2010 Noninvasive SI

8/13/2019 2010 Noninvasive SI

1/26

Supporting Information

Wiley-VCH 2010

69451 Weinheim, Germany

Noninvasive Imaging of Dendrimer-Type N-Glycan Clusters: In VivoDynamics Dependence on Oligosaccharide Structure**

Katsunori Tanaka,* Eric R. O. Siwu, Kaori Minami, Koki Hasegawa, Satoshi Nozaki,

Yousuke Kanayama, Koichi Koyama, Weihsu C. Chen, James C. Paulson, Yasuyoshi Watanabe,

and Koichi Fukase*

anie_201000892_sm_miscellaneous_information.pdf


2/26

1

All commercially available reagents were used without further purification. Dichloromethane were

refluxed over and distilled from CaH2and anhydrous DMF was purchased from Aldrich.1H NMR spectra

were recorded on a JEOL JNM-ECA 500 spectrometer and chemical shifts were represented as !-values

relative to the internal standard TMS. MALDI-TOF-mass spectra were measured on an SHIMADZU

AXIMA-CFR mass spectrometer equipped with a nitrogen laser (!= 337 nm). 1H NMR & mass spectra

and purity of the starting glycans, provided by Otsuka Chemical Co., Ltd., are shown below.

Bis--Neu(2-6)Gal containing N-glycan-Asn (glycan a):1H-NMR (500 MHz, D2O, HOD = !4.65)!

5.03 (s, 1H), 4.97 (d, 1H, J= 9.5 Hz), 4.85 (s, 1H), 4.51 (m, 2H), 4.35 (d, 2H,J= 8 Hz), 4.16 (bs, 1H),

4.09 (bs, 1H), 4.02 (bs, 1H), 2.83 (bd, 1H, J= 17 Hz), 2.77 (dd, 1H,J= 7Hz, 17 Hz), 2.56 (bd, 2H, J=

12Hz ), 1.95-2.07 (multi s, 18H), 1.62 (t, 2H, J= 12Hz). ESI-MS calcd for C88H144N8O64Na (M+Na)+

2359.8, found 2360.0.


3/26

2

Asialo N-glycan-Asn (glycan b):1H-NMR (500 MHz, D2O, HOD = !4.65)!5.02 (s, 1H), 4.9(d, 1H,

J= 9.5 Hz), 4.83 (s, 1H), 4.52 (d, 1H, J= 7.5Hz), 4.49 (d, 2H,J= 8 Hz ), 4.38 (dd, 2H,J= 3Hz, 8 Hz),

4.15 (d, 1H,J= 2 Hz ), 4.10 (s, 1H,J= 3 Hz), 4.02 (s, 1H,J= 3 Hz), 2.83 (dd, 1H,J= 17 Hz), 2.77 (dd,

1H, J = 7Hz, 17 Hz), 1.92-2.02 (multi s, 12H). ESI-MS calcd for C66H110N6O48Na (M+Na)+ 1777.6,

found 1777.6.

Bis--Neu(2-3)Gal containing N-glycan-Asn (glycan c): 1H-NMR (500 MHz, D2O, HOD = !4.65)!

4.85 (s, 1H), 4.82 (d, 1H, J= 9.0 Hz), 4.66 (s,1H ), 4.34 (brs, 1H), 4.29 (brs, 2H), 3.98 (s, 1H), 3.93 (s,1H), 3.85 (brs, 2H), 2.68 (dd, 1H, J= 17 Hz), 2.65 (dd, 1H, J= 7Hz, 17 Hz), 2.48 (bd, 2H, J= 12Hz ),

1.70-1.86 (multi s, 12H), 1.53 (t, 2H, J= 18Hz). ESI-MS calcd for C88H144N8O64Na (M+Na)+2359.8,

found 2359.9.


4/26

3

Mixed -Neu(2-3)Gal & -Neu(2-6)Gal containing N-glycan-Asn (glycan d):1H-NMR (500 MHz,

D2O, HOD = !4.65)!4.98 (s, 1H), 4.92 (d, 1H,J= 9.5 Hz), 4.77 (s, 1H), 4.40 (m, 1H), 4.39 (d, 1H,J=

8 Hz), 4.28 (d, 1H,J= 8 Hz), 4.10 (s, 1H), 4.04 (s, 1H), 3.97 (brs, 1H), 2.78 (dd, 1H,J= 17 Hz), 2.71 (dd,

1H,J= 7Hz, 17 Hz), 2.60 (dd, 1H, J= 4.5 Hz, 12.5 Hz ), 2.51 (dd, 1H,J= 4.5 Hz, 12.5 Hz ), 1.85-1.95

(multi s, 18H), 1.64 (t, 2H, J = 18Hz), 1.56 (t, 2H, J = 18Hz). ESI-MS calcd for C88H144N8O64Na

(M+Na)+2359.8, found 2359.8.


5/26

4

Mixed -Neu(2-6)Gal & -Neu(2-3)Gal containing N-glycan-Asn (glycan e):1H-NMR (500 MHz,

D2O, HOD = !4.65)!5.03 (s, 1H), 4.98 (d, 1H,J= 9.5 Hz), 4.87 (s, 1H), 4.50 (m, 1H), 4.36 (d, 1H,J=

8 Hz), 4.36 (d, 1H,J= 8 Hz), 4.17 (s, 1H), 4.11 (s, 1H), 4.04 (brs, 1H), 2.80 (dd, 1H,J= 4 Hz, 17 Hz),

2.74 (dd, 1H,J= 7Hz, 17 Hz), 2.68 (dd, 1H, J= 4.5 Hz, 12.5 Hz ), 2.59 (dd, 1H, J= 4.5 Hz, 12.5 Hz ),

1.88-2.01 (multi s, 18H), 1.71 (t, 2H, J = 18Hz), 1.64 (t, 2H, J = 18Hz). ESI-MS calcd for

C88H144N8O64Na (M+Na)+2359.8, found 2360.0.


