Antivitamins for Pantothenic Acid

Acta phrmacol., 1946, 1, 207-218.

(From the Carlsberg Laboratory, Copenhagen)

Antivitamins for Pantothenic Acid. BY

Niels Nielsen and Knud Roholt (Received 28 March 1946)

As is well known, pantothenic acid is a very important vitamin (growth substance) to many micro-organisms. It will be of interest therefore to look into the occurrence of antivitamins for pantothenic acid, i. e. , substances which on account of a similar chemical structure are able to abolish the vitamin effect of pantothenic acid - just as sulfonamides are able to abolish the vitamin effect of p-amino-benzoic acid. The occurrence of antivitamins for pantothenic acid is known already. Thus KUHN, WIELAND and MOLLER (1941) and SNELL (1941 a,b) have demonstrated that sulfopantothenic acids (pantoyl-taurine) - i. e., a compound deviating from pantothenic acid by containing taurine instead of @-ahnine - is a strong antivitamin for pantothenic acid. Furthermore, NIELSEN, HARTELIUS and JOHANSEN (1944) have found that methyl pantothenic acid (pantoyl-8-amino-butyric acid) - i. e. , a compound containing @-amino-butyric acid instead of p-alanine - is also a strong antivitamin for pantothenic acid.

For certain micro-organisms, e . g., yeast, @-alanine, one of the components of pantothenic acid, is just as active 8 vitamin as is pantothenic acid itself - evidently because yeast is able readily to form pantothenic acid from @-ahnine. Antivitamins have been demonstrated also for @-ahnine, namely : @-amino- butyric acid, @-phenyl-@-ahnine, isoserine, and most of the a-amino acids (NIELSEN, HARTELIUS and JOHANSEN, 1944). Evidently this antivitamin effect is due to the circumstance that these substances prevent the formation of pantothenic acid from @-ahnine.

208 NIELSEPU’ AND ROHOLT

In the present studies we have looked into the action of a number of pantothenic acid-like substances in order to ascer- tain whether they act as antivitamins for pantothenic acid. Altogether 15 substances were examined. They deviate from pantothenic acid in the substitution of other amino acids for 8-alanine. The antivitamin effect of these substances for pantothenic acid is examined by employment of 3 micro-organisms: Streptobacterium plantaruni, Saccharomyces cerevisiae and Aspergillus niger. These 3 organisms are selected because in their relation to pantothenic acid they represent three different types. Strbm. plantarum requires pantothenic acid for its growth ; j3-alanine, a component of pantothenic acid, has not this effect (MOLLER, 1939). Saccharomyces cerevisiae C. L. 1 requires either pantothenic acid or 8-alanine for a good grou.th ; still it is able to grow, though slowly, also without such an addition, and thus it is able itself to produce a small amount of pantothenic acid (NIELSEN and HARTELIUS, 1945). Further- more, this yeast grows just as well with addition of j3-alanine as with addition of pantothenic acid because it is able rapidly, within a few hours (HARTELIUS, 1944), to synthesize pantothenic acid from an addition of 8-alanine. Finally, the growth of Aspergillus niger is not influenced by any addition of pantothenic acid or j3-alanine; and this is easily explainable as the micro-organism itself forms large amounts of pantothenic acid (NIELSEN and HARTELIUS, 1945).

1. Antivitamins Tested. The substances prepared are amide-like condensation products

of a, y -dioxy-j3,p -dimethyl-butyric acid and various amino acids. The condensation, which in most instances proceeds quite readily, is carried out in aqueous solution between equimolecular amounts of a-oxy-8,p -dimethyl-y-butyrolactone and $he sodium salt of the respective amino acid. As to the aliphatic amino acids the d, 1-form is employed; still, 1-asparagine was used.

In the designation of these compounds, for practical reasons, we have named the radical of a ,y -dioxy-p,/3 -dimethyl-butyric acid “pantoyl” (in the following formulae designated as “R”).

