Intramolecular Triplet Diffusion Facilitates Triplet Dissociation in a Pentacene Hexamer.

Triplet dynamics in singlet fission depend strongly on the strength of the electronic coupling. Covalent systems in solution offer precise control over such couplings. Nonetheless, efficient free triplet generation remains elusive in most systems, as the intermediate triplet pair 1 (T1 T1 ) is prone to triplet-triplet annihilation due to its spatial confinement. In the solid state, entropically driven triplet diffusion assists in the spatial separation of triplets, resulting in higher yields of free triplets. Control over electronic coupling in the solid state is, however, challenging given its sensitivity to molecular packing. We have thus developed a hexameric system (HexPnc) to enable solid-state-like triplet diffusion at the molecular scale. This system is realized by covalently tethering three pentacene dimers to a central subphthalocyanine scaffold. Transient absorption spectroscopy, complemented by theoretical structural optimizations and steady-state spectroscopy, reveals that triplet diffusion is indeed facilitated due to intramolecular cluster formation. The yield of free triplets in HexPnc is increased by a factor of up to 14 compared to the corresponding dimeric reference (DiPnc). Thus, HexPnc establishes crucial design aspects for achieving efficient triplet dissociation in strongly coupled systems by providing avenues for diffusive separation of 1 (T1 T1 ), while, concomitantly, retaining strong interchromophore coupling which preserves rapid formation of 1 (T1 T1 ).

Unraveling the Silent Hydrolysis of Cyclic B-X/C═C Isosteres: The Striking Impact of a Single Heteroatom on the Aromatic, Acidic, and Dynamic Properties of Hemiboronic Phenanthroids.

Although heterocyclic hemiboronic acids are represented in several recently approved drugs, many questions remain unanswered regarding the physical properties and reactivity of these boranol (BOH)-containing compounds in aqueous media. Over the past 60 years, studies on the acidic and aromatic character of 10-hydroxy-10,9-boroxarophenanthrene and its boraza analog have been conflicting. In contradiction with the Lewis acidic behavior of arylboronic acids in aqueous conditions, it has been proposed that the central boroheterocyclic ring of these borophenanthroids confers sufficient aromatic character to compel the boranol unit to behave as a Brønsted acid and favor the boron oxy conjugate base, thereby avoiding the disruption of cyclic resonance that would otherwise occur with a tetravalent boronate anion. These questions are addressed with a combination of physical and spectroscopic characterizations, X-ray crystallographic analysis, and computational studies. Although both oxa and aza derivatives are conclusively shown to behave as Lewis acids in aqueous solutions, according to pKa measurements and MO and NICS calculations, only the boraza derivatives possess an appreciable aromatic character within the boroheterocyclic ring. For the first time, the possibility of dynamic chemical exchange via a reversible hydrolysis of the endocyclic B-heteroatom bond was examined using VT and EXSY NMR with suitable probe compounds. Whereas the boraza analog is static at neutral pH, its oxa analog undergoes a rapid hydrolytic ring opening-closing equilibrium with the transient boronic acid. Altogether, this study will guide the methodical application of these heterocycles as reaction catalysts, in bioconjugation, and as new-drug chemotypes and bioisosteres of pharmaceutically important classes of heterocycles.

Genetically-encoded discovery of proteolytically stable bicyclic inhibitors for morphogen NODAL.

In this manuscript, we developed a two-fold symmetric linchpin (TSL) that converts readily available phage-displayed peptides libraries made of 20 common amino acids to genetically-encoded libraries of bicyclic peptides displayed on phage. TSL combines an aldehyde-reactive group and two thiol-reactive groups; it bridges two side chains of cysteine [C] with an N-terminal aldehyde group derived from the N-terminal serine [S], yielding a novel bicyclic topology that lacks a free N-terminus. Phage display libraries of SX1CX2X3X4X5X6X7C sequences, where X is any amino acid but Cys, were converted to a library of bicyclic TSL-[S]X1[C]X2X3X4X5X6X7[C] peptides in 45 ± 15% yield. Using this library and protein morphogen NODAL as a target, we discovered bicyclic macrocycles that specifically antagonize NODAL-induced signaling in cancer cells. At a 10 µM concentration, two discovered bicyclic peptides completely suppressed NODAL-induced phosphorylation of SMAD2 in P19 embryonic carcinoma cells. The TSL-[S]Y[C]KRAHKN[C] bicycle inhibited NODAL-induced proliferation of NODAL-TYK-nu ovarian carcinoma cells with apparent IC50 of 1 µM. The same bicycle at 10 µM concentration did not affect the growth of the control TYK-nu cells. TSL-bicycles remained stable over the course of the 72 hour-long assays in a serum-rich cell-culture medium. We further observed general stability in mouse serum and in a mixture of proteases (Pronase™) for 21 diverse bicyclic macrocycles of different ring sizes, amino acid sequences, and cross-linker geometries. TSL-constrained peptides to expand the previously reported repertoire of phage-displayed bicyclic architectures formed by cross-linking Cys side chains. We anticipate that it will aid the discovery of proteolytically stable bicyclic inhibitors for a variety of protein targets.

