A Comprehensive Study of the Ca2+ Ion Binding of Fluorescently Labelled BAPTA Analogues

European Journal of Organic Chemistry

Accepted Article

Title: A comprehensive study of the Ca2+ ion binding of fluorescently

labelled BAPTA analogues

Authors: Attila Csomos, Bence Kontra, Attila Jancsó, Gábor Galbács,

Ruth Deme, Zoltán Kele, Balázs József Rózsa, Ervin Kovács,

and Zoltán Mucsi

This manuscript has been accepted after peer review and appears as an

Accepted Article online prior to editing, proofing, and formal publication

of the final Version of Record (VoR). This work is currently citable by

using the Digital Object Identifier (DOI) given below. The VoR will be

published online in Early View as soon as possible and may be different

to this Accepted Article as a result of editing. Readers should obtain

the VoR from the journal website shown below when it is published

to ensure accuracy of information. The authors are responsible for the

content of this Accepted Article.

To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202100948

Link to VoR: https://doi.org/10.1002/ejoc.202100948

01/2020

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A comprehensive study of the Ca²⁺ion binding of fluorescently

labelled BAPTA analogues

Attila Csomos^[a], Bence Kontra^[a], Attila Jancsó^[b], Gábor Galbács^[b], Ruth Deme^[c], Zoltán Kele^[d], Balázs

József Rózsa^[e,f], Ervin Kovács*^[a,g], and Zoltán Mucsi*^[a,h]

This article is dedicated to Professor Ferenc Fülöp.

[a]

A. Csomos, B. Kontra, Dr. E. Kovács, Dr. Z. Mucsi

Department of Chemistry, Femtonics Ltd.

H-1094 Budapest, Hungary

E-mail: ervin.kovacs@femtonics.eu, zmucsi@femtonics.eu

Dr. A. Jancsó, Dr. G. Galbács

Department of Inorganic and Analytical Chemistry, University of Szeged

Szeged H-6720, Hungary

[b]

[c]

[d]

[e]

Dr. R. Deme

Department of Organic Chemistry, Semmelweis University,

Budapest H-1092, Hungary

Dr. Z. Kele

Institute of Medical Chemistry, University of Szeged

Szeged H-6720, Hungary

Dr. B. J. Rózsa

Two-Photon Measurement Technology Research Group,

The Faculty of Information Technology, Pázmány Péter Catholic University

Budapest H-1083, Hungary

[f]

Dr. B. J. Rózsa

Laboratory of 3D Functional Imaging of Neuronal Networks and Dendritic Integration,

Institute of Experimental Medicine

Budapest H-1083, Hungary

[g]

[h]

Dr. E. Kovács

Polymer Chemistry Research Group,

Research Centre for Natural Sciences

Budapest H-1117, Hungary

Dr. Z. Mucsi

Faculty of Materials Science and Engineering, University of Miskolc

Miskolc H-3515, Hungary

Supporting information for this article is given via a link at the end of the document.

Introduction

Abstract

Calcium ions play an essential role in multiple physiological

processes e.g in muscles, various organs and parts of the

nervous system. Examples include signal transduction, gene

transcription and blood coagulation, among others.^[1]Furthermore,

the omnipresence of Ca²⁺ions has contributed substantially to the

success of imaging over the last 40 years. Understanding cellular

processes by monitoring the presence of Ca²⁺ions at the cellular

level was made widely available by the introduction of the visually

Since

its

development,

the

ionophore

BAPTA

has

(1,2-bis(2-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid)

been used unchanged in calcium sensing applications. In this work

we present a comprehensive experimental and theoretical study of

novel alterations in the structure of BAPTA, with a focus on the

systematic modification of the chain connecting the two aromatic rings

of the molecule (denoted as “linker”). A bis-(diethylamino)xantene

fluorophore was also attached to the structures in a fixed position and

the structure-fluorescence response relationship of these molecules

was investigated in addition. The effect of the length of the linker, the

number of O atoms in this chain and even the removal of one of the

rings was tested; these all proved to significantly alter the

characteristics of the compounds. For example, it was found that the

second aromatic ring of BAPTA is not essential for the turn-on of the

fluorescence. We also demonstrated that successful sensing can be

realized even by replacing the chain with a single oxygen atom, which

suggests the availability of a new calcium binding mode of the chelator.

The reliable turn-on characteristic, the steep Ca²⁺fluorescence

titration curve and the intense fluorescence emission combine to

make this compound a prospective candidate as a calcium sensing

molecular probe in diagnostic neurobiological applications.

active

ionophore

1,2-bis(2-aminophenoxy)-

ethane-N,N,N’,N’-tetraacetic acid (BAPTA, 1a) in the pioneering

work of Tsien et al.^[2]Since then, a wide range of BAPTA

derivatives were prepared and used in biological research.^[3]

Lately, with the spread of genetically encoded calcium indicators,

the use and the development of small molecular probes were

slightly relegated into the background. Nevertheless, the latter

have several advantages over the genetically encoded indicators

as they offer a wider choice of Ca²⁺affinity, display unique

spectral properties and can even be used simultaneously.^[3]

These advantageous features allow highly customizable

measurements to be executed. In addition to this, synthetic

molecular probes are also suitable for subcellular Ca²⁺imaging,^[4]

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and recent studies also revealed possibilities for using them as

MRI contrast agents^[5,6]or therapeutic compounds.^[7]In

combination with microendoscopes,^[8]fluorescent Ca²⁺chelators

also have a bright future in the fields of human diagnostics and

therapy - which are applications where - the use of genetically

encoded indicators is not a viable option. In spite of their above

highlighted importance, the development of small Ca²⁺sensors

based on fluorescent chelators has received little attention lately.

selected because it exhibits better two-photon absorption than the

tetramethyl variant (TAMRA) which is beneficial for further

biological applications.^[13,14]This set of modified fluorescent

ionophores were subjected to a spectroscopic investigation

combined with fluorescence-monitored Ca²⁺-titrations in order to

study the details of their interaction with Ca²⁺ions. In particular,

the optimal length of the linker and the role of the oxygen atom

with regards to Ca²⁺affinity were studied along with the

photophysical changes of the fluorescent dyes (4b–7b). For

comparative purpose, derivatives of the original and longer

ionophore analogues (BAPTA and BAPPTA, 1b and 3b) were

also prepared and studied in the same way. The photophysical

characteristics of the fluorescent derivatives of AATA (2b) were

not reported in the literature earlier, therefore we investigated our

novel construction as well.^[15]This fluorophore unit lacks the

possibility of regioisomer formation, and it is easily attachable to

the scaffolds.^[16]

For the purpose of fluorescent Ca²⁺imaging, the BAPTA (1a)

ionophore can be coupled to a wide range of fluorophores thereby

producing a large number of different dyes (a few important

examples shown in Figure 1, bottom) with potential uses in

several applications.^[9]In this work, we present our efforts to

optimize the BAPTA ionophore scaffold for fluorescent Ca²⁺

imaging and to discuss the structure-activity relationship of the

modifications on the chelating and spectrophysical properties of

the complexes.

In summary, our aim was the synthesis of seven novel

compounds (1b–7b, Figure 1) to investigate the relationship

between their structure and fluorescence properties induced by

Ca²⁺binding.

The substituent effects on the aromatic rings of BAPTA (1, Figure

[10]

1) were widely investigated earlier by Pethig and co-workers

.

Kimura et al.^[11]have measured the affinity of the ionophores

towards Ca²⁺, which decreased radically with the enlargement of

the binding pocket by extending the linker of the compound (e.g.

3a, BAPPTA in Figure 1). Analogous molecular probes for Ca²⁺

detection by NMR, known as AATA (2a in Figure 1) were also

developed. In this case one of the aromatic rings was replaced

with ethylene, combining the features of BAPTA and EGTA.^[12]

However, in none of these studies was the coupling of the

ionophores to fluorophores accomplished. This motivated us to

investigate the effects of the linker on the Ca²⁺complexation and

the fluorescence property in a comprehensive study.

Results and discussion

Chemical synthesis

Tetraethyl ester derivatives of BAPTA (1a) and BAPPTA (3a)

were prepared as previously reported (see Scheme S1 in the

supplementary information).^[2,11]Tetraethyl ester of AATA was

synthesized by a more productive way compared to the previous

attempts (Scheme 1).^[15]The original azide formation and

reduction steps were replaced by N-alkylation of diethyl

iminodiacetate using compound 8, followed by a reduction and

further alkylation, which produced the desired ester of AATA (11).

This work discusses three known (1a, 2a, 3a) and four novel

chelating scaffolds (4a–7a) containing the same tetraethyl

rhodamine fluorophore (rosamine analogues, summarized in

Figure 1 as 1b–7b). The tetraethyl rosamine fluorophore was

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Figure 1. Top: The concept of the present study. Systematic modification in of the BAPTA (1) structure using shorter linkers (4, 5, 6, 7 = ODATA), than the previously

reported modifications (2) as well as a derivative with a single phenyl group (3, AATA). Bottom: a few selected examples of the available fluorescent dyes based on

BAPTA.^[9]

Scheme 2. Synthetic route to 14. a: 2 equiv. K₂CO₃, DMF, 110°C, 2 h, b: 6 equiv.

Na₂HPO₄, 0.2 equiv. KI, DMF, 90°C,12 h.

Scheme 1. Preparation of the tetraethyl ester of AATA (11). a: 1.5 equiv. K₂CO₃,

DMF, 110°C, 2 h., b: 1.5 equiv. DIPEA, DMF, 110°C, 144 h, c: H₂(10 bar), 10%

Pd/C, EtOH, RT, 2 h. d: 6 equiv. Na₂HPO₄, 0.2 equiv. KI, MeCN, reflux, 10 h.

For the synthesis of the ionophore 5a, the Mitsunobu reaction, a

reduction and an alkylation step proved to be an effective route

Preparation of the ester of 4a was carried out from 2-nitrophenol

providing intermediate 17 with a good yield (Scheme 3).

by an alkylation followed by nitration, and reduction (Scheme 2).

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formyl group was introduced into one of the benzene rings of 11,

14, 17, 20–23, to allow the attachment of the rhodamine moiety

to the molecule,^[19]according to a well-described procedure

(Scheme 5).^[16]

Spectroscopic characterization

Fluorescence spectra of each fluorescent Ca²⁺chelating

candidates (1b–7b) were recorded in samples containing the

compounds in a c < 1 μM concentration and at pH = 7.4 (MOPS

buffer). Spectra were acquired in the absence as well as in the

presence of Ca²⁺ions the concentration of which was set to a high,

but biologically relevant level (37 μM) with a Ca²⁺-buffer, EGTA.

Figure 2 shows that the shape and position of the fluorescence

emission bands of these compounds are very similar, with

significant differences present only in their intensities. All spectra

recorded in the absence of metal ions reflect very weak emissions.

Scheme 3. Synthetic route to the tetraethyl ester 17. a: 1.1 equiv. PPh₃, 1 equiv.

DIAD, abs. THF, 0 °C → RT,1 h., recrystallisation, b: H₂, 10% Pd/C, RT, d: 6

equiv. BrCH₂COOEt, 6 equiv. Na₂HPO₄, 0.2 equiv. KI, DMF, 12 h.

For the preparation of 20, after the O-alkylation (18), PtO₂was

applied to an effective catalyst ^[17]to avoid the unwanted benzylic

cleavage instead of reduction. However, amine 19 is extremely

sensitive to oxidation and was found to decompose at elevated

temperatures during the alkylation. To avoid this, DMF solvent

was used at a lower temperature (70 °C). The desired product

(20) formed with a moderate yield after 15 hours (Scheme 4).

The intensity enhancements observed in the presence of Ca²⁺

ions suggests that all compounds bind Ca²⁺ions to some extent

and that the binding is followed by a change in their photophysical

properties. This turn on change can be explained by Ca²⁺induced

diminishing of the photoinduced electron transfer (PET)

mechanism operating otherwise in the metal ion free form of the

molecules.^[20]The observed fluorescence enhancement factors

(FEF) are between ca. 3.5 and 120 (see Table 1). Although this

confirms that all compounds show some response to Ca²⁺, the

increase in the fluorescence activity covers a broad range. The

observed values reflect that none of these molecular probes are

capable to overperform the BAPTA derivative (1b). Out of the

longer ionophore derivatives, AATA (2b), BAPPTA (3b) and 4b,

2b shows a notably larger fluorescence enhancement. The

probes, based on a shorter ionophore design (5b–7b), display a

rather mixed fluorescence response for Ca²⁺ions. The observed

fluorescence enhancements measured for 4b, 5b and 6b are

rather low (4b) or modest (5b, 6b). The compound containing only

one oxygen as a linker (7b) exhibits the second largest FEF, on

par with 2b, only superseded by the original BAPTA-derivative

(1b).

