Solid State Nuclear Magnetic Resonance 65 (2015) 2–11
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Fine refinement of solid state structure of racemic form of phospho-tyrosine employing NMR Crystallography approach Piotr Paluch a, Tomasz Pawlak a, Marcin Oszajca b, Wieslaw Lasocha b,c, Marek J. Potrzebowski a,n a
Polish Academy of Sciences, Centre of Molecular and Macromolecular Studies, Sienkiewicza 112, PL-90-363 Lodz, Poland Jerzy Haber Institute of Catalysis and Surface Chemistry, PAS, Niezapominajek 8, 30-239 Krakow, Poland c Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland b
art ic l e i nf o
a b s t r a c t
Article history: Received 2 June 2014 Accepted 29 August 2014 Available online 8 September 2014
We present step by step facets important in NMR Crystallography strategy employing O-phospho-DLtyrosine as model sample. The significance of three major techniques being components of this approach: solid state NMR (SS NMR), X-ray diffraction of powdered sample (PXRD) and theoretical calculations (Gauge Invariant Projector Augmented Wave; GIPAW) is discussed. Each experimental technique provides different set of structural constraints. From the PXRD measurement the size of the unit cell, space group and roughly refined molecular structure are established. SS NMR provides information about content of crystallographic asymmetric unit, local geometry, molecular motion in the crystal lattice and hydrogen bonding pattern. GIPAW calculations are employed for validation of quality of elucidation and fine refinement of structure. Crystal and molecular structure of O-phospho-DLtyrosine solved by NMR Crystallography is deposited at Cambridge Crystallographic Data Center under number CCDC 1005924. & 2014 Elsevier Inc. All rights reserved.
Keywords: Powder X-ray diffraction Solid state NMR Very fast MAS 2D HETCOR correlations Inverse detection GIPAW calculations Hydrogen bonding Molecular motions
1. Introduction Protein phosphorylation is of prime importance for enzymatic and cellular regulation [1]. Its mechanism controls protein structure, dynamics, and biological functions and plays a crucial role in metabolic pathways, kinase cascade activation, membrane transport, and gene transcription. Phosphorylation and dephosphorylation involve replacement of the hydroxyl group by a phosphate residue and vice versa in numerous biologically active compounds such as carbohydrates, nucleic acids, and proteins [2]. O-Phosphorylated amino acids are often used as reference samples for testing of advanced analytical methodologies which can be further employed in structural studies of more complicated biological systems [3]. In many cases, NMR spectroscopy in the liquid and/or solid state is the first choice of researchers dealing with analysis of the structure of phosphorylated species on the molecular level. Particularly in this field, recent years have witnessed a number of the spectacular applications of solid state NMR (SS NMR) spectroscopy [4]. Usually, the starting point in developing SS NMR methodologies is correlation between well
n
Corresponding author. Fax: þ48 426847126. E-mail address: [email protected] (M.J. Potrzebowski).
http://dx.doi.org/10.1016/j.ssnmr.2014.08.002 0926-2040/& 2014 Elsevier Inc. All rights reserved.
defined X-ray structure of O-phosphorylated amino acids and structural constraints obtained by NMR measurements. With one exception the crystal and molecular structures of basic phosphorylated amino-acids (serine, threonine and tyrosine) in enantiomeric and racemic form refined by XRD of single crystals were deposited in the Cambridge Crystallographic Data Center (CCDC) [5]. Unfortunately to date the attempts to grow up the single crystals of DL Ophospho-tyrosine with quality suitable for X-ray measurements failed. In this work we present NMR Crystallography approach allowing fine refinement of crystal and molecular structure of racemic form of O-phospho-tyrosine by analysis of powder pattern. NMR Crystallography is an idea which combines analysis of X-ray diffraction (XRD) data, quantum mechanical calculations and NMR measurements [6]. The key technique in this strategy is SS NMR. SS NMR provides rich set of constraints, which are extremely useful in structural analysis of condensed matter [7]. First, spectra provide a ‘fingerprint’ of the local structure and represent the local chemical environment for each nucleus under investigation. NMR responds to the short-range environment of relevant atoms and is not directly influenced by long-range order. Most important NMR parameter, chemical shift gives information about intermolecular interactions. Analysis of principal elements of chemical shift tensor δii provides detailed information about electronic
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Scheme 1. Molecular structure and numbering system for O-phospho-tyrosine.
distribution around each individual nucleus. Inter- and intramolecular hydrogen-bond linkages can be identified. Information on crystallographic asymmetric units is especially readily available, usually merely by counting lines. Polymorphs are usually easily distinguished. Phase transitions can be monitored. Crystallographic disorder is detectable, and distinctions between spatial and temporal disorder can be made. Measurement of dipolar coupling constants yields through-space inter-atomic (i.e. internuclear) distances, though these will be modulated by local mobility. Our experimental PXRD and NMR data are verified by theoretical calculations employing the CASTEP program [8,9]. In order to validate the correctness of refinement the comparative analysis of O-phospho-L-tyrosine 1 and DL racemic form 2 is done. The molecular structure and numbering system used in this work for describing of 1 and 2 is shown in Scheme 1.
2. Experimental 2.1. SSNMR measurements 1
H, 13C CP MAS, 1H–31P and 1H–13C, 1H–1H correlation experiments were carried on a Bruker Avance III 600 spectrometer equipped with 4 mm and 1.3 mm 1H/BB(31P–15N) CP-MAS probeheads with the 1H, 13C resonance frequencies of 600.13 and 150.90 MHz respectively. 1H–31P correlations were measured using Bruker Avance III 500 spectrometer equipped with 1.3 mm 1 H–19F/BB(31P–15N) CP-MAS probe-head with the 1H, 31P 500.13 and 202.46 MHz. In all proton detected experiments sample was spun at 60 kHz, for initial setup 1H X (X ¼ 13C, 31P) CP MAS experiment were run with contact time equal to 2 ms and RF on X channel equal to 160 kHz, for 1H we used ramp shape from 90% to 100% with RF near to 100 kHz. The experiment was carried out with ZQ-1 Hartman–Hahn matching condition. For setup we used 13 C, 15N uniformly labeled histidine hydrochloride or target sample. 1H X inverse detected HETCOR experiment was carried out employing sequence described by Pruski and coworkers [10]. In this case carefully optimization (initial parameters were get from 1 H X CP MAS experiment) of 1H RF during first and second CP transfer was done using target sample. First and second contact time was equal to 2 ms in case 1H–13C correlation. In case 1H–31P first contact time was set to 1 ms and second contact time was set to 50 ms. In the 1H-1H Back-to-Back experiment DQ excitation as well as reconversion time was 33.3 ms. Slow spinning speed (12 kHz and 4 kHz) 13C and 31P CP MAS spectra were measured with 50–100% ramp on 1H channel. Anisotropic parameters (δ11, δ22, δ33) were determined using TopSpin 3.0 software. 2.2. XRD of powdered sample of phospho-DL-tyrosine 2 Powder diffraction experiments were performed using a PANalytical X'Pert PRO MPD powder diffractometer (240 mm goniometer radius) equipped with a sealed copper tube, an elliptic X-ray focusing mirror and a PIXCEL position sensitive detector. Divergence slit of 1/21 and 0.02 rad. Soller slits (in both incident and diffracted beampaths) were applied. Generator settings used
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during the experiment were 40 kV and 30 mA providing an intense incident beam. Powder sample was packed inside a borosilicate glass capillary (0.5 mm diameter) mounted on a goniometric head and rotated (to reduce preferred orientation effects) during a four scan measurement. The registered data range was 3–851 2θ with a step of 0.021 with 3.5 h per scan. Obtained scans were tested for discrepancies between them and summed since no significant difference was observed. 2.3. GIPAW calculations for phospho-L-tyrosine and phospho-DLtyrosine The quantum chemical DFT calculations in periodic boundary conditions were performed using the CASTEP code [8,9,11]. The geometry optimization was performed using the X-ray diffraction crystal structures of phospho-L-tyrosine as an input file [12]. If we take into consideration phospho-DL-tyrosine, the structure was calculated de novo (base on our XRD solution). The generalized density approximation DFT functional PBE [13] was applied. A comparison of the average forces remaining on the atoms after geometry optimization was carried out for proton-only and allatom optimizations by using a maximum plane wave cutoff energy of 550 eV and ultrasoft pseudopotential [14]. The unit cell parameters were taken from the X-ray structures and kept fixed during the optimization of the geometry of the structures, and a Monkhorst–Pack grid [15] was used to sample the Brillouin zone. The NMR chemical shifts were computed using the GIPAW method [16]. When calculating the full crystal structure, a planewave basis set with a maximum cutoff energy of 550 eV was used. Finally, we obtained NMR chemical-shielding values in periodic boundary conditions using two approaches: (i) optimized with only the hydrogen atoms allowed to relax and (ii) optimized so that all atoms were allowed to relax. In all cases, the optimization algorithm was BFSG [17] with line search. All numerical data are presented in the Supplementary information.
