ENZYME-CATALYZED PROCESS FOR PREPARING N-ACYL AMINO ACIDS AND N- ACYL AMINO ACID AMIDES

METHODE CATALYSEE PAR UNE ENZYME POUR LA PREPARATION D'AMINOACIDES N-ACYLES ET D'AMIDES D'AMINOACIDES N-ACYLES

English Abstract

2057041 9014429 PCTABS00002
Compounds of general formula (I) or (II), wherein R is an
optionally substituted alkyl group with 3-23 carbon atoms, and R1 is
hydrogen or an optionally substituted branched or straight-chain,
saturated or unsaturated, aliphatic or aromatic hydrocarbon
group, are prepared by reacting a compound of the general formula
(III): RCOOR2, wherein R2 is H or an alkyl group with 1-6 carbon
atoms, and R is as defined above, with a compound of general
formula (IV), wherein R1 is as defined above, in the presence of an
enzyme capable of catalysing the formation of amide bonds, in
particular a lipase. The amide group may be removed from the
compound (I) by means of a second enzyme capable of selectively
cleaving amide bonds, e.g. a carboxypeptidase, resulting in the compound
(II).

Claims

NEW SUGGESTED CLAIMS
1. A process for preparing a compound of the general formula
I
(I)
<IMG>
or II
(II)
<IMG>
wherein R is an alkyl group with 3 - 23 carbon atoms, op-
tionally substituted by a branched or straight-chain,
saturated or unsaturated, aliphatic or aromatic hydrocarbon
group, and R1 is hydrogen or a branched or straight-chain,
saturated or unsaturated, aliphatic or aromatic hydrocarbon
group, optionally substituted by alkyl with 1 - 20 carbon
atoms, hydroxy, amino or mercapto, or an alkali metal or al-
kaline earth metal salt thereof,
the process comprising
a) reacting a compound of the general formula III
RCOOR2 (III)
wherein R2 is hydrogen or an alkyl group with 1 - 6 carbon
atoms, and R is as defined above, with a compound of the
general formula IV

2
<IMG> (IV)
wherein R1 is as defined above, in the presence of a hydro-
lase, to produce the compound of the general formula I,
b) optionally removing the amide group of the compound of
the general formula I by means of a carboxypeptidase, to
produce the compound of the general formula II, and
c) optionally converting a thus formed compound of the
general formula II to a salt by means of a suitable base.
2. A process according to claim 1, wherein R is an alkyl
group with 6 - 20 carbon atoms.
3. A process according to claim 2, wherein RCO- is selected
from the group consisting of hexanoyl, heptanoyl, octanoyl,
nonanoyl, decanoyl, dodecanoyl, tetradecanoyl, hexadecanoyl,
octadecanoyl, eicosanoyl, docosanoyl, cis-9-octadecanoyl and
9,12-octadecadienoyl.
4. A process according to claim 1, wherein R1 is a methyl,
ethyl, propyl, isopropyl, methylthio, -CH2-CH2-S-CH3,
benzyl, hydroxybenzyl, indolyl or alkylguanidine group.
5. A process according to claim 1, wherein the compound of
the general formula IV is an amino acid amide selected from
the group consisting of alanine amide, leucine amide,
phenylalanine amide, phenylglycine amide, lysine amide,
glycine amide, valine amide, tryptophan amide, arginine
amide, histidine amide, cysteine amide, iso-leucine amide,
glutamine amide, asparagine amide, aspartic acid amide, glu-
tamic acid amide and ornithine amide in racemic or optically
active form.

6. A process according to any of the preceding claims for
preparing compounds of the general formula I as defined in
claim 1, which compounds are selected from the group con-
sisting of N-hexanoyl alanine amide, N-octanoyl alanine
amide, N-decanoyl alanine amide, N-dodecanoyl alanine amide,
N-tetradecanoyl alanine amide, N-hexadecanoyl alanine amide,
N-octadecanoyl alanine amide, N-hexanoyl leucine amide, N-
octanoyl leucine amide, N-decanoyl leucine amide, N-dodeca-
noyl leucine amide, N-tetradecanoyl leucine amide, N-hexa-
decanoyl leucine amide, N-octadecanoyl leucine amide, N-
hexanoyl phenylalanine amide, N-octanoyl phenylalanine
amide, N-decanoyl phenylalanine amide, N-dodecanoyl phenyl-
alanine amide, N-tetradecanoyl phenylalanine amide, N-hexa-
decanoyl phenylalanine amide, N-octadecanoyl phenylalanine
amide, N-hexanoyl phenylglycine amide, N-octanoyl phenyl-
glycine amide, N-decanoyl phenylglycine amide, N-dodecanoyl
phenylglycine amide, N-tetradecanoyl phenylglycine amide, N-
hexadecanoyl phenylglycine amide, N-octadecanoyl phenyl-
glycine amide, N-hexanoyl lysine amide, N-octanoyl lysine
amide, N-decanoyl lysine amide, N-dodecanoyl lysine amide,
N-tetradecanoyl lysine amide, N-hexadecanoyl lysine amide,
N-octadecanoyl lysine amide, N-hexanoyl glycine amide, N-
octanoyl glycine amide, N-decanoyl glycine amide, N-dodeca-
noyl glycine amide, N-tetradecanoyl glycine amide, N-hexa-
decanoyl glycine amide, N-octadecanoyl glycine amide, N-
hexanoyl valine amide, N-octanoyl valine amide, N-decanoyl
valine amide, N-dodecanoyl valine amide, N-tetradecanoyl
valine amide, N-hexadecanoyl valine amide, N-octadecanoyl
valine amide, N-hexanoyl tryptophan amide, N-octanoyl tryp-
tophan amide, N-decanoyl tryptophan amide, N-dodecanoyl
tryptophan amide, N-tetradecanoyl tryptophan amide, N-
hexadecanoyl tryptophan amide, N-octadecanoyl tryptophan
amide, N-hexanoyl arginine amide, N-octanoyl arginine amide,
N-decanoyl arginine amide, N-dodecanoyl arginine amide, N-
tetradecanoyl arginine amide, N-hexadecanoyl arginine amide,
N-octadecanoyl arginine amide, N-hexanoyl histidine amide,
N-octanoyl histidine amide, N-decanoyl histidine amide, N-

