Design, synthesis and evaluation of enzyme-responsive fluorogenic probes based on pyridine-flanked diketopyrrolopyrrole dyes

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 248 (2021) 119179

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular and Biomolecular

Spectroscopy

journal homepage: www.elsevier.com/locate/saa

Design, synthesis and evaluation of enzyme-responsive ﬂuorogenic

probes based on pyridine-ﬂanked diketopyrrolopyrrole dyes

Sébastien Jenni ^a^,^,¹, Flavien Ponsot , Pierre Baroux , Lucile Collard , Takayuki Ikeno , Kenjiro Hanaoka ,

⇑

a,1

a

b

a

a,

⇑

Valentin Quesneau , Kévin Renault , Anthony Romieu

a

Institut de Chimie Moléculaire de l’Université de Bourgogne, UMR 6302, CNRS, Univ. Bourgogne Franche-Comté, 9, Avenue Alain Savary, 21000 Dijon, France

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan

b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

ꢀ

DPP-based ﬂuorescent probes with

self-immolative 4-aminobenzyl

quaternary pyridinium were

synthesized.

ꢀ

DPP-based ﬂuorescent probes based

on fully quaternized 3,6-bis-(4 -

0

pyridyl)-DPP are not stable in aq.

solution.

ꢀ

DPP-based ﬂuorescent probes for

hypoxia detection are not reactive

towards AzoR and NTR.

A DPP-based ratiometric ﬂuorescent

probe for penicillin G acylase

detection was developed.

Activation mechanism of PGA-

sensitive probe was deciphered by

RP-HPLC-ﬂuorescence/MS.

a r t i c l e i n f o

a b s t r a c t

Article history:

The ever-growing demand for ﬂuorogenic dyes usable in the rapid construction of analyte-responsive ﬂu-

orescent probes, has recently contributed to a revival of interest in the chemistry of diketopyrrolopyrrole

Received 28 September 2020

Received in revised form 30 October 2020

Accepted 1 November 2020

Available online 17 November 2020

(DPP) pigments. In this context, we have explored the potential of symmetrical and unsymmetrical DPP

derivatives bearing two or one 4-pyridyl substituents acting as optically tunable group(s). The unique ﬂu-

orogenic behavior of these molecules, closely linked to N-substitution/charge state of their pyridine unit

(

i.e., neutral pyridine or cationic pyridinium), has been used to design DPP-based ﬂuorescent probes for

Keywords:

detection of hypoxia-related redox enzymes and penicillin G acylase (PGA). In this paper, we describe

synthesis, spectral characterization and bioanalytical validations of these probes. Dramatic differences

in terms of aqueous stability and enzymatic ﬂuorescence activation were observed. This systematic study

enables to delineate the scope of application of pyridine-ﬂanked DPP ﬂuorophores in the ﬁeld of enzyme

biosensing.

Diketopyrrolopyrrole

Enzyme detection

Fluorogenic probe

Hypoxia

Penicillin G acylase

Reductase

⇑

Corresponding authors.

E-mail addresses: sebstien.jenni@u-bourgogne.fr (S. Jenni), anthony.romieu@u-bourgogne.fr (A. Romieu).

These authors contributed equally to this work.

1

https://doi.org/10.1016/j.saa.2020.119179

1

Sébastien Jenni, F. Ponsot, P. Baroux et al.

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 248 (2021) 119179

1

. Introduction

hydroxyl, . . .) as is frequently the case for reaction-based ﬂuores-

cent probes using coumarins, cyanines or xanthenes as photoactive

reporters [6]. In an effort to address this gap, the Hua group has

recently proposed an unsymmetrical 3,6-diaryl-DPP core structure

Over the past decade, intensive research efforts have been

devoted to develop novel high-performance visible ﬂuorescent

organic dyes based on the diketopyrrolopyrrole (DPP) framework

which was used previously only as basic structural element of pig-

ment for paints, coatings and printing inks industry [1]. The high

degree of interest for these photoactive bis-lactam-based small

molecules is related to their numerous valuable features including

facile synthetic accessibility, ease of structural modiﬁcation, out-

standing chemical and thermal robustness and attractive spectral

properties [2]. However, compared with common classes of

organic-based ﬂuorophores (i.e., BODIPY, coumarin, cyanine and

xanthene dyes) [3], the main limitation currently associated with

the ﬂuorescent DPP dyes is the lack of simple and effective struc-

tural alteration strategies to readily tune their emission capability,

especially after their speciﬁc interaction or reaction with a biolog-

ical and/or reactive species to be detected. Modulation of funda-

mental photophysical processes as the ﬁnal result of such

activity-based sensing approaches, is the key to the design of

analyte-responsive ﬂuorescent probes applied to sensing/imaging

applications [4]. Nevertheless, there are some interesting achieve-

ments focusing on ﬂuorogenic detection of reactive analytes (e.g.,

0

bearing a single 4 -pyridyl unit acting as ﬂuorogenic reactive cen-

ter [7] (Fig. 1). Indeed, depending on the charge state of this N-

heterocycle (i.e., pyridinium cation bearing N-alkyl substituent

and neutral pyridine molecule), internal charge transfer (ICT) pro-

cess is operative or completely abolished. Thus, a ratiometric

detection strategy of enzymes (esterase activity and

glutamyltranspeptidase -GT)) [7a,7c] and reactive oxygen

species (ROS) such as superoxide radical anion (O

c-

(c

ꢀ

ꢁ

2

) [7b],

through an effective biocompatible domino reaction triggered by

0

the targeted bioanalyte and leading to N-dealkylation of 4 -

pyridinium moiety, has been devised and applied to living cells

and in vivo context. To the best of our knowledge, these

contributions are the only examples of enzyme-responsive DPP

probes that, in addition, highlight the potential of these less

common activatable ﬂuorophores for biological imaging

applications. However, curiously no attention has been paid to

optimization of their physicochemical properties (e.g., water

solubility) [8] for better performances in aq. media whereas some

effective strategies for imparting polarity and possibly bioconjuga-

tion ability to DPP derivatives are readily available in the literature

[9]. In pursuit of novel structurally simple ﬂuorophores possessing

both biocompatibility and ﬂuorogenic reactivity, the chemistry of

pyridine-ﬂanked DPP dyes deserves to be revisited. Furthermore,

this will enable to assess the level of versatility of this emerging

class of ﬂuorescent platforms, particularly illustrated by the range

2

Hg(II) cations, H S and biothiols) both in vitro and in living cells

through irreversible and stoichiometric organic reactions. How-

ever, as illustrated by the selected examples shown in Fig. 1 [5],

the corresponding DPP-based ﬂuorescent chemodosimeters are

not constructed from a common and structurally simple ﬂuoro-

genic scaffold bearing an optically tunable group (e.g., amino,

O

S

C

N

8

H

17

NO₂

O

NO

2

O

C

N

4 9

H

O

reactive site

for H S & biothiols

2

O

O N

2

O

reactive site

for Cys

N

C

O N

2

H

17

O

8

N

C

reactive site

2

Ph N

4

H

9

for H S & biothiols

2

"

OFF-ON" probe responsive to biothiols

2

"OFF-ON" probe responsive to H S & biothiols

(

phenol deprotection through S Ar & SO release)

(NO

2

reduction or Michael addition)

N

2

SR

R

N

-

Y

N

C

N

6 13

H

SR

+

O

reactive site

for Hg(II)

O

reactive site

for Hg(II)

1, X = O, P = Ac (esterase)

-

2, X = O, P = Ph P(O) (O ° )

2

O

X

P

3, X = NH, P = L-glutamyl moiety ( -GT)

RS

N

R

O

N

C H

6

13

O

R =

3

Ratiometric probes responsive to hydrolases or ROS

aniline or phenol deprotection followed by 1,6-elimination)

(

Ratiometric probe responsive to Hg(II) cations

thioacetal deprotection mediated by Hg(II))

(

H

N

H

N

R

N

_P_y+

N⁺

O

+

O

Y

-

6, Ar = Py , R = CH

2

CO

2

H, P-X = NO

CO H, P-X = NO

2

7

, Ar = p-Br-Ph, R = CH

2

8

, Ar = p-Br-Ph, R = Me, P-X = p-DMA-N

2

X

P

O

N

H

N

H

Ar

O

9, Ar = p-Br-Ph, R = Me, P-X = NO

2

N

R

CO

Br

10, Ar = p-Br-Ph, R = Me, P-X = PhAc-NH

11, Ar = p-Br-Ph, R = C H , P-X = PhAc-NH

6 13

symmetrical

,6-bis-(4'-Py)-DPP 4

unsymmetrical

mono-(4'-Py)-DPP 5

3

R = -CH

2

H or Me

Fig. 1. Selected examples of DPP-based ﬂuorescent chemodosimeters (top) [5,7]; structures of pyridine-ﬂanked DPP pigments and related enzyme-responsive ﬂuorogenic

probes studied in the present work (bottom) (Cys = L-cysteine, Ac = acetyl,

c

-GT =

c

-glutamyltranspeptidase, Py = pyridyl, p-Br-Ph = p- or 4-bromophenyl, p-DMA-N

2

= 2-[4-

ꢁ

2

(

dimethylamino)phenyl]diazenyl), PhAc = phenylacetyl, for compounds 1–3, the nature of counter-ion Y was not speciﬁed by the authors, for 6–11, Y = CF

3

CO

).

2

Sébastien Jenni, F. Ponsot, P. Baroux et al.

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 248 (2021) 119179

of different enzymes (both peptidases and reductases) which could

be detected.

performed with an Hettich Universal 320 instrument (with the fol-

1

13

19

lowing parameters: 15 min, 3000 rpm). H-, C- and F NMR

spectra were recorded on Bruker spectrometers: Avance Neo

500 MHz equipped with a 5 mm BBOF iProbe and Avance III HD

The present Article comes within this topic. Indeed, we report

the synthesis of six different enzyme-responsive ﬂuorogenic DPP

0

probes rationally designed from symmetrical 3,6-bis-(4 -pyridyl)-

600 MHz equipped with a 5 mm BBOF N

2

cryoprobe. NMR spec-

0

DPP pigment 4 or an unsymmetrical analog namely mono-(4 -

troscopy chemical shifts are quoted in parts per million (ppm) rel-

1

13

(for F). For ¹H and

19

13

pyridyl)-DPP pigment 5. They differ in both the type of their bio-

metabolizable recognition moiety with the aim of targeting

hypoxia-related reductases (azo- and nitroreductase, AzoR and

NTR respectively) or penicillin G acylase (PGA) [10], and the nature

of their N-lactam substituents (i.e., carboxymethyl, methyl and

hexyl) directly impacting the solubility and the photophysical

behavior of the probes in aq. environments (Fig. 1). However, the

common feature of all probes related to their activation mecha-

ative to TMS (for H, and C) and CFCl

3

C

spectra, calibration was made by using residual signals of partially

deuterated solvent summarized by Fulmer et al. [13]. J values are

1

3

expressed in Hz. C substitutions of some compounds (16 and

18) were determined with JMOD experiments, differentiating sig-

nals of methyl and methine carbons pointing ‘‘up” (+) from methy-

lene and quaternary carbons pointing ‘‘down” (-) [14]. IR spectra

were recorded with a Bruker Alpha FT-IR spectrometer equipped

with an universal ATR sampling accessory. The bond vibration fre-

nism, is the transient formation of

a highly reactive 4-

ꢁ

1

aminobenzyl quaternary pyridinium intermediate readily prone

to self-immolation [11]. The ﬂuorogenic behavior as well as enzy-

matic activation and aq. stability of these DPP probes were studied

through in vitro ﬂuorescence assays and HPLC-ﬂuorescence/-MS

analyses. In the light of results and data produced, we will identify

suitable combination of structural elements to achieve conversion

of early industrial DPP pigments to high-performance ﬂuorescent

probes for tracking enzymatic activities in living systems [10,12].

