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Article

Predicting the Crystal Structure of Decitabine byPowder NMR Crystallography: Influence of Long-

range Molecular Packing Symmetry on NMR ParametersJiri 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

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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

aInstitute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky sq. 2, 162 06 Prague 6, Czech Republic. bUniversity 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 (ss-NMR) chemical shifts through comparison with the NMR param-eters calculated for DFT-optimized, computer-generated crystal structure predictions. In particular, we discuss the previously unconsidered influence of long-range molecular packing sym-metry on the NMR parameters and subsequent selection of the correct crystal structure. Symmetry operations considerably influ-enced the global molecular packing and unit cell parameters of the predicted crystal structures, while the conformations and short-range molecular arrangements were practically identical. Conse-quently, 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) 1H-13C HETCOR and 1H-1H DQ/SQ NMR correlation spectra. By determining the covariance, which provides a quantitative meas-ure of the differences between the experimental and calculated resonance frequencies of the correlation signals, the set of NMR-consistent 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 epi-genetic cancer therapy.1-3 However, despite its efficacy, the thera-peutic administration of DAC is limited by its hydrolytic lability, which causes a decrease in the plasma circulation time.4 To cir-cumvent this problem, we recently developed a new biodegrada-ble formulation based on a dispersion of the active compound in a crystalline matrix of poly(sebacic acid-co-1,4-cyclohexanedicarboxylic acid).5 However, structural characteriza-tion 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 dif-fraction patterns from the two crystal forms of the applied poly-mer 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

The term NMR crystallography11-20, usually refers to an ap-

proach combining X-ray diffraction data analysis with the meas-urement of NMR parameters in order to refine structures of mo-bile 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 com-plex 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, 1H NMR isotropic chemical shifts were demonstrated to be suffi-ciently 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.

4

2 6

1’

2’

3’ 4’

5’

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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, hydro-gen-bonded NH and OH protons must be excluded from the spec-tral analysis probably because of their unpredictable thermal receptivity.9 This powder NMR crystallography approach also failed for a low-molecular-weight compound, theophylline, prob-ably 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 small-er 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

It is thus clear that the widespread application of powder NMR crystallography still requires experimental verification, particular-ly 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 discrimina-tion 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-

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 transmis-sion 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 record-ed 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 ortho-rhombic crystal system; P212121 space group; and lattice parame-ters of a=5.6547(2), b=7.1471(4), c=24.935(2) Å. Structure solu-tion 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 prob-lem 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 parame-ter were refined. As the last step, the R-factor and lattice parame-ters 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 13C CPPI/MAS NMR experiments,43,44 iv) 2D 1H-13C FSLG HETCOR experiments,45 v) 2D NOESY-type 1H-1H spin-diffusion experiments with DUMBO homo-decoupling in both detection periods, 46 and vi) 2D DQ/SQ 1H-1H MAS NMR exper-iment47 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.

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 con-formations, 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 Mate-rial Studio 4.4 by torsion angles modification.51 The conformers B and C represent significant conformational changes of DAC mol-ecule. These changes are induced by the gauche-trans jump in the O-C5’-C4’-O segment (conformer B), and by the 180°rotation of

Structure selection

1H and

13C NMR

1H-

13C HETCOR

1H-

1H CRAMPS

13C-

13C INADEQUATE

Signal assignment

ss-NMR (Solid-state NMR)

CSP (Crystal structure prediction)

Polymorph Predictor

Generation of CSPs

DFT calculations (Density functional theory)

Measurement of NMR

parameters

Computation of NMR

parameters from CSP

r.m.s.d. (

1H), r.m.s.d. (

13C)

Comparison of experimental isotropic chemical shifts

and DFT calculations for predicted structures

GIPAW

Conformer A Conformer B Conformer C

2

1’

C2-N-C1’-O: -165.8°

O-C4’-C5’-O: -72.8°

5’ 4’

2

1’

5’ 4’

2

1’

5’ 4’

