Simple azo‐based salicylaldimine as colorimetric and fluorescent probe for detecting anions in semi‐aqueous medium

A series of novel, highly sensitive, and selective azo‐based anion sensors 1–3 have been designed and synthesized from the condensation reaction between 4‐amino azo benzene and three different aldehydes. The structure of the sensors 1–3 were confirmed by IR, HRMS, ¹H NMR, and ¹³C NMR spectroscopic methods. Colorimetric naked‐eye analysis revealed the anion detection by receptors 2 and 3 as color changes from yellow to pink and yellow to orange, respectively. Anion sensing ability of all receptors was further investigated by ¹H NMR titration, UV‐vis experiment, and fluorescence titration. UV‐vis measurements highly indicate the selective recognition of fluoride and acetate ions in 9:1 dimethyl sulfoxide–H₂O (v/v) for receptors 2 and 3. Binding constant value showed among all receptors, receptor 3 has strong affinity toward F^? and AcO^? in semi‐aqueous medium, which is due to the presence of chromogenic signaling unit in it. The F^? ion detection property of receptor 2 in organic medium was also extended in the real sample like toothpaste. Copyright © 2013 John Wiley & Sons, Ltd.     

Paramagnetic relaxation enhancement‐assisted structural characterization of a partially disordered conformation of ubiquitin

Nuclear magnetic resonance (NMR) is a powerful tool to study three‐dimensional structures as well as protein conformational fluctuations in solution, but it is compromised by increases in peak widths and missing signals. We previously reported that ubiquitin has two folded conformations, N₁ and N₂ and plus another folded conformation, I, in which some amide group signals of residues 33–41 almost disappeared above 3 kbar at pH 4.5 and 273 K. Thus, well‐converged structural models could not be obtained for this region owing to the absence of distance restraints. Here, we reexamine the problem using the ubiquitin Q41N variant as a model for this locally disordered conformation, I. We demonstrate that the variant shows pressure‐induced loss of backbone amide group signals at residues 28, 33, 36, and 39–41 like the wild‐type, with a similar but smaller effect on CαH and CβH signals. In order to characterize this I structure, we measured paramagnetic relaxation enhancement (PRE) under high pressure to obtain distance restraints, and calculated the structure assisted by Bayesian inference. We conclude that the more disordered I conformation observed at pH 4.0, 278 K, and 2.5 kbar largely retained the N₂ conformation, although the amide groups at residues 33–41 have more heterogeneous conformations and more contact with water, which differ from the N₁ and N₂ states. The PRE‐assisted strategy has the potential to improve structural characterization of proteins that lack NMR signals, especially for relatively more open and hydrated protein conformations.     

Solution structure for an Encephalitozoon cuniculi adrenodoxin‐like protein in the oxidized state

Encephalitozoon cuniculi is a unicellular, obligate intracellular eukaryotic parasite in the Microsporidia family and one of the agents responsible for microsporidosis infections in humans. Like most Microsporidia, the genome of E. cuniculi is markedly reduced and the organism contains mitochondria‐like organelles called mitosomes instead of mitochondria. Here we report the solution NMR structure for a protein physically associated with mitosome‐like organelles in E. cuniculi, the 128‐residue, adrenodoxin‐like protein Ec‐Adx (UniProt ID Q8SV19) in the [2Fe‐2S] ferredoxin superfamily. Oxidized Ec‐Adx contains a mixed four‐strand β‐sheet, β2‐β1‐β4‐β3 (↓↑↑↓), loosely encircled by three α‐helices and two 3₁₀‐helices. This fold is similar to the structure observed in other adrenodoxin and adrenodoxin‐like proteins except for the absence of a fifth anti‐parallel β‐strand next to β3 and the position of α3. Cross peaks are missing or cannot be unambiguously assigned for 20 amide resonances in the ¹H‐¹⁵N HSQC spectrum of Ec‐Adx. These missing residues are clustered primarily in two regions, G48‐V61 and L94‐L98, containing the four cysteine residues predicted to ligate the paramagnetic [2Fe‐2S] cluster. Missing amide resonances in ¹H‐¹⁵N HSQC spectra are detrimental to NMR‐based solution structure calculations because ¹H‐¹H NOE restraints are absent (glass half‐empty) and this may account for the absent β‐strand (β5) and the position of α3 in oxidized Ec‐Adx. On the other hand, the missing amide resonances unambiguously identify the presence, and immediate environment, of the paramagnetic [2Fe‐2S] cluster in oxidized Ec‐Adx (glass half‐full).     

