Download Prevention of Tryptophan Oxidation During Iodination of Tyrosyl

Prevention of Tryptophan Oxidation During Iodination
of Tyrosyl Residues in Peptides
G. M ourier, L. M oroder, and A. Previero*
C e n tre de R e ch e rch e IN S E R M , 60 rue N avacelles, 34100 M o n tp e llier (F ra n ce )
an d *M ax-P lanck-Institut fü r B iochem ie, D-8000 M artin sried (G e rm a n y )
Z . N a tu rfo rsch .
39b,
1 0 1 -1 0 4 (1984); received July 20, 1983
Io d in a tio n o f T yrosyl R esid u es, O xidative D e g rad a tio n o f T ry p to p h a n ,
N in-F orm yl T ry p to p h a n
S tu d ies on m odel system s clearly revealed oxidative d e g rad a tio n o f try p to p h a n u n d e r th e usual
co n d itio n s o f io d in a tio n o f tyrosine residues in p e p tid e s, w h ereb y th e ra te o f fo rm e r re ac tio n w as
fo u n d to be fa ster th a n the io dination itself. N in-form ylation o f try p to p h a n brings a b o u t an
efficient in d o le p ro tec tio n against this oxidative d e g rad a tio n .
Introduction
Experimental
The conversion of tyrosyl residues into iodo-derivatives can be brought about under different ex­
perim ental conditions [1 -3 ] and is essentially an
electrophilic substitution by iodine at the phenolic
ring of tyrosine. This reaction has been perform ed
for different purposes in protein chemistry, e.g. the
iodination of tyrosine has been extensively used in
the preparation of labelled proteins and peptidic h or­
mones by using radioactive iodine isotopes [4 -6 ].
U nfortunately this simple and rather popular reac­
tion is not highly selective. Secondary reactions may
involve to some extent histidine [7] and sulfur con­
taining amino acids [8 ] yielding side products, which
m ust be elim inated by chrom atographic purification
of the labelled derivative. A more severe drawback is
the oxidative degradation of tryptophan, when p re­
sent, which interfers with a clean iodination of ty­
rosine in peptides.
For exam ple, iodination of somatostatin using all
available iodinating procedures gave in all cases an
heterogeneous family of derivatives each containing
an oxidized tryptophan residue [9].
These undesired products can induce erroneous in­
terpretation in receptor studies and radioim m unoas­
says. In this paper we propose the use of Nin-formyl
[ 1 0 ] as tryptophan protecting group during tyrosine
iodination.
Tyrosine, 3-iodo-tyrosine, 3,5-diiodo-tyrosine,
tryptophan, chloramine T and all chemicals were
pure commercial grade. Nin-formyl-tryptophan was
prepared as already described [11]. The peptides
H —T rp —G ly—O H [12], H - G l y - T r p - O H [13] and
P y r - G l y - P r o - T r p - L e u - O H [14] were prepared
according to known procedures; H - P h e - V a l —
A s n - T r p - L e u - L e u - O H was obtained by catalytic
hydrogenation of the corresponding Na-benzyloxycarbonyl derivative, an interm ediate of the Gastric
inhibitory polypeptide (G IP) synthesis [15] [m.p.
220-222 °C (dec.); [a ß 0 : -5 1 .7 ° and [a]52£,: -6 1 .8 °
(c = 1 , in dim ethylform am ide); amino acid analysis
of the HCl-hydrolysate: Asp 1.06(1) Val 0.94(1)
Leu 2.00(2) Phe 0.99(1) Trp 0.87(1)]; H - L e u T rp -M e t-A rg -P h e -O H
was
prepared
by
condensation of B oc—L e u —T rp —M et—OH and
H - A r g ( H B r ) - P h e - O B u r via dicyclohexylcarbodiimide/N-hydroxysuccinimide to B oc—L eu —T rp —
M et—A rg(H B r)—P h e—O B uf followed by deprotec­
tion with trifluoroacetic acid and exchange of the
counterions by acetate [amino acid analysis of the
H Cl-hydrolysate: Leu 1.00( 1) Trp 0.94( 1) M et 0.98( 1)
Arg 1.02(1) Phe 1.01(1)].
* R e p rin t re q u e sts to Prof. D r. A . Previero.
A b b revia tio n s
S ta n d ard a b b rev iatio n s for am ino acid and p ep tid e d e ­
rivatives are used as re co m m e n d e d by the IU P A C -IU B
com m ission on biochem ical n o m en c latu re. T he am ino
acids are o f L -configuration.
