Crystal Structures of Amylose and Its Derivatives - ACS Symposium

28 Crystal Structures of Amylose and Its Derivatives A Review

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A N A T O L E SARKO Department of Chemistry, State University of New York, College of Environmental Science and Forestry, Syracuse, N Y 13210 PETER ZUGENMAIER Institute of Macromolecular Chemistry, University of Freiburg, D-7800 Freiburg i.Br., West Germany

Amylose, a l i n e a r , high molecular weight (1->4)-α-D-glucan, i s one of the p r i n c i p a l polysaccharides of starch. Because of the longstanding utility of starch as a raw material, and its wide spread botanical availability, its structure and properties have been studied for centuries. Since the more recent r e a l i z a t i o n that almost all varieties of starch are composed of two polysac charides - the l i n e a r amylose and the branched amylopectin - a significant share of interest has shifted to the study of these components. Of particular interest has been the observation that both components occur naturally in c r y s t a l l i n e form in the starch granule. Structural studies of amylose have, in turn, revealed a wide range of c r y s t a l l i n e polymorphy, both in chain conformation and i n c r y s t a l l i n e packing. An example is the group of V-amyloses that exist as complexes with small organic molecules, water, or iodine. The l a t t e r complex is p a r t i c u l a r l y interesting because it displays an intense blue color. The V-amyloses can be prepared by p r e c i p i tation or drying from solution, and they c r y s t a l l i z e r e a d i l y . Consequently, t h e i r c r y s t a l structures are of interest i n connec t i o n with any regenerated form of starch material. Another group of c r y s t a l l i n e amyloses consists of complexes with ionic substances, for example, a l k a l i or salts such as KBr. As w i l l be shown l a t e r , these c r y s t a l structures differ consider ably from the V-amyloses. The amylose found i n the native starch granule i s , i n many respects, the most fascinating polymorph. I t i s double-helical i n structure, which raises a question how t h i s molecule i s synthe sized and deposited i n such a complex c r y s t a l l i n e form into a l a y ered, r a d i a l l y organized spherulitic granule, a morphology that has not been duplicated i n the laboratory. The complexity does not stop with amylose, as the branched amylopectin almost certain l y possesses the same double-helical structure. Also, there are two different types of c r y s t a l l i n e starch granules: the A-starch of the cereals and the B-starch of the tubers. The two granules possess the same chain conformation, but differ i n c r y s t a l l i n e packing. 0-8412-0589-2/80/47-141-459$06.00/0 © 1980 American Chemical Society French and Gardner; Fiber Diffraction Methods ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


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F i n a l l y , b e c a u s e t h e a m y l o s e m o l e c u l e i s a p o l y a l c o h o l , chemi c a l d e r i v a t i v e s o f i t can b e e a s i l y p r e p a r e d . I n c l u d e d i n t h i s a r e t h e a c e t a t e , m e t h y l , e t h y l and s i m i l a r d e r i v a t i v e s , a l l o f which belong t o a c l a s s of polymers w i t h completely d i f f e r e n t prop e r t i e s f r o m t h e p a r e n t s u b s t a n c e . Many o f t h e s e d e r i v a t i v e s demo n s t r a t e u s e f u l f i l m and f i b e r p r o p e r t i e s , b u t t h e y h a v e n o t reached s i g n i f i c a n t commercial u t i l i z a t i o n . A l l amylose d e r i v a t i v e s c r y s t a l l i z e e a s i l y , and many show i n t e r e s t i n g f e a t u r e s i n their crystalline state. The r e l a t i o n s h i p s b e t w e e n t h e m a i n c l a s s e s o f a m y l o s e p o l y morphs a r e s c h e m a t i c a l l y i l l u s t r a t e d i n F i g . 1. As shown b y a s e l e c t i o n o f x - r a y d i f f r a c t i o n p a t t e r n s i n F i g . 2, a l l c l a s s e s o f amyloses g e n e r a l l y y i e l d good f i b e r d i f f r a c t i o n diagrams. I n add i t i o n , s i n g l e c r y s t a l s o f a number o f a m y l o s e p o l y m o r p h s can be grown f r o m d i l u t e s o l u t i o n s . T h e s e c r y s t a l s , shown i n F i g . 3, a l s o g i v e good e l e c t r o n d i f f r a c t i o n p a t t e r n s . The a n a l y s i s o f t h e r e s u l t i n g x - r a y and e l e c t r o n d i f f r a c t i o n i n t e n s i t i e s , w i t h t h e h e l p o f t h e modern m o d e l i n g t e c h n i q u e s s u c h as d e s c r i b e d i n t h i s v o l u m e ( 1 , 2 ) , has p r o d u c e d s t r u c t u r a l i n f o r m a t i o n o f p r e v i o u s l y unavailable d e t a i l . I n t h e f o l l o w i n g , we r e v i e w t h e s t r u c t u r e s o f amyloses determined i n t h i s f a s h i o n , i n c l u d i n g , however, o n l y t h o s e r e s u l t s t h a t h a v e b e e n o b t a i n e d w i t h t h e h e l p o f t h e more r e cent methods. C l a s s i f i c a t i o n o f Amylose S t r u c t u r e s

and

Their General Features

The u n i t c e l l d i m e n s i o n s o f a l l c r y s t a l l i n e a m y l o s e s t h a t h a v e b e e n d e t e r m i n e d i n some d e t a i l , a r e l i s t e d i n T a b l e I . Also i n c l u d e d a r e some i n t e r m e d i a t e f o r m s b e t w e e n t h e V and V£ amyl o s e s ( 5 ) and some V - a m y l o s e c o m p l e x e s w i t h n - b u t a n o l , w h i c h , a l though not y e t c o m p l e t e l y d e t e r m i n e d , have been added t o i l l u s t r a t e the range o f v a r i a b i l i t y i n u n i t c e l l dimensions. I n the c a s e o f t h e V - B u 0 H c o m p l e x , a d o u b l i n g o f one u n i t c e l l a x i s was detected a f t e r a c a r e f u l study o f e l e c t r o n d i f f r a c t i o n diagrams o f single c r y s t a l s (10). A consequence o f the d o u b l i n g i s t h a t the u n i t c e l l now c o n t a i n s f o u r c h a i n s , i n s t e a d o f t h e two n o r m a l l y found i n amylose s t r u c t u r e s , ( i n a s t r i c t s e n s e , t h e A- and Ba m y l o s e s s h o u l d a l s o be c o n s i d e r e d as f o u r - c h a i n u n i t c e l l s , b u t t h e i r d o u b l e - h e l i c a l s t r u c t u r e s t i l l r e s u l t s i n o n l y two h e l i c e s per c e l l ) ( 1 3 , l U ) . a

a

A l m o s t a l l u n i t c e l l s shown i n T a b l e I a r e e i t h e r o r t h o r h o m b i c or pseudo-orthorhombic, w i t h a m a j o r i t y o f space groups P2i2]_2]_ and P 2 O n l y a few s t r u c t u r e s e x h i b i t h i g h e r symmetry and none shows l o w e r symmetry. A l l s t r u c t u r e s h a v e an a n t i p a r a l l e l p a c k i n g o f c h a i n s ( h o w e v e r , see A- and B - a m y l o s e s ) . On t h e o t h e r hand, a l a r g e v a r i e t y o f h e l i x c h a r a c t e r i s t i c s are e v i d e n t , i n a d d i t i o n t o t h e v a r i a b i l i t y i n t h e u n i t c e l l d i m e n s i o n s . Some o f t h e f e a t u r e s u s e f u l f o r c l a s s i f y i n g a m y l o s e s t r u c t u r e s a r e shown i n T a b l e I I . The d i s t a n c e b e t w e e n t h e two n e a r e s t a n t i p a r a l l e l 1 #

French and Gardner; Fiber Diffraction Methods ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


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ALKALI AMYLOSES AMYLOSE - SALT COMPLEXES

Figure 1.

461

Amylose and Its Derivatives

V- AMYLOSES HYDRATES COMPLEXES IODINE

DERIVATIVES: ACETATE METHYL ETHYL, ttc.