quencies are expressed in reciprocal centimeters (cm ). HPLC-MS

analyses were performed on a Thermo-Dionex Ultimate 3000

instrument (pump + autosampler at 20 °C + column oven at

25 °C) equipped with a DAD (Thermo-Dionex DAD 3000-RS) and

a

MSQ Plus single quadrupole mass spectrometer. HPLC-

ﬂuorescence analyses were performed with the same instrument

but connected to Thermo Scientiﬁc Dionex UltiMate 3000 ﬂuores-

cence detector FLD-3400 RS dual-PMT. UHPLC analyses (imple-

mented during in vitro assays with rat liver microsomes) were

performed on a Waters Acquity UPLC (H class)/QDa quadrupole

MS analyzer (Waters). Puriﬁcations by semi-preparative HPLC

were performed on a Thermo-Dionex Ultimate 3000 instrument

2

. Experimental section

See SI for all experimental details related to the preparation of

compounds 4, 5, 11, 19–21, photophysical characterizations and

(

semi-preparative pump HPG-3200BX) equipped with a RS Vari-

able Detector (VWD-3400RS, four distinct wavelengths within

the range 190–800 nm). Lyophilization operations were performed

with a Christ Alpha 2–4 LD plus. TFA mass content of samples puri-

ﬁed by semi-preparative was determined by ion chromatography

according to a method developed by the PACMSUB staff (see sup-

porting information). Such analyses were performed with an ion

chromatograph Thermo Scientiﬁc Dionex ICS 5000 equipped with

a conductivity detector CD (Thermo Scientiﬁc Dionex) and a con-

ductivity suppressor ASRS-ultra II 4 mm (Thermo Scientiﬁc Dio-

nex). Low-resolution mass spectra (LRMS) were recorded either

on a Thermo Scientiﬁc MSQ Plus single quadrupole equipped with

an electrospray (ESI) source (LC-MS coupling mode), or on a Bruker

Amazon SL instrument equipped with an ESI source (direct intro-

duction mode).

HPLC analyses/puriﬁcations.

2.1. General

Unless otherwise noted below, all commercially available

reagents and solvents were used without further puriﬁcation. TLC

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

The spots were directly visualized and through illumination with

dual wavelength UV lamp (k = 254 and 365 nm). Column chro-

matography puriﬁcations were performed with different types of

silica gel for which speciﬁcations are given throughout the descrip-

tion of synthetic protocols. MeCN and THF were dried over alumina

cartridges immediately prior to use, using a solvent puriﬁcation

system PureSolv PS-MD-5 model from Innovative Technology.

DMF (Fisher Chemicals, extra pure, ꢂ99%, #D/3840/17) was dried

by storage over activated 3 Å molecular sieves and kept under Ar

atmosphere. Anhydrous NMP over 3 Å molecular sieves was pur-

chased from Acros Organics. DMSO (for spectrophotometry,

0

2

.3. Synthesis of symmetrical 3,6-bis-(4 -pyridyl)-DPP dye 13 and

related NTR-sensitive probe 6

0

2

.3.1. Di-tert-butyl 3,6-bis-(4 -pyridyl)-DPP N,N’-diacetate 12

#

D5293) was purchased from TCI Europe. Formic acid (puriss p.

a., ACS reagent, reag. Ph. Eur., ꢂ98%, #33015), NTR (from Escheri-

chia coli, #N9284, 0.1 U/ g, lyophilized enzyme + buffer resus-

0

A suspension of 3,6-Bis-(4 -pyridyl)-DPP pigment 4 (255 mg,

0

.88 mmol, 1.0 equiv.) and tBuOK (247 mg, 2.2 mmol, 2.5 equiv.)

l

in dry NMP (20 mL) was stirred at 75 °C under Ar atmosphere

for 15 min (color changes from dark pink to night blue). Thereafter,

pended in ultrapure water), NADH and LAP (microsomal, from

porcine kidney, #L5006, type IV-S, ammonium sulfate suspension,

tert-butyl bromoacetate (386

and the resulting reaction mixture was stirred at 75 °C for

0 min (color changes from blue to brown). The reaction was

lL, 2.61 mmol, 3 equiv.) was added

1

0–40 U per mg protein) were obtained from Merck-Millipore

(

brand Sigma-Aldrich) and stored at ꢁ20 °C for NTR/NADH and

9

LAP. PGA (from Escherichia coli, EZ50150, 841 U/mL) was provided

by Iris Biotech GmbH. The HPLC-gradient grade MeCN was

obtained from Carlo Erba, Fisher Scientiﬁc or VWR. Aq. buffers

and aq. mobile-phases for HPLC were prepared using ultrapure

water produced by an ELGA PURELAB Ultra system (puriﬁed to

checked for completion by TLC (DCM-EtOAc 8:2, v/v + 1% TEA)

and RP-HPLC (system A). After cooling to RT, toluene (60 mL)

was added and the resulting organic layer was washed with deion-

ized water (2 ꢃ 100 mL) to remove NMP, dried over anhydrous

2 4

Na SO and ﬁnally evaporated to dryness. The resulting residue

1

8.2 MX.cm).

was puriﬁed by column chromatography over silica gel (VWR,

technical grade, 40–63 m, #84814.360, eluent: step gradient of

EtOAc in DCM (+1% TEA) from 0% to 7.5%). Fractions containing

the desired product, were combined, washed with aq. 1.0 M KHSO

(100 mL) and deionized water (2 ꢃ 100 mL) to remove TEA, dried

over anhydrous Na SO and evaporated under reduced pressure.

Compound 12 was obtained as an orange-brown solid (160 mg,

l

2.2. Instruments and methods

4

Centrifugation operations were performed with a Thermo Sci-

entiﬁc Espresso Personal Microcentrifuge instrument. Centrifuga-

tion steps required for isolation of DPP pigments 4 and 5 were

2

4

3

Sébastien Jenni, F. Ponsot, P. Baroux et al.

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 248 (2021) 119179

0

.31 mmol, yield 35%). ¹H NMR (600 MHz, CDCl

3

): d = 8.78

H-H = 5.4 Hz, 4H), 4.39 (s, 4H),

.36 (s, 18H); C NMR (150 MHz, CDCl ): d = 167.1 (2C), 161.6

R

t = 3.1 min (purity 88% at 270 nm and 98% at 500 nm); LRMS

3

2+

+

(

d,

J

H-H = 4.2 Hz, 4H), 7.61 (d,

J

(ESI+, recorded during RP-HPLC analysis): m/z 695.2 [M - H] ,

1

3

+

1

3

27 6

calcd for C34H N O11 695.2; LRMS (ESI-, recorded during RP-

ꢁ

(

2C), 151.0 (4C), 146.7 (2C), 135.0 (C), 122.1 (4C), 111.4 (2C),

HPLC analysis): m/z 693.2 [M - 3H] , calcd for C34H N O11 693.2.

25 6

8

9

k

R

3.5 (C), 44.2 (2C), 28.1 (6C); HPLC (system A): t = 4.7 min (purity

9% at 500 nm); UV–vis (recorded during RP-HPLC analysis):

max = 230, 260 and 477 nm LRMS (ESI+, recorded during

0

2

.4. Synthesis of unsymmetrical mono-(4 -pyridyl)-DPP dye 17 and

related NTR-sensitive probe 7

+

31 4 6

RP-HPLC analysis): m/z = 519.2 [M+H] , calcd for C28H N O 519.2.

0

2

.4.1. Di-tert-butyl mono-(4 -pyridyl)-DPP N,N’-diacetate 16

0

2

.3.2. 3,6-Bis-(4 -pyridyl)-DPP N,N’-diacetic acid 13

A suspension of mono-(4 -pyridyl)-DPP pigment 5 (250 mg,

Di-tert-butyl ester 12 (17.0 mg, 32.8

l

mol, 1.0 equiv.) was dis-

0.68 mmol, 1.0 equiv.) and tBuOK (190 mg, 1.70 mmol, 2.5 equiv.)

in dry NMP (30 mL) was stirred at 75 °C under N atmosphere for

15 min. tert-Butyl bromoacetate (300 L, 2.04 mmol, 3.0 equiv)

was added and the mixture was stirred at 75 °C under N for

90 min. Thereafter, the mixture was cooled down to RT and deion-

ized water was added. The product was extracted with toluene

three times. The combined organic layers were dried over anhy-

solved in a TFA-DCM (1:1, v/v) mixture (2 mL) and the mixture

was stirred at RT for 3 h. The reaction was checked for completion

by RP-HPLC (system B) and the reaction mixture was evaporated

to dryness. The resulting residue was dissolved in a (1:1, v/v) mix-

ture of 0.1% aq. TFA and MeCN (4 mL) and puriﬁed by semi-

2

l

2

R

preparative RP-HPLC (system C, 1 injection, t = 15.8–17.4 min of

collected fractions containing the desired compound). The

product-containing fractions were lyophilized to give 13 as TFA salt

drous Na

reduced pressure. The product was puriﬁed by column chromatog-

raphy over silica gel (VWR technical grade, 40–63 m,

2 4

SO , ﬁltered and the solvent was evaporated under

(

6.4 mg, 11.3

l

mol, yield 34% based on mass percentage of TFA = 28%

l

1

determined by ionic chromatography, 1.4 TFA). H NMR (600 MHz,

#84814.360) using a step gradient of EtOAc in DCM as eluent (from

1

3

DMSO d

6

): d = 8.85 (m, 4H), 7.77 (m, 4H), 4.53 (s, 4H); C NMR

151 MHz, DMSO d ): d = 170.0 (2C), 161.3 (2C), 150.9 (4C), 146.9

2C), 134.9 (2C), 122.5 (4C), 110.5 (2C), 43.5 (2C); F NMR

565 MHz, DMSO d ): d = -74.5 (3F, CF -triﬂuoroacetate); HPLC (sys-

= 4.3 min (purity 94% at 270 nm and 95% at 500 nm); LRMS

0% to 10% + 1% TEA). Compound 16 was obtained as an orange solid

1

(

6

(86.0 mg, 144

l

mol, yield 22%). H NMR (500 MHz, CDCl

3

): d = 8.73

19

(brs, 2H), 7.76 (brs, 2H), 7.61 (m, 4H), 4.38 (s, 2H), 4.32 (s, 2H), 1.34

1

3

(

s, 9H), 1.32 (s, 9H); C NMR (126 MHz, CDCl

3

): d = 167.3, 167.2,

(

6

3

1

62.0, 161.7, 150.8, 149.2, 144.8, 135.3, 132.6 (2C), 130.3 (2C),

tem B): t

R

+

126.8, 126.4, 122.0, 111.5, 109.9, 83.3, 83.2, 44.3, 44.1, 28.0 (6C);

HPLC (system A): t_R= 5.6 min (purity 85% at 260 nm and 95% at

(

ESI+, recorded during RP-HPLC analysis): m/z 407.3 [M+H] , calcd

+

for C20

15 4 6

H N O 407.1. Please note: the poor quality of NMR spectra of

4

70 nm); UV–vis (recorded during RP-HPLC analysis): kmax = 261,

this DPP dye was explained by the presence of a further set of signals

2

99 and 462 nm; LRMS (ESI+, recorded during RP-HPLC analysis):

assigned to the open-chain lactam form 13-RO formed during the

+

1

3 6

m/z 596.2 and 598.2 [M+H] , calcd for C₂₉H₃₁BrN O 596.1 and

freeze-drying process. H NMR (600 MHz, DMSO d

6

): d = 10.26 (bs,

5

98.1.

