Synthesis and high stability of complementary complexes of N-(2

to Improve Nucleic Acids Detection in the Real-Time Snake Polymerase Chain Reaction. Igor V. Kutyavin. ASSAY and Drug Development Technologies 201...
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Bioconjugate Chem. 1992, 3, 4 14-4 19

414

Synthesis and High Stability of Complementary Complexes of N-(2-Hydroxyethy1)phenazinium Derivatives of Oligonucleotides Sergei G. Lokhov, Mikhail A. Podyminogin, Dmitrii S. Sergeev, Vladimir N. Silnikov, Igor V. Kutyavin, Gennadii V. Shishkin, and Valentina P. Zarytova’ Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, Prospect Lavrentjeva, 8 Novosibirsk 630090, Russia. Received June 1, 1992

Two simple methods for the synthesis of oligonucleotides bearing a N-(2-hydroxyethyl)phenazinium (Phn) residue at the 5’- and/or 3’4erminal phosphate groups are proposed. By forming complexes between a dodecanucleotide d(pApApCpCpTpGpTpTpTpGpGpC),a heptanucleotide d(pCpCpApApApCpA), and Phn derivatives of the latter, it is shown that the introduction of a dye at the end of an oligonucleotide chain strongly stabilizes its complementary complexes. The T,, and the thermodynamic parameters (AH, AS, AG) of complex formation were determined. According to these data, coupling of a dye with the 5’-terminal phosphate group is the most advantageous: AG(37 “C) is increased by 3.59 f 0.04 kcal/mol compared to 2.06 f 0.04 kcal/mol for 3’-Phn derivatives. The elongation of the linker, which connects the dye to the oligonucleotide, from a dimethylene up to a heptamethylene usually leads to destabilization of the oligonucleotide complex. The complementary complex formed by the 3’,5’-di-Phn derivative of the heptanucleotide was found to be the most stable among all duplexes investigated. Relative to the unmodified complex the increase in free energy was 4.96 f 0.04 kcal/mol. The association constant of this modified complex at 37 OC is 9.5.106 M-l, whereas the analogous value for the unmodified complex is only 3.103 M-l.

INTRODUCTION

EXPERIMENTAL PROCEDURES

Oligonucleotides bearing dye residues (1-4)are now under scrutinity by many nucleic acid chemists, as they are successfully used for directed effects on structure (5, 6) and functional activity (7-9) of nucleic acids. The oligonucleotide-dye conjugates have remarkable physicochemical and biochemical properties. Compared to unmodified oligomers, oligonucleotides covalently linked to intercalating dyes have a higher affinity for complementary targets (1-4)and are more resistant to cellular nucleases (10,lI). For example, oligodeoxyribonucleotidesbearing an acridine derivative were found to be successful antisense agents for suppression of target nucleic acid expression not only in the in vitro experiments (7)but also in living cells (8,9).According to some data (6,121 4 ) ,the dye may play the role of a reactive agent under particular conditions. In the present paper we describe the synthesis and hybridization properties of oligodeoxyribonucleotidesbearing N- (2-hydroxyethy1)phenaziniumresidues (Phnl) at the 3’- or 5’-end. Oligonucleotides which contain one or more phenazine quaternary salts form strong hybrids with complementary target strands due to stabilization by the phenazine residue. The phenazine residues were introduced by facile oxidative coupling of N-(Bhydroxyethyl)phenazinium chloride with readily prepared aminoalkylsubstituted oligonucleotides, a process which offers advantages of ease and quantitative yield. Based on the results obtained, we recommend Phn-oligonucleotide derivatives as promising starting materials for the design of nucleic acid targeted reagents and probes.

