Synthesis of C-11 linked active ester derivatives of vitamin D3 and their conjugations to 42-residue helix-loop-helix peptides

Derivatives of vitamin D₃ carrying an 8-carbon linker at C-11 terminating in an active ester were synthesized from commercial vitamin D₃ using a disassembly-reassembly strategy. Vitamin D₃ was cleaved at the C6-C7 double bond and the ‘upper’ fragment was converted, via a series of reactions, to derivatives substituted at C-11 with an 8-carbon linker terminating in an ethyl ester. Reassembly with modified ‘lower’ fragments using Horner-Wittig olefination followed by linker ester hydrolysis and re-esterification with p-nitrophenol gave C-11 substituted p-nitrophenyl esters. These vitamin D derivatives were conjugated to 42-amino acid helix-loop-helix peptides by reaction of their p-nitrophenyl esters with lysyl side-chain amino groups on the peptides. The vitamin D—peptide conjugates, being potential specific binder candidates for vitamin D-binding protein, were characterized by mass spectroscopy and CD measurements.     

Directed Evolution of a Thermophilic β-glucosidase for Cellulosic Bioethanol Production

Characteristics that would make enzymes more desirable for industrial applications can be improved using directed evolution. We developed a directed evolution technique called random drift mutagenesis (RNDM). Mutant populations are screened and all functional mutants are collected and put forward into the next round of mutagenesis and screening. The goal of this technique is to evolve enzymes by rapidly accumulating mutations and exploring a greater sequence space by providing minimal selection pressure and high-throughput screening. The target enzyme was a β-glucosidase isolated from the thermophilic bacterium, Caldicellulosiruptor saccharolyticus that cleaves cellobiose resulting from endoglucanase hydrolysis of cellulose. Our screening method was fluorescence-activated cell sorting (FACS), an attractive method for assaying mutant enzyme libraries because individual cells can be screened, sorted into distinct populations and collected very rapidly. However, FACS screening poses several challenges, in particular, maintaining the link between genotype and phenotype because most enzyme substrates do not remain associated with the cells. We employed a technique where whole cells were encapsulated in cell-like structures along with the enzyme substrate. We used RNDM, in combination with whole cell encapsulation, to create and screen mutant β-glucosidase libraries. A mutant was isolated that, compared to the wild type, had higher specific and catalytic efficiencies (k _cat/K _M) with p-nitrophenol-glucopyranoside and -galactopyranoside, an increased catalytic turnover rate (k _cat) with cellobiose, an improvement in catalytic efficiency with lactose and reduced inhibition (K _i) with galactose and lactose. This mutant had three amino acid substitutions and one was located near the active site.     

Photoinduced energy transfer coupled to charge separation in a Ru(II)-Ru(II)-acceptor triad

The bichromophoric system Ru-Ru(C)-PI ([(bpy)3Ru-Ph-Ru(dpb)(Metpy-PI)][PF6]3, where bpy is 2,2'-bipyridine, Hdpb is 1,3-di(2-pyridyl)-benzene, Metpy is 4'-methyl-2,2':6',2' '-terpyridine and PI is pyromellitimide) containing two Ru(II) polypyridyl chromophores with a N6 and a N5C ligand set, respectively, was synthesized and characterized. Its photophysical properties were investigated and compared to those of the monochromophoric cyclometalated complexes Ru(C)-PI ([Ru(dpb)(Metpy-PI)][PF6]), Ru(C)-phi-PI ([Ru(dpb)(ttpy-PI)][PF6], ttpy is 4'-p-tolyl-2,2':6',2' '-terpyridine), Ru(C)-phi ([Ru(dpb)(ttpy)][PF6]), and Ru(C) ([Ru(dpb)(Metpy)][PF6]). Excitation of the Ru(C) unit in the dyads leads to oxidative quenching, forming the Ru(C)(III)-phi-PI*- and Ru(C)(III)-Pl.- charge-separated (CS) states with k(f)(ET) = 7.7 x 10(7) s(-1) (CH3CN, 298 K) in the tolyl-linked Ru(C)-phi-PI and k(f)(ET) = 4.4 x 10(9) s(-1) (CH2Cl2, 298 K) in the methylene-linked Ru(C)-PI. In the Ru-Ru(C)-PI triad, excitation of the Ru(C) chromophore leads to dynamics similar to those in the Ru(C)-PI dyad, generating the Ru(II)-Ru(C)(III)-PI*- CS state, whereas excitation of the Ru unit results in an initial energy transfer (k(EnT) = 4.7 x 10(11) s(-1)) to the cyclometalated Ru(C) unit. Subsequent electron transfer to the PI acceptor results in the formation of the same Ru(II)-Ru(C)(III)-PI*- CS state with k(f)(ET) = 5.6 x 10(9) s(-1) that undergoes rapid recombination with k(b)(ET) = 1 x 10(10) s(-1) (CH2Cl2, 298 K). The fate of the Ru(II)-Ru(C)(III)-PI*- CS state upon a second photoexcitation was studied by pump-pump-probe experiments in an attempt to detect the fully charge-separated Ru(III)-Ru(C)(II)-PI*- state.     

