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Makromol.

Chem. 178,2595-2602

(1977)

Departm ent of Industria l Chem istry, College of Technology,

Seikei

University, M usashino-shi, Tokyo, Japan

Studies on Chitin, 3*)

Preparation of Pure Chitin, Po~y N-acetyl-D-glucosamine),rom the Water-Soluble Chitin

Keisuke Kurita, Takanori Sannan, and Yoshio Iw akura

(Date of receipt: December 28,1976”’)

SUMMARY:

Pure chitin,

poly(N-acetyl-D-glucosamine),

was successfully prepared by acetylating the partially deacety-

lated water-soluble chitin

with

acetic an hydride-pyridine

or

with acetic

acid-dicyclohexylcarbodiimide.

Although enough

selectivity

between amino and hydroxyl groups was

not

shown,

acetylation

with

acetic

anhydride-pyridinegave rise to the complete acetylation of amino groups and thereby

0-,

N-acety-

lated chitin was formed. The ester groups were then

selectively

transformed to hydroxyl groups by

means of either hydrolysis or transesterification to give the pure chitin. Acetic

acid-dicyclohexylcarbodi-

imide system

was

found to enable one step complete and selective acetylation of am ino groups under

very mild

conditions and i t appeared

to

be superior to the others

for

t h e purpose.

Introduction

Chit in is generally considered to consist of N-aCetyl-D-glUCOSamine units , b ut it is well

known that chitin is found in close association with other organic and inorganic materials

such as protein an d calcium carbo nate in crustacean shells. Drastic procedures are hence

required to remove these accompanying substances. However, these methods also degrade

chitin’’, and thus, isolated chitin can not strictly be regarded

as

a “natural” chemical entity,

but has und ergo ne deg radatio n including deacetylation2 In fact chitin isolated by Hackman’s

me thod 3’ is partially deacetylated by hydrolysis, an d we have rep orted that the degree

of

deacetylation was found to be 15 4’. Most studies of chitin have been based on such

a

sample. Hence obtain ing pu re chitin,

poly(N-acetyl-D-glucosamine),

appe ared t o be very signifi-

cant for every field of studies relating to chitin.

Co ncern ing the pur e chitin, the only rep ort found in the literature was that by

McLachlan

et aL5’, wh o claimed th e isolation of the p ur e chitin from a kind of alga by

a

complicated

procedure. N o attempt has been made hitherto to prepare poly(N-acetyl-D-glucosamine) by

the acetylation of the patially deacetylated chitin, although acetylated chitosan was briefly

mentioned in the literature with no detailed information about the extent of acetylation6

Schorigin

et

al.’)

had investigated in detail the acetylation of chitin. They found that it

could not be acetylated in its inner region of the solid by the usual methods except

a

drastic

one with dry hydrogen chloride and acetic anhydride.

As

can be seen from the facts, it has

been by n o mean s easy to acetylate chitin by the conventional heterogeneou s method s.

We recently reported that chitin became water-soluble by an alkaline treatment under appro-

priate conditions and that this interesting solubility phenomenon was observed for only the

I

Part 2 : ~ f . ~ ’ .

f

’

Revised manuscript of February 15,

1977.

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K. Kurita, T. Sannan, and Y. Iwakura

samples with the degree of deacetylation of abo ut 5 0% 4'. Th is water-solubility has enabled

us

to study the acetylation

of

the partially deacetylated chitin under homogeneous

or

almost

homogeneous condit ions to obtain the

po~y(hi-acetyl-D-glucosamine).

Th e present s tudy has sought to p repare

poly(L~-acetyl-D-ghcosamine)

rom th e water-soluble

chitin by acetylation with three different agents:

1

acetic anhydride with pyridine, 2) acetic

acid with dicyclohexylcarbodiimide (DC C), and 3) acetyl chlorid e in interfacial cond ensa tion.

Results and Discussion

Acetylation with acetic anhydride-pyridine

Chitin and chitosan swell little in organic solvents including pyridine, which has been one

of

the barrier s in their acetylation an d m ade it difficult to acetylate uniformly. Th e water-soluble

chitin, however, was found to be able to form a highly swollen gel by pouring its aqueous

solution into pyridine. The gel was considered to be easily accessible for the reaction and

it was, therefore, treated with acetic anhydride at room temperature.

