Tethered derivatives of d-glucose and pentacyclic triterpenes for homo/heterobivalent inhibition of glycogen phosphorylase

Article abstract of DOI:10.1039/b9nj00602h

Propargyl esters of the C-28 carboxylic acids of pentacyclic triterpenes (oleanolic, ursolic, and maslinic acids) were coupled with 2,3,4,6-tetra-O- acetyl-β-d-glucopyranosyl azide as well as N-(ω-azido-[C-2, C-6, and C-11]alkanoyl)-β-d-glucopyranosylamines under conditions of copper(i)-catalyzed azide-alkyne cycloaddition (CuAAC) to give tethered d-glucose-triterpene heteroconjugates. The O-acetyl protecting groups were removed by base-catalyzed hydrolysis. N-(ω-Azido-[C-2, C-6, C-11, and C-16]alkanoyl)-β-d-glucopyranosylamines were also tethered by 1,7-octadiyne under CuAAC conditions to furnish d-glucose homoconjugates. O-Deacetylation was carried out by the Zemplen protocol. The new compounds were assayed against rabbit muscle glycogen phosphorylase (RMGP) a or b enzymes. Some of the heteroconjugates inhibited the enzyme in the low micromolar range (IC ₅₀ values 40-70 μM), while the homoconjugates proved inefficient as inhibitors.

Full text of DOI:10.1039/b9nj00602h

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Tethered derivatives of D-glucose and pentacyclic triterpenes for
homo/heterobivalent inhibition of glycogen phosphorylasew
a
b
a
c
c
´
´
Keguang Cheng, Jun Liu, Hongbin Sun,* Eva Bokor, Katalin Czifrak,
Balint Konya, Marietta Toth, Tibor Docsa, Pal Gergely and Laszlo Somsak*
c
c
d
e
c
´
´
´
´
´
´
´
Received (in Montpellier, France) 27th October 2009, Accepted 25th January 2010
First published as an Advance Article on the web 9th March 2010
DOI: 10.1039/b9nj00602h
Propargyl esters of the C-28 carboxylic acids of pentacyclic triterpenes (oleanolic, ursolic, and
maslinic acids) were coupled with 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl azide as well as
N-(o-azido-[C-2, C-6, and C-11]alkanoyl)-b-D-glucopyranosylamines under conditions of
copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) to give tethered D-glucose–triterpene
heteroconjugates. The O-acetyl protecting groups were removed by base-catalyzed hydrolysis.
N-(o-Azido-[C-2, C-6, C-11, and C-16]alkanoyl)-b-D-glucopyranosylamines were also tethered by
1,7-octadiyne under CuAAC conditions to furnish D-glucose homoconjugates. O-Deacetylation
was carried out by the Zemplen protocol. The new compounds were assayed against rabbit
´
muscle glycogen phosphorylase (RMGP) a or b enzymes. Some of the heteroconjugates inhibited
the enzyme in the low micromolar range (IC50 values 40–70 mM), while the homoconjugates
proved inefficient as inhibitors.
and the new allosteric sites,6,9 as well as the recently discovered
benzimidazole site.10
Introduction
Type 2 diabetes mellitus has become a widespread disease
afflicting a very large proportion of the population all over the
world.1–3 The diseased state is associated with disorders in
glucose metabolism by the liver and periphery resulting in
elevated blood glucose levels which, in turn, are responsible
for fatal long-term complications.1,4 An ideal anti-diabetic
agent should be capable of lowering blood glucose in both
fed and fasted states. Control of the hepatic glycogen metabolism
is one of the key events through which insulin maintains blood
glucose homeostasis. Among other means for influencing
glucose production in the liver, inhibition of glycogen
phosphorylase (GP), the rate-limiting enzyme of glycogen
degradation, has been regarded as a promising therapeutic
approach to the treatment of type 2 diabetes.5,6 Some GP
inhibitors have shown efficacy in lowering blood glucose in
animal models and clinical trials.7,8 In the liver and muscle
isoforms of GP enzymes, six binding sites have been identified
by X-ray crystallographic studies of enzyme–inhibitor complexes:
the catalytic, the inhibitor, the allosteric, the glycogen storage,
Among the large variety of compounds tested as GP
inhibitors, the most populated class is that of D-glucose
derivatives,11,12 which bind primarily to the catalytic site of
the enzyme, as proven by several X-ray crystallographic
investigations.9 These glucose analogue inhibitors of GP
are characterized by maintaining an intact hexopyranoid
sugar ring with the full OH substitution pattern of D-gluco
configuration, thus resembling the non-reducing end of the
natural substrate glycogen. The modifications are located at
the anomeric centre as spirocycles, as well as b-NHCOR,
b-NHCONHCOR, and b-C-heterocyclic substituents, just to
mention the most efficient ones.5,6
Pentacyclic triterpenes like 1–3 and related compounds
have been reported to represent a new class of glycogen
phosphorylase inhibitors.13–15 X-Ray crystallographic studies
revealed the molecular basis of their inhibitory effect, demon-
strating that pentacyclic triterpenes such as asiatic and maslinic
acids bind to GP at the allosteric site.16 Oleanolic acid (1, OA),
ursolic acid (2, UA) and maslinic acid (3, MA) have recently
attracted much attention due to their broad biological
activities such as protection of the liver against toxic injury,
anti-inflammation, anti-HIV, antitumor, antioxidation, anti-
hyperglycemia and cardiovascular activities.17
a Center for Drug Discovery, College of Pharmacy, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China
b Jiangsu Center for Drug Screening, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, China
Inhibitors having the potential to bind to more than one site
of an enzyme may be significantly more efficient than those
with a single binding group (for some tentatively selected
examples of bi- or trivalent enzyme inhibitors see
ref. 18–23). This principle is well known in the interactions
of multivalent carbohydrate derivatives with various proteins,
and is frequently called the glycoside cluster effect in that
field.24 Trivalent glucose analogues have very recently been
tested for GP inhibition to show a slightly better effect than
that of derivatives with a single sugar unit.25 Homobivalent
c Department of Organic Chemistry, University of Debrecen, POB 20,
H-4010 Debrecen, Hungary
d Cell Biology and Signaling Research Group of the Hungarian
Academy of Sciences at the Department of Medical Chemistry,
Medical and Health Science Centre, University of Debrecen,
´
Egyetem ter 1, H-4032 Debrecen, Hungary
e Department of Medical Chemistry, Medical and Health Science
´
Centre, University of Debrecen, Egyetem ter 1, H-4032 Debrecen,
Hungary
w Electronic supplementary information (ESI) available: Copies of
1H NMR and 13C NMR spectra. See DOI: 10.1039/b9nj00602h
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indolcarboxamide26 as well as cinnamic acid27,28 derivatives
proved very efficient inhibitors of GP.
synthetic step furnished the corresponding product in very
good yield.
With these preliminaries in mind, we envisaged conjugation
of triterpenes and D-glucose in such a way that both could
bind to the site to which they bind on their own, thus
providing the first potentially heterobivalent inhibitors of
GP. The recently reported triterpene–glucose conjugates were
not capable of this because the sugar parts were attached to
the triterpene via the C-6 position.29 Furthermore, some new
bivalent glucose derivatives are also reported.
To perform the CuAAC, an alkyne 4–6 and an azide 7 or
12–14 each were dissolved in CH2Cl2–H2O, followed by the
addition of a catalytic amount of sodium L-ascorbate and
CuSO4ꢁ5H2O. The ‘click reactions’ proceeded very well at
room temperature to afford b-D-glucopyranosyl-1,2,3-triazoles
16–18 (Scheme 3) and the tethered compounds 22–30
(Scheme 4) in good to excellent yields. The O-acetyl groups
were cleaved with 4 N NaOH/MeOH to give the corresponding
deprotected compounds 19–21 (Scheme 3) and 31–37,
respectively (Scheme 4). During deprotection of 28 the desired
compound was not obtained; instead compound 38 could be
isolated as a result of cleavage of the glucosylamide bond.
For bivalent glucose derivatives the N-(o-azidoalkanoyl)-b-
D-glucopyranosylamines 12–15 were reacted with 1,7-octadiyne
(43) under CuAAC conditions (Scheme 5). The reactions
proceeded smoothly to give good to excellent yields of the
coupled derivatives 44–47, which were deprotected under
Results and discussion
Syntheses
The new triterpene glycoconjugates were designed to include
oleanolic, ursolic, and maslinic acids (1–3) on one hand and
N-acyl-b-D-glucopyranosylamines on the other, by connecting
them via linker chains of different length. The Cu(I)-catalyzed
azide–alkyne cycloaddition30 (CuAAC) was chosen as the
linking methodology. The syntheses are summarized in
Schemes 1–5.
Zemplen conditions to give compounds 48–51 in similarly
´
good yields. In order to make comparisons with monovalent
glucose derivatives, azides 12 and 13 were deprotected by the
Zemplen protocol to 39 and 41, respectively, which were
´
Direct esterification of oleanolic acid 1, ursolic acid 2, and
maslinic acid 3 with propargyl bromide (Scheme 1) afforded
alkynes 4,29 5, and 6, respectively, in excellent yields.
N-Acyl-b-D-glucopyranosylamines with a terminal azide
group were synthesized from per-O-acetylated-b-D-gluco-
pyranosyl azide32 7 (Scheme 2). o-Bromoalkanoyl derivatives
8–11 were obtained by a ‘Staudinger reaction’ of 7 with
PMe3, resulting in an intermediate phosphinimine which,
without being isolated, was reacted33 with the corresponding
o-bromoalkanoic acid. Subsequent substitution with NaN3 in
DMF gave compounds 12–15, respectively. Practically each
further reduced to the o-amino compounds 40 and 42.
Glycogen phosphorylase inhibition
The above-synthesized derivatives were evaluated in enzyme
inhibition assays described previously35,36 against rabbit
muscle glycogen phosphorylase a (RMGPa) or b (RMGPb)
which shared considerable sequence similarity with human
liver GP (Schemes 1 and 3–5, and Charts 1 and 2). As we
found previously,36 inhibitions of a and b forms of GP showed
acceptable similarity.
Scheme 1 Reagents and conditions: (a) K2CO3, propargyl bromide, DMF, rt. Inhibition of RMGPa (IC50 [mM], values are means of three
experiments).
Scheme 2 Reagents and conditions: (a) PMe3, Br–(CH2)n–COOH, CH2Cl2, rt; (b) NaN3, DMF, rt.
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Scheme 3 Reagents and conditions: (a) CuSO4, sodium L-ascorbate, CH2Cl2–H2O, rt; (b) NaOH, MeOH, rt. Inhibition of RMGPa (IC50 [mM],
values are means of three experiments).
Scheme 4 Reagents and conditions: (a) CuSO4, sodium L-ascorbate, CH2Cl2–H2O, rt; (b) NaOH, MeOH, rt. Inhibition of RMGPa (IC50 [mM],
values are means of three experiments).
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Scheme 5 Reagents and conditions: (a) cat. NaOMe, MeOH rt.; (b) RANEYs-Ni, H2, MeOH, 70 1C; (c) CuSO4, L-ascorbic acid, CH2Cl2–H2O,
rt. Inhibition of RMGPb.
The assay results showed that propargylation of the C-28
carboxyl depressed the GPa enzyme inhibitory activity (compare
1–3 to 4–6 in Scheme 1). This observation is similar to the
effect of other esterifications of OA (52–54, Chart 1) resulting
in a significant loss of activity.14
In the sugar-coupled series (Schemes 3 and 4) the activities
of OA derivatives were generally better than those of the
derivatives of UA and MA (19 vs. 20 and 21; 22 vs. 25 and
28; 23 vs. 26 and 29; 32 vs. 34 and 36). Deprotection of the
sugar part in the 1-b-D-glucopyranosyl-1,2,3-triazole series
(Scheme 3) gave better inhibitors with OA (16 vs. 19) and
MA (18 vs. 21), while no significant change was observed with
UA (17 vs. 20). Appending the sugar to the triazole via the C-6
position as in 55 (Chart 1) gave a very good inhibitor,
although the O-peracetylated analogue had no activity at all.29
The effect of the length of the linker between the sugar and
the triterpene parts was studied in the o-triazolylalkanoyl-
amide series (Scheme 4): with OA (22 vs. 23 and 24) and
MA (28 vs. 29 and 30) derivatives the one-carbon linkage
was significantly better than the longer ones, while among
the UA compounds an opposite effect (27 vs. 25 and 26)
was observed. Removal of the O-acetyl protecting groups in
the o-triazolylalkanoyl-amide series (Scheme 4, 31–37)
brought about no obvious difference. Comparison of 19 with
the hydroxymethyl-triazole 56 shows that the presence of the
OA moiety makes the inhibition somewhat better. However,
56 binds to the catalytic site,37 while 19 can be expected to
occupy the allosteric site.29 Thus, the comparable inhibitory
Chart 1 Literature data for some inhibitors of RMGP.
Chart 2 Comparison of inhibition assay results against RMGPa (abbreviations: derivatives of maslinic acid (MA), ursolic acid (UA) and
oleanolic acid (OA); ni = no inhibition).
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activities may not be directly relevant, except in the as-yet
unproven case of a dual binding mode which could be
expected to occur between two enzyme dimers.25 Similar
considerations may apply to a comparison of 31 and 57.
In cases of bivalent glucose derivatives 48–51 (Scheme 5) no
inhibition could be observed. Study of analogous monovalent
compounds revealed that with an azide as endgroup (39, 41)
the inhibitory activity was moderate and decreased with the
length of the linker. Bivalent compound 48 can also be
compared with the monovalent triazole 5738 (Chart 1) to
show that the dimeric structures seem to be too large to
occupy the catalytic site, and no other interactions exist with
the enzyme. In the presence of amine endgroups (40, 42) the
inhibition was much weaker, and with the longer linker chain
no effect was detected.
K2CO3 (4.4 mmol). The reaction mixture was stirred at
rt for 18 h, then concentrated. The residue was diluted with
EtOAc (50 mL), washed successively with 1 N HCl, water,
satd. aq. NaHCO3, water and brine, dried (MgSO4), filtered
and concentrated. The residue was purified by column
chromatography.
Propargyl 3b-hydroxyolean-12-en-28-oate (4)29. Prepared
from 1 (1 g, 2.2 mmol) and propargyl bromide (0.27 mL,
2.4 mmol) according to General procedure I. The residue was
purified by column chromatography (EtOAc–hexane, 1 : 6).
Yield: 1.05 g, 97%, white solid, mp 121–122 1C; Rf = 0.33
(EtOAc–hexane, 1 : 4); [a]D = +67.9 (c = 0.50, CH2Cl2). IR
(KBr, cmꢂ1): 3308, 2945, 2866, 1731, 1157, 1032, 739;
1H NMR (300 MHz, CDCl3): d 0.74, 0.77, 0.92, 0.98, 1.13
(5 s, each 3H, 5 ꢃ CH3), 0.90 (s, 6H, 2 ꢃ CH3), 0.71–2.04 (m,
22H), 2.41 (t, 1H, J = 2.6 Hz, CH), 2.87 (dd, 1H, J = 4.1, 9.5
Hz, H-18), 3.21 (dd, 1H, J = 5.1, 10.7 Hz, H-3), 4.56 (dd, 1H,
J = 2.6, 15.4 Hz, CO2CH2), 4.68 (dd, 1H, J = 2.6, 15.4 Hz,
CO2CH2), 5.30 (t, 1H, J = 3.5 Hz, H-12); 13C NMR (75 MHz,
CDCl3): d 15.3, 15.6, 17.1, 18.3, 23.0, 23.4, 23.6, 25.8, 27.2,
27.7, 28.1, 30.7, 32.2, 32.8, 33.1, 33.8, 37.0, 38.5, 38.8, 39.4,
41.3, 41.7, 45.9, 46.8, 47.6, 51.6, 55.2, 74.4, 78.1, 79.0, 122.63,
143.4, 176.8. ESI-MS (positive mode) m/z: 517.3 [M + Na]+.
