## 3 Results and Discussion
In our preliminary experiments, we found the disulfide bridge formed with the primary thiol was clearly more labile than those formed with the secondary and tertiary thiols, when it was cleaved with DTT (see Supplementary Materials S4 for details). Under the conditions of disulfide cleavage used here (TCEP, over 20 h), this stability difference could not been observed, since cleavage from support **A** and **D**, with either 12-mercaptoethanoldodecanoic acid or 2-mercaptoethanol acid as linker was completed after a rather long incubation time. Nevertheless, based on our previous results with DTT, we decided to use 2-mercaptoethanolpropanoic acid as linker for our further studies, because of the higher stability of the formed disulfide bridge and, hence, less risky trinucleotide assembly. Thus, in addition to T, the 5'-O-DMT-3'-O-MTM-functionalized derivatives of dA, dC, and dG (**3a-c**, Scheme 1), were also  
Figure 2: Overview of supports loaded with starting nucleoside. linked to 2-mercaptoethanolpropionic acid followed by immobilization on amino-functionalized polystyrene (**D**) (Table 1). Before use of the loaded supports, free amino groups were capped by treatment with acetic anhydride. Then, trinucleotides were synthesized by coupling of either \(\beta\)-cyanoethyl- or methyl-protected phosphoramidites of \(N\)-acyl-5\({}^{\prime}\)-O-DMT-protected 2\({}^{\prime}\)-deoxynucleosides. Previously, we had used \(\beta\)-cyanoethyl-protected phosphoramidites for trinucleotide assembly in solution and found that the \(\beta\)-cyanethyl group was suitable for phosphate protection. It was stable throughout trinucleotide assembly and phosphitylation, provided that particular attention was paid to reaction and purification conditions, in particular to the pH of reaction media, solvents,luents, and buffers. In order to circumvent these challenges and to allow easier handling of the fully protected trinucleotides during purification and in further reactions, we later switched to the more stable methyl group for phosphate protection. Altogether, ten different trinucleotides were synthesized basically following the standard protocol for DNA synthesis [10]. For initial testing, syntheses were carried out at 1 \(\upmu\)mol scale. Three trimeric oligodeoxyribonucleotides were assembled from \(\beta\)-cyanoethyl phosphoramidites, and seven were assembled from methyl phosphoramidites (Table 2). For oxidation of the phosphite, a diluted solution of iodine (0.02 M) was used, because of the previously observed partial cleavage of the disulfide bond when treated with 0.1 M iodine solution [18; 22]. Upon cleavage from the support with a buffered solution of DTT, the quality of fully protected trinucleotides TTT, TTA, and TTC carrying the \(\beta\)-cyanoethyl group at the phosphates was confirmed by HPLC and MALDI-TOF MS (Table 2 and Supplementary Materials S5). However, when using methyl-protected phosphoramidites, partial loss of the methyl group was observed upon removal of trinucleotides from the support with DTT, due to nucleophilic attack of the DTT-SH group onto the methyl group carbon (Supplementary Materials S6). On the contrary, when trinucleotides were removed by disulfide cleavage with TCEP under the conditions mentioned above, the methyl protection at the phosphates remained intact (Supplementary Materials S7). Upon cleavage from the support, fully protected trinucleotides were precipitated from water, dried, and stored under vacuum. Trinucleotides should be phosphitylated directly prior to use in DNA synthesis. Particular care has to be taken when \(\beta\)-cyanoethyl-protected trinucleotides are phosphitylated, as we have described recently [6]. Here, the ability of the \(\beta\)-cyanoethyl group under basic conditions requires careful choice of the phosphitylation reagent and control of reaction conditions. The same optimized conditions can be applied to methyl-protected phosphoramidites, although the methyl group at the phosphorous is less labile.