Biologically Active Compounds Nina A. Kasyanenko Faculty of Physics - - PowerPoint PPT Presentation

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Biologically Active Compounds Nina A. Kasyanenko Faculty of Physics - - PowerPoint PPT Presentation

Mar 02, 2024 147 likes •417 views

AFM Study of DNA Complexes with Biologically Active Compounds Nina A. Kasyanenko Faculty of Physics St.-Petersburg State University Biologically Active Compounds Metal Ions Trivalent: Coordination compounds NH 3 Cl Fe3+ (FeCl 3)

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SLIDE 1

Nina A. Kasyanenko Faculty of Physics St.-Petersburg State University

AFM Study of DNA Complexes with Biologically Active Compounds

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SLIDE 2

Biologically Active Compounds

  • Metal Ions
  • Coordination

compounds

  • Polyamines
  • Polycations

Trivalent:

  • Fe3+ (FeCl3)
  • La3+ (LaCl3)
  • Al3+

Divalent:

  • Mg2+, Ba2+, Ca2+
  • Mn2+, Ni2+, Cu2+

Spermidine

NH2 – (CH2)3 – NH –(CH2)4 – NH2

Spermine

NH2 – (CH2)3 – NH –(CH2)4 – NH –(CH2)2-NH2

N O C H2 C H2 N O C H2 C H2 O C H2 N C H3 + I- C H3 + I- C H N C H2 C H C C H3 C H2 C O C C H3 C O C H2 C C H3 C H2 C O C C H3 C H2 C C H2 C H2 N C H3 C H3 C H3 C H3 C H3 C H3 C H3 Alc n n n n m n H 3+ H 3+ ( ) 2 ( O N 2 ) O C H2 C H2 C C C C O NH C H2 n C H3 C H3 O O C H2O H H O H H O H n H O H H C H2)2 O C H3 N C H3 (

Coordination compounds Platinum Palladium Cobalt Ruthenium

NH3 NH3 Cl Cl Pt

Pt NH3 Cl NH3 Cl

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SLIDE 3

It is over 100 years since Paul Ehrlich envisioned the development of a “Magic Bullet” a dye carrying a toxic heavy metal which would target disease causing agents while leaving healthy tissue unharmed In his Nobel Lecture on December 11th, 1908 Paul Ehrlich layed out the foundation of chemotherapy and the use of drugs to cure patients.

Paul Ehrlich 1854–1915

Chemotherapy based on toxic metals

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SLIDE 4

N N N N O C H3 O H3C Pt O H NH3 Cl O H Pt Cl O H NH3 NH3 NH3 O H Cl

Pt Cl N NH N C H3 O O H3C N NH3 NH3 Cl

Pt

C l NH 3 NH 3 N C C C H NH C R

O O

Pt

NH 3 NH 3 N C C C H NH C R

O O

HN HC C C N C R

O O

R=Br,H,N O 2

N N N N Pt NH3 NH3

Cl2

H H

Pt

Prdam NH3 NH3 Cl

Pt

Cl NH3 NH3 Cl2

N N Pt Pt NH3 NH3 Cl NH3 NH3 Cl

Cl2 N N

O O C H3 C H3 N N Pt Pt NH3 NH3 Cl NH3 NH3 Cl N N Pt Pt NH3 Cl NH3 Cl NH3 NH3

N N

O O C H3 C H3 Cl Cl Cl N N N N O C H3 O H3C Pt O H NH3 NH3 O H Pt NH3 O H Cl NH3 Cl O H

NH3 NH3 Cl Cl Pt

Pt NH3 Cl NH3 Cl

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SLIDE 5

Palladium Coordination Compounds

.