Conclusions
Copper(I)-catalyzed azide–alkyne cycloaddition
– ‘click
chemistry’ – proved suitable for the synthesis of conjugates
of pentacyclic triterpenes and D-glucose derivatives as new,
potentially heterobivalent inhibitors of glycogen phosphorylase.
Compounds 17 (IC50 = 51 mM), 19 (IC50 = 26 mM), 20
(IC50 = 45 mM), 22 (IC50 = 68 mM), 27 (IC50 = 65 mM) and
25 (IC50 = 78 mM) were the most potent inhibitors of
RMGPa. Homobivalent glucose derivatives proved inefficient
in RMGPb inhibition assays. The monovalent analogues of
both triterpenes and glucose derivatives proved generally more
efficient than the bivalent compounds.
Propargyl 3b-hydroxyurs-12-en-28-oate (5). Prepared from 2
(2.0 g, 4.4 mmol) and propargyl bromide (0.54 mL, 4.8 mmol)
according to General procedure I. The residue was purified by
column chromatography (EtOAc–hexane, 1 : 5). Yield: 2.0 g,
93%, white solid, mp 129–131 1C; Rf = 0.48 (EtOAc–hexane,
1 : 5). IR (KBr, cmꢂ1): 3309, 2927, 2871, 1729, 1454, 1383,
1221, 1167, 1139, 1106, 1032, 996, 757, 667; 1H NMR
(300 MHz, CDCl3): d 0.76, 0.77, 0.91, 0.95, 0.98, 1.08 (6 s,
each 3H, 6 ꢃ CH3), 0.87 (d, 3H, J = 6.4 Hz, CH3), 0.75–2.10
(m, 22H), 2.26 (d, 1H, J = 11.3 Hz, H-18), 2.41 (t, 1H,
J = 2.4 Hz, CH), 3.20 (dd, 1H, J = 5.1, 10.7 Hz, H-3), 4.57
and 4.65 (dd, each 1H, J = 2.5, 15.6 Hz, COOCH2), 5.27 (1H,
t, J = 3.6 Hz, H-12); 13C NMR (75 MHz, CDCl3): d 5.5, 15.6,
17.0, 17.2, 18.3, 21.1, 23.3, 23.5, 24.6, 27.3, 28.0, 28.1, 30.6,
33.1, 36.4, 37.0, 38.67, 38.75, 38.8, 39.1, 39.6, 42.1, 47.6, 48.2,
51.6, 52.8, 55.3, 74.3, 78.1, 79.0, 125.9, 137.8, 176.6. ESI-MS
(positive mode) m/z: 495.4 [M + H]+.
Experimental
General methods
All commercially available solvents and reagents were used
without further purification. Melting points were measured on
a RY-1 or on a Kofler hot-stage melting point apparatus.
Column chromatography was carried out on E. Merck Silica
Gel 60 (230–400 mesh), on silica gel (200–300 mesh, Qindao
Ocean Chemical Company, China), or Kieselgel 60 (Merck,
particle size 0.063–0.200 mm). IR spectra were recorded on
Shimadzu FTIR-8400S spectrometer. 1H- and 13C-NMR spectra
were measured on Bruker AV-300 (300/75 MHz for 1H/13C),
Bruker 360 (360/90 MHz for 1H/13C) or Avance DRX 500
(500/125 MHz for 1H/13C) spectrometers. Chemical shifts are
reported as values from an internal tetramethylsilane standard.
TLC was performed on DC-Alurolle Kieselgel 60 F254 (Merck),
and the plates were visualised under UV light and by gentle
heating. Mass spectral data were obtained on Agilent 1100
LC/DAD/MSD or Q-Tof Micro MS/MS spectrometers. Optical
rotations were measured using a Perkin-Elmer 141 or a Perkin-
Elmer 241 polarimeters at rt. PMe3 (1 M solution in toluene) and
1,7-octadiyne were purchased from Sigma-Aldrich.
Propargyl 2a,3b-dihydroxyolean-12-en-28-oate (6). Prepared
from 3 (1.4 g, 3.0 mmol) and propargyl bromide (0.37 mL,
3.3 mmol) according to General procedure I. The residue was
purified by column chromatography (EtOAc–hexane, 1 : 5).
Yield: 1.3 g, 87%, white solid, mp 233–234 1C; Rf = 0.69
(EtOAc–hexane, 1 : 5). IR (KBr, cmꢂ1): 3394, 3309, 2946,
1729, 1463, 1388, 1364, 1259, 1217, 1157, 1121, 1049, 1033,
995, 758, 669, 633; 1H NMR (300 MHz, CDCl3): d 0.74, 0.82,
0.90, 0.92, 0.98, 1.03, 1.13 (7 s, each 3H, 7 ꢃ CH3), 0.75–2.01
(m, 20H), 2.41 (t, 1H, J = 2.4 Hz, CH), 2.87 (dd, 1H, J = 4.3,
13.7 Hz, H-18), 3.01 (d, 1H, J = 9.5 Hz, H-3), 3.65–3.73
(m, 1H, H-2), 4.57 and 4.69 (dd, each 1H, J = 2.4, 15.6 Hz,
COOCH2), 5.31 (t, 1H, J = 3.5 Hz, H-12); 13C NMR
(75 MHz, CDCl3): d 16.6, 16.7, 17.2, 18.4, 23.0, 23.5, 23.6,
25.9, 27.7, 28.6, 30.7, 32.2, 32.7, 33.1, 33.9, 38.3, 39.2, 39.5,
41.3, 41.8, 45.9, 46.5, 46.8, 47.6, 51.6, 55.4, 69.0, 74.4, 78.1,
Syntheses
General procedure I for the propargylation of oleanolic acid,
ursolic acid or maslinic acid
To a solution of a carboxylic acid (1 or 2 or 3, 2.2 mmol) in
DMF (5 mL), was added propargyl bromide (2.4 mmol) and
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84.0, 122.5, 143.5, 176.8. ESI-MS (positive mode) m/z: 533.4
[M + Na]+.
(594.50): C 50.51, H 6.78, N 2.36. Found: C 50.64, H 6.91,
N 2.49.
N-(16-Bromohexadecanoyl)-2,3,4,6-tetra-O-acetyl-b-D-gluco-
pyranosylamine (11). Prepared from 7 (0.50 g 1.34 mmol)
according to General procedure II. The residue was purified
by column chromatography (EtOAc–hexane, 1 : 1). Yield:
0.68 g, 76%, white crystalline product, mp 91–93 1C;
[a]D = +10 (c = 0.20, CHCl3); 1H NMR (360 MHz, CDCl3):
d(ppm) 1.28 (m, 18H, 9 ꢃ CH2), 1.36–1.39 (m, 2H, CH2),
1.48–1.52 (m, 2H, CH2), 1.57–1.61 (m, 2H, CH2), 1.84–1.88
(m, 2H, CH2), 2.03, 2.04, 2.06, 2.08 (4 s, 12H, 4 ꢃ OCOCH3),
2.27–2.31 (m, 2H, CH2), 3.36–3.40 (m, 2H, CH2), 3.87 (ddd,
1H, J = 1.2, 2.6, 10.6 Hz, H-5), 4.06 (dd, 1H, J = 1.2,
11.9 Hz, H-6b), 4.35 (dd, 1H, J = 2.6, 11.9 Hz, H-6a), 4.98,
5.22, 5.27, 5.32 (4 pseudo t, 4H, J = 9.2, 10.6 Hz in each, H-1,
H-2, H-3, H-4), 6.50 (d, 1H, J = 9.2 Hz, NH); 13C NMR
(90 MHz, CDCl3): d (ppm) 20.2 (3), 20.3 (4 ꢃ OCOCH3), 23.0,
23.4, 24.8, 25.0, 26.0, 26.3, 28.1, 28.8, 29.0, 29.3, 29.4, 32.0,
33.8, 34.5, 36.2, (16 ꢃ CH2), 61.7 (C-6), 68.1, 71.2, 72.5, 74.3
(C-2, C-3, C-4, C-5), 76.8 (C-1), 169.3, 169.6, 170.4, 170.6
General procedure II for the preparation of N-(x-bromoalkanoyl)-
2,3,4,6-tetra-O-acetyl-b-D-glucopyranosylamines (9–11)
2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl azide (7, 0.10 g,
0.27 mmol) was dissolved in dry CH2Cl2 (3 mL). To the
solution Me3P (1.1 equiv. of a 1 M solution in toluene) was
added in one portion. The mixture was stirred at rt. until
nitrogen evolution had ceased and TLC (EtOAc–hexane, 1 : 1)
had indicated complete transformation of the azide. This
solution was then reacted with an o-bromoalkanoic acid
(1.1 equiv., as indicated with the particular compounds) till
the disappearance of the iminophosphorane (TLC, EtOAc–
hexane, 1 : 1). Then, it was diluted with CH2Cl2 (5 mL) and
washed with satd. aq. NaHCO3 solution (2 ꢃ 5 mL). The
organic phase was dried over MgSO4 and the solvent was
removed under diminished pressure. The crude product was
purified by column chromatography.
N-(6-Bromohexanoyl)-2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl-
amine (9). Prepared from 7 (0.50 g 1.34 mmol) according
to General procedure II. The residue was purified by column
chromatography (EtOAc–hexane, 1 : 1). Yield: 0.65 g, 93%,
colourless oil, Rf = 0.35 (EtOAc–hexane, 1 : 1); [a]D = +22
(c = 0.59, CHCl3); 1H NMR (360 MHz, CDCl3): d(ppm)
1.42–1.47 (m, 2H, CH2), 1.60–1.63 (m, 2H, CH2), 1.83–1.90
(m, 2H, CH2), 2.02, 2.04, 2.06, 2.08 (4s, 12H, 4 ꢃ OCOCH3),
2.21–2.27 (m, 2H, CH2), 3.39–3.43 (m, 2H, CH2), 3.86 (ddd,
1H, J = 1.2, 2.6, 10.6 Hz, H-5), 4.08 (dd, 1H, J = 1.2, 11.9 Hz,
H-6b), 4.32 (dd, 1H, J = 2.6, 11.9 Hz, H-6a), 4.93, 5.06, 5.29,
5.32 (4 pseudo t, 4H, J = 9.2, 10.6 Hz in each, H-1, H-2, H-3,
H-4), 6.63 (d, 1H, J = 9.2 Hz, NH); 13C NMR (90 MHz,
CDCl3): d (ppm) 20.3 (3), 20.5 (4 ꢃ OCOCH3), 23.9, 27.3,
32.0, 33.3, 35.9 (5 ꢃ CH2), 61.5 (C-6), 67.9, 70.4, 72.5, 73.2
(C-2, C-3, C-4, C-5), 77.7 (C-1), 169.3, 169.6, 170.4, 170.5
(4
ꢃ
OCOCH3), 172.3 (NHCO). Anal. calcd. for
C30H50BrNO10 (664.64): C 54.22, H 7.58, N 2.11. Found: C
54.11, H 7.72, N 2.29.
General procedure III for the preparation of N-(x-azidoalkanoyl)-
2,3,4,6-tetra-O-acetyl-b-D-glucopyranosylamines (12–15)
An
N-(o-bromoalkanoyl)-2,3,4,6-tetra-O-acetyl-b-D-gluco-
pyranosylamine (9–11) was dissolved in dry DMSO
(15 mL/mmol). To the solution NaN3 (2 equiv.) was added
in one portion. The mixture was stirred at rt until the
disappearance of the starting bromide (TLC EtOAc–hexane
1 : 1). The solution was diluted with water (150 mL), washed
with Et2O (5 ꢃ 25 mL) and water (1 ꢃ 25 mL), dried over
MgSO4 and the solvent was removed under diminished
pressure. The crude product was purified by column chromato-
graphy or crystallisation.
(4
ꢃ
OCOCH3), 172.8 (NHCO). Anal. calcd. for
C20H30BrNO10 (524.37): C 45.81, H 5.77, N 2.67. Found: C
45.64, H 5.94, N 2.59.
N-Azidoacetyl-2,3,4,6-tetra-O-acetyl-b-D-glucopyranosylamine
(12). To a solution of azide 7 (0.20 g, 0.54 mmol) in dry
CH2Cl2 (3 mL), PMe3 (0.54 mL of a 1 M solution in toluene)
was added in one portion. The mixture was stirred at rt until
nitrogen evolution had ceased, and TLC (EtOAc–hexane, 1 : 1)
had indicated complete transformation of 7 (approx. 15 min).
This soln was then reacted with bromoacetic acid (0.082 g,
0.59 mmol). When TLC (EtOAc–hexane, 1 : 1) showed no
more change (conversions were incomplete), the solvent was
evaporated, and the pale yellow oil was dissolved in CH2Cl2
(30 mL) and extracted with satd. aq. NaHCO3 solution
(2 ꢃ 30 mL). The organic phase was dried over MgSO4,
concentrated under diminished pressure, then the residue
(0.20 g) was dissolved in dry DMF (3 ml) and NaN3 (0.056 g,
0.82 mmol) was added. The reaction mixture was stirred at rt
for 2 h (TLC, EtOAc–hexane, 1 : 1). The mixture was then
diluted with water (20 mL) and extracted with Et2O
(5 ꢃ 30 mL). The combined organic phase was dried over
MgSO4 and the solvent was removed under diminished pressure.
The obtained syrup was purified by column chromatography
(EtOAc–hexane, 4 : 6) to give 12 as white crystals: 0.15 g, 63%,
N-(11-Bromoundecanoyl)-2,3,4,6-tetra-O-acetyl-b-D-gluco-
pyranosylamine (10). Prepared from 7 (5.0 g 13.4 mmol)
according to General procedure II. The residue was purified
by column chromatography (EtOAc–hexane, 1 : 1). Yield:
5.13 g, 64%, white crystalline product, mp 61–63 1C; [a]D
=
+15 (c = 0.38, CHCl3); 1H NMR (360 MHz, CDCl3): d(ppm)
1.28 (bs, 10H, 5 ꢃ CH2), 1.40–1.45 (m, 2H, CH2), 1.57–1.50
(m, 2H, CH2), 1.80–1.86 (m, 2H, CH2), 2.02, 2.04, 2.06, 2.08
(4 s, 12H, 4 ꢃ OCOCH3), 2.17–2.21 (m, 2H, CH2), 3.38–3.43
(m, 2H, CH2), 3.85 (ddd, 1H, J = 1.2, 2.6, 10.6 Hz, H-5), 4.07
(dd, 1H, J = 1.2, 11.9 Hz, H-6b), 4.32 (dd, 1H, J = 2.6, 11.9
Hz, H-6a), 4.93, 5.06, 5.27, 5.32 (4 pseudo t, 4H, J = 9.2, 10.6
Hz in each, H-1, H-2, H-3, H-4), 6.51 (d, 1H, J = 9.2 Hz,
NH); 13C NMR (90 MHz, CDCl3): d (ppm) 20.4(3), 20.5
(4 ꢃ OCOCH3), 24.8, 25.0, 27.9, 28.4, 28.8, 29.0, 29.1, 32.5,
33.7, 36.3 (10 ꢃ CH2), 61.5 (C-6), 67.9, 70.4, 72.5, 73.3
(C-2, C-3, C-4, C-5), 77.8 (C-1), 169.3, 169.6, 170.4, 170.6
(4 ꢃ OCOCH3), 173.3 (NHCO). Anal. calcd. for C25H40BrNO10
ꢀc
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calcd for 7; mp 150–152 1C, [a]D = +39 (c = 0.21, CHCl3),
(lit.40 [a]D +4.1 (c = 1, CHCl3)); 1H NMR (360 MHz,
CDCl3): d (ppm) 2.03, 2.04, 2.07, 2.09 (4s, 12H, 4 ꢃ
OCOCH3), 3.85 (ddd, 1H, J = 2.6, 4.0, 9.2 Hz, H-5),
3.93–4.11 (m, 3H, H-6b, CH2), 4.30 (dd, 1H J = 2.6,
13.2 Hz, H-6a), 4.99, 5.08, 5.24, 5.33 (4 pseudo t, 4H, J =
9.2, 10.6 Hz in each, H-1, H-2, H-3, H-4), 7.19 (d, 1H, J = 9.2 Hz,
NH); 13C NMR (90 MHz, CDCl3): d (ppm) 20.6, 20.5
(4 ꢃ OCOCH3), 52.4 (CH2), 61.5 (C-6)) 67.9, 70.3, 72.5, 73.6
(C-2, C-3, C-4, C-5), 78.0 (C-1), 167.5, 169.5, 169.8, 170.5
(4 ꢃ OCOCH3), 170.8 (NHCO). Anal. calcd. for C16H22N4O10
(430.37): C, 44.65; H, 5.15; N, 13.02; Found: C, 44.53; H, 5.22;
N, 13.12.
