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effects in solvolysis reactions’s? and (2) the effect of mum hyperconjugatave loss as that the C-I1 bond, alkyl substitution on hyperconjugation. 3--8 have the proper oraentatzon, nearly parallel, to tiic I-l,l,1-d3 was made via the corresponding alcohol electron dejiczent adjacent orbital6 and (3) that thc from 4,4-dimethyl-2-pentanone and CD3hZgI. I- end methyL groups of the neopentyl group sterically 3,3-d2 was made via the following sequence: ethyl hander the approach of the methylene group to thc pivalate, neopentyl-a-d2 alcohol, neopentyl-a-dz proper oraentataon for maxzmzim hyperconjugatton. iodide, t-butylacetic-a-d2 acid, ethyl t-butylaceAcknowledgment.-This work has been sup tate-a-dz and 2,4,4-trimethyl-2-pentanol-3,3-d2. ported by a grant from the Xational Sciencc The deuterated chlorides were about ninety per Foundation. cent. isotopically pure. The solvolysis rate con- DEPARTMENT OF CHEMISTRY stants were measured conductimetrically in ‘‘SOC,” INDIANA UYIVERSITY \- J SHI?IER, J R aqueous ethanol a t 25.00°. B L O O M I V G TIYDIAVA O~, RECEIVED APRIL 5, 1956
SOLVOLYSIS RATE CONSTANTS Compound
ki (10-4 sec.-I)
2,4,4-Trimethyl-2-chloropentam ( I ) I-l,l,l-da I-3,3-dr ISOTOPE
2.212 1 ,580 2.04
Reproducibility (‘3 or 4 runs)
10,015 3t0.010 10.03
EFFECTOF DEUTERATION, SOLVOLYSIS ALCOHOL AT 25’
Group deuterated
CHaCH1-CHp-CH2-
Compound
&Amyl chlorideIb I &Amyl chlorideIh I
IS
‘‘8057C’’
kH/ki)
1.31 1.10 1.-10 1.08
The importantfact to be noted is that the isotope rate efect of a-deuteration is so much smaller for the neopentyl than f o r the ethyl or methyl groups. Since these isotope rate effects arise from a difference in zero-point energy between the initial and transition states,’,* the conclusion t h a t t h e a-C-H bonds of the neopentyl group are not weakened in the reaction process nearly as much as those of the methyl or ethyl groups seems certain. It follows t h a t the electron loss from the a-C-H bonds of the neopentyl group is also relatively much less. This must be correlated with the fact t h a t the neopentyl group is known t o be a poorer electron releaser than the ethyl group in many reaction^.^^^^^ If this parallelism between the low isotope rate eyed of deuteration of the neopentyl group and its low electron releasing ability is accepted then the following explanations which have been offered for the latter phenomena can be discarded as not predicting the correct trends in the isotope rate effects: steric shielding of the reaction center,I0 second order h y p e r c ~ n j u g a t i o ninductive ,~ effect on h y p e r c ~ n j u g a t i o n ,and ~ steric inhibition of bond contraction.* T h e preferred explanation which apparently best fits the data involves the following points : (1) hyperconjugatizfe loss of electron density from the a-C-H bonds to the reaction center is necessary for the isotope rate eflect and ( 2 ) a condition for maxi(1) V. J. Shiner, Jr., THISJOURNAL, (a) 76, 1803 (1954); (h) 7 5 ’ 2925 (1953). . . (2) E. S. Lewis and C. E. Boozer, ibid.,76, 791 (1954). (3) V. J. Shiner, Jr.. M. J . Boskin a n d M . L. Smith, ibid., 77, 5 5 2 ; (1955). (4) E. Berliner and F. Berliner, ibid., 73, 222 (1950). (5) W. A. Sweeney and W. hl. Schubert, ibid., 76, 2085 (1954). (6) G. Baddeley a n d hl. Gordon, J . Ckem. Soc., 2190 (1952). (7) P. W. Robertson, J. R. Heyes a n d B. E. Swedlund, ibid., 1014 (1954). (8) A . Burawoy and E. Spinner, ibid., 2085 (1!?:5). (9) D. G. Coe, S. R. Landauer and H. N. Rydon, ibid., 2281 (1954). (10) C. C.Price and W . J. Belanger, Tius JOUKSAI., 76, 1014 (1954)
