1

In Vitro Imaging and In Vivo Liver Targeting with Carbohydrate Capped

Quantum Dots
Raghavendra Kikkeri, Bernd Lepenies, Alexander Adibekian, Paola Laurino, and
Peter H. Seeberger*


Laboratory for Organic Chemistry, Swiss Federal Institute of Technology (ETH)
Zurich, Wolfgang-Pauli-Str. 10, 8093 Zurich, Switzerland

Table of Contents:
1. General Information
2. Synthesis of QDs
3. Synthesis of sugars and PEG derivatives
4. Synthesis of glyco-QDs
5. Synthesis of PLL-galactose polymer
6. Photophysical properties of QDs
7. Transmission Electron Microscopy (TEM)
8. Estimation of the concentration of carbohydrates per QD
9. In vitro and in vivo experimental details
10. References
11.
1
H and
13
C-NMR data of all the compounds.








2

1. General Information
All chemicals used were reagent grade and used as supplied except where noted.
Dichloromethane (CH
2
Cl
2
) was purified by a Cycle-Tainer Solvent Delivery System.
Triethylamine was distilled over CaH
2
prior to use. Analytical thin layer chromatography (TLC)
was performed on Merck silica gel 60 F
254
plates (0.25 mm). Compounds were visualized by UV
irradiation or dipping the plate in CAN solution followed by heating. Flash column
chromatography was carried out using force flow of the indicated solvent on Fluka Kieselgel 60
(230-400 mesh).
1
H and
13
C NMR spectra were recorded on a Varian VXR-300 (300 MHz) or
Bruker DRX500 (500 MHz) spectrometer. High-resolution mass spectra (HR MALDI MS) were
recorded using mass Spectrometry service at the Laboratory for Organic Chemistry (ETH
Zurich). IR spectra were recorded on a Perkin-Elmer 1600 FTIR spectrometer. Optical rotation
measurements were conducted using a Perkin-Elmer 241 polarimeter. Absorption spectra were
recorded using a Varian CARY 50 spectrophotometer fitted with Hellma optical fibers (Hellma,
041.002-UV) and an immersion probe made of quartz suprazil (Hellma, 661.500-QX).
Fluorescence emission spectra were recorded on a Perkin-Elmer LS-50B spectrofluorometer.
Poly-L-lysine (mol wt 15000-30000) was purchased from sigma-fine chemicals.

2. Synthesis of QDs
CdSe/ZnS quantum dots with an emission maximum centered at 635 nm were synthesized via
pyrolysis of the organometallic precursors, following published procedure.
1
Cadmium oxide
(0.05 g, 4.0 mmol), trioctylphosphine oxide (TOPO) (2 g, 5.2 mmol), lauric acid (0.22 g, 1.1
mmol), and hexadecylamine (2 g, 8.3 mmol) were mixed and heated to 310

C, under nitrogen
flow, for 30 min. A selenium stock solution, prepared from selenium (0.03 g, 4.0 mmol) in
trioctylphosphine (TOP) (2 mL, 5.4 mmol), was rapidly injected into the reaction mixture at
310

C. The reaction mixture was then immediately cooled to 270

C, the stirring was stopped

after 5 min and the reaction vessel cooled to 60

C. CdSe-QD cores were purified by precipitation

in methanol/CHCl
3
. The CdSe particles were subsequently coated with ZnS layers by dropwise
addition of a mixture of TOP (3 mL), diethylzinc in hexane (2.0 mL), and
hexamethyldisilathiane (0.2 mL) over 1h at 160

C. Following cooling to room temperature, the

3

concentration of the resulting CdSe/ZnS QDs solution was then estimated by using a previously
published procedure.
2
3. Synthesis of Sugars and PEG Derivatives
(ii)
(iii)
(iv)
6
6a
(i)
n
(v)
n
4
4a
S S
O
O
O
O
OH
SHSH
O
O
O
O
OH
HO
O
O
OH
n
N
3
O
O
N
3
n
H
2
N
O
O
NH
2
n
n
n
S S
N
H
O
O
O
NH
2
SHSH
N
H
O
O
O
NH
2
1a
3
(v)

Scheme 1. Reagents and conditions: (i) MsCl, TEA, NaN
3
, 12 h; (ii) Ph
3
P, H
2
O, 12 h; (iii) DL-thioctic acid, DIC,
12 h; (iv) DL-thioctic acid, DCC, DMAP, 12 h; (v) NaBH
4
, MeOH/H
2
O;
(i) Diazido-PEG
2000
(1a). PEG
2000
(10 g, 5.0 mmol) was coevaporated with 50 mL toluene to
remove water, and then 50 mL of THF and methyl sulfonyl chloride ( 1.22 mL. 15.0 mmol) were
added. The solution was stirred at 0C, as triethylamine (2.29 mL, 16.5 mmol) in 20 mL of THF
was added dropwise over 30 min. After 1 h the ice bath was removed and the mixture was stirred
overnight. The formed solid was dissolved by the addition of water (50 mL), forming two layers,
that were cooled on an ice bath. Then, 1N solution of sodium bicarbonate (5 mL) was added
followed by sodium azide (1.3 g, 20.0 mmol). THF was removed and the water layer was
refluxed for 24 h. The aqueous layer was extracted with dichloromethane (4 X 20 mL). Each
dichloromethane layer was washed with an additional 100 mL saturated NaCl solution. The
4

dichloromethane layers were combined, dried over sodium sulfate and concentrated to yield (8.4
g, 84%) diazido-PEG
2000
as 2a white solid flakes.
1
H NMR (300 MHz, CDCl
3
): 3.37 (t, J = 5.1
Hz, 4H); 3.63-3.68 (m, 168H).
13
C NMR (75 MHz, CDCl
3
); 50,7, 67.8, 68.1, 68.9, 69.6, 70.5,
70.6, 71.6, 72.6, 3.0, 73.2.
(ii) Diamino-PEG
2000
(3). Diazido-PEG
2000
1a (8 g, 4.0 mmol) was dissolved in 50 mL THF
and triphenylphosphine (3.14 g, 12.0 mmol) was added. The solution was stirred at room
temperature for 10 h, water (3 mL) was added and stirred overnight. THF was removed in vacuo,
and additional 100 mL of water were added. The triphenyl phosphinoxide precipitate was
removed by filtration and the water was removed in vacuo to yield (5.1 g, 63%) diamino-
PEG
2000
3 as white solid.
1
H NMR (300 MHz, CD
3
OD): 2.77 (t, J = 5.4 Hz, 4H); 3.52 (t, J =
5.4 Hz, 4H); 3.63-3.68 (m, 164H).
13
C NMR (75 MHz, CD
3
OD); 39.7, 68.1, 68.6, 68.8, 69.6,
70.7.
(iii) TA-PEG
2000
-NH
2
(4). DL-thioctic acid (0.3 g, 1.45 mmol) and N-hydroxysuccinimide (0.16
g, 1.6 mmol) were added to diisopropyl carbodiimide (0.27 g, 1.74 mmol) in CH
2
Cl
2
and stirred
at 0