Chemicals. Chemicals used in the present work were from Fluka or Merck; T4-polynucleotide kinase was from Fermentas, [ T - ~ ~ P I A T (specific P radioactivity > 2000 Ci/ mmol) was from Isotop. p-Chlorophenyl N-acyl-3’-0levulinyl nucleoside 5’-phosphates, p-chlorophenyl O-cyanoethyl N-acyl nucleoside 5’-phosphates, 2,4,6-triisopropylbenzenesulfonylchloride, and 4-chlorophenyl phosphorodichloridate were products of the Novosibirsk Institute of Organic Chemistry (Novosibirsk, Russia). Dimethyl sulfoxide was distilled under reduced pressure. Absolute pyridine was prepared by successivedistillations under TPS, KOH, and P z O ~ .2,2’-Dipyridyl disulfide, triphenylphosphine, 4-(dimethylamino)pyridine, polymethylenediamines, and polymethylenamine alcohols were commercially available and used without further purification. Chromatography. Purification of unprotected oligonucleotides and their derivatives was by HPLC with a Beckman-332 system on Polisil SA (10pm) anion-exchange and Lichrosorb RP 18 (5 pm) reverse-phase columns. The Polisil SA column was a gift of Dr. S. I. Yastrebov (Koltsovo, Russia). Physical Measurements. UV-visible absorption spectra of dyes in water and oligonucleotide-dye conjugates in 0.16 M NaC1,0.02 M NazHP04, and 0.1 mM EDTA (pH 7.4) were recorded in the 200-600-nm range on a UV-2100 spectrophotometer (Shimadzu). lHNMR spectra for dyes were run at 20 OC, on a Bruker WP-200 spectrometer; chemical shifts are reported in ppm downfield from Me4Si. Optical melting curves of oligonucleotide complexes were obtained on the UV detector of a Milichrom liquid chromatograph in a thermoregulated cell specially designed for this purpose. Speed of heating was 0.5-0.7 “C/ min. The melting curves were constructed by integrating over 500-600 data points obtained every 10 s from optical and thermal sensors. Optical noise did not exceed fO.OO1 A. The data were collected and processed on an Iskra-226 personal computer. Before the thermodynamic calculation, the melting curves were smoothed by a linear central

1 Abbreviations used: Phn, N-(2-hydroxyethyl)phenazinium residue; TPS, 2,4,6-triisopropylbenzenesulfonyl chloride; MeIm, N-methylimidazole; (Ph)3P, triphenylphosphine; ( P y s ) ~2,2’, dipyridyl disulfide; DMAP, 4-(dimethylamino)pyridine;d(pNpNpN...p N), unblocked oligonucleotide; d ( p N p N p N...JIM, blocked oligonucleotides; T,-, melting temperature of a oligonucleotide duplex determined at the point of curve bending.

0 1992 American Chemical Soclety

Bloconjugate Chem., Vol. 3, No. 5, 1992 415

Phenazlnlum-Stablllzed Complementary Complexes

Table I. Physical Properties and Elemental Analysis Data of the Compounds of General Formula HO(CH2),,$JHCOCFs

elemental analvsis data m

n2OD

2"

3 4 5 a

1.405 1.412 1.417

bp, "C (0.5 mmHg) 97-98 105-106 118-119 127-128

This compound is solid with mp 50-55

C 30.9 35.3 38.8 42.2

% found H

4.12 4.81 5.40 6.02

% calcd

F 35.6 33.7 31.1 28.5

C

H

F

30.6 35.1 38.9 42.2

3.85 4.71 5.44 6.07

36.2 33.3 30.8 28.6

OC.

interpolation treatment of the data using a 2" window with consecutive averagings offset by 0.1". Thermodynamic Calculations. Thermodynamic parameters of complex formation were calculated according to a '%wostates" model by minimization of mean square errors between the calculated and experimental melting curves, where the AH and A S values were varied (15). Furthermore, these parameters were found to be similar to those obtained using the van't Hoff procedure for selected duplexes. The thermodynamic parameters were confirmed by the results of at least two experiments. The mean arithmetical errors of experiments are given in Table 11. The melting temperatures of complexes (T',,) were determined a t the points of curve bending. Molar Extinction of Oligonucleotides. The concentration of the oligonucleotides and their derivatives was measured spectrophotometrically. The molar extinction of unmodified oligonucleotides was determined after complete hydrolysis by venom nuclease as described in ref 16. To determine the molar extinctions of dyeoligonucleotide conjugates, the starting unmodified oligonucleotides (-0.5 pmol) were labeled ( 10-20 pCi) by reaction with T4-polynucleotide kinase, [T:~~PIATP, and ADP according to the technique described in ref 17. After purification of the labeled oligonucleotides by reversephase chromatography, they were used in the synthesis of the corresponding derivatives according to the methods described below. UV-visible absorption spectra of the starting oligonucleotides and then derivatives were recorded in the above buffer. Radioactivity of equal portions of these solutions was determined on an LKB scintillation counter by Cherenkov effect. Molar extinctions of oligonucleotide derivatives (eD260) were calculated using the formula N

