U.S. patent application number 12/711629 was filed with the patent office on 2010-06-17 for production method of capsinoid by dehydrating condensation, stabilizing method of capsinoid, and capsinoid composition.
This patent application is currently assigned to AJINOMOTO CO., INC.. Invention is credited to Yusuke AMINO, Kazuko Hirasawa, Wataru Kurosawa, Takashi Nakano.
Application Number | 20100152291 12/711629 |
Document ID | / |
Family ID | 39161070 |
Filed Date | 2010-06-17 |
United States Patent
Application |
20100152291 |
Kind Code |
A1 |
AMINO; Yusuke ; et
al. |
June 17, 2010 |
PRODUCTION METHOD OF CAPSINOID BY DEHYDRATING CONDENSATION,
STABILIZING METHOD OF CAPSINOID, AND CAPSINOID COMPOSITION
Abstract
Capsinoids of formula (3) may be conveniently prepared in a high
yield, in a short time, without using a dehydrating agent by
esterification of a fatty acid of formula (1) with a
hydroxymethylphenol of formula (2) using an enzyme without a
solvent or in a low-polar solvent. Addition of a fatty acid
represented by formula (4) is effective for stabilizing the ester
compound of formula (3). ##STR00001## wherein each symbol is as
defined in the specification.
Inventors: |
AMINO; Yusuke;
(Kawasaki-shi, JP) ; Kurosawa; Wataru;
(Kawasaki-shi, JP) ; Nakano; Takashi;
(Kawasaki-shi, JP) ; Hirasawa; Kazuko;
(Kawasaki-shi, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, L.L.P.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
AJINOMOTO CO., INC.
Tokyo
JP
|
Family ID: |
39161070 |
Appl. No.: |
12/711629 |
Filed: |
February 24, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11493826 |
Jul 27, 2006 |
7700331 |
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12711629 |
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PCT/JP2006/303343 |
Feb 17, 2006 |
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11493826 |
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60702606 |
Jul 27, 2005 |
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Current U.S.
Class: |
514/552 ;
435/134 |
Current CPC
Class: |
C07C 67/62 20130101;
C07C 67/62 20130101; A61P 3/00 20180101; A61P 3/04 20180101; C12P
7/6436 20130101; C12P 7/62 20130101; C12P 7/6418 20130101; C07C
69/28 20130101 |
Class at
Publication: |
514/552 ;
435/134 |
International
Class: |
A61K 31/23 20060101
A61K031/23; C12P 7/64 20060101 C12P007/64; A61P 3/00 20060101
A61P003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 18, 2005 |
JP |
2005-043154 |
Claims
1. A method of producing an ester compound represented by formula
(3): ##STR00017## wherein R1 is an unsubstituted or substituted
alkyl group having 5 to 25 carbon atoms or an unsubstituted or
substituted alkenyl group having 5 to 25 carbon atoms, and R2 to R6
are each independently a hydrogen atom, a hydroxyl group, an alkyl
group having 1 to 25 carbon atoms, an alkenyl group having 2 to 25
carbon atoms, an alkynyl group having 2 to 25 carbon atoms, an
alkoxy group having 1 to 25 carbon atoms, an alkenyloxy group
having 2 to 25 carbon atoms or an alkynyloxy group having 2 to 25
carbon atoms, wherein at least one of R2 to R6 is a hydroxyl group,
which method comprises: (a) condensing a fatty acid represented by
formula (1): ##STR00018## wherein R1 is as defined above, and a
hydroxymethylphenol represented by formula (2): ##STR00019##
wherein R2 to R6 are as defined above, said condensing occurring in
the presence of an enzyme as a catalyst and without the presence of
a solvent or within a low-polar solvent.
2. The method of claim 1, wherein the low-polar solvent is at least
one solvent selected from the group consisting of heptane; hexane;
pentane; toluene; 4-methyl-2-pentanone; 2-butanone;
1,2-dimethoxyethane; and a mixture thereof.
3. The method of claim 1, wherein said hydroxymethylphenol
represented by formula (2) is vanillyl alcohol.
4. The method of claim 1, wherein said fatty acid represented by
formula (1) is present in a molar excess as compared to said
hydroxymethylphenol represented by formula (2), such that an amount
of said fatty acid represented by formula (1) is contained in the
reaction mixture after said condensing.
5. The method of claim 1, which further comprises: (b) adding a
fatty acid represented by formula (4): ##STR00020## wherein R1' is
an unsubstituted or substituted alkyl group having 5 to 25 carbon
atoms or an unsubstituted or substituted alkenyl group having 5 to
25 carbon atoms, after said condensing of said fatty acid
represented by formula (1) and said hydroxymethylphenol represented
by formula (2).
6. The method of claim 4, which further comprises: (b') after said
condensing, preparatively separating said ester compound
represented by formula (3) as a mixture with said fatty acid
represented by formula (1).
7. The method of claim 5, which further comprises: (b') after the
condensation, preparatively separating said ester compound
represented by formula (3) as a mixture with said fatty acid
represented by formula (4).
8. The method of claim 1, wherein R1 is a group selected from the
group consisting of a hexyl group; a 5-methylhexyl group; a
trans-5-methyl-3-hexenyl group; a heptyl group; a 6-methylheptyl
group; a 5-methylheptyl group; a trans-6-methyl-4-heptenyl group;
an octyl group; a 7-methyloctyl group; a trans-7-methyl-5-octenyl
group; a nonyl group; a 8-methylnonyl group; a 7-methylnonyl group;
a trans-8-methyl-6-nonenyl group; a trans-8-methyl-5-nonenyl group;
a trans-7-methyl-5-nonenyl group; a decyl group; a 9-methyldecyl
group; a trans-9-methyl-7-decenyl group; a trans-9-methyl-6-decenyl
group; an undecyl group; and a dodecyl group.
9. The method of claim 1, wherein said enzyme is lipase.
10. The method of claim 1, wherein said condensing is carried out
at a temperature of 15.degree. C. to 90.degree. C.
11. The method of claim 1, wherein said fatty acid represented by
formula (1) is obtained by a method comprising: (i) hydrolyzing an
ester compound represented by formula (8): ##STR00021## wherein R1
is as defined above, and Rc is a methyl group, an ethyl group, an
isopropyl group, a tert-butyl group, an allyl group, or a benzyl
group; and (ii) subjecting the resulting compound to (A) reacting
the compound with a base to form a salt crystal and converting the
crystal to a free form thereof, and/or (B) distillation.
12. The method of claim 11, wherein said ester compound represented
by formula (8) is obtained by a method comprising: (A) converting a
compound represented by formula (5): Ra--X (5) wherein Ra is an
unsubstituted or substituted alkyl group having 1 to 24 carbon
atoms or an unsubstituted or substituted alkenyl group having 2 to
24 carbon atoms, and X is a halogen atom, to a Grignard reagent
represented by the formula (6): Ra--MgX (6) wherein Ra and X are as
defined above, and (B) cross coupling said Grignard reagent with a
compound represented by formula (7): ##STR00022## wherein Rb is an
unsubstituted or substituted alkyl group having 1 to 24 carbon
atoms or an unsubstituted or substituted alkenyl group having 2 to
24 carbon atoms, provided that the total of the carbon atoms of Ra
and Rb is 5 to 25, Rc is as defined above, and Y is a halogen atom,
a methanesulfonyloxy group, a p-toluenesulfonyloxy group, or a
trifluoromethanesulfonyloxy group.
13. The method of claim 1, wherein said fatty acid represented by
formula (1) is obtained by a method comprising: (i) reacting a
mixture of a fatty acid represented by formula (10): ##STR00023##
wherein Rd and Re are each independently a hydrogen atom or an
alkyl group having 1 to 6 carbon atoms, m is 0 or 1, and n is an
integer of 1 to 5, and a cis isomer thereof with a base to form
salts thereof; (ii) purifying, based on the difference in the
crystallinity or solubility of the formed salts, a salt of the
fatty acid represented by formula (10); and (iii) then converting
said salt to a free form thereof.
14. A method of making a food composition, comprising: (1)
incorporating an ester compound represented by formula (3):
##STR00024## wherein R1 is an unsubstituted or substituted alkyl
group having 5 to 25 carbon atoms or an unsubstituted or
substituted alkenyl group having 5 to 25 carbon atoms, and R2 to R6
are each independently a hydrogen atom, a hydroxyl group, an alkyl
group having 1 to 25 carbon atoms, an alkenyl group having 2 to 25
carbon atoms, an alkynyl group having 2 to 25 carbon atoms, an
alkoxy group having 1 to 25 carbon atoms, an alkenyloxy group
having 2 to 25 carbon atoms or an alkynyloxy group having 2 to 25
carbon atoms, wherein at least one of R2 to R6 is a hydroxyl group,
into a food, wherein said ester compound represented by formula (3)
is prepared by a method according to claim 1.
15. A method of making a nutraceutical composition, comprising: (1)
incorporating an ester compound represented by formula (3):
##STR00025## wherein R1 is an unsubstituted or substituted alkyl
group having 5 to 25 carbon atoms or an unsubstituted or
substituted alkenyl group having 5 to 25 carbon atoms, and R2 to R6
are each independently a hydrogen atom, a hydroxyl group, an alkyl
group having 1 to 25 carbon atoms, an alkenyl group having 2 to 25
carbon atoms, an alkynyl group having 2 to 25 carbon atoms, an
alkoxy group having 1 to 25 carbon atoms, an alkenyloxy group
having 2 to 25 carbon atoms or an alkynyloxy group having 2 to 25
carbon atoms, wherein at least one of R2 to R6 is a hydroxyl group,
into a nutraceutical, wherein said ester compound represented by
formula (3) is prepared by a method according to claim 1.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of International Patent
Application No. PCT/JP2006/303343, filed on Feb. 17, 2006, and
claims priority to U.S. Provisional Application No. 60/702,606,
filed on Jul. 27, 2005, and Japanese Patent Application No.
2005-043154, filed on Feb. 18, 2005, all of which are incorporated
herein by reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to method of producing a
capsinoid compound by dehydrating condensation. The present
invention also relates to a method for stabilizing a capsinoid
compound. The present invention further relates to stabilized
capsinoid compositions.
[0004] 2. Discussion of the Background
[0005] Capsaicin
((E)-N-(4-hydroxy-3-methoxybenzyl)-8-methyl-6-nonenamide), the
pungent ingredient of Capsicum annuum L., has physiological
activities such as the suppression of obesity, promotion of energy
metabolism, and the like. Due to its extremely strong pungent
taste, however, capsaicin can be used only in a limited amount, and
cannot be used as a food additive, a pharmaceutical product, and
the like.
[0006] In recent years, Yazawa et al. have developed and reported a
non-pungent cultivar of Capsicum annuum L., CH-19 Sweet, by fixing
a non-pungent fruit over the years, which was selected from the
fruits of a highly pungent cultivar CH-19, a native of Thailand
(see, e.g., Yazawa, S.; Suetome, N.; Okamoto, K.; Namiki, T. J.
Japan Soc. Hort. Sci., 1989, 58, 601-607).
[0007] CH-19 Sweet contains a large amount of capsinoids, which are
free of a pungent taste. These capsinoids include capsiate,
dihydrocapsiate, and nordihydrocapsiate, in the order of content,
the first being the highest, which have the following
structures.
##STR00002##
[0008] These capsinoids have the same physiological activities as
capsaicin and are free of a pungent taste. Accordingly, they may be
usable as food additives or pharmaceutical products. However,
production of capsinoids with high purity in a large amount from
natural sources is limited, and a novel synthetic method for
conveniently producing such capsinoids in a large amount has been
desired.
[0009] To form an ester bond of a capsinoid, it is a general
practice to condense vanillyl alcohol and a fatty acid
derivative.
[0010] Vanillyl alcohol has two reaction sites of a primary
hydroxyl group and a phenolic hydroxyl group. Since conventional
esterification methods, such as a method of condensing vanillyl
alcohol and an acid chloride of fatty acid in the presence of a
base (see, e.g., Kobata, K.; Todo, T.; Yazawa, S.; Iwai, K.;
Watanabe, T. J. Agric. Food Chem., 1998, 46, 1695-1697), permit
reaction of the acid chloride with both the primary hydroxyl group
and the phenolic hydroxyl group, the yield of the object capsinoid
becomes lower.
[0011] For synthesis of capsinoids by a conventional esterification
method, therefore, the phenolic hydroxyl group of vanillyl alcohol
may be selectively protected. However, this requires protection and
deprotection before and after esterification, thus unpreferably
increasing the number of steps necessary for the production.
Furthermore, such capsinoids are associated with a problem that
they are unstable and easily decomposed during deprotection.
[0012] As a method for selectively reacting the primary hydroxyl
group alone, the Mitsunobu reaction (see, e.g., Appendino, G.;
Minassi, A.; Daddario, N.; Bianchi, F.; Tron, G. C. Organic
Letters, 2002, 4, 3839-3841) and a method involving the use of
LiClO.sub.4 (see, e.g., Bandgar, B. P.; Kamble, V. T.; Sadavarte,
V. S.; Uppalla, L. S. Synlett, 2002, 735-738) can be mentioned. The
former is defective in that triphenylphosphine oxide and reduced
diethyl azodicarboxylate occur as co-products after the reaction,
which makes purification difficult, and the latter did not permit
reproduction of the yield described in the publication, though the
experiment was faithfully repeated by the present inventors.
Accordingly, neither of them are suitable for industrial
practice.
[0013] In the meantime, the primary hydroxyl group alone can be
selectively reacted by an esterification method using an enzyme.
This method is considered to be suitable for industrial practice
from the aspects of easily available reagents and convenient steps.
Specific examples of the method using an enzyme include a method of
condensation of vanillyl alcohol and a fatty acid using an
immobilized enzyme Novozym 435 (manufactured by Novozymes), which
is one kind of lipase, in an acetone solvent (see, e.g.,
JP-A-2000-312598). However, since the reaction using the enzyme is
an equilibrium reaction with water produced during esterification,
the reaction takes a long time and the yield is as low as about
60%. To increase the yield, one of the starting materials may be
used in a large excess to shift the equilibrium toward the
esterification. However, this approach necessitates a step of
separating the starting material remaining after the reaction from
the resultant product, making the step complicated. When molecular
sieves are added as a dehydrating agent, the yield increases, but
only up to about 80%, and the dehydrating agent needs to be removed
by filtration. For reuse of the enzyme, the enzyme and the
dehydrating agent need to be separated from the cake after the
reaction.
