BIOLOGICAL CHEMISTRY.1. INTRODUCTION.IN the past two issues of Annual RePorts the Reporters concerned withbiological chemistry have tended to review their topics rather than to confinethemselves to the immediate past. This policy is continued as we believethat most of our readers are likely to be unfamiliar with the background ofsome interesting current researches. For example, L( +) -ergothionehe,discovered many years ago in ergot and later in mammalian blood, is nowshown to be a widely distributed intracellular component; as yet its bio-logical function can only be guessed at. Most text-books neglect this sub-stance. Antithyroid substances in plants used for food of men and animalshave been known to exist for some time; two powerful chemicals have beenisolated, their structures have been determined, they have been synthesised,and their origins in the neglected class of mustard-oil glycosides ” have beenfairly clearly indicated.But there must, from the evidence, be other anti-thyroid factors in plants which do not contain mustard-oil glycosides. Hereis a little explored field.The plethora of steroids produced by animal tissues has led to extensivestudies on the mechanisms by which hydroxylation takes place in vivo.These studies received a great impetus from demands for cortisone and it isnoteworthy that certain moulds may be used to facilitate the synthesis ofthis clinically important substance because they possess the power of intro-ducing a hydroxyl group at exactly the right place in the perhydrocyclo-pentanophenanthrene system.Natural fatty acids have long been regarded by many as dull, difficultto deal with, and few in number.This Report should dispel such ideas.Chromatography, and improvements in the measurements of physicalcharacteristics and in analytical reactions, to say nothing of the dramaticdiscoveries centering around coenzyme A, have resulted in a new and growinginterest in the fatty acids and their natural relations. This section comple-ments the Report of 1953 and may introduce some to the natural acetylenicbond.Finally, a range of interesting oligosaccharides has been discovered inhuman milk through the nutritional “ fussiness ” of a Lactobacillus. Thiswork was made possible by the intimate collaboration of German carbo-hydrate chemists with microbiologists in the United States of America.D. J.B.2. L(+)-ERGOTHIONEINE.Structure and Synthesis.+ +) -Ergothionehe was first obtained by ex-traction of ergot by Tanret 1 in 1909. Since then it has been the subject ofl C. Tanret, J . Pharm. Chim., 1909, 30, 145; Compt. rend., 1909, 149, 222. Forpreparation from, and levels in, ergot, cf. N. W. Pirie, Biochem. J., 1933, 27, 202;G. Hunter, G. D. Molnar, and N. J. Wight, Canad. J . Res., 1949,27, El 226; G. Hunter,S. G. Fushtey, and D. W. Gee, ibid., p. 240286 BIOLOGICAL CHEMISTRY.periodic interest on account of its discovery in the blood and in other bio-logical situations in various species of animals. At the present time atten-tion is again being paid to this biologically bafing chemical.Its structure(1) is suggestive in view of the catalytic importance recently attributed toSH(3) (4)thiol-bearing cell-components and because of the transferring reactionsundergone by “ labile ” methyl groups carried by quaternary nitrogen atomsof certain metabolites.Ergothioneine was established as a derivative of histidine by Barger andEwins2 in 1911, after the isolation, also from ergot, of histamine (2) byBarger and Dale.3 In view of certain later observations the first two stepsin Barger and Ewins’s work are noted here : The compound, on treatmentwith concentrated (50%) aqueous potassium hydroxide is quantitativelytransformed into the yellow 2-mercaptourocanic acid (3) and trimethylamine.Dilute nitric acid oxidatively removes the sulphur atom from the last acid (3),to give @-4-glyoxalin ylacrylic acid and sulphuric acid.Mild acidic oxidisingagents quantitatively eliminate the thiol group, as sulphuric acid, fromergothioneine itself and from 2-mercaptohistidine : from ergothioneine (1)(by ferric chloride), hercyanine (4) is thus obtained; this substance has beenfound in the fungi Agavatus campestris and Boletus edulis.Barger and Ewins 2 pointed out that the thiol group in ergothioneine isquite different in character from that in cysteine. Not only is it stable toalkali, but also it is readily and quantitatively oxidised to sulphuric acidunder conditions which yield a sulphonic acid from cysteine. That thea-carbon system has the L-configuration arises from the synthesis, albeitaccompanied by some racemisation, through methylation of 2-ethyl-carbonylthio-~-histidine.5 It is unfortunate that this partially racemisedsynthetic ergothioneine has been erroneously described by a reviewer as“ in every respect identical with the natural material.”Analytical Methods.-The first, and most widely used, specific methodwas described by Hunter 7 in 1928, since when it has undergone modificationsonly with respect to concentration of the ergothioneine and elimination ofinterfering substances.It consists essentially in treating ergothioneine withG. Barger and A. J. Ewins, J., 1911, 99, 2336.G. Barger and H. Dale, J., 1910, 97, 2592.H. Heath, A. Lawson, and C. Rimington, J., 1951, 2215.6 Cf.also J. N. Ashley and C. R. Harington, J . , 1930, 2586; C. Tesar and D.Rittenberg, J . BioZ.,Fhem., 1947, 170, 35.6 K. Hofman, Imidazole and its Derivatives, Part I,” Interscience Publ., NewYork and London, 1953.G. Hunter, Biochenz. J . , 1928, 22, 4BELL : L( +)-ERGOTHIONEINE. 287diazotised sulphanilic acid and subsequently adding strong alkali, an intensered-purple colour being produced. It seems that this colour originates fromthe 2-mercaptourocanic acid derivative (cf. 3) and is not given by a numberof other 2-mercaptoglyoxalines examined by Lawson and his collabor-at0rs.*,8,~ So far as is known, 2-mercaptourocanic acid does not occurnaturally; if it does, it will be liable to be confused with ergothioneine in theHunter reaction.Hunter originally used tungstic acid for blood deproteinisation withsuccess.In 1949 he found lo that this reagent was not satisfactory andsuggested that there was some unknown difference between the originaland 1949 samples of tungstate. He has described a simple deproteinisationby boiling acidified diluted blood. In attempts to eliminate “ interferingsubstances ” which Hunter had noted, Lawson, Morley, and Woolf addedthe refinement of concentration of ergothioneine from Hunter’s blood filtrates(and from urine, etc.) by precipitation with potassium tetraiodobismuthate(Kraut’s, or Dragendorff ’s, reagent) or iodobismuthous acid.Baldridge and Lewis loa have applied the Hunter reaction to the deter-mination of ergothioneine in presence of histidine (which is also chromogenicwith the diazo-reagent although the colour produced is different) and uricacid.Touster l1 proposed a determination based on the ease of oxidation, byaqueous bromine, of the 2-mercapto-group of ergothioneine, sulphuric acidbeing formed (cf. ref.2). This method has been used by Mann and Leone l2in parallel with that of Hunter, and also by Heath et aZ.26 in experimentsusing 35S-labelled compounds (see below). This oxidative method may wellalso produce sulphuric acid from mercaptoglyoxalines other than ergo-thioneine. Melville and Horner l3 have devised a modified extractionprocedure for blood and other animal tissues which is sensitive to 10 pg. ofergothioneine per ml. of blood ; in it glutathione and dithionite are added tolaked, diluted erythrocytes, and protein is precipitated by trichloroacetic acidexcess of which, along with other ions, is removed by chloroform extractionand treatment with resins.Using chromatographic concentration on analumina column, Melville, Horner, and Lubschez l4 have detected andmeasured ergothioneine in animal tissues other than blood. Lawson,Morley, and Woolf and Mann and Leone l2 have described detection ofergothioneine and related compounds by paper chromatography.Occurrence.-Sixteen years after the discovery of ergothioneine in ergot,Hunter et aZ.15 reported that satisfactory uric acid determinations could notbe made on Folin-Wu filtrates of whole blood with “ direct ” addition ofBenedict’s reagent.16 They isolated from pig erythrocytes a crystallinesubstance giving the uric acid reaction; because they were unable to detect* H.Heath, A. Lawson, and C. Rimington, J . , 1951, 2218.!a A. Lawson, H. V. Morley, and L. I. Woolf, Biochem. J., 1960, 47, 573.lo G. Hunter, Canad. J . Res., 1949, 27, E, 230; Biochem. J., 1951, 48, 265.loo R. C. Baldridge and H. B. Lewis, J . Biol. Chem., 1953, 202, 169.l1 0. Touster, ibid., 1951, 188, 371.l2 T. Mann and E. Leone, Biochem. J . , 1953, 53, 140.l3 D. B. Melville and W. H. Horner, J . Biol. Chem., 1953, 202, 187.l4 D. B. Melville, W. H. Horner, and R. Lubschez, ibid., 1964, 206, 231.l6 F. M. R. Bulmer, B. A. Eagles, and G . Hunter, ibid., 1925, 65, 17 ; G. Hunter andl6 S. R. Benedict, ibid., 1922, 51, 187.B. A.Eagles, ibid., p. 623288 BIOLOGICAL CHEMISTRY.labile sulphur after boiling it with concentrated alkali they arrived at theempirical formula C,HI1O,N,. Benedict, Newton, and Behre 1 7 likewiseisolated a similar substance but, using metallic-sodium fusion, found that it,in fact, did contain sulphur. Both groups of workers l8 in 1927 showed itto be L(+)-ergothioneine (sometimes, in U.S.A., called " thioneine "). In1930 Gulland and Peters l9 isolated the substance from pigeon blood.In blood, ergothioneine is confined to the erythrocytes. Variousmeasurements have been made on different animals; in some, e.g., the ox,unknown blood constituents interfere with the diazo-reaction and theapparent ergothioneine level is low.? As noted above, Lawson et d9 andMelville et aZ.139 l4 have evolved methods to eliminate this " anti-diazo "-effect.Table I gives some findings.TABLE 1. Ergothioneine in mg./lOO ml. of whole blood.Man ..................Pig ..................ox ..................Rabbit.. .............Guinea pig .........Rat ..................Cat ..................Fowl ...............Chick ...............Hunter, 71928,Toronto,Canada1.2-4-02-26<11 4 . 51-4-1-5-10 -Hunter,lo1951, 1951,Edmonton, London,Canada U.K.2.5-9.5 1.8-4.46.0-9.3 3.3-5.3 - - - - - -2.9-8.8 1.3-3.1 - - - -I -* Germ-free chick.Lawson Melvilleet aZ.,B andLondon, Horner l3U.K.2.0-3.1 -4.3-4.6 -2.2 -4.5-5.0 -<04-2.2 -- -- -- -- 4 & 6 *The recent application of column chromatography to tissue extracts byMelville, Horner, and Lubschez l4 has shown that, in the rat a t least, ergo-thioneine is not confined to the erythrocytes but can be detected by theHunter reaction, as an intracellular component, in various tissues as shownin Table 2.TABLE 2. Ergothioneine (Ung.llO0 g.of fresh tissue) in the 7at.14Skeletal Intes- SeminalLiver Kidney Heart Lung Spleen muscle tine Stomach vesicles13.3 4.3 1.5 1.5 1.1 0.7 0.6 0.4 0-2Brain, testes and plasma contained no detectable amounts.In all these animal tissues ergothioneine occurs only as an intracellularconstituent. Boar seminal-vesicular secretion has been shown by Mannand Leone 12 to be particularly rich in ergothioneine; it is present thereextracellularly in amounts between -30 and 250 mg./100 ml.(average-80 mg.). Boar blood contained (average of three animals) -4.5 mg./100ml., while pig corpora Zutea, adrenals, thyroid, eye vitreous fluid, and foetal1' S. R. Benedict, E. B. Newton, and J. A. Behre, J . Bid. Chem., 1926, 67, 267.l8 B. A. Eagles and T. B. Johnson, J . Amer. Chem. SOC., 1927, 49, 675; E. B.Newton, S. R. Benedict, and H. D. Dakin, J . BioZ. Chem., 1927, 72, 367; G. Hunterand B. A. Eagles, ibid., p. 123.19 J. M. Gulland and R. A. Peters, Biochsm. J., 1930, 24, 91. For extraction fromblood, cf. S. W. Williamson and N. U. Meldrum, ibid., 1932, 26, 815; G. Hunter, G. D.Molnar, and N. J. Wight, Canad. J. Res., 1947, 27, E, 226BELL : L( +)-ERGOTHIONEINE. 289fluids showed no detectable amounts.In ram and human semen onlytraces of ergothioneine, if any, are present.Origin of Ergothionehe in Animal Tissues.-The origin of ergothioneinein the animal is no less obscure than is the reason for its presence in thefungal product, ergot.20 Some recent experiments have yielded apparentlyconflicting results. It is not yet known whether the ergothioneine found inanimal tissues is an essential metabolite of truly endogenous origin or whetherit is elaborated from a precursor in the food. In 1928 Eagles and Vars 21suggested that, in pigs, diet had a significant effect on the blood level whichcould be appreciably raised from low values by addition to a basal diet ofcertain proteins and of, more specifically, maize (US., corn).In 1951Hunter,10 besides commenting on environmental differences in blood ergo-thioneine between groups both of pigs and of men (cf. Table 2), repeated themaize experiment without finding support for Eagles and Vars’s hypothesis.None the less, he found that methanolic extracts of maize contain at leastthree different bodies which give colour reactions, which were atypical ofthe mercaptoglyoxaline system. These, however, he did not consider to benecessarily ergothioneine precursors ; methanol-extracted maize, fed to rats,was equally efficient as the unextracted cereal in maintaining the ergo-thioneine levels within identical ranges.About the same time, Spicer, Wooley, and Kessler 22 showed that theerythrocytes of rabbits fed on a purified diet with casein as sole protein werealmost devoid of ergothioneine.Melville et aZ.14 found similar results withrats fed on casein as sole protein ; in these animals not only were the erythro-cytes affected, but the ergothioneines in other tissues fell to vanishing point,i.e., 0.05 mg.1100 g. of fresh tissue (cf. Table 2). Melville et aZ.24 found that,in the rat, zein, like casein, did not provide an ergothioneine precursor. Onthe other hand, maize could do so; an aqueous acetone extract of maize waslikewise effective. A dietary concentration of as low as 1 in 10,000 parts wassufficient to lead to an accumulation of ergothioneine in the rat erythrocyte.Other workers 23 have also noted the inefficiency of casein in maintainingblood ergothioneine in rabbits and rats, but when rabbits were fed on oats 25and cabbage a “-fold increase was observed after 8-10 weeks.Inwhite rats fed on ground oats, or maize or casein for several weeks, theerythrocytes contained less than 1 mg./100 ml. ofThese casein experiments apparently show that the methionine residues,which bear nearly all the sulphur contained in the protein, are not availablefor ergothioneine synthesis. On the other hand, we have the results ofHeath et aZ.26 who fed a number of 35S derivatives to two adult boars;labelled sulphate, 2-mercaptohistidine, methionine, and ergothioneine wereseparately fed. Measurements of bromine-oxidisable sulphur 11 (afterremoval of inorganic and ester sulphate) were made on alcohol-precipitatedfiltrates of seminal plasma and urine; only p5S]methionine appeared to actte A.St. Garay, Nature, 1956, 177, 91.22 S. S. Spicer, J. G. Wooley, and V. Kessler, PYOC. SOC. Exp. Biol. Med., 1951,77, 418.23 R. G. Bartlett and U. D. Register, ibid., 1953, 83, 708.24 D. B. Melville, C. C. Otken, and V. Kovalenko, J . Biol. Chem., 1955, 216, 325.26 Cf. V. R. Potter and K. W. Franke, J . Nutrition, 1935, 9, 1 .2b R. C. Baldridge, Fed. Proc., 1954, 13, 178; see also ref. 10a.0s IT. Heath, C . Rimington, T. Glover, T. Mann, and E. Leone, Biochem. J., 1953,B. A. Eagles and H. M. Vars, J . Biol. Chem., 1928, 80, 615.64, 606.REP.-VOL. LII 290 BIOLOGICAL CHEMISTRY.as source of ergothioneine sulphur ; the labelled 2-mercaptohistidine wastotally eliminated in the urine.Heath 27 also administered 2-[35S]mercapto-histidine and [35S]ergothioneine to rats for 21 days and found that isotopefrom the latter only was incorporated into the erythrocytes, bone marrow,liver, and kidney, but not into the seminal vesicles. The mercaptohistidine(90%) was recovered unchanged in the urine; it therefore did not act as anergothioneine precursor (Lawson et aL9 found no ergothioneine in human orrat urine).Melville et aL2* fed ~-[~~S]methionine to an %weeks’ old boar, too youngto secrete vesicular fluid. Ergothioneine was isolated from the seminalvesicular tissue (49.5 mg./100 g. wet weight) and from the blood, but in thiscase no evidence for radioisotope incorporation was found. This does notseem to the Reporter an unreasonable finding since it may well be that theturnover of ergothioneine in the young, immature boar may be considerablyslower than it is in the sexually active adult.That the intestinal flora are not responsible for ergothioneine formationin the chick was shown by Melville and HornerI3 when the erythrocytelevels of ordinary and germ-free chicks were compared.The ergothioneinein the former was somewhat below the level of that in the germ-free animals.Suggested Functions of Ergothionehe.-In 1947 Lawson and Rimington 28concluded that ergothioneine had an anti-thyroid action, in rats, similar tothat of thiouracil; their claim was refuted, however, in the instances of ratsand man by Astwood and Stanley29 and of man and monkeys by Wilsonand M ~ G i n t y .~ ~Spicer et found that treatment of washed rabbit erythrocytes withsodium nitrite resulted in oxidation of the haemoglobin to methzmoglobinat a rate inversely proportional to the ergothioneine content. Moreover,added ergothioneine reversed the oxidation. The substance can thereforefunction as a reducing agent.Mann and Leone l2 have shown that ergothioneine can antagonise thiol-inactivators including Cu2+ and o-iodosobenzoate, and suggest, as a possiblefunction, the preservation of thiol groupings against dehydrogenation.In a brief communication Heath and Toennies 31 report that ergothioneinedisulphide is formed slowly by the action of oxygen in 5~-hydrochloric acidor very rapidly by one equivalent of hydrogen peroxide.The reaction canbe followed either spectrophotometrically or by paper chromatography.The disulphide can be rapidly reduced by excess of cysteine or reducedglutathione. Ergothioneine disulphide reacts with cysteine, to form first amixed disulphide ; this with excess of cysteine gives ergothioneine andcyst ine.It is not known if the disulphides containing ergothioneine give theHunter colour test. They should certainly give some colour when coupledwith diazotised sulphanilic acid. The Reporter considers that the followingare possible : (a) that oxidised ergothioneine may occur naturally and bemissed if it fails to give the Hunter test; (b) that oxidised ergothioneine27 H. Heath, Biochem, J., 1953, 54, 689.28 A. Lawson and C. Rimington, Lancet, 1947, 252, 586.20 E.B. Astwood and M. N. Stanley, ibid., 1947, 253, 905.30 M. L. Wilson and D. A. McGinty, Amer. J . Physiol., 1949, 156, 377.31 H. Heath and G. Toennies, 3me Congrb internat. Biochimie, Resume des com-munications, 1955, p. 42-MERCAPTO-A2-1 3-OXAZOLINES (2-THIO-1 : 3-OXAZOLIDINES). 291niay occur in plants and act as the precursor of (reduced) ergothioneine inanimals.Added in Proof.-Melville and Eich 32 have isolated ergothioneine from‘‘ Quaker ” oats. D. J. B. The optical rotation was not recorded.3. 2-MERCAPTO-As-1: 3-OXAZOLINES (Z-THIO-1: 3-OXAZOLIDINES)* ASANTI-THYROID SUBSTANCES FROM VEGETABLE SOURCES.The Discovery of ‘‘ Plant Goitr0gens.”-Following the original observation(U.S.A.) by Chesney, Clawson, and Webster,l a number of investigators havefound that rabbits and rats fed on diets containing a large proportion ofcabbage (Brassica oleracea) developed enlarged thyroid gland^.