Part 3

This was also true for the methods of chemical degradation, carried out in order to find the components of nucleins in their highest state of natural complexity. It was learned for example, that the special kind of carbohydrate present in nucleins was very susceptible to change under the conditions of hydrolysis by acids. Phoebus Aaron Theodor Levine (1869-1940), therefore, used the digestion by a living organism. With E. S. London, he introduced a solution of nucleic acid into, e.g., the gastrointestinal segment of a dog through a gastric fistula and withdrew the product of digestion through an intestinal fistula. Fortunately, the products obtained in such degradations were not new in themselves. The carbohydrate in this nucleic acid proved to be identical with D-ribose, which Emil Fischer had artificially made from arabinose and named ribose to indicate this relationship (1891). The nitrogenous products of the degradation were identical with substances previously prepared in the long study of uric acid. In the course of this study, Emil Fischer established uric acid and a number of its derivatives as having the elementary skeleton of what he called "pure uric acid," abbreviated to purine. Out of Adolf Baeyer's work on barbituric acid came the knowledge of pyrimidine and its derivatives.

From these findings, together with what Oswald Schmiedeberg (1838-1921) had established concerning the presence of four phosphate groups in the molecule (1899), Robert Feulgen (1884-1955) constructed the following scheme of a nucleic acid. Feulgen's formula of 1918 is:

Phosphoric acid--Carbohydrate--Guanine Phosphoric acid--Carbohydrate--Cytosine Phosphoric acid--Carbohydrate--Thymine Phosphoric acid--Carbohydrate--Adenine

Of the four basic components on the right, thymine occurs in the nucleic acid from the thymus gland. Yeast contains uracil instead. The difference between these two bases is one methyl group: thymine is a 5-methyluracil. In all of these basic substances, the structure of urea

NH_{2} / C=O \ NH_{2}

is involved, and they form pairs of oxidized and reduced states:

PURINE PYRIMIDINE

(reduced) Adenine + (oxidized) Thymine (oxidized) Guanine + (reduced) Cytosine

3N = CH4 | | 2H--C CH5 || || 1N--CH6

Pyrimidine

1N==CH6 | | H | | 7/ N==C--NH_{2} 2H--C C--N | | || ||5 \ H--C C--NH || || \ || || \ || || CH8 || || CH || || // || || // 3N--C--N N--C--N 4 9 Adenine Purine

HN--C=O | | NH_{2}--C C--NH N==C--NH_{2} H--N--C=O || || \ | | | | || || CH O=C C--H O=C CH || || // | || | || N--C--N H--N--CH HN--CH

Guanine Cytosine Uracil

The carbohydrate is ribose or deoxyribose.

CHO CHO | | H--C--OH HO--C--H | | HO--C--H HO--C--H | | HO--C--H HO--C--H | | CH_{2}OH CH_{2}OH

Arabinose L-Ribose

Fischer and Piloty, 1891

H \(1)/-----O-----\(4) (5) C CH--CH_{2}OH / \(2) (3)/ HO CH_{2}--HC(OH)

Deoxyribose

The exact position of phosphoric acid was established after long work and verified by synthesis.[33]

A compound of adenine, ribose, and phosphoric acid was found in yeast, blood, and in skeletal muscle of mammals. From 100 grams of such muscle, 0.35-0.40 grams of this compound were isolated. If the muscle is at rest, the compound contains three molecules of phosphoric acid, linked through oxygen atoms. It was named adenosine triphosphate or adenyltriphosphoric acid,[34] usually abbreviated by the symbol ATP. It releases one phosphoric acid group very easily and goes over in the diphosphate, ADP, but it can also lose 2 P-groups as pyrophosphoric acid and leave the monophosphate, AMP.

N==C--NH_{2} | | HC C--N +----O----+ || || \\ | | || || CH | OH OH | H OH || || / | | | | | / N--C--N-----C--C---C--C--C--O--P=O | | | | | \ H H H H H OH \---------/\---------------/\--------/ Adenine D-Ribose Phosphoric acid

This change of ATP was considered to be the main source of energy in muscle contraction by Otto Meyerhof.[35] The corresponding derivatives of guanine, cytosine, and uracil were also found, and they are active in the temporary transfer of phosphoric acid groups in biological processes.

