Cell-targeting peptides (CTPs) are increasingly used in the field of cancer research due to their high affinity and specificity to cell or tissue targets. In the search for novel metal-based drug ...candidates, our research group is particularly focused on bioconjugates by utilizing peptides to increase the selectivity of cytotoxic organometallic compounds. Motivated by the relatively high cytotoxic activity of gold complexes, such as Auranofin (approved to treat rheumatoid arthritis), for the treatment of various diseases, we anticipated that gold peptide bioconjugates would present interesting candidates for novel breast cancer therapies. For this, we investigate the use of the natural compound lipoic acid (Lpa) as a bioconjugation handle to link Au complexes in the oxidation state +III to peptides using the dithiol moiety. Using this strategy, we have synthesized Au(III) complex bioconjugates linked to the linear LTVSPWY peptide and two cyclic DfKRG and KTTHWGFTLG tumor-targeting peptides. Solid-phase peptide synthesis (SPPS) was used to prepare the peptides, with lipoic acid introduced N-terminally as a conjugation handle. After peptide cleavage, the metal complex was introduced in solution by first reducing the internal disulfide bond, followed by reaction with Au(ppy)Cl2 (1, ppy: 2-phenyl-pyridine), to yield the Au(III)–Lpa–peptide bioconjugates. The new bioconjugates were successfully synthesized, purified by semi-preparative HPLC, and characterized by ESI-MS. Au(III)-peptide bioconjugates were tested as cytotoxic agents against two different human breast cancer cell lines (MCF-7 and MDA-MB-231) and normal human fibroblasts cells (GM5657T) and compared to cisplatin, the parent Au(III) dichloride complex, and metal-free peptides. These in vitro data show that the Au(III)-peptide bioconjugate 5, possessing the cyclic integrin-targeting RGD-derived peptide sequence in the structure, exhibits improved activity compared to the parent gold(III) compound Au(ppy)Cl2 (1) as well as to cisplatin or the metal-free peptide. Moreover, the excellent targeting properties of 5 are supported by the fact that a Au(III)-peptide conjugate with the exact same peptide sequence, but a linear rather than the cyclic form of 5 exhibits 10 times lower cytotoxic activity.
Labeling of biomolecules with organometallic moieties holds great promise as a tool for chemical biology and for the investigation of biochemical signaling pathways. Herein, we report a robust and ...reproducible synthetic strategy for the synthesis of ruthenocenecarboxylic acid, giving the acid in 53% overall yield. This organometallic label was conjugated via solid-phase peptide synthesis in near-quantitative yield to a number of different biologically active peptides, using only 1 equiv of the acid and coupling reagents, thereby avoiding wasting the precious organometallic acid. This optimized method of stoichiometric N-terminal acylation was then also successfully applied to conjugating ferrocenecarboxylic acid and a novel organometallic ReI(CO)3 complex, showing the generality of the synthetic procedure.
Evasion of apoptosis is critical for the development and growth of tumors. The pro-survival protein myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family, associated with ...tumor aggressiveness, poor survival, and drug resistance. Development of Mcl-1 inhibitors implies blocking of protein–protein interactions, generally requiring a lengthy optimization process of large, complex molecules. Herein, we describe the use of DNA-encoded chemical library synthesis and screening to directly generate complex, yet conformationally privileged macrocyclic hits that serve as Mcl-1 inhibitors. By applying a conceptual combination of conformational analysis and structure-based design in combination with a robust synthetic platform allowing rapid analoging, we optimized in vitro potency of a lead series into the low nanomolar regime. Additionally, we demonstrate fine-tuning of the physicochemical properties of the macrocyclic compounds, resulting in the identification of lead candidates 57/59 with a balanced profile, which are suitable for future development toward therapeutic use.
Synthetic antimicrobial peptides (SynAMPs) are an interesting class of novel antibiotic agents used to fight infections caused by resistant bacterial strains. Herein we report the synthesis of a ...ferrocene‐containing lysine (FcLys) suitable for standard Fmoc/tBu solid‐phase peptide synthesis. A library of (Arg‐Trp)3‐based antimicrobial peptides have been synthesized in which all the tryptophan residues were replaced systematically by FcLys and their biological activities evaluated. We call this technique a “ferrocene scan”, in analogy with the well‐established “alanine scan” used to investigate crucial interactions in peptides. The FcLys‐peptides showed excellent activity against gram‐positive bacterial strains compared with the metal‐free parent peptide, including a four‐fold increase in activity against the resistant bacterial strain MRSA. Moreover, some members of this family of ferrocenoyl peptides also showed an up to 16‐fold increased activity compared with the parent peptide against the gram‐negative bacterium A. baumannii. This behavior distinguishes these synAMPs from the previously investigated N‐terminally ferrocenoylated peptides, which in general showed a dramatically reduced activity against gram‐negative bacteria. The FcLys‐peptides also showed low hemolytic activity against red blood cells (RBCs), which indicates excellent bacterial selectivity, making them an attractive class of novel peptide‐based organometallic antimicrobial agents.
