# Insights into the Crystallization and Structural Evolution of Glycine Dihydrate by In Situ Solid-State NMR Spectroscopy

Paolo Cerreia Vioglio, Giulia Mollica, Marie Juramy, Colan E. Hughes, P.

Andrew Williams, Fabio Ziarelli, Stephane Viel, Pierre Thureau, Kenneth D.

M. Harris

► To cite this version:

Paolo Cerreia Vioglio, Giulia Mollica, Marie Juramy, Colan E. Hughes, P. Andrew Williams, et al. Insights into the Crystallization and Structural Evolution of Glycine Dihydrate by In Situ Solid-State NMR Spectroscopy. Angewandte Chemie International Edition, 2018, 57 (22), pp.6619–6623. 10.1002/anie.201801114. hal-02091904

HAL Id: hal-02091904

https://amu.hal.science/hal-02091904

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## Crystallization

# Insights into the Crystallization and Structural Evolution of Glycine Dihydrate by In Situ Solid-State NMR Spectroscopy

Paolo Cerreia Vioglio, Giulia Mollica, Marie Juramy, Colan E. Hughes, P. Andrew Williams, Fabio Ziarelli, Stéphane Viel, Pierre Thureau,* and Kenneth D. M. Harris*

**Abstract:** In situ solid-state NMR spectroscopy is exploited to monitor the structural evolution of a glycine/water glass phase formed on flash cooling an aqueous solution of glycine, with a range of modern solid-state NMR methods applied to elucidate structural properties of the solid phases present. The glycine/water glass is shown to crystallize into an intermediate phase, which then transforms to the β polymorph of glycine. Our in situ NMR results fully corroborate the identity of the intermediate crystalline phase as glycine dihydrate, which was first proposed only very recently.

Polymorphism in glycine was first observed, although not fully understood, by Fischer in 1905.[1] Although originally ascribed to possible isomerism, his observations are now attributed to the existence of the α and β polymorphs of glycine.[2] A third polymorph (γ polymorph) was discovered in 1954.[3] Except for three further polymorphs formed at high pressure,[4] no new solid form of glycine was reported until over 60 years later, with the very exciting discovery and structural characterization of a crystalline dihydrate of glycine, reported by Xu et al. in 2017.[5]

In general, crystallization of glycine from pure aqueous solution at neutral pH gives the α polymorph,[6] which transforms to the stable γ polymorph over time.[7] Crystallization from water in nano-confined environments promotes the formation of the β polymorph.[8] In spite of extensive studies, no hydrated crystalline phase of glycine was reported until the recent study by Xu et al.[5] In contrast, hydrate phases of several other amino acids are well known,[9] and interconversion processes between anhydrous and hydrate phases have been studied in detail.[9e,f] For certain amino acids

(e.g., L-lysine and L-arginine), the anhydrous solid form is very hygroscopic and transforms readily to a hydrate phase under normal atmospheric conditions.[10]

The recent work[5] that proved the existence of glycine dihydrate (GDH) relied on a preparation procedure reported by Pyne and Suryanarayanan[11] and later by Surovtsev et al.[12] In this procedure, an aqueous solution of glycine is quench frozen in liquid nitrogen to form a glycine/water glass phase (frozen solution). When this glass phase is warmed to around 209 K and held at this temperature, crystallization occurs to give a new solid phase. Xu et al.[5] prepared the new crystalline phase in situ in a synchrotron powder X-ray diffractometer and determined the structure directly from the powder XRD data, revealing that the new phase is a dihydrate of glycine. It was also shown[5] that, on heating above 250 K, this phase transforms to the β polymorph of glycine with loss of water.

In the present work, the procedure reported previously[5,11,12] has been carried out inside an NMR spectrometer, exploiting in situ solid-state NMR techniques to monitor the structural evolution, as a function of temperature and time, of the glycine/water glass formed after flash cooling an aqueous solution of glycine. In recent years, in situ NMR techniques for monitoring crystallization processes have been applied to a range of systems,[13] including crystallization of glycine from aqueous solution near ambient temperature.[13a,b] A significant advantage of solid-state NMR spectroscopy for in situ studies of such processes is that it allows the identification and characterization of both amorphous and crystalline phases. Thus, the in situ solid-state NMR study presented here offers the prospect of monitoring details of the structural evolution from the glycine/water glass phase to the dihydrate of glycine and then to the β polymorph of glycine. Furthermore, a range of modern solid-state NMR techniques are exploited to elucidate specific structural aspects of the solid phases present at different stages of the process.

