Flow synthesis of a versatile fructosamine mimic and quenching studies of a fructose transport probe

Article abstract of DOI:10.3762/bjoc.9.238

We describe the synthesis of 1-amino-2,5-anhydro-D-mannose ("mannitolamine"), a key intermediate to the 7-nitro-1,2,3- benzadiazole conjugate (NBDM), using commercially available fluidic devices to increase the throughput. The approach is the first example of a flow-based Tiffeneau-Demjanov rearrangement. Performing this step in flow enables a ～64-fold throughput enhancement relative to batch. The flow process enables the synthesis to be accomplished three times faster than the comparable batch route. The high throughput enabled the production of larger quantities of the fluorescent fructose transport probe NBDM, enabling us to measure key photophysical properties that will facilitate future uptake studies.

Full text of DOI:10.3762/bjoc.9.238

Flow synthesis of a versatile fructosamine mimic and
quenching studies of a fructose transport probe
Matthew B. Plutschack1,2, D. Tyler McQuade*1,2, Giulio Valenti2
and Peter H. Seeberger2
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 2022–2027.
1Department of Chemistry and Biochemistry, Florida State University,
Tallahassee, FL 32306, USA and 2Max Planck Institute of Colloids
and Interfaces, Am Mühlenberg 1, 14476 Potsdam, Germany
doi:10.3762/bjoc.9.238
Received: 19 June 2013
Accepted: 03 September 2013
Published: 07 October 2013
Email:
D. Tyler McQuade* - mcquade@chem.fsu.edu
This article is part of the Thematic Series "Chemistry in flow systems III".
Guest Editor: A. Kirschning
* Corresponding author
Keywords:
flow chemistry; fructose mimic; gluts; NBDM
© 2013 Plutschack et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
We describe the synthesis of 1-amino-2,5-anhydro-D-mannose (“mannitolamine”), a key intermediate to the 7-nitro-1,2,3-benzadia-
zole conjugate (NBDM), using commercially available fluidic devices to increase the throughput. The approach is the first example
of a flow-based Tiffeneau–Demjanov rearrangement. Performing this step in flow enables a ~64-fold throughput enhancement rela-
tive to batch. The flow process enables the synthesis to be accomplished three times faster than the comparable batch route. The
high throughput enabled the production of larger quantities of the fluorescent fructose transport probe NBDM, enabling us to
measure key photophysical properties that will facilitate future uptake studies.
Introduction
The impact of dietary fructose on human health is not well- disorders [1]. Abnormal Glut expression is observed in the
understood. A growing body of work suggests that those eating pancreatic islets and hepatic cells of people with diabetes,
diets high in fructose exhibit increased rates of metabolic disor- which may explain glucose insensitivity and the progression of
ders and aggressive cancers [1,2]. Since the landmark reports non-alcoholic fatty liver disease [6,7].
from Warburg [3], researchers have recognized that cancer
cells/tissues consume more carbohydrates than normal tissues, Passive carbohydrate transporters are well-known targets for
fueling rapid growth and proliferation [4]. While cancer cells carbohydrate-based probes [8]. The positron emission tomog-
utilize multiple strategies to increase carbohydrate consump- raphy (PET) tracer 2-deoxy-2-[18F]fluoro-D-glucose (FDG)
tion, one mechanism is to increase passive glucose transporter primarily targets Glut1 [9]. Although FDG is an effective
(Glut) expression [2,5]. Uncharacteristic Glut expression is now tumour probe [10], Glut1 is expressed in every type of tissue
implicated as a hallmark of not just cancer but also metabolic and this prevalence often results in false positive tests [11,12].
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Beilstein J. Org. Chem. 2013, 9, 2022–2027.
