July 5, 1957
3 ti03
COMMUNICATIONS T O T H E EDITOR T H E CLEAVAGE OF 3-TROPANYL CHLORIDE W I T H POTASSIUM CYANIDE
Sir: When 3-tropanyl chloride' (I) was treated with potassium cyanide in alcohol solution a sharpboiling (b.p. 6S-7Oo, 0.2 mm.) mixture of isomers was obtained whose analysis agreed with the formula C9HI4N2 ( I ) ; (Anal. Calcd. for CgH14N2: N , 18.65, NAP,'9.33. Found: N, 18.35; NAP,^ 9.38). The infrared spectrum3 was characterized by the presence of two cyanide bands a t 4.50 1.1 and an intense band a t 6.08 y. When this oil was treated with phenylmagnesium bromide a liquid (b.p. 69-70', 0.1 rnm.), presumably a mixture of isomers also, was obtained, the analysis of which was compatible with the formula C14H19N (111); (Anal. Calcd. for C14H19N: C, 83.53; H , 9.51; N, 6.96. Found: C, 83.70; H, 9.81; N, 6.84). The infrared spectrum showed that bands due to the aromatic ring system and the 6.08 ~1 band were present but the abcorption a t 4.50 p had disapneared. This displacement of a nitrile function by the radical of a Grignard reagent is manifested by a-dialkylaminoacetonitriles.4 The band a t 6.08 y is indicative of a terminal vinyl qroup (confirmatory bands a t 11.00 y 3.27 p). Catalytic hydrogenation resulted in the uptake of one mole of hydrogen and the bases IV (b.p. 111-113', 0.4 mm.). (Anal. Calcd. for C I ~ H ~ I NN,: 6.86. Found: N, 6.90) were isolated. The band a t 6.08 y had disappeared. Structures 1-117 illustrate the course of the reactions. The mixtures which persisted throughout were due to the formation of cis and trans pyrrolidine nitriles in the first step. The bases (IV) were converted to a mixture of methiodides (V), m.p. 126-130', the most readily purified of which melted a t 123-174' after crystallization from acetone (Anal. Calcd. for C15Hz01N: I, 36.1. Found: I, 36.0). Hofmann degradation of the bases corresponding to V furnished an oil VI (b.p. 125-128', 0.4 mm.). (Anal. Calcd. for CISHzvN: N, 6.46. Found: N, 6.44) which showed 253 (log e typical styrene absorption, AFz 4.07). Reduction in the presence of Adams catalyst which furnished 4-dimethylamino-l-phenylheptane, was independently synthesized from l-phenyl-4heptanone via oximation, reduction and methylation. The picrates from both samples melted at 92.5-94" (Anal. Calcd. for C21HZsN407:N, 12.49. Found: N, 12.23) and were otherwise identical in all respects. The 1-phenyl-4-heptanone (b.p. 96-
+
11) hf. Polonovski and M. Polonovski. Bull. soc. chim.. [IV] 46,
305 11929) ( 2 ) Aceticperchloric acid titration of basic nitrogen. Analyses were carried out under the supervision of Mr. K. D. Fleischer. ( 3 ) We are indebted t o Miss C. M. Martini for determination L f t h e spectra reported here. (4) P. Briiylants, Bull. SOL. chim. Belg., 33, 4fi7 (1924); Chcm. Abs., 19, 255 (1925).
c1
''
__
-
2CHzCH3
l C s H ,i\ I - C H i C H l C H a
CsHi\x/-CH I
CHI
IV
--f
Iy,
I
V C~H,CH=CH-CH~-CH(YZ-C~H~) (CH3)z
1.1
I
S(CHj)?
98'; 0.1 mm.; Anal. Calcd. for C13HI80:C, 82.06; H , 9.53. Found: C, 82.07; H , 9.43) required for this synthesis was prepared from y-phenylbutyronitrile and propylmagnesium iodide. S ARCHER STERLING-~'IYTHROP T R LEWIS R E S E A R CIXSTITUTE H REBSSELAER, X-EW YORK BERVARD ZEKITZ RECEIVED M A Y29, 1957
THYMIDINE INCORPORATION INTO DEOXYRIBONUCLEIC ACID OF RAT LIVER HOMOGENATES
Sir: The incorporation of radioactive thymidine into DNA' of cell-free extracts of E . coli was reported in 1936.' Attempts to demonstrate thymidine incorporation in homogenates of chick embryo were unsuccessful, although rabbit thymus nuclei have been reported to be active in this recpect.* In normal or regenerating rat liver homogenates which actively incorporate radioactive orotic acid into RNA, no radioactivity was found in D N A 5 The availability6 of very high specific activity tritium labeled thymidine encouraged us to re-examine DNA synthesis in rat liver homogenates. Our experiments have met with some success, and will be described briefly herein. (1) These abbreviations are used: DNA, deoxyribonucleic acid; R N A , ribonucleic acid; ATP, adenosine triphosphate; and Tris, tris(hydroxymethy1)-aminomethane. (2) A. Kornberg, 1 . R . Lehman and E. S . Simms, Federation Proc., 16, 291 (195ti). ( 3 ) M. Friedkin, D. Tilson and D . Roberts, J . B i d . Cheni., 220, 627 (195R). (4) M. Friedkin and H. Wood, ibid., 220, 639 (1926). ( 5 ) E. Herbert, V. R . Potter and L. 1. Hecht, ibid., 226, 659 (1957). (6) From Schwarz Laboratories, Inc., Mount Vernon. N. Y. Sprcific activity of t h e thymidine is reported t o be 390 p c . / p M . , labeled in position 4; radiochemical purity 100%.