Thus, the study of organic phosphates progressed from the comparatively simple esters connected with fatty substances of organisms to the proteins and the nuclear substances of the cell. The proportional amount of phosphorus in the former was larger than in the latter; the actual importance and function in the life of organisms, however, is not measured by the quantity but determined by the special nature of the compounds.

The study of this function is the newest phase in the history of phosphorus and represents the culmination of the previous efforts. This newest phase developed out of an accidental discovery concerning one of the oldest organic-chemical industries, the production of alcohol by the fermentative action of yeast on sugar. A transition of carbohydrates through phosphate compounds to the end products of the fermentation process was found, and it gradually proved to be a kind of model for a host of biological processes.

Specific phosphates were thus found to be indispensable for life. In reverse, the wrong kind of phosphates can destroy life. As a result, an important part of the new phase in phosphorus history consisted in the study--and use--of antibiotic phosphorus compounds.

Phosphates in Biological Processes

The first indication that phosphorus is important for life came from the experience that plants take it up from the substances in the soil. They incorporate it in their body substance. What makes phosphorus so important that they cannot grow without it? The next insight was that animals acquire it from their plant food. It is then found in bones, in fat and nerve tissue, in all cells and particularly in the cell nuclei. What are its functions there?

The answers to such questions were developed from the study of a long-known process, the conversion of carbohydrates into carbon dioxide and alcohol by yeast. It started with Eduard Buchner's discovery of 1890, that fermentation is produced by a preparation from yeast in which all living cells have been removed. When yeast is dead-ground and pressed out, the juice still has the ability to produce fermentation.

It is strange, but in many ways characteristic for the process of science, that the "riddle" of phosphorus in life was solved by first eliminating life. In such "lifeless" fermentations, Arthur Harden found that the conversion of sugar begins with the formation of a hexose phosphate (1904). The "ferment" of yeast, called zymase, proved to be a composite of several enzymes. Hans von Euler-Chelpin isolated one part of zymase, which remains active even after heating its solution to the boiling point. From 1 kilogram of yeast, he obtained 20 milligrams of this heat-stable enzyme, which he called cozymase and identified as a nucleotide composed of a purine, a sugar, and phosphoric acid.[36] In the years between the two World Wars, zymase was further resolved into more enzymes, one of them the coenzyme I, which was shown to be ADP connected with another molecule of ribose attached to the amide of nicotinic acid, or diphosphopyridine nucleotide:

^ NH_{2} / \\ | / \\ N ^ || |-CONH_{2} //\ / \\ || | | || N \ // | || | N_{+} N--+ | | | \// | | N H--C------+ H--C------+ | | | | H--C--OH | H--C--OH | | O | O H--C--OH | H--C--OH | | | | | H--C------+ O O H--C------+ | || || | CH_{2}--O--P--O--P--O--CH_{2} | | O- OH

Coenzyme I

Its function is connected with the transfer of hydrogen between intermediates formed through phosphate-transferring enzymes. Fermentation proceeds by a cascade of processes, in which phosphate groups swing back and forth, and equilibria between ATP with ADP play a major role.

Many of the enzymes are closely related to vitamins. Thus, cocarboxylase A, which takes part in the separation of carbon dioxide from an intermediate fermentation product, is the phosphate of vitamin B_{1}. Others of the B vitamins contain phosphate groups, for example those of the B_{2} and B_{6} group, and in B_{12}, one lonely phosphate forms a bridge in the large molecule that contains one atom of cobalt: C_{63}H_{90}N_{14}O_{14}PCo. The formation of vitamin A from carotine occurs under the influence of ATP.

The first stages in fermentation are like those in respiration, which ends with carbon dioxide and water. These two are the materials for the reverse process in photosynthesis. When light is absorbed by the chlorophyll of green plants, one of the initial reactions is a transfer of hydrogen from water to a triphosphopyridine nucleotide, which later acts to reduce the carbon dioxide. Under the influence of ATP, phosphoglyceric acid is synthesized and further built up by way of carbohydrate phosphates to hexose sugars and finally to starch. In many starchy fruits, a small proportion of phosphate remains attached to the end product.

