study of complex associates between cationic porphyrins and nucleosides or nucleotides magdalena...

StudyStudy of Complex of Complex Associates Between Associates Between

Cationic Porphyrins and Cationic Porphyrins and Nucleosides or Nucleosides or

NucleotidesNucleotides

Magdalena MakarskaMagdalena Makarska, Stanisław Radzki, Stanisław Radzki

Department of Inorganic ChemistryDepartment of Inorganic Chemistry

Maria Curie-Skłodowska University in LublinMaria Curie-Skłodowska University in Lublin

HH22TMePyPTMePyP5,10,15,20-tetrakis[4-(1-methyl-4-5,10,15,20-tetrakis[4-(1-methyl-4-

pyridyl)]porphyrinpyridyl)]porphyrin

N

N

N

N

N N

NN

CH3

CH3

CH3

CH3

+ +

+

+ H3C

4

H

H

SO3

-

CC4444HH3838NN88 · 4(C · 4(C77HH77SOSO33) = 1363.63 g/mol) = 1363.63 g/mol

violet powder, dark purple in solutionviolet powder, dark purple in solution

HH22TTMePPTTMePP5,10,15,20-tetrakis[4-5,10,15,20-tetrakis[4-

(trimethylamino)phenyl]porphyrin(trimethylamino)phenyl]porphyrin

N N

NN

CH3

+

+

H

H

-H3C

4

SO3

NCH3 CH3

CH3

+

NCH3

CH3

CH3

+

N

CH3

CH3

CH3

N

CH3

CH3

CC8484HH9090NN88OO1212SS44 = 1531.95 g/mol = 1531.95 g/mol

dark red powder, red in solutiondark red powder, red in solution

H2TMePyP, [H2O], cuvette 1 cmH2TTMePP, [H2O], cuvette 1 cm

[nm]200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

A

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

500 550 600 650 700 750

0.00

0.02

0.04

0.06

0.08

0.10

0.12

423

414

520, 556, 586, 643

516, 551, 579, 636

Molar absorbancy index for H2TMePyP423 nm - 1.5680520 nm - 0.1045556 nm - 0.0411586 nm - 0.0463643 nm - 0.0135

Molar absorbancy index for H2TTMePP

414 nm - 1.8646516 nm - 0.0706551 nm - 0.0253

579 nm - 0.02752636 nm - 0.0139

Q bandSoret band

H2TMePyP, [H2O], cuvette 1 cmH2TTMePP, [H2O], cuvette 1 cm

[nm]200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

A

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

500 550 600 650 700 750

0.00

0.02

0.04

0.06

0.08

0.10

0.12

423

414

520, 556, 586, 643

516, 551, 579, 636

Molar absorbancy index for H2TMePyP423 nm - 1.5680520 nm - 0.1045556 nm - 0.0411586 nm - 0.0463643 nm - 0.0135

Molar absorbancy index for H2TTMePP

414 nm - 1.8646516 nm - 0.0706551 nm - 0.0253

579 nm - 0.02752636 nm - 0.0139

Q bandSoret band

Molar absorbancy Molar absorbancy indexesindexes

Cu(ac)2 + H2TMePyP

Cu(ac)2 + H2TTMePP

[nm]

200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4A

550

426

413

540

Cu(ac)2 + H2TMePyP

Cu(ac)2 + H2TTMePP

[nm]

200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4A

550

426

413

540

Spectra of porphyrin complexesSpectra of porphyrin complexes with copper (II)with copper (II)

Protonation of HProtonation of H22TTMePP porphyrinTTMePP porphyrin

H2TTMePP + H

2SO

4 [H2O]

[nm]360 380 400 420 440 460 480 500

A

0,00

0,20

0,40

0,60

0,80

1,00

1,20

H2P + 2 H+ = H4P2+

413 nm

436 nm

H2TTMePP + H

2SO

4 [H2O]

[nm]360 380 400 420 440 460 480 500

A

0,00

0,20

0,40

0,60

0,80

1,00

1,20

H2P + 2 H+ = H4P2+

413 nm

436 nm

dication:

H4P2+ H3P

+ + H+ (K4) monocation:

H3P+ H2P + H+ (K3)

free porphyrin:

H2P HP- + H+ (K2) monoanion:

HP- P2- + H+ (K1)

Four porphyrin acid-base equilibriaFour porphyrin acid-base equilibria

M-P

(H2P)2 (H2P)n

M H2P

(H4P2+ H3P

+ H2P HP-)

M-N H2P-N

N

where: H2P – free porphyrins,M – metals,N – nucleic bases, nucleosides and nucleotides.

