Functionalizable red emitting calcium sensor bearing a 1,4-triazole chelating moiety

Article abstract of DOI:10.1039/c4ra12858c

Herein we developed a functionalizable OFF-ON red emitting fluorescent calcium probe based on a new chelating system formed by CuAAC click chemistry (Huisgen cycloaddition). The pro-sensor 7 which is not sensitive to Ca²⁺, contains an alkyne moiety that, upon the click reaction, forms a chelating group involving the 1,4-triazole. Probe 10 exhibited good sensitivity towards calcium (K_d = 5.8 μM) and zinc (5.6 μM) with a high dynamic range (65 fold fluorescence increase), high quantum yield (0.59) and showed very low fluorescence enhancement in the presence of a high concentration of Mg²⁺. We extended this method and generated two dextran conjugates in order to compare their sensing properties with those of the molecular form of 10.

Full text of DOI:10.1039/c4ra12858c

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Functionalizable Red Emitting Calcium Sensor
Bearing a 1,4-triazole Chelating Moiety
Cite this: DOI: 10.1039/x0xx00000x
Mayeul Collot,*a Christian Wilms,b JeanꢀMaurice Malletc, d, e
Received 00th January 2012,
Accepted 00th January 2012
Herein we developed a functionalizable OFFꢀON red emitting fluorescent calcium probe based
on a new chelating system formed by CuAAC click chemistry (Huisgen cycloaddition). The
DOI: 10.1039/x0xx00000x
proꢀsensor
7
which is not sensitive to Ca2+, contains an alkyne moiety that, upon click reaction,
www.rsc.org/
forms a chelating group involving the 1,4ꢀtriazole. Probe 10 exhibited a good sensitivity
towards calcium (Kd= 5.8 µM) and zinc (5.6 µM) with a high dynamic range (65 fold
fluorescence increase), high quantum yield (0.59) and showed very low fluorescence
enhancement in presence of high concentration of Mg2+. We extended this method and
generated two dextran conjugates in order to compare their sensing properties with those of the
molecular form 10
.
1 Introduction
Ca2+ is an ubiquitous second messenger involved in numerous intracellular signalling cascades. Therefore fluorescent Ca2+ indicators are
indispensable tools for studying spatiotemporal fluctuations of intracellular free Ca2+ concentration ([Ca2+]i).1,2 The first fluorescent Ca2+
probes were introduced by Tsien and colleagues in the midꢀeighties involving the BAPTA (1,2ꢀbis(oꢀaminophenoxy)ethaneꢀN,N,N′,N′ꢀ
tetraacetic acid) chelating moiety.3 Consequently, most Ca2+ indicators, and certainly those that work best, combined BAPTA with
fluorescein derivatives and hence emit yellow/green fluorescence.4,5 However, the increasing use of cells transfected with fluorescent
proteins (FPs), of FP expressing transgenic mice for targeting identified subpopulations of cells, together with the advent of optical
techniques for purposes other than imaging require the development of new red Ca2+ probes.6,7 We recently reviewed the redꢀfluorescent
calcium indicators allowing photoactivation and multiꢀcolor imaging8 and contributed in this field by developing a new family of
functionalizable red emitting calcium sensors based on BAPTA, the CaꢀRubies, with various dissociation constants (Kd).9 Cellular Ca2+
signals cover concentrations from near 100 nM for the basal free [Ca2+]i of most mammalian cells to >100 ꢁM at the peak of Ca2+
microdomains. A Ca2+ probe works best if its dissociation constant is close to the intracellular Ca2+ concentration. However, if the Kd is too
large, Ca2+ measurement will be insensitive; on the other hand, if the calcium concentration is higher or is involved in fast transients, the
probe with small Kd could saturate quickly and results in inaccurate measurement. Therefore, it is of great importance to develop fluorescent
sensors with micromolar range Kd for the measurement of accurate intracellular Ca2+ fast transients. To our knowledge, only few alternatives
to BAPTA systems have been reported as new calcium sensors. APTRA (aminophenol triacetic acid) developed by Levy et al.10 has a poor
selectivity for Ca2+ over other divalent ions. Other systems like MOBHA (2ꢀ(2′ꢀmorpholinoꢀ2′ꢀoxoethoxy)ꢀN,Nꢀbis(hydroxycarbonyl
methyl)aniline)11 or sensors based on PEGylated moieties either display low affinities or aggregate under biological conditions.12 Most of
fluorescent metal sensors tend to compartmentalize in cells du to their hydrophobicity, therefore the capability of functionalization can lead
to much higher hydrophilicity or specificity and thus extend the scope as the probe can be linked for instance to polymer, particles, peptide or
dextran.