3. Results and discussion 3.1. Structural constraints from solid state NMR. Comparative Analysis of phospho-L-tyrosine 1 and phospho-DL-tyrosine 2 3.1.1. 1D 1H, 13C and 31P solid state NMR The important issue in crystal structure prediction for each material employing the NMR Crystallography approach is collecting the possibly large set of NMR constraints which can be further used for validation of refinement. Usually, the starting point in structural analysis is an inspection of content of crystallographic asymmetric units by counting the number of isotropic NMR signals. The comparative analysis of 31P CP/MAS spectra provides straightforward evidence confirming distinction for 1 and 2 (Fig. 1a and b). In the case of 1 we have two resonances which reflect presence of two molecules (A and B) in the asymmetric unit while for 2 only single line is recorded. This conclusion is further supported by inspection of 13 C CP/MAS spectra shown in Fig. 1c and d. Such picture is consistent with single crystal X-ray data for 1. Its structure has been reported by Suga and co-workers [12]. Fig. 2 shows the crystal and molecular packing of sample 1. The size of the unit cell is given in the legend of the Figure. Compound 1 crystallizes in the monoclinic space group P21 with two conformers present in the asymmetric unit cell. A and B molecules are aligned in a head-to-tail orientation (Fig. 2b) It is known, that the 1H isotropic chemical shift is very sensitive to the local electronic environment and provides valuable constraints that can be further used for structure prediction [18].
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Fig. 1. 1D 13C and 31P spectra of O-phospho-L-tyrosine 1 and O-phospho-DL-tyrosine 2. Upper spectra (a and c) represent sample 1 while bottom (b and d) sample 2. Spectra recorded with spinning rate 12 kHz.
Fig. 2. (a) Unit cell of O-phospho-L-tyrosine with labeled size (A ¼ 11.4800 Å, B ¼8.1140 Å, C¼ 11.8820 Å) and (b) alignment of two nonequivalent molecules in unit cell. Coordinates are taken from Ref. [12].
Fig. 3. 1D 1H spectra of O-phospho-L-tyrosine 1 and O-phospho-DL-tyrosine 2. Spectra recorded with spinning rate 60 kHz. Acidic protons are labeled.
Unfortunately, assignment of the 1H resonances and quantitative analysis of spectra using SS NMR spectroscopy is still very challenging due to extremely strong homonuclear dipolar couplings, which in many cases exceed the range of chemical shifts for protons. For true solids, the broadening of proton lines is not removed by slow or medium magic angle spinning without the application of complex pulse sequences [19].
1
H measurements of L and DL-phospho-tyrosine recorded with rotation of 32 kHz were reported elsewhere [20]. Under spinning regime greater than 50 kHz, obtained using commercially available 1.3-mm rotors, the spinning frequency exceeds the strength of homonuclear proton dipolar coupling and is therefore expected to enter a new regime for spin dynamics. Fig. 3 shows the proton spectra of 1 (a) and 2 (b) recorded with
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Fig. 4. (a) 1H–13C HETCOR and (b) 1H–31P HETCOR spectra for O-phospho-L-tyrosine 1. (c) 1H–13C HETCOR and (d) 1H–31P HETCOR spectra for DL-phospho-L-tyrosine 2. All spectra were recorded with 60 kHz spinning rate employing inverse detection mode. Measuring times was 40 h (a), 1 h (b), 18 h (c) and 1 h (d). SWFTPPM decoupling with RF-field equal to 10 kHz was used during 13C and 31P chemical shift evolution period.
Fig. 5. 600 MHz 1H–1H Back-to-Back (BaBa) correlations for 1 (a) and 2 (b). Spectra were recorded with spinning rate 60 kHz. DQ excitation as well as reconversion time was 33.3 ms.
spinning rate 60 kHz. Compared to the previous data [20] the resolution of spectra is dramatically improved. In particular acidic protons (carboxylic and phosphoric) observed in typical region of 9–16 ppm are very well resolved and first time seen as clearly separated resonances. On the other hand for such spectra
the assignment of signals on the base of 1D 1H VF MAS measurement is ambiguous. In the case of sample 1 the crucial question regards the assignment of proton signals to A and B molecules. For sample 2 distinguishing of proton signals for C(O) OH and O–P(O)OH is not trivial.
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3.1.2. 2D 1H–13C, 1H–31P HETCOR and 1H–1H HOMCOR NMR correlations The ambiguities in structure assignment for 1 and 2 can be solved by application of 2D heteronuclear correlated (HETCOR) NMR spectroscopy. As shown in previous section, spinning rate of sample above 40 kHz gives opportunity to obtain proton spectra with good resolution. Therefore, it is not necessary to apply complex homo-decoupling sequences to reduce 1H line width [21–25]. The other important point is possibility to carry out HETCOR correlation employing inverse detection techniques (inv-HETCOR) what greatly improve S/N ratio and reduce the time Table 1 Experimental 31P values of the principal tensor elements (δii) for O-phospho-Ltyrosine 1 and O-phospho-DL-tyrosine 2. 31P δii data are taken from Ref. [20]. O-phosphorylated amino acid
δiso
δ11
δ22
δ33
Ωa
κb
1a 1b 2
5.9 4.4 3.3
61 66 60
3 7 7
82 86 63
143 152 123
0.19 0.22 0.09
a b
Span is expressed as Ω¼ δ11 δ33. Skew is expressed as κ¼ (δ22 δiso)/Ω.
