Radiolabeling and in vitro /in vivo evaluation of N‐(1‐adamantyl)‐8‐methoxy‐4‐oxo‐1‐phenyl‐1,4‐dihydroquinoline‐3‐carboxamide as a PET probe for imaging cannabinoid type 2 receptor

The cannabinoid type 2 (CB₂) receptor plays an important role in neuroinflammatory and neurodegenerative diseases such as multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer's disease and is therefore a very promising target for therapeutic approaches as well as for imaging. Based on the literature, we identified one 4‐oxoquinoline derivative (designated KD2) as the lead structure. It was synthesized, radiolabeled and evaluated as a potential imaging tracer for CB₂. [¹¹C]KD2 was obtained in 99% radiochemical purity. Moderate blood–brain barrier (BBB) passage was predicted for KD2 from an in vitro transport assay with P‐glycoprotein‐transfected Madin Darby canine kidney cells. No efflux of KD2 by P‐glycoprotein was detected. In vitro autoradiography of rat and mouse spleen slices demonstrated that [¹¹C]KD2 exhibits high specific binding towards CB₂. High spleen uptake of [¹¹C]KD2 was observed in dynamic positron emission tomography (PET) studies with Wistar rats and its specificity was confirmed by displacement study with a selective CB₂ agonist, GW405833. A pilot autoradiography study with post‐mortem spinal cord slices from amyotrophic lateral sclerosis (ALS) patients with [¹¹C]KD2 suggested the presence of CB₂ receptors under disease conditions. Specificity of [¹¹C]KD2 binding could also be demonstrated on these human tissues. In conclusion, [¹¹C]KD2 shows good in vitro and in vivo properties as a potential PET tracer for CB₂.

     

HIGH‐RESOLUTION MAGIC ANGLE SPINNING NMR ANALYSIS OF WHOLE CELLS OF CHAETOCEROS MUELLERI (BACILLARIOPHYCEAE) AND COMPARISON WITH 13C‐NMR AND DISTORTIONLESS ENHANCEMENT BY POLARIZATION TRANSFER 13C‐NMR ANALYSIS OF LIPOPHILIC EXTRACTS1

Lipid composition in extracted samples of Chaetoceros muelleri Lemmermann was studied with ¹³C‐NMR and distortionless enhancement by polarization transfer (DEPT) ¹³C‐NMR, resulting in well‐resolved ¹³C‐NMR spectra with characteristic resonance signals from carboxylic, olefinic, glyceryl, methylene, and methyl groups. The application of a DEPT pulse sequence aided in the assignment of methylene and methine groups. Resonance signals were compared with literature references, and signal assignment included important unsaturated fatty acids such as eicosapentaenoic and docosahexaenoic and also phospholipids and glycerols. Results from the extracted samples were used to assign resonance signals in a high‐resolution magic angle spinning (HR MAS) DEPT ¹³C spectrum from whole cells of C. muelleri. The NMR analysis on whole cells yielded equally good information on fatty acids and also revealed signals from carbohydrates and amino acids. Broad resonance signals and peak overlapping can be a problem in whole cell analysis, but we found that application of HR MAS gave a well‐resolved spectrum. The chemical shift of metabolites in an NMR spectrum depends on the actual environment of nuclei during analysis, and some differences could therefore be expected between extracted and whole cell samples. The shift differences were small, and assignment from analysis of lipophilic extract could be used to identify peaks in the whole cell spectrum. HR MAS ¹³C‐NMR therefore offers a possibility for broad‐range metabolic profiling directly on whole cells, simultaneously detecting metabolites that are otherwise not detected in the same analytical set up and avoiding tedious extraction procedures.     

