Predicting the Crystal Structure of Decitabine by Powder NMR

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Predicting the Crystal Structure of Decitabine by Powder NMR Crystallography: Influence of Longrange Molecular Packing Symmetry on NMR Parameters Jiri Brus, Jiri Czernek, Libor Kobera, Martina Urbanova, Sabina Abbrent, and Michal Husak Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.6b01341 • Publication Date (Web): 24 Oct 2016 Downloaded from http://pubs.acs.org on October 26, 2016

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Crystal Growth & Design

Predicting the Crystal Structure of Decitabine by Powder NMR Crystallography: Influence of Long-range Molecular Packing Symmetry on NMR Parameters Jiri Brus,a* Jiri Czernek,a Libor Kobera,a Martina Urbanova,a Sabina Abbrent,a and Michal Husakb a

Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky sq. 2, 162 06 Prague 6, Czech Republic. b University of Chemistry and Technology, Prague, Department of Solid State Chemistry, Technicka 5, 166 28 Prague 6, Czech Republic. Supporting Information Placeholder ABSTRACT: Crystal structure determination in the absence of diffraction data still remains a challenge. In this contribution, we demonstrate a complete reconstruction of the crystal structure of decitabine exclusively from 1H and 13C solid-state NMR (ssNMR) chemical shifts through comparison with the NMR parameters calculated for DFT-optimized, computer-generated crystal structure predictions. In particular, we discuss the previously unconsidered influence of long-range molecular packing symmetry on the NMR parameters and subsequent selection of the correct crystal structure. Symmetry operations considerably influenced the global molecular packing and unit cell parameters of the predicted crystal structures, while the conformations and shortrange molecular arrangements were practically identical. Consequently, the NMR parameters calculated for NMR-consistent candidates were similar and barely distinguishable by the standard deviations of the experimental and calculated 1H and 13C chemical shifts. Therefore, to further refine the crystal structure selection, we simulated and analyzed the entire two-dimensional (2D) 1H13 C HETCOR and 1H-1H DQ/SQ NMR correlation spectra. By determining the covariance, which provides a quantitative measure of the differences between the experimental and calculated resonance frequencies of the correlation signals, the set of NMRconsistent candidates was additionally narrowed down, and the correct crystal structure was finally unambiguously identified. By applying the extended protocol including the comparative analysis of 2D ss-NMR correlation spectra, powder NMR crystallography can thus be used to describe the crystal structures differing in the long-range symmetry of molecular packing for which ss-NMR spectroscopy is otherwise less sensitive. 1. Introduction The DNA methyltransferase inhibitor, 5-aza-2'-deoxy-cytidine (decitabine, DAC, Scheme 1), is an efficient therapeutic for epigenetic cancer therapy.1-3 However, despite its efficacy, the therapeutic administration of DAC is limited by its hydrolytic lability, which causes a decrease in the plasma circulation time.4 To circumvent this problem, we recently developed a new biodegradable formulation based on a dispersion of the active compound in a crystalline matrix of poly(sebacic acid-co-1,4cyclohexanedicarboxylic acid).5 However, structural characterization of the resulting multicomponent system represents a great challenge because traditional diffraction techniques fail due to the overlap of weak reflections from DAC with much stronger diffraction patterns from the two crystal forms of the applied polymer matrix.5 To overcome this problem, we focused our attention

on powder NMR crystallography, which was recently proven to have remarkable potential.6-10 Scheme 1. Chemical Structure of DAC

4 2

6 5’ 4’

3’

1’ 2’

The term NMR crystallography11-20, usually refers to an approach combining X-ray diffraction data analysis with the measurement of NMR parameters in order to refine structures of mobile and partially disordered molecular systems21-23 or complex pharmaceutical solids24 and co-crystals;25,26 to probe local changes induced by dehydration, desolvation or other transformation processes;27-29 to analyze the role of hydrogen bonding30 and other non-covalent interactions;31-33 or to refine the structures of complex polycrystalline and macromolecular systems.34,35 A more specific concept of NMR crystallography developed by Emsley et al. 6-9 however, constitutes a unique protocol of the ab initio establishment of the crystal structure based on the combination of solid-state NMR (ss-NMR) spectroscopy, computer-generated crystal structure prediction (CSP) and density function theory (DFT) chemical shift calculations (Scheme 2). Experimentally, this approach is built on the measurement of 1H and 13C isotropic chemical shifts, parameters that are easily accessible with high accuracy using standard ss-NMR techniques, whereas X-ray diffraction data do not enter into the structure refining process at all. Moreover, as was recently demonstrated in a series of blind tests,36 developments in computational methods for CSP have resulted in the determination of stable phases of a wide range of organic solids. The key point of the powder NMR crystallography approach thus lies in the reliable selection of the correct crystal structure from a number of computer-generated trial coordinates. Recently, 1 H NMR isotropic chemical shifts were demonstrated to be sufficiently indicative for identification of the correct structures of organic crystalline compounds using a value of one standard deviation (referred to as ‘r.m.s.d.’ in the following text) between the experimentally determined and calculated chemical shifts.

