Synthesis of an α-Gal epitope α-D-Galp-(1 → 3)β-D-Galp-(1 → 4)-β-D-Glcp NAc-lipid conjugate

Article abstract of DOI:10.1080/07328300500308113

The synthesis of a neoglycoconjugate containing the Galili epitope trisaccharide connected to a spacer-lipid entity is described. The α-D-Galp-(1 → 3)-β-D-Galp-(1 → 4)-β-D-GlcpNAc trisaccharide, equipped with a 3-aminopropyl spacer, is efficiently assembl

Full text of DOI:10.1080/07328300500308113

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Synthesis of an α‐Gal epitope
α‐D‐Galp‐(1→3)‐β‐D‐Galp‐(1→4)‐β‐D‐Glcp
NAc–lipid conjugate
Remy E. J. N. Litjens a , Peter Hoogerhout b , Dmitri V. Filippov a ,
Jeroen D. C. Codée a , Leendert J. van den Bos a , Richard J. B. H.
N. van den Berg a , Herman S. Overkleeft a & Gijsbert A. van der
Marel a
a Leiden Institute of Chemistry , Leiden University , Leiden, The
Netherlands
b Unit Research and Development , The Netherlands Vaccine
Institute , Bilthoven, The Netherlands
Published online: 20 Aug 2006.
To cite this article: Remy E. J. N. Litjens , Peter Hoogerhout , Dmitri V. Filippov , Jeroen D. C.
Codée , Leendert J. van den Bos , Richard J. B. H. N. van den Berg , Herman S. Overkleeft & Gijsbert
A. van der Marel (2005) Synthesis of an α‐Gal epitope α‐D‐Galp‐(1→3)‐β‐D‐Galp‐(1→4)‐β‐D‐Glcp
NAc–lipid conjugate , Journal of Carbohydrate Chemistry, 24:7, 755-769
To link to this article: http://dx.doi.org/10.1080/07328300500308113
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Journal of Carbohydrate Chemistry, 24:755–769, 2005
Copyright # Taylor & Francis, Inc.
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1080/07328300500308113
Synthesis of an a-Gal
epitope a-D-Galp -(1!3)-b-D-
Galp-(1!4)-b-D-Glcp NAc–
lipid conjugate
Remy E. J. N. Litjens
Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands
Peter Hoogerhout
Unit Research and Development, The Netherlands Vaccine Institute, Bilthoven,
The Netherlands
´
Dmitri V. Filippov, Jeroen D. C. Codee, Leendert J. van den Bos,
Richard J. B. H. N. van den Berg, Herman S. Overkleeft, and
Gijsbert A. van der Marel
Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands
The synthesis of a neoglycoconjugate containing the Galili epitope trisaccharide con-
nected to a spacer-lipid entity is described. The a-D-Galp-(1 ! 3)-b-D-Galp-(1 ! 4)-b-
D-GlcpNAc trisaccharide, equipped with a 3-aminopropyl spacer, is efficiently assembled
from easily accessible building blocks in a one-pot procedure. Global deprotection of the
trisaccharide and ensuing introduction of a bis(palmitamido)- propanamido moiety
afforded title compound 1 as depicted in Scheme 1.
Keywords a-Gal epitope, Glycolipid, Oligosaccharide synthesis
Received March 10, 2005; accepted May 3, 2005.
Address correspondence to Gijsbert A. van der Marel, Leiden Institute of Chemistry,
Leiden University, P.O. Box 9502, 2300, Leiden, The Netherlands. E-mail: marel_g@
chem.leidenuniv.nl
Dedicated to the memory of Prof. Dr. Jacques H. van Boom, our teacher, colleague, and
friend.
755

R .E. J. N. Litjens et al.
756
INTRODUCTION
The carbohydrate structure a-D-Galp-(1 ! 3)-b-D-Galp-(1 ! 4)-b-D-GlcpNAc,
in the biologic literature also referred to as “Galili antigen” or simply “a-Gal,”
is expressed on many cells and tissues of nonprimate mammals and
New World monkeys.[1] However, the Galili epitope trisaccharide is nonself to
Old World monkeys, apes, and humans due to evolutionary inactivation of
the gene encoding a1,3-galactosyltransferase. The Galili epitope is a major
obstacle in the field of xenotransplantation of tissues or organs from pigs to
monkeys (or humans).[2,3] On the other hand, the strong immunologic
response to the Galili trisaccharide could be beneficial in vaccinology or
immune therapy. It was demonstrated that covalent introduction of the Galili
trisaccharide onto hepatitis B virus hemagglutinin[4,5] or tumor cells[6–10]
enhanced the immunogenicity. To this end, the b-D-Galp-(1 ! 4)-GlcNAc
epitopes present on the hemagglutinin or liberated (with neuraminidase) on
the tumor cell surface were modified with uridine diphosphogalactose
(UDP-Gal) and a suitable a1,3-galactosyltransferase. It is obvious that such
an enzymatic modification of antigens will not always be possible or practical.
We envisage that a noncovalent association of the Galili trisaccharide and
the target particle should be possible via lipid anchors attached to the
carbohydrate. With this objective in mind, we prepared the artificial glycolipid
1 as described in this paper.
RESULTS AND DISCUSSION
Our synthetic strategy to target compound 1 comprises the construction of the
hydrophilic trisaccharide 2, followed by the introduction of the lipophilic tails
(Sch. 1). Following this strategy, 3-aminopropyl equipped trisaccharide 2
should be functionalized with diaminopropionic acid, allowing the introduction
of two palmitic acid moieties to give amphiphile 1. Construction of the spacer
containing trisaccharide 2 was envisaged to proceed by either a chemoselective
or an orthogonal coupling sequence of monosaccharide building blocks 3a or
3b, 4, and 5 followed by a global deprotection of the formed trisaccharide.[11,12]
Initially, attention was focussed on the synthesis of the intermediate
thiodisaccharide 6 employing benzylated and acylated galactosides 3a/b
and 4. Based on the findings in armed-disarmed,[13] chemoselective,[14,15] and
orthogonal glycosylations,[16] several condensation conditions were
investigated (Table 1).
Iodonium sym-collidine perchlorate (IDCP)-mediated chemoselective gly-
cosidation of armed phenyl 2,3,4,6-tetra-O-benzyl-b-D-thiogalactoside[17] 3a
with disarmed phenyl 4-O-acetyl-2,6-di-O-benzoyl-b-D-thiogalactoside[18] 4 in
a mixture of dichloroethane and diethyl ether gave a-linked disaccharide 6
in 50% yield as the sole isomer (entry 1). Executing the IDCP protocol in