Fig. 6. Simulated static experimental 31P CSA line shape for O-phospho-L-tyrosine 1 (molecule ‘A’ – blue and molecule ‘B’ – green) and for O-phospho-DL-tyrosine 2 (red). Spectra were computed with 31P δii parameters collected in Table 1. Experimental static spectra for samples 1 and 2 are given in the Supplementary information. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
of measurement. Inv-HETCOR correlation can be done employing two schemes; with typical solid state CP transfer (giving spectra via dipolar interactions) or using INEPT type transfer (giving spectra via J-coupling) [26]. These two approaches provide spectra containing information similar to that obtained employing HMBC and HSQC correlations in the liquid phase. In our work, in order to obtain long range 1H–13C and 1H–31P correlations we applied former approach. Fig. 4 shows appropriate 2D inv-HETCOR correlations for 1 (a and b) and for 2 (c and d). By inspection the cross peaks for all 2D spectra the distinguishing between carboxylic and phosphoric protons is straightforward. The F1 and F2 projections shown assignment of signals according to numbering system given in Scheme 1. Comparative analysis of 1H–31P correlations is in particular interesting. For racemic phosphotyrosine (Fig. 4d) we observe dominating cross-peak reflecting P–OH interaction and weaker signals representing P¼O⋯HO–C ¼ O and P ¼O⋯H3N contacts. For L-form (Fig. 4b) we see P–OH correlations and strong contacts between phosphorus and NH3 protons. Furthermore, weak interactions between phosphorus centers and protons of C (O)OH residues are apparent. Such results clearly prove that for samples 1 and 2 we have slightly different hydrogen bonding network in the crystal lattices. In order to deeply understand the inter-molecular contacts in the crystal lattice for 1 and 2 in the next step we have carried out homo-nuclear 1H–1H DQ 2D measurements. Such experiment allowed us searching of long range contacts and hydrogen bonding pattern for both samples. Fig. 5 presents the 2D Back-to-Back (BaBa) correlations for 1 (a) and for 2 (b). From analysis of correlation peaks the interactions between acidic protons and NH3 group can be concluded for both samples. We assume that amine residue plays a role of donor in the hydrogen bonding interacting with C ¼O and/or P¼ O acceptors. However, it has to be stressed that BABA correlations do not provide straightforward information supporting this hypothesis. On the other hand keeping in mind the cross-peak pattern for HETCOR correlations (Fig. 4) we can presume that for sample 2 amine protons are closer to C ¼O than P ¼O residue. It means that strength of P¼O⋯H3N hydrogen bonding for 2 is much lower compared to 1. It is worthy to note the significant difference in 1H chemical shifts of P-OH protons for 1 and 2 (ca. 5 ppm). The P-OH
Fig. 7. CP–VC experiment after FT for 1 (a) and 2 (b). Spectra are recorded with spinning rate 60 kHz, and RF for 1H ¼100 kHz, and
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C¼ 160 kHz.
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signals of 2 are downfield shifted with respect to 1 what suggests that phosphoryl residue of 2 is involved in stronger O–H⋯O interactions. The strength of C–O–H⋯O interactions in crystal lattices of 1 and 2 is comparable. Finally, relatively weak crosspeaks between P–OH and C–OH protons rather exclude formation of hydrogen bonded hetero dimer as reported for DL-phosphothreonine [27].
3.1.3. Analysis of 31P chemical shift tensors 31 P CST for phosphorylated amino acids have been investigated by several research groups [28,29]. It has been found that CST parameters give precise information about the ionization state of phosphoryl residues and their contribution into hydrogen bonding. The CST values for 1 and 2 obtained by analysis of spinning sidebands pattern under slow rotation were reported elsewhere [20]. The values of principal elements of 31P δii are collected in Table 1. Fig. 6 shows the calculated line shape of static spectra computed with values given in Table 1. At the first glance it is apparent that line shape for 1 and 2 is different. We assume that this distinction is rather due to the differences in the strength of hydrogen bonds than in the ionization state. As proved by Gajda et al. for phosphoryl group the strong hydrogen bond reduces the chemical shift anisotropy (CSA) expressed by span parameter Ω ¼ δ11 δ33 [27]. From comparative analysis of span for 1 and 2 it is seen that Ω for former compound is larger ca. 20–30 ppm. The hydrogen bonding has also influence on values of 31P δ22 parameters shifting them in direction of higher field. This effect is also seen in Fig. 6. Given supra discussion for 31P NMR is consistent with 1H results showing different P–OH hydrogen bonding for 1 and 2.
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3.1.4. Local molecular motions for 1 and 2 It is well known that in the solid state, local molecular dynamics will average NMR tensor parameters such as the CSA, dipolar interactions and quadrupolar interactions [30,31]. The quantum chemical calculations are typically performed using static structures, i.e., at 0 K where zero-point motion is neglected. As shown in recent papers, molecular dynamics can lead to significant discrepancies between computed and experimental data [32]. Solid state NMR offers number of tools allowing detailed understanding of the nature of the molecular motions in condensed matter. Parameters such as 13C and 1H spin-lattice relaxation times (13C T1 and 1H T1), carbon and proton rotating frame relaxation times (13C T1ρ and 1H T1ρ), the C–H cross-relaxation constant (TC–H) and the proton relaxation time in the dipolar state (T1D) exhibit substantial utility for elucidating local dynamics. Another group of techniques is based on the analysis of dipolar interactions. The partial averaging of C–H dipolar couplings gives either geometric information or the amplitude of the motional processes in the solid state. In a recent paper we have shown that a simple cross polarization variable contact (CPVC) experiment performed under fast MAS conditions provides very accurate values of hetero-nuclear (1H–13C, 1H–15N) dipolar couplings, D, which reflect the interatomic distances and/or molecular motions [33]. We have also shown that, as long as only the dipolar peak separations, Δ ¼ D/√2, are used, CPVC methods are very robust with respect to rfinhomogeneity and to offsets. Fig. 7 displays the 2D CPVC spectra for 1 and 2 recorded with spinning rate 60 kHz. For both samples the splitting between
Fig. 8. The correlations of experimental isotropic chemical shift values (δiso) and calculated nuclear shielding values (σiso) of 1H (a and c) and 13C (b and d) for O-phospho-Ltyrosine (a and b) and O-phospho-DL-tyrosine (c and d).
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singularities in aromatic and α-carbon region is found to be ca. 15.4 kHz. These values unambiguously prove that residues under discussion are rigid and do not undergo local molecular motion in the crystal lattice. The spectral pattern for CH2 groups is slightly different, typical for three spin system. However, splitting values also exclude the possibility of local motion of methine carbons. Thus we can conclude that dynamic molecular disorder will not affect the anisotropic parameters making interpretation of computed data straightforward.
obtained R values. Calculated bond length precision in the refined structure model is not too high, which is caused by a rapid decrease in the diffracted intensities with diffraction angle. This precision is still sufficient for a starting model for further quantum-mechanical optimization. The summary of the calculation of the Rietveld refinements is given in the Supplementary information. 3.3. GIPAW calculations for phospho-L-tyrosine 1 and phospho-DLtyrosine 2
3.2. XRD study of powdered sample of O-phospho-DL-tyrosine 2 Experimental diffraction data was indexed in a monoclinic cell applying the successive dichotomy method as implemented in DICVOL04 [34]. Obtained cell parameters accounted for all but two impurity lines among the observed diffraction peaks. Despite numerous attempts at finding an alternative indexing, which would include these maxima, no better solution was detected and the first result was accepted for further stages of structure solution. Based on the systematic absences analysis, space group P21/c was selected and used from this point. Global optimization technique was utilized, as implemented in FOX [35] using a phosphotyrosine molecule model. Multiple runs of calculations were performed with 4,000,000 trials per run and the best obtained solution was selected for refinement. Rietveld method implemented in Jana2006 [36] was used in the refinement step. Final structure model gave a satisfactory fit to the measured diffraction data which is reflected in the difference curve and the
Many methods are currently available for computing of NMR data. Today, in theoretical predictions of parameters for solid materials the gauge-including projector augmented wave (GIPAW) method pioneered by Pickard et al. and Umari et al. represents a landmark development [37]. In our work, the GIPAW approach was employed for computing of NMR shielding for samples 1 and 2. We started calculations with sample 1 for which single crystal X-ray data were deposited [12]. These coordinates, with preserved position of heavy atoms and optimized geometry of hydrogen were taken as an input file for computing. Refinement was tested by observing of average forces AF (given as Cartesian components). For hydrogen and heavy atoms of 1 the AF were found to be 0.010 eV/Å and 0.480 eV/Å, respectively. Such value for heavy atoms suggests that proton-only optimized structures is not the proper model for further computation. For fully optimized structure of 1 the AF are smaller and with similar magnitude for all atomic species, ca. 0.008 eV/Å (protons) and 0.007 eV/Å (heavy
Fig. 9. The upper plots show the correlation of experimental chemical shift tensor values (δii) and calculated nuclear shielding parameters (σii); the bottom plots represent correlations between the experimental span (Ωexp) and calculated span values (Ωcalc). Solid lines indicate ideal correlations with a slope equal to 1. The correlations are shown for sample 1 (a and c) and 2 (b and d). The experimental 13C δii CST values for 1 and 2 are taken from Ref. [19].
