Process for preparing cyclooctaamylose

A process for preparing cyclooctaamylose by enzymatic cleavage of an aqueous preparation of starch in the presence of a complexing agent is disclosed. As a selective complexing agent for cyclooctaamylose, a compound of the formula I is employed. ##STR1## in which A, B, D and E, independently of one another, represent ##STR2## (R=hydrogen, alkyl, hydroxyl, alkoxy or carboxyl radical), and m, n, o and p are within the limits 0 to 20, with the proviso that the number of atoms forming the ring is within the limits from 13 to 24. The compounds prepared by the process of the invention are useful in the medicaments sector, in crop protection and cosmetics, or in the foodstuffs industry.

The present invention relates to a process for preparing cyclooctaamylose 
by enzymatic cleavage of starch in the presence of a complexing agent. 
Cyclooctaamylose is relatively readily water-soluble and has a hydrophobic 
torus of diameter 10.times.10.sup.-10 m, in which guest molecules can be 
included. Due to these properties, cyclooctaamylose is a proven substance 
for use, inter alia, in the medicaments sector, in the areas of crop 
protection and cosmetics, or in the foodstuffs industry. 
According to DE No. 3,317,064 Al, the state of the art is to separate 
cyclooctaamylose from starch hydrolysates by precipitation using 
bromobenzene. Disadvantageously, the complexing agent bromobenzene is not 
selective for cyclooctaamylose, but instead cycloheptaamylose, which can 
only be separated by complicated methods, is also precipitated at the same 
time. 
In DE No. 3,446,080 Al and in the corresponding GB No. 2,151,647 A, phenol 
and benzene derivatives, to which complexing agents for cyclohexaamylose 
are simultaneously added, are mentioned as complexing agents for 
cyclooctaamylose. Separation of cyclooctaamylose, cycloheptaamylose and 
cyclohexaamylose is also necessary in this case. 
Chemical Abstracts 104:128250q, in which Japanese Published Specification 
JP No. 60-227,693 is cited, describes tetracyclic or pentacyclic 
triterpenoids as complexing agents in the preparation of cyclooctaamylose. 
Accordingly, it is an object of the present invention to provide a process 
for preparing cyclooctaamylose in high purity; the process providing 
improved yields while, at the same time, simplifying the isolation of 
cyclooctaamylose. 
The foregoing and related objects are accomplished by the present invention 
which provides a process for preparing cyclooctaamylose by enzymatic 
cleavage of an aqueous preparation of starch in the presence of a 
complexing agent, wherein, as a selective complexing agent for 
cyclooctaamylose, compounds of the formula I R1 ? 
##STR3## 
wherein A, B, D and E, independently of one another, represent 
##STR4## 
(R=a hydrogen, alkyl, hydroxyl, alkoxy or carboxyl radical) and m, n, o 
and p are within the limits 0 to 20, with the proviso that the number of 
atoms forming the ring is within the limits from 13 to 24, are employed. 
Examples of radicals R are hydrogen, methyl, ethyl, n-propyl, iso-propyl, 
n-butyl, sec.-butyl, tert.-butyl, hydroxyl, methoxy, ethoxy and acetoxy 
radicals. The hydrogen and methyl radicals are preferred, in particular 
due to their ready accessibility. 
Examples of compounds of the formula I are: 
(a) macrocyclic unsaturated hydrocarbons, such as cyclotetradeca-1,8-diene, 
cyclopentadeca-1,8-diene, cyclohexadeca-1,9-diene, 
cyclohexa-1,5,9,13-tetraene, 
1,5,9,13-tetramethylcyclohexadeca-1,5,9,13-tetraene, 
cyclotetracosa-1,9,17-triene, cyclohexadeca-1,9-diyne and the monoepoxides 
and polyepoxides thererof, such as 15-oxabicyclo[12.1.0]pentadec-7-ene, 
8,16dioxatricyclo[13.1.0.0.sup.7,9 ]hexadecane, 
17-oxabicyclo[14.1.0]-heptadec-8-ene, 9,18-dioxatricyclo[15.1.0.0.sup.8,10 
]octadecane and 17-oxabicyclo[14.1.0]heptadeca-4,8,12-triene; 
(b) macrocyclic ketones or polyketones and acetals, such as 
cyclotridecanone, cyclotetradec-7-en-1-one, cycloheptadec-9-en1-one, 
cyclohexadec-8-yn-1-one, cyclotetracosane-1,8,16-trione, 
cyclotetracosane-1,8,17-trione, 2-methylcyclotridecan-1-one, 
3-methylcyclopentadecan-1-one, 17,20-dioxaspiro[15,4]cosane and 
1,1-diethoxycyclohexadec-8-ene; 
(c) macrocyclic alcohols, such as cyclotetradec-7-en-1-ol, 
cyclohexadecane-1,8-diol, cyclohexadecane-1,9-diol and 
cyclohexadeca-5,9,13-trien-1-ol; 
(d) macrocyclic alkoxy or carboxyl compounds, such as 
1-acetoxycyclohexadec-8-ene, 1-methoxycyclohexadec-8-ene and 
1,2-dimethoxycyclohexadec-8-ene; 
(e) macrocyclic oxaoxo compounds, such as 
2,5-dioxa-1,6-dioxocyclotetradecane, 2,5-dioxa-1,6-dioxocyclohexadecane, 
2,5-dioxa-1,6-dioxocycloheptadecane, 2,5-dioxa-1,6-dioxocycloeicosane, 
3-methyl-2,5-dioxa-1,6-dioxocyclohexadecane, 
2,8-dioxa-1-oxo-cycloheptadecane and 2,7-dioxa-1-oxo-cycloheptadecane; 
(f) macrocyclic hydroxyimino compounds, such as 
2-oxa-1-oxo6-hydroxyiminocyclohexadecane, 1-hydroxyiminocyclotridecane and 
1-hydroxyiminocyclohexadec-8-ene; and 
(g) macrocyclic monoaza and oligoaza compounds, such as 
2-aza-1-oxocyclotridecane, 2,8-diaza-1,9-dioxocyclohexadecane and 
2-aza-1-oxocyclotetradec-7-ene. 
