Degradation of target oligosaccharides by anthraquinone-lectin hybrids with light switching

Article abstract of DOI:10.1002/asia.201100586

Anthraquinone-lectin hybrids were effectively synthesized using water-soluble anthraquinone derivative 11 with concanavalin A (ConA) and hygrophorus russula lectin (HRL) to give anthraquinone-ConA (16) and anthraquinone-HRL (17) hybrids, respectively. These anthraquinone-lectin hybrids effectively and selectively degraded oligosaccharides containing a mannose residue as a non-reducing terminal sugar, which has affinity for ConA and HRL, under photo-irradiation with long-wavelength UV light without additives and under neutral conditions. In addition, anthraquinone-HRL (17) selectively photo-degraded only Man(α1,6)Man, which has a high affinity for HRL, among several mannosides by recognition of both the type and glycosidic linkage profile of the sugar in an oligosaccharide.

Full text of DOI:10.1002/asia.201100586

DOI: 10.1002/asia.201100586
Degradation of Target Oligosaccharides by Anthraquinone–Lectin Hybrids
with Light Switching
Yukari Imai,[a] Shingo Hirono,[a] Haruka Matsuba,[b] Tomohiro Suzuki,[b]
Yuka Kobayashi,[b] Hirokazu Kawagishi,[b] Daisuke Takahashi,[a] and
Kazunobu Toshima*[a]
Abstract: Anthraquinone–lectin hy-
brids were effectively synthesized using
water-soluble anthraquinone derivative
11 with concanavalin A (ConA) and
hygrophorus russula lectin (HRL) to
give anthraquinone–ConA (16) and an-
thraquinone–HRL (17) hybrids, respec-
tively. These anthraquinone–lectin hy-
brids effectively and selectively degrad-
ed oligosaccharides containing a man-
nose residue as a non-reducing termi-
nal sugar, which has affinity for ConA
and HRL, under photo-irradiation with
long-wavelength UV light without ad-
ditives and under neutral conditions. In
addition, anthraquinone–HRL (17) se-
lectively photo-degraded only Man-
AHCTUNGTRENGN(UN a1,6)Man, which has a high affinity
for HRL, among several mannosides
by recognition of both the type and
glycosidic linkage profile of the sugar
in an oligosaccharide.
Keywords: anthraquinone · lectin ·
oligosaccharides · photochemistry ·
selective-degradation
Introduction
ability was designed and synthesized to enhance the efficien-
cy of the synthesis of anthraquinone–lectin hybrids. In addi-
tion, we found that an anthraquinone–hygrophorus-russula-
lectin (HRL) hybrid selectively degraded an oligosaccharide
by recognition of both type and glycosidic linkage profile of
the sugar in an oligosaccharide.
Oligosaccharides play important roles in numerous biologi-
cal events, including protein folding, cell signaling, fertiliza-
tion, pathogens binding to host tissues, leukocyte trafficking
and the associated inflammatory response, tumor-cell meta-
stasis, and regulation of hormone and enzyme activity.[1]
Therefore, the development of innovative methods for selec-
tively controlling specific functions of certain oligosacchar-
ides has attracted much attention in the fields of chemistry,
biology, and medicine. In this context, we recently found
that certain anthraquinone derivatives could degrade oligo-
saccharides upon photo-irradiation in the absence of any ad-
ditives and under neutral conditions.[2,3] In addition, a de-
signed anthraquinone–lectin (peanut agglutinin; PNA)
hybrid was found to selectively degrade an oligosaccharide,
Results and Discussion
Design and Synthesis of Water-Soluble Anthraquinone
Derivative with Oligosaccharide Photo-Degrading Activity
In our previous study, we found that anthraquinone deriva-
tive 1 could degrade oligosaccharides upon photo-irradia-
tion, in the absence of any additives and under neutral con-
ditions (Scheme 1). Furthermore, a designed and synthe-
sized anthraquinone–lectin (PNA) hybrid (2) was found to
GalACHTUNGTRENNUNG(b1,3)GalNAc, with high affinity for PNA upon photo-ir-
radiation.[2] Herein, we report the application and improve-
ment of this fundamental study: a water-soluble anthraqui-
none derivative with high oligosaccharide photo-degrading
selectively degrade an oligosaccharide, GalACTHNGUTERN(UNG b1,3)GalNAc,
with high affinity for PNA under photo-irradiation condi-
tions.[2] However, it was also revealed that anthraquinone–
lectin hybrid 2 was not effectively synthesized, owing to the
low efficiency of the photoreaction[4] using a photo-reactive
and ligand-tethered diazirine substrate (3) to attach the an-
thraquinone moiety to the sugar pocket of PNA.[2,5] There-
fore, we planned to synthesize anthraquinone–lectin hybrids
using an acyl-transfer reaction using a ligand-tethered dime-
thylaminopyridine (DMAP) substrate reported by Hamachi
et al.[6] instead of the photoreaction of diazirine with lectin.
First, we synthesized anthraquinone-tethered acyl donors
4 and 5 and ligand (mannose)-tethered dimethylaminopyri-
dine (DMAP) 6[6] (for the synthesis, see the Supporting In-
formation), and then carried out an acyl-transfer reaction
using these substrates and concanavalin A (ConA). Howev-
er, the acyl-transfer reaction in 50 mm 4-(2-hydroxyethyl)-1-
[a] Y. Imai, S. Hirono, Dr. D. Takahashi, Prof. Dr. K. Toshima
Department of Applied Chemistry
Faculty of Science and Technology
Keio University
3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522 (Japan)
Fax : (+81)45-566-1576
E-mail: toshima@applc.keio.ac.jp
[b] H. Matsuba, Dr. T. Suzuki, Dr. Y. Kobayashi, Prof. Dr. H. Kawagishi
Department of Applied Biological Chemistry
Faculty of Agriculture, and/or Department of Bioscience
Graduate School of Science and Technology
Shizuoka University
Shizuoka 422-8529 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201100586.