dodecanoyl histidine amide, N-tetradecanoyl histidine amide,
N-hexadecanoyl histidine amide, N-octadecanoyl histidine
amide, N-hexanoyl cysteine amide, N-octanoyl cysteine amide,
N-decanoyl cysteine amide, N-dodecanoyl cysteine amide, N-
tetradecanoyl cysteine amide, N-hexadecanoyl cysteine amide,
N-octadecanoyl cysteine amide, N-hexanoyl glutamine amide,
N-octanoyl glutamine amide, N-decanoyl glutamine amide, N-
dodecanoyl glutamine amide, N-tetradecanoyl glutamine amide,
N-hexadecanoyl glutamine amide, N-octadecanoyl glutamine
amide, N-hexanoyl isoleucine amide, N-octanoyl isoleucine
amide, N-decanoyl isoleucine amide, N-dodecanoyl isoleucine
amide, N-tetradecanoyl isoleucine amide, N-hexadecanoyl
isoleucine amide, N-octadecanoyl isoleucine amide, N-
hexanoyl asparagine amide, N-octanoyl asparagine amide, N-
decanoyl asparagine amide, N-dodecanoyl asparagine amide, N-
tetradecanoyl asparagine amide, N-hexadecanoyl asparagine
amide, N-octadecanoyl asparagine amide, N-hexanoyl aspartic
acid amide, N-octanoyl aspartic acid amide, N-decanoyl as-
partic acid amide, N-dodecanoyl aspartic acid amide, N-
tetradecanoyl aspartic acid amide, N-hexadecanoyl aspartic
acid amide, N-octadecanoyl aspartic acid amide, N-hexanoyl
glutamic acid amide, N-octanoyl glutamic acid amide, N-de-
canoyl glutamic acid amide, N-dodecanoyl glutamic acid
amide, N-tetradecanoyl glutamic acid amide, N-hexadecanoyl
glutamic acid amide, N-octadecanoyl glutamic acid amide, N-
hexanoyl ornithine amide, N-octanoyl ornithine amide, N-de-
canoyl ornithine amide, N-dodecanoyl ornithine amide, N te-
tradecanoyl ornithine amide, N-hexadecanoyl ornithine amide,
N-octadecanoyl ornithine amide, or an alkali metal or alka-
line earth metal salt thereof, each compound being in race-
mic or optically active form.
7. A process according to any of the preceding claims for
preparing compounds of the general formula II defined in
claim 1, the compounds being selected from the group con-
sisting of N-hexanoyl alanine, N-octanoyl alanine, N-
decanoyl alanine, N-dodecanoyl alanine, N-tetradecanoyl ala-

nine, N-hexadecanoyl alanine, N-octadecanoyl alanine, N-
hexanoyl leucine, N-octanoyl leucine, N-decanoyl leucine, N-
dodecanoyl leucine, N-tetradecanoyl leucine, N-hexadecanoyl
leucine, N-octadecanoyl leucine, N-hexanoyl phenylalanine,
N-octanoyl phenylalanine, N-decanoyl phenylalanine, N-
dodecanoyl phenylalanine, N-tetradecanoyl phenylalanine, N-
hexadecanoyl phenylalanine, N-octadecanoyl phenylalanine, N-
hexanoyl phenylglycine, N-octanoyl phenylglycine, N-decanoyl
phenylglycine, N-dodecanoyl phenylglycine, N-tetradecanoyl
phenylglycine, N-hexadecanoyl phenylglycine, N-octadecanoyl
phenylglycine, N-hexanoyl lysine, N-octanoyl lysine, N-deca-
noyl lysine, N-dodecanoyl lysine, N-tetradecanoyl lysine, N-
hexadecanoyl lysine, N-octadecanoyl lysine, N-hexanoyl
glycine, N-octanoyl glycine, N-decanoyl glycine, N-
dodecanoyl glycine, N-tetradecanoyl glycine, N-hexadecanoyl
glycine, N-octadecanoyl glycine, N-hexanoyl valine, N-octa-
noyl valine, N-decanoyl valine, N-dodecanoyl valine, N-
tetradecanoyl valine, N-hexadecanoyl valine, N-octadecanoyl
valine, N-hexanoyl tryptophan, N-octanoyl tryptophan, N-de-
canoyl tryptophan, N-dodecanoyl tryptophan, N-tetradecanoyl
tryptophan, N-hexadecanoyl tryptophan, N-octadecanoyl tryp-
tophan, N-hexanoyl arginine, N-octanoyl arginine, N-decanoyl
arginine, N-dodecanoyl arginine, N-tetradecanoyl arginine,
N-hexadecanoyl arginine, N-octadecanoyl arginine, N-hexanoyl
histidine, N-octanoyl histidine, N-decanoyl histidine, N-
dodecanoyl histidine, N-tetradecanoyl histidine, N-
hexadecanoyl histidine, N-octadecanoyl histidine, N-hexanoyl
cysteine, N-octanoyl cysteine, N-decanoyl cysteine, N-dode-
canoyl cysteine, N-tetradecanoyl cysteine, N-hexadecanoyl
cysteine, N-octadecanoyl cysteine, N-hexanoyl glutamine, N-
octanoyl glutamine, N-decanoyl glutamine, N-dodecanoyl glu-
tamine, N-tetradecanoyl glutamine, N-hexadecanoyl glutamine,
N-octadecanoyl glutamine, N-hexanoyl isoleucine, N-octanoyl
isoleucine, N-decanoyl isoleucine, N-dodecanoyl isoleucine,
N-tetradecanoyl isoleucine, N-hexadecanoyl isoleucine, N-
octadecanoyl isoleucine, N-hexanoyl asparagine, N-octanoyl
asparagine, N-decanoyl asparagine, N-dodecanoyl, asparagine

N-tetradecanoyl asparagine, N-hexadecanoyl asparagine, N-
octadecanoyl asparagine, N-hexanoyl aspartic acid, N-octa-
noyl aspartic acid, N-decanoyl aspartic acid, N-dodecanoyl
aspartic acid, N-tetradecanoyl aspartic acid, N-hexadecanoyl
aspartic acid, N-octadecanoyl aspartic acid, N-hexanoyl glu-
tamic acid, N-octanoyl glutamic acid, N-decanoyl glutamic
acid, N-dodecanoyl glutamic acid, N-tetradecanoyl glutamic
acid, N-hexadecanoyl glutamic acid, N-octadecanoyl glutamic
acid, N-hexanoyl ornithine, N-octanoyl ornithine, N-decanoyl
ornithine, N-dodecanoyl ornithine, N-tetradecanoyl orni-
thine, N-hexadecanoyl ornithine, N-octadecanoyl ornithine,
or an alkali metal or alkaline earth metal salt thereof,
each compound being in racemic or optically active form.
8. A process according to any one of the preceding claims,
wherein the hydrolase is a lipase, peptidase, esterase or
protease.
9. A process according to claim 8, wherein the lipase is one
producible by species of Mucor, Candida, Humicola, or Pseu-
domonas.
10. A process according to claim 9, wherein the lipase is
one produced by Candida antarctica, DSM 3855, DSM 3908 or
DSM 3909, Pseudomonas cepacia, DSM 3959, Humicola
lanuainosa, DSM 3819 or 4109, Humicola brevispora, DSM 4110,
Humicola brevis var. thermoidea, DSM 4111, or Humicola inso-
lens, DSM 1800.
11. A process according to claim 1, wherein the enzyme is an
immobilized enzyme.
12. A process according to claim 1, wherein the carboxypep-
tidase is carboxypeptidase Y.
13. A process according to claim 12, wherein the carboxypep-
tidase is one produced by Saccharomvces cerevisiae.

7
14. A process according to any of the preceding claims,
wherein the reaction of the compound of the general formula
III with the compound of the general formula IV proceeds at
room temperature.
15. A process according to any of the preceding claims,
wherein the reaction of the compound of the general formula
III with the compound of the general formula IV proceeds in
the absence of a solvent.
16. A process according to any of the preceding claims,
wherein the reaction of the compound of the general formula
III with the compound of the general formula IV proceeds at
a low pressure such as a pressure of below about 0.05 bar,
in particular a pressure below about 0.01 bar.