C2-N-C1’-O: -161.3°

O-C4’-C5’-O: 176.9°

C2-N-C1’-O: 41.3°

O-C4’-C5’-O: -66.8°

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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 energy-minimized 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 previ-ous 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 elimi-nate 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 ac-cordingly 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 geome-tries 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 gauge-including 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 calcu-lations 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 ingre-dients.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 agree-ment between theory and experiment for a nucleus Q was quanti-fied by the values of one r.m.s.d. (Q), as described in the Support-ing Information SI4. In order to describe the similarity of the measured and predicted 2D spectra, the approach originally pro-posed 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 proce-dure 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 1H 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 DAC-I can be said to be stable and thermally independent. However, this finding does not exclude existence of the temperature de-pendence of 1H chemical shifts at much lower temperatures.

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 experi-mental regimes. Whereas 1H DUMBO homo-decoupling experi-ment 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).

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 1H-1H DQ/SQ DUMBO NMR spectrum measured with 2 excita-tion 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-bond-attached proton-carbon pairs (Figures 3a and 3b). Sufficient reso-lution in the indirect 1H dimension of these 2D spectra then re-vealed a clear nonequivalence of the CH2 protons of unit no. 5’ denoted in Scheme 1 (3.91 and 3.36 ppm), whereas the CH2 pro-tons in group no. 2’ were almost equivalent, exhibiting a differ-ence in isotropic shifts of only 1.96 and 1.83 ppm as determined

-10-525 20 15 10 5 0 ppm

1H MAS and

1H DUMBO NMR Spectra

1H MAS NMR

1H DUMBO NMR

1H

1H

1H

3H

2H 2H

2H

OH/OH

100150 ppm

ppm

12 10 8 6 4 2 ppm

10

5

ppm

20

15

10

5

4

CH2 CH2

CH

CH

CH

CH

C=O -C=

2'

C1‘/H1’

6,2

C4/NH2*

C4/NH2**

C6/H6

C4‘/H4’

C3‘/H3’

C2‘/H2’

C5‘/H5’

C1’/H6

NH2**/NH2*

NH2**/H6

NH2*/NH2* H6/H6

H6/H1’

H6/H4’

H6/H2’

H2’**/H2’*

H4’/H2’

NH2* NH2**

H6 H2’**

H2’*

H4’ H5’

H5’H3’

H1’

OH

H3’/H2’

H1’/H2’ 1H-

13C HETCOR

(70 µµµµs)

1H-

13C HETCOR

(220 µµµµs)

C6/H6

13C

13C CPPI

1H-

1H SQ/SQ DUMBO

(50 µµµµs)

1H-

1H SQ/DQ

DUMBO

(2 loops)

NH2**/H6

NH2**/NH2*

5' 3'

1'

4'

C3’/OH

C5’/OH

H-C-H

C3’/H2’ C1’/H2’

H6/H1’

1H-

13C HETCOR and

1H-

1H SQ/DQ DUMBO NMR

Correlation Spectroscopy

a)

b)

c)

e)

d)

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by deconvolution of the 1H NMR signal extracted from the 2D 1H-13C HETCOR spectrum. To assign the resonances of CH units no. 4’ and no. 1’, the 1H-13C FSLG HETCOR spectra were meas-ured at longer CP times (150-300 µs, Figure 3c). This way, the medium-range coherence C1’/H2’ was clearly detected. Moreo-ver, 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 nonequiva-lent 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 unam-biguous signal assignment is welcomed. In this regard, recently developed 14N-1H ss-NMR correlation experiments showed ex-tremely high potentiality.14,31

In the chase for the highest possible spectral resolution the 2D 1H-1H correlation spectroscopy has already proven its high capa-bility.65 In this regard, the 1H-1H DQ/SQ NMR correlation tech-nique combined with DUMBO homo-decoupling yielded a partic-ularly 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. No-tably, using the SPC5 recoupling technique, the 1H-1H DQ coher-ences were generated with high efficiency over distances up to ca. 5 Å. These coherences thus provide information about the con-formation as well as the molecular packing in the crystal unit.

