Synthesis of novel thiol-reactive amphiphilic lipids based on cholesterol for protein-liposome coupling

Article abstract of DOI:10.1007/PL00000090

The synthesis of a series of coupling lipids designed for covalently linking proteins to liposomes is described. The new compounds have in common a cholesterylsuccinyl unit as a lipid anchor and a thiol-reactive maleimidobenzoyl unit which are linked by alkyl or (poly)ethylene glycol spacers that differ in length and polarity.

Full text of DOI:10.1007/PL00000090

Monatshefte fuÈr Chemie 129, 319±327 (1998)
Synthesis of Novel Thiol-Reactive Amphiphilic
Lipids Based on Cholesterol for Protein-
Liposome Coupling
Ã
È
Jorg T. Kley, Thomas Fichert, and Ulrich Massing
Tumor Biology Center, Department of Medical Oncology, Division of Medical Research, D-79106
Freiburg, FRG
Summary. The synthesis of a series of coupling lipids designed for covalently linking proteins to
liposomes is described. The new compounds have in common a cholesterylsuccinyl unit as a lipid
anchor and a thiol-reactive maleimidobenzoyl unit which are linked by alkyl or (poly)ethylene glycol
spacers that differ in length and polarity.
Keywords. Maleimides; Polyethylene glycols; Cholesterol derivatives; Synthesis of; Liposomes.
Synthese neuartiger thiol-reaktiver amphiphiler Lipide auf Cholesterolbasis fuÈr die Kopplung
von Proteinen an Liposomen
Zusammenfassung. Die Synthese einer Serie von Kopplungslipiden fuÈr die kovalente Bindung von
Proteinen an Liposomen wird beschrieben. Die neuen Verbindungen haben eine Cholesterylsuccinyl-
Einheit als Lipidanker und eine thiol-reaktive Maleimidobenzoylgruppe gemeinsam, die durch Al-
È È
kyl- oder (Poly)ethylenglykol-Spacer verbunden sind, welche sich in Kettenlange und Polaritat
unterscheiden.
Introduction
For many applications liposomes have proven to be the vehicle of choice for drug
delivery [1, 2]. In vitro and in vivo studies have revealed their potential for the
therapy of cancer, HIV infection, and other applications [3±11]. Liposomes can be
selectively targeted to different tissues by covalently linking antibodies or other
proteins to the liposome surface [1, 7, 11±14]. This linking is usually achieved by
reacting the proteins with thiol- or amino-reactive lipid components of the
preformed liposomes [15±17]. Coupling lipids with thiol-reactive maleimido
groups are widely used because they selectively form stable thioether linkages to
proteins bearing thiol groups. Almost all coupling lipids reported until now are
based on phosphatidylethanolamine as the lipid anchor [5, 13, 18±20]. These
compounds are prepared from heterobifunctional coupling agents and phosphati-
dylethanolamine, thus forming phospholipid derivatives with reactive groups for
* Corresponding author

320
J. T. Kley et al.
protein linkage. In contrast, however, little is reported on coupling lipids based on
cholesterol anchors [10, 21]. This may be due to poor coupling ef®ciencies
observed when using coupling lipids bearing maleimido groups linked to a
cholesterol moiety by short apolar spacer units [21]. We now report the synthesis of
a series of novel coupling lipids with spacers varying in both length and polarity.
The use of long hydrophilic spacers is essential for coupling proteins to sterically
stabilized liposomes with lipid bilayers containing glycolipids or lipid conjugates
with polyethylene glycols [1, 5, 12, 13, 16, 17].
Results and Discussion
To synthesize the desired coupling lipids 7±12, ꢀ,!-diol spacer units (ethylene
glycol, 1, 12-octadecanediol, tetraethylene glycol, polyethylene glycol 400, or
polyethylene glycol 1000) were ®rst esteri®ed with cholesterol hemisuccinate (1)
as the lipid anchor at one end and next with a maleimidobenzoic acid as the thiol
reactive group at the other end. Being sensitive to reactions with nucleophiles,
maleimidobenzoates were introduced in the last step.
7±12 were prepared by a linear reaction sequence starting with cholesterol
hemisuccinate (1) obtained from cholesterol and succinic anhydride by a simpli®ed
version of the synthesis described by ElKihel et al. [22].