Mesophyll CO2 conductance and leakiness are not responsive to short‐ and long‐term soil water limitations in the C4 plant Sorghum bicolor

Breeding economically important C₄ crops for enhanced whole‐plant water‐use efficiency (WUE_plant) is needed for sustainable agriculture. WUE_plant is a complex trait and an efficient phenotyping method that reports on components of WUE_plant, such as intrinsic water‐use efficiency (WUE_i, the rate of leaf CO₂ assimilation relative to water loss via stomatal conductance), is needed. In C₄ plants, theoretical models suggest that leaf carbon isotope composition (δ¹³C), when the efficiency of the CO₂‐concentrating mechanism (leakiness, ?) remains constant, can be used to screen for WUE_i. The limited information about how ? responds to water limitations confines the application of δ¹³C for WUE_i screening of C₄ crops. The current research aimed to test the response of ? to short‐ or long‐term moderate water limitations, and the relationship of δ¹³C with WUE_i and WUE_plant, by addressing potential mesophyll CO₂ conductance (g_m) and biochemical limitations in the C₄ plant Sorghum bicolor. We demonstrate that g_m and ? are not responsive to short‐ or long‐term water limitations. Additionally, δ¹³C was not correlated with gas‐exchange estimates of WUE_i under short‐ and long‐term water limitations, but showed a significant negative relationship with WUE_plant. The observed association between the δ¹³C and WUE_plant suggests an intrinsic link of δ¹³C with WUE_i in this C₄ plant, and can potentially be used as a screening tool for WUE_plant in sorghum.     

Distinct solid and solution state self‐assembly pathways of RADA16‐I designer peptide

Solid state NMR measurements on selectively ¹³C‐labeled RADA16‐I peptide (COCH₃–RADARADARADARADA–NH₂) were used to obtain new molecular level information on the conversion of α‐helices to β‐sheets through self‐assembly in the solid state with increasing temperature. Isotopic labeling at the A4 C_β site enabled rapid detection of ¹³C NMR signals. Heating to 344–363 K with simultaneous NMR detection allowed production of samples with systematic variation of α‐helix and β‐strand content. These samples were then probed at room temperature for intermolecular ¹³C–¹³C nuclear dipolar couplings with the PITHIRDS‐CT NMR experiment. The structural transition was also characterized by Fourier transform infrared spectroscopy and wide angle X‐ray diffraction. Independence of PITHIRDS‐CT decay shapes on overall α‐helical and β‐strand content infers that β‐strands are not observed without association with β‐sheets, indicating that β‐sheets are formed at elevated temperatures on a timescale that is fast relative to the NMR experiment. PITHIRDS‐CT NMR data were compared with results of similar measurements on RADA16‐I nanofibers produced by self‐assembly in aqueous salt solution. We report that β‐sheets formed through self‐assembly in the solid state have a structure that differs from those formed through self‐assembly in the solution state. Specifically, solid state RADA16‐I self‐assembly produces in‐register parallel β‐sheets, whereas nanofibers are composed of stacked parallel β‐sheets with registry shifts between adjacent β‐strands in each β‐sheet. These results provide evidence for environment‐dependent self‐assembly mechanisms for RADA16‐I β‐sheets as well as new constraints on solid state self‐assembled structures, which must be avoided to maximize solution solubility and nanofiber yields. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Lipid interactions of acylated tryptophan‐methylated lactoferricin peptides by solid‐state NMR

