Acetamidomethyl. A Novel Thiol Protecting Group for Cysteine

Veber, Daniel F.; Milkowski, John D.; Varga, Sándor; Denkewalter, Robert G.; Hirschmann, Ralph

doi:10.1021/ja00770a600

Cited by 326 publications

(173 citation statements)

References 4 publications

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“…The synthesized peptides were cleaved and deprotected by using a mixture containing 82.5% trifluoroacetic acid (TFA), 5% phenol, 5% H 2 O, 5% thioanisole, and 2.5% ethanedithiol for 12 to 15 h at room temperature. HNP-1 was synthesized on 4-hydroxymethylphenoxymethyl resin (Applied Biosystems; 0.9 mmol/g) (28) with the cysteines protected by acetamidomethyl (46). The removal of acetamidomethyl was effected by Hg(II) acetate and ␤-mercaptoethanol treatment (28,46) followed by gel filtration on a Biogel P-2 column.…”

Section: Methodsmentioning

confidence: 99%

“…HNP-1 was synthesized on 4-hydroxymethylphenoxymethyl resin (Applied Biosystems; 0.9 mmol/g) (28) with the cysteines protected by acetamidomethyl (46). The removal of acetamidomethyl was effected by Hg(II) acetate and ␤-mercaptoethanol treatment (28,46) followed by gel filtration on a Biogel P-2 column. HNP-1 was then oxidized in 20% dimethyl sulfoxide-water (42) at a peptide concentration of 0.2 to 0.4 mM.…”

Section: Methodsmentioning

confidence: 99%

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Antibacterial Activity of Human Neutrophil Defensin HNP-1 Analogs without Cysteines

Varkey

Nagaraj

2005

Antimicrob Agents Chemother

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The antibacterial activity of human neutrophil defensin HNP-1 analogs without cysteines has been investigated. A peptide corresponding to the HNP-1 sequence without the six cysteines (HNP-1⌬C) exhibited antibacterial activity toward gram-negative and gram-positive bacteria. Truncated analogs wherein the nine N-terminal residues of HNP-1 and the remaining three cysteines were deleted (HNP-1⌬C18) or the G was replaced with A (HNP-1⌬C18A) also exhibited antibacterial activity. Substantial activity was observed for HNP-1⌬C and HNP-1⌬C18 in the presence of 100 mM NaCl, except in the case of Pseudomonas aeruginosa. The linear peptides were active in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), indicating that proton motive force was not essential for killing of bacteria by the peptides. In fact, in the presence of CCCP, the peptides were active against P. aeruginosa even in the presence of 100 mM NaCl. The antibacterial activity of HNP-1⌬C, but not that of the shorter, 18-residue peptides, was attenuated in the presence of serum. The generation of defensins without cysteines would be easier than that of disulfide-linked defensins. Hence, linear defensins could have potential as therapeutic agents.Mammalian defensins are a family of cationic antimicrobial peptides characterized by three disulfide bridges (13,22,35). They are expressed in granulocytes, monocytes, macrophages, and epithelial cells of the skin, intestines, and other tissues (2, 13, 39). Based on the pattern of disulfide bonding, mammalian defensins have been classified into ␣ and ␤ families. In another class named the -defensins, in addition to three disulfide bridges, the amino-and carboxyl-terminal ends are linked via an amide bond (13). In humans, ␣-defensins are produced in neutrophils and Paneth cells of the small intestine, while ␤-defensins are expressed by epithelial cells at various sites (13,22). The -defensins have been identified only in primates (13,19). The expression of defensins is constitutive as well as induced by infection and inflammation (13,22,23). Defensin biosynthesis and release have been observed to be regulated by microbial signals, developmental signals, and cytokines (13,14). Apart from their role as microbicidal agents, defensins have also been implicated as effective immune modulators in adaptive immunity (11, 54). They have potency to enhance phagocytosis, induce the activation and degranulation of mast cells, and indirectly regulate innate antimicrobial immunity by affecting the production of chemokines such as interleukin-8, interleukin-10, and tumor necrosis factor from different cells (3,24,53). Studies have also revealed the role of defensins in adaptive immunity, as reflected by their chemotactic effect on monocytes, T cells, and immature dendritic cells (5,32,35,43,48,54).Human neutrophil defensins (HNP-1 to HNP-4) are stored in azurophilic granules of the neutrophil. They are transferred to phagolysosomes upon phagocytosis to kill the ingested microbes (14, 15). The crystal structure of HNP-3 rev...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Antibacterial Activity of Human Neutrophil Defensin HNP-1 Analogs without Cysteines

