Synthesis of 13C-labeled and functionalized Hyaluronan derivatives for biophysical studies and surface modifications

Article abstract of DOI:10.1016/j.bmc.2012.11.025

A convergent synthesis of a tetrasaccharide partial sequence of ¹³C-labeled Hyaluronan is presented. This tetrasaccharide can be used for biophysical studies as well as for surface modifications. Furthermore, tetrasaccharide 7 can be employed for the synthesis of additionally labeled higher oligomers of Hyaluronan on the basis of the presented methodology.

Full text of DOI:10.1016/j.bmc.2012.11.025

Bioorganic & Medicinal Chemistry 21 (2013) 733–741
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 13C-labeled and functionalized Hyaluronan derivatives
for biophysical studies and surface modifications
⇑
Stephan Rigol, Liang Xia, Athanassios Giannis
Institut für Organische Chemie, Fakultät für Chemie und Mineralogie, Universität Leipzig, Johannisallee 29, 04103 Leipzig, Germany
a r t i c l e i n f o
a b s t r a c t
A convergent synthesis of a tetrasaccharide partial sequence of 13C-labeled Hyaluronan is presented. This
tetrasaccharide can be used for biophysical studies as well as for surface modifications. Furthermore, tet-
rasaccharide 7 can be employed for the synthesis of additionally labeled higher oligomers of Hyaluronan
on the basis of the presented methodology.
Article history:
Received 1 October 2012
Revised 14 November 2012
Accepted 19 November 2012
Available online 1 December 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Hyaluronic acid
Labeling
1. Introduction
cell–cell and cell–ECM interactions, cell trafficking and lymph node
homing and cancer (stem) cell proliferation.4 HA is also important
For a long time the extracellular matrix (ECM) was only consid-
ered as a type of static glue that helps to connect the cells with
each other. This view has completely changed during last years, be-
cause it was found that there is a plethora of specific interactions
between extracellular molecules as well as between ECM mole-
cules and imbedded cells.1 Today the sum of all macromolecules
that can be found outside of the plasma membrane of cells are
count as constituents of the ECM. Mainly, they can be divided into
fibrous proteins and glycosaminoglycans (GAGs).2 GAGs are built
out of disaccharide repeating units consisting of a hexose or a hex-
uronic acid connected with a hexosamine. Examples for these are:
Heparan sulfate, Chondroitin sulfate and Keratan sulfate which can
also form proteoglycans and Hyaluronan (HA) as an example for a
non-proteoglycan forming GAG. Hyaluronan is an unbranched,
exclusively non-sulfated polysaccharide that appears as a polyan-
ion under physiological pH values. HA is built out of disaccharide
in tissue injury and repair.5 Here, the function is correlated to the
size of HA fragments. While short HA chains are immunostimula-
tory longer sequences cause the opposite effect. For all these rea-
sons it is not surprising, that extracellular matrix molecules
represent important targets in pharmacotherapy.6
Well defined HA subunits of different length are required for
such investigations7 and substantial work can already been found
on that field. Flowers and Jeanloz as well as Takanashi et al. suc-
ceeded with the first chemical synthesis of HA disaccharides in
1962.8,9 After this pioneering work also longer fragments were
synthesized.10–12 Usage of different protective groups is serving
the specific needs to suit different reaction conditions. The synthe-
sized fragments, if not bearing a free reducing end, mainly exhibit a
non-functionalized aglycon (e.g., methyl, aryl) or a mono-function-
alized moiety like an alkyl-azide.13 Recently van der Marel and
Codée reported an impressive automated solid phase synthesis of
hepta- to pentadecameric O-1-allyl substituted HA sequences.14
Here, we describe a convergent synthesis of a 13C-labeled HA
tetramer for ongoing biophysical studies.
repeating units of 2-acetamido-2-deoxy-D-glucose and D-glucu-
ronic acid. Compared with other GAGs HA is different: it is non-sul-
fated, is neither produced in the Golgi apparatus nor in the
endoplasmic reticulum but by integral membrane proteins—the
HA synthases (HAS1, HAS2, HAS3). Also the size is different: HAs
molecular weight can reach 105–107 Da and this is two orders of
magnitudes higher compared with other GAGs.3 Altogether, HA
plays an important role as part of the ECM: so it acts as water stor-
age, it affects cell proliferation, migration and differentiation4 and
also serves as ligand for different receptors such as RHAMM, ICAM-
1 and CD44. The latter is a cell-surface glycoprotein involved in
2. Results and discussion
Reaction of glycosyl donor 115 with derivative 216,17 using
TMSOTf as promoter, afforded disaccharide 4 in 90% yield. Simi-
larly, disaccharide 5 was obtained after coupling of trichloracetim-
idate 1 with monosaccharide 318 and subsequent O-TBS group
cleavage with TBAF in modest yield. In both cases only the desired
b-anomers were obtained. Next, both disaccharides were linked by
activating thioglycoside 4 with NIS and trifluoromethanesulfonic
acid, to afford protected tetrasaccharide 6 in a 59% yield. Then,
⇑
Corresponding author.
E-mail address: giannis@uni-leipzig.de (A. Giannis).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.025

734
S. Rigol et al. / Bioorg. Med. Chem. 21 (2013) 733–741
the N-Troc group was cleaved under mild reducing conditions (Zn,
AcOH)19 and subsequently the generated amino group was acety-
lated to obtain tetrasaccharide 8. The removal of the O-TBS as well
as 4,6-benzylidene groups was achieved by treatment with Olah’s
reagent. This reaction was slow and required four days. Last,
Zemplén conditions were applied to cleave all benzoates before
the ester groups of the glucuronic acid moieties were hydrolyzed
by sodium hydroxide to furnish O-1-allyl Hyaluronan tetramer
10. Although, derivative 10 can be obtained in one step by alkaline
hydrolysis of 9 we experienced difficulties to separate it from ben-
zoic acid and therefore we used the described two-step procedure.
For future studies we also prepared the glycosyl acceptor 7 by re-
moval of the TBS group at O-4000. Since TBAF was not working for
cleavage of this protective group we applied again Olah’s reagent
adding an additional amount of pyridine to avoid benzylidene group
cleavage. Derivative 7 was finally obtained in 78% yield.
Synthesis of the 13C-labeled derivative 5 (label on glucuronic
acid moiety) was accomplished analogously with small variations
OMe
O
OMe
O
Ph
O
O
O
Ph
a
O
O
TBSO
TBSO
O
O
BzO
BzO
O
HO
HO
STol
BzO
BzO
AcNH
O
CCl3
Ph
O
CCl3
NH
NHTroc
O
NH
a, b
1
2
1
3
OMe
OMe
Ph
O
O
O
O
O
O
O
O
O
O
HO
O
TBSO
STol
BzO
O
BzO
AcNH
NHTroc
BzO
BzO
O
4
5
c
OMe
OMe
Ph
O
Ph
O
O
O
BzO
O
O
O
O
O
TBSO
O
O
O
O
BzO
NHTroc
AcNH
BzO
BzO
O
6
i
OMe
OMe
Ph
Ph
O
O
O
O
O
O
d,e
O
O
O
HO
O
O
O
O
BzO
BzO
NHTroc
NH
BzO
BzO
O
7
O
OMe
OMe
Ph
Ph
O
O
O
O
O
O
O
O
O
TBSO
O
O
O
O
BzO
BzO
NH
NH
BzO
BzO
O
O
O
8
f
OH
OH
OH
OMe
OMe
O
O
O
O
O
HO
O
O
HO
O
HO
O
BzO
BzO
NH
O
NH
OBz
OBz
O
9
O
g, h
OH
OH
OH
O
O
O
O
O
HO
O
O
HO
O
HO
O
HO
HO
NH
O
NH
OH
OH
O
O
10
Scheme 1. Reagents and conditions: Synthesis of free tetrasaccharide 10. (a) TMSOTf, ꢀ20 °C?rt, 90% (for 4), 62% (for coupling of 1+3); (b) TBAF, THF, 35%; (c) NIS, TfOH, 59%;
(d) AcOH, Zn; (e) Ac2O, pyridine, 43% (over 2 steps); (f) HF-Py, 46%; (g) NaOMe, MeOH, Amberlite 120; (h) NaOH, H2O, Dowex 50, 63% (over two steps); (i) HF-Py, Py, 78%.

S. Rigol et al. / Bioorg. Med. Chem. 21 (2013) 733–741
735
OPMB
OPMB
Ph
Ph
+
*
*
O
*
O
O
O
TBSO
BzO
O
a
O
O
*
O
* *
*
O
STol
OH
O
O
TBSO
BzO
HO
* *
BzO
*
AcNH
AcNH
O
*
*
*
BzO
O
11
12
3
OMe
Ph
O
Ph
O
O
*
* *
O
*
* *
O
b
*
c, d
*
O
TBSO
BzO
O
O
TBSO
BzO
O
AcNH
AcNH
*
*
BzO
*
BzO
O
O
13
14
OMe
Ph
O
HO
BzO
O
O
*
* *
O
e
*
O
O
AcNH
*
*
BzO
O
5
Scheme 2. Reagents and conditions: Synthesis of 13C-labeled disaccharide 5. (a) NIS, AgOTf, 80%; (b) DDQ, CH2Cl2, NaHCO3 sat, 67%; (c) BAIB, TEMPO, CH2Cl2, H2O; (d) MeI,
K2CO3, 90% (over 2 steps); (e) TBAF, THF, 35%.
