Self‐assembly of short peptides composed of only aliphatic amino acids and a combination of aromatic and aliphatic amino acids

The morphology of structures formed by the self‐assembly of short N‐terminal t‐butyloxycarbonyl (Boc) and C‐terminal methyl ester (OMe) protected and Boc‐deprotected hydrophobic peptide esters was investigated. We have observed that Boc‐protected peptide esters composed of either only aliphatic hydrophobic amino acids or aliphatic hydrophobic amino acids in combination with aromatic amino acids, formed highly organized structures, when dried from methanol solutions. Transmission and scanning electron microscopic images of the peptides Boc‐Ile‐Ile‐OMe, Boc‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe and Boc‐Trp‐Ile‐Ile‐OMe showed nanotubular structures. Removal of the Boc group resulted in disruption of the ability to form tubular structures though spherical aggregates were formed. Both Boc‐Leu‐Ile‐Ile‐OMe and H‐Leu‐Ile‐Ile‐OMe formed only spherical nanostructures. Dynamic light scattering studies showed that aggregates of varying dimensions were present in solution suggesting that self‐assembly into ordered structures is facilitated by aggregation in solution. Fourier transform infrared spectroscopy and circular dichroism spectroscopy data show that although all four of the protected peptides adopt well‐defined tertiary structures, upon removal of the Boc group, only H‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe had the ability to adopt β‐structure. Our results indicate that hydrophobic interaction is a very important determinant for self‐assembly and presence of charged and aromatic amino acids in a peptide is not necessary for self‐assembly. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Novel side reaction accompanying cyclization of Glu(R1)‐Glu(R2) dipeptides via lactamization of the Glu(R1) residue

A series of linear peptides with the general formula H‐Glu(R1)‐Glu(R2)‐OH was subjected to cyclization under standard conditions. Formation of respective 2,5‐diketopiperazines was accompanied by transformation of the N‐terminal Glu(R1) to pyroglutamic acid residue. Even in the case R1 is an amino acid residue attached to the N‐terminal γ‐carboxyl group, lactamization leads to its elimination. The observed reaction has not been reported so far in the literature. Correspondingly, an alternative route to Glu(R1)‐Glu(R2)‐containing 2,5‐diketopiperazines was applied to improve the overall yields.     

Peptide array‐based characterization and design of ZnO‐high affinity peptides

Peptides with both an affinity for ZnO and the ability to generate ZnO nanoparticles have attracted attention for the self‐assembly and templating of nanoscale building blocks under ambient conditions with compositional uniformity. In this study, we have analyzed the specific binding sites of the ZnO‐binding peptide, EAHVMHKVAPRP, which was identified using a phage display peptide library. The peptide binding assay against ZnO nanoparticles was performed using peptides synthesized on a cellulose membrane using the spot method. Using randomized rotation of amino acids in the ZnO‐binding peptide, 125 spot‐synthesized peptides were assayed. The peptide binding activity against ZnO nanoparticles varied greatly. This indicates that ZnO binding does not depend on total hydrophobicity or other physical parameters of these peptides, but rather that ZnO recognizes the specific amino acid alignment of these peptides. In addition, several peptides were found to show higher binding ability compared with that of the original peptides. Identification of important binding sites in the EAHVMHKVAPRP peptide was investigated by shortened, stepwise sequence from both termini. Interestingly, two ZnO‐binding sites were found as 6‐mer peptides: HVMHKV and HKVAPR. The peptides identified by amino acid substitution of HKVAPR were found to show high affinity and specificity for ZnO nanoparticles. Biotechnol. Bioeng. 2010;106: 845–851. © 2010 Wiley Periodicals, Inc.     

