Supporting Information Table of contents

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were recorded at the UCSD Chemistry and Biochemistry Mass Spectrometry ... (BocNH)2C=NTf (12 eq)/NaHCO3/dioxane-H2O, rt, 5d, 31%; b) ..... molar ratio.
Supporting Information

Table of contents Materials and Instrumentation

2

Synthesis

3 Scheme S1. Synthesis of guanidinoneomycin transporters

3

Synthesis of compound 2

4

Synthesis of compounds 3, 4, 5, 6 and 7

6

HPLC spectra for compounds 2-7

12

Supporting Figures Figure S1. COSY spectrum for compound 2

14

Figure S2. TOCSY spectrum for compound 2

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Figure S3. HSQC spectrum for compound 2

16

Figure S4. HMBC spectrum for compound 2

17

Figure S5. Expanded HMBC spectrum for compound 2

18

Figure S6. 13C-NMR spectrum for compound 2

19

Figure S7. FRET analysis of Streptavidin conjugates

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Supporting Table S1. DLS analysis of Streptavidin conjugates

21

References

22

1

Materials: Unless otherwise specified, materials obtained from commercial suppliers were used without further purification. Deuterated NMR solvents were purchased from Cambridge Isotope Laboratories. PBS (Dulbecco’s phosphate buffered saline), Dulbecco's Modified Eagle Medium (DMEM, low glucose), Versene (EDTA), and ST-Cy5 were purchased from Life technologies (San Diego, CA, USA). ST-PE was purchased from eBioscience (San Diego, CA, USA). Streptavidin–PECy5 was purchased from BD Biosciences (San Jose, CA, USA). Trypsin/EDTA was purchased from VWR (Mediatech, Manassas, VA, USA). Costar 3524 (Corning) 24-well plates were used. Instrumentation: NMR spectra were recorded on either a Varian Mercury 400 MHz or 500 MHz spectrometers. Mass spectra were recorded at the UCSD Chemistry and Biochemistry Mass Spectrometry Facility by utilizing an LCQDECA (Finnigan) ESI with a quadrupole ion trap. Reversed-phase HPLC purification (CLIPEUS, C18, 5m, 25010 mm, Higgins analytical) and analysis (Eclipse, XDB-C18, 5m, 14.6150 mm) were carried out on an Agilent 1200 series instrument. Flow-cytometry studies were performed on a BD FACSCalibur. Dynamic Light Scattering was measured on a Wyatt Dynapro Nanostar.

2

Synthesis:

Scheme S1. Synthesis of guanidinoneomycin transporters; a) (BocNH) 2C=NTf (5.5 eq)/DCM-MeOH, rt, 7d, 24% or (BocNH)2C=NTf

(12

eq)/NaHCO3/dioxane-H2O,

rt,

5d,

31%;

b)

RNHCH2CH/Cu(OAc)2.H2O/sodium

ascorbate/THF-MeOH-H2O, 38%; c) acyl chloride/NaHCO3/dioxane-H2O, 55% (11), 51% (12), 61% (13); d) Acyl chloride/NaHCO3/dioxane-H2O, 71% (14), 35% (15); e) RNHCH2CCH/CuI/Cs2CO3/MeCN-DCM-MeOH, 76% (16) or RNHCH2CCH/CuSO4.5H2O/sodium ascorbate/tBuOH-H2O, 50% (17); f) iPr3SiH/TFA-DCM, 35% (2), 6% (3), 29% (4), 27% (5), 23% (6), 27% (7)

