This invention relates to a phipathic compounds which are of value as detergents or in other contexts.

Amphipathic compounds, which contain a polar group joined to a non-polar group, find various applications including particularly the use of amphipathic compounds with surface active properties (surfactants) as detergents. Detergents find very many uses and one area in which there has recently been increasing interest in the use of detergents is that of biochemistry, cell biology, immunology, etc. An example of this type of use is the dissoci- ation of cell membranes by means of detergents. The satisfactory dissocation of cell membranes does, however, require detergents which are both non-denaturing and readily removable by dialysis, and these two properties are not usually compatible. Thus, most of the detergents in current usage which are non-denaturing are non-ionic and have a very low critical micellar concentration

(CMC), thereby making removal by dialysis extremely difficult. On the other hand, detergents having a high CMC are mostly of the ionic type and are therefore likely to denature proteins.

Among the detergents which are currently available, those most suited to the dissociation of cell membranes and like uses are cyclic pyranose sugars which are substituted through an oxygen atom by an alkyl group, for example β-D-octylglucoside. Such com¬ pounds are, however, extremely costly to prepare and I have now developed a novel group of compounds which may not only be prepared at considerably less cost but which may be used instead of detergents such as β-D-octylglucoside with results which may often be superior.

According to the present invention an amphipathic compound comprises an aliphatic hydrocarbon chain with a length of at least three carbon atoms linked to a polyhydroxy substituted acyclic aliphatic group through an amide grouping.

As discussed in more detail hereinafter, the compounds according to the present invention may, where desired, contain more than one aliphatic hydrocarbon chain and/or more than one

polyhydroxy substituted acyclic aliphatic group, and these groups may be linked through one or more amide groupings which may be unsubstituted, i.e. being -C0.NH-, or N-substituted. The valuable properties of amphipathic compounds according to the present invention derive in large measure from the use of an amide linkage to join the non-polar or hydrophobic part, i.e. the aliphatic hydrocarbon group or groups, and the polar or hydrophilic part, i.e. the polyhydroxy substituted acyclic aliphatic group or groups, of the molecule. The use of the amide linkage results in a non- ionic, chemically inert compound containing a highly polar poly¬ hydroxy head group. This head group is most effective at breaking interactions of a highly polar nature between proteins and its presence leads to an elevation of the CMC of the compound which not only assists in removal of the compound by dialysis but also increases the solubilizing action of the compound on membranes. The chemical inertness of the compounds of the present invention makes them compatible with all commonly used buffers and heavy metal salts and the compounds can be prepared very readily in a high state of purity through construction of the amide linkage. The polyhydroxy substituted acyclic aliphatic group or groups in compounds according to the present invention will usually contain at least three hydroxy groups, rather than the minimum of two required by the term "poly", and more often will contain four or five hydroxy groups. A convenient source for the polyhydroxy substituted acylic aliphatic group and the nitrogen atom of the amide grouping is provided by amino sugars, which may be readily synthesised by the "reductive alkylation" of ammonia or primary amines with reducing sugars, for example as described by Holley et al, Journal of the American Chemical Society 1950, 72, 5416 and by Karrer and Herkenrath, Helvetica Chemica Acta, 1937, 20, 83. An example of such a reaction is that of D-glucose with methylamine to provide the 1-deoxy-amino sugar, N-methyl-D-glucamine.

Such monosaccharide amino sugars are necessarily acylic since they have lost the carbonyl functional group and the primary or secondary amino group which they contain may be utilised in the attachment of the sugar to an aliphatic hydrocarbon chain through an amide grouping.

A wide variety of sugars may be employed in the formation of amino sugars and thus in the preparation of compounds according to the present invention, including aldoses and ketoses, and trioses, tetroses, pentoses, hexoses and heptuloses. From the point of view of accessibility, the aldoses, especially the aldopentoses and more particularly the aldohexoses, are perhaps of most interest, the naturally occuring sugars and sugars derivable therefrom of course being the most readily available, for example the D-form of various aldoses. Specific examples include mannose, galactose, and particularly glucose.

It will be appreciated, however, that the preparation of compounds according to the present invention is not limited to the use of amino sugars and that any polyhydroxy acyclic aliphatic compound containing a reactive amino group (primary or secondary) may also be used as a ready source of the compounds. Thus for example the compound tris [tris-(hydroxymethyl)-methylamine or 2-amino-2-hydroxymethyl-l,3-dihydroxypropane] may be used. Although much of the following discussion is presented with particular relation to the use of amino sugars, it should be borne in mind, therefore, that other compounds may also be used. More¬ over, although monosaccharide amino sugars may conveniently be used in the preparation of compounds according to the present invention, other alternative saccharide sources do exist. Thus,

OMPI

for example, also of some particular interest are amino sugars derived from disaccharide sugars in which one of the saccharide units is in cyclic non-reducing form through the involvement of its aldehyde (or ketone) group in the linkage formed with the second unit, which is itself in acyclic non-reducing form through involvement of its aldehyde (or ketone) group in the formation of the amine grouping. An example of a disaccharide which may be utilised in this way is maltose. Such compounds will still contain a polyhydroxy substituted acyclic aliphatic group but this will be additionally substituted by a polyhydroxy substituted cyclic aliphatic group, and the principle may be extended to even larger compounds such as trisaccharide sugars and higher poly- saccharides.

