Synthesis of tri-, penta-, and heptasaccharides, functionalized with orthogonally N-protected amino residues at the reducing and non-reducing ends

Article abstract of DOI:10.1016/j.tet.2012.05.118

The synthesis of four bifunctionalized orthogonally N-protected oligosaccharides derived from lactose and mannose, intended as cross-linking derivatives, is described. The aminosugar at the non-reducing end is derivatized with an N-Boc-protected glycine m

Full text of DOI:10.1016/j.tet.2012.05.118

Tetrahedron 68 (2012) 6712e6720
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of tri-, penta-, and heptasaccharides, functionalized with orthogonally
N-protected amino residues at the reducing and non-reducing ends
*
Timmy Fyrner, Stefan C.T. Svensson, Peter Konradsson
€
€
Division of Chemistry, IFM, Linkoping University, SE-581 83 Linkoping, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of four bifunctionalized orthogonally N-protected oligosaccharides derived from lactose
and mannose, intended as cross-linking derivatives, is described. The aminosugar at the non-reducing
end is derivatized with an N-Boc-protected glycine moiety, and further connected to either a mannose
(1/6) disaccharide or (1/3) lactose units (one, two or three) resulting in tri-, penta-, or hepta-
saccharides. All of the synthesized oligosaccharides have an N-benzyloxycarbonyl-aminoethyl residue at
the reducing end. The orthogonal N-Boc/N-Cbz protection group pattern enables further conjugation/
derivatization and results in a hydrophilic cross-linking molecule. It was found that the order of the final
synthetic steps was crucial to avoid acyl migration. A suitable amide coupling protocol has been applied
to introduce the N-Boc-protected glycine moiety in alcoholic solvent. The synthesized oligosaccharides
will provide a model system to investigate the influence of length, structure and flexibility.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 21 February 2012
Received in revised form 7 May 2012
Accepted 28 May 2012
Available online 6 June 2012
Keywords:
Glycosylation
Glycosidation
Oligosaccharide synthesis
Spacer molecules
Orthogonally protected
Carbohydrates
Bifunctionalized
1. Introduction
using a poly(amido)-,21,22 ethylene(glycol)23 or polyether24 back-
bone. Saccharides as building blocks in dendrimers are suitable for
To study biological processes, appropriate cross-linking mole-
cules are often required to circumvent the synthesis of complex ol-
igosaccharides. A challenge within the field of glycobiology and
biotechnology is to achieve controlled immobilization of proteins
onto surfaces, while retaining their biological features and proper-
ties.1,2 The design of bifunctional spacers comprises several aspects
that have to be taken into consideration; (i) the extent of the syn-
thesis, (ii) homo-/heterobifunctionality (identical or different re-
active groups) (iii) length (iv) water-solubility and (v)
conformational flexibility.3e8 One of the most common classes of
compounds used for bioconjugation in various biophysical or bi-
ological systems is the poly- and oligoethylene glycol (PEG/OEG)
chains.9 Both are known for their protein-resistant properties10,11
and occur in numerous research fields.12e15 PEGs and OEGs have
also been used as spacers to form glycoconjugates.16,17 An in-
vestigation on how the spacer length and flexibility influence the
liposomeephagocyte interaction18 confirmed the importance of
tuningthe lengthof the spacerdependingon the biologicalsystem.19
To resemble biomolecules and compensate for weak individual
protein-carbohydrate interactions, glycodendrimers are often used,
facilitating the effect of multivalency.20 Dendrimers are constructed
controlling characteristics, such as flexibility, surface properties and
to impart biocompatibility.25 Several carbohydrate-containing sur-
faces have also shown protein-resistant properties,26e28 i.e., an eli-
gible feature for serving as general cross-linking molecules.
Oligosaccharide-based spacer molecules, comprised of (oligo-)lac-
toses19 appears to be comparable with those containing OEGs, thus
demonstrating possible cross-linking features of oligosaccharides in
biophysical- or biosensing applications. Herein, we present the
synthesis of four oligosaccharides to be used as bifunctionalized
spacer molecules (1e4) (Fig. 1) derived from lactose and mannose,
containing an N-Boc-glycine functionalized aminosugar. The
orthogonal N-Boc/N-Cbz protection group pattern allows for further
conjugation and derivatization depending on the specific target,
thus resulting in a potential cross-linking molecule. The results from
the oligosaccharides synthesized by Schmidt and co-workers,29
suggest that oligo(lactoses) adopts linear rod-like conformations.
Using the selected aminosugar from the corresponding gluco series,
glycosylated in a (1/3) manner (1e3), we hypothesize that the
sameconformationwill be retained, giving fairlyrigidrod-like target
molecules. Using this analogy, we also synthesized a more flexible
mannose (1/6) based trisaccharide (4) with the identical terminal
aminosugar moiety, allowing for investigations and tuning of the
cross-linking property, i.e., length, structure and flexibility.
* Corresponding author. E-mail address: petko@ifm.liu.se (P. Konradsson).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.05.118

T. Fyrner et al. / Tetrahedron 68 (2012) 6712e6720
6713
Fig. 1. The four synthesized orthogonally N-protected oligosaccharides.
2. Results and discussion
circumventing intramolecular migration of acyls, followed by
subsequent reduction of the azido functionality to the corre-
sponding amine using NaBH4 and NiCl2$6H2O34,35 in MeOH. It
was found that the order of addition strongly affects the outcome
of the reaction. The highest yields were obtained when the
NiCl2$6H2O was stirred with the azido sugar for 5 min followed
by addition of the NaBH4 and subsequently quenching the re-
action using DowexÒ-Hþ. The amine was then coupled with N-
Boc-glycine using a combination of methods36,37 with EDC$HCl in
MeOH. Initially, the azido group was reduced prior to the
deprotection using either PPh3 or NiCl2$6H2O, however, the re-
duction exclusively resulted in acyl migration, which is consistent
with the finding made by Lin et al.38 Tetrasaccharide 10 (n¼1)
was obtained by deprotection of the isopropylidene acetal of
compound 8 followed by an NIS/AgOTf-promoted glycosylation
with donor 7 and subsequent benzoylation to afford compound
10 in an overall yield of 69%. Applying the protocol described for
9, tetrasaccharide 10 was used to afford the protected azido
pentasaccharide 11 (n¼2) in 66% yield. Finally, target compound 2
(n¼2) was obtained in 64%, using the previously described syn-
thetic protocol for 1. Product 3 was synthesized in 29% yield from
10, via 12 and 13 (69% and 51% yields, respectively) using the
methods described above. It should be noted that H2O had to be
added in the glycine derivatization due to poor solubility of the
deprotected amino heptasaccharide in MeOH. The H2O, in com-
bination with the increased steric hindrance of the amine, is
probably the reason why a lower yield was obtained for com-
pound 3 (n¼3) (cf. 1 (65%), 2 (64%), and 3 (29%)) (Scheme 2).
The mannoside 1539,40 was obtained in 45% yield by well-known
procedures (i.e., tritylation, benzoylation, deprotection) starting
from the tetraol 14 (Scheme 3).41 However, to the best of our
knowledge the characterization of compound 15 has not been de-
scribed previously. Chloroacetylation of the6-hydroxyl mannoside 15
at ꢀ40 ꢁC gave compound 16 in 82% yield. Conversion to the corre-
sponding bromosugar followed KoenigseKnorr glycosylation using
AgOTf as promotor at ꢀ30 ꢁC gave disaccharide 17 in 87% yield. A NIS/
TfOH-promoted glycosylation at ꢀ30 ꢁC with N-Cbz-2-aminoethanol
afforded 18 in 82% yield. Treatment with thiourea/2,6-lutidine re-
moved the chloroacetyl groupto afford the corresponding 6-hydroxyl
acceptor, which was subsequently glycosylated with azido donor 6
using NIS/AgOTf to give compound 19 in 85% yield. The conditions for
the final three-step conversion were in accordance with the protocol
The azido sugar 6 was synthesized starting from the known
thioglycoside 530 using triflic anhydride followed by NaN3 dis-
placement, giving the azido functionalized derivative 6 as a crys-
talline compound in 85% yield (Scheme 1).
Scheme 1. Reagents and conditions:(i)Tf2O, pyridine,CH2Cl2, 20ꢁC;(ii)NaN3, DMF, 70ꢁC.
The synthesis of the lactosides 1e3 (n¼0, 1, 2) (Scheme 2)
started from the commercially available lactose monohydrate. In
a straightforward six-step manner, i.e., acetylation, formation of the
bromosugar, phase-transfer substitution at the anomeric center,
deacetylation, selective introduction of an isopropylidene group at
the 30,40-position and subsequent protection of the remaining al-
cohols using BzCl, afforded the crystalline building block 731 in 43%
yield over six steps. The phase-transfer conditions for introducing
the phenyl thioglycosides32 and the use of TMSCl/2,2-
dimethoxypropane in acetone to selectively install the 30,40-iso-
propylidene acetal provides a scalable synthetic route to donor 7
without to resort to purification by chromatography.
