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C. Gernert and J. Grotemeyer, Eur. J. Mass Spectrom. 21, 599–608 (2015) Received: 6 May 2015 n Revised: 18 June 2015 n Accepted: 21 June 2015 n Publication: 26 June 2015
EUROPEAN JOURNAL OF MASS SPECTROMETRY Special Issue Celebrating the 20th Anniversary of EJMS—European Journal of Mass Spectrometry
Photodissociation at various wavelengths: fragmentation studies of oxazine 170 using nanosecond laser pulses Claus Gernert and Jürgen Grotemeyer* Christian-Albrechts Universität zu Kiel, Max-Eyth-Straße 1, 24118 Kiel, Germany. E-mail: [email protected]
The fragmentation of oxazine 170, a rhodamine-type dye, has been investigated by means of collisions and photodissociation with visible and ultraviolet radiation in a Fourier transform ion cyclotron resonance mass spectrometer. Because of an improved experimental setup, the photodissociation processes of stored ions are measured with high intensity with respect to the absorbed photons. By isotope labelling and quantum chemical calculations, the various fragmentation mechanisms are investigated. It is shown that the most important intermediate ion structure leading to the various ionic products is an even-electron azarine cation. Several new fragmentation mechanisms have been unveiled for the first time. Keywords: UV photodissociation, photon processes, dyes, fragmentation mechanisms, ICR mass spectrometry
Introduction Organic dyes are widely used in chemistry and physics for a variety of purposes.1 Applications range from use as colorants, markers in chemical reactions and in dye lasers to lightharvesting devices in solar cells. This use is based on intrinsic features like high photostabilities, high quantum yields and very high extinction factors. In analytical applications, various dyes are commonly used in fluorescence studies, especially for biopolymers and other larger molecular systems.2 Because of their common application, their mass spectral behaviour has been thoroughly investigated with respect to their fragmentation reactions.3–5 But it should be noted that several different dyes, commonly of the xanthene type, have found further importance in mass spectrometry, such as in laser desorption studies,6 in cluster ion research7,8 or as chromophoric anchors for photodissociation studies.9–11 In view of the above considerations, the mass spectra of rhodamine B have been intensively investigated in order to understand the fragmentation reactions after electrospray ionization in a Fourier transform ion cyclotron resonance ISSN: 1469-0667 doi: 10.1255/ejms.1368
(FT-ICR) mass spectrometer.12–15 To gain more insight into the observed reactions of rhodamine B and extend the fragmentationin a general form, we investigated a different xanthene-type dye, oxazine 170. This dye is known for several interesting applications in chemistry and biochemistry. It has been successfully used for the induction of a hybrid triplex structure, poly rA:(poly dT)2, which otherwise would not be formed under solution conditions.16 A further application of oxazine 170 is in the investigation of photonic crystals and the energy transfer to it from other dyes.17 Even more important are the photophysical properties of this compound, which are of fundamental interest in spectroscopy. Oxazine 170 is used as an efficient laser dye, emitting light in the region between 600 and 700 nm. 18 In addition, oxazine 170 acts as a donor chromophorein several studies that lead to an understanding of its gas-phase photophysical properties. Additionally, this class of xanthene dyes provides a benchmark for electronic structure calculations, which remain a challenging prospect. © IM Publications LLP 2015 All rights reserved
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The substitution of both amine groups by an ethyl group and the introduction of two further substituents at the aromatic ring system, methyl and annulated phenyl moieties, are the important differences between the oxazines and the rhodamines. While in rhodamine B two ethyl groups doubly substitute both amine groups, oxazine 170 has on both amine groups only a single substitution. Therefore, this compound is particularly suited for the mass spectrometric investigation of its isomerization and fragmentation processes. Due to its absorption spectrum in the neutral and ionic state, activation of the molecular ions by laser radiation should give further insight into the reaction mechanisms leading to the most prominent fragments. By use of various wavelengths, it is possible to control the energy deposited into the molecular system over a certain range. Contrary to the frequently used infrared multiphoton dissociation (IR-MPD),19 photodissociation with nanosecond pulses in the visible and ultraviolet regions is usually a process with only few photons. While in IR-MPD the photon energy is typically 0.1 eV (10.6 µ; CO2 laser), a Nd:YAG laser delivers, depending on the wavelength used, energy between 1.17 eV (1.064 µ) and 4.66 eV (266 nm). As a result, different isomerization and fragmentation products can be expected in general as previously reported.20 In this paper, we report on an investigation of the various fragmentation reactions of oxazine 170 using photodissociation wavelengths of 532, 355 and 266 nm. To perform these investigations we introduced a new experimental setup for the photodissociation in an ICR mass spectrometer, which allows readjustment of the laser beam during the change of the wavelength. This guarantees a high reliability of the measured spectra. Furthermore, due to the challenging task of synchronizing the laser and the experimental pulse timeline, we now can perform photodissociation experiments with a nanosecond-pulsed Nd:YAG laser. With this setup, the various fragmentations of oxazine 170 were investigated and compared to results from quantum chemical calculations. Furthermore, isotopically labelled oxazine 170 was prepared to confirm the proposed reaction mechanisms. This compound, as some other rhodamines, shows an interesting rearrangement and fragmentation mechanism leading to the loss of an alkane from the molecular ion. It will be shown that the most important reactions involve the intermediate formation of radical cations from even-electron molecular ions. The complete reaction sequence leads to a question concerning the appearance of radical cations from even-electron ions and is in contrast to the well-known “evenelectron rule”. This rule has been well known in mass spectrometry for a long time, but several violations and exceptions can be found in the literature.21,22
source. The various substances were ionized with the electrospray ionization method. All samples were dissolved in dimethyl sulfoxide and added to a standard solvent mixture (water– methanol–formic acid, 50:50:0.2) yielding a final concentration in the range 1–100 pmol mL−1. The instrument usually was mass-calibrated with arginine clusters to guarantee a resolution error of under 2 ppm. For the collision-induced dissociation (CID) and photodissociation experiments, the molecular ion peak was isolated in the ICR cell to get isotopic-free spectra. This guarantees a measurement of the different ions without interference either from fragmentation processes or, in the case of deuterated substances, from incomplete labelling. Furthermore, it proves the radical ion character of the observed losses. Fragmentation of the molecular cations was achieved through collisional activation with argon gas using the sustained off-resonance irradiation (SORI) CID technique. After a stored waveform inverse Fourier transform isolation of the ions of interest in the ICR cell, the CID process was induced. The SORI powers were set between 3% and 4.5% and irradiation was applied for 0.1–0.5 s in all experiments. Photodissociation experiments were performed using a Nd:YAG laser (Continuum, Germany) and homemade control software. The different wavelengths were overlaid and then coupled directly into the ICR (Infinity) cell as shown in Figure 1. Thus, it was possible to choose one of the three possible wavelengths (532, 355 and 266 nm) for the fragmentation experiments with nanosecond laser pulses. It should be noted that this setup allows also the simultaneous measurement of two or even three wavelengths. Average laser energies used for the fragmentation were 2.8 mJ at 532 nm, 1.7 mJ at 355 nm and 0.6 mJ at 266 nm. The mass accuracy in the photodissociationand CID spectra was better than 10 mDa in all cases. Oxazine 170 and all solvents were purchased from Sigma Aldrich (Germany) and used without further purification. The deuterated compounds were prepared according to standard literature procedures,23 and therefore only a brief description is given here. The exchange of the amino hydrogens in oxazine 170 was conducted by dissolving a sample in ethanol. The
Experimental All experiments were performed using an Apex III FT-ICR instrument (Bruker Daltoniks, Bremen, Germany) equipped with a 7.05 T superconducting magnet and an Apollo I ESI
Figure 1. ICR setup with laser beam lines of differentwavelengths: 532 nm in green, 355 nm in blue and 266 nm in purple.
