The new histidine derivative 3-[1-[3-(9H-fluoren-9-ylmethoxycarbonylamino)-propyl]-1H-imidazol-4-yl]-2-(3-trimethylsilanyl-ethylcarboxyamino)-propionic acid methyl ester (7) has been prepared via alkylation of the histidine urea derivative (7S)-5,6,7,8-tetrahydro-7-(methoxycarbonyl)-5-oxoimidazo-[1,5-c]-pyrimidine (2) with Fmoc-protected 3-iodopropyl-amine, followed by ring opening with 2-trimethylsilylethanol. After Fmoc cleavage by HNEt2, the histidine amine derivative was coupled to biotin, to the pentapeptide leucine-enkephalin and to Vitamin B12-b-acid by amide formation, employing TBTU as the coupling reagent. In order to make the histidine accessible for labelling, the teoc protecting group was removed by either NBu4F (for the biotin conjugate) or by TFA (for the enkephalin and B12 conjugates). Reaction of a 10(-4) M solution of the bioconjugates with [99mTc(H2O)3(CO)3]+ at 50 degrees C for 30 min led to the formation of one single new peak in the HPLC radiochromatogram in each case, confirming quantitative labelling of the respective biomolecules. To assess the nature of the labelled compounds, the rhenium analogues with Re(CO)3 were also synthesised and similar retention times confirmed the identity with the 99mTc labelled conjugates.
Molecular clusters with archimedean and platonic shape, which in order to build a closed polyhedron, spontaneously eliminate H(2) or voluntarily encapsulate Li(2)O as a "cluster nucleus", result from the dilithiation of primary silylphosphanes and silylarsanes with BuLi. Thus, the first mixed-valent, decameric P(10)Li(16) cluster 1 was obtained from iPr(3)SiPH(2) and tBuLi (molar ratio 1:2) with strict exclusion of LiOH and Li(2)O, whereas partial metalation in the presence of LiOH initially leads to a dodecameric, Li(2)O-containing cluster 2, from which the three-shell cluster 3 with a [Li(6)O](4+) core is obtained.
The Pd-catalyzed Sonogashira coupling of ferrocene alkyne derivatives as metal probes to iodophenylalanine containing peptides is described. 4-Iodophenylalanine was incorporated into dipeptides and the neuropeptide [Leu5]-enkephalin (Enk) by solid phase peptide synthesis, thereby creating a functional group suitable for the Sonogashira coupling. The reaction with two different ferrocene alkynes resulted in the corresponding ferrocene-labeled derivatives, which were obtained in good yield and purity. All new compounds were comprehensively characterized, including elemental analysis, 1D and 2D NMR, EI-, FAB- or ESI-MS, IR and UV-vis spectroscopy, and electrochemistry of the ferrocene label. Unlike well-established conjugation methods for peptide side chains such as lysine and cystein, the phenyl group in Phe is not readily available for derivatization. This work presents a versatile procedure for the regioselective introduction of an organometallic label into biologically relevant peptides as exemplified for enkephalin.
Enkephalin peptides are thought to be suitable vectors for the passage of the blood-brain barrier (BBB). Modifications that do not alter the amino acid sequence are often used to improve the permeation through living membrane systems. As a new type of modification we introduce organometallic compounds, in particular ferrocene carboxylic acid. Derivatives of [Leu5]enkephalin were synthesised and labelled with organometallic compounds by using solid-phase synthesis techniques. All new metal-peptide bioconjugates were comprehensively characterised by HPLC, NMR spectroscopy and mass spectrometry and found to be at least 95% pure. For the first time, permeation coefficients in a BBB model for organometal-peptide derivatives were determined in this work. The uptake and localisation of fluorescein-labelled enkephalins was monitored by fluorescence microscopy on three cancer cell lines. Octanol/H2O partition coefficients of the compounds were measured by HPLC. The introduction of the organometallic moiety enhances the uptake into cells and the permeation coefficient of [Leu5]-enkephalin. This could be due to an increase in lipophilicity caused by the organometallic label. The metal-peptide conjugates were found to be nontoxic up to mM concentrations. The low cytotoxicity encourages further experiments that could take advantage of the selectivity of enkephalin derivatives for opioid receptors.
The synthesis and full characterization of a series of (triphenylphosphine)gold complexes with thiol-containing amino acids and peptides is reported. Boc-Cys-Au(PPh3) was prepared in solution by reaction of Boc-Cys-OH with (Ph3P)AuCl. The related Ac-Cys-Au(PPh3) and an Au derivative of the cysteine-modified neuropeptide enkephalin (Enk), [Cys]5-Au(PPh3)-Enk, were prepared on the resin, using the orthogonal trityl-protecting group on Cys. For comparison, the metal-free [Cys]5-Enk and an S-tert-butyl-protected [Cys]5-Enk were also prepared. Most noteworthy, gold complexation works best when carried out on the resin, and the gold thiolate survives cleavage from the resin under optimized conditions. The new conjugates were comprehensively characterized, including 1D and 2D NMR spectroscopy, and mass spectrometry. Along with characteristic changes in the 1H and 13C NMR spectra, 31P NMR spectra reveal a characteristic downfield shift of ca. 5 ppm upon complexation of the Au(PPh3)-fragment to Cys, which is also pertinent in the Au-labelled [Cys]5-Enk.
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