Scheme 4. Synthetic route to the tetraethyl ester of 6b. a: 1.2 equiv. NaH, DMF,

RT → 90 °C, 6 h, b: 6 equiv. BrCH₂COOEt, 6 equiv. Na₂HPO₄, 0.2 equiv. KI,

DMF, 70°C, 15 h.

The tetraethyl ester of oxydianylinyl N,N,N’,N’-tetraacetic acid

(ODATA, 7a) was prepared by employing a method reported in

the literature (Scheme S2).^[2,18]In the next synthetic step, the

Scheme 5. General synthetic route to achieve the desired fluorescent Ca²⁺molecular probes (1b–7b). a: 1.2 equiv. POCl₃in DMF, 85°C, 3 h. b: 1) 2 equiv. 2-

(diethylamino)phenol in propionic acid, 110°C, 4 h. 2) 0.95 equiv. chloranil in EtOH, RT, overnight, c: 8 equiv. aqueous NaOH 20% in 5 ml EtOH, RT, 30 min.

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Table 1. Measured spectroscopic properties of the prepared compounds without Ca²⁺ion (and in the presence of Ca²⁺ion).^[a]

λ_abs/ nm (Ca²⁺) ε / M^-1× cm^-1(Ca²⁺)

F_max/ AU

FEF^[b]

122

93

QY (Ca²⁺)^[c]

0.001 (0.113)

0.001 (0.117)

0.001 (0.052)

< 0.001 (0.001)

< 0.001 (0.009)

0.002 (0.015)

0.002 (0.077)

λ_em/ nm (Ca²⁺)^[d]

575 (578)

577 (578)

573 (577)

577 (577)

578 (577)

581 (580)

p[Ca²⁺] ^[e]

8.4910⁴(9.3710⁴)

1b

2b

3b

4b

5b

6b

7b

555 (559)

432

6.1

5.6710⁴(5.9110⁴)

553 (559)

273

5.8

7.2210⁴(7.8110⁴)

554 (557)

157

65

5.4

554 (554)

553 (554)

555 (561)

5.4

3.5

14

<< 5 ^[f]

5.7

8.0610⁴(8.4610⁴)

7.5110⁴(7.8810⁴)

9.6310⁴(1.0410⁵)

8.5710⁴(8.8510⁴)

29.3

65.9

281

10

32

^[a]Data published here (pH = 7.20 (MOPS); I = 0.1 M KCl; T = 25 ± 0.1 °C. Fluorescence intensities (F_max) are normalized to c = 1.0 μM concentrations of the studied

compounds. ^[b]Fluorescent enhancement factor: (F_max-F₀)/F₀). F₀and F_maxare the fluorescent intensity observed without (F₀) and in the presence (F_max) of Ca ion.

^[c]Fluorescence quantum yields (QY) were estimated based on the method described by Lakowicz^{[20] [d]}Wavelength of the intensity maximum of the emission bands

in the absence and presence of Ca²⁺ions. ^[e]p[Ca²⁺] values at the half of the overall fluorescence intensity change observed in the Ca²⁺-titration procedure. ^[f]These

values are below the experimental measurement limit and can be significantly smaller due to the assumed incomplete fluorescence change by the end of the titration

process terminating at [Ca²⁺] = 37 M.

concentration scale, suggesting that the Ca²⁺-binding event is

450

No Ca²⁺

1b

37 M Ca²⁺

completed within the concentration range of the titrations and that

the compounds are characterized by Ca²⁺-binding affinities falling

in the pK’_d= 5.6 – 6.2 range (Figure 3). These values, including

the p[Ca²⁺] = 5.7 for the Ca²⁺-binding step of 7b (Table 1) are

rather high and indicate remarkable Ca²⁺-sensitivities of these

sensor candidates. The steeper and more distinguished increase

in the curve of 7b offers better usability in neurobiological

applications, where the binary (active/inactive) signalling is

preferred in contrast to the exact determination of the Ca²⁺

concentration.

400

350

300

250

200

150

100

50

1b

2b

3b

4b

5b

6b

7b

2b

3b

4b

5b

6b

7b

0

565

585

605

625

 / nm

645

665

685

450

400

350

1b

2b

3b

Figure 2. Fluorescence spectra of the prepared compounds in solutions

containing no Ca²⁺ions (dashed lines) and 37 μM free Ca²⁺ions (continuous

lines). Spectra were recorded at pH = 7.4 (MOPS buffer) and normalized to 1.0

M concentrations of the compounds.

300

4b

250

5b

200

6b

150

To gain a more detailed insight into the Ca²⁺ion promoted

fluorescence response of the investigated compounds and about

their affinity towards Ca²⁺ions, Ca²⁺-titrations were performed

with all seven candidate molecules by recording their

fluorescence emission spectra in a series of solutions containing

the studied compound in a constant concentration and Ca²⁺ions

in gradually increasing concentrations. The Ca²⁺concentrations

were adjusted following a widely used protocol using EGTA as a

Ca²⁺-buffer, while the pH was kept at physiologically relevant

value of 7.2 (MOPS) and the ionic strengths at 0.1 M (KCl).^[21,22]

The fluorescence intensity values observed at the maxima of the

emission bands plotted against the concentration of Ca²⁺ion at

logarithmic scale produce classical fluorescence titration curves,

as depicted in Figure 3. The inflection points of such curves may

provide a good estimation of the apparent dissociation constants

of the complexes if the shape of the curve represents a simple

one-step association process completed by the end of the titration

protocol, such as that of the BAPTA-derivative (1b).

7b

100

50

0

-8.3

-7.8

-7.3

-6.8

-6.3

log[Ca²⁺

-5.8

-5.3

-4.8

-4.3

]

Figure 3. Titration curves representing the normalized fluorescence intensities

at the maxima of the emission bands of the studied compounds as a function of

the free Ca²⁺concentrations, set by the Ca²⁺-buffer EGTA according to the

protocol described previously ^[21,22]

.

Interestingly, the observed fluorescence response steps are

rather steep for many of the studied compounds suggesting

differences in their Ca²⁺-binding mechanisms as compared to the

BAPTA-based molecular probe. At the same time, titration curves

of other compounds might indicate that the complexation process

is not complete. Both are interesting phenomena that may initiate

further investigations but are out of the scope of the present study.

For the characterization of the Ca²⁺-binding abilities of the

investigated compounds we used the p[Ca²⁺] values at the

inflection points (where relevant) or at the half of the overall

The titration curves of 1b, 2b and 7b clearly have a sigmoidal

shape with saturating fluorescence intensities at the far end of the

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fluorescence change observed by the end of the titration protocol

(see the last column in Table 1).

Compound 3b displays an intense Ca²⁺-promoted response

around p[Ca²⁺] ~ 5.3 – 5.4, however, the continuous increase of

fluorescence even at the far end of the applied concentration

range suggests a more complex metal ion binding mechanism.

The observed poor performance of 4b, 5b and 6b suggests that

these compounds either possess weak affinity towards Ca²⁺,

preventing to reach fluorescence saturation in the studied

concentration range, or the PET quenching of the fluorophore

moiety, induced by the attached ionophores, is not fully obstructed

upon Ca²⁺-binding.

The main difference between 7b (ODATA) and the other three

novel analogues (4b–6b) is that both aromatic rings in 7b are

connected to an oxygen atom, just like in 1b–3b. In contrast, the

second aromatic ring in 4b–6b is connected to an aliphatic chain.

It would seem rational that only the fluorophore-linked aromatic

ring is responsible for the quenching of fluorescence in the

absence of Ca²⁺ions, and the fluorescence turns on when Ca²⁺

ions are coordinated. However, our data imply that the more

distant ring also contributes to the quenching, which is not

abolished if the potential Ca²⁺-binder, ring-connected oxygen

atom is missing. The question, whether the weak fluorescence

enhancement is the result of weak Ca²⁺-binding or the inefficient

induction of fluorescence via Ca²⁺-coordination (via breaking

down the internal PET) or both, cannot be unambiguously

answered based on these results. It is possible that the

fluorescence of these compounds may further increase at even

higher Ca²⁺concentrations, e.g. via a more complex metal binding

scheme. Nevertheless, too low pK_Dvalues are not relevant for the

intended use of these molecular probes, i.e. in general biological

applications.

Figure 4. Solvent-dependent energy diagram of the binding process. The

absolute enthalpy of the octacoordinated complex was taken as a reference in

all solvents and enthalpies of the hexacoordinate forms were compared to this,

by the implicit solvent model at M062X/6-31G(d,p) level of theory.

In other implicit solvent models presented in Figure 4, however,

the preferred arrangement is the symmetrical and

octacoordinated structure, which finally reaches a significant

enthalpy benefit of 26.3 kJ·mol^–1in water as a solvent (Figure 4).

Using the symmetrical conformation as

a reference, our

hypothesis for the process shown in Figure 4 was investigated for

water, MeCN, THF, benzene and heptane. An exponential

relation between the relative stability of the asymmetrical complex

and the dipole moment of the solvent was calculated (favouring

the symmetric structure in polar solvents). This finding is in

accordance with the experimental results showing a weaker

binding ability of derivatives 4b–7b.

In the next step, structural optimizations were carried out on the

disodium (model for the non-complexed form) as well as

monocalcium complexes of 1a–7a and EGTA separately, to avoid

the change of the overall charges. Despite BAPTA (1a) shares

structural motifs with EGTA, their thermodynamic stability

constants corresponding to Ca²⁺binding have a difference of ca.

four orders of magnitude (i.e. pK_{D, BAPTA}= 6.97 (T = 22 C) versus

pK_{D, EGTA}= 10.97 (T = 20 C).^[23]In order to approach the

theoretical bases of this large deviation in pK_Dfor Ca²⁺-binding

caused by the significant difference in the basicity of the two

amino functions in the two ligands,^[23]we compared the enthalpy

difference (H) of the Ca²⁺binding of BAPTA and EGTA.

According to quantum chemical calculations (Figure 5, top), the

enthalpy difference of complexation is 30.4 kJ mol^–1in favour of

EGTA. This matches the expected value based on the reported

difference of pK_D~ 4, which corresponds well to ca. 25 kJ·mol^–1

Gibbs free energy and approximately 25 kJ·mol^–1enthalpy

difference at room temperature.

Finally, we can draw the conclusion that the molecular probe

based on the original BAPTA (1b) has the highest and optimal

affinity towards Ca²⁺and the largest fluorescence enhancement

factor via Ca²⁺-binding, however, it is closely followed by the new

molecular probes 2b and 7b.

Theoretical studies

A study of the Ca²⁺ion affinity of the prepared chelators (1a–7a)

at M06-2X/6-31G(d,p) level of theory is presented, aiming to shed

light on the causes of the differences in the Ca²⁺binding with

regard to alterations in the structures. Previous computational

works have not addressed BAPTA-based chelators. Our study

revealed that the optimal conformations of complex 1a-Ca²⁺is

different in solvents of different polarity. DFT structure

optimalization

in

vacuo

predicted

an

asymmetrical,

To highlight the structure-activity relationship for the Ca²⁺-binding

of BAPTA and EGTA, we split the overall enthalpy changes into

different sub-effects caused by the various functionalities (A, B

and C). First, the conformational change of the ethylene linkers

was studied (A), which turns from all-trans (38a and 38b) to

gauche (39a and 39b, Figure 5A) in the course of the

complexation process, resulting only a negligible (H_A= –8.4 +

7.8 kJ·mol^–1= –0.6 kJ·mol^–1) difference in favour of EGTA. Second,

hexacoordinated structure for this complex, with a benefit in

enthalpy as large as 14.4 kJ·mol^–1(process shown in Figure 4)

compared to the symmetrical (octacoordinated) arrangement.

This hexacoordinated structure motivated the synthesis of new

derivatives containing one linker O atom.

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a larger difference was assigned to the hybrid state change of the

coordinating nitrogen atom (B, Figure 5B). In the EGTA model

(39a), the nitrogen atom retains its pure sp³state (pyramidity ~

333–335) during the complexation. In contrast, the nitrogen atom

in the BAPTA model (39b) is in a mixed sp²–sp³state due to the

strong conjugation with the benzene ring (pyramidity = 344),

which transforms into an sp³state during the metal ion binding,

while the above-mentioned conjugation vanishes. The

computation resulted in a H_B= –6.3 kJ·mol^–1energy benefit for

the EGTA model, confirming that the effect of conjugation (B) is

one of the major causes of EGTA being a stronger ionophore.

