Guanidinylated Neomycin Mediates Heparan Sulfate-Dependent Transport of
Active Enzymes to Lysosomes
Stéphane Sarrazin‡, Beth Wilson†, William S. Sly§, Yitzhak Tor†, and Jeffrey D. Esko‡
‡
Department of Cellular and Molecular Medicine, Glycobiology Research and Training
Center, University of California, San Diego, La Jolla, California, 92093-0687
§
Department of Biochemistry and Molecular Biology, Saint Louis University, St. Louis,
MO 63104
†
Department of Chemistry and Biochemistry, University of California, San Diego, La
Jolla, California, 92093-0358
Supplementary Material / Methods
To enhance the understanding of the reader, supplementary material is provided and is
organized in five sections such as:
S1.
S2.
S3.
S4.
S5.
General synthesis of NHS-GNeo
Synthesis of guanidinylated neomycin derivatives
Trypsin sensitivity of bound GNeo-QD525.
Purification of GNeo-bGUS by heparin-sepharose chromatography
Colocalization of GNeo-QD525 with Lysotracker Red
S1
S1. General Synthesis of NHS-GNeo
All reagents were obtained with the highest available purity and purchased from the
Aldrich-Sigma Chemical Company. Proton and carbon NMR were recorded at 500 MHz
and 125 MHz, respectively, using an ECA-500 Joel spectrophotometer. The proton and
carbon assignments were based on COSY, TOCSY, and HSQC experiments.
Electrospray ionization (ESI) mass spectrometry was conducted employing a
ThermoFinnigan LCQDECA-MS spectrometer. Infra red spectroscopy was recorded in a
JASCO FT/IR-4100 spectrophotometer.
The attached supplementary Scheme S1 describes the synthesis of NHS-GNeo
S2. Synthesis of guanidinylated neomycin derivatives.
Compound 1 was prepared as previously reported (Kirk, S. R, Luedtke, N. W. and Tor,
Y. J. Am. Chem. Soc. 2000, 122, 980-981.
Compound 2. To a solution of 6-hexynoic acid (26 µL, 0.236 mmol) in 400 µL of CH2Cl2
was added 1-ethyl-3-(3’-dimethylaminopropyl)carbodiimide hydrochloride (45 mg, 0.234
mmol). After 10 min, compound 1 (260 mg, 0.204 mmol) dissolved in 500 µL of CH2Cl2
and N,N’-diisopropylamine (81 µL, 0.468 mmol) were added. The reaction mixture was
stirred at room temperature for 8 h. The reaction mixture was diluted in 200 mL of
CH2Cl2 and extracted with water (2 x 50 mL). The organic layer was washed with brine
(50 mL), dried over sodium sulfate, and concentrated in vacuo. The reaction products
were purified via flash chromatography using silica gel and (0-4% CH3OH in CH2Cl2) as
eluent to afford a white solid (200 mg, 72%). Rf = 0.43 (10% CH3OH in CH2Cl2, ninhydrin
stain). 1H NMR (500 MHz, CD3OD, ppm):  5.37 (b, 1H, H-1’), 5.14 (b, 1H, H-1’’), 4.92
(b, 1H, H-1’’’), 4.23-4.22 (m, 2H, H-2’’ and H-3’’), 4.10-4.07 (m, 1H, H-4’’), 3.90-3.87 (m,
2H, H-5’’’ and H-5), 3.77 (b, 1H, H-4’’’), 3.74-3.71 (m, 1H, H-5’), 3.64-3.52 (m, 3H, H-3’,