10.6 Hz, H-5), 3.29 (1H, dd, J = 1.1, 11.9 Hz, H-6b), 3.52 (dd,
1H, J = 4.0, 11.9 Hz, H-6a), 4.20, 4.25, 4.71, 4.60 (4 pseudo t,
4H, J = 9.2, 10.6 Hz, in each, H-1, H-2, H-3, H-4), 7.48 (d,
1H, J = 9.2 Hz, NH); 13C NMR (90 MHz, CDCl3): d (ppm)
20.4 (3), 20.5 (4 ꢃ OCOCH3), 23.3, 23.4, 3 24.8, 26.0, 27.1,
27.6, 28.2, 28.5, 29.0, 29.7, 30.4, 31.9, 33.8, 36.5, 49.2 (15 ꢃ
CH2), 61.5 (C-6), 68.3, 70.0, 72.1, 72.5 (C-2, C-3, C-4, C-5),
78.7 (C-1), 168.5, 168.6, 168.7, 169.3 (4 ꢃ OCOCH3), 172.1
(NHCO). Anal. calcd. for C30H50N4O10 (626.75): C 57.49,
H 8.04, N 8.94. Found: C 57.23, H 8.24, N 8.82.
General procedures IV for the CuAAC ‘click’ reaction
(a). To a solution of an alkyne (0.27 mmol) and an azide
(0.27 mmol) in CH2Cl2 (2 mL) and H2O (2 mL), was added
CuSO4ꢁ5H2O (0.32 mmol) and Na-L-ascorbate (0.64 mmol).
The resulting solution was stirred for 12 h at rt. The reaction
mixture was diluted with H2O (10 mL), then extracted with
CH2Cl2 (3 ꢃ 10 mL). The combined organic layer was dried
over MgSO4, filtered, and concentrated. The residue was
purified by column chromatography.
N-(6-Azidohexanoyl)-2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl-
amine (13). Prepared from 9 (2.00 g, 3.81 mmol) according
to General procedure III. Yield: 1.57 g, 85%, white crystalline
product, mp 135–137 1C; [a]D = +37 (c = 0.34, CHCl3);
1H NMR (360 MHz, CDCl3): d(ppm) 1.36–1.44 (m, 2H, CH2),
1.57–1.68 (m, 4H, 2 ꢃ CH2), 2.02, 2.04, 2.05, 2.08 (4 s, 12H,
4 ꢃ OCOCH3), 2.18–2.24 (m, 2H, CH2), 3.24–3.29 (m, 2H,
CH2), 3.84 (ddd, 1H, J = 1.2, 2.6, 10.6 Hz, H-5), 4.08 (dd, 1H,
J = 1.1, 11.9 Hz, H-6b), 4.31 (dd, 1H, J = 11.9, 4.0 Hz, H-6a),
4.92, 5.06, 5.28, 5.31 (4 pseudo t, 4H, J = 9.2, 10.6 Hz in each,
H-1, H-2, H-3, H-4), 6.57 (d, 1H, J = 9.2 Hz, NH); 13C NMR
(90 MHz, CDCl3): d (ppm) 20.4 (3), 20.5 (4 ꢃ OCOCH3), 24.4,
26.0, 28.4, 36.1, 51.0 (5 ꢃ CH2), 61.5 (C-6), 68.0, 70.4, 73.4,
72.6 (C-2, C-3, C-4, C-5), 77.9 (C-1), 169.5, 169.7, 170.5, 170.8
(4 ꢃ OCOCH3), 173.0 (NHCO). Anal. calcd. for C20H30N4O10
(486.48): C 49.38, H 6.22, N 11.52. Found: C 49.46, H 6.14,
N 11.59.
(b). An N-(o-azidoalkanoyl)-2,3,4,6-tetra-O-acetyl-b-D-
glucopyranosylamine (0.46 mmol) was added to a 1 : 1 (v/v)
mixture of CH2Cl2 and water (10 mL/mmol). To the solution
1,7-octadiyne (43, 1.0 equiv.), CuSO4ꢁ5H2O (5 mol%) and
L-ascorbic acid (15 mol%) were added. The mixture was
heated at 50 1C until complete transformation of the starting
azide (TLC, EtOAc). The solution was diluted with water
(25 mL) and extracted with CH2Cl2 (5 ꢃ 12 mL). The organic
phase was dried over MgSO4 and the solvent was removed
under diminished pressure. The crude product was purified by
column chromatography or crystallisation.
N-(11-Azidoundecanoyl)-2,3,4,6-tetra-O-acetyl-b-D-gluco-
pyranosylamine (14). Prepared from 10 (1.00 g, 1.69 mmol)
according to General procedure III. Yield: 0.83 g, 88%,
white crystalline product, mp 68–70 1C; [a]D = +13
(c = 0.22, CHCl3); 1H NMR (360 MHz, CDCl3): d(ppm)
1.25–1.31 (m, 14H, 7 ꢃ CH2), 1.46–1.50 (m, 2H, CH2), 2.02,
2.04, 2.05, 2.08 (4 s, 12H, 4 ꢃ OCOCH3), 2.20–2.25 (m, 2H,
CH2), 2.52–2.56 (m, 2H, CH2), 3.17 (ddd, 1H, J = 1.2, 2.6,
10.6 Hz, H-5), 3.31 (dd, 1H, J = 1.1, 11.9 Hz, H-6b), 3.53
(dd, 1H, J = 4.0, 11.9 Hz, H-6a), 4.22, 4.29, 4.56, 4.61
(4 pseudo t, 4H, J = 9.2, 10.6 Hz in each, H-1, H-2, H-3,
H-4), 7.49 (d, 1H, J = 9.2 Hz, NH); 13C NMR (90 MHz,
CDCl3): d (ppm) 20.4 (3), 20.5 (4 ꢃ OCOCH3), 25.0, 26.4,
27.9, 28.0, 28.2, 28.3, 30.1, 32.4, 35.0, 50.2 (10 ꢃ CH2), 60.9
(C-6), 67.1, 69.6, 72.2, 72.4 (C-2, C-3, C-4, C-5), 77.9 (C-1),
168.5, 168.6, 168.7, 169.3 (4 ꢃ OCOCH3), 173.0 (NHCO).
Anal. calcd. for C25H40N4O10 (556.62): C 53.95, H 7.24,
N 10.07. Found: C 53.76, H 7.04, N 10.19.
General procedure V for O-deacetylation
To a solution or suspension of an O-peracetylated compound
(0.081 mmol) in MeOH (3 mL) was added 4 N aq. NaOH
(0.4 mL), then stirred at rt for 1 h, and neutralized with 1 N
HCl (1.8 mL). The mixture was concentrated in vacuo and the
residue was taken up in EtOAc (50 mL), washed successively
with 1 N HCl (3 ꢃ 15 mL), water (3 ꢃ 15 mL) and brine
(3 ꢃ 15 mL), dried (Na2SO4), filtered and concentrated. The
residue was purified by column chromatography.
[1-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-1H-1,2,3-triazol-
4-ylmethyl] 3b-hydroxyolean-12-en-28-oate (16). Prepared
from 4 (0.13 g, 0.27 mmol) and 7 (0.10 g, 0.27 mmol) according
to General procedure IVa. The residue was purified by column
chromatography (EtOAc–hexane, 1 : 1). Yield: 0.13 g, 57%,
white solid, mp 124–125 1C. Rf = 0.13 (EtOAc–hexane, 1 : 2).
IR (KBr, cmꢂ1): 2947, 2869, 1757, 1460, 1369, 1227, 1036, 758;
1H NMR (300 MHz, CDCl3): d 0.66, 0.78, 0.89, 0.90, 0.91,
0.99, 1.13 (7 s, each 3H, 7 ꢃ CH3), 0.67–2.09 (m, 22H), 1.86,
2.03, 2.07, 2.09 (4 s, each 3H, 4 ꢃ OCOCH3), 2.86 (dd, 1H,
J = 3.8, 13.9 Hz, H-18), 3.18–3.23 (m, 1H, H-3), 3.96–4.02 (m,
1H, H-5-Glc), 4.11–4.16 (m, 1H, H-6a-Glc), 4.33 (dd, 1H, J =
4.8, 12.6 Hz, H-6b-Glc), 5.17 (s, 2H, COOCH2), 5.21–5.27 (m,
1H, H-2-Glc), 5.31 (t, 1H, J = 3.3 Hz, H-12), 5.39–5.43 (m,
2H, overlapping, H-3-Glc and H-4-Glc), 5.84–5.87 (m, 1H,
H-1-Glc), 7.81 (s, 1H, NCH); 13C NMR (75 MHz, CDCl3):
N-(16-Azidohexadecanoyl)-2,3,4,6-tetra-O-acetyl-b-D-gluco-
pyranosylamine (15). Prepared from 11 (0.4 g, 0.60 mmol)
according to General procedure III. Yield: 0.30 g, 81%, white
crystalline product, mp 92–94 1C; [a]D = +16 (c = 0.22,
1
CHCl3); H NMR (360 MHz, CDCl3): d(ppm) 1.24–1.28 (m,
18H, 9 ꢃ CH2), 1.36–1.39 (m, 2H, CH2), 1.47–1.50 (m, 2H,
CH2), 1.56–1.61 (m, 2H, CH2), 1.84–1.87 (m, 2H, CH2), 2.02,
2.04, 2.07, 2.08 (4s, 12H, 4 ꢃ OCOCH3), 2.27–2.31 (m, 2H,
CH2), 3.38–3.41 (m, 2H, CH2), 3.21 (ddd, 1H, J = 1.2, 2.6,
ꢀc
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1456 | New J. Chem., 2010, 34, 1450–1464

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d 15.3, 15.6, 16.9, 18.4, 20.1, 20.5, 20.6, 23.0, 23.4, 23.6, 25.9,
27.2, 27.7, 28.1, 29.7, 30.7, 32.2, 32.7, 33.1, 33.9, 37.1, 38.5,
38.8, 39.3, 41.4, 41.8, 45.9, 46.7, 47.6, 55.3, 57.4, 61.5, 67.7,
70.3, 72.6, 75.3, 79.0, 85.9, 122.0, 122.5, 143.7, 144.0, 168.6,
169.3, 169.9, 170.4, 177.4. ESI-MS (positive mode) m/z: 890.8
[M + Na]+.
[a]D = +45 (c = 0.05, MeOH); IR (KBr, cmꢂ1): 3424, 2942,
1712, 1636, 1052, 1033, 1016, 772; 1H NMR (300 MHz,
C5D5N): d 0.82, 0.85, 0.89, 0.93, 1.01, 1.17, 1.21 (7 s, each
3H, 7 ꢃ CH3), 0.82–1.92 (m, 22H), 3.08–3.11 (m, 1H, H-18),
3.39–3.44 (m, 1H, H-3), 4.20–4.22 (m, 1H, H-5-Glc), 4.27–4.41
(m, 3H, overlapping, H-6a-Glc, H-6b-Glc, H-4-Glc), 4.50
(m, 1H, H-2-Glc), 4.79 (t, 1H, J = 8.9, 8.9 Hz, H-3-Glc),
5.40 (s, 1H, H-12), 5.47–5.57 (m, 2H, COOCH2), 6.35 (d, 1H,
J = 9.2 Hz, H-1-Glc), 8.64 (s, 1H, NCH); 13C NMR (75 MHz,
C5D5N): d 15.7, 16.6, 17.4, 18.8, 23.4, 23.6, 23.8, 26.0, 28.1,
28.8, 30.8, 32.7, 33.1, 33.2, 34.0, 37.4, 39.0, 39.4, 39.8, 41.9,
42.1, 46.1, 47.0, 48.1, 55.9, 58.2, 62.4, 71.1, 73.9, 78.2, 79.1,
82.0, 89.6, 123.0, 124.1, 143.5, 144.0, 177.4. ESI-MS (positive
mode) m/z: 744.3 [M + HCOO]+; HRMS (MALDI) m/z =
C39H61N3O8 [M + Na]+ calcd. 722.4356, found 722.4371.
[1-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-1H-1,2,3-triazol-
4-ylmethyl] 3b-hydroxyurs-12-en-28-oate (17). Prepared from 5
(0.13 g, 0.27 mmol) and 7 (0.10 g, 0.27 mmol) according to
General procedure IVa. The residue was purified by column
chromatography (EtOAc–hexane, 1 : 2). Yield: 0.22 g, 96%,
white solid, mp 120–122 1C. Rf = 0.20 (EtOAc–hexane, 1 : 2);
IR (KBr, cmꢂ1): 2932, 2872, 1757, 1456, 1376, 1228, 1104;
1H NMR (300 MHz, CDCl3): d 0.72, 0.81, 0.94, 0.97, 1.01,
1.11 (6 s, each 3H, 6 ꢃ CH3), 0.89 (d, 3H, J = 6.3 Hz, CH3),
0.72–2.13 (m, 22H), 1.89, 2.06, 2.10, 2.13 (4 s, each 3H,
4 ꢃ OCOCH3), 2.25 (d, 1H, J = 10.8 Hz, H-18), 3.25 (dd,
1H, J = 4.3, 10.7 Hz, H-3), 4.00–4.04 (m, 1H, H-5-Glc),
4.14–4.19 (m, 1H, H-6a-Glc), 4.35 (dd, 1H, J = 4.5, 12.7 Hz,
H-6b-Glc), 5.17 and 5.18 (2 d, each 1H, J = 12.8 Hz,
COOCH2), 5.23–5.30 (m, 2H, overlapping, H-12 and H-2-
Glc), 5.40–5.48 (m, 2H, overlapping, H-3-Glc and H-4-Glc),
5.90 (d, 1H, J = 9.2 Hz, H-1-Glc), 7.82 (s, 1H, NCH); 13C
NMR (75 MHz, CDCl3): d 15.5, 15.6, 19.96, 17.0, 18.4, 20.1,
20.5, 20.6, 21.1, 23.3, 23.6, 24.2, 27.3, 28.1, 28.2, 30.6, 33.0,
36.4, 37.0, 38.7, 38.8, 38.82, 39.1, 39.6, 42.1, 47.6, 48.1, 52.9,
55.3, 57.3, 61.5, 67.8, 70.4, 72.7, 75.3, 79.1, 86.9, 122.0, 125.8,
138.1, 144.1, 168.6, 169.3, 169.9, 177.3. ESI-MS (positive
mode) m/z: 890.8 [M + Na]+.