The lipid anchor 1 was esteri®ed with an excess of the ꢀ,!-diol spacer units to
achieve monoacylation (see Scheme 2). Interestingly, when exploring suitable
reaction conditions we found a non-statistical product distribution with 60% of the
diester 2a and only 34% of the desired monoester 2 for the reaction of 1 with a
Scheme 1. Esteri®cation of cholesterol hemisuccinate (1) with ethylene glycol:
product distribution depending on the solvent

Amphiphilic Lipids for Protein-Liposome Coupling
321
Scheme 2. Synthesis of the diol monoesters 2±6
tenfold excess of ethylene glycol and DCC as condensing agent if THF was used as
solvent (see Scheme 1). In contrast, a statistical product distribution was observed
when using toluene/acetonitrile 1:1 as solvent. This could be explained by
aggregate formation of 1 and 2 in THF. In this case, 1 would preferably react with 2
due to high local concentrations. The high bulk concentration of the diol which
would not be present in the superstructure would be ineffective. This explanation is
in accordance with observations that amphiphilic lipids tending to aggregate
formation in one-component solvents can be monomolecularly dissolved by the use
of appropriate solvent mixtures [23]. Therefore, the synthesis of 2±6 was
performed in toluene/acetonitrile. For an easier workup of the upscaled
preparations of 2±6, we avoided the need of separating dicyclohexyl urea by
converting 1 into the respective acid chloride using thionyl chloride in toluene
instead of DCC as condensing agent (Scheme 2). After removal of the excess
thionyl chloride, the resulting solution was added to the respective diol dissolved in
acetonitrile, obtaining the proper solvent mixture (see above). After chromato-
graphic puri®cation, 2±5 were obtained in 56±64% yield. The lower yield (23%) of
6 which contains a PEG 1000 moiety is due to the chromatographic workup and
corresponds to our experience of other long chain polyethylene glycol derivatives.
Esteri®cation of 2±6 with meta- or para-maleimidobenzoic acid chloride was
performed in dichloromethane with triethylamine as base at ambient temperature.
The acid chlorides were freshly prepared from the respective maleimido benzoic
acids (see Schemes 3 and 4). The products were puri®ed by column chromatog-
raphy without aqueous workup in order to avoid reaction of maleimide with
hydroxide ions.
Our results show that the reaction sequence outlined above allows the
preparation of thiol reactive lipids with variations in (i) polarity, (ii) chain length of
the spacer unit, and (iii) the constitution of the thiol-reactive moiety. The in¯uence

322
J. T. Kley et al.
Scheme 3. Synthesis of the coupling lipids 7 and 8 bearing ꢀ,!-alkanediol spacer units
Scheme 4. Synthesis of the coupling lipids 9±12 bearing hydrophilic tetra- or polyethylene glycol
spacer units
of these three parameters on the ef®ciency of coupling proteins to liposomes are
currently under investigation in our group.
Experimental
Cholesterol (from lanolin) was purchased from Fluka (Buchs, CH). (Poly)ethylene glycols were of
reagent grade and purchased from Merck (Darmstadt, FRG). Solvents used for the acylations were
dried with sodium sulfate (ethyl acetate), over molecular sieve 0.4 nm (toluene), or by distillation
from di-phosphorus pentoxide (acetonitrile). Triethylamine was distilled from calcium oxide.
Removal of solvents was carried out in a rotatory evaporator under reduced pressure. Residual water
was removed by azeotropic distillation with ethyl acetate. Chromatographic separations were
performed in glass columns (diameter 4 cm or 2 cm, fraction size 20 ml). Silica gel (Kieselgel 60,
1
0.063±0.100 mm) and TLC plates were obtained from Merck (Darmstadt, FRG). H NMR spectra
were recorded with a Bruker AM 400 spectrometer (400 MHz, ꢁ in ppm, TMS ˆ 0 ppm). Infrared

Amphiphilic Lipids for Protein-Liposome Coupling
323
spectra were measured using a Perkin Elmer 16 PC FT-IR spectrometer. Peaks are given in cmꢀ1and
labelled `ss' (very strong), `s' (strong), and `m' (medium).
