Facile preparation of indoxyl- and nitrophenyl glycosides of lactosamine and isolactosamine

Article abstract of DOI:10.1039/c3ra47128d

The synthesis of the novel indoxyl glycosides of N-acetyl-lactosamine (X-LacNAc) and N-acetyl-isolactosamine (X-LNB) is reported employing glycosyl chlorides in a facile phase transfer glycosylation, followed by mild decarboxylation and finally deacetylat

Full text of DOI:10.1039/c3ra47128d

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Facile preparation of indoxyl- and nitrophenyl
glycosides of lactosamine and isolactosamine†
Cite this: RSC Adv., 2014, 4, 10856
*
Stephan Bottcher and Joachim Thiem
¨
Received 28th November 2013
Accepted 24th January 2014
The synthesis of the novel indoxyl glycosides of N-acetyl-lactosamine (X-LacNAc) and N-acetyl-
isolactosamine (X-LNB) is reported employing glycosyl chlorides in a facile phase transfer glycosylation,
followed by mild decarboxylation and finally deacetylation. Correspondingly the ortho-nitrophenol and
para-nitrophenol glycosides of LacNAc and LNB could be obtained.
DOI: 10.1039/c3ra47128d
www.rsc.org/advances
Introduction
N-Acetyl-lactosamine (LacNAc, Galb1-4GlcNAc) and N-acetyl-
isolactosamine (LNB, Galb1-3GlcNAc) are integral parts of
biologically very important glycostructures. Both represent
essential subunits of complex milk oligosaccharides1,2 as well as
of antigens3–5 and glycoconjugates.6,7
Access to samples or intermediates of complex glyco-
structures is still rather difficult. The chemical synthesis of
complex saccharide structures is loaded with barriers,
regarding protecting group chemistry as well as control of
stereo- and regio-chemistry. Enzymatic syntheses need fewer
steps, are oen highly stereo- and regio-selective and are
therefore superior if available. Thus, these approaches
complement the array of methods, however, oen enzymes are
not available or difficult to isolate, purify or handle.
Fig.
glycosides.
1 Monitoring of glycosidase activity employing indoxyl
used aer washing directly without further workup for phase
transfer glycosylation with the respective nitrophenol acceptor.
The oNP and pNP glycosides were obtained aer Zemplen
´
A convenient method for screening of glycosidases as well as
transglycosidases is to use indoxyl glycosides (Fig. 1). Indoxyl is
released by enzymatic cleavage of the glycosidic linkage and
then rapidly oxidized, e.g. by atmospheric oxygen, to an indigo
type dye. Common substrates are halogenated, as the substi-
tution pattern determines colour and physical properties of the
resulting indigo dye. The most common pattern of indoxyls are
5-bromo-4-chloro, 5-bromo and 5-bromo-6-chloro derivatives.8
Nitrophenol glycosides in turn are widely utilised compounds
for activity measurements and are employed as convenient
donor substrates in enzymatic syntheses.9–12
Here we wish to report convenient syntheses of novel indoxyl
glycosides of N-acetyl-lactosamine (X-LacNAc) and N-acetyl-iso-
lactosamine (X-LNB), as well as the synthesis of para-nitro-
phenyl and ortho-nitrophenyl glycosides of both disaccharides.
The peracetates of LacNAc and LNB were converted into the
corresponding glycosyl chlorides. The crude products could be
deacetylation13 in good overall yields. Preparation of the indoxyl
glycosides followed our recently developed approach. In a phase
transfer glycosylation indoxylic acid allyl esters were used as
acceptors, followed by selective mild silver mediated decar-
14,15
´
boxylation and nally Zemplen deacetylation.
Results and discussion
The synthetic route for the preparation of LNB-oNP/pNP (5a/b)
and LacNAc-oNP/pNP (5c/d) is summarized in Scheme 1.
The peracetates 2a/b were treated with acetyl chloride and
methanol under argon atmosphere to give the glycosyl chlorides
3a/b. These donors were dried and washed to remove acetyl
chloride, and were then subjected to phase transfer glycosyla-
tion without further workup to yield peracetylated LNB-pNP (4a,
40%), LNB-oNP (4b, 34%), LacNAc-pNP (4c, 45%), LNB-oNP (4d,
44%) via two steps. Finally, the acetyl protecting groups were
removed by Zemplen deacetylation.13 In case of 5d the reaction
´
Department of Chemistry, Faculty of Science, University of Hamburg,
Martin-Luther-King-Platz 6, 20146 Hamburg, Germany. E-mail: thiem@chemie.
uni-hamburg.de
mixture needed to be kept at 45 ꢀC overnight to achieve
complete deacetylation. All glycosides crystallised during
deacetylation and were washed aer ltration with a small
amount of cold methanol to give very pure 5a–d in good yields.
† Electronic supplementary information (ESI) available: NMR data for all new
compounds. See DOI: 10.1039/c3ra47128d
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Scheme 1 Synthesis of p/o-nitrophenol glycosides of isolactosamine (5a/b) and lactosamine (5c/d) as well as indoxyl glycosides of iso-
lactosamine (8a) and lactosamine (8b). *Yield via two steps.
The synthesis of the indoxyl glycosides 8a/b was again based and morpholine in THF16 followed by mild silver mediated
on the conversion of the respective peracetate 2a or 2b to the decarboxylation14,15 gave the acetylated glycosides 7a (85%)
13
´
crude glycosyl chlorides 3a/b. Phase transfer glycosylation ad 7b (62%). Finally Zemplen deacetylation was used to give
under common phase transfer conditions gave 6a (46%) and 6b the novel indoxyl glycosides X-LNB in 75% and X-LacNAc in
(45%). Then selective allyl ester deprotection with Pd(PPh3)4 73% yield.
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neutralised with Amberlite IR-120 (H+) resin and concentrated.
The product was dried at 40 C under high vacuum.
Conclusions
ꢀ
General procedure 4. Allyl ester deprotection and subse-
quent decarboxylation. The starting material (1.0 mmol) dis-
solved in THF (15 mL) was treated morpholine (10 equiv.) and
Pd(PPh3)4 (0.1 equiv.). The solution was stirred overnight at
room temperature. Aer removal of the solvent silver acetate
(3 equiv.), potassium carbonate (6–7 equiv.) and acetic anhy-
dride (10 mL) were added. The resulting mixture was heated to
90–95 ꢀC for 15–20 min. Aer cooling to room temperature, the
mixture was diluted with water and DCM. The organic phase
was washed twice with water and once with a diluted aqueous
NaHCO3 solution. Aer drying (Na2SO4) the solvent was
removed under reduced pressure and the crude product was
subjected to column chromatography in the solvent stated.
