AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA...

18
AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR. (From the Departments of Biochemistry and Bacteriology, College of Physicians and Surgeon,s, Columbia University, New York) (Received for publication, August 17, 1945) When Escherichia coli was grown in a synthetic medium containing salts, amino acids, and glucose in the presence of bacteriostatic concentra- tions of sulfonamides, an increase in total diasotizable amine was noted (1). This has nolv been found to be due to the production of a new hetero- cyclic amine, the nature of mhich may ultimately throIv light on the mechanism of sulfonamide bacteriost,asis. The formation of the amine is not limited to one kind of bacterium nor to any specific sulfonamide drug. It lvas noted with a number of different organisms (Table I). Each of the drugs tried, sulfanilamide, sulfadiazine, sulfapyridine, and sulfathiazole, xvas found to be effective, but the actual amount formed varied xvith the concentration and nature of the drug. From the figures in Table II it is seenthat the amount of nelv amine formed Tvas not proportional to the amount of drug used, but the quantity formed was ahvays of the same order of magnitude lvhen bacteriostatic concentrations of different drugs were used. Since the rat,io of amine formed to drug used Was greatest xvith sulfadiazine, this drug lvas selected for preparation of the material for isolation. p-Aminobenzoic acid, in concentrations sufficient to block the action of the sulfonamides, prevented the formation of the amine. None of the amine ‘vvas formed in the absence of sulfonamides or lvhen atabrine, plas- mochin, or penicillin Tvas used in concentrations sufficient to inhibit the growth of the bacteria. The cultures used for chemical studies and for isolation were chiefly those of Escherichia coli lvith 0.5 mg. per cent of sulfadiazine as the bacterio- static agent. The nelvly formed amine, in contrast to sulfonamides, mas not acetylated in neutral aqueous solution by acetic anhydride. It could thus be distinguished conveniently and quantitatively from the sulfonamide in solutions containing both of the amines. Quantitative measurements of diazotizable amine by the Bratton-Marshall method for sulfonamide analysis (2) shelved that the total amount after incubation was equal to * This work was done under the terms of a contract, recommended by the Com- mittee on Medical Research, between the Office of Scientific Research and Develop- ment and Columbia University. 333 by guest on June 17, 2020 http://www.jbc.org/ Downloaded from

Transcript of AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA...

Page 1: AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS*

BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

(From the Departments of Biochemistry and Bacteriology, College of Physicians and Surgeon,s, Columbia University, New York)

(Received for publication, August 17, 1945)

When Escherichia coli was grown in a synthetic medium containing salts, amino acids, and glucose in the presence of bacteriostatic concentra- tions of sulfonamides, an increase in total diasotizable amine was noted (1). This has nolv been found to be due to the production of a new hetero- cyclic amine, the nature of mhich may ultimately throIv light on the mechanism of sulfonamide bacteriost,asis.

The formation of the amine is not limited to one kind of bacterium nor to any specific sulfonamide drug. It lvas noted with a number of different organisms (Table I). Each of the drugs tried, sulfanilamide, sulfadiazine, sulfapyridine, and sulfathiazole, xvas found to be effective, but the actual amount formed varied xvith the concentration and nature of the drug. From the figures in Table II it is seen that the amount of nelv amine formed Tvas not proportional to the amount of drug used, but the quantity formed was ahvays of the same order of magnitude lvhen bacteriostatic concentrations of different drugs were used. Since the rat,io of amine formed to drug used Was greatest xvith sulfadiazine, this drug lvas selected for preparation of the material for isolation.

p-Aminobenzoic acid, in concentrations sufficient to block the action of the sulfonamides, prevented the formation of the amine. None of the amine ‘vvas formed in the absence of sulfonamides or lvhen atabrine, plas- mochin, or penicillin Tvas used in concentrations sufficient to inhibit the growth of the bacteria.

The cultures used for chemical studies and for isolation were chiefly those of Escherichia coli lvith 0.5 mg. per cent of sulfadiazine as the bacterio- static agent. The nelvly formed amine, in contrast to sulfonamides, mas not acetylated in neutral aqueous solution by acetic anhydride. It could thus be distinguished conveniently and quantitatively from the sulfonamide in solutions containing both of the amines. Quantitative measurements of diazotizable amine by the Bratton-Marshall method for sulfonamide analysis (2) shelved that the total amount after incubation was equal to

* This work was done under the terms of a contract, recommended by the Com- mittee on Medical Research, between the Office of Scientific Research and Develop- ment and Columbia University.

333

by guest on June 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Page 2: AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

334 AMINE FROlM BACTERIOSTASIS

the sum of the amount of drug initially present and the amount of diazotiz- able amine remaining after acetylation (Table III). Thus none of the sulfonamide disappeared during the incubation in which the new amine was formed.

The amine has been isolated in a pure state from bacterial culture medium. When precautions were taken to avoid oxidizing conditions,

TABLE I

Ej’ect of Variation of Bacteria

The production of the new amine was compared when various bacteria were used wit.h sulfadiazine as the bacteriostatic agent.

Bacteria

Proteus vulgaris ............................

Shigella dysenteriae (Shiga), ................. Salmonella paratyphi R ......................

Escherichia coli .............................. Eberthella typhosa ........................... Vibrio comma ............................... Shigella sonnei .............................. Hemophilus injlxenzae .......................

“ ~ertussis ........................ Shigellu paradysenteriae ..................... Aerobacler aerogenes .........................