[0014] Furthermore, capsinoids are unstable and are known to be
decomposed by mere dissolution in an organic solvent (see, e.g.,
Sutoh, K.; Kobata, K.; Watanabe, T. J. Agric. Food Chem., 2001, 49,
4026-4030). Therefore, techniques for stable separation and
preservation of the capsinoid after industrial production of the
capsinoid, become necessary.
SUMMARY OF THE INVENTION
[0015] Accordingly, it is one object of the present invention to
provide novel methods of producing a capsinoid.
[0016] It is another object of the present invention to provide
novel methods for producing a capsinoid by esterification using an
enzyme.
[0017] It is another object of the present invention to provide
novel methods for producing a capsinoid which conveniently affords
the capsinoid in a high yield in a short time without using a
dehydrating agent.
[0018] It is another object of the present invention to provide a
method of stably preserving a capsinoid thus produced, by
separating the resultant capsinoid under stable conditions.
[0019] It is another object of the present invention to provide
stabilized compositions which contain a capsinoid.
[0020] These and other objects, which will become apparent during
the following detailed description, have been achieved by the
inventors' discovery that carrying out a condensation reaction
using an enzyme without solvent or in a low-polar solvent
conveniently affords capsinoid in a short time and in a high yield,
because water produced during the condensation is quickly separated
from the reaction mixture to accelerate the reaction even without
using a dehydrating agent. Furthermore, the inventors have also
found that the coexistence of several percent of a fatty acid with
a capsinoid enables the stable separation of the capsinoid, as well
as long-term preservation of the capsinoid.
[0021] Accordingly, the present invention provides the
following:
[0022] (1) A method of producing an ester compound represented by
formula (3):
##STR00003##
wherein R1 is an unsubstituted or substituted alkyl group having 5
to 25 carbon atoms or an unsubstituted or substituted alkenyl group
having 5 to 25 carbon atoms, and R2 to R6 are each independently a
hydrogen atom, a hydroxyl group, an alkyl group having 1 to 25
carbon atoms, an alkenyl group having 2 to 25 carbon atoms, an
alkynyl group having 2 to 25 carbon atoms, an alkoxy group having 1
to 25 carbon atoms, an alkenyloxy group having 2 to 25 carbon atoms
or an alkynyloxy group having 2 to 25 carbon atoms, wherein at
least one of R2 to R6 is a hydroxyl group (hereinafter to be also
referred to as ester compound (3)), which method comprises:
[0023] (a) condensing a fatty acid represented by formula (1):
##STR00004##
wherein R1 is as defined above (hereinafter to be also referred to
as fatty acid (1)), and a hydroxymethylphenol represented by
formula (2):
##STR00005##
wherein R2 to R6 are as defined above (hereinafter to be also
referred to as hydroxymethylphenol (2)), in the presence of an
enzyme as a catalyst without a solvent or in a low-polar
solvent.
[0024] (2) The method of the above-mentioned (1), wherein the
low-polar solvent comprises one or more solvents selected from the
group consisting of heptane, hexane, pentane, toluene,
4-methyl-2-pentanone, 2-butanone, 1,2-dimethoxyethane, and mixtures
thereof.
[0025] (3) The method of the above-mentioned (1) or (2), wherein
the hydroxymethylphenol (2) is vanillyl alcohol.
[0026] (4) The method of any one of the above-mentioned (1) to (3),
wherein the fatty acid (1) is used in excess of the
hydroxymethylphenol (2), such that some fatty acid (1) is contained
in the reaction mixture after the condensation.
[0027] (5) The method of any one of the above-mentioned (1) to (3),
which further comprises:
[0028] (b) adding a fatty acid represented by the formula (4):
##STR00006##
wherein R1' is an unsubstituted or substituted alkyl group having 5
to 25 carbon atoms or an unsubstituted or substituted alkenyl group
having 5 to 25 carbon atoms (hereinafter to be also referred to as
fatty acid (4)), after the condensing of fatty acid (1) and
hydroxymethylphenol (2).
[0029] (6) The method of the above-mentioned (4), further
comprising:
[0030] (b') after the condensation, preparatively separating the
obtained ester compound (3) as a mixture with fatty acid (1).
[0031] (7) The method of the above-mentioned (5), further
comprising:
[0032] (b') after the condensation, preparatively separating the
obtained ester compound (3) as a mixture with fatty acid (4).
[0033] (8) The method of any one of the above-mentioned (1) to (7),
wherein R1 is a group selected from the group consisting of a hexyl
group, a 5-methylhexyl group, a trans-5-methyl-3-hexenyl group, a
heptyl group, a 6-methylheptyl group, a 5-methylheptyl group, a
trans-6-methyl-4-heptenyl group, an octyl group, a 7-methyloctyl
group, a trans-7-methyl-5-octenyl group, a nonyl group, a
8-methylnonyl group, a 7-methylnonyl group, a
trans-8-methyl-6-nonenyl group, a trans-8-methyl-5-nonenyl group, a
trans-7-methyl-5-nonenyl group, a decyl group, a 9-methyldecyl
group, a trans-9-methyl-7-decenyl group, a trans-9-methyl-6-decenyl
group, an undecyl group, and a dodecyl group.
[0034] (9) The method of any one of the above-mentioned (1) to (8),
wherein the enzyme is lipase.
[0035] (10) The method of any one of the above-mentioned (1) to
(9), wherein the condensation is carried out at a temperature of
15.degree. C. to 90.degree. C.
[0036] (11) The method of any one of the above-mentioned (1) to
(10), wherein fatty acid (1) is obtained by a process
comprising:
[0037] (i) hydrolyzing an ester compound represented by the formula
(8):
##STR00007##
wherein R1 is as defined above, and Rc is a methyl group, an ethyl
group, an isopropyl group, a tert-butyl group, an allyl group, or a
benzyl group (hereinafter to be also referred to as ester compound
(8)); and
[0038] (ii) subjecting the resulting compound to (A) reacting the
compound with a base to form a salt crystal and converting the
crystal to a free form thereof, and/or (B) distillation.
[0039] (12) The method of the above-mentioned (11), wherein ester
compound (8) is obtained by a method comprising:
[0040] (A) converting a compound represented by the formula
(5):
Ra--X (5)
wherein Ra is an unsubstituted or substituted alkyl group having 1
to 24 carbon atoms or an unsubstituted or substituted alkenyl group
having 2 to 24 carbon atoms, and X is a halogen atom (hereinafter
to be also referred to as compound (5)), to a Grignard reagent
represented by the formula (6):
Ra--MgX (6)
wherein Ra and X are as defined above (hereinafter to be also
referred to as Grignard reagent (6)); and
[0041] (B) cross coupling Grignard reagent (6) with a compound
represented by the formula (7):
##STR00008##
wherein Rb is an unsubstituted or substituted alkyl group having 1
to 24 carbon atoms or an unsubstituted or substituted alkenyl group
having 2 to 24 carbon atoms (provided that the total of the carbon
atoms of Ra and Rb is 5 to 25), Rc is as defined above, and Y is a
halogen atom, a methanesulfonyloxy group, a p-toluenesulfonyloxy
group or a trifluoromethanesulfonyloxy group (hereinafter to be
also referred to as compound (7)).
[0042] (13) The method of any one of the above-mentioned (1) to
(10), wherein fatty acid (1) is obtained by a method
comprising:
[0043] (i) reacting a mixture of a fatty acid represented by the
formula (10):
##STR00009##
wherein Rd and Re are each independently a hydrogen atom or an
alkyl group having 1 to 6 carbon atoms, m is 0 or 1, and n is an
integer of 1 to 5 (hereinafter to be also referred to as fatty acid
(10)), and a cis isomer thereof with a base to form salts
thereof;
[0044] (ii) purifying, based on the difference in the crystallinity
or solubility of the formed salts, the salt of fatty acid (10);
and
[0045] (iii) then converting the salt to a free form thereof.
[0046] (14) A composition, comprising:
[0047] (A) an ester compound (3):
##STR00010##
wherein R1 is an unsubstituted or substituted alkyl group having 5
to 25 carbon atoms or an unsubstituted or substituted alkenyl group
having 5 to 25 carbon atoms, and R2 to R6 are each independently a
hydrogen atom, a hydroxyl group, an alkyl group having 1 to 25
carbon atoms, an alkenyl group having 2 to 25 carbon atoms, an
alkynyl group having 2 to 25 carbon atoms, an alkoxy group having 1
to 25 carbon atoms, an alkenyloxy group having 2 to 25 carbon atoms
or an alkynyloxy group having 2 to 25 carbon atoms, wherein at
least one of R2 to R6 is a hydroxyl group; and
[0048] (B) a fatty acid represented by the formula (11):
##STR00011##
wherein R1'' is an unsubstituted or substituted alkyl group having
5 to 25 carbon atoms or an unsubstituted or substituted alkenyl
group having 5 to 25 carbon atoms (hereinafter to be also referred
to as fatty acid (11)), provided that the composition is not a fats
and oils extract from a plant.
[0049] (15) The composition of the above-mentioned (14), wherein
fatty acid (11) is contained in a proportion of 0.1 wt % to 30 wt %
relative to the weight of ester compound (3).
[0050] (16) The composition of the above-mentioned (14) or (15),
further comprising, as an extender or a carrier, one or more kinds
of additives selected from the group consisting of a fats and oils
composition, an emulsifier, a preservative, and an antioxidant.
[0051] (17) A method of preparing a composition, comprising a
stabilizing ester compound (3):
##STR00012##
wherein R1 is an unsubstituted or substituted alkyl group having 5
to 25 carbon atoms or an unsubstituted or substituted alkenyl group
having 5 to 25 carbon atoms, and R2 to R6 are each independently a
hydrogen atom, a hydroxyl group, an alkyl group having 1 to 25
carbon atoms, an alkenyl group having 2 to 25 carbon atoms, an
alkynyl group having 2 to 25 carbon atoms, an alkoxy group having 1
to 25 carbon atoms, an alkenyloxy group having 2 to 25 carbon atoms
or an alkynyloxy group having 2 to 25 carbon atoms, wherein at
least one of R2 to R6 is a hydroxyl group, which method comprises
adding, to said composition, at least one fatty acid (4):
##STR00013##
[0052] wherein R1' is an unsubstituted or substituted alkyl group
having 5 to 25 carbon atoms or an unsubstituted or substituted
alkenyl group having 5 to 25 carbon atoms.
[0053] (18) The method of the above-mentioned (17), wherein fatty
acid (4) is added in a proportion of 0.1 wt % to 30 wt % relative
to the weight of ester compound (3).
[0054] According to the present invention, a large amount of
capsinoid can be conveniently produced in a high yield in a short
time using an enzyme. In addition, since a dehydrating agent (e.g.,
a molecular sieves and the like) is not necessary, the enzyme can
be re-used upon simple recovery by filtration. According to the
present invention, moreover, the reaction proceeds in a high yield
with a small amount of enzyme. Therefore, the amount of the enzyme
can be reduced, and the enzyme can be recovered easily.
Furthermore, the resulting capsinoid can be stably obtained by
separation in the coexistence of a fatty acid. In this manner, the
present invention enables industrially advantageous production of
capsinoid.
[0055] According to the stabilizing method of the present
invention, moreover, the capsinoid can be stably preserved by
coexistence of a fatty acid with the capsinoid.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0056] The embodiment of the present invention is explained in the
following.
[0057] The terms used in the present invention are explained in the
following.
[0058] The "alkyl group having 5 to 25 carbon atoms" of the
"unsubstituted or substituted alkyl group having 5 to 25 carbon
atoms" represented by R1 may be linear or branched. Specific
examples include an n-pentyl group, a sec-pentyl group, a
tert-pentyl group, an isopentyl group, an n-hexyl group, an
isohexyl group, a 5-methylhexyl group, a heptyl group, a
6-methylheptyl group, a 5-methylheptyl group, a 4,4-dimethylpentyl
group, an octyl group, a 2,2,4-trimethylpentyl group, a
7-methyloctyl group, a nonyl group, a 8-methylnonyl group, a
7-methylnonyl group, a decyl group, a 9-methyldecyl group, an
undecyl group, a dodecyl group, a tetradecyl group, a hexadecyl
group, an octadecyl group, an icosyl group, a docosyl group, a
pentacosyl group, and the like. Besides these, it includes various
branched-chain isomers thereof. Preferred is an alkyl group having
6 to 12 carbon atoms.
[0059] The "alkenyl group having 5 to 25 carbon atoms" of the
"unsubstituted or substituted alkenyl group having 5 to 25 carbon
atoms" represented by R1 may be linear or branched, and the number
of double bonds may be one or more. Specific examples include a
pentenyl group (e.g., 4-pentenyl group, 3-pentenyl group, etc.), a
hexenyl group (e.g., 2-hexenyl group, 4-hexenyl group, etc.), a
5-methyl-3-hexenyl group, a 5-methyl-4-hexenyl group, a heptenyl
group (e.g., 2-heptenyl group, 3-heptenyl group, 5-heptenyl group,
etc.), a 6-methyl-4-heptenyl group, an octenyl group (e.g.,
3-octenyl group, 6-octenyl group, etc.), a 7-methyl-5-octenyl
group, a nonenyl group (e.g., 3-nonenyl group, 7-nonenyl group,
etc.), a 8-methyl-6-nonenyl group, a 8-methyl-5-nonenyl group, a
7-methyl-5-nonenyl group, a decenyl group (e.g., 8-decenyl group,
etc.), a 9-methyl-7-decenyl group, a 9-methyl-6-decenyl group, an
undecenyl group (e.g., 9-undecenyl group, etc.), a dodecenyl group
(e.g., 10-dodecenyl group, etc.), a tetradecenyl group, a
4,8,12-tetradecatrienyl group, a pentadecenyl group (e.g.,
13-pentadecenyl group, etc.), a hexadecenyl group, a heptadecenyl
group (e.g., 15-heptadecenyl group, etc.), an octadecenyl group
(e.g., 16-octadecenyl group, etc.), a 17-nonadecenyl group, an
icosenyl group (e.g., 18-icosenyl group, etc.), a henicosenyl group
(e.g., 19-henicosenyl group, etc.), a docosenyl group (e.g.,
20-docosenyl group, etc.), a pentacosenyl group, and the like.
