Recebido em 17/9/03; aceito em 27/11/03; publicado na web em 17/6/04 CHEMICAL AND ENZYMATIC PEPTIDE SYNTHESES: BASIC ASPECTS AND APPLICATIONS. This review begins with a brief discussion of the biological importance and chemical features of peptides. A description of the existing synthetic methods follows with emphasis on the basic aspects of the chemical and enzymatic syntheses. Techniques used to purify and characterize the synthesized peptides are also discussed. Finally, a few applications of the final products in chemistry, biochemistry, immunology and medicine are presented, such as identification and quantification of naturally occurring peptides, inspection of structureactivity relationships, therapeutics, development and/or improvement of analytical techniques and search for new vaccines.Keywords: peptides; synthesis; applications of synthetic peptides. A DIVERSIDADE FUNCIONAL E QUÍMICA DOS PEPTÍDEOSOs peptídeos são biomoléculas que contém de dois a dezenas de resíduos de aminoácidos unidos entre si através de ligações peptídicas. Se comparados às proteínas, são quimicamente mais versáteis, pois podem ser amidados ou esterificados em suas carboxilas terminais, acetilados em seus grupos amino terminais, fosforilados ou sulfatados em um ou mais resíduos (serina, treonina ou tirosina), lineares, semicíclicos (geralmente via uma ou mais ligações dissulfeto intra-ou intercadeias peptídicas) ou cíclicos (via ligação entre os grupos amino e carboxila dos aminoácidos terminais). Muitos contêm um ácido piroglutâmico como resíduo N-terminal, outros apresentam Daminoácidos e outros, ainda, possuem aminoácidos não usuais 1 . Os peptídeos são também extremamente diversificados em termos funcionais. Muitos atuam como hormônios ou fatores liberadores destes, enquanto outros são neuropeptídeos, neurotransmissores, toxinas, antibióticos naturais, adoçantes ou substratos de proteases. Na verdade, vários deles fazem parte de nossas conversas sem que nos apercebamos disto: é o caso do aspartame, da insulina, da ocitocina e de diversas drogas comerciais, que consistem em antagonistas de peptídeos naturais ou em inibidores de enzimas envolvidas na sua produção e liberação no organismo 2-4 . A Tabela 1 fornece alguns exemplos desta diversidade funcional e química. Todo este conhecimento começou a ser acumulado principalmente a partir da década de 50, quando vários peptídeos ativos foram descobertos e tiveram as suas estruturas químicas determinadas. Foi o caso de diversos hormônios que controlam o metabolismo animal (glucagon e insulina, p.ex.) e de outros que desempenham papéis específicos em nosso organismo (ocitocina, vasopressina e o hormônio estimulador de melanócitos, p. ex.) 10 . Estas descobertas geraram um enorme interesse por esta classe de compostos e por metodologias para seu isolamento, análise, purificação, identificação e quantificação, as quais passaram a ser sistematicamente estudadas e aprimoradas. Em paralelo, deparou-se com a necessidade de sintetizar estas moléculas e análogos (derivados com modificações p...

Glycine-rich proteins (GRPs) serve a variety of biological functions. Acanthoscurrin is an antimicrobial GRP isolated from hemocytes of the Brazilian spider Acanthoscurria gomesiana. Aiming to contribute to the knowledge of the secondary structure and stepwise solid-phase synthesis of GRPs' glycine-rich domains, we attempted to prepare G(101)GGLGGGRGGGYG(113)GGGGYGGGYG(123) GGY(126)GGGKYK(132)-NH(2), acanthoscurrin C-terminal amidated fragment. Although a theoretical prediction did not indicate high aggregation potential for this peptide, repetitive incomplete aminoacylations were observed after incorporating Tyr(126) to the growing peptide-MBHA resin (Boc chemistry) at 60 degrees C. The problem was not solved by varying the coupling reagents or solvents, adding chaotropic salts to the reaction media or changing the resin/chemistry (Rink amide resin/Fmoc chemistry). Some improvement was made when CLEAR amide resin (Fmoc chemistry) was used, as it allowed for obtaining fragment G(113)-K(132). NIR-FT-Raman spectra collected for samples of the growing peptide-MBHA, -Rink amide resin and -CLEAR amide resin revealed the presence of beta-sheet structures. Only the combination of CLEAR-amide resin, 60 degrees C, Fmoc-(Fmoc-Hmb)Gly-OH and LiCl (the last two used alternately) was able to inhibit the phenomenon, as proven by NIR-FT-Raman analysis of the growing peptide-resin, allowing the total synthesis of desired fragment Gly(101)-K(132). In summary, this work describes a new difficult sequence, contributes to understanding stepwise solid-phase synthesis of this type of peptide and shows that, at least while protected and linked to a resin, this GRP's glycine-rich motif presents an early tendency to assume beta-sheet structures.

