Synthesis of unsymmetrical spin-labelled bolaamphiphiles

Article abstract of DOI:10.1016/j.tetlet.2008.05.125

This work presents the efficient synthesis of a novel spin-labelled bolaamphiphile bearing sugar and cationic polar heads. Preliminary ESR studies were conduced both in CH₂Cl₂ and water and have confirmed that this methodology can be used to study the transversal rotation (flip-flop) dynamic of membrane involving such bolaamphiphiles.

Full text of DOI:10.1016/j.tetlet.2008.05.125

Tetrahedron Letters 49 (2008) 4690–4692
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of unsymmetrical spin-labelled bolaamphiphiles
*
´
Mathieu Berchel, Loïc Lemiègre, Jelena Jeftic, Thierry Benvegnu
UMR CNRS 6226, Equipe ‘Chimie Organique et Supramoléculaire’, Ecole Nationale Supérieure de Chimie de Rennes, Université Européenne de Bretagne,
Avenue du Général Leclerc, 35700 Rennes, France
a r t i c l e i n f o
a b s t r a c t
Article history:
This work presents the efficient synthesis of a novel spin-labelled bolaamphiphile bearing sugar and cat-
ionic polar heads. Preliminary ESR studies were conduced both in CH2Cl2 and water and have confirmed
that this methodology can be used to study the transversal rotation (flip–flop) dynamic of membrane
involving such bolaamphiphiles.
Received 7 May 2008
Revised 26 May 2008
Accepted 28 May 2008
Available online 2 June 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Bolaamphiphile
Archaea
Synthesis
Nitroxide
ESR
Bipolar lipids found in archaebacterial membranes, generally
termed bolaamphiphiles or bolas,1 induce increased stability in
membranes exposed to environments such as acidic conditions,
high temperatures, high salt concentrations and/or absence of oxy-
gen. Several approaches to synthetic bipolar lipids have been per-
formed in order to mimic archaeal membranes.2–8 The flip–flop,
that is, the transversal rotation of lipid units in the membrane is
one of the fundamental processes involved in the membrane
dynamics (Fig. 1).9 Generally, the flip–flop from the exovesicular
to the endovesicular membrane surface (and vice versa) is a rela-
tively slow process, which is due to the high energy barrier in
transferring the polar amphiphilic heads through the lipophilic
membrane.10,11
Figure 1. Flip–flop phenomenon of bolas.
Flip–flop phenomenon can be involved in cell membrane trans-
port mechanisms12 and its rate in this case is strongly dependent
on polar head-group composition and less dependent on the alkyl
chain length.13 Recently, the transbilayer flip–flop of early interme-
diates in the glycosylphosphatidylinositol (GPI) biosynthetic path-
way has been demonstrated using novel fluorescent GPI probes
and a biochemical reconstitution approach.14 The ESR spectroscopy
is also well-adapted for the study of such dynamic as described
previously for monopolar lipids.11 Few studies have been per-
formed on the flip–flop dynamics of bolas4,5,15 but to our knowl-
edge no data are available on unsymmetrical bolas.
Br
H
N
O
N R
N
H
HO
O
O
O
OH
1 : R = CH3
2 : R =
OH
N O
Figure 2. Non-labelled (1) and labelled bolas (2).
Our research group has already studied the self-organisation of
unsymmetrical bolaamphiphiles such as 1 (Fig. 2).7 Indeed 1 self-
assembles into different shapes (discs, vesicles) depending on the
temperature and we assume that the transition between these
two types of organisation is due to flip–flop dynamics within the
membrane. To point out this kind of dynamics we describe herein
the synthesis of a spin-labelled analogue of 1 (2) (Fig. 2).
* Corresponding author. Tel.: +33 022 323 8060; fax: +33 022 323 8046.
E-mail address: thierry.benvegnu@ensc-rennes.fr (T. Benvegnu).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.05.125

