Acridine Mutagen, Anthracycline Antitumor, and ... - ACS Publications

11 D r u g - D N A Interactions in Solution: Acridine Mutagen, Anthracycline Antitumor, and Peptide

Downloaded by EAST CAROLINA UNIV on January 22, 2016 | http://pubs.acs.org Publication Date: November 10, 1980 | doi: 10.1021/bk-1980-0142.ch011

Antibiotic Complexes DINSHAW J. PATEL Bell Laboratories, Murray Hill, NJ 07974 The successful application of high resolution nuclear magnetic resonance (NMR) spectroscopy to monitor the structure and dynamics of the helix-coil transition of oligonucleotide duplexes (1-4) and transfer RNA (5-9) in solution have prompted efforts in our laboratory to extend these investigations to the polynucleotide duplex level in solution (10,11). Proton and phosphorus NMR spectra of DNA in solution show very large line widths and poor spectral resolution despite recent demonstrations of time-averaged flexibility of the double-helical state (12-15). We have circumvented the sequence dependent dispersion of the chemical shifts by investigating synthetic DNA's of defined repeating nucleotide sequence.[1] Thermodynamic and kinetic studies have demonstrated that the alternating purine-pyrimidine polynucleotide poly(dA-dT) folds into smaller duplexes which melt independently of each other in solution (16,17). The NMR spectrum should be considerably simplified since all base pairs are structurally equivalent due to the symmetry of the alternating purine-pyrimidine duplex. Further, the NMR resonances may exhibit moderate line widths in the duplex state due to segmental mobility resulting from the rapid migration of the branched duplexes along the polynucleotide backbone. Finally, since the smaller duplexes melt independently of each other, the duplex dissociation rate constants may be on the NMR time scale, so that the resonances shift as average peaks through the melting transition. These features suggest that well resolved base and sugar resonances may be observable for high molecular weight alternating purine-pyrimidine polynucleotides in the duplex state and this has been confirmed experimentally (18) in studies of the duplex to strand transition of poly(dA-dT) (mol. wt. VL06, 2i2000 base pairs) as a function of temperature.[2] The NMR resonances of the polynucleotide duplexes at the Watson-Crick protons, the base and sugar protons and the backbone phosphates would provide sufficient markers to monitor ligandnucleic acid interactions in solution. Such an approach has great potential in adding to our current knowledge of the interactions 0-8412-0594-9/80/47-142-219$16.40/0 © iJoO American Chemical Society In Polymer Characterization by ESR and NMR; Woodward, Arthur E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


220

POLYMER

CHARACTERIZATION

B Y ESR A N D N M R

of a n t i b i o t i c s and carcinogens w i t h n u c l e i c a c i d s i n s o l u t i o n (19-21) and p r o v i d e s experimental probes to i n v e s t i g a t e the r o l e of symmetry i n such r e c o g n i t i o n processes (22,23). We f i r s t describe the NMR parameters f o r the duplex to strand t r a n s i t i o n of the s y n t h e t i c DNA poly(dA-dT) (18) w i t h o c c a s i o n a l r e f e r e n c e t o poly(dA-dU) (24) and poly(dA-^brdU) and the c o r r e s ponding s y n t h e t i c RNA poly(A-U) (24). This i s f o l l o w e d by a comp a r i s o n of the NMR parameters of the s y n t h e t i c DNA i n the presence of 1 M Na i o n and 1 M tetramethylammonium i o n i n an attempt to i n v e s t i g a t e the e f f e c t of c o u n t e r i o n on the conformation and s t a b i l i t y of DNA. We next o u t l i n e s t r u c t u r a l and dynamical a s pects of the complexes of poly(dA-dT) w i t h the mutagen p r o f l a v i n e (25) and the anti-tumor agent daunomycin (26) which i n t e r c a l a t e between base p a i r s and the p e p t i d e a n t i b i o t i c n e t r o p s i n (27) which binds i n the groove of DNA. S y n t h e t i c DNA.

A l t e r n a t i n g Adenosine-Uridine Sequences

This c o n t r i b u t i o n complements an e a r l i e r review (11) which summarized our NMR research on s y n t h e t i c DNA's and RNA's w i t h a l t e r n a t i n g i n o s i n e - c y t i d i n e and g u a n o s i n e - c y t i d i n e p o l y n u c l e o t i d e s and the s t r u c t u r e and dynamics of e t h i d i u m - n u c l e i c a c i d complexes. Hydrogen Bonding i n Duplex S t a t e : The base p a i r e d duplex s t a t e i n n u c l e i c a c i d s can be r e a d i l y c h a r a c t e r i z e d by m o n i t o r i n g the exchangeable imino protons i n H 2 O s o l u t i o n (28-32). E a r l i e r s t u d i e s have demonstrated that the thymidine H-3 imino p r o t o n of nonterminal base p a i r s i n n u c l e i c a c i d duplexes are i n slow exchange w i t h s o l v e n t H 2 O and resonate between 12 and 15 ppm. The 360 MHz p r o t o n NMR s p e c t r a of poly(dA-dT) i n 0.1 M phosphate, H 2 O s o l u t i o n (6 t o 14 ppm) were recorded between 0° and 55°C and the exchangeable protons i d e n t i f i e d by comparison w i t h the c o r r e s ponding s p e c t r a recorded i n ^ ^ 0 s o l u t i o n . The thymidine H-3 imino hydrogen-bonded resonance i s observed a t 13.0 ppm i n the spectrum of poly(dA-dT) a t 25.5°C (Figure 1A) and i t s chemical s h i f t and l i n e w i d t h dependence a r e p l o t t e d as a f u n c t i o n of temperature i n F i g u r e s IB and 1C r e s p e c t i v e l y . The l i n e a r u p f i e l d s h i f t of the exchangeable resonance w i t h i n c r e a s i n g temperature (Figure IB) r e f l e c t s , i n p a r t , c o n t r i b u t i o n s from a temperature dependent premelting conformational change which i s a l s o observed f o r the nonexchangeable protons (see below). The l i n e width of the exchangeable resonance increases from V70 Hz (20° to 40°C) to VL50 Hz (54°C) (Figure 1C) which suggests t h a t the l i f e t i m e of the thymidine H-3 imino p r o t o n i n the Watson-Crick hydrogen bond decreases w i t h i n c r e a s i n g temperat u r e on approaching the m e l t i n g t r a n s i t i o n r e g i o n (midpoint = 59.1°C) of poly(dA-dT) i n 0.1 M phosphate s o l u t i o n .

