Synthesis of a biotinated lipofullerene as a new type of transmembrane anchor

Article abstract of DOI:10.1002/1099-0690(200004)2000:7<1173::AID-EJOC1173>3.0.CO;2-I

As a prototype for a new class of lipid membrane components, the lipophilic fullerene derivative (so-called lipofullerene) 1 was synthesized and characterized. This mixed [1:5]hexakisadduct consists of ten long alkyl chains within five didodecyl malonate addends and a linker malonate carrying a (+)-biotin unit as part of an amphiphilic spacer. The malonates are attached to the fullerene core in an octahedral addition pattern, which was achieved by two successive cyclopropanation sequences with the functional precursor malonate 8 and dodecyl malonate 10. The final step of the synthesis of 1 was the attachment of activated biotin 5 with the deprotected precursor 11. Binding experiments followed by reflectometric interference spectroscopy [RIfS] proved the capability of 1 to bind specifically the protein streptavidin (SA) through the biotin unit. The amphiphilic behavior of 1 was demonstrated by Langmuir Blodgett (LB) film investigations.

Full text of DOI:10.1002/1099-0690(200004)2000:7<1173::AID-EJOC1173>3.0.CO;2-I

FULL PAPER
Synthesis of a Biotinated Lipofullerene as a New Type of Transmembrane
Anchor
[
a]
[a]
[a]
[a]
[b]
Martin Braun, X. Camps, Otto Vostrowsky, Andreas Hirsch,* Emil Endreß,
[
b]
[c]
[c]
Thomas M. Bayerl, Oliver Birkert, and Günter Gauglitz
Keywords: Biotin / Fullerenes / Membranes / Nanostructures / Reflectometric interference spectroscopy
As a prototype for a new class of lipid membrane compon- panation sequences with the functional precursor malonate
ents, the lipophilic fullerene derivative (so-called lipofuller- 8 and dodecyl malonate 10. The final step of the synthesis of
ene) 1 was synthesized and characterized. This mixed [1:5]- 1 was the attachment of activated biotin 5 with the depro-
hexakisadduct consists of ten long alkyl chains within five
didodecyl malonate addends and a linker malonate carrying
a (+)-biotin unit as part of an amphiphilic spacer. The malon-
ates are attached to the fullerene core in an octahedral addi-
tected precursor 11. Binding experiments followed by re-
flectometric interference spectroscopy [RIfS] proved the ca-
pability of 1 to bind specifically the protein streptavidin (SA)
through the biotin unit. The amphiphilic behavior of 1 was
tion pattern, which was achieved by two successive cyclopro- demonstrated by Langmuir Blodgett (LB) film investigations.
Introduction
advantage of the pronounced noncovalent binding of the
lipofullerenes within the lipid bilayer and to use them as
transmembrane anchors for biologically active molecules
located outside the membrane. As a biological function the
recognition of proteins is considered. This requires a careful
design of the linker connecting the lipofullerene and the
biofunctional group. The linker must be able to span the
lipid layer and must therefore consist of a lipophilic chain
and a subsequent hydrophilic part. It has to be compatible
with the dimensions of the corresponding lecithin DPPC
We have shown recently that T -symmetrical hexakisad-
h
ducts of C60 containing six pairs of long alkyl chains form
entirely new membrane composites with synthetic lecithin
[
1]
bilayers. These lipofullerenes are easily accessible in large
quantities via template mediated cyclopropanation of [6,6]
double bonds with the corresponding dialkyl malonates in
octahedral sites.[2,3] In membrane bilayers unprecedentedly
high degrees of loading of up to 25 mol-% of lipofullerenes
can be achieved. Such intercalated lipofullerenes form new
supramolecular arrangements within the lipid bilayer con-
sisting of rod-shaped nanostructures. At lower temper-
atures, in the gel phase of the bilayer, the rods exhibit long-
range order, whereas at higher temperatures, in the fluid
phase, this spatial correlation is drastically reduced. The bi-
layer itself is stabilized by the lipofullerene rods against de-
formation, e.g. in a magnetic field. This appears to be re-
miniscent of the stabilization of membranes by cytoskel-
etons. However, unlike the location of the cytoskeleton in-
side the cell membrane, the lipofullerene rods are located in
between the bilayer. One implication of these discoveries is
the possibility of a membrane-assisted formation of per-
fectly spherical lipofullerene polymers which we have dem-
(
dipalmitoyl-sn-glycero-3-phosphatidylcholine) used as the
membrane building block. The lipophilic part should match
with the lipophilic fatty acid chains of the glycerides and
the hydrophilic part with the phosphatidyl choline moiety.
Moreover, the spacer chain must be long and flexible
enough to allow for unrestricted molecular recognition
events outside the membrane. As a suitable biofunctional
group we have chosen (ϩ)-biotin because of its strong and
well-established binding interactions with the protein
streptavidin. Streptavidin (SA) is a fungal protein with a
MW of 60 000 Da. An advantage of this approach is that
the lipofullerene anchor inside the bilayer is expected to
prevent the bound protein from extracting the biotinated
component out of the membrane.
[
2f]
onstrated recently.
Another challenging task is to take
We now report on the synthesis and complete character-
ization of the biotinated lipofullerene 1 as the first proto-
type of such a new class of transmembrane anchors. The
dimensions and the design of the biotin linker of 1 was
based on computer-generated models of DPPC-lipofuller-
ene bilayer composites as depicted in Figure 1. We further
report on the first coupling studies of 1 with streptavidin
(SA) and on the assembly of transmembrane systems using
a series of RIfS[ and LB-film experiments.
[
a]
Institut für Organische Chemie der Universität Erlangen-
Nürnberg,
Henkestr. 42, D-91054 Erlangen, Germany
Fax: (internat.) ϩ49–9131/85-26864
E-mail: hirsch@organik.uni-erlangen.de
[
[
b]
c]
Physikalisches Institut EP-5, Universität Würzburg,
Am Hubland, D-97074 Würzburg, Germany
Institut für Physikalische Chemie, Universität Tübingen,
Auf der Morgenstelle 8, D-72078 Tübingen, Germany
4]
Eur. J. Org. Chem. 2000, 1173Ϫ1181

WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
1434Ϫ193X/00/0407Ϫ1173 $ 17.50ϩ.50/0
1173

A. Hirsch et al.
FULL PAPER
Figure 1. Schematic representation (left) and computer generated space-filling model (right) of the biotinated lipofullerene 1 embedded
in a DPPC bilayer; the space-filling model is based on MMϩ force field calculations using the program package HyperChem[
5]
Results and Discussion
(Scheme 3), the malonate spacer is attached to C60 and the
resulting monoadduct is transferred by fivefold addition of
The synthesis of 1 consists of two main parts. The first lipophilic malonates to a mixed [1:5]-hexakisadduct with an
part (Scheme 1 and Scheme 2) comprises the synthesis of octahedral addition pattern. The final step is the coupling
amphiphilic malonate spacers. In the second part reaction with biotin.