Description

W O 90/1~29 ~ .'~ 7~ PCT/DK90/00127
AN ENZYME-CATALYZED PROCESS FOR PREPARING N-ACYL AMINO ACIDS
AND N-ACYL AMINO ACID AMIDES
FIELD OF ~NVENTION
5 The present invention relates to an enzyme-catalyzed pro-
cess for preparing N-acylated amino acid amides and N-acy-
lated amino acids, and a cleaning composition and personal
care composition containing an N-acyl amino acid amide.
;
10 BACKGROUND OF THE INVENTION
Surface-active agents constitute an extremely important
class of indus~rial chemicals which have a wide variety of
uses, for instance as detergents for washing purposes, as
15 emulsifiers in food products and as essential ingredients
in various personal care products such as shampoos, soaps -~
or moisturizing creams.
,
At the molecular level, surface-active agents are sub-
20 stances which are characterized by the presen~e of hydro-
phobic and hydrophilic regions within each individual sur-
factant molecule and which owe their ability to reduce
surface tension to this particular structure. For in-
stance, surface-active agents are able to effect solution
25 of otherwise water-insoluble substances in water by inter-
acting with such substances at the hydrophobic region of
"
the surfactant molecule and with water at the hydrophilic
region of the surfactant molecule.
30 Because of the ready availability of hydrophilic as well
as hydrophobic substances and the well-developed chemical
technologies for combining such substances to form sur-
face-active agents, a large number of surface-active
agents are at present commercially available. Most such
~5 surfactants are based on petrochemically derived products
which are attractive and owe their widespread use to their
.. . ., - - . ., . , , . ,. , . - , , . , ~-

~()9~ 9 PCT/D~90/00127
low cost. However, certain important surface-active agents
are composed of naturally occurring compounds such as
fatty acids and glycerol (commercially available as mono-
and diglycerides), mainly for application as emulsifiers
5 in food products.
The combination of hydrophobic and hydrophilic regions
within the same molecule may be obtained in many different
ways, for instance by combining a sulphonic acid residue,
10 a quaternized ammonium moiety or a glycerol moiety with an
alkyl chain as is the case with the linear alkyl surfac-
tants, the quarternized alkyl amines or the monogly-
cerides, respectively. When designing a surfactant mole-
cule, the detailed molecular architecture of the compounds
15 is a major concern, care being taken to achieve a precise
balance between the hydrophobic and hydrophilic regions of
the surfactant molecules as well as to achieve a favour-
able spatial arrangement of these individual regions of
the molecules. Apart from this, the possibility of pro-
20 ducing surface-active agents by high-yielding processes
and on the basis of inexpensive and abundant raw materials
is always carefully considered. The environmental issues
related to the eventual loading of the surfactant into the
environment are finally a matter of major concern.
As a result of these considerations, efforts have been
made to develop surface-active agents based on naturally
occurring substances. One such class of compounds are a-
cylated amino acids (also known as lipoamino acids) which
30 exhibit surface-active properties due to the hydrophilic
properties of the amino acid moiety of the compounds and
the hydrophobic properties of the fatty acid moiety of the
compounds. The balance between hydrophilic and hydrophobic
properties might be varied by modifying the amino acid
35 and/or the fatty acid by adding one or more substituents.
Acylated amino acids may be prepared from relatively inex-
~ :: : , - :. .
: ~ : . - - ,

~09(~ ") PCr/DK90/00l27
2~
pensive starting materials and have the advantage of being
biodegradable into their naturally occurring constituent
parts so that they do not constitute an environmental haz-
ard. Acylated amino acids are known to be useful as deter-
5 gents and emulsifiers in cosmetics due to their surface-
active properties.
At present, acylated amino acids are prepared by organic
synthesis. One conventional method for producing the com-
10 pounds (briefly referred to in GB 1 483 500) is to acylateamino acids with a higher fatty acid chloride in an aque-
ous alkaline medium. This method is stated to have the
disadvantage that a chloride salt is left in the reaction
mixtu~e which makes it necessary to remove the salt in or-
15 der to preserve a good detergency of the compounds. An-
other method, also disclosed in GB 1 483 500, for pro-
ducing N-acyl amino acids comprises reacting a mixed acid
anhydride of a higher fatty acid and sulphuric acid with
an amino acid in a liquid medium in the presence of a
20 base.
General disadvantages of methods of organic synthesis of
N-acyl amino acids are that they tend to be rather time-
consuming and that there is a considerable risk that un-
25 desirable side products will be formed during the reactionprocess which makes the purification of the desired end
products more difficult. As a result of this, the prepara-
tion of N-acyl amino acids by conventional organic synthe-
sis is rather expensive for which reason acylated amino
30 acids have not found as widespread a commercial applica-
tion as surfactants based on petrochemically derived pro-
ducts.
Amides of N-acyl amino acids are also known surface-active
35 substances for use in cosmetics, and as antioxidants and
antibacterial agents, cf. JP-B 52-18691, according to
: :: - : :., - . . ..
: ::: .-, - . . - : :: ,

0 9()/l~2~ PCT/DK90/00127
;~C`t~7'~ ~
which N-acyl amino acid amides are prepared by heating the
corresponding N-acyl amino acids with ammonia or a primary
amine in the presence of a water-soluble acidic boron com-
pound. The reaction is conducted using a hydrocarbon as
5 solvent. As is the case with the N-acyl amino acids used
as starting materials, the corresponding amides are quite
expensive to produce and, consequently, their commercial
use has not become widespread.
10 It is therefore an object of the present invention to pro~
vide an enzyme-catalysed process for producing N-acyl a-
mino acids and N-acyl amino acid amides which is simpler
and less time-consumlng to carry out than the conventional
processes for preparing such compounds, and which results
15 in satisfactory yields of the acylated amino acids and
amino acid amides.
SUMMARY OF THE INVENTION
20 Accordingly, the present invention relates to a process
for preparing a compound of the general formula I
RCO-NH
~ NH2 (I)
R1 ~
o
or II
RCO-NH
~ OH (II)
Rl 11
o
wherein R is an alkyl group with 3-23 carbon atoms, op-
35 tionally substituted by a branched or straight-chain,
saturated or unsaturated, alipha~ic or aromatic hydrocar-

W090/l~29 PCT/DK90/00127
~ ~ _ 7 ~ ~1
bon group, and Rl is hydrogen or a branched or straight~chain, saturated or unsaturated, aliphatic or aromatic hy-
drocarbon group, optionally substituted by alkyl with 1-20
carbon atoms, -OH, -NH2 or SH, or an alkali metal or al-
5 kaline earth metal salt thereof,
the process comprising
a) reacting a compound of the general formula III
RCOOR2 (III)
wherein R2 is H or an alXyl group with 1-6 carbon atoms,
and R is as defined above, with a compound of the general
15 formula IV :
NH2
l NH2 (IV)
R l ~

wherein Rl is as defined above, in the presence of an en-
zyme capable of catalysing the formation of amide bonds,
to produce the compound of the general formula I,
b) optionally removing the amide group of the compound of
the general formula I by means of a second enzyme capable
of selective cleavage of amide bonds, to produce the com-
pound of the general formula II, and
::
c) optionally converting a thus formed compound of the
general formula II to a salt by means of a suitable base. :
In a further aspect, the invention relates to a cleaning
35 composition which comprises an effective amount of a com-
pound of the general formula I as defined above.