Although glycine-rich antimicrobial peptides (AMPs) are found in animals and plants, very little has been reported on their chemistry, structure activity-relationship, and properties. We investigated those topics for Shepherin I (Shep I), a glycine-rich AMP with the unique amino acid sequence G(1)YGGHGGHGGHGGHGGHGGHGHGGGGHG(28). Shep I and analogues were synthesized by the solid-phase method at 60 °C using conventional heating. Purification followed by chemical characterization confirmed the products' identities and high purity. Amino acid analysis provided their peptide contents. All peptides were active against the clinically important Candida species, but ineffective against bacteria and mycelia fungi. Truncation of the N- or C-terminal portion reduced Shep I antifungal activity, the latter being more pronounced. Carboxyamidation of Shep I did not affect the activity against C. albicans or C. tropicalis, but increased activity against S. cerevisiae. Carboxyamidated analogues Shep I (3-28)a and Shep I (6-28)a were equipotent to Shep I and Shep Ia against Candida species. As with most cationic AMPs, all peptides had their activity significantly reduced in high-salt concentrations, a disadvantage that is defeated if 10 µM ZnCl2 is present. At 100 µM, the peptides were practically not hemolytic. Shep Ia also killed C. albicans MDM8 and ATCC 90028 cells. Fluo-Shep Ia, an analogue labeled with 5(6)-carboxyfluorescein, was rapidly internalized by C. albicans MDM8 cells, a salt-sensitive process dependent on metabolic energy and temperature. Altogether, such results shed light on the chemistry, structural requirements for activity, and other properties of candidacidal glycine-rich peptides. Furthermore, they show that Shep Ia may have strong potential for use in topical application.

This study expands the knowledge on chemical synthesis and properties of Hb40-61a as well as provides results of the first steps given towards knowing how it kills Candida cells. For the first time, this peptide, its all-D analogue (D-Hb40-61a) and its fluorescently labeled analogue (FAM-Hb40-61a) were successfully assembled on resin at 60°C using conventional heating in all steps. Purified and characterized, these peptides exhibited very low toxicity on human erythrocytes. Hb40-61a and D-Hb40-61a were equally active against Candida strains, ruling out sterically specific interactions on their working mechanism. Cell permeabilization assays confirmed progressive damage of the yeast plasma membrane with increasing concentrations of Hb40-61a. While experiment using the fluorescent probe DiBAC4(5) revealed that this synthetic hemocidin alters the yeast plasma membrane potential, test employing DPH indicated that Hb40-61a might affect its dynamics. Exposure of the yeast cells to FAM-Hb40-61a showed that the peptide accumulates in the cell membrane at the ½ MIC, but stains about 97% of the cells at the MIC. Such effect is salt-dependent and partially energy-dependent. These new findings indicate that the central target of Hb40-61a in Candida cells is the plasma membrane and that this synthetic hemocidin should be considered as a potential candidacidal for topic uses.

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The preparation of small-sized protected peptide acids related to cholecystokinin and gomesin was attempted using peptide-Kaiser oxime resins (KOR) as starting materials. For comparison, peptide-2-Cl-trityl resin (CLTR) was also employed. While peptide detachment from KOR was achieved through the oxime ester bond hydrolysis mediated by DBU, hydroxide ion or Ca(+2) ion, peptide release from CLTR was accomplished through acid-catalysed hydrolysis of the peptide-resin ester linkage. Amino acid analysis of the peptide-resins before and after peptide release allowed the calculation of the reaction yields. RP-HPLC and LC/ESI-MS of the resulting crude peptides allowed estimation of their quality. The data collected indicated that: (i) among the procedures used for peptide displacement from KOR, the one employing DBU was the most efficient since it furnished all model protected peptide acids with the highest quality in a very short time; (ii) although slow, Ca(+2)-assisted peptide detachment from KOR was selective and was suitable for generating high-quality protected peptide acids containing up to five residues; (iii) even though the protocols employed for peptide release from CLTR have shown to be appropriate, the one employing 1% TFA/DCM was the most productive; (iv) in terms of product quality, DBU-catalysed peptide detachment from KOR was similar to TFA-catalysed peptide release from CLTR although the latter was more productive. These findings are relevant to peptide chemists in general, but especially to those interested in preparing acyl donors for convergent peptide syntheses by the t-Boc chemistry.

Se ha investigado el contenido proteico y algunas actividades enzimáticas del veneno piente cascabel Crotalus durissus terrificus procedente de la región de Sandia, Puno; p empleó el veneno total así como las fracciones obtenidas mediante cromatografía de f Sephadex G-100. El porcentaje de proteína calculado por el método de Lowry fue de 68 veneno total; habiéndose obtenido 3 picos de proteína durante el fraccionamiento; en el registró actividad proteolítica, en el segundo, las actividades amidolítica, coagulante y fos mientras que en el tercero se detectó otra fracción proteolítica . No se registró la acetilcolinesterasa mientras que la actividad L-aminoácido oxidasa sólo se encontró en total.Palabras clave: Veneno , serpiente cascabel, enzimas, Puno. ABSTRACTThe venom of the rattlesnake Crotalus durissus terrificus from the region of Sandia, Puno investigated for its protein content and some enzymatic activities, using for it the whole ven as the fractions obtained by gel filtration chromatography in Sephadex G-1 OO. The protein calculated by the method of Lowry was of 68,6% for the whole venom; 3 peaks were obta the fractionation; the first showed proteolytic activity, the second, amidolytic, clotting and pase A 2 activities, while the third, another proteolytic activity. Acetylcholinesterase activity wa while L~amino acid oxidase activity was found only in the whole venom.

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