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Photophysical Properties of Dipeptides Containing Substituted 3-(Quinoxalin-6-yl) Alanine. Spectroscopic Studies and Theoretical Calculations Ł. Wis´niewski,† I. Deperasin´ska,*,‡ A. Staszewska,§ P. Stefanowicza,† S. Berski,† P. Lipkowski,| Z. Szewczuka,† and A. Szemik-Hojniak*,† Faculty of Chemistry, UniVersity of Wroclaw, Joliot-Curie 14 st, 50-383 Wroclaw, Poland, Institute of Physics, Polish Academy of Sciences, Al.Lotniko´w 32/46, 02-668 Warsaw, Poland, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul. Pawin´skiego 5a, 02-106, Warsaw, Poland, and Theoretical Chemistry Group, Institute of Physical and Theoretical Chemistry, UniVersity of Technology, Wyb. Wyspian´skiego 27, 50-370 Wrocław, Poland ReceiVed: NoVember 27, 2009; ReVised Manuscript ReceiVed: May 29, 2010
The photophysical properties of excited states of two hybrid dipeptides [N-(3-(2,3-diphenylquinoxaline-6ylo)alanylo) glycine], Pe-DPhQ, and [N-(3-(2,3 (pirydine-2-ylo) quinoxaline-6-ylo)alanylo) glycine], Pe-DPiQ, have been investigated by a combined solution-state study (absorption, emission) and quantum-mechanical (ab initio, DFT) calculations. The RHF and DFT B3LYP/6-31G (d,p) computations of the ground-state isomers of Pe-DPiQ dipeptide (open, half-closed, and closed) indicate that the most stable is the “open”-type structure with approximately equal (-44.43°, -43.05°) dihedral angles describing rotation of the aromatic side rings with respect to the quinoxaline framework. This agrees with the literature findings that synthetic peptides are mostly unfolded. The experiments show that emission of Pe-DPiQ dipeptide is strongly temperature dependent, and at ambient and elevated temperatures the fluorescence is prevailing while the phosphorescence dominated emission spectra are observed at 77 K. On the basis of the decay curves that in the broad temperature range (rt-77 K) are biexponential (2 and 9 ns), it was concluded that at least its two major excited-state conformations may interconvert on the nanosecond time scale. The third component, of a small amplitude (10%) and a long time constant (25 ns), appears only in a new fluorescence band (570 nm) that grows up with the temperature increase. Analysis of the CIS/6-31G(d,p) results of the excited-state isomers of Pe-DPiQ supports the interpretation of experimental emission spectra and enables one to assign two excited-state conformations, demonstrating a tendency to keep one of their two side rings coplanar relative to the central quinoxaline plane, as Pe-DPiQ-I* (41.9°, 6.3°) and Pe-DPiQ-II* (40.1°, 4.5°) isomers contributing to the room temperature (403 nm) and 363 K (570 nm) fluorescence bands, respectively. The calculations also explain the electronic character of the corresponding S1TS0 transitions and show that the state ordering of Pe-DPiQ resembles that of other diazines where the first singlet is of the nπ* character while the S2 and T1 are the ππ* states. The reason for a strong phosphorescence is assigned to an effective spin-orbit coupling of appropriate singlet and triplet states that leads to ISC transitions and in result to population of the T1 state and a phosphorescence from the T1 state. From the present study, it was concluded that incorporation of quinoxaline moiety into the model peptides does not change the useful spectroscopic properties of the fluorophore and allows one to design its new analogues with improved activity and specificity. 1. Introduction Quinoxalines and their derivatives have recently received much interest in a broad variety of fields. Differently substituted quinoxalines have been studied intensively due to their wide spectrum of biological activities such as antibacterial,1 antiviral,2 or anticancer activity3 as well as practical applications such as, for example, copper(I) sensors,4 building blocks for dendrimers,5 ligands in metal complexes of supramolecular devices, or DNA probes.6–9 Significant conformational changes occurring on coordination of some quinoxaline analogues to transition metals10–17 have been the subject of interest in view of their binding to DNA.18 The gas-phase emission studies of the * Corresponding author. Phone: (004871) 3757-366. E-mail: anias@ wchuwr.pl. † University of Wroclaw. ‡ Institute of Physics, Polish Academy of Sciences. § Institute of Biochemistry and Biophysics, Polish Academy of Sciences. | University of Technology.
prototype quinoxaline molecule, 1,4-diazanaphthalene (Q in Figure 1), revealed its fluorescence emission from the 1(n, π*) state19–22 and the phosphorescence emission from the 3(π, π*) state.19–23 Hence, the emissive abilities of quinoxaline-based compounds (quinoxaline/diphenylfluorene) have been explored to develop a new type of electroluminescent materials for organic light-emitting diodes (OLED). In these compounds, a weak transfer of charge occurs upon complexation, while a strong charge transfer on photoexcitation takes place.24,25 The understanding of electronic structure as well as the charge transfer (CT) and proton transfer processes in the short peptides is crucial for explaining fragmentation processes and conformational changes in larger polypeptides and proteins.26 The limited steric hindrance in dipeptides with the short side chains allows for the existence of numerous conformations to occur. Because potential applications of such short peptides continuously increase, the understanding of their characteristics is challenging.
