b Collaborative Innovation Center of Chemical Science and Engineering, Nankai University, Tianjin 300071, ;China
Serine octamer has been extensively studied by both experimentalists and theorists since it was firstly observed in electrospray ionization (ESI) mass spectra by Cooks et al. [1-18]. The cluster exhibits a pronounced preference for homochirality [2-6]. It is very attractive to scientists because the aggregates of amino acids with same chirality might be involved in a sequence of chemical events that are relative to the origin of homochirality in life [2, 4, 6]. Although previous cross section experiments showed that serine octamer had a tightly packed structure [10, 11], other experiments also suggested the existence of multi isomers in the gas phase [12-16]. On the other hand, substitution reactions in the serine octamer can happen readily for many amino acids, such as leucine, proline and so on [4, 10]. In order to study these cluster ions, many experimental methods, including collisional activated dissociation (CAD) mass spectrometry and infrared photodissociation (IRPD) spectroscopy, have been performed [14-18]. For example, Liao et al. investigated the chirality effect on prolinesubstituted serine octamers by IRPD spectroscopy. It was found that chiral recognition could be achieved by comparing their corresponding IRPD spectra .
Threonine has a very close structure to serine. It is characterized by the side chain of CH (OH) CH3. The protonated dimer of Thr2H+ has been studied by IRPD spectroscopy and theoretical calculations [14, 19]. It is found that the IRPD spectrum of Thr2H+ is similar to that of Ser2H+ in the region of 3000-3700 cm-1. The most stable isomer of Thr2H+ is also characterized by a charge-solvated structure, but it's structure is different from that of Ser2H+ [14, 19]. Threonine can also form homochiral octamer ions in the ESI process, which has a much weaker intensity than that of serine [5, 6]. Cooks et al. have found that a mixture of threonine and serine with same chirality can generate a series of octamer ions under sonic spray conditions without electric field applied . Considering the structural similarity between serine and threonine, it is interesting and important to find how the substitution of threonine can affect the structure and homochiral preference of serine octamer. However, due to the structural complexity of the cluster, their structural information is still very lacking.
In this study, we study the IRPD spectra of homo-and heterochiral threonine-substituted serine octamer ions for the first time. The cluster ions were generated by ESI method and selected in the cell of a Fourier-transform ion cyclotron resonance (FT ICR) mass spectrometer for IRPD study. Both chiral differentiation and substitution effects are revealed for those threonine-substituted serine octamer ions.2. Results and discussion
Fig. 1a shows a typical mass spectrum of L-threoninesubstituted L-serine clusters produced by the method of ESI. Substitution effects can be clearly identified in the ESI mass spectrum of the mixed solution of L-serine and L-threonine. The most abundant ion was found to be changed from [L-Ser8]H+ to [L-Ser7 + L-Thr1]H+. Different from the results of other amino acids, such as proline, multi-substituted ions of [L-Ser5 + L-Thr3]H+ and [L-Ser4 + L-Thr4]H+ were also observed besides the di-substituted product ions of [L-Ser6 + L-Thr2]H+. The results are consistent with the previous results observed by Cooks et al. , which can be rationalized by the structural similarity between serine and threonine. When the L-Ser was replaced by D-Thr, only substituted ions of [L-Ser7 + D-Thr1]H+ and [L-Ser6 + D-Thr2]H+ were observed (Fig. 1b), and their intensities were weaker than that of [L-Ser8]H+, indicating the existing homochiral preference for the L-threonine in the substituted L-serine octamer.
|Figure 1. Electrospray ionization mass spectra of mixtures containing 3 mmol/L L-serine in 49/49/2 CH3OH/H2O/CH3COOH, and 0.5 mmol/L of (a) L-threonine, (b) D-threonine, obtained in positive ion mode. The cluster ions of [Sern + Thrm]H+ are abbreviated to SnTmH+.|
Laser irradiation can cause dissociation of the selected cluster ions and produce fragment ions in the mass spectrum if the photons can be absorbed by the precursor ions. For example, the previously reported IRPD spectra of [L-Ser8]H+ showed that there was strong and broad absorption in the range from 2950 to 3150 cm-1, and fragment ions of [L-Sern]H+ (n=2-7) were observed in the corresponding IRPD mass spectra [15, 18]. Here, the IRPD mass spectra of [L-Ser7 + L-Thr1]H+ and [L-Ser7 + DThr1]H+ obtained under 3110 cm-1 were shown in Fig. 2a and 2b, respectively. It can be found that the fragment ions and their distribution are very similar for both clusters. The main fragment ions observed are [Ser6 + Thr1]H+, [Ser5 + Thr1]H+ and [Ser4 + Thr1]H+, indicating that their fragmentation pathways are mainly characterized by the subsequent loss of serine unit for both of them. The results are very similar to those of [L-Ser7 + L/DPro1]H+ . However, there are still some difference for ions of [L-Ser7 + Thr1]H+, since weak signals of [L-Ser6]H+ were also observed in Fig. 2a and 2b, indicating the existence of a different dissociation way characterized by the loss of the unit of threonine. Interestingly, for [Ser6 + Thr2]H+, their dissociation mass spectra (Fig. 2c and 2d) are very different from those of [L-Ser6 + L/DPro2]H+. Fragment ions formed by the loss of serine unit and/or threonine unit, including [Ser2 + Thr2]H+, [Ser2 + Thr1]H+ and [Ser1 + Thr2]H+, were observed. The difference can be explained by the fact that threonine has a proton affinity of 219.5 kcal mol-1, which is 4.3 kcal mol-1 higher than serine, but 5.6 kcal mol-1 lower than proline. The chiral difference can also be reflected in their IRPD mass spectra. For the homochiral cluster ions of [L-Ser6 + L-Thr2]H+, it is found that fragment ions by subsequent loss of serine up to [L-Thr2]H+ were identified in the mass spectrum except for [L-Ser5 + L-Thr2]H+, although that other fragment ions formed by the loss of threonine were also observed. However, for heterochiral cluster ions of [L-Ser6 + D-Thr2]H+, no fragment ion of [Ser4 + Thr1]H+ or [Ser3 + Thr2]H+ was found in the mass spectrum (Fig. 2d).
