Total Synthesis of a Mycolic Acid from Mycobacterium tuberculosis

Article abstract of DOI:10.1002/anie.202000523

In Mycobacterium tuberculosis, mycolic acids and their glycerol, glucose, and trehalose esters (“cord factor”) form the main part of the mycomembrane. Despite their first isolation almost a century ago, full stereochemical evaluation is lacking, as is a scalable synthesis required for accurate immunological, including vaccination, studies. Herein, we report an efficient, convergent, gram-scale synthesis of four stereo-isomers of a mycolic acid and its glucose ester. Binding to the antigen presenting protein CD1b and T cell activation studies are used to confirm the antigenicity of the synthetic material. The absolute stereochemistry of the syn-methoxy methyl moiety in natural material is evaluated by comparing its optical rotation with that of synthetic material.

Full text of DOI:10.1002/anie.202000523

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Accepted Article
Title: Total Synthesis of a Mycolic Acid from Mycobacterium
tuberculosis
Authors: Nabil Tahiri, Peter Fodran, Dhineshkumar Jayaraman, Jeffrey
Buter, Martin D. Witte, Tonatiuh A. Ocampo, D. Branch
Moody, Ildiko Van Rhijn, and Adriaan J. Minnaard
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202000523
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10.1002/anie.202000523
Angewandte Chemie International Edition
RESEARCH ARTICLE
Total Synthesis of a Mycolic Acid from Mycobacterium
tuberculosis
Nabil Tahiri,[a] Peter Fodran,[a] Dhineshkumar Jayaraman,[a] Jeffrey Buter,[a] Martin D. Witte,[a] Tonatiuh
A. Ocampo,[b] D. Branch Moody,[b] Ildiko Van Rhijn,[b],[c] Adriaan J. Minnaard,[a] *
[a]
[b]
[c]
Dr. N. Tahiri, Dr. P. Fodran, Dr. D. Jayaraman, Dr. J. Buter, Prof. Dr. M. D. Witte, Prof. Dr. Ir. A. J. Minnaard
Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands.
E-mail: a.j.minnaard@rug.nl
T. A. Ocampo, Prof. D. B. Moody, MD, Dr. I. Van Rhijn
Brigham and Women's Hospital, Division of Rheumatology, Inflammation, and Immunity, Harvard Medical School. Hale BTM, 60 Fenwood Road, Boston
MA, 02115, USA.
Dr. I. Van Rhijn
Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands.
Supporting information for this article is given via a link at the end of the document.
Abstract: In Mycobacterium tuberculosis, mycolic acids and their glycerol, glucose, and trehalose esters (“cord factor”) form the main part of
the mycomembrane. Despite their first isolation almost a century ago, full stereochemical evaluation is lacking, as is a scalable synthesis
required for accurate immunological, including vaccination, studies. Herein, we report an efficient, convergent, gram-scale synthesis of four
stereo-isomers of a mycolic acid and its glucose ester. Binding to the antigen presenting protein CD1b and T cell activation studies are used to
confirm the antigenicity of the synthetic material. The absolute stereochemistry of the syn-methoxy methyl moiety in natural material is evaluated
by comparing its optical rotation with that of synthetic material.
