Solid-state 13C NRM and X-ray diffraction of dermatan sulfate

Vol.

137,

No.

May

29,

1986

1, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

SOLID-STATE William

NMR AND X-RAY DIFFRACTION

'3C

T. Winter’

, Michael

Edwin

R. Morris4

87-93

OF DERMATAN SULFATE

G. Taylor’,

S. Stevens3, 5

Eugene

, and David

A. Rees

1

Department of Chemistry and Polymer Research Institute, Polytechnic University, Brooklyn, New York 11201 2 Xerox Research Centre of Canada, 2660 Speakman Drive, Mississauga, Ontario L5K 2L1, Canada 3 Department of Chemistry, State University of New York, Binghamton, New York 13901 4

Department of Food Research and Technology, Cranfield Silsoe, Bedford MK45 4DT, England of Technology, Silsoe College,

Institute

5

Received

March

National

24,

Institute Mill Hill,

for Medical Research, The Ridgeway, London NW7 lAA, England

1986

SUbWARY Dermatan sulfate in the solid state has been studied by 13C CP/MAS nmr and X-ray diffraction in order to establish the ring conformation of the Liduronate moiety. The solid state nmr spectrum is similar to the solution spectrum obtained previously, indicating that a ring conformation at least approximating to ‘C LI predominates in the solid state. X-ray powder diffraction data from the same sample indicate the presence of the 8-fold helix form previously observed by fiber diffraction, and interpreted in terms of a ‘C, ring Form. A likely explanation of the results is that a distorted ‘C, Liduronate ring conformation, not considered in the initial X-ray analysis, may emerge to provide a satisfactory interpretation of all available physicalchemical data. D 1986 Academic Press, Inc.

The conformation topic (1)

of considerable around

indicate

residues

demonstrating as in

the

indicate

(Figure the

1)

same (7). in dermatan a small

4Cl ring

L-iduronate

controversy.

the ring

conformation

ate

of the

Small

or a ring On the

other

been resolved

to which

a reaction

angle

for

the

between

(Figure

1).

groups

very

the O(2) aspect

the Curtin-Hammett a molecule

that

exists

coupling

to ‘CQ.

constants

(3)

lC4 ring ’ 3C nmr data

susceptibility

oxidation

This

has been the

as in the

close

large

to periodate

by invoking pathway

hand,

sulfate

nmr vicinal

hydroxyl

conformation

sulfate dihedral

in dermatan

proton

trans-diaxial

conformation

recently

ring

of iduron-

was interpreted

and O(3) hydroxyl

as groups,

of the controversy principle

(4),

as an equilibrium

has

according mix-

Vol. 137, No. 1, 1986

Fig.

BIOCHEMICAL

ring sulfate.

Alternative

1

dermatan

ture

of two conformers

tion

interconversion

Thus,

a minor

tion,

with

compensating

for

conformer

(5)

is

and instead

the

aspect

these the

calls

for

the iduronate amorphous

between

from

conformation

through

the use of high-power heteronuclear angle’

crystal

sharpening Lastly,

with

through the

long

crossed-polarization

respect

typical

state when the

diffraction

evi-

line

(7).

of a

states

ex-

would

difference

resolution

of the ques-

of detecting

differences

in

solution,

to

from

broadening

arising

field

direction

leads

shift

anisotropy

in solids

spec-

techniques.

at the

atoms

88

different

spinning

chemical

(7).

of high-resolution

Secondly,

to the external

(CP) technique

The postulation

energy

on going

ring,

samples.

of three

of carbon

L-iduronate

the

of the small

sulfate

minimizes

of

for

the collection

application

minimization T,‘s

reservoir.

expected

and solution

capable

1 ine

permits

interactions

fiber

an experimental

of dermatan

decoupling

dipolar

of 54.70

the

X-ray

the solid

of techniques

to oriented

solids

oxida-

transition

conformation.

in view

However,

a combination

solids,

conformareactions.

of periodate

in the

‘C4 geometry

and is plausible

ring

chemical

collisions

that

4C, ring

The CP/MAS nmr experiment tra

is

standard

conformation

(6).

