Assignment of reference pH-values to primary standard buffer solutions for standardization of potentiometric sensors in acetonitrile-water mixtures

Standard pH-values pH(PS) for seven primary standard buffer solutions in 0, 10, 30, 40, 50, 70 and 100% (w/w) acetonitrile-water mixed solvents at 298.15 K were determined according to the criteria recently endorsed by IUPAC. The preferential solvation of the ions in acetonitrile-water mixtures was studied in order to clarify the acid-base behaviour of the solutes in such mixtures. The influence of variation in the solvent composition on pH(PS) values was considered with a view to obtaining correlations that can be used to determine the pH(PS) values in any acetonitrile-water mixture up to 70% (w/w) acetonitrile. The pH(PS) values were then correlated with the weight and volume percentages and molar fraction of acetonitrile and with the Kamlet-Taft, pi(*), alpha and beta solvatochromic parameters of the acetonitrile-water mixtures. The equations obtained permit the standardization of potentiometric sensors in these mixtures.     

Calorimetric and computational study of 1,3-dithiacyclohexane 1,1-dioxide (1,3-dithiane sulfone)

The enthalpies of combustion and sublimation of 1,3-dithiacyclohexane 1,1-dioxide (1,3-dithiane sulfone) were measured by a rotating-bomb combustion calorimeter and the Knudsen effusion technique, and the gas-phase enthalpy of formation was determined, Delta(f)H(m)*(g) = -326.3 +/- 2.0 kJ mol(-1). Standard ab initio molecular orbital calculations at the G2(MP2) level were performed, and a theoretical study on molecular and electronic structure of the compound has been carried out. Calculated Delta(f)H(m)*(g) values agree very well with the experimental one. These experimental and theoretical studies support the relevance of the repulsive electrostatic interaction between sulfur atoms in 1,3-dithiane sulfone, that apparently counterbalances any n(S) --> rho(C-SO2)* stabilizing hyperconjugative interaction.     

Sulfur-directed synthesis of enantiopure hydroxy 2-sulfinyl butadienes

The treatment of sulfinyl chlorohydrins with KO-t-Bu in THF generates epoxy vinyl sulfoxides that undergo an efficient base-induced rearrangement to generate enantiopure hydroxy 2-sulfinyl dienes. This novel process takes place with high chemo- and stereoselectivity. The chirality at sulfur effectively controls the geometry of the trisubstituted alkene.     

δ-Nitroalkanols as precursors for the one-pot synthesis of substituted tetrahydrofurans

DBU catalyses the one-pot Michael addition of nitroalkanes to 1,4-dienone derivatives, elimination of nitrous acid, then intramolecular conjugate addition-ring closure to allow the direct formation of a variety of tetrahydrofurans.     

Liquid chromatography-mass spectrometry approach for the characterization and purification of crude synthetic peptide hormones

In this study, conditions for the optimal separation by LC-UV and characterisation by LC-ES-MS of crude mixtures generated during SPPS of several peptide hormones are compiled. The linear solvation energy relationship (LSER) methodology has been used to predict the retention of the target peptides and their side products and then to develop a separation LC methodology with applicability on both the analytical and preparative scale. Identification of these side products by LC-ES-MS analysis has been made on the basis of their calculated molecular masses. This method may be regarded as a key tool for the optimisation of the synthetic procedures and for complying with regulatory agencies' requirements before commercialisation of a safe and effective peptide-based pharmaceutical drug.Awarded a prize as outstanding poster on the occasion of Jornadas de Análisis Instrumental (JAI), November 26 to 29, 2002, Barcelona, Spain     

Rapid on-membrane proteolytic cleavage for Edman sequencing and mass spectrometric identification of proteins

A method for the rapid limited enzymatic cleavage of PVDF membrane-immobilized proteins is described. This method allows the fast characterization of PVDF blotted proteins by peptide mass fingerprinting (Henzel, W. J., Billeci, T. M., Stults, J. T., Wong, S. C., Grimley, C., Wantanabe, C., Proc. Natl. Acad. Sci. USA 1993, 90, 5011-5015), LC-MS/MS, or N-terminal sequencing and has been demonstrated on a range of proteins using a full complement of proteolytic enzymes. This technique allows the generation of proteolytic fragments between 5 and 60 min (depending on the enzyme employed), which is significantly faster than previously reported on-membrane digestion methods. To date, this on-membrane rapid digestion protocol has aided in the identification and confirmation of mutation sites in over 200 recombinant proteins.     

