5944 2.4. Other Attachment Methods 5945 2.4.1. Sonogashira Coupling 5945 2.4.2. Other Methods for Labeling the C-Terminus 5946 2.4.3. Other Methods for Labeling the N-Terminus 5947 2.4.4. Labels for the Thiol Group in Cysteine 5947 2.4.5. Miscellaneous 5947 2.5. Application of Ferrocene Compounds in Peptide Synthesis 5948 2.5.1. The Ferrocenylmethyl (Fem) Group as a Protecting Group 5948 2.5.2. Peptide Synthesis via Four-Component Reactions with Ferrocene Alkylamines 5949 3. Conjugates of Ferrocene with Proteins 5950 3.1. Redox Proteins 5950 3.1.1. Glucose Oxidase (GOD) 5950 3.1.2. D-Amino Acid Oxidase (DAAO) 5953 3.1.3. Cytochrome P450 cam 5953 3.1.4.
Rapidly growing cells show an increased demand for nutrients and vitamins. The objective of our work is to exploit the supply route of vitamin B12 to deliver new derivatives of this vital vitamin to hyperproliferative cells. To date, radiolabeled ( 57 Co and 111 In) vitamin B12 derivatives showed labeling of tumor tissue but also undesired high accumulation of radioactivity in normal tissue. By abolishing the interaction of a tailored vitamin B12 derivative to its transport protein transcobalamin II and therefore interrupting transcobalamin II receptor and megalin mediated uptake in normal tissue, preferential accumulation of a radiolabeled vitamin in cancer tissue could be accomplished. We identified transcobalamin I on tumors as a possible new receptor for this preferential accumulation of vitamin-mediated targeting. The low systemic distribution of radioactivity and the high tumor to blood ratio opens the possibility of a more successful clinical application of vitamin B12 for imaging or therapy.
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.
S‐Alkylated cysteines are used as efficient tridentate N,O,S‐donor‐atom ligands for the fac‐[M(CO)3]+ moiety (M=99mTc or Re). Reaction of (Et4N)2[ReBr3(CO)3] (3) with the model S‐benzyl‐L‐cysteine (2) leads to the formation of [Re(2′)(CO)3] (4) as the exclusive product (2′=C‐terminal anion of 2). The tridentate nature of the alkylated cysteine in 4 was established by X‐ray crystallography. Compound 2 reacts with [99mTc(OH2)3(CO)3]+ under mild conditions (10−4 M, 50°, 30 min) to afford [99mTc(2′)(CO)3] (5) and represents, therefore, an efficient chelator for the labelling of biomolecules. L‐Cysteine, S‐alkylated with a 3‐aminopropyl group (→7), was conjugated via a peptide coupling sequence with Coα‐[α‐(5,6‐dimethyl‐1H‐benzimidazolyl)]‐Coβ‐cyanocobamic b‐acid (6), the b‐acid of cyanocob(III)alamin (vitamin B12) (Scheme 3). More convenient was a one‐pot procedure with a derivative of vitamin B12 comprising a free amine group at the b‐position. This amine 15 was treated with NHS (N‐hydroxysuccinimide)‐activated 1‐iodoacetic acid 14 to introduce an I‐substituent in vitamin B12. Subsequent addition of unprotected L‐cysteine resulted in nucleophilic displacement of the I‐atom by the S‐substituent, affording the vitamin B12 alkylated cysteine fragment 17 (Scheme 4). The procedure was quantitative and did not require purification of intermediates. Both cobalamin–cysteine conjugates could be efficiently labelled with [99mTc(OH2)3(CO)3]+ (1) under conditions identical to those of the model complex 5. Biodistribution studies of the cobalamin conjugates in mice bearing B10‐F16 melanoma tumors showed a tumor uptake of 8.1±0.6% and 4.4±0.5% injected dose per gram of tumor tissue after 4 h and 24 h, respectively (Table 1).
Square-wave voltammograms of the complex Mo(His-N e -C 2 H 4 CO 2 Me)(h-allyl)(CO) 2 (His = N d ,N,O-L-histidinate) 2 recorded at variable temperatures establish the existence of two isomers in solution in both the reduced and the oxidized paramagnetic form of 2; a complete analysis yields thermodynamic parameters such as equilibrium constants, DG and DH for all species.
The acid hydrolysis of natural vitamin B 12 yields several products in which the acetamide or propionamide side chains on the corrin framework are converted into the corresponding acids. These acids can be derivatised with further functionalities. We have separated in particular the b-and the dacid derivatives 1 and 2, respectively, since functionalisation at these positions of the corrin ring generally keeps the affinity for vitamin B 12 transport proteins intact. Although the authenticity of 1 and 2 seemed evident from 1 H NMR investigations, it has not been supported by X-ray structure analysis. The coupling of ethyl N-(3-aminopropyl)-N-(pyridin-2-ylmethyl)glycinate (3) to the carboxylate groups in 1 and 2 by
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