5446 only with vigorous shaking; no color ever developed. The tube was opened and the contents were dissolved in 50 ml of acetone.
ether-pentane eluent, uv developer), and ir, and had a specific rotation of [cx]*~D -100" (c 0.65, chloroform), >go% optically
The solution was filtered, 100 ml of 30-60" petroleum ether was added, and the precipitated polymer was collected, washed well with petroleum ether, and thoroughly dried under vacuum. Isolated was 70 mg (4.2%) of optically inactive white material identical with known polymethyl methacrylate by ir. The polymer was analyzed for sulfur and nitrogen: S, 0.00 i 0.20%; N, 0.00 =k 0.20z;. An average molecular weight of 4620 was determined osmometrically by Galbraith Laboratories. The acetone-petroleum ether soluble portion (contaminated by about 10% of IX). which was pure by proton nmr and glpc. NBD [(C6H5)3P]3RhC1. A mixture of 10 g (108 mmol) of NBD (c). V [(C6H5)3P]3RhC1. A tube containing 150 mg of and 964 mg (1.03 mmol) of [(C6H&P],RhC1 was refluxed under [(C6H5)3P]3RhC1and 300 mg of V was shaken at 95" for 4 days. nitrogen for 4 days. Dilution with hexane precipitated the rhodium Proton nmr of the reaction mixture showed only starting material. complex (295 mg). 36 Concentrating the filtrate and chromatography (d). V [(C6Hj),P]Rh(NBD)Cl. A tube containing 50 mg of [(C6H,)3P]Rh(NBD)Cland 300 mg of V was shaken at 95" for 4 days. Proton nmr of the reaction mixture showed only starting (38) The procedure is modeled on that of D. H. R. Barton and D. material. Elad, J . Chem. Soc., 2085 (1956). (e). XI11 [(C8H1&RhC1I2 (C6Hd3P. A tube containing 27 (39) Prepared as described in ref 19c, m p (vac) 295-300" dec (lit. dec, 195-200°,19a and f95-197"19d). mg of [(CsH14)zRhC1]2, 40 mg of (C6Hj),P, and 162 mg of XI11 was
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Journal of fhe American Chemical Society ] 94.15
July 26, 1972
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5455 were separated by glpc (column A, 240") and are tentatively assigned structures VI11 and XXV, respectively. WII. Ir (neat, cm-') 3054 s, 3012 s, 2942 s, 2880 s, 2870 s, 1610 w, 1587 m, 1489 S, 1459 m, 1352 s, 1327 m, 1317 m, 1306 s, 1278 s, 1259 m, 1219 m, 1207 m, 1190 w, 1169 w, 1073 w, 1026 w, 984 w, 966 w, 957 w, 938 m, 924 w, 914 m, 853 m, 820 m, 800 m, 775 s, 764 s, 748 m, 703 s, 659 w; mass spectrum (75 eV) mje (relative intensity) 41 (26.3),44(18.8), 51 (20.4), 53(10.1), 63(12.1), 65(29.0), 66(14.7), 67(10.4),77(48.2),78(41.0),79(46.0), 80(28.7), 91 (93.2), 92(18.5), 93 (14.6), 103 (19.0),104(39.0), 105(64.2), 106(44.4), 115 (53.5), 116 (38.2), 117 (100.0), 118 (63.8), 119 (22.1), 128 (33.0), 129 (29.5), 130 (28.7), 141 (25.0), 142 (25.0), 143 (22.8), 155 (21.3), 156 (14.0), 169 VI,andVIIIintheratio6:47:43:4. (16.3), 184(69.5), 185(10.2). [(CsH&P1rRhC1 NBD. A tube containing 250 mg (g). V A n d . Calcd for C14H16: C, 91.25; H, 8.75. Found: C, of V, 35 mg of CH~CIZ as internal standard, 94 mg of [(C6H&PI391.03; H, 8.64. RhCI, and 220 mg of NBD was shaken at 85-95" for 4 days. ProIts proton nmr spectrum is identical with that of the product obton nmr integration of the CHzClz and of the 7 3.95 multiplet of V tained by reacting NBD with [(CBH&P],R~CI (see above), gave a ratio of 1 :2.6 before the reaction and 1 :2.9 after. Glpc XXV. Ir (CSz, cm-', Perkin-Elmer Model 137) 3080 w, 2740 s, (column F, 170") of the reaction mixture showed VII, V, VI, and 2840 w, 1320 w, 788 s, 785 s, 768 S , 703 s; mass spectrum (75 eV) VI11 in the ratio of 3 :69:27: 1. These figures mean the amounts of m/e (relative intensity) 41 (14.5), 51 (10.0), 65 (14.0), 77 (23.5), 78 VII, V, VI, and VIII formed are 12,29,109, and 4 mg. The amount (24.0), 79 (20.0), 80 (39.0), 91 (46.31, 92 (17.0), 93 (20.8), 104 (16.7), by which V is low should be no more than 25 mg, or 10% of that put 105 (20.5), 106 (43.4), 115 (22.1), 116 (12.71, 117 (100.0), 118 (33.1), in originally, while 62% of the NBD was consumed. Thus V does 119 (36.5), 128 (15.3), 129 (20.0), 130 (16.0), 141 (15.5), 142 (26.1), not appear t o be a precursor of the other products. 143(32.0), 155(16.0), 156(14.0), 169(13.2), 184(87.4), 185(13,8). XI11 Aqueous HI.40 A tube containing 2.0 g of XIII4I and 3.0 XIX. Bromine (1.1 ml, 16.8 mmol) in 50 ml of CCl, was added in g of 47-50Z aqueous HI was shaken a t 65-70" for 10.75 hr. Workdrops to a stirred solution of 2.35 g (12.8 mmol) of XVI in 100 ml of up (pentane extraction followed by washing with aqueous NaHC03, CC1, maintained at ca. 0". The temperature was allowed to rise to water, then drying) gave 3.2 g of an oil which was chromatographed ca. 20" and the solution washed (25 ml of 0.1 N Na&O,, then HzO) on 610 g of acid-washed alumina, eluting with pentane, then benzene, and dried (MgSOd). Solvent was removed and the product disthen chloroform. The first fractions contained 170 mg (8.5%) of tilled evaporatively at mm between 100 and 200". After two IX. 4 2 Continued elution yielded 462 mg of iodide XVIII, which was recrystallizations from pentane-acetone, 1.70 g (39%) of white recrystallized from cold pentane and sublimed at 65" (10-6 mm), crystals was obtained, mp 102-104.5". The sample for analysis giving 364 mg (10,6%) of material, mp 50-56"; nmr (100 MHz, was sublimed; nmr (100 MHz, CDC13) broad singlet at 7 5.25 CCl,) broad multiplets a t T 6.25 (0.92 H), 7.4-7.7 (1.92 H), 7.8-8.45 (0.95 H), multiplet 6.05 (0.85 H), multiplet 7.41 (1.95 H), multiplet (9.35 H), triplet 8.68 (2.08 H, J = 1 Hz), broad triplet 8.75 (0.92 H, 7.66 (1.80 H), multiplet 8.00 (5.30 H), broad singlet 8.43 (2.05 H), J = 4.6 Hz), broad doublet 9.18 (1.83 H, J = 4.6 Hz); ir (CS2, cm-1) multiplet 8.97 with overlapping doublet 9.15 (3.14 H , J = 5 Hz); 3068 m, 3050 m, 2973 s, 2931 s, 2903 s, 2890 s, 2863 s, 1365 w, 1340 ir (KBr, cm-l) 3039 w, 2972 w, 2941 m, 2929 s, 2851 m, 1436 m, 1352 w, 1318 w, 1303 m, 1285 w, 1275 w, 1255 w, 1248 m, 1238 w, 1221 m, w, 1319 w, 1290 s, 1232 w, 1196 w, 1106 m, 1042 w, 1017 w, 963 m, 1220 m, 1212 s, 1170s, 1147 w, 1140 w, 1125 m, 1108 m, 1063 w, 920 m, 896 w, 803 s, 772 s. 694 w, 639 w; mass spectrum (75 eV) 1025 w, 1003 w, 978 w, 951 w, 937 m, 927 m, 915 w, 902 w, 882 m, mje (relative intensity) 51 (45), 52 (lo), 53 (23), 56 (12), 63 (21), 75 872 m, 830 m, 814 s, 800 s, 767 w, 655 w; mass spectrum (75 eV) (14), 77 (45), 78 (29), 79 (53), 89 (32), 90 (21), 91 ( 4 9 , 92 (16), 94 m/e(relativeintensity)41(15), 64(11), 65 (14.5), 66 (17.2), 77(15.5). (15), 103 ( l l ) , 105 (38), 115 (30). 116 (12), 117 (loo), 118 (38), 119 79(25.0),91(47.0), 105(19.0). 115(12.5), 117(26.0), 119(40.5), 129 (Sl), 128 (22), 129 ( 3 9 , 142 ( l l ) , 145 (IS), 147 (14), 155 (16), 183 (18.0). 