Scheme 1. Stepwise synthesis of the amphiphilic spacer malonate 7 carrying a biotin anchor unit (i: lithium aluminium hydride, THF; ii:
monomethyl malonyl chloride, pyridine, THF; iii: pyridinium dichromate, DMF; iv: boc anhydride, dioxane; v: TFA/CH
2
Cl
2
, CDI)
1174
Eur. J. Org. Chem. 2000, 1173Ϫ1181

Synthesis of a Biotinated Lipofullerene as a New Type of Transmembrane Anchor
FULL PAPER
Scheme 2. Synthesis of the functionalized malonate 8 without a biotin moiety (i: CDI/THF; boc ϭ tert-butyloxycarbonyl)
Molecular modelling investigations (Figure 1) showed, indicates that side reactions of DBU with the amide bonds
that the malonate spacer should have a C16 chain, elongated of 8 are not significant. The product 9 was purified by flash
1
by a bis(2-aminoethyl)ethylene glycol ether moiety in order chromatography and fully characterized by IR, UV/Vis, H-
to have the right length to penetrate the thickness of a and 13C-NMR and mass spectroscopy. The UV/Vis and
DPPC monolayer. The CH chains of the spacer fit well NMR spectra are typical for [6,6]-bridged monoadducts of
2
[8]
with the length of the glyceride fatty acid chains, whereas C . In the UV/Vis spectrum the typical electronic absorp-
the amino ether region of the spacer has the dimension of tion for [6,6] monoadducts appears at 426 nm. The C-
60
[8]
13
the phosphatidyl choline moiety. The biotin terminus pro- NMR spectrum shows 16 signals between δ ϭ 145.3 and
2
trudes from the outer bilayer surface with the right position 138.9 corresponding to 16 different types of sp -C atoms
3
to be recognised by the protein.
and one signal at δ ϭ 71.5 corresponding to the sp -C
As starting material hexadecanedioic acid 2a was taken atoms of 9 which have a local C2v symmetry. SIMS mass
ϩ
and converted into the corresponding 1,16-hexadecanediol spectroscopy (Cs ) shows a molecular ion signal with
2
b by reduction with lithium aluminium hydride, and then m/z ϭ 1321 as well as fragment ion peaks at m/z ϭ 1222
coupled with monomethyl malonyl chloride (Scheme 1). and 720 corresponding to 9 with loss of the boc group and
Malonate 3a was subsequently converted into the carb- the C60 fragment, respectively.
oxylic acid 3b by treatment with pyridinium dichromate.
The transformation of the monoadduct 9 to the mixed
For the introduction of the hydrophilic part of the spacer, hexakisadduct 11 was carried out by template-mediated re-
the diamino ether 4a was converted into the monoprotected action with didodecyl malonate 10 in the presence of CBr
4
compound 4b by treatment with boc anhydride. Subsequent and DBU/DMA at room temperature (Scheme 3).[ After
2]
[
6,7]
Staab coupling
of 4b with the activated biotin derivative purification by flash chromatography and preparative
5
obtained by treatment of (ϩ)-biotin with a slight excess HPLC the hexakisadduct 11 was isolated in 44% yield. The
of carbonyldiimidazole (CDI), afforded the boc protected UV/Vis spectrum shows the absorptions at 317 and 335 nm
amine 6 in good yields. To complete the synthesis of 7, the that are typical for hexakisadducts of C with an octahe-
6
0
biotinated precursor 6 was deprotected by the action of tri- dral addition pattern.[ The C-NMR spectrum of 11 is
2]
13
[2]
fluoroacetic acid (TFA) and subsequently coupled with 3b very characteristic for such mixed hexakisadducts of C .
by activation in situ with CDI (Scheme 1). However, an un- The sp resonances of the fullerene core are separated into
60
2
satisfactory yield of only 18% was obtained in the last de- two groups of closely overlapping signals located at about
protection and coupling step, indicating that the biotin δ ϭ 141 and 145. This is about the peak position of the
2
might be too labile under the conditions of the deprotec- two magnetically different sorts of sp -C atoms within T -
h
tion/coupling procedure.
symmetrical hexakisadducts containing just one type of ad-
In order to improve the overall yield, we decided to use dend.[ The FAB mass spectrum shows the molecular ion
2]
a spacer malonate carrying an amphiphilic spacer without peak and a quasimolecular ion peak at m/z ϭ 3513 and
ϩ
ϩ
the terminal biotin unit for the subsequent cyclopropan- 3647 corresponding to M and [M ϩ Cs] .
ation of C . The coupling of the spacer with biotin was For the synthesis of the biotinated lipofullerene 1 the boc
performed as the very last step. Indeed, Staab coupling of protecting group was cleaved with a mixture of TFA and
b with 4b (Scheme 2) afforded the functional malonate 8 CH Cl (1:1), and the resulting amine coupled with the ac-
60
3
2
2
in much better yield (64%) than was achieved with the reac- tivated (ϩ)-biotin 5 (Scheme 3). This final step proceeds
tion sequence leading to 7. Another important reason to comparatively slowly in a yield of only 55% when 1.5
prefer this route was that we noticed that DBU, which is equivalents of biotin and CDI are used. With an excess of
used in the following cyclopropanation reactions, could three equivalents of activated biotin the yield of 1 can be
promote side reactions with the biotin moiety.
increased to 92% after purification by FC and HPLC. The
Scheme 3 depicts the two step attachment of the spacer UV/Vis spectrum of 1 is almost identical to that of 11 and
malonate 8 followed by the fivefold cyclopropanation with shows the two characteristic absorptions of hexakisadducts
the malonate 10 and the final coupling with the activated with an octahedral addition pattern at 316 and 333 nm. The
(
ϩ)-biotin 5 to give the target molecule 1. The cyclopropan- fullerene part of the 13C-NMR spectrum of 1 is very similar
2
ation of C60 with the spacer malonate 9 was promoted by to that of 11 with two sets of signals for the fullerene sp -
CBr4 and DBU.[
2b]
The nonoptimized yield of 29% of C atoms at δ ϭ 141 and 145. The resonances for the fuller-
3
monoadduct 9 is acceptable for this type of reaction and ene sp C-atoms appear at δ ϭ 69. The FAB mass spectrum
Eur. J. Org. Chem. 2000, 1173Ϫ1181
1175

A. Hirsch et al.
FULL PAPER
Scheme 3. Successive cyclopropanation of C60 leading to the hexakisadduct 11 and final coupling with biotin. (black: front C60 hemisphere,
grey: rear hemisphere; i: C60, CBr , DBU; ii: DMA, CBr , DBU; iii: TFA/CH Cl , (ϩ)-biotin/CDI)
4
4
2
2
shows the molecular ion and quasimolecular ion peaks at Interference spectroscopy (RIfS).[4] With RIfS, an immobil-
ϩ
ϩ
m/z ϭ 3641 and 3773 corresponding to M and [M ϩ Cs] . ized layer of a biologically active haptophoric component
is treated with a potential interaction partner. When the
partner is recognised and adapted, a corresponding change
in optical thickness of the layer is observed. Two different
Reflectometric Interference Spectroscopy (RIfS)
Investigations
The capability of the fullerene-attached biotin unit in 1 to experiments have been carried out, one with immobilized
couple with streptavidin (SA) was proven by Reflectometric biotin, the other with poly-streptavidin (poly-SA). These ex-
1176
Eur. J. Org. Chem. 2000, 1173Ϫ1181

Synthesis of a Biotinated Lipofullerene as a New Type of Transmembrane Anchor
FULL PAPER
periments provide evidence for intense and selective coup-
ling of poly-SA and 1.
