Supplementary Information
Discovery of a Structural-Element Specific G-Quadruplex
“Light-Up” Probe
Liyun Zhang†, Jun Cheng Er†, Krishna Kanta Ghosh, Wan Jun Chung, Jaeduk Yoo,
Wang Xu, Wei Zhao, Anh Tuân Phan and Young-Tae Chang*
Department of Chemistry, National University of Singapore, 3 Science Drive 2,
Singapore 117543, Singapore
Table of contents
A. Experimental ·························································································· 2
B. Supporting Figures and Tables···································································· 10
C. NMR Spectra························································································ 21
D. References··························································································· 35
S1
A. Experimental
Materials
All commercially available reagents, solvents and oligonucleotides (dsDNA, ssDNA and
RNA) were purchased from Sigma Aldrich, Alfa Aesar, Fluka, Merck or Acros, and used
as received unless otherwise stated. CH2Cl2 (Fisher Scientific, analytical grade) was
freshly distilled from P2O5 under nitrogen. Anhydrous THF was purchased from Alfa
Aesar and used without further purification.
Synthesis
N N
B
F F
O
O
6
ethyl
3-(5,5-difluoro-7,9-dimethyl-5H-54,64-dipyrrolo[1,2-c:2',1'-f][1,3,2]diaza
borinin-3-yl)propanoate (6). Ethyl 3-(1H-pyrrol-2-yl)propanoate was synthesized
following the procedure described in reference [2].1, 2 The colorless oil (0.65 g, 3.9 mmol,
1 eq.) was dissolved in DCM (40 mL) and cooled to 0oC. POCl3 (0.39 mL, 4.2 mmol, 1.1
eq.) in DCM was added dropwise and the reaction mixture was stirred at 0oC for 30 min
and RT for an additional 6 h. Upon reaction completion, the reaction mixture was cooled
to 0oC. DIEA (2.57 mL, 14.7 mmol, 3.8 eq.) and BF3.OEt2 (2.04 mL, 16.6 mmol, 4.3 eq.)
S2
was subsequently added and the reaction mixture was stirred at RT for 12 h. The crude
mixture was purified by silica gel column chromatography (hexane/EA) to afford 6 as a
red solid (436 mg, 1.36 mmol, 35% yield). 1H NMR (500 MHz, DMSO-d6) δ 7.70 (s,
1H), 7.08 (d, J = 4.0 Hz, 1H), 6.37 (d, J = 4.0 Hz, 1H), 6.31 (s, 1H), 4.09 (q, J = 7.1 Hz,
2H), 3.11 (t, J = 7.7 Hz, 2H), 2.72 (t, J = 7.7 Hz, 2H), 2.47 (s, 3H), 2.26 (s, 3H), 1.19 (t, J
= 7.1 Hz, 3H);
13
C NMR (500MHz, DMSO-d6) δ 171.8, 159.7, 156.4, 144.5, 134.7,
133.0, 128.7, 125.5, 120. 5, 116.5, 60.1, 32.3, 23.4, 14.5, 14.1, 11.0; HRMS
(C16H19BF2N2NaO2): calc. [M + Na]+: 343.1403, found [M + Na]+: 343.1414.
N N
B
F F
HO
O
7
3-(5,5-difluoro-7,9-dimethyl-5H-54,64-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-3yl)propanoic acid (7). To a solution of 6 (1.5 g, 4.9 mmol) in THF/H2O (3:2) (200 mL)
was added 37 % HCl (8 mL) and refluxed for 6 h. The reaction mixture was cooled to RT
diluted with water. THF was removed in vacuo and the resulting residue extracted with
DCM (3 x 200 mL). The organic layers were collected, dried over anhydrous Na2SO4 and
evaporated in vacuo. The crude product was purified with flash column chromatography
on silica gel (hexane/EA) to afford 7 as a deep red solid (570 mg, 1.95 mmol, 40% yield);
S3
1
H NMR (500 MHz, DMSO-d6) δ 12.29 (br. s, 1H), 7.69 (s, 1H), 7.09 (d, J = 4.0 Hz,
1H), 6.38 (d, J = 4.0 Hz, 1H), 6.30 (s, 1H), 3.08 (t, J = 7.7 Hz, 2H), 2.64 (t, J = 7.7 Hz,
2H), 2.47 (s, 3H), 2.26 (s, 3H);
C-NMR (500MHz, DMSO-d6) δ 173.4, 159.5, 156.9,
13
144.3, 134.6, 133.0, 128.8, 125.4, 120.4, 116.5, 32.3, 23.5, 14.5, 11.0; HRMS
(C14H15BF2N2NaO2): calc. [M + Na]+: 315.1089, found [M + Na]+: 315.1081.
