Perfluoro-tert-butanol for selective on-resin detritylation: a mild alternative to traditionally used methods.

Solid-phase synthesis of cyclic, branched or side-chain-modified peptides typically involves introduction of a residue carrying a temporary side-chain protecting group that undergoes selective on-resin removal. In particular, Nα-Fmoc-Nε-(4-methyltriphenylmethyl) (Mtt)-protected lysine and its shorter analogues are commercially available and extensively used in this context. Nevertheless, rapid reliable methods for on-resin removal of Mtt groups in the presence of tert-butyloxycarbonyl (Boc) groups are needed. Current commonly used conditions involve low concentrations (1-3%) of trifluoroacetic acid (TFA) in dichloromethane, albeit adjustment to each specific application is required to avoid premature removal of Boc groups or cleavage from the linker. Hence, a head-to-head comparison of several deprotection conditions was performed. The selected acids represent a wide range of acidity from TFA to trifluoroethanol. Also, on-resin removal of the N-(4-methoxytriphenylmethyl) (Mmt) and O-trityl groups (on serine) was investigated under similar conditions. The mildest conditions identified for Mtt deprotection involve successive treatments with 30% hexafluoroisopropanol (HFIP) or 30% perfluoro-tert-butanol [(CF3)3COH] in dichloromethane (3 × 5 or 3 × 15 min, respectively), while 30% HFIP, 30% (CF3)3COH, or 10% AcOH-20% trifluoroethanol (TFE) in CH2Cl2 (3 × 5 min) as well as 5% trichloroacetic acid in CH2Cl2 (3 × 2 min) enabled Mmt removal. Treatment with 1% TFA with/without 2% triisopropylsilane added (3 × 5 min), but also prolonged treatment with 30% (CF3)3COH (5 × 15 min), led to selective deprotection of an O-Trt group on a serine residue. In all cases, the sequences also contained N-Boc or O-tBu protecting groups, which were not affected by 30% HFIP or 30% (CF3)3COH even after a prolonged reaction time of 4 h. Finally, the optimized conditions involving HFIP or (CF3)3COH proved applicable also for selective deprotection of a longer resin-bound peptide [i.e., Ac-Gly-Leu-Leu-Lys(Mtt)-Arg(Pbf)-Ile-Lys(Boc)-Ser(tBu)-Leu-Leu-RAM-PS] as well as allowed for an almost complete deprotection of a Dab(Mtt) residue.

Fmoc-Based Assembly of PNA Oligomers: Manual and Microwave-Assisted Automated Synthesis.

Exploration of PNA-peptide conjugates as potential antisense antibiotics necessitates a fast and efficient synthesis protocols for amounts that facilitate determination of structure-activity relationships and in vivo studies in animal infection models. Fmoc/Boc-protected PNA monomers are here used for assembly of oligomers by optimized protocols involving either a manual synthesis method at room temperature or automated microwave-assisted coupling of monomers on a peptide synthesizer.

Facile Preparation of PNA-Peptide Conjugates with a Polar Maleimide-Thioether Linkage.

Conjugation of a delivery peptide containing a thiol functionality (e.g., a cysteine residue) with a PNA oligomer displaying a single unprotected aliphatic primary amine (e.g., the N-terminus or a C-terminal lysine residue) can be achieved via a one-pot modification with a bisfunctional maleimide linker also displaying a reactive N-hydroxysuccinimidyl ester group (e.g., Mal-PEG2-OSu). Here, an optimized protocol with respect to ratios between the reactants as well as recommended reaction times is presented. Formation and conversion of the maleimide-PNA intermediate was followed by analytical HPLC as exemplified by its conjugation to (KFF)3K-Cys-NH2. In addition, the reaction time required for direct conversion of a preformed Mal-(CH2)2-(C=O)-PNA oligomer in the presence of a slight excess of thiol-modified peptide (with a varying degree of sterical hindrance: HS-(CH2)2-CONH-(KFF)3K-NH2, (KFF)3K-hCys-NH2 and (KFF)3K-Cys-NH2) is provided.