Third, the effect of difference in the electronic environment of the

N atom (C, Figure 5C, inset) was investigated as another potential

reason behind the strength of EGTA. In the proposed reaction

exchanges ligands to form the Ca²⁺-tetraaqua-bis-trimethylamine

complex. The enthalpy difference of this process is highly

endothermic (∆∆H_C= –18.7 kJ·mol^–1). By taking everything into

account, the total enthalpy effects sum up to ∆∆H = –31.9

kJ·mol^–1, which corresponds excellently to the value computed for

the two Ca²⁺complexes. In conclusion, we proved in silico, that

the difference between the complexation of BAPTA and EGTA

can be explained by the characteristics of the aliphatic and

aromatic N atoms, i.e. their different basicity. It was shown that an

aliphatic N atom has a larger attraction to Ca²⁺, than an aromatic

one. At pH = 7.2, however, the competing protonation equilibrium

decreases this difference and the conditional stabilities of the

Ca²⁺-binding of EGTA and BAPTA are nearly identical at this

pH.^[23]

scheme

the

Ca²⁺-tetraqua-bis-dimethylaniline

complex

-6.3

-0.6

-18.7

Enthalpy / kJ·mol^-1

-10

-30

0

-20

Σ -31.9 kJ · mol^-1

Figure 5. Complexation process of calcium ion by EGTA in the presence of BAPTA. This process is modelled by the deconvolution of

three main processes as A: conformational changes, B: nitrogen conjugation differences, C: electronic effects. The corresponding

enthalpy values were calculated at M062X/6-31G(d,p) level of theory.

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Table 2. Enthalpy (H in kJ mol^–1), Gibbs free energy (G in kJ mol^–1) and entropy (S in kJ mol^–1K^–1) values for the Ca²⁺complexation

[A], protonation [B] and their sum [A+B=C], of the aliphatic amine EGTA and 2a–7a relative to BAPTA (1a). The calculated pK_Ddata

are compared to experimental values.

Coord.

number

Complexation

Protonation

at pH = 7.4 [B]

Sum

[A+B=C]

p[Ca²⁺] ~

pK_D

experiment

al

pK_D

theoretical theoretical

pK_D

[A]

G_R

H_R

S_R

H_P

G_P

H_R

G_R

1a

EGTA

2a

3a

4a

8

7

0.0

–30.4

–37.9

38.6

25.4

56.3

43.1

60.3

0.0

–24.4

–42.1

27.2

15.1

41.7

26.4

38.8

0.0

–19.9

13.8

38.4

34.5

48.7

55.9

71.9

0.0

4.6

0.0

–0.2

0.0

–0.1

–0.9

4.8

6.1 ^[a]

6.2

7.0

6.1

35.0^[b]

0.0 ^[d]

36.7 ^[b]

0.0 ^[d]

6.7 ^[c]

5.8

–2.9 –5.4

38.6 27.2

25.4 15.1

56.3 41.7

43.1 26.4

60.3 38.8

1.3

5.4

2.7

3.5

<< 5 ^[e]

5a

6a

7.3

4.6

–1.2

1.5

6.8

–0.7

7a

5.7

dimer^[f]

0.1

19.9

–66.5

0.0 ^[d]

0.1

4.9*

0.9

5.2

^[a]experimental value used as reference value; ^[b]according to model calculation, see SI; ^[c]literature data; ^[d]only anilinyl moiety, no

difference in protonation to BAPTA (1a); ^[e]much below the current experimental measurement limit; ^[f]for 7a, two types of complex

formation were considered, a mono complex and a dimer species, see Figure 6.

We estimated the enthalpy difference between the protonation of

EGTA and BAPTA by model calculation (see SI, –35.0 kJ·mol^–1)

which indicates that EGTA can be protonated more easily than

BAPTA (column B in Table 2). The analogous thermodynamic

values (H and G), calculated for the Ca²⁺-complexation of 2a–7a

relative to BAPTA, are listed in Table 2. These were calculated

according to the same equation. Most of the calculated enthalpies

are in a good agreement with the experimental values, except for

those of 3a and 7a.

2.626

2.34-2.36

1a, BAPTA

2.34-2.36

EGTA

2a

2.610

2.34-2.36

3a

4a

5a

2.34-2.36

7a

6a

7a-dimer

Figure 6. Optimized structures of the ionophores EGTA and 1a–7a in the presence of Ca²⁺calculated at M062X/6-31G(d,p) level of

theory. For 7a, two types of complex formation were considered, such as 7a and 7a-dimer, where the dimeric structure was modelled

by one of the two Ca²⁺centres surrounded by the coordinating donor atoms from two ligands. C–N and C–O bond lengths are given in

blue and red numbers (in Ångstrom units), respectively. For details, see Table S1 in the supporting information. Binding cavities are

illustrated by orange clouds.

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Based on the similar equilibrium enthalpies in Table 2 for the Ca²⁺

complexation of EGTA and BAPTA it can be assumed that they

possess the same coordination mode (8-coordination) and one

aliphatic N atom of 2a can produce the same effect as two in 1a.

However, the competing protonation process on the aliphatic N in

EGTA decreases the overall pK_Dsimilar to that of BAPTA.

Interestingly, the sign of the calculated entropy change is the

opposite in these two cases (EGTA and 2a). The computation for

results, we raise the possibility of the existence of a unique

binding model, i.e. the formation of a dimeric complex, in this latter

chelator. This study not only contributes to the clarification of why

BAPTA currently performs so well in Ca²⁺capture, but also opens

up new possibilities for further structural optimizations that target

specific sensing applications.

Experimental Section

the compound bearing

a longer linker (3a) significantly

Reagents and solvents were purchased from Sigma Aldrich in

reagent grade and used as received, while NMR solvents were

purchased from Eurisotop. Thin layer chromatography (TLC) was

performed on commercially available pre-coated TLC plates

(Merck Silica gel 60 F₂₅₄aluminium sheets or Merck Aluminium

oxide 60 F₂₅₄plates). Visualisation was either achieved under UV

light at 254 nm or by exposing the plates to iodine or to the

aqueous solution of (NH₄)₆Mo₇O₂₄, Ce(SO₄)₂and sulphuric acid.

Melting points were determined on a Büchi M-560 capillary

melting point apparatus and are uncorrected. For flash column

chromatography an Interchim PuriFlash XS 420 system was

employed using gradient elution on normal phase (silica or

aluminium oxide column; hexane−EtOAc or DCM/MeOH as

eluents). Preparative HPLC was applied for purification in many

cases using an Armen SPOT Prep II instrument with UV detector

(200-600 nm scan) equipped with a Phenomenex Gemini C18,

250×50.00 mm; 10 µm, 110A column. Gradient elution was

employed using 0.4 g NH₄HCO₃in 1 L water (A) and acetonitrile

(B) or 10 mL TFA in 1 L water (A) and acetonitrile (B) as eluent

systems. NMR spectra were recorded on Varian Unity INOVA

spectrometers operating at an equivalent ¹H frequency of 400 and

underestimates the complexation enthalpy, compared to the

experimental results. The predicted weak binding of 3a to Ca²⁺

could be due to the decrease of the coordination number to 7. In

our in silico model one of the etheric O atoms does not coordinate

to Ca²⁺. Similarly, compounds 4a, 5a and 6a also lack the second

O atom, and thus the largest possible coordination number for

Ca²⁺ion is 7, hence the relative enthalpies of the corresponding

complexation processes are endothermic. Interestingly, the best

fitting among 4a, 5a and 6a was found for 4a that has the same

linker length as BAPTA (Figure 6). For 7a, the calculated

unbeneficial binding enthalpy and the corresponding pK_Dvalues

are in sharp contrast with the experimental observation, as this

compound experimentally displayed efficient Ca²⁺binding ability.

To resolve this contradiction, one can look for alternative binding

possibilities. Supposing a possible dimer complex formation

proposed in Figure 6 resulted in a more beneficial enthalpy value

(Table 2).

1

500 MHz. Notations for the H NMR spectral splitting patterns

Conclusion

include singlet (s), doublet (d), triplet (t), broad (br) and

multiplet/overlapping peaks (m). Chemical shifts of the

resonances are given as δ values in ppm and coupling constants

(J) are expressed in Hertz. Individual resonances were assigned

on the basis of DEPT 135, 1D (NOESY) and 2D NMR (COSY,

HSQC and HMBC) experiments. HRMS spectra were recorded

In this study, a systematic structural modification of the BAPTA

ionophore was performed and seven new fluorescent

BAPTA-analogues were successfully synthesized and subjected

to spectrophysical characterization. The excitation and emission

wavelength values in addition to the absorption and emission

intensities were studied in the presence and absence of Ca²⁺ions.

The affinities of the compounds towards the desired analyte ion

were characterized based on fluorescence-monitored Ca²⁺

titration data. Our structure–activity relationship results suggest

that even a ring at greater distance is able to quench the

fluorescence in the presence of Ca²⁺ions if there are no oxygen

on

a

Q Exactive Plus hybrid quadrupole-orbitrap mass

spectrometer (Thermo Scientific, Waltham, MA, USA) equipped

with a heated electrospray ionization (HESI-II) probe that was

used in positive and negative ion mode. Samples were introduced

with FIA (flow injection analysis) method and the eluent stream

(water and acetonitrile in a 1:1 volume ratio with 0.1 % formic acid)

was provided by a Waters UPLC (Waters, Milford, USA).

UV-Vis absorption spectra were recorded were recorded on a Thermo

Scientific Evolution 220 spectrometer in the wavelength range of 220–

700 nm using a quartz cuvette (pathlength = 5.0 cm). The general

sample preparation protocol involved the dissolution of 1–3 mg of the

studied compounds in 10.0 – 15.0 mL of MOPS buffer (c = 0.03 M, pH

= 7.2) that were diluted further to the concentrations used for the

fluorescence Ca²⁺-titrations by ‘Buffer A’ or ‘Buffer B’ (typically to c_dye

< 1.0 M). ‘Buffer A’ and ‘Buffer B’ are solutions containing the pH-

buffer MOPS (c = 0.03 M), KCl background electrolyte (c = 0.1 M) and

the Ca²⁺-buffer EGTA (c = 0.01 M), adjusted to a pH value of 7.2.

Besides these components, ‘Buffer B’ contains Ca²⁺ions (CaCl₂) in a

concentration identical to that of EGTA (c = 0.01 M). These

background-corrected spectra of the relevant buffers (‘Buffer A’ or

‘Buffer B’) were subtracted from the sample spectra. These

background-corrected spectra were used for determining the

wavelength of absorption maxima (λ_abs) and the absorbances

normalized to 1.0 M concentrations of the dyes (ε). Fluorescence

emission spectra were acquired on a Hitachi F-4500 fluorescence

spectrophotometer using a quartz cell (pathlength = 1.0 cm). Slit

widths were selected to provide 2.5 nm bandpass both for the

excitation and emission beams. Emission data were normalized to the

intensity of an external reference probe body, recorded every day.

Background emission spectra of the relevant buffers (‘Buffer A’ or

atoms directly connected to this ring.

A comprehensive

computational study was executed to reveal further information

regarding the role of the structure in the complexation process.

The optimal structure of the complexes depends on the polarity of

the applied solvent model continuum. In apolar solvents or

vacuum, an asymmetric hexacoordinated form is preferred. On

the other hand, in polar models, the symmetric octacoordinated

arrangement becomes preferable. The structural analysis

confirmed that the hybrid state and the electronic environment of

the coordinating N atom is responsible mainly for the difference in

the affinity of EGTA and BAPTA towards Ca²⁺. In conclusion, an

optimal chain length between the aromatic rings, an exact cavity

volume and advantageous dynamic characteristics are present in

BAPTA, which explains its effectiveness in calcium binding.

These features are similarly present in the fluorescent derivative

of AATA developed in this work. Moreover, the steep Ca²⁺-

induced fluorescence turn-on step observed for 7b (ODATA) is

expected to be the most useful feature in neurobiological

diagnostic applications in which the detection of cellular signals

with a large contrast is demanded. According to the computational

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‘Buffer B’) were subtracted from the spectra of samples. Samples

were excited at the wavelength values of their absorption maxima

(λ_abs) determined during UV-Vis absorption measurements. Quantum

yields were calculated by method published earlier.^[20]

(1.1 mmol, 100 μL, 1.1 equiv.) in 10 ml DMF. After stirring at room

temperature for 2 hours, the mixture was neutralized by the

addition of 10 w/w % aqueous solution of NaHCO₃. The organic

compounds were extracted with EtOAc, the organic layers were

combined and the solvent was removed under reduced pressure.