Besides these, it includes various branched-chain isomers thereof.
Preferred is an alkenyl group having 6 to 12 carbon atoms. The
steric structure of the double bond may be a trans form or a cis
form, with preference given to a trans form.
[0060] The "alkyl group having 5 to 25 carbon atoms" of the
"unsubstituted or substituted alkyl group having 5 to 25 carbon
atoms" represented by R1 and the "alkenyl group having 5 to 25
carbon atoms" of the "unsubstituted or substituted alkenyl group
having 5 to 25 carbon atoms" represented by R1 optionally have 1 to
4 substituents. As the substituent, an alkyl group, a halogen atom,
a haloalkyl group, an amino group, a hydroxyl group, an acyl group,
a nitro group, a cyano group, a mercapto group, and the like can be
mentioned. Of these, an alkyl group having 1 to 4 carbon atoms is
preferable. As the alkyl group having 1 to 4 carbon atoms, a methyl
group, an ethyl group, a propyl group, an isopropyl group, a butyl
group, a tert-butyl group, an isobutyl group and the like can be
mentioned.
[0061] As R1, a hexyl group, a 5-methylhexyl group, a
trans-5-methyl-3-hexenyl group, a heptyl group, a 6-methylheptyl
group, a 5-methylheptyl group, a trans-6-methyl-4-heptenyl group,
an octyl group, a 7-methyloctyl group, a trans-7-methyl-5-octenyl
group, a nonyl group, a 8-methylnonyl group, a 7-methylnonyl group,
a trans-8-methyl-6-nonenyl group, a trans-8-methyl-5-nonenyl group,
a trans-7-methyl-5-nonenyl group, a decyl group, a 9-methyldecyl
group, a trans-9-methyl-7-decenyl group, a trans-9-methyl-6-decenyl
group, an undecyl group, and a dodecyl group are preferable from
the aspect of usefulness of the object ester compound (3) as a
capsinoid.
[0062] While fatty acid (1) may be a single compound or a mixture
of two or more kinds of compounds wherein R1 varies among the
above-mentioned definitions, it is preferred that fatty acid (1) is
a single compound. When using a fatty acid obtained by hydrolysis
of a natural capsaicinoid for synthesis of a capsinoid by
condensation of the fatty acid with vanillyl alcohol, such fatty
acid (1) is mixture of trans-8-methyl-6-nonenoic acid,
8-methylnonanoic acid, 7-methyloctanoic acid, and the like. For
reproduction of a capsinoid composition having a natural abundance
ratio by the use of synthetic substances, and the like, respective
capsinoids independently synthesized by the present method may be
mixed at the same abundance ratio as mentioned above. The object
can also be achieved by performing the present method using a
mixture of the corresponding fatty acids (1) at the same abundance
ratio as mentioned above.
[0063] The "alkyl group having 1 to 25 carbon atoms" represented by
R2 to R6 may be linear or branched. Specific examples include a
methyl group, an ethyl group, a propyl group, an isopropyl group,
an n-butyl group, a tert-butyl group, and the like, and those
similar to the above-mentioned "alkyl group having 5 to 25 carbon
atoms" of the "unsubstituted or substituted alkyl group having 5 to
25 carbon atoms" represented by R1. Preferred is an alkyl group
having 1 to 12 carbon atoms.
[0064] The "alkenyl group having 2 to 25 carbon atoms" represented
by R2 to R6 may be linear or branched, and the number of double
bonds may be one or more. Specific examples include a vinyl group,
an allyl group, a propenyl group, an isopropenyl group, a butenyl
group, and the like, and those similar to the above-mentioned
"alkenyl group having 5 to 25 carbon atoms" of the "unsubstituted
or substituted alkenyl group having 5 to 25 carbon atoms"
represented by R1. Preferred is an alkenyl group having 2 to 12
carbon atoms.
[0065] The "alkynyl group having 2 to 25 carbon atoms" represented
by R2 to R6 may be linear or branched, and the number of triple
bonds may be one or more. Specific examples include an ethynyl
group, a propynyl group, a pentynyl group, a hexynyl group, an
octynyl group, a nonynyl group, and the like. Preferred is an
alkynyl group having 2 to 12 carbon atoms.
[0066] The "alkoxy group having 1 to 25 carbon atoms" represented
by R2 to R6 may be linear or branched, and is exemplified by an
alkoxy group wherein the alkyl moiety is the same as the
above-mentioned "alkyl group having 1 to 25 carbon atoms"
represented by R2 to R6. Preferred is an alkoxy group having 1 to
12 carbon atoms.
[0067] The "alkenyloxy group having 2 to 25 carbon atoms"
represented by R2 to R6 may be linear or branched, and the number
of double bonds may be one or more. Example thereof include an
alkenyloxy group wherein the alkenyl moiety is the same as the
above-mentioned "alkenyl group having 2 to 25 carbon atoms"
represented by R2 to R6. Preferred is an alkenyloxy group having 2
to 12 carbon atoms.
[0068] The "alkynyloxy group having 2 to 25 carbon atoms"
represented by R2 to R6 may be linear or branched, and the number
of triple bonds may be one or more. Example thereof include an
alkynyloxy group wherein the alkynyl moiety is similar to the
above-mentioned "alkynyl group having 2 to 25 carbon atoms"
represented by R2 to R6. Preferred is an alkynyloxy group having 2
to 12 carbon atoms.
[0069] As R2 to R6, a hydrogen atom, a hydroxyl group, a methoxy
group, an ethoxy group, an allyl group, a vinyl group, and a
vinyloxy group are preferable.
[0070] Of R2 to R6, at least one of them is a hydroxyl group, and
R4 is preferably a hydroxyl group. In addition, it is preferable
that only one of R2 to R6 is a hydroxyl group.
[0071] Preferable combination of R2 to R6 is a combination of R2,
R5, and R6 being hydrogen atoms, R3 being a methoxy group, an
ethoxy group, an allyl group, a vinyl group, or a vinyloxy group,
and R4 being a hydroxyl group. Particularly, it is most preferable
that R3 is a methoxy group (i.e., hydroxymethylphenol (2) is
vanillyl alcohol), from the aspect of usefulness of the object
ester compound (3) as a capsinoid.
[0072] While hydroxymethylphenol (2) may be a single compound or a
mixture of two or more kinds of compounds having the
above-mentioned definitions, it is preferred that
hydroxymethylphenol (2) is a single compound.
[0073] The "unsubstituted or substituted alkyl group having 5 to 25
carbon atoms" represented by R1' is exemplified by those similar to
the "unsubstituted or substituted alkyl group having 5 to 25 carbon
atoms" represented by R1.
[0074] The "unsubstituted or substituted alkenyl group having 5 to
25 carbon atoms" represented by R1' is exemplified by those similar
to the "unsubstituted or substituted alkenyl group having 5 to 25
carbon atoms" represented by R1.
[0075] As R1', a hexyl group, a 5-methylhexyl group, a
trans-5-methyl-3-hexenyl group, a heptyl group, a 6-methylheptyl
group, a 5-methylheptyl group, a trans-6-methyl-4-heptenyl group,
an octyl group, a 7-methyloctyl group, a trans-7-methyl-5-octenyl
group, a nonyl group, a 8-methylnonyl group, a 7-methylnonyl group,
a trans-8-methyl-6-nonenyl group, a trans-8-methyl-5-nonenyl group,
a trans-7-methyl-5-nonenyl group, a decyl group, a 9-methyldecyl
group, a trans-9-methyl-7-decenyl group, a trans-9-methyl-6-decenyl
group, an undecyl group, and a dodecyl group are preferable, and
R1' is most preferably the same as the group selected as R1. That
is, R1 of fatty acid (1) and R1' of fatty acid (4) are preferably
the same group.
[0076] The "unsubstituted or substituted alkyl group having 5 to 25
carbon atoms" represented by R1'' is exemplified by those similar
to the "unsubstituted or substituted alkyl group having 5 to 25
carbon atoms" represented by R1.
[0077] The "unsubstituted or substituted alkenyl group having 5 to
25 carbon atoms" represented by R1'' is exemplified by those
similar to the "unsubstituted or substituted alkenyl group having 5
to 25 carbon atoms" represented by R1.
[0078] As R1'', a hexyl group, a 5-methylhexyl group, a
trans-5-methyl-3-hexenyl group, a heptyl group, a 6-methylheptyl
group, a 5-methylheptyl group, a trans-6-methyl-4-heptenyl group,
an octyl group, a 7-methyloctyl group, a trans-7-methyl-5-octenyl
group, a nonyl group, a 8-methylnonyl group, a 7-methylnonyl group,
a trans-8-methyl-6-nonenyl group, a trans-8-methyl-5-nonenyl group,
a trans-7-methyl-5-nonenyl group, a decyl group, a 9-methyldecyl
group, a trans-9-methyl-7-decenyl group, a trans-9-methyl-6-decenyl
group, an undecyl group, and a dodecyl group are preferable, and
R1'' is most preferably the same as the group selected as R1. That
is, R1 of fatty acid (1) and R1'' of fatty acid (11) are preferably
the same group.
[0079] The present invention provides a production method of ester
compound (3), which characteristically comprises condensing fatty
acid (1) and hydroxymethylphenol (2) using an enzyme as a catalyst
without solvent or in a low-polar solvent.
[0080] According to the method of the present invention which is
performed without solvent or in a low-polar solvent (non-miscible
with water or hardly miscible with water, e.g., toluene, etc.),
unlike known methods for accelerating a condensation reaction that
essentially require use of a high-polarity solvent (miscible with
water, e.g., acetone, dioxane, etc.) that can completely dissolve
the hydroxymethylphenol (2) (e.g., vanillyl alcohol), the reaction
is accelerated even without using a dehydrating agent, because the
water produced during the condensation is quickly separated from
the reaction mixture. Therefore, the method of the present
invention is superior to known methods in the following
aspects.
[0081] (i) Since the water produced by the condensation reaction is
rapidly separated from the reaction mixture and removed from the
reaction system, the equilibrium shifts toward the ester production
side and the conversion ratio becomes advantageously high.
Therefore, it is not required to use one of the starting materials
in a large excess, nor an enzyme catalyst in an excess amount of
several times higher weight than the starting material.
[0082] (ii) Since addition of molecular sieves as a scavenger
(i.e., dehydrating agent) of the water produced by the condensation
reaction is not necessary, the enzyme does not need to be separated
from the molecular sieves after filtration, and the enzyme can be
easily reused.
[0083] (iii) Since the conversion ratio (yield) is high and
by-product is absent, a high quality object product can be obtained
by a convenient workup alone without purification by
chromatography, which includes adding a low-polar solvent after the
completion of the reaction to remove the enzyme catalyst by
filtration and concentrating the filtrate, or partitioning after
removal of the enzyme catalyst and concentrating the organic
layer.
[0084] The fatty acid to be used in the present invention may be
commercially available or can be synthesized by a known method
(e.g., method described in Kaga, H.; Goto, K.; Takahashi, T.; Hino,
M.; Tokuhashi, T.; Orito, K. Tetrahedron, 1996, 52, 8451-8470).
[0085] Since most of ester compounds (3) (e.g., capsinoid, etc.),
which are the object compounds, are in an oily state at ambient
temperature, purification by recrystallization cannot be performed.
In view of stability, purification by distillation under reduced
pressure is also difficult. Since the method of purification is
limited as mentioned above, fatty acid (1) having a highest
possible purity is preferable as a starting material for the
production of ester compound (3) having a high purity. Accordingly,
the use of a fatty acid (1) having a purity of at least 97 wt % or
more for the esterification reaction is desirable. To obtain such
fatty acid having a high purity, a fatty acid obtained by a known
method and the like, particularly a fatty acid containing an
impurity such as stereoisomer and the like, is preferably purified
by first forming a salt crystal of the fatty acid and then
converting it to its free form. When a fatty acid is to be
synthesized by a cross coupling method shown by the following
reaction scheme, a fatty acid having a high purity can be obtained
by optimizing the reaction conditions by selection of a catalyst
and the like to suppress production of by-products, by dissolving
the fatty acid in a basic aqueous solution after hydrolysis and
removing the by-product by extraction with an organic solvent, or
by distillation. Optionally, a method of purifying by first forming
a salt crystal of the fatty acid and then converting the crystal to
its free form is also preferable as a method of obtaining a high
purity fatty acid.
[0086] In the following, a method of synthesizing a fatty acid by a
cross coupling method and a method of purifying the fatty acid as
its salt crystal are shown.
[0087] First, a method of synthesizing a fatty acid by a cross
coupling method is explained.
##STR00014##
wherein X is a halogen atom, Ra and Rb are each independently an
unsubstituted or substituted alkyl group having 1 to 24 carbon
atoms, or an unsubstituted or substituted alkenyl group having 2 to
24 carbon atoms (where the total of the carbon atoms of Ra and Rb
is 5 to 25), Rc is a methyl group, an ethyl group, an isopropyl
group, a tert-butyl group, an allyl group, or a benzyl group, Y is
a halogen atom, a methanesulfonyloxy group, a p-toluenesulfonyloxy
group, or a trifluoromethanesulfonyloxy group, and R1 is as defined
above.
[0088] Ra and Rb are each an unsubstituted or substituted alkyl
group having 1 to 24 carbon atoms, or an unsubstituted or
substituted alkenyl group having 2 to 24 carbon atoms, where the
total of the carbon atoms of Ra and Rb is 5 to 25, provided that
when the substituent contains a carbon atom, the carbon atom of the
substituent is excluded.
[0089] As the "alkyl group having 1 to 24 carbon atoms" of the
"unsubstituted or substituted alkyl group having 1 to 24 carbon
atoms" represented by Ra or Rb, an "alkyl group having 1 to 25
carbon atoms" for R2 to R6, wherein the number of carbon atoms is 1
to 24, can be mentioned.