In Polymer Characterization by ESR and NMR; Woodward, Arthur E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


11.

PATEL

0

Drug-DNA

Interactions

10 20 30 40 TEMPERATURE, c #

50

60

in Solution

221

Figure 1. (A) The 360-MHz proton NMR spectrum of the thymidine H-3 proton in polv(dA-dT) in 0.1 M phosphate, JmM EDTA, H>0 at 25.5°C. The (B) chemical shifts and (C) linewidths of this proton in the synthetic DNA in 0.1 M phosphate are plotted as a function of temperature between 0° and 55°C. The poly(dA-dT) duplex exhibits a duplex-tostrand transition midpoint of 59°C.


POLYMER

222

CHARACTERIZATION

B Y ESR A N D N M R

Duplex to Strand T r a n s i t i o n : The temperature dependence of the 360 MHz proton NMR s p e c t r a (9 to 4.5 ppm) of poly(dA-dT) i n 0.1 M phosphate b u f f e r , 2^0, are presented i n F i g u r e 2. P a r t i a l l y r e s o l v a b l e resonances a r e observed i n the duplex s t a t e (48°C) w i t h l i n e widths of ^50 Hz and w e l l r e s o l v e d resonances are observed i n the strand s t a t e (67°C) w i t h l i n e widths of a few Hz. The base resonances can be r e a d i l y assigned from t h e i r chemical s h i f t p o s i t i o n s a t h i g h temperature w h i l e we cannot d e f i n i t i v e l y d i f f e r e n t i a t e between sugar H - l * (and sugar H-3 ) resonances l i n k e d t o the adenosine and the thymidine r e s i d u e s (Figure 2 ) . The duplex to strand c o n v e r s i o n occurs by a c o o p e r a t i v e t r a n s i t i o n a t 59°C w i t h the observable resonances s h i f t i n g as average peaks during the m e l t i n g t r a n s i t i o n . The temperature dependence of the chemical s h i f t s of the base and sugar resonances of poly(dA-dT) i n 0.1 M phosphate b u f f e r i s p l o t t e d i n F i g u r e 3. There are u p f i e l d and d o w n f i e l d s h i f t s a s s o c i a t e d w i t h the noncooperative p r e m e l t i n g t r a n s i t i o n between 5° and 55°C w h i l e only d o w n f i e l d s h i f t s are observed f o r most of the base and sugar protons on r a i s i n g the temperature above 65°C i n the noncooperative p o s t m e l t i n g t r a n s i t i o n temperature range. The c o o p e r a t i v e m e l t i n g t r a n s i t i o n (midpoint, tj^ = 59.0°C) exh i b i t s downfield s h i f t s a t the base and sugar H - l protons w i t h i n c r e a s i n g temperature but not a t a l l the remaining sugar protons (Figure 3 ) .


f

1

Resonance Assignments: The adenosine H-2 and thymidine H-6 resonances of poly(dA-dT) e x h i b i t s i m i l a r chemical s h i f t s around 40°C i n 0.1 M phosphate s o l u t i o n (Figure 2). We have evaluated the s p i n l a t t i c e proton r e l a x a t i o n times, T]_, of the base and sugar H-1 protons of poly(dA-dT) through the p r e m e l t i n g t r a n s i t i o n and observe that the adenosine H-2 resonance e x h i b i t a T^ value which i s 2 to 3 times l o n g e r than that of the remaining resonances. Thus the adenosine H-2 resonance can be r e a d i l y d i f f e r e n t i a t e d from the thymidine H-6 resonance s i n c e i t e x h i b i t s a narrower l i n e w i d t h i n d i c a t i v e of a longer s p i n - s p i n r e l a x a t i o n time, T 2 , and a longer s p i n - l a t t i c e r e l a x a t i o n time T-^ i n the duplex s t a t e i n s o l u t i o n . The experimental data r e q u i r e that the adenosine H-2 and the thymidine H-6 resonances cross over a t 50°C (Figure 3 ) . F u r t h e r r e s o l u t i o n of the 4.5 to 9.0 ppm r e g i o n of poly(dAdT) i n the duplex s t a t e can be achieved by i n v e s t i g a t i n g the r e l a t e d p o l y n u c l e o t i d e f o r which bromine r e p l a c e s the methyl group a t p o s i t i o n 5 of the p y r i m i d i n e r i n g . This i s r e a d i l y obs e r v a b l e on comparing the s p e c t r a of poly(dA-dT) and poly(dA5brdU) duplexes i n the aromatic r e g i o n a t 43°C (Figure 4 ) . The p y r i m i d i n e H-6 proton s h i f t s 0.57 ppm downfield from 7.05 ppm i n poly(dA-dT) t o 7.65 ppm i n poly(dA-^brdU) at t h i s temperature (Figure 4 ) . A s i m i l a r downfield s h i f t of 0.68 ppm i s observed a t the p y r i m i d i n e H-3 exchangeable proton which s h i f t s from 12.95 ppm i n poly(dA-dT) to 13.65 ppm i n poly(dA-5brdU) a t 37°C. T



11.