143(12.5), 185(100.0), 186(16.0), 312(0.8). (73), 184(19), 262(15), 264(15), 342(0.6), 344(1.1), 346(0.6). A d . Calcd for Cl4HI7I: C, 53.86; H, 5.49; I, 40.65. Found: A n d . Calcd for ClaH16Br2: C, 48.87; H, 4.69; Br, 46.45. C, 54.04: H, 5.43; I,40.64. Found: C, 48.99; H,4.95; Br,46.65. Continued elution gave a mixture of isomeric iodides, which was XX. Hexamethylphosphoric triamide (40 ml, freshly distilled rechromatographed on 460 g of acid-washed alumina. eluting with from CaHS) was injected into a 200-ml, round-bottomed flask pentane. Bulb-to-bulb distillation at 80" (10-5 mm) gave 289 mg equipped with reflux condenser, stirrer, and thermometer, and (8.8%) of iodides XXIII and XXIV; nmr (100 MHz, CCl,) broad which contained 790 mg (2.3 mmol) of XIX under a NS atmosphere, multiplets at 7 5.75-6.2 (0.87 H ) and at 7.15-8.90 (16.13 H); ir KO-tert-CaHa (1.1 g, 9.9 mmol) was added, the flask warmed to (neat, cm-l) 3022 w, 2865 s, 2713 s, 1455 m, 1440 s, 1282 s, 1244 m, 1 2 2 0 s , 1 2 0 3 ~ , 1 1 9 1 s , 1 1 5 0 ~ , 1 1 2 0 w , 1 0 9 7 ~ , 9 4 3 ~ , 8 9 6 m , 8 7 065", ~ , and stirred at 50" for 1 hr. After cooling, water was added. Extraction with ether, washing with water, drying (MgS04), and 750 m, 708 w, 692 w; mass spectrum (75 eV) m/e (relative intensity) removal of solvent yielded 576 mg (96%) of white crystals, which 41(14.0),67(20.0~,77(18.5),78(13.5~,79~25.2~,91~34.7~.93~10.0~. were sublimed for analysis; nmr (100 MHz, CDCI,) broad singlet 105 (20.5), 117 (23.31, 119 (31.0), 128 (15.5), 129 (15.0),'143 (10.5), at 7 3.92 (1.94 H), broad singlet 5.16 (0.93 H), multiplet 7.14 (1.92 185(100.0). 186(17.7). 312(0.1). H), broad singlet 7.82 (1.05 H), sharp singlet 7.94 (2.01 H), broad Anal. Calcd for C14H171iC, 53.86; H , 5.49; I, 40.65. Found: singlet 8.03 (1.91 H), broad singlet 8.41 (1.98 H), broad triplet 8.85 C , 54.12; H, 5.78; I,40.40. (1.16 H, J = 5 Hz), broad doublet 9.12 (2.13 H , J = 5 Hz); ir (KBr, V.43 A tube containing 262 mg of iodide XVIII (0.84 mmol) and cm-1)3063 m, 3009 m, 2986 m, 2947 m, 2931 m, 2865 m, 1356s, 220 mg of K O H in 1.4 ml of absolute ethanol was shaken at 80' for 1319 s, 1286 s, 1260 s, 1237 s, 1167 m, 1105 m, 1056 w, 1036 w, 991 25 hr. Work-up (pentane extraction then washing with water) m, 953 m, 896 m, 868 m, 837 s, 815 s, 796 s, 766 s, 714 s, 603 m ; mass followed by bulb-to-bulb distillation at 70" (0.1 mm) gave 120 mg spectrum (75 eV) m/e (relative intensity) 39 (33), 51 (14), 63 ( l l ) , (78 %) of dimer V,5which was pure by proton nmr and glpc. 65 (30), 67 (15), 77 (20), 78 (lo), 79 (24), 91 (38). 103 (lo), 105 (48), VIII and XXV.43 A tube containing 150 mg (0.48 mmol) of 115 (23), 116 (121, 117 (loo), 118 (60), 130 (15), 142 (17), 156 (lo), iodides XXIII and XXIV and 117 mg of KOH in 1 ml of absolute 183 (58), 262 (5.6), 264 (5.5). ethanol was shaken at 80" for 24 hr. After work-up, bulb-to-bulb Anal. Calcd for CldHlSBr: C, 63.89; H, 5.75; Br, 30.37. distillation at 75" (0.3 mm) gave 58 mg (65%) of a 15:85 mixture Found: C, 63.60; H , 5.68; Br, 30.37. (by glpc on column A, 240") of two norbornadiene dimers. These XVII. Sodium metal (in excess, ca. 1 g) was added in small pieces to a stirred solution of 393 mg (1.5 mmol) of XX, 15 ml of tetrahydrofuran, and 3 ml of tert-butyl alcohol under NS and refluxed (40) The procedure is modeled on that of H. A . Bruson and T. W. overnight. The solution was decanted (from N a and NaO-teriRiener, J. Amer. Chem. Soc., 67, 1178 (1945). Bu) and 150 ml of H20 added. The product was extracted with (41) Isolated by distilling the mixture of dimers obtained from norether (3 X 30 ml), washed with HzO(2 X 50 ml), and dried (MgSO,). bornadiene and rhodium on carbon through a Nester and Faust annular Bulb-to-bulb distillation at 70" (0.5 mm) yielded 172 mg (63%) of Teflon spinning band distillation column. In the original preparation XVII, aliquid. the isolation was effected by glpc.5 The proton nmr spectrum is the same as that published else(42) The proton nmr, ir, and melting point are identical with those where;*Z ir (neat, cm-l) 3050 s, 3010 m, 2959 s, 2937 s, 2862 s, 1467 reported.27 (43) The procedure is modeled on that in ref 23b. m, 1357 m, 1320 s, 1286 m, 1244 w, 1168 w, 1016 w, 895 m, 869 m,
