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Reaction of £-2-Acetylamino-2-carbomethoxyethanesulfonyl

Chloride with Ammonia

R O D E R I C H W A L T E R a n d I R V I N G L . S C H W A R T Z

The Mount Sinai Medical and Graduate Schools, New York, New York

and The Dedical Research Center, Brookhaven National Labora-

tory, Upton, New York (Z. Naturforschg. 22 b, 106 [1967] ; eingegangen am 18. Juli 1966)

The conversion of L-2-acetylamino-2-carbomethoxy-ethanesulfonyl chloride (I) to L-2-acetylamino-2-carbo-methoxyethanesulfonamide (II) proceeds with con-siderable variability in yield We therefore investi-gated the nature of the by-product (s) of this reaction in order to prepare II in consistently high yields and with reproducible physical data.

Compound II [m. p. 145°, [a] 54 - 2 8 ° (c 2, 95% ethanol), [ajc1—30° (c 2, water); characteristic ab-sorption bands in the infrared spectrum are located at 3360, 3270, 1725, 1650, and 1320 c m - 1 ] 1 ' 2 was ob-tained in 75% yield (crystallized from water/methanol) when I (12 g) was treated in benzene with dry am-monia for not longer than 4 minutes. In addition, pro-duct II had to be removed from the benzene solution immediately upon termination of the reaction. An

1 H . HEYMANN, T . GINSBERG, Z . R . GULICK, E . A . KONOPKA, and R . L . M A Y E R , J . Amer. chem. Soc. 81, 5 1 2 5 [ 1 9 5 9 ] .

2 All melting points were determined with a Thomas-Hoover capillary melting point apparatus and are corrected. The infrared spectra were measured in pressed discs of potas-sium bromide at an approximate concentration of 0.3 per

increase in the reaction time during the conversion of I to II led to the formation of L-2-acetylamino-2-carbamoylethanesulfonamide (III) [m. p. 186 — 187°, [a] 52 —32° (c 2, water); distinctive absorption bands in the infrared spectrum are located at 3390, 3340, 3205, 1650, 1630 and 1335 cm - 1 ; anal, calcd. for C 5 H N N 3 0 4 S : C, 28.7; H, 5.30; N , 20.1; found: C, 28.7; H, 5.25; N, 19.9. The ratio of cysteic acid to ammonia was found to be 1: 2.1 when a sample was hydrolyzed in 6 N HCl at 110° for 22 hours and then analyzed according to S P A C K M A N , STEIN, and M O O R E 3

for amino acid and ammonia content on a Beckman/ Spinco amino acid analyzer]. When the reaction time was increased to 15 minutes, as much as 20% of the diamide was isolated by crystallization from water/ace-tone, following the extraction of compound II with acetone.

NHCOCH3

I R - S 0 2 - C H 2 - C H - C 0 - R '

I = R —Cl ; R ' - O C H 3

II = R - N H 2 ; R ' - O C H 3

III = R - N H 2 ; R ' - N H 2

This work was supported by U.S. Public Health Service Grant # AM-10080-01 of the N a t i o n a l I n s t i t u t e s o f A r t h r i t i s a n d M e t a b o l i c D i s e a s e s and by the U. S. A t o m i c E n e r g y C o m m i s s i o n . The authors thank Miss PATRICIA HERLING and Mr. Louis TRAUTH of this laboratory for technical assistance.

cent. The optical rotations were determined with a C a r l Zeiss photoelectric precision Polarimeter .005°. Elementary analysis was carried out by Galbraith Laboratories.

3 D. H. SPACKMAN, W . H. STEIN, and S . MOORE , Analytic. Chem. 30, 1190 [1958].

Inhibition of rat spleen proteinases by serum M . F U R L A N a n d A . S U H A R

Nuclear Institute "Jozef Stefan", Ljubljana, Yugoslavia (Z. Naturforschg. 22 b, 106—107 [1967] ; eingeg. am 28. September 1966)

A number of authors have shown evidence of the presence in mammalian blood of antiproteolytic factors which are specific inhibitors of trypsin and plasmin 1 - 3 . The activity of intracellular proteinases in animal tis-sues is usually assayed with organ homogenates. It is possible that the activity of these enzymes might be influenced by the presence of blood in the tissues. The spleen is rich in intracellular proteinases; considerable amounts of blood are contained in this organ. In the present report, the inhibitory effects of rat serum on the activity of rat spleen proteinases were studied.

1 E. E. SALE, S . G . PRIEST , and H . JENSEN, J . biol. Chemistry 227. 83 [1957].

2 J . L . G R A Y , S . G . PRIEST, W . P . BLATT , U . WESTPHAL , a n d H . JENSEN, J . biol. Chemistry 235, 56 [ I960] .

Wistar rats, 6-month-old, were used in experiments. 10% homogenate of spleen in distilled water, and un-diluted serum were used for the determination of PH optima. For the assay of serum antiproteolytic activity, supernatants of 25% spleen homogenate (30 minutes at 10 000 g) were prepared and mixed with different amounts of serum. Proteolytic activity towards hemo-globin was measured according to the method of A N S O N 4 .

