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Robert Bruce Merrifield (1921–2006)

2006; Wiley; Volume: 45; Issue: 33 Linguagem: Inglês

10.1002/anie.200602806

1521-3773

Bernd Gutte,

Enzyme Catalysis and Immobilization

Bruce Merrifield, Professor Emeritus at the Rockefeller University in New York, passed away on May 14, 2006, shortly before his 85th birthday. The significance of his work allows him to be remembered as one of the most important chemists of the 20th century. Bruce Merrifield was born on July 15, 1921, in Fort Worth, Texas. A year later his family moved to California. As a salesman, his father and, consequently, the entire family were heavily afflicted by the great depression that started in 1929. This time made a life-long impression on the young Merrifield. In his autobiography,1 he wrote: “The depression surely affected my life and influenced my future behavior. Stability and security became important, but wealth and social position were not. Steady, hard work and conservative approaches to problems seem to have been superimposed on whatever genetic traits were present.” Merrifield studied chemistry and biochemistry at the University of California at Los Angeles (UCLA). In his doctorate work in the laboratory of M. S. Dunn, he investigated the influence of nucleic acid derivatives on the growth of various strains of Lactobacillus. He completed his PhD on June 19, 1949, married Elizabeth Furlong—PhD student in the Department of Zoology at UCLA—on June 20, 1949, and set off with his wife for New York on June 21, 1949. There, at the Rockefeller University in the laboratory of D. W. Woolley, he took up his first and also his last position. Woolley and Merrifield discovered bacterial-growth properties of peptide fragments of insulin and oxytocin that contained at least one serine or cysteine residue. By preparing these peptides and their analogues, Merrifield learned the labors of classical peptide synthesis in solution, and he began to think about more efficient synthetic methods. Here is an excerpt of an entry in his laboratory note book, dated May 26, 1959:1 “A new approach to the continuous, stepwise synthesis of peptides. There is a need for a rapid, quantitative, automatic method for synthesis of long chain peptides. A possible approach may be the use of chromatographic columns where the peptide is attached to the polymeric packing and added to by an activated amino acid, followed by removal of the protecting group and with repetition of the process until the desired peptide is built up. Finally the peptide must be removed from the supporting medium.” From this idea and through several years of research, the so-called solid-phase peptide synthesis came into being and can be described as follows: The C-terminal amino acid of the target peptide sequence, temporarily protected at the amino group, is bound to a solid organic carrier through an ester linkage. Upon removal of the amino protecting group of the carrier-bound amino acid, the next amino acid of the target sequence, likewise protected at the amino group but with a free carboxy group, is added and linked to the carrier-bound amino acid through a peptide bond by activation of the carboxy group. These steps are repeated until the desired sequence has been assembled on the solid support. Finally, the ester bond between the peptide and the carrier is selectively cleaved, and the peptide can then be purified and characterized in solution. The main advantage of solid-phase peptide synthesis over synthesis in solution is that intermediate peptides do not need to be isolated, which speeds up the synthesis tremendously. In 1963, Merrifield published the first synthesis of a peptide that was assembled by the solid-phase method, the tetrapeptide Leu-Ala-Gly-Val.2 This work has been cited more than 5000 times so far. As complete reactions of the carrier-bound amino acids or peptides were not always achieved, the new method met with skepticism and even disapproval, especially in “old Europe”. These perceptions intensified when the synthesis of the enzyme ribonuclease A was published in 1969. At the time, the synthetic product was certainly not homogeneous, as the carrier material and the synthetic steps were not yet optimized and purification methods such as affinity chromatography and HPLC were in their infancy or still unknown. Yet the conclusion was allowed that biologically active proteins could be built and modified through chemical synthesis starting with discrete amino acids and linking them in the same sequences as found in natural proteins. Very soon after the introduction of the solid-phase method, the Merrifield group synthesized the peptide hormones bradykinin, angiotensin, desamino-oxytocin, insulin, and glucagon, the latter in crystalline form, and in collaboration with his colleague John Stewart and Nils Jernberg, a member of the Rockefeller University mechanical workshop, Merrifield built the first automated synthesizer.3 From that time on, many peptide chemists around the world worked on the fine-tuning and modification of the solid-phase method and were successful in synthesizing, for example, parathyroid hormone (1983), interleukin-3 (1986), and crystalline HIV-1 protease (1988, 1989). Soon thereafter, the concept of synthesis on solid supports was also applied to the preparation of oligonucleotides, carbohydrates, and other organic compounds, and when the Nobel Committee awarded Bruce Merrifield the Nobel Prize in Chemistry in 1984,4 it was “for his development of methodology for chemical synthesis on a solid matrix”. In the following years, the groups of Merrifield and Cecilia Unson published important papers on the mechanism of action of glucagon. Numerous publications resulted also from work on the action of the peptide antibiotics cecropin A and melittin, as well as their D-, retro-, retro-D, and hybrid analogues. The paramount significance of the idea of carrying out syntheses on a solid matrix lies in the speed with which a multitude of useful products can be prepared. Peptides of clinical importance synthesized by the Merrifield method include peptide hormones and a 36mer peptide from the HIV-1 envelope protein gp41, which inhibits HIV infection of T cells. Synthetic peptides that correspond to protein segments are used for the generation of sequence-specific antibodies, and synthetic peptide libraries are potential sources of new peptide drugs. Equally important are oligonucleotides prepared by the Merrifield method. They are the starting materials for the polymerase chain reaction, with which, for example, virus infections and genetic defects are detected and traces of DNA are amplified for the forensic identification of suspects. Presently, synthetic oligonucleotides also find multiple applications as small interfering RNAs for the regulation of gene expression. Bruce Merrifield became a full professor in 1966, was elected to the National Academy of Sciences in 1972, and was named John D. Rockefeller Jr. Professor in 1983. In addition to the Nobel Prize, he received a number of other prestigious awards, including the Albert Lasker Award for Basic Medical Research (1969), the Gairdner Foundation International Award (1970), the Royal Society of Chemistry Medal (1987), and the Ralph F. Hirschmann Award of the American Chemical Society (1990). The list of his honorary doctorates is also long and impressive. The author of this obituary came to the Rockefeller University in the spring of 1967 as Merrifield's first postdoctoral fellow. The five years that followed were an unparalleled time from both a scientific and a personal point of view. Merrifield had a strong inner balance, which also affected life in the laboratory and provided a fertile soil for our work. His clear judgement, modesty, tolerance, and amicable relations with his collaborators made a lasting impression on us. We also appreciated very much that, besides pursuing our main project, we were given the opportunity to engage in collaborations with other research groups at Rockefeller University. To our best memories belong the Thanksgiving dinners and the summer cookouts with the obligatory volleyball games at the Merrifields in New Jersey. Despite his advancing illness (skin cancer), Bruce Merrifield was always in good spirits but finally had to give up his daily rides to the Rockefeller University in Manhattan. He is survived by his wife Elizabeth, who worked with him for many years in the laboratory and devotedly cared for him at the end of his life, as well as by six children, 16 grandchildren, and numerous grateful former collaborators, who apply, all over the world, the Merrifield solid-phase synthesis.