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

Article abstract of DOI:10.1002/chem.201605287

Mycobacterium tuberculosis produces dideoxymycobactin-838 (DDM-838), a lipopeptide that potently activates T cells upon binding to the MHC-like antigen-presenting molecule CD1a. M. tuberculosis produces DDM-838 in only trace amounts and a previous solid-phase synthesis provided sub-milligram quantities. We describe a high-yielding solution-phase synthesis of DDM-838 that features a Mitsunobu substitution that avoids yield-limiting epimerization at lysine during esterification, and amidation conditions that prevent double-bond isomerization of the Z-C20:1 acyl chain, and provides material with equivalent antigenicity to natural DDM-838. Isomers of DDM-838 that varied in stereochemistry at the central lysine and the C20:1 acyl chain were compared for their ability to be recognised by CD1a-restricted T cell receptors (TCRs). These TCRs, derived from unrelated human donors, exhibited a similar spectrum of reactivity towards the panel of DDM-838 isomers, highlighting the exquisite sensitivity of lipopeptide-reactive T cells for the natural DDM stereochemistry.

Full text of DOI:10.1002/chem.201605287

A Journal of
Accepted Article
Title: Total synthesis of the Mycobacterium tuberculosis
dideoxymycobactin-838 and stereoisomers: Diverse CD1a-
restricted T cells display a common hierarchy of lipopeptide
recognition
Authors: Janice Cheng, Ligong Liu, Daniel Pellicci, Scott JJ Reddiex,
Rachel Cotton, Tan-Yun Cheng, David Young, Ildiko Van
Rhijn, Branch Moody, Jamie Rossjohn, David Fairlie, Dale
Godfrey, and Spencer John Williams
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201605287
Link to VoR: http://dx.doi.org/10.1002/chem.201605287
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10.1002/chem.201605287
Chemistry - A European Journal
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Total synthesis of the Mycobacterium tuberculosis
dideoxymycobactin-838 and stereoisomers: Diverse CD1a-
restricted T cells display a common hierarchy of lipopeptide
recognition
Janice M. H. Cheng,†[a,c,f] Ligong Liu,†[b,g] Daniel G. Pellicci,[c,f] Scott J. J. Reddiex,[c,f] Rachel N.
Cotton,[d] Tan-Yun Cheng,[d] David C. Young,[d] Ildiko Van Rhijn,[d,e] D. Branch Moody,[d] Jamie
Rossjohn,*[f,g,h] David P. Fairlie,*[b,h] Dale I. Godfrey,*[c,g] Spencer J. Williams*[a,g]
Abstract:
Mycobacterium
tuberculosis
produces
a cytoplasmic tail that lacks lysosomal targeting motifs. Thus,
CD1a localizes predominantly at the cell surface where it can
promiscuously capture foreign or exogenous lipids. The only
extensively-characterized type of foreign lipidic molecule that
activates CD1a-restricted T cells is a family of lipopeptides
dideoxymycobactin-838 (DDM-838),
a
lipopeptide that potently
activates T cells upon binding to the MHC-like antigen-presenting
molecule CD1a. M. tuberculosis produces DDM-838 in only trace
amounts and
a previous solid-phase synthesis provided sub-
milligram quantities. We describe a high yielding solution-phase
synthesis of DDM-838 that features a Mitsunobu substitution that
avoids yield-limiting epimerization at lysine during esterification, and
amidation conditions that prevent double-bond isomerization of the
Z-C20:1 acyl chain, and provides material with equivalent
antigenicity to natural DDM-838. Isomers of DDM-838 that varied in
stereochemistry at the central lysine and the C20:1 acyl chain were
compared for their ability to be recognised by CD1a-restricted T cell
receptors (TCRs). These TCRs, derived from unrelated human
donors, exhibited a similar spectrum of reactivity towards the panel
of DDM-838 isomers, highlighting the exquisite sensitivity of
lipopeptide-reactive T cells for the natural DDM stereochemistry.
