S1
SUPPORTING INFORMATION
Opening Enediyne Scissors Wider: pH-Dependent DNA Photocleavage by meta-Diyne Lysine Conjugates
Kemal Kaya, Madeleine Johnson and Igor V Alabugin
Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306-4390, USA.
alabugin@chem.fsu.edu
TABLE OF CONTENTS
Figure S1………....................................................………………………………..S2
Figure S2…….........................................................………………………………..S2
Experimental Detalis…………..……….………………………………………..S3-S6
NMR Spectra………………………….………………………………………..S7-S24
S2
1.2E+08
1.0E+08
No DNA
8.0E+07
2.7 microM DNA
6.0E+07
5.4 microM DNA
4.0E+07
10.8 microM
DNA
2.0E+07
0.0E+00
320
340
360
380
400
420
440
Figure S1. Fluorescence titration spectra of the compound 1 (15 µM) with ct DNA (0-10.8 µM/b.p) at pH 7 (excitation at 307nm).
1.2E+08
1.0E+08
No DNA
8.0E+07
2.7 microM
DNA
5.4 microM
DNA
10.8 microM
DNA
6.0E+07
4.0E+07
2.0E+07
0.0E+00
320
340
360
380
400
420
Figure S2. Fluorescence titration spectra of the compound 1 (15 µM) with ct DNA (0-10.8 µM/b.p) at pH 8 (excitation at 307nm).
S3
Synthesis of compounds.
((5-nitro-1,3-phenylene)bis(ethyne-2,1-diyl))dibenzene
A mixture of 1,3-dibromo-5-nitrobenzene (0.56 g, 2.00 mmol), bis(triphenylphosphine)palladium(II) chloride (0.1 g, 0.14 mmol) and
copper(I) iodide (0.027 g, 0.14 mmol) in Et3N was degassed (three times). Phenylacetylene (0.50 g, 4.89 mmol)
was added and mixture was stirred overnight. The reaction mixture was filtered through a celite pad and the pad
was rinsed with CH2Cl2.The filtrate was washed with brine and the organic layer was dried with (Na2SO4), filtered,
and concentrated. The residue was purified by column chromatography (Dichloromethane;Hexane= 1:10) to
afford ((5-nitro-1,3-phenylene)bis(ethyne-2,1-diyl))dibenzene. Solvent was evaporated to afford 0.52 gr (80 %) of the product: 1H NMR
(400 MHz, CDCl3): δ 8.30 (d, J= 1.4 Hz 2H), 7.97 (t, J= 1.4 Hz 1H), 7.57-7.54 (m, 4H), 7.40 (d, J=2.2 Hz, 4H), 7.39 (d, J=1.5 Hz, 2H); 13C NMR
(100 MHz, CDCl3): δ 148.1, 139.6, 131.9, 129.3, 128.6, 125.5, 125.4, 122.0, 92.6, 86.4.
3,5-bis(phenylethynyl)aniline
A solution of SnCl2 (0.96 g, 5.10 mmol) was added slowly (1 h) to the mixture of ((5-nitro-1,3-phenylene)bis(ethyne-2,1-diyl))dibenzene
(0.33 g, 1.02 mmol) and HCl (1 mL) in THF (10 ml). The reaction mixture was stirred at room temperatur for 3
hours. After neutralization with NaOH (1.0 N solution), the product was extracted with EtOAc and dried over
(Na2SO4). Solvent was evaporated to afford 0.26 g (87 %) of the product: 1H NMR (400 MHz, CDCl3): δ 7.55-7.52
(m, 4H), 7.37-7.34 (m, 6H), 7.16 (s, 1H), 6.83 (d, J= 1.0 Hz, 2H), 3.73 (bs, 2H); 13C NMR (100 MHz, CDCl3): δ 146.4,
131.6, 128.4, 125.1, 124.2, 123.1, 117.8, 89.4, 89.0; HRMS (ESI+): calcd for C22H15N1H [M + H] 294.12827, found
294.12858
(S)-di-tert-butyl (6-((3,5-bis(phenylethynyl)phenyl)amino)-6-oxohexane-1,5-diyl)dicarbamate
HOBt (0.22 g, 1.63 mmol) was added to the solution of L-Boc-Lys(Boc)-OH (0.56 g, 1.62 mmol) and EDCI (0.31 g, 1.61 mmol) in
dichloromethane at 00C, then 3,5-bis(phenylethynyl)aniline (0.24 g 0.81 mmol) was added at 00C. The reaction
mixture was stirred for 30 min at 00C and then at room temperature for 18 h. The reaction mixture was quenched
with water and extracted with EtOAc and washed with saturated NaHSO4, then dried with Na2SO4. The solvent
was evaporated in vacuo. The crude product was subjected to chromatography with (EtOAc:Hexane= 2:5).