(Scheme 2). Here, disaccharide 12 was prepared from the corre-
sponding thioglycoside 11 and monosaccharide 3. Then, the PMB
group was cleaved by treatment with DDQ and subsequently the
generated primary alcohol moiety was converted to the carboxylic
acid using the TEMPO/BAIB methodology.20 Next, the carboxy
group of the glucuronic acid was protected as methyl ester by
treatment MeI and potassium carbonate. Last, TBS deprotection
using TBAF furnished disaccharide 5. This derivative was used for
the synthesis of the labeled tetrasaccharide 10 on the basis of the
above described methodology (Scheme 1).
and/or with ceric ammonium molybdate, potassium permanganate
or vanillin staining solution. Chromatographic purification was
performed as flash chromatography on Acros silica gel 35–70,
60 Å, using a forced flow of eluent (method of Still) or as prepara-
tive TLC on Merck silica gel 60 F254 glass plates with concentration
zone. Concentration under reduced pressure was performed by ro-
tary evaporation at 40 °C at the appropriate pressure. NMR spectra
were recorded on a Varian Mercury plus 300 (operating at
300 MHz for 1H and 75 MHz for 13C acquisitions), a Varian Mercury
plus 400 (operating at 400 MHz for 1H, 100 MHz for 13C), and a Bru-
ker Avance-700 (operating at 700 MHz for 1H, 175 MHz for 13C).
Chemical shifts d are reported in ppm with the solvent resonance
3. Conclusion
as internal standard (chloroform-d1: 7.26 (1H NMR), 77.16 (13
C
NMR); methanol-d4: 3.31 (1H NMR), 49.00 (13C NMR). Coupling con-
stants J are given in Hertz (Hz). Multiplicities are classified by the
following abbreviations: s = singlet, d = doublet, t = triplet and com-
binations thereof, or m = multiplet or br = broad signal. High resolu-
tion mass spectra were obtained on a Bruker Daltonics ESI-FT-ICR-
MS APEX II [7 T]. IR spectra were obtained on an ATI/MATTSON Gen-
esis FT-IR as thin film or KBr disk. Absorbance frequencies are re-
ported in reciprocal centimeters (cmꢀ1). Melting points were
measured with a Büchi ‘Melting Point B-540’ and are uncorrected.
Optical rotation data was obtained with a Schmidt + Haensch Polar-
tronic MHZ-8 at the sodium-D line (589 nm) using a 50 mm path-
length cell in the solvent and concentration indicated.
In summary, here we reported the first synthesis of a 13C-labeled
tetrasaccharide sequence of Hyaluronan. In addition the corre-
sponding non-labeled derivative 10 was obtained. Whereas the la-
beled tetrasaccharide can be employed for NMR spectroscopic
investigations (interaction with Interleukin-8 or growth factors)21
the non-labeled derivative can be used for surface modifications.14
Finally, derivative 7 can be used in the context of other oligosaccha-
ride synthesis. The selective removal of the N-Troc group (transfor-
mation from 6 to 8) opens the possibility for the introduction of
different groups covalently attached to the amino function of the
subterminal D-glucosamine moiety of the HA tetrasaccharide.
4. Experimental section
4.1.1. 4-Methylphenyl 4,6-O-benzylidene-2-deoxy-3-O-{2,3-di-
O-benzoyl-4-O-[tert-butyl-(dimethyl)silyl]-6-methyl-b-
glucopyranuronosyl}-1-thio-2-{[(2,2,2-trichloroethoxy)-
carbonyl]amino}-b- -glucopyranoside 4
D-
4.1. General experimental methods
D
All reactions were run under an atmosphere of argon unless
otherwise indicated. Room temperature refers to 22 °C, ambient
pressure to 1013 hPa. Reagents and anhydrous solvents were
transferred via oven-dried syringe or cannula. Flasks were flame-
dried under vacuum and cooled under a constant stream of argon.
Tetrahydrofuran (THF) was distilled under argon from potassium,
dichloromethane from SICAPENT (phosphorus pentoxide on solid
support with indicator). Acetone, acetonitrile and pyridine were
purchased from Acros or Aldrich (anhydrous over molecular
sieves). All other chemicals were purchased from ABCR, Acros, Al-
drich, Alfa Aesar, Fluorochem, TCI Europe and VWR at highest com-
mercially available purity and used as such. Reactions were
monitored by thin layer chromatography using Merck silica gel
60 F254 TLC aluminium sheets and visualized under an UV lamp
OMe
O
Ph
O
O
O
O
TBSO
BzO
S
O
NH
OBz
O
Cl3C
O
Methyl 2,3-di-O-benzoyl-4-O-[tert-butyl(dimethyl)silyl]-1-O-(2,2,
2-trichloroethanimidoyl)- -glucopyranuronate (3.00 g, 4.44
mmol, 1.1 equiv) and 4-methylphenyl 4,6-O-benzylidene-2-deoxy-
1-thio-2-{[(2,2,2-trichloroethoxy)carbonyl]amino}-b- -glucopyran-
oside 2 (2.22 g, 4.04 mmol, 1.0 equiv) were co-evaporated twice with
a
-D
1
D

736
S. Rigol et al. / Bioorg. Med. Chem. 21 (2013) 733–741
toluene (15 ml) and then dried at high vacuum for 16 h. This mixture
was dissolved in dichloromethane (60 ml) and transferred to a flask
containing activated powdered 4 Å molecular sieves (5.50 g). After
stirring at rt for 1 h it was cooled to ꢀ20 °C and TMSOTf (0.146 ml,
0.180 g, 0.808 mmol, 0.2 equiv) was added. Next, the reaction was al-
lowed to slowly warm up to rt during 15 h. Then triethylamine
(0.150 ml) was added to quench the reaction. After removal of sol-
vent and purification by column chromatography disaccharide 4
(3.86 g, 3.64 mmol, 90%) was obtained as white solid.
was allowed to slowly warm up to rt and stirred for 1 h. Then trieth-
ylamine (100 l) was added to quench the reaction. After removal of
solvent and purification by column chromatography disaccharide 12
l
(0.762 g, 0.799 mmol, 80%) was obtained as white solid.
4.1.2.1. Non-labeled.
Rf: 0.25 (n-hexane/ethyl acetate = 1:1 v/v).
1H NMR: (400 MHz, CDCl3) d [ppm] ꢀ0.27 (s, 3H, Si-CH3), ꢀ0.06
(s, 3H, Si-CH3), 0.68 (s, 9H, Si-C(CH3)3), 1.70 (s, 3H, CO-CH3),
3.23–3.27 (m, 1H), 3.39–3.42 (m, 2H), 3.68–3.73 (m, 2H),
3.95–4.00 (m, 2H), 4.03–4.10 (m, 2H), 4.18–4.26 (m, 2H), 3.81
(s, 3H, O-CH3), 4.34 (d, J = 12.0 Hz, pMP-CHaHb), 4.53 (d,
J = 12.0 Hz, pMP-CHaHb), 4.86 (d, J = 4.0 Hz, H-1), 4.99 (d,
J = 8.0 Hz, H-10), 5.12 (dd, J = 7.1, 7.1 Hz), 5.11–5.23 (m, 2H),
5.40 (s, 1H, Ph-CH), 5.41–5.46 (m, 2H), 5.70–5.80 (m, 1H,
CH@CH2), 6.89 (d, J = 8.6 Hz 2H, Har), 7.25–7.27 (m, 2H, Har),
7.30–7.36 (m, 7H, Har), 7.42–7.51(m, 4H, Har), 7.85–7.88 (m,
4H, Har), 8.77 (br, 1H, NH).
Rf: 0.34 (n-hexane/ethyl acetate = 4:1 v/v).
1H NMR: (400 MHz, CDCl3) d [ppm] ꢀ0.23 (s, 3H, Si-CH3), ꢀ0.08
(s, 3H, Si-CH3), 0.70 (s, 9H, Si-C(CH3)3), 2.32 (s, 3H, –CH3), 2.91
(dt, J = 9.8, 7.5 Hz, 1H, H-2), 3.51 (td, J = 9.5, 4.7 Hz, 1H, H-5),
3.56–3.61 (m, 1H, H-4), 3.63 (s, 3H, –O-CH3), 3.74 (t,
J = 10.2 Hz, 1H, H-6a), 3.86 (d, J = 9.0 Hz, 1H, H-50), 4.11 (d,
J = 12.1 Hz, 1H, –CHaHb–CCl3), 4.26 (t, J = 8.8 Hz, 1H, H-40),
4.34 (dd, J = 10.4, 4.8 Hz, 1H, H-6b), 4.42 (t, J = 9.2 Hz, 1H, H-
3), 4.55 (d, J = 12.1 Hz, 1H, –CHaHb–CCl3), 4.90 (d, J = 7.8 Hz,
1H, H-10), 5.01 (d, J = 7.2 Hz, 1H, NH), 5.17 (d, J = 10.3 Hz, 1H,
H-1), 5.34 (dd, J = 9.4, 8.0 Hz, 1H, H-20), 5.46 (t, J = 9.1 Hz, 1H,
H-30), 5.51 (s, 1H, Ph-CH), 7.02 (d, J = 7.8 Hz, 2H, Har), 7.20–
7.25 (m, 2H, Har), 7.30–7.37 (m, 3H, Har), 7.37–7.44 (m, 4H,
Har), 7.44–7.51 (m, 3H, Har), 7.51–7.56 (m, 1H, Har), 7.84–
7.88 (m, 2H, Har), 7.92–7.96 (m, 2H, Har).
13C NMR: (100 MHz, CDCl3) d [ppm] ꢀ4.7 (Si-CH3), ꢀ4.0 (Si-
CH3), 17.9 (Si-C(CH3)3), 25.7, 29.7, 52.7 (C-2), 55.4 (–O-CH3),
62.9, 67.9, 68.7, 69.0, 69.2, 72.9, 74.1, 75.7, 76.3, 76.5, 81.5,
97.1 (C-1), 100.1 (C-10), 101.6 (Ph-CH), 113.9, 117.8 (CH@CH2),
126.3, 128.3, 128.6, 129.2, 129.4, 129.6, 129.82, 129.85,
129.91, 130.4, 133.1, 133.4, 133.5, 137.4, 159.3, 165.3, 166.0,
170.3, 177.6.