H-4’, and H-4), 3.49-3.46 (m, 3H, H-3’’’, H-3 and H-6), 3.43-3.38 (m, 2H, H-), 3.36-3.32
(m, 3H, H-2’’’ and H-6’), 3.29-3.28 (m, 2H, H-6’’’), 3.27-3.26 (m, 1H, H-1), 3.20-3.17 (m,
1H, H-2’), 2.90-2.89 (m, 2H, H-5’’), 2.76 (t, 2H, J = 6.9 Hz, H-), 2.34 (t, 2H, J = 7.4 Hz,
H-), 2.24-2.21 (m, 3H, H- CH alkyne), 1.97-1.95 (m, 1H, H-2 a or b), 1.85-1.79 (m,
2H, H-), 1.46-1.44 (m, 54H, Boc (CH3)3), 1.36-1.33 (m, 1H, H-2 a or b); 13C NMR (125
MHz, CD3OD, ppm): 174.1 (C=ONH), 157.5-156.5 (Boc C=O x 6), 109.8 (C-1’’), 99.2
(C-1’’’), 97.8 (C-1’), 85.7 (C-3’’’), 82.9 (C-alkyne), 81.4 (C-4’’), 80.2 (C-3’’), 79.4-78.7
(Boc Cq x 6), 78.5 (C-6), 74.3 (C-1), 73.9 (C-2’’’), 73.1 (C-5), 71.9 (C-2’’), 71.5 (C-5’),
70.3 (C-5’’’), 68.9 (CH alkyne), 67.5 (C-3), 55.6 (C-3’), 55.5 (C-4’), 52.3 (C-4’’’), 50.8 (C6’), 50.1 (C-4), 41.2 (C-6’’’), 40.4 (C-2’), 38.8 (C-), 34.6 (C-), 34.2 (C-5’’), 31.5 (C-),
27.7 (C-2), 27.5-27.4 (Boc (CH3)3 x 6), 24.6 (C-), 17.4 (C-). LRMS (ESI): calculated for
C61H105N7O25S [M + H]+ 1367.7, found 1368.3.
Compound 3. To a solution of 2 (200 mg, 0.146 mmol) in 2 mL of CH2Cl2 were added
triisopropylsilane (20 µL, 0.50% v/v) and trifluoroacetic acid (2 mL). The reaction mixture
was stirred at room temperature for 2 h. Then, the reaction mixture was concentrated in
vacuo (2 X using toluene to azeotrope the trifluoroacetic acid) and the residue obtained
was dissolved in water (20 mL) and extracted with CH2Cl2 (100 mL). The aqueous
solution was filtered and lyophilized to afford a white solid (205 mg, 97%). 1H NMR (500
MHz, D2O, ppm):  6.04 (d, J = 4 Hz, 1H, H-1’), 5.39 (d, J = 3 Hz, 1H, H-1’’), 5.29 (d, J =
2 Hz, 1H, H-1’’’), 4.42-4.40 (m, 2H, H-2’’ and H-3’’), 4.33-4.29 (m, 2H, H-4’’ and H-4’’’),
4.22-4.20 (m, 1H, H-5’’’), 4.09-4.06 (m, 1H, H-5), 4.01-3.97 (m, 1H, H-5’), 3.93-3.87 (m,
2H, H-3’ and H-4), 3.82-3.81 (m, 1H, H-3’’’), 3.69-3.65 (m, 1H, H-6), 3.58 (b, 1H, H-2’’’),
S2
3.55-3.52 (m, 1H, H-4’), 3.50-3.49 (m, 1H, H-1), 3.47-3.43 (m, 2H, H-6’) 3.42-3.40 (m,
2H, H-), 3.38-3.37 (m, 1H, H-3), 3.36-3.31 (m, 2H, H-6’’’), 3.28-3.24 (m, 1H, H-2’), 3.153.12 (m, 1H, H-5’’ a or b), 2.81-2.70 (m, 3H, H-and H-5’’ a or b), 2.49-2.45 (m, 1H, H-2
a or b), 2.37-2.34 (m, 3H, H-and CH alkyne), 2.24-2.21 (m, 2H, H-), 1.91-184 (m, 1H,
H-2 a or b), 1.80-1.75 (m, 2H, H-); 13C NMR (125 MHz, CD3OD, ppm): 176.4
(C=ONH), 117.6 (C-1’’), 115.3 (C-1’’’), 110.5 (C-1’), 95.7 (C-3’’’), 95.1 (C-alkyne), 85.5
(C-4’’), 84.8 (C-3’’), 80.1 (C-6), 78.6 (C-1), 75.1 (C-2’’’), 73.7 (C-5), 72.5 (C-2’’), 70.7 (C5’), 70.0 (C-5’’’), 69.5 (CH alkyne), 68.0 (C-3), 67.7 (C-3’), 53.7 (C-4’), 50.9 (C-4’’’), 50.9
(C-6’), 49.7 (C-4), 40.6 (C-6’’’), 40.2 (C-2’), 33.9 (C-), 33.8 (C-), 28.1 (C-5’’), 30.7 (C), 28.1 (C-2), 24.6 (C-), 17.1 (C-). LRMS (ESI): calculated for C31H57N7O13S [M + H]+
767.4, found 768.3.