'09n/l~29 PCT/DK90/00127
~ 71~ 6
In a still further aspect, the present invention relates
to a personal care composition comprising a compound of
the general formula I as defined above.
5 DETAILED DISCLOSURE OF THE INVENTION
In the general formulae I and II, R is preferably an un-
substituted alkyl group with 6-20 carbon atoms. Thus, RCO-
may suitably be selected from the group consisting of
10 hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, dode-
canoyl, tetradecanoyl, hexadecanoyl, octadecanoyl, eicosa-
noyl, docosanoyl, cis-9-octadecanoyl and 9,12-octadeca-
dienoyl.
15 Rl is preferably a methyl, ethyl, propyl, isopropyl,
methylthio, -CH2-CH2-S-CH3, benzyl, hydroxybenzyl, indolyl
or alkylguanidine group.
The compound of the general formula IV is preferably an
20 amino acid amide selected from the group consisting of
alanine amide, leucine amide, phenylalanine amide, phenyl-
glycine amide, lysine amide, glycine amide, valine amide,
tryptophan amide, arginine amide, histidine amide,
cysteine amide, iso-leucine amide, glutamine amide, as-
25 paragine amide, aspartic acid amide, glutamic acid amideand ornithine amide. Amino acid amides may be obtained
from the corresponding amino nitriles by hydrolysis. Amino
nitriles may be prepared according to methods known in the
art by means of the Strecker synthesis starting from the
30 appropriate aldehyde cyanide and ammonia.
Thus, some particularly preferred compounds of the general
formula I are selected from the group consisting of N-
hexanoyl alanine amide, N-octanoyl alanine amide, N-deca-
35 noyl alanine amide, N-dodecanoyl alanine amide, N-tetra-
decanoyl alanine amide, N-hexadecanoyl alanine amide, N-
: ., . . . ; - -
. . :

wO9o/~ s ~ C ~7~ 1 r~r/D~90/oot27
octadecanoyl alanine amide, N-hexanoyl leucine amide, N
octanoyl leucine amide, N-decanoyl leucine amide, N-
dodecanoyl leucine amide, N-tetradecanoyl leucine amide,
N-hexadecanoyl leucine amide, N-octadecanoyl leucine
5 amide, N-hexanoyl phenylalanine amide, N-octanoyl phenyl-
alanine amide, N-decanoyl phenylalanine amide, N-dodeca-
noyl phenylalanine amide, N-tetradecanoyl phenylalanine
amide, N-hexadecanoyl phenylalanine amide, N-octadecanoyl
phenylalanine amide, N-hexanoyl phenylglycine amide, N-
10 octanoyl phenylglycine amide, N-decanoyl phenylglycine
amide, N-dodecanoyl phe~ylglycine amide, N-tetradecanoyl
phenylglycine amide, N-hexadecanoyl phenylglycine amide,
N-octadecanoyl phenylglycine amide, N-hexanoyl lysine
amide, N-octanoyl lysine amide, N-decanoyl lysine amide,
15 N-dodecanoyl lysine amide, N-tetradecanoyl lysine amide,
N-hexadecanoyl lysine amide, N-octadecanoyl lysine amide,
N-hexanoyl glycine amide, N-octanoyl glycine amide, N-
decanoyl glycine amide, N-dodecanoyl glycine amide, N-
tetradecanoyl glycine amide, N-hexadecanoyl glycine amide,
20 N-octadecanoyl glycine amide, N-hexanoyl valine amide, N-
octanoyl valine amide, N-decanoyl ~aline amide, N-dodeca-
noyl valine amide, N-tetradecanoyl valine amide, N-
hexadecanoyl valine amide, N-octadecanoyl valine amide, N-
hexanoyl tryptophan amide, N-octanoyl tryptophan amide, N-
25 decanoyl tryptophan amide, N-dodecanoyl tryptophan amide,
N-tetradecanoyl tryptophan amide, N-hexadecanoyl trypto-
phan amide, N-octadecanoyl tryptophan amide, N-hexanoyl
arginine amide, N-octanoyl arginine amide, N-decanoyl
arginine amide, N-dodecanoyl arginine amide, N-tetra-
30 decanoyl arginine amide, N-hexadecanoyl arginine amide, N-
octadecanoyl arginine amide, N~hexanoyl histidine amide,
N-octanoyl histidine amide, N-decanoyl histidine amide, N-
dodecanoyl histidine amide, N-tetradecanoyl histidine
amide, N-hexadecanoyl histidine amide, N-octadecanoyl
35 histidine amide, N-hexanoyl cysteine amide, N-octanoyl
cysteine amide, N-decanoyl cvsteine amide, N-dodecanoyl
~.'

W09~ PCT/DK90/00127
cysteine amide, N-tetradecanoyl cysteine amide, N-
hexadecanoyl cysteine amide, N-octadecanoyl cysteine
amide, N-hexanoyl glutamine amide, N-octanoyl glutamine
amide, N-decanoyl glutamine amide, N-dodecanoyl glutamine
5 amide, N-tetradecanoyl glutamine amide, N-hexadecanoyl
glutamine amide, N-octadecanoyl glutamine amide, N-
hexanoyl isoleucine amide, N-octanoyl isoleucine amide, N-
decanoyl isoleucine amide, N-dodecanoyl isoleucine amide,
N-tetradecanoyl isoleucine amide, N-hexadecanoyl iso-
10 leucine amide, N-octadecanoyl isoleucine amide, N-hexanoyl
asparagine amide, N-octanoyl asparagine amide, N-decanoyl
asparagine amide, N-dodecanoyl asparagine amide, N-tetra-
decanoyl asparagine amide, N-hexadecanoyl asparagine
amide, N-octadecanoyl asparagine amide, N-hexanoyl
15 aspartic acid amide, N-octanoyl aspartic acid amide, N-
decanoyl aspartic acid amide, N-dodecanoyl aspartic acid
amide, N-tetradecanoyl aspartic acid amide, N-hexadecanoyl
aspartic acid amide, N-octadecanoyl aspartic acid amide,
N-hexanoyl glutamic acid amide, N-octanoyl glutamic acid
20 amide, N-decanoyl glutamic acid amide, N-dodecanoyl
glutamic acid amide, N-tetradecanoyl glutamic acid am1de,
N-hexadecanoyl glutamic acid amide, N-octadecanoyl
glutamic acid amide, N-hexanoyl ornithine amide, N-octa-
noyl ornithine amide, N-decanoyl ornithine amide, N-
25 dodecanoyl ornithine amide, N-tetradecanoyl ornithine
amide, N-hexadecanoyl ornithine amide, N-octadecanoyl
ornithine amide, or an alkali metal or alkaline earth
metal salt thereof.
30 Furthermore, some particularly preferred compounds of the
general formula II are selected from the group consisting
of N-hexanoyl alanine, N-octanoyl alanine, N-decanoyl
alanine, N-dodecanoyl alanine, N-tetradecanoyl alanine, N-
hexadecanoyl alanine, N-octadecanoyl alanine, N-hexanoyl
35 leucine, N-octanoyl leucine, N-decanoyl leucine, N-
dodecanoyl leucine, N-tetradecanoyl leucine, N-hexa-
-- .. - . , ~ .
. : - . ...
:. :