Lactoferricin (LfB) is a 25‐residue innate immunity peptide released by pepsin from the N‐terminal region of bovine lactoferrin. A smaller amidated peptide, LfB6 (RRWQWR‐NH₂) retains antimicrobial activity and is thought to constitute the “antimicrobial active‐site” (Tomita, Acta Paediatr Jpn. 1994; 36 : 585–91). Here we report on N‐acylation of 1‐Me‐Trp⁵‐LfB6, Cn‐RRWQ[1‐Me‐W]R‐NH₂, where Cn is an acyl chain having n = 0, 2, 4, 6 or 12 carbons. Tryptophan 5 (Trp⁵) was methylated to enhance membrane binding and to allow for selective deuteration at that position. Peptide/lipid interactions of Cn‐RRWQ[1‐Me‐W ]R‐NH₂ (deuterated 1‐Me‐Trp⁵ underlined), were monitored by solid state ³¹P NMR and ²H NMR. The samples consisted of macroscopically oriented bilayers of mixed neutral (dimyristoylphosphatidylcholine, DMPC) and anionic (dimyristoylphosphatidylglycerol, DMPG) lipids in a 3:1 ratio with Cn‐RRWQ[&1‐Me‐W ]R‐NH₂ peptides added at a 1:25 peptide to lipid ratio. ²H‐NMR spectra reveal that the acylated peptides are well aligned in DMPC:DMPG bilayers. The ²H NMR quadrupolar splittings suggest that the 1‐Me‐Trp is located in a motionally restricted environment, indicating partial alignment at the membrane interface. ³¹P‐NMR spectra reveal that the lipids are predominantly in a bilayer configuration, with little perturbation by the peptides. Methylation alone, in C0‐RRWQ[1‐Me‐W ]R‐NH₂, resulted in a 3–4 fold increase in antimicrobial activity against E. coli. N‐acylation with a C12 fatty acid enhanced activity almost 90 fold. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Mapping interactions of gastric inhibitory polypeptide with GIPR N‐terminus using NMR and molecular dynamics simulations

Glucose‐dependent insulinotropic polypeptide (gastric inhibitory polypeptide, or GIP), a 42‐amino acid incretin hormone, modulates insulin secretion in a glucose‐concentration‐dependent manner. Its insulinotropic action is highly dependent on glucose concentration that surmounts the hypoglycemia side effects associated with current therapy. In order to develop a GIP‐based anti‐diabetic therapy, it is essential to establish the 3D structure of the peptide and study its interaction with the GIP receptor (GIPR) in detail. This will give an insight into the GIP‐mediated insulin release process. In this article, we report the solution structure of GIP(1–42, human)NH₂ deduced by NMR and the interaction of the peptide with the N‐terminus of GIPR using molecular modelling methods. The structure of GIP(1–42, human)NH₂ in H₂O has been investigated using 2D‐NMR (DQF‐COSY, TOCSY, NOESY, ¹H‐¹³C HSQC) experiments, and its conformation was built by constrained MD simulations with the NMR data as constraints. The peptide in H₂O exhibits an α‐helical structure between residues Ser8 and Asn39 with some discontinuity at residues Gln29 to Asp35; the helix is bent at Gln29. This bent gives the peptide an ‘L’ shape that becomes more pronounced upon binding to the receptor. The interaction of GIP with the N‐terminus of GIPR was modelled by allowing GIP to interact with the N‐terminus of GIPR under a series of decreasing constraints in a molecular dynamics simulation, culminating with energy minimization without application of any constraints on the system. The canonical ensemble obtained from the simulation was subjected to a detailed energy analysis to identify the peptide–protein interaction patterns at the individual residue level. These interaction energies shed some light on the binding of GIP with the GIPR N‐terminus in a quantitative manner. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Sequence‐dependent folding and unfolding of ligand‐bound purine riboswitches

Riboswitch regulation of gene expression requires ligand‐mediated RNA folding. From the fluorescence lifetime distribution of bound 2‐aminopurine ligand, we resolve three RNA conformers (C_o, C_i, C_c) of the liganded G‐ and A‐sensing riboswitches from Bacillus subtilis. The ligand binding affinities, and sensitivity to Mg²⁺, together with results from mutagenesis, suggest that C_o and C_i are partially unfolded species compromised in key loop‐loop interactions present in the fully folded C_c. These data verify that the ligand‐bound riboswitches may dynamically fold and unfold in solution, and reveal differences in the distribution of folded states between two structurally homologous purine riboswitches: Ligand‐mediated folding of the G‐sensing riboswitch is more effective, less dependent on Mg²⁺, and less debilitated by mutation, than the A‐sensing riboswitch, which remains more unfolded in its liganded state. We propose that these sequence‐dependent RNA dynamics, which adjust the balance of ligand‐mediated folding and unfolding, enable different degrees of kinetic discrimination in ligand binding, and fine‐tuning of gene regulatory mechanisms. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 953–965, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Water scaffolding in collagen: Implications on protein dynamics as revealed by solid‐state NMR