Utilizing NIS/AgOTf promoted glycosylation of 7 with N-Cbz-2-
aminoethanol afforded the crystalline glycoside 8 (n¼0) in 87%
yield. Deprotection of the isopropylidene acetal of 8 followed by
a regioselective glycosylation with azido sugar 6 and final ben-
zoylation of the 40-hydroxyl group gave the (
b 1/3) azido tri-
saccharide 9 (n¼1) in 81% yield. Although the regioselectivity of
the 3-position on galacto-configured epitopes is known,29,31 we
confirmed this by NMR spectroscopic analysis after benzoylation
at the remaining 40-position. The selectivity depends on the gly-
cosyl donor and promotor used, which was earlier found by the
group of Oscarson.33 This extra benzoylation was in agreement
with the protection group pattern and also used for the synthesis
of compounds 10e13. The target molecule 1 (n¼1) was obtained
by deprotection of the benzoates using NaOMe, thereby

6714
T. Fyrner et al. / Tetrahedron 68 (2012) 6712e6720
OH
OH
O
HO
HO
O
O
HO
OH
OH OH
43% i, ii, iii, iv, v, vi
OBz
OBz
O
O
O
O
O
SPh
BzO
OBz
OBz
7
59-69%
87% vii
O
OBz
OBz
OBz
OBz
OBz
O
O
O
O
O
O
O
viii, ix, x
O
O
BzO
BzO
NHCbz
OBz
OBz
OBz
OBz
8 (n = 0), 10 (n = 1), 12 (n = 2)
OBz
O
N3
BzO
viii
SEt
OBz
6
xi, x
O
51-81%
OBz
OBz
O
OBz
OBz
O
O
N3
BzO
O
O
BzO
NHCbz
OBz
OBz
OBz
9 (n = 1), 11 (n = 2), 13 (n = 3)
29-65% xii, xiii, xiv
O
OH
OH
OH
OH
BocHN
O
O
O
N
H
O
HO
O
O
HO
NHCbz
OH
OH
OH
1 (n = 1), 2 (n = 2), 3 (n = 3)
Scheme 2. Reagents and conditions: (i) Ac2O, HOAc, HClO4, 0 ꢁC; (ii) HBr/HOAc, CH2Cl2, 0 ꢁC; (iii) PhSH, Bu4NHSO4, 1 M Na2CO3 (aq), CH2Cl2; (iv) NaOMe, MeOH; (v) Acetone, DMP,
ꢁ
TMSCl; (vi) BzCl, DMAP, pyridine; (vii) N-Cbz-2-aminoethanol, NIS, AgOTf, 4 A MS, CH2Cl2, 0 C/rt; (viii) TFA (90%), CH2Cl2; (ix) 7, NIS, AgOTf, 4 A MS, CH2Cl2, 0 ꢁC/rt; (x) BzCl,
ꢀ
ꢀ
pyridine; (xi) NIS, AgOTf, 4 A MS, CH2Cl2, 0 ꢁC/rt; (xii) NaOMe, MeOH, CH2Cl2; (xiii) NiCl2$6H2O, NaBH4, MeOH; (xiv) N-(tert-butyloxycarbonyl)-glycine, HOAt, N-methyl-
ꢀ
morpholine, EDC$HCl, MeOH.
previously described for the synthesis of 1, 2, and 3 resulting in the
target trisaccharide 4 in 62% yield.
provide a relevant model system that enables investigations of cross-
linker influence on biological studies.
3. Conclusions
4. Experimental
The function of biologically conjugated motifs for, e.g., biosensing
strongly depends on the cross linker. We have demonstrated the
synthesis of oligosaccharides, with variable lengths and flexibility, as
possible bifunctional cross-linking structures (1e4). Common prob-
lems associated with the synthesis of the traditionally used PEGs/
OEGs are purification and low crystallinity. Efficient synthetic pro-
tocols have been developed to produce the crystalline building blocks
6, 7, and 8. The synthetic pathway developed is straightforward, using
known transformations and crystalline intermediates and may
therefore be applied on larger scale providing alternative compounds
to the commonly used OEGs/PEGs. Furthermore, these oligosaccha-
rides have the potential to serve as well-defined, biocompatible
spacers for biophysical model systems. Finally these oligosaccharides
4.1. General procedure
CH2Cl2 and toluene were distilled over calcium hydride and col-
lected onto pre-activated 4 A MS. Thin layer chromatography (TLC)
was carried out on Merck 60 F254 plates and visualized by UV light
and/or developed with PAA [EtOH (95%, 740 mL), H2SO4 (concn,
28 mL), AcOH (100%, 8.4 mL), p-anisaldehyde (20 mL)]. Flash column
chromatography (FC) was carried out on silica gel Merck 60
ꢀ
(40e63 mm). Reverse phase chromatography (RP) was carried out on
Merck LiChroprepÒ (RP-18). Proton nuclear magnetic resonance (1H
NMR) were recorded on Varian 300 MHz and 600 MHz spectrome-
ters, carbon nuclear magnetic resonance (13C NMR) was recorded on
Varian 300 (75.4 MHz) spectrometer; multiplicities are quoted as

T. Fyrner et al. / Tetrahedron 68 (2012) 6712e6720
6715
O
Cl
OBz
HO
OH
O
OBz
O
HO
HO
O
O
BzO
BzO
45%
i, ii, iii
82%
iv
BzO
HO
BzO
SEt
SEt
SEt
Br
v
14
15
16
87% vi
O
Cl
O
OBz
O
BzO
BzO
OBz
O
O
BzO
BzO
SEt
17
82% vii
O
Cl
OBz
O
O
BzO
BzO
OBz
O
O
BzO
BzO
O
NHCbz
18
85% viii, ix
OBz
O
N3
BzO
O
OBz
O
OBz
BzO
BzO
OBz
O
O
BzO
BzO
19
O
NHCbz
62% x, xi, xii
O
OH
BocHN
O
N
H
O
OH
HO
O
OH
HO
HO
OH
O
O
HO
HO
4
O
NHCbz
Scheme 3. Reagents and conditions: (i) TrCl, pyridine; (ii) BzCl, CH2Cl2, 0 ꢁC; (iii) p-TsOH, CHCl3/MeOH; (iv) Chloroacetyl chloride, pyridine, CH2Cl2, 40 ꢁC; (v) Br2, CH2Cl2; (vi) AgOTf,
ꢁ
ꢁ
4 A MS, CH2Cl2, 30 C; (vii) N-Cbz-2-aminoethanol, NIS, 4 A MS, TfOH, CH2Cl2, 30 C; (viii) 2,6-lutidine, thiourea, CH2Cl2, MeOH; (ix) 6, NIS, AgOTf, 4 A MS, CH2Cl2, 0 ꢁC/rt; (x)
ꢀ
ꢀ
ꢀ
NaOMe, MeOH, CH2Cl2; (xi) NiCl2$6H2O, NaBH4, MeOH; (xii) N-(tert-butyloxycarbonyl)-glycine, HOAt, N-methylmorpholine, EDC$HCl, MeOH.
apparent doublet (ad), apparent singlet (as), broad singlet (bs), sin-
glet (s), doublet (d), doublet of doublets (dd), double doublet of
doublets (ddd), triplet (t) and multiplet (m). If overlaps in carbon
spectra occur (e.g., anomeric- or carbamate carbons) this is noted. In
situations where a doublet of doublets (dd) appears as a triplet (t)
due to resolution, this is denoted as a (dd) with identical couplings
constants. TMS or the resonances of the deuterated solvent was used
4.2. Ethyl 4-deoxy-4-azido-2,3,6-tri-O-benzoyl-b-D-thio-
glucopyranoside (6)
To
a
solution of ethyl 2,3,6-tri-O-benzoyl-b-D-thio-gal-
actopyranoside30 (5) (5.00 g, 9.32 mmol) and dry pyridine
(2.25 mL, 28.0 mmol) in CH2Cl2 (50 mL) under argon was added
Tf2O (2.35 mL, 14.0 mmol) at ꢀ20 ꢁC. After 30 min the solution
was diluted with CH2Cl2 (50 mL), washed with H2O (2ꢂ50 mL),
dried over MgSO4 (s) and evaporated. The crude triflate Rf¼0.72
(toluene/EtOAc 4:1) was dissolved in DMF (50 mL) whereupon
NaN3 (2.42 g, 37.3 mmol) was added and the solution was
heated to 70 ꢁC. After 2 h the solution was diluted with toluene
(100 mL), washed with brine (2ꢂ100 mL), H2O (2ꢂ100 mL),
dried over MgSO4 (s) and evaporated. Crystallization from EtOH
(99.5%) gave the azide 6 (4.44 g, 7.91 mmol, 85%) as white
needles. Rf¼0.78 (toluene/EtOAc 4:1); mp 129e130 ꢁC (EtOH);
as internal standard; CDCl3 (1H NMR,
d
¼7.26; 13C NMR,
¼77.2);
d
CD3OD (1H NMR,
d
¼3.31; 13C NMR,
d
¼49.0); with D2O, CH3OH (1H
NMR,
d
¼3.34; 13C NMR,
d
¼49.5) was used as reference.42 Matrix
assisted laser desorption ionizationdtime of flight (MALDI-TOF)
mass spectrometry was recorded on a Voyager-DE STR Biochemistry
Workstation, in a positive mode, using a-cyano-4-hydroxycinnamic
acid (CHCA) or 2,4,6-trihydroxyacetophenone (THAP) as matrices.
ESI-MS (recorded at Medivir AB, Huddinge, Sweden) was performed
on a Water Synapt HDMS instrument equipped with electrospray
interface. Optical rotation measurements were recorded at 20 ꢁC
with a PerkineElmer 141 polarimeter. FTIR was recorded on a Per-
kineElmer Spectrum 1000 using KBr pellets. Melting points were
recorded on a StuartÒ melting point apparatus.
[
a
]Dþ108 (c 1, CHCl3); 13C NMR (75.4 MHz, CDCl3):
d 14.9, 24.4,
61.0, 63.6, 70.5, 74.9, 76.6, 83.9, 128.2e128.6 (several carbons),
128.9, 129.2, 129.8e130.0 (several carbons), 133.4, 133.6, 165.4,
165.7, 166.2 (note: overlaps occur in aromatic region); 1H NMR

6716
T. Fyrner et al. / Tetrahedron 68 (2012) 6712e6720
(300 MHz, CDCl3):
d
1.26 (t, 3H, J¼7.4 Hz), 2.63e2.81 (m, 2H),
37.7 mmol) and AgOTf (w0.1 equiv) were added and stirred for 4 h,
0 ꢁC/rt. The reaction was quenched with Et3N, diluted with CH2Cl2
(50 mL) and filtered through CeliteÒ, washed with Na2S2O3 (10 wt %,
aq) (2ꢂ50 mL),1 M HCl (aq) (2ꢂ50 mL), NaHCO3 (satd aq) (2ꢂ50 mL),
H2O (2ꢂ50 mL) dried over MgSO4 (s), filtered, and concentrated. FC
(toluene/toluene/EtOAc 6:1) followed by crystallization from
EtOAc/n-heptane gave glycoside 8 (23.5 g, 21.7 mmol, 87%) as white
needles. Rf¼0.68 (toluene/EtOAc 1:1); mp 153e154 ꢁC (EtOAc/n-
3.80 (ddd, 1H, J¼2.3, 4.7, 10.2 Hz), 3.91 (dd, 1H, J¼9.6, 10.2 Hz),
4.60 (dd, 1H, J¼4.7, 12.2 Hz), 4.76 (dd, 1H, J¼2.3, 12.2 Hz), 4.77
(d, 1H, J¼9.8 Hz), 5.44 (dd, 1H, J¼9.6, 9.8 Hz), 5.70 (dd, 1H,
J¼9.6, 9.6 Hz), 7.35e7.40 (m, 4H), 7.46e7.54 (m, 4H), 7.58e7.64
(m, 1H), 7.92e7.97 (m, 4H), 8.08e8.11 (m, 2H); HRMS (ESI):
[MþNH4]þ calcd for C29H31N4O7S, 579.1835; found 579.1833.
