THE

INHIBITION
By DALE

OF MICROBIAL &2-FURYLALANINE*
A. CLARKi of Chemistry, for publication,
AND

GROWTH
DITTMER of Colorado, 10, 1947)

BY

KARL

(From

the Department (Received

University October

Boulder)

The effect of /3-%thienyl-DL-alanine, an isostere of phenylalanine, upon the growth of Saccharomyces cerevisiae, Escherichia coli, and certain other microorganisms has been reported by du Vigneaud and coworkers (1, 2) and by Beerstecher and Shive (3). Thienylalanine was inhibitory toward the growth of these organisms, and the inhibition was counteracted by phenylalanine. These investigators found that as the concentration of phenylalanine in the medium was increased larger amounts of thienylThus, thienylalanine alanine were required to produce inhibition of growth. was shown to act as an antiphenylalanine for these microorganisms. Since furan compounds are isosteres of analogous thiophene compounds (4), it seemed desirable to study the effect of P-2-furylalanine on the growth of microorganisms and to compare its activity with that of P-2-thienylalanine. In this paper the authors wish to report the inhibitory action of p-2furyl-nn-alanine upon the growth of Saccharomyces cerevisiae and Escherichia coli and the effects of naturally occurring amino acids on the toxicity of furylalanine.
EXPERIMENTAL

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&%J$ryl-nn-alanine-The method described by Herz, Dittmer, and Cristol (5) was employed for the preparation of P-2-furyl-nn-alanine which was used in all of the experiments described below. Amino Acids-Most of the amino acids used were obtained from comL-Proline and hydroxy-L-proline were obtained through mercial sources. the courtesy of Dr. E. E. Howe of Merck and Company. Inhibition of Growth of Saccharomyces cerevisiae, Fleischmanns Strain 139 -The technique followed in the yeast growth experiments and the medium and supplement used in this work with yeast were the same as those previously described (2, 6) except that commercial sucrose was used without 6.2 ml. of medium were added to 1.0 ml. of water containing purification.
* This work was supported in part by a research Research. t Present address, Department of Biochemistry, City, Utah. 313 contract University with the Office of Utah, of Naval Salt Lake

314

GROWTH

INHIBITION

BY

FURYLALANINE

the various additions, making a total of 7.2 ml. per assay tube (20 X 150 mm.). The effect of increasing amounts of furyl-nn-alanine upon the growth of Saccharomyces cereuisiae is illustrated in Fig. 1. An average of 140 y of furyl-nn-alanine per 7.2 ml. was required to inhibit growth to 50 per cent of normal. To inhibit yeast growth in the presence of various amounts of DL-phenylalanine, additional amounts of furyl-or,-alanine were necessary. The additional amount of furyl-m,-alanine required to produce 50 per
Normal Growth
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50% Inhibition 14OY Furvlalanine

0 Micrograms
Fm.

300 600 of Furylalanine

per

900
7.2

ml.

1. Inhibition of the growth of yeast by furylalanine

cent inhibition of growth, when related to the amount of nL-phenylalanine present, was a constant ratio over the range of concentrations shown in Table I. To reverse the growth inhibition to 50 per cent of normal inhibition when varying amounts of furyl-nn-alanine were present, increasing amounts of DL-phenylalanine were required. The data in Table [Link] that the ratio of the amount of furyl-DL-alanine present to the amount of or,-phenylalanine required to produce half nullification of the toxicity is nearly constant.

D.

A.

CLARK

AND

K.