|Figure 2. IRPD mass spectra of (a) [L-Ser7 + L-Thr1]H+, (b) [L-Ser7 + D-Pro1]H+, (c) [L-Ser6 + L-Thr2]H+ and (d) [L-Ser6 + D-Thr2]H+. The laser wavelength was 3110 cm-1, and the irradiation time was 20 s.|
The IRPD spectra of threonine-substituted serine octamers in the range of 2700-3600 cm-1 are shown in Fig. 3. In order to make a good comparison, the previously reported IRPD spectra of [LSer8]H+, [L-Ser7 + Pro1]H+ and [L-Ser6 + Pro2]H+ were also shown in the figure . Board absorption in the range of 2800-3250 cm-1 was also observed for all ions, which is in good agreement with the previous results of [L-Ser8]H+ and proline-substituted cluster ions. Chiral differentiation of homo-and hetero-chiral complexes of the threonine-substituted serine octamer can be achieved by comparing their IRPD spectra in the range of 3300-3550 cm-1. Interestingly, substitution effects can also be observed in their IRPD spectra. For example, the peak at 3425 cm-1 that should correspond to the stretching mode of H-bonded aliphatic O-H does not change much for [L-Ser7 + L-Thr1]H+, but broadens very much for D-Thr substituted octamer. This trend becomes clearer when two units of L-Ser were replaced. For [L-Ser6 + L-Thr2]H+, the peak becomes weak but does not shift manifestly, while for [L-Ser6 + D-Thr2]H+, the peak shifts to 3465 cm-1 clearly, which is very similar to the spectra of [L-Ser6 + D-Pro2]H+. Similarly, the results also indicate that some H-bonds in the heterochiral cluster ions are weaker than those in the homochiral cluster ions, making the corresponding vibrational modes to be less red-shifted. On the other hand, absorption at~3335 cm-1 that disappeared for the prolinesubstituted octamer ions, survived for the D-Thr substituted octamer. And there is a big difference between IPRD spectral intensities of [L-Ser6 + L-Thr2]H+ and [L-Ser6 + D-Thr2]H+, which means the dissociation for L-Thr substituted octamers is much harder than that for D-Thr substituted ones. The results are in consistent with the preference of homochirality for the magic clusters, that is, the substituted units with same chirality as serine can form more stable clusters than their enantiomers. However, due to the structural complexity, a better understanding of these results still depends on further study of the magic serine octamer. Theoretical calculations and more experimental approaches, including cross section measurement, should be performed and investigated.
|Figure 3. IRPD spectra of (a) [L-Ser8]H+, (b) [L-Ser7 + D/L-Thr1]H+, (c) [L-Ser6 + D/L-Thr2]H+, (d) [L-Ser7 + L/D-Pro1]H+, and (e) [L-Ser6 + L/D-Pro2]H+. The data of (a), (d) and (e) are taken from Ref. .|
Cluster ions of threonine-substituted serine octamer were generated by ESI method and studied using the FT ICR mass spectrometer. Chiral preference can be reflected by comparing the ESI mass spectra of substituted serine octamers for L-threonine or D-threonine. Experimental IRPD spectra of [L-Ser7 + L/D-Thr1]H+ and [L-Ser6 + L/D-Thr2]H+ were obtained in the range of 2700-3600 cm-1. Chiral differentiation and recognition of threonine substituted octamer ions were achieved by comparing their IRPD spectra and IRPD mass spectra at certain wavenumbers. Substitution effects were also found in the spectra, especially in the range of 3300-3500 cm-1. The changes of the peaks at 3425 and 3465 cm-1 showed that the replacement by D-Thr could loosen the original compact structure of serine octamer, while it did not change much for the replacement by L-Thr. This trend is close to the one previously observed in the proline-substituted octamers , indicating their structural similarity that comes from the wellorganized, but still puzzling structure of serine octamer.4. Experimental
To perform IRPD experiments, an OPO laser (FireFly-IR-A, M squares) was coupled with a 7.0 T FT ICR mass spectrometer (Varian IonSpec Lake Forest, CA, USA). The experimental details can be found in the previous reports [18-21]. Protonated threoninesubstituted serine clusters were prepared by the ESI technique using the mixed solution containing 0.5 mmol/L Thr and 3 mmol/L L-Ser in 49:49:2 H2O:MeOH:AcOH. All mass spectra obtained here were in the positive ion mode. Ions produced by ESI were injected into an open-ended cylindrical Penning trap via a quadrupole ion guide. The precursor ions of interest were further isolated by the method of stored waveform inverse Fourier transform (SWIFT) . IRPD spectra of the selected ions were obtained in the range from 2700 to 3600 cm-1 using the OPO laser. The typical laser output power and linewidth in this experiment are 200 mW and 5 cm-1, respectively. A mechanical shutter (Sigma-Koki, Japan) was used to control the irradiation time (with a typical value of 20 s). The spectral intensity at each wavelength was calculated as I=-ln (Ip/(∑If + Ip)), in which the intensities of parent ions and fragment ions were identified by Ip and If, respectively.Acknowledgment
Financial support from the National Natural Science Foundation of China (No. 21475065) is gratefully acknowledged.
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