in blue tits

Most studies on the interaction between food supply and reproduction in animals have assumed that energy is likely to be the factor limiting egg number and/or size. In this paper, we investigate whether dietary protein proximately constrains egg production in birds. We provisioned breeding blue tits with two food supplements that differed only in the concentration of five essential amino acids. Birds receiving a supplementary diet containing an amino acid balance close to that required for egg protein formation laid significantly larger clutches (18% greater) than control birds, whereas birds receiving an otherwise identical supplementary diet but without a favourable amino acid balance did not increase egg production. To our knowledge, this is the first demonstration that dietary amino acid composition may limit egg production in free-living birds.     

Modularity of the slit protein. Characterization of a conserved carboxy-terminal sequence in secreted proteins and a motif implicated in extracellular protein interactions.

Since our characterization of the slit cDNA sequence, encoding a protein secreted by glial cells and involved in the formation of axonal pathways in Drosophila, we have discovered that the protein contains two additional sequence motifs that are highly conserved in a variety of proteins. A search of the GenPept database with the 73 amino acids at the carboxy terminus of slit revealed that this region contains significant similarity to a carboxy-terminal domain found in six other exported proteins. This observation has allowed us to define a new carboxy-terminal protein motif. In addition, comparisons with a 202 amino acid domain residing between epidermal growth factor (EGF) repeats in slit shows this region to be conserved in laminin, agrin and perlecan and, strikingly, also to lie between EGF repeats in both agrin and perlecan. Our analysis suggests this motif is involved in mediating interactions among extracellular proteins. Consistent with our previous characterization of the slit protein, both new motifs are found only in extracellular proteins. The identification of these two conserved motifs in slit reveals that the entire 1469 amino acids of the protein are made up of modular regions similar to those conserved in other extracellular proteins.     

A new theoretical model for the origin of amino acid homochirality

Amino acid homochirality, as a unique behavior of life, could have originated synchronously with the genetic code. In this paper, phosphoryl amino-acid -5′-nucleosides with P－N bond are postulated to be a chiral origin model in prebiotic molecular evolution. The enthalpy change in the intramolecular interaction between the nucleotide base and the amino-acid side-chain determines the sta-bility of the particular complex, resulting in a preferred conformation associated with the chirality of amino acids. Based on the theoretical model, our experiments and calculations show that the chiral selection of the earliest amino acids for L-enantiomers seems to be a strict stereochemi-cal/physicochemical determinism. As other amino acids developed biosynthetically from the earliest amino acids, we infer that the chirality of the later amino acids was inherited from the precursor amino acids. This idea probably goes far back in history, but it is hoped that it will be a guide for further ex-periments in this area.