Changes in proteolytic activity of rat spleen homo-genate and rat serum with PH are shown in Figs. 1 and 2. It is evident that the activities in spleen (ex-pressed per mg nitrogen) are more than 100 times higher than those in serum, PH optimum of spleen proteinases is at ph 3.1 while serum exerted the opti-mum activity at ph 2.5.

3 G. LUNDBLAD. Acta chem. scand. 16, 975 [1962]. 4 M . L . ANSON , J. g e n . P h y s i o l . 2 0 , 5 6 5 [ 1 9 3 7 ] .

Fig. 1. Variation of spleen proteinase activity with PH: 10% homogenate of rat spleen in distilled water; 2% hemoglobin

substrate in 0.1 M citrate-phosphate buffer.

Fig. 2. Variation of serum proteinase activity with PH: un-diluted rat serum; 2% hemoglobin substrate in 0.1 M citrate-

phosphate buffer.

The rates of hemoglobin hydrolysis by rat spleen proteinases in the presence of serum were plotted against the serum concentrations as proposed by D I X O N 5 (Fig. 3). It is shown that the inhibition is com-petitive. From the plot, the value of [ / ] = — 1 jK\ can be obtained:

l /K = 0.15 ml serum = 1.62 g serum nitrogen/2.4 ml incubation mixture.

This value is the equilibrium constant

of the reaction E - f1 ^ E I.

In our previous experiments, the M i c h a e l i s constant for the hydrolysis of hemoglobin by purified

6,0 Oy

l

t 5,0 - /

3,0 i i 0,1 0,2

[ I ] m l -

Fig. 3. Inhibition of spleen proteinase by serum: proteolytic activity in 0.1 ml supernatant of 25% rat spleen homogenate in the presence of rat serum; hemoglobin substrates in 0.1 M sodium acetate buffer, pn 3.5. v=E 1 5 0 /O . l ml spleen super-

natant. [/J = concentration of serum.

bovine spleen proteinases was determined 6 :

Km= - ^ i ^ =0.19mg hemoglobin nitrogen/

2.4 ml incubation mixture. The comparison of the obtained value of l/K\ to Km suggests that the stability of the complex EHb is con-siderably greater than that of E I. Influence of serum on the proteolytic activity towards hemoglobin has therefore to be taken into account only in the presence of considerable amounts of serum using low hemoglo-bin concentration. Since the proteolytic activity is as-sayed by the use of high substrate concentrations (2% hemoglobin), the competitive inhibition of serum can be neglected. Similar findings were recently reported by P R A G A Y who concluded that the presence of small amounts of blood does not interfere with the assay of proteolytic activity in rat liver homogenate.

This work was supported by the Y u g o s l a v N u c l e a r E n e r g y C o m m i s s i o n , Contract No. 03-3-10.

5 M . DIXON, Biochem. J. 5 5 , 1 7 0 [ 1 9 5 3 ] . 6 M. FURLAN, Ph. D. Thesis, Univ. Ljubljana [1964].

7 D. A. PRAGAY, Experientia [Basel] 18, 116 [1962].

Cytogenetische Untersuchungen am induzierten Plattenepithelcarcinom der Haut bei Mäusen

O . W I E S E R , ADELHEID EMMINGER u n d U . M O H R

Deutsches Krebsforschungszentrum Heidelberg Institut für experimentelle Pathologie

(Z. Naturforschg. 22 b, 107—108 [1967]; eingegangen am 18. Mai 1966)

Numerische und formale Änderungen des Karyotyps an durch 3.4-Benzpyren induzierten Carcinomen der Mäusehaut wurden untersucht. 60 weibliche, ca. gleich-schwere und gleichalte Mäule (NMRI-Stamm) erhiel-ten bis zu 8 Monate 3-mal wöchentlich 0,1 ml 3.4-Benz-pyren (0,2% in Aceton) auf die rasierte Rückenhaut

aufgeträufelt. Die Haut und die bei der Sektion auf-fälligen Organe wurden histologisch untersucht; Fär-bung mit HE. 15 Mäuse mit infiltrierend wachsenden Plattenepithelcarcinomen der Haut wurden für die cytogenetischen Untersuchungen ausgewählt. Vorbe-handlung (4 —10 Stdn.) 0,5 bis 1,0 ml einer Desacetyl-methylcolchicinlösung (1:1000). Die Tiere wurden ge-tötet, die Tumoren entnommen, zerkleinert, 15 — 120 Min. in Trypsin gerührt, mit H a n k scher Salzlösung gewaschen und in C a r e 11 sehen Kulturflaschen mit je 10 ml Kulturmedium (Hühnerembryonalextrakt : Käl-berserum : H a n k s = 2 : 3,4 : 4,5) gebracht. Nach erneuter Zugabe von 0,4 — 0,6 ml Desacetylmethyl-colchicin-Lösung (4-10 Stdn.) und Quellung der Zellen