1

2

H, CO

H), 7.72 (d, J = 5.4 Hz, 2H), 4.12 (s, 2H), 3.75 (s, 2H); HPLC (system

= 1.0 min; UV–vis (recorded during RP-HPLC analysis):

max = 260 and 456 nm; LRMS (ESI+, recorded during RP-HPLC anal-

2

H), 8.84 (m, 2H), 8.66 (d, J = 5.4 Hz, 2H), 7.83 (d, J = 5.4 Hz,

0

2

.4.2. Mono-(4 -pyridyl)-DPP N,N’-diacetic acid 17

B): t

k

R

To a solution of di-tert-butyl ester 16 (16.2 mg, 27

lmol, 1.0

+

equiv) in DCM (2 mL), TFA (2 mL) was added. The mixture was stir-

red at RT for 4 h. Thereafter, the reaction mixture was evaporated

to dryness. The resulting residue was dissolved in a (2:1, v/v) mix-

ture of 0.1% aq. TFA and MeCN (3 mL) and puriﬁed by semi-

ysis): m/z 425.3 [M+H] , calcd for C20

recorded during RP-HPLC analysis): m/z 423.2 [M-H] , calcd for C20

H

17

N

O

4 7

425.1; LRMS (ESI-,

ꢁ

-

ꢁ

15

N

4

O

7

423.1.

R

preparative RP-HPLC (system E, 1 injection, t = 35.0–42.5 min of

collected fractions containing the desired compound). The

product-containing fractions were lyophilized to give TFA salt of

2

.3.3. Open-chain lactam form of probe 14

Di-tert-butyl ester 12 (16.8 mg, 32.4

l

mol, 1.0 equiv.) was dis-

solved in dry MeCN (3 mL), 4-nitrobenzyl bromide (54 mg,

2

1

7 as an orange amorphous powder (7.8 mg, 14

lmol, yield 50%

50 mol, 7.7 equiv.) was added and the resulting reaction mixture

l

based on mass percentage of TFA = 15% determined by ionic chro-

was stirred under reﬂux and Ar atmosphere for 24 h. The progress

of the reaction was checked by RP-HPLC (system B). Thereafter, the

reaction mixture was evaporated to dryness. The resulting residue

was dissolved in a TFA-DCM (1:1, v/v) mixture (2 mL) and stirred at

RT for 3 h. The reaction was checked for completion by RP-HPLC

1

matography, 0.7 TFA). H NMR (500 MHz, DMSO d

6

): d = 13.24 (brs,

3

2

4

H), 8.83 (d, JH-H = 6.1 Hz, 2H), 7.84 (d, JH-H = 8.5 Hz, 2H), 7.75 (m,

H), 4.53 (s, 2H), 4.49 (s, 2H); C NMR (151 MHz, DMSO d ):

6

13

d = 170.1, 161.7, 161.3, 151.0, 149.1, 145.2, 134.9, 132.7, 131.0,

19

1

26.6, 126.1, 122.4, 110.6, 109.1, 56.5, 43.6, 43.5;

565 MHz, DMSO d ): d = -73.9 (3F, CF

-triﬂuoroacetate), ꢁ75.2

0.19F, CF -free TFA); HPLC (system A): t = 3.6 min (purity 94%

F NMR

(

system A) and the mixture was evaporated to dryness. The result-

(

6

3

ing residue was dissolved in a (1:1, v/v) mixture of 0.1% aq. TFA and

MeCN (4 mL) and puriﬁed by semi-preparative RP-HPLC (system C,

(

3

R

at 460 nm); UV–vis (recorded during RP-HPLC analysis): kmax = 260,

07 and 475 nm; LRMS (ESI+, recorded during RP-HPLC analysis):

1

injection, t

desired compound). The product-containing fractions were lyophi-

lized to give 14 as TFA salt (5.2 mg, 5.6 mol, yield 17% based on

R

= 24.5–28.5 min of collected fractions containing the

3

+

m/z 484.1 and 486.1 [M+H] , calcd for C21

3 6

H15BrN O 484.0 and

l

4

86.0.

mass percentage of TFA = 23% determined by ionic chromatogra-

phy, 1.9 TFA). H NMR (500 MHz, DMSO d

1

3

6

): d = 10.18 (t,

J

H-H

=

3

0

5

2

7

2

1

.7 Hz, 1H), 9.15 (d,

J

H-H = 6.8 Hz, 2H), 8.82 (d,

J

H-H = 7.1 Hz,

2.4.3. N-(4-Nitrobenzyl)pyridinium mono-(4 -pyridyl)-DPP N,N’-

3

H), 8.33 (m, 4H), 8.07 (d,

JH-H = 6.8 Hz, 2H), 7.88 (d,

J

H-H

=

diacetic acid 7

3

.1 Hz, 2H), 7.82 (d,

J

H-H = 8.8 Hz, 2H), 7.76 (d,

J

H-H = 8.8 Hz,

To a solution of di-tert-butyl ester 16 (34 mg, 57

equiv.) in dry MeCN (5 mL), 4-nitrobenzyl bromide (99 mg,

456 mol, 8.0 equiv.) was added. The mixture was reﬂuxed under

2

N atmosphere overnight. Thereafter, the reaction mixture was

lmol, 1.0

3

H), 6.02 (s, 2H), 5.83 (s, 2H), 4.20 (s, 2H), 3.82 (d,

JH-H = 5.7 Hz,

1

3

H); C NMR (151 MHz, DMSO d

6

): d = 178.7, 171.3, 170.6,

l

66.0, 164.7, 159.2, 148.0, 147.9, 147.7, 144.6, 142.8, 141.6,

41.3, 134.9, 134.8, 130.2 (2C), 129.9 (2C), 129.6, 127.0, 126.7,

25.7, 125.4, 124.4 (2C), 124.3 (2C), 124.2, 123.8, 123.3, 121.2,

01.8, 62.0, 61.7, 60.7, 42.5, 41.1, 36.5, 16.9; HPLC (system A):

evaporated to dryness. The resulting residue was dissolved in

DCM (2 mL) and TFA (2 mL) was added dropwise. The mixture

was stirred at RT for 3 h, then evaporated to dryness. The resulting

4

Sébastien Jenni, F. Ponsot, P. Baroux et al.

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 248 (2021) 119179

1

residue was dissolved in a (2:3, v/v) mixture of 0.1% aq. TFA and

MeCN (4 mL) and puriﬁed twice by semi-preparative RP-HPLC

687, 639, 600, 583, 553, 511, 496, 469, 434, 413; H NMR

3

(500 MHz, CDCl

3 H-H

): d = 9.20 (d, JH-H = 6.4 Hz, 2H), 8.57 (d, J =

3

(

system D, 1 injection, t

R

= 46.0–50.0 min of collected fractions

6.4 Hz, 2H), 7.89 (m, 4H), 7.81 (d,

JH-H = 8.7 Hz, 2H), 7.70

JH-H = 8.2 Hz, 2H), 6.75 (d, JH-H =

3

containing the desired compound). The product-containing frac-

(d, JH-H = 8.7 Hz, 2H), 7.62 (d,

tions were lyophilized to give TFA salt of 7 as a purple solid

9.2 Hz, 2H), 6.02 (s, 2H), 3.48 (s, 3H), 3.36 (s, 3H), 3.11 (s, 6H);

C NMR (151 MHz, CD CN): d 162.1, 161.1, 153.9, 153.3, 153.3,

3

1

3

(

4.9 mg, 6.7

l

mol, yield 12% based on mass percentage of

1

TFA = 17% determined by ionic chromatography, 1.1 TFA).

NMR (500 MHz, DMSO d

6

2

H

=

144.7, 143.1, 143.0, 139.3, 133.4, 132.2, 131.3, 130.4, = 126.8,

126.5, 126.1, 125.1, 122.7, 115.5, 111.6, 109.7, 63.9, 39.5,

3

6

): d 13.34 (brs, 2H), 9.36 (d,

J

H-H

3

H-H = 6.5 Hz, 2H), 8.33 (d, ³

29.2, 29.1; ¹⁹F NMR (565 MHz, CD

.5 Hz, 2H), 8.47 (d,

H), 7.88 (m, 2H), 7.82 (m, 4H), 6.04 (s, 2H), 4.59 (s, 2H), 4.53 (s,

H); ¹⁹F NMR (565 MHz, DMSO d

-TFA); full

J

H-H = 8.1 Hz,

3

CN)

-free TFA); HPLC (system

R

= 4.9 min (purity 97% at 260 and 98% at 460 nm); UV–vis

d = -75.9 (3F,

CF

3

-triﬂuoroacetate), ꢁ76.5 (0.05F, CF

3

2

6

): d = ꢁ73.5 (3F, CF

3

A): t

1

3

spectroscopic characterization of this probe through C NMR mea-

surements was not completed immediately, and an unexpected degra-

(recorded during RP-HPLC analysis): kmax = 280 and 452 nm; LRMS

(ESI+, recorded during RP-HPLC analysis): m/z 633.4 and 635.4

1

3

+

dation occurring at the solid state has prevented us to record C NMR

spectrum. HPLC (system A): t = 3.8 min (purity 98% at 260 nm and

00% at 520 nm); UV–vis (recorded during RP-HPLC analysis):

max = 271, 330 and 518 nm; LRMS (ESI+, recorded during RP-

[M] , calcd for C34

6

H30BrN O

2

633.2 and 635.2.

R

1

k

0

2

.5.3. N-(4-Nitrobenzyl)pyridinium mono-(4 -pyridyl)-DPP N,N’-

dimethyl 9

+

4 8

HPLC analysis): m/z 619.2 and 621.2 [M] , calcd for C28H20BrN O

To a solution of 18 (20.0 mg, 51 mmol, 1.0 equiv.) in dry MeCN

3 mL) was added 4-nitrobenzyl bromide (32.7 mg, 150 mmol, 2.9

6

19.1 and 619.2.

(

equiv.). The resulting reaction mixture was stirred at 75 °C under

overnight and the disappearance of the starting material was

0

2

.5. Synthesis of unsymmetrical mono-(4 -pyridyl)-DPP dye 18 and

N

2

related AzoR- and NTR-sensitive probes 8 and 9

checked by RP-HPLC (system A). Thereafter, the mixture was evap-

orated under reduced pressure, re-dissolved in a (1:3, v/v) mixture

of 0.1% aq. TFA and MeCN (4 mL) and puriﬁed by semi-preparative

0

2

.5.1. Mono-(4 -pyridyl)-DPP N,N’- dimethyl 18

0

A suspension of mono-(4 -pyridyl)-DPP pigment 5 (300 mg,

.81 mmol, 1.0 equiv.) and tBuOK (229 mg, 2.04 mmol, 2.5 equiv.)

R

RP-HPLC (system F, 1 injection, t = 43.0–50.0 min of collected frac-

0

tions containing the desired compound). The product-containing

fractions were lyophilized to give TFA salt of 9 as a purple solid

(8.3 mg, 12 mmol, yield 24% based on mass percentage of

TFA = 22% determined by ionic chromatography, 1.3 TFA). IR

in dry NMP (30 mL) was stirred at RT under nitrogen for 15 min.

