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Late-stage functionalisation of alkyne-modiﬁed
phospha-xanthene dyes: lysosomal imaging using
an oﬀ–on–oﬀ type of pH probe†
Hiroaki Ogasawara, ‡a Yoshiki Tanaka,‡a Masayasu Takiand Shigehiro Yamaguchi *ab*b

Near-infrared (NIR) ﬂuorescent molecules are of great importance for the visualisation of biologicalprocesses. Among the most promising dye scaﬀolds for this purpose are P]O-substituted phosphaxanthene (POX) dyes, which show NIR emission with high photostability. Their practical utility for in vitro
and in vivo imaging has recently been demonstrated. Although classical modiﬁcation methods have beenused to produce POX-based ﬂuorescent probes, it is still a challenge to introduce additional functionalgroups to control the localisation of the probe in cells. Herein, we report on the development of POXs
that bear a 4-ethynylphenyl group on the phosphorus atom. These dyes can subsequently be
Received 25th March 2021
Accepted 30th April 2021

functionalised with azide-tagged biomolecules via a late-stage Cu-catalysed azide/alkyne cycloaddition(CuAAC) reaction, thus achieving target-selective labelling. To demonstrate the practical utility of the

DOI: 10.1039/d1sc01705efunctionalised POXs, we designed a sophisticated NIR probe that exhibits a bell-shaped oﬀ–on–oﬀ pH-

rsc.li/chemical-science

response and is able to assess the degree of endosomal maturation.

Introduction
The visualisation of specic cellular events at an organelle levelcan provide valuable information that aids in the understanding of complicated biological systems. Small-moleculebased uorescent probes, which exhibit stimuli-induced uorescence switching, are important chemical tools in the eld ofuorescence imaging. They enable the acquisition of multidimensional images of biological functions occurring in living
cells with a high spatiotemporal resolution.1–6 Fluorescentprobes that have absorption and emission bands in the far-red
to near-infrared (NIR) region are particularly useful because
they exhibit negligible light-induced photodamage, show low
interference with autouorescence, and engage in minimal
cross-talk with other dyes and uorescent proteins.7,8 Accordingly, various NIR uorescent probes based on uorophores
such as cyanine, aza-BODIPY, and xanthene have been developed.9–14 In most cases, these probes can be localised,depending on the intrinsic properties of the uorophore, to onea

Department of Chemistry, Graduate School of Science, Integrated ResearchConsortium on Chemical Sciences (IRCCS), Nagoya University, Furo, Chikusa,
Nagoya 464-8602, Japan. E-mail: yamaguchi@chem.nagoya-u.ac.jpbInstitute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo,
Chikusa, Nagoya 464-8601, Japan. E-mail: taki@itbm.nagoya-u.ac.jp† Electronic supplementary information (ESI) available: Experimental details,
photophysical properties, theoretical calculations, determination of the aciddissociation constant, cell imaging and NMR spectra for all new compounds.
See DOI: 10.1039/d1sc01705e‡ These authors contributed equally to this work.

7902 | Chem. Sci., 2021, 12, 7902–7907

or more organelles in a cell. For instance, anionic dyes such asuorescein diﬀuse into the cytosol, while cationic uorophores
such as rhodamine and cyanine tend to accumulate in the
mitochondria.15–17 However, due to a low synthetic diversity in
NIR uorophores, it is still a challenge to develop biocompatible NIR probes with specic target localisation. This isa particular problem for those that use multiple uorescentswitching units for analysing complex and cooperative biological events.18,19 The ideal probe-design is one that allows theintroduction of various organelle-targeting groups into a uorophore skeleton during the later stages of synthesis without
any perturbation of its properties, especially its sensing capabilities. To meet the aforementioned requirements, phosphaxanthene uorophores shown in Fig. 1a, which have beendeveloped as a new class of NIR dyes, are potentially suitable
candidates.20–29 These dyes replace the endocyclic oxygen atomof the classical xanthene scaﬀold with an electron-withdrawing
phosphine oxide (>P(]O)R) moiety. Phospha-xanthene dyes
have been used as NIR-labelling reagents in both in vitro and invivo optical imaging.22,27,29 Moreover, following molecular
design strategies that have been established for classicalxanthene dyes, several phospha-xanthene-based NIR uorescent probes for the detection of H2O2,30 Cu+,31 Ca2+,32,33 pHchanges,34 and membrane potentials35 have been reported. In
this context, we have successfully developed a series of >P(]O)
Ph-substituted dyes, POXs (Fig. 1a), including phosphauoresceins (POFs),20 phospha-rhodols (PORLs),21 andphospha-rhodamines (PORs).22,23 These POXs exhibit highbrightness with absorption and emission maxima (labs/lem) at© 2021 The Author(s). Published by the Royal Society of ChemistryView Article Online

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the highly acidic luminal pH in the lysosomes. As a showcase of
the practical utility of this probe, we have successfully visualised
the luminal pH heterogeneity during endosome maturation inliving cells.

