Biotin model studies

The elegant, enantiospecific synthesis of biotin (1) by Hoffmann-La Roche1 is based on a strategy that takes advantage of the powerful intramolecular nitrone-olefin cycloaddition reaction. Our analysis begins with model studies in which the straightforward conversion of L-cysteine (2) into aldehyde 3 (see Scheme 1) constitutes... 

Scheme 1. Model studies for the total synthesis of biotin (1).

Another possibility within this class of mechanism involves the initial reaction of biotin with ATP, forming ADP and a phosphorylated biotin species. It has been proposed, based on model studies, that such a species would be O-phosphobiotin (52,53). This reacts with bicarbonate to produce iV-carboxybiotin and inorganic phosphate (Scheme 20). The transfer of oxygen from bicarbonate occurs in the second step in this case. Models for the 0-phosphorylation of biotin suggest that such a process can occur readily. 

Both the AFM rupture experiments as well as our simulation studies focussed on the streptavidin-biotin complex as a model system for specific ligand binding. Streptavidin is a particularly well-studied protein and binds its ligand biotin with high affinity and specificity [51]. Whereas previous experiments (see references in Ref. [49]) and simulation studies [52] referred only to bound/unbound states and the associated kinetics, the recent AFM... 

Recently, SETA BioMedicals has developed a new near-infrared squaraine-based label Seta-633, which can be used to study the interaction between low-molecular-weight analytes and proteins using fluorescence lifetime as the readout parameter [19]. This label exhibits lower quantum yields and shorter fluorescence lifetimes when free in solution, but these values substantially increase upon interaction with proteins, which is contrary to tracers like Cy5 or Alexa 647. It was demonstrated in a model assay that a biotinylated Seta-633 binds to anti-biotin with high specificity. Importantly, the lifetime of Seta-633-biotin increases about 2.76 fold upon binding to a specific antibody (anti-biotin, MW =160 kDa), while the titration with BSA or nonspecific antibody does not result in a noticeable change in lifetime (Fig. 13). The label is compatible with readily available light sources (635 nm or 640 nm lasers) and filter sets (as for Cy5 or Alexa 647) and its... 

An elegant study was presented by Thomas et al. who employed selective solid-phase extraction by immunoaffinity CEC (IACEC) to enhance detection limits. A model compound, fluorescein isothiocyanate biotin, was electrokinetically applied to a capillary column packed with an immunoaffinity stationary phase. The analyte was first selectively bound to the stationary phase, then eluted, migrated by zone electrophoresis, and detected by LIF [79],... 

So in a sense, these experiments suggest that Uposomes, as they were used here, are not a siutable model system for systematic studies on cell-surface interactions by QCM, mostly due to the unavoidable rupture of the Uposome when surface attraction becomes too strong. Nevertheless, for intermediate biotin concentrations these experiments did provide important information since they indicate that an aqueous compartment surrounded... 

The formation of A -carboxybiotin from bicarbonate and biotin on an enzyme involves the stoichiometric hydrolysis of ATP to produce ADP and inorganic phosphate (Scheme 17). This is particularly significant because the hydrolysis of ATP is coupled to formation of the carbon-carbon bond (45, 46) but is not explicitly involved in the apparent stoichiometry of the biosynthetic process. Therefore, the function of ATP is cryptic, as it is in most processes in which ATP hydrolysis accompanies a biosynthetic process. Although biotin-dependent reactions are not a general model for other ATP-dependent processes, the patterns that emerge from the study of such a mechanism guides one in thinking about the other processes. In the biotin-dependent case, the reaction with ATP... 

Further support for this model comes from experiments with the surface forces apparatus. Here, two functionalized, curved mica sheets (in a geometry of crossed cylinders) are approaching each other, and the mutual force is measured as a function of the surface separation down into the Angstrom-range. Three basic architectures were studied as schematically sketched in Fig. 8 (a) is the biotin-biotin case which... 

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