Photoinduced charge separation in ...

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www.rsc.org/pps | Photochemical & Photobiological Sciences

Photoinduced charge separation in pyrenedicarboxamide-linked DNA hairpins†‡ Pierre Daublain,a Karsten Siegmund,a Mahesh Hariharan,a Josh Vura-Weis,a Michael R. Wasielewski,a Frederick D. Lewis,*a Vladimir Shafirovich,*b Qiang Wang,c Milen Raytchevc and Torsten Fiebig*c Received 12th August 2008, Accepted 7th October 2008 First published as an Advance Article on the web 27th October 2008 DOI: 10.1039/b813995d The synthesis and photophysical properties of the dihydroxypropylamide derivative of pyrene-1,6-dicaboxamide, its aniline dyad, and DNA conjugates are reported. The dicarboxamide serves as a hairpin linker for bis(oligonucleotide) conjugates having short base pair stems. The dihydroxypropyl derivative has a large fluorescence quantum yield and long singlet decay time, as determined by fluorescence and time-resolved broad band pump–probe spectroscopy. The aniline dyad undergoes exergonic charge separation with formation of a radical ion pair which decays via charge recombination. The highly characteristic transient absorption spectrum of the pyrene anion radical is used to monitor the dynamics of its formation and decay. The dicarboxamide-linked hairpin conjugates undergo charge separation with adjacent guanine and adenine bases. Charge separation with guanine is accompanied by efficient pyrene fluorescence quenching. In contrast, reversible charge separation with adenine results in multiple exponential fluorescence decay. The energetics and dynamics of charge separation are compared with those of other arenedicarboxamide DNA hairpin linkers.

Introduction Pyrene-oligonucleotide conjugates have been widely employed in studies of molecular assembly, fluorescence energy transfer, and photoinduced electron transfer in DNA.1,2 Several approaches to the introduction of the pyrene chromophore into duplex DNA have been developed. These include the use of pyrenemodified nucleosides in which the chromophore is located in the major or minor groove3–6 and nucleoside analogs in which pyrene replaces the nucleobase.7 Pyrene derivatives have also been attached to the 5¢- or 3¢-terminus of oligonucleotides using short flexible tethers8 and used as linkers for the formation of bisoligonucleotide conjugates which can form hairpin, duplex, and triplex structures.9,10 Studies of the electronic interactions between pyrene and nucleobases have established that singlet pyrene can serve as an electron acceptor in interactions with purines and as an electron donor with pyrimidines.3,11 Selective oxidation of guanine in DNA duplexes by pyrene radical cations derived from twophoton ionization of covalently-linked pyrenyl residues has also been reported.12 Our initial studies of the mechanism and dynamics of photoinduced charge separation and hole transport in DNA conjugates employed stilbenedicarboxamide (Sa, Chart 1) chromophores as hairpin linkers.13,14 A continuing search for chromophores with a Departments of Chemistry, Northwestern University, Evanston, IL, 602083113. E-mail: [email protected] b Boston College, Chestnut Hill, MA, 02467. E-mail: [email protected] c New York University, New York, NY, 10003. E-mail: fi[email protected] † This paper was published as part of the themed issue in honour of Nicholas Turro. ‡ Electronic supplementary information (ESI) available: MALDI-TOFMS data for pyrene conjugates (Table S1), 1 H NMR spectrum of Pyd-4G (Figure S1) and transient absorption spectra for Pyd-2G (Figure S2) and Pyd-3G (Figure S3). See DOI: 10.1039/b813995d

Chart 1

Arene dicarboxamide hairpin linkers.

longer singlet lifetimes and enhanced photochemical stability has led us to investigate electronic interactions in hairpins possessing diphenylacetylene, naphthalene, phenanthrene, and perylene diamide as well as naphthalene and perylene diimide chromophores as linkers (Chart 1).15–18 The use of dicarboxamide and diimide hairpin linkers has also been investigated by other research groups.19–22 We report here the results of our collaborative investigation of the synthesis, spectroscopy, and photoinduced charge transfer processes in DNA bis(oligonucleotide) conjugates derived from the hydroxypropyl derivatives of pyrene-1,6-dicarboxamide (Pyd-H) (Chart 2). The 1,6-dicarboxamide was selected for these studies rather than the 1,8-dicarboxamide employed by Langenegger and Häner9 because of the linear symmetry of the 1,6-isomer. The singlet excited state of the Pyd conjugates undergoes irreversible charge separation with adjacent guanine and reversible charge separation with neighboring adenine. The behavior of Pyd-H is compared with that of other arene dicarboxamide hairpin linkers.