DITTMER

315

medium used in the work Inhzbition of Growlh of Escherichia coli-The with E. COW was the same as that described by MacLeod (7), except that the pH was adjusted to 7.2. As in the work with yeast, G.2 ml. of medium were added to 1.0 ml. of water containing the various additions. The technique of inoculation of the tubes with the E. coli culture and of measurRelationship between Furylalanine I
Furyl-nl-alanine TABLE I and Phenylalanine

for Inhibition

of Growth of Yeast
Furylalanine Phenylalanine
molar ratio

DL-Phenylalanine added per 7.2 ml. 7

added per 7.2 ml. for 50 per cent inhibition of growth

7 18 158 216 325 525 18 76 185 385

0 10 50 100 200

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1.9 1.6 2.0 2.0

* Values in this column are the amounts of furylalanine that had t,o be added above the amount (140 y) required to produce 50 per cent inhibition without added phenylalanine. Since 140 y of furylalanine were required to inhibit growth to 50 per cent of normal, this value is subtracted from the amounts of furylalanine required to give 50 per cent inhibition in the presence of added phenylalanine. This gives a true relationship between the amounts of phenylalanine added and the extra furylalanine required to produce 50 per cent inhibition again. These amounts are used to calculate the molar ratios of furylalanine to phenylalanine.
TARLE

Half Nullification
Furyl-c&-alanine present per 7.2 ml. 7

of Toxicity

II of Furylalanine

by Phenylalanine

nL-Phenylalanine required for 50 per cent nullification of toxicity 7

Furylalanine Phenylalanine
molar ratio

200 500 1000 2000

150 290 575 1180

1.42 1.33 1.85 1.82

ing the growth was the same as that employed by Dittmer

and coworkers

A typical curve illustrating the inhibition of growth of Escherichia coli is shown in Fig. 2. An average of 12.4 y of furyl-DL-alanine per 7.2 ml. was
* The Escherichia coli culture was an unidentified strain obtained from Professor Vincent du Vigneaud and was the same as that used in the studies with thienylalanine (2).

316

OROWTH INHIBITION Normal

BY FURYLALANINE Growth

ifi _ zi 2 m608 .P ;t: d 0 piti 2 8I l WIx)44 0 _

--, 50% Inhibition 12.4YFurylalanine

[Link] of
FIG.

lQrylalanine

per

7.2 ml. coli by furylalanine

2. Inhibition
of

of the growth

of Escherichia

Inhibition

of Growth

Escherichia
Furyl-DL-alanine Y

TABLE III coli by Furylalanine

in

Presence of Phenylalanine
Furylalanine Phenylalanine

ox,-Phenylalanine added pe1 1.2 ml.

added per 7.2 ml. for 50 per cent inhibition of growth Y

0.5 1.0 2.0 5.0 10 20

13.2 30.8 49.5 60.6 170 845 1770

17.6 36.3 47.4 157 832 1757

38 39 26 33 88 93 that had to be added without added phenyl-

* Values in this column are the amounts of furylalanine above that amount required to produce 50 per cent inhibition alanine.

required to produce 50 per cent inhibition of growth. When inhibition was obtained in the presence of various amounts of nn-phenylalanine, additional furyl-nn-alanine was required to produce 50 per cent inhibition of growth. The ratio of additional furylalanine to phenylalanine present was not constant at the levels tested, as is shown in Table III,

D.

A.

CLARK

AND

K.

DITTMER

317

With increasing amounts of furyl-DL-alanine, larger amounts of DLphenylalaninewere required to nullify by half the toxicity of the furylalanine; the ratio of these quantities was nearly constant at higher levels of furylalanine, as is shown in Table IV.
TABLE Nullijkation by

Phenylalanine

IV of Inhibition

of

Escherichia

coli Growth
Furylalanine Phenyiam molar ratio

by Furylalanine
Furyl-m-alanine Y per 7.2 ml. m-Phenylalanine required for 50 per cent nullification of toxicity

25 250 500 1000 1500 2500

cl.36

4.2 6.5 12.7 18.0 31.8

74 64 82 82 88 84

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Level of Growth Inhipition with 200 Microgratxs of FurylalanLne Milligrams

of Adno

Acids

per

4 7.2 ml.

