Synopsis
The synthesis of Leu-enkephalin selectively l70-enriched in Gly' and Gly3 is reported. The 170-nm chemical shifts of Leu5]-and [170-Gly3, Le~~l-enkephalins in H,O are almost identical and independent of the pH. Since hydrogen bonding is the dominant factor governing the chemical shifts of the peptide oxygen, it can be concluded that the hydration state of both oxygens is identical end independent of the pH. The 170 chemical shifts of the [170-Leu5]enkephalin terminal carboxyl group at pH -1.9 and 5.6 are very different in H,O but very similar in CH,CN/DMSO (4 : 1) solution. This suggests that the protonation state of the carboxyl group at both pH values in CH,CN/DMSO solution is the same and consequently that Leu-enkephalin exists in the neutral form at pH -5.6. In this organic mixed solvent system both Gly2 and Gly3 oxygen resonances exhibit a significant shift to high frequency by the same extent (A8 -30 ppm). It is concluded that both peptide oxygens are not hydrogen bonded to an appreciable extent and that no specific 2 + 5 hydrogen bonding exists to an appreciable extent.
This conclusion is in agreement with the energy of activation for molecular rotation, as determined from TI measurements, which was found to be almost identical for both [170-Gly2, Leu5]and [170-Gly3, Leu5]-enkephaLins in CH3CN/DMS0 (4 : 1) mixed solvent.