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atoms). It clearly indicates that these structures are much closer to optimal geometry. In similar way sample 2 was tested. In the first approach, coordinates defined by XRD of powdered sample were taken as an input file. The AF value for heavy atoms was found to be 0.920 eV/ Å. This result, two times higher compared to sample 1, clearly prove that PXRD offers only coarse structure solution. After full optimization of geometry of 2, AFs are in range of 0.007 eV/Å both for hydrogen and heavy atoms. In the next step the experimental and theoretical NMR parameters were compared. The GIPAW calculated 1H, 13C and 31P shielding parameters are given in the Supplementary information. Fig. 8 shows the correlations of 1H and 13C isotropic chemical shifts δiso versus isotropic shielding σiso for both molecules. The calculated 1H and 13C nuclear isotropic shieldings for optimized structures show excellent agreement with the experimental results (Fig. 8). As one can see the correlations are very good with high R2 values what confirm quality of GIPAW refinement. It is worthy to express that employing the theoretical approach we were in position to assign the NMR signals to A and B molecules of sample 1. It is known that more precise information about molecular structure of compounds compared to isotropic values, provides analysis of principal elements of shielding and chemical shift parameters. Fig. 9a and b shows the correlations of 13C σii versus 13 C δii for samples 1 and 2. As in case of plots shown in Fig. 8 very good agreement between experimental and theoretical data is apparent. The plot of the experimental and calculated span values (Ω ¼ δ11 δ33) for 1 and 2 is displayed in Fig. 9c and d. For an ideal correlation, the slope is equal to 1 (represented by solid line in figures). As we reported in our previous paper the scatter of experimental points reflects the local molecular motion of individual residues [38]. Lack of scattering seen in Fig. 9 (bottom plots) unambiguously proves that molecules 1 and 2 are rigid and do not undergo dynamic processes in the crystal lattice. This conclusion is consistent with results discussed in Section 3.1.4. With this knowledge, having full set of coordinates we are able to present most reliable molecular packing and molecule structure in the unit cell for sample 2 (Fig. 10). In the final part of this section we wish to discuss the problem of hydrogen bonding, analyzed via inspection of 31P σii parameters. As reported elsewhere these parameters are very sensitive to change in strength of bonds and ionization state [29,39]. The proper localization of hydrogen atoms in the crystal lattice is always the challenge, even for X-ray crystallography of monocrystals. The PXRD provides only coarse information for these
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interactions. For sample 2, our study we started with different localizations of hydrogen on the phosphoryl residues and computing NMR parameters after minimization of energy system. On the course of these calculations it was found that dimer with proton between carboxyl and phosphorus groups is the most energetically stable form. Fig. 11 shows how sensitive is 31P line shape on change of ionization state. When two hydrogen atoms are bonded to phosphoryl residue (red line) the span value Ω is equal to 288 ppm. For monoanion with hydrogen located between phosphoryl and carboxyl group (green line) Ω is found to 138 ppm. This value very well correlates with experimental data. Table 2 shows the selected interatomic distances for atoms involved in the hydrogen bonding for samples 1 and 2. Fig. 12 displays environment of POOH groups. As seen phosphoryl group of 2 (enlarge III) is involved in more strong contacts than 1 (enlarge I and II). The nonequivalence of phospho-L-tyrosine molecules is due to difference in the hydrogen bonding. Molecule denoted as ‘a’ contributes in two hydrogen PO⋯H⋯OOC bonding, molecule ‘b’ is involved in one interaction of this type. This conclusions very well correlate with NMR data presented in Section 3.1.
Fig. 11. (a) Simulated static 31P CSA line shape for O-phospho-DL-tyrosine 2 for two different hydrogen patterns. Red line corresponds to model when hydrogen atoms are located on phosphoryl residues, green line is for monoanion with hydrogen located between phosphoryl and carboxyl group. Models are displayed in (b). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 2 Selected interatomic distances for (underline) atoms involved in hydrogen bonds. Distances are given in Angstroms.
Fig. 10. (a) Unit cell and molecular packing for O-phospho-DL-tyrosine 2 (A ¼10.9010 Å, B¼ 5.9342 Å, C¼ 16.8050 Å); b) molecule in the asymmetric unit. Structure obtained by NMR Crystallography (CCDC 1005924).
Molecule
Phospho-L-Tyr (a)
Phospho-L-Tyr (b)
Phospho-DL-Tyr
P–OH⋯O–P P–OH⋯OOC P–O⋯(H)OOC P–O⋯NH3 COO(H)⋯NH3
2.66 2.61 2.54 2.85 2.87
2.66 4 3.50 2.58 2.80 2.70
2.49 43.50 2.52 2.74 2.89
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Fig. 12. Plot of structure showing selected hydrogen bonding patterns for (a) O-phospho-L-tyrosine and (b) O-phospho-DL-tyrosine with enlarged three parts of systems and indicated selected hydrogen bonds.
4. Conclusions In this work we showed how NMR Crystallography approach can be used for fine refinement of crystal and molecular structure of compound for which it is not possible to grow up single crystals with quality suitable for X-ray Diffraction study. This paper has tutorial character and presents step by step facets important in NMR Crystallography strategy. The significance of three major techniques being components of this approach; SS NMR, PXRD, GIPAW is highlighted. Each experimental technique provides different set of structural constraints. From the PXRD measurement the size of the unit cell, space group and roughly refined molecular structure is established. SS NMR provides information about content of crystallographic asymmetric unit, local geometry, molecular motion in the crystal lattice and hydrogen bonding pattern. GIPAW calculations are employed for validation of quality of elucidation and fine refinement of structure. If all elements give the consistent picture the solved structure can be deposited in the crystallographic data bases. In the case of DL-phosphotyrosine 2 NMR Crystallography data are deposited in Cambridge Crystallographic Data Center under number (CCDC 1005924).
Acknowledgment The authors are grateful to the Polish National Science Centre (NCN) for financial support under Grant 2011/01/B/ST4/02637. The computational resources were partially provided by the Polish Infrastructure for Supporting Computational Science in the European Research Space (PL-GRID) and ACK CYFRONET AGH Grant no. MNiSW/IBM_BC_HS21/CBMMPAN/029/2011.
