Langmuir 1996,11, 4217-4221
4217
CisSlk.ans Isomerization in Azobenzene-Chain Liposomes Robert A. MOSS*and Weiguo Jiang Department of Chemistry, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903 Received July 21, 1995@
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Pseudoglyceryl cationic azobenzene-chain lipid 1 undergoes efficient trans cis and cis trans photoisomerization,as well as smooth cis trans thermal isomerizationin hololiposomes and in coliposomes with dioctadecyloxylipid 2. Rate constants and activation parameters for the cis trans intraliposomal thermal isomerization of 1 are reasonably comparable to those measured in homogeneous solution. Azobenzene lipid domain formation in coliposomes of 1and 2 is exploredby differentialscanningcalorimetry and U V spectroscopy.
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During the past 15 years, photoresponsive supramolecular azobenzene (AB) systems have been intensively studied because of their potential as high-density optical memory elements and molecular switches.’S2 Kunitake has been especially innovative in correlating the molecular structure of AB lipids with the morphology and spectroscopic properties of the derived aggregate^,^^^ particularly bilayer a ~ s e m b l i e s .Others ~ have focused on AB-functionalized polymers,l” monolayers,1a,2 micelles,‘j liposomes,’ Langmuir-Blodgett (LB)films,8lipid-coatednylon capsule^,^ and H-bonded complexes.10 The utility of the AB derivatives stems from their facile and reversible trans * cis photoisomerization, and cis trans thermal relaxation;1,2eq (1).The spatial demands,
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one exception,s the few double chain example^'^,^^,^ were employed as reporters of aggregate structure, rather than as subjects of isomerization studies. Perhaps this bias toward single chain AB lipids arose from the observation that their trans cis photoisomerization was subject to steric inhibition if there was insufficient room available for the spatially more demanding cis isomer,lar4 As Kunitake noted, “mobility of the chromophores is crucial for efficient isomerization,” 4~11so that double chain AB lipids might unneccessarily complicatephotoisomerization within molecular assemblies. In view of our ongoing interest in the relation between lipid molecular structure and intraliposomal dynamics,12J3 we prepared the pseudoglyceryl cationic lipid 1, which incorporates a 4,4‘-dioxoazobenzene residue in one of its two chains. Mindful of Kunitake’s cautionary report,ll
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Me3N*CH2FHCH20(CH2)40O
N
0
O(CH2)17CH3 N ‘ O(CH2)4CHs absorption spectra, and dipole moments of the trans and cis AB isomers differ markedly, so that the pair can be 1 considered a molecular “switch” with two available positions. Assemblies (or membranes) of AB-derived monoMe3N+CH2?HCH20(CH2)1 7CH3 mers can function as control elements to modulate such properties as permeability,l” surface potential,2 fluoresO(CHP)I~CHB cence and a b ~ o r b a n c e ,and ~ aggregation.‘j Moreover, 2 because the AB UV absorption maximum is sensitive to the “stacking” of adjacent AB c h r o m o p h o r e ~ it, ~can ~~~~~ we wanted to know (1)whether leaflets ofbilayer liposomal “report” on the relative distribution, orientation, and 1 would provide sufficient room to permit efficient trans interdigitation of AB monomers within aggregate^.