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Autobiography
AUTOBIOGRAPHY Durey
H. Peterson,
Ph.D.
It was the winter of 1917. I was eight years old, confined to a bed with rheumatic fever in the cold, drafty hayloft of the newly built barn. Our parents and six of their eight children had moved that fall with two lumber wagons and our horses and cows 50 miles across the prairie from a homestead in arid country to a new farm in the irrigated land north of Denver. Our house would not be built until the following summer. As I listened to the wind, felt the cold, and heard the horses and cows below us, I thought, "When I grow up, I'll never be a farmer." become a scientist, part of the Never did I dream that some day I would "Golden Age" of science. Insufficient rainfall, hail, and poor crops seemed to follow us from farm to farm. We were taught early on how to contend with the hazards of nature--how to shoot a gun to kill coyotes that preyed on our livestock, as well as how to shoot rabbits and pheasants for food. I also trapped muskrats and skunks for their fur. We moved six times when I was a child, and I had to adjust to six different grade schools. While my parents, both immigrants from Sweden, never financial success, they gave me something money couldn't philosophy of life that included hard work and honesty. especially, felt that there was always a way to accomplish using one's physical and mental resources.
January 1985
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achieved great buy--a My mother, a goal by
Volume 45, Number 1
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In 1923, my father gave up farming, moved the family to Denver, and found work as a carpenter in the railroad yards repairing box cars, providing for us the bare necessities of life. Each of us children found jobs outside our home to cover personal needs. Changing from a country school to the high school in a big city was a difficult Because I was so interested in chemistry and did well adjustment. during my junior year, my chemistry teacher, Mr. Beattie, appointed me his assistant in the chemistry lab during my senior year. It was he who encouraged and inspired me to continue my interest in chemistry by studying at The University of Denver under Dr. R. G. Gustavson, who we affectionately called "Gus" and who proved to be an inspiring and stimulating professor. Because I did well in chemistry at the university, I was made a chemistry assistant in my junior and senior years and head chemistry assistant the year I did my Masters work. "Gus" became like a second father to me. My major hobby during high school and college was baseball. I was an all-conference pitcher for three years and helped win four consecutive championships, as well as playing semi-professional baseball during the summer. The latter served to help me financially through college. After receiving my Master's degree in 1932, I was awarded a buition scholarship to The University of Chicago. It was during the depression, and outside jobs were essentially nonexistent. "Gus, " along with other professors, collected a $500 loan to pay for room, board, and expenses. My graduate work was done under Dr. F. C. Koch, then the world authority on the sex hormones, paticularly the male hormone. Financial problems for my existence required that I spend over one-half of my time on and off the campus on jobs that paid 501# an hour. One of my jobs was working in a gold mine at 12,000 feet above sea level in Colorado in exchange for my baseball ability. However, the five years I spent at the university also had its virtues. I was exposed to some of the outstanding professors in the world from 1932-1937; namely, Koch, Carlson, Moore, Steiglitz, Karasch, and others. LIFE MAGAZINE of 1934 rated The University of Chicago as having an outstanding faculty equal to any in the world. Aside from this exposure, other important conditions that greatly influenced my life were present during and directly after my five years on the campus. No reports were given at the end of a course--only if one flunked. One did not have to attend classes; however, I found it stimulating to do so. The program developed an independence that rapidly placed one on his own. After receiving my Ph.D. in 1937, I began working for Bauer & Black in Chicago; and this allowed me to continue part time research at the university. This also gave me the opportunity to observe the presence of at least eight Nobel Prize winners in a program that I didn't recognize at the time as "Development of the Atomic Age." Among these workers were Fermi, the Italian Nobel Laureate in Physics, who was really responsible for the key experiment that triggered the atomic age. He harnessed atomic energy by building a carbon brick pile in a handball court under the stadium where I had played games many times. The
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stadium is long gone, but the laboratory site contains a famous memorial indicating the birth of the atomic age. Fermi's work was absolutely necessary for the development of the atomic bomb at Los Alamos, New Approximately 20 years later, Fermi died of leukemia due to Mexico. excess radiation exposure. R. I.
At the university, I shared a laboratory Dorfman, both outstanding in the field
with T. F. Gallagher of steroid metabolism.
and
The significance of steroids as hormones became important when F. C. Koch (1) discovered the presence of the male hormone in bull testes, while Allan and Doisy (2) first found the female hormones in body fluids. It is interesting that I am one of the few earlier workers remaining who had the unique distinction of working on the steroids before their structures were elucidated in 1933. Although my B.S. and M.S. were obtained in Chemical Engineering, my great interest in biochemistry was spurred by "GUS" and my thesis was concerned with "The Route of Excretion of the Female Sex Hormone in the Hen." One of the routes was through the bile duct. A conclusion was never reached because, on the addition of an active concentrate from human pregnancy urine, most of the problem was concerned with the development of a good method of extraction from chicken bile. My Ph.D. thesis consisted of two parts: "I. The Effect of Acid Hydrolysis on the Yield of Androgenic and Estrogenic Substances from Human Urine (3); II. The Daily Urinary Excretion of Androgenic and Estrogenic Substances from Human Urine in Normal Men and Women" (4). Such studies required the use of capons in the case of androgens and ovariectomized rats in the case of estrogens. As a result, I performed approximately 7,000 operations during my research in order to sustain a sufficient number of rats and capons. Each assay required one week, and these studies took three years to complete. In Part I, it had been known for some time that it was necessary to boil human urine with acid before the estrogenic material could be recovered with immiscible solvents. This work showed that a shorter acid hydrolysis period of 15 minutes released the maximum androgenic activity. Further hydrolysis resulted in lower yields. Since urine without acid treatment does not yield any androgenic activity using immiscible solvents for extraction, it was concluded that this activity In 1938, I was able to extract a is excreted as an inactive complex. concentrated inactive complex using N-butanol from unhydrolyzed urine. I concluded that the complex was a glucuronide of the male hormone constituents. This concentrate could then be boiled with hydrochloric acid to produce androgenic material extractable with benzene. It was at this point that I became interested in microbiological In attempting to find out if the complex was conversion of steroids. an a- or B-glucuronide, an incubation mixture became contaminated with an unknown microorganism; and the resulting ether extract was more active in androgenic activity than an acid hydrolyzed sample from the
same concentrated glucuronide complex. This led me to believe that the androstenedione was converted to testosterone by reduction to the 17BThe latter compound was never hydroxyl group that is testosterone. isolated in human urine but was considered to be the natural hormone found in human blood at that time and produced by the testes. My program to continue to study the microbiological transformation of steroids was interrupted for the next 11 years. I was then able to resume this project at The Upjohn Company, after the significance of the cortical hormones became apparent in the rheumatoid arthritis study done at The Mayo Foundation by Wench and Kendall 1949 (5). The second part of my thesis, "The Daily Urinary Excretion of Estrogenic and Androgenic Substances by Normal Men and Women," consisted of a long and extensive study on urine from four normal men and four From this normal women and collected for 27 to 45 consecutive days. study, it was shown that there are marked fluctuations in the daily urinary excretion of androgens and estrogens in normal men and women. There is no evidence to support a monthly cycle in the excretion of either androgens or estrogens in normal men. In normal women, the excretion of estrogens is characteristically low during the menstrual flow and rises during the intermenstruum, with a double peak in certain instances. The average daily excretions of estrogens, calculated as gammas of estrone, are 9-12 for men and 18-36 for women. The rates of excretion of both hormones do not seem to bear any relationship to each other in either sex. Following my graduate work at The University of Chicago I joined Bauer & Black as a biochemist and developed the use of nylon as a nonabsorbable surgical suture in 1941. Nylon produces no tissue reactions and is processed as either twisted, braided, or single filament form. The latter is especially good for skin and other special suturing purposes and is still used by surgeons today. In 1943, I decided to join Raymond Laboratories in St. Paul, Minnesota, in a venture to initiate the development of drugs and enter the pharmaceutical field. Instead, we were successful in the cold hair waving business with a product called "Toni." I received a patent for the use of potassium bromate as a setting solution. About one-half of my time was spent on this project and the other half on control work for the Army in World War II on signal shells of various colors for airport landing and takeoff of military planes on dark airfields. Our company also made one plastic part for the atomic bomb. Failure to develop drugs for the pharmaceutical industry after three years led me to join G. F. Cartland and his group at The Upjohn Company in 1946 to work on antibiotics. This was an exciting new field, following the dramatic development and production of penicillin. My first contribution was the isolation and purification of a polypeptide type of antibiotic called Circulin (6), which contained leucine, threonine, a,b-diaminobutyric acid, and an optically active pellargonic acid. The components were followed by chromatographic paperstrip techniques based on previous work with the amino acids.