Introduction
Mycobacterium tuberculosis (Mtb), the causative agent of
tuberculosis, is by far the most lethal member of the family of
Mycobacteriaceae, causing around 10 million new infections
yearly.[1] The difficult diagnosis and treatment of tuberculosis is
reflected in high mortality rates (1.5 million in 2018, which makes
it the leading cause of death by a single infectious agent
worldwide.[1] This difficult treatment is in part caused by the
presence of the mycomembrane in the cell envelope of Mtb, which
consists largely of α-alkyl, β-hydroxy long-chain (C70–C90) fatty
acids, known as mycolic acids.[2,3] These highly lipophilic fatty
acids are categorized according to the presence of unsaturations
or cyclopropyl groups (“-mycolic acid“), a methoxy, or a keto
function in the main chain. As the number of methylene units
between these substituents is variable, mycolic acids are found
as an inseparable mixture of homologues. Mycolic acids are found
either as the free acids or esterified to the arabinogalactan layer,
trehalose, glucose (glucose monomycolate; GMM) or glycerol
The overall molecular structure of mycolic acids has been
elucidated in the late 1960s,[7–9] and several members have been
prepared by the group of Baird[10–13] including two methoxy
mycolic acid diastereomers (Figure 2, 1c and 1d). The complete
elucidation of their stereochemistry, however, has so far not been
achieved. Moody and co-workers demonstrated that R,R
stereochemistry of the α-alkyl, β-hydroxy carboxylic acid is crucial
those of reference compounds.[7] For this, the authors applied the
principle of optical superposition, originally hypothesized by Van ’t
Hoff.[15] This hypothesis states that stereocenters that are
remotely located do not influence each other’s optical rotation,
which therefore can be added up to provide the overall optical
rotation. The analysis was supported by the synthesis work of
Baird et al., but it remained uncertain whether this hypothesis was
valid for mycolic acids as the optical rotation of the cis-cyclopropyl
ring in model systems was immeasurably small, making the
determination of its absolute stereochemistry using this strategy
impossible. Due to the lack of other viable analytical techniques,
the stereochemistry of the cyclopropyl ring still remains to be
determined.
Figure 1. The structure of the major methoxy mycolic acid homologue.
Methoxy mycolic acids (Figure 1) appear, with some
exceptions, to be present only in pathogenic mycobacteria[3] and
are antigenic in serological assays[4] and T cell assays.[5,6] Upon
binding to the antigen-presenting protein CD1b, mycolic acids and
their glucose mycolates activate T cells.[16,17] For glucose
monomycolates the type and length of the mycolate does not
influence the T cell stimulatory capacity.[17] Crystallography of the
trimolecular complex of CD1b, glucose monomycolate and T cell
for
T
cell recognition of glucose monomycolate.[14] The
stereochemistry of the α-methyl methoxy moiety in methoxy
mycolic acids was inferred by Asselineau et al. to be S,S by
comparison of molar optical rotations of natural samples with
1
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10.1002/anie.202000523
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RESEARCH ARTICLE
receptor showed that the glucose forms the T cell epitope on top
of the CD1b protein and stabilizes the complex.[18] No
crystallographic data of CD1b-mycolic acid are available, but it is
known that the type of functional groups in the chain and its length
determine the level of T cell activation.[5,6] We hypothesized that
the stereochemistry of the cyclopropyl and the α-methyl methoxy
moiety of mycolic acid might influence its capacity to bind CD1b
and activate T cells.
The development of mycolic acids and glucose
monomycolates as potential antigens in vaccine development and
diagnostics requires synthetic material strictly free from
immunologically-active impurities. This was our incentive to
undertake the total synthesis of four stereoisomers of methoxy
mycolic acid (Scheme 1, so S,S and R,R-stereochemistry in the
α-methyl methoxy moiety plus R,S and S,R-stereochemistry in the
cis-cyclopropyl moiety) and their glucose esters. This endeavour
The α-branched β-hydroxy acid moiety in fragment A was
installed via an anti-selective Abiko–Masamune asymmetric aldol
reaction[20] between aldehyde 6 and chiral ester 9 (Scheme 2a).
The synthesis of 6 started with a DIBAL reduction of commercially
available 2 to the corresponding lactol 3, directly followed by a
Wittig olefination with 4[21] resulting in 5 in 70% yield over these
two steps. The α,β-unsaturated thioester 5 was then silylated,
saturated, and reduced to the aldehyde in a single step in 82%
yield by a Fukuyama reduction. Esterification of chiral auxiliary 8
with the acyl bromide of 7 gave 9 in nearly quantitative yield. The
subsequent Abiko–Masamune aldol reaction of 9 and 6 resulted
in a rewarding dr of 9 : 1, and partial hydrolysis of the TES ether.