PATEL

Drug-DNA

Interactions

in Solution

223

A(H-2)

Figure 2. The 360-MHz proton NMR spectra (4.5 to 9.0 ppm) of poly(dA-dT) in 0.1M phosphate, ImM EDTA, H,0, ptl 6.3 in the duplex state (48°C), during the melting transition (61 °C) and in the strand state (67°C) 2



224

POLYMER

CHARACTERIZATION

B Y ESR A N D N M R

Figure 3. The temperature dependence (5° to 95°C) of the base and sugar proton chemical shifts of poly(dA-dT) in 0.1M phosphate, ImM EDTA, H.O, pH 7.0 2



11.

PATEL

Drug-DNA

Interactions

225

in Solution

A(H-2) T(H-6) Poly(dA-dT) (Η-Ι')

(Η-Ι')

(Η-Ι')

(Η-Γ)

A(H-8)

Poly(dA-brdU)

A(H-2) brU(H 6) A

A(H-8)

1 9

/

Λ

1

1

I

I

8

7

6

5

Figure 4. The 360-MHz proton NMR spectra of poly(dA-dT) in 0.1M phosphate, ImM EDTA, H>0, pH 7, 43°C and poly(dA- brdU) in 0.1M NaCl, WmM phos phate, IIΌ, pH 8.1,43°C 2

5

2


226

POLYMER

CHARACTERIZATION

B Y ESR A N D N M R


The temperature dependence of the chemical s h i f t s of the base resonances i n poly(dA-dT) and poly(dA-5brdU) a r e p l o t t e d i n F i g u r e 5. These data demonstrate that the adenosine H-8 and H-2 protons e x h i b i t very s i m i l a r behavior over the e n t i r e temperature range and are not perturbed by the s u b s t i t u t i o n on the p y r i m i d i n e 5 position. Base P a i r Overlaps: The chemical s h i f t s i n the duplex s t a t e , 6^, and the chemical s h i f t d i f f e r e n c e between duplex and strand s t a t e s , Δ6, a t t i ^ of the m e l t i n g t r a n s i t i o n , ( f o l l o w i n g c o r r e c t i o n of the temperature dependent chemical s h i f t s a s s o c i a t e d w i t h the premelting and p o s t m e l t i n g t r a n s i t i o n s ) are summarized i n Table I . The adenosine and thymidine base protons s h i f t u p f i e l d t o d i f f e r e n t extents on poly(dA-dT) duplex formation (Table I , F i g u r e 3 ) . These u p f i e l d s h i f t s r e f l e c t the base p a i r overlap geometries i n the duplex s t a t e and r e s u l t predominantly from r i n g current c o n t r i b u t i o n s due to nearest and next-nearest neighbor base p a i r s (33,34). These c o n t r i b u t i o n s can be computed f o r the B-DNA overlap geometry and a r e compared w i t h the experimental up f i e l d chemical s h i f t s on duplex formation of poly(dA-dT) and the r e l a t e d s y n t h e t i c DNA poly(dA-dU) i n Table I I . There i s good agreement between the experimental and c a l c u l a t e d values at the adenosine H-2, u r i d i n e H-5 and H-3 p o s i t i o n s f o r which the experimental chemical s h i f t changes are >0.5 ppm (Table I I ) . By c o n t r a s t , the agreement i s poor a t the adenosine H-8 and p y r i m i d i n e H-6 p o s i t i o n s , f o r which s m a l l ex p e r i m e n t a l s h i f t s were observed (Table I I ) . The adenosine H-8 and p y r i m i d i n e H-6 protons a r e d i r e c t e d towards the sugarphosphate backbone and f a c t o r s i n a d d i t i o n to the r i n g c u r r e n t e f f e c t s may c o n t r i b u t e t o the observed s h i f t s . Duplex D i s s o c i a t i o n Rates: The adenosine H-8 resonances e x h i b i t a l i n e width of V30 Hz at 55°C, s h i f t as an average resonance during the m e l t i n g t r a n s i t i o n of poly(dA-dT) i n 0.1 M phosphate (Δδ a t t ^ = 0.184 ppm) and narrow t o t h e i r value i n the s t r a n d s t a t e above 65°C ( F i g u r e 6 ) . By c o n t r a s t , the adenosine H-2 resonance (Δ6 a t = 0.899 ppm) e x h i b i t s u n c e r t a i n t y broad ening c o n t r i b u t i o n s i n the f a s t exchange r e g i o n d u r i n g the m e l t i n g t r a n s i t i o n , w i t h observed l i n e widths of >100 Hz at the midpoint of the t r a n s i t i o n (Figure 2 ) . The excess l i n e w i d t h c o n t r i b u t i o n to the adenosine H-2 resonance ( r e l a t i v e t o the adenosine H-8 resonance) a t a given temperature during the m e l t i n g t r a n s i t i o n , can be combined w i t h the chemical s h i f t d i f f e r e n c e a s s o c i a t e d w i t h the m e l t i n g t r a n s i t i o n , Δν, i n Hz, and the p o p u l a t i o n of duplex ( f ) and s t r a n d ( f = 1-f^) s t a t e s , t o y i e l d the d i s s o c i a t i o n r a t e constant, k^, (= τ^^") , f o r conversion from duplex t o strands a

g

-

excess width = 4 π ί f ( Δ ν ) ( τ + T d ) . The c a l c u l a t e d d i s s o c i a t i o n r a t e constants f o r poly(dA-dT) i n 0.1 M phosphate b u f f e r s o l u t i o n at f ^ = 0.47, 0.24 and 0.009 are presented i n Table I I I w i t h magnitudes of VL.5 x 103 sec 1 i n the v i c i n i t y of the midpoint of the m e l t i n g t r a n s i t i o n . 2

g

2

d

2

8



11.

