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The Solid Phase Synthesis of Ribonuclease A by Robert Bruce Merrifield

      The Synthesis of Ribonuclease A (Gutte, B., and Merrifield, R. B. (1971) J. Biol. Chem. 246, 1922–1941)
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      Robert Bruce Merrifield. Photo courtesy of the National Library of Medicine.
      Robert Bruce Merrifield (1921–2006) was born in Fort Worth, Texas. Two years later, he and his family moved to California. Merrifield became interested in chemistry in high school and was a runner up in the annual science contest during his senior year. He attended Pasadena Junior College but transferred to the University of California at Los Angeles at the end of his second year. At UCLA he worked in Max S. Dunn's laboratory assisting in the synthesis of the complex amino acid dihydroxyphenylalanine (DOPA).
      After receiving a B.A. in chemistry in 1943, Merrifield went to work at the Philip R. Park Research Foundation as a chemist. During his stay at the laboratory, he assisted in growth experiments feeding test animals a synthetic amino acid diet. The experience lasted only a year, but it was enough to convince Merrifield that to pursue his goals he would need to return to school. Fortunately, an Anheuser-Busch Inc. fellowship allowed him to continue his studies in the chemistry department at UCLA. He served as a chemistry instructor from 1944 until 1947 and then returned to Dunn's laboratory as a research assistant. Merrifield's work with Dunn included the development of microbiological methods for the study of yeast purines and the pyrimidines. In 1949, he received his Ph.D. in biochemistry and accepted an appointment as an assistant chemist at the Rockefeller University, then known as the Rockefeller Institute for Medical Research, in New York City. He remained at the institute for his entire career, becoming a John D. Rockefeller Jr. Professor, the institution's highest academic rank, in 1984.
      At Rockefeller, Merrifield first worked as an assistant to Dilworth W. Woolley. By this time, Merrifield had recognized that proteins were the key components of all living things, and he chose to focus on a dinucleotide growth factor he discovered in graduate school and on peptide growth factors that Woolley had discovered earlier. This research required Merrifield to isolate biologically active peptides and synthesize their analogues. He soon realized that the peptide synthesis methods pioneered by Emil Fischer and his students were difficult and time-consuming and that a new approach was needed. Fischer's process involved blocking the carboxyl group of one amino acid and the amino group of the second amino acid. Then, by activation of the free carboxyl group, the peptide bond could be formed, and selective removal of the two protecting groups would lead to the free dipeptide. The peptide was then separated from the by-products and unreacted starting material, and the process was repeated.
      Merrifield came up with an idea of how more efficient peptide synthesis could be achieved. The plan was to assemble a peptide chain in a stepwise manner while it was attached at one end to a solid support that could easily be removed by the proper solvents. Not only did this solid phase method facilitate one of the most cumbersome steps in Fischer's process, the need to purify the intermediate product prior to the addition of another bond, it also allowed excess reagents to be used that helped drive the reactions to completion.
      It soon became apparent to Merrifield that the solid phase technique should be applicable to units other than amino acids, and he extended it to the synthesis of depsipeptides while other laboratories succeeded in synthesizing polyamides, polynucleotides, and polysaccharides. Merrifield also felt that the technique lended itself nicely to a mechanized and automated process. Working in the basement of his house, he devised the first prototype of an automated peptide synthesizer by 1965.
      With the automation of the technique, Merrifield felt the time was right to attempt the total synthesis of an enzyme, which is the subject of the Journal of Biological Chemistry (JBC) Classic reprinted here. Working with Bernd Gutte, Merrifield selected bovine pancreatic ribonuclease A because it was a small stable protein with a known amino acid sequence and three-dimensional structure. It had also been established that the denatured, reduced form of ribonuclease A could be reoxidized and refolded into a protein that possessed full enzymatic activity. These characteristics of ribonuclease A were the subjects of JBC Classic papers by Stanford Moore and William H. Stein (

      JBC Classics: Hirs, C. H. W., Moore, S., Stein, W. H., (1960) J. Biol. Chem. 235, 633–647; Spackman, D. H., Stein, W. H., and Moore, S., with the assistance of Zamoyska, A. M. (1960) J. Biol. Chem. 235, 648–659; Crestfield, A. M., Stein, W. H., and Moore, S. (1963) J. Biol. Chem. 238, 2413–2420 (http://www.jbc.org/cgi/content/full/280/50/e4)