4.3. Phenyl 2,6-di-O-benzoyl-3,4-di-O-isopropylidene-
b-
D-
heptane); [
a
]Dþ39 (c 1, CHCl3); 13C NMR (75.4 MHz, CDCl3):
d 26.1,
galactopyranosyl-(1/4)-2,3,6-tri-O-benzoyl-1-thio-
b-
D-
27.4, 40.9, 62.4, 62.8, 66.4, 69.4, 71.3, 71.9, 72.2, 73.1, 73.2, 73.6, 75.2,
77.1, 100.1, 101.3, 110.8, 127.9e128.6 (several carbons), 129.1e129.8
(several carbons), 132.9, 133.1, 133.2, 133.3, 136.5, 156.2, 164.8, 165.3,
glucopyranoside (7)31
To a stirred solution of lactose monohydrate (75.0 g, 208 mmol),
Ac2O (190 mL, 2.01 mol), HOAc (375 mL), was added two drops of aq
HClO4 (60%, aq) with 15 min interval at 0 ꢁC (exothermic!). After 1 h
the mixture containing the per-acetylated lactose (Rf¼0.52, toluene/
EtOAc1:1)wasdiluted withCH2Cl2 (200 mL)whereupon HBrinHOAc
(250 mL, 33 wt %) was added (over 30 min) at 0 ꢁC and stirred for an
additional 3 h. The solution was diluted with CH2Cl2 (200 mL) and
washed with ice/H2O (2ꢂ300 mL), NaHCO3 (satd aq) (2ꢂ300 mL),
dried over MgSO4 (s), filtered and concentrated. The crude bromo-
sugar (Rf¼0.58, toluene/EtOAc 1:1) was dissolved in CH2Cl2 (250 mL)
whereupon PhSH(50.9mL, 496mmol), Bu4NHSO4 (56.1g,165mmol),
and 1 M Na2CO3 (aq) (250 mL) were added. The mixture was stirred
vigorously overnight. The mixture was separated and the organic
phase waswashed with 1 M NaOH (aq) (2ꢂ100 mL), H2O (2ꢂ200 mL),
dried over MgSO4 (s), filtered and concentrated. To a mixture of the
crude thioglycoside (Rf¼0.56, toluene/EtOAc 1:1) in MeOH (200 mL)
was added NaOMe (8.92 g, 165 mmol) and stirred overnight, where-
upon the solution was neutralized with DOWEX-Hþ and filtered. The
solution was washed with n-heptane (200 mL) followed by evapo-
ration and co-concentration with toluene. To a mixture of the
deprotected thioglycoside (Rf¼0.24, chloroform/MeOH 7:2) in ace-
tone (400 mL)and 2,2-dimethoxypropane(150mL)wasadded TMSCl
(150 mL,1.18 mol) and stirred for 2 h. To the reaction mixture, EtOAc/
n-heptane (500 mL, 1:1) was added and the precipitate was filtered
and washed with additional n-heptane (200 mL). To a solution of
pyridine (500 mL), DMAP (1.00 g, 8.19 mmol) and BzCl (112 mL,
1.24 mol) was added (over 30 min) the crude isopropylidene-
protected derivative (Rf¼0.60, chloroform/MeOH 7:2) at 0 ꢁC and
stirred overnight at rt. The reaction mixture was diluted with CH2Cl2
(300 mL) and washed with 1 M HCl (aq) (6ꢂ1 L), NaHCO3 (satd aq)
(2ꢂ2 L), H2O (2ꢂ2 L), dried over MgSO4 (s), filtered, and concentrated.
Crystallization followed by re-crystallization from EtOH (4 L, 99.5%, v/
v) gave thiosugar 7 as white crystals (89.2 g, 89.7 mmol, 43%). Rf¼0.73
165.5, 165.8, 165.9; 1H NMR (300 MHz, CDCl3):
d 1.25 (s, 3H), 1.52 (s,
3H), 3.24e3.32 (m, 2H), 3.56e3.63 (m, 1H), 3.69e3.85 (m, 4H), 4.09
(dd, 1H, J¼2.1, 5.6 Hz), 4.17 (dd,1H, J¼9.5, 9.5 Hz), 4.22e4.28 (m, 2H),
4.45(dd,1H, J¼4.3,12.1Hz), 4.58e4.64(m, 3H),4.89(d,1H, J¼12.3Hz),
4.96 (d, 1H, J¼12.3 Hz), 5.09 (bs, 1H), 5.15 (dd, 1H, J¼6.8, 7.7 Hz), 5.37
(dd,1H, J¼7.8, 9.7 Hz), 5.71 (dd,1H, J¼9.4, 9.4 Hz), 7.22e7.37 (m,13H),
7.42e7.60 (m, 7H), 7.91e8.02 (m, 8H), 8.07e8.11 (m, 2H); HRMS (ESI):
[MþNa]þ calcd for C60H57NNaO18, 1102.3395; found 1102.3372.
4.5. 2-(N-Benzyloxycarbonyl)-aminoethyl (4-N-azido-4-
deoxy-2,3,6-tri-O-benzoyl-
tri-O-benzoyl- -galactopyranosyl)-(1/4)-2,3,6-tri-O-
benzoyl- -glucopyranoside (9)
b-D -glucopyranosyl)-(1/3)-(2,4,6-
b-D
b-D
Compound 8 (3.50 g, 3.24 mmol) was added to a solution of
CH2Cl2/TFA (90% v/v, aq) (50 mL, 4:1) and stirred for 1 h. The
mixture was diluted with CH2Cl2 (50 mL) and washed with NaHCO3
(satd aq) (2ꢂ200 mL), dried over MgSO4 (s), filtered, and concen-
trated. The crude acceptor (Rf¼0.24 (toluene/EtOAc 2:1)) was dis-
solved in CH2Cl2 (50 mL) whereupon 6 (2.00, 3.56 mmol) and pre-
ꢀ
activated 4 A MS were added and stirred for 10 min. NIS (1.09 g,
4.86 mmol) and AgOTf (w0.1 equiv) were added and stirred for 1 h,
(0 ꢁC/rt). The reaction was quenched with Et3N whereupon pyr-
idine (10 mL) and BzCl (1.5 mL, 13 mmol) were added. After 2 h, the
mixture was diluted with CH2Cl2 (50 mL) and washed with 1 M HCl
(aq) (2ꢂ100 mL), NaHCO3 (satd aq) (2ꢂ100 mL), H2O (2ꢂ100 mL),
dried over MgSO4 (s), filtered, and concentrated. FC (tolue-
ne/toluene/EtOAc 2:1) gave trisaccharide 9 (3.99 g, 2.62 mmol,
81%) as a colorless syrup. Rf¼0.62 (toluene/EtOAc 2:1); [
a
]Dþ75 (c
0.1, CHCl3); 13C NMR (75.4 MHz, CDCl3):
d
40.8, 60.1, 62.0, 62.3, 62.7,
66.4, 69.3, 69.4, 71.2, 71.6, 71.9, 72.4, 72.5, 73.0, 73.4, 75.3, 78.2,
100.6, 101.2, 101.3, 127.8e128.7 (several carbons), 129.0e130.1
(several carbons), 132.6e133.4 (several carbons), 156.2, 164.1, 164.4,
165.2, 165.3, 165.4, 165.7, 165.8, 165.9. (Note: overlaps occur in
(toluene/EtOAc 1:1); mp 189e191 ꢁC (EtOH); [
NMR (75.4 MHz, CDCl3):
a
]Dþ34 (c 1, CHCl3); 13
C
d
26.3, 27.6, 62.9, 63.0, 70.6, 71.5, 73.3, 73.8,
spectra); 1H NMR (600 MHz, CDCl3):
d
3.16 (dd, 1H, J¼8.1, 11.4 Hz),
74.0, 75.5, 77.3, 77.3, 86.1, 100.4, 111.2, 128.2e130.0 (several carbons),
129.4e130.0 (several carbons), 132.0, 133.1e133.5 (several carbons),
165.0, 165.3, 165.7, 166.0, 166.1, (note: overlaps occur in aromatic re-
3.19e3.24 (m, 1H), 3.28e3.31 (m, 1H), 3.57e3.60 (m, 1H), 3.62e3.65
(m, 2H), 3.67 (dd, 1H, J¼5.1, 7.7 Hz), 3.74e3.77 (m, 1H), 3.78 (dd, 1H,
J¼10.1, 10.1 Hz), 3.86 (dd, 1H, J¼4.7, 11.6 Hz), 4.00 (dd, 1H, J¼3.3,
10.0 Hz), 4.06 (dd, 1H, J¼9.5 Hz), 4.31 (dd, 1H, J¼4.5, 12.0 Hz),
4.42e4.44 (m, 1H), 4.54e4.56 (m, 2H), 4.60e4.63 (m, 2H), 4.79 (d,
1H, J¼7.7 Hz), 4.87 (d, 1H, J¼12.3 Hz), 4.93 (d, 1H, J¼12.3 Hz), 5.08
(bs, 1H), 5.18 (dd, 1H, J¼7.7, 9.6 Hz), 5.36 (d, 1H, J¼8.0, 9.8 Hz), 5.44
(dd, 1H, J¼9.6, 9.6 Hz), 5.51 (dd, 1H, J¼8.3, 9.5 Hz), 5.63e5.66 (m,
2H), 6.78e6.83 (m, 2H), 7.01e7.06 (m, 2H), 7.12e7.57 (m, 32H),
7.68e7.77 (m, 5H), 7.88e7.95 (m, 7H), 8.08e8.10 (m, 2H); HRMS
(ESI): [MþH]þ calcd for C91H79N4O26, 1643.4903; found 1643.4865.
gion); 1H NMR (300 MHz, CDCl3):
d 1.25 (s, 3H), 1.52 (s, 3H), 3.68 (dd,
1H, J¼7.4,11.4 Hz), 3.80e3.91 (m, 2H), 4.09 (dd,1H, J¼2.1, 5.6 Hz), 4.12
(dd,1H, J¼9.6, 9.6 Hz), 4.21e4.26 (m, 2H), 4.47 (dd,1H, J¼5.0,12.1 Hz),
4.60 (d, 1H, J¼7.7 Hz), 4.66 (dd, 1H, J¼1.9, 12.1 Hz), 4.88 (d, 1H,
J¼9.9 Hz), 5.13 (dd,1H, J¼6.8, 7.7 Hz), 5.40 (dd,1H, J¼9.6, 9.6 Hz), 5.73
(dd,1H, J¼9.2, 9.2 Hz), 7.07e7.12 (m, 2H), 7.17e7.20 (m,1H), 7.25e7.62
(m, 17H), 7.91e8.01 (m, 8H), 8.05e8.08 (m, 2H); MALDI-TOF (CHCA):
[MþNa]þ calcd for C56H50NaO15S, 1017.27; found 1017.30.
