Synthesis of an α-kojibiosyl substituted glycerol teichoic acid hexamer

Article abstract of DOI:10.1016/j.bmc.2010.03.071

In this paper the synthesis of an Enterococcus Faecalis teichoic acid (TA) hexamer is presented. The key kojibiosyl-glycerol phosphoramidite building block was obtained by condensation of thioglucose donors, provided with various protecting groups at the

Full text of DOI:10.1016/j.bmc.2010.03.071

Bioorganic & Medicinal Chemistry 18 (2010) 3668–3678
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of an
a-kojibiosyl substituted glycerol teichoic acid hexamer
*
Wouter F. J. Hogendorf, Leendert J. Van den Bos, Herman S. Overkleeft, Jeroen D. C. Codée ,
*
Gijsbert A. Van der Marel
Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
In this paper the synthesis of an Enterococcus Faecalis teichoic acid (TA) hexamer is presented. The key
kojibiosyl-glycerol phosphoramidite building block was obtained by condensation of thioglucose donors,
provided with various protecting groups at the C2 hydroxyl function with an orthogonally protected glyc-
erol acceptor. After selective deprotection, the resulting 1,2-cis-linked pseudodisaccharide acceptor was
Received 18 January 2010
Revised 23 March 2010
Accepted 26 March 2010
Available online 31 March 2010
coupled to an a-directing thioglucose donor, giving the corresponding pseudotrisaccharide, which is then
transformed to a phosphoramidite synthon. The kojibiosyl-glycerol phosphoramidite in combination
with a glycerolphosphoramidite, an aminohexylphosphoramidite and dibenzylglycerol were coupled to
a fully protected glycerol TA hexamer, using chemistry that can be amended for future automated syn-
thesis. Global deprotection afforded the target hexamer kojibiosyl-glycerol containing TA (1).
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Phosphoramidite
Teichoic acid
Glycosylation
Carbohydrate
1. Introduction
with the advent of vancomycin-resistant strains an increasing
amount of E. faecalis infections is being reported.6
Teichoic acids (TAs) are major constituents of the cell wall of
most Gram-positive bacteria. TAs are polyanionic glycopolymers
that can be divided in wall teichoic acids, which are covalently
linked to the peptidoglycan layer, and lipoteichoic acids, which
are anchored in the bacterial membrane.1 The backbone of these
anionic polymers is mostly composed of repeating alditol (glycerol,
ribitol) phosphates. These monomers are often (randomly) deco-
Because of the repetitive nature of the TA structure, we elected
to aim for a solution-phase synthetic strategy that in the future can
be translated to an automated solid-phase synthesis.7 We here
rated with D
-alanine or carbohydrate residues.2 Situated on the
exterior of the bacterial cell wall, many teichoic acids are sus-
pected or proven points of recognition for both the mammalian
adaptive and the innate immune systems.1–3
In the elucidation of the molecular mode of action of TAs in
effecting an immune response, pure and well-defined fragments
would be valuable tools. We therefore set out to develop a syn-
thetic methodology to efficiently assemble this type of glycopoly-
mers. In this respect it is of interest to note that structure and
activity studies of synthetic derivatives of Staphylococcus aureus
lipoteichoic acid (LTA) fragments led to the discovery of ligands
for the human TLR-2 receptor.4 Our first attention was directed
at the TA of Enterococcus faecalis, which is build up from 1,3-
poly(glycerolphosphate) randomly decorated at the C-2 positions
with
D
-alanine, kojibiose (
a-D-glucopyranosyl-(1?2)-a-D-glucose),
or 6,60-di-alanyl-
a
-kojibiose as depicted in Figure 1.5 E. faecalis is a
commensal bacterium and generally of low virulence. However,
* Corresponding authors. Tel.: +31 71 527 4275; fax: +31 71 527 4307 (J.D.C.C.).
E-mail addresses: jcodee@chem.leidenuniv.nl (J.D.C. Codée), marel_g@chem.lei-
denuniv.nl (Gijsbert A. Van der Marel).
Figure 1. E. Faecalis TA general structure.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.071

W. F. J. Hogendorf et al. / Bioorg. Med. Chem. 18 (2010) 3668–3678
3669
present the construction of an E. faecalis kojibiosyl-TA hexamer uti-
lising synthons that are coupled with phosphoramidite chemistry.
phosphitylation using 2-cyanoethyl-N,N-diisopropylchlorophosph-
oramidite 11 provided building block 3.
With the first two building blocks in hand we directed our
attention to the synthesis of the
a-kojibiosyl-glycerol synthon
2. Results and discussion
(4), containing two 1,2-cis glucosidic linkages. Although tremen-
dous progress has been made in the stereoselective construction
of glycosidic bonds, the instalment of two 1,2-cis-glucosyl linkages
still presents a significant challenge. Recently Boons and co-work-
ers revealed an elegant approach to this long standing problem, by
As retrosynthetically depicted in Scheme 1, TA hexamer 1 can
be assembled from four synthons: dibenzyl glycerol 2, which will
be the starting building block; glycerol phosphoramidite 3 and
kojibiosyl-glycerol phosphoramidite 4, which will be used for
chain elongation and aminohexylphosphoramidite 5, which will
be used as the terminal building block. The primary amino group
of the hexyl spacer presents a chemoselective ligation handle for
attachment of visualization tags, (micro)array slides or carrier pro-
teins. The hydroxyl functions and the amino group of the amin-
ohexyl spacer are masked with benzyl type protecting groups,
which facilitates global deprotection in the final stage of the hexa-
mer assembly by hydrogenolysis. As a temporary protecting group
we selected the dimethoxytrityl (DMTr) group, which is commonly
employed in contemporary DNA and RNA synthesis protocols and
can be cleaved under mildly acidic conditions.8
The synthesis of the four building blocks is depicted in Scheme
2. Dibenzylglycerol 2 was synthesized according to literature pro-
cedures from solketal 6 (Scheme 2A).4b,e,9 Solketal 6 also served as
starting compound in the synthesis of glycerolphosphoramidite 3.
Allylation of the free hydroxyl function, acidic hydrolysis of the iso-
propylidene and subsequent selective silylation of the primary
alcohol yielded partially protected glycerol 7 (Scheme 2B).4b The
remaining hydroxyl in compound 7 was benzylated to provide
the fully protected glycerol in excellent yield. The product was con-
taminated with a minor amount (ꢀ3%) of 1-O-benzyl-2-O-tert-
butyldiphenylsilyl-3-O-allyl-sn-glycerol, which resulted from silyl
migration during the basic etherification and could not be removed
by silica gel chromatography. To liberate the C3 hydroxyl, the allyl
ether was isomerized using Ir(COD)(Ph2MeP)2PF6.10 Subsequent
enol ether cleavage using iodine and aqueous NaHCO3 afforded
glycerol 8 in 71% over two steps. Dimethoxytritylation of the pri-
mary alcohol furnished glycerol 9, which was desilylated to pro-
vide alcohol 10. At this stage the product could be separated by
silica gel chromatography from its regioisomeric impurity. Finally,
installing
a-directing S-ethylmandelate or (1S)-phenyl(thio-
phenyl)ethyl ethers on the 20 hydroxyl of glucose donors.11 Unfor-
tunately, the assembly of the kojibiose-glycerol pseudotri
saccharide requires the glycosylation of the first glucose residue
at the C-2 hydroxyl and the latter ether protecting groups cannot
be removed selectively while keeping the other benzyl ethers in-
tact. Alternative methods which have been used to construct 1,2-
cis glucosidic linkages include Lemieux’s in situ anomerization pro-
tocol12 and the employment of large substituents such as the DMTr
group at the glucosyl C-6 hydroxyl.13 Given the relatively low reac-
tivity of the secondary alcohol function which has to be glucosylat-
ed we deemed the former approach unsuitable. We dismissed the
latter approach because this would exclude the use of trityl groups
in the glycerol part of the pseudotrisaccharide, which was a prere-
quisite in the design of our protecting group strategy (vide supra).
An alternative approach to the formation of 1,2-cis glucosidic link-
ages has been described by Crich et al. using benzylidene protected
thioglucosyl donors.14 We explored three 4,6-O-benzylidene func-
tionalized glucosyl donors, having either a C2 para-methoxybenzyl
(PMB, 13a), a C2 para-azidobenzyl (PAB, 13b) or a C2 trimethylsilyl
(TMS, 13c) protecting group (Scheme 2C). The installment of the
para-methoxylbenzyl group in phenyl 3-O-benzyl-4,6-O-benzyli-
dene-b-D
-thioglucose 1215 led to benzylidene glucoside 13a in
88%. This thioglycoside was then condensed with glycerol acceptor
7 using the Ph2SO/Tf2O activator system16 to provide the interme-
diate pseudodisaccharide in good yield and excellent selectivity (a/
b = 10/1). Subsequent treatment of the fully protected adduct with
DDQ gave alcohol 14 in moderate yield (56%), resulting in a 37% to-
tal yield of
a-glucosyl glycerol 14 from thioglucoside 12. A similar
series of reactions was employed to prepare pseudodisaccharide 14
Scheme 1. Structures of TA hexamer 1 and (phosphoramidite) synthons 2, 3, 4 and 5.

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W. F. J. Hogendorf et al. / Bioorg. Med. Chem. 18 (2010) 3668–3678
Scheme 2. Synthesis of A: dibenzylglycerol 2, B: glycerol phosphoramidite 3, C: kojibiosyl-glycerol synthon 4, and D: aminohexyl building block 5.
with the use of the para-azido benzyl ether17 instead of the para-
methoxy benzyl group as a non-participating group at C-2. In this
case (13b), the benzylation reaction proceeded uneventfully and
the ensuing glycosylation reaction provided the cis-coupled prod-
the transformation of 3-O-allyl-2-O-benzyl-1-O-TBDPS-sn-glycerol
into phosphoramidite 3. Briefly, the allyl ether in 16 was removed
by iridium catalyzed isomerisation and iodine mediated cleavage
of the intermediate enol ether. The DMTr group was installed, after
which the TBDPS ether was removed by fluoride treatment and the
primary alcohol was phosphitylated to complete the synthesis of
the kojibiosyl-glycerol 4. Finally, benzyloxycarbonyl protected
aminohexanol 20 was treated with DIPEA and chlorophosporami-
dite 11 to complete the set of target synthons with phosphorami-
dite 5 (Scheme 2D).
With all necessary building blocks in hand the assembly of the
hexamer was undertaken (Scheme 3). In the first step dibenzyl-
glycerol 2 and glycerol phosphoramidite 3 were coupled with the
use of 4,5-dicyanoimidazole (DCI) in acetonitrile. After oxidation
of the intermediate phosphite (I2 in THF/pyridine 4/1), the crude
intermediate was detritylated using dichloroacetic acid and trieth-
ylsilane in dry DCM, giving dimer 21 in 80% yield. Repetition of this
reaction sequence led to the consecutive formation of trimer 22 in
82% from 21, tetramer 23 in 81% from 22 and pentamer 24 in 62%
from 23. Next, kojibiosyl-glycerol phosphoramidite 4 was coupled
to the glycerolphosphate chain and after iodine mediated oxida-
tion hexamer 25 was obtained in excellent yield (86%). This indi-
cates that the relatively bulky kojibiosyl substituent did not have
uct in good selectivity (62%,
a/b = 7/1). Unfortunately, liberation
of the C2 hydroxyl via a reduction–oxidation sequence provided
target alcohol 14 in only 39%, to complete the four step sequence
in a total yield of 22% of 14 from building block 12. The last non-
participating group we tried was the trimethylsilyl ether (TMS).