13C NMR: (100 MHz, CDCl3) d [ppm] ꢀ5.1 (Si-CH3), ꢀ4.3 (Si-
CH3), 17.8 (Si-C(CH3)3), 21.3 (–CH3), 25.5 (Si-C(CH3)3), 52.5
(–O-CH3), 56.7 (C-2), 68.7 (C-6), 70.5 (C-5), 70.9 (C-40), 72.5
(C-20), 74.0 (CH2-CCl3), 75.2 (C-30), 76.6 (C-50), 78.7 (C-3), 80.1
(C-4), 84.9 (C-1), 95.5 (CCl3), 101.2 (Ph-CH, C-10), 126.2 (Car),
127.2 (Car), 128.4 (Car), 128.5 (Car), 128.7 (Car), 129.1 (Car),
129.2 (Car), 129.6 (Car), 129.8 (Car), 129.9 (Car), 130.0 (Car),
133.2 (Car), 133.5 (Car), 133.9 (Car), 137.3 (Car), 138.9 (Car),
153.5 (NH-CO), 165.0 (CO), 165.6 (CO), 168.3 (C-60).
HR-MS: (ESI positive, MeOH) Calcd for [C52H63NO14SiNa]+:
[M+Na]+ 976.39100. Found: 976.39087.
IR: (KBr) m
max = 3439 cmꢀ1, 2951, 2930, 2858, 1713, 1514, 1275,
1109, 1093, 711.
Optical rotation: ½a D23
ꢁ
ðdeg ꢂ cm3 ꢂ gꢀ1 ꢂ dmꢀ1Þ + 96.3 (c 1.0, CHCl3).
Melting point: 67–70 °C.
4.1.2.2. 13C-labeled.
1H NMR: (400 MHz, CDCl3) d [ppm] ꢀ0.27 (s, 3H), ꢀ0.06 (s, 3H),
0.68 (s, 9H), 1.70 (s, 3H), 3.24 (d, J = 144 Hz, 1H), 3.41 (d,
J = 140 Hz, 2H), 3.68–3.73 (m, 2H), 3.81 (s, 3H), 3.99–4.03
(m,1H), 4.05–4.26 (m, 4H), 4.34 (d, J = 2.8,12 Hz, 1H), 4.53 (dd,
J = 4.8, 12 Hz, 1H), 4.86 (d, J = 4 Hz, 1H), 4.99 (dd, J = 160 Hz, 1H),
5.11–5.20 (m, 2H), 5.40 (s, 1H), 5.43 (d, J = 149 Hz, 2H), 5.70–
5.79 (m, 1H), 6.89 (d, 2H), 7.25 (d, 2H), 7.25–7.27 (m, 2H), 7.30–
7.36 (m, 7H), 7.42–7.51(m, 4H), 7.85–7.88 (m, 4H), 8.77 (br, 1H).
13C NMR: (100 MHz, CDCl3) d [ppm] ꢀ4.8, –4.1, 17.9, 22.9, 25.7,
52.7, 55.4, 62.9, 67.9 (d, J = 45 Hz), 68.7, 69.0, 69.2 (dd, J = 41.4,
41.4 Hz), 73.0, 74.1 (dd, J = 48.5, 39.5 Hz), 76.0–75.8 (m), 81.6,
97.1, 100.2 (d, J = 47 Hz), 101.6, 113.9, 117.8, 126.3, 128.3,
128.6, 129.2, 129.4, 129.6, 129.8, 129.87, 129.92, 130.4, 133.1,
133.4, 137.4, 159.3, 165.4, 166.0, 170.2, 177.4.
HR-MS: (ESI positive, MeOH) Calcd for [C50H56Cl3NO14SSi-
Na]+: [M+Na]+ 1082.21486. Found: 1082.21395.
IR: (KBr)
m
max = 3437 cmꢀ1, 2953, 2928, 2857, 1735, 1272,
1094, 840, 710.
Optical rotation: ½a D23
ꢁ
ðdeg ꢂ cm3 ꢂ gꢀ1 ꢂ dmꢀ1Þ +38.1 (c 1.0, CHCl3).
Melting point: 146–148 °C.
4.1.2. Prop-2-en-1-yl 2-(acetylamino)-4,6-O-benzylidene-2-
deoxy-3-O-{2,3-di-O-benzoyl-4-O-[tert-butyl(dimethyl)silyl]-6-
O-(4-methoxybenzyl)-b-D-glucopyranosyl}-a-D-glucopyranoside
and prop-2-en-1-yl 2-(acetylamino)-4,6-O-benzylidene-2-deoxy-
3-O-{2,3-di-O-benzoyl-4-O-[tert-butyl(dimethyl)silyl]-6-O-(4-
methoxybenzyl)-b-
D
-(13C6)glucopyranosyl}-
a-D-glucopyranoside
12
HR-MS: (ESI positive, MeOH) Calcd for [C4613C6H63NO14SiNa]+:
[M+Na]+ 982.41168. Found: 982.41101
OPMB
4.1.3. Prop-2-en-1-yl 2-(acetylamino)-4,6-O-benzylidene-2-
deoxy-3-O-{2,3-di-O-benzoyl-4-O-[tert-butyl(dimethyl)silyl]-b-
Ph
O
O
O
O
TBSO
BzO
O
D
-glucopyranosyl}-
(acetylamino)-4,6-O-benzylidene-2-deoxy-3-O-{2,3-di-O-
benzoyl-4-O-[tert-butyl(dimethyl)silyl]-b-
-(13C6)
a-D-glucopyranoside and prop-2-en-1-yl 2-
NH
OBz
O
D
O
glucopyranosyl}-
a-
D
-glucopyranoside 13
4-Methylphenyl 2,3-di-O-benzoyl-4-O-[tert-butyl(dimethyl)silyl]-
6-O-(4-methoxybenzyl)-1-thio-b- -glucopyranoside 11 (0.729 g,
1.00 mmol, 1.0 equiv) and prop-2-en-1-yl 2-(acetylamino)-4,6-O-
benzylidene-2-deoxy- -glucopyranoside 2 (0.349 g, 1.00 mmol,
1.0 equiv) were co-evaporated three times with toluene (5 ml) and
then dried at high vacuum for 1 h. This mixture was dissolved in
dichloromethane (20 ml) and transferred to a flask containing acti-
vated powdered 4 Å molecular sieves (150 mg). After cooling the
reaction mixture to 0 °C NIS (0.0247 g, 1.10 mmol, 1.1 equiv) and
AgOTf (0.0514 g, 0.200 mmol, 0.2 equiv) was added and the reaction
OH
D
Ph
O
O
O
O
TBSO
O
a-D
BzO
NH
OBz
O
O
To an aluminum foil covered flask containing a solution of
disaccharide 12 (0.670 g, 0.702 mmol, 1.0 equiv) in dichlorometh-
ane (7.00 ml) was added sat. sodium bicarbonate solution

S. Rigol et al. / Bioorg. Med. Chem. 21 (2013) 733–741
737
(0.350 ml), followed by 2,3-dichloro-5,6-dicyano-1,4-benzoqui-
none (0.207 g, 0.913 mmol, 1.3 equiv). The mixture was stirred at
rt for 5 h, then washed with sat. sodium bicarbonate solution.
The aqueous phase was extracted with dichloromethane for three
times. The combined organic phases were washed with brine and
dried over sodium sulfate. After removal of solvent and purification
by column chromatography disaccharide 13 (0.392 g, 0.471 mmol,
67%) was obtained as white solid.
Method A: To a solution of disaccharide 13 (0.388 g, 0.464 mmol,
1.0 equiv) in dichloromethane (17.0 ml) water (8.50 ml) was added,
followed by bis(acetoxy)iodobenzene (0.374 g, 1.16 mmol,
2.5 equiv) and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (0.0145 g,
0.0928 mmol, 0.2 equiv). The mixture was stirred at rt for 7 h and
quenched by addition of sat. sodium thiosulfate solution. The aque-
ous phase was extracted with dichloromethane for three times. The
combined organic phases were washed with brine and dried over
sodium sulfate. After removal of solvent, the residue was dissolved
in DMF (10.0 ml) followed by the addition of methyl iodide (0.260
ml, 0.593 g, 4.176 mmol, 9.0 equiv) and potassium carbonate
(0.641 g, 4.64 mmol, 10.0 equiv). The mixture was stirred at rt for
2 h. Excess potassium carbonate was removed by filtration. After
solvent removal under reduced pressure and purification by column
chromatography disaccharide 14 (0.361 g, 0.419 mmol, 90%) was
obtained as white solid.
4.1.3.1. Non-labeled.
Rf: 0.10 (dichloromethane/ethyl acetate = 9:1 v/v).
1H NMR: (300 MHz, CDCl3) d [ppm] ꢀ0.23 (s, 3H, Si-CH3), 0.05
(s, 3H, Si-CH3), 0.74 (s, 9H, Si-C(CH3)3), 1.79 (s, 3H, CO-CH3),
3.03 (br, 1H), 3.36–3.41 (m, 1H), 3.54–3.58 (m, 1H), 3.76–3.97
(m, 6H), 4.04–4.13 (m, 2H), 4.24–4.27 (m, 1H), 4.45–4.51 (m,
1H), 4.79 (d, J = 3.6 Hz, 1H, H-1), 5.02 (d, J = 8.0 Hz, 1H), 5.15–
5.26 (m, 3H), 5.50–5.55 (t, J = 9.4 Hz, 2H), 5.59 (s, 1H, Ph-CH),
5.74–5.83 (m, 1H, CH@CH2), 7.22–7.26 (m, 2H), 7.29–7.36 (m,
5H), 7.42–7.47(m, 4H), 7.78–7.87 (m, 2H).
