Single crystals of dextran: 1. Low temperature polymorph

Single crystals of dextran: 1. Low temperature polymorph H. Chanzy and C. Guizard Centre de Recherches sur les Macromol~cules V~ggtales, Laboratoire propre du CNRS associ~ l'Universit~ Scientifique et M~dicale de Grenoble, 53X, 38041 Grenoble Cedex, France

and A. Sarko Department of Chemistry, State University of New York, College of Environmental Science and Forestry, Syracuse, New York 13210, USA

(Received 14 December 1979) Lamellar single crystals of a synthetic, linear dextran and a slightly branched, bacterial dextran were 9rown at 95°C, by seeding their metastable solutions in mixtures of polyethylene glycol and water. The crystals gave well-resolved electron diffraction diagrams from which the unit cell parameters a ~ 2.563 nm and b ~ 1.021 nm were determined. The patterns displayed Pgg symmetry in a b projection. A comparison of the electron and X-ray powder dif.fraction diagrams indicated either 0.81 or 1.62 nm as the probable value Jbr the c parameter of the unit cell.

Introduction The determination of the shape and conformation of polysaccharides is important for the understanding of their properties and biological function 1. For regular polysaccharides, information on the molecular conformation can be obtained in several ways. An experimental approach, in the form of diffraction analysis, is possible if the polysaccharide can be crystallized. If an oriented Xray fibre diffraction diagram can be recorded, a detailed experimental determination of the molecular and crystal structure can be expected and the results may be compared with the theoretical predictions. Often, it is found that the predicted and actual conformations are in relatively close agreement and reside in the same potential energy minimum. In the case of dextran, the linear (1 ~6)-a-D-glucan, conformational analysis has shown that a large number of nearly equally probable conformations can be obtained as a function of the three rotations, ~o, ~ and o (see Figure 1)2'3 . It has not been possible, however, to obtain X-ray fibre patterns to confirm any of these conformations. Although dextran appears to crystallize in a series of polymorphs 4, only X-ray powder diagrams have been obtained to date 4- lo The present work describes how single crystals of dextran, suitable for electron diffraction analysis, can be prepared from a native, bacterial dextran as well as a synthetic dextran polymerized according to the method of Schuerch and Uryu 11. Synthetic dextran has the advantage of being unbranched and of a high degree of stereoregularity. It is, therefore, the material of choice for crystallization studies. The dextran polymorph described here is the 'low temperature polymorph' obtained at temperatures below 120°C. Above that, a 'high temperature polymorph' is obtained, which will be described in a future communication ~2 0141 8130/80/030149-05502.00 © 1980 IPC Business Press

Expeiimental Materials

The bacterial dextran fraction (originally from Pharmacia) was provided by Dr E. D. T. Atkins of the University of Bristol. It had a 57'/wof 19 900, or a degree of polymerization, DP, of ~ 120. It was slightly branched with approximately 5~o of the material in the branches. The synthetic dextran was prepared by cationic polymerization as outlined below. Polymerization and characterization o f the synthetic dextran. The procedure of Schuerch and Uryu was

followed in all details ~1. It involved the preparation of a dry monomer, 1,6-anhydro-2,3,4-tri-O-benzyl-fl-Dglucopyranose, the catalyst PF 5, by thermal decom-

NO~

O\

•

~0.

o~(I-6)Glucan(Dextran) Figure ! The structure of dextran. Note that three interresidue rotations, ~o, ¢ and o~, are possible

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Single crystals of dextran (1): H. Chanzy et al. position of 4-chlorobenzene diazonium hexafluorophosphate (Aldrich), and the purification of methylene chloride (Fluka, puriss grade), by refluxing with oleum prior to neutralization, followed by distillation over Call 2 and storage o v e r P205. The polymerization was conducted with 4 g of monomer, 8 cm 3 of methylene chloride and 10~o of PF 5 (w/w of monomer). The polymerization temperature was maintained at - 60°C and total gelation occurred within three hours. The yield was 3.6 g (90~o) of 2,3,4-tris-O-benzyl dextran which was recovered and debenzylated according to Schuerch and Uryu ~1. The final yield was 1.3 g of dextran (80~o), after freeze-drying from a water solution. The 2,3,4-tri-O-benzyl dextran contained 2% of/~-(1 --,6) linkages, as indicated by n.m.r, spectroscopy*. Its intrinsic viscosity in chloroform at 30°C was 0.365 dl g- 1, corresponding to N/L,= 110000 6, and its optical rotation in chloroform [-c~]~)5= +94". After debenzylation, the intrinsic viscosity in water at 25~C was 0.119 dl g 1. This gave a DP of around 60 assuming that the viscosity law [ q ] = 4 . 9 3 x 10 4 Mn0.6o derived by Gekko et al. 13'14 from branched dextran can apply to linear dextran.