One of the most characteristic 1H-1H SQ/DQ coherences detect-ed for DAC-I (Figure 3e) is represented by the autocorrelation signal H6/H6 at 8.3/16.6 ppm, showing specific medium- or long-range 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 corre-lations 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 O-H…O hydrogen-bonding motifs.

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

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.

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 struc-tures 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, includ-ing a representative set of DAC conformations.

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. Hori-zontal lines represent the high limits of the r.m.s.d. considered as the values indicating the correct crystal structure.

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 calculat-ed 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

-200

-100

0

100

200

0

5

10

15

20

25

30

35

0 25 50 75 100 125

Distribution of Dihedral Angles

Relative Lattice Energies

Dih

ed

ral

an

gle

, d

eg

.

∆∆ ∆∆E

, k

J.m

ol-1

No. of CSPs

Conformer A

Conformer C

Conformer B

a)

b)

1H

r.m

.s.d

., p

pm

1H

r.m

.s.d

., p

pm

1

3C

r.m

.s.d

., p

pm

13C and

1H Chemical Shift Standard Deviations (r.m.s.d.’s)

a)

b)

c)

* * * *

*

*

2.0

ppm

0.5

ppm

0.4

ppm

Conformer B Conformer C Conformer A

* * * * * * *

* * * * * * *

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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 predict-ed crystal structures that are inconsistent with the experimental data. Furthermore, it also follows from the comparison of struc-tures 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 varia-tion in the methoxyl substituent. Specifically, an increase of ca. 1-2 ppm was found for predictions with a trans conformation of the O-C5’-C4’-O fragment (B conformers and prediction A03). Op-positely, 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 previous-ly 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 re-maining candidates for the sought crystal structure (A01 and A04, Figure 5, red bars).

Interestingly, by excluding the exchangeable NH and OH pro-tons from the evaluation of the proton chemical shifts, the 1H r.m.s.d.’s of both these candidates decreased, with a slightly high-er 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 exchange-able 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.

NMR similarity parameters

A01 A04

r.m.s.d. (13C), ppm 1.79 1.97

r.m.s.d. (1H, all), ppm 0.50 0.47

r.m.s.d. (1H, OH/NH excl.), ppm 0.43 0.31

covariance for 1H-13C pairs, ppm2 0.74 0.36

covariance for 1H-1H pairs, ppm2 0.17 0.12

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 com-parable values of the statistical data (r.m.s.d.(1H) and r.m.s.d. (13C)), the corresponding unit cell parameters differed considera-bly (Table 2). To understand this apparent disagreement, we analyzed the crystal structures of both of these preferred predic-tions 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 NMR-

Consistent CSPs and the XRPD-refined Reference Struc-

ture 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 --

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

Prediction A01 Prediction A04

a)

b)

C3’ C3’

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As demonstrated in Figure 6a, the conformations of the DAC molecules in the NMR-consistent CSPs, A01 and A04, are basi-cally identical, and distinguishable differences were found only in the relative orientations of the C3’-OH hydroxyl groups. Moreo-ver, 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 simi-larities, the periodic arrangement of DAC molecules in the pre-dicted 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).

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

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 (rota-tion and shift by two-fold screw axis) generated a group of mole-cules 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.

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 suffi-ciently indicative to identify the correct CSP (Figure 9a). Howev-er, 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. Consequent-ly, in addition to the expected improvements in the DFT predic-tions 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 pre-dicted NMR data61.

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 com-paring 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 re-mained undistinguishable by using r.m.s.d.(1H) 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 cou-plings. As a consequence, we obtained pairs of resonance fre-quencies 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

Prediction

A01 Prediction A04

Prediction A01 Prediction A04 XRPD reference

80100120140160 ppm 15 10 5 0 ppm

1H NMR Spectra

XRPD Patterns

13C NMR Spectra

DAC Form I

A01

A04

DAC Form I

A01

A04

a)

b)

c)

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13C 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 reso-nances considerably reduce the number of possible combinations when unassigned experimental NMR resonances are compared to the calculated ones. This way, many unrealistic signal assign-ments are excluded from statistical analysis.

In the 2D 1H-13C HETCOR spectra of DAC, even visual com-parison 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).