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solution was added to five aliquots of D2O. This mixture was stirred for 10 min and renewed three times. The solution was directly transferred to the FT-ICR mass spectrometer. For the current work, density functional theory calculations using the Gaussian 09 program24 were employed to study the reaction intermediates as well as the various reaction mechanisms. All calculations involved geometry optimization of various reaction intermediates and transition states. Transition state optimizations were performed using the Berny algorithm.25 For most cases, an initial estimated structure of the transition state was obtained through relaxed potential energy surface scans using an appropriate internal coordinate. Vibrational frequencies were calculated to confirm that the reaction intermediates all have positive frequencies and species in the transition states have only one imaginary frequency. Intrinsic reaction coordinate calculations 26 were also performed to ensure that a transition state connects two appropriate local minima in the reaction paths. For all calculations the B3LYP functional with the Gaussian basis set 6-311 + G(2p,2d) was used. 27,28 Cartesian coordinates, electronic energies and vibrational frequencies for all of the
optimizedstructures are available upon request. All values given in the following sections are corrected with respect to the zero point vibration corrected energies (DEzpe).
Results and discussion Figure 2 shows the three photodissociation mass spectra of oxazine 170 obtained with wavelengths of 266, 355 and 532 nm as well as the CID mass spectrum. Although similar main fragmentation signals are found in all four mass spectra, the intensity and the mass range of fragmentation products vary with the activation wavelength. This is due to the energy and the number of photons absorbed in the molecular ion as well as the absorption cross section in the visible and the ultraviolet regions of the spectrum. A comparison of the spectra shown in Figure 2 demonstrates that at the wavelengths of 266 and 532 nm the mass spectra show some additional fragmentation products. Since the laser irradiance is kept at a constant level in the ICR cell, the differences in the mass spectra reflect the absorption spectrum of the molecular ion.
Figure 2. Fragmentation spectra of oxazine 170. Top left: CID spectrum; photodissociation spectra using 266 nm (top right), 355 nm (bottom left) and 532 nm (bottom right).
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Figure 3. Fragmentation spectrum of oxazine 170 using photodissociation with a wavelength of 532 nm.
The comparison clearly shows that photodissociation with 355 nm delivers the same mass spectrum as the CID. The use of 266 nm photons changes the fragmentation pattern clearly, attributed not only to a better signal-to-noise ratio but also to further fragment signals in the lower m/z area. These signals are also found when the wavelength of 532nm is used for photodissociation. Comparing the 266 nm photodissociation with that at 532 nm, the latter shows the most intense fragment signals as well as the largest number of fragmentation signals. Thus, the photodissociation investigations with a wavelength of 532 nm give the best results for obtaining fragmentation products as seen in Figure 3. All photodissociation mass spectra are dominated by reactions that stem from direct bond breaking processes as well as some larger rearrangements. As seen in the mass spectra, the molecular ion is observed at mass 332.173 according to an elementary composition of C21H22N3O+. In addition to the expected neutral loss of methane (m/z 316.142) there are
several further fragmentations of high interest. The signal at m/z 303.134 represents the loss of an ethyl radical. Since the molecular ion is an even-electron system, this signal is due to a violation of the well-known “even-electron” rule.21,22 It is known that there are several exceptions29 to this rule, especially in the case of nitrogen-containing compounds30 or even in negative ion mass spectra.31 Additionally, the products of two further fragmentation reactions are observed at masses 288.111 (C18H16N3O+) and 274.095 (C17H12 N3O+). These signals are due to a formal loss of propane and butane, respectively. However, both signals can only be formed through an extensive rearrangement reaction, which must incorporate distant groups of the molecule. Looking at the structure of the molecule, it is obvious that these fragments cannot be formed in a single-step reaction. Either a rearrangement process consisting of a transfer of one ethyl group to the opposite amine function or a two-step bond-breaking process involving the formation of two ethyl radicals or a methyl and an ethyl radical must be responsible for these two signals. Again the latter sequential reaction would be a violation of the even-electron rule. Indirect proof of this reaction sequence is obtained through an MS3 experiment. This will be discussed in detail later. In the lower mass range, two nitrogenous fragments are detected. At m/z 261.100, the loss of C4H9N, and at m/z 247.085, the loss of C5H11N, as well as some further fragments stemming from the aromatic ring system give rise to signals with low intensities. To gain further insight into photodissociation processes, the energy dependence of the fragmentation processes at 532 nm was studied. Since a pulsed laser was used, the irradiance and the pulse energy applied to the stored molecule ions can be measured as shown in Figure 4. The left-hand panel of Figure 4 shows the intensity of the main fragmentation products with increasing irradiance. This value reflects the number of single laser shots applied. The Nd:YAG laser used has a pulse width of 3 ns and a repetition rate of 20 Hz. Therefore, at least 19 laser pulses are applied at an irradiance of 1 s. With increasing irradiance, the molecular ion signal decreases to a
Figure 4. Fragmentation intensities resulting from changes in the irradiation time (number of laser pulses) (left) and the laser energy used (right).
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Figure 5. Enlargement of the right-hand panel of Figure 4. With the changing intensity of the various fragments it is now possible to identify several fragmentation pathways and timescales, e.g. the formation of C2H5· (blue) and C4H10 (purple). On the right-hand side, a double plot of fragment intensity versus laser energy displays the different energy amounts in photons that are needed for fragmentation.
constant level at time greater than 1.8 s, while in general the fragment ion intensities increase to a more or less constant value. This level is reached at irradiances larger than 0.8 s. The investigation of the laser power results in a slightly different picture. Again, with increasing pulse energy of the laser radiation applied, the molecular ion intensity decreases while the fragment ion intensities increase up to a laser power of approximately 45 mJ. Beyond this value, the signal intensities of the measured fragmentation reactions decrease again. At the highest powers applied, all signals vanish. The reason for this behaviour is due to very fast reactions of the various ions either to smaller fragments or to an interference of the laser radiation with the cyclotron motion of the ions within the ICR cell. As seen from an enlarged view (Figure 5) the fragments behave differently and are separated into two different groups. The first group consists of the loss of methane (–CH 4) and nitrogen-containing fragments (–C4H9N and –C5H11N). These fragmentation reactions show nearly the same low and almost constant intensity with increasing laser power. The second group, involving loss of alkyls (–C2H5·, –C3H8 and –C4H10), show a completely different behaviour. Starting at a laser power of approximately 15 mJ the intensity increases until a maximum is reached between 40 and 50 mJ. Comparing these three curves, it is obvious that laser energy for the loss of C4H10 is shifted to higher value while the two other curves are closely parallel. This difference is easily explained through the photon processes necessary to induce the fragmentation. Following basic photophysical laws for multiphoton processes, 32,33 the number of photons can be estimated through a double-logarithmic plot of the laser power versus the measured ion intensity which results in a straight line. The slope then yields the number photons in the photophysical process, here the various fragmentations. Figure 5 shows these double-logarithmic plots for all investigated fragmentation reactions.
