Association between N-Terminal Pyrenes Stabilizes the Collagen Triple Helix.

Collagen model peptides featuring the fluorophore pyrene at their N-termini have been synthesized, and their thermal denaturation has been examined using circular dichroism (CD) and fluorescence spectroscopies. Flanking the (Pro-Hyp-Gly)7 core of the peptide monomers at positions 1 and/or 23 in the primary sequence, Lys residues were introduced to ensure water solubility. Triple helices derived from such peptides show a broad excimer emission at ∼480 nm, indicative of interaction between the pyrene units. CD experiments show that the fluorophores enhance helix stability primarily through entropic effects. Unfolding temperatures (Tm) increase by up to 7 °C for systems with N-terminal lysine residues and by up to 21 °C for systems in which the first-position Lys is replaced by Ala. Tm values derived from fluorescence measurements (at 50 µM) typically lie within ∼1 °C of those obtained using CD (at 200 µM). Computational modeling in a water continuum using B3LYP-GD3 and M06-2X functionals predicts that face-to-face association of fluorophores can occur while H-bonding within the [(POG)n]3 assembly is retained. Such parallel stacking is consistent with hydrophobically driven stabilization. Labeling collagen peptides with pyrene is a synthetically simple way to promote triple helicity while providing a means to obtain Tm data on relatively dilute samples.

Synthesis of redox-active fluorinated 5-hydroxytryptophans as molecular reporters for biological electron transfer.

Fluorinated 5-hydroxytryptophans (Fn-5HOWs) were synthesized in gram scale quantities and incorporated into a ß-hairpin peptide and the protein azurin. The redox-active Fn-5HOWs exhibit unique radical spectroscopic signatures that expand the function of as probes for biological electron transfer.

Side-Chain Protonation States of a Fluorescent Arginine.

An arginine derivative with a fluorescent side-chain, Boc-Arg(Nap)-OH, was prepared by palladium(0)-catalyzed coupling of Boc-Arg-OH with a 4-bromonaphthalimide. The presence of the fluorophore lowers the pKa of the side-chain guanidinium group by several orders of magnitude, to 9.0 (±0.1), allowing the derivative to access an electrically neutral protonation state that is not generally available to arginine itself. Computational modeling (DFT) predicts that protonation takes place at the side-chain C═N atom that bears the fluorophore. Calculated electronic absorptions for the protonated (356 nm) and neutral species (440 nm) are in good agreement with experiment. When irradiated with light, excited-state proton transfer (ESPT) occurs from cationic side-chains to suitably basic solvents, resulting in fluorescence emission from the neutral species. Arg(Nap) can be incorporated into peptides with sterically accessible N-termini using specially adapted conditions of solid-phase peptide synthesis.

Decarboxylative and dehydrative coupling of dienoic acids and pentadienyl alcohols to form 1,3,6,8-tetraenes.

Dienoic acids and pentadienyl alcohols are coupled in a decarboxylative and dehydrative manner at ambient temperature using Pd(0) catalysis to generate 1,3,6,8-tetraenes. Contrary to related decarboxylative coupling reactions, an anion-stabilizing group is not required adjacent to the carboxyl group. Of mechanistic importance, it appears that both the diene of the acid and the diene of the alcohol are required for this reaction. To further understand this reaction, substitutions at every unique position of both coupling partners was examined and two potential mechanisms are presented.

Incorporation of fluorophore-cholesterol conjugates into liposomal and mycobacterial membranes.

Fluorescently-labeled steroids that emit intense blue light in nonpolar solvent (λem (CH2Cl2)≈440nm, ΦF=0.70) were prepared by treating cholesteryl chloroformate with 4-amino-1,8-naphthalimides. The lipid portion of the conjugates embeds into liposomal membrane bilayers in minutes, leaving the fluorophore exposed to the external aqueous environment. This causes a 40-nm red-shift in λem and significant quenching. DFT optimizations predict the conjugates to be about 30Å long when fully extended, but rotation about the linker group can bring the compounds into an 'L'-shape. Such a conformation would allow the cholesteryl anchor to remain parallel to the acyl chains of a membrane while the fluorescent group resides in the interfacial region, instead of extending beyond it. When incubated with Mycobacterium smegmatis mc2 155, a bacterial species known to use natural cholesterol, the labeled steroids support growth and can be found localized in the membrane fraction of the cells using HPLC. These findings demonstrate stable integration of fluorescent cholesterols into bacterial membranes in vivo, indicating that these compounds may be useful for evaluating cholesterol uptake in prokaryotic organisms.