It is also possible to consider other additional forms of substitution of the polyhydroxy substituted acyclic aliphatic group (apart from that used in the attachment of the hydrocarbon chain), for example through etherification of one or more of the hydroxy groups of a sugar, but the preservation of as many of the sugar's hydroxy groups as possible is usually to be desired. Specific alternatives to naturally occurring saccharides include monosaccharide sugars which are modified through the attachment via an ether linkage formed at the end of the chain remote from the amide grouping of a further monosaccharide sugar also in acyclic form. The chain can, if desired, be extended to any number of such further groups to produce analogues of the naturally occurring disaccharide and higher polysaccharide sugars described above.

It will be appreciated, therefore, that most usually the amphipathic compound will contain an acyclic aliphatic group which is either a hydrocarbon group or a group containing carbon, hydrogen and oxygen (the latter often in ether form), which group is poly¬ hydroxy substituted. In many cases, a polyhydroxy substituted acylic aliphatic group which is a polyhydroxy substituted hydro¬ carbon group, for example one derived from a monosaccharide aldose, will be quite suitable.

In the following discussion particular reference is made to aldoses. This is partly because the use of aldose sugars is preferred and partly because the reactions involved and the formulae of the compounds obtained are more readily represented in general terms in the case of aldoses. It will be appreciated, however, that similar reactions may generally be effected with ketoses and that where the formyl group of an aldose is converted to a grouping -CH -N for example, the carbonyl group of a ketose may similarly be converted to a grouping^CH-N T As indicated above, when using aldose sugars in the prepara¬ tion of compounds according to the present invention the formyl group is readily replaceable by an aminomethyl or N-substltuted aminomethyl group (-CH ₂ NHR wherein R is hydrogen or an organic grouping) to give an amino sugar which is then linked to an aliphatic hydrocarbon group through the formation of an amide linkage (-CH„NHR —» -CH_NR.C0-). It is possible, however, to prepare from the aldose sugars compounds in which the amide linkage is oriented in the opposite sense without initially preparing an amino sugar, i.e. through the conversion -CH0 — > -C0NR-, but this is less readily achieved. Thus, it requires conversion of the formyl group of the selected sugar to a group such as an ester which may be reacted with an amine which is N-substituted by the desired aliphatic hydrocarbon group (and any organic grouping R which is present) to produce the amide linkage. On the other hand, a compound containing a single aliphatic hydrocarbon group and polyhydroxy acyclic aliphatic group linked through an amide group of the form -CH-NR.CO- described above may very readily be prepared through acylation of the selected amino sugar containing the group -CH„NHR with an acylating agent containing the desired aliphatic hydrocarbon group. Such an acylation reaction is conveni¬ ently effected using a suitable derivative, especially an activated derivative, of the carboxylic acid containing the desired aliphatic hydrocarbon group, for example an anhydride or particularly a mixed anhydride such as that formed by reaction with ethyl chloro- formate or other activated derivatives of this type which are

described in the art, particularly in the context of peptide synthesis.

The simplest compounds according to the present invention produced by the procedures just described may be represented by the formula R _j -K- _o wherein R. is an acyl group consisting of an aliphatic hydrocarbon group of at least 3 carbon atoms linked to a carbonyl group and R_ is a polyhydroxy substituted acyclic aliphatic group linked to R. through an amine function -NR-, wherein R is hydrogen or an organic grouping, for example » being a mono- saccharide amino sugar residue such as a D-glucamine residue or an N-substituted D-glucamine residue. Often, 2 will be derived from an amino sugar which comprises an aldose modified at the l-posltion thereof and which may optionally have one or more of Its hydroxy groups modified through the formation of an ether linkage, and the compound will then be of formula R*C0N(R)CH ₂ R" or R"C0N(R)R* wherein R is hydrogen or an organic grouping, R' is an aliphatic hydrocarbon group of at least 3 carbon atoms, for example a group¬ ing R"'CH in which R"* is an aliphatic hydrocarbon group of at least 2 carbon atoms, and R" is the residue of the aldose, I.e. that part thereof excluding the formyl group at the l-positlon.

As indicated above, the amino sugars may conveniently be prepared from the corresponding sugar by heating this under pressure with ammonia when the amine function has the form -NH_ and by reductive alkylation with the approprate base when it is -NHR, for example methylamine being used to prepare amino sugars containing an amine function -NH(CH-). A modification of this procedure may be used to prepare one of the various forms of compound according to the present invention which contain more than one aliphatic hydrocarbon chain and/or polyhydroxy substi- tuted acyclic aliphatic group. Thus, by using a proportion of 2 equivalents of an aldose sugar to one equivalent of ammonia, instead of the usual 1:1 proportion, it is possible to prepare amino sugars of the formula (R"CH_)_NH and thus compounds of formula 'C0N CH ₂ R") ₂ , R' and R" being as defined above. Related compounds of this type containing two polyhydroxy substituted

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acyclic aliphatic groups may also be prepared from amino compounds such as bis-(2-hydroxymethyl-l,3-dihydroxy-prop-2-yl) amine.

