(±)-2′-Phenyl­cyclo­hexa­nespiro-4′-(aze­pano[1,2-b]isoxazolidine)

In the crystal structure of the racemic title isoxazolidine, C19H27NO, the relative stereochemistry between the phenyl group and the bridgehead H atom is shown to be syn. There are two mol­ecules in the asymmetric unit, one of which is the 7R*,13R* enanti­omer, and one of which is the 7S*,13S* enanti­omer. These enanti­omers adopt different orientations of the phenyl ring with respect to the isoxazolidine ring, with C—C—C—C torsion angles of 63.6 (4) and 86.8 (4)°, respectively. In both enanti­omers, the six-membered ring adopts a chair conformation, while the seven-membered ring adopts a twist-chair conformation

Synthesis and Biological Characterization of a New Norbormide Derived Bodipy FL-Conjugated Fluorescent Probe for Cell Imaging

Background: Norbormide (NRB) is a selective rat toxicant endowed with vasoconstrictor activity confined to the rat peripheral arteries. In a recent work we used a fluorescent derivative of NRB (NRB-AF12), obtained by coupling the NBD fluorophore to the parent molecule via a linker, in order to gain information about the possible site of action of the unlabeled compound. We found that NRB-AF12 labeled intracellular organelles in both NRB-sensitive and -insensitive cells and we accordingly proposed its use as a scaffold for the development of a new class of fluorescent probes. In this study, we examined the fluorescent properties of a BODIPY FL-conjugated NRB probe (MC009) developed: (A) to verify if NRB distribution could be influenced by the attached fluorophore; (B) to improve the fluorescent performance of NRB-AF12. Methods: MC009 characteristics were investigated by confocal fluorescence microscopy, in freshly isolated rat caudal artery myocytes (FIRCAM) and in LX2 cells, representative of NRB-sensitive and insensitive cells, respectively. Main results: In both FIRCAM and LX2 cells MC009 stained endoplasmic reticulum, mitochondria, Golgi apparatus and lipid droplets, revealing the same intracellular distribution as NRB-AF12, and, at the same time, had both improved photostability and gave a more intense fluorescent signal at lower concentrations than was possible with NRB-AF12, which resulted in a better and finer visualization of intracellular structures. Furthermore, MC009 was effective in cellular labeling in both living and fixed cells. At the concentration used to stain the cells, MC009 did not show any cytotoxic effect and did not affect the regular progression of cell cycle and division. Conclusions: This study demonstrates that the distribution of fluorescently labeled NRB is not affected by the type of fluorophore attached to the parent compound, supporting the idea that the localization of the fluorescent derivatives may reasonably reflect that of the parent compound. In addition, we observed a marked improvement in the fluorescent properties of BODIPY FL-conjugated NRB (MC009) over its NBD-derived counterpart (NRB-AF12), confirming NRB as a scaffold for the development of new, high performance, non-toxic fluorescent probes for the labeling of intracellular structures in both living and fixed cells

(1R,6R,13R,18R)-(Z,Z)-1,18-Bis[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]-3,16-dimethyl­ene-8,20-diaza­dispiro­[5.6.5.6]tetra­cosa-7,19-diene

The crystal structure of the title compound, C34H54N2O4, has been solved in order to prove the relative and absolute chirality of the newly-formed stereocentres which were established using an asymmetric Diels–Alder reaction at an earlier stage in the synthesis. This unprecedented stable dialdimine contains a 14-membered ring and was obtained as the minor diastereoisomer in the Diels–Alder reaction. The absolute stereochemistry of the stereocentres of the acetal functionality was known to be R based on the use of a chiral (R)-tris­ubstituted dienophile derived from enanti­opure (S)-glyceraldehyde. The assignment of the configuration in the dienophile and the title di-aldimine differs from (S)-glyceraldehyde due to a change in the priority order of the substituents. The crystal structure establishes the presence of six stereocentres all attributed to be R. The 14-membered ring contains two aldimine bonds [C—N = 1.258 (2) and 1.259 (2) Å]. It adopts a similar conformation to that proposed for trans–trans-cyclo­tetra­deca-1,8-dienes

(±)-Cyclo­hexane-1,2-diyl bis­(4-nitro­benzoate)

The crystal structure of the title compound, C20H18N2O8, has been investigated to establish the relative stereochemistry between the ester groups. The cyclo­hexane ring adopts a chair conformation, in which the two ester groups occupy the adjacent equatorial positions in a trans relationship with each other. The mol­ecules assemble in the crystal as chains along the c axis via C—H⋯π inter­actions between the cyclo­hexane ring and a pair of nitro­phenyl rings of the neighbouring mol­ecule. Also observed are π–π stacking inter­actions between the nitro­phenyl rings of neighbouring chains, with a perpendicular distance between these rings of 3.409 Å and a slippage of 0.969 Å

