Electrostatic vs. inductive effects in phosphine ligand donor properties and reactivity.

Enhanced rates and selectivity in enzymes are enabled in part by precisely tuned electric fields within active sites. Analogously, the use of charged groups to leverage electrostatics in molecular systems is a promising strategy to tune reactivity. However, separation of the through space and through bond effects of charged functional groups is a long standing challenge that limits the rational application of electric fields in molecular systems. To address this challenge we developed a method using the phosphorus selenium coupling value (J P-Se) of anionic phosphine selenides to quantify the electrostatic contribution of the borate moiety to donor strength. In this analysis we report the synthesis of a novel anionic phosphine, PPh2CH2BF3K, the corresponding tetraphenyl phosphonium and tetraethyl ammonium selenides [PPh4][SePPh2CH2BF3] and [TEA][SePPh2CH2BF3], and the Rh carbonyl complex [PPh4][Rh(acac)(CO)(PPh2(CH2BF3))]. Solvent-dependent changes in J P-Se were fit using Coulomb's law and support up to an 80% electrostatic contribution to the increase in donor strength of [PPh4][SePPh2CH2BF3] relative to SePPh2Et, while controls with [TEA][SePPh2CH2BF3] exclude convoluting ion pairing effects. Calculations using explicit solvation or point charges effectively replicate the experimental data. This J P-Se method was extended to [PPh4][SePPh2(2-BF3Ph)] and likewise estimates up to a 70% electrostatic contribution to the increase in donor strength relative to SePPh3. The use of PPh2CH2BF3K also accelerates C-F oxidative addition reactivity with Ni(COD)2 by an order of magnitude in comparison to the comparatively donating neutral phosphines PEt3 and PCy3. This enhanced reactivity prompted the investigation of catalytic fluoroarene C-F borylation, with improved yields observed for less fluorinated arenes. These results demonstrate that covalently bound charged functionalities can exert a significant electrostatic influence under common solution phase reaction conditions and experimentally validate theoretical predictions regarding electrostatic effects in reactivity.

Reversible Switching of Organic Diradical Character via Iron-Based Spin-Crossover.

Organic diradicals are uncommon species that have been intensely studied for their unique properties and potential applicability in a diverse range of innovative fields. While there is a growing class of stable and well-characterized organic diradicals, there has been recent focus on how diradical character can be controlled or modulated with external stimuli. Here we demonstrate that a diiron complex bridged by the doubly oxidized ligand tetrathiafulvalene-2,3,6,7-tetrathiolate (TTFtt2-) undergoes a thermally induced Fe-centered spin-crossover which yields significant diradical character on TTFtt2-. UV-vis-near-IR, Mössbauer, NMR, and EPR spectroscopies with magnetometry, crystallography, and advanced theoretical treatments suggest that this diradical character arises from a shrinking TTFtt2- π-manifold from the Fe(II)-centered spin-crossover. The TTFtt2--centered diradical is predicted to have a singlet ground state by theory and variable temperature EPR. This unusual phenomenon demonstrates that inorganic spin transitions can be used to modulate organic diradical character.

pH-dependent cross-linking of catechols through oxidation via Fe3+ and potential implications for mussel adhesion.

The mussel byssus is a remarkable attachment structure that is formed by injection molding and rapid in-situ hardening of concentrated solutions of proteins enriched in the catecholic amino acid 3,4-dihydroxy-L-phenylalanine (DOPA). Fe3+, found in high concentrations in the byssus, has been speculated to participate in redox reactions with DOPA that lead to protein polymerization, however direct evidence to support this hypothesis has been lacking. Using small molecule catechols, DOPA-containing peptides, and native mussel foot proteins, we report the first direct observation of catechol oxidation and polymerization accompanied by reduction of Fe3+ to Fe2+. In the case of the small molecule catechol, we identified two dominant dimer species and characterized their connectivities by nuclear magnetic resonance (NMR), with the C6-C6 and C5-C6 linked species as the major and minor products, respectively. For the DOPA-containing peptide, we studied the pH dependence of the reaction and demonstrated that catechol polymerization occurs readily at low pH, but is increasingly diminished in favor of metal-catechol coordination interactions at higher pH. Finally, we demonstrate that Fe3+ can induce cross-links in native byssal mussel proteins mefp-1 and mcfp-1 at acidic pH. Based on these findings, we discuss the potential implications to the chemistry of mussel adhesion.