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Scheme 1. Anthraquinone derivative 1, anthraquinone–PNA hybrid 2, and ligand-tethered diazirine 3.
piperazineethanesulfonic acid (HEPES) buffer (pH 8.0) at
258C for 3 hours did not afford the desired anthraquinone–
ConA hybrid 7. At this stage, based on acyl donor 8, report-
ed by Hamachi and co-workers which contained a water-
soluble fluorescein moiety,[6] we believed that the problem
was the low water solubility of the anthraquinone-tethered
acyl donor 4 or 5.
In order to overcome this problem, we designed water-
soluble anthraquinone derivative 9, which possessed a sul-
fonic acid group (Scheme 2). Thus, a water-soluble anthra-
quinone-tethered acyl donor 9 was synthesized as summar-
ized in Scheme 3. Commercially available anthraquinone 2-
carboxylic acid 10 was exposed to 30% SO3/H2SO4 with mi-
crowave irradiation to give 11 in 90% yield in a 1:1 mixture
of 2,6- and 2,7-disubstituted isomers. Thus obtained 11 was
amidated by 12 in the presence of 4-(4,6-dimethoxy-1,3,5-tri-
azin-2-yl)-4-methylmorpholinium chloride (DMT-MM) and
(N-ethylmorpholine) NEM to afford 13, whose tBu ester
was then removed using TFA to give 14 in 94% overall
yield. Finally, thioesterification of 14 using PhSH (15) in the
presence of DMT-MM and NEM furnished the desired an-
thraquinone-tethered acyl donor 9 with high water solubility
in 40% yield.
cyclodextrin (CD) as an oligosaccharide.[2,7] As shown in
Figure 1, both regioisomers of 11 showed a high photo-de-
grading ability against b-CD, and no significant difference
between the 2,6- and 2,7-isomers of 11 was observed. There-
fore, 9 was used as a mixture of isomers for further steps.
Design and Synthesis of Anthraquinone–ConA and
Anthraquinone–HRL Hybrids
Using the water-soluble anthraquinone-tethered acyl donor
9, we next conducted an acyl-transfer reaction with ConA,
and mannose (Man)-tethered DMAP 6 (Scheme 4a). Thus,
ConA (20 mm), Man-tethered DMAP 6 (100 mm), and an-
thraquinone-tethered acyl-donor 9 (100 mm) were mixed in
50 mm HEPES buffer (pH 8.0) at 258C for 3 hours to give
the desired anthraquinone–ConA hybrid (16) in 85% yield.
The formation of hybrid 16 was clearly confirmed by both
MALDI-TOF MS and UV spectroscopy (see the Supporting
Information), and purification of 16 was successfully per-
formed by affinity chromatography using Sephadex-100 with
d-glucose.
In contrast, anthraquinone–HRL hybrid 17 was not
obtained by a similar procedure using HRL, Man-tethered
DMAP 6, and anthraquinone-tethered acyl-donor 9. At this
stage, considering the affinity of HRL to several mannosides
At this stage, the oligosaccharide photo-degrading ability
of 11 possessing a sulfonic acid group was examined using b-
Scheme 2. Anthraquinone or fluorescein-tethered acyl donors 4, 5, 8, and 9, ligand-tethered dimethylaminopyridine (DMAP) 6, and anthraquinone–con-
canavalin A (ConA) hybrid 7.
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Degradation of Target Oligosaccharides by Anthraquinone–Lectin Hybrids with Light Switching
Scheme 3. Synthesis of anthraquinone-tethered acyl donor 9. a) 30% SO3/H2SO4, microwave (300 W), 1708C, 15 min, 90%; b) DMT-MM, NEM, MeOH/
H2O 3:1, RT, 19 h, 94%; c) TFA, CH2Cl2, RT, 3 h, 100%; d) DMT-MM, NEM, MeCN, RT, 5 h, 40%. TFA=trifluoroacetic acid.
reported by Kawagishi et al.,[8] we believed that the affinity
of Man-tethered DMAP 6 was insufficient for effective
recognition of HRL. Therefore, we decided to prepare
Man
ACHTUNGTERNN(UNG a1,6)Man-tethered DMAP 27 possessing a Man-
AHCTUNGTRENNUNG
and to use it in the acyl-transfer reaction instead of Man-
tethered DMAP 6.
The synthesis of ManACTHUNGTRENNG(U a1,6)Man-tethered DMAP 27 is
summarized in Scheme 5. The primary alcohol in the known
mannoside 18[9] was protected with a tert-butyldiphenylsilyl
(TBDPS) group to afford 19. The remaining free hydroxy
groups were protected with benzoyl groups to give 20, which
Figure 1. Photo-degradation of b-CD using 11. b-CD (333 mm) was incu-
bated with 2,6-disubstituted or 2,7-disubstituted 11 (100 mm) in H2O
(30 mL) at 258C for 2 h under irradiation with a UV lamp (365 nm, 100
W) placed at 10 cm from the mixture, and was analyzed by HPLC
(TSKgel Amide-80, 4.6ꢁ250 mm; MeCN/H2O=3:2; flow rate=
1.0 mLminꢀ1; 308C; IR detection). The percentage degradation was de-
termined based on the peak area of b-CD. Lane 1: b-CD with UV in the
absence of 11; lane 2: b-CD+11 without UV irradiation; lane 3: b-
CD+2,6-disubstituted 11 under UV irradiation; lane 4: b-CD+2,7-disub-
stituted 11 under UV irradiation.
