COMMUNICATIONS C,,N+ and C,,N+ : Isoelectronic Heteroanalogues of C,, and C,,,** Iris L a m p a r t h , Berthold N u b e r , Georg Schick, Andreas Skiebe, Thomas Grosser, and Andreas Hirsch* For the structural variation of fullerenes three strategies can in principle be considered: 1) the synthesis of exohedral adducts, 2) the formation of heterofullerenes in which one or several C atoms of the fullerene core are replaced by, for example, N or B atoms, and 3) the generation of endohedral complexes. Whereas both exohedral and endohedral derivatives can be synthesized and isolaled by ;I variety of methods.['] it has not been possible s o far to produce macroscopic amounts of heterofullerenes.r'l Even the mass spectrometric detection of heterofullerenes was accomplished only for boron fullerenes C,,_,,B,+ (n = 1 -6), which were gcnerated in situ by laser vaporization of boron/ graphite composite rods.131We now report for the first time on the formation of the nitrogen heterofullerenes C,,N+ and C,,N+, which are isoelectronic with C,, and C,,. These ions are formed by fragmentation (FAB-MS) of regioselectively synthesized iminofullerene derivatives. The specific arrangement of the nitrogen addends tacilitates the removal of a carbon atom from the fullerene core and the incorporation of a nitrogen atom. Bisarafullci.oid 3[4'served as a precursor for compounds leading to C,,,N'. and we synthesized for the first time the bisadducts 2. which directly fragment to form this heterofullerene ion (Scheme 1 . Table 1). The C;-symmetric compounds 2, which are corresponding six-membered ring have double-bond character. According to AM1 calculations the open structure I1 is 3 kcalmol-' lower in energy even though a double bond has to be introduced formally into each of the three five-membered rings. As a precursor for molecules suitable for the formation of C,,N+ we synthesized for the first time the C,-symmetric bisazafulleroid 4. This C,, analogue o f 3 is the major product from the Dc @- E W I / 3 R=CH,COOMe 4 -nBu) 5 R=CH,COOMe 6 N R=CH,COOMe R=CH,COOMe /R twofold reaction of C,, with azide. The preferred formation of 4 shows that the regioselective formation of bisazaf~lleroids[~~ is la,b I 2a,b II Schemc I . a R = ('OOEt. b: R = COOrBu. Reaction conditions: a ) N , R , 1chloronaphth;iI~ii~. 60 C: b) toluene, reflux. green in solution, are the first examples of dicyclo adducts with addends bound in cis-/ The adducts 2 are formed regioselectively from the reaction of 1lS1with two equivalents of azidoformate i n I-chloronaphthalene at 60°C and subsequent thermal extrusion of N, from the triazoline intermediate (Scheme 1 ) . The structure of 2 was determined by "C NMR spectroscopy and was proven unambiguously by the use of IOO'X, "N-labeled 2a and 2a in which only one imino bridge is i5N-labeled (Table I ) . The signals of C-1 and C-4 at 6 = I 2 8 ( ' J , ( , = 14 H z ) and of C-2 and C-3 at S = 114 ('Jo, ,iN)= 15 Hz) exhibit a remarkable downfield shift. This shows that the bridged 6-6 bonds are open and that the 5-6 bonds in the 15Ni [*I Prof. 111..A Hirsch. 