Antonio Deriu Dipartimento di Fisica e Scienze della Terra ... Houches... · to that of granular...

Les Houches - April 15 – 26 2013 WATSURF 2013 1

Antonio Deriu

Dipartimento di Fisica e Scienze della Terra “M. Melloni”Università di Parma - Italy


Outline:

Carbohydrates an overview

Low hydration systems: hydrated powders and fibers

High hydration systems: polysaccharide gels


Charboydrates are ubiquitous in nature:

Structure and textures of terraqueous plants are dominated by polysaccharides ( cellulose,

mannan, alginate and xylan).

Chitin plays a major role in the structure and organisation of insect cuticle

In animal tissues carbohydrate substances, ( hyaluronic acid, chondroitin and dermatan

sulphates) function as lubricants, and provide viscoelastic properties

Carbohydrates are covalently attached to proteins to form proteoglycans and glycoproteins

which serve as vital functional operators in molecular biology

Cell surfaces are decorated with carbohydrate substances, for example heparan sulphate,

which influences cell adhesion and recognition

The polysaccharide heparin suppresses blood clotting and certain glycoproteins act as

antifreeze agents in the blood of polar fish.

…………………………………………………


Simple and complex Carbohydrates


Classification of carbohydratesa) Monosaccharides (monoses or glycoses)

Trioses, Tetroses, Pentoses, Hexoses

b) OligosaccharidesDi, tri, tetra, penta, up to 9 or 10 Most important are the disaccharides

c) Polysaccharides or glycansa) Homopolysaccharidesb) Heteropolysaccharides


Classification of carbohydratesa) Monosaccharides (monoses or glycoses)

Trioses, Tetroses, Pentoses, Hexoses

b) OligosaccharidesDi, tri, tetra, penta, up to 9 or 10 Most important are the disaccharides

c) Polysaccharides or glycansa) Homopolysaccharidesb) Heteropolysaccharides

Glycogen

Starch

Cellulose

Maltose = Glucose + GlucoseLactose = Glucose + GalactoseSucrose = Glucose + Fructose


Polysaccharides or glycans

1) Homoglycans (Starch, Cellulose, Glycogen, Dextrinsor Inulin)

2) Heteroglycans (Mucopolysaccharides)

Characteristics: polymers (MW from 200,000) white and amorphous products (glassy) not sweet not reducing; do not give the typical aldose or ketose reactions form colloidal solutions or suspensions


Homopolysaccharides

Homopoysaccharides are polymers composed of a single type of sugar monomers

Homo polysaccharides

Fructosane.g. Inulin

Glucosanse.g. Starch

GlycogenCellulose

Galactosane.g. Agar


Glucosans /Glucans1) Glycogen (Storage Polysaccharide)

Also known as animal starch Stored in muscle and liver Present in cells as granules (high MW) Contains both α(1,4) links and α (1,6) branches at every 8 to 12 glucose units Complete hydrolysis yields glucose Hydrolyzed by both α and β-amylases and by glycogen phosphorylase


2) Starch (Storage Polysaccharide)

Most common storage polysaccharide in plants Composed of 10 – 30% Amylose and 70-90% Amylopectin depending

on the source

a) Amylose is a linear polymer of α-D-glucose, linked together by α 1→4glycosidic linkages. It is soluble in water, its MW ranges between50, 000 – 200, 000.

b) Amylopectin is a highly branched polymer, insoluble in water. Its MWranges between 70,000 – 1,000,000. Branches are composed of 25-30glucose units linked by α 1→4 glycosidic linkage in the chain and by α1→6 glycosidic linkage at the branch point.


Amylopectin

Amylose


StarchAmylose is simply poly-(1-4)glucose so it is a straight chain. Infact the chain is floppy, andsuspensions of Amylose tend tocoil up into a helical conformation

Iodine (I2) can insert in the middleof the Amylose helix to give a bluecolor that is characteristic anddiagnostic for starch


Agarose (Galactosan)

Agar is a galactose polymer Obtained from the cell walls of some species of red algae or

seaweeds (Sphaerococcus Euchema ) and species of Gelidium Dissolved in hot water and cooled, agar becomes gelatinous; Its chief use is as a culture medium for microbiological work. Other uses are as a laxative, A vegetarian gelatin substitute, A thickener for soups, in jellies, ice cream and Japanese desserts, As a clarifying agent in brewing, and for sizing fabrics.


