Determining the Crystal Structures of Peptide Analogs of Boronic

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Determining the crystal structures of peptide analogs of boronic acid in the absence of single crystals: Intricate motifs of ixazomib citrate revealed by XRPD guided by ss-NMR Michal Husak, Alexandr Jegorov, Jan Rohlicek, Andrew N. Fitch, Jiri Czernek, Libor Kobera, and Ji#í Brus Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b00402 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on May 2, 2018

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Crystal Growth & Design

Cover Page

Determining the crystal structures of peptide analogs of boronic acid in the absence of single crystals: Intricate motifs of ixazomib citrate revealed by XRPD guided by ss-NMR Michal Hušák,a Alexandr Jegorov,b Jan Rohlíček,c Andrew Fitch,d Jiří Czernek,e Libor Kobera,e and Jiří Brus*e a)

Department of Solid State Chemistry, University of Chemistry and Technology, Technicka 5, 166 28 Prague 6, Czech Republic. b) Teva Pharmaceuticals CR, s.r.o, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic. c) Department of Structural Analysis, Institute of Physics of the Czech Academy of Sciences, Na Slovance 2, Praha 8, 182 21, Czech Republic. d) ESRF- The European Synchrotron, CS40220, F-38043 Grenoble Cedex 9, France. e) Department of NMR Spectroscopy, Institute of Macromolecular Chemistry CAS, Heyrovsky sq. 2, 162 06 Prague 6, Czech Republic. ABSTRACT: Uncertainties in the structure determination of peptide analogs of boronic acid, exacerbated by the many coordination modes of boron, represent an obstacle in understanding their role in living organisms and thus also in developing the next generation of anticancer drugs. For that reason, we present here a general experimentalcomputational strategy allowing structure determination of complex boronic acid derivatives with extensive conformational variability. We demonstrate successful solution of the crystal structures of two non-solvated polymorphs of ixazomib citrate directly from synchrotron powder diffraction data, which is challenging because the two molecules in the asymmetric unit cell exhibit 32 degrees of conformation freedom push the limits of current solution procedures. We used a novel two-step Rietveld refinement based on DFT-D restraints to improve information quality derived from powder diffraction data to be comparable with that of single-crystal solutions. NMR crystallography was applied to verify the crystal structures, and the high potential value of using 11B NMR parameters toward the solution of unknown structures was demonstrated. Evolution of 11B-11B double-quantum coherences allows probing of interatomic distances up to 7 Å. Overall, we present an integrated approach that applies several techniques in conjunction to provide otherwise unavailable structural information.

For Table of Contents Use Only

Synopsis: General experimental-computational strategy for exploring the assembly of peptidic boronic acid derivatives in microcrystalline state is demonstrated. By combining the simulated annealing and two-step Rietveld refinement of XRPD data with the comparative analysis of NMR parameters the crystal structures of two non-solvated polymorphs of ixazomib citrate, each consisting of two symmetry-independent molecules in unit cell with 32 free parameters, were solved and refined.

Corresponding Author J.B. *Email: [email protected] http://www.imc.cas.cz/en/umch/o_nmr.htm

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Determining the crystal structures of peptide analogs of boronic acid in the absence of single crystals: Intricate motifs of ixazomib citrate revealed by XRPD guided by ss-NMR Michal Hušák,a Alexandr Jegorov,b Jan Rohlíček,c Andrew Fitch,d Jiří Czernek,e Libor Kobera,e and Jiří Brus*e a)

Department of Solid State Chemistry, University of Chemistry and Technology, Technicka 5, 166 28 Prague 6, Czech Republic.

b)

c)

Teva Pharmaceuticals CR, s.r.o, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic.

Department of Structural Analysis, Institute of Physics of the Czech Academy of Sciences, Na Slovance 2, Praha 8, 182 21, Czech Republic. d)

e)

ESRF- The European Synchrotron, CS40220, F-38043 Grenoble Cedex 9, France.

Department of NMR Spectroscopy, Institute of Macromolecular Chemistry CAS, Heyrovsky sq. 2, 162 06 Prague 6, Czech Republic.

KEYWORDS: XRPD • NMR crystallography • peptide and boronic acid analogs • ixazomib citrate.

ABSTRACT: Uncertainties in the structure determination of peptide analogs of boronic acid, exacerbated by the many coordination modes of boron, represent an obstacle in understanding


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their role in living organisms and thus also in developing the next generation of anticancer drugs. For that reason, we present here a general experimental-computational strategy allowing structure determination of complex boronic acid derivatives with extensive conformational variability. We demonstrate successful solution of the crystal structures of two non-solvated polymorphs of ixazomib citrate directly from synchrotron powder diffraction data, which is challenging because the two molecules in the asymmetric unit cell exhibit 32 degrees of conformation freedom push the limits of current solution procedures. We used a novel two-step Rietveld refinement based on DFT-D restraints to improve information quality derived from powder diffraction data to be comparable with that of single-crystal solutions. NMR crystallography was applied to verify the crystal structures, and the high potential value of using 11

B NMR parameters toward the solution of unknown structures was demonstrated. Evolution of

11

B-11B double-quantum coherences allows probing of interatomic distances up to 7 Å. Overall,

we present an integrated approach that applies several techniques in conjunction to provide otherwise unavailable structural information.