isolated
from
Mycobacterium
tuberculosis,
termed
dideoxymycobactins (DDMs).[3]
The initial discovery of the DDMs was achieved through
bioassay-guided fractionation using a single T cell clone (CD8-2)
obtained through stimulation and expansion with
a
chloroform/methanol M. tuberculosis extract.[4]
A
three-
dimensional X-ray structure of CD1a with a silylated, DDM-like
lipopeptide showed the fatty acyl chain buried in the A' pocket of
CD1a with the peptide protruding to the surface of CD1a, where
it presumably contacts the TCR.[5] The recent development of
CD1a tetramers and dextramers that bind directly to polyclonal T
cells in the ex vivo setting has allowed the detection and
isolation of DDM-reactive T cell clones from small cohorts of
tuberculosis patients.[6] This suggests that CD1a–DDM reactive
T cells occur in unrelated individuals and that CD1a–DDM
tetramers may be useful reagents for the ex vivo diagnosis of M.
tuberculosis infection. Further, the existence of polyclonal T cells
in small human cohorts raises the possibility that DDMs could be
used to immunize or stimulate T cell responses in tuberculosis
Introduction
Humans express four antigen-presenting proteins (CD1a, CD1b,
CD1c and CD1d) belonging to the CD1 family, which present
[f]
Prof. J. Rossjohn
Infection and Immunity Program, Department of Biochemistry
[a]
Dr. J. M. H. Cheng, Prof. S. J. Williams
patients.[6] However, the key obstacle to these lines of new
lipid-based molecules to T cells via their T cell receptors
School of Chemistry and Bio21 Molecular Science and
(TCRs).[1] CD1a differs from the other CD1 molecules based on
and Molecular Biology, Biomedicine Discovery Institute, Monash
Biotechnology Institute, University of Melbourne, Parkville, Victoria
medical technology development is the absence of an abundant
University, Clayton, VIC 3800 (Australia)
its con3s0ti1tu0t(iAveus,trahliaig)h-density expression on dendritic cells,
supply of DDM.
Email: jamie.rossjohn@monash.edu
[2]
E-mail: sjwill@unimelb.edu.au
termed Langerhans cells, in the skin and mucosa. Moreover,
DDMs are extremely potent, but comprise less than 10
[g]
Dr. J. M. H. Cheng, Dr. D. G. Pellicci, S. Reddiex, Prof. D. I.
[b]
Dr. L. Liu, Prof. David P. Fairlie
Division of Chemistry and Structural Biology, Institute for Molecular
distinct from the other human CD1 molecules, CD1a possesses
parts per million of the cell wall mass of M. tuberculosis, a
Godfrey, Prof. S. J. Williams
Australian Research Council Centre of Excellence for Advanced
Molecular Imaging, University of Melbourne,
Parkville, Victoria 3010 (Australia)
Bioscience, The University of Queensland, Brisbane, Queensland
4072 (Australia)
Email: d.fairlie@imb.uq.edu.au
[h]
[i]
Dr. L. Liu, Prof. David P. Fairlie
[c]
Dr. J. M. H. Cheng, Dr. D. G. Pellicci, S. Reddiex, Prof. D. I.
Godfrey
Department of Microbiology and Immunology, Peter Doherty
Institute for Infection and Immunity, University of Melbourne,
Parkville, Victoria 3010 (Australia)
Australian Research Council Centre of Excellence for Advanced
Molecular Imaging, The University of Queensland,
Brisbane, Queensland 4072 (Australia)
Prof. J. Rossjohn
Email: godfrey@unimelb.edu.au
Australian Research Council Centre of Excellence for Advanced
Molecular Imaging, Monash University, Clayton, Victoria 3800
(Australia)
[d]
[e]
R. N. Cotton, Dr. T.-Y. Cheng, Dr. D. C. Young, Dr. I. Van Rhijn,
Prof. D. B. Moody
Division of Rheumatology, Immunology and Allergy,
Brigham and Women’s Hospital, Harvard Medical School,
Boston, MA 02115 (USA)
[j]
Prof. J. Rossjohn
Institute of Infection and Immunity, Cardiff University School of
Medicine, Cardiff CF14 4XN (UK)
Dr. I. Van Rhijn
[†]
These authors contributed equally.
Department of Infectious Diseases and Immunology,
School of Veterinary Medicine, University of Utrecht, The
Netherlands
Supporting information for this article is given via a link at the end of the
document.
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10.1002/chem.201605287
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FULL PAPER
pathogen, whose laboratory cultivation is slow and cumbersome
because it must be grown in biosafety level 3 conditions. 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 e-amino group of a lysine residue,
and an unusual a-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.
peptide and lipid stereochemistry for T cell receptor recognition
and T cell activation.
Results and Discussion
Total synthesis of DDM-838
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.