N-Methylated Analogs of hIAPP Fragments 18–22, 23–27, 33–37 Inhibit Aggregation of the Amyloidogenic Core of the Hormone

Amylin (hIAPP) aggregation leads to the formation of insoluble deposits and is one of the factors in the development of type II diabetes. The aim of this research was to find N-methylated analogs of the aggregating amylin fragments 18–22, 23–27, and 33–37, which would not themselves be susceptible to aggregation and would inhibit the aggregation of the amyloidogenic cores of the hormone. None of the analogs of fragment 18–22 containing one or two N-methylated amino acid residues showed any tendency to aggregate. Only the peptide H−F(N−Me)GA(N−Me) IL−OH ( 6 ) derived from the 23–27 hIAPP hot spot did not form fibrous structures. All analogs of the 33–37 amylin fragment were characterized by the ability to form aggregates, despite the presence of N-methylated amino acids in their structures. N-Methylated peptides 1 – 5 demonstrated inhibitory properties against the aggregation of fragment 18–22. Aggregation of the amyloidogenic core of 23–27 was significantly inhibited by N-methylated peptides 1 – 3 derived from the (18–22) H−HSSNN−OH fragment and by the H−F(N-Me)GA(N−Me)IL−OH ( 6 ) fragment derived from the 23–27 amylin hot spot. Fragment (33–37) H−GSNTY−NH₂ was found to be inhibited in the presence of N-methylated peptides 1 – 3 derived from the 18–22 fragment and by the double methylated peptide H−F(N−Me)GA(N−Me)IL−OH ( 6 ). Research on the possibility of using N-methylated analogs of amyloidogenic amylin cores as inhibitors of hormone aggregation is ongoing, with a focus on finding the minimum concentration of N-methylated peptides capable of inhibiting the aggregation of hIAPP hot spots.     

Incorporation of N‐amidino‐pyroglutamic acid into peptides using intramolecular cyclization of α‐guanidinoglutaric acid

N‐terminal modification of peptides by unnatural amino acids significantly affects their enzymatic stability, conformational properties and biological activity. Application of N‐amidino‐amino acids, positively charged under physiological conditions, can change peptide conformation and its affinity to the corresponding receptor. In this article, we describe synthesis of short peptides, containing a new building block—N‐amidino‐pyroglutamic acid. Although direct guanidinylation of pyroglutamic acid and oxidation of N‐amidino‐proline using RuO₄ did not produce positive results, N‐amidino‐Glp‐Phe‐OH was synthesized on Wang polymer by cyclization of α‐guanidinoglutaric acid residue. In the course of synthesis, it was found that literature procedure of selective Boc deprotection using TMSOTf/TEA reagent is accompanied by concomitant side reaction of triethylamine alkylation by polymer linker fragment. It should be mentioned that independently from cyclization time and coupling agent (DIC or HCTU), the lactam formation was incomplete. Separation of the cyclic product from the linear precursor was achieved by HPLC in ammonium formate buffer at pH 6. HPLC analysis showed N‐amidino‐Glp‐Phe‐OH stability at acidic and physiological pH and fast ring opening in water solution at pH 9. The suggested method of N‐amidino‐Glp residue formation can be applied in the case of short peptide chains, whereas synthesis of longer ones will require fragment condensation approach. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Evidence of π‐stacking interactions in the self‐assembly of hIAPP22‐29

The role aromatic amino acids play in the formation of amyloid is a subject of controversy. In an effort to clarify the contribution of aromaticity to the self‐assembly of human islet amyloid polypeptide (hIAPP)_22‐29, peptide analogs containing electron donating groups (EDGs) or electron withdrawing groups (EWGs) as substituents on the aromatic ring of Phe‐23 at the para position have been synthesized and characterized using turbidity measurements in conjunction with Raman and fluorescence spectroscopy. Results indicate the incorporation of EDGs on the aromatic ring of Phe‐23 virtually abolish the ability of hIAPP_22‐29 to form amyloid. Peptides containing EWGs were still capable of forming aggregates. These aggregates were found to be rich in β‐sheet secondary structure. Transmission electron microscopy images of the aggregates confirm the presence of amyloid fibrils. The observed difference in amyloidogenic propensity between peptides containing EDGs and those with EWGs appears not to be based on differences in peptide hydrophobicity. Fluorescence and Raman spectroscopic investigations reveal that the environment surrounding the aromatic ring becomes more hydrophobic and ordered upon aggregation. Furthermore, Raman measurements of peptide analogs containing EWGs, conclusively demonstrate a distinct downshift in the ? C?C? ring mode (ca. 1600 cm^?1) upon aggregation that has previously been shown to be indicative of π‐stacking. While previous work has demonstrated that π‐stacking is not an absolute requirement for fibrillization, our findings indicate that Phe‐23 also contributes to fibril formation through π‐stacking interactions and that it is not only the hydrophobic nature of this residue that is relevant in the self‐assembly of hIAPP_22‐29. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Fenton chemistry. Amino acid oxidation