^-^ Wintercabbage seemed to be more potent than plants grown in summer. Tadpoles(Rana pipiens), fed with cabbage, attained maturity more quickly and hadlarger thyroids than controls fed with spinach.5aOther Brassica t species and members of other orders can provide goitro-genic factors : Kohlrabi leaves (Brassica oZeracea var.gongyZoides),6 rape orcolza (B. rapa oleifera), wild radish (Raphanus raphanistrum) ,7 Brassica seeds(rape, swede, soft and hard turnip),8 turnip roots,s Legumhosz seeds(soya and other beans, peas, lentils), ground nuts (Arachis hy$ogea),lo andalpine chestnuts.11 On the other hand, New Zealand winter cabbage 99 l2was poor in goitrogen.Rape seed oil meal, widely used in animal feeding, hasbeen shown to cause thyroid enlargement in poultry 13 and thyroid, liver,and kidney enlargement in pigs.14 Soya bean also gives goitre in chicks.15A number of researches point to the pituitary gland as the site of attackof the goitrogen, and not the thyroid itself. Chesney, Clawson, and Webster52 D. B. Melville and S. Eich, J . Biol. Chew., 1956, 218, 647.A. M. Chesney, T. A. Clawson, and B. Webster, Bull. Johns Hopkins Hosp., 1928,R. McCarrison, G. Sankaran, and K. B. Madhava, Indian J . Med. Res., 1933, 20,F. Blum, Endokrinologie, 1937,19,19; Schweiz. med. Wochenschr., 1941,71,1612;G. C. Bianchi, Beitr.pathol. Anat. allgem. Pathol., 1933, 90, 539.43, 61: B. Webster, T. A. Clawson, and A. 35. Chesney, ibid., 1928, 43, 278.23; D. Marine, E. J. Baumann, and A. Cipra, Proc. SOC. Exp. Biol., 1929, 26, 822.1943, 73, 1046; B. Webster, Endocrinology, 1932, 16, 617.6 N. D. Judina, J . Med., Ukraine, 1939, 9, 801.J. R. Borland, J . Exp. Zool., 1943, 94, 115.6 0. Stiner, Mitt. ges. Lebensmitteluntersuh. Hyg., 1933, 24, 1.N. D. Judina, J . Med., Ukraine, 1940,10, 71. * ( a ) T. H. Kennedy and H. D. Purves, Brit. J . Exp. Path., 1941,22,241; ( b ) W. E.Griesbach, T. H. Kennedy, and H. D. Purves, ibid., p. 249; (c) E. Maschmann, Natur-wiss., 1942, 30, 261.lo F. Blum, Schweiz. med. Wochenschr., 1942, 72, 1301, 1329; R. McCarrison, IndianJ . Med. Res., 1934, 21, 179.l1 B.S. Barton, “ A Memoir Concerning the Disease of Goitre as it prevails inDifferent Parts of North America,” Way and Groff, Philadelphia, 1800.12 C. E. Hercus and H. A. A. Aitken, J . Hyg., 1933, 33, 6 5 ; cf. I. T. Zwecker, Amer.J . Path., 1932, 8, 235.l3 R. M. Blakely and R. W. Anderson, Sci. Agric., 1948,28,393 ; C. W. Turner, PoultvySci., 1946, 25, 186; 1948, 27, 118; C. E. Allen and D. S. Dow, Sci. Agric,. 1952,82,403.l4 S . Nordenfeldt, N. Gellerstadt, and S. Falkmer, Acta Pathol. Mzcrobiol. Scand.,1954, 35, 217.l5 G. R. Sharpless and E. M. Hopson, Endocrinology, 1940, 27, 129; H. S. Wilgus,F. X. Gassner, A. R. Patton, and R. G. Gustavson, J . Nutrition, 1941, 22, 34. * The nomenclature of these substances in the literature shows a number of vari-ations from the customary rules.Th,: nomenclature of these plants is confused ; cf. J.Percival, ” AgriculturalBotany, Duckworth, London, 1945.C. E. Hercus and H. D. Purves, J . Hyg., 1936, 36, 182292 BIOLOGICAL CHEMISTRY.showed that the enlarged thyroid glands of their rabbits were due to ahyperplasia of the epithelial elements of the gland and not to an increase in“ colloid ” ; the latter tissue contains the active secretion of the thyroid.At the same time the animals suffered an enlargement of the suprarenalglands which paralleled the “ goitre.” Similar enlargements were found inrats * along with delay in development of immature ovaries and histologicalchanges in the pituitary. The latter were also seen by Sharpless and Hop-son l5 in rats fed with soya beans, and by Griesbach l6 in rats fed with rapeseed.Griesbach, Kennedy, and Purves 8b found that, in rats, hyperplasiaof the thyroid, previously induced by Brassica seeds, rapidly regressed afterremoval of the pituitary gland and did not return despite continued feedingof the seeds. Colloid formation and hormone storage took place afterhypophysectomy ; this was not prevented by feeding with goitrogenicvegetables. Purves,17 Whitehead,18 and Griesbach and Purves l9 concludedthat the goitrogenic factor acted by interfering with the synthesis of thyroxineand that the thyroid hyperplasia was due to increased production of thyro-tropic hormone by the pituitary gland.It seems reasonable to assume with Salter 2o “ that all diets are at leastmildly goitrogenic ” and that a balance should exist between the amounts ofavailable iodine and of goitrogens in the food.Webster and Chesneyalfound that extra dietary iodine protected rabbits against ‘‘ cabbage goitre,”and this was confirmed by Bianchi? Blum (1941, 1943),3 and Purves.17Blum22 has shown that iodine fertilisation of cabbage, linseed, and soyaplants apparently nullifies their goitrogenic properties.The agronomic significance of goitrogens in rape seed 23 has been reviewedby J. M. Bell.=Antithyroid Chemicals from Plants.-Klein and Farkass 25 found evidencefor the presence of thiourea in Laburnum; no confimatory work is knownto the Reporter. Thiourea is an antithyroid substance.26Thiocyanates, as the I‘ mustard oil gIycosides,” have long been known asconstituents of the Crucifera, to which order the Brassica genus belongs.The thiocyanate ion has antithyroid activity ; 27 it is a normal constituent ofthe saliva of many animals and it is metabolised by thyroid tissue, the sulphurbecoming bound in organic combination.This binding is inhibited by pre-treatment of the animal with another antithyroid substance, 2-thiouracil.It has been suggested that the thiocyanate ion may act by inhibiting com-petitively the enzyme which oxidises iodide ion to elementary iodine.2816 W. Griesbach, Brit. J . Exp. Path., 1941, 22, 246.17 H. D. Purves, ibid., 1943, 24, 171.18 V. I . E. Whitehead, ibid., p. 192.19 W. E. Griesbach and H. D. Purves, ibid., p.174.20 W. T. Salter, “ The Hormones,” Academic Press, New York, 1953, VoI. 11, p. 325.21 B. Webster and A. M. Chesney, Amer. J . Path., 1930, 6, 275.Sa F. Blum, Sckweiz. wed. Wochenschr., 1950, 80, 142.23 J. Matet, R. Montagne, and A. Buchy, Oleangheux, 1949, 303, 145.24 J. M. Bell, Canad. J. Agric. Sci., 1955, 35, 242.25 G. Klein and E. Farkass, osterr. botan. Z . , 1930, 79, 107.26 E. B. Astwood and M. M. Stanley, Trans. Amer. Assoc. Study Goiter, 1947, 216;M. M. Stanley and E. B. Astwood, Endocrinology, 1947, 41, 66.87 Cf. M. H. Wald, H. A. Lindberg, and M. H. Barker, J . Amer. Med. Assoc., 1939,112, 1120; R. W. Rawson, S. Hertz, and J. H. Means, Ann. Internal Med., 1943, 19,829; R. W. Robinson and J. P. O’Hare, New Engl. J .Med., 1939, 221, 964.28 J. L. Wood and E. F. Williams, J . Biol. Chem., 1949, 137, 592-MERCAPTO-A2-1 : 3-OXAZOLINES (2-THIO-1 : 3-OXAZOLIDINES) . 293Mustard oils, tested on rats, could not be shown to have any activity towardsthe thyroid.In 1938, Hopkins 29 isolated, by chloroform extraction of a water extractof seeds of hare's ear mustard (Coringia orientalis), a crystalline substance,C,H,ONS, m. p. 108.5". This was identified as 2-mercapto-5 : 5-dimethyl-A2-1 : 3-oxazoline (2a), which had just previously been synthesised by Brusonand Easte~,~* by treating l-amino-2-methylpropan-2-ol (1) with carbondisulphide and alkali. It seems to the Reporter that the substance may wellalso exist in the thione form (2b) as 5 : 5-dimethyl-2-thio-1 : 3-oxazolidine.This substance (2) was subsequently 31 shown to have antithyroid activityabout one-fifth of that of 2-thiouracil (3a) (2-mercaptouracil, 3b).H,C-CMe, ~ C S L Hs7-FMe2 ~ H2CrM42 I I I IHN\ PI F H,N OH -"" "\'c/oIn 1948 Greer and Astwood32 fed 61 different foodstuffs to humanvolunteers and assessed any antithyroid action by measuring the diminutionof 131 I-uptake by their thyroids.Of the foods tested the Swedish turnip(Brassica napobrassica) (U.S.A., rutabaga) was the most active. Materialsfrom the following plant orders were also markedly potent : Chenopodiacea,Compositz, Cruciferae, Cupuli ferae, Juglandacez, Leguminosz, RosaceE,and Umbelliferze. In addition, cow's milk, beef liver, and oysters showedsome activity.The active principle of swedes could be extracted into ether from anaqueous extract of the fresh plants and was contained in the water-solublefraction of the evaporated ether phase.In some instances, e.g., swedes andpears, the antithyroid activity is lost on cooking; in others, e.g., peas andground-nuts the antithyroid activity is then retained.33 In 1949, Astwood,Greer, and Ettlinger isolated an active material from aqueous extracts offresh swedes. These extracts gave a pink or purple colour with the Grotenitroprusside reagent 35 and a strong ultraviolet absorption maximum at240 mp ; both these characteristics closely paralleled the antithyroid activity.The active substance was obtained crystalline by high-vacuum distillation,followed by chromatography on alumina; it had m.p. 50", [ a ] ~ -70" inMeOH, and the elementary composition C,H,ONS, No compound of thisformula and having the above properties had been previously described.The structure (4a), (-)-2-thio-5-vinyl-l : 3-oxazolidine, was ascribed to itfrom chemical and physical evidence. It should be noted that, like its6 : 6-dimethyl analogue (2), it may exist also as (4b) 2-mercapto-5-vinyl-A2-oxazoline. The ultraviolet spectrum of the 5-vinyl compound is virtuallyidentical 54 with that of its 5 : 5-dimethyl analogue. (Astwood, Greer, andEttlingera state that the spectrum of the latter given by Hopkins29 isXI C. Y. Hopkins, Canad. J. Res., 1939, 16, B, 341.80 H. A. Bruson and J . W. Eastes, J. Amer. Chem. SOC, 1937, 59, 2011.81 E. B.Astwood, A. Bissel, and A. M. Hughes, Endocrinology, 1945, 37, 456.8% M. A. Greer and E. B. Astwood, ibid., 1948, 43, 105.88 M. A. Greer, M. G. Ettlinger, and W. B. Astwood, J. Clin. Endocrinol., 1949, 9,84 E. B. Astwood, M. A. Greer, and M. G. Ettlinger, J. Biol. Chem., 1949, 181, 121.88 I. W. Grote, ibid., 1931, 93, 25; B. H. Chase and J . Walker, J., 1955, 4443.1069294 BIOLOGICAL CHEMISTRY.incorrect.) It seems to the Reporter that it has not been finally settledwhether these two compounds, when crystalline, exist as thiols (2a and 4b)H HH @C 0 HC"'C 0 H7C-C H. c H: cH, H 2C -CH* C H: C H,I ! I I A ' 'Qn i SH(3 0 ) (3 6) (4 0) (46)HN\ /NH N + / J H H N , p - ior thiones (2b and 4a). It is to be regretted that (-)-2-thio-5-vinyl-l : 3-oxazolidine is now appearing in the literature with the designation " L "since no evidence of the configuration of the asymmetric centre at hasso far been presented (the error is improper use of L for the I originally usedto indicate laevorotation).(-)-2-Thio-5-vinyl-l : 3-oxazolidine was synthesised by Ettli~~ger.~G3 : 4-Epoxybut-1-ene (5), on ammonolysis, gave 37 a mixture of l-amino-but-3-en-2-01 (6) and 2-aminobut-3-en-1-01 which were separated by meansof their hydrogen oxalates. The former, on treatment with carbon disulphideand alkali in 45% dioxan,3O yielded the racemic compound (an or b) whichwas resolved, to afford its (-)-enantiomorph, by (+)-a-bromocamphor-x-sulphonic acid.This synthesis has been improved by Raciszewski et aZ.38who have also recorded analyses for rape seed meal.- (44 H zC,-,CH.CH:CHa NHJ_ H z ~ - ~ . ~ ~ : ~ ~ z CszI IHaN OH 0(5) ( 6 )Astwood et aZ.,34 using the absorption maximum at 240 mp, assayed anumber of Brassica species and obtained the results in the annexed Table;they assumed the absorbing material to be the thione (4a). No such absorp-Material (g./kg.) absorbing at 240 mp :Plant giving extract range meanSwede ....................................... 0.8-8.6 2.5Turnip .................................... 0.3-2.5 1.0Cabbage .................................... 0.24.7 1.5Rape ....................................... 1.8-2.1 1.9Brussels sprouts ........................ 0.5-0.8 0.7Kale ....................................... 0.9-6.3 4.4Broccoli .................................... 1.6Kohlrabi ....................................0.7-1.4 1.1Cauliflower & mustard .................. - 0tion was found in plant extracts from Raphanus (radish), Lobularia,Matthiola, Iberis, Nasturtium, Lepidum, Avabis, Cheiranthus, and Lunaria.The possibility that acetylenic and ethylenic constituents (cf. section onNatural Long-chain Fatty Acids) of plants might have an antithyroidaction through their capacity to bind iodine should not be overlooked.The natural occurrence of nitriles is another possibility since some nitrilesare known to be goitrogenic.Origin of Plant Heterocyclic Goitrogens.-It seems that neither compound(2) nor compound (4) exists as such in the plant's tissues. On the other341 M.G. Ettlinger, J . Amer. Chem. SOC., 1950, 72, 4793.37 R. G. Kadesah, i b i d . , 1946, 88, 41.38 2. M. Raciszeweski, E. Y. Spencer, and L. W.Trevoy, Canad. J . Tech., 1955,33,129~-MERCAPTO-A~-~ : 3-OXAZOLINES (2-THIO-1 : 3-OXAZOLIDINES) . 295hand, plants, especially members of the Cruciferze, are known to contain" mustard oil glycosides " (" thioglycosides "),39 which are isothiocyanatederivatives of the general structure (7). The mustard oils apparently donot exist free in the plant's tissues, but arise by enzymic breakdown of theglycosides. R is usually ethylenic or arylalkyl. E.g., compounds areknown in which R is ally1 and crotonyl, and a butenyl isothiocyanate was firstobtained by Sjollema40 in 1901 who suggested that it was the but-3-enylderivative.Ter Meulen 41 proposed the name '' gluconapin " for the hypo-thetical parent glycoside but did not obtain the substance in a state ofpurity. " Gluconapin " 26 is frequently met in literature relating to mustardoils, but, as far as the Reporter can find, the substance has never been isolated.Sjollema's isothiocyanate is also referred to as a " crotonyl " d e r i ~ a t i v e , ~ ~a name which would ordinarily be applied to the but-2-enyl compound.The inference by J. M. Bellx that Matet, Montagne, and Buchy26 haveisolated " gluconapin " from rape seed is incorrect. It has therefore beensuggested that the 2-thio-1 : 3-oxazolidines (or 2-mercapto-A2-1 : 3-oxazol-ines) might originate by cyclisation of alkenyl isothiocyanates liberated onhydrolysis of the parent glycosides.hop kin^,^^ assuming the existence of a2-methylallyl isothiocyanate glycoside, giving the mustard oil (8), hassuggested the annexed mechanism for the formation of 2-mercapto-5 : 5-(7) (8)dim et hyl- A2- 1 : 3-oxazoline. However, since ( + ) -sec. -but yl isot hiocyanat ehas been obtained from plant ~ o u r c e s , ~ ~ ~ ~ ~ this substance might form theprecursor of (2a or b) by oxidative ring-closure (see below). Liebermann(personal communication to Pitt-Rivers) 44 has suggested an analogous,but oxidative, ring closure of but-3-enyl isothiocyanate (9) to (-)-2-thio-5-vinyl-1 : 3-oxazolidine. This mechanism produces an asymmetric centre atSince the chemical mechanisms in biological systems frequently followpaths quite different from those of classical organic chemistry there is afairly strong possibility that the above suggestions may prove to be invalid.Synthetical Oxazoline Derivatives of Monosaccharides.-It will be ofinterest if the 2-mercapto-oxazoline derivatives of sugars 45 have anti-thyroid activity, since their solubilities in oily tissues should be much lessthan those of the compounds (2) and (4).The sulphur atom in these mono-saccharides, and presumably also in the thio-oxazolines (2) and (4), isremoved by mild ~ x i d a t i o n . ~ ~it therefore appears to require an enzyme to carry it out.D. J. B.39 Cf. A. L. Raymond, Adv. Carbohydrate Chem.. 1945,l. 129.40 B. Sjollema, Rec. Trav. chim., 1901, 20, 237.4 1 H.ter Meulen, ibid.. 1905, 24, 444.42 Cf. E. Andre and M. Kogan-Charles, Comfit. rend., 1944, 218, 1002; 1946, 222,43 W. Bottomley and D. E. White, Roy. Austral. Chem. Inst. J . Proc., 1950, 17, 31.44 R. Pitt-Rivers, Physiol. Rev., 1950, 30, 194.4L J. C. P. Schwarz, J., 1954, 2644.4 8 G. ZemplCn, A. Cerecs, and M. Rados, Ber., 1936, 60, 748.201 ; E. Andre and P. Delaveau, ibid., 1950, 231, 872; cf. refs. 26, 32296 BIOLOGICAL CHEMISTRY.4. NATURAL LONG-CHAIN FATTY ACIDS: SATURATED AND UNSATURATED.Though aspects of the chemistry of natural long-chain fatty acids arereferred to annually in the “ Organic Chemistry ” section of these Reports,and fatty-acid metabolism in animal tissues was reviewed two years ago,lthe topic as a whole has not been treated in these Reports for many years.While attention is focused on progress during the past year, it has beennecessary to make many references to earlier work, particularly that of thepreceding five years.Progress towards a more precise knowledge of complexlipids depends on building a sound foundation of chemical information oncomponent fatty acids and on the perfection of rapid and accurate methodsfor separation, identification, and estimation. The present report concen-trates particularly on these aspects and includes reference to some of themost recent papers on the metabolism and function of long-chain fatty acids.Recent Literature.-Three volumes of the series ‘‘ Progress in theChemistry of Fats and Other Lipids ” and a second volume of Deuel’s newwork3 are now to hand.Vegetable oils and fats have been dealt withunder a biological clas~ification.~ Meara discusses fats and other lipids ;a small but admirable volume by Lovern is available, as is Piskur’s compre-hensive annual review of the literature on fats.’ Progress in fatty acidchemistry was reviewed by Hilditch in 1953. Hutt proposes a classifica-tion scheme for lipids.General Methods and Separation Techniques.-Fractional distillation ofesters lo and low-temperature crystallisation l1 continue to be’much usedas analytical and preparative tools, and useful information on fatty acidsolubilities at low temperature is available. l2 Urea complexes for segre-gation, and protection from oxidation, of fatty acids and glycerides havebeen reviewed l3 and are widely emp10yed.l~ Fractionation of fatty acidcyclohexyl esters as their thiourea complexes is feasible.15 Adducts of1 4nn.Reports, 1953, 50, 301.2 Progress in the Chemistry of Fats and Other Lipids,” Pergamon Press, London,a H. J. Deuel, “ The Lipids. Their Chemistry and Biochemistry,” Interscience Publ.4 E. W. Eckey,6 M. L. Meara i:“‘ Modern Methods of Plant Analysis,” Springer, Berlin, 1954.6 J. A. Lovern,7 M. M. Piskur, J . Amer. Oil Chemists’ SOG., 1955, 32, 255, 319.8 T. P. Hilditch, Awn. Rev. Biochem., 1953, 22, 125.s H. H. Hutt, Nature, 1955, 175, 303.10 K. E. Murray, ref. 2, Vol. 111, p. 243; E. W. Jones and M. A. Maclean, J . Amer.11 J. B. Brown and D. K. Kolb, ref. 2, Vol. 111, p. 57.1.2 D. K.Kolb and J. €3. Brown, J . Amer. Oil Chemists’ SOC., 1955, 52, 357.l3 H. Schlenk, ref. 2, Vol. 11, p. 243.14 E.g.. J. S. Heckles and L. H. Dunlap, J . Amer. Oil Chemists’ SOG., 1955, 32, 224;0. E. McElroy, W. Jordan, J. McLaughlin, and M. E. Freeman, ibid., p. 286; C. Domart,D. T. Miyauchi, and W. N. Sumenvell. ibid., p. 481 ; Y. D. Karkhanis and N. G. Magar,ibid., p. 492; W. F. Shipe, J . Assoc. OBc. Agvzc: Chemzsts, 1955,38, 156; J. M. Martinez-Moreno, F. Mazuelos, and C. Janer, Fette u. Sezfen, 1955, 57, 652; T. N. Mehta, C. V. N.Rao, B. Y. Rao, and K. S. Rao, J . Indian Chem. SOC., I n d . News, 1954,17, 177; T. N.Mehta, B. Y . Rao, G. S. Prabhu, and G. S . Sihota, ibid., p. 182; T. N. Mehta, B. Y.Rao, and S. M. Abhyankar, ibid., 1955,18, 1 ; K.D. Pathak, and J. S. Aggarwal, J . Sci.I n d . Res., India, 1955, 14, B, 229; K. T. Achaya, B. P. Baliga, S. A. Saletore, andS. H. Zaheer, ibid., p. 348; R. Rigamonti and W. Riccio, Gazzetta, 1955, 85, 521.16 H. Schlenk, J. A. Tillotson, and B. G. Lamp, J . Amer. Chem. SOG., 1955, 77,5437.Vol. I, 1952; Vol. 11, 1954; Vol. 111, 1955.Inc., New York, Vol;,I, 1951 ; Vol. 11, 1955.Vegetable Fats and Oils,” Reinhold, New York, 1954.The Chemistry of Lipids of Biochemical Significance,” Methuen,London, 1954.Oil Chemists’ SOG., 1954, 31, 473CROMBIE : NATURAL LONG-CHAIN FATTY ACIDS. 