Development of a benzophenone and alkyne functionalised trehalose probe to study trehalose dimycolate binding proteins

Article abstract of DOI:10.1039/c2ob27257a

Trehalose dimycolates (TDMs) are the most abundant glycolipids found in the cell wall of Mycobacterium tuberculosis (M. tb). TDMs play an important role in the pathogenesis of M. tb yet the only known receptor for TDM is the macrophage inducible C-type lectin (mincle). To understand more about the interaction of TDMs with immune cells, affinity based proteome profiling (AfBPP) can be used to determine receptors that bind TDMs. To this end, we present the synthesis of the first AfBPP-TDM probe and report on its ability to activate macrophages. By doing so, we establish that the AfBPP-TDM probe appears to be a suitable substrate for future proteomic profiling experiments.

Full text of DOI:10.1039/c2ob27257a

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Development of a benzophenone and alkyne
functionalised trehalose probe to study trehalose
Cite this: Org. Biomol. Chem., 2013, 11,
881
dimycolate binding proteins†
Ashna A. Khan,a,b Faustin Kamena,c Mattie S. M. Timmer*a and Bridget L. Stocker*b
Received 20th November 2012,
Accepted 6th December 2012
DOI: 10.1039/c2ob27257a
www.rsc.org/obc
Trehalose dimycolates (TDMs) are the most abundant glycolipids
found in the cell wall of Mycobacterium tuberculosis (M. tb). TDMs
play an important role in the pathogenesis of M. tb yet the only
known receptor for TDM is the macrophage inducible C-type
lectin (mincle). To understand more about the interaction of TDMs
with immune cells, affinity based proteome profiling (AfBPP) can
be used to determine receptors that bind TDMs. To this end, we
present the synthesis of the first AfBPP-TDM probe and report on
its ability to activate macrophages. By doing so, we establish that
the AfBPP-TDM probe appears to be a suitable substrate for future
proteomic profiling experiments.
Trehalose dimycolates (TDMs 1, Fig. 1) are the most abundant
extractable lipids from M. tuberculosis (M. tb) and since their
isolation in the early 1950s,1 they have attracted much atten-
tion due to their involvement in the pathogenesis of M. tb.
TDMs form a thick waxy coating on the outside of the bacteria
and thereby confers protection to the bacteria against harsh
environmental conditions.2–4 TDMs also have important
immunomodulatory functions such as preventing phagosome–
lysosome fusion, which allows the bacteria to survive inside
the host macrophage,5–8 and have been found to play a pivotal
role in granuloma formation.9–14 In 2009, TDMs and the
simpler C22 analogue, trehalose dibehenate (TDB, 2) were
found to activate macrophages via the Macrophage Inducible
C-type Lectin (mincle),15–17 with mincle thought to bind both
the ester and carbohydrate groups in the trehalose deriva-
tives.15,18 Of other linear trehalose diesters, lipid lengths of
greater than 18 carbons were required for macrophage acti-
vation.19 Aside from mincle, however, there are no other
Fig. 1 Trehalose dimycolate (TDM) 1 and trehalose dibehenate (TDB) 2.
known receptors for TDMs. This does not mean that other
TDM-receptors do not exist and indeed the recent conflicting
results in the literature about the role of mincle and TDMs in
the development of tuberculosis in murine models illustrates
the potential presence of multiple TDM receptors.20,21 More
insight into TDM–protein interactions is thus required to
better understand the pathogenicity of M. tb.
To determine which receptors bind TDMs, we envisioned
developing a trehalose-diester probe for affinity based pro-
teome profiling (AfBPP).22 Generally, an AfBPP probe is
equipped with a recognition group which resembles the
natural substrate of the target protein, a reactive group (‘trap’)
that reacts with amino acids in the protein’s active site thus
forming a covalent bond between the probe and protein, and a
‘tag’ which allows for visualisation and/or enrichment and
purification of the trapped protein.23–25 Accordingly, we
designed trehalose probe 3 (Fig. 2) which contains a benzo-
phenone group as a photoreactive trap [for the labeling of proxi-
mal resides in the proteins active site following irradiation
with UV light (365 nm)],26 and an alkyne tag that can be used
as a purification handle following a ‘click’ reaction27 with a
suitably functionalised (e.g. biotinylated) azide.28 Probe 3 is
the first trehalose glycolipid probe developed thus far and has
been designed to leave the reactive carbohydrate portion of the
aSchool of Chemical and Physical Sciences, Victoria University of Wellington,
PO Box 600, Wellington, New Zealand. E-mail: mattie.timmer@vuw.ac.nz,
bstocker@malaghan.org.nz; Fax: +64 4 463 5237
bMalaghan Institute of Medical Research, PO Box 7060, Wellington, New Zealand
cMax-Planck-Institute for Infection Biology, Department of Parasitology, Charitéplatz 1,
10117 Berlin, Germany
†Electronic supplementary information (ESI) available: Experimental pro-
cedures, spectral and analytical data for all synthesised compounds. See DOI:
10.1039/c2ob27257a
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A much improved yield (85%) of benzophenone ester 7,
however, could be obtained by adding AlCl3 (3 equiv.) at the
beginning of the reaction and a further 3 equiv. after
30 minutes. Purification by silica gel flash column chromato-
graphy then allowed for the isolation of 7 as a single isomer
(Rf = 0.24, Pet. ether/EtOAc, 2/1).
