Formation of highly charged quasi-molecular ions of a polycationic [60]fullerene hexakis-adduct and their fragmentation behavior in the gas phase

Article abstract of DOI:10.1002/ejoc.201500102

A novel polycationic [60]fullerene hexakis-adduct has been synthesized and investigated by electrospray ionization and tandem mass spectrometry. The polycationic ligand system comprises 12 pre-formed positive charges, compensated in the neutral molecule b

Full text of DOI:10.1002/ejoc.201500102

FULL PAPER
DOI: 10.1002/ejoc.201500102
Formation of Highly Charged Quasi-Molecular Ions of a Polycationic
[60]Fullerene Hexakis-Adduct and Their Fragmentation Behavior in the Gas
Phase
Jing Li,[a] Leanne C. Nye,[b] Lennard K. Wasserthal,[c] Chau Vinh,[c] Rolf W. Kirschbaum,[a]
[b]
[c]
[a]
´
´
Ivana Ivanovic-Burmazovic, Andreas Hirsch,* and Thomas Drewello*
Keywords: Fullerenes / Polycations / Electrospray ionization / Mass spectrometry
A novel polycationic [60]fullerene hexakis-adduct has been
synthesized and investigated by electrospray ionization and
tandem mass spectrometry. The polycationic ligand system
comprises 12 pre-formed positive charges, compensated in
the neutral molecule by bromide anions. Stable quasi-mo-
to 12+. Collision-induced dissociation experiments revealed
three fragmentation pathways. A dominant neutral loss
channel leads to daughter ions of the same charge state as
the highly charged precursor ion and singly charged daugh-
ter ions can be expelled from the polycations through two
lecular cations were obtained through the release of the an- distinct dissociation channels involving both the neutral and
ionic counter ions covering a charge-state envelope from 3+
the charge-carrying part of their ligands.
blocks each involving a Th-symmetrical addition pattern. It
contains a total of 60 amines as terminating groups. Thus,
in slightly acidic conditions the entire architecture carries
60 positive charges. The success in synthesizing such large
and highly symmetrical systems is based on our highly re-
gioselective method for generating hexakis-malonates of
C60 with an octahedral and Th-symmetric addition
pattern.[8–10] To generate an oligocationic fullerene deriva-
tive for which the number of positive charges is independent
of pH, as needed for transfectional studies, we targeted in
this study the synthesis of the dodecapyridinium derivative
1 (Figure 1). Given the need to attach the 12 charges in the
last step while being aware of the sensitivity of fullerenes
towards nitrogen nucleophiles, we needed a method that
would allow the reliable creation of each of the 12 charges.
Synthetic progress towards the production of new fuller-
ene-based nanostructures is connected to the successful ap-
plication of modern mass spectrometry.[11,12] However, the
generation of quasi-molecular ions of certain C60 hexakis-
adducts has proven difficult. Nierengarten and co-workers
encountered several examples[6,13] in which the target mo-
lecule was found too unstable for analysis by soft ionization
methods, including electrospray ionization (ESI). Problem
cases that proved inappropriate for analysis included, for
example, hexakis adducts with sugar termini[13] or with pre-
formed cations within the ligands.[6] Clearly, the ESI of such
systems is expected to lead to multiple charging either
through multiple protonation or though the release of
counter anions compensating the positive charges of the
pre-formed polycationic derivative. Coulombic repulsion
may have an additional destabilizing effect in such ions.
Also, the analysis by mass spectrometry of multifunction-
Introduction
The clinical success of gene therapy is highly dependent
upon the development of efficient delivery systems.[1–3] In
the past decades polycations have gained increasing atten-
tion as nonviral gene delivery vectors. The complex (poly-
plex) formed by the interaction of the polycation with DNA
enters the cell/nucleus and releases DNA for gene expres-
sion (transfection).[4] Research into the usage of poly-
cationic lipids and polymers as gene delivery systems has
been accompanied by investigations into the structural
requirements of polycationic fullerene derivatives for ef-
ficient transfection.[5] A recent extension to the fullerene-
based gene delivery systems in the form of a polycationic
[60]fullerene hexakis-adduct has been identified.[6]
We have recently reported on the synthesis of a globular
heptafullerene that is the largest well-defined and monodis-
perse polyelectrolyte known to date.[7] This heptafullerene
consists of one central and six peripheral fullerene building
[a] Physical Chemistry I, Department of Chemistry and Pharmacy,
Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU),
Egerlandstraße 3, 91058 Erlangen, Germany
E-mail: thomas.drewello@fau.de
[b] Bioinorganic Chemistry, Department of Chemistry and
Pharmacy, Friedrich-Alexander-Universität
Erlangen-Nürnberg (FAU),
Egerlandstraße 1, 91058 Erlangen, Germany
[c] Organic Chemistry II, Department of Chemistry and
Pharmacy, Friedrich-Alexander-Universität
Erlangen-Nürnberg (FAU),
Henkestraße 42, 91054 Erlangen, Germany
E-mail: andreas.hirsch@fau.de
http://www.chemie.uni-erlangen.de/hirsch/index.html
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500102.