Synthesis of a Lipid Conjugate
757
Scheme 1: Retrosynthetic analysis of target glycolipid 1.
toluene/dioxane, as advocated by Zhu and Boons, gave a-disaccharide 6 in 54%
(entry 2).[19] Condensation of the same donor and acceptor with the aid of
1-benzenesulfinylpiperidine
(BSP)/trifluoromethanesulfonic
anhydride
(Tf2O),[20] the activation system of the Crich group, followed by quenching
with triethyl phosphite (TEP)[15] (entry 3) resulted in a 52% isolated yield
of 6. Diphenyl sulfoxide (DPS)/Tf2O[21] promoted orthogonal condensation of
galactosyl donor 3b with 4 afforded 6 in 64% yield (entry 4).
Having thiodisaccharide 6 in hand, the elongation with acceptor 5 was
examined.a DPS/Tf2O[15a] mediated condensation of donor disaccharide 6
with acceptor 5 afforded trisaccharide 7 with the expected equatorial
orientation of the newly introduced glycosidic bond in 69% yield (Sch. 2).
On the basis of the above described glycosylation experiments, it was
investigated whether the construction of trisaccharide 7 could be improved
by performing the condensation of 3b, 4, and 5 in a one-pot procedure.[16]
aAcceptor 5 wasreadily prepared in two steps starting from ethyl 3-O-benzyl-4,6-O-benzy-
lidene-2-deoxy-2-phthalimido-1-thio-b-D-glucopyranoside[23] (first BSP/Tf2O-mediated
b-glycosylation of 3-azido-propan-1-ol,[24] then regioselective reductive opening of the
benzylidene in 3-azido-1-propyl 3-O-benzyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-
b-D-glucopyranoside under the agency of TES/TfOH)

R .E. J. N. Litjens et al.
Table 1: Synthesis of thiodigalactoside 6.
758
Yield of
6 (%)
Entry
Donor
Activator
IDCP
Solvent
1
2
3a
3a
DCE/Et2O (1 : 5 v/v)
Toluene/dioxane
(3 : 1 v/v)
50
54
IDCP
3
4
3a
3b
BPS/Tf2O TEP
quench
DPS/Tf2O
DCM
52
64
DCM
Therefore, hemiacetal 3b was activated with DPS/Tf2O followed by adding
acceptor thiogalactoside 4 to the reaction mixture to afford transient dimer 6
with concomitant regeneration of DPS. Ensuing activation of the thio
function in 6 was affected by the addition of another equivalent of triflic
anhydride. Subsequent introduction of acceptor 5 to the reaction vessel led to
the one-pot construction of 7 in 61% yield. The yield of this operation is a
Scheme 2: Stepwise and one-pot construction of trisaccharide 7.

Synthesis of a Lipid Conjugate
759
significant improvement in comparison with the overall yield of 44% from the
stepwise approach.
The introduction of the lipophilic tails started with the global deprotection
of trisaccharide 7 (Sch. 3). First, the phthalimide in 7 was transformed into the
free amine using ethylenediamine[22] (EDA) in refluxing nBuOH under strictly
anhydrous conditions. H was followed by acetylation (Ac2O in pyridine) and
subsequent saponification of the ester moieties by treatment with catalytic
KOtBu in MeOH to give 8 with the 20-O-acetate in the central galactose
residue unaffected. Ensuing treatment with stoichiometric KOtBu in reflux-
ing MeOH resulted in clean removal of the acetate, furnishing the desired
trisaccharide 9 in 94%. Hydrogenolysis of 9 with palladium on carbon
under a hydrogen atmosphere afforded 3-aminopropyl trisaccharide 2 in
quantitative yield. The identity of 2 was ascertained by NMR spectroscopy
and HRMS spectrometry and was in accordance with the reported
data.[11a] The overall yield of trisaccharide 2 starting from monosaccharides
3b, 4, and 5 is 52% over seven steps (based on 4). In a related synthesis by
Hanessian et al., 2 was prepared in 27% over four steps starting from three
monosaccharide building blocks.[11b]
The stage was now set for the introduction of the lipid anchor moiety
(Sch. 4). The free amine in 2 was acylated with bis-Fmoc-diaminopropionic
acid under the influence of benzotriazol-1-yloxytris(dimethylamino)-
phosphonium hexafluorophosphate (BOP reagent) and di-isopropylethylamine
(DiPEA), to afford bis-Fmoc trisaccharide 10, which was purified by a sequence
of trituration steps. Removal of the Fmoc groups in 10 by treatment with piperi-
dine and purification of the resulting diamine proved to be very tedious. This
problem could be circumvented by the following sequence of steps. The
diamine was released by treatment of 10 with 1,8-diazabicyclo[5.4.0]undec-7-
ene (DBU), and the generated fluorene was quenched with ethanethiol to
avoid any side reactions. The reaction mixture was concentrated and the
Scheme 3: i. EDA, nBuOH, reflux; ii. Ac2O, pyridine; iii. KOtBu (cat.), MeOH, 92% over the three
steps; iv. KOtBu (1 eq.), MeOH, reflux, 94%; v. H2, Pd/C, HCl, tBuOH/H2O 11/4 v/v, quant.

R .E. J. N. Litjens et al.
760
Scheme 4: i. 2(S),3-bis-(9H-fluoren-9-ylmethoxycarbonylamino)-propionic acid, BOP, DiPEA,
DMSO/DMF 1 : 2 v/v; ii. DBU, DMSO/DMF 1 : 2 v/v; iii. palmitic acid N-hydroxysuccinimide ester,
DiPEA, DMF/DMSO/CHCl3 2 : 1 : 1 v/v, 35% over the three steps.
residue dissolved in a mixture of DMSO, DMF, and CHCl3. Subsequent treat-
ment with excess palmitic acid N-hydroxysuccinimide ester in the presence of
DiPEA followed by concentration and precipitation from hot methanol
provided homogeneous glycolipid 1 in 35% over three steps. The identity of 1
1
was ascertained by H NMR, LCMS, and HRMS. The purity was determined
from the 1H NMR spectrum and was .95%.
CONCLUSION
Artificial glycolipid 1, designed for incorporation into liposomes and mem-
branes, was assembled in two stages. Aminopropyl spacer containing
trisaccharide 7 was synthesized in a one-pot procedure from easily available
orthogonal protected building blocks. After removal of the protecting groups
in 7, the lipid anchor was introduced via a procedure, in which the number
of chromatographic purification steps was minimized. The application of
neoglycoconjugate 1 in immunologic experiments is currently being examined.
EXPERIMENTAL SECTION
General Methods
Dichloromethane was refluxed with P2O5 and distilled before use.
1-Benzenesulfinylpiperidine (BSP) and tri-tert-butylpyrimidine (TTBP) were