297linoleic acid, linolenic acid, or methyl linolenate formed with “ a-dextrin ”(cyclohexa-amylose), “ p-dextrin ” (cyctohepta-amylose) , or deoxycholicacid are resistant to oxidation l6 and less toxic than urea adducts in feedingexperiments.Protection is due either to the crystal lattice’s offering abarrier to free penetration of oxygen, or to its preventing operation of aradical-chain oxidation.Separations of fatty acids 1’ and related substances by countercurrentextraction were reviewed 18 in 1954 and there is present interest in glyceride~eparati0ns.l~ Chromatography of fatty acids has presented difficulties,but recent progress has been made by methods involving columns or papersheets; column methods may be considered as (a) elution, (b) displacement,or (c) partition chromatography. Some work has been done on frontalanalysis.20 Elution methods 21 applied to preparative separations of fattyacids or esters are of limited value : the coloured 2 : 4-dinitrophenylsulphenylchloride addition products of unsaturated acids can be chromatographed onmagnesium sulphate.22 Methods for displacement and carrier chromato-graphy have been perfected by H ~ l m a n , ~ ~ but, though powerful and welltested, they require expensive ancillary equipment.Nonnal-phase partitionsystems have had marked success (cf. Nijkamp 24), though perhaps the mostuseful at the present time are the reversed-phase partition systems. Anumber of these are based on fundamental work by Howard and Martin 25who use paraffin-loaded non-wetting kieselguhr as the stationary phase, andalcohol-water or acetone-water mixtures as the mobile phase. Rubber 26or Polythene powder 27 may be used as stationary phase (Polythene is usefulfor long-chain acids) : hydroxy-acids are best chromatographed on non-wetting kieselguhr impregnated with castor Reversed-phase partitionmay be employed for saturated even-chain acids from C, to CZ4; the be-haviour of a wide variety of unsaturated acids has also been examined.29This technique is useful for ascertaining the selectivity of hydrogenationduring preparation of cis-long-chain fatty acids, as the acetylenic, ethenoid,and saturated acids are usually separable.30 Combined with hydrogen-ation31 and controlled the method is valuable for analysis ofl6 H.Schlenk, D. M. Sand, and J. A. Tillotson, J . Amer. Chem. SOL, 1955,77,3587.1’ E. H. Ahrens and L. C . Craig, J . Biol. Chem., 1952, 195, 299.18 H.J. Dutton, ref. 2, Vol. 11, p. 292.lQ E. S. Perry and G. Y . Brokaw, J . Amer. Oil Chemists’ SOC., 1955, 39, 191; J. J.Taber, Diss. Abs., 1955, 15, 727.20 Ref. 2, Vol. I, p. 110.21 H. G. Cassidy, J . Amer. Chem. SOC., 1941, 83, 2735; R. W. Riemenschneider,S. F. Herb, and P. L. Nicols, J . Amer. Oil Chemists’ SOC., 1949, 26, 371; H. J. Duttonand C. L. Reinhold, ibid., 1948, 25, 117, 120; M. M. Graff and E. L. Skau, Ind. Eng.Chem. Analyt., 1943, 15, 340.22 R. 0. Simmons and F. W. Quackenbush, J . Amer. Oil Chemists’ Soc., 1953,30,614.as R. T. Holman, ref. 2, Vol. I, p. 104.2c H. J. Nijkamp, Nature, 1953, 172, 1102; Analyt. Chim. A d a , 1954, 10, 448.25 G. A. Howard and A. J. P. Martin, Biochem. J., 1950, 48, 532.27 T. Green, F. 0.Howitt, and R. P. Preston, Chem. and Ind., 1955, 591.28 P. Savary and P. Desnuelle, Bull. SOL chirn. France, 1953, 939.aQ W. M. L. Crombie, R. Comber, and S. G. Boatman, Natare, 1954, 174, 181;Biochem. J.. 1955. 59, 309.so L. Crombie, J., 1955, 3510.s1 G. Popjak and A. Tietz, Biochem. J., 1954, 56, 46; M. H. Silk and H. H. Hahn,ibid., 1954, 67, 677; J. Boldingh in “ Biochemical Problems of Lipids,’’ Internat.Coll., Brussels, 1953.J. A. Boldingh, Experientia, 1948, 4, 270; Rec. Trav. chim., 1950, 69, 247TABLE 1. Column chromatography.Methoddetection Ref. Class22 AStationary phaseA1,0, (40)-" Celite " (7)MgSO,SiO,-MeO*[CHJ ,*OH-H,O (9 : 1)Si0,-furfuryl alcohol-2-aminopyridineSi0,-MeOHCellulose powder-hypophaseKieselguhr-liq. paraffinMobile phaseC,H6-Etz0 (95 : 5 )n-C,H 1Z-EtzOSkellysolve B-Bun,On-C6H 14" isoOctane "Species chromatd.derivs.of 2 : 4-( NO,),C,HS*SHalMe estersAcidI, (salt)ColourWeighingTitrationI,Bromothymol-(on column)Fe (C10,) 3Titration32 A33 N.P.P.34 N.P.P.24 N.P.P. 8 1Hydroxamic acidAcid35 N.P.P. EpiphaseCOMe,- or EtOH-C OMe,-H ,OH2O25 R.P.P.28 R.P.P. Kieselguhr-liq. paraffinKieselguhr-castor oilKieselguhr-liq. paraffinor -cycZohexaneI 8 I,I,28 R.P.P. I J I ,36 R.P.P.29 R.P.P.,II,MeOH<OMe,-C,H 6-COMe,-H,OHZO26 R.P.P.27 R.P.P.23, 37 D.A.23,38 D.A.23,39 C.S.Rubber powder" Polythene " powderDzrco G. 60 tubon (1) :Supercel (2)I,,I Interferometry EtOHI-Hab, etc.E t OH-H 2OEtOH-H,O (95 : 5) +carrier (fatty acidesters)8 1I D I, 8 1a A, adsorption ; N.P.P., normal phase partition ; R.P.P., reversed phase partition ; D.A., displacementb Be., behenolic; Bs., brassidic; El., elaidic ; Er., erucic; Es., elaeostearic; Ey., erythrogenic;leic ; LInc., linolenic ; Oc., octadec-2-enoic ; Ol., oleic ; Ps., petroselinic ; Pto., palmitoleic ; Rl.,stearic ; Un., undecylenic ; Xm., ximenynic ; C,-C,4 denotes saturated even-chain acids of chainc From water-acetic acid-methanol-hexane ( 5 : 1 : 50 : 50).Also dihydroxy- and dibromoTABLE 2. Paper chromatography.Ref.2641424344454647484950515253545569Stationary phase +paper Mobile phaseRubber latex MeOH-COMe, (1 : 1)Petroleum MeOH-H,OPetroleum (b.p. 190- AcOH-H,O (9 : 1)Paraffin Pentanol, hexanol, orOlive oil (or tristearin,chloronaphthalene,etc. )Paraffin AcOHParaffin (Nu jol) MeOH or EtOH-H,OPetroleum (b. p. 140- MeOH-petrol or -ace-Footnote 1 EtOH-tetrahydrofuran-Olive oil EtOH-H,O (3: 1) orParaffin AcOH-H,O (9 : 1)Footnote 2 CC1,-MeOH-NH,Footnote 3Tetralin or petroleum 90% MeOH-AcOH-2200)octanolLower alcohols(9 : 1)170") tone (3 : 1)-petrolH,O (0.6 : 3 : 5 )Pr'OH-H,O (4 : 1)(81 : 18 : 1) (epiphase)MeOH-H,O (4 : 2 to 9 : 1)satd. with decalintetralin (60 : 20 : 11) orMeOH-AcOH- petro-leum(b. p. 140-170")Petroleum (b. p. 190- AcOH-H,O (9 : 1)Paraffin or rubber MeOH-H,O ( 4 : 1 tolatex 19 : l ) , MeOH-H,O(4 : 1) saturated withcyclohexane220O)Silicone AcOH-H,O (17 : 3)Species chromatd.EstersAcidJ .NH,Me or NH,EtsaltAcid,I t? JHydroxamic acidAcidJ JJ JI ,AcOHg-derivAcidAcid or esterSudan IVCu(OAc), + eriochrom-Rhodamine B, Cu(OAc),-IndicatorsDetection of spotcyanin, etc.K,Fe(CN),, etc.AgN0,-NH,-(NH,),Scuso,AgN03-NH3-(NH4)Cu(OAc),-K,Fe(CN),Bromocresol-green,Ferric saltAgNO, (renewedAgN03-(NH4)2SNa,S, etc.)Cu (OAc) ,-K,Fe (CN)Rhodamine BBromothymol-blueMeO*[CH,] ,*OHDiphenylcarbazone-0.05N-HNOSCu(OAc),-K,Fe(CN),Pb(OAc),-H,S or rhodi-zonic acid, or bromo-th ymol-bluePb(OAc), or iodinea-Dextrin-iodine or0 Unsaturated acids also examined as hydroxylated compounds or iodine monobromide derivatives.b See footnote to Table 1.1, Acetylated paper. 2, Alum-treated paper + hypophase. 3, Cellulos300 BIOLOGICAL CHEMISTRY.natural mixtures of saturated and unsaturated acids. The more importantcolumn chromatographic methods are summarised in Table 1.Vapour-phase partition chromatography has been successfully applied 4*to even-numbered saturated fatty esters from C,, to Czz; it should beapplicable to some unsaturated fatty esters but it remains to be seen whetherit is suitable for the sensitive polyunsaturated compounds.Paper chromatography of fatty acids is rapidly developing and itsimportance need hardly be stressed. Methods are summarised in Table 2.Most of them involve a reversed-phase system with filter paper impregnatedwith liquid paraffin, petroleum hydrocarbon, silicone, olive oil, rubber,tristearin, etc., as the stationary phase.Baker 52 uses an octadecyloxy-methyl ether of cellulose, supporting decalin. Spot development is not easyand a number of methods have been employed : besides those in the Table,alkaline permanganate will detect 1 pg. of unsaturated acid, osmium tetr-oxide 5 pg., and Kaufmann’s foam test 56 5-10 pg. (the acid is convertedinto its copper salt and a foam is produced when it is treated with hydrogenperoxide in ammonia). If the paper is treated with 6oCo ions, these are fixedby the acid spots which can then be detected by a radi~autograph.~’ Re-tention analysis is successful with larger quantities (0-2-1.0 mg.) of acid.s8Photometric determination of fatty acids on paper, using the copper acetate-potassium ferrocyanide method for spot-development, has been achieved.54Recently the use of ‘‘ a-dextrin ” followed by treatment with iodine has beenrecommended for locating saturated and unsaturated acids or esters.59The reagent gives an inclusion compound with the fatty acid spot which,unlike free “ a-dextrin,” gives no colour with iodine. Lead tetra-acetate oriodine vapour may be used to detect unsaturated acids.59 Inouye and his32 D. R. Howton, Science, 1955, 121, 704.33 V. Zbinovsky, Analyt. Chem., 1955, 27, 764.34 L. L. Ramsey and I. Patterson, J . Assoc. O@c. Agric. Chemists, 1948, 31, 441.35 J. B. Davenport, Chem. and Ind., 1955, 705.36 M. H. Silk and H. H. Hahn, Biochem.J . , 1954, 56, 406.37 R. T. Holman and L. Hagdahl, J . Biol. Chem., 1950, 182, 421.38 R. T. Holman and W. T. Williams, J . Amer. Chem. Soc., 1951, 73, 5285.30 R. T. Holman, J . Amer. Chem. SOC., 1951, 73, 1261.40 F. R. Cropper and A. Heywood, Nature, 1953, 172, 1101;G. Dijkolra, J. G. Keppler, and J. A. Schols, Rec. Trav. chim., 1955, 74, 805.41 H. P. Kaufmann, J. Budwig, and C. W. Schmidt, Fette u. Seifen, 1952, 54, 10.42 H. P. Kaufmann and W. €3. Nitsch, ibid., 1954, 56, 154; 1955, 57, 473.43 L. A. Liberman, A. Zafarronia, and E. Stotz, Fed. Proc., 1951, 10, 216.44 G. Nunez and J. Spiteri, Compt. rend., 1952, 234, 2603; Bull. SOC. Chim. biol.,45 J. Spiteri, ibid., 1954, 36, 1355.46 P. F. Ceccaldi, R. Wegmann, and J. Biez-Charreton, ibid., p.415; Fette u.47 Y. Inouye and M. Noda, J . Agric. Chem. SOC. Japan, 1952,26, 634; 1953, 27, 50.48 F. Micheel and H. Schweppe, Angew. Chem., 1954, 66, 136.49 V. Kobrle and R. Zahradnik, Chem. Listy, 1954, 48, 1187, 1703.60 0. Perila, Acta Chem. S a n d . , 1955, 9, 864.61 A. Holasek, Angew. Chem., 1954, 66, 330.63 R. G. Baker, Biochem. J . , 1953, 54, xxxix.63 Y. Inouye, M. Noda, and 0. Hirayama, J . Amer. Oil Chemists’ SOL, 1955, 32, 132.64 H. Wagner, L. Abisch, and K. Bernhard, Helv. Chim. Acta, 1955, 38. 1536.65 B. D. Ashley and U. Westphal, Arch. Biochem. Biophys., 1955, 56, 1.66 H. P. Kaufmann and J. Budwig, Fette u. Seifen, 1950, 52, 555.57 Idem, ibid., 1951, 53, 69.68 H. P. Kaufmann, ibid., 1950, 52, 331, 713.69 H. K. Mangold, B.G. Lamp, and H. Schlenk, J . Amer. Chem. SOC., 1955,77, 6070.1954, 174, 1063;1953, 35, 851.Seijen, 1954, 56, 159CROMBIE : NATURAL LONG-CHAIN FATTY ACIDS. 301co-workers have studied the paper chromatography of hydroxamic deriv-atives of unsaturated acids.60 Paper electrophoresis (Table 3) has not beenparticularly useful as yet.61968Ultraviolet spectrophotometric methods 63 for determining (in con-junction with alkaline isomerisation) methylene-interrupted conjugatedfatty acids * have been further examined.65 Infrared spectra arevaluable for estimation and detection of functional groups, but homologousfatty acids are not readily distinguished and different crystal farms havedifferent spectra: 67 the spectra of the sodium salts are sometimes morediagnostic.68 Various X-ray studies are reported.ggTABLE 3. Paper electrophoresis.Ref. Mobile Species Detn. of Approx. of acidTypesClo-Clephase chromatd. spot range (pg.) examined61 Aq. NH, Salt Methyl-red-bromothymol- 80blue62 0-2N-NaOH in glycerol ,, Cu(OAc),-Rhodamine B - c6-c16(90”)Methods for determining lipoperoxides are availableD70 and Bolley 71reports on the chemical determination of unsaturation in fats : Mukherjee 72uses hypochlorous acid for the latter purpose and, with Chowdhury, describesconditions for evaluating a “ true iodine number ” for conjugated acids.73An analytical method for component acids of oils containing epoxy- orhydroxy-acids is available.’* Small quantities of fatty esters (0.2-3.0ymole) may be estimated by treatment with alkaline hydroxylamine andferric perchlorate, followed by photometric measurement at 520 mp.75Reviews of the autoxidation of unsaturated fatty acids and related* Methylene-intzrrupted conjugation refers to systems of type *CH:CH*CH,*CH:CH*.6o Y.Inouye and M. Noda, J . Agric. Chem. SOC. Japan, 1950, 23, 368; 1952, %,61 A. J. G. Barnett and D. K. Smith, Nature, 1954, 174, 659; J . Sci. Food Agric.,62 0. Perila, Acta Chem. Scand., 1955, 9, 1231.ti3 R. T. O’Connor, J . Amer. Oil Chemists’ SOG., 1955, 32, 616, 624.64 W. J. Gensler and A. P. Mahadevan, J . Amev. Chem. SOC., 1955, 77, 3076.6s S. F. Herb, J. Amer. Oil Chemists’ Soc., 1955, 32, 153; J. Moretti and R. .I.Cheftel, Bull. SOC. Chim. biol., 1955, 37, 699; D. Firestone, J .Assoc. Ofic. Agrzc.Chemists, 1955, 38, 657; K. A. Narayan and B. S. Kulkami, J . Indian Chem. SOC., Ind.News Edn., 1964, 17, 79.a6 D. H. Wheeler, ref. 2, Vol. 11, p. 268.6 7 R. G. Sinclair, A. F. McKay, and R. N. Jones, J . Amer. Chem. Soc., 1952, 74,2570, 2575; E. von Sydow, Acta Chem. Scand., 1956, 9, 1119.68 E. Childers and G. W. Struthers, Analyt. Chem., 1955, 27, 737.6Q E. von Sydow, Acta Cryst., 1955, 8, 557; J. Fridrichsons, Austral. J . Chem.,1955,8, 339; A. R. Verma, Proc. Roy. SOG., 1955, A , 227, 34; D. Swern, L. P. Witnauer,S. A. Fusari, and J. B. Brown, J . Amer. Oil Chemists’ SOC., 1955,32, 539; T. R. Lomer,Nature, 1955, 175, 653; T. Malkin, ref. 2, Vol. I, p. 1.70 J. Glavind and S . Hartmann, Acta Chem. Scand., 1955, 9, 497; A.M. Siddiqi andA. L. Tappel, Chemist-Analyst, 1955, 44, 6 2 ; C. B. Kenaston, K. M. Wilbur, A. Otto-lenghi, and F. Bernheim, J . Amer. Oil Chemists’ Sot., 1955, 52, 33.71 D. S. Bolley, ibid., p. 235.72 S. Mukherjee, ibid., p. 351.73 R. B. Chowdhury and S. Mukherjee, ibid., p. 484.74 K. E. Bharucha and F. D. Gunstone, J. Sci. Food Agric., 1955, 6, 373.75 M. H. Hack, Arch. Biochew. Biophys., 1955, 58, 19; R. Nailor, F. C. Bauer, andGensler et al. 64 use491 (Chem. Abs., 1952, 48, 6408).1955, 6, 63.skipped double bonds.”E. F. Hirsch, ibid., 1955,5;4, 201302 BIOLOGICAL CHEMISTRY.substances are available; 76 while it is not possible here to deal adequatelywith this subject there is much current intere~t.~' Monohydroxystearicacids can be obtained by catalytic hydrogenation of peroxidised methyloleate and oleic acid.78 Under certain conditions of hydrogenation, migra-tion of the double bond of oleic acid occurs equally in each direction to givea 1 : 2-cis-trans-equilibrium mixture of positional isomers : 79 a partial-hydrogenation-dehydrogenation theory is used to explain the results.Sodamide in liquid ammonia reacts with methyl linoleate producing acomplex mixture of conjugated and non-conjugated esters and amides :the conjugated compounds are cis-trans with a little trans-trans.80 Linolenategives the cis-trans-diene and stereoisomeric trienes.Long-chain Saturated Fatty Acids.-Until recently it was assumedthat unbranched acids with odd-numbered chains rarely occurred in mam-malian or fish lipids.A number of examples have now come to light,though the acids occur only in small amount and failure to detect them has,at least in part, been due to inadequate techniques for separation. n-Nona-decanoic acid can be obtained from hydrogenated ox perinephric fat.81rt-Heptadecanoic (margaric) and pentadecanoic acid are found in shark liveroil : 8, butter fat also contains the latter acid, together with n-tridecanoicand rt-undecanoic acids. 83 Hydrogenated mutton fat has yielded rt-hepta-decanoic and pentadecanoic acid,s4 and odd-numbered acids from C, to C,can be detected in hydrogenated ox tallow.85 An unsaturated odd-numberedacid, n-heptadec-9-enoic acid occurs in lamb-caul fat. 86Besides the even- and odd-numbered straight-chain acids, branched-chain acids having iso- (Me,*CH*CH,*) or anteiso- (MeEt-CHCH,.) endgroups are probably widely distributed in animal fats, usually in only smallconcentrations.Weitkamp's pioneer work 87 on wool wax resulted in theisolation of thirty-two acids from this source, ten being iso-acids, eleven(+)-artteiso-, and the remainder hydroxy- and normal acids. His work hasstimulated synthetic investigations in these groups.8876 W. Kern and H. Willersinn, Angew. Chem., 1955, 67, 573; R. T. Holman, ref. 2,Vol. 11, p. 51.77 Intev alia, N. A. Khan, J . Chem. Phys., 1954, 22, 2090; Biochim. Biophys. Ada,1955, 16, 159; N. A. Khan, W. E. Tolberg, D. H. Wheeler, and W. 0. Lundberg, J .Amer. Oil Chemists' SOC., 1954, 31, 460; D.H. Saunders, C. Ricciuti, and D. Swern,ibid., 1955, 32, 79; J. E. Coleman, H. B. Knight, and D. Swern, ibid., p. 135; A. L.Tappel, ibid., p. 252; S. S. Kalbag, K. A. Narayan, S. S. Chang, and F. A. Kummerow,ibid., p. 271 ; 0. S. Privett, C. Nickell, W. 0. Lundberg, and P. D. Boyer, ibid., p. 505;W. Kern, A. R. Heinz, and J. Stallmann, Makromol. Chem., 1955, 16, 21; W. Kernand H. Willersinn, ibid., 1955, 15, 1, 15, 36; Y . Toyama and K. Suzuki, J . Chem. SOC.Japan, Ind. Chem. Sect., 1955, 58, 52.'8 J. E. Coleman and D. Swern, J . Amer. Oil Chemists' SOC., 1955, 32, 221.79 R. R. Allen and A. A. Kiess, ibid., p. 400.81 R. P. Hansen, F. B. Shorland, and N. J. Cooke, Nature, 1955, 176, 882.82 I. M. Morice and F. B. Shorland, Biochem. J ., 1955, 81, 453.83 F. B. Shorland, T. Gerson, and R. P. Hansen, ibid., 1955, 59, 350; R. P. Hansen,84 Idem, Biochem. J . , 1954, 58, 513, 516.85 R. P. Hansen and A. G. MacInnes, Nature, 1954, 173, 1093.86 F. B. Shorland and A. S. Jessop, ibid., 1955, 176. 737.87 A. W. Weitkanip, J . Amer. Chem. Soc., 1945, 67, 447; S. F. Velick, ibid., 1947,J. R. Nunn, J., 1951, 1740; F. W. Hougen, D. Ilse, D. A. Suttpn, and J. P.A. M. Abu-Nasr and R. T. Holman, ibid., p. 414.F. B. Shorland, and N. J. Cooke, Chem. and Ind., 1955, 92.69, 2317.de Villiers, J . , 1953, 98; A. H. Milburn and E. V. Truter, J . , 1954, 3344CROMBIE : NATURAL LONG-CHAIN FATTY A4CIDS. 303Hansen, Shorland, and their collaborators have made extensive searchesfor branched-chain acids in a number of animal fats.By use of low-temper-ature crystallisation, distillation, chromatography, and hydrogenation,butter fat has been shown 89 to contain (+)-12-methyltetradecanoic,* 13-methyltetradecanoic, and 12-methyltridecanoic acid, two acids isomeric withheptadecanoic acid (one apparently of the iso- and the other the anteiso-series), and other branched-chain acids. The isolation of these acidsinvolved a hydrogenation step, but objections that the acids may be artefactsderived from unsaturated or cyclopropane acids have been met in part byisolation of (+)-12-methyltetradecanoic (0.43y0), 13-methyltetradecanoic(0.37y0), and n-pentadecanoic acid (O-82y0) by distillation and chromato-graphy on1y.m (+)-lO-Methyldodecanoic (0.01 %) and 1 l-methyldodecanoicacid (0.05%) also occur in butter fat.91Carcass fats of sheep yield small amounts of (+)-14-methylhexadecanoic(+)-l Z-methyltetradecanoic, 13-methyltetradecanoic, 10-methyldodecanoic,and a liquid saturated branched C,, acid.92 Ox perinephric fat gives (+)-14-methylhexadecanoic, 15-methylhexadecanoic (0.06y0),93 and 14-methyl-pentadecanoic a ~ i d .~ 4 Branched-chain acids occur in the preen glands ofducks 95 and in shark liverThe alzteiso-compounds (+)-14-methylpalmitic acid (1 ; n = 12) 97 and(+)-6-methyloctanoic acid (1 ; n = 4) 98 have been related to natural(-)-2-methylbutanol (" active amyl alcohol ") (2) and there is little doubtthat all the natural (+)-alzteiso-acids are configuratively of the " L-"series tand are related to natural isoleucine (3) as shown.99 Little is as yet knownCOiH CO2H --rH CHiOH I+& IMr-C -H Mc-C-H Mr-C-HI I IEt Et Et(441) (-)-(4 (+)-PIof the biosynthesis of the odd-numbered and branched-chain acids, thoughthe .addition of acetate units to propionate (known to be converted intovaleric acid in the rumen) loo would give the former,g0 and addition of acetateunits to isovalerate (or isobutyrate) and (+)-2-methylbutyrate would yield,F.B. Shorland, R. P. Hansen, and N. J. Cooke, Biochent. J . , 1954, 5S, 358;1953,53,374; Chem. and Ixd., 1951,839; R. P. Hansen and F. B. Shorland, Biochem.J . , 1952, 50, 207, 358.F. B. Shorland, T. Gerson, and R. P. Hansen, ibid., 1955, 59, 350.Idem, ibid., 1955, 61, 702.82 F. B. Shorland, R.P. Hansen, and N. J. Cooke, ibid., 1952, 52, 203; 1953, 58,93 Idem, Biochem. J., 1955, 61, 141.95 G. Weitzel and K. Lennert, 2. physiol. Chem., 1951, 288, 251.O 6 I. M. Morice and F. B. Shorland, Chsm. and Ind., 1952, 1267.O 7 S. F. Velick and J. English, J . Biol. Chern., 1946, 160, 473.88 L. Crombie and S . H. Harper, J . , 1950, 2685.loo F. V. Gray, A. F. Pilgrim, H. J. Rodda, and R. A. Weller, Nature, 1951,167, 954. * Geneva nomenclature (C02H = 1) is used in this Report.