Thermochemistry of 1,3-dithiacyclohexane 1-oxide (1,3-dithiane sulfoxide): calorimetric and computational study

The enthalpies of combustion and sublimation of 1,3-dithiacyclohexane 1-oxide (1,3-dithiane sulfoxide, 2) were measured by a rotating-bomb combustion calorimeter and the Knudsen effusion technique, and the gas-phase enthalpy of formation was determined, DeltafH degrees m(g) = -98.0 +/- 1.9 kJ mol(-1). This value is not as large (negative) as could have been expected from comparison with thermochemical data available for the thiane/thiane oxide reference system. High-level ab initio molecular orbital calculations at the MP2(FULL)/6-31G(3df,2p) level were performed, and the optimized molecular and electronic structures of 2 afforded valuable information on (1) the relative conformational energies of 2-axial and 2-equatorial--the latter being 7.1 kJ mol(-1) more stable than 2-axial, (2) the possible involvement of nS --> sigma*(C-S(O)) hyperconjugation in 2-equatorial, (3) the lack of computational evidence for sigma(S-C) --> sigma*(S-O) stereoelectronic interaction in 2-equatorial, and (4) the relevance of a repulsive electrostatic interaction between sulfur atoms in 1,3-dithiane sulfoxide, which apparently counterbalances any nS --> sigma*(C-S(O)) stabilizing hyperconjugative interaction and accounts for the lower than expected enthalpy of formation for sulfoxide 2.     

A traceless linker for aliphatic amines that rapidly and quantitatively fragments after reduction

Reduction sensitive linkers (RSLs) have the potential to transform the field of drug delivery due to their ease of use and selective cleavage in intracellular environments. However, despite their compelling attributes, developing reduction sensitive self-immolative linkers for aliphatic amines has been challenging due to their poor leaving group ability and high pK_a values. Here a traceless self-immolative linker composed of a dithiol-ethyl carbonate connected to a benzyl carbamate (DEC) is presented, which can modify aliphatic amines and release them rapidly and quantitatively after disulfide reduction. DEC was able to reversibly modify the lysine residues on CRISPR–Cas9 with either PEG, the cell penetrating peptide Arg₁₀, or donor DNA, and generated Cas9 conjugates with significantly improved biological properties. In particular, Cas9–DEC–PEG was able to diffuse through brain tissue significantly better than unmodified Cas9, making it a more suitable candidate for genome editing in animals. Furthermore, conjugation of Arg₁₀ to Cas9 with DEC was able to generate a self-delivering Cas9 RNP that could edit cells without transfection reagents. Finally, conjugation of donor DNA to Cas9 with DEC increased the homology directed DNA repair (HDR) rate of the Cas9 RNP by 50% in HEK 293T cell line. We anticipate that DEC will have numerous applications in biotechnology, given the ubiquitous presence of aliphatic amines on small molecule and protein therapeutics.

Reduction sensitive linkers (RSLs) have the potential to transform the field of drug delivery due to their ease of use and selective cleavage in intracellular environments.     

Single-step affinity purification of toxic and non-toxic proteins on a fluidics platform

Single-step fusion-based affinity purification of proteins with pH-controllable linkers was carried out in a fluidic device. The linkers were previously derived from self-splicing protein elements called inteins. Two different linkers were generated to solve two distinct separation problems: one for rapid single-step affinity purification of a wide range of proteins, and the other specifically for the purification of cytotoxic proteins. Scale-down factors of 185 resulted in separations in a 27 microl bed-volume. A rotating CD format was chosen because of its simplicity in effecting fluid movement through centrifugal force without the complications associated with electro-osmosis and other pumping methods. The design and fabrication of the fluidic device and the protein purification process are described. This work, which demonstrates the purification of active proteins by two distinct fluidic separations, is widely applicable to small-scale massively parallel proteomic separations.     

Li(+) ion conductivity in rock salt-structured nickel-doped Li(3)NbO(4)

Two mechanisms of doping Li(3)NbO(4), which has an ordered, rock salt superstructure, have been established. In the "stoichiometric mechanism", the overall cation-to-anion ratio is maintained at 1:1 by means of the substitution 3Li(+) + Nb(5+) --> 4Ni(2+). In the "vacancy mechanism", Li(+) ion vacancies are created by means of the substitution 2Li(+) --> Ni(2+). Solid solution ranges have been determined for both mechanisms and a partial phase diagram constructed for the stoichiometric join. On the vacancy join, the substitution mechanism has been confirmed by powder neutron diffraction; associated with lithium vacancy creation, a dramatic increase in Li(+) ion conductivity occurs with increasing Ni content, reaching a value of 5 x 10(-4) Omega(-1) cm(-1) at 300 degrees C for composition x= 0.1 in the formula Li(3-2x)Ni(x)NbO(4). This is the first example of high Li(+) ion conductivity in complex oxides with rock salt-related structures.