ꢀ
was subjected to desilylation using HF Py to yield primary
alcohol 21. Glycosylation of 21 and mannosyl bromide 22
using AgOTf[10] proceeded smoothly to give disaccharide 23.
The azide moiety of 23 was reduced under H2 in the pres-
ence of Pd/C to afford crude amine 24. Amine 24 was cou-
pled with carboxylic acid 25[6] using 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (EDC) and diiso-
propylethylamine (DIEA) to give protected ManACTHNUTRGENUG(N a1,6)Man-
Scheme 4. Synthesis of anthraquinone-lectin hybrids 16 and 17. a) 50 mm HEPES buffer (pH 8.0), 258C, 3 h, 85% for 16 and 35% for 17.
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K. Toshima et al.
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Scheme 5. Synthesis of ManACHTNUTRGNE(UNG a1,6)Man-tethered dimethylaminopyridine 27. a) TBDPSCl, imidazole, CH2Cl2, RT, 18 h, 84%; b) BzCl, Py, 608C, 17 h,
99%; c) HF-Py, Py, 08C to RT, 7 h, 100%; d) AgOTf, 4 ꢂ M.S., CH2Cl2, 08C to RT, 4 h, 83%; e) H2, Pd/C, MeOH/EtOAc 5:1, RT, 3 h; f) EDC, DIEA,
DMF, RT to 608C, 38 h, 36% (2 steps); g) NaOMe, MeOH, RT, 2 h, 74%. DMF=N,N-dimethyl formamide.
tethered DMAP 26. Finally, deprotection of the benzoyl
groups in 26 using NaOMe yielded the desired Man-
ACHTUNGTRENNUNG
reaction was monitored by HPLC, and the percentage of
degradation was calculated based on the peak area corre-
sponding to each oligosaccharide. The results are summar-
ized in Figure 2.
A
When anthraquinone derivative 11 (without lectin) and 28
were used in the reaction, no degradation was detected
owing to the low concentration of 11 and its low affinity for
the oligosaccharide (Figure 2a). However, when the anthra-
quinone–ConA hybrid 16 was exposed to oligosaccharides
9
ACHTUNGTRENNUNG
anthraquinone-tethered acyl donor 9 (100 mm) were mixed
in 50 mm HEPES buffer (pH 8.0) at 258C for 3 hours to give
the desired anthraquinone–HRL hybrid 17 in 35% yield.
Again, the formation of hybrid 17 was clearly confirmed by
both MALDI-TOF MS and UV spectroscopy (see the Sup-
porting Information), and purification of 17 was successfully
performed by affinity chromatography using Sephadex-100
with d-glucose. Although the yield of hybrid formation in
the synthesis of 17 was lower than that of 16, both synthetic
efficiencies were much higher than that of the previous
method using a photoreaction and diazirine substrate 3.[2]
Man
ACHUTGTNRENNUG(a1,6)Man (28), ManAHCTUNGTREN(NUGN a1,2)Man (29), and Man-
AHCTUNGTRENNUNG
tion took place (degradation yields=65%–69%) (Fig-
ure 2b). This result was in sharp contrast to those for the
oligosaccharides GlcACTHNUTRGENN(GU a1,4)Glc (31) and GalACHTUNGTERN(NUGN b1,4)Glc (32),
which showed less degradation (degradation yields<25%)
under photo-irradiation with 16. These results indicate that
anthraquinone–ConA hybrid 16 selectively degraded the oli-
gosaccharides containing an a-mannoside residue at the
non-reducible end of the oligosaccharides upon photo-irra-
diation without any additives under neutral conditions. Fur-
thermore, the photo-degrading efficiency for these oligosac-
Target-Selective Photo-Degradation of Oligosaccharides by
Anthraquinone–ConA and Anthraquinone–HRL Hybrids
charides (Man
N
ACHTUNGTRENNUNG
We examined the application of both anthraquinone–ConA
(16) and anthraquinone–HRL (17) in the target-selective
photo-degradation of oligosaccharides. Photo-induced deg-
radation of five types of oligosaccharide (60 mm each), Man-
Man(a1,3)Man (30)>Glc
G
E
ACHTUNGTRENNUNG
(32)) by 16 correlated well with the affinity of ConA for
these oligosaccharides.[11]
On the other hand, when the anthraquinone–HRL hybrid
A
E
(a1,3)Man (30),
17 was exposed to the oligosaccharide ManACTHGNUTERNNU(G a1,6)Man (28)
A
R
under photo-irradiation, significant degradation took place
(degradation yield: 20%; Figure 2c). This result was in
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Degradation of Target Oligosaccharides by Anthraquinone–Lectin Hybrids with Light Switching
Figure 2. Photo-degradation of oligosaccharides. a) ManACHTUNTRGNEUNG(a1,6)Man (28) (60 mm) was incubated with and without 11 (18 mm) in phosphate buffer (120 mL,
10 mm, pH 7.4) under the conditions described in Figure 1, and was analyzed by HPLC (Mightysil RP-18 GP 5 mm, 4.6ꢁ150 mm; MeOH/H2O=1:2
(0.1% TFA); flow rate=1.0 mLminꢀ1; 408C; UV detection (210 nm)) after total acetylation of photo-degradation products. Percentage degradation was
determined based on the peak area of 28. Lane 1: 28 alone, lane 2: 28 with UV in the absence of 11, lane 3: 28+11 without UV irradiation, lane 4: 28+11