In this work, we describe an unprecedented system where the proꢀsensor 7 becomes a calcium sensor upon functionalisation via Huisgen
cycloaddition by completing the coordination sphere of calcium involving the Nꢀ3 of the newly formed 1,4ꢀtriazole moiety (Abstract
scheme). These sensors (molecular form and dextran conjugates) based on the PET (Photoinduced Electron Transfer) quenching phenomena
display a bright red fluorescence upon binding to Ca2+ with low micromolar range Kds combined to a strong fluorescence enhancement and a
very low sensitivity towards Mg2+.
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2 Results and Discussion
Huisgen 1,3ꢀdipolar azide–alkyne cycloaddition has become the most popular “click chemistry” type reaction due to its efficiency and the
availability of the reactants.13 1,4ꢀtriazole moieties formed after this ligation have already successfully been used to form complexes with
metals14,15 and also served for the development of fluorogenic sensors.16,17,18,19 To this regard, 7 was designed to provide a functionalisable
fluorophore with a new chelating pattern involving the N,Nꢀdiacetic acid aminophenoxy moiety taken from BAPTA as well as the nitrogenꢀ3
of the newly formed 1,4ꢀtriazole moiety making this system a “2 in 1” approach”. The synthesis starts with 2ꢀaminophenol to obtain
aldehyde 5 in 5 steps with an overall yield of 43%. 5 was then converted into the corresponding Xꢀrhodamine 6 to give, after saponification,
the clickable proꢀsensor 7 (scheme 1).
Scheme 1. Synthesis of proꢀsensor 7. a) Boc2O, MeOH, 80%; b) propargyl bromide; K2CO3, DMF, 80°C, 86%; c) TFA; d) Methyl bromoacetate, DIEA, ACN,
90°C, 94% over 2 steps; e) POCl3, DMF, 80°C, 67%; f) 8ꢀhydroxyjulolidine, PTSA, propionic acid, then chloranil, 13%; g) KOH, MeOH, 76%.
5 was then involved in a Huisgen cycloaddition catalysed by CuI in order to afford the 1,4ꢀtriazole compound 8, the latter was converted into
the Xꢀrhodamine 9 and finally saponified to yield the probe 10 (Scheme 2). In order to study the involvement of the Nꢀ3 of the triazole
moiety, a 1,5ꢀtriazole isomer 13 of the sensor 10 was synthesised. 5 was reacted with methyl 2ꢀazidoacetate in presence of a small amount
(0.04 equivalent) of [RuClCp*(PPh3)2] as a catalyst20 to furnish 11 which was transformed in two steps in 13 (Scheme 2). Isomers 10 and 13
only differ from the availability of a nitrogen atom of the triazole groups. Whereas 10 is able to form a chelate with the Nꢀ3 of its 1,4ꢀ
triazole, the steric conformation of 13 does not allow the participation of any nitrogen atom to complete the chelation ability of the molecule.
Scheme 2. a) Methyl azidoacetate, CuSO4ꢂ5H2O, sodium ascorbate, Dioxane, Water, 83%; b) Methyl azidoacetate, [RuClCp*(PPh3)2], dioxane, 90°C, 90%; c)
8ꢀhydroxyjulolidine, PTSA, propionic acid, then chloranil, 20% for 9, 16% for 12; d) KOH, MeOH, 81% for 10, 76% for 13.
The chelating properties of fluorescent sensors 7, 10 and 13 were then investigated by fluorescence spectroscopy. First, the fluorescence
enhancement (ꢀF/F0) of the sensors in the presence of 3 equivalents of various metals (Ca2+, Cd2+, Cu2+, Fe3+, Hg2+, Mg2+, Mn2+ and Zn2+)
was evaluated (Figure 1).
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50
40
30
20
10
0
10
13
7
EDTA
Ca
Cd
Cu
Fe
Hg
Mg
Mn
Zn
Metals
Figure 1. Fluorescence enhancement ((FꢀF0)/F0, with F0= Fluorescence Intensity in presence of EDTA 1 mM) of 7, 13 and 10 (5 ꢁM, MOPS 30 mM, KCl 100
mM, pH 7.2) in presence of 3 equivalents (15 ꢁM) of various metals.
In the case of the proꢀsensor 7, the majority of tested metals did not trigger the fluorescence or only small enhancements were observed (Zn,
5 fold). Interestingly, 7 displayed a quite significant fluorescence enhancement in presence of Hg2+ with a ꢀF/F0 of 17 fold. This could be
explained by an oxymercuration21,22 reaction of the alkyne moiety of 7 leading to a stabilised organomercury complexe (scheme 3).
Scheme 3. Proposed formation of stabilised mercury chelate via an oxymercuration reaction involving 7.