The calculation of the slope for the six fragmentation reactions results in values of between 1.6 and 3.7. This converts to two-, three- and four-photon processes, respectively. Taking into account that 532 nm photons have been used, the minimum energy to induce any fragmentation is about 4.6 eV for the loss of CH4, C2H5·, C2H6 and C3H8. The formation of the last fragment is at the edge between a two- and a threephoton process with a value for the slope of 2.5. Taking into account the experimental deviation and the error, this value is closer to that for a two-photon process. All other fragmentation reactions need higher activation energies. Therefore, a three- or four-photon process is necessary to induce the fragmentation. In agreement with the intensity plot, the loss of C4H10 has a slope nearly twice as high as that for ethyl radical loss. This supports again a two-step reaction forming this fragment. Higher numbers of photons needed for the fragmentation can also be found in the case of the nitrogenous losses, which can also not be formed in a one-step reaction due to the structure of oxazine 170. From these measurements, the mechanisms for the formation of the various fragments can be deduced as shown in Figure 6. Reaction path A leads to the formation of the signals at masses 288.111 and 275.103. Either a one-step or a twostep mechanism leads to the fragments A2 and A3, as denoted in Figure 6. A one-step mechanism would require a preceding rearrangement reaction in the molecular ion to form either a propane or a butane moiety, which would be fragmented. However, there are no indications for such a rearrangement in the mass spectrum nor are similar reactions known from the literature. In the two-step mechanism, an ethyl radical is lost from the amine function followed by a second loss of either a methyl or an ethyl radical. This reaction has already been described in the literature for several xanthene compounds.34 To gain more information on this two-step mechanism, the fragmentation of the intermediate ion A1 was measured using a MS3 experiment. In the MS3 spectrum (Figure 7), which
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Figure 6. Proposed fragmentation scheme for oxazine 170.
was generated using CID as the fragmentation method, two radical fragments are observed next to the precursor signal at m/z 303.138. As the most intense fragment, the loss of an ethyl radical (–C2H5·) at m/z 274.099 is observed. The loss of a methyl radical (–CH3·) is seen at m/z 288.114. This observation is a strong support for the proposed fragmentation mechanism of the loss of propane as well the loss of the C4H10 moiety.
Figure 7. MS3 spectrum of oxazine 170 after the loss of one C2H5 radical. Two further radical fragments, CH3· and C2H5·, are observed.
As indicated in Figure 6, this reaction sequence finally forms the (Z)-N-(azirino[2,3-i]benzo[a]phenoxazin-5(9H)-ylidene) ethanaminium ion A2 (azarine ion) through the loss of the methyl radical from the aromatic ring. Those azarine ions structures have been already investigated by Sander and co-workers35 through different spectroscopic means. It should be noted that the formation of the methyl radical could stem from a second ethylamino group. However, other investigations of different xanthene dyes showed clearly that the formation of a methyl radical is not observed through fragmentation from the ethylamino function.36 Therefore, the participation of the aromatic methyl group is highly probable. The MS 3 experiment also gives an explanation for the observed C4H10 fragment, which must be a double loss of two ethyl radicals. As indicated in Figure 6, this reaction sequence starts also with the fragmentation of the ethyl radical, followed in the second step by loss of a further ethyl radical from the opposite amine function. The mechanism for this two-step fragmentation is complicated because the first loss of an ethyl radical leads to a cation radical from the molecular ion. The second fragmentation again is radical driven. Therefore a biradical cation would be formed. This cannot be ruled out because labelling experiments showed unambiguously no indications for large rearrangements in the molecular backbone. 36 Biradicals usually have a very short lifetime, 37–39 especially if compared to the measurement time in an ICR experiment. A direct measurement would also be impossible, because, in the proposed mechanism, a proton shift leads
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Figure 8. Mass spectrum of amine-deuterated oxazine 170 using photodissociation with 532 nm radiation.
then to a highly stabilized azarine structure A3, the azirino[2,3i]benzo[a]phenoxazin-5(9H)-iminium ion, as shown in Figure 6. As already discussed above, the photodissociation experiment clearly supports this behaviour. The loss of the first ethyl radical from the molecular ion has a minimum activation energy of 4.6 eV, while the following losses of a methyl radical or a second ethyl radical need another photon of 532 nm wavelength, respectively. As a result, in the case of the total loss of C4H10, the minimum activation energy added to the molecular ion is at least 6.9 eV. With such a high energy involved, a biradical mechanism is possible. Reaction path B leads to the loss of methane (–CH4). In general, this fragment can be formed by various mechanisms. Again, the formation of the pyrrolidine ring system drives this reaction through a ring-closure mechanism.40 It should be noted that any reaction incorporating hydrogen bound directly to amine can be excluded through the deuteration experiments performed. Figure 8 shows the resulting photodissociation mass spectrum of oxazine 170 with both amine hydrogen atoms replaced by deuterium. The main fragmentation products are shifted by two mass units as expected. Neither for the loss of methane (m/z 318.154) nor for the [M − C3H6]+ and the [M − C4H10]+ fragments is participation of these deuterium atoms observed. This rationalizes the proposed fragmentation mechanism. Additional experiments using different dyes with the same principal structure as oxazine 170, but without the methyl group attached to the aromatic ring system, do not show this loss of methane. Therefore, one can assume that this aromatic methyl group is a prerequisite for the loss of methane. Beside these alkyl losses, two nitrogenous fragments resulting in the loss of C4H9N and C5H11N are observed. These are typical rearrangements of secondary aromatic amines as indicated by reaction path C in Figure 6. In both cases, the first step is a g-hydrogen shift to the aromatic ring system followed by an H-ring walk, which removes a vinyl-l2-azane. The second step of the complete mechanism is then reactions
at the second amine function. The loss of ethene is normally the main fragmentation process for molecules with an ethylamine function,31,40 so the combination of both fragments is the explanation for the formation of the C4H9N fragment. In the case of the larger nitrogenous fragment C5H11N, the second step is again a process in which the aromatic methyl group is involved. After the loss of vinyl-l2-azane in the first step, a C3H6 moiety is formed through a cyclic transition state resulting again in an azarine structure. It should be noted that the formation of this fragment cannot be explained without the formation of the azarine structure and is overall another hindrance for the azarine ring formation during the fragmentation of oxazine 170. Additionally the fragmentation of a C3H6 molecule from xanthenes has been reported before.11 Both processes, the loss of C4H9N and C5H11N, are high-energy processes. The photodissociation experiments indicate that both two-step fragmentations require at least three photons or an activation energy of 6.9 eV to take place. It should be noted that again deuterated oxazine 170 shows a mass shift in the resulting fragment ions. Clearly, no indication of a hidden H-rearrangement can be found, which adds some further arguments to the discussed mechanisms. To gain further insight in these different rearrangement and fragmentation reactions, quantum chemical calculations with the B3LYP functional and the Gaussian basis set 6-311 + G(2p,2d) have been performed. From the calculation of the different stable and transition states, the minimum energy reaction path is constructed for the loss of the various alkyl radicals and alkanes, as shown in Figure 9. The calculations show for the loss of a C 3H8 moiety two different mechanisms, which have been discussed above. The stepwise mechanism starts with the fragmentation of the ethyl radical from the amine function, followed by a ring attack of the amine cation and the subsequent loss of a methyl radical. The result of this reaction sequence is the formation of the azarine structure A2. The calculations show that the
Figure 9. Calculated minimum energy reaction path for the loss of C3H8 and C4H10 neutrals. The arrows indicate the energy distributed in the molecular ion through the absorption of 532 nm radiation.
