Peter R. Andreana

Immunological Response from an Entirely Carbohydrate Antigen: Design of Synthetic Vaccines Based on Tn−PS A1 Conjugates

An entirely carbohydrate-based immunogen consisting of a zwitterionic polysaccharide (ZPS) PS A1 and the well-known tumor antigen Tn has been designed, synthesized, and studied for immunological effects. The PS A1 motif was included to act as an MHCII elicitor for a T-cell-dependent immune response with increased immunogenicity against tumor-associated carbohydrate antigens, providing an alternative to carrier proteins. Through the use of C57BL/6 mice, it has been shown that chemical modification of PS A1 does not alter the recognition sequence responsible for an MHCII-mediated, T-cell-dependent immune response. The Tn-PS A1 conjugate construct confers specificity toward the Tn antigen alone, and specific carbohydrate immunoglobulins, namely, IgG3, are generated from intraperitoneal immunizations with or without adjuvant. The properties of the vaccine candidate are attributed to a site-specific linking strategy that incurs significant incorporation of Tn antigen.

A tandem one-pot, microwave-assisted synthesis of regiochemically differentiated 1,2,4,5-tetrahydro-1,4-benzodiazepin-3-ones.

A one-pot, two-step synthesis of the title compounds employs a multicomponent Ugi condensation reaction, microwave irradiation, and Fe(0) as a reductant. Two pathways are accessible; both routes utilize bifunctional, o-nitro-substituted arenes leading to either C2, N4, C5 substitution (A) or C2, N4 substitution (B).

Chemo-enzymatic synthesis of polyhydroxyazepanes

Abstract Galactose oxidase (EC 1.1.3.9, GAO) is an extracellular copper-containing enzyme that utilizes molecular oxygen to convert the C6-primary hydroxyl moiety of d -galactopyranosides to hydrated aldehydes. Subsequent dehydratative coupling with hydroxylamines produces oximes ( 3a – f ), which, when subjected to conditions of hydrogenolysis, give rise to polyhydroxyazepanes ( 11 – 17 ).

Reaction of hemoglobin with HOCl: Mechanism of heme destruction and free iron release

Hypochlorous acid (HOCl) is generated by myeloperoxidase using chloride and hydrogen peroxide as substrates. HOCl and its conjugate base (OCl(-)) bind to the heme moiety of hemoglobin (Hb) and generate a transient ferric species whose formation and decay kinetics indicate it can participate in protein aggregation and heme destruction along with subsequent free iron release. The oxidation of the Hb heme moiety by OCl(-) was accompanied by marked heme destruction as judged by the decrease in and subsequent flattening of the Soret absorbance peak at 405 nm. HOCl-mediated Hb heme depletion was confirmed by HPLC analysis and in-gel heme staining. Exposure of Hb to increasing concentrations of HOCl produced a number of porphyrin degradation products resulting from oxidative cleavage of one or more of the carbon-methene bridges of the tetrapyrrole ring, as identified by their characteristic HPLC fluorescence and LC-MS. A nonreducing denaturing SDS-PAGE showed several degrees of protein aggregation. Similarly, porphyrin degradation products were identified after exposure of red blood cells to increasing concentrations of HOCl, indicating biological relevance of this finding. This work provides a direct link between Hb heme destruction and subsequent free iron accumulation, as occurs under inflammatory conditions where HOCl is formed in substantial amounts.

Carbohydrates in transplantation.

Carbohydrate materials have become increasingly utilized in transplantation and cell/tissue engineering within the past year. This has been well documented in recent applications of immobilized or soluble alpha-galactosyl epitopes (i.e. oligosaccharides with a terminal Galalpha1-3Gal sequence) in preventing hyperacute rejection in pig-to-primate xenotransplantation. In addition, alpha-galactosyl polymers have been shown to exhibit much greater activity (up to 10(4) times) than alpha-galactosyl monomers in inhibiting the binding of anti-galactosyl antibodies to pig kidney epithelial cells and assisting in the prevention of cytotoxicity in human serum.

Targeting nitric oxide to cancer cells: cytotoxicity studies of glyco-S-nitrosothiols.

Glyco-S-nitrosothiols, fructose-2-SNAP and glucose-2-SNAP, were synthesized and found to be much more cytotoxic than SNAP in killing DU-145 human prostate cancer cells in vitro.