MeI (151 mL, 2.44 mmol, 3.0 equiv.) was added and the mixture

was stirred at RT under N

the product was extracted with toluene three times. The combined

organic layers were dried over anhydrous Na SO , ﬁltered and the

2 2

for 1 h. Deionized H O was added and

(ATR):

m = 3046, 2923, 1670, 1630, 1564, 1520, 1487, 1430, 1402,

1374, 1347, 1289, 1197, 1165, 1125, 1072, 1043, 1005, 830, 798,

2

4

1

solvent was evaporated under reduced pressure. Compound 18

759, 736, 718, 705, 686, 619, 521, 489, 441, 411; H NMR

was obtained as a dark-orange solid (141 mg, 0.35 mmol, yield

3

(500 MHz, CD CN): d = 8.84 (d, J = 7.1 Hz, 2H), 8.54 (d, J = 7.1 Hz,

4

1

7

4

2

4%). IR (ATR):

m

= 3046, 2850, 1650, 1587, 1539, 1494, 1468,

2H), 8.30 (d, J = 8.8 Hz, 2H), 7.92 (d, J = 8.7 Hz, 2H), 7.80 (d,

J = 8.7 Hz, 2H), 7.69 (d, J = 8.8 Hz, 2H), 5.85 (s, 2H), 3.37 (s, 3H),

425, 1397, 1363, 1283, 1218, 1098, 1071, 1045, 1006, 824, 757,

40, 729, 701, 687, 647, 582, 554, 535, 519, 496, 469, 434, 423,

1

3

3.30 (s, 3H); C NMR (126 MHz, CD

3

CN): d = 162.9, 161.8, 154.2,

1

3

12, 402; H NMR (500 MHz, CDCl

3

): d = 8.90 (d,

J

H-H = 5.5 Hz,

145.8, 144.0, 140.3, 139.9, 133.0, 132.1, 131.1, 127.6, 127.4,

3

19

H), 8.11 (d,

JH-H = 5.5 Hz, 2H), 7.84 (d,

J

H–H = 8.6 Hz, 2H), 7.75

126.8, 125.1, 63.7, 29.9, 29.9; F NMR (565 MHz, CD

3

CN): d = -

3

13

(

d,

J

H-H = 8.6 Hz, 2H), 3.44 (s, 3H), 3.39 (s, 3H); C NMR

): d = 162.3, 162.0, 150.2 (2C), 149.8, 144.7,

35.5, 132.4 (2C), 130.8 (2C), 126.9, 126.3, 122.5 (2C), 111.4,

09.4, 29.6, 29.5; HPLC (system A): t = 4.7 min (purity 99% at

60 nm and 100% at 460 nm); UV–vis (recorded during RP-HPLC

75.5 (3F, CF -triﬂuoroacetate; HPLC (system A): t = 3.4 min (purity

3

R

(

126 MHz, CDCl

3

99% at 260 nm, 98% at 460 nm and 100% at 520 nm); UV–vis

(recorded during RP-HPLC analysis): k_m_a_x= 264 and 539 nm; LRMS

(ESI+, recorded during RP-HPLC analysis): m/z 531.2 and 533.3

1

2

R

+

[M] , calcd for C₂₆H₂₀BrN O 531.1 and 533.1.

4

analysis): kmax = 264 and 485 nm; LRMS (ESI+, recorded during

+

15

RP-HPLC analysis): m/z 396.1 and 398.1 [M+H] , calcd for C19H -

2

.6. Synthesis of PGA-sensitive probe 10 based on unsymmetrical

+

BrN

3

O

2

396.0 and 398.0.

0

mono-(4 -pyridyl)-DPP 18

2

.5.2. N-(4-[2-[4-(dimethylamino)phenyl]diazenyl]-benzyl)

To a solution of 18 (20.0 mg, 51 mmol, 1.0 equiv.) in dry MeCN

(5 mL) was added benzyl bromide 20 (46.0 mg, 150 mmol, 2.9

equiv.). The resulting reaction mixture was stirred at 75 °C under

0

pyridinium mono-(4 -pyridyl)-DPP N,N’- dimethyl 8

To a solution of 18 (22.0 mg, 56 mmol, 1.0 equiv.) in dry DMF

5 mL), benzyl bromide 19 (53.0 mg, 167 mmol, 3.0 equiv.) was

(

2

N for 12 h. The disappearance of the starting material was checked

added. The resulting mixture was stirred at 75 °C under N

2 h. Thereafter, this mixture was evaporated under reduced pres-

sure and the resulting residue was puriﬁed by column chromatog-

raphy over silica gel (VWR technical grade, 40–63 m, #84814.360,

2

for

by RP-HPLC (system A) and the mixture was evaporated under

reduced presssure. The residue was dissolved in a (1:3, v/v) mix-

ture of 0.1% aq. TFA and MeCN (4 mL) and puriﬁed by semi-

1

l

R

preparative RP-HPLC (system F, 1 injection, t = 47.5–55.5 min of

eluent: a step gradient of MeOH in DCM as eluent from 5% to 15%).

The isolated product was dissolved in a (1:3, v/v) mixture of 0.1%

aq. TFA and MeCN and (4 mL) and subjected to a further puriﬁca-

collected fractions containing the desired compound). The

product-containing fractions were lyophilized to give TFA salt of

10 as a purple solid (9.7 mg, 12.4 mmol, yield 25% based on mass

percentage of TFA = 20% determined by ionic chromatography,

tion by semi-preparative RP-HPLC (system F, 1 injection, t

R

= 55.0–

6

3.0 min of collected fractions containing the desired compound).

1.4 TFA). IR (ATR):

m = 1672, 1631, 1602, 1537, 1515, 1488, 1416,

The product-containing fractions were lyophilized to give TFA salt

of 8 as a purple solid (7.9 mg, 9.5 mmol, yield 17% based on mass

percentage of TFA = 24% determined by ionic chromatography,

1373, 1199, 1131, 1073, 1044, 1006, 833, 798, 755, 719, 625,

517, 433, 412; H NMR (500 MHz, CD CN): d = 8.91 (s, 1H), 8.78

3

(d, J = 7.1 Hz, 2H), 8.46 (d, J = 7.1 Hz, 2H), 7.90 (d, J = 8.7 Hz,

2H), 7.79 (d, J = 8.6 Hz, 2H), 7.70 (d, J = 8.7 Hz, 2H), 7.44 (d,

J = 8.6 Hz, 2H), 7.38–7.29 (m, 4H), 7.28–7.24 (m, 1H), 5.66 (s,

1

.8 TFA). IR (ATR):

m 2923, 1674, 1629, 1599, 1519, 1488, 1429,

1

365, 1200, 1136, 1073, 1043, 1005, 943, 830, 798, 756, 720,

5

Sébastien Jenni, F. Ponsot, P. Baroux et al.

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 248 (2021) 119179

2

H), 3.67 (s, 2H), 3.33 (s, 3H), 3.28 (s, 3H); ¹³C NMR (126 MHz, CD

CN): d = 170.8, 163.0, 162.1, 154.1, 145.5, 143.8, 141.6, 140.3,

3

-

at the corresponding m/z value selected for the SIM detection mode

and then avoid misinterpretations. For RP-HPLC (ﬂuorescence or

MS detection) analyses of mono-(4 -pyridyl)-DPP samples,

1.0 lM solution in PB was prepared and 20 lL was injected into

the HPLC-ﬂuorescence/MS apparatus (systems H and I).

0

1

36.6, 133.1, 132.3, 131.2, 130.2, 129.4, 128.4, 127.8, 127.7,

19

27.4, 127.1, 121.0, 110.6, 64.8, 44.5, 30.1, 30.0;

CN): d = -76.0 (3F, CF -triﬂuoroacetate); HPLC (sys-

= 3.5 min (purity 97% at 260 nm and 100% at 460 nm);

F

NMR

(

565 MHz, CD

3

tem A): t

R

UV–vis (recorded during RP-HPLC analysis): kmax = 256 and

2.7.4. In vitro assays with rat liver microsomes

5

6

34 nm; LRMS (ESI+, recorded during RP-HPLC analysis): m/z

19.3 and 621.3 [M] , calcd for C34H28BrN O 619.1 and 621.1.

4 3

All animal experiments were conducted in accordance with

institutional guidelines. Rats (Wistar, # 6–7 weeks) were pur-

chased from CLEA Japan company. Rats were fasted overnight

and sacriﬁced by exsanguination from the abdominal aorta. The

liver containing 0.15 M KCl (pH 7.4) was homogenized in 3 vol-

umes of the same buffer. Microsomes were prepared according to

the method of Omura and Sato [15]. Microsomes contained

+

2

-

.7. In vitro activation of DPP-based probes 7–11 by PGA or reductases

experimental details

2.7.1. Stock solutions of ﬂuorogenic probes, enzymes and co-factor

5

1.0 mg protein/mL and 0.82 nmol P450/mg protein. They were

diluted with 0.1 M potassium phosphate buffer at pH 7.4 for assay

and the ﬁnal concentration was 170 g/mL. Azo-based ﬂuorogenic

probes 8 and MAR (1.0 M) were incubated in the presence of rat

(

NADH)

Stock solutions of DPP-based ﬂuorogenic probes (1.0 mg/mL)

l

were prepared in DMSO (spectrophotometry grade). Solution NTR

was prepared by dissolving the commercial lyophilized powder

l

liver microsomes (vide supra) under hypoxic conditions. Fluores-

cence measurements were performed in PB (0.1 M, pH 7.4) con-

taining DMSO (0.1%) as a co-solvent and the enzymatic reaction

mixtures were also subjected to UHPLC analyses (see Fig. S88-

in ultrapure water (0.1 U/lL). Stock solution of NADH (140 mM)

was prepared from commercial powder and ultrapure water. Com-

mercial PGA solution (841 U/mL) was directly used without dilu-

tion. Solution of LAP was pepared by dissolving 14.2

commercial suspension in 985.8 L of ultrapure water (0.001 U/

L). Please note: stock solutions of parent DPP ﬂuorophores were

lL of the

S94 form more details). NADPH (50

lM) was added after 5 min.

l

The hypoxic conditions were prepared by bubbling Ar gas into

the reaction solution for 30 min.

l

also prepared in DMSO (1.0 mg/mL) but prolonged sonication is

required to complete solubilize N,N’-dimethyl DPP dye in this polar

aprotic solvent.

3

. Results and discussion

2.7.2. Fluorescence-based assays

In order to simplify the synthesis of ﬂuorogenic pyridyl-based

All assays were performed at 37 °C (using a temperature control

system combined with water circulation and conducted with mag-

netic stirring). For all probes 7–11 (ﬁnal concentration in 3.5 mL

DPP dyes, and contrary to the structure intensively studied by

the Hua group [7], we have chosen to focus ﬁrst on the preparation

of a symmetrical derivative based on the easily-accessible 3,6-bis

0

ﬂuorescence quartz cell: 1.0

l

M, volume: 3.0 mL of PB, pH 7.6),

(4 -pyridyl)-DPP dye, equipped with two identical enzyme-

0

the ﬂuorescence emission of the released mono-(4 -pyridyl)-DPP

dye was monitored at k (Ex./Em.) = 440/555 nm = 5 nm, over time

with measurements recorded every 5 s. For assays conducted with

recognition moieties and hence operating as a dual-reactable

probe. Due to the prominent role of reductases (i.e., AzoR, NTR

and quinone-reductases such as DT-diaphorase) in devising optical

detection/imaging methods of tumor hypoxia (i.e., oxygen-

deprivation state) [16] and bacterial infections [17], we selected

the well-known NTR-4-nitrobenzyl pair for rapid access to DPP-

based ﬂuorogenic enzyme substrates and to facilitate in vitro vali-

dations of their ﬂuorogenicity. To improve aq. solubility of the

overall structure while keeping a neutral state at physiological

pH, the introduction of carboxymethyl arms through N-lactam

alkylation has been also considered.