The oxidation of amino acids by Fenton reagent (H2O2 + Fe(II] leads mainly to the formation of NH+4, alpha-ketoacids, CO2, oximes, and aldehydes or carboxylic acids containing one less carbon atom. Oxidation is almost completely dependent on the presence of bicarbonate ion and is stimulated by iron chelators at levels which are substoichiometric with respect to the iron concentration but is inhibited at higher concentrations. The stimulatory effect of chelators is not due merely to solubilization of catalytically inactive polymeric forms of Fe(OH)3 nor to the conversion of Fe(II) to complexes incapable of scavenging hydroxyl radicals. The results suggest that an iron chelate and another as yet unidentified form of iron are both required for maximal rates of amino acid oxidation. The metal ion-catalyzed oxidation of amino acids is likely a "caged" process, since the oxidation is not inhibited by hydroxyl radical scavengers, and the relative rates of oxidation of various amino acids by the Fenton system as well as the distribution of products formed (especially products of aromatic amino acids) are significantly different from those reported for amino acid oxidation by ionizing radiation. Several iron-binding proteins, peptides, and hemoglobin degradation products can replace Fe(II) or Fe(III) in the bicarbonate-dependent oxidation of amino acids. In view of their ability to sequester metal ions and their susceptibility to oxidation by H2O2 in the presence of physiological concentrations of bicarbonate, amino acids may serve an important role in antioxidant defense against tissue damage.     

Investigation of the Feasibily of an Amide-based Prodrug Under Physiological Conditions

Two different chemical classes of putative amide-based prodrugs of glucagon-like peptide-1 (GLP), one with an amino and the other with α hydroxyl terminal extension have been synthesized and biochemically characterized. The conversion of these terminally-extended peptide hormone analogs to a peptide of much enhanced potency through cyclization of the terminal dipeptide was studied under physiological conditions. The peptides studied demonstrated great stability and little propensity to cyclize to DKP and DMP under physiological conditions. These results stand in contrast to previous reports with model amide-based peptides and indicate that such cleavage is unlikely in larger peptides constituted by naturally coded amino acids. An erratum to this article can be found at     

Quantification of hydrolyzed peptides and proteins by amino acid fluorescence

Reliable quantification of peptides and proteins is essential for drug discovery. We report the successful development and validation of an accurate and broadly applicable high performance liquid chromatography hyphenated to fluorescence detector procedure for the quantitative determination of the aromatic amino acids tyrosine, phenylalanine, and tryptophan, without relying on derivatization chemistry. Using ion‐pair chromatography, fluorescent amino acids were clearly separated within 10 minutes. The hydrolysis of peptides was performed under acidic and heated conditions to yield the monomeric building blocks. Various protecting agents were tested to ensure tryptophan stability. The presented analytical method accurately (>95%) quantifies all fluorescent residues. The power of the method was confirmed by correct quantification of protein reference standard to 98.6% over all fluorescence traces. The method allowed us to identify pre‐analytical differences between the nominal and actual concentrations of 12 peptide solutions. Salt formation, weighing errors, and other pre‐analytical pitfalls resulted in noteworthy differences of up to 85% between the indicated and actual concentration of peptide solutions, subsequently leading to false positive or negative interpretation of activity data. Finally, only one solution is needed to perform quantification as well as UV‐purity tests and can further be used as stock solution for activity testing.     