Relationships and conversion paths between different classes of amylose structures

Figure 2. X-ray fiber diffraction patterns for flop, left to rightj: V -amylose; Vmiso-amylose; KOH-amylose; (bottom, left to rightj: B-amylose, amylose triacetate I, triethylamylose I-nitromethane complex a


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Table I U n i t c e l l dimensions o f d i f f e r e n t polymorphs o f amylose and a m y l o s e d e r i v a t i v e s . A l l "unit c e l l s c o n t a i n 2 c h a i n s e x c e p t V -BuOH a n d A- a n d B - a m y l o s e s , w h i c h c o n t a i n h c h a i n s . a

b

o (fiber y Helix r e p e a t ) ( d e g . ) symme(Angstroms) try


Structure

a Intermediate forms

DMSO

0

P2 2 2

3,h

22. h6

13.30

23.0

90

13.50 13.55 13.7

23.50

23. U5

8.05

90 90 90

6/5

P2 2 2

19.17

19.17

2U.39

90

6/5 i n

P2 2 2

23.7

7.91

90

2 (~6/5) n

5

1 1 1

X

8.13 repeat (per turn) 2 .k2

8.17

90

V -BU0H

26. h

27.0

7.92

90

V -BU0H

13.7

25.8

8.10

90

h

Ref.

12.97

V ^ - i o d i n e 13.60 a

Space Group

6/5

"^1^1

P2 (S)

8

P2 2 2

9,10

1

T i l

9

22. hi

90

6/5

P2 2 2 1 1 1

11

KBr

10.88

10.88

16.52

90

h/3

Pi+^2

12

A

11.90

17.70

10.52

90

2x6/1 i n 21.Oh 1

P2 (S)

13

P3 _21

lU

P2 (S)

16 18

K0H

Q.Qh 12.31

repeat 2x6/1 i n 20.8 1 repeat lU/11

1

B

18.50

18.50

10. hO

120

ATA I

10.87

18.83

52.53

90

TMA

17.2U

15.6k

90

TEA1

16.13

11.66

15. hQ

90

2/K-U/3) P2 2 2 1 1 1 P2 2 2 h/3

TEA3

15.36

12.18

15. h8

90

h/3

P2 2 2

20

TEA1-C1

16.76

lU.28

16.02

90

h/3

P2 2 2

21

TEA1-DCM1 16.52

13.95

16.02

90

h/3

P2 2 2

21

TEA1-N, 1U.70 -C2,-DCM2

1U.70

1 5 . U8

90

h/3

P2 2 2

22

8.70

]

1

1 1 1

1 1 1 1 1 1 1 1 1

T i l


19

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Table I I I n t e r c h a i n and i n t e r s h e e t spacings and r i s e p e r r e s i d u e f o r d i f f e r e n t amyloses and d e r i v a t i v e s . Structure

d

ai Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on June 22, 2016 | http://pubs.acs.org Publication Date: November 17, 1980 | doi: 10.1021/bk-1980-0141.ch028

V

a h DMSO V -iodine KOH KBr A B ATA I TMA TEA1 TEA3 TEA1-C1 TEA1-DCM1 TEA1-N, C 2 , DCM2 v

v

h

12.97 13.69 13.56 13.5^

110

h

(X)

&)

11.23

1.32

11.86 13.56 11.76

1.3U 1.36 1.36

7.58

7.18

3.1k

7.21

7.21

U.10

9.87

3.51 3.hi

10.66 10.68 10.87

9.66 9.95 9.80 11.01

10.81 10.39

9.25 9.hi

7.77 9.^5 9.5^ 10.87 10.66 10.39

3.75 3.91 3.87 3.87 h.01

U.01 3.87

Figure 3. Electron micrograph and electron diffraction diagram of a single crystal of triethylamylose. (The diffraction rings are due to TICl used for calibration.)



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METHODS

packed c h a i n s ( d j ^ ) i s l i s t e d i n t h e f i r s t column. T h i s u s u a l l y r e p r e s e n t s t h e d i s t a n c e between a c o r n e r and a c e n t e r i n an o r t h orhombic, two-chain u n i t c e l l , and i t i n v a r i a b l y determines t h e c l o s e s t contact d i s t a n c e between the chains i n the c r y s t a l s t r u c ture. A s i m i l a r f e a t u r e , t h e Bragg duo s p a c i n g , l i s t e d i n t h e second column, u s u a l l y r e p r e s e n t s t h e s p a c i n g between the sheets o f c l o s e s t - p a c k i n g c h a i n s . I t may b e a n i m p o r t a n t m e a s u r e when adjacent sheets a r e separated by solvent molecules occupying t h e i n t e r s t i t i a l spaces i n the u n i t c e l l . F i n a l l y , the r i s e p e r r e s idue, l i s t e d i n t h e l a s t column o f T a b l e I I , r e f l e c t s t h e e x tension o f the h e l i x along i t s a x i s . A c l e a r d i v i d i n g l i n e separates, t h e amylose s t r u c t u r e s w i t h r e s p e c t t o h: a l l V - t y p e s t r u c t u r e s f a l l on one s i d e w i t h h r a n g i n g f r o m 1.32 t o 1.36 X , l e a v i n g a l l o t h e r s t r u c t u r e s i n a n o t h e r g r o u p w i t h v a l u e s o f h f r o m 3.5 t o U . l X . The i n t e r c h a i n distance a n d t h e dj_±Q s p a c i n g c a n b e u s e d t o f u r t h e r c l a s s i f y the amylose s t r u c t u r e s i n t o f o u r groups. The V - t y p e s t r u c t u r e s , f o r m i n g one g r o u p , e x h i b i t n e a r l y i d e n t i c a l v a l u e s o f d\\ w i t h o n l y V somewhat o u t o f l i n e b y ~0.7 A, w h i c h i s a t t r i b u t a b l e t o the absence o f water molecules i n i t s i n t e r s t i t i a l spaces. Such w a t e r m o l e c u l e s a r e f o u n d i n t h e h y d r a t e d f o r m s o f Vft- a n d V^^go" a m y l o s e s . The ^ P S a l s o i n c r e a s e s upon h y d r a t i o n b y a b o u t t h e same amount, b u t jumps b y 1.7 X- when l a r g e DMSO m o l e c u l e s l o c a t e between the s h e e t s . I n t h e s e c o n d g r o u p o f s t r u c t u r e s , t h e KOH- a n d K B r - a m y l o s e s p o s s e s s t h e l o w e s t v a l u e s f o r b o t h d\\ a n d d^Q. This i snot s u r p r i s i n g b e c a u s e t h e s e a m y l o s e h e l i c e s a r e much more e x t e n d e d t h a n t h e V - t y p e s t r u c t u r e s , w i t h h a t 3.7^ a n d k.10 51, r e s p e c tively. C o n s i d e r i n g t h a t t h e KOH-amylose i s a 6/5 h e l i x a n d K B r a m y l o s e i s a k/3 h e l i x , t h e t w o v a l u e s o f h a r e s u r p r i s i n g l y close. The a m y l o s e d e r i v a t i v e s , f o r m i n g a n o t h e r g r o u p o f s t r u c t u r e s , a r e c h a r a c t e r i z e d b y h i n t h e same r a n g e a s i n KOH- a n d KBr-amyl o s e s , but w i t h b o t h d j ^ a n d d^lO l ^ - 5 because o f t h e space r e q u i r e d f o r t h e s u b s t i t u e n t groups. However, t h a t d^.^ s t a y s w i t h i n ^1,2 A f o r a l l d e r i v a t i v e s , i n c l u d i n g t h e s o l v e n t - c o m p l e x e d s t r u c t u r e s , i s u n e x p e c t e d . A l a r g e r r a n g e i n t h e d]_j_o s p a c i n g i s more i n l i n e w i t h t h e p r e s u m e d e f f e c t o f t h e s o l v e n t . The d o u b l e - h e l i c a l s t r u c t u r e s o f n a t i v e A- a n d B - a m y l o s e s are found i n the f o u r t h group. I t i s i n t e r e s t i n g t h a t i n both h as w e l l a s t h e d\^, a n d d^Q s p a c i n g s , t h e y a r e c o m p a r a b l e w i t h the s t r u c t u r e o f amylose t r i a c e t a t e I (ATAI). I n p a r t , t h i s may a r i s e because the packing o f the b u l k y a c e t a t e s u b s t i t u e n t s i n A T A I i s s i m i l a r t o t h e c l o s e - p a c k i n g o f two a m y l o s e c h a i n s i n t o a double h e l i x . I n t h e l a t t e r , one c h a i n may a c t a s t h e " s u b s t i t u e n t " f o r the o t h e r c h a i n . A t any r a t e , a l l t h r e e s t r u c t u r e s contain similar, cylindrical-shaped helices. Somewhat u n e x p e c t e d l y , t h e d i s t a n c e s d^ a n d dj_10 v e r y c l o s e f o r t h e two n a t i v e polymorphs, even though t h e i r u n i t c e l l s and p a c k i n g a r e 9

a

s

1

1

a c i n

0

n c r e a s e

Q

a

r

e



28.