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Quasi-Molecular Ions of a Polycationic Fullerene Hexakis-Adduct
Figure 1. Structure of the polycationic [60]fullerene hexakis-adduct and a simplified version of it in which the pyridinium cations are
represented by white squares and the bromide anions by black circles.
alized cationic fullerene adducts (the so-called Prato ad- bromide anions. Figure 1 also contains a simplified repre-
ducts) has been found too complex for a meaningful analy- sentation of the compound under study in which the pyrid-
sis.[4]
inium cationic parts are represented by white squares and
In this paper we report on the successful electrospray for- the anionic counter ions by black circles to allow for a
mation of stable, multiply charged ions of a pre-formed po- quick identification of the charge state of the respective
lycationic hexakis-adduct of C60, namely quasi-molecular ions.
ions with 3 to 12 charges. The fragmentation behavior of
the molecular polycations has been studied in collision-in-
duced dissociation (CID) experiments.
Results and Discussion
Figure 1 displays the polycationic fullerene derivative un-
der investigation. The C60 core contains six malonate li-
The initial concepts for the synthesis of Th-symmetrical
gands attached in the well-established octahedral addition hexakis-adducts of C60 that we previously developed[8–10]
pattern (hexakis-adduct).[14] Each end of the malonate li- and were later modified by other groups[15,16] turned out to
gand extends until terminated by cationic pyridinium moie- be inefficient for the synthesis of the dodeca-cationic target
ties. The resulting 12 positive charges are compensated by molecule 1. The quaternization leading to the formation of
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the desired pyridinium had to be the final step because terns, leaves no doubt regarding the proposed identity of
chromatographic purification on silica of such highly polar the molecular polycations. The recorded isotope patterns of
and water-soluble adducts is impossible. For this reason a all the observed quasi-molecular ions can be compared with
comparatively nonpolar precursor hexakis-adduct had to be the simulations in Figure S1 in the Supporting Information
generated. The malonate 4 equipped with two precursor and the assignments can be confirmed.
groups that we reported recently[17] (Scheme 1) could be at-
The dissociation behavior of the polycations is discussed
tached in the desired way, however, chromatographic sepa- in the following. Polycations with the charge states 4+ to
ration from the reaction mixtures obtained by using the pre- 9+ were selected and submitted to collision-induced dissoci-
vious synthetic protocols[7–10] failed because its surface po- ation (CID). The collisions were performed with N2 in a
larity is very similar to those of side-products such as penta- collision cell located between the mass-selecting quadrupole
kis-adducts. To circumvent this problem and to simplify the (q) and the fragment ion-recording time-of-flight (ToF)
purification we first generated the oxazoline-protected part of a qToF mass spectrometer. Figure 3 shows the par-
mono-adduct 5 (Scheme 2), which was subsequently con- tial CID mass spectra of the polycations covering the tetra-
verted into the mixed hexakis-adduct 6 following a new pro- cation (top right) to the nona-cation (bottom left). The CID
cedure that we developed very recently.[18] The chromato- spectra reveal a peculiar pattern in which the number of
graphic separation of 6 was then easy to accomplish. The daughter ion signals corresponds to the number of bromide
protecting group was subsequently removed upon UV irra- anions and thus to the number of charge-compensated
diation to form the pentakis-adduct 7[18] and a sixth addend (neutral) ligands within the precursor ion. That is, the
4 was allowed to bind to the remaining octahedral [6,6]- nona-cation with three neutral ligands shows three daugh-
double bond of 7 by using modified cyclopropanation con- ter ions and the tri-cation possessing nine neutral ligands
ditions.[19] The final steps were the conversion of the sym- shows nine fragment ion signals, which leads to the conclu-
metrical hexakis-adduct 8 into the dodecabromide 9 and sion that the fragmentations occur exclusively within the
the subsequent quaternization with 4-tert-butylpyridine.[17]
neutral ligands. Moreover, the daughter ions retain the
charge state of the dissociating parental polycation. For the
CID experiments the particular parent ion was selected by
covering all its isotopomers so that the daughter ions would
also show the full isotope pattern. The charge state is
straightforwardly calculated from the m/z spacing between
the individual isotope peaks of one ion. The procedure is
shown in Figure 3 (insert a) for the daughter ion signals of
the hexa-cationic parent ion. Also evident is the perfect
match of the recorded (top trace) and calculated (bottom
trace) isotope patterns with the assumed elemental compo-
sition (see below) of a daughter ion that still possesses six
positive charges just like its parent ion. The elemental com-
position of the fragment ion was derived by establishing the
mass of the neutral entity that has been expelled from the
Scheme 1. Synthesis of the malonate ligand precursor 4.
