Synthesis of novel amphiphilic conjugates with a biological recognition function for developing targeted triggered liposomal delivery systems

Article abstract of DOI:10.1016/j.tet.2011.07.089

Several novel amphiphilic lipid derivatives were synthesized consisting of a lipid anchor connected to the hydrophilic moiety via a disulfide or glycoside bond and biotin linked to the hydrophilic part. Disulfide bonds were established by the help of 4-ph

Full text of DOI:10.1016/j.tet.2011.07.089

Tetrahedron 67 (2011) 7763e7774
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of novel amphiphilic conjugates with a biological recognition function
for developing targeted triggered liposomal delivery systems
,
y
a,
Nicolai Brodersen a, Anna Arbuzova b, Andreas Herrmann b, Holger Egger c , Jurgen Liebscher
*
€
a Institute of Chemistry, Humboldt-University Berlin, Brook-Taylor-Str. 2, 12489 Berlin, Germany
b Institute of Biology/Biophysics, Humboldt-University Berlin, Invalidenstr. 42, 10115 Berlin, Germany
c Bayer Technology Services GmbH, Product Design and Nanotechnology, Building E41, D-51368 Leverkusen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 April 2011
Received in revised form 27 July 2011
Accepted 28 July 2011
Available online 3 August 2011
Several novel amphiphilic lipid derivatives were synthesized consisting of a lipid anchor connected to the
hydrophilic moiety via a disulfide or glycoside bond and biotin linked to the hydrophilic part. Disulfide
bonds were established by the help of 4-phenyltriazol-3,5-dione. Dansyl or fluorescein was covalently
linked as fluorescent marker to some of the conjugates, allowing spectroscopic and microscopic de-
tection. The conjugates represent first amphiphilic lipids carrying all four functions, i.e., lipophilic, hy-
drophilic, recognition, and disulfide cleavage group in one molecule, which are necessary for targeted,
triggered drug delivery from phospholipid liposomes on demand.
Dedicated to Prof. Dr. Barry M. Trost on the
occasion of his 70th birthday
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Amphiphile
Lipid
Disulfide
Biological recognition
Drug delivery
Glucoside
Lipids
Membranes
1. Introduction
internal, i.e. (intra)cellular factors, e.g., by disassembly of the su-
pramolecular carriers due to enzymatic cleavage of conjugates and
Targeted and triggered delivery of drugs in nano- and micro-
containers is in the focus of contemporary medical and biomedical
research.1,2 It allows increasing stability of drugs in vivo by en-
capsulation in the carrier thus reducing systemic and local adverse
drugs reactions. Furthermore, multiple drugs can be delivered in
one carrier, the solubility in body fluids can be improved, and drugs
can be efficiently shielded from multidrug resistance transporters
in cancer cells. Amongst the various nano- and micro-containers
applied, liposomes have attracted great interest because they are
formed from biocompatible materials, namely essentially from
phospho-, glycolipids, and sterols.3,4 Also modified lipids were used
for this purpose.5e8
pH changes.5,8,9 These factors can cause instabilities or structural
changes, for example, phospholipases are hydrolyzing phospho-
lipids of liposomes to lipophilic chains and hydrophilic groups.
While the latter redistributes into the aqueous environment, lipo-
philic chains remain in the membrane disturbing the supramolec-
ular arrangement, e.g., by inducing hexagonal phase formation.4,10
This rearrangement leads to cargo release, e.g., drug delivery,
from the liposomes.
In continuation of our research activities in anchoring of
nucleolipids in biocompatible phospholipid membranes,11e16 we
have approached amphiphilic lipids with the potential of triggering
drug release from liposomes by reductive disulfide and enzymatic
glucose cleavage.17 Unlike previously known redox-cleavable
disulfide systems,9 these conjugates bear an additional biological
targeting function linked to the hydrophilic moiety. The latter is
connected to the lipophilic entity by a disulfide moiety and, hence,
can be disconnected on demand by chemical means (Fig. 1). For
fluorescence spectroscopy and microscopy imaging, the hydro-
philic moieties can additionally be labeled with a fluorescence dye.
Release of cargo from liposomes can be spontaneous or can be
triggered by external factors, e.g., hyperthermia, irradiation, or by
* Corresponding author. Fax: þ49 30 2093 7552; e-mail addresses: holger.egger@
bayer.com (H. Egger), liebscher@chemie.hu-berlin.de (J. Liebscher).
y
Fax: þ49 214 30 50262.
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.07.089

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N. Brodersen et al. / Tetrahedron 67 (2011) 7763e7774
triethylenglycol tosylate 2 leading directly to the conjugate 5 in 39%
yield in the final step.
A different strategy was used for the ammonium conjugate 9
(Scheme 2). Here, a quaternary ammonium unit was built up by al-
kylation of N0-biotinylated N,N-dimethyl-1,2-diaminoethane 6 with
bromoacetic acid in an earlier step. The resulting glycine derivative 7
was connected with the disulfide 8 by final amide formation to give
pure conjugate 9 in 22% yield after column chromatography.
Linkage of a quaternary ammonium unit to the disulfide by
amide formation was also used to synthesize conjugate 14 (Scheme
3). In this product the lipophilic part, quaternary ammonium group
and the biotin as recognition function were not arranged in linear,
but in a branched structure. The disulfide 11 derived from 2-amino-
3-mercaptopropan-1-ol 10 was acylated by the known succinate
12. Esterification of the untouched hydroxy group of the resulting
amide 13 by biotin gave the conjugate 14, which was isolated in 35%
yield using column chromatography.
Approaching the synthesis of amphiphilic biotin conjugates
containing an additional fluorescent label, the serine derivative 16
was biotinylated at the hydroxy group affording 17. Selective
cleavage of the methyl ester without touching the biotin ester was
possible in good yields by trimethylstannanol. The resulting acid 18
was esterified with triethylene glycol monotosylate 1 in the pres-
ence of N,N-dicyclohexylcarbodiimide leading to the tosylate 19 in
33% yield. The latter was used as an alkylating reagent for the
thiocholine-derived disulfide 4 to provide the dansyl-labeled am-
phiphilic biotin conjugate 20 in 50% yield after chromatography.
Further we sought to synthesize a dansyl-labeled conjugate,
wherein biotin is situated not at the end of the linker but more in
the middle. Here we started with S,O-diprotected 2-amino-3-
mercaptopropan-1-ol derivative 23, which was obtained from the
corresponding hydroxy compound 21 by Mitsunobu reaction in
two steps. Biotinylation gave quantitative yields of the amide 24,
which was then S-deprotected by treatment with sodium in liquid
ammonia to give the thiol 25 in 55% yield. The mixed disulfide 26
with 2,3-dioctadecyloxypropan-1-thiol 3 was again obtained with
PTAD in 38% yield. After deprotection the resulting alcohol 27 was
esterified with the succinate 29 containing the hydrophilic choline
unit and the dansyl group to give the final product 30 in compar-
atively high yield (55%).
Lipid Anchor
Recognition
Function
Cleavable Group
Hydrophylic
Moiety
Fluorescence
Label (optional)
Fig. 1. Amphiphilic lipids with biological recognition function, a cleavable linker group
and an optional fluorescence label useful for incorporation into liposomes.
2. Results and discussion
There is a variety of components, which can be used to establish
amphiphilic lipid conjugates. We report here on the synthesis of
conjugates wherein a disulfide or glucose moiety serves as a cleav-
able group and biotin as the recognition function. As a hydrophilic
moiety weused eitheran oligoethylenglycol-thiocholine skeleton or
a glucose unit, the latter acting as the cleavable group at the same
time. Since two lipid chains are optimal for stable anchoring in
phospholipid membranes, we chose a glycerol or thioglycerol unit
with two octadecyl ether groups as lipid anchors. Although several
compounds with disulfide or glucose moieties as cleavable groups
have been already studied for drug delivery from liposomes, nev-
ertheless, a targeting recognition function was missing in those
cases.18 For this purpose, we have introduced a biotin moiety. This
allows a flexible attachmentof receptors/ligands, e.g., antibodies, via
streptavidin, and, hence, adaptation to the specific target cell.
It was our strategy to establish the disulfide unit early in the
synthetic scheme because this reaction often gives low yields. Nu-
merous methods for disulfide formation from two thiols are
known.19 Many of them, however, are not suitable for establishing
unsymmetrical disulfides at all or are leading to the formation of
symmetric and unsymmetrical disulfides mixtures. After checking
several methods, we decided to apply phenyltriazolin-3,5-dione
(PTAD) for establishing the disulfide moiety. Using this reagent it
was possible to synthesize the unsymmetrical disulfides 4, 8, 11, and
32 in yields higher than 60%. Although symmetric disulfides were
also formed, they could be completely removed by column
chromatography.
The conjugate 5 with a diethylenglycol-thiocholine moiety as
hydrophilic group was obtained in only three steps starting from
the known triethylenglycol tosylate 1 (Scheme 1). The thiocholine-
In a similar strategy, the synthesis of the fluorescein-labeled
amphiphilic biotin conjugate 35 was followed up. As compared
with the previous dansyl derivative 30 the hydrophilic character
derived disulfide
4
was quaternized by the biotinylated
Ts
O
H
Ts
O
S
(+)-Biotin
43%
O
OH
1.
O
O
NH
O
O
O
O
N
H
HN
O
O
1
2
O
N
N
N
PTAD
CHCl3 , 70 °C,
2 bar, 6 d
O
8
O
S
O
SH
MeCN, DCM, 60 °C-RT
O
S
8
8
O
68%
2.
3
8
N
4
HS
39%
O
8
O
S
H
S
H
S
8
O
O
N
NH
O
O
HN
O
TsO
O
5
Scheme 1. Synthesis of oligoethylene glycol conjugate 5.

N. Brodersen et al. / Tetrahedron 67 (2011) 7763e7774
7765
O
O
O
O
bromoacetic
acid
O
HN
H
S
HN
H
Me2N
N
NH
N
H
NH
HO
N
H
(+)-Biotin
H
S
Br
H
EDC, HOBt, DMF, DIPEA
MeCN, 100 °C
97%
quantitative yield
6
7
O
N
N
N
PTAD
1.
NH2
O
8
O
EDC, HOBt, DIPEA,
S
O
SH
MeCN, DCM, 60 °C-RT
O
8
S
DCM, DMF, 30 °C, 1 d
8
O
70%
2.
3
8
NH2
HS
8
22%
O
NH
O
O
HN
H
N
S
S
O
8
N
H
N
H
S
O
8
Br
H
9
Scheme 2. Synthesis of glycine amide conjugate 9.
OH
O
O
N+
Cl
8
O
O
S
S
OH
O
O
8
HN
O
EDC, HOBt, DIPEA
DCM. DMF, 30 °C
12
+
13
54%
O
O
8
O
S
N+ Cl
S
OH
8
11
NH2
+ 3, PTAD
70%
(+)-Biotin, DCC, DMAP,
DCM, 30 °C, 4 d
35%
HS
OH
NH2
10
O
HN
O
O
H
S
8
NH
O
S
S
O
O
8
H
HN
O
14
O
N+ Cl
Scheme 3. Synthesis of conjugate 14 (hydrophilic group in the side chain).
was reduced in this target molecule because of the missing am-
monium moiety. The thioglycerol-derived disulfide 31 was selec-
tively biotinylated at the terminal hydroxy group in the presence of
DCC providing 43% yield of the pure product 32 after 3 days re-
action time. Esterification of the remaining hydroxy group with the
succinated fluorescein 34 provided 52% of the final product 35.
Finally, we designed a biotin conjugate 42 with a carbohydrate
serving as the hydrophilic part and the cleavable group again by using
the dioctadecylglyceryl unit as a lipid anchor. This synthesis (Scheme
7) started with peracetylated 2-bromoglucose, which was trans-
€
formed into the amphiphilic glucoside 38 by KonigseKnorr method.
After deprotectionwith sodium methoxide in methanol, the resulting
glucose derivative 39 was transformed into the 6-aminoglucose de-
rivative 41 by selective 6-tosylation (formation of 40) and amination
with liquid ammonia. The 6-aminogroup was biotinylated under
EDC-activation providing the final product 42 in 61% yield.

