Synthesis, spectroscopic and computational evaluation of a xanthene-based fluorogenic derivatization reagent for the determination of primary amines

Article abstract of DOI:10.1016/j.dyepig.2021.109798

To detect an analyte, typically at the sub-nanomolar scale, extremely sensitive analytical tools are required. Fluorescence is the spectroscopy of choice to achieve such a level due to its non-invasive nature and efficiency in accurately probing the sub-n

Full text of DOI:10.1016/j.dyepig.2021.109798

Journal Pre-proof
Synthesis, spectroscopic and computational evaluation of a xanthene-based
fluorogenic derivatization reagent for the determination of primary amines
Amalia D. Kalampaliki, Steve Vincent, Suman Mallick, Hoang-Ngoan Le, Guillaume
Barnoin, Yogesh W. More, Alain Burger, Yannis Dotsikas, Evagelos Gikas, Benoît Y.
Michel, Ioannis K. Kostakis
PII:
S0143-7208(21)00664-1
DOI:
https://doi.org/10.1016/j.dyepig.2021.109798
DYPI 109798
Reference:
To appear in: Dyes and Pigments
Received Date: 4 May 2021
Revised Date: 4 September 2021
Accepted Date: 6 September 2021
Please cite this article as: Kalampaliki AD, Vincent S, Mallick S, Le H-N, Barnoin G, More YW, Burger A,
Dotsikas Y, Gikas E, Michel BenoîY, Kostakis IK, Synthesis, spectroscopic and computational evaluation
of a xanthene-based fluorogenic derivatization reagent for the determination of primary amines, Dyes
and Pigments (2021), doi: https://doi.org/10.1016/j.dyepig.2021.109798.
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2021 Published by Elsevier Ltd.

Dyes and Pigments
Research Article
CRediT authorship contribution statement
—
Amalia D. Kalampaliki: Methodology, Formal analysis, Investigation. Steve Vincent: Methodology,
Formal analysis, Investigation. Suman Mallick: Methodology, Investigation, Review. Hoang-Ngoan
Le: Visualization, Project administration. Guillaume Barnoin: Visualization, Validation. Yogesh W.
MORE: Validation, Data curation. Alain BURGER: Funding acquisition, Review. Yannis Dotsikas:
Conceptualization, Investigation. Evagelos Gikas: Funding acquisition, Writing – review & editing.
Benoît Y. MICHEL: Funding acquisition, Writing – review & editing, Supervision. Ioannis K. Kostakis:
Funding acquisition, Writing – review & editing, Supervision.

Graphical Abstract
MAGENTA or RED: select your 1° amine detection color 
Synthesis, spectroscopic and computational
evaluation of a xanthene-based fluorogenic
derivatization reagent for the determination
of primary amines
Leave this area blank for abstract info.
Amalia D. Kalampaliki, Steve Vincent, Suman Mallick, Hoang-Ngoan Le, Guillaume Barnoin, Yogesh W. More,
Alain Burger, Yannis Dotsikas, Evagelos Gikas*, Benoît Y. Michel*, Ioannis K. Kostakis*

1
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Synthesis, spectroscopic and computational evaluation of a xanthene-based
fluorogenic derivatization reagent for the determination of primary amines
a,†
b,†
b
b
b
Amalia D. Kalampaliki , Steve Vincent , Suman Mallick , Hoang-Ngoan Le , Guillaume Barnoin ,
b
b
c
d,*
b,*
Yogesh W. More , Alain Burger , Yannis Dotsikas , Evagelos Gikas , Benoît Y. Michel , Ioannis K.
a,*
Kostakis
a
Department of Pharmacy, Division of Pharmaceutical Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis, Zografou, 157 71,
Athens, Greece.
b
Universite
́
Co
̂
te d’Azur, CNRS, Institut de Chimie de Nice, UMR 7272 − Parc Valrose, 06108 Nice cedex 2, France.
Laboratory of Pharmaceutical Analysis, Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis, Zografou, 157 71,
c
Athens, Greece.
d
Laboratory of Analytical Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis, Zografou, 157 71, Athens,
Greece.
†
These authors contributed equally to this work.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
To detect an analyte, typically at the sub-nanomolar scale, extremely sensitive analytical tools are
required. Fluorescence is the spectroscopy of choice to achieve such a level due to its non-invasive
nature and efficiency in accurately probing the sub-nanomolar concentration range. Here, we
report the design, synthesis and photophysical characterization of a fluorogenic derivatization
reagent with exclusive selectivity for primary amines. This xanthene-based dye owns an
exacerbated fluorogenic character making the derivatized amine absorbing in yellow and emitting
in the vermilion edge. In addition to being fluorogenic, this derivatization method also has the
crucial advantage of being chromogenic (colorless <=> fuchsia). Chemical quantum calculations
give an insight into the dye’s molecular properties, while the development of an LC analytical
method provides proof of concept regarding its application for the analysis of primary amines in
a complex matrix.
Available online
Keywords:
Amine derivatization
Colorimetric detection
Fluorogenic sensing
Chromatographic analysis
Quantum calculations
2
021 Elsevier Ltd. All rights reserved.
a) Classical approach for amine derivatization:
1
. Introduction
-
Fluorescent dye bearing NHS ester
Modern analytical chemistry continuously pushes the limits of
Primary or Secondary
ultrasensitive analysis towards lower levels of detection [1].
Among all sensing methods, fluorescence spectroscopy appears to
be the technique of choice because of its non-destructive character
and its high sensitivity of response, especially for an extremely low
O
O
N
Amines
DYE
O
DYE
N
R
R'
O
R-NH2 or RR'NH
[lack of selectivity 1° vs. 2°]
no colorometric detection]
[
-
9
concentration of analyte (<10 M)[2,3]. Traditionally, there are
two approaches for fluorescence-based sensing. One consists in
turning on the fluorescence in the presence of a specific analyte
while the other is to turn it off. Unequivocally, the turn-on method
is significantly superior due to its high contrast to the background
and the negligible risk of false positives, since most organic
compounds are not intrinsically fluorescent [4].