4-Nitrophenyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,
6-tetra-O-acetyl-b-D-galactopyranosyl)-b-D-glucopyranoside (4a).
Prepared according to general procedures 1 and 2. (1) 2a (3.00 g,
4.43 mmol), acetyl chloride (30 mL), methanol (4.2 mL). TLC
(heptane/acetone 3 : 7, RF: 0.45; starting material 0.40). (2) DCM
(60 mL), aqueous K2CO3 solution (60 mL, 1 M), TBAHS (1.50 g,
4.42 mmol), p-nitrophenol (620 mg, 4.46 mmol). Yield 40%
(1.35 g, 1.78 mmol); colorless solid; mp 129 ꢀC; [a]D25 -27.8 (c 0.50
Within this work we could elaborate facile syntheses of novel
indoxyl glycosides X-LAcNAc and X-LNB as well as the corre-
sponding ortho- and para-nitrophenyl glycosides of LacNAc and
LNB. Both reaction pathways could be carried out in good
yields, employing facile phase transfer glycosylation of the
crude glycosyl chloride donors, straight forward workup and
crystallization.
Experimental section
General remarks
All reagents were purchased from commercial sources and used
as received. TLC was performed on Merck silica gel 60 F254
plates. Compounds were detected by UV and/or by treatment
with EtOH/H2SO4 (9 : 1) and subsequent heating. Column
chromatography was performed with Merck/Fluka silica gel 60
(230–400 mesh). Solvents for column chromatography were
1
distilled prior to use. H and 13C NMR spectra were recorded
with Bruker AMX-400 or Bruker AV-400 spectrometers (400 MHz
for 1H, 101 MHz for 13C) and calibrated using the solvent
residual peak. In CDCl3 TMS was used for calibration. Melting
1
in CHCl3); RF 0.69 (heptane/acetone 3 : 7); H-NMR (400 MHz,
¨
points were measured with an Buchi melting point M-565.
CDCl3) d 8.21–8.16 (2H, m, Harom), 7.10–7.04 (2H, m, Harom),
5.93 (1H, d, J ¼ 7.5 Hz, NH), 5.71 (1H, d, J1,2 ¼ 7.0 Hz, H-1), 5.37–
¨
Optical rotations were obtained using a Kruss Optronic P8000
polarimeter (589 nm). HRMS (ESI) were recorded with a Thermo
Finnigan MAT 95XL mass spectrometer.
5.35 (1H, m, H-40), 5.12 (1H, dd, J1 ,2 ¼ 7.8 Hz, J2 ,3 ¼ 10.4 Hz, H-
0
0
0
0
20), 5.06 (1H, ddꢁvt, H-4), 4.99 (1H, dd, J2 ,3 ¼ 10.4 Hz, J3 ,4 ¼ 3.4
0
0
0
0
0
Hz, H-30), 4.64 (1H, d, J1 ,2 ¼ 7.8 Hz, H-1 ), 4.57 (1H, ddꢁvt, H-3),
4.24 (1H, dd, J5,6a ¼ 6.1 Hz, J6a/b ¼ 12.3 Hz, H-6a), 4.19–4.07 (3H,
0
0
General procedures
General procedure 1. Glycosyl chlorides. A solution of the m, H-6b, H-60a/b), 3.98–3.88 (2H, m, H-5, H-50), 3.67 (1H,
peracetylated disaccharide (1.0 mmol) in acetyl chloride (7.0 dddꢁvdd, H-2), 2.16, 2.09, 20.8, 2.07, 2.00, 1.98 (each 3H, s, C(O)
mL) was kept at 0 C under argon atmosphere. Dry methanol CH3), 2.02 (3H, s, NHC(O)CH3); 13C-NMR (100 MHz, CDCl3) d
ꢀ
(1.0 mL) was added dropwise over a period of 3 hours. Aer nal 170.7, 170.4, 170.1, 170.1, 169.1, 169.1 (C(O)CH3), 161.4
addition, the solution was allowed to warm slowly to room (NHC(O)CH3), 143.0 (Cquaternary), 125.7, 116.5 (CHarom), 100.7 (C-
temperature and stirred until TLC (heptane/acetone 3 : 7) 10), 96.3 (C-1), 76.0 (C-3), 72.4 (C-5), 70.9, 70.8 (C-30, C-50), 69.4
indicated no further reaction. The solvent was removed under (C-20), 68.5 (C-4), 66.8 (C-40), 62.5 (C-6), 61.0 (C-60), 56.1 (C-2),
reduced pressure and the crude product was dissolved in DCM, 23.5 (NHC(O)CH3), 20.8, 20.8, 20.6, 20.6, 20.5 (C(O)CH3); HRMS
concentrated again and washed with of diisopropyl ether (ESI) m/z for C32H40N2O19: [M + Na]+ calcd 779.2123 found
(25 mL). The product was used directly according to general 779.2134.
procedure 2 without further workup.
2-Nitrophenyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,
General procedure 2. Phase transfer glycosylation. The 6-tetra-O-acetyl-b-D-galactopyranosyl)-b-D-glucopyranoside (4b).
crude glycosyl chloride (1.0 mmol), TBAHS (1.0 equiv.) and the Prepared according to general procedures 1 and 2. (1) 2a (3.00 g,
respective acceptor (1.0–1.1 equiv.) were mixed in DCM (10– 4.43 mmol), acetyl chloride (30 mL), methanol (4.2 mL). TLC
15 mL) and an aqueous solution of K2CO3 (12.5 mL, 1 M) was (heptane/acetone 3 : 7, RF: 0.45; starting material 0.40). (2) DCM
added. The reaction mixture was stirred at room temperature (60 mL), aqueous K2CO3 solution (60 mL, 1 M), TBAHS (1.50 g,
until TLC indicated complete consumption of the donor. The 4.42 mmol), o-nitrophenol (620 mg, 4.46 mmol). Yield 34% (1.15
organic phase was separated, dried over Na2SO4, and the g, 1.52 mmol); colorless solid; mp 182–184 ꢀC; [a]D25 -49.6 (c 0.50
1
solvent was removed under reduced pressure. The crude in CHCl3); RF 0.69 (heptane/acetone 3 : 7); H-NMR (400 MHz,
product was subjected to column chromatography in the CDCl3) d 7.80–7.76 (1H, m, Harom), 7.54–7.49 (1H, m, Harom),
solvent stated.