Compound 4. A solution of 3 (150 mg, 0.104 mmol) in 200 µL of CH3OH was treated with
triethylamine (200 µL, 1.44 mmol) and N,N’-diBoc-N’’-triflylguanidine (Baker, T. J.,
Luedtke, N. W., Tor, Y. and Goodman, M. J. Org. Chem. 2000, 65, 9054-9058.) (563
mg, 1.44 mmol) dissolved in 800 µL of CH2Cl2. The reaction mixture was stirred under
argon at room temperature for 36 h. Then, the reaction mixture was concentrated in
vacuo and the residue obtained was diluted with CH2Cl2 (200 mL) and extracted with 5%
aqueous NaHCO3 solution (2 x 50 mL). The CH2Cl2 solution was washed with brine (50
mL), dried over sodium sulfate, and concentrated in vacuo. The product was purified via
flash chromatography using silica gel and (0-2% CH3OH in CH2Cl2) as eluent to afford a
white solid (140 mg, 61%). Rf = 0.54 (5% CH3OH in CH2Cl2, ninhydrin stain). 1HNMR
(500 MHz, CD3OD, ppm):  5.90 (b, 1H, H-1’), 5.06 (b, 2H, H-1’’ and H-1’’’), 4.58-4.54
(m, 1H, H-5), 4.31-4.28 (m, 2H, H-2’’ and H-3’’), 4.31-4.29 (m, 2H, H-4’’’ and H-5’’’),
4.15-4.11 (m, 1H, H-4), 3.99-3.95 (m, 4H, H-3’, H-3’’’, H-4’ and H-4’’), 3.91-3.87 (m, 1H,
H-6), 3.82-3.73 (m, 2H, H-1, H-2’’’ and H-5’), 3.75-3.74 (m, 1H, H-4’), 3.67-3.61 (m, 2H,
H-6’’’), 3.55-3.47 (m, 2H, H-6’), 3.29-3.27 (m, 3H, H-3 and H-), 3.24-3.22 (m, 1H, H-2’),
2.75-2.66 (m, 4H, H-5’’ and H-), 2.33-2.30 (m, 2H, H-), 2.25-2.20 (m, 4H, alkyne CH,
H-2 (a or b) and H-), 1.79-1.76 (m, 3H, H-2 (a or b) and H-), 1.56-1.38 (m, 108H, Boc(CH3)3); 13C NMR (125 MHz, CD3OD, ppm): 173.7 (C=ONH), 163.2-162.9 (NHC=NH x
6) 157.5-152.5 (Boc C=O x 12), 111.3 (C-1’’), 97.7 (C-1’’’), 95.9 (C-1’), 83.4 (C-5’), 83.382.8 (Boc Cq x 6), 81.0 (C-alkyne), 79.3-79.1 (Boc Cq x 6), 78.9 (C-4’’), 78.5 (C-6), 77.9
(C-3’’’), 75.9 (C-3), 75.0 (C-4’’’), 74.5 (C-3’’), 72.8 (C-2’’’), 72.6 (C-4’), 71.9 (C-2’), 70.3
(C-1), 68.6 (CH alkyne), 66.7 (C-5), 55.6 (C-3’), 54.2 (C-5’’’), 51.4 (C-4), 48.2 (C-2’’),
43.0 (C-6’’’), 40.4 (C-6’), 39.2 (C-), 34.7 (C-), 34.5 (C-5’’), 32.09 (C-), 29.1 (C-2),
27.5-27.4 (Boc (CH3)3 x 12), 24.6 (C-), 17.5 (C-). LRMS (ESI): calculated for
C97H165N19O37S [M+ 2H]2+ 2220.1, found 1110.9.
