Synthesis and characterisation of some newN-nitrosodipeptides

摘要

J. CHEM. SOC. PERKIN TRANS. 1 1990 3 103 Synthesis and Characterisat ion of Some New N-Nitrosodipeptides Brian C. Challisardquo;, Jamie R. Milligan,rdquo; and Robert C. Mitchell a Chemistry Department, imperial College, London SW7 2AZ I, Smith, Kline amp; French Research Ltd, The Frythe, Welwyn, Herts AL6 9AR The synthesis of 11 new N-nitrosodipeptides by aprotic nitrosation with N,O, is described for N- (Nrsquo;-acetyl-L-prolyl)glycine, -L-alanine, -L-phenylalanine; and N-phthalimidoacetylglycine peptides and their (benzyl or ethyl) esters. The UV-vis, IR, lsquo;H NMR and MS properties of the new N- nitrosodipeptides are reported and their structural significance is analysed. The nitrosation of proteins and peptides is of interest in connection with dietary-related cancers. Nitrosation reactions proceed in the stomachrsquo; and gastric contents often contain small amounts of unidentified N-nitroso compounds which may well derive from proteins and peptides, the commonest dietary N-compounds.Further, there is a correlation between the incidence of colon cancer and dietary protein intake.3 Diazotisation of the terminal primary amino groups of proteins and peptides by nitrous acid has been known for over 80 years,4 but evidence for the nitrosation of peptide N-atoms to give N-nitroso derivatives has proven more elusive. Bonnett and his colleagues found no I5N NMR evidence for N-nitroso- peptides using aqueous HN02,5 but were able to isolate N-acetyl-N-nitrosomethionine methyl ester.6 The N-nitroso derivatives of N-acyl amino acid esters are readily obtained, however, by aprotic nitrosation.rsquo; Chow et al.prepared some N-acyl-N-nitroso-a-amino acids both aprotically * and in aqueous sol~tion,~but all were too unstable to isolate. Preliminary reports of the synthesis of N-nitrosopeptides by aprotic nitrosation and their characterisation were published in 1984.rsquo;.rsquo;rsquo; These refer to both N-acylpeptides and their ester analogues. Subsequently, it has been shown that one of these compounds, N-(N-acetyl-L-proly1)-N-nitrosoglycine(If), ex-hibits a broad spectrum of genotoxic properties.rsquo; lsquo;-I4 The synthesis and formation kinetics of several N-nitrosoprolyl peptides have also been described, but these compounds are a-substituted N-nitrosamines.rsquo; s Here the synthesis and characterisation of five N-(N-acetyl- L-proly1)-N-nitrosopeptide esters (la-e), three N-(N-acetyl-L- proly1)-N-nitrosopeptides (If-h) and three N-phthalimido-acetyl-N-nitrosoglycine derivatives (2a-c) are described in detail along with three parent un-nitrosated N-(W-acetyl-L- prolyl) peptides (5f-h).Results and Discussion The lapse of 80 years between the first,synthesis of diazopeptides and N-nitrosopeptides suggests that the terminal primary amino groups of peptides nitrosate much more readily than peptide N-atoms. It follows that N-nitrosopeptides are best obtained from terminal N-protected compounds. Preliminary experiments showed that N-nitroso derivatives of glycylglycine, for example, could not be obtained from either protic or aprotic nitrosations which readily produce the N-nitroso derivatives of simple amides.Our initial studies therefore involved the nitrosation of N-phthalimidoacetylglycineesters (3a) and (3b). These readily gave the corresponding N-nitroso derivatives (2a)and (2b)by protic and aprotic nitrosation. Compounds (2a) and (2b) were distinguished by a characteristic visible absorption triplet at A,,, = 38CL-430 nm and shifts in lsquo;H NMR and IR bands due to electron-withdrawal by the N-nitroso (1) 8; R = H, Y = OCHZPh (2) 8; Y = OEt b;R = CH3, Y = OCHZPh b;Y = OCH2Ph C; R = CHzPh, Y = OCHpPh c;Y=OH d; R= H, Y = OEt e; R = CH3, Y = OEt f; R = H, Y = OH 9; R=CH3,Y=OH h; R = CHpPh, Y = OH (3) a; Y = OEt b; Y = OCH2Ph 0 R Q+COY 0AH (5) 8; R = H Y = OCH2Ph b;R = CH3, Y = OCH2Ph C; R = CHzPh, Y = OCH2Ph d; R = H, Y = OEt e; R = CH3, Y = OEt f; R= H, Y =OH 9; R=CHa,Y=OH h; R = CH2Ph, Y = OH substituent.Attempts to remove selectively the N-phthalimide and ester functions from compounds (2a) and (2b) were unsuccessful with one exception. Thus, treatment of esters (2a) and (2b) with various acids and bases and the catalytic hydrogenolysis