Smart tools and orthogonal click-like reactions onto small unilamellar vesicles

Article abstract of DOI:10.1016/j.chemphyslip.2015.03.004

Abstract Click-based reactions were conducted at the surface of small unilamellar vesicles (SUVs) to provide onto-vesicle chemistry with efficient innovative ready-for-use tools. For that purpose, four amphiphilic molecules were designed to insert into bi

Full text of DOI:10.1016/j.chemphyslip.2015.03.004

Chemistry and Physics of Lipids 188 (2015) 27–36
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Smart tools and orthogonal click-like reactions onto small unilamellar
vesicles
Christophe Salomé, Maria Vittoria Spanedda, Benoit Hilbold, Etienne Berner,
**
Béatrice Heurtault, Sylvie Fournel, Benoit Frisch , Line Bourel-Bonnet
*
Laboratoire de Conception et Application de Molécules Bioactives, Equipe de BioVectorologie, UMR 7199 – CNRS/Université de Strasbourg, Faculté de
Pharmacie, 74 route du Rhin, BP 60024, 67401 Illkirch Cedex, France
A R T I C L E I N F O
A B S T R A C T
Article history:
Click-based reactions were conducted at the surface of small unilamellar vesicles (SUVs) to provide onto-
vesicle chemistry with efficient innovative ready-for-use tools. For that purpose, four amphiphilic
molecules were designed to insert into bilayers while presenting a reactive functional head. In this
manner, a dioleylglycero-ethoxy-ethoxy-ethoxy-ethanamine (DOG-PEG4-NH2) was chosen as a common
platform while the reactive amine head was converted into several electrophilic functions. Thus, two
dioleylglycerol-based cyclooctyne anchors were prepared: cyclooct-1-yn-3-glycolic acid-based anchor
(DOG-COA) and 1-fluorocyclooct-2-ynecarboxylic acid-based anchor (DOG-FCOA). The last one differed
from the first one in that a fluorine atom reinforces the electrophilic properties of the unsaturated bond.
In addition, a third dioleylglycerol-based triphenylphosphine (DOG-PPh3) was synthesized for the first
time. These three innovative amphiphilic anchors were designed to react with any azide-based
biomolecule following copper-free Huisgen 1,4-cycloaddition and Staudinger ligation, respectively. A
fourth anchor bearing a 3,4-dibromomaleimide ring (DOG-DBM) was also unprecedentedly synthesized,
to be further substituted by two thiols. Model reactions conducted in solution with either model biotinyl
azide or model biotinyl disulfide gave good to total conversions and excellent isolated yields. The four
new anchors were inserted into SUVs whose formula is classically used in in vivo biology. Stability and
surface overall electrostatic charge were in the expected range and constant over the study. Then, the
functionalized liposomes were ligated to biotin-based reagents and the experimental conditions were
finely tuned to optimize the conversion. The biotinyl liposomes were demonstrated functional and totally
accessible in an affinity test based on biotin scaffold quantification. Finally, DOG-FCOA’s reactivity was
confronted to that of DOG-DBM in a ‘one-pot’ orthogonal reaction. (Biotin-S)2 and TAMRA-N3
(tetramethylcarboxyrhodamine azide) were successively conjugated to the liposome suspension in a
successful manner. These data implement and reinforce the interest of bioorthogonal click-like reactions
onto lipid nanoparticles.
Received 14 January 2015
Received in revised form 5 March 2015
Accepted 24 March 2015
Available online 28 March 2015
Keywords:
Liposome
Copper-free click chemistry
Double ligation
Staudinger
Dibromomaleimide
ã 2015 Elsevier Ireland Ltd. All rights reserved.
Abbreviations: AcOH, acetic acid; CH2Cl2, dichloromethane; Chol, cholesterol; DCC, dicyclohexylcarbodiimide; DIEA, diisopropylethylamine; DLS, dynamic light
scattering; 4-DMAP, 4-N,N-dimethylaminopyridine; DMF, N,N-dimethylformamide; DOG-PEG4-NH2, dioleylglycero-ethoxy-ethoxy-ethoxy-ethanamine; EDCI, N-(3-
dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride; Et3N, triethylamine; EtOA, acetyl acetate; HATU, 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium
hexafluorophosphate; HBTU, O-benzotriazole-N,N,N0,N0-tetramethyl-uronium-hexafluoro-phosphate; HMPA, hexamethylphosphoramide; HOBt, hydroxybenzotriazole;
NHS, N-hydroxysuccinimide; PC, L-a-phosphatidylcholine; PDI, polydispersity index; PEG, polyethylene glycol; PG, L-a-phosphatidylglycerol; PTSA, para-toluene sulfonic
acid; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; SUVs, small unilamellar vesicles; TAMRA-N3, tetramethylcarboxyrhodamine azide;
TBAS, tetrabutylammonium sulfate; TCEP, tris(2-carboxyethyl)phosphine; THF, tetrahydrofurane; rt, room temperature.
* Corresponding author. Tel.: +33 (0)368 854 143; fax: +33 368 854 306.
** Corresponding author. Tel.: +33 (0)368 854 168.
E-mail addresses: frisch@unistra.fr (B. Frisch), line.bourel@unistra.fr (L. Bourel-Bonnet).
http://dx.doi.org/10.1016/j.chemphyslip.2015.03.004
0009-3084/ã 2015 Elsevier Ireland Ltd. All rights reserved.