Desired product was obtained (0.35 g, 70%): 1H NMR (600 MHz, CDCl3): δ 8.87 (bs, 1H), 7.72 (s, 2H), 7.49 (d, J =
3.6 Hz, 4H), 7.41 (s, 1H), 7.33-7.32 (m, 6H), 5.36 (bs, 1H), 4.70 (bs, 1H), 4.27 (s, 1H), 3.18 (q, J=2.5 Hz, 1H), 3.09 (dd, J=12.8 Hz J=6.1 Hz, 1H),
1.97 – 1.92 (m, 1H), 1.73 – 1.69 (m, 1H), 1.55 – 1.51 (m, 2H), 1.47 (s, 11H), 1.44 (s, 9H); 13C NMR (150 MHz, CD3OD): δ 171.0, 156.5, 138.2,
131.8, 130.5, 128.5, 128.4, 124.3, 123.1, 122.5, 90.1, 88.5, 80.7, 79.4, 55.2, 39.7, 31.5, 29.6, 28.6, 28.5, 22.7; HRMS (ESI): calcd for C38H43N3O5Na
[M + Na]+ 644.31004, found 644.30891.
S4
(S)-2,6-diamino-N-(3,5-bis(phenylethynyl)phenyl)hexanamide bis(2,2,2-trifluoroacetate)
(S)-di-tert-butyl (6-((3,5-bis(phenylethynyl)phenyl)amino)-6-oxohexane-1,5-diyl)dicarbamate (0.35 g, 0.56 mmol) was reacted with
neat TFA:CH2Cl2 (1:1) 10mL and the solution was stirred for 5 hours at room temperature. TFA: CH2Cl2
mixture was evaporated and the product (0.34 mg, 93 %) was purified by recrystallization with ethanol: 1H
NMR (400 MHz, CD3OD): δ 7.80 (d, J= 1.4 Hz, 2H), 7.55-7.52 (m, 4H), 7.45 (t, J=1.4 Hz, 1H) 7.41-7.39 (m, 6H),
4.06 (t, J= 6.5 Hz, 1H), 2.96 (t, J=7.7Hz, 2H), 2.07-1.96 (m, 2H), 1.78-1.70 (m, 2H), 1.60-1.50 (m, 2H); 13C NMR (100
MHz, CD3OD): δ 168.7, 163.1 (d, J=34 Hz), 139.6, 132.6, 131.3, 129.9, 129.6, 125.7, 124.0, 123.6, 91.6, 88.7, 54.9,
40.2, 32.1, 28.2, 22.9; HRMS (ESI+): calcd for C28H27N3OH [M + H] 422.22324, found 422.22172.
((5-nitro-1,3-phenylene)bis(ethyne-2,1-diyl))bis(trimethylsilane)
A mixture of 1,3-dibromo-5-nitrobenzene (1.35 g, 4.80 mmol), bis(triphenylphosphine)palladium(II) chloride (0.50 g, 0.71 mmol) and
copper(I) iodide (0.14 g, 0.73 mmol) in Et3N was degassed by freeze/pump/thaw technique (three times).
Trimethylsilylacetylene (1.03 g, 10.08 mmol) was added and mixture was stirred 14 hours. The reaction mixture
was filtered through a celite pad and the pad was rinsed with CH2Cl2. The filtrate was washed with sat. NH4Cl(aq.)
and brine and the organic layer was dried (Na2SO4), filtered, and concentrated. The residue was purified by column
chromatography (EtOAc;Hexane= 1:15) to afford ((5-nitro-1,3-phenylene)bis(ethyne-2,1-diyl))bis(trimethylsilane) (1.01 g, 67% yield):
H NMR (400 MHz, CDCl3): δ 8.20 (d, J=1.0 Hz, 2H), 7.82 (t, J= 1.5 Hz, 1H), 0.26 (s, 18H); 13C NMR (100 MHz, CDCl3): δ 148.0, 140.5, 126.2,
125.2, 101.5, 98.4, -0.2.