NTR/NADH, 1

cell: 47 M) was added after 5 min of incubation of the probe in PB,

and NTR (1 L, 0.1 U) after 5 min of incubation with NADH. For

assays conducted with amidase/peptidase, 1.2 L of PGA solution

1 U) or 2 L of LAP solution (0.002 U) was added after 5 min of

lL of co-factor solution (ﬁnal concentration in quartz

l

(

l

incubation of the probe in PB. Blank experiments to assess the sta-

bility of the probes in PB, were achieved in the same way but with-

out adding the corresponding enzyme. Further blank experiments

were performed with PGA-sensitive probe 10, either under Ar

atmosphere (bubbling of Ar for 15 min prior to incubation in PB,

use of a quartz cell with screw top, sealing cap, Starna Scientiﬁc,

3

.1. Synthesis and aqueous stability of NTR-sensitive probe 6 based on

0

symmetrical 3,6-bis-(4 -pyridyl)-DPP

#

3-Q-10-GL14-C, to keep such Ar atmosphere), or in the presence

of NaN (ﬁnal concentration: 14.3 mM), or in acetate buffer (0.1 M,

pH 5.6).

3

As displayed in Scheme 1, the planned synthetic route towards

the water-soluble NTR-sensitive probe 6 was based on the sequen-

0

tial N-alkylation of lactam and pyridyl moieties of 3,6-bis-(4 -

2

.7.3. RP-HPLC-ﬂuorescence and RP-HPLC-MS (full scan and SIM

pyridyl)-DPP 4 with tert-butyl bromoacetate and 4-nitrobenzyl

bromide respectively. As we had anticipated, the main difﬁculty

associated with this synthesis was the selective N-substitution of

lactam units with the protected carboxymethyl arm. On the basis

of the valuable work of Vakuliuk et al. [18] identifying tBuOK as

the most suited base to promote this challenging N-alkylation

and by conducting a rapid screening of reaction conditions (sol-

vent, heating temperature and duration), we have managed to

optimize this step and the corresponding N,N’-dialkyl-DPP 12

was obtained in an acceptable 35% yield after puriﬁcation by col-

umn chromatography over silica gel. Thereafter, quaternization of

pyridyl moieties with 4-nitrobenzyl bromide was readily achieved

by treatment of 12 with a large excess of this alkylating agent (7.7

equiv.) in reﬂuxing MeCN for 24 h. It is important to specify that

modes) analyses

Enzymatic reaction mixtures from ﬂuorescence-based in vitro

assays were directly analyzed by RP-HPLC-ﬂuorescence after an

incubation time of 60–150 min (injected volume: 20

H). Thereafter, each enzymatic reaction mixture (ca. 3 mL) was

freeze-dried; the resulting white amorphous powder was dissolved

lL, system

in a (1:1, v/v) mixture of ultrapure H

00–1000 L depending on the kinetics). The solution was vor-

texed followed by centrifugation (9 000 rpm, 1 min). 20 L of

supernatant was injected into the HPLC-MS apparatus (system I).

Please note: injection of PB and H O-MeCN (1:1, v/v) were also

2

O and MeCN (total volume =

4

l

2

achieved immediately prior to this latter analysis, especially to conﬁrm

the lack of residual contaminants within the C18 column or ESI probe

6

Sébastien Jenni, F. Ponsot, P. Baroux et al.

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 248 (2021) 119179

tBuO

O

2

C

HO

O

2

C

H

N

+

N

1. R'X, MeCN, Ar, reflux, 24 h

2. TFA-DCM (1:1, v/v), RT, 3 h

N

1. tBuOK, NMP, Ar, 75 °C, 15 min

O

2

. RX, Ar, 75 °C, 90 min, 35%

2

O N

yield after

RP-HPLC purification 17%

CO

2

H

NO

2

O

RX =

O

N

H

N⁺

NH

-

N

3 2

2x CF CO

Br

CO

2

tBu

R'X =

2

CO H

4

OtBu

1

2

1

4 (TFA salt)

Br

2

O N

TFA-DCM (1:1, v/v), RT, 3 h

yield after RP-HPLC purification 34%

HO

O

2

C

HO C

2

N

O

partial conversion

during freeze-drying

2

3 2

x CF CO H

2

CO H

O

(following RP-HPLC

purification)

2x CF

3 2

CO ^-

N

NH

CO

2

H

CO

2

H

1

3 (TFA salt)

13-RO (TFA salt)

0

Scheme 1. Synthesis of water-soluble 3,6-bis-(4 -pyridyl)-DPP dye 13 and open-chain lactam form 14 of targeted NTR-sensitive ﬂuorogenic probe 6 (Ar = argon, NMP = N-

methyl-2-pyrrolidone, RT = room temperature).

0

the addition of an inorganic base such as K

2

CO

3

, in an attempt to

Scheme 1. The key step was the preparation of mono-(4 -pyridyl)-

DPP pigment 5 through a two-step procedure based on literature

protocols (Scheme 2 and supporting information). Contrary to

the DPP structure selected by the Hua group [7a,7c], we have

chosen to work with a pigment bearing the 4-bromophenyl

moiety as the second (hetero)aryl substituent. Indeed, the

availability of a reactive bromine atom will enable a further

functionalization of the probe, through post-synthetic Pd-

catalyzed derivatization, if structural elements (and properties

accelerate this reaction, has led to rapid and complete degradation

of the bis-lactam-fused structure. Fully N-alkylated crude product

was directly subjected to anhydrous acidic conditions TFA-DCM

(

1:1, v/v) for the deprotection of tBu esters, and to obtain the tar-

geted water-soluble ﬂuorogenic probe 6. Unfortunately, all our

attempts to isolate this highly polar compound by semi-

preparative RP-HPLC (different aq. mobile phases with pH in the

range 2.0–6.0) failed owing to its instantaneous conversion into

the mono-lactam ring-opening product 14. Its structure was con-

ﬁrmed by detailed measurements including ESI mass spectrometry

and NMR analyses (see supporting information for the correspond-

ing spectra, Figs. S51-S56). To the best of our knowledge, this

propensity to undergo lactam hydrolysis under acidic or neutral

aq. conditions, has never been reported for DPP dyes. One possible

explanation may be the strong electron-withdrawing effect of both

N-(4-nitrobenzyl) pyridinium moieties that dramatically enhance

electrophilicity of lactam carbonyl and hence its facile reaction

with water molecules. This hypothesis is strengthened by the fact

that the parent DPP ﬂuorophore 13 bearing free pyridyl nitrogen

positions, synthesized according the same overall strategy

associated with) are speciﬁcally required for

a particular

biosensing/bioimaging application. Brieﬂy, 3-ethyloxycarbonyl-2-

pyrrolin-5-one 15 carrying 4-bromophenyl substituent was

reacted with 4-cyanopyridine in the presence of sodium tert-

amylate (t-AmylONa, in situ generation through reaction of tert-

amyl alcohol with metallic sodium) to give DPP pigment 5 in a

satisfying 73% yield. This compound has undergone the N-

alkylation/pyridine

quaternization/tBu

ester

deprotection

sequence to give the NTR-sensitive probe. Puriﬁcation was

achieved by semi-preparative RP-HPLC under acidic conditions

(i.e., linear gradient of MeCN in aq. TFA 0.1%, pH 2.0) that

enabled isolation of 7 (TFA salt) in a pure form (>95%) required

for photophysical studies and enzymatic bioassays. In order to

have the reference for ﬂuorescence assays and HPLC analyses

(see Sections 3.4 and 3.5), the parent DPP ﬂuorophore 17 was

prepared using the same reaction sequence whilst omitting

quaternization of pyridyl moiety with 4-nitrobenzyl bromide.

To expand the range of hypoxia-related reductases detectable

with ﬂuorogenic pyridine-ﬂanked DPP platform 5, a second biore-

ductive recognition moiety, sensitive to AzoR activitiy, was also

considered. Installation of a self-immolative azobenzene was

achieved using 4-[2-[4-(dimethylamino)phenyl]diazenyl]-benzyl

bromide 19 as alkylating agent (see supporting information for

its synthesis) and under the same conditions previously used for

the reactions conducted with 4-nitrobenzyl bromide. Another

structural change has been made and has concerned N-lactam

substituents. Indeed, the methyl group was preferred to the

carboxymethyl moiety in order to obtain a positively-charged

compound more suited to biological models possibly contemplated

to validate ﬂuorogenic activation by AzoR. Indeed, contrary to

NTRs, some of which are commercially available and easy to use

in the context of in vitro enzyme assays (e.g, recombinant NTR

expressed in E. coli), isolated forms of AzoR are not readily avail-

(Scheme 1), was found to be a more stable and isolatable com-

pound. However, we noted that the acidic conditions used for its

RP-HPLC puriﬁcation favored its partial conversion (during the

freeze-drying process) into the corresponding non-ﬂuorescent

ring-opened derivative 13-RO.

This unexpected result allows us to unambiguously state that

0

3

,6-bis-(4 -pyridyl)-DPP 4 is not a suitable photoactive platform

to construct reaction-based ﬂuorescent probes for use in aq. envi-

ronments, whose modulation of output optical signal is based on a

N-pyridyl alkylation-dealkylation process. Perhaps as a result, the

Hua group preferred to use an unsymmetrical analog bearing a

single ﬂuorogenic 4-pyridyl group for designing esterase- and

c-GT-activated ratiometric probes 1 and 3 [7a,7c]. Thus, we have

been focused on this type of DPP pigments for our next

investigations.

3

.2. Synthesis of AzoR- and NTR-sensitive probes based on

0

unsymmetrical mono-(4 -pyridyl)-DPP

The synthetic route towards the unsymmetrical analog of NTR-

sensitive probe discussed above, is very similar to that depicted in

7

Sébastien Jenni, F. Ponsot, P. Baroux et al.

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 248 (2021) 119179

HO

O

2

C

N⁺

N

-

3 2

CF CO

NO

2

O

Br

CO

2

H

7

(TFA salt)

R'X =

1

2

. R'X, MeCN, N , reflux, O/N

. TFA-DCM (1:1, v/v), RT, 3 h

Br

yield after RP-HPLC purification 12%

2

O N

2

tBuO C

1

. Na°, t-Amyl-OH, N

2. 4-cyanopyridine, 50 °C to 110 °C,

, 24 h, 73%

2

, 100 °C

Br

H

N

H

N

1. tBuOK, NMP, N

2

, 75 °C, 15 min

O

N

O

N

2

2. RX, N , 75 °C, 90 min, 22%

2

O

RX =

O

CO tBu

N

H

OEt

Br

O

Br

1

5

Br

2

OtBu

1

6

TFA-DCM (1:1, v/v), RT, 4 h

yield after RP-HPLC purification 50%

HO

O

2

C

N

3 2

CF CO H

O

Br

CO

2

H

1

7 (TFA salt)

0

Scheme 2. Synthesis of water-soluble mono-(4 -pyridyl)-DPP dye 17 and related NTR-sensitive ﬂuorogenic probe 7 (t-AmylOH = tert-amyl alcohol or 2-methylbutano-2-ol,

NMP = N-methyl-2-pyrrolidone, O/N = overnight, RT = room temperature).

able. Synthetic reactions for the preparation of AzoR-sensitive

probe 8 and its parent ﬂuorophore 18 are summarized in Scheme 3.