Whereas 10 exhibits strong responses with Ca2+, Cd2+ and Zn2+ up to 52 folds for Cd2+ and 49 for Ca2+, 13 gave only weak fluorescence
enhancements with Ca2+ and Cd2+ and a ꢀF/F0 of 7.9 fold with Zn2+ (Figure 1). These results suggest that the Nꢀ3 of the 1,4ꢀtriazole is
involved in the stabilisation of a calcium complex. This hypothesis was confirmed by the dissociation constants measurements. 10 exhibited
substantially the same high affinity for Ca2+ and Zn2+ (Kd Ca2+= 5.84 ± 0.21 µM, Kd Zn2+= 5.68 ± 0.14) whereas 7 and 13 have low affinity
towards these metals (table 1). Job’s plot evidenced that 10 formed a 1:1 complex with Ca2+ which displays a bright red fluorescence at 604
nm with an increase of quantum yield of 78 fold and a fluorescence enhancement of 65 fold (Figure 2). In view to these results, 10, which
has a KdCa close our previously published CaRubyꢀF, can be considered as an efficient Ca2+ sensor as the concentration of Zn2+ in cells is
nanomolar, far below its KdZn.23
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A
B
[Ca2+] (ꢁM)
[Ca2+] (ꢁM)
Wavelength [nm]
Wavelength [nm]
1600
C
D
60
50
40
30
20
10
0
1400
1200
1000
800
600
400
200
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
10
100
1000
[Ca2+] (ꢁM)
[Ca2+] / {[Ca2+]+[10]}
Figure 2. (A) UVꢀVisible absorption spectra and (B) emission spectra (λEx= 535 nm) of 10 (5 ꢁM, MOPS 30 mM, KCl 100 mM) at increasing concentration
of Ca2+. (C) Plot of fluorescence enhancement ((FꢀF0)/F0, with F0= Fluorescence Intensity in presence of EDTA 1 mM) of 10 (5 ꢁM, MOPS 30 mM, KCl 100
mM, pH 7.2) vs. increasing concentration of Ca2+. The fit line, according to Hill’s equation, yielded the Kd. (D) Job’s plot of 10 ([Ca2+].[10]= 5 ꢁM)
determined a Ca2+ complex with a 1:1 stoichiometry.
a
a
λAbs
ε. (M-1
λEm
b
ΦEGTA
ΦCa
Φ
Ca/ΦEGTA
KdZn (ꢁM)c
KdCa (ꢁM)c
Compound
(nm)
cm-1)
(nm)
7
577ꢀ582
576ꢀ581
576ꢀ580
48,000
54,000
80,000
606ꢀ608
599ꢀ604
601ꢀ605
0.004
0.06
0.59
0.29
6
205 ± 40
5.6 ± 0.1
N/Ad
10
13
0.007
0.005
78
54
5.8 ± 0.2
448 ± 10
118.91 ± 3.9
a Calcium free [EGTA]= 1 mM and [Ca2+]= 1 mM
b Calcium free [EGTA]= 1 mM
c Obtained by fitting the data with Hill’s equation
d Fitting the data with Hill’s equation provided no result
Table 1. Physicochemical properties of the sensors (5 ꢁM, MOPS 30 mM, KCl 100 mM, pH 7.2)
A fluorescent calcium probe intended for use in cellular assays should fulfil several criteria. First the sensitivity to pH variations met in cells
should not interfere with the signal obtained in response to Ca2+. pH sensitivity of 10 was evaluated and whereas the pKa value was found to
be in the physiological range (5.93 ± 0.07), the fluorescence enhancement from pH 7.5 to pH 4.5 is only 1.9 fold (Figure S2) which is
negligible in regard of the much higher fluorescence increase (65 fold) provoked by Ca2+. A major drawback in molecular Ca2+ probes is that
their hydrophobicity often leads to compartmentalization in organelles such as plasma membrane, ER (Endoplasmic Reticulum),
mitochondria, etc. To avoid this issue, probes can be linked to non toxic and inert dextrans which are effective waterꢀsoluble carriers for dyes
and indicators.24 Dextran conjugates have enhanced hydrophilicity and hence, facilitate the distribution of the probe in the cytosol through
microinjection.25 Therefore, proꢀsensor 7 was linked via click chemistry to two prepared azidoꢀdextran (obtained from dextran of 1,500 MW
and dextran 6000 MW, see SI) (figure 3A) in order to study the influence of the dextran carrier on the Ca2+ sensing properties. Despite
dextran conjugates 1,500 and 6,000 displayed slightly higher KdCa of 16.3 ± 0.6 ꢁM and 18.3 ± 0.8 ꢁM respectively (figure 3B), their
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fluorescence enhancement, respectively 41 and 36 fold, make them efficient calcium sensors. The slight drop of sensitivity (fluorescence
enhancement) could be attributed to the formation of dark Hꢀaggregates due to the proximity of the sensors within the dextran polymer as the
absorbance spectra exhibited a second band at 545 nm (figure 3C). This phenomenon could be reduced by decreasing the number of sensors
per dextran molecules (14 sensors per 100 glucose units). The shift of the KdCa values could be attributed to an effect that we recently
described as a remote control.26 Even though the coordination sphere is not affected by what the probe is linked to, the close environment of
the complex can influence the dissociation constant. While, in the case of 10, a close negative charge (COOꢀ) seems to increase the affinity of
the sensor towards Ca2+, a side PEG chain, in the case of the dextran conjugates, slightly lower this affinity, these results are in accordance to
what we already observed.