In the first experiment poly-SA was preincubated with 1
by mixing it with an excess of 1 in DMF and PBS buffer, in
order to occupy the binding sites of poly-SA. The resulting
solution was rinsed over an already biotinated transducer
surface layer and, after 10 min, the layer was washed with
buffer. No change in optical thickness was observed, indi-
cating a complete blocking of the original poly-SA by 1. As
a control experiment, the transducer layer was washed with
methanol and, subsequently, a solution of poly-SA in buffer
and DMF, but without 1, was rinsed over the same surface.
As a result, an increase in optical thickness of about 3.7 nm
Figure 3. Reflectometric Interference spectroscopy [RIfS] experi-
ment 2: A) an immobilised poly-SA transducer layer is exposed to
1
in buffer solution; because of the small molecular size, almost no
increase in optical thickness is observed; after 3 min the washed
surface is brought into contact with biotinated bovine serum albu-
min [BSA]; again, no binding interactions occur and hence no in-
crease of thickness (solid line); B) control: immobilised poly-SA
surface is exposed to buffer only; an exposure to biotinated BSA
after 3 min results in coupling interactions and in an increase of
optical layer thickness (dashed line)
˚
2
is observed at a molecular area of 305 A and an LE–LC
liquid expanded to liquid condensed) transition is observed
(
˚
2
at 250 A . Further compression causes a transition into an
S-like (solid) phase at 140 A and the maximum compres-
˚
2
˚
2
Figure 2. Reflectometric Interference spectroscopy [RIfS] experi- sion was reached at 130 A (lateral pressure 34 mN/m). The
ment 1: A) poly-Streptavidin [poly-SA], preincubated with 1 to oc-
latter molecular area is a factor of 2.7 higher than that of
DPPC at identical lateral pressure.
cupy its biotin adaptor sites, is exposed to a transducer surface
covered with biotin; no binding interactions occur and hence no
increase of thickness is observed; B) control: nonincubated poly-
SA with free active sites in contact with biotin surface results in
coupling interactions and in an increase of optical layer thickness
Conclusions
(Figure 2) was observed, caused by additional occupation
of the biotin adaptor sites.
We present here for the first time the synthesis of a lipo-
philic fullerene derivative 1 with a biofunctional group,
which can be used as a transmembrane anchor. Biotin was
chosen as the biofunctional group of the lipofullerene, since
it exhibits high affinity towards the protein streptavidin. As
a consequence, complex supramolecular assemblies reminis-
cent of those of biological membranes can be envisaged.
The biotin carrying linker of 1 is thoroughly designed for
lecithin membranes such as DPPC. Due to its ability to
penetrate through a monolayer it does not only provide a
suitable molecular recognition unit on the outer membrane
surface but also guarantees a more homogeneous distribu-
tion of lipofullerenes within the lipid bilayer. This could
lead to membrane morphologies of lecithin/lipofullerene
vesicle composites different from the rod-like aggregation
of plain lipofullerenes observed previously.[ Detailed in-
vestigations of such membrane composites consisting of
In the second experiment, poly-SA was immobilised on
the transducer surface and a solution of 1 in PBS buffer
and DMF was piped over the surface. Because of the rela-
tively small size of 1, no increase should be observed. The
slight decrease in thickness shown in Figure 3 is possibly
due to different refraction indices of poly-SA and the buf-
fer/DMF solution. Subsequently, the surface was rinsed
with buffer solution and incubated with biotinated BSA
(bovine serum albumin). No increase in thickness was ob-
served, the almost constant signal proved that 1 had effec-
tively blocked the biotin binding pockets of the poly-SA
at the transducer layer. The control experiment with buffer
solution and DMF without 1 resulted in a signal increase of
about 1.6 nm on account of the addition of biotinated BSA.
1]
Langmuir-Blodgett Film Experiments
First compression experiments with the target molecule DPPC and target molecule 1 show that the biotinated lipo-
1
spread as a monolayer at the air/water interface of a fullerene indeed penetrates through the membrane of the
Langmuir trough at 20 °C suggest a (reversible) phase be- liposome, as depicted in Figure 1, and binds streptavidin at
havior which is qualitatively similar to that of a DPPC the outer surface. These results will be reported in detail in
monolayer. The first increase of pressure upon compression an upcoming publication.
Eur. J. Org. Chem. 2000, 1173Ϫ1181
1177

A. Hirsch et al.
FULL PAPER
–
1
ϩ
cm . – MS (EI, 80 °C, C H O , calcd. 358.51): m/z ϭ 359 [M ]
(
2
0
38
5
Experimental Section
ϩ
ϩ
1%), 327 [M – CO], 222 (C16
H30, 4%), 119 [M – C16OH]
(
6
100%). – C20 (358.5): calcd. C 67.00, H 10.68; found C
6.84, H 10.34.
38 5
H O
General Remarks: 1H and 13C NMR: JEOL JNM EX 400 and
JEOL JNM GX 400; MS: Micromass Zab Spec (FAB, EI), Varian
MAT 311 A (EI), Micromass Tofspec (MALDI); IR: Bruker FT-
IR IFS88 and FT-IR Vector 22; UV/Vis: Shimadzu UV 3102 PC;
HPLC preparative: Shimadzu Class-LC10, SIL 10A, SPD 10A,
CBM 10A, LC 8A, FRC 10A (Nucleosil, 5 µm, 200 ϫ 4); HPLC
analytical: Shimadzu Class-LC10, SIL 10A, SPD-M10A, CBM
1
5-Carboxypentadecyl Methyl Malonate (3b): To a solution of 16-
hydroxyhexadecyl methyl malonate 3a (820 mg, 2.29 mmol) in
6
8
mL of dry DMF was added pyridinium dichromate (3.01 g,
.0 mmol, 3.5 equiv.) in 6 mL of dry DMF. The mixture was stirred
[10]
for 8 h and filtered by means of a short silica gel column.
The
10A, LC 10AT (Nucleosil, 5 µm, 250 ϫ 21); flash chromatography:
column was rinsed with ethanol and the combined solutions were
concentrated in vacuo. Purification by flash chromatography (SiO
diethyl ether/hexane 2:1) gave 420 mg of product 3b (49%) as a
Merck, silica gel 60, 230–400 mesh; TLC: Riedel-de Ha e¨ n, silica
gel 60 F 254 (0.2 mm), visualization by UV or staining with molyb-
2
,
dophosphoric acid and cerium sulfate in H SO4 (aq) or with
1
2
3
white solid. – H NMR (400 MHz, CDCl ): δ ϭ 11.06 (br, 1 H,
4
KMnO (aq). – Materials and solvents were obtained from com-
mercial suppliers and were used without further purification. Film
COOH), 4.11 (t, J ϭ 6.6 Hz, 2 H, OCH
2
), 3.72 (s, 3 H, OCH
CO), 2.32 (t, J ϭ 7.4 Hz, 2 H, CH COOH),
CH O, CH CH COOH), 1.23 (m, 24 H, CH ). –
C NMR (100.5 MHz, CDCl ): δ ϭ 179.75, 167.07, 166.60 (3 C,
CO), 65.75 (1 C, OCH ), 52.46 (1 C, CH O), 41.38 (1 C,
OCCH CO), 33.98 (1 C, CH COOH), 29.92, 29.83, 29.57, 29.50,
9.44, 29.39, 29.13, 28.40, 25.86, 25.72 (13 C, CH ). – IR (KBr):
ν˜ ϭ 3443, 2964, 2920, 2850, 1749, 1737, 1722, 1699, 1466, 1441,
3
),
3
.36 (s, 2 H, OCCH
.61 (m, 4 H, CH
2
2
balance experiments were performed at 25 °C on a subphase of
1
2
2
2
2
2
3
ultrapure water using a 15 ϫ 45 ϫ 0.5 cm Teflon trough equipped
13
3
with a computer controlled movable Teflon barrier and a Wilhelmy
pressure detection system (accuracy 0.1 mN/m). The barrier speed
was 100 µm/s.
