Scholarly article on 1-Propanol, 3-[(2-nitrophenyl)amino]- 56636-93-2 from European Journal of Organic Chemistry p. 166

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FULL PAPER

DOI: 10.1002/ejoc.201403215

A Synthetic Route to 3-(Heteroaryl)-7-hydroxycoumarins Designed for

Biosensing Applications

[a]

[b,c]

Benoît Roubinet, Arnaud Chevalier, Pierre-Yves Renard, and Anthony Romieu*

Keywords: Fluorescent probes / Fluorescence / Biosensors / Oxygen heterocycles

A straightforward method to synthesize 3-(2-benzimidazol-

yl)-7-hydroxycoumarins, based on the condensation reaction

of 7-acetoxy-3-formylcoumarin with various C- and/or N-

under physiological conditions. The further extension of this

condensation reaction to bis(2-aminophenyl)diselenide en-

abled the first synthesis of 3-(2-benzoselenazolyl)-7-

substituted ortho-phenylenediamine derivatives is pre- hydroxycoumarin. The potential utility of these new 7-

sented. This unusual approach proved particularly effective

for introducing different hydrophilic groups (carboxylic or

sulfonic acids or trimethylalkylammonium moieties) onto the

hydroxycoumarins was demonstrated through the synthesis

and spectroscopic and analyte-responsive behavior of

fluorogenic probes suitable for sensing biologically relevant

heteroaryl scaffold, leading to cyan-green emitting couma- thiols and urokinase, a protease that plays a key role in can-

rins that were both water-soluble and strongly fluorescent

cer invasion and metastasis.

Introduction

maxima of the 7-hydroxycoumarin scaffold by the introduc-

tion of a heteroaryl substituent (mainly 2-benzimidazolyl,

7-Hydroxycoumarin derivatives are popular fluorescent

2-benzothiazolyl, 2-benzoxazolyl, and 2-pyridyl) at the C-3

organic dyes. They are currently used as reporters in various

position, and possibly a cyano group at the C-4 position

biological and/or bioanalytical applications that involve

[6]

of this coumarin moiety. These structural modifications

biosensing or biolabeling operations.^[

1,2]

This widespread

induce a 20–150 nm bathochromic displacement of the

emission band, depending on the substitution pattern, and

this is particularly relevant when considering these respon-

sive fluorogenic reporters for use in various biosensing and

use is particularly due to their valuable and unique photo-

physical properties (especially their broad Stokes’ shift and

good-to-high quantum yields under physiological condi-

tions), mainly in the blue or violet spectral regions, which

are often associated with a good (photo)chemical stability.

Thus, a huge number of “smart” fluorescent (bio)probes

[7]

bioimaging applications. However, such π-extended cou-

marins are often more hydrophobic than the parent umbel-

liferone, they are more prone to self-aggregation, and they

show lower fluorescence quantum yields, especially in aque-

ous media. Furthermore, the lack of a bioconjugatable han-

dle (typically a carboxylic acid) within their core structure

prevents their use as fluorescent labels for biomolecules.

There is thus a real need to design bioconjugatable long-

wavelength 7-hydroxycoumarins that are both soluble and

strongly fluorescent in water. Unfortunately, most current

strategies for enhancing the aqueous solubility of umbelli-

ferone, based on the site-specific introduction of some

hydrophilic groups (carboxylic or sulfonic acids, pyridine

moieties, and PEG-type chains) at the C-3 or C-4 position

(also known as fluorescent chemodosimeters or chemosen-

sors, or profluorophores) based on a 7-hydroxycoumarin

scaffold have been developed and described in the literature

for the detection of various (bio)analytes including en-

zymes, (bio)thiols, polluting chemicals, cations, and

[

3,4]

anions.

Interestingly, 7-O-substituted coumarin com-

pounds have also been extensively investigated in medicinal

chemistry to obtain therapeutic agents effective against

various diseases (e.g., neurodegenerative diseases).

In the early 1980s, the Wolfbeis group carried out de-

tailed work aimed at redshifting the absorption/emission

[5]

[

8,9]

of the oxabenzopyran scaffold,

cannot be applied to 3-

[

a] Normandie Université, COBRA UMR 6014 & FR 3038, UNIV heteroaryl derivatives. Two independent publications have

Rouen; INSA Rouen; CNRS, IRCOF,

described the synthesis of some heterocycle-substituted 7-

hydroxycoumarin dyes equipped with a conjugatable carb-

oxylic acid, and possibly with an anionic solubilizing moi-

1

, rue Tesnières, 76821 Mont-Saint-Aignan Cedex, France

http://ircof.crihan.fr

b] ICMUB, UMR CNRS 6302, Université de Bourgogne,

9

, Avenue Alain Savary, 21078 Dijon CEDEX, France

[10]

ety (Scheme 1). However, the synthetic strategies used for

E-mail: anthony.romieu@u-bourgogne.fr

http://www.icmub.fr

their preparation rely on acid- or base-catalyzed condensa-

tion reactions that are difficult to generalize to a wide range

of 3-(heteroaryl)umbelliferone derivatives (Scheme 1). Al-

ternatively, more sophisticated water-solubilizing ap-

c] Institut Universitaire de France,

1

03 Boulevard Saint Michel, 75005 Paris, France

Supporting information for this article is available on the

WWW under http://dx.doi.org/10.1002/ejoc.201403215.

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B. Roubinet, A. Chevalier, P.-Y. Renard, A. Romieu

FULL PAPER

Scheme 1. Background information about the synthesis of water-soluble and bioconjugatable 3-heteroaryl-7-hydroxycoumarin dyes, and

the synthetic strategy explored in this work.

proaches have recently been explored, especially by our bioconjugatable arm (typically a carboxylic acid), should be

group. Electrophilic aromatic sulfonation of the C-6 posi- considered. The goal would be to impart to 3-(heteroaryl)-

tion of 3-(2-benzothiazolyl)-7-hydroxycoumarin, and Man- umbelliferone derivatives both water solubility and the

nich-type reactions to functionalize the C-8 position with possibility for bioconjugation without compromising the

an aminomethyl arm derived from a hydrophilic secondary (fluorogenic) reactivity of their phenol moiety. Inspired by

[

24]

amine (i.e., iminodiacetic acid, sarcosine, or isonipecotic recent work by Yoon et al.

on the efficient synthesis of

acid) have led to water-soluble 7-hydroxycoumarins with 1,2-disubstituted benzimidazoles through a condensation

unprecedentedly high fluorescence quantum yields under reaction between an ortho-phenylenediamine derivative and

physiological conditions (Scheme 1).^[

11,12]

However, the an aromatic aldehyde, we have devised a concise synthetic

presence of these polar substituents in close proximity to route to functionalized 3-(heteroaryl)-7-hydroxycoumarins

the 7-OH group makes the synthesis of reaction-based using 7-acetoxy-3-formylcoumarin as the common starting

fluorescent probes through chemical modification of the material (Scheme 1).

phenol moiety difficult.^[Furthermore, these water-solubi-

12]

In this paper, we report the practical implementation of

lizing groups negatively influence both the stability and the this new synthetic strategy for the preparation of original

fluorogenic reactivity of the resulting profluorophores in water-soluble and/or bioconjugatable derivatives of 3-(2-

[

13–15]

aqueous buffers. To overcome the difficulties associated benzimidazolyl)-7-hydroxycoumarin.

A further exten-

with these synthetic methods, a substantially different ap- sion of this method to the direct synthesis of the unknown

proach based on the facile functionalization of the 3- 3-(2-benzoselenazolyl)-7-hydroxycoumarin was also ex-

heteroaryl moiety of such long-wavelength coumarins with plored. To demonstrate the beneficial effects of the remote-

anionic or cationic hydrophilic groups (trimethylalkyl- ness of water-solubilizing groups from the phenol moiety

ammonium, carboxylate, or sulfonate), and possibly with a on the reactivity of these new fluorogenic dyes, some thiol-

2

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3-(Heteroaryl)-7-hydroxycoumarins

sensitive profluorophores were synthesized, and their trimethylammonium- or sulfonate-terminated alkyl chain

fluorescence behavior was studied in detail. Finally, the substituent onto one of the two anilines was achieved

bioconjugation ability of this new family of heterocycle- through an S 2 reaction of trimethylamine (TMA) or so-

N

substituted coumarins was also confirmed through the dium sulfite with a mesylate derived from the correspond-

preparation of Förster resonance energy transfer (FRET)- ing primary alcohol (i.e., 1 or 5). To access these alcohols,

based probes suitable either for biothiol sensing or in vitro/ an S Ar reaction between ortho-nitro-fluorobenzene deriv-

N

in cellulo detection of urokinase-type plasminogen activator atives and 3-aminopropan-1-ol was carried out. This reac-

(

uPA), a serine protease known to play an important role tion worked well under mild conditions. The final step of

[16]

in tumor-associated proteolysis.

this synthetic route was the reduction of the nitro group by

catalytic hydrogenation over Pd/C, to install the primary

aniline of the target ortho-phenylenediamines 2, 3, 6, and 7

(overall yields: 52, 55, 20, and 27%, respectively). Concern-

ing the preparation of derivative 6, which bears a free carb-

oxylic acid, a further TFA (trifluoroacetic acid)-mediated

deprotection step was required for the complete removal of

the tert-butyl ester from its nitro precursor. The premature

partial cleavage of this protecting group occurred during

Results and Discussion

Numerous synthetic methods are currently available for

the preparation of 3-(heteroaryl)coumarins [especially 3-(2-

benzimidazolyl) derivatives]. These include Pechmann, Per-

kin, or Knoevenagel reactions,

pling reactions, condensation reactions of 2-chloro-2-ar-

[14,17]

Pd-catalyzed cross-cou-

[

18]

the S 2 reaction with sodium sulfite, and thus we decided

N

ylacetaldehyde with salicylaldehyde catalyzed by N-hetero-

to isolate the free carboxylic acid form (for the synthesis of

the coumarin, vide infra) by chromatography over reverse-

phase silica gel (for synthetic details and analytical data, see

the Supporting Information).

cyclic carbenes (NHC),^[BBr -induced domino reactions

19]

3

starting from homophthalic anhydride and a 2-methoxy-

benzaldehyde derivative,^[

20]

and acid-catalyzed condensa-

tion reactions between an ortho-phenylenediamine deriva-

tive and a 3-(ethoxycarbonyl)coumarin.^[However, most

21]

of these methods are not really suitable for the introduction Synthesis of Water-Soluble 3-(2-Benzimidazolyl)-

of charged functional groups into the heteroaryl moiety, 7-hydroxycoumarins Ϫ Extension to Unknown

and they are not easily transferable to more sensitive 7- 3-(2-Benzoselenazolyl)-7-hydroxycoumarin

hydroxycoumarin derivatives. Since the key step of our syn-

Condensation of ortho-phenylenediamine derivatives, in-

thetic strategy is a condensation reaction between an N-

cluding the N-monosubstituted derivatives described above

monosubstituted aryl diamine and the known 7-acetoxy-3-

and bis-primary anilines such as 3,4-diaminobenzene-

formylcoumarin (Scheme 1), our initial efforts were devoted

[

22]

sulfonic acid 8

acetoxy-3-formylcoumarin

20 °C for 3 h to give six new 3-(2-benzimidazolyl)-7-

and 3,4-diaminobenzoic acid 9, with 7-

to the development of an efficient and versatile synthetic

route to hydrophilic ortho-phenylenediamine derivatives

bearing a hydrophilic substituent.

[

23]

was achieved in DMSO at

1

hydroxycoumarins 10–15 (Scheme 3). The successful syn-

thesis of the 6-sulfonated derivative of 7-acetoxy-3-formyl-

coumarin according to the three-step procedure reported by

Lim et al. (see Scheme 3 and the Supporting Information

for synthetic details) enabled us to achieve the preparation

Synthesis of Hydrophilic ortho-Phenylenediamine

Derivatives

[

23]

The synthesis of ortho-phenylenediamine derivatives 2, 3, of a further water-soluble derivative (compound 16). Except

, and 7 is shown in Scheme 2. The introduction of a single for compounds 10, 11, and 16, this reaction required the

6

Scheme 2. Synthesis of ortho-phenylenediamine derivatives. Reagents and conditions: i) 3-aminopropan-1-ol (1.6 equiv.), DIEA (diiso-

propylethylamine; 1.2 equiv.), CH Cl , room temp., overnight, 80% (for 1) and 58% (for 5); ii) MsCl (1.1 equiv.), TEA (triethylamine;

.2 equiv.), dry CH Cl (0 °C), 1 h, 94–96%; iii) Na SO (5 equiv.), EtOH/H O (2:1, v/v), reflux, 12 h, 72% (for nitro intermediate leading

2

1

2

3

2

to 2) and 37% (for nitro intermediate leading to 6; premature cleavage of the tBu ester occurred during this reaction step); iv) TMA,

THF (1.0 m solution; 5 equiv.), reflux, 12 h, 76% (for nitro intermediate leading to 3) and 51% (for nitro intermediate leading to 7);

v) H

temp., 1 h, between steps (iii) and (v) was used to completely remove the tBu ester.

, Pd/C (10%), MeOH, room temp., 3 h, 97% (for 2, 3, 6, and 7); ^[^a^]A further treatment with CH

Cl /TFA (5:2, v/v), 0 °C to room

2 2 2

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B. Roubinet, A. Chevalier, P.-Y. Renard, A. Romieu

FULL PAPER

Scheme 3. Synthesis of water-soluble 3-(2-benzimidazolyl)-7-hydroxycoumarin dyes. Reagents and conditions: i) oleum (6 equiv.), 0 °C,

0 min, then room temp., 2 h, 54%; ii) diethyl glutaconate (1 equiv.), piperidine (1.2 equiv.), EtOH, reflux, 24 h, 67% (for intermediate

leading to 7-acetoxy-3-formylcoumarin) and 42% (for intermediate leading to 7-acetoxy-3-formylcoumarin-6-sulfonic acid); iii) Ac

60 equiv.), pyridine, room temp., 1 h, 88% (for intermediate leading to 7-acetoxy-3-formylcoumarin) and 97% (for intermediate leading

to 7-acetoxy-3-formylcoumarin-6-sulfonic acid); iv) OsO (4% in water), NaIO (2.9 equiv.), THF, room temp., 4 d, 79% (for 7-acetoxy-

-formylcoumarin) and 65% (for 7-acetoxy-3-formylcoumarin-6-sulfonic acid); v) ortho-phenylenediamine 8 or 9 (1 equiv.), DMSO,

(1 equiv.), DMSO, 120 °C,

3

2

O

(

4

3

1

3

2

2 2 5

20 °C, 3 h, 19% (10), 20% (11), and 25% (16); vi) ortho-phenylenediamine 2, 3, 6, or 7 (1 equiv.), Na S O

h, 23% (12), 21% (13), 24% (14) and 23% (15); vii) DCC (5.3 equiv.), NHS (5.3 equiv.), NMP (N-methyl-2-pyrrolidone), room temp.,

+

h; viii) 11-NHS (1 equiv.), TBA salt of 2-aminoethane-1,1-disulfonic acid (13 equiv.), DIEA (5 equiv.), NMP, 0 °C then room temp.,

overnight, 28%; 3-(2-benzimidazolyl)-7-hydroxycoumarin derivatives were isolated as TEA salts (10–13 and 16) or TFA salts (14 and 15),

except for 17 (acid form, after Dowex H desalting).

