Vol. 188, No. 3, 1992 November 16, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

1060-1066

Crystalline ,L?-Cyclodextrin .12 Hz0 Reversibly Dehydrates to ,B-Cyclodextrin .10.5 HZ0 under Ambient Conditions *

THOMAS STEINER~, GERTRAUD KOELLNER~, SHAUKAT ALIT, DAVID ZAKIM~ AND WOLFRAM SAENGER~

‘Institut

fiir Kristallographie,

Freie Universitat

W-1000 2Division Cornell Received

University 7,

College,

TakustraBe

6,

33, Germany

o f D’g1 estive Diseases,

Medical

September

Berlin

Berlin,

Department

1300 York

of Medicine,

Av., New York,

New York

10021

1992

Summary: In contact with mother liquor, crystalline P-cyclodextrin (/?-CD) hydrate has composition N P-CD + 12H20. If crystals are dried at ambient conditions (18” C, N 50 % humidity), the unit cell volume diminishes N 30 to 50 A”. X-ray structure analysis of a dry crystal (0.89 A resolution, 4617 data, R = 0.059) s h owed the composition P-CD. 10.5 H20, with N 5.5 water molecules in the P-CD cavity (7 partially and 2 fully occupied sites) and N 5.0 between the P-CD molecules. The positions of the P-CD host and of most of the hydration waters are conserved during dehydration, but the occupancies of the waters in the ,&CD cavity diminish. Dry crystals put into solvent re-hydrate to the original form. 0 1992 Academic Press, 1°C. The mechanism of de- and re-hydration is not evident.

The crystalline

cyclodextrin

structural

and dynamic

biological

systems

was determined

(1).

seven dynamically

water

clusters

studied state

with NMR

* Topography 0006-291X/92

Copyright All rights

systems.

(10, 11) and computer

crystal

water

(3, 6, 7), dielectric

are primarily

bonding

of Cyclodextrin

,&CD.

(7), incoherent

neutron

simulation

(12, 13) techniques. temperature

cavity

can be regarded hydate

to study clusters

The crystal

The ,&CD

P-CD

Inclusion

Complexes,

Inc.

reserved.

1060

Part

in

structure contains

as a model

for

was extensively

scattering

(8,9),

solid-

The interpretations

neutron

diffraction

structure

11 H20.

$4.00

0 1992 by Academic Press, of reproduction in any form

of P-CD.

that

system

and of water

For this reason,

based on the room

composition

structure

molecules

model

networks,

(4, 5) d i ff raction.

(2, 3) and neutron

complex

are an important

is the hydrate

disordered

calorimetric

hydrates

of hydrogen

Best known

in more

these studies (4) with

properties

with X-ray

about

(CD)

30. For part

29, see Ref. 17.

of

Vol.

188,

No.

3,

Crystals

BIOCHEMICAL

1992

of P-CD

hydrate

tely disintegrate.

To prevent

on ‘wet’

sealed

- ll),

crystals various

crystals

sample

was dried

not without depend

not only

cracking,

solvent

conditions

were used,

to prevent

artefacts

in the crystal

on the crystallisation

studies

(4), but do not comple(2,4,

5) were performed

For the non-crystallographic

methods

as details

COMMUNICATIONS

RESEARCH

the crystallographic

in glass capillaries.

from

BIOPHYSICAL

tend to crack at ambient

preparation

problems,

AND

but in all cases the surface

due to adhering

structure

method,

but

experiments

bulk

water.

of CD complexes

(6 of the

This

is

may critically

also on the subsequent

history

of the

12 Hz0

occur-

sample. In this study ring under properties

we report

ambient

a slight

but significant

dehydration

This

loss of crystal

water

conditions.

of ‘dry’

crystals

of /?-CD.

must

be considered

if physical

are studied.

