Metabolites of p-aminobenzoic acid II. p-Amenobenzyl alcohol- A metabolite of p-aminobenzoic acid

PRELIMINARY NOTES

511

Metabolites of p-aminobenzoic acid II. p-Aminobenzyl alcohol: A metabolite of p-aminobenzoic acid SLOANE et al. 1 reported that p-aminobenzoic acid was metabolized by acid-fast bacteria. More recently the isolation of two supposed metabolites I and II (fI as the crystalline oxalate) was described 2. These compounds accumulated in the medium during the metabolism of p-aminobenzoie acid. Early investigations to determine whether either of the two compounds could be derived from the other, indicated that I (C2sHa0N40) was a degradation product of II, since acid treatment was found to convert II to I and elemental analysis agreed well with a formulation for II as CaeH,vNsO 4 (oxalate)3. However, contrary to this conclusion, II has now been shown to be p-aminobenzyl alcohol. The crystalline free base was obtained upon evaporation (in vacuo) of a chloroform extract of an aqueous solution of II oxalate containing a small quantity of Na,SzO 4 at pH 7.1. The compound was recrystallized from benzene, m.p. 63-63.5 °. The isolated metabolite II (free base) was identical in all respects with authentic p-aminobenzyl alcohol (which was synthesized by the reduction of p-nitrobenzyl alcohol 3) : (a) Analysis* for CvH9NO. Calc. : C, 68.27 ; H, 7.37 ; N, II.37. Found : C, 68.49 ; H, 7.1o; N, 11.33. (b) The equivalent weight** (HCI04 titration in glacial acetic acid) was 124 and 127 (theory 123). (c) The melting point of an admixture of authentic p-aminobenzyl alcohol and metabolite II (free base) was undepressed. (d) The infrared-absorption spectra of p-aminobenzyl alcohol and metabolite II are identical in all respects I0000 tOO - -

3000

4000 I I

I

I

2000 I [

1600 [

1400 I

Frequency 1200 [

(crn -4) I000 I ]

900 I

700

800 [

t

80-60-40--

$

20-"

°= 100 ~q -~. 80-60-40-20-0

I .__ 2

I

3

[ 4

[ 5

I 6

I 7

I 8 Wovelength

J 9 (rap.)

I I0

[ II

I 12

I 13

I 14

; t5

Fig. I. I n f r a r e d - a b s o r p t i o n s p e c t r a of s y n t h e t i c p - a m i n o b e n z y l alcohol (Curve A) a n d m e t a b o l i t e I I (Curve B) in chloroform solution. * Microanalyses were p e r f o r m e d b y A. BERNHARDT, Mfilheim {Germany) a n d J. Kerns, Mellon Institute. ** T h e t i t r a t i o n d a t a was o b t a i n e d b y J. KERNS.

Biochim. Biophys. Acta, 59 (1962) 511-512

512

PRELIMINARY NOTES

(Fig. i). The previously reported analysis of the metabolite II oxalate 2 is in agreement with (p-aminobenzyl alcohol)2 oxalate, C zeH20N2Os. Calc. C, 57.13; H, 5.99; N, 8.33. Found: C, 56.91; H, 6.21; N, 8.00 (another preparation of the oxalate analyzed: C, 56.92; H, 6.22; N, 8.12). NHz

NH 2

NH~

C00H

CH~0H

OH

Fig. 2. A new p a t h w a y for the metabolism of p-aminobenzoic acid by acid-fast bacteria.

The establishment of the metabolite II as p-aminobenzyl alcohol together with the previously published results ~ demonstrates a new pathway for the metabolism of p-aminobenzoic acid. A metabolic end product of this sequence of reactions is p-hydroxyaniline as shown in Fig. 2. These reactions which occur in resting cells of acid-fast bacteria are not inhibited by chlortetracycline, whereas aniline hydroxylation is inhibited by this antibiotic 2. The structure of I, its relation to p-aminobenzyl alcohol and its role as a cofactor in aniline hydroxylation* will be reported in a future publication.

Mellon Institute, Pittsburgh, Pa. (U.S.A.)

N. H. SLOANE

K. G. UNTCrI 1 N. H. SLOANE, C. CRANE AND t{. L. MAYER, J. Biol. Chem., 193 (I95 I) 4532 •. H. SLOANE, J. Biol. Chem., 236 (1961) 448. 3 0 . FISCHER AND G. FISCHER, Ber., 28 (1895) 879.

Received March I3th, I962 Biochim. Biophys. Acta, 59 (I962) 511-512

T h e effect of urea on lens proteins ~-Crystallin prepared by vertical starch-block electrophoresis 1,2 appeared to be a homogeneous protein as judged by physico-chemical and immunological criteria. When such homogeneous preparations are treated with 7 M urea and submitted to vertical starch-gel electrophoresis in 7 M urea, 12-13 zones migrating towards the anode are observed 3. Splitting into various new bands has also been observed by Fig. I. TRAUTMANplot of ~-crystallin in 7 M urea, o. z M Tris buffer (pH 7.6). × = lO589 r e v . / m i n ; /k = i522o rev./min; • = 2o41o rev./ rain; O = 2598o r e v . / m i n ; [] = 29 500 rev./min.

qna

, .

30

/ 20

10

qm --

mZrm

g/mob g/ml

&Cm = (Cm--Co) m g / m l

-6

-5

-4

-3

-2

-1

Ac m

Biochim. Biophys. Acta, 59 (I962) 512-514