Stabilizing Bifurcated Hydrogen Bond in 8-Aminoquinoline Appended Peptides.

The hydrogen bonding interaction between an amide N-H and the amide N of the preceding residue is prevalent in proline-containing proteins and peptides. However, the N-H⋅⋅⋅N hydrogen bonding interaction is rare in non-prolyl natural peptides due to restricted dihedral angles. Herein, we stabilize this type of interaction in 8-aminoquinoline appended non-prolyl peptides through bifurcated N⋅⋅⋅H⋅⋅⋅N hydrogen bond. The 8-aminoquinoline-incorporated model peptides 2 a-i were designed, synthesized, and the crystal structures of 2 a-c and 2 i were solved. Analysis of crystal data reveals that the amide N-H of aminoquinoline is involved in bifurcated hydrogen bonding interaction with the nitrogen of the preceding amino acid residue and the nitrogen in quinoline. Analysis of crystal packing, Hirshfeld surface and fingerprint plots confirms that the intermolecular O⋅⋅⋅H contacts significantly contribute to stabilizing bifurcated N⋅⋅⋅H⋅⋅⋅N hydrogen bonding interaction. Furthermore, NMR experiments and CD spectroscopy were conducted to examine the preferred conformation in solution, and the data corroborate with the crystal structure conformation.

Oral Administration of a Specific p300/CBP Lysine Acetyltransferase Activator Induces Synaptic Plasticity and Repairs Spinal Cord Injury.

TTK21 is a small-molecule activator of p300/creb binding protein (CBP) acetyltransferase activity, which, upon conjugation with a glucose-derived carbon nanosphere (CSP), can efficiently cross the blood-brain barrier and activate histone acetylation in the brain. Its role in adult neurogenesis and retention of long-term spatial memory following intraperitoneal (IP) administration is well established. In this study, we successfully demonstrate that CSP-TTK21 can be effectively administered via oral gavage. Using a combination of molecular biology, microscopy, and electrophysiological techniques, we systematically investigate the comparative efficacy of oral administration of CSP and CSP-TTK21 in wild-type mice and evaluate their functional effects in comparison to intraperitoneal (IP) administration. Our findings indicate that CSP-TTK21, when administered orally, induces long-term potentiation in the hippocampus without significantly altering basal synaptic transmission, a response comparable to that achieved through IP injection. Remarkably, in a spinal cord injury model, oral administration of CSP-TTK21 exhibits efficacy equivalent to that of IP administration. Furthermore, our research demonstrates that oral delivery of CSP-TTK21 leads to improvements in motor function, histone acetylation dynamics, and increased expression of regeneration-associated genes (RAGs) in a spinal injury rat model, mirroring the effectiveness of IP administration. Importantly, no toxic and mutagenic effects of CSP-TTK21 are observed at a maximum tolerated dose of 1 g/kg in Sprague-Dawley (SD) rats via the oral route. Collectively, these results underscore the potential utility of CSP as an oral drug delivery system, particularly for targeting the neural system.

Metal-free sp2 -C7-H Borylation of Tryptophan Containing Peptides and Late-stage Modification.

The discovery of milder and robust strategies to enable the introduction of organoboronates in peptides remains conspicuously underdeveloped. Herein, we demonstrate an efficient method for the site-selective sp2 -C7-H borylation of tryptophan under metal-free condition using BBr3 directed by pivaloyl group. The versatility of this approach is that gram scale synthesis and C7-borylated N-Phth-Trp(N-Piv)(C7-BPin)-OMe was modified into various C7-substituted derivatives. Moreover, the strategy enables for the peptide elongation and late-stage borylation of peptides, natural product Brevianamide F and drug Oglufanide.

ß-Turn editing in Gramicidin S: Activity impact on replacing proline α-carbon with stereodynamic nitrogen.