Dimerization and anion binding of a fluorescent phospholipid analogue.

A diarylacetylene fluorophore featuring spatially separated urea and phosphocholine (PC) groups forms a macrocyclic "head-to-tail" dimer stabilized by NH(urea)···OP(PC) hydrogen bonds. At concentrations above ~2 × 10(-5) M in CH(2)Cl(2), the emission intensity of the dimer is quenched by HCO(3)(-) and H(2)PO(4)(-) but not by Cl(-) and NO(3)(-). Under more dilute conditions, all four anions are bound unselectively with association constants on the order of 10(5) M(-1).

Binding of carboxylic acids by fluorescent pyridyl ureas.

Fluorescent pyrid-2-yl ureas were prepared by treating halogenated 2-aminopyridines with hexyl isocyanate, followed by Sonogashira coupling with arylacetylenes. The sensors emit light of ∼360 nm with quantum yields of 0.05-0.1 in acetonitrile solution. Addition of strong organic acids (pK(a) < 13 in CH(3)CN) shifts the fluorescence band to lower energy, and clean isoemissive behavior is observed. Fluorescence response curves (i.e., F/F(0) vs [acid](total)) are hyperbolic in shape for CCl(3)COOH and CF(3)COOH, with association constants on the order of 10(3) M(-1) for both acids. (1)H NMR titrations and DFT analyses indicate that trihaloacetic acids bind in ionized form to the receptors. Pyridine protonation disrupts an intramolecular H-bond, thereby unfolding an array of ureido NH donors for recognition of the corresponding carboxylates. Methanesulfonic acid protonates the sensors, but no evidence for conjugate base binding at the urea moiety is found by NMR. An isosteric control compound that lacks an integrated pyridine does not undergo significant fluorescence changes upon acidification.

"Wurster-type" ureas as redox-active receptors for anions.

Four redox-active receptors, 1-4, based on the incorporation of p-phenylenediamine(s) within a urea framework, were synthesized, and the affinities of two for a series of anions were quantified through UV-vis and NMR spectroscopic studies. The structure of 1 was confirmed by X-ray crystallography. For the oxoanions studied, complex stabilities approached 10(6) M(-1) in acetonitrile and decreased with the decreasing basicity of the anion (CH(3)COO(-) > C(6)H(5)COO(-) > H(2)PO(4)(-) > NO(2)(-) > NO(3)(-)). The presence of the urea functionality caused an increase in the oxidation potential of the p-phenylenediamine subunit compared to that of free p-phenylenediamine. Electrochemical studies of the anion complexes revealed two-wave behavior with the appearance of a second oxidation wave cathodic to that in the free receptors and characteristic of the bound anion. Ab initio DFT studies of a representative acetate complex revealed the consequences of host oxidation state on complex structure.

Fluorous effects in amide-based receptors for anions.

Hybrid receptors designed to recognize both the sulfonate headgroup and the fluorous tail of perfluorooctanesulfonate (CF(3)(CF(2))(7)SO(3)(-), "PFOS") were prepared by coupling fluorinated carboxylic acids onto poly(aminomethyl)benzene scaffolds. Binding to PFOS, CF(3)SO(3)(-), p-TsO(-), and Cl(-) was monitored by (1)H NMR and isothermal titration calorimetry (ITC). In chloroform solvent, hydrogen-bonding to anions is accompanied by downfield shifts in the amide NH protons of the fluorinated receptors and by evolution of heat. Association constants for 1:1 complexation (K(assoc)) are >1000 M(-1). An analogous hydrocarbon receptor binds weakly to anionic guests (K(assoc) < 50 M(-1)). Ab initio calculations indicate that the differences in 1:1 binding strengths between fluorous and nonfluorous hosts cannot be ascribed to differences in NH donor acidities.

Hydrogen bonding vs steric gearing in a hexasubstituted benzene.

Treatment of hexakis(bromomethyl)benzene with excess NaN 3, followed by hydrogenation of the resultant polyazide, affords hexamine 3 in high yield. Coupling to six equivalents of nonanoic acid provides hexamide 5 without chromatographic purification. The NH resonance of 5 appears far downfield (approximately 9.7 ppm) in CDCl3 and is unaffected by changes in concentration or by addition of chloride or trifluoromethanesulfonate ions. DFT calculations predict that 5 exists as a bowl, with all six substituents intramolecularly H-bonded together on one side of the plane defined by the anchoring arene.