6/26

5

Solid-phase synthesis of acetylene-containing polylysine dendrimer (template for click reaction, 16-

mer). To a suspension of Fmoc-Lys(Boc)-OH (39 mg, 84 mol) and MSNT (25 mg, 84 mol) in dry

CH2Cl2(1.0 mL) was added 1-methylimidazole (6.7 L, 84 mol). After the solution was stirred at room

temperature for 1 h, the mixture was treated with Wang resin (30 mg, 28 mol), and shaken overnight at

room temperature. The resin was washed with CH2Cl2(5 min x 5) and DMF (5 min x 5).

To the Fmoc-Lys-attached resin was added 20% piperidine in DMF (1.0 mL), and the mixture was

shaken for 20 min. After the resin was washed with DMF (5 min x 5), the success of the Fmoc

deprotection was checked by bromophenolblue (BPB) test. In a separated flask, DIPC (13 L, 84 mol)

was added to a solution of Fmoc-Lys(Fmoc)-OH (49 mg, 84 mol) and HOBt (11 mg, 84 mol) in DMF

(1.0 mL) at 0 C. After the solution was stirred for another 30 min, the mixture was treated with the

mono-Lys-attached resin, obtained above. The resulting mixture was shaken for 5 h and the resin was

washed with DMF (3 min x 5). Fmoc deprotection & acylation cycle was further repeated by using Fmoc-

Lys(Fmoc)-OH (82 mg, 140 mol), Fmoc-Lys(Fmoc)-OH (132 mg, 220 mol), Fmoc-Lys(Fmoc)-OH

(263 mg, 450 mol), Fmoc-His(Bn)-OH (391 mg, 840 mol), and finally by Fmoc-Pra-OH (281 mg, 840

mol).

After deprotection of Fmoc group on Fmoc-Pra-attached resin, the resulting resin was treated with

Ac2O (279 L, 3.79 mmol) and pyridine (279 L, 5.30 mmol) in CH2Cl2(300 L) and the mixture was

shaken for 3 h at room temperature. The resin was washed by THF, MeOH, CH2Cl2(each 5 min x 5), and


7/26

6

finally by Et2O (5 min x 3), and then dried in vacuo for 3 h. 78 Mg out of 159 mg of the dendrimer-

attached resin obtained above, was then treated with 2.0 mL of TFA/TES/H2O (31 : 1 : 1) and shaken for

30 min at room temperature. The mixture was filtered, and the filtrate was lipophilized to give acetylene-

containing polylysine dendrimer (16-mer) as a white solid (50 mg). The dendrimer were analyzed by

reverse phase HPLC (see Fig. SI-3,column: Nacalai Tesque 5C18-AR300, 4.6 x 250 mm; MeCN in H2O

containing 0.1% TFA (10-100% gradient over 40 min, 1 mL/min); UV detection at 250 nm; retention

time: 13.1 min) and gel-filtration HPLC (see Fig. SI-5, column: TSK-Gel G4000PWXL, 7.8 x 300 mm;

0.1 M NaCl at pH 7.2, 1 mL/min; retention time: 13.5 min). 1H-NMR (500 MHz, D2O, HOD = !4.65,

Fig. SI-1): !8.52-8.70 (16H), 6.99-7.37 (m, 96H), 5.24 (brs, 32H), 3.91-4.17 (m, 32H), 2.71-3.18 (m,

32H), 2.36 (s, 16H), 2.25 (m, 32H) 1.85 (s, 3H x 16, Ac), 1.37-1.70 (m, 32H), 0.90-1.37 (m, 64H).

MALDI-TOF-MS m/zcalcd for C416H515N96O65(M+H)+7899.0, found 7899.0 (Fig. SI-2).

Figure SI-1.1H-NMR of acetylene-containing polylysine dendrimer (16-mer) (500 MHz, D2O).

Figure SI-2. MALDI-TOF-MS of acetylene-containing polylysine (16-mer).


8/26

7

Acetylene-containing polylysine dendrimer (4-mer).1H-NMR (500 MHz, D2O, HOD = !4.65): !8.83

(s, 4H), 7.51 (m, 24H), 5.41 (brs, 2H) 4.76 (brs, 4H), 4.67 (brs, 4H), 4.34 (m, 8H), 3.95 (brs, 4H), 3.29 (m,

12H), 2.57 (brs, 4H), 2.44 (m, 8H), 2.07 (s, 3H x 4, Ac), 2.00 (m, 4H), 1.73 (m, 8H), 1.57 (m, 4H), 1.45

(m, 4H), 1.33 (m, 4H); MALDI-TOF-MS m/zcalcd for C104H131N24O17(M+H)+1989.0, found 1989.0.

Acetylene-containing polylysine dendrimer (8-mer).1H-NMR (500 MHz, D2O, HOD = !4.65): !8.87

(s, 8H), 7.52 (m, 48H), 5.37 (s, 16H), 4.80 (brs, 8H) ,4.45 (m, 16H), 4.21 (m, 8H), 2.91-3.32 (m, 16H),

2.49 (brs, 8H), 2.43 (m, 16H), 2.01 (s, 3H x 8, Ac), 2.55 - 280 (m, 16H),1.15 1.50 (m, 32H). MALDI-

TOF-MS m/z calcd for C208H259N48O33(M+H)+3959.0, found 3959.0.

Preparation of azide derivative of bis--Neu(2-6)Gal containing N-glycan (azide a):To a solution ofbis-"-Neu(2-6)Gal containingN-glycan-Asn (provided by Otsuka Chemical Co., Ltd., 3.9 mg, 1.7 "10

-3

mmol) and Et3N (0.55 L) in DMF (100 L) was added 7-azidoheptanoic acid succinimidyl ester (1.4 mg,

5.0 "10-3mmol) and the solution was stirred at room temperature overnight under Ar atmosphere. After

the solvent was removed in vacuo, the residue was purified by HPLC [column: Nacalai Tesque 5C18-

AR300, 4.6 x 250 mm; MeCN in H2O containing 0.1% TFA (10-100% gradient over 40 min, 1 mL/min);

UV detection at 250 nm; retention time: 10.5 min, see Fig. SI-3 (azide awas eluted at 9.4 min at the

gradient over 20 min)]. The fraction containing the desired product was lyophilized to give the azide aas

an amorphous solid (3.9 mg, 94%). 1H-NMR (500 MHz, D2O, HOD = !4.65): !4.93 (s, 1H), 4.84 (d,J=

10 Hz, 1H), 4.79 (s, 1H), 4.76 (s, 1H), 4.62 (s, 1H), 4.56 (m, 3H), 4.40 (d,J= 8 Hz, 2H), 4.25 (brs, 1H),

4.05 (brs, 1H), 3.90 (brs, 1H), 3.26-3.83 (m, 54H), 2.61 (dd, J= 4.5 Hz, 12 Hz, 2H), 2.11 (t, J= 7 Hz,

2H), 1.93 (s, 3H x 6, Ac), 1.42 (m, 5H), 1.53 (dd,J= 12 Hz, 2H), 1.42 (m, 5H), 1.17 (m, 5H); MALDI-

TOF-MS m/zcalcd for C95H158N11O65(M+H)+2492.9, found 2492.9.