^ cis photoisomerization, (2)what constraints the bilayer Most AB lipids previously examined in supramolecular might impose on the thermally driven cis trans assemblies have been single chain molecules.1-6~9J0With reversion, and (3)how the coliposomal inclusion of varying Abstract published inAduanceACSAbstracts, October 15,1995. quantities of diluant lipid 2 might modulate the stacking (1)Reviews: (a)Anzai, J-i.;Osa, T. Tetrahedron 1994,50,4039.(b) and isomerization of 1. Kunitake,T.Angew. Chem., Int. Ed. Engl. 1992,31,709.(c) Shimomura, While our work was in progress, Osa’s group reported M.; Ando, R.; Kunitake, T. Ber. Bunsen-Ges. Phys. Chem. 1983,87, that the head group functionalized AB lipid 3 afforded LB 1134. (2)See Maack, J.; Ahuja, R. C.; Tachibana, H. J . Phys. Chem. 1996, films in which the two “bulky” chains created sufficient 99,9210.Ahuja, R. C.;Maack, J.; Tachibana, H. J . Phys. Chem. 1996, free volume within the membrane for trans cis pho99,9221 and references therein. toisomerization to occur, whereas monoalkyl chain ana(3)Kunitake, T.; Okahata, Y.; Shimomura, M.; Yasunami, S-i.; logues of 3 resisted photoisomerization.8 Our bilayer Takarabe, K. J . Am. Chem. SOC.1981,103,5401. (4)Shimomura, M.; Kunitake, T. J.Am. Chem. SOC.1987,109,5175. experiments with AB chain lipid 1 are, in part, comple( 5 ) (a) Shimomura, M.; Kunitake, T. Chem. Lett. 1981, 1001. (b) mentary to those of Osa. Note added in proof: An Kunitake, T.; Ishikawa, Y.; Shimoumura, M. J . Am. Chem. SOC.1986,
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108,327.(c) Ishikawa, Y.; Nishimi, T.; Kunitake, T. Chem. Lett. 1990, 165. (d)Nishimi, T.; Tachikawa, M.; Maeda, T.; Ishikawa, Y.; Kunitake, T. Chem. Lett. 1994,331. (e) Kimizuka, N.;Kawasaki, T.; Kunitake, T. Chem. Lett. 1994,1399. (0 Ishikawa, Y.; Kuwahara, H.; Kunitake, T. J. Am. Chem. SOC. 1994,116,5579. ( 6 )Higuchi, M.; Minoura, N.; Kinoshita, T. Chem. Lett. 1994,277. (7)Tabushi, I.; Nishiya, T. Tetrahedron Lett. 1986,27,4589. (8)Anzai, J-i.; Sugaya, N.; Osa, T. J . Chem. Soc., Perkin Trans. 2 1994,1897. (9) Okahata, Y. Acc. Chem. Res. 1986,19,57. (10)Rosengaus, J.;Willner, I. J . Phys. Org. Chem. 1996,8, 54.
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(11)Kunitake,T. etal. [Nippon KagakuKaishi 1988,10011reported that, in closely-packed LB membranes, trans cis AB photoisomerization was significantly restricted due to the lack of free volume in the membrane; cited in ref l a , p 4057. See also Yabe, A.; et al. Chem. Lett. 1988,1, And Nishiyama, K.; Fujihara, M. Chem. Lett. 1988,1257, where sufficient room for photoisomerization was created by the provision of cyclodextrin cavities or a polyallylammonium ion subphase. (12)Moss, R.A.Pure Appl. Chem. 1994,66, 851. (13)Moss, R. A.;Ganguli, S.; Okumura, Y.; Fujita, T. J . Am. Chem. SOC.1990, 112,6391.
0743-746319512411-4217$09.0010 0 1995 American Chemical Society
Letters
4218 Langmuir, Vol. 11, No. 11, 1995 Scheme I CH2CHCH2NMe2
I
HO
1
&
OH
CH2CHCH2NMe2
I
Ph3CO
I
b
OH 6 CH2CHCH2NMe2
I
1
Ph3CO OCY8H37 7
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4
(a) Ph3CC1, MesN, DMAF', DMF, room temperature, 12 h, 77%. (b) NaH, THF, room temperature, 1h; C18H37Br, reflux 3 d, 52%. (c) Concentrated HCl, 1:l CHzClZ-MeOH, room temperature, 2 h, 92%.