0 3 4 0 -5 0 8 7 /8 4 /0 1 0 0 -0 1 0 1 /$ 01.00/0
Iodination experim ents
I) A m in o acid mixtures. Suitable amounts of tyro­
sine, tryptophan and Nin-formyl-tryptophan solu­
tions were treated with freshly prepared chloramine
T and N al solutions. All experim ents were carried
out at 20 °C. Ratio between amino acids and rea­
gents as well as reaction times are reported for each
experim ent in Fig. 1, Table I, Table II and Table III.
II) Peptides. Stock solutions of N al (10 ~ 2 M),
chloramine T (10 - 2 M ), Na 2S20 5 (1.5 • 10“~ M) in
0.5 M phosphate buffer pH 7.4 were therm ostatized
at 20 °C. The tryptophan containing peptide
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102
G. M o u rie r et al. • Io d in atio n o f T yrosyl R esidues in P e p tid e s
T able I. Io d in atio n o f T yr alone and in presen ce o f T rp o r
T rp (F o r). [Tyr] = [Trp] = T rp (F o r) = 1.7 x 10~2 M ;
[C hloram ine T] = 6.8 x 10~2 M ; [N al] = 1.7 x 10“2 M ; p H
= 7.4 (0.25 M p h o sp h a te b u ffer); T em p .: 20 °C; R e ac tio n
tim e: 10 sec; R eactio n w as sto p p e d w ith 0.1 vol o f 3 M
N a 2S2O s.
C onversion
p ro d u c t [%]
S tarting com pounds:
recovery [% ]
C om pound
T y r(I)
Tyr
32.6
T y r + T rp
33.9
T y r + T rp (F o r) 30
T y r(2 I)
T yr
T rp
38.6
37.3
38
28.8
28.8
32
0
-
T rp (F o r)
100
T able II. Iodin atio n o f T y r, a lo n e an d in p re sen c e o f T rp o r
T rp (F o r) w ith stoichiom etric a m o u n ts o f C h lo ram in e T.
[Tyr] = [Trp] = T rp (F o r) = 1.7 x 10“2 M ; [C hloram ine T]
= 1.7 x 10“2 M ; [N al] = 6.8 x 10~2 M ; p H - 7.4; T em p .:
20 °C; R eactio n tim e: 20 m in.
C onversion
p ro d u c t [% ]
S tarting c om p ounds:
recovery [% ]
C om pound
T y r(I)
T yr
32
T yr + T rp
T races
T yr + T rp (F o r) 29
T y r(2 I)
Tyr
T rp
T rp (F o r)
29
0
28
38
97
41
52.6
-
96.12
T able III. Io d in atio n o f T y r/T rp a n d T y r/T rp (F o r) m ixtures
w ith p re g e n e ra te d iodine in a b sen ce o f free C h lo ram in e T.
[Tyr] - [Trp] = T rp (F o r) = 1.7 x 10~2 M ; [IO H ] - 3.4 x
10“2 M ; p H = 7.4; T e m p .: 20 °C. R e a c tio n tim e: 20 m in.
C onversion
p ro d u c t [% ]
S tarting c om pounds:
recovery [%]
T yr + T rp
T races
T yr + T rp (F o r) 5.3
T y r(2 I)
T yr
T rp
0
92.5
98
13
T races -
T rp (F o r)
97.4
P ep tid e
//m ole), dissolved in 1 0 /u\ of w ater (as sodium or
hydrochloride salt), was treated in rapid succession
with N al solution (100 //l) and chloramine T solution
(400 pi1). A fter one m inute N a 2S2 0 5 solution (400//l)
was added and the resulting reaction m ixture was
directly applied to a column of Sephadex LH 20,
equilibrated and eluted with 0.5% H C O O H in w ater
(Fig. 2 ).
The fractions with a 280 nm absorbance were lyophilized and subm itted to amino acid analysis after
hydrolysis in 4 M m ethane sulfonic acid containing
tryptam ine (0.2% ), 110 °C, 24 h [17]. N on-iodinated
parent peptides were used as standard reference
compounds. Each peptide was converted into the Nin(1
formyl derivative by dissolving it in H C O O H half
saturated with anhydrous hydrochloric acid ( 2 h at
20 °C) [10]. Aliquots of the formic solution contain­
ing 1 //m ole of N in-formyl-Trp-peptide were evapo­
rated to dryness over KO H pellets and treated as
above described (Table IV).
T able IV . R ecovery of T rp from p e p tid e s tre a te d w ith
C hloram ine T /N al reag en t (see Io d in atio n e x p erim e n ts II).
A ) u n p ro tec te d p ep tid es; B) p e p tid e s p ro te c te d by N m-form ylation o f T rp residue.
C om pound
T y r(I)
eq. iodine
Fig. 1. Io d in atio n of T yr with increasing am o u n ts o f iodine
(C h lo ram in e T )
[Tyr] = 1.7 x 10“2 M ; [N al] = 1.7 x 10"2 M
p H — 7.4 (0.25 M p hosphate buffer)
T em p .: 20 °C; R eaction tim e: 20 m in
( • - • ) : T yr; ( * - * ) : T y r(I); ( A - A ) : T y r(2 I).