-f For the significance of D and L see R. P. Linstead, J. C. Lunt, and B. C. L. Weedon,374; Chem. and Ind., 1953, 516; 1954, 1229.Idem, ibid., p. 547.Idem, Chem. and Ind., 1950, 757; W. Klyne, Biochem. J., 1953, 53, 378.J . , 1950, 3333, and W. Klyne, Chem. and Ind., 1951, 1022304 BIOLOGICAL CHEMISTRY.respectively, the iso- and anteiso-seriesW, lol These acids may arise fromdeamination of the corresponding amino-acids.101An interesting study of human sebum waxes is reported : sebum containslong-chain alcohols of three types-saturated normal, saturated iso-, andunsaturated normal.lo2 Hougen points out that in many natural waxes theacids and alcohols are structurally related, suggesting a common biosyntheticprocess (thus wool wax contains both acids and alcohols of the n-, iso-,artteiso-, and u-hydroxy-series) .lO3 This relation does not exist between thecombined alcohols of sebum and the free fatty acids though the combinedacids have not yet been examined.A useful review of bacterial fattyacids,lo5 and studies of the lipids of typhus and diphtheria bacteria lo6and of a group C Strefitoco~czcs,~~~ are available.Mycoceranic acid fromtubercule bacilli lipids is provisionally regarded as 2 ( 0 ) : 4(0) : 6(D)-tri-methyloctacosanoic acid and the 2 : 4 : 6(D)-compound has been syn-thesised.1O8 In connexion with mycolipenic acid [(+)-2 : 4 : 6-trimethyl-tetracos-2-enoic acid], isolated from the same source, syntheses of a numberof long-chain 2-methylalk-2-enoic acids are described : lo9 1 : 2- and 1 : 3-diglycerides of 2-methyloctadec-2-enoic acid are available.110 Mycolicesters and amides from amino-sugars 111 are of interest in connexion withBloch’s toxic lipid “ cord factor ” 11, and palmitic 113 and mycolic 114 esters ofsugars have been synthesised.There is synthetic interest 115 in mycolicacids of the general formula R*CH,-CH(OH)*CHR*CO,H, and anothersynthesis of (-j-)-tuberculostearic acid has been carried out.116Thioctic (c4poic) acid occurs as a conjugate in the lipid fraction fromScenedesmus obliqztus : l 1 7 several syntheses of this and related compounds,including the preparation of the 35S acid, are reported.118Long-chain Unsaturated Fatty Acids.-Unsaturated fatty acids presentan outstanding problem to the biochemist since little is known about their101 F. €3. Shorland, ref. 2, Vol. 111, p. 276.102 F. W. Hougen, Biochem. J., 1955, 59, 302; see also Shkng-Lieh Liu, J . Chinese103 H . W. Knoll, J . Amer. Oil Cltemists’ SOC., 1954, 31, 59.104 A. W. Weitkamp, A. M. Smiljanic, and S.Rothman, J . Amev. Chem. SOL, 1947,106 J. Asselineau and E. Lederer, “ Fortschritte der Chemie organischer Natur-106 S. Cmelik, 2. physiol. Chem., 1955,299,227; 1955,300,167; 1955,302,20.107 K. Hofmann and F. Tausig, J . Biol. Chem., 1955, 213, 415.108 G. S. Marks and N. Polgar, J., 1955, 3851.10s J. Cason and M. J. Kalm, J . Org. Chem., 1954, 19, 1836, 1947; A. S. Bailey,110 G. I. Fray and N. Polgar, J., 1955, 1802.111 J. Asselineau and E. Lederer, Bull. Soc. chim. France, 1955, 1232.112 H. Bloch, J . Exp. Med., 1950, 91, 197; H. No11 and H. Bloch, J . Bid. Chem.,113 J . Asselineau, Bull. Soc. chim. France, 1955, 937.114 U. Eisner, J. Polonsky, and E;,Lederer, ibid., 1955, 212.115 J. Asselineau and E. Lederer, Experimental Tuberculosis,” Colloquium, Ciba116 M.Sy, Ng. Ph. Buu-Hoi, and Ng. D. Xuong, Compt. vend., 1954, 239, 1813.117 R. C. Fuller, H. Grisebach, and M. Calvin, J . Awzer. Chem. Soc., 1955, 77, 2659.118 E. A. Braude, R. P. Linstead, and K. H. R. Wooldridge, Chem. and Ind., 1955,508; A. Campbell, J., 1955, 4218; L. J. Reed and Ching-I Niu, J . Amer. Chem. SOL,1955, 77, 416; A. F. Wagner, E. Walton, C. H. Hofmann, L. H. Peterson, F. W. Holly,and K. Folkers, ibid., p. 5140; E. Walton, A. F. Wagner, F. W. Bachelor, L. H. Peter-son, F. W. Holly, and K. Folkers, ibid., p. 5144; P. T. Adams, ibid., p. 5357; R. C.Thomas and L. J. Reed, ibid., p. 6446.Chem. Soc. (Formosa), 1954, Ser. 2, 1, 71.69, 1936.stoffe,” Springer, Wien, 1953, Vol. X, p. 170.N. Polgar, F.E. G. Tate, and A. Wilkinson, J., 1955, 1547.1955, 214, 251.Foundation, London, 1955CROMBIE : NATURAL LONG-CHAIN FATTY ACIDS. 305metabolism and much has yet to be learnt about the specific roles which someof them play in biological processes. Synthetic work is of basic interest inthat it offers the means of testing biochemical hypotheses by 14C-labelling atselected points in the chain.Useful anodic routes 119 have been added to existing syntheses of oleicacid : in one, adipic half-ester is cross-coupled with an acetylenic acid andthe product semihydrogenated :MefCH,],GC*[CH2J3*C02H + HO,C*[CH,],*CO,MeC Me*[CH,],*GC-[CH,],*CO,Me Me*[CH2],*CH=CH*[CH2],*C0,H(In such formulz, c denotes cis, and t trans.)Octadec-cis- and -trans-1 l-enoic acid have been prepared by anodicmethods 120 and are of interest in that the cis-acid is the hzemolytic factorfrom horse-brain 121 and is also the principal unsaturated fatty acid inLactobacillus arabinosus and L.casei.122 “ Vaccenic acid ” is believed to bea mixture in which octadec-trans-1 1-enoic acid predominates. Hufrnannand Tausig 123 make the interesting suggestion that the l 1 vaccenic acid ’’ ofanimal fats may arise from absorption of octadec-cis-1 l-enoic acid elabor-ated by intestinal bacteria, stereomutation occurring during absorption ortransport. The recent work of Hartman and his collaborators 1% is relatedto this suggestion; using infrared analysis they find that ruminants havesubstantial amounts (3.5-1 102%) of tram-unsaturated acids in their fatsbut non-ruminants have less than 0.9%.Marsupial fats contain 18.1-2100% of trans-acid. According to Hartman et al. the trans-acids arisemainly from hydrogenation of dietary unsaturated acids by bacteria in therumen, or in the rumen-like stomach of the marsupials.The above synthetic method for octadec-trans-1 I-enoic acid employsprotection of the olefinic linkage as a dihydroxy-derivative. The anodictechnique is used in the anodic synthesis of tetracos-cis- and -trans-15-enoicacid (the cis-compound is nervonic or selacholeic acid) : 125 here the startingmaterials are oleic and elaidic acid. Tariric, petroselinic, erucic, and brassidicacid can be prepared by the anodic route, and natural eicos-ll-enoic acid isshown to have the Synthetic cis- and trans-isomers ofmyristoleic, palmitoleic, gadoleic, octadec-4-enoic, undec-9-enoic acid, aswell as erucic, brassidic, oleic, and elaidic acid, are accessible by Ames andBowman’s general methods.127The gross structure of stillingic acid (deca-2 : 4-dienoic acid) from theseed oil of Sapium sebiferum (stillingia oil) 128 has been confirmed by syn-llo 13.W. Baker, R. P. Linstead, and B. c. L. Weedon, J., 1955, 2218.120 D. G. Bounds, R. P. Linstead, and B. C. L. Weedon, J., 1954, 4219.121 I. D. Morton and A. R. Todd, Biochem. J., 1950, 47, 327.lZ2 K. Hofmann, R. A. Lucas, and S. M. Sax, J . B i d . Chem., 1952, 195, 473; K.l Z 3 K. Hofmann and F. Tausig, ibid., 1955, 213, 425.124 L. Hartman, F. B. Shorland, and I. R. C .McDonald, Biochem. J . , 1955, 61, 603.125 D. G. Bounds, R. P. Linstead, and B. C. L. Weedon, J., 1954, 448,1g6 R. P. Linstead, B. C. L. Weedon, and B. Wladislaw, J., 1955, 1097; B. W. Baker,127 R. E. Bowman, J., 1950,177; B. W. Boughton, R. E. Bowman, and D. E. Arnes,128 A. Crossley and T. P. Hilditch, J., 1949, 3353.Hofmann and S. M. Sax, ibid., 1953, 205, 55.R. W. Kierstead, R. P. Linstead, and B. C. L. Weedon, J., 1954, 1804; 1953, 2393.J., 1952, 671; D. E. Ames and R. E. Bowman, J., 1951, 1079; 1952, 677306 BIOLOGICAL CHEMISTRY.thesis. Four possible stereoisomers have been prepared 129 and the trans-2 : cis-4-acid is identical with the natural acid. Stillingic acid is the firstpolyethenoid acid containing less than sixteen carbon atoms to be isolatedfrom a glyceride.Its homologue, dodeca-2 : 4-dienoic acid (stereochemistryunknown), has recently been found in the seed oil of Sebistiana lingustrina.lmA cis9 : trans-1 1 : trans-13-configuration was recently proposed 131 fora-elzeostearic acid (4) from an infrared study of the maleic anhydride adduct(in conjunction with earlier oxidation work and other evidence). Thisconfiguration, and the structure of the acid, have now been established bythe annexed synthesis132 Isomerisation with iodine and ultraviolet lightgives the all-iruns-(p)-form.Me*[CH,],-CH=CH*CHO + Rr*CH,*CCH -- Me*[CH,],*CH=CH*CH (OH) -CH,*CCHtPBr,.d Me.[CH,],*CH=CH*CH=CH*GCH __tMefCH,] ,*CH=CH*CH=CH*GC*[CH,] ,*C1 -KOHNaI- t t ,-+ Me*[CH,],.CH=CH*CH=CH-GC*[CH,],*CO,HKCN-OH- t t C Me*[CH,],*CH=CH*CH=CH*CH=CH,I,.CO,HUseful methods for marking the 1-carboxy-group of natural unsaturatedfatty acids are available. [l-14C]Oleic acid is prepared by Hunsdiecker(silver salt-bromine) degradation of er-ythro-9 : 10-dihydroxystearic acid(from natural oleic acid) to the nor-1-bromo-compound, followed by[14C]nitrile synthesis and stereospecific elimination to regenerate the cis-9 : 10-double bond.133 Linoleic acid is brominated and the carboxyl replacedby bromine by the Hunsdiecker reaction : debromination removes thecontiguous bromine substituents.The monobromide is then converted intoits Grignard reagent and carboxylated with 14C0,. 134A number of syntheses of the essential fatty acid, linoleic acid, appeareda few years ago 135 and a formal synthesis of linolenic acid has now beena~hieved.1~~ The route is as shown.The hexabromostearic acid ( 5 ) , m. p.180-181", was equated with material derived from natural linolenic acid.It can be debrominated to a-linolenic acid from which the natural all-cis-acidcan be obtained by crystallisation.Evidence is repeatedly obtained for the presence in Nature of unsaturatedfatty acids containing methylene-interrupted conjugation, other than thewell-known examples such as linoleic, linolenic, and arachidonic acid. ThelZg L. Crombie, J., 1955, 1007; Chem. and Iizd., 1952, 1034.130 D. P. Hanks and W. M. Potts, J . Amer. Oil Chemists' SOC., 1951, 28, 292; R. T.Holman and D. P. Hanks, ibid., 1955, 32, 356.131 R.F. Paschke, W. Tolberg, and D. H. Wheeler, ibid., 1953, 30, 97; W. G. Bick-ford, E. F. DuPr6, C. H. Mack, and R. T. O'Connor, ibid., p. 376; N. H. E. Ahlers,R. A. Brett, and N. G. McTaggart, J . Appl. Chem., 1953, 3, 433.l32 L. Crombie and A. G. Jacklin, Chem. and Ind., 1955, 1186.13, S . Bergstrom, K. Paabo, and M. Rottenberg, Acta Chem. Scand., 1952, 8, 1127.134 D. R. Howton, R. H. Davis, and J. C . Nevenzal, J . Amer. Chem. SOC., 1952, 74.1109.135 R. A. Raphael and I;. Sondheimer, J., 1950, 2100; W. J. Gensler and G. R.Thomas. J . Amer. Chem. SOC., 1951, 73, 4601; H. M. Walborsky, R. H. Davis, andD. R. Howton, ibid.. p. 2590; R. A. Raphael, " Acetylenic Compounds in OrganicSynthesis," Butterworths, London, 1955, p. 89.(4)136 S. S . Nigam and B.C. L. Weedon, Chem. and Ind., 1955, 1665CROMBIE : NATURAL LONG-CHAIN FATTY ACIDS. 307acids are highly unstable and difficult to purify but there is little doubt thatthey are biologically important, and intensification of effort in this exactingfield is much to be desired. The techniques employed for isolation ofhexadeca-6 : 9 : 12 : 15-tetraenoic acid from pilchard oil are worthy ofMC.CH~*C=C*CH~B~ + HCaC=CHpPy MC-EH~*C=C]~*CH~P~ &,O-CH2,O-CH2 O-CH2Me *EH2.CIC], .CH2Br ?, Me*[CHI.C~],.[CH,],*C~ I &Me *[CH2-CH+CH], *[CH&-CH I & Me .CtH2gCHBr.CHB33*EH,];C02H'O-CHa ( 5 )Py = tetrahydropyranyloxy.Reagents : 1, CuC1. 2, Hf-PBr,. 3, X.Mg*CZC*[CH,],*CH 'O-p'. 4, H,-Lindlar.5, Br,; oxidn.\O-CH,st~dy.13~ In the past year Klenk and Dreike 138 have found evidencefor eicosa-5 : 8 : 11- and -8 : 11 : 14-trienoic, eicosa-5 : 8 : 11 : 14- and-8 : 11 : 14 : 17-tetraenoiq docosa-7 : 10 : 13 : 16 : 19-pentaenoic, and docosa-4 : 7 : 10 : 13 : 16 : 19-hexaenoicacid, as well as linoleic and linolenic acid, in liver phosphatides.Theglycerophosphatides of brain contain eicosa-5 : 8 : 11 : 14-tetraenoic (ara-chidonic) acid and eicosa-5 : 8 : ll-and -8 : 11 : 14-trienoic acid, togetherwith eicosa-1 1 : 14-dienoic acid.139 Considerable quantities of docosa-4 : 10 : 13 : 16-tetraenoic and docosa-4 : 7 : 10 : 13 : 16 : 19-hexaenoic acidhave been isolated by counter-current distribution from the mixture of C,,polyene-carboxylic acids from glycerophosphatides of brain.140 Smallamounts of docosa-4 : 7 : 10 : 13 : 16-pentaenoic acid, and probably adocosatrienoic acid are present.Natural Hydroxy-acids.-There is current interest in natural hydroxy-acids. 18-Hydroxyoctadec-9-enoic acid is obtainable from cork 141 and anew synthesis of phloionic acid (9 : 10-dihydroxyoctadecanedioic acid), alsoZnMe*[CH,],.CHO + Br*CH,*CZCH __t Me*[CH,],CH(OH)*CH,*C~CH *eicosa-5 : 8 : 11 : 14 : 17-pentaenoic,__t Me.[CH,],*CH(OH)CH,-CZCfCH,],Cl+Me-[CH,],CH (OH) *CH,*CH=CH*[CH,] ,*CO,H (6)C* Hydroxy-group protected as the pyranyloxy-derivative during chain extension.obtainable from cork, has been pub1i~hed.l~~ A series of even-numberedw-hydroxy-acids of chain lengths 18-30 inclusive, together with even-numbered ao-diols of chain lengths 22-28, occur in carnauba wax.143 Fulldetails of a synthesis of (-+)-ricinoleic acid (6) have appeared.lU A second13' M.H. Silk and H. H. Hahn, Biochem. J., 1964, 57, 582.138 E. Klenk and A. Dreike, 2. j?&ysioZ. Chem., 1955, 300, 113.139 E. Klenk and F. Lindlar, ibid., 1955, 301, 156.I4O Idem, ibid., 1955, 299, 74.1 4 1 I. Ribas and E. Seoane, Anales Fis. Quim., 1954, 50, B, 963.14% W. J. Gensler and H. N. Schlein, J . Amer. Chem. SOC., 1955, 77, 4846.143 K. E. Murray and R. Schoenfeld. AustraZ. J. Chem., 1955, 8, 432, 437.144 L. Crornbie and A. G. Jacklin, Chem. and Ind., 1954, 1197; J., 1956, 1740308 BIOLOGICAL CHEMISTRY.synthesis of the racemic acid has been briefly rep0rted.1~~ It seems likelythat natural (+)-ricinoleic has the D-~onfiguration.1~~ As ricinoleic acid issimply converted into octadec-trans-1 l-en-9-ynoic acid,147 these synthesesalso give a total synthesis of ximenynic acid which is obtained from seed fatsof Ximenia and Santalum genera (Olacacea) .148 An isomer of ricinoleicacid, 9-hydroxyoctadec-12-enoic acid, is present in the seed oil of Strofihanthussarmentosus 149 and 8-hydroxyoctadec-trans-1 l-en-9-ynoic acid in that ofXimeniar caffra.150 The structure and stereochemistry of the a- and the@-form of kamlolenic acid from the nuts of Mallotus phiZip+inensis (Euphor-biacea) seems clear : the a-form is 18-hydroxyoctadec-cis-9 : trans-11 : trans-13-trienoic acid and the @-form the all-trans-isomer.151 Vernolic acid fromVernonia anthelmintica (Compositeae) is 12 : 13-epoxyoctadec-9-enoic (anepoxylinoleic) a ~ i d .1 ~ ~cycEoAlkane Acids.-The lipids of the plant pathogen, Agrobacterium(Phytomonas) tumefaciens, contain a liquid fatty acid long thought to be 10-or ll-methylnonadecanoic acid : it is in fact the cyclopropane compoundlactobacillic acid (7) The latter had previously been isolated from Lacto-bacillus arabinosus and L. casei : 122 there is a speculation on its bio-synthesis.153 A . tumefaciens is also the richest natural source of octadec-cis-1 l-enoic acid.Closely related to lactobacillic acid is sterculic acid from the kernel oil ofSterculia fatida. Nunn 154 proposes a cyclopropene structure (8) but thishas been challenged by Indian workers 155 who prefer structure (9).Theseauthors do not explain Nunn’s evidence, obtained by ozonisation (whichgives a 1 : 3-diketone), and some of their criticisms are open to objection.Mat *EHJ7 HC -CH$H,I, * CO2HM ~ + H J ~ * c,=,c EH~],*co~H(7)(8)\ ICH2CH2Me *EH& * HC -CH*CH=CH pHd7 CO2H (9)\ /Chaulmoogric acid (10) can be synthesised by anodically cross-coupling(+)-cyclopent-2-enylacetic acid with methyl hydrogen bra~sylate.1~~ De-gradation of the (+I-cyclopentenylacetic acid (11) gives the (-)-tricarboxylic145 V. G. Kendall, P. B. Lumb, and J. C. Smith, Chem. and Ind., 1954, 1228.146 K. Serck-Hanssen and E. Stenhagen, Acta Chem. Scand., 1955, 9, 866.147 J. Grigor, D. M. MacInnes, J. McLean, and A. J. P. Hogg, J., 1955, 1069.148 S. P. Ligthelm and H. M.Schwartz, J. Amer. Chem. Soc., 1950, 72, 1868; S. P.Ligthelm, H. M. Schwartz, and M. M. von Holdt, J., 1952, 1088; M. H. Hatt and A. 2.Szumer, Chem. and Ind., 1954, 962; F. D. Gunstone and M. A. McGee, ibid., p. 1112.149 F. D. Gunstone, J., 1952, 1274.150 S. P. Ligthelm, Chem. and Ind., 1954, 249.151 Ifiter alia, S. D. Gupta and J. S. Aggarwal, J. Amer. Oil Chemists’ Soc., 1955,32, 501; R. C. Caldenvood and F. D. Gunstone, Chem. and I n d . , 1953, 436; N. H. E.Ahlers and F. D. Gunstone, ibid., 1954, 1291 ; R. C. Calderwood and F. D. Gunstone,J. Sci. Food Agric., 1954, 5, 382; L. Crombie and J. L. Tayler, J., 1954, 2816; J. D.von Mikusch, Deut. Farben. 2.. 1954, 8, 166.lS2 F. D. Gunstone, J., 1954, 1611.153 E . M . Kosower, Science, 1951, 113, 605.J.R. Nunn, J.. 1952, 313; cf. G. DijkstraandH. J. Duin, Nature, 1955,176, 71.1 S 6 1. P. Verma, B. Nath, and J. S. Agganval, ibid., 1955, 175, 84; 1955, 176, 1082.166 K. Mislow and I. V. Steinberg, J. Amer. Chem. Soc., 1955, 77, 3807CROMBIE : NATURAL LONG-CHAIN FATTY ACIDS. 309acid (12) which can be configuratively correlated, through shikimic acid,with glyceraldehyde. The (-)-acetic acid (11) was also correlated withglyceraldehyde, through (+)-Z-ethylglutaric acid, by the displacementprinciple. It seems reasonable to suppose that gorlic acid and the naturallower homologues have the same configuration as chaulmoogric acid, and thatthe correlation with glyceraldehyde is valid for the whole g r 0 ~ p . l ~ ~fA synthesis of (&)-dihydrohydnocarpic acid (13), a s shown, is re~0rted.l~'( -J-)-Dihydrochaulmoogric acid is obtained by a similar route.Alepresticacid has also been ~ynthesised.1~~Reagents : 1, SnC1,-thiophen. 2, Wolff-Kishner. 3, AIC1,-(CH,*CO),O.4, Wolff-Kishner ; Raney Ni.Natural Acetylenic Acids.-Some chemical aspects of this subject havebeen reviewed.159 A group of methyl esters of related acetylenic acids occursin the Compositeae; Sorensen and his school have greatly extended ourknowledge of them. cis-Lachnophyllum ester (14) is found in manyplants of the Erigeron genus and it is believed that all true Erigerons containthis and lor, matricaria ester.161 trans-Lachnophyllum ester is found 162in the root oil of the common daisy (Bellis perennis) and has been synthesisedby Glaser c0up1ing.l~~ Its isomer methyl dec-cis-8-ene-4 : 6-diynoate (15) isconsidered to be present in the flower oil of Matricaria inodora and probablyother plants.164Pr*C-=CH + HEC.CH=CH*CH,-OH ~_t PrGC*EC*CH=CHCH,.OHPr=CGC*CH=CH.CO,Me (14)Two of the four possible stereoisomers of matricaria ester (16) are knownin Nature-the cis : cis- in Matricaria inodora and the cis-2 : trans-8- inM . inodova and Amellus stvigosus.164 The trans-trans- and the trans-2 : cis-8-compound are synthetically available by coupling tram-pentenynol with15' Ng. Ph. Buu-Hoi', M. Sy, and Ng. D. Xuong, Compt. vend., 1955, 240, 785.16* B. Wladislaw, J . , 1955, 4227.lB0 W. W. Wiljams, V. S. Smirnov, and V. P. Goljmov, J. Gen. Chem. (U.S.S.R.),lB1 G.M. Tronvold, M. Nestvold, D. Holme, J . S. Sorensen, and N. A. Sorensen,lBa D. Holme and N. A. Sorensen, ibid., 1954, 8, 280.163 T. Brunn, C. M. Haug, and N. A. Sorensen, ibid., 1950, 4, 851.164 K. S. Baalsrud, D. Holme, hl. Nestvold, J. Pliva, J. S. Sorensen, and N. A.F. Bohlmann, Angew. Chem., 1955,67. 389.1935, 5, 1195; N. A. Sarensen and J. Stene, Annalen, 1941, 549, 80.Acta Chem. Scand., 1953, 7 , 1375.Sorensen, ibid., 1952, 6, 883; P. K. Christiansen and N. A. Sorensen, ibid., p. 893310 BIOLOGICAL CHEMISTRY.trans- and cis-pentenyne.166 Jones and his collaborators 1G6 have recentlyshown that trans : trans-matricaria methyl ester occurs in Polyporus anthra-cophilus cultures : deca-2 : 8-diene-4 : 6-diynedioic acid occurs in the samematerial.A dehydromatricaria ester (17) or (18) is obtainable fromArtemesia vulgaris : 167 investigation of the spectra of model compoundsMe*CH=CH*CC.C=C*CH,*CH,*CO,Me (1 5)Me*CH=CH*GC*CEC*CH=CH*CO,Me (1 6)Me*CH=CH*GC*CEC*CC*CO,Me (17)Me*CC*CEC*CC*CH=CH*CO,Me (18)n-C,H ,,*CH=C=C=CH*CO,Me (19)suggests that it is cis-form (18).lG8 The trans-isomer, synthesised by oxid-ative coupling,169 has also been found in Nature (M. oreades and M.i n o d ~ r a ) . ~ ~ ~ A cumulene ester, believed to have structure (19) occurs in thetMeGC-CCH + HEC*CH=CH.CO,Me __t trans-( 18)essential oil of M. inodora 171 and probably in other C o m p o ~ i t e a . ~ ~ ~ Re-lated acetylenic alcohols, ketones, and hydrocarbons occur in this family butdiscussion is outside the scope of the present Report, The distribution ofacetylenic compounds amongst the tribes of the Compositeze, and species ofthe Erigeron genus is summarised by Sorensen 173 and a study of this aspecthas use in botanical classification.The antibiotic amide agrocybin, recognised by Anchel174 as a poly-yne,occurs in the culture liquid of Agrocybe dura and is known by synthesis 175to have structure (20). From Clytocybe diatreta a half amide (21) is ob-tained : 176 synthesis 176 confirms its structure.The corresponding nitrile(22) also occurs n a t ~ r a l l y . 