Figure 3. (A) Synthesis of Dextran conjugates by Click Chemistry (CuSO4ꢂ5H2O, sodium ascorbate, DMF, water, 76% for dextran 1500, 80% for dextran
6000). (B) Normalized fluorescence enhancement ((FꢀF0)/F0, with F0= Fluorescence Intensity in presence of EDTA 1 mM) of 10 and its dextran conjugates
(MOPS 30 mM, KCl 100 mM, pH 7.2) vs. increasing concentration of Ca2+. The fit lines, according to Hill’s equation, yielded the Kds. (C) Absorbance
spectra and (D) emission spectra (λEx= 535 nm) of Dextran 6000 conjugate (MOPS 30 mM, KCl 100 mM) at increasing concentration of Ca2+.
Finally, since Mg2+ is buffered in the cytoplasm at concentration in the subꢀmillimolar range, it is of extreme importance that the sensor
remains insensitive to Mg2+ at high concentrations to avoid background fluorescence. Figure 4 shows that 10 and its dextran conjugates were
virtually non sensitive to high concentration of Mg2+ (1 mM), moreover the presence of an equimolar concentration of Mg2+ an Ca2+ does not
influence the fluorescence enhancement of the sensors.
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Figure 4. Fluorescence enhancement ((FꢀF0)/F0, with F0= Fluorescence Intensity in presence of EDTA 1 mM) of 7 and its dextran conjugates (MOPS 30 mM,
KCl 100 mM, pH 7.2) in presence of Mg2+ (1 mM), Ca2+ (50 ꢁM) and a mixture of Ca2+ and Mg2+ (50 ꢁM).
The properties of these dextran conjugates are attractive for potential cell biology applications. In the case of measurement of intracellular
calcium ion ([Ca2+]i) transients in myocytes, it was shown that low affinity indicators, like Furaptra (KdCa ∼ 50µM), yielded greater peak
amplitude of stimulated [Ca2+]i transients than those obtained with high affinity indicators (Furaꢀ2 KdCa = 240 nM). This is partially due to
the detrimental buffering effect of high affinity indicators.27 Therefore, the dextran conjugates we present, could offer an interesting
alternative to Furaptra with no sensitivity towards Mg2+, an enhance brightness, redꢀshifted absorption and emission spectra, lower Kds and a
better hydrophilicity brought by the dextran molecules.
3 Conclusion
We developed a new Ca2+ sensor system where the newly formed 1,4ꢀtriazole moiety is involved in the chelation pattern. The proꢀsensor 7 is
insensitive to Ca2+ and thus can be used to functionalise any azide decorated (bio)molecule, polymers or chips without need to separating the
precursor from the product. This system is readily accessible and represents a good alternative to micromolar range BAPTA based sensors.
Moreover, its sensitivity and brightness combined to its functionalisable capability makes this system a valuable tool for potential monitoring
of Ca2+ transients. These new indicators (molecular sensor 10 and dextran conjugates) feature very good properties and we believe that as
red, Mg2+ insensitive, low affinity fluorescent Ca2+ probes, they will strengthen the arsenal of fluorescent Ca2+ sensors.
4 Experimental
4.1 Materials and general methods
All the solvents were of analytical grade. Chemicals were purchased from commercial sources. The salts used in stock solutions of metal ions
1
were CaCl2·2H2O, CdCl2, CuCl2·2H2O, FeCl3·6H2O, HgCl2, KCl, MgCl2·6H2O, MnCl2·4H2O, NaCl, Zn(NO3)2. HꢀNMR and 13CꢀNMR
were measured on a Bruker avance IIIꢀ300 MHz spectrometer with chemical shifts reported in ppm (TMS as internal standard). Mass spectra
were measured on a Focus GC / DSQ II spectrometer (ThermoScientific) for IC and an API 3000 spectrometer (Applied Biosystems, PE
Sciex) for ES. All pH measurements were made with a Mettler Toledo pHꢀMeter. Fluorescence spectra were recorded on a JASCO FPꢀ8300
spectrofluorometer. Absorption spectra were measured on a VARIAN CARY 300 Bio UVꢀVisible spectrophotometer. All measurements
were done at a set temperature of 25°C. The purity of the dyes were checked by RPꢀHPLC Cꢀ18, eluant: ACN 0.1% TFA/Water 0.1% TFA,
method: 20/80 to 100/0 within 20 min then 100/0 for 10 min. detection at λAbs= 254 nm.