C for 2 h. This mixture was added dropwise to diamino-PEG

2000
3 (4.0 g, 2.0 mmol) in 30
mL of THF. The reaction mixture was stirred at room temperature for 12 h and concentrated in
vacuo. The crude residue was purified by flash silica column chromatography using
CH
2
Cl
2
/CH
3
OH (10-15%) as eluent to yield 2.25 g (48%) of TA-PEG
2000
-NH
2
4.
1
H NMR (300
MHz, CDCl
3
): 1.42-1.51 (m, 2H); 1.62-1.70 (m, 4H); 1.89-1.93 (m, 2H); 2.17-2.2 (br.s, 2H);
2.42-2.48 (m, 1H); 2.58-2.62 (m, 1H); 3.12-3.18 (m, 2H); 3.41-3.46 (m 4H); 3.55-3.72 (m,
164H);
13
C NMR (75 MHz, CDCl
3
); 24.5, 24.9, 28.6, 33.8, 34.5, 38.4, 40.1, 61.56, 63.4, 68.5,
68.6, 69.1, 69.7, 70.5, 71.2, 71.3, 72.5, 73.3; HRMS-MALDI (m/z): [M+Na]+ Calculated for
C
82
H
164
O
37
N
2
S
2
Na +(C
2
H
4
O)
n
1856.03466 + (44.026)
n
; Found: 1856.0346 + (44.032)
n
.
(iv) DHLA-PEG
2000
-NH
2
(4a). TA-PEG
2000
-NH
2
4 (2.0 g, 0.5 mmol) was dissolved in an
ethanol/water (1:1) mixture, cooled to 0

C and sodium borohydride (20 mg, 0.54 mmol) was

added and stirred at 0
0
C for 3 h and overnight stirring at room temperature . The solution was
extracted with chloroform, dried over sodium sulfate and concentrated to yield (0.94 g, 47%) of
DHLA-PEG
2000
-NH
2
.
1
H NMR (300 MHz, CD
3
OD): 1.28-137 (m, 2H); 1.48-1.75 (m, 4H);
1.82-1.92 (m, 1H); 2.34 (t, J = 7.5 Hz, 2H); 2.42-2.48 (m, 2H); 2.68-2.672 (m, 2H); 2.84-2.92
5

(m, 1H); 3.55-3.72 (m, 164H);
13
C-NMR (75 MHz, CD
3
OD); 21.6, 24.2, 26.1, 33.5, 38.2,
38.9, 42.5, 61.2, 63.1, 68.9, 69.9, 70.2, 71.2, 71.8, 72.1, 72.5, HRMS-MALDI (m/z): [M+Na]+
Calculated for C
82
H
166
O
37
N
2
S
2
Na +(C
2
H
4
O)
n
: 1858.03466 + (44.026)
n
Found: 1858.0342 +
(44.022)
n
.
(v) TA-PEG
2000
-OH (6). DL-thioctic acid (0.13 g, 0.63 mmol) and PEG
2000
(12.0 g, 6.0 mmol)
were added to dicyclohexyl carbodiimide (0.17 g, 0.83 mmol) and dimethylaminopyridine (0.10
g, 0.8 mmol) in dichloromethane at 0

C, stirred at room temperature for 12 h and concentrated in

vacuo. The crude residue was purified by flash silica column chromatography using
EtOAc/MeOH (2-5%) to yield 1.5 g (12%) of TA-PEG
2000
-OH 6.
1
H NMR (300 MHz, CD
3
OD):
1.42-1.51 (m, 2H); 1.61-1.70 (m, 4H); 1.87-1.95 (m, 2H); 2.35 (t, J = 7.5 Hz, 2H); 2.42-2.48
(m, 1H); 2.58-2.62 (t, J = 6.0 Hz, 1H); 3.12-3.18 (m, 2H); 3.41 (t, J = 6.0 Hz, 1H); 3.55-3.72 (m,
164H); 3.87 (t, J = 6.0 Hz, 1H); 4.24 (t, J = 5.4 Hz, 2H);
13
C-NMR (75 MHz, CD
3
OD); 22.3,
24.6, 26.6, 34.1, 88.7, 39.4, 42.7, 53.4, 61.7, 69.3, 70.5, 71.2, 71.3, 72.2, 72.7; HRMS-MALDI
(m/z): [M+Na]+ Calculated for C
80
H
158
O
38
S
2
Na + (C
2
H
4
O)
n
1813.9770 + (44.026)
n
; Found:
1813.972 + (44.032)
n
.
(vi) DHLA-PEG
2000
-OH (6a). TA-PEG
2000
-OH 6 (1.0 g, 0.5 mmol) was dissolved in an
ethanol/water (1:1) mixture and cooled to 0

C. Sodium borohydride (20 mg, 0.54 mmol) was

added and stirred at 0

C for 3 h. The solution was neutralized with 1 N HCl solution, extracted

with chloroform, dried over sodium sulfate, concentrated and purified by flash column
chromatography using CH
2
Cl
2
/CH
3
OH (2-5%) to yield 0.45 g (46%) of DHLA-PEG
2000
-OH 6a.
1
H NMR (300 MHz, CD
3
OD): 1.28-137 (m, 2H); 1.48-1.75 (m, 4H); 1.82-1.92 (m, 1H); 2.34
(t, J = 7.5 Hz, 2H); 2.42-2.48 (m, 2H); 2.68-2.672 (m, 2H); 2.84-2.92 (m, 1H); 3.55-3.72 (m,
164H); 4.21 (t, J = 6.0 Hz, 1H);
13
C NMR (75 MHz, CD
3
OD); 21.8, 24.1, 26.1, 33.5, 38.3,
38.9, 42.4, 61.2, 63.1, 68.7, 69.9, 70.2, 71.3, 71.8, 72.2, 72.5; HRMS-MALDI (m/z): [M+Na]+
Calculated for C
80
H
160
O
38
S
2
Na + (C
2
H
4
O)
n
: 1815.9770 + (44.026)
n
; Found: 1815.975 +
(44.031)
n
.