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ssnmr.2014.08. 002. References [1] (a) (b) (c) (d) (e)
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Contents lists available at ScienceDirect
Solid State Nuclear Magnetic Resonance journal homepage: www.elsevier.com/locate/ssnmr
Fine refinement of solid state structure of racemic form of phospho-tyrosine employing NMR Crystallography approach Piotr Paluch a, Tomasz Pawlak a, Marcin Oszajca b, Wieslaw Lasocha b,c, Marek J. Potrzebowski a,n a
Polish Academy of Sciences, Centre of Molecular and Macromolecular Studies, Sienkiewicza 112, PL-90-363 Lodz, Poland Jerzy Haber Institute of Catalysis and Surface Chemistry, PAS, Niezapominajek 8, 30-239 Krakow, Poland c Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland b
art ic l e i nf o
a b s t r a c t
Article history: Received 2 June 2014 Accepted 29 August 2014 Available online 8 September 2014
We present step by step facets important in NMR Crystallography strategy employing O-phospho-DLtyrosine as model sample. The significance of three major techniques being components of this approach: solid state NMR (SS NMR), X-ray diffraction of powdered sample (PXRD) and theoretical calculations (Gauge Invariant Projector Augmented Wave; GIPAW) is discussed. Each experimental technique provides different set of structural constraints. From the PXRD measurement the size of the unit cell, space group and roughly refined molecular structure are established. SS NMR provides information about content of crystallographic asymmetric unit, local geometry, molecular motion in the crystal lattice and hydrogen bonding pattern. GIPAW calculations are employed for validation of quality of elucidation and fine refinement of structure. Crystal and molecular structure of O-phospho-DLtyrosine solved by NMR Crystallography is deposited at Cambridge Crystallographic Data Center under number CCDC 1005924. & 2014 Elsevier Inc. All rights reserved.
Keywords: Powder X-ray diffraction Solid state NMR Very fast MAS 2D HETCOR correlations Inverse detection GIPAW calculations Hydrogen bonding Molecular motions
1. Introduction Protein phosphorylation is of prime importance for enzymatic and cellular regulation [1]. Its mechanism controls protein structure, dynamics, and biological functions and plays a crucial role in metabolic pathways, kinase cascade activation, membrane transport, and gene transcription. Phosphorylation and dephosphorylation involve replacement of the hydroxyl group by a phosphate residue and vice versa in numerous biologically active compounds such as carbohydrates, nucleic acids, and proteins [2]. O-Phosphorylated amino acids are often used as reference samples for testing of advanced analytical methodologies which can be further employed in structural studies of more complicated biological systems [3]. In many cases, NMR spectroscopy in the liquid and/or solid state is the first choice of researchers dealing with analysis of the structure of phosphorylated species on the molecular level. Particularly in this field, recent years have witnessed a number of the spectacular applications of solid state NMR (SS NMR) spectroscopy [4]. Usually, the starting point in developing SS NMR methodologies is correlation between well
n
Corresponding author. Fax: þ48 426847126. E-mail address: [email protected] (M.J. Potrzebowski).
http://dx.doi.org/10.1016/j.ssnmr.2014.08.002 0926-2040/& 2014 Elsevier Inc. All rights reserved.
defined X-ray structure of O-phosphorylated amino acids and structural constraints obtained by NMR measurements. With one exception the crystal and molecular structures of basic phosphorylated amino-acids (serine, threonine and tyrosine) in enantiomeric and racemic form refined by XRD of single crystals were deposited in the Cambridge Crystallographic Data Center (CCDC) [5]. Unfortunately to date the attempts to grow up the single crystals of DL Ophospho-tyrosine with quality suitable for X-ray measurements failed. In this work we present NMR Crystallography approach allowing fine refinement of crystal and molecular structure of racemic form of O-phospho-tyrosine by analysis of powder pattern. NMR Crystallography is an idea which combines analysis of X-ray diffraction (XRD) data, quantum mechanical calculations and NMR measurements [6]. The key technique in this strategy is SS NMR. SS NMR provides rich set of constraints, which are extremely useful in structural analysis of condensed matter [7]. First, spectra provide a ‘fingerprint’ of the local structure and represent the local chemical environment for each nucleus under investigation. NMR responds to the short-range environment of relevant atoms and is not directly influenced by long-range order. Most important NMR parameter, chemical shift gives information about intermolecular interactions. Analysis of principal elements of chemical shift tensor δii provides detailed information about electronic
P. Paluch et al. / Solid State Nuclear Magnetic Resonance 65 (2015) 2–11
Scheme 1. Molecular structure and numbering system for O-phospho-tyrosine.
distribution around each individual nucleus. Inter- and intramolecular hydrogen-bond linkages can be identified. Information on crystallographic asymmetric units is especially readily available, usually merely by counting lines. Polymorphs are usually easily distinguished. Phase transitions can be monitored. Crystallographic disorder is detectable, and distinctions between spatial and temporal disorder can be made. Measurement of dipolar coupling constants yields through-space inter-atomic (i.e. internuclear) distances, though these will be modulated by local mobility. Our experimental PXRD and NMR data are verified by theoretical calculations employing the CASTEP program [8,9]. In order to validate the correctness of refinement the comparative analysis of O-phospho-L-tyrosine 1 and DL racemic form 2 is done. The molecular structure and numbering system used in this work for describing of 1 and 2 is shown in Scheme 1.
2. Experimental 2.1. SSNMR measurements 1
H, 13C CP MAS, 1H–31P and 1H–13C, 1H–1H correlation experiments were carried on a Bruker Avance III 600 spectrometer equipped with 4 mm and 1.3 mm 1H/BB(31P–15N) CP-MAS probeheads with the 1H, 13C resonance frequencies of 600.13 and 150.90 MHz respectively. 1H–31P correlations were measured using Bruker Avance III 500 spectrometer equipped with 1.3 mm 1 H–19F/BB(31P–15N) CP-MAS probe-head with the 1H, 31P 500.13 and 202.46 MHz. In all proton detected experiments sample was spun at 60 kHz, for initial setup 1H X (X ¼ 13C, 31P) CP MAS experiment were run with contact time equal to 2 ms and RF on X channel equal to 160 kHz, for 1H we used ramp shape from 90% to 100% with RF near to 100 kHz. The experiment was carried out with ZQ-1 Hartman–Hahn matching condition. For setup we used 13 C, 15N uniformly labeled histidine hydrochloride or target sample. 1H X inverse detected HETCOR experiment was carried out employing sequence described by Pruski and coworkers [10]. In this case carefully optimization (initial parameters were get from 1 H X CP MAS experiment) of 1H RF during first and second CP transfer was done using target sample. First and second contact time was equal to 2 ms in case 1H–13C correlation. In case 1H–31P first contact time was set to 1 ms and second contact time was set to 50 ms. In the 1H-1H Back-to-Back experiment DQ excitation as well as reconversion time was 33.3 ms. Slow spinning speed (12 kHz and 4 kHz) 13C and 31P CP MAS spectra were measured with 50–100% ramp on 1H channel. Anisotropic parameters (δ11, δ22, δ33) were determined using TopSpin 3.0 software. 2.2. XRD of powdered sample of phospho-DL-tyrosine 2 Powder diffraction experiments were performed using a PANalytical X'Pert PRO MPD powder diffractometer (240 mm goniometer radius) equipped with a sealed copper tube, an elliptic X-ray focusing mirror and a PIXCEL position sensitive detector. Divergence slit of 1/21 and 0.02 rad. Soller slits (in both incident and diffracted beampaths) were applied. Generator settings used
3
during the experiment were 40 kV and 30 mA providing an intense incident beam. Powder sample was packed inside a borosilicate glass capillary (0.5 mm diameter) mounted on a goniometric head and rotated (to reduce preferred orientation effects) during a four scan measurement. The registered data range was 3–851 2θ with a step of 0.021 with 3.5 h per scan. Obtained scans were tested for discrepancies between them and summed since no significant difference was observed. 2.3. GIPAW calculations for phospho-L-tyrosine and phospho-DLtyrosine The quantum chemical DFT calculations in periodic boundary conditions were performed using the CASTEP code [8,9,11]. The geometry optimization was performed using the X-ray diffraction crystal structures of phospho-L-tyrosine as an input file [12]. If we take into consideration phospho-DL-tyrosine, the structure was calculated de novo (base on our XRD solution). The generalized density approximation DFT functional PBE [13] was applied. A comparison of the average forces remaining on the atoms after geometry optimization was carried out for proton-only and allatom optimizations by using a maximum plane wave cutoff energy of 550 eV and ultrasoft pseudopotential [14]. The unit cell parameters were taken from the X-ray structures and kept fixed during the optimization of the geometry of the structures, and a Monkhorst–Pack grid [15] was used to sample the Brillouin zone. The NMR chemical shifts were computed using the GIPAW method [16]. When calculating the full crystal structure, a planewave basis set with a maximum cutoff energy of 550 eV was used. Finally, we obtained NMR chemical-shielding values in periodic boundary conditions using two approaches: (i) optimized with only the hydrogen atoms allowed to relax and (ii) optimized so that all atoms were allowed to relax. In all cases, the optimization algorithm was BFSG [17] with line search. All numerical data are presented in the Supplementary information.
