Addition of NaHCO3 to an aqueous solution of (4u) pre- cipitates the red quinonoid diazo compound (5u) [diazo band at 2220 cm-I] in 63 :< yield. In contrast to the diazo- dicyanomethylene compound prepared by Hortzlcr, (5tr) can be recrystallized from methanol by addition of ether and gives good results for its elemental analysis even after 24 h. On heating, it decomposes slowly without melting. The diazo compound (Sb) is much less stable than (5u). I t is not precipitated by NaHC03 but can be extracted from its aqueous solution with methylene chloride or ethyl acetate. It decomposes within a few minutes and has, therefore, not yet been isolated in the solid form. If hydrogen chloride is passed into a fresh extract of (5b) in methylene chloride, (46) is reformed. Compound (56) also couples with 9- naphthol to give an azo dye.

Received, October 12th, 1964 [Z 842/667 IE] German version: Angew. Chem. 76, 954 (1964)

[I] H. D. Hartiler, J . Amer. chem. SOC. 86, 2174 (1964). [2] H. G. Garg, J. Indian chem. SOC. 38, 343 (1961). [3] F. M . Beringer, P . S. Forgione, and M . D. Yudis, Tetrahedron 8, 49 (1960).

Azaflavazole Derivatives ~ New Heterocycles

By Prof. Dr. G. Henseke and Dip1.-Chem. D. Lehmann

Institut fur Organische Chemie der Bergakademie Freiberg/Sachsen (Germany)

We have obtained azaflavazoles by three different routes. 2,3-Diamino-5-bromopyridine condenses with l-phenyl-3- methyl-4,5-dioxopyrazoline in water slightly acidified with acetic acid to give the anil ( I ) , m. p. 218-220 'C (decornp.), as red brown crystals from aqueous acetic acid. When heated briefly in 1 N NaOH, the latter affords an 80 yield of 6- bromo-3-methyl-l-phenyl-4-azal=iavazole (20) [ I I as yellow prisms, m. p. 223-224 "C from ethanol.

k (1) (Za), R = CH3, (2c), R = CO,H

HP 7 (2b), R = -C-C-CHZOH

I OH

The reaction of 2,3-diamino-5-bromopyridine with dehydro- L-ascorbic acid and phenylhydrazine [2] at 25 "C leads to 20 % of the azaquinoxaline (3.) as red prisms, m.p. 281 to 283 "C (decomp.) from methanol; when (3.) is heated with 0.01 N NaOH, it is transformed quantitatively into 6- bromo-3-[~-threu- I ,2,3-trihydroxypropyI]- 1 -phenyl-4-ara- flavazole (Zb), yellow needles, m.p. 214-216 OC (decomp.) from ethanol.

CGHS-NH I H N O H

Dehydrogenative cyclization with phenylhydrazine of 7- bromo-5-azaquinoxaline-2-carboxaldehyde phenylhydrazone (36) in water weakly acidified with mineral acid gives a 50 7(: yield of 5-bromo-1-phenyl-7-azaflavazole (4) as yellow prisms, m.p. 247-248 "C from ethanol.

Oxidation with alkaline peroxide at 30°C of the aldehyde prepared from (26) led to 6-bromo-l -phenyl-4-azaflavazole- 3-carboxylic acid (2c), yellow prisms, m.p. 268-270°C froin dioxan.

Received, October 12th. 1964 [Z 8431668 IE] German version: Angew. Chem. 76. 954 (1964)

[I] Regarding the nomenclature, cf. H . Ohle and C. A . Mel- konian, Chem. Ber. 74, 280 (1941). 121 Cf. G. Henseke and K . Dittrich, Chem. Ber. Y2, 1550 (1959).