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N. Brodersen et al. / Tetrahedron 67 (2011) 7763e7774
If not otherwise mentioned, all compounds reported in Schemes
synthesized according to literature procedures. All the other
starting materials and reagents were purchased from commercial
suppliers.
1e7 are new. Their structures were confirmed by NMR spectros-
copy and HRMS. As it is typical for amphiphilic compounds, their
purification was often not trivial and required repeated column
chromatography.
4.1.1. 1-O-Tosyl-2-O-biotinyl-tetraethylenglycol
(2). (þ)-Biotin
(1.00 g, 4.1 mmol),1-O-tosyl-tetraethylenglycol 1 (1.04 g, 4.1 mmol),
O
O
O
O
2
O
O
HN
HN
H
S
H
TsO
1
NH
NH
O
O
X
O
N
O
O
S
S
H
NH
H
DCC, DMAP, DCM, RT
33%
O
O
S
O
O
17 (X = OH)
19
Me3SnOH
18 (X = OMe)
N
1. (+)-Biotin, DCC, DMAP
4
+
18
DCM, RT ( 78% yield)
CHCl3 ,70 °C,
2 bar, 5 d
2. Me3SnOH, DCE, 70 °C,
(17 78% yield)
50%
O
O
O
MeO
OH
O
HN
O
H
NH
O
S
N
NH
O
O
N
S
O
2
S
O
O
S
NH
H
Ts
S
O
O
16
20
N
81 %
Dansyl chloride, acetone,
aq. NaHCO3, RT
O
MeO
OH
Cl
NH3
15
Scheme 4. Synthesis of dansyl-labeled conjugate 20.
ꢀ
3. Conclusion
N,N-dicyclohexylcarbondimide (DCCI) (1.01 g, 4.9 mmol), and 4-
dimethylaminopyridine (DMAP) (70 mg, 0.57 mmol) were com-
bined in dichloromethane (DCM) (30 ml). The was stirred at rt for
96 h. The solvent was removed under vacuum and the remainder
purified by column chromatography (silica, DCM/MeOH 10:1;
Rf¼0.3) to give product 2 (1.03 g, 1.7 mmol, 43%) as colorless solid.
Seven novel amphiphilic biotin conjugates were developed
consisting of a dioctadecylglycerol-derived unit as a lipophilic part
and different hydrophilic moieties by either disulfide or glycoside
linkage. Three of them contain in addition a fluorescence marker
linked to the hydrophilic site. The conjugates represent the first
examples, in which all four functions (lipophilic, hydrophilic, rec-
ognition, disulfide or glucose cleavable group) are combined. Ex-
amples reported so far lack either a recognition function or
a cleavable group. Our novel conjugates are designed for building
liposomes filled with cargo and directing these liposomes to a bi-
ological/pharmacological target where the disulfide or glycoside
would be cleaved. The cleavage will result in the separation of the
hydrophilic part of the conjugates into the surrounding. As the
remaining lipophilic structure adopts an inverted cone shape per-
turbing the stable bilayer arrangement of the membrane, defects in
and/or reorganization of the liposomes are induced, leading to
a release of the entrapped drug. The results of these investigations
will be published elsewhere.
Mp 130e132 ꢀC. 1H NMR (300 MHz, CDCl3):
d
¼1.30e1.46 (m, 2H,
eCH2e), 1.51e1.73 (m, 4H, eCH2e), 2.31 (t, J¼7.55 Hz, 2H,
eCH2e(C]O)eOe), 2.39 (s, 3H, CareCH3), 2.63e2.90 (m, 2H,
eCH2eSeCHe), 3.03e3.14 (m, 1H, eCH2eSeCHe), 3.50e3.54 (m,
4H, eCH2eOe), 3.54e3.59 (m, 4H, eCH2eOe), 3.59e3.67 (m, 4H,
eCH2eOe), 4.07e4.12 (m, 2H, eCH2eOe), 4.15 (dd, J¼5.76, 3.68 Hz,
2H, eCH2eOe), 4.24 (dd, J¼7.84, 4.82 Hz, 1H, eCHeCHe), 4.44 (dd,
J¼7.65, 4.82 Hz,1H, eCHeCHe), 5.84 (br s,1H, eNHe(C]O)eNHe),
6.32 (s, 1H, eNHe(C]O)eNHe), 7.30 (d, J¼8.50 Hz, 2H, CHar), 7.73
(d, J¼8.31 Hz, 2H, CHar) ppm. 13C NMR (75.5 MHz, CDCl3):
¼21.4
d
(CareCH3), 24.5 (CH2), 27.9 (CH2), 28.1 (CH2), 33.5 (eCH2e(C]O)e
Oe), 40.3 (eCH2eSeCHe), 55.4 (eCH2eSeCHe), 59.9 (eCHeCHe),
61.7 (eCHeCHe), 63.1 (eCH2eOe), 68.4 (eCH2eOe), 68.8
(eCH2eOe), 69.1 (eCH2eOe), 70.2 (eCH2eOe), 70.3 (eCH2eOe),
70.4 (eCH2eOe), 127.7 (CHar), 129.6 (CHar), 132.6 (Car), 144.6 (Car),
163.7 (eNHe(C]O)eNHe), 173.4 (eCH2e(C]O)eOe) ppm.
HRMS (ESI): calcd for C25H39N2O9S2 [MþH]þ 575.2116; found
575.2091.
4. Experimental section
4.1. General remarks
1H NMR and 13C NMR spectra were recorded at 300 and
75.5 MHz, respectively, with a Bruker AC 300 in CDCl3 with TMS as
internal standard. Silica gel (0.04e0.063 mm, Merck) was used for
preparative column chromatography. Starting materials 1,20 3,21 8
(see also preparation of 4),22 12,23,24 28,25 34,26 3727 were
4.1.2. 2-((2,3-Bis(octadecyloxy)propyl)disulfanyl)-N,N-dimethyle-
thanamine 4. PTAD (114 mg, 0.66 mmol) was added to a solution of
thiol 3 (400 mg, 0.66 mmol) in dry dichloromethane (DCM, 14 ml)
and MeCN (5.4 ml) under stirring in an argon atmosphere. After
stirring at rt for 30 min the temperature was elevated to 50e55 ꢀC.

N. Brodersen et al. / Tetrahedron 67 (2011) 7763e7774
7767
1. DIAD, PPh3, Phthalimid, THF, RT:
22 (X=Phthalimido), 68%
2. hydrazine hydrate EtOH, refl. :
23 (X=NH2) 57%
3. (+)-biotin, EDC, HOBt, DIPEA, DMF: RT:
24 (X=Biotinylamino), quant.
4. Na, NH3 l, -78 °C, 55%
O
O
HN
H
S
X
NH
HO
HN
BnS
HS
H
N
H
N
OTBDPS
Dansyl
OTBDPS
25
Br
21 (X = OH)
28
22 (X = Phthalimido)
23 (X = NH2)
24 (X = NH-bioninyl)
Succinanhydride, DMAP, pyridine, 70 °C, 3 d
90%
3
26
1. PTAD, MeCN, DCM, 60 °C - RT: , 38%
2. TBAF, THF, NH4F, MeOH, DCM, RT, 27, 66%
O
O
O
O
HN
H
S
Br
NH
N
O
O
HN
HO
8
NH
Dansyl
O
S
S
H
O
29
8
OR
26
(R = TBDPS)
DCC, DMAP,
DCM, 40 °C,
2 bar, 14 d
27 (R = OH)
(R = OH)
55%
O
O
HN
H
NH
O
HN
8
O
S
S
S
H
O
8
O
O
Br
H
N
N
O
30
O
S
N
Scheme 5. Synthesis of conjugate 30 with a dansyl label in the side chain.
After the color of the mixture had faded (about 2 h) N,N-dimethy-
laminoethanthiol$HCl (243 mg, 1.72 mmol) was added and the
mixture was stirred at rt for 20 h. The solvent was distilled off
and the remainder was purified by column chromatography
(silica; DCM/MeOH, 12:1; Rf¼0.6) giving product 4 (322 mg,
0.45 mmol, 68%) as colorless solid. Mp 42e44 ꢀC.1H NMR (300 MHz,
methane/MeOH, 5:1; Rf¼0.2) giving the product (115 mg,
0.09 mmol, 39%) as yellow solid. Mp >190 ꢀC (decomposition).
C68H127N3O11S4. HRMS: calcd for C61H121N3O8S22þ 559.9152;
found 559.9158.
4.1.4. 2-N-Biotinyl-N,N-dimethylethan-1,2-diamine
thylethylendiamine (195
l, 1.8 mmol) and (þ)-Biotin (437 mg,
1.8 mmol) were dissolved in dry DMF (17 ml). After the addition of
EDC (380 l, 2.1 mmol), HOBt (380 mg, 2.5 mmol) and DIPEA
(310 l, 1.8 mmol) the mixture was stirred for 24 h. After the
6. N,N-Dime-
CDCl3):
d
¼0.77e0.87 (m, 6H, eCH2eCH3), 1.10e1.34 (m, 60H,
m
eCH2e), 1.42e1.59 (m, 4H, eCH2eCH2eOe), 2.25 (s, 6H,
eCH2eNe(CH3)2), 2.61 (t, J¼7.18 Hz, 2H, eSeCH2eCH2e), 2.72e2.94
(m, 4H, eSeCH2eCH2e, eSeCH2eCHe), 3.31e3.54 (m, 6H,eO
eCH2e), 3.55e3.65 (m, 1H, eSeCH2eCHe) ppm. 13C NMR
m
m
addition of toluene (5 ml) all solvent was distilled off. The
remainder was kept under vacuum (5 mbar, 40 ꢀC) for several
hours and was then purified by column chromatography
(silica; DCM/MeOH/Et3N, 7:1:0.5; Rf¼0.4) giving the product
6 (566 mg) as colorless wax in quantitative yield. 1H NMR
(75.5 MHz, CDCl3):
d
¼14.0 (eCH2eCH3), 22.6 (eCH2eCH3), 26.0
(CH2), 26.0 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 29.9
(CH2), 31.8 (CH2), 36.2 (eSCH2eCH2e), 41.2 (eSeCH2eCHe), 45.0
(eCH2eN(CH3)2), 58.4 (eSeCH2eCH2e), 70.3 (eOeCH2e), 71.3
(eOeCH2e), 71.5 (eOeCH2e), 77.4 (eCH2eCHeCH2e) ppm.
(300 MHz, CDCl3):
d
¼1.19e1.33 (m, 2H, eSeCHeCH2e), 1.38e1.62
(m, 4H, eSCHeCH2eCH2eCH2e), 2.05 (s, 6H, eCH2eN(CH3)2), 2.27
(t, J¼5.85 Hz, 2H, eCH2eN(CH3)2), 2.48e2.79 (m, 2H,
eSeCH2eCHe), 2.92e3.05 (m, 1H, eSeCHeCH2e), 3.10e3.19 (m,
2H, eNHCH2e), 4.13 (dd, J¼7.55, 4.91 Hz, 1H, eCHeCHe), 4.34 (dd,
J¼7.55, 4.91 Hz, 1H, eCHeCHe), 6.72 (s, 1H, eNHe(C]O)eNHe),
6.86 (s, 1H, eNHe(C]O)eNHe) ppm. 13C NMR (75.5 MHz, CDCl3):
4.1.3. 2-((2,3-Bis(octadecyloxy)propyl)disulfanyl)-N,N-dimethyl-N-
(2-O-biotinyl-tetraethylenglykol-1-yl)-ethanammonium tosylate 5. A
suspension of disulfide 4 (161 mg, 0.23 mmol) and tosylate 2 in
trichloromethane (1.7 ml) was put into a pressure vessel under-
argon and stirred at 70 ꢀC (pressure around 2 bar) for 7 days.
After cooling to rt the solvent was stripped off and the
residue was purified by column chromatography (silica; dichloro-
d
¼25.3 (CH2), 27.5 (CH2), 28.1 (CH2), 36.5 (eCH2e(C]O)), 44.8
(eCH2eN(CH3)2), 55.6 (eSeCHeCH2e), 57.8 (eCH2eN(CH3)2),

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N. Brodersen et al. / Tetrahedron 67 (2011) 7763e7774
3, PTAD, MeCN,
H2N
OH
O
8
60 °C - RT
HS
OH
O
S
S
OH
OH
72 %
8
OH
O
O
O
32
31
34
O
(+)-Biotin, DCC, DMAP, DCM, 3 d
43%
Succinic anhydride, pyridine, 79 °C
55%
O
O
O
HO
O
HN
H
S
NH
8
OH
O
S
NH
O
S
O
8
H
OH
33
O
O
O
+
O
35
DCC, DMAP, THF, DMF,
70 °C, sealed tube (2 bar), 4 d
52%
O
O
O
8
HN
H
O
S
S
O
NH
8
O
O
S
H
O
HN
OH
36
O
O
O
O
Scheme 6. Synthesis of fluorescein-labeled conjugate 36.
O
8
O
OH
8
37
OAc
OAc
OAc
Ag2O, Ag2CO3, I2,
toluene, RT, - 50 °C
O
O
O
8
NaOMe, MeOH, CHCl3, RT
62%
OAc
OAc
Br
O
O
8
61%
AcO
AcO
OAc
38
OTs
OH
OH
OH
TsCl, pyridine, RT
27%
NH3l, RT, CHCl3, 10 bar
51%
O
O
8
8
O
O
O
O
OH
OH
O
O
8
8
HO
HO
40
39
O
O
HN
H
NH2
OH
OH
(+)-Biotin, EDC, HOBt,
DIPEA, DCM, DMF, RT
NH
NH
OH
O
8
O
S
H
O
8
O
O
O
O
O
61%
8
8
HO
OH
HO
41
42
Scheme 7. Conjugate of glucose containing conjugate 42.

