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

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) NH 3 Cl • Coordination • Pt La3+ (LaCl 3 ) Pt • NH 3 Cl compounds Al3+ Cl NH 3 Platinum Divalent: • Polyamines • Palladium Mg2+, Ba2+, Ca2+ • Polycations • Cobalt Mn2+, Ni2+, Cu2+ Ruthenium C H 3 C H 3 C H 3 C H C H 2 C H C H 2 C H 2 C n n n C C H 2 C H 2 C H 3 + C H 2 N C O n n Spermidine O N H 3 + C O C O C H 2 C H 2 O NH N C H 3 C H 3 H ( C H 2 ) 2 NH 2 – (CH 2 ) 3 – NH – (CH 2 ) 4 – NH 2 O H O H H O H N H H C H 3 C H 3 O C H 3 C H 3 C H 3 Spermine C H 2 C H 2 C C C H 2 C n C H 2 O H n m C O C O C O O O O C H 2 ( C H 2 ) NH 2 – (CH 2 ) 3 – NH – (CH 2 ) 4 – NH – (CH 2 ) 2 -NH 2 ( C H 2 ) 2 2 C H 2 I- I- + N N + N C H 3 C H 3 C H 3 C H 3 C H 3 C H 3 Alc C H 3

Chemotherapy based on toxic metals 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

NH 3 NH 3 Cl Cl Pt NH 3 NH 3 Pt NH 3 NH 3 O Pt O O Pt Cl NH 3 NH 3 Cl C C C HN N C l N NH N NH HC C C C H C C H H N C C O O O C N NH NH 3 R R R N R=Br,H,N O 2 Pt Cl 2 H 3 C N O Cl NH 3 Pt NH 3 Cl N N NH 3 O C H 3 NH 3 NH 3 NH 3 NH 3 H N Cl 2 NH 3 Pt N N Pt NH 3 Pt Cl Prdam Pt Cl 2 Cl Cl Cl NH 3 NH 3 O H O H NH 3 NH 3 Pt N N Pt Cl NH 3 NH 3 NH 3 NH 3 NH 3 NH 3 O H Cl Cl O H Cl Pt N N Pt Cl NH 3 Pt N N Pt NH 3 N O H 3 C NH 3 NH 3 Cl Cl N Cl Cl C H 3 O N O N C H 3 O C H 3 N N O O O H NH 3 O H C H 3 C H 3 NH 3 Pt N N Pt Cl Cl Cl O H NH 3 NH 3 O H N O H 3 C N C H 3 O

Palladium Coordination Compounds NH 3 Cl Cl NH 2 H 2 C Pd Pd H 2 C Cl NH 2 Cl NH 3 . Br Br K 2 Pd Br Br

Coordination Compounds of Cobalt and Ruthenium NH 3 NO 2 NO 2 NH 3 NH 3 NO 2 Na Co Cl 3 Co 3 NO 2 NO 2 NH 3 NH 3 NH 3 NO 2 [Co(NH 3 ) 6 ] 3+ Iztok Turel University of Ljubljana, Faculty of Chemistry and Chemical [Co(NO 2 ) 6 ] 3- -> [Co(NO 2 )(H 2 O) 5 ] 2+ -> [Co(H 2 O) 6 ] 3+ Technology NH 3 NH 3 NH 3 NH 3 NH 3 NH 3 Cl NH 3 NH 3 Ru Cl 3 Ru Cl 2 Ru Cl Cl Cl NH 3 NH 3 NH 3 NH 3 NH 3 NH 3 NH 3 [Ru(NH 3 ) 6 ] 3+ [Ru(NH 3 ) 4 (Cl) 2 ] + [Ru(NH 3 ) 5 (Cl)] 2+

Experimental methods • ATOMIC FORCE MICROSCOPY NanoScope IV, Veeco NanoScope IV, Veeco • DYNAMIC LIGHT SCATTERING PhotoCor, Russia • CIRCULAR DICHROISM Mark IV, Jobin Ivon • LOW GRADIENT UV-VIS SP-56, VISCOMETRY Dichrograph Mark IV, Russia France Zimm-Crozers type viscometer • FLOW BIREFRIGENCE Original technique • UV SPECTROSCOPY • SF 56, Russia • ELECTROPHORESIS Flow Low gradient Birefrigence Rotation viscometer

DNA Calf Thymus DNA (Sigma) DNA pFL 44/EcoI (4,4 kbp) 0.005 M NaCl 5х10 -4 M MgCl 2 1 M NaCl

Variation in DNA charge density with pH DNA is a highly charged polyion _______________________ • negative phosphates (at pH>1,5) DNA interaction with ligands in a • additional positive or negative charges solutions is greatly depended on on nitrogen bases at acid or alkaline pH electrostatic interaction: H + H v - Long-range electrostatic N O N 7 H interactions (responsible for 8 6 5 4 5 9 polyelectrolyte swelling) N HN 1 N 4 6 3 (G ) (C) 2 3 2 1 N N H N - Short-rang electrostatic O H interactions (determine the electrostatic contribution to DNA persistent length) Guanene N7 is the main group for double stranded DNA protonation - Non-electrostatic interaction also O - OH O play an important role. Water-salt C H 3 C H 3 C H 3 HN N N + H+ < > < > solution is a good solvent for native DNA (for single-strand it O N O N O N R R R isn't as good as for double strand) Timine and Guanine can get a negative charge in alkaline area