Compound 5 (over two steps). A stirred solution of 6-bromohexanoic acid (1.00 g, 5.15
mmol) in DMF (5 mL) was treated with sodium azide (5.05 g, 7.78 mmol) and heated to
85 ºC for 3 h. The reaction mixture was concentrated in vacuo and the crude obtained
was diluted in 300 mL of CH2Cl2 and washed with 0.1 N aqueous HCl. The CH2Cl2
solution was washed with brine (50 mL), dried over sodium sulfate, and concentrated in
vacuo to yield an oil (6-aziodohexanoic acid, 730 mg, 90%) that was used in the next
step without any further purification. The 6-azidohexanoic acid (500 mg, 3.18 mmol) and
N-hydroxysuccinimide (366 mg, 3.18 mmol) were dissolved in (CH2Cl2/DMF 9:1 v/v) 10
mL and treated with 1-ethyl-3-(3’-dimethylaminopropyl)carbodiimide hydrochloride (610
mg, 3.18 mmol). The reaction mixture was stirred at room temperature overnight. The
solvent was concentrated in vacuo, the residue was diluted with 300 mL of CH2Cl2 and
washed with water (2 X 50 mL). The organic layer was washed with brine (50 mL), dried
S3
over sodium sulfate, and concentrated in vacuo. The product was purified via flash
chromatography using silica gel and (100% CH2Cl2) as eluent to afford a dark-yellow oil
(650 mg, 80%). Rf = 0.46 (5% CH3OH in CH2Cl2, bromocresol stain). 1HNMR (500 MHz,
DMSO-d6, ppm):  3.33-3.30 (m, 2H, H-), 2.80 (s, 4H, CH2-CH2, NHS), 2.68 (t, 2H, J = 5
Hz, H-), 1.67-1.61 (m, 2H, H-), 1.58-1.49 (m, 2H, H-), 1.43-1.37 (m, 2H, H-); 13C
NMR (125 MHz, DMSO-d6, ppm):  170.3 (OC=O), 168.9 (C=O, NHS), 50.4 (C-30.0
(C-), 27.7 (CH2-CH2, NHS), 25.4 (C-), 25.2 (C-), 23.7 (C-). IR (film), 2099 cm-1 (N3),
1812 and 1782 cm-1 (C=O, NHS). LRMS (ESI): calculated for C10H14N4O4 [M + H]+ 254.1,
found 255.0.
Compound 6. Compounds 4 (100 mg, 0.045 mmol) and 5 (17 mg, 0.067 mmol) were
dissolved in 100 µL of acetonitrile and treated with 10 mol% CuBr (56 µL, 0.080 M
solution in acetonitrile) and 10 mol% (56 µL, 0.080 M solution in acetonitrile) of tris[(1benzyl-1H-1,2,3-triazol-4yl)methyl]amine (ligand) in acetonitrile. The reaction was
allowed to stir at room temperature under an argon atmosphere. After 8 hr, an additional
10 mol% of each CuBr and ligand were added. The reaction mixture was reacted for 24
h, after which flash chromatography using silica gel and (2% CH3OH in CH2Cl2) as
eluent afforded a white solid (81mg, 73%). Rf = 0.44 (5% CH3OH in CH2Cl2, ninhydrin
stain). 1HNMR (500 MHz, DMSO-d6, ppm):  11.5-11.4 (m, 6H, NHOC=O), 8.71 (b, 1H,
NHC=O), 8.52 (m, 3H, NH), 8.24 (m, 3H, NH), 7.81 (b, 1H, CH triazole), 5.64 (b, 1H,
OH), 5.56 (d, J = 3 Hz, 1H, H-1’), 5.53 (b, 1H, OH), 5.54-533 (m, 3H, OH), 5.13 (b, 1H,
H-1’’), 4.97 (b, 1H, H-1’’’), 4.25-4.23 (m, 3H, H-and H-2’’), 4.12-4.09 (m, 1H, H-3’’),
3.89-3.87 (m, 1H, H-4’’), 3.82 (b, 1H, H-5), 3.80-3.78 (m, 2H, H-5’ and H-5’’’), 3.73-3.71
