Regioselective synthesis of folic acid conjugates from diether-type archaeal lipid analogues

Article abstract of DOI:10.1016/j.tet.2008.12.013

A regioselective access to both α- and γ-folic acid conjugates derived from archaeal lipid analogues is described. The synthetic approach is based on conveniently protected glutamates that led first to α- and γ-glutamate derivatives. The final reconstruct

Full text of DOI:10.1016/j.tet.2008.12.013

Tetrahedron 65 (2009) 1455–1460
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Regioselective synthesis of folic acid conjugates from diether-type archaeal
lipid analogues
a
,
b
a
,
b
a
,
,b,
Celine Laine , Clemence Mocquet , Loıc Lemiegre b, Thierry Benvegnu a
¨
*
´
´
´
`
a Ecole Nationale Supe´rieure de Chimie de Rennes, CNRS, UMR 6226, Avenue du Ge´ne´ral Leclerc, CS 50837, 35708 Rennes Cedex 7, France
b Universite´ europe´enne de Bretagne, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A regioselective access to both
described. The synthetic approach is based on conveniently protected glutamates that led first to
g
a
- and
g
-folic acid conjugates derived from archaeal lipid analogues is
- and
-glutamate derivatives. The final reconstruction of the folic acid moiety was achieved through the re-
action of a protected/activated pteroate followed by a simple deprotection step. These - and -folic acid
Received 21 October 2008
Received in revised form
2 December 2008
Accepted 3 December 2008
Available online 10 December 2008
a
a
g
conjugates would permit to establish the importance of a regiocontrolled introduction of folic acid on the
folic acid/folate receptor interaction in the case of a targeted drug/gene delivery.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
that provide highly stable liposomes (archaeosomes).11–14 In fact,
these membrane lipids play a key role in the remarkable capabil-
Efficient drug/gene delivery should reach
a
series of re-
ities of archaebacteria to survive under extreme environmental
conditions (high or low temperature, high pressure, high or low
pH). In particular, diether-type lipids were found to exhibit fuso-
genic membrane properties and the presence of branching methyl
groups hinders the ordered packing of the hydrocarbon tails, which
in turn improves membrane fluidity.15 Our previous synthetic
pathway for the preparation of folate derivatives was based on the
direct coupling of folic acid with an amino group present at the
terminal end of the diether-linked PEGylated chain. Even if this
quirements including targeting capability.1–3 Thus recent develop-
ments are focused on the introduction of cell recognition ligands on
different types of vectorization systems based on nanoparticle
technologies (lipids or polymers). Due to its particular interest in
cancer therapy, folic acid is one of the most attractive ligands. In-
deed folate receptors (FR) are highly expressed on cancer cells
leading to a good differentiation from other types of cells.4–8 Folic
acid is a small molecule bearing two carboxylic acid functions
typically used for its incorporation into the vectorization system.
However, the presence of these two acid functions usually led to
strategy led to a mixture of
a- and g-folates, these novel lipids have
shown significant and ligand-mediated transfection of HeLa cells
a mixture of
Recently, we developed the synthesis of archaeal lipid analogues
bearing cell recognition ligands (sugar9 or folate10
through
a
- and
g-regioisomers (Fig. 1).
(Fig. 2).10 As
g-conjugates have already shown to be better recog-
nized by the FR on KB cells,16 it should be interesting to have the
two regioisomers of our folate conjugates at one’s disposal with the
)
a PEGylated spacer. Archaeal lipids and synthetic analogues exhibit
atypical molecular structures (diether- and tetraether-type lipids)
aim to evaluate the impact of the
the ligand/receptor affinity. Thus, we describe herein the access to
both - and -diether folate derivatives in a regioselective manner.
a- or g-folate introduction on
a
g
2. Results and discussion
O
OH
O
γ
α
Our regioselective strategy is based on a retrosynthetic cleavage
OH
O
N
N
H
at the pteroate/glutamate amide bonds leading to a
mate conjugate from which an regioselective control would be
easier (Scheme 1, only -conjugate shown). Indeed a suitable pro-
tected glutamate could ensure the isolation of well defined - or
coupling products. This retrosynthetic scheme also applies for the
-regioisomer (not shown in Scheme 1). The pteroic acid 2 is
a- or g-gluta-
N
N
O
HN
H2N
N
H
a/g
g
a
g-
Figure 1. Structure of folic acid.
a
readily accessible by enzymatic cleavage of folic acid using car-
boxypeptidase G17 and PEG570-diether derivative 4 was efficiently
prepared as described in our recent paper.10
* Corresponding author. Tel.: þ33 223 238 060; fax: þ33 223 238 046.