[dye excess removal]
[
detrimental intrinsic fluorescence]
b) This work: Fluorogenic sensing
-
Selective reaction towards 1° amines
LG
Primary Amines
Fluorogenic
Agent
Fluorescence
Turn-On
N R
R-NH2
In the case of a turn-on sensing, the simplest strategy is the
fluorescence derivatization, i.e. the transformation of a non-
emissive analyte into a fluorescent molecule, based in most cases
on a chemical reaction [5]. The most frequently used methodology
is the fluorescent labeling corresponding to the covalent
attachment of an emissive dye (Scheme 1). The strong extrinsic
fluorescence – brought by the coupled bright fluorophore – allows
an efficient detection of the analyte with high sensitivity [6].
Scheme 1. State-of-the-art labeling for the amine fluorescent derivatization vs.
rational design for the engineering of the targeted fluorogenic reagent 1. LG
denotes leaving group.
A myriad of fluorescent derivatization reagents has been
synthesized; many of them being employed for quantitative
purposes in analytical chemistry, generally for chromatography-
based separations where exquisite sensitivity is required [7–10].
The derivatization and sensing of amines have been the subject of
intense research as this functional group is found in many

2
Dyes and Pigments
biomolecules including amino acids, proteins and nucleic acids
11–17]. A wide variety of reagents for amine derivatization was
reported, mainly based on the classical families of dyes (coumarin
18–20], Bodipy [21–23], fluorescein [24,25], rhodamine [26],
cyanine [27]) bearing an amine-reactive function such as
succinimyl and sulfosuccinimidyl esters (Scheme 1). To a lower
extent, carbonyl azides, isothiocyanates, sulfonylchlorides and
tetrafluorophenyl esters were proposed as an alternative amine-
reactive group [7].
against several cancer cell lines [47,48]. Interestingly, these
compounds of the general formula PyXa, showed a bright fuchsia
color in contrast to their starting material with a pseudo benzyl
bromide 1 (BrXa), which presents the dramatic advantage of being
completely colorless (Scheme 2). Noticeably, the Bischler-type
reaction employed for the synthesis of PyXa proceeds only with
primary amines, since secondary amines afford the amino-
[
[
substituted compounds NR
2
2
X , with no color and fluorescence
detected.
A major drawback of this technique is the fluorescence cross-
contamination due to the large excess of the derivatization reagent
used to drive the reaction kinetics to completion. Classically, this
excess must either be chromatographically separated or removed
by some pre-treatment procedures, as the intrinsic fluorescence of
the reagent will interfere with both absorption and emission of the
derivatized analyte and may contaminate the resulting fluorescent
signal.
Prompted by these findings, we decided to further explore the
potential of the above-mentioned scaffold as a fluorescent
derivatization reagent for the colorimetric/fluorometric detection
of primary amines. The chromogenic and fluorogenic sensing
method would rely on the in-situ formation of an expanded pyrrole
ring, thus extending the conjugation and concomitantly
introducing a push–pull relationship between the electron-
donating nitrogen of the sensed amine and the electron-
withdrawing nitro group (Scheme 2). Therefore, the imposed
structural changes are expected to shift the absorption maximum
towards the red region of the electromagnetic spectrum, rendering
BrXa an ideal candidate for amine derivatization. Additionally,
the architecture obtained with a Donor-π-Acceptor (D-π-A)
relationship should allow a larger dipole moment to be established
in the excited state, which should be reflected in an enhanced
sensitivity with the increasing polarity, resulting in a λ-shift to the
lower energetic wavelength. This operating redshift should greatly
facilitate amine detection by avoiding potential fluorescent cross-
contamination from the extracts.
To overcome this, the ideal solution would be a “fluorogenic”
reagent, viz. which fluoresces only after the derivatization reaction,
thus avoiding both cross-contamination during the spectroscopic
measurements and the implementation of extensive purification
processes [28–33]. In that respect, innovative reagents such as
quinolizinium [34], thiazolo-isoquinoline [35], DOOB – 2,2-
diphenyl-1-oxa-3-oxonia-2-boratanaphthalene [36], catechol [37],
stilbene trifluoroacetate [38], and ylidenemalononitrile enamines
[
39] were alternatively developed to sense fluorogenically various
types of amines by derivatization. For that purpose, the most
widely used fluorogenic derivatization reagent is ortho-
pthalaldehyde, which intrinsically exhibits no fluorescence [40].
In the presence of 2-mercaptoethanol, it reacts chemoselectively
with primary amines to afford emissive isoindoles [40–42]. The
requirement for a large excess of this thiol additive combined with
the blue fluorescence of the product, severely limits the usage of
this system; especially for derivatization of amino-based
biomolecules accompanied by strong background fluorescence.
Indeed, besides demonstrating negligible fluorescence in the
emission range of the derivatization reaction product, a
fundamental property of a successful reagent should be its ability
to emit an intense signal in the red or NIR region to avoid the auto-
fluorescence from biological media. This could be achieved either
through the significant rearrangement of the reagent scaffold or via
its extension with a conjugate π-system [43]. It is expressly on this
precise point of “fluorogenicity” that our research program has
focused [44–46].
Herein, we report the synthesis of a novel xanthene-based
reagent BrXa for the fluorogenic derivatization of primary amines.
The implementation of this derivatization reaction will be
described, as well as the photophysics of the product i.e. the newly
formed fluorescent pyrroloxanthone. Its application to the
derivatization of cyclopropylamine by the development of an LC
analytical method will be provided. Correlation between the
establishment of a push–pull relationship into the derivatized
amine scaffold and the resulting environment sensitivity will be
further evidenced by employing quantum mechanics calculations.