Cholesterol hemisuccinate (1)
A solution of 38.7 g (100 mmol) Cholesterol, 20.0 g (200 mmol) succinic anhydride, 0.6 g (5 mmol)
DMAP and 51.2 ml (400 mmol) triethylamine in 500 ml ethyl acetate was re¯uxed for 12 h. 100 ml
ethyl acetate and 100 ml methanol were added, and the mixture was extracted with 200 ml 2 N
hydrochloric acid. After addition of further 100 ml ethyl acetate, the organic layer was extracted
twice with 150 ml 0.2 N hydrochloric acid/methanol 2:1. The solvent of the organic layer was
removed and 300 ml methanol were added. The resulting suspension was stirred for 15 min; then
300 ml water were added. The crude product was ®ltered off with suction, washed twice with water,
dried in vacuo, and recrystallized from 200 ml diisopropyl ether to yield 43.4 g (89.2 mmol, 89%) 1
as a colorless powder.
Rf ˆ 0.38 (ethyl acetate); IR (KBr): ꢂ ˆ 2950 (ss) -CH2-, 2870 (m) -CH2-, 1730 (ss) C=O ester,
1710 (ss) C=O acid, 1175 (s) C-O; 1H NMR (CDCI3, 400 MHz): ꢁ ˆ 0.68 (s, 3H, CH3-18), 0.86 and
3
3
0.87 (2d, J ˆ 6.6 Hz, 6H, diastereotopic CH3-26/27), 0.92 (d, J ˆ 6.4 Hz, 3H, CH3-21), 0.94±1.62
(m, 21H) 1.02 (s, 3H, CH3-19), 1.78±1.89 (m, 3H), 1.93±2.05 (m, 2H), 2.31 and 2.33 (2sb, 2H, allyl.
CH2-4), 2.58±2.70 (m, 4H, -OCO-(CH2)2-COO-), 4.59±4.68 (m, 1H, CH-3), 5.35±5.39 (m, 1H,
vinyl. CH-6).
Compounds 2±6 (Esteri®cation of 1)
9.73 g (20 mmol) 1 and 3.63 ml (50 mmol) thionyl chloride in 100 ml toluene were stirred at 80ꢁC for
1 h. The excess of thionyl chloride was distilled off, and the resulting solution was added dropwise to
an ice-cold solution of the corresponding diol and 6.93 ml (50 mmol) triethylamine in 100 ml (for the
preparation of 2, 3, and 4 or 150 ml (for the preparation of 5 and 6) acetonitrile. The mixture was
stirred for further 4 h at ambient temperature and then worked up as follows.
Workup of cholesteryl-(2-hydroxyethyl)-succinate (2)
Diol employed: 11.2 ml (200 mmol) ethylene glycol. After addition of 200 ml 0.25 N hydrochloric
acid, the mixture was extracted twice with 80 ml ethyl acetate. The organic layers were evaporated to
dryness, and the residue was puri®ed on silica gel (150 g) using ®rst diisopropyl ether, then
diisopropyl ether/tert-butylmethyl ether 2:1 as eluent. Yield: 6.42 g (12.1 mmol, 60%) 2 as a wax-
like solid.
Rf ˆ 0.49 (tert-butylmethyl ether); IR (KBr): ꢂ ˆ 2950 (ss) -CH2-, 2905 (m) -CH2-, 2870 (m)
1
-CH2-, 1735 (ss) C=O, 1165 (s) C±O; H NMR (CDCI3, 400 MHz): ꢁ ˆ 0.68 (s, 3H, CH3-18), 0.86
3
3
and 0.87 (2d, J ˆ 6.7 Hz, 6H, diastereotopic CH3-26/27), 0.91 (d, J ˆ 6.7 Hz, 3H, CH3-21), 0.93±
1.64 (m, 21 H), 1.02 (s, 3H, CH3-19), 1.77±1.89 (m, 3H), 1.92±2.04 (m, 2H), 2.16 (t,
3JOH,CH ˆ 6.2 Hz, 1H, OH), 2.31 and 2.32 (2sb, 2H, allyl. CH2-4), 2.60±2.68 (m, 4H, -OCO-
3
3
(CH2)2-COO-), 3.82 (dt, JOH,CH ˆ 6.2 Hz, JCH,CH ˆ 4.7 Hz, -CH2OH), 4.23±4.27 (m, 2H,
diastereotopic -CH2-O-CO), 4.57±4.67 (m, 1H, CH-3), 5.35±5.39 (m, 1 H, vinyl. CH-6).