28
C. Salomé et al. / Chemistry and Physics of Lipids 188 (2015) 27–36
1. Introduction
cyclo-addition – called ‘click chemistry’ by metonymy – to site-
specifically immobilize a mannose-based tree onto preformed
liposomes (Said Hassane et al., 2006; Frisch et al., 2010). The
disadvantage of this method is the in situ generation of a reducing
agent – Cu(I) – in excess. Moreover, the subsequent use of a water
soluble Cu(I) chelator, such as bathophenanthroline disulfonate,
was essential to obtain good yields (80%) in reasonable reaction
times (1 h) while eliminating copper ions reputed toxic for living
organisms. However, as assessed by agglutination experiments
using concanavalin A, the mannose residues were perfectly
accessible at the surface of the targeted vesicles. This first click
chemistry onto preformed vesicles paved the way for further fully
‘bioorthogonal’ reactions. Nowadays, copper-free click chemistry
is an outstanding and popular alternative and an increasing
amount of experiments performed in the field of chemical biology
(Jewett and Bertozzi, 2010; Shelbourne et al., 2012) are now
conducted without copper to get a totally ‘bioorthogonal’ bond as
the semantics puts it (Sletten and Bertozzi, 2009). As the use of
this alternate route remained very rare in the field of chemical
ligation involving liposomes or nanoparticles until very recently
(Jolck et al., 2011; Tarallo et al., 2011; Bostic et al., 2012; Colombo
et al., 2012; Koo et al., 2012; Wu et al., 2012; Emmetiere et al.,
2013; Airoldi et al., 2014), we decided to implement our previous
work by a further study. Here, we have designed four new anchors
(Fig. 1) based on a dioleylglycero-ethoxy-ethoxy-ethoxy-ethan-
amine platform (DOG-PEG4-NH2, (1), whose amine has been
converted into several electrophilic heads: cyclooctyne (DOG-COA,
(2), fluorocyclooctyne (DOG-FCOA, (3), triphenylphosphine
(DOG-PPh3, (4) and dibromomaleimide (DOG-DBM, (5).
These anchors were expected to react following copper-free
Huisgen cycloaddition, Staudinger ligation and substitution
respectively.
The modification of the surface properties of liposomes by
peptides, proteins or carbohydrates that convey either specificity
or targeting has been extensively studied (Sankaram, 1994;
Silvius and l’Heureux, 1994; Hojo et al., 1996; Boeckler et al., 1998;
Schelté et al., 2000). For example, the stable anchorage of
peptides into a phospholipid half-bilayer requires their branching
by
a lipid tail (Shahinian and Silvius, 1995; Epand, 1997)
composed of at least one, or better, two alkyl chains (Martin
and Papahadjopoulos, 1982; Kung and Redemann, 1986). Howev-
er, it is a tedious task due to the poor water solubility of the
resulting amphiphilic compounds. To overcome these difficulties,
alternative strategies have been developed where the compound
to be immobilized is covalently ligated to a functionalized lipid
anchor already inserted in the membrane (Heeremans et al.,1992;
Fleiner et al., 2001). In these strategies and until the mid-2000s,
the chemical bond between anchor and either peptide, protein or
carbohydrate compounds aimed at decorating the liposomes was
chosen among amide bond (Kamps et al., 1996), thioether bond
(Roth et al., 2004; Horak et al., 1999; Nakano et al., 2001), disulfide
bridge (Zalipsky et al., 1995; Elliott and Prestwich, 2000) or imine
bond (Harding et al., 1997). Later on, hydrazone and alpha-oxo
hydrazone bonds were also developed and for the first time fully
‘bioorthogonal’ chemoselective ligations were available (Zalipsky,
1993; Chenevier et al., 2002; Bourel-Bonnet et al., 2005;
Jolimaitre et al., 2007).
Then bioconjugate chemistry knew the boom of ‘click
chemistry’ (Kolb et al., 2001; Torne et al., 2002; Rostovtsev
et al., 2002). As a consequence several thousand papers and
reviews can now be reached when typing these two words on a
relevant research motor. Many authors now consider ‘click
chemistry’ as a powerful tool for chemistry (Meldal and Torne,
2008), pharmaceutical sciences (Hein et al., 2008) and chemical
biology (Speers et al., 2003; Boyce and Bertozzi, 2011; Beal and
Jones, 2012). In this wake, in parallel to another pioneer work
(Cavalli et al., 2006) and before a further work in this field
(Mourtas et al., 2011), we have already proposed a Huisgen [1,3]-
Biotin- and TAMRA-based tools were designed and synthesized
(Fig. 2) to tune and monitor the experimental conditions leading to
the expected decorated liposomes. The reactions could be perfectly
conducted in solution and were transposed to SUVs suspensions.
The biotinyl liposome adducts were proven functional in a
biocompatible environment.
O
R
O
O
O
O
O
R = NH2; DOG-PEG4-NH2,
1
O
O
O
R =
R =
NH
F
NH
DOG-COA,
2
DOG-FCOA,
3
O
O
Br
Br
P
R =
R =
N
O
NH
O
O
DOG-PPh3,
4
DOG-DBM, 5
Fig. 1. Four anchors based on a DOG-PEG4-NH2 platform

C. Salomé et al. / Chemistry and Physics of Lipids 188 (2015) 27–36
29
N
O
N
H
N
O
O
H
HN
HN
O
S
O
H
O
N H
O
O
biotin-N3,
6
O
O
O
N3
O
H
N
O
O
N3
H
TAMRA-N3,
8
HN
HN
S
S
H
H
S
S
N H
NH
H
N
H
(biotin-S)2,
7
O
O
Fig. 2. Molecular tools to study and quantify the bioconjugate chemistry onto liposomes.
2. Material and methods
mentioned activated solution. The resulting mixture was stirred at
room temperature overnight and concentrated under vacuum. The
crude was purified on silica gel using CH2Cl2/MeOH (100/0 to 95.2/
4.8) to give 55 mg (55%) of 2 as a yellow oil; Rf = 0.80 (CH2Cl2/
2.1. Generals
Reagent grade solvents were used without further purification.
Egg yolk -phosphatidylcholine (PC), -phosphatidylglycerol
MeOH: 9.5/0.5); 1H NMR (400 MHz, CDCl3):
d 5.34 (m, 4H), 4.23 (m,
L
-a
L-
a
1H), 4.05 (d, 1H, J = 15.3 Hz), 3.86 (d, 1H, J = 15.3 Hz), 3.65–3.40 (m,
(PG) transesterified from egg yolk PC, cholesterol (Chol) (recrystal-
lized in methanol), polymer supported triphenylphosphine and
anhydrous CH2Cl2 were purchased from Sigma–Aldrich. The PyBOP
was purchased from Novabiochem, the DIEA was from Alfa Aesar
and both were used without further purification. Column
chromatography was carried out on silica gel 60 (Merck, 70–
230 mesh). 1H NMR spectra at either 300 MHz, 400 MHz or
500 MHz and 13C NMR spectra at either 75 MHz, 100 MHz or
133 MHz were recorded on Brucker spectrometers either 300,
400 or 500 respectively with residual undeuterated solvent as
23H), 2.72–1.25 (m, 10H and 56H), 0.87 (t, 6H, J = 7.0 Hz); 13C NMR
(100 MHz, CDCl3): d 14.8, 21.3, 23.3, 26.8, 26.9, 27.9, 29.9, 30.2, 30.3,
30.4, 30.7, 32.6, 33.3, 34.9, 39.2, 42.8, 69.0, 70.9, 71.2, 71.5, 72.0,
72.3, 73.7, 78.5, 91.9, 102.2, 130.5, 130.9, 170.3; HRMS calcd for
C57H105NO8Na 954.7737; found 954.7738.