10.1021/jp911285u 2010 American Chemical Society Published on Web 08/17/2010
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Figure 1. Molecular structures of quinoxaline [Q], diphenyl quinoxaline [DPhQ], diphenyl-iso-quinoxaline [DPiQ], as well as N-(3-(2,3diphenylquinoxaline-6ylo) alanylo) glycine dipeptide [Pe-DPhQ] and N-(3-(2,3(pirydine-2-ylo) quinoxaline-6-ylo)alanylo) glycine dipeptide [Pe-DPiQ].
An ongoing search of compounds with a broad range of biological and toxicological properties resulted in some quinoxaline derivatives being used in the design and synthesis of hybrid-like structures where specific parts of the biomolecules were replaced by the quinoxaline moiety.27 It is well-known that short peptides possess a broad spectrum of biological activity. The introduction of nonproteinaceous amino acid residues into the peptide chains may allow one to design new analogues with improved activity, specificity, and enzymatic stability. A peptide backbone may serve as a scaffold to properly organize the introduced structural motifs. The modifications may also be applied as fluorogenic markers in enzymatic assays, delivery systems, and in the analysis of their structures. Recently, we developed an efficient method of synthesis of peptides containing quinoxaline-derived amino acid side chains.27 The method involved the preparation of a solidsupported peptide containing a β-(4-amino-3-nitrophenyl)alanine residue, reduction with SnCl2, and subsequent reaction of the resultant diamine with various dicarbonyl compounds. The method is compatible with solid-phase peptide synthesis protocols and may allow one to introduce quinoxaline derivative to the side chain of any amino acid residue of the peptide. The synthesized dipeptides contain various analogues of 3-(quinoxalin-6-yl)alanine, including diphenylquinoxaline (DPhQ in Figure 1) and dipyridyl-iso-quinoxaline (DPiQ in Figure 1). Because the fluorophore moiety is incorporated into the peptide during the last step of solid-phase synthesis, this approach gives flexibility in designing fluorescent peptides. The ability to tune the wavelengths and intensities of absorption and emission of fluorophores broadens the range of potential applications. Amino acids containing the quinoxaline-derived fluorophores located in their side chains, introduced to particular positions of the peptide chain, may serve as the sensors of conformational changes and surrounding environment of modified peptide. Additionally, these molecules may serve as fluorogenic substrates for proteases. Herein, we report the results of a combined solution-state study by steady-state (absorption, emission) and time-resolved spectroscopy (emission) as well as ab initio and DFT calculations of newly synthesized hybrid-like dipeptides obtained through incorporation of quinoxaline backbone into model dipeptide molecules and their parent quinoxaline compounds.
Wis´niewski et al. The following pairs of compounds, presented in Figure 1, have been investigated: N-(3-(2,3-diphenylquinoxaline-6-ylo)alanylo) glycinedipeptide,[Pe-DPhQ]27 anddiphenylquinoxaline(DPhQ28,29) pair, as well as N-(3-(2,3(pirydine-2-ylo) quinoxaline-6-ylo)alanylo) glycine dipeptide, [Pe-DPiQ],27 and dipyridyl-iso-quinoxaline (DPiQ30). The aim of this study was to investigate the structural characteristics and photophysical properties of target molecules and to determine whether the quinoxaline analogues conjugated directly to the side chains of model peptides possess the emission characteristics similar to corresponding quinoxaline analogues alone or not. On the basis of temperature dependence of emission spectra, it will be shown that at room temperature conditions, in polar butyronitrile, a single fluorescence band peaks at 403 nm and decays biexponentially (2.0 and 8.9 ns), while at elevated temperatures, a new, strongly red-shifted fluorescence band appears at about 570 nm and decays according to a triple-exponential function (2.1, 9.5, and 25 ns). In the light of these findings, we conclude that emission of Pe-DPiQ dipeptide originates from the major two excited-state conformations. The quantum mechanical calculations performed to elucidate the origin of this band indicate that among excitedstate isomers, a major contribution may be brought specifically by the “half-closed” Pe-DPiQ-II* structure, of which one of the lateral pyridine rings is almost coplanar with the plane of the quinoxaline framework. The conclusions drawn from experimental phosphorescence spectra are confirmed by the results of theoretical calculations, which indicate that this state ordering of Pe-DPiQ facilitates effective intersystem crossing transitions leading to a strong phosphorescence appearance. 