Introduction Boron-containing compounds have long been recognized as potentially active pharmaceutical ingredients.1-3 As recent investigations have resulted in the discovery of many promising pharmaceuticals exhibiting anticancer and antibacterial activity such as bortezomib, MLN978, CEP-18770, and GSK2251052, research on peptidic derivatives with boronic acid fragments is rapidly gaining in intensity.4


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Ixazomib citrate, an active pharmaceutical ingredient (API) consisting of a peptidic boronic acid derivative and citric acid, is a prodrug for the treatment of multiple myeloma (a type of white blood cell cancer). Ixazomib, the active part, is formed by hydrolysis of boronic ester under physiological conditions. However, due to the many coordination possibilities for boron atoms, the structures of ixazomib and its complexes are still under extensive investigation. Several possible coordination schemes for citric acid have been proposed, including those featuring five- or six-membered rings (Scheme 1).5 However, the coordination possibilities of boron are not entirely exhausted by these options; which coordination takes part in each crystalline form of ixazomib citrate is unclear, as is whether the coordination modes are equivalent when several symmetry-independent molecules are present in the crystal unit.

Scheme 1. Expected Structures of Ixazomib Citrate Complexes (C20H23BCl2N2O8).

In this regard, the recently derived single-crystal structures of two solvates indicated that the more preferred arrangement involves a 5-membered ring.6 In practice, however, most crystalline forms do not form single crystals of sufficient quality for crystal structure determination, and only attempting to solve the structure from powder diffraction data remains an option. However, prediction of the complicated solid-state structures of the peptide analogs of boronic acid, which result from a complex pathway involving reversible covalent and non-covalent interactions, is particularly challenging. The future design of new drugs based on boronic acid compounds thus requires new tools for their precise structural characterization.


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Hence, the purpose of our study is to demonstrate an extended experimental-computational strategy for exploring the structure of complex peptide and boronic acid analogs with many degrees of conformational freedom. By combining advanced analysis of powder diffraction data with comparative analysis of NMR parameters and high-level quantum chemical calculations, complete crystal structures of two non-solvated polymorphs of ixazomib citrate (Form II and Form X), each consisting of two symmetry-independent molecules in a unit cell with 32 free parameters, were successfully determined.

Results and Discussion Structure solution and refinement from powder data. In the absence of suitable single crystals allowing for complete X-ray diffraction (XRD) analysis, we focused our attention on the advanced analysis of powder diffraction data (Figure 1). The data were preprocessed by synchrotron beamline-specific software7 as described in experimental section. The reflection positions were determined via DASH software,8 and indexing of all peaks was done via DICVOL06.9 DASH automatic space group determination indicated a P21/c space group for both forms of ixazomib citrate (Form II and Form X), which is inconsistent with the chiral character of the molecule. Detailed investigation of the diffraction record for Form II showed several weak reflections violating the P21/c group extinction, proving that the space group is P21 with two molecules in the asymmetric unit (very probably exhibiting pseudo-centrosymmetric behavior). The diffraction record of Form X did not violate the P21/c group extinction in any way—the correct P21 space group was confirmed during refinement. Both Forms II and X have two molecules of ixazomib citrate in their asymmetric unit.


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X-ray synchrotron powder records a)

Form II

b) Form X

Figure 1. Comparison of the Measured and Calculated Powder Diffractograms for Ixazomib Form II and Form X (a and b, respectively). For Full-size Diffractograms, See Supporting Information, Figure S1.

Correct selection of an initial model is the key step in the structure determination from XRPD data. In the case of ixazomib citrate it was necessary to decide between the two possible structures consisting either of a five-membered ring or the six-membered arrangement (see Scheme 1). Therefore, to select the most suitable initial model for the subsequent structure solution the model of ixazomib citrate complex was taken from the known crystal structure of the ixazomib citrate ethanol solvate.6 However, the in vacuo RI-MP2 optimized models (IXAModel-4 and Model-5, see discussion below) showing the lowest relative potential energies and excellent agreement with experimental 11B NMR isotropic chemical shifts can be also utilized,