[1-(b-D-Glucopyranosyl)-1H-1,2,3-triazol-4-ylmethyl] 3b-hydroxy-
urs-12-en-28-oate (20). Prepared from 17 (0.14 g, 0.17 mmol)
according to General procedure V. The residue was purified by
column chromatography (EtOAc). Yield: 0.11 g, 92%, white
solid, mp 160–162 1C, Rf = 0.16 (EtOAc); [a]D = +23 (c = 0.1,
MeOH); IR (KBr, cmꢂ1): 3381, 2923, 2869, 1723, 1454, 1136,
1098, 1051, 1032, 1016, 772; 1H NMR (300 MHz, C5D5N): d 0.81
(d, 3H, J = 4.7 Hz, CH3), 0.83, 0.85, 0.89, 0.97, 1.06, 1.17 (6 s,
each 3H, 6 ꢃ CH3), 2.34 (d, 1H, J = 11.2 Hz, H-18), 0.75–1.94
(m, 22H), 3.38 (m, 1H, H-3), 4.09–4.17 (m, 1H, H-5-Glc),
4.22–4.36 (m, 3H, H-4-Glc and H-6-Glc), 4.46 (d, 1H, J =
10.8 Hz, H-2-Glc), 4.75 (t, 1H, J = 8.8, 8.8 Hz, H-3-Glc), 5.33
(brs, 1H, H-12), 5.44 (s, 2H, COOCH2), 6.33 (d, 1H, J = 9.2 Hz,
H-1-Glc), 8.58 (s, 1H, NCH); 13C NMR (75 MHz, C5D5N): d
15.8, 16.6, 17.3, 18.8, 19.1, 21.2, 23.7, 23.8, 24.6, 28.2, 28.5, 28.8,
30.8, 33.5, 36.8, 37.3, 39.1, 39.2, 39.3, 39.4, 40.0, 42.4, 48.1, 48.4,
53.4, 55.9, 58.1, 62.4, 71.1, 73.9, 78.2, 79.1, 81.9, 89.6, 124.1,
126.2, 129.3, 138.6, 143.5, 177.1. ESI-MS (positive mode) m/z:
744.5 [M + HCOO]+; HRMS (MALDI) m/z = C39H61N3O8
[M + Na]+ calcd. 722.4356, found 722.4365.
[1-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-1H-1,2,3-triazol-
4-ylmethyl] 2a,3b-dihydroxyolean-12-en-28-oate (18). Prepared
from 6 (0.14 g, 0.27 mmol) and 7 (0.10 g, 0.27 mmol) according
to General procedure IVa. The residue was purified by column
chromatography (EtOAc–hexane, 1 : 1). Yield: 0.20 g, 84%,
white solid, mp 158–160 1C, Rf = 0.17 (EtOAc–hexane, 1 : 1);
IR (KBr, cmꢂ1): 3395, 2948, 1758, 1460, 1368, 1227, 1101,
1037, 758; 1H NMR (300 MHz, CDCl3): d 0.69, 0.85, 0.92,
0.94, 1.00, 1.06, 1.15 (7 s, each 3H, 7 ꢃ CH3), 0.69–2.12 (m,
20H), 1.88, 2.06, 2.10, 2.12 (4 s, each 3H, 4 ꢃ OCOCH3), 2.89
(dd, 1H, J = 3.1, 13.2 Hz, H-18), 3.05 (d, 1H, J = 9.5 Hz,
H-3a), 3.68–3.76 (m, 1H, H-2b), 4.00–4.04 (m, 1H, H-5-Glc),
4.14–4.18 (m, 1H, H-6a-Glc), 4.35 (dd, 1H, J = 4.8, 12.6 Hz,
H-6b-Glc), 5.19 and 5.20 (2 d, each 1H, J = 12.9 Hz,
COOCH2), 5.24–5.30 (m, 1H, H-2-Glc), 5.34 (brs, 1H,
H-12), 5.43–5.46 (m, 2H, H-3-Glc and H-4-Glc), 5.90 (d,
1H, J = 9.2 Hz, H-1-Glc), 7.84 (s, 1H, NCH); 13C NMR
(75 MHz, CDCl3): d 16.9, 17.0, 17.2, 18.6, 20.3, 20.7, 20.9,
23.3, 23.7, 23.9, 26.1, 27.9, 28.8, 30.9, 32.4, 32.9, 33.3, 34.1,
38.6, 39.3, 39.4, 39.7, 41.6, 42.0, 46.1, 46.7, 47.0, 47.9, 55.6,
57.6, 61.8, 68.0, 69.2, 70.6, 72.9, 75.5, 77.4, 84.2, 86.1, 122.2,
122.6, 144.0, 144.3, 168.9, 169.5, 170.1, 170.6, 177.7. ESI-MS
(positive mode) m/z: 906.4 [M + Na]+.
[1-(b-D-Glucopyranosyl)-1H-1,2,3-triazol-4-ylmethyl] 2a,3b-
dihydroxyolean-12-en-28-oate (21). Prepared from 18 (0.13 g,
0.15 mmol) according to General procedure V. The residue
was purified by column chromatography (MeOH–CH2Cl2,
1 : 15). Yield: 0.09 g, 85%, white solid, mp 205–207 1C, Rf =
0.18 (MeOH–CH2Cl2, 1 : 15); [a]D = +27 (c = 0.07, MeOH).
IR (KBr, cmꢂ1): 3461, 2945, 2864, 1720, 1642, 1457, 1051,
1
1031, 1017, 772, 667; H NMR (300 MHz, C5D5N): d 0.82,
0.85, 0.88, 1.03, 1.07, 1.15, 1.25 (7 s, each 3H, 7 ꢃ CH3),
0.82–2.26 (m, 20H), 3.07–3.10 (m, 1H, H-18), 3.29 (d, 1H,
J = 9.3 Hz, H-3a), 4.09 (m, 1H, H-2b), 4.23–4.31 (m, 1H,
H-5-Glc), 4.34–4.42 (m, 3H, overlapping, H-4-Glc and H-6a-Glc,
and H-6b-Glc), 4.52 (d, 1H, J = 11.0 Hz, H-2-Glc), 4.80
(t, 1H, J = 8.9, 8.9 Hz, H-3-Glc), 5.37 (s, 1H, H-12), 5.52
(s, 2H, COOCH2), 6.38 (d, 1H, J = 9.2 Hz, H-1-Glc), 8.65
(s, 1H, NCH); 13C NMR (75 MHz, C5D5N): d 17.0, 17.5, 17.7,
18.9, 23.4, 23.6, 23.9, 26.0, 28.1, 29.3, 30.7, 32.7, 33.1, 33.2,
33.9, 38.6, 39.8, 41.9, 42.1, 46.1, 47.0, 47.8, 48.1, 55.9, 58.2,
62.4, 68.6, 71.1, 73.9, 79.1, 81.9, 83.9, 89.6, 122.9, 124.1, 143.5,
144.0, 177.4. ESI-MS (positive mode) m/z: 716.4 [M + H]+;
HRMS (MALDI) m/z = C39H61N3O9 [M + Na]+ calcd.
738.4306, found 738.4320.
[1-(b-D-Glucopyranosyl)-1H-1,2,3-triazol-4-ylmethyl] 3b-hydroxy-
olean-12-en-28-oate (19). Prepared from 16 (0.07 g,
0.08 mmol) according to General procedure V. The residue
was purified by column chromatography (EtOAc). Yield:
0.05 g, 91%, white solid, mp 178–180 1C, Rf = 0.13 (EtOAc);
ꢀc
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[1-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosylaminocarbonyl-
methyl)-1H-1,2,3-triazol-4-ylmethyl] 3b-hydroxyolean-12-en-
28-oate (22). Prepared from 4 (0.11 g, 0.23 mmol) and 12
(0.10 g, 0.23 mmol) according to General procedure IVa. The
residue was purified by column chromatography (EtOAc–
hexane, 1 : 1). Yield: 0.18 g, 85%, white solid, mp 146–148 1C,
Rf = 0.21 (EtOAc–hexane, 1 : 1); IR (KBr, cmꢂ1): 2947, 2872,
1755, 1552, 1463, 1374, 1230, 1175, 1159, 1045, 759, 667;
1H NMR (300 MHz, CDCl3): d 0.57, 0.78, 0.98, 1.11 (4 s,
each 3H, 4 ꢃ CH3), 0.89 (s, 9H, 3 ꢃ CH3), 0.57–2.10 (m, 22H),
2.01, 2.02, 2.03, 2.08 (4 s, each 3H, 4 xOCOCH3), 2.82–2.85
(1H, m, H-18), 3.20 (dd, 1H, J = 5.0, 10.4 Hz, H-3), 3.78–3.83
(m, 1H, H-5-Glc), 4.07 (dd, 1H, J = 2.1, 12.6 Hz, H-6a-Glc),
4.28 (dd, 1H, J = 4.3, 12.5 Hz, H-6b-Glc), 4.90 (t, 1H,
J = 9.6, 9.6 Hz, H-4-Glc), 4.94–5.32 (m, 8H, overlapping,
NCH2, COOCH2, H-1-Glc, H-2-Glc, H-3-Glc, H-12), 6.78
(d, 1H, J = 8.7 Hz, OH), 7.69 (s, 1H, NCH); 13C NMR
(75 MHz, CDCl3): d 15.4, 15.6, 16.7, 18.3, 20.5, 20.7, 23.0,
23.4, 23.6, 26.0, 27.2, 27.6, 28.1, 30.6, 32.3, 32.7, 33.0, 33.8,
37.0, 38.4, 38.7, 39.3, 41.3, 41.7, 45.8, 46.7, 47.5, 52.5, 55.2,
57.3, 61.5, 68.0, 70.3, 72.4, 73.8, 78.5, 79.0, 122.5, 125.4, 143.5,
143.9, 165.4, 169.4, 169.8, 170.5, 171.0, 177.6. ESI-MS (positive
mode) m/z: 947.6 [M + Na]+. HRMS (MALDI) m/z =
C45H72N4O10 [M + Na]+ calcd. 852.0638, found 851.5158.
(300 MHz, CDCl3): d 0.52, 0.77, 0.87, 0.88, 0.90, 0.98, 1.11
(7 s, each 3H, 7 ꢃ CH3), 0.52–2.07 (m, 38H), 2.02, 2.03, 2.04,
2.07 (4 s, each 3H, 4 ꢃ OCOCH3), 2.13–2.23 (m, 2H, CH2CO),
2.82 (m, 1H, H-18), 3.20 (dd, 1H, J = 5.0, 10.9 Hz, H-3),
3.80–3.85 (m, 1H, H-5-Glc), 4.07 (dd, 1H, J = 1.9, 12.5 Hz,
H-6a-Glc), 4.28–4.34 (m, 3H, overlapping, H-6b-Glc and
NCH2), 4.91 (pseudo t, 1H, J = 9.6, 9.7 Hz, H-4-Glc), 5.06
(pseudo t, 1H, J = 9.6, 9.7 Hz, H-3-Glc), 5.18 (s, 2H,
COOCH2), 5.22–5.34 (m, 3H, overlapping, H-12 and H-1-
Glc, H-2-Glc), 6.30 (d, 1H, J = 9.3 Hz, NH), 7.56 (s, 1H, s,
NCH); 13C NMR (75 MHz, CDCl3): d15.4, 15.6, 16.8, 18.3,
20.5, 20.6, 20.7, 23.0, 23.4, 23.6, 25.1, 25.8, 26.5, 27.2, 27.7,
28.1, 29.0, 29.1, 29.2, 29.3, 29.33, 30.2, 30.7, 32.4, 32.7, 33.0,
33.9, 36.6, 37.1, 38.5, 38.8, 39.4, 41.4, 41.7, 45.9, 46.8, 47.6,
50.4, 55.2, 57.5, 61.7, 68.3, 70.7, 72.8, 73.6, 77.2, 78.2, 79.0,
122.4, 123.9, 143.1, 169.5, 170.0, 170.5, 171.0, 173.3, 177.8.
ESI-MS (positive mode) m/z: 1073.9 [M + Na]+.
[1-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosylaminocarbonyl-
methyl)-1H-1,2,3-triazol-4-ylmethyl] 3b-hydroxyurs-12-en-28-
oate (25). Prepared from 5 (0.11 g, 0.23 mmol) and 12
(0.10 g, 0.23 mmol) according to General procedure IVa.
The residue was purified by column chromatography (EtOAc–
hexane, 1 : 1). Yield: 0.024 g, 11%, white solid, mp 116–118 1C,
Rf = 0.21 (EtOAc–hexane, 1 : 1); [a]D = +29 (c = 0.05,
CHCl3). IR (KBr, cmꢂ1): 3340, 2947, 2871, 1756, 1454, 1377,
[1-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosylaminocarbo-
nylpentyl)-1H-1,2,3-triazol-4-ylmethyl] 3b-hydroxyolean-12-en-
28-oate (23). Prepared from 4 (0.10 g, 0.21 mmol) and 13
(0.10 g, 0.21 mmol) according to General procedure IVa. The
residue was purified by column chromatography (EtOAc–
hexane, 1 : 1). Yield: 0.16 g, 80%, white solid, mp 99–100 1C,
Rf = 0.12 (EtOAc–hexane, 1 : 1); IR (KBr, cmꢂ1): 2947, 2866,
1
1230, 1046, 1033, 997, 758; H NMR (300 MHz, CDCl3): d
0.60, 0.78, 0.91, 0.93, 0.98, 1.06 (6 s, each 3H, 6 ꢃ CH3), 0.83
(d, 3H, J = 6.4 Hz, CH3), 0.60–2.01 (m, 22H), 2.01, 2.03, 2.08
(s, 12 H, 4 ꢃ OCOCH3), 2.20 (d, 1H, J = 11.3 Hz, H-18), 3.22
(dd, 1H, J = 4.9, 10.8 Hz, H-3), 3.78–3.83 (m, 1H, H-5-Glc),
4.08 (dd, 1H, J = 1.9, 12.7 Hz, H-6a-Glc), 4.28 (dd, 1H,
J = 4.3, 12.6 Hz, H-6b-Glc), 4.87 (pseudo t, 1H, J = 9.5,
9.6 Hz, H-4-Glc), 4.99–5.09 (m, 3H, overlapping, NCH2CO
and H-2-Glc), 5.16 (d, 1H, J = 12.7 Hz, COOCH2), 5.19
(d, 1H, J = 12.7 Hz, COOCH2), 5.22–5.23 (m, 2H, over-
lapping, H-1-Glc and H-12), 5.29 (pseudo t, 1H, J = 9.5, 9.6 Hz,
H-3-Glc), 6.75 (d, 1H, J = 8.7 Hz, NH), 7.68 (s, 1H, NCH);
13C NMR (75 MHz, CDCl3): d 15.5, 15.6, 16.9, 17.0, 18.3,
20.5, 20.7, 21.1, 23.3, 23.5, 24.2, 27.3, 28.0, 28.2, 30.6, 33.0,
36.6, 37.0, 38.7, 38.76, 38.84, 39.1, 39.6, 42.1, 47.6, 48.2, 52.6,
52.9, 55.3, 57.2, 61.6, 68.1, 70.4, 72.5, 73.9, 78.5, 79.1, 125.3,
125.8, 138.0, 144.0, 165.3, 169.4, 169.8, 170.5, 171.0, 177.4.
ESI-MS (positive mode) m/z: 947.0 [M + Na]+.
1
1755, 1537, 1463, 1367, 1229, 1176, 1039, 757, 667; H NMR
(300 MHz, CDCl3): d 0.55, 0.77, 0.89, 0.97, 1.11 (5 s, each 3H,
5 ꢃ CH3), 0.88 (s, 6H, 2 ꢃ CH3), 0.55–2.07 (m, 28H), 2.02,
2.03, 2.04, 2.07 (4 s, each 3H, 4 ꢃ OCOCH3), 2.14–2.18
(m, 2H, CH2CO), 2.82–2.85 (m, 1H, H-18), 3.20 (dd, 1H,
J = 4.9, 10.6 Hz, H-3), 3.79–3.84 (m, 1H, H-5-Glc), 4.07 (dd,
1H, J = 2.1, 12.5 Hz, H-6a-Glc), 4.28–4.34 (m, 3H,
overlapping, NCH2 and H-6b-Glc), 4.90 (t, 1H, J = 9.7,
9.7 Hz, H-4-Glc), 5.06 (t, 1H, J = 9.7, 9.7 Hz, H-3-Glc),
5.20 (s, 2H, COOCH2), 5.24–5.34 (m, 3H, overlapping, H-12,
H-1-Glc and H-120), 6.23 (d, 1H, J = 9.2 Hz, NH), 7.56
(s, 1H, NCH); 13C NMR (75 MHz, CDCl3): d 15.3, 15.6, 16.7,
18.3, 20.6, 20.66, 20.7, 22.9, 23.4, 23.6, 24.2, 25.8, 25.9, 27.2,
27.6, 28.1, 29.9, 30.6, 32.3, 32.7, 33.0, 33.8, 36.0, 37.0, 38.4,
38.7, 39.3, 41.3, 41.7, 45.8, 46.7, 47.5, 49.9, 55.2, 57.4, 61.6,
68.1, 70.7, 72.6, 73.6, 77.2, 78.2, 79.0, 122.4, 123.9, 143.1,
143.6, 169.5, 169.8, 170.6, 171.1, 172.7, 177.8. ESI-MS
(positive mode) m/z: 1003.5 [M + Na]+.