Unlike Glut1, the fructose-specific transporter Glut5 is overnight lyophilisation was required to isolate the product.
expressed in fewer tissues [2]. We are developing probes that Because the conditions are not easily integrated into a contin-
are selectively transported by Glut5. Using design principles uous process, we sought alternative approaches.
gleaned from the Holman group [13-15] as well as other fruc-
tose analogue research [16], we synthesized NBDM [17] and
demonstrated that this probe is transported into cancer cells
known to overexpress Glut5 and poorly transported into cells
known to express little Glut5 [2,5,18]. We demonstrated that the
transport is inhibited by fructose but not by other dietary sugars
(glucose, glucosamine) [17]. Furthering our understanding of
fluorescent probes like NBDM will expedite the development of
Glut5-specific PET compounds which could be non-invasive
tools for determining Glut5 expression in vivo and provide a
means for monitoring the onset and progression of metabolic
syndrome and aggressive cancers.
Scheme 1: Batch synthesis of NBDM. a) NaNO2, Amberlite IR-120 H+
(100 mL), 0 °C, water, 4 h. b) NH2OH·HCl, NaOAc, MeOH, rt, 6 h.
c) H2, Pd/C (10%), 4.4% formic acid, MeOH, rt, 10 h.
The promising initial results obtained with NBDM prompted us
to synthesize larger quantities of material. More NBDM is
required to examine uptake across many cell lines and with
access to amine 3, we can prepare analogues with different fluo- Our initial approach was to continue using a resin (Amberlite
rophores or other types of tags. Finally, access to more NBDM IRA-900) supporting nitrite in flow. While the use of supported
will enable assessment of probe photophysical properties as a reagents in flow is now well-established, the use of supported
function of concentration and in the presence of potential nitrite has not been widely adopted [20]. Amberlite IRA-900
quenchers. This increased understanding will be critical when was exchanged by eluting the chloride ion from the resin using
probing uptake into various biological systems where cell a 1 M solution of sodium nitrite until no more chloride was
staining techniques or supplemental amino acids are used. observed via an AgNO3 test. Once the exchange was complete,
Herein, we report an efficient flow synthesis of amine 3 that the column was washed with deionized water. The column was
enabled an increase in scale as well as a reduction in the time first assessed by flowing a 0.2 M aqueous solution of D-(+)-
needed to prepare NBDM. We also present fluorescent prop- glucosamine·HCl into a round bottom flask. Our initial experi-
erties of NBDM under conditions relevant to future cellular ment was performed at room temperature and took two days to
studies.
reach completion. Heating the reaction to 40, 60 or 70 °C
resulted in a clean acceleration in reaction rate.
Results and Discussion
Synthesis: The batch synthesis of 1-amino-2,5-anhydro-D- Although the supported nitrite approach at elevated tempera-
mannitol was reported by Claustre et al (Scheme 1). We used tures provided high yields of the desired product, the process
their basic approach, but improved throughput significantly was slow and throughput was restricted by the resin loading. In
[19]. The synthesis began with a Tiffeneau–Demjanov addition, attempts to increase concentration or use organic
rearrangement of glucosamine·HCl using an acidic resin and co-solvents further reduced the efficiency. Based on these prob-
NaNO2 to make nitrous acid in situ (Scheme 2). The original lems, we turned our attention to Tiffeneau–Demjanov condi-
conditions required neutralization by a basic resin. After rinsing tions using a catalytic amount of acid and sodium nitrite in
both resins, a dilute aqueous solution of 1 resulted and water. Flow conditions enabled the use of high concentrations
Scheme 2: The initial polymer-supported nitrite set up. A solution of glucosamine hydrochloride was passed over the resin into a round bottom flask
and then stirred with heating.
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Beilstein J. Org. Chem. 2013, 9, 2022–2027.
(1.0 M) and temperatures (100 °C) even though the reaction The hydrogenolysis of the oxime illustrates a flow chemistry
evolves large quantities of nitrogen gas. At a flow rate of challenge. The output from the oxime step contains excess
5 mL/min at 100 °C using a 10 mL reactor (2 min residence hydroxylamine and salts carried from the first step that
time), we achieved a throughput of 800 mg/min (Scheme 3). poisoned the packed-bed catalyst we screened. In theory,
Table 1 shows a comparison between previously reported batch continuous purification could remove these materials but
conditions and our continuous flow conditions and illustrates existing strategies do not enable removal of water soluble
that this first step has a throughput more than 63-fold higher byproducts from a water soluble product. Thus, we obtained
than the batch Tiffeneau–Demjanov conditions.