2.2.2. Preparation of 1-fluoro-N-((Z)-14-((Z)-octadec-9-en-1-yloxy)-
3,6,9,12,16-pentaoxatetratriacont-25-en-1-yl) cyclooct-2-
ynecarboxamide (3)
EDCI (58 mg, 0.30 mmol) and NHS (1 mg, 0.03 mmol) were
added to a CH2Cl2 solution (1 mL) of 1-fluorocyclooct-2-ynecar-
boxylic acid (51 mg, 0.30 mmol) (Schultz et al., 2010). The solution
was stirred at room temperature (30 min) and a CH2Cl2 (1 mL)
solution of DOG-PEG4-NH2 (1) (120 mg, 0.15 mmol) was added. The
solution was stirred at room temperature (16 h). The volatiles were
evaporated and the crude was purified by chromatography on silica
gel using cyclohexane/EtOAc (70/30 to 0/100) as eluent to obtain
7 mg (25%) of 3 as a yellow oil; IR (cmꢁ1) 3321, 2926, 2849, 2360,
1623, 1568, 1448, 1435, 1309, 1242, 1087, 640; 1H NMR (400 MHz,
internal reference. All chemical shift values (d), coupling constants
(J) and the multiplicity (s = singlet, d = doublet, t = triplet, m =
multiplet, br = broad) are quoted in ppm and in Hz respectively.
High-resolution mass spectra (HRMS) were obtained using an
Agilent Q-TOF (time of flight) 6520 and low-resolution mass
spectra (LRMS) using an Agilent MSD 1200 SL (ESI/APCI). Analytical
RP-HPLC-MS was performed using a C18 column (30 mm ꢀ 1 mm;
1.9 mm) using the following parameters: (1) the eluent system A
(0.05% TFA in H2O) and B (0.05% TFA in acetonitrile); (2) the linear
gradient t = 0 min with 98% A, t = 5 min with 5% A, t = 6 min with 5%
A, t = 7 min with 98% A, and t = 9 min with 98% A; (3) flow rate of
0.3 mL minꢁ1; (4) column temperature 50 ꢂC; (5) ratio of products
determined by integration of spectra recorded at 210 or 254 nm;
(6) ionization mode ESI. TLC spots were detected by UV irradiation
at 254 nm or with KMnO4 stain. IR spectra were performed on
Nicolet 380 FT-IR, Thermo Instruments.
CDCl3):
d 5.40-5.35 (m, 4H), 3.70-3.43 (m, 25H, 11CH2O, CH2N and
(CH2)2CHO), 2.38-2.28 (m, 4H, 2CH2 cyclooctyne), 2.09-1.98 (m,
10H, 4CH2 and CH2 cyclooctyne), 1.62-1.55 (m, 7H, 4H and 3H
cyclooctyne), 1.25-1.35 (m, 45H, 44H and H cyclooctyne), 0.82 (t,
6H, J = 7.2 Hz, 2CH3); 13C NMR (133 MHz, CDCl3):
d 14.1,14.2, 20.6 (d,
J = 3.0 Hz), 21.1, 22.7, 26.1 (d, J = 4.8 Hz), 27.2, 28.9, 29.3, 29.4, 29.5,
29.6, 29.7, 29.7, 29.8, 30.2 (d, J = 9.3 Hz), 31.9, 32.0, 39.3, 46.4 (d,
J = 25.0 Hz), 60.4, 69.5, 70.4, 70.5, 70.6, 70.6 70.7, 70.8, 70.9, 71.4,
71.7, 77.9, 109.5 (d, J = 10.9 Hz), 127.8, 128.7, 129.9, 130.0, 171.2;
HRMS (ESI) m/z calcd for C53H106FNO7SNa+ 942.7566; found
942.7557.
2.2. Chemical synthesis
2.2.1. Preparation of dioleylglycero-ethoxy-ethoxy-ethanamide
cyclooct-1-yn-3-glycol (2)
To
a
solution of cyclooct-1-yn-3-glycolic acid (20 mg,
2.2.3. Preparation of methyl 2-(diphenylphosphino)-4-(((Z)-14-((Z)-
octadec-9-en-1-yloxy)-3,6,9,12,16-pentaoxa-tetratriacont-25-en-1-
yl) carbamoyl) benzoate (4)
2-(Diphenylphosphino) terephthalic acid 1-methyl 4-penta-
fluorophenyl diester (10 mg, 0.019 mmol) was added to a DMF
(1 mL) solution of DOG-PEG4-NH2 (1) (17 mg, 0.022 mmol) and
0.109 mmol) (Bernardin et al., 2010) in anhydrous CH2Cl2 (2 mL),
DIEA (68 mg, 0.540 mmol) and PyBOP (68 mg, 0.130 mmol) were
added. The resulting mixture was stirred for 10 min for activation.
