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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 Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Creative Commons Namensnennung-Keine Bearbeitung 3.0 Deutschland Lizenz. This work has been digitalized and published in 2013 by Verlag Zeitschrift für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution-NoDerivs 3.0 Germany License. Zum 01.01.2015 ist eine Anpassung der Lizenzbedingungen (Entfall der Creative Commons Lizenzbedingung „Keine Bearbeitung“) beabsichtigt, um eine Nachnutzung auch im Rahmen zukünftiger wissenschaftlicher Nutzungsformen zu ermöglichen. On 01.01.2015 it is planned to change the License Conditions (the removal of the Creative Commons License condition “no derivative works”). This is to allow reuse in the area of future scientific usage. 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. [1] I. Covelli and J. Wolff, Biochemistry 5, 860 (1966). [2] I. Covelli and J. Wolff. Biochemistry 5, 867 (1966). [3] H. Kurebart, A . Coli, L. Vaucheri, and U. Rosa, Biochim. Biophys. Acta 230, 160 (1971). [4] P. H. Springell, Biochim. Biophys. Acta 63, 136 (1962). [5] P. J. Dockray, J. H. Walsh, and M. I. Grossman, Biochem. Biophys. Res. Comm. 69, 339 (1976). [6] P. J. Fraker and J. C. Speek (Jr.), Biochem. Biophys. Res. Commun. 80, 849 (1978). [7] I. Covelli and J. Wolff, J. Biol. Chem. 242, 881 (1967). [8] A. N. Glazer, Am. Rew. Biochem. 39, 101 (1970). [9] R. L. Vandlen. L. Hupe, and K. King, Fed. Proc. 41, 1182 (1982). [10] A. Previero, M. A. Coletti-Previero, and J. C. Cavadore, Biochim. Biophys. Acta 147, 453 (1967). [11] W. A. Remers and R. K. Brown, Heterocyclic Com pounds, Wiley-Interscience, Part I, 153 (1972). [12] D. Stevenson and G. T. Young, J. Chem. Soc. (c) 1969, 2389. [13] E. Wünsch and R. Spangenberg, Chem. Ber. 104, 2427 (1971). [14] E. Wünsch and K.-H. Deimer, Hoppe-Seyler’s Z. Physiol. Chem. 353, 1246 (1972). [15] L. Moroder, A. Hallett, P. Thamm. L. Wilschowitz, W. Göhring, J. C. Brown, and E. Wünsch, in I. Z. Siemion and G. Kupryszewski (eds): Peptides 1978, Wroclaw University Press, Wroclaw, Poland, pp. 5 8 5 -588. [16] E. Wünsch, A. Fontana, and F. Drees, Z. Naturforsch. 22b, 607 (1967). [17] M. A. Coletti-Previero, A. Previero, and E. Zuckerkandl, J. Mol. Biol. 39, 493 (1967). [18] A. Holmgren. Europ. J. Biochem. 26, 528 (1972). [19] R. Magous, J. P. Bali. L. Moroder. and A. Previero, Eur. J. Pharmacol. 77, 11 (1982). [20] M. Ohno. S. Tsukamoto, S. Sato, and N. Izumiya, Bull. Chem. Soc. Jpn. 46n, 3280 (1973). [21] M. Bienert. E. Klauschenz, K. Nicolies, and H. Niedric. Int. J. Peptide Protein Res. 19, 310 (1982). This work was partially supported by a grant of Fondation pur la Recherche M edicale Francaise.