OOO

HO

O

HOHO

HO

O

O

HOO

HO

N

O

HO

HO

OOAcHN

HO

HO OH

HO

COOH

OH

O

N

HO

HO

O O

N

HO

HO

N H

N

COOH

OO

O

AcH

AcHH

AcH

N3

H

O

OHO

HO

HO

O

O

NHO

O

OHO

OH

HO

O

OOAcHN

HO

COOH

HO

OHOH

H

AcH

azidea

OOO

HO

O

HOHO

HO

O

O

HOO

HO

N

O

HO

HO

OOAcHN

HO

HO OH

HO

COOH

OH

O

N

HO

HO

O O

N

HO

HO

HN NH2

COOHO

O

AcH

AcHH

AcH

O

OHO

HO

HO

O

O

NHO

O

OHO

OH

HO

OOOAcHN

HO

COOH

HO

OHOH

H

AcH


9/26

8

Azide derivative of asialo N-glycan (azide b):To a solution of an asialo N-glycan-Asn (1.1 mg, 6.2 "

10-4mmol) and Et3N (0.26 L) in DMF (100 L) was added 7-azidoheptanoic acid succinimidyl ester

(500 g, 1.9 " 10-3 mmol) and the mixture was stirred overnight at room temperature under Ar

atmosphere. After the solvent was removed in vacuo, the residue was purified by HPLC [column: Nacalai

Tesque 5C18-AR300, 4.6 x 250 mm; MeCN in H2O containing 0.1% TFA (10-100% gradient over 20 min,

1 mL/min); UV detection at 250 nm; retention time: 9.5 min]. The fraction containing the desired product

was lyophilized to give the azide bas an amorphous solid (1.2 mg, 96 %). 1H-NMR (500 MHz, D2O,

HOD = !4.65): !4.92 (s, 1H), 4.83 (d,J= 10 Hz, 1H), 4.78 (s, 1H), 4.93 (s, 1H), 4.56 (s, 1H), 4.45 (m,

3H), 4.26 (d,J= 8 Hz, 2H), 4.05 (brs, 1H), 3.99 (brs, 1H), 3.90 (brs, 1H), 3.45-4.01 (m, 58H), 2.59 (dd,J

= 4.5 Hz, 12 Hz, 2H), 2.08 (t, J= 7 Hz, 2H), 1.78-1.95 (s, 3H x 6, Ac), 1.41 (m, 5H), 1.17 (m, 5H);

MALDI-TOF-MS m/zcalcd for C95H158N11O65(M+H)+1910.7, found 1910.7.

azideb

O

OHOHO

HO

OOO

HO

O

HOHO

HO

O

O

HOO

HOO

HO

HO

OH

OH

O

HO

HO

O O

HO

HO

HN

HN

COOH

OO

OH

N3

O

OHO

O

OHO

OH

HO

O

HO

H

NHAc

NHAcNHAc

AcHN

O

OHOHO

HO

OOO

HO

O

HOHO

HO

O

O

HOO

HOO

HO

HO

OH

OH

O

HO

HO

O O

HO

HO

HN NH2

COOHO

OH

O

OHO

O

OHO

OH

HO

O

HO

H

NHAc

NHAcNHAc

AcHN


10/26

9

Azide derivative of bis--Neu(2-3)Gal containing N-glycan (azide c):To a solution of bis-"-Neu(2-

3)Gal containing N-glycan-Asn (900 g, 3.9 " 10-4

mmol) and Et3N (0.26 L) in DMF(100 L) was

added 7-azidoheptanoic acid succinimidyl ester (310 g, 1.2 " 10-3mmol) and the mixture was stirred

overnight at room temperature under Ar atmosphere. After the solvent was removed in vacuo, the residue

was purified by HPLC [column: Nacalai Tesque 5C18-AR300, 4.6 x 250 mm; MeCN in H2O containing

0.1% TFA (10-100% gradient over 20 min, 1 mL/min); UV detection at 250 nm; retention time: 9.3 min].

The fraction containing the desired product was lyophilized to give the azide c as an amorphous solid

(820 g, 85%).1H-NMR (500 MHz, D2O, HOD = !4.65): !4.90 (s, 1H), 4.85 (d,J= 10.5 Hz, 1H), 4.80

(s, 1H), 4.40 (d,J= 7.0 Hz, 2H), 4.30 (m, 3H), 4.05 (brs, 1H), 3.97 (brs, 1H), 3.90 (brs, 2H), 3.20-3.80

(m, 54H), 2.62 (dd,J= 4.5 Hz, 12 Hz, 2H), 2.10 (m, 2H), 1.87 (s, 3H x 6, Ac), , 1.50 (dd,J= 12 Hz, 2H),

1.45 (m, 5H), 1.18 (m, 5H); MALDI-TOF-MS m/zcalcd for C95H158N11O65(M+H)+2492.9, found 2492.9.

azidecO

AcHN

HO

HOOH

HO

HOOC

OAcHN

HO

COOH

HO

OHOH

O

OHOHO

HO

OOO

HO

O

HOHO

HOO

O

HOO

HO

N

O

O

HOOH

OH

O

N

HO

HO

O O

N

HO

HO

HN

HN

COOH

OO

O

AcH

AcHH

AcH

N3

O

O

NHOO

OHO

OH

O

O OHH

AcH

OAcHN

HO

HOOH

HO

HOOC

OAcHN

HO

COOH

HO

OHOH

O

OHOHO

HO

OOO

HO

O

HOHO

HOO

O

HOO

HO

N

O

O

HOOH

OH

O

N

HO

HO

O O

N

HO

HO

HN NH2

COOHO

O

AcH

AcHH

AcH

O

O

NHOO

OHO

OH

O

O OHH

AcH


11/26

10

Azide derivative of mixed -Neu(2-3)Gal & -Neu(2-6)Gal containing N-glycan (azide d): To a

solution of "-Neu(2-3)Gal & "-Neu(2-6)Gal containing N-glycan-Asn (1.0 mg, 4.3 " 10-4mmol) and

