Chemoenzymatic radiosynthesis of 2-deoxy-2-[18F]fluoro-D-trehalose ([18F]-2-FDTre): A PET radioprobe for in vivo tracing of trehalose metabolism

Article abstract of DOI:10.1016/j.carres.2018.11.002

Trehalose analogues bearing fluorescent and click chemistry tags have been developed as probes of bacterial trehalose metabolism, but these tools have limitations with respect to in vivo imaging applications. Here, we report the radiosynthesis of the ¹⁸F-modified trehalose analogue 2-deoxy-2-[¹⁸F]fluoro-D-trehalose ([¹⁸F]-2-FDTre), which in principle can be used in conjunction with positron emission tomography (PET) imaging to allow in vivo imaging of trehalose metabolism in various contexts. A chemoenzymatic method employing the thermophilic TreT enzyme from Thermoproteus tenax was used to rapidly (15–20 min), efficiently (70% radiochemical yield; ≥ 95% radiochemical purity), and reproducibly convert the commercially available radiotracer 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]-2-FDG) into the target radioprobe [¹⁸F]-2-FDTre in a single step; both manual and automated syntheses were performed with similar results. Cellular uptake experiments showed that radiosynthetic [¹⁸F]-2-FDTre was metabolized by Mycobacterium smegmatis but not by various mammalian cell lines, pointing to the potential future use of this radioprobe for selective PET imaging of infections caused by trehalose-metabolizing bacterial pathogens such as M. tuberculosis.

Full text of DOI:10.1016/j.carres.2018.11.002

CarbohydrateResearch472(2019)16–22
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Chemoenzymatic radiosynthesis of 2-deoxy-2-[18F]fluoro-D-trehalose ([18F]-
2-FDTre): A PET radioprobe for in vivo tracing of trehalose metabolism
Santiago Peña-Zalbideaa,b,1, Ashley Y.-T. Huangc,1, Herbert W. Kavunjac, Beatriz Salinasa,b,f
,
Manuel Descoa,b,f,g, Christopher Draked, Peter J. Woodruffe, Juan J. Vaqueroa,b,∗∗
Benjamin M. Swartsc,∗
,
a Dept. Bioingeniería e Ingeniería Aeroespacial, Universidad Carlos III de Madrid, Madrid, Spain
b Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain
c Department of Chemistry and Biochemistry, Central Michigan University, Mount Pleasant, MI, United States
d SOFIE Co, Culver City, CA, United States
e Department of Chemistry, University of Southern Maine, Portland, ME, United States
f Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain
g Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Spain
A B S T R A C T
Trehalose analogues bearing fluorescent and click chemistry tags have been developed as probes of bacterial trehalose metabolism, but these tools have limitations
with respect to in vivo imaging applications. Here, we report the radiosynthesis of the 18F-modified trehalose analogue 2-deoxy-2-[18F]fluoro-D-trehalose ([18F]-2-
FDTre), which in principle can be used in conjunction with positron emission tomography (PET) imaging to allow in vivo imaging of trehalose metabolism in various
contexts. A chemoenzymatic method employing the thermophilic TreT enzyme from Thermoproteus tenax was used to rapidly (15–20 min), efficiently (70% radio-
chemical yield; ≥ 95% radiochemical purity), and reproducibly convert the commercially available radiotracer 2-deoxy-2-[18F]fluoro-D-glucose ([18F]-2-FDG) into
the target radioprobe [18F]-2-FDTre in a single step; both manual and automated syntheses were performed with similar results. Cellular uptake experiments showed
that radiosynthetic [18F]-2-FDTre was metabolized by Mycobacterium smegmatis but not by various mammalian cell lines, pointing to the potential future use of this
radioprobe for selective PET imaging of infections caused by trehalose-metabolizing bacterial pathogens such as M. tuberculosis.
1. Introduction
targets for drug and diagnostic development [6,7]. Many gut bacteria,
some of which can become lethally pathogenic (e.g., Clostridium diffi-
cile), can also utilize trehalose [8–12].
