Light fluorous synthesis of glucosylated glycerol teichoic acids

Article abstract of DOI:10.1016/j.carres.2012.02.023

We here describe the synthesis of glucosylated teichoic acid (TA) fragments using two complementary fluorous scaffolds. The use of a perfluorooctylpropylsulfonylethyl (F-Pse) linker in combination with (glucosyl)glycerol phosphoramidite building blocks al

Full text of DOI:10.1016/j.carres.2012.02.023

Carbohydrate Research 356 (2012) 142–151
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Light fluorous synthesis of glucosylated glycerol teichoic acids
W.F.J. Hogendorf a, A. Kropec b, D.V. Filippov a, H.S. Overkleeft a, J. Huebner b, G.A. van der Marel a,
,
⇑
J.D.C. Codée a,
⇑
a Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
b Division of Infectious Diseases, Department of Medicine, University Medical Center Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 January 2012
Accepted 24 February 2012
Available online 6 March 2012
Keywords:
Teichoic acid
Fluorous
Glycerol
Glucose
Phosphate
Phosphoramidite
We here describe the synthesis of glucosylated teichoic acid (TA) fragments using two complementary
fluorous scaffolds. The use of a perfluorooctylpropylsulfonylethyl (F-Pse) linker in combination with
(glucosyl)glycerol phosphoramidite building blocks allows for the assembly of TA fragments with a ter-
minal phosphate mono-ester, whereas the use of a perfluorooctylsuccinyl spacer delivers TA oligomers
featuring a terminal alcohol functionality. These complementary linker systems have been developed
because the nature of the TA chain terminus can play a role in the biological activity of the synthetic
TAs. A novel
a-glucosylated glycerolphosphoramidite building block is introduced to allow for a robust
light fluorous synthetic protocol.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
permeability and their task as scaffold for various extracytoplasmic
enzymes.1 TAs also mediate extracellular interactions and are rec-
ognized by the mammalian adaptive and innate immune system,
thus functioning as antigenic structures and immunostimulatory
ligands for Toll-like receptors, respectively.2 To establish struc-
ture–activity relationships of teichoic acids we started a pro-
gramme aimed at the development of synthetic strategies to
generate well-defined TA structures to help elucidate their mode
of action at the molecular level.3
Enterococcus faecalis is a commensal, Gram-positive bacterium,
responsible for many hospital related infections and represents a
significant health threat especially to immunocompromised pa-
tients. The advent of strains resistant against multiple antibiotics
is a strong stimulus for the development of alternative prophylac-
tic and therapeutic strategies.4 E. faecalis LTA has been shown to be
a target of protective antibodies, and as such it represents a poten-
tial candidate for future vaccine development.5 To identify potent
The cell walls of pathogenic and non-pathogenic Gram-positive
bacteria are characterized by a thick peptidoglycan layer, in which
polyanionic glycopolymers are present. An important class of these
cell surface polymers is comprised by the teichoic acids (TAs),
which can be divided in wall teichoic acids (WTAs), covalently
linked to the peptidoglycan layer and lipoteichoic acids (LTAs),
which are anchored in the bacterial membrane. The repeating units
of both types of TA are generally alditol (glycerol, ribitol) phos-
phates of which the hydroxyl functions are randomly equipped
with carbohydrate or
functions as exemplified by their role in membrane integrity and
D-alanine residues. TAs perform vital
⇑
Corresponding authors.
E-mail addresses: marel_g@chem.leidenuniv.nl (G.A. van der Marel), jcodee@
chem.leidenuniv.nl (J.D.C. Codée).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2012.02.023

W. F. J. Hogendorf et al. / Carbohydrate Research 356 (2012) 142–151
143
group and concomitantly serve as a fluorous linker in the solution
phase synthesis of TA fragments.3c,8g Therefore we initially ex-
plored the synthesis of hexamer 10, a phosphorylated analogue of
TA-fragment 1, using this linker-system. Thus, F-Pse 3 was elon-
gated in a stepwise manner (Scheme 1) with glycerol phosphorami-
dite 43a in a four-step elongation process, in which: (1) The
phosphoramidite was coupled with the alcohol using dicyanoimi-
dazole (DCI) as an activator in acetonitrile. (2) The resulting phos-
phite intermediate was oxidized using I2 in a mixture of THF/H2O/
pyridine. (3) From the elongated fragment the 4,40-dimethoxytrityl
(DMT) ether was cleaved using dichloroacetic acid (DCA) and tri-
ethylsilane (TES) in DCM. (4) Fluorous solid phase extraction (F-
SPE) was employed to separate the fluorous products from the
non-fluorous side products. Before the F-SPE purification the mix-
ture was partitioned between acetonitrile/water (80/20) and hex-
ane to remove the bulk of TES and DMT-H to simplify the F-SPE
purification, as described previously.3c Repeating this process four
times led to pentamer 5 which was then elongated with benzyli-
dene protected glucosylglycerol phosphoramidite 6 under the
agency of DCI and oxidized. As the 4,6-O-benzylidene moiety is
unstable towards DCA/TES, detritylation of the intermediate hexa-
mer was effected using the milder PPTS/MeOH cocktail.3a,9 The
presence of the lipophilic carbohydrate moiety did not influence
the F-SPE purification and the target compound was obtained
uneventfully in 74% yield. At this stage an aminospacer was intro-
duced to allow the conjugation of the target structure to, for in-
O
P O
ONa
O
P O
O
H3N
1
2
O
HO
HO
O
OH
O
5
HO
HO
HO
HO
O
O
P
O
H3N
O
P O
ONa
O
O
O
O
5
HO
HO
HO
HO
O
Figure 1. Lead structures 1 and 2.
antigenic fragments of the microheterogenic E. faecalis TA we
developed an automated solid phase synthesis approach, in which
well-defined TA fragments were assembled on a commercially
available DNA synthesizer using suitably protected glycerol phos-
phoramidite building blocks.3b Through this methodology we
generated a small library of TA fragments, which allowed us to
identify two potent antigenic TA structures in an opsonophagocytic
killing inhibition assay (OPIA). In this assay compounds are
screened for their ability to inhibit the complement-assisted killing
of bacteria though opsonic antibodies.5 These two compounds, 1
stance,
a carrier protein. Condensation of hexamer 7 and
phosphoramidite 8 was followed by oxidation and F-SPE to give
the fully protected construct 9 in 86% yield. Deprotection of hexa-
mer 9 started by removal of the cyanoethyl (CE) and F-Pse groups
by overnight treatment with aqueous ammonia at 40 °C. The
semi-protected intermediate was separated from the eliminated
and 2 (Fig. 1), are characterized by the presence of an
a-glucosyl
substituent on either of the two terminal glycerolphosphate resi-
dues. Notably, this type of substitution is common in Bacillus sub-
tilis1c,6 and several Staphylococcus species1c,7 but has not been
encountered in native E. faecalis LTA. To further investigate these
antigenic lead compounds we required an amount of these struc-
tures, larger than automated solid phase synthesis could provide
us with and therefore we set out to develop a complementary syn-
thesis strategy based on the use of ‘light fluorous’ chemistry.3c,8
fluorous scaffold (perfluorooctylpropylsulfonylethene) using
a
Et2O/H2O extraction. Subsequently, the benzylidene moiety, benzyl
ethers and benzyl carbamate function were all removed by means
of hydrogenolysis (Pd/H2), leading to the target hexamer 10 in
98% yield. Surprisingly, in sharp contrast to its close analogue 1,
hexamer 10 proved to be inactive in the OPIA inhibition assay, indi-
cating that the presence of the terminal phosphate is detrimental to
the antigenicity of the synthetic TA fragment.10
2. Results and discussion
To allow the light-fluorous assembly of TA fragments without a
terminal phosphate moiety we set out to probe a hydroxyl
We have recently disclosed that perfluorooctylpropylsulfony-
lethanol (F-Pse) can be used effectively as a phosphate protecting
O
BnO
Ph
O
O
1)
BnO
iPr2N
CEO
6
BnO
P O
ODMT
1)
O
iPr2N
Ph
O
BnO
4
O
O
O
BnO
P
O
ODMT
DCI, ACN
O O
S
CEO
DCI, ACN
F-PseO P O
OCE
OH
BnO
F17C8
O
BnO
O
O
OH 2) I2, H2O, pyridine, THF
3) DCA, TES, DCM
4) F-SPE
5
OH
2) I2, H2O, py ridine, T HF
3) PPTS,MeOH
4) F-SPE
F-PseO P
O
O
P O
OCE
3 (F-Pse-OH)
OCE
5
5) repeat steps 1-4 4x
5
7 (74 %)
CbzHN
1) 8, DCI, ACN
2) I2, H2O, pyridine, THF
8
iPr2N
P O
CEO
3) F-SPE
Ph
O
O
O
HO
HO
O
BnO
HO
CbzHN
BnO
NH3
O
O P O
O
O
HO
1) 25% NH4OH,40 oC
2) H2, Pd(0), H2O
O
O
O
BnO
O
O
O
NaO P O
ONa
BnO
O P
O
O
P O
OCE
F-PseO P
O
O
P O
ONa
OCE
OCE
5
9 (86 %)
5
10 (98 %)
Scheme 1. Assembly of TA-fragment 10.

144
W. F. J. Hogendorf et al. / Carbohydrate Research 356 (2012) 142–151
HO
12
OAllyl
R1O
BnO
BnO
RO
BnO
OTBDPS
TMSOTf, Et2O/DCM (4/1), 0 oC
RO
BnO
BnO
O
O
O
BnO
1. Ir (COD)(PPhMe2)2PF6,
THF
R2
BnO
BnO
or
BnO
O
O
TfOH, Et2O, 0 oC
OAllyl
DBU, DCM
OAllyl
2. NaHCO3 (aq), I2
OTBDPS
OTBDPS
11a R1 = Bn, R2 = α/β OC(=NH)CCl3
13a R = Bn (83 %, α/β = 4/1)
11b R1 = FMOC, R2 = α/β OC(=NPh)CF3
14 R = H (94 %)
15 R = Bn (87 %)
13b R = Fmoc (91%, α/β = >9/1)
BnBr, NaH, DMF
BnO
BnO
BnO
BnO
BnO
O
O
BnO
BnO
O
BnO
BnO
1, H2N(CH2)3C8F17, BOP,
BnO
BnO
O
BnO
O
DIPEA, DMF, DCM.
O
succinic anhydride,
Et3N, DCM
OH
ODMTr
OR1
OR2
2. DCA, TES, DCM.
O
O
3. F-SPE
O
O
16 R1 = H, R2 = TBDPS (87 %)
17 R1 = DMT, R2 = H (89 %)
1. DMT-Cl, Et3N,
DCM
O
O
2. TBAF, THF
20 (90 %)
19 (96 %)
HN
OH
( Pr N)P(OCE)Cl,
i
2
Et3N, DCM
BnO
BnO
O
C8F17
BnO
BnO
O
ODMTr
O
P
18 (74 %)
iPr2N
OCE
Scheme 2. Synthesis of phosphoramidite 18 and perfluorooctylpropyl succinyl linked glycosyl glycerol 20.
protecting fluorous linker. Inspired by contemporary DNA synthesis
methods, a succinyl type linker was deemed suitable because of its
stability towards coupling, oxidation and detritylation conditions.11
The base lability of a fluorous succinyl linker allows the same depro-
tection strategy as employed in the synthesis of hexamer 10. At the
same time we were interested in developing a more acid-stable glu-
cosyl glycerol synthon. As described above, the benzylidene acetal
is sensitive to standard detritylation conditions, necessitating the
use of a carefully controlled procedure in the removal of the tempo-
rary DMT group. Introduction of the benzylidene glucosyl glycerol
synthon early on in the synthesis therefore significantly affects
the robustness of the assembly process. We therefore implemented
tetra-O-benzyl glucosyl synthon 18 in our subsequent syntheses (
Schemes 2 and 3). This building block was prepared as described
in Scheme 2. We first explored the use of per benzylated glucosyl
natively, 17 was reacted with succinic anhydride and Et3N in
DCM, giving succinyl ester 19 in 96% yield. Subsequently, 19 was
coupled to perfluorooctylpropylamine, using BOP as a coupling
agent and detritylated to give the crude fluorous glucosylglycerol
20, which was purified by F-SPE to give the pure target compound
in 90% yield. This molecule was elongated in a step-wise manner
with glycerol phosphoramidite 5 using the chemistry described
above leading to hexamer 25. The aminohexylspacer was then
introduced to give the fully protected hexamer 26. Deprotection
by 25% aqueous ammonia (1 h, rt), was followed by hydrogenolysis
to give 40 mg of target compound 2 (92%).
