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Adam J Janczuk

Publications and source records attributed to Adam J Janczuk.

4 recordsLinked to original sources
PubMed2004

Alpha-Galactosyl trisaccharide epitope: Modification of the 6-primary positions and recognition by human anti-alphaGal antibody.

Galactose oxidase (EC 1.1.3.9, GAO) was used to convert the C-6' OH of Galbeta(1 --> 4)Glcbeta-OBn (5) to the corresponding hydrated aldehyde (7). Chemical modification, through dehydratative coupling and reductive amination, gave rise to a small library of Galbeta(1 --> 4)Glcbeta-OBn analogues (9a-f, 10, 11). UDP-[6-(3)H]Gal studies indicated that alpha1,3-galactosyltransferase recognized the C-6' modified Galbeta(1 --> 4)Glcbeta-OBn analogues (9a-f, 10, 11). Preparative scale reactions ensued, utilizing a single enzyme UDP-Gal conversion as well as a dual enzymatic system (GalE and alpha1,3GalT), taking full advantage of the more economical UDP-Glc, giving rise to compounds 6, 15-22. Galalpha(1 --> 3)Galbeta(1 --> 4)Glcbeta-OBn trisaccharide (6) was produced on a large scale (2 g) and subjected to the same chemoenzymatic modification as stated above to produce C-6" modified derivatives (23-30). An ELISA bioassay was performed utilizing human anti-alphaGal antibodies to study the binding affinity of the derivatized epitopes (6, 15-30). Modifications made at the C-6' position did not alter the IgG antibody's ability to recognize the unnatural epitopes. Modifications made at the C-6" position resulted in significant or complete abrogation of recognition. The results indicate that the C-6' OH of the alphaGal trisaccharide epitope is not mandatory for antibody recognition.

Antibodies↗
PubMed2002

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

Alpha-Gal epitopes (also termed as alpha-Gal) are carbohydrate structures bearing the alpha-D-Gal-(1-->3)-beta-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 alpha-Gal were synthesized. Inhibition ELISA using mouse laminin was established to determine the binding affinity of the synthesized alpha-Gal derivatives. 4-Deoxy-alpha-Gal derivative 7 showed a significant reduction in antibody recognition. The IC(50) value was 15-fold poorer than the standard alpha-Gal epitopes alpha-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-beta-D-Glc-NHAc (39) and alpha-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-beta-D-Glc-OBn (40). A similar observation was seen with 2-deoxy-alpha-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-alpha-Gal derivative 8 exhibited similar antibody recognition to both alpha-Gal epitope 39 and alpha-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 alpha-Gal derivatives.

Antibodies↗
PubMed2002

Novel substrates for nitric oxide synthases.

Enzymatic generation of nitric oxide (NO) by nitric oxide synthase (NOS) consists of two oxidation steps. The first step converts L-arginine to N(G)-hydroxy-L-arginine (NOHA), a key intermediate, and the second step converts NOHA to NO and L-citrulline. To fully probe the substrate specificity of the second enzymatic step, an extensive structural screening was carried out using a series of N-alkyl (and N-aryl) substituted-N'-hydroxyguanidines (1-14). Among the eleven N-alkyl-N'-hydroxyguanidines evaluated, N-n-propyl (2), N-iso-propyl (3), N-n-butyl (4), N-s-butyl (5), N-iso-butyl (6), N-pentyl (8) and N-iso-pentyl (9) derivatives were efficiently oxidized by the three isoenzymes of NOS (nNOS, iNOS and eNOS) to generate NO. N-Butyl-N'-hydroxyguanidine (4) was the best substrate for iNOS (K(m)=33 microM) and N-iso-propyl-N'-hydroxyguanidine (3) was the best substrate for nNOS (K(m)=56 microM). When the alkyl substituents were too small (such as ethyl 1) or too large (such as hexyl 10 and cyclohexyl 11), the activity decreased significantly. This suggests that the van der Waals interaction between the alkyl group and the hydrophobic cavity in the NOS active site contributes significantly to the relative reactivity of compounds 3-11. Moreover, five N-aryl-N'-hydroxyguanidines were found to be good substrates for iNOS, but not substrates for eNOS and nNOS. N-phenyl-N'-hydroxyguanidine was the best substrate among them (K(m)=243 microM). This work demonstrates that N-alkyl substituted hydroxyguanidine compounds are novel NOS substrates which 'short-circuit' the first oxidation step of NOS, and N-aryl substituted hydroxyguanidine compounds are isoform selective NOS substrate.

Animals↗