After extensive HPLC purification, this approach yielded only
trace quantities of DDM-838 (~95 µg or 2% overall yield), which
we verified independently. As this synthesis was conducted on
solid phase, it is difficult to identify the yield-limiting steps. In a
solution-phase approach, used for the synthesis of analogues
that lacked the distinguishing a-methyl group, an e-amino-
protected deoxymycobactic acid was coupled to deoxycobactin,
and the depsipeptide was deprotected prior to attaching the fatty
acyl chain. This resulted in low yields for both attachment of the
fatty acyl chain and for formation of the ester linkage. Moreover,
unexpected doubling of signals in NMR spectra of advanced
intermediates was attributed to the presence of rotational
isomers, but could alternatively reflect the formation of undefined
stereoisomers. With the above issues in mind, we initially sought
to develop a solution-based synthesis of DDM-838 broadly
inspired by this earlier study,[3b] but with the intention to fully
characterize all intermediates and identify yield-limiting steps in
order to identify an improved synthetic approach.
The required building block fragments deoxymycobactic
acid 13, deoxycobactin 16, and Z-C20:1 fatty acid 20 were
prepared by optimization of the previously described literature
approaches.[3b] Oxazoline acid 11 was prepared from a-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 CH2Cl2.[8] Saponification
of the ester of 10 with LiOH afforded the O-benzyl-protected acid
11, which was coupled with NH2-L-Lys(Boc)OMe using
Figure 1. DDM-838 (1) and three stereoisomers.
A previous synthetic approach[3b] to DDM-838 used solid-
phase peptide chemistry to provide sufficient material to help
clarify the structure of the natural product, yet the poor overall
yield provided only sub-milligram quantities that are insufficient
for in vivo studies. 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). This
and other problems were overcome by the development of a
new route that creates the challenging ester linkage through a
Mitsunobu coupling and allows the preparation of milligram
quantities of DDM-838 (1). Through the isolation of
stereochemical isomers of DDM-838 (2-4) we have explored
structure-activity relationships in antigen recognition by the
prototypical DDM-reactive T cell clone CD8-2, as well as new
DDM-reactive clones, and demonstrate the importance of
EDC.HCl/HOAt/DMAP.
Saponification
of
12
afforded
deoxymycobactic acid 13. Next, deoxycobactin 16 was prepared
by coupling of (R)-3-hydroxybutyric acid 14 and L-α-amino-ε-
caprolactam
hydrochloride
15
in
the
presence
of
EDC.HCl/HOAt/DMAP (Scheme 1b).[3b] Finally, the Z-C20:1 fatty
acid 20 was synthesized using Ando's reagent,[9] which enables
a
highly Z-selective Horner-Emmons-Wadsworth coupling.
Treatment of octadecanol 17 with PCC in CH2Cl2 afforded
stearylaldehyde 18, which condensed with Ando's reagent[9-10] in
the presence of DBU/NaI to afford a 9:1 Z:E ratio of alkenes 19
(use of the Still-Gennari reagent provided the alkenes in a 3:1
Z:E ratio) (Scheme 1c). These isomers could be separated by
careful chromatography, prior to saponification to afford the acid
20.
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Coupling of deoxymycobactic acid 13 with deoxycobactin
16 was explored under a range of esterification conditions using
assorted peptide coupling reagents. In general, the esterification
reactions were slow and low yielding, and occurred with varying
amounts of epimerization at lysine. The best results were
achieved using DIC/HOAt in the presence of 1.5 eq of DMAP,
which afforded a 1:1 ratio of S-21/R-21 (Scheme 2). Importantly,
the O-benzyl protected epimeric intermediates 21 were
separable by preparative t.l.c., but stereochemical assignment
was not possible at this stage. Each isomer was individually
carried through to the lipopeptide by deprotection of benzyl and
Boc groups (to give S- and R-22), then coupled with the Z-C20:1
i
acid 20 using EDC.HCl/HOAt/DMAP in the presence of Pr2NEt.
Each product was formed as a mixture of E and Z isomers,
which were separated by flash chromatography to afford four
pure DDM isomers 1-4, and in some cases small amounts of a
contaminating trifluoroacetamide. The less polar isomer 21
afforded material whose spectral data matched the natural
product DDM-838 (1). At this stage the stereochemistry of the
central lysine was not known, but established later (vide infra).
Scheme 1. Synthesis of deoxymycobactic acid 13, deoxycobactin 16, and Z-
C20:1 fatty acid 20.
