Kinetics of Oxidation of Tryptophan by Sodium Hypochlorite

Thomas Rausch, Frieder Hofmann, and Willy Hilgenberg*

B o t a n i s c h e s I n s t i t u t d e r J o h a n n - W o l f g a n g - v o n - G o e t h e - U n i v e r s i t ä t ,
S i e s m a y e r s t r a ß e 70, D - 6 0 0 0 F r a n k f u r t

Z . N a t u r f o r s c h . 3 6 b , 3 5 9 - 3 6 1 ( 1 9 8 1 ) ; r e c e i v e d S e p t e m b e r 12, 1980

T r y p t o p h a n , I n d o l e a c e t a l d e h y d e , A m i n o A c i d O x i d a t i o n , S o d i u m H y p o c h l o r i t e , R e a c t i o n K i n e t i c s

T h e o x i d a t i o n o f t r y p t o p h a n t o 3 - i n d o l e a c e t a l d e h y d e w i t h s o d i u m h y p o c h l o r i t e w a s
i n v e s t i g a t e d w i t h 1 4 C labelled D L - t r y p t o p h a n . T h e r e a c t i o n w a s p e r f o r m e d u n d e r p s e u d o
first o r d e r c o n d i t i o n s . F r o m t h e p H d e p e n d e n c e o f t h e r e a c t i o n it w a s c o n c l u d e d t h a t o n l y
t h e u n p r o t o n a t e d t r y p t o p h a n is c o n v e r t e d t o t h e a l d e h y d e . T h e a c t i v a t i o n e n e r g y is
35 ± 2.2 ( S E ) k J x m o l - 1 as d e r i v e d f r o m t h e A r r h e n i u s plot. V a r i i n g t h e p H b e t w e e n 8.5
a n d 11.0 a n d t h e t e m p e r a t u r e in t h e r a n g e f r o m 298 K t o 3 1 8 K d i d n o t alter t h e selectivity
o f t h e r e a c t i o n as c o n f i r m e d b y T L C o f t h e p r o d u c t ( p u r i t y > 9 0 % ) .
A possible r e a c t i o n m e c h a n i s m is p r o p o s e d .

Introduction supplied at one minute intervals throughout the

reaction time. Every 5 min a 0.5 ml sample of the
Several kinetic investigations on amino acid benzene layer is taken for determination of its
oxidation have been performed [1-3]. Depending on radioactivity and replaced by fresh benzene. At the
the nature of the oxidant these authors report end of the reaction the benzene layer is pipetted off.
different products involving different reaction
mechanisms. In this study sodium hypochlorite was Identification and quantification of the reaction
used for the oxidation of the aromatic amino acid product: For determination of purity the benzene
tryptophan. Under slight alkaline conditions 3- phase is dried on Na2S04 and its volume reduced
by RFE. Aliquots equivalent to 50 nmol IAAld are
indoleacetaldehyde is the major oxidation product submitted to TLC in 2 different systems:
[4]. Under our experimental conditions this reaction
1. Silicagel F254 on alufoil (Merck); solvent system
was highly selective, so it was possible to investigate chloroform: methanol (96:4).
the reaction kinetics. 2. Cellulose F254 on alufoil (Merck); solvent system
water: methanol (99:1).
Materials and Methods The product is identified by cochromatography
Materials: DL-tryptophan (side-chain-3-14C), spe- with authentic IAAld and localized by both its
cific activity 48 mCi/mmol (New England Nuclear); fluorescence quench at 254 nm and its colour
3-indolyl-(2-14C)-acetic acid, specific activity 25 reaction with Ehmann's reagent [6]. To determine
to 35 mCi/mmol (CEA); 3-indoleacetaldehyde X the distribution of radioactivity non sprayed
NAHSO3 (Sigma); indole-3-ethanol (Sigma); 3- chromatograms are cut horizontally into 0.5 cm
indoleacetaldoxime, synthesized according to [5]; stripes and their radioactivity is determined by
5.2% NaOCl solution (Merck). liquid scintillation counting.