T rp reco v ery [% ]
G ly -T rp
T rp -G ly
P y r - G ly - P r o - -T rp -L e u
L e u —T r p —M e t- A rg —Phe
P h e —V al —A s n - T r p - L e u —L eu
A
B
6.8
5.0
8.2
10a
6.5
96
94
94
92a
94
a M eth io n in e residue is extensively c o n v erted into m e th io ­
nine sulfoxide [21].
Results and Discussion
Fig. 1 reports the course of tyrosine iodination
with chloramine T as a function of increasing
am ounts of iodine. It can be seen that diiodo-ty-
G. M ourier et al. ■Iodination of Tyrosyl Residues in Peptides
103
Fig. 2. Elution profiles of Pyr—G ly—Pro—Trp—Leu—OH from a column of Sephadex LH 20 (40 x 1 cm) equilibrated with
0.5% HCOOH.
A: starting peptide; A': after iodination; B: N in-formyl-peptide; B': Nin-formyl-peptide after iodination. I: arising from
Chloramine; II: peptide.
rosine can be form ed quantitatively while the m onoiodo-derivative, when desired, needs a critical
am ount of oxidant and its yield will not exceed
30—35%. An analogous pattern of reaction has been
observed during electrolytic iodination of angioten­
sin II [3]. How ever in other labelling experim ents,
reported in the literature, the partial iodination is
mainly pursued by using an excess of reagent and a
very reduced reaction time ( 1 0 sec), rather than a
lack of oxidant.
W hen tyrosine alone or in presence of tryptophan
is treated with one equivalent of N al and an excess of
chloramine T (Table I) tryptophan completely disap­
pears. This reaction does not seem to be iodine-con­
suming since the available iodine is almost quantita­
tively bound to tyrosine. In order to identify the
agent responsible of the tryptophan modification,
separate experiments showed that, even if tryp­
tophan is quite stable towards chloramine T at neutral
pH, its destruction readily takes place in the pre­
sence of catalytic amounts of iodine. Conversely,
N in-formyl-tryptophan shows a much greater stability
towards the chloram ine/Nal system (Table I).
When one equivalent of tryptophan together with
one equivalent of tyrosine are treated with one equi­
valent of chloramine T (N al in excess), the most
reactive amino acid is tryptophan (Table II) showing
an apparent stoichiometry of one mole of chloramine
T for one half mole of tryptophan. Nin-formyl-tryptophan, under this condition as well, behaves as an
inert com pound and is completely recovered to ­
gether with the iodo derivatives of tyrosine.
104
G. M ourier et al. • Iodination of Tyrosyl Residues in Peptides
M oreover, if chloramine T is allowed to react with an
excess of N al before the addition of tyrosine/tryp­
tophan or tyrosine/Nin-formyl-tryptophan mixtures,
again the destruction of tryptophan takes place be­
fore the iodination of tyrosine (as reported in Table
III) and again N in-form yl-tryptophan is unaffected.
Since iodine is the only oxidative agent, the tryp­
tophan destruction com petes, in this case, with the
iodination of tyrosine. Thus, even the use of iodine
generated “in situ” by iodogens [6 ] or by electrolytic
procedures [3] do not eliminate the tryptophan de­
gradation.
The susceptibility of tryptophan to electrophilic re­
agents, such as oxidants, is well known in the field of
indole chemistry [11], The Nin-formylation reduces
the nucleophilic character of the indole moiety,
which becomes more resistant towards oxidants and,
in our experim ental conditions, even less reactive
than the phenolic ring of tyrosine.
The results hitherto presented were obtained on
free amino acids but it is well known that tryptophan
in peptides can show reduced or enhanced reactivity
[16], depending on the surrounding peptide chain.
Experim ents were therefore carried out on a number
of tryptophan-containing peptides of different com ­
position. In all cases their Nin-formyl derivatives are
much more stable towards iodinating reagents than
their N-unsubstituted counterpart (Table IV).
The formylation of tryptophan is a reversible reac­
tion: the formyl group, which is stable at pH values
lower than 9, can be easily and specifically removed
in slightly alkaline solutions. Since the discovery of
this reaction [ 1 0 ] a num ber of applications in the field
of structure/activity studies of proteins [17, 18] and
peptides [19] and in the field of peptide synthesis [20]
have been published. The protection of tryptophan,
via Nin-formylation, during iodination presented in
this work, is another application and offers a simple
procedure to prepare non-oxidized iodo-tyrosine
containing peptides.
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This work was partially supported by a grant of
Fondation pur la Recherche M edicale Francaise.