Synthesis of a Lipid Conjugate
761
synthesized as described by Crich et al.[20,25] Trifluoromethanesulfonic anhy-
dride (Tf2O) was stirred for 3 hr on P2O5 and subsequently distilled. All other
chemicals (Fluka, Acros, Merck, Aldrich, Sigma) were used as received. Hyflo
Super Cel filter aid was purchased from Fluka and used as received. Reactions
were performed under an inert atmosphere under strictly anhydrous con-
ditions unless stated otherwise. Traces of water from reagents used in
reactions that require anhydrous conditions were removed by coevaporation
0
˚
with toluene and dichloroethane. Molecular sieves (3A and 5 ) were flame
dried before use. Column chromatography was performed on Fluka Silica gel
60 (0.04–0.063 mm, 230–400 mesh ASTM). TLC analysis was conducted on
DC-alufoil (Merck, Kieselgel 60 F254). Compounds were visualized by UV
absorption (254 nm) and by spraying with 20% H2SO4 in ethanol, with a
solution of ninhydrin 0.4 g in EtOH (100 mL) containing acetic acid (3 mL) or
.
with a solution of (NH4)6Mo7O24 4H2O 25 g/L, followed by charring at
+1408C. 1H and 13C NMR spectra were recorded with a Bruker DPX 300
(300 and 75.1 MHz), a Bruker AV 400 (400 and 100 MHz), or a Bruker DMX
600 (600 and 125 MHz). NMR spectra were recorded in CDCl3 with chemical
shifts (d) relative to tetramethylsilane unless stated otherwise. Mass spectra
were recorded on a PE/SCIEX API 165 equipped with an Electrospray Inter-
face (Perkin-Elmer) or a Finnigan LTQ-FT (Thermo Electron). Optical
rotations were recorded on a Propol automatic polarimeter. The purity of the
1
synthesized compounds was .95% as judged by H NMR.
Phenyl (2,3,4,6-Tetra-O-benzyl-a-D-galactopyranosyl)-
(1 ! 3)-4-O -acetyl-2,6-di-O -benzoyl-1-thio-b-D-
galactopyranoside (6).
IDCP: To a solution of 3a (190 mg, 0.3 mmol) and 4 (131 mg, 0.25 mmol) in
˚
DCE/Et2O (5 mL, 1 : 5 v/v) containing powdered 5A MS was added IDCP
(280 mg, 0.6 mmol). After stirring for 1 hr, the reaction mixture was diluted
with ethyl acetate and washed with a 10% aq. Na2SO4 solution. The organic
layer was dried (MgSO4), filtered, and concentrated. Column chromatography
of the residue (ethyl acetate/light petroleum, 1 : 9 ! 1 : 5 v/v) afforded 6
(130 mg, 125 mmol, 50%) as a colorless oil.
BSP/Tf2O; (EtO)3P quench: To a solution of thiodonor 3a (0.2 mmol, 1.0
equiv), BSP (46 mg, 0.22 mmol), and TTBP (124 mg, 0.5 mmol) in dichlorome-
thane (5 mL) containing 30 MS at –608C was added trifluoromethanesulfonic
anhydride (37 mL, 0.22 mmol). The reaction mixture was stirred for 5 min,
after which a solution of acceptor thioglycoside 4 (115 mg, 0.22 mmol) in
dichloromethane (2 mL) was added. The mixture was stirred at –608C for
1 hr followed by slowly warming to –108C. The reaction was quenched with
triethyl phosphite (1.0 equiv.) and triethylamine (5 equiv.). Sat. aq. NaHCO3
was added and the organic layer was separated, washed with saturated NaCl

R .E. J. N. Litjens et al.
762
solution, dried (MgSO4), and concentrated. Purification by silica gel chromato-
graphy (light petroleum ! ethyl acetate/light petroleum 1 : 4 v/v) gave
disaccharide 6 (109 mg, 104 mmol, 52%) as an oil.
DPS/Tf2O: To a solution of the 1-hydroxyl donor 3b (140 mg, 0.26 mmol),
DPS (120 mg, 0.57 mmol), and TTBP (140 mg, 0.57 mmol) in DCM (5 mL) was
added trifluoromethanesulfonic anhydride (0.27 mmol, 46 mL) at –608C. The
temperature was raised to –408C, and the reaction mixture was stirred at
this temperature for 1 hr. Then, a solution of acceptor 4 (104 mg, 0.2 mmol) in
DCM (2 mL) was added, and the reaction mixture was allowed to warm to rt.
Dry Et3N (10 equiv to donor) was added, and the reaction mixture was
washed with saturated NaHCO3 and water. After drying (MgSO4) and concen-
tration, the residue was purified by column chromatography (ethyl acetate/
light petroleum) to give disaccharide 6 (134 mg, 128 mmol, 64%) as a colorless
syrup.
6: Rf 0.80 (33% ethyl acetate in light petroleum). [a]2D5 þ90.8 (c ¼ 1). [1]H NMR:
d (ppm) 8.12–7.14 (m, 35 H, CHarom), 5.67 (d, 1H, J ¼ 2.8 Hz, H-4), 5.61 (t, 1H,
J ¼ 9.9 Hz, H-2), 5.22 (d, 1H, J ¼ 3.2 Hz, H-10), 4.81 (d, 1H, J ¼ 10.1 Hz, H-1),
4.76 (d, 1H, J ¼ 11.4 Hz, –CHPh), 4.65 (s, 2H, –CHPh), 4.64 (d, 1H,
J ¼ 12.2 Hz, –CHPh), 4.48 (m, 2H, H-6, –CHPh), 4.36 (m, 4H, H-6, –CHPh),
4.16 (dd, 1H, J ¼ 3.0, 9.7 Hz, H-3), 3.93 (m, 3H, H-20, H-5, H-50), 3.75 (dd, 1H,
J ¼ 2.6, 10.1 Hz, H-30), 3.44 (dd, 1H, J ¼ 7.3, 9.6 Hz, H-60), 3.23 (bs, 1H, H-40),
3.20 (dd, 1H, J ¼ 5.2, 9.6 Hz, H-60), 1.89 (s, 3H, Ac). 13C NMR: d (ppm) 170.3,
165.9, 164.8 (C55O), 138.6, 138.5, 138.3 (Cq Bn), 133.4 (Cq SPh), 129.5, 129.4
(Cq Bz), 133.2–127.4 (CHarom), 93.3 (C-10), 87.0 (C-1), 78.7 (C-30), 75.5 (C-20),
74.8 (C-50), 74.7 (C-40), 74.3, 74.2, 73.1, 73.0 (CH2 Bn), 72.7 (C-3), 69.9 (C-5),
69.4 (C-60), 68.9 (C-2), 65.1 (C-4), 62.7 (C-6), 20.4 (CH3 Ac). ESI-MS: m/z
1068.1 [M þ Na]þ.
3-Azidopropyl 3,6-Di-O-benzyl-2-deoxy-2-phthalimido-b-D-
glucopyranoside (5).
A solution of ethyl 3-O-benzyl-4,6-O-benzylidene-2-deoxy-2-phthalamido-
1-thio-b-D-glucopyranoside (3.7 g, 7 mmol), BSP (1.77 g, 8.5 mmol), and TTBP
˚
(3.8 g, 15.4 mmol) containing 3A MS in DCM (50 mL) at –608C was
treated with Tf2O (1.44 mL, 8.5 mmol) for 10 min, after which 3-azidopropan-
1-ol (2.12 g, 21 mmol) in DCM (10 mL) was added. The mixture was allowed
to warm to 08C, and Et3N (2 mL) was added. The reaction mixture was
filtered and washed with sat. aq. NaHCO3. The organic layer was dried
(MgSO4), filtered, and concentrated in vacuo. Column chromatography of the
residue (light petroleum ! ethyl acetate/light petroleum 1 : 5 v/v) afforded
3-azidopropyl 3-O-benzyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-b-D-gluco-
pyranoside (3.35 g, 5.9 mmol, 84%) as a white foam. 1H NMR: d (ppm)