4.2 Synthesis
1 was synthesized according to a published protocol.28
2. To a solution of 1 (3.200 g, 15.31 mmol) in DMF (30 mL) were added propargyl bromide 80 wt. % in toluene (2.52 mL, 22.96 mmol, 1.5
eq) and K2CO3 (3.168 g, 22.96 mmol, 1.5 eq). The solution was heated at 80°C for 5h before being cooled down to room temperature. The
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solvents were evaporated and the product was extracted with EtOAc washed with water (3 times) and brine (2 times). The organic phase was
dried over MgSO4, filtered and evaporated. The crude was purified by column chromatography on silica gel (Cyclohexane/EtOAc : 9/1) to
obtain 3.28 g of 2 (86%) as a yellowish syrup. Rf=0.69 (Cyclohexane/EtOAc, 8/2). 1HꢀNMR (300 MHz, CDCl3): δ 8.13 (d, J = 5.3 Hz, 1H,
HAr), 7.10 (s, 1H, NH), 7.02ꢀ7.00 (m, 3H, H Ar), 4.78 (d, J = 2.4 Hz, 2H, CH2), 2.58 (t, J = 2.4 Hz, 1H, CH), 1.56 (s, 9H, tBu). 13CꢀNMR
(75 MHz, CDCl3): δ 152.72 (CO Boc), 145.54 (Cq Ar), 128.64 (Cq Ar), 122.21 (CH Ar), 122.15 (CH Ar), 118.53 (CH Ar), 111.74 (CH Ar),
80.42 (Cq tBu), 78.20 (C≡CH), 76.07 (C≡CH), 56.47 (CH2), 28.40 (tBu). MS (CI), calcd for C14H17NO3 [M]+ 247.1, found 247.1, HRMS
(CI), C14H17NO3 [M]+ 247.1208, found 247.1195.
4. To a cooled (0°C) solution of 2 (3.280 g, 13.28 mmol) in DCM (20 mL) was added TFA (5 mL). The solution was allowed to stir at room
temperature overnight. TFA was neutralized by addition of a saturated solution of NaHCO3 to reach a pH of 8. The product was extracted
with DCM and the solution was dried over MgSO4, filtered and evaporated to obtain 3. Rf=0.35 (Cyclohexane/EtOAc, 8/2). To a solution of
3 (1.923 g, 13.06 mmol) in acetonitrile (26 mL) were added methyl bromoacetate (3.69 mL, 39.84 mmol, 3 eq) and DIEA (6.92 mL, 39.84
mmol, 3 eq) before being warmed up to 90°C overnight. 2 more equivalent of both methyl bromoacetate and DIEA were then added to
complete the reaction. The solution was stirred at 90°C over 6h. The solvents were evaporated, the product was extracted with DCM and
washed with water. The organic phase was dried over MgSO4, filtered and evaporated. The crude was purified by column chromatography
on silica gel (Cyclohexane/EtOAc : 9/1) to obtain 3.64 g of 4 (94%) as a yellowish syrup. Rf=0.28 (Cyclohexane/EtOAc, 8/2). 1HꢀNMR (300
MHz, CDCl3): δ 6.95 (m, 4H, H Ar), 4.72 (d, J = 2.4 Hz, 2H, OCH2), 4.18 (s, 4H, NCH2), 3.75 (s, 6H, OMe), 2.52 (t, J = 2.4 Hz, 1H, CH).
13CꢀNMR (75 MHz, CDCl3): δ 171.83 (CO esters), 149.38 (C Ar), 139.73 (C Ar), 122.55 (CH Ar), 122.42 (CH Ar), 119.56 (CH Ar), 115.37
(CH Ar), 78.80 (C≡CH), 75.37 (C≡CH), 56.81 (OCH2), 53.77 (NCH2), 51.82 (OMe). MS (ES+), calcd for C15H17NO5Na [M + Na]+ 314.1,
found 314.4. HRMS (ES+), calcd for C15H18NO5 [M + H]+ 292.1179, found 292.1197.
5. To a solution of 4 (3.06 g, 10.51 mmol) in DMF (10 mL) was slowly added POCl3 (7.82 mL, 84.08 mmol, 8 eq). The mixture turned black
and was allowed to stir at 80°C for 3 hours before being cooled down to room temperature. The mixture was then poured in water (1L) and
the product was extracted with EtOAc (3 times) and washed with brine twice. The organic phase was dried over MgSO4, filtered and
1
evaporated to 50 mL EtOAc. The product precipitated under cooling and was filtered to obtain 2.262 g of 5 (67%) as a beige powder. Hꢀ
NMR (300 MHz, CDCl3): δ 9.83 (s, 1H, CHO), 7.48ꢀ7.43 (m, 2H, H Ar), 6.80 (d, J = 8.2 Hz, 1H, H Ar), 4.74 (d, J = 2.2 Hz, 2H, OCH2),
4.25 (s, 4H, NCH2), 3.81 (s, 6H, OMe), 2.56 (t, J = 2.1 Hz, 1H, CH). 13CꢀNMR (75 MHz, CDCl3): δ 190.45 (CHO), 171.18 (CO esters),
148.33 (C Ar), 145.36 (C Ar), 129.95 (C Ar), 127.12 (CH Ar), 116.91 (CH Ar), 112.81 (CH Ar), 77.72 (C≡CH), 76.10 (C≡CH), 56.71
(OCH2), 54.08 (NCH2), 52.16 (OMe). MS (CI), calcd for C16H18NO6 [M+H]+ 320.1, found 320.1, HRMS (CI), C16H18NO3 [M+H]+ 320.1129,
found 320.1191.
