<i>In situ</i> neutralization in Boc‐chemistry solid phase peptide synthesis

Schnölzer, Martina; Alewood, Paul F.; Jones, Ashley; Alewood, Dianne; Kent, Stephen B. H.

doi:10.1111/j.1399-3011.1992.tb00291.x

Cited by 1,000 publications

(251 citation statements)

References 26 publications

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“…Mitchell et al developed a more acid stable PAM resin support [48][49][50] and Kent and coworkers improved the synthetic protocols used with PAM resins and introduced in situ neutralization into SPPS using Boc/Benzyl chemistry for the rapid, efficient synthesis of difficult sequences. 51 In addition, several side reactions were examined and eliminated. The improved chemistry and protocols were utilized in the synthesis of the L and D enantiomers of the 99 residue HIV-1 protease (1-99).…”

Section: )''mentioning

confidence: 99%

Bruce Merrifield and solid‐phase peptide synthesis: A historical assessment

Mitchell

2008

Biopolymers

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Bruce Merrifield, trained as a biochemist, had to address three major challenges related to the development and acceptance of solid‐phase peptide synthesis (SPPS). The challenges were (1) to reduce the concept of peptide synthesis on a insoluble support to practice, (2) overcome the resistance of synthetic chemists to this novel approach, and (3) establish that a biochemist had the scientific credentials to effect the proposed revolutionary change in chemical synthesis. How these challenges were met is discussed in this article. © 2008 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 175–184, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Section: )''mentioning

confidence: 99%

Bruce Merrifield and solid‐phase peptide synthesis: A historical assessment

Mitchell

2008

Biopolymers

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“…The concentrations of peptide samples were determined spectrophotometrically using an extinction coefficient of 5875 M Ϫ1 cm Ϫ1 at 280 nm. Synthesis of Lysine Mutants-The linear precursors of 23 lysine mutants of kalata B1 containing an N-terminal Cys residue were synthesized using manual solid-phase peptide synthesis with an in situ neutralization/HBTU protocol (35) for Boc chemistry on a 0.5 mM scale, using a procedure described previously (27,33). Precursor peptides were assembled on PAM resin via a thioester linker and cleaved from the resin with hydrogen fluoride.…”

Section: Methodsmentioning

confidence: 99%

Lysine-scanning Mutagenesis Reveals an Amendable Face of the Cyclotide Kalata B1 for the Optimization of Nematocidal Activity

Huang

Colgrave

Clark

et al. 2010

Journal of Biological Chemistry

105

148

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Cyclotides are a family of macrocyclic peptides that combine the unique features of a head-to-tail cyclic backbone and a cystine knot motif, the combination of which imparts them with extraordinary stability. The prototypic cyclotide kalata B1 is toxic against two economically important gastrointestinal nematode parasites of sheep, Haemonchus contortus and Trichostrongylus colubriformis. A lysine scan was conducted to examine the effect of the incorporation of positive charges into the kalata B1 cyclotide framework. Each of the non-cysteine residues in this 29-amino acid peptide was successively substituted with lysine, and the nematocidal and hemolytic activities of the suite of mutants were determined. Substitution of 11 residues within kalata B1 decreased the nematocidal activity dramatically. On the other hand, six other residues that are clustered on the surface of kalata B1 were tolerant to Lys substitution, and indeed the introduction of positively charged residues into this region increased nematocidal activity. This activity was increased further in double and triple lysine mutants, with a maximal increase (relative to the native kalata B1) of 13-fold obtained with a triple lysine mutant (mutated at positions Thr-20, Asn-29, and Gly-1). Hemolytic activity correlated with the nematocidal activity of all lysine mutants. Our data clearly highlight the residues crucial for nematocidal and hemolytic activity in cyclotides, and demonstrate that the nematocidal activity of cyclotides can be increased by incorporation of basic amino acids.Gastrointestinal nematodes cause major losses to livestock industries worldwide. The control of these pests, until now, has been via synthetic anthelmintics, including benzimidazoles (e.g. thiabendazole), nicotinic acetylcholine receptor agonists (e.g. levamisole), and macrocyclic lactones (e.g. ivermectin and moxidectin). However, resistance of sheep and cattle nematodes to these broad-spectrum anthelmintics is a serious issue for livestock production globally (1-3). Thus, the development of novel agents with potent anthelmintic activity is of great economic and agricultural importance.Cyclotides are circular miniproteins discovered originally in plants of the Rubiaceae, Violaceae, and Cucurbitaceae families (4, 5). Kalata B1, the first cyclotide to be structurally characterized, is abundant in the tropical African plant Oldenlandia affinis (6, 7) but has also been reported in the European Sweet Violet (Viola odorata) (8) and several other Viola species (9 -13). Members of the cyclotide family possess a cyclic peptide backbone and a cystine knot motif, which is formed by three disulfide bonds at the core of their three-dimensional structure. The sequence and structure of kalata B1 is illustrated in Fig. 1, which highlights the six backbone loops between the six conserved cysteine residues that make up the cystine knot (14). The cyclic cystine knot motif is thought to be responsible for the extraordinary enzymatic and chemical stability (15) In a recent study (30), the anthel...

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“…The amino acid sequence of HNP1 is 1 ACYCRIPACIAG-ERRYGTCIYQGRLWAFCC 30 . Machine-assisted solid phase chemical synthesis of HNP1 and some of its analogs, using the 2-(1H-benzotriazolyl)-1,1,3,3-tetramethyluronium hexafluorophosphate activation/N,N-diisopropylethylamine in situ neutralization protocol developed by Kent and co-workers for Boc chemistry (37), was previously reported (38). Most Alasubstituted analogs of HNP1 were prepared essentially as described for the wild type defensin, except for R5A-HNP1, E13A-HNP1, and F28A-HNP1.…”

Section: Methodsmentioning

confidence: 99%

Trp-26 Imparts Functional Versatility to Human α-Defensin HNP1

Wei

Pazgier

Leeuw

et al. 2010

Journal of Biological Chemistry

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We performed a comprehensive alanine scan of human ␣-defensin HNP1 and tested the ability of the resulting analogs to kill Staphylococcus aureus, inhibit anthrax lethal factor, and bind human immunodeficiency virus-1 gp120. By far, the most deleterious mutation for all of these functions was W26A. The activities lost by W26A-HNP1 were restored progressively by replacing W26 with non-coded, straight-chain aliphatic amino acids of increasing chain length. The hydrophobicity of residue 26 also correlated with the ability of the analogs to bind immobilized wild type HNP1 and to undergo further self-association. Thus, the hydrophobicity of residue 26 is not only a key determinant of the direct interactions of HNP1 with target molecules, but it also governs the ability of this peptide to form dimers and more complex quaternary structures at micromolar concentrations. Although all defensin peptides are cationic, their amphipathicity is at least as important as their positive charge in enabling them to participate in innate host defense.

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In situ neutralization in Boc‐chemistry solid phase peptide synthesis

Cited by 1,000 publications

References 26 publications

Bruce Merrifield and solid‐phase peptide synthesis: A historical assessment

Bruce Merrifield and solid‐phase peptide synthesis: A historical assessment

Lysine-scanning Mutagenesis Reveals an Amendable Face of the Cyclotide Kalata B1 for the Optimization of Nematocidal Activity

Trp-26 Imparts Functional Versatility to Human α-Defensin HNP1

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