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Typically, the correct structure is characterized by an r.m.s.d.(1H) smaller than 0.5 ppm.6-9 However, some precautions still need to be taken when applying this approach. Predominantly, explicit signal assignment is required. In addition, in some cases, hydrogen-bonded NH and OH protons must be excluded from the spectral analysis probably because of their unpredictable thermal receptivity.9 This powder NMR crystallography approach also failed for a low-molecular-weight compound, theophylline, probably because of its small number of structurally significant proton species.9 Furthermore, 13C chemical shifts were found to be less sensitive during the crystal structure determination. Nevertheless, the r.m.s.d.(13C) of the correct crystal structure was always smaller than 2.5 ppm, although other authors reported the r.m.s.d.(13C) to be below 1.0 ppm.37 Scheme 2. Schematic Representation of the Powder NMR Crystallography Methodology.6-9 ss-NMR (Solid-state NMR)

CSP (Crystal structure prediction)

Measurement of NMR parameters

Generation of CSPs

1

H and 13C NMR

Signal assignment 1

13

H- C HETCOR H-1H CRAMPS 13 13 C- C INADEQUATE 1

Polymorph Predictor

DFT calculations (Density functional theory) Computation of NMR parameters from CSP GIPAW

Structure selection Comparison of experimental isotropic chemical shifts and DFT calculations for predicted structures r.m.s.d. (1H), r.m.s.d. (13C)

It is thus clear that the widespread application of powder NMR crystallography still requires experimental verification, particularly because only a few examples have been studied to date. The importance of this requirement is further increased for DAC, a small compound in which the exchangeable NH and OH protons involved in hydrogen bonding amount to one-third of all proton species. Consequently, the main purpose of this study was to determine the threshold limits for the NMR parameters vital to the reliable determination of the complete crystal structure of DAC in the absence of X-ray diffraction data. In this regard, we focused on complementing the traditionally used r.m.s.d.’s of the linear regression of the predicted and measured isotropic chemical shifts by assessing the differences in the resonance frequencies of the correlation signals detected in two-dimensional (2D) 1H-13C HETCOR and 1H-1H double-quantum/single-quantum (DQ/SQ) NMR spectra. In this way, we expect a strengthened discrimination between candidates exhibiting similar local structural motifs, which should lead to the unambiguous identification of the correct crystal structure. 2. Experimental Section Materials. Powdered DAC polymorphic form I (DAC-I) was purchased from Sigma-Aldrich (Czech Republic). Before struc-

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tural characterization, the sample was recrystallized according to the patent literature.38 The reference crystal structure of DAC-I was determined through structure solution from laboratory-source X-ray powder diffraction (XRPD) data. Laboratory-source XRPD analysis. The sample of DAC-I was ground and placed into a 0.3 mm borosilicate glass capillary. Its diffraction pattern was measured at room temperature in transmission mode on a PANalytical Empyrean powder diffractometer from 3° to 80° 2θ with Cu Kα1,2 radiation (λ= 1.54184 Å, focusing mirror, step size of 0.013° 2θ). The Kα2 component of the recorded data was stripped out in X'Pert Hi Score Plus software.39 The final solution and refinement was performed in the DASH 3.2 software package.40 First, indexation was performed using DICVOL06 software. The intensity statistic indicated an orthorhombic crystal system; P212121 space group; and lattice parameters of a=5.6547(2), b=7.1471(4), c=24.935(2) Å. Structure solution in DASH software was performed by simulated annealing. The known geometry of DAC as found in DAC hydrate41 (CSD structure SOBBUG) was used as the starting fragment. The problem was easy, with 8 degrees of freedom, and all runs thus yielded the same solution. The structural refinement was performed in DASH software as well, and all bonds lengths and angles were fixed. The torsion, fragment position and global thermal parameter were refined. As the last step, the R-factor and lattice parameters were re-refined by Rietveld refinement with fixed molecular geometry in X'Pert Hi Score Plus software39 (Rp 5.9%, Rwp 10.2%). ss-NMR Spectroscopy. ss-NMR spectra were measured at 11.7 T on a Bruker Avance III HD 500 US/WB NMR spectrometer (Karlsruhe, Germany, 2013). The following measurements were taken: i) one-dimensional (1D) 1H MAS NMR experiments with and without DUMBO 1H homo-decoupling,42 ii) 13C CP/MAS and 13 C CPPI/MAS NMR experiments,43,44 iv) 2D 1H-13C FSLG HETCOR experiments,45 v) 2D NOESY-type 1H-1H spindiffusion experiments with DUMBO homo-decoupling in both detection periods, 46 and vi) 2D DQ/SQ 1H-1H MAS NMR experiment47 with an SPC5 DQ recoupling period48 and DUMBO homo-decoupling in both detection periods. Frictional heating49,50 of the spinning samples was compensated for by active cooling, and temperature calibrations were performed with Pb(NO3)2. For all experimental details, see the Supporting Information SI1. Conformer A

Conformer B

2

2

1’ 5’

4’

C2-N-C1’-O: -165.8° O-C4’-C5’-O: -72.8°

Conformer C

2

1’ 5’

4’

C2-N-C1’-O: -161.3° O-C4’-C5’-O: 176.9°

1’ 5’

