Vincent du Vigneaud’s synthesis of oxytocin, a small polypeptide hormone from the pituitary gland, provided the scientific and medical communities with hope that the more complex polypeptides and proteins could be synthesized and used in medicine.
In England in 1895, physician George Oliver
In addition to the pressor and antidiuretic activities in the posterior pituitary, two additional effects were found in 1909. Sir Henry Hallett Dale, an English physiologist, was able to show that the extracts could cause the uterine muscle to contract (oxytocic effect), and Isaac Ott
Following the discovery of these various effects, attempts were made to concentrate and isolate the substance or substances that were responsible. John Jacob Abel was able to concentrate the pressor activity at Johns Hopkins University, using heavy metal salts and extraction with organic solvents. The results of the early work, however, were varied, primarily because most investigators used only one biological assay method to study the progress of the purification. Some investigators came to the conclusion that only one substance was responsible for all of the activities, while others concluded that two or more substances were likely to be involved.
In 1928, Oliver Kamm and his colleagues, at the drug firm of Parke, Davis and Company
The problem of these hormones and their nature interested Vincent du Vigneaud at the George Washington University School of Medicine. Du Vigneaud became interested in hormones and their chemistry in 1923 after hearing a lecture on insulin given by his biochemistry teacher William Rose, who had just returned from Toronto, Canada, where Sir Frederick Grant Banting and Charles H. Best had reported success in the treatment of diabetes mellitus with preparations of the hormone.
While pursuing his Ph.D. studies at the University of Rochester School of Medicine, du Vigneaud worked with John R. Murlin, who was interested in insulin. Du Vigneaud was able to improve the method Abel had devised for crystallizing insulin and was able to show that all the sulfur content of insulin was a result of the amino acid cystine. Following his graduate work, du Vigneaud had a fellowship that allowed him to do research on insulin at Johns Hopkins University with Abel. He spent the next year in Germany at the Kaiser Wilhelm Institute working in Max Bergmann’s laboratory on peptide synthesis.
Shortly after arriving at George Washington University, du Vigneaud collaborated with Kamm on the investigation of the latter’s posterior pituitary fractions. He was able to show that the sulfur content of both the oxytocin and vasopressin fractions was a result of, as with insulin, the amino acid cystine. This helped strengthen the concept that they were polypeptide or proteinlike substances. Du Vigneaud and colleagues next tried to find a way of purifying oxytocin and vasopressin. This required not only the separation of the hormones themselves but also the separation from other impurities present in the preparations. Electrophoresis, a method used to separate proteins from one another based on the differences in their charges, served to resolve them and give some additional purification.
In 1938, du Vigneaud and several colleagues left to work at Cornell University Medical College in New York. At about this time, du Vigneaud had to put aside the hormone studies to participate with other scientists in work related to World War II. Du Vigneaud was part of the team of English and American scientists who were asked to work on the chemistry of penicillin. The goal was to determine the structure of the antibiotic so that, if possible, it could be produced more efficiently through synthesis rather than fermentation.
During the war years and shortly thereafter, other techniques were developed that would give du Vigneaud the tools he needed to complete the job of purifying and characterizing the two hormonal factors. One of the most important was the countercurrent distribution method of Lyman C. Craig at the Rockefeller Institute
Du Vigneaud and his group next used a new chromatographic method developed by William H. Stein and Stanford Moore at the Rockefeller Institute. This method not only separated amino acids but also determined them quantitatively. The procedure involved adding the amino acids derived from a protein to the top of a column of starch held in a glass tube. The amino acids were washed down the column with a solvent and came off the end separated from one another. Each of the amino acids was then determined quantitatively. Thus, the Stein and Moore analysis identified the amino acids in a mixture and also determined the amount of each. Du Vigneaud was able to break the pure oxytocin down into the component amino acids by heating it with acid.
The mixture was analyzed by the Stein and Moore method. The analysis showed that the oxytocin was made up of one unit of eight different amino acids. In addition to the cystine detected earlier, du Vigneaud found aspartic acid, glutamic acid, glycine, isoleucine, leucine, proline, and tyrosine. Also released from the oxytocin on breakdown were three units of ammonia. This seemed to indicate that oxytocin was an octapeptide with an amide group, rather than a free carboxylic acid group, and that the aspartic acid and glutamic acid were derived from their respective amides, asparagine and glutamine.
The next task was to find out in what sequence the amino acids were joined together. The eight amino acids of oxytocin could be connected together in 181,440 different sequences, assuming that the hormone was a peptide with a structure similar to a small protein. It should be noted that the structure of cystine is such that it acts as two amino acids would in forming peptides. The two halves are connected together by two sulfur atoms. Oxytocin actually could be considered a nonapeptide (a peptide with nine amino acids), but historically it has been referred to as an octapeptide.
