Preparation of trinucleotide synthons for the synthesis of gene libraries
Over the past decades, the interest in efficient methods for site-directed mutagenesis in protein engineering has strongly grown. Oligonucleotide-directed mutagenesis is the favoured method for preparation of large peptide or protein libraries. Among a host of methods, the use of mixtures of pre-formed trinucleotide blocks representing codons for the 20 canonical amino acids stands out as allowing fully controlled partial (or total) randomization at any arbitrarily chosen codon positions of a given gene. Such trinucleotide synthons, however, are not easy to come by. Several strategies have been suggested making use of various 3′-O- and phosphate protecting groups. However, most of these methods suffer from one or more limitations, such as di- and mononucleotide side products, loss of protecting groups, bad coupling yields or false linked nucleotides. More recent work has successfully overcome these limitations. Herein we review the strategies for preparation of fully protected trinucleotide synthons.
Gene libraries are most efficiently synthesized by the use of trinucleotide phosphoramidites, each representing the codon of a specific amino acid.
The use of trinucleotide synthons allows full control over the nature of randomization and in consequence, preparation of a gene library with the largest amount possible of promising candidates.
Key to trinucleotide synthesis is the definition of a suitable set of orthogonal protecting groups.
The generation of novel proteins (enzymes, pharmaceutic drugs etc.) with experimentally pre-defined properties has been a long-standing goal in the field of molecular biotechnology. Over the past years, this has been achieved in a number of cases, although rather rudimentary, due to methodological progress in combinatorial and evolutionary protein engineering, which is based on the generation of highly diverse libraries of partially randomized genes (1). Gene libraries are expressed into protein libraries, which subsequently can ...