Drug-DNA

PATEL

Poly(dA-dT) I

I

I

I

I

Interactions

in Solution

227

Poly(dA-brdU) I

I

I

I

I

I

I

I

I I 1 1 1 1 i I 1 I I 11 0 20406080 100 0 20 40 6080 IC TEMPERATURE, °C

Figure 5. The temperature dependence of the adenosine H-2, pyrimidine H-6, and adenosine H-8 protons in polyfdAdT) in 0.1M phosphate, ImM EDTA, H.O, pH 7, and poly(dA- brdU) in 0.1 M NaCl, WmU phosphate, ImM EDTA, H 0, pH 8.1 2

5

2

2


POLYMER

228

CHARACTERIZATION

B Y ESR A N D N M R

TABLE I


Chemical S h i f t Parameters Melting Transition Poly(dA-dT)

A s s o c i a t e d With The ( t ^ = 59°C) of

i n 0.1 M Phosphate S o l u t i o n

m

Δό,ppm

A(H-8)

8.085

0.184

A (H-2)

7.139

0.899

Τ(H-6)

7.052

0.194

T(CH -5)

1.300

0.359

c

5.592

0.343

d(H-l )c

6.118

0.148

Çd?PP

3

u(H-l') T

Chemical s h i f t i n the duplex s t a t e , 6 , i s defined as the e x t r a p o l a t i o n of the temperature dependent premelting s h i f t to i t s v a l u e at the t r a n s i t i o n midpoint. d

^The duplex to strand t r a n s i t i o n chemical s h i f t change Δ 6 , i s defined as the chemical s h i f t d i f f e r e n c e f o l l o w i n g e x t r a p o l a t i o n of the temperature dependent premelting and postmelting s h i f t s to t h e i r values at the t r a n s i t i o n midpoints. The Δ6 values only approximate the t o t a l s h i f t change on proceeding from a stacked duplex to unstacked s t r a n d s . T

T

u ( H - l ) and d ( H - l ) represent the u p f i e l d and downfield sugar protons r e s p e c t i v e l y .


11.

PATEL

Drug-DNA

Interactions

229

in Solution

TABLE I I

A Comparison o f the Experimental And Downloaded by EAST CAROLINA UNIV on January 22, 2016 | http://pubs.acs.org Publication Date: November 10, 1980 | doi: 10.1021/bk-1980-0142.ch011

C a l c u l a t e d U p f i e l d S h i f t s A s s o c i a t e d With Poly(dA-dT)

and Poly(dA-dU) Duplex Formation

3

Experimental poly(dA-dU) poly(dA-dT)

Calculated^ poly(dA-dU)

A(H-8)

0.17

0.18

0.04

A(H-2)

0.92

0.90

1.06

U/T(H-6)

0.34

0.19

0.06

U/T(H-3)

1.9

U(H-5)

C

0.68

1.7

C

1.88 0.62

The s t r a n d to duplex t r a n s i t i o n u p f i e l d chemical s h i f t change Δδ, i s defined as the chemical s h i f t d i f f e r e n c e f o l l o w i n g e x t r a p o l a t i o n of the temperature dependent premelting and p o s t m e l t i n g s h i f t s t o t h e i r values at the t r a n s i t i o n midpoints. These Δδ values only approximate the t o t a l s h i f t change on proceeding from a stacked duplex to unstacked s t r a n d s . ^The computed u p f i e l d r i n g c u r r e n t s h i f t s are based on nearest neighbor, next-nearest neighbor and c r o s s - s t r a n d r i n g c u r r e n t c o n t r i b u t i o n s t a b u l a t e d by A r t e r and Schmidt.(34) The experimental p y r i m i d i n e H-3 u p f i e l d s h i f t represents the d i f f e r e n c e between the observed resonance chemical s h i f t i n the p o l y n u c l e o t i d e duplex e x t r a p o l a t e d to the t r a n s i t i o n midpoint and the 14.7 ppm i n t r i n s i c p o s i t i o n f o r an i s o l a t e d base p a i r .



230

POLYMER

CHARACTERIZATION

B Y ESR A N D N M R

Figure 6. The temperature dependence (5° to 95°C) of the downfield H-V (A) and the adenosine H-8 (O) linewidths of poly(dA-dT) in 0.1M phosphate, ImM EDTA, H 0, pH 7.0 2

2


11.

PATEL

Drug-DNA

Interactions

in

Solution

231


By c o n t r a s t , the s y n t h e t i c 30 base p a i r DNA b l o c k polymer duplex (dCi5dAi5)·(dTi5dGi5) e x h i b i t s slow exchange between duplex and strand s t a t e s during the m e l t i n g t r a n s i t i o n (35). The d i s s o c i a t i o n r a t e f o r DNA d e n a t u r a t i o n i s p r e d i c t e d to be prop o r t i o n a l to the i n v e r s e square of the length of the duplex to be unwound and f o r molecular weights of 10^ would be s e v e r a l orders of magnitude slower (36) than the VLO^ sec"^- r a t e constants e v a l uated f o r the poly(dA-dT) m e l t i n g t r a n s i t i o n i n s o l u t i o n . The NMR r e s u l t s support the e a r l i e r c o n c l u s i o n s of Baldwin and coworkers that poly(dA-dT) u n f o l d s by the opening of short branched duplex regions which melt independently of each other (16,17). Premelting T r a n s i t i o n (37*38)» The n u c l e i c a c i d base and sugar resonances of poly(dA-dT) (Figures 3 and 5) and poly(dA- brdU) (Figure 5) e x h i b i t non-cooperative chemical s h i f t changes with temperature i n the duplex s t a t e . Both u p f i e l d and downfield s h i f t s are observed during t h i s premelting t r a n s i t i o n at the nonexchangeable protons (Figures 3 and 5) while an u p f i e l d s h i f t i s observed at the exchangeable thymidine H-3 proton (Figure 1) with i n c r e a s i n g temperature. The observed premelting t r a n s i t i o n may r e f l e c t a conformational change i n the p o l y n u c l e o t i d e duplex with temperat u r e . The adenosine H-2 resonance l o c a t e d i n the minor groove s h i f t s to high f i e l d while the thymidine group l o c a t e d i n the major groove remains unchanged on lowering the temperature i n the premelting t r a n s i t i o n r e g i o n (Figure 3). This suggests that the base p a i r s may t i l t r e l a t i v e to each other on r a i s i n g the temperature i n the premelting r e g i o n with a kink s i t e opening i n t o the minor groove (39). The adenosine H-8 and u r i d i n e H-6 protons which are d i r e c t e d towards the phosphodiester backbone, and the two sugar H-1 protons, which are s e n s i t i v e to v a r i a t i o n s i n the g l y c o s i d i c t o r s i o n angles, a l l s h i f t downfield with decreasing temperature (Figure 3). This suggest that the premelting t r a n s i t i o n may a l s o i n v o l v e conformational changes i n the sugarphosphate backbone and g l y c o s i d i c t o r s i o n angles. Baldwin and coworkers have demonstrated that the degree of branching i n the poly(dA-dT) duplex v a r i e s s i g n i f i c a n t l y with temperature (16,17). T h e r e f o r e , the premelting t r a n s i t i o n chemic a l s h i f t changes may a l s o r e f l e c t , i n p a r t , the conversion from a h i g h l y branched duplex at temperatures j u s t below the m e l t i n g t r a n s i t i o n (40) to a l e s s branched duplex s t r u c t u r e on lowering the temperature. There i s no i n f o r m a t i o n to our knowledge on premelting t r a n s i t i o n s i n RNA duplexes. We have t h e r e f o r e i n v e s t i g a t e d the NMR parameters f o r the r e l a t e d s y n t h e t i c RNA duplex poly(A-U) as a f u n c t i o n of temperature. T y p i c a l 360 MHz aromatic proton region spectra f o r the poly(A-U) duplex i n 0.1 M phosphate ( t ^ = 66.5°C) at 8.2° and 44.5°C are presented i n F i g u r e 7. I t i s c l e a r that the chemical s h i f t s of the base protons vary with 5