under UV irradiation. b) ManCAHTUNGTREN(NGUN a1,6)Man (28), ManACUTHTGNRENNU(GN a1,2)Man (29), ManACHTUNRTGENN(GUN a1,3)Man (30), GlcAHTCNUGTREN(NUNG a1,4)Glc (31), or GalACHTUNTGERNUG(N b1,4)Glc (32) (60 mm each) was incu-
bated with 16 (18 mm) in phosphate buffer (120 mL, 10 mm, pH 7.4) under the conditions described in Figure 1, and was analyzed by the method indicated
in Figure 2a). Percentage degradation was determined based on the peak area of each oligosaccharide. Lane 1: 28+16 without UV irradiation, lane 2:
28+16 with UV irradiation, lane 3, 29+16 without UV irradiation, lane 4: 29+16 with UV irradiation, lane 5: 30+16 without UV irradiation, lane 6:
30+16 with UV irradiation, lane 7: 31+16 without UV irradiation, lane 8: 31+16 with UV irradiation, lane 9: 32+16 without UV irradiation, lane 10:
32+16 with UV irradiation. c) ManACHTUNGERTN(NUGN a1,6)Man (28), ManACTHUNGTRENN(GUN a1,2)Man (29), ManACHTUNTRGEG(NNUN a1,3)Man (30), GlcAHTCUNGTRENN(UNG a1,4)Glc (31), or GalACTHUNTGNRUEGN(b1,4)Glc (32) (60 mm each) was
incubated with 17 (18 mm) in phosphate buffer (120 mL, 10 mm, pH 7.4) under the conditions described in Figure 1, and was analyzed by the method indi-
cated in Figure 2a). Percentage degradation was determined based on the peak area of each oligosaccharide. Lane 1: 28+17 without UV irradiation,
lane 2: 28+17 with UV irradiation, lane 3: 29+17 without UV irradiation, lane 4: 29+17 with UV irradiation, lane 5: 30+17 without UV irradiation,
lane 6: 30+17 with UV irradiation, lane 7: 31+17 without UV irradiation, lane 8: 31+17 with UV irradiation, lane 9: 32+17 without UV irradiation,
lane 10: 32+17 with UV irradiation.
sharp contrast to those for the oligosaccharides Man-
(a1,2)Man (29), Man(a1,3)Man (30), Glc(a1,4)Glc (31), and
Gal(b1,4)Glc (32), which showed much-less degradation
(degradation yield<7%) under photo-irradiation with 17.
These results indicate that anthraquinone–HRL hybrid 17
selectively degraded the target oligosaccharide, Man-
gosaccharide by photo-irradiation using anthraquinone–
lectin hybrids under neutral conditions. The target selectivity
of the hybrids is dependent on the recognition profile of the
lectin attached. The results presented here will contribute to
the molecular design of novel and artificial oligosaccharide
photo-degradation agents. We hope that this method will
provide a means of controlling specific functions of certain
oligosaccharides.
G
E
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG(a1,6)Man (28), upon photo-irradiation without any addi-
tives and under neutral conditions. These results also indi-
cate that anthraquinone–HRL 17 selectively photo-degraded
ManACHTUNGTRENNUNG(a1,6)Man, which has a high affinity for HRL, among
several mannosides by recognition of both type and glycosi-
dic linkage profile of the sugar in an oligosaccharide.
Experimental Section
General Methods
Concanavalin A (ConA) and anthraquinone 2-carboxylic acid (10) were
purchased from Funakoshi Chem. Co. and Sigma–Aldrich Co., respec-
tively, and used without further purification. Melting points were deter-
mined on a micro hot-stage (Yanako MP-S3) and were uncorrected. Op-
tical rotations were measured on a JASCO DIP-370 photo-electric polar-
imeter. NMR spectra were recorded on a JEOL JNM-Lambda 300
Conclusions
In the present study, we have developed an efficient and
general method for the selective degradation of a target oli-
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K. Toshima et al.
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(300 MHz for 1H) or a JEOL ECA-500 (500 MHz for 1H, 125 MHz for
13C) spectrometer using trimethylsilane as the internal standard, unless
otherwise noted. ESI-TOF Mass spectra were measured on a Waters
LCT premier XE. Matrix-assisted laser desorption ionization time-of-
flight mass spectrometry (MALDI-TOF MS) was conducted using a
Bruker Ultraflex mass spectrometer with detection in linear mode. Sina-
pinic acid was used as the matrix, with positive ionization mode. Silica
gel TLC, column chromatography and reverse phase column chromatog-
raphy were performed using Merck TLC 60F-254 (0.25 mm), Silica Gel
60 N (spherical, neutral) (Kanto Chemical Co., Inc.), and Wakosil 40C18
(Wako), respectively. High performance liquid chromatography (HPLC)
was performed on a JASCO apparatus using a TSKgel Amide-80 or a
Mightysil RP-18. Air- and/or moisture-sensitive reactions were carried
out under an argon atmosphere using oven-dried glassware. In general,
organic solvents were purified and dried using appropriate procedures,
and evaporation and concentration were carried out under reduced pres-
sure below 308C, unless otherwise noted.
