ch3 ome ch2ohfgdfg 1 1 c:\bruker\topspin3.1\examdata! s29! 13c nmr (100 mhz, cdcl 3) h3c meo n h o...

$: CH3 OMe CH2OHfgdfg 1 1 C:\Bruker\TopSpin3.1\examdata! S29! 13C NMR (100 MHz, CDCl 3) H3C MeO N H O OH HOOH H N CH3 OMe 3b 47.589 180 160 140 120 100 80 60 40 20 ppm Plotname: --Not$
S12

1H NMR (400 MHz, CDCl3)

CH3

OMe

CH2OH

4

Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2017

S13

13C NMR (100 MHz, CDCl3)

CH3

OMe

CH2OH

4

S14


CH3

OMe

CHO

5

S15


CH3

OMe

CHO

5

S16

1H NMR (400 MHz, CDCl3) H3C

MeONH

HN

CH3

OMe

1a

S17

13C NMR (100 MHz, CDCl3) H3C

MeONH

HN

CH3

OMe

1a

S18


H3C

MeONH

1b

NH

CH3

OMe

S19


H3C

MeONH

1b

NH

CH3

OMe

S20


MeONH

1c

HN

CH3

OMe

S21


MeONH

1c

HN

CH3

OMe

S22


1d

H3C

MeONH

N3

2.923.806.34

3.135.69

9.006.076.809.18

ppm12345678

Plotname: --Not assigned--

S23

1d

H3C

MeONH

N3

156.751

127.124

133.584

134.237

126.988

123.496

121.340

118.532

102.530

77.367

77.048 76.737

55.111

53.471

49.835

46.936

19.950

ppm20406080100120140160180200


S24

1H NMR (400 MHz, CDCl3) 2a

H3C

MeONH

HN

CH3

1.023.85

4.992.00

2.983.91

5.92

ppm12345678


S25


ppm20406080100120140160180200


2a

H3C

MeONH

HN

CH3

S26


[ppm] 8 6 4 2

[rel

] 0

5

1

0 1

5 2

0 2

5

1.00

00

2.13

671.

0009

1.00

402.

0486

2.03

601.

0058

2.86

302.

4345

1.93

38

13.1

665

2.46

50

fdsfesdf 1 1 C:\Bruker\TopSpin3.1\examdata

N3O

OBn

BnO OBn

N3

8

S27


ppm20406080100120140160180200


N3O

OBn

BnO OBn

N3

8

S28


MeONH

O

OH

HO OH

HN

CH3

OMe

3b

[ppm] 8 6 4 2

[rel

] 0

5

1

0 1

5

2.00

00

5.54

112.

3869

2.15

63

2.26

96

6.26

04

3.15

14

1.33

91

6.36

72

1.64

88

2.04

22

1.29

681.

3528

1.79

94

1.53

471.

0759

fgdfg 1 1 C:\Bruker\TopSpin3.1\examdata

S29


MeONH

O

OH

HO OH

HN

CH3

OMe

3b

47.589

ppm20406080100120140160180


S30

DNA Binding Studies

Materials: CT-DNA was purchased from CALBIOCHEM. Solutions of CT-DNA were prepared in 10mM Tris-EDTA buffer at pH 5.48 (as described in Jenkins: Jenkins, T.C. Optical Absorbance and Fluorescence Techniques for Measuring DNA-Drug Interactions. In Methods in Molecular Biology, Drug-DNA Interaction Protocols; Fox, K.R., Ed. Humana: Totowa, 1997; Vol 90, pp.195-217) and gave a 1.83:1 absorbance ratio at 260 nm and 280 nm. DNA and ligand concentrations were determined using 8452A HP Diode Array Spectrophotometer: CT-DNA, e260 = 6600 M-1 cm-1 bp-1 (1.82:1 absorbance ratio at 260 nm and 280 nm); poly(dG)•polyd(C), e253 = 7400 M-1 cm-1 bp-1; poly (dA) • poly (dT), e260 = 6000 M-1 cm-1 bp-1; poly(dA-dT)•poly(dA-dT), e262 = 6600 M-1 cm-1 bp-1; Lambda phage DNA: 5 units dissolved in 500 mL buffer provides a solution of 0.76 mM/bp. Methyl Green, e631 = 85, 300 M-1 cm-1; netropsin, e296 21,500 M-1cm-1; Hoechst 33342, e340 = 47,000 M-1 cm-1; Ethidium Bromide, e480 = 5450 M-1 cm-1; 1a-1c, 3b, e336 = 6089 M-1 cm-1; 1d, e336 = 9134 M-1 cm-1; 2a, e336 = 3045 M-1 cm-1. EB competitive experiments Constant concentrations of CT-DNA (poly(dG)•polyd(C), poly (dA)•poly (dT), poly(dA-dT)•poly(dA-dT), or Lambda phage DNA, 10 µM) and EtBr (10 µM) were titrated with increasing concentrations of the ligands (from 1 mM and 100 µM stock solutions), in the presence or absence of fixed concentrations of NaCl or the competitors methyl green or netropsin. The maximum emission wavelength was 490 nm when the excitation wavelength was 520 nm. Fluorescence titrations were recorded from 520 nm to 692 nm after an equilibration period of 3 min. Ex Slit (nm) = 10.0; Em Slit (nm) = 10.0; Scan Speed (nm/min) = 200. Viscosity Studies Viscosity experiments were performed with an Ostwald viscometer in a constant water bath at 23.0 ± 1°C. Solutions of constant DNA concentrations and varying ligand concentrations in Tris-EDTA buffer were incubated for 30 minutes. A digital stopwatch was used to record the flow time. The relative viscosity was calculated as from the following equation: 𝜂 = !!!!