Donor 13c was obtained quantitatively from 12, and used in the
subsequent glycosylation reaction without purification. The cou-
pling of TMS protected thioglucose 13c and glycerol 7 was imme-
diately followed by treatment of the resulting pseudodisaccharide
(ꢀ6:1 mixture of
a and b-anomers) with sodium carbonate in
methanol to yield 14 in 46% over the three steps, using a single
chromatographical purification step, making this the most efficient
route.
With the first 1,2-cis glycosidic linkage in place, we continued
our synthesis with the condensation of dibenzyl benzylidene thi-
oglucoside 15 and alcohol 14. Gratifyingly, the pseudotrisaccharide
(16) was obtained as a single diastereoisomer in 82% yield. This tri-
saccharide was transformed into the phosphoramidite building
block 4 following the same sequence of reactions as described for

W. F. J. Hogendorf et al. / Bioorg. Med. Chem. 18 (2010) 3668–3678
3671
Scheme 3. Elongation towards fully protected hexamer 27 and subsequent deprotection yielding TA 1.
an adverse effect on the formation of the mixed phosphotriester.
To avoid unwanted acidolysis of the benzylidene functionalities
in 25 we used the mild pyridinium para-toluenesulphonate
(PPTS)/MeOH cocktail for the cleavage of the DMTr group.18 Alco-
hol 26 was obtained in 70% yield when 1 mg/ml PPTS in MeOH/
DCM (9/1) was employed. Subsequent coupling of spacer phospho-
ramidite 5 and oxidation gave the fully protected target compound
27 in 77% yield.
The deprotection sequence started with ammonolysis of the
cyanoethyl groups in 27, giving the hexameric phosphodiester
quantitatively. Finally, all benzyl ethers, the two benzylidene ace-
tals and the benzyloxycarbamate were removed using H2/Pd(0) in
water/dioxane/AcOH. After size-exclusion chromatography, re-
peated lyophilization, and ion exchange the target E. faecalis TA
hexamer (1) was obtained as the per sodium salt in 76% yield.
ried out dry, under an argon atmosphere, at ambient temperature,
unless stated otherwise. Tf2O was distilled over P2O5 using flame-
dried glass-ware. 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 140 °C. Some unsatu-
rated compounds were visualized by spraying with a solution of
KMnO4 (2%) and K2CO3 (1%) in water. Optical rotation measure-
ments (½a 2D0
ꢂ
) 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 recorded on a
Shimadzu FT-IR 8300. 31P, 1H, and 13C NMR spectra were recorded
with a Bruker AV 400 (161.7, 400 and 100 MHz, respectively) or a
Bruker DMX 600 (600 and 150 MHz, respectively). NMR spectra
were recorded in CDCl3 with chemical shift (d) relative to tetra-
methylsilane, unless stated otherwise. High resolution mass spec-
3. Conclusion
In summary, we have described the synthesis of a kojibiose con-
taining Enterococcus faecalis teichoic acid hexamer. The teichoic
acid fragment was constructed by coupling of suitably protected
tra were recorded by direct injection (2 ll of a 2 lM solution in
water/acetonitrile; 50/50; v/v and 0.1% formic acid) 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 250 °C) with resolution
R = 60,000 at m/z 400 (mass range m/z = 150–2000) and dioc-
tylphthalate (m/z = 391.28428) as a lock mass. The high resolution
mass spectrometer was calibrated prior to measurements with a
calibration mixture (Thermo Finnigan). TLC-MS (ESI) spectra of
phosphoramidites 3, 4 and 5 were obtained by analysis of TLC-
plates (Schleicher & Schuell, treated with Et3N before applying
the phosphoramidites) with a CAMAG TLC-MS interface coupled
to a Perkin Elmer Sciex API 165 mass instrument.
glycerol and
The key kojibiosyl synthon was obtained via a sequence of reac-
tions involving two successive stereoselective -glucosylations.
a-kojibiosyl substituted glycerol phosphoramidites.
a
The use of a benzylidene thioglucose donor, temporarily protected
at the C2-hydroxyl with a TMS-group proved to be the most effi-
cient in the synthesis of the kojibiosyl-glycerol pseudotrisaccha-
ride. The chemistry used for the assembly of the teichoic acid
hexamer is compatible with an automated solid-phase approach,
and we are currently studying the efficiency of this in the solid-
phase synthesis of differentially substituted TAs.
4. Experimental
4.1. General
4.2. Experimental procedures
4.2.1. 1-O-(tert-Butyldiphenylsilyl)-2-O-benzyl-sn-glycerol (8)
Silyl ether 7 (22.8 g, 61.5 mmol) was dissolved in DMF (200 ml)
and cooled to 0 °C. After the addition of benzyl bromide (11.1 ml,
All chemicals (Acros, Fluka, Merck, Schleicher & Schuell, Sigma–
Aldrich, Genscript) were used as received and reactions were car-

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W. F. J. Hogendorf et al. / Bioorg. Med. Chem. 18 (2010) 3668–3678
92.5 mmol) and heptane flushed NaH (60% dispersion in mineral
oil, 3.70 g, 92.5 mmol), the mixture was allowed to stir for 1.5 h.
H2O (50 ml) was added, and after addition of Et2O (1.0 l), the mix-
ture was washed with H2O (2 ꢃ 200 ml) and brine (200 ml). The
organic layer was dried over MgSO4 and concentrated in vacuo. Re-
peated co-evaporation of the residue with H2O (5 ꢃ 100 ml) and
toluene (5 ꢃ 100 ml), followed by column chromatography (tolu-
ene/PE) resulted in the benzylated adduct (28.3 g, 61.4 mmol,
100%, containing ꢀ3% 1-O-benzyl-2-O-tert-butyldiphenylsilyl-3-
7.60–7.62 (m, 4H, Harom); 13C NMR (100 MHz): d = 19.1 (Cq t-butyl),
26.8 (3 ꢃ CH3 TBDPS), 55.1 (2 ꢃ OMe) 63.7 (CH2 glycerol), 63.9
(CH2 glycerol), 72.3 (CH2 Bn), 79.3 (CH glycerol), 86.0 (Cq DMTr),
113.0, 113.6 (CHarom), 126.1–130.3 (CHarom), 133.4, 133.5 (2 ꢃ Cq
phenyl), 136.3 (CHarom), 136.4, 138.9, 145.1, 158.3 (Cq Bn, 5 ꢃ Cq
DMTr); HRMS: C47H50O5Si+Na+ requires 745.3320, found 745.3322.
4.2.3. 2-O-Benzyl-3-O-(4,40-dimethoxytrityl)-sn-glycerol (10)
To a solution of glycerol derivative 9 (25.5 g, 35.3 mmol) in THF
(200 ml), was added TBAF (1.0 M solution in THF, 53 ml). After stir-
ring for 3 h at room temperature, the solvent was removed in va-
cuo. The residue was purified by column chromatography
(EtOAc/PE/Et3N), affording primary alcohol 10 (16.4 g, 33.8 mmol,
O-allyl-sn-glycerol) as
a
colourless oil. (analytical data:
½ ꢂ
a 2D0
(CHCl3): ꢄ10.4; IR (neat): 737, 924, 1103, 1427, 2856 cmꢄ1
;
1H
NMR (400 MHz): d = 1.05 (s, 9H, 3 ꢃ CH3 TBDPS), 3.55–3.71 (m,
3H, CH glycerol, 2 ꢃ CHH glycerol), 3.77–3.80 (m, 2H, 2 ꢃ CHH
glycerol), 3.99 (dt, 2H, J = 1.5 Hz, 1.5 Hz, 5.5 Hz, OCH2–CH = CH2),
4.64 (s, 2H, CH2 Bn), 5.16 (ddd, 1H, J = 1.4 Hz, 3.1 Hz, 10.4 Hz, –
CH@CHH), 5.26 (ddd, 1H, 1,7 Hz, 3.4 Hz, 17.3 Hz, –CH@CHH),
5.86–5.93 (m, 1H, –CH@CH2), 7.18–7.43 (m, 11H, Harom), 7.66–
7.71 (m, 4H, Harom); 13C NMR (100 MHz): d = 19.2 (Cq t-butyl),
26.8 (3 ꢃ CH3 TBDPS), 63.5 (CH2 glycerol), 70.2 (CH2 glycerol),
72.2, 72.3 (OCH2–CH@CH2, CH2 Bn), 78.8 (CH glycerol), 116.8 (–
CH@CH2), 127.4, 127.7, 128.2, 129.6 (CHarom), 133.5 (2 ꢃ Cq phe-
nyl), 134.8 (–CH@CH2), 135.6 (CHarom), 138.8 (Cq Bn)). A solution
of the fully protected intermediate (9.21 g, 20.0 mmol) in freshly
distilled THF (125 ml) was stirred under argon atmosphere for
30 min. After addition of (1,5-cyclooctadiene)-bis-(methyldiphe-
96%) as colourless oil. ½a D20
ꢂ
(MeOH): +10.6; IR (neat): 829, 1034,
1177, 1250, 1508, 1607, 2359 cmꢄ1
;
1H NMR (400 MHz): d = 2.02
(br s, 1H, OH), 3.21–3.31 (m, 2H, 2 ꢃ CHH glycerol), 3.63–3.67
(m, 2H, CH glycerol, CHH glycerol), 3.70–3.74 (m, 7H, 2 ꢃ OMe,
CHH glycerol), 4.53 (d, 1H, J = 11.6 Hz, CHH Bn), 4.66 (d, 1H,
J = 12.0 Hz, CHH Bn), 6.80 (ad, 4H, J = 8.8 Hz, Harom), 7.16–7.38
(m, 12H, Harom), 7.44 (d, 2H, J = 7.6, Harom); 13C NMR (100 MHz):
d = 55.0 (2 ꢃ OMe), 63.0 (CH2 glycerol), 63.3 (CH2 glycerol), 72.0
(CH2 Bn), 78.9 (CH glycerol), 86.4 (Cq DMTr), 113.2 (CHar-
om),126.7–129.9 (CHarom), 135.9, 138.2, 144.7, 158.4 (Cq Bn,
5 ꢃ Cq DMTr); HRMS: C31H32O5+Na+ requires 507.3142, found
507.3135.