Structure dissection of zebrafish progranulins identifies a well‐folded granulin/epithelin module protein with pro‐cell survival activities

The ancient and pluripotent progranulins contain multiple repeats of a cysteine‐rich sequence motif of ∼60 amino acids, called the granulin/epithelin module (GEM) with a prototypic structure of four β‐hairpins zipped together by six inter‐hairpin disulfide bonds. Prevalence of this disulfide‐enforced structure is assessed here by an expression screening of 19 unique GEM sequences of the four progranulins in the zebrafish genome, progranulins 1, 2, A and B. While a majority of the expressed GEM peptides did not exhibit uniquely folded conformations, module AaE from progranulin A and AbB from progranulin B were found to fold into the protopypic 4‐hairpin structure along with disulfide formation. Module AaE has the most‐rigid three‐dimensional structure with all four β‐hairpins defined using high‐resolution (H–¹⁵N) NMR spectroscopy, including 492 inter‐proton nuclear Overhauser effects, 23 ³J(HN,H_α) coupling constants, 22 hydrogen bonds as well as 45 residual dipolar coupling constants. Three‐dimensional structure of AaE and the partially folded AbB re‐iterate the conformational stability of the N‐terminal stack of two beta‐hairpins and varying degrees of structural flexibility for the C‐terminal half of the 4‐hairpin global fold of the GEM repeat. A cell‐based assay demonstrated a functional activity for the zebrafish granulin AaE in promoting the survival of neuronal cells, similarly to what has been found for the corresponding granulin E module in human progranulin. Finally, this work highlights the remaining challenges in structure‐activity studies of proteins containing the GEM repeats, due to the apparent prevalence of structural disorder in GEM motifs despite potentially a high density of intramolecular disulfide bonds.     

A Lithium Amide‐Borohydride Solid‐State Electrolyte with Lithium‐Ion Conductivities Comparable to Liquid Electrolytes

High ionic conductivity of up to 6.4 × 10^?3 S cm^?1 near room temperature (40 °C) in lithium amide‐borohydrides is reported, comparable to values of liquid organic electrolytes commonly employed in lithium‐ion batteries. Density functional theory is applied coupled with X‐ray diffraction, calorimetry, and nuclear magnetic resonance experiments to shed light on the conduction mechanism. A Li₄Ti₅O₁₂ half‐cell battery incorporating the lithium amide‐borohydride electrolyte exhibits good rate performance up to 3.5 mA cm^?2 (5 C) and stable cycling over 400 cycles at 1 C at 40 °C, indicating high bulk and interfacial stability. The results demonstrate the potential of lithium amide‐borohydrides as solid‐state electrolytes for high‐power lithium‐ion batteries.     

Efficient synthesis of polypeptide‐α‐thioester by the method combining polypeptide expression and chemical activation for the semi‐synthesis of interferon‐γ having oligosaccharides

In order to synthesize interferon‐γ glycoform having an oligosaccharide at the 97 position by a semi‐synthetic method, interferon‐γ‐polypeptide‐(1–94)‐α‐hydrazide was prepared by the specific Cys‐cyanylation of polypeptide‐(1–94)‐Cys‐His₆ expressed from E. coli and subsequent hydrazinolysis in 22% yield (two steps). This polypeptide‐α‐hydrazide was then converted into corresponding polypeptide‐α‐thioester under NaNO₂/acid conditions followed by thiolysis in 83% yield. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Further crystallographic evidence of NH· · ·π (system) and CO· · ·π (system) interactions: The structures of bis(diarylhydrazonecarbonyl)methylene derivatives [{ArPhCNNH—C(O)}2CH2] (Ar = Ph, 2‐C5H4N, 2‐C4H3S)