6

(ii)-(iii)
14
15
16
(vi)
O
AcO
AcO
OAc
12
(iv)
O
AcO
AcO
N
3
SePh
OAc
13
O
AcO
AcO
N
3
O
OAc
O
O
Cl
(v)
O
HO
HO
N
3
O
OH
O
O
SH
O
HO
HO
H
2
N
O
OH
O
O
SH
O
AcO
AcO
OAc
OAc
OAc
O
AcO
AcO
OAc
O
OAc
O
O
Cl
O
HO
HO
OH
O
OH
O
O
SH
(i)
8
9
(ii)-(iii)
O
OAc
AcO
OAc
OAc
OAc
O
OAc
AcO
OAc
OAc
O
O
O
Cl
O
OAc
AcO
OAc
OAc
O
O
O
Cl
(ii)-(iii)
(i)
10
11

Scheme 2. Reagents and conditions: (i) Bis(2-chloroethoxy)ethanol, BF
3
.
Et
2
O, 12 h; (ii) KSAc, DMF, 12 h; (iii)
NaOMe, MeOH; Amberlite-H
+
; (iv) Trimethylsilyl azide, bis(acetoxy)iodobenzene, diphenyl diselenide, 12 h; (v)
bis(2-chloroethoxy)ethanol, N-Iodosuccinimide, TfOH; (vi) PMe
3
, H
2
O, 12 h.

(i) General procedure A. The acetylated sugar (1 eq.) and bis(2-chloroethoxy)ethanol (3 eq.)
were dissolved in 10 mL CH
2
Cl
2
and cooled to 0
o
C. BF
3
.
Et
2
O (4 eq.) was added and stirred at
room temperature for 48 h. The reaction was quenched with triethylamine and extracted with
7

dichloromethane/water mixture. The organic layer was concentrated and purified by silica
column flash chromatography to yield sugar-PEG-Cl.
(ii) General procedure B. Sugar-PEG-Cl (1 eq.) and potassium thioacetate (3 eq.) were
dissolved in 10 mL of anhydrous DMF and stirred at room temperature for 12 h. Ethyl acetate
was added and the organic layer was washed five times with water. Purification by flash silica
column chromatography afforded sugar-PEG-SAc.
(iii) General Procedure C. Sugar-PEG-Sac (1 eq) and sodium methoxide (10 mol%) were
dissolved in 10 mL of methanol and stirred at room temperature for 15 min. The reaction mixture
was neutralized with Amberlite-H
+
and solvent was evaporated to yield the free thiol.
(iv) 2-(2-(2-Chloroethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl--D-mannopyranoside (8).
General procedure A with 1,2,3,4,6-penta-O-acetyl-D-mannopyranoside (1.5 g, 3.8 mmol),
bis(2-chloroethoxy) ethanol (1.92 mL, 11.4 mmol) and BF
3
.
Et
2
O (2.43 mL, 15.2 mmol),
followed by purification by flash silica column chromatography using hexane/ethyl acetate
(40:60) yielded 1.23 g (64%) of 2-(2-(2-chloroethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl--D-
mannopyranoside 8. []
D
r.t
= +0.17 (c = 1.0, CHCl
3
);
1
H NMR (300 MHz, CDCl
3
): 1.91 (s,
3H); 1.96 (s, 3H); 2.01 (s 3H); 2.07 (s 3H); 3.53-3.72 (m, 11H); 4.02-4.04 (m, 2H); 4.18 (dd, J

=
5.4, 4.2 Hz, 1H); 4.79 (d, J = 2.1 Hz, 1H); 5.17- 5.26 (m, 4H);
13
CNMR (75 MHz, CDCl
3
);
20.5, 42.7, 61.6, 66.4, 66.2, 67.3, 68.3, 68.5, 69.1, 69.4, 69.6, 70.3, 71.3, 72.5, 97.6, 97.7, 169.6,
169.8, 169.9, 170.5; HRMS-MALDI (m/z): [M]
+
Calculated for C
20
H
31
O
12
Cl: 498.1504; Found:
498.1501.
(v) 2-(2-(2-Thioacetylethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl--D-mannopyranoside.
General procedure B with 2-(2-(2-chloroethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl--D-
mannopyranoside (1.0 g, 2.0 mmol) and potassium thioacetate (0.58 g, 6.0 mmol), followed by
purification by flash column chromatography using hexane/ethylacetate (30:70) yielded 0.86 g
(79%) of 2-(2-(2-thioacetylethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl--D-mannopyranoside.
[]
D
r.t
= +0.19 (c = 1.0, CHCl
3
);
1
H NMR (300 MHz, CDCl
3
): 1.98 (s, 3H); 2.03(s, 3H); 2.10 (s
3H); 2.15 (s 3H); 2.33 (s, 3H); 3.11 (t, J = 6.6 Hz, 2H); 3.58-3.68 (m, 9H); 4.08-4.15 (m, 2H);
4.31 (dd, J
1
= 5.4, 4.2 Hz, 1H); 4.87 (d, J = 2.1 Hz, 1H); 5.25- 5.38 (m, 4H);
13
C NMR (75 MHz,
CDCl
3
); 20.7, 21.1, 28.8, 30.5, 30.7, 60., 62.2, 62.4, 66.2, 67.4, 68.3, 68.5, 68.9, 69.5, 69.7,
8