1
4,4'-((5-nitro-1,3-phenylene)bis(ethyne-2,1-diyl))bis(2,3,5,6-tetrafluoropyridine)
Pentafluoropyridine (1.64 g, 9.64 mmol) was added to the mixture of ((5-nitro-1,3-phenylene)bis(ethyne-2,1-diyl))bis(trimethylsilane)
(1.01 g, 3.21 mmol) and CsF (1.46 g, 9.61 mmol) in DMF (10 mL) slowly. The reaction mixture was stirred
overnight. Brine (30 mL) and EtOAc (50 mL) were added. Organic phase was separated and washed with
water. Solvent was evaporated by rotary evaporation and the residue was chromatographed (EtOAc:Hexane
= 1:10) to provide the desired product in 35 % yield (0.53 gr): 1H NMR (600 MHz, CDCl3): δ 8.54 (d, J=1.2 Hz,
2H), 8.16 (t, J=1.3 Hz, 1H); 13C NMR (150 MHz, CDCl3): δ 148.5, 143.8 (dm, J=239 Hz), 142.1 (dm, J=286 Hz),
140.6, 128.4, 123.4, 115.9 (t, J=16 Hz), 101.3, 76.7 (t, J=4 Hz).
S5
3,5-bis((perfluoropyridin-4-yl)ethynyl)aniline
SnCl2 (0.40 g, 2.13 mmol) was added slowly (1 h) to the solution of 4,4’-((5-nitro-1,3-phenylene)bis(ethylene-2,1-diyl))bis(2,3,5,6tetrafluoropyridine (0.20 g, 0.42 mmol) and HCl (1 mL) in THF (10 ml). The reaction mixture was stirred for
3 hours. The reaction mixture was basified till pH>9 with NaOH (1.0 N solution), the product was extracted
with EtOAc and dried over (Na2SO4). Solvent was evaporated to afford 0.17 g (95 %) of the product: 1H NMR
(600 MHz, CDCl3): δ 7.27 (s, 1H), 7.00 (d, J=1.0 Hz 2H), 3.95 (s, 2H); (150 MHz, CDCl3): δ 147.0, 143.6 (dm, J =262
Hz), 141.9 (dm, J=253 Hz), 126.0, 122.2, 120.1, 117.1 (t, J=8 Hz), 105.2 (t, J=2 Hz), 73.8 (t, J=2 Hz); HRMS (ESI+):
calcd for C20H5F8N3H [M + H] 440.04340, found 440.04462.
(S)-di-tert-butyl (6-((3,5-bis((perfluoropyridin-4-yl)ethynyl)phenyl)amino)-6-oxohexane-1,5-diyl)dicarbamate
L-Boc-Lys(Boc)-OH (0.16 g, 0.46 mmol) was dissolved in 1 ml of pyridine. The solution was cooled to –25 oC and phosphorus
oxychloride (0.07 g, 0.46 mmol) was added dropwise with vigorous stirring. After stirring for 15 min. at –25
o
C, 3,5-bis((perfluoropyridin-4-yl)ethynyl)aniline (0.10 g, 0.23 mmol) in pyridine (1 ml) was added slowly.
The reaction mixture was stirred for 0.5h at –25 oC and then at room temperature for 12 h. The reaction mixture
was quenched with ice/water and extracted with EtOAc. The organic layer was washed with sat. NaHSO4 three
times, dried with Na2SO4 and was concentrated in vacuo. The crude product was subjected to chromatography
with (EtOAc:Hexane= 1:5) and the product was obtained in 22 % yield: 1H NMR (600 MHz, CDCl3): δ 9.50 (s,
1H), 7.80 (s, 2H), 7.43 (s, 1H), 5.48 (s, 1H), 4.68 (s, 1H), 4.31 (s, 1H), 3.16 (bs, 1H), 3.10 (bs, 1H), 1.90 (bs, 1H), 1.74 (bs, 1H), 1.50 (s, 11H), 1.44
(s, 11H); 13C NMR (150 MHz, CDCl3): δ 171.7, 157.0, 156.6, 144.4 (dm, J=227 Hz), 142.8 (dm, J=242 Hz), 139.3, 130.8, 124.5, 121.8, 116.8 (t,
J=16 Hz), 104.4, 81.2, 79.6, 74.2, 55.5, 39.5, 31.4, 29.8, 28.6, 28.5, 22.7; HRMS (ESI): calcd for C36H33F8N5O5Na [M + Na]+ 790.22516, found
790.22536.
