Anti-glycophorin single-chain Fv fusion to low-affinity mutant erythropoietin improves red blood cell-lineage specificity.

The presence of erythropoietin (Epo) receptors on cells besides red blood cell precursors, such as cancer cells or megakaryocyte precursors, can lead to side effects during Epo therapy including enhanced tumor growth and platelet production. It would be ideal if the action of Epo could be limited to erythroid precursors. To address this issue, we constructed single-chain variable fragment (scFv)-Epo fusion proteins that used the anti-glycophorin 10F7 scFv amino-terminal to Epo analogues that would have minimal activity alone.

Enhancement of cell type specificity by quantitative modulation of a chimeric ligand.

Evolution modulates the quantitative characteristics of protein interactions and often uses combinations of weak interactions to achieve a particular specificity. We addressed how quantitative optimization might be used in the design of multidomain proteins, using a chimera containing epidermal growth factor (EGF) as a cell targeting element and interferon-alpha-2a (IFNalpha-2a) to initiate signal transduction. We first connected EGF and IFNalpha-2a via a linker that allows both ligands to bind to their receptors on a cell surface and then incorporated a series of mutations into the IFNalpha-2a portion that progressively decrease both the on rate and the dissociation constant of the IFNalpha-2a-IFNalpha receptor 2 (IFNAR2) interaction.

Genetically encoded short peptide tags for orthogonal protein labeling by Sfp and AcpS phosphopantetheinyl transferases.

Short peptide tags S6 and A1, each 12 residues in length, were identified from a phage-displayed peptide library as efficient substrates for site-specific protein labeling catalyzed by Sfp and AcpS phosphopantetheinyl transferases (PPTases), respectively. S6 and A1 tags were selected for useful levels of orthogonality in reactivities with the PPTases: the catalytic efficiency, kcat/Km of Sfp-catalyzed S6 serine phosphopantetheinylation was 442-fold greater than that for AcpS. Conversely, the kcat/Km of AcpS-catalyzed A1 labeling was 30-fold higher than that for Sfp-catalyzed A1 labeling.

Solid-phase chemistry in the total synthesis of non-peptidic natural products.

Solid-phase chemistry, first introduced for peptide synthesis in the 1960's, has become an integral part of organic synthetic methodology. Presented herein is an overview of recent examples of the use of solid-phase in the preparation of non-peptidic natural products and related compounds, encompassing on-resin total syntheses as well as the use of polymer-supported reagents in solution.

Solid-phase syntheses of furopyridine and furoquinoline systems.

[reaction: see text] Syntheses of 2-substituted furo[3,2-b]pyridines and furo[3,2-h]quinolines have been achieved for the first time in the solid-phase mode. The central enabling steps involved concomitant deprotection/cyclization promoted by the mild base K(2)CO(3). Reactions were monitored "in situ" in real time by a variety of spectroscopic techniques, which allowed full and accurate control of progress in these syntheses.

1H NMR spectroscopy with internal and external standards for the quantification of libraries.

A convenient and easy method based on 1H NMR spectroscopy with both external and internal standards is described for the quantification of members of libraries.

Solid-phase total synthesis of the pentacyclic system lamellarins U and L.

[reaction: see text] A total solid-phase synthesis of lamellarins U and L has been achieved. The conversion of an aldehyde group into a formate by a Baeyer-Villiger reaction and a intramolecular [3 + 2] cycloaddition of a 3,4-dihydroisoquinolinium salt over a triple bond comprise the key steps of the process. Each transformation has been controlled with the proper spectroscopic and analytical methods.