An entirely fmoc solid phase approach to the synthesis of daptomycin analogs.

Daptomycin is an important Ca2+ -dependent cyclic lipodepsipeptide antibiotic used to treat serious gram-positive infections. The search for daptomycin analogs with improved activity and their application as tools for studying its mechanism of action has prompted us to develop an entirely Fmoc solid phase approach to the synthesis of daptomycin analogs. Key to the success of this approach was the development of conditions that allowed for the formation of the ester bond on resin-bound peptides consisting of residues 1-10 and the decanoyl lipid tail. The esterification reaction proceeded more efficiently on Tentagel resin as opposed to standard polystyrene resin. This approach was used to synthesize a series of analogs in which each position of Dap-E12-W13, a relatively active daptomycin analog, was individually substituted by alanine. Only positions 2, 6, and 11 were found to be amenable to substitution by alanine in that the corresponding alanine analogs were only 1.5- to 4-fold less active than Dap-E12-W13. We also found that the daptomycin analog, Dap-K6-E12-W13, exhibits in vitro activity approaching that of daptomycin at physiological Ca2+ concentration. Studies with Dap-K6-E12-W13 and model liposomes indicate that this analog interacts with membranes by the same mechanism as daptomycin. This analog is currently being used as a lead for the development daptomycin analogs with improved activity.

α-Azido Acids in Solid-Phase Peptide Synthesis: Compatibility with Fmoc Chemistry and an Alternative Approach to the Solid Phase Synthesis of Daptomycin Analogs.

α-Azido acids have been used in solid phase peptide synthesis (SPPS) for almost 20 years. Here we report that peptides bearing an N-terminal α-azidoaspartate residue undergo elimination of an azide ion when treated with reagents that are commonly used for removing the Fmoc group during SPPS. We also report an alternative solid-phase route to the synthesis of an analog of daptomycin that uses a reduced number of α-azido amino acids and without elimination of an azide ion.

α-Azido Esters in Depsipeptide Synthesis: C-O Bond Cleavage during Azido Group Reduction.

α-Azido esters, when treated with dithiothreitol (DTT)/diisopropylethylamine (DIPEA), undergo both azido group reduction to give α-amino esters and C-O bond cleavage to give triazoles. The extent of triazole formation depends upon leaving group ability. Some C-O bond cleavage via triazole formation was also found to occur when a resin-bound peptide, which contained a terminal α-azido ester group, was treated with DTT/DIPEA. C-O bond cleavage also took place when this peptide was treated with PPh3, PBu3, or PMe3; however, in these cases, C-O bond cleavage occurred via either triazole formation and/or hydrolysis of the ester bond in the iminophosphorane intermediate to give betaines. The mechanism that dominated for C-O bond cleavage depended upon the phosphine that was used for azido group reduction. C-O bond cleavage during reduction of the azido group in the peptide was minimized by performing the reduction with PBu3 in the presence of a symmetric anhydride derived from an amino acid in dry THF followed by the addition of water. Surprisingly, these conditions provided the amine as the major product, while the expected amide was formed as a minor product. These conditions were employed in an improved synthesis of an analogue of the cyclic lipodepsipeptide antibiotic daptomycin.

Solid-phase synthesis and in vitro biological activity of a Thr4→Ser4 analog of daptomycin.

Daptomycin is a Ca(+2)-dependent cyclic lipodepsipeptide antibiotic used clinically to treat serious infections caused by Gram-positive bacteria. The recent appearance of daptomycin-resistant strains, daptomycin's lack of activity in the presence of lung surfactant, and its incompletely understood mechanism of action underscores the need for establishing detailed structure-activity relationships. Here we report a solid-phase synthesis of a daptomycin analog in which Thr4, 3-MeGlu12 and Kyn13 in daptomycin were replaced with Ser, Glu and Trp residues, respectively (Dap-S4-E12-W13). The Thr4 to Ser4 substitution was detrimental to activity, as Dap-S4-E12-W13 was at least 20-fold less active at physiological Ca(+2) concentration than Dap-E12-W13. Much of its activity could be recovered at high (100 mM) Ca(+2) concentration, suggesting that the residue at position 4 affects Ca(+2) binding and, consequently, biological activity.

Fluorescent dendron-cyclodextrin nanotubes with surface peptide spacer as a recyclable sensory platform.

Nanotube sensory platform: Dendron-cyclodextrin nanotubes with a surface coumarin unit attached by a GH dipeptide spacer were constructed by a combination of molecular recognition and self-assembly. These unique fluorescent nanotubes can serve as a recyclable metal ion sensory platform with high selectivity and sensitivity (see scheme).

A methionine-based turn-on chemical sensor for selectively monitoring Hg2+ ions in 100% aqueous solution.

Dansyl-labeled methionine is synthesized by solid-phase synthesis, and found to be a highly sensitive and selective sensor for Hg(2+). The sensor sensitively detects Hg(2+) ions in aqueous solution by a turn-on response; however, the sensor detects Hg(2+) ions by a turn-off response in organic and mixed aqueous-organic solutions. We investigated the binding stoichiometry, binding constant, and binding mode of the sensor under various solvent conditions. In 100% aqueous solution, 2 : 1 complexation of the sensor with Hg(2+) ions is more favorable than 1 : 1 complexation, whereas the sensor preferentially forms a 1 : 1 complex in 100% CH(3)CN or in 50% CH(3)CN-aqueous solutions. Results reveal that the stoichiometry of the sensor-Hg(2+) complex plays an important role in the type of response to Hg(2+) ions, and that 2 : 1 complexation is required for a turn-on response to Hg(2+) ions in aqueous solution.