7.35–7.32 (1H, m, Harom), 7.21–7.26 (1H, m, Harom), 5.99 (1H, bs,
General procedure 3. Zemplen deacetylation. The starting NH), 5.62 (1H, d, J1,2 ¼ 8.2 Hz, H-1), 5.37–5.34 (1H, m, H-40), 5.08
´
material (1.0 mmol) was dissolved in MeOH (15–20 mL) and was (1H, dd, J1 ,2 ¼ 7.8 Hz, J2 ,3 ¼ 10.4 Hz, H-20), 5.03–4.97 (1H, m,
0
0
0
0
0
0
0
0
0
treated with a catalytic amount of sodium methoxide. The H-4), 4.98 (1H, dd, J2 ,3 ¼ 10.4 Hz, J3 ,4 ¼ 3.8 Hz, H-3 ), 4.75 (1H,
solution was stirred until TLC indicated complete consumption ddꢁvt, H-3), 4.58 (1H, d, J1 ,2 ¼ 7.8 Hz, H-10), 4.25 (1H, dd, J5,6a
of the starting material. If the product has precipitated during ¼ 5.7 Hz, J6a/b ¼ 12.3 Hz, H-6a), 4.19 (1H, dd, J5,6b ¼ 2.5 Hz, J6a/b
this time it was ltered off, otherwise the solution was ¼ 12.3 Hz, H-6b), 4.14–4.09 (2H, m, H-60a/b), 3.92–3.85 (2H, m,
0
0
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H-5, H-50), 3.45–3.37 (1H, m, H-2), 2.15, 2.10, 2.08, 2.07, 2.06, (ESI) m/z for C32H40N2O19: [M + Na]+ calcd 779.2123 found
1.98 (each 3H, s, C(O)CH3), 2.05 (3H, s, NHC(O)CH3); 13C-NMR 779.2122.
(100 MHz, CDCl3): d ¼ 171.9 (NHC(O)CH3), 170.5, 170.4, 170.2,
4-Nitrophenyl
2-acetamido-2-deoxy-3-O-(b-D-galactopyr-
170.1, 169.3, 169.1 (C(O)CH3), 149.7, 141.1 (Cquaternary), 134.0, anosyl)-b-D-glucopyranoside (5a). Prepared according to general
125.1, 123.4, 119.4 (CHarom), 100.8 (C-10), 98.4 (C-1), 76.1 (C-3), procedure 3. 4a (1.20 g, 1.56 mmol), MeOH (25 mL), cat.
72.4 (C-5), 71.0 (C-30), 70.7 (C-50), 69.4 (C-20), 68.9 (C-4), 66.9 (C- NaOMe. Yield 88% (706 mg, 1.40 mmol); colorless solid; mp
40), 62.3 (C-6), 61.0 (C-60), 58.2 (C-2), 23.6 (NHC(O)CH3), 20.9, 160 ꢀC dec (lit.,18 184–186); [a]D25 -13.4 (c 0.35 in H2O) (lit.,18 [a]2D4
20.8, 20.7, 20.6, 20.6, 20.5 (C(O)CH3); HRMS (ESI) m/z for -14 (c 0.5 in H2O)); 1H-NMR (400 MHz, D2O) d 8.29–8.24 (2H, m,
C
32H40N2O19: [M + Na]+ calcd 779.2123 found 779.2139.
H
arom), 7.25–7.20 (2H, m, Harom), 5.39 (1H, d, J1,2 ¼ 8.3 Hz, H-1),
0
0
0
4-Nitrophenyl 2-acetamido-3,6-di-O-acetyl-2-deoxy-4-O-(2,3,4, 4.52 (1H, d, J1 ,2 ¼ 7.6 Hz, H-1 ), 4.22 (1H, dd, J1,2 ¼ 8.3 Hz, J2,3
¼
6-tetra-O-acetyl-b-D-galactopyranosyl)-b-D-glucopyranoside (4c). 10.4 Hz, H-2), 4.03–3.95 (3H, m, H-3, H-40, H-6a), 3.89–3.69 (6H,
Prepared according to general procedures 1 and 2. (1) 2b (3.00 g, m, H-4, H-5, H-50, H-6b, H-60a/b), 3.69 (1H, dd, J2 ,3 ¼ 10.1 Hz,
0
0
4.43 mmol), acetyl chloride (30 mL), methanol (4.2 mL). TLC J3 ,4 ¼ 3.5 Hz, H-30), 3.59 (1H, dd, J1 ,2 ¼ 7.6 Hz, J2 ,3 ¼ 10.1 Hz,
(heptane/acetone 3:7, RF: 0.52; starting material 0.43). (2) DCM H-20), 2.05 (3H, s, NHC(O)CH3); 13C-NMR (100 MHz, D2O) d
(60 mL), aqueous K2CO3 solution (60 mL, 1 M), TBAHS (1.50 g, 174.5 (NHC(O)CH3), 161.7, 142.7 (Cquaternary), 126.1, 116.5
4.42 mmol), p-nitrophenol (620 mg, 4.46 mmol). Yield 45% (CHarom), 103.5 (C-10), 98.4 (C-1), 81.8 (C-3), 75.8, 75.3 (C-5, C-50),
0
0
0
0
0
0
(1.59 g, 2.10 mmol); colorless solid; mp 156–157 ꢀC (lit.,17 155– 72.5 (C-30), 70.7 (C-20), 68.6, 68.4 (C-4, C-40), 61.0 (C-60), 60.5 (C-
25
156 C); [a]D -36.2 (c 0.5 in CHCl3) (lit.,17 [a]D ꢂ36.7 (c 1.3 in 6), 54.3 (C-2), 22.1 (NHC(O)CH3); HRMS (ESI) m/z for
ꢀ
CHCl3)); RF 0.47 (heptane/acetone 3 : 7); 1H-NMR (400 MHz,
C
20H26N2O13: [M + Na]+ calcd 527.1489 found 527.1488.