Trehalose, a non-reducing disaccharide composed of two glucose
units linked via a 1,1-α,α-glycosidic bond (Fig. 1A), has diverse and
important roles in biology and human health. It is abundant in bacteria,
yeast, fungi, plants, and invertebrates, where it fulfills functions in
stress protection, energy storage, and pathogenesis [1,2]. Although
trehalose is not produced by mammals, it is of high significance to
human health. Trehalose's ability to preserve biomaterials has led to its
inclusion in thousands of pharmaceutical, food, and cosmetics products
[3]. In addition, trehalose induces autophagy and mitigates disease
burden in multiple murine models, which has led to studies on its
mechanism(s) of action and therapeutic potential [4,5]. Trehalose is
also critical to many human-resident microbes. Numerous bacterial
pathogens require trehalose for viability and virulence [2]. For ex-
ample, Mycobacterium tuberculosis (Mtb) has unique trehalose metabolic
pathways that are essential for pathogenesis and represent valuable
The ability to image trehalose metabolism in vivo is expected to
stimulate basic and applied research in various areas of biomedical
interest. First, because many bacteria possess dedicated trehalose up-
take and processing pathways that are absent from humans [2], tre-
halose-based probes may selectively accumulate in bacteria and thus
enable targeted imaging of bacterial infections. Such tools are urgently
needed in the clinic [13] given the massive health burdens of increas-
ingly drug-resistant nosocomial pathogens such as C. difficile, S. aureus,
and P. aeruginosa (722,000 cases, 75,000 deaths in the US in 2011 [14])
as well as global pathogens such as Mtb (10.0 million cases, 1.3 million
deaths estimated in 2017 [15]). Infection imaging tools will enable
pathophysiology and antibiotic development studies in animals, and
they could revolutionize clinical infection management by enhancing
diagnosis, staging, and therapy monitoring [13,16,17]. Targeting
∗ Corresponding author.
∗∗ Corresponding author. Dept. Bioingeniería e Ingeniería Aeroespacial, Universidad Carlos III de Madrid, Madrid, Spain.
E-mail addresses: jjvaquer@ing.uc3m.es (J.J. Vaquero), ben.swarts@cmich.edu (B.M. Swarts).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.carres.2018.11.002
Received 2 October 2018; Received in revised form 24 October 2018; Accepted 3 November 2018
Availableonline04November2018
0008-6215/©2018PublishedbyElsevierLtd.

S. Peña-Zalbidea et al.
CarbohydrateResearch472(2019)16–22
with the established capabilities and broad/expanding accessibility of
PET.
A major challenge to the development of 18F-FDTre probes is that
the short half-life (109.7 min) of fluorine-18 requires rapid synthetic
methods capable of generating the radioprobe from the radionuclide in
∼2 h or less. Radiosynthesis using SN2 reactions (e.g., between sugar
triflates and nucleophilic 18F-fluoride) can be considered, but these
routes have lengthy and inefficient precursor syntheses due to the
challenges associated with trehalose's 1,1-α,α-glycosidic bond and C2
symmetry [46]. Moreover, “cold” 19F-fluorinations of protected treha-
lose intermediates are slow and have moderate yields [21,49,50]. A
significantly more streamlined and efficient approach would be to ac-
cess 18F-FDTre from the corresponding 18F-FDG, which would be par-
ticularly attractive for the 2-position isomer, as 18F-2-FDG is widely
commercially available (over a million 18F-2-FDG–PET scans are done
annually in the US). Given that naturally occurring trehalose biosyn-
thetic pathways utilize glucose or glucose phosphates as acceptor sub-
strates to generate trehalose, there is an opportunity to adapt these
pathways—if they are sufficiently substrate tolerant—to enable the
biocatalytic conversion of 18F-FDG analogues to 18F-FDTre analogues
[34]. This concept was originally explored in 2011, when a che-
moenzymatic method inspired by the OtsAB trehalose biosynthetic
pathway was used to convert non-radioactive 19F-2-FDG into 19F-2-
FDTre (Scheme 1A) [21]. While this method can potentially be adapted
to the radiosynthesis of 18F-2-FDTre, it used three enzymes and was
limited to the production of only one regioisomer [21]. In 2014, we
developed a one-step chemoenzymatic method for trehalose analogue
synthesis enlisting the substrate-permissive trehalose synthase (TreT)
from Thermoproteus tenax [51,52], and in 2016 we applied this method
to the synthesis of non-radioactive 19F-2-, 19F-3-, and 19F-6-FDTre
analogues from the corresponding 19F-FDG analogues (Scheme 1B)
[53]. These reactions featured quantitative conversion of substrate to
product in ≤60 min, suggesting that TreT may be suitable for radio-
synthesis [53]. Despite these advances, to date the radiosynthesis of
18F-2-FDTre has not be reported in the literature. Here, we describe the
successful application of TreT catalysis to the rapid one-step radio-
synthesis of 18F-2-FDTre, as well as preliminary uptake evaluation of
this radioprobe in mammalian and mycobacterial cells.