1. Lainpurth. B. Nuber. G. Schick. A. Sktebe. T. Grosser I n \ t i l u l f i r Organibche Chemie dcr Univcrsitit Kichaid-Will\iiitter-Aliee 2. D-7613 I Karlsruhe (Germany) .rcicl;lx: 1t1t [**I Thi\ work +( n i ) h ~ 3 0 s C O ~ C \&;I\ supported by the Bundesministerium fur Bildung und For- schung ( B M B F ) . Hoechrt AG. and the Dr. O t t o Rohm Gedichtntsstiftung. also possible with C,,. In analogy to the ' H N M R spectrum of 314] we observe a quartet for the diastereotopic protons of the methylene groups in 4 (Table 3 ) . Whereas the mass spectra of the bisadducts 3 and 4 show the typical characteristics of fullerene adducts (a relatively small M + peak. the peak of the fullerene fragment ions C& or C,: at nzjr =720 or 840 as the most intense signals). completely new behavior was observed with the bisadducts 2 (Fig. l a ) . In this case the peak at mjr =722 is the most intense signal, which is due to the formation of the heterofullerene ion CgqN+.["The signal of "C,f, at nijz = 720, which arises by the usual fragmentation, is less intense. After isotope correction. the spectrum of 2 b exhibits an intensity ratio of 5:4 for the ions C:,,N+ and C;,. The assignment of the peak at n i / ; =722 to I'C,,'4N+ is also confirmed by the observation that after the first "shrink-wrapping" the signal at in/; = 698 (12C,,'4N+)is still more intense than that at tnji = 696 ("C,,N'). Shrink-wrapping is the fragmentation of the fullerene core by successive elimination of C, units with retention of the fullerene structure.'"'. 'I This process requires such high energy ( > 40 eV)[7b1that all exohedrally bound groups are removed first. Thus the peaks at m:: =722 and 698 cannot be due to exohedral fullerene derivatives such as dihydrofullerenes. Trisadducts, which are formed by the reaction of azidoformates and 1 or of the corresponding 5-6-bridged azafulleroids having a mixed 5-6/6-6 addition pattern. show fragmentation behavior similar to that of 2.['] The observation that heterofullerene ions are formed under these conditions from 2 and mixed 5-616-6 adducts but not from the bisadducts 3 and 4 or the 5-6 or 6-6 monoadducts such as I [4J COMMUNICATIONS Table 1. Selected 5pecti-oscopic data of the ncw compounds 2 and 4. ["N]-2a: ' H N M R (CS2~10"/b CDVI,. 250MHs. 25 C): d = 4 . 1 1 (dq. J = 1 . 2 . 7.0 Hz). 1.25 (t. J =7.1 H<): " C N M R (CS,:lO"A, CDCI,. 62.9 MH7. 25 C): 6 =152.57 (d. 'J(C.N) = 27.6 H r ) . 146.46 (1 C). 145.91. 145.55. 145.33. 144.53, 144.42 (IC ) . 144.25. 143.73. 143.18. 143.09. 143 02, 142.84. 142.78. 142.76. 142.58. 140.96. 140.83. 140.76 tic), 140.71. 140.28. 139.95. 139.31 id. 'J(c.N) = 2 ~ 7 ) 139.14 (1 C). 338.96. 137.74. 137.1X (d. 'J(C.N) = 1 H7). 134.92 (d. 'J(C.N) = 3 Hr). 128.09 (d. 'J(C.N) = I 4 Hz). 114.73 (dd, 'J(C.N) = I S . 'J(C.N) = 3 Hz). 62.65. 14.32; UV;Vis (CH,CI,): i.,, [nm] , ( (26000). 549 (1000): FAB-MS (NBA): / t i : : : 896 ( M '. 2 IOO'Yo), 720 (CA,, 9 5 % ) . ["'N,''N]-2a (C,,,,("NCOOEt)('JN<'OOEt)): " C N M R (CS2;10% C'DC'I,. 62.9 MHz, 25 C): 6 =152.57 is. d. ' J ( C , N ) = 27.6 Hz). 146.46 ( I C ) , 145q1. 145.55. 145.33. 144.53. 144.42 (1 C ) . 144.25. 143.73. 143.18. 143.09, 143.02. 142.84. 142.7X. 142.76. 142.58. 140.96. 140.X3. 140 76 ( 1 C), 140.71, 140.28, 139.95. 139.31 ( h i , 139.14 ( I C ) . 138.96. 137.74. 137.18 ( h ) , 134.92 (b). 128.09 (s. d. 'J(C.N) = 1 4 H r ) . 114.73 id. d. 'J(C.N) =15. 'J(C,N) = 3 Hz), 62.6.5, 14.32 2b: ' H N M R (CS2;I0% CDCI,. 250MHz. 25 C ) : ii =1.44 (5): I3CNMR (CS,: 10% CDC'I,. 62.9MHr. 2 5 . C ) : =151.43. 146.35 ( l < ' ) , 145.81. 145.40. 14526. 144.48. 144.31 ( I C ) . 144.18. 