COMMUNICATIONS TO THE EDITOR
June 5 , 1 9 3
2653
progesterone t o their respective C-21hydroxylated derivatives was also demonstrated and the products characterized b y paper chromatography with several modified Bush solvent systems. The conversion of 17a-hydroxyprogesterone t o Substance S is the model 2 1-hydroxylation reaction described in this report, but the characteristics of the system were the same for all progesterone derivatives tested. Microsomes and 10t5,000g supernatant fractions of beef adrenals were found t o be inactive separately but 21-hydroxylation occurred after recombination :
et al.,4 for the oxidation of drugs with liver microsomes and T P N H and with certain aspects of cholesterol s y n t h e s i ~ . ~The same fundamental mechanisms may be operative in all these cases.
TABLE I Conditions: Microsomes (20 mg. protein) and/or supernatant (94 mg. protein) were incubated in phosphate buffer pH 6.8 for one hour a t 37' in air with 10 p M . D P S , 10 p M . ATP, 15 pM. niacinamide, 3 p M . 17-hydroxyprogesterone in a total of 5 cc. Incubation mixtures were extracted as described, Porter-Silber chromogen developed and measured at 410 mp.
NOVOBIOCIN. IV. SYNTHESIS OF DIHYDRONOVOBIOCIC ACID AND CYCLONOVOBIOCIC ACID
Yield. aM.
Fraction
1 brashed microsomes 2 105,OOOg supernatant 3 Recombination of 1 and 2
0.00 0.00
0.64
The 105,OOOg supernatant fraction from r a t liver was found t o be as effective as adrenal supernatant fraction when combined with the adrenal microsomes, but r a t liver microsomes were inactive in recombination experiments. Treatment of the supernatant fraction with an anion exchange resin (Dowex 1-acetate form) rendered the recombined system inactive unless D P N and ,4TP, or T P N were added. Niacinamide also increased activity. Glucose-6-phosphate, Zwischenferment and T P N could substitute for the supernatant. Finally, T P N H in substrate amounts was equally effective and appears t o be the specific coenzyme (Table 11). D P N H appeared t o be about one-fifth as active. Oxygen was required but cyanide and azide had no effect on the hydroxylation. Moreover, hydroxylation proceeded in the presence of catalase.
FROM THE MEDICAL LABORATORIES OF THE COLLISP. HUSTINGTON MEMORIAL HOSPITALOF HARVARD USIVERSITYAT THE MASSACHUSETTS GENERAL HOSPITAL A N D THE DEPARTMENT OF BIOLOGICAL CHEMISTRY, KENNETH J. R Y A N ~ * HARVARD MEDICALSCHOOL, BOSTOX,MASSACHUSETTS LEWISL ENGEL RECEIVED MARCH12, 1956 (11) Fellow
1
2
3
4
105,OOOg supernatant T P S (0.3 p M . / c c ) Zwischenferment and glucose-6phosphate (8 p M ./ c c . ) D P S H (3 pM./cc) T P S H (3 ~ M . / c c ) 17,21 - Dihydroxyprogesterone 0 . 9 1 1.05 0.00 0.00 formed, p M .
+
+
6
+
+
+
5
+
+ 0.26
+ 1.41
I t is interesting t o note the similarity of this adrenal system with t h a t described b y Brodie,
Cancer Research of the American Cancer Society.
Sir: The structure of novobiocin has been elucidated %-e have now synby degradative studies. thesized dihydronovobiocic acid (VII) I and cyclo(VI) ; these syntheses confirm novobiocic the structure assigned t o the aglycon moiety of novobiocin. Condensation of 2-methylresorcinol with ethyl cyanoacetate in the presence of zinc chloride and hydrogen chloride gave 7-hydroxy-3-imino-8-methyl-2-oxochroman (I), m.p. > 350'. 1t2.3*4
0 11.