Repurposing Azithromycin and Rifampicin Against Gram-Negative Pathogens by Combination With Peptidomimetics.

Synthetic peptidomimetics may be designed to mimic functions of antimicrobial peptides, including potentiation of antibiotics, yet possessing improved pharmacological properties. Pairwise screening of 42 synthetic peptidomimetics combined with the antibiotics azithromycin and rifampicin in multidrug-resistant (MDR) Escherichia coli ST131 and Klebsiella pneumoniae ST258 led to identification of two subclasses of α-peptide/ß-peptoid hybrids that display synergy with azithromycin and rifampicin (fractional inhibitory concentration indexes of 0.03-0.38). Further screening of the best three peptidomimetics in combination with a panel of 21 additional antibiotics led to identification of peptidomimetics that potentiated ticarcillin/clavulanate and erythromycin against E. coli, and clindamycin against K. pneumoniae. The study of six peptidomimetics was extended to Pseudomonas aeruginosa, confirming synergy with antibiotics for five of them. The most promising compound, H-(Lys-ßNPhe)8-NH2, exerted only a minor effect on the viability of mammalian cells (EC50 ≥ 124-210 µM), and thus exhibited the highest selectivity toward bacteria. This compound also synergized with rifampicin and azithromycin at sub-micromolar concentrations (0.25-0.5 µM), thereby inducing susceptibility to these antibiotics at clinically relevant concentrations in clinical MDR isolates. This peptidomimetic lead and its analogs constitute promising candidates for efficient repurposing of rifampicin and azithromycin against Gram-negative pathogens.

Microwave-assisted solid-phase synthesis of antisense acpP peptide nucleic acid-peptide conjugates active against colistin- and tigecycline-resistant E. coli and K. pneumoniae.

Recent discovery of potent antibacterial antisense PNA-peptide conjugates encouraged development of a fast and efficient synthesis protocol that facilitates structure-activity studies. The use of an Fmoc/Boc protection scheme for both PNA monomers and amino acid building blocks in combination with microwave-assisted solid-phase synthesis proved to be a convenient procedure for continuous assembly of antisense PNA-peptide conjugates. A validated antisense PNA oligomer (CTCATACTCT; targeting mRNA of the acpP gene) was linked to N-terminally modified drosocin (i.e., RXR-PRPYSPRPTSHPRPIRV; X = aminohexanoic acid) or to a truncated Pip1 peptide (i.e., RXRRXR-IKILFQNRRMKWKK; X = aminohexanoic acid), and determination of the antibacterial effects of the resulting conjugates allowed assessment of the influence of different linkers as well as differences between the L- and D-forms of the peptides. The drosocin-derived compound without a linker moiety exhibited highest antibacterial activity against both wild-type Escherichia coli and Klebsiella pneumoniae (MICs in the range 2-4 µg/mL ∼ 0.3-0.7 µM), while analogues displaying an ethylene glycol (eg1) moiety or a polar maleimide linker also possessed activity toward wild-type K. pneumoniae (MICs of 4-8 µg/mL ∼ 0.6-1.3 µM). Against two colistin-resistant E. coli strains the linker-deficient compound proved most potent (with MICs in the range 2-4 µg/mL ∼ 0.3-0.7 µM). The truncated all-L Pip1 peptide had moderate inherent activity against E. coli, and this was unaltered or reduced upon conjugation to the antisense PNA oligomer. By contrast, this peptide was 8-fold less potent against K. pneumoniae, but in this case some PNA-peptide conjugates exhibited potent antisense activity (MICs of 2-8 µg/mL ∼ 0.3-1.2 µM). Most interestingly, the antibacterial activity of the D-form peptide itself was 2- to 16-fold higher than that of the L-form, even for the colistin- and tigecycline-resistant E. coli strains (MIC of 1-2 µg/mL ∼ 0.25-0.5 µM). Low activity was found for conjugates with a two-mismatch PNA sequence corroborating an antisense mode of action. Conjugates containing a D-form peptide were also significantly less active. In conclusion, we have designed and synthesized antisense PNA-drosocin conjugates with potent antibacterial activity against colistin- and tigecycline-resistant E. coli and K. pneumonia without concomitant hemolytic properties. In addition, a truncated D-form of Pip1 was identified as a peptide exhibiting potent activity against both wild-type and multidrug-resistant E. coli, P. aeruginosa, and A. baumannii (MICs within the range 1-4 µg/mL ∼ 0.25-1 µM) as well as toward wild-type Staphylococcus aureus (MIC of 2-4 µg/mL ∼ 0.5-1.0 µM).