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Photochem. Photobiol. Sci., 2008, 7, 1501–1508 | 1501

Chart 2 Structures of the Pyd-linked hairpins.

Experimental General procedures UV absorption, fluorescence, and circular dichroism spectra were obtained as previously described on samples contained in 1 cm pathlength cells.23 Quantum yields for fluorescence were determined using quinine sulfate in sulfuric acid as a reference standard.24 CD spectra are the average of 10 scans with a data interval of 0.5 nm and a time interval of 2 s per point. The base lines are corrected by subtraction of the spectrum of the buffer (10 mM phosphate, 0.1 M NaCl). Electrochemical measurements were performed using a CH Instruments Model 660A electrochemical workstation. The solvent was dimethylsulfoxide containing 0.1 M tetra-n-butylammonium perchlorate electrolyte. A 1.0 mm diameter platinum disk electrode, platinum wire counter electrode, and Ag/Agx O reference electrode were employed. Ferrocene/ferrocinium (Fc/Fc+ , 0.52 vs. SCE) was used as an internal reference for all measurements. All samples for kinetic spectroscopic measurements were prepared in 10 mM sodium phosphate, pH 7.2, buffer with 0.1 M NaCl (standard buffer). Samples were stirred during these measurements and absorption spectra checked for the occurrence of decomposition. Hairpin concentrations were adjusted to provide an absorbance of 0.2 at 354 nm in a 1 cm path-length cuvette for fluorescence measurements and 0.27 in a 1 mm path-length cell for pump–probe measurements. Fluorescence decay measurements, nanosecond transient absorption measurements, and femtosecond broadband pump–probe spectra were obtained as previously described.25,26

Materials N,N¢-Bis(3-hydroxypropyl)pyrene-1,6-dicarboxamide (Pyd-H). Friedel–Crafts reaction of pyrene with chloroacetyl chloride following the procedure of Vollmann et al.27 afforded a mixture of 1,6- and 1,8-bis(chloroacetyl)pyrene which was separated by fractional crystallization from acetic acid. The 1,6-derivative was converted to pyrene-1,6-dicarboxylic acid by refluxing with NaOH in pyridine following the method of Teramoto, et al.28 The 1,6dicarboxylic acid was converted to its diacid chloride which was reacted with 3-hydroxypropylamine by the method of Letsinger and Wu19 to provide Pyd-H. 1 H NMR 500 MHz, DMSO-d6 8.72 (2H, t, 5.5 Hz), 8.50 (2H, d, 9.5 Hz), 8.37 (2H, d, 8 Hz), 8.29 (2H, d, 9.5), 8.14 (2H, d, 8 Hz), 8.56 (2H, t, 5 Hz), 3.58 (4H, q, 6 Hz), 3.47 (4H, q, 6.5 Hz), 1.80 (4H, m). 1502 | Photochem. Photobiol. Sci., 2008, 7, 1501–1508

N,N¢-Bis(N -phenyl-N -methyl-3-aminopropyl)pyrene-1,6-dicarboxamide (Pyd-An). Reaction of the diacid chloride of pyrene-1,6-dicarboxylic acid with excess N-(3-aminopropyl)-Nmethylbenzenamine and triethylamine in tetrahydrofuran afforded Pyd-An as a white solid. 1 H NMR, DMSO-d6 8.83 (2H, t, 2.5 Hz), 8.51 (2H, d, 9.3 Hz), 8.39 (2H, d, 7.9 Hz), 8.29 (2H, d, 9.3 Hz), 8.16 (2H, d, 7.9 Hz), 7.17 (4H, dd, 7.3 Hz) 6.8 (4H, d, 8.2 Hz), 6.61 (2H, t, 7.2 Hz), 3.48 (8H, m), 2.94 (6H, s), 1.89 (4H, m). Hairpin conjugates. The diol Pyd-H was converted to its mono-DMT derivative in 35% yield following the method of Letsinger and Wu.19 The mono-DMT derivative was converted to its cyanoethyl-N,N-diisopropyl phosphoramidite derivative immediately prior to use in the preparation of conjugates (Chart 2) using conventional solid-supported phosphoramidite synthesis. The conjugates were first isolated as trityl-on derivatives by RP HPLC and then detritylated in 80% acetic acid for 30 min and repurified by HPLC. Structures confirmed by MALDI-TOF-MS (Table S1‡)29 and the purities were assured by analytical HPLC.