In this study are reported the syntheses of three bis(diarylhydrazonecarbonyl)methylene derivatives [{ArPhCNNH C(O)}₂CH₂] [Ar = 2 C₅H₄N (5), C₆H₅ (6), and 2‐C₄H₃S (7)], obtained by condensation of corresponding hydrazones with carbon suboxide, C₃O₂. The solid‐state self‐assembly of these carbonyl derivatives, giving rise to polymeric and dimeric networks, is described. In the formation of these structural features, in addition to N—H· · ·OC intermolecular hydrogen bonds, stabilizing intramolecular NH· · · π (systems) and intermolecular CO· · ·π (systems) interactions also seem to play an important role. Solution ¹H‐nmr data of compounds 5–7 indicate that the polymeric and dimeric structures are not maintained in solution and show the occurrence of keto‐enolic equilibria. © 1999 John Wiley & Sons, Inc. Biopoly 49: 541–549, 1999     

High-level expression and purification of Cys-loop ligand-gated ion channels in a tetracycline-inducible stable mammalian cell line: GABAA and serotonin receptors

The human neuronal Cys‐loop ligand‐gated ion channel superfamily of ion channels are important determinants of human behavior and the target of many drugs. It is essential for their structural characterization to achieve high‐level expression in a functional state. The aim of this work was to establish stable mammalian cell lines that enable high‐level heterologous production of pure receptors in a state that supports agonist‐induced allosteric conformational changes. In a tetracycline‐inducible stable human embryonic kidney cells (HEK293S) cell line, GABA_A receptors containing α1 and β3 subunits could be expressed with specific activities of 29–34 pmol/mg corresponding to 140–170 pmol/plate, the highest expression level reported so far. Comparable figures for serotonin (5‐HT_3A) receptors were 49–63 pmol/mg and 245–315 pmol/plate. The expression of 10 nmol of either receptor in suspension in a bioreactor required 0.3–3.0 L. Both receptor constructs had a FLAG epitope inserted at the N‐terminus and could be purified in one step after solubilization using ANTI‐FLAG affinity chromatography with yields of 30–40%. Purified receptors were functional. Binding of the agonist [³H]muscimol to the purified GABA_AR was enhanced allosterically by the general anesthetic etomidate, and purified 5‐hydroxytryptamine‐3A receptor supported serotonin‐stimulated cation flux when reconstituted into lipid vesicles.     

Toward engineering efficient peptidomimetics. Screening conformational landscape of two modified dehydroaminoacids

Effective peptidomimetics should posses structural rigidity and appropriate interaction pattern leading to potential spatial and electronic matching to the target receptor site. Rational design of such small bioactive molecules could push chemical synthesis and molecular modeling toward faster progress in medicinal chemistry. Conformational properties of N‐t‐butoxycarbonyl‐glycine‐(E/Z)‐dehydrophenylalanine N′,N′‐dimethylamides (Boc‐Gly‐(E/Z)‐ΔPhe‐NMe₂) in chloroform were studied by NMR and IR spectroscopy. The experimental findings were supported by extensive calculations at DFT(B3LYP, M06‐2X) and MP2 levels of theory and the β‐turn tendency for both isomers of the studied dipeptide were determined in vacuum and in solution. The theoretical data and experimental IR results were used as an additional information for the NMR‐based determination of the detailed solution conformations of the peptides. The obtained results reveal that N‐methylation of C‐terminal amide group changes dramatically the conformational properties of studied dehydropeptides. Theoretical conformational analysis reveals that the tendency to adopt β‐turn conformations is much weaker for the N‐methylated Z isomer (Boc‐Gly‐(Z)‐ΔPhe‐NMe₂), both in vacuum and in polar environment. On the contrary, N‐methylated E isomer (Boc‐Gly‐(E)‐ΔPhe‐NMe₂) can easily adopt β‐turn conformation, but the backbone torsion angles (φ₁, ψ₁, φ₂, ψ₂) are off the limits for common β‐turn types. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 28–40, 2014.     

