Acanthoscurrin fragment 101–132: Total synthesis at 60°C of a novel difficult sequence

Abstract

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.

Ctenidins: antimicrobial glycine-rich peptides from the hemocytes of the spider Cupiennius salei

“…No structures of truly glycinerich peptides are available, making it difficult to predict a target or functional mechanism of such peptides. While a recent report suggests a b-sheet structure for the C-terminal part of acanthoscurrin [43], the cationic plasticins, glycineleucine-rich peptides from frog skin, were shown to go through multiple conformational transitions, including helix states, b-structures, and disordered states [44]. We had to measure the combined MIC of the three ctenidin isoforms as not enough material was present to separate the peptides on a larger scale to obtain enough material for separate tests.…”

Hemocidinas derivadas da hemoglobina: estruturas, propriedades e perspectivas

“…Embora comprovadamente certos AMPs sejam seletivos aos microrganismos, 14 muitos são altamente hemolí-ticos (ativos frente aos eritrócitos), como é o caso da melitina que foi isolada do veneno da abelha Apis mellifera e é muito utilizado como controle positivo em ensaios hemolíticos ou de dano na membrana celular, mas também como agente antimicrobiano. 22 Vários AMPs foram isolados de fontes naturais diversas e estão sendo ampla e eficientemente estudados por diferentes grupos de pesquisa brasileiros, 9,23-28 incluindo o nosso [além de realizar pesquisa em química de peptídeos, [29][30][31] colaboramos com alguns desses grupos em estudos da migalina 32 e da gomesina, 9,33 bem como vimos estudando o fragmento C-terminal de acanthoscurrina, 31 hemocidinas sintéticas 34 e a cheferina I (dados em publicação)].…”

Difficult Peptides

“…Although it needs further studies before being used routinely, SPPS at high temperature has been effectively used for the preparation of peptides, glycopeptides, and peptoids under a variable range of conditions that employ conventional heating [35,64] or microwave irradiation [36,65]. Although it needs further studies before being used routinely, SPPS at high temperature has been effectively used for the preparation of peptides, glycopeptides, and peptoids under a variable range of conditions that employ conventional heating [35,64] or microwave irradiation [36,65].…”

“…on: (i) methods, procedures, and protocols that may furnish comparatively higher quality products (free or surface-bound) with higher yields in less time or, at least, overcome problems that are inherent in existing technologies [35,36]; and (ii) new uses [37,38].…”