4.4. 2-(N-Benzyloxycarbonyl)-aminoethyl (2,6-O-benzoyl-3,4-
4.6. 2-(N-Benzyloxycarbonyl)-aminoethyl (4-deoxy-4-N-tert-
O-isopropylidene-
b-D
-galactopyranosyl)-(1/4)-2,3,6-tri-O-
butyloxycarbonyl-glycyl- -glucopyranosyl)-(1/3)-(
b
-
D
b-D-
benzoyl- -glucopyranoside (8)
b
-
D
galactopyranosyl)-(1/4)-b-D-glucopyranoside (1)
Compound 7 (25.0 g, 25.1 mmol) and N-Cbz-2-aminoethanol
Compound 9 (1.03 g, 0.679 mmol) was added to a solution of
CH2Cl2/MeOH (10 mL, 1:4) whereupon NaOMe (660 mg,
12.2 mmol) was added. After 3 h the solution was neutralized
(9.80 g, 50.3 mmol) were dissolved in CH2Cl2 (50 mL) and pre-
ꢀ
activated 4 A MS were added and stirred for 10 min. NIS (8.48 g,

T. Fyrner et al. / Tetrahedron 68 (2012) 6712e6720
6717
with DowexÒ-Hþ, filtered, and evaporated. The crude debenzoy-
4.8. 2-(N-Benzyloxycarbonyl)-aminoethyl (4-N-azido-4-
deoxy-2,3,6-tri-O-benzoyl- -glucopyranosyl)-(1/3)-(2,4,6-
tri-O-benzoyl- -galactopyranosyl)-(1/4)-(2,3,6-tri-O-
benzoyl- -glucopyranosyl)-(1/3)-(2,4,6-tri-O-benzoyl-b-D-
galactopyranosyl)-(1/4)-2,3,6-tri-O-benzoyl-b-D-
glucopyranoside (11)
lated trisaccharide was dissolved in MeOH (30 mL), and washed
with n-heptane (3ꢂ50 mL), and concentrated. Without further
purification, the compound was dissolved in MeOH (10 mL) and
NiCl2$6H2O (w1 mg) was added and stirred for 5 min followed by
the addition of NaBH4 (51 mg, 1.4 mmol). After 15 min, the
mixture was neutralized with DowexÒ-Hþ, filtered and concen-
trated. The crude amine was dissolved in MeOH (10 mL) followed
by the addition of N-(tert-butyloxycarbonyl)-glycine (125 mg,
b-D
b-D
b
-D
Compound 10 (4.00 g, 1.97 mmol) was added to a solution of
CH2Cl2/TFA (90%, v/v, aq) (50 mL, 4:1). After 30 min the solution was
diluted with CH2Cl2 (50 mL) and washed with NaHCO3 (satd aq)
(2ꢂ150 mL), dried over MgSO4 (s), filtered, and concentrated. The
crude acceptor (Rf¼0.22 (toluene/EtOAc 2:1)) was dissolved in
CH2Cl2 (30 mL) whereupon 6 (1.28 g, 2.17 mmol) and pre-activated
0.713 mmol), HOAt (0.5 M in DMF, 0.14 mL, 68
mmol), 4-
methylmorpholine (79 L, 0.71 mmol) and stirred for 10 min
m
whereupon EDC$HCl (0.137 g, 0.713 mmol) was added. After 3 h
the mixture was concentrated. RP chromatography (H2O/MeOH/
H2O 1:1) gave the title compound 1 (0.370 g, 0.443 mmol, 65%) as
ꢀ
4 A MS were added and the reaction mixture was stirred for
a white solid. Rf¼0.80 (chloroform/MeOH/H2O 7:4:1); [
a
]Dꢀ8 (c
10 min. NIS (890 mg, 3.90 mmol) and AgOTf (w0.1 equiv) were
0.1, H2O); 13C NMR (75.4 MHz, CD3OD):
d
27.3, 40.6, 41.5, 43.5,
added and stirred for 1.5 h, (0 ꢁC/rt). The reaction was quenched
51.1, 51.8, 60.6, 61.1, 66.1, 68.2, 68.7, 70.2, 73.2, 73.3, 74.5, 74.8,
75.0, 75.3, 79.4, 79.5, 83.1, 102.9, 103.3, 104.2, 127.5, 127.6, 128.1,
with Et3N, whereupon pyridine (5 mL) and BzCl (460 mL, 3.90 mmol)
were added. After an additional 1.5 h, the mixture was diluted with
CH2Cl2, (w50 mL) filtered through CeliteÒ and washed with 1 M HCl
(aq) (2ꢂ50 mL), NaHCO3 (satd aq) (2ꢂ100 mL), H2O (2ꢂ200 mL),
dried over MgSO4 (s), filtered, and concentrated. FC (tolue-
ne/toluene/EtOAc 4:1) gave pentasaccharide 11 (3.35 g,1.29 mmol,
136.9, 157.1, 157.5, 172.1; 1H NMR (300 MHz, CD3OD):
d 1.44 (s,
9H), 3.23e3.46 (m, 6H), 3.53e3.93 (m, 17H), 4.1 (ad, 1H, J¼2.6),
4.32 (d, 1H, J¼7.8 Hz), 4.44 (d, 1H, J¼7.4 Hz), 4.58 (d, 1H,
J¼7.59 Hz), 5.07 (s, 2H), 7.27e7.35 (m, 5H); HRMS (ESI): [MþH]þ
calcd for C35H56N3O20, 838.3379; found 838.3335.
66%) as a colorless syrup. Rf¼0.64 (toluene/EtOAc 2:1); [
a
]Dþ66 (c
0.1, CHCl3); 13C NMR (75.4 MHz, CDCl3):
d
40.9, 60.2, 61.8, 62.0, 62.3,
62.7, 66.5, 69.3, 69.4, 69.5, 71.1, 71.4, 71.6, 71.7, 71.9, 72.0, 72.4, 72.5,
72.8, 73.1, 73.5, 74.7, 75.3, 78.2, 78.2, 100.6, 100.7, 101.3, 101.4, 126.3,
127.7e128.8 (several carbons), 129.1e130.1 (several carbons),
132.5e133.4 (several carbons),136.6,156.3,164.1,164.1,165.2,165.2,
164.4,164.5,165.3,165.3,165.5,165.7,165.7,165.7,165.9,165.9. (note:
4.7. 2-(N-Benzyloxycarbonyl)-aminoethyl (2,6-di-O-benzoyl-
3,4-di-O-isopropylidene-
tri-O-benzoyl- -glucopyranosyl)-(1/3)-(2,4,6-tri-O-
benzoyl- -galactopyranosyl)-(1/4)-2,3,6-tri-O-benzoyl-
-glucopyranoside (10)
b-D-galactopyranosyl)-(1/4)-(2,3,6-
b-D
several overlaps occur in spectra); 1H NMR (600 MHz, CDCl3):
d 2.92
b-D
b-
(dd, 1H, J¼8.0, 11.5 Hz), 3.07 (dd, 1H, J¼8.1, 11.2 Hz), 3.16e3.31 (m,
2H), 3.46 (d,1H, J¼5.1, 7.8 Hz), 3.51e3.62 (m, 5H), 3.72e3.81 (m, 4H),
3.86e3.91 (m, 2H), 3.95 (dd, 1H, J¼9.4, 9.4 Hz), 4.03 (dd, 1H, J¼9.5,
9.5 Hz), 4.25e4.33 (m, 3H), 4.40e4.43 (m, 2H), 4.50e4.60 (m, 4H),
4.65 (d, 1H, J¼7.6 Hz), 4.74 (d, 1H, J¼7.6 Hz), 4.86 (d, 1H, J¼12.3 Hz),
4.93 (d, 1H, J¼12.3 Hz), 5.05 (bs, 1H), 5.15 (dd,1H, J¼7.7, 9.5 Hz), 5.19
(dd,1H, J¼7.7, 9.8 Hz), 5.33 (dd,1H, J¼8.0, 9.7 Hz), 5.39e5.43 (m, 3H),
5.47 (dd, 1H, J¼8.5, 9.6 Hz), 5.50 (ad, 1H, J¼3.3 Hz), 5.51 (ad, 1H,
J¼3.3 Hz), 5.62 (dd, 1H, J¼9.4, 9.4 Hz), 6.64e6.67 (m, 2H), 6.74e6.77
(m, 2H), 6.98e7.58 (m, 52H), 7.61e7.91 (m, 20H), 7.94e7.98 (m, 2H),
8.02e8.05 (m, 2H); MALDI-TOF (THAP): [MþNa]þ calcd for
C145H122N4NaO42, 2613.74; found 2613.77.
