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

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

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 segment lengths vary greatly.

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