Fig. 1. Structures of (A) trehalose and (B) regioisomeric 18F-labeled FDTre
analogues as PET probes proposed for in vivo imaging of trehalose metabolism.
This study focuses on the radiosynthesis of 18F-2-FDTre.
bacterial carbohydrate metabolism for the development of infection
imaging tools is an attractive approach, as recently exemplified by the
development of maltose-, maltohexaose-, and sorbitol-based in vivo
imaging probes [18–20]. In this context, trehalose-based probes are
attractive candidates for bacterial infection imaging, a notion which
was first put forth in the context of Mtb imaging by the Barry and Davis
groups [21]. Second, trehalose has gained traction as a human ther-
apeutic due to its ability to induce autophagy and alleviate disease
burden in models of neurodegeneration and non-fatty alcoholic liver
disease, but the current in vivo mechanistic understanding of these
phenomena is incomplete [5,22–33]. The ability to directly trace tre-
halose metabolism in vivo will overcome a critical barrier to mechan-
istic inquiry in these fields and may inform optimal administration
route and dosing as trehalose advances to clinical utility.
To date, several probes have been developed that permit tracking of
trehalose metabolism in vitro, but pose problems for in vivo imaging
[34]. 14C-labeled trehalose is commercially available and has been used
to probe trehalose metabolism in diverse biological systems [35–42],
but it cannot be used for imaging. Fluorescently-tagged [21,43–45] and
clickable [46,47] trehalose analogues have been introduced over the
past several years and used to image trehalose metabolism in cells.
However, these probes are not suitable for non-invasive in vivo imaging
due to disadvantages such as the size of the detectable moiety, the need
for a secondary labeling step, and reliance on optical imaging. In ad-
dition, the multi-step chemical syntheses of these probes are inefficient
and require synthetic expertise, limiting their accessibility. Positron
emission tomography (PET) imaging employing deoxy-[18F]fluoro-D-
trehalose (18F-FDTre) probes (structures shown in Fig. 1B) is an ideal
approach for in vivo imaging of trehalose metabolism to support the
applications described above. PET is a non-invasive, clinically estab-
lished technique for rapid, quantitative, 3-D in vivo imaging of (often
disease-associated) metabolic processes deep within the body. The most
extensively used radionuclide for PET is fluorine-18, whose beneficial
nuclear and physical properties have led to its incorporation into a
broad range of PET probes, most notably 2-deoxy-[18F]fluoro-D-glucose
2. Results and discussion
2.1. Kinetic analysis of TreT-catalyzed synthesis of 19F-2- FDTre from 19F-
2-FDG
Our prior work demonstrated that Thermoproteus tenax TreT, which
is a thermostable enzyme that couples glucose and UDP-glucose to form
trehalose [54], is substrate tolerant and can be employed to efficiently
convert various glucose analogues into the corresponding trehalose
analogues in high yield (with the main exception being 4-position-
modified analogues) [51,52]. We subsequently showed that TreT is
active on 19F-FDG analogues and can generate 19F-FDTre analogues
[53]. However, we did not analyze the kinetic properties of these
(
18F-2-FDG), which is widely available and the standard probe for a
range of research and clinical PET imaging applications [48]. 18F-
FDTre–PET is a logical merger of the trehalose-based probe concept
Scheme 1. (A) OtsAB-inspired three-step
synthesis of 2-FDTre using hexokinase, treha-
lose-6-phosphate synthase (TPS), and alkaline
phosphatase. (B) TreT-catalyzed one-step
synthesis of FDTre regioisomers. Both
methods can potentially convert the widely
commercially available PET probe 18F-2-FDG
into the corresponding trehalose probe 18F-2-
FDTre.