Scheme 2. Preparation of DDM-838 (1) and stereoisomers 2-4. Slow = higher polarity product; fast = lower polarity product. a yield over 3 steps from (S)- or (R)-21.
b
analysis of 1H NMR of crude mixture revealed the formation of 1:2:trifluoroacetamide byproduct at a ratio of 6:3:4, presumably derived from residual
trifluoroacetate anion carried over from step iii.
The above work, and an alternative approach utilizing a
different order of assembly that gave improved yields yet also
suffered from similar problems (see SI for details), highlighted
two key problems that likely contributed to the low yield of the
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prior solid- and solution-based syntheses of DDM-838.[3b] The
first is the difficulty of effecting the esterification of 3-
hydroxybutyramide, which occurred at best in only moderate
yield and was complicated by epimerization at the central lysine.
The difficulty of this coupling likely arises from the sterically-
congested nature of the linkage, between an a-branched acid,
and a secondary alcohol. The propensity for epimerization at the
central lysine during esterification of the 3-hydroxybutyramide
group means that the stereochemistry at this position had not
been unambiguously secured, although the biosynthetic
relationship of DDM-838 to the mycobactins allows assumption
of the L-configuration.[11] The second problem was the
unexpected isomerization of the unsaturated fatty acyl chain,
over Pd/C gave enantiomerically-pure amine S-22, which was
isolated as a free base and then coupled with the Z-C20:1 acid
20, this time using only DIC, and afforded DDM-838 1 as a pure
Z isomer in 49% yield over two steps. The spectral data were in
agreement with the natural product. By this approach we
unambiguously established the stereochemistry of DDM-838 (1)
as SSRS, as well as the configuration of the three new
stereoisomers (2–4) (Schemes 2 and SI).
and sometimes contamination with
byproduct.
a
trifluoroacetamide
We initially focussed on solving the problem experienced
with the unexpected formation of E and Z isomers upon
acylation of S- and R-22 with Z-C20:1 acid 20, and the formation
of the trifluoroacetamide by-product. By conversion of the
trifluoroacetate salt of compound 22 to the free base, prior to
acylation, we were able to avoid trifluoroacetamide formation.
i
Treatment of a pure Z-C20:1-Lys model with Pr2NEt did not
result in double bond isomerization, suggesting that
isomerization was occurring to the activated acyl-intermediate.
Attempts to acylate the model amine (Bz-L-Lys-OMe) using
DIC/HOAt/DMAP, without iPr2NEt, or with pyridine, still led to
double bond isomerization, suggesting that base-catalyzed
isomerization is faster than acylation of lysine. Hocking has
shown that double bond isomerization of cis-crotonoyl chloride
during reaction with ammonia does not occur when ammonia is
passed over the surface of an ethereal solution of the acid
chloride.[12] Inspired by this precedent, we examined pre-forming
the activated ester by combining DIC and Z-C20:1 20 in CH2Cl2,
followed by addition of a CH2Cl2 solution of Bz-L-Lys-OMe,
which afforded the cis-amide as the sole product.
To overcome the problem with epimerization at the central
lysine, a Mitsunobu reaction was explored to form an ester bond
and interchange the roles of the acid and alcohol to nucleophile
and electrophile, respectively, thereby potentially alleviating the
steric demands in forming this linkage. In related work, Miller
and co-workers applied the Mitsunobu reaction in the synthesis
of Mycobactins S and S2 through coupling of cobactin and
mycobactic acid fragments in 49 and 50% yield, respectively.[13]
In a recent article, Tsakos et al. reviewed a large number of
challenging esterifications, and suggested that performing
difficult esterification reactions on smaller fragments can often
avoid yield-limiting side reactions by reducing the number of
potentially interfering functional groups.[14]
Based on these considerations we devised a second linear
approach to DDM-838. Coupling of (S)-3-hydroxybutyric acid
((S)-14) (rather than (R)-14) to L-α-amino-ε-caprolactam 15
using EDC.HCl/HOAt/DMAP and afforded the deoxycobactin 23
(Scheme 3).[3b] Mitsunobu coupling of 23 with Fmoc-L-Lys(Z)-OH
in the presence of Ph3P/DIAD afforded the ester 24 with the
stereochemistry of 3-hydroxybutyrate inverted, and an
elimination by-product 25 inseparable by flash chromatography.
While the yield of this reaction was modest, no epimerization at
lysine was observed. Removal of the Fmoc group with piperidine
allowed separation of the alkene 25 by chromatography and
afforded pure amine 26 in 26% yield over two steps.
