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Ingallinella et al. 10.1073/pnas.0901007106

SI Materials and Methods trifluoroacetica acid, 5% phenol, 2% triisopropylsilane, and 5%
Peptide Synthesis. All peptides were synthesized by solid phase water for 1.5 h at room temperature. Each resin was filtered, and
using Fmoc/tBu chemistry on a peptide synthesizer ABI433A the solution was added to cold methyl-t-butyl ether to precipitate
(Applied Biosystems). For each peptide 0.75 g of resin Fmoc- the peptide. After centrifugation, the peptide pellets were
Linker AM-Champion, 1% cross-linked (Biosearch Technolo- washed with fresh cold methyl-t-butyl ether to remove the
gies) was used. The acylation reactions were performed for 60 organic scavengers. The process was repeated twice. Final pellets
min with 4-fold excess of activated amino acid over the resin-free were dried, resuspended in H2O and 20% acetonitrile, and
lyophilized.
amino groups. The amino acids were activated with equimolar
The crude peptides were purified by reverse-phase HPLC
amounts of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
using semipreparative Waters RCM Delta-Pak C4 cartridges
hexafluorophosphate (HBTU; Novabiochem) and a 2-fold molar (25 ⫻ 200 mm, 15 ␮m) and using as eluents (A) 0.1% triflu-
excess of N,N-diisopropylethylamine (DIEA; Sigma–Aldrich) in oroacetic acid in water and (B) 0.1% trifluoroacetic acid in
dimethylformamide (DMF). acetonitrile, flow rate 30 mL/min. Analytical HPLC was per-
The side chain protecting groups were: tert-butyl for Asp, Glu, formed on a Phenomenex Jupiter C4 column (150 ⫻ 4.6 mm, 5
Ser, and Thr; trityl for Asn, Cys, His, and Gln; tert-butoxy-carbonyl ␮m), flow rate 1 mL/min. The purified peptides were charac-
for Lys, Tyr, and Trp; and 2,2,4,6,7-pentamethyldihydrobenzofu- terized by liquid chromatography–mass spectrometry using a
ran-5-sulfonyl for Arg. Lys(N␧-palmitoyl) (Bachem) was used as Waters–Micromass LCZ platform.
C-terminal residue for the synthesis of C34-Pam. N-terminal acet-
ylation was performed at the end of the peptide assembly by Synthesis of Cholest-5-en-3-yl Bromoacetate (1). The synthetic
reaction with a 10-fold excess of acetic anhydride in DMF. scheme for the preparation of the precursor for chemoselective
At the end of the synthesis, the dry peptide–resins were incorporation of cholesterol into cysteine-containing peptides is
treated individually with 20 mL of the cleavage mixture, 88% outlined below.
H H
OH
DIPC, DMAP
Br + H H
O
H H
O 48h r.t. Br
HO O 73%
Bromoacetic acid Cholesterol Cholest-5-en-3-yl bromoacetate (1)
A mixture of 100 mg of cholesterol (Sigma–Aldrich) and 40 mg resulting cholesteroylated peptide was purified by reverse-phase
of bromoacetic acid (1.1 eq) (Sigma–Aldrich) was dissolved in 10 HPLC with semipreparative Waters RCM Delta-Pak C4 car-
mL of anhydrous CH2Cl2. Then 44 ␮L (1.1 eq) of N,N- tridges (25 ⫻ 200 mm, 15 ␮m), using as eluents (A) 0.1%
diisopropylcarbodiimide (DIPC; Sigma–Aldrich) and 1.5 mg trifluoroacetic acid in water and (B) 0.1% trifluoroacetic acid in
(0.05 eq) of 4-dimethylaminopyridine (DMAP; Novabiochem) acetonitrile, and an isocratic step at 50% (B) for 5 min followed
were added. The solution was left stirring at room temperature by the linear gradient: 50–70% (B) in 20 min to 80% (B) in 3 min,
for 48 h and analyzed by TLC (30% CH2Cl2 in hexane). washing step at 80% (B) for 3 min, flow 30 mL/min. The purified
Afterward, the solvent was removed under vacuum, and the peptide was characterized by HPLC/MS on a Waters–Micromass
crude was purified by flash column chromatography on silica gel LCZ platform (theoretical molecular mass, 5,020.7 Da; found,
(BIOTAGE) with a gradient of 10–30% CH2Cl2 in petroleum 5,020.0 Da).
ether to obtain 95 mg of the desired compound (1) as colorless
oil (yield: 73%). The purified product was analyzed by NMR. Synthesis of Chol-C34. Similarly to the synthesis of C34-Chol,
Chol-C34 was prepared by conjugation between the Cys-peptide
Synthesis of C34-Chol. C34-Cholesterol was prepared by chemo- precursor (Ac-CGSGWMEWDREINNYTSLIHSLIEESQN-
selective thioether conjugation between the Cys-peptide precur- QQEKNEQELL) and the cholesterol derivative (1). In partic-
sor (Ac-WMEWDREINNYTSLIHSLIEESQNQQEKNEQ- ular, 6.5 mg of purified Cys-peptide precursor (1.41 ␮mol) was
ELLGSGC) and the cholesterol derivative (1). In particular, 12 dissolved in 400 ␮L of DMSO, and 0.9 mg of (1) (1.77 ␮mol, 1.2
mg of purified Cys-peptide precursor (2.61 ␮mol) was dissolved eq), dissolved in 150 ␮L of THF, was added. Then 5 ␮L (1% by
in 600 ␮L of DMSO, and 1.59 mg of (1) (3.13 ␮mol, 1.2 eq), volume) of DIEA was added to the mixture, which was left
dissolved in 100 ␮L of THF, was added. Then 7 ␮L (1% by stirring at room temperature.
volume) of DIEA was added to the mixture, which was left After a 1-h incubation, the reaction was complete, and the
stirring at room temperature. The reaction was monitored by resulting cholesteroylated peptide was purified as described
liquid chromatography–mass spectrometry using a Waters– above. The purified peptide was characterized by HPLC/MS
Micromass LCZ platform with a Phenomenex Jupiter C4 column (theoretical molecular mass, 5,020.7 Da; found, 5,020.3 Da).
(150 ⫻ 4.6 mm, 5 ␮m) using as eluents (A) 0.1% trifluoroacetic
acid in water and (B) 0.1% trifluoroacetic acid in acetonitrile, Synthesis of T20-Chol. Similarly to the synthesis of C34-Chol,
and the following linear gradient: 30–70% (B) in 20 min to 80% T20-Chol was prepared by conjugation between the Cys-peptide
(B) in 3 min, washing step at 80% (B) for 3 min, flow 1 mL/min. precursor (Ac-YTSLIHSLIEESQNQQEKNEQELLELDK-
After a 1-h incubation, the reaction was complete, and the WASLWNWFGSGC) and the cholesterol derivative (1). In
Ingallinella et al. www.pnas.org/cgi/content/short/0901007106 1 of 4