!!

where t0 and t are the flow time in the absence and presence of the ligand. η is the viscosity in the presence of the ligand and η0 is the viscosity in the absence of the ligand. The data were graphed as (η/η0)1/3 vs. [ligand]/[DNA]. Circular Dichroism Studies. Small aliquots (0.6-5.0 μL) of a concentrated 1d solution (1 mM) were added to a solution (2 mL, 100 mM KCl, 10 mM SC, 0.5 mM EDTA, pH 6.8) of CT-DNA (80 μM/bp), inverted twice, and incubated for 5 min at 20 °C. The CD spectra were then recorded as an average of three scans from 220 to 310 nm and data recorded in 0.1 nm increments with an averaging time of 2 s.

1H(400 MHz) and 13C NMR (100 MHz) spectra

S31

Compound 1a Ethidium displacement studies; Kapp=2.1x106M-‐1 x 10/C50

Titration of CT DNA (10 µM) and ethidium (10 µM) with 1a: 0.005, 0.01, 0.02, 0.04, 0.05, 0.1, 0.2, 0.33, 0.45, 0.56, 0.63, 0.71, 0.83, 1.00, 1.25, 1.67,

2.50, 3.30, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0, 60.0, 100.0, 200.0 µM

Trial 1: Kapp= 6.17 x 106 M-1; Trial 2: Kapp= 8.40 x 106 M-1 ; Trial 3: Kapp= 7.00 x 106 M-1

Average Kapp=7.19 ± 0.92 x 106 M-‐1

Average data for Trials 1-3

Nor

mal

ized

Flu

ores

cen

ce

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

log([1a])-9 -8 -7 -6 -5 -4 -3

-9 -8 -7 -6 -5 -4 -3

C(50)=3.09±0.02x10-6M

Plot of Δ Fluorescence vs. rbd for 1a

Δ Fl

uor

esce

nce

-0.08

-0.07

-0.06

-0.05

-0.04

-0.03

-0.02

-0.01

0

0.01

-0.08

-0.07

-0.06

-0.05

-0.04

-0.03

-0.02

-0.01

0

0.01

rbd0 5 10 15 20 25 30 35

0 5 10 15 20 25 30 35

lines intersect at rbd=4.2

[1a]=0.005 µM

[1a]=200 µM

Titration of CTDNA•ethidium with 1a

Nor

mal

ized

Flu

ores

cen

ce

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

log([1a])-9 -8 -7 -6 -5 -4

-9 -8 -7 -6 -5 -4

0mM NaCl, C(50)=3.23x10-6M10mM NaCl, C(50)=8.70x10-6M100mM NaCl, C(50)=12.6x10-6M

S32

Effect of increasing amount of EtBr (▲) and 1a (●) on relative viscosity of CT-‐DNA. EB (▲): [CT-‐DNA] = 300 µM, [EB]= 4, 26, 70, 113, 160, and 200 µM; 1a (●): [CT-‐DNA] = 300 µM, [1a]: 8, 52, 140, 226, 320, and 400 µM.