Synthesis of a Lipid Conjugate
763
7.74–6.89 (m, 14H, Harom), 5.62 (s, 1H, –CHPh), 5.20 (d, 1H, J ¼ 8.8 Hz, H-1),
4.80 (d, 1H, J ¼ 12.4 Hz, –CHPh), 4.50 (d, 1H, J ¼ 12.4 Hz, –CHPh), 4.43
(m, 2H, H-2, H-3), 4.20 (dd, 1H, J ¼ 11.0, 8.8 Hz, H-2), 3.82 (m, 3H, H-4, 2 ꢀ
H-6), 3.72 (m, 1H, H-5), 3.41 (m, 2H, O–CH2–CH2), 3.12 (m, 2H, CH2N3),
1.71 (m, 2H, CH2–CH2–CH2). 13C NMR: d (ppm) 167.1, 137.4, 136.9, 133.4,
131.6, 130.8, 128.5, 128.3, 127.6, 127.4, 126.8, 125.5, 124.5, 122.7, 100.5,
98.3, 82.3, 74.0, 73.3, 68.0, 65.7, 65.5, 60.5, 58.5, 55.2, 47.2, 28.1. ESI-MS: m/
z 593.2 [M þ Na]þ.
3-Azidopropyl 3-O-benzyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-b-D-glu-
copyrano-side (2.85 g, 5 mmol) was treated with TfOH (1.27 mL, 15.0 mmol)
in the presence of triethylsilane (2.6 mL, 16.6 mmol) in DCM (50 mL) at –
788C. After 20 min the reaction was quenched by the subsequent addition of
MeOH and triethylamine. After the reaction mixture was washed with satu-
rated aqueous NaHCO3, dried, and concentrated, the residue was purified by
column chromatography (ethyl acetate/light petroleum, 1 : 20 ! 1 : 4 v/v) to
1
provide compound 5 (2.0 g, 3.55 mmol, 71%) as a slightly yellow oil. H NMR:
d (ppm) 7.79–6.92 (m, 14 H, Harom), 5.14 (d, 1H, J ¼ 8.3 Hz, H-1), 4.75
(d, 1H, J ¼ 12.2 Hz, –CHPh), 4.64 (d, 1H, J ¼ 12.0 Hz, –CHPh), 4.58 (d, 1H,
J ¼ 12.0 Hz, –CHPh), 4.53 (d, 1H, J ¼ 12.2 Hz, –CHPh), 4.24 (dd, 1H,
J ¼ 8.4, 10.8 Hz, H-3), 4.15 (dd, 1H, J ¼ 8.4, 10.8 Hz, H-2), 3.81 (m, 4H, H-4,
H-6, O–CH2–CH2), 3.66 (m, 1H, H-5), 3.45 (m, 1H, H-6), 3.11 (m, 2H,
CH2N3), 1.66 (m, 2H, CH2–CH2–CH2). [13]C NMR: d (ppm) 138.0, 137.6 (Cq
Bn), 133.8, 128.4, 128.0, 127.7, 127.6, 127.3, 123.2 (CHarom), 131.4 (Cq, Phth),
98.2 (C-1), 78.6 (C-3), 74.2 (CH2 Bn), 74.2 (C-5), 74.0 (C-4), 73.6 (CH2 Bn),
70.3 (O–CH2–CH2), 68.1 (C-6), 55.2 (C-2), 47.8 (CH2N3), 28.7 (OCH2–CH2–
CH2). ESI-MS: m/z 595.3 [M þ Na]þ.
3-Azidopropyl (2,3,4,6-Tetra-O-benzyl-a-D-
galactopyranosyl)-(1 ! 3)-(4-O-acetyl-2,6-di-O-benzoyl-
b-D-galactopyranosyl)-(1 ! 4)-3,6-di-O-benzyl-2-deoxy-
2-phtalimido-b-D-glucopyranoside (7).
Stepwise procedure: A solution of disaccharide 6 (209 mg, 0.2 mmol), DPS
(81 mg, 0.4 mmol), and TTBP (124 mg, 0.45 mmol) was treated with Tf2O
(37 mL, 0.22 mmol) for 10 min at –608C. Then, acceptor 5 (179 mg, 0.3 mmol)
was added and the reaction mixture was slowly warmed to 08C. Standard
workup and purification gave trisaccharide 9 (211 mg, 138 mmol, 69% yield)
as a colorless oil.
One-pot procedure: 2,3,4,6-Tetra-O-benzyl-a,b-D-galactopyranose 3b
(1.36 g, 2.5 mmol), DPS (1.02 g, 5.0 mmol), and TTBP (1.86 g, 7.5 mmol)
˚
were dissolved in DCM (50 mL), powdered 3A MS (500 mg) was added, and