N. Brodersen et al. / Tetrahedron 67 (2011) 7763e7774
7769
59.9 (eCHeCHe), 61.6 (eCHeCHe), 164.3 (eNHe(C]O)eNH),
173.3 (eNHe(C]O)eCH2e) ppm.
disulfide 11 (141 mg, 0.20 mmol) and the acid 12 (47 mg, 0.20 mmol)
were suspended in dry DCM/DMF (1:1, 4 ml). After the addition of EDC
(37 ml, 0.23 mmol), HOBt (42 mg, 0.27 mmol) and DIPEA (34 ml,
4.1.5. 2-N-Biotinyl-2-amino-N-(carboxymethyl)-N,N-dimethyletha-
nammonium bromide 7. Bromoacetic acid (323 mg, 2.3 mmol) was
added to a solution of the biotin amide 6 (566 mg, 1.8 mmol) in
dry MeCN (3 ml). After heating the mixture to 100 ꢀC for 30 h the
solvent was removed by a rotary evaporator and the remainder
was recrystallized from MeOH/Et2O (1:3) providing the product 7
(808 mg, 1.78 mmol, 97%) as colorless semicrystalline solid. TLC
Rf¼0.0 (dichloromethane/MeOH/Et3N, 7:1:0.5). HRMS: calcd for
C16H29N4O4Sþ 373.1904; found 373.1908.
0.20 mmol) the mixture was stirred at 30 ꢀC for 20 h. The solvent was
distilled off and the remainder purified by column chromatography
affording a diastereomeric mixture of the product 13 (100 mg,
0.11 mmol, 54%) as colorless foam. Mp 63e65 ꢀC. 1H NMR (300 MHz,
CDCl3):
d¼0.78e0.89 (m, 6H, eCH2eCH3), 0.94e1.33 (m, 60H, eCH2e),
1.42e1.52 (m, 4H, eCH2eCH2eO), 2.40e2.73 (m, 4H, e(C]O)e
CH2eCH2e(C]O)e), 2.75e2.92 (m, 4H, eCH2eSeSe, eCHe
CH2eSeSe), 3.23e3.52 (m,15H, eCH2eN(CH3)3, eCH2eOe), 3.58 (dt,
J¼9.82, 4.91 Hz, 1H, eCHeOeCH2e), 3.64e3.75 (m, 2H, eCH2eOH),
3.82e4.03 (m, 2H, eCH2eOe(C]O)e), 4.06e4.17 (m, 1H, eCHeNH),
4.40e4.67 (m, 2H, eCH2eN(CH3)3) ppm. 13C NMR (75.5 MHz, CDCl3):
4.1.6. 2-(2-((2,3-Bis(octadecyloxy)propyl)disulfanyl)ethylamino)-
N,N-dimethyl-2-oxo-N-(biotinylethyl)ethanammonium
bromide
l,
d
¼14.0 (eCH2eCH3), 22.6 (eCH2eCH3), 26.0 (CH2), 29.3 (CH2), 29.4
9. EDC (97 l, 0.55 mmol), HOBt (98 mg, 0.64 mmol), and DIPEA (76
m
m
(CH2), 29.6 (CH2), 30.0 (CH2), 30.6 (CH2), 31.8 (CH2), 39.5
(eCH2eSeSe), 40.8 (eCH2eSeSe), 51.2 (eCHeNHe), 54.3
(eCH2eN(CH3)3), 58.1(eCH2eN(CH3)3), 62.6 (eCH2eOH), 64.8(e(C]
O)eOeCH2e), 70.4 (eOeCH2e), 71.5 (eOeCH2e), 71.6 (eOeCH2e),
77.3 (eCHeOeCH2e),171.8 (e(C]O)eOe),172.1 (e(C]O)eOe) ppm.
HRMS: calcd for C51H103N2O6S2 [MꢁCl]þ 903.7252; found 903.7240.
0.46 mmol) were added to a suspension of the disulfide 8 (158 mg,
0.23 mmol) and the carboxylic acid 7(208 mg, 0.46 mmol) in dry DCM/
DMF (1:1, 8 ml). The yellowish solution was stirred at 30 ꢀC for 3 h.
After distilling off the solvent the remainder was purified by column
chromatography (ALOX, Typ-T, neutral; dichloromethane/MeOH,10:1;
Rf¼0.5) giving product 9 (56 mg, 0.05 mmol, 22%) as a colorless solid.
The diastereomers were not separated. Mp 68e70 ꢀC. 1H NMR
4.1.9. 1-O-Biotinyl-(2R)-2-amino-2-N-(2-O-succinyl-choline)-3-
((2,3-bis(octadecyloxy) propyl)disulfanyl)propan-1-ol chloride 14. A
suspension of (þ)-biotin (104 mg, 0.42 mmol) and the alcohol 13
(58.88 mg, 0.42 mmol) in dichloromethane (3.5 ml) and DMAP
(19 mg, 0.15 mmol) was stirred at 30 ꢀC for 4 days. The mixture was
concentrated with a rotary evaporator and the remainder was pu-
rified by column chromatography (ALOX, Type-T, neutral; CHCl3/
MeOH, 10:1; Rf¼0.3) affording a diastereomeric mixture of the
product 14 (45 mg, 0.04 mmol, 35%) as colorless solid. Mp 51e53 ꢀC.
(300 MHz, CDCl3):
d¼0.81e0.94 (m, 6H, eCH2eCH3), 1.16e1.36 (m,
60H, eCH2e), 1.40e1.87 (m, 10H, eCH2eCH2eO, eCH2e), 2.25e2.47
(m, 2H, eCH2e(C]O)eOe), 2.77e2.98 (m, 4H, eCH2eSeSeCH2e),
3.15 (td, J¼7.36, 4.91 Hz, 1H, eCH2eSeCHe), 3.34e3.67 (m, 15H,
eCH2eN(CH3)2e, eCH2eOe, e(C]O)eNHeCH2e, eCH2eCHeCH2
e), 3.69e3.98 (m, 4H, e(C]O)eNHeCH2eCH2e), 4.33e4.61 (m, 4H,
eCHeCHe, e(C]O)eCH2eN(CH3)2e), 5.88e6.15 (m, 1H, NHe(C]
O)eNH), 6.67e6.87 (m, 1H, eNHe(C]O)eCH2e) ppm. 13C NMR
(75.5 MHz, CDCl3):
d¼14.1 (eCH2eCH3), 22.7 (eCH2eCH2), 26.1 (CH2),
1H NMR (300 MHz, CDCl3):
d
¼0.81e0.91 (m, 6H, eCH2eCH3),
27.9(CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2), 30.0 (CH2), 30.1
(CH2), 31.9 (CH2), 33.9 (eCH2e(C]O)eOe), 36.8 (eNHeCH2e), 38.4
(eCH2eSeSe), 41.1 (eCH2eSeSe), 41.3 (eCH2eSeCHe), 46.0
(eNHeCH2e), 50.8 (eN(CH3)2e), 52.8 (eCHe), 56.0 (eCHe), 62.0
(eCHe), 70.5 (CH2), 71.5 (CH2), 71.7 (CH2), 71.8 (CH2), 77.5
(eCHeOeCH2e), 164.1 (eNHe(C]O)eNHe), 173.5 (eCH2e(C]O)e
NHe), 174.6 (eCH2e(C]O)eNHe) ppm. HRMS: calcd for
C57H112N5O5S3þ1042.7820; found 1042.7837.
1.00e1.34 (m, 60H, eCH2e), 1.36e1.78 (m, 10H, eCH2eCH2eO,
eCH2e), 2.24e2.43 (m, 2H, eCH2e(C]O)eOe), 2.44e2.80 (m, 4H,
e(C]O)eCH2eCH2e(C]O)e), 2.80e2.96 (m, 4H, eCH2eSeSe,
eCH2eSeCHe), 3.07e3.20 (m, 2H, eCH2eSeSe), 3.33e3.56 (m,
15H, eCH2eN(CH3)3, eCH2eOe), 3.57e3.67 (m, 1H, eCHeOCH2e),
3.96e4.60 (m, 9H, eCH2eOH, eCH2eOe(C]O)e, eCHeNH,
eCH2eN(CH3)3, eCHeCHe), 5.52.5.75 (m, 1H, NHe(C]O)eNH),
6.03e6.18 (m, 1H, NHe(C]O)eNH) ppm. 13C NMR (75.5 MHz,
CDCl3):
d
¼14.1 (eCH2eCH3), 22.6 (eCH2eCH3), 24.7 (CH2), 26.1
4.1.7. (2R)-2-Amino-3-((2,3-bis(octadecyloxy)propyl)disulfanyl)
propan-1-ol 11. PTAD (114 mg, 0.66 mmol) was added to a solution
of the octadecyl ether 3 (400 mg, 0.66 mmol) in dry DCM (14 ml),
and dry MeCN (5.4 ml) under stirring in an argon atmosphere. The
mixture was stirred for 30 min at rt and was then heated to
50e55 ꢀC until the color had faded (about 2 h). (R)-cysteinol
(180 mg, 1.68 mmol) and MeOH (4 ml) were added and the
resulting mixture was stirred at rt for 20 h. After the solvent was
stripped off the remainder was purified by column chromatography
(silica gel; CHCl3/MeOH, 10:1; Rf¼0.4). The colorless waxy product
11 was obtained as a diastereomeric mixture (332 mg, 0.46 mmol,
(CH2), 28.1 (CH2), 29.2 (CH2), 29.3 (CH2), 29.5 (CH2), 29.6 (CH2), 30.0
(CH2), 30.8 (CH2), 31.9 (CH2), 33.6 (eCH2e(C]O)eOe), 39.8
(eCH2eSeSe), 40.6 (eCH2eSeSe), 41.2 (CH2), 48.1 (eCHeNHe),
54.3 (eCH2eN(CH3)3), 55.5 (eCHe), 58.2 (eCH2eN(CH3)3), 60.1
(eCHe), 61.8 (eCHe), 64.3 (CH2), 64.7 (e(C]O)eOeCH2e), 70.5
(eOeCH2e),
71.5
(eOeCH2e),
71.7
(eOeCH2e),
77.3
(eCHeOeCH2e), 163.9 (eNHe(C]O)eNHe), 171.4 (e(C]O)eOe),
172.2 (e(C]O)eOe), 173.8 (eCH2e(C]O)eNHe) ppm. HRMS:
calcd for C61H117N4O8S3 [MꢁCl]þ 1129.8028; found 1129.8017.
4.1.10. 2-N-Dansyl-serine methyl ester 16. A solution of serine
methyl ester$HCl (311 mg, 2 mmol) in satd aqueous NaHCO3 (6 ml)
was added drop wise into a mixture of dansyl chloride (540 mg,
2 mmol) and acetone/water (2:1, 24 ml) in a brown colored flask.
After the mixture was stirred at rt for 20 h the solvent was stripped
off under vacuum. The remainder was put into a mixture of water
(100 ml) and EtOAc (140 ml). The organic layer was separated, dried
over MgSO4 and concentrated with a rotary evaporator. The re-
mainder was put under vacuum for a view hours (4 mbar, 40 ꢀC)
giving the yellow solid (568 mg, 1.6 mmol, 81%), which was used in
the next step without further purification.
70%). Mp 206e207 ꢀC. 1H NMR (300 MHz, CDCl3):
d
¼0.82e0.92 (m,
6H, eCH2eCH3), 1.00e1.40 (m, 60H, eCH2e), 1.45e1.69 (m, 4H,
eCH2eCH2eO), 2.24 (br s, 2H, eCHeNH2), 2.50e3.03 (m, 4H,
eCH2eSeSeCH2e), 3.12e3.25 (m, 1H, eCHeNH2), 3.35e3.75 (m,
9H, eOeCH2eCHe, eCHeCH2eOH, eCH2e, eOeCH2eCHe) ppm.
13C NMR (75.5 MHz, CDCl3):
d
¼14.1 (eCH2eCH3), 22.7 (CH2eCH3),
26.1 (CH2), 29.3 (CH2), 29.5 (CH2), 29.7 (CH2), 30.0 (CH2), 31.9 (CH2),
41.3 (eCHeCH2eSe), 43.1 (NH2eCHeCH2eSe), 51.5 (eCHeNH2),
65.4 (eCH2eOH), 70.5 (OeCH2eCH2), 71.3 (CHeCH2eO), 71.7
(OeCH2eCH2), 77.4 (eOeCHeCH2eO) ppm. HRMS: calcd for
C42H88NO3S2 [MþH]þ 718.6200; found 718.6192.
4.1.11. 3-O-Biotinyl-2-N-dansyl-serine 17. Me3SnOH (1 g, 5.5 mmol)
was added to a suspension of the methyl ester 18 (725 mg,
1.3 mmol) in 1,2-dichloroethane (10 ml) under stirring and argon
4.1.8. (2R)-2-Amino-2-N-(2-O-succinyl-choline)-3-((2,3-
bis(octadecyloxy)propyl)disulfanyl) propan-1-ol chloride 13. The

