Robert C. Sheppard (1932‐2019), In Memoriam

Robert C. Sheppard
(1932‐2019)

Robert (Bob) Sheppard, former Head of the Sub‐Division of Peptide Chemistry at the MRC Laboratory of Molecular Biology, Cambridge, UK, passed away at his home on 15th January 2019, aged 87. His legacy to peptide science is a substantial body of work on the development of novel solid supports and orthogonal Fmoc/t‐butyl‐based chemistry that enable solid phase peptide synthesis to be carried out under mild conditions. These were also adapted to novel automated instruments designed to operate under efficient continuous flow conditions. The chemical synthesis methods remain in widespread use today by both academia and industry.

Bob received a BA in Natural Sciences (Class I) from the University of Cambridge in 1953 and then a PhD under the supervision of Professor George Kenner on the “Studies in the degradation of peptides” in 1957. In the same year, he was a Salters’ Company Postdoctoral Research Fellow at Harvard University under the direction of Professor R.B. Woodward where he focused on the synthesis of tetracyclic and pentacyclic triterpines. In 1958, he returned to the United Kingdom to work with Professor Kenner who had been appointed to the Department of Organic Chemistry, Liverpool University. Bob was appointed as a lecturer, senior lecturer, and then tutor. During this time, he made significant contributions to the Liverpool group’s leading research including its isolation, primary structure elucidation, and chemical synthesis of the peptide hormone, gastrin.

In 1971, the then‐chairman of Cambridge’s MRC Laboratory of Molecular Biology, Dr Max Perutz, made the far‐reaching decision to establish a peptide chemistry laboratory with the goal of using chemical peptide synthesis, and in the longer term, protein synthesis, to contribute to the important protein folding problem upon which theoretical studies were already underway. The critically important roles that peptide synthesis was later to play in molecular biology was yet to be conceived. Bob was appointed Head of the new Sub‐Division of Peptide Chemistry in the same year. The aim was to research and develop new methods for the partial and total synthesis of peptides and proteins. Together with his assembled research team that included Eric Atherton, Bob undertook a comprehensive assessment of the solid phase peptide synthesis methodology as brilliantly developed by R.B. Merrifield (who subsequently received the Nobel Prize in Chemistry in 1984) a few years earlier to determine if improvements could be made and implemented to enhance its capabilities.1 To address the issue of inadequate solvation within the resin matrix that was recognized to contribute to failed peptide syntheses, it was hypothesized that the inherently polar peptides would be more compatible with, and better assembled upon, polar solid supports rather than the originally developed non‐polar polystyrene‐based supports. This led to the development of beaded polydimethylacrylamide which swelled far more in polar solvent medium such as dimethylformamide.2 This support contributed to improved yields of several synthetic peptides including the opioid, β‐endorphin. Importantly, Bob recognized that these modifications could be applied to the assembly of oligonucleotides which was subsequently developed within his team by Mike Gait.

It also soon became apparent to Bob that an inherent limitation of the Merrifield technique was the use of a range of differentially acid‐labile protecting groups for the terminal amino group and side chain protection and for the linkage to the solid support, the latter requiring the use of highly corrosive liquid hydrogen fluoride and specialized handling apparatus for its cleavage. Together with Eric Atherton, he recognized the potential advantages for solid phase peptide synthesis to be conferred by a strategy that enabled a combination of both acid‐ and base‐labile protecting groups. Critical to this was the adoption and use of the novel, recently developed amino acid amino‐terminal protecting group, fluorenylmethoxycarbonyl (Fmoc) that was developed by Louis Carpino and which is cleaved by, typically, secondary amines such as piperidine. From this work, the Fmoc/t‐butyl strategy emerged. This combination of Fmoc group and mild acid‐labile t‐butyl groups for side chain protection provided a system which was highly efficient, chemically mild, and truly orthogonal.3,4 Importantly, it also subsequently facilitated the development of efficient and economical continuous flow methods and of real‐time UV monitoring via the Fmoc chromophore of both acylation and deprotection reactions.5 This technology was adopted in commercially available automated continuous flow peptide synthesizers from manufacturers such as LKB, MilliGen, and Protein Technologies. His extraordinarily productive laboratory also developed novel side chain protecting groups, active ester acylation chemistry, peptide‐resin linkers, multiple peptide synthesis, composite solid supports, and improved fragment condensation coupling methods (for example, previous works6-8). All of these helped significantly advance the conduct of solid phase peptide synthesis to enable the acquisition of ever‐larger peptides in generally greater yields and purity than previously possible.

