Observation of the rare chrysene excimer †

Chrysene (Fig. 1) is an alternant polycyclic aromatic hydrocarbon (PAH). Compared to other PAHs such as pyrene, benzo [a]pyrene, or linear acenes, it has met rather limited interest as a substrate for biological or electronic applications or for use in the materials sciences. While extensive reviews on organic electronics are available for, e.g., pyrene and linear acenes, none exist for chrysene, even though it has recently been explored for organic light-emitting diode (OLED) applications. One of the main photophysical characteristics of PAHs is their ability to form excimers in solution, in the solid state (e.g. polymers, crystals) and in organized assemblies (e.g. membranes, micelles, LB lms). Excimers (excited dimers)


Introduction
Chrysene (Fig. 1) is an alternant polycyclic aromatic hydrocarbon (PAH). 1 Compared to other PAHs such as pyrene, benzo [a]pyrene, or linear acenes, it has met rather limited interest as a substrate for biological or electronic applications or for use in the materials sciences. 2While extensive reviews on organic electronics are available for, e.g., pyrene 3 and linear acenes, 2 none exist for chrysene, even though it has recently been explored for organic light-emitting diode (OLED) applications. 4ne of the main photophysical characteristics of PAHs is their ability to form excimers in solution, 5 in the solid state (e.g.polymers, crystals) and in organized assemblies (e.g.membranes, micelles, LB lms).Excimers (excited dimers) can be easily observed in the uorescence emission spectrum 6 and were rst described by Förster in 1954 for pyrene. 7Since then, excimers have been observed with many PAHs, the limiting factor being primarily the low solubility of larger PAHs at relatively high concentrations ($10 À2 M) that are needed for excimer formation. 8The excimer band exhibits a broad, Gaussian shape that lacks vibrational structure.It is strongly red-shied with respect to the monomer emission band. 5,8lthough the photophysical properties of chrysene have been extensively studied, [9][10][11] one question remains unanswered: can chrysene form an excimer in solution under standard conditions?The history of the chrysene excimer is noteworthy.Early attempts to detect it in solution failed, leading J. Birks himself to postulate that perhaps chrysene is the only PAH that cannot form an excimer. 12Chrysene excimer emission could not be detected in LB lms, 13 nor was it observed in pure single crystals. 14,15So far the only experimental evidence for its formation has been obtained from a high pressure study of chrysene microcrystals. 16Furthermore, a possible excimer component was observed in chrysene containing vinyl copolymers. 17NA has been successfully applied as a supramolecular scaffold for organizing and studying organic chromophores.  We h previously reported on the introduction of PAHs into DNA, which leads to the appearance of remarkable spectroscopic and electronic effects, such as the formation of excimers, 46,47 exciplexes, 48 J-and H-aggregates, 49 energy transfer 50 or aggregation-induced uorescence. 515][56][57][58][59] Here, we report on the formation and characterization of the chrysene excimer in single and double stranded DNA (Fig. 1).
Fig. 1 Illustration of structural organization of chrysene molecules in a DNA supramolecular scaffold (HyperChem, minimized with amber force field). 60ompound 2 was then coupled with 5-hexyn-1-ol under Sonogashira conditions giving the dialkynyl diol 3 in a 42% yield.Finally mono-DMT protection (/ 4) and phosphitylation gave the phosphoramidite 5.The latter was incorporated into DNA by standard solid-phase synthesis procedures to furnish oligonucleotide strands with one (6 and 7) or two (8 and 9) chrysene incorporations per strand (Table 1).

