AU636894B2 - An enzyme-catalyzed process for preparing n-acyl amino acids and n-acyl amino acid amides - Google Patents

Description

OPI DATE 18/12/90

PCT

AOJP DATE 07/02/91

INTERNATIONAL

APPLN. ID 57485 PCT NUMBER PCT/DK90/00127

(PCT)

(51) International Patent Classification 5 (11) International Publication Number: WO 90/14429 C12P 13/02, A61K 7/075, 7/15 Al A61K 7/16, 7/48, C11D 3/32 (43) International Publication Date: 29 November 1990(29.1190) (21) International Application Number: PCT/DK90/00127 (74) Common Representative: NOVO NORDISK A/S; Patent Department, Novo Alle, DK-2880 Bagsvaerd (DK).

(22) International Filing Date: 22 May 1990 (22.05.90) (81) Designated States: AT (European patent), AU, BE (Euro- Priority data: pean patent), CA, CH (European patent), DE (Euro- 2558/89 25 May 1989 (25.05.89) DK pean patent)*, DK (European patent), ES (European patent), FI, FR (European patent), GB (European patent), IT (European patent), JP, KR, LU (European patent).

(71) Applicant (for all designated States except US): NOVO NL (European patent), NO, SE (European patent), US.

NORDISK A/S [DK/DK]; Novo Alle, DK-2880 Bagsvaerd (DK).

Published (72) Inventors; and With international search report.

Inventors/Applicants (for US only) GODTFREDSEN, Sven, Erik [DK/DK]; Smedegade 15B, DK-3500 Varlose BJORKLING, Frederik [SE/SE]; Hbvitsmansgatan 2, S-252 37 Helsingborg 6 3 6 8 9 4 (54)Title: AN ENZYME-CATALYZED PROCESS FOR PREPARING N-ACYL AMINO ACIDS AND N-ACYL AMINO ACID AMIDES

RCO-NH

R1NH 2 0

RCO-NH

1

OH

0

RCOOR

2

(III)

NH

2 R1

NH

2

R

1 0

(II)

(IV)

(57) Abstract Compounds of general formula or wherein R is an optionally substituted alkyl group with 3-23 carbon atoms, and

R

1 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 RCOOR 2 wherein R 2 is H or an alkyl group with 1-6 carbon atoms, and R is as defined above, with a conmpound of general formula wherein RI 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 by means of a second enzyme capable of selectively cleaving amide bonds, e.g. a carboxypeptidase, resulting in the compound (II).

See back of page WO 90/14429 PCT/DK90/0127 AN ENZYME-CATALYZED PROCESS FOR PREPARING N-ACYL AMINO ACIDS AND N-ACYL AMINO ACID AMIDES FIELD OF INVENTION The present invention relates to an enzyme-catalyzed process for preparing N-acylated amino acid amides and N-acylated amino acids, and a cleaning composition and personal care composition containing an N-acyl amino acid amide.

BACKGROUND OF THE INVENTION Surface-active agents constitute an extremely important class of industrial chemicals which have a wide variety of uses, for instance as detergents for washing purposes, as 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 substances which are characterized by the presence of hydrophobic and hydrophilic regions within each individual surfactant molecule and which owe their ability to reduce surface tension to this particular structure. For instance, surface-active agents are able to effect solution of otherwise water-insoluble substances in water by interacting with such substances at the hydrophobic region of the surfactant molecule and with water at the hydrophilic region of the surfactant molecule.

Because of the ready availability of hydrophilic as well as hydrophobic substances and the well-developed chemical technologies for combining such substances to form surface-active agents, a large number of surface-active agents are at present commercially available. Most such surfactants are based on petrochemically derived products which are attractive and owe their widespread use to their WO 90/14429 PCDK90/00127 2 low cost. However, certain important surface-active agents are composed of naturally occurring compounds such as fatty acids and glycerol (commercially available as monoand diglycerides), mainly for application as emulsifiers 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, a quaternized ammonium moiety or a glycerol moiety with an alkyl chain as is the case with the linear alkyl surfactants, the quarternized alkyl amines or the monoglycerides, respectively. When designing a surfactant molecule, the detailed molecular architecture of the compounds is a major concern, care being taken to achieve a precise balance between the hydiophobic and hydrophilic regions of the surfactant molecules as well as to achieve a favourable spatial arrangement of these individual regions of the molecules. Apart from this, the possibility of producing 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 acylated amino acids (also known as lipoamino acids) which exhibit surface-active properties due to the hydrophilic properties of the amino acid moiety of the compounds and the bydrophobic properties of the fatty acid moiety of the compounds. The balance between hydrophilic and hydrophobic properties might be varied by modifying the amino acid and/or the fatty acid by adding one or more substituents.

Acylated amino acids may be prepared from relatively inex- WO 90/14429 PCr/DK90/00127 3 pensive starting materials and have the advantage of being biodegradable into their naturally occurring constituent parts so that they do not constitute an environmental hazard. Acylated amino acids are known to be useful as detergents and emulsifiers in cosmetics due to their surfaceactive properties.

At present, acylated amino acids are prepared by organic synthesis. One conventional method for producing the compounds (briefly referred to in GB 1 483 500) is to acylate amino acids with a higher fatty acid chloride in an aqueous alkaline medium. This method is stated to have the disadvantage that a chloride salt is left in the reaction mixture which makes it necessary to remove the salt in order to preserve a good detergency of the compounds. Another method, also disclosed in GB 1 483 500, for producing 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 base.