Concentration of sulfadiazine

P

_ _

Lpparent yield of diazo- tizable amine*

mg. *er cent ng. $3‘3 cent

0.67 0.80 4.95 0.56 0.67 0.28 0.67 0.11 4.95 1.00 4.95 1.02 4.95 0.26 4.95 0.56 1.25 1.54 4.95 1.60 4.95 0.22 4.95 0.23 4.95 0.52

* Measured by diazotization after acetylation of the amino group of the drug. The quantity of diaxotizable amine was measured in all cases by the Bratton- Marshall method of sulfonamide dct.erminaticn (2) and the color read on a Coleman universal spwtrophotomcter at X 540 mp. For convenience in calculation and comparison it was assumed that the molecular weight of the unknown amine was the same as that, of sulfadiazinc: and that the compounds gave mole for mole the same amount of color on being tlirtxot izctl and coupled.

a total of about 2 gm. of a picratc of the amine \vas obtained from about 200 liters of culture. By essentially the same isolation procedure, starting with a cult,urc in which sulfapyridine replaced sulfadiazine as the bac- teriostat,ic agent, the identical picratc was obtained. The nature of the amine does not depend upon the substituent on the amide group of t.he sulfonamide.

The picrate gives analytical values for the formula C&N40. CcH3N307. 2H&. From it a number of other derivatives have been prepared, none of which is as stable as the picrate. Analyses of the free amine with and without water of crystallization are in good agreement) with the empirical

by guest on June 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Page 3: AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

M. R. STETTEN AND C. L. FOX, JR. 335

TABLE II

.Yfect of Varialiorl of Bacterioslatic Agent

The production of the new amine was compared when various concentrations of different sulfonamides served as the bacteriostatic

Drug

Sulfanilamide

Sulfapyridine

Sulfathiazole

Sulfadiazine

Conce~tr$ion of r

mg. per cent

35.0 4.77 4.98 4.62 2.49 0.97 4.9 4.0 3.1 2.4 2.0 1.5 1.0 0.55 0.92 0.57 0.45

.v - 11

;ent.

vxease of diazotiz- able amine*

mg. per cent

0.78 0.27 0.54 0.77 0.49 0.40 1.05 1.13 1.13 0.99 0.74 0.81 0.57 0.56 1.27 1.27 1.14

- Per cent increase

2.2 5.7

11 17 20 41 21 28 36 41 37 54 57

102 138 223 253

* Measured by diazotization after acetylation of the amino groups of the drug. See the foot-note to Table I.

TABLE III

Concentration of Non-Acetylated Amine

The increase in the concentration of total diazotizablesubstances after incubation has been compared with the concent,ration of the non-acetylated component.. In this experiment all samples were diluted in the same way for analysis and the color- imeter readings were converted to concentrations on the basis of a calibration curve.

Drug

Sulfadiazine

Sulfapyridinc

Total concentra- Increase in di- Concentration tion of diazotiz- azotizable

of drug able amine after amine during incubation incubation

(a) (b) (b - a)

?ng. per cd ??q. per cent mg. per cent o.n2 2.20 1.25 0.45 l.Gl 1.16 0.52 1.25 0.73 4.98 5.49 0.51 2.49 2.97 0.48

Concentration of non-acetyl-

ated diazo- tizable amine

mg. per cen1*

1.27 1.14 0.69 0.54 0.49

* See the foot-note to Table I.

by guest on June 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Page 4: AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

336 AMINE FROM BACTERIOSTASIS

formulae GHsN40.HzO and C4HeNhO. Samples of a chloroplatinate, (C4HeN40)2.HzPtCls.HCI, a sulfate, C4HsN40.H2SOd, and a monohydro- chloride, C4HsN40. HCl, have also been prepared. The amine probably also forms a dihydrochloride which has not been obtained free of the

Y= CH N- c=o

O=C Hb-NH2 H: Hi-NH,

I: = ~-NM I

iv = &NH,

N / \\ HO-C ‘C-NH~ o-c ‘C- NH2

~-NH,

HO-C

H-Z

or

J

/“‘a H o=c CHN\

/N, H o=c yN,

I I N I I c=o H2c ,&

‘N H

monohydrochloride. Although it is not acetylated in aqueous solution, the amine can be acetylated by acetic anhydride in glacial acetic acid.

The coupling ability of the diazotized solution diminishes on standing, on being heated, or when more concentrated nitrite or acid is used. The disappearance of the diazonium compound was not accompanied by

by guest on June 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Page 5: AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

M. R. STETTEN AND C. 1,. FOX, JR. 337

liberation of nitrogen. The product of prolonged action of nitrite was isolated; the empirical formula of C4H3N50.Hz0 suggested that cyclization to a triazole (IV) had occurred. The related assumption that our com- pound is an orthodiamine was supported by the ease with which the amine undergoes oxidative deamination and by the formation of a compound, C&&N402 (V), on fusion with urea, a reaction that has been used to differ- entiate ortho- from other diamines in the pyrimidine series (3, 4). An attempt to prepare a derivative with phenanthraquinone was unsuc- cessful.

An indication that the oxygen is phenolic was obtained by observing that the amine could be coupled with diazobenzenesulfonic acid to give an orange-yellow color in alkaline solution.