Crystal structure of a 3B3 variant—A broadly neutralizing HIV‐1 scFv antibody

We present the crystal structure determination of an anti‐HIV‐1 gp120 single‐chain variable fragment antibody variant, 3B3, at 2.5 Å resolution. This 3B3 variant was derived from the b12 antibody, using phage display and site‐directed mutagenesis of the variable heavy chain (V_H) complementary‐determining regions (CDRs). 3B3 exhibits enhanced binding affinity and neutralization activity against several cross‐clade primary isolates of HIV‐1 by interaction with the recessed CD4‐binding site on the gp120 envelope protein. Comparison with the structures of the unbound and bound forms of b12, the 3B3 structure closely resembles these structures with minimal differences with two notable exceptions. First, there is a reorientation of the CDR‐H3 of the V_H domain where the primary sequences evolved from b12 to 3B3. The structural changes in CDR‐H3 of 3B3, in light of the b12‐gp120 complex structure, allow for positioning an additional Trp side chain in the binding interface with gp120. Finally, the second region of structural change involves two peptide bond flips in CDR‐L3 of the variable light (V_L) domain triggered by a point mutation in CDR‐H3 of Q100eY resulting in changes in the intramolecular hydrogen bonding patterning between the V_L and V_H domains. Thus, the enhanced binding affinities and neutralization capabilities of 3B3 relative to b12 probably result from higher hydrophobic driving potential by burying more aromatic residues at the 3B3‐gp120 interface and by indirect stabilization of intramolecular contacts of the core framework residues between the V_L and V_H domains possibly through more favorable entropic effect through the expulsion of water.     

Isostructural unbranched alkyl‐chains as tools for stabilizing β‐turn structure

To investigate the structural role played by isostructural unbranched alkyl‐chains on the conformational ensemble and stability of β‐turn structures, the conformational properties of a designed model peptide: Plm‐Pro‐Gly‐Pda ( 1 , Plm: H₃C—(CH₂)₁₄—CONH—; Pda: —CONH— (CH₂)₁₄—CH₃) have been examined and compared with the parent peptide: Boc‐Pro‐Gly‐NHMe ( 2 , Boc: tert‐butoxycarbonyl; NHMe: N‐methylamide). The characteristic ¹³C NMR chemical‐shifts of the Pro C^β and C^γ resonances ascertained the incidence of an all‐trans peptide‐bond in low polarity deuterochloroform solution. Using FTIR and ¹H NMR spectroscopy, we establish that apolar alkyl‐chains flanking a β‐turn promoting Pro‐Gly sequence impart definite incremental stability to the well‐defined hydrogen‐bonded structure. The assessment of ¹H NMR derived thermodynamic parameters of the hydrogen‐bonded amide‐NHs via variable temperature indicate that much weaker hydrophobic interactions do contribute to the stability of folded reverse turn structures. The far‐UV CD spectral patterns of 1 and 2 in 2,2,2‐trifluoroethanol are consistent with Pro‐Gly specific type II β‐turn structure, concomitantly substantiate that the flanking alkyl‐chains induce substantial bias in enhanced β‐turn populations. In view of structural as well as functional importance of the Pro‐Gly mediated secondary structures, besides biochemical and biological significance of proteins lipidation via myristoylation or palmytoilation, we highlight potential convenience of the unbranched Plm and Pda moieities not only as main‐chain N‐ and C‐terminal protecting groups but also to mimic and stabilize specific isolated secondary and supersecondary structural components frequently observed in proteins and polypeptides. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 419–426, 2013.     

Amide-linkage formed between ammonia plasma treated poly(D,L-lactide acid) scaffolds and bio-peptides: enhancement of cell adhesion and osteogenic differentiation in vitro

