Supporting Information
Chemicals and materials. Fmoc-amino acids were obtained from GL Biochem. (Shanghai). All the other
starting materials were obtained from Alfa (Beijing). Commercially available reagents and solvents were
used without further purification, unless noted otherwise.
General methods: The synthesized compounds were characterized by 1H NMR (Bruker ARX-300) using
DMSO-d6 as the solvent. HPLC was conducted at LUMTECH HPLC (Germany) system using a C 18 RP
column with MeOH (0.05% of TFA) and water (0.05% of TFA) as the eluents. LC-MS was conducted at
the LCMS-20AD (Shimadzu) system.
Synthesis of β-alanine conjugated NBD-Cl (Scheme S-1, also see Ref. 17): To a solution of β-alanine
(98
mg,
1.1
mmol)
and
K2CO3
in
MeOH
and
H2 O
under
N2 ,
5
mL
of
4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (200 mg, 1 mmol) solution was injected slowly. The reaction
was completed 5 hours later at room temperature. The reaction mixture was concentrated by vacuum to
remove the MeOH, then the pH of the resulted solution was adjusted to 1~2 by HCl (2 mol/L). The
aqueous mixture was extracted with diethyl ether (50 mL * 2) and dichloromethane (50 mL * 2), the
organic layer was combined and concentrated to give crude 200 mg, which was directly reacted for next
step without purification.
O
O N
N O
N
O
+
Cl
H2N
K2CO3
OH
O
O N
N O
N
MeOH/H2O
O
N
H
OH
NBD--Alanine
Scheme S1. The synthetic route for NBD--Alanine
Peptide synthesis: The peptide derivative was prepared by solid phase peptide synthesis (SPPS) using
2-chlorotrityl chloride resin and the corresponding N-Fmoc protected amino acids with side chains
properly protected by a tert-butyl group . 20% piperidine in anhydrous N,N’-dimethylformamide (DMF)
was used during deprotection of Fmoc group. Then the next Fmoc-protected amino acid was coupled to
the free amino group using O-(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluroniumhexafluorophosphate
(HBTU) as the coupling reagent. The growth of the peptide chain was according to the established Fmoc
SPPS protocol. In the last coupling step, NBD--Alanine was used to produce NBD-peptides. After the
last coupling step, excessive reagents were removed by a single DMF wash for 5 minutes (5 mL per gram
1
of resin), followed by five steps of washing using DCM for 2 min (5 mL per gram of resin). The peptide
derivative was cleaved using 95% of trifluoroacetic acid with 2.5% of trimethylsilane (TMS) and 2.5% of
H2O for 30 minutes. 20 mL per gram of resin of ice-cold diethylether was then added to cleavage reagent.
The resulting precipitate was centrifuged for 10 min at 4 0C at 10,000 rpm. Afterward the supernatant was
decanted and the resulting solid was dissolved in DMSO for HPLC separation using MeOH and H2O
containing 0.05% of TFA as eluents.
O
O N
N O
N
N
H
COOH
O
N
H
H
N
O
O
N
H
H
N
O
O
N
H
OH
H
N
O
O
N
H
H
N
O
OH
O
OH
Compound 1: NBD-FFETIGGY
Scheme S2. Chemical structure of compound 1
NBD-FFETIGGY (1): 1H NMR (300 MHz, DMSO-d6) δ 9.25 (d, J = 42.4 Hz, 2H), 8.49 (d, J = 8.6 Hz,
1H), 8.38 – 8.15 (m, 4H), 8.10 (d, J = 8.1 Hz, 1H), 7.98 (t, J = 5.8 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.77
(d, J = 8.3 Hz, 1H), 7.62 – 7.20 (m, 5H), 7.13 (dt, J = 28.1, 13.5 Hz, 5H), 7.00 (d, J = 8.5 Hz, 2H), 6.65 (d,
J = 8.5 Hz, 2H), 6.34 (d, J = 8.6 Hz, 1H), 4.54 (dd, J = 9.4, 4.0 Hz, 2H), 4.47 – 4.38 (m, 1H), 4.40 – 4.28
(m, 2H), 4.25 – 4.16 (m, 1H), 4.04 – 3.95 (m, 1H), 3.80 – 3.61 (m, 4H), 3.55 (d, J = 24.8 Hz, 3H), 3.18 (s,
1H), 3.05 (d, J = 10.2 Hz, 1H), 3.00 – 2.89 (m, 2H), 2.86 – 2.73 (m, 2H), 2.71 – 2.63 (m, 1H), 2.54 (d, J =
9.6 Hz, 1H), 2.31 – 2.22 (m, 2H), 1.94 (s, 1H), 1.86 – 1.52 (m, 3H), 1.45 (s, 1H), 1.39 – 1.08 (m, 2H),
1.03 (d, J = 6.2 Hz, 3H), 0.83 (dd, J = 12.6, 7.1 Hz, 6H). HR-MS: calc. M+ = 1166.47, obsvd. (M-H)- =
1165.47.