Solid‐state NMR studies of collagen samples of various origins confirm that the amplitude of collagen backbone and sidechain motions increases significantly on increasing the water content. This conclusion is supported by the changes observed in three different NMR observables: (i) the linewidth dependence on the ¹H decoupling frequency; (ii) ¹³C CSA changes for the peptide carbonyl groups, and (iii) dephasing rates of ¹H‐¹³C dipolar couplings. In particular, a nearly threefold increase in motional amplitudes of the backbone librations about C‐C^α or N‐C^α bonds was found on increasing the added water content up to 47 wt%D₂O. On the basis of the frequencies of NMR observables involved, the timescale of the protein motions dependent on the added water content is estimated to be of the order of microseconds. This estimate agrees with that from wideline T₂ ¹H NMR measurements. Also, our wideline ¹H NMR measurements revealed that the timescale of the microsecond motions in proteins reduces significantly on increasing the added water content, i.e., an ～15‐fold increase in protein motional frequencies is observed on increasing the added water content to 45 wt% D₂O. The observed changes in collagen dynamics is attributed to the increase in water translational diffusion on increasing the amount of added water, which leads to more frequent “bound water/free water” exchange on the protein surface, accompanied by the breakage and formation of new hydrogen bonds with polar functionalities of protein. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 246–256, 2014.     

Structural and dynamical characterization of tubular HIV‐1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy

The wild‐type HIV‐1 capsid protein (CA) self‐assembles in vitro into tubular structures at high ionic strength. We report solid state nuclear magnetic resonance (NMR) and electron microscopy measurements on these tubular CA assemblies, which are believed to contain a triangular lattice of hexameric CA proteins that is similar or identical to the lattice of capsids in intact HIV‐1. Mass‐per‐length values of CA assemblies determined by dark‐field transmission electron microscopy indicate a variety of structures, ranging from single‐wall tubes to multiwall tubes that approximate solid rods. Two‐dimensional (2D) solid state ¹³C? ¹³C and ¹⁵N? ¹³C NMR spectra of uniformly ¹⁵N,¹³C‐labeled CA assemblies are highly congested, as expected for a 25.6 kDa protein in which nearly the entire amino acid sequence is immobilized. Solid state NMR spectra of partially labeled CA assemblies, expressed in 1,3‐¹³C₂‐glycerol medium, are better resolved, allowing the identification of individual signals with line widths below 1 ppm. Comparison of crosspeak patterns in the experimental 2D spectra with simulated patterns based on solution NMR chemical shifts of the individual N‐terminal (NTD) and C‐terminal (CTD) domains indicates that NTD and CTD retain their individual structures upon self‐assembly of full‐length CA into tubes. 2D ¹H‐¹³C NMR spectra of CA assemblies recorded under solution NMR conditions show relatively few signals, primarily from segments that link the α‐helices of NTD and CTD and from the N‐ and C‐terminal ends. Taken together, the data support the idea that CA assemblies contain a highly ordered 2D protein lattice in which the NTD and CTD structures are retained and largely immobilized.     

Conformational studies of a bicyclic,lactam‐constrained parathyroid hormone‐related protein‐derived agonist