3

Synthesis of compound 2 Compound 9

Condition A: To a solution of compound 8[1] (1 g, 0.755 mmol) in methanol (1 mL) was added triethylamine (4.21 mL, 30.2 mmol), dichloromethane (10 mL), and N,N’-di-tert-butoxycarbonyl-N’’-trifluoromethanesulfonylguanidine (1.63 g, 4.15 mmol) at ambient temperature under an argon atmosphere. The mixture was stirred for 7 days. The mixture was partitioned between dichloromethane and 10% citric acid. The organic layer was washed with brine, dried over anhydrous MgSO4, filtered, and the filtrate was evaporated. Silicagel column chromatography (dichloromethane-methanol, a linear gradient of methanol from 0 to 7%) afforded the desired product as a colorless amorphous solid (340 mg, 24%). See below for analytical data. Condition B: To a mixture of compound 8[1] (200 mg, 0.151 mmol) and NaHCO3 (190 mg, 2.27 mmol) in dioxane-water (3:1, 4 mL) was added N,N’-di-tert-butoxycarbonyl-N’’-trifluoromethanesulfonylguanidine (710 mg, 1.81 mmol) at ambient temperature. The mixture was stirred for 5 days. The mixture was partitioned between dichloromethane and brine. The organic layer was collected, dried over anhydrous MgSO4, filtered, and the filtrate was evaporated. Silica gel column chromatography (dichloromethane-methanol, a linear gradient of methanol from 0 to 7%) afforded the desired product as a colorless amorphous solid (87.7 mg, 31%). H-NMR (400MHz, CD3OD):  1.29-2.22 (90H, m), 2.24 (1H, m), 2.92 (1H, m), 3.27-2.59 (10H, m),

1

3.62-3.73 (2H, m), 3.81-3.88 (2H, m), 3.96-4.05 (4H, m), 4.11 (1H, m), 4.23-4.27 (2H, m), 4.33 (1H, m), 5.07 (1H, d, J = 1.6 Hz), 5.12 (1H, d, J = 3.6 Hz), 5.97 (1H, d, J = 4.0 Hz). MS (ESI) m/z: exact mass calcd. for C78H135N19O32 + Na+: 1872.94, found 1872.54 [M + Na]+

4

Compound 10

To a solution of compound 9 (18.4 mg, 0.00994 mmol) and compound 18[2] (11.8 mg, 0.0299 mmol) in methanol-tetrahydrofuran-water (1–1–0.4, 2.4 mL) was added sodium ascorbate (5.9 mg, 0.03 mmol) and Cu(OAc)2 monohydrate (6.0 mg, 0.030 mmol) at ambient temperature. The mixture was sonicated for 30 min (3 cycles of each 10 min with 5 min intervals). The mixture was partitioned between dichloromethane–methanol and satd. aq. ammonium chloride. The organic layer was dried over anhydrous MgSO4, filtered, and the filtrate was evaporated. Silica gel column chromatography (dichloromethane–methanol, a linear gradient of methanol from 0 to 9%) afforded the desired product as a colorless amorphous solid (8.4 mg, 38%). H-NMR (400MHz, CD3OD):  1.29-1.78 (112H, m), 2.18-2.27 (5H, m), 2.71 (1H, d, J = 12.8 Hz), 2.93

1

(1H, dd, J = 4.8 and 12.8 Hz), 2.98 (1H, m), 3.13-4.67 (30H, m), 5.01 (1H, d, J = 2.0 Hz), 5.09 (1H, d, J = 4.0 Hz), 5.75 (1H, d, J = 4.0 Hz), 7.88 (1H, s). MS (ESI) m/z: exact mass calcd. for C97H165N23O35S: 2244.16, found 1123.25 [M + 2H]2+, 1134.07 [M + H + Na]2+ Compound 2

To a solution of compound 10 (27.0 mg, 0.0120 mmol) in dichloromethane (0.5 mL) was added triisopropylsilane (0.037 mL, 0.18 mmol) followed by trifluoroacetic acid (0.5 mL) at ambient temperature. The mixture was stirred for 13 h. The volatiles were evaporated off. The residue was azeotroped with toluene (three times), dissolved in water and washed with dichloromethane. The aqueous solution was lyophilized to give a colourless amorphous (29.8 mg). HPLC (C18, acetonitrile-water conrtaining 0.1% trifluoroacetic acid, a linear gradient of acetonitrile from 0 to 50% over 20 min) afforded the purified product (Rt = 12.25 min) as a colorless amorphous solid (8.7 mg, 35%).