+

addition of sodium metabisulfite (Na S O ) to take place. thesis involving ortho-phenylenediamine 7, these reaction

2

5

As suggested by Yoon et al.,^[this unusual additive may conditions also led to the removal of the tert-butyl protect-

favor the in situ formation of the more reactive bisulfite ing group to reveal the carboxylic acid. Due to the high

adduct of the aromatic aldehyde derived from umbelli- polarity of the resulting 3-heteroaryl-coumarins, all reac-

ferone. Furthermore, it is important to point out that the tion mixtures were purified by semipreparative RP (reverse-

temporary protection of the 7-OH group of 3-formyl-um- phase) HPLC or using an automated flash-purification sys-

belliferone as an acetate ester was also found to be benefi- tem equipped with an RP-C₁₈cartridge, with aqueous tri-

cial to enhancing the electrophilic reactivity of the aldehyde, ethylammonium hydrogen carbonate buffer (TEAB; 50 mm,

24]

even if its premature cleavage occurred during the reaction pH 7.5) and CH CN as eluents to recover compounds 10–

3

due to the severe conditions of heating. For coumarin syn- 13 and 16 in a pure form. Indeed, our first attempts at puri-

4

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3-(Heteroaryl)-7-hydroxycoumarins

fication by reverse-phase chromatography using a more to be equal to or above 95%, and thus suitable for an accu-

conventional aqueous mobile phase [TFA (0.1% aq.), pH rate and reliable determination of their photophysical prop-

2.0] partly failed due to nonspecific adsorption of these po- erties.

lar compounds over the stationary phase under the acidic Next, we wanted to extend this promising strategy to the

conditions. The use of this TEAB buffer was found to be preparation of 3-(2-benzoselenazolyl)-7-hydroxycoumarin

the only way to achieve acceptable yields of the desired (Scheme 4), especially to assess the effects of the heteroatom

water-soluble 3-(heteroaryl)-7-hydroxycoumarins. On the on the spectral properties of the 3-(heteroaryl)-7-hydroxy-

other hand, compounds 14 and 15 could be recovered using coumarin scaffold. By analogy with the recent work of Con-

aq. TFA and CH CN as eluents. However, all the isolated ley et al. on the synthesis and bioluminescence properties

3

[

26]

yields were modest (in the range 20–25%), which is in con- of a selenium analog of firefly d-luciferin,

we hypothe-

trast to the good conversions (in the range 70–80%), as de- sized that this atom replacement [Se instead of N/S of al-

termined by RP-HPLC analysis of the crude reaction mix- ready known 3-(heteroaryl)-7-hydroxycoumarins] would re-

tures (data not shown). This is related to the loss of material sult in a redshift of the absorption/emission maxima of the

during the RP chromatographic purification and lyophiliza- fluorescent heterocyclic scaffold. Although there is growing

tion steps.

interest in the synthesis of organoselenium-based molecular

To dramatically improve the solubility of 3-(2-benzimid- dyes and their applications, particularly in photodynamic

azolyl)-7-hydroxycoumarin 11 in aqueous media, and to therapy (PDT) or as fluorescent probes for the detection of

check the reactivity of its benzoic acid moiety, we next in- various endogenous (bio)analytes in physiological sys-

vestigated a “post-synthetic” sulfonation process through tems,^[²⁷^]this selenium-containing coumarin dye has never

amidification of its carboxylic acid with 2-aminoethane-1,1- been reported in the literature. Commercially available

[

25]

disulfonic acid. This synthesis involves the initial conver- bis(2-aminophenyl)diselenide was treated with 7-acetoxy-3-

sion of the carboxylic acid of 11 into the corresponding N- formylcoumarin under the same conditions as described

hydroxysuccinimidyl (NHS) active ester by treatment with above, to give the target 3-(2-benzoselenazolyl)-7-

an excess of DCC (N,NЈ-dicyclohexylcarbodiimide) and N- hydroxycoumarin (i.e., 18), which was isolated by conven-

hydroxysuccinimide. Despite its lower reactivity, this rea- tional silica gel column chromatography in a modest 14%

gent was preferred over more effective uronium- or phos- yield. In this case, Na S O promoted the Se–Se bond cleav-

2

5

·

–

phonium-based coupling agents [i.e., TSTU (2-succinimido- age, probably through the radical anion SO generated

,1,3,3-tetramethyluronium tetrafluoroborate), PyBOP from S O₅by heating.

2

2–

[28]

1

2

(benzotriazol-1-yl-oxytripyrrolidinophosphonium

hexa-

fluorophosphate), or PyBrOP (bromo-tris-pyrrolidinophos-

phonium hexafluorophosphate)] because these latter rea-

gents are known to readily react with the phenol moiety of

umbelliferone derivatives (7-hydroxycoumarins, vide infra,

Scheme 8). After this activation step, subsequent reaction

Photophysical Properties of Water-Soluble 3-(2-

Benzimidazolyl)-7-hydroxycoumarin Dyes

All the synthesized coumarin dyes (except 18) have excel-

with 2-aminoethane-1,1-disulfonic acid (tributylammon- lent water solubility (greater than 10 mm). Thus, their pho-

+

ium, TBA salt) led to the desired disulfonated 7- tophysical properties were evaluated in phosphate-buffered

hydroxycoumarin (i.e., 17), which was purified by flash saline (PBS; pH 7.5), which mimics physiological condi-

chromatography over reverse-phase silica gel using aqueous tions, and the results are compiled in Table 1 (see the Sup-

TEAB and CH CN as eluents, followed by desalting with porting Information for the corresponding spectra). In or-

3

+

Dowex H resin. The structures of all of the hydrophilic 3- der to compare their spectral behavior in aqueous and or-

(2-benzimidazolyl)-7-hydroxycoumarins were confirmed by ganic media, further measurements in DMSO were also

detailed measurements, including ESI-HRMS and NMR carried out. As previously reported by Deligeorgiev et

[

15]

spectroscopic analysis (see the Supporting Information for al.,

the introduction of a 2-benzimidazolyl substituent

the corresponding spectra). Furthermore, the purity of each onto the C-3 position of umbelliferone led to a long-wave-

compound (determined by RP-HPLC analysis) was found length 7-hydroxycoumarin with absorption/emission max-

Scheme 4. Synthesis of 3-(2-benzoselenazolyl)-7-hydroxycoumarin.

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Table 1. Spectral properties of water-soluble 3-(2-benzimidazolyl)- groups prevent the aggregation of the dye molecules and/or

7

-hydroxycoumarin derivatives at 25 °C.

the formation of nonemitting dimers in aqueous solutions.

Similar trends were observed in DMSO, although larger

Stokes’ shifts were obtained. This set of results clearly

shows that a water-solubilizing approach based on the func-

tionalization of the benzimidazolyl moiety with hydrophilic

groups is effective for obtaining long-wavelength 7-hydroxy-

coumarins with strong cyan-green emission in physiological

media.

Some photophysical properties of the parent 3-(2-benz-

imidazolyl)-7-hydroxycoumarin and its thio and seleno ana-

logs [i.e., 3-(2-benzothiazolyl)-7-hydroxycoumarin and 18,

respectively] were also determined both in phosphate buffer

Dye

Solvent Absorption λmax Emission λmax

Φ

F

[%]

nm]^[

a]

[nm]

[b]

[

_-₍₂_-_B_Z_M₎_-₇_-_O_H_[c]

3

pH 7.0

pH 8.0

PBS

DMSO

PBS

420

480

22

61

57

78

66

60

59

3-(2-BZM)-7-OH

417

390

420

389

421

477

493

476

484

478

3-(2-BZM)-7-OH

1

0

1

0

DMSO

PBS

11

Tris·HCl

pH 8.0

DMSO

PBS

DMSO

PBS

DMSO

PBS

DMSO

PBS

11

424

478

69

1

389

392

350

394

350

395

350

396

351

426

389

426

388

474

467

478

466

471

467

480

468

459

481

474

477

474

70

78

79

78

84

77

78

73

83

57

76

67

72

1

2

(PB, pH 7.4) and DMSO, and the results are given in

1

2

1

3

Table 2. In DMSO, a 14 nm bathochromic shift for the

benzothiazole derivative, and a 19 nm bathochromic shift

for 18 at the wavelength of the absorption maximum can

be observed as the heteroatom becomes larger. The Stokes’

shift is of the same order of magnitude (75–80 nm) for 18

and 3-(2-benzothiazolyl)-7-hydroxycoumarin, but that for

the benzimidazolyl derivative is higher. As the chalcogen

atom becomes larger, spin-orbit (heavy-atom) effects pro-

1

3

1

4

1

4

1

5

1

5

DMSO

PBS

DMSO

PBS

1

6

1

6

1

7

17

DMSO

mote triplet formation over fluorescence, and Φ dramati-

F

[

a] Most of the dyes were not obtained in sufficient amounts for

highly accurate measurements of molar absorption coefficients.

b] Determined at 25 °C by using 7-hydroxycoumarin [Φ = 76%

in phosphate buffer (PB), pH 7.4, λex = 390 nm] as standard.

[29]

cally decreases from 81 to 13%. Despite a low solubility

in water (limited to the micromolar concentration range),

the spectral characteristics of 18 were next measured in PB.

A very broad absorption spectrum that does not match with

the corresponding excitation curve was obtained (see the

Supporting Information). This trend may suggest the pres-

[

F

[50]

[

c] BZM = benzimidazolyl, spectral properties reported by Deli-

[

15]

georgiev et al.

ima close to 420/480 nm in PBS, and 390/495 nm in DMSO. ence of aggregates rather than the prevalence of the weakly

Further functionalization of the heteroaryl scaffold with a emitting phenol form of 18. Indeed, the pK of the phenol

a

carboxylic or sulfonic acid group has no significant effect moiety of 18 was evaluated by monitoring the fluorescence

on the position of the absorption/emission bands (see com- emission of this fluorophore as a function of the environ-

pounds 10 and 11), and good fluorescence quantum yields mental pH, and it was found to be slightly different from

in the range 60–70% (both in PBS and DMSO) are re-

tained. Contrary to our previous observations with 3-(2-

Table 2. Spectral properties of 3-(heteroaryl)-7-hydroxycoumarin

benzothiazolyl)-7-hydroxycoumarin,^[ortho-sulfonation of dyes at 25 °C.

12]

the phenol moiety does not lead to a significant increase of

the quantum yield (see compound 16). The presence of the

Dye

Solvent

Absorption λmax Emission λmax

Φ

F

[%]

[a]

[b]

[nm]

more hydrophilic benzimidazole heterocycle (compared to

a benzothiazolyl ring) is probably a major factor in the re-

sistance to aggregation-induced fluorescence quenching of 18

this 3-heteroaryl-7-hydroxycoumarin scaffold. The post-

3

-(2-BZM)-7-OH PBS

417

430

439

477

486

492

57

43

–

[b]

3-(2-BT)-7-OH

PBS

PB

PB + 5%

BSA

2

H O + 2.5%

[c]

18

473

400

495

475

60

5

synthetic derivatization of the carboxylic acid of 11 with

-aminoethane-1,1-disulfonic acid leads to compound 17, 18

which also shows a good fluorescence quantum yield under

physiological conditions, close to 70%. Interestingly, N-alk- 3-(2-BT)-7-OH

2

[d]

SDS

-(2-BZM)-7-OH DMSO

3

383

397

478

472

477

473

78

81

13

11

[b]

DMSO

EtOH

[

e]

1

8

402

ylation of the benzimidazolyl moiety with a propyl chain

terminated by a trimethylammonium group or a sulfonic

acid induces hypsochromic shifts of ca. 30 nm for the ab-

sorption maximum and ca. 10 nm for the emission maxi-

mum of the 3-heteroaryl-7-hydroxycoumarin (see com-

pounds 12–15). Furthermore, both of these polar N-substit- relationship between fluorescence emission and absorption at

uents have a positive impact on the fluorescence efficiency 390 nm was obtained, and this prevented an accurate determi-

of the resulting hydrophilic coumarinic scaffold. Indeed, a

18

399^[

e]

[

a] Determined at 25 °C by using 7-hydroxycoumarin (Φ

F

= 76%

in PB, pH 7.4, λex = 390 nm) as standard,^[except for measure-

50]

ment in PB + 5% BSA [standard: fluorescein in NaOH (0.1 n), Φ

=

F

[51]

95%, λex = 440 nm]. [b] BT = benzothiazolyl. [c] A nonlinear

nation of the relative quantum yield in PB. [d] pH = 6.9, measure-

ments with this additive were performed in water due to its poor

solubility in PB. [e] Molar absorption coefficients: 36880 m cm

in DMSO, and 35125 m cm in EtOH; SDS = sodium dodecyl

3

3% increase in quantum yield compared to parent 3-(2-

benzimidazolyl)-7-hydroxycoumarin was observed, which

can partly explained by the fact that such ionized/ionizable sulfate buffer.

–1

6

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3-(Heteroaryl)-7-hydroxycoumarins

that of its benzimidazolyl and benzothiazolyl analogs (6.7 and sensing ability of thiol-sensitive profluorophores. These

compared with 6.9 and 7.0, see the Supporting Infor- probes are based on the reversible alkylation of the 7-OH

mation). This hypothesis was further supported by the fact group with quenching moieties that may incur photo-in-

that a nonlinear relationship between the fluorescence emis- duced electron transfer (PeT) or strongly influence the in-

sion and the absorption value (at the wavelength used for ternal charge transfer (ICT) state of the fluorophore.^[

excitation) was observed, which prevented the determi- As an illustrative example, phenol-based fluorophore 14

nation of the fluorescence quantum yield. However, the was converted into a thiol-sensitive fluorogenic probe using

strong cyan-green fluorescence of 18 was recovered by add- 7-nitro-2-oxa-1,3-diazole (NBD) as the quenching moiety

ing 5% (w/v) of bovine serum albumin (BSA), an additive (Scheme 5). NBD ethers have recently emerged as electro-

often used in buffers to mimic body fluids (Φ_F= 60%). philic triggers for reaction-based probes; they allow the

Indeed, this protein is known to enhance the emission of dual colorimetric and fluorogenic detection of the gaso-

31,32]

[

32,33,34]

many fluorophores owing to a combination of rigidifica- transmitter hydrogen sulfide (H S).