2
. Experimental section
2
.1. Material and methods
All commercially available chemicals and solvents were
purchased from Alfa Aesar and used as received without any
further purification. Melting points were determined on a Büchi
1
13
apparatus and were uncorrected. All NMR spectra ( H, C, 2D)
were recorded on 200, 400 or 600 MHz Bruker spectrometers
respectively AC, Avance™ DRX and III instruments (Bruker
1
BioSpin GmbH – Rheinstetten, Germany). H-NMR (200, 400 and
1
3
1
6
00 MHz) and C{ H}NMR (50, 101 and 151 MHz, recorded
with complete proton decoupling) spectra were obtained with
samples dissolved in CDCl or DMSO‐d with the residual solvent
signals used as internal references: 7.26 ppm for CHCl , and 2.50
H)S(O) regarding H-NMR experiments; 77.2
3
6
3
1
ppm for (CD
ppm for CDCl
NMR experiments [49,50]. Chemical shifts (δ) are given in ppm
3
)(CD
2
1
3
3
and 39.4 ppm for (CD ) S(O) concerning C-
3 2
1
13
to the nearest 0.01 ( H) or 0.1 ppm ( C). The coupling constants
J) are given in Hertz (Hz). The signals are reported as follows: (s
singlet, d = doublet, t = triplet, m = multiplet, br = broad).
Assignments of H and C-NMR signals were unambiguously
achieved with the help of D/H exchange and 2D techniques:
COSY, NOESY, HMQC, and HMBC experiments. Systematic
xanthene nomenclature is used below for the assignment of each
(
=
Scheme 2. Fluorogenic sensing using chemoselective reactivity of the
derivatization reagent 1 towards primary amines. Donor and acceptor groups
involved in the push–pull relationship are depicted in blue and red,
respectively.
1
13
Previously, we reported the synthesis and biological activities
of a series of pyrroloxanthones exhibiting substantial cytotoxicity

3
+
spectrum. Flash chromatography was performed on Merck silica
gel (40–63 μm) with the indicated solvent system using gradients
of increasing polarity in most cases (Merck KGaA – Darmstadt,
Germany) [51]. The reactions were monitored by analytical thin-
layer chromatography (Merck pre-coated silica gel 60 F254 TLC
plates, 0.25-mm layer thickness). Compounds were visualized on
TLC plates by both UV radiation (254 and 365 nm) and spraying
155.9 (C-1’), 157.9 (C-2), 165.6 (CO). MS (ESI , MeOH): m/z:
+
+
257.1 [M+H] ; HRMS (ESI ): m/z calcd for C16
H
17
O
3
: 257.1172
+
[M+H] ; found 257.1177.
2
.4. Ethyl 2-(3-methyl-4-nitrophenoxy)benzoate (6b)
To a suspension of ester 5 (39.46 g, 0.15 mol) in acetic
anhydride (93.4 ml, 0.99 mol), previously cooled down to 0 °C,
was added dropwise a solution of fuming HNO (6.7 ml, 0.16 mol)
in acetic anhydride (23.3 ml, 0.25 mol). The resulting mixture was
stirred at rt for 24 h. After completion of the reaction, the mixture
was poured into ice-water bath, basified with 15% NaOH aq.
(3 x 30 mL). The
SO and
evaporated to dryness. The residue was purified by column
chromatography on silica gel, using petroleum ether/EtOAc
30:1), to afford 6a as the major regioisomer (29.28 g, 63 %) and
b (9.76 g, 21 %) as the minor one. Ethyl 2-(5-methyl-2-
4
with a staining agent (vanillin, PMA, KMnO or ninhydrin)
3
followed by subsequent warming with a heat gun. All solvents for
absorption and fluorescence experiments were of spectroscopic
grade. Absorption spectra were recorded on a Cary 100 Bio UV–
®
Vis spectrophotometer (Varian/Agilent) using Suprasil quartz
solution (pH ~ 9) and extracted with CH
2
Cl
2
cuvettes with 1-cm path length. Stock solutions of investigated
dyes were prepared using dimethylformamide. The samples used
for spectroscopic measurements contained ≈ 0.2% v/v of solvents
of the stock solution. Fluorescence spectra were recorded on a
FluoroMax 4.0 spectrofluorometer (Jobin Yvon, Horiba) with a
thermostatically controlled cell compartment at 20 ± 0.5 °C with
slits open to 2 nm and were corrected for Raman scattering, lamp
fluctuations and instrumental wavelength-dependent bias.
Excitation wavelengths were set at the absorption maxima except
when mentioned in the corresponding experiments. A Waters
Acquity UPLC system (Waters Corporation – Manchester, UK),
combined organic layers were dried over anhydrous Νa
2
4
(
6
1
nitrophenoxy)benzoate 6a: oil. H-NMR (400 MHz, CDCl
3
): δ
), 4.22 (q, J =
3
), 6.58 (s, 1H, H-6΄), 6.95 (d, J = 8.3 Hz, 1H,
1
7
.16 (t, J = 7.1 Hz, 3H, CH
.1 Hz, 2H, CH CH
2 3 3
CH ), 2.30 (s, 3H, CH
2
H-4΄), 7.11 (d, J = 8.2 Hz, 1H, H-3), 7.33 (td, J = 8.2, 2.1 Hz, 1H,
H-5), 7.58 (td, J = 8.2, 2.1 Hz, 1H, H-4), 7.92 (d, J = 8.3 Hz, 1H,
H-3΄), 8.03 (dd, J = 8.2, 2.1 Hz, 1H, H-6). C-NMR (50 MHz,
13
comprising
a solvent manager module connected to an
CDCl
3
): δ 13.8 (CH
2 3 3 2 3
CH ), 21.6 (CH ), 61.2 (CH CH ), 118.5 (C-
autosampler, was employed for the analytical chromatography.