Separation of the components were realized by using powdered cellulose and a solvent, a mixture of 25% acetic acid:Xl% N-butanol:H20--as was used in the paperstrip technique. Since the solvent had to move slowly this chromatography required over the column for proper resolution, three months to resolve and to obtain the pure components. My most important contribution in this field was the first successful development of a paper chromatographic technique for differentiating the streptomyces type of antibiotics using solvents similar to the ones used for separation of amino acids mentioned above (7). Using this mixture of solvents and others, this technique has long been in use by all laboratories and today still forms the basis in the This discovery was important also since screening of new antibiotics. it formed the idea for following micro amounts of steroids, when I later became active in the cortisone field. During World War II, U.S. intelligence sources picked up a rumor that Nazi Luftwaffe pilots were being "hopped up" with a fabulous drug that enabled them to function more efficiently aloft. The Nazis supposedly were buying large quantities of adrenal glands of cattle from Argentina and extracting them to give the airmen a drug with these unusual powers. Eight years earlier, cortisone and the more active hydrocortisone were isolated in pure form, along with other different biologically active steroids, from bovine adrenals by Reichstein, Mason et al, Kuizenga and Cartland, and Wintersteiner and Pfiffner (8). The emphasis shifted to the Mayo Clinic where a physician, Dr. Hench, noticed that women with severe rheumatoid arthritis had remissions during pregnancy. Hench,working with Kendall, knew that the adrenal glands produced large amounts of these hormones during pregnancy, so he asked Sarett of the Merck Company for cortisone (I), now called the "Wonder Drug." Based on the earlier U.S. government request and support (during the Nazi rumor period), Sarett had worked out a 35-step chemical process for making the drug from desoxycholic acid obtained from cattle bile (9). In order to move the oxygen at carbon-12 to the biologically it was necessary to carry out many required oxygen at carbon-11, chemical steps at a high cost. The process ended up with a very small yield since the the procedure consumed so many steps. Hench then asked Merck for enough cortisone to treat a 29-year-old woman (who looked 50) with rheumatoid arthritis. This patient had stiff, swollen joints that were very tender and could walk only with a She was bedridden almost all the time with weird shuffling gait. After a one-week treatment by Hench, not only could she severe pain. walk normally but she even left the hospital for a three-hour shopping spree! With great excitement, Hench attempted to obtain more cortisone from Sarett; but none was available. Sarett renewed his supplied the Mayo Clinic
bile acid syntheses and in several months with enough cortisone to treat 14 other
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rheumatoid arthritics. In April 1949, Hench revealed the results and showed an audience of physicians who sat astounded as they watched before and after films of the patients given cortisone (5). These severely crippled people walked up and down stairs after treatment and even danced. The dancing arthritics made headlines all over the world, and a sudden surge of demand arose for cortisone. However, the headlines were deceiving because there was no cortisone available for anyone except a few, well-chosen patients. The clinical work at Mayo triggered some of the most intensive and Every extensive chemical and biological research work ever undertaken. major drug company and many university laboratories all over the world became interested in joining the adventure to find a satisfactory, economical industrial process for making cortisone. After assessing the great technical difficulties and Merck's lead, only a few firms, including The Upjohn Company, continued in the race. Mr. Gilmore, president of Upjohn, gave this notice to the company's scientists: "Don't spare the horses; we want cortisone!" A blow torch was the symbol chosen to keep the scientists busy and alert. Six different approaches were pursued by Upjohn researchers in an attempt to solve this important problem; namely: (a) a chemical approach using ergosterol as the starting steroid; (b) a chemical approach involving some of Sarett's methods; (c) a chemical approaeh using total synthesis; (d) investigating the presence of naturally occurring 11-oxygenated raw materials in seeds; and (e) the microbiological approach initiated by myself, together with H. C. Murray, a microbiologist. Our approach was considered a long-term possibility and was given the least chance for success. The dramatic clinical findings by Hench and Kendall had opened the way for one of the most fascinating episodes in the history of medicine and chemistry. Since that time the use of cortisone and hydrocortisone and other active derivatives has been extended to many other inflammatory diseases such as rheumatic fever, asthma, eye infections, ulcertive colitis, Addison's disease, and various allergies. Based on the biological law that "ontology recapitulates phylogeny," it was my idea that lower microorganisms might also contain enzymes similar to the adrenal glands of humans for making cortisone and hydrocortisone. With this in mind, I suggested the use of the microbiological approach as a solution to introducing the biologically required oxygen at carbon-11. It was this step in particular that required the chemist to carry out the difficult and lengthy chemical steps to make cortisone (I) and hydrocortisone (II). My proposal was questioned by many outstanding organic chemists who believed it couldn't be done. My response was that "the microorganisms do not know this" and experiments would have to be done to determine the answer. The first Hechter et al,
biological synthesis of hydrocortisone was that who perfused 17a-hydroxy-11-desoxycorticosterone
of through
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Cortisone
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Hydrocortisonc
(I)
Figure
(II)
1
beef adrenal glands and obtained hydrocortisone. It was obvious that this method could not be economical and I had already initiated the microbiological appproach when this work was published in 1950 (lo). I firmly believed we should not proceed with any work of this until a micromethod of following the transformation of the steroid was available, sensitive to 10 mcg or less. It becomes obvious that the Project must be designed as a deliberate attempt to introduce the strategic oxygen at carbon-11 in a single step.
natWe
The author, together with H. 11, 1949. I attempted to develop micro afIIOUntS Of steroids, when a Zaffaroni (11). We contacted him essentially his method.
C. Murray, began this work on November a paperstrip method for identifying publication in this area appeared by and returned to Kalamazoo using
Our first successful microbiological conversion involved the transformation of desoxycorticosterone to a 20EGhydroxy derivative by Reichstein's reduction of the 20-ketone group using a Streptomyces x. earlier work had involved five steps to prepare this compound (12). Since Hey1 and Herr (13) had developed an economical method for Murray and I first making the progestational hormone, progesterone, looked at this material as a potential substrate. An agar plate placed in a window sill of the oldest and dirtiest laboratory at The Upjohn Company by Murray yielded a Rhizopus so of a fungus which was then Five milligrams of the latter was incubated with progesterone. fermented in a test tube containing 20 ml of medium in February 1950 for 24 hours. The methylene chloride extract was concentrated and an equivalent of 10 gamma examined by paperstrip chromatography. The uvsensitive spots showed that the progesterone had almost disappeared. However, larger amounts of a slower-moving compound as well as an even The latter eventually proved to slower-moving component were present. be 68,lla-dihydroxyprogesterone. This fermentation was scaled up to 500 mg of progesterone as a After papergram analysis showed a change similar to that of substrate. methylene chloride the screening sample described above, a concentrated extract was chromatographed over an aluminum oxide column and the
fractions examined by papergram analyses. The various fractions were crystallized and the prevailing fraction (next to the slowest moving one) furnished physical constants different from progesterone and different from 116-hydroxyprogesterone. This compound was acetylated and found to have a single hydroxyl group. Oxidation of the hydroxyl group produced a ketone derivative, 11-ketoprogesterone, identical in all respect with the physical constants reported in the literature and identical with known ll-ketoprogesterone obained from T. F. Gallagher of studies. the Sloan-Kettering Institute in ir, uv, and X-ray diffraction This meant that progesterone (III) had been enzymatically converted to a new compound, the previously undescribed lla-hydroxyprogesterone (IV) , as shown in Figure 2 (14).
CH3 I c=o
Progesterone
CH3 I c=o
H%,, 9xP 0 ’ Ila-Hydroxyprogesterone
(III)
Figure
(IV)
2
The introduction of oxygen at carbon-11 by a microorganism was actually achieved in five months, even though the structure was not definitely established for another two months. Obtaining a solution to such a major project was a remarkable accomplishment and it was not due to luck alone. The deliberate attempt to solve this problem involved carefully planned microtechniques used in an orderly fashion, thus speeding up the whole project. This set the stage for an integrated study involving of the microbiological step and a variety of new chemical challenged the organic chemists to an architecturally and sound solution to the production of cortisone and all the naturally occurring anti-inflammatory steroids, including superior analogs.
a combination reactions that economically other new and
After a tremendous amount of work, an integrated and successful process was capably accomplished by J. A. Hogg and his colleagues. For the microbiological conversion resulting in the one-step introduction of oxygen at carbon-11, the author received the Upjohn Prize in 1951 and Outstanding Alumni Awards in Science from The University of Chicago in 1976 and The University of Denver in 1983. Seventy-four patents were issued and 54 scientific articles published the extensions of this work.
on
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It is important that this microbiological conversion proved the concept that substitution of an inactivated carbon atom could be The value of achieved by an enzymatic attack using a microorganism. microorganisms in chemical synthesis thus was dramatically enhanced. This success triggered massive programs in which many thousands of molds and bacteria were tested for their usefulness in the modification of As a result, many types of oxidation and reduction various steroids. reactions, as well as combinations of such changes, were discovered. It also spurred similar investigations of other substances using microbiological conversions and many reports have since been published on straight chain and cyclic hydrocarbons, aromatic terpenes and alkaloids as well as pesticides and antibiotics. A formal announcement of the oxygenation of steroids was made in a note sent to J. AM. CHEM. SOC. on February The CHEMICAL ENGINEERING NEWSedition following announcement:
of April
at carbon-11 14, 1952 (14).
7, 1952,
made the
"In the neatest trick of the year Upjohn has come up with a single-step process for the microbiological oxidation of steroids at carbon 11. Two Upjohn scientists, D. H. Peterson and H. C. Murray, announced the discovery in the April 5, 1952, J.A.C.S. A variety of steroids may serve as starting material for conversion to the cortical hormones such as cortisone and possible Compound F (hydrocortisone) and Compound B (corticosterone). Using common molds the sex hormone, progesterone, is transformed to llahydroxyprogesterone, only a few steps away from cortisone etc., etc . 1' The importance numerous scientists published in almost
of this discovery was not only acknowledged by and drug companies but the announcement was every newspaper of any size in the world.
It was interesting that this announcement was made at the Gordon Conference at Mt. Tremblant, Canada, in 1953 and the author was inadvertently assigned Cabin 11. To top this off at the next year's meeting, I announced the microbioloical introduction of the 17a-hydroxy group and was assigned Cabin 17, an absolutely incredible occurrence! In order to reduce the cost of production of all these cortical hormones by microbiological means, it was necessary to introduce the remaining necessary chemical groups such as the 17a-hydroxy and 21hydroxy groups. This was accomplished by several organic chemists at The Upjohn Company, led by Dr. John A. Hogg and coworkers. The whole character of the Upjohn project and the company changed immediately, and the emphasis shifted to the microbiological approach for the production of hydrocortisone. Approximately 150 scientists and assistants zeroed in on the venture. The necessary
patent
applications
were prepared.