Mechanism of hypochlorous acid mediated heme destruction and free iron release

Here, we show that hypochlorous acid (HOCl), a potent neutrophil-generated oxidant, can mediate destruction of free heme (Ht) and the heme precursor, protoporphyrin IX (PPIX). Ht displays a broad Soret absorbance peak centered at 365 and 394 nm, indicative of the presence of monomer and μ-oxo-dimer. Oxidation of Ht by HOCl was accompanied by a marked decrease in the Soret absorption peak and release of free iron. Kinetic measurements showed that the Ht-HOCl reaction was triphasic. The first two phases were HOCl concentration dependent and attributable to HOCl binding to the monomeric and dimeric forms. The third phase was HOCl concentration independent and attributed to Ht destruction with the release of free iron. HPLC and LC-ESI-MS analyses of the Ht-HOCl reaction revealed the formation of a number of degradation products, resulting from the cleavage or modification of one or more carbon-methene bridges of the porphyrin ring. Similar studies with PPIX showed that HOCl also mediated tetrapyrrole ring destruction. Collectively, this work demonstrates the ability of HOCl to modulate destruction of heme, through a process that occurs independent of the iron molecule that resides in the porphyrin center. This phenomenon may play a role in HOCl-mediated oxidative injury in pathological conditions.

The synthesis of deoxy-α-Gal epitope derivatives for the evaluation of an anti-α-Gal antibody binding

Abstract α-Gal epitopes (also termed as α-Gal) are carbohydrate structures bearing the α- d -Gal-(1→3)-β- d -Gal terminus 1 and are known to be the antigen responsible for antibody-mediated hyperacute rejection in xenotransplantation. Terminal 2-, 3-, 4-, and 6-deoxy-Gal derivatives of α-Gal were synthesized. Inhibition ELISA using mouse laminin was established to determine the binding affinity of the synthesized α-Gal derivatives. 4-Deoxy-α-Gal derivative 7 showed a significant reduction in antibody recognition. The IC 50 value was 15-fold poorer than the standard α-Gal epitopes α- d -Gal-(1→3)-β- d -Gal-(1→4)-β- d -Glc-NHAc ( 39 ) and α- d -Gal-(1→3)-β- d -Gal-(1→4)-β- d -Glc-OBn ( 40 ). A similar observation was seen with 2-deoxy-α-Gal derivative 5 , whose IC 50 value was nearly tenfold higher than the standards. Interestingly, substitution at the terminal 3-position resulted in only a fourfold decrease in antibody recognition, suggesting a possible point of future derivation. Finally, 6-deoxy-α-Gal derivative 8 exhibited similar antibody recognition to both α-Gal epitope 39 and α-Gal epitope 40 . This strongly suggests that derivatization at the 6-position can be accomplished without loss of antibody recognition. These findings can be utilized for the future design of other α-Gal derivatives.

Efficient chemoenzymatic synthesis of globotriose and its derivatives with a recombinant α-(1→4)-galactosyltransferase

A truncated alpha-(1-->4)-galactosyltransferase (LgtC) gene from Neisseria meningitidis was cloned. The recombinant glycosyltransferase was expressed in Escherichia coli BL21 (DE3) strain with high specific activity (5 units/mg protein). Its acceptor specificity was carefully characterized. Then the purified enzyme was utilized in highly efficient syntheses of globotriose and a variety of alpha-(1-->4)-galactosylated derivatives as potential antibacterial agents.

Biocatalytic and chemical routes to all the stereoisomers of methionine and ethionine sulfoxides

Abstract Biotransformations of the N-phthaloyl derivatives of d - and l -methionine and of d - and l -ethionine by Beauveria bassiana ATCC 7159 or Beauveria caledonica ATCC 64970 produce the corresponding (SS) sulfoxides in good yield and diastereomeric excess. Pure (SSSC) diastereomers can be obtained from l -series substrates by crystallisation of the biotransformation extract, and the corresponding (SSRC) products obtained from d -series substrates by chromatography of the biotransformation extract. Hydrogen peroxide-catalysed oxidation of the N-phthaloyl derivatives of d - and l -methionine and of d - and l -ethionine gives diastereomeric mixtures from which the (SSSC) and (RSRC) diastereomers can be obtained by crystallisation, and the (SSRC) and (RSSC) diastereomers obtained by chromatography. N-Cbz- and N-t-Boc methionines are also converted to sulfoxides with predominant (SS) configuration by both B. bassiana and B. caledonica, but the isolated yields and d.e. of products were generally lower than those obtained from the N-phthaloyl substrates. Removal of the N-phthaloyl group from diastereomerically pure methionine and ethionine sulfoxides gave the corresponding amino acid sulfoxides in high yield; removal of N-Cbz and N-t-Boc groups from protected methionine sulfoxides was also achieved without loss of configuration at sulfur.