M. Berchel et al. / Tetrahedron Letters 49 (2008) 4690–4692
4691
O
H
O
O
N
(CH2)12 NH2
O(CH2)7CH3
BnO
BnO
O
OOct
HO
i, ii
iii
O
O
O
O
OH
OH
OBn
3
4
5
OBn
iv
OH
H
H
N
H
N
H
N
O
O
N
(CH2)12
OOct
(CH2)12
OOct
N
N
R
Br
HO
BnO
v, vi
O
O
O
OH
O
OH
Br
6
7 : R = H
2 / 7
OH
OBn
vii
: R = O / H
Scheme 1. First strategy for the preparation of labelled bola 2. Reagents and conditions: (i) 1-octanol, BF3ꢀEt2O, THF, reflux, 70%; (ii) Ag2O, BnBr, CH2Cl2, 70%; (iii) 1,12-
diaminododecane, MeOH, 60%; (iv) BrC(O)CH2Br, pyridine, 65%; (v) 4-dimethylamino-2,2,6,6-tetramethylpiperidine, DMF, 59%; (vi) H2, Pd/C, MeOH, 70%; (vii) oxone, acetone,
nBu4NHSO4, CH2Cl2, phosphate buffer pH 7, 76%.
(CH2Cl2/phosphate buffer) using acetone, oxone and phase-transfer
catalyst Bu4NHSO4 led to a mixture of nitroxide 2 and amine 7.
Unfortunately, neither a complete oxidation of 7 nor an efficient
separation of 2 from the remaining amine 7 could be performed,
leading to a mixture of non-labelled and spin-labelled products
in 76% yield after chromatography on silica gel and gel filtration
on Sephadex G-10.
O
O
H
O
O
O
N
(CH2)12 NH2
O(CH2)7CH3
HO
HO
HO
OOct
i
ii
O
O
O
OH
OH
OH
3
8
9
OH
OH
iii
To circumvent the troublesome drawback of the precedent
strategy, we then considered a more convergent synthetic pathway
involving this time a readily available pure-labelled piperidine
PhO
iv
N
N O
N
N O
O
10
11
Br
nitroxide (Scheme 2). Unprotected octyl-b-D-glucofuranosidurono-
6,3-lactone 8 was converted without protection to the glucofur-
anosiduronamide 9 by a condensation of 1,12-diaminododecane
in MeOH (70% yield).7 A nucleophilic substitution between amine
1016,17 and phenyl bromoacetate furnished the nitroxide ammo-
nium 11,18 which was enough reactive to induce, in a next step,
the acylation of the glucofuranosiduronamide 9 in DMF (50%
yield).
H
H
N
O
N
(CH2)12
OOct
N
N O
HO
O
O
Br
OH
2
OH
Scheme 2. Efficient synthesis of pure-labelled bola 2. Reagents and conditions: (i)
1-octanol, BF3ꢀEt2O, THF, reflux, 70%; (ii) 1,12-diaminododecane (1.5 equiv), MeOH,
70%; (iii) BrCH2COOPh (1.0 equiv), THF, 75%; (iv) 11 (1.5 equiv), DMF, rt, 50%.
Attempts with other activated esters, instead of the phenyl
derivative, such as more reactive 2,2,2-trifluoroethyl,19 and more
stable 3,30-dimethoxyphenyl ester did not improve the yield of
the final step. This strategy, removing protection (benzylation)
and deprotection (hydrogenolysis) steps, has afforded, in moderate
Bolaamphiphile 2 has been synthesised according to two strat-
egies which involve the introduction of a spin-active nitroxide
either in a final step (oxidation) (Scheme 1) or through a readily
available nitroxide piperidine (Scheme 2).
The first procedure involves a strategy similar to the one
-Glucofuranurono-6,3-lactone 3 was
converted into the corresponding octyl-b- -glucofuranosidurono-
6,3-lactone. Protection of the free hydroxyl groups was performed
under mild conditions by reaction of the glycoside with Ag2O and
BnBr in CH2Cl2 in order to prevent the ring lactone opening. Careful
treatment of the protected lactone 4 with 1.5 equiv of 1,12-diami-
nododecane at room temperature in MeOH provided the glucofur-
anosiduronamide 5 resulting from the monoacylation of the
diamine in 60% yield (the separable diacylated product was formed
in 18% yield). N-Acylation of amine 5 was performed with bromo-
acetyl bromide in pyridine. The protection of the 5-OH as a benzyl-
oxy group found its importance at this stage as it inhibited the
competitive O-acylation at this position, observed with the unpro-
tected counterpart. Quaternarisation of 4-dimethylamino-2,2,6,6-
tetramethyl-piperidine with the alkyl bromide derivative 6 in
DMF resulted in a clean reaction at the tertiary amine group lead-
ing to the corresponding ammonium derivative 7 after hydrogenol-
ysis of the benzyloxy groups. The conversion of the secondary
amine to the corresponding nitroxide 2 under biphasic conditions
overall yield but pure-labelled bolaamphiphile 220 from
furanurono-6,3-lactone 3 (25%, 3 steps).
D-gluco-
Preliminary ESR studies were conduced in CH2Cl2 and in water
(c = 10 mg/mL). The plain-line spectrum shows a narrow signal
from the bolaamphiphile 2 dispersed in CH2Cl2, where no aggrega-
tion occurs (Fig. 3). The triplet observed in the ESR spectrum is due
to the nitroxide signal with the characteristic constants a = 15.55 G
already-described for bola 1.7
D
D
Figure 3. ESR spectra of spin-labelled bolaamphiphile 2; in CH2Cl2 (plain line); in
H2O (dashed line). (All the experiments were performed at room temperature.)