4’ C2-N-C1’-O: 41.3° O-C4’-C5’-O: -66.8°

Figure 1. Initial DFT-optimized conformations of DAC used for the crystal structure predictions. Torsion angles accounting for the most significant conformation differences are listed below each structure. CSP. The crystal structures of DAC-I were predicted by global lattice energy minimizations. We generated 3 representative conformations, A, B, and C (Figure 1). Conformer A corresponded to the known conformation of DAC in the form of DAC hydrate41. From conformer A, conformers B and C were generated in Material Studio 4.4 by torsion angles modification.51 The conformers B and C represent significant conformational changes of DAC molecule. These changes are induced by the gauche-trans jump in the O-C5’-C4’-O segment (conformer B), and by the 180°rotation of


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the triazine ring along the C1’-N bond (conformer C). Other, less dramatic conformational changes in the molecule of decitabine were generated spontaneously during the procedure of crystal structure prediction. The geometry of all conformers was energyminimized by the Dmol3 DFT module of Materials Studio, and the atomic charges based on ESP fitting were calculated. Crystal structures were generated with the Polymorph Predictor module of the Materials Studio Package51 using Monte Carlo packing simulation to search for the global minimum and possible structure generation. The decitabine molecule is chiral and the crystal must be enantiomerically pure therefore the search was performed only in the most common Sohncke space groups not generating opposite enantiomer by symmetry operation (namely P1, P21, C2, and P212121). The Dreiding force field was used with the atomic charge values calculated by Dmol3 DFT in the previous step. The precise description of the Polymorph Predictor setup for structures generation can be found in supplementary materials (Supporting Information SI2). The resulting structures were lattice-energy minimized using the empirical Dreiding force field with ESP-fitted charges again. The default setting of the Polymorph Predictor program was used for the rest of parameters. In this way, three separate sets of CSPs representing three initial conformations were generated. To eliminate unrealistic crystal structures, only candidates featuring low relative crystal-lattice energies within 0-10 kJ.mol-1 of the global minimum were retained for further analysis. DFT Calculations. A total of 32 structures (12, 10, and 10 accordingly for “Conformers A”, “B”, and “C”; see Figure 1) were subjected to plane-wave (PW), DFT-based, structural refinement using the method described in references52-54 and implemented in the CASTEP 6.1 software package.54 Thus, the predicted unit cell parameters remained fixed, and all the internal coordinates were optimized with respect to the lattice energy estimated by the PBE55 DFT exchange-correlation functional. The obtained geometries are provided in the Supporting Information files and were the input for the NMR chemical shielding predictions, which were carried out by combining the PBE functional with the gaugeincluding projector augmented wave (GIPAW) method,56,57 as implemented in the CASTEP-NMR module.54 In all the above calculations (the geometrical optimizations followed by the calculations of the NMR parameters), the corresponding “Fine” level of the CASTEP settings was adopted. Additional computational details are provided in the Supporting Information SI3. This GIPAW-PBE PW DFT approach is very well established58,59 and was successfully applied in investigations of the structural and NMR spectroscopy characteristics of active pharmaceutical ingredients.60-63 The GIPAW-PBE chemical shielding data sets were evaluated as follows. Without any attempts at referencing the predicted NMR isotropic chemical shielding,64 the level of agreement between theory and experiment for a nucleus Q was quantified by the values of one r.m.s.d. (Q), as described in the Supporting Information SI4. In order to describe the similarity of the measured and predicted 2D spectra, the approach originally proposed in [ref.60] and validated in [ref.61] was adopted. In brief, this approach approximates the values of the theoretical chemical shifts, ε(X) and ε(Y), for nuclei X and Y and applies the two linear regressions to their measured counterparts, δ(X) and δ(Y), to arrive at the value of their covariance, sXY, quantifying the similarity between the 2D spectra (the description of this procedure is given in the Supporting Information SI4). 3. Results and Discussion Signal Assignment and Characteristic Structural Features. As previous investigations8,9 revealed that hydrogen atoms are the most vital species to reflect changes in the crystal structure of

organic solids, special precautions were taken when recording the 1 H NMR parameters. Specifically, the 1H NMR spectra were measured at various temperatures to probe the thermal receptivity of the hydrogen-bonded fragments. However, as the differences in the chemical shifts measured within a temperature range of 0-40 °C were ca. ± 0.05 ppm, the hydrogen-bonded structures in DACI can be said to be stable and thermally independent. However, this finding does not exclude existence of the temperature dependence of 1H chemical shifts at much lower temperatures. 1

1

H MAS and H DUMBO NMR Spectra 3H 2H 2H 1H 2H 1

1H

H MAS NMR

1H 1

25

20

15

10

5

0

-5

H DUMBO NMR

-10

ppm

Figure 2. 1H MAS (32 kHz) and DUMBO (10 kHz) NMR spectra of DAC-I with indicated signals intensities. The 1H NMR spectra were also measured under two experimental regimes. Whereas 1H DUMBO homo-decoupling experiment at 10 kHz provided considerably better spectral resolution, the single-pulse 1H MAS NMR spectrum measured at 32 kHz was used to verify the referencing of the 1H NMR chemical shifts and to refine the scaling factor of the DUMBO sequence (Figure 2). 1

13

1

1

H- C HETCOR and H- H SQ/DQ DUMBO NMR Correlation Spectroscopy H1’

a)

