“Free of Base” Sulfa-Michael Addition for Novel o-Carboranyl-DL-Cysteine Synthesis

The sulfa-Michael addition reaction was applied for the two-step synthesis of o-carboranyl cysteine 1-HOOCCH(NH2)CH2S-1,2-C2B10H11 from the trimethylammonium salt of 1-mercapto-o-carborane and methyl 2-acetamidoacrylate. To avoid the decapitation of o-carborane into its nido-form, the “free of base” method under mild conditions in a system of two immiscible solvents toluene-H2O was developed. The replacement of H2O by 2H2O resulted in carboranyl-cysteine containing a deuterium label at the α-position of the amino acid 1-HOOCCD(NH2)CH2S-1,2-C2B10H11. The structure of the protected o-carboranyl cysteine was determined by single-crystal X-ray diffraction. The obtained compounds can be considered as potential agents for the Boron Neutron Capture Therapy of cancer.

Model Substrate/Inactivation Reactions for MoaA and Ribonucleotide Reductases: Loss of Bromo, Chloro, or Tosylate Groups from C2 of 1,5-Dideoxyhomoribofuranoses upon Generation of an α-Oxy Radical at C3

We report studies on radical-initiated fragmentations of model 1,5-dideoxyhomoribofuranose derivatives with bromo, chloro, and tosyloxy substituents on C2. The effects of stereochemical inversion at C2 were probed with the corresponding arabino epimers. In all cases, the elimination of bromide, chloride, and tosylate anions occurred when the 3-hydroxyl group was unprotected. The isolation of deuterium-labeled furanone products established heterolytic cleavage followed by the transfer of deuterium from labeled tributylstannane. In contrast, 3-O-methyl derivatives underwent the elimination of bromine or chlorine radicals to give the 2,3-alkene with no incorporation of label in the methyl vinyl ether. More drastic fragmentation occurred with both of the 3-O-methyl-2-tosyloxy epimers to give an aromatized furan derivative with no deuterium label. Contrasting results observed with the present anhydroalditol models relative to our prior studies with analogously substituted nucleoside models have demonstrated that insights from biomimetic chemical reactions can provide illumination of mechanistic pathways employed by ribonucleotide reductases (RNRs) and the MoaA enzyme involved in the biosynthesis of molybdopterin.

Stereochemical Studies on the Addition of Allylsilanes to Aldehydes. The SE’ Component

Model compounds ul-1 and lk-1 have been studied to determine both the position of the silicon electrofuge and the relative orientation of the double bonds in the transition structure of the allylmetal-aldehyde condensation. The use of the deuterium label allows an unbiased assessment of the syn versus anti SE’ pathways. The synthesis of configurational proof of model systems ul-1 and lk-1 are discussed as well as the cyclization of the model system. Cyclization of model 1 was found to proceed with high selectivity via an anti SE’ pathway regardless of the proximal/distal ratio for all Lewis acids studied. Reactions promoted by fluoride ion favored the proximal product, but both syn and anti pathways were observed.

Hydrogen-deuterium exchange coupled to top- and middle-down mass spectrometry enables high-resolution measurements of histone tail dynamics before and after nucleosome assembly

Until recently, a major limitation of hydrogen deuterium exchange mass spectrometry (HDX-MS) was that resolution of deuterium localization information was limited to the length of the peptide generated during proteolysis. Recently, however, it has been demonstrated that electron transfer dissociation (ETD) allows for preservation of deuterium label in the gas phase and therefore can be used to obtain more resolved information. To date, this technology has remained mostly limited to single, small, already well-characterized model proteins. Here, we optimize, expand, and adapt HDX-MS/MS capabilities to accommodate histone and nucleosomal complexes on top-down (TD) HDX-MS/MS and middle-down (MD) HDX-MS/MS platforms and demonstrate that near site-specific resolution of deuterium localization can be obtained with high reproducibility. We are able to study histone tail dynamics in unprecedented detail, which have evaded rigorous analysis by traditional structural biology techniques for decades, revealing important novel insights into chromatin biology. This work represents the first heterogeneous protein complex and protein-DNA complex to be analyzed by TD- and MD-HDX-MS/MS, respectively. Together, the results of these studies highlight the versatility, reliability, and reproducibility of ETD-based HDX-MS/MS methodology to interrogate large protein and protein/DNA complexes.