2 of the 4 nitrogen atoms in the compound appear to form part of a ring structure. From its empirical formula and many of its chemical properties the compound was at first suspect.ed to be a monohydroxy- diaminopyrimidine. Of the various hydroxydiaminopyrimidines (5) only I and II, which contain ortho amino groups, come into consideration. 2-Hydroxy-4,5-diaminopyrimidine (I) which resembles our product in several respects (6, 7), was found to differ from it in t,hat it decomposes at about 265” instead of melting at 170”, does not give the characteristic red color on diazotization and coupling, and shows an entirely different ab- sorption curve in the ultraviolet (Fig. 1). Johns (7) described the forma- tion of isobarbituric acid from 2-hydroxy-4,5-diaminopyrimidine when it was heated at 140.-150” in a bomb tube with 20 per cent HzS04, whereas our bacterial product gave glycine under similar conditions. The urea fusion product, 2, Glihydroxypurine, prepared from 2-hydroxy-4,5- diaminopyrimidine gave a positive murexide test but an entirely different ultraviolet absorption curve from t)hat of the corresponding derivative of our amine.

6, S-Dihydroxypurine, the product obtained by fusion of 6-hydroxy- 4,5-diaminopyrimidine (II) with urea, crystallizes in long, narrow, shiny leaves and does not give a positive murexide test (8), whereas our product precipitates as a powder and gives a positive murexide test, carried out according to Fischer. A sample of 6-hydroxy-4 ) 5diaminopyrimidine itself, which has recently been synthesized (9), was found to give no color on being diazotized and coupled and to have a very different melting point from that of our amine?

By vigorous hydrolysis with concentrated HCl in a bomb tube at 150” the ring of our amine was split and good yields of NH, and glycine re- covered. This fact we believe to contraindicate meta- and orthodiazine formulation, and to suggest, that t)he ring structure is that of a paradiazine,

1 We wish to thank Dr. B. 0. Goblin, Jr., for this sample.

by guest on June 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Page 6: AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

338 rZMIXE FROM BACTERIOSTASIS

a pyrazine. We believe the most probable constitution to be the unknown 2-hydroxy-5, G-diaminopyrazine (III). This compound can readily be pictured as giving glycine and NH3 in good yields on acid hydrolysis and its properties would resemble those of the isoln.td amine.

1.8r

1.6

240 260 280 300

FIG. 1. Ultraviolet absorption spectra. The ordinate scale represents readings in log IO/I (1 cm.) ; the abscissa, wave-length in mr. Left-hand section, from isolated

amine, 1.17 mg. per cent, at pH 7 (Curve I), at pH I1 (Curve 2), at pH 3 (Curve 3); 2-hydroxy-4,5-diaminopyrimidine, 1.40 mg. per cent in dilute acid (Curve 4). Right- hand section, from isolated amine, 1.27 mg. per cent of triazole (Curve l), 2.28 mg. per cent of imidazolone, pH 11 (Curve 2), 1.93 mg. per cent of 2,8-dihydrosypurine, pH 11 (Curve 3).

A number of as yet unsuccessful attempts have been made to synthesize this compound or its urea fusion product. The only approach which has yielded any encouraging results has been the condensation of cyanogen with glycinamide, analogous to the formation of diaminobenzpyrazine from o-phenylenediamine (10). Treatment of glycinamide in a water- alcohol solution with cyanogen gave among other products minute amounts of a product which behaved like our isolated amine with regard to the

by guest on June 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Page 7: AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

RI. I?.. STET’l%:K AKD C. L. FOX, JR. 339

diazo test and the difficulty of acetylation. Unfortunately it has not yet been found possible to isolate t,his product in a pure state.

The biological r61e of the product is as yet obscure. In tests carried out so far the compound fails to exhibit striking effects on bacterial growth. It is not removed from the medium by normally growing Escherichia coli.

Rings of the paradiazine type are not unknown in nature, and in some instances occur in substances of striking biological activity. They are found in such condensed ring systems as alloxazine and in the pterins, which may be related to “folic acid”2 (11). Dutcher and Wintersteiner (12) have recently shown that aspergillic acid is probably a substituted pyrazine.

The isolation of what is probably 2-hydroxy -5,Gdiaminopyrazine from culture media in which the normal proliferation of bacteria has been pre- vented by addition of sulfonamides suggests a hit,herto unsuspected type of action of this group of bacteriostatic agents. As the product does not arise directly from the sulfonamide used, it must be pictured as having been produced by action of the bacteria on other constituents of the medium. It may be either a normal intermediate in some metabolic reaction which is blocked by the drug or an abnormal product formed by the bacteria under the influence of the drug.

Pro&don of the Amirle3-The basal medium used was a modified Long’s medium containing in each liter 5.0 gm. of NH1 citrate, 3.0 gm. of KHZPOI, 3 gm. of Na2C03, 2 gm. of NaCI, 0.2 gm. of MgSO4, 0.05 gm. of ferric am- monium citrate, 0.005 gm. of nicotinic acid, 2 gm. of glucose, and 10 cc. of a 5 per cent, solution of a hydrolysate of either gelatin or casein. The effects of va,rious concentrations of different, sulfonamides are given in Table II.

Using Es&G&a coli, we observed that the yield was increased by expanding the surface area exposed to the air, but was greatly decreased when air was slomly bubbled through the growing culture. Substitution of equivalent amounts of pyruvate, lactate, or succinate for the glucose

2 We wish to thank Dr. W. 1-I. Sebrell of the National Institute of Health for his assay of our compound for “folic acid” activity. He reported : “The results of the ‘folic acid’ assay of your compound are as follows: Rat assay, no activity at 250 y per rat per day (5 y of ‘folic acid’ would give a positive t.est). AS’. Zactis R, tested in concentrations up to 560 y per tube. Less activity than 2.5 X IO-4 y of ‘folic acid.’ L. casei, tested in concentration up to 210 y per tube. Less activity than 3 X 10-d y of ‘folic acid.’ In view of these results, I t.hinlc we can say that the compound has no ‘folic acid’ activity.”