Spectroscopic elucidation of the inhibitory mechanism of Cys2His2 zinc finger transcription factors by cobalt(III) Schiff base complexes.

Transcription factors are key regulators in both normal and pathological cell processes. Affecting the activity of these proteins is a promising strategy for understanding gene regulation and developing effective therapeutics. Co(III) Schiff base complexes ([Co(acacen)(L)2](+) where L=labile axial ligands) have been shown to be potent inhibitors of a number of zinc metalloproteins including Cys2His2 zinc finger transcription factors. Inhibition by [Co(acacen)(L)2](+) of the target protein is believed to occur through a dissociative exchange of the labile axial ligands for histidine (His) residues essential for function. Here, we report a series of spectroscopic investigations with model peptides of zinc fingers that elucidate the interaction between [Co(acacen)(L)2](+) complexes and zinc finger transcription factors. Observed changes in NMR chemical shifts and 2D (1)H-(1)H NOESY NMR spectra demonstrate the preference of [Co(acacen)(L)2](+) complexes to coordinate His residues over other amino acids. The conformation of [Co(acacen)(L)2](+) upon His coordination was characterized by (1)H NMR spectroscopy, near-UV CD, and electronic absorption. These studies reveal that the resulting His-coordinated [Co(acacen)(L)2](+) complex possesses an octahedral structure. The effects of [Co(acacen)(L)2](+) complexes on the zinc-finger structure were assessed by the degree of hydrogen bonding (probed by 2D NMR spectroscopy) and secondary-structure profiles measured by far-UV CD. These structural studies demonstrate the ability of [Co(acacen)(L)2](+) complexes to disrupt the ßßα structure of zinc fingers, resulting in primarily random-coil conformations. A mechanism is described wherein [Co(acacen)(L)2](+) complexes inhibit zinc finger transcription factor activity through selectively coordinating His residues in the zinc finger by dissociative ligand exchange and disrupting the ßßα structural motif required for gene regulation.

Antifouling glycocalyx-mimetic peptoids.

The glycocalyx of the cell is composed of highly hydrated saccharidic groups conjugated to protein and lipid cores. Although components of the glycocalyx are important in cell-cell interactions and other specific biological recognition events, a fundamental role of the glycocalyx is the inhibition of nonspecific interactions at the cell surface. Inspired by glycoproteins present in the glycocalyx, we describe a new class of synthetic antifouling polymer composed of saccharide containing N-substituted polypeptide (glycopeptoid). Grafting of glycopeptoids to a solid surface resulted in a biomimetic shielding layer that dramatically reduced nonspecific protein, fibroblast, and bacterial cell attachment. All-atom molecular dynamics simulation of grafted glycopeptoids revealed an aqueous interface enriched in highly hydrated saccharide residues. In comparison to saccharide-free peptoids, the interfacial saccharide residues of glycopeptoids formed a higher number of hydrogen bonds with water molecules. Moreover, these hydrogen bonds displayed a longer persistence time, which we believe contributed to fouling resistance by impeding interactions with biomolecules. Our findings suggest that the fouling resistance of glycopeptoids can be explained by the presence of both a 'water barrier' effect associated with the hydrated saccharide residues as well as steric hindrance from the polymer backbone.

Mechanism of cis-inhibition of polyQ fibrillation by polyP: PPII oligomers and the hydrophobic effect.