Condensation of amine 26 and oxazoline acid 11 afforded the
depsipeptide 27. Global deprotection of depsipeptide 27 with H2
Scheme 3. Mitsunobu route to DDM-838 (1).
Immunological investigation of CD1a-restricted
clones
T cell
We explored the ability of our synthetic compounds to
mediate binding interactions between CD1a and TCRs using a
series of immunological assays that measure either the
biological potency of compounds in cellular assays (through the
expression of activation marker CD69) or the mechanistic basis
of TCR binding to CD1a-lipopeptide complexes (through
tetramer staining and flow cytometry). We studied several CD1a-
restricted TCR+ clones including autoreactive clones that
recognize CD1a alone, and those recognizing CD1a presenting
foreign DDM and related antigens. Initially, we compared the
biological activity of synthetic DDM-838 (1) prepared here, with
natural DDM-838 isolated from M. tuberculosis, and material
synthesized through the solid phase route of Young and
coworkers.[3b] Titration of lipids into cultures of the native CD8-2
T cell clone with all three materials and quantification of IFNg
release by ELISA provided dose-response curves for the two
synthetic compounds that were essentially identical (Figure 2).
The slightly lower potency of natural DDM can be explained by
the presence of small amounts of naturally occurring variants in
acyl chain length and saturation, which are known to be less
potent than DDM-838.[3] This result established the chemical
identity of the synthetic compounds, and concordant activity with
natural DDM-838.
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GnT1– cells used for expression of CD1a monomers). By
contrast, staining of Jurkat.BK6 cells was partially or completely
inhibited by all four DDM analogs, suggesting that DDM lipids
were loaded into CD1a and affected the TCR binding site. In
contrast, Jurkat cells transfected with any of three TCRs derived
from DDM-reactive T cell clones (CD8-2;[4] and Clone 8 and
Clone 15, isolated from two different subjects with latent
tuberculosis infection) stained more brightly with CD1a tetramers
loaded with synthetic DDM-838, compared to CD1a-endo
tetramers, indicating that synthetic lipopeptide enhances the
interaction between CD1a and these TCRs. In comparing the
heirarchy of CD8-2 staining, CD1a tetramers loaded with
compound 2 (containing the E-C20:1 acyl lipid) stained less
intensely than compound 1. Compound 3 (bearing the opposite
stereochemistry at the central lysine residue compared to DDM-
838), and compound 4 (with unnatural stereochemistry at lysine
and the acyl lipid) showed minimal, if any staining of the DDM-
838-reactive T cell clones. A negative control cell line expressing
the MR1-restricted TCR, Jurkat.M33,[18] provided only
background staining levels with all of the CD1a tetramers,
indicating that the interactions observed with CD1a-DDM
tetramers were due to specific TCR interactions. These data
indicate a role of peptide and stereochemistry in T cell response,
and the highly similar patterns seen among 3 distinct TCRs
Figure 2. Synthetic and natural DDM-838 possess equivalent ability to
activate the native CD8-2 T cell clone. Compound 1 prepared herein (solution
phase); according to Young and co-workers (solid phase);[3b] and isolated from
M. tuberculosis H37Ra, were incubated with the human T cell clone CD8-2
and CD1a-expressing K562 myeloid cells[15] over 24 h and IFNg release
measured by ELISA. DDM-838 isolated from M. tuberculosis contained 25% of
the saturated analogue DDM-840,[3a] which is less stimulatory than DDM-838.
Error bars indicate standard deviation among triplicates. Data representative
of three independent experiments. Controls at
0 nM DDM-838 are for
experiments including T cells alone, K562 cells alone, and both cell lines.
The BK6 clone[16] recognizes cellular CD1a when loaded
with endogenous lipids and does not require DDM.[17] Jurkat
cells expressing the BK6 TCR were stained brightly by CD1a
loaded with endogenous lipids (endo, Figure 3) (these
endogenous lipids are derived from the mammalian HEK293
suggests
a
conserved mechanism of DDM recognition.
Figure 3. Reactivity of CD1a-restricted TCR+ Jurkat cell clones to DDM isomers. (a) Flow cytometry (left) of BK6, CD8-2, Clone-8, Clone-15 and M33 TCR
expressing cells labeled with CD1a-endo tetramers or CD1a tetramers loaded with DDM-838 (cis-SSRS) 1, DDM trans-SSRS 2, DDM (cis-SRRS) 3 and DDM
(trans-SRRS) 4, or endogenous lipid antigen (Endo). Numbers in the bottom right corner indicate geometric mean fluorescence intensity (gMFI) of staining with
CD1a-DDM or CD1a-endo. (b) gMFI of each T cell line against endo and DDM isomers 1-4. Data are representative of three independent experiments.