7770
N. Brodersen et al. / Tetrahedron 67 (2011) 7763e7774
atmosphere in a brown colored flask. After stirring at 70 ꢀC for 1.5 h
the solvent was removed by a rotary evaporator. The remainder was
combined with EtOAc (150 ml) and the organic layer was washed
with (0.01 N) KHSO4 (three times 150 ml). After drying over MgSO4
the solvent was removed and the remainder was put under vacuum
(3 mbar, 40 ꢀC) for a few hours providing the product 17 (558 mg,
0.99 mmol, 79%) as yellow solid. TLC (DCM/MeOH, 10:1); Rf¼0.4. 1H
(75.5 MHz, CDCl3):
d
¼21.5 (CareCH3), 24.4 (CH2), 27.9 (CH2), 28.1
(CH2), 33.1 (eCH2e(C]O)eOe), 40.4 (eCH2eSeCHe), 45.3
(eN(CH3)2), 55.0 (eCHeNHeSO2e), 56.1 (eCH2eSeCHe), 60.3
(eCHeCHe), 61.6 (eCHeCHe), 64.3 (eCH2eOe(C]O)e), 64.6
(eCH2eOe), 68.3 (eCH2eOe), 68.5 (eCH2eOe), 69.2 (eCH2eOe),
70.3 (eCH2eOe), 70.6 (eCH2eOe), 115.1 (CHar), 119.3 (CHar), 123.1
(CHar), 127.8 (CHar), 128.1 (CHar), 128.8 (CHar), 129.5 (Car), 129.6 (Car),
129.7 (CHar), 130.3 (CHar), 132.8 (Car), 135.5 (Car), 144.7 (Car), 151.5
(Car), 164.5 (eNHe(C]O)eNHe), 168.8 (eCHe(C]O)eOe), 173.0
(eCH2e(C]O)eOe) ppm.
NMR (300 MHz, CDCl3):
d
¼0.97e1.32 (m, 2H, eCH2e), 1.38e1.62
(m, 4H, eCH2e), 2.02e2.17 (m, 2H, eCH2e(C]O)eOe), 2.53e2.66
(m, 1H, eCH2eSeCHe), 2.71e2.92 (m, 7H, eCH2eSeCHe,
eN(CH3)2), 2.98e3.14 (m, 1H, eCH2eSeCHe), 4.06e4.36 (m, 4H,
eCHeCH2eOe, eCHeCHe), 4.37e4.47 (m, 1H, eCHeCHe), 7.12 (d,
J¼7.74 Hz, 1H, CHar), 7.37e7.58 (m, 2H, CHar), 8.20 (d, J¼7.18 Hz, 1H,
CHar), 8.28 (d, J¼8.50 Hz, 1H, CHar), 8.47 (d, J¼8.31 Hz, 1H, CHar)
4.1.14. 2-((2,3-Bis(octadecyloxy)propyl)disulfanyl)-N,N-dimethyl-N-
(2-O-(3-O-biotinyl-2-N-dansyl-serin-1-yl)-tetraethylenglykol-1-yl)-
ethanammonium tosylate 20. A solution of the tosylate 19 (292 mg,
0.33 mmol) and disulfide 4 (80 mg, 0.11 mmol) in CHCl3 (1 ml) was
stirred under argon in a sealed flask to 70 ꢀC (2 bar) for 5 days. The
solvent was stripped of by a rotary evaporator and the remainder was
purified by column chromatography (silica, dichloromethane/MeOH,
5:1; Rf¼0.2). The product was obtained as diastereomeric mixture in
50% yield (88 mg) as yellow solid. Mp 55e57 ꢀC. 1H NMR (400 MHz,
ppm. 13C NMR (75.5 MHz, CDCl3):
d
¼24.7 (CH2), 27.7 (CH2), 33.4
(eCH2e(C]O)eOe), 40.1 (eCH2eSeCHe), 45.3 (eN(CH3)2), 55.0
(eCHeNHeSO2e), 57.8 (eCH2eSeCHe), 60.3 (eCH2eOe(C]O)e),
60.4 (eCHeCHe), 62.0 (eCHeCHe), 115.2 (CHar), 119.1 (CHar), 123.1
(CHar), 128.2 (CHar), 129.1 (CHar), 129.6 (Car), 129.6 (CHar), 130.5 (Car),
135.0 (Car),151.4 (Car),164.8 (eNHe(C]O)eNHe),172.2 (eCHe(C]
O)eOe), 172.9 (eCH2e(C]O)eOe) ppm.
CDCl3/CD3COD, 7:2):
d
¼0.66e0.71 (m, 6H, eCH2eCH3), 0.81e1.17 (m,
60H, eCH2e), 1.17e1.53 (m, 10H, eCH2eCH2eO, eCH2e), 1.76 (t,
J¼7.28 Hz, 2H, eCH2e(C]O)eOe), 2.17 (s, 3H, CareCH3), 2.49e2.59
(m,1H, eCH2eSeCHe), 2.67e2.76 (m, 7H, eCH2eSeCHe, eN(CH3)2),
3.20e3.55 (m, 27H, eCH2eSeSeCH2e, eCH2eOe, eCH2eSeCHe,
eN(CH3)2), 3.70e3.77 (m, 2H, eCH2e), 3.78e3.85 (m, 1H,
eCHeOeCH2e), 3.87e4.11 (m, 10H, eCHeCH2eOe, eCHeCHe,
eCH2e), 4.24e4.33 (m,1H, eCHeCHe), 7.00 (d, J¼8.03 Hz, 2H, CHar),
7.05 (d, J¼7.53 Hz, 1H, CHar), 7.31e7.45 (m, 2H, CHar), 7.53 (d,
J¼8.03 Hz, 2H, CHar), 8.04 (dd, J¼7.28, 1.25 Hz, 1H, CHar), 8.12 (d,
J¼8.53 Hz, 1H, CHar), 8.36 (d, J¼8.53 Hz, 1H, CHar) ppm. 13C NMR
4.1.12. 3-O-Biotinyl-2-N-dansyl-serine methyl ester 18. (þ)-Biotin
(394 mg, 1.6 mmol), DCCI (399 mg, 1.9 mmol), and DMAP (28 mg,
0.23 mmol) were added to a solution of N-dansylserine 16 (568 mg,
1.6 mmol) in dry dichloromethane (12 ml) under stirring in a brown
colored flask. The suspension was stirred at rt for 9 days. After dis-
tilling off the solvent the remainder was purified by column chro-
matography (silica gel; dichloromethane/MeOH, 20:1; Rf¼0.5)
affording product 18 (725 mg, 78% yield) as yellow solid. 1H NMR
(300 MHz, CDCl3):
d¼1.22e1.46 (m, 2H, eCH2e), 1.44e1.78 (m, 4H,
eCH2e), 2.06e2.31 (m, 2H, eCH2e(C]O)eOe), 2.57e2.79 (m, 2H,
eCH2eSeCHe), 2.82 (s, 6H, eN(CH3)2), 3.02e3.12 (m, 1H,
eCH2eSeCHe), 3.36 (s, 3H, e(C]O)eOeCH3), 4.13e4.33 (m, 4H,
eCHeCH2eOe, eCHeCHe), 4.39e4.48 (m, 1H, eCHeCHe), 6.58 (s,
1H, C]OeNH), 7.13 (d, J¼7.55 Hz, 1H, CHar), 7.19 (s, 1H, C]OeNH),
7.44 (dd, J¼8.40, 7.46 Hz,1H, CHar), 7.48e7.57 (m,1H, CHar), 7.91e8.05
(m, 1H, C]OeNH), 8.21 (dd, J¼7.36, 0.94 Hz, 1H, CHar), 8.34 (d,
J¼8.69 Hz, 1H, CHar), 8.47 (d, J¼8.50 Hz, 1H, CHar) ppm. 13C NMR
(75.5 MHz, CDCl3/CD3COD, 7:2):
d¼13.6 (eCH2eCH3), 20.7 (CareCH3),
22.3 (eCH2eCH3), 25.7 (CH2), 27.8 (CH2), 27.9 (CH2), 28.9 (CH2), 29.1
(CH2), 29.2 (CH2), 29.2 (CH2), 29.3 (CH2), 29.5 (CH2), 29.6 (CH2), 31.5
(CH2), 32.7 (eCH2e(C]O)eOe), 39.9 (eCH2eSeSe), 40.8
(eCH2eSeCHe), 45.0 (eN(CH3)2), 51.6 (eN(CH3)2), 54.7
(eCHeNHeSO2e), 55.2 (eCH2eSeCHe), 59.8 (eCHeCHe), 61.7
(eCHeCHe), 62.9 (CH2), 63.2 (CH2), 64.3 (CH2), 64.6 (CH2), 68.2 (CH2),
69.8 (CH2), 69.9 (CH2), 69.9 (CH2), 70.1 (CH2), 70.2 (CH2), 70.9 (CH2),
71.4 (CH2), 76.9 (eCH2eOeCHe), 115.0 (CHar), 119.0 (CHar), 123.0
(CHar),125.4 (CHar),127.9 (CHar),128.5 (CHar),128.9 (CHar),129.2 (Car),
129.3 (Car), 130.1 (CHar), 135.0 (Car), 140.2 (Car), 141.2 (Car), 151.0 (Car),
168.7 (eCH2e(C]O)eOe), 172.8 (eCH2e(C]O)eOe) ppm. HRMS:
calcd for C76H136N5O12S4 (Mꢁtosylate)þ 1438.9063; found1438.9057.
(75.5 MHz, CDCl3):
d
¼24.4 (CH2), 27.8 (CH2), 28.1 (CH2), 33.0
(eCH2e(C]O)eOe), 40.4 (eCH2eSeCHe), 45.2 (eN(CH3)2), 52.3
(e(C]O)eOeCH3), 54.8 (eCHeNHeSO2e), 56.1 (eCH2eSeCHe),
60.2 (eCHeCHe), 61.5 (eCHeCHe), 64.2 (eCH2eOe(C]O)e), 115.0
(CHar),119.1 (CHar),122.9 (CHar),128.0 (CHar),128.8 (CHar),129.5 (Car),
129.5 (CHar), 130.2 (Car), 135.3 (Car), 151.5 (Car), 164.6 (eNHe(C]O)e
NHe), 169.2 (eCHe(C]O)eOe), 173.0 (eCH2e(C]O)eOe) ppm.
4.1.15. 1-(Benzylthio)-3-(tert-butyldiphenylsilyloxy)propan-2-ol
21. Imidazole (2.55 g, 37.8 mmol) and TDPS chloride (8.5 g,
31 mmol) were added to a solution of 1-benzylthio propan 2,3-diol
(5.75 g, 29 mmol) in dry DMF (140 ml). The mixture was stirred at
rt for 72 h. Ethanol (30 ml) was added and all solvents were distilled
off. The remainder was dissolved in CHCl3 (200 ml) and washed with
water (2ꢂ100 ml). The organic layer was dried over MgSO4 and
concentrated by a rotary evaporator. The remainder was purified by
column chromatography (silica gel; CHCl3/MeOH, 30:1; Rf¼0.8)
providing the product 21 (12.37 g, 96% yield) as yellow oil. 1H NMR
(300 MHz, CDCl3): d¼1.16 (s, 9H, CqeCH3), 2.66e2.77 (m, 2H,
SeCH2eCH), 3.70e3.82 (m, 4H, CHeCH2eO, SeCH2ePh), 3.88 (dd,
J¼7.27, 5.00 Hz, 1H, CH2eCHeCH2), 7.24e7.39 (m, 5H, CHar),
7.42e7.57 (m, 6H, CHar), 7.69e7.82 (m, 4H, CHar) ppm. 13C NMR
(75.5 MHz, CDCl3): d¼19.1 (Cq), 26.8 (eCqeCH3), 34.5 (SeCH2eCH),
36.4 (SCH2ePh), 66.4 (CHeCH2eO), 70.4 (CHeCH2eO), 127.0 (CHar),
127.7 (CHar),128.4 (CHar),128.8 (CHar),129.7 (CHar),132.9 (Car),135.4
(CHar), 138.0 (Car) ppm.
4.1.13. 3-O-Biotinyl-2-N-dansyl-serin-(1-O-tosyl-tetraethylenglykol)
ester 19. Tosylate 1 (345 mg, 0.99 mmol), DCCI (245 mg, 1.2 mmol),
and DMAP 20 mg (0.16 mmol) wereadded toa solution of the acid 17
(558 mg, 0.99 mmol) in dry dichloromethane (8 ml) under stirring
in a brown colored flask. The mixture was stirred at rt for 72 h. Then
the solvent was removed by a rotary evaporator and the remainder
purified by column chromatography (Silica gel; dichloromethane/
MeOH, 13:1; Rf¼0.4) affording product 19 (292 mg, 0.33 mmol, 33%
yield) as yellow solid. 1H NMR (300 MHz, CDCl3):
d¼1.26e1.76 (m,
6H, eCH2e), 2.03e2.31 (m, 2H, eCH2e(C]O)eOe), 2.40 (s, 3H,
CareCH3), 2.56e2.81 (m, 2H, eCH2eSeCHe), 2.83 (s, 6H, eN(CH3)2),
3.00e3.15 (m, 1H, eCH2eSeCHe), 3.42 (t, J¼4.82 Hz, 2H,
eCH2eOe), 3.45e3.60 (m, 8H, eCH2eOe), 3.61e3.66 (m, 2H,
eCH2eOe), 3.91e4.05 (m, 2H, eCH2eOeSO2e), 4.09e4.15 (m, 2H,
eCH2eOe(C]O)e), 4.18e4.34 (m, 4H, eCHeCHe, eCH2eCHe(C]
O)eOe), 4.38e4.48 (m, 1H, eCHeCHe), 6.28 (s, 1H, eNHeSO2e),
7.08e7.17 (m, 2H, CHar), 7.27e7.34 (m, 2H, CHar), 7.39e7.60 (m, 2H,
CHar), 7.73e7.80 (m, 1H, CHar), 8.19e8.24 (m, 1H, CHar), 8.34 (d,
J¼8.69 Hz, 1H, CHar), 8.48 (d, J¼8.50 Hz, 1H, CHar) ppm. 13C NMR
4.1.16. 2-(1-(Benzylthio)-3-(tert-butyldiphenylsilyloxy)propan-2-yl)
isoindoline-1,3-dione 22. Diisopropyl azodicarboxylate (DIAD)