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 demon-strated in Figure 11. In this case, we considered medium- and long-range 1H-1H dipolar contacts up to ca. 4.0 Å,68 including exchangeable OH and NH species. For this reason, the experi-mental 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-

lapped or unresolved correlations were excluded from the analy-sis.

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 particular-ly clear from the reduced scatter of the correlation signals involv-ing 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 deter-mined 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.

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 struc-ture 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 chem-ical shifts by simulating the entire 2D 1H-13C HETCOR and 1H-1H DQ/SQ NMR correlation spectra. The experimentally deter-mined 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

0

2

4

6

8

10

0255075100125150175

0

2

4

6

8

10

0255075100125150175δδδδ(

13C), ppm

δδ δδ(

1H

), p

pm

Experimental data

Predicted values

Prediction A04

Prediction A01

δδδδ( 13

C), ppm

δδ δδ(

1H

), p

pm

Experimental data

Predicted values

1H-

13C HETCOR NMR Correlation Spectroscopy

C2‘/H2’*

C2‘/H2’**

C5‘/H5’*

C5‘/H5’**

C3‘/H3’

C1‘/H1’

C4‘/H4’

C6/H6

C2‘/H2’*

C2‘/H2’**

C5‘/H5’*

C5‘/H5’**

C3‘/H3’

C1‘/H1’

C4‘/H4’

C6/H6

0

5

10

15

20

024681012

0

5

10

15

20

024681012

1H-

1H SQ/DQ DUMBO Correlation Spectroscopy

SQ δδδδ( 1H), ppm

DQ

δ δ δ δ(

1H

), p

pm

SQ δ δ δ δ( 1H), ppm

D

Q δ δ δ δ

( 1H

), p

pm

Prediction A04

Prediction A01

Experimental data

Experimental data

Predicted values

Predicted values

H2’**/H2’*

H4’/H2’ H3’/H2’

H1’/H2’

H6/H1’

H6/H3’

H6/H2’

NH2**/NH2*

NH2**/H6 H6/H6

H6/H4’

H2’**/H2’*

H4’/H2’ H3’/H2’

H1’/H2’

H6/H1’ H6/H3’

H6/H2’

NH2**/NH2*

NH2**/H6 H6/H6

H6/H4’

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also revealed the characteristic structural features that were help-ful for selection of the correct crystal structures. Moreover, these pairs of directly coupled resonance frequencies defining the corre-lation of NMR signals allowed for a more comprehensive compar-ison 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 iden-tified. 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 other-wise less sensitive, and expands the capabilities of NMR crystal-lography 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, 13C chemical shift data for which the r.m.s.d. of the fit between theory and experiment exceeds ca. 3 ppm clearly represents pre-dicted 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 ap-proach 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 multi-component 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 da-ta_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

AUTHOR INFORMATION

Corresponding Author

*Email: brus@imc.cas.cz

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 finan-cial support. We thank Jan Rohlicek from FZU AV ČR for meas-urement of the DAC-I powder diffraction record.

REFERENCES

(1) Bryan, J.; Kantarjian, H.; Jabbour, E. Exp. Opin. Orphan Drugs 2013, 1, 661-673.

(2) Gnyszka, A.; Jastrzebski, Z.; Flis, S. Anticancer Res. 2013, 33, 2989-2996.

(3) Singh, V.; Sharma, P.; Capalash, N. Curr. Cancer Drug Tar-gets 2013, 13, 379-399.

(4) Rogstad, D. K.; Herring, J. L.; Theruvathu, J. A.; Burdzy, A.; Perry, C. C.; Neidigh, J. W.; Sowers, L. C. Chem. Res. Toxicol. 2009, 22, 1194-1204.

(5) Hruby, M.; Agrawal, K.; Policianova, O.; Brus, J.; Skopal, J.; Svec, P.; Otmar, M.; Dzubak, P.; Stepanek, P.; Hajduch, M. Biomedical Papers-Olomouc 2016, 160, 222-230.