Crystallization A seeding technique was used throughout this work. The initial crystallization provided the seeds which were used to induce crystallization in metastable dextran solutions. The quality of the crystals was improved by repeating the crystallization as many times as was necessary.

Initial crystallization. The initial crystallization was achieved from a 0.05~o solution (w/v) of freeze-dried dextran in water, An amount of poly(ethylene glycol) (PEG 300 from Prolabo, freshly purified by high speed centrifugation) was added to this solution at room temperature to bring the final concentration to 40/60 (v/v of PEG/water). The solution became turbid and was heated in a sealed container to 160°C, maintaining it at this temperature for one hour. It was then quenched at 95°C where crystallization occurred within two hours. The crystals were collected, washed by repeated centrifugation and stored in methanol. Seedin9 of metastable dextran solutions. Metastable dextran solutions were prepared as outlined above but with a PEG/water ratio of 45/55 (v/v). The turbid solutions were heated at 180°C in a sealed container for one hour. The resulting clear solutions had apparently lost all memory of crystallization, a s they remained clear indefinitely at as low as room temperature. Crystallization could be achieved by seeding these metastable solutions at 95°C with nuclei of dextran crystals. The seeds were prepared by sonicating a suspension of the initial crystals in methanol. The sonication was continued for 10 min with a Branson B12 Sonifier equipped with a microtip probe, at intensity 5. Drops of suspension containing the broken crystals were filtered through a sintered glass filter of porosity 4, directly into the metastable dextran solutions thermostatted at 95°C. Total crystallization took place within two hours. This procedure was repeated several times, with the crystals of each succeeding batch being used as nuclei for the next one. Suitable dextran single crystals were finally *

Courtesy of Dr M. Vignon, using a 250 MHz Cameca.

150 Int. J. Biol. Macromol., 1980, Vol 2, June

obtained after eight generations. The crystals were washed by repeated centrifugation and stored in methanol.

Electron microscopy Drops of the crystal suspension in methanol were deposited on carbon coated grids. The crystals were observed using a Philips EM 300 electron microscope operating at 100 kV for diffraction and 80 kV for imaging. The crystal preparations designed for imaging were shadowed with a W/Ta alloy, using a Balzers electron gun. The thickness of the crystals was computed from the shadowcast length, using latex spheres as calibration. No special technique 15 was required to record the electron diffraction patterns which were stable in the vacuum of the electron microscope. The patterns were calibrated with gold diffraction lines.

X-ray diffraction Powder X-ray diagrams were recorded either with a Warhus flat-film vacuum camera or a Debye-Scherrer camera for higher precision, using CuK~ radiation. Some of the patterns were recorded with the sample maintained in a controlled humidity atmosphere. The patterns were calibrated with diffraction lines from calcite.

Results and discussion Growth of the crystals Following the work of Kochetkov et al. 9, it was confirmed that mixtures of water and PEG were suitable for solubilizing dextran at high temperatures and crystallizing it upon cooling. As expected, it was found that the crystallization temperature varied directly with the P E G content of the solution. In the present work, the optimum mixture was 40/60 PEG/water (v/v), with a crystallization temperature of 95°C. With another specimen of different DP, not considered here, the PEG/water ratio had to be modified in order to achieve the crystallization at this temperature. 95°C represented a compromise between a high crystallization temperature which favoured good crystal geometry, and a preparation without the presence of the high temperature polymorph. A fraction of the latter was persistently obtained at crystallization temperatures exceeding 95°C. The critical step in the preparation of dextran single crystals remained the providing of suitable crystallization nuclei. Only in their presence could rapid and controlled crystallization be achieved at the desired temperature. In the absence of such nuclei, the solutions remained metastable for months, even when an excess of P E G was added and they were cooled far below their normal crystallization temperature. In this respect, dextran resembled such polymers as polyethylene and poly(oxyethylene), which can be crystallized with elegant seeding and selfseeding techniques 16- i~. In contrast, however, dextran could only be crystallized with its own crystal fragments as seeds, and not with heterogeneous nuclei. It was also important that the solution be freed of all previous crystalline nuclei prior to seeding; for this reason, it was necessary to autoclave the solutions at 180°C for one hour. At higher temperatures, thermal decomposition of dextran may occur 1s. The repetition of the crystallization sequence was also necessary to obtain well-formed single crystals.