17

S. Peña-Zalbidea et al.
CarbohydrateResearch472(2019)16–22
Fig. 2. Michaelis-Menten plots of initial velocity (μM UDP min−1) versus the concentration of (A) glucose or (B) 19F-2-FDG (mM) for T. tenax TreT. The concentration
of UDP-glucose was held constant at 1 mM. Data are representative of two independent trials.
reactions, which would provide useful information for predicting/un-
derstanding reaction outcomes and optimizing reaction conditions if
needed. Here, we obtained the kinetic parameters of TreT-catalyzed
conversion of 19F-2-FDG to 19F-2-FDTre analogues by employing a
commercially available luminescence-based glycosyltransferase assay
(UDP-Glo, Promega). TreT reactions were run in 50 mM Tris-HCl buffer
with the donor (UDP-glucose) held at a saturating concentration of
1 mM and the acceptor (glucose or 19F-FDG isomers) varied in con-
centration from 0 to 10 mM for durations of 0–10 min. UDP released in
the reactions was quantified by the luminescence assay and Michae-
lis–Menten analyses were performed (Fig. 2). The kinetic parameters
Radio-HPLC analysis employing an aminopropyl column was
used to monitor reactions, showing that (i) 18F-2-FDG was fully
converted to 18F-2-FDTre in only 15 min; and (ii) the 18F-2-FDTre
radiochemical purity of the product was high (Fig. 3A). Injection of
18F-2-FDG alone gave a single peak with a retention time (tR) of
14 min. When the TreT reaction was analyzed following 10 min,
some unreacted 18F-2-FDG (tR = 14 min) was observed along with a
major new peak for 18F-2-FDTre (tR = 27 min). At 15 min reaction
time, the 18F-2-FDG was completely consumed and a single peak
corresponding to the 18F-2-FDTre product was present. The non-
decay corrected radiochemical yield and purity for this reaction were
70% and > 99%, respectively. The tR trends for the starting material
and product were in agreement with those previously observed for
non-radioactive compounds using a similar HPLC method [51]. To
confirm the identity of the radio-HPLC peak at tR = 27 min as 18F-2-
FDTre, we performed a modified radiochemical reaction with addi-
tional “cold” 19F-2-FDG doped in, then separated the mixture by
HPLC and collected the peak of interest (tR = 27 min). After the
radioactivity dissipated, the sample was analyzed by ESI-MS and
confirmed to be 19F-2-FDTre (Fig. 3C; base peaks observed: positive
mode, [M+Na]+ = 367.0; and negative mode, [M+Cl]– = 379.1).
The chromatogram shown in Fig. 3A is representative of several re-
action trials, which all had non-decay corrected 18F-2-FDTre radio-
chemical yields of approximately 70% and radiochemical purities of
≥95%. The radionuclidic purity of synthetic 18F-2-FDTre was es-
tablished by confirming that the decay time and photopeak (511 keV)
of the product matched fluorine-18. The chemical stability of 18F-2-
FDTre was also confirmed by incubating newly-synthesized material
in phosphate-buffered saline (PBS) at 37 °C with shaking and re-
analyzing the sample by radio-HPLC, which in the chromatograms
showed no changes over 6 h (data not shown).
obtained for the natural acceptor glucose (Km = 0.31
0.02 mM;
V
max = 0.80
0.22 μM min−1) were similar to those observed when
19F-2-FDG
was
employed
as
the
acceptor
substrate
(Km = 1.45
0.20 mM; Vmax = 0.35
0.01 μM min−1). Although
19F-2-FDG exhibited moderately higher Km and lower Vmax values than
glucose, these data suggested that TreT utilizes 2-FDG with comparable
efficiency to glucose, potentially allowing conversion of 18F-2-FDG to
the corresponding 18F-labeled trehalose analogue.
2.2. TreT-catalyzed radiosynthesis of 18F-2-FDTre from 18F-2-FDG
Encouraged by our promising results using non-radioactive substrates
reported previously [53] and in this study, we next sought to develop a
protocol for the rapid radiosynthesis of 18F-2-FDTre from commercially
available 18F-2-FDG. The molar concentration of 18F-2-FDG in a com-
mercial batch is typically in the picomolar range—much lower than the
10 mM concentration we used for semi-preparative scale TreT-catalyzed
synthesis of 19F-2FDTre. While unlabeled 19F-2-FDG can be added to
radiolabeled 18F-2-FDG to increase the molar concentration of substrate,
this would decrease the specific activity of the tracer, which is often un-
desirable for PET imaging applications. Thus, the most significant change
in adapting the established TreT method to 18F-2-FDTre radiosynthesis
involved the usage of extremely dilute acceptor substrate. Fortunately, this
did not pose a problem, as the optimized synthetic method allowed rapid
and quantitative conversion of unaltered commercial 18F-2-FDG to 18F-2-
FDTre. Briefly, starting from commercial 18F-2-FDG (IBA Molecular, Ma-
drid), UDP-glucose and MgCl2 were added to final concentrations of
40 mM and 20 mM, respectively. Then, the reaction was initiated by ad-
dition of TreT to a final concentration of ∼10 μM in 50 mM Tris-HCl
buffer (pH 8). The reaction was incubated for 15 min at 70 °C—an elevated
temperature that was enabled by the high thermostability of T. tenax TreT.
