Supporting information

Metal complex synthesis

Rh

Cl

3

N

Rh

H

N

H

HO

O

3+

N

+

OTF

N

Rh

TFO

3+

NH

3

NH

3

N

Rh

H

3

N

H

3

N

3+

CF

3

SO

3

H

NH

3

3+

O

N

O

OH

3+

NH

3

NH

3

N

Rh

H

N

H

3+

Cl

N

Rh

Cl

Scheme 1: Synthesis of the carboxylic acid modified Rh(III) complex

[Rh(phen)(chrysi)(NH

3

)

2

]

3+

was synthesized in four steps from rhodium trichloride,

phenantholine and 5,6-chrysenequinone, and purif

ied as described by Mürner, Jackson and

Barton [1]. Propionic acid modified

bipyridine was then reacted with

[Rh(phen)(chrysi)(NH

3

)

2

]

3+

in a 50% water, ethanol mixtur

e by refluxing it for 20h. The crude

[Rh(phen)(chrysi)(bpy3C)]

3+

was purified on a Sephadex SP C25 column (eluated with 0,05-

0,5M MgCl

2

) and the product fractions we

re isolated on a Sep Pak C

18

Cartridge, washed with

copious amounts of water, and th

en eluted from the cartridge with 50% acetonitrile/water,

acidified with 0,1% TFA.

Characterization:

Esi-MS:

M

calc

: 779.6 (M-2H)

+

M

found

:

778.9

+

UV-Vis:

200

300

400

500

600

700

0,0

0,1

0,2

0,3

0,4

Absorption

Wavelength

[1]

H. Mürner, B. A, Jackson, J. K. Barton,

Inorg. Chem.

1998

,

37

, 3007-3012

S 1

1

H-NMR, D

2

O:

8.95 (m, 2H); 8.83 (m, 1H); 8.82 (m, 1H); 8,48 (m, 3H); 8,31 (d, 1 H); 8,27

(s, 1H); 8,21 (s, 1H); 7.99 (m, 2H); 7.78 (m, 3H); 7.53 (m, 2H); 7.41 (m, 1H); 7.31 (m, 1H);

3.17 (m, 1H); 3.00 (m, 1H); 2.75 (m, 1 H); 2.63 (m, 2H); 2.42 (m, 2H); 1.85 (s, 1H)

Coupling of the carboxylic acid modified

metal complex to the peptide and

addition of the fluorophores

Scheme 2: Synthesis of the Rh(III)

complex and fluorophore modified peptides

Solid phase bound and protected (arginine wa

s protected as its 2,2,4,6,7-pentamethyldihydro-

benzofuran-5-sulfonyl (Pbf) derivative, lysine as its methyltr

ityl (Mtt) derivative) peptides

were purchased from AnaSpec. The acid modified metal complex or the acid modified

fluorophores were coupled to the free amines

of the peptide by standard HOBT/HBTU

activated coupling reactions. Th

e MTT-group was selectively de

protected by treatment with

3% TFA in dichloromethane for 10 minutes. The

free amino group could then be acylated as

described above, or treated with fluoresceini

sothiocyanate in dimethylformamide. The

peptides were cleaved from the resin us

ing 95% trifluoroacetic acid (TFA), 2.5%

Lys(Mtt)Arg(Pbf)

8

NH

2

N

H

N

H

N

H

N

H

O

H

N

O

H

N

O

HN

OH

O

H

2

NNH

NH

H

2

N

NH

H

2

N

NH

H

2

N

HN

NH

2

HN

NH

2

HN

NH

2

HN

NH

2

HN

HOBT/HBTU

R

1

NH

S

O

OH

O

CO

2

H

R

2

N

Rh

N

H

R

2

=

R

1

=

O

Rh

FITC

Rh

1

2

3

4

H

Ac

O

N

+

S

N

TO

H

5

TO

Rh

Ac = Acetyl

R

1

R

1

Lys(Mtt)Arg(Pbf)

8

NH

TFA/DCM

R

1

R

1

LysArg(Pbf)

8

NH

LysArg(Pbf)

8

NH

R

2

R

2

HOBT/HBTU,

or + FITC

TFA/TIS/H

2

O

3+

S 2

triisopropylsilane and 2.5% wate

r for 3h at room temperature. In all cases, the final products

were obtained in analytical purity using a

HP1100 HPLC system fitted with a C18-packed

reverse phase column and analyzed by ES

I- and/or Maldi-Tof mass spectrometry. All

conjugates employed in this study were used

as their corresponding tr

ifluoroacetate salts

bearing a full complement of counterions.