70.0, 70.3, 70.6, 97.7, 169.5, 169.7, 169.8, 170.5. HRMS-MALDI (m/z): [M]
+
Calculated for
C
22
H
34
O
13
S: 538.1723; Found: 538.1721.
(vi) 2-(2-(2-Thioethoxy)ethoxy)ethoxy--D-mannopyranoside (9). General procedure C
with 2-(2-(2-thioacetylethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl--D-mannopyranoside (0.6
g, 1.11 mmol) and sodium methoxide (60 mg, 10%) yielded 0.29 g (80%) of 2-(2-(2-
thioethoxy)ethoxy)ethoxy--D-mannopyranoside 9. []
D
r.t
= +7.82 (c = 1.0, MeOH);
1
H NMR
(300 MHz, CD
3
OD): 2.67 (t, J = 6.6 Hz, 2H); 3.58-3.71 (m,12H); 3.81-3.85 (m, 4H) 4.92 (s,
1H);
13
C NMR (75 MHz, CD
3
OD); 21.9, 60.2, 65.1, 65.8, 68.5, 68.6, 68.8, 69.3, 69.4, 69.8,
71.4, 71.8, 98.9; HRMS-MALDI (m/z): [M]
+
Calculated for C
12
H
24
O
8
S 328.1192; Found:
328.1192.
(vii) 2-(2-(2-Chloroethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl--D-galactopyranoside
(10). General procedure A with 1,2,3,4,6-penta-O-acetyl-D-galactopyranoside (1g, 2.5 mmol),
bis(2-chloroethoxy) ethanol (1.28 mL, 7.6 mmol) and BF
3
.
Et
2
O (1.6 mL, 10.1 mmol), followed
by purification by flash silica column chromatography using hexane/ethyl acetate (40:60) yielded
0.88 g (68%) of 2-(2-(2-chloroethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl--D-galacto-
pyranoside 8. []
D
r.t
= +6.8 (c = 1.0, CHCl
3
);
1
H NMR (300 MHz, CDCl
3
): 1.96 (s, 9H); 2.02
(s, 9H); 2.04 (s, 9H); 2.14 (s, 9H); 3.58- 3.84 (m, 25H); 4.08 -4,12 (m, 6H); 4.49 (d, J = 8.0 Hz,
1H); 4.99 (dd, J = 10.9, 3.4 Hz, 1H); 5.37 (d, J = 3.5 Hz, 1H); 5.29-5.43 (m, 4H);
13
C NMR (75
MHz, CDCl
3
): 20.6, 28.3, 36.4, 37.0, 39.1, 41.7, 45.6, 59.7, 61.2, 66.9, 67.0, 67.2, 68.7, 69.1,
70.6, 101.1, 169.5, 169.6, 169.7, 169.9, 170.1, 170.2; HRMS-MALDI (m/z): [M]
+
Calculated for
C
20
H
31
O
12
Cl: 498.1504; Found: 498.1501.
(viii) 2-(2-(2-Thioacetylethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl--D-galactopyranoside.
General procedure B with 2-(2-(2-chloroethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl--D-
mannopyranoside (0.5 g, 1.0 mmol) and potassium thioacetate (0.29 g, 3.0 mmol), followed by
purification by flash column chromatography using hexane/ethylacetate (40:60) yielded 0.51 g
(84%) of 2-(2-(2-thioacetylethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl--D-galactopyranoside.
[]
D
r.t
= +11.5 (c = 1.0, CHCl
3
);
1
H NMR (300 MHz, CDCl
3
): 1.96 (s, 9H); 2.02 (s, 9H); 2.04
(s, 9H); 2.14 (s, 9H); 3.58- 3.84 (m, 25H); 4.08- 4.12 (m, 6H); 4.49 (d, J = 8.0 Hz, 1H); 4.99 (dd,
J = 10.9, 3.4 Hz, 1H); 5.37 (d, J = 3.5 Hz, 1H); 5.29-5.43 (m, 4H);
13
C NMR (75 MHz,
9

CDCl
3
): 20.6, 28.3, 36.4, 37.0, 39.1, 41.67, 45.65, 59.7, 61.2, 66.9, 67.0, 67.2, 68.7, 69.1, 70.6,
101.1, 169.5, 169.6, 169.7, 169.9, 170.1, 170.2; HRMS-MALDI (m/z): [M]
+
Calculated for
C
22
H
34
O
13
S: 538.1723; Found: 538.1721.
(ix) 2-(2-(2-Thioethoxy)ethoxy)ethoxy--D-galactopyranoside (11). General procedure C
with 2-(2-(2-thioacetylethoxy)ethoxy)ethoxy-2,3,4,6-tetra-O-acetyl--D-mannopyranoside (0.2
g, 0.33 mmol) and sodium methoxide (20 mg, 10%) yielded 0.11 g (87%) of 2-(2-(2-
thioethoxy)ethoxy)ethoxy--D-galactopyranoside 9. []
D
r.t
= +0.22 (c = 1.0, MeOH);
1
H NMR
(300 MHz, CD
3
OD): 2.67 (t, J = 6.6 Hz, 2H); 3.56-3.67 (m,12H); 3.81-3.88 (m, 4H); 4.26 (d, J
= 7.2 Hz, 1H);
13
C NMR (75 MHz, CD
3
OD); 21.9, 60.2, 65.1, 65.8, 68.5, 68.6, 68.8, 69.3,
69.7, 71.4, 72.3, 72.9, 102.5; HRMS-MALDI (m/z): [M]
+
Calculated for C
12
H
24
O
8
S 328.1192;
Found: 328.1192.
(x) Phenyl seleno-2-azido-3,4,6-tri-O-acetyl--D-galactopyranoside (13). Tri-O-acetyl-D-
galactal 10 (1.5 g, 5.51 mmol) was dissolved in 30 mL CH
2
Cl
2
and diphenyl diselenide was
added (1.72 g, 5.51 mmol). The solution was cooled to -30C and bis(acetoxy)iodobenzene was
added (1.77 g, 5.51 mmol). After addition of trimethylsilyl azide (1.45 ml, 11.02 mmol) the
reaction was stirred for 12 h while warming to rt. The solvent was removed and the residue was
purified by silica column chromatography (10% to 30% EtOAc in cyclohexanes) to give
galactosamine 11 as -anomer (2.51 g, 5.35 mmol, 97%). The analytical data were in agreement
with those reported in the literature.
3

