Determination of site selectivity of different carcinogens for preferential mutational hot spots in oligonucleotide fragments by ion-pair reversed-phase nano liquid chromatography tandem mass spectrometry.

Ion-pair reversed-phase nano liquid chromatography coupled with nanospray ion trap mass spectrometry was used to investigate site selectivity of the known carcinogens N-acetoxy-2-acetylaminofluorene, N-hydroxy-4-aminobiphenyl and (+/-)-anti-benzo[a]pyrene diol epoxide with the synthetic double-strand 14-mer long oligonucleotide fragment of the p53 gene containing two mutational hot-spot codons (5'-P-ACC155 CGC156 GTC157 CGC158 GC/5'-GCG CGG ACG CGG GT). The investigation was performed using a monolithic polystyrene divinylbenzene capillary column and triethylammonium bicarbonate as an ion-pair reagent. The exact location of the carcinogen on the modified oligonucleotide backbone was determined using characteristic collision-induced dissociation fragmentation patterns obtained under negative-ion mode ionization. In all these cases, the adducted, isomeric oligonucleotides formed were chromatographically resolved and structural identification was performed without any prior deoxyribonucleic acid cleavage or hydrolysis. The knowledge of the site specificity of a carcinogen, especially at purported mutational hot spots, is of paramount importance (1) in establishing the identity of biomarkers for an early risk assessment of the formed DNA adducts, (2) developing repair mechanisms for the formed carcinogen adducted DNA, and (3) understanding the nature of the covalent bond formed and mapping the frequency of the adduction process.

Marangoni-driven deterministic formation of softer, hollow microstructures for sensitivity-enhanced tactile system.

Microengineering the dielectric layers with three-dimensional microstructures has proven effective in enhancing the sensitivity of flexible pressure sensors. However, the widely employed geometrical designs of solid microstructures exhibit limited sensitivity over a wide range of pressures due to their inherent but undesired structural compressibility. Here, a Marangoni-driven deterministic formation approach is proposed for fabricating hollow microstructures, allowing for greater deformation while retarding structural stiffening during compression. Fluid convective deposition enables solute particles to reassemble in template microstructures, controlling the interior cavity with a void ratio exceeding 90%. The hollow micro-pyramid sensor exhibits a 10-fold sensitivity improvement across wider pressure ranges over the pressure sensor utilizing solid micro-pyramids, and an ultra-low detect limit of 0.21 Pa. With the advantages of facilitation, scalability, and large-area compatibility, such an approach for hollow microstructures can be expanded to other sensor types for superior performance and has considerable potential in robotic tactile and epidermal devices.

A Snakeskin-Inspired, Soft-Hinge Kirigami Metamaterial for Self-Adaptive Conformal Electronic Armor.

A majority of soft-body creatures evolve armor or shells to protect themselves. Similar protection demand is for flexible electronics working in complex environments. Existing works mainly focus on improving the sensing capabilities such as electronic skin (E-skin). Inspired by snakeskin, a novel electronic armor (E-armor) is proposed, which not only possesses mechanical flexibility and electronic functions similar to E-skin, but is also able to protect itself and the underlying soft body from external physical damage. The geometry of the kirigami mechanical metamaterial (Kiri-MM) ensures auxetic stretchability and meanwhile large areal coverage for sufficient protection. Moreover, to suppress the inherent but undesired out-of-plane buckling of conventional Kiri-MMs for conformal applications, soft hinges are used to form a distinct soft (hinges)-rigid (tiles) configuration. Analytical, computational, and experimental studies of the mechanical behaviors of the soft-hinge Kiri-MM E-armor demonstrate the merits of this design, i.e., stretchability, conformability, and protectability, as applied to flexible electronics. Deploying a conductive soft material at the hinges enables facile wiring strategies for large-scale circuit arrays. Functional E-armor systems for controllable display and sensing purposes provide simple examples of a wide spectrum of applications of this concept.

The Conformal Design of an Island-Bridge Structure on a Non-Developable Surface for Stretchable Electronics.

Conformal design of the island-bridge structure is the key to construct high-performance inorganic stretchable electronics that can be conformally transferred to non-developable surfaces. Former studies in conformal problems of epidermal electronics are mainly focused on soft surfaces that can adapt to the deformation of the electronics, which are not suitable for applications in hard, non-developable surfaces because of their loose surface constraints. In this paper, the conformal design problem for the island-bridge structure on a hard, non-developable surface was studied, including the critical size for island and stiffness and the demand for stretchability for the bridge. Firstly, the conformal model for an island on a part of torus surface was established to determine the relationship between the maximum size of the island and the curvatures of the surface. By combining the principle of energy minimization and the limit of material failure, a critical non-dimensional width for conformability was given for the island as a function of its thickness and interfacial adhesion energy, and the ratio of two principal curvatures of the surface. Then, the dependency of the tensile stiffness of the bridge on its geometric parameters was studied by finite element analysis (FEA) to guide the deterministic assembly of the islands on the surface. Finally, the location-dependent demands for the stretchability of the bridges were given by geometric mapping. This work will provide a design rule for stretchable electronics that fully conforms to the non-developable surface.