of (2a) invariably removed the N-nitroso group more rapidly than the protection or caused massive decomposition. Catalytic hydrogenolysis of ester (2b),however, gave acid (2c)in good yield, which showed that hydrogenolysis of the benzyl ester was more facile than the N-nitroso group. Other initial studies involving peptides bearing more labile terminal N-protection 1e.g.trityl compound (4) resulted in extensive removal of the N-protection and subsequent di- * Current address: Chemistry Department, The Open University,Walton Hall, Milton Keynes MK7 6AA. azotisation in the course of both protic and aprotic nitrosation. These findings suggested that the synthesis of authentic N-nitrosopeptides could be achieved for substrates (5a-c) where diazotisation is blocked and the benzyl ester facilitates solubility in organic solvents for aprotic nitrosation yet is removable by hydrogenolysis under mild, neutral conditions. The successful procedure for the synthesis of compounds (lf-h) is outlined in the Scheme. The peptide benzyl esters (5a-c) were synthesised by conventional procedures. Nitrosation was carried out aprotically at -10 "C using liquid N204in CH,Cl, with a 4-fold excess of NaOAc to neutralise HNO, by-product.Other solvents were investigated, but CH2Cl, proved best for reasons of solubility and volatility. After an appropriate aqueous work-up, the N-nitroso benzyl esters (la-c) were obtained as chromatographically homogenous, yellow oils in yields 90 and typically quantitative (see Experimental section). They were debenzylated by hydrogenolysis without further purification. (5a-c) N20JNaOAc (if -h) (la -c) Scheme. In contrast to the N-nitrosation step, the hydrogenolysis of (la-c) required precise conditions for success. Removal of the benzyl group without appreciable N-NO cleavage could only be achieved in either EtOH or AcOEt with palladium (5-10 on charcoal) catalyst. Hydrogenolysis was slow unless relatively large amounts of catalyst were used, and it was advantagous to follow the extent of reaction by the uptake of hydrogen to minimise unintentional N-NO cleavage.Polar solvents (e.g., HOAc, H20) not only increased the rate of hydrogenolysis but also decomposition of the N-nitrosopeptide product, as did Pd-BaSO,. Homogeneous catalysts were not examined be- cause the work-up procedures seemed likely to decompose the N-nitrosopeptides. The major impurity after hydrogenolysis was the parent dipeptide, which exemplifies the lability of the N-NO bond. After appropriate work-up, the N-nitrosopep- tides were purified by recrystallisation (see Experimental) and obtained as yellow crystalline solids in yields of 40-50.Compounds (If) and (Ig) were microanalytically pure, but (lh) could not be separated from ca. 10 of the parent dipep- tide. Apart from the elemental analyses, compounds (If-h) were charactzrised by UV-vis spectrophotometry, IR and 'H NMR spectrometry and their FAB (+ve ion) mass spectra. These results are best analysed by comparison with the parent (unnit rosated) di pept ide. UV-vis Spectra-Compounds (If-h) in EtOH showed triplet J. CHEM. SOC. PERKIN TRANS. I 1990 absorbance bands between A,,, 380 and 430 nm (log E ca. 2) which are characteristic of N-nitrosoamides (but absent in the parent peptides) and related to forbidden n-n* transitions.This property has been noted by Shuker et ~1.'~and is consistent with data for N-nitroso-a-amino acid esters reported by Djerassi et a/.' and Bonnett and Nicolaidou.6 Compounds (If-h) also showed absorbances at h,,, 238 nm (log E cu. 3.7-3.8) corresponding to an allowed n-n* transition. These are consistent with the N-nitrosopeptide structure but not charac- teristic because the Ph substituent of acid (lh) should also absorb strongly at this wavelength. IR Spectra-As Nujol mulls, compounds (If-h) show no peptide NH stretch and absence of the amide CO and I1 bands compared with the parent dipeptides. The C==Obands of N-nitrosamides are generally found at 1 730 cm-l,'* which coincides with the carboxylic acid C=O of compounds (If-h); this contrasts with an absorption at 1 660 cm-' for the parent dipeptides and manifests extensive electron withdrawal by the N-NO group.The N=O absorption is apparent at 1 510 cm-' for compounds (lf-h), which is also typical of N-nitroso- amides '* compared to 1 500 cm-'for N-nitrosoamines." The shift to higher frequencies also reflects electron withdrawal, in this instance by the C==O function. The N-acetyl C=O stretch is observed at 1 590-1 610 cm-' in compounds (If-h) and the parent dipep tides. 