To broaden the palette of TA fragments, and further explore the
effectiveness of the light fluorous chemistry, we continued with
the assembly of hexamer 29, containing two glucosyl moieties
(Scheme 4). Pentamer 24 was coupled to glucosylglycerol phos-
phoramidite 18, resulting in bis-glucosylated hexamer 27 in 87%.
Also this compound was uneventfully purified by F-SPE. After
introduction of the spacer, the resulting hexamer 28 was deprotec-
ted using the aforementioned conditions, to yield the bis-glucosyl
TA fragment 29 in 96% yield.
imidate 11a12 for the construction of the crucial
Condensation of this donor with glycerol acceptor 12 in DCM led to
formation of product 13a with poor selectivity ( /b = 2: 1). The use
of ether as co-solvent improved the /b-ratio, but the anomeric
a-glucosyl linkage.
a
a
mixture proved to be inseparable. To circumvent this problem, we
explored the use of a glucosyl donor bearing a bulky Fmoc protect-
ing group on the C6 hydroxyl, as these are known to favour the for-
3. Conclusion
mation of the
with glycerol 12 using Et2O as a solvent, led to the formation of 13b
in high selectivity (
/b ꢀ 10/1). The major amount of b-glucoside
a
-product.13 Coupling 6-O-Fmoc glucosyl imidate 11b
We have described the development of two complementary flu-
orous linker systems for the assembly of TA fragments. The first lin-
ker, perfluorooctylpropylsulfonylethyl, is used as a phosphate
protecting group and allows the assembly of TA fragments featuring
a terminal phosphate monoester. The second linker, a perfluorooc-
tylpropyl succinyl system, is used as a hydroxyl protecting function-
ality and leads to the formation of TA structures terminating in an
alcohol functionality. Light fluorous chemistry is an efficient means
for the assembly of TA fragments and allows the construction of
pure TA oligomers in multi milligram quantities, sufficient for most
initial biochemical studies. Full immunochemical evaluation of the
compounds is ongoing and will be reported in due course.
a
was removed by column chromatography, affording glucoside 13b
in 91% yield (containing <3% b-adduct, based on 1H NMR analysis).
Compound 13b was then treated with DBU in DCM, and benzylation
of the intermediate alcohol 14 led to tetrabenzylglucosyl derivative
15 in 87% yield. In the next step the allyl ether was removed by irid-
ium catalysed isomerization, followed by oxidative cleavage of the
intermediate enol ether, giving alcohol 16 in 87% yield. Installation
of the DMT ether and desilylation led to building block 17, which
was transformed into the phosphoramidite synthon 18 using N,N-
diisopropyl-2-cyanoethyl-chlorophosphoramidite and Et3N. Alter-

W. F. J. Hogendorf et al. / Carbohydrate Research 356 (2012) 142–151
145
24
20
1 .4, DCI, ACN
1. 18, DCI, ACN
2. I2, H2O, pyridine, THF
3. DCA, TES, DCM
4. F-SPE
2. I2, H2O, pyridine, THF
3. DCA, TES, DCM
4. F-SPE
O
O
H
N
O
P O
OCE
H
N
O
P
OCE
O
P
OCE
F17C8
F17C8
O
O
OH
O
O
O
O
O
OH
O
O
OBn
O
O
OBn
O
n
4
BnO
BnO
BnO
BnO
BnO
BnO
21 n = 1 (92 %)
22 n = 2 (90 %)
23 n = 3 (92 %)
24 n = 4 (93 %)
25 n = 5 (90 %)
BnO
BnO
BnO
BnO
BnO
BnO
O
O
O
27 (87 %)
1. 8, DCI, ACN
2. I2, H2O, pyridine, THF
3. F-SPE
O
O
H
1. 8, DCI, ACN
N
C8F17
O
2. I2, H2O, pyridine, THF
3. F-SPE
ZHN
O
O
O
O
P
OCE
O
P
O
OCE
O
P
O
O
O
O
H
N
O
O CbzHN
OBn
OCE
O
F17C8
O
O
P O
OCE
O
P O
OCE
4
BnO
BnO
O
O
OBn
BnO
BnO
BnO
BnO
BnO
BnO
5
O
O
28 (89 %)
BnO
26 (89 %)
BnO
BnO
1. 25 % NH4OH, RT, 1h
2. H2, Pd(0), H2O
O
1. 25 % NH4OH, RT, 1h
2. H2, Pd(0), H2O
BnO
O
P
ONa
O
P
ONa
O
P
O
H3N
O
P O
ONa
O
P O
O
H3N
HO
O
O
O
O
O
O
O
HO
O
O
OH
O
O
OH
4
HO
HO
5
HO
HO
HO
HO
HO
HO
HO
O
O
29 (96 %)
HO
HO
2 (92 %)
O
HO
Scheme 4. Light fluorous assembly of 29.
Scheme 3. Light fluorous assembly of 2.
NMR spectra were recorded with chemical shift relative to TMS
(external standard). High resolution mass spectra (HRMS) were
recorded by direct injection (2 ll of a 2 lM solution in water/ace-
4. Experimental section
4.1. General
tonitrile; 50/50; v/v and either 0.1% formic acid or 10 mM ammo-
nium formate for the oligomers) on a mass spectrometer (Thermo
Finnigan LTQ Orbitrap) equipped with an electrospray ion source
in positive mode (source voltage 3.5 kV, sheath gas flow 10,
capillary temperature 275 °C) with resolution R = 60,000 at m/z
400 (mass range m/z = 150–2000) and dioctylphthalate (m/z =
391.28428) as a lock mass. The high resolution mass spectrometer
was calibrated prior to measurements with a calibration mixture
(Thermo Finnigan).
All chemicals (Acros, Fluka, Merck, Schleicher & Schuell, Sigma–
Aldrich, Genscript) were used as received and reactions were car-
ried out dry, under an argon atmosphere, at ambient temperature,
unless stated otherwise. Column chromatography was performed
on Screening Devices silica gel 60 (0.040–0.063 mm). TLC analysis
was conducted on HPTLC aluminium sheets (Merck, silica gel 60,
F245). Compounds were visualized by UV absorption (245 nm),
by spraying with 20% H2SO4 in ethanol or with a solution of
(NH4)6Mo7O24ꢁ4H2O 25 g/l and (NH4)4Ce(SO4)4ꢁ2H2O 10 g/l, in
10% aqueous H2SO4 followed by charring at +/ꢂ 140 °C. Some
unsaturated compounds were visualized by spraying with a solu-
tion of KMnO4 (2%) and K2CO3 (1%) in water. Optical rotation mea-
4.1.1. General procedure for phosphoramidite coupling,
oxidation, detritylation and F-SPE
Starting alcohol was dissolved in ACN (0.1 M). DCI (0.25 M solu-
tion in CH3CN, 2 equiv compared to phosphoramidite) was added,
together with freshly activated MS 3 Å and the mixture was stirred
under argon for 15 min. Phosphoramidite (0.175 M in ACN, 1.3–
4.0 equiv) was added and the reaction was stirred until TLC analy-
sis revealed full conversion of the starting material into a higher
running spot (ꢀ1 h). Added were, respectively, H2O (ꢀ1 ml) and
I2 (0.2 M in THF/pyr 4/1), and the mixture was stirred for an addi-
tional 5 min. The mixture was diluted with EtOAc (ꢀ50 ml) and
washed with, respectively, satd aq Na2S2O3 (ꢀ20 ml), 0.5 M KHSO4
(ꢀ20 ml) and a 1/1 mixture of satd aq NaHCO3 and brine (ꢀ20 ml).
The organic layer was dried over Na2SO4 (s) and concentrated
under reduced pressure. The residue was coevaporated once with
surements (½a 2D0
ꢃ
) were performed on a Propol automated
polarimeter (Sodium D-line, k = 589 nm) with a concentration of
10 mg/ml (c = 1), unless stated otherwise. Infrared spectra were re-
corded on a Shimadzu FT-IR 8300. 31P, 1H and 13C NMR spectra
were recorded with a Bruker AV 400 (161.7, 400 and 125 MHz,
respectively) or a Bruker DMX 600 (600 and 150 MHz, respec-
tively). NMR spectra were recorded in CDCl3 with a chemical shift
(d) relative to tetramethylsilane, unless stated otherwise. When
D2O was used, 1H NMR spectra were recorded with chemical shift
relative (d) to HDO (4.755 ppm), 31P spectra were measured with
chemical shift relative to 85% H3PO4 (external standard) and 13C

146
W. F. J. Hogendorf et al. / Carbohydrate Research 356 (2012) 142–151
toluene (10 ml) before it was redissolved in DCM. Triethylsilane
and dichloroacetic acid were added and the mixture was stirred
until the bright orange colour fully disappeared (ꢀ30 min). DCM
(ꢀ40 ml) was added and the organic layer was washed with a 1/
1 mixture of satd aq NaHCO3 and brine (ꢀ20 ml, check if pH >7),
before it was dried over Na2SO4 and concentrated in vacuo. The
residue was taken up in 4/1 ACN/H2O (10 ml) and washed with
hexane (50 ml). The hexane layer was extracted twice with 4/1
ACN/H2O (2 ꢄ 10 ml) and the combined ACN/H2O layers were con-
centrated under reduced pressure in a 100 ml pear shaped flask.
The residue was taken up in 0.5 ml ACN and applied to a small col-
umn containing FluoroFlashÒ fluorous silica (4 g) which was pree-
luted with 1/1 ACN/H2O. The column was eluted with 1/1 ACN/
H2O until all the non-fluorous byproducts (DMTr, phosphates,
DCI) were removed. Subsequently the fluorous product was eluted
from the column with, respectively, CH3CN and acetone.
4.87 (d, 1H, J = 11.6 Hz, CHH Bn), 4.91 (d, 1H, J = 3.7 Hz, H-1), 4.95
(d, 1H, J = 11.3 Hz, CHH Bn), 5.55 (s, 1H, CH benzylidene), 7.25–
7.40 (m, 38H,
H
arom), 7.44–7.48 (m, 2H, Harom); 13C NMR
(100 MHz): d = 13.3 (F17C8CH2CH2CH2SO2–), 19.2–19.5 (12 ꢄ CH2
cyanoethyl), 29.2 (t, J = 22 Hz, F17C8CH2CH2CH2SO2–), 53.0, 53.1
(F17C8CH2CH2CH2SO2–, –OCH2CH2SO2–), 60.9 (CH2 glycerol), 61.3
(–OCH2CH2SO2–), 62.0–62.4 (6 ꢄ CH2 cyanoethyl), 62.8 (C-5),
65.4–66.0 (10 ꢄ CH2 glycerol), 67.2 (CH2 glycerol), 68.7 (C-6),
72.0–72.1 (5 ꢄ CH2 Bn), 74.3 (CH2 Bn), 75.0 (CH2 Bn), 75.2–75.4
(5 ꢄ CH glycerol), 78.6–78.8 (C-2, C-3, CH glycerol), 82.0 (C-4),
98.6, 98.9 (C-1), 100.9 (CH benzylidene), 116.6–116.7 (6 ꢄ Cq cya-
noethyl), 125.7 (CHarom), 127.6–128.9 (CHarom), 137.1–137.4
(7 ꢄ Cq Bn), 138.4 (Cq benzylidene); HRMS: C111H127F17N6O38
þ
P6S + NH4 requires 2710.6403, found 2710.6393.
4.1.4. Glucosyl-2-O-benzylglycerol phosphate hexamer-amino
hexyl spacer (9)
4.1.2. Global deprotection and purification of oligomers
The fully protected oligomer was treated with a 9/1 mixture of
28% NH4OH (aq)/1,4-dioxane at a concentration of 5 mg/ml at 40–
45 °C overnight in a sealed flask or tube in case of 10. In the synthe-
sis of oligomers 1 and 29, the corresponding protected hexamers
(26 and 28, respectively) were treated with a 9/1 mixture of 28%
NH4OH (aq)/1,4-dioxane at a concentration of 5 mg/ml at room
temperature for 2.5 h. Next, in all cases, the mixture was washed
with Et2O (equal volume) and the ether layer was extracted twice
with H2O. The aqueous layer was concentrated under reduced
pressure after which NMR and HRMS analysis confirmed full con-
version to the semiprotected intermediate. The intermediate was
then treated with Pd (0)/H2 in a slightly acidic (pH ꢀ2.7) mixture
of dioxane/water (1/4, containing ꢀ1% AcOH). After stirring for
three days the mixture was filtered and concentrated in vacuo.