D
To a solution of CH2Cl2/TFA (90% v/v, aq) (50 mL, 4:1) compound 8
(15.0 g, 13.9 mmol) was added. After 2 h the mixture was diluted with
CH2Cl2 (50 mL) and washed with NaHCO3 (satd aq) (2ꢂ100 mL), dried
over MgSO4 (s), filtered, and concentrated. The crude acceptor (Rf¼0.23
(toluene/EtOAc 2:1)) was dissolved in CH2Cl2 (50 mL) whereupon 7
ꢀ
(15.2 g, 15.3 mmol) and pre-activated 4 A MS was added and the re-
action mixture was stirred for 10 min. NIS (4.69 g, 20.8 mmol) and
AgOTf (w0.1 equiv) were added and the reaction was stirred for 1.5 h,
(0 ꢁC/rt). The reaction was quenched with Et3N, whereupon pyridine
(30 mL) and BzCl (6.45 mL, 55. 6 mmol) were added. After an additional
1.5 h the mixture was diluted with CH2Cl2, (50 mL) filtered through
CeliteÒ and washed with 1 M HCl (aq) (2ꢂ200 mL), NaHCO3 (satd aq)
(2ꢂ100 mL), H2O (2ꢂ100 mL), dried over MgSO4 (s), filtered, and
concentrated. FC (toluene/toluene/EtOAc 2:1) gave tetrasaccharide
10 (19.4g, 9.54mmol, 69%)asacolorlesssyrup. Rf¼0.63 (toluene/EtOAc
4.9. 2-(N-Benzyloxycarbonyl)-aminoethyl (4-deoxy-4-N-tert-
butyloxycarbonyl-glycyl-
galactopyranosyl)-(1/4)-(
galactopyranosyl)-(1/4)-
b
-
b
D
-glucopyranosyl)-(1/3)-(
-glucopyranosyl)-(1/3)-(
-glucopyranoside (2)
b-D-
b-D-
-
D
b
-D
2:1); [
a
]Dþ38 (c 1, CHCl3); 13C NMR (75.4 MHz, CDCl3):
d
26.1, 27.4, 40.9,
Compound 11 (0.782 g, 0.302 mmol) was added to a solution of
CH2Cl2/MeOH (10 mL,1:4) whereupon NaOMe (0.489 g, 9.05 mmol)
was added and stirred overnight. The solution was neutralized with
DowexÒ-Hþ, filtered and evaporated. The crude debenzoylated
derivative was dissolved in MeOH (30 mL), and washed with n-
heptane (3ꢂ50 mL), and concentrated. Without further purification
the compound was dissolved in MeOH (10 mL) and NiCl2$6H2O
(w1 mg) was added and stirred for 5 min followed by the addition
of NaBH4 (23 mg, 0.60 mmol). After 15 min, the mixture was neu-
tralized with DowexÒ-Hþ, filtered and concentrated. The crude
amine was dissolved in MeOH (10 mL) followed by the addition of
N-(tert-butyloxycarbonyl)-glycine (55 mg, 0.32 mmol), HOAt (0.5 M
62.0, 62.3, 62.7, 66.4, 69.4, 69.4, 71.2, 71.3, 71.6, 71.7, 72.0, 72.4, 72.5,
72.9, 73.0, 73.2, 73.6, 74.6, 75.3, 77.1, 78.2, 100.1, 100.7, 101.4, 110.8,
127.8e130.0 (several carbons), 132.5e133.4 (several carbons), 136.6,
156.2, 164.1, 164.4, 164.8, 165.2, 165.3, 165.3, 165.4, 165.7, 165.7, 165.8,
165.8. (Note: overlaps occur in spectra); 1H NMR (600 MHz, CDCl3):
d
1.18 (s, 3H),1.44 (s, 3H), 3.10 (dd,1H, J¼8.0,11.4 Hz), 3.19e3.31 (m, 2H),
3.49 (dd,1H, J¼7.4,11.4 Hz), 3.56e3.67 (m, 4H), 3.73e3.76 (m, 2H), 3.87
(dd, 1H, J¼4.6, 11.6 Hz), 3.91 (dd, 1H, J¼3.4, 10.1 Hz), 3.97 (dd, 1H, J¼2.1,
5.5 Hz), 4.03e4.07 (m, 2H), 4.11e4.16 (m, 2H), 4.29 (dd, 1H, J¼4.5,
11.9 Hz), 4.40e4.48(m, 4H), 4.55(d,1H, J¼7.8 Hz), 4.58 (d, 1H, J¼7.9 Hz),
4.71 (d, 1H, J¼7.6 Hz), 4.87 (d, 1H, J¼12.3 Hz), 4.93 (d, 1H, J¼12.3 Hz),
5.04(dd,1H, J¼7.3,7.3Hz),5.07(bs,1H),5.22(dd,1H,J¼7.6, 9.5 Hz), 5.34
(dd, 1H, J¼7.9, 9.8 Hz), 5.48 (dd, 1H, J¼9.4, 9.4 Hz), 5.50 (dd, 1H, J¼8.0,
9.8 Hz), 5.54 (ad, 1H, J¼3.4 Hz), 5.63 (dd, 1H, J¼9.5, 9.5 Hz), 6.76e6.78
(m, 2H), 7.02e7.05 (m, 2H), 7.11e7.53 (m, 36H), 7.66e7.68 (m, 4H),
7.77e7.89 (m, 9H), 7.93e8.10 (m, 7H); MALDI-TOF (CHCA): [MþNa]þ
calcd for C114H101NNaO34, 2050.63; found 2050.61.
in DMF, 60 mL, 32 mmol), 4-methylmorpholine (35 mL, 0.32 mmol)
and stirred for 10 min whereupon EDC$HCl (61 mg, 0.32 mmol) was
added. After 3 h the mixture was concentrated. RP chromatography
(H2O/MeOH 95:5/MeOH/H2O 50:50) gave the title compound 2
(0.223 g, 0.192 mmol, 64%) as a white solid. Rf¼0.45 (chloroform/
MeOH/H2O 7:4:1); [
a
]Dþ74 (c 0.1, H2O); 13C NMR (75.4 MHz, D2O):

6718
T. Fyrner et al. / Tetrahedron 68 (2012) 6712e6720
ꢀ
d
28.2, 41.1, 42.6, 44.1, 52.1, 53.3, 60.5, 60.7, 61.3, 61.5, 67.5, 68.8,
0.377 mmol) and pre-activated 4 A MS were added and the reaction
mixture was stirred for 10 min. NIS (0.154 g, 0.685 mmol) and AgOTf
(w0.1 equiv) were added and stirred for 1.5 h, (0 ꢁC/rt). The reaction
was quenched with Et3N, whereupon pyridine (5 mL) and BzCl
(0.160 mL, 1.37 mmol) were added. After an additional 1.5 h, the
mixture was diluted with CH2Cl2 (25 mL), filtered through CeliteÒ and
washed with 1 M HCl (aq) (2ꢂ50 mL), NaHCO3 (satd aq) (2ꢂ50 mL),
H2O (2ꢂ50 mL), dried over MgSO4 (s), filtered, and concentrated. FC
(toluene/EtOAc 9:1) gave heptasaccharide 13 (0.615 g, 0.174 mmol,
69.6, 70.6, 70.7, 73.4, 73.6, 74.7, 74.9, 75.2, 75.3, 75.6, 78.6, 78.9, 82.2,
82.6, 82.7, 102.9, 103.1, 104.2, 104.2, 128.3, 129.0, 129.4, 137.0, 158.4,
158.9, 173.7. (Note: overlaps occur in region 52.1e104.2); 1H NMR
(300 MHz, D2O):
d 1.43 (s, 9H), 3.24e3.37 (m, 4H), 3.39e3.46 (m,
2H), 3.48e3.96 (m, 28H), 4.16e4.17 (m, 2H), 4.40 (d, 1H, J¼7.5 Hz),
4.47 (d, 1H, J¼7.5 Hz), 4.50 (d, 1H, J¼7.4 Hz), 4.65e4.68 (m, 2H)
(overlap with HDO), 5.09 (s, 2H), 7.35e7.45 (m, 5H); HRMS (ESI):
[MþH]þ calcd for C47H76N3O30, 1162.4436; found 1162.4419.
51%) as a colorless syrup. Rf¼0.64 (toluene/EtOAc 2:1); [
a
]Dþ55 (c 0.1,
4.10. 2-(N-Benzyloxycarbonyl)-aminoethyl (2,6-di-O-benzoyl-
CHCl3); 13C NMR (75.4 MHz, CDCl3):
d
40.8, 60.1, 61.8, 61.9, 62.2, 62.7,
3,4-di-O-isopropylidene-
tri-O-benzoyl- -glucopyranosyl)-(1/3)-(2,4,6-tri-O-
benzoyl- -galactopyranosyl)-(1/4)-(2,3,6-tri-O-benzoyl-
-glucopyranosyl)-(1/3)-(2,4,6-tri-O-benzoyl-
galactopyranosyl)-(1/4)-2,3,6-tri-O-benzoyl-b-D-
glucopyranoside (12)
b
-
D
-galactopyranosyl)-(1/4)-(2,3,6-
66.5, 69.2, 69.2, 69.3, 69.5, 71.0, 71.2, 71.3, 71.5, 71.6, 71.9, 71.9, 72.3,
72.5, 72.7, 72.7, 73.0, 73.4, 74.6, 74.6, 75.3, 77.2, 78.1, 78.1, 100.5, 100.6,
101.2, 101.3, 101.4, 127.6e128.8 (several carbons),129.1e130.0 (several
carbons), 132.4e133.3 (several carbons), 136.5, 156.2, 163.9, 164.0,
164.3, 164.4, 165.1, 165.1, 165.1, 165.2, 165.2, 165.3, 165.4, 165.5, 165.6,
165.6, 165.8, 165.9. (note: several overlaps occur in spectra); 1H NMR
b-D
b-D
b-
D
b-D-
(600 MHz, CDCl3):
d
2.84 (dd, 1H, J¼8.2, 11.3 Hz), 2.90 (dd, 1H, J¼8.0,
To a solution of CH2Cl2/TFA (90% v/v, aq) (50 mL, 4:1) compound
10 (9.25 g, 4.81 mmol) was added. After 1.5 h, the mixture was di-
luted with CH2Cl2 (w50 mL) and washed with NaHCO3 (satd aq),
dried over MgSO4 (s), filtered and concentrated. The crude acceptor
(Rf¼0.24 (toluene/EtOAc 2:1)) was dissolved in CH2Cl2 (50 mL) fol-
lowed by the addition of 7 (5.26 g, 5.29 mmol) and pre-activated
11.5 Hz), 3.06 (dd, 1H, J¼8.1, 11.1 Hz), 3.16e3.31 (m, 2H), 3.36 (dd, 1H,
J¼5.0, 7.6 Hz), 3.43 (dd,1H, J¼5.3, 7.4 Hz), 3.48e3.63 (m, 6H), 3.67 (dd,
1H, J¼4.4,11.6 Hz), 3.71e3.78 (m, 5H), 3.84e3.94 (m, 4H), 4.01 (dd,1H,
J¼9.4, 9.4 Hz), 4.22e4.28 (m, 5H), 4.35e4.40 (m, 4H), 4.49e4.64 (m,
7H), 4.71 (d, 1H, J¼7.6 Hz), 4.86 (d, 1H, J¼12.3 Hz), 4.92 (d, 1H,
J¼12.3 Hz), 5.04 (bs, 1H), 5.12e5.17 (m, 3H), 5.30e5.50 (m, 8H), 5.60
(dd, 1H, J¼9.5, 9.5 Hz), 6.58e6.68 (m, 4H), 6.73e6.78 (m, 3H),
6.94e7.56 (m, 71H), 7.60e8.03 (m, 32H); MALDI-TOF (THAP):
[MþNa]þ calcd for C199H166N4NaO58, 3562.01; found 3562.06.