2. Experimental Section 2.1. Compounds Studied. The synthesis, purification, and identification of N-(3-(2,3-diphenylquinoxaline-6-ylo)alanylo) glycine dipeptide [Pe-DPhQ] and the N-(3-(2,3(pirydine-2-ylo) quinoxaline-6-ylo)alanylo) glycine dipeptide [Pe-DPiQ] were described elsewhere.27 The related quinoxaline derivative DPhQ of the former dipeptide was obtained according to the literature.28 2.2. Electronic Absorption Spectra. Electronic absorption spectra in solution were recorded on CARY-50 UV-vis (Varian) spectrometer at the concentration of about 10-5 M. The solvents used in absorption and emission experiments (cyclohexane, butyronitrile, and propanol) were of spectroscopic grade and used fresh as purchased (Merck, Uvasol). Experimental data for diphenyl-iso-quinoxaline [DPiQ] have been taken from ref 30. 2.3. Steady-State Emission Spectroscopy. Emission spectra were recorded on a FLS920 combined fluorescence lifetime and steady-state spectrometer (Edinburgh Instruments Ltd.) using as the excitation source a Xe900, 450 W steady-state xenon lamp (ozone free) with computer-controlled excitation shutter and with a spectral bandwidth of e5 nm for both excitation and emission spectra. Luminescence was detected using a red sensitive (185-850 nm) single photon counting photomultiplier tube (R928-Hamamatsu) in a Peltier cooled housing. For emission spectra, the optical density was kept at ∼0.2 (path length 1 cm) to avoid reabsorption and inner filter effects. Spectra were corrected for detector response and excitation source. The concentration of the solutions was of the order 10-5 M. 2.4. Time-Resolved Fluorescence Spectroscopy. Fluorescence lifetimes were measured in solution (10-5 M) using the time-correlated single photon counting (TCSPC) option of FSL920 setup (Edinburgh Instruments Ltd.). Excitation was
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TABLE 1: Dihedral Angles Di (See Figure 2) and Dipole Moments of Studied Molecules in Their Ground-State Minimum Energy Structures DPhQ torsion angles D1(1,2,3,4) D2(6,5,4,3)
RHF/6-31G(d,p) DFT B3LYP/6-31G(d,p) experiment29
dipole moment µ [D]
RHF/6-31G(d,p) DFT B3LYP/6-31G(d,p) TD DFT B3LYP/6-31G(d,p)
excitation energy [cm-1] and oscillator strength S0 f S1 (nπ*) S0 f S2 (ππ*) first maximum in absorption spectrum [cm-1]
experiment
provided by a nF900 nitrogen-filled nanosecond flashlamp under computer control, with typical pulse width 1 ns, and pulse repetition rate typical 40 kHz and the possibility of measuring decays from 100 ps to 50 µs. Data acquisition ensured plug-in PC card model TCC900 with maximum count rate 3 MHz, time channels per curve up to 4096, and minimum time per channel 610 fs. A Hamamatsu (R928-Hamamatsu) in Peltier cooled housing was used as detector. Phosphorescence lifetimes were measured using the excitation source provided by a µF900 microsecond flashlamp under computer control with pulses 1.5-3 µs, an average power >60 W up to 100 Hz, and the possibility of measuring decays from 1 µs to 10 s. Phosphorescence decay measurements were performed in multichannel scaling mode (MCS) and required a TCC900 fast counter plug-in PC Card. The PG900 microsecond photomultiplier gating option was used to fix the gate width and gate delay. 2.5. Theoretical Calculations. In this work, the Gaussian 03W, revision E0131 package of programs was used in the calculations. The RHF and DFT B3LYP/6-31G(d,p) methods were used for the optimization of the ground state (S0) and the CIS/6-31G(d,p) method for the optimization of the excited state (S1) of investigated molecules. The energies and oscillator strengths of the electronic transitions between S0 and S1 states were calculated by the TD DFT B3LYP/6-31G (d,p) method, and the QST3 method was applied to obtain the barriers separating different energy minima. The full set of positive frequencies was obtained for the optimized structures, and the energies have been corrected for the zero-point energy. Such choice of methods inspired us, among others, by the results obtained by RHF and DFT B3LYP methods for the diphenyl-iso-quinoline [DPiQ] structure.30 It was shown that these methods were able to generate geometries that are consistent with experiment.