Our in situ solid-state ¹³C NMR study used a sample of glycine ¹³C-labelled in the carboxylate group (1-¹³C-glycine; Scheme 1); thus, we focus on the carboxylate region of the ¹³C NMR data. An aqueous solution (1.8 M) of 1-¹³C-glycine

<figure><img src="image_2.png" /><figcaption>**Scheme 1.** Molecular structure of glycine (the asterisk indicates the labelled ¹³C site in 1-¹³C-glycine).</figcaption></figure>

[*] P. Cerreia Vioglio, Dr. G. Mollica, M. Juramy, Prof. S. Viel, Dr. P. Thureau
Aix Marseille Univ, CNRS, ICR
Marseille (France)
E-mail: pierre.thureau@univ-amu.fr
Dr. C. E. Hughes, Dr. P. A. Williams, Prof. K. D. M. Harris
School of Chemistry, Cardiff University
Park Place, Cardiff, Wales, CF10 3AT (UK)
E-mail: HarrisKDM@cardiff.ac.uk
Dr. F. Ziarelli
Aix Marseille Univ, CNRS, Centrale Marseille, FSCM
Marseille (France)
Prof. S. Viel
Institut Universitaire de France
Paris (France)

<figure><img src="image_3.png" /></figure>

Supporting information and the ORCID identification number(s) for

the author(s) of this article can be found under:

https://doi.org/10.1002/anie.201801114.
prepared at ambient temperature was flash cooled in a few seconds to ca. 145 K inside the NMR spectrometer. The frozen glycine/water solution was then warmed slowly (2 K min⁻¹) and ¹H→¹³C CPMAS NMR spectra were recorded (MAS frequency, 8 kHz[¹⁴]) at several (fixed) temperatures during warming (Figure 1). At lower temper-

<figure><img src="image_4.png" /><figcaption>**Figure 1.** ¹H→¹³C CPMAS NMR spectra for an aqueous solution of 1-¹³C-glycine following flash cooling to 145 K and then slow warming to 215 K. The spectra were recorded at: (a) 165 K, (b) 175 K, (c) 210 K, (d) 211 K (recorded immediately on reaching this temperature), (e) 211 K (recorded after 3 min at this temperature) and (f) 215 K (recorded after 25 min at this temperature).</figcaption></figure>

atures (e.g., 165 K; Figure 1a), a broad signal centered at 173.9 ppm is observed and is attributed to the glycine/water glass phase. However, on warming to 211 K, a significantly sharper peak appears at 173.5 ppm, with gradual loss of the broad peak due to the glass phase. The position of the sharp peak does not match the ¹³C NMR spectra of the α, β, or γ polymorphs of glycine under similar operating conditions (see Figure S1 and Table S1; Supporting Information).

Similar behavior is observed in in situ ¹H→¹⁵N CPMAS NMR spectra for an aqueous solution (1.8 M) of ¹⁵N-labelled glycine subjected to the same temperature schedule (Figure S3). The isotropic ¹⁵N chemical shift (30.3 ppm) of the crystalline phase that emerges at 211 K is clearly distinct from those of the α, β and γ polymorphs of glycine (Figure S2 and Table S2).[¹⁵]

The results from another in situ solid-state ¹³C NMR study at a different set of temperatures are shown in Figure 2. After flash cooling the aqueous solution of 1-¹³C-glycine and then warming to 210 K, the sharp peak due to the new crystalline phase again appears at 173.5 ppm (Figure 2a). However, after a total of 375 min at 210 K, a new peak emerges at 175.3 ppm, characteristic of the β polymorph (Figure 2b). The transformation to the β polymorph continues even on reducing the temperature to 205 K (Figure 2c,d) and continues more rapidly at higher temperatures (Figure 2e,f). By the end of

the experiment (at 222 K), the transformation to the β polymorph is virtually complete.