3 The authors wish to thank Mr. Saul Frances for assisting with some of thr bacteriological work.

by guest on June 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Page 8: AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

340 AMINE FROM BACTERIOSTASIS

of the medium resulted in increments of 27, 5, and 7 per cent respectively a,s compared with about 200 per cent when glucose was used.

Inocula of 1 to 3 million Escherichia coli per cc. of medium were found to produce satisfactory amounts of the amine. A lo-fold increase of inoculum resulted in no increased amine format,ion, while reduction of the inoculum to one-tenth greatly decreased the yield. 18 hours were found to be the optimum time of incubation.

In addition to Eschel-ichia coli a number of other bacteria which could be grown in our media were tested, and the production of diazotizable amine in the presence of sulfonamide noted. The medium used in these experiments consisted of the usual basal medium containing casein hydrol- ysate and glucose, to each liter of which were added 50 mg. of cysteine hydrochloride and 0.2 mg. of thiamine hydrochloride. Each solution was inoculated with 1 million bacteria per cc. and incubated for 18 hours at 37”. The bacteria were then removed and the quantity of diazotizable amine was measured before and after incubation and after acetylation of the incubated solution. The results are given in Table I.

Strains of sulfonamide-fast Staphylococcus aureus (F, and F14 Landy) (13) were grown in our medium with added thiamine and cysteine with no drug present. The amine which accumulated, thought by Landy to be p-aminobenzoic acid, although diazotizable, was unlike our amine in being readily acetylated. It was noted that the specific soil bacillus used by Mirick to oxidize p-aminobenzoic acid (14) destroyed the amine formed by F, and F14 but did not destroy our amine.*

The large scale cultures from which the amine was isolated were grown in 4 liter Pyrex glass bottles. 2 liters of culture medium containing 0.5 mg. per cent of sulfadiazine, but no glucose, were placed in each bottle and the solution adjusted so that after autoclaving the pH was between 7.0 and 7.6. 2 gm. of glucose per liter were added and the medium inoculated :vith 1 cc. of a 24 hour culture of Escherichia coli per liter, the resulting initial bacterial concentrat.ion being about 1 million bacteria per cc. The bottles were laid on their sides to increase the surface area and mere in- cubated for 18 hours at 37”.

Properties of l3ilute Solutions of the Amine from Bacterial Cultwes- After removal of the bacteria from the incubated solution by means of a Berkefeld filter or a Sharples centrifuge, the properties of the amine in the resulting solution, containin, c about 1 mg. per cent of the amine, were studied. In this crude mixture the amine was relatively stable in boiling acidic, alkaline, or neutral solution. The amine was diazotizable and its

4 We wish to thank Dr. RI. Landy for providing the subcultures of his sulfonamide- resistant StaphyZococcus aureus and Dr. G. S. Mirick for subcultures of his soil bacil- lus which were used in these experiments.

by guest on June 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Page 9: AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

M. R. STETTEN AND C. L. FOX, JR. 341

diaeo derivative, when coupled with N-(1-naphthyl)ethylenediamine dihydrochloride, gave a colored compound which had nearly the same absorption maximum in the visible region as the corresponding products obtained from any of the sulfonamides. In contrast to the sulfonamides, the amine was not acetylated by 2 per cent acetic anhydride in neutral aqueous solutions. This property proved very useful as a means of quanti- tatively measuring the compound in the presence of the drug and of fol- lowing its separation from the drug. In practice these determinations mere made by the Bratton-Marshall method (2) before and after half an hour’s treatment with 2 per cent acetic anhydride at room temperature, as de- scribed by Rosenthal (15). The diazotizable amino group of each of the sulfonamides was readily and completely blocked by acetylation, while that of the unknown amine was not blocked (Table III). Excellent agreement was obtained between the total amount after incubation and the sum of the initial amount of drug used plus the amount remaining after acetylation of the incubated solution. Thus the sulfonamide was not lost during the incubation period in which the new amine was formed. In order to get this agreement it was found necessary to dilute all samples in the same way and to calculate the concentrations from the calorimeter readings on the basis of a calibration curve.

Unlike sulfadiazine the amine was not removed from a water solut,ion at pH 4 to 6 by continuous ether extraction. The amine could be removed from the dilute water solution slowly by continuous extraction with butanol or by extraction with ethanol of a water solution saturated with K&OS. It,s extraction with acetone from an aqueous solution saturated with CaC& was accompanied by considerable loss of material. Chloroform, benzene, and ethyl acetate did not extract the amine.

The amine in solution was apparently readily destroyed by oxidizing agents. Short exposure to Hz02 in acidic, alkaline, or neutral solution or to AgzO, Cl;, or KMnOl resulted in total loss of diazotizability. Even aeration resulted in gradual disappearance of diazotizable amine from solution. The diazotizable properties were unaffected by Zn and HCl.

No adsorption was observed on A&OS, MgO, Johns-Manville Standard Super-Cel, CaO, talc, SiOz, or MgHP04 when each of these was shaken with a neutral solution of the amine. The material disappeared from solution when shaken with charcoal but could not be eluted from the charcoal by acid, ammonia, or alcohol. It could be adsorbed on fullers’ earth from acid solution and eluted with concentrated ammonia in 50 per cent alcohol.