crude 2 by simple work up: neutralization, concentration,
precipitation of salts using tetrahydrofuran, filtration, concentra-
tion and dissolution in 10:1 ethanol/acetic acid. This solution
was converted to amine 3 using an H-Cube (commercially
available from Thales). With a flow rate of 1 mL/min at 100 bar
and 70 °C using a 10% Pd/C CatCart, a throughput of 8 mg/min
was achieved. The crude reaction solution was pure by NMR
and was used without further purification. As shown in Table 1,
the hydrogenolysis represents a bottleneck in the synthesis of
compound 3 because the throughput drops down to only 2-fold
enhancement relative to batch. We predict that this throughput
could be significantly improved if the wider range of catalysts
were screened.
Table 1: Batch versus flow comparison.
Time (h) Compound
Batch yield (g)
Flow yielda (g)
4
6
1
2
3
3.06
2.63
2.21
194.4
63.8
4.8
10
aFlow yield is based off of throughput for the equivalent amount of time
the batch conditions required. Throughput was estimated by the quan-
tity of starting material pumped through the reactor. No percent yields
were obtained because the crude mixtures could be used in the subse-
quent steps.
The concentrated Tiffeneau–Demjanov reaction not only The fructose analog probe NBDM was then produced by
increased the throughput but also enabled oxime formation combining the concentrated output from the H-Cube in satu-
without the removal of water. Using excess hydroxylamine rated sodium bicarbonate (0.4 M) with a 0.4 M solution of
hydrochloride (4 equiv) in methanol resulted in full conversion NBD-Cl. This step can be conducted in flow as well as in batch
to compound 2. At a total flow rate of 5 mL/min at 60 °C using with no significant difference in yield or productivity. The low
a 20 mL reactor (4 minutes residence time), we achieved a yield (20–30%) of NBDM may be the result of competitive re-
throughput of 177 mg/min (24-fold improvement relative to activity between the NBD-Cl and the hydroxy groups present
batch).
on 3. When 1 M solutions of sodium bicarbonate are used
Scheme 3: Continuous flow synthesis of the key intermediate 1-amino-2,5-anhydro-D-mannose (3).
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instead of saturated sodium bicarbonate, the resulting TLCs
indicate the formation of more byproducts and the lower
isolated yields of NBDM (<20%) also support this hypothesis.
We are confident that a completely continuous high-throughput
process to 3 could be realized with improvements in continuous
extraction techniques. That being said, the semi-continuous ap-
proach we have defined here results in significant improve-
ments compared to the original batch conditions.
Many fluorophores and biologically relevant tags have been
developed for conjugation to amines. For this reason, amine 3
was of particular interest. Likely, 3 will be a key branch-point
for the synthesis of numerous biologically active conjugates and
our improved production of 3 will provide significantly greater
quantities of conjugates. In particular, we can now produce
NBDM using this system in a single day which is a 3-fold
improvement relative to the original process. This rapid access
to more material enabled us to begin to assess the fluorescent
properties as a function of typical quenchers used in cell-
staining as well as intrinsic quenchers found in cells. Without
easy access to NBDM, the use of this compound in biological
experiments would supersede the investigation of its fluores-
cent properties.
Figure 1: Normalized NBDM absorption and emission, 40 µM and
2 µM.
Fluorescence: Fluorescence in biological systems is often
complicated by fluorophore quenching. Alexa fluorophores can
be quenched by certain amino acids and NBD is known to self-
quench at high concentrations [21,22]. Trypan blue is routinely
used to quench autofluorescence in confocal fluorescence
microscopy and fluorescence activated cell sorting (FACS)
[23,24], and dyes like Bromophenol Blue, Brilliant Blue R, and
Methylene Blue have been applied to colorimetric cytotoxicity
assays as well as in vitro staining [25-28].
Figure 2: NBDM fluorescence from 1–40 µM (PBS buffer). The data
set was plotted in OriginPro 8.6 and fitted using a self-quenching
model,
with direct weighting.