2-aminothiazole-derived opioids. Bioisosteric replacement of phenols.

A series of aminothiazole-derived morphinans, benzomorphans, and morphine were synthesized. Although their affinities were somewhat lower than their phenol prototypes, one compound (9a, ATPM) has been identified possessing high affinity and selectivity at the kappa receptor. Functional assays showed that 9a was a full kappa but partial mu agonist; the efficacy at kappa was significantly greater than at mu receptors. This novel compound may be valuable for the development of long-acting analgesics and drug abuse medication.

Design and synthesis of novel dimeric morphinan ligands for kappa and micro opioid receptors.

A novel series of morphinans were synthesized, and their binding affinity at and functional selectivity for micro, delta, and kappa opioid receptors were evaluated. These dimeric ligands can be viewed as dimeric morphinans, which were formed by coupling two identical morphinan pharmacophores (cyclorphan (1) or MCL 101 (2)) with varying connecting spacers. Ligands 6 and 7 with alkyl spacers on the nitrogen position and ligands 8 and 9 in which the two morphinan pharmacophores were coupled by ether moieties at the 3-hydroxyl positions showed significant decrease in affinity at all three opioid receptors. An improvement in the affinity was achieved by introducing an ester moiety as the spacer in the dimeric morphinans. It was observed that the affinity of these ligands was sensitive to the character and length of the spacer. Compound 13 (MCL-139) with a 4-carbon ester spacer, compound 17 (MCL-144) containing a 10-carbon spacer, and compound 19 (MCL-145) with the conformationally constrained fumaryl spacer were the most potent ligands in this series, displaying excellent affinities at micro and kappa receptors (K(i) = 0.09-0.2 nM at micro and K(i) = 0.078-0.049 nM at kappa), which were comparable to the parent compound 2. Ligand 12, a compound containing only one morphinan pharmacophore and a long-chain ester group, had affinity at both micro and kappa receptors almost identical to that of the parent ligand 2. In the [(35)S]GTPgammaS binding assay, ligands 13, 17, and 19 and their parent morphinans 1 and 2 stimulated [(35)S]GTPgammaS binding mediated by the micro and kappa receptors. Compounds 13 and 17 were full kappa agonists and partial micro agonists, while compound 19 was a partial agonist at both micro and kappa receptors. These novel ligands, as well as their interesting pharmacological properties, will serve as the basis for our continuing investigation of the dimeric ligands as potential probes for the pharmacotherapy of cocaine abuse and may also open new avenues for the characterization of opioid receptors.

10-Ketomorphinan and 3-substituted-3-desoxymorphinan analogues as mixed kappa and micro opioid ligands: synthesis and biological evaluation of their binding affinity at opioid receptors.

A series of 10-ketomorphinan analogues were synthesized, and their binding affinity at all three opioid receptors was investigated. In most cases, high affinity at micro and kappa receptors, and lower affinity at delta receptor was observed, resulting in good selectivity for micro and kappa receptors. A wide range of substituents can be accommodated on the nitrogen position. The N-(S)-tetrahydrofurfuryl analogue 11 displayed the highest affinity at all three receptors. The N-cyclobutylmethyl analogue 13 gave both high affinity and selectivity at kappa receptor, and N-2-phenylethyl analogue 18 exhibited good affinity and selectivity at micro receptor. Further modifications of the 3-substituent indicated that one H-bond donor was an essential requirement for good affinity at micro and kappa receptors. Similar modifications were investigated at the 3-OH group of morphinans: levorphanol (2a), cyclorphan (2b), and MCL-101 (2c) lacking the 10-keto group. The 3-amino bioisosteric analogues (40 and 41) displayed reasonably good affinity at micro and kappa receptors. The 3-carboxamido replacement (compounds 46-48) in the morphinan subseries resulted in similar affinities comparable to their corresponding 3-OH congeners. The high affinity of these carboxamido analogues, along with their greater lipophilicity and metabolic stability, make them promising candidates for further pharmacological investigation.

Highly enantioselective alkynylation of aldehydes catalyzed by a readily available chiral amino alcohol-based ligand.

A new inexpensive chiral amino alcohol-based ligand, (1S,2S)-2-N,N-dimethylamino-1-(p-nitrophenyl)-3- (t-butyldimethylsilyloxy)propane-1-ol, was developed for the asymmetric alkynylation of aliphatic and aromatic aldehydes, to prepare the corresponding propargylic alcohols in high yields with up to 99% ee.

