Amide cis trans isomerization (CTI) in methyl 2-deoxy-2-acylamido-D-glucopyranosides was investigated by H-1 and C-13 NMR spectroscopy. Singly C-13-labeled methyl 2-deoxy-2-formamido-D-glucopyranoside (MeGlcNFm) anomers provided standard H-1 and C-13 chemical shifts and H-1-H-1 and C-13-C-13 spin-coupling constants for cis and trans amides that are detected readily in aqueous solution. Equipped with this information, doubly C-13-labeled methyl 2-deoxy-2-acetamido-o-glucopyranoside (MeGlcNAc) anomers were investigated, leading to the detection and quantification of cis and trans amides in this biologically important aminosugar. In comparison to MeGIcNFm anomers, the percentage of cis amide in aqueous solutions of MeGIcNAc anomers is small (similar to 23% for MeGIcNFm versus similar to 1.8% for MeGlcNAc at 42 degrees C) but nevertheless observable with assistance from C-13-labeling. Temperature studies gave thermodynamic parameters Delta G degrees, Delta H degrees, and Delta S degrees for cis-trans interconversion in MeGlcNFm and MeGlcNAc anomers. Cis/trans equilibria depended on anomeric configuration, with solutions of alpha-anomers containing less cis amide than those of beta-anomers. Confirmation of the presence of cis amide in MeGlcNAc solutions derived from quantitative C-13 saturation transfer measurements of CTI rate constants as a function of solution temperature, yielding activation parameters E-act, Delta G degrees double dagger, Delta H degrees double dagger and Delta S degrees double dagger for saccharide CTI. Rate constants for the conversion of trans to cis amide in MeGIcNFm and MeGIcNAc anomers ranged from 0.02 to 3.59 s(-1) over 31-85 degrees C, compared to 0.24-80 s(-1) for the conversion of cis to trans amide over the same temperature range. Energies of activation ranged from 16-19 and 19-20 kcal/mol for the cis --> trans and trans --> cis processes, respectively. Complementary OFT calculations on MeGlcNFm and MeGlcNAc model structures were conducted to evaluate the effects of an acyl side chain and anomeric structure, as well as C2-N2 bond rotation, on CTI energetics. These studies show that aqueous solutions of GlcNAc-containing structures contain measurable amounts of both cis and trans amides, which may influence their biological properties.