Ion-spray tandem mass spectrometry in peptide synthesis: Structural characterization of minor by-products in the synthesis of ACP(65-74)

Ion-spray triple quadrupole mass spectrometry was used to investigate the products from the solid phase synthesis of the decapeptide (H)-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-(OH) [acyl carrier protein(65-74)]. The target sequence was assembled in stepwise fashion from the C-terminal using Boc chemistry on a Gly-OCH-Pam-copoly(styrenedivinylbenzene) resin. The product was deprotected and cleaved from the resin by treatment with HF p-cresol for 1 h at 0°C. The crude product was analyzed by reverse-phase HPLC and contained a single major peptide component, one significant minor (late-eluting) component and several trace-level peptide by-products. The components were separated by HPLC and the fractions directly analyzed by mass spectrometry and tandem mass spectrometry. The major product was confirmed as the desired ACP(65-74). The significant minor component was apparently from incomplete deprotection of Asp, an artifact of this particular experiment. The trace by-products were found to arise from succinimide formation at Asp, succinimide formation at Asn, acylation of the Tyr side chain phenolic hydroxyl leading to a branched heptadecapeptide, and tert-butylation of the decapeptide. The possible origins of these by-products are discussed in light of known peptide chemistry. Also notable was the absence, to very low detection levels, of by-products frequently reported to occur in peptide synthesis, illustrating the high degree of refinement and the accuracy of currently used synthetic methods

In situ neutralization in Boc‐chemistry solid phase peptide synthesis: Rapid, high yield assembly of difficult sequences

Simple, effective protocols have been developed for manual and machine‐assisted Boc‐chemistry solid phase peptide synthesis on polystyrene resins. These use in situ neutralization [i.e. neutralization simultaneous with coupling], high concentrations (> 0.2 M) of Boc‐amino acid‐OBt esters plus base for rapid coupling, 100% TFA for rapid Boc group removal, and a single short (30 s) DMF flow wash between deprotection/coupling and between coupling/deprotection. Single 10 min coupling times were used throughout. Overall cycle times were 15 min for manual and 19 min for machine‐assisted synthesis (75 residues per day). No racemization was detected in the base‐catalyzed coupling step. Several side reactions were studied, and eliminated. These included: pyrrolidonecarboxylic acid formation from Gln in hot TFA‐DMF; chain‐termination by reaction with excess HBTU; and, chain termination by acetylation (from HOAc in commercial Boc‐amino acids). The in situ neutralization protocols gave a significant increase in the efficiency of chain assembly, especially for “difficult” sequences arising from sequence‐dependent peptide chain aggregation in standard (neutralization prior to coupling) Boc‐chemistry SPPS protocols or in Fmoc‐chemistry SPPS. Reported syntheses include HIV‐1 protease(1–50,Cys.amide), HIV‐1 protease(53–99), and the full length HIV‐1 protease(1–99)

Kinetic properties of HIV-1 protease produced by total chemical synthesis with cysteine residues replaced by isosteric L-α-amino-n-butyric acid

Human immunodeficiency virus-1 protease, produced by total chemical synthesis with the cysteine residues replaced by L-α-amino-n-butyric acid ([Aba] HIV-1 PR), has been used extensively for the X-ray crystallographic structural analysis of the enzyme and its complexes utilized in drug design. Here we report kinetic studies on the synthetic enzyme. The pH optimum is 5.5 at ionic strengths of 0.1 and 1.0. The acid pH optimum is due to a decrease in binding affinity at higher pH values rather than to a reduction in catalytic efficiency. Activity is markedly increased by high ionic strength, although the major effect is on K and not K. The effect of pH and ionic strength on the kinetic constants determined for substrates and inhibitors is demonstrated and attention is drawn to the need for assay conditions to be explicitly reported in studies on inhibitor activity. The effect of a number of inhibitors has been measured against the synthetic enzyme and a recombinant HIV-1 PR. This work shows that [Aba] HIV-1 PR has full enzymatic activity and normal kinetic properties

Observations optiques d'etoiles jeunes enfouies dans leurs nuages moleculaires progeniteurs

SIGLEINIST T 75684 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Total chemical synthesis and biophysical characterization of the minimal isoform of the KChIP2 potassium channel regulatory subunit

The potassium channel accessory subunit KChIP2 associates with Kv4.2 channels in the cardiac myocyte and is involved in the regulation of the transient outward current (Ito) during the early phase of repolarization of the action potential. As a first step to biophysically probe the mechanism of KChIP2, we have chemically synthesized its minimal isoform, KChIP2d, using Boc chemistry solid phase peptide synthesis in conjunction with native chemical ligation. The synthetic KChIP2d protein is primarily alpha-helical as predicted and becomes more structured upon binding calcium as assessed by 1H-NMR and CD spectroscopy. Synthetic KChIP2d is in a monomer-dimer equilibrium in solution, and there is evidence for two monomer binding sites on an N-terminal peptide of Kv4.2. Planned future studies include the incorporation of fluorescent and spin labeled probes in KChIP2d to yield structural information in parallel with electrophysiologic studies to elucidate KChIP2d's mechanism of action

NMR structure of a minimized human agouti related protein prepared by total chemical synthesis

AbstractThe structure of the chemically synthesized C-terminal region of the human agouti related protein (AGRP) was determined by 2D 1H NMR. Referred to as minimized agouti related protein, MARP is a 46 residue polypeptide containing 10 Cys residues involved in five disulfide bonds that retains the biological activity of full length AGRP. AGRP is a mammalian signaling molecule, involved in weight homeostasis, that causes adult onset obesity when overexpressed in mice. AGRP was originally identified by homology to the agouti protein, another potent signaling molecule involved in obesity disorders in mice. While AGRP’s exact mechanism of action is unknown, it has been identified as a competitive antagonist of melanocortin receptors 3 and 4 (MC3r, MC4r), and MC4r in particular is implicated in the hypothalamic control of feeding behavior. Full length agouti and AGRP are only 25% homologous, however, their active C-terminal regions are ∼40% homologous, with nine out of the 10 Cys residues spatially conserved. Until now, 3D structures have not been available for either agouti, AGRP or their C-terminal regions. The NMR structure of MARP reported here can be characterized as three major loops, with four of the five disulfide bridges at the base of the structure. Though its fold is well defined, no canonical secondary structure is identified. While previously reported structural models of the C-terminal region of AGRP were attempted based on Cys homology between AGRP and certain toxin proteins, we find that Cys spacing is not sufficient to correctly determine the 3D fold of the molecule