The acetylation reaction appeared to proceed rapidly and almost homogeneously as the

gel remained in

a

highly swollen state during the reaction. Fig. 1 shows the IR spectra

of the acetylated chitin samples. All the spectra

of

the samples obtained after various length

of t ime indicated the presence of 0-acetyl g roup in ad dition t o N-acetyl group. T he ab sorption

bands d ue to the 0-acetyl group at 1735 and 1235 cm -' were found even in the spectrum

of the sample obtained after 3min reaction. As it is well known that the amino group is

much mo re reactive than the hydroxyl gro up tow ards acetylation, 3 min was considered to

be sufficient to complete the acetylation at the amino group. However, the enough selectivity

to acetylate only at the amino group was not attained under these conditions, and it was

found t o be necessary to remove the 0-acetyl g roup selectively without harming the N -acetyl

group from the resulting acetylated chitin to obtain the pure

poly(N-acetyl-D-glucosamine).

These acetylated chitin samples were highly swollen in reaction mixture after the reaction,

an d even the dried samples showed good swelling property in polar solvents such as N,N-dimeth-

ylformamide (D M F),N,N -dim ethyla cetam ide DM Ac), N-methyl-2-pyrrolidone, m-cresol, benzyl

alcohol, and pyridine, although they became insoluble in water and diluted hydrochloric acid

which were goo d solvents for the water-soluble chitin. Th us, hydrolysis a nd transesterification

under the swollen state were employed to regenerate the hydroxyl group.

Ac

=

COCH3)

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Studies on C hitin, 3

\

\

\

\ I

597

Fig.

1. IR

spectra

of

the water-solu-

ble chitin (A) an d the acetylated chi t in

samples prepared by acetylating the

water-soluble

chitin with acetic

anhydride-pyridine

for

3 min (B), 10

rnin (C), and

24

h

(D)

L ’

L I

Wave number

i n cm-’

1 I

1800 1600 14 1200 900

Hydro lys i s

Th e hydrolysis of the ester gr ou p was carried out with a weak base such as sodium hydrogen

carbonate in order

to

avoid the unfavorable effect on the amide group. The swollen acetylated

sample, which was obtained after 3min reaction, was added to an aqueous sodium hydrogen

carbon ate solution. The mixture was kept at room temp erature for a given time a nd the extent

of

the hydrolysis of the ester grou p was followed by IR spectroscopy.

Ac = COCH3)

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K . Kurita, T. Sannan, and

Y.

Iwakura

The absorbances of the bands at 1735 and 235 cm -' decreased gradually as the reaction

progressed and the change was shown in Fig. 2. In the spectrum of the sample obtained

after 32 h, these ban ds became small shoulders. Finally even these should ers disapp eared comple-

tely in the spectrum

of

the sample hydrolyzed over 141 h. T he spectrum

of

the resulting

Wave num ber in cm -

1 1 * j

1800

16 14

12 900

Fig. 2.

IR

spectra of the samples

obtained by hydrolysis of the acety-

lated chitin in sat. aq. sodium hydro-

gen carbonate solution: Samples hyd-

rolyzed for 17 h (A), 32 h (B), and 141 h

(C)

sample showed strong bands at

1650

and 15 50 cm -' of the amide an d a distinct peak at

1l lOcm-' as shown in Fig. 2. The assignment

of

t he peak a t l l lO cm - ' w as not clear,

but i t was found to have something to do with the content

of

the acetylamino groups or

the degree of deacetylation (DD): a dist inct peak ( D D 90 ).

The shape of the peak, therefore, appeared to be a

rough

indicator for the degree of deacetylation and in the pure chitin, it should be a distinct one

instead of a shoulder.

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Studies

on

Chitin, 3 2599

T he samples obtained after hydrolyzing for

141

h were insoluble in water an d diluted hydro-

chloric acid. Their degrees of deacetylation were determined by Elek and

Harte s

method')

and they were found

to

be 0 a n d

1 %

for the two different samples, revealing that these

samples were the

poly(N-acetyl-D-glucosamine).