Workup of cholesteryl-(12-hydroxydodecyl)-succinate (3)
Diol employed: 40.5 g (200 mmol) 1,12-dodecanediol. After addition of 200 ml 0.25 N hydrochloric
acid, the mixture was extracted with 80 ml ethyl acetate. The organic layer was evaporated to
dryness, and the residue was stirred with 80 ml diisopropyl ether at 45ꢁC for 1 h. The undissolved
diol was ®ltered off with suction and the ®ltrate again evaporated to dryness. Puri®cation of the

324
J. T. Kley et al.
residue on silica gel (150 g) using cyclohexane/diisopropyl ether 1:1 as eluent yielded 7.45 g
(11.1 mmol, 56%) 2 as a wax-like solid.
Rf ˆ 0.31 (diisopropyl ether; IR (KBr): ꢂ ˆ 2930 (ss) -CH2-, 2850 (s) -CH2-, 1735 (s) and 1725 (s)
1
C=O, 1170 (s) C-O; H NMR (CDCl3, 400 MHz): ꢁ ˆ 0.68 (s, 3H, CH3-18), 0.86 and 0.87 (2d,
3J ˆ 6.7 Hz, 6H, diastereotopic CH3-26/27), 0.92 (d,3J ˆ 6.7 Hz, 3H, CH3-21), 0.93±1.71 (m, 25H),
1.02 (s, 3H, CH3-19), 1.27 (s, 16H, O-(CH2)2-(CH2)8-(CH2)2-O), 1.78±1.89 (m, 3H), 1.92±2.04 (m,
2H), 2.30 and 2.32 (2sb, 2H, allyl. CH2-4), 2.56±2.64 (m, 4H, -OCO-(CH2)2-COO-), 3.60±3.67 (m,
2H, -CH2-OH), 4.08 (t, 3J ˆ 6.7 Hz, 2H, -CH2-O-CO), 4.57±4.67 (m, 1H, CH-3), 5.35±5.38 (m, 1H,
vinyl. CH-6).
Workup of mono-O-(3-cholesteryloxycarbonyl)propionyl tetraethylene glycol (4)
Diol employed: 34.5 mmol (200 mmol) tetraethylene glycol. Workup as described for 2 using ethyl
acetate as eluent yielded 8.10 g (12.2 mmol, 61%) 4 as a wax-like solid.
Rf ˆ 0.27 (ethyl acetate); IR (KBr): ꢂ ˆ 2940 (ss) -CH2-, 2905 (m)-CH2-, 2870 (m) -CH2-, 1730
(ss) C=O, 1160 (m) C±O; 1H NMR (CDCl3, 400 MHz): ꢁ ˆ 0.68 (s, 3H, CH3-18), 0.86 and 0.87 (2d,
3J ˆ 6.6 Hz, 6H, diastereotopic CH3-26/27), 0.91 (d, 2J ˆ 6.7 Hz, 3H, CH3-21), 0.93±1.64 (m, 21H),
1.02 (s, 3H, CH3-19), 1.77±1.89 (m, 3H), 1.92±2.04 (m, 2H), 2.30 and 2.32 (2sb, 2H, allyl. CH2-4),
2.47±2.54 (m, 1H, OH), 2.57±2.68 (m, 4H, -OCO-(CH2)2-COO-), 3.59±3.75 (m, 14H, HO-
3
(C2H4O)3-CH2-), 4.25 (t, J ˆ 4.8 Hz, 2H, -CH2-O-CO), 4.57±4.67 (m, 1H, CH-3), 5.35±5.38 (m,
1H, vinyl. CH-6).
Workup of mono-O-(3-cholesteryloxycarbonyl)propionyl polyethylene glycol 400 (5)
Diol employed: 80.0 g (200 mmol) polyethylene glycol 400. Workup as described for 2 using ®rst
ethyl acetate, then ethyl acetate/methanol 9:1 as eluent yielded 11.2 g (12.8 mmol, 64%) 5 as a soft
slime.
Rf ˆ 0.1±0.5 (ethyl acetate/methanol 8:1); IR (KBr): ꢂ ˆ 2935 (ss) -CH2-, 2905 (m) -CH2-, 2870
1
(m)-CH2-, 1735 (ss) C=O, 1165 (m) C-O, 1115 (s) C±O; H NMR (CDCl3, 400 MHz): ꢁ ˆ 0.68 (s,
3
3
3H, CH3-18), 0.86 and 0.87 (2d, J ˆ 6.7 Hz, 6H, diastereotopic CH3-26/27), 0.92 (d, J ˆ 6.7 Hz,
3H, CH3-21), 0.93±1.64 (m, 21H), 1.02 (s, 3H, CH3-19), 1.77±1.89 (m, 3H), 1.92±2.04 (m, 2H), 2.30
and 2.32 (2sb, 2H, allyl. CH2-4), 2.57±2.68 (m, 4H, -OCO-(CH2)2-COO-), 3.59±3.73 (m, approx.