The residue was purified by size exclusion chromatography
(Sephadex HW40, eluent: 0.15 M NH4OAc). After repeated lyophi-
lization, the purified product was eluted through a small column
containing Dowex Na+ cation-exchange resin (type: 50WX4-200,
stored on 0.5 M NaOH in H2O, flushed with H2O and MeOH before
use). Lyophilization gave the fully deprotected oligomer of which
the integrity and purity was confirmed by HRMS and NMR (1H,
13C, 31P) analysis.
Hexamer 7 (272 mg, 101 lmol) was coupled to spacer phospho-
ramidite 8 (4 equiv), oxidized and purified (FSPE) using the general
procedure as described above. Fully protected hexamer 9 (267 mg,
87.2 lmol, 86%) was obtained as a
colourless oil. 31P NMR
(161.7 MHz): d = ꢂ1.9, ꢂ1.8 (1P), ꢂ1.3 to ꢂ1.0 (6P); 1H NMR
(400 MHz): d = 1.27–1.37 (m, 4H, 2 ꢄ CH2 hexylspacer), 1.42–1.51
(m, 2H, CH2 hexylspacer), 1.60–1.68 (m, 2H, CH2 hexylspacer),
2.09–2.34 (m, 4H, F17C8CH2CH2CH2SO2–, F17C8CH2CH2CH2SO2–),
2.39–2.69 (m, 14H, 7 ꢄ CH2 cyanoethyl), 3.05–3.17 (m, 4H,
F
17C8CH2CH2CH2SO2–, CH2–N hexylspacer), 3.24–3.33 (m, 2H, –
OCH2CH2SO2–), 3.57–3.72 (m, 3H, H-2, H-4, H-6), 3.75–3.85 (m,
5H, 5 ꢄ CH glycerol), 3.94–4.33 (m, 44H, H-3, H-5, H-60, CH glyc-
erol, 12 ꢄ CH2 glycerol, 7 ꢄ CH2 cyanoethyl, CH2–O hexylspacer),
4.43–4.50 (m, 4H, –OCH2CH2SO2–, CH2 Bn), 4.53–4.64 (m, 10H,
5 ꢄ CH2 Bn), 4.71–4.77 (m, 2H, CH2 Bn), 4.81, (d, 1H, J = 11.6 Hz,
CHH Bn), 4.92, (d, 1H, J = 11.6 Hz, CHH Bn), 4.99–5.12 (m, 4H, H-
1, NH CBz, CH2 CBz), 5.55 (s, 1H, CH benzylidene), 7.26–7.38 (m,
43H, Harom), 7.44–7.48 (m, 2H, Harom); 13C NMR (100 MHz):
d = 13.3 (F17C8CH2CH2CH2SO2–), 19.2–19.5 (7 ꢄ CH2 cyanoethyl),
24.8, 25.9 (2 ꢄ CH2 hexylspacer), 29.3–29.9 (2 ꢄ CH2 hexylspacer,
F
17C8CH2CH2CH2SO2–), 40.7 (CH2–N hexylspacer), 53.0, 53.1
(F17C8CH2CH2CH2SO2–, –OCH2CH2SO2–), 61.3 (–OCH2CH2SO2–),
61.8–62.4 (7 ꢄ CH2 cyanoethyl), 62.8 (C-5), 65.3–66.0 (11 ꢄ CH2
glycerol), 66.3 (CH2 CBz), 68.4–68.6 (C-6, CH2 glycerol), 72.0–72.2
(5 ꢄ CH2 Bn), 73.4–73.5 (CH2 Bn), 75.0 (CH2 Bn), 75.2–75.5
(6 ꢄ CH glycerol), 78.0–78.1 (C-3), 78.9 (C-2), 81.7–81.8 (C-4),
97.4–97.7 (C-1), 100.8 (CH benzylidene), 116.6–116.7 (7 ꢄ Cq cya-
noethyl), 125.7 (CHarom), 127.5–128.9 (CHarom), 136.6 (Cq Bn),
137.1–137.3 (6 ꢄ Cq Bn), 137.9 (Cq Bn), 138.5 (Cq benzylidene),
4.1.3. Glucosyl-2-O-benzylglycerol phosphate hexamer (7)
Glycerol phosphate pentamer 5 (286 mg, 139
lmol) and DCI
(0.25 M solution in CH3CN, 2.22 ml, 556 mol) were dissolved in
l
CH3CN (2.0 ml) together with freshly activated MS 3 Å and stirred
for 15 min under argon. Subsequently, glucosyl-glycerol phospho-
þ
ramidite 6 (0.1 M in CH3CN, 2.30 ml, 230
lmol) was added and
156.3 (Cq CBz); HRMS: C128H150F17N8O43P7S + NH4 requires
the mixture stirred for 30 min at rt. H2O (1.0 ml) was added after
which the oxidation step was performed according to the general
procedure. The crude intermediate was redissolved in a 1/1 mixture
of DCM and MeOH (40 ml) and treated with PPTS (40 mg,
0.16 mmol) for 8 h under gentle stirring. The mixture was diluted
with DCM (80 ml) and washed with a 1/1 mixture of satd aq NaH-
CO3 and brine (50 ml). The organic layer was dried (Na2SO4) and
concentrated in vacuo, after which the crude product was purified
with FSPE, according to the general procedure. Glucosylated hexa-
3076.7748, found 3076.7789.
4.1.5. Glucosyl-glycerolphosphate hexamer (10)
Protected hexamer 9 (99.5 mg, 32.5 lmol) was treated with
aqueous ammonia as described above. Additionally, the compound
was eluted through a small column containing Dowex Na+ cation-
exchange resin (type: 50WX4-200, stored on 0.5 M NaOH in H2O,
flushed with H2O and MeOH before use) and, subsequently, lyoph-
ilized, yielding the intermediate semiprotected hexamer (75.2 mg,
mer 7 (277 mg, 103
l
mol, 74%) was obtained as a colourless oil.
32.5 lmol, 100%) as an amorphous white solid. Analytical data
31P NMR (161.7 MHz): d = ꢂ1.9, ꢂ1.8 (1P), ꢂ1.3 (2P), ꢂ1.1 to ꢂ0.9
(3P); 1H NMR (400 MHz): d = 2.09–2.35 (m, 4H, F17C8CH2CH2CH2
SO2–, F17C8CH2CH2CH2SO2–), 2.47–2.77 (m, 13H, 6 ꢄ CH2 cyano-
ethyl, CH2–OH), 3.05–3.13 (m, 2H, F17C8CH2CH2CH2SO2–), 3.24–
3.34 (m, 2H, –OCH2CH2SO2–), 3.51–3.72 (m, 5H, H-2, H-4, H-6,
CH2 glycerol), 3.77–3.88 (m, 6H, 6 ꢄ CH glycerol), 3.98–4.34 (m,
37H, H-3, H-5, H-60, 11 ꢄ CH2 glycerol, 6 ꢄ CH2 cyanoethyl), 4.42–
4.50 (m, 2H, –OCH2CH2SO2–), 4.56–4.65 (m, 10H, 5 ꢄ CH2 Bn),
4.70 (d, 1H, J = 11.6 Hz, CHH Bn), 4.80 (d, 1H, J = 11.3 Hz, CHH Bn),
intermediate: 31P NMR (161.7 MHz, D2O): d = 0.9–1.1 (6P), 2.9
(1P, phosphomonoester); 1H NMR (400 MHz, D2O): d = 0.95–1.24
(m, 6H, 3 ꢄ CH2 hexylspacer), 1.34–1.44 (m, 2H, CH2 hexylspacer),
2.85–2.94 (m, 2H, CH2–N hexylspacer), 3.51–4.15 (m, 38H, H-2, H-
3, H-4, H-5, H-6, H-60, CH2–O hexylspacer, 6 ꢄ CH glycerol,
12 ꢄ CH2 glycerol), 4.29–4.41 (m, 10H, 5 ꢄ CH2 Bn), 4.47–4.58
(m, 4H, 2 ꢄ CH2 Bn), 4.89 (s, 2H, CH2 CBz), 5.29 (d, 1H, J = 3.6 Hz,
H-1), 5.47 (s, 1H, CH benzylidene), 6.98–7.37 (m, 45H, Harom);
HRMS: [C94H120NO41P7 + 2H]2+ requires 1068.7822, found

W. F. J. Hogendorf et al. / Carbohydrate Research 356 (2012) 142–151
147
1068.7828. A portion of the intermediate (75.1 mg, 32.5
deprotected with Pd (0)/H2 using the standard procedure. Mono-
glucosylated hexamer 10 (45.5 mg, 31.7 mol, 98%) was obtained
l
mol) was
Bn), 75.7 (CH2 Bn), 75.8 (CH glycerol), 77.1 (C-4), 79.5 (C-2), 81.7
(C-3), 95.7 (C-1), 116.9 (CH2 allyl), 125.1, 125.2 (CHarom), 127.1–
128.6 (CHarom), 129.7 (CHarom), 133.1, 133.2 (Cq phenyl), 134.6
(CH allyl), 135.5 (CHarom), 138.1, 138.2, 138.7, 138.8 (3 ꢄ Cq Bn),
l
as an amorphous white solid. 31P NMR (161.7 MHz, D2O): d = 0.9
(1P), 1.2–1.3 (4P), 1.4 (1P), 4.7 (1P, phosphomonoester); 1H NMR
(600 MHz, D2O): d = 1.38–1.43 (m, 4H, 2 ꢄ CH2 hexylspacer),
1.60–1.68 (m, 4H, 2 ꢄ CH2 hexylspacer), 2.97 (t, 2H, J = 7.5 Hz,
CH2–N hexylspacer), 3.37 (at, 1H, J = 9.6 Hz, H-4), 3.49 (dd, 1H,
J = 3.8 Hz, 9.9 Hz, H-2), 3.71–3.77 (m, 3H, H-3, H-6, CHH glycerol),
3.79–4.04 (m, 32H, H-5, H-60, 5 ꢄ CH glycerol, 11 ꢄ CH2 glycerol,
CHH glycerol, CH2–O hexylspacer), 4.06–4.09 (m, 1H, CH glycerol),
5.14 (d, 1H, J = 3.7 Hz, H-1); 13C NMR (150 MHz, D2O): d = 25.4,
26.1, 27.6 (3 ꢄ CH2 hexylspacer), 30.4 (d, J = 6.8 Hz, CH2 hexylsp-
acer), 40.4 (CH2–N hexylspacer), 61.5 (C-6), 65.2 (d, J = 6.0 Hz,
CH2 glycerol), 65.7 (d, J = 4.5 Hz, CH2 glycerol), 66.1 (d, J = 5.2 Hz,
CH2 glycerol), 67.1–67.3 (8 ꢄ CH2 glycerol, CH2–O hexylspacer),
67.7 (d, J = 5.5 Hz, CH2 glycerol), 70.5 (t, J = 7.7 Hz, 4 ꢄ CH glycerol),
70.7 (C-4), 71,3 (t, J = 7.3 Hz, CH glycerol), 72.5 (C-2), 72.8 (C-5),
73.9 (C-3), 76.4 (t, J = 8.0 Hz, CH glycerol), 98.7 (C-1); HRMS:
141.2, 141.2 (2 ꢄ Cq FMOc), 143.2, 143.4 (2 ꢄ Cq FMOc), 155.0
þ
(C@O FMOc); HRMS:
C
64H68O10Si + NH4 requires 1042.4920,
found 1042.4933.
4.1.8. 3-O-Allyl-2-O-(2,3,4-tri-O-benzyl-
O-(tert-butyldiphenylsilyl)-sn-glycerol (14)
To a solution of compound 13b (3.40 g, 3.32 mmol) in DCM
(65 ml) was added DBU (165 l, 1.10 mmol). After stirring for
a-D-glucopyranosyl)-1-
l
15 min the solvent was removed under reduced pressure and the
residue purified by silica gel column chromatography (EtOAc/PE)
giving alcohol 14 (2.50 g, 3.11 mmol, 94%) as a colourless oil.