where A = optical density (260 nm) of the solution of starting oligonucleotide ( A S )and its derivative ( A D )and R = radioactivity of the aliquots of the corresponding solutions. N-(2-Hydroxyethyl)phenazinium Chloride. Ethylene oxide (50 mL) was added dropwise to a solution of phenazine (10 g) in 100 mL of glacial acetic acid at 10 "C. After 6 days of incubation in a closed flask at 20 "C, 10 mL of concentrated HC1 was added and the reaction mixture was evaporated in vacuo. The dark-brown gum was twice treated with a mixture of toluene-heptane (1:l) and evaporated. Diethyl ether (150 mL) was added to form a suspension and to precipitate the product over 12 h. The precipitate was filtered and washed by acetone and diethyl ether. After the additional reprecipitation, 10.5 g of N-(2-hydroxyethyl)phenazinium chloride was obtained in a yield of 72%. Mp: 340-350 "C dec. UVvis, ,,A, nm (log E ) : 262 (4.89), 387 (4.391, 440 (3.47). lH NMR, 6, ppm (CD30D): 7.93-9.03 (arom H, 8 H, m), 5.455.95 (CHzN+,2 H, m), 4.20-4.44 (-CH20-, 2 H, m). Anal.

Calcd for C14H13ClN20: C, 64.5; H, 5.0; N, 10.7. Found: C, 64.5; H, 5.1; N, 10.5. 2- [ (6-Aminohexy1)aminol-10- (2-hydroxyethy1)phenazinium Chloride. N-(2-Hydroxyethyl)phenazinium chloride (1g) was dissolved in a solution of 2 g of l,&hexanediamine in 5 mL of dried methanol, and the reaction mixture was incubated at 20-23 "C for 1day. The product was precipitated with diethyl ether (10 mL), reprecipitated from methanol, washed with diethyl ether, and dried. The yield of dye was 0.95 g (66%). To obtain a preparation of analytical grade, the dye was converted from the chloride to the perchlorate form by treatment of its concentrated aqueous solution with 10% aqueous LiC104 up to a 2-fold excess. The precipitated dye was filtered and crystallized from water. Mp: 123-125 "C. UV-vis, Am, nm (log e): 237 (4.53), 295 (4.54), 380 (4.00), 394 (4.02),530 (4.20). 'H NMR, 6, ppm ((CD3)zSO): 7.94 (arom H, 6 H, m), 6.83 (arom H, 1H, s), 5.10 (CHzN, 2 H, t),4.05 (CH20,2 H, t),3.58 (CH2NH2, 2 H, m), 2.81 (CH2HNAr, 2 H, m), 1.45 (-(CH2)4-, 8 H, m). Anal. Calcd for C20H26C1N405.1/2C032-: C, 53.7; H, 6.20; N, 11.9; C1,8.08. Found: C, 53.7; H, 6.15; N, 12.0; C1, 7.94. Synthesis of HO(CHz),NHCOCFa (m= 2-5). The compounds of general formula HO(CH2),NHCOCF3 ( m = 2-5) were prepared by treatment of the corresponding polymethylenamine alcohol (1mol) with the ethyl ester of trifluoroacetic acid (1.3 mol) at 20 "C for 2 h. The desired product was isolated from the reaction mixture after two stages of distillation under low pressure (0.3-0.5 mmHg) with a yield of 50-70 % Some physical properties and elemental analysis data are summarized in Table I. Blocked Oligonucleotides. The synthesis of blocked oligonucleotides was performed in chloroform by the triester method (18) starting with 5'-p-chlorophenyl N-acyl-3'-O-levulinyl nucleoside 5'-phosphates and p chlorophenyl 8-cyanoethyl N-acyl nucleoside 5'-phosphates. TPS and MeIm (1:2) were used as condensing reagents. The 3'-hydroxyl groups of the OH components were blocked by levulinate. The base residues were protected as in ref 18. At all stages the P and OH components were used at 0.2 M concentration and at a ratio close to equimolar. A 3-fold excess of the condensing reagents over the P components was used. The cyanoethyl groups were removed by a mixture of dry acetonitrile and triethylamine (1:l)(18). After the removal of the levulinate residue (19) the OH components were isolated by chromatography on silica gel in a gradient of methanol in chloroform. Unblocked Oligonucleotides. The unblocked oligonucleotides were prepared from the corresponding products of the solid-phase phosphoramidite procedure using an automated Victoria 4M synthesizer (Novosibirsk, Russia) (20). Terminal phosphates were introduced by conversion of uridine 5'-phosphate residues by periodate oxidation and subsequent &elimination as described (21). To prepare oligonucleotides containing these residues at the 3'- and 5'-termini, ( ~ P ~ ) ~ N - P ( O C H B ) O Uwas ( Bused Z)~

.

