Reaction of tryptophan derivatives with nitrite

摘要

062 J.C.S. Perkin IReaction of Tryptophan Derivatives with NitriteBy Raymond Bonnett and Robin Holleyhead, Department of Chemistry, Queen Mary College, Mile EndTreatment of N-acyltryptophan derivatives with sodium nitrite under mildly acidic conditions furnishes the nitros-amine with the nitroso-group located at the indolic nitrogen.Road, London E l 4NSSINCE certain nitrosamines are carcinogenic,l it ispertinent to enquire to what extent such compounds canarise by the interaction of nitrite and organic nitro-genous compounds which may be ingested together.2The interaction with proteins and peptides is of especialinterest : N-terminal prolyl peptide derivatives areknown to be nitrosated at the secondary aininef ~ n c t i o n . ~ However, although the reaction of nitrousacid with peptides has been extensively used analytically(van Slyke estimation) the nature of the organic productshas often been overlooked.This appears to be the casefor tryptophan derivatives. Philpot and Small * ob-served a rapid reaction between tryptophan and nitrousacid but were not able to detect a nitroso-derivative suchas they had observed with tyrosine. Demyaiiov andPutokhin reported the formation of a brick-red powderwhich they regarded as nitroso- p-indolylacrylic acid.I n order to avoid trivial deamination of the a-amino-function, we have examined the ,V-acetyl derivatives(I)-(111) (which are, incidentally, more satisfactorymodels for the peptide situation) and have shown that* J. M. Barnes and P. N.Magee, Brat. J . Iizd. illed., 1964, 11,167; P. N. Magee, Biochem. J . , 1966, 64, 676.* For recent reviews see D. H. K. Lee, Enoiron. Res., 1970, 3,484; J. Sander and F. Schweinsberg, Zent. Baht. Hyg. I Abt.Orig. B, 1972, 156, 290.N1-nitroso-compounds (1V)-(VI) are readily forinedunder mild conditions.NHAc N HAcH NO(I) X = OMe (IV) X = OMe(II 1 X = OH ( y ) X = O H (m) X = NH.CH2COzEt (YI) X = NH.CH2C02 EtThus treatment of N-acetyltryptophan methyl ester(I) with sodium nitrite in aqueous acetic acid at roomtemperature gave yellow crystals. ,4 similar reactionoccurred with the peptide ester (111): with (11) thereaction occurred in water, 2.e. in the absence of addedacid. Nitrite under acidic conditions is expected toyield an electrophilic nitrosating species (NO+, or itsequivalent, e.g.H2N02+, N203) and attack at the five-membered ring (at positions 1, 2, or 3) is likely, although3 F. H. C. Stewart, Austral. J . Chem., 1969,22,2461.J. S . L. Philpot and P. A. Small, Biochem. J . , 1938, 82, 634,6 N. Y. Demyanov and N. I. Putokhin, Conapt. rend. Acad.542.Sci. U.R.S.S., 1936, 2, 390 (Chewz. .4bs., 1935, sL9, 0889)1974reaction in the benzenoid ring or at the amide functionmay also need to be considered.The assignment of the structures of the isolated yellowderivatives may be illustrated by reference to the productfrom (I). Elemental analysis and mass spectrometryof this product accorded with the molecular formula(C1,Hl,N,O,) of a mono-nitroso-derivative. That thenitroso-group was located at N-1 was inferred from thefollowing evidence: (i) The i.r.spectrum of (I) possesseda sharp absorption at 3380 cm-l attributed to the indoleK-H stretching mode. This band was missing in thespectrum of the nitroso-derivative. A band a t 1480cm-' appeared in the latter and is assigned to theN-N=O function (1430-1500 cm-1).6(ii) The aromatic region of the n.m.r. spectrum of thenitroso-compound integrated for five protons : oneproton (presumably that at C-7) was considerably de-shielded (6 8.1).