Then previously prepared DOG-PEG4-NH2 (1, Supplementary
information, Data in Brief) (100 mg, 0.130 mmol) solubilized in
2 mL of anhydrous dichloromethane was added to the above-
Et3N (13 mL, 0.095 mmol). The solution was stirred at room

30
C. Salomé et al. / Chemistry and Physics of Lipids 188 (2015) 27–36
temperature (16 h). The reaction mixture was concentrated under
vacuum and the crude was purified by chromatography on silica
gel using cyclohexane/EtOAc (100/0 to 50/50) as eluent to obtain
15 mg of DOG-PPh3 (4) as a colorless oil (62%); 1H NMR (400 MHz,
was mixed with an aqueous solution (1 mL) containing cystamine
dihydrochloride (107 mg, 0.47 mmol) and Et3N (309 L,
m
2.10 mmol). The reaction proceeded at room temperature over-
night with stirring. The white solid was filtered and washed with
water and EtOH to obtain N,N'-(disulfanediylbis(ethane-2,1-diyl))
bis(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentana-
mide) (7) as a white solid (250 mg; 87%); 1H NMR (400 MHz,
CDCl3): d 8.10 (dd, 1H, J = 8.0, 3.6 Hz, H of C6H3), 7.81 (dd, 1H, J = 8.0,
1.5 Hz, H of C6H3), 7.48-7.44 (m,1H, H of C6H3), 7.38-7.24 (m,10H, H
of C6H5), 6.65-6.59 (br s, 1H, NH), 5.41-5.32 (m, 4H), 3.76 (s, 3H,
OCH3), 3.68-3.41 (m, 25H, 11CH2O, CH2N and (CH2)2CHO), 2.08-
1.94 (m, 8H, 4CH2), 1.62-1.51 (m, 4H), 1.38-1.20 (m, 44H), 0.81 (t,
DMSO-d6):
d 8.08-8.04 (br m, 2H), 6.48 (s, 2H), 6.43 (s, 2H), 4.39-
4.34 (m, 2H), 4.22-4.17 (m, 2H), 3.42-3.35 (m, 2H), 3.19-3.14 (m,
2H), 2.88-2.81 (m, 6H), 2.64 (d, J = 12.9 Hz, 2H), 2.13 (t, J = 7.3 Hz,
4H), 1.72-1.53 (m, 8H), 1.40-1.34 (m, 4H); 13C NMR (100 MHz,
6H, J = 7.0 Hz, 2CH3); 13C NMR (100 MHz, CDCl3):
d 14.1 (2CH3), 22.7,
27.2, 29.3, 29.5, 29.5, 29.6, 29.6, 29.7, 29.8, 30.1, 32.6 (29CH2), 39.8
(CH2CH2N), 52.2 (OCH3), 69.6 (11CH2O), 70.3, 70.5, 70.6, 70.7, 70.9,
71.5, 71.7, 77.9 (CHCH2O), 126.7, 126.8, 128.6, 128.7, 129.0, 129.8,
129.9, 132.8, 133.8, 134.0 (10CH), 137.2, 137.3, 137.4, 141.3, 142.6
(4C), 158.7 (C(O)NH), 166.4 (C(O)OMe); HRMS (ESI) m/z calcd for
DMSO-d6):
d 25.7, 28.5, 28.7, 35.6, 37.8, 38.3, 40.3, 55.9, 59.7, 61.5,
163.2, 172.7.
2.2.7. N-(2-(2-(2-(2-azidoethoxy) ethoxy) ethoxy) ethyl)-5-(6)-
(tetramethylcarboxyrhodamine (TAMRA-N3,8)
C
68H108NO9PH+ 1114.7834; found 1114.7856.
To a solution of 5-(6)-TMCR (43 mg, 0.10 mmol) in anhydrous
2.2.4. Preparation of 3,4-dibromo-1-((Z)-14-((Z)-octadec-9-en-1-
yloxy)-3,6,9,12,16-pentaoxatetratriacont-25-en-1-yl)-1H-pyrrole-
2,5-dione (5)
CH2Cl2 (3 mL), DIEA (87 mL, 0.50 mmol) and PyBOP (63 mg,
0.12 mmol) were added. The resulting mixture was stirred for
10 min to get the corresponding activated ester. Then 11-azido-
Dibromomaleic acid (2.5 mg, 9.11
m
mol) and DCC (2 mg,
3,6,9-trioxaundecan-1-amine (22 mL, 0.11 mmol) solubilized in
9.54 mol) were stirred in CH2Cl2 (2.5 mL) at room temperature
m
anhydrous CH2Cl2 (3 mL) were added to the above-mentioned
solution. The resulting mixture was stirred at room temperature
(3 h) protected from light. Then the solution was concentrated
under vacuum and the crude was purified on silica gel using
CH2Cl2/MeOH (100/0 to 80/20) to give 53 mg (84%) of 8 as a purple
(1 h) for activation. A CH2Cl2 solution of DOG-PEG4-NH2 (1) (7 mg,
0.01 mmol) was added and the solution was stirred at room
temperature (16 h). The volatiles were evaporated and acetic acid
(4 mL) was added. Then, the solution was stirred at reflux (16 h).
Acetic acid was evaporated under vacuum and the residue was
purified by chromatography on silica gel using cyclohexane/EtOAc
(100/0 to 50/50) as eluent to obtain 7 mg (60%) of DOG-DBM (5) as
solid; 1H NMR (300 MHz, CDCl3):
d 8.52-8.48 (br s, 0.4H), 8.19-8.09
(m, 0.8H), 8.03-7.98 (m, 0.4H), 7.70-7.64 (br m, 1.2H), 7.54-7.50 (br
m, 1H), 7.21-7.15 (m, 2.2H), 6.79 (t, J = 10.3 Hz, 1H), 6.51-6.45 (m,
a colorless oil; 1H NMR (400 MHz, CDCl3):
d
5.35-5.32 (m, 4H), 3.79
3H), 3.72-3.58 (m, 14H), 3.37-3.33 (m, 2H), 3.05-3.07 (m, 12H); 13
C
(t, 2H, J = 5.2 Hz), 3.67-3.38 (m, 21H), 2.03-1.95 (m, 8H), 1.53-1.49
NMR (75 MHz, DMSO-d6), d (ppm): 29.7, 40.0, 40.1, 40.4, 50.6, 50.7,
(m, 4H), 1.42-1.15 (m, 44H), 0.86 (t, 6H, J = 6.7 Hz); 13C NMR
69.6, 69.7, 69.8, 69.9, 70.0, 70.1, 70.2, 70.3, 70.41, 70.46, 70.53,
70.56, 70.6, 97.2, 97.4, 111.0, 111.13, 111.14, 111.19, 111.30, 111.34,
111.35, 117.9, 124.4, 125.3, 125.90, 125.96, 126.0, 126.96, 126.98,
127.9, 128.2, 128.3, 128.6, 130.0, 130.3, 132.0, 136.1, 142.2, 154.5,
154.6, 155.1, 154.2, 166.4, 169.3, 169.4; LRMS calcd for C33H39N6O7
631.3; found 631.3.
(100 MHz, CDCl3): d 14.1, 16.8, 22.7, 26.1, 26.2, 27.2, 29.3, 29.5, 29.5,
29.6, 29.7, 29.8, 30.1, 31.9, 32.6, 39.0, 67.5, 70.1, 70.6, 70.6, 70.6, 70.7,
70.8, 70.9, 71.5, 71.7, 77.9, 129.4, 129.8, 129.9; HRMS (ESI) m/z calcd
for C51H91Br2NO8H+ 1004.5 [79Br, 79Br], 1006.5 [79Br, 81Br], 1008.5
[
[
81Br, 81Br]; found 1004.4 [79Br, 79Br], 1006.4 [79Br, 81Br], 1008.4
81Br, 81Br].