Pe-DPhQ
-48.4 -48.4 -41.2 -41.2 -37.1 -53.3 0.65 0.39 28 215 (0.0025) 29 269 (0.2101)
-48.6 -48.5 -40.7 -41.6
2.24 2.46 28 302 (0.0034) 29 126 (0.2572)
29 150
28 490
of molecules studied in this work. A brief comparison of theoretical and experimental results for DPhQ and Pe-DPhQ is reported in Table 1. In this comparison, different parameters and properties of studied molecules are concerned, and, among them, two dihedral angles (D1 and D2), described in Figure 2, are of significant importance. They characterize the location of the flanking aryl appendages in quinoxaline and in peptide molecules with respect to the planar quinoxaline framework. One may notice that the isolated (non interacting with medium) DPhQ molecule is symmetrical (C2 symmetry group) with D1 ) D2 ) D. It is seen from Table 1 that the angle D, calculated by the RHF method (-48.4°), is slightly larger than the D value calculated by the DFT B3LYP (-41.2°) method, although both of them are comparable with experimental values (-37.1°; -53.3°) obtained from the X-ray studies of DPhQ crystals.29 Therefore, the intermolecular interactions in crystal lead to the symmetry breaking. This symmetry is also broken by linking the dipeptide chain (Pe) to the DPhQ moiety. Next, in the supramolecular Pe-DPhQ structure, D1 * D2, although this effect is minor (changes of Di are smaller than 1°). Incorporation of the peptide chain into the parent DPhQ molecule results in asymmetry of dipeptide, reflected by the
3. Results and Discussion This section begins with presentation of the results obtained for the target molecules optimized in the ground electronic (S0) state. In the next step, we compare the experimental absorption spectra with their simulations derived from theoretical calculations, and the results of excited-state (S1) optimization are thoroughly discussed. Finally, experimental emission spectra of studied molecules in different temperatures are presented and analyzed. 3.1. Structures and Spectra of Molecules in the Ground S0 State. 3.1.1. DPhQ and Pe-DPhQ in the Ground State. As we mentioned above, the results obtained30 for DPiQ indicate that RHF and DFT B3LYP methods can be used for description
Figure 2. Scheme of the Pe-DPiQ dipeptide molecule, the quinoxaline derivative substituted both by the peptide chain (indicated by the oval) and by the two pyridine rings. Orientation of these rings with respect to the central quinoxaline is described by the two dihedral angles D1(1,2,3,4) and D2(6,5,4,3). Herein, the analogue of Pe-DPiQ without the peptide chain is called DPiQ, while equivalents of both of these compounds with phenyl rings are called Pe-DPhQ and DPhQ, respectively.
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TABLE 2: Comparison of the Ground-State Properties of DPiQ Isomersa
a D1 and D2, dihedral angles; δE, energy difference with respect to the DpiQ(in-in); δE includes zero-point energy (ZPE) correction; µ, dipole moment; excitation energies and oscillator strengths for the S0 f S1 and S0 f S1 transitions. b Energy of transition state for DPiQ(in-in) f DPiQ(in-out) process is 2318 cm-1 (6.6 kcal/mol).
significantly increased value of its dipole moment relative to the parent quinoxaline. Its value as calculated by both methods is similar (∼2.2 and 2.4 D). It is reported in Table 1 that TDDFT B3LYP calculated vertical transition energies for S0 f S1 electronic excitation are similar for DPhQ and Pe-DPhQ molecules (28 215 and 28 302 cm-1, respectively) and are also similar to the values obtained from experiment (29 150 and 28 490 cm-1, respectively). 3.1.2. DPiQ and Pe-DPiQ in the Ground State. The two ground-state optimized isomers of DPiQ are illustrated in Table 2. A difference between them is related to the position of the two nitrogen atoms of the side pyridine rings described by the corresponding dihedral angles (D1, D2). The most stable is the DPiQ(in-in) structure. It is only slightly polar (µ ) 1.08 D), and its two pyridine rings are symmetrically twisted along the C-C bond in such a way that both pyridine nitrogen atoms are vis a vis each other (in-in) and both dihedral angles are identical [D1 ) D2 ) -43.7°].
Wis´niewski et al. The nitrogen atoms of the two lateral pyridine rings of the second isomer, DPiQ (in-out), are asymmetrically placed one (in) to another (out), both dihedral angles are different (D1 ) 36.5°, D2 ) -121.9°), and the dipole moment is significant (3.6 D). The energy difference between these two isomers is 1225 cm-1 (3.5 kcal/mol), while the energy barrier height for the DPiQ(in-in) f DPiQ(in-out) process is 2318 cm-1 (6.6 kcal/ mol). This means that at ambient temperatures, the population of the DPiQ(in-out) isomer should be small. Such situation, however, can be changed in solvents of larger polarity. In such conditions, the latter, as characterized by a larger dipole moment relative to the DPiQ(in-in) isomer, should be strongly stabilized.32,33 When the parent quinoxaline molecule is connected to the dipeptide chain, more conformational degrees of freedom are made available. Thus, even more various ground-state isomers of Pe-DPiQ dipeptide, differing in dihedral (D1, D2) angles, can be envisaged than in the case of the quinoxaline, DPiQ, molecule. The structures of three of them are illustrated in Table 3. They do not exclude, however, all possibilities but solely show examples of isomers being described only by the two above-mentioned factors, for example, the peptide chain orientation and the side ring rotation with respect to the quinoxaline framework. The most stable among the simulated isomers is the “open”, Pe-DPiQ-I, structure, which is relatively polar (2.06 D), and its conformation is described by a set of dihedral angles (about 40°) similar to that in the isomer of its parent, quinoxaline [DPiQ(in-in)] molecule. The next geometry, denoted here as the Pe-DPiQ-II or the “half-closed” isomer, is slightly more polar (2.479 D), and still keeps the conformation of DPiQ(in-in), although its peptide chain is now approximately in the half distance from the pyrazine nitrogen atom. Because the energy difference and height of the barrier separating both conformations are small [0.3 and 3.2 kcal/mol, respectively, for the Pe-DPiQ-I f PeDPiQ-II transition], their interconversion at ambient temperatures is highly probable.