2
3
2
2
2
2
1,16-Hexadecanediol (2b): A solution of the commercially available
1
8
418, 1336, 1300, 1285, 1245, 1200, 1154, 1039, 1022, 984, 954, 933,
hexadecanedioic acid 2a (2.1 g, 7.34 mmol) in 75 mL of dry of THF
was added dropwise to a suspension of lithium aluminium hydride
–
1
36 6
62, 808, 724, 684, 613, 588, 545, 511 cm . – MS (EI, C20H O ,
ϩ
ϩ
ϩ
calcd. 372.50): m/z ϭ 373 [M ] (27%), 354 [M – H
2
O], 341 [M
CO], 255 [M – C16OH], 237, 119 [M – C16OH], 98 (80%), 29
100%). – C20 (372.5): calcd. C 64.49, H 9.74; found C 64.44,
H 9.69.
–
(
4.3 g, 113 mmol) in 40 mL of dry THF at room temperature under
ϩ
ϩ
[
9]
nitrogen. The mixture was refluxed for 1 day and stirred at ambi-
ent temperature for 4 days. TLC control showed complete conver-
sion. To destroy the excess of LAH, a mixture of methanol and
(
36 6
H O
diethyl ether (1:5) was added slowly. Subsequently dilute H
2
SO
4
2-{2-[Amino-2-(N-tert-butyloxycarbonyl)ethyloxy]ethyloxy}-
ethylamine (4b): Boc anhydride (4.37 g, 0.02 mol) in 30 mL of diox-
ane was added dropwise to a solution of aminoethyloxyethyloxy
ethylamine 4a (17.8 mL, 0.122 mol) in 85 mL of dioxane within
was added until two phases formed. The phases were separated and
the aqueous phase was extracted three times with 50 mL of diethyl
ether. The combined organic phase was washed with 5% aqueous
NaHCO
3
solution and dried with MgSO
4
. After evaporation of the 5 h. After additional stirring for 5 h the solution was concentrated
in vacuo. The resulting yellow oil was dissolved in 40 mL of water
2 f
O/hexane 2:1, R ϭ 0.4). – H NMR (400 MHz, and washed four times with 50 mL of CH Cl . The combined or-
solvent pure product 2b was obtained as a white solid (1.75 g, 92%).
1
TLC: (Et
]DMSO, 25 °C): δ ϭ 4.31 (br t, J ϭ 4.6 Hz, 2 H, OH), 3.34 (dt, ganic phases were washed four times with 30 mL of saturated saline
ϭ 5.9 Hz, J ϭ 8.8 Hz, 4 H, CH OH), 1.38 (tt, J ϭ J ϭ 6.6 Hz,
solution and dried with Na SO . TLC control (DCM/ethanol 9:1)
H, CH CH OH), 1.23 (m, 24 H, CH
). – 13C NMR (100.5 MHz, showed almost no bis-protected diamine. The yellow oil (4.33 g,
]DMSO, 25 °C): δ ϭ 60.69 (2 C, CH
9.09, 29.03, 28.96 (10C, CH ), 25.45 (2 C, γ-CH
2
2
[D
6
J
1
2
2
1
2
2
4
4
2
2
2
[D
6
2
OH), 32.52 (2 C, β-CH
2
), 87% with respect to boc O) was used without further purification. –
2
1
2
2
2
). – IR (KBr):
H NMR (400 MHz, CDCl ): δ ϭ 5.18 (br, 1 H, NH), 3.51 (s, 4
3
ν˜ ϭ 3419, 3360, 2923, 2849, 1481, 1462, 1363, 1348, 1316, 1057,
H, OCH CH O), 3.44, 3.41 (2t, J ϭ 5.4, 4 H, CH O), 3.21 (dt, 2
2
2
2
–1
1
036, 1012, 995, 968, 728, 613 cm . – MS (EI, 80 °C, C16
H
34 2
O ,
2 2 2 2
H, CH NH), 2.77 (t, J ϭ 5.1, 2 H, CH NH ), 1.42 (br, 2 H, NH ),
ϩ
13
calcd. 258.44): m/z ϭ 259 [M ], 222 (C16
H
30), 194, 166, 138, 124,
3 3
1.34 (s, 9 H, CH ). – C NMR (100.5 MHz, CDCl ): δ ϭ 155.84
1
1
09, 96, 82, 69, 55 (100%). – C16
3.26; found C 73.92, H 13.20.
H
34
O
2
(258.4): calcd. C 74.36, H
3 3 2
(1 C, CO), 78.88 [1 C, C(CH ) -CO], 73.27 (1 C, CH O), 70.02 (2 C,
CH O), 66.88 (1 C, CH O), 41.56 (1 C, CH NH ), 40.15 (1 C,
2
2
2
2
CH
2
NH), 28.24 (3 C, CH
3
). – IR (KBr): ν˜ ϭ 3369, 2975, 2931,
1
6-Hydroxyhexadecyl Methyl Malonate (3a): To a mixture of hexa-
2
9
868, 1713, 1521, 1456, 1391, 1366, 1274, 1251, 1174, 1123, 1042,
16, 872, 781, 509, 495, 482 cm . – MS (EI, 60 °C, C11H N O ,
24 2 4
decanediol 2b (1.22 g, 4.72 mmol) and pyridine (0.382 mL,
.72 mmol) in 60 mL of dry THF at room temperature under a
–1
4
ϩ
ϩ
calcd. 248.32): m/z ϭ 248 [M ], 219 [M – CH
2
NH
] (2%), 175 [M – tBuO] (27%), 163 (28%),
50 [M – boc] (17%), 119, 106, 88. – C11 (248.3): calcd.
2
] (2%), 206
protective atmosphere was added malonyl chloride monomethyl es-
ter (0.50 mL, 4.72 mmol) in 20 mL of THF from a dropping funnel.
After stirring for 2 days the solution was concentrated and separ-
ϩ
ϩ
[
1
M
2 2 2
– CH CH NH
ϩ
24 2 4
H N O
C 53.20, H 9.74; found C 52.73, H 9.40.
