Single Crystal Growth and Structure Determination of the Natural

Dec 20, 2011 - *(M.U.) Address: The Coca-Cola Company, P.O. Box 1734, Atlanta, GA 30301, USA. E-mail: [email protected]. Tel.: +1-404-676-2923. Fa...
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Single Crystal Growth and Structure Determination of the Natural “High Potency” Sweetener Rebaudioside A Mani Upreti,*,† Jared P. Smit,*,‡ Eric J. Hagen,‡ Valeriya N. Smolenskaya,‡ and Indra Prakash† †

The Coca-Cola Company, Atlanta, Georgia 30301, United States SSCI, a Division of Aptuit, West Lafayette, Indiana 47906, United States

‡

S Supporting Information *

ABSTRACT: The single crystal growth and structure determination of the natural high potency sweetener rebaudioside A is reported for the first time. The single crystal structure of a tetrahydrated monomethanolated form of rebaudioside A was determined to confirm the molecular structure. Three additional crystal forms were also identified by X-ray powder diffraction, and their powder patterns were indexed.

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INTRODUCTION Rebaudioside A (‘reb A’, Figure 1) is an ent-kaurene diterpene glycoside that is one of the major steviol glycosides derived

manufacturers in providing low to noncaloric food and beverage product options. The use of reb A is now widespread in many countries beyond South America and in Asia. Reb A sweetened products such as table-top sweeteners Truvia (Cargill, Wayzata, MN, USA) and PureVia (Whole Earth Sweetener LLC, South Bend, IN, USA) are rapidly gaining popularity and acceptance in the American marketplace by consumers. The isolation of reb A from S. rebaudiana leaves and its molecular structural characterization was first carried out in 1976 by Kohda et al.6 NMR studies have also been carried out to investigate the conformations of reb A,7 but no crystal structures of reb A are known to date. From rebaudioside purification of crude stevia (80% reb A) using aqueous alcoholic solutions, we have previously identified highly pure reb A (99+% pure) as a hydrated form (Form I) with poor aqueous solubility and as a variable solvated form (Form II) with higher aqueous solubility.8 As part of our research in the purification, structural characterization, and properties of natural noncaloric sweeteners, we have determined by single-crystal X-ray diffraction (SCXRD) the first three-dimensional crystal framework of reb A. The single crystal structure corresponds to a novel crystal form of reb A (designated Form III) and is being reported here for the first time: C45H82O28 [C44H70O23, 4(H2O), CH4O]. Additionally, a likely anhydrous crystal form of reb A (designated Form IV) has been identified as a single phase, and the unit cell parameters for Forms I, II, and IV have been

Figure 1. The molecular structure of Rebaudioside A. OH groups involved in intramolecular hydrogen bonding are shown in red.

from the leaves of Stevia rebaudiana (Bertoni) Bertoni (Asteraceae), a plant native to Brazil and Paraguay. Reb A has a clean sweet taste that is 200−300 times sweeter than sugar and is superior in taste qualities compared to the other sweet tasting steviol glycosides.1 The stability and metabolic pathway of reb A have been studied extensively, and the safety of high purity reb A has been successfully documented.2,3 Crude stevia extract has been available as a dietary supplement in United States since the mid-1990s, and in 2008 pure reb A was for the first time self-affirmed Generally Recognized as Safe (GRAS) status with a no-objection letter from the FDA.4,5 As consumers seek to reduce their caloric intake, high potency sweeteners such as reb A are critical ingredients for © 2011 American Chemical Society

Received: November 7, 2011 Revised: December 19, 2011 Published: December 20, 2011 990

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software v. 2.2b. Indexing was performed using either DICVOL0414 or proprietary SSCI software.

determined by indexing the X-ray powder diffraction (XRPD) patterns. Form IV is suggested to be anhydrous based on the method of preparation and the unit cell volume when compared with Forms I, II, and III.