nylphosphine)
iridium(I)
hexafluorophosphate
(423 mg,
2.5 mol %), the solution turned red. The solution was then purged
with H2 (g), until the colour disappeared (ꢀ1.5 min), and was al-
lowed to stir for 2 h under argon atmosphere. The solution was di-
luted with THF (250 ml) and, after addition of satd aq NaHCO3
(300 ml) and I2 (7.61 g, 30.0 mmol), the mixture was allowed to stir
overnight. The mixture was diluted with EtOAc (1.5 l) and washed
with sat. aq. NaS2O3 (300 and 200 ml) and brine (200 ml). The or-
ganic layer was dried over MgSO4 and concentrated in vacuo. Puri-
fication of the residue, using column chromatography (EtOAc/PE),
yielded 8 (6.26 g, 14.9 mmol, 75%, containing ꢀ3% 1-O-benzyl-2-
4.2.4. 1-([N,N-Diisopropylamino]-2-cyanoethylphosphite)-2-O-
benzyl-3-O-(4,40-dimethoxytrityl)-sn-glycerol (3)
To a solution of 10 (3.19 g, 6.59 mmol) and DIPEA (1.72 ml,
9.88 mmol) in DCM (65 ml) were added activated MS 3 Å and
(N,N-diisopropylamino)-2-cyanoethyl-chlorophosphite (11, 1.82 g,
7.90 mmol). After stirring for 2 h, the reaction was quenched with
H2O (5.0 ml) and washed with H2O (50 ml) and brine (50 ml),
respectively. The organic layer was dried over Na2SO4 and concen-
trated in vacuo. Column chromatography (EtOAc/heptane/Et3N)
gave phosphoramidite 3 (3.74 g, 5.34 mmol, 81%, mixture of dia-
stereoisomers) as colourless oil. 31P NMR (161.7 MHz, CD3CN):
d = 149.2; 1H NMR (400 MHz, CD3CN): d = 1.08 (t, 6H, J = 6.5 Hz,
2 ꢃ CH3 isopropylamino), 1.14 (dd, 6H, J = 1.8 Hz, 6.8 Hz, 2 ꢃ CH3
isopropylamino), 2.51–2.56 (m, 2H, CH2 cyanoethoxy), 3.17–3.24
(m, 2H, 2 ꢃ CH isopropylamino), 3.50–3.58 (m, 2H, 2 ꢃ CHH glyc-
erol), 3.64–3.81 (m, 11H, CH glycerol, 2 ꢃ CHH glycerol, CH2 cyano-
ethoxy, 2 ꢃ OMe), 4.61–4.68 (m, 2H, CH2 Bn), 6.83 (d, 4H,
J = 8.9 Hz, Harom), 7.19–7.39 (m, 12H, Harom), 7.45–7.47 (m, 2H,
Harom); TLC-MS: C40H49N2O6P+H+ requires 685.34, found 685.5.
O-tert-butyldiphenylsilyl-sn-glycerol) as slightly yellow oil. ½a D20
ꢂ
(CHCl3): ꢄ22.0; IR (neat): 739, 824, 1113, 1427, 2361,
2858 cmꢄ1
;
1H NMR (400 MHz): d = 1.06 (s, 9H, 3 ꢃ CH3 TBDPS),
2.05 (t, 1H, J = 6.2 Hz, OH), 3.60–3.82 (m, 5H, CH glycerol,
2 ꢃ CH2 glycerol), 4.51 (d, 1H J = 11.7 Hz, CHH Bn), 4.63 (d, 1H,
J = 11.7 Hz, CHH Bn), 7.25–7.45 (m, 11H, Harom), 7.66–7.68 (m,
4H, Harom); 13C NMR (100 MHz): d = 19.2 (Cq t-butyl), 26.8
(3 ꢃ CH3 TBDPS), 62.8 (CH2 glycerol), 63.5 (CH2 glycerol), 72.1
(CH2 Bn), 79.6 (CH glycerol), 127.7, 128.4, 129.8 (CHarom), 133.1,
133.2 (2 ꢃ Cq phenyl), 135.6 (CHarom), 138.3 (Cq Bn); HRMS:
C26H32O3Si+Na+ requires 443.2013, found 443.2013.
4.2.5. Phenyl 2-O-(4-methoxybenzyl)-3-O-benzyl-4,6-O-benzyl-
idene-1-thio-b-D-glucopyranoside (13a)
4.2.2. 1-O-(tert-Butyldiphenylsilyl)-2-O-benzyl-3-O-(4,40-dime-
thoxytrityl)-sn-glycerol (9)
To a cooled (0 °C) solution of thioglucose 12 (18.3 g, 40.5 mmol)
in THF (250 ml) were added, respectively, p-methoxybenzyl chlo-
ride (6.03 ml, 44.5 mmol) and NaH (60% dispersion in mineral oil,
1.78 g, 44.5 mmol). After stirring for 5 h, the mixture was
quenched with MeOH (5.0 ml), diluted with EtOAc (1.0 l) and
washed with, satd aq NaHCO3 (400 ml) and brine (400 ml). The or-
ganic layer was dried with MgSO4 and concentrated in vacuo, after
which crystallization (EtOAc/PE) yielded fully protected thioglu-
cose derivative 13a (20.4 g, 35.8 mmol, 88%) as white solid. Mp:
Alcohol 8 (6.18 g, 14.7 mmol) was dissolved in DCM (100 ml)
and cooled to 0 °C. Added were Et3N (3.05 ml, 22.0 mmol) and
4,4’-dimethoxytrityl chloride (DMTr–Cl, 5.47 g, 16.2 mmol),
respectively, and the mixture was allowed to stir overnight at room
temperature. The reaction was quenched by the addition of H2O
(3 ml), and stirred for 15 min. After washing with H2O (30 ml)
and brine (30 ml), the organic layer was dried over Na2SO4 and
concentrated in vacuo. The resulting oil was further purified by col-
146–147 °C; ½a 2D0
ꢂ
(CHCl3): ꢄ11.2; IR (neat): 748, 818, 1030, 1088,
umn chromatography (EtOAc/PE/Et3N), giving
9
(10.3 g,
1250, 1516 cmꢄ1 1H NMR (400 MHz): d = 3.45 (m, 1H, H-5), 3.50
;
14.2 mmol, 97%, containing ꢀ3% 1-O-benzyl-2-O-tert-butyldiphen-
(dd, 1H, J = 8.4 Hz, 9.7 Hz, H-2), 3.69 (at, 1H, J = 9.4 Hz, H4), 3.76–
3.84 (m, 5H, OMe, H-3, H-6), 4.37 (dd, 1H, J = 5.0 Hz, 10.5 Hz, H-
6), 4.73–4.81 (m, 4H, 3 ꢃ CHH Bn, H-1), 4.94 (d, 1H, J = 11.2 Hz,
CHH Bn), 5.57 (s, 1H, CH benzylidene), 6.85–6.88 (m, 2H, Harom),
7.26–7.39 (m, 13H, Harom), 7.46–7.54 (m, 4H, Harom); 13C NMR
(100 MHz): d = 55.3 (OMe), 68.7 (C-6), 70.2 (C-5), 75.3, 75.5 (CH2
Bn, CH2 PMB), 80.1 (C-2), 81.4 (C-4), 83.0 (C-3), 88.3 (C-1), 101.1
ylsilyl-3-O-(4,4’-dimethoxytrityl)-sn-glycerol) as colourless oil. ½a D20
ꢂ
(MeOH): +2.0; IR (neat): 827, 1036, 1113, 1250, 1508, 1609, 2359,
2930 cmꢄ1
;
1H NMR (400 MHz): d = 0.97 (s, 9H, 3 ꢃ CH3 TBDPS),
3.23–3.31 (m, 2H, 2 ꢃ CHH glycerol), 3.71–3.78 (m, 9H, 2 ꢃ OMe,
2 ꢃ CHH glycerol, CH glycerol), 4.66 (add, 2H, J = 12.2 Hz, 13.4 Hz,
CH2 Bn), 6.74–6.83 (m, 4H, Harom), 7.00–7.45 (m, 20H, Harom),

W. F. J. Hogendorf et al. / Bioorg. Med. Chem. 18 (2010) 3668–3678
3673
(CH benzylidene), 113.8 (CHarom), 126.0–129.9 (CHarom), 130.2 (Cq
S-phenyl), 132.2 (CHarom), 133.2, 137.2 138.3 (Cq Bn, Cq PMB, Cq
benzylidene), 159.4 (Cq PMB); HRMS: C34H34O6S+H+ requires
571.2149, found 571.2147.
78.2 (C-3, C-4), 78.6 (CH glycerol), 82.1 (C-2), 97.2 (C-1), 101.2
(CH benzylidene), 113.7 (CHarom), 116.9 (–CH@CH2), 126.1–129.7
(CHarom),130.4, 133.2, 133.2, 134.7, 135.5, 137.5, 138.9 (CHarom, –
CH@CH2, 2 ꢃ Cq phenyl, Cq PMB, Cq Bn, Cq benzylidene), 159.2 (Cq
PMB); HRMS: C50H58O9Si+Na+ requires 853.3742, found
853.3745). A combination of batches of the fully protected pesud-
odisaccharide (8.81 g, 10.6 mmol) was dissolved in DCM (100 ml)
and cooled to 0 °C. After adding H2O (5.0 ml) and 2,3-dichloro-
5,6-dicyano-1,4-benzoquinone (DDQ, 2.88 g, 12.7 mmol), respec-
tively, the mixture was stirred vigorously for 4 h, before it was
quenched by the addition of satd aq NaHCO3 (25 ml). The mixture
was diluted with EtOAc (500 ml) and washed with satd aq NaHCO3
(3 ꢃ 200 ml) and brine, respectively, (3 ꢃ 150 ml) in order to re-
move the bulk of DDQH2. The organic layer was dried over MgSO4
and concentrated in vacuo. The crude product was purified by col-
umn chromatography (EtOAc/PE). Alcohol 14 (4.23 g, 5.95 mmol,
56%) was obtained as pale yellow oil.
4.2.6. Phenyl 2-O-(4-azidobenzyl)-3-O-benzyl-4,6-O-benzyl-
idene-1-thio-b-
D-glucopyranoside (13b)
To cooled (0 °C) solution of thioglucose 12 (895 mg,
a
1.99 mmol) in DMF (20 ml) were added p-azidobenzyl bromide
(634 mg, 2.99 mmol) and NaH (60% dispersion in mineral oil,
127 mg, 3.18 mmol), respectively. After stirring for 1.5 h, MeOH
(2.0 ml) was added before the mixture was diluted with Et2O
(80 ml) and washed with H2O (20 ml) and brine (20 ml). The or-
ganic layer was dried with MgSO4 and concentrated in vacuo.
The residue was taken up in EtOAc (5 ml) and crystallized upon
addition of PE, yielding 13b (1.07 g, 1.84 mmol, 93%) as ochreous
solid. Mp: 150–155 °C (decomp.); ½a D20
ꢂ
(CHCl3): +3.0; IR (neat):
991, 1088, 1508, 2112 cmꢄ1
;
1H NMR (400 MHz): d = 3.44–3.51
From 13b: solution of donor 13b (145 mg, 0.250 mmol), TTBP
(155 mg, 0.625 mmol) and Ph2SO (60.7 mg, 0.300 mmol) in freshly
distilled DCM (8.0 ml), together with activated MS 3 Å, was stirred
under argon at rt for 30 min. Subsequently, the mixture was cooled
to ꢄ78 °C and stirred for another 15 min. After the addition of Tf2O
(m, 2H, H-2, H-5), 3.70 (at, 1H, J = 9.4 Hz, H4), 3.78–3.84 (m, 2H,
H-3, H-6), 4.39 (dd, 1H, J = 5.2 Hz, 10.4 Hz, H-6), 4.73–4.82 (m,
4H, 3 ꢃ CHH Bn, H-1), 4.95 (d, 1H, J = 11.2 Hz, CHH Bn), 5.59 (s,
1H, CH benzylidene), 6.96–7.00 (m, 2H, Harom), 7.26–7.40 (m,
13H, Harom), 7.45–7.53 (m, 4H, Harom); 13C NMR (100 MHz):
d = 68.7 (C-6), 70.2 (C-5), 75.1, 75.3 (CH2 Bn, CH2 p-N3Bn), 80.3
(C-2), 81.5 (C-4), 82.9 (C-3), 88.2 (C-1), 101.1 (CH benzylidene),
118.9 (CHarom), 126.0–132.2 (CHarom), 133.0, 134.8, 137.2, 138.2,
139.5 (Cq S-phenyl, Cq Bn, 2 ꢃ Cq p-N3Bn, Cq benzylidene); HRMS:
C34H31N3O5S+H+ requires 582.2057, found 582.2056.