NH2 NH2 C H2 C H2

Pd

Cl Cl

NH3 Cl NH3 Cl Pd

Pd Br Br Br Br K 2

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SLIDE 6

Coordination Compounds of Cobalt and Ruthenium

Co NH3 NH3 NH3 NH3 NH3 NH3 Cl 3 Cl 3 NH3 NH3 NH3 NH3 NH3 NH3

Ru

Co

3

Na NO 2 NO 2 NO 2 NO 2 NO 2 NO 2 Cl 2

Ru

NH3 NH3 NH3 NH3 NH3 Cl

Ru

Cl NH3 NH3 NH3 NH3 Cl Cl

[Ru(NH3)6] 3+ [Co(NH3)6] 3+ [Co(NO2)6] 3- -> [Co(NO2)(H2O)5] 2+ -> [Co(H2O)6] 3+ [Ru(NH3)4(Cl)2] + [Ru(NH3)5(Cl)] 2+ Iztok Turel University of Ljubljana, Faculty of Chemistry and Chemical Technology

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SLIDE 7

Experimental methods

  • ATOMIC FORCE

MICROSCOPY NanoScope IV, Veeco

  • DYNAMIC LIGHT

SCATTERING PhotoCor, Russia

  • CIRCULAR DICHROISM

Mark IV, Jobin Ivon

  • LOW GRADIENT

VISCOMETRY Zimm-Crozers type viscometer

  • FLOW BIREFRIGENCE

Original technique

  • UV SPECTROSCOPY
  • SF 56, Russia
  • ELECTROPHORESIS

NanoScope IV, Veeco

Dichrograph Mark IV, France UV-VIS SP-56, Russia

Low gradient Rotation viscometer

Flow Birefrigence

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SLIDE 8

Calf Thymus DNA (Sigma)

DNA pFL 44/EcoI (4,4 kbp)

DNA

0.005 M NaCl 5х10-4 M MgCl2 1 M NaCl

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SLIDE 9

DNA is a highly charged polyion

_______________________ DNA interaction with ligands in a solutions is greatly depended on electrostatic interaction:

  • Long-range electrostatic

interactions (responsible for polyelectrolyte swelling)

  • Short-rang electrostatic

interactions (determine the electrostatic contribution to DNA persistent length)

  • Non-electrostatic interaction also

play an important role. Water-salt solution is a good solvent for native DNA (for single-strand it isn't as good as for double strand)

H + v

1 2 3 4 5 6 1 2 3 4 5 6 7 8 9

(C) (G )

N N

O N

HN N

O N H H

N N

H H

  • negative phosphates (at pH>1,5)
  • additional positive or negative charges
  • n nitrogen bases at acid or alkaline pH
R > < > <
  • HN
N O O C H 3 N N O C H 3 OH R N N O C H 3 O R

+ H+

Timine and Guanine can get a negative charge in alkaline area Guanene N7 is the main group for double stranded DNA protonation

Variation in DNA charge density with pH

slide-10
SLIDE 10 3 4 5 6 7 8 9 10 11 0,0 0,5 1,0 1,5 0,023 M NaCl 0,1 M NaCl ([n]/[ ])pH ([n]/[ ])o pH

pH=6.2 pH=11.3 pH=9.5

1 – рН=6,2 2 – рН=11,3 3 – рН=9,5 4 – рН=2,55

2 4 6 8 10 12 10 20 30 40 0,1 M N aC l 0,023 M N aC l [ ], дл/г pH 240 260 280 300
  • 1
1 4,5 4,7

0,005 M NaCl

4,9 4,8 5,3 5,4 6,3

, нм

0,15 M pK=3,1 0,023 pK=4,0 0,005 pK=4,7 Kasyanenko N. et al. Colloids and Surfaces A, 148/1-2 p. 121.(1999)

n g n kT n n

g r s s 2 2 1 2

1 4 45 2

n S

i

LA M

3 2 3 /

H + v 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 (C) (G ) N N O N HN N O N H H N N H H

(1 m)

) ( ) (

2 1 II

S

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SLIDE 11

1M NaCl 0,005 M NaCl

T2 DNA in NaCl solution with Yo-Yo dye Axiolab (Carl Zeis ) Abramchuk S.S. MSU (α ≈ 1.6)