E-mail address: thierry.benvegnu@ensc-rennes.fr (T. Benvegnu).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.12.013

1456
C. Laine´ et al. / Tetrahedron 65 (2009) 1455–1460
α/γ-FA-PEG-tetraether : X = O, full molecule
HO
γ
O
O
α/γ-FA-PEG-diether : X = CH2, without dashed part
H
N
α
O
N
H
O
O
N
N
H
NH
10
O
O
N
N
O
NH2
OH
HN
O
O
O
O
X
Figure 2. Folate equipped archaeal lipid analogues (diether or tetraether) (only g-derivative is represented).
2.1. Synthesis of the
a
- and
g-glutamate diether conjugates
presence of Pd/C in THF/MeOH (1:1). Deprotected glutamates
a-3
and -3 were thus obtained in 67% and 59% yields, respectively
g
The reaction of readily or commercially available protected
glutamates ( -5) with the PEGylated diether 4 was per-
-518 and
(Scheme 4).
a
g
formed in the presence of TBTU/DIEA to furnish the corresponding
amide compounds in moderate to good yields (50–76%)
2.2. Reconstruction of the folate moiety
(Scheme 2). It is noteworthy that if the
a
-glutamate reacted at 40 ꢀC
Having the two regiocontrolled glutamates in hand, we envis-
aged the final reconstruction of the folate moiety by a coupling
reaction with an activated pteroate. The reaction conditions de-
scribed by Nomura et al.21 efficiently provided imidazolide 9
(Scheme 5). It is noteworthy that carbonyl diimidazole (CDI) reac-
ted in two reaction sites (carboxylic acid and guanidine sites)
leading to the protected derivative 9 after reaction with trime-
thylsilyl ethanol.
The crucial step in the preparation of the two folate
regioisomers involved the coupling reaction between 9 and gluta-
mates 3, which was carried out in the presence of tetrame-
thylguanidine in refluxing chloroform. Unprecedentedly to our
knowledge, the corresponding products were deprotected by using
(DCM reflux), the -glutamate required higher reaction tempera-
g
ture (CHCl3 reflux). This difference can be explained by the in situ
formation of the pyroglutamate 719 after the action of the TBTU/
DIEA on
In fact this side reaction (less feasible in the case of
prevent the formation of
-6 as we and others20 have shown that
g-5, which was isolated from the crude mixture.
a-5) did not
g
pyroglutamates can be opened by primary amines such as ben-
zylamine (Scheme 3) at room temperature in several days. These
results suggest that 7 could play a role as a reaction intermediate to
the formation of g-6.
The simultaneous deprotection of both the primary amine and
the carboxylic acid groups was achieved by hydrogenation in the
O
OH
γ
O
O
H
N
α
O
10
O
O
N
H
O
N
N
H
O
N
N
N
H
HN
H2N
O
γ-1
γ-FA-PEG570-diether
O
OH
γ
O
H
α
N
O
10
O
O
H2N
N
H
O
O
+
γ-3
O
OH
O
N
N
HN
H2N
H
N
N
pteroic acid 2
O
H2N
O
10
O
O
N
H
+
O
OPG
O
4
γ
α
OH
PGHN
O
PG : Protecting group
Scheme 1. Retrosynthetic scheme for the preparation of the
g
-regioisomer.

C. Laine´ et al. / Tetrahedron 65 (2009) 1455–1460
1457
O
H2N
O
10
O
O
N
H
O
4
O
OH
α-5
γ
α
OBn
TBTU, DIEA
CBzHN
CH2Cl2, Δ, 48h
O
O
OBn
γ-5
γ
α
OH
76%
CBzHN
O
O
H
N
CHCl3, Δ, 24h
O
O
10
O
O
OBn
N
H
γ
α
CBzHN
O
α-6
O
50%
O
OBn
O
H
N
γ
α
O
10
O
O
O
CBzHN
N
H
O
γ-6
Scheme 2. Regioselective synthesis of protected
a- and
g
-glutamate derivatives.
cesium fluoride. These conditions applied to
uncompleted coupling reaction and afforded after deprotection
a
-3 and
g
-3 led to an
PEG570-diether compounds were prepared and would be involved
in comparative biological assays.
(CsF) a mixture of folate derivative 1, accompanied by its corre-
sponding glutamate 3 (a-1/a-3 15:85; g-1/g-3 36:64) (Scheme 6).