The first
to
appear was a 1951 South African patent specification. Interestingly, the latter was noticed by the Syntex Corporation quite early and this group then used our fermentation techniques to prepare cortisone in relatively fewer reactions. The latter was published in 1952 (15). However, the Upjohn research group (although not published) had proceeded in a similar fashion and was in pilot plant production in late 1951. Before the Syntex publication appeared, an enormously detailed and classical U.S. patent issued July 8, 1952, to Murray and Peterson (16). This patent was written in the style of J. AM. CHEM. SOC. and contained a variety of substrates and hydroxylated steroid products using the molds Mucoralis sp for conversion. The Franklin Pierce Law Center conducted a study for the National Science Foundation in 1974 and concluded that this patent was one of the A publication by most significant technological advances in 25 years. Chemtech in November, 1978, published the entire patent as one of the most outstanding, well-written, significant, and unique examples in the On July 9, 1968, the Wall Street Journal (17) covered this industry. work under the title of "Innovators." This, along with the work of other innovators, was highlighted in a book in 1968 (18). The history of this important microbial conversion is also covered in detail in a book by Leonard Engel (19). For the introduction of the necessary oxygen at carbon-11 by the mold to be commercially successful, the process had to be financially feasible. To accomplish this: (a) the substrate must be readily available and economically produced; (b) the reaction must proceed in high yield; (c) the substrate level must be high; and (d) the product must be obtained and purified in high yields. All of these requirements were fulfilled by this transformation. One of the most interesting aspects of such conversions is the fact that steroids are ordinarily essentially insoluble in aqueous media. It is for this reason that early workers in this field were undoubtedly discouraged. The steroid substrates can be added to the culture medium in very small amounts of acetone, where the solvent plays no'role, or as a micronized solid in large concentrations. Small amounts of the substrate are apparently adsorbed to the surface of the cell where enzymatic conversion occurs. The converted product then moves into solution and is replaced by the substrate. More substrate moves to the surface of the cell, etc., etc. Microbial enzymes effect a wide variety of chemical changes in steroids. These changes include the introduction of nuclear and sidechain hydroxyl groups; cleavage of carbon-carbon linkages in the side chain as well as in rings A and D; dehydrogenation of ring hydroxyl groups as well as rings A and 8; opening of an oxide ring; reduction of ring double bonds and of ketone groups on a ring or side-chain; hydrolysis of steroid esters as well as many combinations of the above reactions. Of these changes, the most common one is hydroxylation, and only the 18-C atom has not yet been hydroxylated. Our laboratory has hydroxylated eleven positions: 6D-, 7D-, 8D-, 9a-, lo&, lla-, ll&, 14a-, I5a-, I56-, 17a-; while other laboratories have reported
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la-, 26-, 7a-, 12b-, 19-, and 21-. In tissues are able to effect hydroxylations at 6a-, 66-, llb-, 17a-,18, 19, 20, and from other tissue at 2a-, 2B-, 6a-, 6B-, 75-, and 16a-. The range of positions affected may be seen by inspection of Figure 3. hydroxylation at six positions: contrast, enzymes from adrenal
Figure
3
Although these changes are prominantely fungi, some reactions take place to a lesser actinomycetes, and even protozoa.
carried extent
out by filamentous by bacteria, yeasts,
The rapid development and progress in this field was due mainly to the use of microtechniques such as paper chromatography, uv light, and ir spectroscopy. Paper chromatography was especially useful since it allowed the rapid examination of a wide variety of reactions by many microorganisms within a short period of time. The general methods used consisted of the following sequence of steps: (a) the microorganism is first grown under suitable aerobic conditions in submerged media for 24-48 hours; (b) the steroid to be tested is then added in a water-soluble solvent such as acetone; (c) after a conversion period of several hours to several days (average about 24-72 hours), the transformation products are recovered in pure form by extraction with a water insoluble solvent, purification, using direct crystallization or column chromatography or countercurrent distribution; (d) structure work by the usual methods of classical organic chemistry, viz: by conversion to known structures. In some cases the use of microbial methods has also been most useful in structure work. In a continuing effort to increase the anti-inflammatory activity and decrease the side effects of corticosteroids, it was found by several workers that the introduction of the 1,2-double bond did indeed accomplish this goal. In our laboratory we used Septomyxa affinis to introduce the 1,2-double bond into anti-inflammatory steroids, which are sold by The Upjohn Company, namely, prednisone (V), prednisolone (VI), and methylprednisolone (VII), products used extensively in the medical field, Figure 4.
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CH20H
CH20H iH20H
o&H
o&H
o#H
I= CH3 Prednisone
(VI
Prednisolone
Figure
(VI)
Ga-Methylprednisolone
(VII)
4
After the important discovery of microbiological introduction of oxygen at carbon-11 in a single step, a whole host of biological transformations were conducted in our laboratories both before and after the extension of these findings by other workers who discovered similar and additional important and interesting reactions. In almost all cases, the steroids formed were new compounds. However, these will not be discussed since the purpose of this manuscript is to describe my own contributions to this field. A particularly extensive study was carried out on the IIa-hydrofty compounds and other products derived from numerous steroid substrates Rhizopus nigricans and Rhizopus arrhizus q. These transformations
are briefly
described
below
(12).
Progesterone (III) is converted by Rhizopus arrhizus to lla-hydroxyprogesterone (IV) as well as to 68,lladihydroxyprogesterone in rather large amounts. It was then found that Rhizopus nigricans with progesterone (III) produced essentially lla-hydroxyprogesterone (IV) with traces of 66,lla-dihydroxyprogesterone and, in addition, 76-hydroxyprogesterone. Rhizopus nigricans also converts Compound S (17a,21-dihydroxy-4-pregnene3,20-dione), desoxycorticosterone, 17ahydroxyprogesterone, 3-keto-bisnor-4-cholen-22-01, 16dehydroprogesterone, 16a,l7a-oxidoprogesterone, pregnane3,20-dione, allopregnane-3,20-dione, androstene-3,17dione, testosterone, 17a-methyltestosterone, and 19nortestosterone to the lla-hydroxy as well as to the 66hydroxy derivatives and in the C-21 steroidal substrates mentioned above, to the 6B,lla-dihydroxy derivatives. As one can obseve, many variations occur. Oesoxycorticosterone, as indicated above, is converted to the lla-hydroxy' derivative. Selective acetylation of the product results in the El-acetoxy derivative. Oxidation of this compound with chromic acid affords the ll-keto-21-acetoxy derivative or Compound A acetate. Hydrolysis of the acetate can easily afford one of the naturally occurring adrenal hormones, Kendall's
by
S Compound A or the desoxycorticosterone.
11-keto
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derivative
of
Compound S (17a,21-dihydroxy-4-pregnene-3,20-dione) is converted to the lla-hydroxy derivative, also called epi-F. The latter compound was converted to the 21acetate and the lla-hydroxy group oxidized with chromic acid to the 11-ketone, Compound E acetate (cortisone acetate). Hydrolysis then produces cortisone (I) in good yields. Interesting byproducts in small amounts were isolated when Rhizopus niqricans was the microorganism. Progesterone (III) yields lla-hydroxy-5a-pregnane-3;20dione, while use of Compound S yields lla,17 -dihydroxy5B-pregnane-3,20-dione. The 6B,lla,l4a-trihydroxy derivative is also produced. An interesting and unusual transformation was that of 16-dehydroprogesterone by Rhizopus niqricans. The product formed is lla-hydroxy-17-iso-progesterone, where reduction occurs at the 16,17-double bond and a unique 17a-side chain is formed. The normal side chain has one 178 configuration. When 36-hydroxy-5a-pregnan-20-one was incubated with Rhizopus arrhizus, the 78-hydroxy derivative was isolated. In addition, pregnenolone was conv.erted by the same fungus to 36,11a-dihydroxy-5-pregnene-7,20dione. From a similar transformation using Rhizopus niqricans, 36,76-dihydroxy-5a-pregnan-20-one was formed. Instead of the expected 66-hydroxylation, species of . Rhizopus have produced 76-hydroxy steroids with pregnenolone. It seems reasonable to ascribe this to the influence of the 36-hydroxy group or of the 5,6-double bond, since with neither the 3-keto-pregnenes nor 3-ketoallopregnanes was there any evidence of 7-hydroylation. The 9a-hydroxy derivative of desoxycorticosterone was formed when the latter substrate was fermented with Helicostylum piriforme. When Compound S (17ahydroxydesoxycorticosterone) was used as the substrate, the product appeared to be the 8B-hydroxy derivative. Rhizopus niqricans transformed 19-nortestosterone to the lO@hydroxynortestosterone in small quantitites, as well as to the expected llaand 6B-hydroxy derivatives. Hydroxylation at carbon-14 was achieved when it was found that genera of the Mucorales, v&, Mucor, Cunninqhamella, and Helicostylum could carry out 14ahydroxylation of such steroids as progesterone, desoxycorticosterone, Compound S, and testosterone.