The product was purified using preparative HPLC.

Fluorescence-monitored Ca2+-titrations were executed by using

a solution of the selected dye molecule containing the Ca2+-

buffer EGTA in a concentration of 0.01 M but no Ca2+ ions. These

starting solutions were made by the dilution of the stock solution

of the dye into ‘Buffer A’. Another solution, containing the dye in

the same concentration, was also prepared by using ‘Buffer B’.

This latter solution contained the Ca2+-buffer EGTA and Ca2+

ions at identical, 0.01 M concentration, setting the free Ca2+-level

to 37 M. For the measurement of the first (0) point of the titration

curve 2.0 mL of the sample made with ‘Buffer A’ was used. In the

titration process various (typically increasing) volume fractions of

the sample were replaced by the same volumes of the solution

made with ‘Buffer B’. Fine details of this fluorescence titration

protocol have been described previously.^[21,22]

[16]

General procedure 4 for coupling to the fluorophore

.

Formylated compound (1 mmol, ca. 500 mg) was dissolved in 5

ml propionic acid and 3-diethylaminophenol (363,5 mg, 2.2 mmol,

2.2 equiv.) was added to the mixture. This solution was stirred

under microwave irradiation in a sealed tube at 80 °C for 2-4 hours,

then cooled to room temperature. A suspension of chloranil (233

mg, 0.95 mmol, 0.95 equiv.) in 5 ml DCM/EtOH 1:1 was added to

the reaction mixture and it was stirred at room temperature for

overnight to promote the oxidative ring closure. The reaction

mixture was concentrated and purified by preparative HPLC to

afford the desired product.

General procedure 5 for hydrolysis of the ethyl esters. Ethyl ester

(0.1 mmol) was dissolved in 5 ml EtOH and 20 w/w% NaOH

solution (200 mg, ca. 200 μl, 1 mmol, 10 equiv.) was added to the

solution and stirred for 20 minutes at room temperature. The

reaction mixture was injected to preparative HPLC. The collected

fractions were lyophilized to yield the desired final products.

Density-functional theory (DFT) computation was performed with

the Gaussian 16 program package (G16) ^[24], using convergence

criteria of 3.0 × 10^–4, 4.5 × 10^–4, 1.2 × 10^–3and 1.8 × 10^–3, for the

gradients of the root mean square (RMS) force, maximum force,

RMS displacement, and maximum displacement vectors,

respectively. Computation was carried out at M062X/6 31G(d,p)

level of theory. ^[25]The vibrational frequencies were computed at

the same levels of theory, as used for geometry optimization, in

order to properly confirm that all structures reside at minima on

their potential energy hypersurfaces (PESs). Thermodynamic

functions, such as energy (U), enthalpy (H), Gibbs free energy (G),

and entropy (S) were computed for 298.15 K, using the quantum

chemical, rather than the conventional thermodynamic reference

state.

Preparation of the fluorescent derivatives 1b and 3b (See

Figure S1 in Supplementary Information)

The synthesis of BAPTA ethyl ester (21) and BAPPTA ethyl ester

(22) were carried out following the procedure described in

references ^[2,11]with minor modifications. K₂CO₃(7.3 g, 53 mmol,

1.5 equiv.) was suspended in 40 ml DMF and a solution of 2-

nitrophenol (4.9 g, 35.3 mmol, 1.0 equiv.) in 10 ml DMF was

added under stirring. To this red solution 8.8 ml (15.2 g, 106.5

mmol, 3.0 equiv.) of 1-bromo-2-chloroethane was added in the

case of BAPTA ethyl ester (21), while 9.8 ml (16.8 g, 106.5 mmol,

3 equiv.) of 1-bromo-2-chloropropane in the case of BAPPTA

ethyl ester (22). 880 mg (5.3 mmol, 0.1 equiv.) of KI was added

to each mixture and the solutions were stirred at 90 °C for 3 hours,

then the solvents were removed under reduced pressure and the

Synthesis and characterization

General procedure 1 for reduction. The nitro compound was

suspended in EtOH and 5% of Pd/C (10 w/w % Pd in carbon) was

added to the suspension. The mixture was flushed with argon in

an autoclave, then filled with H₂(10 bar pressure) and stirred

overnight at room temperature. The suspension turned into yellow, solid residues were dissolved in EtOAc and extracted with

it was filtered and concentrated in vacuo to yield the desired

product, which was used in the next step without further

purification.

saturated Na₂CO₃, then with brine. The organic phase fractions

were dried over MgSO₄, then the solvent was removed under

reduced pressure. The resulting brown oil was dissolved in 30 ml

DMF containing 7.3 g of suspended K₂CO₃(53 mmol, 1.5 equiv.).

880 mg (5.3 mmol, 0.1 equiv.) of KI and 4.9 g 2-nitrophenol (35.3

mmol, 1 equiv.) was added and the mixture was stirred at 110 °C

for 2 hours. The brownish mixture was poured on crushed ice.

The white precipitate was filtered from the orange mother liqueur

and washed with water, then dried overnight. Carrying out the

reaction in two steps resulted in higher product purity. The

products 1,2-bis(2-nitrophenoxy)ethane (S2) (beige powder,

yield: 9.66 g, 90%, melting point: 158 °C, ref 167 °C), and 1,2-

bis(2-nitrophenoxy)propane (S5) (beige powder, yield: 14.4 g,

90%, melting point: 108 °C, ref 106 °C) were used without further

purification.

General procedure 2 for N-alkylation. 27 mmol of the amino

compound was dissolved in 20 ml MeCN, then Na₂HPO₄(23 g,

162 mmol, 6 equiv.), KI (450 mg, 2.7 mmol, 0.1 equiv.) and 17.9

ml ethyl bromoacetate (27 g, 162 mmol, 6 equiv.) were added to

the reaction mixture before stirring at 90°C for 10-18 hours. The

complete conversion to the tetraalkyl derivative was assured and

confirmed by HPLC-MS, since the removal of underalkylated

derivatives is difficult. The solvent was removed under reduced

pressure. The resulting solids were dissolved in 150 ml water and

100 ml EtOAc was added. The layers were separated and the

aqueous phase was washed with 2×100 ml EtOAc. The combined

organic phases was washed with water, then with brine and dried

over MgSO₄. The solvent was removed under reduced pressure

and the resulting oil was suspended in hexane, then cooled to

5 °C. The pure product precipitated as brownish crystals. If the

crystallization did not occur (11, 14), flash chromatography was

used for purification on silica gel using a gradient elution (0% →

20% EtOAc in hexane). For compound 11, the quantity of ethyl

bromoacetate and the base was halved.

Hydrogenations were carried out under pressure (10 bar),

compared to the previous methods,^[2]using general procedure 1.

described in S3.1.1. starting from the quantities from the quantity

given above. The products 1,2-bis(2-aminophenoxy)ethane (S3)

(yield: 7.13 g, 92%) and 1,2-bis(2-aminophenoxy)propane (S6)

(yield: 10.72 g, 94%) were isolated as yellow oils.

Alkylations were performed based on general procedure 2,

starting from 6.58 g and 6.96 g of amine. The Ethyl ester products

21 (yield: 14.45 g, 91%, melting point: 99 °C, ref 95-97 °C) and 22

[19]

General procedure 3 for formylation.

2 mmol of the ethyl

esters in 5 ml DMF were added to a cool (0 °C) solution of POCl₃

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(yield: 14.62 g, 90%, melting point: 77 °C, ref 76 °C) were isolated

as brown crystals. The spectroscopic properties of all above

compounds are in agreement with previously published data.^[2,11]

melting point: decomposed at 116 °C.) were isolated as red

powders. The spectroscopic properties of compound 1b: ¹H NMR

(400 MHz, [D₆]DMSO) δ = 12.55 (bs, 4H, C[19-22]OO-H), 7.50 (d,

J = 8.2 Hz, 2H, C[25, 34]-H), 7.19 (d, J = 1.9 Hz, 1H, C[5]-H), 7.16

(dd, J = 8.2, 2.2 Hz, 2H, C[26, 33]-H), 7.11 – 6.72 (m, 8H, C[2, 3,

10-13, 28, 31]-H), 4.31 (t, J = 7.0 Hz, 4H, C[7, 8]-H), 4.24 (s, 4H,

C[15-16]-H), 4.02 (s, 4H, C[17-18]-H), 3.96 (q, J = 7.1 Hz, 8H,

C[36-39]-H), 1.20 (t, 12H, J = 7.0 Hz, C[19-22]-H). ¹³C {¹H} NMR

(101 MHz, [D₆]DMSO) δ = 172.5, 172.3, 157.4, 156.7, 154.9,

149.4, 148.4, 141.3, 139.3, 132.2, 123.9, 122.8, 121.5, 121.1,

118.2, 116.8. 116.1, 114.8, 114.2, 112.5. 96.0, 67.5, 67.0, 53.7,

53.5, 45.3, 12.5. HRMS (ESI+): m/z calcd for C₄₃H₄₉N₄O₁₁⁺([M]⁺):

797.33923, found: 797.33812.

The formylations were performed according to general procedure

3 from 1 mmol of starting materials. The aldehyde compounds 24

(yield: 262 mg, 42.5%, melting point: 109 °C; ref 139 °C.) and 26

(yield: 296 mg, 47%, melting point: 83 °C.) were isolated as brown

solids. The spectroscopic properties of compound 24 are in

agreement with data previously reported.^[19]The spectroscopic

1

properties of compound 26: H NMR (400 MHz, [D₆]DMSO) δ =

(s, 1H, C[32]–H), 7.39 (dd, J = 8.2, 1.8 Hz, 1H, C[3]-H), 7.33 (d, J

= 1.9 Hz, 1H, C[5]-H), 6.89 (dd, J = 7.8, 1.9 Hz, 1H, C[14]-H), 6.85

– 6.76 (dt, J = 14.9, 7.3, 1.8 Hz, 2H, C[12-13]-H), 6.71 – 6.65 (m,

2H, C[2, 11]-H), 4.19 (s, 4H, C[16-17]-H), 4.12 – 4.02 (m, 16H,

C[7,9,18-19,20-23]-H), 2.05 (t, J = 6.2 Hz, 2H, C[8]-H), 1.15 (dt, J

= 14.1, 7.1 Hz, 12H, C[24-27]-H). ¹³C {¹H} NMR (101 MHz,

[D₆]DMSO) δ = 190.7, 170.8, 170.3, 149.7, 148.7, 144.5, 138.7,

128.8, 125.5, 121.3, 120.8, 118.1, 115.9, 113.2, 111.4, 65.5, 65.1,

60.4, 60.1, 53.7, 53.3, 28.5, 14.1. HRMS (ESI+): m/z calcd for

C₃₂H₄₃N₂O₁₁⁺([M+H]⁺): 631.28614, found: 631.28611.

The spectroscopic properties of compound 3b: ¹H NMR (400 MHz,

[D₆]DMSO) δ = 12.55 (s, 4H, C[20-23]OO-H), 7.49 (d, J = 9.6 Hz,

2H, C[26, 35]-H), 7.11 (dd, J = 9.6, 2.5 Hz, 2H, C[27, 34]-H), 7.06

(d, J = 2.0 Hz, 1H, C[5]-H), 7.00 (dd, J = 8.2, 1.9 Hz, 1H, C[3]-H),

6.94 (d, J = 2.5 Hz, 2H, C[29, 32]-H), 6.91 – 6.86 (dd, J = 9.6, 2.5

Hz, 1H, C[14]-H), 6.83 (d, J = 8.3 Hz, 1H, C[2]-H), 6.76 (dt, J =

7.1, 6.4, 3.6 Hz, 2H, C[12-13]-H), 6.66 – 6.59 (m, 1H, C[11]-H),

4.16 (s, 4H, C[16-17]-H), 4.11 (t, J = 6.4 Hz, 2H, C[9]-H), 4.07 (t,

J = 5.9 Hz, 2H, C[7]-H), 3.92 (s, 4H, C[18-19]-H), 3.64 (q, J = 7.0

Hz, 8H, C[37-40]-H), 2.19 – 2.10 (m, 2H, C[8]-H), 1.21 (t, J = 7.0

Hz, 12H, C[41-44]-H). ¹³C {¹H} NMR (101 MHz, [D₆]DMSO) δ =

172.6, 172.3, 157.4, 156.8, 154.8, 149.5, 148.7, 141.0, 138.9,

132.1, 123.4, 122.8, 120.9, 120.7, 117.5, 116.4, 114.7, 114.2,

113.0, 112.4, 95.9, 65.7, 64.9, 53.6, 53.4, 45.2, 28.7, 12.5. HRMS

The couplings to the fluorophores were carried out using the

general procedure 4 using the quantities given above. The

tetraethyl ester compounds 31 (yield: 119 mg, 20 %, melting point:

decomposed) and 33 (yield: 205 mg, 22 %, melting point:

decomposed) were isolated as red powders.