Gramicidin S, natural antimicrobial peptide is used commercially in medicinal lozenges for sore throat and Gram-negative and Gram-positive bacterial infections. However, its clinical potential is limited to topical applications because of its high red blood cells (RBC) cytotoxicity. Given the importance of developing potential antibiotics and inspired by the cyclic structure and druggable features of Gramicidin S, we edited proline α-carbon with stereodynamic nitrogen to examine the direct impact on biological activity and cytotoxicity with respect to prolyl counterpart. Natural Gramicidin S (12), proline-edited peptides 13-16 and wild-type d-Phe-d-Pro ß-turn mimetics (17 and 18) were synthesized using solid phase peptide synthesis and investigated their activity against clinically relevant bacterial pathogens. Interestingly, mono-proline edited analogous peptide 13 showed moderate improvement in antimicrobial activity against E. coli ATCC 25922 and K.pneumoniae BAA 1705 as compared to Gramicidin S. Furthermore, proline edited peptide 13 exhibited equipotent antimicrobial effect against MDR S. aureus and Enterococcus spp. Analysis of cytotoxicity against VERO cells and RBC, reveals that proline edited peptides showed two-fivefold lesser cytotoxicity than the counterpart Gramicidin S. Our study suggests that introducing single azPro/Pro mutation in Gramicidin S marginally improved the activity and lessens the cytotoxicity as compared with the parent peptide.

Design and synthesis of N-acyl and dimeric N-Arylpiperazine derivatives as potential antileishmanial agents.

The current regime for leishmaniasis is associated with several adverse effects, expensive, parenteral treatment for longer periods and the emergence of drug resistance. To develop affordable and potent antileishmanial agents, a series of N-acyl and homodimeric aryl piperazines were synthesized with high purity, predicted druggable properties by in silico methods and investigated their antileishmanial activity. The in vitro biological activity of synthesized compounds against clinically validated intracellular amastigote and extracellular promastigote form of Leishmania donovani parasite showed eight compounds inhibited 50% amastigotes growth below 25 µM. The half maximal inhibitory concentration (IC50) and cytotoxicity assessment of eight active compounds, 4a, 4d and 4e demonstrated activity with an IC50 2.0 - 9.1 µM and selectivity index 10 - 42. Compound 4d (IC50 2.0 µM, SI = 42) found to be the best among them with four-folds more potent and eight-folds less toxic than the control drug miltefosine. Overall, results demonstrated that compound 4d is a promising lead candidate for further development as antileishmanial drug.

Design and synthesis of novel halogen rich salicylanilides as potential antileishmanial agents.

The available therapeutic treatment for leishmaniasis is inadequate and toxic due to side effects, expensive and emergence of drug resistance. Affordable and safe antileishmanial agents are urgently needed and toward this objective, we synthesized a series of 32 novel halogen rich salicylanilides including niclosamide and oxyclozanide and investigated their antileishmanial activity against amastigotes of Leishmania donovani. In vitro data showed fifteen compounds inhibited intracellular amastigotes with an IC50 of below 5 µM and selectivity index above 10. Among 15 active compounds, 14 and 24 demonstrated better activity with an IC50 of 2.89 µM and 2.09 µM respectively and selectivity index is 18. Compound 24 exhibited significant in vivo antileishmanial efficacy and reduced 65% of the splenic parasite load on day 28th post-treatment in the experimental visceral leishmaniasis golden hamster model. The data suggest that 24 can be a promising lead candidate possessing potential to be developed into a leishmanial drug candidate.

Influence of Proline Chirality on Neighbouring Azaproline Residue Stereodynamic Nitrogen Preorganization.

We report herein the first systematic crystal structural investigation of azaproline incorporated in homo- and heterochiral diprolyl peptides. The X-ray crystallography data of peptides 1-5 illustrates that stereodynamic nitrogen in azaproline adopted the stereochemistry of neighbouring proline residue without depending on its position in the peptide sequence. Natural bond orbital analysis of crystal structures indicates OazPro -C'Pro of peptides 4 and 5 participating in n→π* interaction with stabilization energy about 1.21-1.33 kcal/mol. Density functional theory calculations suggested that the endo-proline ring puckering favoured over exo-conformation by 6.72-7.64 kcal/mol. NBO and DFT data reveals that the n→π* interactions and proline ring puckering stabilize azaproline chirality with the neighbouring proline stereochemistry. The CD, solvent titration, variable-temperature and 2D NMR experimental results further supported the crystal structures conformation.

Chemical synthesis of Torenia plant pollen tube attractant proteins by KAHA ligation.