Synthesis of 9
To a solution of 14 (26.2 mg, 62.8 mmol) in MeCN (1.60 mL) were added
DMT-MM (43.4 mg, 157 mmol) and NEM (13.8 mL, 126 mmol), and the
reaction mixture was stirred for 15 min at room temperature. And then,
thiophenol (15) (27.7 mL, 0.251 mmol) was added dropwise to the mixture
at 08C. After being stirred for 5 h at room temperature, the reaction mix-
ture was concentrated in vacuo. The residue was subjected to column
chromatography on silica gel (39:1:1 to 32:8:1 CHCl3/MeOH/HCO2H) to
give
9
(12.9 mg, 40% yield) as
a
pale-yellow oil; TLC
(CHCl3:MeOH:AcOH, 3:1:0.1 v/v/v): Rf =0.50; 1H NMR (300 MHz,
[D6]DMSO): d=9.03 (1H, m), 8.69 (1H, m), 8.43–8.21 (4H, m), 8.12–
8.09 (1H, m), 7.44 (5H, m), 3.38–3.34 (2H, m), 2.83 (2H, t, J=7.4 Hz),
1.92 ppm (2H, quint, J=6.8 Hz); HRMS (ESI-TOF) (m/z): [M]ꢀ calcd.
for C25H18NO7S2, 508.0525; found, 508.0548.
Synthesis of 19
To a solution of 18 (91.3 mg, 0.271 mmol) in CH2Cl2 (4.50 mL) were
added imidazole (55.3 mg, 0.812 mmol) and TBDPSCl (106 mL,
0.408 mmol) at 08C. After the reaction mixture was stirred for 18 h at
room temperature, H2O (5.0 mL) was added to the reaction mixture. The
resulting solution was extracted with two portions of CHCl3. The com-
bined extracts were washed with brine, dried over anhydrous Na2SO4, fil-
tered, and concentrated in vacuo. The residue was subjected to column
chromatography on silica gel (toluene/acetone, 9:1 to 3:2) to give 19
(132 mg, 84% yield) as a colorless oil; TLC (toluene/EtOAc, 3:2 v/v):
Rf =0.47; ½aꢁ2D5 =ꢀ12.88 (c=0.48, CHCl3); 1H NMR (500 MHz, CDCl3):
d=7.69–7.67 (4H, m), 7.46–7.38 (6H, m), 4.86 (1H, br-d), 3.95–3.80 (6H,
m), 3.69–3.57 (10H, m), 3.37 (2H, t, J=4.9 Hz), 2.97 (1H, m), 2.68 (1H,
d, J=3.7 Hz), 2.37 (1H, d, J=4.6 Hz), 1.07 ppm (9H, s); 13C NMR
(125 MHz, CDCl3): d=135.66, 132.98, 132.91, 129.96, 127.87, 99.60, 71.67,
70.99, 70.76, 70.38, 70.29, 70.12, 66.58, 65.15, 50.72, 26.89, 19.26 ppm;
HRMS (ESI-TOF) (m/z): [M+Na]+ calcd. for C28H41N3O8NaSi, 598.2561;
found, 598.2583.
Synthesis of 11
Anthraquinone 2-carboxylic acid (10; 205 mg, 0.814 mmol) was dissolved
in 30% SO3/H2SO4 (1.50 mL), and the resulting solution was stirred
under microwave irradiation (300 W) for 15 min at 1708C. After cooling,
the reaction mixture was added dropwise to ice-cold water (40 mL), and
then NaCl (6.0 g) was added to the resulting mixture. Then, the thus-
formed precipitate was filtered, and the resulting solid was washed with
10% NaCl (aq.) and EtOH, and dried in vacuo to give 11 (244 mg, 90%
yield, 2,6-disubstituted 11/2,7-disubstituted 11=1/1) as a dark orange
solid; TLC (CHCl3/MeOH/AcOH, 3:1:0.1 v/v/v): Rf =0.13; m.p.>3008C;
HRMS (ESI-TOF) (m/z): [M]ꢀ calcd. for C15H7O7S, 330.9912; found,
330.9910; 2,6-disubstituted 11: 1H NMR (500 MHz, [D6]DMSO): d=8.69
(1H, d, J=6.4 Hz), 8.43 (1H, m), 8.41 (1H, dd, J=1.8, 8.0 Hz), 8.34 (1H,
d, J=8.0 Hz), 8.24 (1H, d, J=8.1 Hz), 8.13 ppm (1H, d, J=8.1 Hz);
13C NMR (125 MHz, [D6]DMSO): d=182.50, 182.13, 166.52, 154.23,
136.36, 136.22, 134.91, 133.93, 133.55, 133.46, 131.92, 127.99, 127.94,
127.73, 124.32 ppm. 2,7-disubstituted 11: 1H NMR (500 MHz,
[D6]DMSO): d=8.70 (1H, d, J=1.5 Hz), 8.42 (1H, d, J=2.0 Hz), 8.41
(1H, m), 8.33 (1H, d, J=8.1 Hz), 8.22 (1H, d, J=8.0 Hz), 8.11 ppm (1H,
dd, J=1.5, 8.0 Hz); 13C NMR (125 MHz, [D6]DMSO): d=182.35, 182.29,
166.52, 154.34, 136.36, 136.15, 134.95, 133.98, 133.53, 133.45, 131.89,
127.96, 127.71, 127.31, 124.31 ppm.