(50.5
ll, 0.300 mmol), the mixture was stirred for 45 min at ꢄ50 °C
before it was cooled down to ꢄ78 °C. After the addition of a solution
of glycerol acceptor 7 (111 mg, 0.300 mmol) in DCM (2.5 ml), the
mixture was stirred at ꢄ75 °C for 1 h, before it was allowed to grad-
ually (ꢀ3 h) warm to room temperature. The reaction was
quenched by the addition of Et3N (0.2 ml, 1.4 mmol) and stirred
for 30 min. The mixture was diluted with DCM (30 ml) and after
washing with satd aq NaHCO3 (15 ml) and brine (15 ml), the organ-
ic layer was dried over MgSO4 and concentrated in vacuo. Purifica-
tion of the resulting oil by size-exclusion chromatography
(sephadex LH-20, MeOH/DCM 1/1), followed by column chroma-
tography (EtOAc/PE) gave the fully protected intermediate
4.2.7. 1-O-(tert-Butyldiphenylsilyl)-2-O-(3-O-benzyl-4,6-O-ben-
zylidene-a-D-glucopyranosyl)-3-O-allyl-sn-glycerol (14)
From 13a: A solution of donor 13a (2.85 g, 4.99 mmol), TTBP
(2.86 g, 11.5 mmol) and Ph2SO (1.11 g, 5.50 mmol) in freshly dis-
tilled DCM (100 ml), together with activated MS 3 Å, was stirred
under argon at rt for 30 min. Subsequently, the mixture was cooled
to ꢄ75 °C and stirred for another 15 min. After the addition of Tf2O
(0.93 ml, 5.5 mmol), the mixture was stirred for 45 min at ꢄ75 °C
and, subsequently, glycerol acceptor 7 (2.22 g, 5.99 mmol) in
DCM (10 ml) was added. After stirring for 60 min at ꢄ75 °C, the
mixture was allowed to, gradually, warm to room temperature
(ꢀ3 h). The reaction was quenched upon addition of Et3N (2.0 ml,
14 mmol) and stirred for 30 min. After washing the mixture with
satd aq NaHCO3 (30 ml) and brine (30 ml), the organic layer was
dried over MgSO4 and concentrated in vacuo. The resulting oil
was dissolved in pyridine (30 ml) and, after the addition of Ac2O
(7 ml), stirred for 2 h. After removal of the solvents in vacuo, the
residue was dissolved in EtOAc (200 ml) and washed with satd
aq NaHCO3 (2 ꢃ 60 ml) and brine (60 ml). The organic layer was
dried over MgSO4 and the solvent removed under reduced pres-
sure. Purification of the resulting oil by column chromatography
(EtOAc/PE) gave the fully protected product (3.16 g, 3.80 mmol,
(131 mg, 0.156 mmol, 62%,
a
/b mixture, ꢀ7/1), as pale yellow oil.
Analytical data: 1H NMR
a-product (400 MHz): d = 1.06 (s, 9H,
3 ꢃ CH3 TBDPS), 3.49–4.04 (m, 13H, CH glycerol, 2 ꢃ CH2 glycerol,
H-2, H-3, H-4, H-5, 2 ꢃ H-6, OCH2–CH@CH2), 4.68 (d, 1H,
J = 12.0 Hz, CHH Bn), 4.74 (d, 1H, J = 12.0 Hz, CHH Bn), 4.80 (d, 1H,
J = 12.0 Hz, CHH Bn), 4.88 (d, 1H, J = 11.6 Hz, CHH Bn), 5.17 (dd,
1H, J = 1.2 Hz, 10.4 Hz, –CH@CHH), 5.23–5.28 (m, 2H, H-1, –
CH@CHH), 5.50 (s, 1H, CH benzylidene), 5.88 (ddd, 1H, J = 5.6 Hz,
10.6 Hz, 22.8 Hz, –CH@CH2), 6.93 (d, 2H, J = 8.4 Hz, Harom), 7.22–
7.46 (m, 18H, Harom), 7.64–7.68 (m, 4H, Harom); 13C NMR
a-product
(100 MHz): d = 19.2 (Cq t-butyl, 26.8 (3 ꢃ CH3 TBDPS), 62.4 (C-5),
63.9 (CH2 glycerol), 68.8 (C-6), 70.9 (CH2 glycerol), 71.8, 72.3, 75.2
(OCH2–CH@CH2, CH2 p-N3Bn, CH2 Bn), 76.7, 78.2 (C-3, C-4), 79.0
(CH glycerol), 82.2 (C-2), 97.2 (C-1), 101.2 (CH benzylidene),
116.9 (–CH@CH2), 118.8 (CHarom) 126.1–129.7 (CHarom), 133.1,
133.2 (2 ꢃ Cq phenyl), 134.6 (–uH@CH2), 135.1 (Cq p-N3Bn), 135.5
(CHarom), 137.5, 138.8, 139.3 (Cq Bn, Cq benzylidene Cq p-N3Bn)). A
part of the intermediate (58 mg, 0.069 mmol) was dissolved in a
mixture of dioxane/water (9/1, 10 ml) and treated with PMe3
(1 M solution in toluene, 0.34 ml). After stirring for 1 h, the mixture
was concentrated in vacuo and coevaporated three times with diox-
ane (portions of 5 ml) before the mixture was redissolved in moist
DCM (10 ml) and DDQ (24 mg, 0.10 mmol) was added. After stirring
vigorously for 4 h, the reaction was quenched by the addition of
satd aq NaHCO3 (2.5 ml). The mixture was then diluted with EtOAc
(40 ml) and washed with, respectively, satd aq NaHCO3 (3 ꢃ 15 ml)
and brine (3 ꢃ 15 ml) in order to remove the bulk of DDQH2. The or-
ganic layer was dried over MgSO4 and concentrated in vacuo. The
crude product was purified by column chromatography (EtOAc/
76%,
a
/b mixture, ꢀ10/1), as colourless oil. IR (neat): 737, 1088,
1369, 1454, 1751, 2855, 2924 cmꢄ1
;
1H NMR
a-product
(400 MHz): d = 1.05 (s, 9H, 3 ꢃ CH3 TBDPS), 3.51–3.65 (m, 4H, CH
glycerol, CHH glycerol, H-2, H-6), 3.69–3.80 (m, 6H, OMe, 3 ꢃ CHH
glycerol), 3.89 (m, 1H, H-5), 3.96–4.03 (m, 5H, OCH2–CH@CH2, H-3,
H-4, H-6), 4.66 (d, 1H, J = 11.7 Hz, CHH Bn), 4.71 (d, 1H, J = 11.7 Hz,
CHH Bn), 4.79 (d, 1H, J = 11.2 Hz, CHH Bn), 4.85 (d, 1H, J = 11.3 Hz,
CHH Bn), 5.17 (dd, 1H, J = 1.6 Hz, 10.4 Hz, –CH@CHH), 5.20 (d, 1H,
J = 3.8 Hz, H-1), 5.26 (dd, 1H, J = 1.7 Hz, 17.2 Hz, –CH@CHH), 5.49
(s, 1H, CH benzylidene), 5.89 (ddd, 1H, J = 5.5 Hz, 10.7 Hz,
22.7 Hz, –CH@CH2), 6.83 (d, 2H, J = 8.6 Hz, Harom), 7.24–7.47 (m,
18H, Harom), 7.65–7.68 (m, 4H, Harom); 13C NMR
a-product
(100 MHz): d = 19.2 (Cq t-butyl), 26.8 (3 ꢃ CH3 TBDPS), 55.2
(OMe), 62.4 (C-5), 63.8 (CH2 glycerol), 68.9 (C-6), 70.7 (CH2 glyc-
erol), 72.3 (OCH2–CH@CH2), 72.3, 75.2 (CH2 Bn, CH2 PMB), 76.6,
PE), giving alcohol 14 (19.0 mg, 26.7 lmol, 39%) as pale yellow oil.
From 12 (via 13c): To a solution of thioglucose 12 (225 mg,
0.500 mmol) in DCM (5.0 ml) were added Et3N (0.69 ml, 5.0 mmol),

3674
W. F. J. Hogendorf et al. / Bioorg. Med. Chem. 18 (2010) 3668–3678
TMS-Cl (0.32 ml, 2.50 mmol) and a drop of pyridine. After stirring
for 2 h, the reaction was diluted with EtOAc (40 ml) and washed
with H2O (15 ml) and brine (15 ml), respectively. The organic layer
was dried with MgSO4 and concentrated in vacuo. Crude 13c,
together with Ph2SO (121 mg, 0.600 mmol) and TTBP (323 mg,
1.30 mmol) were coevaporated with toluene (three times 5 ml)
and together with activated MS 3 Å dissolved in dry DCM
(10 ml). After stirring for 15 min, the mixture was cooled to
1H, J = 11.8 Hz, CHH Bn), 4.74–4.83 (m, 4H, CH2 Bn), 4.90 (d, 1H,
J = 11.8 Hz, CHH Bn), 5.12 (dd, 1H, J = 1.1 Hz, 10.4 Hz, –CH@CHH),
5.19 (dd, 1H, J = 1.6 Hz, 17.3 Hz, –CH@CHH), 5.27 (d, 1H,
J = 3.5 Hz, H-1), 5.40 (d, 1H, J = 3.6 Hz, H-1), 5.52 (s, 1H, CH benzyl-
idene), 5.54 (s, 1H, CH benzylidene), 5.82 (ddd, 1H, J = 5.6 Hz,
10.7 Hz, 22.7 Hz, –CH@CH2), 7.06–7.11 (m, 2H, Harom), 7.19–7.39
(m, 25H, Harom), 7.44–7.48 (m, 4H, Harom), 7.64–7.68 (m, 4H, Harom);
13C NMR (100 MHz): d = 19.1 (Cq t-butyl), 26.8 (3 ꢃ CH3 TBDPS),
62.5 (C-5), 62.8 (C-5), 63.5 (CH2 glycerol), 68.8 (C-6), 68.9 (C-6),
69.7 (CH2 glycerol), 72.1 (OCH2–CH@CH2), 72.4 (CH2 Bn), 75.0
(CH2 Bn), 75.3 (C-2), 75.3 (CH2 Bn), 75.9 (CH glycerol), 76.7 (C-3),
78.1 (C-3), 79.0 (C-2), 82.2 (2 ꢃ C-4), 95.4 (C-1), 95.5 (C-1), 101.2
(2 ꢃ CH benzylidene), 117.0 (–CH@CH2), 126.1–129.7 (CHarom),
133.2 (2 ꢃ Cq phenyl), 134.5 (–CH@CH2), 135.5 (CHarom), 137.5,
137.6, 138.3, 138.6 (3 ꢃ Cq Bn, 2 ꢃ Cq benzylidene); HRMS:
C69H76O13Si+Na+ requires 1163.4947, found 1163.4962.