34H, HO-(C2H4O)8
ꢂon average†
CH-3), 5.35±5.38 (m, 1H, vinyl. CH-6).
-CH2-), 4.25 (t, 3J ˆ 4.8 Hz, 2H, -CH2-O-CO), 4.57±4.67 (m, 1H,
Workup of mono-O-(3-cholesteryloxycarbonyl)propionyl polyethylene glycol 1000 (6)
Diol employed: 100 g (100 mmol) polyethylene glycol 1000. After addition of 200 ml 0.25 N
hydrochloric acid the mixture was extracted twice with 120 ml ethyl acetate/methanol 2:1. The
combined organic layers were evaporated to dryness. Puri®cation of the residue on Silica gel (150 g)
using ®rst diisopropyl ether/methanol 2:1 to eluate the apolar by-products, then ethyl acetate/
methanol 2:1 as eluent yielded 6.86 g (4.67 mmol, 23%) 6 as an amorphous solid.
Rf ˆ 0.2±0.3 (ethyl acetate/tetrahydrofurane/methanol 1:1:1); IR (KBr): ꢂ ˆ 2940 (s) -CH2-, 2890
(ss) -CH2-, 1730 (s) C=O, 1115 (ss) C-O; 1H NMR (CDCl3/CD3OD/D2O (1:4:1), 400 MHz): ꢁ ˆ 0.71
(s, 3H, CH3-18), 0.87 and 0.88 (2d, 3J ˆ 6.7 Hz, 6H, diastereotopic CH3-26/27), 0.94 (d, 3J ˆ 6.7 Hz,
3H, CH3-21), 0.95±1.69 (m, 21H), 1.05 (s, 3H, CH3-19), 1.81±1.94 (m, 3H), 1.95±2.08 (m, 2H), 2.32
and 2.34 (2sb, 2H, allyl. CH2-4), 2.61±2.70 (m, 4H, -OCO-(CH2)2-COO-), 3.67 (s, approx. 90H, HO-
(C2H4O)22 -CH2-), 4.24±4.28 (m, 2H, -CH2-O-CO), 4.56±4.65 (m, 1H, CH-3), 5.38±5.41
ꢂon average†
(m, 1H, vinyl. CH-6).

Amphiphilic Lipids for Protein-Liposome Coupling
325
O-(3-Cholesteryloxycarbonyl)propionyl-O0-m-maleimidobenzoyl ethylene glycol (7)
236 mg (1.0 mmol) m-maleimidobenzoic acid and 218 ml (3.0 mmol) thionyl chloride in 5 ml toluene
were stirred at 80ꢁC for 1 h. The volatile components were distilled off to obtain crude m-
maleimidobenzoyl chloride as a light yellow solid. 637 mg (1.2 mmol) 2 and 139 ml (1.0 mmol)
triethylamine dissolved in 5 ml dichloromethane were added, and the mixture was stirred at ambient
temperature for 2 h. After evaporation to dryness the residue was puri®ed on silica gel (35 g) using
cyclohexane/ethyl acetate 4:1 as eluent. Yield: 431 mg (0.59 mmol, 59%) 7 as a solid.
Rf ˆ 0.40 (cyclohexane/ethyl acetate 2:1); IR (KBr): ꢂ ˆ 2950 (ss) -CH2-, 2905 (m) -CH2-, 2865
(m) -CH2-, 2850 (m) -CH2-, 1725 (ss) C=O, 1590 (m) arom, 1490 (m) arom, 1165 (ss) C-O, 755 (m)
1
3
arom; H NMR (CDCl3, 400 MHz): ꢁ ˆ 0.67 (s, 3H, CH3-18), 0.85 and 0.88 (2d, J ˆ 6.7 Hz, 6H,
diastereotopic CH3-26/27), 0.91 (d, 3J ˆ 6.7 Hz, 3H, CH3-21), 0.93±1.64 (m, 21H), 1.02 (s, 3H, CH3-
19), 1.77±1.89 (m, 3H), 1.92±2.04 (m, 2H), 2.30 and 2.32 (2sb, 2H, allyl. CH2-4), 2.56±2.72 (m, 4H,
-OCO- (CH2)2-COO-), 4.40±4.48 (m, 2H, -CH2-O-CO-CH2), 4.50±4.56 (m, 2H,-CH2-O-CO-aryl),
4.55±4.70 (m, 1H CH-3), 5.32±5.40 (m, 1H, vinyl. CH-6), 6.89 (s, 2H, OC-CH=CH-CO), 7.50±7.62
(m, 2H, arom. H), 8.00±8.11 (m, 2H, arom. H).