[0090] As the "alkenyl group having 2 to 24 carbon atoms" of the
"unsubstituted or substituted alkenyl group having 2 to 24 carbon
atoms" represented by Ra or Rb, an "alkenyl group having 2 to 25
carbon atoms" for R2 to R6, wherein the number of carbon atoms is 2
to 24, can be mentioned.
[0091] The "alkyl group having 1 to 24 carbon atoms" of the
"unsubstituted or substituted alkyl group having 1 to 24 carbon
atoms" represented by Ra or Rb and the "alkenyl group having 2 to
24 carbon atoms" of the "unsubstituted or substituted alkenyl group
having 2 to 24 carbon atoms" represented by Ra or Rb may have 1 to
4 substituents. As the substituent, substituents similar to those
that the "alkyl group having 5 to 25 carbon atoms" of the
"unsubstituted or substituted alkyl group having 5 to 25 carbon
atoms" represented by R1 may have, and the like can be
mentioned.
[0092] The group represented by Ra and the group represented by Rb
are bonded by a cross coupling reaction to become a group
represented by R1 (i.e., an unsubstituted or substituted alkyl
group having 5 to 25 carbon atoms, or an unsubstituted or
substituted alkenyl group having 5 to 25 carbon atoms). Therefore,
Ra and Rb are appropriately determined by the structure of R1.
[0093] As the halogen atom represented by X or Y, a fluorine atom,
a chlorine atom, a bromine atom, and an iodine atom can be
mentioned, with preference given to a bromine atom.
[0094] In the cross coupling method, compound (5) is first
converted to Grignard reagent (6), which is then subjected to a
cross coupling reaction with compound (7) to give ester compound
(8), which is then hydrolyzed to give fatty acid (1).
[0095] The compound (5) and compound (7) can be obtained by
synthesis according to a known method and the like, and when they
are commercially available, commercial products can be used as they
are.
[0096] The compound (5) can be converted to Grignard reagent (6) by
reacting compound (5) with magnesium according to a conventional
method.
[0097] The cross coupling reaction between Grignard reagent (6) and
compound (7) can be carried out, for example, by reacting Grignard
reagent (6) and compound (7) in an amount of 1 to 3 equivalents
relative to Grignard reagent (6) in a solvent in the presence of a
copper catalyst at a low temperature (preferably at a reaction
mixture temperature of -20.degree. C. to 15.degree. C., more
preferably -5.degree. C. to 10.degree. C., particularly preferably
-3.degree. C. to 5.degree. C.) for 15 minutes to 3 hours.
[0098] As the solvent, ethers such as tetrahydrofuran (THF),
diethyl ether, tert-butyl methyl ether, 1,2-dimethoxyethane, and
the like; N-methylpyrrolidone (NMP);
1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidine (DMPU); etc. and
mixed solvents of these can be used.
[0099] As the copper catalyst, Li.sub.2CuCl.sub.4, CuI, CuBr, CuCl,
CuBr.Me.sub.2S, and the like can be mentioned. The copper catalyst
is used in an amount of 0.5 to 20 mol %, preferably 1 to 3 mol %,
relative to compound (7). CuBr is more preferable as a catalyst,
because it produces a fewer by-products.
[0100] For smooth progress of the reaction, additives such as
trimethylchlorosilane and the like may be used in an amount of 0.5
to 4 equivalents (preferably 1 to 2 equivalents) relative to
compound (7).
[0101] The hydrolysis of ester compound (8) obtained by the
above-mentioned coupling reaction can be carried out by a known
method (method using an acid, method using an alkali, etc.).
[0102] The fatty acid (1) obtained by hydrolyzing ester compound
(8) is dissolved in a basic aqueous solution and extracted with an
organic solvent such as ether, t-butyl methyl ether, hexane,
heptane, and the like to efficiently remove by-products such as
ketone, alcohol, and the like.
[0103] Now, a method of purifying a fatty acid by first obtaining
the fatty acid as a salt crystal, and then converting the salt
crystal to its free form is explained.
[0104] Impurities can be removed from a fatty acid obtained by a
known method and the like, or fatty acid (1) obtained by the
above-mentioned hydrolysis, by forming a salt crystal with a base.
While the purification method of fatty acid (1) is explained in the
following for the sake of convenience of the explanation, the
method explained in the following is similarly applicable to the
fatty acid obtained by a known method and the like.
[0105] The salt crystal can be formed, for example, by stirring
fatty acid (1) and a base in a solvent.
[0106] As the base, inorganic bases (e.g., hydroxides, carbonates,
hydrogencarbonates, etc. of lithium, sodium, potassium, calcium,
magnesium, barium, etc.), organic amines (e.g., ethylenediamine,
1,3-diaminopropane, 1,3-diamino-2-propanol, cyclohexylamine,
4-methoxybenzylamine, ethanolamine, (S)- or (R)-phenylglycinol,
(S)- or (R)-phenylalaminol, cis-2-aminocyclohexanol,
trans-4-aminocyclohexanol, (1S,2R)-cis-1-amino-2-indanol, L-lysine,
L-arginine, etc.), ammonia, and the like can be mentioned. The
amount of the base to be used is 0.8 to 1.2 equivalents, preferably
0.9 to 1.1 equivalents, relative to fatty acid (1).
[0107] As the solvent, for example, water; alcohols such as
methanol, ethanol, isopropanol, and the like; acetates such as
ethyl acetate, isopropyl acetate, and the like; ethers such as
diethyl ether, tert-butyl methyl ether, THF, and the like;
hydrocarbons such as hexane, heptane, and the like; ketones such as
acetone and the like; halogenated hydrocarbons such as chloroform
and the like, and mixed solvents of these can be used.
[0108] By forming a salt crystal of fatty acid (1) with a base as
mentioned above and, where necessary, recrystallization, the
reaction by-products other than fatty acid (1), such as alcohol,
ketones and the like can be efficiently removed with ease.
[0109] Then, the obtained salt crystal is added to an acidic
aqueous solution (e.g., hydrochloric acid, aqueous citric acid
solution, etc.), the mixture is extracted with an organic solvent
(e.g., hexane, heptane, etc.), and the organic solvent is
evaporated to give the object fatty acid (1) at a high purity.
[0110] When the fatty acid (1) is a mixture of a compound
represented by the formula (10):
##STR00015##
wherein Rd and Re are each independently a hydrogen atom or an
alkyl group having 1 to 6 carbon atoms, m is 0 or 1, and n is an
integer of 1 to 5, and a cis isomer thereof; the mixture can be
reacted with a base to form salts thereof, and the salt of fatty
acid (10) can be separated from the salt of its cis isomer based on
the difference in the crystallinity or solubility of the salts
formed.
[0111] Examples of the "alkyl group having 1 to 6 carbon atoms"
represented by Rd or Re include a methyl group, an ethyl group, a
propyl group, an isopropyl group, a butyl group, an isobutyl group,
a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl
group, a neopentyl group, a tert-pentyl group, a hexyl group, and
the like, with preference given to a methyl group for both Rd and
Re.
[0112] m is 0 or 1, preferably 0.
[0113] n is an integer of 1 to 5, preferably 3 or 4, more
preferably 4.
[0114] For separation of fatty acid (10) from its cis isomer, the
salts thereof can be formed in the same manner as in the
aforementioned formation of the salt crystal of fatty acid (1) with
a base.
[0115] As a method for separation of the salt of fatty acid (10)
from the salt of its cis isomer based on the difference in the
crystallinity or solubility of the salts formed, crystal
precipitation, slurry washing, recrystallization, and the like can
be mentioned.
[0116] One example of the separation of fatty acid (10) from its
cis isomer is now shown. In the case of a mixture (trans form 88%,
cis form 12%) of trans-8-methyl-6-nonenoic acid and its cis isomer
(cis-8-methyl-6-nonenoic acid), salts of the isomers therein are
formed using cis-2-aminocyclohexanol as a base, and the salt of cis
isomer is removed by two or three times of crystal precipitation of
the salts of the isomers, whereby the ratio of the
trans-8-methyl-6-nonenoic acid can be increased to not less than
97%.
[0117] The obtained salt crystal is added to an acidic aqueous
solution (e.g., hydrochloric acid, aqueous citric acid solution,
etc.), the mixture is extracted with an organic solvent (e.g.,
hexane, etc.), and the organic solvent is evaporated to give fatty
acid (10).
[0118] By the application of such a purification method by
formation of a salt crystal of fatty acid (1) with a base, neutral
substances such as ketone, alcohol, and the like, as well as fatty
acid (acidic substance) other than the object product, which occur
as a by-product, can be simultaneously removed.
[0119] The above-mentioned separation and purification method of
fatty acid (10) and its cis isomer is not limited to the fatty acid
obtained by the aforementioned coupling reaction, but is similarly
applicable as a purification method of fatty acid (10) obtained by
a known method.
[0120] The hydroxymethylphenol (2) to be used in the present
invention can be obtained by synthesis according to a known method,
and when it is commercially available, a commercial product can be
used.
[0121] The operation of condensation is not particularly limited as
long as the condensation reaction of fatty acid (1) and
hydroxymethylphenol (2) proceeds. For example, fatty acid (1),
hydroxymethylphenol (2), and enzyme are added to a reaction vessel,
where necessary, a low-polar solvent is added and, where necessary,
the mixture is heated. Alternatively, fatty acid (1) and
hydroxymethylphenol (2) are dissolved in a low-polar solvent, an
enzyme is added and, where necessary, the mixture may be
heated.
[0122] As the enzyme to be used in the present invention, any can
be used without particularly limitation as long as it can mediate
the condensation reaction of fatty acid (1) and hydroxymethylphenol
(2), and an esterase is representatively used. As the esterase,
lipase is generally used, and one originated from microorganism,
one originated from animal, or one originated from plant can be
also used. Of those, lipase originated from a microorganism is
preferable. Specifically, lipases originated from the genus Candida
(e.g., Candida antarctica, Candida cylindracea, etc.), the genus
Pseudomonas (e.g., Pseudomonas fluorescens, Pseudomonas sp.,
Pseudomonas cepacia, etc.), the genus Alcaligenes (e.g.,
Alcaligenes sp., etc.), the genus Aspergillus (e.g., Aspergillus
niger, etc.), and the genus Rhizopus (e.g., Rhizopus delemar,
Rhizopus oryzae, etc.) can be mentioned. While these lipases can be
obtained by culture of the microorganisms capable of producing
them, commercial products can also be used preferably. As such
commercially available lipase, lipase PS "Amano", lipase AK
"Amano", lipase AS "Amano", lipase AYS "Amano" (all manufactured by
Amano Enzyme Inc.), Lipozyme CALB L (Novozymes), and the like can
be mentioned.
[0123] Each of these enzymes can be used alone or as a mixture
thereof.
[0124] While the enzymes can be used in any form as long as they
can be added to a reaction solution, use of an immobilized enzyme
is preferable since recovery of the enzyme and the like are
facilitated. As the immobilized enzyme, immobilized enzymes of
lipase, such as lipase PS-C "Amano" I (immobilized on ceramic),
lipase PS-C "Amano" II (immobilized on ceramics), and lipase PS-D
"Amano" I (immobilized on diatomaceous earth) (all manufactured by
Amano Enzyme Inc.), Novozym 435, Lipozyme RM IM, and Lipozyme TL IM
(all manufactured by Novozymes A/S), and the like can be used. Of
these, lipase PS "Amano" and Lipozyme CALB L are desirable in view
of the low cost, and immobilized enzymes of lipase such as lipase
PS-C "Amano" and the like are desirable in view of recyclability.
Use of lipase PS-C "Amano" or lipase PS-D "Amano" I may result in
slight coloration of the reaction mixture. In view of the absence
of coloration, Novozym 435 is desirable.
[0125] While the amount of the enzyme to be added varies depending
on the activity of enzyme and the amount of the solvent and the
starting materials to be added, it can be selected from the range
of 0.01 to 60 wt %, desirably 0.1 to 30 wt %, of fatty acid (1). In
addition, the enzyme may be further added during the reaction for
use in excess.
[0126] The reaction is carried out without solvent or in a
low-polar solvent.
[0127] Here, the low-polar solvent means a low-polarity solvent
which is hardly miscible with water. Specific examples include one
kind of solvent selected from heptane, hexane, pentane, toluene,
4-methyl-2-pentanone, 2-butanone, and 1,2-dimethoxyethane, and a
mixed solvent of two or more kinds thereof. It is preferable to
carry out the reaction without solvent, from the aspects of short
reaction time, convenient operation and cost reduction. The
reaction mixture can be stirred more efficiently by the use of
toluene or the minimum amount of heptane or hexane.
[0128] When a low-polar solvent is used, the amount of the solvent
to be added is appropriately determined in consideration of the
kind of solvent, the activity of the enzyme to be used, the amount
of the starting materials, concentration of each reagent and the
like, and in view of yield and the like, it is generally 0.05 to
100 ml, preferably 0.3 to 50 ml, per 1 g of fatty acid (1).
[0129] When the reaction is carried out without solvent,
hydroxymethylphenol (2) (e.g., vanillyl alcohol) is not
sufficiently dissolved in oily fatty acid (1) and the reaction
system is non-uniform. However, the stirring operation is not
affected, and the reaction system becomes homogeneous as the
reaction proceeds.
[0130] Of course, the condensation reaction of fatty acid (1) with
hydroxymethylphenol (2) may be carried out in the presence of a
small amount of high-polarity solvent, as long as the high polarity
solvent is present in such a low amount so that it does not impede
the condensation reaction. Thus, the condensation reaction may be
carried out in a reaction mixture which is substantially free of
any high-polarity solvent. By the term "substantially free" it is
meant that the reaction mixture contains a total amount of
high-polarity solvent of less than 50 wt %, preferably less than 25
wt %, more preferably less than 10 wt %, even more preferably less
than 5 wt % of any high-polarity solvent, based on the total weight
of the reaction mixture. These amounts refer to the amount of
high-polarity solvent at the beginning of the condensation and do
not include the amount of water formed during the condensation.