ANTIVITAMINS FOR PANTOTHENIC ACID. 209

CHS I

I CH,

Pantoyl- (“R”) HOHZC-C-CHOH-CO-

The following substances were examined :

R-NH-CH 2-C 0 0 H

R-NH-CHz-( CH,),-COOH Pantoyl- 8-amino-valeric acid

R-NH-CHz-(CHz)4-COOH Pantoyl-c-amino-caproic acid

Pantoyl-glycine

CH3 I

CH, I

Pantoyl-alaninc R-NH--CH-COOH

Pantoyl-fi-amino-butyric acid R-NH-CH-CHZ-COOH

(CH,), I I

Pantoyl-amino-iso-butyric acid R-NH-CH-COOH

I R-NH-CH-COOH

CH-(CH,h I

R-NH-CH-COOH

IGNH-CHZ-CHOH-COOH

CH,OH I

R-NH-CH-COOH

CHz-COOH I

R-NH-CH-COOH

R--NH--CH~-CH~-SO~H

R-NH?-/ I7-C0OH

Aota pharmacologica vol. 1, fasc. 2

Pantoyl-amino-valeric acid

Pantoyl-dine

Pantoyl-isoserine

Pantoyl-serine

Pantoyl-asparagine

Pantoyl-taurine

Pantoyl-p-amino-benzoic acid

18

210 NIELSEN AND ROHOLT

R-NH-CH-COOH

2. Experimental a. Streptobacterium plantarum.

Pantoyl-sulfanilamide

Pantoyl-phenylalanine

Technique.

The antivitamin activity of the various substances was examined by determination of the effect on the growth of the micro -organism.

The culture medium employed contained per liter: 4 g. ammonium acetate - 2 g. KH,PO, - 0.4 g. MgSO,, 7 H,O - 10 mg. ferricitrate - 10 mg. MnCl,, 4 H,O - 10 g. glucose - 100 y thiamine - 250 cc. casein hydrolysate. The pH of the medium was adjusted to 7.0. The casein hydrolysate which provides the solution with the amino acids required, was prepared by boiling 1 kg. casein for 6 hours with 7.6 liters 5 n sulfuric acid; then the excess of sulfuric acid was removed with a Ba(OH), solution. The pantothenic acid originally present in the casein is destroyed by the protracted boiling in strong acid solution. After this treatment the casein hydrolysate will still contain 8-alanine, but this substance is inactive as to Strbm. plantarum (MBLLER, 1939). The other vitamins, biotin, etc., are present in the casein hydrolysate in such large amounts that it is not necessary to add them to the solution.

The substances to be assayed for antivitamin effect were dissolved in water, and 5 cc. of the solutions was transferred by means of a pipette to test-tubes (200x25 mm.). Then a suitable portion of the culture media was inoculated with a culture of Strbm. plantarum, and 10 cc. of the inoculated medium was pipetted off into each tube. The tubes thus contain altogether 15 cc. of mixed solution. The tubes were incub- ated at 25". The tests were not carried out sterilly, which would have been unnecessary, as the experimental period was only 24 hours. After 24 hours the growth in the tubes was measured nephelometrically by means of a Zeiss-Pulfrich photometer. The bacterial concentration in the tubes was calculated from the nephelometer readings obtained by means of a standard curve.

Two test series were set up. In one series 26 y d-Ca-pantothenate per liter was added to the culture medium besides the above-mentioned substances ; this amount is just optimal.


In the other series 250 y d-Ca-pantothenate per liter was added to the culture medium, that is, a strongly overoptimal amount. If one of the substances examined acts a8 an antivitamin for pantothenic acid, this will be revealed by the fact that the growth in the series in which just an optimal amount of pantothenic acid was added to the medium becomes weaker or stops completely. Such an inhibition of the growth, however, may be due both to antivitamin activity and to an ordinary inhibitory unspecific effect. Whichever may be the case can be decided by the test in the second series, in which a strongly optimal amount of pantothenic acid is added to the medium. For, if the inhibition of growth is due to a true antivitamin effect on pantothenic acid, it will be abolished in this series or at any rate suppressed to a great extent. If the inhibition is equally pronounced in both series it is not a matter of a antivitamin activity against pantothenic acid but an entirely different inhibitory effect.

b. Saccharomyces cerevisiae C . L. 1. The culture medium contained per liter: 1 g. KH,PO, - 0.7 g. MgSOd,

7 H,O - 0.5 g. NaCl - 0.4 g. CaCl,, 6 H,O - 1 g. (NHJ, SO, - 5 mg. FeCI,, 6 H,O - 50 g. saccharose - 0.2 y biotin - 50 y thiamine - 200 mg. glutamic acid. Otherwise the technique was quite the same as described for Strbm. plantarum.

3 test series were set up. In 2 of them, respectively 25 y and 250 y d-Ca-pantothenate per liter ww added to the culture medium. These amounts are just optimal and strongly overoptimal respectively, and the purpose of the employment was the same as in the tests with Strbm. plantarum: to decide whether or not a possibly inhibitory effect of a substance is an antivitamin effect for panthothenic acid.