ꢄ78 °C and stirred for 5 min. After the addition of Tf2O (101
ll,
0.600 mmol), the mixture was allowed to slowly warm up to
ꢄ60 °C (ꢀ30 min), after which the mixture was cooled down to
ꢄ78 °C and glycerol acceptor 7 (278 mg, 0.750 mmol, dissolved in
2.0 ml DCM) was added. After stirring for 1 h at ꢄ78 °C, the mix-
ture was allowed to warm up to rt overnight. After the addition
of a few drops of H2O the mixture was diluted with MeOH
(20 ml). K2CO3 (10 g) was subsequently added and, after stirring
for 5 h, the mixture was diluted with EtOAc (100 ml). The organic
layer was washed with H2O (40 ml) and brine (40 ml) and dried
with MgSO4. After removal of the solvents in vacuo, the residue19
was purified by column chromatography (EtOAc/PE), affording 14
4.2.9. 1-O-(tert-Butyldiphenylsilyl)-2-O-(2-[2,3-di-O-benzyl-4,6-
O-benzylidene-
a-D-glucopyranosyl]-3-O-benzyl-4,6-O-benzyl-
idene- -glucopyranosyl)-sn-glycerol (17)
a
-D
(165 mg, 0.232 mmol, 46%, only
a
) as pale yellow oil. ½a D20
ꢂ
(CHCl3):
A solution of 16 (3.18 g, 2.79 mmol) and freshly activated MS
3 Å in freshly distilled THF (35 ml) was stirred under argon for
30 min. After the addition of Ir(COD)(Ph2MeP)2PF6 (236 mg,
10 mol %) the solution turned red and the mixture was purged with
H2 (g) until the solution turned colourless again (ꢀ1 min). After
stirring under argon for 4 h, the solution was diluted with THF
(30 ml) and satd aq NaHCO3 (100 ml). After the addition of I2
(1.06 g, 4.19 mmol), the mixture was allowed to stir overnight at
room temperature. The mixture was diluted with EtOAc (250 ml)
and washed with satd aq NaS2O3 (2 ꢃ 80 ml) and brine (80 ml),
respectively. The organic layer was dried over MgSO4 and concen-
trated in vacuo. Column chromatography (EtOAc/toluene) afforded
+33.6; IR (neat): 741, 1040, 1072, 2343, 2361 cmꢄ1
;
1H NMR
(400 MHz): d = 1.06 (s, 9H, 3 ꢃ CH3 TBDPS), 2.92 (br s, 1H, OH),
3.54–3.79 (m, 8H, 2 ꢃ CH2 glycerol, H-2, H-3, H-4, H-6), 3.85–
3.95 (m, 2H, H-5, CH glycerol), 4.00 (ad, 2H, J = 5.6 Hz, OCu2–
CH@CH2), 4.05 (dd, 1H, J = 4.9 Hz, 10.2 Hz, H-6), 4.85 (d, 1H,
J = 11.8 Hz, CuH Bn), 4.91 (d, 1H, J = 11.8 Hz, CHH Bn), 5.06 (d,
1H, J = 3.8 Hz, H-1), 5.20 (dd, 1H, J = 1.1 Hz, 10.4 Hz, –CH@CHH),
5.28 (dd, 1H, J = 1.5 Hz, 17.2 Hz, –CH@CHH), 5.51 (s, 1H, CH benzyl-
idene), 5.88 (ddd, 1H, J = 5.5 Hz, 10.8 Hz, 16.0 Hz, –CH@CH2), 7.23–
7.47 (m, 16H, Harom), 7.64–7.68 (m, 4H, Harom); 13C NMR
(100 MHz): d = 19.2 (Cq t-butyl, 26.8 (3 ꢃ CH3 TBDPS), 63.0 (C-5),
63.8 (CH2 glycerol), 68.8 (C-6), 69.8 (CH2 glycerol), 72.3 (OCH2–
CH@CH2), 73.1 (C-2), 74.6 (CH2 Bn), 79.0 (CH glycerol), 79.1, 81.5
(C-3, C-4), 99.8 (C-1), 101.2 (CH benzylidene), 117.5 (–CH@CH2),
126.1–129.8 (CHarom), 133.0, 133.1 (2 ꢃ Cq phenyl), 134.2
(–CH@CH2), 135.5 (CHarom), 137.5, 138.8 (Cq Bn, Cq benzylidene);
HRMS: C42H50O8Si+Na+ requires 734.3201, found 734.3203.
17 (2.05 g, 1.86 mmol, 67%) as pale yellow foam. ½a D20
ꢂ
(CHCl3):
+29.4; IR (neat): 748, 997, 1028, 1074, 1371, 2360, 2858 cmꢄ1
;
1H NMR (400 MHz): d = 1.07 (s, 9H, 3 ꢃ CH3 TBDPS), 3.44 (dd, 1H,
J = 5.3 Hz, 8.1 Hz, OH), 3.50–3.68 (m, 8H, 2 ꢃ CHH glycerol, 2 ꢃ H-
2, 2 ꢃ H-4, 2 ꢃ H-6), 3.76–3.91 (m, 5H, 2 ꢃ CHH glycerol, CH glyc-
erol, H-5, H-6), 3.96 (dd, 1H, J = 4.9 Hz, 10.2 Hz, H-6), 4.02 (at, 1H,
J = 9.3 Hz, H-3), 4.16 (at, 1H, J = 9.3 Hz, H-3), 4.13–4.22 (m, 1H, H-
5), 4.73–4.86 (m, 5H, H-1, 2 ꢃ CH2 Bn), 4.94 (d, 1H, J = 11.1 Hz,
CHH Bn), 4.97 (d, 1H, J = 10.7 Hz, CHH Bn), 5.09 (d, 1H, J = 3.6 Hz,
H-1), 5.48 (s, 1H, CH benzylidene), 5.50 (s, 1H, CH benzylidene),
7.12–7.15 (m, 3H, Harom), 7.27–7.47 (m, 28H, Harom), 7.66–7.68
(m, 4H, Harom); 13C NMR (100 MHz): d = 19.1 (Cq t-butyl), 26.8
(3 ꢃ CH3 TBDPS), 62.4 (CH2 glycerol), 62.5 (C-5), 62.8 (C-5), 63.7
(CH2 glycerol), 68.7 (2 ꢃ C-6), 74.0 (CH2 Bn), 75.1 (CH2 Bn), 75.2
(CH2 Bn), 76.4 (C-3), 77.2 (C-2), 77.7 (C-2), 78.8 (C-3), 80.7 (CH
glycerol), 78.1 (C-3), 79.0 (C-2), 82.5 (C-4), 82.8 (C-4), 97.6 (C-1),
98.6 (C-1), 101.2 (CH benzylidene), 101.3 (CH benzylidene),
126.0–129.8 (CHarom), 133.0, 133.1 (2 ꢃ Cq phenyl), 135.5 (CHarom),
137.2, 137.5, 137.6, 138.0, 138.5 (3 ꢃ Cq Bn, 2 ꢃ Cq benzylidene);
HRMS: C66H72O13Si+Na+ requires 1123.4634, found 1123.4648.
4.2.8. 1-O-(tert-Butyldiphenylsilyl)-2-O-(2-[2,3-di-O-benzyl-4,6-
O-benzylidene-
a-D-glucopyranosyl]-3-O-benzyl-4,6-O-benzyl-
idene- -glucopyranosyl)-3-O-allyl-sn-glycerol (16)
a-D
A solution of glucose donor 15 (130 mg, 0.240 mmol), TTBP
(149 mg, 0.600 mmol) and Ph2SO (57 mg, 0.28 mmol) in freshly
distilled DCM (5.0 ml), together with activated MS 3 Å, was stirred
under argon at rt for 30 min. The mixture was cooled to ꢄ78 °C and
stirred for another 15 min before Tf2O (47 ll, 0.28 mmol) was
added and the mixture stirred for another 45 min at ꢄ70 °C. Subse-
quently, alcohol 14 (142 mg, 0.200 mmol, dissolved in 2.5 ml DCM)
was added and after stirring for 60 min at ꢄ75 °C, the mixture was
allowed to, gradually, warm to room temperature (ꢀ3 h). The reac-
tion was quenched by the addition of Et3N (0.14 ml, 1.0 mmol) and
stirred for 30 min. The mixture was diluted with EtOAc (30 ml) and
washed with satd aq NaHCO3 (10 ml) and brine (10 ml). The organ-
ic layer was dried over MgSO4 and concentrated in vacuo. The
resulting oil was purified by size-exclusion chromatography
(sephadex LH-20, MeOH/DCM 1/1) and, subsequently, column
chromatography (EtOAc/pentane). Kojibiose derivative 16
4.2.10. 1-O-(tert-Butyldiphenylsilyl)-2-O-(2-[2,3-di-O-benzyl-
4,6-O-benzylidene-
zylidene-
-glucopyranosyl)-3-O-(4,40-dimethoxytrityl)-sn-
glycerol (18)
a-D-glucopyranosyl]-3-O-benzyl-4,6-O-ben-
a-D
Alcohol 17 (480 mg, 0.436 mmol) was dissolved in DCM (20 ml)
and cooled to 0 °C. After the addition of DIPEA (0.23 ml, 1.3 mmol)
and DMTr–Cl (295 mg, 0.872 mmol), respectively, the mixture was
allowed to stir at room temperature overnight. The reaction was
quenched upon addition of H2O (0.2 ml), and stirred for 15 min.
The mixture was diluted with DCM (20 ml) and, after washing with
H2O (10 ml) and brine (10 ml), the organic layer was dried over
Na2SO4 and concentrated in vacuo. The resulting oil was purified
by size-exclusion chromatography (sephadex LH-20, MeOH/DCM
(187 mg, 0.164 mmol, 82%, pure
a) was obtained as white foam.