To determine if synthetic DDMs could also activate cells,
we next investigated if compounds 1–4 to could cause TCR-
transduced Jurkat cells to express an activation marker, CD69
(Figure 4). Titration of the DDM-reactive TCR+ CD8-2 cell line
with compounds 1-4 resulted in a concentration-dependent
increase in activation with the same pattern of potency as was
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10.1002/chem.201605287
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observed in tetramer-based binding assays. CD1a-autoreactive
complexes,[3] predict that the peptide moiety might block
BK6 cells showed no sign of activation by any of the compounds, approach of the BK6 TCR to CD1a, based on the known docking
and even showed
a
concentration-dependent reduction in
mode of BK6 with CD1a.[17] This prediction is supported by our
results, which also provides evidence that all four DDM analogs
bind to CD1a. Furthermore, Jurkat cells expressing the CD8-2,
Clone 8 and Clone 15 TCRs fail to bind to CD1a tetramers
loaded with self lipids that are permissive to CD1a–BK6 TCR
binding. This suggests an alternative TCR recognition mode
compared to BK6 in which the peptide moiety likely protrudes
from the groove to facilitate rather than block TCR binding.
activation. These data are consistent with lipopeptide loading
into CD1a but reduced binding of CD1a-lipopeptide to the CD8-2
TCR for compounds 2-4. Titration of the DDM-reactive TCR+
clone 8 and clone 15 cell lines with 1-4 showed limited activation
(data not shown).
Conclusions
Our data reveal a conserved spectrum of T cell recognition
for the panel of DDM isomers for a set of three unrelated CD1a-
DDM-reactive TCRs. The natural form, DDM-838 1 is the most
potent, while compound 2, which varies in the lipid
stereochemistry, maintained significant, albeit reduced, activity
relative to 1. This result is reminiscent of the effect seen upon
saturation of the acyl chain of DDM-838 to generate DDM-
840,[3a] demonstrating the importance of the cis-D2-unsaturation
of DDM-838 for its potent antigenicity. The stereochemistry and
location of this unsaturation (at C2) is unusual and is generated
by mycobactin synthase N (MbtN).[19] While the biochemical
function of the unsaturation is unknown, this work confirms its
importance for antigen recognition.
Figure 4. Activation of Jurkat cells expressing BK6 and CD8-2 TCRs by C1R
cells transduced to express CD1a, measured by expression of the activation
marker CD69. BK6 and CD8-2 expressing cells were co-cultured overnight
with CD1a-expressing C1R cells in the presence of DDM isomers 1-4 at
varying concentrations (0.036, 0.12, 0.36, 1.2, 3.6 nM). For BK6 and CD8-2
expressing cells with a high TCR to GFP ratio, the level of CD69 expression
was measured as mean fluorescence intensity (MFI). Data is representative of
three independent experiments.
Discussion
The inversion of stereochemistry at the lysine of the
depsipeptide backbone compromises the activity of lipopeptide
and this was reflected in the low reactivity of the DDM-reactive
TCRs to CD1a-compound 3 tetramers, similar to prior studies of
DDM-838 varying at either the oxazoline or butyrate moieties
(positions a and c, Figure 1).[3b] The strong attenuation of TCR
binding and T cell activation by compound 4, which is both
epimerized at the central lysine and contains the trans acyl chain,
reinforces these structure-activity relationships. Collectively,
these results reveal that the stereochemistry of the depsipeptide
backbone is important for DDM-recognition by CD1a-restricted
TCRs, and that variation in stereochemistry or saturation in the
lipid chain leads to partial attenuation of reactivity. The
recognition of DDM-838 1 by three distinct T cell clones supports
the biomedical development of these compounds to target the
naturally occurring polyclonal T cells present in latent TB
patients.[6] The ability to synthesize larger quantities of DDM-838
by the solution-phase scheme described here makes possible
structural and functional studies of TCR recognition of CD1a–
DDM-838 complexes, as well as in vivo studies that use DDM as
a vaccine component or recall antigens for immunodiagnosis of
TB.