416

at the last step and (DMTr)U(Ac)at the first step of the oligonucleotide synthesis. Some products of triester synthesis were used to prepare unblocked oligonucleotidesbearing 5’-terminal phosphate groups by treatment with 0.3 M tetrabutylammonium fluoride in 50% aqueous pyridine at 20 “C over 1 2 h followed by treatment with concentrated ammonia (2 days at 20 “C) (22). The triester approach was also used to produce oligonucleotides bearing two 3’- and 5’-terminal phosphate groups. In this case the 3‘-hydroxyl of the oligonucleotideswas phosphorylated before the deblocking step according to ref 22. Upon complete removal of the protecting groups, the unblocked oligonucleotides were isolated by ion-exchange and reverse-phase chromatography. The sequences of oligonucleotides were confirmed by the Maxam-Gilbert procedure (24). 5’-PhnDerivatives of Oligonucleotides. Approach A. A 0.2-0.5 pmol portion of a cetyltrimethylammonium salt of oligonucleotide (25)bearing a 5’-terminalphosphate group was dried in vacuo and dissolved in dry dimethyl sulfoxide (30pL) with (PyS)2(13pmol), (Ph)SP (13.5pmol), and DMAP (13 pmol). After 10 min of incubation at 2023 “C, 30 p L of 0.2 M NH2(CH2hNH2 ( n = 2-7) in dimethyl sulfoxide was added and the mixture was incubated at the same temperature for 10 min. The product was precipitated by 2% LiC104 in acetone (1-1.5 mL) and dissolved in 80 pL of 0.05 M N-(2-hydroxyethyl)phenazinium chloride in 0.1-0.2 M aqueous Na2C03. After 10 min of incubation, the product was precipitated and isolated stepwise by anion-exchange and reverse-phase chromatography in a yield of 80-90 76. 3’-PhnDerivatives of Oligonucleotides. Approach B. For the synthesis of 3’-Phn derivatives of oligonucleotides, final products of the triester method were used. After removal of the levulinic group (19),the 3’-hydroxyls of blocked oligonucleotides were phosphorylated as described (23). The product of phosphorylation was hydrolyzed by an equimolar amount of water and isolated by gel filtration on a Sephadex LH-20 column using the elution mixture of chloroform-ethanol (1:l). After isolation, the oligonucleotide material (- 10pmol) was dissolved in 100 pL of absolute pyridine with one of the blocked polymethylenamine alcohols (30 pmol) of general formula HO(CH2),NHCOCF3 ( m = 2-5). TPS (30 pmol) and MeIm (90 pmol) were added to the reaction mixture. In 40-60 min the product of reaction was chromatographed on silica gel using a methanol gradient (0 15%) in chloroform. Deblocking was performed as discussed above. The amino-containing oligonucleotide was successively isolated by anion-exchange and reversephase chromatography. Further introduction of the Phn residue and isolation of the product was performed as in the case of 5’-Phn derivatives of oligonucleotides. 3’,5’-Di-PhnDerivatives of Oligonucleotides were obtained using approach A starting with oligonucleotides bearing both 3’- and 5’-terminal phosphate groups or the 3’-Phn derivatives containing a 5’-terminal phosphate group. The yield of di-Phn derivatives was 40-60% regardless of the starting oligonucleotide.

-

RESULTS AND DISCUSSION

The peculiarity of phenazine quaternary salts is that oxidizing addition of primary or secondary amines to the second position of the dye heterocyclic system is very easy (26).Thus, at the first stage of synthesis, oligonucleotide derivatives with primary amino groups were prepared. Two methods of synthesis were used (Scheme I, parts A and

B).

Lokhov et ai.