Spectrophotometric detection was performed using an Acquity
PDA detector. Chromatographic separation was achieved on a C18
BEH column (Waters Acquity, 50 mm × 2.1 mm, 1.7 m). Mass
spectra were recorded on a hybrid LTQ™ Orbitrap Discovery XL
instrument (Thermo Fisher Scientific – Bremen, Germany),
coupled to an Accela HPLC system (Thermo Fisher Scientific)
equipped with a binary pump, an autosampler, and Xcalibur 2.1 as
a software.
6
3
1
΄), 122.1 (C-3), 123.1 (C-4΄), 124.0 (C-1), 125.2 (C-5), 125.8 (C-
΄), 132.3 (C-6), 133.9 (C-4), 137.8 (C-2΄), 145.9 (C-5΄), 151.7 (C-
΄), 153.8 (C-2), 164.9 (CΟ). 6b: white solid, m.p. = 63–64 °C
1
(
Et
Hz, 3H, CH
CH CH ), 6.75 (d, J = 8.2 Hz, 1H, H-6΄), 6.8 (s, 1H, H-2΄), 7.15
d, J = 8.3 Hz, 1H, H-3), 7.36 (t, J = 8.3 Hz, 1H, H-5), 7.62 (t, J =
2
O/n-hexane). H-NMR (400 MHz, CDCl
3
): δ 1.18 (t, J = 7.3
2
CH
3
), 2.56 (s, 3H, CH
3
), 4.20 (q, J = 7.3 Hz, 2H,
2
3
(
8
.3 Hz, 1H, H-4), 8.03 (d, J = 8.3 Hz, 1H, H-6), 8.05 (d, J = 8.2
13
Hz, 1H, H-5΄). C-NMR (50 MHz, CDCl
CH ), 61.2 (CH CH ), 113.9 (C-6΄), 119.4 (C-2΄), 123.1 (C-3),
24.5 (C-1), 125.7 (C-5), 127.4 (C-5΄), 132.3 (C-6), 134.1 (C-4),
137.0 (C-3΄), 143.1 (C-4΄), 153.4 (C-2), 162.0 (C-1΄), 164.7 (CΟ).
3 2 3
): δ 14.0 (CH CH ), 21.5
(
1
3
2
3
2
.2. Ethyl 2-iodobenzoate (3)
A suspension of 2-iodobenzoic acid 2 (50 g, 0.20 mol) and
+
+
+
conc. H
2
SO
4
(4.5 mL) in ethanol (200 mL) was refluxed for 24 h.
MS (ESI , MeOH): m/z: 302.1 [M+H] ; HRMS (ESI ): m/z calcd
for C16
+
The progress of the reaction was monitored using TLC. After
completion of the reaction, volatiles were removed under reduced
pressure and the resulting mixture was diluted with
H16NO
5
: 302.1023 [M+H] ; found 302.1019.
2
.5. 2-(3-Methyl-4-nitrophenoxy)benzoic acid (7)
dichloromethane (500 mL) and neutralized with 10% Na
mL). The organic layer was separated using a separating funnel,
then dried over Νa SO and evaporated to dryness to afford 52 g
94 %) of the ester 3, which was used without further purification
2 3
CO (50
To a solution of ester 6b (9.4 g, 30 mmol) in ethanol at rt, a cold
5% NaOH aq. solution (10 mL, 100 mmol) was added dropwise.
The reaction mixture was stirred at rt for 1.5 h, before being poured
into water and acidified with 6M HCl aq. solution (pH ~ 2). The
resulting white precipitate was filtered and dried over phosphorus
pentoxide (glass dessicator) to afford 7 (8.13 g, 96 %), which was
1
2
4
(
+
+
for the next reaction. MS (ESI , MeOH): m/z: 277.0 [M+H] .
2
.3. Ethyl 2-(3-methylphenoxy)benzoate (5)
used for the next step without any further purification. M.p. = 144–
1
A suspension of m-cresol 4 (35.55 g, 0.33 mol), 3 (45.43 g, 0.17
145 °C (EtOAc). H-NMR (400 MHz, CDCl
3
): δ 2.43 (s, 3H, CH
3
),
mol), K
2
CO
3
(45.33 g, 0.33 mol) and CuCl (6.45 g, 0.028 mol) in
6.90 (s, 1H, H-2΄), 6.94 (d, J = 8.2 Hz, 1H, H-6΄), 7.15 (d, J = 8.1
Hz, 1H, H-3), 7.26 (t, J = 8.1 Hz, 1H, H-5), 7.57 (td, J = 8.1, 1.9
Hz, H-4), 8.04 (d, J = 8.2 Hz, 1H, H-5΄), 8.22 (dd, J = 8.1, 1.9 Hz,
dry pyridine (250 mL) was heated at 120 °C for 24 h under argon
atmosphere. After completion of the reaction as indicated by TLC,
volatiles were removed under reduced pressure and the residue
1
3
3 3
H-6). C-NMR (50 MHz, CDCl ): δ 21.7 (CH ), 120.2 (C-6΄),
was diluted in CH
subsequently with 3M aq. HCl solution (3 x 50 mL), water (3 x 50
mL), brine (3 x 50 mL), dried over Νa SO , and evaporated to
2
Cl
2
(100 mL). The organic layer was washed
120.7 (C-2’), 121.4 (C-3), 124.7 (C-1), 124.8 (C-5), 126.1 (C-5΄),
133.3 (C-6), 135.0 (C-4), 138.6 (C-3΄), 146.6 (C-4΄), 150.1 (C-2),
+
+
2
4
155.6 (C-1’), 168.9 (CO). MS (ESI , MeOH): m/z: 274.1 [M+H] ;
+
+
dryness. Flash chromatography on silica gel using a mixture of
HRMS (ESI ): m/z calcd for C14
H12NO
5
: 274.0710 [M+H] ; found
cyclohexane/EtOAc (20:1, v/v) afforded 5 as an oil (40.04 g,
274.0713.