S33

Compound 1b Ethidium displacement studies; Kapp=2.1x106M-‐1 x 10/C50

Titration of CT DNA (10 µM) and ethidium (10 µM) with 1b: 0.005, 0.01, 0.02, 0.04, 0.05, 0.1, 0.2, 0.33, 0.45, 0.56, 0.63, 0.71, 0.83, 1.00, 1.25, 1.67, 2.50, 3.30, 5.0, 10.0, 20.0, 30.0, 40.0, 60.0, 80.0, 100.0 µM

Trial 1: Kapp=2.35 x 106M-1 ; Trial 2: Kapp=3.31 x 106M-1; Trial 3: Kapp=3.80 x 106M-1

3 trials: Average Kapp=3.15(±0.60)x106M-1

Plot of Δ Fluorescence vs rbd for 1b

Δ Fl

uor

esce

nce

-0.14

-0.12

-0.1

-0.08

-0.06

-0.04

-0.02

0

-0.14

-0.12

-0.1

-0.08

-0.06

-0.04

-0.02

0

rbd0 5 10 15 20 25 30 35

0 5 10 15 20 25 30 35


Titlration of CTDNA•ethidium with 1b

Nor

mal

ized

Flu

ores

cen

ce

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

log([1b])-9 -8 -7 -6 -5 -4 -3

-9 -8 -7 -6 -5 -4 -3

C(50)=5.89x10-6M

[1b]=0.005 µM

[1b]=100 µM

S34

Effect of increasing amount of EtBr (▲) and 1b (●) on relative viscosity of CT-‐DNA. EtBr (▲): [CT-‐DNA] = 301 µM, [EtBr]= 1.06, 2.12, 3.71, 9.54, 18.02, and 25.97 µM; 1b (●): [CT-‐DNA] = 298 µM, [1b]: 1.08, 2.17, 3.79, 9.76, 17.88, and 26.02 µM

S35

Compound 1c Ethidium displacement studies; Kapp=2.1x106M-‐1 x 10/C50

Titration of CT DNA (10 µM) and ethidium (10 µM) with 1c: 0.005, 0.01, 0.02, 0.04, 0.05, 0.1, 0.2, 0.33, 0.45, 0.56, 0.63, 0.71, 0.83, 1.00, 1.25, 1.67, 2.50, 3.30, 5.0, 10.0, 20.0, 30.0, 40.0 µM

Trial 1: Kapp=4.64 x 106M-1; Trial 2: Kapp=4.71 x 106M-1; Trial 3: Kapp=5.65 x106M-1

3 trials: Average: Kapp=5.00 (±0.46) x 106M-1

Titration of CTDNA•ethidium with 1c

Nor

mal

ized

Flu

ores

cen

ce

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

log([1c])-8.5 -8 -7.5 -7 -6.5 -6 -5.5 -5 -4.5 -4

-8.5 -8 -7.5 -7 -6.5 -6 -5.5 -5 -4.5 -4

C(50)=4.79x10-6M

Plot of Δ Fluorescence vs. rbd for 1c

Δ Fl

uor

esce

nce

-0.14

-0.12

-0.1

-0.08

-0.06

-0.04

-0.02

-0.14

-0.12

-0.1

-0.08

-0.06

-0.04

-0.02

rbd0 5 10 15 20 25 30 35

0 5 10 15 20 25 30 35


[1c]=0.005 µM

[1c]=40 µM

S36

Effect of increasing amount of EtBr (▲) and 1c (●) on relative viscosity of CT-‐DNA. EB (▲): [CT-‐DNA] = 300 µM, [EB]= 4, 26, 70, 113, 160, and 200 µM; 1c (●): [CT-‐DNA] = 300 µM, [1c]: 1, 2, 4, 10, 18, and 26 µM.

S37

Compound 1d Ethidium displacement studies; Kapp=2.1x106M-‐1 x 10/C50

Titration of CT DNA (10 µM) and ethidium (10 µM) with 1d: 0.005, 0.01, 0.02, 0.04, 0.05, 0.1, 0.2, 0.33, 0.45, 0.56, 0.63, 0.71, 0.83, 1.00, 1.25, 1.67, 2.50, 3.30, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0, 60.0, 100.0, 200.0 µM

Trial 1 Kapp=26.45 x 106 M-1; Trial 2 Kapp=24.11 x 106 M-1; Trial 3 Kapp=22.00 x 106 M-1