3. Results and discussion 3.1. Structural constraints from solid state NMR. Comparative Analysis of phospho-L-tyrosine 1 and phospho-DL-tyrosine 2 3.1.1. 1D 1H, 13C and 31P solid state NMR The important issue in crystal structure prediction for each material employing the NMR Crystallography approach is collecting the possibly large set of NMR constraints which can be further used for validation of refinement. Usually, the starting point in structural analysis is an inspection of content of crystallographic asymmetric units by counting the number of isotropic NMR signals. The comparative analysis of 31P CP/MAS spectra provides straightforward evidence confirming distinction for 1 and 2 (Fig. 1a and b). In the case of 1 we have two resonances which reflect presence of two molecules (A and B) in the asymmetric unit while for 2 only single line is recorded. This conclusion is further supported by inspection of 13 C CP/MAS spectra shown in Fig. 1c and d. Such picture is consistent with single crystal X-ray data for 1. Its structure has been reported by Suga and co-workers [12]. Fig. 2 shows the crystal and molecular packing of sample 1. The size of the unit cell is given in the legend of the Figure. Compound 1 crystallizes in the monoclinic space group P21 with two conformers present in the asymmetric unit cell. A and B molecules are aligned in a head-to-tail orientation (Fig. 2b) It is known, that the 1H isotropic chemical shift is very sensitive to the local electronic environment and provides valuable constraints that can be further used for structure prediction [18].
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Fig. 1. 1D 13C and 31P spectra of O-phospho-L-tyrosine 1 and O-phospho-DL-tyrosine 2. Upper spectra (a and c) represent sample 1 while bottom (b and d) sample 2. Spectra recorded with spinning rate 12 kHz.
Fig. 2. (a) Unit cell of O-phospho-L-tyrosine with labeled size (A ¼ 11.4800 Å, B ¼8.1140 Å, C¼ 11.8820 Å) and (b) alignment of two nonequivalent molecules in unit cell. Coordinates are taken from Ref. [12].
Fig. 3. 1D 1H spectra of O-phospho-L-tyrosine 1 and O-phospho-DL-tyrosine 2. Spectra recorded with spinning rate 60 kHz. Acidic protons are labeled.
Unfortunately, assignment of the 1H resonances and quantitative analysis of spectra using SS NMR spectroscopy is still very challenging due to extremely strong homonuclear dipolar couplings, which in many cases exceed the range of chemical shifts for protons. For true solids, the broadening of proton lines is not removed by slow or medium magic angle spinning without the application of complex pulse sequences [19].
1
H measurements of L and DL-phospho-tyrosine recorded with rotation of 32 kHz were reported elsewhere [20]. Under spinning regime greater than 50 kHz, obtained using commercially available 1.3-mm rotors, the spinning frequency exceeds the strength of homonuclear proton dipolar coupling and is therefore expected to enter a new regime for spin dynamics. Fig. 3 shows the proton spectra of 1 (a) and 2 (b) recorded with
P. Paluch et al. / Solid State Nuclear Magnetic Resonance 65 (2015) 2–11
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Fig. 4. (a) 1H–13C HETCOR and (b) 1H–31P HETCOR spectra for O-phospho-L-tyrosine 1. (c) 1H–13C HETCOR and (d) 1H–31P HETCOR spectra for DL-phospho-L-tyrosine 2. All spectra were recorded with 60 kHz spinning rate employing inverse detection mode. Measuring times was 40 h (a), 1 h (b), 18 h (c) and 1 h (d). SWFTPPM decoupling with RF-field equal to 10 kHz was used during 13C and 31P chemical shift evolution period.
Fig. 5. 600 MHz 1H–1H Back-to-Back (BaBa) correlations for 1 (a) and 2 (b). Spectra were recorded with spinning rate 60 kHz. DQ excitation as well as reconversion time was 33.3 ms.
spinning rate 60 kHz. Compared to the previous data [20] the resolution of spectra is dramatically improved. In particular acidic protons (carboxylic and phosphoric) observed in typical region of 9–16 ppm are very well resolved and first time seen as clearly separated resonances. On the other hand for such spectra
the assignment of signals on the base of 1D 1H VF MAS measurement is ambiguous. In the case of sample 1 the crucial question regards the assignment of proton signals to A and B molecules. For sample 2 distinguishing of proton signals for C(O) OH and O–P(O)OH is not trivial.
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3.1.2. 2D 1H–13C, 1H–31P HETCOR and 1H–1H HOMCOR NMR correlations The ambiguities in structure assignment for 1 and 2 can be solved by application of 2D heteronuclear correlated (HETCOR) NMR spectroscopy. As shown in previous section, spinning rate of sample above 40 kHz gives opportunity to obtain proton spectra with good resolution. Therefore, it is not necessary to apply complex homo-decoupling sequences to reduce 1H line width [21–25]. The other important point is possibility to carry out HETCOR correlation employing inverse detection techniques (inv-HETCOR) what greatly improve S/N ratio and reduce the time Table 1 Experimental 31P values of the principal tensor elements (δii) for O-phospho-Ltyrosine 1 and O-phospho-DL-tyrosine 2. 31P δii data are taken from Ref. [20]. O-phosphorylated amino acid
δiso
δ11
δ22
δ33
Ωa
κb
1a 1b 2
5.9 4.4 3.3
61 66 60
3 7 7
82 86 63
143 152 123
0.19 0.22 0.09
a b
Span is expressed as Ω¼ δ11 δ33. Skew is expressed as κ¼ (δ22 δiso)/Ω.