Having synthesised the benzophenone trap, we then set out
to prepare the alkyne tag (Scheme 2). To this end, 1,4-butane-
diol (8) was reacted with hydrogen bromide under Dean–Stark
conditions to form the corresponding mono-substituted
bromide (40% yield),34 which was subsequently reacted with
3,4-dihydropyran and pyridinium p-toluenesulfonate (PPTS) to
give THP ether 9 in excellent (97%) yield.35 With THP ether 9
in hand, the next step was to couple this to the appropriate
lipophilic alkyne. Accordingly, bromo-hexadecane (10) was
first reacted with sodium acetylide to give 1-octadecyne (11) in
98% yield. Here, the concentration of the reaction mixture
proved critical, with DMF at 1.1 mL/mmol 36 giving optimal
yields. Alkyne 11 was then treated with BuLi followed by THP
ether 9 to give the lipophilic alkyne 12 in high (83%) yield.
Subsequent removal of the THP protecting group using
p-toluenesulfonic acid (p-TsOH) and isomerisation of the
internal alkyne in product 13 by way of a zipper reaction37
then proceeded smoothly to provide alcohol 14, as evidenced
Fig. 2 Proposed trehalose diester AfBPP probe 3.
TDM unfunctionalised and includes a trap and tag system that
is small and hydrophobic in character and therefore a good
mimic of the original TDM.
To prepare trehalose probe 3, we first set out to synthesise
the benzophenone trap equipped with a carboxylic acid for
coupling to the trehalose core and a hydroxyl to allow for
installation of the lipophilic alkyne tag (Scheme 1). To this
end, terephthalic acid 4 was converted to its dimethyl ester via
reaction with thionyl chloride and MeOH and one ester was
then hydrolysed using potassium hydroxide (1.1 equiv.) to give
monoacid 5 in good (87%) yield over two steps.29 Acid 5 was
then reacted with SOCl2 to form the corresponding acid chlor-
ide, which was immediately used in the subsequent AlCl3-
mediated Friedel–Crafts acylation of anisole. While a literature
procedure reported the addition of AlCl3 (1 equiv.) in one
portion to give benzophenone 6 in 42% yield,30 we noted that
this yield could be significantly improved to 70% via the
portion-wise addition of AlCl3 (2 equiv.) over the course of the
reaction. About 5% of the expected ortho-substituted product
was also formed, which was inseparable from the para-substi-
tuted product at this stage in the synthesis (Rf = 0.76, EtOAc,
for both isomers). Several attempts were then made to
demethylate 6, initially via the use of TMSI (1.1 equiv.)31 or
BCl3 (4 equiv.),32 though in both instances only starting
material was recovered. AlCl3 (4 equiv.) and elevated temp-
eratures were then used33 which resulted in approximately 30%
of the desired product along with unreacted starting material.
4
by the presence of a peak at δ 1.94 ppm (t, J20,22 = 2.6 Hz, 1H,
H-22) in the 1H NMR which corresponds to the terminal
alkyne proton in 14. Finally, transformation of the hydroxyl to
an iodide gave 21-iodo-docosyne (15) ready for coupling to the
benzophenone core.
Several attempts were then made to couple benzophenone
7 and alkyne 15 (Table 1). First, NaH (1.1 equiv.) was used as a
base, though this gave only 10% of the alkylated product 16
and both starting materials were reisolated (entry 1). Increas-
ing the amount of NaH to 2.2 equiv. and warming the reaction
from room temperature to 70 °C did not lead to any significant
Scheme 1 Synthesis of benzophenone 7.
Scheme 2 Synthesis of the alkyne tag.
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Table 1 Alkylation of benzophenone 7
Alkylating
Entry agent
Yield 16
Conditions
(%)a
1
2
15
15
NaH (1.1 equiv.), toluene, r.t.
NaH (2.2 equiv.), toluene, r.t.
−70 °C
10
12
3
4
5
6
7
8
9
15
15
15
17
17
17
17
K2CO3 (1.1 equiv.), 18-crown-6,
toluene, 70 °C
12
16
24
30
30
56
64
Cs2CO3 (1.1 equiv.), 18-crown-6,
toluene, 70 °C
Cs2CO3 (1.1 equiv.), 18-crown-6
DMF : toluene 1 : 1, 50 °C
NaH (1.1 equiv.), DMF : toluene
1 : 1, r.t. −50 °C
Cs2CO3 (1.1 equiv.), DMF : toluene
1 : 1, 50 °C
NaH (1.1 equiv.), 18-crown-6,
DMF : toluene, r.t. −70 °C
Cs2CO3 (1.1 equiv.), 18-crown-6,
DMF : toluene, r.t. −70 °C
Scheme 3 Synthesis of trehalose AfBPP probe 3.