(E)-3-(5,5-difluoro-7-(4-methoxystyryl)-9-methyl-5H-54,64-dipyrrolo[1,2-c:2',1'f][1,3,2]diazaborinin-3-yl)propanoic acid (4). To a solution of 7 (500 mg, 1.9 mmol, 1
eq.) in 10 mL ACN, 4-methoxybenzaldehyde (1.03 g, 7.5 mmol, 4 eq.) and pyrrolidine
(541 mg, 7.5 mmol, 4 eq.) and acetic acid (452 mg, 7.5 mmol, 4 eq.) were added and
heated at 80oC for 15 min. The resulting solution was cooled to RT and ACN was
evaporated in vacuo. The residue was purified with flash column chromatography on
silica gel (DCM/MeOH) to afford 4 as a deep purple solid (231 mg, 0.56 mmol, 30%
yield); 1H NMR (500 MHz, DMSO-d6) δ 12.31 (br. s, 1H), 7.66 (d, J = 16.5 Hz, 1H),
7.62 (s, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 16.4 Hz, 1H), 7.06 (d, J = 3.5 Hz, 1H),
7.04 (s, 1H), 7.03 (d, J = 7.7 Hz, 2H), 6.39 (d, J = 3.6 Hz, 1H), 6.39 (d, J = 3.6 Hz, 1H),
S4
3.81 (s, 3H), 3.13 (t, J = 7.6 Hz, 2H), 2.67 (t, J = 7.6 Hz, 2H), 2.30 (s, 3H);
13
C NMR
(125 MHz, DMSO-d6) δ 173.5, 160.8, 156.6, 156.0, 143.5, 139.2, 136.1, 133.3, 129.2,
128.4, 127.7, 123.2, 116.9, 116.3, 115.4, 114.8, 55.4, 32.4, 23.6, 11.1; HRMS
(C22H20BF2N2O3): calc. [M - H]-: 409.1544, found [M - H]-: 409.1527.
(E)-3-(5,5-difluoro-7-(4-methoxystyryl)-9-methyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',
1'-
f][1,3,2]diazaborinin-3-yl)-N-isopropyl-N-(isopropylcarbamoyl)propanamide
(1,
GQR) To a solution of 4 (10 mg, 24 μmol, 1 eq.) in 2 mL DMF, was added DIC (19 mg,
0.15 mmol, 6 eq.) and DMAP (0.3 mg, 2.4 μmol, 10 mol%) and stirred for 12 h at RT.
The reaction was quenched with water (20 mL) and extracted with DCM (3 x 20 mL),
dried over anhydrous Na2SO4 and evaporated in vacuo. The crude product was purified
by flash column chromatography on silica gel (DCM/MeOH) to afford GQR as a deep
purple solid (10 mg, 19.2 μmol, 80% yield); 1H NMR (500 MHz, DMSO-d6) δ 8.30 (d, J
= 7.7 Hz, 1H), 7.66 (d, J = 16.3 Hz, 1H), 7.62 (s, 1H), 7.58 (d, J = 8.8 Hz, 2H), 7.30 (d, J
= 16.3 Hz, 1H), 7.06 (d, J = 4.0 Hz, 1H), 7.04 (s, 1H), 7.03 (d, J = 8.7 Hz, 2H), 6.32 (d, J
= 4.0 Hz, 1H), 4.41 – 4.32 (m, 1H), 3.81 (s, 3H), 3.81 – 3.73 (m, 1H), 3.15 (t, J = 7.5 Hz,
S5
2H), 2.71 (t, J = 7.5 Hz, 2H), 2.30 (s, 3H), 1.17 (d, J = 6.8 Hz, 6H), 1.09 (d, J = 6.6 Hz,
6H); 13C NMR (125 MHz, DMSO-d6) δ 168.8, 160.8, 156.5, 156. 5, 153.4, 143.5, 139.1,
136.1, 133.3, 129.2, 129.0, 128.5, 127.8, 123.2, 116.9, 116.1, 115.4, 114.8, 113.2, 55.4,
45.3, 42.5, 32.8, 23.8, 21.7, 20.4, 11.1; HRMS (C29H35BF2N4O3Na): calc. [M + Na]+:
559.2668, found [M + Na]+: 559.2692.