Results and discussion
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Synthesis of alkyne modied phospha-xanthene (POX) dyes

Fig. 1 (a) Functionalisation of phospha-xanthene ﬂuorophores shownin a dashed box. A phenyl ring on the phosphorus atom of the parent
POX dyes is modiﬁed with an ethynyl group. The alkynylated phosphaxanthene dyes thus obtained can be converted into the corresponding
triazole compounds via a copper-catalysed azide–alkyne cycloaddition (CuAAC) at the late stage of synthesis. (b) POX-based organellemarkers obtained from alkynylated-POXs (POX-CCHs) and azide
compounds used in this work.

633/660, 670/700, and 712/740 nm in aqueous solution at pH
7.4, respectively. We have also shown that they exhibit exceptionally high stability to photo-irradiation, arising from theelectron-withdrawing character of the P]O group, anddemonstrated their practical utility for the long-term imaging of
biological events.22 However, even in these POXs, as with theclassical xanthene dyes, the modiable sites are limited to the 9aryl group and/or the O- and N-termini.36,37 Once these positions
have been functionalised to become the sensing sites, it isdiﬃcult to subsequently introduce an additional functionalgroup to enable control over the intracellular localisation.
To solve this problem, we propose a new series of NIR
phospha-xanthene dyes, in which a 4-ethynylphenyl group is
installed on the phosphorus atom (Fig. 1a). The terminal alkyne
can be readily modied with a variety of azide-containing
molecules of interest at the late stage of synthesis via
a copper-catalysed azide–alkyne cycloaddition (CuAAC) without
perturbation of the photophysical properties.38–40 We herein
report the development of alkynylated POX dyes, POX-CCHs,
and their functionalisation by CuAAC with various azide
compounds (Fig. 1b). Regardless of the type of xanthene dyeused, the subcellular localisation of the POX dyes can be
precisely controlled using organelle-targeting labelling groups.
Moreover, we have designed a NIR emissive pH probe with two
pKa values which exhibits an “oﬀ–on–oﬀ” uorescence responsewhen exposed to a change from neutral pH in the medium to© 2021 The Author(s). Published by the Royal Society of Chemistry

A method for the synthesis of the POX-CCH dyes is shown in
Scheme 1, where P-ethynylphenyl-substituted phosphaxanthone 5 is a common key intermediate. This compound
was readily synthesised from (4-bromophenyl)triisopropylsilylacetylene (1) in four steps. The lithiation of 1 with tBuLi, followedbytreatmentwithbis(diisopropylamino)
chlorophosphine produced phosphanylphenylacetylene 2.
Subsequent treatment of the crude mixture with dry HCl gas
yielded moisture-sensitive dichloroarylphosphine 3, which wasused without purication in the next reaction with dilithiatedbis(4-diethylamino-2-bromophenyl)methane. Further oxidation
with an aqueous solution of H2O2 aﬀorded P]O-bridged diarylmethane 4 in 54% overall yield over three steps. The benzylic
position was then oxidised under an oxygen atmosphere ina basic solution to give phospha-xanthone 5. A nucleophilic
addition of 2,6-dimethoxyphenyllithium, followed by treatment
with an aqueous solution of HCl aﬀorded the corresponding
silyl-protected phospha-rhodamine, which was then deprotected with CsF to give POR-CCH. Importantly, POR-CCH could
be transformed into the corresponding rhodol PORL-CCH and

Scheme 1 Synthesis of POX-CCHs and POX-SO3Hs. Reagents andconditions: (a) for POF-CCH: 1 M NaOH, H2O/CH3OH (1 : 1, v/v), 72 h;
for PORL-CCH: 0.1 M NaOH, H2O/CH3OH (9 : 1, v/v), 3 h. TIPS ¼
triisopropylsilyl. THPTA ¼ tris[(hydroxypropyl-1H-1,2,3-triazol-4-yl)
methyl]amine.