Scheme of interactions between porphyrins systemsScheme of interactions between porphyrins systems and nucleic bases and their derivativesand nucleic bases and their derivatives

Nucleic bases and their derivativesNucleic bases and their derivatives

adenineadenine

adenosineadenosine

ATPATP

guanineguanine

guanosineguanosine

GTPGTP

Nucleic bases and their derivativesNucleic bases and their derivatives

thyminethymineuraciluracil

uridineuridine

UTPUTP CTPCTP

cytosinecytosine

cytidinecytidinethymidinethymidine

The sites of metal fixation in nucleic basesThe sites of metal fixation in nucleic bases

adenineadenine guanineguanine

thyminethyminecytosinecytosine

uraciluracil

nL

2L P(N)...P(N) PN N+P

N][]P(N)[]P(N)[

K1-n

nn

]P[[N]...KKK...[N]KK+[N]K1

[N]...KKK...]N[KK]N[K=A n

n212

211

nn21n

2212110

Association constantsAssociation constants

A - absorbancy;A - absorbancy;εε0 0 - molar absorbancy index of initial porphyrin concentration;- molar absorbancy index of initial porphyrin concentration;

εε11 and K and K11, ..., etc. - molar absorbancy indexes and gradual stability constants, ..., etc. - molar absorbancy indexes and gradual stability constants

for complexes with stoichiometry 1:1, 1:2, ..., etc;for complexes with stoichiometry 1:1, 1:2, ..., etc;[N] - molar nucleic base (ligand) concentration;[N] - molar nucleic base (ligand) concentration;[P] - molar porphyrin concentration.[P] - molar porphyrin concentration.

Titration experiments (pH=12.4)Titration experiments (pH=12.4)

H2TTMePP [Tris 0.025 M, pH =12.4] + Tris

[nm]350 375 400 425 450 475 500 525 550 575 600 625 650 675 700

A

0.0

0.1

0.20.3

0.4

0.50.6

0.70.8

0.9

1.0

1.1

1.21.3

1.4

1.5

1.6

1.7

1.8

Q band10 x414

421

516Soret band

560


[nm]350 375 400 425 450 475 500 525 550 575 600 625 650 675 700

A

0.0

0.1

0.20.3

0.4

0.50.6

0.70.8

0.9

1.0

1.1

1.21.3

1.4

1.5

1.6

1.7

1.8

Q band10 x414

421

516Soret band

560

H2TTMePP [Tris 0.025 M, pH =12.4] + uracil

[nm]350 375 400 425 450 475 500 525 550 575 600 625 650 675 700

A

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

Q band10 x

414

422

516

Soret band 560

H2TTMePP [Tris 0.025 M, pH =12.4] + uracil

[nm]350 375 400 425 450 475 500 525 550 575 600 625 650 675 700

A

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

Q band10 x

414

422

516

Soret band 560

H2TTMePP [Tris 0.025 M, pH =12.4] + uridine

[nm]350 375 400 425 450 475 500 525 550 575 600 625 650 675 700

A

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

Q band10 x

414

422

516Soret band

560

H2TTMePP [Tris 0.025 M, pH =12.4] + uridine

[nm]350 375 400 425 450 475 500 525 550 575 600 625 650 675 700

A

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

Q band10 x

414

422

516Soret band

560

H2TTMePP [Tris 0.025 M, pH =12.4] + UTP

[nm]350 375 400 425 450 475 500 525 550 575 600 625 650 675 700

A

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

Q band10 x

414

422

516

Soret band

560


[nm]350 375 400 425 450 475 500 525 550 575 600 625 650 675 700

A

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

Q band10 x

414

422

516

Soret band

560

Titration experiments (pH=12.7)Titration experiments (pH=12.7)

H2TTMePP [Tris 0.025 M, pH =12.69] + Uracil

[nm]350 375 400 425 450 475 500 525 550 575 600 625 650 675 700

A

0.00.10.20.30.40.50.60.70.80.91.01.11.21.31.41.51.61.71.81.92.0

Q band10 x

414

422

516

Soret band

560

H2TTMePP [Tris 0.025 M, pH =12.69] + Uracil

[nm]350 375 400 425 450 475 500 525 550 575 600 625 650 675 700

A

0.00.10.20.30.40.50.60.70.80.91.01.11.21.31.41.51.61.71.81.92.0

Q band10 x

414

422

516

Soret band

560

H2TTMePP [Tris 0.025 M, pH =12.69] + Uridine

[nm]

350 375 400 425 450 475 500 525 550 575 600 625 650 675 700

A

0.00.10.20.30.40.50.60.70.80.91.01.11.21.31.41.51.61.71.81.92.0

Q band10 x

414

422

516

Soret band

560

H2TTMePP [Tris 0.025 M, pH =12.69] + Uridine

[nm]