4 A MS and stirred for 10 min at 0 ꢁC. NIS (1.62 g, 7.21 mmol) and
ꢀ
AgOTf (w0.1 equiv) were added and stirred for 2 h, (0 ꢁC/rt). The
reaction was quenched with Et3N followed by the addition of pyri-
dine (30 mL) and BzCl (2.23 mL, 19.2 mmol). After 2 h the mixture
was diluted with CH2Cl2 (50 mL), filtered through CeliteÒ and
washed with 1 M HCl (aq) (2ꢂ200 mL), NaHCO3 (satd aq)
(2ꢂ150 ml), H2O (2ꢂ200 mL), dried over MgSO4 (s), filtered, and
concentrated. FC (toluene/toluene/EtOAc 4:1) gave hex-
asaccharide 12 (8.45 g, 2.84 mmol, 59%) as a white syrup. Rf¼0.62
4.12. 2-(N-Benzyloxycarbonyl)-aminoethyl (4-deoxy-4-N-tert-
butyloxycarbonyl-glycyl-b-D-glucopyranosyl)-(1/3)-(b-D-
galactopyranosyl)-(1/4)-(
galactopyranosyl)-(1/4)-(
galactopyranosyl)-(1/4)-(
b
b
b
-
-
-
D
D
D
-glucopyranosyl)-(1/3)-(
-glucopyranosyl)-(1/3)-(
-glucopyranoside) (3)
b
-
D
D
-
-
b-
(toluene/EtOAc 2:1); [
CDCl3):
a
]Dþ32 (c 1, CHCl3); 13C NMR (75.4 MHz,
d
26.1, 27.4, 40.8, 61.7e62.0 (several carbons), 62.2, 62.3, 62.7,
Compound 13 (0.500 g, 0.141 mmol) was added to a solution of
CH2Cl2/MeOH (10 mL, 1:4) whereupon NaOMe (0.320 g, 5.92 mmol)
was added and stirred overnight. The solution was neutralized with
DowexÒ-Hþ, filtered and evaporated. The crude debenzoylated hep-
tasaccharide was dissolved in MeOH (20 mL), and washed with n-
heptane (2ꢂ30 mL), and concentrated. Without further purification
the compound was dissolved in MeOH (10 mL) and NiCl2$6H2O
(w1 mg) was added and stirred for 5 min followed by the addition of
NaBH4 (11 mg, 0.28 mmol). After 15 min, the mixture was neutralized
with DowexÒ-Hþ, filtered and concentrated. The crude amine was
dissolved in MeOH/H2O (10 mL, 1:1) followed by the addition of N-
(tert-butyloxycarbonyl)-glycine (26 mg, 0.15 mmol), HOAt (0.5 M in
66.5, 69.3, 69.5, 71.0, 71.1, 71.3, 71.6, 71.7, 71.9, 72.3, 72.5, 72.7, 72.8,
72.9, 73.0, 73.1, 73.5, 73.6, 74.5, 74.6, 75.3, 100.1, 100.5, 100.6, 101.3,
101.4, 110.8, 127.6e128.8 (several carbons), 129.1e123.0 (several
carbons), 132.5e133.0 (several carbons), 136.6, 156.2, 164.0, 164.3,
164.4,164.8,165.1,165.1,165.3,165.3,165.4,165.6,165.6,165.7,165.8,
165.8. (note: several overlaps occur in spectra); 1H NMR (600 MHz,
CDCl3):
d
1.18 (s, 3H), 1.43 (s, 3H), 2.89 (dd, 1H, J¼8.0, 11.5 Hz),
3.02e3.10 (m, 2H), 3,17e3.31 (m, 2H), 3.39 (dd, 1H, J¼4.8, 8.1 Hz),
3.45e3.80 (m, 11H), 3.85 (dd, 1H, J¼3.5, 9.9 Hz), 3.90e4.05 (m, 5H),
4.09e4.14 (m, 3H), 4.19e4.31 (m, 4H), 4.34e4.44 (m, 5H), 4.30 (dd,
1H, J¼3.7, 7.8 Hz), 4.64 (dd,1H, J¼6.9 Hz), 4.46 (d,1H, J¼12.3 Hz), 4.92
(d,1H, J¼12.3 Hz), 5.01 (dd,1H, J¼7.4, 7.4 Hz), 5.04 (bs,1H), 5.15e5.18
(m, 2H), 5.31 (dd,1H, J¼8.0, 9.9 Hz), 5.36e5.48 (m, 4H), 5.60 (dd,1H,
J¼9.5, 9.5 Hz), 6.60e6.65 (m, 2H), 6.74e6.80 (m, 3H), 6.94e7.58 (m,
58H), 7.61e8.05 (m, 27H); MALDI-TOF (THAP): [MþNa]þ calcd for
C168H145NNaO50, 2998.87; found 2999.03.
DMF, 28 mL, 14 mmol), 4-methylmorpholine (16 mL, 0.15 mmol) and
stirred for 10 min whereupon EDC$HCl (28 mg, 0.15 mmol) was added
and stirred overnight followed by concentration. RP chromatography
(H2O/MeOH 95:5/MeOH/H2O 75:25) gave the title compound 3
(61 mg, 41
H2O 7:4:1); [
m
a
mol, 29%) as a white solid. Rf¼0.25 (chloroform/MeOH/
]Dþ17 (c 0.1, H2O); 13C NMR (75.4 MHz, D2O):
d 28.2, 41.1,
4.11. 2-(N-Benzyloxycarbonyl)-aminoethyl (4-N-azido-4-
42.6, 44.1, 52.1, 53.3, 60.5, 60.7, 61.3, 61.6, 62.1, 67.5, 68.9, 69.6, 70.7,
73.4, 73.6, 73.7, 73.9, 74.8, 74.8, 74.9, 75.2, 75.3, 75.4, 75.6, 78.6, 78.8,
82.3, 82.6, 82.7, 102.9, 103.2, 104.2, 104.2, 104.3, 128.4, 129.0, 129.4,
137.1, 159.0, 173.8. (Note: several overlaps occur in spectra); 1H NMR
deoxy-2,3,6-tri-O-benzoyl-
tri-O-benzoyl- -galactopyranosyl)-(1/4)-(2,3,6-tri-O-
benzoyl- -glucopyranosyl)-(2,4,6-tri-O-benzoyl-
galactopyranosyl)-(1/4)-(2,3,6-tri-O-benzoyl-
glucopyranosyl)-(1/3)-(2,4,6-tri-O-benzoyl-
b-D-glucopyranosyl)-(1/3)-(2,4,6-
b-D
b-D
b-D-
b-
D-
(300 MHz, D2O): d 1.43 (s, 9H), 3.27e3.46 (m, 8H), 3.49e3.96 (m, 37H),
b
-D-
4.17e4.18 (m, 3H), 4.43e4.51 (m, 4H), 4.63e4.67 (m, 3H), 5.12 (s, 2H),
galactopyranosyl)-(1/4)-2,3,6-tri-O-benzoyl-
b-D
-
7.37e7.47 (m, 5H); HRMS (ESI): [MþH]þ calcd for C59H96N3O40
,
glucopyranoside (13)
1486.5492; found 1486.5562.
Compound 12 (1.02 g, 0.343 mmol) was added to a solution of
CH2Cl2/TFA (90%, v/v, aq) (10 mL, 4:1). After 30 min the solution was
diluted with CH2Cl2 (25 mL) and washed with NaHCO3 (satd aq)
(2ꢂ100 mL), dried over MgSO4 (s), filtered, and concentrated. The
crude acceptor was dissolved in CH2Cl2 (20 mL) whereupon 6 (0.212 g,
4.13. Ethyl 2,3,4-tri-O-benzoyl-1-thio-a-D-mannopyranoside
(15)
To a solution of 1441 (24.9 g,111 mmol) in pyridine (100 mL), TrCl
(46.5 g, 167 mmol) was added. After 16 h, the reaction mixture was

T. Fyrner et al. / Tetrahedron 68 (2012) 6712e6720
6719
diluted with CH2Cl2 (200 mL) and BzCl (58.4 mL, 503 mmol) was
added dropwise (over 30 min) at 0 ꢁC. After 4.5 h, the solution was
washed with ice/H2O (2ꢂ500 mL), 1 M HCl (aq) (2ꢂ200 mL),
NaHCO3 (satd aq) (2ꢂ200 mL), H2O (2ꢂ200 mL), dried over MgSO4
(s), filtered, and concentrated. Without further purification, the
crude monosaccharide was dissolved in CHCl3/MeOH (300 mL, 2:1)
and p-TsOH (10.5 g, 55.4 mmol) was added. After 3 h, the reaction
mixture was diluted with CH2Cl2 (100 mL), washed with NaHCO3
(satd aq) (2ꢂ150 mL), H2O (2ꢂ150 mL), dried over MgSO4 (s), fil-
tered, and concentrated. FC (petroleum ether/EtOAc 4:1/EtOAc)
gave 15 (27.0 g, 50.3 mmol, 45%) as a colorless oil. Rf¼0.30 (toluene/
J¼15.0 Hz), 4.09e4.15 (m, 1H), 4.23e4.37 (m, 3H), 4.79e4.85 (m, 1H),
5.12 (d,1H, J¼1.8 Hz), 5.63 (d,1H, J¼1.1 Hz), 5.74 (dd,1H, J¼1.8, 3.1 Hz),
5.84e5.92 (m, 3H), 5.96 (dd, 1H, J¼3.1, 10.3 Hz), 6.04 (dd, 1H, J¼9.9,
9.9 Hz), 7.15e7.63 (m, 18H), 7.81e7.86 (m, 4H), 7.97e8.02 (m, 4H),
8.06e8.09 (m, 2H), 8.16e8.19 (m, 2H); HRMS (ESI): [MþNH4]þ calcd
for C58H55ClNO17S, 1104.2879; found 1104.2859.