shaken at 100" for 4 days. Proton nmr of the reaction mixture showed only XI11 and cyclooctene. (f). XI11 [(CsH&PI3RhCl NBD. A tube containing 240 mg of XIII, 35 mg of CHzClz as internal standard, 83 mg of [(Cd-I&P],RhCI, and 200 mg of NBD was shaken at 85-95' for 4 days. Proton nmr integration of the CHzClz and of the 7 9.65 doublet of XI11 gave a ratio of 1 :2.9 before the reaction and 1 :3.0 after, indicating no loss of XIII. The ratio of the intensity of the resonance at T 9.65 and of all olefinic protons was 2.2. Glpc (column F, 170") of the reaction mixture showed VII, V, VI, XI11 (not resolved), and VI11 in the ratio 2.4:20:76:1.6. These figures imply that while no loss of XI11 occurred, 88% of the NBD was converted into VII, V,
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Katz, et al. / Dimerization and Trimerization of Norbornadiene
5456 809 s, 715 s : mass spectrum (75 eV) m/e (relative intensity) 66 (18), 91 (18). 115(11). 117(100), 118(81), 184(16). X-Ray Diffraction. Needle crystals of XVb suitable for X-ray diffraction studies were obtained from methanol. The intensity data on which the refinement was carried out were collected on a needle (along b axis) with dimensions 0.07 X 0.05 mm and length 0.5 mm, mounted on a Picker FACS-1 diffractometer with Cu KCYradiation and using the 8 - 28 scan technique. The intensities were measured with a scintillation counter with background counts being made at each limit of the scan. A total of 2759 reflections was considered nonzero out to a 28 limit of 130" using the criteria that the net count be greater than 0.06 times the total background count and/or 50 counts whichever is greater. No corrections were made for absorption. The range of transmission coefficients was calculated as 0.26-0.36. Full-matrix least-squares refinement, varying positional and isotropic thermal parameters for the nonhydrogen atoms, gave values o ~0.182 z ] ' ~and ~ )0.166. of R and Rz (RP = [ Z w ( / F, / - / F c ~ ) z / Z w ~ Fof All reflections were given unit weights and the quantity minimized was 2w(IFo1 Least-squares refinement on positional and
anisotropic thermal parameters for the nonhydrogen atoms reduced R and RZ to 0.085 and 0.084. A difference map calculated at this point revealed several positive peaks in the range 0.2-0.4 electron/ A 3 , some of which could represent hydrogen atoms. As the location of all of the hydrogen atoms could not be made with certainty, however, we decided to neglect the contribution of the hydrogen atoms and to terminate refinement. The list of / I , k , I , F, and F, values will appear in the microfilm edition. z9 The atomic scattering curves used for Br, C, and 0 were those tabulated by Cromer, et a/.4 4
Acknowledgments. We are grateful to the National Science Foundation for its support under grants GP7809 and GP-30669X and to the National Institutes of H e d t h for a predoctoral traineeship t o J. K. F. under grant GM722. (44) D. T. Cromer and J. B. Mann, Acta Crystallogr., Sect. A , 24, 321 (1968).