Studies on acid stability and solid-phase block synthesis of peptide-peptoid hybrids: ligands for formyl peptide receptors.

α-Peptoids as well as peptide/α-peptoid hybrids and peptide/ß-peptoid hybrids constitute major classes of proteolytically stable peptidomimetics that have been extensively investigated as mimetics of biologically active peptides. Representatives of lipidated peptide/ß-peptoid hybrids have been identified as promising immunomodulatory lead compounds, and hence access to these via protocols suitable for gram-scale synthesis is warranted to enable animal in vivo studies. Recent observations indicated that several byproducts appear in crude mixtures of relatively short benzyl-based peptide/ß-peptoid oligomers, and that these were most predominant when the ß-peptoid units displayed an α-chiral benzyl side chain. This prompted an investigation of their stability under acidic conditions. Simultaneous deprotection and cleavage of peptidomimetics containing either α-chiral α- or ß-peptoid residues required treatment with strong acid only for a short time to minimize the formation of partially debenzylated byproducts. The initial work on peptide/ß-peptoid oligomers with an alternating design established that it was beneficial to form the amide bond between the carboxyl group of the α-amino acid and the congested amino functionality of the ß-peptoid residue in solution. To further simplify oligomer assembly on solid phase, we now present a protocol for purification-free solid-phase synthesis of tetrameric building blocks. Next, syntheses of peptidomimetic ligands via manual solid-phase methodologies involving tetrameric building blocks were found to give more readily purified products as compared to those obtained with dimeric building blocks. Moreover, the tetrameric building blocks could be utilized in automated synthesis with microwave-assisted heating, albeit the purity of the crude products was not increased.

Repurposing azithromycin and rifampicin against Gram-negative pathogens by combination with peptide potentiators.

Gram-negative bacterial pathogens are intrinsically resistant to several antibiotics that are not able to penetrate the cell envelope barrier. The aim of this study was to identify peptides that at low concentrations induce susceptibility to these antibiotics in multidrug-resistant (MDR) Gram-negative bacterial strains of clinical relevance. Pairwise screening of 34 diverse peptides and four antibiotics (erythromycin, linezolid, rifampicin and vancomycin) with primary activity against Gram-positive bacteria identified 4 peptides that at submicromolar concentrations conferred susceptibility to rifampicin or erythromycin in Escherichia coli ATCC 25922. The identified peptides exhibited synergy with azithromycin and potentiated clindamycin in MDR E. coli ST131 and Klebsiella pneumoniae ST258. The low cytotoxicity toward eukaryotic cells (IC50 > 50 µM) observed for two of these peptides (KLWKKWKKWLK-NH2 and GKWKKILGKLIR-NH2) prompted synthesis and evaluation of the corresponding all-d analogues (D1 and D2), which retained similar synergistic antibacterial profiles. Low concentrations of D1 and D2 in combination with azithromycin and rifampicin inhibited growth of most clinical E. coli, K. pneumoniae and Acinetobacter baumannii strains tested. These data demonstrate that combinatorial screening at low peptide concentrations constitutes an efficient approach to identify clinically relevant peptide-antibiotic combinations. In vivo pharmacokinetic/pharmacodynamic and toxicity studies are needed to further validate the use of the peptides identified in this study for repurposing azithromycin and rifampicin against Gram-negative pathogens.

Identification and characterization of a new antifungal peptide in fermented milk product containing bioprotective Lactobacillus cultures.

