Lipopeptides, their preparation and their application as emulsifiers

The lipopeptides according to the invention are amphiphatic and consist of a hydrophobic chain containing from about 8 to 24 carbon atoms and of a peptide chain which is hydrophilic or has been rendered hydrophilic. They are applied, in particular, to emulsions of immiscible media and to the preparation of lyotropic liquid crystals.

FIELD OF THE INVENTION 
The present invention relates to the synthesis of lipopetides, and more 
particularly of amphiphatic lipopeptides, and to the application of these 
compounds as emulsifiers or as liquid crystals. 
SUMMARY OF THE INVENTION 
The invention relates firstly to a new class of lipopeptides which are 
amphiphatic lipopeptides composed of a hydrophobic chain containing at 
least about 8 carbon atoms and preferably from about 8 to 24 carbon atoms, 
and of a peptide chain which is hydrophilic or has been rendered 
hydrophilic. 
The lipopeptides according to the invention can be defined by the general 
formula: 
EQU CnPP 
in which Cn represents a hydrophobic chain having at least about 8 carbon 
atoms and preferably about 8 to 24 carbon atoms, n denoting the number of 
carbon atoms, and PP represents a polypeptide obtained from natural 
aminoacids or their derivatives (having the 1 or d configuration). In 
practice, the polypeptide PP is formed of one or more aminoacids, 
depending on the chosen degree of polymerization, which can be 1 or more. 
The nomenclature of some of the most common peptide sequences which can be 
used is summarized later in Table I. 
The term "hydrophobic chain" is understood as meaning preferably, but not 
exclusively, an optionally substituted, aliphatic hydrocarbon chain having 
the indicated number of carbon atoms. 
These compounds have been obtained by a technique which is itself novel. 
The invention therefore also relates to a process for the preparation of 
amphiphatic lipopeptides as defined above, which basically consists in: 
(1) producing a peptide linkage between a fatty amine and an N-protected 
aminoacid to give a lipopeptide whose peptide chain has a degree of 
polymerization of 1, and, if it is desired to obtain a degree of 
polymerization of 2 or 3 for the peptide chain, producing a further 
peptide linkage between an N-protected aminoacid and the product whose 
degree of polymerization is 1 lower, or 
(2a) carrying out the polymerization of the N-carboxyanhydride of the 
aminoacid by initiating it with the fatty amine CnNH.sub.2 to give 
lipopeptides whose peptide chains have a degree of polymerization which 
will depend on the operating conditions chosen, and 
(2b) if desired, fractionating the lipopeptides from step (2a) in respect 
of their composition, and 
(3) except in the case where the peptide chain of the product from step (1) 
and/or (2a) or (2b) is directly a hydrophilic chain, unblocking the side 
chains of the hydrophobic peptide chain in order to render them 
hydrophilic. 
To obtain lipopeptides having a degree of polymerization of 1, 2 or 3, a 
possible procedure is to produce a peptide linkage between the fatty amine 
of the formula CnNH.sub.2, where Cn is as defined above, and the aminoacid 
whose amino nitrogen atom is protected, for example by the 
tert.-butoxycarbonyl group (abbreviated to Boc), and this amino nitrogen 
atom is unblocked to give the product having a degree of polymerization of 
1, which will be coupled again with the blocked aminoacid to give the 
product having a degree of polymerization of 2, after unblocking of the 
terminal nitrogen, and so on. 
To obtain the lipopeptides according to the invention, it is also possible 
to polymerize the N-carboxyanhydride (abbreviated to NCA) of the 
aminoacid, the polymerization being initiated by the fatty amine 
CnNH.sub.2. If desired, fractionation in respect of composition is then 
carried out (the lipid sequence being monodisperse) by selective 
precipitation of the lipopeptides, and a series of lipopeptides are 
obtained which differ in their peptide composition. 
The monomeric peptides used are commercial products or are prepared in a 
known manner. 
The monomer used for the peptide part of the lipopeptides according to the 
invention is not therefore the aminoacid itself but its cyclic derivative, 
namely the aminoacid N-carboxyanhydride (NCA), obtained by reacting 
phosgene with the aminoacid, according to the equation: 
##STR1## 
or with the aminoacid N-protected by one of the groups normally used in 
peptide synthesis. 