TABLE 3: Comparison of the Ground-State Properties of Pe-DPiQ Isomersa
a D1 and D2, dihedral angles; δE, energy difference with respect to the Pe-DPiQ-I form; δE includes zero-point energy (ZPE) correction; µ, dipole moment; excitation energies and oscillator strengths for the S0 f S1 and S0 f S1 transitions. b Energy of transition state for Pe-DPiQ-I f Pe-DPiQ-II is 1132 cm-1 (3.2 kcal/mol). c Energy of transition state for Pe-DPiQ-II f Pe-DPiQ-III is 1610 cm-1 (4.6 kcal/mol).
Dipeptides Containing Substituted 3-(Quinoxalin-6-yl) Alanine
Figure 3. The calculated and experimental (cyclohexane, red line) absorption spectra of the studied molecules. The calculation results are presented in two ways: as the lines of an appropriate height corresponding to oscillator strength versus the calculated transition energies and also in the form of simulated spectra when each of the calculated lines is convoluted with Gaussian distribution of a width of 1500 cm-1.
In the third conformation, denoted as Pe-DpiQ-III or the “closed” isomer, the peptide chain is approaching the quinoxaline part in such a way that a close contact through the carboxyl group either with the pyrazine or with the pyridine nitrogen atom might lead to the hydrogen-bonding formation. The calculated OH · · · N distances between the OH group of the carboxyl terminal group of the peptide chain and the pyrazine or pyridine nitrogen atom of the quinoxaline part are 2.779 and 2.164 Å, respectively. This isomer is higher in energy than the “half-closed” Pe-DPiQ-II, and in a fact it may be formed through the Pe-DPiQ-II f Pe-DPiQ-III transition, although the corresponding barrier (4.6 kcal/mol) is slightly higher than that for the former interconversion process. The supramolecular structure of Pe-DPiQ-III may be directly compared to that of the asymmetric parent quinoxaline DPiQ(in-out) isomer where the pyridine nitrogens are also reversely placed each to other, [(in) and (out)], and both (D1 and D2) dihedral angles are totally different. In Pe-DPiQ-III, they are -131.35° and 37.88°, respectively. It is the most polar (3.720 D) of the three simulated conformations, and in polar media, apart from the OH · · · N and (CdO)COOH · · · H-C (2.541 Å) hydrogen bonds, it may be additionally stabilized through dipole-dipole interactions.
J. Phys. Chem. A, Vol. 114, No. 35, 2010 9409 3.1.3. Absorption Spectra of Studied Quinoxalines and Dipeptides. The overlapped TD DFT simulated and experimental absorption spectra of DPhQ and Pe-DPhQ as well as DPiQ and Pe-DPiQ molecules are illustrated in Figure 3. It is obvious that they match very well in a large spectral range (25 000-45 000 cm-1), which makes our calculated results highly reliable. Because all of them originate from the same quinoxaline chromophore, they are resembling each other with respect to both the band positions and the oscillator strength values. In all studied molecules, likewise in parent quinoxaline [1,4diazanaphthalene, Q in Figure 1],34,35 the S0 f S1 transition is of the nπ* type and the S0 f S2 transition carries the signature of the ππ* state. The calculated oscillator strength values of the former state are small and as exemplified by DPhQ or PeDPiQ molecules are 0.0025 and 0.0043, respectively. For the S0 f S2 transition, these values are significantly larger (0.21 and 0.19, respectively). It is known that in diazines, the energy gap between the two lower lying electronically excited states depends strongly on the type of substituents.35 Hence, in the molecules under consideration, it is clearly lower (1000-2000 cm-1) than in the parent quinoxaline chromophore (Q) where it exceeds 5000 cm-1.34,35 From this reason, the nπ* state in both peptides and in parent compounds is superimposed on the more intense π f π* state. 3.2. Structures and Spectra of Molecules in the Excited S1 State. 3.2.1. Excited-State Optimized Structures. Upon electronic excitation, the charge distribution on the molecule changes and in consequence the excited-state energy minimum may correspond to altered molecular geometry in comparison to that in the ground state. The most important data characterizing the structures optimized in the excited state are collected in Table 4. It may be observed that, in the case of symmetric molecules [DPhQ and DPiQ(in-in)], the values of Di angles in the excitedstate optimized structures are slightly smaller (ca. 6°) than those in the ground state. On the other hand, a tendency to larger changes manifest the symmetryless geometries [DPiQ(inout), Pe-DPhQ, Pe-DPiQ-I, and Pe-DPiQ-II ]. It is interesting to note that in these structures, the S1 f S0 transition energy is smaller than in symmetric isomers, and a coplanarity of one of the aromatic side rings relative to the central quinoxaline plane is achieved. Furthermore, the energy differences between the energy minima of particular isomers in the excited state are smaller in comparison to those in the ground state. Nevertheless, taking into consideration that the excited-state optimization and transition energy calculations have been performed by use of the two methods [RCIS and TDDFT], the results obtained for
TABLE 4: Computational Results for the Excited Molecules Optimized in the S1 Statea DPiQ D 1, D 2 δE* [cm-1 (kcal/mol)] ∆E [cm-1] f
Pe-DPiQ
DPhQ
in-in*
in-out*
Pe-DPhQ
I*
II*
III*
-42.6 -42.6
-36.7 -36.7 0 24 375 0.0036
133.3 -1.2 689 (2.0) 21 726 0.0311
-36.5 -12.3
41.9 6.3 0 23 711 0.0921
40.1 4.5 266 (0.8) 21 662 0.0465
43.5 -144.4 1196 (3.4) 22 791 0.0021
24 324 0.0024
24 695 0.1944
a D1 and D2, dihedral angles [deg]; δE*, energy of excited isomers with respect to the most stable structure in this state; ∆E and f, transition energy and oscillator strength of the S1 f S0 transition. Differences between the I*, II*, and III* isomers of Pe-DPiQ dipeptide are the same as in Table 3. The structures that manifest a tendency where one of the two side rings aspires to achieve a coplanarity with the central quinoxaline framework are highlighted.