Single crystals of dextran (1): H. Chanzy et al.

Figure 2 Single crystals ofdextran obtained at 110C in a solution containing 50'~',~iwater and 50'~i (v/v) of PEG. The solutions were heated to 180°C and seeded at 110~C with fourth generation crystals of synthetic dextran. (a) Synthetic dextran; (b) bacterial dextran

Figure 3 Single crystals of synthetic dextran obtained at 95C in a solution containing 55~0 water and 45°J0 (v/v) PEG. The solution was heated to 180'~Cand seeded with eighth generation crystalline seeds. Shadowcast with W/Ta alloy

Typical fourth generation crystals of both synthetic and bacterial dextran are shown in Figure 2. The crystals of synthetic dextran (Figure 2a) are in the form of thick lozenges roughly 2/~m in their longest diagonal and an acute angle close to 75 °. They are a few hundred nm thick, as estimated from their lack of transparency in the 100 kV electron beam. All crystals have roughly the same size and shape as is expected with self-seeding techniques. The crystals of bacterial dextran (Figure 2b) resemble those of synthetic dextran. Their overall shape, however, is more oval than lozenge-like and their thickness and size vary

greatly, with the latter in the range between one and several microns. The synthetic dextran crystals reached their final shape and perfection after the eighth generation (Figure 3), beyond which further improvement was not obtained. The crystals are nearly square (the acute angle is now ~ 85 °) and their edge dimension is ~ 1 ~m. They consist of stacks of about 10 to 12 lamellae, grown through screw dislocation. Each lamella has a thickness of the order of 6 to 7 nm, a value often encountered with unsubstituted polysaccharides 19'2°. As is usual for polymer single crystals 21, the chain axes are presumed to be oriented perpendicular to the plane of the lamellae. Such crystal geometry is consistent with a chain folding mechanism during the growth of the crystals. The folding of chains which is likely in view of the apparent flexibility of the dextran molecule 2 cannot be concluded decisively since the crystals presented above have been prepared with dextran of relatively low DP and only a few monolamellar crystals are present. With thicker muitilamellar crystals as found here, the chains may very well extend from one lamella to the next without going through a chain folding mechanism.

Crystallographic parameters When a crystal such as that shown in Figure 3 was subjected to electron diffraction, a good quality diagram could be obtained without difficulty (Figure 4). The diffraction in Figure 4b contains over 300 reflections symmetrically distributed about the two axes a* and b* oriented along the diagonals of the crystal; this gives a set

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Single crystals of dextran (1): H. Chanzy et al.

,@,

t

o

L

0,s

Figure 4 (a) Single crystal of synthetic dextran obtained as in Figure 2; (b) the corresponding electron diffraction diagram. Orientations of the crystal and the diagram are indicated