(iii) The Inass spectrum showed a weak molecular ion(m/e 289, 0.5) together with strong fragments corre-sponding t o losses of NO and (NO + C0,Me). The basepeak at m/e 130 corresponded to the P-indolyl methylcarbonium ion (or its isomer).(iv) The electronic spectrum showed two maxima at267 and 274, and a broad but less intense band at335 nm.This spectrum is not that expected for asimple 3H-indole chromophore (such as might resultfrom attack at C-3): however, it does resemble thespectra reported for N-nitrososkatole A,, (hexane) 264and 329-334 nm and for the nitroso-derivative ofZ-methylindole,8 which exists predominantly as thehydroxyimino-tautomer (VII) and has A,,.(EtOH) 242,255, and 315 nm. Such a 3-hydroxyimino-structuremight arise during the nitrosation of (I) if an acid-catalysed C-3-C-2 rearrangement occurred. The elec-tronic spectrum of the 3-hydroxyimino-compound (VII)underwent a profound change (to A,,,,,. 245, 285, andOH345 nin) when the mesomeric anion was formed byadding dilute sodium hydroxide to the solution.Similarbasification of the solution of the nitroso-derivativefrom (I) caused no change in the spectrum, and hencethe possibility of a 3-hydroxyimino-structure is elimin-ated.I t is concluded that the nitrosation product of N -acetyltryptophan methyl ester (I) is the nitrosaminederivative (IV). The products from (11) and (111) areformulated analogously as (V) and (VI). These con-clusions are supported by 16N n.m.r. observations.@The N1-nitrosamine bond in the tryptophan derivativesL. J . Bellamy, ' The Infra-red Spectra of Complex Mole-H. F. Hodson and G. F. Smith, J. Chem. SOC., 196i, 3546;cules,' Methuen, London, 1968, p. 306.cf. I:. Fischcr, A+zwalen, 1886, 286, 126.is cleaved with mineral acids.Thus after brief treat-ment of the N1-nitroso-carboxylic acid (V) with HBr-HOAc the acid (11) was detected by paper chromato-graphy as the major product. The acid (V) was alsophotolabile, so much so that the electronic spectrumcould not be accurately measured. Thus irradiation ofan ethanolic solution with white light (100 W tungstenlamp at 15 cm for 30 min) caused complete loss of the335 nm peak: under these conditions the correspondingester (IV) was little affected.These results show that under mild conditions oftemperature and pH sodium nitrite reacts with simpletryptophan derivatives, including an N-acetyl dipeptide,to give nitroso-derivatives in which the nitroso-group issubstituted a t N-1 of the indole system.Such sub-stitution at tryptophan units might occur when proteinsare treated with nitrite, although it would be expectedto be accompanied by reaction a t other sites (e.g.,histidine, tyrosine). These reactions are under activeinvestigation.EXPERIMENTALThe following spectroscopic facilities were eniployed :electronic spectra, Unicam SP 800B, calibrated withholmium glass and reported as A/nm (E) for maxima; i.r.spectra, Perkin-Elmer 225, KBr disc, reported as v,,/cm-l;n.m.r. spectra, Varian A60, with tetramethylsilane asinternal reference, reported as 6 values; mass spectra,A.E.I. MS902, ionising voltage 70 eV, direct insertion,probe temperature indicated, reported as m/e values withrelative abundance in parentheses ().Petroleum refersto that fraction of light petroleum with b.p. 60-80".Solvent ratios refer to volumes.DL-N-Acetyl-N1-nitrosotryptophan Methyl Ester.