Synthesis of 20
To a solution of 19 (19.7 mg, 34.2 mmol) in pyridine (1.00 mL) was added
BzCl (23.9 mL, 0.206 mmol) at 08C. After the reaction mixture was
stirred for 17 h at 608C, 1 m HCl (aq.) was added to the reaction mixture
at 08C to adjust the pH between 4 and 5. The resulting solution was ex-
tracted with two portions of CHCl3. The combined extracts were washed
with brine, dried over anhydrous Na2SO4, filtered, and concentrated in
vacuo. The residue was subjected to column chromatography on silica gel
(9:1 to 3:2 n-hexane/EtOAc) to give 20 (30.1 mg, 99% yield) as a color-
less oil; TLC (n-hexane/EtOAc, 3:2 v/v): Rf =0.47; ½aꢁ2D5 =ꢀ15.48 (c=
0.50, CHCl3); 1H NMR (500 MHz, CDCl3): d=8.13–7.26 (25H, m), 6.16
(1H, t, J=10.1 Hz), 5.85 (1H, dd, J=1.7, 10.3 Hz), 5.72 (1H, m), 5.20
(1H, d, J=1.7 Hz), 4.20–4.17 (1H, m), 3.94–3.87 (2H, m), 3.79–3.69
(10H, m), 3.37 (2H, t, J=5.4 Hz), 1.05 ppm (9H, s); 13C NMR
(125 MHz, CDCl3): d=171.95, 165.69, 165.37, 135.81, 135.63, 133.87,
133.48, 133.25, 133.16, 133.04, 130.30, 130.09, 128.59, 128.46, 128.36,
127.69, 97.85, 71.46, 71.02, 70.82, 70.33, 70.21, 67.36, 66.72, 62.56, 50.78,
26.72, 19.30 ppm; HRMS (ESI-TOF) (m/z): [M+Na]+ calcd. for
C49H53N3O11NaSi, 910.3347; found, 910.3308.
Synthesis of 13
To a solution of 11 (177 mg, 0.535 mmol) in MeOH (3.30 mL) and H2O
(1.10 mL) were added DMT-MM (231 mg, 0.834 mmol) and NEM
(47.6 mL, 0.433 mmol) at room temperature. The reaction mixture was
stirred for 15 min, and then 12 (53.6 mg, 0.337 mmol) was added to the
mixture. After being stirred for 19 h at room temperature, the reaction
mixture was concentrated in vacuo. The residue was subjected to silica
gel column chromatography (3:1:1:0.1 CHCl3/acetone/MeOH/AcOH) to
give 13 (150 mg, 94% yield) as a light-yellow solid; TLC (CHCl3/MeOH/
AcOH, 3:1:0.1 v/v/v): Rf =0.39; m.p.>3008C; 1H NMR (500 MHz,
CD3OD): d=8.91 (1H, m), 8.69 (2H, m), 8.35 (m, 2H), 8.26 (m, 2H),
3.46 (2H, dt, J=6.6, 12.5 Hz), 2.34 (2H, t, J=7.5 Hz), 1.91 (2H, quint,
J=7.2 Hz), 1.44 ppm (9H, s); HRMS (ESI-TOF) (m/z): [M]ꢀ calcd. for
C23H22NO8S, 472.1048; found, 472.1066.
Synthesis of 21
Synthesis of 14
To a solution of 20 (139 mg, 0.157 mmol) in pyridine (4.20 mL) was
added HF-Py (0.400 mL) at 08C. After the reaction mixture was stirred
at room temperature for 7 h, saturated aq. NaHCO3 (20 mL) was added
to the reaction mixture to quench the reaction. The resulting mixture was
extracted with two portions of CHCl3. The combined organic layers were
washed with brine, dried over anhydrous Na2SO4, and concentrated in
vacuo. The residue was subjected to column chromatography on silica gel
(toluene/EtOAc=1:1) to give 21 (101 mg, quant.) as a colorless oil; TLC
(n-hexane/EtOAc, 1:4 v/v): Rf =0.54; ½aꢁ2D8 =ꢀ109.98 (c=0.50, CHCl3);
1H NMR (500 MHz, CDCl3): d=8.10 (2H, m), 7.97 (2H, m), 7.81 (2H,
m), 7.63–7.37 (7H, m), 7.27–7.23 (2H, m), 5.99 (1H, dd, J=3.4, 10.2 Hz),
5.82 (1H, t, J=10.2 Hz), 5.70 (1H, dd, J=1.7, 3.4 Hz), 5.16 (1H, d, J=
1.7 Hz), 4.16 (1H, ddd, J=2.3, J=3.9, J=9.8 Hz), 3.94 (1H, ddd, J=3.9,
To a solution of 13 (140 mg, 0.296 mmol) in CH2Cl2 (4.20 mL) was added
TFA (4.20 mL) at room temperature. After being stirred for 3 h at the
same temperature, the reaction mixture was concentrated in vacuo, and
the residue was subjected to reverse-phase column chromatography on
silica gel (H2O/MeOH, 1:0 to 4:1) to give 14 (123 mg, quant.) as a pale-
yellow solid; TLC (CHCl3/MeOH/AcOH, 3:1:0.1 v/v/v): Rf =0.22; m.p.>
3008C; 1H NMR (500 MHz, D2O): d=8.14 (1H, d, J=5.2 Hz), 7.99–7.78
(5H, m), 3.30 (2H, dt, J=1.9, 5.6 Hz), 2.34 (2H, t, J=7.2 Hz), 1.82 ppm
(2H, quint, J=6.4 Hz); HRMS (ESI-TOF) (m/z): [M]ꢀ calcd. for
C19H14NO8S, 416.0440; found, 416.0441.