N. Brodersen et al. / Tetrahedron 67 (2011) 7763e7774
7771
(2.09 g, 10.3 mmol) was slowly added to a solution of the alcohol 21
(3.72 g, 8.5 mmol), triphenylphosphane (2.41 g, 9.2 mmol), and
phthalimid (1.39 g, 9.4 mmol) in dry THF (68 ml) under stirring at rt
under argon within 30 min. After stirring for 24 h the solvent was
distilled off and the remainder purified by column chromatography
(silica gel; dichloromethane/cyclohexane, 1:2; Rf¼0.4) affording
product 22 (3.26 g, 68% yield) as colorless oil. 1H NMR (300 MHz,
(eCH]CHeCH2e), 25.6 (eCH2e), 26.0 (eCH2e), 27.6 (Cq(CH3)3),
28.1 (eCH2e), 28.2 (eCH2e), 28.7 (eCHeCH]CHe), 36.0
(eCH2e(C]O)eOe), 40.4 (eCH2eSeCHe), 42.2 (eCH2eCHeCH2e),
43.8 (eCH2eSH), 55.6 (eCH2eSeCHe), 60.2 (eCHeCHe), 61.7
(eCHeCHe), 67.6 (eCH2eOe), 122.1 (eCH]CHe), 122.2 (eCH]
CHe),125.4 (eCH]CHe),125.5 (eCH]CHe),164.2 (eNHe(C]O)e
NHe), 173.4 (eCH2e(C]O)eNHe) ppm.
CDCl3):
d
¼1.12 (s, 9H, Cq(CH3)3), 3.15e3.28 (m, 1H,
eCH2eCHeCH2e), 3.49e3.74 (m, 2H, eCHeCH2eSe), 3.75e3.98
(m, 3H, eSeCH2eCar, eOeCH2e), 4.07e4.20 (m, 1H, eOeCH2e),
7.09e7.21 (m, 5H, CHar), 7.36e7.50 (m, 6H, CHar), 7.64e7.79 (m, 6H,
CHar), 7.79e7.89 (m, 2H, CHar) ppm. 13C NMR (75.5 MHz, CDCl3):
4.1.19. 1-(tert-Butyl(cyclohexa-2,5-dienyl)phenylsilyloxy)-2-N-bio-
tinyl-3-((2,3-bis(octadecyloxy)propyl)disulfanyl)-2-aminopropane
26. PTAD (26 mg, 0.15 mmol) was added to a solution of the thiol 3
(91 mg, 0.15 mmol) in dry dichloromethane (3.4 ml) DCM and dry
MeCN under argon and stirring. After stirring at rt for 1.5 h the
temperature was elevated to 60 ꢀC. After the solution had become
colorless the biotin derivative 25 (95 mg, 0.17 mmol) was added
and stirring continued at rt for 24 h. The solvent was removed by
distillation and the residue purified by column chromatography
(silica gel; gradient: dichloromethaneꢃdichloromethane/MeOH,
10:1; Rf¼0.5). The product 26 (76 mg, 38% yield) was obtained as
diastereomeric mixture of a colorless solid. 1H NMR (300 MHz,
d
¼19.0 (Cq(CH3)3), 26.6 (Cq(CH3)3), 35.5 (CareCH2eSe), 39.2
(eSeCH2e), 45.8 (eCH2eCHeCH2e), 65.7 (eCH2eOe),123.1 (CHar),
126.7 (CHar), 127.6 (CHar), 128.1 (CHar), 128.6 (CHar), 129.6 (CHar),
131.9 (Car), 133.0 (Car), 133.6 (CHar), 135.5 (CHar), 135.6 (CHar), 138.2
(Car), 167.9 (e(C]O)eNHe) ppm.
4.1.17. 1-(Benzylthio)-3-(tert-butyldiphenylsilyloxy)propan-2-amine
23. Hydrazine hydrate (51%, 0.4 ml, 6.6 mol) was added to a solu-
tion of the phthalimid 22 (3.2 g, 5.66 mmol) in 96% EtOH (48 ml)
under stirring. After refluxing for 2.5 h the mixture was stirred at rt
for 3 days. After distilling off the solvent the remainder was purified
by column chromatography (silica gel; dichloromethane/MeOH,
25:1; Rf¼0.3) affording the product 23 (1.40 g, 57% yield) as color-
CDCl3):
d
¼0.83e0.92 (m, 6H, eCH2eCH3), 0.92e1.11 (m, 9H,
Cq(CH3)3), 1.11e1.36 (m, 60H, eCH2e), 1.36e1.78 (m, 10H, eCH2e,
eCH2eCH2eOe), 1.98e2.32 (m, 2H, eCH2e(C]O)eOe), 2.65e2.99
(m, 6H, eCH2eCH]CH, eCHeCH]CH, eCH2eSeCHe), 3.02e3.18
(m, 2H, eCH2eSe), 3.33e3.66 (m, 9H, eCH2eSe, eCH2eOe,
eCH2eCHeCH2e), 3.71e4.17 (m, 2H, eCH2eOe), 4.19e4.33 (m, 1H,
eCHeCHe), 4.41e4.48 (m, 1H, eCHeCHe), 5.46e5.97 (m, 4H,
eCH]CHe), 7.29e7.70 (m, 5H, CHar) ppm. 13C NMR (75.5 MHz,
less oil. 1H NMR (300 MHz, CDCl3):
d¼1.04e1.16 (m, 9H, Cq(CH3)3),
2.64e2.76 (m, 1H, eCH2eCHeCH2e), 2.80e3.14 (m, 2H, eCHe
CH2eSe), 3.63e3.88 (m, 4H, eSeCH2eCar, eOeCH2e), 7.21e7.36
(m, 5H, CHar), 7.37e7.54 (m, 6H, CHar), 7.65e7.77 (m, 4H, CHar) ppm.
CDCl3):
d
¼14.1 (eCH2eCH3), 19.3 (Cq), 22.6 (eCH2e), 25.6 (eCH2e),
13C NMR (75.5 MHz, CDCl3):
d
¼19.1 (Cq(CH3)3), 26.8 (Cq(CH3)3),
26.1 (eCH2e), 26.8 (Cq(CH3)3), 28.1 (eCH2e), 29.3 (eCH2e), 29.5
(eCH2e), 29.6 (eCH2e), 30.0 (eCH2e), 31.9 (eCH2e), 36.0
(eCH2e(C]O)eOe), 40.4 (eCH2eSe), 42.3 (eCH2eSe), 42.5
(eCH2eSe), 53.0 (eNHeCHe), 55.5 (eCH2eSeCHe), 60.2
(eCHeCHe), 61.7 (eCHeCHe), 65.4 (eCH2eOe), 70.5 (eCH2eOe),
71.4 (eCH2eOe), 71.7 (eCH2eOe), 77.6 (eCHeOe), 122.2 (eCH]
CHe), 125.7 (eCH]CHe), 127.8 (CHar), 129.9 (CHar), 132.9 (Car),
133.0 (Car), 135.6 (CHar), 169.2 (eNHe(C]O)eNHe), 173.2
(eCH2e(C]O)eNHe) ppm.
35.6 (CareCH2eSe), 42.8 (eSeCH2e), 50.8 (eCH2eCHeCH2e),
64.9 (eCH2eOe), 126.9 (CHar), 127.7 (CHar), 128.4(CHar), 128.7
(CHar), 129.7 (CHar), 133.2 (Car), 133.3 (Car), 135.5 (CHar), 138.5
(Car) ppm.
4.1.18. 1-Mercapto-3-(tert-butyldicyclohexa-2,5-dienylsilyloxy)
propan-2-biotinylamide 25. EDC (38 ml, 0.21 mmol), HOBt (38 mg,
0.25 mmol), and DIPEA (31 ml, 0.18 mmol) were added to a solution
of the amine 23 (77 mg, 0.18 mmol) and (þ)-biotin (40 mg,
0.16 mmol) in dry DMF. After stirring at rt for 24 h toluene (5 ml) was
added and all solvents removed by distillation. The remainder was
kept under vacuum (5 mbar, 40 ꢀC) and then dissolved in CHCl3
(20 ml). The organic layer was washed with water (3ꢂ20 ml) and
dried over MgSO4. The solvent was removed by a rotary evapo-
rator leaving behind 1-(benzylthio)-3-(tert-butyldiphenylsilyloxy)
propan-2-biotinylamide 24 (124 mg, quantitative yield). Liquid
ammonia is condensed into a solution of the benzylthioether 24
(200 mg, 0.3 mmol) in a dry-ice bath (ꢁ78 ꢀC) under argon. Sodium
(100 mg, 4.3 mol) was added in small pieces under a currentof argon
and stirring. The dark blue solution was stirred at (ꢁ78 ꢀC) for 2.5 h
NH4Cl (500 mg) were added and ammonia was removed from the
colorless suspension bya continuous stream of argon. Water (40 ml)
was added tothe residue and the suspensionwas adjusted to pH 3 by
adding 2 N aqueous HCl. The mixture was extracted with tri-
chloromethane (100 ml). The organic layer was separated and
concentrated with a rotaryevaporator. The reminder was purified by
column chromatography (silica gel; dichloromethane/MeOH, 10:1;
Rf¼0.3) affording a diastereomeric mixture of product 26 (95 mg,
4.1.20. 2-N-Biotinyl-3-((2,3-bis(octadecyloxy)propyl)disulfanyl)-2-
aminopropanol 27. Ammonium fluoride (50 mg, 1.35 mol) was
added to a solution of the silyl ether 26 (76 mg, 0.064 mmol) in
THF (2 ml) under stirring. 1 M solution of TBAF in THF (1 ml,
1 mmol) was added and this suspension was drop wise. After the
mixture had been stirred at rt for 3 days, the solvent was distilled
of under vacuum and the remainder purified by column chroma-
tography (silica gel; gradient: dichloromethaneꢃdichloromethane/
MeOH, 12:1; Rf¼0.3). The colorless solid product 27 (40 mg) was
obtained as diastereomeric mixture in 66% yield. Mp 112e114 ꢀC.
1H NMR (300 MHz, CDCl3):
d
¼0.82e0.92 (m, 6H, eCH2eCH3),
0.96e1.37 (m, 60H, eCH2e), 1.38e1.76 (m, 10H, eCH2e,
eCH2eCH2eOe), 2.25 (t, J¼7.46 Hz, 2H, eCH2e(C]O)eOe),
2.67e2.96 (m, 4H, eCH2eSeCHe, eCH2eSe), 3.00e3.08 (m, 1H,
eCHeNHe), 3.09e3.16 (m, 1H, eCH2eSeCHe), 3.38e3.59 (m, 8H,
eCH2eSe, eCH2eOe), 3.59e3.69 (m, 1H, eCH2eCHeCH2e),
3.69e3.81 (m, 2H, eCH2eOe), 4.27e4.36 (m, 1H, eCHeCHe),
4.47e4.56 (m, 1H, eCHeCHe), 5.79 (d, J¼7.93 Hz, 1H, e(C]O)e
NHe), 6.81 (d, J¼8.50 Hz, 1H, e(C]O)eNHe), 6.90e7.06 (m, 1H,
55% yield) as colorless oil. 1H NMR (300 MHz, CDCl3):
d¼0.89e1.10
e(C]O)eNHe) ppm. 13C NMR (75.5 MHz, CDCl3):
(eCH2eCH3), 22.7 (eCH2eCH3), 25.8 (CH2), 26.1 (CH2), 28.0 (CH2),
29.3 (CH2), 29.5 (CH2), 29.6 (CH2), 29.6 (CH2), 29.7 (CH2), 30.0
(CH2), 31.9 (CH2), 35.8 (eCH2e(C]O)eOe), 39.2 (eCH2eSe), 40.5
d
¼14.1
(m, 9H, Cq(CH3)3), 1.34e1.50 (m, 2H, eCH2e), 1.55e1.85 (m, 4H,
eCH2e), 2.20 (t, J¼7.36 Hz, 2H, eCH2e(C]O)eOe), 2.61e2.91 (m,
6H, eCH2eSeCHe, eCH]CHeCH2e), 3.02e3.29 (m, 3H, HSeCH2e
CHe, eCH2eSeCHe), 3.60e3.76 (m, 1H, HSeCH2eCHe), 3.80e4.09
(m, 2H, eCH2eOe), 4.19e4.32 (m,1H, eCHeCHe), 4.41e4.53 (m,1H,
eCHeCHe), 5.48e5.59 (m, 4H, eCH]CHe), 5.73e5.83 (m, 4H,
(eCH2eSe),
42.1
(eCH2eSe),
52.8
(eNHeCHe),
55.7
(eCH2eSeCHe), 60.2 (eCHeCHe), 61.4 (eCH2eOH), 61.8
(eCHeCHe), 70.5 (eCH2eOe), 71.4 (eCH2eOe), 71.8 (eCH2eOe),
77.4 (eCHeOe), 164.3 (eNHe(C]O)eNHe), 174.9 (eCH2e(C]O)e
eCH]CHe) ppm. 13C NMR (75.5 MHz, CDCl3):
d¼20.0 (Cq), 21.7