超分子多肽自组装在生物医学中的应用

近年来,自组装多肽纳米技术因其可形成规则有序的结构、具有多样的功能而备受关注。研究发现自组装多肽能在特定的条件下形成具有确定结构的聚集体,这种聚集体具备生物相容性好、稳定性高等优点,表现出不同于单体多肽分子的特性和优势,因此其在药物传递、组织工程、抗菌等领域具有良好的应用前景。文中介绍了自组装多肽形成的分子机理、类型、影响因素,综述了自组装多肽形成的纤维肽基水凝胶与自组装抗菌肽的最新进展,并提出目前多肽自组装技术所存在的问题及展望。     

Quantitative analysis of the relationship between structure and antioxidant activity of tripeptides

Peptides from enzymatic hydrolysates of food proteins exhibit significant antioxidant activity. Several studies have attempted to determine the factors contributing to the antioxidant activity of peptides; however, the physicochemical properties and factors essential for the antioxidant activity of peptides are still unclear. In this study, in order to clarify the factors important for peptide antioxidant activity based on the properties of component amino acids, 55 tripeptides were synthesized from 20 natural amino acids and their antioxidant activity was measured using the Trolox equivalent antioxidant capacity (TEAC) assay system. The tripeptides were divided into two data sets: a training set comprising 50 compounds and a validated set comprising five compounds. The structure‐activity relationship of the training set was then analyzed using classical quantitative structure‐activity relationship (QSAR) analysis. The study findings demonstrate that the presence of a cysteine residue at any position, an aromatic amino acid at the C‐terminus, higher hydrophobicity of the N‐terminal residue, and smaller HOMO‐LUMO energy gap of the middle residue can significantly enhance the antioxidant activity. The activities of the five validated compounds were predicted using the constructed QSAR model, and a good correlation between measured and predicted activities was observed. The information obtained from the QSAR model could be useful for effective production of antioxidant peptides from food proteins such as egg white proteins.     

Effect of SXWS/WSXWS peptides on chemotaxis and adhesion of the macrophage‐like cell line J774

WSXWS motif is a conserved amino acid sequence that is present in type I cytokine receptors. This motif that can be found both in the ligand binding chains and signal transducer molecule of the receptors with different amino acids at the position “X” plays a role in the receptor folding, ligand binding and signal transduction as well. Structural analysis proved that WSEWS motif of IL‐6R is located in a highly accessible location in the protein. Structural properties and chemotaxis of a tetrapeptide library with SXWS sequence, where X was the 19 proteinogenic amino acids except cystein were systematically studied earlier. It has been proved that C‐terminal amidation and the identity of amino acid X had a pronounced influence on the chemotactic properties but less of the structure of the peptides. Here, we present our findings on the effect of a tetrapeptide and a pentapeptide library with the sequence of SXWS and WSXWS on the chemotaxis and adhesion of J774 murine macrophage cell line. We studied the effect of the presence/absence of N‐terminal tryptophan and the different amino acids at the X position on these physiological responses. Results indicated that amino acid X had a marked influence on chemotaxis, adhesion as well as on proliferation induced by (W)SXWS peptides. Elongation of SXWS sequence with a tryptophan at the N terminus also altered pronouncedly all the physiological responses of the cells studied. A good correlation could be observed between the chemotaxis and the proliferation and physicochemical parameters of the amino acid X. Copyright © 2015 John Wiley & Sons, Ltd.     