O-(3-Cholesteryloxycarbonyl)propionyl-O0-m-maleimidobenzoyl 1,12-dodecanediol (8)
204 mg (0.87 mmol) m-maleimidobenzoic acid were converted into m-maleimidobenzoyl chloride as
described in the preparation of 7. 487 mg (0.73 mmol) 3 and 100 ml (0.73 mmol) triethylamine
dissolved in 5 ml dichloromethane were added, and the mixture was stirred at ambient temperature
for 2 h. After evaporation to dryness the residue was puri®ed on silica gel (30 g) using cyclohexane/
ethyl acetate 6:1 as eluent. Yield: 290 mg (0.33 mmol, 46%) 8 as a solid.
Rf ˆ 0.22 (cyclohexane/ethyl acetate 4:1); IR (KBr): ꢂ ˆ 2935 (ss) -CH2-, 2870 (m) -CH2-,
2850(m) -CH2-, 1725 (ss) C=O, 1165 (ss) C-O, 755 (m) arom; 1H NMR (CDCl3, 400 MHz):
3
ꢁ ˆ 0.67 (s, 3H, CH3-18), 0.85 and 0.87 (2d, J ˆ 6.7 Hz, 6H, diastereotopic CH3-26/27), 0.92 (d,
3J ˆ 6.7 Hz, 3H, CH3-21), 0.93±1.71 (m, 25H), 1.01 (s, 3H, CH3-19), 1.20 (m, 16H, O-(CH2)2-
(CH2)8-(CH2)2-O), 1.40 (m, 4H, O-CH2-CH2-(CH2)8-CH2-CH2-O), 1.78±1.89 (m, 3H), 1.92±2.04
(m, 2H), 2.30 and 2.32 (2sb, 2H, allyl. CH2-4), 2.56±2.64 (m, 4H, -OCO-(CH2)2-COO-), 4.08 (t,
3J ˆ 6.7 Hz, 2H, -CH2-O-CO-CH2), 4.32 (t,3J ˆ 6.7 Hz, 2H, -CH2-O-CO-aryl), 4.54±4.70 (m, 1H,
CH-3), 5.33±5.40 (m, 1H, vinyl. CH-6), 6.89 (s, 2H, OC-CH=CH-CO), 7.51±7.58 (m, 2H, arom. H),
8.00±8.09 (m, 2H, arom. H).
O-(3-Cholesteryloxycarbonyl)propionyl-O0-m-maleimidobenzoyl tetraethylene glycol (9)
200 mg (0.92 mmol) m-maleimidobenzoic acid were converted into m-maleimidobenzoyl chloride as
described in the preparation of 7. 502 mg (0.74 mmol) 4 and 127 ml (0.92 mmol) triethylamine
dissolved in 5 ml dichloromethane were added, and the mixture was stirred at ambient temperature
for 2 h. After evaporation to dryness the residue was puri®ed on silica gel (20 g) using cyclohexane/
ethyl acetate 1:1 as eluent. Yield: 432 mg (0.50 mmol, 68%) 9 as a wax-like solid.