Varkey

Nagaraj

2005

Antimicrob Agents Chemother

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“…The cysteines were deprotected with mercury (II) acetate in the ratio of 2 equivalents for each equivalent of cysteine. Mercuric sulfide salts formed were precipitated with 20 eq of ␤-mercaptoethanol (24). The peptides were then desalted and the disulfide bridges were formed with 20% dimethyl sulfoxide (25).…”

Section: Peptide Synthesismentioning

confidence: 99%

Tigerinins: Novel Antimicrobial Peptides from the Indian FrogRana tigerina

Korrapati¹,

Jagannadham²,

Vairamani³

et al. 2001

Journal of Biological Chemistry

105

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Four broad-spectrum, 11 and 12 residue, novel antimicrobial peptides have been isolated from the adrenalinestimulated skin secretions of the Indian frog Rana tigerina. Sequences of these peptides have been determined by automated Edman degradation, by mass spectral analysis and confirmed by chemical synthesis. These peptides, which we have named as tigerinins, are characterized by an intramolecular disulfide bridge between two cysteine residues forming a nonapeptide ring. This feature is not found in other amphibian peptides. Conformational analysis indicate that the peptides tend to form ␤-turn structures. The peptides are cationic and exert their activity by permeabilizing bacterial membranes. Tigerinins represent the smallest, nonhelical, cationic antimicrobial peptides from amphibians.Antimicrobial peptides constitute a very important component of the innate immune system in organisms across the evolutionary scale (1-8). Amphibians being the first group of organisms forming a connecting link between land and water are forced to adopt and survive in a variety of conditions laden with pathogenic microbes. Thereby, they are endowed with an excellent chemical defense system composed of pharmacological and antimicrobial peptides (9). Bombinins were the first antimicrobial peptides characterized from the skin of Bombina variegata in 1969 (10). The discovery of magainins from the skin secretions of Xenopus laevis in 1987 (11) triggered extensive search and characterization of antimicrobial peptides from amphibians (12)(13)(14). Antimicrobial peptides from genus Rana share an interesting structural motif composed of a disulfidebridged cationic heptapeptide segment at the COOH-terminal end. Peptides with this motif include brevinins and esculentins which are composed of 24 and 46 amino acids, respectively (14). The primary structures of large number of peptides belonging to this family have been determined. Another group of short peptides composed of 13 residues called temporins, which do not contain this COOH-terminal ring, have also been characterized from frogs of genus Rana (15). However, considering the large variety of amphibian species in nature, antimicrobial peptides from only a small number of them have been characterized, that too only with respect to primary structure. Also, studies directed toward determining structure-function relationships have been confined to magainins (16) and dermaseptins (17, 18). Hence, characterizing host-defense peptides from other species would be of interest and could conceivably result in the identification of new structural motifs which would be useful in designing peptides for therapeutic applications. Rana tigerina is the predominant species of frogs found in India (19). The skin of these frogs have been used traditionally by some tribal communities to heal both open and burn wounds and the antimicrobial components could possibly contribute to the wound healing process (20). In this study, we have described the isolation and characterization of antimicrobial peptides from...

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“…Peptides a2GPRCG, a2GPRRC, a,GPCRG, and a4GPRRC were synthesized on a Milligen 9050 automated peptide synthesizer with their C-termini blocked as acarboxamide on PAL (Milligen) resin. The Cys residues were blocked with Acm protecting groups (Veber et al, 1972). All peptides were acetylated at the N-terminus by treatment with acetic anhydride and diisopropylethylamine before cleavage from the resin.…”

Section: Peptide Synthesis and Purificationmentioning

confidence: 99%

Disulfide crosslinks to probe the structure and flexibility of a designed four‐helix bundle protein

et al. 1994

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The introduction of disulfide crosslinks is a generally useful method by which to identify regions of a protein that are close together in space. Here we describe the use of disulfide crosslinks to investigate the structure and flexibility of a family of designed 4-helix bundle proteins. The results of these analyses lend support to our working model of the proteins' structure and suggest that the proteins have limited main-chain flexibility.

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Acetamidomethyl. A Novel Thiol Protecting Group for Cysteine

Cited by 326 publications

References 4 publications

Antibacterial Activity of Human Neutrophil Defensin HNP-1 Analogs without Cysteines

Antibacterial Activity of Human Neutrophil Defensin HNP-1 Analogs without Cysteines

Tigerinins: Novel Antimicrobial Peptides from the Indian FrogRana tigerina

Disulfide crosslinks to probe the structure and flexibility of a designed four‐helix bundle protein

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