In the third series 50 y j?-alanine per liter was added to the medium, this amount being just optimal. For the yeast grows just as well with 8-alanine in the medium as with pantothenic acid. This must be due to the circumstance that the yeast itself is able easily t o form pantothenic acid from the added j?-alanine.


As mentioned before, however, this synthesis of pantothenic acid may be inhibited by various amino acids, and by means of this third test series it was practicable to decide whether the pantoyl amino acids were just as inhibitory as the corresponding amino acids. Finally, this test series afforded a control of the purity of the compounds prepared. For if they contained any essential amounts of the amino acids employed for their preparation they would have 'an inhibitory effect in the test series with 8-alanine added to the medium.

c . Aspergillus niger .

technique.

The culture medium contained per liter: 1 g. KH,POI - 0.7 g. MgSO,. 7 H,O - 4 g. (NH,),SO, - 6 mg. FeCl,, 6 H,O - 60 g. saccharose. No vitamins were added to the medium, as they would be of no signific- ance to the growth. Into Erlenmeyer flasks of 300 cc. were pipetted 5 cc. of solutions of the substances to be tested and then 60 cc. of the culture medium, SO that each flask contained 65 cc. of mixed solution. After sterilization, each flask was inoculated with 2 drops of a conidial suspension. It was necessary to carry out these tests sterilly, as it took 5 days' incubation a t 32" before the growth could be measured. The growth was determined by filtration of the mycelium on 1G3 Jena glass filter crucibles, with following washing and drying to constant weight at 105", whereafter the dry matter of mycelium was determined by weighing.

For these test it was necessary to employ an entirely different

With this micro-organism only one test series was set up, without addition of pantothenic acid.

3. Experimental Results. From Table 1 it is evident that of the 15 substances examined

6 act as antivitamins for pantothenic acid with regard to Strbm. plantarum. These 6 compounds are : pantoyl-8-amino-butyric acid, pantoyl- S-amino-valeric acid, pantoyl-6-amino-caproic acid, pantoyl-isoserine, pantoyl-glycine and pantoyl-taurine. Of these 6 substances, 2, pantoyl-p-amino-butyric acid and pantoyl-


taurine, have been recogniqed previously as antivitamins for pantothenic acid. That these substances actually are antivitamin for pantothenic acid is evident from the circumstance that their inhibitory effect is abolished or at any rate greatly reduced in the test series in which a great excess of pantothenic acid is added to the culture medium. Also pantoyl-p-amino- benzoic acid has an inhibitory effect. This inhibition is equally great in both series, however, and as an excess of pantothenic acid does not lower the inhibition, the effect observed here is not an antivitamin effect against pantothenic acid but some other inhibitory activity. Perhaps pantoyl-p-amino-benzoic acid is antivitamin for some other vitamin, possibly for p-amino- benzoic acid.

Table 1. Antivitamin Effect with Strbm. plantarum for Test Organism.

Relative growth Growth without antivitamin: 100

25y d-Ca-pantothenate 260y d-Ca-pantothenatc Addition per 15 cc. of medium per liter of medium per liter of medium

.5 nig. Pantoyl-glycine 55 100 3 - 50 98 1 - 64 98 0.3 - 90 94 0 . 1 - 98 100

3 - 24 43 1 - 30 78 0.3 - 57 94 0 . 1 - 83 100 0.03 - 94 100 0.01 - 100 98 5 mg. Pantoyl-~-aniino-cnproic acid 24 25 3 - 21 31 1 - 18 49 0.3 - 24 95 0.1 - 44 93 0.03 - 78 100 0.01 - 97 97

5 mg. Pantoyl-6-amino-valeric acid 31 39

5 mg. Pantoyl-alanine 102 98


(Table 1 continued) Relative growth

Growth without antivitamin: I00 25.1 d-Ca-pantothenate 260.1 d-Ca-pantothenat Addition per 15 cc.

of medium per liter of niediiun per liter of medium 5 mg. Pantoyl-8-amino-butyric acid 3 1 0.3 - 0.1 - 0.03 - 0.01 - 5 mg. Pantoyl-amino-iso-butyric acid 5 mg. Pantoyl-amino-valeric acid 5 rng. Pantoyl-valine 5 mg. Pantoyl-iso-serine 3 - 1 0.3 - 0.1 - 5 mg. Pantoyl-mrine 3 - 1 0.3 - 0.1 - 0.03 - 6 mg. Pantoyl-asparagine 6 mg. Pantoyl-taurine 3 - 1 0.3 - 0.1 - 0.03 - 6 mg. Pantoyl-p-amino-benzoic acid 3 1 - 0.3 - 0.1 - 6 mg. Pantoyl-sulfanilamide 6 mg. Pantoyl-phenylalanine