'H NMR Spectra.-The lack of a suitable, common solvent complicates the comparison of the 'H NMR spectra of the N-nitroso compounds (lf-h) with the parent dipeptides. Thus, compounds (If-h) were too unstable in (CD,),SO to record their 'H NMR spectra, whereas CDCl, and (CD,),CO were suitable.Conversely, the parent dipeptides were insoluble in CDCl, and (CD,),CO, but dissolved in (CD3)2S0. CDCIJ was a suitable common solvent, however, for the 'H NMR measurements of the benzyl and ethyl esters (5a-e) and their N-nitroso derivatives (la-e). Results summarised in the Table show that the protons a to the peptide CO are deshielded by ca. 1 ppm on N-nitrosation and the protons a to the peptide N-atom are deshielded by ca. 0.6 ppm. For a few results in the Table where overlapping signals are unresolved at 60 MHz, the uncertainties in the chemical shift differences are as high as 0.3 ppm. In most cases, however, the uncertainty was eliminated by correlating signal multiplicities.A pair of compounds, where precise assignment proved impossible, was N-(N'-acetyl-L-proly1)-N-nitroso-L-phenylalaninebenzyl ester (lc) and its parent peptide (5).Nonetheless, the results in the Table show that 'H NMR deshielding effects in compounds (la-h) relative to the parent dipeptide are in good agreement with data for a-amino acid esters reported by Shuker et ~1.'~and by Bonnett and Nicolaidou.6 Muss Spectra-In common with simple N-nitrosamines, the molecular ion is unobservable in the electron-impact spectra of the N-nitrosopeptides (If-h) because of the ready fragmentation of the N-NO bond. Good results were obtained for compounds (If) and (lg),however, by fast atom bombardment (FAB) tech- niques in the positive ion mode using a glycerol-water matrix.The most important features from a structural con- firmation standpoint were relatively weak (7) MH' ions but stronger ions resulting from the loss of both the NO and CH2C0 groups. Four other ions with m/z 140, 112, 70 (loo), and 43 were prominent in both the FAB (positive ion) mass spectra of acids (If) and (lg) and the FAB and electron impact spectra of the parent dipeptides and their ester derivatives. These are assigned the structures (6) to (9), respectively. Our findings agree with recently published results 2o for compound (lf).19 J. CHEM. SOC. PERKIN TRANS. 1 1990 3 105 Table. Etrect of N-nitrosationon the 'HNMR deshielding of protons adjacent to the peptide C--O group (6HA) and N-atom (6HB): A6H = 6H (N-nitrosopeptide) -6H (peptide).Compound Solvent 6HA 6HB A6HA A6HB (CD3)2S0 4.1 14.39 (CD3)2C-* 5.5-5.9 CDCl, 4.33-4.63 CDCl, 5.49-5.78 CDCI, 4.5-4.8 CDCl, 5.6-5.8 (CD,),SO 4.0-4.4 CDCl 5.29 CDC1, 4.57 CDCI, 5.70 CDC1, 4.1-4.8 CDCI, 5.58 (CD3)2S0 3.98-4.70 CDCl, 5.31-5.78 CDCI, 4.31-4.94 CDCI, 5.30-5.69 (6) mlz 140 (7) mlz 112 (8)mlz70 CHamp; O+ (9)rntz43 Experimental M.p.s were measured on a Gallenkamp hot-stage and are uncorrected. IR spectra were recorded on a Perkin-Elmer 298 grating spectrometer and calibrated against polystyrene. 'H NMR spectra were recorded on Varian EM-360A and JEOL FX-90Q spectrometers in the solvent indicated, with tetra- methylsilane as internal standard. UV and visible spectra were recorded on Pye-Unicam SP8-500, Cecil CE599, and Perkin- Elmer 555 spectrophotometers.Mass spectra were obtained with a VG7070 instrument. Amino acids, peptides, benzyl chloroformate, ethyl chloroformate, acetic anhydride, and toluene-4-sulphonic acid were obtained from commercial sources and used as supplied. N204 (99 Matheson) was also used without further purification. Other reagents and solvents were purified by standard procedures.21 N-Acetyl-L-proline (lO).