102
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Chem. Asian J. 2012, 7, 97 – 104

Degradation of Target Oligosaccharides by Anthraquinone–Lectin Hybrids with Light Switching
J=5.1, J=10.8 Hz), 3.85–3.68 (11H, m), 3.37 (2H, t, J=5.1 Hz),
2.67 ppm (1H, dd, J=6.0, J=8.1 Hz); 13C NMR (125 MHz, CDCl3): d=
166.58, 165.59, 165.55, 133.72, 133.61, 133.24, 130.00, 129.97, 129.77,
129.40, 129.24, 128.88, 128.71, 128.58, 128.36, 97.97, 71.06, 70.90, 70.84,
70.71, 70.36, 70.18, 69.73, 67.69, 67.52, 61.54, 50.76 ppm; HRMS (ESI-
TOF) (m/z): [M+Na]+ calcd. for C33H35N3O11Na, 672.2169; found,
672.2167.
71.40, 71.31, 70.79, 70.70, 70.23, 70.04, 69.93, 69.16, 67.28, 67.24, 66.34,
66.11, 61.57, 48.52, 39.18, 36.51, 33.25 ppm; HRMS (ESI-TOF) (m/z):
[M+H]+ calcd. for C27H46N3O14, 636.2980; found, 636.2968.
Preparation of Anthraquinone–ConA Hybrid 16
To a solution of 100 mm ConA in 50 mm HEPES buffer (pH 8.0, 5.90 mL)
was slowly added 250 mm Man-tethered DMAP 6 in 50 mm HEPES
buffer (pH 8.0, 11.8 mL), followed by addition of 250 mm acyl donor 9 in
the same buffer (11.8 mL; final concentrations of ConA, 6 and 9 are
20 mm, 100 mm and 100 mm, respectively). After incubation for 3 h at 258C
in the dark, the reaction mixture was dialyzed against H2O, and the re-
sulting solution was lyophilized. The residue was subsequently purified
by affinity chromatography (Sephadex G100, 2.6 cmꢁ12 cm), eluted with
a linear gradient of 10 mm acetate buffer (pH 5.0) containing d-glucose
(from 0 mm to 40 mm), followed by dialysis against H2O, and the resulting
solution was lyophilized to afford anthraquinone–ConA hybrid 16 in
85% yield.
Synthesis of 23
A suspension of 21 (73.1 mg, 0.113 mmol), 22 (222 mg, 0.338 mmol) and
4 ꢂ M.S. (36.5 mg, 50 wt% to 21) in CH2Cl2 (11.0 mL) was stirred for
30 min at 08C, and then AgOTf (105 mg, 0.409 mmol) was added to the
suspension. After the suspension was stirred at room temperature for 4 h,
triethylamine (1.0 mL) was added to the reaction mixture to quench the
reaction, and then saturated aq. NaHCO3 was added to the resulting so-
lution. The resulting mixture was extracted with two portions of CHCl3.
The combined organic layers were washed with brine, dried over anhy-
drous Na2SO4, and concentrated in vacuo. The residue was subjected to
column chromatography on silica gel (n-hexane/EtOAc=4:1 to 3:2) to
give disaccharide 23 (115 mg, 83% yield) as a colorless oil; TLC (n-
hexane/EtOAc, 7:5 v/v): Rf =0.48; ½aꢁ2D5 =ꢀ20.38 (c=0.46, CHCl3);
1H NMR (300 MHz, CDCl3): d=8.18–7.28 (35H, m), 6.15–5.94 (4H, m),
5.79 (2H, m), 5.19 (1H, br-d), 5.16 (1H, br-d), 4.52–4.12 (6H, m), 3.85–
3.69 (10H, m), 3.38 ppm (2H, t, J=4.9 Hz); 13C NMR (125 MHz,
CDCl3): d=166.12, 165.74, 165.63, 165.57, 165.32, 133.60, 133.52, 133.22,
133.09, 130.12, 129.91, 129.79, 129.39, 129.24, 129.13, 129.05, 128.87,
128.65, 128.52, 128.38, 98.04, 97.79, 70.97, 70.83, 70.62, 70.36, 70.20, 69.50,
69.00, 67.67, 67.14, 66.74, 66.70, 50.78 ppm; HRMS (ESI-TOF) (m/z):
[M+Na]+ calcd. for C67H61N3O20Na, 1250.3746; found, 1250.3691.
Preparation of Anthraquinone–HRL Hybrid 17
To a solution of 100 mm HRL in 50 mm HEPES buffer (pH 8.0, 5.90 mL)
was slowly added 250 mm ManACTHNUTRGENU(GN a1–6)Man-tethered DMAP 27 in 50 mm
HEPES buffer (pH 8.0, 11.8 mL), followed by addition of 250 mm acyl
donor 9 in the same buffer (11.8 mL; final concentrations of HRL, 27,
and 9 are 20 mm, 100 mm, and 100 mm, respectively). After incubation for
3 h at 258C in the dark, the reaction mixture was dialyzed against H2O,
and the resulting solution was lyophilized. The residue was subsequently
purified by affinity chromatography (Sephadex G100, 2.6 cmꢁ12 cm),
eluted with a linear gradient of 10 mm phosphate buffer (pH 8.0) contain-
ing d-mannose (from 0 mm to 300 mm), followed by dialysis against H2O,
and the resulting solution was lyophilized to afford anthraquinone–HRL
hybrid 17 in 35% yield.
