Table 2.
Synthetic Data for Nucleotide Derivatives, Including Structural Verification Using High-Resolution Mass Spectroscopy and Purity Verification Using HPLC
| HPLC (rt; min)a |
||||||
|---|---|---|---|---|---|---|
| no. | formula | FAB (M − H+) |
system 1 (purity) (%) |
system 2 (purity) (%) |
yield (%)e |
|
| calcd | found | |||||
| 3a | C12H18N5O12P3 | 516.0087 | 516.0085 | 4.289 (98) | 17.009d (98) | 14 |
| 4a | C12H16N5O6P | 356.0760 | 356.0765 | 4.178 (99) | 8.145d (98) | 65 |
| 5a | C11H17N2O14P3 | 492.9814 | 492.9832 | 1.737 (99) | 6.764b (96) | 22 |
| 6a | C11H16N2O11P2 | 413.0151 | 413.0151 | 2.130 (98) | 4.531c (97) | 24 |
| 7a | C11H15N2O8P | 333.0488 | 333.0506 | 3.235 (99) | 3.466c (98) | 58 |
| 8a | C12H16N5O6P | 356.0760 | 356.0765 | 5.057 (97) | 11.091d (99) | 60 |
| 9a | C12H18N5O12P3 | 516.0087 | 516.0085 | 5.709 (97) | 17.517d (97) | 30 |
Purity of each derivative was ≥ 95%, as determined using HPLC with two different mobile phases. Purity of all compounds in solvent 1 and compounds 5a, 6a, and 7a in solvent 2 was checked using an SMT OD-5-60 RP-C18 analytical column (250 × 4.6 mm; Separation Methods Technologies, Inc., Newark, DE) in two solvent systems. Compounds 3a, 4a, 8a, and 9a were checked in solvent 2 using a Phenomenex Luna 5µ C18(2) analytical column (250 × 4.6 mm). All gradients were completed in 20 min. System 1: gradient of 0.1 M TEAA/CH3CN from 95/5 to 40/60. System 2: as specified below.
Gradient of 5 mM TBAP/CH3CN from 80/20 to 40/60.
Gradient of 0.1 M TEAB/CH3CN from 100/0 to 90/10.
Gradient of 5 mM TBAP/CH3CN from 95/5 to 40/60.
The percent yields refer to overall yield for the entire phosphorylation sequence.
3a, MRS2340; 5a, MRS 2341.