Zn(ODf)2: preparation and application in asymmetric alkynylation of aldehydes.

A new Lewis acid, Zn(ODf)2, was first prepared from commercially available 3,3,4,4-tetrafluoro[1,2]oxathietane 2,2-dioxide in four steps with 56% yields and also was applied to catalyze highly enantioselective alkynylation of aldehydes in the presence of ligand (1S,2S)-3-(tert-butyldimethylsilyloxyl)-2-N,N-dimethylamino-1- (p-nitrophenyl)-propane-1-ol or ligand (-)-N-methylephedrine to afford the corresponding propargylic alcohols in high yields with up to 99% ee.

A convenient stereoselective synthesis of trifluoromethyl-substituted polyfunctionalized cyclopropane: synthesis of (+/-)-trans-trifluoronorcoronamic acid.

Trifluoromethylated polyfunctionalized cyclopropanes were obtained in high stereoselectivity by reacting 2-bromo-3,3,3-trifluoropropene (BrTFP) with active methylenes. This novel method was further applied to the synthesis of (+/-)-trans-trifluoronorcoronamic acid.

The influence of cytosine methylation on the chemoselectivity of benzo[a]pyrene diol epoxide-oligonucleotide adducts determined using nanoLC/MS/MS.

Benzo[a]pyrene is a major carcinogen implicated in human lung cancer. Almost 60% of human lung cancers have a mutation in the p53 tumor suppressor gene at several specific codons. An on-line nanoLC/MS/MS method using a monolithic nanocolumn was applied to investigate the chemoselectivity of the carcinogenic diol epoxide metabolite, (+/-)-(7R,8S,9S,10R)-benzo[a]pyrene 7,8-diol 9,10-epoxide [(+/-)-anti-benzo[a]pyrene diol epoxide (BPDE)], which was reacted in vitro with a synthesized 14-mer double stranded oligonucleotide (5'-ACCCG5CG7TCCG11CG13C-3'/5'-GCGCGGGCGCGGGT-3') derived from the p53 gene. This sequence contained codons 157 and 158, which are considered mutational 'hot spots' and have also been reported as chemical 'hot spots' for the formation of BPDE-DNA adducts. In evaluating the effect of cytosine methylation on BPDE-DNA adduct binding, it was found that codon 156, containing the nucleobase G5 instead of the mutational hot spot codons 157 (G7) and 158 (G11), was the preferential chemoselective binding site for BPDE. In all permethylated cases studied, the relative ratio for adduction was found to be G5 >> G11 > G13 > G7. Permethylation of CpG dinucleotide sites on either the nontranscribed or complementary strand did not change the order of sequence preference but did enhance the relative adduction level of the G11 CpG site (codon 158) approximately two-fold versus the unmethylated oligomer. Permethylation of all CpG dinucleotide sites on the duplex changed the order of relative adduction to G5 >> G7 > G11 > G13. The three- to four-fold increase in adduction at the mutational hot spot codon 157 (G(7)) relative to the unmethylated or single-stranded permethylated cases suggests a possible relationship between the state of methylation and adduct formation for a particular mutation site in the p53 gene. Using this method, only 125 ng (30 pmol) of adducted oligonucleotide was analyzed with minimal sample cleanup and high chromatographic resolution of positional isomers in a single chromatographic run.

Separation and sequencing of isomeric oligonucleotide adducts using monolithic columns by ion-pair reversed-phase nano-HPLC coupled to ion trap mass spectrometry.

An ion-pair reversed-phase nano-high-performance liquid chromatography (IP-RP-nano-HPLC) method using a monolithic poly(styrene-divinylbenzene) (PS-DVB) column coupled to nanoelectrospray ionization mass spectrometry (nano-ESI-MS) was evaluated to separate and identify isomeric oligonucleotide adducts derived from the covalent binding of (+/-)-anti-7r,8t-dihydroxy-9t,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+/-)-anti-BPDE] to double-stranded (ds) 5'-PO4--ACCCGCGTCCGCGC-3'/5'-GCGCGGGCGCGGGT-3' oligonucleotide. The influence of three different nanospray emitters on electrospray signal was evaluated in terms of analyte ion sensitivity. The best nanoelectrospray performance for the oligonucleotides was observed with the distal metal-coated emitter. The performance of three different stationary phases was also investigated. The chromatographic separation performance of the polymeric monolithic PS-DVB stationary phase significantly surpassed that of columns packed with the microparticulate sorbents C18 or PS-DVB. Different mobile phase organic solvents and ion-pairing reagents were also evaluated. An optimized mobile phase consisting of methanol and 25 mM triethylammonium bicarbonate resulted in the best chromatographic resolution and increased MS sensitivity of the oligonucleotides. By using a monolithic PS-DVB stationary phase fabricated in a nanocolumn, four positional isomeric (+/-)-BPDE-oligonucleotide adducts were separated and identified. In addition to four of the possible five positional isomers, three positional isomers were also resolved to several diastereoisomers, although their stereostructures could not be identified in the absence of reference standards.