General disadvantages of methods of organic synthesis of N-acyl amino acids are that they tend to be rather timeconsuming and that there is a considerable risk that undesirable side products will be formed during the reaction process which makes the purification of the desired end products more difficult. As a result of this, the preparation of N-acyl amino acids by conventional organic synthesis is rather expensive for which reason acylated amino acids have not found as widespread a commercial application as surfactants based on petrochemically derived products.

Amides of N-acyl amino acids are also known surface-active substances for use in cosmetics, and as antioxidants and antibacterial agents, cf. JP-B 52-18691, according to WO 90/14429 PCr/K90/00127 4 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 compound. The reaction is conducted using a hydrocarbon as 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.

It is therefore an object of the present invention to provide an enzyme-catalysed process for producing N-acyl amino acids and N-acyl amino acid amides which is simpler and less time-consuming to carry out than the conventional processes for preparing such compounds, and which results in satisfactory yields of the acylated amino acids and amino acid amides.

SUMMARY OF THE INVENTION Accordingly, the present invention relates to a process for preparing a compound of the general formula I

RCO-NH

RNH

2 (I) 0 or II

RCO-NH

K OH (II) 0 wherein R is an alkyl group with 3-23 carbon atoms, optionally substituted by a branched or straight-chain, saturated or unsaturated, aliphatic or aromatic hydrocar- WO 90/14429 PCT/DK90/00127 bon group, and R 1 is hydrogen or a branched or straightchain, saturated or unsaturated, aliphatic or aromatic hydrocarbon group, optionally substituted by alkyl with 1-20 carbon atoms, -OH, -NH 2 or SH, or an alkali metal or alkaline earth metal salt thereof, the process comprising a) reacting a compound of the general formula III

RCOOR

2

(III)

wherein R 2 is H or an alkyl group with 1-6 carbon atoms, and R is as defined above, with a compound of the general formula IV

NH

2

NH

2

(IV)

0 wherein R 1 is as defined above, in the presence of an enzyme 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 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.

In a further aspect, the invention relates to a cleaning composition which comprises an effective amount of a compound of the general formula I as defined above.

WO 90/14429 PC/DK90/00127 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.

DETAILED DISCLOSURE OF THE INVENTION In the general formulae I and II, R is preferably an unsubstituted alkyl group with 6-20 carbon atoms. Thus, RCOmay suitably be selected from the group consisting of hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, dodecanoyl, tetradecanoyl, hexadecanoyl, octadecanoyl, eicosanoyl, docosanoyl, cis-9-octadecanoyl and 9,12-octadecadienoyl.

R

1 is preferably a methyl, ethyl, propyl, isopropyl, methylthio, -CH 2

-CH

2

-S-CH

3 benzyl, hydroxybenzyl, indolyl or alkylguanidine group.

The compound of the general formula IV is preferably 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, glutamic acid amide and 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 appropriate aldehyde cyanide and ammonia.

Thus, some particularly preferred compounds of the general formula I are selected from the group consisting of Nhexanoyl alanine amide, N-octanoyl alanine amide, N-decanoyl alanine amide, N-dodecanoyl alanine amide, N-tetradecanoyl alanine amide, N-hexadecanoyl alanine amide, N- WO 90/14429 PCT/DK9O/0O1 27 7 octadecanoyl alanine amide, N-hexanoyl leucine amide, Noctanoy. leucine amnide, N-decanoyl leucine amnide, Ndodecanoyl leucine amide, N-tetradecanoyl leucine amnide, N-hexadecanoyl leucine amide, N-octadecanoyl leucine amnide, N-hexanoy2. phenylalanine amide, N-octanoyl phenylalanine amide, N-decanoyl pheriylalanine amide, N-dodecanoyl phenylalanine amide, N-tetradecanoyl phenylalanine amide, N-hexadecanoy. phenylalanine amnide, N-octadecanoy.

phenylalaniie amide, N-hexanoyl phenyiglycine amide, Noctanoyl phenyiglycine amnide, N-decanoyl phenyiglycine amide, N-dodecanoyl phenyiglycine amide, N-tetradecanoyl phenyiglycine amide, N-hexadecanoyl phenyiglycine amide, N-octadecanoyl phenyiglycine amide, N-hexancj1 lysine amnide, N-octanoy2. lysine amide, N-decanoyl lysine amnide, N-dodecanoyl lysine amide, N-tetradecanoyl lysine amnide, N-hexadecanoyl lysine amide, N-octadecanoyl lysine amide, N-hexanoyl glycine amiide, N-octanoyl glycine amide, Ndecanoyl glycine amride, N-dodecanoyl glycine amide, Ntetradecanoyl glycine amnide, N-hexadecanoyl glycine amide, N-octadecanoy. glycine amuide, N-hexanoyl valine amnide, Noctanoyl valine amide, N-decanoy. valine amide, N-dodecanoyl valine amide, N-tetradecanoyl valirie arnids, Nhexadecanoyl valine amide, N-octadecanoy. valine amide, Nhexanoyl tryptophan amide, N-octanoy. tryptophan amnide, Ndecanoyl tryptophai amide, N-dodecanoyl tryptophan amnide, N-tetradecanoyl tryptophan amide, N-hexadecanoy. tryptophan amnide, N-o'ctadecanoyl tryptophan amide, N-hexanoy.