~()9()/1~29 ~ - 7~ PCT/DK90/OOt27
decanoyl leucine, N-octadecanoyl leucine, N-hexanoyl phe-
nylalani.ne, N-octanoyl phenylalanine, N-decanoyl phenyl-
alanine, N-dodecanoyl phenylalanine, N-tetradecanoyl
phenylalanine, N-hexadecanoyl phenylalanine, N-octa-
5 decanoyl phenylalanine, N-hexanoyl phenylglycine, N-
octanoyl phenylglycine, N-decanoyl phenylglycine, N
dodecanoyl phenylglycine, N-tetradecanoyl phenylglycine,
N-hexadecanoyl phenylglycine, N-octadecanoyl
phenylglycine, N-hexanoyl lysine, N-octanoyl lysine, N-
10 decanoyl lysine, N-dodecanoyl lysinP, N-tetradecanoyl
lysine, N-hexadecanoyl lysine, N-octadecanoyl lysine, N-
hexanoyl glycine, N-octanoyl glycine, N-decanoyl glycine,
N-dodecanoyl glycine, N-tetradecanoyl glycine, N-
hexadecanoyl glycine, N-octadecanoyl glycine, N-hexanoyl
15 valine, N-octanoyl valine, N-decanoyl valine, N-dodecanoyl
~aline, N-tetradecanoyl valine, N-hexadecanoyl valine, N-
octadecanoyl valine, N-hexanoyl tryptophan, N-octanoyl
tryptophan, N-decanoyl tryptophan, N-dodecanoyl
tryptophan, N-tetradecanoyl tryptophan, N-hexadecanoyl
20 tryptophan, N-octadecanoyl tryptophan, N-hexanoyl
arginine, N-octanoyl arginine, N-decanoyl arginine, N-
dodecanoyl arginine, N-tetradecanoyl arginine, N-
hexadecanoyl arginine, N-octadecanoyl arginine, N-hexanoyl
histidine, N-octanoyl histidine, N-decanoyl histidine, N-
25 dodecanoyl histidine, N-tetradecanoyl histidine, N-
hexadecanoyl histidine, N-octadecanoyl histidine, N-
hexanoyl cysteine, N-octanoyl cysteine, N-decanoyl
cysteine, N-dodecanoyl cysteine, N-tetradecanoyl cysteine,
N-hexadecanoyl cysteine, N-octadecanoyl cysteine, N-
30 hexanoyl glutamine, N-octanoyl glutamine, N-decanoyl
glutamine, N-dodecanoyl glutamine, N-tetradecanoyl
glutamine, N-hexadecanoyl glutamine, N-octadecanoyl
glutamine, N-hexanoyl isoleucine, N-octanoyl isoleucine,
N-decanoyl isoleucine, N-dodecanoyl isoleucine, N-
35 tetradecanoyl isoleucine, N-hexadecanoyl isoleucine, N-
octadecanoyl isoleucine, N-hexanoyl asparagine, N-octanoyl
: . , ., . -. ; . . .
... , . -~- , .
. i: . . . : -

,3, PCT/DK9n/00127
asparagine, N-decanoyl asparagine, N-dodecanoyl aspara-
gine, N-tetradecanoyl asparagine, N-hexadecanoyl
asparagine, N-octadecanoyl asparagine, N-hexanoyl aspartic
acid, N-octanoyl aspartic acid, N-decanoyl aspartic acid,
5 N-dodecanoyl aspartic acid, N-tetradecanoyl aspartic acid,
N-hexadecanoyl aspartic acid, N-octadecanoyl aspartic
acid, N-hexanoyl glutamic acid, N-octanoyl glutamic acid,
N-decanoyl glutamic acid, N-dodecanoyl glutamic acid, N-
tetradecanoyl glutamic acid, N-hexadecanoyl glutamic acid,
10 N-octadecanoyl glutamic acid, N-hexanoyl ornithine, N-
octanoyl ornithine, N-decanoyl ornithine, N-dodecanoyl
ornithine, N-tetradecanoyl ornithine, N-hexadecanoyl
ornithine, N-octadecanoyl ornithine, or an alkali metal or
alkaline earth metal salt thereof.
The starting amino acid amides (IV) as well as the re-
sulting compounds (I) or (II) may be in the form of a
racemic mixture or in optically active form.
20 Enzymes which may be useful as catalysts in the process of
the invention should be selected according to the follow-
ing criteria: (a) their ability to catalyse the formation
) of an amide bond; (b) their ability to use a fatty acid or
fatty ester as substrate; and (c) their ability to use an
25 amino acid amide as the nucleophile. More specifically,
suitable enzymes for the process of the invention are
those which catalyse the hydrolysis of amide bonds or the
reverse synthesis reaction, e.g. hydrolases.
30 An enzyme catalysing the hydrolysis of N-long chain acyl
amino acids is described by Y. Shintani et al., J.
Biochem. 96, 1984, pp. 637-643, who denote it "N-long
chain acyl aminoacylase". It is briefly suggested that
this enzyme may also catalyse the synthesis of lipoamino
35 acids from fatty acids and amino acids. However, there is
no indication of the reaction conditions under which the
; ' ~'. : '
,
:
. , .

~09()/1~_9 ~ ~7~ PCT/DK90/U0127
11 ~' '
synthesis takes place, nor is there any indication of the
reaction times or the final yield of lipoamino acids pro-
vided by the synthesis. Additionally, it appears that the
enzyme described by Shintani et al. is specific to lipo-
5 amino acids containing L-glutamate so that, apparently,
it cannot be used generally in a process for producing
lipoamino acids containing several different amino acid
residues.
10 EP 298 796 discloses the use of acyl transferases, includ-
ing lipase, to catalyse a process for preparing N-substi-
tuted fatty amides from fatty acids and amines (including
amino acids though no examples are actually given of
this). Apart from resulting in a different end product,
15 the present process is distinguished from the process de-
scribed in EP 298 796 by using amino acid amides as start-
ing materials which are monoionic compounds and as such
chemically distinct from amino acids which are zwitter-
ionic compounds. There would be no reason to expect that
20 an enzymatic process using amines as starting materials
might also be employed using another, chemically distinct,
starting material. Moreover, it was surprisingly found
that the process of the present invention is selective,
i.e. that only one of the amino groups in the amino acid
25 amide is N-acylated. In an industrial context, the present
process is attractive because the starting amino acid
amide may be produced on a large scale from synthetic
starting materials, and because amino acid amides are more
easily soluble than amino acids in organic solvents, re-
30 sulting in higher yields of the N-acylated end products.
Hydrolytic enzymes for use in the present process may be
lipases, peptidases (in particular non-specific pepti-
dases), esterases or proteases, in particular lipases
35 which may be defined as enzymes catalyzing reactions in-
volving ester bonds, e.g. hydrolysis, synthesis and/or ex-
-.- , : - . : ,- . . :