[1-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosylaminocarbonyl-
pentyl)-1H-1,2,3-triazol-4-ylmethyl] 3b-hydroxyurs-12-en-28-
oate (26). Prepared from 5 (0.10 g, 0.21 mmol) and 13
(0.10 g, 0.21 mmol) according to General procedure IVa.
The residue was purified by column chromatography (EtOAc–
hexane, 1 : 1). Yield: 0.10 g, 50%, white solid, mp 102–104 1C,
Rf = 0.56 (EtOAc–hexane, 2 : 1); IR (KBr, cmꢂ1): 3352, 2940,
[1-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosylaminocarbonyl-
decyl)-1H-1,2,3-triazol-4-ylmethyl] 3b-hydroxyolean-12-en-28-
oate (24). Prepared from 4 (0.09 g, 0.18 mmol) and 14
(0.10 g, 0.18 mmol) according to General procedure IVa.
The residue was purified by column chromatography (EtOAc–
hexane, 1 : 1). Yield: 0.12 g, 64%, white solid, mp 90–92 1C,
Rf = 0.22 (EtOAc–hexane, 1 : 1); IR (KBr, cmꢂ1): 3359,
1
2870, 1754, 1534, 1455, 1377, 1228, 1043, 756, 666; H NMR
(300 MHz, CDCl3): d 0.59, 0.77, 0.90, 0.93, 0.98, 1.06 (6 s, each
3H, 6 ꢃ CH3), 0.83 (d, J = 6.4 Hz, 3H, CH3), 0.59–2.00 (m,
28H), 2.02, 2.03, 2.04, 2.08 (4 s, each 3H, 4 ꢃ OCOCH3),
2.14–2.23 (m, 3H, overlapping, H-18 and CH2CON), 3.21 (dd,
1H, J = 4.0, 10.1 Hz, H-3), 3.79–3.84 (m, 1H, H-5-Glc), 4.07
(dd, 1H, J = 2.1, 12.7 Hz, H-6a-Glc), 4.29–4.34 (m, 3H,
1
2922, 2860, 1744, 1693, 1364, 1220, 1049, 1032, 772; H NMR
ꢀc
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1458 | New J. Chem., 2010, 34, 1450–1464

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overlapping, H-6b-Glc and NCH2), 4.90 (pseudo t, 1H, J =
9.5, 9.7 Hz, H-4-Glc), 5.06 (pseudo t, 1H, J = 9.5, 9.7 Hz,
H-3-Glc), 5.14 and 5.15 (2 s, each 1H, COOCH2), 5.21–5.34
(m, 3H, overlapping, H-12, H-1-Glc, H-2-Glc), 6.24 (d, 1H,
J = 8.8 Hz, NH), 7.54 (s, 1H, NCH); 13C NMR (75 MHz,
CDCl3): d 15.5, 15.6, 16.9, 17.0, 18.3, 20.6, 20.67, 20.7, 21.1,
23.3, 23.5, 24.2, 24.22, 25.9, 27.2, 28.0, 28.1, 29.9, 30.6, 33.0,
36.0, 36.6, 37.0, 38.6, 38.7, 38.8, 39.1, 39.5, 42.1, 47.5, 48.1,
49.9, 52.8, 55.2, 57.4, 61.6, 68.2, 70.7, 72.6, 73.6, 77.2, 78.2,
79.0, 123.8, 125.6, 138.0, 143.2, 169.5, 169.8, 170.6, 171.1,
172.7, 177.5. ESI-MS (positive mode) m/z: 1003.6 [M + Na]+.
(75 MHz, CDCl3): d 16.6, 16.7, 18.3, 20.5, 20.7, 23.0, 23.4,
23.6, 25.8, 27.6, 28.6, 30.6, 32.3, 32.6, 33.0, 33.8, 38.3, 39.2,
39.4, 41.3, 41.8, 45.8, 46.5, 46.7, 52.6, 55.3, 57.4, 61.6, 68.1,
68.9, 70.4, 72.4, 73.9, 78.5, 83.9, 122.3, 125.3, 143.6, 144.0,
165.3, 169.4. ESI-MS (positive mode) m/z: 963.7 [M + Na]+,
979.7 [M + K]+.
[1-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosylaminocarbonyl-
pentyl)-1H-1,2,3-triazol-4-ylmethyl] 2a,3b-dihydroxyolean-12-
en-28-oate (29). Prepared from 6 (0.11 g, 0.21 mmol) and 13
(0.10 g, 0.21 mmol) according to General procedure IVa. The
residue was purified by column chromatography (EtOAc–
hexane, 2 : 1). Yield: 0.15 g, 71%, white solid, mp 160–162 1C,
Rf = 0.06 (EtOAc–hexane, 1 : 1); IR (KBr, cmꢂ1): 3369, 2947,
[1-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosylaminocarbonyl-
decyl)-1H-1,2,3-triazol-4-ylmethyl]
3b-hydroxyurs-12-en-28-
oate (27). Prepared from 5 (0.09 g, 0.18 mmol) and 14
(0.10 g, 0.18 mmol) according to General procedure IVa.
The residue was purified by column chromatography (EtOAc–
hexane, 1 : 2). Yield: 0.14 g, 74%, white solid, mp 92–93 1C.
Rf = 0.30 (EtOAc–hexane, 1 : 1); IR (KBr, cmꢂ1): 3369, 2928,
2857, 1756, 1693, 1537, 1455, 1376, 1223, 1141, 1102, 1043,
996, 761, 666; 1H NMR (300 MHz, CDCl3): d 0.57, 0.78, 0.90,
0.93, 0.99, 1.06 (6 s, each 3H, 6 ꢃ CH3), 0.83 (d, 3H, J =
6.4 Hz, CH3), 0.57–2.02 (m, 38H), 2.02, 2.03, 2.04, 2.07 (4 s,
each 3H, 4 ꢃ OCOCH3), 2.13–2.24 (m, 3H, overlapping, H-18
and CH2CON), 3.21 (dd, 1H, J = 5.0, 11.0 Hz, H-3),
3.79–3.85 (m, 1H, H-5-Glc), 4.07 (dd, 1H, J = 2.0, 12.5 Hz,
H-6a-Glc), 4.29–4.38 (m, 3H, overlapping, H-6b-Glc and
NCH2), 4.92 (pseudo t, 1H, J = 9.6, 9.7 Hz, H-4-Glc), 5.06
(pseudo t, 1H, J = 9.6, 9.7 Hz, H-3-Glc), 5.16 (s, 2H,
COOCH2), 5.21–5.23 (m, 1H, H-1-Glc), 5.26–5.34 (m, 2H,
overlapping, H-12 and H-2-Glc), 6.30 (d, 1H, J = 9.4 Hz,
NH), 7.55 (s, 1H, NCH); 13C NMR (75 MHz, CDCl3): d15.5,
15.7, 16.9, 17.0, 18.3, 20.5, 20.6, 20.7, 21.1, 23.3, 23.5, 24.2,
25.1, 26.5, 27.3, 28.0, 28.2, 29.0, 29.1, 29.2, 29.3, 29.34, 30.2,
30.7, 33.0, 36.6, 37.0, 38.7, 38.8, 38.84, 39.1, 39.6, 42.1, 47.6,
48.2, 50.4, 52.9, 55.2, 57.4, 60.1, 61.7, 68.3, 70.8, 72.8, 73.6,
77.2, 78.2, 79.0, 123.8, 125.6, 138.1, 143.1, 169.5, 169.8, 170.5,
171.0, 173.3, 177.5. ESI-MS (positive mode) m/z: 1073.5
[M + Na]+.
1
1794, 1745, 1364, 1228, 1175, 1160, 1046, 757, 666; H NMR
(300 MHz, CDCl3): d 0.55, 0.82, 0.89, 0.90, 0.95, 1.02, 1.11
(7 s, each 3H, 7 ꢃ CH3), 0.81–2.02 (m, 26H), 2.02, 2.03, 2.04,
2.08 (4 s, each 3H, 4 ꢃ OCOCH3), 2.14–2.20 (m, 2H,
CH2CON), 2.85 (dd, 1H, J = 3.7, 10.3 Hz, H-18), 2.98
(d, 1H, J = 9.5 Hz, H-3a), 3.64–3.71 (m, 1H, H-2b),
3.80–3.85 (m, 1H, H-5-Glc), 4.07 (dd, 1H, J = 2.0, 12.5 Hz,
H-6a-Glc), 4.28–4.34 (m, 3H, overlapping, NCH2 and H-6b-
Glc), 4.92 (t, 1H, J = 9.6, 9.6 Hz, H-4-Glc), 5.06 (t, 1H, J =
9.7, 9.7 Hz, H-3-Glc), 5.17 (s, 2H, COOCH2), 5.21–5.34 (m,
3H, overlapping, H-12 and H-1-Glc, H-2-Glc), 6.23 (d, 1H,
J = 9.3 Hz, NH), 7.55 (s, 1H, NCH); 13C NMR (75 MHz,
CDCl3): d 16.6, 16.7, 16.8, 18.3, 20.5, 20.6, 20.7, 23.0, 23.5,
23.6, 24.3, 25.8, 25.9, 27.6, 28.6, 29.9, 30.6, 32.4, 32.6, 33.0,
33.8, 36.0, 38.3, 39.1, 39.4, 41.3, 41.8, 45.8, 46.4, 46.7, 47.5,
49.9, 55.3, 57.6, 61.7, 68.2, 68.9, 70.8, 72.7, 73.6, 76.7, 77.2,
78.2, 83.9, 114.6, 120.2, 121.8, 122.2, 123.7, 143.2, 143.5, 143.7,
169.5, 169.8, 171.1, 172.6, 177.7. ESI-MS (positive mode) m/z:
1019.4 [M + Na]+.
[1-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosylaminocarbonyl-
decyl)-1H-1,2,3-triazol-4-ylmethyl] 2a,3b-dihydroxyolean-12-
en-28-oate (30). Prepared from 6 (0.09 g, 0.18 mmol) and 14
(0.10 g, 0.18 mmol) according to General procedure IVa. The
residue was purified by column chromatography (EtOAc–
hexane, 1 : 1). Yield: 0.16 g, 82%, white solid, mp 110–112 1C,
Rf = 0.23 (EtOAc–hexane, 1 : 1); IR (KBr, cmꢂ1): 3374, 2931,
2858, 1795, 1753, 1536, 1461, 1364, 1220, 1175, 1160, 1049,
1034, 770, 667; 1H NMR (300 MHz, CDCl3): d 0.50, 0.81,
0.89, 0.90, 0.95, 1.02, 1.11 (7 s, each 3H, 7 ꢃ CH3), 0.81–2.02
(m, 36H), 2.02, 2.03, 2.04, 2.08 (4 s, each 3H, 4 ꢃ OCOCH3),
2.18–2.29 (m, 2H, CH2CON), 2.84 (dd, 1H, J = 3.5, 13.3 Hz,
H-18), 2.97 (d, 1H, J = 9.5 Hz, H-3a), 3.64–3.71 (m, 1H,
H-2b), 3.80–3.85 (m, 1H, H-5-Glc), 4.07 (dd, 1H, J = 2.0,
12.5 Hz, H-6a-Glc), 4.28–4.34 (m, 3H, overlapping, NCH2 and
H-6b-Glc), 4.92 (pseudo t, 1H, J = 9.6, 9.7 Hz, H-4-Glc), 5.06
(pseudo t, 1H, J = 9.6, 9.7 Hz, H-3-Glc), 5.16 and 5.17 (2 s,
each 1H, COOCH2), 5.22–5.34 (m, 3H, overlapping, H-12 and
H-1-Glc, H-2-Glc), 6.32 (d, 1H, J = 9.3 Hz, NH), 7.56 (s, 1H,
NCH); 13C NMR (75 MHz, CDCl3): d 16.7, 16.79, 16.84, 18.4,
20.5, 20.6, 20.7, 23.0, 23.5, 23.6, 25.1, 23.8, 26.6, 27.6, 28.6,
29.0, 29.1, 29.2, 29.3, 30.3, 30.7, 32.5, 32.6, 33.0, 33.9, 36.6,
38.3, 39.2, 39.4, 41.3, 41.8, 45.9, 46.4, 46.7, 47.5, 50.3, 55.3,
57.6, 61.7, 68.3, 68.9, 68.4, 70.8, 72.8, 73.6, 78.2, 83.9, 122.2,
[1-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosylaminocarbonyl-
methyl)-1H-1,2,3-triazol-4-ylmethyl] 2a,3b-dihydroxyolean-12-
en-28-oate (28). Prepared from 6 (0.12 g, 0.23 mmol) and 12
(0.10 g, 0.23 mmol) according to General procedure IVa. The
residue was purified by column chromatography (EtOAc–
hexane, 1 : 1). Yield: 0.13 g, 57%, white solid, mp 178–180 1C,
Rf = 0.39 (EtOAc–hexane, 2 : 1); IR (KBr, cmꢂ1): 3345, 2947,
1755, 1556, 1460, 1371, 1230, 1175, 1160, 1048, 1034, 759;
1H NMR (300 MHz, CDCl3): d 0.58, 0.83, 0.89, 0.90, 0.97,
1.03, 1.12 (7 s, each 3H, 7 ꢃ CH3), 0.83–2.01 (m, 20H), 2.01,
2.02, 2.03, 2.08 (4 s, each 3H, 4 ꢃ OCOCH3), 2.84 (dd, 1H,
J = 4.1, 9.7 Hz, H-18), 2.99 (d, 1H, J = 9.5 Hz, H-3a),
3.68–3.71 (m, 1H, H-2b), 3.78–3.83 (m, 1H, H-5-Glc),
4.06–4.13 (m, 1H, H-6a-Glc), 4.28 (dd, 1H, J = 4.3, 12.5
Hz, H-6b-Glc), 4.87 (t, 1H, J = 9.6, 9.6 Hz, H-4-Glc),
4.95–5.07 (m, 3H, overlapping, NCH2CO and H-3-Glc),
5.15–5.21 (m, 3H, overlapping, COOCH2 and H-2-Glc),
5.25–5.32 (m, 3H, overlapping, H-1-Glc and H-12), 6.72
(d, 1H, J = 8.7 Hz, NH), 7.69 (s, 1H, NCH); 13C NMR
ꢀc
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1
1463, 1386, 1158, 1123, 1050, 1032, 1012, 756, 697; H NMR
123.8, 143.1, 169.5, 169.8, 171.0, 173.4, 177.8. ESI-MS
(positive mode) m/z: 1089.6 [M + Na]+.