The NCA compounds are prepared in THF by reacting a solution of phosgene in 
tetrahydrofuran (THF) with the aminoacid. This method is a modified 
version of the method of Fuller, Verlander and Goodman (Biopolymers, 15, 
1869/1976), in which the solvent for the phosgene is benzene. 
The fatty amine CnNH.sub.2 is a commercially available amine, or an amine 
obtained from the fatty acid having one carbon atom more by means of 
Schmidt's degradation reaction using sodium azide in a strong acid medium 
[Indian J. Technol., 5, 262 (1967)], or an amine obtained by coupling 
acryloyl or methacryloyl chloride with a primary diamine, or an 
N-protected aminoalcohol. The choice of a chain Cn having from about 8 to 
24 carbon atoms is not critical but results solely from the greater 
availability of the corresponding products. This hydrophobic chain Cn can 
be any hydrocarbon chain, but it can also contain substituents and/or 
heteroatoms provided that they do not have an unfavorable influence on the 
synthesis process. 
The preparation of hydrophobic lipopeptides and their conversion to 
amphiphatic lipopeptides, and the direct preparation of amphiphatic 
lipopeptides, are described successively below, with reference to the 
nomenclature given in Table I.

A-1 Synthesis of Hydrophobic Lipopeptides (CnEb.sub.p, CnDb.sub.p and 
CnKt.sub.p) 
The fatty amine (CnNH.sub.2) is dissolved in chloroform, the NCA of the 
appropriate aminoacid is then added and the mixture is left to polymerize 
at ambient temperature for two days, with agitation. This gives the 
lipopeptides CnEb.sub.p possessing a peptide sequence of poly(benzyl 
glutamate), CnDb.sub.p possessing a peptide sequence of poly(benzyl 
aspartate) and CnKt.sub.p possessing a peptide sequence of 
polytrifluoroacetyllysine. 
By way of example, lipopeptides C17Kt.sub.p are synthesized using 
heptadecylamine, C.sub.17 H.sub.35 NH.sub.2, obtained from stearic acid 
(see Indian J. Technol., 5, 262 (1967)). More precisely, to obtain 
C17Kt.sub.10, having a degree of polymerization of 10 (DP=10), 27 g (0.1 
mole) of trifluoroacetyllysine NCA are added to a solution of 2.55 g (0.01 
mole) of C17 amine in 150 ml of chloroform, and the mixture is left to 
polymerize at ambient temperature for several hours, with agitation. The 
chloroform is then evaporated off, the residue is taken up in methanol and 
the polymer is precipitated in water and then filtered off, washed and 
dried. 
A-2 Conversion of Hydrophobic Lipopeptides to Amphiphatic Lipopeptides 
1. Lipopeptides CnK.sub.p 
The lipopeptides CnK.sub.p possessing a hydrophilic sequence of polylysine 
(K) were prepared from the lipopeptides CnKt.sub.p by the method of Sela 
et al. [Biopolymers, 1, 517 (1963)]. A solution of the lipopeptides 
CnKt.sub.p in THF is treated firstly with a solution of piperidine in 
methanol and then with a solution of piperidine in water. 
As an example illustrating a conversion of C17Kt.sub.p to C17K.sub.p, 5 g 
of C17Kt.sub.p are dissolved in 150 ml of a molar solution of piperidine 
in methanol at ambient temperature. After 2 hours, 100 ml of a molar 
solution of piperidine in water are added and the mixture is left for 48 
hours at ambient temperature. The methanol is removed in an evaporator and 
the aqueous solution is passed through a column of anion exchange resin 
(Duolite A 102D, OH.sup.- form) in order to remove the trifluoroacetate 
anions therefrom, and the eluate is lyophilized to recover the C17K.sub.p. 