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Figure 4. Total emission spectrum of Pe-DPiQ in n-PrOH at 77 K (exc 340 nm).
Figure 6. Temperature effect (above 298 K) on the fluorescence intensity ratios at particular wavelength of the lower and higher energy emission bands, that is, IF(613 nm/IF(400 nm) and IF(613 nm)/IF(385 nm) of Pe-DPiQ dipeptide (upper part), as well as the fluorescence spectra of Pe-DPiQ in butyronitrile (exc 340 nm) in the temperature range 298-363 K (lower part).
Figure 5. Diagram of electronic excited states of Pe-DPiQ molecule obtained from TD DFT B3LYP/6-31G(d,p) calculations. Aside, the shapes of the molecular orbitals contributing to electronic configurations describing the lowest excited states are demonstrated.
the excited states should be understood only as preliminary, and the final conclusions may be only regarded as indication. 3.2.2. Emission Spectra of Pe-DPiQ Dipeptide. 3.2.2.a. Low Temperature Emission. The low temperature emission spectrum of Pe-DPiQ in n-PrOH at 77 K is presented in Figure 4. As is readily seen, this spectrum is phosphorescence dominated, although a very weak fluorescence at 24 690 cm-1 (405 nm) is also observed. The phosphorescence band is split into two submaxima at 20 202 cm-1 (495 nm) and 19 066 cm-1 (524 nm), and its decay curve shows a biexponential character. The estimated lifetimes in the whole range of the phosphorescence band (470-650 nm) show the constant values, approximately, of 1.0 and 9.0 µs with constant amplitudes (30% and 70%, respectively) regardless of emission wavelength. Similarly, the low-temperature fluorescence also decays biexponentially (2.5 and 28.2 ns), but in this case the corresponding amplitudes are reversed (70% and 30%, respectively). The character of the low temperature emission of Pe-DPiQ may be better explained by the diagram in Figure 5 where electronic excited states, as derived from our computational results, are presented.
The excited-state ordering, shown in the diagram, resembles the corresponding system of quinoxaline (Q, see Figure 1) where likewise to other diazines a considerable temperature-dependent vibrational interaction between the closely lying nπ* and ππ* states (proximity effect) leads to their thorough mixing followed by an increased probability of the nonradiative excitation energy dissipation processes.34,35 The lowest singlets of Pe-DPiQ alternate as the corresponding nπ* (S1) and the ππ* (S2) states, while the first triplet state (T1) has the ππ* character. According to El Sayed rules,36 an effective mixing of almost isoenergetic S1 (nπ*) and T4 (ππ*) states as well as the T5 (nπ*), T6 (nπ*), and the S2 (ππ*) states of Pe-DPiQ should lead to a strong spin-orbit coupling and to effective nonradiative intersystem crossing (ISC) transitions resulting in strong phosphorescence from the T1 state. This indeed is observed, and the phosphorescence is much stronger than the low temperature fluorescence. The TD DFT calculated T1 f S0 transition energy (21 495 cm-1) also correctly reproduces experimental phosphorescence maxima (20 202; 19 066 cm-1) (see Figure 4). 3.2.2.b. Fluorescence at Ambient and EleVated Temperatures. The room temperature experimental fluorescence spectra of PeDPiQ are complex and strongly temperature dependent. Contrary to that at 77 K, the emission spectrum is fluorescence dominated, as presented in Figure 6. The fluorescence maximum in butyronitrile, for example, is at 24 795 cm-1 (403 nm), and the fluorescence decays according to the biexponential function with the time constants 2.0 ns (91%) and 8.9 ns (9%). With temperature increase, however, the in-growth of a new structureless, red-shifted fluorescence band (Figure 6) is observed. At 363 K, its maximum is at 17 750
Dipeptides Containing Substituted 3-(Quinoxalin-6-yl) Alanine cm-1 (570 nm), and apart from the two above-mentioned lifetimes [in this case, 2.0 ns (13%) and 9.5 (77%) ns], an additional species with a drastically longer lifetime of 25 ns (10%) appears. In general, the time-resolved data obtained for Pe-DPiQ at different temperatures show that two major lifetimes are found with amplitudes that are temperature dependent. The shorter time constant, regardless of different conditions, is almost invariable (2 ns), while the longer one (∼9 ns) elongates more than 3 times (∼28 ns) at 77 K. The third component, of a minor population (10%), appears only at elevated temperatures (363 K), and surprisingly its time constant (25 ns) is almost as long as that of the second lifetime at 77 K. In the light of these findings, one may conclude that emission of Pe-DPiQ dipeptide originates from the major two excitedstate