of about 80 independent reflections in each quadrant of the diagram. From measurements on calibrated diagrams, the lengths of the two perpendicular lattice parameters are a=2.563+0.005 nm and b=1.021_+0.005 nm, and reflections are seen to extend down to 0.18 nm. Along the a* and b* axes only even-order reflections appear, suggesting Pgg two-dimensional symmetry in projection down the chain axis. With bacterial dextran, similar electron diffraction patterns could be recorded, as shown in Figure 5. The resolution of the diagram is lower, with the spots slightly arced, and no reflections appearing lower than 0.2 nm. However, these diagrams indicate that even slightly branched bacterial dextran can be crystallized when properly nucleated, with the diagrams yielding good diffraction data. This confirms the findings of previous reports in the literature'* 10 When the diffraction patterns were obtained from thicker crystals, the patterns contained two arced and rather broad reflections which did not fall on the hkO reciprocal lattice grid. Such a diagram, reproduced in Figure 5b, is schematically drawn in Figure 5c. The extra reflections lie along a* at 0.590 + 0.008 nm and along b* at 0.430 + 0.005 nm. They originate from hk116lanes and are due to the curvature often found in thick crystals 22"z3. This curvature brings planes from the upper levels with low l indices (/= 1 or 2) into diffracting position. The indexing of the spot at 0.59 nm along a*, which falls between 400 and 500, must be of the hOl type with h < 5. For the spot at 0.43 nm along b*, the same reasoning implies that its index is Okl with k = I or 2. Taken together, these observations suggest that the indexing of the 0.59 nm reflection is either 301 or 302, whereas the reflection at 0.43 nm is either 021 or 022. The c parameter of the unit cell is then likely to be either 0.81 or 1.62 nm. Higher multiples of 8.1, i.e. 2.43 nm, 3.24 nm etc., are theoretically possible, but not likely. The data from the electron diffraction diagrams can be confirmed with X-ray diagrams obtained from a powder of the dry crystals. The spacings on the powder X-ray diagrams, listed in Table 1, reveal two new reflections, not observed by electron diffraction. They are located at 0.862 nm (very weak) and 0.397 nm (weak). The former one

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cannot be indexed with c = 0.81 nm but can be indexed (as 011) ire = 1.62 nm. The latter can be indexed either as 002 or 004, depending on the choice ofc. Two additional very w e a k reflections at 0,279 and 0.240 nm are also best indexed as 531 and 242, respectively, with c=1.62 nm. However, the accuracy of the measurement for these reflections is relatively poor. In the light of these data, the value for c = 1.62 nm is favoured. However, since this choice is based principally on the observation and measurement of a very weak diffraction line, a firm assignment of a value to the c parameter cannot be made at this time. The diffraction data obtained here are consistent with a number of probable chain models for dextran. Since the crystals have a density of 1.67 g/cm 3 (measured by flotation in bromoform-ethanol mixture), the number of glucose residues per unit cell may be either 12 or 24, corresponding to c=0.81 or 1.62 nm. As suggested previously 1°, this gives either a model with two chains per unit cell and six residues per chain (12 residues for the second choice of the c parameter), or a model with 4 chains per unit cell with 3 residues per chain (6 residues in the second case). In each case, up to 1 water of crystallization per glucose residue may be present in the unit cell. Larger amounts of water are unlikely, because no changes in the X-ray diffraction patterns are observed under different humidity conditions and because the crystals are unaffected by the vacuum of the electron microscope. The X-ray diffraction pattern shown here is identical with that previously obtained by Stipanovic 1° and similar to the patterns of bacterial dextrans L~ and L~ by Jeanes et al. 4. On the other hand, our diagrams differ markedly from those of other authors 5- 9. This is a strong indication that dextran crystallizes in several polymorphic forms. It

b"

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-

Figure 5 (a) Electron diffraction diagram of a bacterial dextran crystal such as in Figure 2b. (b) Electron diffraction diagram of a thick synthetic dextran crystal such as in Figure2a. tc) Schematic representation of the diffraction diagram of (b), indicating the extraneous reflections

S i n g l e c r y s t a l s o f d e x t r a n (1): H. C h a n z y et al. Table 1 I n t e r p l a n a r d-spacings from the X-ray powder diagr a m of dextran, indexed with unit cell p a r a m e t e r s from electron diffraction ( a = 2 . 5 6 3 , b = 1.02l, c = 1.616 nm) Observed d'spacing (nm) 1.300 0.862 0.806 0.656 0.592 0.557 0.514 0.474 0.440 0.397 0.359 0.336 0.319 0.306 0.279 0.256 0.240 0.229

0.211

Calculated d-spacing (nm)

h

k

Ia

Visual intensity c

1.282 0.863 0.799 0.655 0.587 0.543 0.511 0.474 0.438 0.432 0.427 0.404 0.362 0.337 0.329 0.316 0.306 0.301 0.279 0.255 0.239 0.229 0.229 0.227 0.214 0.212 0.209 0.197