The preparation

2

tion, a reduction in the polarity of the dye’s microenviron- of phenolic ether 19 was achieved by reaction of 14 with

ment (binding in the hydrophobic BSA pocket), and deag- NBD chloride (NBDCl) in the presence of TEA in dry

[

30]

gregation.

BSA also had a marked effect on both the DMF at room temperature (Scheme 5). This NBD probe

shape and the position (redshifted) of the absorption band was recovered in a pure form and in good yield (73%) by

of this fluorescent phenol. A more comprehensive study of semipreparative RP-HPLC. This NBD ether is essentially

spectral properties of 3-(2-benzoselenazolyl)-7-hydroxycou- nonfluorescent in both PBS and DMSO (see the Support-

marin aimed at assessing its potential as a fluorogenic dye ing Information), and its sensing response to thiols [Cys,

–

will be carried out, and the results will be reported in due thiophosphate (ThioPi), and HS , the predominant form of

course.

H S at pH 7.5] was also studied through time-dependent

2

UV/Vis absorption and fluorescence analysis (Figure 1).

Surprisingly, thiophosphate anion appeared to be the most

reactive sulfhydryl analyte towards the NBD electrophilic

Water-Soluble Profluorophore for Thiol Sensing

In order to both demonstrate the potential utility of the site, since a plateau for fluorescence intensity at 480 nm was

new hydrophilic 3-(2-benzimidazolyl)-7-hydroxycoumarin reached within 30 min, compared to 60 min for Cys (Fig-

derivatives and assess the possible benefits of the larger dis- ure 1, B). The fluorescence enhancement estimated by the

tance between the phenol and water-solubilizing moieties emission intensity ratio of 19 at 480 nm in the presence and

on their fluorogenic reactivity, we explored the synthesis absence of added thiols, I/I , was 100 for ThioPi and 65.4

0

Scheme 5. Synthesis of 7-O-NBD fluorogenic ether 19.

Figure 1. (A) Time-dependent changes in the UV/Vis spectrum of NBD probe 19 (8.6 μm) in PBS (100 mm phosphate + 150 mm NaCl,

pH 7.5), in the presence of ThioPi (Na salt, 5 equiv.) at 25 °C. (B) Time-dependent fluorescence intensity of NBD probe 19 (1.1 μm) at

–

4

80 nm (λex = 390 nm) in the presence of sulfhydryl analytes: Cys (5 equiv.), ThioPi (Na salt, 5 equiv.) and HS (Na salt, 200 equiv.),

recorded in PBS at 25 °C; a further kinetics curve for Pi (K salt, 5 equiv.) was recorded under the same conditions.

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FULL PAPER

for Cys. The thiolysis of NBD probes such as 19 proceeds Water-Soluble FRET Probe for Thiol Sensing

by an S Ar mechanism (addition-elimination process). In

N

this case, this was supported by changes observed in the

Since our synthetic method also gave access to water-

UV/Vis spectrum of 19 upon addition of ThioPi (Figure 1, soluble 3-(2-benzimidazolyl)-7-hydroxycoumarins bearing a

A). The appearance of two strong absorption bands at 395 bioconjugatable handle (i.e., a carboxylic acid), we also ex-

and 550 nm was observed, which were assigned to free 7- plored the preparation of thiol-sensitive fluorogenic probes

hydroxycoumarin 14 and the NBD-ThioPi adduct, respec- based on the covalent association of a fluorophore of this

tively. Kinetic fluorescence measurements achieved with type with a dark quencher chromophore (i.e., a nonfluores-

NaHS (200 equiv.) and nonnucleophilic phosphate anion cent acceptor), together making up a FRET pair,^[

35]

(

Pi; 50 equiv., used as a negative control) revealed that through a thiol-reactive linker (namely a disulfide bridge).

NBD probe 19 shows a moderate reactivity toward H₂S In this way, we demonstrate the utility and reactivity of this

I/I = 52, but only after a prolonged incubation time and additional functional group.

(

0

at much higher concentrations), and is not prone to hydrol-

We focused on the preparation of FRET probe 24. In

ysis at physiological pH. This preliminary biosensing appli- this compound, coumarin-carboxylic acid 11 is linked to an

cation has enabled us to demonstrate that water-soluble 3- original water-soluble analog of azo-based quencher DAB-

(2-benzimidazolyl)-7-hydroxycoumarins unsubstituted at CYL {4-([4-(dimethylamino)phenyl]azo)benzoic acid}, i.e.,

their C-6 and/or C-8 positions can easily be converted into 21 (Δλ₁_/₂_m_a_xvalue of 112 nm, corresponding to a quench-

reaction-based fluorescent probes through chemical modifi- ing range 394–506 nm, and fully compatible with the

cation of their 7-OH group with various chemical moieties emission of 11, centered at 478 nm), through a cleavable

that can act as both the quencher and the fluorescence trig- disulfide spacer (Scheme 6). The synthesis of azo-based

ger.

quencher 21 was achieved as follows: in situ formation of

Scheme 6. Synthesis of thiol-sensitive FRET-based probe 24. Reagents and conditions: i) a) NOBF

b) N-(2-azidoethyl)-N-methylaniline (1.2 equiv.), CH CN, 0 °C, 30 min, 86% (for the two steps a and b); ii) a) TSTU (1.3 equiv.), DIEA

3 equiv.), NMP, room temp., 30 min; b) TBA salt of 2-aminoethane-1,1-disulfonic acid (10 equiv.), DIEA (3 equiv.), 0 °C then room

4 3

(1.1 equiv.), CH CN, 0 °C, 15 min;

3

+

(

temp., 2 h, 61% (for the two steps a and b); iii) DCC (5.3 equiv.), NHS (5.3 equiv.), NMP, room temp., 2 h; iv) alkyne-functionalized

cystamine 22 (8.5 equiv.), NMP, DIEA (5 equiv.), overnight, 25% (for the two steps iii and iv); v) 21 (1.1 equiv.), 23 (1 equiv.), Cu⁰

(

0.5 equiv.), CuSO

4 2

(0.1 equiv.), DMSO/H O (2:1, v/v), 50 °C, 2 h, 48%; compounds were isolated as TEA salt (24), TFA salt (23) or in

+

acid form after Dowex H desalting (21).

8

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3-(Heteroaryl)-7-hydroxycoumarins

the diazonium salt of para-aminobenzoic acid was readily was obtained within 15 min of incubation. Conversely,

achieved using the bench-stable, commercially available 90 min was not enough time to obtain complete reaction

nitrosating agent nitrosonium tetrafluoroborate (NOBF ) when the ThioPi anion was used as reductant in a three-

4

[

36]

in dry CH CN.

Subsequent reaction with an electron- times larger amount. This is consistent with the lower oxi-

3

rich tertiary aniline, namely N-(2-azidoethyl)-N-methylanil- dation–reduction potential of ThioPi (EЈ = 0 V at pH 7.0)

0

[

40]

ine, led to azido-acid azo dye 20. “Post-synthetic” sul- compared to that of Cys (EЈ = –0.32 V at pH 7.0). Once

0

fonation through amidification of its carboxylic acid with again, no increase in fluorescence was observed for the reac-

2

-aminoethane-1,1-sulfonic acid was achieved under condi- tion involving the Pi anion, which proves the stability of

tions similar to those used for the synthesis of disulfonated this FRET probe at pH 8.0.

-hydroxycoumarin 17, except that the activation step in-

7

volved the use of TSTU/DIEA in NMP, to give water-solu-

ble “clickable” DABCYL analog 21 in a good overall yield

uPA-Sensitive FRET-Based Probe

(

53%). On the other hand, a coupling reaction between 3-

As illustrated above, 3-(2-benzimidazolyl)-7-hydroxycou-

marins functionalized with a carboxylic acid on their het-

eroaryl moiety are suitable reagents for covalent labeling.

Furthermore, their cyan-green fluorescence can be effec-

tively switched off through the design of FRET probes in-

volving a DABCYL derivative as the quencher. To expand

the scope of these new bioconjugatable umbelliferone deriv-

atives to more biologically relevant fluorescent probes, we

next explored the preparation and in vitro validation of a

FRET substrate for the detection of a serine protease,

(2-benzimidazolyl)-7-hydroxycoumarin 11 and alkyne-func-

[37]

tionalized cystamine 22 was carried out under conditions

previously optimized for the synthesis of 17 (vide supra), to

give, after semipreprative RP-HPLC, the second molecular

partner (i.e., 23) for the copper-catalyzed azide–alkyne 1,3-

dipolar cycloaddition (CuAAC). This latter reaction was

achieved using microsized Cu in a mixture of DMSO/H O

[2]

[38]

0

2

[

39]

(2:1, v/v) at 50 °C.

The resulting disulfide-based FRET

probe 24 was isolated in pure form by semipreparative RP-

HPLC (yield 48%, purity 98%). Its structure was unambig-

uously confirmed by ESI-HRMS and NMR spectroscopic

analysis (see the Supporting Information). The quenching

efficiency (QE) of DABCYL analog 21 was measured as the

difference in fluorescence (area under the emission curve) of

FRET probe 24 before and after reductive cleavage of its

disulfide bridge with a large excess of DTT (100 equiv.) in

phosphate buffer (PB, pH 7.5), and expressed as a percen-

tage (QE = 100ϫ [1 – I (em)/I(em)]). An excellent value of

0

98% was found. The fluorogenic reactivity of FRET probe

24 was next examined at pH 8.0 (Tris·HCl buffer) because

the reductive disulfide bond cleavage mediated by Cys is

known to be favored under slightly alkaline conditions (Fig-

ure 2, see also the Supporting Information for details of the

same experiment performed in PB, demonstrating the poor

fluorogenic reactivity of 24 towards Cys in this buffer). As

expected, the optical response of 24 towards Cys is very

fast, and a plateau for the fluorescence emission at 480 nm

Scheme 7. Synthesis of uPA-sensitive FRET-based probe 29. Rea-

4 3

gents and conditions: i) NOBF (1.1 equiv.), CH CN, 0 °C, 15 min,

then maleimide-terminated aniline 25, 0 °C, 30 min, then NaOAc

Figure 2. Time-dependent fluorescence intensity of disulfide-based

FRET probe 24 (2.15 μm) at 480 nm (λex = 390 nm) in the presence buffer (0.1 m, pH 4.0), 74%; ii) 16 (1.1 equiv.), PyBOP (1.1 equiv.),

of sulfhydryl analytes: Cys (50 equiv.), and ThioPi (Na salt, DIEA (5 equiv.), NMP, room temp., 2 h; iii) DTT (10 equiv.),

50 equiv.), recorded in Tris·HCl buffer (100 mm, pH 8.0) at 25 °C; NaHCO (0.1 m aq.; pH 8.5), room temp., 1 h, 31% (for the two

1

3

a further kinetics curve for Pi (K salt, 50 equiv.) was recorded under

the same conditions.

steps ii and iii); iv) NMP/NaHCO

room temp., 1 h, 62%.

3

(0.1 m aq.; pH 8.15), 2:1, v/v,

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FULL PAPER

Scheme 8. Comparative reactivity of bioconjugatable 3-(2-benzimidazolyl)-7-hydroxycoumarin 11 and 16 towards a phosphonium-based

coupling reagent, namely PyBOP (generalized to all peptide coupling agents involving a basic catalysis: phosphonium and uronium salts).

For clarity, only one of the three structures known for HOBt (hydroxybenzotriazole) ester^[is shown.

52]

namely urokinase-type plasminogen activator (uPA). This cant changes in the fluorescence signal were observed in the

protease plays a critical role in malignancies, and its over- absence of protease.

expression has been linked to poor clinical prognosis in

[

41]

breast cancer. The ability to serially map uPA expression

as a biomarker and through fluorogenic assays would thus

have significant potential to improve new cancer therapies.

Heptapeptide H-Ser-Gly-Arg-Ser-Ala-Asn-Ala-OH (also

SGRSANA) has been reported to be a highly potent sub-

strate of uPA (cleavage site between Arg and Ser resi-

[

42]

dues);

therefore, we decided to label this peptide with a

FRET pair consisting of 3-(2-benzimidazolyl)-7-hydroxy-

coumarin 16 as the fluorophore, and nitro-DABCYL deriv-

ative 26 as the quencher (Scheme 7). To achieve chemoselec-

tive labeling reactions, cysteine and lysine residues were in-

corporated at the N- and C-termini, respectively, as handles

for the introduction of maleimide-DABCYL 26 and the

amine-reactive fluorophore. Thus, peptide Ac-Cys(StBu)-

Ser-Gly-Arg-Ser-Ala-Asn-Ala-Lys-NH 27 was readily ob-

2

tained by standard solid-phase peptide synthesis (SPPS)

techniques, and was doubly labeled according to a three-

step procedure previously reported by us.^[Interestingly,

36]

the use of a 6-sulfonated 7-hydroxycoumarin 16 whose

phenol group is weakly reactive,^[enabled us to carry out

12]

the acylation of the ε-amino group of 27 with this fluores- Figure 3. In vitro fluorescence-based assay of uPA-sensitive FRET-

based probe 29: (A) Fluorescence emission spectrum (λex

=

cent carboxylic acid by using a phosphonium-based cou-

pling reagent (PyBOP) and DIEA, which is in contrast to

the amidification reactions involving 7-hydroxycoumarin 11

4

00 nm) of probe 29 in PBS before (dashed line) and after (solid

line) incubation with uPA; (B) Time-dependent fluorescence inten-

sity at 480 nm (λex = 400 nm) of probe 29 (1.0 μm) with and without

(

vide supra and Scheme 8). The resulting uPA fluorogenic uPA from human urine (0.6 U, incubation time 25 min) recorded

in PBS at 37.5 °C.

substrate (i.e., 29) was isolated in pure form by semiprepar-

ative RP-HPLC (overall yield 20%, purity 100%). Its struc-

ture was unambiguously confirmed by ESI-MS analysis.

The results from the fluorogenic cleavage assay with com-

mercial uPA (from human urine) are summarized in Fig-

ure 3. Almost complete quenching (QE = 98%) was found

Conclusions

In this paper, we have reported the design, synthesis, and

for 29 until it was cleaved by uPA, which caused a 32-fold some biolabeling applications of a new family of water-

increase in fluorescence at λ_e_m= 480 nm over time (a pla- soluble cyan-green emitting fluorophores based on the 3-(2-

teau was reached within 120 min). As expected, no signifi- benzimidazolyl)-7-hydroxycoumarin scaffold. An original

10

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3-(Heteroaryl)-7-hydroxycoumarins

synthetic pathway whose key step is a condensation reac- disulfonic acid (tributylammonium salt, TBA salt),^[²⁵^]alkyne-

+

[

37]

functionalized cystamine 22,

TFA salt of 3,4-diaminobenz-

tion between 7-acetoxy-3-formylcoumarin and prefunction-

alized ortho-phenylenediamine derivatives has, for the first

time, enabled the introduction of both water-solubilizing

moieties and a bioconjugatable handle onto the 3-hetero-

aryl substituent of these 7-hydroxycoumarin derivatives.

enesulfonic acid,^[N-(2-azidoethyl)-N-methylaniline,

22]

[36]

maleim-

ide-terminated aniline 25,^[sodium thiophosphate (NaThioPi),

46]

[47]

and peptide Ac-Cys(StBu)-Ser-Gly-Arg-Ser-Ala-Asn-Ala-Lys-NH

2

[36]

27.