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loss of the aromatic methyl radical leading to ion A4 has an activation energy of 442 kJ mol−1, which is slightly higher than that for the formation of ion A1 with an activation energy of 325 kJ mol−1. Consequently, the loss of the aromatic methyl group in the first stage of the reaction sequence leading to ion A1 is therefore unlikely. Furthermore, the formation of this ion would require a s-type ion, in which the radical is perpendicular to the aromatic electron system. The second reaction is the loss of either the other ethyl or methyl radical. This leads to a total energy of 856 kJ mol−1 for the successive stepwise fragmentation mechanism and the formation of the product ion A2 or A3. One should keep in mind that the energies given are the minimum requirements for the reaction. The actual energies might be considerably higher, since the real transition states are difficult to calculate. These will be published in a forthcoming paper.36 The experimental data from the photodissociation experiments show a twophoton process for the formation of the product ion A2. With 532 nm photons, this would lead to a total energy applied through the absorption of photons of 443 kJ mol−1. Even if the precursor ion absorbs three photons, which leads to an energy of 665 kJ mol−1, this would be too little to induce the stepwise fragmentation process. Alternatively, the loss of the C3H8 moiety can occur via a concerted mechanism. Such a reaction has been proven already in the fragmentation of rhodamine B, another xanthene-type dye.11 This reaction sequence is induced by a nucleophilic attack of the charged nitrogen atom on the ortho position of the aromatic ring system, which has the methyl group attached. By formation of the azarine system, the methyl and ethyl groups are expelled as propane. The result of this reaction is again the formation of ion A2. The calculations show for this concerted reaction sequence a minim energy requirement of 478 kJ mol−1, which is very close to the measured and estimated energy for a two-photon process which deposits 443 kJ mol−1 in the precursor ion. This adds further proof to the proposed reaction scheme. In addition to the loss of C3H8, the mass spectra formally show also a loss of a C4H10 moiety. It is obvious that this reaction can only occur through a two-step reaction, since both amine groups are involved. The photodissociation experiments show for the formation of the resulting fragment ion A3 a fourphoton process. This converts to an energy of at least 9.2 eV or 878 kJ mol−1. The calculations of various ions in reaction path A yield again an energy of 325 kJ mol−1 for the loss of the ethyl radical, while the loss of a second ethyl group formed from ion A1 requires between 303 and 468 kJ mol−1 depending on the final state of ion A3. This amounts to a total energy for this sequence of between 628 and 793 kJ mol−1. The experimentally measured energy is smaller than this calculated one. Therefore, this adds again proof to the proposed fragmentation mechanism of oxazine 170. It should be noted that the other proposed reaction pathways could also be proven through calculation, which will be reported in a forthcoming paper.
Conclusions The fragmentation reactions of oxazine 170 have been investigated in a FT-ICR mass spectrometer using photodissociation experiments using for the first time nanosecond laser pulses. It is shown that fragment intensities are noticeably improved compared to previous CID measurements. This leads to the detection of several new fragmentation mechanisms. With the help of energy studies as well as deuteration and MS3 experiments it is possible to suggest reasonable fragmentation mechanisms for the observed fragments. The crucial point is that different fragmentation pathways lead to azarine ring structures. The primary dissociation pathways consist of a set of reactions that start in general at one of the amine groups and incorporate the second amine group or ring substituents. Utilizing the physical basics of multiphoton processes, the photon processes and thus the energies leading to the different fragments could be measured. The minimum energy requirements for the various fragmentations are deduced and compared to those from quantum chemical calculations. These calculations are in full agreement with the measured energetics of the various fragmentations.
Acknowledgements The authors acknowledge gratefully the support through the Schleswig–Holstein Fonds for purchase of the Fourier transform ion cyclotron mass spectrometer. The laser was funded by the Deutsche Forschungsgemeinschaft (GR 917/13-3).
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dye oxazine 170 as a function of environment and pH”, J. Phys. Colloques 48, C7-471–C7-473 (1987). doi: http:// dx.doi.org/10.1051/jphyscol:19877112 19. N.C. Polfer, “Infrared multiple photon dissociation spectroscopy of trapped ions”, Chem. Soc. Rev. 40, 2211–2221 (2011). doi: http://dx.doi.org/10.1039/c0cs00171f 20. J.P. Reilly, “Ultraviolet photofragmentation of biomolecular ions”, Mass Spectrom Rev. 28, 425–447 (2009). doi: http://dx.doi.org/10.1002/mas.20214 21. M. Karni and A. Mandelbaum, “The ‘even-electron rule’”, Org. Mass Spectrom. 15, 53–64 (1980). doi: http://dx.doi. org/10.1002/oms.1210150202 22. A. Knoop, Ol. Krug, M. Vincenti, W. Schänzer and M. Thevis, “In vitro metabolism studies on the selective androgen receptor modulator (SARM) LG121071 and its implementation into human doping controls using liquid chromatography-mass spectrometry”, Eur. J. Mass Spectrom. 21, 27–36 (2015). doi: http://dx.doi.org/10.1255/ ejms.1328 23. A.F. Thomas, Deuterium Labeling in Organic Chemistry. Appleton-Century-Crofts, New York (1971). 24. M.J. Frisch, G.W. Trucks, H.B. Schlegel et al., Gaussian 09. Gaussian, Wallingford, CT (2009). 25. H.B. Schlegel, “Optimization of equilibrium geometries and transition structures”, J. Comput. Chem. 3, 214–218 (1982). doi: http://dx.doi.org/10.1002/jcc.540030212 26. C. Gonzalez and H.B. Schlegel, “Reaction path following in mass-weighted internal coordinates”, J. Phys. Chem. 94, 5523–5527 (1990). doi: http://dx.doi.org/10.1021/ j100377a021 27. W.J. Hehre, R. Ditchfield and J.A. Pople, “Self-consistent molecular orbital methods. XII. Further extensions of Gaussian-type basis sets for use in molecular orbital studies of organic molecules”, J. Chem. Phys. 56, 2257– 2261 (1972). doi: http://dx.doi.org/10.1063/1.1677527 28. T. Clark, J. Chandrasekhar, G.W. Spitznagel and P.V.R. Schleyer, “Efficient diffuse function augmented basis sets for anion calculations. III. The 3-21 + G basis set for first-row elements, Li–F”, J. Comput. Chem. 4, 294–301 (1983). doi: http://dx.doi.org/10.1002/jcc.540040303 29. K. Chen, N. Rannulu, Y. Cai, P. Lane, A. Liebl, B. Rees, C. Corre, G. Challis and R. Cole, “Unusual odd-electron fragments from even-electron protonated prodiginine precursors using positive-ion electrospray tandem mass spectrometry”, J. Am. Soc. Mass Spectrom. 19, 1856–1866 (2008). doi: http://dx.doi.org/10.1016/j. jasms.2008.08.002 30. R. Bowen and R. Harrison, “Loss of methyl radical from some small immonium ions: unusual violation of the even-electron rule”, Org. Mass. Spectrom. 16, 180–182 (1981). doi: http://dx.doi.org/10.1002/oms.1210160408 31. Y. Cai, Z. Mo, N. Rannulu, B. Guan, S. Kannupal, B. Gibb and R. Cole, “Characterization of an exception to the ‘even-electron rule’ upon low-energy collision induced decomposition in negative ion electrospray tandem
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mass spectrometry”, J. Mass Spectrom. 45, 235–240 (2010). doi: http://dx.doi.org/10.1002/jms.1706 32. W. Dietz, H.J. Neusser, U. Boesl and E.W. Schlag, “A model for multiphoton ionisation mass spectroscopy with application to benzene”, Chem. Phys. 66, 105–127 (1982). doi: http://dx.doi.org/10.1016/03010104(82)88011-7 33. C. Weickhardt, F. Moritz and J. Grotemeyer, “Multiphoton ionization mass spectrometry: principles and fields of application”, Eur. Mass Spectrom. 2, 151–160 (1996). 34. J. Peters, Dissertation, Christian-Albrechts University at Kiel (2015). 35. D. Grote, C. Finke, S. Kossmann, F. Neese and W. Sander, “3,4,5,6-Tetrafluorophenylnitren-2-yl: a ground-state quartet triradical”, Chem. Eur. J. 16, 4496–4506 (2010). doi: http://dx.doi.org/10.1002/chem.200903285