OH T'
CH8
CH3
TABLE I1 Conditions: additions were incubated with microsomes (34 mg. protein in case 1; 17 mg. protein in others) in Tris buffer a t pH 7.2 for one hour a t 37' in air with 1.0 pM./cc. 17-hydroxyprogesterone ; total volume 3 cc. Incubation mixtures were extracted as described and Porter-Silber chromogen developed and measured a t 410 mp. The quantitative data are supported by chromatographic analysis. Additions
In
1-11
Hydrolysis of I in 50% sulfuric acid afforded 2,7-dihydroxy-8-methylchromone(11), m.p. 270" (Calcd. for CIOHR04:C, 62.60; H , 4.20. Found: C,62.70;H , 4.45). Treatment of I1 with nitrous acid yielded 2,4-dioxo-7-hydroxy-8-methyl-3-oximinochroman (111), which was hydrogenated to give the amine hydrochloride IV. This compound has also been obtained by degradation. Acetylation (1) C. H. Shunk, C. H. Stammer, E. A . Kaczka, E. Walton, C. F. Spencer, A. N.Wilson, J. W. Richter, F. W. Holly and K. Folkers, THISJOURNAL, 78, 1770 (1956). (2) J. W. Hinman, H. Hoeksema, E. L. Caron and W. G. Jackson, ibid.,7 8 , 1072 (1956). (3) E.A. Kaczka, C. H. Shunk. J. W. Richter, F. J. Wolf, M. Gasser and K. Folkers, ibid., in press. (4) H. Hoeksema, J. L. Johnson and J. W. Hinman, ibid., 77, 6710
(1955).
2636
COMhIUNIC.3TIONS TO T H E
EDITOR
Vol.
is
: CHS-CH.2. (I) ,CHX-CHr: (la) of IV with acetic anhydride in pyridine yielded IT, m.p. 263-263° (Calcd. for C14H&06: C, 57.73; \Vith excess of monomer both ends of I or Ia H, 4.50; N, 4.81. Found: C, 3T.43; H, 4.12; N, 5.10). The infrared absorption spectrum and propagate polymerization, each by a different melting point of the synthetic product and of the mechanism, one end growing as a radical, the other compound obtained by degradation of novobiocin as a carbanion. After addition of the first monomeric unit t o either end, structures I or I a become were identical. 2,2-Dimethylchroman-6-carboxylic a ~ i dwas ~ , converted ~ with thionyl chloride perfectly legitimate, i.e., a true separation of elecinto 2,2-dimethylchroman-6-carbonyl chloride, n1.p. trons takes place and species like I1 are formed. 95-97'. Treatment of the amine hydrochloride : CHS--CH?-CHX-CH?. or IV with this acid chloride in pyridine gave cyclo:CHX-CH?-CH?-CHX. (11) novobiocic acid (VI), m.p. 280-2884". Aimixture of this product and VI obtained by degradation of The radical ends do not long exist. At low novobiocin melted a t 280-2SG0, and the infrared temperature they dimerise, and consequently absorption spectra of the two were identical. species I11 are formed .l-Xcetoxy-3-(3-methylbutyl) -benzoic acid,4 m.p. : CHS--CH2--CH?-CHX : (111) 147-1849', was converted with thionyl chloride into 4 - acetoxy - 3 - (3 - methylbutyl) - benzoyl chloride. Inspection of species I11 suggests t h a t they do Upon treatment of IV with this acid chloride in not terminate, and thus propagation should pyridine, followed by hydrolysis, dihydronovobiocic continue until all the monomer is consumed, and acid (VII) was obtained, m.p. 23'7-2339". ;Z mix- eventually a "living" polymer is produced. ture of this product and VI1 obtained by degradaThe correctness of all these assumptions has been tion of novobiocin melted a t 237-239". The iden- tested. Polymerization, if carried out correctly, tity was confirmed by the infrared absorption spec- always goes to completion. The green color of tra. naphthalene- ion instantaneously changes into deep CLACDE F. SPESCER red on addition of styrene, the latter color due CHARLES H. STAMMER MERCKSHARP & DOHME Xfter completion of polymerizaRESEARCH