Scheme 2 H
~
N
H
A
~
-
C ~ H ~ , O ~ N Hb A ~
C 5 H 1 , O e N = N o O H
5
10
(a)CSHllBr, NaOH, EtOH, reflux 5 h, 89%. (b)Concentrated HC1, EtOH, reflux 5 h; concentratedHC1,50%aqueousacetone, NaN02. (c) PhOH, NaOH, Na2CO3, HzO, room temperature, 12 h, then HOAc, 81%. (d) Br(CHABr, KOH, EtOH, reflux 6 h, 68%.
important set of experiments with AB phospholipids has just appeared; see Song, X.; Perlstein, J.;Whitten, D. G. J.Am. Chem. SOC. 1995,117, 7816. 0
Lipid Synthesis. AB lipid 1 was synthesized by the reaction of backbone fragment 4 with AB derivative 5, followed by quaternization with MeBr; cf. eq 2. PreparaCH2CHCH2NMe2
4
5
tive sequences for reactants 4 and 5 appear in Schemes 1 and 2, respectively. Fragment 4 was generated from 1,2-dihydroxy-3-(dimethy1amino)propanevia a three-step, 37% sequence that featured protective tritylation a t the C1 hydroxyl group, yielding 6; formation of the Czoctadecyl ether, 7,by reaction with NaH and octadecyl bromide; and HC1-catalyzed detritylation to 4. AB synthon 5 was prepared fromp-hydroxyacetanilide in a four-step, 49% sequence: First, pentylation afforded 4-(pentyloxy)acetanilide, 8 , which was deacetylated and diazotized to 9. Next, the diazonium ion was coupled to phenol, affording azophenol 10,which, upon reaction with 4-fold excess 1,4-dibromobutane, gave the desired AB derivative 5 (mp 113- 115 "C), characterized by NMR and elemental analysis. To complete the preparation of 1, fragment 4 was converted to the alkoxide with NaH in THF (reflux, 1h) and then refluxed with 5 for 3 days under nitrogen, yielding
the dimethylamino analogue of 1 (mp 38-40 "C). This was purified by chromatography on silica gel with CHZC12 (42% yield),characterized by NMR and elemental analysis, and finally quaternized (MeBr, acetone, room temperature, 10 h), affording 1, mp 115-119 "C, in 80% yield (34% from 4 and 5) after trituration with acetone. The structure of 1 was secured by a n appropriate lH NMR spectrum14 and a satisfactory elemental analysis (C, H, N). Other intermediates in Schemes 1 and 2 (except diazonium ion 9) were each isolated and characterized by NMR. The preparation of lipid 2 was readily achieved from 1,2-dihydroxy-3-(dimethylamino)propaneby bis-octadecylation (NaH, THF, 1h, then excess C18H37Br,reflux, 3 d, 48%), followed by quaternization with MeBr (acetone, 25 "C, 10 h, 97%). The structure was supported by an appropriate NMR spectrum and satisfactory elemental analysis (C, H, N). Liposomes. Hololiposomes of 1 and coliposomes of 1 and 2 were created by immersion probe sonication (60 W, 5 min, 58 "C) of CHCls-evaporated lipid or colipid films in 0.01 M pH 8.0 aqueous Tris buffer, 0.01 M in KC1. After sonication, liposome solutions were cooled to 25 "C and filtered through 0.8 pm Millex filters. Hydrodynamic diameters of these liposomes were determined by dynamic light scattering15 on 5 x M lipid preparations. The mean liposomal diameters were relatively independent of 112 composition, ranging from 330 to 450 A. The small sizes were consistent with unilamellar species. Differential scanning calorimetry (DSC) revealed domain formation or lipid sorting within the liposomes. DSC traces appear in Figure 1 and are summarized in Table 1. Liposome samples (prepared as above) were 5-10 mM in lipids and were scanned from - 10 to 80 "C a t 1deglmin with a Microcal MC-1 scanning calorimeter. The van't Hoff enthalpies were calculated from the DSC curves as described by Marky and Breslauer.16 At high AB lipid compositions(80- 100mol % of 1,traces 1-3), the liposomes consist mainly of "low-melting" AB domains, T, 28-30 "C, in which the lipids' AB chains are stacked (see spectroscopy, below) in face-to-face orientations;lC cf. Figure 2. Even in pure liposomal 1, however, there appears to be some contribution of faceto-back lipid domains (Figure 2), visible in Figure 1as the small feature a t 62 "C in trace 1. With increasing admixture of lipid 2, the coliposomes display increasing contributions of high-melting domains (Tc,50-56 "C), in which the AB chains of 1 are "isolated either by neighboring chains of 2 or front-to-back alignments with 1. The broadness or shoulders of the main transitions in traces 3-6 suggest that 2 or more lipid domains may coexist throughout the midrange coliposomal compositions, a conclusion consistent with the positions of the corresponding AB UV maxima (see below). The relative (AH) and van't Hoff (AHW)enthalpies of the thermal transitions change as a function of lipid composition (Table l), tracing the evolution of the stacked AB domains, present a t high mol % of 1, into isolated AB domains in coliposomes that are rich in 2. The stacked AB domains are associated with high molecular cooper-
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(14) NMR of 1 (200 MHz, 6,CDC13): 0.88 (m, 6 H, ~ C H ~ C H Z 1.2), 1.5 [s + m, 36 H, C H ~ ( C H Z )CH3(CHz)zl, ~~ 1.80(m, 6 H, OCHz(CH&CHzO-azobenzene-OCHZCHz), 3.4-3.7 (s + m, 16 H,(CH3)3NA+ CHOCHz + CHZOCHZ), 4.0-4.2(m, 6H,CHzN++ CHZO-azobenzeneOCHz), 6.96,7.83 (2 x AzBz, J = 9 Hz, 8 H, aromatic). (15) See Moss, R. A,; Bhattacharya, S.; Chattejee, S. J.Am. Chem. SOC.1989,111, 3680 for experimental details. (16)Marky, L.A,; Breslauer, K. J. Biopolymers 1987,26,1601.See also Breslauer, K. J.; Freire, E.; Straume, M. Meth. Enzymol. 1992,
+
211, 533.
Letters
Langmuir, Vol. 11, No. 11, 1995 4219
Figure 2. Schematic renderings of face-to-face(left)and faceto-back (right)orientationswithin liposomal 1 . For simplicity, only 1 leaflet of the bilayer is shown. case
1 2 3 4 5
6 7 8
Table 2. W Data for AB Liposomes molar ratio 1:2 I,,, nm % cis-la rel. isom. eff.b 1o:o 336 48 1.00 3:1 344 53 0.97 2:1 356 59 1.06 1:1 358 60 1.24 1:2 360 60 1:3 362 62 1:6 364 62 1:9 364 65 0.92'
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At photostationary state after irradiation; see text and note 19. Relative efficiency of trans cis photoisomerization as a function of the number of laser pulses; see text. ' The molar ratio of 1:2 was 1:lO.
10
20
30
40
50
60
70
EO
Temperature ("C)
Figure 1. Differential scanning calorimetry traces of 1/2 coliposomes in 10 mM Tris buffer, pH 8, p = 0.01 (KC1). See text for preparative details. The trace numbers are collated with the derived data in Table 1. Table 1. Differential Scanning Calorimetry of AB Liposonies
tracea 1 2 3 4 5 6 7 8
98
molar ratio, 1:2
1o:o 9:1 4:1 2:1 1:1 1:2 1:4 1:9 0:lO
Tc, "Cb 30,62 28,54 28,56 52 50 52 53 55 56
AH, AHvli, kcal/molc kcallmold 3.97,1.08 343,196 3.94,0.89 258,f 2.46,1.49 199,143 6.7 186 4.3 244 5.2 251 8.8 278 9.3 288 9.5 378
coop:
86,182 65,f 81,96 28 57 48 32 31 40
a See Figure 1. Critical or phase transition temperatweb); errors, f0.5"C. Heat absorbed in transition,calculated from areas
beneath heat absorptions and calibrated against an internal calibration circuit. Estimated errors, f5-10%. van't Hoff enthalpy, calculated from the DSC traces as described in ref 16. Estimated errors, &lo%. e Cooperativity = AHw/AH,a measure of how many lipid molecules are involved in a typical phase transition. Estimated error, f10-15%. f The feature was too broad to permit accurate calculation.