conformations. The character of the in-growth of the band at 570 nm is additionally illustrated in the upper part of Figure 6. It is seen that dependence describing the intensity ratio between the long and the short-wavelength fluorescence band [ln(I2/I1)] versus 1/T may be approximated by the two quasi linear relationships. The first two, almost linear and horizontal upper curves, in the upper part of the figure show that in the wavelength range of either of the single emission bands, the intensity ratio is temperature independent. On the other hand, the two lower lines of varying slope are found for two different temperature ranges (300-330 and 320-370 K). A strong overlap of both emission bands together with small temperature range, which was possible to achieve in experiment, does not allow for a full kinetic analysis of excited-state reactions to be performed. It seems, however, that we are able to observe these processes characterized by different energetics and with participation of different isomers of Pe-DPiQ dipeptide. The calculated, excited-state isomers, presented above, should be regarded only as representatives of possible, real geometries because in solution many conformational degrees of freedom for the solvated dipeptide are available. Furthermore, the energetic relations in solution can be also modified. Taking into consideration that the energy gaps between particular isomers, especially in the excited states, are not too large, the changes in population of different isomers with temperature increase are probable. The results of our calculations on the excited states of molecules under consideration indicate a tendency to form the conformations where one of the lateral pyridine rings is almost coplanar relative to the quinoxaline framework (compare Table 4, DPiQ*[in-out], Pe-DPhQ*, Pe-DPiQ-I*, and Pe-DPiQ-II*). The energy of the S1 f S0 transition, that is, the fluorescence of such conformers, strongly depends on the value of the corresponding dihedral angles (compare the cases of Pe-DPiQI* and Pe-DPiQ-II*). As easily noted, the smaller is the Di angle, the smaller becomes the energy. We conclude that a contribution to the room temperature fluorescence spectrum may be brought by the Pe-DPiQ-I* isomer with a D2 dihedral angle of 6.5°, while at elevated temperatures a possible contribution to the long wavelength fluorescence band at 570 nm (butyronitrile) may originate from a “half-closed” conformation of Pe-DPiQII* isomer with lower S1 f S0 transition energy and lower dihedral angle (4.5°). Taking into consideration that the oscillator strength of the S1 f S0 transition in the Pe-DPiQ-II* structure is lower in comparison to the rest of the excited-state isomers, this form can be poorly marked in the fluorescence spectrum. Nevertheless, as was already mentioned, the temperature dependence of
J. Phys. Chem. A, Vol. 114, No. 35, 2010 9411 the fluorescence spectra indicates the presence of at least three contributing forms. Regarding the longwavelength band at 570 nm, the literature data on similar geometry changes in other donor-acceptor substituted quinoxalines refer them to the excited-state photoinduced electron transfer process.37 On the basis of our results, we may conclude that the mechanism of such structural changes in quinoxaline derivatives may be treated in a broader perspective. A more precise description, however, requires further theoretical calculations and detailed experimental studies on temperature and solvent effect on the emission spectra of Pe-DPiQ to be undertaken. 4. Conclusions From the present study, we conclude that standard investigations of absorption spectra are not sensitive enough for identification of peptide isomers, because the incorporation of quinoxaline moiety into the model peptides does not change the electronic structure of the fluorophore. Nevertheless, a good compatibility of computational results for their most stable isomers with experimental data indicates that spectroscopic methods should not be excluded. It shows that structural differences in new supramolecular structures of Pe-DPhQ and Pe-DPiQ dipeptides (with flexible peptide chain and more asymmetric and polar than the quinoxalines alone) may be revealed by following the changes in emission spectra as caused by the temperature and polarity effect of the medium. We have observed that among the RHF- and DFT B3LYP/ 6-31G(d,p)-generated ground-state isomers of Pe-DPiQ dipeptide (open, half-closed, and closed), the most stable is the “open”-type structure with approximately equal (D1, D2) dihedral angles (-44.43°, -43.05°) describing rotation of the aromatic side rings with respect to the quinoxaline