The evolution of the solid crystalline phases observed in our in situ solid-state NMR studies mirrors the behavior reported in the powder XRD study of Xu et al.[⁵] In their work, using the same procedure[¹¹,¹²] of flash cooling and an aqueous solution of glycine below 145 K followed by slow warming to 209 K, a new powder XRD pattern was observed and the crystal structure of the new crystalline phase was determined from the powder XRD data as a dihydrate of glycine (GDH). On further warming of the sample to 250 K, the powder XRD pattern of GDH disappeared and was replaced by that of the β polymorph of glycine.[⁵,¹⁶] Given the similarity between our results and those of Xu et al.[⁵] concerning the evolution of solid phases starting from the glycine/water glass phase, progressing to a new crystalline phase and then to the β polymorph of glycine, we conclude that the new crystalline phase giving the ¹³C NMR signal at 173.5 ppm and the ¹⁵N NMR signal at 30.3 ppm in our work is GDH.

We now consider specific structural aspects of GDH inferred from our in situ NMR studies and results from additional NMR experiments, which further support this assignment.

First, the reported crystal structure of GDH[⁵] has one molecule of glycine in the asymmetric unit (Z' = 1). Our solid-state NMR results are fully consistent with this assignment, as the ¹H→¹³C and ¹H→¹⁵N CPMAS NMR spectra of GDH contain only one isotropic peak for each of the CO₂⁻, CH₂ and NH₃⁺ environments.

Second, a significant feature of the reported crystal structure of GDH[⁵] is that glycine molecules are hydrogen-bonded only to water molecules, with no direct glycine-

<figure><img src="image_5.png" /><figcaption>**Figure 2.** ¹H¹³C CPMAS NMR spectra for an aqueous solution of 1-¹³C-glycine following flash cooling to 145 K and then slow warming to 210 K. The spectra were recorded according to the following time/temperature sequence: (a) after 150 min at 210 K, (b) after a further 225 min at 210 K, (c) after 360 min at 205 K, (d) after a further 285 min at 205 K, (e) after 50 min at 210 K and then 50 min at 212 K, and (f) after 50 min at 222 K.</figcaption></figure>

<figure><img src="image_6.png" /></figure>
glycine hydrogen-bonding. To investigate whether the material assigned as GDH in our in situ NMR study is consistent with this feature, we have carried out 2D ¹H-¹³C and ¹H-¹⁵N HETCOR experiments at 212 K (Figures 3 and S4, respec-

tively) on this material and the material (assigned as the β polymorph) to which it transforms over time. In the ¹H-¹³C HETCOR spectrum (Figure 3), a strong correlation peak is observed between the CO₂⁻ group of glycine (¹³C) and water molecules (¹H), consistent with glycine and water molecules in intimate proximity in a crystalline phase. In contrast, for the phase (β polymorph) into which GDH transforms over time, no correlation peak is observed between the carboxylate group of glycine (¹³C) and water molecules (¹H), fully consistent with the assignment that this phase is not a hydrate phase of glycine. Together with the other evidence presented above, these observations are fully consistent with the assignment that the initially formed crystalline phase is GDH, which transforms over time to the (anhydrous) β polymorph of glycine. We note that, for the β polymorph, no correlation peak is observed between ¹H in the NH₃⁺ group and ¹³C in the CO₂⁻ group as a consequence of rotational motion of the NH₃⁺ group. As discussed in SI, three resonances are observed for the NH₃⁺ group in the ¹H NMR spectrum of the β polymorph at 200 K, but coalescence occurs in the temperature regime around 212 K due to rotation of the NH₃⁺ group, producing a very broad peak that is not seen in the contour plot in Figure 3. We note that rotational dynamics of NH₃⁺ groups has been observed for several amino acids in the solid state,[17] including the α and γ polymorphs of glycine.[17a,d]

The corresponding ¹H-¹⁵N HETCOR spectrum (Figure S4) also shows a correlation peak between the NH₃⁺

group of glycine (¹⁵N) and water molecules (¹H), further supporting our assignment that the initially formed crystalline phase contains glycine and water molecules in intimate proximity and consistent with the assignment of this phase as GDH.