½
a 2D0 (CHCl3): +33.6; IR: 737, 1026, 1072, 1454, 2928; 1H NMR
ꢃ
(400 MHz): d = 1.03 (s, 9H, t-Bu TBDPS), 3.48–3.80 (m, 9H, H-2,
H-4, H-5, H-6, H-60, 2 ꢄ CH2 glycerol), 3.97–4.04 (m, 4H, H-3, CH
glycerol, CH2 allyl), 4.61 (d, 1H, J = 10.8 Hz, CHH Bn), 4.68 (d, 1H,
J = 12.0 Hz, CHH Bn), 4.76 (d, 1H, J = 12.0 Hz, CHH Bn), 4.78 (d,
1H, J = 10.4 Hz, CHH Bn), 4.86 (d, 1H, J = 11.2 Hz, CHH Bn), 4.97
(d, 1H, J = 10.8 Hz, CHH Bn), 5.16 (dd, 1H, J = 1.6 Hz, 10.4 Hz, CHH
allyl), 5,24–5.28 (m, 2H, H-1, CHH allyl), 5.88 (ddd, 1H, J = 5.5 Hz,
10.7 Hz, 17.1 Hz, CH allyl), 7.24–7.42 (m, 21H, Harom), 7.64–7.67
(m, 4H, Harom); 13C NMR (100 MHz): d = 19.1 (Cq t-Bu), 26.8
(3 ꢄ CH3 TBDPS), 61.4 (C-6), 63.8 (CH2 glycerol), 70.7 (C-5), 70.9
(CH2 glycerol), 72.2 (CH2 allyl), 72.3 (CH2 Bn), 74.9 (CH2 Bn), 75.6
(CH2 Bn), 76.0 (CH glycerol), 77.1 (C-4), 79.6 (C-2), 81.6 (C-3),
96.0 (C-1), 116.9 (CH2 allyl), 127.5–128.4 (CHarom), 129.7 (CHarom),
133.1, 133.2 (Cq phenyl), 134.6 (CH allyl), 135.5 (CHarom), 138.2,
138.3, 138.8 (3 ꢄ Cq Bn); HRMS: C49H58O8Si + Na+ requires
825.3793, found 825.3784.
C
30H68NO39P7 + H+ requires 1284.1605, found 1284.1610.
4.1.6. 3-O-Allyl-2-O-(2,3,4,6-tetra-O-benzyl- ,b- -glucopyran
a
D
osyl)-1-O-(tert-butyldiphenylsilyl)-sn-glycerol (13a)
To a cooled (0 °C) solution of donor 11a (171 mg, 0.250 mmol)
and semiprotected glycerol 12 (111 mg, 0.300 mmol) in a 4/1 mix-
ture of Et2O/DCM (5.0 ml) was added TMSOTf (2.25 ll, 12.4 lmol).
After stirring for 40 min, Et3N (3 drops) was added and the mix-
ture diluted with DCM (10 ml). After washing once with a 1/1
mixture of satd aq NaHCO3 and brine (10 ml), the organic layer
was dried (Na2SO4) and concentrated in vacuo. Purification of
the residue by silica gel column chromatography (EtOAc/PE) gave
pseudodisaccharide 13a (168 mg, 0.188 mmol, 75%) as an insepa-
rable mixture of anomers (
a
/b ratio of ꢀ4/1, based on 1H NMR
analysis. This was ꢀ2/1 when the reaction was performed in pure
4.1.9. 3-O-Allyl-2-O-(2,3,4,6-tetra-O-benzyl-a-D-glucopyran
osyl)-1-O-(tert-butyldiphenylsilyl)-sn-glycerol (15)
DCM). For analytical data of the pure
a-isomer see the synthesis of
compound 15.
A solution of alcohol 14 (2.521 g, 3.14 mmol) together with
BnBr (0.94 ml, 7.85 mmol) in DMF (20 ml) was stirred for 5 min
at 0 °C, after which NaH (60% dispersion in mineral oil, 0.314 g,
7.85 mmol) was added. The resulting mixture was stirred for
75 min and allowed to slowly warm up to rt, before MeOH
(5.0 ml) was added. After stirring for 15 min, H2O (30 ml) was
added and the mixture was extracted with Et2O (50 ml). The organ-
ic layer was washed twice with H2O (20 ml) and once with brine
(20 ml) before it was dried (Na2SO4) and concentrated in vacuo.
Purification of the residual oil by silica gel column chromatography
(EtOAc/PE) furnished perbenzylglucosyl glycerol derivative 15
4.1.7. 3-O-Allyl-2-O-(2,3,4-tri-O-benzyl-6-O-[9-fluorenylmethyl
oxycarbonyl]- -glucopyranosyl)-1-O-(tert-butyldiphenyl
silyl)-sn-glycerol (13b)
To a cooled (0 °C) solution of donor 11b (7.51 g, 8.90 mmol) and
semiprotected glycerol 12 (3.96 g, 10.7 mmol) in Et2O (180 ml) was
added TfOH (157 ll, 1.78 mmol). After stirring for 25 min, satd aq
a
-D
NaHCO3 (75 ml) was added and the layers separated. The ether
layer was washed once with brine (50 ml) before it was dried
(Na2SO4) and concentrated in vacuo. Purification of the residue
by silica gel column chromatography (EtOAc/PE) gave pseudodi-
saccharide 13b (8.30 g, 8.09 mmol, 91%) as a colourless oil contain-
ing a minor amount (<3%, based on 1H NMR analysis) of the
(2.429 g, 2.72 mmol, 87%) as a colourless oil. ½a D20
ꢃ
(CHCl3): +31.8;
IR: 737, 1026, 1069, 1454, 2928; 1H NMR (400 MHz): d = 1.05 (s,
9H, t-Bu TBDPS), 3.36 (dd, 1H, J = 1.7 Hz, 10.6 Hz, H-6), 3.54–3.84
(m, 8H, H-2, H-4, H-5, H-60, 2 ꢄ CH2 glyc), 3.95–4.00 (m, 3H, CH2
allyl, H-3), 4.06 (m, 1H, CH glycerol), 4.36 (d, 1H, J = 12.4 Hz, CHH
Bn), 4.43 (d, 1H, J = 10.8 Hz, CHH Bn), 4.55 (d, 1H, J = 12.0 Hz,
CHH Bn), 4.69 (d, 1H, J = 12.0 Hz, CHH Bn), 4.74–4.82 (m, 3H, CH2
Bn, CHH Bn), 4.97 (d, 1H, J = 10.8 Hz, CHH Bn), 5.15 (dd, 1H,
J = 1.4 Hz, 10.6 Hz, CHH allyl), 5.24–5.29 (m, 2H, CHH allyl, H-1),
5.88 (ddd, 1H, J = 5.5 Hz, 10.7 Hz, 17.1 Hz, CH allyl), 7.08–7.11
(m, 2H, Harom), 7.20–7.38 (m, 24H, Harom), 7.64–7.67 (m, 4H, Harom);
13C NMR (100 MHz): d = 19.1 (Cq t-Bu), 26.8 (3 ꢄ CH3 TBDPS), 63.8
(CH2 glycerol), 68.0 (C-6), 70.1 (C-5), 70.5 (CH2 glycerol), 72.2 (CH2
allyl), 72.2 (CH2 Bn), 73.3 (CH2 Bn), 74.8 (CH2 Bn), 75.5 (CH2 Bn),
76.0 (CH glycerol), 77.4 (C-4), 79.5 (C-2), 81.8 (C-3), 96.1 (C-1),
116.7 (CH2 allyl), 127.4–128.2 (CHarom), 129.6 (CHarom), 133.1,
133.3 (Cq phenyl), 134.6 (CH allyl), 135.5 (CHarom), 137.9, 138.3,
b-product. ½a 2D0
ꢃ
(CHCl3): +32.0; IR: 1007, 1072, 1254, 1450, 1748,
2859; 1H NMR (400 MHz): d = 1.05 (s, 9H, t-Bu TBDPS), 3.58–3.63
(m, 3H, H-2, H-4, CHH glycerol), 3.69–3.80 (m, 3H, CHH glycerol,
CH2 glycerol), 3.94–4.14 (m, 6H, H-3, H-5, CH glycerol, CHH FMOc,
CH2 allyl), 4.20–4.26 (m, 2H, CHH FMOc, CH FMOc), 4.31–4.40 (m,
2H, H-6, H-60), 4.55 (d, 1H, J = 10.8 Hz, CHH Bn), 4.69 (d, 1H,
J = 11.6 Hz, CHH Bn), 4.76–4.79 (m, 3H, 1 ꢄ CH2 Bn, CHH Bn),
4.88 (d, 1H, J = 10.8 Hz, CHH Bn), 5.00 (d, 1H, J = 10.8 Hz, CHH
Bn), 5.16 (d, 1H, J = 10.8 Hz, CHH allyl), 5.25 (dd, 1H, J = 1.4 Hz,
17.4 Hz, CHH allyl), 5.32 (d, 1H, J = 3.6 Hz, H-1), 5.87 (ddd, 1H,
J = 5.5 Hz, 10.7 Hz, 17.1 Hz, CH allyl), 7.22–7.40 (m, 25H, Harom),
7.58 (d, 1H, J = 7.5 Hz, Harom), 7.61 (d, 1H, J = 7.5 Hz, Harom), 7.66
(d, 4H, J = 7.1 Hz, Harom), 7.75 (d, 2H, J = 7.6 Hz, Harom); 13C NMR
(100 MHz): d = 19.2 (Cq t-Bu), 26.8 (3 ꢄ CH3 TBDPS), 46.6 (CH
FMOc), 63.8 (CH2 glycerol), 66.2 (CH2 FMOc), 68.5 (C-5), 69.9 (C-
6), 70.6 (CH2 glycerol), 72.2 (CH2 allyl), 72.3 (CH2 Bn), 75.0 (CH2
þ
138.4, 138.8 (4 ꢄ Cq Bn); HRMS: C56H64O8Si + NH4 requires
910.4709, found 910.4718.

148
W. F. J. Hogendorf et al. / Carbohydrate Research 356 (2012) 142–151
4.1.10. 2-O-(2,3,4,6-Tetra-O-benzyl-
(tert-butyldiphenylsilyl)-sn-glycerol (16)
a
-
D
-glucopyranosyl)-1-O-
4.1.12. 1-O-([N,N-Diisopropyl]-2-cyanoethyl-phosphoramidite)-
2-O-(2,3,4,6-tetra-O-benzyl-
-glucopyranosyl)-3-O-(4,40-
a-D
A solution of glycoside 15 (2.37 g, 2.65 mmol) in freshly dis-
tilled THF (18 ml) was stirred under argon for 30 min. After the
addition of Ir(COD)(Ph2MeP)2PF6 (112 mg, 0.133 mmol) the solu-
tion was purged with H2 (g) for ꢀ15 s. After stirring under argon
for 2 h, the mixture was diluted with THF (20 ml) and satd aq NaH-
CO3 (20 ml). Upon addition of I2 (1.01 g, 3.98 mmol), the mixture
was allowed to stir for 1.5 h at room temperature. The mixture
was then diluted with EtOAc (100 ml) and washed with, respec-
tively, satd aq NaS2O3 (30 ml) and brine (40 ml). The organic layer
was dried over Na2SO4 and concentrated in vacuo. Column chro-
matography (EtOAc/PE) afforded 16 (1.97 g, 2.31 mmol, 87%) as a
dimethoxytrityl)-sn-glycerol (18)
To a cooled (0 °C) solution of alcohol 17 (801 mg, 0.873 mmol)
and Et3N (0.19 ml, 1.4 mmol) in DCM (6.0 ml) was added 2-cyano-
ethyl-N,N-diisopropylchlorophosphoramidite (258 mg, 1.09 mmol).