5

H-NMR (500MHz, D2O):  1.22-1.77 (13H, m), 2.22 (2H, t, J = 7.2 Hz), 2.28 (2H, t, J = 7.5 Hz), 2.33 (1H,

1

m), 2.74 (1H, d, J = 13.2 Hz), 2.96 (1H, dd, J = 5.2 and 13.2 Hz), 3.09-3.18 (2H, m), 3.31 (1H, m), 3.37-3.42 (2H, m), 3.48-3.67 (7H, m), 3.72-3.89 (6H, m), 4.13-4.18 (2H, m), 4.29-4.33 (2H, m), 4.38-4.48 (4H, m), 4.57-5.06 (3H, m), 5.03 (1H, m), 5.07 (1H, m), 5.94 (1H, d, J = 4.0 Hz), 7.86 (1H, s) MS (ESI) m/z: exact mass calcd. for C47H85N23O15S: 1243.63, found 1244.71 [M + H]+, 1266.65 [M + Na]+, High resolution mass found 622.8233 [M + 2H]2+, calcd. 622.8231 Synthesis of compounds 3, 4, 5, 6 and 7 Compounds 11, 12 and 13

Compound 11 To an ice-chilled mixture of compound 10 (27.2 mg, 0.0121 mmol) in 1 M NaHCO3 (0.485 mL, 0.485 mmol) was added a solution of n-decyloxyacetyl chloride (19.9 mg, 0.0848 mmol) in dioxane (1 mL). The mixture was stirred for 20 h at ambient temperature. The mixture was partitioned between dicloromethane and brine. The organic layer was collected, dried over anhydrous MgSO4, filtered, and the filtrate was evaporated. Silica gel column chromatography (dichloromethane-methanol, a linear gradient methanol from 0 to 6%) afforded the desired product as a colorless amorphous solid (16.2 mg, 55%). H-NMR (400MHz, CD3OD-CDCl3):  0.868 (3H, t, J = 6.6 Hz), 1.25-1.72 (119H, m), 2.16-2.26 (5H, m),

1

2.71 (1H, d, J = 12.8 Hz), 2.90 (1H, dd, J = 4.8 and 12.8 Hz), 3.15-4.57 (33H, m), 4.92 (1H, m), 5.04 (1H, d, J = 3.6 Hz), 5.58 (1H, d, J = 3.6 Hz), 7.13 (1H, d, J = 10.0 Hz), 7.67 (1H, s). MS (ES) m/z: exact mass calcd. for C109H187N23O37S: 2442.32, found 1233.01 [M + H + Na]2+, 1244.28 [M + 2Na]2+ Compound 12 To an ice-chilled mixture of compound 10 (21.7 mg, 0.00966 mmol) in 1 M NaHCO3 (0.193 mL, 0.193 mmol) was added a solution of n-tetradecyloxyacetyl chloride (8.4 mg, 0.029 mmol) in dioxane (1 mL). The mixture was stirred for 15 h at ambient temperature. The mixture was partitioned between dichloromethane and brine. The organic layer was collected, dried over anhydrous MgSO4, filtered, and the filtrate was evaporated. Silica gel column chromatography (dichloromethane-methanol, a linear gradient methanol from 0 to 6%) afforded the desired product as a colorless amorphous solid (12.3 mg, 51%). H-NMR (CD3OD):  0.90 (3H, t, J = 6.6 Hz), 1.29-1.76 (127H, m), 2.18-2.28 (5H, m), 2.71 (1H, d, J =

1

6

12.8 Hz), 2.93 (1H, dd, J = 5.2 and 12.8 Hz), 3.16-4.65 (33H, m), 5.01 (1H, m), 5.06 (1H, d, J = 3.6 Hz), 5.64 (1H, d, J = 3.6 Hz), 7.25 (1H, d, J = 10.0 Hz), 7.85 (1H, s). MS (ESI) m/z: exact mass calcd. for C113H195N23O37S: 2498.38, found 1250.33 [M + 2H]2+, 1261.29 [M + H + Na]2+, 1272.12 [M + 2Na]2+ Compound 13 To an ice-chilled mixture of compound 10 (23.2 mg, 0.0103 mmol) in 1 M NaHCO3 (0.207 mL, 0.207 mmol) was added a solution of n-octadecyloxyacetyl chloride (12.9 mg, 84.0% purity, 0.0311 mmol) in dioxane (1 mL). The mixture was stirred for 15 h at ambient temperature. The mixture was partitioned between dichloromethane and brine. The organic layer was collected, dried over anhydrous MgSO4, filtered, and the filtrate was evaporated. Silica gel column chromatography (dichloromethane-methanol, a linear gradient methanol from 0 to 6%) afforded the desired product as a colorless amorphous solid (16.1 mg, 61%). 1H-NMR (CD3OD-CDCl3):  0.90 (3H, t, J = 6.8 Hz), 1.29-1.76 (135H, m), 2.18-2.28 (5H, m), 2.71 (1H, d, J = 12.4 Hz), 2.93 (1H, dd, J = 5.2 and 12.8 Hz), 2.86-4.65 (33H, m), 5.00 (1H, m), 5.06 (1H, d, J = 3.6 Hz), 5.64 (1H, d, J = 4.0 Hz), 7.25 (1H, d, J = 7.6 Hz), 7.85 (1H, s). MS (ESI) m/z: exact mass calcd. for C117H203N23O37S: 2554.44, found 1278.51 [M + 2H]2+, 1289.25 [M + H + Na]2+ Compounds 14 and 15