-L-Proline (5.76 g, 50 mmol) and toluene-4-sulphonic acid monohydrate (10.46 g, 55 mmol) were suspended in a mixture of benzene (30 cm3) and benzyl alcohol (30 cm3). On heating, a pale yellow colour developed. The solution was heated under reflux using a Dean and Stark trap until azeotropic removal of water was complete (ca.10 h). The remaining solution was diluted with ether (300 cm3), the oily lower phase was isolated by decantation, and dissolved in CH2C12 (70 cm3) containing Et3N (5.06 g, 50 mmol). The solution was cooled to -5 OC, treated with a solution of Ac,O (5.10 g, 50 mmol) in CH,C12 (10 cm3), and then washed successively with dilute HCl(0.01~; 20 cm3), satd. aq. NaHCO, (20 cm3), water (20 cm3) and saturated brine (20 cm3) and then dried (MgSO,). Removal of solvent under reduced pressure left N-acetyl-L-proline benzyl ester (11) as a mobile 3.55-3.82 1.45 0.92 4.6 3.8 1-3.92 1.16 0.54 4.4 3.95-4.10 1.05 0.58 4.6 404.4 1.09 0.32 4.52 4.43 1.13 0.76 5.19 4.1-4.8 1.13 0.74 5.19 3.98-4.70 1.21 0.26 4.37-4.83 4.31-4.94 0.87 0.87 5.30-5.69 yellow oil (10.1 g, 82) which was purified by vacuum distillation, b.p.100 OC at 7 x mmHg (Found: C, 67.9; H, 7.2; N, 5.65. Cl4HI7NO3 requires C, 68.00, H, 6.93; N, 5.66); v,,,(CCl,) 1750 (ester CO) and 1660 cm-' (amide CO); GH(CDC1,) 2.02 (4 H, m, 3-H2 and 4-H2), 2.10 (3 H, s, Ac), 3.60 (2 H, m, 5-H2), 4.50 (1 H, m, 2-H), 5.18 (2 H, s, CH,Ph), and 7.29 (5 H, s, Ph); m/z (electron impact) 247 (9, M+), 112 (61, M-C02CH2Ph), 91 (17, C7H7+) and 70 (100, M -AcC0,-CH2Ph). A solution of benzyl ester (11) (9.40 g, 38 mmol) in absolute EtOH (40 cm') containing 5 Pd-C (100 mg) was stirred under 760 mmHg of hydrogen.After cessation of H, uptake (ca. 1 h), the catalyst was removed by filtration, and the filtrate concentrated under vacuum to give a white solid, which was recrystallized from water to give N-acetyl-L-proline (10) (4.68 g, 78); m.p. 89-90deg;C (Found: C, 53.05; H, 6.95; N, 9.2. C7H,,N03 requires C, 53.49; H, 7.04; N, 8.91); v,,,(Nujol) 3 000-2 500 (CO,H), 1 730 (acid CO), and 1 610 cm-I (amide CO);amp;(CDC13) 2.10 (4 H, m, 3-H2 and 4-H2), 2.19 (3 H, S, Ac), 3.55 (2 H, m, 5-H,), 4.60 (1 H, m, 2-H), and 9.21 (1 H, br s, C0,H). N-(N'-Acetyl-L-proly1)-a-amino Acid Benzyl Esters.-All were prepared from N-acetyl-L-proline (10) and the appropriate a-amino acid benzyl ester by mixed anhydride coupling. The following procedure for N-(N-acetyl-L-proly1)glycine benzyl ester (5a) is exemplary.N-(N'- Acetyl-L-proly1)glycine Benzyl Ester (5a).-A solution of N-acetyl-L-proline (lo), 1.57 g, 10 mmol and Et3N (1.12 g, 10 mmol) in dry THF (30 cm3) at -10 "C was treated with a solution of ethyl chloroformate (0.87 g, 8 mmol) in dry THF (6 cm3). The resulting suspension was stirred at -10deg;C for 30 min, then treated with glycine benzyl ester (1.32 g, 8 mmol) and allowed to reach room temperature. After removal of solvent under reduced pressure, the residue was dissolved in CH2C12 (60 cm3) and washed successively with dilute HCl (0.01~; 20 cm3), saturated aqueous NaHCO, (20 cm3), water (20 cm3), and saturated brine (20 cm3), and then dried (MgS04). Removal of the solvent under reduced pressure gave N-(W-acetyl-L-proly1)glycine benzyl ester (5a) as a colourless oil (2.15 g, 71); v,,,(CCl,) 3 305 (NH), 1 750 (ester CO), 1 690 (amide co),and 1620 cm-' (amide co);GH(CDC1,) 2.10 (3 H, s, Ac), 1.7-2.6 (4 H, m, 3-H2 and 4-H,), 3.3-3.9 (2 H, m, 5-H,), 3.95 and 4.10 (2 H, s, CH,CO,CH,Ph), 4.5-4.8 (1 H, m, 2-H), 5.10 (2 H, s, CH,Ph), 7.30 (5 H, s, Ph), and 7.60 (1 H, br s, NH).N-(N'-Acetyl-L-proly1)glycine Ethyl Ester (5d). As for (5a) using glycine ethyl ester in place of the benzyl ester. N-(N-Acetyl-L-proly1)glycine ethyl ester (Sa) was obtained as a colourless oil in 81 yield; v,,,(CCI,) 3 300 (NH), 1 750 (ester CO), 1 690 (amide CO), and 1 640 cm-' (amide CO); G,(CDC13) 1.20 (3 H, t, J 8 Hz, C02CH,CH3), 2.06 (3 H, S, Ac), 1.62-2.51 (4 H, m, 3-H2 and 4-H,), 3.20-3.77 (2 H, m, 5-H,),3.81 and 3.92 (2 H, S, CH,CO,Et), 4.08 (2 H, 9, J 8 Hz, COZCH,CH3), 4.33- 4.63 (1 H, m, 2-H), and 7.35 (1 H, br s, NH); m/z (electron impact) 242 (5, M') 197 (2, M -OEt), 185 (3,175 (4), 167 (2), 142 (20, M -NHCH,COzEt), 112 (86, M -CONHCHZ-CO,Et), 102 (70), 92 (21), 70 (100, M -AC -CONHCHZ-CO,Et), and 43 (47, Ac).N-(N'-Acetyl-~-prolyl)-~-alanineBenzyl Ester (Sb). From N-acetyl-L-proline (lo); 0.19 g, 1.2 mmol and L-alanine benzyl ester (0.08 g, 1 mmol) as for (5a) above. N-(N-Acetyl-L- proly1)-L-alanine benzyl ester (Sb)was isolated as a white solid after recrystallisation from ethyl acetate (270 mg, 85); m.p. 113 "C (Found