CDCl3) d 8.21–8.17 (2H, m, Harom), 7.10–7.05 (2H, m, Harom),
2-Nitrophenyl
2-acetamido-2-deoxy-3-O-(b-D-galactopyr-
6.19 (1H, d, J ¼ 8.8 Hz, NH), 5.40–5.38 (1H, m, H-40), 5.30 (1H, d, anosyl)-b-D-glucopyranoside (5b). Prepared according to general
0
0
J1,2 ¼ 6.0 Hz, H-1), 5.21 (1H, ddꢁvt, H-3), 5.14 (1H, dd, J1 ,2 ¼ 7.9 procedure 3. 4b (750 mg, 991 mmol), MeOH (15 mL), cat.
0
0
0
0
0
0
0
Hz, J2 ,3 ¼ 10.4 Hz, H-2 ), 5.02 (1H, dd, J2 ,3 ¼ 10.4 Hz, J3 ,4 ¼ 3.5 NaOMe. Yield 80% (400 mg, 793 mmol); colorless solid; mp 203–
25
1
Hz, H-30), 4.51 (1H, d, J1 ,2 ¼ 7.9 Hz, H-10), 4.47 (1H, dd, J5,6a
¼
204 C; [a]D -35.7 (c 0.41 in H2O); H-NMR (400 MHz, D2O) d
ꢀ
0
0
4.1 Hz, J6a/b ¼ 12.0 Hz, H-6a), 4.43–4.37 (1H, m, H-2), 4.20–4.08 7.90–7. 86 (1H, m, Harom), 7.70–7.65 (1H, m, Harom), 7.49–7.45
(3H, m, H-6b, H-60a/b), 3.97–3.90 (2H, m, H-5, H-50), 3.89–3.85 (1H, m, Harom), 7.31–7.26 (1H, m, Harom), 5.25 (1H, d, J1,2 ¼ 8.5
0
0
0
(1H, m, H-4), 2.14, 2.13, 2.10, 2.06, 2.01, 1.99 (each 3H, s, C(O) Hz, H-1), 4.46 (1H, d, J1 ,2 ¼ 7.6 Hz, H-1 ), 4.18 (1H, ddꢁvt, H-2),
CH3), 2.02 (NC(O)CH3); 13C-NMR (100 MHz, CDCl3) d 170.3, 4.02–3.97 (1H, m, H-6a), 3.94–3.88 (2H, m, H-3, H-40), 3.85 (1H,
170.3, 170.2, 170.1, 170.0, 169.9, 169.8 (C(O)CH3), 161.3, 142.8 dd, J5,6b ¼ 4.7 Hz, J6a/b ¼ 12.3 Hz, H-6b), 3.82–3.62 (5H, m, H-4,
(Cquaternary), 125.7, 116.4 (CHarom), 100.7 (C-10), 97.8 (C-1), 74.0 H-5, H-50, H-60a/b), 3.65 (1H, dd, J2 ,3 ¼ 10.4 Hz, J3 ,4 ¼ 3.8 Hz,
(C-4), 72.9 (C-5), 71.0 (C-50), 70.4 (C-3, C-30), 69.0 (C-20), 66.6 (C- H-30), 3.55 (1H, ddꢁvt, H-20), 2.03 (3H, s, NHC(O)CH3); 13C-NMR
40), 62.5 (C-6), 60.8 (C-60), 51.1 (C-2), 23.1 (NC(O)CH3), 20.9, 20.7, (100 MHz, D2O) d 174.8 (NHC(O)CH3), 149.3 (Cquaternary), 134.9,
20.6, 20.6, 20.5, 20.5 (C(O)CH3); HRMS (ESI) m/z for 125.2, 123.6, 118.2 (CHarom), 103.5 (C-10), 100.2 (C-1), 81.8 (C-3),
0
0
0
0
C
32H40N2O19: [M + Na]+ calcd 779.2123 found 779.2133.
75.9, 75.3 (C-5, C-50), 72.5 (C-30), 70.7 (C-20), 68.5, 68.4 (C-4, C-40),
2-Nitrophenyl 2-acetamido-3,6-di-O-acetyl-2-deoxy-4-O-(2,3,4, 61.0 (C-60), 60.5 (C-6), 54.1 (C-2), 22.1 (NHC(O)CH3); HRMS
6-tetra-O-acetyl-b-D-galactopyranosyl)-b-D-glucopyranoside (4d). (ESI) m/z for C20H26N2O13: [M + Na]+ calcd 527.1489 found
Prepared according to general procedures 1 and 2. (1) 2b (3.00 g, 527.1490.