Fig. 6. Simulated static experimental 31P CSA line shape for O-phospho-L-tyrosine 1 (molecule ‘A’ – blue and molecule ‘B’ – green) and for O-phospho-DL-tyrosine 2 (red). Spectra were computed with 31P δii parameters collected in Table 1. Experimental static spectra for samples 1 and 2 are given in the Supplementary information. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
of measurement. Inv-HETCOR correlation can be done employing two schemes; with typical solid state CP transfer (giving spectra via dipolar interactions) or using INEPT type transfer (giving spectra via J-coupling) [26]. These two approaches provide spectra containing information similar to that obtained employing HMBC and HSQC correlations in the liquid phase. In our work, in order to obtain long range 1H–13C and 1H–31P correlations we applied former approach. Fig. 4 shows appropriate 2D inv-HETCOR correlations for 1 (a and b) and for 2 (c and d). By inspection the cross peaks for all 2D spectra the distinguishing between carboxylic and phosphoric protons is straightforward. The F1 and F2 projections shown assignment of signals according to numbering system given in Scheme 1. Comparative analysis of 1H–31P correlations is in particular interesting. For racemic phosphotyrosine (Fig. 4d) we observe dominating cross-peak reflecting P–OH interaction and weaker signals representing P¼O⋯HO–C ¼ O and P ¼O⋯H3N contacts. For L-form (Fig. 4b) we see P–OH correlations and strong contacts between phosphorus and NH3 protons. Furthermore, weak interactions between phosphorus centers and protons of C (O)OH residues are apparent. Such results clearly prove that for samples 1 and 2 we have slightly different hydrogen bonding network in the crystal lattices. In order to deeply understand the inter-molecular contacts in the crystal lattice for 1 and 2 in the next step we have carried out homo-nuclear 1H–1H DQ 2D measurements. Such experiment allowed us searching of long range contacts and hydrogen bonding pattern for both samples. Fig. 5 presents the 2D Back-to-Back (BaBa) correlations for 1 (a) and for 2 (b). From analysis of correlation peaks the interactions between acidic protons and NH3 group can be concluded for both samples. We assume that amine residue plays a role of donor in the hydrogen bonding interacting with C ¼O and/or P¼ O acceptors. However, it has to be stressed that BABA correlations do not provide straightforward information supporting this hypothesis. On the other hand keeping in mind the cross-peak pattern for HETCOR correlations (Fig. 4) we can presume that for sample 2 amine protons are closer to C ¼O than P ¼O residue. It means that strength of P¼O⋯H3N hydrogen bonding for 2 is much lower compared to 1. It is worthy to note the significant difference in 1H chemical shifts of P-OH protons for 1 and 2 (ca. 5 ppm). The P-OH
Fig. 7. CP–VC experiment after FT for 1 (a) and 2 (b). Spectra are recorded with spinning rate 60 kHz, and RF for 1H ¼100 kHz, and
13
C¼ 160 kHz.
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signals of 2 are downfield shifted with respect to 1 what suggests that phosphoryl residue of 2 is involved in stronger O–H⋯O interactions. The strength of C–O–H⋯O interactions in crystal lattices of 1 and 2 is comparable. Finally, relatively weak crosspeaks between P–OH and C–OH protons rather exclude formation of hydrogen bonded hetero dimer as reported for DL-phosphothreonine [27].
3.1.3. Analysis of 31P chemical shift tensors 31 P CST for phosphorylated amino acids have been investigated by several research groups [28,29]. It has been found that CST parameters give precise information about the ionization state of phosphoryl residues and their contribution into hydrogen bonding. The CST values for 1 and 2 obtained by analysis of spinning sidebands pattern under slow rotation were reported elsewhere [20]. The values of principal elements of 31P δii are collected in Table 1. Fig. 6 shows the calculated line shape of static spectra computed with values given in Table 1. At the first glance it is apparent that line shape for 1 and 2 is different. We assume that this distinction is rather due to the differences in the strength of hydrogen bonds than in the ionization state. As proved by Gajda et al. for phosphoryl group the strong hydrogen bond reduces the chemical shift anisotropy (CSA) expressed by span parameter Ω ¼ δ11 δ33 [27]. From comparative analysis of span for 1 and 2 it is seen that Ω for former compound is larger ca. 20–30 ppm. The hydrogen bonding has also influence on values of 31P δ22 parameters shifting them in direction of higher field. This effect is also seen in Fig. 6. Given supra discussion for 31P NMR is consistent with 1H results showing different P–OH hydrogen bonding for 1 and 2.
7
3.1.4. Local molecular motions for 1 and 2 It is well known that in the solid state, local molecular dynamics will average NMR tensor parameters such as the CSA, dipolar interactions and quadrupolar interactions [30,31]. The quantum chemical calculations are typically performed using static structures, i.e., at 0 K where zero-point motion is neglected. As shown in recent papers, molecular dynamics can lead to significant discrepancies between computed and experimental data [32]. Solid state NMR offers number of tools allowing detailed understanding of the nature of the molecular motions in condensed matter. Parameters such as 13C and 1H spin-lattice relaxation times (13C T1 and 1H T1), carbon and proton rotating frame relaxation times (13C T1ρ and 1H T1ρ), the C–H cross-relaxation constant (TC–H) and the proton relaxation time in the dipolar state (T1D) exhibit substantial utility for elucidating local dynamics. Another group of techniques is based on the analysis of dipolar interactions. The partial averaging of C–H dipolar couplings gives either geometric information or the amplitude of the motional processes in the solid state. In a recent paper we have shown that a simple cross polarization variable contact (CPVC) experiment performed under fast MAS conditions provides very accurate values of hetero-nuclear (1H–13C, 1H–15N) dipolar couplings, D, which reflect the interatomic distances and/or molecular motions [33]. We have also shown that, as long as only the dipolar peak separations, Δ ¼ D/√2, are used, CPVC methods are very robust with respect to rfinhomogeneity and to offsets. Fig. 7 displays the 2D CPVC spectra for 1 and 2 recorded with spinning rate 60 kHz. For both samples the splitting between
Fig. 8. The correlations of experimental isotropic chemical shift values (δiso) and calculated nuclear shielding values (σiso) of 1H (a and c) and 13C (b and d) for O-phospho-Ltyrosine (a and b) and O-phospho-DL-tyrosine (c and d).
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singularities in aromatic and α-carbon region is found to be ca. 15.4 kHz. These values unambiguously prove that residues under discussion are rigid and do not undergo local molecular motion in the crystal lattice. The spectral pattern for CH2 groups is slightly different, typical for three spin system. However, splitting values also exclude the possibility of local motion of methine carbons. Thus we can conclude that dynamic molecular disorder will not affect the anisotropic parameters making interpretation of computed data straightforward.
obtained R values. Calculated bond length precision in the refined structure model is not too high, which is caused by a rapid decrease in the diffracted intensities with diffraction angle. This precision is still sufficient for a starting model for further quantum-mechanical optimization. The summary of the calculation of the Rietveld refinements is given in the Supplementary information. 3.3. GIPAW calculations for phospho-L-tyrosine 1 and phospho-DLtyrosine 2
3.2. XRD study of powdered sample of O-phospho-DL-tyrosine 2 Experimental diffraction data was indexed in a monoclinic cell applying the successive dichotomy method as implemented in DICVOL04 [34]. Obtained cell parameters accounted for all but two impurity lines among the observed diffraction peaks. Despite numerous attempts at finding an alternative indexing, which would include these maxima, no better solution was detected and the first result was accepted for further stages of structure solution. Based on the systematic absences analysis, space group P21/c was selected and used from this point. Global optimization technique was utilized, as implemented in FOX [35] using a phosphotyrosine molecule model. Multiple runs of calculations were performed with 4,000,000 trials per run and the best obtained solution was selected for refinement. Rietveld method implemented in Jana2006 [36] was used in the refinement step. Final structure model gave a satisfactory fit to the measured diffraction data which is reflected in the difference curve and the
Many methods are currently available for computing of NMR data. Today, in theoretical predictions of parameters for solid materials the gauge-including projector augmented wave (GIPAW) method pioneered by Pickard et al. and Umari et al. represents a landmark development [37]. In our work, the GIPAW approach was employed for computing of NMR shielding for samples 1 and 2. We started calculations with sample 1 for which single crystal X-ray data were deposited [12]. These coordinates, with preserved position of heavy atoms and optimized geometry of hydrogen were taken as an input file for computing. Refinement was tested by observing of average forces AF (given as Cartesian components). For hydrogen and heavy atoms of 1 the AF were found to be 0.010 eV/Å and 0.480 eV/Å, respectively. Such value for heavy atoms suggests that proton-only optimized structures is not the proper model for further computation. For fully optimized structure of 1 the AF are smaller and with similar magnitude for all atomic species, ca. 0.008 eV/Å (protons) and 0.007 eV/Å (heavy
Fig. 9. The upper plots show the correlation of experimental chemical shift tensor values (δii) and calculated nuclear shielding parameters (σii); the bottom plots represent correlations between the experimental span (Ωexp) and calculated span values (Ωcalc). Solid lines indicate ideal correlations with a slope equal to 1. The correlations are shown for sample 1 (a and c) and 2 (b and d). The experimental 13C δii CST values for 1 and 2 are taken from Ref. [19].