The spectroscopic properties of compound 31: ¹H NMR (400 MHz,

[D₆]DMSO) δ = 7.46 (d, J = 9.6 Hz, 2H, C[33, 42]-H), 7.16 – 7.09

(m, 3H, C[5, 34, 41]-H), 7.03 (dd, J = 8.2, 1.9 Hz, 1H, C[3]-H),

6.94 (dd, J = 15.3, 2.2 Hz, 3H, C[13, 36, 39]-H), 6.89 – 6.81 (m,

3H, C[2, 11-12]-H), 6.68 (dd, J = 7.3, 2.3 Hz, 1H, C[10]-H), 4.26 –

4.13 (m, 8H, C[7-8, 15-16]-H), 4.06 (s, 4H, C[17-18]-H), 3.96 (dq,

J = 21.7, 7.1 Hz, 8H, C[44-47]-H), 3.67 (s, 8H, C[19-22]-H), 1.21

(t, J = 7.0 Hz, 12H, C[48-51]-H), 1.06 (td, J = 7.1, 5.3 Hz, 12H,

C[23-26]-H). ¹³C {¹H} NMR (101 MHz, [D₆]DMSO) δ = 170.8,

170.7, 157.4, 156.8, 154.9, 149.5, 148.8, 140.7, 138.8, 132.0,

123.4, 123.3, 121.3, 121.1, 117.9, 116.9, 114.8, 114.2, 113.1,

112.6, 96.0, 67.5, 66.8, 60.4, 60.1, 53.3, 53.1, 45.3, 13.9, 13.9,

12.5. HRMS (ESI+): m/z calcd for C₅₁H₆₅N₄O₁₁⁺([M]⁺): 909.46444,

found: 909.46217.

+

(ESI+): m/z calcd for C₄₄H₅₁N₄O₁₁([M]⁺): 811.35488, found:

811.35354.

Preparation of the fluorescent dye 2b. (See Figure S2 in

Supplementary Information)2-nitrophenol (10 g, 72 mmol, 1.0

equiv.) was dissolved in DMF (150 ml), then K₂CO₃(14.9 g,

108 mmol, 1.5 equiv.), and KI (1.19 g, 7.2 mmol, 0.1 equiv.) were

added. The mixture was stirred at 110 °C, 2-chloroethyl ether

(30.8 g, 25.3 ml, 216 mmol, 3.0 equiv.) was added and stirred for

2 hours at 110 °C. The solvents were removed under reduced

pressure and the solid residue was dissolved in EtOAc and water.

The layers were separated, the organic phase was extracted with

saturated aqueous Na₂CO₃and brine. The mixture was dried over

MgSO₄, the solvent was removed under reduced pressure.

Mononitro compound 8 (brown oil, yield: 21.7 g, 92%) was used

in the next step without further purification. The spectroscopic

properties of 8 are in good agreement with those published

previously.^[26]

The spectroscopic properties of compound 33: ¹H NMR (400 MHz,

[D₆]DMSO) δ = 7.48 (d, J = 9.6 Hz, 2H, C[24, 43]-H), 7.16 – 7.05

(m, 3H, C[5, 35, 42]-H), 6.97 (dd, J = 19.3, 5.2 Hz, 3H, C[2, 37,

40]-H), 6.89 – 6.83 (m, 2H, C[3, 14]-H), 6.77 (dt, J = 13.9, 7.0 Hz,

2H, C[12, 13]-H), 6.61 (dd, J = 7.6, 2.1 Hz, 1H, C[11]-H), 4.23 (s,

4H, C[16-17]-H), 4.18 – 3.93 (m, 16H, C[7, 9, 18-19, 20-23]-H),

3.64 (d, J = 7.1 Hz, 8H, C[45-48]-H), 2.05 (p, J = 6.2 Hz, 2H, C[8]-

H), 1.21 (td, J = 7.0, 4.4 Hz, 18H, C[24-25, 49-52]-H), 1.09 (t, J =

7.1 Hz, 6H, C[26-27]-H). ¹³C {¹H} NMR (101 MHz, [D₆]DMSO) δ =

170.9, 170.7, 158.2, 157.9, 157.4, 156.8, 154.9, 149.5, 148.8,

140.6, 138.6, 132.1, 123.3, 121.3, 120.8, 117.9, 114.7, 114.2,

113.0, 112.5, 104.6, 96.0, 65.7, 60.4, 60.1, 53.5, 53.2, 45.3, 28.6,

14.2, 14.1, 12.5. HRMS (ESI+): m/z calcd for C₅₂H₆₇N₄O₁₁⁺([M]⁺):

923.48009, found: 923.47866.

11.5 g (37.5 mmol, 1.0 equiv.) of 8 was dissolved in 100 ml DMF

and 10.1 g DIPEA (77.9 mmol, 13.6 ml, 2.0 equiv.). 12. 8 g diethyl

iminodiacetate (67.5 mmol, 12.1 ml, 1.8 equiv.) was added and

the mixture was stirred at 120 °C for 2 days. Additional 7.09 g

(37.5 mmol, 6. 7 ml, 1.0 equiv.) diethyl iminodiacetate and 2 g (12

mmol, 0,32 equiv.) of KI were added, then stirred for 4 days. The

solvents were removed under reduced pressure, the solid residue

was dissolved in EtOAc and the mixture was extracted with

saturated aqueous Na₂CO₃, then with brine. The organic phase

was dried over MgSO₄, then the solvent was removed under

reduced pressure. Product 9 was isolated as a brown oil (yield:

21.8 g, 80%). ¹H NMR (400 MHz, [D₆]DMSO) δ = 7.86 (dd, J=8.1,

The hydrolysis of the ethyl esters were carried out using the

general procedure 5. using the quantities given above. The

fluorescent dyes 1b (red powder, yield: 38 mg, 62 %, melting

point: decomposed at > 110 °C.) and 3b (yield: 52 mg, 85 %,

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1.7 Hz, 1H, C[2]-H), 7.64 (ddd, J=8.8, 7.4, 1.7 Hz, 1H, C[3]-H),

7.37 (dd, J=8.6, 1.1 Hz, 1H, C[5]-H), 7.12 (dt, J=8.3, 7.4, 1.1 Hz,

1H, C[4]-H), 4.26 (t, J=5.2 Hz, 2H, C[7]-H), 4.08 (q, J=7.1 Hz, 4H,

C[13-14]-H), 3.80 (s, 4H, C[11-12]-H), 3.76 (t, J=5.2 Hz, 2H, C[8]-

H), 3.67 (t, J=5.2 Hz, 2H, C[9]-H), 3.10 (t, J=5.2 Hz, 2H, C[10]-H),

1.16 (t, J=7.1 Hz, 6H, C[15-16]-H). ¹³C {¹H} NMR (101 MHz,

[D₆]DMSO) δ = 169.1, 151.3, 139.6, 134.4, 125.0, 120.7, 115.3,

69.0, 68.5, 68.1, 60.6, 54.9, 53.7, 13.9. HRMS (ESI+): m/z calcd

for C₁₈H₂₇N₂O₈⁺([M+H]⁺): 399.17620, found: 399.17621.

The hydrolysis of this ethyl ester was carried out using the

General procedure 5 staring from the quantity given above. The

fluorescent dye 2b (yield: 19 mg, 64%) was isolated as blue

1

crystals. Melting point: decomposed at > 110 °C. H NMR (400

MHz, [D₆]DMSO) δ = 7.49 (d, J=9.6 Hz, 2H, C[21, 30]-H), 7.14

(dd, J=9.6, 2.4 Hz, 2H, C[22, 29]-H), 7.09 (d, J=2.0 Hz, 1H, C[5]-

H), 7.02 (d, J=8.3 Hz, 1H, C[2]), 6.95 (d, J=2.4 Hz, 2H, C[24, 27]-

H), 6.85 (d, J=8.3 Hz, 1H, C[3]-H), 4.18 (s, 4H, C[11-12]-H), 4.07

(t, J=4.5 Hz, 2H, C[7]-H), 3.82 (s, 4H, C[13-14]-H), 3.66 (m, 12H,

C[8-9, 32-35]-H), 3.18 (s, 2H, C[10]-Hf), 1.21 (t, J=7.0 Hz, 12H,

C[36-39]-H). ¹³C {¹H} NMR (101 MHz, [D₆]DMSO) δ = 172.2,

170.1, 157.4, 156.8, 154.9, 148.5, 141.0, 132.1, 123.6, 122.7,

116.6, 115.3, 114.2, 112.5, 96.0, 68.6, 68.0, 55.0, 54.3, 53.6, 45.3,

Reduction of 9 was carried out according to general procedure 1

starting from 26 mmol of staring material. The amine compound

1

10 (yield: 9,3 g, 97%) was isolated as a brown oil. H NMR (400

+

36.5, 12.5. HRMS (ESI+): m/z calcd for C₃₉H₄₉N₄O₁₁([M]⁺):

MHz, [D₆]DMSO) δ = 7.21 – 7.11 (m, 3H, C[2-3, 5]-H), 6.96 (dt,

J=8.3, 7.3, 1.1 Hz, 1H, C[4]-H), 4.17 (t, J=5.2 Hz, 2H, C[7]-H),

4.08 (q, J=7.1 Hz, 4H, C[13-14]-H), 3.74 (t, J=5.5 Hz, 2H, C[8]-H),

3.69 (s, 4H, C[11-12]-H), 3.63 (t, J=5.5 Hz, 2H, C[9]-H), 2.98 (t,

J=5.5 Hz, 2H, C[10]-H), 1.18 (t, J=7.1 Hz, 6H, C[15-16]-H). ¹³C

{¹H} NMR (101 MHz, [D₆]DMSO) δ = 170.0, 150.2, 121.6, 121.3,

117.3, 114.4, 113.5, 68.8, 68.7, 68.3, 60.2, 55.1, 53.3, 14.0.

749.33923, found: 749.33765.

Preparation of the fluorescent dye 4b.

To perform the initial alkylation shown in Scheme 2, 2-nitrophenol

(10 g, 72 mmol, 1.0 equiv.) was dissolved in dry DMF (20 ml), then

K₂CO₃(19.9 g, 144 mmol, 2.0 equiv.) was added. The mixture

was stirred at 110 °C for 2 hours after the addition of

(3-bromopropyl)benzene (15.8 g, 11.7 ml, 79.2 mmol, 1.1 equiv.).

The formed solids were filtered out. Mononitro compound 12

(yield: 17.4 g, 94%) was isolated as brown oil. The spectroscopic

properties of the compound are in good agreement with the ones

reported in the literature.^[27]

+

HRMS (ESI+): m/z calcd for C₁₈H₂₉N₂O₆([M+H]⁺): 369.20202,

found: 369.20183.

Alkylation of 10 was carried out according to general procedure 2.

starting from 9.3 mmol of starting compound to yield 11. The

quantity of ethyl bromoacetate was halved compared to other

compounds. The ethyl ester compound 11 (yield: 3,5 g, 70%) was

isolated as a brown oil. The spectroscopic properties of 11 are in

good agreement with those published.^[12]The formylation was

performed according to general procedure 3 starting from 2.5

mmol staring material. The aldehyde compound 25 (yield: 470 mg,

To achieve precursor 13, 9.5 g of 12 (37 mmol, 1.0 equiv.) was

dissolved in 30 ml 65% HNO₃(7.5 equiv.) and 1 ml of conc. H₂SO₄

was added. The mixture was stirred at 110 °C for 1 hour, then

poured on 100 ml icy water. The aqueous phase was extracted

with 3x50 ml EtOAc. The organic layer was washed with 30 ml of

saturated aqueous Na₂CO₃, then washed with brine, dried over

MgSO₄and concentrated to give 9.1 g of yellow oil. The reduction

step was carried out according to general procedure 1. starting

from the mixture of isomers present in the crude product. The

isomers were separated at the end of reduction by preparative

HPLC. The amine compound 13 (yield: 3.13 g, 35 % for the overall

process) was isolated as an off-white powder. Melting point:

1

35 %) was isolated as a brown viscous oil. H NMR (400 MHz,

[D₆]DMSO) δ = 9.75 (s, 1H, C[27]-H), 7.41 (dd, J=8.3, 1.8 Hz, 1H,

C[3]-H), 7.35 (d, J=1.8 Hz, 1H, C[5]-H), 6.75 (d, J=8.3 Hz, 1H,

C[2]-H), 4.25 (s, 4H, C[11-12]-H), 4.17 – 4.02 (m, 10H, C[7, 15-

18]-H), 3.67 (s, 6H, C[8, 13-14]-H), 3.59 (t, J=5.6 Hz, 2H, C[9]-H),

2.98 (t, J=5.5 Hz, 2H, C[10]-H), 1.18 (dt, J=14.8, 7.1 Hz, 12H,

C[19-22]-H). ¹³C {¹H} NMR (101 MHz, [D₆]DMSO) δ = 190.4,

170.0, 169.9, 148.6, 144.4, 128.7, 125.3, 116.1, 112.4, 68.6, 68.3,

67.9, 60.2, 60.0, 55.0, 53.7, 53.4, 13.9, 13.9. HRMS (ESI+): m/z

calcd for C₂₇H₄₁N₂O₁₁⁺([M+H]⁺): 569.27049, found: 569.27001.

