Nucleobase-Specific Quenching of Fluorescent Dyes. 1. Nucleobase

Nov 30, 1995 - derivatives have been determined in aqueous media. One common sequence of the quenching efficiency has been found for the nucleobases...
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J. Phys. Chem. 1996, 100, 5541-5553

5541

Nucleobase-Specific Quenching of Fluorescent Dyes. 1. Nucleobase One-Electron Redox Potentials and Their Correlation with Static and Dynamic Quenching Efficiencies Claus A. M. Seidel* Max-Planck-Institut fu¨ r Biophysikalische Chemie, Abt. 010, POB 2841, D-37018 Go¨ ttingen, Germany

Andreas Schulz and Markus H. M. Sauer Physikalisch Chemisches Institut, UniVersita¨ t Heidelberg, Im Neuenheimer Feld 253, D-69120 Heidelberg, Germany ReceiVed: May 30, 1995; In Final Form: NoVember 30, 1995X

Intermolecular static and dynamic fluorescence quenching constants of eight coumarin derivatives by nucleobase derivatives have been determined in aqueous media. One common sequence of the quenching efficiency has been found for the nucleobases. The feasibility of a photoinduced electron transfer reaction for the nucleobasespecific quenching of fluorescent dyes is investigated by the calculation of the standard free energy changes with the Rehm-Weller equation. A complete set of one-electron redox potential data for the nucleobases are determined electrochemically in aprotic solvents for the first time, which are compared with values obtained by various other methods. Depending on the redox properties of the fluorescent dyes, the sequences of the quenching efficiencies can be rationalized by the orders of electrochemical oxidation potentials (vs NHE) of nucleosides (dG (+1.47 V) < dA < dC ≈ dT < U (g +2.39 V)) and reduction potentials (dG (< -2.76 V) < dA < dC < dT < U (-2.07 V)). The correlation between the intermolecular dynamic quenching constants and the standard free energy of photoinduced electron transfer according to the classical Marcus equation indicates that photoinduced electron transfer is the rate-limiting step. However, an additional, water-specific gain of free energy between -0.5 and -0.9 eV shows that additional effects, like a coupled proton transfer and a hydrophobic effect, have to be considered, too. Furthermore, the capability of the nucleobases to form ground state complexes with fluorescent dyes is influenced by their redox potentials. The relevance of these observations to current efforts for DNA sequencing with a detection by laser-induced fluorescence and their application to other dyes are discussed.

1. Introduction DNA sequence analysis and DNA detection play important roles in modern biology, medicine, and biotechnology.1-3 Thus, many efforts are made to improve the current technologies for automated DNA sequencing. One very successful technique of DNA detection is the use of laser-induced fluorescence.4-9 The laser-induced fluorescence analysis is of broad interest because of the exceedingly low detection limits achievable with this technique.10-14 In this way, the detection of a single fluorescent molecule has been realized10-12 by fluorescence correlation spectroscopy, time-resolved fluorescence spectroscopy, and confocal fluorescence microscopy. Until now, only the spectral properties of the dyes are used for the nucleobase identification. Two new concepts of fluorescence detection, based on time-resolved fluorescence spectroscopy, have been developed for DNA sequencing: four “multiplex” dyes15 or one “intelligent” dye.16,17 Multiplex dyes have nearly identical absorption and fluorescence emission spectra, but differ in their fluorescence lifetimes, which are used to characterize the nucleobases. Using several sets of multiplex dyes with different excitation wavelengths, a simultaneous multianalyte analysis may be possible. An intelligent dye makes use of, usually unwanted, excited state interactions between the fluorescent dye and the DNA to characterize the neighboring nucleobase. By use of one coupled, “intelligent” dye with nucleobase-specific fluorescence lifetimes, * Corresponding author. X Abstract published in AdVance ACS Abstracts, February 15, 1996.

0022-3654/96/20100-5541$12.00/0

a one-lane/one-dye concept for DNA sequencing may be realized. A dye which is appropriate for this purpose is Coumarin-120 (C-120). If coupled to nucleotides, this dye has four nucleobase-specific fluorescence lifetimes, which range from 5.3 to 1.9 ns (see below). The fluorescence quantum yield of the label in the DNA environment has to be optimized to achieve low detection limits for DNA sequencing with detection by laser-induced fluorescence.14 In this contribution we will present investigations which may help to solve two problems: (I) avoidance of dyeDNA interactions; (II) design of dyes with nucleobase-specific interactions. The interactions between nucleobase derivatives and fluorescent labels have been studied by various groups.18-28 But in many cases questions on the mechanisms of the quenching reactions remained open, because the redox properties of the nucleobases are still not completely known. Using the nucleobase-specific quenching of Coumarin-120 (C-120) as an example, we want to present our investigations on the molecular aspects of this quenching reaction, which is useful for understanding the dye-DNA interactions of many other fluorescent dyes, such as rhodamines, pyrene, fluoresceine, naphthalimides, stilbenes, and 1,3,4-oxadiazoles. Nucleobase-specific quenching by Fo¨rster fluorescence resonance energy transfer is ruled out by the lack of appropriate spectral properties. Furthermore, nucleobases contain no heavy atoms, and the nucleobase quenching efficiencies do not correlate with their pKa values (see below). Therefore, it becomes evident that photoinduced electron transfer and protoncoupled electron transfer (H atom transfer) have to be considered © 1996 American Chemical Society

5542 J. Phys. Chem., Vol. 100, No. 13, 1996 for these molecules. In an additional paper of this series we will provide direct evidence, by transient absorption spectroscopy, that radicals of the Carbostyryl-124 are formed by nucleobase fluorescence quenching. In this paper we discuss the fundamental properties of the nucleobase-specific quenching of coumarins in four sections. Intermolecular and intramolecular quenching constants of nucleobases are discussed qualitatively in the first section using one-electron redox potentials. Several classes of coumarin dyes were used to cover a wide range of rate constants. In the next section the correlation of the free energy ∆G0ET with the intermolecular quenching rate constant kq is analyzed by the classical Marcus equation taking a water-specific free energy shift into account. In the third part we discuss possible reasons of this free energy shift. Besides a detailed analysis of the redox potentials of the nucleobases and of the coumarins, we consider coupled reactions and the hydrophobic effect. Finally, we will show in the fourth part that the ground state complex formation between coumarins and nucleobases correlates with their redox properties, too. 2. Experimental Section The fluorescent dyes (Lambda Physik (Go¨ttingen, FRG) and Lambda Probes & Diagnostics (Graz, Austria)) were used without further purification. Coumarin-39 was a kind gift of Prof. Drexhage (Gesamthochschule Siegen, FRG). Nucleosides, 2′-deoxynucleosides, and nucleotides were used as received from Sigma. Conjugates (C-120/N) between C-120 and nucleotides were synthesized from the corresponding nucleoside-R-thiomonophosphates and 7-amino-N-(2-ethylaminocarbonyliodomethyl)4-methylcoumarin (I-C-120).30 The purity of the conjugates was proved by HPLC (Beckman System Gold) and mass spectrometry.16d The coumarin chromophore of I-C-120 was synthesized from 3-aminophenol in four steps.31-33 The obtained 7-amino-N-(2-ethylamino)-4-methylcoumarin was treated with iodoacetic acid 4-nitrophenyl ester in dry diethyl ether to give I-C-120. Nucleoside-R-thiomonophosphates of Ade and Gua were a kind gift of R. Goody (Max-Planck-Institut fu¨r Molekularbiologie, Dortmund, FRG). The 2′-deoxynucleosideR-thiomonophosphates of Cyt and Thy were synthesized according to Goody.34 All experiments were done at 25 °C. Absorption measurements were performed with the UV spectrometer Lambda 7 (Perkin-Elmer). Fluorescence emission spectra and steady state quenching experiments were monitored on the spectrometer Smart 8000 (SLM). The absorption and fluorescence measurements were performed in a buffered aqueous solution (0.05 M K2HPO4/KH2PO4, pH 7), which was not degassed. Fluorescence lifetimes of the conjugates were measured by a DFDL-UV fluorescence spectrometer as described previously.16a,35 To exclude polarization effects, the fluorescence of the probe was observed in a conventional 90° setup with a polarizer set to the magic angle (54.7°). The total fluorescence emission was detected; scattered laser light was eliminated by a cutoff filter (Schott glass filter, GG-400; 2 mm). To determine intermolecular dynamic quenching constants by Stern-Volmer experiments, time-resolved fluorescence spectroscopy was performed with the time-correlated single-photoncounting fluorescence spectrometer LS 100 (Photon Technology, Wedel, FRG). The samples were excited with a nitrogen-filled flashlamp at 337 or 358 nm monitoring the fluorescence maxima of the different dyes. For time-resolved fluorescence spectroscopy the dye concentrations were adjusted to provide an absorbance of approximately 0.005-0.01 at the excitation wavelength.