WO~()/1l~2s PCT/DK90/00127
~ 12
change of ester bonds. Lipases which may be employed in
the present process may be porcine pancreatic lipase or
microbial lipases produced, for instance, by strains of
Asperaillus, Enterobacterium, Chromobacterium, Geotricium
5 or Penicillium. Preferred lipases for use according to the
invention are those produced by species of Mucor (e.g.
LipozymeTM, produced by Mucor miehei), Humicola, Pseudo-
monas or Candida (such as Candida antarctica or Candida
cylindracea).
Particularly preferred lipases are those produced by the
following strains of microorganisms, all of which have
been deposited in the Deutsche Sammlung von Mikroorga-
nismen in accordance with the provisions of the Budapest
15 Treaty on the International Recognition of the Deposit of
Microorganisms for the purposes of Patent Procedure:
Candida antarctica, deposited on 29 September 1986, with
- the number DSM 3855, and on 8 December 1986, with the num-
bers DSM 3908 and DSM 3909.
20 Pseudomonas cePhacia, deposited on 30 January 1987, with
the number 3959.
Humicola lanuainosa, deposited on 13 August 1986 and 4
May, with the deposit numbers 3819 and 4109, respectively.
Humicola brevis~ora, deposited on 4 May 1987, with the de-
25 posit number DMS 4110,
Humicola brevis var. thermoidea, deposited on 4 May 1987,with the deposit number DSM 4111, and
Humicola insolens, deposited on 1 October 1981, with the
deposit number DSM 1800.
Currently preferred lipases are those produced by Candida
antarctica, DSM 3855, DSM 3908 and DSM 3909. These enzymes
may be produced by the process disclosed in WO 88/02775.
Briefly, the Candida strains in question are cultivated
35 under aerobic conditions in a nutrient medium containing
assimilable carbon and nitrogen sources as well as essen-
,
.. .

~090/1~4 9 PCT/DK90/00127
13
tial minerals, trace elements etc., the medium being com-
posed according to established practice. After cultiva-
tion, liquid enzyme concentrates may be prepared by re-
moving insoluble materials, e.g. by filtration or centri-
5 fugation, after which the broth is concentrated by evap-
oration or reverse osmosis. Solid enzyme preparations may
be prepared from the concentrate by precipitation with
salts or water-miscible solvents, e.g. ethanol, or by dry-
ing such as spray-drying in accordance with well-known
10 methods.
Additional lipases may be obtained from the following
strains which are publicly available without restriction
from the Centraalbureau voor Schimmelculturen (CBS),
15 American Type Culture Collection ~ATCC), Agricultural Re-
search Culture Collection (NRRL) and Institute of Fermen-
tation, Osaka (IFO) with the following deposit numbers:
Candida antarctica, CBS 5955, ATCC 34888, NRRL Y-8295, CBS
6678, ATCC 28323, CBS 6821 and NRRL Y-7954; Candida
20 tsukubaensis, CBS 6389, ATCC 24555 and NRRL Y-7795;Candida
auriculariae, CBS 6379, ATTC 24121 and IFO 1580; Candida
humicola, CBS 571, ATCC 14438, IFO 0760, CBS 2041, ATCC
9949, NRRL Y-1266, IFO 0753 and IFO 1527; and Candida
follorum, CBS 5234 and ATCC 18820. -
:
It is known to produce lipase by recombinant DNA tech-
niques, cf. for instance EP 238 023. Recombinant lipases ~ ~
may also be employed for the present purpose. -
N-acyl amino acid amides produced by the process of the
invention may be employed as such as surface-active
agents. If, however, it is desired to produce N-acyl amino
acids, it has been found possible to cleave off the NH2
35 group selectively by an enzyme-catalysed process by means
of an enzyme which is able to cleave amide bonds. An
,, . . , . - . . . . .
.: ~ . .' .: - - -'
. .
.. .. , . ~ :. :.. . : : - : -
~ . . , . . :

~)9(~ 29 PCT/DK90/00127
2C`c~ 14
example of such an enzyme is carboxypeptidase such as car-
boxypeptidase Y, which is produced by Saccharomyces
cerevisiae.
5 When employed in step a) or b) of the process of the in-
vention, the enzymes may be in a soluble state. It is,
however, preferred to immobilize either or both enzymes in
order to facilitate the recovery of the N-acyl amino acids
or N-acyl amino acid amides produced by the present pro-
lO cess. Immobilization procedures are well known (cf. forinstance K. Mosbach, ed., "Immobilized Enzymes", Methods
in Enzymoloay 44, Academic Press, New York, 1976) and in-
clude cross-linking of cell homogenates, covalent coupling
to insoluble organic or inorganic supports, entrapment in
15 gels and adsorption to ion exchange resins or other adsor-
bent materials. Coating on a particulate support may also
be employed (cf. for instance A.R. Macrae and R.C.
Hammond, Biotechnoloay and Genetic Engineerina Reviews 3,
1985, p. 193. Suitable support materials for the immo-
20 bilized enzyme are, for instance, plastics ~e.g. poly-
styrene, polyvinylchloride, polyurethane, latex, nylon,
teflon, dacron, pol~vinylacetate, polyvinylalcohol or any
siutable copolymer thereof), polysaccharides (e.g. agarose
or dextran), ion exchange resins (both cation and anion
25 exchange resins), silicon polymers (e.g. siloxane) or
silicates (e.g. glass).
It is preferred to immobilize the enzymes on an ion ex-
change resin by adsorbing the enzymes to the resin or by
30 cross-linking it to the resin by means of glutaraldehyde
or another cross-linking agent in a manner known per se. A
particularly preferred resin is a weakly basic anion ex-
change resin which may be a polystyrene-, acrylic- or
phenol-formaldehyde-type resin. Examples of commercially
35 available polyacrylic-type resins are Lewatit E 1999/85
(registered trademark of Bayer, Federal Republic of Ger-
., .:
,,. ~ : :~, :- .. : ,,
: .
.
- ' ' :. ~ , :- ~:

~()9~1/11~29 ~t ~7'~ PCT/D~90/~0127
many) and Duolite ES-568 (registered trademark of ~ohm &
Haas, Federal Republic of Germany). Immobilizatlon of en-
zymes to this type of resin may be carried out according
to EP 140 542~ Immobilization to phenyl-formaldehyde-type
5 resins may be conducted as described in DK 85/878.
Another convenient material for immobilizing enzymes is an
inorganic support, such as a silicate. The enzymes may be
attached to the support by adsorption or by covalent
coupling, eg. as described in K. Mosbach, ed., op.cit.
The reaction of the compound of the general formula III
with the compound of the general formula IV in step a) of
the process of the invention may advan~ageously proceed at
a low pressure such as a pressure below about 0.05 bar, in
15 particular below about O.Ol bar. The reaction temperature
is not critical, but is conveniently in the range of about
20-100C, preferably about 30-80~C. For the reaction of
short-chain fatty acids with amino acid amides, the reac-
tion may suitably proceed at room temperature.
The choice of solvent in which the reaction of the com-
pound (III) with the compound (IV) is of some importance.
Firstly, the polarities of the enzyme substrate (the fatty
acid or fatty ester III) and the nucleophile (the amino
25 acid amide IV) differ widely. Secondly, water-soluble or-
ganic compounds may inactivate the enzyme used in the pro-
cess. In a preferred embodiment of the process of the in-
vention, the reaction of the compound (III) with the com-
pound (IV) proceeds in a substantially non-aqueous medium,
30 e.g. a suitable organic solvent (such as ethylmethyl
ketone), or substantially in the absence of a solvent
which is to say that the compound (III) acts as a solvent
for the compound (IV). In this case, an excess of the com-
pound (III) may be added to the reaction mixture. It
35 should be noted that a minor amount of water may be
present bound to the enzyme to ensure a satisfactory reac-
,,: - , : , ~-
~ . ~ ' , r , ' "
' ' ' ~ ,
. ~ . ' .