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d i f f e r e n t . A s shown b e l o w , i n t h e s t r u c t u r e f o r B - a m y l o s e t h e s h e e t s o f a n t i p a r a l l e l - p a c k e d d o u b l e h e l i c e s a r e i n t e r r u p t e d bychannels o f w a t e r , w h i l e i n A-amylose t h e s h e e t s a r e i n t a c t and the w a t e r i s l o c a t e d i n t h e i n t e r s t i t i a l s p a c e s b e t w e e n t h e d o u b l e helices. O r i g i n a l l y , space groups were n o t a s s i g n e d t o ATAI and t h e A- a n d B - a m y l o s e s , a n d t h e i r s t r u c t u r e s w e r e s o l v e d i n t h e t r i c l i n i c s p a c e g r o u p P I . I t was l a t e r o b s e r v e d ( P . Z u g e n m a i e r , u n p u b l i s h e d w o r k ) t h a t t h e same c h a i n p o s i t i o n s r e s u l t e d when b o t h ATAI a n d A - a m y l o s e w e r e a s s i g n e d t h e s p a c e g r o u p P 2 ^ , b u t w i t h t h e 2-]_ s c r e w a x i s p o s i t i o n e d p e r p e n d i c u l a r t o t h e h e l i x a x i s ( d e n o t e d here as P 2 i ( S ) ) . L i k e w i s e , B-amylose c o u l d be a s s i g n e d t h e t r i g o n a l s p a c e g r o u p P3]_21 ( P . Z u g e n m a i e r , u n p u b l i s h e d w o r k ) . The c o n s e q u e n c e o f a s s i g n i n g s p a c e g r o u p P2]_(£) t o A T A I i s t h a t t h e c o m p l e t e l U / 1 1 h e l i x must b e c o n s i d e r e d a n a s y m m e t r i c u n i t . Simi l a r l y , t h r e e g l u c o s e r e s i d u e s , o r o n e - h a l f t u r n o f t h e h e l i x , now form t h e asymmetric u n i t o f A-amylose. S p a c e g r o u p P 3 ^ 2 1 f o r Ba m y l o s e r e q u i r e s an a s y m m e t r i c u n i t o f t w o g l u c o s e r e s i d u e s , o r one-third of the helix turn. I n t h e l i g h t o f t h e s e o b s e r v a t i o n s , i t i s c l e a r t h a t t h e amyl o s e c h a i n can adopt a v a r i e t y o f c o n f o r m a t i o n s . P r e d i c t i o n s o f the most p r o b a b l e c o n f o r m a t i o n s b a s e d on i s o l a t e d c h a i n s , w h e t h e r u s i n g hardsphere contact c r i t e r i a (15) o r p o t e n t i a l energy f u n c t i o n s ( 1 7 ) , a r e not capable o f p r o d u c i n g t h e whole range o f observed conformations. Furthermore, i t i s apparent t h a t t h e molecu l a r symmetry - s u c h as t h e f o u r - o r s i x - f o l d h e l i c e s - does n o t n e c e s s a r i l y c o i n c i d e w i t h t h e symmetry o f t h e c r y s t a l l i n e p a c k i n g or t h e s p a c e g r o u p . The l a t t i c e f o r c e s a r e t h u s p r o b a b l y a s i m p o r t a n t as t h e i n t r a m o l e c u l a r f o r c e s i n d e t e r m i n i n g t h e conformat i o n s o f c r y s t a l l i n e amyloses. The f o u r g r o u p s o f a m y l o s e s a r e d e s c r i b e d i n d i v i d u a l l y i n more d e t a i l i n t h e f o l l o w i n g s e c t i o n s . V-Amyloses The s t r u c t u r e s o f t h e V - a m y l o s e s w e r e among t h e f i r s t p o l y s a c c h a r i d e s on w h i c h t h e modern methods o f c o n f o r m a t i o n a n d p a c k ing r e f i n e m e n t were t e s t e d . The s o p h i s t i c a t i o n o f t h e computer p r o g r a m s h a s i m p r o v e d s i g n i f i c a n t l y o v e r t h e y e a r s , a s have some of t h e e a r l y i d e a s , p a r t i c u l a r l y t h o s e c o n c e r n i n g hydrogen bonding. I n t h e e a r l y s t a g e s , c o n f o r m a t i o n a l a n a l y s i s was g e n e r a l l y s e p a r a t e d from t h e p a c k i n g r e f i n e m e n t , and b o t h , i n t u r n , were conducted s e p a r a t e l y from t h e x-ray a n a l y s i s . I n such modeling approaches, t h e conformation o f t h e glucose r i n g remained i n v a r i ant, a n d t h e p a c k i n g r e f i n e m e n t was sometimes c o m p l e t e l y o m i t t e d . For t h i s r e a s o n , t h e e a r l i e r work has n o t been i n c l u d e d i n t h i s review. A l l V - a m y l o s e s t r u c t u r e s shown i n T a b l e I h a v e i n common a l e f t - h a n d e d , s i x - r g s i d u e h e l i x , w i t h h i n a very narrow range f r o m 1.32 t o 1.36 A, a n d a n 0-2...0-3(2) i n t r a m o l e c u l a r h y d r o g e n