Insights into the Mechanism of Action of the Two-Peptide Lantibiotic Lacticin 3147.

Lacticin 3147 is a two peptide lantibiotc (LtnA1 and LtnA2) that displays nanomolar activity against many Gram-positive bacteria. Lacticin 3147 may exert its antimicrobial effect by several mechanisms. Isothermal titration calorimetry experiments show that only LtnA1 binds to the peptidoglycan precursor lipid II, which could inhibit peptidoglycan biosynthesis. An experimentally supported model of the resulting complex suggests that the key binding partners are the C-terminus of LtnA1 and pyrophosphate of lipid II. A combination of in vivo and in vitro assays indicates that LtnA1 and LtnA2 can induce rapid membrane lysis without the need for lipid II binding. However, the presence of lipid II substantially increases the activity of lacticin 3147. Furthermore, studies with synthetic LtnA2 analogues containing either desmethyl- or oxa-lanthionine rings confirm that the precise geometry of these rings is essential for this synergistic activity.

Methylammonium lead chloride: A sensitive sample for an accurate NMR thermometer.

A new solid-state nuclear magnetic resonance (NMR) thermometry sample is proposed. The 207Pb NMR chemical shift of a lead halide perovskite, methylammonium lead chloride (MAPbCl3) is very sensitive to temperature, 0.905±0.010ppmK-1. The response to temperature is linear over a wide temperature range, from its tetragonal to cubic phase transition at 178K to >410K, making it an ideal standard for temperature calibrations in this range. Because the 207Pb NMR lineshape for MAPbCl3 appears symmetric, the sample is ideal for calibration of variable temperature NMR data acquired for spinning or non-spinning samples. A frequency-ratio method is proposed for referencing 207Pb chemical shifts, based on the 1H and 13C frequencies of the methylammonium cation, which are used asan internal standard. Finally, this new NMR thermometer has been used to measure the degree of frictional heating asa function of spinning frequency for a series of MAS rotors ranging in outer diameter from 1.3 to 7.0mm. As expected, the largest diameter rotors are more susceptible to frictional heating, but lower diameter rotors are subjected to higher frictional heating temperatures as they are typically spun at much higher spinning frequencies.

Identification and three-dimensional structure of carnobacteriocin XY, a class IIb bacteriocin produced by Carnobacteria.

In this study, we report that CbnX (33 residues) and CbnY (29 residues) comprise a class IIb (two-component) bacteriocin in Carnobacteria. Individually, CbnX and CbnY are inactive, but together act synergistically to exert a narrow spectrum of activity. The structures of CbnX and CbnY in structure-inducing conditions were determined and strongly resemble other class IIb bacteriocins (i.e., LcnG, PlnEF, PlnJK). CbnX has an extended, amphipathic α-helix and a flexible C terminus. CbnY has two α-helices (one hydrophobic, one amphipathic) connected by a short loop and a cationic C terminus. CbnX and CbnY do not appear to interact directly and likely require a membrane-bound receptor to facilitate formation of the bacteriocin complex. This is the first class IIb bacteriocin reported for Carnobacteria.

Instantaneous Functionalization of Chemically Etched Silicon Nanocrystal Surfaces.

Remarkable advances in surface hydrosilylation reactions of C=C and C=O bonds on hydride-terminated silicon have revolutionized silicon surface functionalization. However, existing methods for functionalizing hydride-terminated Si nanocrystals (H-SiNCs) require long reaction times and elevated temperatures. Herein, we report a room-temperature method for functionalizing H-SiNC surfaces within seconds by stripping outermost atoms on H-SiNC surfaces with xenon difluoride (XeF2 ). Detailed analysis of the reaction byproducts by in situ NMR spectroscopy and GC-MS provided unprecedented insight into NC surface composition and reactivity as well as the complex reaction mechanism of XeF2 activated hydrosilylation.

Solution Structures of Phenol-Soluble Modulins α1, α3, and ß2, Virulence Factors from Staphylococcus aureus.