4. Experimental
4.1. General
Even if this coupling reaction was not complete, a dialysis pu-
rification allowed to isolate pteroic acid free samples, which is re-
quired for their further biological evaluation. Only small amounts of
ligand are needed and recommended to exhibit effective ligand/
Commercially available chemicals were used without further
purification, and solvents were carefully dried and distilled prior to
use. Unless otherwise noted, nonaqueous reactions were carried
out under a nitrogen atmosphere. Analytical TLC was performed on
Merck 60 F254 silica gel nonactivated plates. A solution of 5% H2SO4
receptor interactions,22 the
a/g differences would therefore be
evaluated in binding tests for instance. It is noteworthy that our
experience in the synthesis of such folate derivatives has shown
that the use of a large excess of pteroate or folate derivatives, which
could increase the completion of the coupling step, would lead to
additional difficulties in its dialysis removal (loss of product during
long dialysis).10 However, the application of the methodology de-
scribed here to other lipid systems that are easily retained during
the dialysis process, would allow the use of a large excess of 9 and
in EtOH was used to develop the plates. Merck 60 H (5–40 mm) silica
gel was used for column chromatography. 1H and 13C NMR spectra
were recorded at 400 and 100 MHz, respectively, on a Bruker
Avance III spectrometer.
would furnish the corresponding
higher yields.
a- and g-folate derivatives in
4.2. Synthetic procedures
4.2.1. CBzGlu-
To mixture of the protected glutamate
0.380 mmol,1.2 equiv) and TBTU (132 mg, 0.410 mmol,1.3 equiv) in
20 mL of dry CH2Cl2 was added DIEA (68 L, 0.410 mmol, 1.3 equiv)
under nitrogen atmosphere. After stirring for 20 min at room
temperature under nitrogen atmosphere, the mixture was added to
the PEG570-diether derivative 4 (360 mg, 0.316 mmol, 1 equiv). The
resulting mixture was refluxed for 48 h under nitrogen atmo-
sphere. Aqueous HCl (1 N) was added and the organic phase was
washed with water. The combined organic layers were dried with
MgSO4 and concentrated under reduced pressure. The product was
a-PEG570-diether a-6
3. Conclusion
a
a-5 (140 mg,
We described herein the synthesis of regiocontrolled folate
equipped archaeal lipid analogues (diether) based on a retro-
synthetic cleavage of the folate group into pteroate and glutamate
derivatives. The functionalization of appropriately protected glu-
tamates permitted to achieve this goal. The reconstruction of the
folate moiety involved an imidazolide pteroate derivative and a fi-
m
nal deprotection step. Therefore regioncontrolled
a
- and g-FA-
purified by flash chromatography on silica gel (CH2Cl2/MeOH 96:4).
20
O
N
OBn
Yield: 76%; [
a
]
ꢁ3.3 (c 1, CH2Cl2); Rf (CH2Cl2/MeOH 96:4) 0.17. IR
D
O
OBn
O
NH2
CH2Cl2, 4d
(cmꢁ1): 3424, 3322, 3033, 2925–2855, 1731, 1673, 1529, 1456, 1377,
O
H
N
1348, 1250, 1110, 951, 737–667. 1H NMR (CDCl3, 400 MHz)
d
(ppm):
BnO
BnO
N
H
0.83–0.88 (m,18H),1.00–1.54 (m, 52H),1.92–1.99 (m, 1H), 2.12–2.21
(m, 1H), 2.39–2.54 (m, 2H), 3.42–3.47 (m, 6H), 3.52–3.55 (m, 6H),
3.57–3.63 (m, 40H), 3.74–3.75 (m,1H), 3.77–3.78 (m, 1H), 3.88–3.90
O
O
7
8
Scheme 3. Ring opening of the pyroglutamate by benzylamine.

1458
C. Laine´ et al. / Tetrahedron 65 (2009) 1455–1460
O
H
N
O
O
10
O
O
OBn
N
H
γ
α
CBzHN
O
α-6
O
H2, Pd/C
THF/MeOH
67%
O
H
N
O
O
10
O
O
OH
N
H
γ
α
H2N
O
α-3
O
O
OBn
γ
O
H
α
N
O
10
O
O
CBzHN
N
H
O
O
γ-6
H2, Pd/C
THF/MeOH
59%
O
OH
γ
O
H
N
α
O
10
O
O
H2N
N
H
O
O
γ-3
Scheme 4. Deprotection of
a- and
g
-glutamate derivatives.
