The DNA Radical Code. Resolution of Identity in Dissociations of Trinucleotide Codon Cation Radicals in the Gas Phase.

Sixty DNA trinucleotide cation radicals covering a large part of the genetic code alphabet were generated by electron transfer in the gas phase, and their chemistry was studied by collision-induced dissociation tandem mass spectrometry and theoretical calculations. The major dissociations involved loss of nucleobase molecules and radicals, backbone cleavage, and cross-ring fragmentations that depended on the nature and position of the nucleobases. Mass identity in dissociations of symmetrical trinucleotide cation radicals of the (XXX+2H)+• and (XYX+2H)+• type was resolved by specific 15N labeling. The specific features of trinucleotide cation radical dissociations involved the dominant formation of d2+ ions, hydrogen atom migrations accompanying the formation of (w2+H)+•, (w2+2H)+, and (d2+2H)+ sequence ions, and cross-ring cleavages in the 3'- and 5'-deoxyribose moieties that depended on the nucleobase type and its position in the ion. Born-Oppenheimer molecular dynamics (BOMD) and density functional theory calculations were used to obtain structures and energies of several cation-radical protomers and conformers for (AAA+2H)+•, (CCC+2H)+•, (GGG+2H)+•, (ACA+2H)+•, and (CAA+2H)+• that were representative of the different types of backbone dissociations. The ion electronic structure, protonation and radical sites, and hydrogen bonding were used to propose reaction mechanisms for the dissociations.

Resolution of Identity in Gas-Phase Dissociations of Mono- and Diprotonated DNA Trinucleotide Codons by 15N-Labeling and Computational Structure Analysis.

Dissociations of DNA trinucleotide codons as gas-phase singly and doubly protonated ions were studied by tandem mass spectrometry using 15N-labeling to resolve identity in the nucleobase loss and backbone cleavages. The monocations showed different distributions of nucleobase loss from the 5'-, middle, and 3'-positions depending on the nucleobase, favoring cytosine over guanine, adenine, and thymine in an ensemble-averaged 62:27:11:<1 ratio. The distribution for the loss of the 5'-, middle, and 3'-nucleobase was 49:18:33, favoring the 5'-nucleobase, but also depending on its nature. The formation of sequence w2+ ions was unambiguously established for all codon mono- and dications. Structures of low-Gibbs-energy protomers and conformers of dAAA+, dGGG+, dCCC+, dTTT+, dACA+, and dATC+ were established by Born-Oppenheimer molecular dynamics and density functional theory calculations. Monocations containing guanine favored classical structures protonated at guanine N7. Structures containing adenine and cytosine produced classical nucleobase-protonated isomers as well as zwitterions in which two protonated bases were combined with a phosphate anion. Protonation at thymine was disfavored. Low threshold energies for nucleobase loss allowed extensive proton migration to occur prior to dissociation. Loss of the nucleobase from monocations was assisted by neighboring group participation in nucleophilic addition or proton abstraction, as well as allosteric proton migrations remote from the reaction center. The optimized structures of diprotonated isomers for dAAA2+ and dACA2+ revealed combinations of classical and zwitterionic structures. The threshold and transition-state energies for nucleobase-ion loss from dications were low, resulting in facile dissociations involving cytosine, guanine, and adenine.

Low-scale syringe-made synthesis of 15 N-labeled oligonucleotides.

Fast and reasonable low-scale (200 nmol) syringe-made synthesis of 15 N-labeled oligonucleotides representing DNA trinucleotide codons is communicated. All codons were prepared by solid-phase controlled pore glass synthesis column technique via the phosphoramidite method. Twenty-four labeled oligonucleotides covering the DNA genetic code alphabet were prepared using commercially available reagents and affordable equipment in a reasonably short period of time, with acceptable yields and purity for direct applications in mass spectrometry.

Tritium hydrogen-isotope exchange with electron-poor tertiary benzenesulfonamide moiety; application in late-stage labeling of T0901317.