Seidel et al. The fluorescence decay curves were fitted by a least squares fit using Marquart’s algorithm36 in terms of a sum of exponentials (eq 1). If a monoexponential decay was inadequate to describe the data, we used two exponentials, where the amplitudes a1 and a2 correspond to the fluorescence quantum yield weighted proportion of excited fluorophores (a1 + a2 ) 1) and τ1 and τ2 to the lifetime of the species.

(

( )

( ))

1 -t 1 -t I(t) ) I0 a1 exp + a2 exp τ1 τ1 τ2 τ2

(1)

The quality of the fit was judged by the weighted residuals,37 the autocorrelation function,38 and the reduced χ2.37 The average lifetime τav is defined by eq 2.

τav ) a1τ1 + a2τ2

(2)

Redox potentials were determined by differential pulse polarography (Pg) and cyclic voltammetry (CV) in N,N-dimethylformamide (DMF) and acetonitrile (AN). DMF (water-free, under nitrogen) and AN (water-free, under nitrogen) were purchased from Aldrich. Tetra-n-butylammonium hexafluorophosphate (TBA PF6) was used as received from Merck (Darmstadt, FRG), dried at 110 °C, and stored under argon. Further purification of the solvent system (0.1 M TBA PF6 in DMF or AN) was reached by flash chromatography with aluminum oxide (Alumina N-Super I; ICN Biomedicals, Eschwege, FRG) according to Kiesele.39 Dried argon was bubbled through the test solution for 5 min prior to and passed over the solution during the measurements. The aqueous saturated calomel electrode (SCE) was used as the reference electrode. The reported potentials are cited against the normal hydrogen electrode (NHE) by adding 0.24 V to the measured potential. In electrochemical measurements typical concentrations of the nucleosides and of the fluorescent dyes were 1 mM for cyclic voltammetry and 0.1 mM for differential pulse polarography. Cyclic voltammetry was performed with a potentiostat (Bank LB 75L), voltage scan generator (Bank VSG 72), digital multimeter (Keithley 177 DMM), and a x-y recorder (Metrawatt, RE 551). The scan speed was 100 mV/s. A threeelectrode arrangement in a single cell was used for measurements: a thin Pt sheet as auxiliary electrode, a Luggin capillary with reference electrode, and a glassy carbon electrode (Methrom 628, surface 18.8 mm2) as working electrode, which was polished with Al2O3 powder in water after each measurement. The positive and negative voltage limits are +1.84 and -2.76 V vs NHE in DMF and +2.44 and -2.56 V vs NHE in AN. Ferrocene had a half-wave potential of +690 mV vs NHE in DMF and +650 mV vs NHE in AN. Differential pulse polarography was made with a polarograph (E506) in a single cell with the electrode (VA-663) from Metrohm. DMF was used as solvent, with positive and negative voltage limits of 0.24 and -2.66 V vs NHE. 3. Results and Discussion 3.1. Dynamic Quenching Efficiencies and Redox Properties of Nucleobase Derivatives. Fluorescence quenching of coumarins by nucleosides and nucleotides was investigated by steady state and time-resolved fluorescence spectroscopy in aqueous media. In many cases these nucleobase derivatives are effective quenchers. The molecular structures of eight coumarin derivatives are given in Figure 1a. The quenching efficiency depends on the dyes and on the nucleobases. Systematic sequences of the intermolecular quenching efficiencies have been found for the nucleobases in Stern-Volmer plots

Nucleobase-Specific Quenching of Fluorescent Dyes

J. Phys. Chem., Vol. 100, No. 13, 1996 5543 TABLE 1: Intermolecular Quenching Constants kq of Nucleobase Derivativesa,d nucleosides: kq(109 M-1 s-1) dye

τ0 (ns)

dG

dA

dC

dT

U

P

Ca-124 C-120 C-39 C-102 C-307

4.9 5.0 5.6 5.6 4.5

bi 3.6b + + 2.0

1.3 + + + 0

1.5 1.6 1.1 1.5 0

2.3 2.8b 1.6 2.7b 0

2.3 3.0 n.d. 2.9 n.d.

4.5 4.3 n.d. 4.0 n.d.

nucleotides: kq (109 M-1 s-1) dye C-3H C-3Es C-3CN

τ0 (ns) 1.5 1.9 2.6

dGMP 2.5/5.9 5.3 4.7

dAMP

dCMP

dTMP

UMP

P

3.5 4.2 3.9

0.6 1.3 2.3

1.7 2.5 1.4

n.d. bi 1.2

bi n.d. n.d.

c

a 3σ-error estimation: 3% for τ0, 10% for kq. b Double-exponential decays (τ2 < τ1 < τ0), kq is determined from τ1. c Second value: nucleoside dG. d Symbols: (n.d.) not done, (+) increase of fluorescence lifetime due to the “quencher”, (bi) non-single-exponential decays with τ1 < τ0 < τ2 and τav ≈ τ0.