Rf ˆ 0.65 (ethyl acetate); IR (KBr): ꢂ ˆ 2945 (ss) -CH2-, 2905 (m) -CH2-, 2870 (m) -CH2-, 1720
(ss) C=O, 1490 (m) arom, 1165 (m) C-O, 1145(m) C-O, 755 (m) arom; 1H NMR (CDCl3, 400 MHz):
3
ꢁ ˆ 0.67 (s, 3H, CH3-18), 0.85 and 0.88 (2d, J ˆ 6.6 Hz, 6H, diastereotopic CH3-26/27), 0.91 (d,
3J ˆ 6.7 Hz, 3H, CH3-21), 0.93±1.64 (m, 21H), 1.01 (s, 3H, CH3-19), 1.77±1.89 (m, 3H), 1.92±2.04
(m, 2H), 2.30 and 2.32 (2sb, 2H, allyl. CH2-4), 2.55±2.69 (m, 4H, -OCO-(CH2)2-COO-), 3.59±3.75
3
(m, 10H,-OCH2CH2O-(C2H4O)2-C2H4O-CO-aryl), 3.84 (t, J ˆ 4.8 Hz, 2H, -CH2-CH2-O-CO-aryl),
3
3
4.24 (t, J ˆ 4.8 Hz, 2H, -CH2-O-CO-CH2), 4.50 (t, J ˆ 4.8 Hz, 2H, -CH2-O-CO-aryl), 4.55±4.70
(m, 1H, CH-3), 5.35±5.40 (m, 1H, vinyl. CH-6), 6.89 (s, 2H, OC-CH=CH-CO), 7.61±7.53 (m, 2H,
arom. H), 8.03±8.05 (m, 2H, arom. H).

326
J. T. Kley et al.
O-(3-Cholesteryloxycarbonyl)propionyl-O0-p-maleimidobenzoyl tetraethylene glycol (10)
200 mg (0.92 mmol) p-maleimidobenzoic acid were converted into p-maleimidobenzoyl chloride
according to the procedure described in the preparation of 7. 502 mg (0.74 mmol) 4 and 127 ml
(0.92 mmol) triethylamine dissolved in 5 ml dichloromethane were added, and the mixture was
stirred at ambient temperature for 2 h. After evaporation to dryness the residue was puri®ed on silica
gel (20 g) using cyclohexane/ethyl acetate 1:1 as eluent. Yield: 448 mg (0.52 mmol, 71%) 10 as a
wax-like solid.
Rf ˆ 0.67 (ethyl acetate); IR (KBr): ꢂ ˆ 2945 (ss) -CH2-, 2905 (m) -CH2-, 2870 (m)-CH2-, 1720
(ss) C=O, 1610 (m) arom, 1515 (m), arom, 1145 (m), C-O, 755 (m) arom; 1H NMR (CDCl3,
400 MHz): ꢁ ˆ 0.67 (s, 3H, CH3-18), 0.85 and 0.87 (2d, 3J ˆ 6.6 Hz, 6H, diastereotopic CH3-26/27),
3
0.91 (d, J ˆ 6.7 Hz, 3H, CH3-21), 0.93±1.64 (m, 21H), 1.01 (s, 3H, CH3-19), 1.77±1.89 (m, 3H),
1.92±2.04 (m, 2H), 2.30 and 2.32 (2sb, 2H, allyl. CH2-4), 2.56±2.67 (m, 4H, -OCO-(CH2)2-COO-),
3
3.61±3.72 (m, 10H, -OCH2CH2O-(C2H4O)2-C2H4O-CO-aryl), 3.84 (t, J ˆ 4.8 Hz, 2H, -CH2-CH2-
O-CO-aryl), 4.23 (t, 3J ˆ 4.8 Hz, 2H, -CH2-O-CO-CH2), 4.49 (t, 3J ˆ 4.8 Hz, 2H, -CH2-O-CO-aryl),
4.55±4.70 (m, 1H, CH-3), 5.35±5.40 (m, 1H, vinyl. CH-6), 6.88 (s, 2H, OC-CH=CH-CO), 7.49±7.52
(m, 2H, arom. H), 8.13±8.17 (m, 2H, arom. H).
O-(3-Cholesteryloxycarbonyl)propionyl-O0-m-maleimidobenzoyl polyethylene glycol 400 (11)
326 mg (1.5 mmol) m-maleimidobenzoic acid were converted into m-maleimidobenzoyl chloride as
described in the preparation of 7. 870 mg (1.0 mmol) 5 and 139 ml (1.0 mmol) triethylamine dissolved
in 10 ml dichloromethane were added, and the mixture was stirred at ambient temperature for 2 h.
After evaporation to dryness the residue was puri®ed on silica gel (20 g) using ®rst cyclohexane/ethyl
acetate 1:1, then ethyl acetate as eluent. Yield: 357 mg (0.33 mmol, 33%) 11 as a wax-like solid.
