[CH2:1]([OH:5])[CH2:2][CH2:3][CH3:4].N1C=C(C)C=C(C)C=1.[CH3:14][P:15](Cl)(Cl)=[O:16].C[C@H]1[C@@]2(O)O[C@H](C[C@H](OC)C(C)=CC=CC=C[C@@H](C)C[C@@H](C)C([C@H](OC)[C@H](O)C(C)=C[C@@H](C)C(C[C@@H]([C@@H](C[C@H]3C[C@@H](OC)[C@H](O)CC3)C)OC([C@H]3N(C(C2=O)=O)CCCC3)=O)=O)=[O:36])CC1.C([O-])(O)=O.[Na+]>C(Cl)Cl.CCOC(C)=O.N1C=NN=N1>[CH2:1]([O:5][P:15]([CH3:14])(=[O:16])[OH:36])[CH2:2][CH2:3][CH3:4]To a flask containing 1H-tetrazole (˜0.002 g, 0.028 mmol) was added a solution of n-butanol (0.041 g, 0.55 mmol) in 0.33 mL of DCM, followed by a solution of 3,5-lutidine (0.090 g, 0.84 mmol) in 0.33 mL of DCM. The resulting clear solution was cooled to 0° C. then added, under an atmosphere of N2, a solution of methylphosphonic dichloride (0.073 g, 0.55 mmol) in 0.33 mL of DCM. The resulting white suspension was stirred to ambient temperature overnight. To a cooled (0° C.) solution of rapamycin (0.1 g, 0.11 mmol) in 0.5 mL of DCM was added a solution of 3,5-lutidine (0.090 g, 0.84 mmol) in 0.5 mL of DCM, followed immediately by the phosphorylating reagent (yellow solution with white precipitate) and a 1.0 mL DCM wash. The resulting yellow solution was stirred at 0° C. for 1.0 h (followed by MS). The cold (0° C.) reaction solution was diluted with ˜20 mL EtOAc then transferred to a separatory funnel containing EtOAc (120 mL) and saturated NaHCO3 (100 mL). Upon removing the aqueous layer, the organic layer was washed successively with ice cold 1N HCl (1×100 mL), saturated NaHCO3 (3×100 mL), and brine (1×100 mL), then dried over MgSO4 and concentrated. The crude product was purified by silica gel flash chromatography (eluted with 0.25:10:3:3 then 0.5:10:3:3 MeOH/DCM/EtOAc/hexane) then RP HPLC (85% MeOH/H2O) to provide 0.063 g of a white solid (˜2:1 diastereomeric mixture): 1H NMR (300 MHz, CDCl3) 4.15 (m, 1Ha, 1Hb), 4.11-3.89 (m, 3Ha, 3Hb), 3.04 (m, 1Ha, 1Hb); 31P NMR (121 MHz, CDCl3) □32.1, 29.9; 1071 m/z (M+Na).
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