36. C. Gernert, M. Clemen and J. Grotemeyer, in preparation. 37. R.A. Caldwell, in Kinetics and Spectroscopy of
Carbenes and Biradicals, Ed by M.S. Platz. Springer Science + Business Media, New York (1990). doi: http:// dx.doi.org/10.1007/978-1-4899-3707-0 38. J.A. Berson, in Reactive Intermediate Chemistry, Ed by R.A. Moss, M.S. Platz and M. Jones Jr. John Wiley, Hoboken, NJ, pp. 165–204 (2004). 39. M. Fu, S. Li, E. Archibold, M.J. Yurkovich, J.J. Nash and H.I. Kenttämaa, “Reactions of an aromatic s,s-biradical with amino acids and dipeptides in the gas phase”, J. Am. Soc. Mass Spectrom. 21, 1737–1752 (2010). doi: http:// dx.doi.org/10.1016/j.jasms.2010.06.010 40. F.W. McLafferty and F. Turecek, Interpretation of Mass Spectra, 4th Edn. University Science Books, Mill Valley, CA (1993).
C. Gernert and J. Grotemeyer, Eur. J. Mass Spectrom. 21, 599–608 (2015) Received: 6 May 2015 n Revised: 18 June 2015 n Accepted: 21 June 2015 n Publication: 26 June 2015
EUROPEAN JOURNAL OF MASS SPECTROMETRY Special Issue Celebrating the 20th Anniversary of EJMS—European Journal of Mass Spectrometry
Photodissociation at various wavelengths: fragmentation studies of oxazine 170 using nanosecond laser pulses Claus Gernert and Jürgen Grotemeyer* Christian-Albrechts Universität zu Kiel, Max-Eyth-Straße 1, 24118 Kiel, Germany. E-mail: [email protected]
The fragmentation of oxazine 170, a rhodamine-type dye, has been investigated by means of collisions and photodissociation with visible and ultraviolet radiation in a Fourier transform ion cyclotron resonance mass spectrometer. Because of an improved experimental setup, the photodissociation processes of stored ions are measured with high intensity with respect to the absorbed photons. By isotope labelling and quantum chemical calculations, the various fragmentation mechanisms are investigated. It is shown that the most important intermediate ion structure leading to the various ionic products is an even-electron azarine cation. Several new fragmentation mechanisms have been unveiled for the first time. Keywords: UV photodissociation, photon processes, dyes, fragmentation mechanisms, ICR mass spectrometry
Introduction Organic dyes are widely used in chemistry and physics for a variety of purposes.1 Applications range from use as colorants, markers in chemical reactions and in dye lasers to lightharvesting devices in solar cells. This use is based on intrinsic features like high photostabilities, high quantum yields and very high extinction factors. In analytical applications, various dyes are commonly used in fluorescence studies, especially for biopolymers and other larger molecular systems.2 Because of their common application, their mass spectral behaviour has been thoroughly investigated with respect to their fragmentation reactions.3–5 But it should be noted that several different dyes, commonly of the xanthene type, have found further importance in mass spectrometry, such as in laser desorption studies,6 in cluster ion research7,8 or as chromophoric anchors for photodissociation studies.9–11 In view of the above considerations, the mass spectra of rhodamine B have been intensively investigated in order to understand the fragmentation reactions after electrospray ionization in a Fourier transform ion cyclotron resonance ISSN: 1469-0667 doi: 10.1255/ejms.1368
(FT-ICR) mass spectrometer.12–15 To gain more insight into the observed reactions of rhodamine B and extend the fragmentationin a general form, we investigated a different xanthene-type dye, oxazine 170. This dye is known for several interesting applications in chemistry and biochemistry. It has been successfully used for the induction of a hybrid triplex structure, poly rA:(poly dT)2, which otherwise would not be formed under solution conditions.16 A further application of oxazine 170 is in the investigation of photonic crystals and the energy transfer to it from other dyes.17 Even more important are the photophysical properties of this compound, which are of fundamental interest in spectroscopy. Oxazine 170 is used as an efficient laser dye, emitting light in the region between 600 and 700 nm. 18 In addition, oxazine 170 acts as a donor chromophorein several studies that lead to an understanding of its gas-phase photophysical properties. Additionally, this class of xanthene dyes provides a benchmark for electronic structure calculations, which remain a challenging prospect. © IM Publications LLP 2015 All rights reserved
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The substitution of both amine groups by an ethyl group and the introduction of two further substituents at the aromatic ring system, methyl and annulated phenyl moieties, are the important differences between the oxazines and the rhodamines. While in rhodamine B two ethyl groups doubly substitute both amine groups, oxazine 170 has on both amine groups only a single substitution. Therefore, this compound is particularly suited for the mass spectrometric investigation of its isomerization and fragmentation processes. Due to its absorption spectrum in the neutral and ionic state, activation of the molecular ions by laser radiation should give further insight into the reaction mechanisms leading to the most prominent fragments. By use of various wavelengths, it is possible to control the energy deposited into the molecular system over a certain range. Contrary to the frequently used infrared multiphoton dissociation (IR-MPD),19 photodissociation with nanosecond pulses in the visible and ultraviolet regions is usually a process with only few photons. While in IR-MPD the photon energy is typically 0.1 eV (10.6 µ; CO2 laser), a Nd:YAG laser delivers, depending on the wavelength used, energy between 1.17 eV (1.064 µ) and 4.66 eV (266 nm). As a result, different isomerization and fragmentation products can be expected in general as previously reported.20 In this paper, we report on an investigation of the various fragmentation reactions of oxazine 170 using photodissociation wavelengths of 532, 355 and 266 nm. To perform these investigations we introduced a new experimental setup for the photodissociation in an ICR mass spectrometer, which allows readjustment of the laser beam during the change of the wavelength. This guarantees a high reliability of the measured spectra. Furthermore, due to the challenging task of synchronizing the laser and the experimental pulse timeline, we now can perform photodissociation experiments with a nanosecond-pulsed Nd:YAG laser. With this setup, the various fragmentations of oxazine 170 were investigated and compared to results from quantum chemical calculations. Furthermore, isotopically labelled oxazine 170 was prepared to confirm the proposed reaction mechanisms. This compound, as some other rhodamines, shows an interesting rearrangement and fragmentation mechanism leading to the loss of an alkane from the molecular ion. It will be shown that the most important reactions involve the intermediate formation of radical cations from even-electron molecular ions. The complete reaction sequence leads to a question concerning the appearance of radical cations from even-electron ions and is in contrast to the well-known “evenelectron rule”. This rule has been well known in mass spectrometry for a long time, but several violations and exceptions can be found in the literature.21,22