1 ~ ~ A remarkable acid, mycomycin (23), has beenisolated from the culture fluid of Norcardia acidophilus : on treatment withdilute alkali an allene-acetylene rearrangement sets in to give the isomer(24),178 the structure of the latter having been confirmed by synthesis :179HEC-Li + OHC*CH=CH*CH=CHa HEC*CH(OH)CH=CH*CH=CH,PBr, CU-HC5C*CH=CHCH=CH*CH2Br HCEX*CH=CH.CH=CH*CHa.CNHCNHCzCGC-Me MeOH--w MeGC-CZC*EC*CH=CH*CH.CH,.CN __Q (24)H+~~ ~165 T.Bruun, P. K. Christiansen, C. M. Haug, J. Stene, and N. A. Sorensen, Acla.166 J. D. Bu’Lock, E. R. H. Jones, and W. B. Turner, Chem. and Ind., 1955, 686.187 K. Stavholt and N. A. Sorensen, Aclu Chem. Scand., 1950, 4, 1567.168 F. Bohlmann and H.-J. Mannhardt, Chem. Ber., 1955, 88, 429.P. K. Christiansen and N. A. Sorensen, Acta Chem. Scand., 1952, 6, 602.170 J. S. Sorensen, T. Bruun, D. Holme, and N. A. Sorensen, ibid., 1954, 8, 26.1 7 1 N. A. Sorensen and K. Stavholt, ibid., 1950, 4, 1080.1 7 2 Idem, ibid., p. 1575.1 7 3 N.A. Sorensen, Chem. and Ind., 1953, 240.174 M. Anchel, J . Amer. Chem. SOC., 1952, 74, 1588.1 7 5 E. R. H. Jones and J. D. Bu’Lock, J., 1953, 3719; J. D. Bu’Lock, E. R. H.Jones, G. H. Mansfield, J. W. Thompson, and M. C. Whiting, Chem. and Ind., 1954, 990.1 7 6 M. Anchel, J . Amer. Chem. SOC., 1953, 75, 4621; M. Anchel and M. P. Cohen,J . Biol. Chem., 1954, 208, 319.1 7 7 M. Anchel, Trans. N . Y . Acad. Sci., 1954, 16, 337; Science, 1955, 121, 607.1 7 8 W. D. Celmer, and I. A. Solomons, J . Amer. Chem. Soc., 1952, 74, 1870, 2245,3838; 1953, 75, 1372.1711 F. Bohlmann and H. G. Viehe, Chem. Bey., 1954, 87, 712.Chem. Scand., 1951, 5, 1244CROMBIE : NATURAL LONG-CHAIN FATTY ACIDS. 31 1Nemotinic acid is found to be the hydroxy-acid (25) ; 180 it occurs togetherwith the corresponding lactone, nemotin.HOCH ,-CC* C-=C.C-=C *CO*NH ,HO,C*CH=CH*CXGC.CO.NH,HO,C*CH=CH*GC-CCCNC tHC-CGC*CH=C=CH*CH=CH-CH=CH.CH,-CO,H (23)t tMeC*EC*CC*CEC*CH=CH CH=CH *CH,*CO,H (24)HEC*EC*CH=C=CH*CH (OH) -CH,*CH,*CO,H (25)Besides these acetylenic acids, others occur as glycerides in the seed fatsof higher plants : tariric acid (26) was in fact the first acetylenic substanceto be recognised in Nature.181 Its structure is confirmed by twosyntheses.182.126 Ximenynic and 8-hydroxyoctadec-trans-1 l-en-9-ynoic acidhave already been referred to (p.308). The structure for the conjugateddiyne, erythrogenic (isanic) acid (27), found in the seed fats of Onguekoaspecies, is now on a firm basis since its synthesis 183 by cross-coupling ofdec-9-ynoic acid with octa-l-ene-7-yne.Natural erythrogenic acid is knownto contain an incompletely described enediyne impurity named bolekicacid.184 An acetylenic acid, isanolic acid, has been isolated 185 from thesame oil ; the ascribed structure (28) 186 requires confirmation.MefCH,] lo*~C~[CH2],*C02H (26)CH,=CH.[CH2]4*CC*CC*[CHz],*C0,H (27)Pr~CH=CH*CC*G~C*CH,*CH(OH)*[CH,],*CO,H (28)In the past, acetylenic acids have tended to be regarded as mere curiosi-ties. The steadily mounting list, and their occurrence in the highest andlowest groups of plants, indicate that more serious biochemical attentionmust be paid to them. In particular, work on the metabolism of theacetylenic linkage is overdue and its relation, if any, to the olefinic linkage, isin need of study.Natural isoButy1amides.-A distinct group of plant acids occur asisobutylamides : the latter have insecticidal and sialogogue properties buttheir role in plant metabolism is not known. Jacobson 187 has shown thatsynthetic N-isobutyldeca-tram-2 : trans-6 : trans-8-trienamide is identicalwith the isomerisation product of affinin (from HeZzoPsis Zongipes) whichmust be one of the stereoisomers (29).The structure originally proposed 188for herculin (from Zanthoxylzcm claraherculis bark), N-isobutyldodeca-1 : 8-dienamide (30), is incorrect as all four stereoisomers have been synthesisedand none is identical with the natural product.la9 neoHerculin (probably a180 J.D. Bu’Lock, E. R. H. Jones, and P. R. Leeming, J., 1955, 4270.181 A. Amaud, Compt. rend., 1892, 114, 79; Bull. SOC. cham. France, 1892, 7 , 233.182 P. B. Lumb and J. C. Smith, J., 1952, 5032.183 H. K. Black and B. C. L. Weedon, J., 1953, 1785.184 E. R. H. Jones, M. C. Whiting, J. B. Armitage, C. L. Cook, and N. Entwistle,185 H. P. Kaufmann, J. Baltes, and H. Herminghaus, Fette u. Seifen, 1951, 53, 537.l a 6 A. Seher, Annalen, 1054, 589, 222.187 M. Jacobson, J . Amer. Chem. Sot., 1954, 76, 4606; 1955, 77, 2461.189 R. A. Raphael and F. Sondheimer, J., 1950, 115; 1951, 2693; L. Crombie, J.,Nature, 1951, 168, 900.Idem, ibid., 1948, 70, 4234.1952, 2997; N. A. Dobson and R. A. Raphael, J., 1955,3558312 BIOLOGICAL CHEMISTRY.purer specimen of herculin) has been isolated from the same source andstructure (31) proposed.lW Echinacein (from Echinacea angustifolia) maybe similar to, or identical with, neoherculin.191 The proposed lg2 structure(32) for pellitorine from Anacyclus Pyrethrum root has also been disproved lg3by synthesis of the four stereoisomers.It is a mixture of at least threedifficultly separable substances of the type R*CH=CHCH=CH*CO*NHBui inwhich R is a C,, C,, and C, saturated or unsaturated unit.lgP A dienediyneisobutylamide anacyclin (33), isolated from the same r00t,194 is not insecti-cidal but becomes so on partial hydrogenation of the diyne system. Thecompound N-isobutyldeca-trans-2 : trans-4-dienamide is insecticidally activeagainst Musca domestica but the other three stereoisomers are less thanone-tenth as active.129, lg5t tMe*[CH=CH] aCH2CHa*CH=CH CO *NH Bu~Pm*CH=CH*[CH,] ,CH=CH *CO*NHBdMe*[CH=CH]s*CHaCHa*CH=CHCO*NHBu~Prn*CH=CH.[CH,]a.CH=CH*CO.NHBulPrn*[EC],*CH,*CH,*[CH=CH] ,*CO*NHBd (33)Pm.CH=CH CH,*CH,* [CHZCH] ,*CO*NHBu* (34)Me,CH*CH=CH*[CH,],*CO*NH*CH,.C,H,(OH)~OMe-l : 4 : 3 (35)Since N-isobutyldeca-trans-2 : trans-4 : trans-8- and -trans4 : trans-4 : cis-8-trienamide (34) have been synthesised,lg5 there is no doubt that sanshool I(from ZanthoxyZum pi$erit.um) does not possess the structure proposed byAiharalg6 or has never been obtained in a state approaching purity.Asynthesis of the branched-chain vanillin derivative capsaicin (39, the activeprinciple of red peppers, is reported.lQ7Analysis of Component Acids of Fats and Waxes.-This work occupiesinvestigators in many parts of the world and, apart from its commercialinterest, yields information useful for correlation with biological classification:if carried out critically it can lead to detection of new types of fatty acids.However, there is scope for the employment of newer and more searchingmethods of investigation.Chromatography seems promising and has beenapplied with good results to palm-kernel oil and watermelon-seed oil.29Hydrogenation, in conjunction with a chromatographic method, is anexcellent way of determining the distribution of chain lengths in a mixtureof acids.s1 Unsaturated acids may be separated and determined by hydr-oxylation with performic acid, followed by reversed-phase chromatographyon a rubber 1g8 or castor-oil column.199recently investigated are those from Hedera japonica Among plant oils190 L.Crombie, J., 1955, 995.1 9 1 M. Jacobson, Science, 1964, 120, 1028.192 Idem, J . Amer. Chem. Sac., 1949, 71, 366.193 R. A. Raphael and F. Sondheimer, J., 1950, 120; L. Crombie, J., 1952, 4338.104 Idem, J., 1955, 999.195 L. Crombie and J. D. Shah, J., 1955, 4244.197 L. Crombie, S. H. Dandegaonker, and K. B. Simpson, J., 1955, 1025.108 K. Hofmann, C.-Y. Yuan Hsiao, D. B. Henis, and C. Panos, J . Bid. Chew.,109 S . Bergstrom and K. Paabo, A d a Chem. Scand., 1954, 8, 1486.zoo See also T. Kashimoto, J . Chern. Sac. Japan, 1954, 75, 1110.T. Aihara, J . Pharm. SOC. Japan, 1950, 70, 405, 409.1955, 217, 49CROMBIE : NATURAL LONG-CHAIN FATTY ACIDS.313fruits,201 acorns,2o2 coconuts,2~ palm-kernelsJm and the seeds of AnamirtaC O C C U ~ U S , ~ ~ Mucuna @wiens,206 Courupita g u i a n e n ~ i s , ~ ~ Culophyllumwightianum ,208 Elletaria cardarnom~m,~~~ Hippopha rhamnoidesJ210 Hal-optella integrifolia,211 Desmodium gangeticum,212 Bombax s e ~ s i l e , ~ l ~ andLupinus terrnis.213 Analysis of oils from Aspergillus nidulans,214 fresh-waterplants,215 and seeds of the Crucifer= 216 and Cucurbitace~~~l~ as well as twowild Arachis species,218 are reported and there is new information on waxes(~arnauba,l*~ j~te,~19 hinoki leaf ,220 espa.rto,221 and onion-bulb 222).Acids from ckloroplast lipids have been investigated.2BReports are available on fats of the chimpanzee,224 puma,225 tiger,225snake,226 and and the rnesenteric fats of Leporinus o@nis 228and Pimelodus a l b i ~ a n s .~ ~ ~ Lipids of Ascaris lumbricoides,230 as well asshark the body fats of some marine fish,232 Clupea Pilchard~s,~3~Corbicula ~ a n d a i , ~ ~ ~ Lamonema r n o ~ o s u m , ~ ~ ~ and a number of aquaticinvertebratesJB6 have been investigated.and the ovarian dermoid cyst have been examined.238Lipids of globe-fish ovariesG. Kurono, T. Sakai, and I. Seki, Report. Fac. Pharm. Kanazawa Univ., 1954,4, 1.202 N. F. de Castro, Diss. Abs., 1955, 15, 44.203 A. P. Dale and M. L. Meara, J . Sci. Food Agric., 1955, 6, 162.204 Idem ibid., p. 166.205 2’. R. Kasturi and B. H. Iyer, J . Indian Chem.SOC., 1954, 31, 623.206 P. V. Nair and K. S. M. Pillai, Bull. Central Res. Inst., Univ. Travancore, 1954, A ,207 N. S. Bai, ibid., p. 114.208 K. V. Nair and N. S. Varier, ibid., p. 161.209 T. V. Kasturi and B. H. Iyer, J . Indian Inst. Sci., 1955, 37, 106.210 H. P. Kaufmann and A. V. Roncero, Grasas y Aceites, 1955, 6, 81, 129.211 M. 0. Farooq and M. S. Siddiqui, Fette u. Seijen, 1955, 57, 389.B. K. Avasthi and J. D. Tewari, Arch. Pharm., 1955, 288, 272.213 D. N. Grindley and A. A. Akour, J . Sci. Food Agric., 1955, 6, 461.214 J. Singh, T..K. Walker, and M. L. Meara, Biochem. J., 1955, 61, 85.215 M. I. Chechinkin, BioRhiuniya, 1955, 20, 249.a16 E. Andre and M. Maille, Compt. rend., 1954, 239, 1531.217 M. M. Chakrabarty, D. K. Chowdhury, and B.K. Mukherji, Naturwiss., 1955,42, 344; J . Amer. Oil Chemists’ SOG., 1955, 32, 384.218 T. A. Pickett. ibid., p. 521.219 W. G. Macmillan, A. B. Sen Gupta, and A. Roy, J . Indian Chew. Soc., 1955,32,79.220 T. Kariyone and K. Isoi, J . Pharm. SOC. Japan, 1955, 75, 316.221 A. SoIer and G. GuzmAn, Publ. Inst. Quim. Alonso BaYba, 1954, 8, 245.222 M. Okajima, J . Sci. Res. Inst., Tokyo, 1954, 48, 281.223 K. L. Zirm, A. Pongratz, and W. Polesofsky, Biochenz. Z., 1955, 326, 405.224 F. D. Gunstone, Biochem. J., 1955, 59, 454.225 Idem, ibid., p. 455.226 Y. D. Karkhanis and N. G. Magar, ibid., 1955, 60, 565.227 S. P. Pathak and G. D. Pande, J . Sci. Food Agric., 1955, 6, 48.228 R. R. Brenner, S. A. Quaglia, and P. Cattaneo, Anales Asoc. quim.argentina,229 R. R. Brenner, W. H. E. Reinke, and P. Cattaneo, ibid., 1955, 43, 67.230 D. Fairbairn, Canad. J . Biochem. Physiol., 1955, 33, 31, 122.231 S. P. Pathak and G. D. Pande, J . Amer. Oil Chemists’ SOC., 1955, 32,.7; S. P.Pathak and P. N. Suwal, ibid., p. 229; G. G. Kanath and N. G. Magar, J . Indzan Chem.SOC., 1955, 32, 455.232 Y. D. Karkhanis and N. G. Magar, J . Amer. Oil Chemists’ SOC., 1955, 32, 492.233 R. I. Cheftel, J. Moretti, and J. Polonovski, Bull. SOC. Chim. biol., 1955, 37, 709.234 T. Hori, J . Chem. SOG., Japan, 1954, 75, 1144.235 S. Komori and T. Agawa, Technol. Reports Osaka Univ., 1954, 4, 405.236 Y. Toyama and T. Takagi, J . Chem. SOC. Japan, 1955, 76, 237, 240, 243.as7 R. Umezawa, J . Pharm. SOC. Japan, 1955, ‘95, 494.238 Y .Takata, T. Matsuda, and Y . Takata, ibid., 1954 .74, 1401.3, 83.1954, 42, 1923 14 BIOLOGICAL CHEMISTRY.Metabolism and Function of Long-chain Fatty Acids.*-Excellent reviewsof recent advances in fatty-acid metabolism are available; 239 only a fewnew developments will be mentioned. Agreed systematic names for enzymesinvolved in the fatty-acid cycle are listed.239dThe sequence of events in the Lipmann-Lynen '' cycle " has been dupli-cated,m irreversibly, without the use of enzymes, but the chemical methodsemployed are remote from those possible under physiological conditions.They are summarised below. Steps in the (irreversible) chemical sequence12R*C0.NH*CH,*CH2-S.COMe z, R*CO*NH-CH,*CHz*S*CO*CH9*COMeR*CO.NHCH,*CH,*S.COCH,CH,Me[ + R-CO*NH*CH,*CH,*SH]3R*CO-NHCH,CH,*S*CO*CH=CHMe .% R*CO-NH*CH,CH,*S*CO*CH,*CHMe*OH[R = Me] are : 1, Claisen condensation with mesitylmagnesium bromide ;2, sodium borohydride ; 3, thermal dehydration on distillation ; 4, catalytichydrogenation.Enzymes of (reversible) biological " cycle " [R = APP(3-adenylpyrophosphoryl-4'-pantothenyl)] are : 1, p-ketoacylthiolase ; 2, p-hydroxyacyldehydrogenase ; 3, crotonase ; 4, acyldehydrogenase. (Donorsand acceptors are omitted.)A sequence of reactions analogous to the reversible CoA cycle, butinvolving pantetheine thioesters in place of those of CoA, has been an-n0unced.~~1 An interesting feature is the slow speed of hydration of trans-or cis-crotonylpantetheine under the influence of crystalline liver crotonase(0.013% and 0.0023% respectively of the rate for the corresponding reactionsinvolving CoA derivatives).Unlike the CoA cycle, the pantetheine cycle isnot directly linked to the citric acid cycle : it may be concerned in isoprenoidsynthesis.More information about the stereospecificity of two steps in the fatty-acidcycle is available. According to Stem and his colleagues,N2 crystallinecrotonase hydrates both cis- and trans-crotonylCoA to (+)-3-hydroxy-butyrylCoAc (as identified by enzymic assay) in the step :A stereospecific dehydrogenase from pig liver or rat heart oxidises (+)- butnot (-)-3-hydroxybutyrylCoA according to :Me*CH(OH)CH,*CO*CoA + DPN+ e, Me-COCH,*CO*CoA + DPNH + H+However, a racemase occurring in ox and rat liver can racemise (-)-3-hydr-oxybutyrylCoA, and the (+)-isomer is then oxidised.Crystalline crotonase239 ( a ) Ref. 1 ; (b) D. E. Green, Biol. Rev. Cambridge Phil. Soc., 1954, 29, 330;(c) F. Lynen, Ann. Rev. Biochem., 1955, 24, 653; ( d ) idem, Angew. Chem., 1955, 67,463; (e) E. R. Stadtman. Record Chem. Progress (Kresge-Hooker Sci. Lib.), 1954, 15, 1.Z4O J. C. Sheehan and C. W. Beck, J . Amer. Chem. SOC., 1955, 77, 4875.241 J. R. Stern, ibid., p. 5195.242 J. R. Stern, A. del Campillo, and A. L. Lehninger, ibid., p. 1073. * Abbreviations used are : CoA. coenzyme A ; AcCoA, S-acetyl-coenzyme A ;ATP. adenosine triphosphate ; DPN+ and DPNH, oxidised and reduced coenzyme I ;TPNH reduced coenzyme 11.MeCH=CH*CO*CoA + HzO MeCH(OH)*CH,-CO*CoA (+)-(36)( +)- (36CROMBIE : NATURAL LONG-CHAIN FATTY ACIDS.315can dehydrate (+)- but not (-)-(36) : the latter must first be treated withracemase. On the other hand, Wakil 243 reports that a fraction from beef-liver mitochondria (A) catalyses a DPN+ specific oxidation of (-)-3-hydroxy-butyrylCoA, and formulates the apparent racemisation as :A (-)-(36) + DPN+- - Me*CO*CH,*CO*CoA + DPNH + Hf(+)-(36) + DPN+ <B is the dehydrogenase specific for (+)-isomers.The hydration product of trans-crotonylCoA is oxidised by DPN+ in thepresence of B, whilst the hydration product of cis-crotonylCoA is not.However, in the presence of A, the latter product is oxidised by DPN+.Popj&k and Tietzw report that a soluble enzyme system, involvingneither mitochondria1 nor microsomal fractions, can be obtained from themammary glands of lactating rats; it effects synthesis of long- and short-chain fatty acids from acetate, has an absolute requirement for ATP, and isstimulated by oxaloacetate, a-oxoglutarate, succinate, and, particularly,malonate.The effect of poisons has been investigated and presence of apyruvic oxidase is inferred. A similar system synthesising fatty acids hasbeen obtained 245 from rabbit mammary glands where the rate-limitingprocess is one or both of the steps (i) from 3-hydroxyacylCoA to un-saturated acylCoA, (ii) from unsaturated acylCoA to saturated acylCoA.Experiments with a rat-liver preparation indicate that, in the bio-synthesis of fatty acids, TPNH may be needed as a specific electron donor inthe reduction of unsaturated acylCoA derivativesa6An enzyme preparation (solubilised by sodium choleate) from peanutcotyledon microsomes, having the same co-factor requirements as themicrosomal enzymes in sit%, has been made." No co-factor is needed forproduction of 14C0, from [l-14C]palmitic acid, but DPN+ is required forproduction of 14C0, from internally labelled palmitic acid.The oxidativemechanism does not involve CoA or the tricarboxylic acid cycle. Heat-stable substances, from cotyledons of germinating seeds, which catalyse theoxidation of [l-14C]palmitic acid to 14C0,, are identified as glycollic andL-lactic acid (the D-acid is inactive).248 The system is equally active forlabelled stearic, palmitic, and myristic acid, but weakly active for lauric,and inert for lower acids.Carboxy-labelled 2-hydroxypalmitic acid isunaffected, but the 2-oxo-acid is rapidly attacked. Geyer and his co-workers 249 now state that in non-enzymic experiments [l-14C]-palmitic and-stearic acid are converted, under mild conditions, by ascorbic acid andoxygen into 14C0,. Short-chain acids are not responsive. The authorscomment that the ascorbic acid content of germinating seeds reaches >60 mg. 1100 g. of tissue a few days after germination starts.243 S. J. Wakil, Biochim. Biophys. Acta, 1955, 18, 314.244 G. PopjAk and A. Tietz, Biochem. J., 1955, 60, 147, 155.246 P. Hele and G. Popjbk, Biochim. Biophys. Acta, 1955, 18, 294; Biochem. J.,246 R . G. Langdon, J . Amer.Chem. Soc., 1955, '77, 5191.247 T. E. Humphreys and P. K. Stumpf, J. Biol. Chewz., 1955, 213, 941.248 P. Castelfranco, P. K. Stumpf, and R. Contopoulou, ibid., 1955, 214, 567.24a R. P. Geyer, L. Marshal1,M. Ryan, and S. Westhaver, Arch. Biochem. Biophys.,1955, 80, xxxii.1955, 56, 549316 BIOLOGICAL CHEMISTRY.Positive evidence for fatty acid oxidation occurring in tumour mito-chondria has now been obtained.250 Speculative schemes for the formationof plant waxes through acylCoA intermediates are prop0sed.~51More has been published on the effects of the enzyme lipoxidase whichcan be obtained crystalline from soya-bean : it catalyses the peroxidation oflinoleic, linolenic, and arachidonic acid and their e~ters.25~ The principalproducts are optically active cis-trans-conjugated monomeric hydroperoxidesat 0” or 26” in either the dark or daylight; some optically active polymer isalso f0rmed.~53 The enzyme is believed to be involved in the formation ofeach peroxide molecule : 254 it is inactive towards the cis-9 : trans-12- andtrans-9 : trans-12-stereoisomers of linoleic acid.A coupled oxidation ofglutathione and an unsaturated acid of the linoleic type, under the influenceof lipoxidase (two reactions at least are involved, one cyanide-sensitive andone insensitive), has been reported 255 for germinating peas. Extracts fromungerminated peas show no lipoxidase activity but it is initiated by smallamounts of alcohol. The lipoxidase enzyme and substrate are associatedwith the soluble part of the cytoplasm and are absent from the mitochondria.Lipoxidase activity has been studied 256 in maize seedlings at pH 5.0 and 7-2.Recent work shows that linoleic, linolenic, and lauric acid are consider-ably more active as plant-wound hormones than traumatic acid : theiraction is enhanced by cytochrome-C, CoA, and ascorbicL.c.5. THE ENZYMIC HYDROXYLATION OF STEROIDS.Some aspects of the biochemistry of steroid hormones were reviewed inthese Reports 1 in 1951. Advances in this field continue to be so varied andnumerous that a too general survey is liable to be superficial : so this Reportdeals solely with enzymic hydroxylations of steroids.0x0- and hydroxy-groups are the principal substituents in the steroids ofanimal origin. Almost all of them have an oxygen substituent at position 3.In addition, hydroxyl groups are commonly found at positions 7, 11, 12,16, 17, 20, and 21, and less commonly at 6, 18, and 19.Certain physio-logically active plant steroids and toad-venom steroids have hydroxylgroups at other positions, notably 5 and 14; nothing is known regarding thebiological introduction of such groups.Hydroxyl groups may apparently be introduced directly into the mole-cule, as in the formation of 17a-hydroxyprogesterone from progesterone, orthey may arise by reduction of ketones formed on the removal of side chainsat position 21 or 17: such hydroxyl groups will not be considered.1 1 -Hydroxylation.