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Reversibility of the Dissociation of Glutamate Dehydrogenase from Beef Liver into Its

Polypeptide Chains [ l]

By Dr. H . Sund

Chemisches Laboratorium der Universitat Freiburg/Breisgau (Germany)

In its native, associated state, glutamate dehydrogenase from beef liver is a prolate particle with a particle weight of 2x 106. If it is considered as an ellipsoid of revolution, then its major and minor axes are about 930 and 70 A long, respec- tively [2]. This particle is in a reversible, spontaneously attained equilibrium with subunits having particle weights of I .: 106, 0 . 5 ~ 106, and 0.25~ 106, all of which exhibit enzymatic activity. Further cleavage into the polypeptide chains occurs in the presence of urea or long-chain alkyl sulfates or at p H values above 10 or below 4; this change is accompanied by loss of the native chain conformation and the enzymatic activity, and so far has been found to be always irreversible [2-71. We have now succeeded in showing that this dissociation, too, is in fact reversible. Incubation of glutamate dehydro- genase in the presence of mercaptoethanol at p H 2.0-2.2 leads to complete inactivation of the enzyme. Subsequent dialysis against mercaptoethanol at p H 7.6 effects extensive reactivation: specific activity [8] before denaturation with acid: 6200 min-1, after denaturation: 10 min-1, after re- naturation by dialysis at p H 7.6: 4500 min-1. These results show that glutamate dehydrogenase from beef liver contains in its amino-acid sequence the information required for the chain conformation of the native, catalytically active en- zyme protein.

Received, October 12th, 1964 [Z 841/669 IE] German version: Angew. Chem. 76, 954 (1964)

[ I ] Reported at the Meeting of West-German Chemistry Lec- turers in Freiburg/Breisgau, April 29th, 1964. [2] H . Sund, Acta chem. Scand. 17, S 102 (1963) and unpublished results. [3] B. Jirgensons, J . Amer. chem. SOC. 83, 3161 (1961). [4] H . F. Fisher, L. L. McGregor. and D. G. Cross, Biochim. biophysica Acta 65, 175 (1962). [5] J . Wolf , J. biol. Chemistry 237, 230 (1962). [6] C. Frieden, J.bioI.Chemistry237, 2396 (1962); 238, 146 (1963). [7] H . Sund in : Mechanismen enzymatischer Reaktionen (14th Mosbacher Colloquium of the Gesellschaft fur Physiologische Chemie). Springer-Verlag, Berlin-Gottingen-Heidelberg 1964, p. 318. [8] H. Sund and A. Akeson, Biochem. Z. 340, 421 (1964).

Explosion during the Oxidation of Tetralin with Hydrogen Peroxide in Acetone (Comments)

By Prof. Dr. W. Treibs, Heidelberg (Germany)

H. Seidl recently reported [l] on an explosion which occurred during the preparation of tetralone from tetralin in acetone using hydrogen peroxide. Since publication of the original procedure [2], this method has been used in numerous experiments for oxidizing open-

802 Angew. Chem. internut. Edit. Vol. 3 (1964) / Nu. 12

chain and cyclic olefins and ketones, as well as terpenes and sesquiterpenes without any accidents. Since the description of the original procedure was rather terse,detailed instructions are given here for the production of cc-tetralone.

One gram of vanadium pentoxide is placed in a beaker and a total of 30 ml of 10 % hydrogen peroxide is added in portions. Strong effervescence occurs and heat is liberated. The product is a green-brown voluminous mass, which is dissolved in 100 ml of a 20:SO v/v mixture of 10 % H202 and acetone. The solution is filtered and added to a solution of 500 g of tetralin in 5 1 of acetone. The reaction mixture is stirred or shaken and 30-40 % H202 is added in portions of 50 ml, keeping the temperature at 30-40 ‘ C ; before each new addition, the color of the mixture should be allowed to change from yellow-red to green or brown-green. If the mixture separates into two phases, enough acetone is added to make the solution homogenous again. The oxidation is complete when the color change occurs only slowly. A little fresh tetralin is then added, and the whole is boiled for 1 h under reflux. The solvent is then distilled off, and the residue is separated in a separating funnel. The lower, aqueous phase is extracted with ether, and the extract is added to the upper, organic phase, which is then shaken with 100 ml of 2 N sodium hydroxide and washed thrice with 100 ml por- tions of distilled water. The ether is removed, and the residue is fractionated in YUCUO on an oil bath.

The acetone used as solvent in the reaction is distilled before- hand from KMn04. Pure methanol can also be used as solvent, but after dilution with H202 it has less dissolving power than acetone. Minernl acids must not be preAent, not even i n troces.

Received, October 15th, 1964 [Z 856/681 I E I German version: Angew. Chem. 76, 990 (1964)

. . ..

