The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate alkynyl amino acids such as para-propargyloxyphenylalanine into proteins produced in a eubacteria host such as E. coli. The invention provides novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing alkynyl amino acids, and cellular translation systems.