143.62. 143.17. 142.99. 142.97. 142.9.;. 142.86. 142.76. 142.74. 142.48. 142.42. 140.93. 140 76. 140.58, 140.42(IC). 140.31, 139.83. 1.19.83. 139.48 ( I C ) . 139.41. 138.96. 137.58. 137.28. 134.74. 128.09. 114.60. 82.16. 27.62; UVWis (CH,CI,): i,,,,, [nm] ( I : ) = 259 (90700). 337 (26000). 555 (1000): FAB-MS (NBA): 1 1 1 : : : 950 (hi'*. 20%). 722 (C,,N ' , 100%). 720 (C,',,,9 5 % ) . 698 (C,-N '. 15%) 4 : ' H NMR (CS,:20% CDCI,. 250 MH7. 2 6 5.34 (tl), 4.57 (d). 3.93 (s): " C NMR (CS2;20% CDCI,, 62.9 MHz. 25 =169.61. 154.82, 152.23, 150.55. 150.50. 148.89. 148.66. 148.34. 14X.01. 147.83. 147.29. 147.21. 146.85. 146.54. 146.38, 145.55. 145.25. 145.16. 144.66. 144.63, 144.21. 144.11. 143.54. 142.74. 140.93. 138.55, 136.98. 135.81. 134.4s. 133.03, 132.53. 130.76. 129.85. 129.52. 128.61. 124.76. 52.79, 52.23 (h). 51.86. 51.70 (h): UV:Vis (cyclohexane): i , , [nm] , (I.) = 212 (71 000). 237 (76000). 328 (17000). 393 (12600). 458 (11 500): FAB-MS (NBA): w:: 1015 ( M ' . 20%). 928 ( M i - NCHJOOMc, 15%). 840 (C,(,. 100%) 2 prompted us to further modify the fullerene core in 3 and 4 regioselectively and synthesize precursor molecules that are also able to fragment to give heterofullerene ions. According to AM1 calculations bonds a and b are the most polarized and attack of a nucleophile should take place there preferably. Hence, we treated 3 and 4 with one equivalent of butylamine in the presence of DBU. The initially formed precipitate became soluble upon the addition of acetic acid. The products are the monohydroamination products 5 and 6. which are mixtures of regioisomers (attack at bond u or h ) having identical R, values. In analogy to dissolved 2 the solution of the hydroamination product 5 in CHCI, is green. Under the conditions of FAB mass spectrometry 5 and 6 indeed fragment efficiently with formation of the ions C,,N+ and C,,Nt (Fig. Ib). which give rise to the most intense signals in the spectra. In the range of ions with cluster size 58. which are generated by shrink-wrapping, the same fragmentation features as those of 2 were observed. The signals of C,,N+ and C6.,N+ are in general less intense than those of the corresponding ions with cluster size 60 and 70 (Fig. I), which indicates that C N is eliminated more efficiently than C, . The high-resolution signals of. for example, the fragment ions of 5 at 721.997 (-1-0.005)and of 6 at 841.997 (k0.006) clearly prove that these signals d o not correspond to '*C,,HL; (722.0157) and "C,,H-; (842.01 57), because their calculated values are well outside the margin of error. The calculated masses of all possible combinations of '2C,,13C, and 12C5q14N range between 722.0031 and 722.0067 and therefore very close to the range of the experimental values. The ions with cluster size 70 behave analogously. To finally prove unequivocally that a nitrogen atom is incorporated into the fullerene core under these conditions, we treated I00 Yo ,Nlabeled 3 and 4 with unlabeled butylamine to convert them into the corresponding hydroamination products. Like in the FAB spectrum of [I5N]-2athe most intense fragmentation signals are - 4 b,100 842 680 d) 100 690 .3 700 710 720 730 843 816 I Fig. I FAB inass spectrum (Finnigan MAT YO; C s gun, emission current 2 pA. acceleration voltage 20 kV, dctection by secondary electron multiplier. matrix: 3-nitrohenryl alcohol) in the