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RESULTS AND DISCUSSION The Crystal Structure of Rebaudioside A Form III. Relevant crystallographic parameters for the single crystal structure of reb A Form III are listed in Table 1. The

EXPERIMENTAL SECTION

Single Crystal Growth of Form III. Reb A 97+% pure was used to grow crystals suitable for SCXRD analysis. The crystal was prepared from slow evaporation of a solution of reb A in HPLC-grade methanol. The container was left with a loose cover in a laboratory fume hood at ambient temperature. Evaporation was not taken to dryness, leaving crystals in their mother liquor. Form IV. Reb A samples which appeared to be mixtures of Form II and Form IV by XRPD were subjected to vacuum drying at 120 °C continuously for 2 days, and precaution was taken to avoid any exposure to air before conducting the XRPD analysis. Several Form IV samples originally prepared by vacuum drying at 120 °C appeared to contain an unknown peak at 4.6° 2θ by XRPD. This peak was present in the starting material and the postdried material and represents an unknown phase, possibly a chemical impurity. Forms I and II. Pure Forms I and II were present in the commercially available 97+% pure reb A samples. These forms were also obtained by aqueous ethanolic crystallizations of lower purity reb A material.8 Techniques. Single Crystal Analysis. A colorless needle of reb A Form III, C45H82O28 [C44H70O23, 4(H2O), CH4O] having approximate dimensions of 0.20 × 0.11 × 0.03 mm, was mounted on a fiber in random orientation. Preliminary examination and data collection were performed with Cu Kα radiation (λ = 1.54184 Å) on a Rigaku Rapid II diffractometer equipped with confocal optics. Refinements were performed using SHELX97.9 Cell constants and an orientation matrix for data collection were obtained from least-squares refinement using the setting angles of 27 063 reflections in the range 2° < θ < 70°. The space group was determined by the program XPREP.10 From the systematic presence of the following conditions: 0k0 k = 2n, and from subsequent least-squares refinement, the space group was determined to be P21 (No. 4). The data were collected to a maximum 2θ value of 140.35°, at a temperature of 150 ± 1 K. Frames were integrated with DENZO-SMN.11 A total of 27 063 reflections were collected, of which 7181 were unique. Lorentz and polarization corrections were applied to the data. The linear absorption coefficient is 0.992 mm−1 for Cu Kα radiation. An empirical absorption correction using SCALEPACK was applied.11 Transmission coefficients ranged from 0.796 to 0.971. Intensities of equivalent reflections were averaged. The agreement factor for the averaging was 6.9% based on intensity. The structure was solved by direct methods using SIR2004.12 The remaining atoms were located in succeeding difference Fourier syntheses. Hydrogen atoms were included in the refinement but restrained to ride on the atom to which they are bonded. The weight w is defined as 1/[σ2(Fo2) + (0.1026P)2 +(1.5848P)], where P = (Fo2 +2Fc2)/3. Scattering factors were taken from the International Tables for Crystallography.13 Of the 7181 reflections used in the refinements, only the reflections with Fo2 > 2σ(Fo2) were used in calculating R. A total of 6456 reflections were used in the calculation. XRPD Analysis. High resolution XRPD patterns were collected with a PANalytical X’Pert PRO MPD diffractometer using an incident beam of Cu radiation produced using an Optix long, fine-focus source. An elliptically graded multilayer mirror was used to focus Cu Kα X-rays through the specimen and onto the detector. Prior to the analysis, a silicon standard (NIST SRM 640d) was analyzed to verify the observed position of the Si 111 peak was consistent with the NISTcertified position. A specimen of the sample was sandwiched between 3 μm thick films and analyzed in transmission geometry. A beam-stop and short antiscatter extension were used to minimize the background generated by air. Soller slits for the incident and diffracted beams were used to minimize broadening from axial divergence. Diffraction patterns were collected using a scanning position-sensitive detector (X’Celerator) located 240 mm from the specimen and Data Collector

Table 1. Crystallographic Information for Rebaudioside A Form III, C44H70O23·4(H2O)·CH4O formula formula weight space group a, Å b, Å c, Å β, deg V, Å3 Z dcalc, g cm−3 μ, mm−1 (Cu Kα) R(Fo) [I > 2σ(I)], [all data] Rw(Fo2) [I > 2σ(I)], [all data]