Heteropolysaccharides/ Mucopolysaccharides

Mucopolysaccharides or Glycosaminoglycans are carbohydrates containing a repeating disaccharide.

The disaccharide usually contains an acid sugar and an amino sugar.

Acid sugar is generally D- Glucuronic acid or its C-5 epimer Iduronicacid, while amino sugar is either D- Glucosamine or D-Galactosamine, amino group is generally acetylated eliminating its positive charge.

Carboxyl groups of acid sugars together with sulfate groups give Glycosaminoglycans strongly negative nature.


Specific Glycosaminoglycans of Physiological Significance

Hyaluronic acid-(D-glucuronate + N-acetyl-D-glucosamine)n Occurrence: synovial fluid, ECM of loose connective tissue. Serves as a

lubricant and shock absorber. Hyaluronic acid does not contain any sulfate and is not found covalently

attached to proteins. It forms non-covalently linked complexes with Proteoglycans in the ECM. Hyaluronic acid polymers are very large (100 - 10,000 kDa) and can displace

a large volume of water.

N-acetylglucosamine

~ 10 Å

Glucuronic acid


Gel formation

16

• Change temperature• Change solvent quality• Change ionic environment


Gel formation

17


Studies of starch granules Starch granules: semicrystalline structures with a lamellar

arrangement of the two main constituent biopolymers (amylose andamylopectin).

SAXS from hydrated native starches shows a broad scattering peak,from which the average thickness of the lamellar repeat unit(crystalline plus amorphous region) can be calculated (~ 47 Å)

SANS provides the additional ability to quantify the distribution ofwater within the granule so that the gelatinisation behaviour can beanalysed following the location of water during the swelling of thegranule

Resistant starch (RS) is a fraction of starch that is not digested in thesmall intestine and arrives at the colon where it may be fermentedinto short chain fatty acids. The latter molecules are beneficial forthe correct functioning of the bowel and for disease prevention.

A detailed SANS + SAXS study of RS has been performed by Lopez-Rubio et al. (Biomacromolecules, 8 (2007) 1564-1572). The adoptedmodel adopted incorporates parameters that yield the degree ofcrystallinity, the characteristic dimension and the scattering contrastbetween the crystalline and amorphous phases.

From the fits, it was possible to determine that the contrastmatchpoint occurs for a solvent containing 58.6% D2O, very similarto that of granular starch, indicating that the scattering lengthdifferences of amorphous and crystalline phases are identical innative starch and its resistant starch fractions.

TEM image of a starch granule showing thealternating crystalline and amorphous lamellae

SANS patterns of RS. Four solvent contrasts havebeen used: along with an effective fifth contrast fromSAXS. Data were simultaneously fitted with a power lawand two phase non-particulate model (solid lines).


Energy landscape accessible to neutrons


Rayleigh Scattering of Mössbauer RadiationEγ = 14.41 KeVλ = 0.8602 Å

ΔEγ = 70 neVΔE/E = 10-12 !!!


Crystalline B form of Amylose (from potato tubers)

saccharide chains are organised in left-handed double helices with six glucoseunits per turn

The two parallel sixfold single strands are repeated along the c crystallographicaxis (hexagonal unit cell, space group P61, a = b = 18.5 Å, c = 10.4 Å).


Crystalline B form of Amylose (from potato tubers)

hexagonal superstructures, kept together by interchain hydrogen bonds (HBs),with a central pore with 11Å average diameter

H2O molecules gradually fill the pores up to a saturation point : of ~ 0.27 gwater/g dry amylose ( ~ 36 water molecules per unit cell; 3 water moleculesper glucose unit).