0,1 M NaCl 0,005 М NaCl

20 40 60 1 2 3 4 3 1 I
  • 1/2
at /a0 10 20 30 1 2 3 4 5

I

  • 1/2
3 0,001 I 0,003 I 0,01

I, M

2 1 [ ]/[ ] 0, дл/г

Calf thymus DNA

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SLIDE 12

DNA Complexes with Metal Ions

0,0 0,2 0,4 0,6 0,8 1,0 20 40 60

[ ], дл/г I(Me2+) I

2 4 6 8 10 0,6 0,8 1,0 ( - )/( - )0 CMex10 4,M 1 2 3 4 5 10 20 30 40 50 60 [ ], dl/g C(FeCl3) x 10 5, M 0,0 0,5 1,0 1,5 0,0 0,5 1,0 1,5 1 2

( )/( )o

CFe.10 5, M

Mg2+ Mn2+ Fe3+ Fe3+ Mg2+ Mn2+

1 M NaCl

1 2 3 4 5 10 20 30 40 50 60 70 80 90 [ ], дл/г 0,005 M NaCl 1 M NaCl C(FeCl3) x 10 5, M 0,0 0,2 0,4 0,6 0,8 1,0 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 ( )/( )0 Mg2+(a) Mg2+(b) Ba2+(a) Ba2+(b) Mn2+(a) Mn2+(b) Ni2+(a) Ni2+(b) Cu2+(b) I(Me2+) I 0,0 0,2 0,4 0,6 0,8 1,0 20 40 60 80 [ ], dl/g I(Me2+) I
  • 1a
  • 2a
  • 3a
  • 4a
  • 5a
  • 1b
  • 2b
  • 3b
  • 4b
  • 1c
  • 2c

Fe3+ Me2+ Me2+

0,005 M NaCl

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SLIDE 13

Mn2+ С(alc)=0 %

20 40 60 80 0,0 0,4 0,8 1,2 1,6

sp / sp

С

сп%,v/v Mn Mg DNA Co

Mn2+ С(alc)=20 % Mg2+ С(al)=20 % Mg2+ С(al)=75 %

Mn2+ С(alc)=35 % Mg2+ С(alc)=55 %

0.005 M NaCl

С(Me2+)= 5х10-4М

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SLIDE 14 2 4 6 0,00 0,05 0,10 0,15 0,20 N/N о Z 10 6 , м. O N + C H N (C H2)3 N + (C H2)3 C H3 N + O C H N (C H2)3 N + C H3 C H3 C H3 C H3 I I I C H3 I

DNA 0,005 M NaCl DNA + La 0,005 M NaCl

0,0 0,5 1,0 1,5 2,0 0,0 0,5 1,0 1,5 [n]/[ ] ([n]/[ ])0 Fe3+, 1 M NaCl Fe3+ 0,005 M NaCl La3+, 0,005 M NaCl La3+, 1 M NaCl Al3+, 0,005 M NaCl Co[(H 2O )6] 3+, 0,003 M NaCl Co[(NH 3)6] 3+, 0,003 M NaCl

C(M e3+) x 10

5, M

Co NH3 NH3 NH3 NH3 NH3 NH3 Cl 3

1 2 3 4 5 10 20 30 40 50 60 [ ], dl/g C(FeCl3) x 10 5, M 0,0 0,5 1,0 1,5 0,0 0,5 1,0 1,5 1 2 ( )/( )o CFe.10 5, M

n S

i

n g n kT n n

g r s s 2 2 1 2

1 4 45 2

LA M

3 2 3 /

Bogdanov A. , Abramchuk S.S. MSU

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SLIDE 15

ДНК/LaCl3 в 0.005 М NaCl СДНК=0.0001%, СLaCl3 = 4*10-6 М.