5 on

FIG. 3. The effect of various amino acids on the toxicity the growth of Saccharomyces cerevisiae.

of 200 y of furylalanine

Eject of Amino Acids Other Than DL-Phenylalanine in Nullifying Furylalanine Toxicify-A level of 200 y of furyl-nn-alanine,per 7.2 ml. was chosen as a reference point at which to study the effect of amino acids other than phenylalanine on the furylalanine toxicity on yeast. The growth curves in Fig. 3 illustrate the results obtained with the amino acids which showed appreciable activity in counteracting the toxicity of furylalanine.

318

GROWTH

INHIBITION

BY

FURYLALANINE

50% Reversal 8 30 riil d 15 2 3 5 0 1

of Inhibition

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3 4 5 6 7 8 2 Micrograms of Amino Acids per 7.2 ml.

9 on the tox-

FIG. 4 . The effect of DL-phenylalanine, L-tyrosine, and m-tryptophan coli. city of 20 y of furylalanine on the growth of Escherichia

,105 ii 90
k

Norms1 Grc-^ DL-i sol euci ne L-cysteine-IX1 DL-Methionine O$ Reversal. of Inhibition

cl

k Q

licrogr&xs

300

of Aaino

Acids

900 1203 per 7.2 ~1.

1500 of 20 y of furylala-

FIG. 5. The effect of [Link] active amino acids on the toxicity nine on the growth of Escherichia coli.

For a similar study with Escherkhiu co&, a level of 20 y of furylalanine per 7.2 ml. was selected. The results obtained are illustrated by the curves in Figs. 4 and 5. The effects of most of the naturally occurring amino acids on the toxicity

D. A.

CLARK

AND

K.

DITTMER

319

of furylalanine on both Saccharmyces cerevisiae and Escherichia coli are shown in Table V. Those amino acids which counteracted the growth inhibition of either organism are listed first, with their relative effectiveness compared with DL-phenylalanine taken as 100 per cent. The comparisons were made at the levels of amino acids which were necessary to obtain growth at a level half way between normal growth and the level of inhibited growth due to

Nullijication

of Purylalanine
For S. cerevisiae

Inhibition of Growth of Saccharomyces cerevisiae and Escherichia coli by Various Amino Acids

--rE
Amino acid

For E. coli Amount reuired for 50 pe cent nullification of inhibition by 20 y furylalanine Y

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Amino

acid

Amount i-euired for 50 pe cent nullification of inhibition by 200 y furylalanine Y

:ffectiveness on Ill&I basis

per cent 100

per cent 00

nn-Phenylalanine n-Leucine nn-Isoleucine nn-Tryptophan nn-Methionine nn-Valine nn-Alanine

90

200 550 950 2300 2200 6000

36 13 12 3.5 2.9 <l.O

DL-Phenylalanine n-Tyrosine nn-Tryptophan nn-Methionine nn-Isoleucine L-Leucine L-Cysteine HCI n-Proline n-Histidine HCl. HZ0 Inactive: nn-valine

0.34 0.68 1.0 50 68 225 420 1950 4280

55 42 0.61 0.40 0.12 0.06* 0.05* 0.05*

Inactive: n-tyrosine, proline, n-histidine

_____ Inactive for both organisms: glycine, nn-serine, r,-asparagine, n-glutamic acid, hydroxy-n-proline, of contamination

n-cysteine HCl

HCl,

L-

and nn-alanine acid,

--___ nL-threonine, n-aspartic L-lysine HCl, L-cystine

* The possibility for these results.

with more active amino acids is not ruled out

the presence of 200 y of furylalanine in the case of yeast and 20 y with Escherichia coli. This is the growth level which represents 50 per cent nullification of the toxicity of furylalanine.
DISCUSSION

It will be noted from the data of Table V and Figs. 3, 4, and 5 that phenylalanine is the most active amino acid for the reversal of the furylalanine inhibition of both yeast and Escherichia coli growth. This would