An alternative form of compound containing two aldose sugar residues may be prepared through a modification of the procedure described above for the preparation of compounds of the formula R ^l C0N(R)CH_R". Instead of reacting the aldose amino sugar with a compound containing an activated derivative of a mono-carboxylic acid containing the desired aliphatic hydrocarbon group in a 1:1 proportion, 2 equivalents of the amino sugar are reacted with an activated derivative of the corresponding α,o-dicarbox lic acid. Thus, for example, instead of nonanoic acid, nonane-1,1-dioic acid may be used. This procedure results in the formation of a compound of formula R"'CH(C0N(R)CH ₂ R") ₂ wherein R,R" and R ^, are as defined above. In an exactly analogous manner, reaction of 3 equivalents of the aldose amino sugar with an activated derivative of the corresponding α,α,α-tricarboxylic acid, for example nonane 1,1,1- trioic acid, results in the formation of a compound of formula R"'C(C0N(R)CH ₂ R") ₃ wherein R,R" and R"* are as defined above. The dioic and trioic acids required in this procedure may conveniently be obtained by preparing sodiomalonic ester and reacting this with a compound containing the desired group R"' attached to a suitable leaving group such as bro o, tosylate etc. to form a dioic acid in the form of the ethyl diester, the sodio derivative of this diester being further reacted with ethyl chloroformate to form the corres- ponding trioic acid in ethyl triester form where the trioic acid is required. For reaction with the amino sugar the dioic or trioic acid ester is first hydrolyzed and the free acid reacted with ethyl chloroformate to provide a mixed di- or tri-anhydride, the mixed anhydride then being reacted with the amino sugar. It will be appreciated that the compounds of formula R"C0N(R)R' described above may contain a group R which is also of the form R', the two groups R' containing either the same or different aliphatic hydrocarbon groups with a chain of at least 3 carbon atoms. Moreover, compounds of formula R ^, C0N(R)CH„R" may contain a group R which is a polyhydroxy substituted acyclic aliphatic group, this

group more usually differing from the group CHJK.". Thus, for example, reaction of D-glucose with tris Instead of methylamlne in the reaction illustrated hereinbefore will give N-(tris-hydroxy- methyl)methyl-D-glucamine which can be acylated at the nitrogen atom by the several procedures described herein.

In another modification of the procedures described above compounds may be prepared which contain more than one aliphatic hydrocarbon group. The first group of compounds of this type is obtained by reacting an activated derivative of an acid containing two aliphatic hydrocarbon groups, for example an α,c_-dialkylacetic acid such as α,α-dioctadecylacetic acid, with an equivalent of an aldose amino )CH ₂ R" wherein R, R"and R ^,M are as defined above and R"" is also an aliphatic hydrocarbon group of at least 2 carbon atoms which may be the same as or different from the group represented by R"*. The c_,α-dialkylacetic acids may conveniently be obtained by preparing a dialkymalonic acid ester as described hereinafter, and saponifying and decarboxylating this to give the corresponding acetic acid. This acid may then be reacted in activated form, for example as a p-nltrophenyl or other activated ester, with one equivalent of the amino sugar. An alternative group of compounds according to the present Invention containing more than one aliphatic hydrocarbon group is obtainable by reacting one equivalent of an activated derivative of a dioic acid containing two such groups, for example an α,c.-dialkylmalonic acid such as α,α -dioctylmalonic acid, with two equivalents of an aldose amino sugar in order thereby to obtain compounds of formula „„^C(C0N(R)CH ₂ R")_ wherein R, R", R"' and R"" are as defined above. The dioic acids may conveniently be obtained by preparing sodiomalonic ester (the sodium derivative of diethylmalonate) and reacting this with a compound containing the desired group R"' attached to a suitable leaving group such as bromo, tosylate etc. to form a dioic acid, in the form of the diethyl ester, containing an aliphatic hydro¬ carbon group R ^,M . The sodio derivative of this diester is then reacted in a similar manner with a similar type of compound

containing a group R"", this often being the identical compound to that used before although it can contain a group R"" different from the aliphatic hydrocarbon group R"' if desired, to produce a dioic acid containing two groups, R'" and R"", again in ethyl diester form. For reaction with the amino sugar this dioic acid ester is first hydrolyzed and the free acid reacted with ethyl chloroformate to provide a mixed di-anhydride, the mixed anhydride then being reacted with two equivalents of the amino sugar.

It will be appreciated that the methods described above are not the only ones suitable for the preparation of compounds accord¬ ing to the present invention and that other alternative approaches may be used which will be apparent to these skilled in the art.

From the foregoing description it will be appreciated that in a preferred embodiment the present invention comprises a compound which contains one or more acyclic amino sugar residues attached through an amide function or functions incorporating the nitrogen atom of the amino sugar residue or residues to one or more aliphatic hydrocarbon chains of at least three carbon atoms. In a further preferred embodiment the present invention comprises a compound of the general formula (I)

in which R is an aliphatic hydrocarbon group of at least 2 carbon

2 atoms, R is an acyclic amino sugar residue linked to a carbonyl group through which it is attached to the central carbon atom,

3 4 and R and R are each separately hydrogen, an aliphatic hydro- carbon group of at least 2 carbon atoms or an acyclic amino sugar residue linked to a carbonyl group. (Compounds of the type R'C0N(CH ₂ R") ₂ described above may be regards as containing an acyclic amino sugar residue R of the form -N(CH_R") ₂ which itself contains two aldose residues R") .