(dd, J¼2.5, 5.9 Hz, 1H), 4.22–4.28 (m, 1H), 5.08 (s, 2H), 5.09 (s, 2H),
3582, 3419, 2924–2854, 1674, 1526, 1462, 1377, 1349, 1258, 1114,
922, 804–666; 1H NMR (CDCl3, 400 MHz)
(ppm): 0.83–0.89 (m,
5.73–5.75 (m, 1H), 6.89 (m, 1H), 7.03–7.04 (m, 1H), 7.29–7.35 (m,
d
10H); 13C NMR (CDCl3, 100 MHz)
d
(ppm): 14.11, 19.58, 19.66, 19.73,
18H), 1.00–1.54 (m, 52H), 1.79–1.83 (m, 1H), 1.93–2.02 (m, 1H),
2.32–2.43 (m, 2H), 3.40–3.47 (m, 6H), 3.50–3.56 (m, 6H), 3.60–3.64
(m, 40H), 3.74–3.75 (m, 1H), 3.77–3.78 (m, 1H), 3.88–3.90 (dd,
J¼2.5, 5.9 Hz, 1H), 4.27–4.32 (m, 1H), 7.02–7.07 (m, 2H); 13C NMR
22.61, 22.65, 22.67, 22.71, 24.36, 24.46, 24.78, 25.52, 26.03, 27.95,
28.41, 29.34, 29.46, 29.52, 29.63, 29.68, 29.85, 30.25, 32.77, 37.26,
37.36, 37.39, 37.44, 38.57, 38.68, 39.35, 54.04, 66.45, 66.88, 69.52,
69.73, 69.83, 70.45–70.56, 70.22, 70.29, 71.50, 71.69, 80.49, 128.01,
128.11, 128.20, 128.24, 128.48, 128.53, 135.71, 136.22, 156.07, 170.57,
170.98, 172.93. ESI-MS (m/z) calcd for C83H147O19N3 [MþNa]þ:
1513.0527, found: 1513.0505; [MþK]þ: 1529.0266, found:
1529.0268. Anal. Calcd for C83H147O19N3$2H2O: C, 65.28; H, 9.97; N,
2.75. Found: C, 65.11; H, 9.80; N, 2.80.
(CDCl3, 100 MHz)
d (ppm): 14.11, 19.58, 19.68, 19.74, 22.63, 22.69,
22.73, 24.38, 24.49, 24.79, 25.28, 26.05, 27.96, 29.35, 29.54, 29.59,
29.65, 29.70, 29.86, 31.91, 32.79, 37.28, 37.38, 37.41, 37.47, 37.53,
38.57, 38.70, 39.34, 39.35, 49.21, 69.74, 69.86–70.46, 71.53, 71.71,
80.48, 170.58, 170.65, 179.80. ESI-MS (m/z) calcd for C68H135O17N3
[MþNa]þ: 1288.9689, found: 1288.9698.
4.2.2. Glu-
a
-PEG570-diether
a
-3
4.2.3. CBzGlu-
To mixture of the protected glutamate
0.026 mmol, 1.2 equiv) and TBTU (9.1 mg, 0.028 mmol, 1.3 equiv) in
1 mL of dry CHCl3 was added DIEA (4.7 L, 0.028 mmol, 1.3 equiv)
under nitrogen atmosphere. After stirring for 20 min at room
temperature under nitrogen atmosphere, the mixture was added to
the PEG570-diether derivative 4 (24.7 mg, 0.022 mmol, 1 equiv). The
resulting mixture was refluxed for 24 h under nitrogen
g-PEG570-diether g-6
To a solution of
a
-6 (90 mg, 0.060 mmol,1 equiv) in 4 mL of THF/
a
g-5 (9.7 mg,
CH3OH (1:1) was added palladium on activated carbon (15 mg,
17%). After stirring for 5 h 30 min under hydrogen atmosphere, the
suspension was filtered over CeliteÒ. The solvent was evaporated
under reduced pressure and the product was purified by flash
m
chromatography on silica gel (CH2Cl2/MeOH/Et3N 9:1:0.1). Yield:
20
67%; [
a
]
þ4.6 (c 1, CH2Cl2); Rf (CH2Cl2/MeOH 9:1) 0.14. IR (cmꢁ1):
D
O
O
O
N
N
N
O
N
OH
N
N
1) CDI, 40 °C, 5h
2) r.t., 5h
O
HN
N
H
N
N
HN
H2N
N
H
TMS
O
N
9
H
OH
TMS
2
Scheme 5. Preparation of the pteroate imidazolide.

C. Laine´ et al. / Tetrahedron 65 (2009) 1455–1460
1459
O
H
N
O
O
10
O
O
OH
N
H
γ
α
H2N
O
α-3
O
1) 9, TMG 2) CsF,
Dialysis purification
(MWCO 1000)
DMF
Δ, 9h
100%
CHCl3
Δ, 24h
67%
O
H
N
O
O
10
O
O
OH
O
N
H
γ
α
N
H
O
N
O
O
N
N
α-1/α-3 : 15:85
N
H
HN
H2N
O
OH
O
H
N
γ
α
O
10
O
O
H2N
N
H
O
O
γ-3
1) 9, TMG 2) CsF,
Dialysis purification
(MWCO 1000)
DMF
Δ, 9h
100%
CHCl3
Δ, 24h
93%
O
OH
O
O
H
γ
α
N
O
N
O
O
N
H
O
N
10
H
O
N
N
N
H
HN
H2N
O
γ-1/γ-3 : 36:64
Scheme 6. Preparation of a-1 and g-1.