14
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The 15a-hydroxy derivative was formed dissimilation product of progesterone (III) with Penicillium urticae.
as a incubated
Tertiary hydroxylation at carbon-17 was achieved along with lla-hydroxylation when progesterone (II) was incubated with Cephalothecium roseum. Since the 17ahydroxyl group can be efficiently introduced by chemical methods, there is no advantage in using the microbiological method in this case. The introduction of the 1,2-double bond in steroids by Septomyx affinis is unique in that it is the only microorganism that accomplishes this when the substrate has a saturated ring A. All other dehydrogenating microorganisms introduce both the l,?-and 4,5-double bonds simutaneously when exposed to ring A saturated steroids. These types of reactions were discussed earlier in connection with the preparation of more potent anti-inflammatory steroids. Using progesterone as the substrate, Streptomyces lavendulae and Septomyxa affinis affords the following sequence of degradation, namely, 1-dehydroprogesterone plus 1-dehydroandrostenedione plus ldehydrotestololactone. Penicillium lilacinum metabolizes progesterone successively to the 206hydroxy derivative, testosterone, androstenedione, and testololactone, of which testosterone and androstenedione were readily lla-Hydroxyprogesterone (IV) was interconvertible. transformed in a similar manner. When progesterone was incubated with Gliocladium cantenulatum, 66-hydroxyandrostenedione was obtained as This unique reaction was the well as androstenedione. only one of this type reported at that time (1963). In addition, progesterone can be converted to only 66hydroxyprogesterone by Penicillium urticae. Furthermore, androstenedione was transformed to 66hydroxyandrostenedione as well as to llahydroxyandrostenedione by Rhizopus nigricans. Septomyxa affinis degrades the side chain of the saturated ring A steroids Sa- or 5B-pregnane-3,11,20trione to a 17-ketone but in addition introduces only the 1,2-double bond. Gliocladium 2, Penicillium, and Asperqilli convert many of the C-21 steroids such as progesterone and Compound S to androstenedione as well as to hydroxylated steroids and testololactone.
S
15
=EDEOIDrn
The mechanism of side-chain degradation has been investigated. It was found that Cylindrocarpon radicicola oxidized progesterone to androstenedione and With some modification, this pathway is testololactone. generally used by microorganisms. The reduction of 3-keto-bisnor-4-cholen-22-al was accomplished with Penicillium lilacinum and Rhizopus niqricans to the corresponding 22-01 derivative. However, with the latter fungus, lla- as well as 6Bhydroxylation also occurred. The difficulty of isolating adequate amounts of the adrenal, estrogenic and androgenic hormones after their early discoveries impressed me profoundly when I worked in this field in the early 1930s. It was possible to isolate only a few milligrams of each from large quantities of tissue or tissue fluids. In contrast today, large amounts of each estrogenic hormone can be easily prepared from adequate amounts of starting material using microbiological methods! Preparation of the androgenic hormones is easily achieved in high yield using mirobiological, enzymatic conversions of readily available substrates. Androstenedione and testosterone were prepared by the author and associates (12) from several C-21 steroids, such as progesterone, by incubation with Asperqillus, Penicillium, and Gliocladium. Byproducts such as 6f3-hydroxyandrostenedione and testololactone were also produced. The preparation of the estrogenic been accomplished using microorganisms. A is aromatic in these hormones.
hormones and derivatives It should be recalled
has also that ring
Using 19-nortestosterone as the substrate and Septomyxa affinis the fungus, estrone was obtained in 90% yield and estradio? inmeld. Spontaneous aromatization occurs ater the 1,2-double bond has been introduced.
as
The 2-methyl and 4-methyl derivatives of estrone was obtained when E-methyl and 4-methyl-19-nortestosterone were incubated with Septomyxa affinis. Reduction of these compounds with sodium borohydride produced the E-methyl and 4-methyl estradiols, respectively, in high yields. An interesting dehydroandrostenedione
conversion was that of progesterone and 1-dehydrotestosterone using
to lFusarium
solani.
Preparation of 19-hydroxycorticosterone was achieved in the following manner. By the action of the fungus Cunninqhamella blaskesleeana, 19-hydroxydesoxycorticosterone* was converted into a crystalline compound interpreted to be 19-hydroxycorticosterone. Contrary to expectations, the latter compound gave a mixture of products, from which as the main component the crystalline llB,19,21triacetate was isolated. Benzoylation of 19-hydroxycorticosterone gave the crystalline 19,21-dibenzoate (20).
The mechanism of lla-hydroxylation at C-11 of steroids was investigated with Hayano et al (21) by one method and with Corey -et al (22) by a another method. %5110 et al showed that there is a direct exchange of a hydroxyl group with lla-3H,12a-3H (VIII). The hydroxylated derivative, lla-hydroxypregnane-3,20-dione-12a-3H (IX), was prepared by incubation of VIII with Rhizopus nigricans. Furthermore, progesterone-lla-3H (X) was'converted by the adrenal enzymes to The second method corticosterone-lla-3H (XI) without loss of tritium. described similar results using pregnane-3,20-dione-9a,lla-d2 (XII) and pregnane-3,20-dione-llT3-d (XIII) as the substrates. Derivatives XII and XIII were converted to their respective lla-hydroxy compounds XIII and XIV using the same microorganism as above, namely, Rhizopus niqricans. The loss of deuterium was then measured in the transformation of Compound XII to XIII. These results also indicated a direct replacement of the hydrogen at C-11 with the a-hydroxyl group. The origin of,the hydrogen in the.hydroxyl group is unknown. In spite of the exciting hope that cortisone and other antiinflammatory steroids would cure rheumatoid and osteoarthritis, unforseen serious side effects occurred and this goal was never achieved. However, these drugs still remain an important armentariumn in medicine for the treatment of the many medical conditions mentioned earlier. With the great number of unusual different and new chemical reactions described here and elsewhere performed by enzymes of many microorganisms on steroids, one cannot help but be impressed with the fantastic ability of these tiny creatures to equal or even outdo the organic chemists in many respects in this field. The author would be derelict if he were not to mention the unique and fortunate privilege of having lived exactly during the golden age of science. There will undoubtedly be many important discoveries in the future, but I doubt whether there will be as many during such a small span of history. One can cite the following advances as fantastic developments and achievements during this period: the airplane, electricity, radio, video movies, the telephone and telegraph, the structures of important natural products such as vitamins and hormones, radar landing of planes, jet engines, antibiotics starting with penicillin, various physical and chemical methods, development of atomic energy for fuel and for bombs, a vaccine for poliomyelitis, various new treatments for cancer and other diseases such as bypasses for the heart, heart replacements, genetics, space travel to the moon and launching of various satellites for communications, television, and--last, but not least--computer technology. ACiNOWLEDGEMENT For the opportunity and privilege of being able to carry out the microbiological approach to the solution of the cortisone problem, I am grateful to D. I. Weisblat who directed the successful project with his assistant, R. H. Levin.
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REFERENCES ::
3. 4. 5. 6. ii: 1’0: 11. 12. :::
15. 16. 17.
18. 19. 20. 21. 22.
Koch, F.C. and Gallagher, T.F., J. BIOL. CHEM. 84, 495 (1929) Allan, E. and Ooisy, E.A., JAMA 51, 819 (1923). Peterson, O.H., J. EIOL. CHEM. Il9, 185 (1937). Peterson, O.H., J. EIOL. CHEM. =,695 (1937). Hench, P.S., Kendall, E.C., Slocumb, C.H. and Polley, H.F., PROC. MAYO CLINIC 24; 181 (1949). Peterson, D.H. and Reineke, L.M., J. BIOL. CHEM. I8l, 95 (1949). Peterson, D.H. and Reineke, L-M., J. EIOL. CHEM. 72, 3598 (1950). Peterson, D.H., RESEARCH6, 309 (1953). Sarett, L.H., J. EIOL. CHEM. I&, 601 (1945). Hechter, O., Jacobsen, R.P., Jeanloz, R., Levy, H., Marshall, C.W., Pincus, G. and Schenker, V., ARCH. EIOCHEM. I.5, 457 (1950). Zaffaroni, A., Burton, R. 8. and Keutmann, E. H., SCIENCE 111, 6 (1950). Peterson, D.H., in: Industrial Microorganisms (Rainbow, C. and Rose, A.H., Editors), Academic Press, London (1963), pp. 537-606. Heyl, F.W. and Herr, M.E., J. AM. CHEM. SOC. 72, 2617 (1950). Peterson, D.H. and Murray, H.C., J. AM. CHEM. SOC. 71, 1871 (1952). Mancero, O., Zaffaroni, A., Rubin, B.A., Sondheimer, F., Rosenkranz, G. and Djerassi, C., J. AM. CHEM. SOC. 74, 3711 (1952). Murray, H.C., and Peterson, D.H., inventors; The Upjohn Co., assignee. Oxygenation of steroids by Mucorales fungi. U.S. Patent 2,602,769. 1952 July 8. Innovators, The. New Processes Impact Sometimes Can Match That of New Products. Successful Search for Way to Synthetic Cortisone was Key to Medical Gain. The Wall Street Journal, 1952 Jul. 9: Sect. 1 (col. 1). Gottschalk Jr., E., in: The Innovators (Wall Street Journal, Editor), Dow Jones and Co. Books (1968), pp. 76-88. Engel, L., in: Kalamazoo (Leonard Engel, Editor), McGraw Book Co., New York (1961), pp. 128-155. Barber, G.W., Peterson, D.H. and Eherenstein, M., J. ORG. CHEM. 25, 1168 (1960). Hayano,M.,Gut, M., Dorfman, R.I., Sebek, O.K. and Peterson, D.H., JACS 80, 2336 (1958). Corey, E.J., Gregoriou, G.A. and Peterson, D.H., JACS 80, 2338
(1958).