Their puriﬁcation was achieved by semi-preparative RP-HPLC. The

NTR-sensitive probe 9 based on the same ﬂuorogenic N,N’-

dimethyl-DPP dye was also prepared using a similar synthetic

route, to allow a comprehensive and relevant comparison in terms

of enzymatic reactivity. All spectroscopic data (see supporting

information for the corresponding spectra, Figs. S1-S5, S8-S14,

S16-S22, S60-S65 and S67-S72), especially NMR and ESI mass spec-

trometry, were in agreement with the ﬁve structures assigned. The

purity of 7–9, 17 and 18 was conﬁrmed by RP-HPLC analysis with

UV–vis detection at different wavelengths, and mass percentage of

TFA was determined by ionic chromatography (see supporting

information, Figs. S6, S15, S23 and S66).

3.3. Synthesis of PGA-sensitive probes based on unsymmetrical mono-

0

(4 -pyridyl)-DPP

Due to its many positive attributes including commercial avail-

ability at a reasonable cost, stability especially under an immobi-

lized form and structurally simple substrate (phenylacetyl

moiety, PhAc), penicillin G acylase (PGA) is widely used as biocat-

alyst in the synthesis of b-lactam antibiotics [19], for kinetic reso-

lution of racemates and as reagent for the removal of PhAc-based

H

N

O

N

H

Br

5

1

. tBuOK, NMP, N2, RT, 15 min

. MeI, N2, RT, 60 min, 44%

2

N⁺

N

O

19, MeCN, N2, reflux, O/N

R'X, MeCN, N2, reflux, O/N

CF3CO2^-

yield after RP-HPLC purification 17%

yield after RP-HPLC purification 24%

_C_F₃_C_O₂-

N

NO2

O

N

Br

R'X =

Br

8

(TFA salt)

NMe₂

Br

9 (TFA salt)

18

N

O2N

Me2N

1

9

Scheme 3. Synthesis of N,N’-dimethyl-DPP dye 18 and related AzoR- and NTR-sensitive ﬂuorogenic probes 8 and 9 (NMP = N-methyl-2-pyrrolidone, O/N = overnight).

8

Sébastien Jenni, F. Ponsot, P. Baroux et al.

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 248 (2021) 119179

protecting groups. PGA is often regarded as a valuable model pro-

tease to perform in vitro validations of self-immolative molecular

systems used as diagnostic probes, molecular ampliﬁers or drug

delivery systems [20]. Furthermore, several ﬂuorescent probes

were recently developed to image endogenous bacterial PGA and

thus help microbial diagnostics both in health area and food indus-

try [21]. Thus, there is a real interest for the developement of novel

ﬂuorogenic PGA substrates with superior performances.

As illustrated by numerous examples documented in the litera-

ture, the most universal and frequently used PGA-sensitive trigger-

ing unit is para-(phenylacetamido)benzyl moiety which is readily

introduced through a carbamate or an ether linkage depending

on the nature and reactivity of optically tunable group to be

masked [20,22]. In the present case, the availability of N-

alkylating agent para-(phenylacetamido)benzyl bromide 20 (see

supporting information for its optimized synthesis) has enabled

us to use again the synthetic route initially devised for the two

other probes based on N,N’-dimethyl DPP 18, and hence to easily

obtain the targeted compound 10 (Scheme 4).

ratiometric probes 4 and 6, intended to be used in aq. media. As

was the case for reductase-sensitive probes, the structure of each

ﬂuorogenic PGA substrate was conﬁrmed by detailed

spectroscopic measurements including NMR and ESI-MS analyses

(see supporting information for the corresponding spectra,

Figs. S25-S39 and S80-S85).

3.4. Photophysical behavior of enzyme-sensitive DPP-based probes

and parent ﬂuorophores

The spectral properties of the novel pyridine-ﬂanked DPP ﬂuo-

rophores and the related enzyme-sensitive DPP-based probes were

evaluated and collected parameters are summarized in Table 1 (see

Figs. 2 and 3 for the absorption/ﬂuorescence spectra of 8, 10, 13, 17

and 18, and Figs. S7, S24, S40-S41, S86-S87 for 7, 9, 11 and 21). In

contrast to photophysical characterizations performed by the Hua

group on DPP derivatives 1–3, we have deliberately chosen phos-

phate buffer (PB, 0.1 M, pH 7.6) alone and not mixed with 20%

DMSO as solvent for these measurements. Indeed, this allowed to

mimic the real operating conditions for these ﬂuorogenic com-

pounds, to draw any relevant conclusions concerning their resis-

tance to aggregation and their stability at physiological pH. As

expected for conventional 3,6-diaryl-DPP dyes, and in line with

Finally, to facilitate comparison with results obtained by the Hua

group [7a,7c], we have also prepared the analog 11 for which the

ﬂuorogenic DPP platform is N,N’-substituted by hexyl moieties,

even if we were highly sceptical about the relevance of this choice

0

done for the construction of their esterase- and

c

-GT-activated

the spectral parameters of several N,N’-dialkyl mono-(4 -pyridyl)-

C

N

6 13

H

N

O

1. tBuOK, NMP, N

2

, 75 °C, 15 min

O

2

. C 13Br, N , 75 °C, 90 min, 4%

6

H

2

O

N

C

Br

H

5

6 13

H

2

1

O

Br

13

2

1. tBuOK, NMP, N , RT °C, 15 min

20, MeCN, N

2

, reflux, O/N

2. MeI, N

2

, RT, 60 min, 44%

N

H

2

yield after RP-HPLC purification 6%

0

N⁺

N

O

N

C

N

6

H

O

_N+

20, MeCN, N

2

, reflux, O/N

yield after RP-HPLC purification 25 %

-

O

3 2

CF CO

NHPhAc

O

-

N

CF

O

3 2

CO

NHPhAc

Br

O

Br

N

C

10 (TFA salt)

Br

18

N

H

2

6

H

13

0

1

1 (TFA salt)

Scheme 4. Synthesis of PGA-sensitive ﬂuorogenic probes 10 and 11 and N,N’-dihexyl-DPP dye 21 (NMP = N-methyl-2-pyrrolidone, O/N = overnight).

Table 1

Photophysical properties of pyridine-ﬂanked DPP ﬂuorophores and related enzyme-sensitive ﬂuorogenic probes studied in this work, determined in PB (0.1 M, pH 7.6) at 25 °C.

Fluorophore or probe^a

k Abs (nm)^b

k Em. (nm)

e

(M cm

ꢁ1

)

Stokes’ shift (cm

ꢁ1

)

U

F

c

d

7

8

9

1

2

516

460

529

526

531

467

484

471

669

-

14 050

20 500

16 250

9 300

4 432

-

e

d

675

679

613

555

4 089

4 284

2 519

3 395

d

0

1

3

4

7

8

1

f

d

-

g

9 200

0.28

e

-

13 100

13 650

9 900^g

12 250

-

553

556

555

3 148

3 156

2 558

0.50

0.74

h

439 (473)

486

f

d

-

^aStock solutions (1.0 mg/mL) of ﬂuorophores and probes were prepared in spectroscopic grade DMSO.

b

Only 0–0 band of the S0 ? S1 transition is reported.

c

For experimental conditions used for such determination, see supporting information.

d

Non-linear relationship between area of Em. curve and Abs value at Ex. wavelength due to aq. instability or persistence of aggregates in diluted aq. solutions, has prevented

determination of relative ﬂuorescence quantum yield.

e

Non-ﬂuorescent.

f

Spectral measurements were achieved in PB-DMSO (8:2, v/v, pH 8.5) due to poor solubility in pure phosphate buffer.

g

Sample of 13 used for these photophysical characterizations contains less than 5% of non-ﬂuorescent species 13-RO.

h

ꢁ7

ꢁ8

Formation of H-type aggregates that is disrupted for concentrations in the range 10 –10 M used for the recording of Em./Ex. spectra. Ex. maximum is centered at

73 nm and Stokes’ shift was calculated using this latter value.

4

9

Sébastien Jenni, F. Ponsot, P. Baroux et al.

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 248 (2021) 119179

Fig. 3. Normalized absorption (blue), excitation (Em. 700 nm only for 10, green)

and emission (Ex. 465 nm, red) spectra of enzyme-sensitive ﬂuorogenic probes 8 (A,

target enzyme: AzoR) and 10 (B, targeted enzyme: PGA) in PB at 25 °C. Please note:

probe 8 is not ﬂuorescent and only the peak assigned to Raman scattering (553 nm) was

observed.

disrupted for concentrations in the range 10 ⁷-10^ꢁ⁸M (against

ꢁ

ꢁ5

ꢁ6

1

0

-10

M for absorption measurements), typically used for

recording emission/excitation spectra and determination of the

relative ﬂuorescence quantum yield. Thus, whatever N-lactam sub-

0

stituents (i.e., carboxymethyl or methyl) of mono-(4 -pyridyl)-DPP

scaffold, an intense ﬂuorescence emission centered in the range

5

53–556 nm, was observed with the three pyridine-ﬂanked DPP

0

dyes (Ex. at 465 nm). Like N,N’-dialkyl mono-(4 -pyridyl)-DPP dyes

being spectrally characterized in organic media [23], emission

bands of 13, 17 and 18 in PB are not structured and large Stokes’

shift values are obtained. Very satisfactory values of ﬂuorescence

quantum yield in pure aq. medium have been determined. This is

a valuable feature that enables to compensate the relatively low

molar extinction coefﬁcients of DPP ﬂuorophores, in the perspec-

tive of practical applications in biological contexts. Obviously, this

set of results also conﬁrms that the best way to readily convert 3,6-

diaryl-DPP pigments into ﬂuorophores both soluble and strongly

emissive in water, should be based on N-alkylation of their lactam

groups with hydrophilic pendant arms. Indeed, this strategy leads

to DPP molecules with a bolaamphiphilic character (i.e., hydrophi-

lic substituents located on both sides of the hydrophobic bis-

lactam fused core) [24] not prone to micelle formation unlike

amphiphilic dyes that are often non-emissive species. Unsurpris-

ingly, the spectral behavior of N,N’-dihexyl derivative 21 in aq. buf-

fer is dramatically different and unsatisfactory: weak ﬂuorescence

emission and persistence of aggregates even at low concentrations

that prevents determination of relative ﬂuorescence quantum yield

in PB (Fig. S86). This latter result explains why the Hua group had

been chosen a PBS-DMSO (8:2, v/v) mixture for the spectral

Fig. 2. Normalized absorption (blue), excitation (Em 625 nm for 13 or 600 nm for

1

7 and 18, green) and emission (Ex. 465 nm, red) spectra of pyridine-ﬂanked DPP

ﬂuorophores 13 (A), 17 (B) and 18 (C) in PB at 25 °C. Please note: sample of 13 used

for these photophysical characterizations contains less than 5% of non-ﬂuorescent

species 13-RO.

DPP dyes already determined in pure organic solvents (e.g., DMF

and MeCN) by Erbas and Alp [23], the three ﬂuorophores 13, 17

and 18 are characterized by a broad absorption within the blue-

violet spectral range, centered at 467 nm, 471 nm and 439 nm

respectively. The hypsochromic shift of the absorption wavelength

of N,N’-dimethyl derivative 18 and the dramatic change in the

shape of its absorption band (loss of symmetry and appearance

of a shoulder) are attributed to the formation of non-emissive H-

type aggregates. This hypothesis was supported by recording the

excitation spectrum, which does not match the absorption

spectrum (Fig. 2C). However, these aggregates are assumed to be

10

Sébastien Jenni, F. Ponsot, P. Baroux et al.

