Biologically active peptides labeled with

It is demonstrated that polymorphs of the crystalline biologically-active peptides, Leu- and Met-enkephalins, are also easily distinguished by NMR on the basis of displacements of chemical shifts [80, 81]. Recently, we showed that high resolution solid-state NMR approach is a very useful mean to delineate local conformational change from a-helix to j8-sheet associated with fibril formation of selectively C-labeled human calcitonin [82]. In addition, we also demonstrated that chemical shift data were very conveniently used as an initial constraints to construct three-dimensional structures of Leu-enkephalin, based on accurately determined interatomic distances by REDOR [6, 83]. This aspect of work was already described in Chapter 2. 

Heterofunctionalized phosphines, with different denticities and/or donor sets, were successfully applied, at macroscopic and noncarrier added level, to the stabilization of the [M=0]3+, trans-[M02]+, [M=N]2+ and/ac-[M(CO)3]+ cores. The unique electronic and/or stereochemical properties of the tridentate PNS phosphine allowed the synthesis of 3+1 mixed-ligand complexes with an unprecedented in vivo robustness. Phosphines as bifunctional chelate ligands have been successfully applied for labeling different biologically active substrates, namely peptides and CNS-receptor avid molecules. 

In mitochondrial research the phosphate-transporting protein from rat liver mitochondria has been labeled with ° Hg-mersalyl At For protein labeling with astatine (alpha emitter) the following procedures may be u reaction of the protein with p-astatobenzoic acid < ndensation reaction with peptide bond and protein acetylation While labeling by the above procedures seems to be sufficiently stable a remarkable instability of the At-label was obserwd when astatinated protein was prepared electrophoretically 202) jjjg results of these authors indicate that the tyrosine-astatine bond is unstable. The conclusion of Vau an et al. that astatinated proteins lore as much as 50% of their biological activity and, in addition, are extremely toxic, is very serious. 

Conjugation reagents bind to protein functional groups NHj-, SH-, OH-, phenol, etc. Reagents which react with the amino groups are usually preferred. The utilization of SH-groups could be risky because they can be included in the catalytic site of an enzyme or participate in some other way in the biological activity of protein to be labeled protein cannot occur. The presence of a radiolabel outside of the peptide active site. 

Gel filtration in its various forms is an efficient tool for preparations in protein chemistry. It can also be used to test the supposed homogeneity of a protein or peptide preparation. A gel column calibrated with respect to its molecular-sieving properties may also be used to estimate the molecular size of biologically active or otherwise labeled substances before their isolation has been accomplished. Needless to say, the result of such an estimation must be cautiously interpreted. 

Bombesin was derivatized at the C terminus with bidentate chelators (Smith et al. 2003 a, b). The labeled peptide fully retained the biological activity and was stable in vitro and in vivo. Since the bidentate coordination is not optimal with respect to stability (pharmacokinetics), the coordination sphere of the metal tricarbonyl core has been saturated with a highly hydrophilic phosphine. This additional coordination is an example of the 2-Hl approach mentioned in the previous section. The mixed-ligand approach resulted in significantly higher hydrophilicity of the radioconjugates and an improved biodistribution labeled with Tc-99m and also with Re-188 (Smith et al. 2003). 

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