TABLE 3

NMR chemical shift assignments for 1,20,25(OH)₃D3 analysis of its two-dimensional NMR (solvent: methanol-d₄)

The assignments are compared with that for its dihydroxy derivative prior to hydroxylation by CYP27B1.

C Atom	20,25(OH)₂D3		1,20,25(OH)₃D3		Structure
C Atom	¹H	¹³C	¹H	¹³C	Structure
1	2.12α, 2.41β	33.5	4.35	71.3
2	1.97α, 1.54β	36.6	1.89	43.6
3	3.76	70.5	4.13	67.2
4	2.54α, 2.19β	47	2.53α, 2.27β	46
5	NA	136	NA	135.4
6	6.22	122.6	6.33	124.8
7	6.03	119.4	6.08	119.2
8	NA	141.1	NA	NI
9	1.69α, 2.86β	29.7	1.69α, 2.87β	29.8
10	NA	145.9	NA	149.4
11	1.54α, 1.68β	22.7	1.58α, 1.69β	24.2
12	1.39α, 2.10β	42.2	1.38α, 2.11β	42
13	NA	45.4	NA	46.9
14	2.02	57.8	2	57.7
15	1.78	22.9	1.77	22.8
16	1.68α, 1.56β	24.4	1.70, 1.48	22.7
17	1.7	59.9	1.7	59.8
18	0.7	14	0.72	14
19	4.74, 5.04	112.64	4.90, 5.30	111.8
20	NA	74.4	NA	75.9
21	1.26	26.1	1.26	25.9
22	1.48, 1.35	45.4	1.47, 1.37	45.1
23	1.4	19.9	1.41	19.8
24	1.41	45.5	1.41	45.2
25	NA	70	NA	71.1
26	1.19	29.2	1.18	29.1
27	1.19	29.2	1.18	29.1

NA, not applicable (ternary carbons); NI, not identifiable (due to low resolution).