½
a 2D0
(CHCl3): +34.4; IR (neat): 746, 997, 1028, 1074, 1369, 1454,
ꢂ
2860 cmꢄ1
;
1H NMR (400 MHz): d = 1.05 (s, 9H, 3 ꢃ CH3 TBDPS),
3.54 (dd, 1H, J = 5.9 Hz, 10.5 Hz, CHH glycerol), 3.57–3.69 (m, 7H,
2 ꢃ CHH glycerol, H-2, 2 ꢃ H-4, 2 ꢃ H-6), 3.74 (dd, 1H, J = 5.3 Hz,
10.4 Hz, CHH glycerol), 3.80 (dd, 1H, J = 3.7 Hz, 9.4 Hz, H-2), 3.88
(add, 2H, J = 1.1 Hz, 5.5 Hz, OCH2–CH@CH2), 3.91–4.00 (m, 2H, CH
glycerol, H-5), 4.04–4.21 (m, 5H, 2 ꢃ H-3, H-5, 2 ꢃ H-6), 4.70 (d,

W. F. J. Hogendorf et al. / Bioorg. Med. Chem. 18 (2010) 3668–3678
3675
1/1) and, subsequently, column chromatography (EtOAc/pentane/
Na2SO4 and concentrated in vacuo. After purification of the resi-
due by column chromatography (EtOAc/pentane/Et3N), phospho-
ramidite 4 (1.15 g, 0.842 mmol, 72%) was obtained as colourless
oil. 31P NMR (161.7 MHz, CD3CN): d = 148.5, 148.7 (diastereoiso-
mers); 1H NMR (400 MHz, CD3CN): d = 1.10–1.17 (m, 12H, CH3
isopropylamino), 2.46–2.52 (m, 2H, CH2 cyanoethoxy), 3.12–
3.16 (m, 1H, CH isopropylamino), 3.32–3.39 (m, 1H, CH isopro-
pylamino), 3.51–4.15 (m, 24H, 2 ꢃ OMe, 2 ꢃ CH2 glycerol, CH
glycerol, 2 ꢃ H-2, 2 ꢃ H-3, 2 ꢃ H-4, 2 ꢃ H-5, 3 ꢃ H-6, CH2 cyano-
ethoxy), 4.29 (dd, 1H, J = 4.2 Hz, 10.0 Hz, H-6), 4.39 (d, 1H,
J = 11.4 Hz, CHH Bn), 4.46 (d, 1H, J = 11.3 Hz, CHH Bn), 4.60–
4.70 (m, 2H, 2 ꢃ CHH Bn), 4.81 (s, 2H, 2 ꢃ CHH Bn), 5.12 (d,
1H, J = 3.3 Hz, H-1), 5.30 (d, 0.5H, J = 3.3 Hz, H-1 diastereomer
1), 5.34 (d, 0.5H, J = 3.3 Hz, H-1 diastereomer 2), 5.60 (s, 1H,
CH benzylidene), 5.66 (s, 1H, CH benzylidene), 6.82 (d, 4H,
J = 8.6 Hz, Harom), 7.16–7.50 (m, 34H, Harom); TLC-MS: C80H89N2-
O16P+H+ requires 1365.60, found 1365.6.
Et3N), giving DMTr-ether 18 (601 mg, 0.428 mmol, 98%) as white
foam. ½a 2D0
ꢂ
(CHCl3): +35.6; IR (neat): 752, 1030, 1074, 1250, 1508,
2361, 2858 cmꢄ1
;
1H NMR (400 MHz): d = 0.97 (s, 9H, 3 ꢃ CH3
TBDPS), 3.08 (dd, 1H, J = 5.7 Hz, 10.1 Hz, CHH glycerol), 3.44 (dd,
1H, J = 3.4 Hz, 9.2 Hz, H-2), 3.51 (dd, 1H, J = 2.9 Hz, 10.1 Hz, CHH
glycerol), 3.55–3.71 (m, 12H, 2 ꢃ OMe, 2 ꢃ CuH glycerol, 2 ꢃ H-4,
2 ꢃ H-6), 3.77 (dd, 1H, J = 3.4 Hz, 9.4 Hz, H-2), 3.93–4.02 (m, 3H,
CH glycerol, H-3, H-5), 4.07–4.17 (m, 4H, H-3, H-5, 2 ꢃ H-6), 4.30
(d, 1H, J = 11.5 Hz, CuH Bn), 4.36 (d, 1H, J = 11.4 Hz, CuH Bn), 4.56
(d, 1H, J = 11.4 Hz, CuH Bn), 4.72 (d, 1H, J = 11.4 Hz, CHH Bn),
4.78 (d, 1H, J = 11.0 Hz, CHH Bn), 4.88 (d, 1H, J = 11.0 Hz, CHH
Bn), 4.98 (d, 1H, J = 3.4 Hz, H-1), 5.39 (d, 1H, J = 3.4 Hz, H-1), 5.53
(s, 1H, CH benzylidene), 5.55 (s, 1H, CH benzylidene), 6.74 (d, 4H,
J = 8.7 Hz, Harom), 7.08–7.48 (m, 40H, Harom), 7.55 (d, 2H,
J = 7.1 Hz, Harom), 7.59 (d, 2H, J = 6.7 Hz, Harom); 13C NMR
(100 MHz): d = 19.1 (Cq t-butyl), 26.8 (3 ꢃ CH3 TBDPS), 55.1
(2 ꢃ OMe), 62.5 (C-5), 62.8 (C-5), 63.3 (CH2 glycerol), 64.3 (CH2
glycerol), 68.8 (C-6), 68.9 (C-6), 72.4 (CH2 Bn), 75.0 (CH2 Bn),
75.4 (CH2 Bn), 75.7 (C-2), 76.8, 76.9 (C-3, CH glycerol), 78.1 (C-3),
79.4 (C-2), 82.1 (C-4), 82.3 (C-4), 86.4 (Cq DMTr), 95.2 (C-1), 95.6
(C-1), 101.2 (CH benzylidene), 101.3 (CH benzylidene), 113.1 (CHar-
om), 126.1–130.0 (CHarom), 133.1, 133.2 (2 ꢃ Cq phenyl), 135.5
(CHarom), 137.5, 137.7, 138.1, 138.2, 138.7, 144.9, 158.4 (3 ꢃ Cq
Bn, 2 ꢃ Cq benzylidene, 5 ꢃ Cq DMTr); HRMS: C87H90O15Si+Na+ re-
quires 1425.5941, found 1425.5961.
4.2.13. Benzyl 6-([N,N-diisopropylamino]-2-cyanoethyl-phos-
phite)-hexyl-1-carbamate (5)
To a cooled (0 °C) solution of 6-(benzyloxycarbonylamino)-1-
hexanol20 (3.02 g, 12.0 mmol) and DIPEA (2.51 ml, 14.4 mmol)
in DCM (60 ml) was added chlorophosphite 11 (2.90 g,
12.2 mmol). After stirring for 4 h, the reaction was washed with
H2O (20 ml) and brine (20 ml), respectively. The organic layer
was dried over Na2SO4 and concentrated in vacuo. After purifica-
tion of the residue by column chromatography (EtOAc/PE/Et3N),
phosphoramidite 5 (3.97 g, 8.79 mmol, 73%) was obtained as col-
ourless oil. 31P NMR (161.7 MHz, CD3CN): d = 148.2; 1H NMR
(400 MHz, CD3CN): d = 1.18 (d, 6H, J = 2.7 Hz, 2 ꢃ CH3 isopropyla-
mino), 1.19 (d, 6H, J = 2.7 Hz, 2 ꢃ CH3 isopropylamino), 1.30–1.43
(m, 4H, 2 ꢃ CH2), 1.48 (m, 2H, CH2), 1.59 (m, 2H, CH2), 2.65 (t,
2H, J = 6.0 Hz, CH2 cyanoethoxy), 3.10 (dd, 2H, J = 6.7 Hz,
13.2 Hz, CH2–N hexyl), 3.57–3.68 (m, 4H, CH2–O hexyl), 2 ꢃ CH
isopropylamino), 3.73–3.85 (m, 2H, CH2 cyanoethoxy), 5.06 (br
s, 2H, CH2 benzylcarbamate), 5.64 (br s, 1H, NH), 7.31–7.42 (m,
5H, Harom); TLC-MS: C23H38N3O4P+H+ requires 452.27, found 452.1.
4.2.11. 2-O-(2-[2,3-Di-O-benzyl-4,6-O-benzylidene-
anosyl]-3-O-benzyl-4,6-O-benzylidene- -glucopyranosyl)-3-
O-(4,40-dimethoxytrityl)-sn-glycerol (19)
a-D-glucopyr
a-D
To a solution of kojibiose derivative 18 (575 mg, 0.410 mmol) in
THF (10 ml), was added TBAF (1.00 M solution in THF, 1.20 ml).
After stirring for 48 h at rt, the solvent was removed in vacuo.
The residue was purified by column chromatography (EtOAc/pen-
tane/Et3N), affording compound 19 (435 mg, 0.373 mmol, 91%) as
white foam. ½a 2D0
ꢂ
(CHCl3): +38.0; IR (neat): 1030, 1074, 1508,
2341, 2361 cmꢄ1
;
1H NMR (400 MHz): d = 2.52 (br s, 1H, OH),
3.02 (dd, 1H, J = 4.1 Hz, 9.9 Hz, CHH glycerol), 3.44–3.49 (m, 2H,
CHH glycerol, H-2), 3.55–3.78 (m, 14H, 2 ꢃ OMe, 2 ꢃ CHH glycerol,
CH glycerol, H-2, 2 ꢃ H-4, 2 ꢃ H-6), 3.96 (at, 1H, J = 9.3 Hz, H-3),
4.04–4.16 (m, 4H, H-3, 2 ꢃ H-5, H-6), 4.31 (dd, 1H, J = 4.8 Hz,
10.2 Hz, H-6), 4.49–4.55 (m, 3H, 3 ꢃ CHH Bn), 4.73 (d, 1H,
J = 11.4 Hz, CHH Bn), 4.83 (d, 1H, J = 11.0 Hz, CHH Bn), 4.90 (d,
1H, J = 3.3 Hz, H-1), 4.93 (d, 1H, J = 11.0 Hz, CHH Bn), 5.17 (d, 1H,
J = 3.5 Hz, H-1), 5.52 (s, 1H, CH benzylidene), 5.58 (s, 1H, CH ben-
zylidene), 6.75 (d, 2H, J = 8.8 Hz, Harom), 6.76 (d, 2H, J = 8.8 Hz, Har-
om), 7.16–7.50 (m, 34H, Harom); 13C NMR (100 MHz): d = 55.1
(2 ꢃ OMe), 63.0 (2 ꢃ C-5), 63.8 (CH2 glycerol), 64.3 (CH2 glycerol),
68.7 (C-6), 68.9 (C-6), 73.5 (CH2 Bn), 75.0 (CH2 Bn), 75.4 (CH2 Bn),
76.9 (C-3), 77.0 (C-2), 78.4 (C-3), 79.0 (C-2), 79.8 (CH glycerol), 82.3
(C-4), 82.5 (C-4), 86.3 (Cq DMTr), 97.1 (C-1), 97.2 (C-1), 101.2 (CH
benzylidene), 101.3 (CH benzylidene), 113.1 (CHarom), 126.0–
129.9 (CHarom), 135.7, 135.9, 137.2, 137.6, 137.8, 138.1, 138.6,
144.7, 158.4 (3 ꢃ Cq Bn, 2 ꢃ Cq benzylidene, 5 ꢃ Cq DMTr); HRMS:
C71H72O15+Na+ requires 1187.4763, found 1187.4782.