Bioconjugate Chem., Vol. 3, No. 5, 1992

Scheme I Approach

A

(PYS), d(pNpNpN., ,pN)

(Ph) P z -NL-’C”-:-d(NpNpN. H3C

DMAP

H,N ( C H , ) ,NH, n=2-7

+

~~~

H,N(CH,),NH-PO,

,pN)

LJ

H3C

-d(NpNpNp

pN)

CH,CH,OH HN ( C H , ) , N H

Phn*CI

-PO,

- d (NpNpNp

pN)

_____-

Approach

B 0

d(pNpNpN

. p N ) - O H --=---d(pNpNpN

TPS,Melm H O ( CH,),

~

d(pNpNpN

m=2-5

F

I

pN) - 0 - P - 0

0

-COCF,

-~

~~

,

p N ) - 0 - P - 0 - (CH,),NH-COCF,

o,+\

‘./c I

0

NH, d(pNpNpN

I pN)-0-P.0-(CH,),NH,

0

A254

0 100 200 300 mL Figure 1. Elution profile on a Polisil SA anion-exchange column (8 X 250 mm) of crude d(pCpCpApApApCpAp)-OCH&H2NH2 (highest peak). Eluted with a linear gradient from 0 to 0.3 M KzHPOl (pH 6.5) in 30% acetonitrile at a flow rate of 8 mL/min.

In the first case (approach A), a polymethylenediamine residue was attached to the terminal phosphate group of an unprotected oligonucleotide which had been preliminarily activated to form a reactive 4-(dimethylamino)pyridine derivative. The yield of each stage of the synthesis was usually quantitative. In the second case (approach B), a polymethylenamine residue was introduced into the terminal phosphate group of the oligonucleotide via a phosphoroester bond at the last step of the triester synthesis. Approach B is somewhat more laborious than A, but gives an equally good yield of the final product (Figure 1). In alkaline medium, where the primary amino group is unprotonated, the reaction with the quaternary phenazine salt goes quickly (- 10 min). Oligonucleotides bearing a Phn residue were isolated from the reaction mixture in quantitative yield (Figures 2 and 3).

Bloconjugate Chem., Voi. 3, No. 5, 1992 417

Phenarinium-Stabilized Complementary Complexes A254 3.2

[H3CCNI,%

1

100

50

13Om

Figure 2. Results of isolation of Phn-NH(CHZ)zNH-d(pCpCpApApApCpA) (2) and Phn-NH(CH&NH-d(pCpCpApApApCpA) (3)from the reaction mixture by a Lichrosorb RP 18HPLC column (4X 250 mm). Profile 1 is an analysis of the starting unmodified heptanucleotide d(pCpCpApApApCpA). The flow rate was 3 mL/min with a linear gradient of acetonitrile from 0 to 20% in 0.05 M LiC104.

200 300 400 500 600 nm Figure 4. UV-visible spectra of 2-[(6-aminohexyl)amino]-10-

(2-hydroxyethy1)phenazinium chloride (l),d(pCpCpApApApCPA) (2), d(pCpCpApApApCpAp)-OCHZCHZNH-Phn(3), and

Phn-NH(CHz)zNH-d(pCpCpApApApCpAp)-CHzCH2NH-Ph (4) in 0.16 M NaC1,0.02 M Na2HP04, and 0.1 mM EDTA (pH 7.4). Scheme I1

A254

3.2

3'

5

r

3

d ( p A p C p A p A p A p C p C p ) .NH (CH,)*N,H 5 ' b ( I l l , n=2-7)

-

y

CH,CH,OH N h

CH,CH,OH H N ( C H , ) p O - d ( p A p C p A p A P A P C P C p ) - N H ( C H , ) 2N\n 3

5 ( V )

0

20

40

60

A

CH,CH,OH W

N

h

WNW

s(kru.

Figure 3. Results of isolationof d(pCpCpApApApCpAp)-OCH2CH2NH-Phn (2) and Phn-NH(CH2)zNH-d(pCpCpApApApCpAp)-OCHzCH2NH-Phn (3) from the reaction mixture by a reverse-phase column. Profile 1 is analysis of the starting nucleotide d(pCpCpApApApCpAp)-OCHzCHzNH2.The flow rate was 2 mL/min; other conditions of isolation were the same as in Figure 2.

It is possible to prepare conjugates of an oligonucleotide with two Phnresidues. The choice of a synthetic approach depends on whether protected or unprotected oligonucleotides are used as starting material. With unprotected oligonucleotides bearing two phosphate groups on the 3'and 5'-end, we used approach A (Scheme I). Also for this purpose the 3'-Phn derivatives of oligonucleotides (final products of the approach B) were chosen. In contrast to the mono-Phn derivatives of oligonucleotides, the yields of the 3',5'-di-Phn derivatives (Figure 3) were considerably lower. The structures of all oligonucleotide derivatives prepared were confirmed by