A Palette of Minimally Tagged Sucrose Analogues for Real-Time Raman Imaging of Intracellular Plant Metabolism

Article abstract of DOI:10.1002/anie.202016802

Sucrose is the main saccharide used for long-distance transport in plants and plays an essential role in energy metabolism; however, there are no analogues for real-time imaging in live cells. We have optimised a synthetic approach to prepare sucrose analogues including very small (≈50 Da or less) Raman tags in the fructose moiety. Spectroscopic analysis identified the alkyne-tagged compound 6 as a sucrose analogue recognised by endogenous transporters in live cells and with higher Raman intensity than other sucrose derivatives. Herein, we demonstrate the application of compound 6 as the first optical probe to visualise real-time uptake and intracellular localisation of sucrose in live plant cells using Raman microscopy.

Full text of DOI:10.1002/anie.202016802

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How to cite: Angew. Chem. Int. Ed. 2021, 60, 7637–7642
International Edition: doi.org/10.1002/anie.202016802
German Edition: doi.org/10.1002/ange.202016802
Imaging Agents
A Palette of Minimally Tagged Sucrose Analogues for Real-Time
Raman Imaging of Intracellular Plant Metabolism
Fabio de Moliner, Kirsten Knox, Doireann Gordon, Martin Lee, William J. Tipping,
Ailsa Geddis, Anke Reinders, John M. Ward, Karl Oparka, and Marc Vendrell*
Abstract: Sucrose is the main saccharide used for long-
distance transport in plants and plays an essential role in energy
metabolism; however, there are no analogues for real-time
imaging in live cells. We have optimised a synthetic approach to
prepare sucrose analogues including very small ( ꢀ 50 Da or
less) Raman tags in the fructose moiety. Spectroscopic analysis
identified the alkyne-tagged compound 6 as a sucrose analogue
recognised by endogenous transporters in live cells and with
higher Raman intensity than other sucrose derivatives. Herein,
we demonstrate the application of compound 6 as the first
optical probe to visualise real-time uptake and intracellular
localisation of sucrose in live plant cells using Raman
microscopy.
sucrose are essential processes in plant metabolism, there are
no optical probes that can image its cell uptake and local-
isation with high spatiotemporal resolution.
Several different techniques have been employed to
investigate the distribution of sucrose in plants.[7] HPLC-MS
is commonly used but limited by the lack of spatial resolution
and its destructive nature.[8] Assays with sucrose bearing
3
radioactive isotopes (e.g., 14C and H)[9] suffer from limited
spatial and temporal resolution as well as intrinsic drawbacks
including the need for safety precautions. Coinciding with the
emergence of optical probes as inexpensive tools for the non-
invasive monitoring of biological events,[10] sucrose analogues
including fluorescent labels have been recently reported.[11]
These molecules recapitulate some biological features of the
native metabolites, but their large size hampers their appli-
cation for monitoring intracellular localisation and metabo-
lism in live cells.
S
ucrose metabolism is one of the main processes that
regulates the development, growth and functioning of higher
plants.[1] However, imaging studies of sucrose metabolism are
scarce compared to other saccharides, such as glucose,
fructose, sialic acid or lignin components.[2] Sucrose is used
for energy storage in plant cells[3] and for the long-distance
transport of carbon fixed during photosynthesis.[4] As a result,
sucrose is the most abundant sugar in plants, with concen-
trations in the phloem around 100 mM,[5] and is pivotal in
energy metabolism, signalling and gene expression.[6]
Although the mobilisation and intracellular trafficking of
Stimulated Raman scattering (SRS) enables the detection
of chemical groups in their native environment.[12] The
increased sensitivity of SRS has been used to visualise
metabolites and small-molecule drugs in live cells and
plants.[13] Among others, SRS imaging has been reported to
study glucose[2c,14] and cholesterol[15] metabolism, protein
synthesis and degradation,[16] DNA synthesis,[17] lipid dynam-
ics in cell membranes[18] and the chemical composition of
bacterial cell surfaces (Figure 1).[19] Furthermore, chemical
labels for SRS are smaller than fluorophores, causing minimal
disruption in the molecular recognition properties of native
metabolites. Given the capabilities of SRS imaging for
monitoring the distribution of small metabolites in live cells,
we have designed a chemical strategy to synthesize Raman-
active sucrose analogues for direct metabolic imaging in live
plant cells. Herein we report a collection of derivatives
including very small vibrational tags (Figure 1), obtained with
good yields in three synthetic steps. Following their spectro-
scopic evaluation for biological SRS imaging, we have
characterised the transport of an alkyne-tagged sucrose in
genetically modified yeast cells. Finally, we have used our
alkyne-tagged sucrose to image for the first time how sucrose
translocates in live plant cells.