Rf ˆ 0.22±0.6 (ethyl acetate/tetrahydrofurane 4:1); IR (KBr): ꢂ ˆ 2940 (ss) -CH2-, 2905 (m)
-CH2-, 2870 (m) -CH2-, 1720 (ss) C=O, 1590 (m) arom, 1490 (m) arom, 1145 (m) C-O, 755 (m)
1
3
arom; H NMR (CDCl3, 400 MHz): ꢁ ˆ 0.69 (s, 3H, CH3-18), 0.85 and 0.88 (2d, J ˆ 6.7 Hz, 6H,
diastereotopic CH3-26/27), 0.92 (d, 3J ˆ 6.7 Hz, 3H, CH3-21), 0.93±1.64 (m, 21H), 1.03 (s, 3H, CH3-
19), 1.77±1.89 (m, 3H), 1.92±2.04 (m, 2H), 2.30 and 2.32 (2sb, 2H, allyl. CH2-4), 2.57±2.70 (m, 4H,
-OCO-(CH2)2-COO-), 3.60±3.75 (m, approx. 30H, -OCH2CH2O-(C2H4O)7
-C2H4O-CO-
ꢂon average†
3
3
aryl), 3.86 (t, J ˆ 4.8 Hz, 2H,-CH2-CH2-O-CO-aryl), 4.23 (t, J ˆ 4.9 Hz, 2H, -CH2-O-CO-CH2),
4.50 (t, 3J ˆ 4.9 Hz, 2H, -CH2-O-CO-aryl), 4.53±4.69 (m, 1H, CH-3), 5.35±5.40 (m, 1H, vinyl.
CH-6), 6.90 (s, 2H, OC-CH=CH-CO), 7.55±7.61 (m, 2H, arom. H), 8.04±8.12 (m, 2H, arom. H).
O-(3-Cholesteryloxycarbonyl)propionyl-O0-m-maleimidobenzoyl polyethylene glycol 1000 (12)
195 mg (0.9 mmol) m-maleimidobenzoic acid were converted into m-maleimidobenzoyl chloride as
described in the preparation of 7. 930 mg (0.6 mmol) 6 and 83 ml (0.6 mmol) triethylamine dissolved
in 10 ml dichloromethane were added, and the mixture was stirred at ambient temperature for 2 h.
After evaporation to dryness the residue was puri®ed on silica gel (20 g) using ®rst ethyl acetate, then
ethyl acetate/tetrahydrofurane 1:1 as eluent. Yield: 420 mg (0.24 mmol, 40%) 12 as a solid.
Rf ˆ 0.43±0.67 (ethyl acetate/tetrahydrofurane/methanol 1:1:1); IR (KBr):ꢂ ˆ 2950 (ss) -CH2-,
2890 (m) -CH2-, 1720 (ss) C=O, 1145 (m) C-O, 755 (m) arom; 1H NMR (CDCl3, 400 MHz):
3
ꢁ ˆ 0.68 (s, 3H, CH3-18), 0.85 and 0.87 (2d, J ˆ 6.7 Hz, 6H, diastereotopic CH3-26/27), 0.92 (d,
3J ˆ 6.7 Hz, 3H, CH3-21), 0.95±1.69 (m, 21H), 1.01 (s, 3H, CH3-19), 1.80±1.93 (m, 3H), 1.95±2.09
(m, 2H), 2.32 and 2.34 (2sb, 2H, allyl. CH2-4), 2.55±2.70 (m, 4H, -OCO-(CH2)2-COO-), 3.55±3.75
(m, approx. 86H, -OCH2CH2O-(C2H4O)21
-C2H4O-CO-aryl), 3.83 (t, 3J ˆ 4.9 Hz, 2H,
ꢂon average†
3
3
-CH2-CH2-O-CO-aryl), 4.25 (t, J ˆ 4.9 Hz, 2H, -CH2-O-CO-CH2), 4.49 (t, J ˆ 4.9 Hz, 2H, -CH2-
O-CO-aryl), 4.56±4.70 (m, 1H, CH-3), 5.34±5.40 (m, 1H, vinyl. CH-6), 6.90 (s, 2H, OC-CH=CH-
CO), 7.54±7.58 (m, 2H, arom. H), 8.04±8.09 (m, 2H, arom. H).

Amphiphilic Lipids for Protein-Liposome Coupling
327
Acknowledgements
We thank Dr. Felix Kratz for the generous supply of maleimidobenzoic acids.
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Received October 20, 1997. Accepted October 25, 1997