4.43 mmol), acetyl chloride (30 mL), methanol (4.2 mL). TLC
4-Nitrophenyl 2-acetamido-2-deoxy-4-O-(b-D-galactopyranosyl)-
(heptane/acetone 3 : 7, RF: 0.52; starting material 0.43). (2) DCM b-D-glucopyranoside (5c). Prepared according to general proce-
(60 mL), aqueous K2CO3 solution (60 mL, 1 M), TBAHS (1.50 g, dure 3. 4c (1.40 g, 1.85 mmol), MeOH (35 mL), cat. NaOMe. Yield
4.42 mmol), o-nitrophenol (620 mg, 4.46 mmol). Yield 44% (1.49 80% (750 mg, 1.49 mmol); colorless solid; mp 213 ꢀC (lit.,19
25
25
g, 1.97 mmol); colorless solid; mp 118 C; [a]D -12.0 (c 0.55 in 213 ꢀC); [a]D -22.2 (c 0.5 in H2O) (lit.,19 [a]D25 -18.4 (c 1 in H2O)); 1H-
ꢀ
CHCl3); RF 0.40 (heptane/acetone 3 : 7); 1H-NMR (400 MHz, NMR (400 MHz, D2O) d 8.28–8.20 (2H, m, Harom), 7.23–7.18 (2H,
0
0
CDCl3) d 7.78 (1H, dd, Harom), 7.54–7.48 (1H, m, Harom), 7.36– m, Harom), 5.37 (1H, d, J1,2 ¼ 8.6 Hz, H-1), 4.55 (1H, d, J1 ,2 ¼ 7.8
7.32 (1H, m, Harom), 7.17–7.12 (1H, m, Harom), 6.22 (1H, d, J ¼ Hz, H-10), 4.16–4.10 (1H, m, H-2), 4.10–4.04 (1H, m, H-6a), 3.98–
8.8 Hz, NH), 5.40–5.35 (2H, m, H-1, H-40), 5.25 (1H, ddꢁvt, H-3), 3.96 (1H, m, H-40), 3.96–3.86 (4H, m, H-3, H-4, H-5, H-6b), 3.85–
5.16 (1H, dd, J1 ,2 ¼ 7.8 Hz, J2 ,3 ¼ 10.4 Hz, H-20), 5.03 (1H, dd, 3.76 (3H, m, H-50, H-60a/b), 3.72 (1H, dd, J2 ,3 ¼ 9.8 Hz, J3 ,4 ¼ 3.5
0
0
0
0
0
0
0
0
J2 ,3 ¼ 10.4 Hz, J3 ,4 ¼ 3.5 Hz, H-3 ), 4.54 (1H, dd, J5 ,6a ¼ 3.8 Hz, Hz, H-30), 3.61 (1H, dd, J1 ,2 ¼ 7.8 Hz, J2 ,3 ¼ 9.8 Hz, H-20), 2.06
0
0
0
0
0
0
0
0
0
0
0
J6a/b ¼ 11.6 Hz, H-6a), 4.52 (1H, J1 ,2 ¼ 7.8 Hz, H-10), 4.32–4.22 (3H, s, NHC(O)CH3); 13C-NMR (100 MHz, D2O) d 174.9 (C(O)CH3),
(2H, m, H-2, H-6b), 4.20–4.28 (2H, m, H-60a/b), 3.96–3.88 (3H, 161.7, 142.6 (Cquaternary), 126.8, 116.5 (CHarom), 102.9 (C-10), 98.5
m, H-4, H-5, H-50), 2.16, 2.16, 2.08, 2.04, 1.98, 1.98 (each 3H, s, (C-1), 78.1 (C-4), 75.4, 75.1 (C-5, C-50), 72.5 (C-30), 72.0 (C-3), 71.0
C(O)CH3), 2.05 (3H, s, NC(O)CH3); 13C-NMR (100 MHz, CDCl3) d (C-20), 68.6 (C-40), 61.6 (C-60), 59.8 (C-6), 54.9 (C-2), 22.1 (NHC(O)
170.5, 170.3, 170.1, 170.0, 169.9, 169.7 (C(O)CH3), 149.2, 141.2 CH3); HRMS (ESI) m/z for C20H26N2O13: [M + Na]+ calcd 527.1489
(Cquaternary), 133.7, 125.1, 122.9, 119.0 (CHarom), 100.6 (C-10), found 527.1496.
0
0
0
98.4 (C-1), 73.7 (C-4), 72.9 (C-5), 71.0 (C-50), 70.5 (C-30), 69.4 (C-3),
69.0 (C-20), 66.6 (C-40), 62.4 (C-6), 60.8 (C-60), 50.9 (C-2) 23.1 b-D-glucopyranoside (5d). Prepared according to general proce-
(NC(O)CH3), 20.8, 20.7, 20.6, 20.6, 20.6, 20.5 (C(O)CH3); HRMS dure 3. 4d (1.30 g, 1.72 mmol), MeOH (25 mL), cat. NaOMe,
2-Nitrophenyl 2-acetamido-2-deoxy-4-O-(b-D-galactopyranosyl)-
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45 ꢀC. Yield 80% (698 mg, 1.38 mmol); colorless solid; mp 209– 6.08–6.01 (1H, m, –O–CH2–CH]CH2), 5.49–5.45 (1H, m, O–CH2–
212 ꢀC; [a]2D2 -27.0 (c 0.35, H2O); 1H-NMR (400 MHz, D2O) d 7.89– CH ¼ CH2a), 5.41–5.38 (1H, m, O–CH2–CH]CH2b), 5.35–
7.86 (1H, m, Harom), 7.69–7.64 (1H, m, Harom), 7.47–7.44 (1H, m, 5.33 (1H, m, H-40), 5.15–5.07 (3H, m, H-1, H-20, H-3), 4.96 (1H, dd,
0
0
0
0
H
arom), 7.30–7.25 (1H, m, Harom), 5.23 (1H, d, J1,2 ¼ 8.5 Hz, H-1), J2 ,3 ¼ 10.5 Hz, J3 ,4 ¼ 10.5 Hz, H-30), 4.89–4.81 (2H, m, O–CH2–
0
CH]CH2), 4.53 (1H, d, J1 ,2 ¼ 7.6 Hz, H-10), 4.46–4.41 (1H, m, H-
0
0
0
0
4.51 (1H, d, J1 ,2 ¼ 7.9 Hz, H-1 ), 4.08 (1H, dd, J1,2 ¼ 8.5 Hz, J2,3
¼
10.0 Hz, H-2), 4.06–4.02 (1H, m, H-6a), 3.95–3.89 (2H, m, H-40, 2), 4.31 (1H, dd, J5,6a ¼ 2.4 Hz, J6a,6b ¼ 11.8 Hz, H-6a), 4.15–4.09
H-6b), 3.88–3.73 (6H, m, H-3, H-4, H-5, H-50, H-60a/b), 3.68 (1H, (2H, m, H-60a/b), 3.93 (1H, ddꢁvt, H-4), 3.90–3.86 (1H, m, H-50),
dd, J2 ,3 ¼ 10.0 Hz, J3 ,4 ¼ 3.5 Hz, H-30), 3.56 (1H, dd, J1 ,2 ¼ 7.9 3.46–3.42 (1H, m, H-5), 2.14, 2.12, 2.06, 1.98 (each 3H, s, C(O)
0
0
0
0
0
0
Hz, J2 ,3 ¼ 10.0 Hz, H-20), 2.04 (s, 3H, NH(C(O)CH3)); 13C-NMR CH3), 1.99 (3H, s, NHC(O)CH3), 1.95 (6H, s, 2C(O)CH3); 13C-NMR
(100 MHz, D2O) d 174.7 (C(O)CH3), 149.3, 140.3 (Cquaternary), (100 MHz, CDCl3) d 170.7, 170.6, 170.3, 170.2, 170.1, 170.0, 169.1
134.9, 125.1, 123.5, 118.2 (CHarom), 102.9 (C-10), 100.2 (C-1), 77.9 (C(O)CH3), 159.4 (C(O)O–CH2–), 141.3, 133.2 (Cquaternary), 131.4
(C-4), 75.4, 75.2 (C-5, C-50), 72.5 (C-30), 72.1 (C-3), 70.9 (C-20), 68.5 (O–CH2–CH]CH2), 131.0 (CHarom), 127.4, 120.7 (Cquaternary),
(C-40), 61.0 (C-60), 59.8 (C-6), 54.6 (C-2), 22.1 (NH(C(O)CH3)); 120.1 (O–CH2–CH]CH2), 116.1, 115.7 (Cquaternary), 111.5
HRMS (ESI) m/z for C20H26N2O13: [M + Na]+ calcd 527.1489 (CHarom), 103.2 (C-1), 101.2 (C-10), 74.1 (C-3), 73.3 (C-4), 72.8 (C-5),
0
0
found 527.1491.