Phenol-soluble modulins (PSMs) are peptide virulence factors produced by staphylococci. These peptides contribute to the overall pathogenicity of these bacteria, eliciting multiple immune responses from host cells. Many of the α-type PSMs exhibit cytolytic properties and are able to lyse particular eukaryotic cells, including erythrocytes, neutrophils, and leukocytes. In addition, they also appear to contribute to the protection of the bacterial cell from the host immune response through biofilm formation and detachment. In this study, three of these peptide toxins, PSMs α1, α3, and ß2, normally produced by Staphylococcus aureus, have been synthesized using solid-supported peptide synthesis (SPPS) (PSMα1 and PSMα3) or made by heterologous expression in Escherichia coli (PSMß2). Their three-dimensional structures were elucidated using nuclear magnetic resonance spectroscopy. PSMα1 and PSMα3 each consist of a single amphipathic helix with a slight bend near the N- and C-termini, respectively. PSMß2 contains three amphipathic helices, which fold to produce a "v-like" shape between α-helix 2 and α-helix 3, with α-helix 1 folded over such that it is perpendicular to α-helix 3. The availability of three-dimensional structures permits spatial analysis of features and residues proposed to control the biological activity of these peptide toxins.

Nuclear Magnetic Resonance Solution Structures of Lacticin Q and Aureocin A53 Reveal a Structural Motif Conserved among Leaderless Bacteriocins with Broad-Spectrum Activity.

Lacticin Q (LnqQ) and aureocin A53 (AucA) are leaderless bacteriocins from Lactococcus lactis QU5 and Staphylococcus aureus A53, respectively. These bacteriocins are characterized by the absence of an N-terminal leader sequence and are active against a broad range of Gram-positive bacteria. LnqQ and AucA consist of 53 and 51 amino acids, respectively, and have 47% identical sequences. In this study, their three-dimensional structures were elucidated using solution nuclear magnetic resonance and were shown to consist of four α-helices that assume a very similar compact, globular overall fold (root-mean-square deviation of 1.7 Å) with a highly cationic surface and a hydrophobic core. The structures of LnqQ and AucA resemble the shorter two-component leaderless bacteriocins, enterocins 7A and 7B, despite having low levels of sequence identity. Homology modeling revealed that the observed structural motif may be shared among leaderless bacteriocins with broad-spectrum activity against Gram-positive organisms. The elucidated structures of LnqQ and AucA also exhibit some resemblance to circular bacteriocins. Despite their similar overall fold, inhibition studies showed that LnqQ and AucA have different antimicrobial potency against the Gram-positive strains tested, suggesting that sequence disparities play a crucial role in their mechanisms of action.

Solution structure of acidocin B, a circular bacteriocin produced by Lactobacillus acidophilus M46.

Acidocin B, a bacteriocin produced by Lactobacillus acidophilus M46, was originally reported to be a linear peptide composed of 59 amino acid residues. However, its high sequence similarity to gassericin A, a circular bacteriocin from Lactobacillus gasseri LA39, suggested that acidocin B might be circular as well. Acidocin B was purified from culture supernatant by a series of hydrophobic interaction chromatographic steps. Its circular nature was ascertained by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and tandem mass spectrometry (MS/MS) sequencing. The peptide sequence was found to consist of 58 amino acids with a molecular mass of 5,621.5 Da. The sequence of the acidocin B biosynthetic gene cluster was also determined and showed high nucleotide sequence similarity to that of gassericin A. The nuclear magnetic resonance (NMR) solution structure of acidocin B in sodium dodecyl sulfate micelles was elucidated, revealing that it is composed of four α-helices of similar length that are folded to form a compact, globular bundle with a central pore. This is a three-dimensional structure for a member of subgroup II circular bacteriocins, which are classified based on their isoelectric points of ∼7 or lower. Comparison of acidocin B with carnocyclin A, a subgroup I circular bacteriocin with four α-helices and a pI of 10, revealed differences in the overall folding. The observed variations could be attributed to inherent diversity in their physical properties, which also required the use of different solvent systems for three-dimensional structural elucidation.

Accessing zinc monohydride cations through coordinative interactions.

We present isolable examples of formal zinc hydride cations supported by N-heterocyclic carbene (NHC) donors, and investigate the dual electrophilic and nucleophilic (hydridic) character of the encapsulated [ZnH](+) units by computational methods and preliminary hydrosilylation catalysis.

Solution structures of the linear leaderless bacteriocins enterocin 7A and 7B resemble carnocyclin A, a circular antimicrobial peptide.