Therefore, the crude was treated with aqueous HCl in THF and
subsequently purified by column chromatography to provide diol
10 in 55% yield and dr > 97 : 3. Bis-TBS protection of 10, followed
by a Suzuki–Fu coupling reaction[19] with 1-hexadecene resulted
in 11 in good yields, and effectively installed the required α-chain.
Selective deprotection of the primary TBS ether with pyridine·HF,
followed by DMP oxidation of the primary alcohol to the
corresponding aldehyde resulted in fragment A.
The synthesis of fragment B started with the alkyne zipper
reaction[23] applied to commercial 12, followed by protection of the
alcohol to provide terminal alkyne 13 in 86% yield (Scheme 2b).
Deprotonation of 13 with n-BuLi at 0 °C followed by addition to
paraformaldehyde resulted in 14 in 70% yield. In our hands,
Lindlar reduction of 14[24] to cis allylic alcohol 15 resulted in a
mixture with the trans isomer, inseparable on silica and Ag-
impregnated silica. Although this problem was avoided by
subjecting 14 to a P-2 nickel reduction,[25] this time 1H-NMR
indicated concomitant over-reduction. Fortunately, separation of
15 from approximately 15% of the aliphatic alcohol succeeded
with a single run on a Ag-impregnated silica column in 73% yield.
Alkene 15 was cyclopropanated with 95% ee (see SI), mediated
by dioxaborolane 16a or 16b according to Charette’s procedure
yielding the enantiomeric alcohols 17a and 17b respectively.[26]
The alcohols 17a and 17b were subsequently converted into their
pivaloyl esters, and desilylated using TBAF to yield both
enantiomers of 18 in excellent yield over three steps,[22] and
bromination resulted in both enantiomers of fragment B.
Fragment B decomposed upon extended storage (longer than
one month) at 0 °C, and was therefore used within one week after
synthesis.
required
a drastic decrease in the number of synthetic
transformations compared to the previous synthesis by Baird et
al.[11] Therefore we opted for incorporation of the Suzuki–Fu
cross-coupling on several places in the design. Although the
Suzuki–Fu cross-coupling[19] has hardly been applied in natural
product synthesis, we advocate the use of this Pd-catalysed sp3-
sp3 cross-coupling reaction as it avoids the various functional
group transformations and basic reaction conditions required for
Wittig-type and Julia-type reactions.
In order to gain access to the four diastereomers in a
convergent manner and on gram scale, we retrosynthetically
dissected the molecule in three fragments, each containing a
chiral segment (Scheme 1). Both enantiomers of fragments B and
C had to be prepared, and combined with the natural enantiomer
of fragment A. Whereas fragment B and C were planned to be
connected via a Suzuki–Fu cross-coupling reaction, unification of
C-B to A using this reaction could not be achieved since, as model
studies showed, with a cyclopropylmethyl bromide the reaction
failed. A Julia–Kocienski olefination was projected instead.
The synthesis of fragment C (Scheme 3a) started with a
diastereoselective conjugate addition of MeLi to 19a and its
enantiomer 19b.[11,27] Then, LiAlH4 reduction followed by
bromination of 21 resulted in bromide 22 in good yields over two
steps. Chain extension was achieved by subjecting 22 to a
Suzuki-Fu cross-coupling[19] with 1-hexadecene. Acid hydrolysis
of the acetal, followed by a one-pot tosylation of the primary
alcohol and intramolecular SN2 reaction went smoothly, and
yielded the corresponding epoxide 25 in excellent yield over two
steps. Epoxide opening with Grignard reagent 26 using catalytic
CuCl minimized the formation of halohydrin by-products[28] and
afforded the secondary alcohol 27 in good yield. Finally,
methylation of the secondary hydroxyl using an excess of MeI and
NaH resulted in both enantiomers of fragment C in nearly
quantitative yields.
Scheme 1. Retrosynthetic analysis.