Compounds of the formula I in which the number of atoms forming the ring is 
within the limits 13 to 18 are preferably used. These are, in particular, 
cyclotridecanone, cyclotetradecanone, cyclotetradec-7-en-1-one, 
15-oxabicyclo[12.1.0]-pentadec-7-ene, 8,16-dioxatricyclo[13.1.0.0.sup.7,9 
]hexadecane, cyclotetradecane-1,8-dione, cyclopentadec-8-en-1-one, 
cyclopentadecane-1,8-dione, 16-oxabicyclo[13.1.0]hexadec-6-ene, 
8,17-dioxatricyclo[14.1.0.0.sup.7,9 heptadecane, cyclohexadec-8-en-1-one, 
cyclohexadecane-1,9-dione, cyclohexadecane-1,8-dione, 
cyclohexadeca-1,9-diene, 17-oxabicyclo[14.1.0]cycloheptadec-8-ene, 
9,18-dioxatricyclo[15.1.0.0.sup.8,10 ]cyclooctadecane, 
cycloheptadec-9-en-1-one, cycloheptane-1,9-dione, 
18-oxabicyclo-[15.1.0]cyclooctadec-8-ene, 
2,5-dioxa-1,6-dioxocyclohexadecane, 2,5-dioxa-1,6-dioxocycloheptadecane, 
2,8-dioxa-1-oxocycloheptadecane, 1,7-dioxa-1-oxocycloheptadecane, 
2-oxa-1-oxocyclotetradecane, 2-oxa-1-oxocycloheptadecane and 
2-oxa-1-oxo-cyclopentadecane. 
In principle, any type of starch, including native starch or starch partial 
hydrolysates, can be employed. Examples are potato starch, maize starch, 
manioc starch and maltodextrins having a dextrose equivalent&lt;15. 
The aqueous preparations of starch employed can be all aqueous preparations 
used to date, for enzymatic cleavage of starch. These are, in particular, 
4 to 40% by weight aqueous solutions of gelled starch. In the simplest 
case, they are obtained by boiling appropriate amounts of starch in water. 
For enzyme stabilization, the preparations mentioned usually contain small 
amounts of calcium chloride, in particular 5-10 mmole/1. 
The known enzyme cyclodextrin glycosyltransferase is now added to the 
starch preparations mentioned. The source for this enzyme is 
microorganisms such as Bacillus macerans (Zentr. Bakteriol, Parasitenk., 
Dept. II, 14, 722 (1905), Bacillus stearothermophilus (U.S. Pat. No. 
3,988,206), Bacillus subtilis No. 313 (Agric. Biol. Chem 50, 8, 2161-2162 
(1986), Bacillus circulans (U.S. Pat. No. 4,477,568), Bacillus ohbensis 
(JP No. 52-31949), Bacillus megaterium (U.S. Pat. No. 3,812,011), Bacillus 
spec. No. 17-1 (U.S. Pat. No. 3,923,598), Klebsiella pneumoniae M5 a L 
(Arch. Microbiol. 111, 271 (1977), Micrococcus luteus or Micrococcus 
varians (both EP No. 0,017,242). 
The enzyme is preferably added in amounts such that the enzyme to starch 
weight ratio is 1:2000 to 1:50,000, in particular, 1:5000 to 1:20,000. 
In the process according to the invention, the complexing agent is 
preferably added immediately after addition of the enzyme in amounts of, 
preferably, 1-20% by weight, in particular 8-15% by weight, relative to 
the weight of the starch employed. The pH of the starch preparation is 
preferably 4.0 to 11.0, in particular 6.0 to 9.5. 
The cleavage reaction is preferably carried out at temperatures of 
30.degree.-60.degree. C., in particular 40.degree.-50.degree. C., with 
stirring; the reaction time preferably being 10-48 hours. The reaction can 
be monitored, for example, by sampling and chromatographic analysis (HPLC 
method, Agric. Biol. Chem. 49, 4, 1189-1191 (1985). 