P. Paluch et al. / Solid State Nuclear Magnetic Resonance 65 (2015) 2–11
atoms). It clearly indicates that these structures are much closer to optimal geometry. In similar way sample 2 was tested. In the first approach, coordinates defined by XRD of powdered sample were taken as an input file. The AF value for heavy atoms was found to be 0.920 eV/ Å. This result, two times higher compared to sample 1, clearly prove that PXRD offers only coarse structure solution. After full optimization of geometry of 2, AFs are in range of 0.007 eV/Å both for hydrogen and heavy atoms. In the next step the experimental and theoretical NMR parameters were compared. The GIPAW calculated 1H, 13C and 31P shielding parameters are given in the Supplementary information. Fig. 8 shows the correlations of 1H and 13C isotropic chemical shifts δiso versus isotropic shielding σiso for both molecules. The calculated 1H and 13C nuclear isotropic shieldings for optimized structures show excellent agreement with the experimental results (Fig. 8). As one can see the correlations are very good with high R2 values what confirm quality of GIPAW refinement. It is worthy to express that employing the theoretical approach we were in position to assign the NMR signals to A and B molecules of sample 1. It is known that more precise information about molecular structure of compounds compared to isotropic values, provides analysis of principal elements of shielding and chemical shift parameters. Fig. 9a and b shows the correlations of 13C σii versus 13 C δii for samples 1 and 2. As in case of plots shown in Fig. 8 very good agreement between experimental and theoretical data is apparent. The plot of the experimental and calculated span values (Ω ¼ δ11 δ33) for 1 and 2 is displayed in Fig. 9c and d. For an ideal correlation, the slope is equal to 1 (represented by solid line in figures). As we reported in our previous paper the scatter of experimental points reflects the local molecular motion of individual residues [38]. Lack of scattering seen in Fig. 9 (bottom plots) unambiguously proves that molecules 1 and 2 are rigid and do not undergo dynamic processes in the crystal lattice. This conclusion is consistent with results discussed in Section 3.1.4. With this knowledge, having full set of coordinates we are able to present most reliable molecular packing and molecule structure in the unit cell for sample 2 (Fig. 10). In the final part of this section we wish to discuss the problem of hydrogen bonding, analyzed via inspection of 31P σii parameters. As reported elsewhere these parameters are very sensitive to change in strength of bonds and ionization state [29,39]. The proper localization of hydrogen atoms in the crystal lattice is always the challenge, even for X-ray crystallography of monocrystals. The PXRD provides only coarse information for these
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interactions. For sample 2, our study we started with different localizations of hydrogen on the phosphoryl residues and computing NMR parameters after minimization of energy system. On the course of these calculations it was found that dimer with proton between carboxyl and phosphorus groups is the most energetically stable form. Fig. 11 shows how sensitive is 31P line shape on change of ionization state. When two hydrogen atoms are bonded to phosphoryl residue (red line) the span value Ω is equal to 288 ppm. For monoanion with hydrogen located between phosphoryl and carboxyl group (green line) Ω is found to 138 ppm. This value very well correlates with experimental data. Table 2 shows the selected interatomic distances for atoms involved in the hydrogen bonding for samples 1 and 2. Fig. 12 displays environment of POOH groups. As seen phosphoryl group of 2 (enlarge III) is involved in more strong contacts than 1 (enlarge I and II). The nonequivalence of phospho-L-tyrosine molecules is due to difference in the hydrogen bonding. Molecule denoted as ‘a’ contributes in two hydrogen PO⋯H⋯OOC bonding, molecule ‘b’ is involved in one interaction of this type. This conclusions very well correlate with NMR data presented in Section 3.1.
Fig. 11. (a) Simulated static 31P CSA line shape for O-phospho-DL-tyrosine 2 for two different hydrogen patterns. Red line corresponds to model when hydrogen atoms are located on phosphoryl residues, green line is for monoanion with hydrogen located between phosphoryl and carboxyl group. Models are displayed in (b). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 2 Selected interatomic distances for (underline) atoms involved in hydrogen bonds. Distances are given in Angstroms.
Fig. 10. (a) Unit cell and molecular packing for O-phospho-DL-tyrosine 2 (A ¼10.9010 Å, B¼ 5.9342 Å, C¼ 16.8050 Å); b) molecule in the asymmetric unit. Structure obtained by NMR Crystallography (CCDC 1005924).
Molecule
Phospho-L-Tyr (a)
Phospho-L-Tyr (b)
Phospho-DL-Tyr
P–OH⋯O–P P–OH⋯OOC P–O⋯(H)OOC P–O⋯NH3 COO(H)⋯NH3
2.66 2.61 2.54 2.85 2.87
2.66 4 3.50 2.58 2.80 2.70
2.49 43.50 2.52 2.74 2.89
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P. Paluch et al. / Solid State Nuclear Magnetic Resonance 65 (2015) 2–11
Fig. 12. Plot of structure showing selected hydrogen bonding patterns for (a) O-phospho-L-tyrosine and (b) O-phospho-DL-tyrosine with enlarged three parts of systems and indicated selected hydrogen bonds.
4. Conclusions In this work we showed how NMR Crystallography approach can be used for fine refinement of crystal and molecular structure of compound for which it is not possible to grow up single crystals with quality suitable for X-ray Diffraction study. This paper has tutorial character and presents step by step facets important in NMR Crystallography strategy. The significance of three major techniques being components of this approach; SS NMR, PXRD, GIPAW is highlighted. Each experimental technique provides different set of structural constraints. From the PXRD measurement the size of the unit cell, space group and roughly refined molecular structure is established. SS NMR provides information about content of crystallographic asymmetric unit, local geometry, molecular motion in the crystal lattice and hydrogen bonding pattern. GIPAW calculations are employed for validation of quality of elucidation and fine refinement of structure. If all elements give the consistent picture the solved structure can be deposited in the crystallographic data bases. In the case of DL-phosphotyrosine 2 NMR Crystallography data are deposited in Cambridge Crystallographic Data Center under number (CCDC 1005924).
Acknowledgment The authors are grateful to the Polish National Science Centre (NCN) for financial support under Grant 2011/01/B/ST4/02637. The computational resources were partially provided by the Polish Infrastructure for Supporting Computational Science in the European Research Space (PL-GRID) and ACK CYFRONET AGH Grant no. MNiSW/IBM_BC_HS21/CBMMPAN/029/2011.
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