(6) Elena, B.; Emsley, L. J. Am. Chem. Soc. 2005, 127, 9140-9146. (7) Salager, E.; Day, G. M.; Stein, R. S.; Pickard, C. J.; Elena, B.;

Emsley, L. J. Am. Chem. Soc. 2010, 132, 2564-2566. (8) Baias, M.; Dumez, J. N.; Svensson, P. H.; Schantz, S.; Day, G.

M.; Emsley, L. J. Am. Chem. Soc. 2013, 135, 17501-17507. (9) Baias, M.; Widdifield, C. M.; Dumez, J. N.; Thompson, H. P.

G.; Cooper, T. G.; Salager, E.; Bassil, S.; Stein, R. S.; Lesage, A.; Day, G. M.; Emsley, L. Phys. Chem. Chem. Phys. 2013, 15, 8069-8080.

(10) Leclaire, J.; Poisson, G.; Ziarelli, F.; Pepe, G.; Fotiadu, F.; Pa-ruzzo, F. M.; Rossini, A. J.; Dumez, J. N.; Elena-Herrmann, B.; Emsley, L. Chem. Sci. 2016, 7, 4379-4390.

(11) NMR crystallography; Harris, R.K., Wasylishen, R.E., Duer, M.J., Eds.; John Wiley & Sons, Ltd. 2009.

(12) Fernandes, J. A.; Sardo, M.; Mafra, L.; Choquesillo-Lazarte, D.; Masciocchi, N. Cryst. Growth Des. 2015, 15, 3674-3683.

(13) Jaworska, M.; Pawlak, T.; Kruszynski, R.; Cwiklinska, M.; Krzeminski, M. Cryst. Growth Des. 2012, 12, 5956-5965.

(14) Reddy, G. N. M.; Cook, D. S.; Iuga, D.; Walton, R. I.; Marsh, A.; Brown, S. P. Solid State Nucl. Magn. Reson. 2015, 65, 41-48.

(15) Harris, R. K.; Cadars, S.; Emsley, L.; Yates, J. R.; Pickard, C. J.; Jetti, R. K. R.; Griesser, U. J. Phys. Chem. Chem. Phys. 2007, 9, 360-368.

(16) Webber, A. L.; Emsley, L.; Claramunt, R. M.; Brown, S. P. J. Phys. Chem. A 2010, 114, 10435-10442.

(17) Harris, R. K.; Hodgkinson, P.; Zorin, V.; Dumez, J.-N.; Elena-Herrmann, B.; Emsley, L.; Salager, E.; Stein, R. S. Magn. Reson. Chem. 2010, 48, S103-S112.

(18) Abraham, A.; Apperley, D. C.; Gelbrich, T.; Harris, R. K.; Griesser, U. J. Can. J. Chem. 2011, 89, 770-778.

(19) Heider, E. M.; Harper, J. K.; Grant, D. M. Phys. Chem. Chem. Phys. 2007, 9, 6083-6097.

(20) Watts, A. E.; Maruyoshi, K.; Hughes, C. E.; Brown, S. P.; Har-ris, K. D. M. Cryst. Growth Des. 2016, 16, 1798-1804.

(21) Brus, J.; Jegorov, A. J. Phys. Chem. A 2004, 108, 3955-3964.

Page 8 of 10

ACS Paragon Plus Environment

Crystal Growth & Design

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

(22) Arcos-Ramos, R.; Rodriguez-Molina, B.; Gonzalez-Rodriguez, E.; Ramirez-Montes, P. I.; Eugenia Ochoa, M.; Santillan, R.; Farfan, N.; Garcia-Garibay, M. A. RSC Advances 2015, 5, 55201-55208.

(23) Aguilar-Granda, A.; Perez-Estrada, S.; Roa, A. E.; Rodriguez-Hernandez, J.; Hernandez-Ortega, S.; Rodriguez, M.; Rodriguez-Molina, B. Cryst. Growth Des. 2016, 16, 3435-3442.

(24) Geppi, M.; Mollica, G.; Borsacchi, S.; Veracini, C.A. Appl. Spectrosc. Rev. 2008, 43, 202-302.