Acetamidomethyl. A Novel Thiol Protecting Group for Cysteine Daniel F. Veber, John D . Milkowski, Sandor L. Varga, Robert G . Denkewalter, and Ralph Hirschmann* Contribution f r o m the Merck Sharp & Dohme Research Laboratories, Diuision of Merck & Co., Inc., Rahway, New Jersey 07065. Receioed November 2, 1971
Abstract: The acetamidomethyl group has been found to be a useful protecting group for the thiol of cysteine. It was added to the thiol of cysteine and to the eight cysteines of the reduced S-protein of ribonuclease under acidic conditions. It is stable under the conditions commonly used in peptide synthesis and is removed by mercuric ion under mild conditions. The mercuric ion may be removed by treatment with H,S for small peptides and by gel filtration in the presence of mercaptoethanol for large peptides and proteins.
I
n a preliminary communication' we have reported a novel derivative of cysteine in which the sulfur is protected by the acetamidomethyl (Acm) group. 2 , 3 This protecting group has been found to be useful both in the synthesis of peptides and proteins and in the reversible protection of a natural protein. In this report we describe in greater detail the preparation, properties, and utility of this derivative of cysteine. Preparation of S-Acm-cysteine and Octa-S-Acmoctahydao-S-protein. S-Acm-Cys (111) was conveniently prepared in about 50% yield by treatment of cysteine with a 10 % excess of N-hydroxymethylacetamide (I) in aqueous medium at p H 0.5. A minor byproduct, thiazolidine-2-carboxylic acid (IV), was removed by recrystallization. I11 was found to be stable at p H 0.5. We believe, therefore, that IV arose via the decomposition of I to formaldehyde and acetamide. Scheme I indicates that anhydrous conditions should shift the equilibrium between I and I1 to the right, and thus minimize the formation of IV. Indeed, when cysteine was allowed to react with a 2 0 x excess of I in anhydrous H F at O " , I11 was formed in nearly quantitative yield, and IV was not detectable (1) D. F. Veber, J. D. Milkowski, R . G . Denkewalter, and R. Hirschmann, Tetrahedron Lett., 3057 (1968). ( 2 ) A related class of protecting groups has been reported by F. Weygand, W. Steglich, I. Lengyel, F. Fraunberger, A. Maierhofer, and W. Oettmeier, Chem. Ber., 99, 1944 (1966). (3) All amino acids are of the L configuration. Abbreviations used: Acm = acetamidomethyl; Boc = tert-butyloxy carbonyl.
Journal of the American Chemical Society 1 94:15
Scheme I CHnSH
I
+NH,CHCOnH C H ~ NH SdH,
I
CH,
I
+NH,CHCO, H III
0
li
CH,CNHCHzOH
I
It 0
I
CH,CNH,
-k
in the reaction mixture. More importantly, the use of liquid H F as solvent proved to be particularly useful when we applied it to the S-alkylation of reduced Sp r ~ t e i n since , ~ acetamidomethylation of this protein in anhydrous but not in aqueous medium led to a product (4) (a) F. Haber and C. B. Anfinsen, J . Biol. Chem., 236, 422 (1961); (b) S-protein is the protein obtained o n treatment of ribonuclease-A with subtilisin (F. M. Richards, Proc. Nar. Acad. Sci. U . S., 44, 162 (1958)).
July 26, 1972