Method B: Methyl 2,3-di-O-benzoyl-4-O-[tert-butyl(dimethyl)-
silyl]-1-O-(2,2,2-trichloroethanimidoyl)-
(2.42 g, 3.58 mmol, 1.0 equiv) and prop-2-en-1-yl 2-(acetyl-
amino)-4,6-O-benzylidene-2-deoxy- -glucopyranoside 3
a-D-glucopyranuronate 1
13C NMR: (100 MHz, CDCl3) d [ppm] ꢀ4.6 (Si-CH3), ꢀ4.1 (Si-
CH3), 17.9 (Si-C(CH3)3), 23.3, 25.7, 52.9 (C-2), 61.3, 63.4, 68.7,
68.9, 69.0, 75.8, 76.0, 79.3, 97.6, 98.9, 100.0 (C-1), 101.6 (Ph-
CH), 104.9 (C-10), 114.5, 118.0 (CH@CH2), 126.1, 128.39,
128.45, 129.3, 129.5, 129.8, 129.9, 132.1, 133.1, 133.2, 133.4,
137.1, 165.6 (CO), 166.0 (CO), 170.3 (CO).
a
-D
(1.50 g, 4.29 mmol, 1.2 equiv) were co-evaporated twice with tolu-
ene (15 ml) and then dried at high vacuum for 16 h. This mixture
was dissolved in dichloromethane (50 ml) and transferred to a
flask containing activated powdered 4 Å molecular sieves
(4.00 g). After stirring at rt for 1 h it was cooled to ꢀ20 °C and
TMSOTf (0.130 ml, 0.159 g, 0.716 mmol, 0.2 equiv) was added.
Next, the reaction was allowed to slowly warm up to rt during
15 h. Then triethylamine (0.140 ml) was added to quench the reac-
tion. After removal of solvent and purification by column chroma-
tography disaccharide 14 (1.91 g, 2.22 mmol, 62%) was obtained as
white solid.
HR-MS: (ESI positive, MeOH) Calcd for [C44H55NO13SiNa]+:
[M+Na]+ 856.33369. Found: 856.33313, Calcd for [C44H55NO13-
SiK]+: [M+K]+ 872.30782. Found: 872.30673.
IR: (KBr) m
max = 3437 cmꢀ1, 2952, 2929, 2858, 1732, 1665, 1275,
1093, 854, 839, 711.
Opticalrotation:½a D23
ꢁ
ðdeg ꢂ cm3 ꢂ gꢀ1 ꢂ dmꢀ1Þ + 100.1 (c1.0, CHCl3).
Melting point: 209–211 °C.
4.1.4.1. Non-labeled.
4.1.3.2. 13C-labeled.
Rf: 0.30 (n-hexane/ethyl acetate = 1:1 v/v).
1H NMR: (300 MHz, CDCl3) d [ppm] ꢀ0.23 (s, 3H), 0.05 (s, 3H),
0.74 (s, 9H), 1.79 (s, 3H), 3.14 (br, 1H), 3.34 (m, 1H), 3.54 (m,
1H), 3.76–4.22 (m, 8H), 4.26 (m, 1H), 4.48 (m, 1H), 4.79 (d,
J = 3.6 Hz, 1H), 5.02 (d, J = 127 Hz, 1H), 5.15–5.26 (m, 3H), 5.52
(t, J = 9.4 Hz, 2H), 5.59 (s, 1H), 5.78 (m, 1H), 7.24 (m, 2H), 7.31
(m, 5H), 7.45 (m, 4H), 7.83 (m, 2H),
1H NMR: (300 MHz, CDCl3) d [ppm] ꢀ0.23 (s, 3H, Si-CH3), ꢀ0.07
(s, 3H, Si-CH3), 0.70 (s, 9H, Si-C(CH3)3), 1.68 (s, 3H, CO-CH3),
3.62–3.65 (m, 1H), 3.70 (s, 3H, CO-O-CH3), 3.74–4.30 (m, 8H),
4.81 (d, J = 3.6 Hz, 1H), 5.09–5.14 (m, 2H), 5.23–5.31 (m, 2H),
5.47 (t, J = 9.1 Hz, 1H), 5.58 (s, 1H), 5.63–5.78 (m, 1H, CH@CH2),
7.32–7.53 (m, 11H), 7.87–7.90 (m, 4H).
13C NMR: (100 MHz, CDCl3) d [ppm] ꢀ4.6, –4.1, 17.9, 23.3, 25.7,
51.8, 52.9, 61.6 (dd, J = 48, 48 Hz), 62.8, 63.4, 68.4–69.8 (m),
72.0–73.8 (m), 75.2–78.2 (m), 79.3, 97.5, 100.0 (dd, J = 64,
151 Hz), 101.6, 104.9, 114.5, 118.0, 118.3, 126.1, 128.4, 128.4,
129.3, 129.5, 129.6, 129.8, 129.9, 130.0, 133.1, 133.2, 133.3,
133.5, 133.4, 137.1, 165.6, 166.0, 169.9, 170.3.
13C NMR: (100 MHz, CDCl3) d [ppm] ꢀ5.1 (Si-CH3), ꢀ4.3 (Si-
CH3), 17.9 (Si-C(CH3)3), 22.9, 25.5, 52.4, 52.6, 63.0, 68.7, 69.0,
70.7, 73.9, 75.3, 76.7, 76.9, 81.7, 97.2 (C-1), 100.9, 101.4,
117.8, 126.1, 126.2, 128.4, 128.5, 128.7, 129.0, 129.19, 129.22,
129.6, 129.8, 129.9, 133.3, 133.4, 133.6, 137.3, 165.3, 165.8,
168.2, 170.0.
HR-MS: (ESI positive, MeOH) Calcd for [C3813C6H55NO13SiNa]+:
HR-MS: (ESI positive, MeOH) Calcd for [C45H55NO14SiNa]+:
[M+Na]+ 884.32895. Found: 884.32832.
[M+Na]+
862.35381.
Found:
862.35324,
Calcd
for
[C3813C6H55NO13SiK]+: [M+K]+ 878.31810. Found: 878.32545
IR: (KBr)
1092, 1029, 841, 711.
m
max = 3438 cmꢀ1, 2953, 2929, 2858, 1734, 1271, 1112,
Optical rotation:
CHCl3).
½
a 2D3
ꢁ
ðdeg ꢂ cm3 ꢂ gꢀ1 ꢂ dmꢀ1
Þ
+ 90.4 (c 1.0,
4.1.4. Prop-2-en-1-yl 2-(acetylamino)-4,6-O-benzylidene-2-
deoxy-3-O-{2,3-di-O-benzoyl-4-O-[tert-butyl(dimethyl)silyl]-6-
Melting point: 193–194 °C.
methyl-b-
1-yl 2-(acetylamino)-4,6-O-benzylidene-2-deoxy-3-O-{2,3-di-O-
benzoyl-4-O-[tert-butyl(dimethyl)silyl]-6-methyl-b-
-(13C6)
D-glucopyranosyl}-a-D-glucopyranoside and prop-2-en-
4.1.4.2. 13C-labeled.
D
1H NMR: (300 MHz, CDCl3) d [ppm] ꢀ0.23 (s, 3H), ꢀ0.07 (s, 3H),
0.70 (s, 9H), 1.68 (s, 3H), 3.60–3.67 (m, 1H), 3.70 (d, J = 3.6 Hz,
3H), 3.74–4.30 (m, 8H), 4.81 (d, J = 3.6 Hz, 1H), 4.84–5.38 (m,
6H), 5.37 (d, J = 7.5 Hz, 1H), 5.58 (s, 1H), 5.65–5.78 (m, 1H),
7.32–7.53 (m, 11H), 7.87–7.89 (m, 4H).
glucopyranosyl}-
a-D-glucopyranoside 14
OMe
Ph
O
O
O
O
O
13C NMR: (100 MHz, CDCl3) d [ppm] ꢀ5.1, –4.3, 17.9, 22.9, 25.5,
52.4, 52.6, 63.0, 68.7, 68.9, 70.7 (m), 73.9 (m), 75.3 (m), 76.9
(m), 81.7, 97.2, 100.9 (dt, J = 46.8, 6.0 Hz), 101.4, 117.8, 126.1,
TBSO
O
BzO
NH
OBz
O
O

738
S. Rigol et al. / Bioorg. Med. Chem. 21 (2013) 733–741
128.4, 128.5, 128.7, 129.0, 129.18, 129.22, 129.6, 129.8, 129.9,
13C NMR: (100 MHz, CDCl3) d [ppm] 22.9 (CO-CH3), 52.2 (C-2),
52.9 (CO-OCH3), 62.9 (C-5), 68.7 (-O-CH2-), 69.1 (C-6), 70.0–
70.8 (m, C-40), 72.4–73.3 (m, C-20), 74.0–74.7 (m, C-50), 74.7–
75.5 (m, C-30), 76.9 (C-3), 81.8 (C-4), 97.2 (C-1), 100.6–101.8
(m, C-10), 101.8 (Ph-CH), 117.9 (CH@CH2), 126.0 (2 ꢃ Car),
128.48 (2 ꢃ Car), 128.51 (2 ꢃ Car), 128.8 (2 ꢃ Car), 129.17 (Cq),
129.18 (Cq), 129.4 (Car), 129.9 (2 ꢃ Car), 130.0 (2 ꢃ Car), 133.4
(CH@CH2), 133.5 (Car), 133.7 (Car), 137.3 (Cq), 165.1 (C = O),
166.5 (C = O), 168.8–170.0 (m, C-60), 170.0 (-NH-CO-).
133.3, 133.4, 133.6, 137.3, 168.2 (m), 170.0.
HR-MS: (ESI positive, MeOH) Calcd for [C3913C6H55NO14SiNa]+:
[M+Na]+ 890.34873. Found: 890.34822.
4.1.5. Prop-2-en-1-yl 2-(acetylamino)-4,6-O-benzylidene-2-
deoxy-3-O-(2,3-di-O-benzoyl-6-methyl-b- -glucopyranosyl}-
-glucopyranoside and prop-2-en-1-yl 2-(acetylamino)-4,6-O-
benzylidene-2-deoxy-3-O-(2,3-di-O-benzoyl-6-methyl-b-
13C6)glucopyranosyl}-
-glucopyranoside 5
D
a-
D
HR-MS: (ESI positive, MeOH) Calcd for [C3313C6H41NO14Na]+:
[M+Na]+ 776.26211. Found: 776.26107.