if

as an unreactive

assistance

controversy

with

conformers

as in

species.

the alternative

results,

the course solely

(l-4)

abundance

subsequent

of productive

reactive

of the

with

acting

linked

relative

determine

by steric

frequency

incompatible

in ring

between

could

explained

the low

favors

difference

tion

is

is

A second

plain

in comparison

(4C,)

of iduronate

of their

the major conformer (‘C4) rate

dence

conformations

independent

is rapid

conformer

The reaction

minor

is

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

First,

from

static

‘magic to a further

effects

can be overcome

(8). by the

BIOCHEMICAL

Vol. 137, No. 1. 1986

In the parison study

with of the

diffraction

present solution

we have

applied

nmr results,

same solid pattern

MATERIALS

work

state

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

coupled

with

for

direct

sample,

of oriented

solid-state

‘3C nmr,

an X-ray

for

powder

comparison

direct

com-

diffraction

with

the X-ray

samples.

AND METHODS

A 100 mg sample of dermatan sulfate (porcine skin, Na+ salt, Sigma Lot 64F0403) was used in the 13C CP/MAS nmr experiments as received ?dry” sample) or after equilibration with distilled water for 24 hours prior to packing in the In each case a small piece of NBS polyethylene standard rotor (“wet” sample). reference material (No. 1475) was added as an internal reference. The nmr spectra were taken on a Bruker CXP200 spectrometer equipped with a Doty probe and operating at 50.3 MHz. The chemical shift of polyethylene relative to TMS in the solid state at this field strength is 32.9 ppm (9). The spectral window was 20000 Hz with 2K data points used in the acquisition of typically 9000 transients per spectrum. Spinning rates were 3-4 kHz and a contact time of 2 ms was employed. The acquisition time was 51.2 ms and the repetition rate was 6s. The X-ray data were acquired on Osray M-3 (Agfa-Cevaert) film filtered copper radiation from a Phillips fine focus tube operated 15mA. Samples of the humidified dermatan sulfate were removed from rotor immediately after completion of that experiment, sealed into ments of quartz capillary tubes and the tubes were then dusted with amount of NaF (d = 0.2319 nm) as an external cal ibrant.

using Niat 35kV and the nmr short sega small

RESULTS 13C CP/MAS spectra the chemical tion

shifts

are

observed

by Hamer and Perlin

(2),

bon atoms can be identified. to the U-6 carbon nance

unambiguously

atom

[C(6)

assigned

2

in Figure

in the solid by which The

175

Fig.

shown

with

Table

I contains

chemical

shifts

the chemical

shifts

ppm resonance

174.7

of the

2.

uranic

to the U-l

acid

carbon

125

75

obtained

of the

moiety] atom.

and the The broader

of

in solu-

iduronate

is unambiguously 104.1

car-

assigned ppm reso-

carbonyl

reso-

25

‘SC CP/MAS nmr spectrum of dermatan sulfate, relative to TMS. NBS polyethylene standard Assignments from Ref. 2. 89

a comparison

“dry” sample, ppm reference is at 32.9 ppm.

Vol. 137, No. 1, 1986

I.

Table

‘3C

BIOCHEMICAL

CP/MAS

chemical

shifts

nith

solution

data

compared

AND BIOPHYSICAL

(ppm) and

of

dermatan

assignments

sulfate of

Chemical

RESEARCH COMMUNICATIONS

in

Hamer

Shift

and

the

solid

state

Perlin(2)

(ppm)

Assignment Solution

(2 1

“wet”

sample

(broad

component)*

“dry”

A-Ac(C=O)

176.3

176.5

u-6

174.6

174.7

175.1

U-l

103.5

104.1

103.7

A-l

102.6

102.6

176.7

or

80.2

81.5

85.5

A-4

76.7

72-79

72-79

u-4

76.7

72-79

72-79

A-5

75.8

72-79

72-79

O-3

72.0

72-79

72-79

u-2

70.4

68.8

68.6

u-5

70.4

68.E

68.6

A-6

62.5

61.6

A-2

53.4

52.9

A-CH3

24.5

23.8

nance

alpha

to nitrogens

centered

effects

at 176.7

at 68.6 ppm is

resonances

of

those

contributes

chemical

cannot

On the basis between U-5).

the

to those moiety heparin, heparln

after but not

large

taking

not

resolved

resonance

into

weak feature

in dermatan

(2).

at

Independent 90

linewidth.