Although CD1a is broadly expressed on human
Langerhans cells and myeloid dendritic cells, efforts to study
foreign lipid-reactive CD1a-restricted T cells have been limited
by the availability of the few known bacterial ligands. Although
DDM-838 has been bound to CD1a and used in small-scale
studies of tuberculosis patients,[6] this depsipeptidolipid has only
been available in trace quantities from either natural sources or
the reported synthetic route. The present work has investigated
several synthetic approaches to DDM-838; demonstrated a new
approach that markedly improves yield; and provides insight into
a conserved mechanism of DDM-recognition by CD1a-restricted
T cells. The new method utilizes the Mitsunobu reaction to
construct the challenging ester linkage and provides access to
milligram quantities of this important mycobacterial antigen.
Notably, unlike direct esterification methods that caused
epimerization at the central lysine, this approach generates the
ester linkage of the depsipeptide in a substitution reaction
without epimerization permitting an explicit assignment of the L-
lysine stereochemistry of DDM-838.
The preparation of three diastereomers 2-4 of DDM-838 1
permitted investigation of structure-activity relationships for
recognition by DDM-838 reactive CD1a-restricted TCRs, as well
as the CD1a-autoreactive BK6 TCR. The BK6 TCR+ cell line is
brightly stained by CD1a loaded with permissive self lipids,[17]
but less brightly stained with CD1a tetramers treated with
compounds 1-4. Structural studies have revealed that the BK6
TCR binds to the A' roof of CD1a, whereas non-permissive
ligands such as sphingomyelin and sulfatide protrude from the A'
cleft and disrupt the BK6 TCR-CD1a contact zone.[17] An X-ray
structure of a silylated mycobactin analogue revealed the alkyl
chain inserted deep within the A' pocket of the antigen-binding
groove, with the peptide folded over in the F' pocket and with
both the caprolactam and salicyl moieties extending outward to
the predicted plane of the TCR contact.[5] This binary structure,
as well as molecular modelling studies of CD1a–DDM-838
Experimental Section
Expression and purification of CD1a
CD1a was expressed by recombinant methods in
a mammalian
expression system by cloning of constructs encoding CD1a and b2-
microglobulin in a pHLsec vector[20] as carboxyterminal fusions with the
leucine zippers Jun (CD1a) and Fos (b2-microglobulin) and a ‘post-zipper’
BirA-His6 tag for CD1a. Fusion proteins were expressed by
cotransfection of HEK293 GnT1– cells with the plasmids pHLsec-CD1a-
Jun-BirA-His6 and pHLsec-b2m-Fos.[20] Purification of CD1a-b2-
microglobulin heterodimers was achieved by immobilized nickel affinity
followed by size-exclusion chromatography.
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10.1002/chem.201605287
Chemistry - A European Journal
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Solubilization of DDM isomers
human IFN
g
antibody clones 2G2 (Thermo-Fisher), biotin-conjugated
B133.5 (Thermo-Fisher) and streptavidin-horse radish peroxidase (BD
Pharmingen). Absorbance was measured at 405 nm using a VersaMax
plate reader (Molecular Devices) and SoftMaxPro software. IFNg
concentrations were extrapolated from a standard curve in GraphPad
Prism 6.
DDM SSRS Z-C20:1 1, DDM SSRS E-C20:1 2, DDM SRRS Z-C20:1 3
and DDM SRRS E-C20:1 4 were solubilized in a solution of 0.5%
tyloxapol (Sigma) in Tris-buffered saline, pH 8.0 at 1 mg/mL. To ensure
each lipopeptide was completely solubilized, 12 rounds of sonication (1 h,
water bath temperature 25–45°C), vortex (10 sec) and freezing at –20°C
(20 mins) were performed until the mixture was
suspension.
a homogenous
Acknowledgements
Production of CD1a-restricted Jurkat 76 cell lines
This work was supported by the Australian Research Council
(DP130102763, DP160100597, FT130100103, CE140100011
and LE110100106). DGP is supported by an NHMRC ECF
fellowship (1054431); DPF and DIG are supported by NHMRC
Senior Principal Research Fellowships (1027369, 1020770); JR
is supported by an NHMRC Australia Fellowship (AF50); DBM is
supported by the Bill and Melinda Gates Foundation Vaccine
Accelerator Award and NIH (AI R01049313 and U19111224).