4692
M. Berchel et al. / Tetrahedron Letters 49 (2008) 4690–4692
6. Moss, R. A.; Fujita, T. Tetrahedron Lett. 1990, 31, 7559–7562.
7. Guilbot, J.; Benvegnu, T.; Legros, N.; Plusquellec, D.; Dedieu, J. C.; Gulik, A.
Langmuir 2001, 17, 613–618.
8. Brard, M.; Richter, W.; Benvegnu, T.; Plusquellec, D. J. Am. Chem. Soc. 2004, 126,
10003–10012.
9. Smith, B. D.; Lambert, T. N. Chem. Commun. 2003, 2261–2268.
10. Moss, R. A.; Wilk, B.; Krogh-Jespersen, K.; Blair, J. T.; Westbrook, J. D. J. Am.
Chem. Soc. 1989, 111, 250–258.
11. McConnell, H. M.; Kornberg, R. D. Biochemistry 1971, 10, 1111–1120.
12. Sasaki, Y.; Shukla, R.; Smith, B. D. Org. Biomol. Chem. 2004, 2, 214–219.
13. Andrich, M. P.; Vanderkooi, J. M. Biochemistry 1976, 15, 1257–1261.
14. Vishwakarma, R. A.; Menon, A. K. Chem. Commun. 2005, 453–455.
15. Moss, R. A.; Li, J. M. J. Am. Chem. Soc. 1992, 114, 9227–9229.
16. Nakatsuji, S.; Mizumoto, M.; Ikemoto, H.; Akutsu, H.; Yamada, J.-i. Eur. J. Org.
Chem. 2002, 1912–1918.
and g = 2.00585. The dashed-line ESR spectrum is much broader
and shows the signal of bolaamphiphile 2 molecules in H2O (Fig.
3), where a self-organisation can occur. The widening of the signal
comes from the superposition of signals of nitroxide from different
environments.
In conclusion, we have synthesised a pure-labelled unsymmet-
rical bolaamphiphile bearing sugar and cationic polar heads. Note
that phenyl ester 11 would be easily used for the preparation of
other cationic-labelled molecules. The preliminary ESR measure-
ments have confirmed its potential for the characterisation of
flip–flop dynamic of such bolas in water. A full physicochemical
characterisation of the self-organised aggregates is now under
investigation and ESR kinetic studies would provide useful infor-
mation on the membrane dynamic of relevant systems.
17. Nakatsuji, S.; Takai, A.; Nishikawa, K.; Morimoto, Y.; Yasuoka, N.; Suzuki, K.;
Enoki, T.; Anzai, H. Chem. Commun. 1997, 275–276.
18. For the synthesis of 11: To a solution of 10 (439 mg, 2.20 mmol, 1.0 equiv) in
anhydrous THF (10 mL) was added phenyl bromoacetate (444 mg, 2.20 mmol,
1.0 equiv) under nitrogen atmosphere. The reaction mixture was stirred for 8 h
at rt and Et2O (20 mL) was added. The resulting suspension was filtered and
extensively washed with Et2O to afford 11 as an orange solid (675 mg,
Acknowledgements
1.63 mmol, 75%). Mp = 185 °C; IR
m
(cmꢁ1): 1770, 1377; HRMS calcd for
M.B. is grateful to the Ministère de l’Education Nationale et de la
Recherche (France) for a PhD grant. J.-Y. Thépot (Université de
Rennes, France) is acknowledged for his help in ESR measurements.
C
19H30N2O3: 334.2251; found, 334.2250.
19. Lemiègre, L.; Stevens, R. L.; Combret, J.-C.; Maddaluno, J. Org. Biomol. Chem.
2005, 3, 1308–1318.
20. For the synthesis of bolaamphiphile 2: To a solution of 9 (100 mg, 0.20 mmol,
1.0 equiv) in anhydrous DMF (1 mL) was added 11 (88 mg, 0.22 mmol,
1.1 equiv) under nitrogen atmosphere. The reaction mixture was stirred at
30 °C for 3 days and the solvent was removed under reduced pressure. The
residue was purified by flash chromatography on silica gel (EtOAc/i-PrOH/H2O/
NH4OH: 6:3:1:0.5) to afford 2 as an orange solid (83 mg, 0.10 mmol, 50%).
References and notes
1. Fuhrhop, J. H.; Wang, T. Chem. Rev. 2004, 104, 2901–2938.
2. Brard, M.; Lainé, C.; Réthoré, G.; Laurent, I.; Neveu, C.; Lemiègre, L.; Benvegnu,
T. J. Org. Chem. 2007, 72, 8267–8279.
3. Menger, F. M.; Peresypkin, A. V. J. Am. Chem. Soc. 2003, 125, 5340–5345.
4. Moss, R. A.; Fujita, T.; Okumura, Y. Langmuir 1991, 7, 2415–2418.
5. Moss, R. A.; Li, G.; Li, J.-M. J. Am. Chem. Soc. 1994, 116, 805–806.
Rf = 0.2 (EtOAc/i-PrOH/H2O/NH4OH: 6:3:1:0.5); IR
m
(cmꢁ1): 3330, 1674, 1548;
13C NMR (CD3OD, 100 MHz) d (ppm): 14.4, 26.9, 27.8–30.5, 32.7, 40.2, 40.8,
58.1, 69.3, 71.8, 77.1, 81.3, 83.7, 109.7, 163.5, 173.8; HMRS calcd for
C
39H76N4O8: 728.5663; found, 728.5661.