atmosphere. Aqueous HCl (1 N) was added and the organic phase
was washed with water. The combined organic layers were dried
with MgSO4 and concentrated under reduced pressure. The product
suspension was filtered over CeliteÒ. The solvent was evaporated
under reduced pressure and the product was purified by flash
chromatography on silica gel (CH2Cl2/MeOH/Et3N 9:1:0.1). Yield:
20
was purified by flash chromatography on silica gel (CH2Cl2/MeOH
59%; [
a
]
D
þ10.1 (c 1, CH2Cl2); Rf (CH2Cl2/MeOH 9:1) 0.3. IR (cmꢁ1):
20
96:4). Yield: 50%; [
a
]
D
ꢁ4.1 (c 1, CH2Cl2); Rf (CH2Cl2/MeOH 96:4)
3582, 3424, 2924–2854,1668,1527,1463, 1377,1349,1251,1111, 951,
0.2. IR (cmꢁ1): 3414, 3335, 2924–2854, 1722, 1667, 1531, 1463, 1377,
754–666. 1H NMR (CDCl3, 400 MHz)
d (ppm): 0.82–0.88 (m, 18H),
1357, 1257, 1112, 700–666. 1H NMR (CDCl3, 400 MHz)
d
(ppm):
1.03–1.70 (m, 52H), 1.84–1.99 (m, 2H), 2.27–2.55 (m, 2H), 2.93–2.98
(q, J¼7.2 Hz, 2H), 3.39–3.49 (m, 6H), 3.51–3.56 (m, 4H), 3.57–3.63
(m, 40H), 3.77–3.74 (m, 1H), 3.76–3.77 (m, 1H), 3.87–3.89 (dd,
J¼2.5, 5.9 Hz, 1H), 4.01–4.08 (m, 1H), 7.02–7.05 (m, 2H); 13C NMR
0.83–0.88 (m, 18H), 1.00–1.54 (m, 52H), 1.98–2.06 (m, 1H), 2.16–
2.27 (m, 3H), 3.37–3.47 (m, 6H), 3.49–3.56 (m, 6H), 3.57–3.68 (m,
40H), 3.74–3.75 (m, 1H), 3.77–3.78 (m, 1H), 3.88–3.90 (dd, J¼2.5,
5.9 Hz, 1H), 4.35–4.40 (m, 1H), 5.09 (s, 2H), 5.16 (s, 2H), 5.93–5.95
(m, 1H), 6.46–6.47 (m, 1H), 7.03–7.04 (m, 1H), 7.30–7.38 (m, 10H);
(CDCl3, 100 MHz)
d (ppm): 14.10, 19.56, 19.65, 19.72, 22.60, 22.65,
22.66, 22.70, 24.34, 24.45, 24.76, 25.42, 26.02, 27.93, 29.45, 29.51,
29.61, 29.66, 29.78, 29.83, 31.88, 32.75, 37.24, 37.34, 37.37, 37.42,
37.48, 38.67, 39.32, 44.87, 55.77, 69.70, 69.81, 69.95–70.53, 71.49,
71.67, 80.45, 170.59, 170.60, 177.43.
13C NMR (CDCl3, 100 MHz)
d (ppm): 14.12, 19.58, 19.66, 19.73, 22.61,
22.65, 22.71, 22.67, 24.37, 24.47, 24.79, 25.61, 26.03, 27.95, 29.35,
29.47, 29.52, 29.64, 29.69, 29.85, 31.90, 32.78, 37.26, 37.37, 37.40,
37.46, 37.51, 38.57, 38.68, 39.34, 53.79, 66.90, 67.16, 69.51, 69.68,
69.74, 69.83, 70.14, 70.23, 70.29, 70.50–70.57, 71.49, 71.70, 80.48,
128.07, 128.10, 128.27, 128.41, 128.46, 128.58, 135.26, 136.20, 170.59,
170.61, 171.87, 171.91. ESI-MS (m/z) calcd for C83H147O19N3
[MþNa]þ: 1513.0527, found: 1513.0505; [MþK]þ: 1529.0266,
found: 1529.0268. Anal. Calcd for C83H147O19N3$H2O: C, 66.06; H,
9.95; N, 2.78. Found: C, 65.92; H, 10.15; N, 2.76.