[*] Dr. F. de Moliner, D. Gordon, A. Geddis, Prof. M. Vendrell
Centre for Inflammation Research, The University of Edinburgh (UK)
E-mail: marc.vendrell@ed.ac.uk
Dr. K. Knox, Prof. K. Oparka
Institute of Molecular Plant Sciences, The University of Edinburgh
(UK)
Dr. M. Lee
Cancer Research (UK) Edinburgh Centre, The University of Edin-
burgh (UK)
Dr. W. J. Tipping, A. Geddis
EaStCHEM School of Chemistry, The University of Edinburgh (UK)
Dr. W. J. Tipping
Centre for Molecular Nanometrology, University of Strathclyde (UK)
Dr. A. Reinders, Prof. J. M. Ward
Department of Plant and Microbial Biology, University of Minnesota
(USA)
Label-free SRS imaging can resolve cellular structures by
exploiting inherent vibrational groups of target molecules.[20]
Alternatively, chemical tags can be incorporated to improve
detectability and sensitivity. Groups vibrating within the
silent window of the Raman spectrum (i.e., from 2000 to
2500 cmÀ1) are optimal to minimise background interference
from cellular components (e.g., proteins, nucleic acids, fatty
acids).[21] Different chemical groups (e.g., alkynes[22] and
nitriles[23]) have been reported for Raman imaging (Figure 1).
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202016802.
ꢀ 2021 The Authors. Angewandte Chemie International Edition
published by Wiley-VCH GmbH. This is an open access article under
the terms of the Creative Commons Attribution License, which
permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
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which has been reported for successful sucrose functionalisa-
tion,[26,27] and also a single protecting group (i.e., benzoyl)
orthogonal to the different Raman tags. First, we activated the
free hydroxyl group in compound 1 as a triflate to render
common reactive intermediate 2. Nucleophilic substitution
with sodium azide and final deprotection in basic media
afforded the azido-functionalised sucrose 4 with good overall
yields (> 50% over 3 steps). Next, we investigated the
installation of an alkyne tag. Initially, we evaluated acetylide
as the smallest alkyne group, but its introduction proved
unfeasible. In fact, trimethylsilyl acetylene failed to react with
the activated triflate 2, both under Sonogashira coupling
conditions and after treatment with a strong base. Attempts to
generate the alkyne by homologation with the Ohira-Best-
mann reagent were also unsuccessful (Supplementary
Figure 1). Therefore, we employed propargylamine as an
alkyne-containing nucleophile using an activation/substitu-
tion/deprotection sequence to obtain the sucrose analogue 6.
Finally, the synthesis of a nitrile-functionalised sucrose
analogue was designed. Nucleophilic substitutions between
the triflate intermediate 2 and sources of cyanide anion (e.g.,
trimethylsilyl cyanide, potassium cyanide) were attempted
but unsuccessful (Supplementary Figure 2). Alternatively, we
oxidised compound 1 with Dess–Martin periodinane and the
resulting aldehyde 7 was reacted with hydroxylamine to
produce the oxime 8. The dehydration step afforded the
protected nitrile-functionalised sucrose 9 but the final depro-
tection under different reaction conditions did not afford
compound 10 due to the poor stability of the tertiary nitrile
group (Supplementary Table 1). Therefore, we included
a short spacer between sucrose and the nitrile tag by reaction
between the reactive intermediate 2 and the commercial
aminopropionitrile. Importantly, this strategy afforded the
nitrile 12 in high purity upon deprotection of the benzoyl
groups.
Figure 1. Previous and current work for labelling of small metabolites
using vibrational tags for SRS imaging of intracellular metabolism.
However, none of these have been reported for the deriva-
tisation of sucrose or for in situ metabolic imaging in live
plants. In this work, we synthesized a family of sucrose
analogues with different bioorthogonal vibrational tags that
could retain the properties of sucrose and be used to image
intracellular mobilisation under physiological conditions by
SRS microscopy.
