Cell viability studies of HT29 colon cancer cells treated with the CO-releasing compound [Mn(CO)(3)(tpm)]PF(6) revealed a significant photoinduced cytotoxicity comparable to that of established agent 5-fluorouracil (5-FU), while controls kept in the dark were unaffected at up to 100 microM.
By combining organometallic groups and peptides, a large number of conjugates with interesting new biological properties can be prepared. Especially, attachment to cell-penetrating peptides (CPP) that act as efficient cell delivery vehicles has come to the fore. However, the presence of the metal moiety in such systems can interfere with standard conjugate synthesis procedures which therefore need to be optimized for every new compound. In this work, we report on the preparation of six new cymantrene-sC18 peptide bioconjugates that were prepared by solid phase peptide synthesis (SPPS) techniques. The cymantrene complexes were chosen for their different linker to the peptide, to study the influence of the linker group on cellular uptake and cell viability of the conjugates. Interestingly, the attachment of the metal complex leads to a non-standard cleavage of the Rink amide linker used in the SPPS protocol under trifluoroacetic acid (TFA) treatment, resulting in peptide amides that are N-alkylated at the C-terminus. Furthermore, we found that depending on the type of cymantrene moiety attached, the formation of reactive carbocations which result from decomposition of the resin linker is facilitated and can alkylate the metal complex moiety. Both effects were analyzed by MS/MS studies and cleavage mixtures for efficient elimination of this byproduct formation were identified. Moreover, initial biological testing of the cytotoxicity of one of the bioconjugates gave promising results. Concentration-dependent cell viability studies of Cym1-sC18 on human MCF-7 breast adenocarcinoma cells gave an IC(50) value of 59.8 (+/- 6.7) microM and demonstrate their potential in anticancer chemotherapy.
The conjugation of cymantrene CpMn(CO)(3) to cell-penetrating peptide hCT(18-32)-k7 alters the intracellular distribution in MCF-7 cells compared to the unmodified peptide, as visualized by fluorescence microscopy, and leads to an increased nuclear accumulation; the peptide and cymantrene compound themselves are not toxic, but the bioconjugate shows a significant cytotoxicity with an IC(50) value of 36 micromol l(-1).
Due to an enhanced demand for amino acids, the l-type
amino acid transporter 1 (LAT1) is overexpressed in many tumor cell
lines. LAT1 represents therefore an attractive target for cancer therapy
and diagnosis. On the basis of our reported aqueous synthesis of [(Cp-R)99mTc(CO)3]-type complexes,
−
we describe the preparation of unnatural amino acid
analogues [(Cp-CH2CH(NH2)COOH)Mn(CO)3] and [(Cp-CH(NH2)COOH)M(CO)3] (M = Mn, Re, 99mTc). Starting from fully protected HC5H5-aa (aa = amino acid), [(Cp-aa)99mTc(CO)3]
complexes are accessible in quantitative yields and in a one-step
synthesis from [99mTcO4]−.
The rhenium and manganese analogues were prepared and structurally
characterized to confirm the authenticity of the 99mTc
complex. The inhibition constant of natural phenylalanine (phe) for
LAT1 is in the range 70 ± 10 μM. The K
i value of [(Cp-CH(NH2)COOH)Mn(CO)3] (1a) is 53 ± 11 μM, whereas K
i for the “true” phe analogue [(Cp-CH2CH(NH2)COOH)Mn(CO)3] (2) was surprisingly high at 277 ± 37 μM. Complex 1a caused efflux when exposed to cells, underlining its active
transport by LAT1 into the cell. 99mTc analogues of small
biological lead structures such as amino acids are generally not recognized
anymore by their targets, in particular by trans-membrane transporters.
The bioorganometallic analogues presented here are, however, actively
transported and corroborate the importance of organometallic complexes
as mimics of organic lead structures in life sciences.
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