To better understand the behaviour of NBDM, we measured
fluorescence at various concentrations and in the presence of
various dyes, amino acids and sugars. The absorption and emis- curvature that exists suggests that only modest self-quenching
sion spectra are shown in Figure 1. The quenching experiments occurs (See Supporting Information File 1 for fitting data).
were carried out by measuring emission intensity at 546 nm
(ex. 472 nm) as a function of NBDM concentration or quencher Often multiple dyes are used to locate or identify cells by selec-
concentration. For each quenching experiment, 3–6 replicate tive staining. The fluorescence data for NBDM in the presence
fluorescence measurements were taken using a 96-well plate of four commonly used dyes is shown in Figure 3. These data
and a plate reader.
do not fit a simple Stern–Volmer relationship (see Supporting
Information File 1), which indicates that dynamic quenching is
In our initial publication describing the uptake of NBDM into not the only mechanism. The data do, however, fit well to a
MCF-7 cells, we measured uptake as a function of concentra- composite Stern–Volmer model that accounts for both dynamic
tion over a range of 1–40 μM (Figure 2) [17]. While we did not and static quenching.
expect to observe significant self-quenching over this range, we
measured the fluorescent intensity of NBDM from 1–40 μM Free amino acids at high concentrations can also quench fluo-
(1X phosphate buffer solution). As expected, NBDM does not rophores. To access the propensity for NBDM to be quenched
exhibit significant self-quenching over this range. The slight by amino acids, we measured fluorescence in the presence of
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Beilstein J. Org. Chem. 2013, 9, 2022–2027.
Carbohydrate–carbohydrate and carbohydrate–aromatic ring
interactions are well-known [29,30]. Based on these interac-
tions, we hypothesized that abnormal fluorescence behavior
may be exhibited at high carbohydrate concentrations. This
issue is significant because sugar uptake studies are often
performed in the presences of added non-labeled sugars such as
glucose, fructose or glucosamine. To assess our hypothesis, we
measured the fluorescence of NBDM in the presence of
glucose, fructose and glucosamine. No NBDM quenching was
observed even at sugar concentrations as high as 100 mM.
Conclusion
In conclusion, we report the flow synthesis of the fluorescent
fructose mimic NBDM. While we demonstrated for the
first time that resin-supported nitrite ions can facilitate Tiffe-
neau–Demjanov rearrangements, we found that solution phase
rearrangements were superior resulting in throughput gains
>63-fold relative to batch conditions. We also demonstrated that
the output of the Tiffeneau–Demjanov rearrangement reactor
could be telescoped into the oxime reactions. The oxime reac-
tion was very efficient exhibiting throughput gains as high as
24-fold over batch. In addition, we identified extraction and
Figure 3: Comparison of quenching 2 µM NBDM, as measured by
fluorescence intensity of Trypan Blue, Bromophenol Blue, Brilliant
Blue R, and Methylene Blue. Each data set was plotted in OriginPro
8.6 and fitted using a polynomial fit (order = 2) with direct weighting.
varying concentrations of alanine, glutamine, lysine, tyrosine, hydrogenation bottlenecks. Despite these limitations, the syn-
methionine and histidine. As expected, the amino acids lacking thesis of NBDM can now be achieved in one day as opposed to
functionality known to quench fluorophores (alanine, gluta- three days in batch. The access to larger quantities to NBDM
mine and lysine) did not quench NBDM at concentrations as enabled us to assess the probe’s quenching properties as a func-
high as 50 mM. Interestingly, tyrosine did not quench NBDM tion of concentration and in the presence of various quenchers.
fluorescence even at concentrations as high as 2 mM (solubility These data are critical for future uptake studies that use
limit for tyrosine). Methionine and histidine, however, did confocal fluorescence microscopy or FACS strategies.
quench NBDM via a dynamic mechanism (Figure 4).
Supporting Information
Supporting Information File 1
Experimental part.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-9-238-S1.pdf]
Acknowledgements
Financial support from the Max Planck Society and the
National Science Foundation (USA; CHE-1152020) is grate-
fully acknowledged.
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