W09~ 29 PCT/D~91)/00127
7'~ 1 l6
tivity and half-life of the enzyme. By continuously remov-
ing water or alcohol by azeotropic distillation or, if no
solvent is used, in vacuo, it is possible to shift the
equilibrium in the reaction of the compound (III) with the
5 compound (IV) towards formation of the compound (I), thus
improving the yield of the compound (I).
The yield of the end product (I) has also been found to be
dependent on the concentration of the enzyme used in step
lO a) of the present process in that the yield increases with
increasing amounts of enzyme added to the reaction mix-
ture. An advantageous enzyme concentration for the present
purpose is in the range of 1-50% w/w.
15 Suitable salts of the N-acylated amino acids produced by
the process of the invention may be prepared in a manner
known per se, such as by reacting a compound (II) with an
appropriate base, e.g. an alkali metal or alkaline earth
metal hydroxide. Examples of such salts are the sodium,
20 potassium, calcium and magnesium salts, in particular the
sodium salt.
Compounds of the general formulae I and II may convenient-
ly be included in cleaning compositions which may be for-
25 mulated in any convenient way, such as a liquid, powder,etc. Typical examples of cleaning compositions are laundry
detergents, e.g. heavy-duty or light-duty detergents,
dishwash detergents and hard surface cleaners.
30 The cleaning composition may comprise one or more other
surface-active agents, such as anionic surfactants (e.g.
linear alkyl benzene sulfonates, fatty alcohol sulfates,
fatty alcohol ether sulfates, ~-olefin sulfonates or
soaps), non-ionic surfactants (e.g. alkyl polyethylene
35 glycol ethers, nonylphenol polyethylene glycol ethers,
fatty acid esters of sucrose and glucose, alkyl glycosides
:: ~ . : . - ............................... . : .
: -... .. . . - :

~()9~ '9 ~ 7,~ PCT/DK90/00127
or esters of polyox~ethylated alkyl glycosides), cationic
surfactants and/or zwitterionic surfactants.
Liquid and powder detergents may be formulated substan-
5 tially as described in "Frame formulations for liquid/pow-
der heavy-duty detergents" in J. Falbe, Surfactants in
Consumer Products. Theorv TechnoloaY and Ap~lication,
Springer Verlag, 1987, by replacing all or part of the
surfactant in the detergent formulation by one or more N-
lO acyl amino acid amides as described above.
Thus, as described in Falbe, op. cit., a liquid heavy-duty
detergent may comprise anionic surfactants, non-ionic sur-
factants, suds controlling agents, enzymes, foam boosters,
15 builders, formulation aids, optical brighteners, stabi-
lizers, fabric softeners, fragrances, dyestuffs and water. -
Similarly, a powder heavy-duty detergent may comprise
anionic surfactants, non-ionic surfactants, suds con-
trolling agents, foam boosters, chelating agents, ion ex-
20 changers, alkalis cobuilders, bleaching agents, bleach ac-
tivators, bleach stabilizers, fabric softeners, anti-
redeposition agents, enzymes, optical brighteners, anti-
corrosion agents, fragrances, dyestuffs and blueing
agents, formulation aids, fillers and water.
Compounds (I) and (II~ prepared by the process of the in-
vention may advantageously be employed in personal care
compositions of the invention are shampoos, toothpastes,
shaving creams, liquid soaps, skin creams or body lotions.
A shampoo composition of the invention (e.g. a hair or
body shampoo) may contain the compound (I) or (II) as the
main or sole surfactant, in which case it i5 usually pres-
ent in an amount of 1-25% by weight of the composition.
35 However, the composition may further comprise an anionic
surfactant in an amount of 5-35%, in particular 10-25%, by
. .
:: . - . -., :: . -
- . .
~- .-:: - . - :- :
. .,, ~ ~ . . .:

09~ 29 PCT/~K90/00127
~ 18
weight of the composition.
Examples of suitable anionic surfactants for inclusion in
shampoos are alkyl ether sulphonates, alkyl sulphates
5 (e.g. with 10-22 carbon atoms in the alkyl chain), alkyl
polyethoxy sulphonates (e.g. with 10-18 carbon atoms in
the alkyl chain), ~-olefin sulphonates (e.g. with 10-24
carbon atoms), ~-sulphocarboxylates (e.g. with 6-20 carbon
atoms) and esters thereof (prepared with, e.g., Cl-C14 al-
10 cohols), alkyl glyceryl ether sulphonates (e.g. with lO-
18 carbon atoms), fatty acid monoglyceride sulphates and
sulphonates, alkyl phenol polyethoxy ether sulphates (e.g.
with 8-12 carbon atoms in the alkyl chain), 2-acyloxy-1-
sulphonates (e.g. with 2-9 carbon atoms in the acyl group
15 and 9-22 carbon atoms in the alkane moiety) and ~-alkyloxy ~-
alkane sulphonates (e.g. with 1-3 carbon atoms in the
alkyl group and 8-20 carbon atoms in the alkane moiety).
The shampoo composition of the invention may additionally
20 comprise a foam booster, for instance a fatty acid di-
alkanol amide, an N-acyl amino acid or a betain derivative
in an amount of 0.1-20~ by weight of the composition.
If a higher viscosity of the shampoo composition is de-
25 sired, it is possible to include a suitable thickener such
as, for instance, carboxy methyl cellulose or, if the
anionic surfactant is an alkyl ether sulphonate, the vis-
cosity may be regulated by means of a salt, e.g. NaCl.
30 When the N-acyl amino acids or amides prepared by the pro-
cess of the invention are included in toothpaste composi~
tion, it may contain the compounds in an amount of 1-20~
by weight, in addition to conventional ingredients such as
gelling agents, thickeners, abrasives, bulk agents and the
35 like.
. ~

~'090/1~29 PCTIDK90/00127
7')~
1.9
When the compounds tI) or (II) prepared by the process of
the invention are included in a liquid soap composition,
it may contain the surface-active compounds (I) or (II) in
an amount of 1~20%, in addition to conventional ingredi-
5 ents such as anionic surfactants, foam boosters and thelike.
Similarly, a shaving cream composition may contain 1-20%
by weight of the compounds (I) or (II) in addition to con-
10 ventional ingredients.
A skin cream or body lotion may contain 0.1-10% by weight
of the compounds (I) or (II) in addition to conventional
ingredients such as oils, fatty acids and esters thereof,
15 fatty alcohols, water, perfume, and an additional emul-
sifier.
The invention is further illustrated by the following
examples which are not in any way intended to limit the
20 scope or spirit of the invention.
Examples
25 General procedures
Satisfactory lH and 13C NMR-spectra were obta-ined for all
compounds. The spectra were recorded on a Bruker WM 400
Spectrometer with TMS as internal standard. Prèparative
30 liquid chromatography was performed on SiO2 with a
gradient of n-pentane, ethylacetate and methanol as
eluent.
. . .. ... ... .