N N
B
F F
N
O
O
2
(E)-3-(5,5-difluoro-7-(4-methoxystyryl)-9-methyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',
1'-
f][1,3,2] diazaborinin-3-yl)-N,N-diethylpropanamide (2): To a solution of 4 (10 mg, 24
μmol, 1 eq.) in 2 mL DMF/THF (1:1), was added HBTU (30 mg, 85 μmol, 3.5 eq.) and
stirred at RT for 10 min. Diethylamine (4 μL, 37 μmol, 1.5 eq.) and triethylamine (11 μL,
85 μmol, 3.5 eq.) were subsequently added and the reaction mixture further stirred at RT
for 3 h. Upon completion, the reaction was quenched with water (20 mL), extracted with
DCM (3 x 20 mL), dried over anhydrous Na2SO4 and evaporated in vacuo. The crude
product was purified by flash column chromatography on silica gel (5:1 DCM/EA) to
afford 2 as a deep purple solid (10 mg, 22 μmol, 92% yield); 1H NMR (500 MHz,
DMSO-d6) δ 7.65 (d, J = 16.3 Hz, 1H), 7.61 (s, 1H), 7.59 (d, J = 8.8 Hz, 2H), 7.31 (d, J =
S6
16.3 Hz, 1H), 7.07 (d, J = 4.0 Hz, 1H), 7.04 (s, 1H), 7.04 – 7.01 (m, 2H), 6.40 (d, J = 4.0
Hz, 1H), 3.81 (s, 3H), 3.30 – 3.27 (m, 4H), 3.12 (d, J = 7.4 Hz, 2H), 2.70 (dd, J = 8.9, 6.8
Hz, 2H), 2.31 (s, 3H), 1.08 (t, J = 7.1 Hz, 3H), 1.04 (t, J = 7.1 Hz, 3H); 13C NMR (125
MHz, DMSO-d6) δ 160.7, 157.2, 156.2, 143.2, 138.8, 135.9, 133.3, 129.6, 129.1, 128.5,
127.9, 123.1, 117.0, 116.7, 115.4, 114.7, 55.4, 41.2, 31.2, 22.0, 14.1, 13.1, 11.0; HRMS
(C26H30BFN3O2): calc. [M - F]-: 446.2414, found [M - F]-: 446.2431.
(E)-3-(5,5-difluoro-7-(4-methoxystyryl)-9-methyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',
1'-
f][1,3,2] diazaborinin-3-yl)-N-isopropylpropanamide (3): To a solution of 4 (10 mg, 24
μmol, 1 eq.) in 2 mL DMF/THF (1:1), was added HBTU (30 mg, 85 μmol, 3.5 eq.) and
stirred at RT for 10 min. Isopropylamine (6 μL, 37 μmol, 1.5 eq.) and triethylamine (11
μL, 85 μmol, 3.5 eq.) were subsequently added and the reaction mixture further stirred at
RT for 3 h. Upon completion, the reaction was quenched with water (20 mL), extracted
with DCM (3 x 20 mL), dried over anhydrous Na2SO4 and evaporated in vacuo. The
crude product was purified by flash column chromatography on silica gel (5:1 DCM/EA)
to afford 3 as a deep purple solid (9 mg, 20 μmol, 83% yield); 1H NMR (500 MHz,
S7
DMSO-d6) δ 7.78 (d, J = 7.5 Hz, 1H), 7.65 (d, J = 16.4 Hz, 1H), 7.61 (s, 1H), 7.59 (d, J =
8.7 Hz, 2H), 7.31 (d, J = 16.3 Hz, 1H), 7.07 (d, J = 4.0 Hz, 1H), 7.04 (d, J = 5.7 Hz, 2H),
7.02 (s, 1H), 6.35 (d, J = 3.9 Hz, 1H), 3.91 – 3.81 (m, 1H), 3.81 (s, 3H), 3.11 (t, J = 7.7
Hz, 2H), 2.46 (t, J = 7.8 Hz, 2H), 2.30 (s, 3H), 1.06 (d, J = 6.6 Hz, 6H); 13C NMR (125
MHz, DMSO-d6) δ 160.8, 157.0, 156.3, 143.3, 138.9, 136.0, 133.3, 129.6, 129.2, 128.5,
127.9, 123.1, 116.8, 116.4, 115.4, 114.8, 55.4, 40.3, 33.9, 24.1, 22.4, 11.1; HRMS
(C25H28BFN3O2): calc. [M - F]-: 432.2258, found [M - F]-: 432.2269.
