Strategy for the chemical synthesis of large peptides; synthesis of angiogenin as an example.

Although large peptides are now being synthesized by recombinant DNA technology, chemical synthesis remains important for clarifying their chemical and biological properties. In 1969, two notable reports were published on the chemical synthesis of enzymes: one described solid-phase synthesis of ribonuclease A by Gutte & Merrifield [ 1, 21; and the other, solution synthesis of ribonuclease S-protein by a group at Merck Sharp & Dohme [3]. Gutte & Merrifield demonstrated that their product exhibited ribonuclease activity, but their efforts to isolate a fully active product were unsuccessful. Since then, the techniques for solid-phase synthesis have continuously been improved and many effective purification techniques have been introduced. In 1989, Kent attempted synthesis of HIV-1 acid protease, a 99-residue enzyme, by applying the Boc-strategy to an improved solidphase method and, as a result of which, the product was successfully isolated as crystals [4, 51. In the solution synthesis of ribonuclease S-protein, Mercks group demonstrated that their product exhibited enzymic activity in the presence of excess S-peptide, but the amount of their final product was so small that they could not characterize it. Solution synthesis of ribonuclease A was achieved by Fujii & Yajima in 198 1 [6, 71. They assembled the fully protected peptide from 30 segments by the azide procedure and, after deprotection followed by air-oxidation, they succeeded in isolating the product as crystals via affinity chromatography. Since 197 1, we have been deeply involved in a project for supplying a variety of biologically active peptides to researchers for use as standards. To supply reliable peptides, we concluded that all should be synthesized by the segment condensation method in solution, with standardization of the strategy and techniques. After several trials, we designed a maximum protection strategy as follows [8,9]: ( a ) A water-soluble carbodi-imide, 1 -ethyl-3-( 3-dimethylaminopropy1)-carbodi-imide, is used as a reagent for forming peptide bonds in the presence of HOBt or HOOBt. This procedure is better than the dicyclohexylcarbodi-imide (DCC)/HOBt or DCC/HOOBt method because the urea or acylurea formed, if any, is far more easily removable from the reaction products than when DCC is used. ( h ) Each segment is synthesized by stepwise elongation of Boc-amino acids starting from amino acid phenacyl esters: if the C’-terminus of the objective peptide has a free acid or amide, the C-terminal segment is elongated from the terminal amino acid benzyl ester or amide. The side-chain protected amino acids used are: Asp(cHx), Glu(cHx), Arg(Tos), Ser( Bzl). Thr(Bzl), Cys(MeB) or Cys(Acm), Tyr(BrZ), Lys(CIZ), His(Tos) or His( Bom) and Trp( For). ( c ) When the protected segment, Boc-peptide phenacyl ester, is used as an acid component, the terminal phenacyl ester is removed by treatment with zinc powder in acetic acid. When the segment is used as an amino component, it is treated with trifluoroacetic acid. During these deprotection reactions, all side-chain protecting groups remain intact. ( d ) After completion of all coupling reactions, the final fully protected product is deprotected by the HF method. SHIN-ICHIRO KUMAGAYE, HISAYA KURODA,