350 375 400 425 450 475 500 525 550 575 600 625 650 675 700

A

0.00.10.20.30.40.50.60.70.80.91.01.11.21.31.41.51.61.71.81.92.0

Q band10 x

414

422

516

Soret band

560


[nm]350 375 400 425 450 475 500 525 550 575 600 625 650 675 700

A

-0.10.00.10.20.30.40.50.60.70.80.91.01.11.21.31.41.51.61.71.81.92.0

Q band10 x

414

422

516Soret band

560


[nm]350 375 400 425 450 475 500 525 550 575 600 625 650 675 700

A

-0.10.00.10.20.30.40.50.60.70.80.91.01.11.21.31.41.51.61.71.81.92.0

Q band10 x

414

422

516Soret band

560


[nm]

350 375 400 425 450 475 500 525 550 575 600 625 650 675 700

A

0,00,10,20,30,40,50,60,70,80,91,01,11,21,31,41,51,61,71,8

Q band10 x

414

422

516Soret band 560


[nm]

350 375 400 425 450 475 500 525 550 575 600 625 650 675 700

A

0,00,10,20,30,40,50,60,70,80,91,01,11,21,31,41,51,61,71,8

Q band10 x

414

422

516Soret band 560

The absorbance changes The absorbance changes in the functionin the function

of porphyrin concentrationof porphyrin concentration

cM H2TTMePP * 1062.5 3.0 3.5 4.0

A

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

H2TTMePP - normalized plotpH = 12.7+ tris+ uracil+ uridine+ UTP

cM H2TTMePP * 1062.5 3.0 3.5 4.0

A

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8


cM H2TTMePP * 1062.5 3.0 3.5 4.0 4.5

A

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0


cM H2TTMePP * 1062.5 3.0 3.5 4.0 4.5

A

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0


Association constantsAssociation constants

H2TMePyP CuTMePyP H2TTMePP CuTTMePP

pH=9.07 (titrant cM = 10-3)