3'

4'

6,2 4 13

C=O

-C=

b)

H6

2'

13

1

C CPPI

CH

CH

CH

5 CH2

H-13C HETCOR (70 µs) C4‘/H4’

CH2

C5‘/H5’ C2‘/H2’

c)

1

H-13C HETCOR (220 µs)

C1’/H2’

OH/OH H6/H1’ H6/H6

NH2**/H6 NH2**/NH2*

20

ppm

C5’/OH C4/NH2*

1

C1’/H6

5 H6/H1’ NH2**/NH2*

10

NH2**/H6

C4/NH2**

150

d)

H-1H SQ/SQ DUMBO (50 µs)

C3’/OH

C6/H6

e)

H6/H2’ H6/H4’ NH2*/NH2*

C3’/H2’

H4’ H5’ H2’** H2’*

H1’/H2’

10

C1‘/H1’ H-C-H

H3’ H5’

H2’**/H2’* H-1H SQ/DQ DUMBO H4’/H2’ (2 loops) H3’/H2’

15

C3‘/H3’ C6/H6

OH

ppm NH2** NH2*

C

CH 1

5'

1'

100

ppm

12

10

8

6

4

2

ppm

Figure 3. Representative ss-NMR spectra of DAC-I: 1D 13C CP/MAS and CPPI/MAS NMR spectra (a); 2D 1H-13C FSLG HETCOR NMR spectra measured with a CP mixing time of 70 and 220 µs, (b) and (c), respectively; 2D 1H-1H SQ/SQ NMR spectrum measured with a 50 µs spin-diffusion period (d); and 2D 1 H-1H DQ/SQ DUMBO NMR spectrum measured with 2 excitation and recoupling loops (80 µs) (e). Subsequently, in combination with 13C CPPI/MAS NMR, the 2D 1H-13C FSLG HETCOR NMR spectra measured with short cross-polarization (CP) periods (50-70 µs) showed one-bondattached proton-carbon pairs (Figures 3a and 3b). Sufficient resolution in the indirect 1H dimension of these 2D spectra then revealed a clear nonequivalence of the CH2 protons of unit no. 5’ denoted in Scheme 1 (3.91 and 3.36 ppm), whereas the CH2 protons in group no. 2’ were almost equivalent, exhibiting a difference in isotropic shifts of only 1.96 and 1.83 ppm as determined



Relative Lattice Energies 35 30 25 20 15 10 5 0

Conformer C Conformer B

13

1

C and H Chemical Shift Standard Deviations (r.m.s.d.’s)

* *

Conformer A

Conformer B

* *

*

* *

Conformer C

2.0 ppm

13

C r.m.s.d., ppm

a)

*

*

*

*

*

0.5 ppm

1

H r.m.s.d., ppm

b)

c)

*

**

*

*

*

*

* 0.4 ppm

Conformer A

0

25

50 75 No. of CSPs

100

125

Distribution of Dihedral Angles

b)

Prediction of DAC Crystal Structures. Due to the rigidity of the DAC molecule, the CSP can be initiated from only three distinctly different conformations (Figure 1), although the general search for crystal structures would require more a sophisticated procedure.8 In spite of this restriction, however, the crystal structures predicted for each initial conformer exhibited significant differences in their relative lattice energies, which reached up to 25 kJ.mol-1 (Figure 4a). In addition, as demonstrated in Figure 4b, the distribution of several dihedral angles covered a relatively broad range as well. Notably, besides the nearly unrestricted relative orientations of the hydroxyl groups, the pentofuranosyl ring puckering, as represented by torsions N-C1’-C2’-C3’ and C1’-O-C4’-C3’, also varied considerably. Consequently, the generated crystal structures represent a number of the crystal packing arrangements sufficient for meaningful analysis, including a representative set of DAC conformations.

1

∆ E, kJ.mol

-1

a)

A, B and C. The ∆E values calculated for the crystal structures of initial conformers B and C were artificially increased by factors of +5 and +10 kJ.mol-1, respectively. (b) Distribution of selected dihedral angles in the DAC molecule determined for candidates featuring the lowest relative energy.