particular, 10.1 mg of purified Cys-peptide precursor (2.11 ␮mol) SLIEESQNQQEKNEQELLGSGC) with iodoacetamide. In
was dissolved in 500 ␮L of DMSO, and 1.18 mg of (1) (2.53 ␮mol, particular, 10.2 mg of purified Cys-peptide precursor was dis-
1.1 eq), dissolved in 260 ␮L of THF, was added. Then 8 ␮L (1% solved in 1 mL of 6 M guanidine chloride, 0.25 M Tris, 2 mM
by volume) of DIEA was added to the mixture, which was left EDTA (pH 7.5). Then 6.0 mg of iodoacetamide (10 eq), dis-
stirring at room temperature. solved in 200 ␮L of DMSO, was added.
After a 1-h incubation, the reaction was complete, and the After a 1-h incubation, the product was purified by reverse-
resulting cholesteroylated peptide was purified as described phase HPLC. The purified peptide was characterized by
above. The purified peptide was characterized by HPLC/MS HPLC/MS (theoretical molecular mass, 4,651.1 Da; found,
(theoretical molecular mass, 5,223.0 Da; found, 5,221.6 Da).
4,650.0 Da).
Synthesis of C34-Acm. C34-Acm was prepared through reaction of
the Cys-peptide precursor (Ac-WMEWDREINNYTSLIH-

Fig. S1. Mechanism of action of C34-Chol. (Upper) Cells are incubated with C34 or C34-Acm and then washed thoroughly before addition of the virus. In this
condition, most of the inhibitor is removed before initiation of the infection and cannot interfere with transition of the hairpin intermediate to the 6-helix bundle
postfusion structure. (Lower) C34-Chol binds to the raft comparments of the membrane during the incubation, and because of its high affinity for this
compartment, it is not removed during the washing step, remaining available for inhibition.

Table S1. Pharmacokinetic parameters of C34-Acm and C34-Chol in mice
C34-Acm C34-Chol
Parameter s.c. i.v. s.c. i.v.
Cmax, ␮M 1.0 2.1

Tmax, h 0.8 3.0
AUC(03⬁), mM䡠h 1.4 0.7 13.9 18.9
t1/2, h 0.8 0.6 6.5 3.0
Vd, L/kg 0.21 0.03
Cl, mL/min/kg 5.7 0.2
In addition to the subcutaneous (s.c., 3.5 mg/kg) administration shown in Fig. 2, intravenous (i.v., 1 mg/kg)
administration was also performed. The vehicle was 10 mM glycine buffer (pH 10.2), 10% ethanol.

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Supporting Information

Ingallinella et al. 10.1073/pnas.0901007106

Bromoacetic acid Cholesterol Cholest-5-en-3-yl bromoacetate (1)

Ingallinella et al. www.pnas.org/cgi/content/short/0901007106 1 of 4

Ingallinella et al. www.pnas.org/cgi/content/short/0901007106 2 of 4

Ingallinella et al. www.pnas.org/cgi/content/short/0901007106 3 of 4

Parameter s.c. i.v. s.c. i.v.

Cmax, ␮M 1.0 2.1

Ingallinella et al. www.pnas.org/cgi/content/short/0901007106 4 of 4

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