The surface characteristics of scaffolds for bone tissue engineering must support cell adhesion, migration, proliferation, and osteogenic differentiation. In the study, poly(D,L ‐lactide acid) (PDLLA) scaffolds were modified by combing ammonia (NH₃) plasma pretreatment with Gly‐Arg‐Gly‐Asp‐Ser (GRGDS)‐peptides coupling technologies. The x‐ray photoelectron spectroscopy (XPS) survey spectra showed the peak of N1s at the surface of NH₃ plasma pretreated PDLLA, which was further raised after GRGDS conjugation. Furthermore, N1s and C1s in the high‐resolution XPS spectra revealed the presence of ? C?N (imine), ? C? NH? (amine), and ? C?O? NH? (amide) groups. The GRGDS conjugation increased amide groups and decreased amine groups in the plasma‐treated PDLLA. Confocal microscope and high performance liquid chromatography verified the anchored peptides after the conjugation process. Bone marrow mesenchymal stem cells were co‐cultured with scaffolds. Fluorescent microscope and scanning electron microscope photographs revealed the best cell adhesion in NH₃ plasma pretreated and GRGDS conjugated scaffolds, and the least attachment in unmodified scaffolds. Real‐time PCR demonstrated that expression of osteogenesis‐related genes, such as osteocalcin, alkaline phosphatase, type I collagen, bone morphogenetic protein‐2 and osteopontin, was upregulated in the single NH₃ plasma treated and NH₃ plasma pretreated scaffolds following GRGDS conjugation. The results show that NH₃ plasma treatment promotes the conjugation of GRGDS peptides to the PDLLA scaffolds via the formation of amide linkage, and combination of NH₃ plasma treatment and peptides conjugation may enhance the cell adhesion and osteogenic differentiation in the PDLLA scaffolds. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 682–694, 2011.     

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 ¹H‐, ¹³C‐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. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 66–77, 2014.     

Structural studies of the tethered N‐terminus of the Alzheimer's disease amyloid‐β peptide

Alzheimer's disease is the most common form of dementia in humans and is related to the accumulation of the amyloid‐β (Aβ) peptide and its interaction with metals (Cu, Fe, and Zn) in the brain. Crystallographic structural information about Aβ peptide deposits and the details of the metal‐binding site is limited owing to the heterogeneous nature of aggregation states formed by the peptide. Here, we present a crystal structure of Aβ residues 1–16 fused to the N‐terminus of the Escherichia coli immunity protein Im7, and stabilized with the fragment antigen binding fragment of the anti‐Aβ N‐terminal antibody WO2. The structure demonstrates that Aβ residues 10–16, which are not in complex with the antibody, adopt a mixture of local polyproline II‐helix and turn type conformations, enhancing cooperativity between the two adjacent histidine residues His13 and His14. Furthermore, this relatively rigid region of Aβ (residues, 10–16) appear as an almost independent unit available for trapping metal ions and provides a rationale for the His13‐metal‐His14 coordination in the Aβ_1–16 fragment implicated in Aβ metal binding. This novel structure, therefore, has the potential to provide a foundation for investigating the effect of metal ion binding to Aβ and illustrates a potential target for the development of future Alzheimer's disease therapeutics aimed at stabilizing the N‐terminal monomer structure, in particular residues His13 and His14, and preventing Aβ metal‐binding‐induced neurotoxicity.Proteins 2013; 81:1748–1758. © 2013 Wiley Periodicals, Inc.     

Determination of an unusual secondary structural element in the immunostimulating tetrapeptide rigin in aqueous environments: insights via MD simulations, 1H NMR and CD spectroscopic studies

An immunomodulating tetrapeptide, rigin (H‐Gly‐Gln‐Pro‐Arg‐OH), has been examined for its secondary structure preferences through combined use of high‐temperature unrestrained MD simulations in implicit water and 1D and 2D ¹H NMR spectroscopy. The distribution of backbone torsion angles revealed the predominance of trans conformers across the Xaa‐Pro peptide bond. The results of MD simulations revealed that of the five predicted families A–E, the predominant families, family A (92 structures), family C (63 structures) and family D (31 structures), could be complemented by extensive 1D and 2D ¹H NMR parameters acquired in aqueous PBS solution. A survey of specific inter‐ and intraresidue NOEs substantiated the predominance of an unusual type VII β‐turn structure, defined by two torsion angles, i.e. ψ_Gln ～ 155° and ?_Pro ～ ? 65° across the Gln‐Pro segment. The proposed semi‐folded kinked topology precluded formation of any intramolecular interaction, i.e. hydrogen bond or electrostatic interaction. Far‐UV CD spectral characteristics of rigin in aqueous PBS solution and non‐aqueous structure‐promoting organic solvents, TFE and TMP, revealed its strong solvent dependence. However, in aqueous PBS solution, the presence of a weak negative shoulder at nm could be ascribed to a small population with ordered, semi‐folded topology. We propose that the plausible structural attributes may be exploited for design and rigidification of the bioactive conformation of this immunomodulator toward improved immunopharmacological properties. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