D-
(
a-D
OMe
Ph
O
O
4.1.6. Prop-2-en-1-yl 2,3-di-O-benzoyl-4-O-[tert-
O
O
O
O
HO
butyl(dimethyl)silyl]-6-methyl-b-
4,6-O-benzylidene-2-deoxy-2-{[(2,2,2-trichloroethoxy)
carbonyl]-amino}-b- -glucopyranosyl-(1?4)-2,3-di-O-benzoyl-
6-methyl-b- -glucopyranuronosyl-(1?3)-2-(acetylamino)-4,6-
O-benzylidene-2-deoxy- -glucopyranoside and prop-2-en-1-
yl 2,3-di-O-benzoyl-4-O-[tert-butyl(dimethyl)silyl]-6-methyl-b-
-glucopyranuronosyl-(1?3)-4,6-O-benzylidene-2-deoxy-2-
{[(2,2,2-trichloroethoxy)carbonyl]-amino}-b- -glucopyranosyl-
(1?4)-2,3-di-O-benzoyl-6-methyl-b-
-(13C6)
glucopyranuronosyl-(1?3)-2-(acetylamino)-4,6-O-
D-glucopyranuronosyl-(1?3)-
BzO
NH
BzO
O
D
D
O
a-D
To a solution of disaccharide 14 (2.16 g, 2.51 mmol, 1 equiv) in THF
(100 ml) was added a 1 m solution of TBAF in THF (2.51 ml, 0.656 g,
2.51 mmol, 1 equiv) at rt. Then the reaction was stirred at rt for 2 h.
Removal of solvent and purification by column chromatography
furnished disaccharide 5 (0.656 g, 0.878 mmol, 35%) as white solid.
D
D
D
benzylidene-2-deoxy-
a
-D
-glucopyranoside 6
4.1.5.1. Non-labeled.
OMe
Ph
OMe
Ph
O
O
O
O
O
Rf: 0.30 (n-hexane/ethyl acetate = 2:3 v/v).
O
O
O
O
O
TBSO
O
O
1H NMR: (400 MHz, CDCl3) d [ppm] 1.66 (s, 3H, CO-CH3), 3.41
(br, 1H, -OH), 3.76 (s, 3H, CO-OCH3), 3.78–3.83 (m, 1H, H-5),
3.83–3.91 (m, 3H, -O-CHaHb-, H-4, H-6a), 4.02 (dd, J = 10.1,
8.6 Hz, 1H, H-3), 4.05–4.15 (m, 2H, -O-CHaHb-, H-40), 4.21–
4.30 (m, 2H, H-2, H-6b), 4.82 (d, J = 3.6 Hz, 1H, H-1), 5.04 (d,
J = 7.6 Hz, 1H, H-10),5.11–5.14 (m, 1H, CH@CHaHb), 5.17 (ddd,
J = 7.9, 1.5, 1.4 Hz, 1H, CH@CHaHb), 5.23 (d, J = 8.6 Hz, 1H, NH),
5.32 (dd, J = 9.6, 7.7 Hz, 1H, H-20), 5.41 (dd, J = 9.4, 9.4 Hz, 1H,
H-30), 5.60 (s, 1H, Ph-CH), 5.75 (dddd, J = 16.7, 10.5, 6.0,
5.5 Hz, 1H, CH2-CH@CH2), 7.31–7.45 (m, 7H, Har), 7.45–7.51
(m, 3H, Har), 7.51–7.59 (m, 1H, Har), 7.90–8.00 (m, 4H, Har).
13C NMR: (100 MHz, CDCl3) d [ppm] 22.9 (CO-CH3), 52.3 (C-2),
52.9 (CO-OCH3), 62.9 (C-5), 68.7 (-O-CH2-), 69.1 (C-6), 70.4 (C-
40), 72.9 (C-20), 74.6 (C-50), 75.1 (C-30), 76.9 (C-3), 81.8 (C-4),
97.2 (C-1), 100.9 (C-10), 101.8 (Ph-CH), 117.9 (CH@CH2), 126.0
(2 ꢃ Car), 128.48 (2 ꢃ Car), 128.51 (2 ꢃ Car), 128.8 (2 ꢃ Car),
129.17 (Cq), 129.18 (Cq), 129.4 (Car), 129.9 (2 ꢃ Car), 130.0
(2 ꢃ Car), 133.4 (CH@CH2), 133.5 (Car), 133.7 (Car), 137.3 (Cq),
165.1 (c O), 166.5 (c O), 169.1 (C-60), 170.0 (-NH-CO-).
HR-MS: (ESI positive, MeOH) Calcd for [C39H41NO14Na]+:
BzO
O
BzO
NH
NH
BzO
BzO
O
O
Cl3C
O
O
Thioglycoside 4 (0.515 g, 0.485 mmol, 1.5 equiv) and alcohol 5
(0.242 g, 0.323 mmol, 1.0 equiv) were co-evaporated twice with tol-
uene (5 ml) and then dried at high vacuum for 16 h. This mixture
was dissolved in dichloromethane (10 ml) and transferred to a flask
containing activated powdered 4 Å molecular sieves (0.900 g). After
stirring at rt for 1 h NIS (0.116 g, 0.517 mmol, 1.6 equiv) and TfOH
(0.0114 ml, 0.0194 g, 0.129 mmol, 0.4 equiv) were added. Then it
was stirred for 16 h at rt before the reaction was quenched with tri-
ethylamine (0.050 ml). After removal of solvent and purification by
column chromatography tetrasaccharide 6 (0.321 g, 0.191 mmol,
59%) was obtained as white solid.
4.1.6.1. Non-labeled.
Rf: 0.35 (n-hexane/ethyl acetate = 1:1 v/v).
1H NMR: (400 MHz, CDCl3) d [ppm] ꢀ0.25 (s, 3H, Si-CH3), ꢀ0.09
(s, 3H, Si-CH3), 0.69 (s, 9H, Si-C(CH3)3), 1.71 (s, 3H, CO-CH3),
2.25–2.38 (m, 1H), 2.48–2.60 (m, 1H), 3.10–3.19 (m, 1H),
3.24–3.34 (m, 2H), 3.54 (br, 1H, NH), 3.62 (s, 3H, –O-CH3),
3.64 (s, 3H, –O-CH3), 3.69–3.93 (m, 8H), 3.93–4.09 (m, 2H),
4.16–4.30 (m, 5H), 4.45 (d, J = 11.7 Hz, 1H), 4.61 (br, 1H, NH),
4.67–4.91 (m, 3H, H-1), 5.02 (d, J = 7.2 Hz, 1H), 5.13 (d,
J = 10.3 Hz, 1H), 5.16 (s, 1H, Ph-CH), 5.22–5.47 (m, 4H), 5.56
(s, 1H, Ph-CH), 5.67–5.80 (m, 1H, CH@CH2), 7.28–7.43 (m,
15H, Har), 7.43–7.55 (m, 7H, Har), 7.81–7.99 (m, 8H, Har).
13C NMR: (100 MHz, CDCl3) d [ppm] ꢀ5.1 (Si-CH3), ꢀ4.3 (Si-
CH3), 17.8 (Si-C(CH3)3), 23.0 (CO-CH3), 25.5 (Si-C(CH3)3), 52.3,
52.5, 53.1, 58.3, 62.9, 66.0, 67.7, 68.7, 68.9, 70.8, 72.4, 72.5,
73.2, 74.57, 74.63, 75.3, 76.60, 76.64, 77.4, 79.4, 81.4, 95.6
(CCl3), 97.3 (C-1), 100.0, 100.8, 100.9, 101.1, 101.3, 118.0
(CH@CH2), 126.0, 126.2, 128.3, 128.4, 128.47, 128.52, 128.6,
128.7, 128.9, 129.1, 129.2, 129.3, 129.7, 129.82, 129.89,
129.92, 130.0, 133.2, 133.33, 133.37, 133.41, 133.7, 137.2,
[M+Na]+
[C78H82N2O28Na]+: [2 M+Na]+ 1517.49518. Found: 1517.49246.
IR: (KBr)
max = 3434 cmꢀ1, 2917, 2868, 1731, 1383, 1279, 1122,
770.24193.
Found:
770.24193,
Calcd
for
m
1092, 1029, 712.
Optical rotation: ½a D22
ꢁ
ðdeg ꢂ cm3 ꢂ gꢀ1 ꢂ dmꢀ1Þ + 109.5 (c 1.0,
CHCl3).
Melting point: 137–139 °C.
4.1.5.2. 13C-labeled.
1H NMR: (400 MHz, CDCl3) d [ppm] 1.66 (s, 3H, CO-CH3), 3.29
(br, 1H, –OH), 3.56 (br, 0, 5H, 13CH) 3.76 (d, J = 3.8 Hz, 3H,
⁄
CO-OCH3), 3.79–4.11 (m, 6.5H) ⁄, 4.23–4.29 (m, 2H, H-2, H-
6b), 4.82 (d, J = 3.6 Hz, 1H, H-1), 5.13–5.25 (m 4H), 5.60 (s,
1H, Ph-CH), 5.75 (dddd, J = 16.7, 10.5, 6.0, 5.5 Hz, 1H, CH2–
CH@CH2), 7.31–7.45 (m, 7H, Har), 7.45–7.51 (m, 3H, Har),
7.51–7.59 (m, 1H, Har), 7.90–8.00 (m, 4H, Har). ⁄both multiplets
contain one half of a doublet representing one proton.

S. Rigol et al. / Bioorg. Med. Chem. 21 (2013) 733–741
739
137.3, 153.9 (COTroc), 165.1 (CO), 165.2 (CO), 165.6 (CO), 167.9
(CO), 168.3 (CO), 170.0 (CO), 177.4 (CO).