The synthesis of secreted cysteine-rich proteins (CRPs) is a long-standing challenge due to protein aggregation and premature formation of inter- and intramolecular disulfide bonds. Chemical synthesis provides reduced CRPs with a higher purity, which is advantageous for folding and isolation. Herein, we report the chemical synthesis of pollen tube attractant CRPs Torenia fournieri LURE (TfLURE) and Torenia concolor LURE (TcLURE) and their chimeric analogues via α-ketoacid-hydroxylamine (KAHA) ligation. The bioactivity of chemically synthesized TfLURE protein was shown to be comparable to E. coli expressed recombinant protein through in vitro assay. The convergent protein synthesis approach is beneficial for preparing these small protein variants efficiently.

Reactivity vs. selectivity of quinone methides: synthesis of pharmaceutically important molecules, toxicity and biological applications.

Quinone methides (QMs) are considered to be highly reactive intermediates because of their aromatization both in chemical and biological systems. Being highly accessible, quinone methides (QMs) have been widely exploited and their concurrent use has been manifested for the synthesis of tertiary and quaternary carbon centers of bioactives, drugs and drug-like molecules. In this feature article, the synthetic routes, structure-reactivity relationships and synthetic applications of quinone methides are discussed. Formation of the intermediates during bioactivation of different chemical entities and possible chemical manifestations leading to their toxicity in biological systems are also covered.

Bio-evaluation of fluoro and trifluoromethyl-substituted salicylanilides against multidrug-resistant S. aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA) are primary causes of skin and soft tissue infections worldwide. To address the emergency caused due to increasing multidrug-resistant (MDR) bacterial infections, a series of novel fluoro and trifluoromethyl-substituted salicylanilide derivatives were synthesized and their antimicrobial activity was investigated. MIC data reveal that the compounds inhibited S. aureus specifically (MIC 0.25-64 µg/mL). The in vitro cytotoxicity of compounds with MIC < 1 µg/mL against Vero cells led to identification of four compounds (20, 22, 24 and 25) with selectivity index above 10. These four compounds were tested against MDR S. aureus panel. Remarkably, 5-chloro-N-(4'-bromo-3'-trifluoromethylphenyl)-2-hydroxybenzamide (22) demonstrated excellent activity against nine MRSA and three VRSA strains with MIC 0.031-0.062 µg/mL, which is significantly better than the control drugs methicillin and vancomycin. The comparative time-kill kinetic experiment revealed that the effect of bacterial killing of 22 is comparable with vancomycin. Compound 22 did not synergize with or antagonize any FDA-approved antibiotic and reduced pre-formed S. aureus biofilm better than vancomycin. Overall, study suggested that 22 could be further developed as a potent anti-staphylococcal therapeutic.

Design, synthesis, and biological evaluation of stable ß6.3-Helices: Discovery of non-hemolytic antibacterial peptides.

Gramicidin A, a topical antibiotic made from alternating L and D amino acids, is characterized by its wide central pore; upon insertion into membranes, it forms channels that disrupts ion gradients. We present helical peptidomimetics with this characteristic wide central pore that have been designed to mimic gramicidin A channels. Mimetics were designed using molecular modeling focused on oligomers of heterochiral dipeptides of proline analogs, in particular azaproline (AzPro). Molecular Dynamics simulations in water confirmed the stability of the designed helices. A sixteen-residue Formyl-(AzPro-Pro)8-NHCH2CH2OH helix was synthesized as well as a full thirty-two residue Cbz-(AzPro-Pro)16-OtBu channels. No liposomal lysis activity was observed suggesting lack of channel formation, possibly due to inappropriate hydrogen-bonding interactions in the membrane. These peptidomimetics also did not hemolyze red blood cells, unlike gramicidin A.

Design and Synthesis of Simplified Largazole Analogues as Isoform-Selective Human Lysine Deacetylase Inhibitors.

Selective inhibition of KDAC isoforms while maintaining potency remains a challenge. Using the largazole macrocyclic depsipeptide structure as a starting point for developing new KDACIs with increased selectivity, a combination of four different simplified largazole analogue (SLA) scaffolds with diverse zinc-binding groups (for a total of 60 compounds) were designed, synthesized, and evaluated against class I KDACs 1, 3, and 8, and class II KDAC6. Experimental evidence as well as molecular docking poses converged to establish the cyclic tetrapeptides (CTPs) as the primary determinant of both potency and selectivity by influencing the correct alignment of the zinc-binding group in the KDAC active site, providing a further basis for developing new KDACIs of higher isoform selectivity and potency.