It was found that the amine was not readily precipitated from the dilute solution by lead acetate, Ca(OH)s and CUE or tannic acid, but was readily precipitated by phosphotungstic acid and by mercuric ion from a slightly alkaline solution.

by guest on June 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Page 10: AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

342 AMINE FROM I3.ACTERIOSTASIS

Isolation Procedure-After removal of the bacteria the pH was adjusted to 7 to 7.5 with NaOH and a freshly prepared water solution containing 1 gm. of mercuric acetate was added to each lit,er. The flocculent white precipitate which immediately began to settle contained almost all of the amine as well as considerable NH, and some amino acids. After the precipitate had settled overnig,ht, the supernatant was discarded and t,he precipitate washed well several times with mater by centrifugation. A relatively large volume of 2 N HCl was required to dissolve most of the mercury-amine compound. The solution became progressively deeper red-brown in color and a caramel-like odor was noted. A large amount of insoluble material was filtered off and the mercury was removed wibh H&3. After removal of H,S by boiling, the pH was adjusted to 4 to 6 and the solution was extracted continuously with peroxide-free ether5 for 2 days to remove sulfadiazine and other ether-soluble impurities. All of the diazotizablc amine in t,he ether solution after this procedure was acetylatable with acetic anhydridc in neutral water solution, whereas nearly all of that remaining in the water solution after extraction was non-acetylatable under the same conditions.

The water solution was then evaporated at reduced pressure to a small volume in an atmosphere of nitrogen at room temperature. It was ob- served that whenever air was passed through the solution for any length of t’ime the amine was decomposed. To remove ammonia the solution was made alkaline to phenolpl~thalein with 13at(OI-I)Z and evaporated. After removal of the barium, the pH of the pale yellow solution was ad- justed to 7 to 7.5 and t,he amine reprecipitated with mercuric acetate. This second mercury precipitation in t,hc absence of carbonate effected a further separation of the amine from amino acids. The mercury was again removed, the acidic solution evaporated almost to dryness *in vacua under nitrogen, and the residue estrnct,ed several times with aldchyde-free ethanol. Most of the solvent, was removed, the residue l.akcn up in water, and a small amount of cthanolic picric acid solution added. The well formed yellow needles which precipitated from the solution after it had stood in the refrigerator overnight were filtered off, mashed with cold water, and recrystallized twice from very dilute hot alcohol or from a larger volume of hot water. 78 mg. of the crystalline picrstc were isolated from 25 liters of culture. From 82 liters of culture 1.0 gm. of picrate of the amine was obtained and in a third preparation 0.89 gm. of picratc ~vas isolated from 67 liters of culture.

Picrate----An air-dried sample was dried at 100” in NKUO over 2’& and

5 Due to the ease of osidative destruction of the amine, all evapor2tt.ions were carried out under nitrogen and all solvents mere carefully frectl of oritlixing cou- tsminants.

by guest on June 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Page 11: AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

RI. R. STETTEE; A?jD C. I,. FOX, JR. 343

lost 9.2 per cent of water. The theory for GH&&. C&N,O~~2II~O is 9:2 per cent, of water. 1T)ecomposition point, 240”.

C~H,N~C)~CaH,N;O~. Theory. C 33.S, H 2.53, N 27.6, picric acid 64.5 Found. 1st’ preparation. “ 33.1, “ 2.82, “ 26.3,* S 0, picric acid 64.2

2nd “ (( 33.5, (‘ 2.97, “ 25.9

* The microanalyses were performed by Mr. William Saschek. The nitrogen analysts were carried out by the standard Pregl micro-Dumas method and were in some cases somewhat lower t,han the theory for the formula which we propose. Some heterocyclic nitrogen compounds are known to give low values for nitrogen by this method (16).

Picric acid was determined grarimetrically by precipitation of nitron picrate (17).

-4mine Isolaied from Culture in Which Xulfapyridine ‘Was Used As Bac- feriosfafic Agent-The compound was also isolated from cultures in which growrth was inhibited by 6 mg. per cent of sulfapyridine instead of 0.5 mg. per cent of sulfadiazine, all other conditions being kept the same. The same method of isolation was followed, though several additional precipita- tions with mercuric acetate were necessitated by the larger relative amounts of the drug present. 0.2 gm. of picrate was obt,ained from 32 liters of culture. The picrate isolated was ident’ical with t,hat previously obtained. Found C 33.4, II 2.53, K 26.6.

The ultraviolet, absorption curve of the amine from this preparation was identical nith that of the compound isolated from t’he cultures con- taining sulfadiazinc (Fig. 1).

Chloroplalinnle-165 mg. of anhydrous picrate were suspended in a small volume of concentrated HCl and the picric acid extracted w&h pcroside-free &her. After the solution had been evaporated t’o about 3 cc., 300 mg. of II~l’tCI~~(iH~O dissolved in a few drops of water were added to the n-aim concc~ntrated acid solution of t.he amine. Shiny orange plates of the chloroplatinate of the amine precipitated out on cooling. The crystals \I-crc filtered cold, washed with a very small volume of con- centrated HCl, then with a lit,tle cold water, and dried to constant weight in vacno ovtr l’& and I<011 at) room temperature. 133 mg. were obtained. It was foucd that the chloroplatinabe could not conveniently be recrystal- lized because the amine is a sufficiently strong reducing agent to precipitate metallic platinum from a hot water solution of the chloroplatinate. M.p. 210-215” with &composition.

(C4HsiV40)z.HzPtCls.HCl. Theory. C 13.7, H 2.15, N 16.0, Pt 28.0, Cl 35.6 Found. “ 13.8, “ 2.33, “ 15.3, “ 27.9, “ 33.5

28.2

by guest on June 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Page 12: AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

344 AMINE FROM BACTERIOSTASIS

A sample of the chloroplatinate, after the removal of platinum with silver by the method of Dudley (18), gave a solution which on titration with NaOH was found to contain 3 equivalents of acid per mole of chloro- platinate.