ated by flash chromatography (SiO
.3) to give a white solid (820 mg, 48%). – H NMR (400 MHz,
CDCl ): δ ϭ 4.10 (t, J ϭ 6.7 Hz, 2 H, OCH ), 3.71 (s, 3 H, OCH ),
.59 (t, J ϭ 6.6 Hz, 2 H, CH OH), 3.35 (s, 2 H, OCCH
tt, 2 H, CH CH O), 1.52 (tt, 2 H, CH CH OH), 1.45 (br, 1 H, equiv.) in 4 mL of DMF was added the imidazolide activated biotin
OH), 1.22 (m, 24 H, CH ): δ ϭ 5 (2.05 mmol). The mixture was stirred for 6 h. Separation by flash
). – 13C NMR (100.5 MHz, CDCl
67.03, 166.60 (2 C, CO), 65.73 (1 C, OCH ), 63.01 (1 C, CH OH), chromatography (SiO ; Ethanol/CH Cl 2:1, R ϭ 0.44) gave
2.48 (1 C, CH O), 41.36 (1 C, OCCH CO), 32.77 (1 C, 651 mg of an oil (67% with respect to biotin). – H NMR (400
CH OH), 29.92, 29.83, 29.57, 29.50, 29.44, 29.39, 29.13, 28.40, MHz, CDCl ): δ ϭ 7.32, 6.88 (br, 1 H, cis/trans-CONH), 6.71 (br,
5.86, 25.72 (13 C, CH 1 H, biotin–NH), 6.04 (br, 1 H, biotin–NH), 5.51, 5.20 (br, 1 H,
). – IR (KBr): ν˜ ϭ 3453, 3317, 3225, 2966,
917, 2849, 1736, 1476, 1463, 1439, 1409, 1356, 1279, 1218, 1168, cis/trans-CONH), 4.40, 4.21, (2m, 2 H, biotin–CH), 3.52 (s, 4 H,
2 f
, hexane/ethyl acetate 3:2, R ϭ
1
0
19-[(3aS,4S,6aR)-2-Oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]-
2,2-dimethyl-4,15-dioxo-3,8,11-trioxa-5,14-diazanonadecane (6): To
3
2
3
3
(
2
2
CO), 1.60 a solution of monoprotected diamine 4b (750 mg, 3.02 mmol, 1.5
2
2
2
2
2
3
1
5
CH
2
2
2
2
2
f
1
3
2
2
2
3
2
2
1
2
154, 1065, 1030, 995, 969, 927, 851, 731, 720, 689, 611, 581
OCH
2
2
CH O), 3.47, 3.45 (2t, J ϭ 5.3 Hz, 4 H, CH
2
O), 3.33 ( dt,
1178
Eur. J. Org. Chem. 2000, 1173Ϫ1181

Synthesis of a Biotinated Lipofullerene as a New Type of Transmembrane Anchor
FULL PAPER
J
(
1
ϭ J
m, 1 H, biotin–CH), 2.80 (dd, J
CHHexoS), 2.66 (d, J ϭ 12.7 Hz, 1 H, CHHendoS), 2.13 (t, J ϭ 7.6,
2
ϭ 5.4 Hz, 2 H, CH
2
NH), 3.20 (dt, 2 H, CH
ϭ 4.9 Hz, J ϭ 12.7 Hz, 1 H, δ ϭ 173.19 (1 C, CONH), 166.97, 166.50 (2 C, COO), 155.89 (1 C,
CONHboc), 79.23 [1 C, C(CH ], 70.09, 69.91, 65.65 (5 C, OCH ),
52.36 (1 C, OCH ), 41.30 (1 C, OCCH CO), 40.22, 39.02 (2 C,
): δ ϭ CH NH), 36.62 (1 C, CH CO), 29.54, 29.44, 29.40, 29.29, 29.23,
73.41, 164.20 (2 C, CO), 155.94 (1 C, CO boc), 79.06 (1 C, CCH ), 29.08 (12 C, CH ), 28.31 [3 C, C(CH ], 25.66 (1 C, CH ). – IR
9.95, 69.88 (4 C, CH
O), 61.65, 60.06 (2 C, CH), 55.59, (1 C, (KBr): ν˜ ϭ 3346, 3319, 3090, 2918, 2850, 1754, 1735, 1686, 1642,
NH), 38.96 (1 C, CH S), 35.78 (1 C, 1542, 1472, 1441, 1392, 1366, 1279, 1251, 1165, 1027, 971, 933,
), 28.15, 27.93, 25.48 (3 C, CH ). – IR 869, 782, 720, 689, 606, 461, 405 cm . – MS (FAB/NBA,
KBr): ν˜ ϭ 3298, 3088, 2974, 2930, 2869, 1706, 1646, 1552, 1530,
2 3 2 3
NHboc), 3.04 H, CCH
), 1.19 (m, 22 H, CH ):
). – 13C NMR (100.5 MHz, CDCl
1
2
3
)
3
2
2
1
1
6
2
H, CH CO), 1.51–1.67 (m, 4 H, biotin–CH
2
), 1.35 (s, 9 H, CH
3
),
3
2
.32 (m, 2 H, biotin–CH
2
). – 13C NMR (100.5 MHz, CDCl
3
2
2
3
2
3
)
3
2
2
CHS), 40.31, 40.15 (2 C, CH
CH CO), 28.26 (3 C, CH
(
2
2
–
1
2
3
2
ϩ
ϩ
C H
31 58
N
2
O
9
, calcd. 602.80): m/z ϭ 603 [M ] (21%), 503 [M
461, 1392, 1366, 1280, 1249, 1170, 1142, 1122, 859, 653, 603 boc] (100%). – C31 (602.8): calcd. C 61.77, H 9.70, N 4.65;
found C 61.22, H 9.67, N 5.14.
–
1
58 2 9
H N O
–1
38 4 6
cm . – MS (EI, 200 °C, C21H N O S, calcd. 474.62): m/z ϭ 474
ϩ
ϩ
ϩ
ϩ
[
M ], 401 [M – tBuO] (4%), 374 [M – boc] (17%), 332 [M
CH CH NHboc] (10%), 227 [biotinyl] (14%), 59 [C O] (90%),
1 (100%). – C21 S (474.3): calcd. C 53.14, H 8.07, N 11.80,
–
2
2
3 7
H
[
1
(
Methoxycarbonyl(29,29Ј-dimethyl-16,27-dioxo-20,23,28-trioxa-
7,26-diazatriacontyloxycarbonyl)]methano-1,2-dihydro[60]fullerene
9): To a mixture of malonate 8 (290 mg, 0.48 mmol), C60 (446 mg,
4
38 4 6
H N O
S 6.76; found C 52.83, H 7.94, N 12.20, S 5.76.
0
.62 mmol, 1.25 equiv.) and CBr
4
(160 mg, 0.48 mmol, 1 equiv.) in
Methyl {31-[(3aS,4S,6aR)-2-Oxohexahydro-1H-thieno[3,4-d]imid-
azol-4-yl]-16,27-dioxo-20,23-dioxa-17,26-diazahentriacont-1-yl}
Malonate (7): Complete deprotection of 6 (160 mg, 0.337 mmol)
2
50 mL of toluene was added DBU (82 µL, 0.53 mmol, 1.1 equiv.)
and the resulting mixture stirred for 12 h. After evaporation of the
solvent, separation by flash chromatography (SiO ; toluene/ethanol
:1, R ϭ 0.30) gave 185 mg of monoadduct 9 as a reddish brown
solid (29%). – H NMR (400 MHz, CDCl
H, cis-/trans-NH), 5.32, 4.97 (br, 1 H, cis-/trans-NH), 4.48 (t, J ϭ
.4 Hz, 2 H, OCH ), 4.07 (s, 3 H, OCH ), 3.58 (m, 4 H, OCH2-
CH O), 3.53 (t, J ϭ 5.0 Hz, 4 H, CH O), 3.43 (dt, J ϭ J ϭ 5 Hz,
H, CH NH), 3.30 (dt, 2 H, CH NHboc), 2.15 (t, J ϭ 7.5 Hz, 2
CO), 1.81 (m, 2 H, OCH CH ), 1.59 (m, 2 H, CH CH CO),
; s, 9 H, C(CH ], 1.33 (m, 2 H, CH ), 1.25 (m,
). – C NMR (100.5 MHz, CDCl ): δ ϭ 173.28, 164.11,
2
was achieved with 1.5 mL of TFA in 1.5 mL of CH
After evaporation to remove the excess acid, CH
2
Cl
Cl
2
after 2 min.
was added.