Crystallography Crystal size N 0.4 x 0.2 x 0.15 mm3, Enraf-Nonius Turbo-CAD4 diffractometer (Nifiltered Cu K, radiation, X = 1.542 A), T = 18” C. Wet crystals mounted in glass capillaries with some mother liquor, dry crystals glued on glass pins. Diffraction data sets for two dry crystals (Al and B4, see below) measured to X/2 sin I!&~ = 0.89 A [Al: 4673 unique reflections, 4577 with F, > o(F,); B4: 4686 unique reflections, 4617 with F0 > o(F,); g-scan method absorption correction]. Initial phasing with atomic coordinates of the @-CD molecule of the original X-ray study (2), water molecules located from difference Fourier maps. Anisotropic refinement with SHELX76 (16), final R = 0.064 for Al and R = 0.059 for B4 [space group P2i, one P-CD per asymmetric unit, C-H hydrogen atoms calculated in ideal positions, O-H hydrogen atoms not located]. Both structures indicate a water content of N 10.5 Hz0 per &CD molecule. Results (a) The unit

cell volume

The composition degree controversial: was reported ‘Form

P-CD.

in the first

I’, 12.3 Hz0

11 Hz0

were counted

@CD X-ray

cell volume

of ‘wet’ hydrate

work,

for a slightly located

a water

smaller

in the refined differences

analysis,

In a subsequent

12.1 Hz0

hydrate.

in contact

with mother

content

for V = 3067 (1) A3, but challenged

(‘ undecahydrate’).

due to inaccuracies

P-CD

volume

the substance

V = 3059(3)

study

between

of disordered

the single 1061

crystals

f0.5

per P-CD

study is usually

(5), however,

The discrepancies water

in (3)

(4), 10.9 Hz0

A, and in a further

for V = 3055 (1) A (15). occupancies

is to some

1. This was verified

in the neutron

neutron

liquor

of 11.9 Hz0

V = 3073 (6) A3 (2), Table

A. E ver since the latter

lysis we recently

to compositional

content

of crystalline

for a unit

for V = 3066(2)

and water

regarded

11.6 Hz0 X-ray

ana-

are partly

sites, but probably

used in the diffraction

as

also

studies.

Vol.

188,

No.

Comparing P-CD.

3,

(b) In contact

stable

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

the above

12 Hz0

The

1992

cited

(‘dodecahydrate’)

present

study

weeks under

were

suitable

for X-ray

They

showed

significantly

determination

1).

(which

ambient

analysis.

12 Hz0

different

cell constants

and a volume

(18” C, N 50 % humidity) cracked

and optically

as expected,

clear.

Unit

the same values

Table

crystal Ref. (2) Ref. (3) Ref. (3)

state

Ref.

(Bl

lying

‘%orm I” “Form II”

21.271

(5)

10.321

21.085

(7)

(4)b

wet

21.261

(6)

Ref.

(5)”

wet

21.268(11)

Ref.

(15)

wet for weeks

A2

dry

for weeks

Bl B2

wet

that

but some

for two of them.

10.4 HzO.

conventionally solution

1) showed

of the smaller determined

10.33

from

cell con-

Overnight,

the

< 0.5 mm remained crystals

by - 35 A”

(=

P / deg.

respect

n&o

3073

(6)

11.9

(3)

3067

(1)

12.3

(3)

3026

(2)

11.2

112.3 (5)

3066

(2)

10.9

112.40(3)

3059

(3)

11.6

(1)

15.104

112.34

10.212

(2)

15.123(5)

111.66

10.306

(3)

15.123

10.314

(6)

15.082(8)

(4)

with

Vol / A3

112.3

(5)

1.1%)

hydrates

15.10 (2)

(5)

(7)

10.268

(1)

15.110

(5)

112.40

(1)

3055

(1)

12.1

(7)

10.174

(2)

15.181

(5)

111.36

(2)

3026

(2)

10.4

21.001(10)

10.160

(2)

15.171

(2)

111.23

(2)

3017

(2)

-

21.286(10)

10.323

(2)

15.084

(8)

112.46

(2)

3063

(2)

-

10.198 10.119

(2) (1)

15.149 15.150

(6) (2)

111.60 111.58

(3) (1)

3029 3029

(2) (1)

-

for 3/4 h. for 2 d.

