<i>C</i>‑Terminal Acetylene Derivatized Peptides <i>via</i> Silyl-Based Alkyne Immobilization

A new Silyl-based Alkyne Modifying (SAM)-linker for the synthesis of <i>C</i>-terminal acetylene-derivatized peptides is reported. The broad scope of this SAM2-linker is illustrated by manual synthesis of peptides that are side-chain protected, fully deprotected, and disulfide-bridged. Synthesis of a 14-meric (KLAKLAK)₂ derivative by microwave-assisted automated SPPS and a one-pot cleavage click procedure yielding protected 1,2,3-triazole peptide conjugates are also described

Ligand Modifications on a Cp(quinolate)Ru Catalyst to Improve Its Stability in a Bio-orthogonal Deprotection Reaction

The deprotection or activation of substances in biological systems is of particular interest as this method can be used to activate prodrugs in a site- and time-specific manner, thus minimizing possible side effects. Investigations of the literature-known Ru catalyst [RuCp(QL)(η3-allyl)PF6] (with Cp = η-cyclopentadienide, QL= 5-(methoxycarboyl)-8-quinolinolate, 5c) revealed stability issues of the dissolved catalyst in air. We surmised that a more stable catalyst would perform better under biologically relevant conditions and that classical modifications in the ligand set would affect such improved properties. In this work, a systematic study is reported to modify the Cp ligand by using Cp* (Cp* = η-pentamethyl-cyclopentadienide), trimethylsilyl Cp, or t-butyl Cp instead and on the allyl ligand by introducing a methyl group at the middle carbon of 5c. Periodical 1H NMR measurements in DMSO-d6 were performed to monitor the stability of the complexes for longer periods in air, and the catalytic activity of the synthesized compounds was investigated by the deprotection of an alloxycarbonyl (alloc)-protected fluorescent coumarin dye, as monitored by an increase in fluorescence intensity. Modification of the allyl ligand had no effect on the stability, but modification of the Cp ligand was shown to affect the stability of the dissolved complex and, in the case of Cp*, significantly prolong it. As expected, the more stable catalysts are catalytically active for a longer period, but as the reaction rate is not as fast, slightly lower or similar overall yields as compared to the original complex were achieved. Preliminary MTT testing of the obtained complexes revealed IC50 values in the low micromolar range

Efficient Reagent-Saving Method for the N‑Terminal Labeling of Bioactive Peptides with Organometallic Carboxylic Acids by Solid-Phase Synthesis

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 Re^I(CO)₃ complex, showing the generality of the synthetic procedure

Efficient Reagent-Saving Method for the N‑Terminal Labeling of Bioactive Peptides with Organometallic Carboxylic Acids by Solid-Phase Synthesis

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 Re^I(CO)₃ complex, showing the generality of the synthetic procedure

Effects of Enzymatic Activation on the Distribution of Fluorescently Tagged MMP-2 Cleavable Peptides in Cancer Cells and Spheroids

A peptide tagged at the N-terminus with FITC, at the C-terminus with coumarin-343, and incorporating a sequence selectively cleaved by the matrix metalloproteinase, MMP-2, was synthesized to investigate the effect of peptide cleavage on both cellular accumulation and distribution in cancer cell spheroids. The peptide was shown by HPLC and mass spectroscopy to be cleaved in the presence of MMP-2 at the expected site. The cellular and spheroid distribution of each of the fragments was monitored using confocal fluorescence microscopy. The intact peptide had minimal accumulation in 2D-cultured DLD-1 cells that do not express MMP-2 in these conditions. Following addition of serum containing MMP-2 to the cell media, the cleaved C-terminal fragment was seen to enter the cells, while the N-terminal fragment remained extracellular, evidently blocked by the presence of the FITC group. 3D culture of DLD-1 cells as spheroids resulted in measurable MMP-2 activity. Different distribution patterns of the two fluorophores were seen in spheroids treated with the intact peptide, consistent with cleavage occurring. Different rates of accumulation of each of the fragments were observed within the spheroid over time, which is attributed to the extent of accumulation and sequestration of the fragments by cells residing in the periphery of the spheroids. The outcomes suggest that tumor-associated enzymes have the potential to modify the distribution of peptides and peptide fragments in solid tumors by modifying the cellular uptake of those peptides

Silyl-Based Alkyne-Modifying Linker for the Preparation of C‑Terminal Acetylene-Derivatized Protected Peptides

A novel linker for the synthesis of C-terminal acetylene-functionalized protected peptides is described. This SAM1 linker is applied in the manual Fmoc-based solid-phase peptide synthesis of Leu-enkephalin and in microwave-assisted automated synthesis of Maculatin 2.1, an antibacterial peptide that contains 18 amino acid residues. For the cleavage, treatment with tetramethylammonium fluoride results in protected acetylene-derivatized peptides. Alternatively, a one-pot cleavage-click procedure affords the protected 1,2,3-triazole conjugate in high yields after purification

A Ruthenocene–PNA Bioconjugate  Synthesis, Characterization, Cytotoxicity, and AAS-Detected Cellular Uptake

Labeling of peptide nucleic acids (PNA) with metallocene complexes is explored herein for the modulation of the analytical characteristics, as well as biological properties of PNA. The synthesis of the first ruthenocene–PNA conjugate with a dodecamer, mixed-sequence PNA is described, and its properties are compared to a ferrocene-labeled analogue as well as an acetylated, metal-free derivative. The synthetic characteristics, chemical stability, analytical and thermodynamic properties, and the interaction with cDNA were investigated. Furthermore, the cytotoxicity of the PNA conjugates is determined on HeLa, HepG2, and PT45 cell lines. Finally, the cellular uptake of the metal-containing PNAs was quantified by high-resolution continuum source atomic absorption spectrometry (HR-CS AAS). An unexpectedly high cellular uptake to final concentrations of 4.2 mM was observed upon incubation with 50 μM solutions of the ruthenocene–PNA conjugate. The ruthenocene label was shown to be an excellent label in all respects, which is also more stable than its ferrocene analogue. Because of its high stability, low toxicity, and the lack of a natural background of ruthenium, it is an ideal choice for bioanalytical purposes and possible medicinal and biological applications like, e.g., the development of gene-targeted drugs