D* + N f D•(+/-) + N•(-/+) Figure 1. (a) Fluorescent dyes used for quenching experiments. (b) Conjugates (C-120/N) between C-120 and nucleotides.

the standard free energy changes ∆G0ET for the formation of solvent-separated radical ion pairs (SSIP) must be evaluated by the Rehm-Weller equation (eqs 4a,b).40 Since it is not known a priori whether the excited dye reacts as electron donor and the nucleobase as electron acceptor (eq 4a) or vice versa (eq 4b), both possibilities are considered.

nucleobase-reduction: ∆G0ET ) E0(D•+/D) - E0(N/N•-) E0,0(D) + ∆G0() (4a) nucleobase-oxidation: ∆G0ET ) E0(N•+/N) - E0(D/D•-) E0,0(D) + ∆G0() (4b) Figure 2. Typical Stern-Volmer plots of the dye C-120 with the quenching nucleotides U (F0/F (4); τ0/τ (2)) and dC (F0/F (O); τ0/τ (b). In steady state measurements C-120 was excited at the isosbestic point (360 nm) and its fluorescence was detected at the emission maximum (443 nm).

(Figure 2). Table 1 shows the dynamic quenching constants kq which were determined by the Stern-Volmer equation (eq 3),

τ0/τ ) 1 + kqτ0[Q]

(3)

where τ0 is the fluorescence lifetime in the absence of a quencher and τ is the lifetime in the presence of a quencher Q with the concentration [Q]. For the nucleobases there exists one limiting sequence of the quenching efficiency: dG, dA, dC, dT, U. In the case of Ca-124 the efficiency increases, and in the case of C-3CN the efficiency decreases. The intermediate sequences of the efficiency obtained for the other dyes are rationalized by a gradual change of the reaction pathway, resulting in a mixing of the increasing and decreasing sequence. Therefore, we assume a quenching mechanism which is common for all dyes investigated here. In the following we present experimental results gaining some insight into the molecular aspects of nucleobase specific quenching. To check the feasibility of an electron transfer pathway for a quenching reaction of a fluorescent dye (D) by a nucleobase derivative (N) according to

∆G0(), defined in eq 5,41 contains two correction terms based on the Born equation: (I) the interaction energy of radical ions with radius rIon in a complex with a center to center distance aEC; (II) the difference of ion solvation between a solvent of interest, in this case water with a dielectric constant W ) 78, and an organic solvent O.

∆G0() )

[(

)

]

1 e2 1 1 1 4π0 rIon aEC W rIonO

(5)

Using the standard parameters rIon ) 3 Å and aEC ) 7 Å,41 a solvent term ∆G0() ≈ -0.1 eV can be estimated for a quenching reaction in water. If the possibility of an electron transfer pathway for an intermolecular excited state reaction with a series of quenchers is tested by a correlation of quenching rates and redox properties, it must be kept in mind that the electron transfer rate is governed by electronic and nuclear barriers. Classical theories assume electron transfer to be adiabatic to the extent that the electronic transition probability is virtually unity and the rate-determining factor is the nuclear reorganization. For intermolecular quenching experiments this assumption implies a comparable structure of the encounter complexes between the various reactants; that is, it is assumed in this case that the electronic transition probability is not drastically influenced by orientation and symmetry of orbitals and separation distance.42,43 This hypothesis has been verified by many authors (e.g. ref 42 with 29 examples and refs 40, 44, 45).

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TABLE 2: Experimental Oxidation Potentials (V vs NHE) in Acetonitrile with Reaction Types (I-III) and Calculated One-Electron Standard Oxidation Potentialsa E0(N•+/N) of the Nucleobase Derivatives: Nucleobase (Nb), Nucleoside (N), Nucleobase Derivative As Described in Ref (Nd) base

EO,CV(Ox)b

E0O,CV(N•+/N)c

guanine

1.49 (N; I)

1.47

adenine

1.96 (N; I)

1.94

cytosine

2.14 (N; I)

2.12

thymine uracil

2.11 (N; I) g2.39 (N; I)

2.09 g2.37

E0W,CV(N•+/N)

EW,Pu(Ox)d (pH: 13)

1.53 (Nb; III) 1.19 (N; III)f 1.16 (N; III)g 1.63 (Nb; III)e 1.52 (N; III)f 1.47 (N; III)g 1.86 (Nb; III)e

1.67 1.34 1.31 g1.94 g1.75 g1.70 g2.01

1.18 (N; II)

1.73 (Nb; III)e

1.90

EW,CV(Ox) (pH: 6.5) e

E0W,Pu(N•+/N) 1.56

1.28 (N; III)

g1.99

≈1.35(Nb; III) ≈1.60 (Nd; III)h ≈1.33 (Nb; II) ≈1.60 (Nd; III)i

g1.86 g2.11 1.74 2.01

a Calculation of E0(N•+/N) with eqs 13-15; the following pK values were used:52a,68 (G/G(-H)-) ) 10.0, (G•+/G(-H)•) ) 3.9, (A/A(-H)-) g a 14, (A•+/A(-H)•) e1, (C/C(-H)-) g13, (C•+/C(-H)•) ≈ 4, (T/T(-H)-) ) 10.5, (T•+/T(-H)•) ) 3.6, pKa values of U ≈ T. b Estimated to be c accurate to (0.05 V. Solvation energy of -0.02 eV for the solvent change DMF/H2O is estimated using the Born equation. d Reference 48, estimated to be accurate to (0.02 V, except for 1-methyl-pyrimidines; the potential of the reference tryptophan is shifted from 0.57 V vs NHE to 1.05 V vs NHE.52a,69 e Nucleobases; ref 57. f The pH dependence of EW,CV(Ox) for G (1.49-0.046 pH) and for A (1.73-0.032 pH); ref 58. g Reference 56b. h 1-Methylcytosine. i 1-Methyluracil.

TABLE 3: Experimental Reduction Potentials (V vs NHE) with Reaction Types (I-III) in Dimethylformamide and Calculated One-Electron Standard Reduction Potentialsa E0(N/N•-) of Nucleobase Derivativesb base

EO,CV(Red)c

EO,Pg(Red)d

E0O,PG(N/N•-)e

EW,Pg(Red) (pH: 3)

guanine adenine cytosine thymine uracil purine

CMP ≈ TMP > UMP > P), which is equivalent to the order of nucleobase oxidation potentials. On the basis of the quenching constants of Table 1, we conclude that the quenching pathway is stepwise switched from a nucleobase reduction to a nucleobase oxidation on going from Ca-124 to C-3CN (see Figure 4). A more detailed view of the magnitude of the rate constants kq, given in Table 1, reveals additional features of the nucleobase-specific quenching reaction. Besides activation- or diffusion-controlled fluorescence quenching,66 an increase of the fluorescence lifetimes is observed in the presence of nucleobase derivatives, where free energy calculations by eqs 4a,b (see above) indicate that quenching by electron transfer is unfavorable; but there are nevertheless dye-nucleobase interactions. Furthermore, there are some dye-nucleobase combinations (e.g. Ca-124/dG or C-3Es/UMP) where biexponential fluorescence decays are observed with one lifetime smaller and the other larger than τ0, giving evidence for an excited state equilibrium with a free energy close to zero.67 Finally, there is a third group of dye/quencher mixtures (C102/dT, C-120/dT, and C-120/dG) having fluorescence decay