Et3N (0.26 L) in DMF (100 L) was added 7-azidoheptanoic acid succinimidyl ester (340 g, 1.3 "10-3

mmol) and the mixture was stirred overnight at room temperature under Ar atmosphere. After the solvent

was removed in vacuo, the residue was purified by HPLC [column: Nacalai Tesque 5C18-AR300, 4.6 x

250 mm; MeCN in H2O containing 0.1% TFA (10-100% gradient over 20 min, 1 mL/min); UV detection

at 250 nm; retention time: 9.4 min]. The fraction containing the desired product was lyophilized to give

the azide das an amorphous solid (940 g, 88%).1H-NMR (500 MHz, D2O, HOD = !4.65): !4.97 (s,

1H), 4.93 (d,J= 9.5 Hz, 1H), 4.81 (s, 1H), 4.71 (s, 1H), 4.60 (s, 1H), 4.50 (m, 3H), 4.37 (d,J= 8 Hz, 2H),

4.17 (brs, 1H), 4.11 (brs, 1H), 4.04 (brs, 2H), 3.38 3.92 (m, 54H), 2.82 (dd, J= 5 Hz, 12 Hz, 2H), 2.67

(dd,J= 4.5 Hz, 12Hz, 1H), 2.59 (dd,J= 4.5 Hz, 12Hz, 1H), 2.11 (m, 2H), 1.90-2.02 (s, 3H x 6, Ac), 1.73

(dd, J= 12 Hz, 1H), 1.63 (dd, J= 12 Hz, 1H), 1.42 (m, 5H), 1.20 (m, 5H); MALDI-TOF-MS m/zcalcd

for C95H158N11O65(M+H)+2492.9, found 2493.0.

azided

OAcHN

HO

COOH

HO

OHOH

OOO

HO

O

HOHO

HO

O

O

HOO

HO

N

O

HO

HO

OO

AcHN

HO

HO OH

HO

COOH

OH

O

N

HO

HO

O O

N

HO

HO

HN

HN

COOH

OO

O

AcH

AcHH

AcH

N3

O

OHO

HO

HO

O

O

NHO

O

OHO

OH

O

OH OH

AcHOAcHN

HO

COOH

HO

OHOH

OOO

HO

O

HOHO

HO

O

O

HOO

HO

N

O

HO

HO

OOAcHN

HO

HO OH

HO

COOH

OH

O

N

HO

HO

O O

N

HO

HO

HN NH2

COOHO

O

AcH

AcHH

AcH

O

OHO

HO

HO

O

O

NHO

O

OHO

OH

O

OH OH

AcH

Azide derivative of mixed -Neu(2-6)Gal & -Neu(2-3)Gal containing N-glycan (azide e): To a

solution of "-Neu(2-6)Gal & "-Neu(2-3)Gal containing N-glycan-Asn (1.0 mg, 4.3 " 10-4 mmol) and

Et3N (0.26 L) in DMF (100 L) was added 7-azidoheptanoic acid succinimidyl ester (340 g, 1.3 "10-3

mmol) and the mixture was stirred overnight at room temperature under Ar atmosphere. After the solvent

was removed in vacuo, the residue was purified by HPLC [column: Nacalai Tesque 5C18-AR300, 4.6 x

250 mm; MeCN in H2O containing 0.1% TFA (10-100% gradient over 20 min, 1 mL/min); UV detection

at 250 nm; retention time: 9.4 min]. The fraction containing the desired product was lyophilized to give

the azide eas an amorphous solid (900 g, 84%).1H-NMR (500 MHz, D2O, HOD = !4.65): !4.98 (s,

1H), 4.92 (d,J= 9.5 Hz, 1H), 4.77 (s, 1H), 4.70 (s, 1H), 4.58 (s, 1H), 4.45 (m, 3H), 4.30 (d, J= 7.5 Hz,

2H), 4.10 (brs, 1H), 4.04 (brs, 1H), 3.97 (brs, 2H), 3.30-3.92 (m, 54H), 2.75 (dd, J= 4.5 Hz, 12 Hz, 2H),

2.60 (dd, J= 4 Hz, 12Hz, 1H), 2.50 (dd, J= 4 Hz, 12Hz, 1H), 2.05 (m, 2H), 1.82-1.95 (s, 3H x 6, Ac),

1.65 (dd, J= 12 Hz, 1H), 1.56 (dd, J= 12 Hz, 1H), 1.39 (m, 5H), 1.17 (m, 5H); MALDI-TOF-MS m/z

calcd for C95H158N11O65(M+H)+2492.9, found 2492.0.


12/26

11

azideeO

AcHN

HO

HOOH

HO

HOOC

O

OHOHO

HO

OOO

HO

O

HO

HOHO

O

O

HOO

HO

N

O

O

HO OH

OH

O

N

HO

HO

O O

N

HO

HO

HN

HN

COOH

OO

O

AcH

AcHH

AcH

N3

O

O

NHO

O

OHO

OH

HO

O

OOAcHN

HO

COOH

HO

OHOH

H

AcH

OAcHN

HO

HOOH

HO

HOOC

O

OHOHO

HO

OOO

HO

OHOHO

HO

O

O

HOO

HO

N

O

O

HO OH

OH

O

N

HO

HO

O O

N

HO

HO

HN NH2

COOHO

O

AcH

AcHH

AcH

O

O

NHO

O

OHO

OH

HO

O

OOAcHN

HO

COOH

HO

OHOH

H

AcH


13/26

12

Preparation of glycocluster 3a. CuSO4(64 g, 3.2 x 10-4 mmol), sodium L-ascorbate (238 g, 1.2 x 10

-3

mmol), and diisopropylethylamine (74 nL) were added to a solution of acetylene-containing polylysine

dendrimer (16-mer, 158 g, 2.0 x 10-5 mmol) andN-glycan azide a(1.0 mg, 4.0 x 10

-4 mmol) in DMF (50

L) and H2O (50 L) at room temperature under Ar atmosphere. After the mixture was stirred for 40 min

at this temperature, DOTA (1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid, 647 g, 1.56 x 10-3

mmol) was added, and the resulting solution was stirred for another 40 min. Low-molecule weight

compounds were filtered using the Microcon (centrifugal filter YM-10, 10,000 cut, Millipore), and the

resulting aqueous solution was lipophilized to give glycocluster 3aas amorphous solid (960 g, quant).