(m, 1H, H-5’’ a or b), 3.63-3.56 (m, 2H, H-3’ and H-3’’’), 3.54-3.53 (m, 1H, H-4’’’), 3.483.46 (m, 3H, H-1, H-2’’’ and H-4’), 3.45-3.41 (m, 3H, H-4 and H-6’’’), 3.31-3.27 (m, 5H,
H-3, H-6 and H-6’), 3.22-3.18 (m, 2H, H-), 3.13-3.10 (m, 1H, H-2’), 2.77 (s, 4H, CH2CH2, NHS), 2.75-2.72 (m, 1H, H-5’’ a or b), 2.63 (t, 2H, J = 7.42 Hz, H-), 2.55-2.50 (m,
2H, H-), 2.26-2.23 (m, 1H, H-a or b), 2.15-2.11 (m, 2H, H-), 1.95-1.93 (m, 1H, H-2 a
or b), 1.79-1.75 (m, 4H, H- and H-), 1.64-1.58 (m, 1H, H-a or b) 1.52-1.48 (m, 2H, H), 1.46-1.38 (m, 108H, Boc (CH3)3); 1.36-1.31 (m, 1H, H-2 a or b), 1.30-1.23 (m, 2H, H); 13C NMR (125 MHz, DMSO-d6, ppm):  175.7 (C=ONH), 170.3 (OC=O), 168.9 (C=O,
NHS), 163.7-163.1(Boc NHOC=O x 6), 156.4-155.8 (Boc NOC=O x 6), 153.1-152.8
(C=N x 6) 111.4 (C-1’’), 97.8 (C-1’’’), 96.5 (C-1’), 82.8 (Boc Cq x 6), 79.3-79.1 (Boc Cq x
6), 80.4 (C-5’), 78.8 (C-3’’), 77.9 (C-3’’’), 72.2 (C-4’’), 72.8 (C-5), 72.6 (C-4’), 71.9 (C-2’),
71.6 (C-2’’’), 73.2 (C-2’’), 72.2 (C-4’’’), 70.3 (C-1), 69.5 (C-5’’’), 66.6 (C-6), 66.7 (C-3),
55.6 (C-3’), 54.2 (C-6’), 51.4 (C-4), 43.0 (C-6’’’), 39.2 (C-), 34.7 (C-), 34.5 (C-5’’), 50.4
(C-32.09 (C-), 30.0 (C-), 29.1 (C-2), 27.7 (CH2-CH2, NHS), 28.5-27.9 (Boc (CH3)3 x
12), 24.6 (C-) 25.4 (C-), 25.2 (C-), 23.7 (C-), 22.6 (C-). LRMS (ESI): calculated for
C107H179N23O41S [M + 2Na]2+ 2474.2, found 1260.3.
Compound 7. To a solution of 6 (81 mg, 0.033 mmol) in 2 mL of CH2Cl2 were added
triisopropylsilane (20 µL, 0.50% v/v) and trifluoroacetic acid (2 mL). The reaction mixture
was stirred at room temperature for 2 h. Then the reaction mixture was concentrated in
vacuo (2 X using toluene to azeotrope the trifluoroacetic acid) and the residue obtained
was dissolved in cold water (5 mL) and filtered. The aqueous solution was filtered and
lyophilized to afford a white solid (60 mg, 94%). 1HNMR (500 MHz, DMSO-d6, ppm): 
8.12 (b, 1H, NHC=O), 8.04 (m, 2H, NH), 7.81 (b, 1H, CH triazole), 7.63 (b, 1H, NH), 7.46
-7.02 (m, 32, NH2), 6.82 (d, J = 8.6 Hz, 1H, NH) 5.91 (b, 1H, OH), 5.83 (b, 1H, OH), 5.69
(d, J = 5 Hz, 1H, H-1’), 5.56 (b, 2H, OH), 5.38 (b, 1H, OH), 4.95 (b, 1H, H-1’’), 4.82 (b,
1H, H-1’’’), 4.26-4.20 (m, 3H, H-and H-2’’), 4.12-4.09 (m, 1H, H-3’’), 3.89-3.87 (m, 1H,
H-4’’’), 3.81 (b, 1H, H-5’’’), 3.82-3.75 (m, 1H, H-4’’), 3.63-3.61 (m, 1H, H-5), 3.57-3.56
S4
(m, 1H, H-5’), 3.54-3.52 (m, 2H, H-3’ and H-4), 3.50-3.46 (m, 1H, H-3’’’), 3.45-3.42 (m,
2H, H-2’’’ and H-4’), 3.41-3.33 (m, 1H, H-1), 3.31-3.27 (m, 4H, H-6 and H-6’), 3.25-3.17
(m, 5H, H-3, H-6’’’ and H-3.19-3.15 (m, 1H, H-2’), 2.77 (s, 4H, CH2-CH2, NHS), 2.752.72 (m, 1H, H-5’’a or b), 2.65-2.63 (m, 2H, H-), 2.61-2.50 (m, 5H, H-, H-, and H-5’’a
or b), 2.14-2.11 (m, 2H, H-), 1.96-1.93 (m, 1H, H-2 a or b), 1.81-1.72 (m, 4H, H- and