-The dramatic clinical effects of cortisone discovered250 P. Emellot and C.J. Bos, Experientia, 1955, 11, 188.351 G. G. Wanless, W. H. King, and J. J. Ritter, Biochem. J., 1955, 59, 684.2S2 R. T. Holman and S. Bergstrom in J. B. Sumner and K. Myrback’s “TheEnzymes,” Vol. 11, Academic Press, New York, 1951.253 0. S. Privett, C. Nickell, W. 0. Lundberg, and P. D. Boyer, J . Amer. Oil Chemists’Suc., 1955, 32, 505; 0. S. Privett, F. J. Pusch, and R. T. Holman, Arch. Biuchem.Biophys., 1955, 57, 156.zs4 A. L. Tappel, P. D. Boyer, and W. 0. Lundberg, J . B i d . Chern., 1952, 199, 267.255 L. W. Mapson and E. M. Moustafa, Biochem. J., 1955, 60, 71.25~3 G. Fritz and H. Beevers, Arch. Biochem. Biophys., 1955, 55, 436.257 A. J. Haagen-Smit and D. R. Viglierchio, Rec. Trav. chim., 1955, 74, 1197.1 Ann. Reports, 1951, 48, 297GRANT : THE ENZYMIC HYDROXYLATION OF STEROIDS.317by Hench and his co-workers2 in 1949 provided a powerful stimulus toorganic chemists interested in natural steroids. Especial efforts weredirected towards the production of steroids having an 11-oxygen function.Apart from the purely chemical approaches (reviewed by Rosenkranz et d3),biochemical methods have been extensively investigated. As a result moreis known about hydroxylation at the 11-position than at any other. Usingorgan perfusion, Hechter et aL4 demonstrated 11 P-hydroxylation of a numberof steroids by ox adrenals. The authors were careful to point out that theywere concerned to find only whether a reaction could take place in the adrenaland, if so, at what rate. They were less interested in the physiologicalstate of the adrenal glands, which actually gave greater yields of steroidafter rough handling, and they did not claim that the reactions observedwere of physiological importance.These limitations have not always beenborne in mind when the results obtained have been discussed by others inrelation to the biosynthesis of adrenocortical steroids. Ascorbic acid,which disappears from the adrenal glands during steroid hormone secretion,and the adrenocorticotrophic hormone (ACTH) were added to the perfusionmedium in order that the gland might be working at maximum capacity.Neither substance, however, had any effect on llp-hydroxylation of thesteroids investigated, and the opinion was expressed that ACTH influencesRI coR’H(1) R = CH,*OH; (4) R = :O;R’ = H.(6) R = -OH, R’ = * OH.(2) R = CH,*OH ; . * * H . (5) R = :O;R’ = OH. (10) R = -OH, R’ = -OH.(7) R =Me; (9) R’ = :O;R‘ = H. (11) R = -CH(OH)*CH,*OH, R = -CO*CH,*OH.(8) R =Me:R’ = OH.(3) R = :O.- - - CHXH,.* - * OH.[Throughout this section, rings omitted from fornuke are those of the first formulaabove.]adrenocortical steroid biosynthesis at some point before the 11 P-hydroxyl-ation reaction. Crystalline 11 8-hydroxy-derivatives were isolated andcharacterized after perfusion of 1 1-deoxycorticosterone 49 (l), 17a-hydroxy-deoxycorticosterone 4y (2), androst-4-ene-3 : 17-dione (3), androsterone 4P. S. Hench, E. C. Kendall, C . H. Slocomb, and H. F. Polley, Proc. Mayo CZin.,1949, 24, 181.3 G.Rosenkranz, F. Sondheimer, A. Mancera, J. Pataki, H. J. Ringolo, J. Romo,C . Djerassi, and G. Stork, Recent Progr. Hormone Research, 1953, 153, 8, 1.4 0. Hechter, A. Zaffsroni, R. P. Jacobsen, H. Levy, R. W. Jeanloz, V. Schenker, andG. Pincus, ibid., 1951, 6, 215.5 H. Levy, R. W. Jeanloz, C. W. Marshall, R. P. Jacobsen, 0. Hechter, V. Schenker,and G. Pincus, J . Bid. Chem., 1953, 203, 433.Q R. W. Jeanloz, H. Levy, R. P. Jacobsen, 0. Hechter, V. Schenker, and G. Pincus,ibid., p. 453318 BIOLOGICAL CHEMISTRY.(4), epiandrosterone (5), testosterone * (6), progesterone (7), l7a-hydroxy-progesterone (8), and 21-hydroxy-5a-pregnane-3 : 20-dione (9) through oxadrenals. The transformation of progesterone was very poor (<l%). Noreaction occurred with vinyltestosterone (10) or with 17a : 20 : 21-trihydr-oxypregn-4-en-3-one (1 1).11 p-Hydroxylation did not occur when steroidswere perfused through human placenta or through rat liver.* The questionof 11 P-hydroxylation by the liver has given rise to some controversy. Kahntand Wettstein lo claimed to have obtained 1-5% conversion of 17~~-hydr-oxydeoxycorticosterone into cortisol (1 1 p : 17% : 21-trihydroxypregn-4-ene-3 : 20-dione) (12) with homogenates of rabbit or ox liver or kidney : Dorf-man et aL6> l1 and Grant l2 have not been able to confirm this. A claim tohave converted 17a-hydroxydeoxycorticosterone into cortisol with liver wassubsequently withdrawn by Hubener et al. ; l3 the substance confused withcortisol was 17a : 20 : 21-trihydroxypregn-4-en-3-one (11).This is anRI coexcellent example of the result(12) R = CH,*OH; R’ = OH.(13) R = CH,*OH; R‘ = H.(14) R =Me; R’ = H.of inadequate characterisation of a steroidtransformation-product, a fault to be giarded against in facile, uncriticaluse of paper chromatography.Following Hechter et aL4 several groups have made detailed investigationsof 11 p-hydroxylation. Hayano et aZ.14 incubated ox adrenal slices, minces,and homogenates with deoxycorticosterone and observed an increasedglycogenic activity which they ascribed to the formation, from deoxy-corticosterone, of an 11 p-hydroxy-steroid, later identified l5 as corticosterone(13). Subsequently they obtained a residue on centrifuging adrenal homo-genates at 5000 g ; after washing this with saline or water, they referred toit as a purified llp-hydroxylase ~reparati0n.l~ Using this residue theydemonstrated the “ specificity and absolute necessity for fumarate andmagnesium ions and the stimulating capacity of adenosine triphosphate(ATP) and diphosphopyridine nucleotide (DPN+) ” for 11 P-hydroxylation ofsteroids.They suggested that fumarate might play some part in an energy-yielding system for the regeneration of ATP or might function as a hydrogenacceptor in steroid hydroxylation. Dorfman et al. reviewed this work andreported a number of llp-hydroxylations with the “ washed residues.’’ l17 A. S. Meyer, 0. G. Rogers, and G. Pincus, Endocrinology, 1953, 53, 245.L. R. Axelrod and G. Arroyave, J . Amer.Chem. Soc., 1953, 75, 5729.A. S. Meyer, unpublished work cited by 0. Hechter and G. Pincus, Physiol. Rev.,10 F. W. Kahnt and A. Wettstein, Helv. Chim. Acta, 1951, 34, 1790.11 R. I. Dorfman, M. Hayano, R. Haynes, and K. Savard, Ciba Foundation Colloquial 3 H. J. Hiibener and J. Schmidt-Thorn& 2. physiol. Chem., 1955, 299, 126.14 M. Hayano, R. I. Dorfman, and E. Y. Yamada, J . Biol. Chem., 1951,193, 175.l5 M. Hayano and R. I. Dorfman, ibid., 1953, 201, 175.1954, 34, 459.on Endocrinology, 1953, Vol. VII, p. 191.J. K. Grant, Unpublished workGRANT THE ENZYMIC HYDROXYLATION OF STEROIDS. 319Similar results were obtained by others.lO, 16~ 1 7 9 l8 has shown thatthe production of corticosterone and cortisol by 11 p-hydroxylation in hogadrenal homogenates is possible on a commercial scale. After a detailedstudy of co-factor requirements Kahnt et aZ.19 concluded that 11 p-hydroxyl-ation involved synthesis of ATP linked to oxidation of members of theKrebs tricarboxylic acid cycle.Credit for showing that 11 p-hydroxylatingenzymes are associated with adrenal-cell particles, which by their method ofpreparation were mainly mitochondria, is due to Sweat.20 Brownie andGrant 21 drew attention both to the unsatisfactory nature of the enzymepreparations and to the methods of steroid analysis used in earlier studies.Employing ox-adrenal cortical mitochondria, with careful exclusion of othercell components, and improved methods of steroid analysis, they confirmedthe requirement for concurrent oxidative phosphorylation for which addedcitric acid cycle intermediates are oxidisable substrates.It was not neces-sary to add DPN+, ATP, or Mg2+ to such mitochondrial preparations. a-OXO-glutarate was the best “activator” of llp-hydroxylation, and it wasdemonstrated that the “ intact ” mitochondria showed no specific fumaraterequirement for 11 P-hydroxylation. This disagreement with Hayano et aZ.15was explained when Brownie and Grant 22 showed that their mitochondrialpreparations developed a requirement for fumarate when treated withhypotonic media. Such preparations were unable to use a-oxoglutarate tosupport ” 11S-hydroxylation and, in addition to fumarate, required tri-phosphopyridine nucleotide (TPN+) or DPN+ + ATP. (The requirementfor TPN+ had also been shown by Hayano et u Z ., ~ ~ using glands stored a t 0”for long periods.) This suggested that Mg2+ and ATP are required for thephosphorylation DPN _+ TPN in the “ damaged ” particles of Hayano’senzyme preparation. The requirement for oxidative phosphorylation tosupport steroid hydroxylation, as observed by Brownie and Grant withintact ” mitochondria, is not, however, explained. The latter workershave suggested that entry of deoxycorticosterone into mitochondria may bean ‘‘ active ” energy-requiring process ; with the disappearance of penne-ability barriers (protein films ?) on washing of the mitochondria, this energyprovision would become unnecessary.The role of TPN+ and fumarate has been further investigated by Sweatand Lipscomb24 and by Grant and Brownie.25 The former showed thatglucose 6-phosphate and its dehydrogenase together can replace fumarate inan adrenal enzyme system which will 11 p-hydroxylate deoxycorticosteronein presence of TPN’.They concluded that the role of added members ofthe Krebs tricarboxylic acid cycle was to maintain TPN+ in the reducedstate (TPNH) according to the reactions (1) fumarate I) malate, (2)HainesD. A. McGinty, G. N. Smith, M. L. Wilson, and C. S. Worrel, Science, 1950, 112,K. Savard, A. Green, and L. A. Lewis, Ejzdocrinology, 1950, 47, 418.W. J. Haines, Recent Progr. Hormone Res., 1952, 7, 255.F. W. Kahnt, C. Meystre, R. Neher, E. Vischer, and A. Wettstein, Experientia,506.1952, 8, 422.2o M. L. Sweat, J . Amer. Chem.SOC., 1951, 73, 4056.21 A. C. Brownie and J. K. Grant, Biochenz. J . , 1954, 57, 255.22 Idem, ibid., 1956, 62, 29.23 M. Hayano, M. Weiner, and M. C. Lindberg, Fed. PTOC., 1953, 12, 216.24 M. L. Sweat and M. D. Lipscomb, J . Amer. Chem. SOC., 1955, ‘77, 5185.2 6 J. K. Grant and A. C. Brownie, Biochim. Biophys. Acta, 1955, 18, 433320 BIOLOGICAL CHEMISTRY.malate + DPN+ -+ oxaloacetate + DPNH, DPNH + TPN+ --+DPN+ + TPNH , involving the transdehydrogenase described by SanPietro, Kaplin, and Colowick.26 Grant and Brownie 25 produced evidencefor the reduction of TPN+ in a soluble enzyme system extracted from acetone-dried ox-adrenal mitochondria according to reactions (1) and (4) malate +TPN+ + pyruvate + TPNH + CO, or (5) isocitrate + TPN+ _+ a-oxoglutarate + TPNH, and they observed the ll @-hydroxylation of deoxy-corticosterone in dialysed preparations of the soluble enzyme to which2-amino-2-hydroxymethylpropane-1 : S-diol(" Tris ") buffer and TPNH werethe only additions.Hayano et have prepared a soluble enzyme, byextracting acetone-dried adrenal preparations, which requires TPN+ butnot fumarate for 11 p-hydroxylation. Sweat and Lipscomb 24 confirmed thisbut remarked that the slight activity observed in such extracts, withoutfumarate, represents the same small degree of activity observed in mito-chondrial preparations without added fumarate. Soluble enzyme prepar-ations are unlikely to be less dependent upon co-factor-generating systemsthan more highly organised particulate preparations. It may be concludedthat TPNH and oxygen alone are required for llp-hydroxylation of steroids.The availability of TPNH in cells in physiological steady states has beendemonstrated by Chance and Williams ,** and the high TPNH concentrationsin adrenal tissue found by Glock and McLean 29 may be of some significance.The requirements for TPNH and oxygen are remarkably similar to thosefound necessary by Brodie et aL30 for the hydroxylation of certain drugs byliver microsomes.These particles contain no cytochrome oxidase, but willoxidise TPNH via the autoxidation of flavoproteins, a reaction which resultsin the production of hydrogen peroxide. Brodie has suggested that hydrogenperoxide is the hydroxylating agent but was unable 31 to replace TPNH byenzyme systems generating hydrogen peroxide.32 He suggests that theH,02 formed by such systems does not enter the micros0mes.~1 Grant 33has shown independently that hydrogen peroxide generated by the uricacid-uricase system in the presence of his soluble adrenal-enzyme preparationis unable to replace TPNH for 11P-hydroxylation of deoxycorticosterone ;this system contains no permeability barriers to hydrogen peroxide.More-over an increase in oxygen concentration in the reaction mixture increasesthe rate of oxidation of TPNH but inhibits llp-hydroxylation. It thereforeappears unlikely that TPNH is required for production of hydrogen peroxide.A consideration of possible mechanisms for 11 p-hydroxylation of steroidsmay help to throw light on the role of TPNH.The proposal that the hydroxyl is introduced in the hindered Ilkposition of an ll-deoxysteroid (A) by way of the lla-hydroxy- (B) andll-oxo-compound (C) is discounted by the failure to reduce ll-oxo- to11 p-hydroxy-steroids by adrenal enzymes.% By analogy with the reactions,26 A.San Pietro, N. 0. Kaplan, and S. P. CoIowick, J . Bid. Chew., 1955, 212, 941.27 M. Hayano, R. I. Dorfman, and E. Rosenberg, Fed. Pruc., 1955, 14, 224.28 B. Chance and G. R. Williams, Nature, 1955, 176, 250.20 G. E. Glock and P. RlcLean, Biochem. J., 1955, 61, 388.30 B. B. Brodie, J. Axelrod, J. R. Cooper, L. Gaudette, B. N. LaDu, C. Mitoma, and31 B. B. Brodie, personal communication.32 D. Keilin and E. F. Hartree, Proc. Roy. Soc., 1038, B, 125, 171.33 J.K. Grant, unpublished work.34 A. Meyer, J . B i d . Chew., 1953, 203, 469.(3)S. Udenfriend, Science, 1955, 121, 603GRANT : THE ENZYMIC HYDROXYLATION OF STEROIDS. 321succinate + fumarate _.t malate, it has been suggested that steroid11 p-hydroxylation might proceed via an ethenoid intermediate (D). Dorf-man et aZ.11 and Brownie and Grant 22 produced evidence that the A9(11)- and(A 1 ( c )All-compounds are not intermediates. The former suggested that thesecompounds may give small amounts of the parent deoxy-steroid and thatthis will undergo 11 p-liydroxylation and so account for the report by Miescheret ~ 1 . ~ 5 that ethenoid compounds can act as intermediates. g-Jq or )pHOq(0)Fieser 36 proposed the reaction sequence (E) _j (F) , for which there is,Levy et aL5 suggested a mechanism involving however, as yet no evidence.R R0: (yp o:@(F)free hydroxyl radicals in the direct oxidation of deoxycorticosterone. Oneradical would remove and combine with the lla-hydrogen atom, and anotherwould enter the a-position and subsequently undergo inversion to the @-con-figuration.Again there is no evidence in support of this. Hayano andDorfman 37 showed that deuterium does not enter a stable position in thesteroid molecule when the 11P-hydroxylation reaction is carried out in 95%D20. Subsequently they38 and Sweat and Mason39 found that 1 8 0 didnot enter the ll@-hydroxyl group when the hydroxylation was performed inH21*0, but did enter there when the gas phase contained 1802.Theseobservations support the hypothesis that a steroid does not undergo dehydro-genation during 11 p-hydroxylation but is directly oxidised by molecular35 K. Miescher, A. Wettstein, and F. W. Kahnt, Acta Physiol. Latinoarnerican, 1953,3, 144.S c L. F. Fieser, Ciba Foundation Colloquia on Endocrinology, 1953, Vol. VII, p. 288.37 M. Hayano and R. I. Dorfman, J . Biol. Chem., 1954, 211, 227.38 M. Hayano and R. I. Dorfman, personal communication.39 M. L. Sweat and H. S. Mason, personal communication.REP.-VOL. LII 322 BIOLOGICAL CHEMISTRY.oxygen. Such a direct oxidation can occur non-enzymically at C(7) incholesterol, catalysed by traces of heavy metals. It has been formulatedby Bergstrom and Wintersteiner 40 as (G) _+ (H), (I), and (J).It is thusHo [“o&:.J -::&o (H 1L/\ (G 1HO &* (1 1 “omw ( J )Cholesterol derivatives.possible that steroid 11 p-hydroxylation proceeds via a peroxide (K).are, however, few analogies between the two formulations.cholesterol is presumably activated by the neighbouring double bond.TherePosition 7 inNOsuch activation is available for position 11 in deoxycorticosterone. Hydr-oxylation of deoxycorticosterone does not occur if enzymic activity isdestroyed by heat. Transformation of adrenocortical steroids to more polarsubstances when they are shaken in air with solutions of ferrous sulphate,ascorbic acid, or ethylenediaminetetra-acetate has been reported *l butthere is no indication that such reactions involve 11 p-hydroxylation.In seeking an explanation of the requirement for TPNH it may be ofinterest that certain phenolases show high selectivity towards hydrogendonors required for the formation of water in the reactions which they~atalyse.*~ It is possible that the llp-hydroxylase, which appears to catalysethe direct introduction of oxygen into the steroid molecule, requires TPNHfor the subsequent elimination of one oxygen atom as water.Thus the final details of the mechanism of llp-hydroxylation remainunsettled.The interesting possibility has, however, arisen that this reaction,which is probably the last step in the biosynthesis of adrenal hormones, isinfluenced by those processes 43 which control the level of reduced pyridinenucleotides in the cell.No general rules have been evolved regarding the type of steroid whichmay undergo 1 lp-hydroxylation in adrenal tissue.Neither the 17-sidechain nor the A4-3-oxo-grouping appears to be essential since androsterone(4) and ePiandrosterone 7 (5) have been transformed into the corresponding11 fi-hydroxy-derivatives by adrenal perfusion. Hydroxylation is , however,rather less readily achieved with these reduced steroids.40 S. Bergstrom and 0. Wintersteiner, J . Biol. Chem., 1941, 141, 597; 1942, 143,ti03 : 1942, 145, 309, 327.41 A. S . Keston and M. Carsiotis, Arch. Biochem. Biophys., 1954, 52, 282.42 H. S. Mason, Adv. Enzymol., 1965, 16, 105.43 B. Chance, “ Mechanisms of Enzyme Action,” Johns Hopkins Press Baltimore,1954, p. 399GRANT THE ENZYMIC HYDROXYLATION OF STEROIDS.323Clinical demands for cortisone led to an intensive study of the trans-formation of steroids by micro-organisms , which has proved both interestingand rewarding; this has been reviewed by Fried et aL4 and by Wettstein.&Of special interest are the observations which led to a valuable combinedmicrobiological and chemical synthesis of cortisone from readily availableprogesterone. Peterson and Murray 45 found that Rhizofius spp. convertedprogesterone (7) into the lla-derivative in yields up to 85-95%, it beingoften possible to recover the product by direct crystallisation from mycelialextracts. It was particularly fortunate that the lla-epimer was formed,since the A4-3-oxo-group of this substance may be reduced in good yield toa compound of the normal (5p) series suitable for subsequent introductionof 17- and 21-hydroxyl groups.Steroids with l l p - or ll-keto-groups giveaZZo(5a)-reduction products, and these are less readily reoxidised to A4-3-oxo-steroids in the final stages of the synthesis.46 Later 47 it was found possibleto obtain e+i( 11 a)-cortisol in good yield from 17a-hydroxydeoxycortico-sterone (2) by fermentation with Rh. nigricans, thus avoiding the necessityof eliminating and re-introducing the A4-3-oxo-group. It was also found 48that this versatile organism showed a high specificity for hl6-progesterone (la),readily available from steroid sapogenins such as diosgenin, giving 1 la-hydr-oxy-17a-progesterone (16) and thus unexpectedly giving a compound withthe side-chain in the t hermodynamically labile a-position.@ COMc ---+ HO.@-coMe fl(‘61(15)HO(14)Nothing is known of the co-factor requirements or enzymic mechanismsof these mould hydroxylations.Closely related species show remarkablespecificity with regard to direction of attack on the steroid nucleus.49 Sub-stituents in the nucleus have a profound effect on the nature of the trans-formation products. The steroids transformed are not natural substratesfor the mould enzymes, and it is possible that the reactions are attempts at‘‘ detoxication.” It is sometimes necessary to limit the amount of steroidsubstrate or to use deoxycorticosterone acetate on account of inhibitoryeffects of the free alcohol.4917a- and 21-Hydroxylation.