Synthesis of Compound 6

(Fmoc)Lys(Mtt)

Piperidine

Lys(Mtt)

NH

2

HOBT/HBTU, TO

Lys(Mtt)

NH

N

+

S

N

O

Lys

NH

N

+

S

N

O

TFA/DCM

H

N

+

S

N

O

HN

N

Rh

3+

N

NH

HN

O

Rh-complex

HOBT/HBTU

HO

H

N

+

S

N

O

HN

N

Rh

3+

N

NH

HN

O

TFA/TIS/H

2

O

6

Solid phase bound and protected lysine (methy

ltrityl (Mtt) and 9-fluorenylmethyloxycarbonyl

(Fmoc) protecting group) amino acid was purch

ased from novabiochem. The acid modified

metal complex or the acid modified thiazole or

ange were coupled to the free amines of the

peptide by standard HOBT/HBTU activat

ed coupling reactions. The MTT-group was

selectively deprotected by treatment with 3%

TFA in dichloromethane for 10 minutes. The

free amino group could then be acylated as de

scribed above. The compound was cleaved from

the resin using 95% trif

luoroacetic acid (TFA),

2.5% triisopropylsilane

and 2.5% water for 3h

at room temperature. The fina

l products was obtained in anal

ytical purity using a HP1100

HPLC system fitted with a C18-packed reve

rse phase column and analyzed by ESI- and

Maldi-Tof mass spectrometry. The conjugate employed in this study was used as its

corresponding trifluoroacetat

e salt bearing a full comp

lement of counterions.

S 3

Characterizations:

Compound 1

:

1638.0

1888.2

2138.4

2388.6

2638.8

2889.0

Maldi-Tof-MS

: M

calc

: 2546.76(M-2H)

+

M

found

: 2546.45; M

calc

: 2366.55(M-Phen-2H)

+

M

found

:

2367.55; M

calc

: 2290.46(M-Chrysi-2H)

+

M

found

: 2292.80; M

calc

: 2110.25(M-Chrysi-Phen-2H)

+

M

found

: 2111.32; M

calc

: 2010.34(M-Rh-Chrysi-Phen+H)

+

M

found

: 2011.13

ESI-MS

: M

calc

: 637.44 (M+4H)

4+

M

found

: 637.33; M

calc

: 666.20 (M+3H+TFA)

4+

M

found

:

665.83; M

calc

: 693.95 (M+2H+2TFA)

4+

M

found

: 694.34; M

calc

: 722.46 (M+H+3TFA)

4+

M

found

:

722.84; M

calc

: 510.35 (M+5H)

5+

M

found

: 510.04

Compound 2

:

Maldi-Tof-MS

: M

calc

: M

calc

: 2157.38 (M-2H)

+

M

found

: 2157.66; M

calc

: 1977.17 (M-Phen-2H)

+

M

found

: 1979.50; M

calc

: 1901.08 (M-Chrysi-2H)

+

M

found

: 1903.48; M

calc

: 1720.87 (M-Chrysi-

Phen-2H)

+

M

found

: 1722.56; M

calc

: 1620.96 (M-Rh-Chrysi-Phen+H)

+

M

found

: 1621.67

ESI-MS:

M

calc

: 947.83 (M+6TFA)

3+

M

found

: 947.62; M

calc

: 909.83 (M+5TFA)

3+

M

found

:

909.62; M

calc

: 653.62 (M+H+4TFA)

4+

M

found

: 654.05; M

calc

: 625.11 (M+H-3TFA)

4+

M

found

:

625.55; M

calc

: 596.61 (M+H+2TFA)

4+

M

found

: 597.05; M

calc

: 568.1 (M+H+TFA)

4+

M

found

:

568.54

905.0

1260.2

1615.4

1970.6

2325.8

2681.0

Mass

(

m/z

)

