Abstract: Synthetic L-pyrrolysine of formula I, and derivatives thereof, chemical methods of preparing these amino acids and derivatives, and methods of derivatizing these amino acids after incorporation into a peptide or protein. Also provided are derivatives and methods of preparing derivatives, including addition of substituents at any substitutable position; modifications to the lysine alkyl chain; modifications of the pyrroline ring. Also provided are methods of adding labels to the pyrrolysine or derivative thereof.

Abstract: Provided herein is a method for preparing a cell that, when exposed to the non-canonical amino acid, pyrrolysine or a derivative thereof and transformed or transfected with a polynucleotide comprising an in-frame UAG or TAG codon incorporates the pyrrolysine residue or derivative thereof into the protein or polynucleotide into the protein or polypeptide encoded by the polynucleotide. The method comprises introducing a first polynucleotide comprising a sequence that encodes a protein having pyrrolysyl-tRNA synthetase activity and a second polynucleotide comprising a sequence that encodes a pyrrolysine specific tRNA into the cell and maintining the cell under conditions that permit expression of the first and second polynucleotide. Also provided herein are modified cells produced in accordance with the present method and methods of making peptides, polypeptides, and proteins which utilize such modified cells. Also provide are kits comprising pyrrolysine or a pyrrolysine or both.

Abstract: Vitamin B.sub.12 -active substances are produced by the fermentation of an aqueous medium consisting essentially of methanol as the main source of assimilable carbon, a source of assimilable nitrogen, cobalt ions and essential growth factors and minerals with a vitamin B.sub.12 -activity substance producing organism capable of growing under anaerobic conditions.