curves with two fluorescence lifetimes, τ1 and τ2, which are smaller than τ0. The lifetimes τ2 are in the subnanosecond region (400-700 ps) and do not change with the quencher concentration [Q] within the error limits, whereas the lifetime τ1 decreases linearly with an increasing quencher concentration [Q], as expected for an intermolecular quenching experiment. Because fluorescent contaminations of the nucleobase derivatives and dyes could be excluded by HPLC analysis, only a new fluorescent species, formed between the quencher and the dye, can explain the observed behavior. In view of the observed static quenching, which will be discussed in section 3.5, we suggest the existence of weakly fluorescent ground state complexes. Thus, the lifetime τ1 was used for the approximation of the intermolecular quenching constant kq. The quenching properties of nucleosides and nucleotides are compared for a 7-amino-coumarin, C-120, and for a 7-methoxycoumarin, C-3Es. Table 5 shows the quenching rate constants of nucleosides and nucleotides, which are equal within the error limits or quite similar for most nucleobases. There are significant differences between the quenching rates of nucleosides and nucleotides in the case of the nucleobase Gua for both dyes, which may be due to steric reasons. Only for Gua the orientation of the purine ring with respect to the ribose moiety (syn/anti) does depend on the phosphate group.68 As discussed above, steric influences can reduce the maximum electronic transition probability. Therefore, nucleosides and nucleotides are investigated in cases of doubt. If there is a significant difference in their quenching efficiencies, the larger value is used in the latter calculations to reduce artifacts by steric effects. 3.1.4. Intramolecular Quenching Constants in Coumarin/ Nucleobase Conjugates. To compare the inter- and intramolecular fluorescence quenching efficiency, we synthesized conjugates (C-120/N) between a C-120 derivative and nucleoside phosphorothioates, which are shown in Figure 1b. The conjugates have a long flexible linker so that the donor-acceptor pair can adopt geometries which are optimal for the interactions. Table 6 gives fluorescence lifetimes in different aqueous media: (I) 500 mM aqueous KH2PO4/K2HPO4, pH 7; (II) 6 M urea as a denaturing agent; (III) 6% polyacrylamide gel with 6 M urea (100 mM Tris/PO4, pH 7); these conditions are equivalent to those used for DNA sequencing. There are no differences between intra- and intermolecular nucleobase quenching efficiencies. But, the intramolecular rate constants kq(intra) ()1/τ - 1/τ0) of the C-120 conjugates, ranging from 0.02 × 109 to 0.25 × 109 s-1, are at least 1 order of magnitude smaller. Under the assumption of a through-space mechanism, this indicates that chain dynamics decreases the rate of formation of collision complexes appropriate for a successful excited state reaction, but the rate is still determined by the quenching reaction. As in intermolecular quenching experiments of C-120, the conjugates of C-120 with T and G exhibit biexponential fluorescence decays, as in the intermolecular studies. In 6 M urea and in the polyacrylamide gel the

Nucleobase-Specific Quenching of Fluorescent Dyes

J. Phys. Chem., Vol. 100, No. 13, 1996 5547

TABLE 6: Fluorescence Lifetimes of C-120/Nucleobase Derivative Conjugates in Different Aqueous Mediaa solvent

C-120 τ (ns)

τav (ns)

KH2PO4/K2HPO4 6 M urea 6% polyacrylamide gel

4.9 4.9 5.2

1.9 2.3 2.9

a

C-120/G τ1 (ns) τ2 (ns) 2.2 2.4 3.0

0.5 0.5 0.5

a2

C-120/A τ (ns)

C-120/C τ (ns)

τav (ns)

0.16 0.06 0.03

5.3 5.1 5.5

4.4 4.3 4.8

2.3 3.0 3.4

C-120/T τ1 (ns) τ2 (ns) 2.6 3.1 3.5

0.7 0.7 0.7

a2 0.15 0.04 0.02

Excitation wavelength 386 nm; 3σ-error estimation: 3% for single-exponential decays and 5% for double-exponential decays.

fluorescence lifetimes increase slightly for the nucleobases C, T, and G. Furthermore, the amplitude of the short living species decreases by at least 60%, because urea weakens intramolecular interactions of fluorescent ground state complexes. To conclude, the nucleobase-specific quenching of C-120 is maintained in all aqueous media. Another series of conjugates containing the nucleobases Ade, Thy, and Ura, which are linked to 7-methoxycoumarin (C-3H) via a propylene bridge, was synthesized and characterized by Wenska and Paszyc.74 They found a sequence of nucleobase quenching efficiency, Ade > Thy > Ura, which is in agreement with our results of intermolecular quenching of C-3H. 3.2. Free Energy Changes of Nucleobase-Specific Quenching. 3.2.1. Marcus Relation. After the prediction of the quenching pathway, we will correlate the intermolecular quenching constants with the standard free energy of photoinduced electron transfer according to the classical Marcus equation. Before doing this, we consider an additional feature of nucleobase-specific quenching in aqueous media. We investigated the solvent dependence of fluorescence quenching of the C-120/ nucleobase conjugates, but we could not detect any quenching of the coumarin fluorescence in the organic solvents investigated so far (e.g. methanol, ethanol, acetic acid ethyl ester, DMF, AN, propylene carbonate, and formamide). A similar solvent dependence is reported by Wenska and Paszyc74 for C-3H/ nucleobase conjugates. Because of this behavior, the question arises of whether special effects occur in water, which have to be taken into account for the free energy balance. If the standard free energy of radical ion pair formation ∆G0ET is calculated for the C-120/nucleobase conjugates in DMF by eqs 4a,b, the standard free energy of electron transfer ∆G0ET(DMF) is endergonic for all four nucleobases ranging from +0.59 eV for Ade to +0.12 eV for Gua; that is, no quenching should be possible, which is indeed the case in all nonaqueous solvents. If the solvation energy in water ∆G0() ≈ -0.1 eV (eq 5) is taken into account, ∆G0ET(W) is still positive, which stands in contradiction to the observed effective quenching in water. To circumvent the problem of calculating ∆G0ET for aqueous media, the Rehm-Weller equation (eqs 4a,b) is extended. In eq 6 it is now assumed that the standard free energy of fluorescence quenching ∆G0q(W) is a sum of the driving force for electron transfer ∆G0ET(DMF) and of the average free energy in water ∆G0av(W) (see section 3.3).

∆G0q(W) ) ∆G0ET(DMF) + ∆G0av(W)

(6)

Because this additional gain in free energy is not a priori known for nucleobase-specific quenching of coumarins, ∆G0av(W) will be taken as an additional variable in the correlation of kq with ∆G0ET(DMF). In the “normal” Marcus region the intermolecular quenching constant kq depends on two steps: the diffusion-controlled formation/decay of an encounter complex with the rate constants kd and k-d and the activation-controlled rate of electron transfer kET.42,43,44 If the equilibrium constant of the encounter complex KEC is defined by KEC ) kd/k-d and a steady state concentration

Figure 5. Correlation between the intermolecular quenching constants kq in water (Table 1) and the standard free energy of electron transfer ∆G0ET(DMF). For the calculation of the classical Marcus equation we assume kd ) 5.6 × 109 M-1 s-1,66 νN ) 1 × 1011 s-1, KEC ) 0.86 M-1, and ∆G0() ) 0. The dyes are specified by different gray shadings of the symbols, and the quenching nucleobase derivatives are written above the data points. So for equations and fit parameters, see text.

of the encounter complex used, we obtain eq 7 for the experimentally observed rate constant kq.