arginine amnide, N-octanoyl arginine amide, N-decanoyl arginine amide, N-dodecanoyl arginine amide, N-tetradecanoyl arginine amide, N-he~adecanoyl arginine amide, Noctadecanoyl arginine amide, N4-hexanoy. histidine amide, N-octanoyl histidine amnide, N--decanoyl histidine amide, Ndodecanoyl histirline amide, N-tetradecanoyl histidine amnide, N-hexadecanoyl histidine amide, N-octadecanoyl histidine amide, N-hexanoyl cysteine amide, N-octanoyl cysteine amide, N-decanoyl cysteine amide, N-dodecanoyl WO 90/14429 WO 9014429PCT/DK9O/0O1 27 8 cysteine amide, N-tetradecanoyl cysteine amide, Nhexadecanoyl 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, Nhexanoyl isoleucine amide, N-octanoyl isoleucine amide, Ndecanoyl isoleucine amide, N-dodecanoyl isoleucine amide, N-tetradecanoyl isoleucine amide, N-hexadecaioyl isoleucine amide, N-octadecanoyl isoleucine amide, N-hexanoyl asparagine amide, N-octanoyl asparagine amide, N-decarioyl asparagine amide, N-dodecanoy. asparagine amide, N-tetradecanoyl asparagine amide, N-hexadecanoyl asparagine amide, N-octadecanoyl asparagine amide, N-hexanoyl aspartic acid amide, N-octanoyl aspartic acid amide, Ndecanoyl aspartic acid amnide, N-dodecanoy-I 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 gilutamic acid amide, N-decanoyl glutamic acid amnide, N-dodecanoy.

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-decanoyl ornithine axmide, Ndodecanoyl ornithine amride, N-tetradecanoyl ornithine amide, N-hexadecanoyl ornithine amide, N-octadecanoyl ornithine amide, or an alkali metal or alkaline earth metal salt thereof.

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, Nhexadecanoyl alanine, N-octadecanoyl alanine, N-hexanoyl leucine, N-octanoyl leucine, N-decanoyl leucine, Ndodecanoyl leucine, N-tetradecanoyl leucine, N-hexa- WO 90/14429 WO 9014429PCTr/DK90/00i 27 9 decaricyl leucine, N-octadecanoyl leucine, N-hexanoy. phenylalanine, N-octanoyl phenylalanine, N-decanoyl phenylalanine, N-dodecanoyl phenylalanine, N-tetradecanoyl phenylalanine, N-hexadecanoyl phenylalanine, N-octadecanoyl phenylalanine, N-hexanoyl phenyiglycine, Noctanoyl phenyiglycine, N-decanoyl phenyiglycine, Ndodecanoyl phenyiglycine, N-tetradecanoyl phenyiglycine, N-hexadecanoyl phenyiglycine, N-octadecanoyl phenyiglycine, N-hexanoyl lysine, N-octanoyl lysine, Ndecanoyl lysine, N-dodecanoyl lysine, N-tetradecanoyl lysine, N-hexadecancyl lysine, N-octadecanoyl lysine, Nhexanoyl glycine, N-octanoyl glycine, N-decanoyl glycine, N-dodecanoyl glycine, N-tetradecanoyl glycine, Nhexadecanoyl glycine, N-octadecanoy. glycine, N-hexanoyl valine, N-octanoy. valine, N-decanoyl valine, N-dodecaioyl valine, N-tetradecanoyl valine, N-hexadecanoyl valine, Noctadecanoyl valine, N-hexanoy3. tryptophan, N-octanoyl tryptophan, N-decanoyl tryptophan, N-dodecanoyl tryptophan, N-tetradecanoyl tryptophan, N-hexadecanoyl tryptophan, N-octadecanoyl tryptophan, N-hexanoyl arginine, N-octanoyl arginiie, N-decanoyl arginine, Ndodecanoy. arginiie, N-tetradecarioyl arginine, Nhexadecanoyl arginine, N-octadecanoy. arginine, N-hexanoyl histidine, N-octanoyl histidine, N-decanoyl histidine, Ndodecanoyl histidine, N-tetradecanoyl histidine, Nhexadecanoyl histidine, N-octadecanoy. histidine, Nhexanoyl cysteine, N-octanoyl cysteine, N-decanoyl cysteine, N-dodecanoyl cysteine, N-tetradecanoyl cysteine, N-hexadecanoyl cysteine, N-octadecanoyl cysteine, Nhexanoyl glutamine, N-octanoyl glutainine, N-decanoyl glutarnine, N-dodecanoyl glutamine, N-tetradecanoyl glutamine, N-hexadecanoyl glutamine, N-octadecanoyl glutamine, N-hexanoyl isoleucine, N-octanoyl isoleucine, N-decarnoyl isoleucine, N-dodecanoy. isoleucine, Ntetradecanoyl isoleucine, N-hexadecanoyl isoleucine, Noctadecanoyl isoleucine, N-hexanoyl asparagine, N-octanoyl WO 90/14429 PCr/DK90/00127 asparagine, N-decanoyl asparagine, N-dodecanoyl asparagine, N-tetradecanoyl asparagine, N-hexadecanoyl asparagine, N-octadecanoyl asparagine, N-hexanoyl aspartic acid, N-octanoyl aspartic acid, N-decanoyl aspartic acid, 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, Ntetradecanoyl glutamic acid, N-hexadecanoyl glutamic acid, N-octadecanoyl glutamic acid, N-hexanoyl ornithine, Noctanoyl 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 resulting compounds or (II) may be in the form of a racemic mixture or in optically active form.

