Highly Stereocontrolled Total Synthesis of β‑d‑Mannosyl Phosphomycoketide: A Natural Product from <i>Mycobacterium tuberculosis</i>

β-d-Mannosyl phosphomycoketide (<b>C</b>_<b>32</b><b>-MPM</b>), a naturally occurring glycolipid found in the cell walls of <i>Mycobacterium tuberculosis</i>, acts as a potent antigen to activate T-cells upon presentation by CD1c protein. The lipid portion of <b>C</b>_<b>32</b><b>-MPM</b> contains a <b>C</b>_<b>32</b><b>-mycoketide</b>, consisting of a saturated oligoisoprenoid chain with five chiral methyl branches. Here we develop several stereocontrolled approaches to assemble the oligoisoprenoid chain with high stereopurity (>96%) using Julia–Kocienski olefinations followed by diimide reduction. By careful choice of olefination sites, we could derive all chirality from a single commercial compound, methyl (2<i>S</i>)-3-hydroxy-2-methylpropionate (>99% ee). Our approach is the first highly stereocontrolled method to prepare <b>C</b>_<b>32</b><b>-MPM</b> molecule with >96% stereopurity from a single >99% ee starting material. We anticipate that our methods will facilitate the highly stereocontrolled synthesis of a variety of other natural products containing chiral oligoisoprenoid-like chains, including vitamins, phytol, insect pheromones, and archaeal lipids

Characterization of soluble PC protein.

<p><b>A.</b> Size exclusion chromatography (SEC) on HCMV PC purified from cell supernatant with a 12E2 IgG resin. <b>B.</b> SDS-PAGE analysis of PC 12E2 affinity purification eluate and SEC fractions collected in (A) under reducing conditions. kDa = kilo Dalton; FT = flow through, T = test eluate, P = final prep eluate. <b>C.</b> ELISA binding of PC- and gH-specific NAb to the purified PC. NAb targeting different epitopes of the UL128/130/131A subunits (1B2, 54E11, 21F6, 13B5) or gH (18F10, 62–11, 62–100, AP86) were tested in saturating amounts to bind the purified PC protein via ELISA. Error bars represent difference in binding of three independent experiments. <b>D.</b> Inhibition of EC entry by purified PC protein. Serial dilutions of purified PC protein or, anti-PC NAb 1B2, or anti-gH NAb 62–11 were pre-incubated with HCMV TB40/E and subsequently evaluated via microneutralization assay using ARPE-19 EC to determine the protein/antibody concentration at which 50% HCMV infection was neutralized (NT50). <b>E and F.</b> Antibody depletion by purified PC. Commercially available serum products of HCMV seropositive (Seracare 71 and 60) and seronegative (Seracare neg.) individuals or intravenous hyperimmune globulins (IvIg) were serially incubated in ELISA plate wells coated with the purified PC or with mock (1% BSA/PBS). Depleted and mock-depleted samples were tested by ELISA and microneutralization to determine PC-specific binding antibodies (E) and NAb titer (NT50) that block TB40/E infection of ARPE-19 EC (F), respectively.</p

Homologous and heterologous vaccination with MVA-PC and purified PC.

<p>BALB/c mice (n = 5) were i.p. immunized with either MVA-PC alone (5x10⁷ PFU), purified PC protein alone (1 μg, admixed with AddaVax), or the indicated prime-boost regimen. Shown are NAb titers (NT50) following two <b>(A and C)</b> or three <b>(B and C)</b> immunizations that were measured against HCMV TB40/E on ARPE-19 EC. Black triangles indicate immunizations. Bars represent 95% confidence interval of the geometric mean. Floating bars in C represent minimum, maximum, and mean NT50 titers measured on EC at week 16 in the two- and three-immunization regimens. Shown is multiple t-test statistical analysis of NAb titers. (** = p≤0.01, * = p≤0.05).</p

Immunogenicity of purified PC protein to induce NAb and binding antibodies in mice.

<p>Balb/c mice (n = 4) were immunized three-times by intramuscular (i.m.), intraperitoneal (i.p.), or subcutaneous (s.c.) route with 1 μg of purified PC protein admixed with AddaVax adjuvant. Control animals (n = 3) were immunized i.p. using AddaVax alone. Serum NAb titers (NT50) were evaluated against TB40/E on ARPE-19 EC <b>(A)</b> and MRC-5 FB <b>(B)</b> by microneutralization assay at multiple time points over a period of 28 weeks. Black triangles indicate immunizations. Bars represent 95% confidence interval of the geometric mean. PC-specific binding antibody titers (EC50) at week 11 and 28 <b>(C)</b> were analyzed by ELISA. Differences between groups were evaluated using multiple t-test (* = p≤0.05, ** = p≤0.01).</p

2CC core antibody repertoire in patient EB179.