2. Lipopeptides CnE.sub.p and CnD.sub.p 
The lipopeptides CnE.sub.p possessing a hydrophilic sequence of 
polyglutamic acid (E) and CnD.sub.p possessing a hydrophilic sequence of 
polyaspartic acid (D) are obtained from the lipopeptides CnEb.sub.p and 
CnDb.sub.p by treating these lipopeptides with HCl and HBr at ambient 
temperature [J. Am. Chem. Soc., 80, 4631 (1958)]. 
3. Lipopeptides CnEp.sub.p 
The lipopeptides CnEp.sub.p possessing a hydrophilic sequence of 
polyhydroxypropylglutamine (Ep) are prepared by treating the lipopeptides 
CnEb.sub.p with aminopropanol at 60.degree. C., in solution in dioxane 
[Biopolymers, 3, 625 (1965)]. 
4. Lipopeptides CnEe.sub.p 
The lipopeptides CnEe.sub.p possessing a hydrophilic sequence of 
polyhydroxyethylglutamine (Ee) are prepared by treating the lipopeptides 
CnEb.sub.p with ethanolamine at 60.degree. C., in solution in dioxane 
[Biopolymers, 9, 717 (1970)]. 
B Direct Synthesis of Amphiphatic Lipopeptides 
The fatty amine CnNH.sub.2 is dissolved in chloroform, the aminoacid NCA is 
then added and the mixture is left to polymerize at ambient temperature 
for 2 days, with agitation. This gives the lipopeptides CnSar.sub.p 
possessing a peptide sequence of polysarcosine. 
In order to give a more concrete illustration of the process for the 
synthesis of lipopeptides according to the invention, the synthesis of the 
amphiphatic lipopeptides C17Sar.sub.p, formed of an aliphatic chain 
containing 17 carbon atoms (C17) and of a polysarcosine chain (Sar).sub.p, 
and the synthesis of C12Sar.sub.20 and C18Sar.sub.11, are described below. 
1. Synthesis of lipopeptides having a degree of polymerization of more than 
3 
(a) Synthesis of C17Sar.sub.20 
Heptadecylamine (C17NH.sub.2), obtained from stearic acid [see Indian J. 
Technol., 5, 262 (1967)], is first dissolved in chloroform, and the amount 
of sarcosine NCA calculated to give the chosen degree of polymerization is 
then added to the solution. For example, if it is desired to obtain 
C17Sar.sub.10, having a degree of polymerization of 10 (DP: 10), 11.5 g of 
sarcosine NCA (0.1 mole) are added to a solution of 2.55 g (0.01 mole) of 
amine, C.sub.17 H.sub.35 NH.sub.2, in 100 ml of chloroform, and the 
mixture is left to polymerize at ambient temperature for 48 hours, with 
agitation. 
The lipopeptides C17Sar.sub.p are fractionated by fractional precipitation 
using dimethylformamide as the solvent and acetone as the precipitating 
agent. 
(b) Synthesis of C12Sar.sub.20 
23 g (0.2 mole) of sarcosine NCA are added to a solution of 1.85 g (0.01 
mole) of dodecylamine in 100 ml of chloroform, and the mixture is left to 
polymerize at ambient temperature for 48 hours, with agitation. The 
lipopeptides C12Sar.sub.p can be fractionated by fractional precipitation 
using the precipitating solvents dimethylformamide/acetone. 
This gives xg of the desired white solid product, the degree of 
polymerization of which was measured by determining the terminal amine 
group after the degree of purity had been checked by chromatography. 
(c) Synthesis of C18Sar.sub.11 
C18Sar.sub.11 is prepared by applying the same operating conditions as 
under (b), with 0.11 mole of sarcosine NCA (12.65 g) and 0.01 mole of 
C18NH.sub.2 (2.69 g) in 100 ml of chloroform, and is obtained with a yield 
of more than 80%; its infrared spectrum in KBr is the subject of FIG. 1. 
2. Synthesis of lipopeptides having degrees of polymerization of 1, 2 and 3 
To obtain lipopeptides having degrees of polymerization of 1, 2 and 3, a 
possible procedure is to produce a peptide linkage between the fatty amine 
and the aminoacid N-protected by the tert.-butyloxycarbonyl (Boc) group. 