(xi) 2-(2-(2-chloroethoxy)ethoxy)ethoxy-3,4,6-tri-O-acetyl--D-galactosamine (14). D-
galactosamine selenoglycoside 12 (522 mg, 1.1 mmol) was dissolved in 3 mL CH
2
Cl
2
and 3 mL
Et
2
O and cooled to 0C. (2-Chloroethoxy)bis ethanol (561 mg, 3.3 mmol) was added, followed
by addition of N-iodosuccinimide (1.24 g, 5.5 mmol) and triflic acid (18 L, 0.2 mmol). The
mixture was stirred overnight while warming to r.t. The reaction was quenched with NEt
3
and
washed with sat. aq. sodium thiosulfate solution. The organic layer was separated and dried over
Mg
2
SO
4
. The solvent was evaporated and the residue was purified by silica column
chromatography (30% to 60% EtOAc in cyclohexanes) to give galactosamine 12 as a separable
/-mixture (1:1) (376 mg, 0.78 mmol, 71%). R
f
0.3 (cyclohexanes/ethyl acetate = 1:1); []
D
rt
=
42.1 (c = 1, CHCl
3
); IR (thin film on NaCl): = 2874, 2112, 1747, 1434, 1371, 1228, 1127, 1046
10

cm
-1
;
1
H NMR (300 MHz, CDCl
3
): 5.43-5.29 (m, 4H), 5.06 (d, J = 3.5 Hz, 1H), 4.74 (dd, J =
10.9, 3.4 Hz, 1H), 4.48 (d, J = 8.0 Hz, 1H), 4.30-3.98 (m, 6H), 3.84-3.58 (m, 25H), 2.12 (s, 3H),
2.11 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H);
13
C NMR (75 MHz, CDCl
3
):
170.2, 170.1, 169.9, 169.7, 169.6, 169.5, 102.4, 98.1, 71.3, 70.9, 70.8, 70.7, 70.6, 70.5, 70.4,
70.1, 69.3, 68.1, 67.7, 67.5, 66.5, 66.3, 61.5, 61.2, 60.7, 57.3, 42.8, 42.7, 21.0, 20.9, 20.8, 20.7,
20.6 20.5; MALDI-HRMS (m/z): Calculated for C
18
H
28
ClN
3
O
10
Na
+
: 504.1355, Found: 504.1361
[M+Na]
+
.
(xii) 2-(2-(2-Thioacetylethoxy)ethoxy)ethoxy-2-azido-3,4,6-tetra-O-acetyl--D-galacto-
pyranoside. General procedure B with 2-(2-(2-chloroethoxy)ethoxy)ethoxy-2-azido-3,4,6-tetra-
O-acetyl--D-galactopyranoside (0.1 g, 0.21 mmol) and potassium thioacetate (60 mg, 0.63
mmol), followed by purification by flash column chromatography using hexane/ethyl acetate
(20:80) yielded 85 mg (78%) of 2-(2-(2-thioacetylethoxy)ethoxy)ethoxy-2-azido-3,4,6-tetra-O-
acetyl--D-galactopyranoside. []
D
r.t
= +12.3 (c = 1.0, CHCl
3
);
1
H NMR (300 MHz, CDCl
3
):
2.02 (s, 9H); 2.12 (s, 3H); 2.32 (s, 3H); 3.07 (t, J = 6.6 Hz, 2H); 3.58-3.69 (m, 9H); 3.99-4.16
(m, 1H); 4,46 (d, J = 7.5 Hz, 1H); 4.74 (dd, J = 3.3, 7.5 Hz, 1H); 5.07 (d, J = 3.6 Hz, 1H); 5.28-
5.41 ( m, 1H);
13
C-NMR (75 MHz, CDCl
3
); 20.5, 28.7, 30.5, 42.6, 7.3, 60.6, 61.4, 66.3, 67.6,
68.1, 69.3, 70.0, 70.2, 70.4, 70.8, 71.2, 98.1, 169.7, 169.9, 170.2; HRMS-MALDI (m/z): [M]
+

Calculated for C
20
H
31
O
11
N
3
S: 521.1679; Found: 521.1678.
(xiii) 2-(2-(2-Thioethoxy)ethoxy)ethoxy-2-azido--D-galactopyranoside (15). General
procedure C with 2-(2-(2-thioacetylethoxy)ethoxy)ethoxy-2-azido-3,4,6-tetra-O-acetyl--D-
galactopyranoside (80 mg, 0.17 mmol) and sodium methoxide (10 mg, 0.17 mmol) yielded (45
mg, 80%) of 2-(2-(2-thioethoxy)ethoxy)ethoxy-2-azido--D-galactopyranoside 13. []
D
r.t
=
+0.96 (c = 1.0, MeOH);
1
H NMR (300 MHz, CD
3
OD): 2.67 (t, J = 6.6 Hz, 2H); 3.58-3.71
(m,12H); 3.81-3.85 (m, 4H); 4.34 (d, J = 7.2 Hz, 1H); 4.96 (d, J = 3.6 Hz, 1H);
13
C NMR (75
MHz, CD
3
OD); 21.9, 59.7, 66.9, 67.5, 67.6, 68., 6.7, 69.7, 71.3, 72.1, 73.9, 102.3; HRMS-
MALDI (m/z): [M]
+
Calculated for C
12
H
23
O
7
N
3
S: 353.1257; Found: 353.1255.
(xiv) 2-(2-(2-Thioethoxy)ethoxy)ethoxy-2-amino--D-galactopyranoside (16). Trimethyl
phosphine (0.1 mL of a 10 M solution) was added to 2-(2-(2-thioethoxy)ethoxy)ethoxy-2-azido-
-D-galactopyranoside (40 mg, 0.11 mmol) in anhydrous THF. The solution was stirred at room
11

temperature for 3 h before addition of water (1 mL) and stirring overnight. THF was removed in
vacuo, and the residue was dissolved in 10 mL water. The water layer was washed with
chloroform (4 X 5 mL). Water was lyophilized to yield 30 mg (81%) of 2-(2-(2-
thioethoxy)ethoxy)ethoxy-2-amino--D-galactopyranoside 14. []
D
r.t
= +1.25 (c = 1.0, MeOH);
1
H NMR (300 MHz, CD
3
OD): 2.66 (t, J = 6.6 Hz, 2H); 3.45-3.75 (m,14H); 3.99-4.04 (m, 1H);
4.26 (d, J = 7.2 Hz, 1H);
13
C NMR (75 MHz, CD
3
OD); 21.9, 59.7, 66.9, 67.5, 67.6, 68.4, 69.7,
69.7, 71.3, 72.1, 73.9, 102.3; HRMS-MALDI (m/z): [M]
+
Calculated for C
12
H
25
O
7
NS:
327.1352; Found: 327.1351.
4. Synthesis of Glyco-QDs.
ZnS
CdSe
TOPO/TOP
ZnS
CdSe
S S
O
O
O
O
OH
ZnS
CdSe
S S
N
H
O
O
O
NH
2
ZnS
CdSe
S S
N
H
O
O
O
NH N
O
O
O
1, 2 or 3
(i)
(ii)
(iii)
7
n n
n
5
5a
(i)

Scheme 3. Reagents and conditions: (i) 4a or 6a, CdSe/ZnS/TOP/TOPO, EtOH/CHCl
3
; (ii) 3-
maleimidopropanoic acid N-hydroxysuccinimide ester, borate buffer pH 8.5; (iii) 9, 11 or 16, borate buffer pH 7.5.