STEROID 2 1-HYDROXYLATION BY A D m N A L MICROSOMES AND REDUCED TRIPHOSPHOPYRIDINE NUCLEOTIDE’
Sir: Biological reactions involving liver microsomes include proteinz and cholesterol3 syntheses and drug oxidation.4 The enzymatic step for the 21hydroxylation of 17a-hydroxyprogesterone has now been shown to occur in beef adrenal microsomes. Plager and Samuelsj and Hayano and Dorfmanfi have previously reported A T P and DPK dependent steroid 21-hydroxylation in beef adrenal extracts. I n the present study, beef adrenal fractions were prepared by differential centrifugation as described by Schneider and Hogeboom.7 X 1 : 1 homogenete was prepared in 0.25 JI sucrose, 0.1 -11phosphate buffer a t p H 6.8 and 0.04 1f niacinamide. The microsomal fractions were obtained between 20,000g and 105,OOOg, washed and recentrifuged. T h e 105,OOOg supernatant was further purified by discarding protein precipitated a t pH 5. Steroids were dissolved in propylene glycol and added to tissue fractions which were then incubated for one hour a t 37” with air as the usual gas phase. 111cubation mixtures were extracted with methylene chloride, evaporated and the residues partitioned between 95yo methanol and hexane. The aqueous methanol was evaporated and the enzymatic conversion of lia-hydroxyprogesterone to I T , ? 1dihydroxyprogesterone (Reichstein’s Substance S) was assayed by development of the Porter-Silber chromogen8. The product was identified as l i a , ? l dihydroxyprogesterone by Bush paper chromatogr a p h ~ counter-current ,~ distribution and infrared spectroscopy.I0 The conversion of progesterone, 1l&hydroxyprogesterone and 11&17a-dihydroxy(1) This is Publication h’o. 864 of t h e Cancer Commission of Harvard University. This work W a s supgorted by Research Grant No. C 1393-(C4) from t h e S a t i o n a l Cancer Institute of the Kational Institutes of Health, Public Health Service, a grant from t h e American Cancer Society and a grant from t h e Jane Coffin Childs hlemorial Fund for Rfedical Research. ( 2 ) J. W. Littlefield, E. B. Keller, J . Gross and P. C. Zamecnik, J . Bio!. Chem., 217, 1 1 1 (1955). (3) S . L. R . Bucher and K. SlcGarrahan, ibid., in press. (4) B. B. Brodie, e l ai., Science, 1 2 1 , 603 (1955). ( 5 ) E. J . Plager and I,. T . Samuels, Federatio?t Proc., 12, 357 (1953). ( f i ) 3 f . Hayano and R . I . Dorfman, Aich. Bzorizem. a n d Biophrs., 3 6 , 237 (1952). (7) %’. C . Schneider and G. H. Hogeboom, J . Bio!. Ciiem., 183, 123 (1950). 18) C. C. Porter and R . H. Silber, ibid., 1 8 5 , 201 (1950). 19) I. E. Bush, Biochrin. J . , 50, 370 (1951). (IO) Infrared analyhib WLIT carried out b y 1 I r s . D. \Vheeler.
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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, HARVARD MEDICALSCHOOL, KENNETH J. R Y A N ~ * BOSTOX,MASSACHUSETTS LEWISL ENGEL RECEIVED MARCH12, 1956 (11) Fellow
1
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 formed, p M . 0.91
+
+
2
3
5
6
+
+
+
4
+
1.05 0.00 0.00
+ 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 by degradative studies. %-e have now synthesized dihydronovobiocic acid (VII) I and cyclonovobiocic (VI) ; these syntheses confirm 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 p H 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 a n d 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 a n d J. W. Hinman, ibid., 77, 6710
(1955).