Leaderless bacteriocins are a class of ribosomally synthesized antimicrobial peptides that are produced by certain Gram-positive bacteria without an N-terminal leader section. These bacteriocins are of great interest due to their potent inhibition of many Gram-positive organisms, including food-borne pathogens such as Listeria and Clostridium spp. We now report the NMR solution structures of enterocins 7A and 7B, leaderless bacteriocins recently isolated from Enterococcus faecalis 710C. These are the first three-dimensional structures to be reported for bacteriocins of this class. Unlike most other linear Gram-positive bacteriocins, enterocins 7A and 7B are highly structured in aqueous conditions. Both peptides are primarily α-helical, adopting a similar overall fold. The structures can be divided into three separate α-helical regions: the N- and C-termini are both α-helical, separated by a central kinked α-helix. The overall structures bear an unexpected resemblance to carnocyclin A, a 60-residue peptide that is cyclized via an amide bond between the C- and N-termini and has a saposin fold. Because of synergism observed for other two-peptide leaderless bacteriocins, it was of interest to probe possible binding interactions between enterocins 7A and 7B. However, despite synergistic activity observed between these peptides, no significant binding interaction was observed based on NMR and isothermal calorimetry.

Base-catalyzed bifunctional addition to amides and imides at low temperature. A new pathway for carbonyl hydrogenation.

Mono- or dideprotonation at the N-H groups of the Noyori ketone hydrogenation catalyst trans-[RuH2((R)-BINAP)((R,R)-dpen)] (1a) yields trans-M[RuH2((R,R)-HNCH(Ph)CH(Ph)NH2)((R)-BINAP)], where M = K(+)(8-K) or Li(+) (8-Li), or trans-M2[RuH2((R,R)-HNCH(Ph)CH(Ph)NH)((R)-BINAP)], where M = Li(+) (8-M'2), which have unprecedented activity toward the hydrogenation of amide and imide carbonyls at low temperatures in THF-d8. Details of the origins of the enantioselection for the desymmetrization of meso-cyclic imides by hydrogenation with 8-K are also described herein.

Experimental investigations of a partial Ru-O bond during the metal-ligand bifunctional addition in Noyori-type enantioselective ketone hydrogenation.

The transition state for the metal-ligand bifunctional addition step in Noyori's enantioselective ketone hydrogenation was investigated using intramolecular trapping experiments. The bifunctional addition between the Ru dihydride trans-[Ru((R)-BINAP)(H)(2)((R,R)-dpen)] and the hydroxy ketone 4-HOCH(2)C(6)H(4)(CO)CH(3) at -80 °C exclusively formed the corresponding secondary ruthenium alkoxide trans-[Ru((R)-BINAP)(H)(4-HOCH(2)C(6)H(4)CH(CH(3))O)((R,R)-dpen)]. Combined with the results of control experiments, this observation provides strong evidence for the formation of a partial Ru-O bond in the transition state.

NMR conformational analyses on (des-bromo) neuropeptide B [1-23] and neuropeptide W [1-23]: the importance of alpha-helices, a cation-pi interaction and a beta-turn.

The preferred conformations of the orphan G-protein coupled receptor agonists (des-bromo) neuropeptide B [1-23] and neuropeptide W [1-23], referred to as NPB and NPW, have been determined by (1)H NMR, CD, and molecular modeling. The sequences of NPB and NPW are WYKPAAGHSSYSVGRAAGLLSGL and WYKHVASPRYHTVGRAAGLLMGL, respectively. These are hypothalamic peptides that exert their biological actions on GPR7 and GPR8 receptors. Micellar solutions using the membrane mimetic, sodium dodecylsulphate-d(25) (SDS), were used to mimic a physiological environment for the peptides. The secondary structure of NPB consists of a type II beta-turn involving residues Lys(3) to Ala(6). The C-terminal region of NPB exists in a conformational equilibrium between different secondary structures, including an alpha-helix from residues Arg(15) to Ser(21), and a 3(10)-helix from residues Ser(12) to Ser(21). The N-terminus of NPW exhibits a cation-pi interaction between the Lys(3) side chain and the quadrupole moment of the Trp(1) indole group. At the C-terminus of NPW, a well-defined alpha-helical conformation exists from Arg(15) to Met(21). As NPB and NPW have 91% sequence homology from residues Val(13) to Leu(23), with only residue 21 differing between the two peptides, the similar C-terminal secondary structures of these two peptides are consistent with the sequences. This is supported by the similar CD spectra. The different secondary structures at the N-termini for NPB and NPW point to the importance of the N-terminus in receptor binding. This is consistent with the work of Fujii et al. [J. Biol. Chem. 277, 34010-34016 (2002)] who observed that iodination of the NPB Tyr(2) resulted in decreased agonistic activity at GPR7. In addition, Tanaka et al. [Proc. Natl. Acad. Sci. USA 100, 6251-6256 (2003)] showed that deletion of Trp(1) from NPB or NPW drastically decreased activity at GPR7 for NPB and GPR7 and GPR8 for NPW. Therefore, we postulate that the N-terminus is involved in membrane recognition and receptor binding.