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because in all cases the structures featuring five-membered rings were preferred. Consequently, in the absence of single crystal data, the ab initio optimized models featuring lowest potential energy represent quite suitable initial approximations. Since the second axial chiral center on boron is created during the crystallization, predicting whether the structure contains R,R, R,S enantiomers (as in the ethanol solvate), R,R, R,R enantiomers or the R,S, R,S combination (where the first chiral center corresponds to ixazomib and the second is generated on citric acid as a consequence of its coordination to boronic acid) was not possible. Testing all three possible combinations was required. Each molecule of ixazomib citrate has 16 DOF (degrees of freedom). Two molecules of ixazomib citrate in the asymmetric unit give 32 DOF, which is a complex problem whose solution is on the edge of current powder-solution computational methodology. The three possible combinations of conformations were each used 1000 times as a starting point for DASH simulated annealing runs, and 2.0e+7 steps were used per run. The computation was executed on 16 CPU cores in parallel, and the required equivalent single CPU execution time was approximately 1 year for each form. Finding the correct torsion angles was accelerated by statistical bias based on similar molecules’ torsion angles as found in the Cambridge Structural Database (CSD). Three identical solutions were found for Form II, all for the R,R, R,S conformation combination with the lowest cost functions. Fourteen identical solutions were found for Form X, all for the R,R, R,S conformations.


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Table 1. Crystal Data for Form II and Form X, Both Restrained by the DFT-Calculated Molecular Geometry. Form II

Form X

Formula

C20H23BCl2N2O9

M.w.

517.119

Crystal system

Monoclinic

Monoclinic

Space group, Z

P21 (No. 4), 4

P21 (No. 4), 4

a, Å

16.0169(2)

16.1521(2)

b, Å

18.26999(15)

17.03683(12

c, Å

8.52827(6)

8.51817(6)

β, (°)

104.9730(7)

94.1479(8)

V, Å3

2410.88(4)

2337.90(4)

T(K)

293

293

Absorption coefficient (mm-1)

0.08

0.08

Wavelength (Å)

0.399978(2)

0.399963(3)

θ range for data processing (°)

0.502 - 22.498

0.502 - 22.498

Rp; Rwp

0.0442; 0.0643

0.0361, 0.0535

Goof

6.06

4.23

Parameters refined

254

246

Number of restraints and constraints

180, 266

180, 266

March-Dollase correction and direction

1.107(1 0 0)

1.045(1 0 0)

Software used for structure solution, DASH, DICVOL06 DASH, DICVOL06, indexation, interpretation.

refinement

and Jana2006, 2005

MCE Jana2006,

MCE

2005

The final refinement of the structure was done in Jana2006 software.10 Both structural models were checked and corrected by placing atoms via the difference Fourier maps in MCE software.11 Positions of all non-hydrogen atoms were refined together using one isotropic


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displacement parameter shared by carbon, nitrogen, oxygen and boron atomic types; one harmonic displacement parameter shared by all chlorine atoms; unit cell parameters; background (36 points in the case of Form II and 28 points in the case of Form X); zero shift and the MarchDollase parameter. Hydrogen atoms were kept in their positions calculated from the geometry. In the first step of refinement, the bond-length and bond-angle restraints based on the ixazomib citrate isopropyl alcohol solvate6 were used. The resulting model was checked by DFT calculations (described below). In the final refinement, bond-length and bond-angle restraints based on the DFT-calculated geometry were used. All restraints were kept at the same initial weight (w = 1/σ2) with expected deviations of σ = 0.001 for bond-lengths and σ = 0.01 for bondangles from the average. For details of refinement, see Table 1. The accuracy of the X-ray powder diffraction (XRPD) structures of both Forms II and X was first tested by periodic DFT-D geometry optimization using CASTEP12 with the GGA PBE functional, TS dispersion correction, "Fine" settings, and an energy cut-off of 520 eV. "Fine" setting corresponds to mapping k-points with 0.07 Å-1 in reciprocal space - the Monkhorst-Pack parameters are derived to give the specified separation between neighboring grid points. The optimization was done in a two-step manner as suggested by van de Streek and Neumann.13 The geometry of the structure was first optimized, followed by the cell parameters. The maximum number of optimization steps was limited to 100. The convergence to predefined criteria is summarized in Supporting Information Table S1. After the cell optimization, the "Fine" convergence criteria were satisfied for Form X. However, for Form II, we were able to fully satisfy only the "Coarse" criteria for maximal displacement. Better results for Form II were unobtainable, so we used the data satisfying the "Coarse" criteria for maximum displacement, "Medium" criteria for maximal force and "Fine"


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criteria for the rest. (For further details defining “Coarse”, “Medium” and “Fine” criteria see Supporting Information S2). The issues with DFT calculation convergence can be related to a very flat energy minimum. The flat minimum hypothesis corresponds well with the occurrence of two similar polymorphs. To compare the original and DFT-D-optimized geometries, rootmean-square (r.m.s.) Cartesian displacement (RMSCD) as previously defined13 was calculated in Crystal CMP software.14 The results are summarized in Supporting Information Table S2.

Ixazomib citrate Form II

Ixazomib citrate Form X

Figure 2. DFT-Optimized Refined XRPD Crystal Structures of Ixazomib Citrate Forms II and X.