Rz+HzPtCla.HCl + 4Ag+ Pt + 4AgCI + 2R + 3HC1

Isolation of Free Amine-Picric acid was removed from 100 mg. of the piorate by ether extraction from a suspension in aqueous H&Sod. The bulk of the sulfate was removed with Ba(OH)% and the slightly acidic filtrate evaporated to a volume of about 2 cc. The remaining sulfate was quantitatively removed with BaC03 and the solution freed of water by repeated evaporation from alcohol-benzene. The residue was taken up in about 1 cc. of alcohol, a small amount of insoluble material was filtered off, and 5 cc. of benzene were added to the filtrate. The large colorless needles which formed on cooling were centrifuged off, washed with benzene, and recrystallized from alcohol-benzene. The recrystallized sample, weighing 8 mg., was dried in vacua over P205 without heat; m.p. 168-170” with decomposition.

C,HeNhO*H~O. Theory, C 33.3, H 5.6, N 38.9; found, C 33.3, H 5.4, N 38.7

A deep purple precipitate appeared in the mother liquor after several days and in a second preparation the entire sample was lost as a purple product.

Isolation of the free amine from a very small volume of water was found to be more convenient than from alcohol-benzene. A water solution of the amine, equivalent to 56 mg., prepared by removal of picric acid and sulfate as previously described, was carefully evaporated to a volume of less than 1 cc. The yellowish tan crystals when formed on cooling were washed with a small amount of cold water. The sample was recrystallized from 1 cc. of water without undue heating and small colorless needles were obtained. Yield, 18 mg.

Water Determination-l 1.53 mg. dried to constant weight at room temper- ature and atmospheric pressure over CaCh lost 1.39 mg. when dried at room temperature over PZ05 in vucuo. When dried at 110” over PZOF, in vacua, only 0.09 mg. more was lost.

ClHBN40-Hz0 Theory, Hz0 12.5; found, 12.8 C4H,Na0. Theory, C 38.1, H 4.8, N 44.4; found, C 38.2, H 5.1, N 43.8

Spot Tests-A few drops of a solution of the free amine gave a positive biuret test with concenkated NaOH and dilute CUSOJ. In a short time the lavender color was masked by the formation of a black precipitate.

by guest on June 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Page 13: AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

M. R. STETTEN AND C. L. FOX, JR. 345

1 mg. of the amine gave a deep blue color with NaCN in dilute NH,OH and lithium arsenotungstate reagent (19).

Negative results were obtained when murexide and Sakaguchi tests were carried out on solutions of the amine.

When the amine was treated with p-diazobenzenesulfonic acid in alkaline solution, a deep yellom-orange color appeared. Phenol similarily treated gave a deep yellow color, whereas no color was produced with either aniline or o-phenylenediamine.

Hydrochlorids-Difficulties were encountered in the preparation of the amine hydrochloride. After removal of picric acid from a suspension of the picrate in HCl, a tan crystalline product could be obtained by con- centration of the aqueous layer, by saturation with HCI gas, or by addition of alcohol. On analysis it appeared to be a mixture of the mono- and di- hydrochlorides of the amine.

C~H~NIO*HC~. Theory. Cl 22.2, N 34.4 C4HhN40.2HC1. Theory, Cl 35.6, N 28.1; found, Cl 26.9, N 31.2

The monohydrochloride was finally purified by precipitation from a large volume of absolute alcohol by the addition of several volumes of benzene. The white needles which slowly appeared in the solvent mixture decomposed at 210-215”.

GH6N40.HCI. Theory. C 29.5, H 4.3, N 34.4, Cl 22.2 Found. ” 29.9, ” 4.2, ” 32.3, ” 22.2

Sulfate-The sulfate, like the hydrochloride, proved difficult to obtain in pure form. After removal of picric acid from a suspension of the picrate in dilute HzS04, the solution was evaporated to a small volume and the sulfate precipitated as an oil by the addition of alcohol-ether. After long cooling the oil solidified to a tan powder which appeared to deteriorate on attempted recrystallization.

C4HaN40-H2S04. Theory, C 21.4, H 3.57; found, C 21.2, H 3.66

Investigation of Properties of Amino Groups-The intensity of color obtained when the amine was diazotized and coupled with N-( I-naphthyl) - ethylenediamine was greatly influenced by the conditions. The greatest amount of azo dye was obtained when the amine was dissolved in acetic acid and treated with dilute NaN02. When the conditions of the Bratton- Marshall method (2) of sulfonamide analysis, dilute HCI and dilute NaNO*, were used, only slightly less color was obtained. Increasing the concentra- tion of either the KC1 or the NaNOz resulted in appreciably less dye forma- tion. A large portion of the diazonium salt disappeared on standing at room temperature for half an hour and the disappearance was almost com-

by guest on June 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Page 14: AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

plete if the solution ~was heated for half an hour on the steam bath. No nitrogen was evolved Tvhen the amine n-as t,reated lvith 30 per cent NaNOZ and either glacial acetic acid or 3 N HCl and shaken for half an hour at room temperature in a T’an Slykc apparatus (20). Under similar conditions 97 per cent of the diazoninm salt had been shojvn calorimetrically to have disappeared.