[I] H . Seidl, Angew. Chem. 76, 719 (1964); Angew. Chem. internat. Edit. 3, 640 (1964). [2] W. Treibs, Ber. dtsch. chem. Ges. 72, 7. I194 (1939); Angeu.. Chem. 52,698 (1939); Brennstoffchemie 20,358 (1939); W. Treibr, G. Friritke, G. Leichsenrinx, and H . Roeder, Chem. Ber. 86, 616 (1953).

Formation of l-Benzyl-2,3-diphenylindanes from Stilbene in the Presence of Tin Tetrachloride under the Influence of y-Radiation

By Dr. M. Salzwedel, V. Werner, and Priv.-Doz. Dr. D. Schulte-Frohlinde

Strahlenchemisches Laboratorium, Kernforschungszentrum Karlsruhe (Germany)

Irradiation of cis- or trans-stilbene in the presence of tin tetra- chloride with y-rays from 6OCo [ l ] led to trimers plus three isolable dimers (1)-(3) :

/ I ) I 360 1 99-100 1 274 (21 360 114-115 275 (31 1 360 1 147-148 I 281

The G value for the consumption of the stilbene in an equi- molar mixture with tin tetrachloride was 105; the G value for the formation of the mixture of isomers was calculated to be

31.5 on complete reaction. The ultraviolet, infrared, mass and proton magnetic resonance spectra of the dimers as well as the products of their oxidative degradation [benzoic acld, o- benzoylbenzoic acid, anthraquinone, 2,3-diphenyI-Az-inden- 1-one, and (o-benzoy1phenyl)benzoyl benzylmethane] indicate that they are the stereoisomers (1)-(3) of l-benzyl-2,3-di- phenylindane.

Received, October 13th, 1964 [Z 850/676 IEI German version: Angew. Chem. 76, 989 (1964)

[I] Dosage rate of the “Co source (10000 Curie): 7 . 7 ~ l o 5 r /h .

Damped Oscillation of the Enzyme Synthesis in Yeast

By Dr. W. Bernhardt, cand. med. K. Panten, and Prof. Dr. H. Holzer [ I ]

Biochemisches lnstitut der Universitat Freiburg/Breisgau (Germany)

When a disturbance enters a feedback system, the transition from the regulator’s initial output to the new level consists of either a sudden jump, an asymptotic change, or a damped oscillation, depending on the nature of the feedback system.

While studying the regulation of DPN-dependent glutamate dehydrogenase (DPN-GluDH) [2] in yeast, we found that damped oscillations of the rate of enzyme synthesis can occur. Synthesis of the enzyme is repressed by ammonium ions [3,4]. When repressed cells are transferred to a medium containing glutamic acid as source of nitrogen, DPN-GluDH is syn- thesized as a result of “derepression”. After some time, a new stationary content of DPN-GluDH, which is five to ten times higher, is attained. As is shown in Figure 1, the transition

200 /lo,

Fig. I . Rateof changeinthespecific activityof DPN-dependent glutamate dehydrogenase in logarithmically growing cells of Saccharomyccs rere- visiae R 59. The cells were grown on a medium with NH: as sole source of nitrogen (for details see [4]), depleted of nitrogen and glucose for 3 h, and then administered L-glutamic acid and glucose at time zero.

Ordinate: Change in the specific activity pe; hour [activity units x (mg protein) 1 x h-11. Fo; definition of activity units seo [4].

Abscissa: Time [hl

from the low to the high rate of synthesis of the enzyme pro- ceeds by oscillations. These oscillations probably occur be- cause the enzyme synthesis is part of a feedback system i n which the ammonium ion acts as “co-repressor”. The transi- tion does not occur in a sudden jump or asymptotically be- cause the feedback system has a time lag. This time lad is due to mainly two factors: 1. NH,O is released only slowly from the glutamic acid corresponding to the slow accumula- tion of DPN-GluDH; 2. free ammonium ions accumulate in the medium only after the NH?-dependent nitrogen re- serves have been replenished. This interpretation is supported by our finding that oscillations occur only when nitrogen- deficient yeast cells are used for the experiments.

While the frequencies of oscillations observed for DPNH [5,61 and fermentation metabolites [7] are of the order of 1 min-1, that for the enzyme synthesis is about 0.1-0.2 h-1. Oscilla-

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