Gratifyingly, high fluorescence quantum yields under Instruments and Methods: The synthesis of peptide 27 was carried

out with an Applied Biosystems 433A synthesizer using standard

physiological conditions were obtained. The versatility of

this method was also demonstrated through the preparation

of the unknown 3-(2-benzoselenazolyl)-7-hydroxycoumarin

Fmoc/tBu chemistry.^[

48] 1

H and C NMR spectra were recorded

13

with either a Bruker DPX 300 or a Bruker Avance III 500 spec-

trometer (Bruker, Wissembourg, France). Chemical shifts are ex-

pressed in parts per million (ppm), and the residual solvent peak

18, whose spectroscopic and biological properties are cur-

rently under investigation. In contrast to more conventional

long-wavelength 7-hydroxycoumarins bearing a hydrophilic

substituent at their C-6 or C-8 position, the lack of polar

[49]

was used for calibration. J values are expressed in Hz. Infrared

(

IR) spectra were recorded with a universal ATR sampling access-

ory with a Perkin–Elmer FTIR Spectrum 100 spectrometer. Bond

–

1

groups close to the phenol facilitates the preparation of vibration frequencies are expressed in reciprocal centimeters (cm ).

thiol-sensitive profluorophores in good yields through Analytical HPLC was carried out with a Thermo Scientific Sur-

etherification of 7-OH moiety with NBDCl. The high veyor Plus instrument equipped with a PDA detector. Semiprepar-

ative HPLC was carried out with a Thermo Scientific SPECTRA-

“fluorogenic” reactivity of “smart” fluorescent probe 19 to-

SYSTEM liquid chromatography system (P4000) equipped with a

UV/Vis 2000 detector. Automated flash purifications on RP-C18

cartridges were carried out with a Biotage Isolera™ One (ISO-

ward Cys, ThioPi, and H S was proved by fluorescence-

2

based in vitro assays. The reported water-solubilization

strategy can be easily extended to other fluorescent phenols

prefunctionalized with a formyl group, and should be par-

ticularly useful for developing biocompatible fluorogenic

probes targeting nucleophilic (bio)analytes.^[³^,⁴³^]Further-

1EW) system. Ion-exchange chromatography (for desalting disul-

fonated 7-hydroxycoumarin 17) was carried out with an Econo-

®

Pac disposable chromatography column (Bio-Rad, #732–1010)

®

filled with an aq. suspension of Dowex 50WX8–400 (Alfa Aesar,

more, the introduction of a carboxylic acid within the ca. 5 g for 15 mg of dye, 15ϫ50 mm bed), which was regenerated

benzimidazolyl substituent of these 7-hydroxycoumarins using HCl (10% aq.), and equilibrated with deionized water. Low-

has allowed us to develop new coumarin-based reagents for resolution mass spectra (LRMS) were obtained with a Finnigan

LCQ Advantage MAX (ion trap) apparatus equipped with an elec-

fluorescent labeling of biomolecules, as demonstrated by

trospray ionisation (ESI) source. High-resolution mass spectra

the preparation of FRET probes reactive towards biothiols

or proteases.

(

HRMS) were recorded either with a Thermo LTQ Orbitrap XL

apparatus equipped with an ESI source or with an LCT Premier

XE benchtop orthogonal acceleration time-of-flight (oa-TOF)

mass spectrometer (Waters Micromass) equipped with an ESI

source. UV/Vis absorption spectra were obtained with a Varian

Cary 50 scan spectrophotometer using a rectangular quartz cell

(Varian, standard cell, Open Top, light path 10ϫ10 mm, chamber

volume: 3.5 mL). Fluorescence spectroscopic studies (emission/ex-

citation spectra) were carried out with a Varian Cary Eclipse spec-

trophotometer with a semi-micro quartz fluorescence cell (Hellma,

Experimental Section

General Remarks: Flash column chromatography was carried out

_o_n_G_e_d_u_r_a_n® Si 60 silica gel (63–200 μm) from Merck. TLC were

carried out on Merck DC Kieselgel 60 F-254 aluminium sheets.

The spots were visualized by illumination with a UV lamp (λ =

4

254/365 nm) and/or staining with KMnO solution. Unless other-

104F-QS, light path: 10ϫ4 mm, chamber volume: 1400 μL) or an

wise noted, all chemicals were used as received from commercial

sources without further purification. Bis(2-aminophenyl)diselenide

was purchased from Fluorochem. All solvents were dried by stan-

ultra-micro quartz fluorescence cell (Hellma, 105.251-QS, light

path: 3ϫ3 mm, chamber volume: 45 μL) for uPA assays. All ab-

sorption spectra (of 3-benzimidazolyl-7-hydroxycoumarin deriva-

tives, fluorogenic probes, and DABCYL-based quenchers) were re-

corded (220–700 nm or 300–700 nm) at 25 °C in the selected sol-

vents (mainly PB, PBS, and DMSO). Excitation/emission spectra

were recorded under the same conditions after emission/excitation

at 390/510 (or 600 or 650) nm (excitation filter: auto and emission

filter: open, excitation and emission slit: 5 nm). Fluorescence quan-

tum yields were measured at 25 °C by a relative method using 7-

dard procedures [CH

2

Cl

2

: distillation from P

2

O

5

; CH

3

CN: distil-

4

SO ,

lation from CaH ; absolute EtOH: storage over anhydrous Na

2

THF: distillation from sodium benzophenone diketyl; and triethyl-

amine (TEA): distillation from KOH and storage over BaO]. Pept-

ide synthesis grade NMP and anhydrous DMF were purchased

from Carlo Erba, and stored over 4 Å molecular sieves. Peptide

synthesis grade DIEA was provided by Iris Biotech GmbH. HPLC

gradient grade acetonitrile (CH

Aqueous buffers [PB, PBS, and aqueous 0.1 m acetate and borate

buffers used for the pK determination of 3-(heteroaryl)-7-

3

CN) was obtained from VWR.

hydroxycoumarin (Φ

5% in 0.1 n NaOH) as a standard.^[⁵⁰^,⁵¹^]The following equation

was used to determine the relative fluorescence quantum yield:

F F

= 76% in PB, pH 7.4) or fluorescein (Φ =

9

a

hydroxycoumarins] and mobile phases for HPLC were prepared

with water purified using a MilliQ system (purified to 18.2 MΩcm).

Triethylammonium acetate (TEAA; 2.0 m) and triethylammonium

hydrogen carbonate (TEAB; 1.0 m) buffers were prepared from dis-

tilled TEA and glacial acetic acid or CO

following starting materials were synthesized according to literature

₎2 _Φ

F

(s)

Φ

F

(x) = (A

S

/A

X

)(F

X

/F

S

)(n

X

/n

S

where A is the absorbance (in the range of 0.01–0.1 AU), F is the

area under the emission curve, n is the refractive index of the sol-

vents (at 25 °C) used in measurements, and the subscripts s and x

represent the standard and the unknown, respectively. The follow-

ing refractive index values were used: 1.362 for EtOH, 1.479 for

2

gas, respectively. The

[

15]

procedures: 3-(2-benzimidazolyl)-7-hydroxycoumarin,

7-acet-

oxy-3-formylcoumarin,^[

23]

3-[(2-nitrophenyl)amino]-1-propanol,

[44]

4

-fluoro-3-nitrobenzoic acid tert-butyl ester,^[⁴⁵^]2-aminoethane-1,1- DMSO, and 1.337 for PB, PBS, and H

O + 2.5% SDS.

2

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General Procedure for in-vitro Thiolysis of Fluorogenic Probes Ϫ (0.1% aq.) as eluents [100% aq. TFA (5 min), then linear gradient

FULL PAPER

–

1

Fluorescence-Based Assays: Stock solutions (1.0 mgmL ) of water-

soluble fluorophores 10–17 and azido-DABCYL 21 were prepared

in ultrapure water, and a stock solution of profluorophore 19 was

from 0 to 40% (60 min) CH

with the following gradient: [100% TEAB (5 min), then linear gra-

dient from 0 to 100% (100 min) CH CN]. System J: as for system

E, but with the following gradient: [100% aq. TFA (5 min), then

linear gradient from 0 to 100% (105 min) CH CN]. Visible detec-

3

CN]. System I: as for system D, but

3

prepared in H

2

O/DMSO (9:1, v/v). A stock solution of 3-(2-benzo-

selenazolyl)-7-hydroxycoumarin 18 was prepared in DMSO. Stock

solutions (10 mgmL ) of analytes (l-cysteine, NaThioPi, and

PO ) used for thiolysis of water-soluble profluorophores were

3

–1

tion was achieved at 430 nm. System K: semipreparative RP-HPLC

(Thermo Hypersil GOLD C18 column, 5 μm, 10.0ϫ250 mm) with

K

3

4

prepared in ultrapure water. A micromolar solution (for each pro-

fluorophore) was obtained by dilution of the stock solution with

PB + 45% DMSO (pH 8.3) for 24, with PBS [phosphate (100 mm)

CH

3

CN and TFA (0.1% aq.) as eluents [100% aq. TFA (5 min),

CN]

at a flow rate of 4.0 mLmin . Visible detection was achieved at

followed by linear gradient from 0 to 100% (60 min) CH

3

–

1

+

NaCl (150 mm), pH 7.5] for 19, and with Tris·HCl (100 mm, pH 430 nm.

8

.0) for 24. Depending on the reactivity of the probe (i.e., thiolysis

Synthesis: For the detailed synthetic procedures for ortho-phenyl-

enediamine derivatives 2, 3, 5–7, and 7-acetoxy-3-formylcoumarin-

kinetics) and the fluorescence efficiency of the released fluoro-

phore, a concentration between 1.1 and 8.6 μm was used. Stock

solutions (100 μm) of the analytes (Cys, NaThioPi, K PO , and

3 4

6

-sulfonic acid, see the Supporting Information.

Most of 3-(2-benzimidazolyl)-7-hydroxycoumarins and related

fluorogenic probes were found to be soluble in D O, but poor qual-

NaHS) used for the fluorescence-based assay of 19 were prepared

in PBS buffer. A portion (3 mL) of this solution was transferred

into a quartz fluorescence cell (Varian, fluorescence cell, open top,

light path: 10ϫ10 mm, chamber volume: 3.5 mL), and thermostat-

ted at 25 °C. The required number of equivalents of analyte (5, 50,

or 150 equiv.) was added, and the resulting mixture was homoge-

nized through magnetic stirring for 2 min. The fluorescence emis-

sion of the released fluorophore was monitored at λ = 480 nm

2

ity spectra were obtained (i.e., broad and poorly resolved peaks).

Thus, all NMR spectra of water-soluble derivatives were recorded

in [D ]DMSO.

6

General Procedure A (GP A) for the Preparation of 3-(2-Benzimid-

azolyl)-7-hydroxycoumarin Derivatives: A mixture of 7-acetoxy-3-

formylcoumarin (1 equiv.) and ortho-phenylenediamine derivative

(emission slit = 5 nm) upon excitation at λ = 390 nm (excitation

(

1 equiv.) in DMSO was stirred for 3 h at 120 °C. Thereafter, the

slit = 5 nm; excitation/emission filters: auto) over time, with mea-

surements recorded every 1 s.

mixture was diluted with aq. TEAB buffer (pH 7.5), and purified

on an RP-C18 cartridge using an automated flash purification sys-

General Procedure for in-vitro Peptide Cleavage by uPA: A solution tem. The product-containing fractions were lyophilized three times

(1.0 μm) of fluorogenic peptide 29 was prepared in PBS (45 μL),

to give the TEA salt of the 3-(2-benzimidazolyl)-7-hydroxycouma-

and transferred into the ultra-micro quartz fluorescence cell. uPA

rin derivative as a yellow amorphous powder.

solution [5 μL, 0.6 U; prepared from uPA (25 μg) in buffer

General Procedure B (GP B) for the Preparation of 3-(2-Benzimid-

azolyl)-7-hydroxycoumarin Derivatives: A mixture of 7-acetoxy-3-

(100 μL): Tris·HCl (500 mm), NaCl (1.0 m), PEG 6000 (1%), man-

nitol (2.0 m)] was added, and the resulting mixture was incubated

at 37.5 °C. After excitation at λ = 400 nm, the fluorescence emis-

sion of the released fluorophore at 480 nm was monitored over

time, with measurements recorded every 5 s.

formylcoumarin derivative (1 equiv.), Na

tho-phenylenediamine derivative (1 equiv.) in DMSO was stirred for

h at 120 °C. Thereafter, the mixture was diluted with aq. TEAB

2 2 5

S O (1 equiv.), and or-

3

buffer (pH 7.5), and purified on an RP-C18 cartridge using an auto-

mated flash purification system. The product-containing fractions

were lyophilized three times to give the TEA salt of the 3-(2-benz-

HPLC Separations: Several chromatographic systems were used for

the analytical experiments and purification steps (by semiprepar-

ative HPLC or automated flash purification system). System A: imidazolyl)-7-hydroxycoumarin derivative as a yellow amorphous

RP-HPLC (Thermo Hypersil GOLD 18 column, 5 μm, powder.

CN and trifluoroacetic acid (0.1% aq.; pH

.0) as eluents [100% aq. TFA (5 min), then linear gradient from 0

C

2

.1ϫ100 mm) with CH

3

General Procedure C (GP C) for the Activation of the Carboxylic

Acid of Bioconjugatable 3-(2-Benzimidazolyl)-7-hydroxycoumarin

Derivatives: The 3-(2-benzimidazolyl)-7-hydroxycoumarin deriva-

tive (1 equiv.) was dissolved in NMP (0.1 mL), and a solution of

N,NЈ-dicyclohexylcarbodiimide (DCC) in NMP (5.3 equiv. in

–

1

3

to 100% (45 min) CH CN] at a flow rate of 0.25 mLmin . Triple

UV/Vis detection was achieved at 220, 260, and 380 nm, and with

the “Max Plot” (i.e., chromatogram at absorbance maximum for

each compound) mode (220–650 nm). System B: as for system A

but with TEAA buffer (25 mm, pH 7.0) as aq. mobile phase [100%

TEAA buffer (5 min), then linear gradient from 0 to 100% (45 min)

CH

50 mL) and a solution of N-hydroxysuccinimide (NHS) in NMP

(

5.3 equiv. in 50 mL) were added sequentially. The resulting reac-

tion mixture was stirred at room temp. for 2 h. Thereafter, the crude

mixture was centrifuged to remove the DCU precipitate, and the

supernatant (containing the corresponding NHS ester) used in the

next coupling step without further purification.