R .E. J. N. Litjens et al.
764
the reaction mixture was cooled to –608C. Tf2O (440 mL, 2.6 mmol) was
added, and the reaction mixture was brought to –408C. Stirring was conti-
nued for 1 hr at this temperature, after which phenyl 4-O-acetyl-2,6-di-O-
benzoyl-1-thio-b-D-galactopyranoside 4 (1.04 g, 2.0 mmol in 5 mL DCM) was
added. The reaction mixture was kept at –408C for 1 hr after which it was
slowly warmed to 08C. After 30 min at 08C the reaction mixture was cooled
to –608C, and Tf2O (400 mL, 2.4 mmol) was added. After the reaction was
kept at –608C for 10 min, 3-azidopropyl 3,6-di-O-benzyl-2-deoxy-2-phtal-
imido-b-D- glucopyranoside 5 (1.64 g, 3 mmol) was added. The mixture was
slowly warmed to 08C, after which Et3N (2 mL) was added. Standard
workup and purification gave compound 7 (1.85 g, 1.23 mmol, 61%) as a color-
less oil. Rf 0.60 (33% ethyl acetate in light petroleum). [a]2D5 þ39.2 (c ¼ 0.75,
CHCl3). 1H NMR: d (ppm) 8.10–6.83 (m, 44H, Harom), 5.54 (m, 2H, H-20,
H-40), 5.14 (d, 1H, J ¼ 3.3 Hz, H-100), 5.00 (d, 1H, J ¼ 8.5 Hz, H-1), 4.91
(d, 1H, J ¼ 12.4 Hz, –CHPh), 4.78 (d, 1H, J ¼ 8.1 Hz, H-10), 4.74 (d, 1H,
J ¼ 11.4 Hz, –CHPh), 4.63 (s, 2H, –CHPh), 4.62 (d, 1H, J ¼ 11.8 Hz,
–CHPh), 4.55 (d, 1H, J ¼ 12.0 Hz, –CHPh), 4.51 (d, 1H, J ¼ 12.4 Hz,
–CHPh), 4.44 (d, 1H, J ¼ 11.8 Hz, –CHPh), 4.42 (d, 1H, J ¼ 11.8 Hz, –CHPh),
4.35 (d, 1H, J ¼ 12.0 Hz, –CHPh), 4.29 (m, 3H, 2 ꢀ –CHPh, H-3), 4.22 (dd,
1H, J ¼ 6.5 Hz, J ¼ 11.3 Hz, H-60), 4.13 (m, 2H, H-60, H-2), 4.05 (dd, 1H,
J ¼ 8.5 Hz, J ¼ 9.9 Hz, H-4), 3.99 (dd, 1H, J ¼ 3.4 Hz, J ¼ 10.2 Hz, H-30), 3.91
(dd, 1H, J ¼ 3.3, 10.2 Hz, H-200), 3.85 (bt, 1H, J ¼ 6.9 Hz, H-500), 3.75 (m, 1H,
O–CHH–CH2), 3.67 (m, 2H, H-50, H-6), 3.58 (m, 2H, H-6, H-300), 3.41 (m, 1H,
H-5), 3.38 (m, 2H, H-600, O-CHH-CH2), 3.25 (dd, 1H, J ¼ 1.2 Hz, J ¼ 2.6 Hz,
H-400), 3.21 (dd, 1H, J ¼ 5.9 Hz, J ¼ 9.4 Hz, H-600), 3.08 (m, 2H, CH2–CH2–
N3), 1.81 (s, 3H, Ac), 1.64 (m, 2H, CH2–CH2–;CH2). 13C NMR: d (ppm) 170.2,
166.0, 164.6 (C55O), 138.7, 138.4, 138.1, 138.0 (Cq Bn), 131.5 (Cq Phth),
129.3, 129.7 (Cq Bz), 133.7–123.2 (CHarom), 100.8 (C-10), 98.3 (C-1), 94.1
(C-100), 78.8 (C-300), 78.3 (C-4), 76.9 (C-3), 75.5 (C-200), 74.8 (C-400), 74.7 (C-5),
74.5, 73.5, 73.3, 73.2, 73.1 (CH2 Bn), 72.3 (C-30), 71.4 (C-20), 71.0 (C-50), 69.8
(C-500), 69.2 (C-600), 67.8 (C-6), 65.9 (O–CH2–CH2), 65.0 (C-40), 61.7 (C-60),
55.7 (C-2), 48.0 (CH2–CH2–N3), 28.8 (CH2-CH2-CH2), 20.4 (Ac). ESI-MS:
m/z 1529.8 [M þ Na] þ . ESI-HRMS calcd for C87H86N4O20 (M þ H):
1507.5914. Found: 1507.5908.
3-Azidopropyl (2,3,4,6-Tetra-O-benzyl-a-D-
galactopyranosyl)-(1 ! 3)-(2-O-acetyl-b-D-
galactopyranosyl)-(1 ! 4)-2-acetamido-3,6-di-O-
benzyl-2-deoxy-b-D-glucopyranoside (8).
To a solution of 7 (320 mg, 0.21 mmol) in dry nBuOH (2 mL) was added
ethylene diamine (1 mL), and the mixture was heated to 908C and stirred