kq )

kd 1 + (kd/kETKEC)

(7)

The electron transfer rate constant kET can be expressed in an Arrhenius form, and the activation energy is given by Marcus’s outer-sphere electron transfer theory. By these means, eq 8 becomes

kq )

kd 1 + (kd/νNKEC) exp[(∆G0q + λ)2/4λkT]

(8)

In eq 8 the pre-exponential Arrhenius factor is equal to an effective solution collision frequency νN, and λ is the reorganization energy reflecting intramolecular bond length changes and solvent reorganization. In the following data analysis we assume kd ) 5.6 × 109 M-1 s-1 66 and use the standard parameters νN ) 1 × 1011 s-1 and KEC ) 0.86 M-1.42 ∆G0() is set to 0, because all solvent effects are taken into account by the term ∆G0av(W). The correlation between the intermolecular quenching constants kq (Table 1) and the standard free energy of electron transfer ∆G0ET(DMF) (eqs 4a,b) is shown in Figure 5, where log(kq) is plotted vs ∆G0ET(DMF). In Figure 5 the dependence of kq on ∆G0ET(DMF) shows a typical behavior: (I) diffusion-limited rate constants at large negative ∆G0ET(DMF) values; (II) curvature changes in the transition region between the horizontal and inclined branches of the correlation; similar rate constants are observed for a ∆G0ET(DMF) range of approximately 0.4 eV, but the expected dependence on the redox properties within a series of quenchers is evident for each dye; (III) a displacement along the

5548 J. Phys. Chem., Vol. 100, No. 13, 1996

Seidel et al.

∆G0ET(DMF) axis. To illustrate this free energy shift ∆G0av(W), the theoretical Marcus curve is given in Figure 5 (dotted curve). The free energy shift is specific for a dye and does not depend on the quenchers used in the Stern-Volmer experiment. Due to the large free energy shift ∆G0av(W), most of the dye/ quencher combinations have a positive ∆G0ET(DMF). In data analysis it is useful to distinguish between two main groups representing the extremes of the free energy shift ∆G0av(W). The first group (CLS), squares in Figure 5, consists of coumarins with a large free energy shift ∆G0av(W): C-39, C-102, C-120, and C-3H. The second group (CSS), shown as triangles, contains dyes with a small free energy shift ∆G0av(W): Ca-124, C-307, C-3Es, and C-3CN. The theoretical curves of eq 8 were calculated by use of Marquart’s least squares algorithm with the variables λ and ∆G0av(W). A minimum χ2 could be obtained for both data sets with a reorganization energy λ ) 1.2 eV (117 kJ/mol) and free energy shifts ∆G0av(W) of -0.91 eV for the group CLS (solid curve in Figure 5) and -0.55 eV for the group CSS (dashed curve in Figure 5). The fitted reorganization energy is in agreement with results determined by intermolecular quenching experiments of other authors42,44 and theoretical calculations (see eq 9). In intermolecular quenching reactions of organic molecules the solvent reorganization energy λs is the main contribution to the reorganization energy λ. A value of 1.1 eV for λs may be calculated by eq 9,43 where nD is the refractive index and the other symbols have their meaning as defined in eq 5.

λs )

(

)(

)

1 ∆e2 1 1 1 4π0 rIon aEC (n )2 w D

(9)

3.3. Additional Gain of Free Energy in Aqueous Solution. Because the additional gain of free energy ∆G0av(W), which ranges from -0.55 to -0.91 eV, is clearly larger than the solvation energy ∆G0() of the reactants in water, we have to prove whether our ∆G0ET calculation is appropriate for water. The used calculation of the singlet energy E0,0 ) (Emax(Abs) + Emax(Flu))/2 may overestimate E0,0,65,73,75c,76 because coumarins and Carbostyryl-124 are fluorescent dyes with a considerable change of the dipole moment ∆µ upon excitation; for example, ∆µ ≈ 4.5 D for Coumarin-153.77 The evaluation of the magnitude of entropic losses mainly due to solvation effects of the excited state78 shows that the approximation E0,0 ) ∆H00,0 ≈ ∆G00,0 is still justified for our case. Therefore, the question arises of whether the calculation of ∆G0ET may contain systematic errors caused by wrong data on the redox potentials or whether additional effects on the reactions have to be considered. 3.3.1. Redox Potential Shifts Induced by Chemical Followup Reactions. As mentioned in section 3.1, the redox potentials of the nucleobases and of the coumarins, used for the calculation of ∆G0ET(DMF), are obtained from electrochemical measurements with irreversible redox behavior even though aprotic solvents have been used. In the following we will show that despite the irreversibility, the uncertainty of the redox potentials is very small. The irreversible redox behavior indicates that chemical follow-up reactions of the electrochemically generated radicals take place at the electrode-solvent interface, shifting the redox equilibrium. For the limiting case of large ratios of the typical radical reaction rates vs the scan rates V the following equations describe the shift of the observed cathodic peak potential Epc with respect to the standard potential E0c.79 The shifts caused by a pseudo-first-order decomposition with the rate constant kf

or by a dimerization with the rate constant kdim and the bulk concentration c0 are given by eq 10 or 11, respectively, where n is the number of electrons, T the temperature, R the gas constant, and F the Faraday constant.

Epc - E0c )

(( ) ) (( ) )

kf RT RT ln - 1.56 2nF V nF

(10)

kdimc0 RT RT ln - 3.12 3nF V nF

(11)

Epc - E0c )

Hence, the observed anodic peak potentials in Table 2 are lower limits and the cathodic peak potentials in Table 3 are upper limits of the one-electron redox properties; that is, the observed shift of the free energy ∆G0ET would be even larger if reversible redox potentials could be used for the calculation of ∆G0ET. For two examples the uncertainty of the redox potentials is calculated by eqs 10 and 11: (I) Taking the reduction potentials of uridine (U) and 2′-deoxythymidine (dT), a potential shift of 50-150 mV is calculated by the use of kf and kdim values measured by Bresnahan55 and Cummings.54 (II) Using transient absorption spectroscopy, the lifetime of Ca-124 radical cations in DMF and water was determined to be larger than 10 µs, which corresponds to an upper limit of 140 mV for a potential shift (eq 10). Furthermore, we can exclude nucleobase-specific artifacts of the nucleobase oxidation potentials by the good correlation with ionization potentials (see inset, Figure 3).47,80,81 The agreement of the redox potentials with the effects of X-ray radiation on DNA82 is given as an additional example of the biological relevance of the electrochemical redox potentials: Electron holes are trapped at the most easily oxidizable nucleobase Gua, and electrons are predominantly trapped at the most easily reducible nucleobases Thy and Cyt. Therefore, the one-electron redox potentials in organic solvents EO,CV(Ox) and EO,Pg(Red), given in Tables 2 and 3, should be able to explain the nucleic acid base specific quenching efficiency in any solvent if an electron transfer mechanism is the dominant quenching process. The Influence of Protic Equilibria on Redox Potentials. For the further use of the redox potentials it is important to analyze and to compare the data obtained in protic and aprotic solvent systems. Because a one-electron reduction induces pKa changes of the acid (N)z+1 and the corresponding base (N(-H))z with the charge z, it is crucial in aqueous systems to take protonations and deprotonations of the radical ions into account. The resulting additional gain of free energy of a proton transfer reaction (PT) ∆G0PT in a proton-coupled electron transfer equilibrium is described by the “Michaelis cycle”75,83 (eq 12), where ∆G0PT is equal to the difference between the standard redox potentials (vs a reference potential) of the acid with the potential E0(Nz+1/Nz) and the base with the potential E0(N(H)z/N(-H)z-1).