VCD Robustness of the Amide‐I and Amide‐II Vibrational Modes of Small Peptide Models

The rotational strengths and the robustness values of amide‐I and amide‐II vibrational modes of For(AA)_nNHMe (where AA is Val, Asn, Asp, or Cys, n = 1–5 for Val and Asn; n = 1 for Asp and Cys) model peptides with α‐helix and β‐sheet backbone conformations were computed by density functional methods. The robustness results verify empirical rules drawn from experiments and from computed rotational strengths linking amide‐I and amide‐II patterns in the vibrational circular dichroism (VCD) spectra of peptides with their backbone structures. For peptides with at least three residues (n ≥ 3) these characteristic patterns from coupled amide vibrational modes have robust signatures. For shorter peptide models many vibrational modes are nonrobust, and the robust modes can be dependent on the residues or on their side chain conformations in addition to backbone conformations. These robust VCD bands, however, provide information for the detailed structural analysis of these smaller systems. Chirality 27:625–634, 2015 © 2015 Wiley Periodicals, Inc.     

Pyrazole amino acids: hydrogen bonding directed conformations of 3‐amino‐1H‐pyrazole‐5‐carboxylic acid residue

A series of model compounds containing 3‐amino‐1H‐pyrazole‐5‐carboxylic acid residue with N‐terminal amide/urethane and C‐terminal amide/hydrazide/ester groups were investigated by using NMR, Fourier transform infrared, and single‐crystal X‐ray diffraction methods, additionally supported by theoretical calculations. The studies demonstrate that the most preferred is the extended conformation with torsion angles ? and ψ close to ±180°. The studied 1H‐pyrazole with N‐terminal amide/urethane and C‐terminal amide/hydrazide groups solely adopts this energetically favored conformation confirming rigidity of that structural motif. However, when the C‐terminal ester group is present, the second conformation with torsion angles ? and ψ close to ±180° and 0°, respectively, is accessible. The conformational equilibrium is observed in NMR and Fourier transform infrared studies in solution in polar environment as well as in the crystal structures of other related compounds. The observed conformational preferences are clearly related to the presence of intramolecular interactions formed within the studied residue. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Chromatographic profiles of tryptophan and kynurenine enantiomers derivatized with (S)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole using LC–MS/MS on a triazole‐bonded column

d ‐ and l ‐Tryptophan (Trp) and d ‐ and l ‐kynurenine (KYN) were derivatized with a chiral reagent, (S )‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS), and were separated enantiomerically by high‐performance liquid chromatography (HPLC) equipped with a triazole‐bonded column (Cosmosil HILIC) using tandem mass spectrometric (MS/MS) detection. Effects of column temperature, salt (HCO₂NH₄) concentration, and pH of the mobile phase in the enantiomeric separation, followed by MS detection of (S )‐DBD‐PyNCS‐d ,l ‐Trp and ‐d ,l ‐KYN, were investigated. The mobile phase consisting of CH₃CN/10 mM ammonium formate in H₂O (pH 5.0) (90/10) with a column temperature of 50–60 °C gave satisfactory resolution (R s) and mass‐spectrometric detection. The enantiomeric separation of d ,l ‐Trp and d ,l ‐KYN produced R s values of 2.22 and 2.13, and separation factors (α) of 1.08 and 1.08, for the Trp and KYN enantiomers, respectively. The proposed LC–MS/MS method provided excellent detection sensitivity of both enantiomers of Trp and KYN (5.1–19 nM).     