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bond ( c f . F i g . 5). Although the s i x - f o l d , right-handed h e l i x appears t o be e q u a l l y p r o b a b l e from c o n f o r m a t i o n a l a n a l y s i s ( I T , 2 3 ) , i t h a s n o t b e e n s e e n i n a n y V - a m y l o s e . The same h y d r o g e n b o n d a l s o o c c u r s i n t h e m a l t o s e c r y s t a l s t r u c t u r e (2U,25,26) a n d i n cyclohexaamylose (27,28). I tl i m i t s thev a r i a b i l i t y o f the 0-U 0-U(2) d i s t a n c e ~ T t h e virtual bond) t o a r a n g e o f a b o u t U.05 t o k.k A. When t h i s h y d r o g e n b o n d i s n o t p r e s e n t , a s i n a v a r i e t y of s m a l l - m o l e c u l e c r y s t a l s t r u c t u r e s c o n t a i n i n g a-D-glucose, t h e l e n g t h o f t h e virtual bond e x t e n d s a s h i g h a s k.6 X ( 2 9 ) . Other i n t r a c h a i n h y d r o g e n b o n d s a l s o f o r m , p r i m a r i l y i n v o l v i n g t h e 0-6 h y d r o x y l and o c c u r r i n g between t h e t u r n s o f t h e h e l i x . For example, i n V D M S Q - ^ 9 t h e r e i s a n 0-6(i+7) gt.. . 0 - 2 ( i + l ) h y d r o gen b o n d ( £ = 0 , 1 , 2 . . . ) , a n d p o s s i b l y a n a d d i t i o n a l 0-6(£+7) gt... 0 - 3 ( i + 2 ) h y d r o g e n b o n d t h r o u g h e i t h e r DMSO o r w a t e r . (For a des c r i p t i o n o f t h e gt gg, tg r o t a m e r t e r m i n o l o g y , c o n s u l t R e f . ^ l ) . The i n t r a m o l e c u l a r h y d r o g e n - b o n d i n g i n v o l v i n g 0-6 i s s l i g h t l y d i f f e r e n t i n V - (6) a n d V ^ - i o d i n e - a m y l o s e s (J3), b e c a u s e a l l 0-6 a r e i n t h e gg p o s i t i o n i n t h e s e s t r u c t u r e s . N o n e t h e l e s s , h y d r o g e n b o n d s s t i l l f o r m b e t w e e n 0-6(£+7) a n d 0 - 2 ( i + l ) . I n V -amylose, a l l t h r e e r o t a t i o n a l s i t e s f o r 0-6 a r e f o u n d i n s u c c e s s i v e r e s i dues (_3), g i v i n g r i s e t o a l l h y d r o g e n b o n d s p o s s i b l e f o r 0-6 i n gt gg a n d tg p o s i t i o n s ( c f . F i g . h). L a r g e l y b e c a u s e t h e 0-6 h y d r o x y l s o f a l l s i x r e s i d u e s o f one h e l i x t u r n a r e i n equivalent p o s i t i o n s i n the V , V ^ - i o d i n e and D M S 0 s t r u c t u r e s (gg i n V a n d V - i o d i n e , gt i n V J ^ Q ) , t h u s f o r m ing symmetric i n t r a m o l e c u l a r hydrogen bonds, a l l r e s i d u e s i n t h e s e s t r u c t u r e s a r e e q u i v a l e n t . However, i n V - a m y l o s e , w i t h i t s m i x t u r e o f 0-6 p o s i t i o n s , m o l e c u l a r s i x f o l d symmetry i s n o t p r e s e n t i n t h e h e l i x a n d i n s t e a d , a 2^ s c r e w a x i s a l o n g t h e h e l i x a x i s e x i s t s , t h u s c o m b i n i n g t h r e e r e s i d u e s o f one h a l f - t u r n i n t o t h e asymmetric u n i t . N o n e t h e l e s s , t h e h e l i x backbone s t i l l resembles a six-fold helix. Because h i s s m a l l f o r t h e V-amyloses, a wide-diameter h e l i x is c h a r a c t e r i s t i c o f these structures. Complexing a g e n t s , such as DMSO, i o d i n e , o r w a t e r , a r e f o u n d i n s i d e t h e h e l i x c h a n n e l . F o r e x a m p l e , i n Yj^Q-amylose s i x DMSO m o l e c u l e s a r e accommodated i n s i d e t h e c h a n n e l w i t h i n one c r y s t a l l g g r a p h i c r e p e a t , w h i c h c o n s i s t s o f t h r e e h e l i x t u r n s {.c=2k.39 A ) . T h i s f i b e r r e p e a t i s n o t the r e s u l t o f t h e i n t r a c h a n n e l DMSO b u t i s c a u s e d s o l e l y b y t h e p a c k i n g o f t h e i n t e r s t i t i a l DMSO. A non-commensurable f i b e r r e peat f o r t h e amylose h e l i x and t h e i n t r a h e l i c a l i o d i n e i s o b s e r v e d in V^-iodine: a p p r o x i m a t e l y t h r e e i o d i n e s occupy t h e h e l i x chann e l w i t h i n one f i b e r r e p e a t , b u t t h e i o d i n e s form an a l m o s t l i n e a r p o l y i o d i d e chain o f an undetermined l e n g t h . I n this respect, the s t r u c t u r e s o f t h e V ^ - i o d i n e complex a n d t h e a - c y c l o d e x t r i n - i o d i n e complex ( 3 0 ) a r e s i m i l a r . Even though t h e i n s i d e o f t h e h e l i x channel o f V-amyloses i s p r i m a r i l y hydrophobic i n c h a r a c t e r , i n t r a h e l i c a l water has been f o u n d i n a l l o f t h e s t r u c t u r e s o f c o m p l e x e s s t u d i e d t o d a t e . The same was f o u n d t o b e t h e c a s e i n s i n g l e c r y s t a l s o f h y d r a t e d c y 3 2 1

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Figure 5.

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Va-amylose in ab projection. Positions of water molecules are shown by circles and hydrogen bonds are shown by dashed lines.


28.

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clohexaamylose (31), i n which the water, s i t u a t e d w i t h i n the c y c l i c s t r u c t u r e , i s h y d r o g e n - b o n d e d t o t h e 0-6 gt h y d r o x y l . I n t h e V - a m y l o s e , a l l 0-6 a r e i n t h e gg p o s i t i o n a n d c a n n o t h y d r o gen b o n d t o t h e w a t e r i n t h e c h a n n e l , b u t w a t e r i s s t i l l f o u n d i n i t , along w i t h the iodine. W a t e r i s p r e s e n t e v e n i n t h e "anhydrous" V -amylose, although i t i s s i t u a t e d a t a p e r i p h e r a l s i t e i n t h e h e l i x c h a n n e l a n d i s h y d r o g e n - b o n d e d t o a n 0-6 tg h y d r o x y l ( c f . F i g . U ) . (The tg p o s i t i o n h a s n o t y e t b e e n o b s e r v e d i n monomer o r o l i g o m e r c a r b o h y d r a t e s i n g l e c r y s t a l s t r u c t u r e s , b u t h a s b e e n f o u n d i n t h e h e l i c e s o f s e v e r a l a m y l o s e d e r i v a t i v e s , a s shown l a t e r i n t h i s review. I t has a l s o been found i n the polymorphic structures o f cellulose). I t i s p o s s i b l e that the r e l a t i v e l y empty h e l i x o f t h e V - a m y l o s e , i n c o m p a r i s o n w i t h o t h e r V-comp l e x e s , c a n l o w e r i t s e n e r g y o n l y b y d i s t o r t i o n o f t h e 6/5 h e l i x i n t o a 2/1 h e l i x . The same a p p a r e n t l y o c c u r s i n t h e c y c l o h e x a a m y l o s e h e x a h y d r a t e , a l s o a r e l a t i v e l y empty s t r u c t u r e i n compari s o n w i t h o t h e r eyelohexaamylose complexes ( 3 1 ) . The V - a m y l o s e s t r u c t u r e s g e n e r a l l y e x h i b i t s i m i l a r f e a t u r e s of c r y s t a l l i n e packing. A l l are orthorhombic, a n t i p a r a l l e l , twoc h a i n u n i t c e l l s , w i t h one c h a i n a t t h e c o r n e r a n d t h e o t h e r a t t h e c e n t e r o f t h e c e l l ( c f . F i g . 5 ) , a n d a l l e x c e p t one a r e i n s p a c e g r o u p P2j2^2±. The e x c e p t i o n i s V - i o d i n e w h e r e t h e p o l y i o d i d e c h a i n d i s t u r b s t h e 2^ s c r e w a x i s a l o n g t h e c h a i n , a n d t h e space group P 2 ( S ) appears t o f i t t h e s t r u c t u r e . The V - , V" - a n d V^-iodine-amyloses are a l s o very c l o s e t o hexagonal packing, as i n d i c a t e d b y t h e r a t i o o f t h e u n i t c e l l a x e s a/b b e i n g a l m o s t e x a c t l y 1//3. Some i n t e r m o l e c u l a r h y d r o g e n - b o n d i n g s t a b i l i z e s t h e p a c k i n g o f the chains i n a l l V - s t r u c t u r e s . I n V - a m y l o s e , two hydrogen bonds a r e formed between t h e c o r n e r a n d c e n t e r c h a i n s , and one h y d r o g e n b o n d f o r m s b e t w e e n t w o c o r n e r c h a i n s a l o n g t h e a axis. I n V - and V - i o d i n e - a m y l o s e s , a d d i t i o n a l hydrogen bonds form through i n t e r s t i t i a l water m o l e c u l e s . As each water molecule can p a r t i c i p a t e i n up t o f o u r hydrogen bonds, an e x t e n s i v e h y d r o gen b o n d n e t w o r k may b e p r e s e n t i n h y d r a t e d s t r u c t u r e s . Because t h e h y d r o g e n atoms c a n n o t b e d e t e c t e d i n p o l y m e r x - r a y c r y s t a l s t u d i e s , some i n d e t e r m i n a c y i n t h e h y d r o g e n - b o n d i n g scheme c a n n o t be a v o i d e d . The p r e s e n c e o f a h y d r o g e n b o n d i s b a s e d s o l e l y o n t h e o x y g e n - o x y g e n d i s t a n c e a n d , i n some c a s e s , o n t h e b o n d a n g l e s about t h e presumed hydrogen bond. The p a c k i n g o f t h e V Q M S O s t r u c t u r e ^ a t exceptional i n t h a t i t shows f e a t u r e s n o t f o u n d i n o t h e r V - a m y l o s e s i n c l u d e d i n T a b l e I . T h e i n t e r s t i t i a l DMSO m o l e c u l e s p r e v e n t a n y i n t e r a c t i o n between t h e c o r n e r c h a i n s o f t h e u n i t c e l l , b u t a l l o w hydrogen b o n d s t o f o r m b e t w e e n t h e c o r n e r a n d c e n t e r c h a i n s . The l o c a t i o n o f t h e DMSO m o l e c u l e s b e t w e e n t h e p a r a l l e l - p a c k i n g c o r n e r c h a i n s r e s u l t s i n a p s e u d o t e t r a g o n a l u n i t c e l l , b u t the space group i s s t i l l P2-L2221i n t e r s t i t i a l DMSO m o l e c u l e s a r e a l s o c l e a r l y responsible f o r the three-turn f i b e r repeat. h


