To amach 27 to the remainder of the PI5P scaffold, phosphoramidite 34 was required. To prevent racemiza,on at the glycerol sn-2 posi,on, two protec,ng group strategies were inves,gated, however, here we detail the more robust strategy that resulted in no racemisa,on (Scheme 1). Details of the alterna,ve strategy can be found in the supplementary informa,on (Supplementary Scheme S2). Both routes started by reac,ng (R)-(-)-2,3-isopropylidene-sn-glycerol with palmi,c acid and DCC to form the palmitate 29 in a 77% yield. The method used for the opening of the acetonide, which was first reported by Rychnovsky et al. in the total synthesis of (-)-Roxa,cin and then more recently used by Painter et al. in the synthesis of a stearoyl-based glyceride, minimises the risk of racemisa,on. Briefly, acetonide 29 was treated with TESOTf, followed by sodium bicarbonate and iodine, which resulted in the TES-protected glyceride 30 (18% yield). The free alcohol was then esterified with the previously synthesized analogue of palmi,c acid, 27, to give the TES-protected diglyceride 31 in a yield of 85%. The TES group was removed using FeCl3, giving the desired diglyceride 32 in a 93% yield (>99% e.e.). e.e. was determined using 1 H NMR analysis, by deriva,zing the obtained alcohol with either (+)-or (-)-methoxyphenylace,c acid. Details are provided in the supplementary informa,on (Supplementary Figure ). The enan,omerically pure glyceride 32 was then reacted with phosphoramidite 33 and 1H-tetrazole, to furnish the diglyceral phosphoramidite 34 (96%), ready to be coupled with inosi,de 19.