B4

dry

for 7 d.

21.070

(4)

10.191

(1)

15.153

(4)

111.51

(1)

3027

(1)

10.5

B5

wet

again

21.277

(4)

10.319

(1)

15.099

(3)

112.39

(1)

3065

(1)

-

a: Space group P21. b: Partially deuterated. c: Partially For A and B: T = 18°C. 1062

(B3

of 3029 (1) and 3027 (1) A3 respectively,

21.295

(9) (3)

in

and exposed

for two different

c/A (1)

unit

the solvent

crystals

of ,&CD

were taken

the usual

from

grown

21.037

21.085 21.084

of

and kept for

dry dry

B3

is

solution

cracks,

one by one on a glass plate.

volumes

21.29

dry

with

a saturated

in Table

b/A (2)

showed

flask over - 3 days. All crystals

wet

Al

were performed

A, and reduced

alA

vacuum,

N P-CD.

Cell constantsa of @cyclodextrin

1:

an aqueous

under

the composition

cell constants

as for sample

hydrate

[3026 (2) and 3017 (2) A3], and the structure

but several

and B4) after two and seven days showed

from

crystals

A. C r y s t a 1s were taken

of V = 3063(2)

10.5H20.

of a P-CD

were determined

1). For crystallisation,

A wet test crystal

i.e.

Most

(sample

B in Table

to N /?-CD.

dried

experiments

tube.

intact,

grown

were

these observations,

the same batch

specimen

Crystals

yielded

N 70°C in a Dewar

larger

dehydrates analysis

volumes

from

to atmosphere

l,%‘lr,o

conditions.

Hz0 was cooled

stants

(2).

by the X-ray

Unit

composition

to the approximate

did not cocrystallize),

of the 3026 A3 crystal

To verify P-CD.

P-CD.

A in Table

varying

to return

for wet crystals

was initiated

and benzo[a]pyrene

several

we suggest

with the atmosphere,

in air (sample

P-CD

results,

deuterated, room temperature

values.

Vol.

188,

No.

3,

to the volumes diminish,

of the ‘wet’

whereas

A structure

BIOCHEMICAL

1992

c slightly

analysis

corresponding hydrated

of crystal

To observe

after

(d) The dehydration

mounted

were taken again.

disintegrated

mounted

in wet state showed

from

unit

and the changes

in unit

by N 0.9” (Table

the composition

per P-CD

with

time,

the ‘dry’

from

molecule

a crystal

volume

the solvent,

After

P-CD.

with

(B2)

1).

10.5 HzO,

respect

When

was taken

the unit

to fully

from

the

cell constants

of 3029 A3 was already

left at atmosphere

two days, they

the surface, reduced

but

could

cell constants

were found

still suitable

diffraction

power

be determined clearly

of zero humidity,

Originally

(f)

yielded

a and b

reached.

indicate

for 10 days, and put

slightly

for X-ray

opaque,

work.

compared

to ‘dry’

(Table

The

1).

a complete

with

A crystal or original

volume

re-hydration

tiny (B5) ‘wet’

of 3065A3 to the initial

12 HZO.

(e) In atmosphere

hydrate

angle p reduces

on the diffractometer.

cell constats

P-CD.

intact were

extensive

shows that

water

N 45 minutes,

solvent

two days they

isotropically:

is ‘fast’.

particles

indicating

B4 (see below)

COMMUNICATIONS

is reversible.

Some crystals

composition

the monoclinic

the loss of crystal

were determined

but

cell does not reduce

of N 1.5 Hz0

water

and immediately

specimen,

RESEARCH

12 HzO.

(c) The loss of crystal

into saturated

BIOPHYSICAL

The unit

increases;

to a dehydration

P-CD.

solvent

crystals.

