The synthesis of biomimetic thioesters for enzymatic studies of ketoreductase (KR) domains from polyketide synthases is described. A TBS-protected dihydroxyalkene fragment was synthesised by a sequence containing Nagao acetate aldol reaction, Mukaiyama propionate aldol reaction and methylene Wittig olefination. Fragment coupling to N-acetylcysteamine (SNAC) (E)-3-hydroxyhex-4-enethioates using olefin cross-metathesis (OCM) and deprotection gave the potential KR product stereoisomers. Analogous OCM with a SNAC (E)-3-ketohex-4-enethioate did not give the desired KR precursor, but could successfully be substituted by a Horner-Wadsworth Emmons olefination between a SNAC 3-ketothioesterphosphonate and a TBS-protected dihydroxyaldehyde. After deprotection, an intramolecular cyclisation was observed that needs to be considered as a spontaneous side reactivity in the enzymatic assays.