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For both Forms II and X, the RMSCD values calculated without H atoms (cell fixed as well as cell optimized) are less than 0.25 Å, which is typical for correctly solved structures. The RMSCD values calculated with H atoms are ca. 0.30 Å, which indicates slightly incorrect hydrogen placement based on only single-crystal restraints. The introduction of DFT-D-based restraints decreased both the r.m.s. deviation (r.m.s.d.) and the R factor. The refined crystal structures of both Forms II and X (Figure 2) are deposited in the Cambridge Crystallographic Data Centre (CCDC) with provisional numbers CCDC 1822510 and 1822511.

Independent validation of the crystal structures. As discussed above, determining the crystal structures of complex systems consisting of multiple molecules with unknown conformations directly from powder XRD data represents a great challenge. Independent verification of the refined XRPD structures is thus of particular interest. In this regard, NMR crystallography displays a remarkable potential because of the synergy of experimental and computational approaches.15-25 In general, solid-state NMR data can be used in two ways to complement crystal structure refinement. One approach is based on the systematic comparison of the NMR parameters calculated using periodic DFT methods on the refined XRPD structure with the corresponding experimentally determined values. Agreement between the calculated and experimental values then validates the crystal structure. The second, more straightforward route utilizes measurements of dipole-dipole interactions because they provide direct information on specific internuclear distances, molecular conformations and bonding arrangements.15-25 Specifically, we first focused on the analysis of

11

B NMR isotropic chemical shifts and

11

B

quadrupolar coupling constants to validate the refined XRPD structures of ixazomib citrate


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Forms II and X. In this manner, we investigated the coordination geometry of the boron centers. Subsequently, we measured

11

B-11B and

1

H-1H dipolar interactions to probe long-range

interatomic distances and intermolecular contacts. Finally, we compared the DFT-calculated and experimentally determined 1H and

13

C NMR isotropic chemical shifts to determine the overall

consistency of the global architecture of both polymorphs of ixazomib citrate derived from XRPD data.

Determination of coordination geometry of boron centers. NMR parameters for the

11

B

nucleus such as the isotropic chemical shift (δiso) and the quadrupolar coupling constant (QC) represent receptive probes of the coordination geometries around boron atoms.26-31 Although these parameters cover relatively narrow ranges (0 - 40 ppm and 0 - 4 MHz, respectively), the resonance frequencies and line shapes made discriminating a variety of boron species possible, as illustrated in Figure 3.


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B MAS NMR Spectra

symmetric BIV with 6-/6- rings deformed BIV OH 5-/6- rings B deformed BIV B B 5-/5- rings -

-

-

symmetric BIII

B-

HO B OH

HO

deformed BIII R B OH

HO

35

Figure 3.

11

30

25

20

15

10

5

0

-5

ppm

B MAS NMR Lineshapes of Model Compounds Simulated using Experimental

Parameters Found in the Literature:30,32 (2-methylpropyl)boronic acid (δiso = 32.7 ppm, QC = 3.1 MHz and η = 0.44; red line); boric acid (δiso = 19.2 ppm, QC = 2.1 MHz and η = 0.17, blue line); bortezomib Form I (δiso = 10.5 ppm, QC = 1.65 MHz and η = 0.82, purple line); the tetrahedral site in borax (δiso = 2.8 ppm, QC = 0.97 MHz and η = 0.90, green line) and the 6-membered cyclic borate diesters (δiso = 0.4 ppm, QC = 0.99 MHz and η = 0.70, violet line). For direct comparison, the

11

B MAS NMR spectrum of ixazomib citrate Form II simulated from the

experimentally determined parameters (δiso = 12.4 ppm, QC = 1.66 MHz and η = 0.38, black line) is also included.

For ixazomib citrate, the 2D

11

B triple-quantum (TQ)/MAS NMR spectra (Figure 4a)

demonstrate that the structural sensitivity of

11

B resonances is high enough to distinguish

different polymorphs with similar crystal structures. Moreover, in accord with quantum chemical calculations discussed in detail below, the obtained quadrupolar coupling constants (QC = 1.671.75 MHz) and isotropic chemical shifts (δiso = 12.4-13.5 ppm) (Tables 2 and 3) indicate that


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boron atoms in both polymorphs adopt 4-fold coordination with significantly perturbed tetrahedral symmetry.

Table 2. Experimental

11

B NMR Isotropic Chemical Shifts and

11

B Quadrupolar Coupling

Constants (Qcc) of Boron Species in Ixazomib Citrate Forms II and X. Experimental 11B NMR parameters

δ(11B), ppm

Qc, MHz

η

Site 1

Site 2

Site 1

Site 2

Site 1

Site 2

F-II

12.45

12.45

1.67

1.67

0.39

0.39

F-X

13.11

13.54

1.75

1.74

0.45

0.48

Table 3. Calculated

11

B NMR Isotropic Chemical Shifts and

11

B Quadrupolar Coupling

Constants (Qc) of Boron Species in Ixazomib Citrate Forms II and X. The calculated chemical shifts were estimated from the GIPAW-PBE computed chemical shielding using the conversion factor.22 Calculated 11B NMR parameters