UV-vis absorption and uorescence spectra
In general, ethynyl substitution on PAHs causes a red shi in the absorption and emission bands as well as an increase in the uorescence quantum yield. 63,64The UV-vis absorption spectra of the chrysene diol 3 (Fig. 2) reveal pronounced conjugation effects on the absorption and emission properties upon triple bond addition.As a result of a reduction in symmetry, the forbidden S 0 / S 1 transition of chrysene becomes allowed, which is reected by an increase in the absorption coefficient (3). 65The emission maximum of 3 is red-shied by 23 nm compared to chrysene (1).Furthermore, the uorescence quantum yield (F F ) increases from 0.14 for chrysene to 0.41 for the dialkynyl derivative 3 (Table 2), also as a result of the aforementioned symmetry decrease.Similar effects have been reported for pyrene, where the symmetry-forbidden S 0 -S 1 transition also becomes allowed upon ethynyl disubstitution. 63he UV-vis spectra of the different oligomers and hybrids are shown in Fig. 3.The absorption by chrysene is seen between 320 and 390 nm.As can be seen (Fig. 3, inset), the relative heights of the 0-0 and the 0-1 vibronic bands (370 and 350 nm, respectively) in oligomers 6 and 7 are the same as those of compound 3 (Fig. 2).][68] The UV-vis spectra of duplexes 6*7 and 8*9 are also shown in Fig. 3. Again, an inversion of the relative heights of the vibronic bands is observed aer hybridisation of single strands 6 and 7 to form duplex 6*7, whereas little change occurs upon hybridisation of 8 and 9.The latter indicates that adjacent chrysenes are already p-stacked in single strands 8 and 9.
The temperature-dependent UV-vis spectra of the duplexes are shown in Fig. 4. As duplex 6*7 is heated from 20 to 90 C, there is a slight blue shi (4 nm) accompanied by inversion of vibronic band heights indicating breakdown of intermolecular p-stacking interactions.A minor blue shi (2 nm) is observed for duplex 8*9 upon heating and there is almost no change in    the vibronic band structure further indicating that, although the interstrand interaction is removed, considerable intramolecular p-stacking between neighbouring chrysenes still exists within each DNA single strand.
Fluorescence spectra (Fig. 5) provide clear evidence for chrysene excimer formation.Single strands 6 and 7 exhibit only monomer uorescence.However, hybridisation of the two strands results in a complete change of the spectrum showing exclusively an excimer emission centered around 471 nm, which originates from the formation of an excimer via interstrand stacking of the two chrysene molecules.Intrastrand excimer formation can also be seen in single strands 8 and 9 which possess two neighbouring chrysene units.A shoulder on the le side of the excimer emission band in oligomer 8 is attributable to residual monomer uorescence.The maxima of the emission bands are at 446 and 455 nm, respectively.Hybridisation of these two strands leads to a stack of four chrysene units and a red-shi of the excimer band with a maximum at the same wavelength as the one in hybrid 6*7.
A similar emission band with a maximum of 450 nm was observed by Offen in chrysene crystals at 17 kbar pressure, but not at atmospheric pressure (1 atm). 16Since chrysene forms a type A crystal in which the molecules are not packed face-toface, 70 the author attributed the excimer emission band to defects formed within the lattice at extremely high compressions.Chiellini et al. have studied optically active chrysenebased vinyl copolymers, 17 and have described an excimer-like emission band.However, the reported band is complex, consisting of several superimposed components, two distinct maxima and a non-Gaussian shape, therefore its exact identity remains uncertain.
F F increases signicantly upon hybridisation of 6 and 7 (Table 2).The nucleobase type next to the chrysene has a signicant effect on the uorescence intensity 71 (Fig. 5) and quantum yield (Table 2).The lower F F value of duplex and 8*9 (0.16) relative to duplex 6*7 (0.30) remains unclear, but was also observed in the dialkynyl pyrene case. 52pon heating and thermal denaturation of hybrid 6*7 (Fig. 6A), the intensity of the excimer band decreases with a slight blue shi of its maximum (12 nm, 20 / 75 C) while the emission of the chrysene monomer (l max 391 nm) increases with increasing temperature.A distinct isoemissive point is present at 415 nm, which indicates that there are only two emitting species present, i.e., chrysene monomer and excimer. 72he temperature-dependent uorescence spectra of duplex 8*9 (Fig. 6B) show a behaviour that differs in one major way from    hybrid 6*7: as the temperature is increased from 20 C and the strands begin to dissociate, the excimer uorescence band also gradually becomes less intense and blue shied until $70 C (the approximate T m , see Table 2) is reached.On further rise of the temperature, the intensity of the excimer band increases again with progressive blue shiing to reach a maximum around 450 nm at 90 C, which coincides with the value obtained also for the single strands (Fig. 5).Such behaviour is consistent with the initial dissociation of the intermolecular excimer (l max 470 nm) in the duplex, followed by the formation of an intrastrand (l max 450 nm) excimer between neighbouring chrysenes in a DNA single strand.We have previously observed a similar blue shi upon transition from interstrand to intrastrand excimer in DNA sequences with a pyrene uorophore. 73n general, a more red-shied excimer band is correlated with greater stability and stronger p-p interactions. 74Thus, the present nding indicates that the intrastrand chrysene excimer is less stable than the interstrand excimer.