Selective transformations involving one single position of
a saccharide are a major challenge in carbohydrate chemis-
try[24] because of the presence of multiple hydroxyl groups
with similar reactivity.[25] Bearing this in mind, we designed
a general synthetic approach to introduce different chemical
labels (e.g., azides, alkynes, nitriles) into sucrose with the
minimal number of synthetic steps (Scheme 1). For this
purpose, the identification of a single and accessible common
precursor was crucial. We selected the perbenzoylated com-
pound 1 as a suitable starting material because it possesses
a free hydroxyl group in the position 1 of the fructose moiety,
Before measuring the Raman properties of our sucrose
analogues [i.e., compound 4 (azide), compound 6 (alkyne)
and compound 12 (nitrile)], we acquired the Raman spectrum
of pellets from the widely used tobacco BY-2 plant cells.[28] As
shown in Figure 2, the spectrum of BY-2 cells featured peaks
found in many macromolecules (i.e., 1000 cmÀ1 for phenyl-
alanine ring breathing, 1655 cmÀ1 for amide C O stretching
=
Scheme 1. General synthetic strategy for the synthesis of Raman-active sucrose analogues.
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cells (SEY6210) that expressed sucrose transporters from
different plant species (i.e., StSUT1, OsSUT1 and
AtSUC2).[31] For this purpose, we optimised an optical assay
to measure the amount of compound 6 that had been
internalised by each yeast line via derivatisation with the
fluorogenic 3-azido-7-hydroxycoumarin, which produces
bright fluorescence emission around 460 nm after the cyclo-
addition reaction (Figure 3a). This assay allowed us to rapidly
compare the uptake of compound 6 through different trans-
porters without the need for specialised equipment as in
radioactivity experiments.[9b,32] As shown in Figure 3b, we
observed much higher uptake of compound 6 in cells that
expressed OsSUT1 and StSUT1 transporters over those
expressing AtSUC2 transporters. Experiments in the same
yeast cells transformed with the vector control pDR196 but
lacking any SUT1 transporters instead showed very little
uptake of compound 6 (Supplementary Figure 5). Whereas
these results do not exclude the possibility that other trans-
Figure 2. a) Schematic illustration of a plant cell and Raman spectrum
of BY2 cells. b) Representative Raman spectra (3 independent experi-
ments) of sucrose analogues (4, 6, 12 and 13) in water. Wavenumber
and maximum intensity of the highest Raman peaks are highlighted in
black and red, respectively. c) Summary of physicochemical properties
of native sucrose and the synthesized sucrose analogues. *Differences
in molecular weight between the synthesized analogues and native
sucrose.
porters found in plant cells (e.g., SWEET transporters[33]
)
may recognise compound 6, they show that alkyne-modified
sucrose can be taken up by type I (StSUT1) as well as type II
(OsSUT1) transporters. To the best of our knowledge,
compound 6 represents the first optical probe to monitor
sucrose uptake in monocots (e.g., rice, maize, wheat), which
express type II sucrose transporters. Furthermore, we ana-
lysed the degradation and functionality of probe 6 by
measuring reactivity against sucrose invertase and cellular
influx of Ca2+ induced by sucrose uptake. Our results show
that probe 6 is only partially degraded by sucrose invertase
and 2845 cmÀ1 for C H stretching from lipids and proteins)
À
and a silent region between 2000 cmÀ1 and 2500 cmÀ1 (Fig-
ure 2a). This observation confirmed the utility of Raman
microscopy for detecting specific labels in plant cells with
minimal background signal and maximum sensitivity. Next,
we acquired the spectra of all sucrose analogues and
compared them to the commercially available deuterated
sucrose (13, Figure 2b). We acquired the spontaneous Raman
spectra of all derivatives in water and under the same
experimental conditions to compare the wavenumbers and
relative intensities of their Raman peaks. Notably, all four
compounds displayed detectable peaks within the silent
region (Figure 2b), which were not present in native sucrose
(Supplementary Figure 3). Deuterated sucrose showed
a broad peak around 2115 cmÀ1, consistent with the literature
data for carbon-deuterium bonds.[29] The azide derivative 4
showed a weak peak at 2113 cmÀ1, likely due to the reduced
polarizability of the nitrogen-nitrogen triple bonds.[30] The
Raman peak for the nitrile-functionalised sucrose 12 was
observed around 2250 cmÀ1
. Finally, the alkyne-tagged
sucrose 6 displayed a strong Raman peak at 2116 cmÀ1,
showing the highest intensity among all analogues. Com-
pound 6 also showed a lower limit of detection in water
(29 mM) than the commercial compound 13, which is
> 100 mM (Figure 2c and Supplementary Figure 4). Given
these results and the chemical similarities between compound
6 and native sucrose [i.e., 342 Da vs. 379 Da (6), clogP À3.75
vs. clogP À3.52 (6), Figure 2c], we proceeded to investigate
its application as an optical probe for imaging sucrose uptake
and intracellular localisation in live cells.