H. Peterson,
Ph.D.
It was the winter of 1917. I was eight years old, confined to a bed with rheumatic fever in the cold, drafty hayloft of the newly built barn. Our parents and six of their eight children had moved that fall with two lumber wagons and our horses and cows 50 miles across the prairie from a homestead in arid country to a new farm in the irrigated land north of Denver. Our house would not be built until the following summer. As I listened to the wind, felt the cold, and heard the horses and cows below us, I thought, "When I grow up, I'll never be a farmer." become a scientist, part of the Never did I dream that some day I would "Golden Age" of science. Insufficient rainfall, hail, and poor crops seemed to follow us from farm to farm. We were taught early on how to contend with the hazards of nature--how to shoot a gun to kill coyotes that preyed on our livestock, as well as how to shoot rabbits and pheasants for food. I also trapped muskrats and skunks for their fur. We moved six times when I was a child, and I had to adjust to six different grade schools. While my parents, both immigrants from Sweden, never financial success, they gave me something money couldn't philosophy of life that included hard work and honesty. especially, felt that there was always a way to accomplish using one's physical and mental resources.
January 1985
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achieved great buy--a My mother, a goal by
Volume 45, Number 1
2
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In 1923, my father gave up farming, moved the family to Denver, and found work as a carpenter in the railroad yards repairing box cars, providing for us the bare necessities of life. Each of us children found jobs outside our home to cover personal needs. Changing from a country school to the high school in a big city was a difficult Because I was so interested in chemistry and did well adjustment. during my junior year, my chemistry teacher, Mr. Beattie, appointed me his assistant in the chemistry lab during my senior year. It was he who encouraged and inspired me to continue my interest in chemistry by studying at The University of Denver under Dr. R. G. Gustavson, who we affectionately called "Gus" and who proved to be an inspiring and stimulating professor. Because I did well in chemistry at the university, I was made a chemistry assistant in my junior and senior years and head chemistry assistant the year I did my Masters work. "Gus" became like a second father to me. My major hobby during high school and college was baseball. I was an all-conference pitcher for three years and helped win four consecutive championships, as well as playing semi-professional baseball during the summer. The latter served to help me financially through college. After receiving my Master's degree in 1932, I was awarded a buition scholarship to The University of Chicago. It was during the depression, and outside jobs were essentially nonexistent. "Gus, " along with other professors, collected a $500 loan to pay for room, board, and expenses. My graduate work was done under Dr. F. C. Koch, then the world authority on the sex hormones, paticularly the male hormone. Financial problems for my existence required that I spend over one-half of my time on and off the campus on jobs that paid 501# an hour. One of my jobs was working in a gold mine at 12,000 feet above sea level in Colorado in exchange for my baseball ability. However, the five years I spent at the university also had its virtues. I was exposed to some of the outstanding professors in the world from 1932-1937; namely, Koch, Carlson, Moore, Steiglitz, Karasch, and others. LIFE MAGAZINE of 1934 rated The University of Chicago as having an outstanding faculty equal to any in the world. Aside from this exposure, other important conditions that greatly influenced my life were present during and directly after my five years on the campus. No reports were given at the end of a course--only if one flunked. One did not have to attend classes; however, I found it stimulating to do so. The program developed an independence that rapidly placed one on his own. After receiving my Ph.D. in 1937, I began working for Bauer & Black in Chicago; and this allowed me to continue part time research at the university. This also gave me the opportunity to observe the presence of at least eight Nobel Prize winners in a program that I didn't recognize at the time as "Development of the Atomic Age." Among these workers were Fermi, the Italian Nobel Laureate in Physics, who was really responsible for the key experiment that triggered the atomic age. He harnessed atomic energy by building a carbon brick pile in a handball court under the stadium where I had played games many times. The
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stadium is long gone, but the laboratory site contains a famous memorial indicating the birth of the atomic age. Fermi's work was absolutely necessary for the development of the atomic bomb at Los Alamos, New Approximately 20 years later, Fermi died of leukemia due to Mexico. excess radiation exposure. R. I.
At the university, I shared a laboratory Dorfman, both outstanding in the field
with T. F. Gallagher of steroid metabolism.
and
The significance of steroids as hormones became important when F. C. Koch (1) discovered the presence of the male hormone in bull testes, while Allan and Doisy (2) first found the female hormones in body fluids. It is interesting that I am one of the few earlier workers remaining who had the unique distinction of working on the steroids before their structures were elucidated in 1933. Although my B.S. and M.S. were obtained in Chemical Engineering, my great interest in biochemistry was spurred by "GUS" and my thesis was concerned with "The Route of Excretion of the Female Sex Hormone in the Hen." One of the routes was through the bile duct. A conclusion was never reached because, on the addition of an active concentrate from human pregnancy urine, most of the problem was concerned with the development of a good method of extraction from chicken bile. My Ph.D. thesis consisted of two parts: "I. The Effect of Acid Hydrolysis on the Yield of Androgenic and Estrogenic Substances from Human Urine (3); II. The Daily Urinary Excretion of Androgenic and Estrogenic Substances from Human Urine in Normal Men and Women" (4). Such studies required the use of capons in the case of androgens and ovariectomized rats in the case of estrogens. As a result, I performed approximately 7,000 operations during my research in order to sustain a sufficient number of rats and capons. Each assay required one week, and these studies took three years to complete. In Part I, it had been known for some time that it was necessary to boil human urine with acid before the estrogenic material could be recovered with immiscible solvents. This work showed that a shorter acid hydrolysis period of 15 minutes released the maximum androgenic activity. Further hydrolysis resulted in lower yields. Since urine without acid treatment does not yield any androgenic activity using immiscible solvents for extraction, it was concluded that this activity In 1938, I was able to extract a is excreted as an inactive complex. concentrated inactive complex using N-butanol from unhydrolyzed urine. I concluded that the complex was a glucuronide of the male hormone constituents. This concentrate could then be boiled with hydrochloric acid to produce androgenic material extractable with benzene. It was at this point that I became interested in microbiological In attempting to find out if the complex was conversion of steroids. an a- or B-glucuronide, an incubation mixture became contaminated with an unknown microorganism; and the resulting ether extract was more active in androgenic activity than an acid hydrolyzed sample from the
same concentrated glucuronide complex. This led me to believe that the androstenedione was converted to testosterone by reduction to the 17BThe latter compound was never hydroxyl group that is testosterone. isolated in human urine but was considered to be the natural hormone found in human blood at that time and produced by the testes. My program to continue to study the microbiological transformation of steroids was interrupted for the next 11 years. I was then able to resume this project at The Upjohn Company, after the significance of the cortical hormones became apparent in the rheumatoid arthritis study done at The Mayo Foundation by Wench and Kendall 1949 (5). The second part of my thesis, "The Daily Urinary Excretion of Estrogenic and Androgenic Substances by Normal Men and Women," consisted of a long and extensive study on urine from four normal men and four From this normal women and collected for 27 to 45 consecutive days. study, it was shown that there are marked fluctuations in the daily urinary excretion of androgens and estrogens in normal men and women. There is no evidence to support a monthly cycle in the excretion of either androgens or estrogens in normal men. In normal women, the excretion of estrogens is characteristically low during the menstrual flow and rises during the intermenstruum, with a double peak in certain instances. The average daily excretions of estrogens, calculated as gammas of estrone, are 9-12 for men and 18-36 for women. The rates of excretion of both hormones do not seem to bear any relationship to each other in either sex. Following my graduate work at The University of Chicago I joined Bauer & Black as a biochemist and developed the use of nylon as a nonabsorbable surgical suture in 1941. Nylon produces no tissue reactions and is processed as either twisted, braided, or single filament form. The latter is especially good for skin and other special suturing purposes and is still used by surgeons today. In 1943, I decided to join Raymond Laboratories in St. Paul, Minnesota, in a venture to initiate the development of drugs and enter the pharmaceutical field. Instead, we were successful in the cold hair waving business with a product called "Toni." I received a patent for the use of potassium bromate as a setting solution. About one-half of my time was spent on this project and the other half on control work for the Army in World War II on signal shells of various colors for airport landing and takeoff of military planes on dark airfields. Our company also made one plastic part for the atomic bomb. Failure to develop drugs for the pharmaceutical industry after three years led me to join G. F. Cartland and his group at The Upjohn Company in 1946 to work on antibiotics. This was an exciting new field, following the dramatic development and production of penicillin. My first contribution was the isolation and purification of a polypeptide type of antibiotic called Circulin (6), which contained leucine, threonine, a,b-diaminobutyric acid, and an optically active pellargonic acid. The components were followed by chromatographic paperstrip techniques based on previous work with the amino acids.
