The Early Years—Across the Bench From Bruce (1963–1966) The Early Years—Across the Bench From Bruce (1963–1966) Garland R. Marshall1,2 1Department of Biochemistry and Molecular Biophysics, Center for Computational Biology, Washington University, St. Louis, MO 63110 2Department of Biomedical Engineering, Center for Computational Biology, Washington University, St. Louis, MO 63110 Received 14 July 2007; revised 20 September 2007; accepted 5 October 2007 Published online 16 October 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20870 a Nobel Laureate, Chairman of the Department of Biology at ABSTRACT: Caltech and a member of the National Academy of Science, and was still willing to recommend me for graduate studies This personal reflection on the author’s experience as at Rockefeller. Bruce Merrifield’s first graduate student has been I was convinced at the time that I was chosen to study adapted from a talk given at the Merrifield Memorial neurophysiology, having failed miserably to isolate the acetyl- Symposium at the Rockefeller University on November choline receptor from denervated rabbit muscle as an under- graduate at Caltech. The outstanding neurophysiologists at 13, 2006. # 2007 Wiley Periodicals, Inc. Biopolymers Rockefeller including H. Keffer Hartline, Nobel Laureate, (Pept Sci) 90: 190–199, 2008. were more interested, however, in the wiring diagrams of the Keywords: solid phase synthesis; Merrifield; DNA synthe- eye of the horseshoe crab2 than in how a small molecule sis; combinatorial chemistry could trigger the action potential. Thus, my first laboratory experience at Rockefeller was with Prof. Henry Kunkel, a This article was originally published online as an accepted prominent immunologist.3 Prof. Kunkel suggested that I try preprint. The ‘‘Published Online’’ date corresponds to the preprint to develop a radioimmunoassay for angiotensin II, an eight- version. You can request a copy of the preprint by emailing the residue vasoactive peptide hormone, based on the seminal Biopolymers editorial office at [email protected] work of Berson and Yalow who had devised a radioimmuno- assay for insulin4 (for which Yalow shared a Nobel Prize). Karl Landsteiner, the prominent immunochemist who had explored antibodies against a variety of small-molecule hapt- INTRODUCTION ens,5 was still a legend at Rockefeller. The bottleneck for the he Rockefeller Institute for Medical Research1 was a experiments planned was a source of angiotensin II and a formidable institution in 1962, the year I was strategy for selectively attaching it to a protein carrier before accepted for admission with an undergraduate degree injecting rabbits. Henry suggested in early 1963 that I go see T in biology from the California Institute for Technol- Bruce Merrifield (Figure 1), a young faculty member in the ogy. In those days, admission effectively required a Woolley group, whose reputation indicated some experience letter of recommendation from a Nobel Laureate, from a in peptide/protein chemistry, a Rockefeller dominated area chairman of a department at a ‘‘recognized’’ university, or of research (Figure 2). Little did I know this suggestion from a member of the National Academy of Science. Rockef- would change the direction of my thesis research as well as eller President Detlev Bronk controlled the admission pro- dominate the rest of my career. cess, and candidates were personally interviewed before ac- ceptance. I was fortunate in that Prof. George W. Beadle was THE WOOLLEY GROUP First, I need to explain about the hierarchical system in place Correspondence to: Garland R. Marshall, Department of Biochemistry and Molecu- at Rockefeller at the time. Each research group was organized lar Biophysics, Center for Computational Biology, Washington University, 700 S. under the leadership of a Professor in a pyramidal/Prussian Euclid Ave., St. Louis, MO 63110, USA. e-mail: [email protected] or [email protected] manner. Prof. D. Wayne Woolley was a founder of the field of 6,7 VC 2007 Wiley Periodicals, Inc. antimetabolites and had two junior faculty working in his 190 PeptideScience Volume 90 / Number 3 The Early Years—Across the Bench From Bruce 191 FIGURE 1 Bruce Merrifield with new manual solid phase shaker as seen from across the lab bench in 1963. This picture appears on page 115 of Merrifield’s autobiography and on the cover of this special issue of Biopolymers: Peptide Science in honor of Professor Bruce Merrifield. group as well as postdocs and technicians. He was a powerful as there were few formal courses at Rockefeller in those days intellect as well as blind at 26 from diabetic retinopathy (only 12 graduate students were admitted in 1962). The title shortly after receiving his Ph.D. in biochemistry from the of the Institute was changed during my tenure to ‘‘The Rock- University of Wisconsin and moving to Rockefeller. Prof. efeller University.’’ It was a joke among the students that Woolley not only worked daily in the lab doing organic President Detlev Bronk sent an announcement to all the out- chemistry with assistance from his technician, but also had a standing universities in the world welcoming them to the ‘‘photographic’’ memory of the chemical literature. His wife same status as Rockefeller. and others spent hours each day reading the latest journals to Prof. Woolley to keep him up to date. I remember with amazement, when during a discussion of the mechanism of SOLID PHASE PEPTIDE SYNTHESIS cleavage of a benzyl ester by hydrogen bromide, Prof. Wool- Bruce had gotten his Ph.D. developing bacterial bioassays for ley cited the volume, page number, and location on the page amino acids8 in the lab of Prof. Max Dunn at UCLA and was of an experimental graph in an old JACS issue that was an extending this work on other growth factors,9 including pep- essential part of the evidence. I was terrified that he and tides under Dr. Woolley’s direction. But the synthetic tedium others in the lab would realize that my synthetic chemical and repetitiveness of solution peptide synthesis was problem- background was inadequate at best. Bruce had been recruited atic. With his characteristic insight, Bruce had decided that from UCLA, and John M. Stewart from the University of Illi- there had to be a better way to synthesize peptides and, ulti- nois and both had progressed to faculty appointments in mately, proteins. Thus, the famous entry ‘‘A New Approach Woolley’s group. John’s Ph.D. was in synthetic organic chem- to the Continuous, Stepwise Synthesis of Peptides’’ in his istry under the legendary Roger Adams, and I and a few other notebook of May 26, 1959 (Figure 3). Questions regarding students got John to tutor us in advanced organic chemistry the four years of research effort that Bruce had expended Biopolymers (Peptide Science) 192 Marshall technician, had already started to explore benzyl ester linkage to the polymer and t-butyloxycarbonyl (BOC) amino protec- tion (Figure 7) recently disclosed by Carpino.11 Angela related later that Bruce had jokingly suggested the use of a hammer to remove the peptide from the nitrobenzyl polymer due to possible overnitration of the polystyrene and potential production of analogs of TNT. Even though my interest in synthetic chemistry was nascent, I immediately realized the power of Bruce’s invention and never completed the radioimmunoassay project in the Kunkel lab. Having a graduate student in his lab allowed Bruce the luxury of having a new shaker designed and built (shown proudly with Bruce in Figure 1). His old reciprocal-arm shaker, noisy and slinging droplets of oil, was relocated across the bench from Bruce, and I began my life as a peptide chemist apprenticed to a future Nobel Laureate. As Bruce had no teaching responsibilities per se and Rockefeller scientists did not apply for grants at that time, I had the daily privilege of working across the lab bench from Bruce and Angela, while trying hard not to expose my ig- norance of science in general and chemistry in particular. Being treated as an intellectual equal from day one was a great incen- tive for me to learn as quickly as possible. Once Bruce’s first FIGURE 2 The Rockefeller heritage in peptide/protein chemistry note on SPPS appeared in 1963, a steady stream of prominent was very strong. Bergmann, who was trained in Emil Fisher’s lab scientists visited the lab. Almost each one mentioned at some where the first peptide bond was synthesized, and Zervas were refu- point in the visit how he had thought of using a filterable poly- gees from Germany. Bergmann trained most of the next generation meric support as a protecting group (the essence of solid phase of protein chemists [Stanford Moore, William Stein, Joseph Fruton synthesis), but, of course, none had spent the years exploring (Yale), Klaus Hoffman (Pittsburg), etc.] Lyman Craig brought ex- pertise in chemistry of unusual natural products including peptides alternative approaches until a practical solution was found. I as well as separation science to the Institute. (Copied from a picture particularly remember the visit of Sir Robert Robinson, the in the hallway of the Merrifield lab). British Nobel Laureate in Chemistry, who pulled me aside and told me how lucky I was to be Bruce’s student. He said that he exploring numerous alternative supports, linkages, amino was going to nominate Bruce for the Nobel Prize in Chemistry. protection, etc. was never discussed to any extent during my If I remember correctly that was in 1964; I listened every Octo- days across the bench. One could not buy functionalized ber for 20 years to learn that R. Bruce Merrifield had finally polymeric supports per se, for example, but had to modify been recognized by the Nobel Committee. Obviously, the delay the polystyrene-divinylbenzene beads (Figure 4) to allow for was due in some part to the reluctance of the entrenched solu- covalent attachment of the first amino acid.