Results Synthesis and properties of pyrenecarboxamides and their aniline dyads The bis(hydroxypropyl)amide Pyd-H (Chart 2) was prepared by means of reaction of the diacid chloride with 3hydroxypropylamine. The 1 H NMR spectrum of Pyd-H (see Experimental section) consists of four aromatic doublets and three methylene multiplets, in accord with its symmetric structure. The absorption and fluorescence spectra of Pyd-H in methanol are shown in Fig. 1 and its fluorescence quantum yield and decay time in methanol are reported in Table 1. The fluorescence decay of Pyd-H determined using stroboscopic detection with a time resolution of ca. 0.5 ns is best fit as single exponential. The cyclic voltammogram for Pyd in dimethyl formamide (DMF) displays an irreversible reduction wave having a peak potential at - 1.75 V (referenced to SCE using ferrocene as an internal redox standard). Reaction of the acid chloride of pyrene-1,6-dicarboxylic acid with N-phenyl-N-methyl-1,3-diaminopropane provided the pyrene-aniline dyad Pyd-An (Chart 2). The long-wavelength region of the UV spectra of the dyad Pyd-An (l > 300 nm) is superimposable on that of Pyd-H, indicative of the absence of ground state electronic interaction between the pyrene and aniline chromophores in methanol. The dyad Pyd-An is very weakly fluorescent (Uf = 0.003); however its spectrum retains the vibronic structure observed for Pyd-H (Fig. 1).


Table 1 Fluorescence quantum yields and decay times for Pyd derivatives Pyd derivativea

Ufl b

t s, ns (amp)

Pyd-H Pyd-1G Pyd-2G Pyd-3G Pyd-4G Pyd-AT

0.38 0.008 0.044 0.26 0.38 0.38

19 0.044 (90), 4.8 (5), 11 (5) 0.48 (97), 6.0 (3) 0.017 (63), 2.9 (22), 6.7 (16) 0.010 (63), 4.1 (20), 8.3 (17) 0.010 (70), 4.5 (16), 8.7 (14)

a

Structures shown in Chart 2. b Fluorescence quantum yields and singlet decay times for Pyd-H in methanol and for Pyd conjugates in aqueous buffer (ca. 5 mM conjugate in 10 mM sodium phosphate, pH 7.2 with 0.1 M NaCl). Decay times for Pyd-H determined with ca. 0.5 ns time resolution and for other Pyd derivatives with ca. 35 ps instrument response function.

Fig. 2 Structure of Pyd-2G obtained using Macromodel. Fig. 1 Absorption ( ) and fluorescence (---) spectra of Pyd-H in methanol.

Synthesis and properties of the DNA conjugates The Pyd-bis(oligonucleotide) conjugates (Chart 2) were prepared using the method previously employed for Sa-linked conjugates.13,19,30 Pyd-H was first mono-protected by reaction with dimethoxytrityl chloride to provide the mono-DMT derivative and then converted to its phosphoramidite by reaction with 2-cyanoethyl diisopropylchlorophosphoramidite. The activated derivative was incorporated into oligonucleotide conjugates (Chart 2) by means of conventional phosphoramidite chemistry using an Expedite synthesizer. The conjugates were purified by HPLC and characterized by means of their electronic spectra and mass spectrometry. The structure of hairpin Pyd-4G obtained using Marcromodel31 is shown in Fig. 2. The pyrene adopts a co-facial relationship with the adjacent A-T base pairs and the base pair domain adopts a normal B-DNA structure, similar to that previously obtained from the X-ray crystal structure of a stilbenediether-linked hairpin.32 This structure is consistent with the 1D NMR spectrum obtained in D2 O (Figure S1‡).29 Salient features of the NMR spectrum include the five thymine methyl singlets. One of these is upfield of the other four, consistent with its location 5¢ with respect to guanine.33 The absence of shielding for the thymine methyl adjacent to the Pyd linker is consistent with the structure shown for Fig. 2 in which the thymine methyl lies above guanine and outside the anticipated pyrene shielding region. The aromatic region displays significant line broadening and multiple peaks tentatively assigned to the pyrene linker (unlike the simple spectrum observed for Pyd-H) indicative of the presence of multiple conformations for the hairpin linker. The absorption and fluorescence spectra of the Pyd hairpins (Fig. 3) in aqueous buffer are broadened and red-shifted by

Fig. 3 Fluorescence spectra of Pyd-conjugates in aqueous buffer.