Examples of high-polarity solvents include, water, acetone,
tetrahydrofuran, dioxane, and acetonitrile.
[0131] The reaction system may be placed in a mildly reduced
pressure, or an inert gas may be flown on the surface of the
reaction mixture; whereby the produced water can be efficiently
removed and the reaction can be accelerated. When toluene is used
as a solvent, concentration is carried out under reduced pressure
utilizing the azeotropic phenomenon with water, whereby the
dehydrating reaction can be accelerated.
[0132] The fatty acid (1) and hydroxymethylphenol (2) (e.g.,
vanillyl alcohol, etc.) used for the reaction may be used at a
molar ratio affording ester compound (3) (e.g., capsinoid, etc.) in
the highest yield. Those of ordinary skill in the art can determine
the ratio of fatty acid (1) and hydroxymethylphenol (2)
corresponding to the object ester compound (3) by a simple
preliminary test. For example, the ratio of fatty acid (1):vanillyl
alcohol can be appropriately selected from the range of 0.8:1 to
1.2:1, most desirably the range of 1:1 to 1.1:1. Under such a
reaction condition, ester compound (3) (i.e., capsinoid) containing
a by-product at such a low level that obviates purification by
chromatography can be produced by the use of fatty acid in a small
excess. It is of course possible to further add one of the starting
materials while monitoring the progress of the reaction.
[0133] As the reaction temperature, a temperature at which the
enzyme to be used most efficiently catalyzes the reaction can be
selected, and those of ordinary skill in the art can set the
temperature by a simple preliminary test. Since the optimal
temperature varies depending on the enzyme to be used, it cannot be
completely said but the temperature is generally 15.degree. C. to
90.degree. C., more desirably 35.degree. C. to 65.degree. C. For
example, when Novozym 435 or lipase PS "Amano" is used as lipase,
the reaction is accelerated by heating to about 50.degree. C. It is
also desirable to heat to about 50.degree. C. to promote separation
of water and sufficiently melt the fatty acid.
[0134] The reaction time is appropriately determined in
consideration of the activity of the enzyme to be used, the amount
of starting materials, concentration of each reagent, and the like,
and in view of the yield and the like. It is generally 3 to 90
hours, preferably 10 to 30 hours.
[0135] After the completion of the reaction, ester compound (3) can
be separated according to the conventional method. For example, an
organic solvent (e.g., hexane, heptane, etc. when
hydroxymethylphenol (2) is vanillyl alcohol), in which
hydroxymethylphenol (2) is insoluble, is added to allow
precipitation of unreacted hydroxymethylphenol (2), thereby
filtrating hydroxymethylphenol (2) and the enzyme. And then, for
example, 5 to 10% aqueous citric acid solution is added to
partition the filtrate, and the organic layer is concentrated under
reduced pressure to give ester compound (3) (ester compound (3)
having a purity of not less than 99 area % by HPLC analysis can be
obtained in a high yield of not less than 90%). To obtain ester
compound (3) having a still higher purity, separation and
purification can be performed by silica gel column
chromatography.
[0136] When the enzyme is to be reused, the enzyme alone needs to
be filtrated. When the enzyme is contaminated with
hydroxymethylphenol (2) at that time, the mixture can be used for
the next reaction. It is possible to remove hydroxymethylphenol (2)
alone by dissolving it in an organic solvent and the enzyme alone
can be used for the next reaction.
[0137] The obtained ester compound (3) can be stabilized by the
coexistence with fatty acid (4).
[0138] When ester compound (3) is separated and purified by column
chromatography, fatty acid (1) present in excess in the reaction
mixture has an Rf value similar to that of ester compound (3);
therefore, the separation and purification of ester compound (3) is
associated with difficulty. The present inventors tried separation
and purification of fatty acid (1) remaining in the reaction
mixture when added in excess and ester compound (3) by column
chromatography, and the obtained pure ester compound (3) was found
to be easily decomposed. For example, when vanillyl decanoate was
synthesized from decanoic acid and vanillyl alcohol, separated and
purified from decanoic acid by silica gel column chromatography to
give pure vanillyl decanoate, which was dissolved in acetonitrile
and analyzed by HPLC. As a result, the purity of vanillyl decanoate
was 95.6 area %. However, when the sample was reanalyzed 62 hours
later, the purity decreased to 82.0 area %. This result means that
vanillyl decanoate decomposed. Vanillyl decanoate is considered to
have become unstable upon separation from decanoic acid. Therefore,
fatty acid (1), difficult to be separated from ester compound (3),
is preferably left coexisted rather than being separated, from the
aspect of stability of ester compound (3).
[0139] Capsinoid obtained by extraction from a plant containing
capsinoid is known to be comparatively stable in an oil base used
for extraction, but a method for stabilizing capsinoid obtained by
synthesis has not been known.
[0140] The present inventors have found that ester compound (3)
obtained by preparative separation together with fatty acid, rather
than separation without fatty acid, when purifying ester compound
(3) by silica gel chromatography, is stable, namely, the
coexistence of the fatty acid contributes to stabilize ester
compound (3), and completed the stabilizing method of the present
invention. For example, dihydrocapsiate was synthesized using a
small excess of fatty acid, and dihydrocapsiate obtained by
preparatively separating together with the excess fatty acid
remaining in about 2 wt % relative to dihydrocapsiate was analyzed
by HPLC. As a result, the purity was not less than 99 area %, and
it was found that dihydrocapsiate could be stably preserved in
hexane at 5.degree. C. for at least 30 days without
decomposition.
[0141] Therefore, ester compound (3) can be obtained in a stable
state by using fatty acid (1) in more excess than
hydroxymethylphenol (2) for the condensation reaction and, during
the purification step after condensation, preparatively separating
ester compound (3) as a mixture with fatty acid (1) contained in
the reaction mixture.
[0142] Alternatively, ester compound (3) can be obtained in a
stable state by condensing fatty acid (1) and hydroxymethylphenol
(2), adding fatty acid (4) thereto, and, during the purification
step, preparatively separating ester compound (3) as a mixture with
fatty acid (4). The fatty acid (4) can be added after condensation
of fatty acid (1) and hydroxymethylphenol (2) and before the
purification step.
[0143] As a method for obtaining fatty acid (4), a synthesis method
based on the above-mentioned cross coupling method and a
purification method based on distillation or crystallization of the
fatty acid salt are preferable.
[0144] The method of preparative separation is not particularly
limited as long as a mixture of ester compound (3) and the fatty
acid (fatty acid (1) or fatty acid (4)) can be obtained by
separation from other component and, for example, silica gel
chromatography using silica gel as a stationary phase can be
carried out.
[0145] In one example, when preparative separation by silica gel
chromatography is carried out, the conditions thereof include a
column packed with 10 g of silica gel per 1 g of a crude product
and a mixed solvent of diethyl ether:hexane=15:85 as an eluent,
whereby ester compound (3) and the fatty acid are eluted almost
simultaneously. The eluted fractions are collected and concentrated
under reduced pressure to give a mixture of ester compound (3) and
a small amount of the fatty acid.
[0146] By preparative separation of ester compound (3) as a mixture
with the fatty acid in this manner, the amount of the silica gel to
be used for purification can be small, and the obtained ester
compound (3) can have higher stability as compared to isolation of
ester compound (3) alone.
[0147] When ester compound (3) is individually isolated or
preparatively separated as a mixture with the fatty acid in an
amount insufficient for stabilization (in these cases, ester
compound (3) may be obtained by a production method other than the
method of the present invention), ester compound (3) can be
stabilized by adding fatty acid (4) to ester compound (3).
[0148] For example, when 9.1 wt % of decanoic acid was added, in
acetonitrile, to pure vanillyl decanoate separated from decanoic
acid, the purity of vanillyl decanoate was 97.6 area % even after
19.5 hours and the purity was maintained.
[0149] Vanillyl decanoate is considered to be, for example, in the
following equilibrium state.
##STR00016##
[0150] As mentioned above, the present inventors have found that
ester compound (3) is extremely stable when a small excess amount
of fatty acid is coexistent, but ester compound (3) shows lower
purity over time once separated from fatty acid. This is considered
to be attributable to the fact that quinonemethide produced by the
decomposition of ester compound (3) sequentially reacts not only
with fatty acid but also with a phenolic hydroxyl group of vanillyl
decanoate as mentioned above. It is considered, therefore, that,
due to the coexistence with fatty acid, the equilibrium shifts
toward the ester compound (3) production side, decomposition of
ester compound (3) is prevented and stabilization can be
realized.
[0151] The present inventors have also found that further addition
of a small excess of the corresponding fatty acid (4) to ester
compound (3) partly decomposed by separation from fatty acid and
the like prevents decomposition of ester compound (3) and leads to
stabilization thereof, which in turn increases (recovers) purity.
This is considered to be attributable to the addition of the
corresponding fatty acid (4), which results in the equilibrium
between the ester compound (3) and the decomposed products having
shifted toward the ester compound (3) production side, due to the
same mechanism as mentioned above.
[0152] While fatty acid (4) to be added is appropriately selected
depending on the use of the composition containing ester compound
(3) and fatty acid (4), R1' of fatty acid (4) is the same group as
R1 of ester compound (3), particularly fatty acid (1), is most
preferable.
[0153] The fatty acid only needs to be present in an amount within
the range of 0.1 wt % to 30 wt %, preferably 1 wt % to 5 wt %,
relative to the weight of ester compound (3). When an excess amount
of fatty acid (1) is used for condensation, therefore, the amount
of fatty acid (1) to be used should be controlled such that the
excess fatty acid is contained in the reaction mixture in the
above-mentioned range. When fatty acid (4) is added after
condensation, and when fatty acid (4) is added after isolation of
ester compound (3), both for stabilization, the fatty acid is
preferably added such that it is present within the above-mentioned
range.
[0154] The composition of the present invention comprises ester
compound (3) and fatty acid (11). This composition is a composition
artificially obtained, for example, by the above-mentioned method,
rather than an extract of fats and oils obtained from plants, and
has physiological activities such as the suppression of obesity,
promotion of energy metabolism, and the like and can be used as
food additives and pharmaceutical products.
[0155] The fatty acid (11) is a component that contaminates ester
compound (3) and, for example, derived from fatty acid (1)
remaining due to addition in an excess amount than
hydroxymethylphenol (2) in the above-mentioned production method,
separately added fatty acid (4) and the like.
[0156] In this composition, fatty acid (11) is preferably contained
within the range of 0.1 wt % to 30 wt %, more preferably 1 wt % to
5 wt %, of ester compound (3).
[0157] This composition may further contain one or more kinds of
additives selected from the group consisting of compositions of
fats and oils, emulsifiers, preservatives, and antioxidants. It is
needless to say that when these additives are contained, too, the
coexistence of fatty acid (11) is effective for the stabilization
of ester compound (3).
[0158] As the composition of fats and oils, for example,
medium-chain triglycerides, vegetable fats and oils such as canola
oil and the like, animal fats and oils such as fish oil and the
like, and the like can be mentioned.
[0159] As the emulsifier, for example, glycerine fatty acid esters,
sucrose fatty acid esters, sorbitan fatty acid esters, and the like
can be mentioned.
[0160] As the preservative, for example, udo extract, extract of
Japanese styrax benzoin, rumput roman extract, and the like can be
mentioned.
[0161] As the antioxidant, for example, vitamin E, vitamin C,
lecithin, rosemary extract, and the like can be mentioned.
[0162] The composition of the present invention containing ester
compound (3) and fatty acid (11) can be stably preserved for a
long-term without decomposition and is extremely useful, because it
permits long-term stable preservation in the form of a high
concentration bulk which can be used for preparing a supplement or
external agent of an ester compound obtained by synthesis.
[0163] Other features of the invention will become apparent in the
course of the following descriptions of exemplary embodiments which
are given for illustration of the invention and are not intended to
be limiting thereof.
EXAMPLES
[0164] In the following Examples, the structures of the synthesized
compounds were identified by nuclear magnetic resonance spectrum
(Bruker AVANCE400 (400 MHz)). GC-MS was measured using
5890SERIESII, 5972SERIES, 7673CONTROLLER, all HEWLETT PACKARD. The
free fatty acid content was calculated from the peak integral value
of nuclear magnetic resonance spectrum, or analyzed using a fatty
acid analysis kit (YMC).
[0165] The HPLC measurement conditions of capsinoid are as follows.
HPLC conditions:
[0166] column: Inertsil C8 3 u .mu.m (diameter 4.0 mm.times.100
mm)
[0167] eluent: A mixed solvent of eluents A, B shown below and a
buffer was eluted by gradient elution method.
[0168] buffer: 30 mM KH.sub.2PO.sub.4 (pH=2.0, H.sub.3PO.sub.4)
[0169] eluent A: CH.sub.3CN:buffer=80:20
[0170] eluent B: CH.sub.3CN:buffer=0:100
[0171] gradient conditions: 0 minute: A/B=(20/80); 15 minutes:
A/B=(70/30); 30 minutes: A/B=(100/0); 45 minutes: A/B=(100/0); 45.1
minutes: A/B=(20/80); 50 minutes: A/B=(20/80)
[0172] detection: UV210 nm
[0173] temperature: room temperature
Example 1
Synthesis of 8-Methylnonanoic Acid (Example of Cross Coupling
Method)
[0174] Under an argon atmosphere, Mg turnings (6.12 g, 252 mmol)
were suspended in THF (10 ml). 200 mg from isopentyl bromide (34.6
g, 229 mmol) was added at room temperature, and exothermic heat and
foaming were confirmed. THF (50 ml) was added, and a solution of
the entire remainder of the isopentyl bromide in THF (65 ml) was
slowly added dropwise at room temperature over 1 hour, and the
mixture was stirred for 2 hours. At this time, mild refluxing state
was achieved. The reaction solution was filtered through cotton
plug while washing with THF to give a solution (total amount 180
ml) of isopentylmagnesium bromide in THF.
[0175] Under an argon atmosphere, copper (I) chloride (426 mg, 4.30
mmol) was dissolved in NMP (55.2 ml, 575 mmol). The reaction vessel
was cooled to 0.degree. C. (ice bath), and a solution of ethyl
5-bromovalerate (30.0 g, 144 mmol) in THF (35 ml) was added
dropwise over 10 minutes. The THF solution of isopentylmagnesium
bromide prepared in advance was slowly added dropwise at 0.degree.