range of the major fragmentation signals of a) 2 b (line spectrum). h ) 6. c) ["NI-S. and d) ["NI-6 (profile mode) due to the cluster ions 12C5915Ni (mi: =723) and 'ZC69'5Nt ( r n / = = 843) (Fig. Zc, Id). The parent ions C,,N+ and C,,N+ are isoelectronic with C,, and C,,,. AM1 calculations show that C,,N' has almost the COMMUNICATIONS same structure a s C,,, (Fig. 2). Also in this case there is bond length a1ternation"l between 5-6 and 6-6 bonds. Like all 6-6 bonds in thc molecule, the 6-6 C-Nbond in C,,N' has doublebond character ( I .37 A) and is even shorter than the C - C double bonds ( I .38 The pyrainidalization at the N atom is slightly stronger than that at the C atoms. The highest positivc charge density of0.159 is located at C-2 and the next lowest at C-4 (C-I I ) and C-6 (C-9). Therefore, the resonance structures I - IV (Fig. 2 ) . in particular I and 11. are the most important. A). (0.159) IV Ill Fig 2 C'Ii:ir~tcii\tic hoiid lengths in A. Mulliken charges m o s t import:int rooniincc structures of C,,N (in brackets). and the Based on this new observation on the generation of the free ions C , , N + kind C,,N' we are now working on the synthesis and isolation of heterofullerene derivatives such as C,,NR in inacroscopic amounts. Received May 31. 1995 [ZX0471E] German version- .4iigi,i4. C'lrvtir. 1995. /07. 247.1 2476 Keywords: fullcrenes . heterofullerenes * mass spectrometry Synthesis of the Palmitoylated and Farnesylated C-Terminal Lipohexapeptide of the Human N-Ras Protein by Employing an Enzymatically Removable Urethane Protecting Group** Herbert Waldmann* and Edgar Niigele Dcdicaied i o Profl)ssor F r a Eifc~nhrrger ~ on ilu, occctsiori of' his h5rlz h i r t l i d u ~ ~ RLISproteins form a class of membrane-bound lipoproteins that play ii central role in the growth-factor-mediated transduction of extracellular signals across the cell membrane into the interior of the cell. By the so-called R m pathway of signal transduction, organisms as diverse as mammals, flies. worms. and yeast regulate cell growth and proliferation."' If this regulation is disturbed or interrupted an uncontrolled proliferation may occur, which ultimately may result in the establishment of cancer. This is highlighted by the fact that point mutations in the respective rtis-oncogenes may result in malignant transformation of the cell. A remarkable observation is that in about 40% of all human cancers such a point mutation is found, and in some of the major malignancies like lung, colon, and pancreas cancer this figure rises to 85%.[" To study the biological phenomena associated with the functions of the Rtrs proteins. model peptides that embody the characteristic structural elements of their parent lipoproteins are urgently needed.f31 All Rnsproteins terminate in an S-farnesylated cysteiiie methyl ester and, except for Ki-Ras. towards the N-termintis one o r more S-palmitoylated cysteines are present (see. for instance, the Cterminal lipohexapeptide part I of the CH3 human N - R o s protein ; Fig. 1) .141 The presence of these sensitive sidechain functionalities excludes the use of classical acid- and base-labile protecting groups in the assembly of Rasrelated lipopeptides. Thus, we found that -Cys(Pal)-Met-Gly-Leu-Pro-Cys(Far)OMe 1 upon acid-mediated Fig. I . Structure 01' thc ch.ii-acteristic C-terminal removal of the Boc Iipohexapeptide ( I ) d r h c Iitiiiiiin N-R<i.