Manual Solid–Phase Peptide Synthesis of Metallocene–Peptide Bioconjugates

Kirin, Srećko I.; Noor, Fozia; Metzler‐Nolte, Nils; Mier, Walter

doi:10.1021/ed084p108

Cited by 61 publications

(69 citation statements)

References 9 publications

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“…Specific conditions had to be chosen concerning the low stability of 7 against TFA [32], a commonly used cleavage reagent and thus the base labile HMBA (4-hydroxymethylbenzoic acid) linker was used so as to avoid the need for acid treatment altogether. Enkephalin was synthesized according to standard Fmoc-SPPS methods [5]. After removal of the 2-chloro-trityl (2-Cl-Trt) protecting group from tyrosine, Cp iPr Ru(p-(CO 2 H)C 6 H 4 Tp) (7) was coupled.…”

Section: Synthesis and Characterization Of Mixed Ligand Sandwich Biocmentioning

confidence: 99%

“…Consequently, there is a steady quest for compounds that are air-and waterstable and allow the facile attachment of biomolecules. One class of compounds that has been intensively investigated and employed in this area is that of the original metallocene Cp 2 Fe and its heavier congener Cp 2 Ru and their derivatives [4,5]. Notably, the analogues of these metallocenes, whether homoleptic or heteroleptic, have not been studied to any serious extent.…”

Section: Introductionmentioning

confidence: 99%

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Ruthenium-based bioconjugates: Synthesis and X-ray structure of the mixed ligand sandwich compound RuCpiPr(p-(CO2H)C6H4Tp) and labelling of amino acids and the neuropeptide enkephalin

Zagermann

Kuchta

Merz

et al. 2009

Journal of Organometallic Chemistry

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Section: Synthesis and Characterization Of Mixed Ligand Sandwich Biocmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Ruthenium-based bioconjugates: Synthesis and X-ray structure of the mixed ligand sandwich compound RuCpiPr(p-(CO2H)C6H4Tp) and labelling of amino acids and the neuropeptide enkephalin

Zagermann

Kuchta

Merz

et al. 2009

Journal of Organometallic Chemistry

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“…The synthesis of ferrocene-peptide systems has garnered attention by several groups and has led to a library of asymmetric peptide-ferrocene systems. 46,[48][49][50][51][52] The work described herein details the synthesis of the first asymmetric ferrocene bio-conjugates containing biotin-ferrocene-cysteine. This compound serves as a model through which other small molecule receptors could be considered for detection of biologically relevant molecules.…”

Section: Discussionmentioning

confidence: 99%

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Scarborough

González

Rodich

et al. 2015

JoVE

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Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of "point of care" techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician's office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules 2 , methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocenecysteine system that can be immobilized on a gold surface. Video LinkThe video component of this article can be found at

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“…The peptide sequences (NLS or NLS scr ) were synthesised either on a computer-controlled peptide synthesiser or manually by syringe techniques. [43] Wang resin that was preloaded with Fmoc-Val-OH (0.63 mmol g À1 ) was used for NLS and the truncated metal-dipeptides. For the NLS scr sequence, Wang resin Scheme 1.…”

Section: Synthesis and Characterisationmentioning

confidence: 99%

Enhanced Cellular Uptake and Cytotoxicity Studies of Organometallic Bioconjugates of the NLS Peptide in Hep G2 Cells

et al. 2009

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SPACE INVADERS: Organometallic fragments such as the ferrocenyl group (shown in red in the picture) help to enhance cellular entry of NLS peptides. Eventually, these nontoxic conjugates find their way to the cellular nucleus as shown by fluorescence microscopy studies in this work. Intracellular delivery to biomolecular targets is still a major challenge in molecular and cell biology. We recently found that attaching an organometallic group, namely the cobaltocenium cation, to the SV 40 large T antigen nuclear localisation signal (NLS) greatly enhances cellular uptake of the conjugate (Noor et al., Angew. Chem. Int. Ed. 2005, 45, 2429). In addition, nuclear localisation of the conjugate was observed. In this work, we present a thorough investigation of this novel cellular delivery system with respect to the nature of the metal complex and the peptide sequence. A number of ferrocene ((Fe(II)), neutral metal complex) and cobaltocenium ((Co(III)), cationic metal complex) bioconjugates with both the NLS wild-type sequence PKKKRKV and a scrambled sequence (NLS(scr), KKVKPKR) were prepared by solid-phase peptide synthesis (SPPS). Cellular and nuclear uptake of these bioconjugates was studied by fluorescence microscopy on living Hep G2 cells. In addition, cytotoxicity screening on the conjugates was carried out, as the toxic effects of several simple metallocenes have been noted previously. Rapid cellular uptake as well as nuclear localisation was observed for the metal-NLS conjugates, but not for any dipeptide controls, the metal-NLS(scr) conjugates or any metal-free conjugates. It thus appears that the presence of a metallocene, but not its charge, and the correct NLS sequence is essential for cellular uptake. Fluorescence microscopy co-localisation studies did not reveal a significant endosomal entrapment of the conjugates. The metallocene not only provides a hydrophobic handle for membrane translocation but also facilitates the localisation and distribution of the conjugate in the cytoplasm. The NLS peptide on the other hand is responsible for the nuclear localisation of the bioconjugate. Finally, none of the conjugates were found to be toxic up to the highest concentrations that was tested (1 mM) against the Hep G2 cells that were used in this study. In conclusion, this work supports metallocene-NLS bioconjugates, in particular with the very robust cobaltocenium group, as a simple but potent, nontoxic system for cellular uptake and nuclear delivery. Concurrently, our finding is relevant to the still-unresolved question of cytotoxicity of metallocenes because it excludes binding and/or damage to the DNA as a mechanism of metallocene cytotoxicity. This finding is confirmed by a combined yeast cytotoxicity/genotoxicity assay, which also shows very little toxic effects for all organometal-NLS conjugates that were tested.

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Manual Solid–Phase Peptide Synthesis of Metallocene–Peptide Bioconjugates

Cited by 61 publications

References 9 publications

Ruthenium-based bioconjugates: Synthesis and X-ray structure of the mixed ligand sandwich compound RuCpiPr(p-(CO2H)C6H4Tp) and labelling of amino acids and the neuropeptide enkephalin

Ruthenium-based bioconjugates: Synthesis and X-ray structure of the mixed ligand sandwich compound RuCpiPr(p-(CO2H)C6H4Tp) and labelling of amino acids and the neuropeptide enkephalin

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Enhanced Cellular Uptake and Cytotoxicity Studies of Organometallic Bioconjugates of the NLS Peptide in Hep G2 Cells

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