7772
N. Brodersen et al. / Tetrahedron 67 (2011) 7763e7774
NHe) ppm. HRMS: calcd for C52H102N3O5S3 [MþH]þ 944.6976;
Rf¼0.5) affording the colorless solid product 32 (186 mg) as di-
astereomeric mixture in 72% yield. As a by-product the symmetric
disulfide of 3 was isolated in 20% yield. 1H NMR (300 MHz, CDCl3):
found 944.6985.
4.1.21. 2-Dansylamido-N-(2-O-succinyl-ethyl)-N,N-dimethyletha-
nammonium bromide 29. A suspension of the choline derivative 8
(1 g, 2.24 mmol), succinic anhydride (540 mg, 5.4 mmol), and DMAP
(15 mg, 0.12 mmol) in dry pyridine (10 ml) was heated to 70 ꢀC
under argon in a brown flask for 3 days. After cooling to rt toluene
(20 ml) was added and all solvents were distilled off under vacuum.
The remainder was purified by column chromatography (silica gel;
dichloromethane/MeOH/formic acid, 5:1:0.2; Rf¼0.3) affording
product 29 (1.1 g) in 90% yield as ayellowish green hygroscopic solid.
d
¼0.81e0.95 (m, 6H, eCH2eCH3), 1.14e1.48 (m, 60H, eCH2e),
1.50e1.63 (m, 4H, eCH2e), 2.70e3.02 (m, 4H, eCH2eSeSeCH2e),
3.38e3.82 (m, 9H, HeOeCH, eCH2eOeCH2, eCH2eOeCH,
CHeCH2eOH), 3.90e4.08 (m, 1H, eCH2eOeCH) ppm. 13C NMR
(75.5 MHz, CDCl3):
d
¼14.1 (eCH2eCH3), 22.7 (eCH2eCH3), 26.0
(eCH2e), 29.3 (eCH2e), 29.4 (eCH2e), 29.5 (eCH2e), 29.6
(eCH2e), 29.7 (eCH2e), 30.0 (eCH2e), 31.9 (eCH2e), 41.1
(CH2eSeSe), 42.0 (CH2eSeSe), 65.1 (CH2eOeCH2eCH), 70.2
(CHeOeCH2e), 70.5 (eCH2eCH2eOe), 71.2 (HOeCH2eCH), 71.8
(eCH2eCH2eOe), 77.5 (CHeOH) ppm.
1H NMR (300 MHz, CD3OD):
d
¼2.47e2.56 (m, 4H, e(C]O)e
CH2eCH2e(C]O)e), 2.78e2.89 (m, 6H, CareN(CH3)2), 3.09e3.19 (m,
6H, eN(CH3)2), 3.24e3.33 (m, 2H, eCH2eNHe), 3.53 (t, J¼6.23 Hz,
2H, e(CH3)2NþeCH2e), 3.65e3.72 (m, 2H, e(CH3)2NþeCH2e),
4.39e4.54 (m, 2H, eCH2eOe(C]O)e), 7.25 (d, J¼7.36 Hz, 1H, CHar),
7.51e7.66 (m, 2H, CHar), 8.18e8.24 (m, 1H, CHar), 8.27 (d, J¼8.50 Hz,
1H, CHar), 8.55 (d, J¼8.50 Hz, 1H, CHar) ppm. 13C NMR (75.5 MHz,
4.1.24. 1-O-Biotinyl-3-((2,3-bis(octadecyloxy)propyl)disulfanyl)
propan-1,2-diol 33. A suspension of (þ)-biotin (135 mg, 0.56 mmol),
disulfide 32 (264 mg, 0.37 mmol), DCC (152 mg, 0.74 mmol), and
DMA (30 mg, 0.24 mmol) was stirred at rt for 3 days. The solvent
was removed by a rotary evaporator and the remainder purified
by column chromatography (silica gel; CHCl3/MeOH, 25:1; Rf¼0.3)
affording the colorless solid product 33 (150 mg) as diastereomeric
mixture in 43% yield. Mp 62e64 ꢀC. 1H NMR (300 MHz, CDCl3):
CD3OD):
d
¼30.7 (e(C]O)eCH2eCH2e(C]O)e), 38.2 (eCH2eNHe),
45.9 (CareN(CH3)2), 52.9 (eN(CH3)2), 58.8 (eCH2eOe(C]O)e), 64.7
(e(CH3)2NþeCH2e), 65.1 (e(CH3)2eCH2e), 116.8 (CHar), 120.2
(CHar), 124.6 (CHar), 129.7 (CHar), 130.8 (CHar), 130.9 (Car), 131.4 (Car),
131.9 (CHar), 135.8 (Car), 153.6 (Car), 173.9 (eCH2e(C]O)eOe) ppm.
HRMS: calcd for C22H32N3O6S [MꢁBr]þ 466.2006; found 466.2006.
d¼0.79e0.90 (m, 6H, eCH2eCH3), 0.99e1.34 (m, 60H, eCH2e),
1.37e1.48 (m, 2H, eCH2e), 1.49e1.60 (m, 4H, eCH2e), 1.61e1.75 (m,
4H, eCH2e), 2.37 (t, J¼7.55 Hz, 2H, eCH2eCH2e(C]O)eOe),
2.67e2.98 (m, 6H, eCHeCH2eSeSe, eCH2eSeCHe), 3.04e3.16 (m,
1H, eCHeCHeCH2e), 3.37e3.46 (m, 2H, eCH2eCH2eOe),
3.47e3.57 (m, 4H, eCH2eCH2eOe, eCHeCH2eOe), 3.58e3.67 (m,
1H, eCHeCH2eOeCH2e), 3.98e4.23 (m, 3H, eCHeOH,
eCH2eOe(C]O)e), 4.24e4.33 (m, 1H, eCHeCHe), 4.43e4.54 (m,
1H, eCHeCHe), 5.91 (d, J¼10.01 Hz, 1H, NHe(C]O)eNH),
6.72e6.83 (m, 1H, NHe(C]O)eNH) ppm. 13C NMR (75.5 MHz,
4.1.22. 1-O-(2-Dansylamido-N,N-dimethylethanammonium bromid-
N-(ethyl-O-succinyl))-2-N-biotinyl-3-((2,3-bis(octadecyloxy)propyl)
disulfanyl)-2-aminopropanol 30. DCC (181 mg, 0.88 mmol) and
DMAP (37 mg, 0.30 mmol) were added to a solution of succinate 29
(46 mg, 0.084 mmol) and the biotin amide 27 (40 mg, 0.042 mmol) in
dichloromethane (1.1 ml). The mixture was stirred in a sealed tube at
40 ꢀC (2 bar) for 14 days. The solvent was distilled off and the re-
mainder purified by column chromatography (silica gel; dichloro-
methane/MeOH, 5:1; Rf¼0.2). The solid yellow product (34 mg) was
CDCl3):
d¼14.0 (eCH2eCH3), 22.6 (CH2eCH3), 24.7 (CH2), 26.0
(CH2), 28.0 (CH2), 28.2 (CH2), 29.3 (CH2), 29.4 (CH2), 29.6 (CH2), 29.6
(CH2), 30.0 (CH2), 31.8 (CH2), 33.6 (eCH2e(C]O)eOe), 40.5
(eCH2eSeSe), 41.1 (eCH2eSeSe), 42.1 (eCH2eSeCHe), 55.7
(eCH2eSeCHe), 60.2 (eCH2eCHeCHe), 61.7 (eCH2eCHeCHe),
66.8 (eCH2eOe), 68.0 (eCHeOH), 70.5 (eCH2eOe), 71.3
(eCH2eOe), 71.7 (eCH2eOe), 77.4 (eCHeOeCH2e), 164.2
(eNHe(C]O)eNHe), 173.7 (e(C]O)eOe) ppm. HRMS: calcd for
C52H101N2O6S3 (MþH)þ 945.6826; found 945.6816.
1
obtained in 55% yield as diastereomeric mixture. Mp 42e44 ꢀC. H
NMR (300 MHz, CDCl3):
d¼0.81e0.92 (m, 6H, eCH2eCH3), 1.01e1.37
(m, 60H, eCH2e), 1.44e1.73 (m, 10H, eCH2e, eCH2eCH2eOe),
1.71e1.93 (m, 4H, eCH2e(C]O)eOe), 2.45e2.59 (m, 2H, eCH2e(C]
O)eOe), 2.68e2.78 (m, 2H, eCH2eSeCHe), 2.80e2.88 (m, 8H,
CareN(CH3)2, eCH2eSe), 3.09e3.24 (m, 6H, eN(CH3)2), 3.28e3.53 (m,
10H, eCH2eSeCHe, eCH2eSeCHe, eSO2eNHeCH2e, eNHeCHe,
eCH2eOe(C]O)e), 3.53e3.63 (m, 2H, eCH2eOe), 3.76e3.95 (m, 5H,
eCH2eOe, eCHeOe, eCH2eN(CH3)2), 3.99e4.09 (m, 2H, eCH2eOe),
4.47e4.59 (m, 2H, eCHeCHe), 7.13 (d, J¼7.37 Hz, 1H, CHar), 7.41e7.64
(m, 2H, CHar), 8.07e8.20 (m,1H, CHar), 8.25e8.36(m,1H, CHar), 8.48 (d,
4.1.25. 5-N-Succinyl-5-aminofluorescein methyl ester 35. A sus-
pension of 5-aminofluorescein-methyl ester (292 mg, 0.81 mmol)
and succinic anhydride (350 mg, 3.5 mmol) in dry pyridine (9 ml)
was stirred under argon for 26 h. After cooling to rt toluene
(20 ml) was added and all solvents were distilled off under vac-
uum. The remainder was purified by column chromatography
(silica gel; dichloromethane/MeOH, gradient: 10:1ꢃ4:1; Rf¼0.3)
giving product 35 (205 mg) as yellow solid in 55% yield.
J¼8.50 Hz, 1H, CHar) ppm. 13C NMR (75.5 MHz, CDCl3):
d¼14.1
(eCH2eCH3), 22.6(eCH2eCH3), 24.9(CH2), 25.3 (CH2), 25.9(CH2), 26.0
(CH2), 29.6 (CH2), 30.7 (CH2), 31.9 (CH2), 32.2 (CH2), 37.7 (CH2), 40.0
(CareN(CH3)2), 40.3 (eNHeCHe), 45.4 (eN(CH3)2), 50.4
(eCHeSeCH2e), 52.5 (eCHeCHe), 54.6 (eCHeCHe), 57.8 (eCH2e),
63.5 (eCH2e), 64.0 (eCH2e), 70.6 (eCH2e), 71.7 (eCH2e), 77.2
(eCH2eCHeCH2e),115.4 (CHar),119.2 (CHar),123.2 (CHar),128.6 (CHar),
128.9 (CHar),129.4 (Car),129.8 (Car),130.5 (CHar),134.2 (Car),151.7 (Car),
170.0 (eCH2e(C]O)eOe), 172.4 (eCH2e(C]O)eNHe) ppm. HRMS:
calcd for C74H129N6O10S4 [Mꢁ2HꢁBr]ꢁ 1389.8653; found 1389.7842.
4.1.26. 1-O-Biotinyl-2-O-(5-aminofluorescein methyl ester-5-N-suc-
cinyl)-3-((2,3-bis(octadecyloxy)propyl)disulfanyl)propan-1,2-diol
36. TMSCl (6 ml, 0.05) was added to a suspension of succinate 35
(23 mg, 0.05 mmol) and DMAP (6 mg, 0.05 mmol) in dry pyridine
(1 ml). After stirring under argon at rt for 1 h DCC (26 mg,
0.13 mmol), DMAP (8 mg, 0.065 mmol), and THF (2 ml) were added.
The mixture was heated in a sealed tube at 70 ꢀC under argon for 4
days. After removing the solvent by a rotary evaporator the re-
mainder was purified by column chromatography (silica gel;
dichloromethane/MeOH, 10:1; Rf¼0.5) affording a diastereomeric
mixture of product 36 (36 mg) as yellow solid in 52% yield. Mp
4.1.23. 3-((2,3-Bis(octadecyloxy)propyl)disulfanyl)propan-1,2-diol
32. PTAD (64 mg, 0.36 mmol) was added to a solution of the di-
ether 3 (220 mg, 0.36 mmol) in dry dichloromethane (6 ml) and dry
MeCN (3 ml) under stirring at rt. The resulting red solution was
stirred under argon for 1.5 h and afterward at 55 ꢀC for about 2 h
until the mixture became colorless. A solution of 1-thioglycerol
(44 mg, 0.40 mmol) was added and the mixture was stirred at rt
for 20 h. The solvent was distilled off and the remainder purified by
column chromatography (silica gel; dichloromethane/MeOH, 10:1;
152e154 ꢀC. 1H NMR (300 MHz, CDCl3):
eCH2eCH3), 0.93e1.41 (m, 60H, eCH2e), 1.39e1.72 (m, 10H,
eCH2eCH2eOe, eCH2e), 1.92e2.47 (m, 2H, eCH2e(C]O)eOe),
2.57e3.17 (m, 14H, eCH2eSeSeCH2e, eCH2eSeCHe, e(C]O)e
d
¼0.86 (t, J¼6.12 Hz, 6H,