LABORATORIES J O H N OTTORODIY to styrene- ends. tion the red color persists, indicating no reverse DIVISIONOF MERCK& Co , Ixc EDWARD ~I'ALTOX RAHWAY, SEW JERSEY FREDERICK II' HOLLY of reaction (2). If an additional amount of styrene KARLFOLKERS is added after completion of the polymerization of RECEIVED MAY9, 1956 the first portion, the polymerization starts again, and the reaction goes again to completion. The viscosity of the polymer solution can be POLYMERIZATION INITIATED BY ELECTRON A NEW METHOD OF measured without withdrawing the solution out of TRANSFER TO MONOMER. FORMATION OF BLOCK POLYMERS' the reaction vessel. Xddition of further amounts of Sir: styrene and of solvent, quantities of which are *iromatic hydrocarbons react with metallic adjusted in such a way t h a t the ratio styrene: sodium in suitable solvents yielding colored and solvent remains unaltered, leads to an increase in soluble complexes of composition 1 S a to 1 hydro- the viscosity of the solution. For example, carbon*which initiate polymerization of conjugated styrene (9.2 g.) was added to 60 cc. of tetrahydromole of sodium napholefinic hydrocarbons.3 Their structure has been furan containing 3.3 X elucidated by IVeissman and Lipkin,l who showed thalene. Polymerization was carried out a t -80" them t o be composed of negative aromatic hy-dro- and after completion the viscosity of the solution carbon ions and K a + ions. They have shown also was determined a t room temperature (1.2-1 ..? that the negative hydrocarbon ions act as electron sec.). The solution was recooled to - S O " , and an additional '7.7 g. of styrene in 30 cc. of tetrahydrotransfer agents, e.g. furan was added. Xfter completion of the reaction SaphthalenePhenanthrene J_ the viscosity was found to increase to 18-20 sec. a t Phenanthrene- + Saphthalene (1) room temperature. The final yield was IO.(; g. of \Ve postulate the same type of electron-transfer polystyrene, i.e., about 100T conversion. This process t o be responsible for the initiation of poly- proves conclusively the existence of living ends in these polymers, and suggests a n interesting and novel merization by sodium-naphthalene complex, e.g. method for preparation of block polymers. .After Saphthalene- + Styrene -+ completion of the first polymerization process, StyretieSaphthalene (2) a second Inonoiner is added t o the still living The negative monomer ions formed by reaction polymers formed from the first monomer. Thus, ..I block polymers of the type .A .A 1.R .R ., .B .*LA., (2) may be represented formally by I or 1a.j are produced. For example, tyrene (5.7 g . ) (1) This research was partially supported by a grant from the Office was polymerized by 0.3 millimole of catalyst in the of Naval Research Contract Sonr-131091. .6 g. of isoprene was added. usual way and the (2) N. D. Scott, J . 1'. \Valkrr and V. I.. Hansley, THIS~ouxvsl., Xfter precipitation g. of polymer was obtained. 68, 2442 (1930). The polymer solution in toluene could not be pre(3) N. D. Scott, U. S.Patent 2 , 1 8 1 , 7 7 1 ( I 9 3 Q ) . (1)D. E Paul, D. Lipkin and S. I . IVeisiman, T H I S J O I J R S A I . , 78, cipitated by isooctane, proving the absence of pure 1 1 R (1950). polystyrene. On the other hand, the polymer is ( 5 ) This is only a formal notation. Styrene- should be visualized not extractable by isoSctane, proving the absence a s a species possessing an additional electron in t h e lowest li orhital of pure polyisoprene. Thus, the nature of the which is unoccupied in t h e ordinary molecule of stl-rene.
+
+