®

3

CN]. System C: automated flash purification (Biotage SNAP

cartridge KP-C18-HS, 120 g) with aq. TEAB (50 mm, pH 7.5) as

aq. mobile phase [100% TEAB (5 min), then linear gradient from

–

1

0

to 40% (50 min) CH

UV-detection was achieved at 220 and 365 nm. System D: semipre-

parative RP-HPLC (Varian Kromasil 18 column, 10 μm,

1.2ϫ250 mm) with the following gradient [100% TEAB (5 min),

then linear gradient from 0 to 40% (70 min) CH CN] at a flow rate

3

CN] at a flow rate of 35.0 mLmin . Dual

Sulfonated 3-(2-Benzimidazolyl)-7-hydroxycoumarin (10): Com-

pound 10 was synthesized from 7-acetoxy-3-formylcoumarin and

ortho-phenylenediamine 8 according to GP A, and purified with an

automated flash purification (system C). Isolated yield, 19%. IR

(ATR): ν˜ = 3330, 2972, 1688, 1596, 1611, 1453, 1235, 1175, 1082,

C

2

3

–1

of 20.0 mLmin . UV detection was achieved at 365 nm. System E:

as for system D, but with TFA (0.1% aq.). System F: as for system

–

1 1

6

1028 cm . H NMR (300 MHz, [D ]DMSO) [spectrum recorded

D, but with the following gradient: [100% TEAB (5 min), then lin- with the first batch of 10, purified under nonoptimized conditions

ear gradient from 0 to 30% (75 min) CH CN]. System G: as for (aq. TFA 0.1% as the aq. mobile phase)]: δ = 11.38 (s, 1 H, OH),

system C, but with CH CN and TFA (0.1% aq.) as eluents [100%

aq. TFA (5 min), then linear gradient from 0 to 50% (100 min)

CH CN]. System H: as for system C, but with CH CN and TFA 2.0 Hz, 1 H) ppm. C NMR (75 MHz, [D

3

9.01 (s, 1 H), 8.21 (s, 1 H), 7.82 (d, JH,H = 8.6 Hz, 1 H), 7.76 (m,

3

H,H

J J =

H,H = 2.0 Hz, 8.6 Hz, 1 H), 6.91 (d, ³

2 H), 6.97 (dd,

1

3

6

]DMSO) [spectrum re-

12

www.eurjoc.org

Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O43215

/KAP1

Date: 20-11-14 16:47:38

Pages: 18

3-(Heteroaryl)-7-hydroxycoumarins

corded with the second batch of 10 (TEA salt), purified under opti-

mized conditions (TEAB buffer as the aq. mobile phase)]: δ =

toxy-3-formylcoumarin and ortho-phenylenediamine 3 according to

GP B, and purified by semipreparative RP-HPLC (system E). Iso-

lated yield, 24% (TFA salt). IR (ATR): ν˜ = 3315, 2991, 1672, 1597,

1

8

63.0, 161.3, 160.5, 159.5, 147.1, 144.5, 143.6, 143.4, 131.4, 129.7,

21.0, 114.3, 113.2, 111.6, 111.0, 102.1, 45.8 (N-CH -CH , TEA), NMR

6

TEA) ppm. HRMS (ESI ): calcd. for (300 MHz, [D ]DMSO): δ = 8.50 (s, 1 H), 7.85 (d, JH,H = 8.7 Hz,

–

1

2

3

1461, 1373, 1322, 1233, 1180, 1123, 1020 cm .

H

–

3

.6 (N-CH

2

–

-CH

3

,

–

3

C

16

H

9

N

2

O

6

S [M – H] 357.01758; found 357.01817. HPLC (sys-

= 17.4 min, purity = 96%.

1 H), 7.75 (m, 2 H), 7.40 (m, 2 H), 6.93 (dd, JH,H = 2.1 Hz, 8.7 Hz,

3

tem A): t

R

1 H), 6.89 (d, JH,H = 2.1 Hz, 1 H), 4.31 (t, JH,H = 6.0 Hz, 2 H),

3

2

1

.33 (t, JH,H = 3.0 Hz, 2 H), 2.51 (s, 9 H), 2.26 (qt, JH,H = 6.0 Hz,

H) ppm. C NMR (75 MHz, [D ]DMSO): δ = 163.4, 159.2,

6

56.3, 148.6, 147.7, 139.9, 134.4, 131.2, 123.8, 123.3, 118.3, 114.2,

11.6, 111.6, 111.0, 102.2, 62.6, 52.4, 52.3, 52.3, 41.9, 22.7 ppm.

HRMS (ESI ): calcd. for C22

3-(2-Benzimidazolyl)-7-hydroxycoumarincarboxylic Acid (11): Com-

1

3

pound 11 was synthesized from 7-acetoxy-3-formylcoumarin and

ortho-phenylenediamine 9 according to GP A, and purified with an

automated flash purification (system C). Isolated yield, 20%. IR

+

+·

H

24

N

3

O

3

[M] 378.18122; found

(

1

ATR): ν˜ = 3329, 2981, 1678, 1613, 1562, 1305, 1266, 1231, 1198,

130 cm . H NMR (300 MHz, [D ]DMSO) [spectrum recorded

6

378.18059. HPLC (system A): t

R

= 16.4 min, purity = 98%.

–1 1

with the first batch of 11, purified under nonoptimized conditions

aq. TFA 0.1% as the aq. mobile phase)]: δ = 11.10 (s, 1 H, OH),

3

-(2-Benzimidazolyl)-7-hydroxycoumarin N-(NЈ,NЈ,NЈ-Trimethyl)-

(

propylammonium Carboxylic Acid (15): Compound 15 was synthe-

sized from 7-acetoxy-3-formylcoumarin and ortho-phenylenediam-

ine 7 according to GP B, and purified by semipreparative RP-

HPLC (system E). Isolated yield, 23% (TFA salt). IR (ATR): ν˜ =

3

9

1

2

.09 (s, 1 H), 8.28 (s, 1 H), 7.85 (m, 2 H), 7.73 (d, JH,H = 8.7 Hz,

H), 6.92 (dd, ³

JH,H = 2.0 Hz, 8.7 Hz, 1 H), 6.86 (d, J =

3

H,H

13

6

.1 Hz, 1 H) ppm. C NMR (75 MHz, [D ]DMSO) [spectrum re-

corded with the second batch of 11 (TEA salt), purified under opti-

mized conditions (TEAB buffer as the aq. mobile phase)]: δ =

3

1

8

1

340, 2974, 1675, 1599, 1485, 1442, 1305, 1231, 1177, 1123,

009 cm . H NMR (300 MHz, [D ]DMSO): δ = 8.48 (s, 1 H),

6

–1

1

67.7, 163.5, 159.4, 155.9, 148.5, 144.6, 140.9, 137.1, 131.6, 125.2,

3

.28 (s, 1 H), 7.97 (d, JH,H = 8.6 Hz, 1 H), 7.85 (d, JH,H = 8.6 Hz,

–

24.1, 117.1, 115.2, 114.5, 111.5, 110.3, 102.1 ppm. HRMS (ESI ):

3

H), 7.73 (d, JH,H = 8.6 Hz, 1 H), 6.90 (m, 2 H), 4.29 (t, JH,H

=

–

[M – H]^–321.05060; found 321.05040.

calcd. for C17

H

9

N

2

O

5

3

6

.0 Hz, 2 H), 3.32 (t, JH,H = 3.0 Hz, 2 H), 3.00 (s, 9 H), 2.26 (qt,

HPLC (system A): t

R

= 20.4 min, purity = 96%.

3

13

J

6

H,H = 6.0 Hz, 2 H) ppm. C NMR (75 MHz, [D ]DMSO): δ =

1

4

67.7, 163.2, 159.5, 156.3, 150.4, 148.3, 142.0, 138.2, 131.1, 125.2,

3

-(2-Benzimidazolyl)-7-hydroxycoumarin-Substituted N-Propanesul-

24.2, 121.0, 114.2, 112.8, 111.1, 111.0, 102.2, 62.7, 52.3, 52.3, 52.2,

fonic Acid (12): Compound 12 was synthesized from 7-acetoxy-3-

formylcoumarin and ortho-phenylenediamine 2 according to GP B,

and purified by semipreparative RP-HPLC (system D). Isolated

yield, 23%. IR (ATR): ν˜ = 3332, 2980, 1680, 1615, 1564, 1315,

+

+·

1.8, 22.9 ppm. HRMS (ESI ): calcd. for C23

H

24

N

3

O

5

[M]

22.17105; found 422.17056; HPLC (system A): t

R

= 17.4 min,

purity = 98%.

–

1 1

1276, 1232, 1199, 1134 cm . H NMR (300 MHz, [D

6

]DMSO): δ

Sulfonated 3-(2-Benzimidazolyl)-7-hydroxycoumarincarboxylic Acid

3

=

10.91 (s, 1 H, OH), 8.40 (s, 1 H), 7.72 (m, 2 H), 7.68 (d, JH,H

=

(

16): Compound 16 was synthesized from 7-acetoxy-3-formylcou-

0.0 Hz, 1 H), 7.31 (t, ³

J

H,H = 10.0 Hz, 1 H), 7.25 (t, J

H,H

3

1

marin-6-sulfonic acid and ortho-phenylenediamine 9 according to

GP A, and purified with an automated flash purification system

1

0.0 Hz, 1 H), 6.87 (dd, ³

J

H,H = 5.0 Hz, 10.0 Hz, 1 H), 6.84 (d,

3

J

H,H = 5.0 Hz, 1 H), 4.33 (t, JH,H = 5.0 Hz, 2 H), 3.09 (q, JH,H

(

system C). Isolated yield, 25%. IR (ATR): ν˜ = 3299, 2987, 2712,

3

=

1

6.0 Hz, 6 H, N-CH

2

-CH

.98 (qt, JH,H = 5.0 Hz, 2 H), 1.16 (t, JH,H = 6.0 Hz, 9 H, N-

CH -CH ]DMSO): δ = 162.6,

, TEA) ppm. ¹³C NMR (75 MHz, [D

59.2, 156.1, 148.5, 147.2, 142.5, 135.3, 130.9, 122.7, 122.0, 119.1,

13.8, 113.8, 111.2, 111.1, 102.1, 48.2, 45.7 (N-CH -CH , TEA),

, TEA) ppm. HRMS (ESI ): calcd. for

S [M – H] 399.07453; found 399.06508. HPLC (sys-

= 17.5 min, purity = 99%.

3

, TEA), 2.35 (t, JH,H = 4.0 Hz, 2 H),

–1

1

1711, 1610, 1520, 1389, 1216, 1160, 1119, 1076, 1010 cm .

H

3

NMR (300 MHz, [D

H), 8.27 (s, 1 H), 8.18 (s, 1 H), 7.83 (d, JH,H = 8.3 Hz, 1 H), 7.71

6

]DMSO): δ = 12.70 (s, 1 H, OH), 9.14 (s, 1

2

3

6

3

1

4

3

(

d, JH,H = 8.3 Hz, 1 H), 6.93 (s, 1 H), 2.98 (q, JH,H = 3.0 Hz, 10

2

3

H, N-CH

CH , 1.66 TEA) ppm. C NMR (75 MHz, [D

59.4, 158.6, 155.6, 143.8, 129.8, 129.0, 125.1, 125.0, 123.7, 123.5,

20.2, 117.9, 114.8, 112.6, 111.5, 103.0, 45.7 (N-CH -CH , TEA),

TEA) ppm. HRMS (ESI ): calcd. for

2 3 2

-CH , 1.66 TEA), 1.12 (t, JH,H = 3.0 Hz, 14 H, N-CH -

–

3.4, 25.8, 8.6 (N-CH

2

-CH

3

13

3

6

]DMSO): δ = 168.0,

–

19 15 2 6

C H N O

tem B): t

1

9

C

B): t

R

2

3

–

N-Propylsulfonated 3-(2-Benzimidazolyl)-7-hydroxycoumarincarb-

oxylic Acid (13): Compound 13 was synthesized from 7-acetoxy-3-

formylcoumarin and ortho-phenylenediamine 6 according to GP B,

and purified by semipreparative RP-HPLC (system D). Isolated

yield, 21%. IR (ATR): ν˜ = 3325, 2961, 1675, 1623, 1574, 1316,

2 3

.1 (N-CH -CH ,

–

17

H

9

N

2

O

8

S [M – H] 401.00741; found 40100841. HPLC (system

R

= 16.8 min, purity = 95%.

Disulfonated 3-(2-Benzimidazolyl)-7-hydroxycoumarin (17): Com-

pound 11-NHS (12 mg, 0.0284 mmol, 1 equiv.) was synthesized ac-

cording to GP C. Then, the NMP solution containing this active

ester was added dropwise to a precooled (0 °C) solution (0.5 m) of

–

1 1

1269, 1233, 1189, 1124 cm . H NMR (500 MHz, [D

6

]DMSO): δ

3

=

8.44 (s, 1 H), 8.24 (s, 1 H), 7.92 (d, JH,H = 10.0 Hz, 1 H), 7.78

3

(

d, JH,H = 10.0 Hz, 1 H), 7.72 (d, JH,H = 10.0 Hz, 1 H), 6.88 (dd,

J

2

0

2

-aminoethane-1,1-disulfonic acid (TBA⁺salt) in NMP (748 μL,

.374 mmol, 13 equiv.) containing DIEA (2.0 m solution in NMP;

00 μL, 5 equiv.), over a period of 15 min. The resulting mixture

3

H,H = 5.0 Hz, 10.0 Hz, 1 H), 6.83 (d, JH,H = 5.0 Hz, 1 H), 4.37

3

(t, JH,H = 5.0 Hz, 2 H), 2.90 (q, JH,H = 6.0 Hz, 8 H, N-CH

2

-CH

3

,

.33 TEA), 2.33 (t, JH,H = 4.0 Hz, 2 H), 1.98 (br. t, ³JH,H = 5.0 Hz,

H), 1.10 (t, JH,H = 6.0 Hz, 12 H, N-CH

3

1

2

was stirred at room temp. overnight. This amidification reaction

was checked for completion by RP-HPLC (system B). The reaction

was quenched by the addition of glacial acetic acid (100 μL), and

then the mixture was diluted with aq. TEAB (ca. 8.0 mL), and

purified by RP-HPLC (system F). The product-containing frac-

tions were lyophilized three times to give the TEA salt of com-

3

2

-CH

]DMSO): δ = 168.1, 162.9, 159.2, 156.2,

50.4, 147.5, 142.2, 138.3, 131.0, 125.4, 123.9, 120.9, 114.0, 113.4,

11.1, 110.9, 102.2, 48.1, 45.7 (N-CH -CH , TEA), 43.6, 25.8, 9.4

3

, 1.33 TEA) ppm.

