含有络合功能基的非天然氨基酸的设计、合成及在生物活性肽中的应用

设计合成了一类侧链带有络合基团的非天然氨基酸, 即侧链带有N,N-二羧甲基氨甲基、N,N-二酰胺甲基氨甲基和N,N-二羟乙基氨甲基的苯丙氨酸衍生物, 并将这类非天然氨基酸用于促性腺激素释放激素(LHRH)类似物的固相合成. 高效液相色谱分析结果表明, 粗肽的纯度较好, 易于纯化; 用电喷雾质谱测定了多肽的分子量. 这些非天然氨基酸可作为其它肽类药物合成的构建单元.     

Enzyme-Driven,Switchable Catalysis Based on Dynamic Self-Assembly of Peptides

Covalent regulatory systems of enzymes are widely used to modulate biological enzyme activities. Inspired by the regulation of reactive-site phosphorylation in organisms, we developed peptide-based catecholase mimetics with switchable catalytic activity and high selectivity through the co-assembly of nanofibers comprising peptides and copper ions (Cu²⁺). Through careful design and modification of the peptide backbone structure based on the change in the free energy of the system, we identified the peptide with the most effective reversible catalytic activity. Kinase/phosphatase switches were used to control the reversible transition of nanofiber formation and depolymerization, as well as to modulate the active-site microenvironment. Notably, the self-assembly and disassembly processes of nanofibers were simulated using coarse-grained molecular dynamics. Furthermore, theoretical calculations confirmed the coordination of the peptide and Cu²⁺, forming a zipper-like four-ligand structure at the catalytically active center of the nanofibers. Additionally, we conducted a comprehensive analysis of the catalytic mechanism. This study opens novel avenues for designing biomimetic enzymes with ordered structures and dynamic catalytic activities.     

5-Amino-3-methyl-Isoxazole-4-carboxylic Acid as a Novel Unnatural Amino Acid in the Solid Phase Synthesis of α/β-Mixed Peptides

The hybrid peptides consisting of α and β-amino acids show great promise as peptidomimetics that can be used as therapeutic agents. Therefore, the development of new unnatural amino acids and the methods of their incorporation into the peptide chain is an important task. Here, we described our investigation of the possibility of 5-amino-3-methyl-isoxazole-4-carboxylic acid (AMIA) application in the solid phase peptide synthesis. This new unnatural β-amino acid, presenting various biological activities, was successfully coupled to a resin-bound peptide using different reaction conditions, including classical and ultrasonic agitated solid-phase synthesis. All the synthesized compounds were characterized by tandem mass spectrometry. The obtained results present the possibility of the application of this β-amino acid in the synthesis of a new class of bioactive peptides.     

Structure-based design and optimization of antihypertensive peptides to obtain high inhibitory potency against both renin and angiotensin I-converting enzyme

The human renin–angiotensin system (RAS) plays an essential role in regulating blood pressure and systemic vascular resistance. Renin and angiotensin I-converting enzyme (ACE) are two key enzymes in RAS and have long been recognized as attractive antihypertensive targets. Here, a synthetic strategy was proposed integrating quantitative structure–activity relationship (QSAR), molecular dynamics (MD) simulation and binding free energy analysis to discover novel dual renin and ACE peptidic inhibitors. With the strategy a number of candidates were generated virtually, from which eight promising peptides were selected and synthesized for biological assay. Consequently, three peptides (RYLP, YTAWVP and YRAWVL) were successfully identified to have satisfactory inhibitory profile against both renin and ACE with IC₅₀ values of <1 mM and <10 μM, respectively. Structural analysis and energetic dissection revealed different binding modes of peptide to renin and ACE; a peptide only inserts its C-terminus into the active site of ACE, whereas the whole peptide packs tightly against renin. In addition, when limited to structural diversity it is hard to reconcile the renin and ACE inhibitory activities of short peptides such as dipeptides. These findings can be used to guide peptide optimization with improved biological activity.     

A One‐Pot Chemically Cleavable Bis‐Linker Tether Strategy for the Synthesis of Heterodimeric Peptides

Heterodimeric peptides linked by disulfide bonds are attractive drug targets. However, their chemical assembly can be tedious, time‐consuming, and low yielding. Inspired by the cellular synthesis of pro‐insulin in which the two constituent peptide chains are expressed as a single‐chain precursor separated by a connecting C‐peptide, we have developed a novel chemically cleavable bis‐linker tether which allows the convenient assembly of two peptide chains as a single “pro”‐peptide on the same solid support. Following the peptide cleavage and post‐synthetic modifications, this bis‐linker tether can be removed in one‐step by chemical means. This method was used to synthesize a drug delivery‐cargo conjugate, TAT‐PKCi peptide, and a two‐disulfide bridged heterodimeric peptide, thionin (7‐19)‐(24‐32R), a thionin analogue. To our knowledge, this is the first report of a one‐pot chemically cleavable bis‐linker strategy for the facile synthesis of cross‐bridged two‐chain peptides.     