Synthesis of 26
A suspension of 23 (57.4 mg, 46.7 mmol), 10% Pd/C (5.00 mg), and
EtOAc (1.10 mL) in MeOH (5.70 mL) was stirred under H2 atmosphere
(balloon). After the suspension was stirred for 3 h at room temperature,
it was filtered through a pad of celite. The filtrate was concentrated in
vacuo to afford crude 24. The residue was used in the next step without
further purification. To a solution of the crude 24 and 25 (33.7 mg,
0.187 mmol) in DMF (5.60 mL) were added DIEA (24.0 mL,
0.140 mmol) and EDC (13.4 mg, 70.1 mmol) at 08C. After the reaction
mixture was stirred for 38 h at 608C, H2O (5.0 mL) was added to the re-
action mixture. The resulting mixture was extracted with three portions
of CHCl3. The combined organic layers were washed with brine, dried
over anhydrous Na2SO4, and concentrated in vacuo. The residue was sub-
jected to column chromatography on silica gel (CHCl3/MeOH, 4:1) to
give 26 (22.9 mg, 36% yield) as a colorless oil; TLC (CHCl3/MeOH, 4:1
v/v): Rf =0.50; ½aꢁ2D5 =ꢀ21.58 (c 0.51, CHCl3); 1H NMR (300 MHz,
CDCl3): d=8.16–7.16 (37H, m), 6.50 (3H, m), 6.11–5.91 (4H, m), 5.78–
5.75 (2H, m), 5.19 (1H, br d), 5.15 (1H, br d), 4.54–4.04 (6H, m), 3.83–
3.63 (10H, m), 3.54 (2H, t, J=4.9 Hz), 3.46 (2H, t, J=5.1 Hz), 2.94 (3H,
s), 2.44 ppm (2H, t, J=6.8 Hz); 13C NMR (125 MHz, CDCl3): d=170.77,
166.14, 165.85, 165.78, 165.63, 165.52, 165.37, 153.44, 148.96, 133.68,
133.58, 133.69, 133.26, 133.14, 130.08, 129.90, 129.78, 128.91, 128.67,
128.54, 128.44, 106.69, 97.95, 97.73, 70.72, 70.56, 70.41, 70.19, 69.79, 69.59,
69.02, 62.59, 47.89, 39.49, 37.68, 33.72 ppm; HRMS (ESI-TOF) (m/z):
[M+H]+ calcd. for C76H74N3O21, 1364.4815; found, 1364.4786.
Photo-Degradation of Oligosaccharides
Each oligosaccharide (60 mm) was incubated with the anthraquinone–
lectin hybrid (18 mm) in phosphate buffer (120 mL, 10 mm, pH 7.4) at
258C for 2 h under irradiation with a UV lamp (365 nm, 100 W, Blak-ray
(B-100A), UVP, Inc.) placed at 10 cm from the mixture. After photo-irra-
diation, the mixture was dialyzed with
a 30 kDa molecular cut off
Amicon Ultradialysis column. The resulting solution was lyophilized, and
the residue was acetylated using Ac2O in pyridine at room temperature.
After incubation for 22 h, the reaction mixture was concentrated, and
then the residue was subjected to column chromatography on silica gel
(7:3 to 0:1 n-hexane/EtOAc) and analyzed by HPLC (Mightysil RP-18
GP-5 mm, 4.6ꢁ150 mm; 408C; UV detection (210 nm)).
Acknowledgements
This research was supported by the 21st Century COE Program “Keio
Life-Conjugated Chemistry”, the High-Tech Research Center Project for
Private Universities: Matching Fund Subsidy, 2006–2011, and the Scien-
tific Research (B) (No. 20310140 and 23310153) from the Ministry of
Education, Culture, Sports, Science and Technology of Japan (MEXT).
Synthesis of 27
To a solution of 26 (18.8 mg, 13.8 mmol) in MeOH (1.90 mL) was added
0.2m NaOMe (96.6 mL, 19.3 mmol). After being stirred for 2 h at room
temperature, the reaction mixture was concentrated in vacuo. The resi-
due was subjected to reverse-phase column chromatography on silica gel
(H2O/MeOH, 1:0 to 1:1) to give 27 (6.50 mg, 74% yield) as a colorless
oil. Reverse-phase TLC (H2O/MeOH, 1:1 v/v): Rf =0.30; ½aꢁ2D5 =ꢀ18.68
(c 0.38, MeOH); 1H NMR (300 MHz, CD3OD): d=8.06 (2H, d, J=
6.6 Hz), 6.69 (2H, dd, J=1.4, 3.5 Hz), 4.81 (1H, d, J=1.7 Hz), 4.77 (1H,
d, J=1.5 Hz), 3.84–3.57 (22H, m), 3.47 (2H, t, J=4.9 Hz), 3.34–3.29 (2H,
m), 3.02 (3H, s), 2.49 ppm (2H, t, J=6.8 Hz); 13C NMR (125 MHz,
CD3OD): d=172.56, 154.03, 148.08, 106.74, 100.49, 99.98, 73.07, 71.75,
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Received: July 1, 2011
Published online: October 13, 2011
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Chem. Asian J. 2012, 7, 97 – 104