1

3

C NMR (126 MHz, [D

6

1

2

3

–

(

[

N-CH

2 3 15 2 8

-CH , TEA) ppm. HRMS (ESI ): calcd. for C20H N O S

M – H]^–443.05436; found 443.05331. HPLC (system B): t

R

=

+

pound 17 as a mixture with TBA salts. Desalting by ion-exchange

chromatography (followed by lyophilisation) gave compound 17

15.6 min, purity = 99%.

3

-(2-Benzimidazolyl)-7-hydroxycoumarin N-(NЈ,NЈ,NЈ-Trimethyl)- (4.0 mg, 28%) as a yellow amorphous powder. IR (ATR): ν˜ = 3321,

–

1

propylammonium (14): Compound 14 was synthesized from 7-ace- 3053, 1703, 1601, 1590, 1462, 1171, 1060, 1015 cm . H NMR

Eur. J. Org. Chem. 0000, 0–0

www.eurjoc.org

13

Job/Unit: O43215

/KAP1

Date: 20-11-14 16:47:38

Pages: 18

B. Roubinet, A. Chevalier, P.-Y. Renard, A. Romieu

FULL PAPER

(

300 MHz, [D

6

]DMSO): δ = 9.53 (s, 1 H), 8.66 (s, 1 H, NH), 8.08

glacial acetic acid (50 μL), and the mixture was purified by semi-

3

s, 1 H), 8.02 (d, JH,H = 9.0 Hz, 1 H), 7.85 (d, JH,H = 9.0 Hz, 1 preparative RP-HPLC (system H). The product-containing frac-

3

H), 7.77 (d, JH,H = 9.0 Hz, 1 H), 6.94 (dd, JH,H = 9.0 Hz, 3.0 Hz,

tions were lyophilized to give the TFA salt of compound 23 (5 mg,

25%) as a yellow amorphous powder. IR (ATR): ν˜ = 3240, 2920,

2854, 2105 (-CϵC-), 1712, 1613, 1570, 1504, 1439, 1301, 1232,

3

1

H), 6.88 (d, JH,H = 3.0 Hz, 1 H), 4.01 (m, 1 H), 3.92 (m, 2 H)

ppm. ¹³C NMR (126 MHz, [D

]DMSO): δ = 165.3, 164.2, 158.1,

56.6, 149.5, 145.6, 133.5, 132.9, 132.4, 131.2, 124.0, 114.9, 114.2,

13.1, 111.2, 104.5, 102.1, 73.4, 39.5 (masked by DMSO signal)

6

–

1 1

1

6

1189, 1127, 1024 cm . H NMR (300 MHz, [D ]DMSO): δ = 11.08

3

(s, 1 H, OH), 9.07 (s, 1 H), 8.90 [t, JH,H = 5.3 Hz, 1 H, C(O)NH],

8.67 [m, JH,H = 5.3 Hz, 1 H, C(O)NH], 8.17 (s, 1 H), 7.84 (d, ³JH,H

= 8.6 Hz, 1 H), 7.71 (m, 2 H), 6.91 (dd, J = 2.2 Hz, 8.5 Hz, 1 H),

–

3

ppm. HRMS (ESI ): calcd. for C19

5

rity = 95%.

H

14

N

3

O

10

S

2

[M – H]

08.01151; found 508.01263. HPLC (system B): t = 18.5 min, pu-

R

3

6.86 (d, JH,H = 2.2 Hz, 1 H), 4.14 (s, 1 H), 3.40 (m, 4 H, masked

3

by water signal), 2.95 (t,

J

H,H = 6.6 Hz, 2 H), 2.83 (t,

H,H

J =

3

-(2-Benzoselenazolyl)-7-hydroxycoumarin (18): 7-Acetoxy-3-form-

ylcoumarin (200 mg, 0.86 mmol, 1 equiv.) was dissolved in DMSO

20 mL), and Na (163 mg, 0.86 mmol, 1 equiv.) and bis(2-

13

6

1

3

.6 Hz, 2 H) ppm. C NMR (75 MHz, [D ]DMSO): δ = 166.8,

63.3, 159.5, 155.8, 151.8, 148.1, 144.2, 131.6, 128.9, 122.0, 114.4,

11.5, 110.8, 102.1, 78.1, 75.9, 39.5 (masked by DMSO signal),

6

(

2 2 5

S O

aminophenyl)diselenide (235 mg, 0.69 mmol, 0.8 equiv.) were se-

quentially added. The resulting reaction mixture was stirred at

8.7, 37.2, 36.6 ppm. Not enough of this compound was obtained

13

for it to be well-characterized by C spectroscopy (four carbons

1

20 °C under an Ar atmosphere for 48 h. Thereafter, the mixture

was diluted with satd. aq. NH Cl, and extracted with EtOAc. The

combined organic phases were dried with anhydrous MgSO , and

concentrated under reduced pressure. The resulting residue was

purified by flash column chromatography on silica gel (CH Cl

EtOAc with a step gradient from 100:0 to 95:5) to give seleno-dye

–

are missing). LRMS (ESI ): calcd. for C24

H

19

–

N

4

O

5

S

2

507.07 [M –

H] ; found 507.07 and 620.60 [M + TFA – H] . HPLC (system A):

= 21.9 min, purity = 99%.

4

–

4

t

R

Azido-DABCYL Dye 20: Solid NOBF

.1 equiv.) was added to a precooled solution of para-aminobenzoic

acid (200 mg, 1.46 mmol, 1 equiv.) in dry CH CN (10 mL). The

resulting mixture was stirred at 0 °C for 15 min. Then, a solution

of N-(2-azidoethyl)-N-methylaniline (306 mg, 1.74 mmol,

4

(187 mg, 1.6 mmol,

2

/

1

(

8 (53 mg, 14%) as an orange solid. IR (ATR): ν˜ = 1709, 1596,

586, 1562, 1446, 1255, 1233, 1193, 1063 cm .

3

–1

1

H NMR

300 MHz, [D

6

]DMSO): δ = 11.11 (s, 1 H, OH), 9.12 (s, 1 H), 8.18

3

1.2 equiv.) in CH CN (1 mL) was added dropwise, and the resulting

dd, JH,H = 7.9 Hz, 0.7 Hz, 1 H), 8.07 (d, JH,H = 7.6 Hz, 1 H),

3

mixture was stirred at 0 °C for 30 min. Thereafter, the product was

precipitated by the addition of NaOAc buffer (0.1 m; pH 4.0). The

orange solid was recovered by filtration, washed with a mixture

7

.93 (d, JH,H = 8.6 Hz, 1 H), 7.53 (t, JH,H = 7.2 Hz, 1 H), 7.35

3

(

t, JH,H = 8.0 Hz, 1 H), 6.94 (dd, JH,H = 8.6 Hz, 2.2 Hz, 1 H), 6.86

d, JH,H = 2.1 Hz, 1 H) ppm. C NMR (75 MHz, [D

3

13

6

]DMSO): δ

of CH

DABCYL 20 (404 mg, 86%) as an orange amorphous powder. IR

(ATR): ν = 2531, 2095, 1672, 1596, 1515, 1419, 1377, 1286, 1137,

3 2

CN/H O (1:1, v/v), and finally lyophilized to give azido-

=

1

163.5, 163.4, 160.6, 155.7, 153.9, 141.4, 139.0, 132.2, 126.4, 125.3,

24.8, 123.8, 116.6, 114.6, 111.5, 102.1 ppm. HRMS (ESI ): calcd.

+

for C16

(

H

10NO

3

Se [M + H] 343.9805; found 343.9821. HPLC

˜

–

1 1

9

J

45, 862, 814 cm . H NMR (300 MHz, [D

6

]DMSO): δ = 8.08 (d,

H,H = 8.6 Hz, 2 H), 7.83 (dd, JH,H = 2.3 Hz, 8.6 Hz, 4 H), 6.91

d, 3_JH,H = 9.2 Hz, 2 H), 3.69 (t, 3_JH,H = 5.9 Hz, 2 H), 3.55 (t,

system A): t = 28.3 min, purity = 99%.

R

3

N-(NЈ,NЈ,NЈ-Trimethyl)propylammonium NBD Ether (19): Phenol

4 (10 mg, 0.020 mmol, 1 equiv.) was dissolved in dry DMF, and

NBDCl (6 mg, 0.023 mmol, 1.5 equiv.) and TEA (15 μL,

.115 mmol, 5.8 equiv.) were sequentially added. The resulting re-

(

1

3

13

J

6

H,H = 5.9 Hz, 2 H), 3.08 (s, 3 H) ppm. C NMR (75 MHz, [D ]-

DMSO): δ = 167.0, 155.0, 151.8, 143.0, 131.2, 130.5, 125.3, 121.8,

0

–

1

[

=

11.7, 50.6, 48.4, 38.4 ppm. HRMS (ESI ): calcd. for C16

H

15

N

6

O

2

action mixture was stirred at room temp. overnight. Thereafter, the

crude mixture was directly purified by semipreparative RP-HPLC

–

M – H] 323.1262; found 323.1259. UV/Vis (DMSO, 25 °C): λmax

–1

446 nm; ε (446 nm) = 21000 m cm ; Δλ1/2 max = 370–500

(system G). The product-containing fractions were lyophilized to

(

130 nm).

give the TFA salt of NBD ether 19 (11.0 mg, 72%) as a yellow

amorphous solid. IR (ATR): ν˜ = 3430, 3050, 1726, 1675, 1614,

Water-Soluble Azido-DABCYL Dye 21:

–

1

538 (NO

NMR (300 MHz, [D

.65 (s, 1 H), 8.10 (d, JH,H = 8.6 Hz, 1 H), 7.78 (m, 3 H), 7.53

2

), 1457, 1332 (NO

2

), 1261, 1167, 1116, 997 cm . H

Azido-DABCYL 20 (40 mg, 0.123 mmol, 1 equiv.) and TSTU

3

6

]DMSO): δ = 8.74 (d, JH,H = 8.3 Hz, 1 H),

(

48 mg, 0.16 mmol, 1.3 equiv.) were dissolved in NMP (0.5 mL),

and the solution was stirred under an Ar atmosphere. Then, DIEA

2.0 m solution in NMP; 123 mL, 0.246 mmol, 3 equiv.) was added,

3

8

3

(dd, JH,H = 2.3 Hz, 8.6 Hz, 1 H), 7.35 (m, 2 H), 7.11 (d, JH,H =

(

8

2

(

.3 Hz, 1 H), 4.30 (t, J = 7.4 Hz, 2 H), 3.35 (q, J = 2.1 Hz, 7.4 Hz,

and the reaction mixture was stirred at room temp. for 30 min.

The resulting NHS active ester was used in the next step without

purification.

3

13

H), 3.03 (s, 9 H), 2.28 (br. t, JH,H = 2.1 Hz, 2 H) ppm. C NMR

]DMSO): δ = 158.8, 158.2, 157.8, 157.1, 155.3, 151.2,

75 MHz, [D

6

1

2

47.1, 145.5, 144.6, 142.2, 135.1, 131.7, 131.6, 123.4, 122.6, 119.3,

17.9, 117.4, 116.9, 112.9, 111.2, 108.4, 62.7, 52.4, 52.3, 52.3, 41.7,

2

-Aminoethane-1,1-disulfonic acid (TBA⁺salt; 0.5 m solution in

+

+·

NMP; 2.5 mL, 1.25 mmol, 10 equiv.) and DIEA (2.0 m solution in

NMP; 0.184 mL, 184 μL, 3 equiv.) were mixed, and the solution

was cooled to 0 °C. Then, the solution of the crude NHS active

2.9 ppm. HRMS (ESI ): calcd. for C28

H

25

N

6

O

6

[M] 541.18301;

found 541.18150. HPLC (system A): t

R

= 21.4 min, purity = 100%.

Alkyne-Functionalized

3-(2-Benzimidazolyl)-7-hydroxycoumarin ester was added dropwise, and the resulting reaction mixture was

(

1

23): The NHS ester of carboxylic acid 11 (14 mg, 0.033 mmol,

equiv.) was synthesized according to GP C. Then, the NMP solu-

stirred at room temp. for 2 h. The reaction was checked for comple-

tion by RP-HPLC (system B), and the mixture was purified by

semipreparative RP-HPLC (system I). The product-containing

fractions were lyophilized to give the TEA salt of compound 21 as

tion of this active ester was added dropwise to a precooled (0 °C)

solution (0.57 m) of alkyne-functionalized cystamine 24 (TFA salt;

9

0 mg, 0.284 mmol, 8.5 equiv.) in NMP (0.5 mL) containing DIEA a mixture with TBA⁺salts. Desalting by ion-exchange chromatog-

(

2.0 m solution in NMP; 83 μL, 5 equiv.), over a period of 15 min.

raphy (followed by lyophilisation) gave water-soluble azido-DAB-

CYL dye 21 (38 mg, 61%) as a brown amorphous powder. IR

(ATR): ν˜ = 3359, 2107, 1616, 1597, 1538, 1388, 1265, 1219, 1183,

The resulting reaction mixture was stirred at room temp. overnight.

This amidification reaction was checked for completion by RP-

HPLC (system A). The reaction was quenched by the addition of

–

1

3

1141, 1016, 826 cm . H NMR (300 MHz, D

2

O): δ = 7.37 (d, JH,H

14

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Pages: 18

3-(Heteroaryl)-7-hydroxycoumarins

=

8.6 Hz, 2 H), 7.23 (d, ³

J

H,H = 9.5 Hz, 2 H), 7.05 (d, ³JH,H

=

394.1510; found 394.1509. UV/Vis (DMSO, 25 °C): λmax = 502 nm;

ε (502 nm) = 39000 m cm ; Δλ1/2 max(nm) = 438–553 (115 nm).