The Boc-aminoacids are prepared from the aminoacid and di-tert.-butyl 
dicarbonate by the method of Morsder et al. [Hoppe-Seyler's Z. Physiol. 
Chem., 357, 1651 (1976)]. 
(a) Synthesis of C17Sar.sub.1 
(.alpha.) C17BocSar.sub.1 : the product C17BocSar.sub.1 is obtained by 
coupling heptadecylamine with BocSar.sub.1 in the presence of 
dicyclohexylcarbodiimide (DCCI). 3.78 g (0.02 mole) of BocSar and 2.06 g 
(0.01 mole) of DCCI are mixed cold (at 0.degree. C.) in 100 ml of 
chloroform. A copious precipitate of dicyclohexylurea (DCU) is formed. The 
mixture is left for 30 minutes at 0.degree. C., with agitation, and 2.55 g 
(0.01 mole) of heptadecylamine are added. The reaction is left to proceed 
for 20 hours. The precipitate is filtered off and washed, the filtrate is 
recovered, the chloroform is evaporated off, the residue is taken up in 50 
ml of THF, the mixture is cooled to 0.degree. C. and filtered to remove 
the maximum amount of dicyclohexylurea, and the precipitate is washed with 
the minimum amount of cold THF. 
(.beta.) C17Sar.sub.1.HCl: 20 ml of a 5N solution of HCl in THF are added 
to the filtrate, and the mixture is left for 24 hours at ambient 
temperature, with agitation. A copious precipitate of C17Sar.sub.1.HCl is 
formed, which is filtered off and washed with THF. 
(.gamma.) C17Sar.sub.1 : the precipitate is taken up in 100 ml of THF, the 
mixture is heated to 50.degree. C., 2 ml of triethylamine are then added 
and the mixture is left to stand for 2 hours. It is cooled to 0.degree. 
C., the precipitate of triethylamine hydrochloride is filtered off, the 
THF is evaporated off and the C17Sar.sub.1 is recrystallized from acetone. 
This gives 2.1 g of C17Sar.sub.1 (yield: 65%). 
Melting point=58.degree. C. 
(b) Synthesis of C17Sar.sub.3 
To obtain C17Sar.sub.3, the same procedure is followed except that 
C17Sar.sub.2 and BocSar are used as the starting materials. 
Melting point=83.degree. C. 
(c) Synthesis of C17Sar.sub.2 
To obtain C17Sar.sub.2, the procedure is the same except that C17Sar.sub.1 
and BocSar are used as the starting materials. 
Melting point=74.degree. C. 
(d) Synthesis of C12Sar.sub.1 
Preparation of C12SarBoc: 1.85 g (0.01 mole) of dodecylamine, 1.89 g (0.01 
mole) of SarBoc and 1.15 g (0.01 mole) of N-hydroxysuccinimide are 
dissolved in 100 ml of chloroform, 2.06 g of dicyclohexylcarbodiimide are 
then added, with agitation, and the agitation is maintained for 24 hours. 
The precipitate of dicyclohexylurea is then removed, the solvent is 
evaporated off from the filtrate and the residue is taken up in 100 ml of 
acetone in order to remove the remaining solid dicyclohexylurea. The 
desired product precipitates when a volume of water is added to the 
filtrate. After the precipitate has been washed with an acetone/water 
mixture, x.sub.g of C12SarBoc are obtained. 
Preparation of C12Sar hydrochloride: The product obtained above is 
dissolved in 80 ml of THF; 20 ml of a 5N solution of hydrochloric acid in 
diethyl ether are added and the mixture is left to stand for 24 hours at 
ambient temperature, during which time the final hydrochloride 
precipitates. It is isolated by filtration at 0.degree. C., washed with 
ice THF and dried in vacuo. This gives C12Sar.HCl. 
isolation of C12Sar.sub.1 : The salt obtained above is dissolved in 50 ml 
of methanol; 100 ml of a 0.1N aqueous solution of sodium hydroxide are 
added and the solvents are evaporated off in vacuo at ambient temperature 
to a volume of about 25 ml. This solution is poured into 100 ml of water 
and the aqueous phase is extracted with 100 ml and then 50 ml of ethyl 
acetate. The organic phases are combined and dried over sodium sulfate. 
