Azasugar inhibitors as pharmacological chaperones for Krabbe disease

Krabbe disease is a devastating neurodegenerative disorder characterized by rapid demyelination of nerve fibers. This disease is caused by defects in the lysosomal enzyme β-galactocerebrosidase (GALC), which hydrolyzes the terminal galactose from glycosphingolipids. These lipids are essential components of eukaryotic cell membranes: substrates of GALC include galactocerebroside, the primary lipid component of myelin, and psychosine, a cytotoxic metabolite. Mutations of GALC that cause misfolding of the protein may be responsive to pharmacological chaperone therapy (PCT), whereby small molecules are used to stabilize these mutant proteins, thus correcting trafficking defects and increasing residual catabolic activity in cells. Here we describe a new approach for the synthesis of galacto-configured azasugars and the characterization of their interaction with GALC using biophysical, biochemical and crystallographic methods. We identify that the global stabilization of GALC conferred by azasugar derivatives, measured by fluorescence-based thermal shift assays, is directly related to their binding affinity, measured by enzyme inhibition. X-ray crystal structures of these molecules bound in the GALC active site reveal which residues participate in stabilizing interactions, show how potency is achieved and illustrate the penalties of aza/iminosugar ring distortion. The structure–activity relationships described here identify the key physical properties required of pharmacological chaperones for Krabbe disease and highlight the potential of azasugars as stabilizing agents for future enzyme replacement therapies. This work lays the foundation for new drug-based treatments of Krabbe disease.     

An ESR study of some group IVB free radical adducts of diethylketomalonate

The formation of the compound RSnX(acac)₂ (acac = 2,4-pentanedionato) by reaction of bis(2,4-pentanedionato)tin(II) on a halide RX with R = CH₃, C₂H₅, C₄H₉, C₆H₅, CH₂I, (C₆H₅)₃SnCH₂, (C₂H₅)₃SnCH₂ and X = I, Br has been studied by polarography. At 25°C, it is in fact an equilibrium whose constant has been measured. The intermediate formation of the ion-pair [RSn(acac)₂⁺X^?] has allowed us to explain the experimental results.     

Application of the chiral base desymmetrisation of imides to the synthesis of the alkaloid jamtine and the antidepressant paroxetine

The synthesis of the alkaloid jamtine and the antidepressant paroxetine have been addressed by a strategy involving asymmetric desymmetrisation of prochiral imides by a chiral lithium amide base. A short reaction sequence, starting with a cyclohexane fused succinimide, led to the structures originally reported for the alkaloid jamtine and its derived N-oxide. The structures synthesised are shown not to correspond with those originally reported. A second sequence involves desymmetrisation of a 4-arylglutarimide, and provides a short enantioselective synthesis of the drug substance paroxetine.     

Conversion of clavulanic acid into thiadeoxa nuclear analogues

1,4-Addition of sulphur nucleophiles to the diene ($\text{12}$) derived $\text{via}$ the pen-2-em ($\text{5}$) from clavulanic acid provides the thiadeoxa analogues ($\text{14}$–$\text{15}$). X-ray analysis of the ester ($\text{14}$) shows the thermodynamically stable isomers to have the same relative stereochemistry as clavulanic acid.     

Comparative inhibition by substrate analogues 3-methoxy- and 3-hydroxydesaminokynurenine and an improved 3 step purification of recombinant human kynureninase.

Background  

Kynureninase is a key enzyme on the kynurenine pathway of tryptophan metabolism. One of the end products of the pathway is the neurotoxin quinolinic acid which appears to be responsible for neuronal cell death in a number of important neurological diseases. This makes kynureninase a possible therapeutic target for diseases such as Huntington's, Alzheimer's and AIDS related dementia, and the development of potent inhibitors an important research aim.     

Asymmetric alkylation of diarylmethane derivatives. Improved results using methoxyethoxy substituent

Alkylation of 2-methoxyethoxyphenyl phenyl methane using sec-BuLi and (−)-sparteine has been carried out in excellent yields and up to 94% ee. The best results were obtained in allylation reactions but methylation, ethylation, benzylation and trimethylsilylation have all been carried out with acceptable ee.     

Capillary isoelectric focusing-mass spectrometry: analysis of protein mixtures from human body fluids

Isoelectric focusing within a fused silica capillary (cIEF) has proved to be a powerful and practical method for high-resolution separation of analytes from complex biological mixtures. This technique overcomes many of the problems of isoelectric focusing within slab gel media. However current cIEF systems commonly utilize UV detection which limits the detail of analyte structural information that is obtained during analysis. The use of mass spectrometry (MS) as the detection system provides much greater structural information about the detected analytes allowing accurate relative molecular mass (M(r)) determination for proteins and polypeptides. We have constructed a cIEF-MS interface and compared the separation of standard proteins analyzed by cIEF-UV with cIEF-MS. This allowed rapid optimization of the cIEF-MS system performance. Further we have demonstrated the use of MS as a detection system provides accurate M(r) information and can provide analyte modification details. These factors increase the likelihood of absolute identification for physiological proteins within complex in vivo-derived mixtures. To demonstrate the value of cIEF-MS in such analyses we have undertaken an examination of cerebrospinal fluid (CSF), and tentatively identified a number of constituent proteins. We have also analyzed whole blood from control and diabetic patients. We show that glycated alpha- and beta- chains of hemoglobin are found in almost equal abundance in diabetic patient blood. From these results we suggest cIEF-MS is an efficient and useful tool for the separation and examination of in vivo-derived analytes within physiological fluids.     

Benzocyclobutenes-x : Synthesis of cyclobuta[l]phenanthrene derivatives from [2 + 2]photoadducts of chlorinated ethenes and phenanthrene

UV irradiation of phenanthrene and 1,1-dichloro-, 1,2-dichloro-, and 1,1,2-trichloroethenes resulted m [2+2]cycloaddition to give cyclobutane derivatives. Treatment of the 1,2-dichloro-adducts with N-bromosuccinimide resulted in aromatisation to yield cis- and tras-l,2-dichloro-1,2- dihydrocyclobuta[l)phenanthrene, whereas the 1,1-dichloro-adduct resulted in aromatisation and hydrolysis to give cyclobuta[l]phenanthrene-l(2H)-one. The trichloroethene adducts gave 1,2-dichloro-2a,10b-dihydrocyclobuta [l)phenanthrene upon treatment with base, and reaction with N- bromosuccinimide resulted in bromine substitution in the cyclobutane ring. The stereochemistry of the adducts and the stereoselectivity of the bromination reactions are discussed.