(25) Luedeker, D.; Gossmann, R.; Langer, K.; Brunklaus, G. Cryst. Growth Des. 2016, 16, 3087-3100.

(26) Putra, O. D.; Yoshida, T.; Umeda, D.; Higashi, K.; Uekusa, H.; and Yonemochi, E. Cryst Growth Des 2016, 16, 5223−5229.

(27) Dudek, M. K.; Pawlak, T.; Paluch, P.; Jeziorna, A.; Potrze-bowski, M. J. Cryst. Growth Des. 2016, 16, 5312–5322.

(28) Chattah, A. K.; Zhang, R.; Mroue, K. H.; Pfund, L. Y.; Longhi, M. R.; Ramamoorthy, A.; Garnero, C. Mol. Pharm. 2015, 12, 731-741.

(29) Costantino, F; Sassi, P.; Geppi, M.; Taddei, M. Cryst. Growth Des. 2012, 12, 5462-5470.

(30) Dudenko, D. V.; Yates, J. R.; Harris, K. D. M.; Brown, S. P. CrystEngComm 2013, 15, 8797-8807.

(31) Reddy, G. N. M.; Marsh, A.; Davis, J. T.; Masiero, S.; Brown, S. P. Cryst. Growth Des. 2015, 15, 5945-5954.

(32) Carignani, E.; Borsacchi, S.; Bradley, J. P.; Brown, S. P.; Geppi, M. J. Phys. Chem. C 2013, 117, 17731-17740.

(33) Bradley, J. P.; Velaga, S. P.; Antzutkin, O. N.; Brown, S. P. Cryst Growth Des 2011, 11, 3463-3471.

(34) Matejicek, P.; Brus, J.; Jigounov, A.; Plestil, J.; Uchman, M.; Prochazka, K.; Gradzielski, M. Macromolecules 2011, 44, 3847-3855.

(35) Brus, J.; Zhigunov, A.; Czernek, J.; Kobera, L.; Uchman, M.; Matejicek, P. Macromolecules 2014, 47, 6343-6354.

(36) Bardwell, D. A.; Adjiman, C. S.; Arnautova, Y. A.; Bartashe-vich, E.; Boerrigter, S. X. M.; Braun, D. E.; Cruz-Cabeza, A. J.; Day, G. M.; Della Valle, R. G.; Desiraju, G. R.; van Eijck, B. P.; Facelli, J. C.; Ferraro, M. B.; Grillo, D.; Habgood, M.; Hofmann, D. W. M.; Hofmann, F.; Jose, K. V. J.; Karamertzanis, P. G.; Kazantsev, A. V.; Kendrick, J.; Kuleshova, L. N.; Leusen, F. J. J.; Maleev, A. V.; Misquitta, A. J.; Mo-hamed, S.; Needs, R. J.; Neumann, M. A.; Nikylov, D.; Orendt, A. M.; Pal, R.; Pantelides, C. C.; Pickard, C. J.; Price, L. S.; Price, S. L.; Schera-ga, H. A.; van de Streek, J.; Thakur, T. S.; Tiwari, S.; Venuti, E.; Zhitkov, I. K., Towards crystal structure prediction of complex organic compounds - a report on the fifth blind test. Acta Crystallogr. B 2011, 67, 535-551.

(37) Kalakewich, K.; Iuliucci, R.; Harper, J. K. Cryst. Growth Des. 2013, 13, 5391-5396.

(38) Redkar, S.; Joski-Hangal, R. Compositions and formulations of decitabine polymorphs and methods of use thereof. US200600149149 A1, January 19, 2006.

(39) X'Pert HighScore Plus 2.2.5, 2009 PANalytical B. V. (40) David, W. I. F.; Shankland, K.; van de Streek, J.; Pidcock, E.;

Motherwell, W. D. S.; Cole, J. C. J. Appl. Cryst. 2006, 39, 910-915. (41) Davies, M. J.; Jenkins, P. R.; Prouse, L. J. S.; Russell, D. R.;

Devos, D. Acta Crystallogr. C 1991, 47, 1418-1420. (42) Sakellariou, D.; Lesage, A.; Hodgkinson, P.; Emsley, L. Chem.