Results and Discussion
Synthesis of the Fragments
2
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Scheme 2a. The synthesis of fragment A. a) DIBAL, CH2Cl2, -78 °C, 1 h; b) 4, THF/toluene (2:1), 75 °C, 16 h, (70% over two steps); c) Et3SiH, Pd/C, acetone, 0 °C
to rt, 6 h, (82%); d) (COBr)2, 8, pyridine, CH2Cl2, 0 °C to rt, 3 h (94%); e) Cy2BOTf, NEt3, THF, -78 °C, then 6, 16 h; f) 2 M HClaq, THF, rt, 2 h (55% over two steps,
67% yield brsm); g) TBSOTf, 2,6-lutidine, CH2Cl2, 0 °C to rt, 3 h (94%); h) 1-hexadecene, 9-BBN, Pd(PCy3)2 (10 mol%), K3PO4·H2O, THF, rt, 16 h (87%); i)
pyridine·HF, THF, 0 °C to rt, 4 h (95%); j) DMP, CH2Cl2, rt 1 h (96%). Scheme 2b. Synthesis of fragment B. a) NaH, 1,3-diaminopropane, 70 °C, 1.5 h; b) TBDPSCl,
CH2Cl2, 0 °C to rt, 16 h (86% over two steps); c) n-BuLi, paraformaldehyde, THF, 0 °C to rt, 16 h (70%); d) NaBH4, Ni(OAc)2·4H2O, H2, ethylenediamine, EtOH, rt,
15 min (73%); e) 1,2-dimethoxyethane, diethylzinc, diiodomethane, 16, CH2Cl2, -15 °C to rt, 16 h; f) PivCl, DMAP, pyridine, CH2Cl2, 0 °C to rt, 16 h; g) TBAF, THF,
0 °C to rt, 16 h ( 97% over three steps); h) CBr4, PPh3, CH2Cl2, 0 °C to rt, 3 h (90%).[22] DIBAL = DiisoButylALuminium hydride, THF = TetraHydroFurane, TBSOTf
= Tert-Butyldimethylsilyl trifluoromethanesulfonate, 9-BBN = 9-borabicyclo(3.3.1)nonane, DMP = Dess-Martin Periodinane, TBDPSCl = Tert-ButylDiphenylSilyl
Chloride, PivCl = Pivaloyl Chloride, TBAF = TetraButylAmmonium Fluoride.
Endgame of the synthesis
point we suffered from irreproducible yields, even when we
applied starting materials that had provided satisfactory yields
earlier. We could circumvent this problem by using an excess of
lithiated 29, which was nearly quantitatively recovered. With the
complete backbone in place, we subjected 30 to a diimide
reduction[30] of the olefin, followed by TBS deprotection with
pyridineHF in excellent yield. The use of n-BuOH as cosolvent
proved again to be essential for acceptable conversion rates. As
we were reluctant to store large quantities of the mycolic acids
because of the potentially sensitive hydroxy acid moiety, the final
step was carried out portion-wise at sub-gram scale. Removal of
the chiral auxiliary with LiOH, LiOH/H2O2 or hydrogenolysis were
unsuccessful, but hydrolysis with 2 equiv. of tetrabutylammonium
hydroxide (n-Bu4NOH) in THF at rt resulted in complete and clean
conversion, and yielded the desired mycolic acids in sufficient
With all three fragments in hand, we initiated the endgame
by yet another Suzuki-Fu cross-coupling to connect the fragments
B and C (Scheme 3b). Reductive removal of the pivaloate, and a
subsequent Mitsunobu reaction with 1-phenyl-1H-tetrazole-5-thiol
afforded the thio-ether in good yields over three steps.
Unfortunately, ammonium heptamolybdate/H2O2 oxidation to
sulfone 29 proved to be very sluggish due to limited solubility of
the starting material in EtOH/THF, whereas oxidation with m-
CPBA resulted in a complex mixture of unidentifiable products.