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S a l t Complexes

A s e r i e s o f a l k a l i - a m y l o s e c o m p l e x e s can be o b t a i n e d d u r i n g t h e s o l i d - s t a t e d e a c e t y l a t i o n o f a m y l o s e t r i a c e t a t e , as f i r s t des c r i b e d b y S e n t i and W i t n a u e r (32). The u n i t c e l l s o f t h e i n d i v i d u a l members o f t h e s e r i e s o f L i O H - , KOIH NH3OH-, CsOH and g u a n i d i n i u m h y d r o x i d e - a d d u c t s o f a m y l o s e a p p e a r e d t o f i t an i s o morphous s e r i e s b a s e d on t h e s p a c e g r o u p P2-^2^2^ and a t e n t a t i v e c r y s t a l s t r u c t u r e was p r o p o s e d (32). The d e t a i l e d s t r u c t u r e o f t h e KOH-amylose c o m p l e x has now b e e n d e t e r m i n e d ( l l ) and t h e o v e r a l l s t r u c t u r e i s s i m i l a r t o that proposed e a r l i e r . It i s , t h e r e f o r e , l i k e l y t h a t a l l members o f t h e s e r i e s a r e i s o m o r p h o u s . The s t r u c t u r e , as shown i n F i g . 6, i s b a s e d on a l e f t - h a n d e d , 6/5 h e l i x . The l e f t - h a n d e d c o n f o r m a t i o n i s c o n s i s t e n t w i t h t h a t o f amylose t r i a c e t a t e from w h i c h i t i s d e r i v e d by a s o l i d s t a t e t r a n s f o r m a t i o n , as w e l l as w i t h t h a t o f V - a m y l o s e i n t o w h i c h i t c a n be e a s i l y c o n v e r t e d , l i k e w i s e i n t h e s o l i d s t a t e . As shown i n Table I I , i n extension the h g l i x i s very s i m i l a r to t h a j of a m y l o s e t r i a c e t a t e I (h = 3.7*+ A f o r KOH-amylose and 3.75 A for ATA I).. I n t r a m o l e c u l a r h y d r o g e n b o n d s a r e n o t p o s s i b l e i n t h i s e x tended conformation, which suggests that the forces r e s p o n s i b l e for the conformation o f V-amyloses are not dominating here. As i n d i c a t e d by t h e ,ij; a n g l e s o f t h e KOH-amylose c h a i n ( c f . F i g . 7), i t s c o n f o r m a t i o n does n o t c o i n c i d e w i t h t h e c o n f o r m a t i o n a l m i n i mum o f t h e map. The i n t e r m o l e c u l a r h y d r o g e n b o n d s t o w a t e r and t h e c o o r d i n a t i o n o f t h e K i o n apparently determine the energy minimum f o r t h i s s t r u c t u r e . B e c a u s e t h e r e i s one KOH and t h r e e w a t e r m o l e c u l e s f o r e a c h h a l f - t u r n o f t h e h e l i x , t h e asymmetric u n i t of the c r y s t a l s t r u c t u r e c o n s i s t s of three glucose residues which d i f f e r s l i g h t l y i n conformation, p a r t i c u l a r l y i n g l y c o s i d i c b o n d a n g l e s and t h e r o t a t i o n a l p o s i t i o n s o f t h e h y d r o x y m e t h y l g r o u p s . T h e r e f o r e , s i x - f o l d m o l e c u l a r symmetry i s n o t p r e s e n t , w h i c h i s i n agreement w i t h t h e P2\2-£\ s p a c e g r o u p . I n t h i s r e s p e c t , t h e KOH- and V - a m y l o s e s a r e s i m i l a r .


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+

a

B e c a u s e o f t h e good x - r a y d a t a ( a t o t a l o f 99 i n t e n s i t i e s were a v a i l a b l e f o r r e f i n e m e n t ) , d i f f e r e n c e F o u r i e r t e c h n i q u e s , s u c h as d e s c r i b e d b y W i n t e r i n t h i s v o l u m e (33), c o u l d be u s e d t o l o c a t e t h e KOH and w a t e r m o l e c u l e s i n t h i s c r y s t a l s t r u c t u r e . As shown i n F i g . 6, t h e K* i o n c o o r d i n a t e s w i t h f o u r o x y g e n s o f t h e a m y l o s e c h a i n and two w a t e r m o l e c u l e s . A l l three water molecules p a r t i c i p a t e i n hydrogen bonds, but the i n t e r m o l e c u l a r hydrogenb o n d i n g p a t t e r n i s not e x t e n s i v e . This probably accounts f o r the w a t e r - s o l u b i l i t y o f the complex. A s i m i l a r s e r i e s o f s a l t c o m p l e x e s o f a m y l o s e were a l s o des c r i b e d e a r l i e r b y S e n t i and W i t n a u e r (3^J_). A s a l t c o m p l e x , s u c h as K B r - a m y l o s e , i s o b t a i n e d f r o m KOH-amylose b y n e u t r a l i z a t i o n o f the a l k a l i . A l t h o u g h K B r - a m y l o s e has b e e n s t u d i e d s i n c e t h e i n i t i a l d e s c r i p t i o n of the s e r i e s , a d e f i n i t i v e c r y s t a l s t r u c t u r e d e t e r m i n a t i o n b y M i l l e r and B r a n n o n a p p e a r s i n t h i s volume (12). I t i s c l e a r t h a t i n i t s l e f t - h a n d e d c o n f o r m a t i o n and i n t h e e x -



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Figure 6. KOH-amylose in ac projection. The coordination of the K* ion (shown as (&) is indicated by dashed lines. Water and OH~ oxygens are denoted by filled, numbered circles (\\).



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Figure 7. A section of the , \p map for left-handed conformations of amylose, calculated with glycosidic bridge angle of 115° and a virtual bond length of 4.50 A. The bold contours indicate nonbonded conformational energy in kcalf mol, the thin lines indicate the number of residues per turn (n), and the dashed lines indicate the axial rise per residue (h), in Angstroms. The position of the conformation of the KOH-amylose is shown in the map by a filled circle (11).


28.



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t e n s i o n o f t h e h e l i x , KBr-amylose resembles b o t h amylose t r i a c e t a t e I a n d KOH-amylose.