After stirring for 30 min, the reaction was quenched by the addition
of H2O (1.0 ml), diluted with DCM (20 ml) and washed with a 1/1
mixture of satd aq NaHCO3 and brine (20 ml). The aqueous layer
was extracted with DCM (2 ꢄ 10 ml) and the combined organic lay-
ers were dried over Na2SO4 and concentrated in vacuo. Purification
of the residue by column chromatography (EtOAc/PE/Et3N) gave
phosphoramidite 18 (722 mg, 0.646 mmol, 74%) as a colourless
oil. IR: 1030, 1250, 1508, 1605, 2928; 31P NMR (161.7 MHz, CD3CN):
d = 149.0, 149.4 (diastereoisomers); 1H NMR (400 MHz, CD3CN,
mixture of diastereoisomers): d = 1.04–1.12 (m, 12H, 4 ꢄ CH3 iso-
propylamino), 2.40–2.42 (m, 2H, CH2 cyanoethyl), 3.16–3.25 (m,
2H, CH2 glycerol), 3.44–3.55 (m, 4H, H-2, H-4, 2 ꢄ CH isopropylami-
no), 3.60–3.88 (m, 13H, H-3, H-6, H-60, 2 ꢄ OMe, CH2 glycerol, CH2
cyanoethyl), 3.91–4.02 (m, 2H, H-5, CH glycerol), 4.48–4.61 (m,
5H, CH2 Bn), 4.72–4.80 (m, 2H, C2 Bn), 4.86–4.90 (m, 1H, CHH Bn),
5.15–5.18 (m, 1H, H-1), 6.80 (d, 4H, J = 8.9 Hz, Harom), 7.12–7.36
(m, 27H, Harom), 7.44–7.47 (m, 2H, Harom); 13C NMR (100 MHz):
d = 24.9–25.1 (4 ꢄ CH3 isopropylamino), 43.7–43.8 (2 ꢄ CH isopro-
pylamino), 55.8 (2 ꢄ OMe), 59.3–59.6 (CH2 glycerol), 64.3–64.4
(CH2 glycerol, CH2 cyanoethyl), 69.9 (C-6), 71.4–71.5 (C-5), 72.8–
72.9, 73.9, 75.4–75.5, 76.0 (4 ꢄ CH2 Bn), 77.5–77.7 (CH glycerol),
78.8 (C-4), 81.0 (C-2), 82.5 (C-3), 87.2 (Cq DMTr), 97.1–97.3 (C-1),
114.0 (CHarom), 127.7–129.3 (CHarom), 131.0 (CHarom), 136.9, 139.5,
139.5, 139.7, 139.8, 140.1, 146.1, 159.6 (4 ꢄ Cq Bn, 5 ꢄ Cq DMTr,
Cq cyanoethyl); HRMS: C67H77N2O11P + H+ requires 1117.5338,
found 1117.5337.
colourless oil. ½a D20
ꢃ
(CHCl3): +21.4; IR: 737, 1026, 1069, 1454,
1724, 2928, 3449; 1H NMR (400 MHz): d = 1.05 (s, 9H, t-Bu TBDPS),
3.11 (bs, 1H, CH2OH), 3.32 (dd, 1H, J = 1.5 Hz, 10.6 Hz, H-6), 3.54–
3.57 (m, 2H, H-2, H-60), 3.62–3.70 (m, 3H, H-4, 2 ꢄ CHH glycerol),
3.77–3.86 (m, 4H, H-5, CH glycerol, 2 ꢄ CHH glycerol), 3.97 (t,
1H, J = 9.3 Hz, H-3), 4.33 (d, 1H, J = 12.2 Hz, CHH Bn), 4.44 (d, 1H,
J = 10.9 Hz, CHH Bn), 4.52 (d, 1H, J = 12.2 Hz, CHH Bn), 4.66 (d,
1H, J = 11.6 Hz, CHH Bn), 4.77–4.85 (m, 3H, CH2 Bn, CHH Bn),
4.90–4.93 (m, 2H, H-1, CHH Bn), 7.08–7.12 (m, 2H, Harom), 7.20–
7.39 (m, 24H, Harom), 7.62–7.64 (m, 4H, Harom); 13C NMR
(100 MHz): d = 19.0 (Cq t-Bu), 26.7 (3 ꢄ CH3 TBDPS), 62.6 (CH2
glycerol), 63.7 (CH2 glycerol), 67.8 (C-6), 70.4 (C-5), 73.3, 73.8,
74.7, 75.5 (4 ꢄ CH2 Bn), 77.4 (C-4), 79.4 (C-2), 80.8 (CH glycerol),
82.0 (C-3), 98.4 (C-1), 127.4–128.4 (CHarom), 129.6 (CHarom),
132.9, 133.0 (Cq phenyl), 135.4 (CHarom), 137.4, 137.6, 138.1,
138.5 (Cq Bn); HRMS: C53H60O8Si + Na+ requires 875.3950, found
875.3946.
4.1.11. 2-O-(2,3,4,6-Tetra-O-benzyl-
a-D-glucopyranosyl)-3-O-
(4,40-dimethoxytrityl)-sn-glycerol (17)
To a cooled (0 °C) solution of alcohol 16 (1.85 g, 2.17 mmol) and
Et3N (0.45 ml, 3.3 mmol) in DCM (11 ml) was added DMTr-Cl
(881 mg, 2.60 mmol). The mixture was stirred for 2.5 h before
MeOH (1.0 ml) was added. After stirring for an additional 15 min
the reaction mixture was diluted with DCM (40 ml) and washed
with a 1/1 mixture of satd aq NaHCO3 and brine (30 ml). The aque-
ous layer was extracted with DCM (2 ꢄ 10 ml) and the combined
organic layers were dried (Na2SO4) and concentrated under re-
duced pressure. The residual oil was redissolved in THF (15 ml)
and, subsequently, TBAF (1 M solution in THF, 7.8 ml) was added.
The mixture was stirred for 3 h after which the volatiles were re-
moved in vacuo and the residual oil was purified by silica gel col-
umn chromatography (EtOAc/PE/Et3N) yielding mono-alcohol 17
4.1.13. 2-O-(2,3,4,6-Tetra-O-benzyl-a-D-glucopyranosyl)-3-O-
(4,40-dimethoxytrityl)-1-O-succinyl-sn-glycerol (19)
To a cooled (0 °C) solution of alcohol 17 (914 mg, 0.997 mmol)
and Et3N (1.52 ml, 11.0 mmol) in DCM (10 ml) was added succinic
anhydride (498 mg, 4.98 mmol). After stirring for 1 h the mixture
was concentrated under reduced pressure, after which column
chromatography (EtOAc/PE/Et3N) gave succinyl ester 19 (966 mg,
0.963 mmol, 96%) as a pale yellow oil. ½a D20
ꢃ
(CHCl3): +36.6; IR:
1030, 1153, 1246, 1508, 1609, 1713, 1736, 2928; 1H NMR
(400 MHz, CD3CN): d = 2.49 (s, 4H, 2 ꢄ CH2 succinyl), 3.22–3.31
(m, 2H, CH2 glycerol), 3.50 (dd, 1H, J = 3.5 Hz, 9.7 Hz, H-2), 3.57
(at, 1H, J = 9.5 Hz, H-4), 3.68–3.77 (m, 8H, H-6, H-60, 2 ꢄ OMe),
3.90 (at, 1H, J = 9.3 Hz, H-3), 3.93–3.98 (m, 1H, H-5), 4.01–4.08
(m, 1H, CH glycerol), 4.23–4.32 (m, 2H, CH2 glycerol), 4.49–4.61
(m, 5H, CHH Bn, 2 ꢄ CH2 Bn), 4.78 (d, 1H, J = 11.1 Hz, CHH Bn),
4.83 (d, 1H, J = 11.1 Hz, CHH Bn), 4.91 (d, 1H, J = 11.1 Hz, CHH
Bn), 5.12 (d, 1H, J = 3.5 Hz, H-1), 6.84 (d, 4H, J = 8.9 Hz, Harom),
7.16–7.39 (m, 27H, Harom), 7.48 (d, 2H, J = 7.4 Hz, Harom); 13C
NMR (100 MHz, CD3CN): d = 29.8, 30.3 (2 ꢄ CH2 succinyl), 56.5
(2 ꢄ OMe), 64.1 (CH2 glycerol), 65.9 (CH2 glycerol), 70.5 (C-6),
72.2 (C-5), 73.7, 74.5, 76.1 (3 ꢄ CH2 Bn), 76.2 (CH glycerol), 76.6
(CH2 Bn), 79.4 (C-4), 81.5 (C-2), 82.9 (C-3), 87.9 (Cq DMTr), 97.6
(C-1), 114.7 (CHarom), 128.4–129.9 (CHarom), 131.6 (CHarom),
137.3, 137.4, 139.9, 140.0, 140.2, 140.7, 146.6, 160.2 (5 ꢄ Cq DMTr,
4 ꢄ Cq Bn), 173.5, 174.9 (2 ꢄ C@O succinyl); HRMS:
(1.77 g, 1.93 mmol, 89%) as a colourless oil. ½a D20
ꢃ
(CHCl3): +40.6;
IR: 737, 1030, 1065, 1250, 1508, 1609, 2927, 3487; 1H NMR
(400 MHz): d = 2.85 (at, 1H, J = 5.0 Hz, CH2OH), 3.21 (dd, 1H,
J = 6.1 Hz, 9.6 Hz, CHH glycerol), 3.35 (dd, 1H, J = 5.5 Hz, 9.6 Hz,
CHH glycerol), 3.53–3.57 (m, 2H, H-2, H-4), 3.61–3.67 (m, 3H, H-
6, H-60, CHH glycerol), 3.71 (s, 6H, 2 ꢄ OMe), 3.76–3.82 (m, 1H,
CHH glycerol), 3.86 (m, 1H, CH glycerol), 3.96–4.04 (m, 2H, H-3,
H-5), 4.46 (d, 1H, J = 10.8 Hz, CHH Bn), 4.47 (d, 1H, J = 12.4 Hz,
CHH Bn), 4.58 (d, 1H, J = 11.6 Hz, CHH Bn), 4.63 (d, 1H,
J = 12.0 Hz, CHH Bn), 4.80 (d, 1H, J = 10.4 Hz, CHH Bn), 4.82 (d,
1H, J = 10.4 Hz, CHH Bn), 4.96 (d, 1H, J = 10.8 Hz, CHH Bn), 4.99
(d, 1H, J = 3.6 Hz, H-1), 6.78–6.81 (m, 4H, Harom), 7.11–7.13 (m,
2H, Harom), 7.18–7.36 (m, 25H, Harom), 7.46 (d, 2H, J = 7.4 Hz, Har-
om); 13C NMR (100 MHz): d = 55.0 (2 ꢄ OMe), 63.4 (CH2 glycerol),
63.9 (CH2 glycerol), 68.5 (C-6), 70.5 (C-5), 72.6, 73.4, 75.0, 75.6
(4 ꢄ CH2 Bn), 77.7 (C-4), 79.5 (C-2), 80.0 (CH glycerol), 81.8 (C-3),
86.3 (Cq DMTr), 96.8 (C-1), 113.0 (CHarom), 126.7–129.0 (CHarom),
130.0 (CHarom), 135.8, 137.5, 137.9, 138.0, 138.6, 144.7, 158.4
(4 ꢄ Cq Bn, 5 ꢄ Cq DMTr); HRMS: C58H60O10 + Na+ requires
939.4079, found 939.4090.
C
62H64O13 + Na+ requires 1039.4239, found 1039.4237.