1
9
CH
6
(
2 %). H-NMR (400 MHz, CDCl
3
): δ 1.21 (t, J = 7.1 Hz, 3H,
), 4.25 (q, J = 7.1 Hz, 2H, CH CH ),
.72–6.75 (m, 2H, H-2΄, H-6΄), 6.86 (d, J = 7.6 Hz, 1H, Η-4΄), 6.98
dd, J = 8.3, 0.8 Hz, 1H, Η-3), 7.16 (m, 2H, H-5΄, H-5), 7.43 (td, J
2
.6. 1-Methyl-2-nitro-9H-xanthen-9-one (8)
3
CH
2
), 2.29 (s, 3H, CH
3
2
3
A suspension of acid 7 (6.20 g, 0.023 mol) in polyphosphoric
acid (15 mL) was stirred at 100 °C for 2 h. After cooling, the
mixture was poured into crushed-ice bath. The resulting precipitate
was filtered, washed with 10% Na
air-dried. Flash chromatography on silica gel, using a mixture of
cyclohexane/CH Cl (12:1 → 5:1, v/v) afforded 8 as a white solid
1
3
=
8.3, 1.8 Hz, H-4), 7.9 (dd, J = 7.8, 1.8 Hz, 1H, H-6). C-NMR
): δ 14.1 (CH CH ), 21.4 (CH ), 61.0 (CH CH ),
(50 MHz, CDCl
3
3
2
3
3
2
2 3
CO solution and water, and
1
1
14.9 (C-6’), 118.5 (C-2’), 121.2 (C-3), 123.6 (C-5), 123.7 (C-4’),
23.9 (C-1), 129.4 (C-5’), 131.8 (C-6), 133.5 (C-4), 139.7 (C-3’),
2
2

4
Dyes and Pigments
(
5.4 g, 92 %). M.p. = 206–207 °C (EtOH) (lit. 205–207 °C) [47].
(151 MHz, CDCl
29.3 (dodecyl), 29.5 (dodecyl), 29.5 (dodecyl), 29.7 (2 x dodecyl),
29.7 (dodecyl), 31.0 (dodecyl), 32.0 (dodecyl), 50.5 (N-CH ), 98.2
3 3
): δ 14.23 (CH ), 22.8 (dodecyl), 26.8 (dodecyl),
1
H-NMR (400 MHz, DMSO-d
7
7
6
): δ 2.85 (s, 3H, CH
.9, 0.8 Hz, 1H, H-7), 7.64 (d, J = 9.0 Hz, 1H, H-4), 7.68 (d, J =
.9 Hz, 1H, H-5), 7.88 (td, J = 7.9 Hz, 0.8 Hz, 1H, H-6), 8.13 (dd,
3
), 7.50 (td, J =
2
(C-5), 115.3 (C-2a), 116.5 (C-2), 118.5 (C-10a), 118.8 (C-7),
1
3
1
J = 7.9, 0.8 Hz, 1H, H-8), 8.23 (d, J = 9.0 Hz, 1H, H-3). C-NMR
50 MHz, DMSO-d ): δ 16.9 (CH ), 118.0 (C-5), 118.1 (C-4),
20.1 (C-9a), 122.2 (C-8a), 125.2 (C-7), 126.4 (C-8), 129.6 (C-3),
35.4 (C-1), 136.0 (C-6), 147.6 (C-2), 154.5 (C-10a), 158.5 (C-
119.4 (C-10b), 120.6 (C-2a ), 120.6 (C-10), 125.4 (C-9), 128.1 (C-
(
6
3
8), 130.2 (C-4), 132.1 (C-3), 153.2 (C-6a), 157.7 (C-5a). MS
+
+
+
1
1
4
(ESI , MeOH): m/z: 421.2 [M+H] ; HRMS (ESI ): m/z calcd for
+
C
26
H
33
N
2
O
3
: 421.2486 [M+H] ; found 421.2490.
+
+
a), 177.7 (CO). MS (ESI , MeOH): m/z: 256.1 [M+H] ; HRMS
2
.10. 1-cyclopropyl-3-nitro-1H-chromeno[4,3,2-cd]isoindole
+
+
4
(ESI ): m/z calcd for C14H10NO : 256.0604 [M+H] ; found
(11)
2
56.0609.
This compound 11 was synthesized following a similar
2
.7. 1-(bromomethyl)-2-nitro-9H-xanthen-9-one (1 – BrXa)
procedure as described for 9, using cyclopropylamine as the proper
1
A suspension of xanthone 8 (2.86 g, 11 mmol), NBS (1.96 g,
amine of the reaction. Yield: 93 %. H-NMR (400 MHz, CDCl
3
):
1
1 mmol) and BPO (dibenzoyl peroxide, 286 mg, 1.18 mmol) in
δ 1.31–1.41 (m, 4H, CH ), 3.80 (m, 1H, CH-cyclopropyl), 6.18 (d,
2
CCl
fluorescent lamp – CFL 150 W). After completion of the reaction,
the mixture was washed with 5% NaHCO aq. solution (3 x 20
aq. solution (3 x 20 mL), water (3 x 50 mL),
SO and evaporated to dryness. Flash
mixture of
(10:1 → 5:1, v/v) as the eluent afforded 1
4
(100 mL) was refluxed for 8 h under UV irradiation (compact
J = 8.3 Hz, 1H, H-5), 7.20–7.33 (m, 3H, H-7, H-9, H-8), 7.54 (s,
1H, H-2), 8.04 (dd, J = 7.4, 1.8 Hz, 1H, H-10), 8.15 (d, J = 8.3 Hz,
1H, H-4). C-NMR (101 MHz, CDCl ): δ 8.9 (CH ), 31.3 (CH-
3 2
1
3
3
mL), 5% Na
2
S
2
O
4
cyclopropyl), 98.1 (C-5), 114.7 (C-2a), 115.7 (C-2), 118.3 (C-
1
dried over Νa
2
4
,
10a), 118.3 (C-7), 120.1 (C-10b), 121.5 (C-2a ), 121.8 (C-10),
chromatography on silica gel using
cyclohexane/CH Cl
3.01 g, 82 %). M.p. = 179–181 °C (EtOH) [47]. H-NMR (400
a
125.0 (C-9), 128.0 (C-8), 130.5 (C-4), 131.9 (C-3), 153.0 (C-6a),
+
+
2
2
157.7 (C-5a). MS (ESI , MeOH): m/z: 293.1 [M+H] ; HRMS
1
+
+
(
(ESI ): m/z calcd for C17
H
13
N
2
O
3
: 293.0921 [M+H] ; found
MHz, CDCl
3
): δ 5.29 (s, 2H, CH
2
), 7.41–7.48 (m, 2H, H-7, H-5),
293.0923.