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 248 (2021) 119179

measurements and in vitro validations of esterase- and

c

-GT-

to contain a wide range of redox enzymes including azoreductases

[27]. Since these reductases are overexpressed and readily active

only under hypoxic conditions, incubations with rat liver micro-

somes have been achieved with argon-saturated phosphate buffer.

Control experiments (positive control) with ﬂuorogenic azo-

rosamine MAR [27a], whose bioreductive activation leads to the

release of green-emitting rosamine 2Me RG, have also been

achieved to conﬁrm the functionality of this in vitro biological

model which will not involve the use of an isolated enzyme (see

supporting information).

sensitive probes 1 and 3 [7a,7c]. The same medium was used for

spectral characterization of 21 (Table 1, entry 10 and Fig. 87) but

it is necessary to specify that addition of 20% DMSO in phosphate

buffer changes greatly the pH value (8.5 vs. 7.6).

When the same characterization methodology was applied to

N-alkylpyridinium-based probes 7–11, some common features

have been noted and conﬁrm that a strong ICT process operates

within these DPP molecules: (1) broad structureless absorption

and emission proﬁles with full-width at half maximum (FWHM)

D

k

1/2max in the range 100–115 nm and 125–130 nm respectively,

We ﬁrst examined the three DPP-based probes rationally

designed to be reactive towards hypoxia-related reductases

(Fig. 4 and Figs. S88-S92). Somewhat surprisingly, incubation of

N-(4-nitrobenzyl)pyridinium derivatives 7 and 9 with NTR/NADH

at 37 °C did not produce a signiﬁcant and gradual increase of ﬂuo-

rescence emission centered at 555 nm (Ex. at 440 nm) indicating

that the reduction of nitro group and the subsequent 1,6-benzyl

(

2) huge Stokes’ shift values, and (3) NIR-I emission with a very

low ﬂuorescence quantum yield in aq. media (see Fig. 3B for the

absorption/ﬂuorescence spectra of 10 illustrating these spectral

features). However, we did not manage to accurately determine

this latter parameter due to the marked instability of 7 and 9–11

highlighted in PB and at low concentration (see Section 3.5). The

major consequence of the lack of a zero background signal for

these ﬂuorogenic probes, is that the detection of enzyme activity

will be achieved through a ratiometric output signal and not an

intensometric ‘‘OFF-ON” response [4b]. This will not be the case

with the probe 8 that is functionalized with an azobenzene

moiety knowing to act as an effective dark quencher through

photochemical isomerization of its -N=N- bridge [16b,25]. The

lack of emission for 8 was borne out by ﬂuorescence spectrum

shown in Fig. 3A.

0

elimination to release the mono-(4 -pyridyl)-DPP ﬂuorophore 17

or 18, is not triggered by this reductase (Fig. 4). We ruled out the

hypothesis that NTR and/or its co-factor NADH are degraded by

performing further kinetics highlighting rapid and effective biore-

ductive activation of 4-nitrobenzyl ether derived from a known

NIR-I emitting phenol-based dihydroxanthene-hemicyanine dye

(data not shown) [28]. The lack of reactivity of both probes towards

NTR should be linked to their size and molecular geometry driven

by the unique structure of DPP scaffold, that may prevent a favor-

able binding of 7 and 9 in the active site of this reductase. In our

view, the presence of a positively-charged N-alkylpyridinium in

close proximity to the enzyme-recognition moiety (i.e., 4-

nitrobenzyl) does not necessarily have an adverse effect on probe

recognition by such reductase because several functioning NTR-

responsive ﬂuorescent probes bearing a bioreductive structural

3.5. Fluorogenic reactivity of enzyme-sensitive DPP-based probes. In

vitro enzymatic assays

Due to commercial availability of NTR (recombinant, expressed

in E. coli) and PGA (from E. coli), the ﬂuorogenic reactivity of DPP-

based probes

ﬂuorescence-based in vitro assays involving time-course measure-

ments and following reliable protocols previously used by us [26].

For azo-based ﬂuorogenic probe 8, a quite similar methodology

was employed but using rat liver microsomes which are known

unit that is quite similar (i.e.,

a 4-pyridyl substituent N-

7

and 9–11 was readily studied through

quaternized with (5-nitro-2-furyl)methyl group), have already

been reported in the literature [29].

A similar negative result was also obtained when AzoR-

sensitive DPP probe 8 was incubated with rat liver microsomes

Fig. 4. Fluorescence emission time course (Ex./Em. 440/555 nm, slit 5 nm) of NTR-sensitive ﬂuorogenic probes 7 (A) and 9 (B), concentration: 1.0

0.1 U) and NADH (ﬁnal concentration: 47 M) in PB at 37 °C. (C) Fluorescence emission time course (Ex./Em. 440/675 nm, slit 5 nm) of probe 9, concentration: 1.0

alone at 37 °C. (D) UV–vis absorption spectra of probe 9 before and after incubation in PB (12 h at 37 °C, concentration: 15 M).

l

M, in the presence of NTR

(

l

lM, in PB

l

11

Sébastien Jenni, F. Ponsot, P. Baroux et al.

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 248 (2021) 119179

and NADH under simulated hypoxic conditions. Indeed, no

increase in green-yellow ﬂuorescence intensity at 560 nm (Ex. at

Fortunately, the sensing performances of probe 10 toward

hydrolase PGA have been much more encouraging. Indeed, when

this phenylacetamide derivative was incubated with the com-

mercial amidase, a moderately rapid and gradual increase of ﬂu-

orescence emission at 555 nm (Ex. at 440 nm) was observed

(Fig. 5A) and the plateau was reached within 2 h of incubation.

Interestingly, a direct comparison of the ﬂuorescence emission

spectra of PGA-sensitive DPP probe 10 recorded before and after

reaction with enzyme (Ex. at 440 nm, Fig. 5B), highlighted a

spectral blue-shift of 120 nm allowing a proper ratiometric

detection of PGA.

In order to unambiguously conﬁrm that the intense green-

yellow ﬂuorescence signal detected was due to the release of

pyridine-ﬂanked DPP dye 18, this enzymatic reaction mixture

and related blanks (i.e., PB alone for HPLC column blank and 18

in PB alone) were subjected to RP-HPLC-ﬂuorescence analyses

4

65 nm) was observed, thus suggesting the inertness of azoben-

zene moiety towards these bioreductive conditions (Figs. S88 and

S89). This was further supported by incubation of 8 with a large

excess of sodium dithionite (Na S O ), a bio-compatible and effec-

2 2 4

tive reducing agent for diazo bridges highlighted by the Wagner

group for applications in chemical biology [30], that did not pro-

duce the expected ﬂuorescent ‘‘OFF-ON” response (Figs. S95 and

S96). In addition to this lack of enzymatic activation, we noted a

strange spectral behavior of these N-alkylpyridinium based probes

when they are incubated in PB alone at 37 °C. A slow but gradual

decrease of their NIR-I emission was observed, especially for 9.

As illustrated in Fig. 4C, ﬂuorescence signal was signiﬁcantly

reduced by a factor of 17 within 115 min of incubation. Following

this blank kinetics, the recording of absorption spectrum revealed

the formation of a novel non-emissive species characterized by a

blue-shifted maximum wavelength (-35 nm compared to starting

probe 9, Fig. 4D). Additional experiments have been done to gain

insight into the mechanism of formation of this unknown product

that may complicate the practical implementation of ﬂuorogenic

probes based on a pyridine-ﬂanked DPP platform (vide infra). Curi-

ously, this poor aq. stability observed in the absence of targeted

enzyme, was not mentioned by the Hua group whereas their

R

(Fig. 6). As expected, a single peak (t = 4.4 min) was detected

and assigned to the expected ﬂuorophore (as proven by co-

injection analysis with an authentic sample of 18). Furthermore,

this pyridine-ﬂanked DPP was not detected in the blank reaction

mixture (Figs. S109 and S110) thereby conﬁrming that green-

yellow ﬂuorescence activation of the probe is solely triggered by

PGA. It is important to point out that under our RP-HPLC condi-

tions, N-alkypyridinium probe 10 and parent DPP ﬂuorophore 18

esterase- and

c

-GT-activated ratiometric probes 1 and 3 share

R

have the same retention time (t = 4.4 min). However, they can

the same structural features than our probes [7a,7c]. That point

deserves further checking. All these disappointing results led us

to unambiguously state that ﬂuorogenic DPP dyes bearing a

pyridyl moiety as optically tunable group are not really suitable

candidates for designing bioreductive ﬂuorescent imaging agents

intended for hypoxia detection or bacterial monitoring.

be readily differentiated according to the Ex./Em. channel used

for ﬂuorescence detection (i.e., 520/675 nm for 10 and

475/550 nm for 18). Thus, we were able to highlight the conversion

of the probe 10 into a non-ﬂuorescent compound when it was

subjected to a prolonged incubation in PB alone at 37 °C, because

no peak was detected on both channels (Figs. S109 and S110).

Fig. 5. (A) Fluorescence emission time course (Ex./Em. 440/555 nm, slit 5 nm) of PGA-sensitive ﬂuorogenic probe 10, concentration: 1.0

lM, in the presence of PGA (1 U) in PB

at 37 °C. (B) Fluorescence emission spectra (Ex. at 440 nm, slit 5 nm) of ﬂuorogenic probe 10 before and after incubation with PGA (1 U, 150 min of incubation) in PB at 37 °C.

12

Sébastien Jenni, F. Ponsot, P. Baroux et al.

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 248 (2021) 119179

Fig. 6. RP-HPLC elution proﬁles (ﬂuorescence detection Ex./Em. 475/550 nm) of PGA-sensitive ﬂuorogenic probe 10 (A), enzymatic reaction mixture of probe 10 with PGA,

57 min of incubation at 37 °C (B), authentic sample of ﬂuorophore 18 (C) and co-injection of enzymatic reaction mixture and 18 (D). Please note: minor peak at 6.8 min is

1

present in the blank sample run.

The corresponding kinetic curve obtained for this blank experi-

ment, also emphasizes this non-enzymatic transformation, that

appears to occur whatever the benzyl-like substituent carried by

speciﬁc quencher of singlet oxygen [31], also led to the formation

of these degradation products (Figs. S115 and S116). Furthermore,

the dramatic change in the absorption proﬁle of probe 10, already

noted during in vitro bioassays of NTR-sensitive probe 9, (vide

surpa), allowed us to conclude that the DPP chromophore is missing

and probably degraded through hydrolytic ring-opening. In the

light of these observations, we propose an hypothetical mechanism

for the formation of furo[2,3-c]pyridinium derivatives 22 and 23

(Fig. 9), even if an additional, comprehensive study involving the

independent synthesis and spectroscopic characterization of these

unusual heterocyclic compounds, will be required. Once again, we

are truly surprised that the Hua group did not discuss the hydroly-

0

the 4 -pyridyl unit of DPP (vide supra, with NTR-sensitive probe 9).

These investigations were completed by RP-HPLC-MS analyses

with the aim both of conﬁrming the reaction between

phenylacetamide-based probe 10 and PGA and of identifying the

unknown product(s) arising from degradation of probes 9 and 10

in the lack of enzyme (Fig. 7 and Figs. S111-S116). First, the disap-

pearance of the probe 10 and the formation of pyridine-ﬂanked

DPP 18 were clearly observed (Fig. 7), and the structure and integ-

rity of this ﬂuorophore were supported by UV–vis and MS-

ESI + data (both in ‘‘full scan” and single ion monitoring (SIM)

sis problem of esterase- and c-GT-sensitive probes 1 and 3 that is

expected to occur in the same manner in PBS-DMSO (8:2, v/v) mix-

ture whose pH is greater than 7.5 (vide supra) [7a,7c].