2.3. Liposomes preparation and characterization
2.2.5. Preparation of N-(2-(2-(2-(2-azidoethoxy) ethoxy) ethoxy)
ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)
2.3.1. SUVs formation
pentanamide (Biotin-N3, (6)) (Kottani et al., 2006)
Liposomes were prepared by mixing, in a round-bottom flask,
phospholipids (PC, PG), and cholesterol (80/20/50 molar ratio) in
chloroform/methanol 9/1 with the appropriate amount of anchors
(1, 2.5, 5 and 10 mol% for the initial experiments, then 5 mol% for
further experiments with an adjustment of the PC concentration).
After solvent evaporation under high vacuum (45 min), the
resulting dried lipid film was hydrated by vortex mixing (5 min)
after addition of 1 mL of buffer (100 mM NaCl, 10 mM Hepes, pH
Biotin-OSuc (14) (187 mg, 0.55 mmol) was completely dissolved
in DMF (5 mL) with gentle warming. After slowly cooling the
solution to room temperature without recurring precipitation, the
11-azido-3,6,9-trioxaundecan-1-amine (Borcard et al., 2011)
(109 mg, 0.50 mmol) was added. The reaction proceeded at room
temperature overnight with stirring and the reaction products
were concentrated under vacuum. The remaining solid was
triturated in CH2Cl2/Et2O (30/70) solution and the white solid
was filtered to obtain N-(2-(2-(2-(2-azidoethoxy) ethoxy) ethoxy)
ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) penta-
7.4) to get a final concentration of 10 mmol of total phospholipids
per mL. The resulting suspension was sonicated at 25 ꢂC for 45 min
under a continuous flow of argon, using a 3 mm diameter probe
sonicator (Vibra Cell, Sonics and Material Inc., Danbury, CT) at
300 W. The small unilamellar vesicle (SUV) suspensions were
finally centrifuged for 10 min to remove the titanium dust
originating from the probe. Blank liposomes were only composed
of PC/PG/Chol (80/20/50 molar ratio) without anchor.
namide (6) (219 mg; quant.); 1H NMR (300 MHz, DMSO-d6):
d 6.43-
6.38 (s, 1H, NH), 6.37-6.34 (s, 1H, NH), 4.33-4.25 (m, 1H), 4.12-4.09
(m, 1H), 3.62-3.34 (m, 12H), 3.12-3.08 (m, 1H), 2.89-2.78 (m, 4H),
1.55-1.19 (m, 6H); 13C NMR (100 MHz, CDCl3):
d 25.6, 28.0, 28.1,
35.9, 39.2, 40.5, 50.7, 55.5, 60.3, 61.8, 70.0, 70.1, 70.4, 70.5, 70.6,
163.9, 173.5; LRMS (ESI) m/z calcd for C18H32N6O5SH+ 445.2; found
445.2.
2.3.2. PC assay
The PC content of liposome suspension was quantified using an
2.2.6. Preparation of N,N0-(disulfanediylbis(ethane-2,1-diyl))bis(5-(2-
oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanamide) (7)
In accordance to a described procedure (Wang et al., 2012),
Biotin-OSuc (14) (300 mg, 0.87 mmol) was completely dissolved in
DMF (3 mL) with gentle warming (40 ꢂC). After slowly cooling the
solution to room temperature without recurring precipitation, it
enzymatic assay with a commercial test kit (AssayTM Phospholipid,
Wako Chemicals GmbH, Neuss, Germany). 5 or 10
suspensions were distributed in triplicate in a 96-well microplate
before adding 250 L of phospholipid B reagent solution, prepared
mL of liposome
m
according to the manufacturer’s instructions. The plates were then
incubated at 37 ꢂC for 10 min. A blue color appeared, in presence of

C. Salomé et al. / Chemistry and Physics of Lipids 188 (2015) 27–36
31
choline, due to the formation of a phenol-4-aminoantipyrine
complex. Choline chloride was used for calibration curve.
1 h, 6 h, 24 h or 48 h. After the coupling step, the unbound probe
and TCEP derivative were removed by exclusion chromatography
on a 1 ꢀ20 cm Sephadex G-100 column equilibrated with the same
buffer as that used in the coupling step.
Absorbance was measured at
l= 595 nm using a microplate reader
(Bio-Rad model 550, Marnes-la-Coquette, France). It is postulated
that a loss in PC is directly correlated to a loss in anchor within the
same proportions.
2.4.5. ‘One pot’ orthogonal double ligation
Liposomes were prepared by mixing in a round-bottom flask,
phospholipids (PC, PG) and cholesterol (75/20/50 molar ratio) in
chloroform/methanol 9/1 with the DOG-FCOA anchor (2.5% based
on phospholipid quantity,10 mM in chloroform/methanol 9/1) and
the DOG-DBM anchor (2.5% based on phospholipid quantity,
10 mM in chloroform/methanol 9/1). After solvent evaporation
under high vacuum (45 min), the resulting dried lipid film was
hydrated by vortex mixing (5 min) after addition of 1.5 mL of buffer
(150 mM NaCl,10 mM Hepes, pH 7.4), to get a final concentration of
2.3.3. Particle-size measurements
The particle size distribution was measured by dynamic light
scattering (DLS) using a Nano ZS (Malvern Instrumentation, Inc.,
Orsay, France) with an angle of detection of 173 degrees, measured
at 25 ꢂC and in intensity. Each sample was diluted with the Hepes
buffer until the expected particle concentration was achieved, i.e.,
20 mL of a 10 mM liposome suspension and 980 mL of buffer. Each
measurement was performed in triplicate.