∆G0PT ) -E0(Nz+1/Nz) + E0(N(-H)z/N(-H)z-1) (12) Table 7 gives three main categories of redox potentials for coupled electron transfer and proton transfer reactions which must be distinguished for various protonation states of nucleobase N. To compare the midpoint potential of the half-cell Em obtained by various methods under different conditions in Tables 2 and 3, we have converted these data into the potentials of interest (reaction type I), the one-electron oxidation potential E°(N•+/N) and the one-electron reduction potential E0(N/N•-). The pH dependence of the half-cell potential Em for a reduction is described by eqs 13-15, where Ka is the prototropic

Nucleobase-Specific Quenching of Fluorescent Dyes

J. Phys. Chem., Vol. 100, No. 13, 1996 5549

TABLE 7: Correlations between Midpoint Em(I-III) and Standard Potentials E0 type (I) (II) (III)

reaction neutral nucleobase: oxidation of N reduction of N deprotonated or protonated nucleobase: oxidation of N(-H)reduction of N(H)+ coupled electron and proton transfer: oxidation of N reduction of N

equilibrium constant of the oxidized (Ox) or the reduced (Red) species and (H+) is the H+ activity.

Em ) E0(N/N•-) +

(

)

+ RT (H ) + Ka(Red) ln F Ka(Red)

(

)

+ RT (H ) + Ka(Ox) (13) ln F Ka(Ox)

RT ln((H+) + Ka(Red)) F RT ln((H+) + Ka(Ox)) (14) F

Em ) E0(NH+/NH•) +

Em ) E0(N,H+/NH•) +

RT ln((H+) + Ka(Red)) F + RT (H ) + Ka(Ox) (15) ln F Ka(Ox)

(

)

Three rules are crucial for the subsequent analysis of the redox potentials in Tables 2 and 3. (I.) Classification of the reaction type (I-III in Table 7) by solvent and pH is necessary. (II.) For electrochemical redox reactions in aqueous solution, a detailed mechanism of electrochemical and chemical follow up reactions is necessary to understand the potential shift. In many cases a combined loss/gain of two electrons and protons (2e-/2H+ reaction) takes place.56,71,72 An appropriate description for the first reaction step of the nucleobase (N) may be the coupled loss/gain of an electron (e-) and a proton (H+), which is equivalent to the reaction type III in Table 7. Therefore, the calculated standard redox potentials (eqs 13-15) are the lower/ upper limits for one-electron redox potentials. (III.) One-electron redox potentials are obtained in aprotic solvents for neutral nucleobase derivatives, because no protons are involved in the reaction balance (limitations, see eqs 10 and 11). Comparison of Oxidation Properties in Organic SolVents and Aqueous Solution. Table 2 gives the midpoint potentials with the corresponding reaction type I-III and the calculated standard one-electron oxidation potentials E0(N•+/N) for the oxidation of nucleobase derivatives obtained by (i) cyclic voltammetry in acetonitrile (EO,CV(Ox)); (ii) cyclic voltammetry in aqueous solution at pH 6.5 (EW,CV(Ox));56-58 (iii) pulse radiolysis equilibrium analysis in aqueous solution at pH 13 (EW,Pu(Ox)).48 The comparison of the calculated one-electron standard potentials in Table 2 yields two main results. (I) The order of the oxidation potentials EO,CV(Ox) g EW,CV(Ox) g EW,Pu(Ox) is observed for all nucleobases, which corroborates the classification of the reaction types I-III. (II) The agreement between the one-electron standard oxidation potentials obtained by the various methods is satisfactory for the nucleobases Gua, Ade, and Cyt. In agreement with MO calculations47,60 our

standard potential

N•+ + e- f N N + e- f N•-

E0(N•+/N) E0(N/N•-)

N(-H)• + e- f N(-H)N(H)+ + e- f N(H)•

E0(N(-H)•/N(-H)-) E0(N(H)+/N(H)•)

N(-H)• + e- + H+ f N N + e- + H+ f N(H)•

E0(N(-H)•, H+/N) E0(N, H+/N(H)•)

results for U and T clearly show that the potentials obtained from pulse radiolysis E0W,Pu(N•+/N) are too small. Comparison of Reduction Properties in Organic SolVents and Aqueous Solution. Less experimental data on one-electron reduction potentials exist, because the electron affinities of nucleobases are quite low and no electrochemical reductions of neutral nucleobase species were achieved due to the limited accessible potential range (background discharge potential ≈ -1.7 V vs NHE). This agrees with our results in aprotic solvents, where the nucleobases have quite negative reduction potentials ranging from -1.9 to -2.74 V vs NHE. In Table 3 midpoint potentials with the corresponding reaction type I-III and the calculated standard one-electron reduction potentials E0(N/N•-) are given: (i) polarographic reduction potentials in water at pH 3 (EW,Pg(Red));53,56b,71,72 (ii) polyarographic reduction potentials in DMF (EO,Pg(Red)); (iii) cyclovoltammetric reduction potentials in DMF (EO,CV(Red)); (iv) reduction potentials of pyrimidine bases obtained by pulse radiolysis equilibrium analysis at pH 8.5 (EW,Pu(Red)).51 There is no satisfactory agreement between the standard reduction potentials E0W,Pg(N/N•-), E0O,Pg(N/N•-), and E0W,Pu(N/ N•-). The induced anodic potential shift of E0W,Pg(Red) due to fast follow-up reactions is especially large.56b,71,72 Furthermore, there is a discrepancy of about 1 V between cyclic voltammetry and pulse radiolysis for the same redox couple (N/N•-) of U and T. Our result, that the reduction potentials of thymine derivatives are in the range of -2 V vs NHE, is supported by several other methods: (I) fluorescence quenching experiments in acetonitrile;84 (II) that T cannot be electrochemically reduced in aqueous media; (III) MO calculations.47,60 Summary. The above comparison shows that for these important molecules the electrochemical potentials in aprotic solvents, EO,CV(Ox) and EO,Pg(Red), are the only appropriate data set of standard one-electron redox potentials for the description of photoinduced electron transfer reactions at this moment. Due to the complexity of follow up reactions in water, the uncertainty of the redox potentials and the sources of errors are too large for some bases. 3.3.2. Proton-Coupled Electron Transfer and Hydrophobic Effect. The discussion in section 3.3.1 shows that the effects of possible systematic errors on ∆G0ET are much smaller than the observed free energy shift in water. Hence, other effects like the influence of irreversible follow up reactions85,86 or solvent-specific aspects must be considered to explain the free energy shift allowing to operate an endergonic electron transfer. In connection to section 3.3.1, one should be aware that electron transfer and proton transfer can be intimately interrelated in aqueous media.20,22c,52,75 For several electron-donoracceptor systems a sequential or simultaneous proton transfer is suggested to explain the observed effective quenching with ∆G0ET being close to zero, e.g. methylene blue intercalated in DNA,22c acridine quenched by phenols,40 benzo[a]pyrene quenched by nucleosides,29 and tyrosine in peptides,87 but no initially produced protonated or deprotonated radical ion could