70.9 (C-30), 70.7 (C-50), 69.2 (C-20), 66.6 (C-40), 66.0 (O–CH2–CH]
(5-Bromo-4-chloro-indox-3-ylic acid allyl ester) 2-acet- CH2), 61.2 (C-6), 60.8 (C-60), 54.3 (C-2), 23.3 (NHC(O)CH3), 20.9,
amido-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-b-D- 20.6, 20.6, 20.6, 20.5 (C(O)CH3); HRMS (ESI) m/z for
galactopyranosyl)-b-D-glucopyranoside (6a). Prepared according C38H44BrClN2O19: [M + Na]+ calcd 969.1308 found 969.1288.
to general procedures 1 and 2. (1) 2a (1.00 g, 1.47 mmol), acetyl
(N-Acetyl-5-bromo-4-chloro-indox-3-yl) 2-acetamido-4,6-di-O-
chloride (15 mL), methanol (1.5 mL). TLC (heptane/acetone 3 : 7, acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)-
RF: 0.52; starting material 0.43). (2) DCM (17 mL), aqueous K2CO3 b-D-glucopyranoside (7a). Prepared according to general
solution (17 mL, 1 M), TBAHS (520 mg, 1.53 mmol), 5-bromo-4- procedure 4. 6a (520 mg, 0.548 mmol), THF (10 mL), morpho-
chloro-indoxylic acid allyl ester (500 mg, 1.51 mmol). Yield 46% line (500 mL, 5.74 mmol), Pd(PPh3)4 (60 mg, 0.052 mmol), 15
(635 mg, 0.670 mmol); colorless solid; mp 152–155 ꢀC; [a]D23 -24.2 min, 90–95 ꢀC. Yield 85% (420 mg, 0.463 mmol), colorless solid;
(c 0.5, CHCl3); RF 0.39 (hexane/acetone 1 : 1); 1H-NMR (400 MHz, mp 157–159 ꢀC; [a]2D3 -44.0 (c 0.5 in CHCl3); RF 0.36 (hexane/
CDCl3) d 8.69 (1H, s, NH), 7.49 (1H, d, J ¼ 8.9 Hz, Harom), 7.10 (1H, acetone 1 : 1); 1H-NMR (400 MHz, CDCl3) d 8.25 (1H, d, J ¼ 8.9
d, J ¼ 8.9 Hz, Harom), 6.37 (1H, d, J ¼ 7.6 Hz, C2–NH), 6.09–6.01 Hz, Harom), 7.55 (1H, d, J ¼ 8.9 Hz, Harom), 7.29 (1H, s, ]CH–N),
(1H, m, –O–CH2–CH]CH2), 5.47–5.43 (1H, m, O–CH2–CH] 5.87 (1H, d, J ¼ 7.8 Hz, C2–NH), 5.41 (1H, d, J1,2 ¼ 7.9 Hz, H-1),
CH2a), 5.40–5.35 (3H, m, H-1, H-40, O–CH2–CH]CH2b), 5.09 (1H, 5.37–5.35 (1H, m, H-40), 5.10 (1H, dd, J1 ,2 ¼ 7.8 Hz, J2 ,2 ¼ 10.5
0
0
0
0
0
0
dd, J1 ,2 ¼ 7.9 Hz, J2 ,3 ¼ 10.4 Hz, H-2 ), 5.03–4.98 (2H, m, H-3 , H- Hz, H-20), 5.03 (1H, ddꢁvt, H-4), 4.99 (1H, dd, J2 ,3 ¼ 10.5 Hz,
0
0
0
0
0
0
4), 4.70 (1H, d, J1 ,2 ¼ 7.9 Hz, H-10), 4.90–4.83 (2H, m, O–CH2– J3 ,4 ¼ 3.4 Hz, H-30), 4.66–4.59 (1H, m, H-3), 4.62 (1H, d, J1 ,2
¼
0
0
0
0
0
0
CH]CH2), 4.35 (1H, ddꢁvt, H-3), 4.10 (1H, dd, J5,6a ¼ 4.9 Hz, 7.8 Hz, H-10), 4.37 (1H, dd, J5,6a ¼ 2.7 Hz, J6a/b ¼ 12.4 Hz, H-6a),
J6a,6b ¼ 12.1 Hz, H-6a), 4.00 (1H, dd, J5,6b ¼ 3.0 Hz, J6a,6b ¼ 12.1 4.18 (1H, dd, J5,6b ¼ 5.5 Hz, J6a/b ¼ 12.4 Hz, H-6b), 4.14–4.08 (2H,
Hz, H-6b), 3.99–3.94 (1H, m, H-2), 3.92–3.88 (1H, m, H-50), 3.62 m, H-60a/b), 3.92–3.84 (2H, m, H-5, H-50), 3.66–3.57 (1H, m, H-2),
(1H, ddd, J4,5 ¼ 9.4 Hz, J5,6a ¼ 4.9 Hz, J5,6b ¼ 3.0 Hz, H-5), 2.15, 2.60, 2.16, 2.09, 2.09, 2.08, 2.06, 2.05, 1.98 (each 3H, s, C(O)CH3);
2.13, 2.06, 2.03, 1.97, 1.95 (each 3H, s, C(O)CH3), 1.99 (3H, s, 13C-NMR (100 MHz, CDCl3) d 171.1, 170.5, 170.4, 170.2, 170.1,
NHC(O)CH3); 13C-NMR (100 MHz, CDCl3) d 171.2, 170.8, 170.6, 169.3, 169.1, 168.2 (C(O)CH3), 140.0, 133.4 (Cquaternary), 130.5
170.4, 169.6, 169.4 (C(O)CH3), 159.9 (C(O)O–CH2–), 140.3, 133.5 (CHarom), 124.7, 122.6, 118.4 (Cquaternary), 116.3 (CHarom), 112.4
(Cquaternary), 131.7 (O–CH2–CH]CH2), 131.7 (CHarom), 127.3, 120.9 (]CH–N), 100.8 (C-10), 99.4 (C-1), 76.2 (C-3), 72.6 (C-5), 71.0 (C-
(Cquaternary), 119.9 (O–CH2–CH]CH2), 116.9, 115.7 (Cquaternary), 50), 70.7 (C-30), 69.4, 69.0 (C-20, C-4), 66.9 (C-40), 62.4 (C-6), 61.0
111.7 (CHarom), 102.1 (C-1), 100.9 (C-10), 77.6 (C-3), 72.3 (C-5), 71.4, (C-60), 57.4 (C-2), 23.8, 23.8, 20.9, 20.8, 20.8, 20.7, 20.6, 20.5
69.4 (C-30, C-4), 70.6 (C-50), 69.3 (C-20), 67.1 (C-40), 66.1 (O–CH2– (C(O)CH3); HRMS (ESI) m/z for C36H42BrClN2O18: [M + Na]+
CH]CH2), 62.1 (C-6), 61.1 (C-60), 57.3 (C-2), 23.7 (NHC(O)CH3), calcd 927.1202 found 927.1194.
