**Characteristic Conformational Behaviors of Representative Mycolic Acids in the Interfacial Monolayer**

Masumi Villeneuve *Graduate School of Science and Engineering, Saitama University Japan*

### **1. Introduction**

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> Mycobacterial mycolic acid (MA) are long chain 2-alkyl branched, 3-hydroxy fatty acid with two intra-chain groups in the so-called meromycolate chain. On the basis of the nature of the functional groups in the meromycolate chains, MAs are categorized into three major groups: *α*-MA with no oxygen-containing intra-chain groups, methoxy-MA (MeO-MA) in which the distal group has a methoxy gorup and Keto-MA in which the distal group has a carbonyl group (Fig. 1) (Watanabe et al., 2001; 2002). MAs are characteristic components of mycobacterial cell envelopes, where a major proportion are covalently bonded to the underlying cell wall arabinogalactan (Goren & Brennan, 1979; McNeil et al., 1991; Minnikin, 1982).

> In the structural models of the mycobacterial cell envelope proposed previously (Minnikin, 1982; Rastogi, 1991), MAs covalently linked to penta-arabinosyl residues of cell wall arabinogalactan are arranged perpendicular to the cell wall, forming a highly structured monolayer. Recent computer simulation work supported such arrangement of MAs as proposed in the model (Hong & Hopfinger, 2004). This outer leaflet of mycobacterial cell envelope is considered to provide the cells with a special permeability barrier responsible for various physiological and pathogenic features of mycobacterial cells (Daffé et al., 1999). There are various other lipids in the mycobacterial cell envelope and they may also take part in the permeability function of the cell envelope as suggested (Minnikin, 1982; Puech et al., 2001; Rastogi, 1991). Recently, a *Mycobacterium tuberculosis* (*M. tb*) mutant whose MA comprises only *α*-MA (Dubnau et al., 2000), a recombinant mutant having over-produced MeO-MA with no Keto-MA (Yuan et al., 1998) and a mutant having 40 % less cell wall mycolate (Daffé et al., 1999) have been described. These results show that *M. tb* can be viable with highly modified mycolic acid composition and that its pathogenicity may be related to the types of MAs. Those papers also suggest that MAs on the cell envelope have determining effect on the permeability barrier function of the cell wall outer hydrophobic layer barrier and different MAs may contribute to the cell wall permeability barrier functions in different ways.

> In very early studies (Staellberg-Stenhagen & Stenhagen, 1945), the multi-component nature of mycolic acids was not yet known, but it was shown that the total MA formed a stable monolayer on the water surface. It was concluded that MA had extended linear structures, a feature later confirmed by structural analysis (Minnikin, 1982; Minnikin et al., 2002; Rastogi,

**3. Structural features of representative mycolic acid samples**

Characteristic Conformational Behaviors

of Representative Mycolic Acids in the Interfacial Monolayer

methyl ester and subsequent acetonide formation (yield 74%).

compositions of the MAs studied are summarized in Fig. 1 and Table 1.

segment lengths vary greatly.

**4. Experimental details**

remove the byproduct epimer.

4.0.0.2 Other reagents

4.0.0.1 Preparation of the mycolic acid samples

The structures of MAs have been characterized (Watanabe et al., 2001; 2002) and MAs are grouped into three major groups, i.e., *α*-MAs in which X and Y in Fig. 1 are two cyclopropyls or one cyclopropyl and one double bond, MeO-MAs in which X is a methoxy group with a methyl group at the adjacent distal carbon, and Keto-MAs in which X is a keto group with a methyl group at the distal adjacent carbon. In *Mycobacterium avium-intracellulare* complex (MAC), further oxidized wax-ester MA is also known. Further, natural mixtures have both

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The stereochemistry of the proximal carbon and that of the distal carbon of the *cis*-cyclopropyl group have been determined to be *R* and *S*, respectively, according to the knowedge that the *cis*-cyclopropyl group is derived from the same biosynthetic intermediate of the known stereochemistry (Al Dulayymi et al., 2005). The absolute configurations of the hydroxy-bearing carbon and the carboxyl-bearing carbon in -CH2-CH(COOH)-CH(OH)-CH2 are both *R* as reported (Asselineau & Asselineau, 1966; Tocanne & Asselineau, 1968) and as demonstrated by us by easy preparation of its stable chair form acetonide by reduction of MA

The non-oxygenated MA samples assayed were so-called type-1 *α*-MAs (*α*1-MAs, X and Y both *cis*-cyclopropyls) from *M. tb* (strain Aoyama B), *M. kansasii* (strain 304) and MAC (strain KK41-24) and so-called type-3 *α*-MAs (*α*3-MAs), from BCG (strain Tokyo 172) (X *cis*-double bond, Y *cis*-cyclopropyl in Fig. 1) and from MAC (X *cis*-cyclopropyl, Y *cis*-double bond). Their intrachain groups are either *cis*-cyclopropyl or *cis*-double bond but the methylene chain

The oxygenated MA samples were type-1 MeO-MA and Keto-MAs from *M. tb* (strain Aoyama B) and *Mycobacterium bovis* BCG (strain Tokyo 172). The structural characteristics and

MA samples used in our study were prepared by hydrolysis of purified relevant *α*-MA, MeO-MA and Keto-MA methyl esters. The procedures for separation and purification of the methyl esters including argentation thin-layer chromatography (TLC) to remove minor components with double bonds and the analytical details are described elsewhere (Watanabe et al., 2001; 2002). Hydrolysis was performed by heating a sealed tube containing MA methyl ester (70 mg), powdered KOH (200 mg) and 2-propanol (2 ml) in an oil bath kept at 80-85 ◦C for 2 hours with stirring. The hydrolysate was acidified with 2 N H2SO4 and treated with hexane, and the mycolic acid obtained was purified by TLC with hexane/AcOEt (4:1, v/v) to

Distilled reagent grade chloroform (Wako chemicals) was used as the spreading medium.

Water was distilled once and deionized by Milli-Q Plus (resistance 18.2 MΩ cm).

*cis*- and *trans*-cyclopropane rings, the latter having an adjacent methyl group (Fig. 1).

1991). Both in the monolayer on the water surface and in the proposed cell envelope lipid structure models, MA is considered to take the same structural arrangement, with the hydrophilic 3-hydroxy and 2-carboxyl groups touching the hydrophilic surface and with the aliphatic chains stretching out in parallel, and normal to the hydrophilic surface. Therefore, detailed studies on the artificial MA layers on water surface should help elucidation of the roles and the nature of actual mycolate layers in the mycobacterial cell envelope.

Recently, limited Langmuir monolayer studies have been performed on a selection of MA (Hasegawa et al., 2000; 2002; Hasegawa & Leblanc, 2003; Hasegawa et al., 2003). Those studies reported that, in a compressed monolayer, *α*-mycolic acid from *Mycobacterium avium*, apparently took a conformation with three parallel chains, and on further compression, an extended structure, but that the corresponding *M. tb* mycolate appeared to take an extended conformation. As regards the MeO and Keto MAs from *M. tb*, they were reported to take triple chain folded conformations (Hasegawa & Leblanc, 2003; Hasegawa et al., 2003). Regrettably, their monolayer experiments were limited at a single temperature of 25 ◦C whereas temperature is one of the important factors that influence biological activities of the living cells.

In this chapter, the temperature effect on the Langmuir monolayer packing of all three *α*-, Keto-, and MeO-MAs from representative slow growing mycobacteria are analyzed to elucidate the conformational behavior of MAs in the monolayer and to understand their roles in the mycobacterial cell envelope.