Although oxidation with RuO4 was possible,[29] concomitant
oxidation of the methoxy function to the ketone was hard to
suppress. Fortunately, acceptable rates in the ammonium
heptamolybdate/H2O2 oxidation were achieved by addition of n-
BuOH as co-solvent, resulting in nearly quantitative yields of the
sulfone. Subsequently, we applied a Julia–Kocienski olefination
for the final coupling of 29 with fragment A. Although initially
excellent yields (85-90%) were achieved in this reaction, at some
purity and quantitative yields after
a
straightforward
pentane/acetonitrile extraction. Overall, the total synthesis of
mycolic acid 1a was achieved in a longest linear sequence of just
17 steps, in 15% yield. The corresponding glucose esters (Figure
3
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RESEARCH ARTICLE
2, 32a-d) were obtained in two steps using a method developed
by Prandi[31] (see SI for experimental details and yields).
Scheme 3a. The synthesis of fragment C. a) MeLi·LiBr, Et2O, -78 °C, 4 h (92%); b) LiAlH4, THF, 0 °C, 1 h (quant.); c) CBr4, PPh3, CH2Cl2, 0 °C to rt, 4 h (84%); d)
9-BBN, 1-hexadecene, K3PO4, cat. Pd(PCy3)2, THF, 16 h (84%); e) 2 M HClaq, THF, 95 °C, 16 h; f) NaH, tosylimidazole, THF, rt, 16 h (90% over two steps); g) 26,
CuCl (5 mol%), THF, -10 °C, 2 h (85%); h) MeI, NaH, THF, rt, 16 h (95%).[22] Scheme 3b. The endgame synthesis. a) 9-BBN, 1-hexadecene, fragment B, K3PO4,
Pd(PCy3)2 (7 mol%), THF, 16 h; b) LiAlH4, THF, 0 °C, 15 min (78% over two steps); c) phenyl-1H-tetrazole-5-thiol, PPh3, DIAD, THF, 0 °C to rt, 1 h (96%); d) H2O2
(30%), (NH4)6Mo7O28·4H2O, THF, EtOH, n-BuOH, rt, 5 d (99%); e) LiHMDS, fragment A, -35 °C, 1 h (90%); f) flavine cat.[30], hydrazine hydrate, THF, EtOH, n-BuOH,
rt, 5-7 d (98%); g) HF·pyridine, CHCl3, 0 °C, 2 h (99%); h) n-Bu4NOH, (1.5 M in water), THF, rt, 16 h (95%).[22] Tosylimidazole = p-Toluenesulfonylimidazole, DIAD
= Diisopropyl AzoDicarboxylate, LiHMDS = Lithium HexaMethylDiSilazane.
The stereochemistry of natural methoxy mycolic acid
respectively, and comparable with the sum of the values of the
[Φ]D of the α-methyl methoxy and the β-hydroxy acid segments
prepared earlier by Asselineau (+90°). Therefore, it is reasonable
to conclude that the remotely located stereocenters in methoxy
mycolic acid do not influence each other’s optical rotation. The
principle of optical superposition is therefore applicable here and
the assignment of the absolute stereochemistry by Asselineau is
correct, although the stereochemistry in the cyclopropyl ring
remains to be determined (vide infra).
With all four diastereomers in hand, it was now possible to
prove or disprove the ascription of the absolute stereochemistry
(S,S) of the syn-methoxy methyl moiety by Asselineau et al.[7] An
aliquot of the carboxylic acids was treated with
(trimethylsilyl)diazomethane in toluene/methanol to provide the
methyl esters (see SI). The [α]D of both methyl esters with S,S
configuration in the α-methyl methoxy segment (1a and 1d) was
0°, which corresponds with the specific rotation of the natural
sample reported before.[7] Consequently, also the specific molar
rotations [Φ]D were 0°. This supports the previous assignment of
the stereochemistry by Asselineau, and Baird (for 1d). The [α]D
for the diastereomers with R,R configuration in the α-methyl
methoxy segment was +7.8° for 1b and +7.9° for 1c and similar
to those reported by Baird and co-workers for (synthetic)
diastereomer 1c.[11] The [Φ]D of 1b and 1c was +99° and +100°,
4
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10.1002/anie.202000523
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RESEARCH ARTICLE
Figure 3. Biological validation of synthetic glucose methoxy mycolates (a, c)
and methoxy mycolic acids (b, d). Methods are provided in the Supplemental
Info.