Fullerene‐based inhibitors of HIV‐1 protease

A series of Fmoc‐Phe(4‐aza‐C₆₀)‐OH of fullerene amino acid derived peptides have been prepared by solid phase peptide synthesis, in which the terminal amino acid, Phe(4‐aza‐C₆₀)‐OH, is derived from the dipolar addition to C₆₀ of the Fmoc‐Nα‐protected azido amino acids derived from phenylalanine: Fmoc‐Phe(4‐aza‐C₆₀)‐Lys₃‐OH ( 1 ), Fmoc‐Phe(4‐aza‐C₆₀)‐Pro‐Hyp‐Lys‐OH ( 2 ), and Fmoc‐Phe(4‐aza‐C₆₀)‐Hyp‐Hyp‐Lys‐OH ( 3 ). The inhibition constant of our fullerene aspartic protease PRIs utilized FRET‐based assay to evaluate the enzyme kinetics of HIV‐1 PR at various concentrations of inhibitors. Simulation of the docking of the peptide Fmoc‐Phe‐Pro‐Hyp‐Lys‐OH overestimated the inhibition, while the amino acid PRIs were well estimated. The experimental results show that C₆₀‐based amino acids are a good base structure in the design of protease inhibitors and that their inhibition can be improved upon by the addition of designer peptide sequences. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Antiestrogenic and anticancer activities of peptides derived from the active site of alpha‐fetoprotein

Cyclo[EKTOVNOGN] (AFPep), a cyclic 9‐amino acid peptide derived from the active site of alpha‐fetoprotein, has been shown to prevent carcinogen‐induced mammary cancer in rats and inhibit the growth of ER⁺ human breast cancer xenografts in mice. Recently, studies using replica exchange molecular dynamics predicted that the TOVN region of AFPep might form a dynamically stable putative Type I beta‐turn, and thus be biologically active without additional amino acids. The studies presented in this paper were performed to determine whether TOVN and other small analogs of AFPep would inhibit estrogen‐stimulated cancer growth and exhibit a broad effective‐dose range. These peptides contained nine or fewer amino acids, and were designed to bracket or include the putative pharmacophoric region (TOVN) of AFPep. Biological activities of these peptides were evaluated using an immature mouse uterine growth inhibition assay, a T47D breast cancer cell proliferation assay, and an MCF‐7 breast cancer xenograft assay. TOVN had very weak antiestrogenic activity in comparison to AFPep's activity, whereas TOVNO had antiestrogenic and anticancer activities similar to AFPep. OVNO, which does not form a putative Type I beta‐turn, had virtually no antiestrogenic and anticancer activities. A putative proteolytic cleavage product of AFPep, TOVNOGNEK, significantly inhibited E₂‐stimulated growth in vivo and in vitro over a wider dose range than AFPep or TOVNO. We conclude that TOVNO has anticancer potential, that TOVNOGNEK is as effective as AFPep in suppressing growth of human breast cancer cells, and that it does so over a broader effective‐dose range. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of stable C‐linked ferrocenyl amino acids and their use in solution‐phase peptide synthesis

Incorporation of ferrocenyl group to peptides is an efficient method to alter their hydrophobicity. Ferrocenyl group can also act as an electrochemical probe when incorporated onto functional peptides. Most often, ferrocene is incorporated onto peptides post‐synthesis via amide, ester or triazole linkages. Stable amino acids containing ferrocene as a C‐linked side chain are potentially useful building units for the synthesis of ferrocene‐containing peptides. We report here an efficient route to synthesize ferrocene‐containing amino acids that are stable and can be used in peptide synthesis. Coupling of 2‐ferrocenyl‐1,3‐dithiane and iodides derived from aspartic acid or glutamic acid using n‐butyllithium leads to the incorporation of a ferrocenyl unit to the δ‐position or ε‐position of an α‐amino acid. The reduction or hydrolysis of the dithiane group yields an alkyl or an oxo derivative. The usability of the synthesized amino acids is demonstrated by incorporating one of the amino acids in both C‐terminus and N‐terminus of tripeptides in solution phase. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Covalently attached fatty acyl chains alter the aggregation behavior of an amyloidogenic peptide derived from human β2‐microglobulin