(E)-3-(5,5-difluoro-7-(4-methoxystyryl)-9-methyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',
1'-
f][1,3,2] diazaborinin-3-yl)propanamide (5): To a solution of 4 (10 mg, 24 μmol, 1 eq.)
in 2 mL DMF/THF (1:1), was added HBTU (30 mg, 85 μmol, 3.5 eq.) and stirred at RT
for 10 min. Ammonia (500 μL, 7M in MeOH, 3.5 mmol, 146 eq.) and triethylamine (11
μL, 85 μmol, 3.5 eq.) were subsequently added and the reaction mixture further stirred at
RT for 6 h. Upon completion, the reaction was quenched with water (20 mL), extracted
with DCM (3 x 20 mL), dried over anhydrous Na2SO4 and evaporated in vacuo. The
crude product was purified by flash column chromatography on silica gel (4:1 DCM/EA)
S8
to afford 5 as a deep purple solid (5 mg, 12 μmol, 50% yield); 1H NMR (500 MHz,
CDCl3) δ 7.56 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 16.3 Hz, 1H), 7.31 (d, J = 16.3 Hz, 1H),
7.04 (s, 1H), 6.92 (d, J = 8.8 Hz, 2H), 6.86 (d, J = 3.9 Hz, 1H), 6.71 (s, 1H), 6.32 (d, J =
3.9 Hz, 1H), 5.75 (br. s, 1H), 5.44 (br. s, 1H), 3.85 (s, 3H), 3.32 (t, J = 7.5 Hz, 2H), 2.72
(t, J = 7.5 Hz, 2H), 2.29 (s, 3H);
13
C NMR (125 MHz, CDCl3) δ 161.2, 157.7, 156.0,
143.2, 139.0, 136.9, 133.8, 129.6, 129.0, 127.3, 121.8, 117.2, 116.8, 116.6, 114.6, 114.0,
55. 6, 35.5, 24.9, 11.5; HRMS (C22H22BF2N3NaO2): calc. [M + Na]+: 432.1669, found [M
+ Na]+: 432.1686.
S9
B. Supporting Figures and Tables
Table S1. Binding constants of dye to G4 measured by fluorescence titration. λex: 360
nm. Values are represented as means (n = 3). Measurements were taken at RT. Buffer: 20
mM KH2PO4/K2HPO4, 100 mM KCl, pH 7.0.
G-Quadruplex
Compounds
Kd (M)
93del
GQR
25.18±0.02
J19
GQR
44.47±0.05
T95
GQR
47.46±0.04
T95-2T
GQR
52.37±0.03
Table S2. Binding parameters of 93del with GQR derived from autodocking.
Binding Site
Binding Energy (kcal/mol) Calculated Kd at RT (M)
Groove A
-5.70
66. 3
Groove B
-5.48
95.9
Ending A
-1.32
1.1×105
Ending B
-1.25
1.2×105
S10
Figure S1. Schematic flowchart for the discovery of GQR. Compound structures
included in this screening can be found in the diversity-oriented fluorescent libraries
reported.3-9
S11
Figure S2. Job plot analysis.10 GQR was mixed with 93del at different ratios while
maintaining total concentration at 20 M in buffer (20 mM K2HPO4/KH2PO4, 100 mM
KCl, pH 7.0). ex: 360 nm, em: 597nm. Values are represented as means (n = 3).