δ(11B), ppm

Qc, MHz

η

Site 1

Site 2

Site 1

Site 2

Site 1

Site 2

F-II

14.07

14.02

1.96

2.00

0.37

0.38

F-X

15.23

15.75

2.05

2.06

0.45

0.48


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2D B TQ/MAS NMR Ixazomib citrate Form X

a) Ixazomib citrate Form II ppm

ppm

14

14

15

15

16

16 14

12

10

8

6

14

ppm

12

10

8

6

ppm

13

C CP/MAS NMR Ixazomib citrate Form X

b) Ixazomib citrate Form II C-OH (citric acid)

150

CH, CH3 (IXA)

100

50

C-OH (citric acid)

CH, CH3 (IXA)

150

ppm

100

50

ppm

1

H CRAMPS NMR

c) Ixazomib citrate Form II

Ixazomib citrate Form X COOH(2)

COOH(2) COOH(1)

COOH(1)

14

Figure 4. 2D

11

B TQ/MAS,

12

13

10

8

6

4

2

ppm

14

12

10.0 ppm

10

8

6

4

2

ppm

C CP/MAS and 1H CRAMPS NMR Spectra of Ixazomib Citrate

Forms II and X (Red and Blue Lines, Respectively). Spectral regions containing the C-OH and CH3 resonances of citric acid and ixazomib molecules are expanded in boxes above the fullrange spectra. When considering sources of deformation of tetrahedral symmetry, we first analyzed

11

B

isotropic chemical shifts. Literature data show that tetra-coordinated boron atoms shared by two 6-membered rings in spirocyclic borate diesters exhibit very low isotropic chemical shifts ranging from 4 to -2 ppm30 and that boron atoms shared by a 6-membered ring and a 5membered ring have chemical shifts of 9-11 ppm.32-34 Boron atoms shared by two 5-membered rings resonate at higher frequencies (12-14 ppm).27


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As exactly the same trends are reproduced by high-level quantum chemical calculations of 11

B NMR parameters,30-35 we optimized the geometries of a set of different conformations of

ixazomib citrate complexes by the RI-MP2/TZVP method and calculated their isotropic chemical shifts (Figure 5). We found that the boron atoms shared by two 5-membered rings in ixazomib citrate complexes resonate at ca. 13±0.5 ppm, depending on the global conformation. In contrast, the presence of a 6-membered ring considerably decreases the calculated isotropic chemical shifts, to ca. 10±0.5 ppm. Boron atoms in ixazomib citrate Forms II and X are therefore incorporated into spirocyclic borate fragments consisting of two 5-membered rings.

IXA-Model-1 5-/5-membered


δι(11B) = 12.9 ppm

δι(11B) = 11.0 ppm

δι(11B) = 12.7 ppm

δι(11B) = 13.2 ppm

δι(11B) = 14.2 ppm

∆E = 19.9 kJ/mol

∆E = 11.8 kJ/mol

∆E = 16.0 kJ/mol

∆E = 0.0 kJ/mol

∆E = 8.9 kJ/mol




Figure 5. Typical Examples of RI-MP2/TZVP Optimized Geometries of Ixazomib Citrate Complexes with the 5-/5- and 5-/6-Membered Cyclic Borate Fragments. The corresponding values of calculated

11

B NMR isotropic chemical shifts (δi(11B)) and the relative energies with

respect to the most stable conformer (∆E) are also included.

In the subsequent step, we calculated absolute total energies, which allowed us to estimate relative stabilities for each conformer as demonstrated in Figure 5. Notably the most stable structure, IXA-Model-4, is fairly similar to the geometry of one of the crystallographically


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independent ixazomib citrate complexes clipped out of the unit cell of the experimentally determined structure described in the file IXA Form II (the r.m.s.d. of aligned positions is 1.664 Å), whereas the second most stable structure, IXA-Model-5, is similar to the geometry of one such complex from IXA Form X, with an r.m.s.d. of aligned positions of 1.379 Å. It thus appears that in this particular case the most stable configurations consisting of two 5-membered rings are favored. Moreover, the structures IXA-Model-4 and IXA-Model-5 are also clearly suitable models for structure refinement of XRPD data.

Measurements of long-range intermolecular distances.

11

B nuclei, owing to their high

gyromagnetic ratio and high natural isotopic abundance, are particularly suited to probe intermolecular dipolar contacts. As demonstrated previously, the evolution of

11

B-11B double-

quantum (DQ) coherences allow determining 11B…11B distances up to ca. 4 Å.32 In the case of ixazomib citrate, the build-up of 11B-11B DQ coherence shows that the maximum signal intensity is reached at ca. tm = 5.6 ms for both polymorphic forms (Figure 6). By applying the previously derived relation,28 r = 0.23tm0.38, where r represents the 11B…11B interatomic distance and tm is the recoupling time for reaching maximum signal intensity, the obtained tm values indicate that the shortest

11

B…11B interatomic distances are typically 6.3-6.5 Å. Although this estimation is

approximate, the obtained distances correspond well with the values of 6.2-6.4 Å derived from the refined XRPD crystal structures (Figure 2).