4.1.14. 2-O-(2,3,4,6-Tetra-O-benzyl- -glucopyranosyl)-1-O-(N-
a-D
[3-perfluorooctylpropyl]-succinamidyl-sn-glycerol (20)
To a solution of compound 19 (351 mg, 0.350 mmol), perfluo-
rooctylpropylamine (119 mg, 0.250 mmol) and N,N-diisopropyleth-
ylamine (0.366 ml, 2.10 mmol) in a 2/1 mixture of DCM/DMF
(5.0 ml) was added BOP (310 mg, 0.700 mmol). The mixture was

W. F. J. Hogendorf et al. / Carbohydrate Research 356 (2012) 142–151
149
stirred for 1.5 h before it was diluted with EtOAc (100 ml) and,
subsequently, washed with satd aq NaHCO3 (2 ꢄ 50 ml), H2O
(2 ꢄ 50 ml) and brine (50 ml). The organic layer was dried (Na2SO4)
and concentrated under reduced pressure, after which the residue
was taken up in DCM (5.0 ml). After the addition of, respectively,
triethylsilane (0.605 ml, 3.75 mmol) and dichloroacetic acid
(0.308 ml, 3.75 mmol) the mixture was stirred for 30 min and, sub-
sequently, diluted with DCM (40 ml) and washed with a 1/1 mix-
ture of satd aq NaHCO3 and brine (20 ml). The organic layer was
dried (Na2SO4) and concentrated in vacuo, after which the residue
was partitioned between 80/20 acetonitrile/water and hexane and
purified by FSPE as described in the general procedure. Fluorous
compound 20 (263 mg, 0.224 mmol, 90%) was isolated as an amor-
(1P);
1H
NMR
(400 MHz):
d = 1.69–1.77
(m,
2H,
F17C8CH2CH2CH2N), 1.98–2.12 (m, 2H, F17C8CH2CH2CH2N), 2.17–
2.69 (m, 9H, 2 ꢄ CH2 succinyl, 2 ꢄ CH2 cyanoethyl, CH2OH),
3.18–3.26 (m, 2H, F17C8CH2CH2CH2N), 3.55–3.60 (m, 1H, H-2),
3.61–3.74 (m, 6H, H-4, H-6, H-60, CH glycerol, CH2 glycerol),
3.76–3.82 (m, 1H, CH glycerol), 3.86–3.95 (m, 2H, H-3, H-5),
4.01–4.31 (m, 15H, CH glycerol, 5 ꢄ CH2 glycerol, 2 ꢄ CH2 cyano-
ethyl), 4.44–4.49 (m, 2H, 2 ꢄ CHH Bn), 4.56–4.65 (m, 5H, CHH
Bn, 2 ꢄ CH2 Bn), 4.69–4.72 (m, 2H, 2 ꢄ CHH Bn), 4.77–4.83 (m,
2H, 2 ꢄ CHH Bn), 4.91–4.95 (m, 1H, CHH Bn), 5.01–5.04 (m, 1H,
H-1), 6.16–6.22 (m,1H, NH), 7.12–7.15 (m, 2H, Harom), 7.25–7.37
(m, 28H, Harom); 13C NMR (100 MHz): d = 19.2–19.4 (2 ꢄ CH2 cya-
noethyl), 20.6 (F17C8CH2CH2CH2N), 28.2 (t, J = 22 Hz, F17C8CH2CH2-
CH2N), 29.3, 30.6 (2 ꢄ CH2 succinyl), 38.4 (F17C8CH2CH2CH2N),
60.4, 60.5 (CH2 glycerol), 62.0–62.2 (2 ꢄ CH2 cyanoethyl), 63.0
(CH2 glycerol), 65.5–66.7 (4 ꢄ CH2 glycerol), 68.3 (C-6), 70.8 (C-
5), 72.0 (CH2 Bn), 72.1, 72.2 (CH2 Bn), 73.0, 73.1 (CH2 Bn), 73.4
(CH2 Bn), 73.8–74.1 (CH glycerol), 75.1 (CH2 Bn), 75.2–75.4 (CH
glycerol), 75.5 (CH2 Bn), 77.4–77.5 (C-4, CH glycerol), 79.6 (C-2),
81.5 (C-3), 96.9, 97.0 (C-1), 116.5–116.6 (2 ꢄ Cq cyanoethyl),
127.5–128.5 (CHarom), 137.1, 137.7–138.0, 138.5 (6 ꢄ Cq Bn),
171.5, 172.3 (2 ꢄ C@O succinyl); HRMS: C78H84F17N3O20P2 + Na+
requires 1790.4744, found 1790.4744.
phous solid. ½a 2D0
ꢃ
(CHCl3): +23.8; IR: 1026, 1065, 1146, 1200, 1547,
1644, 1736, 2924; 1H NMR (400 MHz): d = 1.71–1.79 (m, 2H,
F
17C8CH2CH2CH2N), 1.99–2.13 (m, 2H, F17C8CH2CH2CH2N), 2.38 (t,
2H, J = 6.7 Hz, CH2 succinyl), 2.63 (t, 2H, J = 6.7 Hz, CH2 succinyl),
3.24 (dd, 2H, J = 6.8 Hz, 13.3 Hz, F17C8CH2CH2CH2N), 3.54–3.75 (m,
6H, H-2, H-4, H-6, H-60, CH2 glycerol), 3.83–3.89 (m, 1H, CH glyc-
erol), 3.91–3.96 (m, 1H, H-5), 4.00 (at, 1H, J = 9.4 Hz, H-3), 4.13–
4.16 (m, 2H, CH2 glycerol), 4.46–4.50 (m, 2H, 2 ꢄ CHH Bn), 4.59
(d, 1H, J = 12.1 Hz, CHH Bn), 4.67 (d, 1H, J = 11.6 Hz, CHH Bn),
4.80–4.90 (m, 4H, H-1, 3 ꢄ CHH Bn), 4.95 (d, 1H, J = 11.0 Hz, CHH
Bn), 5.85 (t, 1H, J = 5.9 Hz, NH), 7.12–7.15 (m, 2H, Harom), 7.24–
7.37 (m, 18H,
H
arom); 13C NMR (100 MHz): d = 20.8 (F17C8
4.1.17. Glucosyl glycerol phosphate tetramer (23)
CH2CH2CH2N), 28.3 (t, J = 22 Hz, F17C8CH2CH2CH2N), 29.3, 30.7
(2 ꢄ CH2 succinyl), 38.5 (F17C8CH2CH2CH2N), 61.8 (CH2 glycerol),
64.1 (CH2 glycerol), 68.4 (C-6), 70.9 (C-5), 73.5, 74.2, 75.1, 75.6
(4 ꢄ CH2 Bn), 77.7 (C-4), 78.8 (CH glycerol), 79.5 (C-2), 82.1 (C-3),
98.6 (C-1), 127.6–128.6 (CHarom), 137.4, 137.7, 138.0, 138.5
(4 ꢄ Cq Bn), 171.5, 172.6 (2 ꢄ C@O succinyl); HRMS: C52H52F17
NO10 + Na+ requires 1196.3212, found 1196.3210.
Trimer 22 (157 mg, 88.7 lmol) was coupled to glycerol phos-
phoramidite 4 (1.5 equiv), oxidized, detritylated and purified (FSPE)
using the general procedure as described above. Tetramer 23
(169 mg, 82.0 l
mol, 92%) was obtained as a colourless oil. 31P
NMR (161.7 MHz): d = ꢂ1.4 to ꢂ1.2 (2P), ꢂ0.9, ꢂ0.9, ꢂ0.9 (1P); 1
H
NMR (400 MHz): d = 1.68–1.76 (m, 2H, F17C8CH2CH2CH2N), 1.98–
2.19 (m, 3H, F17C8CH2CH2CH2N, CH2OH), 2.35–2.67 (m, 10H,
2 ꢄ CH2 succinyl, 3 ꢄ CH2 cyanoethyl), 3.18–3.25 (m, 2H,
4.1.15. Glucosyl glycerol phosphate dimer (21)
F17C8CH2CH2CH2N), 3.54–3.60 (m, 1H, H-2), 3.61–3.75 (m, 6H, H-
Monomer 20 (133 mg, 113
phoramidite 4 (1.5 equiv), oxidized, detritylated and purified (FSPE)
l
mol) was coupled to glycerol phos-
4, H-6, H-60, CH glycerol, CH2 glycerol), 3.76–3.83 (m, 2H, 2 ꢄ CH
glycerol), 3.85–3.95 (m, 2H, H-3, H-5), 4.01–4.30 (m, 21H, CH glyc-
erol, 7 ꢄ CH2 glycerol, 3 ꢄ CH2 cyanoethyl), 4.44–4.49 (m, 2H,
2 ꢄ CHH Bn), 4.56–4.66 (m, 7H, CHH Bn, 3 ꢄ CH2 Bn), 4.69–4.72
(m, 2H, 2 ꢄ CHH Bn), 4.76–4.83 (m, 2H, 2 ꢄ CHH Bn), 4.91–4.95
(m, 1H, CHH Bn), 5.01–5.04 (m, 1H, H-1), 6.12–6.19 (m,1H, NH),
7.10–7.15 (m, 2H, Harom), 7.23–7.38 (m, 33H, Harom); 13C NMR
(100 MHz): d = 19.2–19.5 (3 ꢄ CH2 cyanoethyl), 20.6 (F17C8CH2
CH2CH2N), 28.2 (t, J = 22 Hz, F17C8CH2CH2CH2N), 29.3, 30.6
(2 ꢄ CH2 succinyl), 38.4 (F17C8CH2CH2CH2N), 60.4, 60.5 (CH2 glyc-
erol), 62.0–62.2 (3 ꢄ CH2 cyanoethyl), 63.1 (CH2 glycerol), 65.5–
66.6 (6 ꢄ CH2 glycerol), 68.3 (C-6), 70.8 (C-5), 72.0 (CH2 Bn),
72.1–72.2 (2 ꢄ CH2 Bn), 73.0, 73.1 (CH2 Bn), 73.4 (CH2 Bn), 73.7–
74.2 (CH glycerol), 75.1 (CH2 Bn), 75.2–75.4 (2 ꢄ CH glycerol),
75.5 (CH2 Bn), 77.4–77.5 (C-4, CH glycerol), 79.6 (C-2), 81.6 (C-3),
96.8, 97.0 (C-1), 116.6–116.7 (3 ꢄ Cq cyanoethyl), 127.6–128.5
(CHarom), 137.2, 137.7–138.0, 138.5 (7 ꢄ Cq Bn), 171.4, 172.3
(2 ꢄ C@O succinyl); HRMS: [C91H100F17N4O25P3 + 2Na]2+ requires
1055.2701, found 1055.2705.
using the general procedure as described above. Dimer 21 (153 mg,
104 lmol, 92%) was obtained as a
colourless oil. 31P NMR
(161.7 MHz): d = ꢂ0.9, ꢂ0.9 (1P); 1H NMR (400 MHz): d = 1.70–
1.78 (m, 2H, F17C8CH2CH2CH2N), 1.99–2.12 (m, 2H, F17C8CH2CH2
CH2N), 2.26–2.67 (m, 7H, 2 ꢄ CH2 succinyl, CH2 cyanoethyl,
CH2OH), 3.22 (dd, 2H, J = 6.7 Hz, 13.0 Hz, F17C8CH2CH2CH2N),
3.54–3.60 (m, 1H, H-2), 3.61–3.73 (m, 6H, H-4, H-6, H-60, CH glyc-
erol, CH2 glycerol), 3.84–3.95 (m, 2H, H-3, H-5), 4.01–4.31 (m, 9H,
CH glycerol, 3 ꢄ CH2 glycerol, CH2 cyanoethyl), 4.44–4.49 (m, 2H,
2 ꢄ CHH Bn), 4.56–4.73 (m, 5H, CHH Bn, 2 ꢄ CH2 Bn), 4.78–4.83
(m, 2H, 2 ꢄ CHH Bn), 4.92–4.96 (m, 1H, CHH Bn), 5.01–5.03 (m,
1H, H-1), 6.01–6.05 (m,1H, NH), 7.12–7.14 (m, 2H, Harom), 7.25–
7.37 (m, 23H, Harom); 13C NMR (100 MHz): d = 19.3–19.4 (CH2 cya-
noethyl), 20.7 (F17C8CH2CH2CH2N), 28.3 (t, J = 22 Hz, F17C8CH2
CH2CH2N), 29.4, 30.7 (2 ꢄ CH2 succinyl), 38.5 (F17C8CH2 CH2CH2N),
60.5 (d, J = 5 Hz, CH2 glycerol), 62.0 (d, J = 5 Hz, CH2 cyanoethyl),
63.0 (CH2 glycerol), 66.1–66.6 (2 ꢄ CH2 glycerol), 68.3 (C-6), 70.9
(C-5), 72.0, 72.0 (CH2 Bn), 73.1, 73.2 (CH2 Bn), 73.5 (CH2 Bn),
73.9–74.1 (CH glycerol), 75.1, 75.5 (2 ꢄ CH2 Bn), 77.4–77.6 (C-4,
CH glycerol), 79.6, 79.7 (C-2), 81.5 (C-3), 96.9, 97.1 (C-1), 116.4,
116.5 (Cq cyanoethyl), 127.6–128.5 (CHarom), 137.6–137.7, 137.9,
138.6 (5 ꢄ Cq Bn), 171.5, 172.3 (2 ꢄ C@O succinyl); HRMS:
4.1.18. Glucosyl glycerol phosphate pentamer (24)
Tetramer 23 (165 mg, 79.9 lmol) was coupled to glycerol phos-
phoramidite 4 (1.8 equiv), oxidized, detritylated and purified
(FSPE) using the general procedure as described above. Pentamer
C
65H68F17N2 O15P + Na+ requires 1493.3978, found 1493.3978.