Separation of the components were realized by using powdered cellulose and a solvent, a mixture of 25% acetic acid:Xl% N-butanol:H20--as was used in the paperstrip technique. Since the solvent had to move slowly this chromatography required over the column for proper resolution, three months to resolve and to obtain the pure components. My most important contribution in this field was the first successful development of a paper chromatographic technique for differentiating the streptomyces type of antibiotics using solvents similar to the ones used for separation of amino acids mentioned above (7). Using this mixture of solvents and others, this technique has long been in use by all laboratories and today still forms the basis in the This discovery was important also since screening of new antibiotics. it formed the idea for following micro amounts of steroids, when I later became active in the cortisone field. During World War II, U.S. intelligence sources picked up a rumor that Nazi Luftwaffe pilots were being "hopped up" with a fabulous drug that enabled them to function more efficiently aloft. The Nazis supposedly were buying large quantities of adrenal glands of cattle from Argentina and extracting them to give the airmen a drug with these unusual powers. Eight years earlier, cortisone and the more active hydrocortisone were isolated in pure form, along with other different biologically active steroids, from bovine adrenals by Reichstein, Mason et al, Kuizenga and Cartland, and Wintersteiner and Pfiffner (8). The emphasis shifted to the Mayo Clinic where a physician, Dr. Hench, noticed that women with severe rheumatoid arthritis had remissions during pregnancy. Hench,working with Kendall, knew that the adrenal glands produced large amounts of these hormones during pregnancy, so he asked Sarett of the Merck Company for cortisone (I), now called the "Wonder Drug." Based on the earlier U.S. government request and support (during the Nazi rumor period), Sarett had worked out a 35-step chemical process for making the drug from desoxycholic acid obtained from cattle bile (9). In order to move the oxygen at carbon-12 to the biologically it was necessary to carry out many required oxygen at carbon-11, chemical steps at a high cost. The process ended up with a very small yield since the the procedure consumed so many steps. Hench then asked Merck for enough cortisone to treat a 29-year-old woman (who looked 50) with rheumatoid arthritis. This patient had stiff, swollen joints that were very tender and could walk only with a She was bedridden almost all the time with weird shuffling gait. After a one-week treatment by Hench, not only could she severe pain. walk normally but she even left the hospital for a three-hour shopping spree! With great excitement, Hench attempted to obtain more cortisone from Sarett; but none was available. Sarett renewed his supplied the Mayo Clinic
bile acid syntheses and in several months with enough cortisone to treat 14 other
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rheumatoid arthritics. In April 1949, Hench revealed the results and showed an audience of physicians who sat astounded as they watched before and after films of the patients given cortisone (5). These severely crippled people walked up and down stairs after treatment and even danced. The dancing arthritics made headlines all over the world, and a sudden surge of demand arose for cortisone. However, the headlines were deceiving because there was no cortisone available for anyone except a few, well-chosen patients. The clinical work at Mayo triggered some of the most intensive and Every extensive chemical and biological research work ever undertaken. major drug company and many university laboratories all over the world became interested in joining the adventure to find a satisfactory, economical industrial process for making cortisone. After assessing the great technical difficulties and Merck's lead, only a few firms, including The Upjohn Company, continued in the race. Mr. Gilmore, president of Upjohn, gave this notice to the company's scientists: "Don't spare the horses; we want cortisone!" A blow torch was the symbol chosen to keep the scientists busy and alert. Six different approaches were pursued by Upjohn researchers in an attempt to solve this important problem; namely: (a) a chemical approach using ergosterol as the starting steroid; (b) a chemical approach involving some of Sarett's methods; (c) a chemical approaeh using total synthesis; (d) investigating the presence of naturally occurring 11-oxygenated raw materials in seeds; and (e) the microbiological approach initiated by myself, together with H. C. Murray, a microbiologist. Our approach was considered a long-term possibility and was given the least chance for success. The dramatic clinical findings by Hench and Kendall had opened the way for one of the most fascinating episodes in the history of medicine and chemistry. Since that time the use of cortisone and hydrocortisone and other active derivatives has been extended to many other inflammatory diseases such as rheumatic fever, asthma, eye infections, ulcertive colitis, Addison's disease, and various allergies. Based on the biological law that "ontology recapitulates phylogeny," it was my idea that lower microorganisms might also contain enzymes similar to the adrenal glands of humans for making cortisone and hydrocortisone. With this in mind, I suggested the use of the microbiological approach as a solution to introducing the biologically required oxygen at carbon-11. It was this step in particular that required the chemist to carry out the difficult and lengthy chemical steps to make cortisone (I) and hydrocortisone (II). My proposal was questioned by many outstanding organic chemists who believed it couldn't be done. My response was that "the microorganisms do not know this" and experiments would have to be done to determine the answer. The first Hechter et al,
biological synthesis of hydrocortisone was that who perfused 17a-hydroxy-11-desoxycorticosterone
of through
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Hydrocortisonc
(I)
Figure
(II)
1
beef adrenal glands and obtained hydrocortisone. It was obvious that this method could not be economical and I had already initiated the microbiological appproach when this work was published in 1950 (lo). I firmly believed we should not proceed with any work of this until a micromethod of following the transformation of the steroid was available, sensitive to 10 mcg or less. It becomes obvious that the Project must be designed as a deliberate attempt to introduce the strategic oxygen at carbon-11 in a single step.
natWe
The author, together with H. 11, 1949. I attempted to develop micro afIIOUntS Of steroids, when a Zaffaroni (11). We contacted him essentially his method.
C. Murray, began this work on November a paperstrip method for identifying publication in this area appeared by and returned to Kalamazoo using
Our first successful microbiological conversion involved the transformation of desoxycorticosterone to a 20EGhydroxy derivative by Reichstein's reduction of the 20-ketone group using a Streptomyces x. earlier work had involved five steps to prepare this compound (12). Since Hey1 and Herr (13) had developed an economical method for Murray and I first making the progestational hormone, progesterone, looked at this material as a potential substrate. An agar plate placed in a window sill of the oldest and dirtiest laboratory at The Upjohn Company by Murray yielded a Rhizopus so of a fungus which was then Five milligrams of the latter was incubated with progesterone. fermented in a test tube containing 20 ml of medium in February 1950 for 24 hours. The methylene chloride extract was concentrated and an equivalent of 10 gamma examined by paperstrip chromatography. The uvsensitive spots showed that the progesterone had almost disappeared. However, larger amounts of a slower-moving compound as well as an even The latter eventually proved to slower-moving component were present. be 68,lla-dihydroxyprogesterone. This fermentation was scaled up to 500 mg of progesterone as a After papergram analysis showed a change similar to that of substrate. methylene chloride the screening sample described above, a concentrated extract was chromatographed over an aluminum oxide column and the
fractions examined by papergram analyses. The various fractions were crystallized and the prevailing fraction (next to the slowest moving one) furnished physical constants different from progesterone and different from 116-hydroxyprogesterone. This compound was acetylated and found to have a single hydroxyl group. Oxidation of the hydroxyl group produced a ketone derivative, 11-ketoprogesterone, identical in all respect with the physical constants reported in the literature and identical with known ll-ketoprogesterone obained from T. F. Gallagher of studies. the Sloan-Kettering Institute in ir, uv, and X-ray diffraction This meant that progesterone (III) had been enzymatically converted to a new compound, the previously undescribed lla-hydroxyprogesterone (IV) , as shown in Figure 2 (14).
CH3 I c=o
Progesterone
CH3 I c=o
H%,, 9xP 0 ’ Ila-Hydroxyprogesterone
(III)
Figure
(IV)
2
The introduction of oxygen at carbon-11 by a microorganism was actually achieved in five months, even though the structure was not definitely established for another two months. Obtaining a solution to such a major project was a remarkable accomplishment and it was not due to luck alone. The deliberate attempt to solve this problem involved carefully planned microtechniques used in an orderly fashion, thus speeding up the whole project. This set the stage for an integrated study involving of the microbiological step and a variety of new chemical challenged the organic chemists to an architecturally and sound solution to the production of cortisone and all the naturally occurring anti-inflammatory steroids, including superior analogs.
a combination reactions that economically other new and
After a tremendous amount of work, an integrated and successful process was capably accomplished by J. A. Hogg and his colleagues. For the microbiological conversion resulting in the one-step introduction of oxygen at carbon-11, the author received the Upjohn Prize in 1951 and Outstanding Alumni Awards in Science from The University of Chicago in 1976 and The University of Denver in 1983. Seventy-four patents were issued and 54 scientific articles published the extensions of this work.
on
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It is important that this microbiological conversion proved the concept that substitution of an inactivated carbon atom could be The value of achieved by an enzymatic attack using a microorganism. microorganisms in chemical synthesis thus was dramatically enhanced. This success triggered massive programs in which many thousands of molds and bacteria were tested for their usefulness in the modification of As a result, many types of oxidation and reduction various steroids. reactions, as well as combinations of such changes, were discovered. It also spurred similar investigations of other substances using microbiological conversions and many reports have since been published on straight chain and cyclic hydrocarbons, aromatic terpenes and alkaloids as well as pesticides and antibiotics. A formal announcement of the oxygenation of steroids was made in a note sent to J. AM. CHEM. SOC. on February The CHEMICAL ENGINEERING NEWSedition following announcement:
of April
at carbon-11 14, 1952 (14).
7, 1952,
made the
"In the neatest trick of the year Upjohn has come up with a single-step process for the microbiological oxidation of steroids at carbon 11. Two Upjohn scientists, D. H. Peterson and H. C. Murray, announced the discovery in the April 5, 1952, J.A.C.S. A variety of steroids may serve as starting material for conversion to the cortical hormones such as cortisone and possible Compound F (hydrocortisone) and Compound B (corticosterone). Using common molds the sex hormone, progesterone, is transformed to llahydroxyprogesterone, only a few steps away from cortisone etc., etc . 1' The importance numerous scientists published in almost
of this discovery was not only acknowledged by and drug companies but the announcement was every newspaper of any size in the world.
It was interesting that this announcement was made at the Gordon Conference at Mt. Tremblant, Canada, in 1953 and the author was inadvertently assigned Cabin 11. To top this off at the next year's meeting, I announced the microbioloical introduction of the 17a-hydroxy group and was assigned Cabin 17, an absolutely incredible occurrence! In order to reduce the cost of production of all these cortical hormones by microbiological means, it was necessary to introduce the remaining necessary chemical groups such as the 17a-hydroxy and 21hydroxy groups. This was accomplished by several organic chemists at The Upjohn Company, led by Dr. John A. Hogg and coworkers. The whole character of the Upjohn project and the company changed immediately, and the emphasis shifted to the microbiological approach for the production of hydrocortisone. Approximately 150 scientists and assistants zeroed in on the venture. The necessary
patent
applications
were prepared.