Although the group R may have as few as two carbon atoms in compounds according to the present invention employed for certain purposes, it is usual for the more routine uses as surface active agents for the compounds to contain a group R of at least four

05 and preferably five or six carbon atoms. As regards the upper limit for R , in some circumstances as discussed hereinafter this may be as high as fourteen, sixteen, eighteen or nineteen carbon atoms, or even as high as twenty-two, twenty-four or twenty-six carbon atoms, but for the more routine uses as surface active

10 agents groups of up to (and including) twelve carbon atoms, preferably of up to ten carbon atoms and especially of up to eight or nine carbon atoms, are of most interest. Although an increase in surface active properties generally results from an increase in the number of carbon atoms this advantage is to some extent offset

15 by the accompanying decrease in solubility and in consequence compounds having a group R in a relatively narrow range of six to eight carbon atoms, particularly seven or eight carbon atoms, often represent the most useful compounds of their particular type in the context of conventional use as a surface active agent.

3 4 20 Similar comments as to size apply equally to the groups R and R when these represent an aliphatic hydrocarbon group. Moreover, such comments also apply to the particular types of compound according to the present invention described earlier with appro¬ priate adjustment for the fact that when R is of six carbon 25 atoms, for example, then, together with the central carbon atom of formula (I) the compound contains an aliphatic hydrocarbon group of one more carbon atom than this, i.e. of seven carbon atoms, and

2 together with the central carbon atom and the carbonyl group of R the compound contains an acyl group of two more carbon atoms than

30 this, i.e. of eight carbon atoms. A preferred overall range of size for aliphatic hydrocarbon chains in compounds according to the present invention thus corresponds to a chain length of C_ - C»_, particularly C_ - C _2Q . The aliphatic hydrocarbon groups contained in compounds according to the present invention (including any

35 such group comprising R) will most usually be saturated and also

will often be unbranched, particularly in the case of the lipid compounds discussed hereinafter, although branched alkyl groups rather than straight chain alkyl groups may be of interest in some particular applications. Moreover, it will be appreciated that branching within the whole aliphatic hydrocarbon chain present in the amphipathic compound may arise from the presence of groupings such as (R ^, )-C- and that such a grouping may constitute an aliphatic hydrocarbon chain containing a total of more than twenty or twenty- seven carbon atoms even though the length of the chain, i.e. the longest sequence of carbon atoms therein, may not exceed twenty or twenty-seven carbon atoms.

2 The preferred types of group R present in compounds of formula (I) have already been discussed, for example groups derived from aldoses in which the original formyl group has been replaced by a group -CONH(R)- in which R is hydrogen or an organic grouping, or more particularly by a group -CH„NH(R)C0-, examples of specific

2 groups R being those derived from amino sugars obtained from

D-mannose, D-galactose and D-glucose, such as D-glucamine, etc., and N-substituted derivatives thereof. Although R may be hydrogen it Is often an organic grouping, and this may be an aliphatic hydrocarbon group, for example, of 1 to 19, conveniently of 1 to 12 or 1 to 8, and preferably of 1 to 3 carbon atoms. Alternatively, the organic grouping may, for example, be a phenyl group, or a substituted C. - C, alkyl or phenyl group, the substitutent(s) being, for example one or more hydroxy, sulphydryl or carboxy groups or other polar organic residues. Hydrophilic groups R such as hydroxymethyl can be of interest for the properties they confer on the compounds and certain substituted phenyl groups such as p-hydroxyphenyl are of particular interest in the context of specific uses of the compounds as described hereinafter. However preferred organic groups R are unsubstituted C. - C, alkyl groups, for example n-propyl, ethyl or especially methyl.

2

The comments made in relation to R generally apply also

3 4 to R and R when these groups are acyclic amino sugar residues linked to a carbonyl group. Most commonly R and R are both

hydrogen but compounds in which one is hydrogen and the other an acyclic sugar residue, or in which both are such a residue, more usually the same residue, can be of value in maintaining the hydrophile-lipophile balance where one or more longer aliphatic hydrocarbon groups are present, for example in an amphipathic compound having a group R of C.-, C.., or C.,. Similar comments apply in the case of compounds of the specific type R'C0N(CH R") ₉ .

4 1

Compounds in which R , as well as R , is an aliphatic hydrocarbon

3 group, often the same one, with R being an acyclic amino sugar residue or hydrogen, are of particular interest for use as synthetic lipids as discussed hereinafter.

Compounds of the present invention are of general interest for use as non-ionic surface active agents. Non-ionic surface active agents are currently used in a wide variety of industrial and other contexts including cosmetic and drug products, for example shampoos, lotions, acne creams, eye ointments and contra¬ ceptive formulations. Another very Important use of non-Ionic surface active agents is as emulsifiers and dispensing agents for medicinal products designed for internal use and the compounds of the present invention are particularly suited to such uses which require surface active agents of high purity, low toxicity and good biodegradability.

The recent increasing interest in the use of detergents in biochemistry, cell biology, immunology, etc., has already been referred to. Compounds according to the present invention are also of particular value in this context in view of a high CMC in conjunction with a non-denaturing character. Examples of such applications where the compounds of the present invention may be used with advantage are the solubilization of membranes, whole cells and other tissue samples including particularly situations where reconstitution of solubilized membranes, etc., is required, and the preparation of liposomes.

