C-13 and P-31 NMR studies of 13CN-cyanocobalamin in sulfuric acid-water mixtures

Abstract In a previous study [1] we observed, in agreement with Satterlee [2, 3] an upfield shift of 31.4 Hz. of the chemical shift of the phosphorous atom of the nucleotide loop of cyanocobalamin (CNCbl) upon displacement of the axial benzimidazole ligand by excess cyanide (Eqn. 1). However, we observed no change in chemical shift for methylcobalamin (CH3-Cbl) upon displacement of axial benzimidazole by protonation (Eqn. 2, pKa = 2.89) although further lowering the pH causes an upfield shift of the phosphorous resonance due to protonation of the phosphodiester [1]. We consequently have attempted to determine if a change in phosphorous chemical shift occurs upon displacement of the axial base of CNCbl by protonation (Eqn. 3,pKa = 0.1 [4]). In sulfuric acid-water mixtures the 13C NMR spectrum of 13CNCbl shows two resonances at all H values [5–7] between +1.0 ([H2S04] = 0.0732 M)o and −0.5 ([H2S04] = 1.196 M), one at 111.77 ppm (w 1 2 = 10.4 Hz) and one at 121.40 ppm (w 1 2 =22.2 Hz). As the intensity of the former increases while that of the latter decreases with increasing acidity, they are assigned to the base-off and base-on species, respectively. Similarly, the 31P NMR spectrum of CNCbl shows two well defined resonances (w 1 2 ca. 2.0 Hz) separated by about 30 Hz with the downfield member decreasing in intensity (base-on) while the upfield member increases in intensity (base-off) with increasing acidity in this acidity range. However, in this case, the chemical shifts of both resonances move upfield with increasing acidity due to phosphodiester protonation. Plots of the 31P-chemical shift of the base-off species vs. Ho clearly show evidence of two sequential protonations which must be assigned to the first (Eqn. 4) and second (Eqn. 5) phosphodiester pKa's. The complete ionization scheme for CNCbl thus includes eight microscopic species and 12 microscopic pKa's from which analytical equations for the dependence of the 31P-chemical shifts of the base-off and base-on species as well as the dependence of the fraction of base-on species present (i.e., αbase-on, evaluated from the relative integrals of the 31P and/or 13C resonances) upon acidity may be derived. A non-linear least squares fit of the base-off 31P-chemical shifts to such an equation using Ho gives a poor fit, particularly at higher acidities. Although the fit is considerably improved by use of the HA (amide) acidity function [8–10] which is known to be applicable to many compounds which protonate at doubly bonded oxygen [11–17], an exact fit can be obtained by use of the Cox and Yates [18] generalized acidity function treatment (Eqn. 6, where CH+ is the concentration of hydrogen ion and X is the ‘excess acidity’)-H = m*X + log CH+ (6) using m* = 0.217. This treatment gives values for the macroscopic pKa's for phosphodiester deprotonation of the base-off species of -1.57 and -0.04 and chemical shifts of -335.81 Hz and -37.40 Hz for the base-off phosphodiester protonated and deprotonated species (II in Eqn. 3) respectively. A similar treatment of the 31P chemical shifts of the base-on species does not yield reliable values for the phosphodiester pKa's as this species can only be observed at acidities less than 3.14 M in HaSO (αbase-on is 0.033 at this acidity). Only the chemical shift of the base-on, phosphodiester deprotonated species (I in Eqn. 3) is reliably determined to be -1.72 Hz. Hence an upfield shift of 35.7 Hz is seen for the 31P-chemical shift upon displacement of the axial base of CNCbl by protonation (Eqn. 3). A similar attempt to correlate (αbase-on with acidity to determine the three macroscopic pKa's for the overall system fails as the standard deviation of the fit shows little or no variation with m* (Eqn. 6) and the final fit parameters at a given m* are sensitive to the inital guesses indicating that they are poorly determined by the data. Interestingly, at m* = 0.919 (Eqn. 6) the obaseon data strongly resembles the titration of a single protonable group, a plot of log ( α 1 − α) vs. H (m* = 0.919) giving a good straight line with a slope of 0.993 ± 0.013 and an apparent pKa of 0.11 ± 0.01. This may indicate that the base-on-base-off pKa is fairly insensitive to the state of protonation of the phosphodiester and that the literature value of 0.1 [4] is a good estimate for this pKa. The lack of an upfield shift of the phosphorus resonance of CH3Cbl upon displacement of the axial base by protonation, in contrast to CNCM, must surely be due to differences in the magnetic environments of the phosphorus atoms of the base-on species, as the chemical shifts of the two base-off species are essentially identical. There is a considerable difference in affinity of the free-base benzimidazole ligand for the cobalt atom in these two cobalamins. Using a value of 5.56 for the pKa of α-ribazole (the detached benzimidazole ribonucleoside) [19] the equilibrium constants for formation of the base-on species (Eqn. 7) may be calculated to be 467 for CH3Cbl and 2.88 × 105 for CNCbl. As phosphodiester 31P-chemical shifts are known to be sensitive to OPO bond angles and COPO torsion angles [20-22] it seems likely that the geometry about the phosphodiester phosphorus atom is distorted, relative to the base-off species in the tightly coordinated base-on CNCbl but it is not distorted in the much more loosely coordinated base-on CH3Cbl. Existing X-ray crystal structures of base-on CNCbl [23] and 5′-deoxyadenosylcobalamin [24] (the CH3Cbl structure has not been determined) support a difference in phosphodiester geometry between CNCbl and an alkylcobalamin.