The first
to
appear was a 1951 South African patent specification. Interestingly, the latter was noticed by the Syntex Corporation quite early and this group then used our fermentation techniques to prepare cortisone in relatively fewer reactions. The latter was published in 1952 (15). However, the Upjohn research group (although not published) had proceeded in a similar fashion and was in pilot plant production in late 1951. Before the Syntex publication appeared, an enormously detailed and classical U.S. patent issued July 8, 1952, to Murray and Peterson (16). This patent was written in the style of J. AM. CHEM. SOC. and contained a variety of substrates and hydroxylated steroid products using the molds Mucoralis sp for conversion. The Franklin Pierce Law Center conducted a study for the National Science Foundation in 1974 and concluded that this patent was one of the A publication by most significant technological advances in 25 years. Chemtech in November, 1978, published the entire patent as one of the most outstanding, well-written, significant, and unique examples in the On July 9, 1968, the Wall Street Journal (17) covered this industry. work under the title of "Innovators." This, along with the work of other innovators, was highlighted in a book in 1968 (18). The history of this important microbial conversion is also covered in detail in a book by Leonard Engel (19). For the introduction of the necessary oxygen at carbon-11 by the mold to be commercially successful, the process had to be financially feasible. To accomplish this: (a) the substrate must be readily available and economically produced; (b) the reaction must proceed in high yield; (c) the substrate level must be high; and (d) the product must be obtained and purified in high yields. All of these requirements were fulfilled by this transformation. One of the most interesting aspects of such conversions is the fact that steroids are ordinarily essentially insoluble in aqueous media. It is for this reason that early workers in this field were undoubtedly discouraged. The steroid substrates can be added to the culture medium in very small amounts of acetone, where the solvent plays no'role, or as a micronized solid in large concentrations. Small amounts of the substrate are apparently adsorbed to the surface of the cell where enzymatic conversion occurs. The converted product then moves into solution and is replaced by the substrate. More substrate moves to the surface of the cell, etc., etc. Microbial enzymes effect a wide variety of chemical changes in steroids. These changes include the introduction of nuclear and sidechain hydroxyl groups; cleavage of carbon-carbon linkages in the side chain as well as in rings A and D; dehydrogenation of ring hydroxyl groups as well as rings A and 8; opening of an oxide ring; reduction of ring double bonds and of ketone groups on a ring or side-chain; hydrolysis of steroid esters as well as many combinations of the above reactions. Of these changes, the most common one is hydroxylation, and only the 18-C atom has not yet been hydroxylated. Our laboratory has hydroxylated eleven positions: 6D-, 7D-, 8D-, 9a-, lo&, lla-, ll&, 14a-, I5a-, I56-, 17a-; while other laboratories have reported
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la-, 26-, 7a-, 12b-, 19-, and 21-. In tissues are able to effect hydroxylations at 6a-, 66-, llb-, 17a-,18, 19, 20, and from other tissue at 2a-, 2B-, 6a-, 6B-, 75-, and 16a-. The range of positions affected may be seen by inspection of Figure 3. hydroxylation at six positions: contrast, enzymes from adrenal
Figure
3
Although these changes are prominantely fungi, some reactions take place to a lesser actinomycetes, and even protozoa.
carried extent
out by filamentous by bacteria, yeasts,
The rapid development and progress in this field was due mainly to the use of microtechniques such as paper chromatography, uv light, and ir spectroscopy. Paper chromatography was especially useful since it allowed the rapid examination of a wide variety of reactions by many microorganisms within a short period of time. The general methods used consisted of the following sequence of steps: (a) the microorganism is first grown under suitable aerobic conditions in submerged media for 24-48 hours; (b) the steroid to be tested is then added in a water-soluble solvent such as acetone; (c) after a conversion period of several hours to several days (average about 24-72 hours), the transformation products are recovered in pure form by extraction with a water insoluble solvent, purification, using direct crystallization or column chromatography or countercurrent distribution; (d) structure work by the usual methods of classical organic chemistry, viz: by conversion to known structures. In some cases the use of microbial methods has also been most useful in structure work. In a continuing effort to increase the anti-inflammatory activity and decrease the side effects of corticosteroids, it was found by several workers that the introduction of the 1,2-double bond did indeed accomplish this goal. In our laboratory we used Septomyxa affinis to introduce the 1,2-double bond into anti-inflammatory steroids, which are sold by The Upjohn Company, namely, prednisone (V), prednisolone (VI), and methylprednisolone (VII), products used extensively in the medical field, Figure 4.
S
12
TBEOltDI
CH20H
CH20H iH20H
o&H
o&H
o#H
I= CH3 Prednisone
(VI
Prednisolone
Figure
(VI)
Ga-Methylprednisolone
(VII)
4
After the important discovery of microbiological introduction of oxygen at carbon-11 in a single step, a whole host of biological transformations were conducted in our laboratories both before and after the extension of these findings by other workers who discovered similar and additional important and interesting reactions. In almost all cases, the steroids formed were new compounds. However, these will not be discussed since the purpose of this manuscript is to describe my own contributions to this field. A particularly extensive study was carried out on the IIa-hydrofty compounds and other products derived from numerous steroid substrates Rhizopus nigricans and Rhizopus arrhizus q. These transformations
are briefly
described
below
(12).
Progesterone (III) is converted by Rhizopus arrhizus to lla-hydroxyprogesterone (IV) as well as to 68,lladihydroxyprogesterone in rather large amounts. It was then found that Rhizopus nigricans with progesterone (III) produced essentially lla-hydroxyprogesterone (IV) with traces of 66,lla-dihydroxyprogesterone and, in addition, 76-hydroxyprogesterone. Rhizopus nigricans also converts Compound S (17a,21-dihydroxy-4-pregnene3,20-dione), desoxycorticosterone, 17ahydroxyprogesterone, 3-keto-bisnor-4-cholen-22-01, 16dehydroprogesterone, 16a,l7a-oxidoprogesterone, pregnane3,20-dione, allopregnane-3,20-dione, androstene-3,17dione, testosterone, 17a-methyltestosterone, and 19nortestosterone to the lla-hydroxy as well as to the 66hydroxy derivatives and in the C-21 steroidal substrates mentioned above, to the 6B,lla-dihydroxy derivatives. As one can obseve, many variations occur. Oesoxycorticosterone, as indicated above, is converted to the lla-hydroxy' derivative. Selective acetylation of the product results in the El-acetoxy derivative. Oxidation of this compound with chromic acid affords the ll-keto-21-acetoxy derivative or Compound A acetate. Hydrolysis of the acetate can easily afford one of the naturally occurring adrenal hormones, Kendall's
by
S Compound A or the desoxycorticosterone.
11-keto
13
WDEOXDI
derivative
of
Compound S (17a,21-dihydroxy-4-pregnene-3,20-dione) is converted to the lla-hydroxy derivative, also called epi-F. The latter compound was converted to the 21acetate and the lla-hydroxy group oxidized with chromic acid to the 11-ketone, Compound E acetate (cortisone acetate). Hydrolysis then produces cortisone (I) in good yields. Interesting byproducts in small amounts were isolated when Rhizopus niqricans was the microorganism. Progesterone (III) yields lla-hydroxy-5a-pregnane-3;20dione, while use of Compound S yields lla,17 -dihydroxy5B-pregnane-3,20-dione. The 6B,lla,l4a-trihydroxy derivative is also produced. An interesting and unusual transformation was that of 16-dehydroprogesterone by Rhizopus niqricans. The product formed is lla-hydroxy-17-iso-progesterone, where reduction occurs at the 16,17-double bond and a unique 17a-side chain is formed. The normal side chain has one 178 configuration. When 36-hydroxy-5a-pregnan-20-one was incubated with Rhizopus arrhizus, the 78-hydroxy derivative was isolated. In addition, pregnenolone was conv.erted by the same fungus to 36,11a-dihydroxy-5-pregnene-7,20dione. From a similar transformation using Rhizopus niqricans, 36,76-dihydroxy-5a-pregnan-20-one was formed. Instead of the expected 66-hydroxylation, species of . Rhizopus have produced 76-hydroxy steroids with pregnenolone. It seems reasonable to ascribe this to the influence of the 36-hydroxy group or of the 5,6-double bond, since with neither the 3-keto-pregnenes nor 3-ketoallopregnanes was there any evidence of 7-hydroylation. The 9a-hydroxy derivative of desoxycorticosterone was formed when the latter substrate was fermented with Helicostylum piriforme. When Compound S (17ahydroxydesoxycorticosterone) was used as the substrate, the product appeared to be the 8B-hydroxy derivative. Rhizopus niqricans transformed 19-nortestosterone to the lO@hydroxynortestosterone in small quantitites, as well as to the expected llaand 6B-hydroxy derivatives. Hydroxylation at carbon-14 was achieved when it was found that genera of the Mucorales, v&, Mucor, Cunninqhamella, and Helicostylum could carry out 14ahydroxylation of such steroids as progesterone, desoxycorticosterone, Compound S, and testosterone.