C45H82O28 1071.14 P21 (No. 4) 16.4358(9) 8.0564(4) 19.5073(11) 100.120(4) 2542.8(2) 2 1.399 0.992 0.058; 0.064 0.157; 0.162

asymmetric unit contains one molecule of reb A, four water molecules, and one methanol molecule. One of the water molecules is disordered, refining to a 31%/69% ratio over two sites. Hydrogen bonding in the crystal structure of reb A Form III is very complex. There are 14 free hydroxyl groups among the 4 glucose rings, and each of them is involved in at least one hydrogen bond. The reb A molecule folds back on itself, with intramolecular hydrogen bonds between both O33 and O97 to O41, shown in Figures 1 and 2. These are the only

Figure 2. The intramolecular hydrogen bonding in the crystal structure of rebaudioside A Form III.

intramolecular hydrogen bonds in the structure. O32 is also connected to O41 through H-bonding with a water molecule, also shown in Figure 2. By folding back on itself, all of the hydrophilic glucose groups are located in close proximity. The water and methanol molecules reside along the crystallographic c axis (Figure 3) and are surrounded by the hydrophilic glucose groups. The hydrophobic steviol backbones of the reb A molecules are aggregated in between the solvent layers. XRPD Indexing and Crystallographic Unit Cell Parameters of Forms I, II, and IV. Representative XRPD 991

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Figure 3. Crystal packing diagram of rebaudioside A Form III showing the location of the water and methanol molecules.

Figure 4. XRPD patterns of rebaudioside A crystal forms. Top to bottom: Form I, Form II, Form III (calculated from the single crystal data), Form IV.

(1271.4 Å3 at 150 K) are similar and much larger than the volume per formula unit of Form IV (1192.6 Å3), consistent with the presence of solvent in Forms I, II, and III. Significant differences in XRPD peak positions are observed between various patterns of Form II. However, all of the Form

patterns for the four different crystal forms of reb A are shown in Figure 4, and their unit cell parameters are listed in Table 2. The unit cell parameters of Forms I, II, and IV have been determined from powder pattern indexing. The volume per formula unit of Forms I (1293.5 Å3), II (1253.6 Å3), and III 992

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CONCLUSION The first single crystal structure of reb A has been determined to confirm the molecular structure and corresponds to the tetrahydrated, monomethanolated crystal form. Additionally, three other crystal forms have been isolated, and their crystallographic unit cells have been determined. Forms I, II, and III are solvated crystal forms, and Form IV is likely an anhydrous form of reb A.

Table 2. Crystallographic Unit Cell Parameters for Rebaudioside A Crystal Forms form crystal system space group a (Å) b (Å) c (Å) α (°) β (°) γ (°) volume (Å3) source temperature

I monoclinic P21 (#4) 26.266 7.530 27.340 90 106.89 90 5174.1 powder indexing ambient

IIa monoclinic C2 (#5) 33.664 8.013 19.624 90 108.69 90 5014.4 powder indexing ambient

III monoclinic P21 (#4) 16.4358 8.0564 19.5073 90 100.12 90 2542.8 single crystal 150K

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IV triclinic P1 (#1) 7.958 17.527 17.919 95.92 99.38 102.25 2385.1 powder indexing ambient

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ASSOCIATED CONTENT

S Supporting Information *

The single crystal CIF is supplied. This material is available free of charge via the Internet at http://pubs.acs.org.

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a

AUTHOR INFORMATION

Corresponding Author

Form shows variable unit cell up to: a = 34.346 Å, b = 8.095 Å, c = 19.596 Å, β = 110.15°.

*(M.U.) Address: The Coca-Cola Company, P.O. Box 1734, Atlanta, GA 30301, USA. E-mail: [email protected]. Tel.: +1-404-676-2923. Fax: +1-404-598-2923. (J.S.) Address: SSCI, a Division of Aptuit, 3065 Kent Avenue, West Lafayette, IN 47906, USA. E-mail: [email protected]. Tel.: +1-765-4630112 x 6241. Fax: +1-765-463-4772.

