Location of Fatty Acids in Lipid A Obtained from Lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023”

Monophosphoryl lipid A (MLA) obtained from the lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023 was initially purified by silicic acid column chromatography to yield a single major pentaacyl MLA fraction. This fraction was methylated and further purified by reverse-phase high performance liquid chromatography to yield three prominent peak fractions. Laser desorption mass spectrometry of these three fractions allowed us to complete the important structural analysis of lipid A from this source. Three structurally distinct forms of dimethyl MLA were identified where M, = 1447, 1449, and 1451 atomic mass units. These forms differed only by the presence or absence of unsaturation and keto group in the fatty acids. We established that the acyloxyacyl group (either A’-tetradecenoyloxytetradecanoate or tetradecanoyloxytetradecanoate) and the 3-ketotetra-decanoate or hydroxytetradecanoate the 2’-and 2-positions of the glucosamine disaccharide, re-spectively. Analysis of several minor fractions suggests

Lipopolysaccharide (LPS)' is an amphipathic macromolecule found on the outer surface of the outer membrane of Gram-negative bacteria (1). It consists of the hydrophilic polysaccharide and lipophilic lipid A moieties (2). The lipid A component has been shown to have numerous beneficial biological properties, i.e. antitumor activity, protection against x-irradiation, and protection against bacterial infection (3), but because of its characteristic high toxicity, its beneficial properties have not been utilized clinically.
The toxicity of Salmonella LPS was attenuated considerably by an appropriate mild acid treatment to yield the MLA (4, 5 ) . Such a preparation retained the beneficial biological properties (6, 7). Strittmatter et al. (8) reported the isolation of LPS from Rhodopseudomonas sphaeroides ATCC 17023, which was naturally nontoxic and yet had structural similarities in the lipid A region to the toxic LPS from the Salmonella strain. This group has continued their study of the LPS from this source in an effort to determine the structural basis for this surprising lack of toxicity (9, 10). Although much is now known, the structural analysis of the lipid A from this source is still incomplete.
We examined the HPLC-purified dimethyl MLA fractions obtained from the LPS of R. sphaeroides ATCC 17023 by LDMS and report that the main structural ambiguities have now been resolved. We have established the precise locations of the fatty acids in the major lipid A fractions from this source.

Growth of Bacteria and Preparation of LPS-The microaerophilic R. sphaeroides ATCC 17023 (American Type Culture
Collection, Washington, D. C.) was grown photoheterotrophically in medium 550 as recommended by the American Type Culture Collection a t 34 "C, harvested a t late-log growth, and washed with 0.9% saline. The cell paste (569 g) was extracted by the method described by Strittmatter et al. (8) to yield 2.36 g of crude LPS. We further extracted this crude LPS preparation with chloroform/methanol (2:1, v/v) to yield 543 mg of the rough-type LPS.
HPLC Fractionation-HPLC was performed on the dimethyl MLA as described by Qureshi et al. (13). An 8 mm X 10 cm Cls-bonded silica cartridge (4-pm Nova Pak, Millipore Corp., Waters Chromatography Division, Milford, MA) was used with a linear gradient of 20-80% isopropyl alcohol/water (93:7, v/v) in acetonitrile/water (93:7, v/v) over a period of 60 min at a flow rate of 2.0 ml/min. The wavelength of the detector was set a t 210 nm.

RESULTS AND DISCUSSION
LPS obtained from R. sphaeroides was hydrolyzed under mild acid conditions to yield crude MLA. This preparation was then fractionated on a silicic acid column to yield the purified pentaacyl MLA designated fraction D. This fraction was methylated with diazomethane to yield the dimethyl pentaacyl MLA and subjected to reverse-phase HPLC (Fig.  1). Each of the three major fractions thus obtained was analyzed by LDMS, and the resulting spectra are shown in Fig. 2. LDMS of peak 1 gave a molecular ion M+K' at mlz 1486.
Cleavage of OHC,, at the sugar-oxygen linkage yielded M+K'-OHC,, ion at mlz 1298. Previously, we showed that the reducing-end sugar of dimethyl MLA from LPS of Nekseria gonorrhoeae can undergo three major types of fragmentations on LDMS (14,16). These were simultaneous cleavages of (A) C1-0 and C2-C3, (B) C,-CP and C3-C4, and (C) C4-C5 and C,-O, which resulted in the appearance of (distal unit + reducing unit) fragment + K' ions. Such fragmentations allowed us to determine the fatty acid distribution within the distal and reducing-end sugars. Cleavages A, B, and C of the reducing-end sugar of peak 1 occurred to give fragment + K' ions a t mlz 1204, 1032, and 972 (calculated mass, 972.5), respectively. These results showed that the M , = 1447 atomic mass units, R, = OHClo, Rz = A7-c1,0c1,, RB = OHC,", and R4 = 3KC,, (Table I). This was supported by the results of plasma desorption mass spectrometry (data not shown). We observed an oxonium ion of C46Hs5NP012 at m/z 874, which is diagnostic of the distal sugar fragment (11) and consistent with the fatty acid assignments.   Silicic acid column fraction D was methylated and subjected to HPLC. * Refer to Fig. 3 for structure. atom bombardment mass spectrometry.
LDMS of the minor peak 4 (data not shown) gave molecular ion M+K' a t m / z 1577 (MI = 1538 atomic mass units).
Cleavages of the reducing-end sugar allowed us to suggest a fatty acid distribution of R1 = OHC,,, R, = hydroxylated c14oc14, RB = OHC14, R4 = OHC14. There appeared to be an additional methyl group at an undetermined site on the distal portion of the molecule.
A complex mixture of multiple structural forms of hexaccyl dimethyl MLA was found in the minor silicic acid column fraction B. The size of the molecular ions and the fragmentation patterns by LDMS suggested the presence of two acyloxyacyl groups on the distal sugar (data not presented). This is similar to the MLA from the Salmonella strain (17). However, these MLA appeared to have either hydroxylated, methoxylated, or keto acyloxyacyl groups. This type and extent of structural heterogeneity is reminiscent of the MLA from the LPS of N . gonorrhoeae (14). The extra methyl groups found in some fractions might be due to the diazomethane derivatization reaction.
Work done by Salimath et al. (9) on the structural analysis of the unfractionated free lipid A obtained from LPS of R.  Table I and the  text for the other fatty acid distributions. taken this study further by establishing the structures of the small polysaccharide region of this LPS.
We have now assigned the A7-C140C14 or c14Oc14 to the nitrogen of C-2' (RP) and 3KC14 or to the nitrogen of C-2 (R4). We also showed considerable structure heterogeneity by the presence of three distinct and prominent structural forms of MLA as well as other minor components, which might be highly relevant to the question of why the LPS/lipid A from this source is nontoxic. Both Strittmatter et al. (8) and Salimath et al. (9) have inferred that this lack of toxicity is caused by the presence of unsaturation in A7-Cl40Cl4 and keto group in 3KC14. However, other factors might also be involved. This study completes the structural analysis of the important features of free lipid A obtained from the LPS of R. sphaeroides. The structures as the dimethyl derivatives are given in Fig. 3. A study of the precise nature of the structureto-function relationship of this lack of toxicity of LPS from this source is planned.