(i) QD
635
-DHLA-PEG
2000
-NH
2
(5). To 0.1 mL of QDs in chloroform (O.D > 50 at 400 nm) 50
L of DHLA-PEG
2000
-NH
2
4a in 50 L of ethanol were added. The mixture was stirred at 60

C
for 12 h. In between, another 50 L of ethanol were added. PEGylated-QDs were separated by
12

precipitation using ethanol/chloroform/hexane (1:0.5:5). Centrifugation at 3000 rpm for 3 min
yielded a clear pellet-like suspension. This pellet was separated and dissolved in distilled water
(1 mL), centrifuged at 3000 rpm for 3 min to yield a clear solution. This solution was lyophilized
and pure PEGylated-QD was obtained from NAP-1 column chromatography using 1.0 M
borate-buffer pH 7.5 as the eluent. Concentration of the QDs was estimated using a previously
published procedure.
2

(ii) QD
635
-DHLA-PEG
2000
-maleimide (5a). QD
635
-DHLA-PEG
2000
-NH
2
(0.05 M in 1 mL of
1.0 M borate-buffer pH 8.5) was added to 3-maleimido propanoic acid N-hydroxysuccinimide
ester (13 mg, 49.0 M) and stirred at room temperature for 2 h. The aqueous layer was washed
with chloroform (3 x 1 mL) to remove unreacted maleimido derivative. The aqueous layer was
taken as such for the next step without further characterization.
(iii) QD
635
-DHLA-PEG
2000
-mannose (1). QD
635
-DHLA-PEG
2000
-NH
2
(0.02 M in 1 mL of 1.0
M borate-buffer pH 7.3) was added to 2-(2-(2-thioethoxy)ethoxy)ethoxy--D-mannopyranoside
(10 mg, 30.5 M) and stirred at room temperature for 3 h. The crude product was purified by
NAP-1 column using distilled water as the eluent. The final concentration of the sample was
estimated by using a previously published procedure.
2

(iv) QD
635
-DHLA-PEG
2000
-galactosamine (2). QD
635
-DHLA-PEG
2000
-NH
2
( 0.02 M in 1 mL
of 1.0 M borate-buffer pH 7.3) was added to 2-(2-(2-thioethoxy)ethoxy)ethoxy-2-amino--D-
galactopyranoside ( 10 mg, 30.5 M) and stirred at room temperature for 3 h. The crude product
was purified by NAP-1 column using distilled water as the eluent. The final concentration of the
sample was estimated by using a previously published procedure.
2
(v) QD
635
-DHLA-PEG
2000
-galactose (2a). QD
635
-DHLA-PEG
2000
-NH
2
( 0.02 M in 1 mL of 1.0
M borate-buffer pH 7.3) was added to 2-(2-(2-thioethoxy)ethoxy)ethoxy-2-amino--D-
galactopyranoside ( 10 mg, 30.5 M) and stirred at room temperature for 3 h. The crude product
was purified by NAP-1 column using distilled water as the eluent. The final concentration of the
sample was estimated by using a previously published procedure.
2
(vi) QD
635
-DHLA-PEG
2000
-OH

(7). To 0.1 mL of QDs in chloroform (O.D > 50 at 400 nm) 50
L of DHLA-PEG
2000
-OH 6a in 50 L of ethanol were added. The mixture was stirred at 60

C
13

for 12 h. In between, another 50 L of ethanol were added. PEGylated-QD was separated by
precipitation using ethanol/dichloromethane/hexane (3:1:5). Centrifugation at 3000 rpm for 3
min yielded brownish suspension. This suspension was separated and dissolved in distilled water
(1 mL), centrifuged at 3000 rpm for 3 min to yield a clear solution. This solution was lyophilized
and pure PEGylated-QD was obtained from NAP-1 column chromatography using distilled
water as the eluent. Concentration of the QDs was estimated by using a previously published
procedure.
2
5. Synthesis of PLL-galactose polymer.
H
2
N
O
NH
NH
2
O
OH
H
2
N
n
H
2
N
O
NH
NH
O
OH
HN
n
O
N
O
O
O
N
O
O
H
2
N
O
NH
NH
O
OH
HN
n
O
N
O
O
O
N
O
O
O
OH
HO
OH
O
OH
O
HO
HO
OH
O
OH
(i)
(ii)
17
18

Scheme 4. Reagents and conditions: (i) 3-maleimidopropanoic acid N-hydroxysuccinimide ester, pH 8.0, 24 h;
(ii) Hg Lamp, Hanovia, 450W, allyl galactose, 20 mins.
(i) PLL-maleimido (16). Poly-L-Lysine hydrobromide (10 mg, 0.5 mol) was added to 3-
Maleimidopropionic acid-N-hydrosuccinimide ester (133 mg, 0.5 mmol) in 6 mL of nano pure
water. The solution was basified with 1 N KOH solution and shaked at room temperature. The
aqueous layer was washed with dichloromethane (3 x 7 mL) to remove unreacted maleimido
14