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11

B- B DQ coherence build-ups

I, %

2.5 Å Borax

IXA Form-II

3.6 Å H3BO3

IXA Form-X

t, µs 0.38

r, Å

r = 0.23tm

IXA Form-X IXA Form-II

6.4-6.5 Å 3.6 Å 2.5 Å 1.7 Å

tmax, µs

Figure 6. 11B-11B DQC Build-Ups Recorded for Ixazomib Form II (Red Line) and IXA Form X (Black Line). DQ coherence build-ups for short

11

B…11B pairs (3.6 Å and 2.5 Å) are also

displayed for comparison (upper panel). The relation between the recoupling time for maximum DQ coherence intensity and the interatomic 11B…11B distance is displayed in the bottom panel.

Furthermore, when looking for additional markers of the crystal structures, hydrogen atoms located at molecular peripheries represent sensitive receptors of intermolecular contacts, which can be traced in 1H-1H correlation spectra.36 For ixazomib citrate, 1D 1H DUMBO NMR spectra reveal the existence of two types of COOH protons in citric acid residues (Figure 4c). In the corresponding 2D 1H-1H single-quantum (SQ)/DQ MAS NMR correlation spectra (Figure 7), the high-frequency carboxyl protons (COOH(1)) resonating at 12.3 and 10.7 ppm for Forms II and X, respectively, exhibit clear autocorrelation signals, indicating the presence of strongly hydrogen-bonded COOH(1)…(1)HOOC pairs. In contrast, the low-frequency carboxyl protons (COOH(2)) show clear dipolar contact with the aromatic protons, thus revealing secondary


18

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hydrogen bonding of citric acid residues with amide C=O groups attached to aromatic rings. These experimental findings are in excellent accord with the proposed crystal structures (Figure 8). 1

1

H- H SQ/DQ DUMBO MAS NMR



CH3

Ar(2) COOH(1)

COOH(2)

B-CH

Ar(1) NH

ppm

COOH(2) COOH(1) NH

ppm

-5

Ar(2) Ar(1)

B-CH

CH3

CH2

N-CH

0

0

5

COOH(1)…CH3

5

10

COOH(1)…CH3

10

15

COOH(2)…Ar(1)

15

ssb

20

20

COOH(2)…Ar(1) 25

COOH(1)…HOOC(1)

25

15 14 13 12 11 10

9

8

7

6

5

4

3

2

1

1

0

-1

COOH(1)…HOOC(1) 15 14 13 12 11 10

ppm

9

8

7

6

5

4

3

2

1

ppm

CH3

CH-OH

CH-OH

COOH(2)

-1

13

H- C HETCOR MAS NMR

Ar(1)

COOH(1)

0

Ar(1)

COOH(1)

ppm

ppm

0

0

CH3

2

2 4

CH-OH

4

=C-H(Ar1)

6

6

8

=C-H(Ar2)

8

10

COOH(2) 12

COOH(2) 10

COOH(1)

14 180

160

140

120

100

80

60

40

20

ppm

COOH(1)

12 180

160

140

120

100

80

60

40

20

ppm

Figure 7. 1H-1H DQ/SQ DUMBO MAS NMR and 1H-13C HETCOR MAS NMR Spectra of Ixazomib Citrate Form II and X.


19



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Weak H-bonding COOH(2)…O=C(Ar)

Strong H-bonding COOH(1)…(1)HOOC

Figure 8. Hydrogen-Bonding Motifs in Ixazomib Citrate Forms II and X.

Verifying the global molecular arrangement. As shown recently, solid-state NMR spectra also represent fingerprints from which complete crystallographic data can be extracted.37-39 First, solid-state NMR spectra, via the site-specific structural receptivity, allow for identification of different polymorphic forms and determination of the number of symmetry independent molecules in the unit cell. In our particular case, the signal splitting observed in

13

C CP/MAS

NMR spectra (Figure 4b; and Supporting Information S1, Figure S2) shows that the unit cells of both polymorphic modifications consist of two inequivalent ixazomib molecules and two inequivalent molecules of citric acid. The 11B TQ/MAS NMR spectra (Figure 4a) then reveal that the nonequivalence of boron centers is relatively small for Form II, whereas much more substantial differences in the coordination geometries exist in ixazomib citrate Form X. These findings thus clearly support the generated structural models. Second, systematic comparison of experimentally determined 1H NMR isotropic chemical shifts with such shifts calculated with DFT from the corresponding crystal structure models


20

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allows selection of the most suitable candidates.37-39 Agreement between theory and experiment was analyzed using linear regression when the quantitative measure was the r.m.s.d. From the literature data, the correct structure is clearly characterized by the smallest 1H r.m.s.d., less than 0.5 ppm. Although 13C chemical shifts were found to be less sensitive, the 13C r.m.s.d. indicating the correct crystal structure was always smaller than 2.5 ppm. Therefore, we used the same approach to validate refined XRPD crystal structures in the next step. First, the 1H and