The synthesis of proteins is under the control of deoxyribonucleic acid or ribonucleic acid, abbreviated by the symbols DNA and RNA. The genes in the nucleus are parts of a giant DNA molecule. RNA is a universal constituent of all living cells. Where protein synthesis is intense, the content in RNA is high. Thus, the spinning glands of silkworms are extraordinarily rich in RNA.[37]

In his research on the radioactive isotope P^32, George de Hevesy gained some insight into the surprising mobility of phosphates in organisms: "A phosphate radical taken up with the food may first participate in the phosphorylation of glucose in the intestinal mucose, soon afterwards pass into the circulation as free phosphate, enter a red corpuscle, become incorporated with an adenosine triphosphoric-acid molecule, participate in a glycolytic process going on in the corpuscle, return to circulation, penetrate into the liver cells, participate in the formation of a phosphatide molecule, after a short interval enter the circulation in this form, penetrate into the spleen, and leave this organ after some time as a constituent of a lymphocyte. We may meet the phosphate radical again as a constituent of the plasma, from which it may find its way into the skeleton."[38] Much has been added in the last 30 years to complete this picture in many details and to extend it to other biochemical processes, including even the changes of the pigments in the retina in the visual process, or in the conversion of chemical energy to light by bacteria and insects.

Medicines and Poisons

In the delicate balance of these processes, disturbances may occur which can be remedied by specific phosphate-containing medicines. Thus, adenosine phosphate has been recommended in cases of angina pectoris and marketed under trade names like sarkolyt, or in compounds named angiolysine. A considerable number of physiologically active organic phosphates can be found in the patent literature.[39] Yeast itself is considered to be a valuable food additive.

On the other hand, there are phosphate compounds that act as poisons. One group of such compounds was discovered in 1929 by W. Lange, who wrote: "Of interest is the strong action of mono-fluorophosphate esters on the human body--the effect is produced by very small quantities."[40] Diisopropyl fluorophosphate has since become a potential agent for chemical warfare. It inactivates an enzyme which controls the transmission of nerve impulses to muscle, acetylcholine esterase.

Organic esters of phosphoric acids are used as insecticides. The hexa-ethylester of tetraphosphoric acid, prepared by Gerhard Schrader by heating triethylphosphate with phosphorus oxychloride,[41] actually contains tetraethylpyrophosphate (TEPP) among others. Bayer's Dipterex, the dimethyl ester of 2,2,2-trichloro-1-hydroxyethyl-phosphonate, has been modified to dimethyl-2,2-dichlorovinyl-phosphate and is especially active against the oriental fruit fly.[42]

Cl H O | | || OCH_{3} | | ||/ Cl--C--C--P Bayer's L 13/59 | | \ (Dipterex) | | OCH_{3} Cl OH

(CH_{3})_{2}N O O N(CH_{3})_{2} \|| ||/ P--O--P Schradan / \ (CH_{3})_{2}N N(CH_{3})_{2}

Octamethylpyrophosphoramide

The story of phosphorus, which began 300 years ago, has acquired new importance in this century. Many scientists have contributed to it: 13 of them have received Nobel Prizes for work directly bearing on the chemical and biological importance of phosphorus compounds. In chronological order, they are: Eduard Buchner, Albrecht Kossel, Otto Meyerhof, Arthur Harden, Hans von Euler-Chelpin, George de Hevesy, Carl F. Cori, Gerty T. Cori, Fritz Lipmann, Lord Alexander Todd, Arthur Kornberg, Severo Ochoa, and Melvin Calvin. The developers of industrial production and commercial utilization of phosphate compounds have had other rewards.

Some impression of the continuing growth in this field[43] can be gained from the following data.

PHOSPHATE ROCK

annually "sold or used by producer" in the United States in million long tons (2,240 lbs.)

1880 0.2 1890 0.5 1900 1.5 1910 2.655 1920 4.104 1930 3.926 1940 4.003 1945 5.807 1950 11.114 1955 12.265 1955 (world: about 56) 1960 17.202 1962 19.060

Sources: U.S. Bureau of the Census. _Historical Statistics of the United States 1789-1945_ (1949); _Statistical Abstract of the United States._

ELEMENTAL PHOSPHORUS

annually produced in the United States in short tons (2,000 lbs.)

