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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. Using this strategy, we designed chimeric proteins in which the activation of the IFNalpha receptor in HeLa, A431, and engineered Daudi cells depends on the presence of EGF receptor on the same cell. The mutant chimeric proteins also inhibited proliferation of IFNalpha-sensitive cells in an EGF receptor-dependent manner. These results provide insights into the quantitative requirements for specific binding to multisubunit receptors and illustrate the value of a quantitative approach in the design of synthetic-biological constructs.

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. S6 and A1 peptide tags can be fused to N- or C-termini of proteins for orthogonal labeling of target proteins in cell lysates or on live cell surfaces. The development of the orthogonal S6 and A1 tags represents a significant enhancement of PPTase-catalyzed protein labeling, allowing tandem or iterative covalent attachment of small molecules of diverse structures to the target proteins with high efficiency and specificity.

An effective solid-phase preparation of the pharmaceutically interesting 4 H -2-(3-hydroxy-4-methoxyphenyl)-naphtho[1,2- b ]pyran-1-one system from an anchored bisarylacetylene is described.

[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.

The efficacy, limitations and scope of different spectroscopic techniques to monitor solid-phase syntheses are discussed. The total solid-phase synthesis of a natural product derivative, 3,17-di-O-acetyllamellarin U, has been monitored by gel-phase NMR and HR-MAS NMR as appropriate. FT-IR and colorimetric tests also proved to be valuable tools for monitoring the progress of reactions. While the data obtained by FT-IR provided valuable information on some functional groups, the NMR data provided more specific structural information that allowed the progress of reactions to be monitored. After several reaction steps the "aging" of the resin only allowed the product covalently anchored to the resin to be investigated using an HR-MAS probe, a situation that severely limited the depth of analysis available.

[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.

3,4-Di-O-benzyl-1-O-methyl-L-galactitol (3) has been synthesized in a seven step sequence starting from 1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranose (4). The synthesis involves the methyllation of HO-6 of 4, followed by methanolysis to the mixture of the corresponding methyl 6-O-methyl-alpha-D-galactopyranoside (6, major product) and the beta-furanoside analog (8). Compound 6 was converted into the 3,4-O-isopropylidene derivative 9, and the free HO-group was protected as the methoxyethoxymethyl (MEM) ether. Chemoselective removal of the acetonide by hydrolysis, followed by benzylation gave the methyl 3,4-di-O-benzyl-2-O-methoxyethoxymethyl-6- O-methyl-alpha-D-galactopyranoside (12). Simultaneous acid hydrolysis of the methyl glycoside and MEM group of 12 led to 13, which was then reduced with sodium borohydride to the target molecule 3. 3,4-Di-O-benzil-1-O-metil-L-galactitol (3) foi sintetizado em 7 etapas a partir de 1,2:3,4-di-O-isopropilideno-alfa-D-galactopiranose (4). A síntese envolve a metilação do HO-6 de 4, seguida de metanólise para a mistura do 6-O-metil-alfa-D-galactopiranosídeo de metila (6, produto principal) e do análogo beta-furanosídico 8. O composto 6 foi convertido no derivado 3,4-O-isopropilidênico 9 e o grupo OH livre foi protegido na forma do éter metoxietoximetílico (MEM). A remoção quimiosseletiva do acetonídeo por hidrólise, seguida de benzilação forneceu o 3,4-di-O-benzil-2-O-metoxietoximetil-6-O-metil-alfa-D-galactopiranosídeo de metila (12). A hidrólise ácida simultânea do glicosídeo metílico e do grupo MEM de 12 levou a 13 que foi então reduzido com boroidreto de sódio à molécula alvo 3.

The title compound, C11H13N2O5, was obtained during the preparation of 3,4-dihydro-6,7-dimeth­oxy-8-nitro­isoquinoline from 3,4-dihydro-6,7-dimethoxy­isoquinoline using NO2BF4 as a source of NO2+. 1H/13C NMR and X-ray analysis confirm the presence of a hydr­oxy group at position 1. The structure consists of layers of mol­ecules parallel to the (12\overline{1}) plane.

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. We introduced the Epo mutations N147A, R150A and R150E, which progressively weakened receptor affinity in the context of Epo alone, as defined by cell proliferation assays using TF-1 or UT-7 cells. Fusion of these mutant proteins to the 10F7 scFv significantly rescued the activity of the mutant proteins, but had a relatively small effect on wild-type Epo. For example, fusion to the 10F7 scFv enhanced the activity of Epo(R150A) by 10- to 27-fold, while a corresponding fusion to wild-type Epo enhanced its activity only up to 2.7-fold. When glycophorin was blocked by antibody competition or reduced by siRNA-mediated inhibition of expression, the activity of 10F7 scFv-Epo(R150A) was correspondingly reduced, while such inhibition had essentially no effect on the activity of 10F7 scFv-Epo(wild-type). In addition, potent stimulation of Epo receptors by 10F7 scFv-Epo(R150A) was observed in long-term proliferation and viability assays. Taken together, these results indicate that a combination of targeting and affinity modulation can be used to engineer forms of Epo with enhanced cell-type specificity.

Publisher Summary This chapter provides an overview on lamellarins—an important group of natural products isolated from marine invertebrates, such as sponges, mollusks, and tunicates. Lamellarins are a family of sea natural products with a broad therapeutic profile and are characterized for possessing important biological activities. More than 30 lamellarins have been characterized since the isolation of the first group of lamellarins. Several of lamellarins are in advanced pre-clinical phase testing for the treatment of different tumors. Syntheses of this family of molecules have been possible only by the use of the most modem synthetic protocols, mainly cross-coupling reagents, and cycloadditions. This chapter also discusses the structure of lamellarin.

The synthetic routes used for the preparation of marine alkaloids variolin B and lamellarins are described.

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 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.