The purity of the product was evaluated by reverse phase HPLC [column: Nacalai Tesque 5C18-AR300,

4.6 x 250 mm; MeCN in H2O containing 0.1% TFA (10-100% gradient over 40 min, 1 mL/min); UV

detection at 250 nm; retention time of 3a: 12.4 min, see Fig. SI-3]. Molecular weight was evaluated by

MALDI-TOF-MS (Fig. SI-6) and size-partitioning gel filtration analysis as shown in Fig. SI-5 (column:

TSK-Gel G4000PWXL, 7.8 x 300 mm; 0.1 M NaCl at pH 7.2, 1 mL/min; retention time: 9.0 min):1H-

NMR (500 MHz, D2O)!8.71 (brs, 1H x 16, His-aromatic protons), 7.67 (s, 1H x 16, triazole proton),

7.12-7.37 (m, 6H x 16, Bn- and His-aromatic protons), 5.26 (brs, 2H x 16, benzylic protons of Bn), 5.10

(s, 1H x 16), 5.02 (s, 1H x 16), 4.95 (s, 1H x 16), 4.56 (m, 2H x 16), 4.43 (m, 3H x 16), 4.22 (s, 1H x 16),

4.17 (s, 1H x 16), 4.09 (s, 1H x 16), 4.20-4.30 (m, "-protons of Lys), 3.44-3.98 (m, characteristic vicinal

protons of sugar hydroxyls), 2.86-3.23 (m, #-protons on His), 2.74-2.82 (m, #-protons of Asn), 2.62-2.67

(m, H-3eqNeu

), 2.24-2.28 (m, #-protons of triazolic Ala), 1.92-2.06 (multiple s, Ac), 1.70 (dd, J = 12Hz,

12Hz, H-3axNeu), 1.54-1.60 and 1.30-1.33 (m, part of acylpentane-linker protons), 1.82-1.91, 1.65-1.79,

1.39-1.48, 1.26-1.38, and 0.82-1.24 (m, $- and !-protons of Lys).


14/26

13

Figure SI-3. Reverse phase HPLC of acetylene-containing polylysine dendrimer (16-mer), N-glycan

azide a, click reaction mixture, and purified glycocluster 3aafter Microcon filtration [column: Nacalai

Tesque 5C18-AR300, 4.6 x 250 mm; MeCN in H2O containing 0.1% TFA (10-100% gradient over 40 min,

1 mL/min); UV detection at 250 nm].

Figure SI-4.1H-NMR of 3a(500 MHz, D2O).


15/26

14

Glycocluster 1a. CuSO4 (51 g, 3.2 x 10-4 mmol), sodium L-ascorbate (192 g, 9.6 x 10

-3 mmol), and

diisopropylethylamine (60 nL) were added to a solution of acetylene-containing polylysine dendrimer (4-

mer, 160 g, 8.0 x 10-5 mmol) andN-glycan azide a(800 g, 3.2 x 10

-4 mmol) in DMF (50 L) and H2O

(50 L) at room temperature under Ar atmosphere. After the mixture was stirred for 40 min at this

temperature, DOTA (520 g, 1.2 x 10-3 mmol) was added, and the resulting solution was stirred for

another 40 min. Low-molecule weight compounds were filtered using the Microcon (centrifugal filter

YM-10, 10,000 cut, Millipore), and the resulting aqueous solution was lipophilized to give glycocluster

1aas amorphous solid (760 g, quant). The purity of the product was evaluated by reverse phase HPLC

[column: Nacalai Tesque 5C18-AR300, 4.6 x 250 mm; MeCN in H2O containing 0.1% TFA (10-100%

gradient over 40 min, 1 mL/min); UV detection at 250 nm; retention time: 12.2 min]. Molecular weight

was evaluated by gel filtration analysis as shown in Fig. SI-5 (column: TSK-Gel G4000PW XL, 7.8 x 300

mm; 0.1 M NaCl at pH 7.2, 1 mL/min; retention time: 11.9 min): 1H-NMR (500 MHz, D2O, HOD = !

4.65)!8.74 (brs, 1H x 4, His-aromatic protons), 7.65 (s, 1H x 4, triazole protons), 7.15-7.50 (m, 6H x 4,

Bn- and His-aromatic protons), 5.29 (brs, 2H x 4, benzylic protons of Bn), 5.09 (s, 1H x 4), 5.02 (brs, 1H

x 4), 4.96 (s, 1H x 4), 4.56 (s, 1H x 4), 4.45 (m, 2H x 4), 4.22 (m, 3H x 4), 4.17 (s, 1H x 4), 4.09 (s, 1H x

4), 4.10-4.30 (m, "-protons of Lys), 3.40-4.02 (m, characteristic vicinal protons of sugar hydroxyls), 2.89-

3.25 (m, #-protons on His), 2.75-2.85 (m, #-protons of Asn), 2.60-2.65 (m, H-3eqNeu), 2.26-2.30 (m, #-

protons of triazolic Ala), 1.92-2.05 (multiple s, Ac), 1.68 (dd,J = 12Hz, 12Hz, H-3axNeu

), 1.54-1.59 and

1.30-1.35 (m, part of acylpentane-linker protons), 1.80-1.91, 1.65-1.79, 1.36-1.48, 1.22-1.38, and 0.85-

1.20 (m, $- and !-protons of Lys).