5550 J. Phys. Chem., Vol. 100, No. 13, 1996 be experimentally detected so far. On the other hand, there is some experimental evidence for sequential electron and proton transfer reactions. For example, (I) an unusual deuterium isotope effect29 is observed for fluorescence quenching of benzo[a]pyrene by nucleosides; (II) in fluorescence quenching experiments of an excited ruthenium complex by the nucleotide GMP20 the formation of GMP(-H)• of the initially formed GMP•+ could be demonstrated; (III) Mataga and co-workers88 could show in pyrene-amine systems that a “slow” proton transfer follow up reaction in the nanosecond time scale is one of the depopulation reactions of the rapidly formed charge transfer exciplex state. If the pKa values, given in Tables 2 and 3,52,68 are used to calculate the average free energy changes due to a coupled proton transfer of a nucleobase ∆G0PT at pH 7 by eqs 13-15, an average value of approximately -0.3 eV is obtained for a nucleobase oxidation and reduction. Considering a protoncoupled reaction of the nucleobase and the solvation energy in water ∆G0() ≈ -0.1 eV, one obtains a free energy shift in water of approximately -0.4 eV, which is quite close to the observed shift (-0.55 eV) of the coumarin group CSS (triangles in Figure 5). On the other hand, the shift of the 7-aminocoumarins CLS (squares in Figure 5) of -0.9 eV is clearly not met, which may indicate protic reactions of 7-amino-coumarin radicals, too. Unfortunately, the pKa values of coumarin radicals are not known (the pKa value of the parent compound C-120 is ≈289). On the basis of these experimental facts further work is in progress to find more experimental proof of a proton-coupled electron transfer mechanism responsible for nucleobase-specific quenching in aqueous media. On the other hand, the good correlation of the quenching efficiency with the redox properties of the reactants indicate that the rate-limiting step of the quenching reaction is determined by electronic barriers. Furthermore, steric effects on electron transfer resulting in multiple Rehm-Weller plots90 cannot explain the differences between the two dye groups CSS and CLS, because the variations of the molecular structure in one group (see Figure 1a) have no consequences for the quenching rate constants. In electron transfer theory the influence of solvent polarity on photoinduced transfer rates is introduced by eqs 5 and 9,91 but water-specific effects cannot be rationalized by these equations. Therefore, Abuin and Lissi92 suggested that a hydrophobic effect93 may be responsible for the strongly enhanced intermolecular quenching rates of organic molecules in water. The molecular aspect behind this suggestion is a reduced water-solute interface; that is, the distance in an encounter complex aEC decreases in water. Using eq 5, eq 15,94 and the exponential distance dependence of the electron transfer rate constant, the distance aFC in an encounter complex may be reduced with the consequence of increased quenching rate and lowered activation energy. This explanation is supported by the effect of urea on the fluorescence quantum yield of the C-120/nucleotide conjugates (C-120/N). Urea can be called a “salting-in” agent, because the water solubility of organic molecules is enhanced. Thus, the reduced quenching efficiency (see Table 6) and the reduced ground state complexation in 6 M urea solution are consistent with the idea of reduced hydrophobic interactions. Hydrophobic interactions are especially important for the cases of weak quenching, as described in section 3.1.3 (see Tables 1 and 6). They may induce a gradual change of the quenching pathway from radical formation to exciplex formation:96 that is, new excited state species are formed with new properties (red-shifted fluorescence spectra and increase of lifetimes).97 Now, the value of the apparent quenching rate constant is controlled by the lifetime of the

Seidel et al. TABLE 8: Static Stern-Volmer Constants KS and Dynamic Stern-Volmer Constants KD of Nucleosides for the Dye C-120 Obtained by Eqs 3, 16, and 17 nucleoside

∆G0ET(DMF) (eV)

KD (M-1)a,b

KS (M-1)a,c

fluorescence decays

dA dC dT U dG P

0.59 0.37 0.28 0.18 0.12 -0.03

increase of F 8 14 15 18 21

increase of τ 0d 9 11 23 28

monoexp. monoexp. doubleexp. monoexp. doubleexp. monoexp.

a Excitation wavelength at the isosbestic point: 360 nm. Error estimation: (10-15%. b Calculated from the data of Table 1 with eq 16. This value was used for the calculation of KS. c In the nonlinear least squares fit using eq 17, KD was kept constant.96 d The SternVolmer constants, obtained in steady state and time-resolved fluorescence quenching experiments, are identical within the error limits. Therefore, it is assumed that the static quenching efficiency of dC is below the detection limit.

exciplex and the equilibrium constant of its formation,98 resulting in more efficient electronic interactions than expected for pure radical formation. 3.4. Ground State Complexes. As already mentioned, static quenching is another important aspect of nucleobase-specific quenching, which becomes apparent in many Stern-Volmer plots of this work by the discrepancy between the quenching determined in steady state and time-resolved measurements (see Figure 1). In addition to this, ground state complexation becomes obvious by a bathochromic shift of the absorption and fluorescence excitation spectra of the dyes in the presence of nucleosides with a static quenching efficiency (data not shown). The quenching of the dye C-120 by U is shown in Figure 1 as an example of a nonfluorescent ground state complex. Intermediate cases are excited ground state complexes of C-120/T and C-120/G, with slightly red-shifted fluorescence emission spectra and lifetimes ranging from 400 to 700 ps (see Table 6). The static quenching effects have been analyzed for the dye C-120 in detail. The dynamic Stern-Volmer constant KD is defined by eqs 3 and 16.

KD ) kqτ0

(16)

In steady state Stern-Volmer experiments observing the fluorescence intensity F, the static Stern-Volmer constant KS can be calculated by eq 17, where KS is equal to the association constant of a nonfluorescent 1:1 ground state complex.

F0/F ) (1 + KD[Q])(1 + KS[Q])

(17)

The dynamic and static Stern-Volmer constants KD and KS of all nucleobases along with ∆G0ET are listed in Table 8 for the dye C-120. Table 8 shows that the dynamic Stern-Volmer constant and the static Stern-Volmer constant, as well, increase with the increasing driving force of photoinduced electron transfer. The correlation between the dynamic and static quenching efficiency, demonstrated in Figure 6, is another proof of the importance of charge transfer interactions in nucleobasespecific quenching. If ∆G0ET is unfavorable, the static quenching efficiency is zero, as demonstrated for the nucleosides of Ade and Cyt (see Figure 1). For comparison the association constant was also determined by absorption spectroscopy. The association constant KS and the extinction coefficient DQ of the complex may be determined by eq 1899 if the absorbance  of a solution with the dye D is monitored as a function of the quencher concentration [Q] and if the extinction coefficient of the dye D at a given wavelength is known.

Nucleobase-Specific Quenching of Fluorescent Dyes

J. Phys. Chem., Vol. 100, No. 13, 1996 5551

Figure 6. Correlation between the static Stern-Volmer constants KS and the dynamic Stern-Volmer constants KD of nucleosides for the dye C-120.