Enzymes which may be useful as catalysts in the process of the invention should be selected according to the following criteria: their ability to catalyse the formation of an amide bond; their ability to use a fatty acid or fatty ester as substrate; and their ability to use an 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.

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 acid-s from fatty acids and amino acids. However, there is no indication of the reaction conditions under which the WO 90/14429 P(7/DK90/00127 11 synthesis takes place, nor is there any indication of the reaction times or the final yield of lipoamino acids provided by the synthesis. Additionally, it appears that the enzyme described by Shintani et al. is specific to lipoamino 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.

EP 298 796 discloses the use of acyl transferases, including lipase, to catalyse a process for preparing N-substituted 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, the present process is distinguished from the process described in EP 298 796 by using amino acid amides as starting materials which are monoionic compounds and as such chemically distinct from amino acids which are zwitterionic compounds. There would be no reason to expect that 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 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, resulting 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 peptidases), esterases or proteases, in particular lipases which may be defined as enzymes catalyzing reactions involving ester bonds, e.g. hydrolysis, synthesis and/or ex- WO 90/14429 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 Aspergillus, Enterobacterium, Chromobacterium, Geotricium or Penicillium. Preferred lipases for use according to the invention are those produced by species of Mucor (e.g.

Lipozyme

TM

produced by Mucor miehei), Humicola, Pseudomonas 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 Mikroorganismen in accordance with the provisions of the Budapest 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 numbers DSM 3908 and DSM 3909.

Pseudomonas cephacia, deposited oi 30 January 1987, with the number 3959.

Humicola lanuginosa, deposited on 13 August 1986 and 4 192, May with the deposit numbers 3819 and 4109, respectively.

Humicola brevispora, deposited on 4 May 1987, with the deposit 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 under aerobic conditions in a nutrient medium containing assimilable carbon and nitrogen sources as well as essen- WO 90/14429 PCT/DK90/00127 13 tial minerals, trace elements etc., the medium being composed according to established practice. After cultivation, liquid enzyme concentrates may be prepared by removing insoluble materials, e.g. by filtration or centrifugation, after which the broth is concentrated by evaporation 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 drying such as spray-drying in accordance with well-known methods.

Additional lipases may be obtained from the following strains which are publicly available without restriction from the Centraalbureau voor Schimmelculturen (CBS), American Type Culture Collection (ATCC), Agricultural Research Culture Collection (NRRL) and Inscitute of Fermentation, 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 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 foliorum, CBS 5234 and ATCC 18820.

It is known to produce lipase by recombinant DNA techniques, 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 NH 2 group selectively by an enzyme-catalysed process by means of an enzyme which is able to cleave amide bonds. An WO 90/14429 PCFDK90/00127 14 example of such an enzyme is carboxypeptidase such as carboxypeptidase Y, which is produced by Saccharomvces cerevisiae.

When employed in step a) or b) of the process of the invention, 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 process. Immobilization procedures are well known (cf. for instance K. Mosbach, ed., "Immobilized Enzymes", Methods in Enzymoloqy 44, Academic Press, New York, 1976) and include cross-linking of cell homogenates, covalent coupling to insoluble organic -r inorganic supports, entrapment in gels and adsorption to ion exchange resins or other adsorbent materials. Coating on a particulate support may also be employed (cf. for instance A.R. Macrae and R.C.

Hammond, Biotechnology and Genetic Engineering Reviews 3, 1985, p. 193. Suitable support materials for the immobilized enzyme are, for instance, plastics polystyrene, polyvinylchloride, polyurethane, latex, nylon, teflon, dacron, polyvinylacetate, polyvinylalcohol or any siutable copolymer thereof), polysaccharides agarose or dextran), ion exchange resins (both cation and anion exchange resins), silicon polymers siloxane) or silicates glass).

It is preferred to immobilize the enzymes on an ion exchange resin by adsorbing the enzymes to the resin or by 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 exchange resin which may be a polystyrene-, acrylic- or phenol-formaldehyde-type resin. Examples of commercially available polyacrylic-type resins are Lewatit E 1999/85 (registered trademark of Bayer, Federal Republic of Ger- WO 90/14429 PCT/DK90/00127 many) and Duolite ES-568 (registered trademark of Rohm Haas, Federal Republic of Germany). Immobilization of enzymes to this type of resin may be carried out according to EP 140 542. Immobilization to phenyl-formaldehyde-type 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 advantageously proceed at a low pressure such as a pressure below about 0.05 bar, in particular below about 0.01 bar. The reaction temperature is not critical, but is conveniently in the range of about 20-100°C, preferably about 30-80°C. For the reaction of short-chain fatty acids with amino acid amides, the reaction may suitably proceed at room temperature.

The choice of solvent in which the reaction of the compound (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 acid amide IV) differ widely. Secondly, water-soluble organic compounds may inactivate the enzyme used in the process. In a preferred embodiment of the process of the invention, the reaction of the compound (III) with the compound (IV) proceeds in a substantially non-aqueous medium, 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 In this case, an excess of the compound (III) may be added to the reaction mixture. It should be noted that a minor amount of water may be present bound to the enzyme to ensure a satisfactory reac- NNIO 90/14429 PCT/DK90/00127 16 tivity and half-life of the enzyme. By continuously removing 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 compound (IV) towards formation of the compound thus improving the yield of the compound The yield of the end product has also been found to be dependent on the concentration of the enzyme used in step a) of the present process in that the yield increases with increasing amounts of enzyme added to the reaction mixture. An advantageous enzyme concentration for the present purpose is in the range of 1-50% w/w.