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Chemical Scienceuorescein POF-CCH derivatives via a selective hydrolysis, thecourse of which depends on precise control over the basic
conditions, as we have reported previously.21

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Functionalisation of alkyne modied POX dyes by CuAAC
As an example of the functionalisation of the POX-CCH dyes, we
conjugated them with 3-azide-1-propanesulfonic acid by CuAAC
in the presence of CuSO4, tris[(hydroxypropyl-1H-1,2,3-triazol-4yl)methyl]amine (THPTA), and sodium ascorbate. The photophysical properties of the obtained POX-SO3H molecules are
summarised in Table S1,† together with those of the parent
POXs. Regardless of whether the triazole ring is present at thepara position of the phenyl group or not, the absorption andemission spectra are almost identical under physiologicalconditions (50 mM HEPES, pH 7.4). This result indicates thatthe substituents on the P-phenyl group do not perturb theelectronic structures of the phospha-xanthene skeletons. Thisnding was also supported by theoretical calculations on a triazole-derivative of POR-CCH, where the triazole ring has nodirect participation in either the HOMO or the LUMO of thiscompound (Fig. S4†). It should be noted that the excellent
photostabilities of the parent POXs are retained even aer
conversion into the triazole derivatives (Fig. S5†).21,22
With the intention of labelling lysosomes, mitochondria,
and cell nuclei, we synthesised a series of POX-Z dyes functionalised with the organelle-targeting groups such as morpholine (Mor), triphenylphosphonium (TPP), or bis-benzimide(Hoechst) (Fig. 1b). The imaging results obtained by using POFTPP, PORL-Mor, and POR-Hoechst are shown in Fig. 2, together
with those observed with the corresponding parent POXs. The
staining patterns with functionalised POX-Z dyes show selective
labelling of the target organelles (Fig. S17 and S18†), while theparent POX dyes diﬀuse or non-specically accumulate in thecells. This reveals that, regardless of the inherent properties ofthe uorophores, the subcellular localization of the dyes can benely controlled by the functional groups introduced on the Pphenyl moiety. These results highlight the eﬃcacy of late-stage
functionalisation of the P-phenyl group for the development ofFig. 2 Fluorescence images of A431 cells stained with the original
POXs (top) and with the POX-Zs functionalised with TPP, Mor, or
Hoechst (bottom). POF, PORL, and POR are shown in yellow, magenta,
and red, respectively; scale bar ¼ 20 mm.

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a variety of POX-based uorescent probes to monitor biologicalevents at the subcellular level.

Design and photophysical properties of the oﬀ–on–oﬀ type ofpH probe
To achieve a detailed examination of biological phenomena, it
is desirable to furnish NIR-uorescent probes with a complex
uorescence response, such as an “oﬀ–on–oﬀ” switching.
However, in contrast to simple turn-on “oﬀ–on” probes, suchuorescent probes require a more sophisticated molecular
design based on a multiple switching mechanism. This is hardto achieve due to the limited number of modiable sites inordinary xanthene skeletons. Indeed, although several types of
bio-conjugate probe for monitoring pH changes in target
subcellular regions have been reported, most of them are of the“oﬀ–on” or ratiometric type.3,41–43 While several pH sensors
show an oﬀ–on–oﬀ response,44–47 they have critical limitations,such as an unsuitable pH sensing range, the need for UV-light
excitation, and the lack of a site suitable for conjugation.
Therefore, it remains a great challenge to develop pH-sensitive
uorescent sensors that satisfy all the aforementioned
requirements. To solve this problem, we designed Et2NPOFCCH, based on the POF uorophore skeleton, with two uorescence switching sites (Fig. 3a). In this system, the 9-arylgroup, which contains a diethylamino (Et2N) moiety, acts as anelectron donor in a photo-induced electron transfer (PET)mechanism, resulting in uorescence quenching over a highpH range that prevents protonation of the Et2N group. Theuorescein OH group serves as the other site capable ofswitching in response to a pH change. Namely, the deprotonated POF exhibits an absorption band around 630 nm. When
this band is excited, only the species in which the Et2N group isprotonated and the uorescein OH group is deprotonated canuoresce at 660 nm. As the pH decreases, no uorescence isobserved following excitation around 630 nm, since theprotonated POF only has an absorption band around 480 nm.
Thus, the combination of the PET mechanism with the pHdependence of the uorescein skeleton furnishes an “oﬀ–on–oﬀ” uorescence response upon pH changes (Fig. 3b).
To conrm the pH responsiveness of this uorophoresystem, Et2NPOF, a compound without an alkyne group was
examined (Fig. 3a). As expected, upon excitation at 633 nm, bothan acidic (pH 3.7) and a neutral (pH 7.4) solution of this modelcompound showed weak uorescence (Fig. 3c), while a 14-foldincrease in emission intensity was observed at pH 5.5. The
presence of biological nucleophiles such as glutathione in
the mM range did not interfere with the pH response of Et2NPOF (Fig. S6†). The microscopic and macroscopic protonation
equilibria of the amino and hydroxy groups of Et2NPOF are
shown in Fig. 3d, where RH2+, RH, and R represent the cation(Form I), neutral species (Forms II and III), and anion (Form IV),
respectively. In prototropic equilibria, Form II is the dominantemissive species when excited at 633 nm, while the fully
protonated Form I also uoresces following excitation at around
480 nm (right side of Fig. 3b). Using UV-vis absorption (Fig. S7†)and uorescence (Fig. S8†) spectroscopic titration experiments© 2021 The Author(s). Published by the Royal Society of ChemistryView Article Online