PolyQ peptides teeter between polyproline II (PPII) and beta-sheet conformations. In tandem polyQ-polyP peptides, the polyP segment tips the balance toward PPII, increasing the threshold number of Gln residues needed for fibrillation. To investigate the mechanism of cis-inhibition by flanking polyP segments on polyQ fibrillation, we examined short polyQ, polyP, and tandem polyQ-polyP peptides. These polyQ peptides have only three glutamines and cannot form beta-sheet fibrils. We demonstrate that polyQ-polyP peptides form small, soluble oligomers at high concentrations (as shown by size exclusion chromatography and diffusion coefficient measurements) with PPII structure (as shown by circular dichroism spectroscopy and (3)J(HN-C alpha) constants of Gln residues from constant time correlation spectroscopy NMR). Nuclear Overhauser effect spectroscopy and molecular modeling suggest that self-association of these peptides occurs as a result of both hydrophobic and steric effects. Pro side chains present three methylenes to solvent, favoring self-association of polyP through the hydrophobic effect. Gln side chains, with two methylene groups, can adopt a conformation similar to that of Pro side chains, also permitting self-association through the hydrophobic effect. Furthermore, steric clashes between Gln and Pro side chains to the C-terminal side of the polyQ segment favor adoption of the PPII-like structure in the polyQ segment. The conformational adaptability of the polyQ segment permits the cis-inhibitory effect of polyP segments on fibrillation by the polyQ segments in proteins such as huntingtin.

Versatile cyclic templates for assembly of axially oriented ligands.

In this paper, we describe two novel types of planar cyclic peptide templates for the facile addition of ligands that extend axially from the plane of the template ring. The first uses beta-amino acids of alternating D- and L-chirality, since the insertion of the additional methylene group in the peptide backbone was predicted and subsequently shown by NMR and molecular modeling, to reorient ligands attached to amino acid side chain axially with respect to the template ring. A second contains alternating D- and L-amino acids with an achiral Gly residue interposed between each chiral amino acid. The inserted Gly residues also tend to reorient side chains axially rather than radially, as was demonstrated by NMR and molecular modeling. The axial orientation of attached ligands is intended to foster or allow interactions among attached ligands in situations in which this is desired. Two such situations that we consider are (1) development of immunological reagents with avidity effects and (2) modeling of oligomers in fibril-forming peptides. Toward the first of these goals, we demonstrated that these templates are suitable for attaching macromolecules, by incorporating two types of protein, neutravidin and trypsinogen. Toward the second goal, we demonstrate the attachment of two different fibril-forming peptides to the template. The templates described herein thus have many of the desirable traits of such molecules, i.e., (1) multivalency for the attachment of multiple ligands, (2) suitable chemical functions for facile attachment of ligands, (3) versatility as to the number and spacing of ligand attachment sites, (4) sufficient rigidity so that the attached ligands can be similarly oriented with respect to the template, and (5) sufficient flexibility to allow even large ligands, such as proteins, to attach and interact.

Encapsulation and NMR on an aggregating peptide before fibrillogenesis.