24 (176 mg, 74.3 l
mol, 93%) was obtained as a colourless oil. 31P
NMR (161.7 MHz): d = ꢂ1.4 to ꢂ1.1 (3P), ꢂ0.9, ꢂ0.9, ꢂ0.9 (1P);
4.1.16. Glucosyl glycerol phosphate trimer (22)
Dimer 21 (149 mg, 101 mol) was coupled to glycerol phospho-
1H NMR (400 MHz): d = 1.68–1.76 (m, 2H, F17C8CH2CH2CH2N),
l
1.98–2.12 (m, 2H,
F17C8CH2CH2CH2N), 2.26–2.67 (m, 13H,
ramidite 4 (1.5 equiv), oxidized, detritylated and purified (FSPE)
2 ꢄ CH2 succinyl, 4 ꢄ CH2 cyanoethyl, CH2OH), 3.18–3.26 (m, 2H,
using the general procedure as described above. Trimer 22
F
17C8CH2CH2CH2N), 3.54–3.60 (m, 1H, H-2), 3.61–3.75 (m, 6H, H-
(161 mg, 91.0
l
mol, 90%) was obtained as a colourless oil. 31P
4, H-6, H-60, CH glycerol, CH2 glycerol), 3.76–3.83 (m, 3H, 3 ꢄ CH
NMR (161.7 MHz): d = ꢂ1.4, ꢂ1.3, ꢂ1.3, ꢂ1.3 (1P), ꢂ1.0, ꢂ0.9
glycerol), 3.86–3.95 (m, 2H, H-3, H-5), 4.00–4.30 (m, 27H, CH

150
W. F. J. Hogendorf et al. / Carbohydrate Research 356 (2012) 142–151
glycerol, 9 ꢄ CH2 glycerol, 4 ꢄ CH2 cyanoethyl), 4.43–4.49 (m, 2H,
2 ꢄ CHH Bn), 4.56–4.67 (m, 9H, CHH Bn, 4 ꢄ CH2 Bn), 4.69–4.71
(m, 2H, 2 ꢄ CHH Bn), 4.76–4.83 (m, 2H, 2 ꢄ CHH Bn), 4.91–4.95
(m, 1H, CHH Bn), 5.01–5.04 (m, 1H, H-1), 6.14–6.20 (m,1H, NH),
7.10–7.15 (m, 2H, Harom), 7.23–7.39 (m, 38H, Harom); 13C NMR
(100 MHz): d = 19.3–19.4 (4 ꢄ CH2 cyanoethyl), 20.6 (F17C8CH2CH2
CH2N), 28.2 (t, J = 23 Hz, F17C8CH2CH2CH2N), 29.3, 30.6 (2 ꢄ CH2
succinyl), 38.4 (F17C8CH2CH2CH2N), 60.4, 60.5 (CH2 glycerol),
62.0–62.2 (4 ꢄ CH2 cyanoethyl), 63.0 (CH2 glycerol), 65.5–66.6
(8 ꢄ CH2 glycerol), 68.3 (C-6), 70.8 (C-5), 71.9 (CH2 Bn), 72.1–
72.2 (3 ꢄ CH2 Bn), 73.0, 73.1 (CH2 Bn), 73.4 (CH2 Bn), 73.7–74.0
(CH glycerol), 75.1 (CH2 Bn), 75.1–75.4 (3 ꢄ CH glycerol), 75.5
(CH2 Bn), 77.4–77.5 (C-4, CH glycerol), 79.6 (C-2), 81.5 (C-3),
96.8, 96.9 (C-1), 116.6–116.7 (4 ꢄ Cq cyanoethyl), 127.5–128.4
(CHarom), 137.2, 137.7–138.0, 138.5 (8 ꢄ Cq Bn), 171.4, 172.3
(2 ꢄ C@O succinyl); HRMS: [C104H116F17N5O30P4 + 2Na]2+ requires
1204.3101, found 1204.3100.
5.01–5.13 (m, 4H, H-1, CH2 Cbz, NH Cbz), 6.11–6.18 (m,1H, NH),
7.11–7.14 (m, 2H, Harom), 7.22–7.39 (m, 48H, Harom); 13C NMR
(100 MHz): d = 19.2–19.5 (6 ꢄ CH2 cyanoethyl), 20.6 (F17C8CH2CH2
CH2N), 24.8 (CH2 hexylspacer), 25.9 (CH2 hexylspacer), 28.2 (t,
J = 22 Hz, F17C8CH2CH2CH2N), 29.3 (CH2 succinyl), 29.6 (CH2 hex-
ylspacer), 29.9 (d, J = 7 Hz, CH2 hexylspacer), 30.5 (CH2 succinyl),
38.3 (F17C8CH2CH2CH2N), 40.7 (CH2–N hexylspacer), 61.8–62.1
(6 ꢄ CH2 cyanoethyl), 63.0 (CH2 glycerol), 65.5–66.4 (11 ꢄ CH2
glycerol, CH2 Cbz), 68.3–68.4 (C-6, CH2–O hexylspacer), 70.8
(C-5), 72.1–72.2 (6 ꢄ CH2 Bn), 72.9, 73.0 (CH2 Bn), 73.4 (CH2 Bn),
73.7–74.0 (CH glycerol), 75.0 (CH2 Bn), 75.3–75.5 (5 ꢄ CH glycerol),
77.4 (C-4), 79.6 (C-2), 81.5 (C-3), 96.8, 96.9 (C-1), 116.5–116.7
(6 ꢄ Cq cyanoethyl), 127.5–128.5 (CHarom), 136.6, 137.2, 137.7–
138.0, 138.5 (9 ꢄ Cq Bn, Cq Cbz), 156.3 (C@O Cbz), 171.3, 172.2
(2 ꢄ C@O succinyl); HRMS: [C134H155F17N8O40P6 + 2Na]2+ requires
1535.9156, found 1535.9153.
4.1.21. Glucosyl-glycerol phosphate hexamer (2)
4.1.19. Glucosyl glycerol phosphate hexamer (25)
Pentamer 24 (149 mg, 63.1 lmol) was coupled to glycerol phos-
phoramidite 4 (2.0 equiv), oxidized, detritylated and purified
Protected hexamer 26 (139 mg, 46.3
aqueous ammonia as described above. The intermediate hexamer
(98.5 mg, 44.1 mol, 95%) was obtained as an amorphous white so-
lmol) was treated with
l
(FSPE) using the general procedure as described above. Hexamer
lid. Analytical data intermediate: 31P NMR (161.7 MHz, D2O):
d = 0.9–1.1 (5P), 1.2 (1P); 1H NMR (400 MHz, D2O): d = 0.80–1.10
(m, 6H, 3 ꢄ CH2 hexylspacer), 1.21–1.36 (m, 2H, CH2 hexylspacer),
2.59–2.78 (m, 2H, CH2–N hexylspacer), 3.24–4.05 (m, 38H, H-2, H-
3, H-4, H-5, H-6, H-60, 12 ꢄ CH2 glycerol, 6 ꢄ CH glycerol, CH2–O
hexylspacer), 4.16–5.02 (m, 22H, H-1, 9 ꢄ CH2 Bn, CH2 Cbz, NH
Cbz), 6.66–7.14 (m, 50H, Harom); HRMS: [C101H127NO38P6 + 2NH4]2+
requires 1092.3586, found 1092.3590. A portion of the intermedi-
25 (151 mg, 56.8 l
mol, 90%) was obtained as a colourless oil. 31P
NMR (161.7 MHz): d = ꢂ1.4 to ꢂ1.1 (4P), ꢂ0.9, ꢂ0.9, ꢂ0.9 (1P);
1H NMR (400 MHz): d = 1.68–1.76 (m, 2H, F17C8CH2CH2CH2N),
1.98–2.12 (m, 2H,
2 ꢄ CH2 succinyl, 5 ꢄ CH2 cyanoethyl, CH2OH), 3.17–3.25 (m, 2H,
17C8CH2CH2CH2N), 3.54–3.59 (m, 1H, H-2), 3.61–3.74 (m, 6H, H-
F17C8CH2CH2CH2N), 2.31–2.67 (m, 15H,
F
4, H-6, H-60, CH glycerol, CH2 glycerol), 3.76–3.84 (m, 4H, 4 ꢄ CH
glycerol), 3.85–3.94 (m, 2H, H-3, H-5), 4.00–4.31 (m, 33H, CH glyc-
erol, 11 ꢄ CH2 glycerol, 5 ꢄ CH2 cyanoethyl), 4.44–4.48 (m, 2H,
2 ꢄ CHH Bn), 4.57–4.66 (m, 11H, CHH Bn, 5 ꢄ CH2 Bn), 4.68–4.71
(m, 2H, 2 ꢄ CHH Bn), 4.77–4.82 (m, 2H, 2 ꢄ CHH Bn), 4.90–4.95
(m, 1H, CHH Bn), 5.01–5.04 (m, 1H, H-1), 6.17–6.23 (m,1H, NH),
7.11–7.14 (m, 2H, Harom), 7.23–7.38 (m, 43H, Harom); 13C NMR
(100 MHz): d = 19.2–19.4 (5 ꢄ CH2 cyanoethyl), 20.6 (F17C8CH2
CH2CH2N), 28.2 (t, J = 22 Hz, F17C8CH2CH2CH2N), 29.3, 30.5
(2 ꢄ CH2 succinyl), 38.4 (F17C8CH2CH2CH2N), 60.4, 60.5 (CH2 glyc-
erol), 62.0–62.2 (5 ꢄ CH2 cyanoethyl), 63.1 (CH2 glycerol), 65.5–
66.6 (10 ꢄ CH2 glycerol), 68.3 (C-6), 70.8 (C-5), 71.9 (CH2 Bn),
72.1–72.2 (4 ꢄ CH2 Bn), 73.0, 73.0 (CH2 Bn), 73.4 (CH2 Bn), 73.8–
74.0 (CH glycerol), 75.1 (CH2 Bn), 75.2–75.5 (4 ꢄ CH glycerol),
75.5 (CH2 Bn), 77.4–77.6 (C-4, CH glycerol), 79.6 (C-2), 81.5 (C-3),
96.8, 96.9 (C-1), 116.6–116.8 (5 ꢄ Cq cyanoethyl), 127.5–128.5
(CHarom), 137.2, 137.7–138.0, 138.5 (9 ꢄ Cq Bn), 171.4, 172.3
(2 ꢄ C@O succinyl); HRMS: [C117H132F17N6O35P5 + 2Na]2+ requires
1352.8484, found 1352.8479.
ate (34.5 mg, 15.4
lmol) was deprotected with Pd (0)/H2 using the
standard procedure. Glucosylated hexamer 2 (19.7 mg, 15.0
lmol,
97%) was obtained as an amorphous white solid. 31P NMR
(161.7 MHz, D2O): d = 1.2 (1P), 1.2–1.3 (4P), 1.3 (1P); 1H NMR
(600 MHz, D2O): d = 1.39–1.44 (m, 4H, 2 ꢄ CH2 hexylspacer),
1.61–1.70 (m, 4H, 2 ꢄ CH2 hexylspacer), 2.99 (t, 2H, J = 7.5 Hz,
CH2–N hexylspacer), 3.39 (at, 1H, J = 9.6 Hz, H-4), 3.52 (dd, 1H,
J = 3.9 Hz, 9.9 Hz, H-2), 3.71–3.76 (m, 4H, H-3, H-6, CH2 glycerol),
3.80–4.05 (m, 32H, H-5, H-60, 6 ꢄ CH glycerol, 11 ꢄ CH2 glycerol,
CH2–O hexylspacer), 4.14–4.17 (m, 1H, CH glycerol), 5.15 (d, 1H,
J = 3.8 Hz, H-1); 13C NMR (150 MHz, D2O): d = 25.3, 26.0, 27.5,
30.3 (4 ꢄ CH2 hexylspacer), 40.3 (CH2–N hexylspacer), 61.4 (C-6),
62.2 (CH2 glycerol), 65.2 (d, J = 6 Hz, CH2 glycerol), 66.9–67.1
(CH2–O hexylspacer, 11 ꢄ CH2 glycerol), 70.3–70.5 (5 ꢄ CH glyc-
erol,C-4), 72.4 (C-2), 72.9 (C-5), 73.8 (C-3), 77.7 (d, J = 8 Hz, CH
glycerol), 98.7 (C-1); HRMS:
1204.1941, found 1204.1948.
