The synthetic strategy to access [ 18 F]2 first required synthesis of the aldehyde [ 18 F]1, which could be subsequently activated for reaction with acetic anhydride. A nucleophilic aromatic substitution (SNAr) was performed on N-(4-(1,3-dioxolan-2-yl)benzyl)-N-ethyl-6-chloronicotinamide 4 with [ 18 F]KF/K222 in DMSO at 150 °C for 25 min followed by an acid mediated acetal-cleavage step to furnish [ 18 F]1 in 44 ± 7% radiochemical yield (RCY; n = 5; for further details see Supplementary Figures &). [ 18 F]1 was stirred with acetic anhydride and BF3•Et2O at room temperature for 5 min to produce [ 18 F]2 in 80 ± 4% isolated RCY (n = 4) with a molar activity of up to 20 GBq/µmol (Scheme 1). Reformulation of [ 18 F]2 in PBS ready for biological evaluation was achieved with high radiochemical purity (>98 %) and stability (>95 % radiochemical purity after for 4 h; see Supplementary Figure ). Having shown that [ 18 F]2 successfully undergoes esterase-mediated cleavage to the aldehyde [ 18 F]1, we assessed whether [ 18 F]2 could provide a readout of ALDH activity in tumour cells grown in culture. For this, we chose two ovarian cancer lines, SKOV3-ip1 and SKOV3-TRip2, that had been previously passaged intraperitoneally in mice to select for cells with enhanced tumourgenicity. The SKOV3-TRip2 cell line was generated through progressive exposure to paclitaxel , whereas the SKOV3-ip1 cell line remained untreated. Similar to previous findings with paxlitaxel , resistance to cisplatin was increased by over three orders of magnitude in SKOV3-TRip2 cells compared to SKOV3-ip1 (EC50, 0.88 nM vs. 2.6 µM, respectively; n = 3-5; Fig. ). In SKOV3-TRip2 cells, ALDH1A1 mRNA (Fig. ) and protein expression (Fig. ) were substantially increased in comparison to SKOV3-ip1 cells. Importantly, ALDH activity, as measured by the Aldefluor assay, was also significantly increased in SKOV3-TRip2 cells compared to SKOV3-ip1 (n = 3; P < 0.01; Fig. ). ALDH-specific cellular retention of [ 18 F]2 was examined 20 min after radiotracer addition to SKOV3-ip1 and SKOV3-TRip2 cell lines, as previously optimised with [ 18 F]1. 21 [ 18 F]2 was rapidly taken up and trapped in the high ALDH1A1-expressing SKOV3-TRip2 cells, which was ~2-fold higher than in SKOV3-ip1 cells, at 11.9 ± 5.1 % activity/mg protein and 5.7 ± 2.4 % activity/mg protein, respectively (n = 5; P < 0.05; Fig. ). Intracellular liberation of a reactive aldehyde may allow for substitution for cellular nucleophiles, resulting in trapping of the substrate independent of ALDH activity. To assess ALDH-specific trapping of [ 18 F]2, cells were co-incubated with the pan-ALDH inhibitor N,N-diethyl amino benzaldehyde (DEAB), which significantly reduced intracellular radioactivity in SKOV3-TRip2 by 77%, falling to 2.7 ± 0.3 % activity/mg protein (n = 3; P < 0.05), ruling out non ALDH-mediated mechanisms of trapping. To confirm the identity of the intracellular radioactive species present, we performed radio-HPLC analysis of SKOV3-TRip2 cell lysates. 20 min after radiotracer addition, enzymatic deprotection of [ 18 F]2 to [ 18 F]1 and subsequent conversion to the corresponding carboxylic acid [ 18 F]3 was observed, confirmed using non-radioactive fluorine-19 isotopologues (see ESI for details). With DEAB treatment of cells, cleavage to [ 18 F]1 occurred as expected, however, subsequent oxidation to [ 18 F]3 was prevented (Fig. ). Taken together, this suggests that intracellular production of the carboxylate [ 18 F]3 is mediated by ALDH, the enzymatic activity of which determines total intracellular radiotracer retention. [ 18 F]2 therefore provides a sensitive measure of ALDH activity in tumour cells. Aldehyde protection and carrier-added formulation improve radiotracer blood stability Given that [ 18 F]1 had previously undergone complete oxidation when incubated for 2 min in blood , likely due to high ALDH1 expression in erythrocytes , we next examined whether geminal diacetate protection afforded improved blood stability. Whilst [ 18 F]2 was still present in murine blood at 2 min, rapid cleavage and oxidation to the carboxylate [ 18 F]3 was observed (80 ± 12 % activity; n = 6), increasing to 94 ± 1 % activity (n = 3) at 5 min and negligible amounts of the aldehyde [ 18 F]1 and alcohol [ 18 F]4 seen (Fig. and Supplementary Fig. ). Interestingly when the molar activity (MA) of [ 18 F]2 was lowered from 8.4 ± 6.9 GBq/µmol (0.36 nmol 2 injected) to 0.11 ± 0.002 GBq/µmol (25 nmol 2 injected) its blood metabolism was noticeably altered (Fig. ). With low MAs, the aldehyde [ 18 F]1 was now the predominant species at early time points (33 ± 18 % activity at 2 min; n = 3). Whilst esterase-mediated cleavage of [ 18 F]2 occurred at similar rates to high MA preparations, oxidation of [ 18 F]1 to the carboxylate was substantially reduced over the time course. Indeed, enough [ 18 F]1 remained in the blood at low MAs for its reduction to the alcohol [ 18 F]4 to occur, albeit at a slower rate of conversion than to the carboxylate [ 18 F]3.