The improved methods for solid phase peptide synthesis caused substantial worldwide interest, and the following period was enlivened by short visits from many notable or new peptide chemists. They each contributed to the research work of Bob’s group during the few weeks or months that they stayed. All greatly enjoyed the vibrant, stimulating environment and camaraderie that Bob successfully created within his laboratory. Today, the Fmoc/t‐butyl solid phase peptide synthesis methodology remains in widespread use throughout academia and industry and continues to have a major impact on many fields of science including medicine, immunology, and structural biology. This is testament to not only Merrifield’s original, pioneering development but also Bob’s outstanding refinements. The methods also formed the basis for the success of a local company, Cambridge Research Biochemicals (CRB), which was the first to offer Fmoc‐amino acid derivatives and other reagents for commercial sale. In recognition of this, the MRC Laboratory of Molecular Biology and CRB jointly received the Queen’s Award for Technological Achievement in 1989. The impact of Bob’s significant, sustained contribution to solid phase peptide synthesis was further recognized by his receipt of the European Peptide Society’s premier award, the Josef Rudinger Memorial Medal, in Braga, Portugal, in 1994.

After his official retirement in April 1992, Bob remained actively involved in the peptide community being both a co‐opted member of the European Peptide Society Council and Treasurer of the European Peptide Society (both to 1998) as well as continuing to consult for industry. He was a loving and much‐loved husband, father, and grandfather and is survived by his wife of nearly 60 years, Maureen, three children, and six grandchildren. Bob was a superb chemist with a great capacity to devise simple solutions to seemingly complex peptide chemistry questions and to foster the development of positive outcomes by his team members. He was a wonderful mentor and friend to many and will be greatly missed by all who knew him and who had the great privilege and pleasure of working with him.

Eric Atherton1
John D. Wade2
1Tyn Twll Cottage, Glan Conwy, UK
2Florey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, Victoria, Australia

REFERENCES
1. Sheppard RC. Solid phase peptide synthesis—An assessment of the present position. Proceedings of the 11th European Peptide Symposium, Vienna. Amsterdam: North Holland Publishing Co; 1971:111.
2. Atherton E, Clive DLJ, Sheppard RC. Polyamide supports for polypeptide synthesis.
J. Amer. Chem. Soc. 1975;97:6584.
3. Atherton E, Fox H, Harkiss D, Logan CJ, Sheppard RC, Williams BJ. A mild procedure for solid phase peptide synthesis: use of fluorenylmethoxycarbonyl‐amino‐acids.
J. Chem. Soc., Chem Commun. 1978;537.
4. Atherton E, Fox H, Harkiss D, Sheppard RC. Application of polyamide resins to polypeptide synthesis: an improved synthesis of β‐endorphin using fluorenylmethoxycarbonyl‐amino acids.
J. Chem. Soc., Chem. Commun. 1978;539.
5. Dryland A, Sheppard RC. Peptide synthesis. Part 8. A system for solid phase synthesis under low pressure, continuous flow conditions.
J. Chem. Soc., Perkin Trans. I. 1986;125.
6. Sheppard RC, Williams BJ. Acid‐labile resin linkage agents for use in solid phase peptide synthesis.
Int. J. Peptide Protein Res. 1982;20:451.
7. Atherton E, Sheppard RC. Solid phase peptide synthesis using N‐alpha‐fluorenylmethoxycarbonyl amino acid pentafluorophenyl esters.
J. Chem. Soc., Chem. Commun. 1985;165.
8. Bedford J, Hyde C, Johnson T, et al. Amino acid structure and ‘difficult sequences’ in solid phase peptide synthesis.
Int. J. Peptide Protein Res. 1992;40:300.

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