The

N a t i v e A, B and C Polymorphs

As i s w e l l known, s t a r c h g r a n u l e s a r e n a t u r a l l y c r y s t a l l i n e and most s t a r c h v a r i e t i e s e x h i b i t e i t h e r t h e A o r t h e B d i f f r a c t i o n p a t t e r n , f i r s t d e s c r i b e d i n 1930 b y K a t z a n d V a n l t a l l i e (35.). The A p a t t e r n i s g e n e r a l l y s e e n i n c e r e a l s t a r c h e s , w h e r e a s t h e s t a r c h e s o f t u b e r s a n d r o o t s e x h i b i t t h e B p a t t e r n . The much l e s s common C p a t t e r n i s g i v e n b y t h e s t a r c h e s o f some b e a n s a n d b a n a na. D e s p i t e t h e f a c t t h a t t h e s e p a t t e r n s h a d b e e n known f o r nearl y 50 y e a r s , a d e t a i l e d a n a l y s i s o f n a t i v e s t a r c h p o l y m o r p h s was n o t c o m p l e t e d u n t i l 1978 ( l 3 , l A ) . U n d o u b t e d l y , t h e f a c t t h a t o n l y powder x - r a y p a t t e r n s a r e o b t a i n a b l e f r o m o r d i n a r y s t a r c h granules has been, i n p a r t , r e s p o n s i b l e f o r t h i s l o n g d e l a y , a l t h o u g h K r e g e r , i n 1951 b y u s e o f a n i n g e n i o u s m i c r o c a m e r a a n d v e r y l a r g e s t a r c h g r a n u l e s , was a b l e t o r e c o r d a " f i b e r " p a t t e r n o f a B - s t a r c h (36). W e l l - r e s o l v e d f i b e r p a t t e r n s d u p l i c a t i n g t h e n a t i v e A-, B- a n d C - s t a r c h p a t t e r n s w e r e f i n a l l y o b t a i n e d ( 1 7 , 3 8 ) , u s i n g t h e method o f S e n t i a n d W i t n a u e r ( 3 2 ) , a n d t h e s t r u c t u r e a n a l y s i s was s u c c e s s f u l l y c o m p l e t e d (l3.,l^_) f o l l o w i n g t h e s u g g e s t i o n b y K a i n u m a a n d F r e n c h (37) t h a t t h e n a t i v e p o l y m o r p h s w e r e d o u b l e - s t r a n d e d h e l i c e s . T h e s e s t r u c t u r e a n a l y s e s showed t h a t b o t h A- a n d B - a m y l o s e s w e r e n e a r l y i d e n t i c a l i n m o l e c u l a r c o n f o r mation, while d i f f e r i n g considerably i n the c r y s t a l l i n e packing o f t h e d u p l e x h e l i c e s . T h e C - p o l y m o r p h was shown t o b e s i m p l y a m i x t u r e o f t h e A- a n d B - p o l y m o r p h s ( 3 8 ) . 5

The m a i n d i f f i c u l t y i n a l l p r i o r a t t e m p t s t o d e v i s e m o l e c u l a r m o d e l s c o n s i s t e n t w i t h t h e A o r B x - r a y d a t a stemmed f r o m t h e f a c t t h a t s u c h m o d e l s w e r e s o u g h t i n a ,^ c o n f o r m a t i o n a l map f o r a s i n g l e - s t r a n d e d s t r u c t u r e . Although s u i t a b l e models r e s i d i n g i n e n e r g y m i n i m a a p p e a r e d t o e x i s t , none c o u l d b e p a c k e d i n t o t h e i n dicated unit cells. N o t u n t i l d o u b l e - s t r a n d e d (|),^ maps w e r e c a l c u l a t e d f o r a m y l o s e w i t h d i f f e r e n t virtual bond l e n g t h s , w e r e s u i t a b l e m o l e c u l a r models found. A comparison o f such s i n g l e - and d o u b l e - s t r a n d e d maps i s shown i n F i g . 8. Once a g a i n , t h i s i l l u s t r a t e s t h a t r e l i a n c e on o n l y maps i n a t t e m p t i n g c r y s t a l s t r u c t u r e a n a l y s i s may sometimes b e m i s l e a d i n g . The m o l e c u l a r c o n f o r m a t i o n a n d i n t e r s t r a n d h y d r o g e n b o n d i n g o f t h e A- a n d B - a m y l o s e s a r e shown i n F i g ^ 9- B o t h s t r a n d g o f t h e d u p l e x a r e r e l a t i v e l y e x t e n d e d {h = 3.^7 A f o r B a n d 3-51 A f o r A ) , a l t h o u g h n o t n e a r l y as extended as t h e a l k a l i o r s a l t a m y l o s e s , o r some o f t h e d e r i v a t i v e . s t r u c t u r e s . The e x t e n s i o n o f t h e h e l i x prevents t h e formation o f t h e i n t r a m o l e c u l a r 0-2...0-3(2) hydrogen bond t h a t occurs i n V-amyloses. C o n v e r s e l y , t h e extended c o n f o r m a t i o n p e r m i t s t h e f o r m a t i o n o f t h e i n t e r s t r a n d h y d r o g e n bonds t h a t appear i n s t r u m e n t a l i n t h e s t a b i l i z a t i o n o f t h e s t r u c t u r e . The t w o most i n t e r e s t i n g f e a t u r e s o f t h e d u p l e x h e l i x a r e t h a t t h e s t r a n d s a r e r i g h t - h a n d e d , as opposed t o t h e l e f t - h a n d e d conforma-



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Figure 8. A comparison of the single-stranded (top) and double-stranded (bottom) , \f/ maps for the right-handed conformations of amylose fsee caption of Figure 7 for details) (14)




475

Figure 9. Side view of the double helix of A- and B-amyloses. Interstrand hydrogen bonds are shown by dashed lines (38,).