Enantioselective anion exchangers based on cinchona alkaloid‐derived carbamates: Influence of C8/C9 stereochemistry on chiral recognition

Four diastereomeric chiral stationary phases (CSPs) based on quinine, quinidine, epiquinine, and epiquinidine tert‐butyl carbamate selectors were synthesized and evaluated under ion exchange HPLC conditions with a set of racemic N‐acylated and N‐oxycarbonylated α‐amino acids as selectands. The enantioseparation potential of quinine‐ and quinidine‐derived CSPs proved to be far superior to that of their C₉‐epimeric congeners. The absolute configuration of C₉ stereogenic center of the cinchonan backbone of these selectors was identified as the structural feature controlling the elution order. Guided by an X‐ray structure of a most favorable selector–selectand complex and the observed chromatographic enantioseparation data, a chiral recognition model was advanced. The contributions of ion‐pairing, π–π donor–acceptor, hydrogen bonding and steric interactions were established as crucial factors. Chirality 11:522–528, 1999. © 1999 Wiley‐Liss, Inc.     

Characterization of a type of β‐bend ribbon spiral generated by the repeating (Xaa–Yaa–Aib–Pro) motif: The solution structure of harzianin HC IX,a 14‐residue peptaibol forming voltage‐dependent ion channels

The three‐dimensional solution structure of harzianin HC IX, a peptaibol antibiotic isolated from the fungus Trichoderma harzianum, was determined using CD, homonuclear, and heteronuclear two‐dimensional nmr spectroscopy combined with molecular modeling. This 14‐residue peptide, Ac Aib¹ Asn² Leu³ Aib⁴ Pro⁵ Ala⁶ Ile⁷ Aib⁸ Pro⁹ Iva¹⁰ Leu¹¹ Aib¹² Pro¹³ Leuol¹⁴ (Aib, α‐aminoisobutyric acid; Iva, isovaline; Leuol, leucinol), is a main representative of a short‐sequence peptaibol class characterized by an acetylated N‐terminus, a C‐terminal amino alcohol, and the presence of three Aib‐L ‐Pro motifs at positions 4–5, 8–9, and 12–13, separated by two dipeptide units. In spite of a lower number of residues, compared to the 18/20‐residue peptaibols such as alamethicin, harzianin HC IX exhibits remarkable membrane‐perturbing properties. It interacts with phospholipid bilayers, increasing their permeability and forming voltage‐gated ion channels through a mechanism slightly differing from that proposed for alamethicin. Sequence‐specific ¹H‐ and ¹³C‐nmr assignments and conformational nmr parameters (³J_NHCαH coupling constants, quantitative nuclear Overhauser enhancement data, temperature coefficients of amide and carbonyl groups, NH–ND exchange rates) were obtained in methanol solution. Sixty structures were calculated based on 98 interproton distance restraints and 6 Φ dihedral angle restraints, using high temperature restrained molecular dynamics and energy minimization. Thirty‐seven out of the sixty generated structures were consistent with the nmr data and were convergent. The peptide backbone consists in a ribbon of overlapping β‐turns twisted into a continuous spiral from Asn² to Leuol¹⁴ and forming a 26 Å long helix‐like structure. This structure is slightly amphipathic, with the three Aib–Pro motifs aligned on the less hydrophobic face of the spiral where the Asn² side chain is also present, while the more hydrophobic bulky side chains of leucines, isoleucine, isovaline, and leucinol are located on the concave side. The repetitive (Xaa–Yaa–Aib–Pro) tetrapeptide subunit, making up the peptide sequence, is characterized by four sets of (Φ,Ψ) torsional angles, with the following mean values: Φ_i = −90°, Ψ_i = −27°; Φ_i+1 = −98°, Ψ_i+1 = −17°; Φ_i+2 = −49°, Ψ_i+2 = −50°; Φ_i+3 = −78°, Ψ_i+3 = +3°. We term this particular structure, specifically occurring in the case of (Xaa–Yaa–Aib–Pro)_n sequences, the (Xaa–Yaa–Aib–Pro)‐β‐bend ribbon spiral. It is stabilized by 4 → 1 intramolecular hydrogen bonds and differs from both the canonical 3₁₀‐helix made of a succession of type III β‐turns and from the β‐bend ribbon spiral that has been described in the case of (Aib–Pro)n peptide segments. © 1999 John Wiley & Sons, Inc. Biopoly 50: 71–85, 1999