Bio-inspired, side-on attachment of a ruthenium photosensitizer to an iron hydrogenase active site model

The first ruthenium-diiron complex [(mu-pdt)Fe2(CO)5{PPh2(C6H4CCbpy)}Ru(bpy)2]2+ 1 (pdt = propyldithiolate, bpy = 2,2'-bipyridine) is described in which the photoactive ruthenium trisbipyridyl unit is linked to a model of the iron hydrogenase active site by a ligand directly attached to one of the iron centers. Electrochemical and photophysical studies show that the light-induced MLCT excited state of the title complex is localized towards the potential diiron acceptor unit. However, the relatively mild potential required for the reduction of the acetylenic bipyridine together with the easily oxidized diiron portion leads to a reductive quenching of the excited state, instead. This process results in a transiently oxidized diiron unit which may explain the surprisingly high light sensitivity of complex 1.     

Absence of reverse anomeric effect in furanosides

A series of conformationally restricted N-"furanosides" has been synthesized, where the carbons of the tetrahydrofuran ring are kept in one plane by a rigid norbornane skeleton, permitting only the ring oxygen to move above or below the tetrahydrofuran ring plane. This causes the substituents of the anomeric carbon to occupy a pseudoaxial or a pseudoequatorial position. On protonation of these "norbornane-furanosides" with trifluoromethanesulfonic acid, all three compounds exhibited decreasing coupling constants for the anomeric proton, indicating a shift toward the pseudoaxial conformation. The coupling constant measurements were supported by volume integration of NOESY cross-peaks, which also showed a change toward the pseudoaxial conformation upon protonation of the nitrogen. These results provide no evidence for the so-called reverse anomeric effect; on the contrary they are in full agreement with a small normal anomeric effect.     

Noncovalent interaction between amyloid-β-peptide (1–40) and oleuropein studied by electrospray ionization mass spectrometry

Beta amyloid peptide (Abeta) is the major proteinaceous component of senile plaques formed in Alzheimer's disease (AD) brain. The aggregation of Abeta is associated with neurodegeneration, loss of cognitive ability, and premature death. It has been suggested that oxidative stress and generation of free radical species have implications in the fibrillation of Abeta and its subsequent neurotoxicity. For this reason, it is proposed that antioxidants may offer a protective or therapeutic alternative against amyloidosis. This study is the first report of the formation of the noncovalent complex between Abeta or its oxidized form and the natural derived antioxidant oleuropein (OE) by electrospray ionization mass spectrometry (ESI MS). ESI MS allowed the real time monitoring of the complex formation between Abeta, OE, and variants thereof. Several experimental conditions, such as elevated orifice potential, low pH values, presence of organic modifier, and ligand concentration were examined, to assess the specificity and the stability of the formed noncovalent complexes.     

A simple and robust conductive graphite coating for sheathless electrospray emitters used in capillary electrophoresis/mass spectrometry.

A graphite-polyimide mixture was used as a conductive coating for sheathless electrospray emitters. The coating procedure described is simple and inexpensive compared to previously described methods. An investigation of the stability of the conductive coating carried out by electrochemical methods revealed good performances during oxidative stress. In addition, no decrease in emitter performance was seen during continuous electrospray in the positive electrospray mode for two weeks. Fast capillary electrophoresis with attomole sensitivity demonstrated the excellent performance of the described sheathless interface when used in conjunction with an orthogonal time-of-flight mass spectrometer. The overall simplicity, stability and low cost of this type of sheathless emitter make the described approach highly suitable for any on-column coupling of low flow rate separation techniques to a mass spectrometer.     

New mass spectrometric approaches to structural analysis in mixtures

Structural analysis of minor components in mixtures is a vital requirement in the development of any pharmaceutical compound. Mass spectrometry is uniquely able to give this kind of information on the trace amounts of material present as minor impurities in a drug substance. In this study we show that a combination of mass spectrometric analysers with different characteristics is an even more powerful approach with a higher chance of establishing a potential structure. In particular the advent of analysers capable of accurate mass measurement on small amounts of material has enabled structures to be proposed in situations where previously no real conclusions could be made. Copyright 1999 John Wiley & Sons, Ltd.     

Rational matrix approximation with a priori error bounds for non-symmetric matrix Riccati equations with analytic coefficients

In this paper the exact solution of the non-symmetric matrixRiccati equation with analytic coefficients is approximatedby a rational matrix function with a prefixed accuracy. Thisrational matrix function is locally defined as the exact solutionof a Riccati problem with matrix polynomial coefficients obtainedby truncation of the Taylor expansions of the matrix coefficientsof the original problem.