8. To a solution of 5 (500 mg, 1.567 mmol) and methyl 2ꢀazido acetate (360 mg, 3.134 mmol, 2 eq) in dioxane (16 mL) was added an
heterogeneous solution of CuSO4·5H2O (195 mg, 0.783 mmol, 0.5 eq) and sodium ascorbate (217 mg, 1.097 mmol, 0.7 eq) in water (1 mL).
The mixture was stirred at 50°C overnight before being extracted with DCM and washed successively with water and brine. The organic
phase was dried over MgSO4, evaporated and the crude was purified by column chromatography on silica gel (EtOAc) to obtain 571 mg of 8
(83%) as a yellowish syrup. Rf=0.40 (100% EtOAc). 1HꢀNMR (300 MHz, CDCl3): δ 9.72 (s, 1H, CHO), 7.75 (s, 1H, H triazol), 7.42 (d, J =
1.8 Hz, 1H, H Ar), 7.33 (dd, J = 8.2, 1.8 Hz, 1H, H Ar), 6.71 (d, J = 8.2 Hz, 1H, H Ar), 5.18 (s, 2H, CH2COOMe), 5.12 (s, 2H, OCH2), 4.12
(s, 4H, NCH2), 3.74 (s, 3H, OMe), 3.60 (s, 6H, OMe). 13CꢀNMR (75 MHz, CDCl3): δ 190.52 (CHO), 171.15 (CO esters), 166.53(COOMe),
149.08 (C Ar), 145.37 (C Ar), 143.31 (C Ar), 130.11 (CH Ar), 126.62 (CH triazol), 124.92 (CH Ar), 117.04 (CH Ar), 112.79 (CH Ar), 62.52
(CH2COOMe), 53.90 (NCH2), 53.13 (OMe), 52.00 (2 OMe), 50.77 (OCH2). MS (CI), calcd for C19H23N4O8 [M + H]+ 435.1, found 435.0.
HRMS (ES+), calcd for C19H23N4O8 [M + H]+ 435.1510, found 435.1516.
11. To a solution of 5 (409 mg, 1.282 mmol) and methyl 2ꢀazido acetate (294 mg, 2.564 mmol, 2 eq) in dioxane (15 mL) was added
Cp*RuCl(PPh3)2 (40 mg, 0.05 mmol, 0.04 eq). The solution was allowed to stir at 90°C. The solutions turned quickly black and a monitoring
of the reaction by TLC revealed that the starting material was consumed. The solvent was then evaporated and the crude was purified by
column chromatography on silica gel (Cyclohexane/EtOAc : 4/6) to obtain 582mg of 11 (90%) as a yellowish syrup. Rf=0.16
(Cyclohexane/EtOAc : 5/5). 1HꢀNMR (300 MHz, CDCl3): δ 9.83 (s, 1H, CHO), 7.82 (s, 1H, H triazol), 7.48ꢀ7.46 (m, 2H, H Ar), 6.84 (d, J =
8.6 Hz, 1H, H Ar), 5.34 (s, 2H, OCH2), 5.19 (s, 2H, CH2COOMe), 4.12 (s, 4H, NCH2), 3.79 (s, 3H, OMe), 3.60 (s, 6H, 2 OMe). 13CꢀNMR
(75 MHz, CDCl3): δ 190.25 (CHO), 170.94 (CO esters), 166.96 (CO ester), 148.78 (C Ar), 145.54 (C Ar), 134.82 (CH triazol), 132.47 (C
Ar), 130.27 (C Ar), 127.69 (CH Ar), 117.43 (CH Ar), 112.24 (CH Ar), 59.57 (CH2COOMe), 53.65 (NCH2), 53.08 (OMe), 52.05 (2 OMe),
49.62 (OCH2). MS (CI), calcd for C19H23N4O8 [M + H]+ 435.1, found 435.2. HRMS (ES+), calcd for C19H23N4O8 [M + H]+ 435.1510, found
435.1526.