H r.m.s.d., ppm

by deconvolution of the 1H NMR signal extracted from the 2D 1 H-13C HETCOR spectrum. To assign the resonances of CH units no. 4’ and no. 1’, the 1H-13C FSLG HETCOR spectra were measured at longer CP times (150-300 µs, Figure 3c). This way, the medium-range coherence C1’/H2’ was clearly detected. Moreover, these coherences, generally representing C…H distances of ca. 2.1-2.6 Å, also evolved for the exchangeable NH and OH protons. The 1H doublet detected for the H2N-C4 unit at 10.81 and 9.38 ppm (in the 1H dimension) revealed magnetically nonequivalent NH2 protons, while the 1H resonances of the OH groups C3’OH and C5’-OH were found at nearly the same position (5.85 5.95 ppm). Explicit signal assignment is a necessary prerequisite for the successful crystal structure determination from NMR parameters, and thus any experimental technique allowing unambiguous signal assignment is welcomed. In this regard, recently developed 14N-1H ss-NMR correlation experiments showed extremely high potentiality.14,31 In the chase for the highest possible spectral resolution the 2D 1 H-1H correlation spectroscopy has already proven its high capability.65 In this regard, the 1H-1H DQ/SQ NMR correlation technique combined with DUMBO homo-decoupling yielded a particularly rich correlation pattern with a number of well-resolved signals, in comparison to the NOESY-type 1H-1H SQ/SQ NMR spectrum. Consequently, the recorded correlation spectra (Figures 3d and 3e) serve to not only refine the isotropic chemical shifts and signal assignments but also obtain valuable spatial data. Notably, using the SPC5 recoupling technique, the 1H-1H DQ coherences were generated with high efficiency over distances up to ca. 5 Å. These coherences thus provide information about the conformation as well as the molecular packing in the crystal unit. One of the most characteristic 1H-1H SQ/DQ coherences detected for DAC-I (Figure 3e) is represented by the autocorrelation signal H6/H6 at 8.3/16.6 ppm, showing specific medium- or longrange dipolar contact between these parts of the DAC molecules. Interestingly also, the exchangeable protons exhibit strong NH2/NH2 and OH/OH autocorrelation signals, whereas the correlations between the OH and NH2 groups are missing. This finding thus indicates preferential interactions between the proton species of the same type and the exclusive formation of N-H….N and OH…O hydrogen-bonding motifs.

Dihedral angle, deg.

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200 100

Figure 5. Comparison of the completely assigned experimental and calculated chemical shifts, including all carbon and hydrogen atoms represented as 13C and 1H r.m.s.d. (a) and (b), respectively. The 1H r.m.s.d. calculated after the exclusion of OH and NH species are shown in graph (c). Predicted structures are ordered by increasing relative lattice energy for each initial conformer. Horizontal lines represent the high limits of the r.m.s.d. considered as the values indicating the correct crystal structure.

0 -100 -200

Figure 4. (a) Relative lattice energies ∆E calculated with respect to the most stable prediction for all predicted structures as a function of the structure number. The relative energies were calculated separately for each group of CSPs derived from initial conformers

The crystal structure geometries of the most stable predictions featuring relative crystal-lattice energies within 0-10 kJ.mol-1 of the global minimum were subsequently DFT-optimized, and the corresponding 1H and 13C isotropic chemical shifts were calculated by employing the GIPAW-PBE approach. In this context, it is worthy to note that approximately half of known polymorph pairs are within 2 kJ.mol-1.66 The completely assigned experimental and calculated 13C NMR chemical shifts represented by the r.m.s.d.’s are compared in Figure 5a. The 1H r.m.s.d.’s shown in Figure 5b


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were calculated for all assigned protons, including exchangeable NH and OH species, whereas Figure 5c shows the 1H r.m.s.d.’s calculated after excluding the exchangeable protons from the set of 1H NMR isotropic chemical shifts. As indicated in Figure 5a, the r.m.s.d.’s higher than ca. > 3 ppm calculated for 13C chemical shifts clearly discriminate the predicted crystal structures that are inconsistent with the experimental data. Furthermore, it also follows from the comparison of structures exhibiting the lowest 13C r.m.s.d.’s that the most significant impact on the 13C NMR chemical shifts of DAC can be attributed to the rotation of the triazine ring along the C1’-N bond. The 180° flip of the triazine ring induces an increase in the 13C r.m.s.d.’s of ca. 4 ppm (C conformers). A slightly weaker but still systematic increase in r.m.s.d.(13C) was observed for the conformation variation in the methoxyl substituent. Specifically, an increase of ca. 12 ppm was found for predictions with a trans conformation of the O-C5’-C4’-O fragment (B conformers and prediction A03). Oppositely, all predictions characterized by 13C r.m.s.d.’s < 2 ppm adopted conformations that are consistent with the correct crystal structure, as was verified using XRPD data (discussed later). In contrast, no such systematic relationship was found between the local conformations and 1H r.m.s.d.’s (Figure 5b), where several predictions (6-7) with distinctively low r.m.s.d.(1H) values (< 0.65 ppm) were identified. However, by applying the previously defined criteria,8,9 including r.m.s.d.(1H) < 0.5 ppm and r.m.s.d. (13C) < 2.0 ppm, the predictions representing the inconsistent conformers B and C could be eliminated, leaving only two remaining candidates for the sought crystal structure (A01 and A04, Figure 5, red bars). Interestingly, by excluding the exchangeable NH and OH protons from the evaluation of the proton chemical shifts, the 1H r.m.s.d.’s of both these candidates decreased, with a slightly higher preference for prediction A04 (Table 1). However, such a decrease in the 1H r.m.s.d.’s was observed for all predictions, and for many of them, the resulting r.m.s.d.(1H) values dropped into the zone (< 0.4 ppm) that would otherwise indicate the correct crystal structure (Figure 5c). This fact thus signifies the vital position of the 1H NMR chemical shifts of the exchangeable NH and OH protons in the reliable selection of the correct crystal structures of DAC. The 1H NMR chemical shifts of the exchangeable protons should be taken into account; otherwise, artificial results may appear (Figure 5c, predictions marked by asterisks). This fact is in accord with the key role that hydrogen bonding possesses in the formation of crystal structures. Consequently it is clear, that a single parameter such as r.m.s.d.(1H) is not sufficient to unambiguously identify the correct crystal structure. Rather additional analysis need to be performed and a combination of more statistical parameters must be used.