1939 43,000 1944 85,679 1950 153,233 1956 312,200 1958 335,750 1959 366,350 1960 409,096 1961 430,617 1962 451,970

Source: U.S. Department of Commerce.

FOOTNOTES:

[1] WILHELM HOMBERG, _Memoires Academie, 1666-1699_ (Paris, 1730), vol. 10, under date of April 30, 1692, pp. 57-61.

[2] FORTUNIO LICETUS, _Lithiophosphorus sive de lapide Bononiensi_ (Venice, 1640).

[3] Cited in PETER JOSEPH MACQUER _Chymisches Woerterbuch_, 2nd ed. (Leipzig: Weidmann, 1789), vol. 4, p. 508, footnote "c" as "Kletwich (de phosph. liqu. et solid. 1689, Thes. II)."

[4] FERDINAND HOEFER, _Histoire de la Chimie_ (Paris, 1843), vol. 1, p. 339.

[5] G. W. VON LEIBNIZ, _Memoires Academie_ (Paris, 1682); _Akademie der Wissenschaften, Miscellanea Berolinensia_ (Berlin, 1710), vol. 1, p. 91.

[6] JEAN HELLOT, _Memoires Academie 1737_ (Paris, 1766), under date of November 13, 1737, pp. 342-378.

[7] MACQUER, op. cit. (footnote 3), p. 551.

[8] A. S. MARGGRAF, _Akademie der Wissenschaften, Miscellanea Berolinensia_ (Berlin, 1743), vol. 7, 342 ff.; see also WILHELM OSTWALD _Klassiker der Exakten Naturwissenschaften_ (Leipzig: Engelmann, 1913), no. 187.

[9] G. HANCKEWITZ, [Hankwitz], _Philosophical Transactions of the Royal Society of London_, 1724-1734, abridged (London, 1809), vol. 7, pp. 596-602.

[10] ANTOINE LAURENT LAVOISIER, "Sur la Combustion du Phosphore de Kunckel, Et sur la nature de l'acide qui resulte de cette Combustion," _Memoires Academie 1777_, (Paris, 1780), pp. 65-78.

[11] GUYTON DE MORVEAU and others, _Methode de Nomenclature Chimique_, Proposee par MM. de Morveau, Lavoisier, Bertholet, & de Fourcroy (Paris, 1787), plate 9.

[12] MACQUER, op. cit. (footnote 3), p. 513.

[13] MARIE BOAS, _Robert Boyle and Seventeenth Century Chemistry_ (New York: Cambridge University Press, 1958), p. 226; see also WYNDHAM MILES, "The History of Dr. Brand's Phosphorus Elementarus," _Armed Forces Chemical Journal_ (November-December 1958), p. 25.

[14] ARCHIBALD CLOW and NAN L. CLOW, _The Chemical Revolution_ (London: Batchworth Press, 1952), p. 451.

[15] EMILE KOPP, _Comptes-rendus hebdomadaires des Seances de l'Academie des Sciences, Paris_ (1844), vol. 18, p. 871; WILHELM HITTORF, _Annalen der Chemie und Pharmazie_, suppl. to vol. 4, p. 37; ANTON SCHROeTTER, _Annales de Chimie et de Physique_, series 3, vol. 24 (1848), p. 406; see also Schroetter's report on "Phosphor und Zuendwaaren" in A. W. VON HOFMANN, _Bericht ueber die Entwicklung der Chemischen Industrie_ (Braunschweig: Vieweg, 1875), pp. 219-246.

[16] R. GLAUBER, _Furni Novi Philosphici_ (Amsterdam, 1649), vol. 2, pp. 12 ff.

[17] HERMANN SCHELENZ, _Geschichte der Pharmazie_ (Berlin: Springer, 1904), p. 598.

[18] J. PERSONNE, _Comptes-rendus ..._, Paris (1869), vol. 68, pp. 543-546.