C. (ice bath) over 1.5 hours. After further stirring at the same
temperature for 45 minutes, the reaction was carefully quenched
with saturated aqueous ammonium chloride solution (200 ml), and the
mixture was extracted twice with heptane (200 ml). The combined
heptane layer was washed with saturated aqueous ammonium chloride
solution (100 ml), water (100 ml) and saturated brine (100 ml),
dried over anhydrous magnesium sulfate, filtered and concentrated
under reduced pressure to give a pale-yellow oil (30.8 g). 29.6 G
therefrom was distilled under reduced pressure (1.2 mmHg,
69-71.degree. C.) to give ethyl 8-methylnonanoate (20.6 g, yield
74.7%) as a colorless transparent oil.
[0176] .sup.1H-NMR (CDCl.sub.3, .delta.): 0.860 (d, 6H, J=6.63 Hz),
1.13-1.33 (m, 11H), 1.48-1.64 (m, 3H), 2.28 (t, 2H, J=7.55 Hz),
4.12 (q, 2H, J=7.13 Hz).
[0177] .sup.13C-NMR (CDCl.sub.3, .delta.): 14.60, 22.98, 25.36,
27.56, 28.30, 29.54, 29.89, 34.75, 39.31, 60.47, 174.2.
[0178] From the obtained ethyl 8-methylnonanoate, 19.20 g was
dissolved in ethanol (72.0 ml) and 2M NaOH aqueous solution (72.0
ml) was slowly added at 0.degree. C. (ice bath). The mixture was
heated with stirring using an oil bath at 60.degree. C. for 1 hour,
the reaction vessel was returned to room temperature and ethanol
was evaporated under reduced pressure. 2M NaOH (30 ml) and water
(30 ml) were added to the solution, and the solution was washed
with tert-butyl methyl ether (100 ml). The aqueous layer was washed
with tert-butyl methyl ether (100 ml) once again. The aqueous layer
was carefully acidified with 2M HCl aqueous solution (150 ml), and
the mixture was extracted twice with heptane (150 ml). The combined
heptane layer was washed with water (100 ml) and then saturated
brine (100 ml), dried over anhydrous magnesium sulfate, filtered
and concentrated under reduced pressure to give 8-methylnonanoic
acid (15.9 g, crude yield 96.6%) as a crude product of a
pale-yellow oil. As a result of the GCMS analysis, it contained
structurally unidentified impurities A (0.01%), B (0.03%), C
(0.04%), and D (0.07%), and the purity of 8-methylnonanoic acid was
99.6%.
[0179] .sup.1H-NMR (CDCl.sub.3, .delta.): 0.862 (d, 6H, J=6.64 Hz),
1.14-1.17 (m, 2H), 1.26-1.35 (m, 6H), 1.48-1.65 (m, 3H), 2.35 (t,
2H, J=7.52 Hz).
[0180] .sup.13C-NMR (CDCl.sub.3, .delta.): 22.95, 25.04, 27.55,
28.12, 29.47, 29.88, 34.51, 39.31, 181.0. GC-MS: M=172.
Example 2
Purification of 8-Methylnonanoic Acid by Formation of
Cyclohexylamine Salt Thereof (Example of Purification by Fatty Acid
Salt Crystal)
[0181] From the 8-methylnonanoic acid crude product obtained in
Example 1, 8.00 g was dissolved in heptane (30 ml). Cyclohexylamine
(6.91 ml, 60.4 mmol) was slowly added dropwise at 0.degree. C. (ice
bath), and the mixture was stirred at room temperature for 20
minutes. The reaction mixture was filtered to give 8-methylnonanoic
acid cyclohexylamine salt (15.7 g).
[0182] .sup.1H-NMR (CDCl.sub.3, .delta.): 0.81-0.85 (m, 6H),
1.11-1.20 (m, 3H), 1.24-1.35 (m, 10H), 1.46-1.68 (m, 4H), 1.73-1.81
(m, 2H), 1.96-2.02 (m, 2H), 2.15-2.19 (t, 2H), 2.77-2.88 (m, 1H).
melting point: 70.1 to 70.6.degree. C.
[0183] 10% Aqueous citric acid solution (50 ml) and heptane (50 ml)
were added to the salt (15.6 g therefrom) to allow partitioning.
The aqueous layer was extracted with heptane (50 ml), and the
combined heptane layer was washed with 10% aqueous citric acid
solution (50 ml), water (50 ml) and saturated brine (50 ml). The
heptane layer was dried over anhydrous magnesium sulfate and
filtrated, and the filtrate was concentrated under reduced pressure
to give 8-methylnonanoic acid (7.69 g) as a colorless transparent
oil.
[0184] 7.18 g therefrom was distilled under reduced pressure (1.1
mmHg, 103.degree. C.) to give 8-methylnonanoic acid distillation
product (6.80 g, yield from 8-methylnonanoic acid crude product,
91.0%). As a result of the GCMS analysis, the aforementioned
impurities A, B, C, and D were below detection limit, and the
purity of 8-methylnonanoic acid was 99.7%.
Example 3
Resolution of Trans Form And Cis Form of 8-Methyl-6-Nonenoic Acid
by Cis-2-Aminocyclohexanol Salt Thereof (Example of Purification
Method by Formation of Fatty Acid Salt Crystal)
[0185] 8-Methyl-6-nonenoic acid (isomer ratio trans:cis=88:12, 800
mg, 4.70 mmol) obtained by a known method (J. Org. Chem., 1989, 54,
3477-3478) was dissolved in chloroform (10 ml), and a solution of
cis-2-aminocyclohexanol (460 mg, 4.00 mmol) in chloroform (5 ml)
was added dropwise at room temperature. The reaction mixture was
concentrated under reduced pressure, the residue was again
dissolved in chloroform (4 ml), and hexane (12 ml) was added
dropwise. The reaction mixture was stirred at room temperature for
3 days, and the precipitated crystals were collected by filtration.
Hexane (10 ml) was added to the obtained crystals, and the mixture
was washed three times with 10% aqueous citric acid solution (8 ml)
and once with saturated brine (10 ml), and dried over anhydrous
magnesium sulfate. Magnesium sulfate was removed by filtration, and
the filtrate was concentrated under reduced pressure to give
8-methyl-6-nonenoic acid (isomer ratio trans:cis=29:1, 408 mg, 2.40
mmol).
[0186] The obtained 8-methyl-6-nonenoic acid (isomer ratio
trans:cis=29:1, 408 mg, 2.40 mmol) was again dissolved in
chloroform (10 ml), and a solution of cis-2-aminocyclohexanol (249
mg, 2.16 mmol) in chloroform (5 ml) was added dropwise at room
temperature. The reaction mixture was concentrated under reduced
pressure, the residue was again dissolved in chloroform (3 ml), and
hexane (12 ml) was added dropwise. The reaction mixture was stirred
overnight at room temperature, and the precipitated crystals were
collected by filtration. Hexane (15 ml) was added to the obtained
crystal, and the mixture was washed three times with 10% aqueous
citric acid solution (10 ml) and once with saturated brine (10 ml),
and dried over anhydrous magnesium sulfate. Magnesium sulfate was
removed by filtration, and the filtrate was concentrated under
reduced pressure to give trans-8-methyl-6-nonenoic acid (250 mg,
1.47 mmol, purity 98.8%, yield 35.1%).
[0187] .sup.1H-NMR (CDCl.sub.3, .delta.): 0.96 (d, 6H, J=6.8 Hz),
1.38-1.46 (m, 2H), 1.60-1.70 (m, 2H), 1.95-2.05 (m, 2H), 2.18-2.38
(m, 1H), 2.35 (t, 2H, J=7.4 Hz), 5.28-5.42 (m, 2H).
Example 4
Synthesis of 8-Methylnonanoic Acid (Method to Synthesize at High
Purity Using CuBr as Catalyst)
[0188] A 500 ml three-neck flask equipped with a thermometer was
filled with argon, and CuBr (481 mg, 3.36 mmol) was added. NMP
(43.1 ml, 449 mmol) was added and dissolved at room temperature,
and the reaction vessel was cooled to -20.degree. C. THF (10 ml)
was added and ethyl 6-bromo-n-hexanoate (25.0 g, 112 mmol) was
added dropwise (inside temperature -8.degree. C.). After stirring
for 10 minutes, a solution (160 ml) of isobutylmagnesium bromide in
THF separately prepared was slowly added dropwise over 60
minutes.
[0189] At 90 minutes after completion of the dropwise addition, 10%
aqueous ammonium chloride solution (120 ml) was slowly added
dropwise to quench the reaction, and the mixture was extracted with
n-hexane (120 ml). The n-hexane layer was washed with 10% aqueous
ammonium chloride solution (100 ml), water (100 ml) and saturated
brine (50 ml), dried over anhydrous magnesium sulfate and
filtrated, and the filtrate was concentrated under reduced pressure
to give a crude product 24.2 g of ethyl 8-methylnonanoate as a
pale-yellow oil. The purity measured by GC-MS was 97.5%.
[0190] From the obtained ethyl 8-methylnonanoate, 22.2 g was placed
in a 500 ml eggplant-type flask, and dissolved in ethanol (77 ml).
2M NaOH aqueous solution (77 ml, 154 mmol) was added dropwise at
room temperature over 5 minutes. After the completion of the
dropwise addition, the mixture was heated with stirring in an oil
bath at 60.degree. C. for 90 minutes. After confirmation of
disappearance of the starting material by TLC, the mixture was
cooled to room temperature.
[0191] Ethanol was evaporated under reduced pressure. Water (40 ml)
was added to the solution, and the solution was washed with t-butyl
methyl ether (80 ml). The aqueous layer was further washed with
t-butyl methyl ether (80 ml). Then the aqueous layer was acidified
with 2M aqueous HCl solution (120 ml), and the mixture was
extracted with n-hexane (80 ml). The n-hexane layer was washed with
water (80 ml), water (40 ml), and saturated brine (40 ml), dried
over anhydrous magnesium sulfate and filtered, and the filtrate was
concentrated under reduced pressure to give 17.3 g of
8-methylnonanoic acid as a pale-yellow oil. 15.3 g therefrom was
distilled under reduced pressure to give 12.7 g of 8-methylnonanoic
acid as a pale-yellow oil. The purity measured by GC-MS was not
less than 99.9%. Total yield from ethyl 6-bromo-n-hexanenoate,
81%.
Example 5
Synthesis of Dihydrocapsiate--1
[0192] 8-Methylnonanoic acid (1.00 g, 5.80 mmol), vanillyl alcohol
(851 mg, 5.52 mmol), and Novozym 435 (50 mg) were measured and
placed in a flask (25 ml). The mixture in the flask free of a plug
was heated with stirring in an oil bath at 50.degree. C. for 20
hours. After 2 to 3 hours of stirring with heating, attachment of
water on the wall of the upper part of the flask was observed. The
reaction mixture was returned to room temperature, hexane (25 ml)
was added, and Novozym 435 and a small amount of precipitated
vanillyl alcohol were removed by filtration. Hexane (25 ml) was
added to the filtrate, and the mixture was washed with 5% aqueous
citric acid solution (25 ml) and saturated brine (25 ml), and dried
over anhydrous magnesium sulfate. Magnesium sulfate was removed by
filtration, and the filtrate was concentrated under reduced
pressure to give a mixture (1.66 g) of dihydrocapsiate and
8-methylnonanoic acid as a colorless oil. As a result of the
analysis, the yield of dihydrocapsiate was 89.7%, and the purity
was 99.5 area % by HPLC. The mixture contained 8.0 wt % of
8-methylnonanoic acid relative to dihydrocapsiate.
[0193] .sup.1H-NMR (CDCl.sub.3, .delta.): 0.86 (d, 6H, J=6.60 Hz),
1.12-1.37 (m, 8H), 1.46-1.64 (m, 3H), 2.32 (t, 2H, J=7.56 Hz), 3.89
(s, 3H), 5.02 (s, 2H), 5.63 (br, 1H), 6.83-6.90 (m, 3H).
Example 6
Synthesis of Capsiate
[0194] trans-8-Methyl-6-nonenoic acid (1.00 g, 5.87 mmol), vanillyl
alcohol (1.085 g, 7.04 mmol), and Novozym 435 (100 mg) were
measured and placed in a flask (25 ml). The mixture in the flask
free of a plug was heated with stirring in an oil bath at
50.degree. C. for 16 hours. After 2 to 3 hours of stirring with
heating, attachment of water on the wall of the upper part of the
flask was observed. The reaction mixture was returned to room
temperature, hexane (25 ml) was added, and Novozym 435 and
precipitated vanillyl alcohol were removed by filtration. Hexane
(25 ml) was added to the filtrate, and the mixture was washed with
5% aqueous citric acid solution (25 ml) and saturated brine (25
ml), and dried over anhydrous magnesium sulfate. Magnesium sulfate
was removed by filtration, and the filtrate was concentrated under
reduced pressure. Since production of polar impurity other than
vanillyl alcohol was confirmed by TLC, the residue was dissolved in
50 ml of hexane and passed through a short column packed with 1.5 g
of silica gel, and the silica gel was sufficiently washed away with
a mixed solvent of hexane and ethyl acetate (volume ratio 10:1).
The above-mentioned impurity was not detected in the eluent by TLC.
The eluent was concentrated under reduced pressure to give capsiate
(1.56 g, yield 86.6%) as a colorless oil. This capsiate contained a
trace amount of trans-8-methyl-6-nonenoic acid.
[0195] .sup.1H-NMR (CDCl.sub.3, .delta.): 0.95 (d, 6H, J=6.74 Hz),
1.33-1.40 (m, 2H), 1.59-1.67 (m, 2H), 1.94-1.99 (m, 2H), 2.18-2.23
(m, 1H), 2.33 (t, 2H, J=7.52 Hz), 3.89 (s, 3H), 5.02 (s, 2H),
5.26-5.39 (m, 2H), 5.63 (br, 1H), 6.83-6.90 (m, 3H).