derivative gave 19 mg of pure yellow oil (98%). []
D
25
= -1.2 (c=1.5, MeOH/H
2
O 1:1);
1
H NMR
(300 MHz, MeOD) 2.2-2.4 (m, 10 H); 3.5 (t, J = 7 Hz, 2H); 5.8 (d, J = 13 Hz, 1H); 6.3 (d, J =
13 Hz, 1H);
13
C NMR (300 MHz, MeOD) 26.2. 37.8, 38.5, 125.5, 138.4, 167.5, 174.6, 178.6,
179.9.
(ii) PLL-galactose (17). 3- Maleimidopropionic acid-N-poly-L-Lysine (10 mg, 0.2 mol) and
allyl-galactose (88 mg, 0.4 mmol) were dissolved in H
2
O/CH
3
CN (1:1), and flushed in a
continuous flow reactor for 20 minutes under irradiation (medium pressure Hg Lamp, Hanovia,
450W). To the mixture 2-mercaptothiol (6.45 L, 0.725 mol) was added and stirred for 4 h.
The crude product was further purification with sephadex G-10 to yield 10 mg of PLL-galactose
(77%). []
D
25
= +82.4 (c=1.5, MeOH/H
2
O 1:1);
1
H NMR (300 MHz, D
2
O) 1.70-2.01 (m, 2 H);
2.91 (t, J = 6 Hz, 2H); 3.50-4.15 (m, 16H); 5.05 (s, 1H);
13
C NMR (300 MHz, MeOD) 27.6.
27.9, 28.1, 28.6, 28.9, 33.1, 39.7, 39.9, 40.1, 58.8, 59.1, 59.4, 59.9, 60.2, 60.3, 60.8, 61.1, 66.3,
66.5, 66.6, 68.0, 68.3, 68.6, 68.9, 69.2, 60.5, 70.6, 70.9, 98.1.

6. Photophysical Properties of QDs
Absorbance measurements of QD solutions were performed by a Cary 100 UV-visible scan
spectrophotometer (Varian, Australia). Fluorescence emission spectra were recorded on a Perkin-
Elmer LS-50B spectrofluorometer. The emission spectra of the QDs are shown in Figure 1. Upon
excitation at the 400 nm, a maximum emission at 635 nm was observed. Quantum yields have
been calculated using the equation,

comp
/
ref
=A
comp

*
[C]
ref
/ A
ref

*
[C]
comp

where [C] refers to the concentration of the samples and A to the area of the emission spectra.
Here, Ru(bipy)
3
(Cl)
2
was used as a reference compound of quantum yield
ref
= 0.062.
5
UV-
visible spectra and fluorescent spectra before and after sugar functionalized QDs showed no
significant difference in the
max
.
15


Figure 1. (a) Uv-visible spectrum of QD 2 (b) Fluorescent spectra of the QD 1 (solid line) and 2
(dotted line)
Compound max (nm) QY ( )
1
2
3
19
CdSe/ZnS/TOP/TOPO
635
635
634
635
635
0.33
0.36
0.35
0.34
0.32


7. Transmission Electron Microscopy (TEM).
A drop of the 0.1 M concentration sample was deposited on a 400 mesh copper grids coated
with nitrocellulose followed by carbon evaporation. The grids were observed in a Philips CM
120 at an accelerating voltage of 200 kV. Figure 1 shows typical TEM images of monodispersed
QD
635
-DHLA-PEG
2000
-Mannose. The size of the QDs measured was about 15-20 nm, which fits
well with the expected actual size.
(a) (b)
16


Figure 2 : TEM images of QD
635
-DHLA-PEG
2000
-mannoside and size distribution histogram.

8. Estimation of the Concentration of Carbohydrates per QD.
The concentration of mannose sugar on CdSe-ZnS was determined by the phenol-sulfuric acid
method using a microtiter plate. A sugar functionalized-QD solution (10 L, 1.5 mol),
concentrated sulfuric acid (75 L, 100%) and aqueous phenol solution (5% w/v, 10 L) were
added to the microtiter plate and heated to 80

C. After 5 min, the plate was cooled to room

temperature and the absorbance coefficient at 490 nm was measured. A Carbohydrate-QD
solution and sulfuric acid were used as a control. The mannose concentration was estimated by
comparing the absorption of the sample with a standard curve. The number of sugar molecules
per QD particle was calculated from the ratio of the concentration of sugar and the concentration
of CdSe-ZnS QD particles. QDs 1 and 2 showed 82-85 sugar molecules per each quantum dots.

9. In-vitro and In-vivo experimental details
Uptake of QDs in vitro
Hepatocellular carcinoma cell line HepG2 was plated in 24-Well plates and cultivated in DMEM
culture medium. When cells were 70 % confluent different concentrations of either PEG
2000
-
capped QDs, Man-PEG
2000
QDs, Gal-PEG
2000
QDs or GalN-PEG
2000
QDs were added to the
cells. After incubation times between 2 h and overnight cells were washed three times with cold
PBS. Then, the incubation medium was replaced with 1 mL PBS and cells were collected using a
17

cell scraper. Uptake of QDs was measured by flow cytometry. Excitation wavelength in the
FACS experiments was 488 nm. Cells were gated on living cells and fluorescence channel FL-3
was used to detect HepG2 cells that had incorporated QDs. All data were acquired on a
FACSCanto flow cytometer (Becton Dickinson, Mountain View, CA) and analyzed with the
FlowJo software (Tree Star Inc., Ashland, OR).
In order to inhibit receptor-mediated endocytosis HepG2 cells were preincubated either with a
polymer with numerous galactose residues on its surface for 30 min (for inhibition of Gal-QDs
uptake) or an excess of free D-galactosamine (100 g/ml, for inhibition of GalN-QDs uptake).
Afterwards the Gal-QDs or GalN-QDs were added to the cells and binding and/or uptake of QDs
into the cells was measured as described above.










Figure 3: Partial inhibition of the uptake of GalN-capped QDs by preincubation with free GalN.

Knockdown of ASGP-R1
For knockdown of the ASGP-R1 in HepG2 cell line pre-designed Silencer

Select siRNAs were

used that were synthesized by Applied Biosystems (Foster City, CA). siRNA ID numbers were
s1662 and s224050 (ASGP-R1, Homo sapiens). HepG2 cells were grown in 6-well plates until
70 % confluency and were then transfected with the siRNAs (concentration range between 5
pmol and 50 pmol). Transfection was performed using GeneSilencer siRNA Transfection
Reagent obtained by Genlantis (San Diego, CA) according to the manufacturers instructions.
Cells were cultured for 48 h after transfection and were then harvested. Cells were lysed using
18

lysis buffer (50 mM Tris, pH 6,8; 1% Triton-X 100, protease inhibitors, DNase), Laemmli buffer
was added to the cell lysates and the samples were heated at 95 C for 5 min. Cell lysates were
subjected to SDS-PAGE and afterwards ASGP-R1 protein levels were detected by Western Blot.
Briefly, goat-anti ASGP-R1 Ab (N-18, Santa Cruz Biotechnology, Santa Cruz, CA) was used as
the primary antibody according to the manufacturers instructions and HRP-labeled anti-goat Ab
was used as secondary antibody for detection. ASGP-R1 was detected as a band at the molecular
weight of 46 kDa. Expression of the housekeeping gene actin served as control to allow a
semiquantitative analysis of siRNA-mediated knockdown of ASGP-R1 expression.
48 h after siRNA transfection into the HepG2 cells 20 nmol Gal-QDs were added to the cells (or
to wild-type HepG2 cells) for 2 h and binding/endocytosis was measured by flow cytometry as
described above.