1

183 °C. H NMR (400 MHz, [D₆]DMSO) δ = 7.24 – 6.91 (m, 8H,

C[2-5, 11-14]-H), 4.07 (t, J = 6.1 Hz, 2H, C[7]-H), 2.77 (t, J = 6.1

Hz, 2H, C[9]-H), 2.03 (q, J = 7.9 Hz, 2H, C[8]-H). ¹³C {¹H} NMR

(100 MHz, [D₆]DMSO) δ = 150.1, 137.4, 130.2, 130.0, 127.2,

126.0, 125.1, 123.1, 121.3, 120.9, 119.7, 112.6, 67.6, 28.6, 26.4.

HRMS (ESI+): m/z calcd for C₁₅H₁₉N₂O⁺([M+H]⁺): 243.14919,

found: 243.14890.

The coupling to the fluorophore was carried out using the general

procedure 4 starting from 0.6 mmol of formyl compound. The

tetraethyl ester compound 32 (yield: 80 mg, 15%) was isolated as

1

blue crystals. Melting point: decomposed at > 145 °C. H NMR

(400 MHz, [D₆]DMSO) δ = 7.47 (d, J=9.6 Hz, 2H, C[29, 38]-H),

7.20 – 7.06 (m, 3H, C[5, 30, 37]-H), 7.01 (dd, J=8.2, 1.9 Hz, 1H,

C[2]-H), 6.94 (d, J=2.5 Hz, 2H, C[32, 35]-H), 6.92 (d, J=8.2 Hz,

1H, C[3]-H), 4.27 (s, 4H, C[11-12]-H), 4.16 (q, J=7.1 Hz, 4H, C[15-

16]-H), 4.09 (t, J=5.8 Hz, 2H, C[7]-H), 4.03 (q, J=7.1 Hz, 4H, C[17-

18]-H), 3.66 (m, 10H, C[8, 40-43]-H), 3.54 (t, J=5.8 Hz, 2H, C[9]-

H), 3.51 (s, 4H, C[13-14]-H), 2.85 (t, J=5.8 Hz, 2H, C[10]-H), 1.23

(t, J=7.2 Hz,18H, C[19-20, 44-47]-H), 1.14 (t, J=7.1 Hz, 6H, C[21-

22]). ¹³C {¹H} NMR (101 MHz, [D₆]DMSO) δ = 170.5, 170.2, 164.1,

157.3, 154.8, 148.8, 140.6, 131.8, 123.2, 117.2, 115.8, 114.1,

112.4, 95.9, 69.3, 68.4, 68.2, 60.0, 59.5, 55.0, 53.4, 53.0, 45.1,

Alkylation of 13 was carried out according to general procedure 2.

from 4 mmol of amine to yield 14. The ethyl ester compound 14

(yield: 0.94 g, 40%) was isolated as brown crystals. ¹H NMR (400

MHz, [D₆]DMSO) δ = 7.25-7.15 (m, 2H, C[11, 14]-H), 7.12 (td, J =

7.6, 1.7 Hz, 1H, C[13]-H), 7,02 (td, J = 7.5, 1.6 Hz, 1H, C[12]-H),

6.91 – 6.76 (m, 3H, [3-5]-H), 6.69 (dd, J = 7.2, 2.3 Hz, 1H, [2]-H),

4.18 – 3.90 (m, 18H, C[7, 16-23]–H), 2.78 (t, J = 7.9 Hz, 2H, C[9]–

H), 2.02 (t, J = 11.1 Hz, 2H, C[8]–H), 1.20 – 1.08 (m, 14H, C[24-

27]–H). ¹³C {¹H} NMR (101 MHz, [D₆]DMSO) δ = 170.8, 170.5,

170.3, 149.9, 148.3, 138.6, 136.9, 129.4, 126.3, 124.2, 123.2,

121.3, 120.7, 118.2, 113.3, 68.2, 60.1, 60.0, 54.9, 54.4, 53.4, 29.2,

+

44.7, 13.9, 13.8, 12.2. HRMS (ESI+): m/z calcd for C₄₇H₆₅N₄O₁₁

([M]⁺): 861.46444, found: 861.46278.

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+

26.9, 14.1, 14.1, 14.0. HRMS (ESI+): m/z calcd for C₃₁H₄₃N₂O₉

suspension was let to warm up to room temperature for an hour.

After this step it was cooled to 0 °C again, then 5 ml (6 g, 36 mmol,

1.0 equiv.) of 2-nitro-phenethyl-alcohol in 5 ml THF was added

dropwise into the solution. After stirring for 12 hours at room

temperature, the solvent was removed under reduced pressure,

and the solid residue was dissolved in 70 ml DCM and extracted

with 2x 40 ml 10 % NaOH solution, then with brine. The organic

phase fractions were dried over Na₂SO₄, then the solvent was

removed under reduced pressure. The formed yellow oil was

purified with flash chromatography on a silica column with

Hexane:EtOAc = 10:3 eluent. The resulting fractions were

combined, and the solvent was removed under reduced pressure.

The nitro compound 15 (yield: 8.54 g, 83%) was isolated as white

crystals. Melting point: 99 °C. ¹H NMR (400 MHz, [D₆]DMSO) δ =

7.98 (dd, J = 8.1, 1.4 Hz, 1H, C[13]-H), 7.84 (dd, J = 8.0, 1.7 Hz,

1H, C[2]-H), 7.72 – 7.57 (m, 3H, C[4, 10, 12]-H), 7.52 (td, J = 8.7,

7.4, 1.6 Hz, 1H, C[11]-H), 7.37 (dd, J = 8.0, 1.5 Hz, 1H, C[5]-H),

7.11 (td, J = 8.7, 7.4, 1.6 Hz, 1H, C[3]-H), 4.44 (t, J = 6.4 Hz, 2H,

C[7]-H), 3.32 (t, J = 6.4 Hz, 2H, C[8]-H). ¹³C {¹H} NMR (101 MHz,

[D₆]DMSO) δ = 150.9, 149.3, 139.5, 134.5, 133.3, 133.2, 132.3,

128.3, 125.0, 124.6, 120.8, 115.1, 68.7, 31.7. HRMS (ESI+): m/z

calcd for C₁₄H₁₆N₃O₅⁺([M+NH₄]⁺): 306.10845, found: 306.10837.

([M+H]⁺): 587.29631, found: 587.29669.

The formylation was performed according to general procedure 3

from the quantity given above. The aldehyde compound 27 (yield:

350 mg, 28%) was isolated as brown crystals. ¹H NMR (400 MHz,

[D₆]DMSO) δ = 9.73 (s, 1H, C[32]-H), 7.39 (dd, J = 8.3, 1.8 Hz,

1H, C[3]-H), 7.31 (d, J = 1.8 Hz, 1H, C[5]-H), 7.20 (td, J = 6.1, 5.7,

3.1 Hz, 2H, C[11, 14]-H), 7.12 (td, J = 7.6, 1.7 Hz, 1H, C[13]-H),

7.02 (td, J = 7.6, 1.7 Hz, 1H, C[12]-H), 6.70 (d, J = 8.3 Hz, 1H,

C[2]-H), 4.23 (s, 4H, C[16-17]-H), 4.13 – 3.87 (m, 14H, C[7, 18-

19, 20-23]-H), 2.77 (t, J = 7.7 Hz, 2H, C[9]-H), 2.04 (t, J = 7.7 Hz,

2H, C[8]-H), 1.12 (dt, J = 16.2, 7.1 Hz, 12H, C[24-27]-H). ¹³C {¹H}

NMR (101 MHz, [D₆]DMSO) δ = 190.7, 170.3, 170.3, 148.8, 148.3,

144.5, 136.7, 129.3, 128.8, 126.4, 125.3, 124.2, 123.2, 115.9,

111.6, 68.5, 60.4, 60.1, 54.9, 53.8, 28.8, 26.7, 14.0. HRMS

(ESI+): m/z calcd for C₃₂H₄₃N₂O₁₀([M+H]⁺): 615.29123, found:

+

615.29158.

The coupling to the fluorophore was carried out using the general

procedure 4 from the quantity given above. The tetraethyl ester

compound 34 (yield: 75 mg, 15%) was isolated as purple powder.

Melting point: decomposed at > 120 °C. ¹H NMR (400 MHz,

[D₆]DMSO) δ = 7.45 (d, J = 9.5 Hz, 2H, C[34, 43]-H), 7.28 – 6.91

(m, 10H, C[3, 5, 11-14, 35, 37, 40, 42]-H), 6.86 (d, J=8.3 Hz, 1H,

C[2]-H), 4.25 (s, 4H, C[16-17]-H), 4.12 (q, J = 7.2 Hz, 4H, C[20-

21]-H), 3,98 – 3.88 (m, 10H, C[7, 18-19, 22-23]-H), 3.65 (q, J =

7.2 Hz, 8H, C[45-48]-H), 2.75 (t, J = 7.6 Hz, 2H, C[9]-H), 2.04 (d,

J = 9.4 Hz, 2H, C[8]-H), 1.24 – 1.03 (m, 24H, C[24-27, 49-52]-H).

¹³C {¹H} NMR (101 MHz, CDCl₃) δ = 171.3, 171.0, 158.2, 157.9,

155.4, 150.2, 148.8, 141.3, 137.1, 132.5, 129.5, 126.8, 125.0,

124.4, 124.4, 123.2, 117.8, 114.3, 113.8, 113.4, 96.7, 69.0, 61.2,

60.7, 55.0, 53.9, 46.2, 29.4, 27.1, 14.4, 14.3, 12.7. HRMS (ESI+):

The reduction step was carried out according to general

procedure 1. starting from the quantities given above. The

resulting yellow oil was dissolved in 90 ml EtOAc, and 50 ml water

was added. The layers were separated, and the aqueous phase

was extracted with 2x60 ml EtOAc, Then the united organic phase

fractions were washed with brine, and the EtOAc was removed

under reduced pressure. The resulting brownish oil still contained

some DIAD, but after putting the oil into the fridge overnight, the

product precipitated as crystals. The crystals were washed with

water, filtered, then dried. The amine compound 16 (yield: 5,82 g,

87%) was isolated as a beige powder. Melting point: 71 °C.¹H

NMR (400 MHz, [D₆]DMSO) δ = 7.04 (dd, J = 7.5 Hz, 1.6 Hz, 1H,

C[10]-H), 6.93 (td, J = 7.6, 1.6 Hz, 1H, C[12]-H), 6.76 (dd, J = 8.0,

1.4 Hz, 1H, C[5]-H), 6.70 – 6.59 (m, 3H, C[2, 4, 11]-H), 6.56 –

6.46 (m, 2H, C[3, 13]-H), 4.81 (d, J = 87.8 Hz, 4H, C[1, 14]N-H),

4.07 (t, J = 6.6 Hz, 2H, C[7]-H), 2.95 (t, J = 6.6 Hz, 2H, C[8]-H).

¹³C {¹H} NMR (101 MHz, [D₆]DMSO) δ = 146.7, 145.6, 137.8,

130.3, 127.1, 121.9, 121.0, 116.3, 116.3, 114.9, 113.8, 111.7,

67.3, 30.8. HRMS (ESI+): m/z calcd for C₁₄H₁₇N₂O⁺([M+H]⁺):

229.13354, found: 229.13332.

m/z calcd for C₅₂H₆₇N₄O₁₀([M+H]⁺): 907.48517, found:

+

907.48551.