adenine 7 221 29 1 226 16 19 054 28 6 734 10

adenosine 5 271 11 1 281 6 60 240 24 2 690 18

di-Na-ATP 6 912 5 9 346 4 224 460 25 32 440 18


adenine 1.89 3 2 077 15 285 6 129 5

adenosine 17.30 4 15 120 4 535 8 736 8

di-Na-ATP 13.09 3 12 125 8 4 250 9 3 119 9


guanine 57 884 2 26 326 20 177 500

7590 18 029 14

guanosine 2 581 4 4 255 25 147 500

3460 27 980 21

tri-Na-GTP 1 746 8 25 152 13 22 320 460 41 810 7


guanine 10-6 347 26 23 630 640 1 232 22

guanosine 7 8 1 109 8 21 620 840 643 4



adenine 7 221 29 1 226 16 19 054 28 6 734 10

adenosine 5 271 11 1 281 6 60 240 24 2 690 18

di-Na-ATP 6 912 5 9 346 4 224 460 25 32 440 18


adenine 1.89 3 2 077 15 285 6 129 5

adenosine 17.30 4 15 120 4 535 8 736 8

di-Na-ATP 13.09 3 12 125 8 4 250 9 3 119 9


guanine 57 884 2 26 326 20 177 500

7590 18 029 14

guanosine 2 581 4 4 255 25 147 500

3460 27 980 21

tri-Na-GTP 1 746 8 25 152 13 22 320 460 41 810 7


guanine 10-6 347 26 23 630 640 1 232 22

guanosine 7 8 1 109 8 21 620 840 643 4



uracil 1 890 5 5 354 38 95 250 21 9 407 30

uridine 3 756 23 23 733 27 10 989 33 30 123 14

tri-Na-UTP 5 265 4 60 078 20 71 475 16 57 123 13


uracil 441 5 1 634 30 10 935 14 3 363 17

uridine 6 083 6 10 195 8 3 021 20 1 191 23

tri-Na-UTP 6 776 14 46 410 11 5 538 20 4 021 13


thymine 1 289 4 10 111 4 32 527 23 5 967 26

thymidine 3 330 2 19 896 3 9 078 3 10 207 3


thymine 811 2 1 226 20 1 582 4 2 055 3

thymidine 554 2 3 614 19 2 142 19 1 043 4


cytosine 15 128 2 1 680 5 32 340 8 59 733 15

cytidine 4 141 4 4 670 4 21 214 13 22 399 3

tri-Na-CTP 8 326 2 5 418 3 60 664 10 13 149 2


cytosine 849 2 1 229 2 771 2 4 933 12

cytidine 3 716 3 5 124 1 1 042 3 605 3

tri-Na-CTP 1 543 6 9 389 6 6 873 11 1 863 13



uracil 1 890 5 5 354 38 95 250 21 9 407 30

uridine 3 756 23 23 733 27 10 989 33 30 123 14

tri-Na-UTP 5 265 4 60 078 20 71 475 16 57 123 13


uracil 441 5 1 634 30 10 935 14 3 363 17

uridine 6 083 6 10 195 8 3 021 20 1 191 23

tri-Na-UTP 6 776 14 46 410 11 5 538 20 4 021 13


thymine 1 289 4 10 111 4 32 527 23 5 967 26

thymidine 3 330 2 19 896 3 9 078 3 10 207 3


thymine 811 2 1 226 20 1 582 4 2 055 3

thymidine 554 2 3 614 19 2 142 19 1 043 4


cytosine 15 128 2 1 680 5 32 340 8 59 733 15

cytidine 4 141 4 4 670 4 21 214 13 22 399 3

tri-Na-CTP 8 326 2 5 418 3 60 664 10 13 149 2


cytosine 849 2 1 229 2 771 2 4 933 12

cytidine 3 716 3 5 124 1 1 042 3 605 3

tri-Na-CTP 1 543 6 9 389 6 6 873 11 1 863 13

ConclusionsConclusions11. The big differences in the results for particular nucleic agents, and first of all for different pH . The big differences in the results for particular nucleic agents, and first of all for different pH values indicate the diversification ofvalues indicate the diversification of interaction level of investigated systems. Such results can be interaction level of investigated systems. Such results can be

the proof ofmutualthe proof ofmutual interactions porphyrin - nucleic ligand.interactions porphyrin - nucleic ligand.

2. Interactions of H2. Interactions of H22TTMePP with nucleic bases are much stronger than interactions of TTMePP with nucleic bases are much stronger than interactions of

HH22TMePyP. Such effect is caused by the aniline group, much bigger than pyridyl group - TMePyP. Such effect is caused by the aniline group, much bigger than pyridyl group -

“stacking” (specific interactions consisting in forming of associates by reacting molecules) between “stacking” (specific interactions consisting in forming of associates by reacting molecules) between porphyrin and nucleic ligands is much more stronger for bigger compounds.porphyrin and nucleic ligands is much more stronger for bigger compounds.

3. The results show that the more stable associates are formed at lower pH value. Moreover, the 3. The results show that the more stable associates are formed at lower pH value. Moreover, the strength ofstrength of the obtained associates increases in series:nucleic base the obtained associates increases in series:nucleic base nucleoside nucleoside nucleotide, nucleotide,

although many departures from the rule are observed, because of high diversity and different pH although many departures from the rule are observed, because of high diversity and different pH values of investigatedvalues of investigated systems.systems.

4. The association constants for interactions between porphyrins and nucleic agents let us suppose 4. The association constants for interactions between porphyrins and nucleic agents let us suppose that by means of adequate porphyrin substituent groups and metal ion in porphyrin cave it would that by means of adequate porphyrin substituent groups and metal ion in porphyrin cave it would be possible to control the level of interactions between porphyrins and complicated DNA systems be possible to control the level of interactions between porphyrins and complicated DNA systems

and, probabely, introduce metalloporphyrins to the desirable place of DNA chain.and, probabely, introduce metalloporphyrins to the desirable place of DNA chain.

5. However, the substitution reactions proceeding on the metallic centres connected with nucleic 5. However, the substitution reactions proceeding on the metallic centres connected with nucleic acids are much more complicated, what is caused by the interactions of ligands with other groups, acids are much more complicated, what is caused by the interactions of ligands with other groups, as well as conformational changes in a macromolecule. Therefore all data obtained during study of as well as conformational changes in a macromolecule. Therefore all data obtained during study of porphyrin - DNA interactions should be particularly considered, because some porphyrin systems porphyrin - DNA interactions should be particularly considered, because some porphyrin systems

can join and interact with DNA in different ways, depending on neighbouring chemical can join and interact with DNA in different ways, depending on neighbouring chemical compounds. Very often the manner of interactions between reagents is determined by the kind of compounds. Very often the manner of interactions between reagents is determined by the kind of

reaction buffer and ionic strength connected with environment of investigated system.reaction buffer and ionic strength connected with environment of investigated system.

Metalloporphyrin molecule incorporated in DNA chainMetalloporphyrin molecule incorporated in DNA chain

study of complex associates between cationic porphyrins and nucleosides or nucleotides magdalena...

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