4.16. 2-(N-Benzyloxycarbonyl)-aminoethyl (2,3,4-O-benzoyl-
6-O-chloroacetyl-a-D-mannopyranosyl)-(1/6)-2,3,4-tri-O-
benzoyl- -mannopyranoside (18)
a-D
EtOAc 9:1); [
a
]Dꢀ82 (c 1, CHCl3); 13C NMR (75.4 MHz, CDCl3):
d 15.0,
Compound 17 (1.99 g, 1.83 mmol) and N-Cbz-2-aminoethanol
25.8, 61.5, 67.6, 70.2, 71.6, 72.5, 82.6, 128.4, 128.7, 128.8, 128.9, 129.2,
129.5, 129.8, 130.1, 133.4, 133.7, 133.8, 165.5, 165.6, 166.7, (Note:
(720 mg, 50.0 mmol) were dissolved in CH2Cl2 (50 mL) and pre-
ꢀ
activated 4 A MS were added, and the reaction mixture was stir-
overlap occur aromatic region); 1H NMR (300 MHz, CDCl3):
d 1.37 (t,
red for 10 min. The mixture was cooled to ꢀ30Cꢁ whereupon NIS
(0.82 g, 3.7 mmol) and TfOH (w0.1 equiv) were added under ni-
trogen. After 2 h, the reaction was quenched with Et3N, filtered
through CeliteÒ, and washed with NaHCO3 (satd aq) (100 mL), H2O
(100 mL), dried over MgSO4 (s), filtered and concentrated. FC (tol-
uene/EtOAc 9:1) gave compound 18 (1.83 g, 1.50 mmol, 82%) as
3H, J¼7.4 Hz), 2.65e2.83 (m, 2H), 3.81e3.83 (m, 2H), 4.42e4.47 (m,
1H), 5.58 (d, 1H, J¼1.5 Hz), 5.79 (dd, 1H, J¼1.5, 2.9 Hz), 5.85e5.94
(m, 2H), 7.22e7.28 (m, 2H), 7.36e7.64 (m, 7H), 7.80e7.84 (m, 2H),
7.97e8.01 (m, 2H), 8.09e8.13 (m, 2H); MALDI-TOF (CHCA):
[MþNa]þ calcd for C29H28NaO8S, 559.14; found 559.18.
a colorless syrup. Rf¼0.32 (toluene/EtOAc 6:1); [
a
]Dꢀ82 (c 1, CHCl3);
13C NMR (75.4 MHz, CDCl3):
d
40.5, 40.7, 64.1, 66.6, 66.7, 66.8, 67.1,
4.14. Ethyl 2,3,4-tri-O-benzoyl-6-O-chloroacetyl-1-thio-a-D-
mannopyranoside (16)
67.6, 68.8, 69.8, 70.1, 70.3, 70.5, 97.4, 98.0, 128.0e129.3 (several
carbons), 129.8e130.0 (several carbons), 133.2, 133.3, 133.6, 133.6,
133.7, 156.6, 165.3, 165.5, 165.6, 165.6, 165.7, 165.9. (Note: several
To a solution of ethyl-2,3,4-tri-O-benzoyl-a-D-thio-mannopyr-
overlaps occur in spectra); 1H NMR (300 MHz, CDCl3):
d 3.64e3.86
anoside 15 (2.28 g, 4.25 mmol) in CH2Cl2/Pyridine (30 mL, 14:1),
chloroacetyl chloride (1.70 mL, 21.3 mmol) was added at ꢀ40 ꢁC.
After 1 h, the mixture was diluted with CH2Cl2 (50 mL) and washed
with 1 M HCl (aq) (2ꢂ50 mL), NaHCO3 (satd aq) (100 mL), H2O
(200 mL), dried over MgSO4 (s), filtered and concentrated. FC (tol-
uene/toluene/EtOAc 6:1) gave chloroacetate 16 (2.15 g,
3.50 mmol, 82%) as a colorless syrup. Rf¼0.57 (toluene/EtOAc 9:1);
(m, 4H), 3.96 (d, 1H, J¼15.0 Hz), 3.99 (d, 1H, J¼15.0 Hz), 4.01e4.14
(m, 2H), 4.25e4.46 (m, 4H), 5.06e5.18 (m, 4H), 5.66 (bs, 1H), 5.75
(dd, 1H, J¼1.8, 3.0 Hz), 5.80 (dd, 1H, J¼1.6, 2.9 Hz), 5.90e6.04 (m,
4H), 7.13e7.62 (m, 23H), 7.82e7.87 (m, 4H), 7.97e8.01 (m, 4H),
8.07e8.11 (m, 2H), 8.15e8.18 (m, 2H); HRMS (ESI): [MþH]þ calcd
for C66H59NO20Cl, 1220.3241; found 1220.3234.
[
a
]Dꢀ57 (c 1, CHCl3); 13C NMR (75.4 MHz, CDCl3):
d 14.9, 25.7, 40.6,
64.2, 67.1, 69.9, 70.4, 72.1, 82.5, 128.4e128.9 (several carbons),
129.7e130.0 (several carbons), 133.3, 133.6, 133.7, 166.4, 165.4,
165.6, 166.9, (Note: several overlaps occur in spectra); 1H NMR
4.17. 2-(N-Benzyloxycarbonyl)-aminoethyl (4-N-azido-4-
deoxy-2,3,6-tri-O-benzoyl-
tri-O-benzoyl- -mannopyranosyl)-(1/6)-2,3,4-tri-O-
benzoyl- -mannopyranoside (19)
b-D-glucopyranosyl)-(1/6)-(2,3,4-
(300 MHz, CDCl3):
d
1.34 (t, 3H, J¼7.4 Hz), 2.64e2.83 (m, 2H), 4.09
a-D
(d, 1H, J¼15.0 Hz), 4.14 (d, 1H, J¼15.0 Hz), 4.40e4.45 (m, 1H), 4.55
(dd, 1H, J¼4.8, 12.2 Hz), 4.76e4.82 (m, 1H), 5.60 (as, 1H), 5.85e5.89
(m, 2H), 6.04 (dd, 1H, J¼9.7, 9.7 Hz), 7.18e7.23 (m, 2H), 7.31e7.36
(m, 3H), 7.44e7.49 (m, 3H), 7.55e7.60 (m, 1H), 7.81e7.84 (m, 2H),
7.96 (m, 2H), 8.11e8.14 (m, 2H); HRMS (ESI): [MþNH4]þ calcd for
C31H33ClNO9S, 630.1565; found 630.1595.
a-D
To a solution of 18 (500mg, 410
mmol)inCH2Cl2/MeOH(10mL,1:1)
was added 2,6-lutidine (380 L, 3.28 mmol) and thiourea (440 mg,
m
5.73 mmol). After4 days, themixturewasdiluted withCH2Cl2 (20 mL)
and washed with 1 M HCl (aq) (30 mL), NaHCO3 (satd aq) (2ꢂ50 mL),
H2O (50 mL), dried over MgSO4 (s), filtered and concentrated. FC
(toluene/toluene/EtOAc 2:1) gave 2-(N-Benzyloxycarbonyl)-ami-
4.15. Ethyl (2,3,4-tri-O-benzoyl-6-O-chloroacetyl-a-D-
mannopyranosyl)-(1/6)-2,3,4-tri-O-benzoyl-
a
-D-
noethyl (2,3,4-tri-O-benzoyl-a-D-mannopyranosyl)-(1/6)-2,3,4-tri-
mannopyranoside (17)
O-benzoyl- -mannopyranoside (370 mg, 330 m
a
-
D
mol, 80%). This ac-
ceptor was dissolved in CH2Cl2 (20 mL) followed by the addition of 6
ꢀ
To a solution of compound 16 (5.36 g, 8.75 mmol) in CH2Cl2
(50 mL), Br2 (0.52 mL, 10.1 mmol) was added. After 30 min, the
mixturewasevaporatedandco-concentratedwithtoluene. Thecrude
bromosugar was dissolved in CH2Cl2 (50 mL) whereupon 15 (3.75 g,
(0.20 g, 0.36 mmol) and pre-activated 4 A MS. NIS (0.11 g, 0.49 mmol)
and AgOTf (w0.1 equiv) were added and stirred for 1 h, (0 ꢁC/rt).
After 1 h, the reaction was quenched with Et3N, filtered through
CeliteÒ and washed with NaHCO3 (satd aq) (50 mL), H2O (50 mL),
dried over MgSO4 (s), filtered, and concentrated. FC (tolue-
ꢀ
6.99 mmol) and pre-activated 4 A MS were added. AgOTf (4.49 g,
17.5 mmol) was added at ꢀ30 ꢁC and the solution was stirred for
30min. ThereactionwasquenchedwithEt3N,filteredthroughCeliteÒ
and washed with 1 M HCl (aq) (50 mL), NaHCO3 (satd aq) (100 mL),
H2O (100 mL), dried over MgSO4 (s), filtered, and concentrated. FC
(toluene/toluene/EtOAc 12:1) gave disaccharide 17 (5.82 g,
5.35 mmol, 77%) as a colorless syrup. Rf¼0.53 (toluene/EtOAc 9:1);
ne/toluene/EtOAc 6:1) gave the trisaccharide 19 (460 mg, 280
85%) as a colorless syrup. Rf¼0.27 (toluene/EtOAc 9:1); [ ]Dꢀ34 (c 0.1,
CHCl3); 13C NMR (75.4 MHz, CDCl3):
40.6, 61.0, 63.4, 65.9, 66.8, 67.2,
67.4, 69.4, 69.8, 70.3, 70.4, 70.4, 71.7, 72.5, 73.7, 97.0, 97.8, 101.7,
127.9e128.9 (several carbons), 129.1, 129.1, 129.1, 129.3, 129.4, 129.5,
129.7e129.9 (several carbons), 130.1, 133.1, 133.1, 133.3, 133.4, 133.5,
133.5, 136.6, 156.5, 165.1, 165.3, 165.4, 165.5, 165.5, 165.6, 165.6, 166.0.
(Note: several overlaps occur in spectra); 1H NMR (600 MHz, CDCl3):
mmol,
a
d
[a
]Dꢀ82 (c 1, CHCl3); 13C NMR (75.4 MHz, CDCl3):
d 15.0, 25.6, 40.6,
64.2, 66.8, 66.9, 67.4, 68.7, 69.9, 70.0, 70.4, 70.7, 72.3, 82.3, 97.7,
128.3e129.2 (several carbons), 129.3, 129.4, 129.5, 129.8e130.1 (sev-
eral carbons),133.3,133.3,133.6,133.7,133.7,133.7,165.3,165.4,165.5,
165.7,165.7,165.8,165.9, (Note: several overlaps occur in spectra); 1H
d
3.26e3.28 (m, 1H), 3.61e3.77 (m, 7H), 3.84 (dd, 1H, J¼5.7, 10.7 Hz),
3.95e3.99 (m, 2H), 4.20e4.23 (m, 2H), 4.48 (dd, 1H, J¼4.7, 12.0 Hz),
4.63 (dd, 1H, J¼2.0, 12.1 Hz), 4.67e4.70 (m, 2H), 5.06e5.07 (m, 2H),
5.13 (d,1H, J¼12.2 Hz), 5.29 (dd,1H, J¼8.8, 8.8 Hz), 5.58e5.64 (m, 3H),
5.74 (as,1H), 5.85 (dd,1H, J¼3.2,10.1 Hz), 5.89 (dd,1H, J¼3.3,10.1 Hz),
NMR (300 MHz, CDCl3):
3.76 (dd, 1H, J¼1.9, 10.8), 3.91 (d, 1H, J¼15.0 Hz), 3.96 (d, 1H,
d
1.47 (t, 3H, J¼7.4 Hz), 2.73e2.95 (m, 2H),

6720
T. Fyrner et al. / Tetrahedron 68 (2012) 6712e6720
3. Devouge, S.; Conti, J.; Goldsztein, A.; Gosselin, E.; Brans, A.; Voue, M.; De
Coninck, J.; Homble, F.; Goormaghtigh, E.; Marchand-Brynaert, J. J. Colloid In-
terface Sci. 2009, 332, 408e415.