Example 7
Synthesis of Vanillyl Decanoate--1
[0196] Decanoic acid (1.00 g, 5.80 mmol), vanillyl alcohol (880 mg,
5.71 mmol), and Novozym 435 (25 mg) were measured and placed in a
flask (25 ml), and hexane (0.5 ml) was added. The mixture in the
flask free of a plug was heated with stirring in an oil bath at
50.degree. C. for 48 hours. After 2 to 3 hours of stirring with
heating, attachment of water on the wall of the upper part of the
flask was observed. The flask was returned to room temperature,
hexane (25 ml) was added to the reaction mixture, and Novozym 435
and a small amount of precipitated vanillyl alcohol were removed by
filtration. Hexane (25 ml) was added to the filtrate, and the
mixture was washed with 5% aqueous citric acid solution (25 ml) and
saturated brine (25 ml), and dried over anhydrous magnesium
sulfate. Magnesium sulfate was removed by filtration, and the
filtrate was concentrated under reduced pressure to give a mixture
(1.69 g) of vanillyl decanoate and decanoic acid as a colorless
oil. As a result of the analysis, the yield of vanillyl decanoate
was 93.1%. The mixture contained 2.9 wt % of decanoic acid relative
to vanillyl decanoate.
[0197] .sup.1H-NMR (CDCl.sub.3, .delta.): 0.87 (t, 3H, J=7.1 Hz),
1.18-1.30 (m, 12H), 1.55-1.65 (m, 2H), 2.33 (t, 2H, J=7.7 Hz), 3.90
(s, 3H), 5.03 (s, 2H), 5.64 (br, 1H), 6.80-6.90 (m, 3H).
Example 8
Synthesis of Vanillyl Decanoate--2 (Repeated Use of Enzyme)
[0198] Decanoic acid (2.00 g, 11.61 mmol), vanillyl alcohol (1.74
g, 11.27 mmol), and Novozym 435 (100 mg) were measured and placed
in a flask (25 ml). The mixture in the flask free of a plug was
heated with stirring in an oil bath at 50.degree. C. for 20 hours.
After 2 to 3 hours of stirring with heating, attachment of water on
the wall of the upper part of the flask was observed. The reaction
mixture was returned to room temperature, hexane (50 ml) was added,
and Novozym 435 and a small amount of precipitated vanillyl alcohol
were removed by filtration. The filtrate was washed with 5% aqueous
citric acid solution (25 ml) and saturated brine (25 ml), and dried
over anhydrous magnesium sulfate. Magnesium sulfate was removed by
filtration, and the filtrate was concentrated under reduced
pressure to give a mixture (3.41 g) of vanillyl decanoate and
decanoic acid as a colorless oil. As a result of the analysis, the
yield of vanillyl decanoate was 94.1%. The mixture contained 6.0 wt
% of decanoic acid relative to vanillyl decanoate.
[0199] The above-mentioned operation was repeated using, as a
catalyst, a mixture recovered by the above-mentioned operation,
which contained Novozym 435 and a small amount of vanillyl alcohol.
A mixture (3.42 g) of vanillyl decanoate and decanoic acid was
obtained as a colorless oil. As a result of the analysis, the yield
of vanillyl decanoate was 95.5%. The mixture contained 3.2 wt % of
decanoic acid relative to vanillyl decanoate.
[0200] The above-mentioned operation was repeated using, as a
catalyst, a mixture recovered by the above-mentioned operation,
which contained Novozym 435 and a small amount of vanillyl alcohol.
A mixture (3.47 g) of vanillyl decanoate and decanoic acid was
obtained as a colorless oil. As a result of the analysis, the yield
of vanillyl decanoate was 94.8%. The mixture contained 5.1 wt % of
decanoic acid relative to vanillyl decanoate.
[0201] The above-mentioned operation was repeated using, as a
catalyst, a mixture recovered by the above-mentioned operation,
which contained Novozym 435 and a small amount of vanillyl alcohol.
A mixture (3.46 g) of vanillyl decanoate and decanoic acid was
obtained as a colorless oil. As a result of the analysis, the yield
of vanillyl decanoate was 95.4%. The mixture contained 4.1 wt % of
decanoic acid relative to vanillyl decanoate.
Example 9
Synthesis of Dihydrocapsiate--2
[0202] 8-Methylnonanoic acid (1.50 g, 8.70 mmol), vanillyl alcohol
(1.34 g, 8.70 mmol), and lipase PS "Amano" (375 mg) were measured
and placed in a flask (25 ml). The mixture in the flask free of a
plug was heated with stirring in an oil bath at 55.degree. C. for
45 hours. After 2 to 3 hours of stirring with heating, attachment
of water on the wall of the upper part of the flask was observed.
The flask was returned to room temperature, heptane (10 ml) was
added to the reaction mixture, and the mixture was stirred for 10
minutes. Lipase PS "Amano" and a small amount of precipitated
vanillyl alcohol were removed by filtration. The filtrate was
concentrated under reduced pressure and the obtained oil (2.48 g)
was analyzed by HPLC to find that dihydrocapsiate was contained in
94.0 area %. The mixture was partitioned with heptane (15 ml) and
10% aqueous citric acid solution (15 ml), and the aqueous layer was
further extracted with heptane (15 ml). The combined heptane layer
was washed with saturated brine (15 ml) and dried over anhydrous
magnesium sulfate. Magnesium sulfate was removed by filtration, and
the filtrate was concentrated under reduced pressure to give a
mixture (2.45 g) of dihydrocapsiate and 8-methylnonanoic acid as a
colorless oil. As a result of the analysis, the yield of
dihydrocapsiate was 80.9%, and the purity was 97.4 area % by HPLC.
The mixture contained 12.6 wt % of 8-methylnonanoic acid relative
to dihydrocapsiate.
Example 10
Synthesis of Dihydrocapsiate--3
[0203] 8-Methylnonanoic acid (1.50 g, 8.70 mmol), vanillyl alcohol
(1.34 g, 8.70 mmol), and lipase PS-C "Amano" I (enzyme immobilized
on ceramic: 375 mg) were measured and placed in a flask (25 ml).
The mixture in the flask free of a plug was heated with stirring in
an oil bath at 55.degree. C. for 45 hours. After 2 to 3 hours of
stirring with heating, attachment of water on the wall of the upper
part of the flask was observed. The flask was returned to room
temperature, heptane (10 ml) was added to the reaction mixture, and
the mixture was stirred for 10 minutes. The immobilized enzyme and
a small amount of precipitated vanillyl alcohol were removed by
filtration. The filtrate was concentrated under reduced pressure,
and the obtained oil (2.68 g) was analyzed by HPLC to find that
dihydrocapsiate was contained in 92.9 area %. The mixture was
partitioned with heptane (15 ml) and 10% aqueous citric acid
solution (15 ml), and the aqueous layer was further extracted with
heptane (15 ml). The combined heptane layer was washed with
saturated brine (15 ml) and dried over anhydrous magnesium sulfate.
Magnesium sulfate was removed by filtration, and the filtrate was
concentrated under reduced pressure to give a mixture (2.61 g) of
dihydrocapsiate and 8-methylnonanoic acid as a colorless oil. As a
result of the analysis, the yield of dihydrocapsiate was 95.5%, and
the purity was 97.1 area % by HPLC. The mixture contained 1.97 wt %
of 8-methylnonanoic acid relative to dihydrocapsiate.
Example 11
Synthesis of Dihydrocapsiate--4
[0204] 8-Methylnonanoic acid (1.65 g, 9.59 mmol), vanillyl alcohol
(1.34 g, 8.70 mmol), and lipase PS-C "Amano" I (enzyme immobilized
on ceramic: 335 mg) were measured and placed in a flask (25 ml).
The mixture in the flask free of a plug was heated with stirring in
an oil bath at 45.degree. C. for 37.5 hours. After 2 to 3 hours of
stirring with heating, attachment of water on the wall of the upper
part of the flask was observed. The flask was returned to room
temperature, heptane (10 ml) was added to the reaction mixture, and
the mixture was stirred for 10 minutes. The immobilized enzyme and
a small amount of precipitated vanillyl alcohol were removed by
filtration. The filtrate was concentrated under reduced pressure,
and the obtained oil was analyzed by HPLC to find that
dihydrocapsiate was contained in 95.7 area %. The mixture was
partitioned with heptane (20 ml) and 10% aqueous citric acid
solution (20 ml), and the aqueous layer was further extracted with
heptane (20 ml). The combined heptane layer was washed with
saturated brine (15 ml) and dried over anhydrous magnesium sulfate.
Magnesium sulfate was removed by filtration, and the filtrate was
concentrated under reduced pressure to give a mixture (2.50 g) of
dihydrocapsiate and 8-methylnonanoic acid as a colorless oil. As a
result of the analysis, the yield of dihydrocapsiate was 73.1%, and
the purity was 99.3 area % by HPLC. The mixture contained 27.4 wt %
of 8-methylnonanoic acid relative to dihydrocapsiate.
Example 12
Synthesis of Dihydrocapsiate--5
[0205] 8-Methylnonanoic acid (1.54 g, 8.95 mmol) and vanillyl
alcohol (1.34 g, 8.70 mmol) were measured and placed in a flask (25
ml) and dissolved in heptane (0.5 ml). Lipase PS-C "Amano" I
(enzyme immobilized on ceramic: 335 mg) was added and the mixture
was heated with stirring in an oil bath at 55.degree. C. for 13.5
hours. After 2 to 3 hours of stirring with heating, attachment of
water on the wall of the upper part of the flask was observed. The
flask was returned to room temperature, heptane (5 ml) was added to
the reaction mixture, and the mixture was stirred for 10 minutes.
The immobilized enzyme and a small amount of precipitated vanillyl
alcohol were removed by filtration. The filtrate was concentrated
under reduced pressure, and the obtained oil (2.42 g) was analyzed
by HPLC to find that dihydrocapsiate was contained in 97.2 area %.
The mixture was partitioned with heptane (15 ml) and 10% aqueous
citric acid solution (15 ml), and the aqueous layer was further
extracted with heptane (15 ml). The combined heptane layer was
washed with water (10 ml) and saturated brine (10 ml) and dried
over anhydrous magnesium sulfate. Magnesium sulfate was removed by
filtration, and the filtrate was concentrated under reduced
pressure to give a mixture (2.42 g) of dihydrocapsiate and
8-methylnonanoic acid as a colorless oil. As a result of the
analysis, the yield of dihydrocapsiate was 72.3%, and the purity
was 99.6 area % by HPLC. The mixture contained 24.8 wt % of
8-methylnonanoic acid relative to dihydrocapsiate.
Example 13
Synthesis of Dihydrocapsiate--6
[0206] 8-Methylnonanoic acid (1.54 g, 8.95 mmol), vanillyl alcohol
(1.34 g, 8.70 mmol), and lipase PS-C "Amano" I (enzyme immobilized
on ceramic: 335 mg) were measured and placed in a flask (25 ml).
The mixture in the flask free of a plug was heated with stirring in
an oil bath at 55.degree. C. for 13.5 hours. After 2 to 3 hours of
stirring with heating, attachment of water on the wall of the upper
part of the flask was observed. The flask was returned to room
temperature, heptane (5 ml) was added to the reaction mixture, and
the mixture was stirred for 15 minutes. The immobilized enzyme and
a small amount of precipitated vanillyl alcohol were removed by
filtration. The filtrate was concentrated under reduced pressure,
and the obtained oil (2.73 g) was analyzed by HPLC to find that
dihydrocapsiate was contained in 96.3 area %. The mixture was
partitioned with heptane (15 ml) and 10% aqueous citric acid
solution (15 ml), and the aqueous layer was further extracted with
heptane (15 ml). The combined heptane layer was washed with water
(10 ml) and saturated brine (10 ml) and dried over anhydrous
magnesium sulfate. Magnesium sulfate was removed by filtration, and
the filtrate was concentrated under reduced pressure to give a
mixture (2.67 g) of dihydrocapsiate and 8-methylnonanoic acid as a
colorless oil. As a result of the analysis, the yield of
dihydrocapsiate was 95.5%, and the purity was 99.3 area % by HPLC.
The mixture contained 4.18 wt % of 8-methylnonanoic acid relative
to dihydrocapsiate.
Example 14
Synthesis of Vanillyl Decanoate--3
[0207] Decanoic acid (25.0 g, 145 mmol), vanillyl alcohol (21.7 g,
141 mmol), and Novozym 435 (723 mg) were measured and placed in a
flask (25 ml). The mixture in the flask free of a plug was heated
with stirring in an oil bath at 50.degree. C. for 48 hours. After 2
to 3 hours of stirring with heating, attachment of water on the
wall of the upper part of the flask was observed. The flask was
returned to room temperature, hexane (100 ml) was added to the
reaction mixture, and the mixture was stirred for 1 hour. The
immobilized enzyme and a small amount of precipitated vanillyl
alcohol were removed by filtration. Hexane (100 ml) and 10% aqueous
citric acid solution (200 ml) was added to the filtrate to allow
partitioning. The aqueous layer was further extracted with hexane
(150 ml), and the combined hexane layer was washed with 10% aqueous
citric acid solution (100 ml), water (100 ml) and saturated brine
(100 ml). The hexane layer was dried over anhydrous magnesium
sulfate. Magnesium sulfate was removed by filtration, and the
filtrate was concentrated under reduced pressure to give the
mixture (43.7 g) of vanillyl decanoate and decanoic acid. As a
result of the analysis, the yield of vanillyl decanoate was 97.0%
and the purity was 98.6 area % by HPLC. The mixture contained 3.94
wt % of decanoic acid relative to vanillyl decanoate.
Example 15
Synthesis of Dihydrocapsiate--7
[0208] 8-Methylnonanoic acid (1.54 g, 8.95 mmol), vanillyl alcohol
(1.34 g, 8.70 mmol), and Novozym 435 (67.0 mg) were measured and
placed in a flask (25 ml). The mixture in the flask free of a plug
was heated with stirring in an oil bath at 55.degree. C. for 16
hours. After 2 to 3 hours of stirring with heating, attachment of
water on the wall of the upper part of the flask was observed. The
flask was returned to room temperature, heptane (5 ml) was added to
the reaction mixture, and the mixture was stirred for 10 minutes.