source. The various substances were ionized with the electrospray ionization method. All samples were dissolved in dimethyl sulfoxide and added to a standard solvent mixture (water– methanol–formic acid, 50:50:0.2) yielding a final concentration in the range 1–100 pmol mL−1. The instrument usually was mass-calibrated with arginine clusters to guarantee a resolution error of under 2 ppm. For the collision-induced dissociation (CID) and photodissociation experiments, the molecular ion peak was isolated in the ICR cell to get isotopic-free spectra. This guarantees a measurement of the different ions without interference either from fragmentation processes or, in the case of deuterated substances, from incomplete labelling. Furthermore, it proves the radical ion character of the observed losses. Fragmentation of the molecular cations was achieved through collisional activation with argon gas using the sustained off-resonance irradiation (SORI) CID technique. After a stored waveform inverse Fourier transform isolation of the ions of interest in the ICR cell, the CID process was induced. The SORI powers were set between 3% and 4.5% and irradiation was applied for 0.1–0.5 s in all experiments. Photodissociation experiments were performed using a Nd:YAG laser (Continuum, Germany) and homemade control software. The different wavelengths were overlaid and then coupled directly into the ICR (Infinity) cell as shown in Figure 1. Thus, it was possible to choose one of the three possible wavelengths (532, 355 and 266 nm) for the fragmentation experiments with nanosecond laser pulses. It should be noted that this setup allows also the simultaneous measurement of two or even three wavelengths. Average laser energies used for the fragmentation were 2.8 mJ at 532 nm, 1.7 mJ at 355 nm and 0.6 mJ at 266 nm. The mass accuracy in the photodissociationand CID spectra was better than 10 mDa in all cases. Oxazine 170 and all solvents were purchased from Sigma Aldrich (Germany) and used without further purification. The deuterated compounds were prepared according to standard literature procedures,23 and therefore only a brief description is given here. The exchange of the amino hydrogens in oxazine 170 was conducted by dissolving a sample in ethanol. The
Experimental All experiments were performed using an Apex III FT-ICR instrument (Bruker Daltoniks, Bremen, Germany) equipped with a 7.05 T superconducting magnet and an Apollo I ESI
Figure 1. ICR setup with laser beam lines of differentwavelengths: 532 nm in green, 355 nm in blue and 266 nm in purple.
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solution was added to five aliquots of D2O. This mixture was stirred for 10 min and renewed three times. The solution was directly transferred to the FT-ICR mass spectrometer. For the current work, density functional theory calculations using the Gaussian 09 program24 were employed to study the reaction intermediates as well as the various reaction mechanisms. All calculations involved geometry optimization of various reaction intermediates and transition states. Transition state optimizations were performed using the Berny algorithm.25 For most cases, an initial estimated structure of the transition state was obtained through relaxed potential energy surface scans using an appropriate internal coordinate. Vibrational frequencies were calculated to confirm that the reaction intermediates all have positive frequencies and species in the transition states have only one imaginary frequency. Intrinsic reaction coordinate calculations 26 were also performed to ensure that a transition state connects two appropriate local minima in the reaction paths. For all calculations the B3LYP functional with the Gaussian basis set 6-311 + G(2p,2d) was used. 27,28 Cartesian coordinates, electronic energies and vibrational frequencies for all of the
optimizedstructures are available upon request. All values given in the following sections are corrected with respect to the zero point vibration corrected energies (DEzpe).
Results and discussion Figure 2 shows the three photodissociation mass spectra of oxazine 170 obtained with wavelengths of 266, 355 and 532 nm as well as the CID mass spectrum. Although similar main fragmentation signals are found in all four mass spectra, the intensity and the mass range of fragmentation products vary with the activation wavelength. This is due to the energy and the number of photons absorbed in the molecular ion as well as the absorption cross section in the visible and the ultraviolet regions of the spectrum. A comparison of the spectra shown in Figure 2 demonstrates that at the wavelengths of 266 and 532 nm the mass spectra show some additional fragmentation products. Since the laser irradiance is kept at a constant level in the ICR cell, the differences in the mass spectra reflect the absorption spectrum of the molecular ion.
Figure 2. Fragmentation spectra of oxazine 170. Top left: CID spectrum; photodissociation spectra using 266 nm (top right), 355 nm (bottom left) and 532 nm (bottom right).
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Figure 3. Fragmentation spectrum of oxazine 170 using photodissociation with a wavelength of 532 nm.
The comparison clearly shows that photodissociation with 355 nm delivers the same mass spectrum as the CID. The use of 266 nm photons changes the fragmentation pattern clearly, attributed not only to a better signal-to-noise ratio but also to further fragment signals in the lower m/z area. These signals are also found when the wavelength of 532nm is used for photodissociation. Comparing the 266 nm photodissociation with that at 532 nm, the latter shows the most intense fragment signals as well as the largest number of fragmentation signals. Thus, the photodissociation investigations with a wavelength of 532 nm give the best results for obtaining fragmentation products as seen in Figure 3. All photodissociation mass spectra are dominated by reactions that stem from direct bond breaking processes as well as some larger rearrangements. As seen in the mass spectra, the molecular ion is observed at mass 332.173 according to an elementary composition of C21H22N3O+. In addition to the expected neutral loss of methane (m/z 316.142) there are
several further fragmentations of high interest. The signal at m/z 303.134 represents the loss of an ethyl radical. Since the molecular ion is an even-electron system, this signal is due to a violation of the well-known “even-electron” rule.21,22 It is known that there are several exceptions29 to this rule, especially in the case of nitrogen-containing compounds30 or even in negative ion mass spectra.31 Additionally, the products of two further fragmentation reactions are observed at masses 288.111 (C18H16N3O+) and 274.095 (C17H12 N3O+). These signals are due to a formal loss of propane and butane, respectively. However, both signals can only be formed through an extensive rearrangement reaction, which must incorporate distant groups of the molecule. Looking at the structure of the molecule, it is obvious that these fragments cannot be formed in a single-step reaction. Either a rearrangement process consisting of a transfer of one ethyl group to the opposite amine function or a two-step bond-breaking process involving the formation of two ethyl radicals or a methyl and an ethyl radical must be responsible for these two signals. Again the latter sequential reaction would be a violation of the even-electron rule. Indirect proof of this reaction sequence is obtained through an MS3 experiment. This will be discussed in detail later. In the lower mass range, two nitrogenous fragments are detected. At m/z 261.100, the loss of C4H9N, and at m/z 247.085, the loss of C5H11N, as well as some further fragments stemming from the aromatic ring system give rise to signals with low intensities. To gain further insight into photodissociation processes, the energy dependence of the fragmentation processes at 532 nm was studied. Since a pulsed laser was used, the irradiance and the pulse energy applied to the stored molecule ions can be measured as shown in Figure 4. The left-hand panel of Figure 4 shows the intensity of the main fragmentation products with increasing irradiance. This value reflects the number of single laser shots applied. The Nd:YAG laser used has a pulse width of 3 ns and a repetition rate of 20 Hz. Therefore, at least 19 laser pulses are applied at an irradiance of 1 s. With increasing irradiance, the molecular ion signal decreases to a
Figure 4. Fragmentation intensities resulting from changes in the irradiation time (number of laser pulses) (left) and the laser energy used (right).