H-), 1.64-1.58 (m, 2H, H-), 1.32-1.28 (m, 2H, H-), 1.21-1.18 (m, 1H, H-2 a or b); 13C
NMR (125 MHz, DMSO-d6, ppm):  173.2 (C=ONH), 170.3 (OC=O), 168.9 (C=O, NHS),
157.8-1.56.8 (C=NH x 6), 121.9 (CH triazole), 111.0 (C-1’’), 97.2 (C-1’’’), 95.3 (C-1’),
86.7 (C-5), 80.8 (C-4’’), 79.1 (C-3’’), 76.1 (C-5’), 73.2 (C-2’’), 74.7 (C-6’’’), 73.2 (C-1),
72.2 (C-4’’’), 70.5 (C-3’), 70.2 (C-2’), 69.3 (C-5’’’), 66.7 (C-3), 66.6 (C-6), 55.4 (C-2’’’),
53.6 (C-3’’’), 52.6 (C-4’), 51.7 (C-4), 50.1 (C-6’), 49.7 (C-39.4 (C-), 34.6 (C-5’’), 34.7
(C-), 35.8 (C-), 34.1 (C-2), 30.4 (C-), 30.1 (C-), 26.1 (CH2-CH2, NHS), 25.6 (C-)
23.7 (C-), 25.5 23.7 (C-), 25.4 (C-). LRMS (ESI): calculated for C47H83N23O17S [M +
H]+ 1273.6, found 1274.4.
S3. Trypsin sensitivity of bound GNeo-QD525.
The sensitivity of membrane-bound QDots to trypsin and Versene (EDTA) was assessed
after incubation of cells with GNeo-QD525 at 4oC and 37oC (Fig. S1). Treatment of cells
with EDTA or trypsin released the cells efficiently from the plates under both conditions.
Cells treated with Versene at 4oC retained quantum dots (mean fluorescence = 23,
background = 3), which were released by trypsin. At 37oC, the level of uptake of GNeoQD525 exceeded that observed at 4oC, as measured by the increase in average
fluorescence after treatment with EDTA (average fluorescence = 48). About one-quarter
of this material resised trypsin release (average fluorescence = 11), consistent with its
location in an internal compartment.
S4. Purification of GNeo-bGUS by heparin-sepharose chromatography.
GNeo-bGUS conjugate was prepared by reacting bovine GUS (bGUS) with GNeo-NHS
as described in the manuscript and purified by chromatography on an heparin-sepharose
column. The recovery of protein was assessed in the flow through fractions and after step
elution with 0.3, 0.6, and 0.9 M NaCl (Fig. S2a). Enzyme activity was assayed before
and after reaction and in the various fractions (Fig. S2b).
S5. Colocalization of GNeo-QD525 with Lysotracker Red.
To better illustrate the colocalization between GNeo-QD525 and lysotracker red
presented in Fig. 2, samples were imaged in multiple planes and a 3D reconstruction
was generated. Supplementary Movie 1 shows a fly-through rendering which
demonstrates colocalization of GNeo-QD525 with lysotracker.
CHO cells were
incubated with GNeo-QD525 for 30 min and rinsed. After 2.5 hr, cells were rinse again,
labeled with Hoechst dye and Lysotracker Red. Images were captured with a
DeltaVision Restoration microscope system (AppliedPrecision) seated on an Olympus
IX70. Optical sections were acquired using 100x oil immersion objective in 0.2-m steps
in the z-axis Images were deconvolved on a softWoRx workstation and 3D reconstituted
using Volocity software (Improvision).
S5