10 mmol of total lipids per mL. The resulting suspension was
2.4. Chemistry on liposomes
sonicated at 25 ꢂC for 45 min under a continuous flow of argon,
using a 3 mm diameter probe sonicator (Vibra Cell, Sonics and
Material Inc., Danbury, CT) at 300 W. The SUV preparations were
finally centrifuged for 10 min to remove the titanium dust
originating from the probe. TCEP (100 mM in water, 2.5%) and
(Biotin-S)2 (5 mM in water, 2.5%) were added successively and the
buffer suspension was shaken at room temperature for 1 h to react
2.4.1. Absorbance measurement
The absorbance was measured on
a Safas Xenius XM
spectrometer at
l= 551 nm in a quartz cell. The background
absorbance of the blank liposomes was subtracted from the
recorder absorbance of the liposome.
on anchor 5. Then, one third (1/3) of the suspension (500 mL) was
2.4.2. Biotin coupling and determination of its functionality
The amount of biotin bound on liposomes was quantified using
a commercial kit (Quant*TagTM Biotin Kit Vector Laboratories, Inc.,
purified on SEPHADEX-100, lyophilized and re-solubilized (DMSO/
H2O) for biotin quantification and yield determination. One
equivalent of TAMRA-N3 8 (10 mM in water, 2.5%) was added to
the remaining suspension (2/3; 1 mL) and the mixture was shaken
for 23 h. After the coupling step, the unbound probe was removed
Burlingame, CA). 5
m
L of liposome suspensions were distributed in
L of reagent
triplicate in a 96-well microplate before adding 100
m
solution, prepared according to the manufacturer’s instructions.
The plates were then incubated at 25 ꢂC for 30 min. A yellow–
orange color appeared in presence of biotin. Free biotin was used
by exclusion chromatography on
a
1 ꢀ20 cm Sephadex
G-100 column equilibrated with the same buffer as the one used
in the coupling step. After that purification, lyophilization and
re-solubilization (DMSO/H2O), the rhodamine was quantified by
UV/visible absorption spectrometry at 551 nm (after a standard
curve was obtained with a DMSO/H2O solution of compound 8 –
TAMRA-N3).
for calibration. Absorbance was measured at
l= 535 nm using a
microplate reader (Bio-Rad model 550, Marnes-la-Coquette,
France).
2.4.3. Liposomes ligation: copper-free click
To a suspension of functionalized liposomes in buffer, biotin-N3
6 (10 mM in water, 5%) was added. The resulting mixture was
gently stirred under argon at 25 ꢂC for 1 h, 6 h, 24 h or 48 h. After the
coupling step, the unbound probe was removed by exclusion
chromatography on a 1 ꢀ20 cm Sephadex G-100 column equili-
brated with the same buffer as that used in the coupling step.
3. Results and discussion
3.1. Chemistry
First of all, a key amphiphilic intermediate dioleylglycero-
ethoxy-ethoxy-ethoxy-ethanamine (DOG-PEG-NH2, 1) was syn-
thesized as a racemic mixture in five steps starting from glycerol,
tetraethylene glycol and oleyl alcohol according to a previously
described procedure (Espuelas et al., 2003). Considering the low
overall yield of the reported procedure (16%), we optimized the
oleyl alcohol alkylation and the azide reduction to reach 27%
overall yield. For the first step, we found the modified phase-
2.4.4. Liposomes ligation: thiol-dibromomaleimide
To a suspension of functionalized liposomes in buffer, (biotin-
S)2 7 (1 eq. based on the anchor quantity, 5 mM in water) and TCEP
(1 eq. based on the anchor quantity, 100 mM in water) were added.
The resulting mixture was gently stirred under argon at 25 ꢂC for
Scheme 1. Synthesis of DOG-PEG4-NH2: (a) Trityl chloride, DMAP cat., Et3N, THF, rt (22 h); (b) 10, TBAS 0.1%, NaOH, H2O, 65 ꢂC; (c) PTSA, THF/MeOH (1/1), rt (16 h); (d) 12, NaH
(60%), THF, HMPA; (e) Polymer-bound PPh3, H2O, THF, 50 ꢂC (16 h).

32
C. Salomé et al. / Chemistry and Physics of Lipids 188 (2015) 27–36
transfer-catalyzed procedure (TBAS, H2O, NaOH), already de-
scribed (Bartsch et al., 1989), very efficient and it allowed us to
increase the yield to 80%. In the same way, the azide reduction was
optimized by using polymer-supported triphenylphosphine thus
helping for the purification stage by a simple filtration of a
triphenylphosphine oxide derivative immobilized on the resin
(Scheme 1). Thus the chemical route toward 1 was improved and
the work-up was simplified.
Embarked on the study of new ways of coupling biomolecules of
interest to the surface of preformed liposomes, we designed two
dioleyl cyclooctyne-based anchors (DOG-COAs, 2 and 3) and one
dioleyl phosphine-based anchor (DOG-PPh3, 4) capable of reacting
selectively and successfully on azides (Brase and Barnert, 2010;
Zhang et al., 2009; Vabbilisettya and Sun, 2014) and one further
dioleyl-dibromomaleimide-based anchor (DOG-DBM, 5) suitable
for reacting selectively and efficaciously on thiols (Smith et al.,
2010; Schumacher et al., 2011). This last one shows very interesting
benefits, as its reversible modification is possible. Advantageously,
dibromaleimide/thiol bioorthogonal ligation is a way to double
anchor proteins onto lipid vesicles. As a consequence, an extremely
efficient disulfide bridging bioconjugation could be obtained. For
example, it was showed that conjugation proceeds rapidly and
efficiently to give the expected adducts while maintaining the
disulfide bridge and thus peptide conformation (Jones et al., 2012).
The amphiphilic properties of the target molecule allow it to be
incorporated in the phospholipid bilayer of liposomes while
exposing a constrained and reactive head equally distributed on
the outer and the inner membrane of the vesicle.
A first DOG-COA, dioleylglycero-ethoxy-ethoxy-ethoxy-etha-
namide cyclooct-1-yn-3-glycole (2) was prepared: the free amine 1
reacted with active hydroxybenzotriazolyl ester of cyclooct-1-yn-
3-glycolic acid (Bernardin et al., 2010), obtained with PyBop in the
presence of DIEA in CH2Cl2, to give the alkyne anchor 2 with a
satisfactory overall isolated yield (55%) (Scheme 2). The second
cyclooctyne anchor was then prepared: substituted by a fluorine, it
was chosen to react more efficiently on azides due to the electron
withdrawing effect of the halogen as recently shown (Schultz et al.,
2010; Gold et al., 2013). As previously, the amine 1 was coupled
with the 1-fluorocyclooct-2-ynecarboxylic acid in DMF, in the
presence of EDCI and Et3N with only 25% yield. Attempts to
increase the yield by changing the coupling agent failed. DCC,
HBTU, HATU were successively tried without any success.