Compound 14 To a mixture of compound 9 (38.9 mg, 0.0210 mmol) and NaHCO3 (35.3 mg, 0.420 mmol) in dioxane-water (2-1; 1.5 mL) was added a solution of n-pentyloxyacetylchloride (17.3 mg, 0.105 mmol) in dioxane (1 mL). The mixture was stirred for 15 h at ambient temperature. The mixture was partitioned between dichloromethane and brine. The organic layer was collected, dried over anhydrous MgSO4, filtered, and the filtrate was evaporated. Silica gel column chromatography (dichloromethane-methanol, a linear gradient of methanol from 0 to 6%) afforded the desired product as a colorless amorphous solid (29.4 mg, 71%). H-NMR (400MHz, CD3OD):  0.90 (3H, t, J = 7.0 Hz), 1.15-1.62 (97H, m), 2.19 (1H, m), 3.23-4.33 (26H,

1

m), 5.05 (1H, d, J = 1.6 Hz), 5.11 (1H, d, J = 3.2 Hz), 5.86 (1H, d, J = 4.0 Hz), 7.25 (1H, br, d, J = 9.6 Hz). MS (ES) m/z: exact mass calcd. for C85H147N19O34: 1978.04, found 990.45 [M + 2H]2+, 1001.47 [M + H + Na]2+, 1979.48 [M + H]+

7

Compound 15 To a mixture of compound 9 (50.0 mg, 0.0270 mmol) and NaHCO3 (45.4 mg, 0.540 mmol) in dioxane–water (1 mL-0.5 mL) was added a solution of n-dodecyloxyacetyl chloride (37.4 mg, 94.8% purity, 0.135 mmol) in dioxane (1 mL). The mixture was stirred for 15 h at ambient temperature. The mixture was partitioned between dichloromethane and brine. The organic layer was collected, dried over anhydrous MgSO4, filtered, and the filtrate was evaporated. Silica gel column chromatography (dichloromethane-methanol, a linear gradient of methanol from 0 to 6%) afforded the desired product as a colorless amorphous solid (19.8 mg, 35%). H-NMR (400MHz, CD3OD):  0.90 (3H, t, J = 6.8 Hz), 1.22-1.74 (111H, m), 2.19 (1H, m), 3.23-4.33 (26H,

1

m), 5.06 (1H, d, J = 1.6 Hz), 5.11 (1H, d, J = 3.2 Hz), 5.86 (1H, d, J = 3.6 Hz), 7.23 (1H, br, d, J = 10.0 Hz). MS (ESI) m/z: exact mass cacld. for C92H161N19O34: 2076.15, found 1039.24 [M + 2H]2+, 1050.38 [M + H + Na]2+, 1061.16 [M + 2Na]2+ Compound 16 and 17

Compound 16 To a mixture of compound 14 (29.4 mg, 0.0149 mmol), compound 18[2] (46.9 mg, 0.119 mmol) and Cs2CO3 (4.8 mg, 0.015 mmol) in dichloromethane (1 mL) was added an acetonitrile solution of CuI (2.8 mg, 0.015 mmol in 0.2 mL) at ambient temperature under an argon atmosphere. The mixture was stirred for 15 h (trace of product formation, the mixture being very cloudy and viscous). The mixture was stirred further for 24 h after adding a suspension of CuI (2.8 mg, 0.015 mmol) in methanol (0.3 mL). By adding methanol, the mixture became clearer. The mixture was directly applied to silica gel column chromatography (dichloromethane-methanol, a linear gradient of methanol from 0 to 10%) afforded a partially purified product as a colorless amorphous solid (26.7 mg). The product was used for the next reaction without further purification. H-NMR (400MHz, CD3OD):  0.91 (3H, t, J = 6.8 Hz), 1.40-1.58 (109H, m), 2.18-2.32 (5H, m), 2.71 (1H,