4.2.5.
a
-Benzyl-
mixture of the pyroglutamate
L, 0.040 mmol, 1.3 equiv) in 250 m
L
-glutamate-
g
-amidobenzyl 8
(10.4 mg, 0.029 mmol,
L of
A
7
1 equiv) and benzylamine (4
m
dry CH2Cl2 was stirred for 4 days at room temperature under ni-
trogen atmosphere. The solvent was evaporated under reduced
pressure to afford crude 8. 1H NMR (CDCl3, 400 MHz)
d (ppm): 1.17–
1.24 (m, 1H), 1.90–1.97 (m, 1H), 2.12–2.18 (m, 2H), 4.38–4.40 (d,
J¼5.5 Hz, 2H), 5.08 (s, 2H), 5.14–5.17 (m, 1H), 5.30 (s, 2H), 5.72–5.74
(d, J¼7.5 Hz, 1H), 6.05–6.07 (m, 1H), 7.23–7.37 (m, 15H); 13C NMR
4.2.4. Glu-
g
-PEG570-diether
g-3
To a solution of
g-6 (53 mg, 0.036 mmol,1 equiv) in 4 mL of THF/
CH3OH (1:1) was added palladium on activated carbon (20 mg,
37%). After stirring for 18 h under hydrogen atmosphere, the
(CDCl3, 100 MHz)
d (ppm): 28.7, 32.4, 43.8, 53.6, 67.2, 67.4, 127.6–
138.2, 156.4, 171.7, 171.8.

1460
C. Laine´ et al. / Tetrahedron 65 (2009) 1455–1460
4.2.6. (6-([4-(Imidazole-1-carbonyl)-phenylamino]-methyl)-4-oxo-
3,4-dihydro-pteridin-2-trimethylsilylethyl)-carbamic acid ethyl
ester 921
400 MHz) d (ppm): 0.06–0.07 (m, 9H), 0.83–0.89 (m, 18H), 1.03–
1.71 (m, 54H), 2.05–2.21 (m, 2H), 2.26–2.39 (m, 2H), 3.38–3.49 (m,
6H), 3.52–3.56 (m, 6H), 3.58–3.66 (m, 40H), 3.74–3.75 (m, 1H),
3.77–3.78 (m, 1H), 3.88–3.90 (dd, J¼2.4, 6.0 Hz, 1H), 4.02–4.19 (m,
1H), 4.37–4.41 (m, 2H), 4.70–4.72 (d, J¼6.4 Hz, 2H), 6.65–6.67 (d,
J¼8.8 Hz, 2H), 7.06 (m, 2H), 7.33–7.34 (m, 1H), 7.87–7.89 (d,
A mixture of pteroic acid (26 mg, 0.088 mmol, 1 equiv), CDI
(54 mg, 0.333 mmol, 4 equiv), and dry triethylamine (75
0.333 mmol, 4 equiv) in 430 L of dry DMSO was stirred for 5 h at
40 ꢀC under nitrogen atmosphere. At room temperature, trime-
thylsilyl ethanol (95 L, 0.666 mmol, 8 equiv) was added under
mL,
m
J¼8.8 Hz, 2H), 8.86 (s, 1H); 13C NMR (CDCl3, 100 MHz)
d (ppm):
m
14.11, 19.60, 19.67, 19.74, 22.62, 22.66, 22.68, 22.72, 24.38, 24.49,
24.79, 26.05, 27.97, 29.35, 29.49, 29.55, 29.65, 29.70, 29.84, 29.88,
32.79, 37.29, 37.39, 37.42, 37.46, 37.48, 37.50, 38.72, 39.25, 39.36,
69.75, 69.85, 70.23–70.54, 71.52, 71.72, 80.52, 170.66, 170.72.
nitrogen atmosphere. After stirring for 5 h at room temperature
under nitrogen atmosphere, the mixture was precipitated in 3 mL
of AcOH/Et2O/H2O (1:20:35). The precipitate was filtered and pu-
rified by flash chromatography on silica gel (CHCl3/MeOH 9:1).
Yield: 55%; 1H NMR (CDCl3, 400 MHz)
d
(ppm): 0.05–0.06 (m, 9H),
4.2.8.2. FA-g-PEG570-diether g-1. A mixture of previous TeocFA-g-
1.02–1.06 (m, 2H), 4.27–4.31 (m, 2H), 4.65–4.67 (m, 2H), 6.77–6.80
(m, 2H), 7.09 (m, 1H), 7.61–7.63 (m, 3H), 8.15 (s, 1H), 8.88 (s, 1H).
ESI-MS (m/z) calcd for C23H26O4N8Si [MþNa]þ: 529.1744, found:
529.1747; [MþK]þ: 545.1483, found: 545.1493.
PEG570-diether (10 mg, 0.003 mmol, 1 equiv) and cesium fluoride
(2 mg, 0.013 mmol, 5 equiv) in 1 mL of dry DMF was refluxed for 9 h
under nitrogen atmosphere. After evaporation under reduced
pressure, the solid was purified by dialysis (MWCO 1000) against
DMSO and lyophilized to afford compounds -1 and
g
g-3 (36:64).