4.74 (d, J = 2.3 Hz, 1H, H-1), 5.03 (d, J = 7.9 Hz, 1H), 5.09–5.35
(m, 7H, Ph-CH), 5.35–5.43 (m, 1H), 5.55–5.64 (m, 2H, Ph-CH),
5.81–5.97 (m, 1H, CH@CH2), 7.27–7.41 (m, 14H), 7.41–7.61
(m, 8H), 7.78–7.94 (m, 8H).
HR-MS: (ESI positive, CHCl3/MeOH) Calcd for [C82H89Cl3N2O28-
SiNa]+: [M+Na]+ 1705.43289 found 1705.43255.
IR: (KBr)
m
max = 3433 cmꢀ1, 2957, 2927, 1732, 1384, 1279, 1178,
13C NMR: (75 MHz, CD3OD) d [ppm] 22.5 (CO-CH3), 52.9, 53.7,
54.5, 58.7, 64.4, 67.0, 68.9, 69.65, 69.73, 71.1, 73.58, 73.61,
73.64, 74.5, 75.3, 75.6, 76.5, 76.7, 77.1, 78.48, 78.50, 78.52,
78.6, 80.1, 81.2, 96.8 (CCl3), 98.2 (C-1), 101.5, 101.6, 102.0,
102.2, 103.5, 118.2 (CH@CH2), 127.3, 129.06, 129.14, 129.40,
129.42, 129.49, 129.6, 129.7, 130.61, 130.69, 130.78, 130.80,
130.9, 131.0, 131.1, 134.35, 134.37, 134.47, 134.54, 135.1,
138.8, 139.2, 156.1 (NH-CO-O–), 166.70 (CO), 166.73 (CO),
167.0 (CO), 167.3 (CO), 169.5 (CO), 170.1 (CO), 173.0 (CO).
1094, 1029, 713.
Optical rotation: ½a D23
ꢁ
ðdeg ꢂ cm3 ꢂ gꢀ1 ꢂ dmꢀ1Þ + 50.8 (c 1.0, CHCl3).
Melting point: 144–145 °C.
4.1.6.2. 13C-labeled.
1H NMR: (400 MHz, CDCl3) d [ppm] ꢀ0.26 (s, 3H, Si-CH3), ꢀ0.10
(s, 3H, Si-CH3), 0.69 (s, 9H, Si-C(CH3)3), 1.72 (s, 3H, CO-CH3⁄),
2.25–2.38 (m, 0.5H) ⁄, 2.48–2.57 (m, 1H), 3.10–3.19 (m, 1.5H)
,
HR-MS:
(ESI
positive,
EtOAc/MeOH)
Calcd
for
Found:
[C76H75Cl3N2O28Na]+:
1591.34759.
[M+Na]+
1591.34641.
3.24–3.34 (m, 2H), 3.54 (br, 1H, NH), 3.62 (s, 3H, –O-CH3),
3.64 (d, J = 3.8 Hz, 3H, –O-CH3), 3.69–4.30 (m, 13H), 4.45 (d,
J = 11.7 Hz, 1H), 4.67–4.91 (m, 3H), 5.13 (m, 1H), 5.16 (s, 1H,
Ph-CH), 5.22–5.47 (m, 4H), 5.56 (s, 1H, Ph-CH), 5.67–5.80 (m,
1H, CH@CH2), 7.28–7.43 (m, 15H, Har), 7.43–7.55 (m, 7H, Har),
7.81–7.99 (m, 8H, Har). ⁄both multiplets contain one half of a
doublet representing one proton.
IR: (KBr)
1093, 712.
Optical rotation:
CH3OH).
m
max = 3434 cmꢀ1, 2954, 2926, 1735, 1643, 1384, 1278,
½
a 2D3
ꢁ
ðdeg ꢂ cm3 ꢂ gꢀ1 ꢂ dmꢀ1
Þ
+ 6.9 (c 1.0,
Melting point: 182–184 °C.
13C NMR: (100 MHz, CDCl3) d [ppm] ꢀ5.1 (Si-CH3), ꢀ4.3 (Si-
CH3), 17.8 (Si-C(CH3)3), 23.0 (CO-CH3), 25.5 (Si-C(CH3)3), 52.3,
52.5, 53.1, 58.3, 62.9, 66.0, 67.7, 68.7, 68.9, 70.8, 72.1–73.7
(m, 13C), 74.0–75.1 (m, 13C), 76.5–77.4 (m, 13C), 81.8, 96.0
(CCl3), 97.3 (C-1), 100.3–101.7 (m, 13C), 118.0 (CH@CH2),
126.0, 126.1, 128.36, 128.48, 128.52, 128.6, 128.7, 128.9,
129.1, 129.2, 129.3, 129.7, 129.82, 129.89, 129.92, 130.0,
133.2, 133.37, 133.43, 133.47, 133.7, 137.2, 137.3, 153.8
(COTroc), 165.0 (CO), 165.2 (CO), 165.3 (CO), 166.6, 167.3–
168.8 (m, 13C), 169.0, 170.0.
4.1.8. Prop-2-en-1-yl 2,3-di-O-benzoyl-4-O-[tert-
butyl(dimethyl)silyl]-6-methyl-b- -glucopyranuronosyl-(1?3)-2-
(acetylamino)-4,6-O-benzylidene-2-deoxy-b- -glucopyranosyl-
(1?4)-2,3-di-O-benzoyl-6-methyl-b- -glucopyranuronosyl-
(1?3)-2-(acetylamino)-4,6-O-benzylidene-2-deoxy-
glucopyranoside and prop-2-en-1-yl 2,3-di-O-benzoyl-4-O-[tert-
butyl(dimethyl)silyl]-6-methyl-b- -glucopyranuronosyl-(1?3)-2-
(acetylamino)-4,6-O-benzylidene-2-deoxy-b- -glucopyranosyl-
(1?4)-2,3-di-O-benzoyl-6-methyl-b-
-(13C6)glucopyranuronosyl-
D
D
D
a-D-
D
D
HR-MS: (ESI positive, MeOH) Calcd for [C7613C6H89Cl3N2O28-
SiNa2]2+: [M+2Na]2+ 867.22167. Found: 867.22180.
D
(1?3)-2-(acetylamino)-4,6-O-benzylidene-2-deoxy-
a-D-
glucopyranoside 8
4.1.7. Prop-2-en-1-yl 2,3-di-O-benzoyl-6-methyl-b-
glucopyranuronosyl-(1?3)-4,6-O-benzylidene-2-deoxy-2-{[(2,2,2-
trichloroethoxy)carbonyl]amino}-b- -glucopyranosyl-(1?4)-2,3-
di-O-benzoyl-6-methyl-b- -glucopyranuronosyl-(1?3)-2-
(acetylamino)-4,6-O-benzylidene-2-deoxy-
D-
OMe
OMe
Ph
Ph
O
O
O
O
O
O
O
O
O
O
TBSO
O
O
O
BzO
D
BzO
NH
NH
BzO
BzO
O
D
O
a-D
-glucopyranoside 7
O
OMe
OMe
To
a
solution of Troc-containing tetrasaccharide
6
(195 mg,
Ph
Ph
O
O
O
O
O
O
O
O
O
O
0.116 mmol, 1 equiv) in acetic acid (3.00 ml) was added zinc pow-
der (300 mg) and the mixture was allowed to stir at rt for 3 h. After
excess zinc was filtered off and solvent removal under diminished
pressure the residue was dried at high vacuum for 1 h. The crude
product was dissolved in pyridine (6.00 ml) and acetic anhydride
(3.00 ml) was added dropwise to the reaction before it was allowed
to stir for 16 h at rt. Then it was quenched with sat. NaHCO3 solu-
tion, washed twice with sat. CuSO4 solution and finally with brine.
The organic phase was dried over MgSO4, all volatiles were removed
under reduced pressure and the crude product was purified by col-
umn chromatography to yield derivative 8 (77.3 mg, 0.0498 mmol,
43% over two steps) as white solid.
HO
O
O
O
BzO
BzO
NH
NH
O
BzO
BzO
O
Cl3C
O
O
To
0.0600 mmol, 1 equiv) in THF (404
Olah’s reagent (202 l, 184 mg) and pyridine (404
a
solution of fully protected tetrasaccharide
l) was subsequently added
l) before the
6 (101 mg,
l
l
l
reaction was stirred for 12 h at rt. Then it was diluted with dichlo-
romethane quenched by carful addition of sat. NaHCO3 solution,
washed twice with sat. cupric sulfate solution and brine. The organ-
ic layer was dried over MgSO4, concentrated and the crude product
was subjected to column chromatography for purification to obtain
tetrasaccharide 7 (73.5 mg, 0.0468 mmol, 78%) as white solid.
4.1.8.1. Non-labeled.
Rf: 0.36 (n-hexane/ethyl acetate = 3:7 v/v).
1H NMR: (400 MHz, CDCl3) d [ppm] ꢀ0.25 (s, 3H, Si-CH3), ꢀ0.11
(d, J = 6.6 Hz, 3H, Si-CH3), 0.68 (s, 9H,, Si-C(CH3)3), 1.68 (s, 3H,
CO-CH3), 2.35 (s, 3H, CO-CH3), 2.49 (t, J = 10.2 Hz, 1H), 3.14–
3.27 (m, 2H), 3.54 (s, 3H, CO-O-CH3), 3.56–3.63 (m, 2H), 3.65
(s, 3H, CO-O-CH3), 3.75–3.89 (m, 3H), 3.93–4.10 (m, 3H),
4.14–4.29 (m, 5H), 4.47–4.56 (m, 1H), 4.76–4.83 (m, 3H),
4.83–4.91 (m, 2H), 4.98–5.20 (m, 4H), 5.20–5.49 (m, 5H),
4.1.7.1. Non-labeled.
Rf: 0.25 (n-hexane/ethyl acetate = 1:1 v/v).