AND

‘dry’

crystalline

crystals

found

rugged

(but

were stored with

hydrate

in a silica

cracks.

not complete)

some atmospheric

P-CD

X-ray

is required

gel desiccator

diffraction

disintegration

humidity

disintegrates. at 18” C. After

power

was very

of the crystalline to maintain

structure.

the stability

poor, This

of ‘dry’

P-CD

refined

data

crystals.

Preliminary

X-ray

We briefly

structure

discuss

of

the structure

(R = 0.059 for 0.89 A resolution) B-CD molecular twofold

conformation

orientationally

cavity

(2 fully,

2 partially mutually

occupied, exclusive

The water the interstices,

of the dry crystal

than

(W3A

the structure

Figure

and W3B,

= 5.0).

are in similar

the occupancies

B4, which

is better

The crystal

packing

sites were located,

positions

are reduced.

of the partially W8B

as in P-CD. The

reduction

12 H20,

are

9 in the P-CD

occupied

and W9, W14A

and the groups

= 5.5) and 6 in interstices

S everal

and W8B,

1063

0.

12 HzO. Two @-CD CH,(S)-O(6)

1. 15 water

W8A

i0..!iH2

of Al.

sum of occupancy

sum of occupancy

molecules but

occupied,

P-CD.

crystal:

are as in /?-CD.

disordered,

7 partially

dry

a

(4 fully, sites are

and W14B).

especially

of occupancy

those in concerns

Vol.

188, No. 3, 1992

primarily

BIOCHEMICAL

the molecules

in the cavity:

sums of the occupancies compared

molecules R-value,

H atoms

order

as in /?-CD.

12HzO

‘Form

II’ P-CD. a crystal

molecules.

crystal

asymmetric

and cell volume

this earlier

structure

(‘&-flop are very

not immediately the existence

of a translation

12 Hz0 and P-CD.

program

INSIGHT

This would

are found

(16).

that protude

are not interconnected

exclude

long-range

diffusion

the solvent

Using from

probe

accessible 10.5 Hz0 radii

between of water

neighboring

molecules,

occupancies: -1.0 0.5to1.oQ

co.50

1. One asymmetric crystal unit of P-CD. 10.5 Hz0 (crystal B4). Shown are Two C(6)-O(6) groups are orientationally boundary lines of 50 % probability ellipsoids. disordered [occupancies: 0.55 / 0.45 for O(6)lA / B and 0.36 / 0.64 for 0(6)2A /B]. Water molecules are labelled as those in equivalent positions in P-CD .12 Hz0 (I). W7 is placed ‘below’ the O(6)-rim of the P-CD molecule, and outside the cavity. Water occupancies are: Wl, W2, W4, W5, W6, W7 (l.Oeach); WSA/B (0.83/0.17); W8A/B (0.28/0.57); W9 (0.36); WlO (0.81); W12 (0.44); OW14A /B (0.64 / 0.36). 1064

of

the /?-CD cage to

water

Figure

as

diffusion

in P-CD.

cavities

similar

evident.

packing

molecules,

‘) dis-

has been determined

in the crystal graphics

and

dehydration.

implies

sites, but these cavities units.

orientational

path,

the computer

water

suggesting

1) of the water

of such a diffusion

was inspected

the interstitial

the good resolution

is

are

10.5 HsO.

In the search

molecules

1.4 and 1.3 A for water

water

Despite

at least partially

surface of the P-CD with

of the interstitial

disorder.

that

fast de- and re-hydration

for the water

are 6.9, 7.3 and 6.7, respectively,

(Figure

not be located,

of de- and re-hydration

12 HzO, the

factors

dynamic

(3), we assume

(2, 3, 15) of ,&CD.

temperature

S ince the structure

that had suffered

Relatively path

(3).