14
S
TDEOXDO
The 15a-hydroxy derivative was formed dissimilation product of progesterone (III) with Penicillium urticae.
as a incubated
Tertiary hydroxylation at carbon-17 was achieved along with lla-hydroxylation when progesterone (II) was incubated with Cephalothecium roseum. Since the 17ahydroxyl group can be efficiently introduced by chemical methods, there is no advantage in using the microbiological method in this case. The introduction of the 1,2-double bond in steroids by Septomyx affinis is unique in that it is the only microorganism that accomplishes this when the substrate has a saturated ring A. All other dehydrogenating microorganisms introduce both the l,?-and 4,5-double bonds simutaneously when exposed to ring A saturated steroids. These types of reactions were discussed earlier in connection with the preparation of more potent anti-inflammatory steroids. Using progesterone as the substrate, Streptomyces lavendulae and Septomyxa affinis affords the following sequence of degradation, namely, 1-dehydroprogesterone plus 1-dehydroandrostenedione plus ldehydrotestololactone. Penicillium lilacinum metabolizes progesterone successively to the 206hydroxy derivative, testosterone, androstenedione, and testololactone, of which testosterone and androstenedione were readily lla-Hydroxyprogesterone (IV) was interconvertible. transformed in a similar manner. When progesterone was incubated with Gliocladium cantenulatum, 66-hydroxyandrostenedione was obtained as This unique reaction was the well as androstenedione. only one of this type reported at that time (1963). In addition, progesterone can be converted to only 66hydroxyprogesterone by Penicillium urticae. Furthermore, androstenedione was transformed to 66hydroxyandrostenedione as well as to llahydroxyandrostenedione by Rhizopus nigricans. Septomyxa affinis degrades the side chain of the saturated ring A steroids Sa- or 5B-pregnane-3,11,20trione to a 17-ketone but in addition introduces only the 1,2-double bond. Gliocladium 2, Penicillium, and Asperqilli convert many of the C-21 steroids such as progesterone and Compound S to androstenedione as well as to hydroxylated steroids and testololactone.
S
15
=EDEOIDrn
The mechanism of side-chain degradation has been investigated. It was found that Cylindrocarpon radicicola oxidized progesterone to androstenedione and With some modification, this pathway is testololactone. generally used by microorganisms. The reduction of 3-keto-bisnor-4-cholen-22-al was accomplished with Penicillium lilacinum and Rhizopus niqricans to the corresponding 22-01 derivative. However, with the latter fungus, lla- as well as 6Bhydroxylation also occurred. The difficulty of isolating adequate amounts of the adrenal, estrogenic and androgenic hormones after their early discoveries impressed me profoundly when I worked in this field in the early 1930s. It was possible to isolate only a few milligrams of each from large quantities of tissue or tissue fluids. In contrast today, large amounts of each estrogenic hormone can be easily prepared from adequate amounts of starting material using microbiological methods! Preparation of the androgenic hormones is easily achieved in high yield using mirobiological, enzymatic conversions of readily available substrates. Androstenedione and testosterone were prepared by the author and associates (12) from several C-21 steroids, such as progesterone, by incubation with Asperqillus, Penicillium, and Gliocladium. Byproducts such as 6f3-hydroxyandrostenedione and testololactone were also produced. The preparation of the estrogenic been accomplished using microorganisms. A is aromatic in these hormones.
hormones and derivatives It should be recalled
has also that ring
Using 19-nortestosterone as the substrate and Septomyxa affinis the fungus, estrone was obtained in 90% yield and estradio? inmeld. Spontaneous aromatization occurs ater the 1,2-double bond has been introduced.
as
The 2-methyl and 4-methyl derivatives of estrone was obtained when E-methyl and 4-methyl-19-nortestosterone were incubated with Septomyxa affinis. Reduction of these compounds with sodium borohydride produced the E-methyl and 4-methyl estradiols, respectively, in high yields. An interesting dehydroandrostenedione
conversion was that of progesterone and 1-dehydrotestosterone using
to lFusarium
solani.
Preparation of 19-hydroxycorticosterone was achieved in the following manner. By the action of the fungus Cunninqhamella blaskesleeana, 19-hydroxydesoxycorticosterone* was converted into a crystalline compound interpreted to be 19-hydroxycorticosterone. Contrary to expectations, the latter compound gave a mixture of products, from which as the main component the crystalline llB,19,21triacetate was isolated. Benzoylation of 19-hydroxycorticosterone gave the crystalline 19,21-dibenzoate (20).
The mechanism of lla-hydroxylation at C-11 of steroids was investigated with Hayano et al (21) by one method and with Corey -et al (22) by a another method. %5110 et al showed that there is a direct exchange of a hydroxyl group with lla-3H,12a-3H (VIII). The hydroxylated derivative, lla-hydroxypregnane-3,20-dione-12a-3H (IX), was prepared by incubation of VIII with Rhizopus nigricans. Furthermore, progesterone-lla-3H (X) was'converted by the adrenal enzymes to The second method corticosterone-lla-3H (XI) without loss of tritium. described similar results using pregnane-3,20-dione-9a,lla-d2 (XII) and pregnane-3,20-dione-llT3-d (XIII) as the substrates. Derivatives XII and XIII were converted to their respective lla-hydroxy compounds XIII and XIV using the same microorganism as above, namely, Rhizopus niqricans. The loss of deuterium was then measured in the transformation of Compound XII to XIII. These results also indicated a direct replacement of the hydrogen at C-11 with the a-hydroxyl group. The origin of,the hydrogen in the.hydroxyl group is unknown. In spite of the exciting hope that cortisone and other antiinflammatory steroids would cure rheumatoid and osteoarthritis, unforseen serious side effects occurred and this goal was never achieved. However, these drugs still remain an important armentariumn in medicine for the treatment of the many medical conditions mentioned earlier. With the great number of unusual different and new chemical reactions described here and elsewhere performed by enzymes of many microorganisms on steroids, one cannot help but be impressed with the fantastic ability of these tiny creatures to equal or even outdo the organic chemists in many respects in this field. The author would be derelict if he were not to mention the unique and fortunate privilege of having lived exactly during the golden age of science. There will undoubtedly be many important discoveries in the future, but I doubt whether there will be as many during such a small span of history. One can cite the following advances as fantastic developments and achievements during this period: the airplane, electricity, radio, video movies, the telephone and telegraph, the structures of important natural products such as vitamins and hormones, radar landing of planes, jet engines, antibiotics starting with penicillin, various physical and chemical methods, development of atomic energy for fuel and for bombs, a vaccine for poliomyelitis, various new treatments for cancer and other diseases such as bypasses for the heart, heart replacements, genetics, space travel to the moon and launching of various satellites for communications, television, and--last, but not least--computer technology. ACiNOWLEDGEMENT For the opportunity and privilege of being able to carry out the microbiological approach to the solution of the cortisone problem, I am grateful to D. I. Weisblat who directed the successful project with his assistant, R. H. Levin.
S
TEEOXDI
17
REFERENCES ::
3. 4. 5. 6. ii: 1’0: 11. 12. :::
15. 16. 17.
18. 19. 20. 21. 22.
Koch, F.C. and Gallagher, T.F., J. BIOL. CHEM. 84, 495 (1929) Allan, E. and Ooisy, E.A., JAMA 51, 819 (1923). Peterson, O.H., J. EIOL. CHEM. Il9, 185 (1937). Peterson, O.H., J. EIOL. CHEM. =,695 (1937). Hench, P.S., Kendall, E.C., Slocumb, C.H. and Polley, H.F., PROC. MAYO CLINIC 24; 181 (1949). Peterson, D.H. and Reineke, L.M., J. BIOL. CHEM. I8l, 95 (1949). Peterson, D.H. and Reineke, L-M., J. EIOL. CHEM. 72, 3598 (1950). Peterson, D.H., RESEARCH6, 309 (1953). Sarett, L.H., J. EIOL. CHEM. I&, 601 (1945). Hechter, O., Jacobsen, R.P., Jeanloz, R., Levy, H., Marshall, C.W., Pincus, G. and Schenker, V., ARCH. EIOCHEM. I.5, 457 (1950). Zaffaroni, A., Burton, R. 8. and Keutmann, E. H., SCIENCE 111, 6 (1950). Peterson, D.H., in: Industrial Microorganisms (Rainbow, C. and Rose, A.H., Editors), Academic Press, London (1963), pp. 537-606. Heyl, F.W. and Herr, M.E., J. AM. CHEM. SOC. 72, 2617 (1950). Peterson, D.H. and Murray, H.C., J. AM. CHEM. SOC. 71, 1871 (1952). Mancero, O., Zaffaroni, A., Rubin, B.A., Sondheimer, F., Rosenkranz, G. and Djerassi, C., J. AM. CHEM. SOC. 74, 3711 (1952). Murray, H.C., and Peterson, D.H., inventors; The Upjohn Co., assignee. Oxygenation of steroids by Mucorales fungi. U.S. Patent 2,602,769. 1952 July 8. Innovators, The. New Processes Impact Sometimes Can Match That of New Products. Successful Search for Way to Synthetic Cortisone was Key to Medical Gain. The Wall Street Journal, 1952 Jul. 9: Sect. 1 (col. 1). Gottschalk Jr., E., in: The Innovators (Wall Street Journal, Editor), Dow Jones and Co. Books (1968), pp. 76-88. Engel, L., in: Kalamazoo (Leonard Engel, Editor), McGraw Book Co., New York (1961), pp. 128-155. Barber, G.W., Peterson, D.H. and Eherenstein, M., J. ORG. CHEM. 25, 1168 (1960). Hayano,M.,Gut, M., Dorfman, R.I., Sebek, O.K. and Peterson, D.H., JACS 80, 2336 (1958). Corey, E.J., Gregoriou, G.A. and Peterson, D.H., JACS 80, 2338
(1958).