0

1431.4

0

10

20

30

40

50

60

70

80

90

100

% Intensity

Voyager Spec #1[BP = 540.1, 8979]

1722.56

1621.67

1903.48

2157.66

1979.50

1665.75

1744.56

1924.92

2179.45

1982.76

2259.94

2006.15

Mass

(

m/z

)

0

479.4

0

10

20

30

40

50

60

70

80

90

100

% Intensity

Voyager Spec #1[BP = 540.1, 2902]

2111.32

2292.80

2011.13

1903.65

2080.19

2259.26

2546.45

2367.55

2068.35

2248.33

2589.84

S 4

Compound 3

:

1444

1583

1722

1861

2000

2139

Maldi-Tof MS

: M

calc

: (M+H

+

) 1828.07; M

found

: 1828.59

Compound 4

:

Maldi-Tof-MS

: M

calc

: 1853.93 (M+H

+

) M

found

: 1852.90

Mass

(

m/z

)

0

744.2

0

10

20

30

40

50

60

70

80

90

100

% Intensity

Voyager Spec #1[BP = 1828.6, 744]

1828.59

1832.00

1850.48

1480.44

1673.11

1516.25

1656.18

1834.49

1500.79

1461.16

1872.98

1786.65

1671.20

1630.10

1743.04

1474.03

1518.96

1969.49

1902.29

1694.24

1596.54

1790.46

2006.61

1846.18

1651.91

1532.21

1912.82

1964.46

2056.99

1729.92

2098.70

468.0

1108.2

1748.4

2388.6

3028.8

3669.0

Mass

(

m/z

)

0

2971.7

0

10

20

30

40

50

60

70

80

90

100

% Intensity

Voyager Spec #1[BP = 1852.9, 2972]

1852.90

1874.48

1838.64

2009.03

S 5

Compound 5

:

1208.0

1630.8

2053.6

2476.4

2899.2

3322.0

Maldi-Tof-MS

: M

calc

: 2614.62 (M-2H)

+

M

found

: 2613.57; M

calc

: 2358.32 (M-Chrysi-2H)

+

M

found

: 2359.76; M

calc

: 2178.11 (M-Chrysi-Phen-2H)

+

M

found

: 2179.88; M

calc

: 2078.20 (M-Rh-

Chrysi-Phen+H)

+

M

found

: 2077.63

ESI-MS

: M

calc

: 796.93 (M+5TFA+H)

4+

M

found

: 796.35; M

calc

: 768.43 (M+4TFA+H)

4+

M

found

:

767.85; M

calc

: 739.92 (M+3TFA+H)

4+

M

found

: 739.35; M

calc

: 711.42 (M+2TFA+H)

4+

M

found

:

710.85; M

calc

: 682.91 (M+TFA+H)

4+

M

found

: 683.63

Compound 6:

Maldi-TOF-MS

: 1363.8=M

+

; 1111.06=(M-Chrysi)

+

; 930.33=(M-Chrysi-Phen)

+

; 827.59=(M-

Chrysi-Phen-Rh)

+

; 539.56=(RhChrysiPhen)

+

ESI-MS

: 455.1 (M

3+

); 682.6 (M-H

+

)

2+

499.0

899.4

1299.8

1700.2

2100.6

2501.0

Mass

(

m/z

)

0

3943.8

0

10

20

30

40

50

60

70

80

90

100

% Intensity

Voyager Spec #1[BP = 827.6, 3944]

827.59

539.56

568.61

930.33

767.44

687.71

781.56

884.43

524.58

1111.06

622.49

993.36

1363.80

819.54

1253.40

730.65

Mass

(

m/z

)

0

7363.4

0

10

20

30

40

50

60

70

80

90

100

% Intensity

Voyager Spec #1[BP = 540.2, 9821]

2077.63

2359.76

2179.88

2099.63

1723.60

2243.48

2435.70

2033.18

1915.64

2275.90

2613.57

2133.29

2457.57

S 6

Control experiments for the MALDI-TOF

analysis of the photocleavage reaction

Irradiation without 1 (light control, LC)

3000

4600

6200

7800

9400

11000

1 μM mismatched DNA AB, 50 mM NaCl, 10 mM phosphate buffer pH 7, 15 min irradiation.