A new tri-orthogonal strategy for peptide cyclization

A solid phase tri-orthogonal protection/cleavage strategy that uses acidic, basic, and neutral conditions is described. Strategically protected alpha-azido-gamma-9-fluorenylmethyl-L-glutamate (1) and alpha-azido-epsilon-N-Fmoc-L-lysine (2) were incorporated into growing peptides on Wang resin using a novel azide protection strategy. These residues, separated by 1-3 monomers, were deprotected at the side chains and cyclized via lactam formation. The N-terminus was further functionalized to extend the chain. This method represents a straightforward protocol for peptide cyclization on solid support.     

Solid-Phase Total Synthesis of Cyclosporine Analogues

Syntheses of cyclosporine analogues are reported wherein the peptide couplings were achieved in solid phase. The Wang resin was used as the solid support, and the peptide couplings commenced with the residue 11 of the cyclosporine skeleton. The couplings proceeded in a stepwide manner up to the residue MeBmt¹, using symmetric anhydrides. The peptides were then cleaved off the resin, and the cyclization was achieved in solution using Castro's reagent. The solid-phase synthesis described herein offers a very efficient method for the rapid synthesis of structurally diverse cyclosporine analogues in small quantities. The biological activities of the synthetic cyclosporine analogues were evaluated in two in vitro assays, including the IL-2 reporter gene assay and the cyclophilin binding assay. The structure-activity relationship is discussed.     

CE study of neuroprotective humanin peptide and its derivatives: interactions with phosphate, sulphate, alkylsulphonates and sulphated-beta-CD

Humanin (HN), Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-Ser-Glu-IIe-Asp-Leu-Pro-Val-Lys-Arg-Arg-Ala, recently discovered in the human brain, is an important neuroprotective peptide. Some derivatives of HN show even higher biological activity, for example [G-14]-HN, where Ser at position 14 is replaced with Gly. As structurally related HN peptide derivatives have similar chemical properties, their separation by CE is difficult. In this work, the electrophoretic behaviour of HN derivatives including [G-14]-HN, a tryptophan HN derivative [W-14]-HN, several other HN derivatives and HN fragments was studied. While phosphate buffer was used as the general BGE, the effects of the buffer concentration and various additives were examined, including sulphate, heptane sulphonate, 2-morpholinoethanesulphonic acid N-[tris(hydroxymethyl)methyl]-2-aminoethane sulphonic acid (TES), sulphated-beta-CD and beta-CD. Separation efficiency of 200,000 theoretical plates was achieved in a BGE of 80 mM phosphate at pH 2.5 where seven out of nine major peaks were partially separated. By investigating the influence of concentration of the interrogated ions on peptides migration, the association between positively charged protonated sites of peptides and various anions was proved. Especially a strong interaction with phosphate, sulphate and sulphonate groups was established. Conditional stability constant of the [Pep(z+), (H(2)PO(4)(-))(n)](z - n) ion associate (n = 1) for [G-14]-HN equals to log K approximately 1.78.     

Effect of molecular conformations on the adsorption behavior of gold-binding peptides

Despite extensive recent reports on combinatorially selected inorganic-binding peptides and their bionanotechnological utility as synthesizers and molecular linkers, there is still only limited knowledge about the molecular mechanisms of peptide binding to solid surfaces. There is, therefore, much work that needs to be carried out in terms of both the fundamentals of solid-binding kinetics of peptides and the effects of peptide primary and secondary structures on their recognition and binding to solid materials. Here we discuss the effects of constraints imposed on FliTrx-selected gold-binding peptide molecular structures upon their quantitative gold-binding affinity. We first selected two novel gold-binding peptide (AuBP) sequences using a FliTrx random peptide display library. These were, then, synthesized in two different forms: cyclic (c), reproducing the original FliTrx gold-binding sequence as displayed on bacterial cells, and linear (l) dodecapeptide gold-binding sequences. All four gold-binding peptides were then analyzed for their adsorption behavior using surface plasmon resonance spectroscopy. The peptides exhibit a range of binding affinities to and adsorption kinetics on gold surfaces, with the equilibrium constant, Keq, varying from 2.5x10(6) to 13.5x10(6) M(-1). Both circular dichroism and molecular mechanics/energy minimization studies reveal that each of the four peptides has various degrees of random coil and polyproline type II molecular conformations in solution. We found that AuBP1 retained its molecular conformation in both the c- and l-forms, and this is reflected in having similar adsorption behavior. On the other hand, the c- and l-forms of AuBP2 have different molecular structures, leading to differences in their gold-binding affinities.     

