provides intrinsic drug and stress tolerance to Mycobacterium tuberculosis (Mtb) and related pathogens [7, 8]. Central to mycomembrane biosynthesis is TMM, which is synthesized in the cytoplasm
and transported to the cell envelope [9, 10], then processed by
antigen 85 (Ag85) mycoloyl transferases [11–13] to transfer the
mycoloyl group from TMM onto acceptor molecules (Fig. 1a).
Two classes of complementary in vivo-compatible probes—trehalose-mimicking and TMM-mimicking—have been developed to
hijack these substrate-promiscuous mycomembrane biosynthetic
pathways and metabolically incorporate into cell envelope mycolates, facilitating the study of mycomembrane components and
pathways in a native setting (Fig. 1b) [14]. Because mycoloylation
pathways are specific to mycobacteria, these probes have also generated interest with respect to developing novel mycobacteriatargeting diagnostic and therapeutic strategies [14].
Trehalose-mimicking probes act as mycoloyl acceptors and thus
get incorporated into the mycomembrane as TMM and/or TDM.
Representative trehalose-mimicking probes (Fig. 2a) include
fluorescein-modified trehalose (FITC-Tre) [15], 4-N,N-dimethylamino-1,8-naphthalimide-modified trehalose (DMN-Tre) [16]
and azide-modified trehalose (TreAz) analogs [17]. FITC-Tre,
the first reported trehalose-based probe, enables one-step labeling
and detection of mycobacterial glycolipids [15]. Next-generation
fluorogenic trehalose-based probes, which are designed to generate
fluorescence signal only upon metabolic incorporation, aim to
eliminate wash steps [16, 18, 19]. For example, DMN-Tre features
a solvatochromic dye that permits rapid, no-wash detection of Mtb
via incorporation into the hydrophobic mycomembrane
[16]. However, these and other dye-conjugated trehalose analogs
are limited to fluorescence applications. To provide flexibility for
diverse experimental objectives and applications, “clickable” azido
trehalose analogs, such as 6-azidotrehalose (6-TreAz), were introduced (see Note 1) [17]. TreAz analogs operate via a two-step
process, first metabolic incorporation to generate azide-labeled
TMM/TDM, then selective bioorthogonal “click” reaction with
alkyne-modified secondary reagents via either copper-catalyzed
azide–alkyne cycloaddition (CuAAC) [20] or copper-free strainpromoted azide-alkyne cycloaddition (SPAAC) [21]. In this manner, virtually any type of chemical cargo can be specifically delivered
to azide-labeled trehalose glycolipids in the mycomembrane of live
bacteria.
In contrast to trehalose-mimicking probes, TMM-mimicking
probes can act either as mycoloyl acceptors or donors, depending
on the probe design (Fig. 2b). When bearing an amide-linked acyl
chain, e.g., in amide-linked 6-heptynoyl-modified TMM
(N-AlkTMM-C7), the TMM probe cannot donate its acyl group
and instead only receives native mycoloyl groups through Ag85
activity, thus it forms a labeled version of TDM [22]. On the other
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Nicholas Banahene and Benjamin M. Swarts
and transported to the cell envelope [9, 10], then processed by
antigen 85 (Ag85) mycoloyl transferases [11–13] to transfer the
mycoloyl group from TMM onto acceptor molecules (Fig. 1a).
Two classes of complementary in vivo-compatible probes—trehalose-mimicking and TMM-mimicking—have been developed to
hijack these substrate-promiscuous mycomembrane biosynthetic
pathways and metabolically incorporate into cell envelope mycolates, facilitating the study of mycomembrane components and
pathways in a native setting (Fig. 1b) [14]. Because mycoloylation
pathways are specific to mycobacteria, these probes have also generated interest with respect to developing novel mycobacteriatargeting diagnostic and therapeutic strategies [14].
Trehalose-mimicking probes act as mycoloyl acceptors and thus
get incorporated into the mycomembrane as TMM and/or TDM.
Representative trehalose-mimicking probes (Fig. 2a) include
fluorescein-modified trehalose (FITC-Tre) [15], 4-N,N-dimethylamino-1,8-naphthalimide-modified trehalose (DMN-Tre) [16]
and azide-modified trehalose (TreAz) analogs [17]. FITC-Tre,
the first reported trehalose-based probe, enables one-step labeling
and detection of mycobacterial glycolipids [15]. Next-generation
fluorogenic trehalose-based probes, which are designed to generate
fluorescence signal only upon metabolic incorporation, aim to
eliminate wash steps [16, 18, 19]. For example, DMN-Tre features
a solvatochromic dye that permits rapid, no-wash detection of Mtb
via incorporation into the hydrophobic mycomembrane
[16]. However, these and other dye-conjugated trehalose analogs
are limited to fluorescence applications. To provide flexibility for
diverse experimental objectives and applications, “clickable” azido
trehalose analogs, such as 6-azidotrehalose (6-TreAz), were introduced (see Note 1) [17]. TreAz analogs operate via a two-step
process, first metabolic incorporation to generate azide-labeled
TMM/TDM, then selective bioorthogonal “click” reaction with
alkyne-modified secondary reagents via either copper-catalyzed
azide–alkyne cycloaddition (CuAAC) [20] or copper-free strainpromoted azide-alkyne cycloaddition (SPAAC) [21]. In this manner, virtually any type of chemical cargo can be specifically delivered
to azide-labeled trehalose glycolipids in the mycomembrane of live
bacteria.
In contrast to trehalose-mimicking probes, TMM-mimicking
probes can act either as mycoloyl acceptors or donors, depending
on the probe design (Fig. 2b). When bearing an amide-linked acyl
chain, e.g., in amide-linked 6-heptynoyl-modified TMM
(N-AlkTMM-C7), the TMM probe cannot donate its acyl group
and instead only receives native mycoloyl groups through Ag85
activity, thus it forms a labeled version of TDM [22]. On the other
386
Nicholas Banahene and Benjamin M. Swarts