Circular dichroism
Compared to other PAHs, relatively few CD studies on chrysene derivatives have been published. 17Although studies on DNA alkylated by chrysene metabolites have been reported, the resultant conjugates no longer possess a chrysene aromatic core due to one ring being saturated and thus behaving like a phenanthrene chromophore. 75Fig.7 shows the CD spectrum of duplex 6*7.At 25 C, the 220-300 nm region shows the standard bisignate signal due to double stranded B-DNA.The strong S 0 / S 2 transition of 3 between 250 and 300 nm (cf.Fig. 2), overlaps with the DNA base dichroic absorption.An induced CD (ICD) of the dialkynylchrysene chromophore S 0 / S 1 transition, with three vibronic bands (336, 352 and 362 nm), is clearly visible and coincides well with the absorption band of duplex 6*7 (Fig. 3).Upon heating from 25 to 90 C, all signal intensities of the dichroic effects decrease, and the ICD of the dialkynylchrysene completely disappears aer the 70 to 80 C heating step (Fig. 7, inset), which is in the region of the melting temperature of duplex 6*7.The CD behaviour of duplex 8*9 (ESI †) is similar to that of 6*7.

Conclusion
We have described the rst unambiguous observation of a chrysene excimer formed in solution under standard conditions.Association of individual chrysene units is enabled by a DNA supramolecular scaffold.Alkyne-substituted chrysene building blocks were incorporated into oligodeoxynucleotides.Excimer formation occurs upon duplex formation.Excimer emission is also observed from neighbouring chrysenes in single strands.The uorescence spectrum exhibits the classic red-shied, Gaussian-shaped excimer band between 450 and 470 nm with quantum yields as high as 40%.The high sensitivity of this excimer to spatial proximity opens possibilities for using chrysene as a uorescent label for bioconjugation. 76perimental section

General procedures
All reagents and solvents were purchased from commercial suppliers and used without further purication.Water was taken from a MilliQ system.NMR spectra were obtained on a Bruker AV 300 (300 MHz) spectrometer at 298 K. Mass-spectrometric data were obtained on Thermo Fisher LTQ Orbitrap XL using Nano Electrospray Ionization (NSI).UV-vis spectra were measured on a Cary 100 Bio spectrophotometer.Fluorescence and excitation spectra were measured on a Cary Eclipse spectrouorimeter.CD spectra were measured on a Jasco J-715 spectropolarimeter.Unless otherwise indicated, all experiments were performed in 10 mM sodium phosphate buffer pH 7.0, 0.1 M NaCl.Reference oligonucleotides (refA and refB) were purchased from Microsynth (Switzerland).

Oligonucleotides and duplexes
The oligonucleotides 6-8 were prepared on an Applied Biosystems 394 DNA/RNA synthesizer.A standard cyanoethyl phosphoramidite coupling protocol was used beginning with nucleoside-loaded controlled pore glass (CPG) supports.Commercially available natural nucleoside phosphoramidites were dissolved in CH 3 CN to yield 0.1 M solutions.Compound 5 was dissolved in 1,2-dichloroethane to yield a 0.1 M solution.The CPG-bound oligonucleotides were cleaved and deprotected by treatment with aqueous NH 3 at 55 C for 16 h.The supernatant was collected and the debris was washed three times with 1 ml EtOH-H 2 O 1 : 1. Aer lyophilisation the crude oligonucleotides were puried by reversed phase HPLC (Merck LiChroCART 250-4; LiChrospher 100, RP-18, 5 mm).A gradient starting with 5% up to 50% (within 20 min) CH 3 CN in 0.1 M aqueous triethylammonium acetate was set at a ow rate of 1.0 ml min À1 .The puried oligonucleotides were dissolved in 1 ml H 2 O.Samples of the stock solutions were diluted 50 times and the absorbance at 260 nm was measured.The molar extinction coefficients of the oligonucleotides were calculated using the 3 260 /M À1 cm À1 values of 15 300, 11 700, 7400 and 9000 for A, G, C and T bases, respectively, and 36 653 for the chrysene building block.Formation of duplexes 6*7 and 8*9: equimolar amounts of single strand solutions were combined in buffer and were kept at 90 C for 10 min, before being allowed to cool down overnight.