The N‐terminal 1–34 segments of both parathyroid hormone (PTH) and parathyroid hormone‐related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences in their amino acid sequence. The hypothesis was made that they share the same bioactive conformation when bound to the receptor. A common structural motif in all bioactive fragments of the hormone in water/trifluoroethanol mixtures or in aqueous solution containing detergent micelles is the presence of two helical segments at the N‐ and C‐termini of the sequence. In order to stabilize the helical structures, we have recently synthesized and studied the PTHrP(1–34) analog [(Lys13–As p¹⁷, Lys26–As p³⁰)]PTHrP(1–34)NH₂, which contains lactam‐constrained Lys‐Asp side chains at positions i, i+4. This very potent agonist exhibits enhanced helix stability with respect to the corresponding linear peptide and also two flexible sites at positions 12 and 19 in 1:1 trifluoroethanol/water. These structural elements have been suggested to play a critical role in bioactivity. In the present work we have extended our conformational studies on the bicyclic lactam‐constrained analog to aqueous solution. By CD, 2D‐NMR and structure calculations we have shown that in water two helical segments are present in the region of the lactam bridges (13–18, and 26–31) with high flexibility around Gly¹² and Arg¹⁹. Thus, the essential structural features observed in the aqueous‐organic medium are maintained in water even if, in this solvent, the overall structure is more flexible. Our findings confirm the stabilizing effect of side‐chain lactam constraints on the α‐helical structure. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

ω‐Turn: A novel β‐turn mimic in globular proteins stabilized by main‐chain to side‐chain CH···O interaction

Mimicry of structural motifs is a common feature in proteins. The 10‐membered hydrogen‐bonded ring involving the main‐chain C?O in a β‐turn can be formed using a side‐chain carbonyl group leading to Asx‐turn. We show that the N? H component of hydrogen bond can be replaced by a C^γ‐H group in the side chain, culminating in a nonconventional C? H···O interaction. Because of its shape this β‐turn mimic is designated as ω‐turn, which is found to occur ～three times per 100 residues. Three residues (i to i + 2) constitute the turn with the C? H···O interaction occurring between the terminal residues, constraining the torsion angles ?_{i + 1}, ψ_{i + 1}, ?_{i + 2} and χ′_{1(i + 2)} (using the interacting C^γ atom). Based on these angles there are two types of ω‐turns, each of which can be further divided into two groups. C^β‐branched side‐chains, and Met and Gln have high propensities to occur at i + 2; for the last two residues the carbonyl oxygen may participate in an additional interaction involving the S and amino group, respectively. With Cys occupying the i + 1 position, such turns are found in the metal‐binding sites. N‐linked glycosylation occurs at the consensus pattern Asn‐Xaa‐Ser/Thr; with Thr at i + 2, the sequence can adopt the secondary structure of a ω‐turn, which may be the recognition site for protein modification. Location between two β‐strands is the most common occurrence in protein tertiary structure, and being generally exposed ω‐turn may constitute the antigenic determinant site. It is a stable scaffold and may be used in protein engineering and peptide design. Proteins 2015; 83:203–214. © 2014 Wiley Periodicals, Inc.     

Synthesis,conformational study,and spectroscopic characterization of the cyclic Cα,α‐disubstituted glycine 9‐amino‐9‐fluorenecarboxylic acid

A series of terminally blocked peptides (to the pentamer level) from l ‐Ala and the cyclic C^α,α‐disubstituted Gly residue Afc and one Gly/Afc dipeptide have been synthesized by solution method and fully characterized. The molecular structure of the amino acid derivative Boc‐Afc‐OMe and the dipeptide Boc‐Afc‐Gly‐OMe were determined in the crystal state by X‐ray diffraction. In addition, the preferred conformation of all of the model peptides was assessed in deuterochloroform solution by FT‐IR absorption and ¹H‐NMR. The experimental data favour the conclusion that the Afc residue tends to adopt either the fully‐extended (C₅) or a folded/helical structure. In particular, the former conformation is highly populated in solution and is also that found in the crystal state in the two compounds investigated. A comparison with the structural propensities of the strictly related C^α,α‐disubstituted Gly residues Ac₅c and Dϕg is made and the implications for the use of the Afc residue in conformationally constrained analogues of bioactive peptides are briefly examined. A spectroscopic (UV absorption, fluorescence, CD) characterization of this novel aromatic C^α,α‐disubstituted Gly residue is also reported. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Axially chiral N‐(o‐aryl)‐4‐hydroxy‐2‐oxazolidinone derivatives from diastereoselective reduction of N‐(o‐aryl)‐2,4‐oxazolidinediones: Thermally interconvertible atropisomers via ring‐chain‐ring tautomerization