Phys. Lett. 2000, 319, 253-260.

(43) Schaefer, J.; Stejskal, E. O. J. Am. Chem. Soc. 1976, 98, 1031-1032.

(44) Wu, X. L.; Burns, S. T.; Zilm, K. W. J. Magn. Reson. Ser. A 1994, 111, 29-36.

(45) vanRossum, B. J.; Forster, H.; deGroot, H. J. M. .J Magn. Re-son. 1997, 124, 516-519.

(46) Sakellariou, D.; Lesage, A.; Emsley, L. J. Am. Chem. Soc. 2001, 123, 5604-5605.

(47) Brown, S. P.; Lesage, A.; Elena, B.; Emsley, L. J. Am. Chem. Soc. 2004, 126, 13230-13231

(48) Hohwy, M.; Rienstra, C. M.; Jaroniec, C. P.; Griffin, R. G. J. Chem. Phys. 1999, 110, 7983-7992.

(49) Langer, B.; Schnell, L.; Spiess, H. W.; Grimmer, A. R. J. Magn. Reson. 1999, 138, 182-186.

(50) Brus, J. Solid State Nucl. Magn. Reson. 2000, 16, 151-160. (51) Material Studio 4.4, www.accerlys.com (52) Kresse, G.; Joubert, D. Phys. Rev. B 1999, 59, 1758-1775. (53) Segall, M. D.; Lindan, P. J. D.; Probert, M. J.; Pickard, C. J.;

Hasnip, P. J.; Clark, S. J.; Payne, M. C. J. Phys-Condens Mat. 2002, 14, 2717-2744.

(54) Clark, S. J.; Segall, M. D.; Pickard, C. J.; Hasnip, P. J.; Prob-ert, M. J.; Refson, K.; Payne, M. C. Z Kristallogr. 2005, 220, 567-570.

(55) Perdew, J. P.; Burke, K.; Ernzerhof, M. Phys. Rev. Lett. 1996, 77, 3865-3868.

(56) Pickard, C. J.; Mauri, F. Phys. Rev. B 2001, 63, 245101. (57) Yates, J. R.; Pickard, C. J.; Mauri, F. Phys. Rev. B 2007, 76,

024401. (58) Bonhomme, C.; Gervais, C.; Babonneau, F.; Coelho, C.; Pour-

point, F.; Azais, T.; Ashbrook, S. E.; Griffin, J. M.; Yates, J. R.; Mauri, F.; Pickard, C. J. Chem. Rev. 2012, 112, 5733-5779.

(59) Beran G. J. O. Chem. Rev. 2016, 116, 5567-5613. (60) Czernek, J.; Brus, J. Chem. Phys. Lett. 2013, 586, 56-60. (61) Czernek, J.; Brus, J. Chem. Phys. Lett. 2014, 608, 334-339. (62) Czernek, J.; Pawlak, T.; Potrzebowski, M. J.; Brus, J. Chem.

Phys. Lett. 2013, 555, 135-140. (63) Czernek, J. Chem. Phys. Lett. 2015, 619, 230-235. (64) Harris, R. K.; Hodgkinson, P.; Pickard, C. J.; Yates, J. R.;

Zorin, V. Magn. Reson. Chem. 2007, 45, S174-S186. (65) Brown, S. P. Solid State Nucl. Magn. Reson. 2012, 41, 1-27. (66) Nyman, J.; Day, G. M. CrystEngComm 2015, 17, 5154-5165. (67) Asakura, T.; Ohata, T.; Kametani, S.; Okushita, K.; Yazawa,

K.; Nishiyama, Y.; Nishimura, K.; Aoki, A.; Suzuki, F.; Kaji, H.; Ulrich, A. S.; Williamson, M. P. Macromolecules 2015, 48, 28-36.

(68) Bradley, J. P.; Tripon, C.; Filip, C.; Brown S. P. Phys. Chem. Chem. Phys. 2009, 11, 6941–6952.

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10

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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