~ 09~ '9 PCT/D~9~/00l27
7'3'`~1
Exam le l
Preparation of N-decanoyl phenyl glycine amide:
5 To melted decanoic acid (6.0 g, 34.8 mmol) phenyl glycine
amide (1.0 g, 6.7 mmol) was added. Then immobilized lipase
from Candida antarctica (100 mg) was added and the mixture
stirred for 48 hrs. at 70C. The product was isolated in a
yield of 56~ after purification by preparative chromato-
10 graphy.
H NMR S: 0.87 (3H,t), 1.25 (12H,S), 1.60 (2H,m), 2.23(2H,t), 5.60 (lH,d), 5.82 (lH,S), 6.28 (lH,S), 6.96
(lH,d).
15 Example 2
Preparation of N-decanoyl phenyl glycine amide:
To decanoic acid (5.68 g, 33.0 mmol) in ethylmethyl ketone
20 (50 ml) was phenyl glycine amide (4 g, 26.8 mmol) and
immobilized lipase from Candida antarctica (1.5 g) added.
After 20 h the enzyme was filtered off, the solvent re-
moved in vacuum and the crude product purified by chroma-
tography yielding 1.8 g of product.
Example 3
Preparation of N-hexadecanoyl alanine amide:
3~0 To methylhexadecanoat (0.25 g, 0.9 mmol) in ethylmethyl
ketone (5 ml) was alanine amide hydrobromide (0.5 g, 3.0
mmol) triethylamine (0.44 ml) and immobilized lipase from
Candida _ tarctica (0.2 g) added. The mixture was stirred
for 24 h at room temperature, then filtered free from en-
35 zyme and purified by chromatography yielding 25% of pro-
duct.
-
'. : :-`' ' ` ' ~ ,~' ,
' , ~; ' : :

~C`~ 1. PCI /DK90/00127
Exam~le 4
Preparation of N-decanoyl alanine amide: `
To decanoic acid (0.10 g, 0.59 mmol) in ethylmethyl ketone
(2.5 ml) were alanine amide hydrobromide (0.25 g, 1.5
mmol) triethylamine (0.22 ml) and immobilized lipase from
Candida antarctica (0.1 g) added. The mixture was stirred
10 for 24 h at room temperature, then filtered free from en-
zyme and the yield determined to 25%.
Exam~le 5
15 Preparation of N-decanoyl leucine amide:
To methyldecanoat (0.1 g, 0.59 mmol) in ethylmethyl ketone
(2.5 ml) were leucine amide hydrobromide (0.25 g, 1.45
mmol), triethylamine (0.18 ml) and immobilized lipase from
20 Candida antarctica (0.1 g) added. After (20 -) the lipase
was removed and the yield was determined to 25%.
Example 6
25 Preparation of N-decanoyl phenyl glycine amide:
To methyldecanoat (100 ~1, 0.45 mmol) in ethylmethyl ke-
tone (1 ml) was phenyl glycine amide (0.14 g, 0.9 mmol)
and immobilized lipase from Candida antarctica (0.1 g)
30 added. After 24 h the product was isolated in a 50% yield.
Exam~le 7
Preparation of N-decanoyl alanine:
To a suspension of N-decanoyl alanine amide (1 g, 3.9
: ; . ~ , : , ~ :: , -: . :

~()9(~ PCT/DK90/00127
~ 7~1` i 22
mmol) in phosphate buffer (150 ml, pH=7.0) was carboxy-
peptidase from yeast (9o mg) added. After 24 h no starting
material was left giving a 90% yield of product.
5 Example 8
Sodium salts of N-acylated amino acids were prepared by
dissolving each N-acylated amino acids in the smallest
possible amount of 99% ethanol (about 2 ml per gram). When
10 the compounds were completely dissolved, optionally with a
little heating, an equivalent amount of 6 M NaOH was
added. The resulting sodium salt was precipitated by the
addition of acetone (about 20 ml per gram). The precipi-
tated product was filtered off and dried in vacuo.
Example_9
The ability to reduce the surface tension of water was
tested for the following compounds prepared by the present
20 process:
N-decanoyl alanine, sodium salt (ClOAlaONa)
N-dodecanoyl alanine, sodium salt (Cl2AlaONa)
N-tetradecanoyl alanine, sodium salt (Cl4AlaONa)
25 N-hexadecanoyl alanine, sodium salt (C16AlaONa)
N-octadecanoyl alanine, sodium salt (Cl8AlaONa)
N-decanoyl leucine, sodium salt (ClOLeuONa)
N-dodecanoyl leucine, sodium salt (Cl2LeuONa)
N-tetradecanoyl leucine, sodium salt (Cl4LeuONa)
N-hexadecanoyl leucine, sodium salt (Cl6LeuONa)
5 N-octadecanoyl leucine, sodium salt (Cl8LeuONa)
N-decanoyl phenylalanine, sodium salt (ClOPheONa)
N-dodecanoyl phenylalanine, sodium salt (Cl2PheONa)
N-tetradecanoyl phenylalanine, sodium salt ~C14PheONa)
N-hexadecanoyl phenylalanine, sodium salt (Cl6PheONa)
5 N-octadecanoyl phenylalanine, sodium salt (Cl8PheONa)
,: , . ,. -, .

~09(~ 29 PC~-/DK90/00127
~C~:7'3¢~
23
Reduction in the surface tension of water produced by the
test compounds listed above was measured on a Xruss Digi-
tal tensiometer model K 10 at 25C. The minimum surface
5 tension ( ~ min) was determined as the lowest value
measured for each of the test compounds.
The critical micelle concentration [cmc] (the concentra-
tion at which surface-active compounds begin to form
10 micelles in water; this concentration is indicative of the
concentration of a surface-active compound needed to pro-
duce a detergent effect) of the test compounds was deter-
mined from a plot of the surface tension against the log-
arithm of the molar concentration.
The results are shown in the following table:
Compound ~ min cmc (mol/l)
C1OAlaONa 47.4 4.74 10 2
C12AlaONa 42.5 6.98 10-3
C14AlaONa 40.8 1.63 10 3
C16AlaONa 42.4
C18AlaONa 39.0
C1OLeuONa 37.0 3.17 10-3
C12LeuONa 34.0 2.28 10 3
C14LeuONa 33.8 7.08 10-4
C16LeuONa 35.5
C18LeuONa 38.1
c1OPheONa 36.9 5.17 10 3
C12PheONa 34.5 1.20 10-3
C14PheONa 38.0 4.04 10-4
C16PheONa 37.5 1,12 10-4
C18PheONa 36.9 1.63 10-4
It appears from the table that all the test compounds are
able to reduce the surface tension of water to a consider-
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7~ 1 24
able degree. The minimum surface tension is substantially
the same for compounds containing leucine and phenyl-
alanine residues. The minimum surface tension is generally
higher for compounds containing an alanine residue. The
5 chain length of the acyl group is of little importance to
the minimum surface tension where compounds containing
leucine and phenylalanine residues are concerned as is the
case with compounds containing an alanine residue when the
acyl group has a chain length of 12 carbon atoms or more.
The critical micelle concentration could not be determined
for N-hexanoyl alanine, sodium salt, N-octanoyl alanine,
sodium salt, N-hexanoyl leucine, sodium salt, and N-
octanoyl leucine, sodium salt, as the limit of solubility
15 of these compounds was reached before the cmc. For the
other test compounds, it appears from the table that the
cmc decreases with an increasing acyl group chain length,
except for the N-octanoyl phenylalanine, sodium salt. The
cmc is generally higher for compounds containing an
20 alanine residue, whereas compounds containing a leucine or
phenylalanine residue show substantially the same cmc.
.
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Representative Drawing