3 trials: Average Kapp=24.18 (± 1.5) x 106 M-‐1

Plot of Δ Fluoresence vs. rbd for 1d

Δ Fl

uor

esce

nce

-0.14

-0.12

-0.1

-0.08

-0.06

-0.04

-0.02

0

-0.14

-0.12

-0.1

-0.08

-0.06

-0.04

-0.02

0

rbd0 5 10 15 20 25 30 35

0 5 10 15 20 25 30 35


[1d]=0.005 µM

[1d]=200 µM

Titration of CTDNA•ethidium with 1d

Ave

rag

e N

orm

aliz

ed F

luor

esce

nce

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

log([1d])-8.5 -8 -7.5 -7 -6.5 -6 -5.5 -5 -4.5

-8.5 -8 -7.5 -7 -6.5 -6 -5.5 -5 -4.5

C(50)=8.71x10-7M

S38

UV spectra of 1d (25µM in 10mM Tris-‐EDTA, pH=5.48) in the presence of varying concentrations of CT-‐DNA: 0, 0.25, 0.5, 1.0, 2.0, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 100 µM.

==============================================================================================

==============================================================================================

Method file : <untitled>Information : Default MethodData File : <untitled>

Overlaid Spectra:

Wavelength (nm)200 300 400 500 600 700

Abso

rban

ce (A

U)

0

0.25

0.5

0.75

1

1.25

1.5

1.75

# Name Abs<335nm> Abs<260nm>------------------------------------------------ 1 0 0.19519 0.28519 2 0.25 0.26395 0.38040 3 0.50 0.27064 0.37132 4 1.00 0.26714 0.35623 5 2.00 0.26584 0.35866 6 4.00 0.26093 0.36414 7 5.00 0.25913 0.36742 8 10.00 0.26525 0.41658 9 15.00 0.27602 0.4723210 20.00 0.28812 0.5184211 25.00 0.30500 0.5761912 30.00 0.32296 0.6245413 40.00 0.31963 0.6705814 50.00 0.31213 0.7060215 0.28162 0.86610

Report generated by : Admin Signature: ................

---------------------------------------------------------------------------------------------- *** End Fixed Wavelength Report ***----------------------------------------------------------------------------------------------

Fixed Wavelength Report Date 3/23/2017 Time 17:39:47 Page 1 of 1

[CT DNA]=100 µM

[CT DNA]=0 µM

[CT DNA]=0 µM

[CT DNA]=100 µM

S39

Effect of increasing amounts of 1d (●), ethidium bromide ( ), and netropsin ( ) on the relative viscosity of CT-DNA. R= [DNA(bp)]/[ligand]; 1d (●): [CT-‐DNA] = 300 µM, [1d]: 1, 2, 4, 10, 18, and 26 µM; ethidium bromide ( ): [CT-‐DNA] = 300 µM, [ethidium bromide]= 4, 26, 70, 113, 160, and 200 µM; netropsin ( ): [CT-‐DNA] = 300 µM, [netropsin]= 4, 26, 70, 113, 160, and 200 µM.

Viscosity data comparison for 1d, ethidium, and netropsin

(η/η

o)1/3

0.5

1

1.5

2

2.5

3

0.5

1

1.5

2

2.5

3

1/R0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

1dethidium bromidenetropsin

S40

Affinity of 1d for M. Luteus DNA

Trial 1 Kapp=7.29x106M-1 ;

Trial 2 Kapp=9.50x106M-1 ;

Trial 3 Kapp=6.00x106M-1;

Average Kapp=7.36(±1.40)x106M-1

Affinity of 1d for poly(dA•dT)2

Trial 1 Kapp=1.71 x107M-1;

Trial 2 Kapp=2.00 x 107M-1;