4.2.14. Glycerol phosphate dimer (21)
To a solution of dibenzylglycerol 2 (163 mg, 0.600 mmol) in
acetonitrile (4.0 ml), containing freshly activated MS 3 Å, were
added DCI (0.25 M in acetonitrile, 9.6 ml) and phosphoramidite 3
(0.2 M in acetonitrile, 3.9 ml). After stirring for 2 h, I2 (0.2 M in
THF/pyridine 4/1, 9.0 ml) and H2O (1.0 ml) were added and the
mixture was allowed to stir for 1 h. EtOAc (80 ml) was added and
the mixture was washed with satd aq Na2S2O3 (30 ml), aqueous
KHSO4 (0.5 M, 30 ml), satd aq NaHCO3 (30 ml) and brine (30 ml),
respectively. The organic layer was dried with Na2SO4 and concen-
trated in vacuo, after which the residue was redissolved in DCM
(30 ml), containing freshly activated MS 3 Å. Et3SiH (0.97 ml,
6.0 mmol) and dichloroacetic acid (0.49 ml, 6.0 mmol) were added
and the mixture stirred until the orange colour disappeared (ꢀ1 h),
after which the mixture was diluted with DCM (20 ml) and washed
with satd aq NaHCO3 (20 ml) and brine (20 ml), respectively. The
organic layer was dried with Na2SO4, concentrated in vacuo and
subsequently purified by column chromatography (MeOH/DCM),
yielding dimer 21 (272 mg, 0.478 mmol, 80%, mixture of diastere-
oisomers) as colourless oil. 31P NMR (161.7 MHz): d = ꢄ0.6, ꢄ0.7;
1H NMR (400 MHz): d = 2.50 (t, 1H, J = 6.2 Hz, CH2 cyanoethoxy),
2.54 (t, 1H, J = 6.2 Hz, CH2 cyanoethoxy), 2.66 (br s, 1H, OH),
3.54–3.73 (m, 5H, CH glycerol, 2 ꢃ CH2 glycerol), 3.77–3.82 (m,
1H, CH glycerol), 4.03–4.32 (m, 6H, CH2 cyanoethoxy, 2 ꢃ CH2 glyc-
4.2.12. 1-([N,N-Diisopropylamino]-2-cyanoethyl-phosphite)-2-O
-(2-[2,3-di-O-benzyl-4,6-O-benzylidene-
O-benzyl-4,6-O-benzylidene-
-glucopyranosyl)-3-O-(4,40-
dimethoxytrityl)-sn-glycerol (4)
a-D-glucopyranosyl]-3-
a
-D
To a cooled (0 °C) solution of alcohol 19 (1.37 g, 1.17 mmol)
and DIPEA (0.31 ml, 1.75 mmol) in freshly distilled DCM (12 ml)
was added chlorophosphite 11 (333 mg, 1.41 mmol). After stir-
ring for 4 h, the reaction was quenched with H2O (1.0 ml), di-
luted with DCM (50 ml) and washed with H2O (20 ml) and
brine (20 ml), respectively. The organic layer was dried over
erol), 4.51–4.66 (m, 6H, 3 ꢃ CH2 Bn), 7.25–7.35 (m, 15H, Harom); 13
C
NMR (100 MHz): d = 19.2, 19.3, 19.3 (CH2 cyanoethoxy), 60.6, 60.7
(CH2 glycerol), 61.9 (CH2 cyanoethoxy), 66.3, 66.4 (CH2 glycerol),

3676
W. F. J. Hogendorf et al. / Bioorg. Med. Chem. 18 (2010) 3668–3678
67.4, 67.5, 67.6 (CH2 glycerol), 68.6 (CH2 glycerol), 71.9, 72.0 (CH2
Bn), 72.1 (CH2 Bn), 73.4 (CH2 Bn), 76.2, 76.3 (CH glycerol), 77.3,
77.4, 77.5 (CH glycerol), 116.4 (Cq cyanoethoxy), 127.6–128.4
(CHarom), 137.7, 137.8, 137.8 (Cq Bn); HRMS: C30H37NO8P+Na+ re-
quires 592.2071, found 592.2070.
erol), 4.03–4.30 (m, 18H, 3 ꢃ CH2 cyanoethoxy, 6 ꢃ CH2 glycerol),
4.50–4.65 (m, 10H, 5 ꢃ CH2 Bn), 7.26–7.34 (m, 25H, Harom); 13C
NMR (100 MHz): d = 19.2–19.4 (3 ꢃ CH2 cyanoethoxy), 60.4,
60.5 (CH2 glycerol), 61.8–62.2 (3 ꢃ CH2 cyanoethoxy), 65.5–66.0
(4 ꢃ CH2 glycerol), 66.5–66.7 (CH2 glycerol), 67.4–67.7 (CH2 glyc-
erol), 68.6 (CH2 glycerol), 71.9–72.1 (4 ꢃ CH2 Bn), 73.4 (CH2 Bn),
75.2–75.5 (2 ꢃ CH glycerol), 76.2–76.4 (CH glycerol), 77.4–77.6
(CH glycerol), 116.5 (3 ꢃ Cq cyanoethoxy), 127.6–128.4 (CHarom),
137.2, 137.7–137.9 (5 ꢃ Cq Bn); HRMS: C56H68N3O18P3+Na+ re-
quires 1186.3603, found 1186.3614.
4.2.15. Glycerol phosphate trimer (22)
To a solution of dimer 21 (229 mg, 0.402 mmol) in acetonitrile
(4.0 ml), containing freshly activated MS 3 Å, were added DCI
(0.25 M in acetonitrile, 6.4 ml) and phosphoramidite 3 (0.2 M in
acetonitrile, 2.6 ml). After stirring for 2 h, more 3 (0.2 M in acetoni-
trile, 0.8 ml) was added and the mixture was allowed to react for
another 2 h. I2 (0.2 M in THF/pyridine 4/1, 6.0 ml) and H2O
(1.0 ml) were added and the mixture was stirred for 1 h. EtOAc
(50 ml) was added and the mixture was washed with satd aq
Na2S2O3 (20 ml), aqueous KHSO4 (0.5 M, 20 ml), satd aq NaHCO3
(20 ml) and brine (20 ml), respectively. The organic layer was dried
with Na2SO4 and concentrated in vacuo, after which the residue
was redissolved in DCM (20 ml), containing freshly activated MS
3 Å. Et3SiH (0.65 ml, 4.0 mmol) and dichloroacetic acid (0.32 ml,
4.0 mmol) were added and the mixture stirred until the orange col-
our disappeared (ꢀ1.5 h), after which the mixture was diluted with
DCM (30 ml) and washed with satd aq NaHCO3 (20 ml) and brine
(20 ml), respectively. The organic layer was dried with Na2SO4,
concentrated in vacuo and the residue purified by column chroma-
tography (MeOH/DCM), yielding trimer 22 (286 mg, 0.330 mmol,
82%) as colourless oil. 31P NMR (161.7 MHz): d = ꢄ1.2, ꢄ1.2, ꢄ1.1,
ꢄ1.0, ꢄ0.8, ꢄ0.8; 1H NMR (400 MHz): d = 2.47–2.58 (m, 4H,
2 ꢃ CH2 cyanoethoxy), 2.97 (br s, 1H, OH), 3.53–3.80 (m, 7H,
3 ꢃ CH glycerol, 2 ꢃ CH2 glycerol), 4.03–4.32 (m, 12H, 2 ꢃ CH2
cyanoethoxy, 4 ꢃ CH2 glycerol), 4.48–4.66 (m, 8H, 4 ꢃ CH2 Bn),
7.25–7.33 (m, 20H, Harom); 13C NMR (100 MHz): d = 19.2–19.4
(CH2 cyanoethoxy), 60.5, 60.6 (CH2 glycerol), 61.8–62.1 (2 ꢃ CH2
cyanoethoxy), 65.5–65.9 (2 ꢃ CH2 glycerol), 66.5–66.7 (CH2
glycerol), 67.4–67.7 (CH2 glycerol), 68.6–68.7 (CH2 glycerol),
71.9–72.1 (3 ꢃ CH2 Bn), 73.4 (CH2 Bn), 75.3–75.5 (CH glycerol),
76.3–76.4 (CH glycerol), 77.4–77.6 (CH glycerol), 116.4–116.5
(2 ꢃ Cq cyanoethoxy), 127.6–128.4 (CHarom), 137.2, 137.7–137.8
(4 ꢃ Cq Bn); HRMS: C43H52N2O13P2+Na+ requires 889.2837, found
889.2843.
4.2.17. Glycerol phosphate pentamer (24)
To a solution of tetramer 23 (283 mg, 0.243 mmol) in acetoni-
trile (3.0 ml), containing freshly activated MS 3 Å, were added
DCI (0.25 M in acetonitrile, 4.0 ml) and phosphoramidite
3
(0.2 M in acetonitrile, 2.5 ml). After stirring for 4 h, more 3
(0.2 M in acetonitrile, 1.2 ml) was added and the mixture was al-
lowed to react overnight. I2 (0.2 M in THF/pyridine 4/1, 5.0 ml)
and H2O (1.0 ml) were added and the mixture was stirred for
1 h. EtOAc (30 ml) was added and the mixture was washed with
satd aq Na2S2O3 (15 ml), aqueous KHSO4 (0.5 M, 15 ml), satd aq
NaHCO3 (15 ml) and brine (15 ml), respectively. The organic layer
was dried with Na2SO4 and concentrated in vacuo, after which
the residue was redissolved in DCM (15 ml), containing freshly
activated MS 3 Å. Et3SiH (0.39 ml, 2.4 mmol) and dichloroacetic
acid (0.20 ml, 2.4 mmol) were added and the mixture stirred un-
til the orange colour disappeared (ꢀ3 h), after which the mixture
was diluted with DCM (20 ml) and washed with, respectively,
satd aq NaHCO3 (15 ml) and brine (15 ml). The organic layer
was dried with Na2SO4, concentrated in vacuo and, subsequently,
purified by column chromatography (MeOH/DCM), yielding pen-
tamer 24 (221 mg, 0.151 mmol, 62%) as colourless oil. 31P NMR
(161.7 MHz): d = ꢄ1.3 to ꢄ0.8; 1H NMR (400 MHz): d = 1.67 (br
s, 1H, OH), 2.45–2.64 (m, 8H, 4 ꢃ CH2 cyanoethoxy), 3.55–3.58
(m, 2H, CH2 glycerol) 3.62–3.71 (m, 3H, CH glycerol, CH2 glyc-
erol), 3.75–3.80 (m, 4H, 4 ꢃ CH glycerol), 4.04–4.28 (m, 24H,
4 ꢃ CH2 cyanoethoxy, 8 ꢃ CH2 glycerol), 4.49–4.51 (m, 2H, CH2
Bn), 4.56–4.66 (m, 10H, 5 ꢃ CH2 Bn), 7.26–7.35 (m, 30H, Harom);
13C NMR (100 MHz): d = 19.2–19.4 (4 ꢃ CH2 cyanoethoxy), 60.2–
60.4 (CH2 glycerol), 62.0–62.5 (4 ꢃ CH2 cyanoethoxy), 65.6–66.1
(6 ꢃ CH2 glycerol), 66.7–66.9 (CH2 glycerol), 67.6–67.9 (CH2 glyc-
erol), 68.5 (CH2 glycerol), 71.9–72.2 (5 ꢃ CH2 Bn), 73.4 (CH2 Bn),
75.2–75.4 (3 ꢃ CH glycerol), 76.3–76.4 (CH glycerol), 77.4–77.5
(CH glycerol), 116.6–116.8 (4 ꢃ Cq cyanoethoxy), 127.6–128.5
(CHarom), 137.2–137.3, 137.7–137.8 (6 ꢃ Cq Bn); HRMS:
C69H84N4O23P4+Na+ requires 1483.4369, found 1483.4377.