(300 MHz, C5D5N): d 0.59, 0.86, 1.04, 1.10 (4 s, each 3H,
4 ꢃ CH3), 0.74 (s, 9H, 3 ꢃ CH3), 0.59–1.95 (m, 38H), 2.36
(t, 2H, J = 7.3 Hz, CH2CON), 2.94 (dd, 1H, J = 3.6, 10.0 Hz,
H-18), 3.28 (t, 1H, J = 8.0 Hz, H-3), 3.92–3.93 (m, 2H,
overlapping, H-5-Glc and H-6a-Glc), 4.04–4.10 (m, 3H, m,
overlapping, NCH2 and H-6b-Glc), 4.26–4.38 (m, 3H, m,
overlapping, H-2-Glc, H-3-Glc, and H-4-Glc), 5.26 (s, 1H,
H-12), 5.42 (d, 1H, J = 14.1 Hz, COOCH2), 5.46 (d, 1H,
J = 14.1 Hz, COOCH2), 5.63 (d, 1H, J = 8.6 Hz, H-1-Glc),
8.18 (s, 1H, NCH), 9.67 (d, 1H, J = 8.8 Hz, NH); 13C NMR
(75 MHz, C5D5N): d 15.7, 16.6, 17.3, 18.8, 23.4, 23.7, 23.8,
25.9, 26.0, 26.7, 28.1, 28.8, 29.2, 29.6, 29.64, 29.7, 30.6, 30.8,
32.8, 33.1, 33.2, 34.0, 36.9, 37.4, 39.0, 39.4, 39.8, 41.9, 42.1,
46.1, 47.0, 48.0, 50.3, 55.8, 58.1, 62.7, 71.8, 74.5, 78.1, 79.6,
80.0, 81.4, 122.8, 124.7, 143.4, 144.0, 174.1, 177.4. ESI-MS
(positive mode) m/z: 917.5 [M + Cl]+; HRMS (MALDI)
m/z = C50H82N4O9 [M + Na]+ calcd. 905.5980, found
905.6013.
[1-(b-D-Glucopyranosylaminocarbonylmethyl)-1H-1,2,3-triazol-
4-ylmethyl] 3b-hydroxyolean-12-en-28-oate (31). Prepared
from 22 (0.13 g, 0.14 mmol) according to General procedure
V. The residue was purified by column chromatography
(MeOH–CH2Cl2, 1 : 15). Yield: 0.055 g, 51%, white solid,
mp 197–199 1C, Rf = 0.09 (MeOH–CH2Cl2, 1 : 10); [a]D
=
+37 (c = 0.11, MeOH); IR (KBr, cmꢂ1): 3407, 2943, 2860,
1705, 1556, 1389, 1161, 1059, 1032, 1018, 772; 1H NMR
(300 MHz, C5D5N): d 0.80, 0.90, 1.03, 1.19, 1.24 (5 s, each
3H, 5 ꢃ CH3), 0.88 (s, 6H, 2 ꢃ CH3), 0.80–1.96 (m, 22H), 3.13
(d, 1H, J = 10.3 Hz, H-18), 3.43 (t, 1H, J = 7.8 Hz, H-3),
4.02–4.11 (m, 2H, overlapping, H-5-Glc and H-6a-Glc),
4.25–4.27 (m, 2H, overlapping, H-4-Glc and H-2-Glc), 4.37
(dd, 1H, J = 4.3, 11.8 Hz, H-6b-Glc), 4.49 (d, 1H, J = 11.5
Hz, H-10), 5.43 (s, 1H, H-12), 5.48 (s, 2H, NCH2CO), 5.62 (s,
2H, COOCH2), 5.97 (t, 1H, J = 8.9, 8.9 Hz, H-3-Glc), 8.40 (s,
1H, NCH), 10.60 (d, 1H, J = 8.8 Hz, NH); 13C NMR
(75 MHz, C5D5N): d 15.6, 16.5, 17.1, 18.7, 23.3, 23.6, 23.7,
26.0, 28.0, 28.1, 28.7, 29.9, 30.7, 32.7, 33.05, 33.1, 33.9, 37.3,
38.9, 39.3, 39.7, 41.9, 42.0, 46.1, 46.9, 48.0, 52.7, 55.8, 58.0,
62.4, 71.5, 74.5, 78.1, 79.5, 80.3, 81.5, 123.1, 123.8, 126.7,
135.8, 143.5, 144.0, 150.2, 166.9, 177.3. ESI-MS (negative
mode) m/z: 755.5 [M ꢂ H]+; HRMS (MALDI) m/z =
C41H64N4O9 [M + Na]+ calcd. 779.4571, found 779.4594.
[1-(b-D-Glucopyranosylaminocarbonylpentyl)-1H-1,2,3-triazol-
4-ylmethyl] 3b-hydroxyurs-12-en-28-oate (34). Prepared from
26 (0.065 g, 0.07 mmol) according to General procedure V.
The residue was purified by column chromatography
(MeOH–CH2Cl2, 1 : 20). Yield: 0.053 g, 98%, white solid,
mp 214–216 1C, Rf = 0.67 (MeOH–CH2Cl2, 1 : 15); [a]D
=
+26 (c = 0.1, MeOH). IR (KBr, cmꢂ1): 3386, 2924, 2869,
1724, 1657, 1456, 1392, 1049, 1032, 1017; 1H NMR (300 MHz,
C5D5N): d 0.83, 0.91, 0.94, 1.04, 1.13, 1.23 (6 s, each 3H,
6 ꢃ CH3), 0.88 (d, 3H, J = 3.9 Hz, CH3), 0.83–2.00 (m, 28H),
2.39–2.47 (m, 3H, overlapping, H-18 and CH2CON), 3.44 (dd,
1H, J = 6.1, 9.8 Hz, H-3), 4.04 (m, 1H, H-5-Glc), 4.16 (t, 1H,
J = 9.8, 9.8 Hz, H-4-Glc), 4.24–4.45 (m, 6H, overlapping,
NCH2, H-2-Glc, H-3-Glc and H-6a-Glc, H-6b-Glc), 5.40
(s, 1H, H-12), 5.50 (s, 2H, COOCH2), 5.96 (1H, overlapping,
H-10), 8.13 (s, 1H, NCH), 9.69 (d, 1H, J = 8.2 Hz, NH);
13C NMR (75 MHz, C5D5N): d 15.8, 16.6, 17.3, 18.8, 21.2,
23.7, 23.8, 24.6, 25.1, 26.4, 28.1, 28.4, 28.8, 30.0, 30.3, 30.8,
33.5, 36.4, 37.0, 37.3, 39.1, 39.2, 39.3, 39.4, 40.0, 42.4, 48.0,
48.4, 50.1, 53.4, 55.8, 58.0, 62.7, 71.8, 74.5, 78.2, 79.6, 80.0,
81.3, 124.6, 126.2, 138.6, 143.3, 173.7, 177.1. ESI-MS (positive
mode) m/z: 847.5 [M + Cl]+. HRMS (MALDI) m/z =
C45H72N4O9 [M + Na]+ calcd. 835.5197, found 835.5206.
[1-(b-D-Glucopyranosylaminocarbonylpentyl)-1H-1,2,3-triazol-
4-ylmethyl] 3b-hydroxyolean-12-en-28-oate (32). Prepared
from 23 (0.11 g, 0.11 mmol) according to the general
procedure V. The residue was purified by column chromato-
graphy (MeOH–CH2Cl2, 1 : 15). Yield: 0.076 g, 83%, white
solid, mp 164–166 1C, Rf = 0.25 (MeOH–CH2Cl2, 1 : 10);
[a]D = +42 (c = 0.06, MeOH); IR (KBr, cmꢂ1): 3367, 2939,
2864, 1725, 1663, 1382, 1053, 1032, 1013, 773; 1H NMR
(300 MHz, C5D5N): d 0.81, 0.94, 1.05, 1.20, 1.24 (5 s, each
3H, 5 ꢃ CH3), 0.89 (s, 6H, 2 CH3), 0.81–1.93 (m, 28H), 2.41
(t, 2H, J = 7.3 Hz, CH2CO), 3.15 (d, 1H, J = 13.3 Hz, H-18),
3.45 (brs, 1H, H-3), 4.05–4.13 (m, 2H, H-5-Glc and H-6a-Glc),
4.23–4.30 (m, 4H, H-2-Glc, H-4-Glc and NCH2), 4.37 (dd, 1H,
J = 4.6, 11.8 Hz, H-6b-Glc), 4.48 (d, 1H, J = 11.8 Hz,
H-1-Glc), 5.43 (s, 1H, H-12), 5.53 and 5.54 (2 d, each 1H,
J = 12.6 Hz, COOCH2), 6.01 (t, 1H, J = 9.0, 9.0 Hz, H-3-
Glc), 8.13 (s, 1H, NCH), 9.62 (d, 1H, J = 9.1 Hz, NH);
13C NMR (75 MHz, C5D5N): d 15.6, 16.5, 17.2, 18.8, 23.3,
23.6, 23.8, 25.1, 26.0, 26.4, 28.0, 28.1, 28.8, 30.3, 30.7, 32.8,
33.1, 33.2, 33.9, 36.3, 37.3, 39.0, 39.4, 39.7, 41.9, 42.0, 46.1,
47.0, 48.0, 50.0, 55.8, 58.1, 62.7, 71.8, 74.6, 78.1, 79.7, 80.1,
81.3, 123.0, 124.6, 143.4, 144.0, 173.6, 177.4. ESI-MS (negative
mode) m/z: 811.4 [M ꢂ H]+; HRMS (MALDI) m/z =
C45H72N4O9 [M + Na]+ calcd. 835.5197, found 835.5203.
[1-(b-D-Glucopyranosylaminocarbonyldecyl)-1H-1,2,3-triazol-
4-ylmethyl] 3b-hydroxyurs-12-en-28-oate (35). Prepared from
27 (0.10 g, 0.1 mmol) according to General procedure V. The
residue was purified by column chromatography
(MeOH–CH2Cl2, 1 : 12). Yield: 0.078 g, 93%, white solid,
mp 199–200 1C, Rf = 0.12 (MeOH–CH2Cl2, 1 : 15); [a]D
=
+32 (c = 0.06, MeOH); IR (KBr, cmꢂ1): 3409, 2924, 2861,
1726, 1662, 1453, 1382, 1052, 1032, 1013. 1H NMR (300 MHz,
C5D5N): d 0.81, 0.91, 0.94, 1.03, 1.13, 1.23 (6 s, each 3H,
6 ꢃ CH3), 0.88 (d, 3H, J = 3.6 Hz, CH3), 0.81–1.99 (m, 38H),
2.43–2.49 (m, 3H, overlapping, H-18 and CH2CON), 3.44 (dd,
1H, J = 6.8, 9.7 Hz, H-3), 4.04 (m, 1H, H-5-Glc), 4.14 (t, 1H,
J = 8.8, 8.8 Hz, H-4-Glc), 4.21–4.33 (m, 2H, NCH2),
4.35–4.47 (m, 3H, overlapping, H-3-Glc, H-2-Glc and
H-1-Glc), 5.39 (brs, 1H, H-12), 5.51 and 5.52 (2 d, each 1H,
[1-(b-D-Glucopyranosylaminocarbonyldecyl)-1H-1,2,3-triazol-
4-ylmethyl] 3b-hydroxyolean-12-en-28-oate (33). Prepared
from 24 (0.083 g, 0.08 mmol) according to General procedure
V. The residue was purified by column chromatography
(MeOH–CH2Cl2, 1 : 20). Yield: 0.068 g, 97%, white solid,
mp 195–196 1C, Rf = 0.27 (MeOH–CH2Cl2, 1 : 15); [a]D =
+31 (c = 0.1, MeOH); IR (KBr, cmꢂ1): 3392, 2928, 1727,
ꢀc
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1460 | New J. Chem., 2010, 34, 1450–1464

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J = 12.5 Hz, COOCH2), 8.19 (s, 1H, NCH), 9.62 (d, 1H, J =
9.0 Hz, NH); 13C NMR (75 MHz, C5D5N): d 15.8, 16.6, 17.3,
17.4, 18.8, 21.2, 23.7, 23.8, 24.6, 26.0, 26.8, 28.2, 28.5, 28.9,
29.3, 29.6, 29.67, 29.7, 30.0, 30.6, 30.8, 33.5, 36.9, 37.0, 37.4,
39.2, 39.3, 39.4, 40.0, 42.4, 48.0, 48.4, 50.3, 53.4, 55.9, 58.1,
62.8, 71.9, 74.6, 78.2, 79.7, 80.0, 81.4, 124.7, 126.2,
143.4, 174.1, 177.2. ESI-MS (positive mode) m/z: 927.5
[M + HCOO]+; HRMS (MALDI) m/z = C50H82N4O9
[M + Na]+ calcd. 905.5980, found 905.6004.
2-[4-(2a,3b-dihydroxyolean-12-en-28-carbonyloxymethyl)-1H-
1,2,3-triazol-1-yl] acetic acid (38). Prepared from 28 (0.084 g,
0.09 mmol) according to General procedure V. The residue
was purified by column chromatography (MeOH–CH2Cl2,
1 : 10). Yield: 0.05 g, 91%, white solid, mp 225–227 1C, Rf =
0.06 (MeOH–CH2Cl2, 1 : 15); [a]D = +82 (c = 0.06, MeOH);
IR (KBr, cmꢂ1): 3403, 2940, 2862, 1724, 1450, 1386, 1229,
1
1159, 1050, 1033; H NMR (300 MHz, C5D5N): d 0.84, 1.02,
1.07, 1.13, 1.17 (5 s, each 3H, 5 ꢃ CH3), 0.87 (s, 6H, 2 ꢃ CH3),
0.84–2.22 (m, 20H), 3.12 (dd, 1H, J = 3.7, 13.5 Hz, H-18),
3.37 (d, 1H, J = 9.4 Hz, H-3a), 4.06–4.14 (m, 1H, H-2b), 5.40
(s, 1H, H-12), 5.54 (s, 2H, NCH2CO), 5.64 (s, 2H, COOCH2),
5.67 (s, 2H, COOCH2), 8.45 (s, 1H, NCH); 13C NMR
(75 MHz, C5D5N): d 16.9, 17.3, 17.7, 18.9, 23.4, 23.7, 23.9,
26.0, 28.1, 29.3, 30.0, 30.8, 32.7, 33.1, 34.0, 38.6, 39.8, 39.9,
41.9, 42.1, 46.1, 47.0, 47.8, 48.1, 52.3, 55.9, 58.2, 68.6, 83.9,
122.6, 126.3, 143.5, 144.0, 177.4. ESI-MS (positive mode) m/z:
634.9 [M + Na]+; HRMS (MALDI) m/z = C35H53N3O6
[M + Na]+ calcd. 634.3832, found 634.3845.
[1-(b-D-Glucopyranosylaminocarbonylpentyl)-1H-1,2,3-triazol-
4-ylmethyl] 2a,3b-dihydroxyolean-12-en-28-oate (36). Prepared
from 29 (0.10 g, 0.1 mmol) according to General procedure V.
The residue was purified by column chromatography
(MeOH–CH2Cl2, 1 : 10). Yield: 0.075 g, 93%, white solid,
mp 180–182 1C, Rf = 0.09 (MeOH–CH2Cl2, 1 : 15); [a]D
=
+23 (c = 0.05, MeOH); IR (KBr, cmꢂ1): 3369, 2944, 2873,
1725, 1664, 1546, 1461, 1260, 1159, 1122, 1049, 1033; 1H NMR
(300 MHz, C5D5N): d 0.79, 0.90, 1.02, 1.09, 1.16 (5 s, each 3H,
5 ꢃ CH3), 0.87 (s, 6H, 2 ꢃ CH3), 0.79–2.30 (m, 26H), 2.40
(t, 2H, J = 7.3 Hz, CH2CON), 3.11 (dd, 1H, J = 4.2, 13.5 Hz,
H-18), 3.37 (d, 1H, J = 9.3 Hz, H-3a), 4.10 (m, 1H, H-2b),
4.15–4.49 (m, 6H, overlapping, H-1-Glc, H-2-Glc, H-4-Glc,
H-5-Glc and H-6-Glc), 5.38 (s, 1H, H-12), 5.52 (s, 2H,
COOCH2), 5.99 (t, 1H, J = 9.0, 9.0 Hz, H-3-Glc), 8.11 (s,
1H, NCH), 9.62 (d, 1H, J = 9.0 Hz, NH); 13C NMR (75 MHz,
C5D5N): d 16.9, 17.3, 17.7, 18.9, 23.4, 23.7, 23.9, 25.1, 26.0,
26.5, 28.0, 29.3, 30.0, 30.4, 36.4, 38.6, 39.8, 41.9, 42.1, 46.1,
47.0, 47.8, 48.1, 50.0, 55.9, 58.2, 62.8, 68.6, 71.8, 74.6, 79.7,
80.0, 81.3, 83.8, 122.9, 124.6, 143.3, 144.0, 173.6, 177.4.