The hydrolysis was carried out using the general procedure 5 from

the quantity given above. The fluorescent dye 4b (yield: 20 mg,

54%) was isolated as purple powder. Melting point: decomposed

1

at > 100 °C. H NMR (400 MHz, [D₆]DMSO) δ = 12.68 (bs, 4H,

C[20-23]OO-H), 7.48 (d, J = 9.5 Hz, 2H, C[26, 35]-H), 7.23 – 6.91

(m, 10H, C[3, 5, 11-14, 27, 29, 32, 34]-H), 6.84 (d, J = 8.2 Hz, 1H,

C[2]-H), 4.19 (s, 4H, C[16-17]-H), 3.97 (t, J = 6.5 Hz, 2H, C[7]-H),

3.85 (s, 4H, C[18-19]-H), 3.64 (q, J = 7.2 Hz, 8H, C[37-40]-H),

2.76 (t, J = 7.9 Hz, 2H, C[9]-H), 2.08 (s, 2H, C[8]-H), 1.21 (t, J =

7.0 Hz, 12H, C[41-44]-H). ¹³C {¹H} NMR (101 MHz, [D₆]DMSO) δ

= 172.3, 172.0, 157.4, 156.8, 154.8, 148.8, 148.7, 140.9, 136.4,

132.2, 129.8, 128.2, 126.2, 123.9, 123.6, 122.9, 122.8, 114.9,

114.2, 112.5, 96.0, 68.5, 54.6, 53.7, 45.3, 29.0, 26.9, 12.5. HRMS

Alkylation of 16 was carried out according to general procedure 2.

from 3 mmol of amine to yield 17. The ethyl ester compound 17

(yield: 1.1 g, 63 %) was isolated as a yellow oil. ¹H NMR (400 MHz,

[D₆]DMSO) δ = 7.31-7.23 (m, 2H, C[10, 13]-H), 7.15 (td, J = 7.6,

1.7 Hz, 1H, C[12]-H), 7.02 (td, J = 7.4, 1.3 Hz, 1H, C[11]-H), 6.91

(dd, J = 7.6, 2.0 Hz, 1H, C[5]-H), 6.85 – 6.74 (m, 2H, C[3-4]-H),

6.69 (dd, J = 7.4, 2.1 Hz, 2H, C[2]-H), 4.20 (t, J = 7.4 Hz, 2H ,C[7]-

H), 4.09 – 3.95 (m, 16H,C[15-22]-H), 3.06 (t, J = 7.4 Hz, 2H,C[8]-

H), 1.11 (dt, J = 20.6, 7.1 Hz, 12H, C[23-26]-H). ¹³C {¹H} NMR

(101 MHz, [D₆]DMSO) δ = 170.8, 170.3, 149.8, 148.7, 138.6,

133.8, 130.4, 126.9, 124.5, 123.4, 121.4, 120.8, 118.3, 113.5,

68.1, 60.1, 60.1, 55.0, 53.4, 30.6, 14.1, 14.0. HRMS (ESI+): m/z

calcd for C₃₀H₄₁N₂O₉⁺([M+H]⁺): 573.28066, found: 573.28038.

(ESI+): m/z calcd for C₄₄H₅₁N₄O₁₀([M]⁺): 795.35997, found:

+

795.35955.

Preparation of the fluorescent dye 5b.

5 g (36 mmol, 1.0 equiv.) 2-nitrophenol was suspended in 70 ml

abs. THF, then the solution was cooled to 0 °C and stored under

argon. 10.37 g (40 mmol, 1.1 equiv.) triphenylphosphine and 7.75

ml (8 g, 40 mmol, 1.1 equiv.) of DIAD were added. The

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The formylation was performed according to general procedure 3

starting from the quantities given above. The aldehyde compound

28 (yield: 985 mg, 82%) was isolated as a brown oil. ¹H NMR (400

MHz, [D₆]DMSO) δ = 9.70 (s, 1H, C[31]-H), 7.38 (dd, J = 4.3, 2.6

Hz, 2H, C[3, 5]-H), 7.26 (dd, J = 8.0, 1.5 Hz, 2H, C[10, 13]-H),

7.16 (td, J = 7.5, 1.7 Hz, 1H, C[12]-H), 7.03 (td, J = 7.3, 1.3 Hz,

1H, C[11]-H), 6.70 (d, J = 8.7 Hz, 1H, C[2]-H), 4.26 (t, J = 7.6 Hz,

2H, C[7]-H), 4.19 (s, 4H, C[15-16]-H), 4.10 (q, J = 7.1 Hz, 4H,

C[19-20]-H), 4.02 – 3.95 (m, 8H, C[17-18, 21-22]-H), 3.04 (t, J =

7.5 Hz, 2H, C[8]-H), 1.17 (t, J = 7.1 Hz, 6H, C[23-24]-H), 1.07 (t,

J = 7.1 Hz, 6H, C[25-26]-H). ¹³C {¹H} NMR (101 MHz, [D₆]DMSO)

δ = 190.8, 170.3, 170.2, 148.7, 144.5, 133.3, 130.6, 128.9, 127.1,

125.4, 124.6, 123.4, 116.0, 111.9, 68.3, 60.4, 60.2, 55.0, 53.8,

added dropwise. After stirring for 5 hours at 90 °C, the solution

was cooled to room temperature and poured on crushed ice. The

formed solids were filtered, and dried. The nitro compound 18

(yield: 9.0 g, 90%) was isolated as yellow crystals. Melting point:

153 °C. (Ref: 156 °C) The hydrogenation was carried out

according to general procedure 1, with some modification. Only 5

bar H₂was used, and 2 mol % PtO₂proved to be an effective

catalyst to terminate co-product formation. The amino compound

19 (brown crystal, yield: 3.0 g, 96%) was isolated starting from 4 g

of 18. Melting point: 96 °C. (Ref: 117 °C) The spectroscopic

properties of compounds 18 and 19 were in agreement with those

published previously.^[17,28]

+

30.5, 29.1, 14.1, 14.0. HRMS (ESI+): m/z calcd for C₃₁H₄₁N₂O₁₀

([M+H]⁺): 601.27558, found: 601.27545.

The alkylation was carried out according to general procedure 2,

from 7 mmol of amine. The ethyl ester compound 20 (yield: 8.32

g, 87 %) was isolated as a brown oil. ¹H NMR (400 MHz,

[D₆]DMSO) δ = 7.37 (dd, J = 7.6, 1.6 Hz, 1H, C[12]-H), 7.30 – 7.16

(m, 2H, C[9, 11]-H), 7.06 (td, J = 7.3, 1.4 Hz, 1H, C[10]-H), 6.95

(dd, J = 7.5, 3.2 Hz, 1H, C[5]-H), 6.87 - 6.76 (m, 2H, C[3,4]-H),

6.70 (dd, J = 7.5, 3.2 Hz, 1H, C[2]-H), 5.06 (s, 2H, C[7]-H), 4.07

(s, 4H, C[14,15]-H), 4.04 (s, 4H, C[16,17]-H), 3.96 (q, J = 7.1 Hz,

4H, C[18,19]-H), 3.86 (q, J = 7.1 Hz, 4H, C[20,21]-H), 1.06-1.10

(t, J = 7.1 Hz, 12H, C[22-25]-H). ¹³C {¹H} NMR (101 MHz,

[D₆]DMSO) δ = 170.7, 170.5, 149.7, 149.4, 138.8, 131.0, 130.6,

128.5, 123.4, 122.0, 121.3, 120.8, 118.0, 113.2, 66.7, 60.1, 59.9,

The coupling to the fluorophore was carried out using the General

procedure 4 starting from 1 mmol starting material. The ethyl ester

protected fluorescent dye 35 (yield: 50 mg, 6%) was isolated as a

red powder. Melting point: decomposed at 118 °C. ¹H NMR (400

MHz, CDCl₃) δ = 7.56 (d, J=9.3 Hz, 2H, C[33, 42]-H), 7.49 – 7.09

(m, 4H, C[2, 3, 11, 12]-H), 7.05 – 6.95 (m, 3H, C[5, 10, 13]-H),

6.94 – 6.83 (m, 4H, C[34, 36, 39, 41]-H), 4.35 – 4.29 (m, 8H, C[15-

16, 19-20]-H), 4.14 (t, J=6.6 Hz, 2H, C[7]-H), 4.07 – 3.98 (m, 8H,

C[17-18, 21-22]-H), 3.67 (q, J=7.1 Hz, 8H, C[44-47]-H), 3.25 (t,

J=7.4 Hz, 2H, C[8]-H), 1.38 (td, J=7.2, 1.8 Hz, 18H, C[23-24, 48-

51]-H), 1.20 (t, J=7.1 Hz, 6H, C[25-26]-H). ¹³C {¹H} NMR (101

MHz, CDCl₃) δ = 190.8, 171.3, 170.7, 158.1, 155.4, 150.1, 149.0,

141.3, 133.7, 132.5, 130.8, 127.7, 125.3, 125.2, 124.5, 124.5,

123.2, 117.9, 114.6, 113.7, 113.3, 96.6, 61.2, 60.7, 55.1, 53.9,

46.1, 31.5, 14.4, 14.2, 12.7. HRMS (ESI+): m/z calcd for

C₅₁H₆₅N₄O₁₀⁺([M]⁺): 893.46952, found: 893.46984.

+

54.8, 53.3, 14.0, 13.9. HRMS (ESI+): m/z calcd for C₂₉H₃₈N₂O₉

([M+H]⁺): 559.26501, found: 559.26552.

The formylation was performed according to general procedure 3

using 3 mmol starting material. The aldehyde compound 29 (yield:

1

440 mg, 22 %) was isolated as a yellow oil. H NMR (400 MHz,

[D₆]DMSO) δ = 9.74 (s, 1H, C[30]-H), 7.43 – 7.34 (m, 3H, C[3, 5,

9]-H), 7.27 (td, J = 7.7, 7.3, 1.7 Hz, 1H, C[11]-H), 7.21 (dd, J=8.2,

1.3 Hz, 1H, C[12]-H), 7.07 (td, J = 7.3, 1.3 Hz, 1H, C[10]-H), 6.72

(d, J = 8.2 Hz, 1H, C[2]-H), 5.11 (s, 2H, C[7]-H), 4.14 (s, 4H, C[14-

15]-H), 4.06 (s, 4H, C[16-17]-H), 3.95 (q, J = 7.1 Hz, 4H, C[18-

19]-H), 3.82 (q, J = 7.1 Hz, 4H, C[20-21]-H), 1.07 (td, J = 6.6, 6.1,

2.9 Hz, 14H, C[22-25]-H). ¹³C {¹H} NMR (101 MHz, [D₆]DMSO) δ

= 190.7, 170.6, 170.2, 149.8, 148.7, 144.8, 131.3, 130.2, 128.9,

125.5, 123.5, 122.2, 116.0, 111.6, 67.1, 60.2, 60.2, 56.1, 54.8,

The hydrolysis was carried out using General procedure 5 starting

from the quantities given above. The fluorescent dye 5b (yield: 15

mg, 41%) was isolated as a red powder. Melting point:

1

decomposed at 116 °C. H NMR (400 MHz, [D₄]methanol) δ =

7.57 (d, J = 9.6 Hz, 2H, C[25, 34]-H), 7.28 – 7.23 (m, 2H, C[26,

33]-H), 7.13 – 6.96 (m, 7H, C[2-3, 5, 10-13]-H), 6.91 (d, J = 2.5

Hz, 2H, C[28, 31]-H), 4.38 (t, J = 7.5 Hz, 2H, C[7]-H), 4.23 (s, 4H,

C[15-16]-H), 3.85 (s, 4H, C[17-18]-H), 3.66 (q, J = 7.1 Hz, 8H,

C[36-39]-H), 3.19 (t, J = 7.6 Hz, 2H, C[8]-H), 1.29 (t, J = 7.1 Hz,

14H, C[40-43]-H). ¹³C {¹H} NMR (101 MHz, [D₄]methanol) δ =

175.4, 174.2, 159.7, 159.5, 157.0, 151.2, 150.6, 142.8, 135.1,

133.8, 132.4, 128.5, 126.1, 125.7, 124.9, 124.4, 118.5, 116.9,

115.3, 114.5, 97.4, 70.4, 56.1, 55.4, 46.9, 33.1, 13.0. HRMS

+

53.6, 14.0, 13.9. HRMS (ESI+): m/z calcd for C₃₀H₃₈N₃O₉

([M+H]⁺): 587.25993, found: 587.26038.

The coupling to the fluorophore was carried out using the general

procedure 4 starting from 0.38 mmol formyl compound. The ethyl

ester protected fluorescent dye 36 (yield: 49 mg, 15 %) was

isolated as purple powder. Melting point: decomposed at 121.1 °C.