The reduction of the axially chiral N‐(o‐aryl)‐5,5‐dimethyl‐2,4‐oxazolidinediones by NaBH₄ yielded axially chiral N‐(o‐aryl)‐4‐hydroxy‐5,5‐dimethyl‐2‐oxazolidinone enantiomers having a chiral center at C‐4, with 100% diastereoselectivity as has been shown by their ¹H and ¹³C NMR spectra and by enantioselective HPLC analysis. The resolved enantiomeric isomers were found to interconvert thermally through an aldehyde intermediate formed upon ring cleavage via a latent ring‐chain‐ring tautomerization. It was found that the rate of enantiomerization depended on the size and the electronic effect of the ortho substituent present on the aryl ring bonded to the nitrogen of the heterocycle. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ-THC and related compounds: synthesis of selective ligands for the CB2 receptor

The synthesis and pharmacology of 15 1-deoxy-Δ⁸-THC analogues, several of which have high affinity for the CB₂ receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-Δ⁸-THC (5), 1-deoxy-Δ⁸-THC (6), 1-deoxy-3-butyl-Δ⁸-THC (7), 1-deoxy-3-hexyl-Δ⁸-THC (8) and a series of 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=0–4, 6, 7, where n=the number of carbon atoms in the side chain−2). Three derivatives (17–19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB₁ and CB₂ receptors were determined employing previously described procedures. Five of the 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=1–5) have high affinity (K_i=<20 nM) for the CB₂ receptor. Four of them (2, n=1–4) also have little affinity for the CB₁ receptor (K_i=>295 nM). 3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ⁸-THC (2, n=2) has very high affinity for the CB₂ receptor (K_i=3.4±1.0 nM) and little affinity for the CB₁ receptor (K_i=677±132 nM).

Scheme 3. (a) (C₆H₅)₃PCH₃⁺ Br⁻, n-BuLi/THF, 65°C; (b) LiAlH₄/THF, 25°C; (c) KBH(sec-Bu)₃/THF, −78 to 25°C then H₂O₂/NaOH.     

Novel N‐propylphthalimide‐ and 4‐vinylbenzyl‐substituted benzimidazole salts: Synthesis,characterization, and determination of their metal chelating effects and inhibition profiles against acetylcholinesterase and carbonic anhydrase enzymes

The novel N‐propylphthalimide‐substituted and 4‐vinylbenzyl‐substituted N‐heterocyclic carbene (NHC) precursors were synthesized by N‐substituted benzimidazolium with aryl halides. The novel N‐propylphthalimide‐substituted and 4‐vinylbenzyl‐substituted NHC precursors have been characterized by using ¹H NMR, ¹³C NMR, FTIR spectroscopy, and elemental analysis techniques. They were tested for the inhibition of AChE and hCA enzymes and demonstrated efficient inhibition profiles with K_i values in the range of 351.0–1269.9 nM against hCA I, 346.6–1193.1 nM against hCA II, and 19.0–76.3 nM against AChE. On the other hand, acetazolamide, a clinically used molecule, utilized as CA inhibitor, obtained a K_i value of 1246.7 nM against hCA I and 1407.6 nM against hCA II. Additionally, tacrine inhibited AChE and obtained a K_i value of 174.6 nM.     

Conformational evaluation of labeled C3′‐O‐P‐13CH2‐O‐C4″ phosphonate internucleotide linkage,a phosphodiester isostere