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Figure 5. Enlargement of the right-hand panel of Figure 4. With the changing intensity of the various fragments it is now possible to identify several fragmentation pathways and timescales, e.g. the formation of C2H5· (blue) and C4H10 (purple). On the right-hand side, a double plot of fragment intensity versus laser energy displays the different energy amounts in photons that are needed for fragmentation.
constant level at time greater than 1.8 s, while in general the fragment ion intensities increase to a more or less constant value. This level is reached at irradiances larger than 0.8 s. The investigation of the laser power results in a slightly different picture. Again, with increasing pulse energy of the laser radiation applied, the molecular ion intensity decreases while the fragment ion intensities increase up to a laser power of approximately 45 mJ. Beyond this value, the signal intensities of the measured fragmentation reactions decrease again. At the highest powers applied, all signals vanish. The reason for this behaviour is due to very fast reactions of the various ions either to smaller fragments or to an interference of the laser radiation with the cyclotron motion of the ions within the ICR cell. As seen from an enlarged view (Figure 5) the fragments behave differently and are separated into two different groups. The first group consists of the loss of methane (–CH 4) and nitrogen-containing fragments (–C4H9N and –C5H11N). These fragmentation reactions show nearly the same low and almost constant intensity with increasing laser power. The second group, involving loss of alkyls (–C2H5·, –C3H8 and –C4H10), show a completely different behaviour. Starting at a laser power of approximately 15 mJ the intensity increases until a maximum is reached between 40 and 50 mJ. Comparing these three curves, it is obvious that laser energy for the loss of C4H10 is shifted to higher value while the two other curves are closely parallel. This difference is easily explained through the photon processes necessary to induce the fragmentation. Following basic photophysical laws for multiphoton processes, 32,33 the number of photons can be estimated through a double-logarithmic plot of the laser power versus the measured ion intensity which results in a straight line. The slope then yields the number photons in the photophysical process, here the various fragmentations. Figure 5 shows these double-logarithmic plots for all investigated fragmentation reactions.
The calculation of the slope for the six fragmentation reactions results in values of between 1.6 and 3.7. This converts to two-, three- and four-photon processes, respectively. Taking into account that 532 nm photons have been used, the minimum energy to induce any fragmentation is about 4.6 eV for the loss of CH4, C2H5·, C2H6 and C3H8. The formation of the last fragment is at the edge between a two- and a threephoton process with a value for the slope of 2.5. Taking into account the experimental deviation and the error, this value is closer to that for a two-photon process. All other fragmentation reactions need higher activation energies. Therefore, a three- or four-photon process is necessary to induce the fragmentation. In agreement with the intensity plot, the loss of C4H10 has a slope nearly twice as high as that for ethyl radical loss. This supports again a two-step reaction forming this fragment. Higher numbers of photons needed for the fragmentation can also be found in the case of the nitrogenous losses, which can also not be formed in a one-step reaction due to the structure of oxazine 170. From these measurements, the mechanisms for the formation of the various fragments can be deduced as shown in Figure 6. Reaction path A leads to the formation of the signals at masses 288.111 and 275.103. Either a one-step or a twostep mechanism leads to the fragments A2 and A3, as denoted in Figure 6. A one-step mechanism would require a preceding rearrangement reaction in the molecular ion to form either a propane or a butane moiety, which would be fragmented. However, there are no indications for such a rearrangement in the mass spectrum nor are similar reactions known from the literature. In the two-step mechanism, an ethyl radical is lost from the amine function followed by a second loss of either a methyl or an ethyl radical. This reaction has already been described in the literature for several xanthene compounds.34 To gain more information on this two-step mechanism, the fragmentation of the intermediate ion A1 was measured using a MS3 experiment. In the MS3 spectrum (Figure 7), which
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Figure 6. Proposed fragmentation scheme for oxazine 170.
was generated using CID as the fragmentation method, two radical fragments are observed next to the precursor signal at m/z 303.138. As the most intense fragment, the loss of an ethyl radical (–C2H5·) at m/z 274.099 is observed. The loss of a methyl radical (–CH3·) is seen at m/z 288.114. This observation is a strong support for the proposed fragmentation mechanism of the loss of propane as well the loss of the C4H10 moiety.
Figure 7. MS3 spectrum of oxazine 170 after the loss of one C2H5 radical. Two further radical fragments, CH3· and C2H5·, are observed.
As indicated in Figure 6, this reaction sequence finally forms the (Z)-N-(azirino[2,3-i]benzo[a]phenoxazin-5(9H)-ylidene) ethanaminium ion A2 (azarine ion) through the loss of the methyl radical from the aromatic ring. Those azarine ions structures have been already investigated by Sander and co-workers35 through different spectroscopic means. It should be noted that the formation of the methyl radical could stem from a second ethylamino group. However, other investigations of different xanthene dyes showed clearly that the formation of a methyl radical is not observed through fragmentation from the ethylamino function.36 Therefore, the participation of the aromatic methyl group is highly probable. The MS 3 experiment also gives an explanation for the observed C4H10 fragment, which must be a double loss of two ethyl radicals. As indicated in Figure 6, this reaction sequence starts also with the fragmentation of the ethyl radical, followed in the second step by loss of a further ethyl radical from the opposite amine function. The mechanism for this two-step fragmentation is complicated because the first loss of an ethyl radical leads to a cation radical from the molecular ion. The second fragmentation again is radical driven. Therefore a biradical cation would be formed. This cannot be ruled out because labelling experiments showed unambiguously no indications for large rearrangements in the molecular backbone. 36 Biradicals usually have a very short lifetime, 37–39 especially if compared to the measurement time in an ICR experiment. A direct measurement would also be impossible, because, in the proposed mechanism, a proton shift leads
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Figure 8. Mass spectrum of amine-deuterated oxazine 170 using photodissociation with 532 nm radiation.