ESI-MS (positive mode) m/z: 873.5 [M + HCOO]+; HRMS
(MALDI) m/z = C45H72N4O10 [M + Na]+ calcd. 851.5146,
found 851.5158.
´
General procedure VI for the Zemplen-deacetylation
To a solution of an O-acetyl-protected compound in dry
MeOH 1–2 drops of a B1 M methanolic NaOMe solution
were added, and the reaction mixture was kept at rt until
completion of the transformation (TLC, CHCl3–MeOH, 1 : 1).
Amberlyst 15 (H+ form) was then added to remove sodium
ions, the resin was filtered off, and the solvent removed in
vacuo. If the residue was chromatographically non-uniform it
was purified by column chromatography or crystallisation.
General procedure VII for reduction
An N-(o-azidoalkanoyl)-b-D-glucopyranosyl-amine was dissolved
in dry MeOH (12 mL/mmol). To the solution RANEYs-Ni
(B2 mmol) was added, and H2 gas was bubbled through the
mixture at 70 1C until the complete transformation of the
starting azide TLC (CHCl3–MeOH, 1 : 1). The solution was
filtered over a Celite pad and the solvent was removed in
vacuo.
[1-(b-D-Glucopyranosylaminocarbonyldecyl)-1H-1,2,3-triazol-
4-ylmethyl] 2a,3b-dihydroxyolean-12-en-28-oate (37). Prepared
from 30 (0.11 g, 0.1 mmol) according to General procedure V.
The residue was purified by column chromatography
(MeOH–CH2Cl2, 1 : 15). Yield: 0.065 g, 72%, white solid,
N-Azidoacetyl-b-D-glucopyranosylamine (39). Prepared from
12 (0.10 g, 0.23 mmol) according to General procedure VI.
The residue was purified by column chromatography
(CHCl3–MeOH, 7 : 3). Yield: 0.058 g, 95%, colourless oil, Rf
= 0.34 (CHCl3–MeOH, 7 : 3); [a]D = ꢂ12 (c = 0.22, MeOH),
(lit.40 [a]D = ꢂ61 (c = 1, H2O)); 1H NMR (360 MHz,
CD3OD): d(ppm) 3.29–3.37 (m, 3H, H-3, H-4, H-5), 3.44 (t,
1H, J = 9.2, 9.2 Hz, H-2), 3.68 (dd, 1H, J = 4.0, 11.9 Hz,
H-6b), 3.83–3.99 (m, 3H, CH2, H-6a), 4.95 (d, 1H, J = 9.2 Hz,
H-1). 13C NMR (90 MHz, D2O): d(ppm) 52.8 (CH2), 62.6
(C-6), 71.2, 73.8, 78.8, 79.7 (C-2, C-3, C-4, C-5), 81.0 (C-1),
171.3 (CONH). Analysis: Calcd for C8H14N4O6 (262.22): C,
36.64; H, 5.38; N, 21.37. Found: C, 36.73; H, 5.42; N, 21.25.
mp 163–165 1C, Rf = 0.18 (MeOH–CH2Cl2, 1 : 15); [a]D
=
+33 (c = 0.07, MeOH), IR (KBr, cmꢂ1): 3377, 2923, 2861,
1
1725, 1053, 1032, 1015, 772; H NMR (300 MHz, C5D5N): d
0.79, 1.04, 1.10, 1.18 (4 s, each 3H, 4 ꢃ CH3), 0.89 (s, 9H,
3 ꢃ CH3), 0.79–2.30 (m, 36H), 2.48 (t, 2H, J = 7.5 Hz,
CH2CON), 3.12 (dd, 1H, J = 3.8, 9.9 Hz, H-18), 3.38 (d, 1H,
J = 9.2 Hz, H-3a), 4.05 (m, 1H, H-2b), 4.07–4.51 (m, 8H,
overlapping, H-1-Glc, H-2-Glc, H-4-Glc, H-5-Glc, H-6-Glc
and NCH2), 5.40 (s, 1H, H-12), 5.54 and 5.57 (2 d, each 1H,
J = 12.6 Hz, COOCH2), 6.03 (t, 1H, J = 9.0, 9.0 Hz, H-3-
Glc), 8.12 (s, 1H, NCH), 9.61 (d, 1H, J = 8.9 Hz, NH); 13C
NMR (75 MHz, C5D5N): d 17.0, 17.3, 17.7, 18.9, 23.4, 23.9,
25.95, 26.0, 26.8, 28.0, 29.3, 29.4, 29.6, 29.66, 29.7, 30.0, 30.6,
30.8, 32.8, 33.1, 34.0, 36.9, 38.6, 39.9, 41.9, 42.1, 46.1, 47.0,
47.8, 48.1, 50.3, 55.9, 58.2, 62.8, 68.6, 71.9, 74.7, 79.8, 80.1,
81.4, 83.9, 122.9, 124.7, 143.4, 144.1, 174.0, 177.4. ESI-MS
(positive mode) m/z: 943.6 [M + HCOO]+; HRMS (MALDI)
m/z = C50H82N4O10 [M + Na]+ calcd. 921.5929, found
921.5937.
N-Glycyl-b-D-glucopyranosylamine (40). Prepared from 39
(0.097 g, 0.37 mmol) according to General procedure VII.
Yield: 0.07 g, 79%, amorphous oil, Rf = 0.16 (MeOH); [a]D
=
+34 (c = 0.08, DMSO); 1H NMR (360 MHz, CD3OD):
d(ppm) 3.48–3.61 (m, 6H, H-2, H-3, H-4, H-5, CH2), 3.76 (dd,
1H, J = 5.3, 11.9 Hz, H-6a), 3.90 (dd, 1H, J = 1.2, 11.9 Hz,
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H-6b), 5.04 (d, 1H, J = 9.2 Hz, H-1). 13C NMR (90 MHz,
D2O): d(ppm) 44.1 (CH2), 62.6 (C-6), 69.8, 72.4, 77.1, 78.2
(C-2, C-3, C-4, C-5), 79.9 (C-1), 176.5 (CONH). Analysis:
Calcd for C8H16N2O6 (236.22): C, 40.68; H, 6.83; N, 11.86.
Found: C, 40.75; H, 6.68; N, 11.79.
(EtOAc–MeOH, 95 : 5). Yield: 0.21 g, 96%, colourless oil,
Rf = 0.32 (EtOAc); [a]D = +16 (c = 0.16, CHCl3);
1H NMR (360 MHz, CDCl3): d(ppm) 1.16–1.30 (m, 4H,
2 ꢃ CH2), 1.62–1.74 (m, 4H, 2 ꢃ CH2), 1.85–1.91 (m, 4H,
2 ꢃ CH2), 2.01, 2.03, 2.04, 2.07 (4 s, 24H, 8 ꢃ OCOCH3),
2.18–2.22 (m, 4H, 2 ꢃ CH2), 2.52–2.56 (m, 2H, CH2),
2.72–2.76 (m, 4H, 2 ꢃ CH2), 3.84 (ddd, 2H, J = 1.1, 2.6,
10.6 Hz, 2 ꢃ H-5-Glc), 4.13–4.07 (m, 4H, 2 ꢃ CH2), 4.27–4.31
(m, 6H, 2 ꢃ H-6a-Glc, 2 ꢃ H-6b-Glc, CH2), 5.28, 5.24, 5.06,
4.93 (4 pseudo t, 8H, J = 9.2, 10.6 Hz in each, 2 ꢃ H-1-Glc, 2
ꢃ H-2-Glc, 2 ꢃ H-3-Glc, 2 ꢃ H-4-Glc), 6.57 (d, 2H, J =
7.9 Hz, 2 ꢃ NH), 7.34 (s, 2H, 2 triazole CH); 13C NMR
(90 MHz, CDCl3): d (ppm) 20.3, 20.4, (8 ꢃ OCOCH3), 24.1,
25.1, 25.7, 28.6, 29.7, 35.7, 49.5 (14 ꢃ CH2), 61.6 (2 ꢃ
C-6-Glc), 68.0, 70.4, 72.7, 73.3 (2 ꢃ C-2-Glc, 2 ꢃ C-3-Glc,
2 ꢃ C-4-Glc, 2 ꢃ C-5-Glc), 77.8 (2 ꢃ C-1-Glc), 120.6
(2 triazole C-5), 147.7 (2 triazole C-4), 169.4, 169.6, 170.0,
170.4 (8 ꢃ OCOCH3), 172.9 (2 ꢃ NHCO). Anal. calcd. for
C48H70N8O20 (1079.13): C 53.43, H 6.54, N 10.38. Found: C
53.49, H 6.62, N 10.45.
N-(6-Azidohexanoyl)-b-D-glucopyranosylamine (41). Prepared
from 13 (0.50 g, 1.03 mmol) according to General procedure
VI. The residue was purified by column chromatography
(CHCl3–MeOH, 7 : 3). Yield: 0.31 g (97%) colourless oil, Rf =
0.66 (CHCl3–MeOH, 7 : 3); [a]D = +13 (c = 0.22, MeOH);
1H NMR (360 MHz, MeOD): d(ppm) 1.42–1.49 (m, 2H, CH2),
1.60–1.72 (m, 4H, CH2), 2.25–2.32 (m, 2H, CH2), 3.36–3.24 (m,
5H, H-3, H-4, H-5, CH2), 3.44 (pseudo t, 1H, J = 7.9, 9.2 Hz,
H-2), 3.69 (dd, 1H, J = 5.3, 11.9 Hz, H-6b), 3.85 (dd, 1H, J =
1.2, 11.9 Hz, H-6a), 4.92 (d, 1H, J = 7.9 Hz, H-1); 13C NMR
(90 MHz, MeOD): d (ppm) 26.0, 27.4, 29.6, 36.9, 52.3 (5 ꢃ CH2),
62.6 (C-6), 71.4, 73.9, 79.0, 79.5 (C-2, C-3, C-4, C-5), 80.9 (C-1),
177.0 (NHCO); Anal. calcd. for C12H22N4O6 (318.33): C 45.28,
H 6.97, N 17.60. Found: C 45.36, H 6.84, N 17.49.
N-(6-Aminohexanoyl)-b-D-glucopyranosylamine (42). Prepared
from 41 (0.18 g 0.57 mmol) according to General procedure VII.
1,4-Bis-[1-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosylamino-
carbonyldecyl)-1H-1,2,3-triazol-4-yl)]butane (46). Prepared
from 14 (0.20 g 0.36 mmol) according to General procedure
IVb. The residue was purified by column chromatography
(EtOAc). Yield: 0.136 g, 62%, colourless oil; [a]D = +7
(c = 0.62, CHCl3); 1H NMR (CDCl3, 90 MHz): d (ppm)
1.26 (brs, 24H, 12 ꢃ CH2), 1.56–1.59 (m, 4H, 2 ꢃ CH2),
1.73–1.78 (m, 4H, 2 ꢃ CH2), 1.85–1.89 (m, 4H, 2 ꢃ CH2), 2.02,
2.03, 2.04, 2.08 (4s, 24H, 8 ꢃ OCOCH3), 2.10–2.25 (m, 4H,
2 ꢃ CH2), 2.73–2.76 (m, 4H, 2 ꢃ CH2), 3.83 (m, 2H, J = 2.4,
4.3, 9.9 Hz, 2 ꢃ H-5-Glc), 4.08 (dd, 2H, J = 2.3, 12.3 Hz,
2 ꢃ H-6b-Glc), 4.27–4.34 (m, 6H, 2 ꢃ H-6a-Glc, 2 ꢃ CH2),
4.93, 5.06, 5.27, 5.31 (4 pseudo t, 8 H, J = 9.6, 9.9 Hz in each,
2 ꢃ H-1-Glc, 2 ꢃ H-2-Glc, 2 ꢃ H-3-Glc, 2 ꢃ H-4-Glc), 6.42 (d,
2H, J = 9.2 Hz, 2 ꢃ NH), 7.28 (s, 2H, 2 triazole CH);
13C NMR (90 MHz, CDCl3): d (ppm) 20.4, 20.5, 20.6 (8 ꢃ
OCOCH3), 25.0, 25.3, 26.3, 28.7, 28.8, 28.9, 29.0, 29.1, 29.2,
29.6, 30.2, 50.0, (24 ꢃ CH2), 61.6 (2 ꢃ C-6-Glc), 68.1, 70.5,
72.6, 73.4 (2 C-2-Glc, 2 ꢃ C-3-Glc, 2 ꢃ C-4-Glc, 2 ꢃ C-5-Glc),
78.0 (2 ꢃ C-1-Glc), 120.4 (2 triazole C-5), 147.8 (2 triazole
C-4), 169.5, 169.8, 170.5, 170.8 (8 ꢃ OCOCH3), 173.4 (2 ꢃ
CONH). Anal. calcd. for C58H90N8O20 (1219.38): C, 57.13; H,
7.44; N, 9.19; Found: C, 57.22; H, 7.32; N, 9.30.
Yield: 0.09 g (57%) colourless oil, Rf = 0.05 (MeOH); [a]D
=
1
+11 (c =0.15, MeOH); H NMR (360 MHz, MeOD): d(ppm)
1.34–1.38 (m, 2H, CH2), 1.47–1.51 (m, 2H, CH2), 1.60–1.64 (m,
2H, CH2), 2.21–2.27 (m, 2H, CH2), 2.62–2.68 (m, 2H, CH2),
3.23–3.35 (m, 3H, H-3, H-4,H-5), 3.38 (t, 1H, J = 7.9, 7.9 Hz,
H-2), 3.62 (dd, 1H, J = 5.3, 11.9 Hz, H-6a), 3.81 (dd, 1H, J =
1.2, 11.9 Hz, H-6a), 4.84 (d, 1H, J = 7.9 Hz, H-1); 13C NMR
(MeOD, 90 MHz): d (ppm) 26.2, 27.4, 32.4, 36.9, 41.9 (5 ꢃ CH2),
62.7 (C-6), 71.4, 73.9, 79.0, 79.7 (C-2, C-3, C-4, C-5), 81.0 (C-1),
177.2 (NHCO). Anal. calcd. for C12H24N4O6 (292.33): C 49.30,
H 8.28, N 9.58. Found: C 49.36, H 8.18, N 9.45.