H + H v 0,1 M N aC l N O [ ], дл/г 0,023 M N aC l N H 7 40 8 6 5 4 5 9 N HN 1 N 4 6 3 30 (C) 2 3 (G ) 1 2 N N H N O 20 H 10 0 2 4 6 8 10 12 pH ([n]/[ ]) pH ([n]/[ ]) o 1,5 0,023 M NaCl 1,0 0,1 M NaCl 0,5 pH=6.2 pH=11.3 pH=9.5 (1 m) 0,0 3 4 5 6 7 8 9 10 11 pH 1 – рН=6,2 6,3 2 1 2 5,4 n g 2 – рН=11,3 n 2 n 4 5,3 s g 0 4,9 4,5 3 – рН=9,5 4,8 1 2 4,7 1 45 kT n 4 – рН=2,55 r 0 s 0 0,15 M n 0,005 M NaCl i S pK=3,1 -1 0,023 240 260 280 300 ( ) S ( ) , нм 1 2 II pK=4,0 3 2 / LA Kasyanenko N. et al. Colloids and 0,005 3 Surfaces A, 148/1-2 p. 121.(1999) pK=4,7 M

4 a t /a 0 5 3 [ ]/[ ] 0 , дл/г 2 1 4 2 1 3 1 3 0 20 40 60 -1/2 I 3 2 1 I, M 0,01 0,003 0,001 I I 0 10 20 30 -1/2 I 1M NaCl 0,1 M NaCl T2 DNA in NaCl solution with Yo-Yo dye Axiolab (Carl Zeis ) Abramchuk S.S. MSU 0,005 M NaCl 0,005 М NaCl ( α ≈ 1.6) Calf thymus DNA

1 M NaCl DNA Complexes with Metal Ions [ ], дл/г 60 40 Mg2+ 20 Mn2+ 0 0,0 0,2 0,4 0,6 0,8 1,0 I(Me2+) I 60 [ ], dl/g Fe3+ 50 40 30 20 10 0 0 1 2 3 4 5 5 , M C(FeCl 3 ) x 10 ( - )/( - ) 0 Mg2+ 1,0 Mn2+ 0,8 0,005 M NaCl 0,6 0 2 4 6 8 10 4 ,M C Me x10 Me2+ Me2+ Fe3+ Fe3+ ( )/( ) 0 [ ], дл/г [ ], dl/g 1,4 90 ( )/( ) o 80 1,2 80 0,005 M NaCl 1,5 70 1 M NaCl 1,0 2 60 60 Mg2+(a) 0,8 Mg2+(b) 50 1,0 1 Ba2+(a) 40 Ba2+(b) 0,6 40 Mn2+(a) Mn2+(b) 30 -1a -2a -3a -4a -5a -1b Ni2+(a) 0,4 -2b -3b -4b Ni2+(b) 20 0,5 20 Cu2+(b) -1c -2c 0,2 10 0 0,0 0 0 1 2 3 4 5 0,0 0,2 0,4 0,6 0,8 1,0 0,0 0,2 0,4 0,6 0,8 1,0 0,0 0,0 0,5 1,0 1,5 I(Me2+) 5 , M I(Me2+) C(FeCl 3 ) x 10 5 , M C Fe .10 I I

С( Me 2+)= 5х10 -4 М 1,6 sp / sp 0.005 M NaCl 0 Mn 1,2 Mg DNA Co 0,8 0,4 0,0 Mn2+ С( alc)=20 % Mn2+ С( alc)=0 % 0 20 40 60 80 С сп %,v/v Mg2+ С( al)=20 % Mg2+ С( al)=75 % Mn2+ С( alc)=35 % Mg2+ С( alc)=55 %

NH 3 [n]/[ ] ([n]/[ ]) 0 NH 3 NH 3 Cl 3 Co DNA 1,5 NH 3 NH 3 NH 3 0,005 M NaCl 1,0 Fe3+, 1 M NaCl Fe3+ 0,005 M NaCl La3+, 0,005 M NaCl La3+, 1 M NaCl 0,5 Al3+, 0,005 M NaCl 3+ , 0,003 M NaCl Co[(H 2 O ) 6 ] n 3+ , 0,003 M NaCl Co[(NH 3 ) 6 ] i S 0,0 0,0 0,5 1,0 1,5 2,0 5 , M C(M e3+) x 10 ( )/( ) o 60 [ ], dl/g 1,5 50 2 40 1 1,0 30 20 0,5 10 0 0,0 0,0 0,5 1,0 1,5 0 1 2 3 4 5 5 , M C Fe .10 5 , M C(FeCl 3 ) x 10 DNA + La 2 3 2 / 0,005 M NaCl 2 n g LA n 2 n 4 s g 0 3 1 2 1 45 kT n M r 0 s C H 3 C H 3 + + 0,20 N N I I O O 0,15 C H C H N/N о 0,10 I I N N Bogdanov A. , 0,05 C H 3 C H 3 + + Abramchuk S.S. MSU (C H 2 ) 3 N (C H 2 ) 3 N (C H 2 ) 3 0,00 0 2 4 6 C H 3 C H 3 6 , м. Z 10