The approach that du Vigneaud used for structure determination was similar to that being used by Frederick Sanger in England, who was working on the structure of the larger insulin molecule. This involved breaking down the oxytocin molecule by different methods so as to obtain fragments of smaller peptides. By determining the structures of the smaller peptides, the structure of the oxytocin could be deduced. This approach was successful and completed the first stage of the chemical work. It was du Vigneaud’s goal (having been interested in peptide synthesis for many years) to make synthetic oxytocin by duplicating the structure his group had worked out.
Peptide synthesis is a very complicated procedure because of the chemical nature of the amino acids. Amino acids have two reactive ends. One is referred to as the amino group and the other as the carboxylic acid group. In a peptide, each amino acid can be attached through its two groups to two other amino acids. To synthesize a peptide, it is necessary to carry out the steps in such a manner that only one reaction occurs in any given step. This requires that the first amino acid used in the synthesis be protected on one of its reactive ends so that the second amino acid can react in only one of two possible ways. Also, the product formed in each step of the synthesis must be quite pure before adding the next amino acid. After all the amino acids have been linked, any protective groups must be removed without affecting the rest of the structure. Du Vigneaud’s synthetic oxytocin was obtained and the method published in the Journal of the American Chemical Society on October 5, 1953.
Du Vigneaud wanted to prove that the synthetic and natural forms were identical in every physical, chemical, and biological property. The two forms were tested and found to act identically in every respect. In the final test, the synthetic form was found to induce labor when given intravenously to women about to give birth. Also, when microgram quantities of oxytocin were given intravenously to women who had recently given birth, milk was released from the mammary gland in less than a minute.
Thus, the task of synthesizing a natural, biologically active peptide hormone was completed for the first time. The structure and synthesis of beef vasopressin followed shortly thereafter. Vasopressin also was shown to be a cystine-containing octapeptide amide and differed from oxytocin by only two amino acids, with the rest of the sequence identical. Another interesting fact that du Vigneaud’s group found was that vasopressin from hogs differed from beef vasopressin in that it had the amino acid lysine rather than the amino acid arginine. The synthetic forms of oxytocin and both types of vasopressin have replaced the natural forms for use in medicine.
Du Vigneaud received the Nobel Prize in Chemistry
The work of du Vigneaud and his associates demonstrated for the first time that it was possible to synthesize peptides that have properties identical to the natural ones and that these can be useful in certain medical conditions. Oxytocin has been used in the last stages of labor during childbirth, and vasopressin has been used in the treatment of diabetes insipidus when an individual has an insufficiency of the natural hormone, much as insulin is used by persons having diabetes mellitus.
After receiving the Nobel Prize, du Vigneaud continued his work on synthesizing chemical variations of the two hormones. By making peptides that differed from the oxytocin and vasopressin by one or more amino acids, it was possible to study how the structure of the peptide was related to its physiological activity. It was thought that analogs of the two peptides might be made that possessed only one of the activities associated with the natural peptides. Several other groups participated in this approach. The vasopressin analog desmopressin, which was first synthesized in 1966, has antidiuretic activity but little pressor activity. It is used in medicine.
After the structures of insulin and some of the smaller proteins were worked out, they, too, were synthesized, although with greater difficulty. Other methods of carrying out the synthesis of peptides and proteins have been developed and are used today. The production of biologically active proteins, such as insulin and growth hormone, has been made by the more efficient methods of biotechnology. The genes for these proteins can be put inside microorganisms, which then make them in addition to their own proteins. The microorganisms are then harvested and the useful protein hormones isolated and purified.
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Bricas, E., and P. Fromageot. “Naturally Occurring Peptides.” Advances in Protein Chemistry 8 (1953): 1-125. Covers the naturally occurring peptides of plants and animal origin. Sections on oxytocin and vasopressin cover the literature up to 1952. A good source of early references. -
Chemistry, 1942-1962. River Edge, N.J.: World Scientific, 1999. Exhaustive, voluminous discussion of twenty-one years’ worth of Nobel laureates in chemistry. Du Vigneaud’s life and achievements are set out in detail. -
Du Vigneaud, Vincent. “Hormones of the Posterior Pituitary Gland: Oxytocin and Vasopressin.” In The Harvey Lectures, 1954-1955. New York: Academic Press, 1956. A lecture given after the structure and synthesis of oxytocin had been accomplished; covers the experimental work in some detail. The vasopressin work discussed includes the proposed structures for beef and hog vasopressin and progress on the vasopressin synthesis. One of the best reviews on the subject. -
_______. A Trail of Research in Sulfur Chemistry and Metabolism and Related Fields. Ithaca, N.Y.: Cornell University Press, 1952. Du Vigneaud’s personal account of his chemical work from insulin to the posterior pituitary hormones. Ends shortly before the structure work is completed. One of the few autobiographical accounts by a chemist. -
Malmström, Bo G., and Bertil Andersson. “The Nobel Prize in Chemistry: The Development of Modern Chemistry.” In The Nobel Prize: The First One Hundred Years, edited by Agneta Wallin Levinovitz and Nils Ringertz. River Edge, N.J.: World Scientific, 2001. Overview of the history of the chemistry award, the major laureates, and their effect upon the development of chemical science in the twentieth century.
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