II patterns index to similar unit cell parameters that differ slightly in the crystallographic a parameter and β angle (Table 2), suggesting that Form II represents a continuum of variable solvation states. The largest unit cell volume that has been observed for Form II was obtained from a sample of Form IV that was reanalyzed after 5 years of ambient storage and had converted to Form II. The unit cell parameters of Form II are similar to those of Form III. The planes formed by the b, c, and α parameters are the same in the conventional unit cell of both forms, but the conventional unit cell of Form II is centered and a is doubled. Although similar, Forms II and III appear to be unique crystal forms as the reduced unit cells are a = 8.013 Å, b = 17.302 Å, c = 19.624 Å, α = 108.16°, β = 90°, γ = 103.39° (Form II) and a = 8.0564 Å, b = 16.4358 Å, c = 19.5073 Å, α = 100.12°, β = 90°, γ = 90° (Form III). Hygroscopicity and Dehydration Behavior of Rebaudioside A Forms. Form IV is likely an anhydrous form of reb A, based on the method of preparation and comparison of XRPD indexing results obtained for Forms I, II, III, and IV. Form IV was obtained by vacuum drying Form II at 120 °C for approximately 2 days, and it has the highest aqueous solubility among the known forms.8 The high solubility makes this form of reb A attractive to food and beverage manufacturing applications for which the other forms are less suitable. However, Form IV is difficult to isolate owing to the highly hygroscopic nature of the material, sorbing up to 10% (by weight) in ambient moisture if left exposed to air on the lab bench. This moisture uptake converts Form IV back into Form II and would pose significant challenges in product formulation work using Form IV. When commercial reb A samples 97+% pure were used without any further purification by treatment with methanol, peaks that were inconsistent with the indexed unit cell parameters were often observed in the XRPD patterns. These peaks indicate the presence of an additional crystalline phase or phases, possibly from a chemical impurity. The additional phase was present in commercial samples of Form II before drying and remained present in samples of Form IV that resulted from drying. While the nature of these peaks is unknown, treatment with methanol appeared to remove the impurities and afforded a single crystalline phase of reb A.

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ACKNOWLEDGMENTS Thanks to Dr. Phillip Fanwick at Purdue University for single crystal data collection. REFERENCES

(1) Carakostas, M.; Prakash, I.; Kinghorn, A. D.; Wu, C. D.; Soejarto, D. D. In Alternative Sweeteners, 4th ed.; Nabors, L. B., Ed.; Marcel Dekker: New York, 2011; Chapter 11, pp 159−180. (2) Clos, J. F.; DuBois, G. E.; Prakash, I. J. Agric. Food Chem. 2008, 56, 8507−8513. (3) Roberts, A.; Renwick., A. G. Food Chem. Toxicol. 2008, 46, S31− S39. (4) Carakostas, M. C.; Curry, L. L.; Boileau, A. C.; Brusick, D. J. Food Chem. Toxicol. 2008, 46 (Suppl 7), S1−S10. (5) Cargill Inc. GRN No. 253, 2008 http://www.accessdata.fda.gov/ scripts/fcn/fcnNavigation.cfm?rpt=grasListing (accessed Dec 14, 2011). (6) Kohda, H.; Kasai, R.; Yamasaki, K.; Murakami, K.; Tanaka, O. Phytochemistry 1976, 15, 981−3. (7) Steinmetz, W. E.; Lin, A. Carbohydr. Res. 2009, 344, 2533−8. (8) Prakash, I.; Dubois, G. E.; King, G. A.; Upreti, M. Rebaudioside A composition and method for purifying rebaudioside A U.S. Patent Appl. U.S. 2007/0292582 2007. (9) Sheldrick, G. M. Acta Crystallogr., Sect. A 2008, 64, 112−122. (10) XPREP in SHELXTL, v. 6.12; Bruker AXS Inc.: Madison, WI, USA, 2002. (11) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307− 326. (12) Burla, M. C.; Caliandro, R.; Camalli, M.; Carrozzini, B.; Cascarano, G. L.; De Caro, L.; Giacovazzo, C.; Polidori, G.; Spagna, R. J. Appl. Crystallogr. 2005, 38, 381−388. (13) International Tables for Crystallography; Wilson, A. J. C., Ed; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1992; Vol. C, Tables 4.2.6.8 and 6.1.1.4. (14) Boultif, A.; Louër, D. J. Appl. Crystallogr. 2004, 37, 724−731.

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