For the third anchor, the free amine 1 was simply coupled with
the commercially available (diphenylphosphino) terephthalic acid
1-methyl 4-pentafluorophenyl diester in DMF to yield the expected
compound 4 (62%). Finally, the DOG-DBM anchor 5 was obtained
from the reaction with dibromomaleic acid in DMF using DCC as
coupling agent (60%) (Wilson et al., 2006). The overall yields are in
Scheme 2. Synthesis of the anchors.

C. Salomé et al. / Chemistry and Physics of Lipids 188 (2015) 27–36
33
Scheme 3. Synthesis of the probes. (a) NHS, DCC, DMF; (b) H2N-PEG3-N3, DMF; (c) cystamine. HCl, DMF/H2O (3/1), Et3N; (d) H2N-PEG3-N3, PyBOP, CH2Cl2, DIEA.
good range for amphiphilic compounds whose purification is
reputed challenging: this tedious last operation is usually
responsible for the loss of pure expected final compound and
frequently screens off very good to excellent conversion yields.
liposome preparation procedure) with a polydispersity index
inferior to 0.3. These results show that every anchor is well
tolerated in the liposomal formulation and the diameters are not
significantly different from each other and from blank liposomes.
In the following experiments, we have selected 5% anchor as a
constant concentration to ensure a reliable monitoring if ever a low
conversion yield is obtained in one ligation or another.
3.1.1. Probes’ synthesis
To get the biotin-based probes, biotin succinimidate 14 was
synthesized as a key intermediate following the already described
procedures (Kottani et al., 2006; Susumu et al., 2007). Then, the
two probes could be prepared by coupling 14 with either the
11-azido-3,6,9-trioxaundecan-1-amine (H2N-PEG3-N3) in DMF to
get the azido probe 6 or the cystamine hydrochloride to get the
thiol-probe 7. TAMRA-N3 probe 8 was synthesized starting from
the commercially available 5(6)-carboxytetramethylrhodamine
(15) and 11-azido-3,6,9-trioxaundecan-1-amine (H2N-PEG3-N3)
in the presence of DIEA and PyBOP in CH2Cl2 (Scheme 3). After
purification, the azido fluorescent probe 8 was obtained with 83%
yield. Before conducting further studies, we evaluated the
chemical reactivity of the selected functional groups using in
solution model reactions. In all cases, the expected compounds
were obtained with good to excellent yields (50% to quantitative,
Supplementary information, Data in Brief).
3.3. Click-like chemistry on preformed liposomes
3.3.1. Control ligation
Before performing the ligation reactions, the biotin and TAMRA
probes 6, 7 and 8 respectively were evaluated for their unspecific
affinities with blank liposomes (containing no anchor). The
liposomes were prepared following the above-mentioned general
procedure. Then biotin and TAMRA probes were added and the
suspensions were shaken at room temperature (48 h). After
purification of the liposomes by gel filtration on SEPHADEX-100,
the unspecific binding of biotin was quantified using the biotin kit
and that of TAMRA probe was quantified by fluorescence. For each
probe, less that 1% of unspecific adsorption was observed (data not
shown).
3.2. Liposomes formulation
Table 1
Size of the liposomes (; nm) depending on the type of anchors and their
The liposomes were prepared by integrating the anchors (1%,
2.5%, 5% and 10% molar ratio) within the phosphatidyl choline
(79%, 77.5%, 75% and 70%, respectively), phosphatidyl glycerol
concentration (between parentheses: polydispersity index, PDI).
Anchor type
(20%) and an additional half of cholesterol to get
a final
2
3
4
5
Anchors quantity (%)
concentration of total lipids as high as 10 mM (53.3/13.3/33.3%
molar ratio). After evaporation and lipid film hydratation, the
resulting suspension was sonicated using a probe sonicator to get
unilamellar vesicles. The average diameter was finally measured by
DLS (Table 1). For all the concentrations and anchors, the sizes
were in the expected range (<100 nm which is usual for such a
0
89 (0.25)
61 (0.25)
1
100 (0.29)
101 (0.27)
73 (0.21)
81 (0.17)
86 (0.17)
115 (0.28)
71 (0.24)
90 (0.27)
106 (0.27)
2.5
5
10
43 (0.17)
97 (0.30)
63 (0.24)
87 (0.25)
84 (0.24)
89 (0.22)

34
C. Salomé et al. / Chemistry and Physics of Lipids 188 (2015) 27–36
Table 2
Laboratories, Inc., cat. No. BDK-2000) to calculate the ligation yield
(Table 2).
Yields (%) of the different ligations at four reaction times (1 h, 6 h, 24 h and 48 h).
Molecular tool
Conversiona
3.3.3. Staudinger chemistry on preformed liposomes
1 h
6 h
24 h
48 h
The same strategy was used for the Staudinger ligation. The
liposomes were prepared by integrating 5% (mol/mol) of
DOG-PPh3 4 and biotinyl azide 6. The results are presented in
Table 2 over 1, 6, 24 and 48 h respectively.
2
3
4
5
6
6
6
7
8a
14a
16a
17a
9a
19a
37a
46a
22a
28a
35a
38a
Quant.a,b
Quant.a,b
Quant.a,b
Quant.a,b
a
Yield calculated with the Quant Tag Biotin Kit and on the outer exposed anchors.
Conversion based on the DOG-DBM quantity on the outer exposed anchors.
b
3.3.4. Dibromomaleimide/thiol ligation on preformed liposomes
For this fourth ligation, the procedure was slightly modified.
The liposome formulation was unchanged, but for the ligation step,
1 eq. of 7 was used, leading, after reduction with 1 eq. of TCEP, to
2 eq. of biotin-SH. The incubation mixture was left up 1, 6, 24 and
48 h. Unbound compound 7 was removed by gel filtration on
SEPHADEX-100 and then the biotin was quantified after lyophili-
3.3.2. Azide/alkyne chemistry on liposomes
As there above-mentioned, liposomes were prepared by
integrating 5% (mol/mol) of either DOG-COA 2 or DOG-FCOA 3
to get a 10 mM final concentration. The suspension was sonicated
to get unilamellar vesicles so that half of the alkyne reactive head
functions were exposed on the outer membrane (assuming an
equimolecular repartition of the anchor between outer and inner
membranes). The final liposomes were obtained by simple stirring
of the functionalized liposomes with biotinyl azide 6 (1 eq. toward
total 2 and 3, i.e. 2 eq. toward outer exposed 2 and 3). The
incubation mixture was left up 1, 6, 24 and 48 h. Unbound
compound 6 was removed by gel filtration on SEPHADEX-100. It
was checked by DLS that the size of the SUVs remained unchanged
after chemical ligation and after gel filtration purification steps (;
70–100 nm). Thus, the coupling reaction and the purification steps
do not alter the size of the liposomes. The combined fractions were
zation and re-solubilization in DMSO/H2O (100/200
calculate the ligation yield (Table 2).