21.1, 20.9, 20.8, 20.8, 20.8, 20.7 (C(O)CH3); HRMS (ESI) m/z for
(N-Acetyl-5-bromo-4-chloro-indox-3-yl) 2-acetamido-3,6-di-O-
acetyl-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)-
C38H44BrClN2O19: [M + Na]+ calcd 969.1308 found 969.1293.
(5-Bromo-4-chloro-indox-3-ylic acid allyl ester) 2- b-D-glucopyranoside (7b). Prepared according to general
acetamido-3,6-di-O-acetyl-2-deoxy-4-O-(2,3,4,6-tetra-O-acetyl-b-D- procedure 4. 6b (390 mg, 0.411 mmol), THF (8 mL), morpholine
galactopyranosyl)-b-D-glucopyranoside (6b). Prepared according (350 mL, 4.02 mmol), Pd(PPh3)4 (50 mg, 0.043 mmol), 15 min,
to general procedures 1 and 2. (1) 2b (900 mg, 1.32 mmol), acetyl 90–95 ꢀC. Yield 62% (230 mg, 0.254 mmol); colorless solid; mp
chloride (10 mL), methanol (1.5 mL). TLC (heptane/acetone 3 : 7, 146–148 ꢀC; [a]D23 -38.0 (c 0.25 in CHCl3); RF 0.32 (hexane/acetone
RF: 0.52; starting material 0.43). (2) DCM (15 mL), aqueous K2CO3 1 : 1); 1H-NMR (400 MHz, CDCl3) d 8.17 (1H, d, J ¼ 8.9 Hz,
solution (15 mL, 1 M), TBAHS (414 mg, 1.22 mmol), 5-bromo-4-
H
arom), 7.54 (1H, d, J ¼ 8.9 Hz, Harom), 7.44 (1H, s, ¼CH–N), 6.19
chloro-indoxylic acid allyl ester (400 mg, 1.21 mmol). Yield 45% (1H, d, J ¼ 9.1 Hz, C2–NH), 5.41–5.38 (1H, m, H-40), 5.22–5.15
(562 mg, 0.593 mmol); colorless solid; mp 149–152 ꢀC; [a]D23 -7.6 (2H, m, H-3, H-20), 5.11 (1H, d, J1,2 ¼ 5.4 Hz, H-1), 5.05 (1H, dd,
(c 0.5 in CHCl3); RF 0.41 (hexane/acetone 1 : 1); 1H-NMR (400 J2 ,3 ¼ 10.4 Hz, J3 ,4 ¼ 3.3 Hz, H-30), 4.81 (1H, dd, J5,6a ¼ 4.7 Hz,
0
0
0
0
0
0
0
MHz, CDCl3) d 8.64 (1H, s, NH), 7.48 (1H, d, J ¼ 8.8 Hz, Harom), J6a/b ¼ 11.8 Hz, H-6a), 4.57 (1H, d, J1 ,2 ¼ 8.0 Hz, H-1 ), 4.44–4.39
7.09 (1H, d, J ¼ 8.8 Hz, Harom), 6.58 (1H, d, J ¼ 8.6 Hz, C2–NH), (1H, m, H-2), 4.24 (1H, dd, J5,6b ¼ 5.0 Hz, J6a/b ¼ 11.8 Hz, H-6b),
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4.19 (1H, dd, J5 ,6 a ¼ 6.2 Hz, J6 a/b ¼ 11.3 Hz, H-60a), 4.12 (1H, dd, 72.3 (C-3), 68.2 (C-40), 60.5, 60.4 (C-6, C-60), 54.6 (C-2), 23.1
0
0
0
J5 ,6 b ¼ 7.1 Hz, J6 a/b ¼ 11.3 Hz, H-60b), 3.97–3.87 (3H, m, H-4, H- (C(O)CH3) ppm.
0
0
0
5, H-50), 2.17, 2.09, 1.99 (each 3H, s, C(O)CH3), 2.08, 2.06 (each
6H, s, 2C(O)CH3); 13C-NMR (100 MHz, CDCl3) d 170.4, 170.4,
Acknowledgements
170.3, 170.1, 170.0, 170.0, 169.7, 168.6 (C(O)CH3), 139.4, 133.5
(Cquaternary), 130.4 (CHarom), 118.3 (Cquaternary), 116.3 (CHarom), Support of this work by Glycom A/S, Copenhagen, Denmark, is
111.8 (]CH–N), 100.8 (C-10), 99.7 (C-1), 73.8, 73.1 (C-4, C-5), gratefully acknowledged.
71.0 (C-50), 70.5 (C-30), 70.0 (C-3), 69.1 (C-20), 66.6 (C-40), 62.3 (C-
6), 60.8 (C-60), 50.3 (C-2), 23.9, 23.2, 20.8, 20.7, 20.6, 20.5 (C(O)
References
CH3); HRMS (ESI) m/z for C36H42BrClN2O18: [M + Na]+ calcd
927.1202 found 927.1187.