Shown at 0.5relative size in Figure
1.
ativities during their thermal transitions, whereas domains rich in lipid 2 manifest lower cooperativities. Spectroscopy and Photoisomerization. The absorption maxima of liposomal trans-azo-1appear in Table 2 as a function of lipid composition. The blue-shifted maximum of pure liposomal 1 (336 nm) can be associated with face-to-face stacked AB lipid domain^;^^^^^^ cf. Figure
2. The smooth bathochromic shift of A,, as liposomal 1 is adulterated with increasing quantities of lipid 2 , reflects the changeover to lipid domains in which AB chromophores are i ~ o l a t e dand l ~ ~parallels ~ ~ ~ the DSC results presented above, Figure 3 presents representations of trans- and cis-1 that were constructed by modification of the molecular framework of a n energy-minimized structure of ethylene glycol distearate, whose structure was designed to simulate the crystal packing geometry normally associated with phosph01ipids.l~Geometry optimizations of the modified AB lipids were carried out with the MM2* force field as implemented in MacroModel v. 4.0.18 It is readily apparent that the major geometric changes that accompany trans cis isomerization ought to perturb nearest neighbor packing within the liposomal leaflets, particularly if the "long"AB-containing chain were interdigitated across the bilayer midplane. In this light, and mindful of the AI3 photoisomerization difficulties encountered in LB f i l m ~ , l ~we , ~ were J ~ pleasantly surprised by the smooth photoisomerization that we observed with liposomal 1. Ambient temperature irradiation of liposomal 1 with a XeFz excimer laser at 351 nm (-300 14 ns pulses a t -50 mJlpulse) or a Rayonet reactor (16 8 W, 350 nm lamps, 1 min) afforded photostationary states, in which the absorbance of trans-1 sharply decreased and the n n* and n n* absorptions of cis-1 grew in a t -325 and -450 nm, respectively. Figure 4 illustrates this behavior for undiluted liposomal trans-1. Our observations are in good agreement with those reported by Shimomura and Kunitake for photoisomerizations in bilayers of single chain AB lipid^.^ Importantly, photoisomerization occurred in liposomes of all of the 112 compositions shown in Table 2 , from hololiposomal 1 (Figure 4) to 1:9 coliposomal 112. Very approximate trans-llcis-1 compositions of the photostationary states are indicated in Table 2.19 There may be a mild trend toward increased cis-1 upon greater dilution
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(17)Sauers, R. R. J. Chem. Educ. 1996, in press. (18)Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.; Caufield, C.; Chang, G.; Hendrickson, T.; Still,W. C. J.Comput. Chem. l s e O , l l , 440. (19)The photostationary state compositions are rough estimates derived from the absorbance of trans-1 before and after UVirradiation. Due to overlap with the cis-1 absorbance at 325 nm, these compositions overemphasize the quantity of trans-1.
4220 Langmuir, Vol. 11, No. 11, 1995
Letters
Figure 3.
0
0
m 0
N CC
a Im n
0 0 d
0 Ln d
9 0 L n
0 Ln UI
0 u3 0
WAVELENGTH (nm)
Figure 4. W spectra accompanying laser irradiation at 351 nm of hololiposomal trans-1. The absorption of trans-1 at 336 nm decreases while absorptions of cis-1 increase at 325 and 450 nm. From the highest absorbanceat 336 nm (0 laser shots) downwards, the succeeding absorbances correspond to 20, 50, 90, 140, 200, and 280-380 laser shots, respectively.
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of the liposome with lipid 2,consistent with more “room” for trans cis isomerization amid the less densely functionalized chains of liposomes rich in 2. It will be recalled that these liposomes also displayed lower intramolecular cooperativities during their phase transitions (Table 1). The most notable result, however, is the occurrence of photoisomerization in all cases. Not only was trans cis photoisomerization observed
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across the gamut of 1/2compositions but the efficiency of isomerization (obtained by correlating the incremental decrease of trans-1 absorbance with the number of 351 nm laser pulses) was effectively independent of the 1/2 composition (Table 2). These results were obtained at 25 “C,below the s;2’ of the liposomes, and presumably refer to gel phase domains. As previously observed, irradiation of cis-AB derivatives
Langmuir, Vol. 11, No. 11, 1995 4221
Letters
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Table 3. Thermal Cis Trans Isomerization of Liposomal '1 molar ratio
1:2
10:0
1:l 1:9 b a
in
105k,
s-'"C
10-loA, s-1
1.59 (20) 2.31 3.28(26) 102 1.47(21) 72.9 3.01 (26) 23.3
kcal/mol
E,,
AH+, kcaymol
As ', eu
20.3 22.7 22.6 21.8
19.7 22.1 22.0 21.2
-13.1 -5.59 -6.25 -8.52
Estimated errors a r e f3% i n k , f5% in Ea a n d H ,and f15%
As*.b Solution of 1 in acetonitrile.