1H NMR: (300 MHz, CD3OD) d [ppm] 1.65 (s, 3H, CO-CH3), 2.15–
2.23 (m, 1H), 2.64 (t, J = 10.3 Hz, 1H), 3.08–3.19 (m, 1H), 3.40–
3.50 (m, 2H), 3.65 (s, 3H, CO-O-CH3), 3.67 (s, 3H, CO-O-CH3),
3.76–3.82 (m, 2H), 3.87–4.11(m, 9H), 4.11–4.20 (m, 2H), 4.26
(t, J = 9.0 Hz, 1H), 4.42 (d, J = 10.8 Hz, 2H), 4.46–4.55 (m, 2H),

740
S. Rigol et al. / Bioorg. Med. Chem. 21 (2013) 733–741
subjected to column chromatography for purification to obtain tetra-
saccharide 9 (26.1 mg, 0.0207 mmol, 46%) as white solid.
5.51–5.61 (m, 2H), 5.66–5.90 (m, 2H, CH@CH2), 7.11–7.58 (m,
22H), 7.80–7.99 (m, 8H).
13C NMR: (100 MHz, CDCl3) d [ppm] ꢀ5.1 (Si-CH3), ꢀ4.4 (Si-
CH3), 17.8 (Si-C(CH3)3), 22.9 (CO-CH3), 23.0 (CO-CH3), 25.5 (Si-
C(CH3)3), 52.2, 52.3, 53.0, 62.9, 65.6, 68.6, 70.8, 72.7, 72.8,
72.9, 74.9, 75.3, 76.35, 76.38, 76.7, 77.0, 77.4, 80.2, 81.6, 97.2,
98.8, 100.76, 100.80, 101.0, 101.4, 117.8 (CH@CH2), 125.4,
126.0, 126.12, 126.17, 128.3, 128.38, 128.49, 128.53, 128.6,
128.8, 129.0, 129.09, 129.12, 129.2, 129.3, 129.6, 129.8, 129.9,
133.2, 133.4, 133.46, 133.54, 133.7, 137.3, 165.06 (CO), 165.09
(CO), 165.3 (CO), 165.7 (CO), 167.3 (CO), 168.4 (CO), 170.0
(CO), 170.9 (CO).
4.1.9.1. Non-labeled.
Rf: 0.10 (ethyl acetate).
1H NMR: (400 MHz, CD3OD) d [ppm] 1.38 (s, 3H, CO-CH3), 1.48
(s, 3H, CO-CH3), 3.05–3.12 (m, 2H), 3.41–3.50 (m, 2H), 3.50–
3.62 (m, 3H), 3.64–3.72 (m, 2H), 3.85–3.72 (m, 8H, 2 ꢃ O-
CH3), 3.87–3.98 (m, 2H), 4.03 (t, J = 9.5 Hz, 1H), 4.08–4.23 (m,
2H), 4.29 (d, J = 7.8 Hz, 1H), 4.46 (t, J = 7.8 Hz, 1H), 4.50 (d,
J = 8.2 Hz, 1H), 4.71 (d, J = 3.5 Hz, 1H, H-1), 5.00 (d, J = 7.8 Hz,
1H), 5.07 (d, J = 7.5 Hz, 1H), 5.15 (ddd, J = 10.4, 2.8, 1.2 Hz, 1H,
CH@CHaHb), 5.22–5.31 (m, 3H, CH@CHaHb), 5.55 (t, J = 9.4 Hz,
1H), 5.60 (t, J = 7.7 Hz, 1H), 5.88 (dddd, J = 15.6, 10.4, 6.2,
5.2 Hz, 1H, CH@CH2), 7.31–7.46 (m, 8H, Har), 7.48–7.62 (m,
4H, Har), 7.82–7.96 (m, 8H, Har).
HR-MS: (ESI positive, CHCl3/MeOH) Calcd for [C14H18O6Na]+:
[M+Na]+ 1573.53924. Found: 1573.53976.
IR: (KBr) m
max = 3434 cmꢀ1, 2953, 2929, 2859, 1734, 1697, 1384,
1273, 1093, 1029, 712.
Optical rotation: ½a D23
ꢁ
ðdeg ꢂ cm3 ꢂ gꢀ1 ꢂ dmꢀ1Þ + 67.3 (c 1.0, CHCl3).
13C NMR: (100 MHz, CD3OD) d [ppm] 22.2 (CO–CH3), 23.0 (CO–
CH3), 53.2, 53.5, 54.0, 56.1, 62.6, 62.6, 69.2, 70.4, 70.5, 71.0, 73.5,
73.6, 74.4, 75.8, 76.2, 76.4, 76.6, 77.5, 82.6, 84.5, 97.4 (C-1),
101.9, 102.1, 102.2, 117.9 (CH@CH2), 129.45, 129.54, 130.60,
130.64, 130.67, 130.73, 130.79, 130.9, 131.00, 131.01, 134.42,
134.48, 134.54, 134.59, 135.3, 166.52 (CO), 166.566 (CO),
166.573 (CO), 166.8 (CO), 167.3 (CO), 169.6 (CO), 170.1 (CO),
173.1 (CO), some signals of aromatic carbons are overlapping.
HR-MS: (ESI positive, CDCl3/MeOH) Calcd for [C61H69N2O27]+:
[M+Na]+ 1283.39017. Found: 1283.39095.
Melting point: 172–174 °C.
4.1.8.2. 13C-labeled.
1H NMR: (300 MHz, CDCl3) d [ppm] ꢀ0.25 (s, 3H, Si-CH3), ꢀ0.12
(s, 3H, Si-CH3), 0.68 (s, 9H,, Si-C(CH3)3), 1.40 (s, 3H, CO-CH3),
1.68 (s, 3H, CO-CH3), 2.49 (t, J = 10.2 Hz, 1H),2.69–2.80 (m,
1H) 3.14–3.31 (m, 2H), 3.54 (s, 3H, CO-O-CH3), 3.56–3.63 (m,
2H), 3.65 (d, J = 3.8 Hz, 3H, CO-O-CH3), 3.75–4.29 (m, 11H),
4.49–4.56 (m, 1H), 4.76–4.83 (m, 3H), 4.83–4.91 (m, 2H),
4.98–5.53 (m, 9H), 5.54 (s, 1H), 5.65–5.78 (dddd, 1H, CH@CH2),
7.11–7.58 (m, 22H), 7.80–7.99 (m, 8H).
IR: (KBr) m
max = 3454 cmꢀ1, 2954, 2846, 1730, 1651, 1384, 821.
Optical rotation:
MeOH).
½
a 2D3
ꢁ
ðdeg ꢂ cm3 ꢂ gꢀ1 ꢂ dmꢀ1
Þ
+ 23.2 (c 1.0,
13C NMR: (100 MHz, CDCl3) d [ppm] ꢀ5.1 (Si-CH3), ꢀ4.4 (Si-
CH3), 17.8 (Si-C(CH3)3), 22.9 (CO-CH3), 23.0 (CO-CH3), 25.5 (Si-
C(CH3)3), 52.2, 52.3, 53.0, 62.9, 65.6, 68.6, 70.8, 72.5–77.6 (m,
13C), 80.2, 81.6, 97.2, 98.8, 100.5–101.2 (m, 13C), 101.4, 117.8
(CH@CH2), 125.4, 126.0, 126.12, 126.17, 128.3, 128.38, 128.49,
128.53, 128.6, 128.8, 129.0, 129.09, 129.12, 129.2, 129.3,
129.6, 129.8, 129.9, 133.2, 133.4, 133.46, 133.54, 133.7, 137.3,
165.06 (CO), 165.09 (CO), 165.3 (CO), 165.7 (CO), 166.8–167.7
(m, 13CO), 168.4 (CO), 170.0 (CO), 170.9 (CO).
Melting point: 205–207 °C (decomp).
4.1.9.2. 13C-labeled.
1H NMR: (400 MHz, CD3OD) d [ppm] 1.38 (s, 3H, CO-CH3), 1.48
(s, 3H, CO-CH3), 3.07–3.09 (m, 2H), 3.45–3.70 (m, 7H), 3.42–
3.79 (m, 8H, 2 ꢃ O-CH3), 3.87–4.29 (m, 6H), 4.5–4.71 (m, 3H),
5.00 (d, J = 7.8 Hz, 1H), 5.07–5.60 (m, 7H), 5.88 (dddd, J = 15.6,
10.4, 6.2, 5.2 Hz, 1H, CH@CH2), 7.31–7.46 (m, 8H, Har), 7.48–
7.62 (m, 4H, Har), 7.82–7.96 (m, 8H, Har).
HR-MS: (ESI positive, CDCl3/MeOH) Calcd for [C7513C6H90N2O27-
SiNa]+: [M+Na]+ 1579.55937. Found: 1579.65094
13C NMR: (100 MHz, CD3OD) d [ppm] 22.2 (CO-CH3), 23.0 (CO-
CH3), 53.2, 53.5, 54.0, 56.1, 62.6, 62.6, 69.2, 70.4, 70.5, 71.0,
73.1–76.6 (m, 13C), 77.5, 82.6, 84.5, 97.4 (C-1), 101.6–102.1(m,
13C), 102.2, 117.9 (CH@CH2), 129.45, 129.54, 130.60, 130.64,
130.67, 130.73, 130.79, 130.9, 131.00, 131.01, 134.42, 134.48,
134.54, 134.59, 135.3, 166.52 (CO), 166.566 (CO), 166.573 (CO),
166.8 (CO), 167.3 (CO), 169.3–169.9 (CO), 170.1 (CO), 173.1 (CO).