C H 3 - 5

T

2


232

POLYMER

CHARACTERIZATION

B Y ESR A N D N M R


temperature i n the duplex s t a t e (Figure 7) and the r e s u l t s are p l o t t e d i n F i g u r e 8, I t may be noted that the d i r e c t i o n of the p r e m e l t i n g changes a t i n d i v i d u a l resonances e x h i b i t the same behavior i n the s y n t h e t i c DNA (Figure 3) and the s y n t h e t i c RNA (Figure 7). This suggests that the o r i g i n of the premelting conformation (37,38) i s common f o r s y n t h e t i c RNA and DNA p o l y n u c l e o t i d e duplexes w i t h the same a l t e r n a t i n g p u r i n e - p y r i m i d i n e sequence. Phosphodiester Linkage: The sugar-phosphate backbone of n u c l e i c a c i d s can be probed a t the phosphodiester l i n k a g e by -*lp N M R spectroscopy. There are only two kinds of phosphodiester l i n k a g e s i n a l t e r n a t i n g p u r i n e - p y r i m i d i n e p o l y n u c l e o t i d e s , namely dApdT and dT£dA i n poly(dA-dT). The 145.7 MHz proton n o i s e decoupled 31p NMR s p e c t r a of the poly(dA-dT) duplex i n 10 mM cacodylate b u f f e r between 28° and 54°C are presented i n F i g u r e 9. A broad symmetrical unresolved resonance i s observed a t 28°C. By c o n t r a s t , two r e s o l v e d narrow resonances separated by ^ . 2 ppm have been observed f o r 150 base p a i r long (dA-dT) (41). Thus, though the r e s o l u t i o n of dTp_dA and dApdT phosphodiesters cannot be achieved a t the s y n t h e t i c DNA l e v e l i n s o l u t i o n (18), i t has been observed f o r the same sequence at a s h o r t e r w e l l defined length (41). More r e c e n t l y , two r e solved ^ l p resonances have a l s o been reported i n poly(dA-dT) f i b e r s o r i e n t e d p a r a l l e l to the d i r e c t i o n to the magnetic f i e l d by s o l i d s t a t e 3 l p NMR spectroscopy (42). The P s p e c t r a of poly(dA-dT) e x h i b i t some i n t e r e s t i n g s p e c t r a l changes i n the premelting r e g i o n i n s o l u t i o n . The resonance(s) of poly(dA-dT) e x h i b i t an assymmetric shape w i t h i n c r e a s i n g temperature w i t h the component to higher f i e l d remaining broad compared to the component to lower f i e l d , which narrows cons i d e r a b l y a t 54°C (Figure 9 ) . This suggests that there e x i s t regions i n the p o l y n u c l e o t i d e duplex w i t h increased segmental m o b i l i t y i n the premelting t r a n s i t i o n r e g i o n . The phosphodiester resonance moves downfield and sharpens d r a m a t i c a l l y during the m e l t i n g t r a n s i t i o n and continues t o s h i f t downfield w i t h i n c r e a s i n g temperature i n the p o s t m e l t i n g t r a n s i t i o n r e g i o n (18). n

3 1

Summary : The above r e s u l t s c o n v i n c i n g l y demonstrate t h a t high r e s o l u t i o n proton NMR s p e c t r a can be recorded f o r high molecular weight poly(dA-dT) i n aqueous s o l u t i o n . The base p a i r i n g i n the duplex s t a t e has been v e r i f i e d by monitoring the r i n g imino exchangeable protons i n H 2 O s o l u t i o n . The overlap of adjacent base p a i r s i s r e a d i l y demonstrated by the o b s e r v a t i o n of u p f i e l d s h i f t s on duplex formation and some estimate of the overlap geometries evaluated from the r e l a t i v e magnitudes of the changes a t the d i f f e r e n t proton markers. The o b s e r v a t i o n of average resonances for the nonexchangeable protons during the duplex to strand t r a n s i t i o n r e q u i r e s f a s t i n t e r c o n v e r s i o n between s t a t e s on the NMR time s c a l e consistent w i t h e a r l i e r suggestions that poly(dA-dT) melts by opening of s h o r t e r branched duplex r e g i o n s .


11.

