1️⃣2️⃣Who Was the First to Synthesize Insulin?

Insulin is a peptide hormone consisting of two chains connected by disulfide bonds.

Who?

independently by

Helmut Zahn (1916–2004)
Panayotis G. Katsoyannis (1924–2019)

When?

Helmut Zahn did everything possible to publish his synthesis in 1963, which was released on December 23, 1963, in the journal Zeitschrift für Naturforschung [1].

Panayotis G. Katsoyannis published his synthesis later, in July 1964 [2].

How?

In short, the B-chain and the A-chain were prepared individually and then both chains were combined.

In the Zahn synthesis [1], the B-chain was synthesized by coupling several peptide fragments using dicyclohexylcarbodiimide, followed by treatments such as decarbobenzoxyliation and linking with another peptide to form the protected B-chain. The A-chain synthesis involved similar steps: decarbobenzoxylation and the coupling of peptides to form the partially protected A-chain. Finally, both chains were deprotected and combined in liquid ammonia.

Very Short History of Insulin

Insulin is a polypeptide hormone produced by the β-cells of the pancreas, responsible for regulating blood sugar levels. Diabetes mellitus, a condition resulting from poor blood sugar control, occurs in two types.

Type 1 diabetes usually begins in childhood and is characterized by insufficient insulin production, requiring daily insulin injections for survival.
Type 2 diabetes accounts for nearly 90 % of all cases. Although it is not caused by a lack of insulin, about one-third of patients benefit from insulin therapy [3].

Isolation and Commercialization

Frederick G. Banting (1891–1941) and Charles H. Best (1899–1978), working in the laboratory of John J. R. Macleod (1876–1935) at the University of Toronto, Canada, isolated insulin in 1921. Only twelve months later commercialization started.

The scientists collaborated with Connaught Laboratories, a non-commercial public health entity in Ontario, Canada, (now part of Sanofi Aventis) and Eli Lilly, Indianapolis, IN, USA, for insulin production, and offered royalty-free licenses to several European institutions.

August Krogh (1874–1949), whose wife had diabetes, took this opportunity and founded Nordic Insulin Laboratories (now Novo Nordisk, Bagsværd, Denmark). In 1922, the UK became the first country to approve insulin injections for diabetics, followed by the U.S. granting approval to Eli Lilly in 1923. That same year, Macleod and Banting were awarded jointly the Nobel Prize in Physiology or Medicine “for the discovery of insulin” [3].

Structure and Synthesis

From 1945 to 1955, Frederick Sanger (1918–2013), University of Cambridge, UK, and his team first sequenced all 51 amino acids of insulin (bovine (cow) insulin) through decomposition with acids, showing that insulin consists of two polypeptide chains with defined amino acid sequences linked by three disulfide bonds [4]. It was a tedious and time-consuming process. Insulin was broken into small fragments, and its chains were reassembled by identifying where the amino acids overlapped.

Sanger was awarded the Nobel Prize in Chemistry in 1958 “for his work on the structure of proteins, especially that of insulin”. His achievement laid the foundation for understanding protein structure and genetic coding.

Figure 2. Structure of Insulin [5].

Helmut Zahn, former director of the German Wool Research Institute (today DWI – Leibniz Institute for Interactive Materials), Aachen, Germany, and his team achieved the first chemical synthesis of a protein in 1963. In addition to the first total synthesis of insulin, the scientists were able to make targeted modifications to insulin, which had previously not been possible with molecules of this size. Almost simultaneously, Panayotis G. Katsoyannis, University of Pittsburgh, PA, USA, also synthesized insulin.

Dorothy Crawford Hodgkin (1910–1994), University of Oxford, UK, determined the crystal structure of insulin in the solid state in 1969 after 35 years of work using x-ray crystallography [6]. She published an electron density map for the 788 insulin atoms, providing a clearer understanding of how insulin binds through its spatial structure. In 1934, she had received a 10 mg sample of insulin. It took her a year to grow suitable crystals, and the data analysis to determine the full structural details of insulin took very long in the pre-computer era.

However, insulin is too large to be synthesized in practical quantities using chemical methods. So other methodes needed to be found.

Production—First Genetically Engineered Drug

In 1978, the start-up Genentech in San Francisco, CA, USA, collaborated with researchers from the City of Hope National Medical Center, Los Angeles, CA, USA, to produce human insulin using recombinant DNA technology. Recombinant DNA technology is a method that involves combining genetic material from different sources to create new DNA sequences, often for the production of proteins or other biologically active substances.

The researchers synthesized the insulin gene and inserted it into the genome of Escherichia coli. The bacteria were fermented to produce large amounts of the hormone, which could be extracted using methods similar to those developed for penicillin production.

At the time, producing active peptides through genetic engineering was prohibited, so the company initially produced the two insulin chains separately in different fermenters and chemically linked them after purification [3]. Clinical trials for the drug began in 1980, and in 1982, Humulin^® became the first genetically engineered drug approved in the UK, manufactured by Eli Lilly under a license agreement with Genentech.

This collaboration marked the beginning of a new era in pharmaceutical research, in which established pharmaceutical companies used their infrastructure to support the development and commercialization of discoveries made by smaller, risk-taking biotechnology companies.

References/Sources

[1] Johannes Meienhofer, Eugen Schnabel, Hellmut Bremer, Otto Brinkhoff, Rudolf Zabel, Werner Sroka, Henning Klostermeyer, Dietrich Brandenburg, Toru Okuda, Helmut Zahn, Synthese der Insulinketten und ihre Kombination zu insulinaktiven Präparaten, Zeitschrift für Naturforschung B. 1963, 18, 1120–1121. https://doi.org/10.1515/znb-1963-1223

[2] Panayotis G. Katsoyannis, The Synthesis of the Insulin Chains and Their Combination to Biologically Active Material, Diabetes 1964, 13(4), 339–348. https://doi.org/10.2337/diab.13.4.339

[3] K. C. Nicolaou, Tamsyn Montagnon, Molecules that Changed the World, Wiley-VCH, Weinheim, Germany, 2008. ISBN-13: ‎ 978-3527309832

[4] F. Sanger, E. O. P. Thompson, Ruth Kitai, The amide groups of insulin, Biochem. J. 1955, 59(3), 509–518. https://doi.org/10.1042/bj0590509

[5] Frederick Sanger, The chemistry of insulin, Nobel Lecture, December 11, 1958. (accessed December 12, 2024)

[6] M. J. Adams, T. L. Blundell, E. J. Dodson, G. G. Dodson, M. Vijayan, E. N. Baker, M. M. Harding, D. C. Hodgkin, B. Rimmer, S. Sheat, Structure of Rhombohedral 2 Zinc Insulin Crystals, Nature 1969, 224, 491–495. https://doi.org/10.1038/224491a0