C
30H67NO36P6 + H+ requires
4.1.22. Bis-glucosyl-glycerol phosphate hexamer (27)
Pentamer 24 (22.5 mg, 9.52 mol) was coupled to glucosyl-glyc-
4.1.20. Glucosyl glycerol phosphate hexamer-spacer (26)
l
Hexamer 25 (145 mg, 54.6
l
mol) was coupled to spacer phos-
erol phosphoramidite 18 (3.0 equiv), oxidized, detritylated and
phoramidite 8 (2.5 equiv), oxidized and purified (FSPE) using the
purified (FSPE) using the general procedure as described above.
general procedure as described above. Hexamer 26 (147 mg,
Bis-glucosylated hexamer 27 (25.7 mg, 8.31 lmol, 87%) was ob-
48.7
lmol, 89%) was obtained as
a
colourless oil. 31P NMR
tained as a colourless oil. 31P NMR (161.7 MHz): d = ꢂ1.4 to ꢂ1.3
(161.7 MHz): d = ꢂ1.4 to ꢂ1.1 (6P); 1H NMR (400 MHz): d = 1.25–
1.40 (m, 4H, 2 ꢄ CH2 hexylspacer), 1.44–1.52 (m, 2H, CH2 hexylsp-
acer), 1.60–1.68 (m, 2H, CH2 hexylspacer), 1.68–1.76 (m, 2H,
(2P), ꢂ1.2, ꢂ1.0 (3P); 1H NMR (400 MHz): d = 1.66–1.82 (m, 2H,
F17C8CH2CH2CH2N), 1.97–2.12 (m, 2H, F17C8CH2CH2CH2N), 2.28–
2.68 (m, 15H, 2 ꢄ CH2 succinyl, 5 ꢄ CH2 cyanoethyl, CH2OH), 3.17–
3.28 (m, 2H, F17C8CH2CH2CH2N), 3.51–3.98 (m, 19H, 2 ꢄ H-2,
2 ꢄ H-3, 2 ꢄ H-4, 2 ꢄ H-5, 2 ꢄ H-6, 2 ꢄ H-60, 5 ꢄ CH glycerol, CH2
glycerol), 4.00–4.31 (m, 33H, CH glycerol, 11 ꢄ CH2 glycerol,
5 ꢄ CH2 cyanoethyl), 4.42–4.48 (m, 4H, 4 ꢄ CHH Bn), 4.56–4.64
(m, 10H, 2 ꢄ CHH Bn, 4 ꢄ CH2 Bn), 4.67–4.71 (m, 3H, 3 ꢄ CHH Bn),
4.76–4.84 (m, 5H, 5 ꢄ CHH Bn), 4.90–4.95 (m, 3H, H-1, 2 ꢄ CHH
Bn), 5.01–5.03 (m, 1H, H-1), 6.02–6.09 (m, 1H, NH), 7.09–7.16 (m,
4H, Harom), 7.22–7.38 (m, 56H, Harom); HRMS: [C144H160F17N6O40
P5 + 2Na]2+ requires 1568.9452, found 1568.9454.
F17C8CH2CH2CH2N), 1.98–2.12 (m, 2H, F17C8CH2CH2CH2N), 2.35–
2.67 (m, 16H, 2 ꢄ CH2 succinyl, 6 ꢄ CH2 cyanoethyl), 3.12–3.25
(m, 4H, CH2–N hexylspacer, F17C8CH2CH2CH2N), 3.54–3.59 (m,
1H, H-2), 3.61–3.68 (m, 2H, H-4, H-6), 3.70–3.75 (m, 1H, H-60),
3.76–3.84 (m, 5H, 5 ꢄ CH glycerol), 3.85–3.94 (m, 2H, H-3, H-5),
4.00–4.31 (m, 39H, CH glycerol, 12 ꢄ CH2 glycerol, 6 ꢄ CH2 cyano-
ethyl, CH2–O hexylspacer), 4.44–4.48 (m, 2H, 2 ꢄ CHH Bn), 4.57–
4.65 (m, 11H, CHH Bn, 5 ꢄ CH2 Bn), 4.68–4.71 (m, 2H, 2 ꢄ CHH
Bn), 4.76–4.83 (m, 2H, 2 ꢄ CHH Bn), 4.90–4.95 (m, 1H, CHH Bn),

W. F. J. Hogendorf et al. / Carbohydrate Research 356 (2012) 142–151
151
4.1.23. Bis-glucosyl-glycerol phosphate hexamer-spacer (28)
Hexamer 27 (24.5 mg, 7.92 mol) was coupled to spacer phos-
phoramidite 8 (5 equiv), oxidized and purified (FSPE) using the
76.4 (t, J = 8 Hz, CH glycerol), 77.8 (d, J = 8 Hz, CH glycerol), 98.7,
98.8 (2 ꢄ C-1); HRMS: C36H77NO41P6 + H+ requires 1366.2469,
found 1366.2474.
l
general procedure as described above. Hexamer 28 (24.3 mg,
7.06 lmol, 89%) was obtained as a
colourless oil. 31P NMR
Supplementary data
(161.7 MHz): d = ꢂ1.4 to ꢂ1.0 (6P); 1H NMR (400 MHz): d = 1.26–
1.37 (m, 4H, 2 ꢄ CH2 hexylspacer), 1.41–1.49 (m, 2H, CH2 hexylsp-
acer), 1.59–1.68 (m, 2H, CH2 hexylspacer), 1.68–1.77 (m, 2H,
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2012.02.023.
F
17C8CH2CH2CH2N), 1.97–2.11 (m, 2H, F17C8CH2CH2CH2N), 2.30–
2.66 (m, 16H, 2 ꢄ CH2 succinyl, 6 ꢄ CH2 cyanoethyl), 3.10–3.25
(m, 4H, CH2–N hexylspacer, F17C8CH2CH2CH2N), 3.54–3.94 (m,
18H, 2 ꢄ H-2, 2 ꢄ H-3, 2 ꢄ H-4, 2 ꢄ H-5, 2 ꢄ H-6, 2 ꢄ H-60, 4 ꢄ CH
glycerol), 4.00–4.30 (m, 40H, 2 ꢄ CH glycerol, 12 ꢄ CH2 glycerol,
6 ꢄ CH2 cyanoethyl, CH2–O hexylspacer), 4.40–4.48 (m, 4H,
4 ꢄ CHH Bn), 4.55–4.64 (m, 10H, 2 ꢄ CHH Bn, 4 ꢄ CH2 Bn), 4.68–
4.71 (m, 4H, 4 ꢄ CHH Bn), 4.76–4.83 (m, 4H, 4 ꢄ CHH Bn), 4.90–
4.95 (m, 2H, 2 ꢄ CHH Bn), 5.01–5.13 (m, 5H, 2 ꢄ H-1, CH2 Cbz,
NH Cbz), 6.04–6.11 (m,1H, NH), 7.08–7.15 (m, 4H, Harom), 7.22–
7.38 (m, 61H, Harom); HRMS: [C161H183F17N8O45P6 + 2H]2+ requires
1730.0305, found 1730.0312.
References
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(c) Neuhaus, F. C.; Baddiley, J. Microbiol. Mol. Biol. Rev. 2003, 67, 686–723; (d)
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851.
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2011, 6, e17839; (c) Theilacker, C.; Kropec, A.; Hammer, F.; Sava, I.; Wobser, D.;
Sakinc, T.; Codée, J. D. C.; Hogendorf, W. F. J.; Van der Marel, G. A.; Huebner, J. J.
Infect. Dis. 2012, 205, 1076–1085.
6. Pollack, J. H.; Neuhaus, F. C. J. Bacteriol. 1994, 176, 7252–7259.
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109, 749–795; (f) Zhang, W. Green Chem. 2009, 11, 911–920; For an example of
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2011, 13, 1824–1827.
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10. Complete immunological evaluation studies, including OPA inhibition assays
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4.1.24. Bis-glucosyl-glycerol phosphate hexamer (29)
Protected hexamer 28 (22.5 mg, 6.54
aqueous ammonia as described above. The intermediate hexamer
(17.1 mg, 6.42 mol, 98%) was obtained as an amorphous white so-
lmol) was treated with
l
lid. Analytical data intermediate: 31P NMR (161.7 MHz, D2O):
d = 0.9–1.2 (6P); 1H NMR (400 MHz, D2O): d = 0.76–1.10 (m, 6H,
3 ꢄ CH2 hexylspacer), 1.21–1.34 (m, 2H, CH2 hexylspacer), 2.55–
2.76 (m, 2H, CH2–N hexylspacer), 3.11–4.14 (m, 44H, 2 ꢄ H-2,
2 ꢄ H-3, 2 ꢄ H-4, 2 ꢄ H-5, 2 ꢄ H-6, 2 ꢄ H-60, 12 ꢄ CH2 glycerol,
6 ꢄ CH glycerol, CH2–O hexylspacer), 4.15–4.84 (m, 28H, 2 ꢄ H-1,
12 ꢄ CH2 Bn, CH2 Cbz), 4.96–5.04 (m, 1H, NH Cbz), 6.61–7.17
(m, 65H,
1308.4554, found 1308.4563.
(15.6 mg, 5.85 mol) was deprotected with Pd (0)/H2 using the
standard procedure. Bis-glucosylated hexamer 29 (8.43 mg,
5.71
mol, 98%) was obtained as an amorphous white solid. 31P
H
arom); HRMS: [C128H155NO43P6 + 2NH4]2+ requires
A
portion of the intermediate
l
l
NMR (161.7 MHz, D2O): d = 0.9 (1P), 1.2–1.3 (5P); 1H NMR
(600 MHz, D2O): d = 1.40–1.44 (m, 4H, 2 ꢄ CH2 hexylspacer),
1.62–1.70 (m, 4H, 2 ꢄ CH2 hexylspacer), 2.99 (t, 2H, J = 7.5 Hz,
CH2–N hexylspacer), 3.36–3.41 (m, 2H, 2 ꢄ H-4), 3.48–3.53 (m,
2H, 2 ꢄ H-2), 3.71–3.77 (m, 6H, 2 ꢄ H-3, 2 ꢄ H-6, CH2 glycerol),
3.80–4.05 (m, 33H, 2 ꢄ H-5, 2 ꢄ H-60, 5 ꢄ CH glycerol, 11 ꢄ CH2
glycerol, CH2–O hexylspacer), 4.07–4.10 (m, 1H, CH glycerol),
5.15 (d, 2H, J = 3.4 Hz, 2 ꢄ H-1); 13C NMR (150 MHz, D2O):
d = 25.5, 26.1, 27.6, 30.5 (4 ꢄ CH2 hexylspacer), 40.4 (CH2–N hex-
ylspacer), 61.2 (2 ꢄ C-6), 61.7 (CH2 glycerol), 65.3 (d, J = 5 Hz,
CH2 glycerol), 66.1 (d, J = 5 Hz, CH2 glycerol), 67.1–67.3 (CH2–O
hexylspacer, 10 ꢄ CH2 glycerol), 70.4–70.7 (4 ꢄ CH glycerol,2 ꢄ C-
4), 72.5, 72.5 (2 ꢄ C-2), 72.9, 73.0 (2 ꢄ C-5), 73.9, 74.0 (2 ꢄ C-3),
13. (a) Adinolfi, M.; Barone, G.; Iadonisi, A.; Shiattarella, M. Tetrahedron Lett. 2002,
43, 5573–5577; (b) Adinolfi, M.; Iadonisi, A.; Schiattarella, M. Tetrahedron Lett.
2003, 44, 6479–6482; (c) Walvoort, M. T. C.; Lodder, G.; Overkleeft, H. S.;
Codée, J. D. C.; Van der Marel, G. A. J. Org. Chem. 2010, 75, 7990–8002.