320

GROWTH

INHIBITION

BY

FURYLALANINE

indicate that furylalanine, like thienylalanine, is an antiphenylalanine for these two organisms. However, leucine, isoleucine, and tryptophan also have an appreciable effect on inhibition of yeast growth by furylalanine. For the reversal of the inhibition of the growth of E. coli, tyrosine and tryptophan had a high degree of activity. This is similar to the effect that these amino acids have on the toxicity of thienylalanine. Beerstecher and Shive (3) suggested that tryptophan is a precursor of phenylalanine for the strain of E. coli which they used in their work. Just in what way the
TABLE

VI on Growth
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Comparison

of E$ects

of &%Thienyl-nn-alanine and &@uryl-nn-alanine of Saccharomyces cerevisiae and Escherichia coli

Properties

compared

For S. cerevisiae Thienylalanine Furylalanine

For E. coli

Thienylalanine

Furylalanine

Amount required for 50% inhibition of growth Ratio, antagonist to phenylalanine for inhibition in presence of phenylalanine Ratio, antagonist to phenylalanine for reversal of inhibition

42.5 y 0.83 0.55

140 y 2.0 1.83

1.88 Y per 12.4 y per 60 ml. 7.2 ml. 24* Not constant 22* 34*

Amino acids reversing nn-Phenyialanine n-Tyrosine nn-Tryptophan n-Leucine nn-Isoleucine * Constant at higher t In this comparison, per cent loot Inactive 8.0 13.2 (DL-) 9.8

toxicity per cent loot 140 78 0.15 (DL-) I 0 $C7cent loot 55 42 0.12 (L-) 0.4

per Cenf loot Inactive 12.0 36 (L-) 13

levels only. the activity of phenylalanine

is taken as 100 per cent.

amino acids which showed an appreciable activity in counteracting the antiphenylalanine properties of furylalanine are related to phenylalanine remains to be determined. A comparison of the properties of thienylalanine (2) and those of furylalanine shows that these two metabolite antagonists are acting in identical manners; this can be seen by the comparative data of Table VI, which indicate that p-2-furyl-rm-alanine is approximately 30 per cent as active toward Saccharomycescerevisiae and 15 per cent as active toward Escherichia COG p-2-thienyl-nn-alanine. as

D.

A.

CLARK

AND

K.

DITTMER

321

The authors wish to express their appreciation to Mrs. Hester Purdy McNulty and Mrs. Charmion McMillan for checking some of the microbiological results.
SUMMARY

P-2Furyl-nn-alanine inhibited the growth of Saccharomyces cerevisiae and Escherichia coli and phenylalanine reversed this toxicity. To inhibit growth to 50 per cent of normal required 140 y and 12.4 y per 7.2 ml. for yeast and Escherichia coli, respectively. For Saccharomyces cerevisiae, L-leucine, DL-tryptophan, and nn-isoleucine, although less effective than nn-phenylalanine, also counteracted the toxicity of furylalanine, while nn-valine, nn-methionine, and nn-alanine were slightly effective. For Escherichia coli, L-tyrosine and nrAryptophan were about half as effective as nn-phenylalanine in counteracting the toxicity of furylalanine, while nn-methionine, nn-isoleucine, L-leucine, L-cysteine, L-proline, and Lhistidine were slightly active. The antiphenylalanine properties of p-2-furylalanine were found to be quite similar to those of P-2-thienylalanine.
BIBLIOGRAPHY

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1. du Vigneaud, V., McKennis, H., Jr., Simmonds, S., Dittmer, K., and Brown, G. B., J. Biol. Chem., 169, 385 (1945). 2. Dittmer, K., Ellis, G., McKennis, H., Jr., and du Vigneaud, V., J. Biol. Chem., 164, 761 (1946). 3. Beerstecher, E., Jr., and Shive, W., J. Biol. Chem., 164, 53 (1946). 4. Neuhaus, A., Chemie, 67, 32 (1944). 5. Hem, W., Dittmer, Ii., and Cristol, S. J., J. Biol. Chem., 171, 383 (1947). 6. Snell, E. E., Eakin, R. E., and Williams, R. J., J. Am. Chem. Sot., 62, 175 (1940). 7. MacLeod, C. M., J. Exp. Med., 72, 217 (1940).