Figure 2 displays the mass spectrum that was obtained ion in the dissociation. Figure 3 (insert b) illustrates the
when the title compound was electrospray ionized as a procedure by which the mass of the neutral fragment has
1.7ϫ10–4 molL–1 methanol solution at a capillary entrance been established. Within the isotope pattern of two adjacent
voltage of –4500 V. Although a tremendous amount of frag- fragment ions the peaks that would contain the same iso-
mentation is evident, quasi-molecular ions are clearly ob- topes have been identified by comparing the overall appear-
served in enhanced abundances. Through the loss of brom- ance of both patterns. The mass difference between two
ide anions, the corresponding multiply charged cations are such peaks corresponds to the mass of the released neutral
produced, covering the charge states of 3+ in the high m/z fragment. The peak differences between several of the more
region to the 12+ cation in the low m/z region. The charge abundant signals were used to establish an average value of
states 4+, 5+, 6+, 7+, and 8+ can be observed in abun- the neutral mass, which was obtained as Δm = 135 Da,
dance, whereas the charge states 3+ and 9+ are low in inten- which we have assigned to the loss of 4-tert-butylpyridine.
sity, and those of 10+ to 12+ show only minor abundance. The fragmentation behavior of the parent polycations is il-
The inserts in Figure 2 display enlargements of the quasi- lustrated in Scheme 3. Following the reaction path a, the
molecular ion region of selected polycations to allow com- nitrogen-centered pyridinium cation at the ligand terminus
parison of the recorded (top trace) and simulated signals attracts the electron pair that connects the pyridinium cat-
(bottom trace). The spectrum was recorded with a resolu- ion to the rest of the chain, whereupon it is released as a
tion of approximately 35000 so that the isotope pattern of neutral entity (Δm = 135 Da). The positive charge would be
the signals could be clearly resolved for all the charge states transferred initially to the new terminal carbon atom. That
observed. The corresponding mass accuracy was typically the resulting primary carbenium ion rearranges to a tropyl-
slightly better than 1 ppm, which, together with the excel- ium ion is a speculative assumption that has to be seen in
lent agreement between recorded and simulated isotope pat- the light of recent investigations into the equilibria of sub-
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Quasi-Molecular Ions of a Polycationic Fullerene Hexakis-Adduct
Scheme 2. Synthesis of the polycationic molecule 1.
stituted benzylium/tropylium ions.[20,21] The bromide anion the binding electron pair by the nitrogen (as described pre-
would remain within the ionic fragment so that no change viously) can also be compensated by the bond that is
in the charge state would result and the reaction could oc- formed by the nucleophilic attack of the bromide ion. The
cur as often as there are intact ligands in the ion. Another result of such a substitution reaction would be the forma-
possibility is a substitution mechanism. The withdrawal of tion of a covalent C–Br bond (Scheme 3, reaction aЈ). It
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Figure 2. High-resolution positive-ion ESI mass spectrum of the polycationic [60]fullerene hexakis-adduct. The inserts display the recorded
(top trace) and simulated (bottom trace) isotope patterns of selected charge states of the quasi-molecular ions, together with the mass
accuracy (in ppm) achieved for selected peaks.
should be noted that a second, lower abundant series of
ions is observed in the CID spectra (Figure 3). We attrib-
uted these ions to the uptake of a residual, gaseous neutral
molecule with a mass of 60 Da within the collision cell. This
assignment is based on several circumstantial indications.
These include 1) a much lower mass accuracy compared
with the fragment ions discussed above, 2) the fact that the
extent to which these ions are observed differs greatly when
different instruments of the same type are employed, and
3) for some ions multiple additions of 60 Da could be iden-
tified.
In addition to the loss of the substituted pyridine, two
other reactions occurred during CID leading to the detec-
tion of singly charged ions at m/z = 309 and 389/391. The
signals are shown in Figure 4, which displays the CID spec-
tra of the charge state 6+ quasi-molecular ion obtained at
laboratory collision energies of 150, 180, and 210 eV. Upon
increasing the collision energy, the number of pyridines lost
increases from one molecule at 150 eV to all six at 210 eV,
which underpins the successive nature of these dissociations
Scheme 3. Illustration of the different fragmentation pathways oc-
curring after collisional activation. Reaction a and aЈ: the loss of
and that the singly charged ions are formed more abun-
dantly. The 1:1 isotope pattern of the m/z = 389/391 doublet
4-tert-butylpyridine; reaction b: the formation of a singly charged
indicates the presence of one bromine atom. To form the
species at m/z = 389/391, the complete branch of the ligand
of the malonate group must be released, including the
ion at m/z = 389/391; reaction c: the formation of a singly charged
ion at m/z = 309.
bromide anion. Pathway b in Scheme 3 illustrates this reac- tive charge, a second charge must be created during the
tion. Because the bromide formally compensates one posi- dissociation for m/z = 389/391 to appear as a positively
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Quasi-Molecular Ions of a Polycationic Fullerene Hexakis-Adduct
Figure 3. Partial CID mass spectra of the polycationic quasi-molecular ions covering the charge states 4+ (top right) to 9+ (bottom left).
a) Fragment ion of the charge state 6+ precursor comparing the recorded isotope pattern (top trace) with the simulation (bottom trace).