Drug-DNA

PATEL

Interactions

233

in Solution

8

7

6

5

Figure 7. The temperature dependence (8.2°C and 44.5°C) of the 360-MHz proton NMR spectra (4.5 to 9.0 ppm) of poly(A-U) in 0.1 M phosphate, ImM EDTA, -Η,Ο, pH 7.0

S H I F T , PPM

70

ICAL


(Η-Ι')

Lu χ

• °

-

0

° O O O O O o

; ι I I

7.2 74

78 8.0

\ \

1 U(H-6)

r M > ^ ^

76

UH-6)

-

-oûo^ooooo^^ (Η-1') I»

N c a X X X >

l

W

1

1

—

(Η-Ι')

8.2

A(H-8)

-

—

-oo-o°oc| U(H-5) I

Q

A(H-2)

T f a c o o c

χ>

—

\

Q5ZXDOOO I ιI ιIιIιIι 1

0

20 40

60

80 100

I ι I ι I ι I ι II I 0

20 40

60

80 100

TEMPERATURE, °C Figure 8. The temperature dependence (5° to 95°C) of the base and sugar H-V proton chemical shifts of polv(A-U) in 0.1 M phosphate, ImM EDTA, H 0, pH 7.0 2


2

POLYMER


234

CHARACTERIZATION

B Y ESR A N D N M R

28° C

39° C

49°C

54° C

I

I

I

2

3

I 4

I

ι

5

6

ι

Figure 9. The proton noise decoupled 145.7-MHz P NMR spectra of poly(dAdT) in 0.1 M cacodylate, WmM EDTA, H,0, pH 7.08 between 28° and 54°C (it,, of complex is 60°C). The scale is upfield from standard trimethylphosphate. 31

2


11.

PATEL

Drug-DNA

Interactions

in

235

Solution

The premelting conformational t r a n s i t i o n can be monitored a t the chemical s h i f t s of the exchangeable and nonexchangeable protons and the l i n e shape of the phosphodiester resonance o f poly(dA-dT) and i t s r e l a t e d s y n t h e t i c DNA*s i n s o l u t i o n . The r e s u l t s suggest that the base p a i r s may p a r t i a l l y unstack i n t o the minor groove, accompanied by small changes i n the g l y c o s i d i c t o r s i o n angles and the sugar-phosphate backbone as the temperature i s r a i s e d i n the premelting t r a n s i t i o n r e g i o n .


Counterion

Binding.

Alkylammonium Ions

The research below focusses on the NMR parameters f o r poly(dA-dT) i n 1 M tetramethylammonium c h l o r i d e (TMA ) r e l a t i v e to t h e i r value i n the same c o n c e n t r a t i o n of sodium c h l o r i d e . The methyl groups s h i e l d the charged n i t r o g e n i n the TMA+ i o n and i t was of i n t e r e s t to determine whether conformational changes occur i n the s y n t h e t i c DNA when the counterion was changed from N a to TMA+. The NMR experiments were undertaken on 28 mM ( i n n u c l e o t i d e s ) poly(dA-dT) i n 10 mM cacodylate b u f f e r , to which 1 M s a l t s o l u t i o n s were added. The samples contained i n a d d i t i o n 1 mM and 10 mM EDTA f o r the proton and phosphorus NMR s t u d i e s , r e s p e c t i v e l y . The experimental c o n d i t i o n s are i n d i c a t e d i n order to emphasize that even i n 1 M TMA+ s o l u t ions there are N a ions a s s o c i a t e d with poly(dA-dT), the 10 mM b u f f e r and the EDTA s o l u t i o n s . E q u i l i b r i u m d i a l y s i s s t u d i e s on the r e l a t i v e a f f i n i t i e s of weakly bound c a t i o n s with v a r y i n g base composition DNA have demons t r a t e d that tetraalkylammoniura ions bind more t i g h t l y to dA dT r i c h DNA compared to dG«dC r i c h DNA (43). +

+

+

e

Hydrogen Bonding: The thymidine H-3 proton of poly(dA-dT) can be r e a d i l y monitored i n 1 M TMA+ s o l u t i o n s a t pH 7.5 and pH 9.5 a t 37°C (Figure 10). This demonstrates that the base p a i r s a r e i n t a c t i n the s y n t h e t i c DNA and that t h e i r exchange i s not base c a t a l y z e d up to pH 9.5 at 37°C. These r e s u l t s p a r a l l e l observat i o n s on poly(dA-dT) w i t h Na~*~ as the counterion and i n poly(dA-dT) complexes with i n t e r c a l a t i n g agents. By c o n t r a s t , i n s t e r o i d diamine*poly(dA-dT) complexes which i n v o l v e t i l t e d base p a i r s a t the b i n d i n g s i t e , the exchange of the thymidine H-3 protons are s u s c e p t i b l e to base c a t a l y s i s (44). The l i n e width of the thymidine H-3 proton i s compared w i t h the corresponding v a l u e f o r the adenosine H-8 nonexchangeable proton f o r poly(dA-dT) i n 1 M TMA+ s o l u t i o n , pH 7.5, i n F i g u r e 11. The exchangeable resonance e x h i b i t s the l a r g e r l i n e width i n the duplex s t a t e due to the i n t e r a c t i o n of i t s proton w i t h the d i r e c t l y bonded -^N quadrupolar nucleus. The exchangeable r e s o n ance broadens d r a m a t i c a l l y between 67.5°C ( l i n e width. 0/150 Hz) and 77.5°C ( l i n e width ^250 Hz), a temperature r e g i o n where the duplex i s s t i l l i n t a c t from the nonexchangeable proton data. These


POLYMER


236

I

I 15

Figure 10. The 1M ( H,C),NCl, Spectrum A was ( HjC),NCl was 2

2

I 14

I 13

CHARACTERIZATION

I 12

I 11

B Y ESR A N D N M R

I

360-MHz correlation proton NMR spectra of polv(dA-dT) in WmM phosphate, ImM EDTA, 80% H,0-20% D0, pH 7.95 at 67°C. The chemical shifts are upfield from standard trimethylphosphate. 31