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, potassium, calcium and magnesium salts, in particular the sodium salt.

Compounds of the general formulae I and II may conveniently be included in cleaning compositions which may be formulated 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.

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, a-olefin sulfonates or soaps), non-ionic surfactants alkyl polyethylene glycol ethers, nonylphenol polyethylene glycol ethers, fatty acid esters of sucrose and glucose, alkyl glycosides WO 90/14429 PCT/DK90/00127 17 or esters of polyoxyethylated alkyl glycosides), cationic surfactants and/or zwitterionic surfactants.

Liquid and powder detergents may be formulated substantially as described in "Frame formulations for liquid/powder heavy-duty detergents" in J. Falbe, Surfactants in Consumer Products. Theory, Technology and Application, Springer Verlag, 1987, by replacing all or part of the surfactant in the detergent formulation by one or more Nacyl amino acid amides as described above.

Thus, as described in Falbe, op. cit., a liquid heavy-duty detergent may comprise anionic surfactants, non-ionic surfactants, suds controlling agents, enzymes, foam boosters, builders, formulation aids, optical brighteners, stabilizers, fabric softeners, fragrances, dyestuffs and water.

Similarly, a powder heavy-duty detergent may comprise anionic surfactants, non-ionic surfactants, suds controlling agents, foam boosters, chelating agents, ion exchangers, alkalis cobuilders, bleaching agents, bleach activators, bleach stabilizers, fabric softeners, antiredeposition agents, enzymes, optical brighteners, anticorrosion agents, fragrances, dyestuffs and blueing agents, formulation aids, fillers and water.

Compounds and (II) prepared by the process of the invention 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 a hair or body shampoo) may contain the compound or (II) as the main or sole surfactant, in which case it is usually present in an amount of 1-25% by weight of the composition.

However, the composition may further comprise an anionic surfactant in an amount of 5-35%, in particular 10-25%, by WO 90/14429 PCT/DK90/00127 18 weight of the composition.

Examples of suitable anionic surfactants for inclusion in shampoos are alkyl ether sulphonates, alkyl sulphates with 10-22 carbon atoms in the alkyl chain), alkyl polyethoxy sulphonates with 10-18 carbon atoms in the alkyl chain), a-olefin sulphonates with 10-24 carbon atoms), a-sulphocarboxylates with 6-20 carbon atoms) and esters thereof (prepared with, C 1

-C

14 alcohols), alkyl glyceryl ether sulphonates with 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-lsulphonates with 2-9 carbon atoms in the acyl group and 9-22 carbon atoms in the alkane moiety) and p-alkyloxy alkane sulphonates 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 comprise a foam booster, for instance a fatty acid dialkanol 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 desired, 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 viscosity may be regulated by means of a salt, e.g. NaCl.

When the N-acyl amino acids or amides prepared by the process of the invention are included in toothpaste composition, 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 like.

WO 90/14429 P/DK90/00127 19 When the compounds or (II) prepared by the process of the invention are included in a liquid soap composition, it may contain the surface-active compounds or (II) in an amount of 1-20%, in addition to conventional ingredients such as anionic surfactants, foam boosters and the like.

Similarly, a shaving cream composition may contain 1-20% by weight of the compounds or (II) in addition to conventional ingredients.

A skin cream or body lotion may contain 0.1-10% by weight of the compounds or (II) in addition to conventional ingredients such as oils, fatty acids and esters thereof, fatty alcohols, water, perfume, and an additional emulsifier.

The invention is further illustrated by the following examples which are not in any way intended to limit the scope or spirit of the invention.

Examples General procedures Satisfactory 1 H and 13C NMR-spectra were obtained for all compounds. The spectra were recorded on a Bruker WM 400 Spectrometer with TMS as internal standard. Preparative liquid chromatography was performed on SiO 2 with a gradient of n-pentane, ethylacetate and methanol as eluent.

WO 90/14429 PCT/DK90/00127 Example 1 Preparation of N-decanoyl phenyl glycine amide: 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 70"C. The product was isolated in a yield of 56% after purification by preparative chromatography.

1H NMR 6: 0.87 1.25 (12H,S), 1.60 2.23 5.60 5.82 6.28 6.96 (1H,d).

Example 2 Preparation of N-decanoyl phenyl glycine amide: To decanoic acid (5.68 g, 33.0 mmol) in ethylmethyl ketone (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 removed in vacuum and the crude product purified by chromatography yielding 3.8 g of product.

Example 3 Preparation of N-hexadecAnoyl alanine amide: To methylhexadecanoat (0.25 g, 0.9 mmol) in ethylmethyl ketone (5 ml) was alanine amide hydrobromide (0.5 g, mmol) triethylamine (0.44 ml) and immobilized lipase from Candida antarctica (0.2 g) added. The mixture was stirred for 24 h at room temperature, then filtered free from enzyme and purified by chromatography yielding 25% of product.