General procedure: 500 [A of an aqueous 10 mM
try solution (adjustment to desired pH by either
1 N HCl or 1 N NaOH) including 200 nCi of r e -
labelled try are overlayered with 10 ml of redistilled
benzene. The cylindrical reaction vessel (25 X
100 mm) is fixed in a water bath and the two layers
are thoroughly mixed by a magnetic stirrer. The
reaction is started by the addition of 4 /A of a 0.5%
NaOCl solution of the same pH as the reaction
mixture; the same amount of NaOCl solution is
* R e p r i n t r e q u e s t s t o P r o f .
0 3 4 0 - 5 0 8 7 / 8 1 / 0 3 0 0 - 0 3 5 9 / $
Derivatization of the product: For the conversion
to indole-3-ethanol an aliquot of the dried benzene
phase is evaporated by R F E over 1 ml 1 M NaBH 4
and the reaction is allowed to proceed for additional
10 min. The reaction mixture is extracted twice
with 10 ml benzene and the combined benzene
phases are dried on Na2S04. After volume reduction
by R F E aliquots equivalent to 50 nmol indole-3-
ethanol are chromatographed in the TLC systems
already described. Identification with authentic
indole-3-ethanol and quantification follow the pro-
cedure described for IAAld.
D r . W . H i l g e n b e r g .
The conversion to 3-indoleacetic acid is carried
01.00/0
out by oxidation of IAAld with Ag20. Aliquots of
the dried benzene phase containing additionally
variing amounts of unlabelled IAAld are evaporated
to dryness by R F E . 2 ml 20 mM AgN0 3 of different

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pH values (range 8.0 to 13.0) are added. The reac-
tion time is varied between 0.5 and 90 min. The
reaction is stopped by adjusting the reaction mixture
to pH 2.5 with 1 M H2SO4. The reaction products
are extracted twice with 10 ml of diethylether.
TLC, identification and quantification follow the
procedure described for IAAld.
To form the bisulfite addition product the crude
benzene phase is washed with 0.1 M potassium
phosphate buffer, pH 7.0 and evaporated by R F E
over 1 ml 50 mM NaHS0 3 , pH 7.0. Non-bound
radioactive compounds are reextracted with fresh Fig. 2. D e p e n d e n c e o f initial r a t e o n u n p r o t o n a t e d

benzene. Liberation of IAAld from the bisulfite t r y p t o p h a n c o n c e n t r a t i o n . Initial t r y p t o p h a n c o n -

addition product is performed by adding 1 M sodium c e n t r a t i o n (total) 10 m M . U n p r o t o n a t e d t r y p t o p h a n

carbonate, pH 10.0 followed by extraction with c o n c e n t r a t i o n

t h e p K v a l u e
at
o f
different
t h e a m i n o
p H v a l u e s
g r o u p (9.39).
c a l c u l a t e d f r o m

fresh benzene.
3. Temperature dependence of the reaction

Results and Discussion The selectivity of the reaction was not impaired
when varying the temperature between 298 K and
1. Reaction order 318 K . The activation energy is 35 ± 2 . 2 (SE)
As the oxidant was supplied continuously the k J m o l - 1 as calculated from the Arrhenius plot
reaction order was expected to be first order. The (Fig. 3).
graphical determination of the reaction order con-
firmed this assumption (Fig. 1). For all experiment
variants more than 90% of the reaction products
cochromatographed with authentic IAAld in two
TCL systems demonstrate that the aldehyde is the
only benzene soluble product.

Fig. 3. A r r h e n i u s plot. Initial t r y p t o p h a n c o n c e n t r a -

t i o n : 10 m M ; p H 10.0.