3

–1

8

1

.6 Hz, 2 H), 6.80 (d, JH,H = 9.5 Hz, 2 H), 4.18 (t, JH,H = 5.7 Hz,

3

H), 3.90 (d, JH,H = 5.7 Hz, 2 H), 3.71 (br. s, 2 H), 3.61 (br. s, 2

Urokinase-Sensitive FRET Probe (29):

13

H), 3.17 (s, 3 H) ppm. C NMR (75 MHz, D

2

O): δ = 166.7, 159.1,

The TFA salt of Ac-C(StBu)SGRSANAK-NH

2

27 (9 mg,

1

3

5

43.4, 136.6, 132.7, 130.9, 128.4, 117.3, 117.1, 73.3, 52.8, 48.4, 40.4,

9.0 ppm. HRMS (ESI ): calcd. for C18

10.0871; found 510.0873. UV/Vis (DMSO, 25 °C): λmax = 437 nm;

–

8.8 μmol, 1 equiv.), coumarin 16 (3.9 mg, 9.7 μmol, 1.1 equiv.), and

PyBOP (5.0 mg, 9.7 μmol, 1.1 equiv.) were dissolved in NMP

(100 μL). DIEA (2.0 m solution in NMP; 22 mL, 44 μmol, 5 equiv.)

was added, and the resulting reaction mixture was stirred at room

temp. for 2 h. The reaction was checked for completion by RP-

HPLC (system A). Thereafter, the mixture was diluted with TFA

H

20

N

7

O

7

S

2

[M – H]

–

1

–1

ε (437 nm) = 27000 m cm ; Δλ1/2 max = 394–489 (95 nm). UV/Vis

PBS, 25 °C): λmax ₌₄₆₁_n_m_;_ε₍₄₆₁_n_m₎₌₂₇₀₀₀_m– cm ; Δλ1/2

max = 394–506 (112 nm). HPLC (system B): t = 21.5 min, purity

98%.

1

–1

(

R

=

(

0.1% aq.), and purified by semipreparative RP-HPLC (system J).

Thiol-Sensitive FRET Probe 24: Alkyne-functionalized 7-hydroxy-

The product-containing fractions were lyophilized to give the

coumarin 23 (5.0 mg, 9.8 μmol, 1 equiv.), water-soluble azido- fluorescently-labeled peptide as a yellow amorphous powder.

0

–

DABCYL 21 (5.0 mg, 9.8 μmol, 1 equiv.), and Cu microsized pow-

LRMS (ESI ; CH

3

CN/H

2

O, 2:1, v/v): calcd. for C56

H

78

N

17

O

20

S

3

–

der (0.3 mg, 4.9 μmol, 0.5 equiv.) were suspensed in a mixture of

[M – H] 1404.48; found 1404.27. UV/Vis (PBS, 25 °C): λmax

=

DMSO/H

2

O (2:1, v/v). Then, CuSO

4

·5H

2

O (40 mm solution in

430 nm. Fluorescence (PBS, 25 °C): λem = 481 nm; Φ

HPLC (system A): t = 20.7 min, purity = 97%.

F

= 74%.

(0.1 m

water; 0.24 mg, 0.98 μmol, 0.1 equiv.) was added, and the resulting

mixture was stirred at 50 °C for 2 h. The reaction was checked for

completion by RP-HPLC (system B), and then the mixture was

purified by semipreparative RP-HPLC (system J). The product-

containing fractions were lyophilized three times to give FRET

R

The fluorescently-labeled peptide was dissolved in NaHCO

aq.; pH 8.5; 200 μL), and a solution of DTT (13.5 mg, 88 μmol,

0 equiv.) in NaHCO (0.1 m aq.; 100 μL) was added. The resulting

3

1

3

reaction mixture was stirred at room temp. for 1 h. The reaction

was checked for completion by RP-HPLC (system A). Then, the

mixture was diluted with TFA (0.1% aqueous; 700 μL), and puri-

fied by semipreparative RP-HPLC (system J). The product-con-

taining fractions were lyophilized to give target free-sulfhydryl fluo-

1

probe 24 (4.8 mg, 48%) as a yellow amorphous powder. H NMR

3

(500 MHz, [D

6

]DMSO): δ = 12.60 (d, JH,H = 28.6 Hz, 1 H), 10.96

(

s, 1 H, 1 OH), 9.07 (s, 1 H), 8.82 [m, 2 H, 2 C(O)NH], 8.60 (m, 4

3

H), 8.17 (s, 1 H), 7.79 (m, 8 H), 6.90 (dd, JH,H = 5.0, 10.0 Hz, 1

H), 6.86 (d, JH,H = 5.0 Hz, 1 H), 6.81 (d, JH,H = 10.0 Hz, 2 H),

3

rescent peptide 28 (4.9 mg, 31% over two steps) as a yellow

3

4

(

1

(

1

4

.65 (t, JH,H = 10.0 Hz, 2 H), 3.98 (t, JH,H = 10.0 Hz, 2 H), 3.84

–

amorphous powder. LRMS (ESI ; CH

3

CN/H

2

O, 2:1, v/v): calcd.

3

t, JH,H = 10.0 Hz, 2 H), 3.52 (m, 5 H), 3.10 (q, JH,H = 5.0 Hz,

0 H, N-CH -CH , 1.90 TEA), 2.93 (m, 4 H), 2.85 (s, 3 H), 1.20

t, JH,H = 5.0 Hz, 16 H, N-CH

126 MHz, [D ]DMSO): δ = 164.3, 159.7, 155.7, 153.8, 151.3,

43.0, 142.7, 135.2, 131.3, 128.7, 128.4, 128.3, 127.7, 127.1, 125.0,

21.7, 117.5, 114.2, 112.5, 112.0, 111.6, 101.9, 74.0, 57.8, 51.4, 45.7,

0.0 (N-CH -CH ,TEA), 38.1, 37.9, 37.3, 37.1, 10.9, 8.6 (N-CH

,TEA), 8.5, 7.6 ppm. Not enough of this compound was ob-

–

for C52

H

70

N

17

O

20

S

2

[M – H] 1316.44; found 1316.53. LRMS

ESI ; in CH CN/H O, 2:1, v/v): calcd. for C52H N O S [M +

3 2 72 17 20 2

H] 1318.46; found 1318.27. UV/Vis (PBS, 25 °C): λmax = 433 nm.

Fluorescence (PBS, 25 °C): λem = 481 nm; Φ = 26% (cyan-green

emission of grafted 7-hydroxycoumarin partially quenched par free

thiol). HPLC (system A): t = 17.9 min, purity = 99%.

2

3

+

(

3

13

2

-CH

3

, 1.90 TEA) ppm. C NMR

+

6

F

R

2

3

2

-

Free-sulfhydryl fluorescent peptide 28 (1.0 mg, 0.7 μmol, 1 equiv.)

and solution of maleimide-nitro-DABCYL 26 (0.43 mg,

.77 μmol, 1.1 equiv.) in CH CN (20 mm) were dissolved in a mix-

ture of NMP and NaHCO (0.1 m aq.; pH 8.5) (2:1, v/v; 100 μL).

CH

3

13

a

tained for it to be well-characterized by C spectroscopy (nine

carbons are missing), despite an extended acquisition time (more

than 80000 scans) on a 500 MHz spectrometer (equipped with a

0

3

The resulting reaction mixture was protected from light (with alu-

minum foil), and stirred at room temp. for 1 h. The reaction was

checked for completion by RP-HPLC (system A). Then, the mix-

ture was diluted with TFA (0.1% aq.; 4 mL), and purified by semi-

preparative RP-HPLC (system K). The product-containing frac-

5

mm “BBFO” broadband ATMA gradient z probe four times

more sensitive than a standard 300 MHz spectrometer). HRMS

–

40 11 12 4

(ESI ): calcd. for C42H N O S [M – H] 1018.17352; found

2–

R

1018.17566 and 508.58352 [M – 2 H] . HPLC (system B): t =

2

1.5 min, purity = 98%.

tions were lyophilized to give uPA-sensitive FRET probe 29

+

(

0.8 mg, 62%) as a yellow amorphous powder. LRMS (ESI ;

Maleimide-nitro-DABCYL Dye (26): Solid NOBF

.16 mmol, 1.1 equiv.) was added to a precooled (0 °C) solution of

of para-nitroaniline (20 mg, 0.14 mmol, 1 equiv.) in dry CH CN

1.5 mL). The resulting reaction mixture was stirred at 0 °C for

4

(18.6 mg,

+

CH

1

3

CN/H

2

O, 2:1, v/v): calcd. for C72

H

91

N

22

O

24

+

S

2

[M + H]

0

2

711.60; found 1712.20 and 856.27 [M + 2 H] . HPLC (system

3

A): t = 24.4 min, purity = 100%.

R

(

1

5 min. Then, a solution of maleimide-terminated aniline 25

Supporting Information (see footnote on the first page of this arti-

cle): Details of the synthesis of ortho-phenylenediamine derivatives

(42 mg, 0.17 mmol, 1.2 equiv.) in CH

3

CN (0.2 mL) was added

dropwise, and the resulting mixture was stirred at 0 °C for 30 min.

Thereafter, the product was precipitated by the addition of NaOAc

buffer (0.1 m; pH 4.0). The orange solid was recovered by filtration,

2, 3, 5–7, and 7-acetoxy-3-formylcoumarin-6-sulfonic acid. Analyt-

ical data for all compounds/probes synthesized in this work.

3 2

washed with a mixture of CH CN/H O (1:1, v/v), and finally lyo-

philized to give maleimide quencher 26 (42 mg, 74%) as an orange Acknowledgments

amorphous powder. IR (ATR): ν˜ = 1707, 1601, 1509, 1382, 1334,

–

1 1

1

8

(

6

140, 858, 831, 694 cm . H NMR (300 MHz, [D ]DMSO): δ = This work was partially supported by INSA Rouen, Rouen Univer-

3

.32 (d, JH,H = 8.9 Hz, 2 H), 7.92 (d, JH,H = 7.3 Hz, 2 H), 7.89

sity, Centre National de la Recherche Scientifique (CNRS), Labex

SynOrg (ANR-11-LABX-0029), and Région Haute-Normandie

(CRUNCh network). Financial support from the Région Haute-

Normandie and Fonds Européen de Développement Régional

(FEDER) (TRIPODE, grant number 33883) for Ph. D. grants to

20 5 4

8.7, 35.8, 26.3. HRMS (ESI ): calcd. for C20H N O

[M + H]⁺B. R. and A. C., respectively, is greatly acknowledged. A. R. thanks

3

d, JH,H = 7.3 Hz, 2 H), 7.24 (m, 4 H), 3.60 (t, JH,H = 7.1 Hz, 2

3

H), 3.49 (t, JH,H = 7.1 Hz, 2 H), 3.10 (s, 3 H), 1.97 (qt, JH,H

=

7

1

3

.1 Hz, 2 H) ppm. ¹³C NMR (75 MHz, [D

56.9, 152.4, 147.6, 144.0, 134.4, 126.3, 124.8, 122.8, 111.7, 50.1,

6

]DMSO): δ = 170.8,

+

Eur. J. Org. Chem. 0000, 0–0

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15

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Pages: 18

B. Roubinet, A. Chevalier, P.-Y. Renard, A. Romieu

FULL PAPER

the Institut Universitaire de France (IUF) for financial support,

and the “Plateforme d’Analyse Chimique et de Synthèse

Moléculaire de l’Université de Bourgogne (PACSMUB,

http://www.wpcm.fr) for access to the Bruker Avance III 500 spec-

trometer and the Thermo LTQ Orbitrap XL mass spectrometer.

The authors thank Marie-José Penouilh and Dr. Fanny Picquet

5294; d) P. Kele, X. Li, M. Link, K. Nagy, A. Herner, K. Lor-

incz, S. Beni, O. S. Wolfbeis, Org. Biomol. Chem. 2009, 7, 3486–

3490; e) T. Ehrenschwender, B. R. Varga, P. Kele, H.-A. Wagen-

knecht, Chem. Asian J. 2010, 5, 1761–1764; K. Nagy, E. Orban,

S. Bosze, P. Kele, Chem. Asian J. 2010, 5, 773–777; f) G. B.

Cserep, K. N. Enyedi, A. Demeter, G. Mezo, P. Kele, Chem.

Asian J. 2013, 8, 494–502; g) G. B. Cserep, Z. Baranyai, D.

Komaromy, K. Horvati, S. Bosze, P. Kele, Tetrahedron 2014,

70, 5961–5965.

(University of Burgundy, ICMUB, UMR CNRS 6302) and A.

Marcual (laboratory COBRA, UMR CNRS 6014) for recording

the HRMS spectra, and Patricia Martel (University of Rouen, lab-

oratory COBRA, UMR CNRS 6014) for recording the IR spectra.

[10] a) Z. Diwu, T. Dubrovsky, B. Abrams, J. Liao, Q. Meng

(Becton, Dickinson, and Company, USA), 2010/0255504 A1,

2

010; b) K. K. Sadhu, S. Mizukami, A. Yoshimura, K. Kiku-

chi, Org. Biomol. Chem. 2013, 11, 563–568.

[

1] For comprehensive reviews, see: a) H. E. Katerinopoulos, Curr.

Pharm. Des. 2004, 10, 3835–3852; b) H. Li, L. Cai, Z. Chen,

in: Advances in Chemical Sensors (Ed.: W. Wang), InTech, 2011,

p. 121–150.

[

11] a) M. Adamczyk, M. Cornwell, J. Huff, S. Rege, S. V. T. Rao,

Bioorg. Med. Chem. Lett. 1997, 7, 1989–1988; b) V. Hagen, F.

Kilic, J. Schaal, B. Dekowski, R. Schmidt, N. Kotzur, J. Org.

Chem. 2010, 75, 2790–2797; c) T. Deligeorgiev, T. Tsvetkova, S.

Stanimirov, Color. Technol. 2011, 127, 434–439.

[

2] M. S. T. Gonçalves, Chem. Rev. 2008, 109, 190–212.

3] For comprehensive reviews, see: a) M. Eun Jun, B. Roy, K.

Han Ahn, Chem. Commun. 2011, 47, 7583–7601; b) W. Shi, H.

Ma, Chem. Commun. 2012, 48, 8732–8744.

[

12] B. Roubinet, P.-Y. Renard, A. Romieu, Dyes Pigm. 2014, 110,

270–284.

13] M. H. Elnagdi, S. O. Abdallah, K. M. Ghoneim, E. M. Ebied,

[

4] X. Li, X. Gao, W. Shi, H. Ma, Chem. Rev. 2014, 114, 590–659.

K. N. Kassab, J. Chem. Res. Synop. 1997, 44–45.

14] O. V. Khilya, M. S. Frasinyuk, A. V. Turov, V. P. Khilya, Chem.

Heterocycl. Compd. 2001, 37, 1029–1037.

15] T. Deligeorgiev, T. Tsvetkova, D. Ivanova, I. Timtcheva, Color.

Technol. 2008, 124, 195–203.

[5] a) X.-M. Peng, G. L. V. Damu, C.-H. Zhou, Curr. Pharm. Des.

2013, 19, 3884–3930; b) S. Sandhu, Y. Bansal, O. Silakari, G.

Bansal, Bioorg. Med. Chem. 2014, 22, 3806–3814; c) S. Ham-

ulakova, L. Janovec, M. Hrabinova, K. Spilovska, J. Kora-

becny, P. Kristian, K. Kuca, J. Imrich, J. Med. Chem. 2014, 57,

[

16] L. Tang, X. Han, Biomed. Pharmacother. 2013, 67, 179–182.

17] M. J. Matos, F. Perez-Cruz, S. Vazquez-Rodriguez, E. Uriarte,

L. Santana, F. Borges, C. Olea-Azar, Bioorg. Med. Chem. 2013,

7

073–7084.

