Determination of the tautomeric form of the imidazole ring of L-histidine in basic solution by carbon-13 magnetic resonance spectroscopy

Abstract

°13C shift referred to TMS.respectively.33 The effect of self-association is probably negligible, since the concentration of the solute is low (1 mol %) in polar solvents. Then it is found that the 14N shifts of imidazole in acetone and DMSO (proton acceptors) are shifted downfield with respect to methanol, water, and trifluoroacetic acid (proton donors). The difference of 14N shifts is 6-9 ppm over the range of the experimental error.Similarly upfield 14N shifts of 6 ± 3 ppm and 7.4 ± 3-9.8 ± 5 ppm are observed in pyrazole and 1,2,4-triazole, respectively. These values are recognized as about half of the corresponding values for A-methyl derivatives, taking into account the argument described above.Sometimes difficulty arises in observing 14N shifts in protonated species, because a longer correlation time due to protonation tends to broaden nmr lines and make the two-and three-bond N-H couplings unobservable. Protonation shifts on imidazole and thiazole are obtained by the decoupling of one-bond N-H couplings. The upfield 14N shifts are 41 ± 3 ppm and 123.1 ± 3 (34) H.

Infrared and Raman spectroscopic study of pH-induced structural changes of l-histidine in aqueous environment

“…Indeed, as empirically demonstrated by Ashikawa and Itoh [21,22] and confirmed by others [16,18,31], the 1588 cm À1 band appears to be correlated to the N p -protonated tautomer and the 1571 cm À1 one to the N t -protonated form. From the strong intensity of the latter compared to that of the 1588 cm À1 peak it can be concluded, that protonation of N t is preferred over protonation of N p , which is in line with the results of a temperature dependency studies of histidine by means of NMR [45] and of 4-methyl-imidazole by Raman [16]. The fact that the 1588 cm À1 component is even virtually absent below pH 9 can be interpreted as absence (or largely reduced presence) of the N p -protonated form of H 2 His 0 .…”

“…According to literature [16,[23][24][25]31,32,45,46] histidine can be present in five different pH-dependent ionic forms, which are shown in Fig. 1.…”

Measurement of histidine pKavalues and tautomer populations in invisible protein states

“…1A, up triangle), and 3-methyl L-histidine (Fig. 1A, down triangle), pH 8.2, the latter two compounds serving as mimics of the « and δ tautomeric states (52,58), respectively, with little difference obtained in the coupling values.…”

“…1, Upper). The cationic form is of course dominant at low pH, whereas the « form is favored ∼4:1 over the δ form at high pH, according to observations of the free amino acid in solution (52,53). These conformations are usually indistinguishable in crystal structures, but can be readily identified by heteronuclear multiplebond correlation (HMBC)-type NMR experiments which measure the imidazole ring 15 N chemical shifts (7).…”

“…A promising alternative for studies of rare conformers is to monitor the chemical shifts of the 13 C γ and 13 C δ2 imidazole carbons. Theoretical and experimental studies of model compounds show that the 13 C γ and 13 C δ2 nuclei, but not 13 C e1 , are sensitive to the histidine conformational state, with shift differences as large as 12 and 8 ppm, respectively (7,52,56,57). Although the 13 C e1 chemical shift is insensitive to the imidazole conformational equilibrium, the one-bond 13 C e1 -1 H e1 scalar coupling ( 1 J C«H« ) shows a relatively large variation of ∼12 Hz between neutral and cationic states with little or no difference between neutral tautomers (58,59) (Fig.…”