Delivering Structural Information on the Polar Face of Membrane‐Active Peptides: 19F‐NMR Labels with a Cationic Side Chain

Conformationally constrained non‐racemizing trifluoromethyl‐substituted lysine isosteres [(E)‐ and (Z)‐TCBLys] with charged side chains are presented as a new type of ¹⁹F‐NMR labels for peptide studies. Design of the labels, their synthesis, incorporation into peptides and experimental demonstration of their application for solid state NMR studies of membrane‐active peptides are described. A series of fluorine‐labeled analogues of the helical amphipathic antimicrobial peptide PGLa(Nle) was obtained, in which different lysine residues in the original peptide sequence were replaced, one at a time, by either (E)‐ or (Z)‐TCBLys. Antimicrobial activities of the synthesized analogues were practically the same as those of the parent peptide. The structural and orientational parameters of the helical PGLa(Nle) peptide in model bilayers, as determined using the novel labels confirmed and refined the previously known structure. (E)‐ and (Z)‐TCBLys, as a set of cationic ¹⁹F‐NMR labels, were shown to deliver structural information about the charged face of amphipathic peptides by solid state ¹⁹F‐NMR, previously inaccessible by this method.     

Comparison on effect of hydrophobicity on the antibacterial and antifungal activities of α-helical antimicrobial peptides

HPRP-A1, a 15-mer α-helical cationic peptide, was derived from N-terminus of ribosomal protein L1 (RpL1) of Helicobacter pylori. In this study, HPRP-A1 was used as a framework to obtain a series of peptide analogs with different hydrophobicity by single amino acid substitutions in the center of nonpolar face of the amphipathic helix in order to systematically study the effect of hydrophobicity on biological activities of -helical antimicrobial peptides. Hydrophobicity and net charge of peptides played key roles in the biological activities of these peptide analogs; HPRP-A1 and peptide analogs with relative higher hydrophobicity exerted broad spectrum antimicrobial activity against Gram-negative bacteria, Gram-positive bacteria and pathogenic fungi, but also showed stronger hemolytic activity; the change of hydrophobicity and net charge of peptides had similar effects with close trend and extent on antibacterial activities and antifungal activities. This indicated that there were certain correlations between the antibacterial mode of action and the antifungal mode of action of these peptides in this study. The peptides exhibited antimicrobial specificity for bacteria and fungi, which provided potentials to develop new antimicrobial drugs for clinical practices.     

Oligonucleotides with 2'-O-carboxymethyl group: synthesis and 2'-conjugation via amide bond formation on solid phase

An efficient method for synthesis of oligonucleotide 2'-conjugates via amide bond formation on solid phase is described. Protected oligonucleotides containing a 2'-O-carboxymethyl group were obtained by use of a novel uridine 3'phosphoramidite, where the carboxylic acid moiety was introduced as its allyl ester. This protecting group is stable to the conditions used in solid-phase oligonucleotide assembly, but easily removed by Pd(0) and morpholine treatment. 2'-O-Carboxymethylated oligonucleotides were then efficiently conjugated on a solid support under normal peptide coupling conditions to various amines or to the N-termini of small peptides to give products of high purity in good yield. The method is well suited in principle for the preparation of peptide-oligonucleotide conjugates containing an amide linkage between the 2'-position of an oligonucleotide and the N-terminus of a peptide.     

Construction and activity of a synthetic basement membrane with active laminin peptides and polysaccharides

Cell-adhesive peptides derived from extracellular matrix (ECM) proteins are potential candidates for incorporating cell-binding activities into materials for tissue engineering. We have identified a number of cell adhesive peptides from laminins, which are major components of basement membrane ECM. Our goal is the development of synthetic basement membranes using the peptides on scaffolds. We review peptide–polysaccharide complexes, which were prepared by conjugation of the peptides to chitosan and alginate, and the biological activities of the resulting matrices. The peptide–polysaccharide matrices can also be used as a biomaterial for cell transplantation. These studies suggest that the peptide–polysaccharide complexes have the potential to mimic the multifunctional basement membrane and may be useful for tissue engineering.     

SPED-(styrene-polyethyleneglycol diacrylate-9-decen-1-ol): a novel resin for solid phase peptide synthesis; synthesis and characterization of biologically potent endothelin classes of peptides

Here we present a novel beaded chemically stable, highly permeable hydrophobic/hydrophilic balanced support for solid phase peptide synthesis. The resin (SPED) was prepared by free radical suspension polymerization using monomers styrene and 9-decen-1-ol with amphiphilic cross-linking agent polyethyleneglycol diacrylate (Mn 258). Different cross-linking densities were prepared to check the extent of swelling in different polar and non-polar solvents. The SPED resin was characterized with IR, 13C NMR and surface by SEM. The chemical stability of the support in various peptide synthetic conditions was investigated and monitored by IR spectroscopy. To evaluate the applicability of the new resin in synthetic conditions more challenging peptide sequence of retro-ACP (74-65) was synthesized and compared to commercially available Merrifield resin. The efficiency of SPED was further confirmed by synthesizing biologically important endothelin family of peptides in high yield and purity. The purity of all peptides was checked by RP-HPLC and mass by MALDI-TOF MS.     