1

d, J = 12.8 Hz), 2.92 (1H, dd, J = 5.0 and 12.6 Hz), 3.17-4.65 (33H, m), 5.01 (1H, m), 5.06 (1H, d, J = 3.6 Hz), 5.64 (1H, d, J = 3.6 Hz), 7.27 (1H, br), 7.85 (1H, s). MS (ESI) m/z: exact mass calcd. for C104H177N23O37S: 2372.24, found 1187.48 [M + 2H]+, 1198.35 [M + H + Na]2+, 1209.03 [M + 2Na]2+

8

Compound 17 To a solution of compound 15 (19.8 mg, 0.00953 mmol) and compound 18[2] (5.6 mg, 0.014 mmol) in tert-butanol-water (1:1, 1 mL) was added sodium ascorbate (1.9 mg, 0.0096 mmol) and CuSO4 pentahydrate (0.7 mg, 0.003 mmol) at ambient temperature. The mixture was stirred for 15 h (trace product formation). The mixture was treated with compound 18 (5.6 mg, 0.014 mmol), sodium ascorbate (1.9 mg, 0.0096 mmol), and CuSO4 pentahydrate (1.7 mg, 0.0068 mmol) and was sonicated for 20 min at ambient temperature (4 cycles of each 5 min with 3 min intervals). The mixture was partitioned between dichloromethane-methanol and satd. aq. NH4Cl. The organic layer was dried over MgSO4, filtered, and the filtrate was evaporated. Silica gel column chromatography (dichloromethane-methanol, a linear gradient of methanol from 0 to 9%) afforded the desired product as a colorless amorphous solid (11.8 mg, 50%). H-NMR (CD3OD):  0.94 (3H, t, J = 6.6 Hz), 1.40-1.58 (123H, m), 2.18-2.28 (5H, m), 2.71 (1H, d, J =

1

12.4 Hz), 2.93 (1H, dd, J = 5.0 and 12.6 Hz), 3.26-4.65 (33H, m), 5.01 (1H, m), 5.06 (1H, d, J = 3.6 Hz), 5.64 (1H, d, J = 3.6 Hz), 7.25 (1H, d, br, J = 10.0 Hz), 7.85 (1H, s). MS (ESI) m/z: exact mass calcd. for C111H191N23O37S: 2470.35, found 1247.36 [M + H + Na]2+, 1258.16 [M + 2Na]+ Compounds 3, 4, 5, 6 and 7

Compound 3 To a solution of compound 16 (26.7 mg, 0.0112 mmol) in dichloromethane (0.5 mL) was added triisopropylsilane (0.035 mL, 0.17 mmol) followed by trifluoroacetic acid (0.5 mL) at ambient temperature. The mixture was stirred for 13 h. The volatiles were evaporated off. The residue was azeotroped with toluene (three times), dissolved in water and washed with dichloromethane. The aqueous solution was lyophilized to give a colorless amorphous solid (29.8 mg). HPLC (C18, acetonitrile-water containing 0.1% trifluoroacetic acid, a linear gradient of acetonitrile from 20 to 40% over 20 min) afforded the >99% purified product (4 mg). Additional HPLC purification was done with the same conditions yielded the purified product (Rt = 9.39 min) as a colorless amorphous solid (1.3 mg, 6%). H-NMR (500MHz, D2O):  0.85 (3H, t, J = 6.8 Hz), 1.26-1.72 (19 H, m), 2.05 (1H, m), 2.21 (2H, t, J = 7.2

1

Hz), 2.27 (2H, t, J = 7.5 Hz), 2.73 (1H, d, J = 13.2 Hz), 2.95 (1H, dd, J = 5.2 and 13.2 Hz), 3.12 (2H, m), 3.28-3.33 (2H, m), 3.41 (1H, m), 3.45-3.72 (13H, m), 3.80 (1H, m), 3.99 (1H, d, J = 16.0 Hz), 4.03 (1H, m),