framework. This observation agrees with the literature findings that synthetic peptides are mostly unfolded. The calculated interatomic distances of the most polar (3.72 D), “closed” Pe-DPiQ-III isomer suggest that it can be stabilized by the OH · · · N and N-H · · · OdC (COOH) hydrogen-bonding network. The former may occur between the OH group of the carboxyl terminal group of the peptide chain and the pyrazine (2.779 Å) or pyridine nitrogen (2.164 Å) atom of the quinoxaline part, while the latter may appear in the peptide chain alone. The calculations show that excited-state ordering of Pe-DPiQ resembles that of quinoxaline (Q) and other diazines where the first singlet (S1) is of the nπ* character while the S2 and the T1 states bring the signatures of the symmetry-allowed ππ* state. An effective mixing of almost isoenergetic S1 (nπ*) and T4 (ππ*) states as well as the T5 (nπ*), T6 (nπ*), and the S2 (ππ*) states, according to El Sayed rules, leads to a strong spin-orbit coupling and to effective nonradiative intersystem crossing (ISC) transitions resulting in phosphorescence from the T1 state. Experimental emission spectra of Pe-DPiQ performed in a broad temperature range (363-77 K) confirm it and show that at 77 K a strong phosphorescence prevails (∼20 000 cm-1) while the room temperature spectra are totally fluorescence dominated (25 000 cm-1). Emission of Pe-DPiQ decays according to the biexponential function with the major two amplitudes and the time constants of 2.0 and 8.9 ns for the room temperature fluorescence: 2.5 and 28.2 ns for the fluorescence at 77 K as well as 1.0 and 9.0 µs for the phosphorescence emission. This allowed us to conclude that the two species originate from at least two major interconverting excited-state isomers. The third one (25 ns) is
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a minor component with the population of about 10% and appears only at elevated temperatures where, apart from the fluorescence maximum at about 403 nm, a new, red-shifted fluorescence band grows up at 570 nm when the temperature increases. An interesting conclusion may be also drawn out from the CIS/6-31G(d,p) calculations, which suggest that some excitedstate conformations [DPiQ* (in-out), Pe-DPhQ*, Pe-DPiQ-I*, and Pe-DPiQ-II* ] possess a low S1 f S0 transition energy and demonstrate a tendency to keep one of the two side rings coplanar with respect to the central quinoxaline framework. Analysis of these results leads us to the conclusion that the Pe-DPiQ-I* isomer, with a D2 dihedral angle of 6.5°, contributes to the room temperature fluorescence spectrum, while the “halfclosed” conformation of Pe-DPiQ-II* (with a dihedral angle of about 4.5°) brings in a contribution to the long wavelength fluorescence band that at elevated temperatures appears at about 570 nm. In summary, we conclude that for a more precise description of excited-state behavior of studied dipeptides, further theoretical calculations and detailed experimental studies of the solvent and temperature effect on their emission spectra are required. Acknowledgment. Ł.W. greatly acknowledges the financial support of the Ministry of Science and High Education (Poland, grant Nr N N204 131338). We thank the Interdisciplinary Centre for Mathematical and Computational Modeling in Warsaw for the use of its computational facilities (grant G32-10). References and Notes (1) Seitz, L. E.; Suling, W. J.; Reynolds, R. C. J. Med. Chem. 2002, 45, 5604. (2) Loriga, M.; Piras, S.; Sanna, P.; Paglietti, G. Il Farmaco 1997, 52, 157. (3) Lindsley, C. W.; Zhao, Z.; Leister, W. H.; Robinson, R. G.; Barnett, S. F.; Defeo-Jones, D.; Jones, R. E.; Hartman, G. D.; Huff, J. R.; Huber, H. E.; Duggan, M. E. Bioorg. Med. Chem. Lett. 2005, 15, 761. (4) Stephen, W. I.; Uden, P. C. Anal. Chim. Acta 1967, 39, 357. (5) Mastalerz, M.; Fisher, V.; Ma, Ch.-Q.; Janssen, R. A. J.; Baurele, P. Org. Lett. 2009, 11, 4500. (6) Holmlin, R. E.; Barton, J. K. Inorg. Chem. 1995, 34, 7. (7) Balzani, V.; Campagna, S.; Denti, G.; Juris, A.; Serroni, S.; Venturi, M. Acc. Chem. Res. 1998, 31, 26. (8) Balzani, V.; Gomez-Loez, M.; Stoddart, J. F. Acc. Chem. Res. 1998, 31, 405. (9) Du, M.; Zhao, X.-J. Acta Crystallogr., Sect. C 2003, 59, 403. (10) Krieger, C.; Kocak, A.; Bekaroglu, O. HelV. Chim. Acta 1985, 68, 581. (11) Visser, G. J.; Vos, A. Acta Crystallogr., Sect. B 1971, 27, 1793. (12) Visser, G. J.; Vos, A.; De Groot, A.; Wynberg, H. J. Am. Chem. Soc. 1968, 90, 3253. (13) Woz´niak, K.; Krygowski, T.; Kariuki, B.; Jones, W. Acta Crystallogr., Sect. C 1990, 46, 1946. (14) Woz´niak, K. Acta Crystallogr., Sect. C 1991, 47, 1761.
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