The solvent is then removed in vacuo at 0.degree. C. 
The residue is purified by chromatography on a column of silica gel using, 
as the eluent, a solution of methanol containing 1% by volume of aqueous 
ammonia solution (about 30%). 
This gives 2.05 g of C12Sar.sub.1 having a melting point of 39.degree. C. 
(e) Synthesis of C12Sar.sub.2 and C12Sar.sub.3 
The same process as that described for the synthesis of C12Sar.sub.1 is 
applied, but C12Sar.sub.1 and C12Sar.sub.2, respectively, are used as the 
starting materials. 
The yields are comparable. 
C12Sar.sub.2 : melting point 58.degree. C. 
C12Sar.sub.3 : melting point 66.degree.-67.degree. C. 
(f) Synthesis of C16Sar.sub.1,2,3 
The procedures described under (d) and (e) are applied in order to prepare 
these derivatives, the infrared spectra of which (in KBr) are shown 
respectively in FIGS. 2, 3 and 4. 
(g) Synthesis of C18Sar.sub.2 
2.3 g (0.02 mole) of sarcosine NCA are added in small portions to a 
solution of 2.69 g (0.01 mole) of octadecylamine in 150 ml of chloroform, 
with vigorous agitation. The solvent is then removed, the residue is 
dissolved in diethyl ether and the latter is then removed in vacuo at a 
temperature of the order of 0.degree. C. 
This gives 4 g of a product whose average degree of polymerization, 
measured by determining the terminal amine group with perchloric acid in 
acetic acid, is very close to 2. 
By chromatography on a column of silica gel using, as the eluent, methanol 
containing 1% of concentrated aqueous ammonia solution, C18Sar.sub.1, 
C18Sar.sub.2, C18Sar.sub.3 and C18Sar.sub.4 are separated to give more 
than 60% of C18Sar.sub.2. There is also a small amount of C18Sar.sub.5 and 
a trace of C18Sar.sub.6 with a residue of starting amine. 
The C18Sar.sub.2 thus obtained, even when not separated from its homologs, 
has emulsifying properties comparable to those of the C18Sar.sub.2 
obtained by the SarBoc method. 
The structure of lipopeptides according to the invention was studied by the 
technique of X-ray diffraction. 
It was thus possible to establish that the amphiphatic lipopeptides have 
mesophases of periodic structure in aqueous solution for water 
concentrations of less than about 60%, and that the periodic structure can 
be preserved in the dry state by slow evaporation of the water from the 
mesophase. The amphiphatic lipopeptides according to the invention thus 
constitute a new class of lyotropic liquid crystals and they can have the 
same applications as these liquid crystals. 
The structure of the amphiphatic lipopeptides is now described in greater 
detail with reference to the example of the lipopeptides C17Sar.sub.p, 
consisting of an aliphatic chain possessing 17 carbon atoms and of a 
polysarcosine chain, the degree of polymerization of which was varied from 
1 to 60. 
The lipopeptides C17Sar.sub.p exhibit a dual polymorphism: on the one hand 
as a function of the length of the polypeptide chain and on the other hand 
as a function of their water content. According to their composition, the 
lipopeptides adopt three types of structure: lamellar for degrees of 
polymerization (DP) of less than 9, hexagonal for DP values of between 10 
and about 35, and centered cubic for DP values of more than about 35. 
Furthermore the lipopeptides can exhibit a polymorphism as a function of 
the water content of their mesophases. The addition of water modifies the 
ratio of the volumes of the hydrophilic and hydrophobic sequences and can 
cause the structure to change from lamellar to hexagonal (for DP values of 
between 5 and about 9) or from hexagonal to cubic (for DP values of 
between 17 and about 35). 
The invention therefore also relates to the application of the amphiphatic 
lipopeptides to the composition of lyotropic liquid crystals. 