These results indicate that the amino groups

in the water-soluble chitin were completely acetylated with acetic anhydride-pyridine in the

swollen state under the conditions where a part of the hydroxyl groups were acetylated,

and the ester groups formed were then selectively and fully hydrolyzed to the hydroxyl groups

without affecting the acetylamino groups by the above manner.

7iaiisestelifictrtiorl

The ester interchange reactions of the dried acetylated chitin samples were first attempted

with dry methanol. They were, however, found to be unsuccessful even with the swollen

samples in DMF, DMAc, or rn-cresol as only a little decrease in the absorbances of the

ester bands was observed in the IR spectra of the products. The extent

of

the reaction seemed

to

be quite limited probably because methanol was a poor solvent for the acetylated chitin

an d it suppressed th e swelling of the samples in the po lar solvents.

Benzyl alcohol itself was a good swelling agent and the ester interchange reaction was

then tried in the solvent. T h e dried acetylation p rodu ct was swollen in benzyl alcoho l an d

the reaction was carried out at 100°C for 24h

with the use

of

a small amount of sodium

metal. T h e prod uct isolated was insoluble in water and diluted hyd roch loric acid an d slightly

colored. Its IR spectrum was the same as that

of

the samples obtained by the hydrolysis

me thod . M oreo ver, the degree of deacetylation was determined t o be

4

,

which was considered

to be almost the same value as that obtained by the hydrolysis method within the experimen tal

error. At lower temperature, for example at 50 C, the product h ad weak bands owing to

the ester group, although the coloration was negligible. High temperatures seemed to make

the reaction rapid, but also caused the heavy coloration. Hence the reaction appeared to

be carried out successfully at

IOO'C,

but the slight coloration was always inevitable perhaps

due

to

som e deco mp osition s of chitin a nd /or benzyl alcohol.

Consequently, selective 0-deacetylation was confirmed to be accomplished by either the

hydrolysis method or the transesterification method, but the former was found to be more

clean a nd effective th an the latter.

Acetylation with acetic acid-DCC

DCC

has been widely used in th e prep aration

of

amide com pou nds from amines and carboxylic

acids und er mild cond itions. In a recent paten t,

Yaku

et al.9' reported the acetylation of

chitosa n films with acetic acid an d

DCC

in aqueous DMF, but the acetylation was insufficient

so

as

to

regard the product as pure chitin as the reaction was conducted heterogeneously

on the films. They examined the effect of the constitution

of

the solvent on the extent of

the reaction and found that when th e water con tent was over 40 , excess acetic acid, functioning

as a stro ng acid, stimulated the reverse deacetylation.

Dire ct one step selective conversion of the water-soluble chitin to the pOly(N-aCetyl-D-glUCOS-

am ine) was anticip ated to be achieved by the acetic acid-DC C system, and th e acetyla tion of

the aqueous solution

of

the water-soluble chitin was un dertak en.

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K . Kurita,

T.

Sannan, and Y. Iwakura

The acetylation reaction was carried out

by

adding a solution of twenty-fold excess acetic

acid an d D C C in D M F to the 1 % aqueous solution of the water-soluble chit in a t room

temperature . When the ra t io of water t o D M F was

3 : 2

(water content 60%) or 6:5 (water

content 5 5 ) , the acetylated product was in the solution. The isolated product was insoluble

in water, but soluble in diluted h ydr ochlo ric acid, which insinuated the in com plete acetylation.

This result might be interpreted by the high water content as suggested by Yaku et

aL9).

Then the ratio of water to D M F was reduced to 2 : 3 (water content

40 ).

The reaction

proceeded similarly except the precipitation of an almost homogeneous highly swollen gel.