9

4.08 (1H, d, J = 15.5 Hz), 4.13 (1H, m), 4.17 (1H, m), 4.27-4.30 (2H, m), 4.39 (1H, dd, J = 4.2 and 7.7 Hz), 4.44-4.46 (3H, m), 4.57-4.77 (3H, m), 5.01 (1H, d, J = 1.7 Hz), 5.08 (1H, m), 5.61 (1H, d, J = 4.0 Hz), 7.83 (1H, s). MS (ESI) m/z: exact mass calcd. for C54H97N23O17S: 1371.72, found 458.67 [M + 3H]3+, 687.26 [M + 2H]2+, 1372.63 [M + H]+, High resolution mass found 686.8648 [M + 2H]2+, calcd. 686.8650 Compound 4 To a solution of compound 11 (16.2 mg, 0.00662 mmol) in dichloromethane (0.5 mL) was added triisopropylsilane (0.020 mL, 0.099 mmol) followed by trifluoroacetic acid (0.5 mL) at ambient temperature. The mixture was stirred for 13 h. The volatiles were evaporated off. The residue was azeotroped with toluene (three times), dissolved in water and washed with dichloromethane. The aqueous solution was lyophilized to give a colorless amorphous solid (12.2 mg). HPLC (C18, acetonitrile-water containing 0.1% trifluoroacetic acid, a linear gradient of acetonitrile from 20 to 55% over 20 min) afforded the purified product (Rt = 18.32 min) as a colorless amorphous solid (3.9 mg, 29%). H-NMR (500MHz, D2O):  0.83 (3H, t, J = 6.9 Hz), 1.24-1.73 (29H, m), 2.04 (1H, m), 2.21 (2H, t, J = 7.5

1

Hz), 2.28 (2H, t, J = 7.4 Hz), 2.74 (1H, d, J = 13.2 Hz), 2.95 (1H, dd, J = 4.6 and 13.2 Hz), 3.09-3.17 (2H, m), 3.27-3.33 (2H, m), 3.42 (1H, m), 3.47-3.70 (12H, m), 3.72 (1H, m), 3.80 (1H, m), 3.98 (1H, d, J = 16.1 Hz), 4.02 (1H, m), 4.08 (1H, d, J = 16.1 Hz), 4.14 (1H, m), 4.17 (1H, m), 4.26-4.30 (2H, m), 4.39 (1H, dd, J = 4.6 and 8.1 Hz), 4.41-4.48 (3H, m), 4.58-4.76 (3H, m), 5.00 (1H, m), 5.08 (1H, m), 5.60 (1H, d, J = 3.4 Hz), 7.82 (1H, s) MS (ESI) m/z: exact mass calcd. for C59H107N23O17S: 1441.79, found 722.21 [M + 2H]2+, 1442.68 [M + H]+ , High resolution mass found 721.9042 [M + 2H]2+, calcd. 721.9041 Compound 5 To a solution of compound 17 (11.8 mg, 0.00477 mmol) in dichloromethane (0.5 mL) was added triisopropylsilane (0.015 mL, 0.072 mmol) followed by trifluoroacetic acid (0.5 mL) at ambient temperature. The mixture was stirred for 15 h. The volatiles were evaporated off. The residue was azeotroped with toluene (three times), dissolved in water and washed with dichloromethane. The aqueous solution was lyophilized to give a colorless amorphous solid (8.6 mg). HPLC (C18, acetonitrile-water containing 0.1% trifluoroacetic acid, a linear gradient of acetonitrile from 20 to 60% over 20 min) afforded the purified product (Rt = 16.60 min) as a colorless amorphous solid (2.6 mg, 27%). H-NMR (500MHz, D2O):  0.87 (3H, t, J = 6.6 Hz), 1.29-1.77 (31H, m), 2.09 (1H, m), 2.26 (2H, t, J = 7.2