6] a) O. S. Wolfbeis, J. H. Baustert, J. Heterocycl. Chem. 1985, 22,

215–1218; b) O. S. Wolfbeis, E. Koller, P. Hochmuth, Bull.

[

1

21, 3900–3906.

Chem. Soc. Jpn. 1985, 58, 731–734; c) O. S. Wolfbeis, H. Mar-

hold, Chem. Ber. 1985, 118, 3664–3672.

[

18] a) L. Zhang, T. Meng, R. Fan, J. Wu, J. Org. Chem. 2007, 72,

7

279–7286; b) M. Min, B. Kim, S. Hong, Org. Biomol. Chem.

[

7] For selected examples, see: a) E. Koller, O. S. Wolfbeis, Mon-

atsh. Chem. 1985, 116, 65–75; b) E. Koller, O. S. Wolfbeis, Anal.

Chim. Acta 1985, 170, 73–80; c) O. S. Wolfbeis, E. Koller,

Mikrochim. Acta 1985, 1, 389–395; d) S. T. Huang, Y. X. Peng,

K. L. Wang, Biosens. Bioelectron. 2008, 23, 1793–1798; e) S. T.

Huang, K. N. Ting, K. L. Wang, Anal. Chim. Acta 2008, 620,

2

012, 10, 2692–2698; c) F. Jafarpour, S. Zarei, M. B. Olia, N.

Jalalimanesh, S. Rahiminejadan, J. Org. Chem. 2013, 78, 2957–

2964; d) X. F. Wu, L. Wu, R. Jackstell, H. Neumann, M. Beller,

Chem. Eur. J. 2013, 19, 12245–12248; e) L. G. Meimetis, J. C.

Carlson, R. J. Giedt, R. H. Kohler, R. Weissleder, Angew.

Chem. Int. Ed. 2014, 53, 7531–7534; Angew. Chem. 2014, 126,

1

20–126; f) W. Lin, L. Long, W. Tan, Chem. Commun. 2010,

7661–7664.

46, 1503–1505; g) H.-C. Huang, K.-L. Wang, S.-T. Huang, H.-

[

19] Y. Jiang, W. Chen, W. Lu, Tetrahedron 2013, 69, 3669–3676.

Y. Lin, C.-M. Lin, Biosens. Bioelectron. 2011, 26, 3511–3516;

h) J. O. Moon, J. W. Lee, M. G. Choi, S. Ahn, S.-K. Chang,

Tetrahedron Lett. 2012, 53, 6594–6597; i) K. M. Mahoney, P. P.

Goswami, A. H. Winter, J. Org. Chem. 2013, 78, 702–705.

8] For a selection of relevant references, see: a) W. Baker, C. N.

Haksar, J. F. W. McOmie, J. Chem. Soc. 1950, 170–173; b) H.-

Y. Wang, W.-Y. Leung, F. Mao (Molecular Probes, Inc.), US

20] I. Svinyarov, M. G. Bogdanov, Eur. J. Med. Chem. 2014, 78,

198–206.

[

21] S. C. Tsay, J. R. Hwu, R. Singha, W. C. Huang, Y. H. Chang,

M. H. Hsu, F. K. Shieh, C. C. Lin, K. C. Hwang, J. C. Horng,

E. De Clercq, I. Vliegen, J. Neyts, Eur. J. Med. Chem. 2013, 63,

[

290–298.

22] F. da Silva Miranda, A. M. Signori, J. Vicente, B. de Souza,

J. P. Priebe, B. Szpoganicz, N. S. Gonçalves, A. Neves, Tetrahe-

dron 2008, 64, 5410–5415.

5696157, 1997; c) B. Abrams, Z. Diwu, O. Guryev, S. Aleshkov,

R. Hingorani, M. Edinger, R. Lee, J. Link, T. Dubrovsky, Anal.

Biochem. 2009, 386, 262–269; d) E. Paunescu, L. Louise, L.

Jean, A. Romieu, P.-Y. Renard, Dyes Pigm. 2011, 91, 427–434;

e) A. Romieu, T. Bruckdorfer, G. Clavé, V. Grandclaude, C.

Massif, P.-Y. Renard, Org. Biomol. Chem. 2011, 9, 5337–5342;

f) G. Woronoff, A. El Harrak, E. Mayot, O. Schicke, O. J.

Miller, P. Soumillion, A. D. Griffiths, M. Ryckelynck, Anal.

Chem. 2011, 83, 2852–2857; g) P. Bourbon, Q. Peng, G. Fer-

raudi, C. Stauffacher, O. Wiest, P. Helquist, J. Org. Chem. 2012,

[

23] N. C. Lim, J. V. Schuster, M. C. Porto, M. A. Tanudra, L. Yao,

H. C. Freake, C. Bruckner, Inorg. Chem. 2005, 44, 2018–2030.

24] Y. K. Yoon, M. A. Ali, A. C. Wei, T. S. Choon, H. Osman, K.

Parang, A. N. Shirazi, Bioorg. Med. Chem. 2014, 22, 703–710.

A similar synthetic strategy involving direct condensation–cyl-

ization between ortho-nitroanilines and aldehydes has also been

reported, see: G. Chen, Z. Liu, Y. Zhang, X. Shan, L. Jiang,

Y. Zhao, W. He, Z. Feng, S. Yang, G. Liang, ACS Med. Chem.

Lett. 2013, 4, 69–74.

25] a) A. Romieu, D. Tavernier-Lohr, S. Pellet-Rostaing, M. Le-

maire, P.-Y. Renard, Tetrahedron Lett. 2010, 51, 3304–3308; b)

A. Romieu, C. Massif, S. Rihn, G. Ulrich, R. Ziessel, P.-Y.

Renard, New J. Chem. 2013, 37, 1016–1027.

[26] N. R. Conley, A. Dragulescu-Andrasi, J. Rao, W. E. Moerner,

Angew. Chem. Int. Ed. 2012, 51, 3350–3353; Angew. Chem.

2012, 124, 3406–3409.

[27] J. Fernández-Lodeiro, M. F. Pinatto-Botelho, A. A. Soares-

Paulino, A. C. Gonçalves, B. A. Sousa, C. Princival, A. A.

Dos Santos, Dyes Pigm. 2014, 110, 28–48.

[

7

7, 2756–2762; h) C. Massif, S. Dautrey, A. Haefele, R. Ziessel,

P.-Y. Renard, A. Romieu, Org. Biomol. Chem. 2012, 10, 4330–

336; i) J. Li, C.-F. Zhang, Z.-Z. Ming, W.-C. Yang, G.-F. Yang,

[

4

RSC Adv. 2013, 3, 26059–26065; j) M. Najah, E. Mayot, I. P.

Mahendra-Wijaya, A. D. Griffiths, S. Ladame, A. Drevelle,

Anal. Chem. 2013, 85, 9807–9814; k) H. Surya Prakash Rao,

M. Babu, A. Desai, RSC Adv. 2014, 4, 11064–11072.

[

9] For selected examples of water-soluble red-/far-red-emitting 7-

amino/7-hydroxycoumarins with large Stokes’ shift, see: a) S.

Nizamov, K. I. Willig, M. V. Sednev, V. N. Belov, S. W. Hell,

Chem. Eur. J. 2012, 18, 16339–16348; b) H. Schill, S. Nizamov,

F. Bottanelli, J. Bierwagen, V. N. Belov, S. W. Hell, Chem. Eur.

J. 2013, 19, 16556–16565; c) Q. Zou, Y. Fang, Y. Zhao, H.

Zhao, Y. Wang, Y. Gu, F. Wu, J. Med. Chem. 2013, 56, 5288–

[28] a) C. S. Radatz, D. Alves, P. H. Schneider, Tetrahedron 2013,

69, 1316–1321. For a related reaction mediated by tributyl-

16

www.eurjoc.org

Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O43215

/KAP1

Date: 20-11-14 16:47:38

Pages: 18

3-(Heteroaryl)-7-hydroxycoumarins

phosphine, see: b) C. S. Radatz, D. S. Rampon, R. A. Balaguez,

D. Alves, P. H. Schneider, Eur. J. Org. Chem. DOI: 10.1002/

ejoc.201402808.

[38] a) H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. Int.

Ed. 2001, 40, 2004–2021; Angew. Chem. 2001, 113, 2056–2075;

b) C. W. Tornøe, C. Christensen, M. Meldal, J. Org. Chem.

2002, 67, 3057–3064.

[39] G. Viault, S. Dautrey, N. Maindron, J. Hardouin, P.-Y. Renard,

A. Romieu, Org. Biomol. Chem. 2013, 11, 2693–2705.

[40] H. Neumann, I. Z. Steinberg, E. Katchalski, J. Am. Chem. Soc.

1965, 87, 3841–3848.

[

29] M. R. Detty, P. N. Prasad, D. J. Donnelly, T. Ohulchanskyy,

S. L. Gibson, R. Hilf, Bioorg. Med. Chem. 2004, 12, 2537–2544.

30] J. Pauli, M. Grabolle, R. Brehm, M. Spieles, F. M. Hamann,

M. Wenzel, I. Hilger, U. Resch-Genger, Bioconjugate Chem.

[

2011, 22, 1298–1308.

[

31] For comprehensive reviews, see: a) X. Chen, Y. Zhou, X. Peng,

J. Yoon, Chem. Soc. Rev. 2010, 39, 2120–2135; b) H. S. Jung,

X. Chen, J. S. Kim, J. Yoon, Chem. Soc. Rev. 2013, 42, 6019–

[41] a) J. D. A. Tyndall, M. J. Kelso, P. Clingan, M. Ranson, Recent

Pat. Anti-Cancer Drug Discovery 2008, 3, 1–13; b) B. Law, C.-

H. Tung, Bioconjugate Chem. 2009, 20, 1683–1695.

[42] a) B. Law, A. Curino, T. H. Bugge, R. Weissleder, C.-H. Tung,

Chem. Biol. 2004, 11, 99–106; b) R. Malik, S. Qian, B. Law,

Anal. Biochem. 2011, 412, 26–33.

6031; c) C. Yin, F. Huo, J. Zhang, R. Martinez-Manez, Y.

Yang, H. Lv, S. Li, Chem. Soc. Rev. 2013, 42, 6032–6059.

32] H. Peng, W. Chen, Y. Cheng, L. Hakuna, R. Strongin, B.

Wang, Sensors 2012, 12, 15907–15946.

33] For comprehensive reviews, see: a) A. R. Lippert, J. Inorg. Bio-

chem. 2014, 133, 136–142; b) F. Yu, X. Han, L. Chen, Chem.

Commun. 2014, 50, 12234–12249. c) B. Peng, M. Xian, Asian

J. Org. Chem. 2014, 3, 914–924.

[

[43] J. Chan, S. C. Dodani, C. J. Chang, Nat. Chem. 2012, 4, 973–

984.

[44] L. V. Lafrance, J. D. Leber, M. Li, S. K. Verma, 20100126922

A1, 2010.

[45] C. Qian, X. Cai, H. Zhai, Curis Inc., 2009036051 A1, 2009.

[46] A. Chevalier, P.-Y. Renard, A. Romieu, Org. Lett. 2014, 16,

3946–3949.

[

34] For selected recent examples, see: a) L. A. Montoya, T. F.

Pearce, R. J. Hansen, L. N. Zakharov, M. D. Pluth, J. Org.

Chem. 2013, 78, 6550–6557; b) C. Wei, L. Wei, Z. Xi, L. Yi,

Tetrahedron Lett. 2013, 54, 6937–6939; c) J. Bae, J. Choi, T. J.

Park, S.-K. Chang, Tetrahedron Lett. 2014, 55, 1171–1174; d)

C. Wei, Q. Zhu, W. Liu, W. Chen, Z. Xi, L. Yi, Org. Biomol.

Chem. 2014, 12, 479–485; e) X.-L. Liu, X.-J. Du, C.-G. Dai,

Q.-H. Song, J. Org. Chem. 2014, 79, 9481–9489.

[47] H. Maki, Y. Ueda, H. Nariai, J. Phys. Chem. B 2011, 115,

3571–3577.

[48] G. B. Fields, R. L. Noble, Int. J. Pept. Protein Res. 1990, 35,

161–214.

[49] G. R. Fulmer, A. J. M. Miller, N. H. Sherden, H. E. Gottlieb,

A. Nudelman, B. M. Stoltz, J. E. Bercaw, K. I. Goldberg, Orga-

nometallics 2010, 29, 2176–2179.

[50] K.-I. Setsukinai, Y. Urano, K. Kikuchi, T. Higuchi, T. Nagano,

J. Chem. Soc. Perkin Trans. 2 2000, 2453–2457.

[51] A. M. Brouwer, Pure Appl. Chem. 2011, 83, 2213–2228.

[52] J. Coste, J.-M. Campagne, Tetrahedron Lett. 1995, 36, 4253–

4256.

[

35] K. E. Sapsford, L. Berti, I. L. Medintz, Angew. Chem. Int. Ed.

2006, 45, 4562–4588; Angew. Chem. 2006, 118, 4676–4704.

36] A. Chevalier, C. Massif, P.-Y. Renard, A. Romieu, Chem. Eur.

J. 2013, 19, 1686–1699.

37] M. C. Bryan, F. Fazio, H.-K. Lee, C.-Y. Huang, A. Chang,

M. D. Best, D. A. Calarese, O. Blixt, J. C. Paulson, D. Burton,

I. A. Wilson, C.-H. Wong, J. Am. Chem. Soc. 2004, 126, 8640–

Received: September 15, 2014

8641.

Published Online: ᭿

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Pages: 18

B. Roubinet, A. Chevalier, P.-Y. Renard, A. Romieu

FULL PAPER

Fluorescent Probes

B. Roubinet, A. Chevalier, P.-Y. Renard,

A. Romieu* ..................................... 1–18

A Synthetic Route to 3-(Heteroaryl)-7-

hydroxycoumarins Designed for Biosensing

Applications

Keywords: Fluorescent probes / Fluores-

cence / Biosensors / Oxygen heterocycles

A condensation reaction between 7-acet-

oxy-3-formylcoumarin and various C- and/

or N-monosubstituted ortho-phenylendi-

amine derivatives has led to a new class of

3-(heteroaryl)-7-hydroxycoumarins meet-

ing the requirements for biosensing appli-

cations (bioconjugation ability, water solu-

bility, and high “fluorogenic” reactivity).

18

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Eur. J. Org. Chem. 0000, 0–0

Article Doi

A synthetic route to 3-(heteroaryl)-7-hydroxycoumarins designed for biosensing applications

DOI: 10.1002/ejoc.201403215

Source and publish data:

Authors:

Article abstract of DOI:10.1002/ejoc.201403215

Full text of DOI:10.1002/ejoc.201403215

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