Modified internucleotide linkage featuring the C3′‐O‐P‐CH₂‐O‐C4″ phosphonate grouping as an isosteric alternative to the phosphodiester C3′‐O‐P‐O‐CH₂‐C4″ bond was studied in order to learn more on its stereochemical arrangement, which we showed earlier to be of prime importance for the properties of the respective oligonucleotide analogues. Two approaches were pursued: First, the attempt to prepare the model dinucleoside phosphonate with ¹³C‐labeled CH₂ group present in the modified internucleotide linkage that would allow for a more detailed evaluation of the linkage conformation by NMR spectroscopy. Second, the use of ab initio calculations along with molecular dynamics (MD) simulations in order to observe the most populated conformations and specify main structural elements governing the conformational preferences. To deal with the former aim, a novel synthesis of key labeled reagent (CH₃O)₂P(O)¹³CH₂OH for dimer preparation had to be elaborated using aqueous ¹³C‐formaldehyde. The results from both approaches were compared and found consistent. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 514–529, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The impact of β‐azido(or 1‐piperidinyl)methylamino acids in position 2 or 3 on biological activity and conformation of dermorphin analogues

The synthesis of new dermorphin analogues is described. The (R)‐alanine or phenylalanine residues of natural dermorphin were substituted by the corresponding α‐methyl‐β‐azidoalanine or α‐benzyl‐β‐azido(1‐piperidinyl)alanine residues. The potency and selectivity of the new analogues were evaluated by a competitive receptor binding assay in rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). The most active analogue in this series, Tyr‐(R)‐Ala‐(R)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ and its epimer were analysed by ¹H and ¹³C NMR spectroscopy and restrained molecular dynamics simulations. The dominant conformation of the investigated peptides depended on the absolute configuration around C^α in the α‐benzyl‐β‐azidoAla residue in position 3. The (R) configuration led to the formation of a type I β‐turn, whilst switching to the (S) configuration gave rise to an inverse β‐turn of type I′, followed by the formation of a very short β‐sheet. The selectivity of Tyr‐(R)‐Ala‐(R) and (S)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ was shown to be very similar; nevertheless, the two analogues exhibited different conformational preferences. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of 2‐amino‐3‐cyanopyridine derivatives and investigation of their carbonic anhydrase inhibition effects

The conversion of carbon dioxide (CO₂) and bicarbonate (HCO₃⁻) to each other is very important for living metabolism. Carbonic anhydrase (CA, E.C.4.2.1.1), a metalloenzyme familly, catalyzes the interconversion of these ions (CO₂ and HCO₃⁻) and are very common in living organisms. In this study, a series of novel 2‐amino‐3‐cyanopyridines supported with some functional groups was synthesized and tested as potential inhibition effects against both cytosolic human CA I and II isoenzymes (hCA I and II) using by Sepharose‐4B‐l ‐tyrosine‐sulfanilamide affinity chromatography. The structural elucidations of novel 2‐amino‐3‐cyanopyridines were achieved by NMR, IR, and elemental analyses. K _i values of the novel synthesized compounds were found in range of 2.84–112.44 μM against hCA I and 2.56–31.17 μM against hCA II isoenzyme. While compound 7d showed the best inhibition activity against hCA I (K _i: 2.84 μM), the compound 7b demonstrated the best inhibition profile against hCA II isoenzyme (K _i: 2.56 μM).     

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.     

Enantioselective recognition of carboxylic acids by novel fluorescent triazine‐based thiazoles

Hydrogen bonding and π‐π interactions take special part in the enantioselectivity task. In this regard, because of having both hydrogen acceptor and hydrogen donor groups, melamine derivatives become more of an issue for enantioselectivity. In the light of such information, triazine‐based chiral, fluorescence active novel thiazole derivatives L1 and L2 were designed and synthesized from (S)‐(?)‐2‐amino‐1‐butanol and (1S,2R)‐(+)‐2‐amino‐1,2‐diphenylethanol. The structural establishment of these compounds was made by spectroscopic methods such as FTIR, ¹H, and ¹³C NMR. While the solution of these compounds in DMSO did not show any fluorescence emission, it was observed that the emission increased 44‐fold for L1 and 55‐fold for L2 in 95% water, similar to the aggregation‐induced emission (AIE) characterized compounds. In this regard, enantioselective capabilities of these compounds against carboxylic acids were tested, and in experiments carried out at a ratio of 40/60 DMSO/H₂O, it was determined that R‐2ClMA increased the fluorescence emission of L1 chiral receptor by 2.59 times compared to S‐isomer.