2

40 60 TEMPERATURE,°C Figure 16. The temperature dependence of the thymidine H-3 resonance in poly(dA-dT) (O) and the proflavine · poly(dA-dT) complex Nuc/D = 8 (Φ) in 1M NaCl, WmM cacodylate, ImM EDTA, H*0 at pH 6.53, and pH 7.1 respectively



244

POLYMER

CHARACTERIZATION

B Y ESR A N D N M R

Figure 17. The 360-MHz proton NMR spectra of the proflavine · poly (dA-dT) complex in 7 M NaCl, WmM cacodylate, WmM EDTA, H 0, pH 7. The top spectrum represents the Nuc/D = 24 complex at 78.5°C (t of the proflavine resonances in the complex is 80°C), while the bottom spectrum represents the Nuc/D = 8 complex at 81.4°C (\ of proflavine resonances in complex is 84.3°C). The proflavine resonances are designated by asterisks. 2

2

1/2

i/t



11.

PAT E L

Drug-DNA

Interactions

245

in Solution

Τ

1

1

1

1

Γ

-J 50

ι 60

ι 70

ι 80

I 90

L_ 100

90

100

*C

TEMPERATURE,

50

60

70

80

TEMPERATURE

e

C

Figure 18. The temperature dependence of (A ) the thymidine CH,-5 chemical shift and (B) the adenosine H-8 linewidth in poly(dA-dT) (O), the proflavine · polv(dA-dT) complex, Nuc/D ^24 (A) and Nuc/D = 8 (Φ) in 1M NaCl, WmM cacodylate, WmM EDTA, H,0, pH 7 2


POLYMER

246

POLY (dA-dT) τ—I—'—I—ι

CHARACTERIZATION

C0MPLEX,N/D=24 1— ~H— r

C0MPLEX,N/D=8 1 Π— —I— —I

1

1

1


ΓΊΗ

B Y ESR A N D N M R

J

I 60

ι

I 80

ι

1 1 • ι 100 60

»

» 80

»

Ljj ι 100 60

I 80

»

» 100

TEMPERATURE, °C Figure 19. The temperature dependence of the nucleic acid (O) and proflavine (Φ) chemical shifts between 5.5 and 8.6 ppm for poly(dA-dT) and the Nuc/D = 24 and 8 proflavine · poly(dA-dT) complexes in 1M NaCl, WmM cacodylate, WmM EDTA, HΌ between 50° and 100°C. The poly(dA-dT) concentration was fixed at 12.6mM in phosphates and the proflavine concentration was varied to make the different Nuc/D ratio complexes. 2


11.

Drug-DNA

PATEL

Interactions

in

Solution

247

s y n t h e t i c DNA (Figure 16). The n u c l e i c a c i d complexation s h i f t s r e f l e c t the d i f f e r e n c e i n r i n g current c o n t r i b u t i o n s between the p r o f l a v i n e r i n g (53) and a dA»dT base p a i r (33,34) which i s d i s placed f o l l o w i n g i n t e r c a l a t i o n . N u c l e i c A c i d Sugar Resonances : Decoupling s t u d i e s have c o r r e l a t e d the r e s o l v e d protons on each o f the two sugar r i n g s o f poly(dA-dT) (Table IV) though i t i s not y e t p o s s i b l e t o d e f i n i t i v e l y d i f f e r e n t i a t e between the adenosine and thymidine r e s i d u e s . Several sugar r i n g protons can be monitored i n the p r o f l a v i n e - p o l y (dA-dT) complex and these i n c l u d e H - l p o s i t i o n (Figure 19) and H-3 and H-2 ,2" p o s i t i o n s (Figure 20). Both sugar H - l protons of poly(dA-dT) undergo s m a l l u p f i e l d s h i f t s on p r o f l a v i n e complex formation w i t h a somewhat more pronounced e f f e c t on the H - l a t lower f i e l d , which s h i f t s from 6.1 ppm i n the s y n t h e t i c DNA t o 5.95 ppm i n the Nuc/D = 8 complex (Figure 19). This l a t t e r o b s e r v a t i o n was p r e v i o u s l y r e ported f o r the ethidium bromide poly(dA-dT) complex (11). The sugar H-1 protons predominantly monitor changes i n the g l y c o s i d i c t o r s i o n angle (54) and the r e s u l t s suggest that the generation of the p r o f l a v i n e i n t e r c a l a t i o n s i t e r e q u i r e s changes i n these t o r s i o n angle ( s ) . The r e s u l t i s c o n s i s t e n t w i t h p a r a l l e l observat i o n s on the X-ray s t r u c t u r e s of i n t e r c a l a t i v e drug-dinucleoside m i n i a t u r e duplexes (48-50). The sugar H-3 protons of poly(dA-dT) undergo chemical s h i f t changes of 0 < ?

I 60

^tCDooO

^ck>-o-o-o ι

I 80

ι

1 100 60

80

TEMPERATURE

100 e

1 80

,

1 100

C

Figure 20. The temperature dependence of the nucleic acid H-3' (4.6 to 5.0 ppm) and the H-2', 2" (1.8 to 2.8 ppm) chemical shifts for poly(dA-dT) and the Nuc/D = 24 and - 8 proflavine · poly(dA-dT) complex in 1M NaCl, WmM cacodylate, WmM EDTA, H,O pH 7 between 60° and 100°C 2

t


250

POLYMER

CHARACTERIZATION

B Y ESR A N D N M R

TABLE V

Experimental and C a l c u l a t e d U p f i e l d P r o f l a v i n e Complexation S h i f t s on Formation Downloaded by EAST CAROLINA UNIV on January 22, 2016 | http://pubs.acs.org Publication Date: November 10, 1980 | doi: 10.1021/bk-1980-0142.ch011

of the Proflavine*Poly(dA-dT) Complex

Calculated S h i f t , ppm

Experimental S h i f t , ppm Assignment H a «b H

3

5

Complex*

Free

A6

0

d

7.820

8.740

0.92

0.85

6.930

7.880

0.95

0.8

6.155

7.060

0.905

0.65

5.955

6.840

0.885

0.7

c

The p r o f l a v i n e resonance H , o u t l i n e d i n Reference 25.