+

modes; t

for C19 15BrN

The RP-HPLC elution proﬁle of blank mixture shows the lack of

probe 10 (t = 4.3–4.4 min) and the appearance of a new major

peak (t = 3.2 min) whose MS(ESI+) spectrum (‘‘full scan”) displays

R

= 4.3 min, MS(ESI+): m/z [M+H] = 396.1 and 398.1, calcd

+

H

3 2

O 396.0 and 398.0).

A further control experiment aimed at proving the speciﬁcity of

the enzymatic cleavage of PGA-sensitive probe 10, was achieved.

Incubation with an aminopeptidase (leucine aminopeptidase

(LAP) from porcine kidney [32]) did not produce an increase of

green-yellow ﬂuorescence at 555 nm (Fig. S97), conﬁrming that

this latter protease is not able to cleave the phenylacetamide moi-

ety. Furthermore, a direct comparison of the ﬂuorescence emission

spectra of 10 recorded before and after reaction with LAP (Ex. at

440 nm, Fig. S98) provided evidence once again of poor aq. stability

of this N-alkylpyridinium-based probe.

Finally, to assess the possible inﬂuence of N-lactam substituents

on hydrolytic stability of probes based on N-alkylpyridinium-based

DPP scaffold, more hydrophobic analog 21 bearing hexyl moieties

was studied under the same conditions. As expected, intuitively,

the spectral behavior and enzymatic reactivity of 11 were

negatively impacted by the propensity of this compound to form

aggregates in PB. The alternative use of PB-DMSO (8:2, v/v) as

R

two pairs of ions at m/z = 653.3/655.3 and 671.3/673.0 (Fig. 8).

Compared to pyridine-ﬂanked DPP 18 (vide supra), this species

was found to be much more easily detected in the positive mode,

suggesting the integrity of N-alkylpyridinium moiety within its

structure. If we assign peaks 653.2/655.3 and 671.3/673.3 to

molecular ions the degradation product and its hydrated deriva-

tive respectively, the mass difference of +34/+52 amu with start-

ing probe 10 suggests formal double/triple hydration and loss of

two hydrogen atoms (to promote a ring aromatization?). The

assumption that singlet oxygen, hypothetically formed by triplet

energy transfer from N-alkylpyridinium-based DPP to molecular

oxygen, could play a key role in this degradation pathway, was

rapidly excluded because further blank experiments conducted

under argon atmosphere or in the presence of sodium azide, a

13

Sébastien Jenni, F. Ponsot, P. Baroux et al.

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 248 (2021) 119179

Fig. 7. RP-HPLC elution proﬁles of PGA-sensitive ﬂuorogenic probe 10 before and after incubation with PGA. (A) UV detection at 260 nm of probe 10 in PB alone. (B) UV

detection at 260 nm of probe 10 after enzymatic reaction. (C) ESI-MS detection, positive and SIM mode at m/z = 396.0 ± 0.5 (detection of ﬂuorophore 18). (D) ESI-MS detection,

positive and SIM mode at m/z = 619.1 ± 0.5 (detection of probe 10).

incubation buffer, did not produce better and valuable results in

terms of ratiometric detection of PGA (Figs. S117 and S118).

This conﬁrmed the relevance of using polar or at least small

size N-lactam substituents for ﬂuorogenic DPP dyes used in the

construction of reaction-based ﬂuorescent probes working in bio-

logical media.

14

Sébastien Jenni, F. Ponsot, P. Baroux et al.

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 248 (2021) 119179

Fig. 8. RP-HPLC elution proﬁles of PGA-sensitive ﬂuorogenic probe 10 after incubation in PB (0.1 M, pH 7.6) alone. (A) UV detection at 260 nm and ESI + mass spectrum of

peak at t = 3.7 min (inset). (B) ESI-MS detection, positive and SIM mode at m/z = 673.2 ± 0.5; RP-HPLC elution proﬁle of ﬂuorogenic probe 10 after incubation with PGA,

R

+

ESI-MS detection, positive and SIM mode at m/z = 673.2 ± 0.5 (inset). Please note: complete description of isotopic pattern of [M] of degradation product is omitted for clarity,

m/z = 671.3 (90), 672.3 (30), 673.3 (100) and 674.2 (35).

4

. Conclusion

deprotection strategy (i.e., masking-unmasking of pyridine nitro-

gen) [6]. Thus, we can conclude that pyridine-ﬂanked DPP ﬂuo-

rophores are not well suited to construct ‘‘smart” activity-based

probes for reductase-triggered hypoxia imaging. Conversely, and

as already shown by the Hua group [7a,7c], an effective

ﬂuorogenic ratiometric detection of hydrolases (i.e., esterase and

In an attempt to develop novel bioreductive ﬂuorescent imag-

ing agents with the aim of detecting tumor hypoxia, we assessed

under-utilized photoactive platforms based on a DPP scaffold func-

tionalized with one or two 4-pyridyl units acting as ﬂuorogenic

reactive center(s). We have synthesized four DPP-based ﬂuores-

cent probes functionalized with either 4-nitrobenzyl or 4-[2-[4-(

dimethylamino)phenyl]diazenyl]-benzyl moiety as NTR- or AzoR-

responsive group respectively. In vitro study of all probes has high-

lighted on one hand the aq. unstability of the dual-reactable NTR

c-GT previously studied by the Hua group and PGA in the

present work) was readily achievable through an enzyme-

0

triggered self-immolative N-dealkylation process of 4 -pyridinium

moiety. In this context, we are convinced that structurally and

0

easily functionalizable ﬂuorogenic platforms such as mono-(4 -

0

substrate 6 derived from a symmetrical 3,6-bis-(4 -pyridyl)-DPP

pyridyl)-DPP dyes may be a valuable alternative to more

dye and on the other hand the inability of both reductases (AzoR

and NTR) to activate ﬂuorogenic compounds based on a symmetri-

cal mono-(4 -pyridyl)-DPP dye and designed by the protection-

conventional aniline- or phenol-based ﬂuorophores typically

used in the construction of ﬂuorogenic probes for disease-

relevant hydrolases. However, the issue of slow but effective

0

15

Sébastien Jenni, F. Ponsot, P. Baroux et al.

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 248 (2021) 119179

-

X

N

+

N

O

N

O

2

Nu addition of H O

OH

O

NHPhAc

- HX

NHPhAc

O

N

Br

H

N

fragmentation of

tertiary alcohol^a

N

O

lactam ring-opening

hydration reaction)

O

N

(

O

NHPhAc

CO

2

H

NH

Br

intramolecular

pericyclic reaction

a

Br

aromatization

+ further hydration?)

N⁺

O

N⁺

O

(

N

HN

O

HN

O

NHPhAc

HO

2

C

2

HO C

2

2 (mass calcd. 653.1/655.1, ESI+ m/z 653.2/655.3)

2

3 = 22 + H

2

O (mass calcd. 673.1/671.2, ESI+ m/z 671.3/673.3)

a

Fig. 9. Proposed degradation pathway of PGA-sensitive ﬂuorogenic probe 10 observed in aq. buffers and in the lack of enzyme. For clarity in the drawing of steps namely

‘‘fragmentation of tertiary alcohol” and ‘‘intramolecular pericyclic reaction”, ﬁnal protonation step of postulated carbanion intermediate is omitted. Please note:a similar degradation

pathway may be proposed for NTR-sensitive ﬂuorogenic probe 9.

degradation of N-alkylpyridinium-based DPP dyes in aq. media,

unveiled by the present study, needs to be addressed before

considering further applications in biological media. That is why

we encourage (bio)chemists working in the ﬁeld of enzyme-

responsive ﬂuorescent chemodosimeters to routinely implement

Acknowledgements

This work is part of the project ‘‘Pharmacoimagerie et Agents

Theranostiques”, supported by the Université de Bourgogne and

Conseil Régional de Bourgogne through the Plan d’Actions Régional

pour l’Innovation (PARI) and the European Union through the PO

FEDER-FSE Bourgogne 2014/2020 programs. Financial support

from the French ‘‘Investissements d’Avenir” program, project ISITE

BFC (contract ANR-15-IDEX-0003), especially for the post-doc fel-

lowship of Dr. Sébastien Jenni, Agence Nationale de la Recherche

an analytical methodology involving

a series of RP-HPLC-

ﬂuorescence/MS analyses and along the lines of that used in this

Article, especially for deciphering precisely their activation

mechanism and possible degradation pathway(s), and thus

improving their performances.

(

ANR, AAPG 2018, DetectOP_BChE, ANR-18-CE39-0014 and

LuminoManufac-Oligo, ANR-18-CE07-0045), especially for the

post-doc fellowships of Drs. Valentin Quesneau and Kévin Renault

respectively is also greatly acknowledged. Flavien Ponsot gratefully

acknowledges the French Ministry of National Education, Higher

Education and Research for his Ph. D. grant (2017-2020). This work

was supported in part by JSPS KAKENHI Grant Numbers

JP19H05414, JP20H04767, and JP16H05099 to Associate Prof. Ken-

jiro Hanaoka. GDR CNRS ‘‘Agents d’Imagerie Moléculaire” (AIM)

2037 is also thanked for its interest in this research topic. The

authors thank the ‘‘Plateforme d’Analyse Chimique et de Synthèse

Mol e´ culaire de l’Universit e´ de Bourgogne” (PACSMUB, http://www.

wpcm.fr) for access to analytical instrumentation, Dr. Myriam Laly

CRediT authorship contribution statement

Sébastien Jenni: Conceptualization, Investigation, Formal anal-

ysis, Writing - original draft. Flavien Ponsot: Conceptualization,

Investigation, Formal analysis, Writing - original draft. Pierre Bar-

oux: Investigation. Lucile Collard: Investigation. Takayuki Ikeno:

Investigation. Kenjiro Hanaoka: Formal analysis, Funding acquisi-

tion. Valentin Quesneau: Investigation. Kévin Renault: Investiga-

tion. Anthony Romieu: Conceptualization, Investigation, Formal

analysis, Writing - original draft, Writing - review & editing, Fund-

ing acquisition.

(

University of Burgundy, PACSMUB) for the determination of TFA

content of samples puriﬁed by semi-preparative RP-HPLC, Prof.

Daniel T. Gryko (Institute of Organic Chemistry, Polish Academy

of Sciences, Warsaw, Poland) for his expert advice on the alkylation

of pyridine-ﬂanked DPP pigments, COBRA lab (UMR CNRS 6014),

and Iris Biotech company for the generous gift of some chemical

reagents used in this work. A special note of gratitude is also owed

Declaration of Competing Interest

The authors declare that they have no known competing ﬁnan-

cial interests or personal relationships that could have appeared

to inﬂuence the work reported in this paper.

16

Sébastien Jenni, F. Ponsot, P. Baroux et al.

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 248 (2021) 119179

to all undergraduate or graduate students that occasionally partic-

ipated to this research program: MM. Anthony Lenzi and Sullivan

Guy (2018-2019), MM. Mathieu Bettinger and Tom Charenton

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17

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Design, synthesis and evaluation of enzyme-responsive fluorogenic probes based on pyridine-flanked diketopyrrolopyrrole dyes

DOI: 10.1016/j.saa.2020.119179

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