As an alternative bioassay, mycolic acid and glucose
monomycolate can be loaded into fluorescently labelled CD1b
tetramers in vitro. Tetramerized, fluorescently labelled CD1b-lipid
complexes (“tetramers”) can be used to study glucose mycolate[32]
and mycolic acid[6] specific T cells at single-cell level, in
combination with fluorescently labelled antibodies, in flow
cytometry. The fluorescence level of the cells is an indication of
the strength of the interaction between T cell receptor and
antigenic target, CD1b-mycolate.
All synthetic glucose mycolates, when loaded into CD1b
tetramers, stained LDN5 well (Figure 3c). A minor but noticeable
difference in the Mean Fluorescence Intensity (MFI) is observed
depending on the stereochemistry of the α-methyl methoxy unit.
The MFI with the GMMs with the S,S configuration is twice as high
as in those with the R,R configuration. No noticeable difference is
seen between the tetramers loaded with the glucose mycolates
that differ in the stereochemistry of their cyclopropyl group.
For the mycolic acids, however, more distinct differences are
observed (Figure 3d). Tetramers loaded with mycolic acids with
the S,S-methoxy methyl unit give much higher MFIs than their R,R
counterparts.
Figure 2. Overview of the four diastereomeric mycolic acids (1a-d), glucose
mycolates (32a-d), and methyl esters (33a-d).
Next, we desired to determine whether our synthetic
methoxy mycolic acids are biologically active as antigens for
human T cells. In the presence of CD1b-expressing antigen-
presenting cells, all four synthetic stereoisomers of glucose
monomycolate activated the LDN5 T cell line in a dose-dependent
manner, as determined by release of the cytokine Interleukine-2
(IL-2) (Figure 3a). The level of activation by the four synthetic
diastereomers, the natural long glucose mycolate from
Mycobacterium phlei, and the natural short glucose
monomycolate from Rodococcus equi, were comparable. This
was consistent with previous observations that glucose
monomycolate-specific T cells are insensitive to variations in the
distal parts of mycolic acid, but highly sensitive to changes in or
near the glucose.[17]
As for the T cell activation assays with free mycolic acid, it
is known from studies with natural mycolic acid isolates that a T
cell clone is typically more potently activated by a certain type of
mycolic acid (α-, keto-, or methoxy-) than by another. The pattern
of antigen potency is different for each T cell clone. In the
presence of CD1b-expressing antigen-presenting cells, all four
synthetic mycolic acids stimulated the T cell clone 11 well at the
highest concentration tested, 5 g/mL (Figure 3b). The two
synthetic mycolic acids with the S,S configuration at the α-methyl
methoxy segment, however, were better agonists than those with
R,R configuration, and better than the natural mixture of Mtb-
derived mycolic acids when tested at the suboptimal
concentration of 0.08 g/mL. No significant difference was found
for the stereochemistry at the cyclopropyl group.
In addition to the effects of the configuration of the methoxy
methyl group, mycolic acids with an R,S-cyclopropyl unit have a
moderately increased MFI compared to their S,R counterparts
(MFI compound 1d>1a and compound 1c>1b in Figure 3d). If we
assume that the naturally occurring configuration of the
cyclopropyl provides the best CD1b loading or the highest affinity
for the T cell receptor, this observation suggests the R,S-
stereochemistry of the cyclopropyl group in natural mycolic acids.