, H , and c

assignments are as

Data f o r the p r o f l a v i n e - p o l y ( d A - d T ) complex, Nuc/D = 8 i n 1 M NaCl, 10 mM c a c o d y l a t e , 10 mM EDTA, H 0 , pH 7 a t 69°C. 2

2

Chemical s h i f t of 1.3 mM p r o f l a v i n e i n 0.1 M phosphate, 1 mM EDTA, H 0 , pH 6.6 a t 100°C. 2

2

^The experimental u p f i e l d complexation s h i f t i s the d i f f e r e n c e between the values f o r the i n t a c t complex a t 69°C r e l a t i v e to f r e e p r o f l a v i n e a t 100°C. "The c a l c u l a t i o n s are based on r i n g current and atomic diamagnetic a n i s o t r o p y c o n t r i b u t i o n s (56) based on the i n t e r c a l a t i o n overlap geometry depicted i n the t e x t . The overlap geometry corresponds to i n t e r c a l a t i o n of dT-dA s i t e .


11.

Drug-DNA

PATEL

Interactions

251

in Solution

under c o n d i t i o n s where the drugs acts as a template on which the n u c l e i c a c i d forms a miniature i n t e r c a l a t i v e complex. Our l a b o r atory has extended these i n v e s t i g a t i o n s to the s t a b l e o l i g o n u c l e o t i d e duplex l e v e l with NMR s t u d i e s of the sequence s p e c i f i c i t y of actinomycin D binding to hexanucleotides (64) and t e t r a n u c l e o t i d e s (65) i n aqueous s o l u t i o n . T e t r a n u c l e o t i d e s c o n t a i n i n g dG dC base p a i r s form s t a b l e duplexes at low temperature so that the self-complementary sequences dC-dC-dG-dG [contains d C ( 3 - 5 ) d G but no dG(3 -5 )dC binding s i t e s ] and dG-dG-dC-dC [contains dG(3 -5 )dC but no d C ( 3 - 5 ) d G binding s i t e s ] serve as e x c e l l e n t o l i g o n u c l e o t i d e duplexes f o r d i f f e r e n t i a t i n g p y r i m i d i n e ( 3 - 5 ) p u r i n e specificity from p u r i n e ( 3 - 5 ) p y r i m i d i n e s p e c i f i c i t y a s s o c i a t e d with drug complexation (66,^7). We have monitored the decrease i n the 435 nm actinomycin D absorbance band on gradual a d d i t i o n of t e t r a n u c l e o t i d e i n 0.1 M phosphate at 24.5°C. A comparison of the t e t r a n u c l e o t i d e concent r a t i o n s corresponding to half-maximal change demonstrates stronger binding of actinomycin D to dG-dG-dC-dC compared to dC-dC-dG-dG (Figure 21), e s t a b l i s h i n g a r e l a t i v e sequence s p e c i f i c i t y f o r actinomycin D complex formation at p u r i n e ( 3 - 5 p y r i m i dine s i t e s at the o l i g o n u c l e o t i d e duplex l e v e l (67). By c o n t r a s t , ethidium bromide (Figure 22) and p r o f l a v i n e (Figure 23) e x h i b i t a r e l a t i v e p y r i m i d i n e ( 3 - 5 ) p u r i n e s p e c i f i c i t y at the duplex l e v e l since stronger binding i s observed with dC-dCdG-dG compared to dG-dG-dC-dC i n 0.1 M phosphate s o l u t i o n at 1°C. These r e s u l t s demonstrate that the r e l a t i v e sequence s p e c i f i c i t y f o r drug i n t e r c a l a t i o n at the d i n u c l e o s i d e phosphate l e v e l (^7-63) are a l s o observed at the s t a b l e o l i g o n u c l e o t i d e duplex l e v e l (66,67). The l a t t e r i n v e s t i g a t i o n s have been r e peated i n another l a b o r a t o r y (68,69) with s i m i l a r c o n c l u s i o n s . e

T

f

f

,

T

T

1

l

T


T

T

T

T

f

f

f

Overlap Geometry: A schematic r e p r e s e n t a t i o n of the proposed overlap geometry f o r p r o f l a v i n e i n t e r c a l a t e d i n t o a deoxy pyrimidine(3 -5 )purine s i t e i s presented below w i t h the (o) symb o l s representing the l o c a t i o n of the phenanthridine r i n g protons. The mutual overlap of the two base p a i r s at the i n t e r c a l a t i o n s i t e i n v o l v e s features observed i n the c r y s t a l s t r u c t u r e s of a platinum m e t a l l o i n t e r c a l a t o r ^ m i n i a t u r e dC-dG duplex complex (55) and the more recent proflavine«miniature dC-dG duplex complex (48), as w e l l as f e a t u r e s derived i n a linked-atom conformational c a l c u l a t i o n of the i n t e r c a l a t i o n s i t e i n the proflavine*DNA complex (51). [4] The overlap of p r o f l a v i n e with adjacent base p a i r s was v a r i e d u n t i l there was approximate agreement between the e x p e r i mental u p f i e l d complexation s h i f t s (Table V) and those c a l c u l a t e d from r i n g current and atomic diamagnetic anisotropy c o n t r i b u t i o n s from the base p a i r s (56). The c a l c u l a t e d u p f i e l d s h i f t s are somewhat smaller than the experimental complexation s h i f t s at the p r o f l a v i n e protons i n the s y n t h e t i c DNA complex (Table V ) . This f

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Acridine Mutagen, Anthracycline Antitumor, and ... - ACS Publications

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