La3+ La3+ La3+ C LaCL3 ,M V 1E-4 II IV III 1E-5 I 1E-6 C ДНК ,% 0.000 0.002 0.004 La3+ 100 La3+ La3+ Spd ( ) o 0,005 M NaCl 1,5 80 1 M NaCl [ ], дл/г x 60 x 1,0 0,005 M x x x 40 x x x x 0,5 1 M 20 6 ,M С х 10 0 0,0 0 2 4 6 8 10 7 , M Cx10 0 4 8 12 Spm ( )/( ) 0 Spd La3+ ( )/( ) 1,5 0 Spm 4+ Spd 3+ 1,2 1,0 1,0 0,8 0,6 0,5 0,4 D= (110 ) nm 0,2 0,0 0,0 0,5 1,0 1,5 2,0 2,5 3,0 0,0 0,1 0,2 0,3 0,4 ДНК/ LaCl 3 в 0.005 М NaCl 5 , M 4 , M C(spm)x10 C(spd)x10 С ДНК =0.0001%, С LaCl3 = 4*10 -6 М.

0,20 W R H /(R H ) 0 0,18 0 60 0,16 0,14 0,12 0,10 La3+ 0,08 0,06 1,2 0,04 0,02 0,00 , мкс -0,02 10 -4 10 -2 10 0 10 2 10 4 10 6 10 8 10 10 10 12 (1) 0,8 (2) (3) 0,4 C, M 0,0 -4 -4 1,0x10 2,0x10 0.005 M NaCl NH2 – (CH2)3 – NH – (CH2)4 – NH2 R h /(R h ) 0 C(La)=4*10 -6 M 1,5 CoHex SPD PLL 1,0 1 M NaCl 0,5 C,M 0,0 -4 2,0x10 -4 3,0x10 -4 4,0x10 -4 0,0 1,0x10 -1 ) 1/ (sec 0,20 W 0,18 0 60 0,16 2,5x10 3 et + Mg2+ 0,14 0,12 2,0x10 3 et + Co2+ 0,10 1,5x10 3 0,08 0,06 3 1,0x10 0,04 0,02 2 5,0x10 q 2 (cm -2 ) 0,00 , мкс 0,0 -0,02 0,0 5,0x10 9 1,0x10 10 1,5x10 10 2,0x10 10 2,5x10 10 3,0x10 10 3,5x10 10 4,0x10 10 10 -4 10 -2 10 0 10 2 10 4 10 6 10 8 10 10 10 12 С( et)=65% C(La)=4*10 -6 M C(et)=75% C(et)=75% C(CoCl2)=5 10 C(CoCl2)=5 10-4M 4M C(MgCl C(MgCl2)=5 10 )=5 10 -4 M R=(65 ± 10) nm 1 / 3 D ([ ] M ) 17 1 1 / 3 0 A 3 , 4 10 Дж град моль 0 Kasyanenko et al. Struct.Chem.2007 T

G sp / 0 90 PVA15 0,25 sp0 PAA DNA complexes with polycations 1,5 MAG-DMAEM, 55: 45 моль% 0,20 0,15 0,10 1,0 0,05 ,мкс 0,00 0,5 -3 -1 1 3 5 7 10 10 10 10 10 10 + /P N 0,0 CH 3 0,0 0,5 1,0 1,5 2,0 2,5 H 2 H 2 H 2 C C C C m n C O C O ( 1 - 2 ) 0 NH O ( 1 - 2 ) 0 CH 2 N/P=1,2 CH 2 N/P=3,3 CH 2 N/P=6,6 1,0 CH 2 -2 N/P=0 S N O CH 3 нм H 3 C OH H 3 C C C NH -4 OH 0,5 220 240 260 280 300 CH 2 OH CH 2 OH p O N/P 0,0 0,01 0,1 1 10 N/P=0 N/P=1,0 N/P=1,2 6 N/P=1,3 N/P=,4 4 N/P=2,5 N/P=0,7 2 0 -2 , нм -4 D=115 20 nm 220 240 260 280 300 0 DNA pFL 44 0,7 N + /P N + /P CH 3 H 2 H 2 H C C C C m n C O C O NH O CH 2 CH 2 CH 2 CH 2 S N O H 3 C CH 3 OH H 3 C C C NH OH CH 2 OH CH 2 OH p O 1 1,4 N + /P N + /P 2 1