The use of amphipathic compounds according to the present invention in the preparation of liposomes may involve the solubili- sation of conventional lipσsome-forming lipids such as bovine

O PI sfy, WIPO -

brain lipids, as described hereinafter in Example 8. Alterna¬ tively, in an additional use described in a copending U.K. patent application of even date herewith in the names of Hider and

1 4 Hildreth, compounds of formula (I) in which R and R are each

3 aliphatic hydrocarbon groups, and R is hydrogen, may be used with

1 4 advantage, particularly when R and R each contain 14 to 18 carbon atoms, in place of the lipids more conventionally employed in liposome formation. Certain particular types of compound are of course especially suited in various specific areas of application of the compounds according to the present invention other than the use as surface active agents discussed above. Thus, for the extraction of peripheral proteins, compounds (often of a type

3 4 corresponding to formula (I) with R and R being hydrogen) con¬ taining smaller aliphatic hydrocarbon groups than usual are of especial interest and it is for such a usage that groups of three up to five or six carbon atoms are of most interest, groups of five, six or more carbon atoms being of most interest in relation to the more conventional use of the compounds as surface active agents. Another specific area is in the use, generally, as lipids of compounds containing two aliphatic hydrocarbon groups together with either one or two polyhydroxy substituted acyclic aliphatic

4 groups. Such compounds are those of formula (I) in which R as

1 3 well as R is an aliphatic hydrocarbon group, and R is either hydrogen or an acyclic amino sugar residue attached through a carbonyl group to the central carbon atom. Compounds of this type

4 2 3 may, if desired, contain a group R or groups R and R which comprise an acyclic amino sugar residue linked to a cyclic sugar residue, for example one or both groups being a residue derived from maltose, but preferred groups comprise monosaccharide amino sugar residues. In such lipid compounds the aliphatic hydrocarbon

1 4 groups R and R are conveniently somewhat larger than usual.

Thus it is preferred that they are of at least ten or especially at least twelve or fourteen carbon atoms, whilst a preferred upper limit is twenty-six carbon atoms, or especially twenty-four or twenty-two carbon atoms. Very often, however, a more convenient

upper limit is eighteen or nineteen carbon atoms so that a preferred range of size is 14 to 18 or 19 carbon atoms, for example 14, 16 or 18 carbon atoms. Preferably such lipid compounds contain unbranched groups or groups which are only slightly branched so that these overall sizes for the groups R and R correspond closely to their chain lengths (the length of an aliphatic hydrocarbon chain being the number of carbon atoms in the longest sequence of carbon atoms in the chain so that a branched chain will contain a larger total number of carbon atoms than its length) .

Yet a further specific area involves photographic develop¬ ment. Developer molecules which are amphiphilic in nature are most strongly absorbed to film and paper surfaces and are thus most efficient for electron transfer. Compounds according to the present invention can combine both surface active and developing properties and be of particular value in the photographic area. Such compounds contain an amide grouping in which the nitrogen atom is substituted by a p-hydroxyphenyl group and preferably contain an aliphatic hydrocarbon group of 5 to 8 carbon atoms. A specific example is a compound of formula (I) in which R is a

2 hexyl group, R comprises the residue of the amino sugar obtained from D-glucose by reaction with p-hydroxyaniline and linked through

3 4 a carbonyl group to the central carbon atom, and R and R are hydrogen, the compound thus containing the grouping -N-(p-hydroxy- phenyl)-CO-.

Finally, it may be mentioned that increased attention is currently being paid to amphipathic compounds for use in the formation of thin or molecular films and the compounds of the present invention are also of interest in this context, the invention being of value for the ready preparation of amphipathic compounds containing various types of head group suited to a variety of purposes.

It will be appreciated that the amounts of compound according to the present invention utilised to produce the desired effect will vary from one particular use to another. By way of guidance,

OMP

however, it may be stated that for routine surfactant uses an amount of the compound which gives a final concentration in use which lies In a range of as wide as from 0.01% w/v to 10% w/v is often suitable. A similar range is also often appropriate in other contexts but when used as a lipid a higher concentration of the compound of up to 50% w/w may sometimes be suitable. It will be appreciated that where desired, a mixture of amiphathlc com¬ pounds according to the present invention may be employed instead of a single compound. The invention is illustrated by the following Examples.

EXAMPLES All glassware used is dried at 100 C for 2 hours before use. The commercially obtained starting materials are of the best grade and highest purity available. Example 1 : N-(D-gluco-2,3,4,5,6-pentahydroxyhexyl)-N-methyl octanoic acid amide Crude capryllc acid anhydride (3.5ml) is stirred with N-methyl-D-glucamine (2g) in methanol (50ml) at 50 C for one hour. The solvent is then removed by rotary evaporation under reduced pressure and the residue, which consists of the title amide and octanoic acid, is taken up in ether (50ml). The title amide starts to precipitate immediately and the mixture is allowed to stand on ice for one hour to complete precipitation. The oily solid is collected on a sintered glass funnel and then placed in acetone (50ml). After stirring for 30 minutes at 25 C, the solid is collected on a sintered glass funnel and air dried to give N-(D-gluco-2,3,4,5,6-pentahydroxyhexyl)-N-methyl octanoic acid amide (OMEGA) in 90% yield (2.9g). Capryllc acid anhydride The anhydride starting material is prepared as follows. A mixture of caprylyl chloride (20ml) with NaOH (5g) in distilled water (60ml), together with 1.0M HC1 (1ml) to prevent the formation of sodium carboxylate, Is shaken for 15 minutes at 23°C. The mixture is then extracted with ether (70ml) in a separatory funnel. After vigorous shaking the two phases are allowed to separate over 30 minutes at 23 C. The ethereal phase