1,4-Bis-[1-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosylamino-
carbonylmethyl)-1H-1,2,3-triazol-4-yl)]butane (44). Prepared
from 12 (0.30 g 0.41 mmol) according to General procedure
IVb. The residue was purified by column chromatography
(EtOAc). Yield: 0.10 g, 89%, white crystalline product, mp
197–199 1C, [a]D = +35 (c = 0.20, DMSO); 1H NMR
(360 MHz, DMSO-d6): d(ppm) 1.64 (brs, 4H, 2 ꢃ CH2),
1.93, 1.95, 1.99, 2.00 (4s, 24H, 8 ꢃ OCOCH3), 2.65 (brs, 4H,
2 ꢃ CH2), 3.96–4.16 (m, 6H, 2 ꢃ H-5-Glc, 2 ꢃ H-6a-Glc,
2 ꢃ H-6b-Glc), 5.07 (brs, 4H, 2 ꢃ CH2), 4.86, 4.92, 5.34, 5.42
(4t, 8 H, J = 9.2, 9.2 Hz in each, 2 H-1-Glc, 2 ꢃ H-2-Glc,
2 ꢃ H-3-Glc, 2 ꢃ H-4-Glc), 7.78 (s, 2H, 2 triazole CH), 9.20 (d,
2H, J = 9.2 Hz, 2 ꢃ NH); 13C NMR (90 MHz, DMSO-d6):
d(ppm) 20.3, 20.5, (8 ꢃ OCOCH3), 24.7, 28.4, 51.3 (6 ꢃ CH2),
61.6 (2 ꢃ C-6-Glc), 67.7, 70.5, 72.1, 72.7 (2 C-2-Glc, 2 ꢃ
C-3-Glc, 2 ꢃ C-4-Glc, 2 ꢃ C-5-Glc), 76.8 (2 ꢃ C-1-Glc), 123.4
(2 triazole C-5), 146.4 (2 triazole C-4), 166.4 (2 ꢃ CONH),
169.2, 169.3, 169.5, 170.0 (8 ꢃ OCOCH3). Anal. calcd. for
C40H54N8O20 (966.92): C, 49.69; H, 5.63; N, 11.59; Found: C,
49.59; H, 5.71; N, 11.67.
1,4-Bis-[1-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosylamino-
carbonylpentadecyl)-1H-1,2,3-triazol-4-yl)]butane (47). Prepared
from 15 (0.30 g 0.41 mmol) according to General procedure
IV. The residue was purified by column chromatography
(EtOAc). Yield: 0.28 g, 50%, white crystalline product, mp
144–146 1C; [a]D = +14 (c = 0.24, CHCl3); 1H NMR
(360 MHz, CDCl3): d(ppm) 1.20–1.35 (m, 26H, 13CH2),
1.40–1.50 (m, 8H, 2 ꢃ CH2), 1.60–1.77 (m, 8H, 2 ꢃ CH2),
1.82–1.90 (m, 8H, 2 ꢃ CH2), 2.02, 2.03, 2.05, 2.06 (4 s, 24H,
8 ꢃ OCOCH3), 2.32–2.27 (m, 2H, CH2), 2.60–2.52 (m, 4H,
2 ꢃ CH2), 2.73–2.80 (m, 2H, CH2), 3.70 (ddd, 2H, J = 1.1, 2.6,
10.6 Hz, 2 ꢃ H-5-Glc), 4.11–4.20 (m, 8H, 4 CH2), 4.37–4.44
(m, 4H, 2 ꢃ H-6a-Glc, 2 ꢃ H-6b-Glc), 4.94, 5.00, 5.11,
5.19 (4 pseudo t, 8H, J = 9.2, 10.6 Hz in each, 2 ꢃ H-1-Glc,
1,4-Bis-[1-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosylamino-
carbonylpentyl)-1H-1,2,3-triazol-4-yl)]butane (45). Prepared
from 13 (0.20 g, 0.41 mmol) according to General procedure
IVb. The residue was purified by column chromatography
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2 ꢃ H-2-Glc, 2 ꢃ H-3-Glc, 2 ꢃ H-4-Glc), 6.73 (d, 2H, J = 7.9
Hz, 2 NH), 7.34 (s, 2H, 2 triazole CH); 13C NMR (90 MHz,
CDCl3): d (ppm) 20.4, 20.5 (8 ꢃ OCOCH3). 22.1, 22.5, 23.0,
23.6,24.8, 25.1, 25.8, 26.1, 26.6, 27.0, 27.5, 29.8, 30.7, 31.7, 35.7,
36.7, 50.1 (32 ꢃ CH2), 60.6 (2 ꢃ C-6-Glc), 69.3, 71.0, 72.0, 72.8
(2 ꢃ C-2-Glc, 2 ꢃ C-3-Glc, 2 ꢃ C-4-Clc, 2 xC-5-Glc), 77.6
(2 ꢃ C-1-Glc), 121.0 (2 triazole C-5), 146.6 (2 triazole C-4), 169.4,
169.6, 169.9, 170.1 (8 ꢃ OCOCH3), 171.8 (2 ꢃ HHCO). Anal.
calcd. for C68H110N8O20 (1359.68): C, 60.07; H, 8.15; N, 8.24;
Found: C, 60.16; H, 8.22; N, 8.33.
d (ppm) 24.8, 25.0, 25.9, 28.4, 28.5, 28.9, 29.7, 35.5, 49.2 (24 ꢃ
CH2), 60.9 (2 ꢃ C-6-Glc), 69.9, 72.3, 77.4, 78.4 (2 ꢃ C-2-Glc,
2 ꢃ C-3-Glc, 2 ꢃ C-4-Glc, 2x C-5-Glc), 79.4 (2 ꢃ C-1-Glc),
121.7 (2 triazole C-5), 146.8 (2 triazole C-4), 173.0 (2 ꢃ
CONH). Anal. calcd. for C42H74N8O12 (883.08): C, 57.12;
H, 8.45; N, 12.69; Found: C, 57.23; H, 8.56; N, 12.58.
1,4-Bis-[1-(b-D-glucopyranosylaminocarbonylpentadecyl)-1H-
1,2,3-triazol-4-yl)]butane (51). Prepared from 47 (0.2 g,
0.147 mmol) according to General procedure VI. Yield:
0.12 g, 80%, white crystalline product, mp 158–160 1C;
1
1,4-Bis-[1-(b-D-glucopyranosylaminocarbonylmethyl)-1H-1,2,3-
triazol-4-yl)]butane (48). Prepared from 44 (0.10 g, 0.10 mmol)
according to General procedure VI. Yield: 0.042 g, 65%, white
crystalline product, mp 235–236 1C, [a]D = 42 (c = 0.22,
DMSO);1H NMR (360 MHz, D2O): d (ppm) 1.64 (brs, 4H,
2 ꢃ CH2), 2.70 (brs, 4H, 2 ꢃ CH2), 3.39–3.57 (8H, m, 2 ꢃ
H-2-Glc, 2 ꢃ H-3-Glc, 2 ꢃ H-4-Glc, 2 ꢃ H-5-Glc), 3.71
(dd, 2H, J = 5.3 11.9 Hz, 2 ꢃ H-6b-Glc), 5.01 (d, 2H, J =
9.2 Hz, 2 ꢃ H-1-Glc), 3.85 (dd, 2H, J = o1, 11.9 Hz, 2 ꢃ
H-6a-Glc), 5.24–5.27 (s, 4H, 2 ꢃ CH2), 7.73 (s, 2H, triazole
CH); 13C NMR (90 MHz, DMSO-d6): d (ppm) 24.7, 28.4, 51.5
(6 ꢃ CH2), 60.8 (2 ꢃ C-6-Glc), 69.9, 72.6, 77.3, 78.7 (2 ꢃ
C-2-Glc, 2 ꢃ C-3-Glc, 2 ꢃ C-4-Glc, 2 ꢃ C-5-Glc), 79.7 (2 ꢃ
C-1-Glc), 123.5 (2 triazole C-5), 146.5 (2 triazole C-4), 166.1
(2 ꢃ CONH). Anal. calcd. for C24H38N8O12 (630.62): C, 45.71;
H, 6.07; N, 17.77; Found: C, 45.80; H, 5.97; N, 17.54.
[a]D = +25 (c = 0.20, MeOH); H NMR (360 MHz, D2O):
d(ppm) 1.19–1.32 (m, 30H, 15 ꢃ CH2), 1.49–1.70 (m, 4H,
2 ꢃ CH2), 1.91–1.98 (m, 4H, 2 ꢃ CH2), 2.18–2.38 (m, 8H,
4 ꢃ CH2), 2.51–2.90 (m, 20H, 10 ꢃ CH2), 3.32–3.39 (m, 4H,
2 ꢃ H-3-Glc, 2x H-4-Glc), 3.45 (ddd, 2H, J = 1.2, 5.3, 9.2 Hz,
2 ꢃ H-5-Glc), 3.49 (t, 2H, J = 9.2, 9.2 Hz, 2x H-2-Glc), 3.79
(dd, 2H, J = 5.3, 11.9 Hz, 2 ꢃ H-6b-Glc), 3.87 (2H, dd,
J = 1.2, 11.9 Hz, 2x H-6a-Glc), 4.41–4.47 (m, 4H, 2 ꢃ CH2),
5.01 (d, 2H, J = 9.2 Hz, 2 ꢃ H-1-Glc), 7.67 (s, 2H, 2 triazole
CH); 13C NMR (90 MHz, D2O): d (ppm) 21.7, 22.9, 23.3, 24.1,
24.8, 25.3, 25.8, 27.1, 27.6, 28.1, 29.5, 31.2, 32.0, 33.1, 36.7,
37.4, 51.2 (34 ꢃ CH2), 61.5 (2 ꢃ C-6-Glc), 69.0, 72.1, 76.3, 77.3
(2 ꢃ C-2-Glc, 2 ꢃ C-3-Glc, 2x C-4-Glc, 2 ꢃ C-5-Glc), 78.1
(2 ꢃ C-1-Glc), 121.5 (2 triazole C-5), 146.5 (2 triazole C-4),
179.3 (2 ꢃ NHCO). Anal. calcd. for C52H94N8O12 (1023.38):
C, 61.06; H, 9.26; N, 10.95; Found: C, 61.13; H, 9.36; N, 10.88.
1,4-Bis-[1-(b-D-glucopyranosylaminocarbonylpentyl)-1H-1,2,3-
triazol-4-yl)]butane (49). Prepared from 45 (0.21 g 0.19 mmol)
according to General procedure VI. Yield: 0.09 g, 66%, white
crystalline product, mp: 150–152 1C; [a]D = +14 (c =0.12,
MeOH); 1H NMR (360 MHz, D2O): d(ppm) 1.20–1.25 (m,
4H, 2 ꢃ CH2), 1.52–1.64 (m, 8H, 4 ꢃ CH2), 1.80–1.88 (m, 4H,
2 ꢃ CH2), 2.22–2.28 (m, 4H, 2 ꢃ CH2), 2.66–2.70 (m, 4H,
2 ꢃ CH2), 3.33–3.42 (m, 6H, 2 ꢃ H-3-Glc, 2 ꢃ H-4-Glc, CH2),
3.50 (ddd, 2H, J = 1.2, 5.3, 9.2 Hz, 2 ꢃ H-5-Glc), 3.52 (t, 2H,
J = 9.2, 9.2 Hz, 2 ꢃ H-2-Glc), 3.70 (dd, 2H, J = 5.3, 11.9 Hz,
2 ꢃ H-6b-Glc), 3.85 (dd, 2H, J = 1.2, 11.9 Hz, 2 ꢃ H-6a-Glc),
4.31–4.37 (m, 4H, 2 ꢃ CH2), 4.91 (d, 2H, J = 9.2 Hz, 2 ꢃ H-1-
Glc), 7.82 (s, 2H, 2 triazole CH); 13C NMR (90 MHz, D2O): d
(ppm) 24.0, 24.1, 25.2, 27.8, 29.1, 35.6, 50.4 (14 ꢃ CH2), 60.7
(2 xC-6-Glc) 69.4, 71.9, 76.7, 77.7 (2 ꢃ C-2-Glc, 2 ꢃ C-3-Glc, 2
ꢃ C-4-Glc, 2 ꢃ C-5-Glc), 79.4 (2x C-1-Glc), 123.8 (2 triazole
C-5), 146.2 (2 triazole C-4), 178.1 (2 ꢃ NHCO). Anal. calcd.
for C32H54N8O12 (742.83): C 51.74, H 7.33, N 15.08. Found: C
51.69, H 7.25, N 15.02.
Enzyme assays
(a) Against RMGPa. The inhibitory activity of the prepared
compounds against rabbit muscle glycogen phosphorylase a
(RMGPa) was monitored using microplate reader (BIO-RAD)
based on the published method.35 In brief, GPa activity was
measured in the direction of glycogen synthesis by the release
of phosphate from glucose-1-phosphate. Each prepared
compound was dissolved in DMSO and diluted to different
concentrations for IC50 determination. The enzyme was added
into 100 mL of buffer containing 50 mM Hepes (pH = 7.2),
100 mM KCl, 2.5 mM MgCl2, 0.5 mM glucose-1-phosphate,
1 mg/ml glycogen and the test compound in 96-well micro-
plates (Costar). After the addition of 150 mL of 1 M HCl
containing 10 mg/ml ammonium molybdate and 0.38 mg/ml
malachite green, reactions were run at 22 1C for 25 min, and
then the phosphate absorbance was measured at 655 nm. The
IC50 values were estimated by fitting the inhibition data to a
dose-dependent curve using a logistic derivative equation.
1,4-Bis-[1-(b-D-glucopyranosylaminocarbonyldecyl)-1H-1,2,3-
triazol-4-yl)]butane (50). Prepared from 46 (0.07 g, 0.06 mmol)
according to General procedure VI. Precipitated from the
reaction mixture. Yield: 0.048 g, 95%, white amorphous
product; [a]D = +16 (c = 0.37, DMSO); 1H NMR (360
MHz, DMSO-d6+D2O): d (ppm) 1.20 (brs, 24H, 12 ꢃ CH2),
1.45, 1.60, 1.75, 2.06, 2.60 (5 brs, 20H, 10 ꢃ CH2), 3.00–3.19
(m, 8H, 2 ꢃ H-2-Glc, 2 ꢃ H-3-Glc, 2 ꢃ H-4-Glc, 2 ꢃ H-5-
Glc), 3.38 (dd, 2H, J = 4.6, 11.6 Hz, 2 ꢃ H-6b-Glc), 3.61 (dd,
2H, J o1.0, 11.2 Hz, 2 ꢃ H-6a-Glc), 4.23–4.27 (m, 4H,
2 ꢃ CH2), 4.68 (d, 2H, J = 8.9 Hz, 2 ꢃ H-1-Glc),7.80
(s, 2H, 2 triazole CH); 13C NMR (90 MHz, DMSO-d6+D2O):
(b) Against RMGPb. Glycogen phosphorylase b (RMGPb)
was prepared from rabbit skeletal muscle according to the
method of Fischer and Krebs,41 using dithiothreitol instead of
L-cysteine, and recrystallized at least three times before use.
Kinetic experiments were performed in the direction of glycogen
synthesis using RMGPb as described.36,42 IC50 values were
determined in the presence of 4 mM a-D-glucose-1-phosphate,
1 mM AMP, 1% glycogen and varying concentrations of the
inhibitor.43 Inhibitors were dissolved in dimethyl sulfoxide
(DMSO) and diluted in the assay buffer (50 mM triethanolamine,
1 mM EDTA and 1 mM dithiothreitol) so that the DMSO
concentration in the assay should be lower than 1%. The
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means of standard errors for all calculated kinetic parameters
averaged to less than 10%.
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Acknowledgements
HBS thanks financial support by National Natural Science
Foundation of China (Grants 30672523 and 90713037),
research grants from Chinese Ministry of Education (Grants
706030 and 20050316008) and the program for New Century
Excellent Talents in University (NCET-05-0495). In Hungary
this research was supported by the National Office for
Research and Technology as well as the Hungarian Scientific
Research Fund (Grants NI-61336, NK-68578, and CK-77712).
The authors also thank NORT (Hungary, CHN-25/05) and
the Program for Chinese–Hungarian Scientific and Technological
Cooperation (China, No. CHN-7/2006) for supporting this
bilateral R&D project.
27 S. Furukawa, Y. Tsurumi, K. Murakami, T. Nakanishi,
K. Ohsumi, M. Hashimoto, M. Nishikawa, S. Takase,
O. Nakayama and M. Hino, J. Antibiot., 2005, 58, 497–502.
28 C. Tiraidis, K. M. Alexacou, S. E. Zographos, D. D. Leonidas,
T. Gimisis and N. G. Oikonomakos, Protein Sci., 2007, 16,
1773–1782.
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This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2010
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