with visible light restored the trans-AB isomer. Exposure of the photostationary state cis-lltrans-1 liposomes of Table 2 to visible light at 25 "C afforded the original trans-1 absorbances. Moreover, the apparent "rate constants" for this process ranged from 0.087 to 1.02 min-l over all the liposomal compositions; i.e., there was no compositional dependence. Therefore, both trans cis and cis -trans photoisomerizations of AB lipid 1 are facile and composition independent in gel phase domains of coliposomal 1 and 2 , as well as in hololiposomal 1. Thermal Cis Trans Isomerization. We also examined the kinetics of the thermal cis trans isomerization of AB lipid 1 in acetonitrile solution and in three liposomes of varying 112 composition. Reactions were initiated with photostationary state mixtures of cis- and trans-1, monitoring the growth of trans-1 by UV spectroscopy.20 Excellent first-order isomerization kinetics ( r > 0.99) were obtained over a range oftemperatures (7.750.9 f 0.2 "C); rate constants and derived activation parameters appear in Table 3. Again we observe relatively little dependence of the ease of isomerization on liposome compositionof these gel phase (T < 28 "C) liposomes. The temperature dependencies of the rate constants were assessed by Arrhenius analysis.
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(20) To minimize inadvertent photoisomerization, the monitoring beam of the Hewlett Packard 8,451Adiode array spectrophotometer was activated only for 0.1 s for each absorbance measurement.
Excellent linear correlations ofln k with 11Twere obtained ( r > 0.99), including pure liposomal 1, where the temperature range (19.9-50.9 "C)crossed the phase transition temperature (30 "C). The Lws and AS* values for the cis trans thermal isomerization of 1 in MeCN solution (Table 3) are quite comparable to those reported for the analogous isomerization of 4,4'-dimethoxyazobenzenein benzene (AH'+ = 21.1 kcaumol, AS*= -9.1 eu),21and also similar to the activation parameters for cis-1 isomerizations of 1:1and 1:9 coliposomes of 1 and 2 . In the case of hololiposomal 1 , however, AS* appears to be significantly more negative than the other activation entropies. This might reflect greater intermolecular steric constraints in the hololiposoma1 system, which also appear as greater cooperativity in its thermal transitions (see above). Conclusions. AB-chain lipids with the double chain structure exemplified by 1 undergo efficient photoisomerization and thermal azo isomerization in liposomes of varied composition, presumably via the spatially concise azo inversion mechanism.22 Neighboring chain interactions are more tolerant of isomerization in AB-chain liposomes, relative to AB monolayers or LB f i l m ~ , l ~ , ~ J l and AB-chain as well as AB-head groupa lipids readily isomerize within liposomes.
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Acknowledgment. We are grateful to the U.S. Army Research Office and the Busch Memorial fund of Rutgers University for financial support. We thank Professor Ronald Sauers for the MM2* calculations and Figure 3. We thank Dr. Slawomir Mielewczyk and Professor Kenneth Breslauer for helpful discussions and the use of their microcalorimeter. Professor S. Toby provided a helpful discussion on kinetics. JA950605M (21)Otruba, J. P., 111; Weiss, R. G. J . Org. Chem. 198.3,48,3448. (22) See Ikeda, T.; Tsutsumi, 0. Science 1995,268,1873. Rau, H.; Liiddecke, E. J . Am. Chem.SOC.1982,104,1616and references therein.