then to a highly stabilized azarine structure A3, the azirino[2,3i]benzo[a]phenoxazin-5(9H)-iminium ion, as shown in Figure 6. As already discussed above, the photodissociation experiment clearly supports this behaviour. The loss of the first ethyl radical from the molecular ion has a minimum activation energy of 4.6 eV, while the following losses of a methyl radical or a second ethyl radical need another photon of 532 nm wavelength, respectively. As a result, in the case of the total loss of C4H10, the minimum activation energy added to the molecular ion is at least 6.9 eV. With such a high energy involved, a biradical mechanism is possible. Reaction path B leads to the loss of methane (–CH4). In general, this fragment can be formed by various mechanisms. Again, the formation of the pyrrolidine ring system drives this reaction through a ring-closure mechanism.40 It should be noted that any reaction incorporating hydrogen bound directly to amine can be excluded through the deuteration experiments performed. Figure 8 shows the resulting photodissociation mass spectrum of oxazine 170 with both amine hydrogen atoms replaced by deuterium. The main fragmentation products are shifted by two mass units as expected. Neither for the loss of methane (m/z 318.154) nor for the [M − C3H6]+ and the [M − C4H10]+ fragments is participation of these deuterium atoms observed. This rationalizes the proposed fragmentation mechanism. Additional experiments using different dyes with the same principal structure as oxazine 170, but without the methyl group attached to the aromatic ring system, do not show this loss of methane. Therefore, one can assume that this aromatic methyl group is a prerequisite for the loss of methane. Beside these alkyl losses, two nitrogenous fragments resulting in the loss of C4H9N and C5H11N are observed. These are typical rearrangements of secondary aromatic amines as indicated by reaction path C in Figure 6. In both cases, the first step is a g-hydrogen shift to the aromatic ring system followed by an H-ring walk, which removes a vinyl-l2-azane. The second step of the complete mechanism is then reactions
at the second amine function. The loss of ethene is normally the main fragmentation process for molecules with an ethylamine function,31,40 so the combination of both fragments is the explanation for the formation of the C4H9N fragment. In the case of the larger nitrogenous fragment C5H11N, the second step is again a process in which the aromatic methyl group is involved. After the loss of vinyl-l2-azane in the first step, a C3H6 moiety is formed through a cyclic transition state resulting again in an azarine structure. It should be noted that the formation of this fragment cannot be explained without the formation of the azarine structure and is overall another hindrance for the azarine ring formation during the fragmentation of oxazine 170. Additionally the fragmentation of a C3H6 molecule from xanthenes has been reported before.11 Both processes, the loss of C4H9N and C5H11N, are high-energy processes. The photodissociation experiments indicate that both two-step fragmentations require at least three photons or an activation energy of 6.9 eV to take place. It should be noted that again deuterated oxazine 170 shows a mass shift in the resulting fragment ions. Clearly, no indication of a hidden H-rearrangement can be found, which adds some further arguments to the discussed mechanisms. To gain further insight in these different rearrangement and fragmentation reactions, quantum chemical calculations with the B3LYP functional and the Gaussian basis set 6-311 + G(2p,2d) have been performed. From the calculation of the different stable and transition states, the minimum energy reaction path is constructed for the loss of the various alkyl radicals and alkanes, as shown in Figure 9. The calculations show for the loss of a C 3H8 moiety two different mechanisms, which have been discussed above. The stepwise mechanism starts with the fragmentation of the ethyl radical from the amine function, followed by a ring attack of the amine cation and the subsequent loss of a methyl radical. The result of this reaction sequence is the formation of the azarine structure A2. The calculations show that the
Figure 9. Calculated minimum energy reaction path for the loss of C3H8 and C4H10 neutrals. The arrows indicate the energy distributed in the molecular ion through the absorption of 532 nm radiation.
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loss of the aromatic methyl radical leading to ion A4 has an activation energy of 442 kJ mol−1, which is slightly higher than that for the formation of ion A1 with an activation energy of 325 kJ mol−1. Consequently, the loss of the aromatic methyl group in the first stage of the reaction sequence leading to ion A1 is therefore unlikely. Furthermore, the formation of this ion would require a s-type ion, in which the radical is perpendicular to the aromatic electron system. The second reaction is the loss of either the other ethyl or methyl radical. This leads to a total energy of 856 kJ mol−1 for the successive stepwise fragmentation mechanism and the formation of the product ion A2 or A3. One should keep in mind that the energies given are the minimum requirements for the reaction. The actual energies might be considerably higher, since the real transition states are difficult to calculate. These will be published in a forthcoming paper.36 The experimental data from the photodissociation experiments show a twophoton process for the formation of the product ion A2. With 532 nm photons, this would lead to a total energy applied through the absorption of photons of 443 kJ mol−1. Even if the precursor ion absorbs three photons, which leads to an energy of 665 kJ mol−1, this would be too little to induce the stepwise fragmentation process. Alternatively, the loss of the C3H8 moiety can occur via a concerted mechanism. Such a reaction has been proven already in the fragmentation of rhodamine B, another xanthene-type dye.11 This reaction sequence is induced by a nucleophilic attack of the charged nitrogen atom on the ortho position of the aromatic ring system, which has the methyl group attached. By formation of the azarine system, the methyl and ethyl groups are expelled as propane. The result of this reaction is again the formation of ion A2. The calculations show for this concerted reaction sequence a minim energy requirement of 478 kJ mol−1, which is very close to the measured and estimated energy for a two-photon process which deposits 443 kJ mol−1 in the precursor ion. This adds further proof to the proposed reaction scheme. In addition to the loss of C3H8, the mass spectra formally show also a loss of a C4H10 moiety. It is obvious that this reaction can only occur through a two-step reaction, since both amine groups are involved. The photodissociation experiments show for the formation of the resulting fragment ion A3 a fourphoton process. This converts to an energy of at least 9.2 eV or 878 kJ mol−1. The calculations of various ions in reaction path A yield again an energy of 325 kJ mol−1 for the loss of the ethyl radical, while the loss of a second ethyl group formed from ion A1 requires between 303 and 468 kJ mol−1 depending on the final state of ion A3. This amounts to a total energy for this sequence of between 628 and 793 kJ mol−1. The experimentally measured energy is smaller than this calculated one. Therefore, this adds again proof to the proposed fragmentation mechanism of oxazine 170. It should be noted that the other proposed reaction pathways could also be proven through calculation, which will be reported in a forthcoming paper.
Conclusions The fragmentation reactions of oxazine 170 have been investigated in a FT-ICR mass spectrometer using photodissociation experiments using for the first time nanosecond laser pulses. It is shown that fragment intensities are noticeably improved compared to previous CID measurements. This leads to the detection of several new fragmentation mechanisms. With the help of energy studies as well as deuteration and MS3 experiments it is possible to suggest reasonable fragmentation mechanisms for the observed fragments. The crucial point is that different fragmentation pathways lead to azarine ring structures. The primary dissociation pathways consist of a set of reactions that start in general at one of the amine groups and incorporate the second amine group or ring substituents. Utilizing the physical basics of multiphoton processes, the photon processes and thus the energies leading to the different fragments could be measured. The minimum energy requirements for the various fragmentations are deduced and compared to those from quantum chemical calculations. These calculations are in full agreement with the measured energetics of the various fragmentations.
Acknowledgements The authors acknowledge gratefully the support through the Schleswig–Holstein Fonds for purchase of the Fourier transform ion cyclotron mass spectrometer. The laser was funded by the Deutsche Forschungsgemeinschaft (GR 917/13-3).
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