4.3 Synthesis of X-Rhodamine : typical procedure
Numerotation of Xꢀrhodamines:
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6. To a solution of aldehyde 5 (200 mg, 0.626 mmol) in propionic acid was added 8ꢀhydroxyjulolidine (237 mg, 1.254 mmol, 2 eq) and
PTSA (11 mg, 0.062 mmol, 0.1 eq). The solution was protected from light and stirred at room temperature overnight. To the brown mixture
was added a solution of chloranil (152 mg, 0.626 mmol, 1 eq) in DCM (10 mL), the reaction turned dark and was allowed to stir overnight at
room temperature. The dark purple solution was concentrated to dryness, the residue was dissolved in DCM and purified by column
chromatography on silica gel (gradient of 100% DCM to 9/1 DCM/Methanol) to obtain 58 mg of 6 (∼13%) as a purple solid after
lyophilisation (dioxane/water : 1/1). 1HꢀNMR (300 MHz, CDCl3): δ 7.83 (d, J = 8.1 Hz, 2H, CH Ar PTSA counter ion), 7.05 (d, J = 8.0 Hz,
2H, CH Ar PTSA counter ion), 6.93 (t, J = 7.6 Hz, 5H, Ha, Hb, Hc, H3), 4.70 (d, J = 2.2 Hz, 2H, CH2O), 4.27 (s, 4H, NCH2), 3.83 (s, 6H, 2
OMe), 3.55 (m, 8H, H1, H4), 3.02 (t, J = 6.1 Hz, 4H, H6), 2.73 (t, J = 6.0 Hz, 4H, H3), 2.57 (t, J = 2.1 Hz, 1H, C≡CH), 2.27 (s, 3H, Me PTSA
counter ion), 2.12ꢀ1.98 (m, 8H, H5, H2). 13CꢀNMR (75 MHz, CDCl3): δ 171.73 (CO esters), 154.11 (C Ar), 152.22 (C Ar), 151.04 (C Ar),
148.20 (C Ar), 144.65 (C Ar), 141.08 (C Ar), 138.14 (C Ar), 128.15 (CH PTSA), 126.77 (C7), 126.29 (CH PTSA), 125.35, 124.10 (CH Ar),
123.56, 118.09 (CH Ar), 116.10 (CH Ar), 112.75, 105.43, 78.00 (C≡CH), 76.28 (C≡CH), 56.78 (CH2O), 53.87 (NCH2), 52.15 (2 OMe),
50.95 (C1 or C4), 50.48 (C1 or C4), 27.73 (C3), 21.29 (Me PTSA), 20.73 (C2), 19.96 (C6), 19.77 (C5). MS (ES+), calcd for C40H42N3O6 [M]+
660.3, found 660.7. HRMS (ES+), calcd for C40H42N3O6 [M]+ 660.3068, found 660.3079.
9 was obtained as a purple solid after lyophilisation with ∼20% yield. 1HꢀNMR (300 MHz, CDCl3): δ 8.11 (s, 1H, H triazol), 7.71 (d, J = 8.1
Hz, 2H, 2CH PTSA), 6.95 (dd, J = 4.8, 3.0 Hz, 3H, CH PTSA, 1CH Ar), 6.86ꢀ6.73 (m, 4H, CH Ar), 5.22 (s, 2H, CH2COOMe), 5.11 (s, 2H,
OCH2), 4.16 (s, 4H, NCH2), 3.68 (s, 3H, OMe), 3.65 (s, 6H, 2 OMe), 3.44 (m, 8H, H1, H4), 2.94 (t, J = 6.1 Hz, 4H, H6), 2.69ꢀ2.64 (m, 4H,
H3), 2.18 (s, 3H, Me PTSA), 2.02 (t, J = 5.1 Hz, 4H, H5), 1.93ꢀ1.91 (m, 4H, H2). 13CꢀNMR (75 MHz, CDCl3): δ 171.82 (CO esters), 166.99
(CO ester), 154.70 (C Ar), 152.26 (C Ar), 151.02 (C Ar), 149.12 (C Ar), 144.59 (C Ar), 142.88 (C Ar), 141.13 (C Ar), 138.21 (C Ar), 128.18
(CH PTSA), 127.03 (C7), 126.23 (CH PTSA), 126.09 (CH triazol), 125.36(C Ar), 123.63 (C Ar), 123.48 (CH Ar), 117.91 (CH Ar), 115.93
(CH Ar), 112.88 (C Ar), 105.24 (C Ar), 62.56 (OCH2), 53.82 (NCH2), 52.92 (OMe), 52.03 (2 OMe), 50.96 (C1 or C4), 50.79 (CH2COOMe),
50.44 (C1 or C4), 27.64 (C3), 21.28 (Me PTSA), 20.74 (C2), 19.97(C6), 19.81 (C5). MS (ES+), calcd for C43H47N6O8 [M]+ 775.3, found 776.0.
HRMS (ES+), calcd for C43H47N6O8 [M]+ 775.3450, found 775.3473.