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Chemical ScienceFig. 3 (a) Chemical structure of Et2NPOF-CCH and Et2NPOF. (b) Emissive protonated forms of the probe capable of being excited at 630 nm(left) and 480 nm (right). (c) Absorption and emission spectra (lex ¼ 633 nm) of Et2NPOF in pH-buﬀered solutions at pH 7.4 (blue), 5.5 (red), and3.7 (orange). (d) Macroscopic and microscopic protonation equilibria for Et2NPOF. KT is the equilibrium constant of tautomerisation. (e) A plot of
ﬂuorescence intensity at 660 nm (lex ¼ 633 nm) as a function of the pH value. The solid line is the result of curve ﬁtting using the following pKa
values: pK11 ¼ 5.45, pK12 ¼ 5.05, pK21 ¼ 5.57, and pK22 ¼ 5.96. (f) Calculated species distributions as a function of the pH value.

and nonlinear curve tting of the titration data (for details, see
the ESI†), the four acid dissociation constants (microconstants)
for the prototropic equilibria were determined to be pK11 ¼
5.45, pK12 ¼ 5.05, pK21 ¼ 5.57, and pK22 ¼ 5.96. Based on thesevalues, macroconstants (Ka) and the tautomeric equilibrium
constant (KT) were determined to be pKa1 ¼ 4.91 (Ka1 ¼ K11 +K12), pKa2 ¼ 6.11 (1/Ka2 ¼ 1/K21 + 1/K22), and KT ¼ 0.40 (Table
S4†). Fig. 3e shows a plot of the uorescence intensity upon
excitation at 633 nm as a function of the pH value. The uorescence intensity reaches its maximum at pH 5.5, implying that
Et2NPOF is suitable for detecting physiological pH changes ina range between 4.5 and 6.5. Based on the microconstants, we
calculated the species distribution at each pH value (Fig. 3f) andfound that 19% of the total amount of Et2NPOF is present as theemissive Form II at pH 5.5. It is of note that, owing to the ample
uorescence quantum yield of Form I (FF ¼ 0.19 at pH 3), Et2NPOF would be a potential scaﬀold for the ratiometricmeasurement of pH changes in a dual excitation mode upon
excitation at 488 nm and at the isosbestic point of 534 nm(Fig. 3c and S9†).
Lysosomal imaging using an oﬀ–on–oﬀ type of pH probe
With the sensing capabilities of Et2NPOF-CCH established,dextran, a large-mass biomolecule, was introduced to the oﬀ–
on–oﬀ pH probe via the late-stage functionalisation of the Pphenyl group. This probe was employed to visualise the pHchanges in the cellular acidic compartments formed by© 2021 The Author(s). Published by the Royal Society of Chemistry