1

Hz), 2.32 (2H, t, J = 7.4 Hz), 2.78 (1H, d, J = 13.2 Hz), 3.00 (1H, dd, J = 5.2 and 13.2 Hz), 3.13-3.22 (2H, m), 3.30-3.45 (2H, m), 3.46 (1H, m), 3.52-3.74 (12H, m), 3.77 (1H, m), 3.84 (1H, m), 4.02 (1H, d, J = 16.1 Hz), 4.07 (1H, m), 4.11 (1H, d, J = 16.0 Hz), 4.18 (1H, m), 4.22 (1H, m), 4.32-4.35 (2H, m), 4.44 (1H, dd, J = 4.5 and 8.2 Hz), 4.45-4.53 (3H, m), 4.61-4.78 (3H, m), 5.04 (1H, m), 5.12 (1H, m), 5.63 (1H, d, J = 3.4 Hz), 7.86 (1H, s) MS (ESI) m/z: exact mass calcld. for C61H111N23O17S: 1469.82, found 736.23 [M + 2H]2+, 747.21 [M + H + Na]2+, 1470.89 [M + H]+ , High resolution mass found 735.9195 [M + 2H]2+, calcd. 735.9197

10

Compound 6 To a solution of compound 12 (12.3 mg, 0.00492 mmol) in dichloromethane (0.5 mL) was added triisopropylsilane (0.015 mL, 0.074 mmol) followed by trifluoroacetic acid (0.5 mL) at ambient temperature. The mixture was stirred for 15 h. The volatiles were evaporated off. The residue was azeotroped with toluene (three times), dissolved in water and washed with dichloromethane. The aqueous solution was lyophilized to give a colorless amorphous solid (9.6 mg). HPLC (C18, acetonitrile-water containing 0.1% trifluoroacetic acid, a linear gradient of acetonitrile from 30 to 80% over 20 min) afforded the purified product (Rt = 12.87 min) as a colorless amorphous solid (2.3 mg, 23%). H-NMR (500MHz, D2O):  0.82 (3H, t, J = 6.9 Hz), 1.24–1.71 (37H, m), 2.04 (1H, m), 2.21 (2H, t, J = 7.2

1

Hz), 2.28 (2H, t, J = 7.5 Hz), 2.72 (1H, d, J = 12.6 Hz), 2.95 (1H, dd, J = 5.2 and 13.2 Hz), 3.11-3.14 (2H, m), 3.24-3.30 (2H, m), 3.30 (1H, m), 3.46-3.69 (12H, m), 3.72 (1H, m), 3.80 (1H, m), 3.97 (1H, d, J = 16.0 Hz), 4.01 (1H, m), 4.07 (1H, d, J = 16.0 Hz), 4.13 (1H, m), 4.17 (1H, m), 4.26-4.29 (2H, m), 4.39 (1H, m), 4.41-4.49 (1H, m), 4.58 (1H, dd, J = 4.6 and 8.1 Hz), 4.62-4.85 (3H, m), 4.98 (1H, m), 5.08 (1H, m), 5.58 (1H, d, J = 4.0 Hz), 7.82 (1H, s) MS (ESI) m/z: exact mass calcd. for C63H115N23O17S: 1497.86, found 750.21 [M + 2H]2+, 1498.81 [M + H]+, 1520.80 [M + Na]+ , High resolution mass found 749.9353 [M + 2H]2+, calcd. 749.9354 Compound 7 To a solution of compound 13 (16.1 mg, 0.00630 mmol) in dichloromethane (0.5 mL) was added triisopropylsilane (0.019 mL, 0.094 mmol) followed by trifluoroacetic acid (0.5 mL) at ambient temperature. The mixture was stirred for 15 h. The volatiles were evaporated off. The residue was azeotroped with toluene (three times), dissolved in water and washed with dichloromethane. The aqueous solution was lyophilized to give a colorless amorphous solid (13.8 mg). HPLC (C18, acetonitrile-water containing 0.1% trifluoroacetic acid, a linear gradient of acetonitrile from 50 to 95% over 20 min) afforded the purified product (Rt = 16.00 min) as a colorless amorphous solid (3.6 mg, 27%). H-NMR (500MHz, D2O):  0.84 (3H, t, J = 6.9 Hz), 1.25-1.75 (45H, m), 2.06 (1H, m), 2.23 (2H, t, J = 7.2