Glycocluster 2a. CuSO4 (50 g, 3.2 x 10-4 mmol), sodium L-ascorbate (186 g, 9.4 x 10

-4 mmol), and

diisopropylethylamine (72 nL, 416 nmol) were added to a solution of acetylene-containing polylysine

dendrimer (8-mer, 155 g, 3.9 x 10-5 mmol) andN-glycan azide a(980 g, 3.9 x 10

-4 mmol) in DMF (50


at this temperature, DOTA (630 g, 1.56 x 10-3

mmol) was added, and the resulting solution was stirred

for another 40 min. Low-molecule weight compounds were filtered using the Microcon (centrifugal

filter YM-10, 10,000 cut, Millipore), and the resulting aqueous solution was lipophilized to give

glycocluster 2aas amorphous solid (970 g, quant). The purity of the product was evaluated by reverse

phase HPLC [column: Nacalai Tesque 5C18-AR300, 4.6 x 250 mm; MeCN in H2O containing 0.1% TFA

(10-100% gradient over 40 min, 1 mL/min); UV detection at 250 nm; retention time of 2a: 12.2 min].

Molecular weight was evaluated by gel filtration analysis as shown in Fig. SI-5 (column: TSK-Gel

G4000PWXL, 7.8 x 300 mm; 0.1 M NaCl at pH 7.2, 1 mL/min; retention time: 10.6 min):1

H-NMR (500MHz, D2O, HOD = !4.65)!8.70 (brs, 1H x 8, His-aromatic protons), 7.63 (s, 1H x 8, triazole protons),


16/26

15

7.16-7.48 (m, 6H x 8, Bn- and His-aromatic protons), 5.28 (brs, 2H x 8, benzylic protons of Bn), 5.1 (s,

1H x 8), 5.02 (brs, 1H x 8), 4.95 (s, 1H x 8), 4.56 (s, 1H x 8), 4.43 (m, 2H x 8, 4.23 (m, 3H x 8), 4.15 (s,

1H x 8), 4.10 (s, 1H x 8), 4.08-4.31 (m, "-protons of Lys), 3.39-4.03 (m, characteristic vicinal protons of

sugar hydroxyls), 2.88-3.20 (m, #-protons on His), 2.73-2.80 (m, #-protons of Asn), 2.59-2.67 (m, H-

3eqNeu), 2.28-2.36 (m, #-protons of triazolic Ala), 1.90-2.03 (multiple s, Ac), 1.70 (dd, J = 12Hz, 12Hz,

H-3axNeu), 1.50-1.60 and 1.32-1.38 (m, part of acylpentane linker protons), 1.76-1.91, 1.62-1.75, 1.34-

1.48, 1.25-1.32, and 0.87-1.23 (m, $- and !-protons of Lys).

Glycocluster 3b. CuSO4(120 g, 6.7 x 10-4 mmol), sodium L-ascorbate (400 g, 2.0 x 10

-3 mmol), and

diisopropylethylamine (120 nL, 690 nmol) were added to a solution of acetylene-containing polylysine

dendrimer (16-mer, 331 g, 4.2 x 10-5 mmol) andN-glycan azide b(1.6 mg, 8.4 x 10

-4 mmol) in DMF (50


at this temperature, DOTA (1.4 mg, 3.38 x 10-3

mmol) was added, and the resulting solution was stirred

for another 40 min. Low-molecule weight compounds were filtered using the Microcon (centrifugal

filter YM-10, 10,000 cut, Millipore), and the resulting aqueous solution was lipophilized to give

glycocluster 3bas amorphous solid (1.68 mg, quant). The purity of the product was evaluated by reverse

phase HPLC [column: Nacalai Tesque 5C18-AR300, 4.6 x 250 mm; MeCN in H2O containing 0.1% TFA

(10-100% gradient over 40 min, 1 mL/min); UV detection at 250 nm; retention time: 12.6 min].

Molecular weight was evaluated by MALDI-TOF-MS (Fig. SI-6) and gel filtration analysis as shown inFig. SI-5 (column: TSK-Gel G4000PWXL, 7.8 x 300 mm; 0.1 M NaCl at pH 7.2, 1 mL/min; retention

time: 9.9 min): 1H-NMR (500 MHz, D2O, HOD = !4.65)!8.69 (brs, 1H x 16, His-aromatic protons),

7.64 (s, 1H x 16, triazole proton), 7.10-7.36 (m, 6H x 16, Bn- and His-aromatic protons), 5.26 (brs, 2H x

16, benzylic protons of Bn), 5.01 (s, 1H x 16), 4.91 (brs, 1H x 16), 4.86 (s, 1H x 16), 4.60 (s, 1H x 16),

4.50 (m, 2H x 16), 4.40 (m, 3H x 16), 4.27 (s, 1H x 16), 4.19 (s, 1H x 16), 4.10 (s, 1H x 16), 3.44-3.98 (m,

including characteristic vicinal protons of sugar hydroxyls and "-protons of Lys), 2.85-3.10 (m, #-protons

on His), 2.70-2.80 (m, #-protons of Asn), 2.30-2.35 (m, #-protons of triazolic Ala), 1.98-2.12 (multiple s,Ac), 1.45-1.55 and 1.15-1.26 (m, part of acylpentane linker protons), 1.80-1.90, 1.60-1.78, 1.40-1.55,

1.26-1.36 and 0.90-1.23 (m, $- and !-protons of Lys).

Glycocluster 3c. CuSO4 (42 g, 2.6 x 10-4

mmol), sodium L-ascorbate (156 g, 7.9 x 10-4

mmol), and

diisopropylethylamine (60 nL) were added to a solution of acetylene-containing polylysine dendrimer

(16-mer, 129 g, 1.64 x 10-5 mmol) andN-glycan azide c(820 g, 3.29 x 10-4 mmol) in DMF (50 L) and

H2O (50 L) at room temperature under Ar atmosphere. After the mixture was stirred for 40 min at this



17/26

16



3cas amorphous solid (780 g, quant). The purity of the product was evaluated by reverse phase HPLC



was evaluated by gel filtration analysis (column: TSK-Gel G4000PWXL, 7.8 x 300 mm; 0.1 M NaCl at pH

7.2, 1 mL/min; retention time: 9.0 min):1H-NMR (500 MHz, D2O, HOD = !4.65)!8.73 (brs, 1H x 16,