Measurements were taken at room temperature (RT).
S12
Figure S3. CD signatures of G4-forming oligonucleotides. Oligonucleotides (40 M)
dissolved in buffer (20 mM K2HPO4/KH2PO4, 100 mM KCl, pH 7.0).
S13
Figure S4. Selectivity of GQR (a) Fluorescence and (b) Absorbance response of GQR
(10 μM) upon interaction with various oligonucleotides (40 μM) in buffer (20 mM
K2HPO4/KH2PO4, 100 mM KCl, pH 7.0). λex: 360 nm. Values are represented as means
(n
=
3).
Measurements
were
taken
at
RT.
S14
Figure S5. Absorption spectra of GQR (5, 10, 20, 50 and 100 M) in buffer (20 mM
K2HPO4/KH2PO4, 100 mM KCl, pH 7.0). Values are represented as means (n = 3).
Measurements were taken at room temperature (RT).
S15
Figure S6. TEM image of GQR-aggregates.
S16
Figure S7. DLS measurement of GQR (10 – 150 M) in buffer (20 mM
KH2PO4/K2HPO4, 100 mM KCl, pH 7.0). The different lines indicate the size distribution
in different concentrations. .
S17
Figure S8. Normalized emission spectra of GQR (10 μM) in various MeOH-toluene
mixtures. λex: 360 nm. Values are represented as means (n = 3). Measurements were taken
at
RT.
S18
Figure S9. The absorption spectra of GQR analogues (10 M) in (a) DMSO and (b)
buffer (20 mM K2HPO4/KH2PO4, 100 mM KCl, pH 7.0). λex: 360 nm. Values are
represented as means (n = 3). Measurements were taken at room temperature (RT).
S19
Figure S10. NMR (600 MHz) titrations of GQR to 93del and T95-2T. Imino region of the
1
H NMR spectra of 93del (left) and T95-2T (right) after addition of (a), (a’) 0 eq., (b), (b’)
0.5 eq., (c), (c’) 1 eq. and (d), (d’) 1.5 eq. of GQR with assignment. Conditions: 93del
(100 μM), Buffer: 20 mM KH2PO4/K2HPO4, 70 mM KCl, pH 7.0, RT.
S20
C. NMR Spectra
S21
S22
S23
S24
S25
S26
S27
S28
S29
S30
S31
S32
S33
S34
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3.
M. Vendrell, G. G. Krishna, K. K. Ghosh, D. Zhai, J.-S. Lee, Q. Zhu, Y. H. Yau, S. G. Shochat, H.
Kim, J. Chung and Y.-T. Chang, Chem. Commun., 2011, 47, 8424.
4.
D. Zhai, S.-C. Lee, M. Vendrell, L. P. Leong and Y.-T. Chang, ACS Comb. Sci., 2012, 14, 81-84.
5.
J.-J. Lee, S.-C. Lee, D. Zhai, Y.-H. Ahn, H. Y. Yeo, Y. L. Tan and Y.-T. Chang, Chem. Commun.,
2011, 47, 4508-4510.
6.
J. C. Er, M. K. Tang, C. G. Chia, H. Liew, M. Vendrell and Y. T. Chang, Chem. Sci., 2013, 4,
2168-2176.
7.
J. S. Lee, N. Y. Kang, Y. K. Kim, A. Samanta, S. H. Feng, H. K. Kim, M. Vendrell, J. H. Park
and Y. T. Chang, J. Am. Chem. Soc., 2009, 131, 10077-10082.
8.
Y.-H. Ahn, J.-S. Lee and Y.-T. Chang, J. Am. Chem. Soc., 2007, 129, 4510-4511.
9.
N.-Y. Kang, S.-C. Lee, S.-J. Park, H.-H. Ha, S.-W. Yun, E. Kostromina, N. Gustavsson, Y. Ali, Y.
Chandran, H.-S. Chun, M. Bae, J. H. Ahn, W. Han, G. K. Radda and Y.-T. Chang, Angew. Chem.,
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35