T h e

IR

spectrum of the product isolated showed the stro ng amide I and

I1

bands, the distinct

band at

1

IOOcm-', an d no band s du e to ester grou p. It was identical with those of the

pure chitin samp les obtained by the hydrolysis or the transesterification

of

the 0-,N-acetylated

chitin. T h e sam ple was insoluble in water an d diluted hy dro chlor ic acid. Furtherm ore, the

degrees of deacetylation were found to be

0, 3,

and 4 , respectively, for the three acetylated

samples. These values were considered to be

0

within the experimental error, and hence

it was concluded that the acetic acid-DCC system acetylated only the free amino groups

successfully witho ut acetylating the hydroxyl gro up s t o give the poly(N-acetyl-D-glucosamine)

in one step.

Althoug h the acetylation was co m ple te with twentyfold excess of acetic acid and DC C, smaller

amounts

of

acetic acid and DCC gave rise to the incomplete acetylation. For instance, the

reaction with five-fold excess acetic acid and ten-fold excess DCC gave the products with

the degrees

of

deacetylation of 16 an d

20 by

the two different acetylation runs.

Ac = COCH,)

Tab. 1.

de"'

Sample Carbo diimide Amount of excess Band at Solubilityb ' n DD

Acetylation

of

t h e water-soluble chitin with acetic acid and DCC

or the

water-soluble carbodiim i-

( 1 110 cm - ' i n I

acetic

carbo- water dil. HCI

acid

0, 3, 4

0-fold

20-fold Peak

*

1 6 , 2 0

+

+ +

+ +

d m de

~

~

5-fold 10-fold

'I11

DCC 5-fold

IV

3-fold

Water-soluble

20-fold

V

carbodiimide

20-fold

~

The reactions were carried

out

in a mixed solvent

of

DMF and

water (3 :2 ) (I-IV) or

water (V)

at

room temp. for

44

h.

b,

(-): insoluble; +): partially soluble;

+) :

soluble.

DD=Degree of deacetylation, determined by

Elrk

and Harte's method.

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Studies on Chitin, 3 2601

In place

of

DC C, the water-soluble carbodiimide,

1-ethyl-3- 3-dimethylaminopropyl)carbodi-

imide, was tried as a condensing agent in orde r to carr y ou t the reaction in water instead

of aqueous DMF. The product, however, was soluble in water and almost no acetylation

was found to have proceeded judgin g from its IR spectr um . Th is result indicates that the

acetylation of water-soluble chitin is ham pered by high water conte nt. T h e results are sum ma rized

in Tab.

1.

Interfacial condensation

Cellulose xanthate was reported to be acetylated to some extent by p-nitrobenzoyl chloride

by the interfacial con dens ation method"). In the case of the water-soluble chitin, the free

am ino grou ps in the partially deacetylated chitin were expected to be acetylated by acetyl

chloride quickly. Th e reactions were carried out according to the usual mann er

of

the interfacial

cond ensa tion with the variations of the org anic solvent, the acid accep tor, an d the surface-active

agent. However, the lowest degree ofde acetyla tion obtaine d was 35% an d the extent of acetylation

by this procedure was found to be very low.

Sun et a1.I') exam ined t he esterification of cellulose derivatives by th e interfacial cond en satio n

and found that the limitation in the extent of

the acetylation was attributable to a steric

arrangement of the cellulose molecules at the liquid interface such that only

50

of the

hydroxyl groups were exposed to the organic phase and were thereby made accessible for

the reaction with the acid halide. The sam e reasoning ap peared to be applicable to the present

case, i.e., the results seemed to suggest that only a part of the amino and hydroxyl groups

were exposed t o the o rgan ic phase for the reaction as in the cellulose case.

Conclusion

Pure pOly(N-aCetyl-D-glUCOSamine) was pre pa red from water-soluble chitin by either 1) acetyla-

tion with acetic anhy dride -pyr idine an d subseq uen t hydrolysis or transesterification o r

2)

by

selective acetylation with acetic acid-DCC. Between the two, however, the acetic acid-DCC

system was considered to be superior as the acetylation could be completely and selectively

achieved under mild conditions by one step simple reaction.

Experimental

Part

Chi t i n :

Chitin was isolated from

shells

of

Penneirs japonicus

and

the

water-soluble chitin

with the

degree

of

deacetylation of about

50%

was obtained according to the previously mentioned p rocedures4 '.