4.2.16. Glycerol phosphate tetramer (23)
To a solution of trimer 22 (280 mg, 0.323 mmol) in acetoni-
trile (3.0 ml), containing freshly activated MS 3 Å, were added
DCI (0.25 M in acetonitrile, 5.2 ml) and phosphoramidite
3
(0.2 M in acetonitrile, 2.6 ml). After stirring for 2 h, more 3
(0.2 M in acetonitrile, 1.3 ml) was added and the mixture was al-
lowed to react for another 2 h. I2 (0.2 M in THF/pyridine 4/1,
6.5 ml) and H2O (1.0 ml) were added and the mixture was stirred
for 1 h. EtOAc (50 ml) was added and the mixture was washed
with satd aq Na2S2O3 (20 ml), aqueous KHSO4 (0.5 M, 20 ml), satd
aq NaHCO3 (20 ml) and brine (20 ml), respectively. The organic
layer was dried with Na2SO4 and concentrated in vacuo, after
which the residue was redissolved in DCM (15 ml), containing
freshly activated MS 3 Å. Et3SiH (0.52 ml, 3.23 mmol) and dichlo-
roacetic acid (0.27 ml, 3.23 mmol) were added and the mixture
stirred until the orange colour disappeared (ꢀ2 h), after which
the mixture was diluted with DCM (20 ml) and washed with satd
aq NaHCO3 (15 ml) and brine (15 ml), respectively. The organic
layer was dried with Na2SO4, concentrated in vacuo and, subse-
quently, purified by column chromatography (MeOH/DCM),
yielding tetramer 23 (305 mg, 0.262 mmol, 81%) as colourless
oil. 31P NMR (161.7 MHz): d = ꢄ1.3 to ꢄ0.8; 1H NMR
(400 MHz): d = 2.47–2.61 (m, 6H, 3 ꢃ CH2 cyanoethoxy), 3.20
(br s, 1H, OH), 3.56–3.58 (m, 2H, CH2 glycerol) 3.62–3.71 (m,
3H, CH glycerol, CH2 glycerol), 3.78–3.81 (m, 3H, 3 ꢃ CH glyc-
4.2.18. Hexamer (25)
To a solution of pentamer 24 (150 mg, 0.103 mmol) and koji-
biose-glycerol phosphoramidite 5 (350 mg, 0.257 mmol) in aceto-
nitrile (3.0 ml), containing freshly activated MS 3 Å, was added
DCI (0.25 M in acetonitrile, 2.0 ml). After stirring the mixture
overnight at room temperature, I2 (0.2 M in THF/pyridine 4/1,
2.5 ml) and H2O (1.0 ml) were added and the mixture was stirred
for 1 h. The mixture was diluted with EtOAc (30 ml) and subse-
quently washed with satd aq Na2S2O3 (10 ml), aqueous KHSO4
(0.5 M, 10 ml), satd aq NaHCO3 (10 ml) and brine (10 ml). The
organic layer was dried with Na2SO4 and concentrated in vacuo,
after which the residue was purified by size-exclusion chroma-
tography (sephadex LH-20, THF), followed by column chromatog-
raphy (MeOH/DCM), yielding dimethoxytritylated intermediate
25 (243 mg, 88.6 l
mol, 86%) as white foam. 31P NMR
(161.7 MHz): d = ꢄ1.3 to ꢄ0.9; 1H NMR (400 MHz): d = 2.29–
2.56 (m, 10H, 5 ꢃ CH2 cyanoethoxy), 3.10–4.36 (m, 58H,
5 ꢃ CH2 cyanoethoxy, 6 ꢃ CH glycerol, 12 ꢃ CH2 glycerol,

W. F. J. Hogendorf et al. / Bioorg. Med. Chem. 18 (2010) 3668–3678
3677
2 ꢃ OMe, 2 ꢃ H-2, 2 ꢃ H-3, 2 ꢃ H-4, 2 ꢃ H-5, 4 ꢃ H-6), 4.45–4.89
(m, 18H, 9 ꢃ CH2 Bn), 4.92–4.94 (m, 1H, H-1), 5.10–5.13 (m, 1H,
H-1), 5.52 (s, 1H, CH benzylidene), 5.56 (s, 1H, CH benzylidene),
6.74 (d, 4H, J = 8.6 Hz, Harom DMTr), 7.14–7.48 (m, 64H, Harom);
HRMS: [C143H158N5O40P5+Na]2+ requires 1392.9478, found
1392.9489.
to stir for five days under a hydrogen atmosphere. The mixture
was filtered, concentrated in vacuo, and purified by size-exclusion
chromatography (Sephadex HW40, 0.15 M NH4HCO3). The purified
residue was lyophilized twice before it was dissolved in H2O
(2.0 ml) and eluted through a small column containing Amberlite
Na+ resin. Cryodesiccation of the filtrate yielded deprotected tei-
choic acid 1 (10.6 mg, 6.81 lmol, 76%) as amorphous white solid.
4.2.19. Hexamer (26)
31P NMR (161.7 MHz, D2O): d = 0.9 (1P), 1.1 (1P), 1.2 (3P), 1.3
(1P); 1H NMR (600 MHz, D2O): d = 1.36–1.40 (m, 4H, 2 ꢃ CH2 hex-
ylspacer), 1.59–1.66 (m, 4H, 2 ꢃ CH2 hexylspacer), 2.95 (t, 2H,
J = 7.5 Hz, CH2–N hexylspacer), 3.37–3.41 (m, 2H, 2 ꢃ H-5), 3.51–
3.56 (m, 2H, CHH glycerol, H-2), 3.61–3.64 (m, 2H, CHH glycerol,
H-2), 3.70–4.01 (m, 37H, 5 ꢃ CH glycerol, 11 ꢃ CH2 glycerol, CH2–
O hexylspacer, 2 ꢃ H-3, 2 ꢃ H-4, 4 ꢃ H-6), 4.11–4.14 (m, 1H, CH
glycerol), 5.11 (d, 1H, J = 3.7 Hz, H-1), 5.39 (d, 1H, J = 3.5 Hz,
H-1); 13C NMR (150 MHz, D2O): d = 25.4, 26.1, 27.6 (3 ꢃ CH2
hexylspacer), 30.4 (d, J = 6.7 Hz, CH2 hexylspacer), 40.4 (CH2–N
hexylspacer), 61.4, 61.4 (2 ꢃ C-6), 63.0 (CH2 glycerol), 65.5 (d,
J = 5.1 Hz, CH2 glycerol), 66.0 (d, J = 4.4 Hz, CH2 glycerol), 67.0–
67.3 (9 ꢃ CH2 glycerol, CH2–O hexylspacer), 70.4–70.6 (4 ꢃ CH
glycerol, 2 ꢃ C-5), 71.7 (d, J = 7.8 Hz, CH glycerol), 72.1 (C-2, C-3),
72.7 (2 ꢃ C-4), 73.3 (C-3), 75.1 (C-2), 76.0 (t, J = 8.3 Hz, CH glyc-
erol), 95.6 (C-1), 96.4 (C-1). HRMS: C36H77NO41P6+H+ requires
1366.2469 found 1366.2483.
Intermediate 25 (122 mg, 44.5 lmol) was dissolved in a mix-
ture of DCM (2.5 ml) and MeOH (25 ml). After the addition of
pyridinium para-toluenesulfonate (PPTS, 25 mg, 0.099 mmol), the
solution was allowed to stir overnight. Toluene (50 ml) was added
and the mixture was concentrated partially (leaving ꢀ30 ml) under
reduced pressure. Column chromatography of the solution (MeOH/
DCM) yielded hexamer 26 (76.0 mg, 31.1 lmol, 70%) as colourless
oil. 31P NMR (161.7 MHz): d = ꢄ1.3 to ꢄ0.8; 1H NMR (400 MHz):
d = 1.79 (br s, 1H, OH), 2.44–2.57 (m, 10H, 5 ꢃ CH2 cyanoethoxy),
3.38–4.34 (m, 52H, 5 ꢃ CH2 cyanoethoxy, 6 ꢃ CH glycerol,
12 ꢃ CH2 glycerol, 2 ꢃ H-2, 2 ꢃ H-3, 2 ꢃ H-4, 2 ꢃ H-5, 4 ꢃ H-6),
4.51–4.65 (m, 12H, 6 ꢃ CH2 Bn), 4.76–4.99 (m, 7H, 3 ꢃ CH2 Bn,
H-1), 5.11–5.13 (m, 1H, H-1), 5.50 (s, 1H, CH benzylidene), 5.55
(s, 1H, CH benzylidene), 7.15–7.17 (m, 3H, Harom), 7.26–7.40 (m,
48H, Harom), 7.42–7.47 (m, 4H, Harom); HRMS: [C122H140N5O38P5+-
Na]2+ requires 1241.8824, found 1241.8837.
4.2.20. Hexamer-spacer (27)
Acknowledgements
To a solution of kojibiose substituted hexamer 26 (124 mg,
51.0
l
mol) and Z-aminohexylphosphoramidite
5
(184 mg,
We thank Crucell N.V. and the Netherlands Organization for Sci-
entific Research (NWO) for financial support.
0.408 mmol) in acetonitrile (3.0 ml), containing freshly activated
MS 3 Å, was added DCI (0.25 M in acetonitrile, 2.0 ml). After stir-
ring the mixture for 4 h, acetonitrile (5.0 ml), I2 (0.2 M in THF/pyr-
idine 4/1, 3.1 ml) and H2O (1.0 ml) were added and the mixture
stirred for 1 h. The mixture was diluted with EtOAc (30 ml) and
subsequently washed with satd aq Na2S2O3 (10 ml), aqueous
KHSO4 (0.5 M, 10 ml), satd aq NaHCO3 (10 ml) and brine (10 ml).
The organic layer was dried with Na2SO4 and concentrated in va-
cuo, after which the residue was purified by size-exclusion chro-
matography (sephadex LH-20, MeOH/DCM), followed by column
chromatography (MeOH/DCM), yielding compound 27 (111 mg,
Supplementary data
Supplementary data (1H, 13C, 31P NMR spectra) associated with
this article can be found, in the online version, at doi:10.1016/
j.bmc.2010.03.071.
References and notes
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39.5
l
mol, 77%) as colourless oil. 31P NMR (161.7 MHz): d = ꢄ1.3
to ꢄ0.9; 1H NMR (400 MHz): d = 1.18–1.32 (m, 4H, 2 ꢃ CH2 hex-
ylspacer), 1.35–1.44 (m, 2H, CH2 hexylspacer), 1.53–1.62 (m, 2H,
CH2 hexylspacer), 2.44–2.58 (m, 12H, 6 ꢃ CH2 cyanoethoxy),
3.05–3.13 (m, 2H, CH2–N hexylspacer), 3.56–4.33 (m, 56H,
6 ꢃ CH2 cyanoethoxy, 6 ꢃ CH glycerol, 12 ꢃ CH2 glycerol, CH2–O
hexylspacer, 2 ꢃ H-2, 2 ꢃ H-3, 2 ꢃ H-4, 2 ꢃ H-5, 4 ꢃ H-6),
4.50–4.59 (m, 12H, 6 ꢃ CH2 Bn), 4.74–5.15 (m, 10H, 4 ꢃ CH2 Bn,
2 ꢃ H-1), 5.53 (s, 1H, CH benzylidene), 5.55 (s, 1H, CH benzyli-
dene), 7.16–7.18 (m, 3H, Harom), 7.26–7.39 (m, 53H, Harom), 7.43–
7.48 (m, 4H, Harom); HRMS: [C139H163N7O43P6+Na]2+ requires
1424.9497 found 1424.9512.
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A solution of protected hexamer 27 (51.0 mg, 18.2 lmol) in
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and stirred overnight. The solution was concentrated in vacuo
and the residue redissolved in H2O (2.0 ml). The solution was then
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lyophilized, yielding the partially protected intermediate
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3678
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