Vibrational dynamics of hydration water (Tosca @ ISIS)


Elastic temperature scans on IN13 @ ILL

d

ΔH


Mean square atomic fluctuations (D2O hydrated samples)

Università degli Studi di ParmaINFM

AMYLOSE AMYLOPECTIN


Onset of the dynamic transition

2 2 2 5%a h G GT u u u


Thermodynamics: Van’t Hoff plot

3.0 3.5 4.0 4.5 5.0-5

-4

-3

-2

-1

0

1

1000/T (K-1)

ln(p

1/p2)

Amylopectine h = 40 % Amylopectine h = 32 % Amylopectine h = 12 % Amylose h = 47 % Amylose h = 15 %

2 1ln p p H RT S R


Comparison between RSMR and ENS

Oligosaccharides vs. polysaccharides

0 50 100 150 200 250 300 3500.00

0.05

0.10

< u2 h(T

) > -

(Å

2 )

T (K)

h = 0 (), 0.15 () 0.35 (), 0.47 (), 0.60 ()

Amylose

0 50 100 150 200 250 300 3500.00

0.05

0.10

0.15

0.20

< u2 h(T

) > -

(Å

2 )

T(K)

h = 0 (), 0.25(), 0.50 ()

Glucose

0 .0 0 .1 0 .2 0 .3 0 .4 0 .5 0 .60 .0 0

0 .0 5

0 .1 0

0 .1 5

h ( g D 2 O / g s a c c h a r i d e )

< u2 h(2

97K

) > -

(Å

2 ) Glucose () Amylose () Amylopectin ()

T = 297 K

0 50 100 150 200 250 300 3500.00

0.05

0.10

< u2 h(T

) > -

(Å

2 )

T (K)

h = 0 (), 0.15 () 0.35 (), 0.47 (), 0.60 ()

Amylose

0 50 100 150 200 250 300 3500.00

0.05

0.10

0.15

0.20

< u2 h(T

) > -

(Å

2 )

T(K)

h = 0 (), 0.25(), 0.50 ()

Glucose

0 .0 0 .1 0 .2 0 .3 0 .4 0 .5 0 .60 .0 0

0 .0 5

0 .1 0

0 .1 5

h ( g D 2 O / g s a c c h a r i d e )

< u2 h(2

97K

) > -

(Å

2 ) Glucose () Amylose () Amylopectin ()

T = 297 K

Glucose (triangles) Amylose (open squares) Amylopectin (full squares)


Some conclusions: Marked dependence upon hydration of onset temperature for the

dynamic transition (Ta )

Fluctuations of the hydrogen bond network as basic driving

mechanism

Two states for each hydrogen bond ?

Onset of the dynamical transition when fast H-bond fluctuations

become comparable to vibrational displacements


Water dynamics in Hydrated Hyaluronate fibres: Hyalutonic acid (HA) is made up from wo saccharide heterodimers : β-1,3

linked D-glucuronic acid and β-1,4 linked N-acetyl-D-glucosamine It is a strongly polyanionic molecule due to K+ and NA+ ions complexed

between three or four helical polysaccharide strands HA fibers at moderate hydration show a double helical structure with a variety

of packing arrangements

Ordered biopolymer fibers: the wet spinning technique


Ordered water XRD with Q vector parallel to the

fibers Sharp water peak (WP) at ~ 1.97 Å-1

with hydration dependent intensity In the direction perpendicular to the

fibers the scattering from ordered water is negligible

T = 295 KRH = 75%

Order-disorder transition At about 88% RH a weakening of the

intermolecular bonds takes place The spped of sound (from Brillouin

light scattering) decreases from ~ 4 to 2.5 km/s

At 90% RH the fibers become disordered

HA

DNA


Water Peak: RSMR data

Total (●), elastic (○) and inelastic () scattering in the QII direction

54% RH 75% RH 83% RH

Dependence upon RH of the elastic fraction: f = Iel /(Iel+Iin)

● ● ● ● ● ●●

●


Hyaluronic acid (HYA) based hydrogels:

HYADD™ is a chemically modified hyaluronate with 2–3 % of the carboxylate groups grafted with a hexadecylic amine moiety.

HYADD has been introduced to produce more ‘stiff’ gels improving the viscoelstic properties at low saccharide concentration.