WO 90/14429 PCT/DK(90/00127 21 Example 4 Preparation of N-decanoyl alanine amide: To decanoic acid (0.10 g, 0.59 mmol) in ethylmethyl ketone ml) were alanine amide hydrobromide (0.25 g, mmol) triethylamine (0.22 ml) and immobilized lipase from Candida antarctica (0.1 g) added. The mixture was stirred for 24 h at room temperature, then filtered free from enzyme and the yield determined to Example Preparation of N-decanoyl leucine amide: To methyldecanoat (0.1 g, 0.59 mmol) in ethylmethyl ketone ml) were leucine amide hydrobromide (0.25 g, 1.45 mmol), triethylamine (0.18 ml) and immobilized lipase from Candida antarctica (0.1 g) added. After (20 h) the lipase was removed and the yield was determined to Example 6 Preparation of N-decanoyl phenyl glycine amide: To methyldecanoat (100 tl1, 0.45 mmol) in ethylmethyl ketone (1 ml) was phenyl glycine amide (0.14 g, 0.9 mmol) and immobilized lipase from Candida antarctica (0.1 g) added. After 24 h the product was isolated in a 50% yield.

Example 7 Preparation of N-decanoyl alanine: To a suspension of N-decanoyl alanine amide (1 g, 3.9 WO 90/14429 PCT/DK90/00127 22 mmol) in phosphate buffer (150 ml, pH=7.0) was carboxypeptidase from yeast (90 mg) added. After 24 h no starting material was left giving a 90% yield of product.

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 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 precipitated 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 process: N-decanoyl alanine, sodium salt (C 10 AlaONa) N-dodecanoyl alanine, sodium salt (C 12 AlaONa) N-tetradecanoyl alanine, sodium salt (C 14 AlaONa) N-hexadecanoyl alanine, sodium salt (C 16 AlaONa) N-octadecanoyl alanine, sodium salt (CAlaONa) N-decanoyl leucine, sodium salt (C 10 LeuONa) N-dodecanoyl leucine, sodium salt (Cl2LeuONa) N-tetradecanoyl leucine, sodium salt (C 14 LeuONa) N-hexadecanoyl leucine, sodium salt (C 16 LeuONa) N-octadecanoyl leucine, sodium salt (C 18 LeuONa) N-decanoyl phenylalanine, sodium salt (C 10 PheONa) N-dodecanoyl phenylalanine, sodium salt (C 12 PheONa) N-tetradecanoyl phenylalanine, sodium salt (C 14 PheONa) N-hexadecanoyl phenylalanine, sodium salt (C 16 PheONa) N-octadecanoyl phenylalanine, sodium salt (Cl 8 PheONa) WO 90/14429 PCT/DK90/00127 23 Reduction in the surface tension of water produced by the test compounds listed above was measured on a Kriss Digital tensiometer model K 10 at 25"C. The minimum surface tension y min) was determined as the lowest value measured for each of the test compounds.

The critical micelle concentration [cmc] (the concentration at which surface-active compounds begin to form micelles in water; this concentration is indicative of the concentration of a surface-active compound needed to produce a detergent effect) of the test compounds was determined from a plot of the surface tension against the logarithm of the molar concentration.

The results are shown Compound in the y min following

C

10 AlaONa

C

12 AlaONa

C

14 AlaONa

C

16 AlaONa

C

18 AlaONa

C

10 LeuONa

C

12 LeuONa

C

14 LeuONa

C

16 LeuONa

C

18 LeuONa

C

10 PheONa

C

12 PheONa Cl 4 PheONa

C

16 PheONa

C

18 PheONa 47.4 42.5 40.8 42.4 39.0 37.0 34.0 33.8 35.5 38.3.

36.9 34.5 38.0 37.5 36.9 table: cmc (mol/l) 4.74 10 2 6.98 10 3 1.63 10 3 3.17 10 3 2.28 10 3 7.08 10 4 5.17 10 3 1.20 10 3 4.04 10 4 1,12 10 4 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- WO 90/14429 PCT/DK90/00127 24 able degree. The minimum surface tension is substantially the same for compounds containing leucine and phenylalanine residues. The minimum surface tension is generally higher for compounds containing an alanine residue. The 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 Noctanoyl leucine, sodium salt, as the limit of solubility 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 alanine residue, whereas compounds containing a leucine or phenylalanine residue show substantially the same cmc.

Claims (5)

1. A process for preparing a compound of the general formula I RCO-NH-CH-CONH 2 or II RCO-NH-CH-COOH (II) 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 R 1 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 RCOOR 2 (III) wherein R 2 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 re, I SUBSTITUTE SHEET

26. NH 2 -CH-CONH 2 (IV) R 1 wherein R is as defined above, in the presence of a hydrolase, to produce the compound of the general formula I, 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 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 any one of the preceding claims wherein R is an alkyl group with 6 to 20 carbon atoms. 3. A process according to claim 2, wherein RCO- is hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, dodecanoyl, tetradecanoyl, hexadecanoyl, octadecanoyl, eicosanoyl, docosanoyl, cis-9-octadecanoyl or 9,12-octadecadienoyl. 4. A process according to any one of the preceding claims, wherein R1 is a methyl, ethyl, propyl, isopropyl, methylthio, -CH 2 -CH 2 -S-CH 3 benzyl, hydroxybenzyl, indolyl or alkylguanidine group. A process according to any one of the preceding claims, 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, glutamic acid amide and ornithine amide in racemic or optically active form. 6. A process accordin3 to any one of the preceding claims for preparing compounds of the general formula I as defined in claim 1, which compounds are 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 amida, N-octanoyl leucine amide, N-decanoyl leucine amide, N-dodecanoyl leucine amide, N-tetradecanoyl leucine