13

C isotropic chemical shifts were calculated for both refined XRPD

models (Forms II and X) and then compared with the corresponding experimental data extracted from 2D 1H-13C HETCOR and 1H-1H DQ/SQ DUMBO MAS NMR spectra (Figure 7). Due to the splitting of

13

C NMR resonances caused by the presence of two symmetry-inequivalent

molecules in the unit cells, the signal assignment was made on the basis of an analysis of 1H-13C and 1H-1H correlation signals with the aid of automated prediction of 1H-13C correlation patterns.40 The obtained similarity parameters are summarized in Table 4, while further details are given in Supporting Information SI5.

Table 4. Comparison of Experimental and Calculated 1H and

13

C NMR Parameters for the

Refined XRPD Crystal Structures of Ixazomib Citrate Forms II and X. NMR similarity parameters Form II

Form X

r.m.s.d. (13C, COOH excl.), ppm

1.18

1.85

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

0.47

0.40

r.m.s.d. (13C, all), ppm

1.99

2.55

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

0.65

0.93

covariance for 1H-13C pairs, ppm2

0.24

0.11


21


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While determining the precise location of labile hydrogen atoms is difficult, even from synchrotron powder diffraction data, we excluded these labile species (COOH, OH and NH) from the comparative analysis. We thus obtained similarity parameters that clearly fall below the threshold limits, confirming that the refined XRPD backbone conformations and global molecular packing of ixazomib citrate data are consistent with the experimentally determined solid-state NMR data. However, when the labile species COOH, OH and NH are included in the comparative analysis, the r.m.s.d.s increased, reflecting certain inconsistencies in the locations of hydrogen-bonded protons. Whereas refined XRPD structures predict very strong hydrogen bonding between citric acids (COOH(1)…(1)HOOC), which is typically characterized by high isotropic chemical shifts (reaching ca. 16 ppm; 15.5 and 14.5 ppm were obtained from DFT calculations for Forms II and X, respectively), the corresponding experimental values are considerably lower (12.3 and 10.7 ppm), indicating weaker hydrogen bonding and longer intermolecular distances in these molecular fragments or the presence of thermal motions. Analysis of 1H NMR spectra thus provides additional input which may possibly improve structure determination from XRPD data.

Conclusions Boron-containing compounds have long been recognized as potentially active pharmaceutical ingredients. Uncertainties in the structure determination of these peptide analogs, however, represent an obstacle in developing the next generation of anticancer drugs. Determination of the atomic-resolution structures of these multicomponent solids in the absence of single crystals remains a challenge. When focusing on generally improving structure determination from X-ray powder diffraction data, we considered several aspects of current experimental and


22

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computational approaches. As a result of this analysis, we formulated and successfully applied an extended experimental-computational strategy to explore the crystal structures of complex peptide analogs of boronic acid derivatives, even those exhibiting extensive conformational variability. By combining an advanced analysis of powder diffraction data with a comparative analysis of NMR parameters and high-level quantum chemical calculations, we solved the complete crystal structures of two previously undescribed non-solvated polymorphs of ixazomib citrate. As both polymorphic forms consisted of two molecules in the asymmetric unit cell and thus exhibited 32 degrees of conformational freedom, solving the structure from powder diffraction data represented a challenging problem on the edge of the capabilities of current methodology. Consequently, a novel two-step Rietveld refinement based on DFT-D restraints was used to improve information quality derived from powder diffraction data. NMR crystallography was applied to verify the refined crystal structures. In this regard, the high potential value of 11B NMR parameters was demonstrated. Whereas the evolution of 11B-11B DQ coherences allows probing long-range molecular arrangements with interatomic distances up to ca. 7 Å, a comparative analysis of NMR parameters that were experimentally determined and calculated for the refined crystal structures gives strong support for the accuracy of the XRPDdetermined structures. Overall, an integrated approach that applies several techniques in conjunction, thus providing otherwise unavailable structural information, was developed and successfully demonstrated, resulting in an efficient tool for further advancement of highly active anticancer drugs based on peptidic analogs of boronic acid.