7
8
.53 (d, J = 8.4 Hz, 1H, H-4), 7.77 (td, J = 8.7, 1.7 Hz, 1H, H-6),
.17 (d, J = 8.4 Hz, 1H, H-3), 8.29 (dd, J = 8.7, 1.7 Hz, 1H, H-8).
C-NMR (50 MHz, CDCl ): δ 23.3 (CH Br), 117.6 (C-5), 119.0
3 2
2
.11. 1-((diethylamino)methyl)-2-nitro-9H-xanthen-9-one (12)
1
3
This compound 12 was synthesized following a similar
(
(
(
C-9a), 120.3 (C-4), 122.8 (C-8a), 125.1 (C-7), 127.2 (C-8), 129.8
C-3), 135.3 (C-6), 136.2 (C-1), 146.5 (C-2), 155.5 (C-10a), 159.6
C-4a), 177.8 (CO). MS (ESI , MeOH): m/z: 334.0, 336.0 [M+H] ;
procedure as described for 9, using diethylamine as the proper
1
amine of the reaction. Yield: 89 %. H-NMR (600 MHz, CDCl
δ 0.90 (t, J = 7.1 Hz, 6H, CH CH ), 2.47 (q, J = 7.1 Hz, 4H,
CH CH ), 4.70 (s, 2H, CH ), 7.41 (dd, J = 8.1, 1.1 Hz, 1H, H-5),
3
):
+
+
2
3
+
+
9 4
HRMS (ESI ): m/z calcd for C14H BrNO : 333.9709 [M+H] ;
found 333.9711.
2
3
2
7
7
.44–7.48 (m, 2H, H-7, H-4), 7.74 (dd, J = 8.1, 1.1 Hz, 1H, H-6),
.82 (d, J = 9.0 Hz, 1H, H-3), 8.26 (dd, J = 8.0, 1.1 Hz, 1H, H-8).
2
.8. 1-butyl-3-nitro-1H-chromeno[4,3,2-cd]isoindole (9)
1
3
C-NMR (151 MHz, CDCl
3 2 3 2 3
): δ 11.2 (CH CH ), 46.8 (CH CH ),
To a suspension of compound 1 (35 mg, 0.10 mmol) in dry
50.3 (CH ), 117.6 (C-5), 117.9 (C-9a), 121.0 (C-4), 122.8 (C-8a),
2
MeOH (2 mL), n-butylamine (20 eq.) was added. The resulting
mixture was stirred at rt for 2 h and the progress of the reaction
was monitored by TLC. After the completion of the reaction, the
precipitate was filtered off and then recrystallized with
124.8 (C-7), 127.2 (C-8), 129.2 (C-3), 135.4 (C-6), 141.3 (C-1),
+
148.5 (C-2), 154.9 (C-10a), 157.8 (C-4a), 178.5 (CO). MS (ESI ,
+
+
MeOH): m/z: 327.1 [M+H] ; HRMS (ESI ): m/z calcd for
+
C
18
H
19
N
2
O
4
: 327.1339 [M+H] ; found 327.1334.
CH
(
3
2
Cl
CH Cl
H, CH
CH
Hz, 1H, H-5), 7.14–7.25 (m, 3H, H-7, H-9, H-8), 7.40 (s, 1H, H-
), 7.48 (dd, J = 7.6, 1.5 Hz, 1H, H-10), 8.07 (d, J = 8.4 Hz, 1H,
2
/Et
/Et
), 1.35–1.49 (m, 2H, CH
CH ), 4.40 (d, J = 7.4 Hz, 2H, N-CH
2
O to afford 9 (29 mg, 94 %). M.p. 198–199 °C
2
.12. 3-nitro-1-(2-(pyrrolidin-1-yl)ethyl)-1H-chromeno[4,3,2-
1
2
2
2
O). H-NMR (400 MHz, CDCl
3
): δ 0.94 (t, J = 7.3 Hz,
), 1.85–1.97 (m, 2H,
), 6.11 (d, J = 8.4
cd]isoindole (13)
3
2
CH CH
2
3
CH
2
2
3
2
To a suspension of 1 (70 mg, 0.20 mmol) in abs. EtOH (3 mL),
was added 2-propylaminoethylamine (20 eq.). The reaction
mixture was stirred at rt for 2 h, before being reduced under
vacuum, taken over with CH
Na SO , and filtered. The volatiles were removed in vacuo and the
resulting crude was purified by flash chromatography on silica gel
eluted with CH Cl /MeOH (97:3, v/v) to afford 13 as an oil (66
mg, 94 %) [48]. H-NMR (400 MHz, CDCl ): δ 1.76 (br. s, 4H,
(CH CH ) N-), 2.56 (br. s, 4H, (CH CH ) N), 2.91 (t, J = 7.5 Hz,
2H, (CH CH ) NCH ), 4.45 (t,
2
1
3
H-4). C-NMR (101 MHz, CDCl
CH CH CH ), 32.8 (CH CH CH ), 50.0 (N-CH
15.0 (C-2a), 116.4 (C-2), 118.2 (C-10a), 118.6 (C-7), 119.3 (C-
3
3
): δ 13.7 (CH ), 19.9
2 2 2
Cl , washed with H O, dried over
(
2
2
3
2
2
3
2
), 98.1 (C-5),
2
4
1
1
1
0b), 120.5 (C-2a ), 120.5 (C-10), 125.3 (C-9), 128.0 (C-8), 130.1
2
2
+
1
(C-4), 131.7 (C-3), 152.9 (C-6a), 157.6 (C-5a). MS (ESI , MeOH):
3
+
+
m/z: 309.1 [M+H] ; HRMS (ESI ): m/z calcd for C18
H
17
N
2
O
3
:
2
2 2
2
2 2
+
3
09.1234 [M+H] ; found 309.1233.