N. Brodersen et al. / Tetrahedron 67 (2011) 7763e7774
7773
CH2eCH2e(C]O)e, e(C]O)eOeCH3), 3.35e3.71 (m, 7H,
eCH2eOe, eCH2eOeCHe), 4.14e4.61 (m, 4H, eCHeCHe,
eCH2eOe(C]O)e), 5.60e5.72 (m, 1H, eCHeOe(C]O)e),
6.65e7.01 (m, 7H, CHar), 7.36e7.48 (m, 1H, CHar), 7.73 (d, J¼8.29 Hz,
28.9 (CH2), 29.1 (CH2), 31.4 (CH2), 59.5 (CH2-6), 61.2 (eCH2eOe),
68.4 (eCH2eOe), 68.5 (eCH2eOe), 69.8 (CH-4), 70.2 (eCH2eOe),
71.2 (eCH2eOe), 71.9 (CH-2), 73.1 (CH-3), 75.8 (CH-5), 75.9 (CH-5),
77.4 (eCH2eCHeCH2e), 102.8 (CH-1) ppm.
1H, CHar) ppm. 13C NMR (75.5 MHz, CDCl3/CD3COD, 6:1):
d
¼13.8
(eCH2eCH3), 22.4 (eCH2eCH3), 25.8 (CH2), 28.2 (CH2), 29.1 (CH2),
29.2 (CH2), 29.4 (CH2), 29.7 (CH2), 31.7 (CH2), 33.7 (eCH2e(C]O)e
Oe), 40.1 (eCH2eSeSe), 40.9 (eCH2eSeSe), 46.0 (eCH2eSeCHe),
52.4 (e(C]O)eOeCH3), 55.3 (eCH2eSeCHe), 59.9 (eCHeCHe),
61.8 (eCHeCHe), 63.3 (eCH2eOe), 64.9 (eCH2eOe), 70.3
(eCH2eOe), 71.1 (eCH2eOe), 71.6 (eCH2eOe), 71.8 (eCH2eOe),
77.2 (eCH2eOeCHe), 103.6 (CHar), 114.9 (Car), 121.6 (CHar), 121.6
(CHar), 121.7 (CHar), 128.8 (CHar), 130.0 (CHar), 130.2 (CHar), 130.9
(Car), 131.1 (CHar), 133.2 (Car), 134.3 (Car), 152.4 (Car), 157.1 (Car), 163.8
4.1.29. 6-O-Tosyl-b-1-O-(2,3-bis(octadecyloxy)propyl)-glucose
40. Tetrabutylammonium hydrosulfate (70 mg, 0.21 mmol) and
2 N aqueous NaOH (2 ml, 4 mmol) were added to a solution of
glucose derivative 39 (652 mg, 0.86 mmol) in CHCl3. After cooling
to 13 ꢀC tosyl chloride (210 mg, 1.12 mmol) was slowly added. After
24 h stirring at rt CHCl3 (30 ml) and water (50 ml) were added. The
organic layer was separated and washed with brine (2ꢂ50 ml). The
solvent was removed by a rotary evaporator and the remainder
purified by column chromatography (silica gel; dichloromethane/
MeOH, 20:1; Rf¼0.5). The product 40 (210 mg, 1:1 diastereomeric
mixture) was obtained as colorless solid in 27% yield. 1H NMR
(eNHe(C]O)eNHe),
163.9
(eNHe(C]O)eNHe),
164.4
(eCH2e(C]O)eOe), 172.7 (eCH2e(C]O)eOe), 172.9 (eCH2e(C]
O)eOe), 176.0 (eCH2e(C]O)eOe) ppm. HRMS: calcd for
C77H118N3O13S3 [MþH]þ 1388.7821; found 1388.7485.
(300 MHz, CDCl3):
d
¼0.81e0.91 (m, 6H, eCH2eCH3), 1.14e1.36 (m,
60H, eCH2e), 1.45e1.60 (m, 4H, eCH2eCH2eOe), 2.43 (s, 3H,
CareCH3), 3.34e3.62 (m, 10H, CHe4, eCH2eCHeCH2e, eCH2eOe),
3.72e3.92 (m, 2H, CHe2, CHe3), 4.11e4.22 (m, 3H, CH2-6, CH-5),
4.37e4.46 (m, 1H, CH-1), 7.33 (d, J¼7.93 Hz, 2H, CHar), 7.78 (d,
4.1.27. 2,3,4,6-O-Tetraacetyl-b-1-O-(2,3-bis(octadecyloxy)propyl)-
ꢁ
glucose 38. Molecular sieve (4 A) (3.3 g) was added to a solution of
glycerol-1,2-dioctadecylether (37) (2.96 mg, 5.0 mmol) in dry toluene
(60 ml). Ag2O (2.55 g, 11.0 mmol), Ag2CO3 (1.38 g, 5.0 mmol), iodine
J¼8.31 Hz, 2H, CHar) ppm. 13C NMR (75.5 MHz, CDCl3):
d¼14.0
(eCH2eCH3), 21.6 (CareCH3), 22.6 (eCH2eCH3), 25.9 (CH2), 26.0
(CH2), 29.3 (CH2), 29.4 (CH2), 29.6 (CH2), 29.6 (CH2), 29.9 (CH2), 31.8
(CH2), 66.2 (CH-5), 66.4 (CH-5), 68.4 (-CH2eOe), 68.5 (CH-4), 68.7
(CH-2), 68.7 (eCH2eOe), 69.4 (eCH2eOe), 69.8 (eCH2eOe), 70.2
(eCH2eOe), 70.4 (eCH2eOe), 70.5 (eCH2eOe), 71.0 (CH-3), 71.1
(CH-3), 71.7 (eCH2eOe), 77.4 (eCH2eCHeCH2e), 103.6 (CH-1),
127.9 (CHar), 129.9 (CHar), 132.4 (Car), 145.1 (Car) ppm.
(1.27 g, 5.0 mmol), and 1-bromo-tetraacetyl-D-glucose (4.11 g,
10.0 mmol) were added under stirring and the resulting violet mix-
ture was heated to 50 ꢀC for 24 h. The black precipitate was filtered off
and the slightly yellow filtrate was concentrated by a rotary evapo-
rator. The remainder was purified by column chromatography (silica
gel; dichloromethane/EtOAc/cyclohexane, 8:1:7; Rf¼0.4) giving rise
tothecolorlesssolid product (2.81 g) as1:1 diastereomeric mixture in
61% yield. Mp 57e60 ꢀC.1H NMR (300 MHz, CDCl3): d¼0.80e0.92 (m,
6H, eCH2eCH3), 1.12e1.37 (m, 60H, eCH2e), 1.44e1.58 (m, 4H,
eCH2eCH2eOe),1.99 (s, 3H, CH3e(C]O)eOe), 2.01 (s, 3H, CH3e(C]
O)eOe), 2.02 (s, 1.5H, CH3e(C]O)eOe), 2.03 (s, 1H, CH3e(C]O)e
Oe), 2.07 (s, 3H, CH3e(C]O)eOe), 3.34e3.73 (m, 9H, eCH2eOe, CH-
5, eCH2eCHeCH2e, eOeCH2e), 3.81e3.96 (m, 1H, eOeCH2e),
4.06e4.31 (m, 2H, CH2-6), 4.50e4.62 (m,1H, CH-1), 4.93e5.25 (m, 3H,
CH-2, CH-3, CH-4) ppm. 13C NMR (75.5 MHz, CDCl3): d¼14.1
(eCH2eCH3), 20.5 (CH3e(C]O)eOe), 20.6 (CH3e(C]O)eOe), 20.6
(CH3e(C]O)eOe), 20.7 (CH3e(C]O)eOe), 22.6 (eCH2eCH3), 26.1
(CH2), 26.1 (CH2), 29.3 (CH2), 29.5 (CH2), 29.7 (CH2), 30.0 (CH2), 30.1
(CH2), 31.9 (CH2), 61.9 (CH2-6), 68.4 (CH-4), 69.0 (eCH2eOe), 70.1
(eCH2eOe), 70.3 (eCH2eOe), 70.5 (eCH2eOe), 70.9 (eCH2eOe),
71.3 (CH-2), 71.3 (CH-2), 71.7 (CH-3), 71.7 (eCH2eOe), 71.7 (CH-3),
72.8 (CH-5), 72.9 (CH-5), 77.4 (eCH2eCHeCH2e), 77.9
(eCH2eCHeCH2e), 101.0 (CH-1), 101.0 (CH-1), 169.2 (CH3e(C]O)e
Oe), 169.2 (CH3e(C]O)eOe), 169.4 (CH3e(C]O)eOe), 170.2
(CH3e(C]O)eOe), 170.6 (CH3e(C]O)eOe) ppm.
4.1.30. 6-Desoxy-6-amino-b-1-O-(2,3-bis(octadecyloxy)propyl)-glu-
cose 41. Liquid ammonia (100 ml) and a solution of tosylate 40
(162 mg, 0.18 mmol) in CHCl3 (10 ml) were stirred in an autoclave
at 25 ꢀC for 20 h. The ammonia was allowed to evaporate and the
remainder was washed out of the autoclave by CHCl3 (100 ml).
After filtration the filtrate was dried over MgSO4 and the solvent
removed by a rotary evaporator. The remainder was purified by
column chromatography (silica gel; dichloromethane/MeOH, 5:1;
Rf¼0.3) affording a diastereomeric mixture of product 41 (70 mg) as
colorless wax in 51% yield. 1H NMR (300 MHz, CDCl3):
d¼0.84e0.95
(m, 6H, eCH2eCH3), 1.02e1.41 (m, 60H, eCH2e), 1.51e1.67 (m, 4H,
eCH2eCH2eOe), 2.69e2.90 (m, 2H, eCH2eNH2), 2.91e3.18 (m, 2H,
eCH2eNH2), 3.29e3.37 (m, 1H, CH-4), 3.39e3.69 (m, 9H,
eCH2eOe, CH-2, CH-3, eCH2eCHeCH2e,), 3.80e4.08 (m, 2H,
eCH2eOe), 4.10e4.28 (m, 1H, CH-5), 4.43e4.54 (m, 1H, CH-1) ppm.
13C NMR (75.5 MHz, CDCl3):
d
¼14.1 (eCH2eCH3), 22.7 (eCH2eCH3),
26.0 (CH2), 26.1 (CH2), 29.3 (CH2), 29.5 (CH2), 29.6 (CH2), 29.6 (CH2),
29.7 (CH2), 29.9 (CH2), 30.0 (CH2), 31.9 (eCH2eCH2eCH3), 44.3
(eCH2eNH2), 55.5 (CH), 55.5 (CH), 56.6 (CH), 69.5 (CH), 69.6 (CH),
69.8 (eCH2eOe), 70.1 (eCH2eOe), 70.3 (eCH2eOe), 70.4
(eCH2eOe), 70.5 (eCH2eOe), 70.5 (eCH2eOe), 71.8 (eCH2eOe),
77.5 (eCH2eCHeCH2e), 100.9 (CH-1), 101.2 (CH-1)ppm.
4.1.28.
b
-1-O-(2,3-Bis(octadecyloxy)propyl)-glucose
39. NaOMe
(0.5 N) in MeOH (2 ml, 1 mmol) was slowly added to a solution of
the tetraacetyl derivative 38 (2.81 g, 3.0 mmol) in CHCl3 (10 ml) and
MeOH (10 ml) drop wise under stirring. After 24 h at rt the orange
mixture was neutralized by ion exchange (DOWEX 50ꢂ 400). the
solvent was removed by a rotary evaporator and the residue was
dissolved in CHCl3 (300 ml). After drying over MgSO4 the solvent
was removed by a rotary evaporator. The residue was recrystallized
from affording product 39 (1.41 g, 1:1 diastereomeric mixture) as
voluminous colorless solid in 62% yield; Rf¼0.5 (dichloromethane/
4.1.31. 6-Desoxy-6-N-biotinyl-b-1-O-(2,3-bis(octadecyloxy)propyl)-
glucose 42. EDC (20 ml, 0.11 mmol), HOBt (20 mg, 0.13 mmol), and
DIPEA (31 ml, 0.18 mmol) were added to a solution of the amine 41
(70 mg, 0.09 mmol) and (þ)-biotin (20 mg, 0.08 mmol) in dry DMF
(1 ml) and dry dichloromethane (1 ml). The mixture was stirred at rt
for 24 h and then combined with toluene (5 ml). Solvents were dis-
tilled off, the remainder was kept under vacuum (5 mbar, 40 ꢀC) and
purified by column chromatography (silica gel; dichloromethane/
MeOH, 7:1; Rf¼0.6). The colorless waxy product 42 (52 mg) was
obtained as diastereomeric mixture in 61% yield. Mp 50e52 ꢀC. 1H
MeOH, 5:1). Mp 57e60 ꢀC. 1H NMR (300 MHz, CDCl3):
d
¼0.88 (t,
J¼6.61 Hz, 6H, eCH2eCH3), 1.04e1.38 (m, 60H, eCH2e), 1.46e1.68
(m, 4H, eCH2eCH2eOe), 3.09e3.35 (m, 2H, eCH2eOe), 3.36e3.71
(m, 9H, CHe2, CHe3, CHe4, CHe5, eCH2eCHeCH2e), 3.76e3.88
(m, 2H, eCH2eOe,), 3.89e4.22 (m, 2H, CH2-6), 4.24e4.48 (m, 1H,
NMR (300 MHz, CDCl3):
(m, 60H, eCH2e), 1.36e1.80 (m, 10H, eCH2e, eCH2eCH2eOe),
2.16e2.31 (m, 2H, eCH2e(C]O)eNHe), 2.66e2.93 (m, 2H,
d
¼0.83e0.91 (m, 6H, eCH2eCH3),1.00e1.33
CH-1) ppm. 13C NMR (75.5 MHz, CDCl3/CD3OD, 4:3):
d
¼13.3
(eCH2eCH3), 22.1 (eCH2eCH3), 25.4 (CH2), 25.5 (CH2), 28.8 (CH2),