)

(DQ - D)KS[Q] (1 + KS[Q])

+ D

(18)

A plot according to eq 18 of a spectrophotometric titration of the dye C-120 with the nucleoside dT is shown in Figure 7. The analysis yields an association constant of 7 ( 3 M-1 between Coumarin-120 and thymidine which is in satisfactory agreement with the static Stern-Volmer constant of 9 ( 2 M-1 determined by fluorescence spectroscopy. The obtained association constant is larger than those expected for “pure” charge transfer complexes. This indicates that, besides charge transfer interactions, additional hydrophobic forces are very important for the stabilization energy of the complexes in a polar environment. 4. Conclusion The correlation between the intermolecular dynamic nucleobase quenching constants of coumarin and the standard free energy of photoinduced electron transfer according to the classical Marcus equation indicates that photoinduced electron transfer plays an important role for the quenching mechanism. However, an additional water-specific gain of free energy between -0.5 and -0.9 eV and efficient static quenching show that a coupled proton transfer and hydrophobic interactions allow an endergonic electron transfer to operate. Looking forward to new, highly sensitive DNA-sequencing methods based on single fluorescent label detection, the fluorescence quantum yield and the control of dye-nucleobase interactions are of great importance. After careful examination of all quenching reactions, photoinduced electron transfer and proton-coupled electron transfer (H atom transfer) will be quite often the possible reaction pathways, which have to be taken into consideration. On the basis of the work on coumarins, we are able to explain the often observed fluorescence quenching of other fluorescent dyes by nucleobase derivatives and to predict the sequence of the nucleobase quenching efficiency. If the nucleobases are reduced, the sequence of the quenching efficiency is Gua < Ade < Cyt < Thy eUra < Pur; if the nucleobases are oxidized, the sequence is reversed. If the redox potentials of the dyes of interest are known, the hypothesis of a charge transfer mechanism may be proved by the calculation of the free energy of charge separation ∆G0ET. The dye Rhodamine-6G has an oxidation potential of 1.22 V vs NHE and a reduction potential of -0.6 V. Thus, quenching should be possible only by Gua, which is in accordance with the observed behavior and recent results of Sauer et al.15e obtained for long-wavelength-absorbing rhodamine derivatives. Pyrene has an oxidation potential of 1.44 V and a reduction potential of -1.86 V. Because these redox properties are similar

Figure 7. Spectrophotometric titration of the dye C-120 with the nucleoside dT at the observation wavelength 386 nm. Analysis with eq 18 yields DQ(386) ) 5300 ( 1500 M-1 cm-1 and KS ) 7 ( 3 M-1, if D(386) ) 2300 M-1 cm-1 is used.100

to C-120, comparable reaction pathways for quenching are expected: Gua is oxidized; the other nucleobases, Thy, Cyt, and Ade, are reduced. The observed sequence of the nucleobase quenching efficiency is consistent with this prediction.26b In other cases, the redox potentials of the dyes are not known to the authors, but the quenching efficiency has the typical sequence of the redox properties of the four nucleobases Gua, Thy, Cyt, and Ade, which allows one to estimate the redox properties of the dye. For instance, stilbene-1 is quenched strongly by G and only weakly by dT, whereas the other nucleosides have no effect; Fluorol 7GA100 and fluorescein27100 are only influenced by dG. In sum, nearly all fluorescent dyes are quenched by the nucleobase Gua. The exceptional good quenching efficiency of Gua may be explained by the good electron-donating properties. Acknowledgment. The authors wish to thank J. Wolfrum for the generous support of this work. We thank M. Ko¨llner and A. Orth for their support in the fluorescence lifetime measurements. For their help in the electrochemical measurements we thank M. Stephan, R. Kellerbauer, and A. Schro¨er. We are grateful to K. O. Greulich, K.-H. Grellmann, and S. Steenken for fruitful discussions. Abbreviations Solvents: acetonitrile (AN); N,N-dimethylformamide (DMF). Nucleic acid bases: adenine (Ade); cytosine (Cyt); guanine (Gua); uracil (Ura); thymin (Thy); purine (Pur). Nucleosides: adenosine (A); cytidine (C); guanosine (G); uridine (U); purine riboside (P). 2′-Deoxynucleosides: deoxyadenosine (dA); deoxycytidine (dC); deoxyguanosine (dG); deoxythymidine (dT). Nucleotides: uridylic acid (UMP). 2′-Deoxynucleotides: deoxyadenylic acid (dAMP); deoxycytidylic acid (dCMP); deoxyguanosylic acid (dGMP); deoxythymidylic acid (dTMP). Fluorescent dyes: 7-amino-N-(2-ethylaminocarbonyliodmethyl)-4methyl coumarin (I-C-120); Carbostyryl-124 (Ca-124); Coumarin120 (C-120); Coumarin-307 (C-307); Coumarin-102 (C-102); Coumarin-39 (C-39); 7-methoxycoumarin (C-3H); 3-cyano-7methoxycoumarin (C-3CN); 7-methoxycoumarin-3-carboxylic acid ethyl ester (C-3Es). References and Notes (1) Matthews, J. A.; Kricka, L. J. Anal. Biochem. 1988, 169, 1-25. (2) Trainor, G. L. Anal. Chem. 1990, 62, 418-426. (3) Hunkapillar, T.; Kaiser, R. J.; Koop, B. F.; Hood, L. Science 1991, 254, 59-67. (4) (a) Ansorge, W.; Sproat, B.; Stegemann, J.; Schwager, C. J. Biochem. Biophys. Meth. 1986, 13, 315-323. (b) Ansorge, W.; Zimmer-

5552 J. Phys. Chem., Vol. 100, No. 13, 1996 mann, J.; Schwager, C.; Stegemann, J.; Erfle, H.; Voss, H. Nucleic Acids Res. 1990, 18, 3419-3420. (5) Brumbaugh, J. A.; Middendorf, L. R.; Grone, D. L.; Ruth, J. L. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5610-5614. (6) Prober, J. M.; Trainor, G. L.; Dam, R. J.; Hobbs, F. W.; Robertson, C. W.; Zagursky, R. J.; Cocuzza, A. J.; Jensen, M. A.; Baumeister, K. Science 1989, 238, 336-341. (7) Smith, L. M.; Sanders, J. Z.; Kaiser, R. J.; Hughes, P.; Dodd, C.; Connell, C. R.; Heiner, C.; Kent, S. B. H.; Hood, L. E. Nature 1986, 321, 674-679. (8) Swerdlow, H.; Zhang, J. Z.; Chen, D. Y.; Harke, H. R.; Grey, R.; Wu, S.; Dovichi, N. J. Anal. Chem. 1991, 63, 2835-2841. (9) Karger, A. E.; Harris, J. M.; Gesteland, R. F. Nucleic Acids Res. 1991, 19, 4955-4962. (10) (a) Rigler, R.; Mets, U ¨ .; Widengren, J.; Kask, P. Eur. Biophys. J. 1993, 2, 169-175. (b) Eigen, M.; Rigler, R. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5740-5747. 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