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t i o n s o f a l l V- a n d i o n i c c o m p l e x a m y l o s e s , a n d t h a t t h e p a c k i n g o f t h e two s t r a n d s i s p a r a l l e l . Furthermore, the p a r a l l e l s t r a n d s o f t h e duplex a r e packed i n phase a l o n g t h e i r r e s p e c t i v e h e l i c a l p a t h s , w h i c h causes a l l odd-order l a y e r l i n e s t o be abs e n t i n t h e d i f f r a c t i o n p a t t e r n . T h i s c i r c u m s t a n c e p r e s e n t e d an a d d e d o b s t a c l e i n t h e way o f a n e a r l i e r r e c o g n i t i o n o f t h e t r u e s t r u c t u r e o f t h e s e p o l y m o r p h s . However, t h e p a c k i n g o f t h e d u plexes i n t o the c r y s t a l l a t t i c e i s a n t i p a r a l l e l i n both s t r u c tures. The d i f f e r e n t p a c k i n g o f t h e A- a n d B - a m y l o s e h e l i c e s i s shown i n F i g . 1 0 . B o t h s t r u c t u r e s e x h i b i t h e x a g o n a l p a c k i n g ( a l t h o u g h f o r t h e A-amylose i t i s s l i g h t l y d i s t o r t e d ) ; however, t h e m a i n d i f f e r e n c e b e t w e e n t h e two l i e s i n t h e l o c a t i o n o f t h e water molecules. I n the B-structure, a channel approximately o f t h e same d i a m e t e r a s t h a t o f t h e h e l i x e x i s t s i n t h e c e n t e r o f t h e h e x a g o n o f h e l i c e s . As much as ~30% w a t e r c a n e n t e r t h i s channel. I n t h e A - s t r u c t u r e , on t h e o t h e r h a n d , t h e h e x a g o n i s s l i g h t l y l a r g e r and i t s c e n t e r i s o c c u p i e d by another h e l i x , w h i l e a s m a l l e r amount o f w a t e r i s d i s t r i b u t e d e q u a l l y i n t h e i n t e r s t i a l s p a c e s b e t w e e n t h e h e l i c e s . B e c a u s e t h e r e i s no " h o l e " p r e s e n t i n t h e c e n t e r o f t h e d u p l e x , as t h e r e i s i n t h e s i n g l e h e l i x o f V - a m y l o s e , w a t e r c a n n o t e n t e r t h e h e l i c e s o f t h e A- a n d B-amyloses. F u r t h e r , i n t h e B - s t r u c t u r e , t h e l o c a t i o n o f t h e water i n the channel suggests that t h e water molecules are l o o s e l y h e l d a n d a r e n o n - c r y s t a l l i n e . T h i s i s i n agreement w i t h t h e f a c i l e and r e v e r s i b l e d e h y d r a t i o n - r e h y d r a t i o n o f t h e s t r u c t u r e upon v a c u u m - d r y i n g a n d e x p o s u r e t o h i g h r e l a t i v e h u m i d i t y . The e x p e c t e d changes i n t h e x - r a y d i f f r a c t i o n p a t t e r n s f o l l o w i n g such treatment are observed. I t i s a l s o probable t h a t under proper circumstances a h e l i x c o u l d d i s p l a c e t h e w a t e r i n t h e open c h a n n e l o f t h e B - s t r u c t u r e , thus converting i t t o t h e A - s t r u c t u r e . T h i s c o n v e r s i o n has a l s o b e e n o b s e r v e d . A m i x t u r e o f t h e A a n d B u n i t c e l l s i n t h e same s t r u c t u r e i s a l s o p o s s i b l e and t h i s , i n f a c t , accounts f o r t h e C-polymorph. The d o u b l e h e l i c a l s t r u c t u r e o f t h e A a n d B p o l y m o r p h s i s i n agreement w i t h t h e p h y s i c a l p r o p e r t i e s e x h i b i t e d b y c r y s t a l l i n e s t a r c h and amylose, p a r t i c u l a r l y i n t h e i r i n s o l u b i l i t y i n w a t e r , l a c k o f complexing a b i l i t y w i t h s m a l l molecules o r i o d i n e , and t h e i r g e l l i n g behavior. The h a r d e n i n g a n d c r y s t a l l i z a t i o n o f g e l s o f a m y l o s e a n d s t a r c h upon a g i n g - t h e phenomenon o f " r e t r o g r a d a t i o n " f a m i l i a r t o s t a r c h c h e m i s t s - may o c c u r as a r e s u l t o f t h e formation o f double h e l i c a l " j u n c t i o n zones" i n t h e s o l u t i o n o f amylose, f o l l o w e d b y a g g r e g a t i o n and c r y s t a l l i z a t i o n o f t h e d o u b l e h e l i c e s , a s shown s c h e m a t i c a l l y i n F i g . 1 1 . Because t h e v a r i e t i e s o f s t a r c h t h a t c o n t a i n o n l y amylopect i n a r e a l s o c r y s t a l l i n e , e x h i b i t i n g t h e same d i f f r a c t i o n p a t t e r n s as s t a r c h e s c o n t a i n i n g amylose, t h e r e i s a s t r o n g l i k e l i hood t h a t t h e e x t e n s i v e l y branched amylopectin m o l e c u l e a l s o c r y s t a l l i z e s i n a d o u b l e - h e l i c a l form. I n t u r n , t h i s i m p l i e s t h a t l i n e a r sequences i n a m y l o p e c t i n remain s u f f i c i e n t l y l o n g t o


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Figure 11.

Possible mechanism of crystalline gel formation from aqueous amylose solutions (left: solution; middle: gel; right: crystalline gel) (A2)


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c r y s t a l l i z e i n t h i s fashion, o r that during b i o s y n t h e s i s , l i n e a r sequences c r y s t a l l i z e immediately a f t e r s y n t h e s i s , f o l l o w e d by b r a n c h i n g on t h e s u r f a c e o f t h e c r y s t a l l i t e s .


Amylose D e r i v a t i v e s Three c l a s s e s o f amylose d e r i v a t i v e s have been s t u d i e d : a m y l o s e t r i a c e t a t e I (ATAI) ( l 6 ) , t r i m e t h y l - a m y l o s e (TMA) ( l 8 ) and t r i e t h y l - a m y l o s e ( T E A ) . Two p o l y m o r p h s o f t h e l a t t e r h a v e b e e n o b s e r v e d ( T E A 1 , TEA3) (19,20), a s w e l l a s s e v e r a l o f t h e s o l v e n t c o m p l e x e s o f TEA1 ( 2 1 , 2 2 ) . A l l o f t h e d e r i v a t i v e s possess r e l a t i v e l y extended, Jeft-handed conformations, w i t h h r a n g i n g f r o m 3.75 t o U.05 A. N e a r l y a l l o f t h e s t r u c t u r e a r e f o u r f o l d h e l i c e s , packing i n orthorhombic, a n t i p a r a l l e l , two-chain u n i t c e l l s i n s p a c e g r o u p ~P2-±2\2\. An e x c e p t i o n i s A T A I , w h i c h as a lk/ll h e l i x , h a s U.6T r e s i d u e s p e r t u r n a n d p o s s e s s e s t h e s p a c e g r o u p P2]_(S). Because i n these d e r i v a t i v e s , o n l y van d e r Waals f o r c e s govern b o t h t h e m o l e c u l a r conformation and t h e c h a i n p a c k i n g , i t i s imperative that both are r e f i n e d simultaneously during the s t r u c t u r e a n a l y s i s . A l t h o u g h t h i s was o r i g i n a l l y n o t done w i t h A T A I , i t s s t r u c t u r e was s u c c e s s f u l l y s o l v e d b y s t e r e o c h e m i c a l m e t h o d s , more t h a n 10 y e a r s a g o , t h e f i r s t p o l y s a c c h a r i d e s t r u c t u r e s o l v e d b y t h e s e means. I t s l e f t h a n d e d , n o n - i n t e g r a l h e l i x , w i t h i t s v e r y l o n g f i b e r r e p e a t o f 5 2 . 5 3 X , was e s s e n t i a l l y r e c ognized from t h e s p l i t t i n g o f l a y e r l i n e s i n t h e x-ray f i b e r d i agram. The c a r b o n y l o x y g e n s o f t h e 0-2 a n d 0-3 a c e t y l g r o u p s were f o u n d t o e c l i p s e t h e c o r r e s p o n d i n g r i n g h y d r o g e n s ( c f . F i g . 12) . T h e s e c a r b o n y l p o s i t i o n s were f o u n d s t r i c t l y f r o m c o n f o r m a t i o n a l e n e r g y m i n i m i z a t i o n a n d were t h e f i r s t s u c h p o s i t i o n s s e e n i n acetate structures. Later single c r y s t a l studies o f a c e t y l a t e d oligomes o f sugars confirmed t h e e x i s t e n c e o f t h e e c l i p s e d p o s i t i o n s (39 h0). The 0-6 was f o u n d t o b e i n t h e v i c i n i t y o f t h e tg p o s i t i o n . The p a c k i n g o f t h i s , somewhat u n u s u a l , l U / l l h e l i x i s p s e u d o h e x a g o n a l , w i t h t h e a/b u n i t c e l l a x i s r a t i o v e r y c l o s e t o 1//3. L a t e r r e f i n e m e n t o f t h e s t r u c t u r e c o n f i r m e d t h e e a r l i e r found f e a t u r e s , as w e l l as t h e space group P2]_(S), w i t h t h e s c r e w a x i s p e r p e n d i c u l a r t o t h e c h a i n ( P . Z u g e n m a i e r , unpubl i s h e d w o r k ) . Some o f t h e a c e t y l s u b s t i t u e n t s a l o n g t h e c h a i n were found t o be r o t a t e d s l i g h t l y from t h e i r o r i g i n a l p o s i t i o n s , b u t a l l s h o r t p a c k i n g c o n t a c t s w e r e e l i m i n a t e d as a r e s u l t . I n TMA, t h e r a t i o o f t h e u n i t c e l l d i m e n s i o n s a/b i s a l m o s t 2/1, y e t t h e i n t e r c h a i n d i s t a n c e i s c l o s e t o t h a t o f ATAI a n d e v e n c l o s e r t o b o t h TEA p o l y m o r p h s ( c f . T a b l e Xtj . The t r u e c o n f o r m a t i o n o f TMA, a l t h o u g h n o m i n a l l y a f o u r - f o l d h e l i x ( c f . F i g . 13) , i s g o v e r n e d b y a 2-^ s c r e w a x i s , a s e v i d e n c e d b y a s t r o n g second order m e r i d i o n a l r e f l e c t i o n . The r e a s o n f o r t h e l o w e r symmetry i s t h a t t h e 0-6 m e t h y l g r o u p s o n two s u c c e s s i v e r e s i d u e s a r e i n d i f f e r e n t r o t a t i o n a l p o s i t i o n s , one c l o s e t o tg a n d t h e o t h e r c l o s e t o gt. The d i m e r r e s i d u e i s , t h e r e f o r e , t h e t r u e a s y m m e t r i c u n i t i n a c c o r d a n c e w i t h t h e P2^2]_2^ s p a c e g r o u p . When 1