Conclusion
In conclusion, the synthetic route presented here provides
access to gram amounts of methoxy mycolic acid. The successful
repetitive application of the Suzuki-Fu cross-coupling and the
anti-selective Abiko–Masamune asymmetric aldol reaction in this
natural product synthesis are instrumental to this success. The
availability of all four diastereomers allowed to confirm the S,S
stereochemistry of the methoxy methyl function in natural
methoxy mycolic acid, as inferred by Asselineau and Baird. The
T cell receptor is sensitive to variations in the stereochemistry of
the side chain of free mycolic acid, and not in that of glucose
monomycolate. Although the absolute stereochemistry of the cis-
cyclopropyl function could not be established, the CD1b-free
mycolic acid tetramer staining experiments favour R,S
stereochemistry. The availability of large amounts of fully
synthetic methoxy mycolic acid and glucose mycolate (GMM)
allows further study into their application in vaccine development
and diagnostics.
Acknowledgements
We thank P. van der Meulen, Dr. J. Kemmink, J. L. Sneep and
T. D. Tiemersma-Wegman (University of Groningen) for
assistance on analyses. The Dutch Science Foundation NWO
5
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10.1002/anie.202000523
Angewandte Chemie International Edition
RESEARCH ARTICLE
and the Bill & Melinda Gates Foundation are acknowledged for
funding. Unloaded CD1b monomers were obtained through the
NIH Tetramer Core Facility.
Chem. Soc. 1989, 111, 5335–5340.
[25]
[26]
[27]
C. A. Brown, V. K. Ahuja, J. Org. Chem. 1979, 38, 2226–2230.
A. B. Charette, H. Juteau, J. Am. Chem. Soc. 1994, 116, 2651–2652.
J. Leonard, S. Mohialdin, D. Reed, G. Ryan, P. Swain, Tetrahedron
1995, 51, 12843–12858.
Keywords: Tuberculosis • Mycolic Acid • Total Synthesis • CD1b
• sp3-sp3 cross coupling
[28]
M. Alam, C. Wise, C. A. Baxter, E. Cleator, A. Walkinshaw, Org.
Process Res. Dev. 2012, 16, 435–441.
[1]
[2]
[3]
World Health Organization, Global Tuberculosis Report, 2019.
H. Marrakchi, M.-A. Lanéelle, M. Daffé, Chem. Biol. 2014, 21, 67–85.
C. E. Barry III, R. E. Lee, K. Mdluli, A. E. Sampson, B. G. Schroeder,
R. A. Slayden, Y. Yuan, Prog. Lipid Res. 1998, 37, 143–179.
M. Beukes, Y. Lemmer, M. Deysel, J. R. Al Dulayymi, M. S. Baird, G.
Koza, M. M. Iglesias, R. R. Rowles, C. Theunissen, J. Grooten, G.
Toschi, V. V. Roberts, L. Pilcher, S. Van Wyngaardt, N. Mathebula,
M. Balogun, A. C. Stoltz, J. A. Verschoor, Chem. Phys. Lipids 2010,
163, 800–808.
[29]
[30]
W. Su, Tet. Lett. 1994, 35, 4955–4958.
C. Smit, M. W. Fraaije, A. J. Minnaard, J. Org. Chem. 2008, 73, 9482–
9485.
[31]
[32]
J. Prandi, Carbohydr. Res. 2012, 347, 151–154.
A. G. Kasmar, I. Van Rhijn, K. G. Magalhaes, D. C. Young, T.-Y.
Cheng, M. T. Turner, A. Schiefner, R. C. Kalathur, I. A. Wilson, M.
Bhati, S. Gras, R. W. Birkinshaw, L. L. Tan, J. Rossjohn, J. Shires, S.
Jakobsen, J. D. Altman, D. B. Moody, J. Immunol. 2013, 191, 4499–
4503.
[4]
[5]
E. P. Grant, E. M. Beckman, S. M. Behar, M. Degano, D. Frederique,
G. S. Besra, I. A. Wilson, S. A. Porcelli, S. T. Furlong, M. B. Brenner,
J. Immunol. 2002, 168, 3933–3940.
[6]
[7]
I. Van Rhijn, S. K. Iwany, P. Fodran, T.-Y. Cheng, L. Gapin, A. J.
Minnaard, D. B. Moody, Eur. J. Immunol. 2017, 47, 1525–1534.