Trial 3 Kapp=1.60 x 107M-1

Average Kapp=17.7±1.5 x 106M-1

Affinity of 1d for polydG•polydC

Trial 1: Kapp=18.9x106M-1



Average Kapp=18.4±0.64x106M-1

Titration of polydG•polydC•ethidium with 1d

Ave

rag

e N

orm

aliz

ed F

luor

esce

nce

0.12

0.13

0.14

0.15

0.16

0.17

0.18

0.19

0.2

0.12

0.13

0.14

0.15

0.16

0.17

0.18

0.19

0.2

log([1d])-8.5 -8 -7.5 -7 -6.5 -6 -5.5 -5 -4.5 -4

-8.5 -8 -7.5 -7 -6.5 -6 -5.5 -5 -4.5 -4

C(50)=1.15x10-6M

Titration of (polydA•dT)2•ethidium with 1d

Nor

mal

ized

Flu

ores

cen

ce

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

log([1d])-9 -8 -7 -6 -5 -4

-9 -8 -7 -6 -5 -4

C(50)=1.17x10-6M

Titration of MLuteus DNA•ethidium with 1d

Nor

mal

ized

Flu

ores

cen

ce

0

0.2

0.4

0.6

0.8

1

1.2

0

0.2

0.4

0.6

0.8

1

1.2

log([1d])-8.5 -8 -7.5 -7 -6.5 -6 -5.5 -5 -4.5 -4

-8.5 -8 -7.5 -7 -6.5 -6 -5.5 -5 -4.5 -4

C(50)=2.88x10-6M

S41

Affinity of 1d for Lambda Phage DNA





Affinity of 1d for polydA•polydT





Affinity of netropsin for lambda phage DNA





Titration of Lambda Phage DNA•ethidum with 1d

Nor

mal

ized

Flu

ores

cen

ce

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

log([1d])-9 -8 -7 -6 -5 -4

-9 -8 -7 -6 -5 -4

C(50)=2.53x10-6M

Titration of polydA•polydT•ethidium with 1d

Nor

mal

ized

Flu

ores

cen

ce

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

log([1d])-9 -8 -7 -6 -5 -4

-9 -8 -7 -6 -5 -4

C(50)=1.06x10-6M

Titration of Lambda Phage DNA•ethidium with netropsin

Nor

mal

ized

flu

ores

cen

ce

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

log([netropsin])-9 -8 -7 -6 -5 -4 -3 -2

-9 -8 -7 -6 -5 -4 -3 -2

C(50)=5.95x10-5M

S42

Affinity of netropsin for CT DNA





No competitor, Ka=2.47±1.5 x107 M-‐1; 30µM NP, Ka=1.40±1.2 x107 M-‐1; 30µM MG, 1.05±0.9 x 10-‐7 M-‐1


Nor

mal

ized

Flu

ores

cen

ce

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

log([1d])-9 -8 -7 -6 -5 -4

-9 -8 -7 -6 -5 -4

no competitor, C(50)=0.85 μM 30 μM NP, C(50)= 1.5 μM 30 μM MG, C(50)= 2.0 μM

Titration of CTDNA•ethidium with netropsin

Nor

mal

ized

flu

ores

cen

ce

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

log([netropsin])-9 -8 -7 -6 -5 -4 -3

-9 -8 -7 -6 -5 -4 -3

C(50)=8.6x10-5M

S43

Figure 5. The 220-310 nm region of the CD spectrum of solutions of CT DNA (80 µM) in the absence (black line) and presence of various concentrations of 1d: blue line, 0.15 µM 1d; light green line, 0.30 µM 1d; orange line, 0.45 µM 1d; red line, 1.1 µM 1d; dark green line 2.00 µM 1d

Wavelength (nm)

CD

(mde

g)

!

!!

!! 0.00 µM 1d + 80 µM CTDNA0.15 µM 1d + 80 µM CTDNA0.30 µM 1d + 80 µM CTDNA0.45 µM 1d + 80 µM CTDNA1.10 µM 1d + 80 µM CTDNA2.00 µM 1d + 80 µM CTDNA

S44

10mM NaCl: Kapp=2.25 x 107M-‐1 100mM NaCl: Kapp=2.25 x 107M-‐1 1M NaCl: Kapp=5.30 x 106M-‐1

Titration of CTDNA•ethidium with 1d, 10 mM NaClN

orm

aliz

ed F

luor

esce

nce

0

0.2

0.4

0.6

0.8

1

1.2

0

0.2

0.4

0.6

0.8

1

1.2

log([1d])-8.5 -8 -7.5 -7 -6.5 -6 -5.5 -5 -4.5

-8.5 -8 -7.5 -7 -6.5 -6 -5.5 -5 -4.5

C(50)=9.33 x10-7M

Titration of CTDNA•ethidium with 1d, 100 mM NaCl

Nor

mal

ized

FLu

ores

cen

ce

0

0.2

0.4

0.6

0.8

1

1.2

0

0.2

0.4

0.6

0.8

1

1.2

log([1d])-8.5 -8 -7.5 -7 -6.5 -6 -5.5 -5 -4.5

-8.5 -8 -7.5 -7 -6.5 -6 -5.5 -5 -4.5

C(50)=9.33x10-7M

Titration of CTDNA•ethidium with 1d, 1M NaCl

Nor

mal

ized

Flu

ores

cen

ce

-0.2

0

0.2

0.4

0.6

0.8

1

-0.2

0

0.2

0.4

0.6

0.8

1

log([1d])-8.5 -8 -7.5 -7 -6.5 -6 -5.5 -5 -4.5 -4

-8.5 -8 -7.5 -7 -6.5 -6 -5.5 -5 -4.5 -4

C(50)=3.98x10-6M


Nor

mal

ized

flu

ores

cen

ce

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

log([1d])-9 -8 -7 -6 -5 -4

-9 -8 -7 -6 -5 -4

10mM NaCl, C(50)=0.95x10-6M100mM NaCl, C(50)=0.97x10-6M1M NaCl, C(50)=3.72x10-6M

S45

Model for bis-‐intercalation of ligand 1d in the major groove of DNA sequence 5’-‐ATGCAT-‐3’,