(1R,1′R,3S,3′S)-5,5′,10,10′-Tetra­meth­oxy-1,1′,3,3′-tetra­methyl-3,3′,4,4′-tetra­hydro-1H,1′H-8,8′-bi[benzo[g]isochromene]

In the title compound, C34H38O6, the methyl groups on each pyran ring exhibit 1,3-cis stereochemistry, established during synthesis by pseudo-axial delivery of hydride during a lactol reduction step. In the crystal structure, the mol­ecule lies on a twofold rotation axis and the torsion angle about the central diaryl bond is 41.3 (1)°. The mol­ecules pack in a herringbone arrangement

Structure-activity relationships of the N-terminus of calcitonin gene-related peptide:key roles of alanine-5 and threonine-6 in receptor activation

Background and purpose - The N-terminus of calcitonin gene-related peptide (CGRP) is important for receptor activation, especially the disulphide-bonded ring (residues 1-7). However, the roles of individual amino acids within this region have not been examined and so the molecular determinants of agonism are unknown. This study has examined the role of residues 1, 3-6 and 8-9, excluding Cys-2 and Cys-7. Experimental approach - CGRP derivatives were substituted with either cysteine or alanine; further residues were introduced at position 6. Their affinity was measured by radioligand binding and their efficacy by measuring cAMP production in SK-N-MC cells and ß-arrestin 2 translocation in CHO-K1 cells at the CGRP receptor. Key results - Substitution of Ala-5 by cysteine reduced affinity 270-fold and reduced efficacy for production of cAMP in SK-N-MCs. Potency at ß-arrestin translocation was reduced by 9-fold. Substitution of Thr-6 by cysteine destroyed all measurable efficacy of both cAMP and ß-arrestin responses; substitution with either alanine or serine impaired potency. Substitutions at positions 1, 4, 8 and 9 resulted in approximately 10-fold reductions in potency at both responses. Similar observations were made at a second CGRP-activated receptor, the AMY1(a) receptor. Conclusions and implications - Ala-5 and Thr-6 are key determinants of agonist activity for CGRP. Ala-5 is also very important for receptor binding. Residues outside of the 1-7 ring also contribute to agonist activity

Supramolecular threading of peptide hydrogel fibrils

There is an increasing demand for biocompatible materials in biomedical applications. Herein, we report a modified α-helical decapeptide segment from the cardiac troponin C, which self-assembles into fibers with a secondary β-sheet structure. These fibers cross-link via a novel supramolecular threading mechanism which results in an atypical stiff hydrogel (G′ ≈ 13 kPa). In this work, we provide a first insight into the understanding of such remarkable cross-linking mechanism, which will aid in the development of new biomaterials with unique properties

3-Allyl-2-hydr­oxy-5,6,8-trimethoxy­naphthalene-1,4-dione

In the crystal structure of the title compound, C16H16O6, a pair of naphthoquinone rings are linked via O—H⋯O—C hydrogen bonds in a nearly orthogonal arrangement. This dimeric unit is linked to a neighbouring dimer by π–π stacking inter­actions between the naphthoquinone rings, where the distance between the mean plane of the naphtoquinone backbones is 3.468 Å, and O—H⋯O—C hydrogen bonds

Photoaffinity cross-linking and unnatural amino acid mutagenesis reveal insights into calcitonin gene-related peptide binding to the calcitonin receptor-like receptor/receptor activity-modifying protein 1 (CLR/RAMP1) complex

Calcitonin gene-related peptide (CGRP) binds to the complex of the calcitonin receptor-like receptor (CLR) with receptor activity-modifying protein 1 (RAMP1). How CGRP interacts with the transmembrane domain (including the extracellular loops) of this family B receptor remains unclear. In this study, a photoaffinity cross-linker, p-azido l-phenylalanine (azF), was incorporated into CLR, chiefly in the second extracellular loop (ECL2) using genetic code expansion and unnatural amino acid mutagenesis. The method was optimized to ensure efficient photolysis of azF residues near the transmembrane bundle of the receptor. A CGRP analogue modified with fluorescein at position 15 was used for detection of ultraviolet-induced cross-linking. The methodology was verified by confirming the known contacts of CGRP to the extracellular domain of CLR. Within ECL2, the chief contacts were I284 on the loop itself and L291, at the top of the fifth transmembrane helix (TM5). Minor contacts were noted along the lip of ECL2 between S286 and L290 and also with M223 in TM3 and F349 in TM6. Full length molecular models of the bound receptor complex suggest that CGRP sits at the top of the TM bundle, with Thr6 of the peptide making contacts with L291 and H295. I284 is likely to contact Leu12 and Ala13 of CGRP, and Leu16 of CGRP is at the ECL/extracellular domain boundary of CLR. The reduced potency, Emax, and affinity of [Leu16Ala]-human α CGRP are consistent with this model. Contacts between Thr6 of CGRP and H295 may be particularly important for receptor activation