## 1 Introduction  
A major goal of molecular biotechnology is the evolution/development of enzymes with desired and improved properties for industrial or pharmaceutical application [1; 2]. Often the knowledge about structural details of the enzyme of interest is missing, such that rational design is not an option [2]. On the contrary, directed evolution offers the possibility, via random mutagenesis, of generating large protein libraries, which are screened for functionality, such that detailed structural information is not required [2]. Furthermore, combinatorial gene synthesis offers the option of restricting structural variations to residues of interest, i.e., those that are involved in a certain function such as catalysis. Nevertheless, there are a number of hurdles to be considered. For example, the size of gene libraries resulting from large random mutagenesis would be too big for practical screening [3]. Other challenges are the structure of the genetic code, where some amino acids are encoded by up to six triplets and others by just one, leading to a bias of the mutation, favoring those amino acids with multiple codons [4]. On the methodological side, directed evolution by error-prone PCR is a widely used strategy, which however, generates bias of mutation depending on the polymerase used [4]. Furthermore, the generation of stop codons cannot be prevented, which leads to abortion fragments instead of the full-length enzymes [5]. A possibility to overcome these problems is the use of trinucleotide building blocks (Figure 1), which represent codons of the canonical amino acids and thus permit the controlled synthesis of a fully or partially randomized gene library without stop codons or codon bias [6]. There are several but not easy ways to synthesize such trinucleotide building blocks, either in solution [7] or, as a more recent addition to the field, on soluble support [8; 9]. We have previously developed a strategy for synthesis in solution of fully protected trinucleotide phosphoramidites [6] and, more recently, have succeeded in synthesizing trinucleotides on solid support. The preparation of trinucleotides in solution can be carried out at a larger scale. However, it requires careful design of the strategy and orthogonal protection of individual groups. Thus, a pair of orthogonal protecting groups for the 5'- and 3'-OH functionalities needs to be designed, in order to avoid side reactions during trinucleotide synthesis. Stable protection of the phosphate moieties as well as of the heterocyclic bases throughout trinucleotide synthesis is essential. After each reaction step, isolation and purification of intermediate products is required, making trinucleotide assembly in solution a rather tedious and time-consuming procedure. Syntheses on support are advantageous in terms of efficiency. Products bound to the support and removal of excess reagents by simple washing facilitates the process and saves time. The future may lie in the usage of soluble polymers [8; 9]. Soluble supports combine the advantages of solution chemistry and solid phase methods. However, they require a reaction scheme optimized for liquid phase assembly, which currently is still under investigation. Trinucleotide assembly on solid support can proceed by standard phosphoramidite chemistry and, thus, is a valuable strategy for the preparation of fully protected trinucleotides, until the major hurdles of supported oligonucleotide assembly on soluble polymers have been overcome. Our initial protocol for solid phase trinucleotide assembly has been described recently [10]. We here wish to report our studies regarding the choice of a suitable polymer, efficient conjugation chemistry for linking the starting nucleoside to the support, and detachment of the fully protected trinucleotide.