Figure S1. 1H-NMR of NBD-FFETIGGY
2
Figure S2. MS spectrum of NBD-FFETIGGY
COOH
H
N
H2N
O
H
N
N
H
O
O
HO
O
H
N
N
H
O
O
N
H
H
N
O
OH
O
OH
Scheme S3. Chemical structure of compound 2
Inten.(x1,000,000)
933.60
1.25
1.00
0.75
934.60
0.50
0.25
936.65
0.00
932.0
933.0
934.0
935.0
936.0
938.70
937.0
938.0
939.0
940.0
m/z
Figure S3. MS spectrum of FFETIGGY
Critical micelle concentration (CMC): The CMC values of peptides in water (pH=7.4) were
measured by dynamic light scattering (DLS). The peptides with high concentration were diluted
successively and the light scattering intensities were recorded for each sample. Dynamic Light
Scattering (DLS) was performed on a laser light scattering spectrometer (BI-200SM) equipped with a
digital correlator (BI-9000AT) at 532nm under room temperature (22-25 0C).
3
Figure S4. CMC of NBD-FFETIGGY and FFETIGGY
Transmission electron
microscopy (TEM): TEM sample (0.2mg/mL NBD-FFETIGGY) was
prepared at 25 °C. A micropipet was used to load 5 µL of sample solution to a carbon coated copper grid.
The excess solution was removed by a piece of filter paper. The sample was dried overnight in a desicator
and then conducted on a Tecnai G2 F20 system, operating at 200 kV.
Figure S5. A TEM image of Tris-HCl solution of 1 (0.02 wt%)
4
Figure S6. Emission spectra of Tris-HCl solution of 1 (0.1 wt%, pH = 7.4) in the absence and presence of different metal ions
(150 mM of KCl, CaCl2, NaCl and MgSO4; the concentration of ZnCl2, FeCl2, and CuSO4 was 3 mM, 0.9 mM, and 3 mM,
respectively) (excitation wavelength = 470 nm)
Fluorescence microscopy to determine cellular distribution of nanofibers in different cells.
Fluorescence images were obtained by an inverted fluorescence microscope (Leica DMI 3000 B). The
DMEM solution containing 0.2 mg/mL of 1 was added to the NIH 3T3, HepG2, Cos7 and HeLa cells.
The images were recorded after 2 hours culture. (excitation wavelength = 478-498 nm).
For imaging of living plants with laser scanning confocal microscopy, seven-day-old Arabidopsis
thaliana seedlings were incubated in 500 μM of 1 for 1 hour, and then washed three times with distilled
water before scanning. The root hair zone of the plants was scanned. For imaging of leaf epidermal cells,
four-week-old Nicotianabenthamiana leaves were incubated in 500 μM of 1 for 1 hour, and then washed
three times with distilled water before scanning.
Figure S7. Tandem MS analysis indicates the most abundant hydrogel bound cytosolic proteins by sequence coverage
for band I in Figure 3
5
Figure S8. Tandem MS analysis indicates the most abundant hydrogel bound cytosolic proteins by sequence coverage
for band II in Figure 3
Determination of IC50 values on different cells: Four cells of 3T3, HepG2, Cos7 and Hela were seeded
in a 96-well plate with the density of 1,0000 cells per-well (total medium volume of 100 μL ). 24 hours
post seeding, the solutions with a serial of concentrations (8 concentrations) of the compound in 100 μL
of medium were added to each well (five wells for each concentration). Cells without the treatment of the
compound were used as the control. The MTT assays were performed after an extra culture time of 48
hours. All compounds were removed and 90 μL fresh medium was added for each well, 10 μL of MTT
solution (5 mg/mL) was added and incubated for 4 hours in 370C. Pipette out the spent media, formazon
crystals at the bottom of each well were dissolved in 100 μL DMSO. After 15 minutes at room
temperature, absorbance at wavelength of 490 nm was tested using a microplate reader (BIO-RAD, iMark
TM ). IC 50 values for the inhibition of cell viability were calculated from pharmacological inhibitory
response curves using software Prism 5.0.
Figure S9. Congress curve of cell inhibition assay of 3T3 cell treated with different concentrations of 1
6
Figure S10. Congress curve of cell inhibition assay of HepG2 cell treated with different concentrations of 1
Figure S11. Congress curve of cell inhibition assay of Cos7 cell treated with different concentrations of 1
Figure S12. Congress curve of cell inhibition assay of Hela cell treated with different concentrations of 1
7
Figure S13. Bright field optical images of HepG2 cells left) without and right) with the treatment of nanofibers of 1 (0.25
mg/mL) for 48h
Figure S14. Bright field optical images of Cos7 cells left) without and right) with the treatment of nanofibers of 1 (0.25
mg/mL) for 48h
Figure S15. Bright field optical images of HeLa cells left) without and right) with the treatment of nanofibers of 1 (0.25
mg/mL) for 48h
8