Side reactions in peptide synthesis. 11. Possible removal of the 9-fluorenylmethyloxycarbonyl group by the amino components during coupling

Bodanszky, Miklos; Deshmane, Sharad S.; Martinez, Jean

doi:10.1021/jo01324a008

Cited by 50 publications

(31 citation statements)

References 3 publications

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“…2). 1,5 While both Tsc and Fmoc were completely cleaved within 5 min at room temperature by treatment with 20% (v/v) piperidine in DMF, their cleavage by 0.5 M DIEA in DMF proceeded much slower. Intriguingly, moreover, the difference in their deprotection rates is clearly significant.…”

Section: Chemical and Thermal Stability Of Tsc And Fmoc In The Presenmentioning

confidence: 99%

Orthogonality and compatibility between Tsc and Fmoc amino-protecting groups

et al. 2005

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Section: Chemical and Thermal Stability Of Tsc And Fmoc In The Presenmentioning

confidence: 99%

Orthogonality and compatibility between Tsc and Fmoc amino-protecting groups

et al. 2005

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“…1A, where the peptide was labeled following cleavage from the resin. Care was taken to ensure the peptide reaction with the spin label was at pH 7.5, because the Fmoc group protecting the terminal amine is readily removed under moderately basic conditions (23,24). Because the pK a of the ⑀-amino group is 10.55, the reaction is not very efficient, resulting in a yield of 8% (Fig.…”

Section: Resultsmentioning

confidence: 99%

Probing the Binding Pocket and Endocytosis of a G Protein-coupled Receptor in Live Cells Reported by a Spin-labeled Substance P Agonist

Shafer

Bennett

Kim³

et al. 2003

Journal of Biological Chemistry

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To probe the molecular nature of the binding pocket of a G protein-coupled receptor and the events immediately following the binding and activation, we have modified the substance P peptide, a potent agonist for the neurokinin-1 receptor, with a nitroxide spin probe specifically attached at Lys-3. The agonist properties and binding affinity of the spin-labeled substance P are similar to the native peptide. Using electron paramagnetic resonance (EPR) spectroscopy, the substance P analogue is capable of reporting the microenvironment found in the binding pocket of the receptor. The EPR spectrum of bound peptide indicates that the Lys-3 portion of the agonist is highly flexible. In addition, we detect a slight increase in the mobility of the bound peptide in the presence of a non-hydrolyzable analogue of GTP, indicative of the alternate conformational states described for this class of receptor. The down-regulation of neurokinin-tachykinin receptors is accomplished by a rapid internalization of the activated protein. Thus, it was also of interest to establish whether spin-labeled substance P could serve as a real time reporter for endocytosis. Our findings show the receptor agonist is efficiently endocytosed and the loss of EPR signal upon internalization provides a real time monitor of endocytosis. The rapid loss of signal suggests that endosomal trafficking vesicles maintain a reductive environment. Whereas the reductive capacity of the lysosome has been established, our findings indicate this capacity in early endosomes as well.The neurokinin-1 receptor (Nk1r) 1 is a neuropeptide receptor belonging to the G protein-coupled receptor superfamily (1). The receptor, also designated the substance P or tachykinin 1 receptor, has been implicated in numerous physiological functions, which include modulatory roles in the central nervous system and proinflammatory action (for review see Refs. 2 and 3). Although much debate still exists as to which neurokinin represents the physiological ligand for the receptor, the substance P neurokinin has been identified as the primary ligand (4).Because of the significant pathophysiological role of Nk1r, extensive pharmacological research has been carried out to characterize both agonist and antagonist binding properties of the receptor. Previously, extensive mutagenesis of the receptor identified separate binding sites for substance P and the antagonist CP-96,345 (5-9). These studies indicated that substance P interacts almost exclusively with the NH 2 -terminal extracellular residues, with little involvement by residues located in the membrane-spanning domain. The same studies revealed that the antagonist appeared to bind deeply into the protein, within a pocket formed by the transmembrane helices.Although site-directed mutagenesis identified residues important for ligand binding, it cannot differentiate side chains that have a direct coordination of the ligands from residues involved indirectly, such as through allosteric communication. For example, a Y216A substitution located nea...

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“…Spontaneous cleavage of Fmoc has also been observed [416,417]. A detrimental feature is the favored formation of b-sheets and the propensity for diketopiperazine formation in the dipeptide stage.…”

Section: Side Reactionsmentioning

confidence: 98%

“…This usually leads to prolonged reaction times, and either an increase in the reaction temperature or solvent changes often become necessary; this in turn may favor racemization [256,415] and premature Fmoc cleavage [416,417]. This usually leads to prolonged reaction times, and either an increase in the reaction temperature or solvent changes often become necessary; this in turn may favor racemization [256,415] and premature Fmoc cleavage [416,417].…”

Section: Difficult Sequencesmentioning

confidence: 99%