-Hydroxylations at positions 17a and 21 are44 J.Fried, R. W. Thoma, D. Perlman, J. E. Herz, and A. Borman, Recent Progr.Hormone Res., 1955, 11, 149.44a A. Wettstein, Experientia, 1955, 11, 465.45 D. H. Peterson and H. C . Murray, J . Amer. Chem. SOL, 1952, 74, 1871 ; D. H.Peterson, H. C . Murray, S. H. Eppstein, L. M. Reinelre, A. Weintraub, P. D. Meister,and H. M. Leigh, ibid., p, 5933.46 0. Mancera, A. Zaffaroni, B. A. Rubin, F. Sondheimer, G. Rosenkranz, andC. Djerassi, ibid., p. 3711; T. H. Kritchevsky, D. L. Garmaise, and T. F. Gallagher,ibid., p. 483.47 D. H. Peterson, S. H. Eppstein, P. D. Meister, B. J. Magerlein, H. C . Murray,H. M. Leigh, A. Weintraub, and L. M. Reineke, ibid., 1953, 75, 412.48 P. D. Meister, D. H. Peterson, H.C. Murray, G. B. Spero, S. H. Eppstein, A.Weintraub, L. M. Reineke, and H. M. Leigh, ibid., p. 55.*O S. H. Eppstein, P. D. Meister, D. H. Peterson, H. C. Murray, H. M. Leigh, D. A.Lyttle, L. M. Reineke, and A. Weintraub, ibid., p. 408324 BIOLOGICAL CHEMISTRY.considered together because they are concerned in the formation of theadrenocortical hormone cortisol, and because there is evidence that thepresence of a hydroxyl group in one or other of these positions influences theease with which the other may be introduced. Early observations 50 thatcortisol (12) was formed when progesterone (7) was incubated with wholeox-adrenal homogenates suggested that 17- and 21-hydroxylating enzymeswere removed during the preparation of 1 l-hydroxylating enzymes.Plagerand Samuels 51 confirmed this; the supernatant fluid obtained on centri-fuging ox-adrenal homogenates for 0.5 hr. at 20,000 g contained enzymeswhich catalysed the 17a- and 21-hydroxylation of progesterone and possessedno 11 p-hydroxylating ability. Some 21-hydroxylation occurred on additionof DPN+, but 17a-hydroxylation appeared to require both ATP and DPN+,possibly for the formation of TPN+. Alternatively, ATP might " activate "the steroid molecule by forming a water-soluble phosphate. There is nosatisfactory evidence in favour of either suggestion and it is not clear whetherthe above enzyme preparation contained microsomes. If in fact it did,then ATP might be concerned in overcoming permeability barriers. Sexhad no effect on 21-hydroxylation, as judged by formation of deoxycortico-sterone from progesterone.The enzyme from heifer adrenals was, however,2-3 times more active in the formation of 17a-hydroxydeoxycorticosteronethan that from steer adrenals, and bull adrenals had intermediate effect.Dehydroepiandrosterone (3p-hydroxyandrost-5-en-17-one) (16), addedin vitro, suppressed formation of deoxycorticosterone and increased that ofits 17a-hydroxy-derivative (2) from progesterone (7). Dehydroepiandro-sterone (16) is itself oxidised to androst-4-ene-3 : 17-dione (3) by the sameenzyme preparation, but it would be difficult to explain the effect on 17- and21-hydroxylation by competition for some co-factor.11 p-, 17a-, and 21-Hydroxylation of steroids has been demonstrated withhuman tissues in d r o 52 and in v ~ v o .~ ~The order in which 1 1 p-, 17a-, and 2 1 -1tfydroxyl Groups are introduced.-In the scheme of adrenocortical steroid biogenesis advanced by Hechteret aZ.4 llp-hydroxyprogesterone is shown as a possible intermediate in theformation of corticosterone (13) or cortisol (12) from progesterone (7). Thestatus of 11 P-hydroxyprogesterone (17) as possible intermediate has beendiminished by the following facts. This steroid has been isolated in yieldsonly of less than 1% after perfusion of progesterone through adrenal glands,and has not been found among the products obtained on incubation ofadrenocortical tissue preparations with progesterone. Finally it has notbeen further hydroxylated to corticosterone or cortisol in significant yields.It was therefore suggested 54 that llp-hydroxylation sets the seal of a com-pleted product on the steroid.In view of the negative nature of the evidencethis suggestion may not be of great significance, especially since Brownie,Grant, and Davidson were able to effect llp-hydroxylation of progesteronein fair yield by using adrenal mitochondria. When the ll-hydroxyl group60 J. E. Plager and L. T. Samuels, Fed. Proc., 1952, 11, 383.ti1 Idem, Arch. Biochem. Biophys., 1953, 42, 477; J . Biol. Chem., 1954, 211, 21.62 G. Pincus, Ciba Foundation Colloquia on Endocrinology, 1955, Vol. VIII, p. 97.53 €1. Werbin, G. V. Le Roy, and D. M. Bergenstal, J . Lab. Clin. Med., 1955, 46,64 0. Hechter, Ciba Foundation Colloquia on Endocrinology, 1953, Vol.VII, p. 161.55 A. C. Brownie, J. K. Grant, and D. W. Davidson, Biochem. J.. 1954, 58, 218.964GRANT : THE ENZYMIC HYDROXYLATION OF STEROIDS. 326has the unnatural cc-configuration further hydroxylation at and C&l)can 0ccur.5~Hayano and Dorfman l6 found that washed homogenate residue prepar-ations were unable to use progesterone (7) or 17a-hydroxyprogesterone (8)as substrates for 11 p-hydroxylation and claimed that a 21-hydroxyl groupwas required for 11 P-hydroxylation. The formation of 11 p-hydroxy-progesterone already referred to 55 refutes this idea.In their perfusion studies Hechter et aZ.* noted the conversion of pro-gesterone (7) into corticosterone (13) and cortisol, whereas deoxycortico-sterone (1) gave corticosterone alone.In confirmation of this Plager andSamuels found no 17a-hydroxylation of deoxycorticosterone with theirsupernatan t-fract ion enzyme. Apparently 17 a-hydroxylat ion can onlyprecede 21-hydroxylation.The question of which compounds may or may not be hydroxylatedmust await detailed knowledge of co-factors required and the preparationof pure enzymes. Only in this way can the influence of steroid substrateson reactions supporting hydroxylations be eliminated.The annexed scheme summarises present knowledge of the individualsteps in 11P-, 17a-, and 21-hydroxylation.Progesterone 17a : 2 1 -Dihydroxyprogesterone I2 1 -Hydroxyprogesterone(deoxycorticosterone)1, 2, 3,4 1 11s : 21 -Dihydroxyprogesterone(corticosterone)1, 2, 3, 4 1 I 1 l&Hydroxyprogesterone 118 : 17a : 21-Trihydroxyprogesterone (cortisol)I-+ 1 lg : 17a-Dihydroxyprogesterone.1, By adrenal perfusion.2, By adrenal homogenate.3, By adrenal homogenate washed residue.4, By adrenal mitochondria.5, By supernatant fluid from centrifuged adrenal homogenate.6-HydroxyIation.-The first isolation of a 6p-hydroxylated steroidfrom mammalian tissue was reported by Haines l8 who obtained 6p-hydroxy-deoxycorticosterone (17) on incubation of deoxycorticosterone with hog-66 A.Zaffaroni and J. Hendrichs, Abs. 122nd Meeting, Amer. Chem. SOC., 1952,-1oc326 BIOLOGICAL CHEMISTRY.adrenal homogenate. Subsequently Hayano and Dorfman 57 obtained6p-hydroxycortisol (1 8) and Meyer et aZ.5s obtained 6p-hydroxyandrost-4-ene-3 : 17-dione (19) with ox-adrenal homogenate preparations. YieldsCH,*OHI0(17) R = H(18) R = OHwere increased by addition of ATP and DPN+.Others reported the form-ation of 6p-hydroxy-steroids in adrenal perfusion experiments.4~ 59 6p-Hydroxylation is not, however, confined to the adrenal gland, as 11 p-hydroxyl-ation appears to be. Hayano et aZ.60 obtained 6~-hydroxydeoxycortico-sterone on incubation of deoxycorticosterone with ox corpus luteum homo-genates, and Miller and Axelrod claim to have found 68-hydroxydeoxy-corticosterone among the transformation products of cortisone perfusedthrough cirrhotic rat livers; but, on finding no 6p-hydroxy-steroids on per-fusion of normal rat livers, they suggested that this occurrence of the 6p-hydroxy-compound might be related to the abnormal electrolyte metabolismoccurring during cirrhosis.The 6-hydroxy-steroids, however, have little orno effect on electrolyte metabolism. Although their physiological signi-ficance is obscure, 6-hydroxy-steroids appear to take some part in the steroidmetabolism of intact animals. They have been isolated from the normaland pathological urines of guinea-pigs 62 and human subjects. Grantfound that 6p-hydroxylation of deoxycorticosterone by adrenal preparationsappeared to have the same co-factor requirement as 11 p-hydroxylation ; 64the 11 p-and 613-hydroxy-steroids were formed in the approximately constantratio of 3 : 1. There was no evidence of the formation of 6p : llp-dihydroxy-compounds. Various steroids have given 6p-hydroxy-derivatives on incub-ation with micro-organisms.6516-Hydroxylation.-Various steroids are known to possess 16-hydroxylgroups, almost exclusively in the a-configuration. It is widely held that theintroduction of hydroxyl groups into the steroid nucleus in the positions 68,l l p , and 17a may be regarded as anabolic reactions. By contrast the 16-hydroxy-steroids have been thought to represent stages in the catabolism5 7 M. Hayano and R. I. Dorfman, Arch. Biochem. Bi@hys., 1954, 50, 218.5 8 A. S. Meyer, M. Hayano, M. C. Lindberg, M. Gut, and 0. G. Rogers, Acta Endo-5 o L. R. Axelrod and L. L. Miller, Arch. Biochem. Biophys., 1954, 49, 248.60 M. Hayano, M. C. Lindberg, M. Wiener, H. Rosenkrantz, and R.I. Dorfman,61 L L. Miller and L. R. Axelrod, Metabolism, 1954, 3, 438.62 S. Burstein, unpublished observation cited in ref. 60.133 S. Lieberman, K. Dobriner, B. R. Hill, L. F. Fieser, and C. P. Rhoads, J . Biol.Chem., 1948, 172, 263; S. Burstein, R. I. Dorfman, and E. M. Nadel, Arch. Biochern.Bioplzys., 1954, 53, 307.64 J. K. Grant, unpublished work.6 5 H. C. Murray and D. H. Peterson, U.S. P. 2,602,769/1952.crinol., 1955, 18, 148.Endocrinolog.y, 1954, 55, 326GRANT : THE ENZYMIC HYDROXYLATION OF STEROIDS. 327of steroids, preceding ring opening and the formation of more highly degradedproducts. Pregn-5-ene-3p : 16% : 20a-trio1 66 (20) 5a-pregnane-3p : 16% : 20p-trio1 67 (21), androst-5-ene-3P : 16a : 17P-triol 68 (22), androstane-3p : 16a : 17p-trio1 69 (23), and testane-3a : 16a : 17p-triol 69 (24) are typicalMeIH-C-OHMaIHO-C-HOHHO @--OHof the 16-hydroxy-steroids which have been isolated from urine and maypossibly be derived from adrenal or testicular hormone precursors.Thesuggestion that compound (22) arises from dehydroefiandrosterone (6) issupported by the observed transformation of the compound (6) into (22)in vitro by rabbit-liver slices.70Ofner 71 suggested that 16-hydroxy-steroids may be formed on incubationof testosterone with minced rat liver. It would be useful to have furtherevidence for the formation of 16-hydroxy-steroids in vitro and to investigatethe sites and mechanisms of hydroxylation in order to throw more light onthe origin of the urinary 16-hydroxy-steroids. (Estriol (25) is, of course, animportant 16a-hydroxy-steroid.Marrian and Bauld 72 isolated the 16p -epimer (26) from human pregnancy urine and suggested that ‘‘ 16-oxo-cestra-dio1-17p ” [3 : 17p-dihydroxycestra-1 : 3 : 5(10)-trien-16-one] might be acommon metabolic precursor of cestriol and epicestriol. Watson andMarrian 73 have since reported the detection of the 16-oxo-derivative (27) of6 6 H. Hirschmann and F. B. Hirschmann, J . Biol. Chern., 1950,184, 259.6 7 G. A. D. Haslewood, G. F. Marrian, and E. R. Smith, Biochem. J., 1934, 28, 1316.H. Hirschmann, J . Biol. Chem., 1943, 150, 363; H. L. Mason and E. J. Kepler,69 S. Lieberman, B. Praetz, P. B. Humphries, and K. Dobriner, Abs. 117th Meeting70 J. J. Schneider and H.L. Mason, J . Bid. Chem., 1948, 172, 771.72 G. F. Marrian and W. S. Bauld, Biochem. J.. 1955, 59, 136.73 J. Watson and G. F. Marrian, Biochem. J., 1955, 61, xxiv.ibad., 1945, 160, 265; 1947, 168, 73.Amer. Chem. SOC., 1950, 19c.P. Ofner, Biochem. J., 1955, 61, 287328 BIOLOGICAL CHEMISTRY." cestradiol-178 " in human pregnancy urine. There is no evidence, however,that oxidative attack of the steroid nucleus at position 16 differs from thatat position 6, 11, or 17a, where the primary product appears to be ahydroxy- and not an oxo-steroid.CH2*OH1R (25) R = * * OH, -H.(26) R = -OH, . * . H .(27) R = :O.0: HOProbably because of the lack of satisfactory methods for the extractionand determination of estrogens in tissues, no reliable study of the conversionof cestrone or estradiol into estriol has been reported.Until such methodsare available the nature of the 16-hydroxylation in these compounds willremain obscure.Considerable interest has been shown in the 16-hydroxylations readilyachieved by micro-organisms, since it was erroneously thought 74 at one timethat the adrenocortical hormone aldosterone (28) might be a 16~-hydroxy-deox ycort icost eron e derivative.18- and 19-Hydroxylations.-Stimulated by the discovery of aldosteroneand with growing experience in handling minute traces of steroids present intissues much interest has recently centred on the hydroxylation of theangular 18- and 19-methyl groups. During an investigation of the bio-synthesis of aldosterone Wettstein and his collaborators 75 obtained, amongother products, the 19- (29) and the hitherto unknown 18-hydroxy-derivative(L) of deoxycorticosterone by the action of ox-adrenal homogenates on thissteroid. Final proof of the identity of the 18-hydroxydeoxycorticosterone(L) involved its conversion to the 18-hydroxy-3-oxoeti-4-en-20-oic lactone(M). The homogenates were supplemented with ATP, DPN+, TPN+, andfumarate.No hydroxylation occurred on omission of fumarate. Wett-stein et aZ.75 also concluded that deoxycorticosterone is superior to cortico-sterone (13) as a precursor of aldosterone in their enzyme preparation.Although the oxidation of 18-hydroxydeoxycorticosterone (L) to aldosterone(28) is possible, it cannot be assumed that deoxycorticosterone and its1 8-hydroxy-derivative are the natural precursors of aldosterone.Onedifficulty to be reconciled would be the fact that deoxycorticosterone isderived from steroids, the production of which is stimulated by ACTH. Inthis way ACTH controls the production of further hydroxylated products,74 A. Wettstein, Experientia, 1954, 10, 397.7 5 F. W. Kahnt, R. Neher, and A. Wettstein, Helv. Chim. Acta, 1955, 38, 1237GRANT THE ENZYMIC HYDROXYLATION OF STEROIDS. 329i.e., of the hormones corticosterone and cortisol. It appears, however, thatsecretion of aldosterone in man is uninfluenced by ACTH.76The scale of working necessary to produce 19-hydroxydeoxycortico-sterone (30), which according to Wettstein 75 is found in amounts up to fourtimes that of 18-hydroxydeoxycorticosterone, may be judged from thefollowing reports.Mattox 77 isolated 10 mg. of the 19-hydroxy-derivativefrom 2060 Ib. of ox-adrenals; this was about one-fifth of the amount ofaldosterone obtained. Zaffaroni et al. 7* obtained 365 mg. on incubation of10 g. of deoxycorticosterone with a homogenate of 2.5 kg. of ox adrenals.Small quantities were isolated after perfusion of 90 g. of progesterone through600 ox adrenal^.^^ The best yield was obtained by Hayano and Dorfman 8owho isolated 5 mg. after incubation of 1 g. of deoxycorticosterone withwashed residues from ox adrenal homogenates. The 19-hydroxy-derivativehas only 4% of the activity of deoxycorticosterone in the sodium retentiontest and (2% of the activity of cortisol in the Ingle work test.mby81The biological significance of 19-hydroxy-steroids may, however, lie elsewhere.Meyer 82 has identified 19-hydroxyandrost-4-ene-3 : 17-dione (31) among theproducts obtained on incubating dehydroePiandrosterone (16) with ox0 00: "P 0:oadrenal homogenate, in an excellent piece of work which is a model of itskind.The dione (31) has little biological activity. It readily yields form-aldehyde from the CH,*OH group under mild basic conditions,= to give19-norandrost-4-ene-3 : 17-dione (32) ; this may be of considerable signi-ficance in connection with the production of estrogens by the adrenal gland,the introduction of the 19-hydroxyl group being the first step in the oxid-ative removal of the 19-methyl group, a prerequisite for the aromatisationof ring ~ .~ ~ 9 82* The relative ease of aromatisation of 19-hydroxy-steroidsby chemical means has been shown by Ehren~tein.~~ Meyer 86 recentlydemonstrated the formation of aestrone (33) from 19-hydroxyandrost-4-ene-3 : 17-dione by endocrine tissues. Identification of the product in thiscase rested upon a colour reaction and the absorption spectra of the sub-stance in sulphuric acid. These very interesting findings offer an explanationof the observed conversion of [14C] testosterone (6) into labelled cestradiol-17 p76 For reviews, see: S. A. Simpson and J. F. Tait, Recent Progr. Hormone Res.,1955, 10, 204; R. Grant, A. A. Renzi, and J. J. Chart, J . Clin. Endocrinol. Metabol.,1955, 15, 621.77 V.R. Mattox, Proc. Mayo Clin., 1955, 30, 180.7 8 A. Zaffaroni, V. Troncoso, and M. Garcia, Chem. and Ind., 1955, 634.79 H. Levy and S. Kushinsky, Arch. Biochem. Biophys., 1955, 55, 290.M. Hayano and R. I. Dorfman, ibid., p. 289.8Do Q. B. Deming and J. A. Luetscher, Proc. SOC. Exp. Biol. N.Y., 1950, 73, 171.D. J. Ingle, Endocrinology, 1944, 34, 191.81 G. W. Barber and M. Ehrenstein, J. Amer. Chem. SOC., 1954, 76, 2026.82 A. S. Meyer, Ex$erientia, 1955, 11, 99.83 D. H. R. Barton and P. de Mayo, J . , 1954, 887.84 G. W. Barber and M. Ehrenstein, J . Org. Chem., 1955, 20, 1253.85 M. Ehrenstein, ibid., 1950, 15, 264; 1951, 16, 335.a6 A. S. Meyer, Biochim. Biophys. Acta, 1955, 17, 441330 BIOLOGICAL CHEMISTRY.(34) by human ovarian the isolation of cestradiol-l7@ froma testicular tumour,88 and many similar observations including the biologicalanomaly of the stallion which produces relatively huge amounts of estrogenin its urine.18- and 19-Hydroxylation have not been achieved by micro-organisms.7- and 12-Hydroxylations.-The transformation of cholesterol (35) into(36) R = C02H.(40) R = CH,Pri.(42) R = CO*NH*[CH,],*SO,H.(38) R = OH.(39) R = H.(37) R = OH.(41) R = NH*[CHJ,*SO,H.CHI 9 0 1 1 CH2.W & I o:& Iw)f43) (44)cholic acid (36) was demonstrated in dogs by the classical work of Blochet ~ 1 .~ ~ in 1943. This involved epimerisation of the 3p-hydroxyl group andthe introduction of new 7a- and 12a-hydroxyl groups, among other changes.Similar observations were made later in rats,g0 rabbit~,~l and man.92 Berg-8 7 B.Baggett, L. L. Engel, K. Savard, and R. I. Dorfman, Fed. PYOC., 1955, 14, 175.8 8 M. Marti and H. Heusser, HeZv. Chim. Acta, 1954, 37, 327.8B K. Bloch, B. N. Berg, and D. Rittenberg, J . Bid. Chem., 1943, 149, 511.S. 0. Byers and M. W. Biggs, Arch. Biochem. Biophys., 1952, 39, 301.Dl P. H. Ekdahl and J. Sjovall, Acta Physiol. Scnnd., 1955, 34, 1.s2 R. S. Rosenfeld, L. Hellman, and T. F. Gallagher, Fed. Proc., 1955, 14, 271GRANT THE ENZYMIC HYDROXYLATION OF STEROIDS. 331strom et ~ 1 . ~ 3 found that 7a-hydroxylation of deoxycholic acid (37) occurredin rats. The 12a-hydroxyl group was not, however, readily introduced intochenodeoxycholic acid 94 (38), nor was lithocholic acid (39) converted intocholic acidg5 (36) in rats. It thus appears that the hydroxyl groups ofcholic acid (36) have to be introduced in a certain order.Since Bergstromet aLg6 have shown that coprostane-3a : 7a : 12a-trio1 (40) is rapidly con-verted into cholic acid (36) on intraperitoneal administration to rats withbile fistulz, hydroxylation may occur before degradation of the cholesterolside chain. 7a-Hydroxylation has been achievedg7 in vitro by using rat-liver slices and homogenates. Microsomes and the particle-free supernatantfluid obtained by centrifuging the liver homogenates were together requiredfor the 7a-hydroxylation of taurodeoxycholic (41) to taurocholic acid (42).Only slight activity was found in the supernatant fluid alone in presence ofATP.98 It should be noted, however, that such experiments with traceramounts of radioactive steroid as substrate cannot be expected to indicateco-factor requirements.The small amount of steroid may be transformedwith the help of co-factors already present in undialysed enzyme preparationsor by means of concentrations of coenzymes maintained by substrates in thesupernatant fluid in the presence of microsomal enzymes. Bergstrom 99 hasrecently shown that there is no incorporation of the isotope in a stableposition in the molecule when biological 7a-hydroxylation is performed intritium-labelled water. It is therefore possible that 1 lp- and 7a-hydroxyl-ation are achieved by the same mechanism. From a study of the effects ofX-rays on steroids in solution Weiss loo claims that 7-hydroxylation ofcholesterol may proceed by abstraction of hydrogen and addition of a hydr-oxyl radical, a proposal which is not in agreement with the previous evidence.The conversion of cholesterol into 7-hydroxycholesterol reported byKritmli and Hon-5th,lo1 using Proactinomyces yoseus, is not completely freefrom the doubt that the reaction may have been effected by molecularoxygen without the intervention of enzymes.Kahnt et uZ.lo2 have reported7p-hydroxylation of 3p : 21-hydroxy-5a-pregnan-20-one (43) by an un-identified species of Rhizopus.12a-Hydroxylation has not yet been achieved by microbiological methods.Some interest in such a reaction might be expected since it has been claimedthat 12a : 17a-dihydroxydeoxycorticosterone (44), a position isomer ofcortisol, is an antagonist of the hormone.lO3Hydroxylations at Other Positions in the Steroid Nucleus.