Wurster's crowns: a comparative study of ortho- and para-phenylenediamine-containing macrocyclic receptors.

Two series of isomeric, redox-responsive azacrown ethers based on ortho- and para-phenylenediamine (Wurster's crowns) have been synthesized and their properties explored through 13C NMR spectroscopy, electrospray ionization mass spectrometry, cyclic voltammetry, and X-ray crystallography. These crowns display strong affinity for alkali metal cations while maintaining comparable selectivity profiles to the parent crown ethers from which they are derived. Like Wurster's reagent (N,N,N',N'-tetramethyl-p-phenylenediamine or para-TMPD), the para-Wurster's crowns undergo two reversible one-electron oxidations. The integrity of the alkali metal ion complexes is maintained in the neutral and singly oxidized ligand states but not after removal of two electrons. In contrast, the oxidation of ortho-Wurster's crowns is scan rate dependent, occurring at potentials substantially higher than their para counterparts, with their complexes oxidizing irreversibly. X-ray crystal structures of representative complexes show, in all cases, participation of the redox-active phenylenediamine subunits in complex formation via direct bonding to the guest cation.

A theoretical investigation on the Wurster's crown analogue of 18-crown-6.

An ab initio, quantum mechanical study of the Wurster's crown analogue of 18-crown-6 and its interactions with the alkali metal cations are presented. This study explores methods for accurately treating large, electron-rich species while providing an understanding of the molecular behavior of a representative member of this class of crowns. The molecular geometries, binding energies, and binding enthalpies are evaluated with methods similar to those reported for the analysis of 18-crown-6 and its alkali metal complexes to facilitate direct comparison. Hybrid density functional methods are applied to gauge the effects of electron correlation on the geometries of the electron-rich phenylenediamine moiety present in the Wurster's crowns. While the structure of the crown ether backbone is largely unperturbed by the incorporation of the redox active functionality, the alkali metal binding enthalpies are uniformly stronger for the Wurster's crown complexes, adding 1.8 to 5.1 kcal/mol to the strength of the interaction, depending on cation type. The additional strength, due to the exchange of an oxygen donor atom in the crown ether backbone by a nitrogen donor supplied by the redox group, is tightly coupled to the rotation of the dimethylaminophenyl group with respect to the plane of the macrocycle. Gas-phase selectivities favor the more highly charge-dense cations, while the explicit addition of only a few waters of hydration in the calculations recovers the selectivities expected in solution. The alkali metal binding affinity to the singly oxidized Wurster's crown is significantly diminished, while it is completely eliminated for the doubly oxidized ligand.

Poly(2-thienyl)borates: An Investigation into the Coordination of Thiophene and Its Derivatives.

The synthesis, characterization, and reactivity of a new sulfur-rich tridentate ligand, tetrakis(2-thienyl)borate (1(-)()), are reported along with a molecular orbital analysis of its coordination to a metal center. Unlike the analogous tetrakis((methylthio)methyl)borate (2(-)()), 1(-)() does not coordinate Mo(CO)(3) when reacted with (C(7)H(8))Mo(CO)(3). The sulfur atoms in both ligands are oriented to coordinate the metal in a pyramidal eta(1) sulfur-bound mode. Approximate molecular orbital calculations are used to compare the metal-ligand interactions in these related species, and the results indicate that the magnitude and polarizability of the electronic charge density of the lone pairs on the sulfur atoms dictate the coordination strength of the ligands. Simple Mulliken atomic charges and orbital occupation numbers are used to determine the extent of charge delocalization. While the conjugation of the sulfur lone pair electrons with adjacent pi bonds in the ligands decreases the corresponding Lewis basicity, the contribution from the aromaticity in the thienyl groups is negligible. During the course of these studies, the structure of K[1] was determined by X-ray diffraction. K[1]: monoclinic space group C2/c, with a = 16.00(2) Å, b = 7.680(7) Å, c = 16.22(2) Å, beta = 118.520(7) degrees, V = 1750(3) Å(3), Z = 4, R(F) = 0.0494, and R(w)(F(2)()) = 0.122. The crystal lattice contains one-dimensional chains of 1(-)() bridged by K ions.