• Chemically modified form of HYA: HYADDTM

Chemical modificationHYA

• Native hyaluronic acid: HYA

N-acetylglucosamine

~ 10 Å

Glucuronic acid


Hyaluronic acid based hydrogels:

Gel structure

Dynamics

Diluted gels: 3·10-3, 5·10-3,8·10-3 g/mlSAXS: ID02 @ ESRF

Concentrated gels: 8·10-3, 5·10-2, 0.1 g/ml in D2OSANS: PAXE @ LLB

Polymer dynamics: QENS high resolution• SPHERES @ FRM II - ΔE ~ 0.6 meV (FWHM);

• IN11 @ ILL- Fourier time range: 0.053 – 1.33 ns

Water dynamics: QENS low resolution

• NEAT @ HZB - ΔE ~ 70 meV;

• IRIS @ ISIS - ΔE ~ 17 meV

SAMPLES: HYA and HYADD hydrogels in high ionic strength solvent atconcentrations from 10-3 to 10-1 g/ml and temperatures from 270 to 320 K

ID02 @ ESRF

1E-3 0.01 0.11E-4

1E-3

0.01

0.1

1

I(Q

) (a.

u.)

Q (Å-1)

HYA 0.8 % HYA 0.3 %

Q-1

Q-3 Lp ~ 1000 Å

Q > 0.8 Å-1 I(Q) Q-1 rod-like structures

Q < 0.8 Å-1 I(Q) Q-1.7 random coil

Q > 0.007 Å-1 I(Q) Q-1 rod-like structures

Q < 0.007 Å-1 I(Q) Q-3 3D structure


PAXE @ LLB

( ) ( ) ( )tot D SI Q I Q I Q

22

2 2

1( )1

BD

O S

k TI QK Q

2 3 222 2

1( ) 81

SI QQ

Ornstein-Zernike Debye-Bueche

The first term is due to thermally induced motions of the chains in the solvent and it is governed by the osmotic compression modulus KOS

The second term represents longer range static variations in concentration due to the permanent elastic constraints inside the network. The long correlation length Ξ describes the spatial range of the elastic deformations

0.01 0.10.01

0.1

1

10

100

1000

HYA10% HYADD10%

I (Q

) (a.

u)

Q (Å-1)


QENS experiments (NEAT & IRIS): water dynamics

HYA/HYADD in H2O

(1, 5, 10) · 10-2 g/ml

T=275, 296 e 320 K

Water dynamics

HYA/HYADD in D2O

(1, 5, 10) · 10-2 g/ml

T=275, 296 e 320 K

Polymer dynamics

NEAT IRIS( , ) ( , ) (1 ) ( , ) ( , )poly waterI Q S Q S Q R Q bkg

2 2

( , ) ( , ) ( , )wu Q

water trans rotS Q e S Q S Q

2

22

( 1)( , ) (2 1) ( )( 1)

rotrot l

l rot

l lS Q l j Qal l

2 2

( )1( , )( )

transtrans

trans

QS QQ

2

2( )1

ttrans

t

D QQD Q

a = 0.942 Å (close to O-H distance)Γrot = (350 ± 50) meV, rot = 1.8±0.3 ps(same values as free water)

H2O (squares)HYA10 (circles) HYADD10 (triangles)

H2O

● HYA 10HYADD 10

H2O

● HYA 10HYADD 10

Swater

Spolymer

Swater

Resolution Resolution


2 2

0

0 2 2

( , ) ( ) ( )

( )11 ( )( )

pu Q

po lym er

p

p

S Q e A Q

QA Q

Q

HYA10 T = 320, 296, 275 K from bottom to top

QENS (IRIS): fast polymer dynamics

HHYA10 (open symbols) and HYADD10 (full symbols)T = 320K (triangles), 296K (circles) and 275K (squares)

Confined diffusion in a sphere2

1 00

0

3 ( )( ) (1 )( )j QRA Q f fQR

Spolymer

Swater

Resolution

( , ) ( , ) (1 ) ( , ) ( , )poly waterI Q S Q S Q R Q bkg

Spolymer

Selastic

Squasielastic

275 K 296 K 320 K

275 K 296 K 320 K


QENS (SPHERES @ FRM-II & IN11 @ ILL): slow polymer dynamics

2 2

( , ) ( ) [1 ( )]exp( / );( ,0)