Parental Jurkat 76 cells lack endogenous TCR α-chains and β-chains,[22]
and therefore TCR specificity is conferred by the transgene. CD1a-
restricted BK6, CD8-2, Clone 8, Clone 15 and control MR1-restricted
M33.64[23] Jurkat 76 cells were generated as described[24] using retroviral
transduction whereby green fluorescent protein and an antibody to CD3ε
(anti-CD3ε) (UCHT1; BD Biosciences) signified successful transduction.
These have recently been tested and confirmed to be free of
Mycoplasma.
Keywords: immunology • peptidolipid • antigens • natural
Loading of CD1a, production of CD1a tetramers and staining of
Jurkat 76 cells with CD1a tetramers
product • T cell
[1]
[2]
D. I. Godfrey, A. P. Uldrich, J. McCluskey, J. Rossjohn, D.
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Soluble mammalian CD1a samples were enzymatically biotinylated with
BirA biotin ligase as previously described.[21] Biotinylated CD1a was
loaded overnight with each DDM isomer at a molar ratio of 1:6. CD1a
tetramers were prepared by mixture of streptavidin-phycoerythrin (BD
Biosciences) with DDM-loaded biotinylated CD1a at a molar ratio of 1:4.
CD1a-DDM tetramers (35 µL of stock CD1a-DDM tetramers at 2.5
µg/mL) were incubated overnight with 3 x 104 CD1a-restricted BK6, CD8-
2, Clone 8, Clone 15 and control MR1-restricted M33.64 Jurkat 76 cells,
generated as described above. CD1a tetramer–positive cells were
stained with antibody to CD3ε (anti-CD3ε) (UCHT1; BD Biosciences) and
were analyzed with an LSR Fortessa (BD Biosciences). Data were
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[3]
Assay of activation of the human BK6 and CD8-2 Jurkat 76 cell lines
[4]
[5]
CD1a-restricted BK6 and CD8-2 Jurkat 76 cells were generated as
described above. CD1a-expressing C1R cells were generated as
previously described.[17] 3.33 ´ 103 CD1a-expressing C1R cells were co-
cultured overnight with 2 ´ 104 Jurkat cells (BK6 or CD8-2) and DDM
isomers 1-4 at varying concentrations (0.036, 0.12, 0.36, 1.2, 3.6 nM).
The cells were collected by centrifugation, the supernatant was removed
and the cells were then stained with anti-CD3ε (BV421, UCHT1, BD
Biosciences), anti-CD69 (PE, FN50, BD Biosciences), anti-CD19
(APC/Cy7, SJ25C1, BD Biosciences) and 7-AAD (Sigma Aldrich).
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were assessed by upregulation of CD69. Cells were analyzed with an
LSR Fortessa (BD Biosciences) and data were processed with FlowJo
software (TreeStar).
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and are routinely tested by flow cytometry to confirm surface CD1a
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by electrospray ionization mass spectrometry to be a mixture of DDM-
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natural DDM and of solution-phase DDM-838 were determined by UV
spectroscopy absorbance at 306 nm, normalized to absorbance of 80 µM
solid-phase DDM-838. 5 ´ 104 CD8-2 T cells were incubated with 2.5 ´
104 CD1a-expressing K562 cells and serial dilutions of DDM-838 in 150
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10.1002/chem.201605287
Chemistry - A European Journal
FULL PAPER
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Entry for the Table of Contents
FULL PAPER
Dr. Janice M. H. Cheng,† Dr. Ligong Liu,†
Dr. Daniel G. Pellicci, Scott J.J. Reddiex,
Rachel N. Cotton, Dr. Tan-Yun Cheng, Dr.
David C. Young, Dr. Ildiko Van Rhijn, Prof.
D. Branch Moody, Prof. Jamie Rossjohn,*
Prof. David P. Fairlie,* Prof. Dale I.
Godfrey,* Prof. Spencer J. Williams*
Sensing
stereochemistry:
M.
tuberculosis dideoxymycobactin-838
(DDM-838) is an antigen involved in
cell-mediated immunity. DDM-838 and
stereoisomers
epimeric
in
the
depsipeptide backbone and isomeric
in the C20:1 acyl-chain were
synthesized. T cell receptors derived
Page No. – Page No.
from
unrelated
human
donors
Total synthesis of the Mycobacterium
tuberculosis dideoxymycobactin-838
and stereoisomers: Diverse CD1a-
restricted T cells display a common
hierarchy of lipopeptide recognition
preferentially bind natural DDM-838 in
complex with the antigen presenting
molecule CD1a and display similar
patterns of discrimination for DDM-
838 stereoisomers.
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