C. Asselineau, G. Tocanne, J.-F. Tocanne, Bull. Soc. Chim. Fr. 1970,
1455–1459.
[8]
D. E. Minnikin, N. Polgar, Tetrahedron Lett. 1966, 2643–2647.
D. E. Minnikin, N. Polgar, Chem. Commun. 1967, 1172–1174.
J. R. Al Dulayymi, M. S. Baird, E. Roberts, Chem. Commun. 2003,
228–229.
[9]
[10]
[11]
[12]
[13]
[14]
J. R. Al Dulayymi, M. S. Baird, E. Roberts, M. Deysel, J. Verschoor,
Tetrahedron 2007, 63, 2571–2592.
J. R. Al Dulayymi, M. S. Baird, E. Roberts, Tetrahedron 2005, 61,
11939–11951.
G. Koza, M. Muzael, R. R. Schubert-Rowles, C. Theunissen, J. R. Al
Dulayymi, M. S. Baird, Tetrahedron 2013, 69, 6285–6296.
D. B. Moody, M. R. Guy, E. Grant, T.-Y. Cheng, M. B. Brenner, G. S.
Besra, S. A. Porcelli, J. Exp. Med. 2000, 192, 965–976.
J. H. van ’t Hoff, Bull. Soc. Chim. [2] 1875, 23, 298.
E. M. Beckman, S. A. Porcelli, C. T. Morita, S. M. Behar, S. T. Furlong,
M. B. Brenner, Nature 1994, 372, 691–694.
[15]
[16]
[17]
[18]
[19]
D. B. Moody, B. B. Reinhold, M. R. Guy, E. M. Beckman, D. E.
Frederique, S. T. Furlong, S. Ye, V. N. Reinhold, P. A. Sieling, R. L.
Modlin, G. S. Besra, S. A. Porcelli, Science 1997, 278, 283–286.
S. Gras, I. Van Rhijn, A. Shahine, T.-Y. Cheng, M. Bhati, L. L. Tan,
H. Halim, K. D. Tuttle, L. Gapin, J. Le Nours, D. B. Moody, J.
Rossjohn, Nat. Commun. 2016, 7, 13257.
M. R. Netherton, C. Dai, K. Neuschuetz, G.C. Fu, J. Am. Chem. Soc.
2001, 123, 10099-10100. S. Lou, G. C. Fu, Org. Synth. 2010, 87,
299–309. For a Highlight on sp3-sp3 coupling reactions in the
synthesis of natural products and biologically active molecules, see:
E. Geist, A. Kirschning, T. Schmidt, Nat. Prod. Rep., 2014, 31, 441-
448.
[20]
[21]
[22]
A. Abiko, J. F. Liu, S. Masamune, J. Am. Chem. Soc. 1997, 119,
2586–2587.
G. E. Keck, E. P. Boden, S. A. Mabury, J. Org. Chem. 1985, 50, 709–
710.
See SI for more detailed information regarding the other enantiomers
or diastereomers.
[23]
[24]
H. Huang, C. J. Forsyth, J. Org. Chem. 1997, 62, 8595–8599.
K. C. Nicolaou, C. V. C. Prasad, P. K. Somers, C. Hwang, J. Am.
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10.1002/anie.202000523
Angewandte Chemie International Edition
RESEARCH ARTICLE
Entry for the Table of Contents
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Herein we describe the (gram scale) total synthesis of four methoxy mycolic acid diastereomers. Key synthetic approaches involve
iterative application of the Suzuki-Fu cross-coupling, the anti-selective Abiko-Masamune asymmetric aldol reaction, and an
enantioselective Charette cyclopropanation. The CD1b-free mycolic acid tetramer staining experiments clearly favoured one
diastereomer over the other three.
Institute and/or researcher Twitter usernames: @StratinghInst
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This article is protected by copyright. All rights reserved.