different methods. Thus it was found that the dye is coupled only to the amino groups preliminarily introduced into the oligonucleotidestructure. The unmodified oligonucleotides do not react with N-(2hydroxyethy1)phenaziniumchloride under these conditions. The UV-visible spectra of the Phn derivatives have absorption maxima corresponding to the oligonucleotide domain at 260 nm and to the 2-[ (6-aminohexy1)aminol10-(2-hydroxyethy1)phenaziniumchloride moiety at 237, 290,390, and 530 nm (Figure 4). Having compared spectral profiles of the oligonucleotide and dye with those of the Phn derivatives, we could confirm both the existence and the number of dye residues in the molecular structure. Phn derivatives are violet-pink in color, which makes them easily detectable. The chromatographic data was con-

sistent with the suggested structures (Figures 2 and 3). Naturally, the mobility of the oligonucleotide derivatives during RPC falls with the introduction of the hydrophobic dye residues. As follows from the profile of Figure 2, the length of a polymethylene linker also affects the mobility of the Phn derivatives. A heptanucleotide derivative, PhnNH(CH&NH-d(pCpCpApApApC)pA, was prepared using two methods: approach A (Scheme I) and a reaction between 2-[ (6-aminohexyl)aminol-10-(2-hydroxyethyl)phenazinium chloride and a reactive 5'-phosphoryl4-(dimethy1amino)pyridine derivative of the heptanucleotide. The chromatographic and spectral characteristics of these heptanucleotide Phn derivatives prepared by the two methods were the same. To elucidate the effect of the Phn residue on the stability of oligonucleotide complexes, we examined duplexes formed by dodecanucleotide (I), heptanucleotide (II),and modified heptanucleotide (111, n = 2-7; IV, m = 2-5; and V) derivatives (Scheme 11). By analyzing temperature dependencies we determined that thermal stability of the duplexes increased passing from complex I + I1 to complexes I I11 (n= 2-7) or I IV ( m= 2-5) and further to I V apparently due to the introduction of the Phn residues (see Figure 5). The melting points of all investigated complexes are listed in Table 11. The thermodynamic parameters AH, AS, and AG were calculated on the basis of thermal denaturation curves of the complexes (Figure 5), including those derivatives in which the length of the polymethylene linker between the dye and the terminal phosphate group was varied. It is noteworthy that the introduction of Phn causes large changes in the thermodynamic properties of the complexes. For example,the enthalpy of the complexes formed by mono-Phn derivatives of heptanucleotide I1 is

+

+

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418

Lokhov et

Bioconjugate Chem., Vol. 3, No. 5, 1992 AAzwIAT

A

10

20

30

40

50

60

7OoC

Figure 5. Differential melting curves for complexes of d(pApApCpCpTpGpTpTpTpGpGpC) with d(pCpCpApApApCpA) ( l ) , d(pCpCpApApApCpAp)-OCHZCHZNH-Phn(2), Phn-NH(CHz)zNH-d(pCpCpApApApCpA)(3), and Phn-NH(CH&NHd(pCpCpApApApCpAp)-OCHzCHzNH-Phn(4) in the same buffer as in Figure 4. Table 11. Thermodynamic Parameters and Melting Points of Oligonucleotide Complexes Bearing a n N-(2-Hydroxyethyl)phenaziniumResidue. ASf4 complex AHf 1 AG(37OC) f Tmm* 0.1 OC (nor m) kcal/mol calimol K 0.02 kcalimol -4.93 27.2 I + I1 -37 -104 -8.30 47.4 I + I11 (2) -53 -143 -7.99 45.0 I + I11 (3) -54 -147 47.1 -60 -166 -8.52 I + I11 (4) -7.92 44.5 -54 -150 I + I11 (5) 43.7 -53 -146 -7.76 I + I11 (6) -7.41 41.9 -49 -135 I + I11 (7) 39.6 -49 -134 -6.99 I+IV(2) 37.7 -46 -126 -6.74 I + IV (3) 38.3 -46 -127 -6.84 I + IV (4) -6.75 37.8 I+IV(5) -44 -121 54.8 -67 -183 -9.89 I+V ~

0 The conditions for thermal denaturation of the complementary complexes, from which all thermodynamic parameters and Tmm’s were determined, are described in Figure 4.