C, 64.1; H, 7.0; N, 8.75.C17H2,N204 requires C, 64.13; H, 6.97; N, 8.80); vmax(Nujol) 3 320 (NH), 1 745 (ester CO), 1 660 (amide CO), 1 630 (amide CO), 1 535 (amide 11), 760 and 705 cm-' (Ph); GH(CDC13) 1.36 (2 H, m, 5-H,), 1.6-2.5 (4 H, m, 4-H, and 3-H,), 2.06 (3 H, s, Ac), 3.47 (2 H, m, 5-H,), 4.1-4.8 (2 H, m, 2-H and CHMe), 5.11 (2 H, s, C02CH2Ph), and 7.27 (5 H, s, Ph); m/z (electron impact) 318 (5, M+), 227 (1, M -CHZPh), 210 (l), 183 (2, M -C02CH2Ph), 168 (l), 151 (4), 140 (57, M-NHCHMeC0,- CH,Ph), 112 (57, M -CONHCHMeCO,CH,Ph), 91 (2, C7H7+), 70 (100, M -Ac -CONHCHMeCO,CH,Ph), and 43 (1 6, Ac). N-(N'-Acetyl-L-proly1)-L-alanine Ethyl Ester (5).As for (5b) using L-alanine ethyl ester in place of the benzyl ester. N-(N-Acetyl-L-proly1)-L-alanine ethyl ester (5e) was obtained as a white solid in 79 yield after recrystallisation from EtOAc; m.p.79-80deg;C (Found: C, 56.4; H, 7.9; N, 10.95. C12H20N204 requires C, 56.24; H, 7.86; N, 10.93); vmax(Nujol) 3 320 and 3 260 (NH), 1 740 (ester CO), 1 675 (amide CO), 1 640 (amide co), and 1 540 cm-' (amide 11); GH(CDC13) 1.23 (3 H, t, J 7 Hz, C02CH2CH3), 1.34 (3 H, d, J 7 Hz, CHMe), 2.05 (3 H, s, Ac), 1.8-2.4 (4 H, m, 3-H2 and 4-H,), 3.49 (2 H, m, 5-H,), 4.1 1 (2 H, q, J 7 Hz, CO,CH,Me), 4.43 (1 H, q, J 7 Hz, CHMe), and 4.57 (1 H, m, 2-H); m/z (electron impact) 256 (9, M+), 140 (14, M -NHCHMeCO,Et), 113 (31), 112 (77, M -CONHCHMeCO,Et),70( 100,M -Ac -CONHCHMeC0,-Et), and 43 (1 7, Ac). N-(N'-Acetyl-L-proly1)-L-phenylalanine Benzyl Ester (5c).From N-acetyl-L-proline (6);0.78 g, 5 mmol and L-phenyl- alanine benzyl ester (1.02 g, 4 mmol) as for (5a) above. N-(W-Acet yl-L-proly1)-L-phenylalanine benzyl ester (5c) was obtained as a white solid after recrystallisation from ethyl acetate (1.31 g, 83), m.p. 104deg;C (Found: C, 69.95; H, 6.7; N, 7.05. C23H,,N,04 requires C, 70.03; H, 6.64; N, 7.10); vmax(Nujol) 3 210 (amide NH), 1 745 (ester CO), 1 690 (amide CO), 1 620 (amide I), 1 570 (amide 11), and 755 and 710 cm-' (Ph); G,(CDCI,) 1.96 (3 H, s, Ac), 1.53-2.25 (4 H, m, 3-H, and 4- H2), 2.87-3.46 (4 H, m, 5-H2 and CHCH,Ph), 4.31-4.94 (2 H, m, 2-H and CHCH,Ph), 5.08 (2 H, s, C02CH2Ph), 6.74-7.38 (10 H, m, Ph), and 7.50 (1 H, br s, NH); m/z (electron impact) 394 (2, M+), 319 (30), 140 6, M -NHCH(CH,Ph)C02-CHZPh, 112 60, M -CONHCH(CH2Ph)C02CH,Ph, 108 (13), 91 (25, C7H7+), 77 (16, Ph+), 70 loo, M -AC -CONHCH(CH,Ph)CO,CH,Ph, and 43 (15, Ac).N-(N'-Acefyl-L-prolyf) Peptides (5f-h).-These were pre-pared from the corresponding benzyl esters (5a-c) by hydrogen- olysis in absolute EtOH over 5 Pd-C, as described above for the conversion of (10) into (11). N-("-A cetyl-L-prolyl)gIycine (Sf). After complete uptake of hydrogen (ca. 1 h), the catalyst was filtered off and washed with J. CHEM. SOC. PERKIN TRANS. I 1990 water to dissolve out (50, which is only sparingly soluble in alcohol. The combined filtrate and washings were concentrated by vacuum rotary evaporation and the residue was recrys- tallised from water to give N-(N-acetyl-L-prolylglycine(Sf) as a white, crystalline solid (253 mg, 83); m.p.206-207 "C (decomp.) (Found C, 50.7; H, 6.6; N, 12.95. C9H14N,04 requires C, 50.46; H, 6.59; N, 13.08); vmax(Nujol) 3 320 (NH), 3 200-2 100 (CO,H), 1 740 (acid CO), 1 655 (amide CO), 1 610 (amide CO), and 1 540 cm-I (amide 11); GH(CD3),SO) 1.86 and 1.94 (3 H, s, Ac), 1.60-2.13 (4 H, m, 3-H, and 4-H,), 3.08-3.55 (2 H, m, 5-H,), 3.55-3.82 (2 H, m, CH,CO,H), 4.11-4.39 (1 H, m, 2-H), and 8.19 (1 H, br s, NH). N-(N'-Acetyl-L-proly1)-L-alanine(5g). From N-(W-acetyl-L- proly1)-L-alanine benzyl ester (Sb) (640 mg, 2 mmol) to give, after recrystallisation from EtOH, N-(W-acetyl-L-prolyl)-L-alanine (5g) as a white solid (320 mg, 70); m.p. 255-257 "C (Found: C, 52.65; H, 7.15; N, 12.15.C10H16N204 requires C, 52.62; H, 7.07; N, 12.27); v,,,(Nujol) 3 350 and 3 310 (NH), 3 200-2 400 (CO,H), 1 735 (acid CO), 1 660 (amide CO), 1 610 (amide CO), 1560 cm-' (amide 11); GH(CD3),S0 1.26 and 1.28 (3 H, d, J5 Hz, CHMe), 1.83 and 1.95 (3 H, s, Ac), 1.6-2.3 (4 H, m, 3-H, and 4-H,), 3.1-3.7 (2 H, m, 5-H,), 4.0-4.4 (2 H, m, 2-H and CHMe), 8.1 1-8.35 (1 H, d, J 6 Hz, NH), and 1 1.8- 12.9 (1 H, S, C02H). N-(N'-Acetyl-L-proly1)-L-phenylalanine (5h). From N-(W-acetyl-L-prolyl-L-phenylalaninebenzyl ester (5)(395 mg, 1 mmol) to give, after recrystallisation from a mixture of MeOH and EtOH, N-(N-acetyl-L-proly1)-L-phenylalanine(5h) as a white solid (270 mg, 82); m.p. 201-203 "C (Found: C, 63.1; H, 6.5; N, 9.15.C16H2oN2O4 requires C, 63.14; H, 6.62; N, 9.20); vmax(Nujol)3 320 (NH), 3 100-2 200 (CO,H), 1 735 (acid CO), 1 660 (amide CO), 1 595 (arom. C=C), 1 550 (amide 11), and 705 cm-' (Ph); ~H(CD~),SO 1.41-2.05 (7 H, m, Ac, 3-H2, and 4-H,), 2.70-3.68 (4 H, m, 5-H and CH,Ph), 3.98-4.70 (2 H, m, 2-H and CHCH,Ph), and 7.23 (5 H, s, Ph). N-("-Acetyl-L-proly1)-N-nitroso Peptides (la-h).