2 0 2 3 3 4 0 2 3 0 6 0 5 1 2 3 8 4 5 0 2 10 5 11 12 11 12 12

0 1 1 1 0 1 2 2 2 2 0 0 2 3 3 3 1 3 3 4 4 2 4 1 0 2 1 2

0 1b 0 0 2h 0 0 0

m vw vw m s (broad) vw s s

ib}

vs(broad)

4b 0

w w

0.192 0.183 0.181 0.180

11 14 12 14

3 0 3 1

0.193

0.181

0 0 0

}w (broad)

o

}

0 Ib 0 2b

°00 1

prepare and characterize single crystals of other polymorphic forms of dextran.

Acknowledgements

The authors acknowledge the help of R. Vuong with the electron microscopy. They also thank Dr G. Excoffier for the guidance in preparing the tribenzyl dextran monomer.

References l 2 3 4

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5 6

s

7

vw vw vw

9

vw

10

8

11

i}

vw(brOad)

0

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vw

0 0 0 0

12 13 14 15

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16 17

Zero-level indexing is given only for those reflections that are visible on the electron diffraction diagrams b If c =0.808 rim,0.862 and 0.279 nm reflectionscannot be indexed,302 becomes 301,022 becomes 021,004 becomes 002, and 242 becomes 241 c vs=very strong, s=strong, m=medium, w=weak, vw=very weak °

18 19 20 21

is also likely that a given specimen may contain more than one polymorphic form, rendering it difficult to interpret the diffraction diagrams. Work is presently under way to

22 23

Rees, D. A. in 'Outline Studies in Biology" Chapman and Hall, London, 1977 Rees, D. A. and Scott, W. E. J. Chem. Soc. (B) 1971, 469 Tvaroska, I., P~rez, S. and Marchessault, R. H. Carbohydr. Res. 1978, 61, 97 Jeanes, A., Schieltz, N. C. and Wilham, C. A. J. Biol. Chem. 1948, 176, 617 Ruckel, E. R. and Schuerch, C. J. Am. Chem. Soc. 1966, 88, 2605 Kobayashi, H., MSc Thesis State University of New York, College of Forestry, Syracuse, New York (1968) Barham, P. J., Atkins, E. D. T. and Nieduszynski, I. A. Polymer 1974, 15, 762 Kiselev, V. P., Yu. Tsarevskaya, I., Virnik, A. D. and Rogovin, Z. A. Vysokomol. Soedin (A) 1976, 18, 234 Kochetkov, N. K., Nikitin, I. V., Banatskaya, M. I. and Kozlov, P. V. Dokl. Akad. Nauk, SSSR 1977, 237, 343 Stipanovic, A. J., PhD Thesis State University of New York, College of Environmental Science and Forestry, Syracuse, New York (1978) Schuerch, C. and Uryu, T., in 'Macromolecular Syntheses', John Wiley and Sons, New York, 1972, Vol. 4, p. 151 Chanzy, H., Excoffier, G. and Guizard, C. to be published Gekko, K. and Noguchi, H. Biopolymers 1971 10, 1513 Gekko, K. Makromol. Chem. 1971, 148, 229 Chanzy, H., Guizard, C. and Vuong, R. J. Microsc 1977, t I 1 (1), 143 Kovacs, A.J. andManson, J.A. KolloidZ. Z. Polym. 1966,214,1 Blundell, D. J., Keller, A. and Kovacs, A. J. Polym. Lett. 1966, 4, 481 Gekko, K. Aoric. Biol. Chem. 1978, 42, 1287 Bule~on,A. and Chanzy, H. J. Polym. Sci. (Polym. Phys. Edn) 1978, 16, 833 Atkins, E. D. T., Booy, F. P. and Chanzy, H. 'Proceedings of the EMAG 75 meeting, Bristol', 'Development in electron microscopy and analysis' Academic Press, New York, 1976, p. 319 Geil, P. H. in 'Polymer single crystals' Wiley lnterscience, New York, 1963 Chanzy, H., Dubd, M., Revol, J. F. and Marchessault, R. H. Biopolymers 1979, 18, 887 P~rez, S., Roux, M., Revol, J. F. and Marchessault, R. H. J. Mol. Biol. 1979, 129, 113

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