generated by Autodock Vina using DNA (PDB 1x95) and 1d minimized by Spartan 14 for Macintosh

S46

Additional binding modes for the association of 1d with DNA sequence 5’-‐ATGCAT-‐3’,

generated by Autodock Vina using DNA (PDB 1x95) and 1d minimized by Spartan 14 for Macintosh

S47

Compound 2a Ethidium displacement studies; Kapp=2.1x106M-‐1 x 5/C50

Titration of CT DNA (10 µM) and ethidium (10 µM) with 3b: 0.005, 0.01, 0.02, 0.04, 0.05, 0.1, 0.2, 0.33, 0.45, 0.56, 0.63, 0.71, 0.83, 1.00, 1.25, 1.67, 2.50, 3.30, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0, 60.0, 100.0, 200.0 µM

Trial 1: Kapp= 4.36 x105M-1; Trial 2: Kapp=5.0x105M-1; Trial 3: Kapp= 4.79x105M-1

3 trials: Average Kapp= 4.72±0.27x105M-‐1

Stoichiometry Analysis for 2a

ΔFluorescence

-0.05

-0.04

-0.03

-0.02

-0.01

0

-0.05

-0.04

-0.03

-0.02

-0.01

0

rbd0 5 10 15 20 25 30 35

0 5 10 15 20 25 30 35

Lines intersect at rbd=4.02

Titration of CTDNA•ethidium with 2a

Nor

mal

ized

Flu

ores

cen

ce

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

log([2a])-9 -8 -7 -6 -5 -4 -3

-9 -8 -7 -6 -5 -4 -3

C(50)=3.72x10-5M

[2a]=0.005 µM

[2a]=200 µM

S48

Effect of increasing amount of EtBr (▲) and 2a (●) on relative viscosity of CT-‐DNA. EtBr (▲): [CT-‐DNA] = 300 µM, [EtBr]= 4, 26, 70, 113, 160, and 200 µM; 2a (●): [CT-‐DNA] = 300 µM, [2a]: 1, 2, 4, 10, 18, and 26 µM.

S49

Compound 3b Ethidium displacement studies; Kapp=2.1x106M-‐1 x 10/C50

Titration of CT DNA (10 µM) and ethidium (10 µM) with 3b: 0.005, 0.01, 0.02, 0.04, 0.05, 0.1, 0.2, 0.33, 0.45, 0.56, 0.63, 0.71, 0.83, 1.00, 1.25, 1.67, 2.50, 3.30, 5.0, 10.0, 20.0, 50.0 µM

Trial 1: Kapp=6.88 x106M-1; Trial 2: Kapp=6.99 x106M-1; Trial 3: Kapp=5.55 x106M-1

3 trials: Average Kapp=6.47±0.65 x106M-1

Plot of Δ Fluorescence vs rbd for 3b

Δ Fl

uor

esce

nce

-0.12

-0.1

-0.08

-0.06

-0.04

-0.02

0

-0.12

-0.1

-0.08

-0.06

-0.04

-0.02

0

rbd0 10 20 30 40 50 60

0 10 20 30 40 50 60

lines intersect at rbd=10

Titration of CTDNA•ethidium with 3b

Nor

mal

ized

Flu

ores

cen

ce

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

log(3b])-8.5 -8 -7.5 -7 -6.5 -6 -5.5 -5 -4.5

-8.5 -8 -7.5 -7 -6.5 -6 -5.5 -5 -4.5

C(50)=1.81±01x10-6M

[3b]=0.005 µM

[3b]=50 µM

S50

Effect of increasing amount of EtBr (▲) and 3b (●) on relative viscosity of CT-‐DNA. EtBr (▲): [CT-‐DNA] = 300 µM, [EtBr]= 4, 26, 70, 113, 160, and 200 µM; 3b (●): [CT-‐DNA] = 300 µM, [3b]: 1, 2, 4, 10, 18, and 26 µM.

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