-Introductionof a 3-oxygen atom into the steroid nucleus has excited no comment, possibly93 S.Bergstrom, M. Rottenberg, and J. Sjovall, 2. $hysiol. Chem., 1953, 295, 278.s4 S. Bergstrom and J. Sjovall, Acta Chem. Scand., 1954, 8, 611.95 S. Bergstrom, J. Sjovall, and J. Voltz, Acta Physiol. Scand., 1953,30, 22.s6 S. Bergstrom, K. Paabo, and J. A. Rumpf, ibid., 1954, 8, 1109.O7 S. Bergstrom, A. Dahlquist, and U. Ljungquist, Kgl. Fysiogr. Sallskap. Lund.,1953, Forh., 23, No. 12; S. Bergstrom and U. Gloor, Acta Chem. Scand., 1954, 8, 1373;1955, 9, 34; U. Gloor, Helv. Chim. Acta. 1954, 37, 1927.O 8 S. Bergstrom and U.Gloor, Acta Chem. Scand., in the press.O9 S. Bergstrom, personal communication.lo0 J. Weiss, Ciba Foundation Colloquia in Endocrinology, 1953, Vol. VII, p. 142.l01 A. KrAmli and J. Horv&th, Nature, 1949, 163, 219.lo2 F. W. Kahnt, C. Meystre, R. Neher, E. Vischer, and A. Wettstein, Experientia,lo3 W. J. Adams, B. G. Cross, A. Davies, F. Hartley, D. Patel, V. Petrow, and I. A.1952, 8, 42.Stewart, J . Pharm. Phavmacol., 1953, 6, 861332 BIOLOGICAL CHEMISTRY.because of its ubiquitous occurrence and its apparent association with earlystages of biosynthesis of the steroid nucleus, knowledge of which is of recentorigin. After an early suggestion of Channon’lw Langdon and Bloch105showed that squalene (45) is an efficient precursor of cholesterol in the animalorganism.About the same time Woodward and Bloch lo6 suggested thatsqualene cyclises to an intermediate having the structure of a 4 : 4 : 14-trimethylcholestane derivative (cf. 46), a scheme which would also explainCOMe COMethe formation of lanosterol (47) as an intermediate in the biosynthesis ofcholesterol from ~qualene.10~ Since lanosterol already possesses a 39-hydroxyl group the 3-hydroxylation must have occurred at some earlierstage, possibly before cyclisation. Bucher and McGarrahaw lo* claimedthat DPN+ is essential for the overall reaction of acetate + cholesterol byrat-liver homogenates. Tchen and Bloch lo9 recently found that the super-natant fraction of rat-liver homogenate, supplemented by the microsomalparticles disrupted by ultrasonic vibration, will transform [14C]~qualene intolanosterol, a reaction sequence which includes the hydroxylation step.The authors’ claim that DPN+ had no effect is of limited significance in anexperiment with undialysed enzyme preparation and minute amounts ofradioactively labelled substrate.Microbiological hydroxylations of progesterone in the 14~t-~~O and in thelo4 H.J. Channon, Biochem. J., 1926, 20, 400.lo6 R. B. Woodward and K. Bloch, J. Amer. Chem. SOC., 1953, 75, 2023.lo’ R. B. Clayton and K. Bloch, Fed. PYOC., 1955, 14, 194.lo8 N. L. R. Bucher and K. McGarrahaw, ibid., p. 187.Io9 T. T. Tchen and K. Bloch, J. Amer. Chem. Soc., 1955, 77, 6085.R. G. Langdon and K. Bloch, J. Bid. Chew., 1953, 200, 135.P. D.Meister, S. H. Eppstein, D. H. Peterson, H. C. Murray, H. M. Leigh,A. Weintraub, and L. M. Reineke, Abstr. 123rd Meeting Amer. Chem. SOL, 1953, 5cBELL : OLIGOSACCHARIDES OF MILK. 33315a- ll1 and 15p-position have been achieved. These 14- and 15-hydroxy-derivatives (48, 49) have no known place in mammalian steroid metabolism.J. K. G.6. OLIGOSACCHARIDES OF MILK : THEIR RELATION TO THE “ BIFIDUSFACTOR ” AND TO BLOOD-GROUP SUBSTANCES.The following abbreviations are used in this section :Lb = Lactobacillus bi$dus.LbP = Lactobacillus b<fidus var. Penn.BF = “ Bifidus factor.”BG == Blood-group.The intestinal flora of the normal breast-fed infant is characterised bythe prevalence of Lactobacillus bifidus,1~2~2~ in contrast to the mixed floraof infants fed on cow’s milk.The nutritional requirements of Lb as isolatedfrom the stools of both breast- and bottle-fed infants have been studied byNorris, Flanders, Tomarelli, and G ~ o r g y . ~ During this work,* an apparentlyspecific variant of Lb, named Lactobacillus bifidus var. Pen~z,~ was en-countered ; it showed scant or undetectable growth on the regular Lb medium.If, however, whole or skimmed human milk [containing I ‘ bifidus factor@) ”1was added, vigorous growth followed.Oligosaccharides of Milk.-Recent researches have revealed a number ofhitherto unsuspected and interesting carbohydrates in human milk. Gyorgy,Norris, and Rose 5 showed that BF (virtually absent from cow’s milk) wasthermostable ; a large number of organic compounds including knownmicrobiological growth-factors, yeast extract, vitamins not present in theoriginal Lb growth medium, carbohydrates, and several vegetable extracts,were all ineffective in replacing BF.studied the BF activity of milks of species other than man and found averageactivities highest for human colostrum,* followed, in order, by rat colostrum,human milk, rat milk, and cow colostrum.The milk of ruminants (cow, ewe,goat) on the other hand showed only very slight activity. Somewhat higheractivity appeared in the milk of cat, monkey, dog, rabbit, mare, and sow.High concentrations of BF were shown to be present in various humansecretions, e.g., saliva, semen, amniotic fluid, meconium, and tears. Piggastric mucin, which is a rich source of BG substances, proved to be rich inBF; a number of other nitrogenous polysaccharides, ovomucin, a- andp-heparin, ovomucoid, chondroitin sulphate, urinary glucoprotein, hyaluronicGyorgy, Kuhn, Rose, and ZillikenUnpublished work, cited by J.Fried, ef aZ., ref. 44.H. Tissier, “ Recherches sur la flore intestinale normale et pathologique du nourri-son,” Thesis, Paris, 1900.E. Moro, Wien. klin. Wochenschr., 1900, 13, 114.2a For recent critical remarks on changes in morphological character of Lb see :H. G. Gyllenberg, J . Gen. Microbiol., 19t5:. 13, 394; A. C. Hayward. C . M. F. Hale,and K. A. Bisset, ibid., p. 292 ; E. Olsen, Studies on the Intestinal Flora of Infants,”Ejnar Munksgaard, Copenhagen, 1949.R. F. Norris, T. Flanders, R.M. Tomarelli, and P. Gyorgy, J . Bacteviot., 1950,80, 681.P. Gyorgy, R. Kuhn, R. F. Norris, C. S. Rose, and F. Zilliken, Arner. J . DiseasesChildren, 1952, 84, 848; 1953. 85, 632.P. Gyorgy, R. F. Norris, and C. S. Rose, Arch. Biochem. Biophys., 1954, 48, 193.P. Gyorgy, R. Kuhn, C. S. Rose, and F. Zilliken, ibid., p. 202. * Colostrum is the secretion of the mammary gland which precedes the secretion ofmilk proper in animals commencing lactation334 BIOLOGICAL CHEMISTRY.acid, chitin, ‘‘ 0 ” somatic antigen (Shigella s h i p ) , and gonadotrophin wereall inactive. On the other hand, polysaccharides from Pneumococci typesIV, V, VI, VII, XVIII, and XIX had growth-promoting properties whilethese from types I , 11, 111, and XIV were virtually inactive.Ammoniumsalts in high concentration, N-acetyl-D-glucosamine and N-acetyl-D-galact-osamine were active growth-promoters, the authors suggesting thesesubstances as precursors of BF.then found that the BF activity ofhuman milk and colostrum could be separated into diffusible and non-diffusible fractions and that the BG (A, 13, 0) of the donor had little effecton this fractionation. By adsorption on charcoal or fractional precipit-ation, a mixture of oligosaccharides was obtained from deproteinised,defatted, and desalted human milk; this was resolved into inactive carbo-hydrates, including lactose, and active ones which contained nitrogen ; thelatter may correspond with the “gynolactose” of Polonovski and Les-pagnoLg The allolactose of these authors was not found.The activematerials gave, on hydrolysis, acetic acid, D-glucosamine, L-fucose, D-glucose,and D-galactose. Acetylation gave inactive products from which activenitrogenous oligo- and poly-saccharides could be regenerated. By chromato-graphy on charcoal, and on paper, at least four active components werefound ; all contained N-acetyl-D-glucosamine and were lzvorotatory. Inaddition, a trisaccharide without BF activity was found in human milk(150-300 mg. /l.) ; it is absent from cow’s milk or colostrum. By classicalmethods, Kuhn, Baer, and Gauhe lo have established its structure as ana-L-fucopyranosyl-lactose (1) where the fucosyl radical substitutes position 2of the galactose moiety. The BF-active substances have been structurallyGyorgy, Hoover, Kuhn, and RoseH,O H\ - rHO ’examined by Kuhn and his collaborators; to date they have been char-acterised as follows (where Gal = a D-galactopyranose, G = a D-glUC0-pyranose, GNAc = a N-acetyl-D-glucosamine, and Fuc = a L-fucopyranoseresidue) :(a) Lacto-N-tetraose 8- 119 l 2 ~ l3 (2 ; R = R’ = H) :/&Gal 1-3 /?-GNAc 1-3 /3-Gall-4 G7 P.Gyorgy, J. R. E. Hoover, R. Kuhn, and C. S . Rose, Arch. Biochem. Biophys.,* A. Gauhe, P. Gyorgy, J. R. E. Hoover, R. Kuhn, C. S. Rose, H. W. Ruelius, and9 M. Polonovski and A. Lespagnol, BUZZ SOC. Chim. biol., 1933, 15, 320.lo R. Kuhn, H. H. Baer, and A. Gauhe, Chem. Ber., 1955, 88, 1135.l1 R. Kuhn, A. Gauhe, and H. H. Baer, ibid., 1953, 86, 827.l2 Idem, ibid., 1954, 87, 289.l3 R.Kuhn, Angew. Chem., 1955, 67, 184.1954, 48, 209.F. Zilliken, ibid., p. 214BELL : OLIGOSACCHARIDES OF MILK. 335(b) Lacto-N-fucopentaose I (Morgan-Elson positive) (2 ; R = H,R’ = a-L-fucopyranosyl) :/?-Gall-3 /?-GNAc 1-3 /3-Gall-4 G21a-FucI(c) Lacto-N-fucopentaose I1 (Morgan-Elson negative)l2, 137 (2 :R = a-L-fucopyranosyl, R’ = H) :/?-Gall-3 /?-GNAc 1-3 / ? - G a l l 4 G41a-FucI(a) Lacto-N-difucohexaose l3 (2 ; R = R’ = ct-L-fucopyranosyl) :/?-Gal 1-3 /?-GNAc 1-3 ,%Gal 1-4 G4 2I Ii ia-Fuc a-FucThese four compounds are therefore closely related, the pentaose and thehexaose being formed by addition of a-L-fucosyl radicals at appropriatepositions of the tetraose molecule, which acts as a backbone.Graded hydrolysis of lacto-N-tetraose has yielded two disaccharides andtwo trisaccharides as follows :Lacto-N-biose I : l2* l5 3-U-(/3-~-galactopyranosyl)-N-acetyl-~-glucosamineLacto-N-biose I1 : 12.14 6-GNAc 1-3 GalLacto-N-triose I : 12. l4 P-Gal 1-3 GNAc 1-3 GalLacto-N-triose I1 : 12* l4 p-GNAc 1-3 /?-Gal 1-4 GRecently, two acidic carbohydrates 16 having BF activity have beenadded to the neutral substances mentioned above. These “ acid saccharidesI and 11,” both yield, on hydrolysis, D-galactose, D-glucose, N-acetyl-D-glucosamine, L-fucose, and “ gynaminic acid.” The last is crystalline andgives a deep purple colour with Bial’s reagent.16@have shown the presence of seven carbo-hydrates in addition to lactose on paper chromatograms of charcoal-adsorbedTrucco, Verdier, andl4 R.Kuhn, A. Gauhe, and H. H. Baer, Chenz. Ber., 1954, 87, 1187.l5 R. Kuhn, H. H. Baer, and A. Gauhe, ibid., pp. 1138, 1553.F. Zilliken, G. A. Braun, and P. Gyorgy, Arch. Biochem. Biophys., 1955, 54,564; cf. E. Klenk and H. Langerbeins, 2. physzol. Chem., 1941,270, 185; E. KIenk andK. Lauenstein, ibid., 1952, 291, 147; E. Klrnk acd H. Faillard, ibid., 1954, 298, 230.16a Cf. R. Caputto and R. E. Trucco, Nature, 1952, 189, 1C61; Ciencia e Invest.(Buenos Aires), 1953, 12, 567 ; R. E. Trucco and R. Caputto, J. Biod. Chem., 1954, 206.901 ; R. Heyworth and J. S. D. Bacon, Biochem. J., 1954, 58, xxiv.R. E .Trucco, P. Verdier, and A. Rcga, Biochim. Biophys. Ada, 1954, 15, 582336 BIOLOGICAL CHEMISTRY.material from deproteinised cow’s milk.These substances differ chromato-graphically from the saccharides of human milk; two compounds had BFactivity, two were inactive, and two have not been described. None con-tained L-fucose, but in two instances (BF-active) mannose (presumably theD-isomer) was present. The Table summarises their properties.Substances * Acid hydrolytic products BF activity1 & 2 Galactose, glucose, mannose, N-acetylglucosamine +3 Neuraminic acid, lactose t4 ,# ,D BS 5 Lactose, galactose, glucose -6 & 7 ,D J , 8 8 t-* Numbers indicate relative positions on chromatogram from start-line.f Not described.BF-Active Substances of Non-lacteal Origin: BG Substances.-Theobservation that BF materials from human milk had structural factors incommon with the BG substances, although the molecular sizes of the latterare very much greater than diffusible BF saccharides, led Springer, Rose, andGyorgy l7 to examine the water-soluble BG substances from human andsome animal tissues as growth factors for LbP.BG substances are secretedby “ goblet-cell ” structures and are widely distributed in animals, frommen to molluscs. These authors found that high BG activity was alwaysaccompanied by high BF content. While precipitation by alcohols tendedto destroy BG activity, BF potency remained. Differences in the latterwere noted between different sources of BG substances, Highly purified“ intrinsic factor ” (from gastric mucin) was noted to have high activity ofboth kinds.BG ’polysaccharide materials from the following human sources werefound to have BF activity : colostrum, milk, gall-bladder mucus, mecon-ium ,I* pseudo-mucinous ovarian cyst mucus, cervix uteri mucus, bronchialmucus, nasal mucus, mucocele of appendix, and amniotic fluid.Wateryextracts from the following human tissues were also active : salivary glands,stomach, small intestine, transverse colon, kidney, lung, spleen, pancreas,prostate, and “ intrinsic factor.” Carcinomas sometimes yielded activematerial, sometimes not. Preparations from animal tissues, pig gastricmucin, cattle small intestine and abomasum, frog spawn mucin,lg and wholeoyster tissue contained BF-active material.The above survey indicates the wide distribution in the animal kingdomof material containing fundamentally similar chemical groupings ; itssignificance is therefore wider than is indicated by microbiological study alone.Some Enzyme Activities of LbP Preparations.-Enzymes capable ofinactivating BG substances, as measured by change in their specific sero-logical properties, have been studied from time to time.20 P.Gyorgy et aL2117 G. R. Springer, C. S. Rose, and P. Gyorgy, J . Lab. and Clin. Med., 1954, 43,532; cf. ref. 4.l* D. J. Buchanan and S. Rapport, J . Bio?. Chem., 1951,192, 251.l@ B. F. Folkes, R. A. Grant, and J. K. N. Jones, J., 1950, 2136.2o F. Schiff and G. Weiler, Biochem. Z . , 1931, 235, 454; 1931, 239, 489; F. Schiffand A. Bur6n, Klin. Wochenschr., 1935, 14, 710; F. Schiff, ibid., p. 750; Amer. J.Infect. Diseases, 1939, 65, 127; K. Landsteiner and M. W. Chase, PYOC. SOC. Ex$. BioZ.and Med., N.Y., 1936, 32, 713; W. T. J. Morgan, Natwe, 1946,158, 759; M . V. Stackand W. T. J. Morgan, Brit. J . Exp. Pathol., 1949.80, 470.P. Gyorgy, C. S. Rose, and G. F. Springer, J . Lpb. CEin. Med., 1954,4$, 643BELL OLIGOSACCHARIDES OF MILK. 337found that crude extracts of LbP, culture filtrates of CZostridizm welchii typeB (ATCC No. 7905), and human saliva are all capable of inactivating bothBG and BF activity; no similar enzymes were found in regular strains ofLactobacillus bifidus. BF substances of either low or high molecular weightwere inactivated ; at the same time, N-acetyl-D-glucosamine, L-fucose, andD-galactose were set free. Crude, but not purified, meconium substanceappears to contain an inhibitor to the CZ. welchii enzyme; the salivaryenzyme is not inhibited.Two isomeric crystalline disaccharides were synthesised by a crudeenzyme from LbP acting on lactose and N-acetyl-D-glucosamine, pre-sumably by transgalactosylation.22 One which gave a Morgan-Elsonreaction 23 was slightly BF-active, while the other, which was Morgan-Elsonnegative, was identified with a BF-active substance obtained by Tomarelliet aZ.24 by graded hydrolysis of pig gastric mucin. The constitution of thissugar has now been established as 4-0-((3-~-galactopyranosyl)-N-acetyl-~-gl~cosamine,~~ which has also been obtained from meconium 26 and has beenchemically syn the~ised.~'The following disaccharides of N-acetyl-D-glucosamine are also BF-active,presumably because they are enzymically hydrolysed or have their radicals" transferred " ; 3- 28 and 6-0-( ~-D-galactopyranosyl)-N-acety~-~-glucos-mine ; 22, 29* 3o NN'-diacetylchitobio~e.~~ The last substance was obtainedcrystalline by chromatography of the crude material by gradient elution(waterlethanol) on a charcoal column; it is Morgan-Elson negative with orwithout prior treatment with sodium carbonate (1 mg. was equivalent to30 pg. of N-acetylglucosamine owing to slight alkaline hydrolysis to themonosaccharide). A crude LbP extract 21 hydrolysed the disaccharide togive two mols. of N-acetyl-D-glucosamine.With regard to the Morgan-Elson reaction,= Kuhn, Gauhe, and Baer l5observed that N-acetylglucosamine substituted in position 4 gives no colour.N-Acetylglucosamine gives, with alkali, a chromogen chromatographicallyidentical with anhydro-N-acetylglucosamine. l2 Alkyl glucosaminides giveno colour unless concomitant alkaline hydrolysis liberates a reducing N-acetylglucosamine unsubstituted in position 4.The above-mentioned disaccharides, along with synthetic 6-CQ-2-acetamido-2-deoxy-D-gIucopyranosyl) -D-galactose 32 and 6-0-( P-2-acet-amido-2-deoxy-D-g~ucopyranosy~)-~-g~ucose~~ all possess BF activity,although in differing degrees. LbP enzymes therefore appear to include a@-D-galactopyranosidase and a (3-D-ghcosaminidase. The latter enzyme is22 F. Zilliken, P. N. Smith, C. S. Rose, and P. Gyorgy, J . Biol. Chem., 1954, 208,299. See also ref. 29.2s W. T. J. Morgan and L. A. Elson, Biochem. J., 1934,28,988; D. Aminoff, W. T. J.Morgan, and W. M. Watkins, ibid., 1952, 51, 379.24 R. M. Tomarelli, J. B. Hassinen, E. R. Eckhardt, R. H. Clark, and F. W. Bernhart,Arch. Biochem. Biophys., 1954, 48, 225.25 F. Zilliken, P. N. Smith, R. M. Tomarelli, and P. Gyorgy, ibid., 1955, 54, 398.26 R. Kuhn and W. Kirschenbohr, Chem. Ber., 1954, 87, 560.28 Quoted by P. Gyorgy and C. S. Rose, Proc. SOC. Exp. Biol. Med., N.Y., 1955,2s F. Zilliken, P. N. Smith, C. S. Rose, and P. Gyorgy, J . Biol. Chem., 1955, 217, 79.so R. Kuhn, H. H. Baer, and A. Gauhe, Chem. Bey., 1955, 88. 1713.*l F. Zilliken, G. A. Braun, and P. Gyorgy, J . Amer. Chem. SOC., 1955, '77, 1296.82 R. Kuhn and W. Kirschenbohr, Chem. Ber., 1954, 87, 384.Idem, ibid., p. 1547.90, 219338 BIOLOGICAL CHEMISTRY.capable of hydrolysing a number of alkyl and aryl N-acetyl-p-D-glucos-aminides but not their ~c-anomers.~~ These p-glycosides were found to haveconsiderable, but varied, BF activity. The a-anomers were inactive al-though the activity of methyl N-acetyl-p-D-glucosaminide was greatlyenhanced by addition of its a-anomer. Gyorgy and Rose 33 state that it isnot yet possible to identify the bifidus factor with any specific compound.The dlolactose Question.-Kuhn, Baer, and G a ~ h e , ~ ~ using a crudep-D-galactosidase from E. coli strain VV (ATCC No. 9637), obtained 6-0-(p-~-galactopyranosy1)-D-glucose ( I ' allolactose ") by the action of the enzymeon phenyl p-D-galactopyranoside in presence of glucose. The disaccharidehad previously been synthesised by purely chemical means.35 Kuhn et aL34suggest that the allolactose found by Polonowski et aZ.9936 originated inbacterial contamination ; they were unable to find any allolactose in milk(cf. ref. 3).Lactobacillus bifi dus from Avian Sources-Micro-organisms, classifiedas Lb, have been isolated from the caecal flora of normal 38$ 39 fromthe faeces of turkeys,h* 40 and the czcal flora of turkey poults.39 In the chick,Lb was found in greatest numbers when 16yo of lactose was incorporatedin the diet. Various of these avian strains failed to grow on a chemicallydefined medium ; luxuriant growth was, however, obtained on the additionof a commercial preparation of " papain-hydrolysed casein." This prepar-ation could not be replaced by BG substances A and €3 or by 4-0-(p-D-galactopyranosyl) -N-acetyl-D-glucosamine.D. J. B.D. J. BELL.L. CROMBIE.J. K. GRANT.33 P. Gyorgy el al., see ref. 28.34 R. Kuhn, H. H. Baer, and A. Gauhe, ibid., 1955, 88, 1713.s5 B. Helferich and G. Sparmberg, Ber., 1933, 68, 806.3O M. Polonovski and J. Montreuil, Comtp. rend., 1954, 238, 2263.37 G. L. Romoser, M. S. Short, G. F. Combs, and M. J. Pelczar, Antibiotics and313 R. E. McCarthy, Thesis, Maryland, 1953.3s F. A. Veltre, M. S. Short, and M. J. Pelczar, Proc. SOG. Exp. Riol. Med., N . Y . ,40 A. P. Harrison and P. A. Hansen, J . Bact., 1949, 59, 197.Chemotherapy, 1952, 11, 42.1953, 83, 284