The (2);

the spectrum.

at 75.4 ppm; the present

is expected

to

resonance

be

its

data.

located

at 68.6 ppm (U-2,

71.1 ppm.

the ‘3C chemical

the presence

quadrupolar

appears

in the solid

to those

where

atoms

with

and the

residue

similar

account

which

the U-3 resonance

influence.

in the solution

any more precisely

Furthermore, are

the

to the U-2 and U-5 carbon

the acetamido

sulfate

carbon

to increase

at 75.4 ppm (U-4) to the

for

in solution. in dermatan

data

(broad)*

by quadrupolar

to the amide

expected

be established

resonance

shifts

to be broadened

are also

to the

and may correspond Chemical

are

atoms

of the solution large

52.8 24.2

attributed

carbon

The U-4 carbon shift

62.7

expected

nitrogen

component)*

(broad)

86.8

(broad)*

ppm may be assigned

of the adjacent

resonance

are

(broad

102.6

(broad)

A-3

*Carbons

sample

shifts

of the

are

of the

iduronate

of a sulfate data

state

also iduronate

moiety group

on ‘H chemical

similar

in

at U-2 in shifts,

BIOCHEMICAL

Vol. 137, No. 1, 1986

Table

II.

Comparison

of observed

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

X-ray

spacings

Mitra

for dermatan

sulfate

with

--et a1.(5) Mitra

al.(s)

--et

This Work

32

83

d(nm)

I0a

d(nm)b

hkl

I0’

1.141

M

1.126

103

0.965

m

0.960

0.8630.829

vvs

0.603

0.428

21

hkl

I0c

0.981 0.939

101 002

w vs

0.927

011

w

w

0.601

112

s

w

0.430 0.430

122 014

M

d(nmfb

hkl

I c

d(nm)b

M

1.154

011

M

NOSE

105

M

0.942

102

w

0.896 0.889 0.881 0.871 0.842

110 111 106 112 113

vvs

NONE

Mw

0.613 0.613 0.604

103 00.12 119

M

0.617 0.616

210 104

M

0.432 0.432 0.430 0.429

21.11 10.16 11.15 275

M

0.426

123

a I, for this work is visually medium; S, strong; V, very. b d values for Mitra cates no reflections

estimated

those of

0

from the diffraction

pattern.

W, weak; M,

et al were calculated from their lattic constants. -L are predicted in the vicinity of our observed d.

NONE

indi-

’ I, for Mitra --et al. 83 and 32 conformers was obtained by forming a composite based on their published structure amplitudes. et al was I, for the 21 conformer of Mitra -L visually estimated from the published diffraction pattern.

proton

vicinal

coupling

constants,

effect

of Gd3’

on 13C relaxation,

ring

approximates

to the

fate

solut,ion(2).

The absence

dermatan

sulfate

conclude

that

Our X-ray the sample

on moving this

carbon-proton all

lead

‘Ch (4) conformation

same conformer

must also

is significantly

in

of any significant the aqueous

diffraction

study

heparin

with

to the solid predominate

91

six

that and

change

of the solid

.crystalline,

constants,

to the conclusion

from

powder

coupling

the

iduronate

dermatan

sul-

in chemical

shifts

phase

leads

us to

in the

solid

state,

material

readily

in

and the

indicates

measurable

that maxima

for

Vol. 137, No. 1, 1986

(Table

II).

The most

the range (51,

it

0.81-0.87

crystalline

their

respective

From the

to generate

correspond

(Table

published all

of the

i.e.,

lattices. in the range

AND BIOPHYSICAL

maximum corresponds

polymorphs;

spacings

maxima

intense

run.

is possible

three

with

BIOCHEMICAL

the

interplanar

lattice

constants

possible

spacings

spacings of Mitra for

the 8-fold

0.87-0.82

nm.

to expected

--et al.

packed

form has any diffraction

Similarly,

all

values

for

the

in

each of the

83, 32 and 2, conformers

Only

II)

to

RESEARCH COMMUNICATIONS

maxima

of the other 8-fold

in

observed

form.