27. amide, N-hexadecanoyl leucine amide, N-octadecanoyl leucine amide, N-hexanoyl phenylalanine amide, N-octanoyl phenylalanine amide, N- decanoyl phenylalanine amide, N-dodecanoyl phenylalanine amide, N- tetradecanoyl phenylalanine amide, N-hexadecanoyl phenylalanine amide, N-octadecanoyl phenylalanine amide, N-hexanoyl phenyiglycine amide, N-octanoyl phenyiglycine amide, N-decanoyl phenyiglycine amide, N- dodecanoyl phenyiglycine amide, N-tetradecanoyl phenyiglycine amide, N- hexadecanoyl phenyiglycine amnide, N-octadecanoyl phenyiglycine amide, N- hexanoyl lysine amide, N-octanoyl lysine amide, N-decanoyl lysine amide, N-dodecanoyl lysine amide, N-tetradecanoyl lysine amide, N-hexadecanoyl lys'one amiide, N-octadecanoyl lysine amide, N-hexanoyl glycine amide, N- octanoyl glycine amide, N-decanoyl glycine amide, N-dodecanoyl glycine amiide, N-tetradecanoyl glycine amide, N-hexadecanoyl glycine amilde, N- octadecanoyl g1ycine amide, N-hexanoyl valine amide, N-octanoyl valine amide, N-decanoyl valine amide, N-dodecanoyl valine amide, N-tetra- decanoyl valine amide, N-hexadecanoyl vailne amide, N-octadecanoyl valine amide, N-hexanoyl tryptophan amide, N-octanoyl tryptophan amide, N- decanoyl tryptophan amide, N-dodecanoyl tryptophan amilde, N-tetra- decanoyl tryptophan amide, N-hexadecanoyl tryptophan amide, N-octa- decanoyl tryptophan amide, N-hexanoyl arginine amnide, N-octanoyi arginine amide, N-.decanoyl arginine amide, N-dodecanoyl arginine amide, N-tetra- decanoyl arginine amide, N-hexadecanoyl arginine amide, N-octadecanoyl arginine amide, N-hexanoyl histidine aniide, N-octanoyl histidine amide, N-decanoyl histidine amilde, N-dodecanoyl histidine amide, N-tetradecanoyl histidine amide, N-hexadecanoyl histidine amide, N-octadecanoyl histidine amide, N-hexanoyl cystelne amide, N-octanoyl cystelne amide, N-decanoyl cysteine amide, N-dodecanoyl cystelne amide, N-tetradecanoyl cystelne amide, N-hexadecanoyl cysteine amide, N-octadecanoyl cysteine amide, N- hexaoylgluamin amdeN-ocanol gutamne mid, N-ecaoylgluta- mine amide, N-dodecanoyl glutamine amide, N-tetradecanoyl glutamine amide, N-hexadecanoyl glutamine amide, N-octadecanoyl glutanilne 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 1622a/li

28. amide, N-decanoyl asparagine amide, N-dodecanoyl asparagine amide, N- tetradecanoyl asparagine amide, N-hexadecanoyl asparagln amnide, N-octa- decanoyl asparagine amide, N-hexanoyl aspartic acid amide, N-octanoyi 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 amide, N-decanoyl glutamic acid amide, N-dodecanoyl glutamic acid amide, N-tetradecanoyl glutamic acid amide, N-hexadecanoyl glutamic acid amide, N-octadecanoyl glutanic acid amide, N-hexanoyl ornithine amide, N-octanoyl ornithine amide, N- decanoyl ornithine amide, N-dodecanoyl ornithine amidie, N-tetradecanoyl ornithine amide, N-hexadecanoyl ornithine amide, N-octadecanoyl ornithine amide, or an alkali metal or alkaline earth metal salt thereof, each compound being in racemic 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, which compounds are N-hexanoyl alanine, N-octanoyl alanine, N-decanoyl alanine, N-dodecanoyl alanine, N-tetradecanoyl alanine, 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 phenyl- alanine, N-tetradecanoyl phenylalanine, N-hexadecanoyl phenylalanine, N-octadecanoyl phenylalanine, N-hexanoyl phenylglyclne, N-octanoyl phenylglyclne, N-decanoyl phenyiglycine, N-dodecanoyl phenylglyclne, N-tetradecanoyl phenylglyclne, N-hexadecanoyl phenylglycine, N-octa- decanoyl phenyiglycine, N-hexanoyl lysine, N-octanoyl lysine, N-decanoyl 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-hexa decanoyl glycine, N-octadecanoyl glycine, N-hexanoyl valine, N-octanoyl valine, N-decanoyl valine, N-dodecanoyl valine, N-tetradecanoyl valine, N-hexadecanoyl valine, N-octadecanoyl valine, N-hexanoyl tryptophan, N-octanoyl tryptophan, N-decanoyl tryptophan, N-dodecanoyl tryptophan, 35 N-tetradecanoyl tryptophan, N-hexadecanoyl tryptophan, N-octadecanoyl