4.1.9. Prop-2-en-1-yl 2,3-di-O-benzoyl-6-methyl-b-
D-
glucopyranuronosyl-(1?3)-2-(acetylamino)-2-deoxy-b-
D-
glucopyranosyl-(1?4)-2,3-di-O-benzoyl-6-methyl-b-
D
-
glucopyranuronosyl-(1?3)-2-(acetylamino)-2-deoxy-
a-D-
HR-MS:
(ESI
positive,
EtOAc/MeOH)
Calcd
for
glucopyranoside and prop-2-en-1-yl 2,3-di-O-benzoyl-6-methyl-
b- -glucopyranuronosyl-(1?3)-2-(acetylamino)-2-deoxy-b-
glucopyranosyl-(1?4)-2,3-di-O-benzoyl-6-methyl-b-
-(13C6)
[C5513C6H68N2O27Na]+: [M+Na]+ 1289.41030 found 1289.41046.
D
D-
D
glucopyranuronosyl-(1?3)-2-(acetylamino)-2-deoxy-a-D-
glucopyranoside 9
4.1.10. Prop-2-en-1-yl b-
(acetylamino)-2-deoxy-b-
glucopyranuronosyl-(1?3)-2-(acetylamino)-2-deoxy-
glucopyranoside and prop-2-en-1-yl b- -glucopyranuronosyl-
(1?3)-2-(acetylamino)-2-deoxy-b-
-(13C6)glucopyranosyl-
(1?4)-b- -glucopyranuronosyl-(1?3)-2-(acetylamino)-2-
D
-glucopyranuronosyl-(1?3)-2-
D
-glucopyranosyl-(1?4)-b-
D-
a-D-
OH
OH
OMe
OMe
O
D
O
O
NH
O
O
O
HO
O
O
HO
O
HO
D
O
BzO
BzO
NH
D
OBz
OBz
O
deoxy-
a
-D
-glucopyranoside 10
O
To a solution of tetrasaccharide 8 (69.9 mg, 0.0451 mmol, 1 equiv) in
THF (2.10 ml) was added Olah’s reagent (1.05 ml, 953 mg) before the
reaction was stirred for 4 d at rt. Then it was diluted with dichloro-
methane quenched by carful addition of sat. NaHCO3 solution,
washed twice with sat. cupric sulfate solution and brine. The organic
layer was dried over MgSO4, concentrated and the crude product was
OH
O
OH
OH
OH
O
O
O
O
HO
O
O
HO
O
HO
O
HO
HO
NH
O
NH
OH
OH
O
O

S. Rigol et al. / Bioorg. Med. Chem. 21 (2013) 733–741
741
77.0 (dd, J = 37, 37 Hz), 80.8, 83.0, 96.2, 100.5 (d, J = 48 Hz),
101.5, 103.0, 105.0, 117.9, 133.8, 174.6–177.2 (m).
To a solution of tetrasaccharide 9 (10.6 mg, 0.00837 mmol, 1 equiv)
in methanol (1.00 ml) was added a 0.05 m solution of sodium meth-
oxide in methanol (1.00 ml) and the reaction mixture was stirred
for 14 h at rt. Then Amberlite 120 (H+ form) was added to neutralize
the solution. After removal of the ion exchange resin by filtration all
volatiles were removed under reduced pressure and the crude prod-
uct was partly purified by passage through a bed of RP silica gel
(eluent: water). After lyophilization the residue was dissolved in a
0.1 m NaOH solution (0.50 ml) before the reaction is allowed to stir
for 2 h at rt. Afterwards Dowex 50 (H+ form) was added to neutral-
ize the solution and the solvent was removed by lyophilization. Fi-
nally, purification was achieved by HPLC (column: Nucleosil 120-7
C4, eluent: water) to furnish tetrasaccharide 10 (4.30 mg,
0.00527 mmol, 63%) as white solid.
HR-MS: (ESI positive, D2O/MeOH) Calcd for [C2513C6H49N2O23]+:
[M+H]+
823.29219.
Found:
823.29191,
Calcd
for
[C2513C6H48N2O23Na]+: [M+Na]+ 845.27414. Found: 845.27396
Acknowledgments
This work was supported by the Deutsche Forschungsgemein-
schaft (Transregio 67, project Z3). We thank Dr. Lothar Hennig
for recording NMR spectra and for his help in interpreting the 2D
NMR spectra.
Supplementary data
4.1.10.1. Non-labeled.
Supplementary data (copies of 1H and 13C NMR spectra of all
new compounds) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmc.2012.11.025.
Rf: 0.30 (dichloromethane/methanol/acetic acid = 3:7:1 v/v/v).
1H NMR: (400 MHz, D2O) d [ppm] 1.90 (s, 3H, CO-CH3), 1.92 (s,
3H, CO-CH3), 3.18–3.28 (m, 2H), 3.36–3.49 (m, 5H), 3.59–3.77
(m, 8H), 3.78–3.83 (m, 2H), 3.83–3.85 (m, 1H), 3.91 (dd,
J = 12.9, 6.2 Hz, 1H), 3.97 (dd, J = 10.5, 3.5 Hz, 1H), 4.06–4.13
(m, 1H), 4.36 (t, J = 8.1 Hz, 2H), 4.45 (d, J = 8.4 Hz, 1H), 4.54 (d,
J = 1.6 Hz, 1H, H-1), 4.75–4.78 (m, 2H), 5.10–5.18 (m, 1H,
CH@CHaHb), 5.19–5.27 (m, 1H, CH@CHaHb), 5.85 (dddd,
J = 10.8, 10.1, 5.6, 2.7 Hz, 1H, CH@CH2).
References and notes
1. Kim, S.-H.; Turnbull, J.; Guimond, S. J. Endocrinol. 2011, 209, 139.
2. Gama, C. I.; Hsieh-Wilson, L. C. Curr. Opin. Chem. Biol. 2005, 9, 609.
3. Fraser, J. R. E.; Laurent, T. C.; Laurent, U. B. G. J. Intern. Med. 1997, 242, 27.
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R. R.; Ghatak, S. FEBS J. 2011, 278, 1429.
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2009, 61, 198.
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Jackson, D. G. Nat. Struct. Mol. Biol. 2007, 14, 234.
8. Flowers, H. M.; Jeanloz, R. W. J. Am. Chem. Soc. 1962, 84, 3030.
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84, 3029.
10. Slaghek, T. L.; Nakahara, Y.; Ogawa, T. Tetrahedron Lett. 1992, 33, 4971.
11. Slaghek, T. L.; Hyppönen, T. K.; Ogawa, T. Tetrahedron Lett. 1993, 34, 7939.
12. Huang, L.; Huang, X. Chem. Eur. J. 2007, 13, 529.
13. Dinkelaar, J.; Codée, J. D. C.; van den Bos, L. J.; Overkleeft, H. S.; van der Marel,
G. A. J. Org. Chem. 2007, 72, 5737.
14. Walvoort, M. T. C.; Volbeda, A. G.; Reintjens, N. R.; van den Elst, H.; Plante, O. J.;
Overkleeft, H. S.; van der Marel, G. A.; Codée, J. D. C. Org. Lett. 2012, 14, 3776.
15. Tully, S. E.; Mabon, R.; Gama, C. I.; Tsai, S. M.; Liu, X.; Hsieh-Wilson, L. C. J. Am.
Chem. Soc. 2004, 126, 7736.
13C NMR: (175 MHz, D2O) d [ppm] 22.24, 22.25, 52.1, 54.5, 60.5,
61.0, 68.4, 68.5, 68.6, 71.6, 71.7, 71.9, 72.7, 75.3, 75.4, 76.8, 79.5,
80.6, 82.8, 96.0, 100.3, 101.3, 102.8, 117.7, 133.5, 173.8, 174.8,
both amide carbons are not visible and some carbohydrate
backbone carbons are overlapping.
2ꢀ
HR-MS: (ESI negative, MeOH) Calcd for [C31H46N2O23
]
:
[Mꢀ2H]2ꢀ 407.12512. Found: 407.12503.
IR: (KBr)
712.
m
max = 3442 cmꢀ1, 2958, 2925, 2853, 1635, 1385, 1056,
Optical rotation: ½a D25
ꢁ
ðdeg ꢂ cm3 ꢂ gꢀ1 ꢂ dmꢀ1Þ + 29.5 (c 0.14,
H2O).
Melting point: 288–290 °C (decomp.).
16. Chao, C.-S.; Li, C.-W.; Chen, M.-C.; Chang, S.-S.; Mong, K.-K. T. Chem. Eur. J. 2009,
15, 10972.
4.1.10.2. 13C-labeled.
17. Geng, Y.; Zhang, L. H.; Ye, X. S. Tetrahedron 2008, 64, 4949.
18. Izumi, M.; Fukase, K.; Kusumoto, A. Biosci. Biotechnol. Biochem. 2002, 66, 211.
19. Schulz, M.; Kunz, H. Tetrahedron: Asymmetry 1993, 4, 1205.
20. Vesely, J.; Rydner, L.; Oscarson, S. Carbohydr. Res. 2008, 343, 2200.
21. Pichert, A.; Samsonov, S. A.; Theisgen, S.; Thomas, L.; Baumann, L.; Schiller, J.;
Beck-Sickinger, A. G.; Huster, D.; Pisabarro, M. T. Glycobiology 2012, 22, 134.
1H NMR: (400 MHz, D2O) d [ppm] 2.08 (s, 3H), 2.11 (s, 3H),
3.23–3.66 (m, 6H), 3.68–4.13 (m, 10H), 4.24 (dd, J = 13.1,
5.1 Hz, 1H), 4.35 (d, J = 6.2 Hz, 1H), 4.51 (d, J = 7.6 Hz, 1H),
5.66–5.39 (m, 2H), 5.98 (dddd, J = 17.3, 10.6, 5.9, 5.3 Hz, 1H).
13C NMR: (100 MHz, D2O) d [ppm] 22.5, 52.3, 54.8, 60.8, 61.2,
68.6, 68.7, 68.8, 72.0 (dd, J = 38, 38 Hz), 72.9, 74.9–76.5 (m),