¹H NMR (400 MHz, CDCl₃) δ 7.45 (td, J = 8.2, 7.7, 3.1 Hz, 3H,

C[3, 32, 41]-H), 7.30 – 7.24 (m, 2H, C[11, 12]-H), 7.18 – 7.13 (m,

1H, C[10]-H), 7.01 (d, J = 1.7 Hz, 1H, C[5]-H), 6.91 – 6.86 (m, 2H,

C[35-38]-H), 6.85 – 6.77 (m, 4H, C[2, 9, 33, 40]-H), 5.20 (s, 2H,

C[7]-H), 4.22 (s, 4H, C[14-15]-H), 4.05 – 3.96 (m, 8H, C[16-17,

18-19]-H), 3.93 (q, J = 7.1 Hz, 4H, C[20-21]-H), 3.63 (q, J = 7.1

Hz, 8H, C[43-46]-H), 1.34 (t, J = 7.0 Hz, 12H, C[47-50]-H), 1.23 (t,

J = 7.1 Hz, 6H, C[22-23]-H), 1.14 (t, J = 7.1 Hz, 6H, C[24-25]-H).

¹³C {¹H} NMR (101 MHz, CDCl₃) δ 171.3, 171.0, 158.2, 157.8,

155.3, 149.8, 149.5, 141.7, 132.6, 131.2, 131.0, 129.1, 124.7,

124.1, 123.7, 123.5, 117.4, 115.3, 113.7, 113.2, 96.6, 67.6, 61.1,

+

(ESI+): m/z calcd for C₄₃H₄₉N₄O₁₀([M]⁺): 781.34432, found:

781.34423.

Preparation of the fluorescent dye 6b.

The first step to achieve ethyl ester 20 was carried out following

the previously reported method.^[18]1.72 g (43 mmol, 1.2 equiv.)

NaH (60% in mineral oil) was suspended in 60 ml DMF, then 5 g

(36 mmol, 1.0 equiv.) 2-nitrophenol in 10 ml DMF was added

dropwise to this solution. The yellow solution of 2-nitrophenol

turned into red/orange. The mixture was stirred at room

temperature for 1 hour until the gas formation stopped, then 7.76

g (36 mmol, 1.0 equiv.) 2-nitrobenzyl bromide in 10 ml DMF was

14

10.1002/ejoc.202100948

European Journal of Organic Chemistry

FULL PAPER

60.7, 55.1, 54.1, 46.1, 14.3, 14.2, 12.7. HRMS (ESI+): m/z calcd

for C₅₀H₆₃N₄O₁₀⁺([M]⁺): 879.45387, found: 879.45421.

119.9, 118.2, 116.3, 60.4, 60.2, 56.1, 54.4, 54.0, 14.0, 13.9.

+

HRMS (ESI+): m/z calcd for C₂₉H₃₇N₂O₁₀([M+H]⁺): 573.24428,

found: 573.24412.

The hydrolysis was carried out using general procedure 5 starting

from the quantity given above. The fluorescent dye 6b (yield: 40

mg, 94%) was isolated as a purple powder. Melting point:

decomposed at 141 °C. ¹H NMR (400 MHz, [D₆]DMSO) δ =12.88

– 11.98 (bs, 4H, C[18-21]OO-H), 7.40 (dd, J = 7.8, 1.7 Hz, 1H,

C[5]-H), 7.32 – 7.26 (m, 1H, C[11]-H), 7.16 – 7.08 (m, 4H, C[9, 10,

24, 33]-H), 6.95 – 6.85 (m, 7H, C[2-3, 12, 25, 27, 30, 32]-H), 5.30

(s, 2H, C[7]-H), 4.23 (s, 4H, C[14-15]-H), 3.71 (s, 4H, C[16-17]-H),

3.62 (d, J = 7.4 Hz, 8H, C[35-38]-H), 1.20 (t, J = 7.0 Hz, 14H,

C[39-42]-H). ¹³C {¹H} NMR (101 MHz, [D₆]DMSO) δ = 172.2,

171.8, 157.4, 156.6, 154.6, 148.6, 147.8, 141.1, 131.8, 131.5,

128.2, 127.9, 123.9, 123.6, 123.0, 122.7, 117.0, 116.0, 114.0,

112.2, 96.0, 65.7, 54.4, 53.8, 45.2, 12.5. HRMS (ESI+): m/z calcd

for C₄₂H₄₇N₄O₁₀⁺([M]⁺): 767.32867, found: 767.32667.

The coupling to the fluorophore was carried out using the general

procedure 4 starting from 0.5 mmol of the formyl compound. The

ethyl ester protected fluorescent dye 37 (yield: 104 mg, 24 %) was

isolated as a purple powder. Melting point: decomposed at 120 °C.

161.5-162.4 °C.¹H NMR (500 MHz, [D₆]DMSO) δ = 7.41 (d, J=8.8

Hz, 2H, C[31, 40]-H), 7.12 – 7.04 (m, 3H, C[3, 32, 39]-H), 6.97 (td,

J=8.4, 5.9, 2.9 Hz, 1H, C[10]-H), 6.93 (d, J=8.4 Hz, 1H, C[2]-H),

6.91 – 6.82 (m, 4H, C[8, 9, 34, 37]-H), 6.78 (d, J=2.1 Hz, 1H, C[5]-

H), 6.74 (d, J=8.0 Hz, 1H, C[11]-H), 4.34 (s, 4H, C[13-14]-H), 4.17

(s, 4H, C[15-16]-H), 4.06 (q, J=7.2 Hz, 4H, C[17-18]-H), 3.90 (q,

J=7.1 Hz, 4H, C[19-20]-H), 3.63 (q, J=8.1, 7.6 Hz, 8H, C[42-45]-

H), 1.19 (t, J=7.0 Hz, 12H, C[46-49]-H), 1.12 (t, J=7.0 Hz, 6H,

C[21-22]-H), 1.04 (t, J=7.1 Hz, 6H, C[23-24]-H). APT NMR (126

MHz, [D₆]DMSO) δ = 170.4, 157.8, 155.9, 154.8, 147.0, 146.6,

142.4, 138.9, 132.0, 125.2, 125.0, 122.5, 121.9, 121.3, 120.9,

118.6, 116.9, 115.6, 114.7, 114.0, 112.3, 95.9, 59.7, 54.3, 53.7,

Preparation of the fluorescent dye 7b. (See Figure S2 in

Supplementary Information)

+

45.9, 14.0, 13.9, 12.4. HRMS (ESI+): m/z calcd for C₄₉H₆₁N₄O₁₀

([M]⁺): 865.43822, found: 865.43896.

The first step to achieve ethyl ester 23 was carried out following

the previously reported method.^[18]2.4 g (60 mmol, 1.2 equiv.)

NaH (60% in mineral oil) was suspended in 40 ml DMF, then 8.35

g (60 mmol, 1.2 equiv.) 2-nitrophenol in 30 ml DMF was added

dropwise to this solution. The yellow solution of 2-nitrophenol

turned into red/orange. The mixture was stirred at 60 °C for 30

minutes until the gas formation stopped, then 5.3 ml (7.15 g, 50

mmol, 1.0 equiv.) 2-fluoronitrobenzene was added. After stirring

for 5 hours at 120 °C, the solution was cooled to room

temperature and poured on crushed ice. The formed solids were

filtered. The compound 2,2'-oxybis(nitrobenzene) (S7) (yield: 8.8

g, 68%) was isolated as a beige powder. The hydrogenation and

alkylation steps were carried out according to the previously

described general procedures 1 and 2 (vide supra). The

compound 2,2'-oxybis(aminobenzene) (S8) (brown solid, yield:

5.66 g, 92%) was isolated starting from 8 g of S7. The

spectroscopic properties of compounds S7 and S8 were in

agreement with those published earlier.^[18]The ethyl ester

compound 23 (brown crystals, yield: 11.70 g, 86%) was isolated

starting from 5 g amine (S8). ¹H NMR (400 MHz, [D₆]DMSO) δ =

6.95 (td, J = 8.0, 1.6 Hz, 2H,C[3,10]–H), 6.81 – 6.71 (m, 4H,

C[2,4,9,11]–H), 6.63 (dd, J = 8.0, 1.6 Hz, 2H, C[5,8]–H), 4.17 (s,

8H, C[13-16]–H), 4.00 (q, J = 7.1 Hz, 8H, C[17-20]–H), 1.09 (t, J

= 7.1 Hz, 12H, C[21-24]–H). ¹³C {¹H} NMR (101 MHz, [D₆]DMSO)

δ = 170.6, 148.0, 140.6, 123.7, 120.9, 120.5, 118.2, 60.1, 54.0,

The hydrolysis was carried out using the general procedure 5

using 0.05 mmol starting material. The fluorescent dye 7b (yield:

35 mg, 93 %) was isolated as a red powder. Melting point:

decomposed at 161.5°C. ¹H NMR (400 MHz, CD₃OD) δ = 7.61 (d,

J = 9.5 Hz, 2H, C[23, 32]-H), 7.12 – 6. 90 (m, 6H, C[3, 8-10, 24,

31]-H), 6.87 (dd, J = 2.5, 9.5 Hz, 4H, C[2, 11, 26, 29]-H), 6.77 (d,

J = 1.9 Hz, 1H, C[5]-H), 4.37 (s, 4H, C[13-14]-H), 4.19 (s, 4H,

C[15-16]-H), 3.66 (q, J = 7.1 Hz, 8H, C[34-37]-H), 1.30 (t, J = 7.0

Hz, 12H, C[38-41]-H). ¹³C {¹H} NMR (101 MHz, CD₃OD) δ = 175.1,

174.9, 159.5, 158.5, 156.8, 149.7, 148.4, 143.6, 143.6, 133.8,

126.5, 126.3, 124.9, 123.8, 123.0, 122.6, 119.7, 118.4, 115.0,

114.2, 97.2, 55.9, 55.3, 46.7, 12.8. HRMS (ESI+): m/z calcd for

C₄₁H₄₅N₄O₁₀⁺([M]⁺): 753.31302, found: 753.31355.

Acknowledgements

This project was supported by the National Research,

Development and Innovation Fund of Hungary, financed under

the grant numbers 2018-1.1.2-KFI-2018-00097; 2018-1.3.1-VKE-

2018-00032 and OTKA PD128612. Zoltán Mucsi is grateful to the

Hungarian Academy of Sciences for János Bolyai Research

Fellowship [BO 21/799]. We thank Tamás Gáti (Department of

Organic Chemistry, Semmelweis University, Budapest) for his

assistance in spectroscopy measurements. We also thank Imre

G. Csizmadia for providing critical remarks on the manuscript.

+

14.0. HRMS (ESI+): m/z calcd for C₂₈H₃₇N₂O₉([M+H]⁺):

545.24936, found: 545.25068.

Keywords: Calcium • Chelates • Density functional calculations

The formylation was performed according to general procedure 3

starting from of 1.5 mmol of compound 23. The aldehyde

compound 30 (yield: 427 mg, 50 %) was isolated as a green oil.

¹H NMR (400 MHz, [D₆]DMSO) δ = 9.62 (s, 1H, C[29]-H), 7.49 (dd,

J = 8.5, 2.0 Hz, 1H, C[3]-H), 7.06 – 6.96 (m, 2H, C[5, 9]-H), 6.85

– 6.73 (m, 3H, C[2, 10, 11]-H), 6.62 (dd, J = 8.0, 1.6 Hz, 1H, C[8]-

H), 4.32 (s, 4H, C[13-14]-H), 4.16 (s, 4H, C[15-16]-H), 3.99 (qd, J

= 7.1, 4.0 Hz, 8H, C[17-20]-H), 1.11 – 1.05 (m, 12H, C[21-24]-H).

¹³C {¹H} NMR (101 MHz, [D₆]DMSO) δ = 190.3, 170.6, 170.0,

147.1, 146.9, 145.8, 141.2, 128.3, 127.0, 124.6, 121.1, 120.9,

• Dyes • Fluorescent probes

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10.1002/ejoc.202100948

European Journal of Organic Chemistry

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Novel and effective calcium ion binding molecular fluorescent probes have been developed by systematic structural modification of

BAPTA ionophore. The comprehensive computational studies suggested the role of the coordination number in the chelator moiety.

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Article Doi

A Comprehensive Study of the Ca2+ Ion Binding of Fluorescently Labelled BAPTA Analogues

DOI: 10.1002/ejoc.202100948

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Article abstract of DOI:10.1002/ejoc.202100948

Full text of DOI:10.1002/ejoc.202100948

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