23


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Experimental Section Materials. Ixazomib citrate polymorphs Teva Form II and Teva Form X were obtained from Teva Pharmaceuticals CR (Czech Republic). The applied labeling of the investigated polymorphs strictly follows the terminology of Teva Pharmaceutical Industries Ltd. used in the process of polymorphs screening. Solid-state NMR spectroscopy. Solid-state NMR spectra were measured at 11.7 T using a Bruker Avance III HD 500 US/WB NMR spectrometer (Karlsruhe, Germany, 2013). The following techniques were applied: i) one-dimensional (1D)

11

B and 13C MAS, CP/MAS, and

CPPI/MAS NMR experiments;41 ii) two-dimensional (2D) experiments;42 iii) triple-quantum (TQ) experiments (2D

11

1

H-13C FSLG HETCOR

B TQ/MAS NMR);43 iv)

11

B-11B

double-quantum (DQ) experiments with the BR212 recoupling sequence;44,45 and v) 2D DQ/SQ 1

H-1H MAS NMR experiment46 with SPC5 DQ recoupling47 and DUMBO homodecoupling.48

Frictional heating49,50 of the spinning samples was offset by active cooling, and the temperature calibration was performed with Pb(NO3)2. For all experimental details, see Supporting Information S6. X-ray synchrotron powder data measurement. Powdered samples were placed in a thin-walled 1/1.5 mm capillary (Hilgenberg low-absorption borosilicate glass No. 50). The powder diffraction measurement was made at the ESRF synchrotron radiation source in Grenoble. Data were collected on the high-resolution powder X-ray diffraction beamline ID22 at wavelengths of 0.399978(2) Å (Form II) and 0.399963(3) Å (Form X) and were calibrated against NIST 640c standard silicon. The sample was spun on the axis of the diffractometer to optimize the powder averaging. Measurements were made at room temperature in continuous-scanning mode, from -5 to 35 degrees for 2theta (Form II) and from -5 to 28 degrees for 2theta (Form X) at 10 degrees


24

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per minute, sampling the diffraction pattern every 3 ms (0.0005 degrees). As a precaution against radiation damage, the sample was translated axially by 1.35 mm after each scan to expose fresh sample to the beam. Twenty-six scans were normalized, summed and rebinned into steps of 0.002 degrees, taking account of the exact offsets and efficiencies of the nine channels of detection and the decay in the storage ring current during the acquisition via an incident-beam monitor.7 Computational methods for isolated molecules. The in vacuo potential energy surface (PES) of ixazomib citrate was scanned in the part of the conformational space relevant for the coordination of the boron moiety. Thus, an extensive search for the minima of the PES of ixazomib citrate was performed using the DFT-based B3LYP/6-311G** method by employing numerous starting structures (not shown). This search yielded five minimal geometries that were subject to geometrical optimization at the resolution-of-the-identity (RI) second-order Møller– Plesset (MP2) level of quantum chemical theory. The resulting RI-MP2/TZVP structures were used to approximate their complete basis set (CBS) total energies (see Supporting Information SI3 for details). The CBS-extrapolated MP2 energy was then combined with the zero-point vibrational energy obtained for the corresponding B3LYP/6-311G** minimum to arrive at estimates of the absolute total energy, E. The Gaussian 09 and TURBOMOLE program packages were used for the DFT and MP2 calculations, respectively. Periodic computational methods. The obtained refined XRPD crystal structure geometries for both Forms II and X were the input for the NMR chemical shielding calculations, which were carried out by combining the PBE functional with the gauge-including projector augmented wave (GIPAW) method51,52 as implemented in the CASTEP-NMR module.12 In all calculations (the geometrical optimizations, followed by the calculations of the NMR parameters), the


25


Page 26 of 36

corresponding “Fine” level of the CASTEP settings was adopted. Additional computational details are provided in Supporting Information SI4.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Full-size diffractograms; Computational methods; Details of calculations of the total energy in vacuo; Details of DFT calculations of isotropic chemical shifts; The HETCOR spectra peakassignments aided with DFT calculations; Solid-State NMR Spectroscopy CIF file for XRPD refined crystal structure of Ixazomib Citrate Forms II and X (CSD Code CCDC 1822510 and 1822511). Accession Codes CCDC 1822510 and 1822511 contain 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.

AUTHOR INFORMATION Corresponding Author *Email: [email protected]


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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT The authors thank the Czech Science Foundation (grant nos. GA16-10035S and GA1604109S) and the Ministry of Education, Youth and Sports of the Czech Republic within the National Sustainability Program I (NPU I), Project LO1507 POLYMAT, for their financial support. Computational resources were partially provided under program LM2010005, and through the Centre CERIT Scientific Cloud, part of the Operational Program Research and Development for Innovations, reg. no. CZ.1.05/3.2.00/08.0144.

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For Table of Contents Use Only

Determining the crystal structures of peptide analogs of boronic acid in the absence of single crystals: Intricate motifs of ixazomib citrate revealed by XRPD guided by ss-NMR

Michal Hušák, Alexandr Jegorov, Jan Rohlíček, Andrew Fitch, Jiří Czernek, Libor Kobera, and Jiří Brus*

General experimental-computational strategy for exploring the assembly of peptidic boronic acid derivatives in microcrystalline state is demonstrated. By combining the simulated annealing and two-step Rietveld refinement of XRPD data with the comparative analysis of NMR parameters the crystal structures of two non-solvated polymorphs of ixazomib citrate, each consisting of two symmetry-independent molecules in unit cell with 32 free parameters, were solved and refined.


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Determining the Crystal Structures of Peptide Analogs of Boronic

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