-$UR OM

is collected and washed once with water (20ml) . The ethereal solution of capryllc acid thus obtained is cooled to 0 C and treated with pyridine (20ml) . After stirring briefly, caprylyl chloride (20ml) Is added to initiate the immediate formation of a voluminous white precipitate. The mixture is filtered through a chilled Buchner funnel and the filtrate is placed on a rotary evaporator under reduced pressure until no further solvent can be removed, the resultant residue consisting of crude caprylic acid anhydride (30.3g, 91%). Example 2 : N-(D-gluco-2,3,4,5,6-pentahydroxyhexyl)-K-methyl nonanoic acid amide A solution of nonanoic acid (20ml, 16g) and pyridine (10ml) in ether (100ml) is cooled in ice for 10 minutes and is then treated very rapidly with ethyl chloroformate (11ml). The resulting ethereal solution containing precipitated pyridinium hydrochloride is filtered directly Into a warm solution of N-methyl-D-glucamine (19.5g) in methanol (150ml), the residual hydrochloride being washed with cold ether (50ml) which also passes directly into the methanolic solution. After stirring for 30 minutes at 25 C, the methanol/ether solution is allowed to stand overnight at 4 C and is then filtered to remove crystals of unreacted N-methyl-D- glucamine. The solvents are removed by rotary evaporation under reduced pressure and the residue is poured with stirring into ether (300ml) cooled on ice. After stirring for one hour or longer the solid is collected, washed with ether (100ml) and dried to give N-(D-gluco-2,3,4,5,6-pentahydroxyhexyl)-N-_nethyl nonanoic acid amide (MEGA-9) in 85% yield (28.5g). The amide Is recrystal- lised from warm methanol (lml/g) by adding ether (lOml/g) and allowing to stand overnight at room temperature. Example 3 : N-(D-gluco-2,3,4,5,6-pentahydroxyhexyl)-N-methyl decanoic acid amide • A solution of capric acid (23g) and pyridine (10ml) in ether (100ml) is cooled in ice for 10 minutes and is then treated very rapidly with ethyl chloroformate (13.08g) when the precipitation of pyridinium hydrochloride ^.s immediately initiated. After

_* standing briefly on Ice, the ethereal solution is filtered directly

into a warm solution of N-methyl-D-glucamine (20g) in methanol (150ml). The residual hydrochloride is washed with cold ether (50ml) which also passes directly into the methanolic solution. The methanol/ether solution is stirred for 45 minutes at 50 C and the solvent is then removed by rotary evaporation under reduced pressure. The resultant oily residue is poured into ether (350ml) with stirring and ice cooling. After stirring for one hour or longer the solid is collected, washed with 100ml of ether and dried in vacuo at 23 C to give N-(D-gluco-2,3,4,5,6-pentahydroxy- hexyl)-N-methyl decanoic acid amide (MEGA -10)in 85% yield (29.7g). The amide is recrystallised by dissolving in warm methanol (lml/g) , adding diethyl ether (lOml/g), and allowing the solution to stand overnight at room temperature (68 - 90% recovery) . Example 4 : N-(D-gluco-2,3,4,5,6-pentahydroxyhexyl)-N-methyl- -tetradecyl eicosanoic acid amide

(1) Diethyl α-tetradecylmalonate

Sodium (1.03g) is added to dry ethanol (50ml) and allowed to dissolve completely. Dlethylmalonate (21g) and tetradecyl bromide (27g) are added to the resulting solution of sodium ethoxide and the mixture is refluxed for 18 hours. Water (100ml) is added to the cooled solution, which is then extracted with methylene chloride (3 x 100ml). The methylene chloride extracts are dried with Na^SO, and rotary evaporated (0.1mm Hg) to yield an oil containing pre¬ dominantly diethyl α-tetradecylmalonate in 55% yield, γ 1730, 1745cm ^" .

(2) Diethyl α-tetradecyl-α-octadecylmalonate

Sodium (0.8g) is added to dry ethanol (50ml). When complete dissolution is effected, diethyl α-tetradecylmalonate (lOg) Is added and the solution Is refluxed for 1 hour. After cooling to 40 C, the solution is rotary evaporated to dryness. Xylene

(75ml, sodium dried) is added to the residue together with octadecyl bromide (8.1g). The resulting mixture is refluxed for 24 hours. After cooling, the mixture is washed with water (3 x 100ml) and the resulting xylene solution is dried with Na.SO, . Rotary evaporation (0.1mm Hg) yields a viscous oil containing predominantly diethyl

α-tetradecyl-α-octadecylmalonate In 82% yield, γ 1730cm .