4.2.7. FA-a-PEG570-diether
a-1
Yield: 100 mol %; 1H NMR (CDCl3, 400 MHz)
d (ppm): 0.80–0.82 (m,
18H), 1.04–1.54 (m, 52H), 1.65–1.80 (m, 2H), 2.00–2.02 (m, 2H),
3.16–3.25 (m, 6H), 3.29–3.44 (m, 6H), 3.45–3.56 (m, 41H), 3.65–3.67
(m, 1H), 3.79–3.80 (m, 1H), 4.12–4.17 (m, 1H), 4.52–4.54 (m, 2H),
6.66–6.72 (m, 2H), 7.33–7.34 (m, 2H), 7.56–7.57 (m, 2H), 7.66–7.70
(m, 2H), 8.05–8.11 (m, 2H), 8.31 (s, 1H); 13C NMR (CDCl3, 100 MHz)
4.2.7.1. TeocFA-
0.055 mmol, 1.4 equiv),
TMG (15 L, 0.012 mmol, 3 equiv) in 400
a
-PEG570-diether. A
-3 (50 mg, 0.040 mmol, 1 equiv), and dry
L of dry CHCl3 was
mixture
of
9
(28 mg,
a
m
m
refluxed for 24 h under nitrogen atmosphere. After evaporation
under reduced pressure, the solid was purified by dialysis (MWCO
1000) against DMSO and lyophilized to afford compounds TeocFA-
d
(ppm): 14.17, 19.67, 22.50, 22.67, 24.33, 26.17, 27.83, 28.50, 29.50,
29.67, 31.67, 32.50, 37.17, 38.33, 39.17, 40.17, 69.32, 70.11, 70.80,
71.82, 72.39, 80.45.
a
-PEG570-diether and
a
-3 (15:85). Yield: 67 mol %; 1H NMR (CDCl3,
400 MHz) (ppm): 0.06–0.07 (m, 9H), 0.83–0.88 (m, 18H), 0.99–
d
1.73 (m, 54H), 1.92–2.09 (m, 2H), 2.24–2.43 (m, 2H), 3.38–3.50 (m,
6H), 3.52–3.57 (m, 6H), 3.60–3.69 (m, 40H), 3.74–3.75 (m, 1H),
3.77–3.78 (m, 1H), 3.88–3.90 (dd, J¼2.5, 5.9 Hz, 1H), 4.03–4.14 (m,
1H), 4.32–4.37 (m, 2H), 4.69–4.70 (d, J¼6.4 Hz, 2H), 6.66–6.68 (d,
J¼8.8 Hz, 2H), 7.05 (m, 2H), 7.34–7.35 (t, J¼4.4 Hz, 1H), 7.85–7.87 (d,
Acknowledgements
This work was supported by the Association ‘Vaincre La Muco-
viscidose’ (Paris, France) (grant to C.L.), the ‘Region Bretagne’, and
‘Ouest-Genopole’. We are thankful to S. Pilard (Universite de Pic-
ardie Jules Verne) for recording mass spectra.
´
´
J¼8.4 Hz, 2H), 8.82 (s, 1H); 13C NMR (CDCl3, 100 MHz)
d (ppm): 1.00,
14.11, 16.79, 19.58, 19.67, 19.74, 22.62, 22.68, 22.66, 22.71, 24.34,
24.38, 24.48, 24.79, 26.05, 27.96, 29.18, 29.35, 29.48, 29.51, 29.54,
29.60, 29.64, 29.69, 29.84, 29.88, 31.91, 32.79, 37.28, 37.38, 37.42,
37.46, 37.48, 38.72, 39.25, 39.36, 63.45, 69.67, 69.75, 69.85–70.58,
71.52, 71.72, 80.52.
References and notes
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2. Wasungu, L.; Hoekstra, D. J. Controlled Release 2006, 116, 255–264.
3. Montier, T.; Benvegnu, T.; Jaffre`s, P.-A.; Yaouanc, J.-J.; Lehn, P. Curr. Gene Ther.
2008, 8, 296–312.
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5. Wang, S.; Low, P. S. J. Controlled Release 1998, 53, 39–48.
6. Low, P. S.; Henne, W. A.; Doorneweerd, D. D. Acc. Chem. Res. 2008, 41, 120–129.
7. Reddy, J. A.; Low, P. S. J. Controlled Release 2000, 64, 27–37.
8. Reddy, J. A.; Dean, D.; Kennedy, M. D.; Low, P. S. J. Pharm. Sci. 1999, 88, 1112–
1118.