Establishment of the charge state (z) is also shown. b) Adjacent fragment ion signals obtained from the charge state 7+ precursor.
Establishment of the neutral loss is illustrated. The uptake of a neutral with m = 60 Da corresponds to the signals seen between m/z =
747 and 749.
charged fragment ion. This is suggested to occur by a het-
A more likely pathway to the singly charged ion at m/z
erolytic cleavage of the O–C bond of the ester functionality = 309 proceeds through another unusual decay, this time
of an intact ligand. The resulting carboxylate anion would involving the charged ligand of the polycation, which also
reduce the charge of the polycationic fullerene fragment by undergoes bond cleavage at the ester functionality, however,
one. Therefore the formation of a singly charged ion at m/z accompanied by hydrogen transfer to the polycationic frag-
= 389/391 must be accompanied by the production of a ment ion. Pathway c in Scheme 3 illustrates the reaction.
complementary multiply charged fragment ion carrying one As with the formation of the cation at m/z = 389/391, the
charge less than the precursor ion. Whether the dissociation formation of the singly charged ion at m/z = 309 must also
mechanism proceeds as suggested cannot be answered by be accompanied by the formation of the corresponding po-
these experiments. The reaction may even be facilitated by lycationic fragment ion carrying one charge less than the
the involvement of the bromide anion or proceed through precursor polycation. Unfortunately, in both cases we have
more complex electron movements. The structures of the been unable to trace these polycationic fragment ions,
ions at m/z = 309 and 389/391 shown in Scheme 3 are which are most probably too low in abundance. Although
speculative.
the singly charged ion at m/z = 309 can be formed from any
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Instrumentation: TLC: Merck silica gel 60 F254. Detection: UV
lamp 254 nm; 1% KMnO4 in 1% KOH. Flash chromatography
(FC): Merck silica gel 60 (230–400 mesh, 0.04–0.063 nm, deacti-
vated. NMR spectroscopy: JEOL JNM GX 400, Bruker Avance
300, and Bruker Avance 400 spectrometers. The chemical shifts are
given in ppm relative to TMS. The resonance multiplicities are indi-
cated as s (singlet), d (doublet), t (triplet), q (quartet), and m (mul-
tiplet). UV/Vis spectroscopy: Varian Cary 5000 spectrophotometer.
IR spectroscopy: Bruker Vektor 22 spectrometer with ATR-Di-
Comp-detector. HPLC (preparative): Shimadzu; pump: a single
LC-8 A; column: Nucleosil 100–5. To multicycle the substance, the
outlet could be switched to the pump inlet while excluding the in-
jector circuit. Detector: SPD-10 A; wavelengths detected: 300,
314 nm.
Electrospray ionization: ESI was performed on methanol solutions
of the sample at a concentration of 1.0 gL–1 (1.7ϫ10–4 molL–1)
employing a syringe pump (KDS 100 pump) at a flow rate of
3 mLmin–1 at a source temperature of 180 °C (qToF). High-resolu-
tion mass spectra were obtained with a large-scale quadrupole/
time-of-flight (qToF) instrument (maXis, Bruker) at a mass accu-
racy of typically just better than 1 ppm in MS1 experiments. Colli-
sion-induced dissociations (CID, MS2) were conducted in a colli-
sion cell located between the mass-selecting quadrupole (q) and the
fragment ion-recording time-of-flight (ToF), following mass selec-
tion and preceding the high-resolution daughter-ion analysis in the
ToF analyzer. Nitrogen was used as the collision gas. The labora-
tory collision energy was varied between zϫ10 and zϫ40 eV (z
being the charge state number). Regarding the mechanism of ion
formation, it appears that the title compound has a rather low de-
gree of dissociation into its ionic components when in solution as
the spray and transfer conditions were harsher than under the
“normal” conditions used, for example, for the protonation of
peptides by ESI. The spray and analyzer were interfaced by a dual
funnel system that is capable of transferring ions very gently. We
assume that most ions are formed by Br– loss during the ESI pro-
cess rather than being already present in solution. Furthermore, for
good quality spectra, the sample concentration had to be one to
two orders higher compared with solutions of inorganic salts or
ionic liquids,[22] which are known to be dissociated in solution.
Figure 4. CID mass spectra of the charge state 6+ quasi-molecular
ion at laboratory collision energies of a) 150, b) 180, and c) 210 eV.
of the other ions seen in the CID spectrum, the correspond-
ing multiply charged fragment ion of each dissociation will
have with each intermediate precursor ion a different mass
and thus much lower abundance. Additionally, the greater
number of atoms in the multiply charged fragment ions
means more isotope peaks. Because the ion abundance cor-
relates with the sum of all isotope intensities, the broader
isotope pattern leads to a much lower intensity of each iso-
tope peak.