increased by 7-23 kcal/mol compared to that of the unmodified counterpart. The corresponding change in free energy of complex formation at 37 “C is 1.81-3.59 kcal/mol, which is approximately equivalent to extending the duplex by one G-Cor two A-T pairs (27). It should be pointed out that the contribution of the Phn residue to the enthalpy of the complementary complex I + I11 (n = 4) was more than twice as high as that of the acridine dye (10 kcal/mol) in a duplex formed by polydeoxyriboadenylic acid and a derivatived oligothymidylate (3)* As seen from Table 11,the introduction of a dye into the 5’-terminal phosphate was the most advantageous. In the modified complex I + I11 (n = 4), AG increased by 3.59 kcal/mol (compared to 2.06 kcal/mol for the 3’-Phn derivative). The same effect was observed for DNA duplexes with an unpaired nucleoside residue at the 3’and 5’-terminus (28). Elongation of the polymethylene chain connecting the dye to the oligonucleotide usually leads to destabilization of the hybrid complexes. Yet, in the case of the 5’-Phn derivatives of the heptanucleotide, the enthalpy and entropy of complex formation at first increases and then decreases with elongation of the linker chain from di- to heptamethylenic. The complex I + I11 (n = 4) with a tetramethylene linker demonstrated the greatest stability. Its enthalpy mounted to 60 kcal/mol, whereas for complex I I11 (n= 2) it was 7 kcal/mol lower. The difference between the AG values of these complexes is not as striking (-0.2 kcal/mol) because a pronounced enthalpy effect of the duplex I I11 (n= 4) is compensated by a large entropy effect (23 cal/mol K). It is likely that a tetramethylene linker gives optimal conformational

+

+

al.

freedom to the Phn residue, thus allowing maximal overlap between the heterocyclic system of the dye and the nearest base pair. In contrast to the 5’-Phn derivatives I11 (n = 2-7), the 3’-Phn derivatives of the heptanucleotide do not have a smooth dependence of the energy of complex formation on the length of the polymethylene linker. It may only be stated that the complex I + IV (m= 2) is the most stable on the basis of the AG and T,, data. The analog with a trimethylene linker, on the contrary, is the least stable in the complexes I IV (m = 2-5). Summarizing the thermodynamic data of complexes I I11 (n = 2-7) and I + IV ( m = 2-51, we discovered that complexes with an even number of methylene links are generally more stable than those with an odd number of methylene links. This fact and the expressed dependence of complex stability on the linker length were taken into account when synthesizing DNA-targeted oligonucleotide reagents (29, 30). The complex I V formed by the 3’,5’-di-Phn derivative of heptanucleotide was the most stable among all the duplexes investigated (Figure 3, Table 11). Its melting temperature was 54.8 “C. Relative to the unmodified complex I 11, the increase in free energy was 4.96 kcal/ mol. It is likely that the Phn residues of oligonucleotide V affect the duplex I + V independently, as the value of the free energy change (4.96 kcal/mol) was very similar to the sum of the effects (5.43 kcal/mol) of complexes I + I11 (n = 2) and I IV (m = 2). The association constant of complex I + V at 37 “Cis 9.5.106 M-l, whereas the analogous value for the unmodified complex I I1is 3.103 M-I. Thus it may be possible to equalize affinities of the heptanucleotide I1 and its 3’,5’-di-Phn derivative V during complex formation with dodecanucleotide I, if the di-Phn derivative is used at a concentration 3 orders of magnitude lower. All the data discussed above show that introduction of a phenazine quaternary salt into oligonucleotides is an effective and promising approach for stabilization of nucleic acid complementary complexes and for directed influence on the functional activity of polynucleotide targets. So, it would not be groundless to consider Phn derivatives of oligonucleotides as promising antisence agents and for use as diagnostic probes.