Colorimetric and fluorescent sensing of pyrophosphate in 100% aqueous solution by a system comprised of rhodamine B compound and Al3+ complex.

We developed a simple dual signal (color and 'Off-On' fluorescent change) ensemble system based on the complex between a rhodamine derivative 1 and Al(3+) for the detection of pyrophosphate (PPi) in 100% aqueous solutions. The complex between the rhodamine compound and Al(3+) was utilized as a chemosensing ensemble for the first time. The ensemble showed highly sensitive and selective fluorescent and colorimetric response to pyrophosphate among the anions in 100% aqueous solutions and no interference of the potent biological competitors including ATP, ADP, and phosphate for the detection of PPi in 100% aqueous solutions at pH 7.4.

A highly sensitive and selective detection of Hg(II) in 100% aqueous solution with fluorescent labeled dimerized Cys residues.

A simple design of a ratiometric fluorescent sensor for detecting Hg(II) ion in 100% aqueous solution was demonstrated, based on the structure of dimerized Cys residues with two dansyl fluorophores. The sensor highly sensitively and selectively detected mercury ion (K(d) = 41 nM) in 100% aqueous solution via a turn-on and ratiometric response. The sensor showed no interferences of other metal ions and satisfied for monitoring the maximum allowable level (2 ppb) of mercury ion in drinking water demanded by EPA via a turn-on response.

Facile synthesis of anthracene-appended amino acids as highly selective and sensitive fluorescent Fe3+ ion sensors.

We designed new fluorescent chemical sensors for Fe3+ ion detection, by conjugating amino acids as receptors into an anthracene fluorophore. The conjugates were synthesized in solid phase by Fmoc-chemistry. Fluorescence sensors containing Asp (1) and Glu (2) both had exclusive selectivity for Fe3+ in 100% aqueous solution and in a mixed organic-aqueous solvent system. Other metal ions did not interfere with the detection ability of the sensors for Fe3+. The sensors detect Fe3+ ions via a chelation-enhanced fluorescent quenching effect. The binding affinity, reversible monitoring, and pH sensitivity of the sensors were investigated. In addition, detection of fluoride ion among halide ions was done by a chemosensing ensemble method with 1-Fe3+ and 2-Fe3+ complexes.

Design and synthesis of cyclic disulfide-bonded antibacterial peptides on the basis of the alpha helical domain of Tenecin 1, an insect defensin.

We synthesized cyclic disulfide-bonded (i, i+4) peptides with various net positive charges (+2-+5) from linear peptides derived from the alpha helical domain of Tenecin 1, an insect defensin, and investigated the effect of the intradisulfide bridge (i, i+4) on hydrophobicity, secondary structure, leakage activity and binding activity for large unilamellar vesicles, antimicrobial activity, and hemolytic activity. Intradisulfide bridge formation of the peptides resulted in the increase of amphiphilicity and hydrophobicity. Cyclic forms of the peptides did not deeply penetrate into PG/PC (1:1, mole ratio) large unilamellar vesicles and had a decreased lipid membrane perturbation activity for PG/PC LUVs. When the peptides interacted with PG/CL (2:1, mole ratio) LUVs, cyclic peptides with a high net positive charge (+4-+5) showed similar binding affinities and leakage activities for vesicles to those of linear forms, whereas cyclic peptides with a low net positive charge (+2-+3) exhibited lower leakage activity than their linear forms. CD spectra indicate that the intradisulfide bridge (i, i+4) provided little conformational constraint to linear peptides in buffer solution but resulted in the decrease of alpha helicity of the peptides in lipid membrane mimic conditions. The cyclic peptide with the highest net positive charge had a similar antibacterial activity to that of the linear peptide, whereas the cyclic peptides with a low net positive charge (+3-+4) exhibited lower antibacterial activity than their linear forms. The cyclic peptides of an appropriate net charge showed more potent activities against some bacteria than those of linear forms under high salt conditions.

A Fresh Look at the Staudinger Reaction on Azido Esters: Formation of 2H-1,2,3-Triazol-4-ols from α-Azido Esters Using Trialkyl Phosphines.

Phenyl esters of α-azido acids react with trialkylphosphines in THF/H2O to give 5-substituted 2H-1,2,3-triazol-4-ols in good to excellent yields. In contrast, their reaction with PPh3 in THF/H2O give the amino esters as the major product and no triazoles. Reaction between an α-azido phenyl ester and P(OEt)3 provided the corresponding phosphoramidate in excellent yield, but no triazole was formed.

Solid-phase total synthesis of daptomycin and analogs.

An entirely solid-phase synthesis of daptomycin, a cyclic lipodepsipeptide antibiotic currently in clinical use, was achieved using a combination of α-azido and Fmoc amino acids. This methodology was applied to the synthesis of several daptomycin analogs, one of which did not contain kynurenine or the synthetically challenging amino acid (2S,3R)-methylglutamate yet exhibited an MIC approaching that of daptomycin.

Highly sensitive turn-on detection of Ag+ in aqueous solution and live cells with a symmetric fluorescent peptide.

This communication presents a symmetric fluorescent peptide (K(d) = 17.4 nM) for hypersensitively detecting Ag(+) in 100% aqueous solution by turn-on response. The peptide penetrated live HeLa cells and detected intracellular Ag(+) by turn-on response.