Stefan Mundwiler

New Derivatives of Vitamin B12 Show Preferential Targeting of Tumors

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.

Mono-, bi-, or tridentate ligands? The labeling of peptides with 99mTc-carbonyls†

The labeling of targeting peptides with 99mTc is a useful concept for the diagnosis of various diseases such as cancer. Although in research for at least one decade, only a very few radiopharmaceuticals based on peptides are in clinical use. The difficulty of labeling, and the resulting authenticity of the new vector, is largely responsible for this observation. In this overview, we present an alternate strategy based on the organometallic fac‐[99mTc(CO)3]+ core for introducing 99mTc in biomolecules in general and in peptides in particular. The three coordination sites available in [99mTc(OH2)3(CO)3]+ can be occupied with many different ligand types, pendant to a biomolecule and serving as the anchor group for labeling. This makes the appropriate choice difficult. We intend to present some useful concepts for the practice. Monodentate chelators are robust but bear the risk of multiple binding of biomolecules. Coordinating a bidentate ligand of choice prior to labeling bypasses this problem and enables a systematic drug discovery by variation of the bidentate ligand. Bidentate ligands attached to the biomolecule are stronger but occasionally require protection of the remaining site by a monodentate ligand. Both approaches refer to a mixed‐ligand [2+1] approach. Tridentate chelators are the most efficient but need some protecting group chemistry in order to achieve selectivity for the coupling process. Examples with cysteine and histidine are presented. This article aims to provide versatile and reproducible approaches for the labeling of biomolecules while not focusing on particular systems. It should be left to the readers to derive a strategy for their own peptide.

Conjugation of a novel histidine derivative to biomolecules and labelling with [99mTc(OH2)3(CO)3]+

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.

Structure, Stability, and Biodistribution of Cationic [M(CO)3]+ (M = Re, 99Tc, 99mTc) Complexes with Tridentate Amine Ligands

Bifunctional chelating molecules linking the fac‐[99mTc(CO)3]+ core with targeting biomolecules are required for the development of specific diagnostic radiopharmaceuticals. Diethylenetriamine (1) and N‐(pyridin‐2‐ylmethyl)ethane‐1,2‐diamine (2) both react readily with [99mTc(H2O)3(CO)3]+ in 0.9% saline at micromolar concentrations to form the cationic complexes [99mTc(1)(CO)3]+ (5) and [99mTc(2)(CO)3]+ (6) in quantitative yields. The crystal structures of the corresponding Re or 99Tc complexes were determined and exhibit in particular the small size of 5. Challenging both 99mTc complexes 5 and 6 with a 104 excess of histidine or cysteine showed no decomposition or ligand exchange after 24 hours and both compounds were also stable against reoxidation to [99mTcO4]−. In normal mice, complex 5 revealed a good and fast clearance from the blood, and most organs. Only limited accumulation in the large intestine was visible after 4 hours. Complex 6 was also excreted relatively quickly from the blood but retention was observed in some tissues after 4 h. In order to illustrate the potential of both ligands to be further functionalized, two derivatives containing potentially DNA binding functionalities, N‐(2‐Amino‐ethyl)‐N′‐pyren‐1‐ylmethyl‐ethane‐1,2‐diamine (3) and N‐(quinolin‐2‐ylmethyl)ethane‐1,2‐diamine (4) were synthesized. The respective Re or 99Tc complexes were fully characterized. Based on these results, it appears that functionalization of biomolecules with acyclic triamine ligands is biologically relevant. Complex 5 in particular could be used to mimic a terminal amino group in, e.g., a peptide due to its small size and positive charge. We thank Mallinckrodt Med. BV, Petten, NL for financial support.

Selective Release of Technetium Complexes from a Solid Phase due to C–N Bond Cleavage upon Metal Coordination

A new method for 9 9 m Tc-labeling using solid-phase technology is described. It allows the separation of unlabeled from labeled bioactive molecules by simple filtration. The procedure is based on a site-specific C-N bondcleavage reaction initiated by the formation of [Tc l (CO) 3 L]-complexes.