.9. 1-dodecyl-3-nitro-1H-chromeno[4,3,2-cd]isoindole (10)
This compound 10 was synthesized following a similar
J
=
7.5 Hz, 2H,
2
2 2
2
(CH
2 2 2 2 2
CH ) NCH CH ), 6.00 (d, J = 8.3 Hz, 1H, H-5), 7.15–7.17
2
(m, 3H, H-7, H-9, H-8), 7.32 (s, 1H, H-2), 7.48 (d, J = 7.3 Hz, 1H,
1
3
H-10), 7.97 (d, J = 8.3 Hz, 1H, H-4). C-NMR (50 MHz, CDCl
δ 23.6 ((CH CH NCH CH ), 49.4 ((CH CH NCH CH ), 54.5
((CH CH NCH CH ), 56.3 ((CH CH NCH CH ), 98.0 (C-5),
115.0 (C-2a), 116.4 (C-2), 117.9 (C-10a), 118.5 (C-7), 119.5 (C-
10b), 120.1 (C-2a ), 120.5 (C-10), 125.3 (C-9), 128.0 (C-8), 130.1
(C-4), 131.6 (C-3), 152.8 (C-6a), 157.5 (C-5a). MS (ESI , MeOH):
m/z: 350.1 [M+H] ; HRMS (ESI ): m/z calcd for C20
350.1499 [M+H] ; found 350.1496.
3
):
procedure as described for 9, using dodecacylamine as the proper
amine of the reaction. Yield: 95 %. M.p. 113–114 °C
2
2
)
2
2
2
2
2
)
2
2
2
2
2
)
2
2
2
2
2
)
2
2
2
1
(
CH
2
Cl
H, CH
.44 (p, J = 7.3 Hz, 2H, dodecyl), 1.56–1.66 (m, 2H, dodecyl),
.94–2.01 (m, 2H, dodecyl), 4.44 (t, J = 7.4 Hz, 2H, N-CH ), 6.19
2
/Et
2
O). H-NMR (600 MHz, CDCl
3
): δ 0.87 (t, J = 7.3 Hz,
1
3
1
1
3
), 1.25–1.30 (m, 12H, dodecyl), 1.35 (m, 2H, dodecyl),
+
+
+
H
20
N
3
O
3
:
2
+
(dd, J = 8.6, 1.8 Hz, 1H, H-5), 7.19–7.26 (m, 2H, H-7, H-9), 7.29
(
1
dd, J = 8.1, 1.7 Hz, 1H, H-8), 7.47 (s, 1H, H-2), 7.53 (dd, J = 7.7,
.8 Hz, 1H, H-10), 8.15 (dd, J = 8.6, 1.7 Hz, 1H, H-4). C-NMR
1
3

5
3
. Results and discussion
R
Br
O
N
O
R NH2
(1 eq.)
3
.1. Preparation of the derivatization reagent
Our efforts were initially focused on the development of a
NO2
NO2
DMF, rt
O
reliable procedure for the scalable synthesis of the proposed
reagent BrXa. To overcome previous difficulties such as the
number of steps and overall yield [47,48], we considered a more
convergent approach based on a copper-catalyzed coupling
reaction (Scheme 3).
1
. BrXa
9. BuPyXa: R =
10. DodecPyXa: R =
10
1
1. CypPyXa: R =
Scheme 4. Implementation of the derivatization reaction for primary amines
including aliphatic and saturated cyclic R-NH derivatives.
Commercially available 2-iodobenzoic acid 2 was used as a
starting material which, upon esterification in ethanol, yielded
almost quantitatively the corresponding ethyl ester 3. Ullmann
condensation of m-cresol 4 with the aryl iodide 3 efficiently
afforded the coupling product 5. Nitric acid treatment in acetic
anhydride of the biaryl ether 5, led to a mixture of nitro-isomeric
compounds 6a and 6b, separable by silica gel chromatography and
identifiable by 2D-NMR. More specifically, structural
2
Using DMF as a solvent at room temperature, first attempts on
selected primary amines were carried out in batch. Derivatization
reaction proceeded smoothly on aliphatic amines with side chains
of different sizes (butyl and dodecyl) to quantitatively furnish the
corresponding pyrroloxanthones, 9 BuPyXa and 10 DodecPyXa,
respectively (Scheme 4). Several observations could be made: i) a
clear magenta color appears along the derivatization process,
offering an easy readout ii) a distinct vermilion fluorescence
emerges upon excitation with any type of laser ranging from blue
to yellow, allowing the sensitivity of the reaction to be
substantially increased. As a consequence, these properties make
the detection chromogenic and fluorogenic. Moreover, the
simplicity of the method is clearly an asset, since there is no
requirement of anhydrous conditions, temperature or precise order
in the addition of the reagent.
3
discrimination resulted from the observation of J couplings
between the methyl group and two aromatic CH carbons in ortho
in the case of 6a, against a single correlation for 6b. Subsequently,
saponification under mild conditions of the ester 6b provided the
carboxylic acid 7, which was ring-closed upon treatment with PPA
to give the xanthone 8. The bromomethyl target 1 was finally
obtained from 8 upon free-radical bromination with NBS under
UV irradiation.
