## 3 Results and Discussion  
Due to the obvious advantages of solid phase synthesis over solution chemistry, we sought to develop a strategy that allows the assembly of fully protected trinucleotides on solid support. The key issue is the detachment of the trinucleotide from the support maintaining all protecting groups. Reductive cleavage is a suitable strategy, as it is orthogonal to the acid labile DMT group, as well as to nucleophilic removal of the phosphate and nucleobase protecting groups (Figure 1) [15; 16; 17]. Thus, we took an example from a previously published strategy for the synthesis of RNA using 2\({}^{\prime}\)-_O-tert_-butyldithiomethyl-protected building blocks [18], and developed a protocol for trinucleotide assembly with the starting nucleoside connected to the solid support by a dithiomethyl linkage (Figure 1). Reductive cleavage of the disulfide bond would produce a \(S\),_O_-hemiacetal, which spontaneously decomposes into alcohol and thioformaldehyde, thus yielding the free 3\({}^{\prime}\)-OH group [18], which subsequently can be phosphitylated as described in our earlier work on the preparation of trinucleotide synthons [6]. For immobilization of the starting nucleoside, first the 3\({}^{\prime}\)-OH group needed to be appropriately functionalized (Scheme 1). This was achieved by the site-specific introduction of a methyldithiomethyl group, followed by activation of the obtained 3\({}^{\prime}\)-_O_-methyldithiomethyl (3\({}^{\prime}\)-_O_-MTM) derivatives **3a-d** by treatment with sulfuryl chloride to produce the 3\({}^{\prime}\)-_O_-chloromethyl ether (**4a-d**) in a Pummerer rearrangement [19; 20]. The chloromethylether reacts with potassium thiotosylate forming the reactive compound **5a-d**, which subsequently is linked to the polymer via a disulfide bridge [12; 13]. 5\({}^{\prime}\)-_O_-Dimethoxytritylated nucleosides were used as starting compounds requiring careful control of reaction conditions (pH), in particular during the first step of MTM introduction. 3\({}^{\prime}\)-_O_-MTM derivatives **3a-d** of all four deoxynucleotides were prepared with satisfying yields (52%-68% [10]) and linked either directly to thiol-derivatized polystyrene **B** (**8a-d**) or, via a bifunctional linker, to amino-derivatized polystyrene **C** and **D** (**10a-d**) or amino-derivatized CPG **A** (**9a-d**). As a bifunctional linker, we tested 2-mercaptoethanolpropionic acid or, as a longer variant, 12-mercaptoethanolaci acid. Both linker molecules comprise a thiol group for disulfide formation, however in the shorter variant it is connected to a secondary carbon and in the longer variant it is connected to a primary carbon atom. Thus, the formation of the disulfide bridge, its stability during trinucleotide assembly, and disulfide cleavage can be studied depending on linker lengths and type of the thiol. After attachment of the linker to nucleoside derivatives (**6a-d**, **7a-d**), immobilization to aminopolystyrene or Amino CPG was achieved by amide bond formation upon activation of the linker carboxyl group with TSTU [14]. TCEP was found being more efficient (see Supplementary Materials S3) [21] and, due to the intended use of methyl-protected phosphoramidites (see below), it was the better choice for the cleavage of the synthesized trinucleotides from the support. Overall, highly crosslinked polystyrene showed the best features in terms of nucleoside loading and TCEP-mediated detachment, independent of the linker connecting the starting nucleoside to the amino-functionalized polymer. Thus, hcl polystyrene would be the best choice for trinucleotide assembly, in particular, also when the process needs to be carried out at a larger scale. In previous experiments we had investigated the influence of the nature of the linker thiol being primary, secondary, or tertiary. The stability of a disulfide bridge depends on the nature of the bridge-forming thiol, with a tertiary thiol being most stabilizing due to the electron-donating effect of the tertiary alkyl group.