The mesophases of amphiphatic lipopeptides can also incorporate numerous 
components, both hydrophilic and hydrophobic, such as: alcohols, acids, 
paraffins, carnation oil, ethyl stearate, isopropyl palmitate and the 
like, and can thus give, for example, milks or creams, the viscosity of 
which can easily be varied by altering the structure of the mesophases, 
this structure itself being determined by the respective length of the 
hydrophobic and peptide sequences in the lipopeptides. 
The emulsifying properties of the amphiphatic lipopeptides with respect to 
numerous pairs of immiscible liquids, such as water/hydrocarbons and 
water/base products of the cosmetic industry, were also tested. The type 
and the stability of the emulsions obtained were studied by the method of 
selective staining, the method of dilution, the electrical conductivity, 
the breaking properties of freezing, and electron microscopy. 
To obtain emulsions, about 1% by weight of amphiphatic lipopeptide 
(CnSar.sub.p with n=16, 17 or 18 and p=1, 2 or 3, for example) is added to 
the two immiscible liquids, the mixture is shaken for 10 to 15 minutes and 
the emulsion forms easily. This method was used to prepare emulsions of 
different compositions, from 30 to 70% of each component, with the systems 
water/octane, water/isopropyl myristate, water/isopropyl palmitate, 
water/butyl or ethyl stearate, water/carnation oil, water/vaseline oil, 
water/cosbiol and water/mygliol). The emulsions obtained are very stable 
(several months) and withstand temperature increases up to about 
60.degree. C. The viscosity, compactness and unctuousness of the emulsions 
are modified by varying the lipopeptides content between 1 and 2%. 
The invention therefore also relates to the application of the amphiphatic 
lipopeptides as emulsifiers and to the emulsion incorporating amphiphatic 
lipopeptides as emulsifying agents, present in an amount by weight of the 
order of 1% or more. 
The solubility of the lipopeptides and their affinity for different 
solvents can be varied as desired by modifying the number of carbon atoms 
in the hydrophobic chain and the nature of the peptide chain. It is also 
possible easily to modify the hydrophilic-hydrophobic balance of such 
lipopeptides by modifying the number of carbon atoms in the hydrophobic 
chain and the degree of polymerization of the peptide chain. 
The amphiphatic lipopeptides according to the invention readily give very 
stable emulsions for very low lipopeptide contents (about 1% by weight), 
whereas it is necessary to have 15-16% of the conventional surface-active 
agents. Furthermore, they have the advantage of being produced with 
natural components (lipids and peptides). These amphiphatic lipopeptides 
can be applied as emulsifying agents for immiscible media in a very wide 
variety of fields, for example in the cosmetics industry (moisturising 
creams, antiwrinkle creams, varnishes, solvents for varnishes, and the 
like), in the food industry (mustards, mayonnaises and the like) and in 
the petroleum industry (additives for oils, assisted recovery of 
petroleum), inter alia. 
TABLE I 
______________________________________ 
Nomenclature of the peptide sequences 
Des- 
igna- 
tion Name of the polypeptide 
Formula of the side chain 
______________________________________ 
Eb Poly(benzyl glutamate) 
(CH.sub.2).sub.2COOCH.sub.2C.sub.6 H.sub.5 
Ep Poly(hydroxypropyl- 
(CH.sub.2) .sub.2CONH(CH.sub.2).sub.3 OH 
glutamine) 
Ee Poly(hydroxyethyl- 
(CH.sub.2) .sub.2CONH(CH.sub.2).sub.2 OH 
glutamine) 
E Poly(glutamic acid) 
(CH.sub.2).sub.2COOH 
Db Poly(benzyl aspartate) 
CH.sub.2COOCH.sub.2C.sub.6 H.sub.5 
D Poly(aspartic acid) 
CH.sub.2COOH 
Kt Poly(trifluoroacetyl- 
(CH.sub.2).sub.4NHCOCF.sub.3 
lysine) 
K Polylysine (CH.sub.2).sub.4NH.sub.2 
S Polyserine CH.sub.2OH 
T Polythreonine 
##STR2## 
Sar Polysarcosine (+) 
______________________________________ 
(+) Polysarcosine: 
##STR3##