Table 1. Comparison of Experimental and Calculated 1H and 13C NMR Parameters for the Two Most Preferred CSPs.

Similarities between the NMR-consistent candidates and comparison with XRPD reference data. As shown above in Figure 5, by combining r.m.s.d.’s of the 1H and 13C isotropic chemical shifts, two NMR-consistent candidates, A01 and A04, representing the crystal structure of DAC-I remained in the set of predicted crystal structures (Table 1). However, despite the comparable values of the statistical data (r.m.s.d.(1H) and r.m.s.d. (13C)), the corresponding unit cell parameters differed considerably (Table 2). To understand this apparent disagreement, we analyzed the crystal structures of both of these preferred predictions in detail and compared them to the reference structure of DAC determined by the direct refinement of XRPD data.

Table 2. Unit Cell Parameters Determined for the NMRConsistent CSPs and the XRPD-refined Reference Structure of DAC-I. Unit cell parameters A01 prediction

A04 prediction

XRPD reference

Cell length a, Å

13.35

25.63

24.93

Cell length b, Å

7.51

7.10

7.15

Cell length c, Å

5.46

5.68

5.65

Cell angle α

90°

90°

90°

Cell angle β

106.9°

90°

90°

Cell angle γ

90°

90°

90°

Space group

P21

P212121

P212121

Cell setting

monoclinic

orthorhombic

orthorhombic

Cell volume

546.73

1033.92

1006.92

PXRD similarity

0.951

0.979

--

r.m.s.d., Å

0.382

0.335

--

Prediction A01

Prediction A04

a)

C3’

C3’

b)

NMR similarity parameters A01 A04 r.m.s.d. (13C), ppm

1.79

1.97

1

r.m.s.d. ( H, all), ppm

0.50

0.47

r.m.s.d. (1H, OH/NH excl.), ppm

0.43

0.31

covariance for H- C pairs, ppm

0.74

0.36

2

0.17

0.12

1

13

1

1

covariance for H- H pairs, ppm

2

Figure 6. Comparison of the molecular conformations (a) and hydrogen-bonding motifs (b) of DAC molecules in the NMRconsistent CSPs, A01 and A04.



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As demonstrated in Figure 6a, the conformations of the DAC molecules in the NMR-consistent CSPs, A01 and A04, are basically identical, and distinguishable differences were found only in the relative orientations of the C3’-OH hydroxyl groups. Moreover, in both cases, the NH2 protons preferentially interact with the nitrogen atoms of the triazine rings, whereas the carbonyl oxygen atoms exclusively interact with the hydroxyl protons of the C5’OH and C3’-OH groups. Such arrangements are consistent with the experimental 1H-1H DQ/SQ NMR data. Consequently, in addition to the conformation, the same molecular packing motifs, in which the network of N-H…N hydrogen bonds is separated from the secondary network of O-H…O hydrogen bonds (Figure 6b), also exist in both predictions. As a result of these local similarities, the periodic arrangement of DAC molecules in the predicted unit cells A01 and A04 exhibits similar motifs as well (Figure 7). This fact was quantitatively described by the powder pattern similarity measure (PXRD similarity) and r.m.s.d. of the atomic positions calculated for each prediction relative to the reference XRPD structure (Table 2).

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Figure 8. Global molecular arrangements in CSPs A01 and A04 and the reference XRPD-refined crystal structure of DAC-I. The differences in the molecular arrangements demonstrated in Figure 8 are clearly reflected in the corresponding calculated XRPD patterns, and the reflections at 12.8 and 14.2° 2θ are sufficiently indicative to identify the correct CSP (Figure 9a). However, when comparing the NMR data, the corresponding differences between the simulated and experimental 1H and 13C NMR spectra (Figures 9b and 9c, respectively) were very subtle, and direct identification of the correct CSP was no longer clear. Consequently, in addition to the expected improvements in the DFT predictions of the involved parameters,58,59 the direct determination of the crystal structures from solely NMR data also requires new procedures for the robust comparison of experimental and predicted NMR data61. XRPD Patterns

a)

A04

Prediction A01

Prediction A04

A01

DAC Form I

Figure 7. Overlay of the unit cells of A01 and A04 predictions showing crystal structure fragments with nearly identical molecular arrangements.

b)

13

1

C NMR Spectra

H NMR Spectra

c)

A04

Long-range arrangement. Despite the range of similarities in the local structural features and nearly the same NMR statistical parameters, even simple visual inspection revealed considerable differences between the global arrangement of DAC molecules in the predicted crystal structures A01 and A04 (Figure 8). In the predicted structure A01, all DAC molecules are aligned parallel with respect to the orientation of the triazine ring, whereas in the crystal unit of the A04 prediction, the symmetry operation (rotation and shift by two-fold screw axis) generated a group of molecules oriented nearly perpendicular to the triazine ring, which resulted in zig-zag arrangement. This way, the unit cell parameter a increases to a double value. Prediction A01

