ABSTRACT
A series of self-immolative linkers containing a thiol-reactive group at one end and a hydroxyl- or amine-reactive group at the other were prepared. The utility of these reagents for preparations of bioconjugates was explored by reacting the linkers with appropriately functionalized model drugs and peptides. Degradation studies of a series of conjugates with different linkers reveal that the structure of the linkers has a significant impact on their stability.
Subject(s)
Sulfhydryl Compounds/chemistry , Amines/chemistry , Peptides/chemistry , Pharmaceutical Preparations/chemistryABSTRACT
Within the field of bioprospecting, disulfide-rich peptides are a promising group of compounds that has the potential to produce important leads for new pharmaceuticals. The disulfide bridges stabilize the tertiary structure of the peptides and often make them superior drug candidates to linear peptides. However, determination of disulfide connectivity in peptides with many disulfide bridges has proven to be laborious and general methods are lacking. This study presents a general approach for structure elucidation of disulfide-rich peptides. The method features sequential reduction and alkylation of a peptide on solid phase combined with sequencing of the fully alkylated peptide by tandem mass spectrometry. Subsequently, the disulfide connectivity is assigned on the basis of the determined alkylation pattern. The presented method is especially suitable for peptides that are prone to disulfide scrambling or are unstable in solution with partly reduced bridges. Additionally, the use of small amounts of peptide in the lowest nmol range makes the method ideal for structure elucidation of unknown peptides from the bioprospecting process. This study successfully demonstrates the new method for seven different peptides with two to four disulfide bridges. Two peptides with previous contradicting publications, µ-conotoxin KIIA and hepcidin-25, are included, and their disulfide connectivity is confirmed in accordance with the latest published results.
Subject(s)
Disulfides/chemistry , Peptides/chemistry , Sequence Analysis, Protein/methods , Tandem Mass Spectrometry/methods , Alkylation , Amino Acid Sequence , Oxidation-ReductionABSTRACT
Beta-acyloxy alpha,beta-unsaturated acetylenic ketones have been shown to react with organocuprate reagents and undergo cyclization followed by dehydration to give substituted furans as the final products. The transformation appeared to be versatile, and tri- and tetra-substituted furans were obtained with regiochemical control in moderate to good yields. The best yields were generally obtained when the reactions were performed around -60 degrees C with substrates and cuprates containing sterically demanding substituents. The proposed mechanism for furan formation has been supported by experiments.
ABSTRACT
The molecular aspects and physiological significance of NADP(+)-dependent D-arabinose dehydrogenase (ARA), which is thought to function in the biosynthesis of an analog of ascorbic acid, D-erythroascorbic acid in yeasts, were examined. A large subunit of ARA, Ara1p produced in E. coli, was purified as a homodimer, some of which was degraded at the N-terminus. It showed sufficient ARA activity. Degradation of Ara1p occurs naturally in yeast cells, and the small subunit of ARA previously thought as is, in fact, a naturally occuring degradation product of Ara1p. A deficient mutant of ARA1 lost almost all NADP(+)-ARA activity, but intracellular D-erythroascorbic acid was only halved. This mutant showed increased susceptibility to H(2)O(2) and diamide but not to menadione or tert-butylhydroperoxide. Feeding D-arabinose to mutant cells led to increases in intracellular D-erythroascorbic acid, suggesting the presence of another ARA isozyme. The deficient mutant of ARA1 recovered resistance to H(2)O(2) with feeding of D-arabinose. Our results suggest that the direct contributions of Ara1p both to D-erythroascorbic acid biosynthesis and to oxidative stress resistance are quite limited.