Figure 3. a) Schematic cell-based assay for measuring sucrose uptake
in yeast mutants. 5ꢁ105 cells were incubated with compound 6 for 2 h
at 308C, then centrifuged to obtain cell pellets that were incubated for
20 min with 3-azido-7-hydroxycoumarin (AHC, 1 mM), THPTA (1 mM),
CuSO4 (10 mM) and sodium ascorbate (10 mM). b) Fluorescence
analysis (lexc =400 nm, lem =460 nm) of cell pellets containing SUC2
or SUT1 sucrose transporters after incubation or not with compound 6
(100 mM) and subsequent reaction with AHC. Fold increase relative to
the vector control lacking sucrose transporters presented as
meansÆSD (n=3). P values from one-way ANOVA with multiple
comparisons.
In order to study the uptake of the alkyne-tagged sucrose
analogue 6 in live cells, we utilised genetically modified yeast
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(Supplementary Figure 6) and that its transport leads to
increased levels of intracellular Ca2+, consistently with
previous reports[34] (Supplementary Figure 7).
We finally examined the utility of compound 6 for real-
time imaging of sucrose trafficking in live plant cells. For these
experiments, we incubated BY2 cells with compound 6 before
performing SRS imaging in a multi-photon microscope.
compound 6 to the vacuoles, as shown in Supplementary
Figure 9 and Supplementary Movies 1–2.
Furthermore, BY2 cells are connected to each other
through selective channels called plasmodesmata (PD). Our
SRS images indicate that compound 6 could rapidly trans-
locate across PD-connected cells, like native sucrose does.
This is suggested in Figure 4a, where compound 6 appears to
diffuse from cell 2 to cells 1 and 3, and where the PD-linked
cells 4–7 sequentially display bright intracellular Raman
signals between 1205 and 1520 seconds. Altogether, these
results validate the alkyne-tagged compound 6 as the first
optical probe to visualise in situ plant sucrose transporters
that are prevalent in many crop species in real time.
In summary, we report the first Raman-active sucrose
analogue and its application for real-time imaging of meta-
bolic uptake in live plant cells. We have optimised an efficient
synthetic approach for the generic preparation of sucrose
derivatives including a variety of minimal vibrational tags
(e.g., alkyne, azide, nitrile) providing readouts within the
silent window of the Raman spectrum. Among these, the
alkyne-containing compound 6 showed strong Raman peaks,
with remarkably high intensity and capacity to be recognised
Compound
6 showed no cytotoxicity (Supplementary
Figure 8) and time-lapse images were acquired over 30
minutes with simultaneous monitoring of the alkyne-tagged
compound 6 (2116 cmÀ1) and label-free Raman signatures of
plant cells (2930 and 2855 cmÀ1 for CH3 and CH2 groups,
respectively) (Figure 4). We observed that compound 6 was
gradually taken up by each cell at a time, with sucrose being
initially localised in the cytoplasm. After 30 minutes, all BY2
cells had taken compound 6 up and equilibrated the intra-
cellular sucrose concentration with that found in the media
(i.e., 125 mM). Notably, most cells displayed uniform signal
intensity throughout the cell, including the vacuoles. This
observation indicates that compound 6 can enter the vacuoles,
where plant cells store excess sucrose. Competition experi-
ments with sucrose remarkably reduced the transport of
Figure 4. Representative live-cell SRS images (from 3 independent experiments) of BY2 cells showing the uptake of the alkyne-tagged compound
6 in real time. a) Time-lapse (time in seconds) pseudo-color Raman images (2116 cmÀ1) of BY2 cells upon incubation with compound 6. Squares
defining 7 different regions of interest to monitor the uptake and accumulation of sucrose in cells. Scale bar=50 mm. b) Label-free SRS images at
2930 cmÀ1 (CH3 groups) and 2855 cmÀ1 (CH2 groups) highlighting the structures of BY2 cells, with vacuoles (red v) that confine the cytoplasm
and organelles in the vicinity of the nucleus (yellow arrows). Scale bar=50 mm. c) Time-course Raman intensity in different cells [ROIs in (a)]
showing likely intercellular translocation across plasmodesmata (PD). Scale bar=50 mm.
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The authors acknowledge funding from the Biotechnology
and Biological Sciences Research Council (BB/M025160/1),
the Medical Research Council (MR/R01566X/1), Cancer
Research UK (C157/A25140 and C157/A15703), the Office of
Basic Energy Sciences of the U.S. Department of Energy
(DE-FG02-07ER15886) and the University of Strathclyde.
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(771443).
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Conflict of interest
The authors declare no conflict of interest.
Keywords: imaging · probes · stimulated Raman scattering ·
sugars · transport
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Manuscript received: December 18, 2020
Accepted manuscript online: January 25, 2021
Version of record online: February 26, 2021
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