The multifunctional radioligand [3 H]T0901317 ([3 H]1) has been employed as a powerful autoradiographic tool to target several receptors, such as liver X, farnesoid X, and retinoic acid-related orphan receptor alpha and gamma subtypes at nanomolar concentrations. Although [3 H]1 is commercially available and its synthesis via tritiodebromination has been reported, the market price of this radioligand and the laborious synthesis of corresponding bromo-intermediate potentially preclude its widespread use in biochemical, pharmacological, and pathological studies in research lab settings. We exploit recent reports on hydrogen-isotope exchange (HIE) reactions in tertiary benzenesulfonamides where the sulfonamide represents an ortho-directing group that facilitates CH activation in the presence of homogenous iridium(I) catalysts. Herein, we report a time- and cost-efficient method for the tritium late-stage labeling of compound 1-a remarkably electron-poor substrate owing to the tertiary trifluoroethylsulfonamide moiety. Under a straightforward HIE condition using a commercially available Kerr-type NHC Ir(I) complex, [(cod)Ir (NHC)Cl], the reaction with 1 afforded a specific activity of 10.8 Ci/mmol. Additionally, alternative HIE conditions using the heterogeneous catalyst of Ir-black provided sufficient 0.72 D-enrichment of 1 but unexpectedly failed while repeating with tritium gas.

Tritiodefluorination of alkyl C-F groups.

A straightforward methodology of fluorine substitution by tritium/deuterium is reported. The described method is selective towards the F─C (sp3 ) group and leaves both the aromatic F─C (sp2 ) and F2 ─C (sp3 ) moieties unaffected. Alkylfluorides, readily synthesized from appropriate alcohols by treatment with diethylaminosulfur trifluoride (DAST) reagent in an overall yield up to 76%, undergoes activation with the boron-based Lewis acid B(C6 F5 )3 , and stoichiometric in situ reduction with a tritide/deuteride reagent-the [TMP2(3) H][2(3) HB(C6 F5 )3 ] system of frustrated Lewis pair. This methodology provides an isolated yield of up to 93% of regio-specifically labeled small organic compounds with superior 2 H-enrichment of over 95%. The specific activity of prepared 1-(2-[3 H]-ethyl)naphthalene was determined at 29.0 Ci/mmol. The site selectivity of the Lewis acid/ [TMP2(3) H][2(3) HB(C6 F5 )3 ] approach is orthogonal to currently used methods and allows for isotopic labeling of complementary positions in molecules. Reported labeling methodology proceeds well at ultra-mild reaction conditions (220 mbar of T2 ), allowing very low consumption of the radioactive source (4.2 Ci/156 GBq), and producing limited amount of radioactive waste.

Stereospecific control of peptide gas-phase ion chemistry with cis and trans cyclo ornithine residues.

We report non-chiral amino acid residues cis- and trans-1,4-diaminocyclohexane-1-carboxylic acid (cyclo-ornithine, cO) that exhibit unprecedented stereospecific control of backbone dissociations of singly charged peptide cations and hydrogen-rich cation radicals produced by electron-transfer dissociation. Upon collision-induced dissociation (CID) in the slow heating regime, peptide cations containing trans-cO residues undergo facile backbone cleavages of amide bonds C-terminal to trans-cO. By contrast, peptides with cis-cO residues undergo dissociations at several amide bonds along the peptide ion backbone. Diastereoisomeric cO-containing peptides thus provide remarkably distinct tandem mass spectra. The stereospecific effect in CID of the trans-cO residue is explained by syn-facially directed proton transfer from the 4-ammonium group at cO to the C-terminal amide followed by neighboring group participation in the cleavage of the CO-NH bond, analogous to the aspartic acid and ornithine effects. Backbone dissociations of diastereoisomeric cO-containing peptide ions generate distinct [bn ]+ -type fragment ions that were characterized by CID-MS3 spectra. Stereospecific control is also reported for electron-transfer dissociation of cis- and trans-cO containing doubly charged peptide ions. The stereospecific effect upon electron transfer is related to the different conformations of doubly charged peptide ions that affect the electron attachment sites and ensuing N-Cα bond dissociations.

Synthesis, characterization and in vitro evaluation of substituted N-(2-phenylcyclopropyl)carbamates as acetyl- and butyrylcholinesterase inhibitors.

A serie of O-substituted N-2-phenylcyclopropylcarbamates was prepared and characterized. These carbamates were tested as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). It was found, that these compounds exhibit moderate inhibition activity with values of IC50 in the range of 54.8-94.4 µM (for AChE) and up to 5.8 µM (for BChE). The AChE/BChE selectivity for each carbamate was calculated. These values varied from 0.50 to 9.46, two carbamate derivatives inhibited only AChE selectively. The most promising derivative was prepared in all optically pure forms (four isomers). It was found that individual stereoisomers differed only slightly in the inhibition ability. The cytotoxicity of all carbamates was evaluated using the standard in vitro test with Jurkat cells. With regard to their inhibition activity and cytotoxicity as well as easy preparation, O-substituted N-2-phenylcyclopropylcarbamates can be considered as promising compounds for potential medicinal applications.