His-aromatic protons), 7.65 (s, 1H x 16, triazole proton), 7.10-7.40 (m, 6H x 16, Bn- and His-aromatic

protons), 5.23 (brs, 2H x 16, benzylic protons of Bn), 4.99 (s, 1H x 16), 4.92 (brs, 1H x 16), 4.86 (s, 1H x

16), 4.57 (s, 1H x 16), 4.45 (m, 1H x 16), 4.37 (m, 2H x 16), 4.15 (brs, 1H x 16), 4.05 (brs, 1H x 16), 3.99

(brs, 2H x 16), 3.25-3.96 (m, characteristic vicinal protons of sugar hydroxyls and "-protons of Lys),

2.80-2.95 (m, #-protons on His), 2.55-2.75 (m, #-protons of Asn), 2.45-2.53 (m, H-3eqNeu

), 2.35-2.45 (m,

#-protons of triazolic Ala), 1.80-2.00 (multiple s, Ac), 1.55 (dd,J = 12Hz, 12Hz, H-3axNeu

), 1.60-1.75 and

1.12-1.25 (m, part of acylpentane linker protons), 1.75-1.83, 1.60-1.78, 1.26-1.36 and 0.90-1.25 (m, $-

and !-protons of Lys).

Glycocluster 3d. CuSO4 (48 g, 3.01 x 10-4 mmol), sodium L-ascorbate (179 g, 1.2 x 10

-3 mmol), and


(16-mer, 148 g, 1.9 x 10-5

mmol) andN-glycan azide d(940 g, 3.8 x 10-4

mmol) in DMF (50 L) andH2O (50 L) at room temperature under Ar atmosphere. After the mixture was stirred for 40 min at this




3d as an amorphous solid (900 g, quant). The purity of the product was evaluated by reverse phase

HPLC [column: Nacalai Tesque 5C18-AR300, 4.6 x 250 mm; MeCN in H2O containing 0.1% TFA (10-

100% gradient over 40 min, 1 mL/min); UV detection at 250 nm; retention time: 12.6 min]. Molecularweight was evaluated by gel filtration analysis (column: TSK-Gel G4000PWXL, 7.8 x 300 mm; 0.1 M

NaCl at pH 7.2, 1 mL/min; retention time: 9.0 min):1H-NMR (500 MHz, D2O, HOD = !4.65)!8.70 (brs,

1H x 16, His-aromatic protons), 7.66 (s, 1H x 16, triazole proton), 7.14-7.38 (m, 6H x 16, Bn- and His-

aromatic protons), 5.25 (brs, 2H x 16, benzylic protons of Bn), 5.05 (s, 1H x 16), 4.95 (brs, 1H x 16), 4.79

(s, 1H x 16), 4.50 (m, 2H x 16), 4.42 (m, 3H x 16), 4.10 (s, 1H x 16), 4.03 (s, 1H x 16), 3.95 (s, 1H x 16),

3.95-4.13 (m, "-protons of Lys), 3.26-3.90 (m, characteristic vicinal protons of sugar hydroxyls), 2.70-

3.00 (m, #-protons on His), 2.56-2.70 (m, #-protons of Asn), 2.45-2.49 and 2.50-2.55 (m, H-3eq

Neu

), 2.00-2.10 (m, #-protons of triazolic Ala), 1.80-1.98 (multiple s, Ac), 1.55 and 1.65 (m, H-3ax

Neu), 1.55-1.70


18/26

17

and 1.20-1.35 (m, part of acylpentane linker protons), 1.60-1.78, 1.40-1.55, 1.26-1.36 and 0.87-1.20 (m,

$- and !-protons of Lys).

Glycocluster 3e. CuSO4 (46 g, 2.9 x 10-4 mmol), sodium L-ascorbate (172 g, 8.7 x 10

-4 mmol), and


(16-mer, 142 g, 1.8 x 10-5 mmol) andN-glycan azide e(900 g, 3.6 x 10

-4 mmol) in DMF (50 L) and

H2O (50 L) at room temperature under Ar atmosphere. After the mixture was stirred for 40 min at this

temperature, DOTA (584 g, 1.44 x 10-3

mmol) was added, and the resulting solution was stirred for



3eas amorphous solid (900 g, quant). The purity of the product was evaluated by reverse phase HPLC



was evaluated by gel filtration analysis (column: TSK-Gel G4000PWXL, 7.8 x 300 mm; 0.1 M NaCl at pH

7.2, 1 mL/min; retention time: 9.0 min):1H-NMR (500 MHz, D2O, HOD = !4.65)!8.69 (brs, 1H x 16,

His-aromatic protons), 7.60 (s, 1H x 16, triazole proton), 7.05-7.25 (m, 6H x 16, Bn- and His-aromatic

protons), 5.18 (brs, 2H x 16, benzylic protons of Bn), 4.98 (s, 1H x 16), 4.90 (brs, 1H x 16), 4.78 (s, 1H x

16), 4.56 (m, 2H x 16), 4.30 (m, 3H x 16), 4.09 (s, 1H x 16), 4.02 (s, 1H x 16), 3.96 (s, 1H x 16), 4.10-

4.20 (m, "-protons of Lys), 3.30-3.85 (m, characteristic vicinal protons of sugar hydroxyls), 2.73-2.93 (m,

#-protons on His), 2.56-2.69 (m, #-protons of Asn), 2.56-2.62 and 2.63-2.70 (m, H-3eqNeu), 1.98-2.18 (m,

#-protons of triazolic Ala), 1.80-1.95 (multiple s, Ac), 1.60 and 1.67 (m, H-3 axNeu

), 1.40-1.55 and 1.25-

1.36 (m, part of acylpentane linker protons), 1.69-1.78, 1.40-1.55, 1.06-1.15 and 0.90-1.05 (m, $- and !-

protons of Lys).


19/26

18

Figure SI-5. Molecular weight analysis of glycoclusters 1a, 2a, 3a, and 3b, based on size-partitioning

gel-filtration (column: TSK-Gel G4000PWXL, 7.8 x 300 mm; eluent: 0.1 M NaCl, pH 7.2 at rt; flow rate:

1 mL/min; UV detection at 215 nm). Human serum albumin (HSA, 66 kDa), orosomucoid (44 kDa), and

acetylene-containing polylysine dendrimer (8 kDa) were used as standards. While bis-sialylated clusters

3aand 3c-3eshowed same retention time at 9.0 min, smaller asialo-derivative was eluted at 9.9 min (see

each glycocluster data).