Next, a rapid two-step purification procedure was executed. First, the
enzyme was removed using a centrifugal filter unit, then the reaction
mixture was passed through a mixed-bed ion exchange cartridge to re-
move ionic species, delivering the neutral 18F-2-FDTre product in aqueous
solution. Combined, the entire procedure, including the reaction and two
purifications, was completed in 30 min.
Automation of radiosynthetic protocols is a critical step for com-
mercialization of radioprobes. With this in mind, we sought to de-
monstrate that TreT-catalyzed production of 18F-2-FDTre is amenable to
automation on an ELIXYS FLEX/CHEM automated radiosynthesizer
(SOFIE Co., Culver City). The manual synthesis steps were translated
into an ELIXYS sequence, reflecting the optimized conditions described
above. The sequence was executed, using commercially available 18F-2-
FDG (PetNet solutions, Culver City), and analytical HPLC demonstrated
nearly quantitative conversion of 18F-2-FDG to 18F-2-FDTre in this
format (Fig. 3B). While no further purification was attempted, ELIXYS is
fully compatible with a wide range of cartridge-based purification
protocols; thus automating the purification protocol established for
manual 18F-FDTre radiosynthesis should not pose a significant chal-
lenge. Taken together, our data demonstrate that the TreT catalysis
method is capable of rapidly and reproducibly generating radio-
chemically pure 18F-2-FDTre from commercially available 18F-2-FDG
via manual or automated radiosynthesis.
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S. Peña-Zalbidea et al.
CarbohydrateResearch472(2019)16–22
Fig. 3. Radiosynthesis and characterization of 18F-2-FDTre. (A) Manual radiosynthesis. Representative radio-HPLC analysis of 18F-2-FDG starting material (black)
and the TreT reaction after 10 min (blue dashed) and 15 min (red). (B) Automated radiosynthesis on ELIXYS FLEX/CHEM. Radio-HPLC analysis of reactions per-
formed in the presence (solid line) of TreT enzyme or in the absence of enzyme as a control (dashed line). (C) ESI mass spectra of radio-HPLC fractions corresponding
to the peak at tR = 27 min from (A) in (i) positive mode and (ii) negative mode. (For interpretation of the references to color in this figure legend, the reader is
referred to the Web version of this article.)
2.3. Comparison of 18F-2-FDTre uptake by M. smegmatis and mammalian
cell lines
the tumor cell lines AR42J and HT29 (rat pancreatic and human col-
orectal adenocarcinoma, respectively). Mammalian cells were in-
cubated in PBS containing ∼5 μCi of freshly synthesized 18F-2-FDTre
for 60 min, then washed and assessed for radioactivity. M. smegmatis
cells were subjected to essentially the same procedure, along with
controls using bacteria that were heat-killed and bacteria that were co-
treated with competing excess unlabeled trehalose. As shown in Fig. 4,
while the hMSC, AR42J, and HT29 mammlian cells showed virtually no
probe uptake (< 1% of 18F-2-FDTre in the medium), M. smegmatis ex-
hibited efficient, linear, and time-dependent uptake probe (∼30% up-
take after 3 h). Accumulation of 18F-2-FDTre in M. smegmatis was
abolished if the bacteria were either heat-killed or co-incubated with
excess unlabeled trehalose (< 1% uptake), demonstrating that probe
uptake is active and specific for trehalose metabolism. Together, these
data support our hypothesis that trehalose-metabolizing bacteria such
as M. smegmatis will indeed accumulate 18F-2-FDTre more efficiently
than mammalian cells. Furthermore, at least in the in vitro experimental
conditions employed, even very low (picomolar) concentrations of 18F-
2-FDTre can be taken up by bacteria. Finally, evaluation of 18F-2-FDTre
Because various bacterial pathogens possess dedicated trehalose
transporters but mammals are not known to, we hypothesized that 18F-
FDTre PET radioprobes would be taken up more efficiently by bacteria
than by mammalian cells, and thus may allow for selective imaging of
bacterial infections in vivo. In prior work, we showed that M. smegma-
tis—a frequently used fast-growing non-pathogenic model organism for
the global pathogen Mtb—can uptake three non-radioactive 19F-FDTre
regioisomers, including 19F-2-FDTre, via the trehalose-specific trans-
porter LpqY-SugABC [53]. However, these experiments were performed
with micromolar concentrations of 19F-FDTre analogues, which far
surpasses the trace concentration of 18F-radioprobe that would be
available in vivo. Furthermore, no findings on FDTre uptake by mam-
malian cells have been reported to date.
