Synthesis of Dideoxymycobactin Antigens Presented by CD1a Reveals T Cell Fine Specificity for Natural Lipopeptide Structures

Abstract

Mycobacterium tuberculosis survival in cells requires mycobactin siderophores. Recently, the search for lipid antigens presented by the CD1a antigen-presenting protein led to the discovery of a mycobactin-like compound, dideoxymycobactin (DDM). Here we synthesize DDMs using solution phase and solid phase peptide synthesis chemistry. Comparison of synthetic standards to natural mycobacterial mycobactins by nuclear magnetic resonance and mass spectrometry allowed identification of an unexpected ␣-methyl serine unit in natural DDM. This finding further distinguishes these pre-siderophores as foreign compounds distinct from conventional peptides, and we provide evidence that this chemical variation influences the T cell response. One synthetic DDM recapitulated natural structures and potently stimulated T cells, making it suitable for patient studies of CD1a in infectious disease. DDM analogs differing in the stereochemistry of their butyrate or oxazoline moieties were not recognized by human T cells. Therefore, we conclude that T cells show precise specificity for both arms of the peptide, which are predicted to lie at the CD1a-T cell receptor interface.

Total Synthesis of Mycobacterium tuberculosis Dideoxymycobactin‐838 and Stereoisomers: Diverse CD1a‐Restricted T Cells Display a Common Hierarchy of Lipopeptide Recognition

“…Young and co-workers reported the synthesis of DDM-838 and analogues, emphasizing solid-phase approaches [3b] For assembly of DDM-838 itself, a linear depsipeptide bearing the fatty acyl chain was assembled on Fmoc-Lys Sasrin resin. Following release of the depsipeptide from the resin, the terminal lysine was cyclized to form the caprolactam moiety.…”

“…[3b] Oxazoline acid 11 was prepared from α-methyl- l -serine 5 , by protection as the methyl ester 6 , which was condensed with 2-benzyloxysalicylic acid 8 using COMU to afford amide 9 (Scheme 1a) [7] Cyclization to the oxazoline 10 proceeded smoothly with XtalFluor-M in CH 2 Cl 2 . [8] Saponification of the ester of 10 with LiOH afforded the O -benzyl-protected acid 11 , which was coupled with NH 2 - l -Lys(Boc)OMe using EDC.FICI/FIOAt/DMAP.…”

“…The only extensively-characterized type of foreign lipidic molecule that activates CD1a-restricted T cells is a family of lipopeptides isolated from Mycobacterium tuberculosis , termed dideoxymycobactins (DDMs). [3] …”

“…Thus, isolation in high yield from natural sources remains impractical. [3] The key challenge for chemical synthesis is that naturally occurring DDMs possess a complex depsipeptide backbone bearing a range of saturated and unsaturated fatty acyl groups linked through an amide to the ε-amino group of a lysine residue, and an unusual α-methylserine-derived angular methyl group. The structure of the most reactive compound, DDM-838 ( 1 , Figure 1) was proposed through mass spectrometric and NMR analysis, [3a] as well as the preparation of a range of possible stereoisomers and matching of their spectroscopic data and biological activity to the natural product, [3b] and is consistent with the stereochemistry of the biosynthetically-related mycobactins, iron-chelating hydroxamic acid siderophores.…”

“…Prior work demonstrated that the correct stereochemistry at positions a and c (Figure 1), and the angular methyl group at position a are necessary for activating the DDM-reactive T cell clone CD8-2, and that natural DDMs lacking the unsaturation in the acyl chain are at least 10-fold less potent for T cell activation. [3] In the present work we investigate solution-based peptide-coupling approaches for the preparation of milligram quantities of DDM-838. Through careful isolation of by-products produced in this process, we identify difficulties with the establishment of the key ester linkage that occurs with partial epimerization at lysine, a problem that may have occurred in the previous approach of Young and coworkers ( unpublished ).…”