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Figure 12. A section of the amylose triacetate helix showing acetate positions

Figure 13. Chain conformation of trimethylamylose, shown in 110 projection



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c o m p l e x i n g a g e n t s a r e i n s e r t e d i n t o t h e c r y s t a l l a t t i c e o f TMA, s o l i d s t a t e transformations occur. These r e s u l t i n a l l o f t h e m e t h y l 0-6 g r o u p s r o t a t i n g i n t o one p o s i t i o n a n d a t r u e f o u r fold helix i s established. The TEA d e r i v a t i v e y i e l d s an a b u n d a n t number o f e a s i l y o b t a i n a b l e c r y s t a l l i n e p o l y m o r p h s , most o f w h i c h c o m p l e x w i t h solvent molecules. The c o m p l e x e s t h a t h a v e b e e n s t u d i e d e x h i b i t a f o u r - f o l d , lefthanded h e l i c a l conformation w i t h t h e r o t a t i o n o f t h e e t h y l 0-6 g r o u p a g a i n c l o s e t o tg. I n t r o d u c i n g a s m a l l numb e r o f s o l v e n t m o l e c u l e s (e.g., c h l o r o f o r m o r d i c h l o r o m e t h a n e , one m o l e c u l e p e r two g l u c o s e u n i t s ) , t h e T E A 1 - C 1 a n d TEA1-DCM1 p o l y m o r p h s a r e o b t a i n e d , r e s p e c t i v e l y . The s o l v e n t m o l e c u l e s are l o c a t e d b o t h i n t h e i n t e r s t i t i a l spaces between t h e c h a i n s o f t h e same p o l a r i t y a s w e l l a s b e t w e e n t h e s h e e t s o f a n t i p a r a l lel-packed chains. T h i s r e s u l t s i n an i n c r e a s e i n b o t h and ^ 1 1 0 d i s t a n c e s ( c f . T a b l e I I ) . As s e v e r a l r a t h e r s h o r t C&...0 d i s t a n c e s o c c u r b e t w e e n t h e c o m p l e x i n g a g e n t s a n d t h e TEA h e l i c e s , i t was c o n c l u d e d t h a t s t r o n g d i p o l e i n t e r a c t i o n s f o r c e t h e solvent molecule i n t o the observed p o s i t i o n s (2l). A n o t h e r s e r i e s o f p o l y m o r p h s i s o b t a i n e d when more c o m p l e x i n g a g e n t , s u c h as c h l o r o f o r m , d i c h l o r o m e t h a n e o r n i t r o m e t h a n e , i s a d d e d t o t h e T E A 1 s t r u c t u r e , t o t h e e x t e n t o f one s o l v e n t m o l e c u l e p e r g l u c o s e r e s i d u e . Now t h e s o l v e n t m o l e c u l e s a r e f o u n d a l o n g t h e h e l i x g r o o v e s w i t h t h e i r d i p o l e moments s t a t i s t i c a l l y o r i e n t e d i n s p a c e . The r e s u l t i n g u n i t c e l l i s p s e u d o t e t r a g o n a l , b u t t h e s p a c e g r o u p r e m a i n s P2±2±2± (22_). Conclusions The s t u d y o f c r y s t a l l i n e a m y l o s e s h a s p r o v i d e d c o n s i d e r a b l e s t r u c t u r a l i n f o r m a t i o n , n o t o n l y about t h e d e t a i l s o f s t r u c t u r e , but a l s o about t h e p r i n c i p l e s t h a t appear t o govern t h e c r y s t a l l i z a t i o n o f these polymers. P a r t i c u l a r l y noteworthy i s t h e obs e r v a t i o n t h a t f o r d i v e r s e m o l e c u l a r c o n f o r m a t i o n s , o n l y two space groups account f o r t h e m a j o r i t y o f t h e s t r u c t u r e s . I t seems c l e a r t h a t i n t h e c r y s t a l l i z a t i o n o f t h e s e p o l y s a c c h a r i d e s , energy m i n i m i z a t i o n o f t h e s t r u c t u r e i s not s o l e l y t h e p r o p e r t y o f t h e m o l e c u l a r c o n f o r m a t i o n , as h a d b e e n t h o u g h t n o t t o o l o n g ago. I t i s a l s o n o t e w o r t h y t h a t t h e tg r o t a t i o n a l p o s i t i o n o f the hydroxymethyl group appears r e g u l a r l y . T h i s p o s i t i o n has not y e t been observed i n t h e c r y s t a l s t r u c t u r e s o f g l u c o s e dime r s o r o l i g o m e r s , t h u s one w o n d e r s w h e t h e r t h e o c c u r r e n c e o f t h i s r o t a m e r i n t h e p o l y m e r i s i n some way a f u n c t i o n o f t h e chain structure. The r e f i n e m e n t m e t h o d o l o g y h a s , l i k e w i s e , b e e n f u r t h e r e d b y t h e s t u d y o f c r y s t a l l i n e a m y l o s e s . F o r e x a m p l e , we now know t h a t g i v e n o n l y m i n i m a l l y adequate d i f f r a c t i o n d a t a , b u t h a v i n g on h a n d d e t a i l e d i n f o r m a t i o n f r o m monomer a n d o l i g o m e r c r y s t a l s t r u c t u r e s , a n d h a v i n g t h e c a p a b i l i t y t o r e f i n e m o d e l s w i t h comp l e t e s t e r e o c h e m i c a l f l e x i b i l i t y , s t r u c t u r e s c a n be s o l v e d w i t h



28.



481

h i g h degrees o f r e l i a b i l i t y . W i t h improvements i n i n t e n s i t y measurements o n t h e h o r i z o n , a s a number o f p a p e r s i n t h i s v o l u m e i n d i c a t e , t h e r e l i a b i l i t y o f s t r u c t u r e a n a l y s i s s h o u l d soon approach that o fs i n g l e - c r y s t a l a n a l y s i s . I t i s already impress i v e l y demonstrated b y t h e r e s u l t s o f t h e KBr-amylose s t u d y ( 1 2 ) . S i m i l a r l y , i t i s becoming c l e a r t h a t e l e c t r o n d i f f r a c t i o n w i l l p l a y an i n c r e a s i n g l y i m p o r t a n t r o l e i n f u t u r e s t r u c t u r a l work. And a s one f i n a l o b s e r v a t i o n , t h e s t r u c t u r e s o f t h e n a t i v e a m y l o s e p o l y m o r p h s s u g g e s t once more t h a t s y n t h e t i c p o l y m e r c h e m i s t s h a v e much t o l e a r n f r o m n a t u r a l p o l y m e r p r o c e s s e s . A t t h i s t i m e , o n l y n a t u r e seems t o b e a b l e t o a s s e m b l e s u c h a n e l e gant s u p e r m o l e c u l a r s t r u c t u r e as t h e s t a r c h g r a n u l e , p e r f e c t l y s u i t e d t o i t s needs. Acknowledgement s T h i s work h a s b e e n s u p p o r t e d b y N a t i o n a l S c i e n c e F o u n d a t i o n g r a n t CHE77277^9 ( t o A. S.) a n d a g r a n t f r o m D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t ( t o P. Z . ) . C o o p e r a t i v e e f f o r t s o f t h i s w o r k h a v e a l s o b e e n s u p p o r t e d b y a NATO R e s e a r c h G r a n t No. 1386, t o b o t h authors.

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21,

1980.


Crystal Structures of Amylose and Its Derivatives - ACS Symposium

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