U_.c_.3C

(3) α-Tetradecyl eicosanoic acid

Diethyl α-tetradecyl-α-octylmalonate is added to a solution of KOH (5g) in ethanol (50ml) and the resulting solution is warmed to 45°C for 3 hours with continuous stirring. The solution is then diluted with water (100ml) followed by the addition of concen¬ trated HC1 until the pH of the solution reaches 3.0. The acid solution is extracted with methylene chloride (3 x 100ml) and dried with Na„S0, . Rotary evaporation yields α-tetradecyl eicosanoic acid as (7.5g, 86%), Ύ

TlirtX 1705cm ^"1 .

(4) p-Nitrophenyl α-tetradecyl eicosanoate α-Tetradecyl eicosanoic acid (lg), dlcyclohexyicarbodiimide (0.34g) and p-nitrophenol (022g) are dissolved in methylene dichloride (10ml). The mixture is stirred for 4 hours at room temperature after which time the precipitated dicyclohexylurea is separated by filtration. ^" The CH_C1 ₂ filtrate is washed with saturated aqueous NaHC0_ (3 x 20ml) followed- by water (20ml) . The methlene chloride solution is then dried with Na ₂ S0, and rotary evaporated to yield a yellow paste. Trituration of this paste with ethyl acetate yields a yellow solution, which on evaporation yields a pale yellow oil. Trituration of this oil with methanol gives p-nitrophenyl α-tetradecyl eicosanoate as a pale yellow solid (0.85g, 71%), m.p. 31-34°C; γ 1720cm ^"1 .

(5) N-(D-gluco-2,3,4,5,6-pentahydroxyhexyl)-N-methyl-α-tetradec yl eicosanoic acid amide p-Nitrophenyl α-tetradecyl eicosanoate (0.4g) is dissolved in chloroform (8ml) and added to a solution of N-methyl-D-glucamine (0.6g) in dimethyl formamlde (12ml). The solution is stirred at room temperature for 24 hours and then subjected to rotary evapora- tion. The solid residue is partitioned between water and methylene cloride and the organic phase is washed with saturated aqueous NaHC0_ (2 x 50ml) and dried over Na_S0, . Rotary evaporation yields an oil (0.25g) which is chromatographed on silicic acid (C-44, Mallinkrodt, Camlab), the chromatography column being eluted with an increasing content of ethyl acetate in hexane. The

I

pure ethyl acetate fraction is rotary evaporated to give N-(D-gluco- 2,3,4,5,6-pentahydroxyhexyl)-N-methyl-α-tetradecyl eicosanoic acid amide as a solid (50mg, 10%) .p. 25-27°C; γ 1680cm ^"1 ; δ(CDCl ₃ ) 0.9(m,6H), 1.2(m,60H), 2.2(m,3H), 2.9(s,3H), -3.2-4.3(m,9H) . Example 5 : N-(D-gluco-2,3,4,5,6-pentahydroxyhexyl)-N-methyl-α- tetradecyl hexadecanoic acid amide This compound is obtained as a solid of m.p. 32-34 C in a yield of approximately 10% by the reaction of N-methyl-D-glucamine with p-nitrophenyl α-tetradecyl hexadecanoate in a similar procedure to that described under section 5 of Example 4. The p-nitrophenyl α-tetradecyl hexadecanoate is obtained from diethyl α,α-di-(tetra- decyl)-malonate (obtained from diethyl α-tetradecylmalonate using tetradecyl bromide in an analogous manner to that described in section 2 of Example 4), the formation of this p-nitrophenyl ester being effected by similar procedures to those described in sections 3 and 4 of Example 4.

Example 6 : N-(D-gluco-2,3,4,5,6-pentahydroxyhexyl)-N-methy1-α- octadecyl eicosanoic acid amide This compound is obtained as a solid of m.p. 38-40 C in a yield of approximately 10% by the reaction of N-methyl-D-glucamine with p-nitrophenyl α-octadecyl eicosanoate in a similar procedure to that described under section 5 of Example 4. The p-nitrophenyl α-octadecyl eicosanoate is obtained from diethyl α,α-di-(octadecyl)- malonate (obtained from diethyl α-octadecylmalonate using octadecyl bromide in an analogous manner to that described in sections 1 and 2 of Example 4), the formation of this p-nitrophenyl ester being effected by similar procedures to those described in sections 3 and 4 of Example 4.

Example 7 : Tests on the solubilization of purified plasma membranes with surface active agents

(1) Release of membrane proteins with retention of antigenicity

Plasma membranes purified by standard techniques (Crumpton and Snacy, Contemporary Topics in Molecular Immunology, 1974 3, 27) were solubilized by incubation for 60 minutes at 0 C in phosphate buffered saline (PBS) containing 1% w/v of one of the detergents N-(D-gluco-2,3,4,5,6-pentahydroxyhexyl)-N-methyl octanoic acid

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amide (OMEGA), N-(D-gluco-2,3,4,5,6-pentahydroxyhexyl)-N-methyl nonanoic acid amide (MEGA-9) or N-(D-gluco-2,3,4,5,6-pentahydroxy- hexyl)-N-methyl-decanoic acid amide (MEGA-10) at a detergent to protein ratio of 1:5 w/w. For comparative purposes, a similar procedure was carried out using the detergent octyl glucoside (OG) marketed by Sigma. In each case, the mixture from the incubation was centrifuged at 10 x g for 45 minutes at 4 C to pellet detergent insoluble material. It was noted at this stage that the insoluble material remaining after solubilization with MEGA-10 was a fraction of that remaining with the other detergents.