DISCUSSION The X-ray

diffraction

sistent

with

present

in the

ented

solid

t ion

process.

an 83 helix

state;

of

it

(lo),

recently

‘C,

in both drawn

sulfate

solid

state,

consisting

and amorphous alternative

‘Cb chair

stantial for

degree

conformation studies

(5)

therefore

these

results

sample, (2),

[The

state.

in the oriby the

we have

orientaobtained

indicating

or a somewhat

distorted

study

a

the variant

same conclusion

dichroism

by implying

of crystalline

left

is a distorted involved

domains

iduronate

a biphasic

of the type

moieties

was

of dermatan

system

described

results,

however,

and the CP/MAS experiment

in the

previously

assume a distorted

The X-ray

with

a preference

version

the construction

assume flstandardfl

compression

induced

structure

(11 I.1

films

conformation.

the

form

of the

indicate

a sub-

shows no evidence

domains.

assume 83 or 85 fold steric

state

circular

solid

in which

is not

is con-

Thus,

same as the structure

conformation,

of crystallinity,

two separate We are

rings

domains

experiment (5).

in solution

a vacuum ultraviolet

rationalize

fibers

solid

and the solid

and in

the present

pattern

observed

ring

solution

in solution One might

the

same unoriented

iduronate

from

is

fiber

same as that

in

in oriented

state

the X-ray

the

the

of the

solid

i.e.,

Using

observed

as observed

unoriented

‘3C nmr spectrum presence

pattern

geometries helices

of the

with strictly

for

the conclusion

of the ‘CQ family. of helices

where

(12).

Whether

a fiber

repeat

1C4 model, 92

that

The X-ray

and modeling

the individual

a distorted

pyranose

lC4 structure

of 7.353 nm. while

has yet

the ring

to be addressed.

might

relieving We are

the

BIOCHEMICAL

Vol. 137. No. 1, 1986

informed, however (S. Arnott, analysis

Is currently

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

personal communication), that a more detailed

in progress.

ACKNOWLEDGMENTS This work was partially

supported by NIH Grant GM24862and NATOGrant

One of us (W.T.W.) wishes to thank the Xerox Research Centre of Canada

7OV83.

for support and use of facilities. helpful

We thank Dr. S. Arnott

and Dr. B.Casu for

discussions.

REFERENCES 1.

Perlin, 11,

2. 3. 4. 5. 6.

ii: 9. 10. 11. 72.

A.%, Casu, B., Sanderson, G.R. and Tse, J. 1972 Carbohyd. Res.

123-132.

Hamer, G.K. and Perlin, A.S. 1976 Carbohyd. Res. 2, 37-48. Scott, J.E. and Tigwell, M.J. 1978 Biochem. J. 173, 103-114. Rees, D.A., Morris, E.R., Stoddart, J.F. and Stevens, E.S. 1985 Nature 317, 480. Mitra, A.K., Arnott, S., Atkins, E.D.T. and Isaac, D.H. 1983 J. Mol. Biol. 169, 873-901. Eliel, E.L., Allinger, N.L., Angyal, S.J. and Morrison, G.A. in"Conformational Analysis" 1965 Wiley, New York, 370. Pines, A., Gibby, M.G., and Waugh, J.S. 1973 J. Chem. Pbys. 59, 569-590. Schaefer, J., Stejskal, E.O. 1976 J. Am. Chem. Sot. 2, 1030-?03t. Van der Hart, D. L. 1986 J. Chem. Phys. 2, 1196-1205. Torri, G., Casu, B., Gatti, G., Petitou, M., Choay, J., Jacquinet, J.C., and Sinay, P. 1985 Biochem. Biophys. Res. Commun.2, 134-140. Cziner, D.G., Stevens, E.S., Morris, E.R. and Rees, D.A. 1986 J. Am. Chem. Sot. in press. Arnott, S. and Scott, W.E. 1972 J. Chem. Sot., Perkfn Trans. II, 324-335.

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