A deoxynucleotide derivatization methodology for improving LC-ESI-MS detection.

We have developed a novel LC-UV-MS derivatization method for the analysis of deoxyguanosine monophosphate adducts that demonstrates enhanced signal intensities relative to underivatized analytes in positive ion mode electrospray ionization MS. Detection of DNA nucleotide adducts is normally conducted in negative ion mode, which requires basic mobile phases that make chromatographic separations difficult and reduce MS sensitivity. Utilizing coupling reagents typically employed in peptide synthesis, several different deoxyguanosine nucleotide phosphoramidates and phosphomonoesters were synthesized in high conversion yield and under mild reaction conditions. The derivatives were characterized by MS/MS and reaction conversion yields determined from the DAD-UV traces. The derivatives were evaluated for ionization efficiencies, fragmentation patterns, and reversed-phase chromatographic properties by LC/ESI-MS/MS. Overall, the hydrophobic derivatives showed increases in ionization efficiency and improved peak shape. Rank ordering of the derivatizing agents was initially established using the dGp-modified derivatives. The best derivatizing agent, hexamethyleneimine, showed a 3-4-fold signal enhancement compared to underivatized dGp and was selected for additional evaluation. A model system using the carcinogen, N-acetoxy-2-acetylaminofluorene (AAAF), was used to synthesize a N-acetyl-(2-aminofluorenyl)-guanosine 5'-monophosphate (dGpAAF) adduct, which was subsequently derivatized with hexamethyleneimine. Detection limits for dGphex and dGpAAFhex, purified by HPLC, were 10- and 3-fold higher (S/N) than their respective underivatized analogues. Practical applicability, with similar improvements in sensitivity, was established by derivatizing adducts isolated from calf thymus DNA exposed to AAAF. Our results demonstrate the utility of simple reactions for the enhanced detection of a mononucleotide in positive ion mode ESI MS and the application of this technique for the detection of dGp-DNA adducts at the low-femtomole level.

Characterization of a novel bivalent morphinan possessing kappa agonist and micro agonist/antagonist properties.

Previous research has shown that compounds with mixed kappa and mu activity may have utility for the treatment of cocaine abuse and dependence. The present study characterizes the pharmacological profile of a bivalent morphinan that was shown to be a kappa opioid receptor agonist and a mu opioid receptor agonist/antagonist. MCL-145 [bis(N-cyclobutylmethylmorphinan) fumarate] is related to the morphinan cyclorphan and its N-cyclobutylmethyl derivative MCL-101 [3-hydroxy-N-cyclobutylmethyl morphinan S-(+)-mandelate]. MCL-145 consists of two morphinans connected by a spacer at the 3-hydroxy position. This compound had K(i) values of 0.078 and 0.20 nM for the kappa and mu opioid receptors, respectively, using radioligand binding assays as shown by Neumeyer et al. in 2003. In the guanosine 5'-O -(3-[(35) S]thiotriphosphate) binding assay, MCL-145 produced an E(max) value of 80% for the kappa opioid receptor and 42% for the mu opioid receptor. The EC(50) values obtained for this compound were 4.3 and 3.1 nM for the kappa and mu opioid receptors, respectively. In vivo MCL-145 produced a full dose-response curve in the 55 degrees C warm water tail-flick test and was equipotent to morphine. The agonist properties of MCL-145 were antagonized by the mu-selective antagonist beta-funaltrexamine and the kappa-selective antagonist nor-binaltorphimine. MCL-145 also acted as a mu antagonist, as measured by the inhibition of morphine-induced antinociception.

Structure-based 3-D-QSAR analysis of marine indole alkaloids.

A 3-D-QSAR study has been performed on these indole alkaloid derivatives to correlate their chemical structures with their observed antitumor activity against IGROV1. Due to the absence of information on their active mechanism, comparative molecular field analysis (CoMFA) has been applied. A model able to well correlate the antitumor activity with the chemical structures of mono and bis(indole) alkaloids 1-18 has been developed which is potentially helpful in the design of novel and more potent antitumor agents.

A novel microtubule destabilizing entity from orthogonal synthesis of triazine library and zebrafish embryo screening.

The first orthogonal combinatorial synthesis of a high-purity triazine library was demonstrated. Novel triazine-based microtubule inhibitors were discovered by an efficient zebrafish embryo screening and in vitro microtubule polymerization assay.