CO~IXLTICATIOSS TO THE EDITOR
36U4
Previous work7 demonstrated that following hepatectomy, no DNA is synthesized or labeled by orotic acid for the first 18 hours following the operation. An experiment was therefore designed in which homogenates were prepared from lobes removed at partial hepatectomy, as well as from liver allowed to regenerate 15 hours, and 24 hours and incubated in the presence of radioactive thymidine. The results presented in Table I show the incorporation observed in these preparations,. TABLE 1 INCORPORATION OF THYMIDISE IXTO D S A HOMOCENATES
OF
1
2 I
2 :i
C.p.m./ Homogenate from
Lobes removed a t partial hepatectomy Lobes removed a t partial hepatectomy Liver 15 hours after operatiori Liver 2 1 hours after operatioii Liver 24 hours after operatioii
plate (actual")
D S A a S p . act.mg./ c.p.m./mg. plate DNA
350
0.35
1,000
270
.32
810
360
.30
1,200
12,300
4,iMI
.28 44,000 .%j
cident with an ultraviolet quenching spot, with Rr and spectrum of t h ~ m i n e . ~ (9) Cf.G. R . W y a t t i n E. Chargaff and J. X. Davidson, "The h-ucleic Acids," Vol. I , Academic Press, New York, N . Y., 1955, PP. 243-205. (IO) Postdoctoral Fellow ni t h e Kational Cancer Institute. 'This investigation was also supported b y a grant ( S o . C-646) from t h e National Cancer Institute, National Institutes of Health, Public Health Serrice.
MCARDLEhfEMORIAL I2AB0R.4TOR'2 UNIVERSITYOF \TISCOSSIN MADISOX 6, WISCONSIN RECEIVEDMAY21, 1957
F. J. BULLLJM~" R . POTTER
J-AN
R A T LIVER
Each 50-ml. flask contained: 2 ml. of 2554 r a t liver homogenate; approximately 3 X 106 c.p.m. ( 3 micrograms) of thymidine*: 20 p M . each of K fumarate, K glutamate and I( pyruvate; 5 p M . disodium A T P ; 100 p M . nicotinamide; 12 pM.MgSO,; 12 p M . I; phosphate, pH 7.6; 10 pk1. KCI; 20 p M . Tris:HCI buffer, p H 8; and 500 pkl. of sucrose in a final volume of 2.55 m l . ; incubation, for one hour with shaking, 38', O2 i i i the gas phase. Reaction was stopped b y placing the flasks in ail ice-bath. Sociiurri nucleates extracted and DXA purified as described previously.' A trace of radioactivity associated with RNA4 is attributed t o slight degradation of radioactive D S A during the mild alkaline hydrolysis required t o remove R X A . Rat no.
Vol. 79
FORMATION OF L-XYLULOSE F R O M I.-GULONOLACTONE IN RAT KIDNEY
L-Xylulose, a sugar excreted b y patients with essential pentosuria, is present normally in urine of man,' guinea pig' and rat.2 Conversion of Dglucuronolactone to L-xylulose occurs in and guinea pig.' L-Gulonic acid, which is formed from D-glucuronolactone in the rat and guinea pig,4 has been postulated to be an intermediate in this reaction.' This communication reports the presence of an enzyme system in rat kidney which catalyzes the formation of L-xylulose from L-gulonolactone. I t has been shown previously t h a t L-gulonolactone is converted to L-ascorbic acid in r a t ~ . j - ~ Carboxyl labeled and uniformly labeled Lgulonolactones were incubated with the soluble fraction of rat kidney (Table I).9 Carboxyl labeled L-gulonolactone yielded 30 to BOY', of the added C14 in COz.'o Uniformly labeled L-gulonolactone yielded about one-sixth the amount of CI4 in CO?.
1(\,90(1'
Tritiurn \vas asstyeti i i i winrtowlcss flow counters. Since approsirnatcly the satnc amount ( i f 1 ) S A was placed on each plate iio correction has been made for self-absorption. Crude self absorption curves for tritium, using DN.4 a s absorber, indicate a correction o f about -'-fold could be apColorimetric analj plied to the D S A saniples. the Dische dipheriylamine reagent. This homogenate aged for 2 hours a t 0" before incubation. All others incubated immediately after preparation.
It is apparent that a low level of incorporation is present in the zero hour and 15 hour regenerated preparations, and that the 24 hour regenerating liver shows about a 50-fold increase (Rat no. 2) in ability to incorporate thymidine into DNA. Thus the results with the homogenates are in agreement with previous studies with orotic acid in vivo' and in rat liver slices8 It is our opionion that the marked incorporation seen in the 24-hour livers and the low incorporation in other samples argues against simple exchange reactions, since in all cases the same amount of tissue was incubated with the labeled precursor under identical conditions. T h e distribution of tritium in the isolated DNA was examined by hydrolysis with 989; formic acid for 2 hours a t 165' and chromatography of the bases in 2-propanol : HCl on S. and S. no. 389 filter paper. The chroinatograrn showed only one radioactive spot, coiw 17) I.. I . lIeclil and V I