Synthesis of a Lipid Conjugate
765
overnight. After concentration and coevaporation with toluene (2ꢀ), the
resulting solid was dissolved in pyridine (2 mL), and Ac2O (200 mL,
2.12 mmol) was added. After 3 hr, TLC analysis (ethyl acetate/light petro-
leum, 2 : 1 v/v) showed formation of a single product, and the reaction
mixture was concentrated and then concentrated from toluene (2ꢀ). The
resulting oil was dissolved in dry MeOH (2 mL) and a catalytic amount of
KOtBu was added. After overnight reaction, TLC analysis (ethyl acetate/
light petroleum, 2 : 1 v/v) showed full consumption of the starting material
into one lower running spot. The reaction mixture was neutralized with
Amberlite IR 120 Hþ-resin, filtered, and concentrated. The crude product
was purified by column chromatography (30% ethyl acetate/light petro-
leum ! ethyl acetate), affording the title compound (234 mg, 0.19 mmol,
92%) as a white foam. Rf 0.40 (ethyl acetate/light petroleum, 2 : 1 v/v).
[1]H NMR: d (ppm) 7.28 (m, 30H, Harom), 6.03 (d, 1H, J ¼ 8.5 Hz, H-1), 5.10
(t, 1H, J ¼ 8.3 Hz, H-20), 4.91 (d, 1H, J ¼ 11.4 Hz, –CHPh), 4.86 (d, 1H,
J ¼ 11.5 Hz, –CHPh), 4.74 (bs, 4H, –CHPh), 4.59 (m, 5H, –CHPh, H-100), 4.42
(m, 3H, –CHPh), 4.33 (d, 1H, J ¼ 8.4 Hz, H-10), 4.07 (dd, 1H, J ¼ 9.6, 3.7 Hz,
H-30), 3.90 (m, 2H, H-4, H-600), 3.83 (m, 3H, H-6, H-500, H-3), 3.73 (m, 4H, H-2,
H-200, H-6, H-6), 3.69 (d, 1H, J ¼ 3.3 Hz, H-40), 3.61 (m, 2H, H-60, H-5), 3.50
(m, 3H, H-30, O–CH2–CH2), 3.34 (m, 3H, H-50, CH2–CH2-N3), 2.02 (s, 3H,
Ac), 1.89 (s, 3H, Ac), 1.78 (m, 2H, CH2–CH2–;CH2). ESI-MS: m/z 1233.5
[M þ Na]þ. ESI-HRMS calcd for C67H78N4O17 (M þ H): 1211.5440. Found:
1211.5443.
3-Azidopropyl (2,3,4,6-Tetra-O-benzyl-a-D-
galactopyranosyl)-(1 ! 3)-b-D-galactopyranosyl-
(1 ! 4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-b-D-
glucopyranoside (9).
To a solution of 8 (234 mg, 0.19 mmol) in dry MeOH (2 mL) was added
KOtBu (22 mg, 0.19 mmol) and the mixture was refluxed for 16 hr after
which TLC analysis (MeOH/CHCl3, 1 : 9 v/v) showed full conversion of the
starting material into one lower running spot. The reaction mixture was neu-
tralized by the addition of Amberlite IR 120 Hþ-resin, filtered, and concen-
trated under reduced pressure. Column chromatography gave compound 9
(210 mg, 0.18 mmol, 94%) as a white foam. Rf 0.56 (MeOH/CHCl3, 1 : 9 v/v).
[a]2D5 þ69.1 (c ¼ 0.1, CHCl3). 1H NMR: d (ppm) 7.31 (m, 30H, Harom), 4.90
(m, 3H, –CHPh), 4.85 (d, 1H, J ¼ 8.2 Hz, H-1), 4.74 (s, 2H, –CHPh), 4.67
(d, 1H, J ¼ 11.6 Hz, –CHPh), 4.59 (m, 4H, –CHPh, H-10), 4.47 (m, 3H,
–CHPh), 4.34 (d, 1H, J ¼ 7.6 Hz, H-100), 4.28 (t, 1H, J ¼ 3.3 Hz, H-300), 4.13
(dd, 1H, J ¼ 10.2, 6.0 Hz, H-2), 3.99 (m, 3H, H-3, H-4, H-600), 3.88 (m, 3H,
H-6, H-60, H-6), 3.74 (bs, 1H, H-40), 3.62 (m, 6H, H-5, H-60, H-600, H-500,

R .E. J. N. Litjens et al.
766
O–CH2–CH2), 3.38 (m, 3H, CH2–CH2–N3, H-30), 3.28 (m, 1H, H-50), 1.88
(s, 3H, Ac), 1.80 (m, 2H, CH2–CH2–CH2). 13C NMR: d (ppm) 170.80, 138.18,
138.02, 137.92, 137.76, 137.51, 137.39, 102.25, 100.38, 95.87, 80.14, 79.21,
78.97, 76.22, 75.89, 75.05, 74.69, 74.68, 74.53, 74.35, 73.15, 73.14, 72.53,
71.6, 70.01, 69.65, 68.85, 68.29, 66.19, 65.72, 62.19, 55.19, 47.93, 28.77,
22.82. ESI-HRMS calcd for C65H76N4O16 (M þ Na): 1191.5153. Found:
1191.5141.
3-Aminopropyl a-D-Galactopyranosyl-(1 ! 3)-b-D-
galactopyranosyl-(1 ! 4)-2-acetamido-2-deoxy-b-D-
glucopyranoside (2).
Pd/C (10 wt. % on activated carbon, 100 mg) was added to a solution of 9
(154 mg, 0.13 mmol) in tBuOH/H2O (2 mL, 11 : 4 v/v) and HCl (1 M in H2O,
500 mL), after which H2 was bubbled through the solution for 1 hr followed
by stirring under an H2 atmosphere for 16 hr. TLC analysis (ethyl acetate/
pyridine/AcOH/H2O, 8 : 7 : 1.6 : 1 v/v) showed full transformation of the
starting material into one lower running spot. The mixture was filtered over
Hyflo, concentrated in vacuo, and lyophilized to afford compound 2 (96 mg,
0.13 mmol, quant.) as a white powder. Rf 0.32 (ethyl acetate/pyridine/AcOH/
H2O, 8 : 7 : 1.6 : 1 v/v). [a]D25 þ64.3 (c ¼ 0.5, H2O). 1H NMR (D2O, TSP as
internal standard at d ¼ 0 ppm): d (ppm) 5.15 (bs, 1H, H-100), 4.54 (2H, m,
H-1, H-10), 4.18 (2H, H-100, H-40), 4.03–3.95 (m, 48H), 3.89–3.47 (m, 16H),
3.09 (t, 2H, CH2–CH2–NH2, J ¼ 6.4 Hz), 2.06 (s, 3H, Ac), 1.96 (m, 2H, CH2–
CH2–CH2). 13C NMR (D2O, TSP as internal standard at d ¼ 0 ppm): d (ppm)
176.0, 104.4, 102.7, 97.1, 80.4, 78.9, 76.6, 76.3, 75.2, 73.8, 72.4, 71.2, 70.9,
70.8, 69.8, 69.5, 66.5, 62.6, 61.7, 65.6, 39.2, 28.3, 23.8. ESI-HRMS calcd for
C23H42O16N2 (M þ H): 603.2607. Found: 603.2646.
3-[2(S),3-Bis(palmitamido)]-propanamido]-propyl a-D-
Galactopyranosyl-(1 ! 3)-b-D-galactopyranosyl-
(1 ! 4)-2-acetamido-2-deoxy-b-D-glucopyranoside (1).
To a mixture of 2(S),3-bis-(9H-fluoren-9-ylmethoxycarbonylamino)-propio-
nic acid (26 mg, 46 mmol) and amine 2 (15 mg, 23 mmol) in DMF/DMSO (2/1
v/v, 0.75 mL) were added BOP (46 mmol, 21 mg) and DiPEA (16 mL, 92 mmol),
and the mixture was stirred for 6 hr. Aminoacetaldehyde dimethyl acetal
(5 mL, 46 mmol) was added, and the mixture was stirred for one-half hr, after
which DiPEA (8 mL, 46 mmol) was added, and the reaction mixture was concen-
trated in vacuo. The resulting syrup was triturated with Et2O and centrifuged,
and the supernatant was removed. The precipitate was treated with DCM/
Et2O (1/1 v/v) and centrifuged, and the supernatant removed. The resulting
precipitate was mixed with CHCl3/MeOH/H2O (63/33/4 v/v), and the slurry