4.2.7.2. FA-a-PEG570-diether a-1. A mixture of TeocFA-a-PEG570-
diether (30 mg, 0.018 mmol, 1 equiv) and cesium fluoride (13.5 mg,
0.088 mmol, 5 equiv) in 1 mL of dry DMF was refluxed for 9 h under
nitrogen atmosphere. After evaporation under reduced pressure,
the solid was purified by dialysis (MWCO 1000) against DMSO and
9. Brard, M.; Laine´, C.; Re´thore´, G.; Laurent, I.; Neveu, C.; Lemie`gre, L.; Benvegnu, T.
J. Org. Chem. 2007, 72, 8267–8279.
lyophilized to afford compounds
a
-1 and
a-3 (15:85). Yield:
100 mol %; 1H NMR (CDCl3, 400 MHz)
d
(ppm): 0.83–0.84 (m, 18H),
10. Laine´, C.; Mornet, E.; Lemie`gre, L.; Montier, T.; Cammas-Marion, S.; Neveu, C.;
Carmoy, N.; Lehn, P.; Benvegnu, T. Chem.dEur. J. 2008, 14, 8330–8340.
11. Benvegnu, T.; Lemie`gre, L.; Cammas-Marion, S. Eur. J. Org. Chem. 2008, 4725–
4744.
12. De Rosa, M.; Gambacorta, A.; Gliozzi, A. Microbiol. Rev. 1986, 70–80.
13. Sprott, G. D. J. Bioenerg. Biomembr. 1992, 24, 555–566.
14. Benvegnu, T.; Brard, M.; Plusquellec, D. Curr. Opin. Colloid Interface Sci. 2004, 8,
469–479.
1.06–1.54 (m, 52H), 2.14–2.17 (m, 2H), 2.41–2.46 (m, 2H), 3.20–3.26
(m, 6H), 3.30–3.45 (m, 6H), 3.49–3.53 (m, 40H), 3.59–3.61 (m, 1H),
3.66–3.67 (m, 1H), 3.80–3.82 (m, 1H), 3.97–4.01 (m, 1H), 4.08–4.12
(m, 2H), 6.44–6.46 (m, 2H), 7.54–7.55 (m, 2H), 7.68–7.79 (m, 2H),
8.03–8.04 (m, 1H), 8.26–8.31 (m, 1H), 8.53 (s, 1H); 13C NMR (CDCl3,
100 MHz)
d (ppm): 14.35, 15.08, 22.58, 22.90, 24.19, 25.97, 29.52,
15. Auze´ly-Velty, R.; Benvegnu, T.; Mackenzie, G.; Haley, J. A.; Goodby, J. W.;
Plusquellec, D. Carbohydr. Res. 1998, 314, 65–77.
31.61, 32.42, 37.10, 37.42, 38.55, 39.03, 69.39, 70.00, 70.37, 80.24.
16. Wang, S.; Lee, R. J.; Mathias, C. J.; Green, M. A.; Low, P. S. Bioconjugate Chem.
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17. Luo, J.; Smith, M. D.; Lantrip, D. A.; Wang, S.; Fuchs, P. L. J. Am. Chem. Soc. 1997,
119, 10004–10013.
4.2.8. FA-g-PEG570-diether g-1
18. Desjardins, M.; Garneau, S.; Desgagne´s, J.; Lacoste, L.; Yang, F.; Lapointe, J.;
Cheˆnevert, R. Bioorg. Chem. 1998, 26, 1–13.
19. Baldwin, J. E.; Miranda, T.; Moloney, M.; Hokelek, T. Tetrahedron 1989, 45, 7459–
7468.
4.2.8.1. TeocFA-
0.055 mmol, 1.7 equiv),
TMG (15 L, 0.012 mmol, 3 equiv) in 400
g
-PEG570-diether. A
-3 (42 mg, 0.033 mmol, 1 equiv), and dry
L of dry CHCl3 was
mixture
of
9
(28 mg,
g
m
m
20. Molina, M. T.; del Valle, C.; Escribano, A. M.; Ezquerra, J.; Pedregal, C. Tetrahedron
1993, 49, 3801–3808.
21. Nomura, M.; Shuto, S.; Matsuda, A. J. Org. Chem. 2000, 65, 5016–5021.
22. Ghaghada, K. B.; Saul, J.; Natarajan, J. V.; Bellamkonda, R. V.; Annapragada, A. V.
J. Controlled Release 2005, 104, 113–128.
refluxed for 24 h under nitrogen atmosphere. After evaporation
under reduced pressure, the solid was purified by dialysis (MWCO
1000) against DMSO and lyophilized to afford compounds TeocFA-
g
-PEG570-diether and g
-3 (36:64). Yield: 93 mol %; 1H NMR (CDCl3,