Synthesis and Purification of Lipid Conjugates

The Lipid-dansyl and Lipid-DNP conjugates were synthesized using Fmoc chemistry on solid support using NovaPEG Rink Amide resin as previously described.^{21 (link)} The synthetic scheme is described in Figure S-1. Briefly, protected molecules with terminal acid groups were activated with HBTU and a four-fold molar excess of DIEA for 5 minutes and then conjugated to the resin over 30 minutes. Fmoc was deprotected with 20% piperidine in DMF and IvDdE was deprotected using 2% hydrazine in DMF. Deprotection and coupling steps were monitored with Kaiser tests. Lipid-hapten conjugates were cleaved using a 95/2.5/2.5 TFA/water/TIS solution for 45 minutes. Lipid hapten molecules were purified using 1200 Agilent RP-HPLC using a semi-preparative Zorbax C3 column. A two phase water and 70/20/10 IPA/ACN/water mix was used for purification with a gradient of 60–100% IPA mix over 10 minutes at a flow rate of 3 mL min⁻¹. Hapten-amino acid conjugates were purified using a Zorbax C18 column, using a two phase water/ACN system with a gradient of 20–50 % ACN in 10 minutes. The product was confirmed using a Bruker microTOF II mass spectrometer (Figure S-2). Absorbance peaks at 220nm and 280nm were collected and verified for purity with analytical injections (>95%) (Figure S-3).