To address these questions, we performed an uptake analysis of
radiolabeled 18F-2-FDTre comparing M. smegmatis and several mam-
malian cell lines, including human mesenchymal stem cells (hMSC) and
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S. Peña-Zalbidea et al.
CarbohydrateResearch472(2019)16–22
LpqY-SugABC-mediated transport across the plasma membrane. Given
the lack of radioprobe uptake by various mammalian cell lines (Fig. 4),
there is motivation to perform in vitro uptake studies in additional
trehalose-utilizing bacteria and pursue in vivo imaging studies in re-
levant animal infection models. The chemoenzymatic synthetic
methods reported in this study will facilitate these future directions.
4. Experimental
4.1. Materials and reagents
TreT was expressed and purified from E. coli as previously described
[52]. The UDP-Glo assay kit, including ultra-pure UDP-glucose, were
obtained from Promega. 18F-2-FDG was purchased from IBA Molecular
(Madrid, Spain) or PetNet Solutions (Culver City, CA). Non-radioactive
19F-2-FDG was purchased from CarboSynth. UDP-Glucose was obtained
from Sigma or Abcam. AG1-X8 mixed-bed ion exchange resin and Poly-
Prep columns were purchased from Bio-Rad Laboratories.
Fig. 4. Cellular uptake evaluation of radiolabeled 18F-2-FDTre. (A) Percent of
total 18F-2-FDTre radioactivity in media taken up after 60 min by different
mammalian cell lines (AR42J, HT29, and hMSC) and Msmeg or, as controls,
Msmeg that was heat-killed or incubated in the presence of excess (1 mM) un-
labeled trehalose. Data shown are representative of at least two biological re-
plicates, except the hMSC experiment (n = 1). (B) Percent of total 18F-2-FDTre
radioactivity in media taken up by live Msmeg cells over time.
4.2. Enzyme kinetics
Kinetic properties of TreT-catalyzed synthesis of trehalose and 19F-
2-FDTre were obtained by measuring the production of UDP using the
UDP-Glo luminescence glycosyltransferase assay (Promega), essentially
as previously described [53,55]. TreT (0.8 μg) was incubated in the
presence of 1 mM UDP-glucose, 20 mM MgCl2, 300 mM NaCl, and
50 mM Tris-HCl buffer (pH 8.0) in the absence (negative control) or
presence of acceptor substrate. The acceptors evaluated were glucose
and 19F-2-FDG, each tested over a concentration range of 0–10 mM. For
each concentration, UDP production was assessed at four time points
(immediately, 2, 5, and 10 min) to ensure linearity of the enzymatic
reaction. Reactions were set up in 96 well plates with UDP-glucose,
glucose or 19F-2-FDG, MgCl2, and buffer added to the wells first, then
the reactions were initiated by addition of TreT enzyme to achieve a
final volume of 25 μL at room temperature. The reactions were in-
cubated at room temperature for 0–10 min, then 25 μL UDP detection
reagent were added, which quenched the reaction and coupled UDP
production to a luciferase reaction. After incubation at room tempera-
ture for 60 min, the luminescence signal was recorded using a micro-
plate reader (Tecan Infinite M200 Pro). The luminescence signal was
fitted to a standard curve made from a dilution series of known UDP
concentrations measured in the same 96-well microplate. Relative light
units (RLUs) given by the luminescence reader were converted to UDP
concentration values, which were subsequently processed in GraphPad
Prism v. 6.02 to obtain Michaelis–Menten plots and calculate kinetic
parameters Km and Vmax. Data shown are representative of two in-
dependent trials. Reported Km and Vmax values are the average from the
two trials with standard deviation given.
in a plasma protein binding assay showed that > 99% of the radioprobe
remained unbound to porcine serum proteins (data not shown), in-
dicating that it will not be disadvantageously absorbed by plasma
components during in vivo applications.