Prediction A04

XRPD reference

A01

DAC Form I

160

140

120

100

80

ppm

15

10

5

0

ppm

Figure 9. Calculated and experimental XRPD patterns (a), 13C NMR (b), and 1H NMR spectra obtained for the CSPs of DAC A01 and A04 and the reference XRPD-refined crystal structure of DAC-I. Comparative analysis of the 2D 1H-13C and 1H-1H ss-NMR correlation spectra. As was recently demonstrated, when comparing experimental and predicted isotropic chemical shifts, a primary clue for choosing the correct crystal structure from the set of NMR-consistent candidates is provided by the 1H and 13C r.m.s.d.’s.6-9 However, in some cases, these parameters fail, thus leaving the correct crystal structure unidentified.9 This is also the case of decitabine for which two predictions A01 and A04 re1 mained undistinguishable by using r.m.s.d.( H) and r.m.s.d.(13C) parameters. Therefore, to strengthen the determination of the correct crystal structure, we focused on obtaining additional NMR parameters through comparative analysis of the 2D 1H-13C HETCOR spectra, in which the 1H and 13C chemical shifts are combined and unambiguously coupled by one-bond dipolar couplings. As a consequence, we obtained pairs of resonance frequencies defining the correlation signals, which allowed a more comprehensive comparison of the predicted and experimental NMR data. This way, the local conformation information stored in



13

C NMR chemical shifts and the molecular packing information carried by 1H chemical shifts are analyzed simultaneously for each CHn unit and combined into a single statistical parameter. Moreover, by using short mixing times (max. 70 µs), only the coherences of the CHn groups are effectively evolved, while uncertainties potentially resulting from exchangeable NH and OH protons are successfully suppressed. This approach is particularly valuable for analysis of incompletely assigned NMR spectra because the unambiguously determined pairs of 1H and 13C resonances considerably reduce the number of possible combinations when unassigned experimental NMR resonances are compared to the calculated ones. This way, many unrealistic signal assignments are excluded from statistical analysis. In the 2D 1H-13C HETCOR spectra of DAC, even visual comparison of the experimental and predicted data schematically depicted in Figure 10 indicates better agreement for the crystal structure designated A04, which is quantitatively confirmed by the values of the covariance, which amount to 0.46 and 0.74 ppm2 for structures A04 and A01, respectively. Based on our previous work60-62 and the recent application of this approach by Asakura et al.67 to the signal assignment of complex 1H-13C HETCOR NMR spectra, we consider this difference in the covariance values to be a reliable confirmation of the better match between the experimental and simulated spectra of A04 (and not A01).

lapped or unresolved correlations were excluded from the analysis. As in the previous case, visual inspection of the 1H-1H SQ/DQ DUMBO NMR spectra suggests a better match between the data predicted for the A04 crystal structure and the experimental data, compared to the analogous simulation for A01. This is particularly clear from the reduced scatter of the correlation signals involving the H2’, H1’ and H4’ protons and is also expressed by the low parameter of the covariance (sHH = 0.14 ppm2, Table 1) compared to the prediction for A01 (sHH = 0.17 ppm2). Overall, the NMR similarity measures listed in Table 1 identify CSP A04 as the most suitable structural model of DAC-I. This conclusion clearly agrees with the X-ray diffraction data that were experimentally determined for the investigated sample and is additionally verified by the X-ray data described in patent proceedings.38 As we have described previously, the values of the covariance always need to be considered together with the values of one standard deviation of both of the two underlying regressions.61 As a consequence, no threshold value for the covariance can be set a priori. 1

1

H- H SQ/DQ DUMBO Correlation Spectroscopy Prediction A04 H3’/H2’

10

0 C2‘/H2’* C2‘/H2’** C5‘/H5’* C5‘/H5’**

H6/H6

2 4

C3‘/H3’

6

C1‘/H1’

H6/H2’ H6/H3’

H6/H4’

δ ( 1H), ppm

C4‘/H4’

NH2**/H6

Predicted values

NH2**/NH2*

12

Predicted values

10

20

8

6

4

2

0

SQ δ( 1H), ppm

0

Prediction A01

10 150

125

100

75

50

25

H3’/H2’

0

C2‘/H2’* C2‘/H2’** C5‘/H5’* C5‘/H5’**

4 6

C1‘/H1’

10 150

125

100

75

50

25

H6/H1’

H6/H6 NH2**/H6

0

δ( 13C), ppm

Figure 10. Schematic visualization of the experimental 1H-13C HETCOR NMR spectrum (large dots) and correlation signals calculated for the CSPs of A04 (red small dots, upper spectrum) and A01 (blue small dots, lower spectrum). The same procedure was also applied to analyze the 1H-1H SQ/DQ DUMBO NMR spectra, which is schematically demonstrated in Figure 11. In this case, we considered medium- and 68 long-range 1H-1H dipolar contacts up to ca. 4.0 Å, including exchangeable OH and NH species. For this reason, the experimental 1H-1H SQ/DQ DUMBO NMR spectrum of DAC-I was measured with a relatively long recoupling time (80-120 µs, 2-3 SPC5 loops). For statistical analysis, however, only the clearly resolved correlation signals were taken into account, while over-

15

Predicted values

20

Experimental data NH2**/NH2*

12

10

8

6

4

2

0

1 SQ δ( δ H), ppm

8

Experimental data

H6/H2’ H6/H3’