-These were synthesised by aprotic nitrosation (using N204) of the appropriate ester (Sa-e), followed by removal of the benzyl group by hydrogenolysis over Pd-C catalyst to obtain (If-h). N-(N'-Acetyl-L-proly1)-N-nitrosoglycine(If). To a mixture of N-(N-acetyl-L-proly1)glycinebenzyl ester (Sa) (1.52 g, 5 mmol) and anhydrous sodium acetate (1.64 g, 20 mmol) in dry CH2Cl, (30 cm3) at -10 "C was added N204 (340 p1,5.5 mmol) over 1 min.The suspension was stirred for 30 min at -lOdeg;C, then diluted with water (50 cm3). The organic phase was separated, washed successively with 5 aq. Na,CO, (4 x 20 cm3), water (2 x 20 cm3), and brine (20 cm3), and then dried (MgS04). Removal of the solvent under reduced pressure gave N-(N-acetyl-L-proly1)-N-nitrosoglycinebenzyl ester (la) as a yellow- orange oil (1.62 g, 97); hma,(EtOH) 369 (E 32 dm3 mol-' cm-'), 386 (66), 402 (108), and 421 nm (107); v,,,(CCI,) 1 750 (ester CO and nitrosamide CO), 1 660 (amide CO), and 1 510 cm-' (NO); amp;(CDC13) 2.20 (3 H, S, Ac), 1.7-2.6 (4 H, m, 3-H, and 4-H,), 3.5-3.9 (2 H, m, 5-H,), 4.60 (2 H, s, CH2C02CH2Ph), 5.10 (2 H, s, CH,Ph), 5.6-5.8 (1 H, m, 2-H), and 7.30 (5 H, s, Ph).The benzyl ester (la) (667 mg, 2 mmol) in absolute EtOH (20 cm3) containing 5 Pd-C (50 mg) was stirred at room temperature under 760 mmHg of hydrogen. When the uptake of hydrogen was complete (ca.1 h), the catalyst was removed by filtration and the EtOH removed under reduced pressure and replaced by EtOAc (10 cm3). On addition of light petroleum (35 cm3, b.p. 40-60 "C) a yellow precipitate formed, which was recrystallised from EtOAc at 60 "C to give N-(N-acetyl-L- proly1)-N-nitrosoglycine (If) as a yellow crystalline solid (2 10 mg, 4373, m.p. 110deg;C (Found: C, 44.55; H, 5.3; N, 17.1. C9H13N305 requires C, 44.45; H, 5.39; N, 17.28); h,,,- J. CHEM. SOC. PERKIN TRANS.1 1990 (EtOH) 389 (E 53 dm3 mol-' cm-'), 403 (88), and 426 nm (91); v,,,(Nujol) 3 200-2 300 (CO,H), 1 740 (nitrosamide CO), 1 720 (acid CO), 1590 (amide CO), and 1510 cm-' (NO); ~H(CD,)~CO2.10 (3 H, S, Ac), 1.7-2.6 (4 H, m, 3-H2 and 4- H,), 3.4-3.8 (2 H, m, 5-H2), 4.60 (2 H, s, CH2C02H), and 5.5-5.9 (1 H, m, 2-H); m/z(FAB +ve ion) 244 (774, MH'), 180 (3), 171 (6, M -NO -CH,CO), 158 (23), 140 66, M -N(NO)CH2-COZH, 112 77, M -CON(NO)CH2C02H19 70 loo, M -Ac -CON(NO)CH2CO2H), and 43 (19, Ac). N-(N'-Acetyl-L-proly1)-N-nitroso-L-alanine(lg). From N-(N-acetyl-L-proly1)-L-alaninebenzyl ester (Sb) (159 mg, 0.5 mmol) as for (5a) above to give N-(N-acetyl-L-proly1)-N-nitroso-L-alanine benzyl ester (lb) as a yellow-orange oil (175 mg, 97); h,,,(EtOH) 405 (E 33 dm3 mol-' cm-') and 423 nm (30); v,,,(CCl,) 1 750 (ester CO and nitrosamide CO), 1 660 (amide CO), and 1 510 cm-' (NO); G,(CDCl,), 1.35 (3 H, d, J 7 Hz, CHMe), 2.01 (4 H, m, 3-H, and 4-H,), 2.09 (3 H, s, Ac), 3.61 (2 H, m, 5-H,),5.04 (2 H, s, CO,CH,Ph), 5.19 (1 H, q, J 7 Hz, CHMe), 5.58 (1 H, m, 2-H), and 7.18 (5 H, m, Ph).The benzyl ester group of (lb) (518 mg, 1.5 mmol) was removed by catalytic hydrogenolysis as for (la) above. In this case, however, the product was precipitated from EtOAc by the addition of ether and recrystallised from EtOAc-ether to give N-(N-acetyl-L-proly1)-N-nitroso-L-alanine(lg) as a yellow solid (180 mg, 47); m.p. 109 OC (Found: C, 46.55; H, 5.8; N, 15.65. C,,H,,N,O, requires C, 46.69; H, 5.88; N, 16.33); v,,,(Nujol) 3 3W2 200 (CO,H), 1 750 (acid CO and nitros- amide CO), 1600 (amide CO), and 1510 cm-' (NO); 6,-(CDCI,) 1.26 (3 H, d, J 7 Hz, CHMe), 1.80-2.35 (4 H, m, 3-H, and 4-H,), 2.23 (3 H, s, Ac), 3.40-3.70 (2 H, m, 5-H,), 4.52 (1 H, q, J 7 Hz, CHMe), and 5.29 (1 H, m, 2-H); m/z (FAB +ve ion) 259 (6, MH'), 185 (15, M -NO -CHZCO), 158 (23), 140 57, M -N(NO)CH,CO,H, 112 64, M -CON(N0)-CH,CO,H, 70 loo, M -Ac -CON(NO)CH,CO,H, and 43 (16, Ac).N-(N'-A cetyl-L-proly1)-N-nitroso-L-phenylalanine(1h). From N-(N'-acetyl-L-phenylalanine benzyl ester (5c) (395 mg, 1 mmol) as for (5a) above to give N-(N-acetyl-L-proly1)-N-nitroso-L-phenylalanine benzyl ester (lc) as a thick yellow oil (415 mg, 98); h,,,(EtOH) 391 (E 35 dm3 mol-' cm-'), 406 (48), and 427 nm (47); v,,,(CCl,) 1 745 (ester CO and nitrosamide CO), 1 655 (amide CO), 1 515 (NO), and 700 cm-' (Ph); 6, (CDCl,) 1.33-2.17 (4 H, m, 5-H2 and CHCH,Ph), 5.02 (2 H, s, C02CH2 Ph), 5.30-5.69 (2 H, m, 2-H and CHCH,Ph), and 6.76-7.38 (10 H, m, Ph); m/z (FAB +ve ion) 140 13, A4 -N(NO)CH(CH2Ph)CO,CH2Ph, 112 38, M -CON-(NO)CH(CH2Ph)CO2CH,Ph, 91 (63, C7H7'), 70 97, M -Ac -CON(NO)CH(CH2Ph)C0,CH,Phl, 52 (loo), and 39 (32).The benzyl ester group was removed from (lc) (850 mg, 2 mmol) by catalytic hydrogenolysis as for (la) above.N-(N'-Acetyl-L-proly1)-N-nitroso-L-phenylalanine(1h) was isolated as a yellow solid following recrystallization from EtOAc-light petroleum (225 mg, 34); m.p. 177 "C; v,,,(Nujol) 3 500-2 300 (C02H), 1735 (acid CO and nitrosamide CO), 1610 (amide co), 1 520 (NO), and 705 cm-' (Ph); 6,(CDCI3) 1.52-2.34 (4 H, m, 3-H2 and 4-H2),2.20 (3 H, s, Ac), 2.90-3.78 (4 H, m, CH,Ph and 5-H2),4.374.83 (1 H, m, CHCO,H), 5.31-5.78 (1 H, m, 2- H), 6.767.44 (5 H, m, Ph), and 920 (1 H, br s, C0,H).N-(N'-Acetyl-L-proly1)-N-nitrosoglycine ethyl ester (Id).From N-(N'-acetyl-L-proly1)-N-glycineethyl ester (5d) as for (5a) above to give N-(W-acetyl-L-proly1)-N-nitrosoglycine ethyl ester (la) as a thick yellow oil (70 mg, 98); v,,,(CCl,) 1 740 (ester CO and nitrosamide CO), 1 650 (amide CO), and 1 510 cm-' (NO); 6,(CDCl,), 1.20 (3 H, t, J 7 Hz, CO2- CHZCH,), 2.01 (3 H, s, Ac), 1.69-2.38 (4 H, m, 3-H, and 4-H2), 3.42-3.83 (2 H, m, 5-H2), 4.07 (2 H, 9, J 7 Hz, C02CH2CH3), 4.40 (2 H, s, CH2C02Et), and 5.4-5.78 (1 H, m, 2-H).N-(N'-Acetyl-L-proly1)-N-nitroso-L-alanineethyl ester (le). From N-(N'-acetyl-L-proly1)-L-alanineethyl ester (5e) as for (5a) above to give N-(N-acetyl-L-proly1)-N-nitroso-L-alanine ethyl ester (le) as a thick, yellow oil (68 mg, 98); h,,,(EtOH) 394 (E 44dm3 mol-' cm-'), 407 (70), and 428 nm (72); v,,,(CCl,) 1 745 (ester CO and nitrosamide CO), 1 660 (amide CO), and 1510 cm-I (NO); 6~(cDCl3) 1.18 (3 H, t, J 7 Hz, C02CH2- CH3), 1.32(3H,d,J7Hz,CHMe),2.13(3H,sAc),1.9-2.5(4H, m, 3-H2 and 4-H2), 3.72 (2 H, m, 5-H,), 4.10 (2 H, q, J 7 Hz, C02CH2CH3),5.19 (1 H, q, J 7 Hz, CHMe), and 5.70 (1 H, m, 2-H); m/z (FAB +ve ion) 286 (0.3, MH'), 158 (17), 140 34, M -N(NO)CHMeCO,Et, 112 75, M -CON(N0)-CHMeCO,Et, and 70 loo, A4 -Ac -CON(N0)CHMe-CO,E t .N-Phthalimidoacetyl-N-nitrosoglycineEsters (2a-b).-These were also prepared by the aprotic nitrosation (using N204) of the corresponding ester, which was obtained by coupling phthalimidoacetic acid with glycine ester in the presence of DCC. N-Phthalimidoacetylglycineethyl ester (3a). To a solution of phthalimidoacetic acid (2.05 g, 10 mmol) in dry THF (30 cm3) was added a solution of DCC (2.27 g, 11 mmol) also in dry THF (10 cm3) followed by a solution of glycine ethyl ester (1.03 g, 10 mmol) in dry THF (10 cm3). A white precipitate formed immediately, and the suspension was stirred at room temperature under N, for 4 h. Glacial HOAc (1 cm3) was then added, and the solvent was removed under reduced pressure and replaced by CH,Cl, (50 cm3).The precipitated di-cyclohexylurea was removed by filtration, and the filtrate was washed with water (6 x 10 cm3) and dried (MgSO,). Solvent removal under reduced pressure left a white solid which was recrystallised from EtOH to give N-phthalimodoacetylglycine ethyl ester (3a) (2.75 g, 95); m.p. 192 "C (Found: C, 58.0; H, 4.85; N, 9.55. C1,H,,N2O5 requires C, 57.93; H, 4.86; N, 9.65); vmax(Nujol) 3 290 (NH), 1 725 (imide CO and ester CO), 1 7 15 (imide CO), and 1 645 cm-' (amide CO); 6,(CDCl,) 1.24 (3 H, t, J 7 Hz, C02CH2CH,), 4.01 and 4.07 (2 H, s, NHCH,CO,Et), 4.35 (2 H, q, J 7 Hz, C02CH,CH,), and 7.7-8.0 (5 H, m, Ph and CONH); m/z (electron impact) 290 (13, M+), 217 (15, A4 -CO,Et), 188 (27, M -NHCH,CO,Et), and 161 (100).N-Phthalimidoacetyl-N-nitrosoglycineethyl ester (2a).To a mixture of N-phthalimidoacetylglycineethyl ester (3)(870 mg, 3 