A library of ferrocene‐substituted antimicrobials with one, two, or even three ferrocene residues in lieu of a tryptophan residue have been synthesized. The most potent compound shows a 16‐fold increased activity over the parent, metal‐free (RW)3 peptide against the A. baumannii bacteria. A four‐fold increase in activity was even observed against a multi‐resistant S. aureus MRSA strain.
Browning is one of the most common postharvest changes in button mushrooms, which often results in economic losses. Phenolic compounds, which are associated with browning, were extracted from the ...nonbruised and bruised skin tissue of various button mushrooms with a sulfite-containing solution and analyzed with UHPLC-PDA-MS. In total, 34 phenolic compounds were detected. Only small differences in the total phenolic content between bruising-tolerant and -sensitive strains were observed. The contents of γ-l-glutaminyl-4-hydroxybenzene (GHB) and γ-l-glutaminyl-3,4-dihydroxybenzene (GDHB) correlated with bruising sensitivity; for example, R 2 values of 0.85 and 0.98 were found for nonbruised brown strains, respectively. In nonbruised skin tissue of the strains with brown caps, the GHB and GDHB contents in sensitive strains were on average 20 and 15 times higher, respectively, than in tolerant strains. GHB and GDHB likely participate in the formation of brown GHB–melanin, which seemed to be the predominant pathway in bruising-related discoloration of button mushrooms.
The lipophilic amino acid, (S)-2-aminoundecanoic acid, was synthesized and incorporated at a number of specific positions within the peptide sequence of anoplin. These lipophilic anoplin analogs ...showed to be more active against Escherichia coli and Staphylococcus aureus compared to native anoplin, while the EC50-value of hemolysis was at least one order of magnitude lower than the MIC values. This was in sharp contrast to the N-acylated anoplin derivative, where a gain in activity also led to a complete loss of selectivity. Thus, the incorporation of a lipophilic amino acid residue into anoplin enhanced the antimicrobial activity, while selectivity towards microbial membranes was retained.
Herein, we describe the synthesis, structural characterization, and synthetic use as an advanced intermediate of a cross-stapled alkene-bridged hexapeptide to mimic the DE-ring of the lantibiotic ...nisin. The linear precursor was cyclized by ring-closing metathesis to give the correctly folded bicyclic hexapeptide in a single step, and the four individual diastereoisomers were isolated, structurally assigned and characterized by HPLC, NMR and MS, respectively. The bicyclic hexapeptide was used as a versatile advanced synthon and was modified at its C- and N-terminus, among others, with an azide moiety to access a building block suitable for Cu(I)-catalyzed alkyne-azide cycloaddition-based ligation reactions.
To maintain the integrity of the genome, multiple DNA repair systems exist to repair damaged DNA. Recognition of altered DNA, including bulky adducts, pyrimidine dimers and interstrand crosslinks ...(ICL), partially depends on proteins containing helix-hairpin-helix (HhH) domains. To understand how ICL is specifically recognized by the Fanconi anemia proteins FANCM and FAAP24, we determined the structure of the HhH domain of FAAP24. Although it resembles other HhH domains, the FAAP24 domain contains a canonical hairpin motif followed by distorted motif. The HhH domain can bind various DNA substrates; using nuclear magnetic resonance titration experiments, we demonstrate that the canonical HhH motif is required for double-stranded DNA (dsDNA) binding, whereas the unstructured N-terminus can interact with single-stranded DNA. Both DNA binding surfaces are used for binding to ICL-like single/double-strand junction-containing DNA substrates. A structural model for FAAP24 bound to dsDNA has been made based on homology with the translesion polymerase iota. Site-directed mutagenesis, sequence conservation and charge distribution support the dsDNA-binding model. Analogous to other HhH domain-containing proteins, we suggest that multiple FAAP24 regions together contribute to binding to single/double-strand junction, which could contribute to specificity in ICL DNA recognition.
Functionalization of the lantibiotic nisin with fluorescent reporter molecules is highly important for the understanding of its mode of action as a potent antimicrobial peptide. In addition to this, ...multimerization of nisin to obtain multivalent peptide constructs and conjugation of nisin to bioactive molecules or grafting it on surfaces can be attractive methods for interference with bacterial growth. Here, we report a convenient method for the synthesis of such nisin conjugates and show that these nisin derivatives retain both their antimicrobial activity and their membrane permeabilizing properties. The synthesis is based on the Cu(I)-catalyzed alkyne–azide cycloaddition reaction (CuAAC) as a bioorthogonal ligation method for large and unprotected peptides in which nisin was C-terminally modified with propargylamine and subsequently efficiently conjugated to a series of functionalized azides. Two fluorescently labeled nisin conjugates together with a dimeric nisin construct were prepared while membrane insertion as well as antimicrobial activity were unaffected by these modifications. This study shows that C-terminal modification of nisin does not deteriorate biological activity in sharp contrast to N-terminal modification and therefore C-terminally modified nisin analogues are valuable tools to study the antibacterial mode of action of nisin. Furthermore, the ability to use stoichiometric amounts of the azide containing molecule opens up possibilities for surface tethering and more complex multivalent structures.
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