Condensalion with Nilrozls A&t!; l’riaxolc (IT’)-To a solut’ion of -10 mg. of the amine in 10 cc. of 2 w H2S01, 0.5 gm. of Ba(NO& dissolved in a small amount of lvater was added. After having stood overnight. at room temperature the solution was found to contain only a trace of diazotizable amine. The soWon was lvarmed on a steam bath for 5 minutes, hot Ba(OH)? solution added until a slight excess of sulfate remained, the solution made slightly alkaline &h XaOH, and the BaSO4 filtered off. After acidification lvith T-1$0, the filtrate was cvnporat.ed to a small volume and the pale yellow precipitate Tvhich formed on cooling \vas filtered and xvashed \vith a small amount of cold Ivater. The precipitate rvas purified by solution in NH4OI-I and acidification jvith I-I$X)~. The pale yellow rosettes6 of needles which formed slowly on standing in the refrigerator were jvashed jvith cold water and dried ill vucuo over I’,05 at, 100” . Yield, 27 mg.; the substance began to darken at abollt 150’ and did not melt belolv 2GO”.

CaI13NBO~IIz0. Theory, C 31.1, II 3.2, N 45.1; found, C 31.3, II 3.2, N 43.9

When a dilute solution of the triazole in HCl was treated Jvith the usual coupling compound, no color appeared at first but a faint pink color de- veloped gradually on standing. This color had the same absorption maximum in the visible region as did that obtained from the diazotization and coupling of the amine itself, suggesting reversal of the ring closure.

An unsuccessful attempt was made to prepare a phenanthraquinone derivative of the amine.

Fusion with Urea; Imidazolone (IT)-A sample of the amine was fused with urea by a method used for converting o-diaminopyrimidines into yurines (3, 4). A solution of 40 mg. of the amine in dilute HCI was neu- tralized lvith NHlOH, the solvent evaporated, and 40 mg. of urea added. The dry mixture was heated at 170-180” for 1 hour, during mhich time con- siderable ammonia Ivas evolved. The residue was dissolved in 1 cc. of dilute NaOH, a small amount of slimy material filtered off, and the solution acidified wit.h a drop of concentrated HCI. The white powder -which precipitated ins dashed with Jvater anil prvifietl twice by solution in

6 It was subsequently found that the triaxole could be obtained as white crystals after treatment of a hot water solution wit,h charcoal, but insufficient material rc- maincd for analysis.

by guest on June 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Page 15: AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

hf. R. STETTEN .\IW C. L. FOX, JR. 347

KH4011 and precipitation by acidification with HCl. 21 mg. were ob- tained. The product roquircd drying irb vucz10 at 135” for many hours.

C,I-I,T\‘,O?. Theory, C 39.5, El 2.63, N 36.9; found, C 39.3, Ii 2.86, N 36.6

Acetylntion-Even though the amino groups of our compound mere not acet,ylated under the mild conditions sufficient for the acetylation of sulfon- amides, it xxs found t,hat they could be complet’ely acetylat,ed when more vigorous conditions were used. Several mg. of t.he amine in a drop of water wrc heated on a stcnm bath for 1 hour with 1 cc. of acetic anhydride. A portion of the resulting solution, when treated with nitrite and the usual coupling compound, gave no color, while another portion after being boiled for 5 minutes with dilute HCl and similarily diazotized and coupled gave a tlccg red color.

Oxidc&c I)eq~~ad&ons-A sample of the amine in a dilute Ba(OH)r sohltion containing 2 per cent hydrogen peroxide was boiled gently fol 1 hour. Three-fourths of the I\’ initially present could be account,ed for as MI, e\,ol\-cd during the oxidation. The remaining solution was acidified and the (‘0~ collected in I<a(OH)z. The 13aCOa which formed accounted for about, half of Illc C of the amin,. In 20 per cent NaOH solution, prroxidc comyertcd all of the N of t,hc amine into ammonia. X0 urea could bc isolated from t,hc solutions after oxidation with chlorine, alkaline IiMn04, and chloroplatinic acid.

DqdaLion fo Glycinc cd Ammonitc--- 40 ng. of th: amine in the form of the crude hydrochloride \vere heated in a sealed t,ubc I\-ith 2 cc. of con- centrated IICl for 20 hours at 150”. The resulting yellow solution was craporatcd to a very small volume; 28 mg. of SH&l (55 per cent of theo- retical) were obtained.

The mother liquor was made barely alkaline \vit,h Ca(OH)z in an attempt to identify oxalic acid among the products, but no precipitat,e was formed. The solution was then made strongly alkaline with NaOH and Nt gas was passed through the warmed solution for about an hour to remove the remaining ammonia. The solution wa,s stirred for G hours with 100 mg. of p-toluenesulfonyl chloride in ether. The alkaline solution was extracted with ether to remove excess reagent, acidified with HCI, and the p-tolucne- sulfonyl derivative extracted with ether and crystallized from mater. 44 mg. of p-toluenesulfonylglycinc were obtained (63 per cent of the theoretical). The product was recrysta,llized twice from water and dried in DUCUO at 61” for analysis; m.p. 147-149”. A mixture of the compound with a sample of p-toluenesulfonylglycine showed no depression of melting point.

GHx~O~NS. Theory, N 6:11, S 13.97; found, N 6.09, S 13.80

2-Hydroxy-4,5-diaminopyrimidine (1)-A sample of 2-hydroxy-4,5-

by guest on June 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Page 16: AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

348 AMINE FROM BACTERIOSTASIS

diaminopyrimidine was prepared from cytosine7 by nitration followed by reduct,ion of the nitrocytosine (7). The yellowish needles, dried in vacua over P,Os at room temperature, melted at about 265” with decomposition. On diazotization and coupling with N-(1-naphthyl)ethylenediamine no red color was formed. It was noted that similar treatment after a sample had been boiled with dilute HCl resulted in the appearance of a gradually intensifying pale pink-lavender color.