(1.3 equiv.), itself obtained via the Jones oxidation of
1-docosanol,19 then led to the formation of trehalose mono-
ester 20 in 75% yield. The previously synthesised trap-tag 16
was then treated with LiOH under reflux for 10 min to give carb-
oxylic acid 21 in 80% yield. Although this transformation pro-
ceeded smoothly it should be noted that reflux conditions
a Isolated yield after purification by silica gel column chromatography.
improvement in yield (entry 2) but rather degradation of the were required to ensure complete solubilisation of the starting
alkyl iodide 15 was observed. Use of K2CO3 and 18-crown-6 material and that the reaction must be stopped following com-
ether (entry 3) or Cs2CO3 (entry 4) also did not improve the yield plete consumption of the starting material to prevent product
and it was thought that the low yield could be due to the poor degradation. Trehalose mono-ester 20 was then subjected to a
solubility of benzophenone 7 in toluene. Accordingly, the second esterification using trap-tag carboxylic acid 21. As
solvent system was changed to toluene : DMF (1 : 1) and under anticipated,19 due to the highly lipophilic nature of both start-
these conditions, and with CsCO3 as a base (entry 5), a modest ing materials, this second esterification was slow and only a
(24%) yield of the alkylated product 16 was obtained. In order modest (30%) yield of the desired diester 22 could be obtained
to increase the reactivity of the system, we thus proposed that after refluxing the reaction mixture for 7 days. Treatment of
a triflate or tosylate, rather than an iodide, would be a more diester 22 with Dowex-H+, however, proceeded smoothly and
judicious choice of leaving group as this would make the the desired AfBPP probe 3 was obtained in quantitative yield.
carbon a better electrophile for O-alkylation.38 To this end, Here, the two anomeric signals 5.84 (d, J = 3.6 Hz) and
1
alcohol 14 was reacted with tosyl chloride and pyridine39 to 5.83 ppm (d, J = 3.6 Hz) in the H NMR spectrum highlighted
give tosylate 15 in 97% yield (Scheme 2). Tosylate 15 was then the asymmetrical nature of the trehalose moiety.
subjected to alkylation with benzophenone 7 in the presence
With AfBPP probe 3 in hand, we next needed to determine
of either NaH (entry 6, Table 1) or Cs2CO3 (entry 7) and in if the probe displayed similar immune stimulating properties
both instances a slightly improved but still modest yield (30%) to its parent compound and thus was a suitable chemical tool
of the alkylated product 16 was obtained. The addition of by which to study trehalose glycolipid–protein interactions. To
18-crown-6 ether to the reaction mixture, however, greatly this end, probe 3, and the corresponding mincle-ligand TDB
increased the yield and when NaH was used as the base the (2) (which was synthesised according to previously published
alkylated product 16 was obtained in 56% yield (entry 8). procedures),19 were determined to be endotoxin free41 and
Finally, use of Cs2CO3 and 18-crown-6 ether (entry 9) gave 16 subsequently assayed for their abilities to invoke the in vitro
in a much improved and respectable 64% yield.
production of nitric oxide (NO) by bone marrow derived macro-
Having successfully prepared the required lipophilic trap- phages (BMMs).15,17,19 Known macrophage stimulant lipopoly-
tag 16, our focus then turned to the synthesis of the trehalose saccharide (LPS) and media only were used as controls. As
monoester for the subsequent coupling reaction (Scheme 3). observed (Fig. 3), after a 96 h incubation period,19 probe 3 led
To this end, commercially available α,α′-D-trehalose (18) was to the generation of NO by BMMs in a concentration depen-
per-silylated using N,O-bis-trimethylsilylacetamide (BSA) and dent manner. Although the activity of 3 was lower than that
catalytic TBAF, and the more labile primary TMS ethers then of TDB 2, gratifyingly the probe retained activity and there-
selectively removed via the addition of K2CO3 to give tre- fore appears to be a suitable TDM mimic for future AfBPP
halose diol 19.40 Esterification of diol 19 with behenic acid studies.
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9 For a recent review on the synthesis and biological appli-
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Fig. 3 NO production by BMM treated with AfBP probe 3. BMM were stimu-
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the supernatants were analysed for NO using the Griess assay.42
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Conclusions
In conclusion, we have successfully designed and synthesised
an affinity based trehalose probe for use in proteome profiling.
The overall synthetic route is highly convergent, uses minimal
protecting groups, and illustrates how a benzophenone trap
and alkyne tag can be incorporated into a trehalose diester
recognition moiety, thus enabling the first trehalose glycolipid
probe to be developed. The AfBPP probe 3 can activate macro-
phages, as evidenced by NO production, and would therefore
appear to be a suitable TDM mimic. This supports the validity
of using probe 3 for proteomic studies and we are currently
investigating this avenue of research.
20 L. Heitmann, H. Schoenen, S. Ehlers, R. Lang and
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Acknowledgements
B.L.S. would like to acknowledge the RSNZ Marsden Fund for
financial assistance.
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