Synthesis of a Lipid Conjugate
767
was centrifuged and the liquids removed. The resulting solid was dissolved in
DMF/DMSO (1 mL, 2 : 1 v/v), and DBU (14 mL, 92 mmol) was added. After
10 min, HOBt (16 mg, 115 mmol) was added. This mixture was stirred for
5 min, after which EtSH (15 mL, 200 mmol) was added, and stirring was conti-
nued for 5 min. Subsequently, the mixture was concentrated under vacuum,
and the residue was dissolved in DMF/DMSO/CHCl3 (1 mL, 2 : 1 : 1 v/v).
Palmitic acid N-hydroxysuccinimide ester (40 mg, 115 mmol) and DiPEA
(20 mL, 69 mmol) were added. After stirring for 16 hr, the reaction mixture
was concentrated under reduced pressure. The resulting solid was dissolved
in boiling MeOH, and after cooling down the solution to 08C the formed precipi-
tate was isolated to provide the compound 1 (9.3 mg, 8 mmol, 35% over three
steps) as an amorphous white solid. 1H NMR (DMSO-d6 with internal
standard at 2.54 ppm, T ¼ 303 K): d (ppm) 5.05 (d, 1H, J ¼ 5.2 Hz, H-1), 4.83
(d, 1H, J ¼ 3.5 Hz, H-100), 4.63 (t, 1H, J ¼ 5.1, CH-DAP), 4.64 (d, 1H,
J ¼ 7.7 Hz, H-10), 4.59 (t, 1H, J ¼ 6.1 Hz, H-30), 4.55 (d, 1H, J ¼ 5.0 Hz, H-400),
4.42 (t, 1H, J ¼ 6.0 Hz, H-6), 4.35 (s, 1H, H-60), 4.29 (m, 2H, H-6, H-600), 3.99
(t, 1H, J ¼ 8.1 Hz, H-3), 3.84 (s, 1H, H-60), 3.76 (m, 4H, H-600, O–CH2–CH2,
H-50), 3.63 (m, 2H, H-500, H-300), 3.58 (m, 2H, H-20, H-200), 3.51 (m, 4H, H-4,
CH2–DAP, H-5), 3.37 (m, 2H, H-2, H-50), 3.11 (m, 1H, CH2–CHH–NH2), 3.03
(m, 1H, CH2–CHH–NH2), 2.10 (t, 2H, J ¼ 7.26 Hz, COCH2 palmitoyl), 2.02
(t, 2H, J ¼ 7.20 Hz, COCH2 palmitoyl), 1.80 (s, 3H, Ac), 1.58 (t, 2H,
J ¼ 6.3 Hz, CH2–CH2–CH2), 1.45 (m, 4H, COCH2CH2 palmitoyl), 1.23
(m, 48H, –CH2– palmitoyl), 0.84 (t, 6H, J ¼ 6.7 Hz, –CH3 palmitoyl). ESI-
HRMS calcd for C58H108N4O19 (M þ Na): 1187.7500. Found: 1187.7501.
REFERENCES
[1] Galili, U., Avila, J.L. Alfa-Gal and Anti-Gal; Kluwer Academic/Plenum Publishers:
New York, 1999, 32.
[2] Galili, U. Interaction of the natural anti-Gal antibody with alpha-galactosyl
epitopes—a major obstacle for xenotransplantation in humans. Immunol. Today
1993, 14, 480.
.
.
[3] Galili, U. The alpha-gal epitope a-D-Galp-(1 3)-b-D-Galp-(1 4)-b-D-GlcpNAc in
xenotransplantation. Biochimie 2001, 83, 557.
[4] Galili, U. Enhancement of antigen presentation of influenza virus hemagglutinin
by the natural human anti-Gal antibody. Vaccine 1996, 14, 321.
[5] Henion, T.R.; Gerhard, W.; Anaraki, F.; Galili, U. Synthesis of alpha-gal epitopes on
influenza virus vaccines, by recombinant alpha 1,3galactosyltransferase, enables
the formation of immune complexes with the natural anti-Gal antibody. Vaccine
1997, 15, 1174.
.
[6] Chen, Z.C.; Tanemura, M.; Galili, U. Synthesis of alpha-gal a-D-Galp-(1 3)-b-D-
.
.
Galp-(1 4)-b-D-GlcpNAc-R on human tumor cells by a-(1 3)-galactosyltransferase
produced in Pichia pastoris. Glycobiology 2001, 11, 577.