4. Evidence against a radical mechanism

The oxidant NaOCl is in a pH dependent equili-
brium with HOC1. The latter may readily dispro-
portion to HCl and oxygen radicals. If oxygen or
OH-radicals (which are formed subsequently in
F i g . 1. D e t e r m i n a t i o n o f r e a c t i o n order. Initial t r y p t o -
aqueous solution) were the oxydizing agents the
p h a n c o n c e n t r a t i o n : 10 m M ; t e m p e r a t u r e : 4 5 ° C ;
p H 10.0. a, initial r a d i o a c t i v i t y o f t r y p t o p h a n addition of compounds known to scavenge these
(4.4 x 10 5 d p m ) ; x , a c t u a l r a d i o a c t i v i t y o f indoleacet- radicals should decrease the reaction rate [3].
a l d e h y d e .
However, adding mannitol in excess (1 M) did not
influence the reaction rate significantly (control:
2. pH Dependence of the reaction
rate 320 nmol • min - 1 ; -f 1 M mannitol = rate
Variing the pH in the range from 8.5 to 11.0 did 350 nmol • min -1 ). This result and the high selec-
not alter the selectivity of the reaction as concluded tivity of the reaction suggest that OCL ions are the
from TLC of the dried benzene phase. The initial more likely oxidant.
reaction rate is directly proportional to the con-
centration of the unprotonated try suggesting that 5. Identification of the reaction products
only the unprotonated amino acid is oxidized The identity of the aldehyde was confirmed as
(Fig. 2). follows:

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 a) More than 90% of the radioactivity of the
benzene phase could be reversibly bound to NaHS03 R—l—Nx R-C:
1 H
indicating the formation of the bisulfite addition o'S-
product, -OH
2 OCf (SN) •NHJ-OH
b) in 2 TLC systems more than 90% of the radio- -2 Cf 2H2O
activity cochromatographed with authentic com-
mercially available IAAld, ? 0—H
/U-l H
R-C. n*
H N—0"
c) after derivatisation with NaBH 4 95% of the I
QT '0"
radioactivity cochromatographed with authentic
commercially available indole-3-ethanol, -HJO -C0 2
R
d) the derivatisation to 3-indoleacetic acid was
attempted by oxidation with Ag 2 0 yielding a Vz.
0=C'
variety of labelled compounds one of which co-
chromatographed with authentic 3-indoleacetic acid
(varying the parameters reaction time and pH did
not improve the conversion to 3-indoleacetic acid; conditions. Hence 3-indoleacetaldoxime is a pos-
however, the spectrum of oxidation products was tulated intermediate in the proposed reaction
the same when commercially available unlabelled mechanism.
IAAld or 3-indolyl-(2-14C)-acetic acid were sub- 6. Proposed reaction mechanism
mitted to the same procedure).
The initial step could be a nucleophil substitution
25% of the major impurity (accounting for up to between the unprotonated amino group and 2 OC1-
6% of the radioactivity in the benzene phase) could ions similar to the initial step proposed by Gowda
be converted to IAAld simply by stirring the com- and Mahadevappa [2] who used Chloramine-T as
pound in benzene over sodium carbonate buffer, the oxidant. After dehydration and decarboxylation
pH 10.0 at 45 °C. The compound cochromato- the aldoxime would be formed which under our
graphed with authentic indoleacetaldoxime which experimental conditions hydrolyzes to the al-
is readily hydrolyzed to the aldehyde under these dehyde.

[1] M . B h a r g a v a , B . S e t h u r a m , a n d T . N a v a n e e t h [4] R . A . G r a y , A r c h . B i o c h e m . B i o p h y s . 81, 4 8 0

R a o , I n d i a n J. C h e m . 16 A , 651 (1978). (1959).
[2] N . M . M . G o w d a a n d D . S. M a h a d e v a p p a , [5] A . A h m a d , I. E e l n u r m e , a n d I. D . S p e n s e r , C a n .
M o n a t s h . C h e m . 1 1 0 , 157 (1979). J. C h e m . 38, 2 5 2 3 (1960).
[3] R . Straight a n d J. D . Spikes, P h o t o c h e m . [6] A . E h m a n n , J. C h r o m a t o g r . 132, 267 (1977).
P h o t o b i o l . 27, 5 6 5 (1978).

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