Exploring the consequences of a representative "disallowed" conformation of Aib on a 310-helical fold.

The structural effects of a representative "disallowed" conformation of Aib on the 3(10)-helical fold of an octapeptidomimetic are explored. The 1D ((1)H, (13)C) & 2D NMR, FT-IR and CD data reveal that the octapeptide 1, adopts a 3(10)-helical conformation in solution, as it does in its crystal structure. The C-terminal methyl carboxylate (CO2Me) of 1 was modified into an 1,3-oxazine (Oxa) functional group in the peptidomimetic 2. This modification results in the stabilization of the backbone of the C-terminal Aib (Aib*-Oxa) of 2, in a conformation (ϕ, ψ = 180, 0) that is natively disallowed to Aib. Consequent to the presence of this natively disallowed conformation, the 3(10)-helical fold is not disrupted in the body of the peptidomimetic 2. But the structural distortions that do occur in 2 are primarily in residues in the immediate vicinity of the natively disallowed conformation, rather than in the whole peptide body. Non-native electronic effects resulting from modifications in backbone functional groups can be at the origin of stabilizing residues in natively disallowed conformations.

Steric and electronic interactions controlling the cis/trans isomer equilibrium at X-Pro tertiary amide motifs in solution.

A systematic understanding of the noncovalent interactions that influence the structures of the cis conformers and the equilibrium between the cis and the trans conformers, of the X-Pro tertiary amide motifs, is presented based on analyses of (1)H-, (13)C-NMR and FTIR absorption spectra of two sets of homologous peptides, X-Pro-Aib-OMe and X-Pro-NH-Me (where X is acetyl, propionyl, isobutyryl and pivaloyl), in solvents of varying polarities. First, this work shows that the cis conformers of any X-Pro tertiary amide motif, including Piv-Pro, are accessible in the new motifs X-Pro-Aib-OMe, in solution. These conformers are uniquely observable by FTIR spectroscopy at ambient temperatures and by NMR spectroscopy from temperatures as high as 273 K. This is made possible by the persistent presence of n(i-1) →π(i)* interactions at Aib, which also influence the disappearance of steric effects at these cis X-Pro rotamers. Second, contrary to conventional understanding, the energy contribution of steric effects to the cis/trans equilibrium at the X-Pro motifs is found to be nonvariant (0.54 ± 0.02 kcal/mol) with increase in steric bulk on the X group. Third, the current studies provide direct evidence for the weak intramolecular interactions namely the n(i-1) →π(i)*, the N(Pro) •••H(i+1) (C5a), and the C7 hydrogen bond that operate and influence the structures, stabilities, and dynamics between different conformational states of X-Pro tertiary amide motifs. NMR and IR spectral data suggest that the cis conformers of X-Pro motifs are ensembles of short-lived rotamers about the C'(X)-N(Pro) bond.

Accessing the disallowed conformations of peptides employing amide-to-imidate modification.

Selective modification of the C-terminal amide in peptides to dihydrooxazine (a novel stable imidate isostere) by intramolecular nucleophilic cyclo-O-alkylation of the corresponding N-(3-bromopropyl)amides results in constraining of the C-terminal residue in natively disallowed conformations both in crystals and in solution.

Synthesis and isolation of 5,6-dihydro-4H-1,3-oxazine hydrobromides by autocyclization of N-(3-bromopropyl)amides.

5,6-Dihydro-4H-1,3-oxazine hydrobromides have been synthesized by the nucleophilic autocyclo-O-alkylation of N-(3-bromopropyl)amides under neutral conditions in chloroform. It is found that electron-donating amide α-substituents influence the autocyclization efficiency.

N-{1-[(3-Bromo-prop-yl)amino-carbon-yl]eth-yl}-2-(2-nitro-benzene-sulfonamido)propionamide.

In the title compound, C(15)H(21)BrN(4)O(6)S, all three NH groups are involved in inter-molecular N-H⋯O inter-actions which, together with two inter-molecular C-H⋯O contacts, lead to a continuous anti-parallel ß-sheet structure. There are no π-π inter-actions between mol-ecules, and two C-H⋯π inter-actions primarily govern the linkage between sheets.