Finally, we show that studies of ¹³C-¹³C dipolar coupling[¹⁸] allow a more direct structural assignment of the new crystalline phase as GDH. In this work, experimental ¹³C-¹³C dipolar build-up curves were recorded using the POST-C7 recoupling pulse sequence,[¹⁹] with phase cycling of the reconversion block adjusted to filter zero quantum (ZQF) or double quantum (DQF) signals. The DQF/ZQF intensity ratio provides an experimental dipolar build-up curve that may be compared to the theoretical ¹³C-¹³C dipolar build-up curve calculated for a crystal structure using well-established methods.[¹⁸,²⁰] Figure 4 shows the experimental ¹³C-¹³C dipolar

build-up curve for GDH (containing 1-¹³C-glycine). The dependence of the ¹³C-¹³C dipolar build-up curve on the recoupling time τDQ depends only on the ¹³C-¹³C couplings between labelled ¹³CO₂⁻ sites in this material, which depend on the intermolecular ¹³C···¹³C distances between carboxylate groups. The theoretical ¹³C-¹³C dipolar build-up curve for GDH, calculated from the intermolecular ¹³C···¹³C distances in the reported crystal structure of GDH,[⁵] is also shown in Figure 4. The agreement between experimental and calculated dipolar build-up curves is excellent, giving strong corroboration that the hydrated crystalline phase of glycine observed in our in situ NMR study is indeed GDH.

To illustrate that the ¹³C-¹³C dipolar build-up curve is sensitive to structural properties, Figure 4 also shows experimental and calculated ¹³C-¹³C dipolar build-up curves for 1-¹³C-glycine in a different material (the α polymorph). Again, good agreement is observed between experimental and calculated data. The fact that the DQF/ZQF curve builds up more slowly for GDH than for the α polymorph is consistent with the intermolecular ¹³C···¹³C distances being higher in GDH, as the glycine molecules are separated by intervening water molecules (shortest internuclear ¹³C···¹³C distance: α polymorph, 3.10 Å; GDH, 3.81 Å).

<caption>**Figure 3.** ¹H-¹³C HETCOR spectrum recorded at 212 K after flash cooling an aqueous solution of 1.¹³C-glycine to 145 K and then slow warming to 212 K. Homonuclear ¹H decoupling was applied during t₁ and a mixing time of 0.5 ms was used under Lee-Goldburg conditions. This spectrum was recorded during the in situ study shown in Figure 2 (corresponding to the period of 50 min at which the sample was at 212 K; see caption of Figure 2e). Clearly some transformation of GDH into the β polymorph occurred during measurement of this spectrum (as confirmed from ¹H-¹³C CPMAS NMR spectra recorded before and after the ¹H-¹³C HETCOR experiment). The extracted ¹H NMR spectra of GDH and the β polymorph are shown at the right hand side (¹H chemical shifts of GDH: 2.5 ppm, CH₂; 8 ppm, NH₃⁺; 5 ppm, H₂O).</caption>

<figure><img src="image_7.png" /><figcaption>**Figure 4.** Experimental ¹³C-¹³C DQF/ZQF dipolar build-up curves plotted against recoupling time τDQ for GDH (♦) and the α polymorph (●), in each case containing 1.¹³C-glycine. Theoretical DQF/ZQF dipolar build-up curves calculated for the crystal structures of GDH (grey dashed line) and the α polymorph (black line) are also shown.</figcaption></figure>
Our results demonstrate the advantages of exploiting in situ solid-state NMR techniques to elucidate details of structural evolution during crystallization processes, reporting the first application of such techniques to explore crystallization of an organic material within a glass phase (“frozen solution”) formed on flash cooling a liquid solution. Importantly, our results fully corroborate the existence of GDH as a crystalline intermediate on the transformation pathway from the glycine/water glass phase to the β polymorph, and highlight the complementarity between our in situ solid-state NMR strategy[21] and the in situ powder XRD approach that was first used to identify this new solid phase of glycine very recently.[5] Clearly, a major objective is now to establish whether GDH serves any role in mechanistic pathways for the crystallization of glycine from aqueous solution under ambient conditions.