For work-up, the insoluble cyclooctaamylose complex is removed from the 
other reaction participants by known methods, for example decanting, 
filtering or centrifuging. The cyclooctaamylose complex is subsequently 
again separated into cyclooctaamylose and complexing agent. A suitable 
method for this purpose is treatment with hot water or steam, the 
complexing agent being removed from the mixture by steam distillation. A 
further method includes extracting the water-containing cyclooctaamylose 
complex with an organic solvent. Examples of such solvents are toluene and 
cyclohexane. 
Up to 48% by weight of cyclooctaamylose, relative to the starch employed, 
having a purity up to 95%, is obtained. 
This cyclooctaamylose is preferably treated in a known manner with 
glucoamylase in order to remove entrained traces of starch. An organic 
solvent, such as an alcohol, for example, isopropanol or methanol, or 
acetone, are preferably added subsequently, whereupon cyclooctaamylose of 
a purity of 95 to&gt;99.9% crystallizes out on standing for some time. 
Cyclooctaamylose prepared by the process according to the invention is 
used, inter alia, as a component of plant-protection agents, medicaments, 
cosmetics and foodstuffs. 
The invention will now be described in further detail with reference being 
made to the following examples. It should, however, be recognized that the 
following examples are merely illustrative of the scope of the present 
invention and are not intended to define the limitations thereof.

EXAMPLE 1 
20 g of soluble potato starch were suspended in 200 ml of water containing 
4 mmole of tris(hydroxymethyl)aminomethane hydrochloride (pH 7.2) and 1 
mmole of calcium chloride. The starch was gelled by heating at 95.degree. 
C. for 25 minutes. After cooling to 50.degree. C., 2 mg of cyclodextrin 
glycosyltransferase of Bacillus macerans and 2.5 g of 
cyclohexadec-8-en-1-one were added. The batch was incubated at 50.degree. 
C. for 36 hours with vigorous stirring. By HPLC analysis of an aliquot of 
the reaction mixture, it was apparent that 46% by weight of the starch 
employed had been converted into cyclooctaamylose. 
The insoluble cyclooctaamylose/cyclohexadec-8-en-1-one complex was removed 
by centrifuging. The complex was washed twice by taking up in 200 ml of 
water and subsequent centrifuging. The complex was then taken up in 200 ml 
of water, and the cyclo hexadec-8-en-1-one was removed by distillation as 
an azeotrope with water. The resultant cycloamylose solution contained 
cyclooctaamylose of a purity of 92%. 
In order to remove the traces of starch still present, the cycloamylose 
concentration was set at 40% by weight, and the solution was incubated 
overnight at 55.degree. C. and pH 5 with 0.5 mg of glucoamylase. The batch 
was then cooled to room temperature, an equal volume of isopropanol was 
added, and the mixture was allowed to stand at 4.degree. C. for 4 hours. 
The precipitate formed was separated off, washed with isopropanol and 
dried at 60.degree. C. in a vacuum drying cabinet. The yield of 
cyclooctaamylose was 7.6 g. The purity was&gt;99%. 
EXAMPLE 2 
Example 1 was repeated, however, 1 mg of cyclodextrin glycosyltransferase 
of the alkalophilic Bacillus No. 17-1 was used in place of the enzyme of 
Bacillus macerans. The pH of the reaction batch was set at 9. 8 g of pure 
cyclooctaamylose were obtained. 
EXAMPLE 3 
(comparison experiment) and Examples 4-10 
The following standardized reaction batches were used in Examples 3-10 of 
the following table: 5 g of potato starch were suspended in 50 ml of 
buffer solution (see Example 1) and gelled. 0.5 mg of cyclodextrin 
glycosyltransferase of Bacillus macerans were employed. The reaction 
temperature was 50.degree. C. Incubation times (t) in hours and crude 
yields of cyclooctaamylose (in % relative to the starch employed), which 
were achieved using particular complexing agents, are collated in the 
table. 
TABLE 
______________________________________ 
crude 
Example Complexing Agent t yield 
______________________________________ 
3 Cyclododecanone 48 1.5 
4 Cyclotridecanone 42 42 
5 Cyclotetradec-7-en-1-one 
42 45 
6 Cyclohexadecane-1,9-dione 
48 43 
7 9,18-Dioxatricyclo- 48 38 
[15.1.0.0.sup.8,10 ]cyclooctadecane 
8 2,8-Dioxa-1-oxocycloheptadecane 
48 39 
9 2-Oxa-1-oxocycloheptadec-7-ene 
48 34 
10 2,5-Dioxa-1,6-dioxocyclohexadecane 
48 38 
______________________________________ 
While only several embodiments and examples of the present invention are 
described, it will be obvious to those of ordinary skill in the art that 
many modifications may be made thereunto without departing from the spirit 
and scope of the invention.