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Phenazlnlum-Stablllzed Complementary Complexes

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method a t installation “Victoria-4M”. Izv. Sib. Otd. Akad. Nauk SSSR (Russ.) 1, 119-123. (21) Krynetskaya, N. F., Zayakina, G. V., Oretskaya, T. S., Volkov, E. M., and Shabarova, Z. A. (1986) Oligodeoxyribonucleotide-3’-phosphate synthesis by selective cleavage of 3’terminal uridine. Nucleosides Nucleotides 5, 33-43. (22) Bausk, E. V., Gorn, V. V., and Lebedev, A. V. (1985)General scheme of the solid-phase phosphotriester synthesis of 5’phosphorylated-oligodeoxyribonucleotides on the basis of @-cyanoethylp-chlorophenyl esters of N-acylnucleoside-5’phosphates. Bioorgan. Khimia (Russ.) 11, 815-820. (23) Dobrynin,V. N.,Bystrov,N. S.,Chernov,B.K., Severtsova, I. V., and Kolosov, M. N. (1979) Nucleophilic catalysis of phosphotriazolide-effected phosphorylation in the triester synthesis of oligonucleotides. Bioorgan. Khimia (Russ.) 5, 1254-1256. (24) Ambrose, B. J. B., and Pless, R. C. (1987) DNA sequencing: Chemical methods. Methods Enzymol. 152, 522-538. (25) Knorre, D. G., Vlassov, V. V., Zarytova, V. F., and Karpova, G. G. (1986) Nucleotide and oligonucleotide derivatives as enzymes and nucleic acid targeted irreversible inhibitors. Chemical Aspects. Advances in Enzyme Regulation (G. Weber, Ed.) pp 277-300, Pergamon Press, Oxford. (26) Swan, G. A. (1957) Phenazines. The Chemistry of Heterocyclic Compounds, 11 (A. Weissberger, Ed.) p 21, Interscience Publishers, New York. (27) Breslauer, K. J., Frank, R., Blocker, H., and Marky, L. A. (1986)Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. U.S.A. 83, 3746-3750. (28) Gaffney, B. L., Marky, L. A., and Jones, R. A. (1982) The influence of the purine 2-amino group on DNA conformation and stability. Synthesis and conformationalanalysis of d[T(2aminoA)]~.Nucleic Acids Res. 10, 4351-4361. (29) Zarytova,V. F., Kutyavin, I. V., Silnikov,V.N., andShishkin, G. V. (1986) Modification of nucleic acids in stabilized complementary complexes. I. Synthesis of alkylating derivatives of oligodeoxynucleotides having a 5’-terminal N-(2hydroxyethy1)phenazinium residue. Bioorgan.Khimia (Russ.) 12,911-920. (30) Zarytova, V. F., Ivanova, E. M., Kutyavin, I. V., Sergeev,D. S., Silnikov, V. N., and Shishkin, G. V. (1989) Modification of nucleic acids in stabilized complementary complexes. 111. Synthesis of oligonucleotide derivatives bearing N-(2hydroxyethy1)phenazinium residue at their 3’4erminus. Sib. Otd. Akad. Nauk SSSR (Russ.) 6, 3-9. Registry No. 1.11, 143006-35-3;1.111 n=2, 143036-82-2; 1,111 n=3,143036-84-4;1.111 n=4,143006-37-5;1.111 n=5,143006-39-7; 1,111n=6,143006-41-1;1.111n=7,143006-43-3;1-IVm=2,14300645-5; LIV m=3, 143006-47-7;IeIV m=4, 143006-49-9;1-IVm=5, 143006-51-3; I-V, 143006-53-5; Phn+Cl-, 94496-01-2; (iPr)zNP(OCH3)0U(Bz)z,118934-42-2;(DMTr)U(Ac),142247-31-2;HO(CHz)zNHz,141-43-5;HO(CH2)3NHz, 156-87-6; HO(CH2)4NH2, 13325-10-5; HO(CHz)bNHz, 2508-29-4; HO(CH2)2NHCOCF3, 697429-4; HO(CH2)~COCF~,7800&158; HO(CH2)JWCOCF3, 128238-43-7;HO(CHz)5NHCOCF3,128238-44-8; NHz(CH2)2NH2, 107-15-3; NHz(CHz)3NHz, 109-76-2; NHz(CHz)rNH2, 110-60-1; NHz(CHz)sNHz,462-94-2;NHz(CH&NHz, 124-09-4;NHz(CH2),NHz, 646-19-5;phenazine, 92-82-0;ethylene oxide, 75-21-8;2-[(6aminohexyl)aminol-l0-(2-hydroxyethyl)phenaziniumchloride, 143036-80-0.