( ) Q u

F Q t f Q f Q tF Q

f Q e

τ(ns

)

τ(ns

)


Water diffusivity in Agarose and Hyaluronate gels

Agarose gel

Hyaluronate gelH2O


Thanks to …… My colleagues in Parma: G. Albanese, F. Cavatorta, M.T. Di Bari

in Milano: L.Cantu’, E. Del Favero, P. Brocca

in Roma “Tor Vergata” : G. Paradossi, E. Chiessi

at the ILL: F. Natali, Y. Gerelli, G. Mariani

at the ESRF: T. Narayanan

at the LLB: J. Teixeira

at ISIS: V. Garcia Sakai

at HZB: M. Russina


References1) Cavatorta, F., Albanese, G., Deriu, A., Rupprecht, A., Il Nuovo Cimento D 18, 371 (1996).

2) Deriu, A., Cavatorta, F., De Micheli, T., Rupprecht, A., Langan, P., Physica B 234-236, 215 (1997).

3) Deriu, A., ‘‘Water dynamics in polysaccharide gels’’, in ‘Biological Macromolecular Dynamics’, Adenine Press, N.Y. (1997), p. 135.

4) Di Bari, M., Albanese, G., Cavatorta, F., Deriu, A., Physica B. 276-278, 257 (2000).

5) Di Bari, M., Cavatorta, F., Deriu, A., Albanese, G., Biophysical J. 81, 1190 (2001).

6) Deriu, A, Cavatorta, F., Albanese, G., Hyperfine Int. 141-142, 261 (2002).

7) Cavatorta, F., Angelini, N., Deriu, A., Albanese, G., Appl. Phys. A 74, S504 (2002).

8) Di Bari, M., Deriu, A., Albanese, G., Cavatorta, F., Chem. Phy. 292, 333 (2003).

9) Di Bari, M., Deriu, A., Albanese, G., Cavatorta, F., Phys. Chem. Chem. Phys. 7, 1241 (2005).

10) Sonvico F., Cagnani A., Rossi A., Motta S., Di Bari M.T., Cavatorta F., Alonso M.J., Deriu A., Colombo P. Int. J. Pharm. 324, 67-73 (2006).

11) Sonvico F., Di Bari M.T., Bove L., Deriu A., Cavatorta F., Albanese G.’, Physica B 385, 725–727 (2006).

12) Gerelli Y., Di Bari M.T., Deriu A., Cantù L., Colombo P., Como C., Motta S., Sonvico F., and May R., J. Phys.: Condens. Matter 20, 104211 (2008).

13) Gerelli Y., Barbieri S., Di Bari M.T., Deriu A., Cantu` L., Brocca P., Sonvico F., Colombo P., May R., and Motta S., ’Langmuir 24, 11378-11384(2008).

14) Deriu A., Di Bari, M.T., and Gerelli, Y., Z. Phys. Chem. 224, 227-242 (2010).

15) Gerelli Y., DiBari M.T., Barbieri S., Sonvico F., Colombo P., Natali F., and Deriu A. Soft Matter 6, 685-691 (2010).

16) Gerelli Y., DiBari M.T., Deriu A., Clemens D., Almásy L. Soft Matter 6, 2533-2538 (2010).

17) Gerelli Y., Sakai V.G., Ollivier J., Deriu A. Soft Matter 7, 3929-3835 (2011).

18) Deriu A., Di Bari M.T., Gerelli Y. in "Dynamics of Biological Macromolecules by Neutron Scattering". vol. 1, p. 79-84, Messina:Bentham e-books,ed. S. Magazù & F. Migliardo, (2011) ISBN: 9781608052196.

19) Chiapponi C., Di Bari M. T., Gerelli Y., Deriu A., Chiessi E., Finelli I., Paradossi G., Russina M., Izaola., Z., Garcia Sakai V. (2012). J. Phys. Chem.B, 116, 12915-12921 (2012).

Antonio Deriu Dipartimento di Fisica e Scienze della Terra ... Houches... · to that of granular...

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