The amount of the integral membrane HLA-A,B,C glycoproteins released through the detergent treatment with retention of its native conformation, was determined through an assay based upon the inhibition by the protein of the binding of a radio labelled antibody to the surface of glutaraldehyde-fixed lymphoblastoid cells. The detergent extract obtained after centrifugation was diluted serially twofold using a 1% w/v solution of the detergent in PBS and 50 microlitre samples of the extract diluted at various levels were incubated for 2 hours at 4 C with 30 microlitres of a

125 solution of 1-labelled antibody against the, HLA-Δ,B,C glyco¬ proteins in PBS at a concentration of lyg/ml. 10 fixed cells in 25 microlitres of PBS were then added and the whole mixture incubated for a further period of 45 minutes at 4 C. The cells were then separated by centrifugation at 500 x g, washed twice with PBS containing lOmg/ml of bovine serum albumin (BSA) , and

125 were then assayed for .bound 1-labelled antibody using a gamma counter.

The results obtained are shown in Figure 1 where cell-bound

125 -3

I in cpm x 10 is plotted against level of dilution of extract. The higher the amount of protein released by the detergent, the

125 more I-antibody will be protein-bound and thereby inhibited from binding with the cells. It will be seen, therefore, that MEGA-9 and MEGA-10 are generally superior to the commercial detergent OG in effecting release of intact antigenic protein. In the case of OMEGA, although it is less effective, at the concentra¬ tion used, than the other three detergents in releasing integ'ral

membrane proteins, the compound is very effective in releasing peripheral membrane proteins. OMEGA will also show effective release of integral proteins at higher concentrations. (2) Reconstitution of solubilized membranes with recovery of membrane antigenicity

Plasma membranes purified by standard techniques (Crumpton and Snacy, ibid) (l g) were solubilized by treatment during 30 minutes with a 0.5%w/v solution of MEGA-9 in PBS (1ml) cooled on ice. A control experiment was carried out in which the membranes were incubated for 30 minutes with PBS (1ml) containing no detergent. In both cases, the resulting mixture was dialysed overnight at 4 C against PBS (2 litres) to remove detergent and thereby reconstitute the membranes. The dialysed material was then centrifuged at 10 x g for 45 minutes at 4 C, the supernatant with contained non-reconstituted protein was removed and the resulting membrane pellet was resuspended in PBS (0.5ml). The PBS suspension was subjected to threefold serial dilutions for use in an assay based on the inhibition by the membranes of the binding of monoclonal antibody to glutaraldehyde-fixed lymphoblastoid cells. 0.1ml samples of the diluted PBS suspensions were incubated for one hour at 4 C with 0.05ml of a PBS solution (lμg/ml) of a monoclonal antibody raised against the membrane proteins HLA-A,B,C. The incubation mixtures were then centrifuged at 500 x g for 10 minutes and 50 microlitres of the supernatants were added to 10 fixed cells in 50 microlitres of PBS (duplicate experiments being carried out at each dilution level) and the whole incubated for a further period of one hour at 4 C. The cells were then separated by centrifugation at 500 x g, washed with PBS and incubated for another one hour at 4°C with 0.1ml of a solution in PBS (lyg/ml)

125 of I-labelled antibody specific for the monoclonal antibody described above. The cells were finally washed once more with PBS

125 and were then assayed for bound I-labelled antibody using a gamma counter.

The results obtained are shown in Figure 2 where percentage inhibition of binding is plotted against membrane dilution. .The higher the amount of membranes present in the incubation mixture

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containing the monoclonal antibody, the higher will be the amount of monoclonal antibody which will become bound to the membranes and the lesser the amount which will become bound to the cells, and consequently the higher will be the degree of Inhibition of

125 binding of I-antibody to the cells through attachment to the cell-bound monoclonal antibody. In Figure 2 It Is seen that the level of inhibition is very similar for the membranes reconstituted after detergent treatment and for the control membranes, thereby illustrating the ease with which detergents such as MEGA-9 can be removed after solubilization of membranes with the consequent reconstitution of the membranes.

Example 8 : Use of surface active agents in the preparation of microvesicles Influenza virus haemmagglutinin (75 mlcrograms) purified by standard techniques (Skehel and Schild, Virology, 1971, hh__ 396) in 0.5% w/v aqueous sodium cholate (0.3ml) and bovine brain lipids (Sigma) (300 mlcrograms) in PBS containing 5% w/v MEGA-10 (0.3ml) were mixed in 0.5% w/v MEGA-9 in PBS (1.0ml) to "solubilise" the lipids which cannot be effected with the sodium cholate. The mixture was dialysed for 12 hours at 4°C against PBS (2 litres) and was then centrifuged at 10 x g for 45 minutes (the removal of the MEGA-9 and 10 in such a short time is evidence of their high CMC). The resulting microvesicle pellet was resuspended in PBS (1.0ml) and the suspension tested for haemagglutination activity against one day old chicken red blood cells. These microvesicles showed a titre of greater than 2048 whilst microvesicles prepared in a control experiment in which the haemagglutinin was omitted showed no measurable titre. Examination of the active microvesicles by electron microscopy showed a population of spherical unilamellar vesicles of uniform size (600 - HOOnm).