3. Conclusion
The development of 18F-FDTre–PET technology is motivated by the
prospect of imaging trehalose metabolism in vivo, which is perhaps of
most interest for the selective imaging of infections caused by trehalose-
metabolizing bacterial pathogens (e.g., Mtb). Building on the earlier
successes of fluorescent and clickable trehalose analogues, we recently
applied our TreT catalysis method to produce three non-radioactive 19F-
FDTre regioisomers in high yield. Herein, we translated these findings
to the rapid one-step radiosynthesis of 18F-2-FDTre by exploiting TreT,
which is attractive from numerous standpoints. Most importantly, even
when using the extremely low (picomolar) concentrations of 18F-2-FDG
starting material in commercial preparations, TreT allowed quantitative
conversion of 18F-2-FDG to 18F-2-FDTre under mild conditions in only
15–20 min. This is beneficial because no unlabeled substrate needs to
be added to the reaction to increase substrate concentration, thus
avoiding reduction of the tracer's specific activity. Another advantage
of this process is that the starting material, 18F-2-FDG, is widely com-
mercially available, meaning that the TreT method can tap into existing
infrastructure to make 18F-2-FDTre readily accessible to virtually any
radiopharmacy or PET imaging facility in the world. TreT is a ther-
mostable enzyme, which has practical advantages such as extended
shelf life and heating of reactions to increase rate and avoid microbial
contamination. In addition to a manual synthesis format, we performed
TreT-catalyzed preparation of 18F-2-FDTre on an automated radio-
synthesis module, which should facilitate the adoption of this radiop-
robe and synthetic method by others. Furthermore, we recently re-
ported a bead-immobilized version of TreT [55], which will allow
enzyme reuse and will further simplify the radiosynthesis process by
eliminating the enzyme removal step of the purification. Finally, we
previously showed that TreT catalysis can generate non-radioactive 19F-
3- and 19F-6-FDTre regioisomers, so the corresponding radioactive 18F-
labeled analogues should be accessible through the procedures reported
herein; indeed, the radiosyntheses of the precursors, 18F-3- and 18F-6-
FDG, have been published [56,57] and can easily be coupled to TreT
catalysis. Also in this work, we showed that an avirulent mycobacterial
model organism, M. smegmatis, can uptake trace concentrations of
radiosynthetic 18F-2-FDTre via trehalose-specific metabolism, likely via
4.3. Radiosynthesis and characterization of 18F-2-FDTre
To a 0.5 or 1.5 mL microcentrifuge tube containing a solution of
commercially obtained 18F-2-FDG in 0.9% NaCl, reactants were added
sequentially to achieve final concentrations of 40 mM UDP-glucose,
20 mM MgCl2, ∼10 μM T. tenax TreT enzyme, and enough Tris buffer
(50 mM Tris, 300 mM NaCl, pH 7–8) to achieve the desired volume if
needed. After gently pipetting up and down three times, the tube was
capped and incubated for 15 min at 70 °C with shaking in a Grant Bio
PMHT Thermoshaker inside a cell. The reaction mixture was transferred
to an Amicon Ultra-15 centrifugal filter unit (nominal molecular weight
limit (NMWL) 10 kDa) pre-rinsed 3x with deionized water. The filter
unit was centrifuged at 14,000 rpm for 10 min, then the filtrate was
collected and loaded onto a Bio-Rad Poly-Prep column (0.8 × 4 cm)
pre-packed with pre-equilibrated Bio-Rad AG 501-X8(D) mixed-bed ion
exchange resin (biotechnology grade, 20–50 mesh, H+ + OH− form,
with blue-to-gold indicator dye to monitor resin exchange capacity). A
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CarbohydrateResearch472(2019)16–22
ratio of 75 mg resin per 100 μL solution was used. The initial eluate
containing the void volume was discarded, then the probe-containing
eluate was collected. The total synthesis time including purifications
was 30 min.
mL of bacteria were heat-killed at 80 °C for 60 min prior to performing
uptake experiments as described above. For the M. smegmatis compe-
tition experiment, 108 CFU/mL of bacteria were subjected to uptake
experiments as described above but in the presence of 1 mM unlabeled
native trehalose. For the M. smegmatis uptake time course, 108 CFU/mL
of bacteria were subjected to uptake experiments as described above
but aliquots were taken and analyzed at 30, 60, 90, and 180 min time
points (note: due to the sample washing steps prior to gamma well
counting, there was a ∼15–25 min delay time between the “end point”
and activity measurement, during which some additional uptake could
have occurred). Mammalian cell viability following uptake experiments
was > 90% as determined by trypan-blue staining.