The early stages of peptide and protein aggregation include the formation of soluble oligomers, some of which may be cytotoxic. There is a paucity of structural information on these oligomers, however, because they are temporally unstable and tend to aggregate further into insoluble protofibrils and fibrils. To obtain structural information on soluble oligomers, we have developed a procedure for encapsulating a fibril-forming peptide, Peptide 1 (NH2-SDDYYYGFGSNKFGRPRDD-COOH), in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine single bilayer vesicles (POPC SBVs). We also encapsulated a non-fibril forming peptide, Peptide 2 (NH2-EEWEE-COOH), in POPC SBVs. The nominal concentration of Peptide 1 in the resulting 40 nm diameter SBVs was 2.4 +/- 0.1 mM, well above the concentration at which Peptide 1 forms fibrils. We demonstrated that these peptides had indeed been encapsulated by measuring longitudinal relaxation times (T1) in the presence and absence of a paramagnetic substance, 1 mM Gd-EDTA, by NMR spectroscopy. When the peptides were free in solution, they showed the expected shortening of T1 times and broadening of NMR peaks. In contrast, peptide encapsulated in POPC SBVs were shielded from the effects of Gd-EDTA and showed preservation of T1 values and NMR line widths. To demonstrate that encapsulation inhibits fibril formation, we measured one-dimensional proton (1D-1H) NMR spectra of the peptides in solution, and of the encapsulated peptides immediately after encapsulation, and 4 days after encapsulation, because Peptide 1 forms fibrils within 1 day. A 2.8 mM solution of Peptide 1 shows the loss of NMR signal expected for a fibrillizing peptide. In contrast, the 1D-1H spectra of encapsulated Peptide 1 measured immediately after encapsulation and 4 days after encapsulation were essentially identical, with preservation of line width at 4 days, i.e., well within the time frame of most high-resolution NMR experiments. Encapsulation may provide a means to obtain high-resolution NMR data on unstable soluble oligomers of peptides implicated in amyloidoses such as Alzheimer's Disease and provide the first detailed structural information about these possibly cytotoxic species that have hitherto been inaccessible to analysis.

Long-chain fatty acids increase cellular dopamine in an immortalized cell line (MN9D) derived from mouse mesencephalon.

The lysate of an immortalized monoclonal cell line derived from the striatum (X61) contains a dopaminergic stimulatory activity that is capable of increasing the dopamine content of an immortalized mouse mesencephalic cell line (MN9D) which expresses a dopaminergic phenotype. Purification of an isoamyl alcohol extract of this lysate and subsequent identification by NMR spectroscopic analysis demonstrated that the dopaminergic stimulatory activity contained within the lysate was a mixture of 80-90% cis-9-octadecenoic acid (oleic acid) and 10-20% cis-11-octadecenoic acid (cis-vaccenic acid). The effect of oleic acid on MN9D dopamine is a prolonged event. MN9D dopamine increases linearly over a 48 h period suggesting the induction of an increased dopaminergic phenotype in these dividing cells. The ability to increase MN9D dopamine by oleic and cis-vaccenic acids is shared by a number of other long-chain fatty acids including arachidonic, linoleic, linolenic, palmitoleic, and cis-13-octadecenoic acid. The possibility that oleic or other relatively innocuous fatty acids might affect dopaminergic function in primary neurons is intriguing with respect to possible therapeutic approaches to the treatment of dopaminergic cell loss and the motor sequelae of Parkinson's disease.

NMR structure of lung surfactant peptide SP-B(11-25).

Surfactant protein B (SP-B) is a 79-residue essential component of lung surfactant, the film of lipid and protein lining the alveoli, and is the subject of great interest for its role in lung surfactant replacement therapies. Here we report circular dichroism results and the solution NMR structure of SP-B(11-25) (CRALIKRIQAMIPKG) dissolved in CD(3)OH at 5 degrees C. This is the first report of NMR data related to the protein SP-B, whose structure promises to help elucidate the mechanism of its function. Sequence-specific resonance assignments were made for all observable (1)H NMR signals on the basis of standard 2D NMR methods. Structures were determined by the simulated annealing method using restraints derived from 2D NOESY data. The calculations yielded 17 energy-minimized structures, three of which were subjected to 0.95 ns of restrained dynamics to assess the relevance of the static structures to more realistic dynamic behavior. Our CD and NMR data confirm that this segment is an amphiphilic alpha helix from approximately residue L14 through M21. The backbone heavy-atom RMSD for residues L14 through M21 is 0.09 +/- 0.12 A, and the backbone heavy-atom RMSD for the whole peptide is 0.96 +/- 2.45 A, the difference reflecting fraying at the termini. Aside from the disordered termini, the minimized structures represent dynamic structures well. Structural similarity to the homologous regions of related saposin-like proteins and the importance of the distribution of polar residues about the helix axis are discussed.