- -

-

-

-

-

20 20 28 44 60 82 98 98

102 99 44 49 61 89

102 134 126 123 118 97

103 98 17 17 22 46 73 97 40 53 85 93

100 100 100

42 65 68 88

103 100 101 99

102 100 82 91

103 104 101 131 140 138 134 113 102 96 62 76

101 105 100 96 46 54 76 84 98

102 100

The other compounds examined showed no inhibitory effect. One substance, pantoyl-serine, has shown a rather strong growth- promoting effect. On addition of 1 mg. per 15 cc. of medium


the growth is increased by about 30%, and even an addition of 100 y per 15 cc. shows still a distinct growth-promoting effect. So this substance must tact as a vitamin for Strbm. plantarum. Other experiments show that an addition of pantoyl- serine cannot replace pantothenic acid; and hence the vitamin effect of this substance must differ from that of pantothenic acid. In this connection it may be mentioned that the corresponding amino acid, serine, has no effect.

Of the 6 compounds with antivitamin effect, the most effective are pantoyl-taurine, pantoyl- 6-amino-valeric acid and pantoyl-e- amino-caproic acid, as an addition of 30 y per 16 cc. has a distinct growth-inhibiting effect. Pantoyl-/I-amino-butyric acid has a somewhat weaker effect, as it takes an addition of 300 y per 15 cc. to produce a distinct inhibition of growth.

From the present material it seems impracticable to lay down any definite rule as to which conditions decide whether or not a given substance has an antivitamin effect. All the tested compounds containing a straight unbranched carbon chain have shown antivitamin activity, whereas of the compounds in which the amino acid has a branched carbon chain only pantoyl-/I-amino-butyric acid has an antivitamin effect. Whether this be a rule, however, may be settled by examination of a larger material.

The behavior of these substances towards yeast is quite different from that towards Strbm. plantarum. An antkitamin activity was found only in pantoyl-taurine, pantoyl-amino- valeric acid and pantoyl-alanine. In the test series where /I-alanine is added a corresponding inhibitory effect is observed. Indeed, this was to be expected. Even though the pantoyl- amino acids concerned do not inhibit the formation of pantothenic acid from /I-alanine, they still will exert their inhibitory effect when the pantothenic acid is formed.

Of the three compounds with antivitamin effect on the growth of yeast only one, pantoyl-taurine, shows antivitamin effect on the growth of Strbm. plantarum. On the other hand, the substances which in Strbm. plantarum act as antivitamins are not antivitamins in yeast cultures. In this respect, then,


Table 2. Antivitamin Effect with Yeast for Test Organism.

Relative growth Growth without antivitamin: 100 SOY B- 26y d-Ca- 26Oy d-Ca-

of medium of medium of medium

Addition per 16 00. Alanine pantothenate pantothenate of medium per liter per liter per liter

5 mg. Pantoyl-glycine 5 mg. Pantoyl-6-amino-valeric acid 6 mg. Pantoyl-P-amino-caproic acid 5 mg. Pantoyl-alanine 3 1 0.3 - 5 mg. Pantoyl-8-amino-butyric acid 5 mg. Pantoyl-amino-iso-butyric acid 5 mg. Pantoyl-amino-valeric acid 3 1 0.3 5 mg. Pantoyl-valino 5 mg. Pantoyl-iso-serine 5 mg. Pantoyl-serine 5 mg. Pantoyl-asparagine 5 mg. Pantoyl-taurine 3 1 0.3 - 0.1 - 5 mg. Pantoyl-p-amino-benzoic acid 3 5 mg. Pantoyl-sulfanilamide 5 mg. Pantoyl-phenylalanine

- -

- - -

- -

-

95 105 95 81 90 95

100 96 95 57 65 82 99

110 95

102 94 72 80 91 97

102 80 97

100 100

98 102 100 67 72 79 97

100 96 53 70 80

102 105 104 95

102 60 75 82 88 98 87 104 94 98

104 104 96

108 105 98

100 97

102 88 95

103 98

108 100 100 98

100 96 98

100 95 90 98 97

102

these two micro-organisms behave quite differently even though pantothenic acid is a vitamin for both of them. Something similar is known to apply to the effect of the sulfonamides on p-amino-benzoic acid: the relative antivitamin effect of the various sulfonamides differs with different bacterial strains.