12 was obtained as a purple solid after lyophilisation with ∼16% yield. 1HꢀNMR (300 MHz, CDCl3): δ 7.73 (d, J = 7.9 Hz, 2H, CH PTSA),
7.71 (H triazol) 7.02ꢀ6.83 (m, 7H, 2CH PTSA, Ha, Hb, Hc, 2H7), 5.45 (s, 2H, CH2COOMe), 5.33 (s, 2H, OCH2), 4.21 (s, 4H, NCH2), 3.72 (s,
3H, OMe), 3.70 (s, 6H, 2 OMe), 3.57ꢀ3.51 (m, 8H, H1, H4), 3.02 (t, J = 6.1 Hz, 4H, H6), 2.77ꢀ2.71 (m, 4H, H3), 2.26 (s, 3H, Me PTSA), 2.13ꢀ
2.08 (m, 4H, H5), 2.02ꢀ2.00 (m, 4H, H2). 13CꢀNMR (75 MHz, CDCl3): δ 171.55 (CO esters), 167.27 (CO ester), 154.00 (C Ar), 152.21 (C
Ar), 151.07 (C Ar), 148.62 (C Ar), 144.52 (C Ar), 141.05 (C Ar), 138.23 (C Ar), 134.55 (CH triazol), 133.20 (C Ar), 128.16 (CH PTSA),
126.73 (C7), 126.16 (CH PTSA), 126.05 (C Ar), 124.12 (CH Ar), 123.77 (C Ar), 118.76 (CH Ar), 116.13 (CH Ar), 112.84 (C Ar), 105.30 (C
Ar), 59.91 (OCH2), 53.51 (NCH2), 52.94 (OMe), 52.04 (2 OMe), 50.99 (C1 or C4), 50.47 (C1 or C4), 49.72 (CH2COOMe), 27.64 (C3), 21.29
(Me PTSA), 20.71 (C2), 19.96 (C6), 19.79 (C2). MS (ES+), calcd for C43H47N6O8 [M]+ 775.3450, found 775.7. HRMS (ES+), calcd for
C43H47N6O8 [M]+ 775.3450, found 775.3495.
4.4 Saponification : typical procedure
13. To a solution of 12 (90 mg, ∼0.10 mmol) in methanol (10 mL) were added 700 mg of KOH and water (3 mL), the mixture was stirred
overnight. The product was washed with aq HCl (1M) and extracted with CHCl3 until the aqueous phase become slightly pink. The organic
phase was then dried over MgSO4, filtered and concentrated. The residue was purified on a reverse phase column Cꢀ18 using acetonitrile
(0.1% TFA) and water (0.1% TFA) as eluant (20% ACN to 60%), monitored at 254 nm. The solvents were evaporated and 64 mg of 13
(76%) were obtained as a purple solid after lyophilisation (dioxane/water, 1/1). HRMS (ES+), calcd for C40H41N6O8 [M]+ 733.2980, found
733.3002.
7 was obtained as a purple solid after lyophilisation with ∼74% yield. HRMS (ES+), calcd for C38H38N3O6 [M]+ 632.2755, found 632.2761.
10 was obtained as a purple solid after lyophilisation with ∼81% yield. HRMS (ES+), calcd for C40H41N6O8 [M]+ 733.2980, found 733.3002
4.5 Dextran Conjugates
Dextran 6,000 MW (SigmaꢀAldrich, ref: 31388) and dextran 1,500 MW (SigmaꢀAldrich, ref: 31394) were functionnalised with 1429 (see SI)
using a method described by Nielsen et al.30 The 1HꢀNMR showed that the functionnalised dextrans were alkylated once per glucose unit.
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Final MW Dextran 6,000 : ∼14,500 g.molꢀ1
Final MW Dextran 1,500 : ∼3,600 g.molꢀ1
Conjugation of Dextrans. To a solution of dextranꢀPEGꢀN3 (1,500 or 6,000) (40 mg, ∼100 µmol glucose unit) and 7 (10 mg, 14 µmol) in
DMF (2 mL) was added an heterogeneous solution of CuSO4·5H2O (10 mg, 40 µmol) and sodium ascorbate (10 mg, 50 µmol) in water (1
mL). The solution was allowed to stir in the dark at 50°C overnight. The solvents were evaporated, the crude was dissolved in 1 mL of
EDTA solution (0.1 M) and passed through a Gꢀ25 column to give 40 mg of CaRuꢀDextran 6,000 conjugate (∼80% yield) and 38 mg CaRuꢀ
Dextran 1,500 conjugate (∼76% yield).
Acknowledgements
This work was supported by the French Agence National de la Recherche (ANR P3N, nanoFRET2 grant, to J.M.M.)
Notes and references
a Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie, 74, Route du Rhin, 67401
Illkirch, France. Eꢀmail: mayeul.collot@unistra.fr
b Wolfson Institute for Biomedical Research and Department of Neuroscience, Physiology and Pharmacology, University College London, Gower
Street, London WC1E 6BT, UK
c Laboratory of Biomolecules (LBM), UPMC Université Paris 06, Paris Fꢀ75005, France
d Ecole Normale Supérieure (ENS), Paris Fꢀ75005, France
e CNRS, UMR 7203, Paris Fꢀ75005, France
Supplementary Information
Experimental data: details of the organic synthesis of probes (HPLC, NMR, mass spectra) and measurements of their photophysical properties are available.
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