      ) and Christian Anfinsen (

      JBC Classics: Haber, E., Anfinsen, C. B., (1962) J. Biol. Chem. 237, 1839–1844 (http://www.jbc.org/cgi/content/full/281/14/e11)

      ).
      Merrifield recalled, “The purpose of a chemical synthesis of this 124-residue molecule was, first, simply to demonstrate that a protein with the high catalytic activity and specificity of a naturally occurring enzyme could be synthesized in the laboratory. For the long range, the more important purpose was to provide a new approach to the study of enzymes. We believed it should be possible to modify the structure and to alter the activity and the substrate specificity of the enzyme (
      • Merrifield R.B.
      ).”
      Merrifield and Gutte synthesized the linear polypeptide, purified it, and closed its four disulfide bonds by air oxidation. Their synthetic ribonuclease A was indistinguishable from the natural enzyme by gel filtration, chromatography, and electrophoresis. Moreover, their synthetic enzyme showed the high substrate specificity expected of ribonuclease A. This synthesis of ribonuclease A also provided unambiguous support for the concept that the primary structure of a protein determines its tertiary structure.
      Merrifield's work was recognized when he received the Nobel Prize in Chemistry 1984 “for his development of methodology for chemical synthesis on a solid matrix.” Solid phase synthesis, which has never been patented, either by Merrifield or Rockefeller University, has been used widely and is seen as one of the fundamental techniques of genetic and biochemical research.
      Prior to his death, Merrifield was an adjunct professor at the Oregon Institute of Science and Medicine and an emeritus professor at Rockefeller University. In addition to the Nobel Prize, he received many awards for his work on peptide chemistry including the Lasker Award for Basic Medical Research (1969), the Gairdner Award (1970), the American Chemical Society Award for Creative Work in Synthetic Organic Chemistry (1972), the Nichols Medal (1973), the 2nd Alan E. Pierce Award of the American Peptide Symposium (1979), the Ralph F. Hirschmann Award in Peptide Chemistry from the American Chemical Society (1990), the Josef Rudinger Award (1990), and the Seaborg Medal (1993). The American Peptide Society also offers an annual Merrifield Award for outstanding career achievements in peptide research. Merrifield was elected to the National Academy of Sciences in 1972. Beginning in 1969 he served as editor of the International Journal of Peptide and Protein Research (now the Journal of Peptide Research), and in 1993 he published the semiautobiographical Life during a Golden Age of Peptide Chemistry: the Concept and Development of Solid-Phase Peptide Synthesis (
      • Merrifield R.B.
      ).
      All biographical information on Robert Bruce Merrifield was taken from Refs.
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      and
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      .
      1All biographical information on Robert Bruce Merrifield was taken from Refs.
      • Merrifield R.B.
      and
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      .

      References

      1. JBC Classics: Hirs, C. H. W., Moore, S., Stein, W. H., (1960) J. Biol. Chem. 235, 633–647; Spackman, D. H., Stein, W. H., and Moore, S., with the assistance of Zamoyska, A. M. (1960) J. Biol. Chem. 235, 648–659; Crestfield, A. M., Stein, W. H., and Moore, S. (1963) J. Biol. Chem. 238, 2413–2420 (http://www.jbc.org/cgi/content/full/280/50/e4)

      2. JBC Classics: Haber, E., Anfinsen, C. B., (1962) J. Biol. Chem. 237, 1839–1844 (http://www.jbc.org/cgi/content/full/281/14/e11)

        • Merrifield R.B.
        Nobel Lectures, Chemistry 1981–1990. 1992; (Frängsmyr, T., ed) World Scientific Publishing Co., Singapore
        • Merrifield R.B.
        Life during a Golden Age of Peptide Chemistry: the Concept and Development of Solid-Phase Peptide Synthesis. 1993; (American Chemical Society, Washington, D. C.)
        • Merrifield R.B.
        Les Prix Nobel, The Nobel Prizes 1984. 1985; (Odelberg, W., ed) Nobel Foundation, Stockholm