Novozym 435 and a small amount of precipitated vanillyl alcohol
were removed by filtration. The filtrate was concentrated under
reduced pressure, and the obtained colorless oil (2.74 g) was
analyzed by HPLC to find that dihydrocapsiate was contained in 96.0
area %. The mixture was partitioned with heptane (15 ml) and 10%
aqueous citric acid solution (15 ml), and the aqueous layer was
further extracted with heptane (15 ml). The combined heptane layer
was washed with water (10 ml) and saturated brine (10 ml) and dried
over anhydrous magnesium sulfate. Magnesium sulfate was removed by
filtration, and the filtrate was concentrated under reduced
pressure to give a mixture (2.65 g) of dihydrocapsiate and
8-methylnonanoic acid as a colorless oil. As a result of the
analysis, the yield of dihydrocapsiate was 97.6%, and the purity
was 99.8 area % by HPLC. The mixture contained 1.12 wt % of
8-methylnonanoic acid relative to dihydrocapsiate.
Example 16
Synthesis of Dihydrocapsiate--8
[0209] 8-Methylnonanoic acid (1.54 g, 8.95 mmol), vanillyl alcohol
(1.34 g, 8.70 mmol), and Novozym 435 (8.90 mg) were measured and
placed in a flask (25 ml). The mixture in the flask free of a plug
was heated with stirring in an oil bath at 55.degree. C. for 45
hours. After 2 to 3 hours of stirring with heating, attachment of
water on the wall of the upper part of the flask was observed. The
flask was returned to room temperature, heptane (10 ml) was added
to the reaction mixture, and the mixture was stirred for 30
minutes. Novozym 435 and a small amount of precipitated vanillyl
alcohol were removed by filtration. The filtrate was concentrated
under reduced pressure, and the obtained colorless oil (2.67 g) was
analyzed by HPLC to find that dihydrocapsiate was contained in 97.2
area %. The mixture was partitioned with heptane (15 ml) and 10%
aqueous citric acid solution (15 ml), and the aqueous layer was
further extracted with heptane (15 ml). The combined heptane layer
was washed with water (15 ml) and saturated brine (15 ml) and dried
over anhydrous magnesium sulfate. Magnesium sulfate was removed by
filtration, and the filtrate was concentrated under reduced
pressure to give a mixture (2.67 g) of dihydrocapsiate and
8-methylnonanoic acid as a colorless oil. As a result of the
analysis, the yield of dihydrocapsiate was 95.9%, and the purity
was 99.4 area % by HPLC. The mixture contained 3.86 wt % of
8-methylnonanoic acid relative to dihydrocapsiate.
Example 17
Synthesis of Dihydrocapsiate--9
[0210] 8-Methylnonanoic acid (310 g, 1.80 mol) and Novozym 435 (9.0
g) were placed in a 1 L four-neck flask. The mixture was heated
with stirring in an oil bath at 50.degree. C. Then vanillyl alcohol
(90 g, 0.58 mol) was added, and the mixture was stirred with
heating at the same temperature under reduced pressure (74 mmHg) by
a vacuum pump. A cold trap was included between the vacuum pump and
the flask. Vanillyl alcohol (90 g, 0.58 mol) was added 1 hour later
and 2 hour later each time, and the mixture was reacted with
heating under reduced pressure. The reduced pressure was stopped
after 45 hours from the start of the reaction, and the stirring
with heating was stopped. At this time, the trap contained water.
After confirmation that the reaction mixture returned to room
temperature, n-hexane (465 ml) was added dropwise over 1 hour, and
the mixture was stirred at atmospheric pressure and room
temperature.
[0211] The stirring was stopped 20 hours later, and the mixture was
filtered while washing with n-hexane (155 ml). 10% Aqueous citric
acid solution (775 ml) was added to the filtrate to allow
partitioning. The n-hexane layer was washed with water (775 ml),
water (310 ml), and 15% brine (310 ml), and dried over anhydrous
magnesium sulfate. Magnesium sulfate was removed by filtration, and
the filtrate was concentrated under reduced pressure to give a
mixture (532 g) of dihydrocapsiate and 8-methylnonanoic acid as a
colorless oil. As a result of the analysis, the yield of
dihydrocapsiate was 96% and the purity was 99.2 area % by HPLC. The
mixture contained 3.1 wt % of 8-methylnonanoic acid relative to
dihydrocapsiate.
Example 18
Synthesis of Vanillyl Decanoate--4
[0212] Decanoic acid (10.0 g, 58.1 mmol), vanillyl alcohol (8.05 g,
52.2 mmol), and lipase PS-C "Amano" I (enzyme immobilized on
ceramic: 1.44 g) were measured and placed in a flask (500 ml), and
toluene (200 ml) was added. Under an argon atmosphere, the mixture
was heated with stirring in an oil bath at 40.degree. C. for 2
hours. This reaction mixture was concentrated under reduced
pressure, and dehydration was promoted by azeotropic effect.
Toluene (150 ml) was further added to the concentrate, and the
mixture was heated with stirring in an oil bath at 40.degree. C.
for 20 hours. The reaction mixture was again concentrated under
reduced pressure, and heptane (200 ml) was added. The mixture was
stirred at room temperature for 2.5 hours, and immobilized enzyme
and precipitated vanillyl alcohol were removed by filtration. The
filtrate was concentrated under reduced pressure to give a mixture
(15.8 g) of vanillyl decanoate and decanoic acid. As a result of
the analysis, the yield of vanillyl decanoate was 98% and the
purity was 97.9 area % by HPLC. The mixture contained 8.6 wt % of
decanoic acid relative to vanillyl decanoate.
Example 19
Synthesis of Vanillyl Octanoate
[0213] In the same manner as in Example 5 and using commercially
available octanoic acid, vanillyl octanoate was synthesized at a
yield of 61% (containing 29.9 wt % of octanoic acid).
[0214] .sup.1H-NMR (CDCl.sub.3, .delta.): 0.88 (d, 3H, J=7.10 Hz),
1.20-1.35 (m, 8H), 1.60-1.70 (m, 2H), 2.35 (t, 2H, J=7.40 Hz), 3.90
(s, 3H), 5.03 (s, 2H), 6.83-6.90 (m, 3H).
Example 20
Synthesis of Vanillyl Undecanoate
[0215] In the same manner as in Example 5 and using commercially
available undecanoic acid, vanillyl undecanoate was synthesized at
a yield of 98% (containing 3.3 wt % of undecanoic acid).
[0216] .sup.1H-NMR (CDCl.sub.3, .delta.): 0.88 (d, 3H, J=6.76 Hz),
1.20-1.35 (m, 14H), 1.58-1.68 (m, 2H), 2.35 (t, 2H, J=7.68 Hz),
3.90 (s, 3H), 5.03 (s, 2H), 6.83-6.90 (m, 3H).
Example 21
Synthesis of Vanillyl 9-Methyldecanoate
[0217] In the same manner as in Example 1, 9-methyldecanoic acid
was synthesized from isopentyl bromide and ethyl 6-bromohexanoate
at a yield of 78% (purified by distillation under reduced
pressure), and using this compound, vanillyl 9-methyldecanoate was
synthesized at a yield of 91% (containing 3.1 wt % of
9-methyldecanoic acid) in the same manner as in Example 5.
[0218] .sup.1H-NMR (CDCl.sub.3, .delta.): 0.86 (d, 6H, J=6.64 Hz),
1.12-1.35 (m, 10H), 1.45-1.55 (m, 1H), 1.50-1.60 (m, 2H), 2.34 (t,
2H, J=7.44 Hz), 3.89 (s, 3H), 5.03 (s, 2H), 5.60 (brs, 1H),
6.83-6.90 (m, 3H).
Example 22
Synthesis of Vanillyl 10-Methylundecanoate
[0219] In the same manner as in Example 1, 10-methylundecanoic acid
was synthesized from isopentyl bromide and ethyl 7-bromoheptanoate
at a yield of 81% (purified by distillation under reduced
pressure), and using this compound, vanillyl 10-methylundecanoate
was synthesized at a yield of 98% (containing 8.5 wt % of
10-methyldecanoic acid) in the same manner as in Example 5.
[0220] .sup.1H-NMR (CDCl.sub.3, .delta.): 0.86 (d, 6H, J=6.64 Hz),
1.10-1.40 (m, 12H), 1.50-1.60 (m, 1H), 1.60-1.70 (m, 2H), 2.33 (t,
2H, J=7.68 Hz), 3.90 (s, 3H), 5.03 (s, 2H), 5.63 (s, 1H), 6.83-6.90
(m, 3H).
Example 23
Synthesis of Vanillyl 6-Methyloctanoate
[0221] In the same manner as in Example 1, 6-methyloctanoic acid
was synthesized from 1-chloro-2-methylbutane and ethyl
4-bromobutanoate at a yield of 83% (purified by distillation under
reduced pressure), and using this compound, vanillyl
6-methyloctanoate was synthesized at a yield of 80% (containing 6.7
wt % of 6-methyloctanoic acid) in the same manner as in Example
5.
[0222] .sup.1H-NMR (CDCl.sub.3, .delta.): 0.80-0.90 (m, 6H),
1.05-1.19 (m, 2H), 1.22-1.40 (m, 5H), 1.60-1.70 (m, 2H), 2.34 (t,
2H, J=7.56 Hz), 3.89 (s, 3H), 5.03 (s, 2H), 5.60 (brs, 1H),
6.85-6.91 (m, 3H).
Example 24
Synthesis of Vanillyl 7-Methylnonanoate
[0223] In the same manner as in Example 1, 7-methylnonanoic acid
was synthesized from 1-chloro-2-methylbutane and ethyl
5-bromopentanoate at a yield of 90% (purified by distillation under
reduced pressure), and using this compound, vanillyl
7-methylnonanoate was synthesized at a yield of 93% (containing 6.8
wt % of 7-methyldecanoic acid) in the same manner as in Example
5.
[0224] .sup.1H-NMR (CDCl.sub.3, .delta.): 0.80-0.90 (m, 6H),
1.05-1.20 (m, 2H), 1.20-1.38 (m, 7H), 1.60-1.70 (m, 2H), 2.34 (t,
2H, J=7.72 Hz), 3.90 (s, 3H), 5.03 (s, 2H), 5.60 (brs, 1H),
6.85-6.91 (m, 3H).
Example 25
Synthesis of vanillyl 8-methyldecanoate
[0225] In the same manner as in Example 1, 8-methyldecanoic acid
was synthesized from 1-chloro-2-methylbutane and ethyl
6-bromohexanoate at a yield of 87% (purified by distillation under
reduced pressure), and using this compound, vanillyl
8-methyldecanoate was synthesized at a yield of 88% (containing 9.6
wt % of 8-methyldecanoic acid) in the same manner as in Example
5.
[0226] .sup.1H-NMR (CDCl.sub.3, .delta.): 0.80-0.90 (m, 6H),
1.02-1.20 (m, 2H), 1.20-1.40 (m, 9H), 1.60-1.70 (m, 2H), 2.34 (t,
2H, J=7.72 Hz), 3.90 (s, 3H), 5.03 (s, 2H), 5.60 (brs, 1H),
6.85-6.91 (m, 3H).
Reference Example 1
Stability of Capsinoid in the Non-Existence of Fatty Acid
[0227] Vanillyl decanoate was separately synthesized from vanillyl
alcohol and decanoic acid, and its stability was examined. Decanoic
acid was separated by silica gel column chromatography, and the
purified product was dissolved in acetonitrile and analyzed by
[0228] HPLC to find 95.6 area %. When the sample was reanalyzed 62
hours later, the purity decreased to 82.0 area %, and vanillyl
decanoate was confirmed to have been decomposed.
Example 26
Example of Stabilization by Coexistence of Fatty Acid--1
[0229] Decanoic acid was separated by silica gel column
chromatography, and the purified product, vanillyl decanoate, was
dissolved in acetonitrile and analyzed by HPLC 9 hours later to
find 90.4 area %. To the acetonitrile solution of the purified
product, vanillyl decanoate, was added 9.1 wt % of decanoic acid,
and the mixture was analyzed by HPLC 19.5 hours later to find 97.6
area %, showing increase in the purity as compared to the absence
of decanoic acid addition. Similarly, 16.7 wt %, 28.7 wt % and 44.8
wt % of decanoic acid was added to vanillyl decanoate to result in
higher purities of 98.1 area %, 98.1 area % and 97.9 area % as
compared to the absence of decanoic acid addition.
Example 27
Example of Stabilization by Coexistence of Fatty Acid--2
[0230] Capsiate obtained in the same manner as in Example 5, which
contained 3.2 wt % of fatty acid, was analyzed by HPLC to find the
purity of 97.8 area %. This capsiate was preserved in a hexane
solvent at 5.degree. C. for 30 days and analyzed by HPLC. As a
result, the purity of 97.6 area % was found to have been
maintained.
Example 28
Example of Stabilization by Coexistence of Fatty Acid--3
[0231] Dihydrocapsiate obtained in the same manner as in Example
15, which contained 2.0 wt % of fatty acid, was analyzed by HPLC to
find 99.2 area %. This dihydrocapsiate was preserved in a hexane
solvent at 5.degree. C. for 30 days and analyzed by HPLC. As a
result, the purity of 99.3 area % was found to have been
maintained.
INDUSTRIAL APPLICABILITY
[0232] The method of the present invention is useful for industrial
production of capsinoids, because the capsinoid can be conveniently
synthesized in a high yield in a short time using conventional
techniques and an economical enzyme. Furthermore, the coexistence
of an ester compound (capsinoid) and fatty acid has enabled stable
formation and preservation of conventionally unstable capsinoid.
Therefore, the composition of the present invention, comprising an
ester compound and a fatty acid, can be utilized as a food additive
or a pharmaceutical product.
[0233] Obviously, numerous modifications and variations of the
present invention are possible in light of the above teachings. It
is therefore to be understood that, within the scope of the
appended claims, the invention may be practiced otherwise than as
specifically described herein.
[0234] All patents and other references mentioned above are
incorporated in full herein by this reference, the same as if set
forth at length.
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