1 R. M. Erney, W. T. Malone, M. B. Skelding, A. A. Marcon,
K. M. Kleman-Leyer, M. L. O'Ryan, G. Ruiz-Palacios,
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(5-Bromo-4-chloro-indox-3-yl) 2-acetamido-2-deoxy-3-O-(b-D-
galactopyranosyl)-b-D-glucopyranoside (8a). Prepared according
to general procedure 3. 7a (100 mg, 0.110 mmol), MeOH (3 mL),
cat. NaOMe, rt. Yield 75% (51 mg, 0.083 mmol); colorless solid;
mp 230 ꢀC dec; [a]D25 -100.8 (c 0.25 in 50% DMF/H2O); H-NMR
1
(400 MHz, DMSO-d6) d 11.15 (1H, bs, NH), 7.91 (1H, d, J ¼ 8.3
Hz, C2–NH), 7.36–7.16 (3H, m, 2 Harom, ]CH–N), 4.87 (1H, d,
J1,2 ¼ 8.5 Hz, H-1), 4.83–4.70 (3H, m, 3 OH), 4.66 (1H, bs, OH),
3 J. L. Magnani, Arch. Biochem. Biophys., 2004, 426, 122–131.
4 K. Lau, H. Yu, V. Thon, Z. Khedri, M. E. Leon, B. K. Tran and
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159, 649–664.
0
0
4.50 (1H, bs, OH), 4.33 (1H, bs, OH), 4.15 (1H, d, J1 ,2 ¼ 6.6 Hz,
H-10), 3.85–3.75 (2H, m, H-2, H-6a), 3.66–3.58 (2H, m, H-3, H-40),
3.57–3.49 (3H, m, H-6b, H-60a/b), 3.49–3.31 (5H, m, H-20, H-30,
H-4, H-5, H-50), 1.82 (3H, s, NHC(O)CH3); 13C-NMR (100 MHz,
DMSO-d6) d 169.9 (C(O)CH3), 132.9 (Cquaternary), 125.5 (CHarom),
122.9, 117.6 (Cquaternary), 114.0, 112.3 (CHarom, ¼CH–N), 111.7
(Cquaternary), 104.0 (C-10), 101.7 (C-1), 84.6 (C-3), 76.8, 75.8 (C-5,
C-50), 72.9, 68.9 (C-30, C-4), 70.6 (C-20), 68.2 (C-40), 60.8, 60.6 (C-6,
C-60), 54.2 (C-2), 23.1 (C(O)CH3) ppm.
8 J. A. Kiernan, Biotech. Histochem., 2007, 82, 73–103.
9 J. H. Miller, Experiments in Molecular Genetics, Cold Spring
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(5-Bromo-4-chloro-indox-3-yl) 2-acetamido-2-deoxy-4-O-(b-D- 10 D. Mahrun, Clin. Chim. Acta, 1976, 73, 453–461.
galactopyranosyl)-b-D-glucopyranoside (8b). Prepared according 11 S. Tokutake, N. Yamaji and M. Kato, Chem. Pharm. Bull.,
to general procedure 3. 7b (95 mg, 0.10 mmol), MeOH (3 mL),
1990, 38, 13–18.
cat. NaOMe, rt. Yield 73% (45 mg, 0.073 mmol); colorless solid; 12 K. Clinch, G. B. Evans, R. H. Furneaux, P. M. Rendle,
23
1
ꢀ
mp 230 C dec; [a]D -43.8 (c 0.5 in 50% DMF/H2O); H-NMR
(400 MHz, DMSO-d6) d 11.12 (1H, bs, NH), 7.88 (1H, d, J ¼ 9.2
P. L. Rhodes, A. M. Roberton, D. I. Rosendale, P. C. Tyler
and D. P. Wright, Carbohydr. Res., 2002, 337, 1095–1111.
´
Hz, C2–NH), 7.31 (1H, d, J ¼ 8.8 Hz, Harom), 7.29 (s, 1H, ¼CH– 13 G. Zemplen, Ber. Dtsch. Chem. Ges. B, 1926, 59B, 1254–1266.
¨
N), 7.22 (1H, d, J ¼ 8.8 Hz, Harom), 5.07 (1H, bs, OH), 4.79 (1H, 14 S. Bottcher, M. Hederos, E. Champion, G. Dekany and
bs, OH), 4.75 (1H, t, OH), 4.72 (1H, bs, OH), 4.71 (1H, d, J1,2
¼
J. Thiem, Org. Lett., 2013, 15, 3766–3769.
0
0
¨
8.6 Hz, H-1), 4.67 (1H, t, OH), 4.51 (1H, d, OH), 4.23 (1H, d, J1 ,2 15 S. Bottcher and J. Thiem, Eur. J. Org. Chem., 2014, 564–574.
¼ 7.4 Hz, H-10), 3.93–3.88 (1H, m, H-6a), 3.87–3.81 (1H, m, H- 16 H. Kunz and H. Waldmann, Angew. Chem., Int. Ed. Engl.,
2), 3.68–3.60 (2H, m, H-40, H-6b), 3.56 (1H, ddꢁvt, H-3), 3.54–
1984, 23, 71–72.
3.50 (2H, m, H-60a/b), 3.49–3.42 (1H, m, H-5), 3.40 (1H, ddꢁvt, 17 S. S. Rana, J. J. Barlow and K. L. Matta, Carbohydr. Res., 1983,
H-4), 3.34–3.32 (2H, m, H-20, H-30), 1.82 (3H, s, NHC(O)CH3);
113, 257–271.
13C-NMR (100 MHz, DMSO-d6) d 168.7 (C(O)CH3), 136.9, 132.9 18 H. Ohi, S. Kubota and T. Usui, Carbohydr. Res., 1993, 244,
(Cquaternary), 125.5 (CHarom), 123.0, 117.6 (Cquaternary), 114.0 (] 315–323.
CH–N), 112.3 (CHarom), 111.6 (Cquaternary), 104.0 (C-10), 102.0 19 K. L. Matta and J. J. Barlow, Carbohydr. Res., 1975, 75, 299–
(C-1), 81.4 (C-4), 75.6, 75.4 (C-5, C-50), 73.2, 70.6 (C-20, C-30),
304.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 10856–10861 | 10861