Isotopically labeled sulfur compounds and synthetic selenium and tellurium analogues to study sulfur metabolism in marine bacteria

Members of the marine Roseobacter clade can degrade dimethylsulfoniopropionate (DMSP) via competing pathways releasing either methanethiol (MeSH) or dimethyl sulfide (DMS). Deuterium-labeled [2H6]DMSP and the synthetic DMSP analogue dimethyltelluriopropionate (DMTeP) were used in feeding experiments with the Roseobacter clade members Phaeobacter gallaeciensis DSM 17395 and Ruegeria pomeroyi DSS-3, and their volatile metabolites were analyzed by closed-loop stripping and solid-phase microextraction coupled to GC–MS. Feeding experiments with [2H6]DMSP resulted in the incorporation of a deuterium label into MeSH and DMS. Knockout of relevant genes from the known DMSP demethylation pathway to MeSH showed in both species a residual production of [2H3]MeSH, suggesting that a second demethylation pathway is active. The role of DMSP degradation pathways for MeSH and DMS formation was further investigated by using the synthetic analogue DMTeP as a probe in feeding experiments with the wild-type strain and knockout mutants. Feeding of DMTeP to the R. pomeroyi knockout mutant resulted in a diminished, but not abolished production of demethylation pathway products. These results further corroborated the proposed second demethylation activity in R. pomeroyi. Isotopically labeled [2H3]methionine and 34SO4 2−, synthesized from elemental 34S8, were tested to identify alternative sulfur sources besides DMSP for the MeSH production in P. gallaeciensis. Methionine proved to be a viable sulfur source for the MeSH volatiles, whereas incorporation of labeling from sulfate was not observed. Moreover, the utilization of selenite and selenate salts by marine alphaproteobacteria for the production of methylated selenium volatiles was explored and resulted in the production of numerous methaneselenol-derived volatiles via reduction and methylation. The pathway of selenate/selenite reduction, however, proved to be strictly separated from sulfate reduction.

The fragmentation of 7-norbornyloxychlorocarbene — SNi-like transition states

7-Norbornyloxychlorocarbene (15) fragments mainly to 7-norbornyl chloride (16). A C-2 deuterated analog of 15 (syn-15-d) fragments to labeled chloride 16 with 78% retention and 22% inversion. When the deuterium label is replaced by a syn-exo-2-methyl group, the resultant carbene 22 fragments to syn- and anti-7-chloro-exo-2-methylnorbornane in a ratio of 1:1.1, corresponding to 48% retention and 52% inversion. Computational studies suggest that the fragmentations proceed via competitive SNi-like transition states that lead to either retention or inversion. Key words: carbenes, carbocations, SNi reactions, stereochemistry.

Triple Isotopic Substitution Method in Small-Angle Neutron Scattering: Application to Some Problems of Structural Biology

The triple isotopic substitution (TIS) method is based on the analysis of a scattering curve which is the difference between the scattering of two solutions containing appropriately deuterium-labelled particles. A necessary condition for the application of the method is that the two solutions are identical in all respects except for the extent of the deuterium label. Such an experimental scheme has allowed a number of unique physical experiments to be performed, the main ones being: (1) elimination of the contribution of the interparticle interference; (2) addition of both small- and large-sized foreign particles to those studied without distortions of the structural data; (3) highlighting of individual (quite small) regions in the molecules; (4) suppression of the dimerization contribution to the scattering curve. The application of this method is of special interest for studying the mutual three-dimensional disposition of individual small regions of molecules (3D mapping) and for investigating the geometrical properties of the surfaces of globular proteins. It is evident that TIS has a wide range of experimental possibilities, demonstrating that small-angle neutron scattering is one of the most informative structural methods for low resolution.

The Chemistry of Laurenene. XVI. Investigation of the Mechanism of a Cationic Rearrangement

Some aspects of the mechanism of the acid-induced rearrangement of laurenan-2β-ol have been deduced from deuterium- labelling studies. A key intermediate arises from two pathways, one involving a 1,4- and one a 1,5-hydride shift. Formation of this intermediate is partially suppressed by substitution of the migrating hydrogen by a deuterium. A proposed pathway which involves a 1,3-methyl migration has been disproved, and scrambling and loss of deuterium label has been accounted for in terms of equilibria involving lauren-1-ene and another previously reported rearrangement product. The rearrangement is strongly promoted in dichloromethane, and the product composition is highly solvent-dependent.

Stereochemistry of hydrogen loss during C-21 dehydroxylation of tetrahydrodeoxycorticosterone by Eubacteriumlentum

The loss of hydrogen from the C-21 position of 5β-pregnane-3α,21-diol-20-one (tetrahydrodeoxycorticosterone, THDOC) during reductive removal of the 21-hydroxy group by the anaerobic bacterium Eubacteriumlentum has been shown to be selective for the pro-S position by the use of THDOC labelled with deuterium at the C-21 pro-S and C-21 pro-R positions. The labelled substrates were obtained by using the bacterium Clostridiumparaputrificum to reduce chemically prepared C-21 labelled samples of pregn-4-en-21-ol-3,20-dione (deoxycorticosterone, DOC) at C-3 and C-4 (5). The stereochemistry of deuterium label introduced by chemical means at C-21 of DOC was determined by comparison with a sample of 21-(R)-DOC-21-d1 produced by reduction of the corresponding aldehyde pregn-4-en-21-al-3,20-dione, 21-d by the enzyme 21-hydroxysteroid NAD oxidoreductase from beef liver. Keywords: Eubacterium, Clostridium, steroids, tetrahydrodeoxycorticosteroids.