As mentioned above, many amino acids act as antivitamins for 8-alanine. On the other hand, the corresponding pantoyl- amino acids do not act as antivitamins for pantothenic acid. Thus isoserine, b-amino-butyric acid, glycine, etc. are strong


antivitamins for p-alanine, whereas the corresponding pantoyl- amino acids are inactive. Conversely, on the other hand, taurine is inactive as antivitamin for j3-alanine, whereas pantoyl-taurine is a strong antivitamin for pantothenic acid. This lack of parallel- ism in the action of the substances shows that for pantothenic acid in yeast and presumably in other micro-organisms which are able themselves to form pantothenic acid from j3-alanine there are two distinctly different antivitamin effects. In the first place, the formation of pantothenic acid from j3-alanine may be prevented by substances resembling p-alanine so much that they are able with a,y-dioxy-j3,fl-dimethyl-butyric acid to form pantothenic acid-like inactive compounds. In the next place, however, also pantothenic acid itself may be displayed by chemically similar substances. It is only natural that the pantoyl-amino acids which in the latter case are capable of inhibitory activity need not necessarily contain the amino acids that are inhibitory in the former case.

The experiments with Aspergillus niger gave the result that none of the compounds tested showed any antivitamin effect. As all the compounds were inactive and showed no growth- promoting effect either, no tabulation will be given of the results in these tests.

The reason why Aspergillus niger is not influenced by the substances here examined may be that this fungus itself forms large amounts of pantothenic acid (NIELSEN and HARTELIUS, 1945); and this, of course, will bring about that very large amounts of a substance have to be added if it is to show any antivitamin effect. But the reason may be looked for also in other conditions. Here it may be mentioned that other experiments, which are not t o be dealt with here, have shown that Torula utilis, which likewise forms large amounts of pantothenic acid (NIELSEN and HARTELIUS, 1945) is not inhibited either in its growth by pantoyl-amino acids.

Summary The experiments have shown that a number of pantoy1

amino acids - i. e . , compounds differing from pantothenic


acid by containing other amino acids instead of p-alanine - act as antivitamins for pantothenic acid. Tests with Strepto- bacterium plantarum showed pantoyl 8-amino-butyric acid, pantoyl-8-amino-valeric acid, pantoyl-e-amino-caproic acid, pantoyl-isoserine , pantoyl-glycine and pantoyl-taurine to be strong antivitamins. This applies in particular to pantoyl- 6-amino- d e r i c acid, pantoyl-c-amino-caproic acid and pantoyl-taurine, as 30 y of the compound concerned per 16 cc. of culture medium showed a distinct growth-inhibiting effect. As the culture medium contained 0.26 y d-Ca-pantothenate per 15 cc. these substances thus are active still at the ratio of 120: 1. The 3 other substances are somewhat weaker antivitamins.

In tests with yeast a weak antivitamin effect against pantothenic acid was obtained with pantoyl-alanine, pantoyl-amino- valeric acid and pantoyl-taurine. So the substances acting as antivitamins in tests with Streptobacterium plantarum are not the same as in tests with yeast.

In tests with Aspergillus niger none of the pantoyl-amino- acids examined showed any antivitamin effect. This may perhaps be due to the fact that Aspergillus niger itself forms large amounts of pantothenic acid. Nor did they show any antivitamin effect in tests with Torula utilis which likewise is able itself to form large amounts of pantothenic acid.

In tests with Strbm. plantarum a strong inhibition of growth was obtained with pantoyl-p-amino-benzoic acid. This inhibition, however, is not due to any antivitamin activity against pantothenic acid but must be due to other causes.

REFERENCES Hartelius, V.: Naturwisa., 1944, 32, 81. Kuhn, R., Th. Wieland and E. F. Moller: Ber. dtach. chem. Gee.,

Moller, E. F.: Ztachr. physiol. Chem., 1939, 260, 246. Nielsen, N. and V. Hartelius: C.T. Lab. Carlaberg, Sdr. phyaiol.,

Nielsen, N., V . Hartelius and G. Johansen: C.T. Lab. Carlsberg,

Snell, E. E.: J . Biol. Chem., 1941 a, 139, 975. Snell, E. E.: J . Biol. Chem., 1941 b, 141, 121.

1941, 74, 1612.

1945, 24, 117.

Sdr. physiol., 1944, 24, 39.

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Antivitamins for Pantothenic Acid