Design, synthesis and evaluation of β-lactam antigenic peptide hybrids; unusual opening of the β-lactam ring in acidic media

β-Lactam peptides were envisioned as conformational constraints in antigenic peptides (APs). Three different β-lactam tripeptides of varying flexibility were prepared in solution and incorporated in place of the central part of the altered melanoma associated antigenic peptide Leu(27)-Melan-A(26-35) using solid phase synthesis techniques. Upon TFA cleavage from the solid support, an unexpected opening of the β-lactam ring occurred with conservation of the amide bond. After adaptation of the solid phase synthesis strategy, β-lactam peptides were successfully obtained and both opened and closed forms were evaluated for their capacity to bind to the antigen-presenting class-I MHC HLA-A2 protein system. None of the closed β-lactam peptides bound to HLA-A2, but their opened variants were shown to be moderate to good HLA-A2 ligands, one of them being even capable of stimulating a Melan-A-specific T cell line.     

Revealing protein structures in solid-phase peptide synthesis by 13C solid-state NMR: evidence of excessive misfolding for Alzheimer's β

Solid-phase peptide synthesis (SPPS) is a widely used technique in biology and chemistry. However, the synthesis yield in SPPS often drops drastically for longer amino acid sequences, presumably because of the occurrence of incomplete coupling reactions. The underlying cause for this problem is hypothesized to be a sequence-dependent propensity to form secondary structures through protein aggregation. However, few methods are available to study the site-specific structure of proteins or long peptides that are anchored to the solid support used in SPPS. This study presents a novel solid-state NMR (SSNMR) approach to examine protein structure in the course of SPPS. As a useful benchmark, we describe the site-specific SSNMR structural characterization of the 40-residue Alzheimer's β-amyloid (Aβ) peptide during SPPS. Our 2D (13)C/(13)C correlation SSNMR data on Aβ(1-40) bound to a resin support demonstrated that Aβ underwent excessive misfolding into a highly ordered β-strand structure across the entire amino acid sequence during SPPS. This approach is likely to be applicable to a wide range of peptides/proteins bound to the solid support that are synthesized through SPPS.     

Degree of Ionization in MALDI of Peptides: Thermal Explanation for the Gas-Phase Ion Formation

Degree of ionization (DI) in matrix-assisted laser desorption ionization (MALDI) was measured for five peptides using α-cyano-4-hydroxycinnanmic acid (CHCA) as the matrix. DIs were low 10(-4) for peptides and 10(-7) for CHCA. Total number of ions (i.e., peptide plus matrix) was the same regardless of peptides and their concentration, setting the number of gas-phase ions generated from a pure matrix as the upper limit to that of peptide ions. Positively charged cluster ions were too weak to support the ion formation via such ions. The total number of gas-phase ions generated by MALDI, and that from pure CHCA, was unaffected by the laser pulse energy, invalidating laser-induced ionization of matrix molecules as the mechanism for the primary ion formation. Instead, the excitation of matrix by laser is simply a way of supplying thermal energy to the sample. Accepting strong Coulomb attraction felt by cations in a solid sample, we propose three hypotheses for gas-phase peptide ion formation. In Hypothesis 1, they originate from the dielectrically screened peptide ions in the sample. In Hypothesis 2, the preformed peptide ions are released as part of neutral ion pairs, which generate gas-phase peptide ions via reaction with matrix-derived cations. In Hypothesis 3, neutral peptides released by ablation get protonated via reaction with matrix-derived cations.     

Isolation, structure determination, and synthesis of allo-RA-V and neo-RA-V, RA-series bicyclic peptides from Rubia cordifolia L

Two bicyclic hexapeptides, allo-RA-V (4) and neo-RA-V (5), and one cyclic hexapeptide, O-seco-RA-V (6), were isolated from the roots of Rubia cordifolia?L. Their gross structures were elucidated on the basis of spectroscopic analysis and X-ray crystallography of compound 5. The absolute stereochemistry of compounds 4 and 5 were established by their total syntheses, and the absolute stereochemistry of compound 6 by chemical correlation with deoxybouvardin (3). Comparison of the 3D structures of highly active RA-VII (1) with less-active compounds 4 and 5 suggests that the orientation of the Tyr-5 and/or Tyr-6 phenyl rings plays a significant role in their biological activity. The isolation of peptides 4-6, along with compound 3, and the comparison of their structures seem to indicate that peptide 6 may be the common precursor to bicyclic peptides 3-5 in the plant.