To determine radiochemical yield and radiochemical purity, the
product was analyzed by radio-HPLC using an Agilent 1200 Series
HPLC system equipped with an Imtakt UK-Amino 250 × 46 mm
column, a Raytest gamma detector, and Gina software for monitoring
radioactivity. The HPLC method, which was adapted from our previous
report [51], utilized isocratic elution with a pre-mixed mobile phase of
80% acetonitrile in water at a flow rate of 0.4 mL/min. Radiochemical
yield was determined by dividing the product activity by the starting
activity and multiplying it by the radiochemical purity. Radiochemical
purity was determined by calculating the area under the curves in the
radiochromatogram. The radionuclidic purity of the product was de-
termined by using a portable spectrometer (Hamamatsu Photonics ra-
diation detection module C12137) and calculating decay time using a
PTW Curiementor 4 activimeter.
Acknowledgements
This work was funded by a grant from the National Institutes of
Health, United States (R15 AI117670) to B.M.S. and P.J.W., as well as a
Henry Dreyfus Teacher-Scholar Award from The Camille & Henry
Dreyfus Foundation to B.M.S. (TH-17-034). A.Y.-T.H was supported by
the Mount Holyoke College Lynk program. Dr. Wenyan Xu is thanked
for assistance with kinetic analysis. The research leading to these results
received funding from the Innovative Medicines Initiative, European
Union (www.imi.europa.eu) Joint Undertaking under grant agreement
no. 115337, whose resources comprise funding from EU FP7/2007-
2013 and EFPIA, European Union companies in-kind contribution. This
work was partially supported by the Ministry of Economy and
Competitiveness, Spain TEC2015-73064-EXP and TEC2016-78052-R,
ISCIII-FIS grants PI16/02037, co-financed by ERDF, European Union
(FEDER) Funds from the European Commission, European Union, “A
way of making Europe”.
To confirm product identity by mass spectrometry (MS), a radio-
synthesis of 18F-2-FDTre was performed as described above, except 19F-
2-FDG was added to a final concentration of 0.2 mM to generate suffi-
cient non-radioactive 19F-2-FDTre product to allow MS analysis.
Following the reaction, radio-HPLC was used employing the method
described above to co-isolate the presumed radioactive 18F-2-FDTre and
non-radioactive 19F-2-FDTre species by obtaining eluate from
tR = 27 min. After decay of 18F occurred, the non-radioactive sample
was analyzed by electrospray ionization (ESI) MS in positive and ne-
gative mode using a HCT-Ultra ion trap mass spectrometer.
Automated conversion of 18F-2-FDG to 18F-2-FDTre was performed
on an ELIXYS FLEX/CHEM automated radiosynthesizer (SOFIE Co.,
Culver City, CA) using standard ELIXYS cassettes, reactor vials
(W986259NG, Wheaton; Millville, NJ), magnetic stir bars (SBM-0605-
GLC, stirbars.com; Huntersville, SC), and reagent vials (62413P-2,
Voigt; Lawrence, KN). An automated program (“sequence”) was cre-
ated, based on the optimized manual radiosynthesis, by assembling a
short sequence of intuitive “unit operations” (macros) using a drag-and-
drop interface, and specifying appropriate parameters for each. A “re-
agent mix” (0.55 mL total volume, 50 mM Tris buffer, pH 8.0) con-
taining T. tenax TreT enzyme (450 μL of a 951 μg/mL solution), UDP-
glucose (50 μL of a 0.4 M solution), and MgCl2 (5 μL of a 2 M solution)
was loaded into the ELIXYS cassette prior to starting the sequence. The
sequence commenced with the addition of 18F-2-FDG (433 μCi; ∼20 μL)
to the reactor using the EXTERNAL ADD unit operation. The “reagent
mix” was then added using the ADD unit operation (N.B., ∼100 μL will
not enter the reactor due to the dead volume of the ELIXYS cassette), to
give a solution of 18F-2-FDG, TreT (840 μg/mL), UDP-glucose (40 mM),
MgCl2 (20 mM) in ∼50 mM Tris buffer, which was then heated at 70 °C
for 20 min. The solution was cooled, and a sample withdrawn for
analytical HPLC (method was adapted from that described above for
manual synthesis).
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