## Acknowledgements

We thank Bruker SAS for access to the solid-state NMR spectrometer used here and Dr F. Aussenac and Dr P. Dorffer for experimental assistance. This work was supported by the French National Research Agency grant ANR-13-JS080001 and by Cardiff University. K.D.M.H. is grateful to Dr Tony Hu for stimulating discussions on the discovery of GDH.

## Conflict of interest

The authors declare no conflict of interest.

**Keywords:** crystallization · glycine · polymorphism · solid-state NMR spectroscopy · structural transformation

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<figure><img src="image_8.png" /></figure>
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[21] We note that in situ solid-state NMR allows direct observation (in an isotope-specific manner) of the crystalline and amorphous phases present in the system as a function of time, yielding information on specific structural and dynamic aspects of these phases. By focusing on in situ ¹³C and ¹⁵N NMR, our measurements selectively detect only the phases that contain glycine. Thus, our results are not complicated by the fact that pure ice phases may be present in significant amounts during the processes studied here.

Manuscript received: January 26, 2018

Revised manuscript received: March 1, 2018

Accepted manuscript online: April 6, 2018

Version of record online: ■■■, ■■■■
Communications

<figure><img src="image_9.png" /></figure>

Crystallization

P. Cerreia Vioglio, G. Mollica, M. Juramy, C. E. Hughes, P. A. Williams, F. Ziarelli, S. Viel, P. Thureau,* K. D. M. Harris*

<figure><img src="image_10.png" /></figure>

Insights into the Crystallization and Structural Evolution of Glycine Dihydrate by In Situ Solid-State NMR Spectroscopy

<figure><img src="image_11.png" /></figure>

**Phase Transition:** the . Following flash cooling of an aqueous solution of glycine, in situ solid-state NMR spectroscopy is exploited to study the structural evolution from a glycine/water glass phase to crystalline glycine dihydrate and then to the β polymorph of anhydrous glycine. That way formation and structural properties of a crystalline dihydrate phase of glycine, which was first reported only very recently can be explored.

<figure><img src="image_12.png" /></figure>

Kristallisation

P. Cerreia Vioglio, G. Mollica, M. Juramy, C. E. Hughes, P. A. Williams, F. Ziarelli, S. Viel, P. Thureau,* K. D. M. Harris*

<figure><img src="image_13.png" /></figure>

Insights into the Crystallization and Structural Evolution of Glycine Dihydrate by In Situ Solid-State NMR Spectroscopy

<figure><img src="image_14.png" /></figure>

**Phasenumwandlung:** Nach Blitzkühlung einer wässrigen Glycinlösung wurde mittels In-situ-Festkörper-NMR-Spektroskopie die strukturelle Umwandlung der Glycin/Wasser-Glasphase zu kristallinem Glycindihydrat und dann zum β-Polymorph von wasserfreiem Glycin verfolgt. So konnten die Bildung und strukturellen Eigenschaften einer erst kürzlich beschriebenen kristallinen Dihydratphase von Glycin untersucht werden.

<figure><img src="image_15.png" /></figure>

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Please check that the ORCID identifiers listed below are correct. We encourage all authors to provide an ORCID identifier for each coauthor. ORCID is a registry that provides researchers with a unique digital identifier. Some funding agencies recommend or even require the inclusion of ORCID IDs in all published articles, and authors should consult their funding agency guidelines for details. Registration is easy and free; for further information, see http://orcid.org/.

Paolo Cerreia Vioglio

Dr. Giulia Mollica

Marie Juramy

Dr. Colan E. Hughes

Dr. P. Andrew Williams

Dr. Fabio Ziarelli

Prof. Stéphane Vioglio

Dr. Pierre Thureau

Prof. Kenneth D. M. Harris http://orcid.org/0000-0001-7855-8598