--DL-h'-Acetyltryptophan methyl ester (280 mg; powder; from thecorresponding acid and diazomethane), sodium nitrite (160mg), diethyl ether (5 ml), water (1 ml), and acetic acid (0-2ml) were shaken together (3 h). The yellow crystallinesolid which formed was filtered off and washed with water.Crystallisation from ethyl acetate gave bright yellow rods( 180 mg, 55) of DL-N-acelyZ-Nl-nitrosotryplophu~ trzelhylester, m.p. 145-149" (decomp.) (Found: C, 58.05; H, 5.3;N, 14-7. C14H,amp;W@4 requires C, 58-15; H, 5-25; N,14.55), A (EtOH) 267 (14,000), 274 (11,500), and 335(7300), v 3330, 1750, 1640, 1520, 1480, 1435, 1270, 1115,765, 755, and 750, 6 (CDCI,) 8.1 (lH, m, 7-H), 7.4 (4H, m,.4rH), 6.2br (IH, NH, exchangeable slowly), 5.0 (lH, m,X H - ) , 3-69 (3H, s, OMe), 3-25 (2H, m, Ha-), and 1.98(3H, s, MeCO), mass spectrum (96") 289 (Mf, 0 * 5 ) , 260 (17),259 M - NO, 28), 201 (37), 200 ( M - NO - CO,Me,21), 157 (21), 131 (28), 130 (P-indolyl-CH,+, loo), 129 (42),103 (18), and 102 (23).~~-N-Acetyl-N~-nitrosotryPtophan.- DL-N-Acetyltrypto-phan (264 mg) and sodium nitrite (162 mg) were shaken inwater (10 ml) for 2 h.A yellow precipitate formed. Afteracidification (x-HC1, 5 ml) the organic product was extractedwithout delay into ethyl acetate (30 ml). The organic layerwas separated, dried (NaaSO,), and taken to dryness.Crystallisation of the residue from ethyl acetate gaveW. E.Noland, L. K. Smith, and K. R. Rush, J . Ovg. Chem..1966, SO, 3457. * R. Bonnett, R. Holleyhead, B. L. Johnson, and E. W.Randall, unpublished work964 J.C.S. Perkin IDL-N-acetyZ-N'-nztrosotvyptoPhan ( 159 mg, 53) as yellowplates, m.p. 137-139" (Found: C, 56-4; H, 4.6; N, 14.95.Cl,H,,N,Op requires C, 56.75; H, 4.75; N, 15-3),A (EtOH) 267 (ca. 11,000), 274 (ca. 8900), and 335 (ca. 6100),the spectrum changing with time, v 3310, 1740, 1720, 1600,1540, 1490, 1440, 1275, 1180, 1120, 755, and 750.Brieftreatment with 48 HBr-HOAc regenerated the startingmaterial paper chromatography, Whatman No, 1, irrigatedwith BunOH-H,0-0.880-NH, (270 : 30 : 3) and visualisedwith Erhlich reagent.~~-N-AcetyZ-N~-nitrosot~yPtophyZgZycine Ethyl Ester.-m-N-Acetyltryptophylglycine ethyl ester (760 nig) ,lo sodiumnitrite (597 mg), ethyl acetate (30 ml), water (10 ml), andacetic acid (1 ml) were shaken together (3 h).The ethylacetate layer was separated, the aqueous layer beingextracted with further portions of ethyl acetate (2 x 25The product gave a positive Liebermann reaction.ml). The organic extracts were dried and concentrated,the residue being crystallised from ethyl acetate-petroleumto give yellow rods (431 mg, 52) of DL-N-ucetyZ-Nl-nztvoso-tryptophyZgZycine ethy2 estev, m.p. 147-150" (Found : C,56.8; H, 5-55; N, 15.45. C,,H,,N,O, requires C, 66-65;H, 5.6; N, 1555), h (EtOH) 267 (11,000), 274 (8400), and335 (6300), Y 3240, 1745, 1665, 1625, 1540, 1480, 1425, 1275,1205, 1120, 770, 750, 735, and 710, 6 (C,D,N) 7-8.5 (rn,ArH), 5.5 (lH, m, YH-), ca. 4.25 (2H, m, -NHCH,CO-),4-08 (2H, q, -CH,O-), 3.44 (2H, in, p-indolyl-CH,), 2.02(3H, s, RleCO-), and 1.07 (3H, t, CH,CH,-).We are grateful to the Ministry of Agriculture, Fisheries,and Food for the support of this work.3/2031 Heceived, 3rd October, 19731lo A. Previeio, E. Scoffone, and C. A. Benassi, Gazzetta, 1963,98, 849