7774
N. Brodersen et al. / Tetrahedron 67 (2011) 7763e7774
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eCH2eSeCHe), 3.09e3.16 (m, 1H, eCH2eSeCHe), 3.32e3.72 (m,
12H, eCH2eOe, eCH2eCHeCH2e, eCHe, CH2-6), 3.76e3.81 (m, 1H,
CH-3), 3.88e3.95 (m, 1H, CH-5), 4.08e4.13 (m, 1H, CH-2), 4.28e4.34
(m, 1H, eCHeCHe), 4.39e4.57 (m, 2H, -CHeCHe, CH-1) ppm. 13C
NMR (75.5 MHz, CDCl3):
d¼14.1 (eCH2eCH3), 22.7 (eCH2eCH3), 25.7
(CH2), 26.0 (CH2), 26.1 (CH2), 28.0 (CH2), 28.2 (CH2), 29.3 (CH2), 29.5
(CH2), 29.7 (CH2), 29.8 (CH2), 29.9 (CH2), 29.9 (CH2), 30.0 (CH2), 31.9
(CH2), 36.0 (eCH2e(C]O)eOe), 40.5 (eCH2eSeCHe), 41.1
(eCH2eNHe(C]O)e), 55.6 (eCH2eSeCHe), 60.2 (eCHeCHe), 61.7
(eCHeCHe), 69.4 (CH-5), 69.5 (eCHe), 70.0 (eCH2eOe), 70.1
(eCH2eOe), 70.4 (eCH2eOe), 70.5 (eCH2eOe), 70.5 (eCH2eOe),
70.9 (eCH2eOe), 71.8 (eCH2eOe), 71.9 (eCH2eOe), 73.3 (eCHe),
73.3 (eCHe), 77.4(eCH2eCHeCH2e),101.1 (CH-1),164.2 (eNHe(C]
O)eNHe), 173.6 (eCH2e(C]O)eNHe) ppm. HRMS: calcd for
C55H106N3O9S [MþH]þ 984.7644; found 984.7646.
12. Kurz, A.; Bunge, A.; Windeck, A. K.; Rost, M.; Flasche, W.; Arbuzova, A.;
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Acknowledgements
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Arbuzova, A.; Liebscher, J. Eur. J. Org. Chem. 2008, 1917.
15. Bunge, A.; Loew, M.; Pescador, P.; Arbuzova, A.; Brodersen, N.; Kang, J.; Dahne,
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Liebscher, J.; Herrmann, A.; Huster, D.; Arbuzova, A. Chem. Commun. 2010, 5358.
17. Brodersen, N. Thesis, Humboldt-University Berlin, Germany 2008.
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19. Witt, D. Synthesis-Stuttgart 2008, 2491.
Financial support by Bayer-Technology Services GmbH is
gratefully acknowledged. We thank Bayer Technology Services
GmbH, BASF AG and Sasol GmbH for donation of chemicals.
Supplementary data
20. Shirude, P. S.; Kumar, V. A.; Ganesh, K. N. Eur. J. Org. Chem. 2005, 5207.
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Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2011.07.089. These data in-
clude MOL files and InChIKeys of the most important compounds
described in this article.
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