D= (110 ) nm

0.000 0.002 0.004 1E-6 1E-5 1E-4

V IV

CLaCL3,M CДНК,%

I

II

III

4 8 12 0,0 0,5 1,0 1,5 ( )o 1 M NaCl С х 10 6,M 0,005 M NaCl 2 4 6 8 10 20 40 60 80 100 1 M 0,005 M x x x x x x x x x Cx10 7, M [ ], дл/г 0,0 0,1 0,2 0,3 0,4 0,5 1,0 1,5 ( )/( ) C(spm)x10 5, M Spm 0,0 0,5 1,0 1,5 2,0 2,5 3,0 0,0 0,2 0,4 0,6 0,8 1,0 1,2 C(spd)x10 4, M ( )/( )0 Spd

La3+ La3+ La3+ Spm 4+ Spd 3+ La3+ La3+ La3+ La3+ Spd

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SLIDE 16

C(La)=4*10-6M C(et)=75% C(et)=75% C(MgCl C(MgCl2)=5 10 )=5 10-4M С(et)=65% C(CoCl2)=5 10 C(CoCl2)=5 10-4M 4M

La3+ et + Mg2+ et + Co2+

10
  • 4
10
  • 2
10 10 2 10 4 10 6 10 8 10 10 10 12
  • 0,02
0,00 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16 0,18 0,20 60 , мкс W

0,0 1,0x10

  • 4

2,0x10

  • 4

0,4 0,8 1,2

RH/(RH)0 C, M

(1) (2) (3)

0,0 1,0x10
  • 4 2,0x10
  • 4 3,0x10
  • 4 4,0x10
  • 4
0,0 0,5 1,0 1,5 C,M

Rh/(Rh)0

CoHex SPD PLL

Kasyanenko et al. Struct.Chem.2007

3 / 1 1 17 3 / 1

10 4 , 3 ) ] ([ моль град Дж T M D A

R=(65±10) nm

NH2 – (CH2)3 – NH –(CH2)4 –NH2

0,0 5,0x10 91,0x10 10 1,5x10 10 2,0x10 10 2,5x10 10 3,0x10 10 3,5x10 10 4,0x10 10 0,0 5,0x10 2 1,0x10 3 1,5x10 3 2,0x10 3 2,5x10 3 1/ (sec
  • 1)
q 2 (cm
  • 2)
10
  • 4
10
  • 2
10 10 2 10 4 10 6 10 8 10 10 10 12
  • 0,02
0,00 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16 0,18 0,20 60 , мкс W

1 M NaCl 0.005 M NaCl C(La)=4*10-6M

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SLIDE 17

0,7 N+/P N+/P 1 1,4 1

DNA complexes with polycations

DNA pFL 44

O NH CH2OH OH OH OH C O C CH2 H3C S CH2 CH2 NH C O H C H2 C C H2 C C O O CH2 CH2 N CH3 H3C CH3 p m n O NH CH2OH OH OH OH C O C CH2 H3C S CH2 CH2 NH C O H C H2 C C H2 C C O O CH2 CH2 N CH3 H3C CH3 p m n

N+/P N+/P 2

0,0 0,5 1,0 1,5 2,0 2,5 0,0 0,5 1,0 1,5 PVA15 PAA MAG-DMAEM, 55: 45 моль% sp/ sp0 N +/P 0,01 0,1 1 10 0,0 0,5 1,0 ( 1- 2)0 ( 1- 2)

N/P

220 240 260 280 300
  • 4
  • 2
2 4 6 N/P=0 N/P=1,0 N/P=1,2 N/P=1,3 N/P=,4 N/P=2,5 N/P=0,7

,нм

220 240 260 280 300
  • 4
  • 2
2 N/P=1,2 N/P=3,3 N/P=6,6 N/P=0

нм

10
  • 3
10
  • 1
10 1 10 3 10 5 10 7 0,00 0,05 0,10 0,15 0,20 0,25 G 90 ,мкс

D=115 20 nm

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SLIDE 18 Рисунок 1. АСМ-изображение ДНК pFL44s/EcoRI и ее комплексов с цисДДП и трансДДП. 1- ДНК; 2- ДНК+цисДДП; 3- ДНК+транс-ДДП; 4-(ДНК+цис-ДДП)+транс-ДДП; 5- (ДНК+транс- ДДП)+цис-ДДП; 6- ДНК+цис-ДДП+транс-ДДП.