29. tryptophan, N-hexanoyl arginine, N-octanoyl arginine, N-decanoyl argi- nine, 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 cystelne, N-dodecanoyl cysteine, N-tetradecanoyl cysteine, N-hexadecanoyl cysteine, N-octa- decanoyl cysteine, N-hexanoyl glutarnine, N-octanoyl glutamine, N-decanoyl glutamine, N-dodecanoyl glutamine, N-tetradecanoyl glutamine, N-hexa- decanoyl 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-octanoyl aspartic acid, N-decanoyl aspartic acid, 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, N-octa- decanoyl glutamic acid, N-hexanoyl ornithine, N-oct-anoyl ornithine, N-decanoyl ornithine, N-dodecanoyl ornithine, N-tetradecanoyl ornithine, N-hexadecanoyl ornithine, N-octadecanoyl ornithine, or an alkali metal or alkaline earth metcal 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 Pj,1u1Qoj1nas. 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, OSM 3959, HicolaiI~ lanucginosa, DSM 3819 or 4109, Humi cola brevispora, DSM 4110, Hiii.ia br-Qyj var. thermoidea, DSM 4111, or Humicola insolens, DSM 1800. 11. A process according to any one of the preceding claims, ltj22a/ii wherein the enzyme is an immobilized enzyme. 12. A process according to any one of the preceding claims, wherein the carboxypeptidase is carboxypeptidase Y. 13. A process according to claim 12, wherein the carboxypeptidase is one produced by Saccharomvces cerevisiae. 14. A process according to any one 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. A process according to any one 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 one 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. 17. A process according to claim 16 wherein the reaction proceeds at a pressure below about 0.01 bar. 18. An enzyme-catalyzed process for preparing N-acyl amino acids and N-acyl amino acid amides substantially as hereinbefore described with reference to any one of the Examples. 19. The product of the process of any one the preceding claims. DATED this TWENTY-FOURTH day of FEBRUARY 1993 Novo Nordisk A/S Patent Attorneys for the Applicant SPRUSON FERGUSON 2* 1622a/11 INTERNATIONAL SEARCH REPORT International Application No PCT/DK 90/00127 I. CLASSIFICATION OF SUBJECT MATTER (if several classification symbols apply, indicate According to International Patent Classification (IPC) or to both National Classification and IPC C 12 P 13/02, A 61 K 7/075, 7/15, 7/16, 7/48, C 11 D 3/32 II. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System Classification Symbols C 12 P; A 61 K; C 11 D Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in Fields Searched 8 SE,DK,FI,NO classes as above Ill. DOCUMENTS CONSIDERED TO BE RELEVANT S Category Citation of Document, 11 with indication, where appropriate, of the relevant passages12 Relevant to Claim No. 13 Y J. Biochem., Vol. 96, No. 3, 1984 Yasushi Shintani 1-11 et al.: "Isolation and Characterization of N-Long Chain Acyl Aminoacylase from Pseudomonas diminuta see page 637 page 643 see especially page 643 Y EP, Al, 0298796 (SOCIETE D'EXPLOITATION DE 1-11 PRODUITS POUR LES INDUSTRIES CHIMIQUES 11 January 1989, see especially p. 1 line 46 p.3 line 17 and p. 4 lines 34-40 A GB, A, 1483500 (AJINOMOTO CO) 10 December 1974, 1-11 see the whole document Special categories of cited documents: 10 later document published alter the international filing date A' document delining the general state of the art which Is not or priority date and not in conflict with the applicaloi but A cnstded d to e I ercar r e te a w i cited to understand the principle or theory underlying the considered 1 part cular relevance invention earlier document but published on or after the International document particular relevance the claimed nvention filing date document of particular relevance, the claimed invention cannot be considered novel or cannot be considered to document which may throw doubts pn priority claim(s) or involve an inventive step which is cited to establish the publication date of another d t of p r e citation or other special reason (as specified) Y document o particulr relevance, the aimed i nventon Citation r other special reason s speciied) cannot be considered to involve an inventive step when the me erring t o an or a l disclosure, use, exhibition or document s combined ith one or more other such dou Sdocument reerring to n oral disclosure, Use exhibition or ments, such combination being obvious to a person skilled other means In the art. document published prior to the international filing date but document member of the same patent famy later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 27th August 1990 1990 -08- 3 0 International Searching Authority Signature of Authorized Officer SWEDISH PATENT OFFICE Anna Hedberg Form PCT/ISA/210 (second sheet) (January 1985) International Application No. PCT/DK 90/00127 III, DOCUMENTS CONSIL-ERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) Category Citation of Document, with indication, where appropriate, of the relevant passages Relevant to Claim No A Chemical Abstracts, vol. 82, no. 25, 23 June 1975, 19-20 (Columbus, Ohio, US), Matsuzawa, Yoshimasa et al: "N-Acylamino acid amides", see page 595, abstract 171433r, Japan. Kokai 74,116,005, 6 November 1974 A GB, A, 1445503 (DR KARL THOMAE GMBH) 21-22 3 May 1974, see the whole document Fore PCI/ISA/21u textra, sheet) (January 1985) ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO.PCT/DK 90/00127 This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The members are as contained in the Swedish Patent Office EDP file on 90-08-02 The Swedish Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent document Publication Patent family Publication cited in search report date member(s) date EP-A1- 0298796 89-01-11 FR-A-B- 2616801 88-12-23 GB-A- 1483500 74-12-10 FR-A- 2254554 75-07-11 JP-C- 865556 77-06-23 JP-A- 50088002 75-07-15 JP-B- 51041602 76-11-11 US-A- 3985722 76-10-12 GB-A- 1445503 74-05-03 AT-B- 330761 76-07-26 AU-D- 6849774 75-11-06 BE-A- 814476 74-11-04 CH-A- 604704 78-09-15 DE-A- 2322232 74-11-14 FR-A-B- 2227874 74-11-29 JP-A- 50029754 75-03-25 NL-A- 7405324 74-11-05 US-A- 3948943 76-04-06