9
f
2
2
1
3
): δ ϭ 7.11, 5.98 (br, 1
The solution was neutralised with 120 µL of triethylamine and suc-
cessively washed with saturated NaHCO and a little cold water.
The dried organic phase was added to a mixture of malonate 3b
150 mg, 0.4 mmol) and CDI (195 mg, 1.2 mmol, 3 equiv.) after
3
6
2
3
2
2
1
2
(
2
2
2
stirring for 2 h. After stirring for 2 days at room temperature TLC
control (ethanol) remained unchanged and separation by flash
chromatography (SiO ; ethanol/diethyl ether 1:1) gave 7 (45 mg,
2
H, CH
2
2
2
2
2
1
1
1
1
1
7
.42 [m, 2 H, CH
8 H, CH
63.60, 155.94 (4 C, CO), 145.33, 145.26, 145.17, 145.11, 144.87,
44.67, 144.60, 143.87, 143.06, 142.99, 142.39, 142.19, 141.90,
2
)
3 3
2
1
3
1
2
3
1
6
(
8%) as a white solid. – H NMR (400 MHz, CDCl
.38, 6.37, 5.98 (4br, 4 H, NH), 4.47, 4.28 (2m, 2 H, CHNH), 4.11
), 3.72 (s, 3 H, OCH ), 3.58 (s, 4 H,
O), 3.53, (m, 4 H, OCH ), 3.41 (m, 4 H, CH
CO), 3.13 (m, 1 H, CHS), 2.86 (dd, J
3
): δ ϭ 6.67,
t, J ϭ 6.8 Hz, 2 H, OCH
2
3
2
40.95, 139.10, 138.90 (58 C, C60 sp -C), 79.38 [1 C, C(CH
3
)
3
],
), 53.98 (1 C,
), 52.17 (1 C, methano–C), 40.31, 39.11 (2 C, CH NH), 36.75
1 C, CH CO), 29.68, 29.61, 29.52, 29.41, 29.33, 29.21, 28.55 (11 C,
), 28.40 [3 C, C(CH ], 25.97, 25.75 (2 C, CH ). – IR (KBr):
OCH
2
CH
2
2
2
NH), 3.35
3
1.54 (2 C, C60 sp -C), 70.20, 70.02, 67.54 (5 C, OCH
2
(s, 2 H, OCCH
2
1
ϭ 8.8 Hz,
OCH
(
CH
3
2
J
2
2
2
ϭ 4.2 Hz, 1 H, CHHexoS), 2.71 (d, J ϭ 12.2 Hz, 1 H, CHHendoS),
.16 (2t, J ϭ 7.8 Hz, 2 H, CH CO), 1.59 (m, 8 H, CH ), 1.40 (m,
H, biotin–CH ), 1.22 (m, 24 H, CH
). – 13C NMR (100.5 MHz,
): δ ϭ 173.57, 167.06, 166.59, 156.39 (5 C, CO), 70.06, 70.02,
9.87 (4 C, CH O), 65.73 (1 C, CH OCO), 61.93, 60.35 (2 C,
CHNH), 55.48 (1 C, CHS), 52.42 (1 C, OCH ), 41.37 (1 C,
OCCH CO), 40.35 (1 C, CH S), 39.09 (2 C, CH NH), 36.62, 35.76
2 C, CH CO), 29.62, 29.51, 29.46, 29.38, 29.31, 29.15, 28.40,
8.12, 27.96, 25.73, 25.40 (16 C, CH
); IR (KBr): ν˜ ϭ 3386, 3287,
956, 2921, 2851, 1737, 1702, 1649, 1545, 1516, 1463, 1377, 1366,
2
2
2
2
3
)
3
2
2
2
ν˜ ϭ 3442, 3425, 2923, 2852, 1746, 1709, 1644, 1547, 1532, 1460,
CDCl
6
3
1
5
4
429, 1389, 1365, 1262, 1238, 1174, 1102, 1022, 800, 704, 666, 579,
25 cm . – UV/Vis (CHCl
26 (2900), 481 (1900), 683 nm (260).
2
2
–
1
3
): λmax (ε) ϭ 257 (120000), 326 (37000),
MS (FAB/NBA,
3
–
2
2
2
ϩ
ϩ
C
H
91 56
N
2
O
9
, calcd. 1321.43): m/z ϭ 1321 [M ] (4%), 1222 [M
boc] (27%), 720 [C60] (100%). – C91 (1321.4): calcd. C
2.71, H 4.27, N 2.12; found C 80.96, H 4.56, N 1.78.
–
(
2
56 2 9
H N O
2
2
1
2
8
–
1
331, 1265, 1147, 1028, 881, 815, 750, 720, 699, 587 cm . – MS
ϩ
(MALDI-Tof/CCA, C36
H
64
N
4
O
9
S, calcd. 728.98): m/z ϭ 768 [M
Bis(dodecyl) Malonate 10: A solution of dodecanol (1.86 mg,
10 mmol) and 0.74 mL of pyridine in 50 mL of dry CH Cl was
cooled to 0° C. Malonyl dichloride (0.44 mL, 4.54 mmol) in 5 mL
of dry CH Cl was added dropwise. The mixture was then allowed
to warm to room temperature and stirred for another 6 h. After
ϩ
ϩ K] (100%), 751 [M ϩ Na] (24%); MS (FAB/NBA): m/z ϭ 861
2
2
ϩ
ϩ
ϩ
[M ϩ Cs] (4%), 751 [M ϩ Na] (70%), 729 [M ] (76%).
2
2
Methyl (29,29Ј-Dimethyl-16,27-dioxo-20,23,28-trioxa-17,26-diaza-
triacontyl) Malonate (8): 4-Carbonyldiimidazole (50 mg, 2.8 mmol)
washing with water and drying with MgSO
; CH Cl /hexane 1:1, R
4
separation by flash
was added gradually to a solution of malonate 3b (416 mg, chromatography (SiO
.12 mmol) in dry THF under a nitrogen atmosphere until TLC
2
2
2
f
ϭ 0.37) gave a color-
1
less oil, which crystallised after a few days as a white solid (yield:
1
control (ethyl acetate/hexane 2:1) showed no more starting ma-
terial. To the imidazolide activated acid monoprotected diamine 4b
(
1.73 g, 86%). – H NMR (400 MHz, CDCl
6.7 Hz, 4 H, CH O), 3.37 (s, 2 H, OCCH
CH CH O), 1.26 (m, 36 H, CH ), 0.88 (t, J ϭ 6.6 Hz, 6 H, CH
C NMR (100.5 MHz, CDCl ): δ ϭ 166.71 (2 C, CO), 65.70
(CH O), 41.73 (1 C, OCCH CO), 31.94, 29.67, 29.60, 29.54, 29.38,
29.25, 28.50, 25.82, 22.71 (20C, CH ), 14.12 (2 C, CH ). – IR
(KBr): ν˜ ϭ 3464, 2956, 2926, 2855, 1756, 1739, 1467, 1412, 1380,
3
): δ ϭ 4.14 (t, J ϭ
CO), 1.64 (tt, 4 H,
). –
2
2
334 g, 1.34 mmol, 1.2 equiv.) was added. The mixture was stirred
for 3 days and, after evaporation of the solvent in vacuo, the prod-
uct 8 was separated by flash chromatography (SiO , diethyl ether/
ethanol 10:1) to give 435 mg (64%) of a white solid. – H NMR
400 MHz, CDCl ): δ ϭ 6.03, 5.00 (2br, 2 H, NH), 4.08 (t, J ϭ 6.6
Hz, 2 H, OCH ), 3.69 (s, 3 H, CH O), 3.55 (m, 4 H, OCH CH O), 1330, 1270, 1149, 1010, 893, 722, 685, 583 cm . – MS (EI, 80 °C,
.50 (t, J ϭ 5.2 Hz, 4 H, CH O), 3.40 (dt, J ϭ 5 Hz, 2 H,
NH), 2.12 (45%), 105 [M – C12 – C12] (100%). – C27
O), 1.39 (s, 9 73.59, H 11.89; found C 73.582, H 11.94.