1) 2) 3) 4) 5) 6)

DNA DNA+cis-DDP DNA+trans-DDP (DNA+cis)+trans (DNA+trans)+cis DNA+cis+trans Alexei Bogdanov (poster)

slide-19
SLIDE 19

DNA with cis-DDP

240 260 280 300
  • 1
1 4,50 4,78 4,84 4,90 5,28 5,40 6,3

, nm

220 240 260 280 300

  • 2

2

ДНК при С(Pt)=0 4,86 4,75 4,71 4,65 4,51 4,4 4,23 4,1

, нм

DNA+cis-DDP DNA

220 240 260 280 300 320
  • 4
  • 2
2 141

,нм

Рис. 4.44. Спектры КД ДНК в комплексе с La 3+(C=10
  • 6M) в 0,005M NaCl при разных рН
pH=5,6 pH=4,7 pH=4,5 pH=4,3 pH=4,1 pH=3,9

DNA + La

slide-20
SLIDE 20

DNA Complexes with Palladium Compounds

Pd Cl Cl Cl Cl

(HL)2

1 - 2 - 1 1 1 2 2

slide-21
SLIDE 21

0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0

K2[PdBr4] морфодон эфазол C 104 sp/ sp0 0.0 0.3 0.6 0.9 0.0 0.5 1.0

/

C 104

1 in 0,005 М NaCl 1 in 0,15 М NaCl 2 in 0.005 M NaCl K2[PtCl4]

DNA

1 2 K2[PdCl4]

Pd Cl Cl Cl Cl

(HL)2

1 - 2 -

DNA complexes with Palladium compounds

slide-22
SLIDE 22

0.0 0.5 1.0 1.5 2.0 0.0 0.2 0.4 0.6 0.8 1.0 C1 P2 P1 sp/ sp0

C 10

4

С1 Р2 DNA DNA+С1 DNA+Р1 DNA+Р2 DNA Р2 Р2 Р1 Р1 С1 С1 P1

250 300 350 400 450 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7

250 300 350 400 450 500

  • 3
  • 2
  • 1

1 2

ДНК ДНК + Р1 ДНК + Р2 ДНК + С1

, нм

С1 Р1 Р2

D , нм

slide-23
SLIDE 23

250 300 350 400 450

  • 1.5
  • 1.0
  • 0.5

0.0 0.5 1.0

DNA DNA+P1+P2 (DNA+P1)+P2 DNA+C1

, нм

DNA DNA+C1 (1) DNA+C1 (2) DNA+C1 from(2)

DNA-C1 complex can dissociate after dilution:

DNA-C1 complex can be formed by the addition of P1 and P2 (one by one) into DNA solution

slide-24
SLIDE 24

Participants of researches:

Saint Petersburg State University

Faculty of Physics Students: Elena Levykina, Ludmila Lysialova, Igor Pereviazko, Petr Sokolov, Daria Afanasieva, Ivan Volkov, Zahar Reveguk PhD students: Boris Dribinsky, Dmitry Mukhin Yulia Zaichikova Department of Genetics Dr. V.V. Alenin (circular DNA)

Institute of Macromolecular Compounds RAS, SPB

  • Dr. Olga Nazarova, (synthesis of polycations)
  • Prof. Eugenii Panarin

Institute of Influenza RAMS, SPB

  • Dr. Alexander Slita

Students of SPbU: Nadezda Kovtun Olga Matveeva

St.-PetersburgChemical and Pharmaceutical Academy

  • Dr. Konstantin Yakovlev (synthesis of platinum complexes)

Kurnakov Inst. of General and Inorganic Chemistry

  • Dr. Inessa Efimanko (synthesis of palladium complexes)

St.-Petersburg Technological Institute

  • Dr. Victor Demidov (synthesis of palladium complexes)
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SLIDE 25

Thank you for the attention