Synthesis
Compound 2: 3-Nitrobenzyl methyl ether (25 g, 0.1496 mol) was
dissolved in EtOH (300 mL). Hydrazine hydrate (150 g, 145 mL,
20 equiv.) was added under nitrogen, followed by Pd/C (10%,
300 mg). After 24 h, the solvent was evaporated and the product
purified by simple distillation, yield 99%. 1H NMR (400 MHz,
CDCl3): δ = 7.11 (m, 1 H, aniline-5), 6.71 (m, 1 H, aniline-4), 6.66
(s, 1 H, aniline-2), 6.59 (m, 1 H, aniline-6), 4.36 (2 H, CH2-O),
3.66 (2 H, NH2), 3.36 (3 H, CH3-O) ppm. 13C NMR (100.5 MHz,
CDCl3): δ = 146.5 (1 C, aniline-1), 139.4 (1 C, aniline-3), 129.2 (1
C, aniline-5), 117.8 (1 C, aniline-4), 114.3, 114.2 (2 C, aniline-2,6),
74.6 (1 C, CH2-O), 58.0 (1 C, O-CH3) ppm.
Conclusions
Electrospray ionization of a novel polycationic [60]fuller-
ene hexakis-adduct produces stable quasi-molecular ions
through the loss of its counter anions. Low-energy colli-
sion-induced dissociations involve neutral loss and charge
permutation reactions in which singly charged fragments
are expelled from the polycations.
Experimental Section
Compound 3: Bis[3-(tert-butyloxycarbonyl)propyl] malonate[7] was
dissolved in a quadruple volume of 100% formic acid. The mixture
was stirred at room temperature for 2 d. After evaporation of the
formic acid, traces of formic acid were removed by repeated azeo-
tropic distillation with toluene, and the toluene removed in vacuo,
Materials and Reagents: All chemicals were purchased from chemi-
cal suppliers and used without further purification. CBr4 was pur-
chased as the unstabilized (water-free) form. Except for toluene,
solvents were purified from raw industrial solvents by distillation.
Alcohols were distilled from CaO, methylene dichloride and ethyl
acetate were distilled from K2CO3. For dry conditions, HPLC-
grade solvents were used. Toluene was used as HPLC-grade to
avoid thiophene impurities.
1
yield quantitative. H NMR (400 MHz, CDCl3): δ = 5.28 (s, 2 H,
COOH), 4.20 (m, 4 H, CH2O), 3.36 [2 H, CH2(COO)2], 2.44 (m, 4
H, CH2O), 1.99 (m,
(100.5 MHz, CDCl3): δ = 178.9 (2 C, COOH), 166.3 [2 C,
4
H, CH2CH2CH2) ppm. 13C NMR
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Quasi-Molecular Ions of a Polycationic Fullerene Hexakis-Adduct
C(COO)2], 64.4 (2 C, CH2-O), 41.5 [1 C, CH2(COO)2], 30.4 (2 C, 879 (m), 791 (m), 763 (w), 733 (s), 713 (w), 696 (m) cm–1. UV/Vis
CH2COOH), 29.6 (2 C, CH2CH2CH2) ppm.
(CH2Cl2): λ (ε) = 244 (158000), 277 (53000), 283 (50000), 314
(41000), 334 nm (30000 m–1 cm–1).