Aggregation of a polypeptide chain into highly ordered amyloid aggregates is a complex process. Various factors, both extrinsic and intrinsic to the polypeptide chain, have been shown to perturb this process, leading to a drastic change in the amyloidogenic behavior, which is reflected in the polymorphism of amyloid aggregates at various levels of self‐assembly. In this paper, we have investigated the ability of covalently linked long‐chain fatty acids in modulating the self‐assembly of an aromatic amino acid‐rich highly amyloidogenic sequence derived from the amino acid region 59–71 of human β₂‐microglobulin by thioflavin T (ThT) fluorescence microscopy, circular dichroism, and fluorescence spectroscopy. Our results indicate that under identical conditions of dissolution and concentration, each peptide enhances the fluorescence of ThT. However, the aggregates are morphologically distinct. For the same peptide, the aggregate morphologies are dependent on peptide concentration. Further, an optimum concentration, which varies with solution ionic strength, is required for the formation of fibrillar aggregates. We show that covalent modification of this amyloidogenic sequence, with long‐chain fatty acids, affects the way the higher order amyloid structures assemble from the cross‐β units, in fatty acyl chain‐dependent and position‐dependent manner. Our data indicate that noncovalent interactions leading to amyloid fibril formation can be modulated by the hydrophobicity of covalently attached long‐chain fatty acids resulting in self‐assembly of the peptide chain to form nonfibrillar aggregates. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Design and synthesis of tryptophan containing peptides as potential analgesic and anti‐inflammatory agents

A new series of smaller peptides with tryptophan at C‐terminal and varying N‐protected amino acids/peptides were designed, synthesized and characterized by analytical and spectroscopic techniques. Analgesic and anti‐inflammatory properties of these peptides were carried out in vivo using tail‐flick method and carrageenan‐induced paw edema method, respectively, at different doses and different time intervals. Most of the peptides synthesized displayed enhanced activity, and particularly tetra and hexapeptides 29–31 were found to be even more potent than the reference standards used. Moreover, some peptides have exhibited promising activity even after 24 h of administration, whereas the reference standards were active only up to 3 h. Further, the compounds did not present any ulcerogenic liability. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis, conformational and pharmacological studies on dermorphin N-terminal tetrapeptide analogues

Summary Dermorphin structure-activity relationships toward μ and δ opioid receptors were investigated using a series of synthetic peptides, in which the aromatic residues at positions 1 or/and 3 of the N-terminal tetrapeptide analogue H-Tyr-d-Arg-Phe-β-Ala-NH₂ were replaced by unnatural or constrained amino acids.     

Design and activity of novel lactoferrampin analogues against O157:H7 enterohemorrhagic escherichia coli

Lactoferrampin 265–284 (LFampin 265–284) is a peptide consisting of residues 265–284 of N1‐domain of bovine Lactoferrin (LF). This peptide has several cationic groups in the C‐terminal lobe, exhibiting an antibacterial activity against a wide range of microorganisms. However, LFampin 265–284 exhibits low antimicrobial activity against the O157:H7 enterohaemorrhagic Escherichia coli (EHEC O157:H7) when compared with Lactoferrin chimera and Lactoferricin. Here, we have designed three analogues of LFampin 265–284 based on the distribution of cationic groups, hydrophobicity, size, and sequence. Analogues were synthesized by solid phase chemistry using Fmoc methodology obtaining peptides with 95% purity. All peptides maintain the ability to adopt helical conformations (checked by circular dichroism spectra and molecular simulations). Some of these analogues exhibited a significant increase in antimicrobial activity by counting colony forming units against EHEC O157:H7 compared to native LFampin 265–284, with MIC of 10 and 40 µM for 264G‐D265K and 264G‐D265K/S272R, respectively. The incorporation of a GKLI sequence in the N‐terminal lobe increased dramatically its antibacterial activity, an effect which has been attributed to the addition of cationic groups in the N‐terminal side that may stabilize the helical conformation of the new designed peptides. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 319–328, 2014.