Mold and yeast contamination constitutes a major problem in food commodities, including dairy products, hence new natural preventive measures are in high demand. The aim of the current study is to identify and characterize novel antifungal peptides produced by lactic acid bacteria (LAB) in sour cream. By the use of a newly developed image-based 96-well plate fungal growth inhibition assay targeting Debaryomyces hansenii, combined with a range of analytical tools comprising HPLC-high-resolution mass spectrometry, ultrahigh-performance liquid chromatography-Triple Quadrupole MS and nuclear magnetic resonance spectroscopy, we successfully identified a new antifungal peptide (DMPIQAFLLY; 1211 Da) in sour cream enriched with two bioprotective LAB strains. This peptide represents a fragment of casein, the most abundant protein in milk. Presumably, the proteolytic activity of these bioprotective strains results in the observed 4-fold higher concentration of the peptide during storage. Both bioprotective strains are able to generate this peptide in concentrations up to 0.4 µM, independently of the sour cream starter culture employed. The peptide attenuates the growth rate of D. hansenii at concentrations ≥35 µM, and results in smaller cells and more compact colonies. Hence, the peptide is likely contributing to the overall preserving effect of the investigated bioprotective LAB strains.

Lactam hybrid analogues of solonamide B and autoinducing peptides as potent S. aureus AgrC antagonists.

Emergence of antibiotic-resistant bacteria constitutes an increasing threat to human health. For example, treatment options for Staphylococcus aureus infections is declining with the worldwide spreading of highly virulent community-associated methicillin-resistant S. aureus (CA-MRSA) strains. Anti-virulence therapy has been proposed as an alternative treatment strategy, as it typically involves inhibition of expression of virulence factors rather than direct bacterial killing, thereby attenuating the risk of resistance development. An intriguing target is the agr quorum-sensing system, which is a major inducer of virulence in CA-MRSA upon activation by agr-encoded staphylococcal autoinducing peptides (AIPs). In the present work a previously identified lactam hybrid analogue based on the marine depsipeptide solonamide B and the general structure of AIPs was investigated with respect to structure-function relationships. An array of 27 analogues exploring expansion of ring size, type of side chain, amino acid substitutions, and stereochemistry was designed and tested for AgrC-inhibitory activity. Interestingly, it was found that an analogue derived from the mirror image of the original hit proved to be the hitherto most efficient AgrC inhibitor resembling solonamide B in amino acid sequence. This and closely related compounds were 20- to 40-fold more potent in AgrC inhibition than the starting hit compound.

Combining Elements from Two Antagonists of Formyl Peptide Receptor 2 Generates More Potent Peptidomimetic Antagonists.

Structural optimization of a peptidomimetic antagonist of formyl peptide receptor 2 (FPR2) was explored by an approach involving combination of elements from the two most potent FPR2 antagonists described: a Rhodamine B-conjugated 10-residue gelsonin-derived peptide (i.e., PBP10, RhB-QRLFQVKGRR-OH) and the palmitoylated α-peptide/ß-peptoid hybrid Pam-(Lys-ßNspe)6-NH2. This generated an array of hybrid compounds from which a new subclass of receptor-selective antagonists was identified. The most potent representatives displayed activity in the low nanomolar range. The resulting stable and potent FPR2-selective antagonists (i.e., RhB-(Lys-ßNphe)n-NH2; n = 4-6) are expected to become valuable tools in further elucidation of the physiological role of FPR2 in health and disease.

On-resin N-formylation of peptides: a head-to-head comparison of reagents in solid-phase synthesis of ligands for formyl peptide receptors.

4-Nitrophenyl formate was found to be the most convenient reagent in solid-phase formylation of peptides with a high formylation degree within 20 min to 3 h depending on reaction temperature and length of peptide.

Antibacterial Peptide Nucleic Acid-Antimicrobial Peptide (PNA-AMP) Conjugates: Antisense Targeting of Fatty Acid Biosynthesis.