Animal Experiments
Female C57BL/6 mice (6-10 weeks old) were housed in the HCI rodent center, ETH Zrich, and
were provided food and water ad libitum. All animal experiments were in accordance with local
Animal Ethics Committee regulations.
Mice (n=2) were anesthetized with isoflurane and received 2,5 nmol quantum dots (QDs-PEG,
QDs-Man, QDs-GalN) each via tail vein injection (total volume 100 L). Afterwards 50 L PBS
were injected to flush the tail vein. 2 h after injection mice were sacrificed and livers were
perfused via the portal vein with 4% paraformaldehyde (PFA). Organs were embedded in
paraffin and 10 m sections were prepared. Sequestration of QDs into liver was analyzed by
confocal fluorescence microscopy using Leica TCS-SP1 microscope (Leica, Nussloch,
Germany). Excitation wavelength was 488 nm, detection wavelength was 600-670 nm. The
objective magnification used for the analysis of each liver section was 63x. QD sequestration in
the liver was measured by counting the number of QDs in ten microscopic fields of vision for
each mouse. All microscopic fields used for QD counting had the same size. Statistical analysis
was performed using unpaired Students t-test. All statistical analyses were performed with the
Prism software (Graph Pad Software, San Diego, CA). P<0.05 was considered to be indicative of
statistical significance.
19

GalN-QDs Transmitted light Overlay




Figure 4: Quantification of QD sequestration into liver by analysis of liver sections
For the analysis of liver injury induced by the GalN-capped QDs mice were either injected PBS
buffer (negative control), 2 mg/kg LPS or 2 mg/kg LPS together with 2,5 nmol of QDs (either
Gal-QDs or GalN-QDs). 6 to 8 h after injection blood was taken from the saphenous vein and
blood was allowed to clot to obtain serum. Liver transaminases ALT and AST were quantified in
the serum samples by using a test kit obtained by Hach-Lange GmbH (Berlin, Germany)
according to the manufacturers instructions.

10. References:
1. Peng, Z. A.; Peng, X. J. Am. Chem. Soc. 2001, 123, 183-184.
2. Leutherdale, C. A.; Woo, W.-K.; Mikulec, F. V.; and Bawendi, M. G. J. Phys. Chem. 2002,
106, 7619-7622.
3. Mironov, Y. V.; Sherman, A. A.; Nifantiev, N. E. Tetrahedron Lett. 2004, 45, 9107-9110.
4. Uyeda, H. T.; Medintz, I. L.; Jaiswal, J. K.; Simon, S. M.; Mattoussi, H. J. Am. Chem. Soc.
2005, 127, 3870-3878.
5. Caspar, J. V.; Meyer, T. J. J. Am. Chem. Soc. 1983, 105, 5583.






20


O
AcO
OAc
OAc
O
OAc
O
O
Cl
8

21


O
AcO
OAc
OAc
O
OAc
O
O
SAc
8
a

22


O
HO
OH
OH
O
OH
O
O
SH
9

23


p
p
m

(
f
1
)
2
.
0
3
.
0
4
.
0
5
.
0
6
.
0
7
.
0
8
.
0
O
A
c
O
A
c
O
N
3
O
O
A
c
O
O
C
l
1
5
1
2
O
AcO
OAc
N
3
O
OAc
O
O
Cl
24


1
2
a
O
AcO
OAc
N
3
O
OAc
O
O
SAc

25


1
4
O
HO
OH
H
2
N
O
OH
O
O
SH

26


N
3
O
O
N
3
n
2
a

27


H
2
N
O
O
NH
2
n
3

28


6
n
S
S
O
O
O
O
OH

29


p
p
m

(
f
1
)
1
.
0
2
.
0
3
.
0
4
.
0
5
.
0
6
.
0
7
.
0
8
.
0
5
O
S
H
S
H
O
O
O
O
H
n
6
a
n
HS
HS
O
O
O
O
OH
30


4
n
S
S
HN
O
O
O
NH
2

31


n
4
a
HS
HS
HN
O
O
O
NH
2

32


N
3
O
O
N
3
n
2
a

33


H
2
N
O
O
NH
2
n
3

34


4
n
S
S
HN
O
O
O
NH
2

35


n
4
a
HS
HS
HN
O
O
O
NH
2

36


6
n
S
S
O
O
O
O
OH

37


n
6
a
HS
HS
O
O
O
O
OH

38


O
AcO
OAc
OAc
O
OAc
O
O
Cl
8

39


O
AcO
OAc
OAc
O
OAc
O
O
SAc
8
a

40


O
HO
OH
OH
O
OH
O
O
SH
9

41


p
p
m

(
f
1
)
0
5
0
1
0
0
1
5
0
O
A
c
O
A
c
O
N
3
O
O
A
c
O
O
C
l
1
5
1
2
O
AcO
OAc
N
3
O
OAc
O
O
Cl

42


1
2
a
O
AcO
OAc
N
3
O
OAc
O
O
SAc

43


1
4
O
HO
OH
H
2
N
O
OH
O
O
SH

44


H
2
N
O
NH
NH
O
OH
HN
n
O
N
O
O
O
N
O
O
17

45



H
2
N
O
NH
NH
O
OH
HN
n
O
N
O
O
O
N
O
O
17

46


H
2
N
O
NH
NH
O
OH
HN
n
O
N
O
O
O
N
O
O
O
OH
HO
OH
O
OH
O
HO
HO
OH
O
OH
18

47


H
2
N
O
NH
NH
O
OH
HN
n
O
N
O
O
O
N
O
O
O
OH
HO
OH
O
OH
O
HO
HO
OH
O
OH
18