4. Xue, J.; Zhu, J. M.; Marchant, R. E.; Guo, Z. W. Org. Lett. 2005, 7, 3753e3756.
5. Iwakura, M.; Nakamura, T. Protein Eng. 1998, 11, 707e713.
6. Shiose, Y.; Kuga, H.; Ohki, H.; Ikeda, M.; Yamashita, F.; Hashida, M. Bioconjugate
Chem. 2009, 20, 60e70.
7. Derda, R.; Wherritt, D. J.; Kiessling, L. L. Langmuir 2007, 23, 11164e11167.
8. Shrivastav, T. G.; Chaube, S. K.; Kariya, K. P.; Singh, R.; Kumar, D.; Jain, P.; Karwar,
S.; Uyake, J.; Deshmukh, B. J. Immunoassay Immunochem. 2012, 33, 1e17.
9. Zalipsky, S. Adv. Drug Deliver. Res. 1995, 16, 157e182.
6.01 (dd, 1H, J¼10.2, 10.2 Hz), 7.16e7.62 (m, 32H), 7.73e7.76 (m, 2H),
7.83e7.91 (m, 6H), 7.96e8.04 (m, 8H), 8.17e8.21 (m, 2H); HRMS (ESI):
[MþNH4]þ calcd for C91H82N5O26, 1660.5248; found 1660.5270.
4.18. 2-(N-Benzyloxycarbonyl)-aminoethyl (4-deoxy-4-N-tert-
butyloxycarbonyl-glycyl-
b-
D
-glucopyranosyl)-(1/6)-(
a-D-
mannopyranosyl)-(1/6)-
a
-D-mannopyranoside (4)
10. Li, L. Y.; Chen, S. F.; Zheng, J.; Ratner, B. D.; Jiang, S. Y. J. Phys. Chem. B 2005, 109,
Compound 19 (0.256 g, 0.156 mmol) was added to a solution of
CH2Cl2/MeOH (10 mL, 1:4) whereupon NaOMe (150 mg, 2.80 mmol)
was added and stirred overnight. The solution was neutralized with
DowexÒ-Hþ, filtered, and evaporated. The crude debenzoylated tri-
saccharide was dissolved in MeOH (30 mL) and washed with n-hep-
tane (2ꢂ40 mL) and concentrated. Without further purification the
compound was dissolved in MeOH (10 mL) and NiCl2$6H2O (w1 mg)
was added and the reaction mixture was stirred for 5 min followed by
the addition of NaBH4 (12 mg, 0.31 mmol). After 15 min, the mixture
was neutralized with DowexÒ-Hþ, filtered and concentrated. The
crude amine was dissolved in MeOH (10 mL) followed by the addition
ofN-(tert-butyloxycarbonyl)-glycine(27 mg, 0.16 mmol), HOAt (0.5 M
2934e2941.
11. Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714e10721.
12. Lin, H. Y.; Thomas, J. L. Langmuir 2003, 19, 1098e1105.
13. Harris, J. M.; Zalipsky, S. Poly(Ethylene Glycol) Chemistry: Biotechnical and Bio-
medical Applications; Plenum: New York, NY, London, 1997.
14. Martinez-Avila, O.; Hijazi, K.; Marradi, M.; Clavel, C.; Campion, C.; Kelly, C.;
Penades, S. Chem.dEur. J. 2009, 15, 9874e9888.
15. Kleinert, M.; Rockendorf, N.; Lindhorst, T. K. Eur. J. Org. Chem. 2004,
3931e3940.
16. Andersson, M.; Oscarson, S.; Oberg, L. Glycoconjugate J. 1993, 10, 197e201.
17. Dziadek, S.; Jacques, S.; Bundle, D. R. Chem.dEur. J. 2008, 14, 5908e5917.
18. Engel, A.; Chatterjee, S. K.; Al-arifi, A.; Riemann, D.; Langner, J.; Nuhn, P. Pharm.
Res. 2003, 20, 51e57.
19. Gege, C.; Geyer, A.; Schmidt, R. R. Chem.dEur. J. 2002, 8, 2454e2463.
20. Andre, S.; Ortega, P. J. C.; Perez, M. A.; Roy, R.; Gabius, H. J. Glycobiology 1999, 9,
1253e1261.
21. Yu, G. S.; Bae, Y. M.; Choi, H.; Kong, B.; Choi, I. S.; Choi, J. S. Bioconjugate Chem.
2011, 22, 1046e1055.
22. Sui, G. D.; Micic, M.; Huo, Q.; Leblanc, R. M. Langmuir 2000, 16, 7847e7851.
23. Patel, A.; Lindhorst, T. K. Carbohydr. Res. 2006, 341, 1657e1668.
24. Inoue, K. Prog. Polym. Sci. 2000, 25, 453e571.
in DMF, 31 mL,16 mmol), 4-methylmorpholine (18 mL, 0.16 mmol) and
stirred for 10 min whereupon EDC$HCl (31 mg, 0.164 mmol) was
added and stirred overnight followed by concentration. RP chroma-
tography (H2O/MeOH 95:5/MeOH/H2O 75:25) gave the title com-
pound 4 (81 mg, 97
MeOH/H2O 7:4:1); [
m
a
mol, 62%) as a white solid. Rf¼0.25 (chloroform/
25. Colonna, B.; Harding, V. D.; Nepogodiev, S. A.; Raymo, F. M.; Spencer, N.;
]Dþ29 (c 0.1, H2O); 13C NMR (75.4 MHz, D2O):
Stoddart, J. F. Chem.dEur. J. 1998, 4, 1244e1254.
d
27.5, 40.1, 51.7, 52.7, 60.9, 65.8, 66.4, 66.4, 66.5, 66.8, 68.5, 69.9, 70.0,
26. Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164e1167.
27. Hederos, M.; Konradsson, P.; Liedberg, B. Langmuir 2005, 21, 2971e2980.
28. Fyrner, T.; Lee, H. H.; Mangone, A.; Ekblad, T.; Pettitt, M. E.; Callow, M. E.;
Callow, J. A.; Conlan, S. L.; Mutton, R.; Clare, A. S.; Konradsson, P.; Liedberg, B.;
Ederth, T. Langmuir 2011, 27, 15034e15047.
70.5, 70.7, 71.0, 71.6, 73.1, 73.5, 74.8, 81.7, 99.7, 99.8,102.6,127.6,128.3,
128.8, 136.5, 158.3, 173.2; 1H NMR (300 MHz, D2O):
d
1.36 (s, 9H),
3.26e3.33 (m, 3H), 3.45e3.84(m, 20H), 4.07e4.10 (m,1H), 4.42(d,1H,
J¼8.0 Hz), 4.71e4.71 (m, 2H) (overlap with HDO), 5.02 (d, 1H,
J¼12.3 Hz), 5.08 (d, 1H, J¼12.3 Hz), 7.30e7.41 (m, 5H); HRMS (ESI):
[MþH]þ calcd for C35H56N3O20, 838.3379; found 838.3356.
29. Schneider, M. F.; Mathe, G.; Tanaka, M.; Gege, C.; Schmidt, R. R. J. Phys. Chem. B
2001, 105, 5178e5185.
30. Lindberg, J.; Svensson, S. C. T.; Pahlsson, P.; Konradsson, P. Tetrahedron 2002, 58,
5109e5117.
31. Yan, F. Y.; Wakarchuk, W. W.; Gilbert, M.; Richards, J. C.; Whitfield, D. M. Car-
bohydr. Res. 2000, 328, 3e16.
32. Tropper, F. D.; Andersson, F. O.; Grandmaitre, C.; Roy, R. Synthesis 1991,
734e736.
Supplementary data
33. Sundgren, A.; Lahmann, M.; Oscarson, S. J. Carbohydr. Chem. 2005, 24, 379e391.
34. Dhenin, S. G. Y.; Moreau, V.; Morel, N.; Nevers, M. C.; Volland, H.; Creminon, C.;
Djedaini-Pilard, F. Carbohydr. Res. 2008, 343, 2101e2110.
35. Alpe, M.; Oscarson, S. Carbohydr. Res. 2002, 337, 1715e1722.
36. Pu, Y. J.; Vaid, R. K.; Boini, S. K.; Towsley, R. W.; Doecke, C. W.; Mitchell, D. Org.
Process Res. Dev. 2009, 13, 310e314.
Spectroscopic data, 1H, 13C NMR and IR for all new compounds
are available. Supplementary data associated with this article can
be found in the online version, at http://dx.doi.org/10.1016/
j.tet.2012.05.118. These data include MOL files and InChiKeys of the
most important compounds described in this article.
37. Nozaki, S. J. Pept. Res. 1999, 54, 162e167.
38. Lin, L.; Shen, Q.; Chen, G. R.; Xie, J. Bioorg. Med. Chem. Lett. 2008, 18, 6348e6351.
39. Boons, G. J.; Grice, P.; Leslie, R.; Ley, S. V.; Yeung, L. L. Tetrahedron Lett. 1993, 34,
8523e8526.
40. Sarkar, S.; Dutta, S.; Das, G.; Sen, A. K. Tetrahedron 2011, 67, 4118e4122.
41. Saksena, R.; Zhang, J.; Kovac, P. J. Carbohydr. Chem. 2002, 21, 453e470.
42. Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J. Org. Chem. 1997, 62, 7512e7515.
References and notes
1. Frasconi, M.; Mazzei, F.; Ferri, T. Anal. Bioanal. Chem. 2010, 398, 1545e1564.
2. Wong, L. S.; Khan, F.; Micklefield, J. Chem. Rev. 2009, 109, 4025e4053.