[19] A. WURTZ, _Dictionnaire de Chimie_ (Paris, 1876), vol. 2, part 2, p. 951.

[20] KARL W. SCHEELE, _Nachgelassene Briefe und Aufzeichnungen_, edit. A. E. Nordenskioeld (Stockholm: Norstedt, 1892), pp. 38, 144.

[21] J. J. BERZELIUS, _Lehrbuch_, transl. F. Woehler (Dresden, 1827), vol. 3, part 1, p. 96.

[22] THOMAS GRAHAM, _Philosophical Transactions of the Royal Society of London_ (1833), pp. 253-284.

[23] JUSTUS LIEBIG'S _Annalen der Pharmacie_ (1838), vol. 26, p. 113 ff.

[24] A. WURTZ, _Annales de Chimie et de Physique_, series 3, vol. 16 (1846), p. 190.

[25] CARROLL D. WRIGHT, _The Phosphate Industry in the United States_, sixth special report of the Commissioner of Labor (Washington, 1893).

[26] J. STOKLASA, _Biochemischer Kreislauf des Phosphat-Ions im Boden, Centralblatt fuer Bakteriologie ..._ (Jena: Fischer, March 22, 1911), vol. 29, nos. 15-19.

[27] N. T. GOBLEY, _Comptes-rendus_ ..., Paris (1845), vol. 21, p. 718.

[28] A. WURTZ, _Comptes-rendus_ ..., Paris (1868), vol. 66, p. 772.

[29] L. THUDICHUM, _Die chemische Constitution des Gehirns des Menschen und der Tiere_ (1901); see also H. WITTCOFF, THE PHOSPHATIDES (New York: Reinhold, 1951).

[30] WILLIAM CROOKES, _British Association for the Advancement of Science, Reports_ (1887), sec. B, p. 573.

[31] J. E. COURTOIS and A. LINO, _Progress in the Chemistry of Organic Natural Products_, edit. L. Zechmeister (Vienna: Springer Verlag, 1961), vol. 19, p. 316-373.

[32] A. WURT, _Dictionnaire de Chimie_, supp. part 2, [n.d.] p. 1087; A. KOSSEL, _Zeitschrift fuer physiologische Chemie_, series 3 (1879), p. 284.

[33] ALEXANDER TODD, _Les Prix Nobel en 1957_ (Stockholm).

[34] HANS VON EULER-CHELPIN, _Les Prix Nobel en 1929_ (Stockholm).

[35] O. MEYERHOF and E. LUNDSGAARD, _Naturwissenschaften_ (Berlin, 1930), vol. 18, pp. 330, 787.

[36] K. LOHMANN, _Naturwissenschaften_ (Berlin, 1929), vol. 17, p. 624; C. H. FISKE and Y. SUBBAROW, _Science_ (Washington, 1929), vol. 70, p. 381 f.

[37] J. BRACHET, _Scientia, Revista di Scienza_ (1960), vol. 95, p. 119.

[38] GEORGE DE HEVESY, _Les Prix Nobel en 1940_ (Stockholm). See also EDUARD FARBER, _Nobel Prize Winners in Chemistry_, 2nd ed. (New York: Schuman, 1963), p. 179.

[39] See, e.g., _Chemical Week_, vol. 77 (September 3, 1955), p. 79 f.; J. BOLLE, _Chimie et Industrie_ (1960), vol. 83, p. 252.

[40] W. LANGE, _Berichte der Deutschen Chemischen Gesellschaft_ (Berlin, 1929), vol. 62, p. 793; vol. 65 (1932), p. 1598.

[41] GERHARD SCHRADER, U.S. patent 2,336,302 of 1943 (priority in Germany, 1938); S. A. HALL and M. JACOBSON, _Industrial and Engineering Chemistry_ (1943), vol. 40, p. 694.

[42] A. M. MATTSEN and others, _Journal of Agriculture and Food Chemistry_ (1955), vol. 3, p. 319.

[43] JOHN B. VAN WAZER, _Phosphorus and its Compounds_, 2 vols. (vol. 1, _Chemistry_; vol. 2 _Technology, Biological Functions and Applications_, New York: Interscience, 1958, 1961.

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