H6/H4’

2

C3‘/H3’ Predicted values

5 10

0

Prediction A01

C4‘/H4’

H2’**/H2’* H4’/H2’

H1’/H2’

δ( 13C), ppm

175

H6/H1’

Experimental data

C6/H6

C6/H6

15

Experimental data

8

175

5

H1’/H2’

13

H- C HETCOR NMR Correlation Spectroscopy Prediction A04

0

H2’**/H2’* H4’/H2’

DQ δδ( 1H), ppm

1

δ ( 1H), ppm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

DQ δδ( 1H), ppm

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Figure 11. Schematic visualization of the experimental 1H-1H SQ/DQ DUMBO NMR spectrum (large dots) and correlation signals calculated for the CSPs of A-04 (red small dots, upper spectrum) and A-01 (blue small dots, lower spectrum).

4. Conclusion Crystal structure determination when diffraction data are not available still remains a challenge. In this contribution, we demonstrated the complete determination of the 3D crystal structure of DAC without the assistance of diffraction data, and the NMR-determined crystal structure was subsequently verified by comparing with XRPD-refined crystal structure. For this purpose, we extended the previously introduced approach of powder NMR crystallography based on the analysis of 1H NMR isotropic chemical shifts by simulating the entire 2D 1H-13C HETCOR and 1H1 H DQ/SQ NMR correlation spectra. The experimentally determined 1H-13C and 1H-1H correlation spectra not only provided excellent resolution to obtain accurate 1H isotropic chemical shifts, which are required for crystal structure determinations, but



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also revealed the characteristic structural features that were helpful for selection of the correct crystal structures. Moreover, these pairs of directly coupled resonance frequencies defining the correlation of NMR signals allowed for a more comprehensive comparison of the predicted and experimental NMR data. In this regard, an additional similarity measure was given by the covariance, which can be used to quantify the differences between the experimental and calculated resonance frequencies of the correlation signals. Thus, the NMR-consistent candidates were usefully discriminated, and the correct crystal structure was identified. In this way, we were able to discriminate between the predicted crystal structures in which the molecular conformations and short-range arrangements were basically identical and whose differences in global molecular packing were generated only by different symmetry operations. The proposed procedure thus allows the description of differences in the long-range symmetry of molecular packings, to which ss-NMR spectroscopy is otherwise less sensitive, and expands the capabilities of NMR crystallography to predicting supramolecular structures. In summary, following our experimental data and previously reported results, a combination of several statistical parameters must be used to select the correct crystal structure. Specifically, 13 C chemical shift data for which the r.m.s.d. of the fit between theory and experiment exceeds ca. 3 ppm clearly represents predicted crystal structures that are inconsistent with the experiment. By considering the 1H chemical shift data with r.m.s.d.’s smaller than 0.5 ppm, which were obtained from the measurements and calculations considering all proton species (i.e., including the exchangeable NH and OH units), and by combining these data with the candidates for which the 13C r.m,s.d.’s are smaller than 2.0 ppm, the set of generated crystal structures can be narrowed down to include only the NMR-consistent candidates. In the next step, comparative analysis of the 2D ss-NMR correlation spectra of the remaining candidates then provides a final clue for the refinement and unambiguous selection of the single correct crystal structure. Furthermore, using DAC-I, we verified the validity of this approach and confirmed the previously reported threshold limits for the NMR similarity parameters. These findings have encouraged us to extend this powder NMR crystallography approach to multicomponent polycrystalline drug-delivery systems, in which the active compound directly crystalizes in the interlamellar space of a polymer matrix5.

ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org. ss-NMR experimental parameters; details of CSP setting; details of CASTEP setting; statistical analysis procedure; experimentally determined 1H and 13C isotropic chemical shifts; and CIF files for DFT-optimized NMR-consistent candidates CSPs A01 and A04; CIF file for XRPD refined crystal structure of DAC-I (CSD Code CCDC 1505033). Accession Codes CCDC 1505033 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION Corresponding Author *Email: [email protected]

Author Contributions All the acknowledged authors contributed to the writing of the manuscript. All authors have approved the final version of the manuscript.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS The authors thank the Czech Science Foundation (grant nos. GA14-03636S and GA16-04109S) and the Ministry of Education, Youth and Sports of the CR within the National Sustainability Program I (NPU I), Project LO1507 POLYMAT, for their financial support. We thank Jan Rohlicek from FZU AV ČR for measurement of the DAC-I powder diffraction record.

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For Table of Contents Use Only

Predicting the Crystal Structure of Decitabine by Powder NMR Crystallography: Influence of Long-range Molecular Packing Symmetry on the NMR Parameters Jiri Brus, Jiri Czernek, Libor Kobera, Martina Urbanova, Sabina Abbrent, and Michal Husak

A complete reconstruction of the crystal structure of decitabine exclusively from 1H and 13C solid-state NMR (ss-NMR) chemical shifts through comparison with the NMR parameters calculated for DFT-optimized, computer-generated crystal structure predictions is demonstrated. The previously unconsidered influence of long-range molecular packing symmetry on the NMR parameters and subsequent selection of the correct crystal structure is discussed in detail.


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Predicting the Crystal Structure of Decitabine by Powder NMR

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