A ruthenium-dihydrogen putative intermediate in ketone hydrogenation.

The compound fac-[Ru((R)-BINAP)(H)(2-PrOH)3]+ (6) (BINAP = 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl) reacts with (R,R)-dpen (dpen = 1,2-diphenylethylenediamine) under H2 at -60 degrees C in 2-PrOH-d8/CD2Cl2 to generate the cationic dihydrogen putative intermediate trans-[Ru((R)-BINAP)(H)(eta2-H2)((R,R)-dpen)]+ (2') without H-D exchange between the hydrogen ligands and the solvent. A 1H NMR study concludes that the dihydrogen ligand in 2' does not protonate 2-PrOH to a catalytically significant extent, and that 2' requires an added base or hydride source to be an active catalyst.

NMR conformational studies of micelle-bound orexin-B: a neuropeptide involved in the sleep/awake cycle and feeding regulation.

The preferred conformation of orexin-B, an orphan G-protein coupled receptor agonist (the human sequence is RSGPPGLQGRLQRLLQASGNHAAGILTM-NH(2)) has been determined by (1)H and (13)C 2D NMR spectroscopy and molecular modeling. Orexin-B has been implicated in sleep-wakefulness and feeding regulation. The membrane mimetic, sodium dodecylsulphate-d(25) (SDS), was used to mimic a physiological environment for the peptide. The secondary structure of orexin-B in SDS consists of two helical sections; helix I spans Leu(7) to Ser(18) and helix II spans Ala(22) to Leu(26). Helices I and II are believed to be involved in membrane binding, as is supported by the results of the spin label studies with 5-doxylstearic acid. Lee et al. (Eur. J. Biochem. 266, 831-839 (1999)) determined the [Phe(1)]-orexin-B conformation in water solution by NMR and showed that helix II extends from Ala(23) to Met(28). The C-terminal dipeptide, Thr(27)-Met(28), is unstructured is SDS, whereas in water it forms the end of helix II. The lack of apparent structure for Thr(27)-Met(28) in SDS allows the dipeptide to have conformational freedom to interact with the receptor. The conformation of orexin-B can now be used to explain the Ala substitution mutagenesis experiments and the D-amino acid substitution experiments (S. Asahi et al., Bioorg. Med. Chem. Lett. 13, 111-113, 2003). Asahi et al. have shown that Ala substitution from Gly(24) to Met(28) or D-amino acid substitution from Ala(23) to Met(28) causes a significant reduction in the potency of orexin-B for both OX(1)R and OX(2)R receptors. We postulate that helix II is involved in membrane recognition, and its binding to the membrane is essential for Thr(27)-Met(28) to adopt the correct receptor-binding conformation.

The NMR-derived conformation of orexin-A: an orphan G-protein coupled receptor agonist involved in appetite regulation and sleep.

The conformation of orexin-A, an orphan G-protein coupled receptor agonist has been determined when bound to sodium dodecylsulphate-d(25) (SDS) micelles by (1)H and (13)C NMR and molecular modeling. Orexin-A has been implicated in sleep-wakefulness and feeding regulation. The conformational preference of orexin-A consists of a short helical section, involving Asp(5) to Gln(9) that makes up helix I, followed by a bend from Lys(10) to Ser(13). Residues Leu(16) to Gly(22) make up helix II. The conformation of orexin-A can now be used to explain the results of earlier Ala substitution mutagenesis experiments (J. G. Darker et al., Bioorg. Med. Chem. Lett. 11, 737-740 (2001); S. Ammoun, et al., J. Pharmacol. Expt. Ther. 305, 507-514 (2003)). Darker et al., working with orexin-A (15-33) amide, observed a significant drop in functional potency at the OX(1)R receptor when Leu(16), Leu(19), Leu(20), His(26), Gly(29), Ile(30), Leu(31), Thr(32), and Leu(33) were replaced by Ala. Ammoun et al. identified three areas of interest, which were the same for OX(1)R and OX(2)R receptors, as amino acids 15-17, 20 and 25-26 with the most marked reduction in activity being produced by the replacement of Leu(20) by Ala. We suggest that Leu(16), Leu(19), and Leu(20), which are in helix II, are likely responsible for binding orexin-A to the surface of the micelle.