Fig 1.
Three most common aliphatic polyisocyanates.
Three different polyisocyanates are generated from hexamethylene diisocyanate monomer as shown. The biuret contains one partially hydrolyzed HDI. The 6-member ring structure of the isocyanurate is stable, however one of the internally blocked NCOs of the 4 member ring of the uretdione can react under appropriate conditions.
Fig 2.
LC-MS analysis of GSH reaction products with HDI isocyanurate.
TICs are shown for reaction products generated when HDI isocyanurate was reacted with GSH (A and B) and without GSH (C and D), in solution buffered to pH 7.4 (A and C) or without buffer, pH < 4.0 (B and D). The m/z for the major products recognized as new [M+H]+ ions are labeled, along with peaks reflecting GSH, GSSG. Unlabeled peak eluting ~0.5 min (A and C) is due to sodium phosphate.
Fig 3.
Characterization of GSH reaction product with HDI isocyanurate at physiologic pH.
(A) Mass spec analysis of sample eluting from reverse phase LC column ~3 minutes, note dominance of doubly and triply charged species corresponding to the major product, the 1426.53 m/z [M+H]+ ion. (B) the CID fragmentation spectra of the 1426.53 m/z [M+H]+ ion upon MS/MS. (C) structural model for the major reaction product of GSH with HDI isocyanurate based on exact mass and expected fragmentation pattern (S2 Fig).
Fig 4.
LC-MS analysis of GSH reaction products with HDI biuret.
TICs are shown for reaction products generated when HDI biuret was reacted with GSH (A and B) and without GSH (C and D), in solution buffered to pH 7.4 (A and C) or without buffer, pH < 4.0 (B and D). The m/z for the major products recognized as new [M+H]+ ions are labeled, along with peaks reflecting GSH, GSSG. Unlabeled peak eluting ~0.5 min (A and C) is due to sodium phosphate.
Fig 5.
Characterization of major GSH reaction product with HDI biuret at physiologic pH.
(A) Mass spec analysis of major product eluting from reverse phase LC column ~2.8 minutes, note dominance of doubly and triply charged species corresponding to the 1400.55 m/z [M+H]+ ion. (B) the CID fragmentation spectra of the 1400.55 m/z [M+H]+ ion upon MS/MS. (C) structural model for the major reaction product of GSH with HDI biuret based on exact mass and expected fragmentation pattern (S6 Fig).
Fig 6.
LC-MS analysis of GSH reaction products with HDI uretdione.
TICs (A and B) and A210 chromatograms (C and D) are shown for reaction products generated when GSH was reacted with HDI uretdione (solid black line) or without (dashed red line). The m/z for the major products recognized as new [M+H]+ ions are labeled, along with GSH and GSSG.
Fig 7.
Characterization of major GSH reaction product with HDI uretdione at physiologic pH.
(A) Mass spec analysis of sample eluting from reverse phase LC column ~ 2.1 minutes, note dominance of doubly and triply charged species corresponding to the 1258.44 m/z [M+H]+ ion. (B) the CID fragmentation spectra of the 1258.44 m/z [M+H]+ ion upon MS/MS. (C) structural model for the major reaction product of GSH with HDI uretdione based on exact mass and expected fragmentation pattern (S10 Fig).
Fig 8.
Characterization of minor GSH reaction product with HDI uretdione at physiologic pH.
(A) Mass spec analysis of sample eluting from reverse phase LC column ~ 2.3 minutes, note dominance of doubly and triply charged species corresponding to the 951.36 m/z [M+H]+ ion. (B) the CID fragmentation spectra of the 951.36 m/z [M+H]+ ion upon MS/MS. (C) structural model for the minor reaction product of GSH with HDI uretdione based on exact mass and expected fragmentation pattern (S11 Fig).