endocytosis.34,48 To achieve the conjugation, amino dextran(MW ¼ 10 kDa; Thermo Fisher Scientic) was initially treatedwith azido-PEG3-NHS ester, followed by a CuAAC reaction with
Et2NPOF-CCH to form the target probe Et2NPOF-dex. The
degree of labelling (DOL) in Et2NPOF-dex was calculated to be
1.1. Importantly, Et2NPOF-dex showed almost the same bellshaped pH response as that observed for Et2NPOF, with the
maximum uorescence at pH 5.5 (Fig. 4a and S12†). HeLa cells
were incubated in Dulbecco's modied Eagle's medium(DMEM) containing Et2NPOF-dex (2 mg mL1) for 24 h. Images
were recorded without washing at excitation wavelengths of
640 nm and 488 nm, which correspond to the wavelengths
suitable for the excitation of Form II and Form I, respectively.
Upon excitation at 640 nm, a punctate staining image wasobserved with intense uorescence signals (Fig. 4b), in which
each dot represents weak to moderately acidic compartments,
such as lysosomes and endosomal vesicles, with a luminal pH
value of around 4.5–6.5. The relatively intense spots are likely to
be endosomes with pH  5.5. In contrast, the dots observed
following excitation at 488 nm in Fig. 4c are likely to be moremature endosomes and highly acidic lysosomes with pH below
5.5. The diﬀerence in the staining patterns of these channels,
which show a meaningfully low Pearson's correlation coeﬃcient(R ¼ 0.87, Fig. S22†), arises from the diﬀerence in pH values.
Considering the species distribution shown in Fig. 3f, thedegree of endosomal maturation can be readily discriminatedin the merged image (Fig. 4d).49 For example, a red dot observed

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a platform for additional functionalisation. Various groups forthe targeted labelling of organelles were introduced at a latestage of the synthesis via a copper-catalysed azide–alkyne
cycloaddition (CuAAC) reaction. The obtained NIR-emissive
organelle markers were successfully applied to the selective
staining of lysosomes, mitochondria, and cell nuclei. Functionalisation of the phospha-uorescein POF scaﬀold witha large molecular mass biomolecule, amino dextran, together
with the sophisticated design of an “oﬀ–on–oﬀ” pH probe,
enabled us to produce a probe that can discriminate the degreeof endosomal maturation. This dextran-conjugated probe will
be an important tool for the understanding of important biological events such as autophagy.50,51 Throughout this research
we have demonstrated a unique way of modifying phosphaxanthene dyes, which we believe will open up new avenues tocreate various types of useful NIR-emissive probe.

Author contributions(a) pH-response of Et2NPOF-dex (purple squares) upon excitation at 630 nm shown with that of Et2NPOF (dotted line). (b and c)
Endosomal images in HeLa cells incubated with Et2NPOF-dex. Images
were acquired at 640 nm ((b), quasi-coloured in red) and 488 nm ((c),
quasi-coloured in cyan) without washing the cells; scale bar ¼ 5 mm.
(d) Merged image of the two channels. The areas shown in yellow
boxes are magniﬁed on the right.
Fig. 4

M. T. and S. Y. conceived and led all aspects of the project. H. O.
and Y. T. performed most of the experiments including theorganic synthesis, spectroscopy measurements, and cellimaging experiments. All authors wrote the manuscript.

Conﬂicts of interest
There are no conicts to declare.

in panel A can be attributed to a weakly acidic compartment
with a luminal pH of around 6 to 7. In this pH range, the probe
exists predominantly as Form II, while little Form I is present.
Such dots should represent early endosomes and/or the recycling of endosomes. On the other hand, as the endosome
matures and the luminal acidity increases, the red signal
intensity, attributable to Form II, reaches a peak and eventually
begins to decrease. Conversely, the intensity of the blue signals
resulting from the formation of Form I increases during
maturation. Thus, the punctate staining in white (panel B) andblue (panel C) in Fig. 4d is likely to be late endosomes (pH  5.5)
and lysosomes (pH  4.0), respectively. It is also of great
importance to note that low background uorescence in theimages was acquired without the need to wash the cells. This
stands in sharp contrast to the intense background signals thatarise from the medium observed when POF-dex was used
(Fig. S23†). This is due to the eﬃcient PET uorescencequenching in the incubation solution caused by the oﬀ–on–oﬀ
response of the Et2NPOF dye, a crucial advantage over conventional pH probes.

Conclusions
In summary, we have developed a series of alkyne-modied
phospha-xanthene dyes. A crucial advantage of the introduction of the >P(]O)Ph bridging moiety into the xanthene skeleton is that the phenyl group does not participate in the pconjugation system and therefore this moiety can be used as

7906 | Chem. Sci., 2021, 12, 7902–7907

Acknowledgements
This work was supported by JSPS KAKENHI grants 19H02849
and 19H04858 (MT), and the Mitsubishi Foundation (SY). HO
thanks the JSPS for a Research Fellowship for Young Scientists.
ITbM is supported by the World Premier International ResearchCenter (WPI) Initiative (Japan).

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