20/26

19

Figure SI-6. MALDI-TOF-MS analysis of glycocluster 3aand 3b. Sinapinic acid containing 0.1% TFA

was used as the matrix.


21/26

20

Representative procedures for labeling of glycoclusters. DOTA & fluorescence-labeling was

performed by the previously reported procedure.[8]

Thus, to a solution of glycocluster 3a (210 g, 4.5

nmol) in distilled water (50 L) was added a solution of DOTA-conjugated aldehyde (see Scheme 1)[8]

(62 g, 90 nmol) in water (20 L) at room temperature. After the mixture was stirred at room temperature

for 30 min, the excess reagent was filtered off by using the Microcon (centrifugal filter YM-10, 10,000

cut, Millipore), and the resulting DOTA-glycocluster 3a dissolved in 1 M HEPES buffer was directly

used for the radiometal labeling.

Labeling of DOTA-glycocluster with68

Ga. A TiO2-based, commercially available68Ge/68Ga generator

(10 mCi, Eckert & Ziegler Eurotope, Berlin, Germany) was eluted with 1 N HCl, and 68Ga was purified

by a cation exchange column (AG 50W-X8 resin, 400 Mesh, Bio-Rad Laboratories, Inc., Hercules, CA,

USA). After 68GaCl3was eluted with 3.0 mL of 98% acetone-0.05 M HCl solution, acetone was removed

under a nitrogen stream. DOTA-glycocluster obtained above (4.5 nmol) in 1 M HEPES buffer (400 L)

was added to the eluent, and the pH of this solution was adjusted to 3.5 by adding 1 M HEPES buffer.

After the mixture was incubated at 95 C for 15 min, the resulting [68

Ga]DOTA-glycocluster was isolated

by reverse phase HPLC [Fig. SI-7, column: Nacalai Tesque 5C18-AR300, 4.6 x 250 mm; MeCN in H2O

containing 0.1% TFA (10-100% gradient over 10 min, 1 mL/min); detection by UV at 250 nm and RI;

retention time at 8 min for 3a]. The eluent was evaporated under reduced pressure and the residue was

dissolved in 0.1M PBS buffer (pH=7.4) for PET study. Only 500 pmol of the glycocluster was used for

PET imaging.

Figure SI-7. HPLC purification of [68Ga]DOTA glycocluster 3a(RI detection).


22/26

21

General procedure of [68

Ga]PET imaging. The animal studies were performed according to a protocol

approved by the Ethics Committee of RIKEN. Briefly, the PET study was conducted on male BALB/c

nude mouse weighing 23-27 g at 9-12 weeks of age (Japan SLC, Inc., Hamamatsu, Japan) under

inhalation anesthesia with isoflurane (Escain, Mylan, Osaka, Japan), and was used a small animal PET

scanner, the microPET Focus 220 (Siemens Medical Solutions Inc., Knoxville, TN, USA). The mice were

continuously anesthetized with isoflurane during the experiment. [68Ga]DOTA-glycocluster conjugates

(500 pmol) at a dose of 6.9-12.9 MBq in 100 L were injected via a tail vein, and then the emission data

was collected for 240 min postinjection. Three dimensional dynamic images were reconstructed by

filtered back projection method or ordered subset expectation maximization (OSEM) algorithm with

attenuation correction. In order to analyze the tracer disposition kinetics, another four images were

reconstructed from the summation data of 0 to 5 min, 30 to 60 min, 90 to 120 min, and 210 to 240 min

after injection of [68

Ga]DOTA-glycoclusters. Quantitative analysis was performed using ASIPro VM

version 6.6.2.0 software (Siemens Medical Solutions Inc., Knoxville, TN, USA). Regions of interest

(ROIs) were placed on the tissue region.

Figure SI-8. Standardized uptake value (SUV) of68

Ga-DOTA-Labeled glycoclusters 1a,2a,and3a-cin

normal BALB/c nude mice. SUV was normalized by radioactivity in the tissue region, injected dose, and

weight of the subjects. These values represent the means and S.Ds.

General procedure of fluorescence imaging. Eight week-old mice (BALB/cAJclnu/nu, CLEA Japan,

Inc.) was used for in vivo fluorescence imaging. The cancer model was prepared by subcutaneouslyinjecting the DLD-1 (107 cells / 100 L) to the dorsal division of mouse (BALB/cAJclnu/nu, CLEA


23/26

22

Japan, Inc.). The cancer had grew up to proper size for imaging purposes, after 2~3 weeks from the

injection, and therefore, nine week-old (2 weeks later from injection of DLD-1) was used for imaging.

Cy5-labeled glycoclusters (500 pmol / 100 L in physiological salt solution) were injected from the

caudal vein without anesthesia, and whole body scanning was performed by using eXplore Optix, GE

Healthcare, Bioscience (excitation at 646 nm, emission 663 nm), 45, 60, 120, 180, and 240 min after

injection. Fluorescence images were collected under inhalation anesthesia with Isoflurane; the

concentration of Isoflurane was 4% from 15 min to 60 min after the injection, and then kept at 1.5-2%

during the rest of the measurements.

Figure SI-9.(a)-(e): Dynamic fluorescence imaging of glycoclusters 3a-ein DLD-1 implanted nude mice

(left femoral region) as a cancer model; (a) glycocluster 3b; (b) glycocluster 3c; (c) glycocluster 3a; (d)

glycocluster 3d; (e) glycocluster 3e. Cy5-Labeled glycoclusters 3a-ewere administered from the tail vein

of the mice (n=3, 500 pmol, 100 L/mouse) and whole body scans were performed from the front side by

eXplore Optix, GE Healthcare, Bioscience (excitation at 646 nm, emission 663 nm), 4 h after injection.

Data was normalized. H: heart; L: liver; B: urinary bladder.


24/26

23

Interaction of glycoclusters 3a-3e with B-cell lines.


25/26

24


26/26

25

2010 Noninvasive SI

Documents