Acetyla t ion wi th acet ic

a,ih~cirirle-pyrirlirlc: A 0,30g of the water-soluble

chit in

sample was dissolved

by stirring

with

30g of crushed ice. The

aq.

solution was poured into 200ml

of

pyridine to form

a

highly swollen precipitate. I t was separated by centrifugation, washed well with pyridine repeatedly,

and squeezed

to

about

log.

I t was

then swollen in

20ml

of

fresh pyridine, and

6 0 m l of acetic

anhydride

was added to the mixture. After stirring for

3 m i n at

room temp.,

the 0-.

N-acetylated chitin

was

filtered, washed

successively

with

water, methanol, and acetone, and squeezed.

I t

was

used for the

subsequent hydrolysis

and

transesterification.

Hydrolys i s of 0- N-acety la ted chi t in:

To

a

lOOml of sat. sodium hydrogen carbonate solu tion was

added

the

acetylated chitin obtained above

without

drying. The mixture

was

left standing

at 20°C

for 141

h .

The resulting chitin

was

separated by filtration , and washed

with water ,

methanol, and

acetone.

Yield 0.28

g.

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2602

K. Kuri ta,

T.

San nan , and Y. Iwakura

The sample was strongly hygroscopic and at tempts

to

obtain the rel iable data

of

elemental analysis

were unsuccessful. The degree of deacetylation was determined by Elek and Harte’s method”

on

the

sample dried thoroughly in

a

vacuum oven and weighed in a breakable seal.

I t

was found to be

0 .

Transesterification

of

0 - N-acetglated chitin;

The acetylated chitin obtained above was dried well

i. vac. at room temp., and allowed to swell in 60ml of dry benzyl alcohol for 1 2 h at roo m temp.

A small amount of sodium metal was added to the mixture. After heat ing at 100°C for 24h, the

prod . was filtered an d washed well with metha nol a nd acetone.

I t

weighed 0,32 g. Th e degree

of

deacetyla-

t ion was determined to be 4 % by

EIek

a n d Haire’s method”.

Acerylation with acetic acid-DCC: To an aq. solution of 0,434g of the water-soluble chitin in which

1,2mmol

of

glucosamine units were present in 40 g of water was adde d 1,44 g (2 4m m ol ; 20-fold excess)

of

acetic acid an d 4,94g (24 mm ol; 20-fold excess)

of

DCC in 60ml

of

D M F with st i rr ing. The st irr ing

was continued for 44 h a t room temp., and then the mixture w as taken into

a

sat . aq. sodium hydrogen

carbonate solution for neutralization. The precipitate was filtered. washed with water and methanol,

and dried i. vac. to give 0,432g

of

chitin whose degree

of

deacetylation was O , determined by

Elek

and

Harte’s

method*).

‘

A. B. Foster, J. M. Webber, Adv. Car boh ydr. Che m. 15, 372 (1960)

*) A. G. Richards, “The Integument of Arthropods”, University of Minnesota Press, Minneapolis,

3, R. H. Hac kman , Austr. J . Biol. Sci. 7, 168 (1954)

4, T. Sannan , K. Kuri ta, Y. Iwakura, Makrom ol. Chem . 177, 3589 (1976)

5

J . McLachlan,

A . G.

McInnes, M. F alk, Can . J . Bot.

43,

707 (1965)

P. Karrer , G. V. FranGois, H elv. Ch im . Acta

12,

986 (1929)

’ P.

Schorigin,

E.

Hait, Ber. Dtsch. Chem. Ges.

B 68

971 (1935)

A. Elek, R. A. Harte , Ind . Eng. Chem., Anal. Ed. 8 267 (1936 ); see also A . Steyerm ark, “Qua nti tat ive

Org anic M icroanalysis”, Th e Blackistone Com p., New York 1951, p. 244

Minn. 1951

9 Jpn. P. 731 921 3 (1973), invs.: T. Yaku, I. Yam ashita; C . A.

80,

72291 v (1973)

l o

T.

Sun,

V. K. Chang,

Z.

A. Rogovin, Vysokomol. Soedin.

3,

382 (1961 ); C.

A. 55

27881c(1961)