Compound 4: Bis(3-carboxypropyl) malonate (3; 50 mmol, 13.635 g,
1 equiv.) and 3-methyoxymethylaniline (2; 0.11 mol, 15.1 g,
2.2 equiv.) were dissolved in dichloromethane. After cooling to
0 °C, 1,3-dicyclohexylcarbodiimide (DCC; 45.393 g, 2.2 equiv.) and
N,N-dimethylaminopyridine (10 mmol, 0.252 g, 0.2 equiv.) were
added. The reaction mixture was stirred for 36 h. The precipitate
was removed and the mixture purified by column chromatography
(SiO2; ethyl acetate/hexanes, 3:1). The solvent was evaporated and
traces of ethyl acetate removed in vacuo, yield 30%. 1H NMR
(300 MHz, CDCl3, r.t.): δ = 8.04 (s, 2 H, N-H), 7.49 (s, 2 H, aniline-
Compound 7: The [5,1]-hexakis-adduct
6 (100 mg, 29 μmol,
1 equiv.) was dissolved in dry dichloromethane (50 mL). Maleic an-
hydride (85 mg, 30 equiv., 870 μmol) was added and the mixture
was degassed thoroughly. The mixture was irradiated with a
500 W UV lamp under cooling at 0 °C for 24 h, followed by plug-
filtration (silica; dichloromethane/THF, 1:2) and column
chromatography (silica; toluene/dichloromethane/ethyl acetate/eth-
anol 3.66:2:3.33:0.95). The product is air-sensitive in the presence
of light, yield 90%. 1H NMR (300 MHz, CDCl3): δ = 8.77–8.15
3
3
3
3
2), 7.43 [d, J(H,H) = 9 Hz, 2 H, aniline-6], 7.24 (t, JH,H = 9 Hz,
(m, 5 H, N-H), 7.51 (s, JH,H3 = 8 Hz, aniline-2), 7.33 (d, JH,H
=
3
3
2 H, aniline-5), 7.03 (d, JH,H = 9 Hz, 2 H, aniline-4), 4.37 (s, 4 H,
8 Hz, aniline-6), 7.22 (t, JH,H = 8 Hz, 10 H, aniline-5), 7.03 (t,
3JH,H = 8 Hz, 10 H, aniline-4), 4.36.4.33 (m, 20 H, CH2-O-CH3),
4.24 (m, 20 H, CH2-O-C=O), 3.33 (s, 30 H, CH3), 2.4 (20 H,
CH2C=O), 2.05 (20 H, CH2CH2CH2) ppm. 13C NMR (75 MHz,
CDCl3): δ = 170.9 (10 C, N-C=O), 167.2 (10 C, O-C=O), 148.4,
146.8, 145.9, 145.6, 144.7, 144.5, 144.19, 144.17, 143.8, 143.0,
142.0, 149.8, 139.7 (50 C, C60-sp2), 138.9 (10 C, aniline-1), 138.2
(10 C, aniline-3), 128.9 (10 C, aniline-5), 123.4 (10 C, aniline-4),
119.4, 119.2 (20 C, aniline-2,6), 74.3 (6 C, CH2-O-CH3), 69.8,
69.43, 69.37, 69.1 (10 C, C60-sp3), 65.4 (10 C, CH2-O-C=O), 53.4,
51.0 [5 C, C(C=O)2], 33.1, (10 C, CH2-C=O), 24.1 (10 C,
CH2CH2CH2) ppm. MS (MALDI-ToF): m/z = 3307 [M + Na]+.
UV/Vis (CH2Cl2): λ (ε) = 277 (48000), 282 (46000), 315 (22000),
341 nm (13000 m–1 cm–1).
CH2-O-CH3), 4.23 (m, 4 H, CH2-O-C=O), 3.37 [s, 2 H, CH2-
(C=O)2], 3.33 (s, 6 H, CH3), 2.43 (m, 4 H, H2CC=O), 2.07 (m, 4
H, CH2CH2CH2) ppm. 13C NMR (75 MHz, CDCl3, r.t.): δ = 170.5
(2 C, N-C=O), 166.8 (2 C, O-C=O), 139.1 (2 C, aniline-1), 138.1
(2 C, aniline-3), 129.0 (2 C, aniline-5), 123.4 (2 C, aniline-4), 119.1,
119.0 (4 C, aniline-2,6), 74.3 (2 C, CH2-O-CH3), 64.6 (2 C, CH2-
O-C=O), 58.1 (2 C, CH3), 41.6 [1 C, C(C=O)2], 33.5 (1 C, CH2-
C=O), 24.4 (2 C, CH CH CH ) ppm. FTIR (ATR, r.t.): ν = 3380–
˜
2
2
2
3210 (m), 2990–2800 (m), 1730 (s), 1660 (s), 1610 (m), 1590 (m),
1550 (s), 1490 (m), 1440 (m), 1380 (w), 1330 (m), 1300 (w), 1270
(s), 1190 (s), 1150 (s), 1090 (s), 1030 (s), 960 (w), 920–890 (w), 880
(m), 790 (s), 730 (m), 700 (s), 650–560 (w) 540 (w) cm–1.
Compound 6: Fullerene mono-adduct
5 (200 mg, 225 μmol,
1 equiv.) was dissolved in a dry, degassed mixture of 1,2,4-trimeth-
ylbenzene (20 mL) and chloroform (20 mL). 9,10-Dimethyl-
anthracene (DMA; 557 mg, 2.70 mmol, 12 equiv.) was added and
Compound 8: The pentakis-adduct 7 (37 mg, 11.3 μmol) was dis-
solved in dry dichloromethane (1 mL). Malonate 4 (200 mg,
389 μmol, 35 equiv.) and CBr4 (37.0 mg, 113 μmol, 10 equiv.) were
added, followed by the dropwise addition of P1-tBu (5.3 mg,
22.5 μmol, 2 equiv.) in dichloromethane (1 mL) over 15 min. The
mixture was stirred overnight, plug-filtered (silica; dichlorometh-
ane/THF, 1:2), adsorbed on silica, washed with dichloromethane/
ethyl acetate (1:3), eluted with dichloromethane/THF (1:2), and
the mixture stirred for 4 h. Malonate
4 (2.20 g, 4.28 mmol
19 equiv.) was added to this mixture, followed by the dropwise ad-
dition of CBr4 (895.4 mg, 2.70 mmol, 12 equiv.) and P1-tBu[23]
(679 mg, 734 μL, 12 equiv.) in trimethylbenzene/chloroform (1:1).