mL) to
First of all, as expected, cyclooctyne anchor 3, bearing a fluoride,
was more electrophilic and more reactive than cyclooctyne anchor
2 and greater conversions were reached (46% versus 17% after
48 h). For the Staudinger reaction, surprisingly, the ligation
occurred with only 38% yield that could not be improved. We
hypothesize that the phosphine was oxidized rapidly, leading to an
inactive phosphorous atom in the liposome suspension buffer
(Wang et al., 2012). Next, the ligation with dibromomaleimide was
effected with (biotin-S)2 7. Reducing agent TCEP was chosen for its
solubility in aqueous media, selectivity and inertness toward
liposome constituents. In these conditions, double-biotinated
liposomes were obtained quantitatively after only 1 h. Finally, to
demonstrate the versatility of the construct, we intended to
decorate liposomes with two different species without any buffer
lyophilized. Then 100 mL of DMSO followed by 200 mL of water
were added and stirred to obtain a homogenous solution. Then, the
biotin was quantified using the Quant-Tag Biotin kit (Vector
A
O
O
NH
H
HN
S
H
HN
H
S
NH
H
N
S
O
O
8
H
N
H
O
7
O
S
O
N
O
O
8
7
3
O
Mass calculated for C75H131N7O12S4Na
1472.8630
Found
1472.8627
N
O
B
O
O
N
7
8
H
N
F
O
O
O
-
8
7
H
N
CO2
3
N
O
N
O
N
3
Mass calculated for C89H140FN7O14Na
1573.0334
Found
1573.0341
Fig. 3. Bioconjugate compounds found in the DMSO/H2O solution after destruction of the liposomes.

C. Salomé et al. / Chemistry and Physics of Lipids 188 (2015) 27–36
35
modification. To this, we selected the two best anchors to set up an
orthogonal ligation on liposome.
ligation onto liposomes. These results implement the amount of
bioorthogonal chemical bonds available for liposome chemical
modification.
3.3.5. Double ligation dibromomaleimide/thiol and copper free on the
same liposome suspension
Conflict of interest
For this double ligation, the total anchor's proportion was kept
as high as 5%. Thus 2.5% of each anchor were used in the
formulation. The couple DOG-FCOA 3 and DOG-DBM 5 was chosen
as the best combination for a double ligation without changing
buffer. After liposome formation, 1 eq. of TCEP and 1 eq. of 7 were
added and left in contact for 1 h to react on 5. Then, one third (1/3)
of the solution was purified on SEPHADEX-100, lyophilized and re-
solubilized (DMSO/H2O) for biotin quantification and yield
determination.
One equivalent of TAMRA-N3 8 was added to the remaining
suspension (2/3) and the mixture was shaken for 24 h. After
purification on SEPHADEX-100, lyophilization and re-solubiliza-
tion (DMSO/H2O), the rhodamine was quantified using TAMRA-N3
8 solution as standard curve. To strengthen our double ligation, we
analyzed by mass spectrometry the resulting liposomes and found
evidences (m/z) of both maleimide/biotin conjugate and cyclo-
octyne/TAMRA conjugate (Fig. 3). Finally, to prove that fluorocy-
clooctyne function was totally inert toward the biotin 7, we
prepared a liposome suspension bearing only anchor 3. Then, in
place of biotin-N3 6, we added 1 eq. of biotin disulfide 7 and 1 eq. of
TCEP. After 24 h, the same work-up was performed. After
solubilization in DMSO/H2O (1/2), the biotin was quantified using
the biotin kit and we found that less than 6% of biotin had been
non-specifically anchored.
The results of the double ligation show that the first
conjugation was obtained with quantitative yield following the
above-described quantification procedure. Secondly, the rhoda-
mine quantification demonstrates that the double ligation was
effective too. The coupled compound was obtained with yields
comparable to those observed with the biotin tool (Table 2). These
results demonstrate that a double ligation is possible without
changing the buffer and bring up a very simple method to di-
functionalize liposomes in a ‘one pot’ sequential matter. Finally, we
proved the total stability of the cyclooctyne toward thiol
derivatives. Such additional methodology reinforces the versatility
of the liposome tool, especially when some biomolecules to be
anchored to these vesicles react very differently and therefore
when the use of a statistical mixture is impossible.
The authors report no competing financial interest.
Acknowledgments
The authors thank Philippe Gentine for fruitful scientific
discussions and Patrick Wehrung for technical assistance. The
authors gratefully acknowledge financial support from Université
de Strasbourg and Centre National de la Recherche Scientifique (C.
N.R.S.). This work was also supported by a MRE (Ministère de la
Recherche et de l'Enseignement) fellowship allocated to Line
Bourel and Benoît Hilbold by the President of the University de
Strasbourg (www.unistra.fr).
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
chemphyslip.2015.03.004.
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We have successfully designed and prepared four innovative
anchors and integrated them into liposome bilayers. All ligations
performed on these functionalized anchors were effective. We also
demonstrated that for all four anchors, the conjugated species are
at the surface and still accessible to biomolecules (possibly:
antibodies, proteins, enzymes...). The best result was unprece-
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potentially interesting for peptides bearing disulfide bridges and
allows the incorporation of two thiol derivatives for one anchor
leading to a high atom economy. Another important advantage
would be the possible preservation of the cyclic conformation of
peptides or the possibility to get loops. Furthermore, we
demonstrated that the cyclooctyne derivatives are inert toward
thiols. This last observation allowed us to perform a one pot double
ligation onto liposomes by using two anchors of the type
cyclooctyne and dibromomaleimide without any cross-coupling
reaction. This technics could be of great interest for the
incorporation of epitopes or immunoadjuvants on lipid particles
as ingredients for synthetic vaccines. More widely, those method-
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Pillarsetty, N., Weber, W.A., Lewis, J.S., Reiner, T., 2013. 18F-labeled-
bioorthogonal liposomes for in vivo targeting. Bioconjugate Chem. 24,
1784–1789.
Epand, R.M., 1997. Biophysical studies of lipopeptide-membrane interactions. Pept.
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