Antisense peptide nucleic acid (PNA) oligomers constitute a novel class of potential antibiotics that inhibit bacterial growth via specific knockdown of essential gene expression. However, discovery of efficient, nontoxic delivery vehicles for such PNA oligomers has remained a challenge. In the present study we show that antimicrobial peptides (AMPs) with an intracellular mode of action can be efficient vehicles for bacterial delivery of an antibacterial PNA targeting the essential acpP gene. The results demonstrate that buforin 2-A (BF2-A), drosocin, oncocin 10, Pep-1-K, KLW-9,13-a, (P59→W59)-Tat48-60, BF-2A-RXR, and drosocin-RXR are capable of transporting PNA effectively into E. coli (MICs of 1-4 µM). Importantly, presence of the inner-membrane peptide transporter SbmA was not required for antibacterial activity of PNA-AMP conjugates containing Pep-1-K, KLW-9,13-a, or drosocin-RXR (MICs of 2-4 µM).

Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor.

FFA2 is a G protein-coupled receptor that responds to short chain fatty acids and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds potentially having FFA2 activity and examined the molecular basis of their function. These compounds were confirmed to be potent and selective orthosteric FFA2 agonists. A combination of ligand structure-activity relationship, pharmacological analysis, homology modeling, species ortholog comparisons, and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 and the transmembrane domain regions of FFA2 critical for ligand function. One of these ligands was active with reasonable potency at rodent orthologs of FFA2 and demonstrated the role of FFA2 in inhibition of lipolysis and glucagon-like peptide-1 secretion in murine-derived 3T3-L1 and STC-1 cell lines, respectively. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the binding site of FFA2 that will be invaluable in future ligand development at this receptor.

Mechanistic evidence for intermolecular radical carbonyl additions promoted by samarium diiodide.

In this work, mechanistic studies were performed to understand the SmI2/H2O-mediated coupling of N-acyl oxazolidinones with acrylates and acrylamides, providing gamma-keto esters and amides, respectively. Our results provide experimental evidence that C-C bond formation via intermolecular radical addition reactions to carbonyl substrates can be promoted by samarium diiodide. Coupling reactions with N-cyclopropylcarbonyl-2-oxazolidinone suggest the alpha,beta-unsaturated esters/amides are reduced by the low-valent lanthanide reagent and not the N-acyl oxazolidinones, as originially proposed (J. Am. Chem. Soc. 2005, 127, 6544). Rate measurements support the preferred reduction of an acrylate or acrylamide by SmI2/H2O in the presence of an N-acyl oxazolidinone. In the absence of the N-acyl oxazolidinone, SmI2/H2O promotes dimerization of the acrylates, whereas the C=C bond of the acrylamides is reduced. In addition, coupling of the Pfp ester of Cbz-protected phenylalanine with an acrylamide leads only to reduction of the acrylamide and recovered ester, whereas the same coupling with the N-acyl oxazolidinone derivative provides the gamma-keto amides. These results imply that a pathway involving nucleophilic acyl substitution cannot take place and that a radical mechanism must be invoked to explain the C-C bond formation. We propose that the acrylate/acrylamide is reduced to a conjugated ketyl radical that adds to the exocyclic carbonyl group of the N-acyl oxazolidinone, activated through bidentate coordination to a lanthanide ion.

Can decarbonylation of acyl radicals be overcome in radical addition reactions? En route to a solution employing N-acyl oxazolidinones and SmI2/H2O.

The application of acyl radicals in radical addition reactions in the absence of a CO atmosphere is generally limited to aryl or alpha-unsubstituted alkyl acyl radicals due to competing decarbonylations where the rate constant for this degradation process surpasses 104 s-1. In this work, a potential solution to avoid the problem of decarbonylations is presented employing N-acyl oxazolidinones which are reduced to acyl radical equivalents in the presence of samarium diiodide and water. In the company of an acrylamide, acrylate, or acrylonitrile, the product from a formal acyl radical addition is obtained in yields up to 87%. Examples are given where the decarbonylation rate constants even exceed 108 s-1. It is proposed that the reaction proceeds via a ketyl-like intermediate.