(S)-2,6-diamino-N-(3,5-bis((perfluoropyridin-4-yl)ethynyl)phenyl)hexanamide bis(2,2,2-trifluoroacetate)
(S)-di-tert-butyl
(6-((3,5-bis((perfluoropyridin-4-yl)ethynyl)phenyl)amino)-6-oxohexane-1,5diyl)dicarbamate (0.02 g, 0.26 mmol ) was reacted with TFA:CH2Cl2 (1:1) (2 mL) and the solution was
stirred for 4 hours at room temperature. TFA: CH2Cl2 was evaporated and the product (20 mg, 96 %) was
purified by recrystallization with ethanol: 1H NMR (400 MHz, MeOD): δ 8.08 (s, 2H), 7.66, (s, 1H), 4.11 (t, J=
6.1 Hz, 1H), 2.96 (t, J=7.7 Hz, 2H), 2.12-1.95 (m, 2H), 1.79-1.71 (m, 2H), 1.61-1.56 (m, 2H); 13C NMR (100 MHz,
MeOD): δ 169.1, 163.2, 146.0 (dm, J=241 Hz), 142.1 (dm, J=263Hz), 140.5, 132.0, 125.8, 123.4, 117.4 (t, J=16
Hz), 104.5, (t, J=3 Hz), 75.2 (t, J=4 Hz), 55.0, 40.2, 32.1, 28.2, 22.9; HRMS (ESI+): calcd for C26H17F8N5OH [M +
H] 568.13836, found 568.13709.
S6
((5-nitro-1,3-phenylene)bis(ethyne-2,1-diyl))dibenzene
S7
((5-nitro-1,3-phenylene)bis(ethyne-2,1-diyl))dibenzene
S8
3,5-bis(phenylethynyl)aniline
S9
3,5-bis(phenylethynyl)aniline
S10
(S)-di-tert-butyl (6-((3,5-bis(phenylethynyl)phenyl)amino)-6-oxohexane-1,5-diyl)dicarbamate
S11
(S)-di-tert-butyl (6-((3,5-bis(phenylethynyl)phenyl)amino)-6-oxohexane-1,5-diyl)dicarbamate
S12
(S)-2,6-diamino-N-(3,5-bis(phenylethynyl)phenyl)hexanamide bis(2,2,2-trifluoroacetate)
S13
(S)-2,6-diamino-N-(3,5-bis(phenylethynyl)phenyl)hexanamide bis(2,2,2-trifluoroacetate)
S14
((5-nitro-1,3-phenylene)bis(ethyne-2,1-diyl))bis(trimethylsilane)
S15
((5-nitro-1,3-phenylene)bis(ethyne-2,1-diyl))bis(trimethylsilane)
S16
4,4'-((5-nitro-1,3-phenylene)bis(ethyne-2,1-diyl))bis(2,3,5,6-tetrafluoropyridine)
S17
4,4'-((5-nitro-1,3-phenylene)bis(ethyne-2,1-diyl))bis(2,3,5,6-tetrafluoropyridine)
S18
3,5-bis((perfluoropyridin-4-yl)ethynyl)aniline
S19
3,5-bis((perfluoropyridin-4-yl)ethynyl)aniline
S20
.(S)-di-tert-butyl (6-((3,5-bis((perfluoropyridin-4-yl)ethynyl)phenyl)amino)-6-oxohexane-1,5-diyl)dicarbamate
S21
(S)-di-tert-butyl (6-((3,5-bis((perfluoropyridin-4-yl)ethynyl)phenyl)amino)-6-oxohexane-1,5-diyl)dicarbamate
S22
(S)-2,6-diamino-N-(3,5-bis((perfluoropyridin-4-yl)ethynyl)phenyl)hexanamide bis(2,2,2-trifluoroacetate)
S23
(S)-2,6-diamino-N-(3,5-bis((perfluoropyridin-4-yl)ethynyl)phenyl)hexanamide bis(2,2,2-trifluoroacetate)