R .E. J. N. Litjens et al.
768
[7] Galili, U.; Chen, Z.C.; Manches, O.; Plumas, J.; Preisler, H. Preparation of autolo-
gous leukemia and lymphoma vaccines expressing alpha-Gal epitopes.
J. Hematother. Stem. Cell Res. 2001, 10, 501.
[8] LaTemple, D.C.; Abrams, J.T.; Zhang, S.Y.; Galili, U. Synthesis of alpha-galactosyl
.
epitopes by recombinant a-(1 3)-galactosyltransferase for opsonization of human
tumor cell vaccines by anti-galactose. Cancer Res. 1996, 56, 3069.
[9] Galili, U.; Avila, J.L. Alfa-gal and anti-gal; Kluwer Academic/Plenum Publishers:
New York, 1999361.
[10] LaTemple, D.C.; Henion, T.R.; Anaraki, F.; Galili, U. Increased immunogenicity of
tumor vaccines complexed with anti-Gal: studies in knockout mice for alpha
1,3galactosyltransferase. Cancer Res. 1999, 59, 3417.
[11] See for alternative syntheses of trisaccharide 2: (a) Hanessian, S.; Saavedra, O.M.;
Mascitti, V.; Marterer, W.; Oehrlein, R.; Mak, C.-P. Practical syntheses of B disac-
charide and linear B type 2 trisaccharide-non-primate epitope markers recognized
by human anti-alpha-Gal antibodies causing hyperacute rejection of xenotrans-
plants. Tetrahedron 2001, 57, 3267; (b) Hanessian, S.; Huynh, H.K.; Reddy, G.V.;
Duthaler, R.O.; Katopodis, A.; Streiff, M.B.; Kinzy, W.; Oehrlein, R. Synthesis of
Gal determinant epitopes, their glycomimetic variants, and trimeric clusters—
relevance to tumor associated antigens and to discordant xenografts. Tetrahedron
2001, 57, 3281.
[12] See for other Galili trisaccharide syntheses: (a) Garegg, P.J.; Oscarson, S.
.
A synthesis of 8-methoxycarbonyloct-1-yl O-a-D-galactopyranosyl-(1 3)-O-b-D-
.
galactopyranosyl-(1 4)-2-acetamido-2-deoxy-b-D-glucopyanoside. Carbohydr. Res.
1985, 136, 207; (b) Schaubach, R.; Hemberger, J.; Kinzy, W. Tumor-associated
.
.
antigen synthesis - synthesis of the Gal-a-(1 3)-gal-b-(1 4)-GlucNHAc epitope—
a specific determinant for metastatic progression. Liebigs Ann. Chem. 1991, 607;
(c) Reddy, G.V.; Jain, R.K.; Bhatti, B.S.; Matta, K.L. Synthesis of a-D-galactopyra-
.
.
nosyl-linked oligosaccharides containing the a-Gal-(1 3)-b-Gal-(1 3)-GlcNHAc
sequence employing methyl-2,3,4,6-tetra-O-(4-methoxybenzyl)-1-thio-b-D-galacto-
pyranoside as an efficient glycosyl donor. Carbohydr. Res. 1994, 263, 67;
(d) Vic, G.; Tran, C.H.; Scigelova, M.; Crout, D.H.G. Glycosidase-catalysed syn-
thesis of oligosaccharides: a one step synthesis of lactosamine and of the linear B
.
.
type 2 trisaccharide a-D-Gal-(1 3)-b-D-Gal-(1 4)-b-D-GlcNAcSEt involved in the
hyperacute rejection response in xenotransplantation from pigs to man and as
the specific receptor for toxin A from Clostridium difficile. Chem. Comm. 1997,
169; (e) Nilsson, K.G.I. Glycosidase-catalysed synthesis of di- and trisaccharide
derivatives related to antigens involved in the hyperacute rejection of xenotrans-
plants. Tetrahedron Lett. 1997, 38, 133; (f) Fang, J.; Li, J.; Chen, X.; Zhang, X.;
Wang, J.; Guo, Z.; Zhang, W.; Yu, L.; Brew, K.; Wang, P.G. Higly efficient chemoen-
.
zymatic synthesis of a-galactosyl epitopes with a recombinant a-(1 3)-galactosyl-
transferase. J. Am. Chem. Soc. 1998, 120, 6635; (g) Dahmen, J.; Magnusson, G.;
.
Hansen, H.C. Synthesis of the linear B type 2 trisaccharide a-D-Gal-(1 3)-b-D-
.
Gal-(1 4)-b-D-GlcNAcOTMSEt, and coupling of the corresponding 2-carboxyethyl
b-thioglycoside to Sepharose. J. Carbohydr. Chem. 2002, 21, 189; (h) Wu, X.;
Schmidt, R.R. Solid-phase synthesis of complex oligosaccharides using a novel
capping reagent. J. Org. Chem. 2004, 69, 1853.
[13] Mootoo, D.R.; Konradsson, P.; Udodong, U.; Fraser-Reid, B. Armed and disarmed
n-pentenyl glycosides in saccharide couplings leading to oligosaccharides. J. Am.
Chem. Soc. 1988, 110, 5583.
[14] Veeneman, G.H.; van Boom, J.H. An efficient thioglycoside-mediated formation of
a-glycosidic linkages promoted by iodonium dicollidine perchlorate. Tetrahedron
Lett. 1990, 31, 275.

Synthesis of a Lipid Conjugate
769
´
[15] (a) Codee, J.D.C.; Litjens, R.E.J.N.; den Heeten, R.; Overkleeft, H.S.; van
Boom, J.H.; van der Marel, G.A. Ph2SO/Tf2O: a powerful promotor system in che-
moselective glycosylations using thioglycosides. Org. Lett. 2003, 5, 1519;
´
(b) Codee, J.D.C.; Litjens, R.E.J.N.; van den Bos, L.J.; Overkleeft, H.S.; van
Boom, J.H.; van der Marel, G.A. Chemoselective glycosylations using sulfonium
triflate activator systems. Tetrahedron 2004, 60, 1057.
´
[16] Codee, J.D.C.; van den Bos, L.J.; Litjens, R.E.J.N.; Overkleeft, H.S.; van
Boom, J.H.; van der Marel, G.A. Sequential one-pot glycosylations using
1-hydroxyl and 1-thiodonors. Org. Lett. 2003, 5, 1947.
[17] Garegg, P.J.; Hultberg, H.; Lindberg, C. Synthesis of p-trifluoroacetamidophenyl
.
.
O-a-D-galactopyranosyl-(1 2)-O-a-D-mannopyranosyl-(1 4)-a-L-rhamnopyrano-
side. Carbohydr. Res. 1980, 83, 157.
[18] Zuurmond, H.M.; van der Klein, P.A.M.; van der Marel, G.A.; van Boom, J.H.
A stereospecific approach towards the synthesis of 2-deoxy a- and b-glycosides
based on a 1,2-ethyl (phenyl) thio group migration. Tetrahedron 1993, 49, 6501.
[19] See for the original IDCP mediated synthesis of 6: Zhu, T.; Boons, G.-J. Two-direc-
tional, convergent synthesis of a pentasaccharide that is involved in the hyperacute
rejection response in xenotransplantation from pig to man. J. Chem. Soc. Perkin
Trans. 1 1998, 857.
[20] Crich, D.; Smith, M. 1-benzenesulfinyl piperidine/trifluoromethanesulfonic anhy-
dride: a potent combination of shelf-stable reagents for the low-temperature con-
version of thioglycosides to glycosyl triflates and for the formation of diverse
glycosidic linkages. J. Am. Chem. Soc. 2001, 123, 9015.
[21] Garcia, B.A.; Poole, J.L.; Gin, D.Y. Direct glycosylations with 1-hydroxy glycosyl
donors using trifluoromethanesulfonic anhydride and diphenyl sulfoxide J. Am.
Chem. Soc. 1997, 119, 7597.
[22] Kanie, O.; Crawley, S.C.; Palcic, M.M.; Hindsgaul, O. Acceptor-substrate recog-
nition by N-acetylglucosaminyltransferase-V: critical role of the 400-hydroxyl
.
.
group in b-D-GlcpNAc-(1 2)-a-D-Manp(1 6)-b-D-Glcp-OR. Carbohydr. Res. 1993,
243, 139.
[23] Guenther, W.; Kunz, H. Synthesis of b-D-mannosides from b-D-glucosides via an
intramolecular SN2 reaction at C-2. Carbohydr. Res. 1992, 228, 217.
[24] Boyer, J.H.; Hamer, J. The acid-catalyzed reaction of alkyl azides upon carbonyl
compounds. J. Am. Chem. Soc. 1955, 77, 951.
[25] Crich, D.; Smith, M.; Yao, Q.; Picione, J. 2,4,6-Tri-tert-butylpyrimidine (TTBP):
A cost effective, readily available alternative to the hindered base 2,6-di-tert-butyl-
pyridine and its 4-substituted derivatives in glycosylation and other reactions.
Synthesis 2001, 323.