With the acetonide protected thionoviose derivatives 17a-c in hand, we turned our attention to the semi synthesis of the single atom exchanged Fdx analogue, S-Fdx (3a-C( )), and the corresponding 4-O-acyl derivatives. To perform the substitution reaction and the acetonide deprotection in a single reaction, water was added, the LA loading was increased to 5 mol%, and an elevated reaction time of 14-20 h was applied (Fig. ). Purification via preparative RP-HPLC afforded S-Fdx in a yield of 3% along with the C( )-(10%) and the C(15)-isomer (24%) (Fig. ). Separation of the regioisomers of Fdx derivatives with similar retention times proved difficult and often required several runs of repurification. While cyclopropanoyl was introduced at C(3)-OH of the noviose moiety previously using Shimada catalyst with promising results, the introduction of a pivaloyl group could not be achieved using this method, also not with 1 equiv. of catalyst at 50 °C. Therefore, compound 3c-C(11) is the first Fdx derivative with a pivaloyl substituent on the noviose moiety, allowing us to gain further SAR information.