2
2
2
3
1
3
3
2
2
2
1
2
3
(
3
–
1
2
3
2
2
ϩ
ϩ
3
2
1
ϭ J
2
27 52 4
C H O , calcd 440.70): m/z ϭ 441 [M ϩ H ], 273 [M – C12]
ϩ
2
CH NH), 3.33 (s, 2 H, OCCH
2
CO), 3.27 (dt, 2 H, CH
2
52 4
H O (440.7): calcd. C
2
(t, J ϭ 7.4 Hz, 2 H, CH CO), 1.59 (m, 4 H, CH
CH
2 2
Eur. J. Org. Chem. 2000, 1173Ϫ1181
1179

A. Hirsch et al.
FULL PAPER
{
[Methoxycarbonyl(29,29Ј-dimethyl-16,27-dioxo-20,23,28-trioxa-
(2.0 mL, 26 mmol) to deprotect the boc-protected amino group.
After 1 h stirring at room temperature the mixture was diluted with
1
4
3
7,26-diazatriacontyloxycarbonyl)]methano}-18,36:22,23:27,
5:31,32:55,60-pentakis[di(dodecyloxycarbonyl)methano]-1,2:18,
6:22,23:27,45:31,32:55,60-dodecahydro[60]fullerene (11): A solu-
5 mL of cold CH
added slowly for neutralisation. After washing three times with sat-
urated NaHCO the phases were separated. The organic phase was
washed a few times with a small amount of water and the combined
aqueous phases were extracted with CH Cl . The combined or-
ganic phases were dried with MgSO . To this solution of depro-
2
Cl , and triethylamine (3.62 mL, 26 mmol) was
2
tion of monoadduct 9 (205 mg, 0.15 mmol) and DMA (320 mg,
.55 mmol, 10 equiv.) in 40 mL of dry toluene was stirred for 6 h
at room temperature. After adding 10 equiv. of malonate 10
683 mg), 10 equiv. of CBr (514 mg) and 20 equiv. of DBU (465
µL) in this order, the mixture was stirred for 2 days at room temper-
ature. Separation by flash chromatography (SiO , ethyl acetate/hex-
ane 1:1, R ϭ 0.35; pentakisadducts R
preparative HPLC (SiO , ethyl acetate/hexane 6:4) gave 240 mg
3
1
2
2
(
4
4
tected amine in about 20 mL of DCM was added a mixture of
biotin (39 mg, 0.16 mmol, 3 equiv.) and CDI (26 mg, 0.16 mmol)
2
f
f
Ͻ 0.3) and purification by in 3 mL of dry DMF after 12 h of stirring. The coupling of the
biotin was followed by TLC (toluene/ethanol 3:1, R ϭ 0.36, with
free amino group R ϭ 0.15). After 2 days of stirring at room tem-
), 3.83 (s, 3 H, perature TLC control showed complete reaction. Purification by
O), 3.54 (t, J ϭ 5.2 Hz, 4 H, flash chromatography (SiO ; toluene/ethanol 4:1, increased to 3:1)
NH), 3.31 followed by preparative HPLC (SiO , toluene/ethanol 9:1) gave
2
f
1
(44%) of an amber solid. – H NMR (400 MHz, CDCl
3
): δ ϭ 5.94,
f
4
.97 (br, 2 H, NH), 4.21 (t, J ϭ 6.9 Hz, 22 H, OCH
), 3.59 (m, 4 H, OCH CH
O), 3.44 (dt, J ϭ 5.5 Hz, J
dt, 2 H, CH NH), 2.15 (t, J ϭ 7.7 Hz, 2 H, CH
H, CH ), 1.43 [s, 9 H, C(CH
.6 Hz, 30 H, CH
). – 13C NMR (100.5 MHz, CDCl
63.87 (14 C, CO), 146.01, 145.77, 145.74, 145.49, 141.35, 141.20, biotin–NH), 4.46 (m, 1 H, biotin), 4.27 (m, 1 H, biotin), 4.20 (t,
2
OCH
CH
(
3
2
2
2
2
1
2
ϭ 5.0 Hz, 2 H, CH
2
2
1
2
2
CO), 1.66 (m, 24
hexakisadduct 1 (182 mg, 92%) as an amber solid. – H NMR (400
): δ ϭ 6.66 (br t, J ϭ 5.1 Hz, 1 H, NH), 6.48 (br, 1
3
): δ ϭ 164.30, H, biotin–NH), 6.35 (br t, J ϭ 5.4 Hz, 1 H, NH), 5.53 (br, 1 H,
2
3 3 2 3
) ], 1.24 (m, 202 H, CH ), 0.86 (t, J ϭ MHz, CDCl
6
1
1
7
6
3
2
41.17, 141.14, 140, 83 (48 C, C60 sp -C), 79.38 [1 C, C(CH
3
)
3
],
O), 69.11, 68.99 (12 C, C60 sp -C), 66.98, OCH
OCO), 53.55 (1 C, OCH ), 45.41 (6 C, methano- (m, 1 H, biotin–CHS), 2.86 (dd, J
NH), 37.00 (1 C, CH CO) 31.93 (11 C, CHHexoS), 2.70 (d, J ϭ 13.0 Hz, 1 H, CHHendoS), 2.20 (t, J ϭ 7.6
O), 29.80, 29.68, 29.65, 29.58, 29.42, 29.38, 29.27, 28.50, Hz, 2 H, biotin–CH CO), 2.15 (t, J ϭ 7.6 Hz, 2 H, CH CO), 1.65
8.44 (81 C, CH ), 28.39 [3 C, C(CH ], 25.84, 25.78 (11 C, CH ), (m, 28 H, CH ), 1.40 (m, 2 H, biotin–CH ), 1.26 (m, 202 H, CH ),
2.68 (10C, CH ), 14.11 (10C, CH ). – C NMR (100.5 MHz,
). – IR (KBr): ν˜ ϭ 3415, 3343, 0.84 (t, J ϭ 6.8 Hz, 30 H, CH
925, 2854, 1747, 1679, 1514, 1466, 1365, 1264, 1217, 1125, 1080, CDCl ): δ ϭ 173.48, 173.28, 164.22, 164.00, 163.80, 162.45 (15 C,
J ϭ 6.8 Hz, 22 H, CH
2
O), 3.81 (s, 3 H, OCH
O), 3.41 (dt, 4 H CH
ϭ 7.8 Hz, J ϭ 4.9 Hz, 1 H,
3
), 3.57 (s, 4 H,
3
0.26, 70.14 (4 C, CH
6.86 (11 C, CH
2
2
CH O), 3.54 (2t, 4 H, CH
2
2
2
NH), 3.11
2
3
1
2
C), 39.11 (2 C, CH
CH CH
2
2
2
2
2
2
2
2
2
9
2
2
)
3 3
2
2
2
1
2
3
2
3
2 3
CH
3
–1
2
97, 828, 760, 716, 670, 529 cm . – UV/Vis (CH
45 (114000 ), 271 (88000), 280 (92000), 317 (56000, sh), 335 nm 70.00, 69.95, 69.80 (4 C, CH
29, calcd. 3514.84): 66.90 (11 C, CH OCO), 61.71 (1 C, biotin-CHNH), 60.17 (1 C, bi-