After stirring the biphasic system for 8 d, the mixture was plug-
filtered with dichloromethane/THF (1:1), adsorbed onto silica and
then washed with dichloromethane/ethyl acetate (1:3), eluted with
dichloromethane/THF (1:2), and purified by column chromatog-
raphy (silica; toluene/dichloromethane/ethyl acetate/ethanol
3.66:2:3.33:0.95) and then multicyclic HPLC (12 cycles per run) on
Nucleosil with the same eluent, yield 32%. 1H NMR (300 MHz,
CDCl3): δ = 8.75 (10 H, N-H), 7.52 (10 H, m-aniline-2), 7.38 (d,
3JH,H = 7 Hz, 10 H, aniline-6), 7.17 (t, 3JH,H = 7 Hz, 10 H, aniline-
1
isolated in vacuo, yield 93%. H NMR (400 MHz, CDCl3, r.t.): δ
= 8.66 (s, 12 H, N-H), 7.53 (s, 12 H, aniline-2) 7.36 (m, 12 H,
aniline-6), 7.17 (m, 12 H, aniline-5), 6.99 (m, 12 H, aniline-4), 4.33
(s, 24 H, CH2-O-CH3), 4.26 (m, 24 H, CH2-O-C=O), 3 (s, 36 H,
CH3), 2.36 (m, 24 H, CH2CO), 2.02 (m, 24 H, CH2CH2CH2) ppm.
13C NMR (100.6 MHz, CDCl3, r.t.): δ = 171.2 (12 C, N-C=O),
164.14 [12 C, C(C=O)2], 146.1, 141.6 (48 C, C60-sp2), 139.4 (12 C,
aniline-1), 138.6 (12 C, aniline-3), 129.3 (12 C, aniline-5), 123.9 (12
C, aniline-4), 119.9 (24 C, aniline-2,6), 74.80 (12 C, CH2-O-CH3),
69.6 (12 C, C60-sp3), 66.9 (12 C, H2C-O-C=O), 58.6 (12 C, OCH3),
46.1 [12 C, C(C=O)2], 33.4 (12 C, CH2-C=O), 24.5 (12 C,
CH2CH2CH2) ppm. UV/Vis (CH2Cl2): λ (ε) = 246 (294000), 260
(181000), 278 (101000), 316 (59000), 334 (48000), 378 (12000),
399 nm (8000 m–1 cm–1).
3
5), 6.99 (d, JH,H = 7 Hz, 10 H, aniline-4), 4.30 (20 H, CH2-O-
CH3), 4.16 (20 H, CH2-O-C=O), 3.28 (30 H, O-CH3), 2.36 (20 H,
CH2-C=O), 1.98 (20 H, CH2CH2CH2) ppm. 13C NMR (75 MHz,
CDCl3): δ = 170.87 (10 C, N-C=O), 167.03 (10 C, O-C=O), 154.4
(1 C, dimethylaniline-1) 152.7 (1 C, C=N), 146.68, 146.64, 146.32,
145.92, 145.8, 145.19, 145.15, 145.09, 144.94, 144.06, 143.44,
143.44, 142.91, 142.00, 141.76, 141.68, 141.25, 141.02, 140.14,
139.49, 139.32, 139.18, 138.98 (48 C, C60-sp2), 138.8 (10 C, m-anil- Compound 9: The hexakis-adduct 8 (88 mg, 23.1 μmol) was dis-
ine-1), 138.1 (10 C, m-aniline-3), 135.14 (2 C, dimethylaniline-3),
129.4 (1 C, dimethylaniline-4), 128.7 (10 C, m-aniline-5), 123.2 (10
C, m-aniline-4), 119.3 (2 C, dimethylaniline-2), 119.1 (20 C, m-anil-
ine-2,6), 74.2 (10 C, CH2-O-CH3), 69.74, 69.69, 69.5, 69.1, 67.8,
solved in dichloromethane (3 mL) and a dry, 48% solution of HBr
in acetic acid (3 mL) was added. The mixture was stirred for 3 d.