<p>(A) Pie chart representing ten antibody sequence families sorted using the 2CC core protein as bait. The number in the center of the pie denotes the number of antibodies; slices are proportional to clone size (i.e., frequency) and represent unique clones. The assigned number of each clone is given next to the corresponding slice on the pie chart. The membership of an antibody in a B cell clone is determined by sequence analysis, in particular of the CDR3s and shared V and J genes of paired heavy and light chain genes (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005238#sec002" target="_blank">Methods</a>). (B) Representative variants of each clonal family of sequences were expressed and tested in ELISA for binding to 2CC core and gp140 foldon proteins. The numbers in parentheses correspond to the number of the clone as shown in (A). (C) The table summarizes the different antibodies expressed, the clone they represent and their binding to 2CC core or gp140 YU2 foldon (based on (B)) and their <i>in vitro</i> neutralization IC₅₀ values, as measured in the TZM.bl cell assay.</p

Hu-mice treatment with 179NC75.

<p>(A) Viral loads in six HIV-1_YU2-infected hu-mice when 179NC75 was administered two weeks post-infection (day-0). The red line indicates the geometric mean value. (B) Log change in viral loads in each mouse compared to the day 0 value. (C) Left panel: each horizontal gray bar represents the sequence of a single gp120 clone aligned to HIV-1_YU2. Synonymous and non-synonymous nucleotide substitutions are indicated in green and red, respectively. Sequences are grouped by the mouse from which they were obtained (center), indicated by the vertical black bars. An expanded view of the boxed areas is shown in the right panel. (D) Pie chart showing the recurrent mutations in gp120 compared to the wild-type HIV-1_YU2 sequence. The number in the center of the pie denotes the total number of sequences cloned; the blue, red and purple slices represent the most consistently mutated areas in gp120, i.e., around residues 276 and 460, respectively. The sizes of the slices are proportional to the number of sequences that carried mutations. The original residue, as found in HIV-1_YU2, is indicated on the outside of the pie chart, the various mutations in the cloned sequences are shown on the inside of the chart. “Δ” denotes a deletion of the residue. The purple slice indicates the presence of both the blue (276-area) and red (460-area) mutations. (E) The table shows IC₅₀ values for 179NC75 neutralization of the wild-type HIV-1_YU2 pseudovirus and mutants containing either the gp120 changes most frequently observed after treatment or other mutations that serve as comparators.</p

Autologous viruses from EB179.

<p>(A) Horizontal gray bars represents single gp120 sequences amplified and cloned from patient sera. The vertical rectangles show areas where recurrent mutations were found. (B) Expanded view of the most consistently mutated areas. (C) Pie chart comparing the recurrent mutations in gp120s from the EB179 autologous virus and HIV-1_YU2. The number in the center of the pie denotes the total number of sequences cloned, and the sizes of slices are proportional to the number of sequences that carried the mutations; the color coding is same as used in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005238#ppat.1005238.g005" target="_blank">Fig 5D</a>. (D) The table shows IC₅₀ values for the neutralization of the EB179 autologous culture virus by the autologous patient polyclonal IgG, as well as 179NC75, 3BNC117, 10–1074 and PG16 bNAbs.</p

Mature <i>versus</i> predicted germline antibody binding to BG505 SOSIP.664-D7324 trimers in ELISA.

<p>(A) and (B) Binding of mutated CD4bs bNAbs (“MATURE”) and their reverted germline versions (“GERMLINE”) to BG505 SOSIP,664-D7324 trimers. The MGO.053 antibody [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005238#ppat.1005238.ref048" target="_blank">48</a>] was used as a negative control.</p

179NC75 clonal family variants.

<p>(A) Dendrogram showing heavy chain amino acid sequence alignment of the different 179NC75 variants to their closest germline IGVH3-21. (B) The same plot as in (A), but for the light chains. The heavy and light chains of the variants that were expressed and tested for neutralization are marked with red squares. (C) Neutralization IC₅₀ values for 179NC75 and five variants against an expanded panel of Tier-2 viruses. The phylogenetic relationship, based on heavy chain sequences, between the six variants are shown on the bottom of the table (D) The plot compares the IC₅₀ values for 179NC75 (red circles) and four well characterized CD4bs bNAbs (VRC01 –green diamonds, b12 –yellow squares, CH103 –blue diamonds and HJ16 –white circles) against a panel of 26 clade B Tier-2 viruses.</p

Epitope mapping of 179NC75 variants by ELISA.

<p>(A) Competition ELISA. The plots show the binding of biotinylated 179NC75 (constant concentration of 2 μg/ml) to gp120 in the presence of increasing concentrations of different bNAbs. The black bold line represents 179NC75 binding in the absence of a competitor antibody, the red bold line shows auto-competition by non-biotinylated 179NC75. Upper panel: Competition by CD4bs bNAbs; lower panel competition by non-CD4bs bNAbs. (B) Binding of 179NC75 (red circles), 179NC65 (red triangles) and 179NC1055 (red diamonds) antibodies to the wild-type YU2 gp120 monomer (left upper panel) and variants containing CD4bs-related mutations (D368R+N280Y, right upper panel; D368R, right lower panel; N276D, left lower panel), VRC01 and PGT121 served as control antibodies. (C) 179NC75, 179NC65 and 179NC1055 binding to gp120 monomers from YU2 (clade B) and 93TH057 (clade A/E) before and after deglycosylation with EndoH. VRC01 was used as a control antibody in the experiment involving the YU2 gp120s. (D) Comparison between the neutralizing activity (IC₅₀) of 179NC75 and HJ16 against a cross-clade virus panel of Tier-2 viruses.</p