\protein. protecting group from S-farnesylated cysteinyl peptides, an attack of the acid on the double bonds of the farnesyl residue always occurs, and that the thioesters present in S-palmitoylated lipopeptides hydrolyze spontaneously even at pH 6 -7 in aqueous solution.[" Due to this pronounced lability, up to now a successful sqnthesis of a fully functionalized (that is, an S-farnesylated and S-palmitoylated) Rtis-lipopeptide could not be achieved. For this purpose blocking functions have to be employed that can be selectively removed under the mildest. preferably neutral conditions. Previously we reported the development of enzyme-labile protecting groupsL6]that fulfill these criteria. In particular, the phenyl- A tlirjch. llrc ( ' l i c r i i i s t r y o/ rlii, F I I / / L ~ ~ w LThieme, ~.\. Stuttgurt. 1994. R . K;irlunkel. T. Dressier, A . Hirsch. J. Camp A i d Mol. Dcs.1992. 6 . 521 : b) N Kui-itx K. Kobiiyashi. H. Kumahora. K. Tago. K . Oziwa. Chcin. PAL\. LVII 1992. I Y X . 9 5 ; c i W Andreoni. F. Gyyi. M . Parrinello. h i d 1992. IYO. 159. '11 -1. biio. ( ' . Jin. R. E. Smalley. J P h , ~ s Chen?. . 1991. Y j . 4948: b) V. Chai. T. ( i w . C'. .Iiii. K . E. Hauller. L. P E Chibdnte, 1. Fui-e. L. Wiing. J. M. Alford. R. f,.. Sinalley. h i d . 1991. Y5, 7564; c ) R . E Smalley i n Fiillerwr t'ro/ii'rric,\, iiiiil C ' h e i ? i i \ r r i ~(if1,ar.q. C'irrhon C ' h i v r e r ~( A C S .Si.riip S 141 1 5 0 ) . T Circkcr, M. Prxto. V. Lucchini. A Hirsch. F. Wudl, Atigtw C ' k i i r . 1995. 107. 1362: 4ii,y~11.( ' h i . lnr. E d Etrgl. 1995. 34. 1141 M.R Hanh\. J I . G. C;idogan. I . Gosncy. P. K. Ci. Hodgson. P.R. R. Langridge-Smith. I). W. H. Rainkin. J C ' l i o r i . Soc. Chriii. ( ' o i i ~ r i r i u i .1994. 1365. liidcpendeni o f oui- investigations Mattay ct al. recently shorted that the u i i i i i o i i o - tind ~~/~idiliydrofullercnes C,,NHt and C,,NH,'. respectivelj. cnii he gcnc.r;itctl lroni the 1.?-bridged aziridinofulleretie C,,,NH by using DCI (desorpttxc chcii1ic:iI ioniLation) mass Fpcctrometrj and iinimonia 'is reagent gas. Furtlici- l u i i c t i i ~ i i ; i i i r a t i ~by ~ n nitrogen through the reagent gas seem.\ to be the d c c i c i \ c sicp in this mse. too But this method docs not provide dircct access to the p,ircnt compounds C>,,N ' and Cc,vN' which we achievcd by fragmentation 01 \y\tcm:iiicall!, devigned oligoadducls under conditions of FAB m i i s spect r ~ i i i i cti -y.I Aicrdung. H . Luftmann. 1. Schlachtcr. J. M;itta). Tr~rrrihc~lrlroti 1995. 51. 6071 ;il S C' 0'13ricii. J. II Hcath. R. 1; Curl. R . E.Sm;illey. J. Chcrii. /'hi.s. 1988, NX. 2211: b ) H . Schharr. T. Weiske, D. Hblime. .I.Hrusak i n B ~ i ~ X i r i i i i s r o . / u / i i ~ i c i i i ~ ~ [*) ups. M A. Ciulolini). VCH. Weinheim, 1993. pectruni of a polyimino adduct o f ion ofaiide and subsequent extruyio r'ttion 0 1 '11 Iwst three nitrogen atom\ iiito thc fullcrenc corc: this is particularly (**I ~ & n t Erom thc fragment ions of cluster size 58. a) 11. . Proll Dr. 11. Wnldmann, DiplLCheni. E. Nagele lnstitut fur Organische Chcmie der Universitiit Richai-d-Willstalter-Allee 2. D-761 ZX Karlsruhe (C;eriiimy I Telcth. Int. code t(O721) 608-4825 This work IWS supported by thc Deutsche For\chiin~~gciiieiiisch~ifL m d the Fonda der Chcmischen lnduatrie.