The development of an integrated platform to identify breast cancer glycoproteome changes in human serum

Protein glycosylation represents one of the major post-translational modifications and can have significant effects on protein function. Moreover, changes in the carbohydrate structure are increasingly being recognized as an important modification associated with cancer etiology. In this report, we describe the development of a proteomics approach to identify breast cancer related changes in either concentration and/or the carbohydrate structures of glycoprotein(s) present in blood samples. Diseased and healthy serum samples were processed by an optimized sample preparation protocol using multiple lectin affinity chromatography (M-LAC) that partitions serum proteins based on glycan characteristics. Subsequently, three separate procedures, 1D SDS-PAGE, isoelectric focusing and an antibody microarray, were applied to identify potential candidate markers for future study. The combination of these three platforms is illustrated in this report with the analysis of control and cancer glycoproteomic fractions. Firstly, a molecular weight based separation of glycoproteins by 1D SDS-PAGE was performed, followed by protein, glycoprotein staining, lectin blotting and LC–MS analysis. To refine or confirm the list of interesting glycoproteins, isoelectric focusing (targeting sialic acid changes) and an antibody microarray (used to detect neutral glycan shifts) were selected as the orthogonal methods. As a result, several glycoproteins including alpha-1B-glycoprotein, complement C3, alpha-1-antitrypsin and transferrin were identified as potential candidates for further study.     

Selectfluor-mediated mild oxidative halogenation and thiocyanation of 1-aryl-allenes with TMSX (X = Cl,Br, I,NCS) and NH4SCN

Presence of TMSX (X = Cl, Br, I) unleashes the oxidative character of Selectfluor and provides a mild dihalogenation method for 1-arylallenes. Preference for 2,3-addition was observed with TMSCl in MeCN irrespective of the nature of the substituent on the aryl moiety, whereas 1,2-addition was preferred in [BMIM][BF₄]. With TMSBr and TMSI only products corresponding to 2,3-addition were observed. Reactions carried out with TMSBr in IL solvents gave the corresponding monobromoalkenes as a major product along with the isomeric dibromo-alkenes. Reaction with NH₄SCN provided convenient access to dithiocyanate derivatives. The same products were formed via TMS-NCS/Selectfluor. Formation of common products via TMSNCS and NH₄SCN points to the formation and interplay of SCN⁺/NCS⁺ as incipient electrophiles.     

New protic salts of aprotic polar solvents

Aprotic polar solvents such as DMF, acetonitrile or DMSO can be protonated to form stable triflate salts when treated with triflic acid. The reaction of the same solvents with N,N-bis(trifluoromethanesulfonyl)imide led to the isolation of the corresponding N,N-bis(trifluoromethanesulfonyl)imide salts.     

Acetyl Chloride-Methanol as a Convenient Reagent for: A) Quantitative Formation of Amine Hydrochlorides B) Carboxylate Ester Formation C) Mild Removal of N-t-Boc-Protective Group.

Hydrogen chloride qualitatively generated in situ by the addition of acetyl chloride to alcoholic solutions is a useful reagent for carboxylic acid esterification, N-t-Boc deprotection and phosphoramide solvolysis reactions.     

Enantioselective hydrolysis of a Schiff's base of D,L-Phenylalanine ethyl ester in water-poor media via the reaction catalyzed with α-chymotrypsin immobilized on hydrophilic macroporous gel support

The application of immobilized α-chymotrypsin for the purpose of enantioselective hydrolysis of a Schiff's base of D,L-Phe-OEt (D,L-SBPH) in the mixed water-acetonitrile media with the different content of water is described. The immobilized biocatalyst was prepared by the chemical coupling of the enzyme to poly(vinyl alcohol) (PVA) cryogel—the macroporous hydrogel prepared by means of the freezing-thawing techniques. SBPH is water insoluble, and, therefore, acetonitrile (MeCN) with minor water additives was used as a solvent for the reaction of enantics elective hydrolysis of the racemic substrate. The process was conducted for 96–200 h, and L-Phe with the purity up to 98% e.e. precipitated in both the reaction medium and gel-carrier bulk. Theproduct wasrecovered by washing the organo-insoluble sediment with aqueous ammonia. D-Phe with the purity up to 85% e.e. was recovered from the organic solution of D-ester after its acidic hydrolysis. The PVA-cryogel-attached enzyme was effective in SBPH hydrolysis in MeCN/water mixtures. The immobilized biocatalyst was active for more than 1 mo of application and could be successfully used after another 4 mo storage at +10°C.     

Chemical modification of polymeric microchip devices

Analytical polymeric microchips in both fluidic and array formats offer short analysis times, coupling of many sample processing and chemical reaction steps on one platform with minimal sample and reagent consumption, as well as low cost, minimal fabrication times and disposability. However, the invariable bulk properties of most commercial polymers have driven researchers to develop new modification strategies. This article critically reviews the scope and development of chemical modifications of such polymeric chips since 2003. Surface modifications were based on chemical derivatization or activation of surface layers with reagent solutions, reactive gases and irradiation. Bulk modification of polymer chips used newly incorporation of monomers with selective chemical functionalities throughout the bulk polymer material and integrated the chip modification and fabrication into a single step. Such modifications hold a great promise for establishing a true ‘lab-on-chip’ as can be seen from many novel applications for modulating electroosmosis, suppressing protein adsorption in microchip capillary electrophoretic separations, extraction of analytes and for zone-specific binding of enzymes and other biomolecules.     

Synthesis of novel chiral fluorinating agents, （＋）- and （-）-N-fluoro-3-methyl-3-（4-methylphenyl）-2H-benzo[e] [ 1,2]thiazine- 1,1,4-triones

Treatment of N-t-butylbenzenesulfonamide with an excess of BuLi, followed by the reaction with methyl 2-（4-methylphenyl）propanoate, gave the corresponding 2-carboxybenzenesulfonamide, which underwent a sequence of consecutive N-deprotective cyclization process mediated by TMSCI-NaI-MeCN reagent to afford the N-sulfonylimine. Following the bromination and ring expansion, 3-methyl-3-（4-methylphenyl）-2H-benzo[e][1,2]thiazine-l,l,4-trione was obtained. Optical resolution of the racemic benzosultam using （-）-menthoxyacetyl chloride, furnished the optically p.ure （＋）- and （-）-3-methyl-3-（4-methylphenyl）-2H- benzo[e][1,2]thiazine-1,1,4-triones, which were fluorinated with FC103 to produce the corresponding chiral N-F agents.