11.2HsO

(g) The mechanism

molecules

to 5.0 for P-CD.

indicate could

studies

RESEARCH COMMUNICATIONS

the occupancies

and the high

cavity

hydroxyl

from

compared

occupations

in the ,&CD

water

10.5 HzO, whereas

5.0, 5.0 and 5.4, respectively, The partial

in the X-ray

for the included

to 5.5 in ,&CD.

AND BIOPHYSICAL

and

Vol.

188,

hence

No.

BIOCHEMICAL

3, 1992

the observed

between

adjacent

AND

For probe

dehydration. asymmetric

units

BIOPHYSICAL

radii

are apparent

RESEARCH

COMMLJNlCA-f-IONS

5 1.0 A, however, in the direction

diffusion

channels

of the b-axis.

Discussion The mechanism arrangement

of water

of P-CD

molecules.

water

molecules

with

fusion

of water

molecules

,&CD

glucoses

or of glucose

can travel

through

simulation

of P-CD.

atoms

of around

diffusion

atoms,

12 Hz0

0.4 A. This

“actual

water”

assisted

by the dynamics

can diffuse

lattice.

Disorder

of water

molecules,

in ‘wet’

and ‘dry’

crystals

whether

they were performed

Acknowledgments. und Teclmologie, T32 DK07142

This FKZ

molecules dynamics C and 0

is consistent

We assume

difof the

of the glucose

5 1.0 A, i.

with

e. 0.4 A smaller

our than

that this process

will be

bonds. also of hydroxyl

hydrate.

hydrated

(W.

water

by a molecular

radii

the

to permit

fluctuations

so that

fluctuations

orientations,

For the studies or partially

was supported

03 SA3 FUB

from

since translation

are local

atomic

and probably

on fully

big enough

However,

open paths

lattice.

hydrogen

of ,&CD

study

with

the crystal

of jIZp-jIop

cavity

in fact, is indicated

of local

probes

through

is not obvious

s h ows rms fluctuations

which

water

lattice

that there

temporarily

This,

magnitude

that

in and out.

we conclude which

(12),

the crystal

is no channel-like

to diffuse

is observed,

the crystal

(see (g,) above)

rable

There

1.4 A radius

finding

through

(6 - 11) it is not clear

dehydrated

samples.

by the Bundesministerium

S.) and the National

is compa-

fiir Forschung

Institute

of Health

Grant

(S. A.).

References 1.

Jeffrey, G. A. and Saenger, W. (1991) pp. 309 - 350. Springer Verlag, Berlin.

2.

Lindner,

3.

Fujiwara, T.; Yamazaki, M.; Tomizu, Y.; Tokuoka, R.; Tomita, K.-I.; Suga, H. and Saenger, W. (1983) Nippon Kagaku Kaishi 181-187.

4.

Betzel, Ch.; Saenger, Sot. 106, 7545-7557.

K. and Saenger,

5.

Zabel,

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Hanabata,

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Pathmanathan, 7491-7494. Steiner, Th.; 157, 336-338.

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V.; Saenger,

W. (1982) garbohydr.

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T. and Suga, H. (1987)

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Usha, M. G. and Wittebort,

R. J. (1989)

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Usha, M. G. and Wittebort,

R. J. (1992)

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W. F. (1987)

Eur.

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Koehler, J. E. H.; Saenger, 16, 153-168.

W. and van Gunsteren,

W. F. (1988)

Eur.

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Steiner,

W. and Lechner,

G. M. (1976) of Cambridge,

SHELX UK.

Th. and Saenger, Technologies Th; Koellner,

Inc.

R. E. (1991)

RESEARCH COMMUNICATIONS

9.

15. Steiner,

Th.; Saenger,

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76: Program

W.: unpublished (1991)

INSIGHT

G. and Saenger,

Phys.

72, 1211-1232.

208, 669-678. Sot.

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114, 1541-1548.

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