Compound 1 and mismatched DNA withou

t irradiation (dark control, DC)

1 μM Rh-(Arg)8-FITC (1), 1 μM mismatched DNA AB, 50 mM NaCl, 10 mM phosphate buffer pH 7, without

irradiation.

Compound 1, matched DNA

1 μM Rh-(Arg)8-FITC (1), 1 μM matched DNA AC, 50 mM NaCl, 10 mM phosphate buffer pH 7, 15 min

irradiation.

3000

4600

6200

7800

9400

11000

Mass

(

m/z

)

0

3124.8

0

10

20

30

40

50

60

70

80

90

100

% Intensity

Voyager Spec #1=>NF0.7=>BC=>NF0.7=>MC[BP = 9482.1, 3125]

9482.20

4741.60

9552.73

4777.54

4039.39

4923.35

3000

4600

6200

7800

9400

11000

Mass

(

m/z

)

0

2867.9

0

10

20

30

40

50

60

70

80

90

100

% Intensity

Voyager Spec #1=>BC=>NF0.7=>MC[BP = 9442.1, 2868]

9442.20

9553.49

4721.60

4777.33

4923.00

3365.85

5851.62

9308.66

Mass

(

m/z

)

0

3282.0

0

10

20

30

40

50

60

70

80

90

100

% Intensity

Voyager Spec #1=>BC=>NF0.7=>MC[BP = 9441.6, 3282]

9442.20

9553.11

4721.60

4781.03

4921.20

S 7

Photocleavage experiments of 1 w

ith mismatched D

NA AB; DNA A was

32

P-

labeled; analysis by PAGE

AG CT

0,005-0,75 μM

1,

0,005-0,75 μM mismatched DNA AB, 50 mM NaCl, 10 mM phosphate buffer pH 7, 15 min

irradiation.

S 8

Photocleavage of

5

with mismatched DNA AB; DNA A was

32

P-labeled;

analysis by PAGE

0,001-1

μ

M DNAs and

5

, 50 mM NaCl, 10 mM phosphate buffer pH 7, 15 minutes irradiation.

AG CT

S 9

Photocleavage of

2

with mismatched DNA AB; DNA A was

32

P-labeled;

analysis by PAGE

0,005-1

μ

M mismatch DNA and

2

, 50 mM NaCl, 10 mM phosphate buffer pH 7, 15 min irradiation.

AG CT

S 10

Photocleavage of

6

with mismatched DNA AB; DNA A was

32

P-labeled;

analysis by PAGE

AG CT

0,01-0,5

μ

M DNAs,

6

, 50 mM NaCl, 10 mM phosphate buffer pH 7, 15 min irradiation.

S 11

Temperature dependence of fluorescence of

compound 5 to matched (left, DNA AC) and

mismatched (right, DNA AB) DNA

20

30

40

50

60

70

80

0

1

2

3

4

5

6

500

520

540

560

580

600

620

640

660

0

1

2

3

4

5

6

Fluorescence (a.u.)

Wavelength

Fluorescence at 528 nm (a.u.)

Temperature °C

20

30

40

50

60

70

80

0

1

2

3

4

5

6

7

8

500

520

540

560

580

600

620

640

660

0

1

2

3

4

5

6

7

8

Fluorescence (a.u.)

Wavelength

Fluorescence at 528 nm (a.u.)

Temperature °C

0,1 μM

5

, 0,1 μM DNAs, 50 mM NaCl, 10 mM phosphate buffer pH 7, excitation wavelength: 500 nm.

Salt concentration dependence of fluorescen

ce of 5 with match (left) and mismatch

(right) DNA

0

200

400

600

800

1000

0

2

4

6

8

10

500

520

540

560

580

600

620

640

660

0

2

4

6

8

10

12

14

Intensity (a.u.)

Wavelength/nm

Intensity at 526 nm

mM NaCl

0

200

400

600

800

1000

0

2

4

6

8

10

500

520

540

560

580

600

620

640

660

0

2

4

6

8

10

Fluorescence (a.u.)

W avelength / nm

Fluorescence (a.u.)

NaCl (mM)

0,1 μM

5

, 0,1 μM DNAs, 30-1000 mM NaCl, phosphate buffer pH 7, excitation wavelength: 500 nm.

S 12