1

Hz), 2.30 (2H, t, J = 7.5 Hz), 2.75 (1H, d, J = 13.2 Hz), 2.97 (1H, dd, J = 5.2 and 13.2 Hz), 3.13-3.16 (2H, m), 3.24-3.32 (2H, m), 3.48-3.70 (12H, m), 3.73 (1H, m), 3.82 (1H, m), 3.99 (1H, d, J = 16.1 Hz), 4.03 (1H, m), 4.09 (1H, d, J = 16.1 Hz), 4.15 (1H, m), 4.19 (1H, m), 4.28-4.30 (2H, m), 4.40 (1H, dd, J = 4.6 and 8.1 Hz), 4.42-4.52 (3H, m), 4.59 (1H, m), 4.64-4.76 (3H, m), 4.99 (1H, m), 5.10 (1H, m), 5.59 (1H, d, J = 3.4 Hz), 7.83 (1H, s) MS (ESI) m/z: exact mass calcd. for C67H123N23O17S: 1553.92, found 778.31 [M + 2H]2+, 1554.92 [M + H]+ , High resolution mass found 777.9668 [M + 2H]2+, calcd. 777.9667

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HPLC chromatograms for compounds 2-7 Eluent: MeCN-H2O containing 0.1% TFA. Detection: 220 nm. Compound 2 (a linear gradient of MeCN from 0 to 50% over 20 min)

Compound 3 (a linear gradient of MeCN from 0 to 50% over 20 min)

Compound 4 (a linear gradient of MeCN from 0 to 100% over 20 min)

Compound 5 (a linear gradient of MeCN from 0 to 100% over 20 min)

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Compound 6 (a linear gradient of MeCN from 0 to 100% over 20 min)

Compound 7 (a linear gradient of MeCN from 20 to 95% over 20 min)

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Supporting Figures

Figure S1. COSY spectrum for compound 2.

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Figure S2. TOCSY spectrum for compound 2.

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Figure S3. HSQC spectrum for compound 2.

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Figure S4. HMBC spectrum for compound 2.

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Figure S5. Expanded HMBC spectrum for compound 2. Shown are 2’, 2’’’, 6’, 6’’’, and 1-H (from top to bottom), which are coupled to guanidinium carbons.

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Figure S6. 13C-NMR spectrum for compound 2. MeCN was added as a reference.

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Figure S7. Intermolecular FRET result using ST-PE and ST-Cy5. Cells were incubated at 4°C for 30 min with preformed conjugates of carrier and ST-PE/ST-Cy5 mixture. Carrier:ST-PE:ST-Cy5 was set as 10:1:1 molar ratio. Cells were detached by Versene and analyzed by FACS. Y axis (FL2-H) and X axis (FL3-H) represent fluorescence intensity of PE (em = 560 nm) and PE/Cy5 FRET complex (em = 670 nm), respectively. ST-PE (ST-Cy5) concentration was varied as 1 nM A), 3 nM B). Compound 1 (blue), compound 2 (green), compound 3 (tangerine), compound 4 (cyan), compound 5 (magenta) and cells (red)

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Supporting Table S1 Streptavidin (ST) (0.3 nmol) and the molecular transporters (1.5 nmol) were mixed in a total volume of 100L and incubated for 20 minutes at room temperature. The final concentrations were 3M and 15M respectively in phosphate buffered saline (pH 7.4). All the solutions were filtered through 0.22 m filters before mixing and incubating. DLS was measured at 25 °C.

Conjugate

Average diameter [nm]

STDV

ST

4.13

0.19

ST-1

5.52

0.26

ST-2

6.32

0.18

ST-3

5.52

0.11

ST-4

4.34

0.92

ST-5

3.36

0.45

Table S1. Diameter of Streptavidin/molecular transporter conjugates. Diameter of Streptavidin (ST) and Streptavidin incubated with compound 1 (ST-1), compound 2 (ST-2), compound 3 (ST-3), compound 4 (ST-4) and compound 5 (ST-5).

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References [1] Childs-Disney, J. L., Wu. M., Pushechnikov, A., Aminova, O. and Disney, M. D., ACS Chem. Biol. 2007, 2(11), 745–754

[2] Inoue, M., Tong, W. Esko, J.D., and Tor, Y. ACS Chem. Biol. 2013, 8, 1383–1388.

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