The mixture was poured into water and diluted with dichlorometh-
ane. Traces of HBr were removed with solid potassium hydrogen
67.3 (10 C, C60-sp2), 66.4, 65.8 (2 C, C60-oxazoline), 65.3 (10 C, carbonate. After filtration, the solvent and acetic acid were evapo-
CH2-O-C=O), 57.9 (10 C, O-CH3), 45.4, 44.5, 41.9 [5 C, C(C=O) rated and the sample was washed with water. After addition of first
2], 39.8 (2 C, N-CH3), 32.9 (10 C, CH2-C=O), 24.0 (10 C,
dichloromethane, then toluene and evaporation three times, the
substance was immediately dissolved in dry THF and subjected to
the next step, yield 89%. H NMR (100.6 MHz, [D6]THF, r.t.): δ
CH2CH2CH2) ppm. MS (ESI-ToF): m/z = 1745.6 [M + K]+. FTIR
1
(ATR, r.t.): ν = 3306 (m), 3151 (w), 3087 (w), 2961 (w), 2961 (w),
˜
2926 (m), 2855 (w), 2822 (w), 2360 (m), 2341 (w), 1742 (m), 1664
= 9.33 (12 H, N-H), 7.70 (s, 12 H, aniline-2), 7.53 (m, 12 H, aniline-
(m), 1610 (m), 1596 (w), 1549 (w), 1488 (w), 1442 (w), 1374 (m), 6), 7.18 (12 H, aniline-5), 7.03 (12 H, aniline-4), 4.46 (s, 24 H,
1298 (w), 1260 (s), 1216 (s), 1167 (w), 1088 (s), 1026 (m), 898 (w), CH2Br), 4.34 (m, 24 H, CH2O), 2.88 (24 H, CH2-C=O), 2.06 (24
Eur. J. Org. Chem. 2015, 2282–2290
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2289

A. Hirsch, T. Drewello et al.
FULL PAPER
H, CH2CH2CH2) ppm. 13C NMR (100.6 MHz, [D6]THF, r.t.): δ =
171.5 (12 C, C=O-N), 164.5 (12 C, O-C=O), 147.1, 142.4 (48 C,
C60-sp2), 141.0 (24 C, aniline-1,3), 130.0 (12 C, aniline-5), 125.0 (12
C, aniline-4), 121.1, (12 C, aniline-6), 120.4 (24 C, aniline-2), 70.5
(12 C, C60-sp3), 68.5 (12 C, CH2-O), 46.9 [6 C, C(C=O)2], 34.5 (12
C, CH2-Br), 34.0 (12 C, CH2-C=O), 26.7 (12 C,
CH2CH2CH2) ppm. UV/Vis (CH2Cl2): λ (ε) = 280 (100), 315 (59),
336 nm (46 a.u.).
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Compound 1: The deprotected hexakis-adduct 9 (90 mg, 20 μmol)
was dissolved in dry THF (3 mL). 4-tert-Butylpyridine (34 mg,
0.2464 μmol, 12 equiv.) and dichloromethane (3 mL) were added
and the mixture stirred for 3 d. Dichloromethane (10 mL) was
added and the mixture stirred for a further 3 d. The liquid was
removed and THF and further 4-tert-butylpyridine (34 mg,
0.2464 μmol, 12 equiv.) were added. The supernatant was removed
and the sample was washed with dichloromethane and diethyl
1
ether. H NMR (300 MHz, CD3OD, r.t.): δ = 8.98 (24 H, pyridyl-
3), 8.14 (24 H, pyridyl-2), 7.83 (12 H, aniline-2), 7.63 (12 H, aniline-
6), 7.34 (12 H, aniline-5), 7.20 (12 H, aniline-4), 5.81 (s, 24 H,
CH2N+), 5.52 (s, 12 H, N-H), 4.4, 4.3 (m, 24 H, CH2O), 2.52 (24
H, CH2CO), 2.04 (24 H, CH2C=O), 1.43 (24 H,
CH2CH2CH2) ppm. 13C NMR (100.6 MHz, CD3OD, r.t.): δ =
173.1 (24 C, pyridyl-4, N-C=O), 164.7 (12 C, O-C=O), 147.0 (24
C, C60-sp2), 145.2 (24 C, pyridyl-3), 142.5 (24 C, C60-sp2), 141.0 (12
C, aniline-1), 135.3 (12 C, aniline-3), 131.2 (12 C, aniline-5), 126.9
(12 C, pyridyl-2), 125.3 (12 C, aniline-4), 122.3 (12 C, aniline-2),
121.4 (12 C, aniline-6), 70.5 (12 C, C60-sp3), 67.92 (12 C, CH2O),
64.6 (12 C, Ph-C-N+), 47.2 [6 C, C(C=O)2], 37.6 (12 C, CH2-C=O),
34.2 [12 C, C(CH3)3], 30.3 (36 C, CH3), 25.5 (12 C,
CH2CH2CH2) ppm. UV/Vis (MeOH): λ (ε) = 282 (100), 318 (54),
335 nm (43 a.u.).
Supporting Information (see footnote on the first page of this arti-
cle): UV/Vis, NMR and HRMS spectra of the target compound,
and NMR spectra of the precursor compounds.
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Received: January 21, 2015
Acknowledgments
The authors thank the Deutsche Forschungsgemeinschaft (DFG) –
SFB 953 “Synthetic Carbon Allotropes” for financial support.
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Published Online: February 19, 2015
2290
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© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 2282–2290