several nm, when compared to those of Pyd-H in methanol. The conjugate Pyd-AT has a value of T m = 56 ◦ C, obtained from the derivative of the 260 nm thermal dissociation profile. This value is similar to the value for the analogous Sa-linked hairpin (59 ◦ C).19 The Pyd-nG conjugates (Chart 2) have a G-C base pair in place of one A-T base pair and thus are expected to have higher melting temperatures. Fluorescence quantum yields and decay times for the Pyd hairpins are reported in Table 1. Fluorescence decay times for the Pyd hairpins were determined by time-correlated single-photon counting using a Ti-Sp based laser system having an instrument response function of ca. 35 ps and are best fit as either double or triple exponentials. The circular dichroism spectrum of conjugate Pyd-AT is similar to that of the analogous Sa-linked hairpins,34 having a very weak broad negative band around 350 nm attributed to induced CD of the Pyd chromophore and strong bands between 200–300 nm similar to those for the duplex poly(dA):poly(dT). Pump–probe results for reference compounds Pump–probe measurements employed a Ti-Sp based system with a white-light continuum which provides a usable probe source


Photochem. Photobiol. Sci., 2008, 7, 1501–1508 | 1503

between 350–750 nm with a time resolution of ca. 150 fs and a spectral resolution of ca. 10 nm.6,35 Transient spectra for PydH obtained using pump wavelengths of 346 nm during the time window 0–5 ps are shown in Fig. 4a. The transient spectra display a short-lived band at ca. 575 nm, which is formed during the pump pulse and decays within 1 ps. This band is assigned to pyrene 2 S* which is excited by the laser pulse and decays to 1 S* via rapid internal conversion. The decay of the 575 nm band is accompanied by the rise of bands at 390 and 490 nm within the first ps which are characteristic of the pyrene S1 →Sn absorption. A shoulder at 520 nm grows in with a rise time of ca. 2 ps and intensities of the 390 and 490 nm bands increase slightly on a slower time scale (ca. 100 ps). We are uncertain as to the origin of these changes; however, they may reflect a structural relaxation process of the Pyd-H 1 S* state (such as a decrease in the pyreneamide dihedral angle) or solvent relaxation. The intensity and shape of the 490 nm band do not change appreciably during the ensuing 1.9 ns (Table 2), consistent with the long fluorescence decay time for Pyd-H (Table 1).

The transient spectra for the dyad Pyd-An are shown in Fig. 4b. These spectra are dominated by the formation of a narrow band 535 nm assigned to the anion radical Pyd- ∑ , based on comparisons with the transient spectra observed by Okada et al.36 for alkanelinked pyrene-dimethylaniline dyads and to our studies of urealinked pyrene-aniline diads.37 Simultaneous fitting of the 390 nm and 535 nm transients provides rise and decay times of 16 ps and 950 ps, assigned to charge separation and charge recombination of the Pyd- ∑ /An+ ∑ charge-separated state.

Fig. 4 Transient absorption spectra for (a) Pyd-H (0–5 ps time window) and (b) Pyd-An (0–60 ps time window) in methanol solution. Early spectra are shown in blue/green and late spectra in orange/red. Arrows indicate rising and decaying components.

Fig. 5 Transient absorption spectra of conjugate Pyd-1G in aqueous buffer, (a) 0–6 ps and (b) 6–250 ps. Early spectra are shown in blue/green and late spectra in orange/red. Arrows indicate rising and decaying components.

Pump–probe results for Pyd conjugates Transient spectra for Pyd-1G and Pyd-4G are shown in Fig. 5 and 6. Transient spectra for Pyd-2G and Pyd-3G are shown in Figures S2 and S3 and single wavelength 395 and 540 nm transient decays for Pyd-2G are shown in Figure S4. Except in the case of Pyd-1G, ultrafast rising components observed at both wavelengths and decay of the 575 nm band are attributed to internal conversion from 2 S* to 1 S*. The spectra of Pyd-1G are dominated by a

Table 2 Single wavelength decays and assignments for DNA conjugatesa Pyrene derivative

l 1 /nmb

t decay /ps

l 2 /nmc

t rise /ps

t decay /ps

Pyd-H Pyd-An Pyd-1G Pyd-2G Pyd-3G Pyd-4G Pyd-AT

390 395 395 395 395 395 395

>2000d 16 (78) 2.0 (26), 31 (67) 19 (26), 540 (74)

490 540 540 540 540 540 540

120d 16 (67) 2.0 19 (30) 5.9 (68), 28 (32) 3.9 (59), 36 (41) 3.1 (42), 45 (58)

>2000 950 31 (96) 540 5400e >5000 >5000

a Components attributed to charge separation and charge recombination for Pyd-An and Pyd-conjugates are in plain font and bold font, respectively. Data for Pyd-H, 3G, 4G, and AT obtained from single wavelength decays. Data for Pyd-An, 1G, and 2G obtained from simultaneous fitting of 395 and 540 nm data. Ultrafast 575 nm S2 decaying components (