C4HaN40. Theory, C 38.1, H 4.8; found, C 37.8, H 5.1

Some of I was fused with urea at 180-l 90” and 2,8-dihydroxypurine isolated (7). This purine gave a positive murexide test. The ultraviolet absorp- tion curves of both the pyrimidine and the purine are given in Fig. 1.

attempted Syntheses of &Hydroxy-5,6-diaminopyra2ine (III)-A number of attempts to prepare III by condensation of glycinamide with oxamide were made. Successively chloroform, alcohol, anhydrous HzS04, and acetic anhydride, both cold and hot, were tried unsuccessfully as media for the condensation.

The most promising methods to date were those starting with glycin- amide and cyanogen. An exact equivalent of aqueous NaOH was added to a solution of glycinamide hydrochloride in ethanol and 2 times the theoretical amount of cyanogen, generated from KCN and CuSO4 (21), was passed into the solution. After a few minutes the solution became yellow-tan and then red-brown in color and a brown precipitate formed on standing. A few drops of the solution after diazotization and coupling gave a deep red color of the same intensity both before and after the solution had been treated with dilute acetic anhydride. From the intensity of color we have estimated that at best about 100 mg. of the amine were formed when 5 gm. of glycinamide were used. When cyanogen was passed into a water-alcohol solution containing no glycinamide, the same red-brown color and precipitate were noted but the solution contained no diazotizable amine. About the same small amount of diazotizable amine was formed when either water or ethanol was used as the solvent. When methanol was used instead, less was produced and still less was formed when cyanogen was dissolved in CHsOH prior to addition of the glycinamide. No diazotizable amine was produced with chloroform, dilute HCl, dilute NaOH, or NHkOH as the solvent.

SUMMARY

When the normal growth of certain bacteria is inhibited by bacteriostatic concentrations of sulfonamide drugs, a diazotizable amine is found to accumulate in the medium.

7 We wish to thank Professor T. B. Johnson for the sample of cytosine used in this synthesis.

by guest on June 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Page 17: AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

M. R. STETTEN AND C. L. FOX, JR. 349

From cultures of Escherichia co&i, whose growth was inhibited by either sulfadiazine or sulfapyridine, the amine has been isolated as a picrate and samples of the free amine and a number of derivatives prepared. It is a heterocyclic orthodia,mine having the empirical formula CtH,jN.+O. The possibility of its being a pyrimidine has been eliminated. Its properties indicate that, it is probably 2-hydroxyd, 6-diaminopyrazine.

BIBLIOGRAPHY

1. Fox, C. L., Jr., Proc. Sot. Exp. Biol. and Med., 51, 102 (1942). 2. Bratton, A. C., and Marshall, E. IS., Jr., J. Biol. Chem., 128,537 (1939). 3. Johnson, T. B., and Johns, C. O., J. ilm. Chem. Sot., 36, 545 (1914). 4. Fischer, E., Ber. them. Ges., 47, 2611 (1914). 5. Johnson, T. B., and Hahn, D. A., Chem. Rev., 13, 193 (1933). 6. Johnson, T. B., Johns, C. O., and Heyl, F. W., Am. Chem. J., 36, 160 (1906). 7. Johns, C. O., Am. Chem. J., 45, 79 (1911). 8. Fischer, E., and Ach, L., Ber. them. Ges., 30,220s (1897). Fischer, E., Ber. them.

Ges., 32, 435 (1899). 9. Roblin, R. O., Jr., Lampen, J. O., English, J. P., Cole, &. P., and Vaughan, J. R.,

Jr., J. Am. Chem., Sot., 67, 290 (1945). 10. Bladin, J. A., Ber. them. Ges., 18, 666 (1885). 11. Wright, L. D., and Welch, A. D., Science, 98, 179 (1943). 12. Dutcher, J. D., and Wintersteiner, O., J. Biol. Chem., 165, 359 (1944). 13. Landy, M., Larkum, N. W., Oswald, E. J., and Streightoff, F., Science, 97, 265

(1943). 14. Mirick, G. S., J. Ezp. Med., 78, 255 (1943). 15. Rosenthal, S. M., and Bauer, H., Pub. Health Rep., U. S. P. H. S., 54, 1880 (1939). 16. Spies, J. R., and Harris, T. H., Ind. and Eng. Ckem., Anal. Ed., 9, 304 (1937). 17. van Busch, M., and Blume, G., 2. angew. Chem., 21, 354 (1908). 18. Dudley, H. W., Biochem. J., 23, 1064 (1929). 19. Newton, E. B., J. Biol. Chem., 120, 315 (1937). 20. Van Slyke, D. D., J. Biol. Chem., 23, 407 (1915). 21. Reis, A., Z. phys. Chem., 88, 536 (1914).

by guest on June 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Page 18: AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE … · 2000-02-07 · AN AMINE FORMED BY BACTERIA DURING SULFONAMIDE BACTERIOSTASIS* BY MARJORIE R. STETTEN AND CHARLES L. FOX, JR.

Marjorie R. Stetten and Charles L. Fox, Jr.BACTERIOSTASIS

DURING SULFONAMIDE AN AMINE FORMED BY BACTERIA

1945, 161:333-349.J. Biol. Chem. 

  http://www.jbc.org/content/161/1/333.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  tml#ref-list-1

http://www.jbc.org/content/161/1/333.citation.full.haccessed free atThis article cites 0 references, 0 of which can be

by guest on June 17, 2020http://w

ww

.jbc.org/D

ownloaded from