m/z ϭ 3647 [M ϩ Cs] (1%), 3513 [M ] (5%), 3414 [M – boc] otin-CHNH), 55.46 (1 C, CHS), 53.34 (1 C, OCH ), 45.38, 45.16
11%), 720 [C60] (100%). – C226 29 (3194.7): calcd. C 84.96, (6 C, methano-C), 40.41 (1 C, CH S), 39.07 (2 C, CH NH), 36.59,
H 9.65, N 0.87; found C 84.12, H 9.21, N 0.52. 35.86 (2 C, CH CO), 31.86 (11 C, CH CH O), 29.59, 29.48, 29.30,
9.19, 28.38 (81 C, CH ), 28.00 (2 C, biotin-CH CH ), 25.72 (11 C,
CH ), 25.46 (1 C, biotin-CH ), 22.61 (10C, CH ), 14.02 (10C,
2
Cl
2
): λmax (ε) ϭ
CO), 145.93, 145.71, 145.42, 141.07, 140.76 (48 C, C60 sp -C),
3
2
O), 69.05, 68.94 (12 C, C60 sp -C),
(45000, sh). – MS (FAB/NBA, C226
H
306
ϩ
N
2
O
2
ϩ
ϩ
3
(
H
306
N
2
O
2
2
2
2
2
2
2
2
2
5
-[(3aS,4S,6aR)-4-[5-(1H-imidazol-1-yl)-5-oxopentyl]tetrahydro-1H-
2
2
2
thieno-[3,4-d]imidazol-2(3H)-one (5): Biotin (500 mg, 2.05 mmol) in CH ). – IR (KBr): ν˜ ϭ 3287, 2924, 2853, 1748, 1703, 1652, 1543,
3
2
0 mL of of dry DMF was gently heated to obtain a clear solution.
Carbonyldiimidazole [CDI] (332 mg, 2.05 mmol) was then added. 529 cm . – UV/Vis (CH Cl ): λ
(ε) ϭ 245 (99000), 271 (72000),
max
1466, 1379, 1353, 1264, 1215, 1124, 1079, 998, 826, 761, 716, 670,
–
1
2
2
The mixture was stirred until no more CO
2
formation was observed
281 (76000), 316 (47000, sh), 333 nm (38000, sh). – MS (FAB/NBA,
ϩ
and used without further purification in order to prevent solubility
problems and decomposition. If necessary the imidazole can be
easily removed by sublimation. NMR showed almost complete
C231H312N O S, calcd 3641.02): m/z ϭ 3773 [M ϩ Cs], 3641
4
29
ϩ
[M ], 3309, 720 [C ] (100%). – C231H312 N O S (3641.0): calcd.
6
0
4
29
C 76.20, H 8.64, N 1.54, S 0.88; found C 75.55, H 8.16, N 1.13,
S 0.50.
conversion. – 1H NMR (400 MHz, CDCl
3
): δ ϭ 8.41(s, 1 H,
NCHN), 7.69 (d, J ϭ 1.7 Hz, 1 H, NCH), 7.63 (s, 1 H, NCHN
imidazole), 7.05 (d, J ϭ 1.7 Hz, 1 H, NCH), 7.01, 7.00 (2s, 2 H,
NCH imidazole), 6.47, 6.39 (2br, 2 H, biotin–NH), 4.30, 4.14 (2m,
RIfS Experiments
Experiment 1: poly-SA (50 µg) in 997 µL of PBS buffer was mixed
with a solution of 150 µg of 1 in 3µL of DMF. The mixture was
allowed to stand for 10 minutes with occasional agitation. This
solution was piped for 10 min over a transducer surface covalently
linked to biotin. The transducer layer was rinsed thoroughly with
methanol for 5 min, and the same surface was subsequently ex-
posed to a control solution of 50 µg of poly-SA in 997 µL of PBS
buffer and 3 µL of DMF.
2
2
2
H, CH), 3.11 (m, 1 H, CH), 3.01 (dt, J
H, CH CO), 2.83 (dd, J ϭ 14 Hz, J ϭ 5 Hz, 1 H, CHHexoS),
.57 (d, J ϭ 12.7 Hz, 1 H, CHHendoS), 2.19 (t, J ϭ 7 Hz,
COOH corresponding to 7% nonactivated biotin or decom-
1 2
ϭ 4.7 Hz, J ϭ 2.6 Hz,
2
1
2
CH
posed biotin imidazolide), 1.65, 1.51, 1.41 (3m, 6 H, CH
NMR (100.5 MHz, CDCl ): δ ϭ 170.48, 162.75 (2 C, CO), 137.01
1 C, NCHN), 135.14 (1 C, NCHN imidazole), 130.23 (1 C, CHN),
2
1
3
2
). –
C
3
(
121.71 (2 C, CHN imidazole), 116.43 (1 C, CHN), 61.02, 59.20
(
2
2 C, CH), 55.33 (1 C, CHS), 39.90 (1 C, CHS), 34.16 (1 C, CH
7.99, 27.83, 23.62 (3 C, CH ).
2
2
), Experiment 2: Compound 1 (150 µg) was dissolved in 997 µg of
PBS buffer and 3µL of DMF. This solution was exposed on a trans-
ducer surface carrying immobilized poly-SA with an optical thick-
ness between 2.71 nm and 3.75 nm. The signal showed a slight de-
crease of 0.21 nm. The thickness remained nearly the same during
incubation with BSA. In the two analogous control experiments
with 997 µL of PBS buffer, 3 µL of DMF (without 1) and bi-
2:55,60-dodecahydro[60]fullerene (1): To a solution of hexaadduct otinated bovine serum albumin BSA an increase in layer thickness
{
[Methoxycarbonyl(31-[(3aS,4S,6aR)-2-oxohydro-1H-thieno[3,4-d]-
imidazol-4-yl]-16,27-dioxo-20,23-dioxa-17,26-diazahentriacontyl-
carbonyl)]methano}-18,36:22,23:27,45:31,32:55,60-pentakis-
[
bis(dodecyloxycarbonyl)methano]-1,2:18,36:22,23:27,45:31,
3
1
1 (190 mg, 0.05 mmol) in 2.0 mL of cold CH
Cl
2 2
was added TFA of 2.2 nm and 1.56 nm, respectively, was detected.
1180
Eur. J. Org. Chem. 2000, 1173Ϫ1181

Synthesis of a Biotinated Lipofullerene as a New Type of Transmembrane Anchor
FULL PAPER
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Acknowledgments
2
[
3]
This work was supported by the Deutsche Forschungsgemein-
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1181