mmol) and anhydrous NaOAc (0.98 g, 12 mmol) in dry CH,Cl, (25 cm3) at -10 "C was added liquid N204 (220 pl, 3.5 mmol) over 1 min. The suspension was stirred for 30 min at -10 OC, then diluted with water (40 cm3). The organic phase was separated, washed successively with 5 aq. NaHCO, (3 x 20 cm3), water (2 x 20 cm3), and brine (20 cm3), and then dried (MgSO,). Solvent removal under reduced pressure left a yellow solid, which was recrystallised from ether to give N-phthalimidoacetyl-N- nitrosoglycine ethyl ester (2a) as yellow crystals (805 mg, 84); m.p. 117-1 18 "c(Found: C, 52.55; H, 4.0; N, 13.1.C,,H,,N,O, requires C, 52.67; H, 4.10 N, 13.16); h,,,(Et,O) 404 (E 100 dm3 mol-* cm-') and 423 nm (110); v,,,(Nujol) 1 765 (imide CO), 1 730 (ester CO, nitrosamide CO, and imide CO), and 1 500 cm-' (NO); 6,(CDC13) 1.23 (3 H, t, J7 Hz, C02CH,Me), 4.1 5 (2 H, q, J 7 Hz, CO,CH,Me), 4.49 (2 H, s, CH2C02Et), 5.33 (2 H, s, CH,CON(NO), and 7.78 (4 H, m, ArH). N-Phthalimidoacetylglycine benzyl ester (3b). As for (3a) using glycine benzyl ester (1.65 g, 10 mmol). After recrystal- lization from EtOH, N-phthalimidoacetylglycinebenzyl ester (3b) was obtained as a white solid (2.54 g, 72); m.p. 158 "C (Found: C, 64.9; H, 4.55; N, 7.95. CI9Hl6N5o5 requires C, 64.77; H, 4.58; N, 7.95); vmax(Nujo1) 3 320 (NH), 1 730 (ester CO and imide CO), 1 675 (amide CO), 1 570 (amide II), and 760, 720 and 705 cm-' (Ph); 6,(CDCl,) 3.98 and 4.06 (2 H, s, CHZCOZCHZPh), 4.31 (2 H, S, CHZCONH), 5.11 (2 H, S, C02CH2Ph),7.26 (5 H, s, Ph), and 7.61-7.82 (5 H, m, ArH and CONH).N-Phthalimidoace ty I-N-n itrosoglycine benzy f ester (2b). As for (2a) from the benzyl ester (3b) (96 mg, 300 pmol). After recrystallization from MeOH, N-phthalimidoacetyl-N-nitroso-glycine benzyl ester (2b) was obtained as a yellow solid (97 mg, 92); m.p. 119 "C (Found: C, 59.2; H, 4.2; N, 11.35. CI9Hl5N3O6 requires C, 59.84; H, 3.97; N, 11.02); h,,,(Et,O) 386 (E 67 dm3 mol-' cm-I), 403 (110), and 422 nm (116); v,,,(Nujol) 1 735 (ester CO, nitrosamide CO, and imide CO), 1520 (NO), and 720 cm-' (Ph); G,(CDCl,) 4.47 (2 H, s, CH2C02CH,Ph), 5.03 (2 H, S, CH2CON(NO), 5.28 (2 H, S, C02CH2Ph),7.25 (5 H, s, Ph), and 7.63-7.89 (4 H, m, ArH). N-(N'-7'riphenyfmethyfgfycyf)gfycine Benzyf Ester (4).A suspension of N-glycylglycine (3.30 g, 25 mmol) and toluene-4- sulphonic acid monohydrate (4.95 g, 26 mmol) in a mixture of benzyl alcohol (25 cm3) and benzene (30 cm3) was heated at 60 "C to form a yellow solution. The solution was heated under reflux with a Dean and Stark trap until azeotropic removal of water was complete (ca. 12 h). The resulting solution was allowed to reach ambient temperature, diluted with CH2Cl, (75 cm3), and the resulting white precipitate was collected and air dried, then suspended in CH2C12 (30 cm3) and treated with Et3N (2.5 g, 25 mmol), to form a clear solution.A solution of chlorotriphenylmethane (6.8 g, 25 mmol) in dry CH,CI2 (10 cm3) was then added over 10 min and the resulting solution allowed to stand under argon for 20 h at ambient temperature. The solution was then washed with water (3 x 30 cm3)and dried (Na,SO,). Solvent removal under reduced pressure followed by recrystallization from EtOH gave N-(N-triphenyl- methylglycy1)glycine benzyl ester (4) as a white solid (5.8 g, 50); m.p. 152-153 "C (Found: C, 77.6; H, 6.05; N, 6.0. C30H28NZ03requires C, 77.56; H, 6.07; N, 6.03); vmax(Nujol) 3 320 (NH), 1 740 (ester CO), 1 650 (amide I), and 750,720,710 and 700 cm-' (Ph); GH(CDC13) 2.20 (1 H, br s, D20 exch., Ph,CNH), 2.98 (2 H, s, NHCH,CONH), 4.06 and 4.15 (2 H, s, CH2C02CH,Ph), 5.19 (2 H, s, C02CH2Ph), and 7.1-7.4 (21 H, m, 4 x Ph and CONH); m/z (electron impact) 464 (3, M'), 387 (30, M -Ph), 243 (100, PhSC'), 165 (23, Ph3C' -PhH), and 91 (25, C7H7+).Acknowledgements We thank the SERC and Smith, Kline amp; French Research Ltd., for a CASE studentship to J. R. M. J. CHEM. SOC. PERKIN TRANS. i 1990 References 1 For example, the formation of N-nitrosoproline: H. Ohshima and H. Bartsch, Cancer Res., 1981,41,3658. 2 B. Pignatelli, I. Richard, M-C. Bourgade, and H. 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Perrin, 'Purification of Laboratory Chemicals,' 2nd edition, Pergamon, Oxford, 1980. Paper 0/01298J Received 26th March 1990 Accepted 4th May 1990