Sandwich and Half-Sandwich Derivatives of Platensimycin: Synthesis and Biological Evaluation

The multistep synthesis and biological evaluation of five structurally diverse, chiral and achiral CpMn­(CO)₃ (<b>4</b>, <b>7</b> and <b>8</b>), (η⁶-arene)­Cr­(CO)₃ (<b>5</b>), and [3]­ferrocenophane-1-one (<b>6</b>) containing platensimycin (<b>1</b>) derivatives are described in this report. The structures were inspired by the antibiotic platensimycin. All the chiral compounds presented in this report are racemates. The new compounds were unambiguously characterized by ¹H and ¹³C NMR spectroscopy, mass spectrometry, IR spectroscopy, and elemental analysis and in certain cases by X-ray crystallography (<b>4</b>, <b>16</b>, <b>18</b>, and <b>29</b>). The antibacterial and antitumor activity of selected derivatives was tested. Molecular modeling suggests that the derivatives described here may well fit into the active site of the FabF enzyme, which is the biological target of platensimycin. Hence, the antimicrobial activities of our new bioorganometallices <b>4</b>–<b>8</b> and the protected amide intermediates <b>15</b>, <b>17</b>, <b>18</b>, <b>23</b>, <b>28</b>, <b>29</b>, and <b>31</b> were tested against various Gram-positive and Gram-negative bacterial strains. However, all compounds were inactive up to concentrations of 180 μg/mL. The cytotoxicity of compounds <b>4</b> and <b>6</b> and the protected amide intermediates <b>15</b>, <b>17</b>, <b>18</b>, <b>23</b>, <b>28</b>, <b>29</b>, and <b>31</b> was tested against HepG2 and PT45 mammalian cancer cell lines. Surprisingly, all compounds containing a trimethylsilylethyl ester functionality at the aromatic ring (<b>17</b>, <b>23</b>, <b>29</b>, and <b>31</b>) displayed rather high cytotoxicity between 2 and 9 μM

Sandwich and Half-Sandwich Derivatives of Platensimycin: Synthesis and Biological Evaluation

The multistep synthesis and biological evaluation of five structurally diverse, chiral and achiral CpMn­(CO)₃ (<b>4</b>, <b>7</b> and <b>8</b>), (η⁶-arene)­Cr­(CO)₃ (<b>5</b>), and [3]­ferrocenophane-1-one (<b>6</b>) containing platensimycin (<b>1</b>) derivatives are described in this report. The structures were inspired by the antibiotic platensimycin. All the chiral compounds presented in this report are racemates. The new compounds were unambiguously characterized by ¹H and ¹³C NMR spectroscopy, mass spectrometry, IR spectroscopy, and elemental analysis and in certain cases by X-ray crystallography (<b>4</b>, <b>16</b>, <b>18</b>, and <b>29</b>). The antibacterial and antitumor activity of selected derivatives was tested. Molecular modeling suggests that the derivatives described here may well fit into the active site of the FabF enzyme, which is the biological target of platensimycin. Hence, the antimicrobial activities of our new bioorganometallices <b>4</b>–<b>8</b> and the protected amide intermediates <b>15</b>, <b>17</b>, <b>18</b>, <b>23</b>, <b>28</b>, <b>29</b>, and <b>31</b> were tested against various Gram-positive and Gram-negative bacterial strains. However, all compounds were inactive up to concentrations of 180 μg/mL. The cytotoxicity of compounds <b>4</b> and <b>6</b> and the protected amide intermediates <b>15</b>, <b>17</b>, <b>18</b>, <b>23</b>, <b>28</b>, <b>29</b>, and <b>31</b> was tested against HepG2 and PT45 mammalian cancer cell lines. Surprisingly, all compounds containing a trimethylsilylethyl ester functionality at the aromatic ring (<b>17</b>, <b>23</b>, <b>29</b>, and <b>31</b>) displayed rather high cytotoxicity between 2 and 9 μM

Short Antibacterial Peptides with Significantly Reduced Hemolytic Activity can be Identified by a Systematic l‑to‑d Exchange Scan of their Amino Acid Residues

High systemic toxicity of antimicrobial peptides (AMPs) limits their clinical application to the treatment of topical infections; in parenteral systemic application of AMPs the problem of hemolysis is one of the first to be tackled. We now show that the selectivity of lipidated short synthetic AMPs can be optimized substantially by reducing their hemolytic activity without affecting their activity against methicillin resistant <i>Staphylococcus aureus</i> (MRSA). In order to identify the optimized peptides, two sets of 32 diastereomeric H-^dArg-WRWRW-^lLys­(C­(O)­C_<i>n</i>H_2<i>n</i>+1)-NH₂ (<i>n</i> = 7 or 9) peptides were prepared using a split–split procedure to perform a systematic l-to-d exchange scan on the central WRWRW-fragment. Compared to the all-l C₈-lipidated lead sequence, diastereomeric peptides had very similar antibacterial properties, but were over 30 times less hemolytic. We show that the observed hemolysis and antibacterial activity is affected by both differences in lipophilicity of the different peptides and specific combinations of l- and d-amino acid residues. This study identified several peptides that can be used as tools to precisely unravel the origin of hemolysis and thus help to design even further optimized nontoxic very active short antibacterial peptides