CO2Et
i) conc.
H2SO4
CO2H
I
CO Et
OH
O
2
ii) CuCl, K2CO3
Pyr, reflux
EtOH, reflux
I
2
3
4
5
CO2Et
CO2Et
iv) 40 %
aq. NaOH
iii) fuming
HNO3
O
O
EtOH, rt
Ac2O, 24 h, 0 °C
NO2
O
O2N
6
b
6a
Br
CO2H
O
vi) NBS, BPO
O
v) PPA
NO2 60W-UV lamp
CCl4, 8 h
NO2
1
00 °C
O
NO2
O
7
8
1: BrXa
Scheme 3. Synthetic pathway leading to the derivatization reagent BrXa.
It is worth mentioning that the overall yield of the newly
developed synthesis is >40 %, i.e., thrice higher than the precedent
approach. In a nutshell, an original and straightforward synthetic
approach has been developed for the preparation of the
derivatization reagent BrXa, using practical, inexpensive and
scalable procedures with purification processes remaining simple.
3
.2. Derivatization reaction
To develop suitable conditions for this derivatization in order
to make it an effective detection tool, a certain set of specifications
have to be met. First of all, the reaction must be performed at room
temperature to be truly practical. Another prerequisite is to find a
solvent in which both the derivatization reagent BrXa and the
targeted amines are fully soluble and also remains reactive. Protic
solvents such as methanol and ethanol – typical for this kind of
reaction – revealed to be not enough solvating in the concentration
range routinely used for spectroscopic measurements. The chosen
Scheme 5. Negative (top) and positive (bottom) control experiments performed
for the engineering of the derivatization reaction.
Then, to establish the selectivity towards the order (1°, 2° & 3°)
of the amines to be derivatized, different types of amines were
chosen. Triethylamine was first screened as a negative control, and
as expected, no reaction was observed with this tertiary amine
N
solvent has to be polar to allow the first step (S 2) to be conducted,
but the pseudo benzylic bromide – known to be reactive – must be
stable during the reaction time. Dimethylformamide proved to be
the one that ticks all the boxes.
(Scheme 5). This chemical inertia bodes well for the development
of analytical methods with amine-based buffers. Thereafter, the
reactivity of secondary amines was tested using diethylamine. As
suspected, the first S
N
2-type step worked normally to provide 12,
but no further cyclization could take place due to the formation of

6
Dyes and Pigments
an unstable ammonium intermediate (Scheme S1).
Advantageously, NEt Xa has an electronic π-scaffold similar to
2
that of the reagent, meaning therefore that it will be both colorless
and devoid of fluorescence. Thus, derivatized secondary amines
should not cross-contaminate the colorimetric detection of the
amines of interest during the derivatization of a complex matrix.
Eventually, using 2-(pyrrolidinyl)ethylamine explicitly showed
both the lack of reactivity of tertiary amines and the versatility of
2
primary R-NH for this derivatization procedure. Thence, this test
Fig. 2. UV–Vis spectra of the peaks, respectively assigned to the derivatization
reagent BrXa (top) and the derivatized cyclopropylamine CypNXa (bottom).
Snapshots collected by the PDA detector.
exclusively produced the pyrrolidinyl-pyrroloxanthone 13,
PyrroPyXa. Furthermore, a competition experiment using N-
methylethylenediamine demonstrated the superior reactivity of the
1
° amine at the expense of the 2° one towards BrXa (Fig. S7–9).
Absorbance spectra – illustrated in Fig. 2 – confirmed this
increase of conjugation occurring through the derivatizing
reaction, even in presence of a chromophore-free amine. Indeed,
an intense and broad absorption band centered around 540 nm
clearly appears, as a result of the important electronic conjugation
being established between the pyrrolo donor (D) and nitro acceptor
(A) groups. On another note, even in case of contamination by
traces of reagent, these observables substantiate that no cross-
excitation can take place given the λ-shift of ca. 200 nm offered by
the derivatized product. This bodes well for the photophysical
characterization of the sensing reaction.
3
.3. Chromatographic and mass spectrometry analysis
In order to apply the novel derivatization reagent to
chromatographic analysis, cyclopropylamine was selected as a
model analyte. The derivatization of the saturated cyclic primary
amine with BrXa proceeded smoothly in acetonitrile to yield 11
CypPyXa, as the derivatized product (Scheme 4). Triethylamine
was used as a base to scavenge HBr formed in situ during the
reaction process.
An analytical method was developed to separate the product
from the excess of the derivatization reagent, using H
2
O and
Transferring this liquid chromatographic analysis to an MS
detector provides a more reliable characterization. Both analytes
were precisely identified in the presented MS spectra obtained
from the respective chromatographic peaks (Fig. 3). Typically, the
two molecular ion isotopes observed with similar abundance were
attributed to the presence of a bromine in the reagent structure.
acetonitrile, as solvents A & B, respectively. The total analysis
time, including column equilibration, was 5 min per injection and
the flow rate was set at 0.5 mL/min. The gradient elution program
used was as follows: from 10% to 100% B in 3.0 min => 0.5 min
at 100% B => from 100% to 10% B in 0.2 min => equilibration at
1
0% B for 1.3 min. The injection volume was adjusted to 5 μL and
the column temperature was maintained at 40 °C throughout all
experiments.
Fig. 3. Mass spectrograms of the peaks assigned to the derivatization reagent
(
top) and derivatized cyclopropylamine (bottom). The expected theoretical m/z
+
values for the [M+H] molecular ions of both compounds are respectively:
BrXa (C14 BrN) = 333.971 & 335.969; and CypPyXa (C17 ) =
H
8
O
4
12 2 2
H O N
2
93.092.
3
.4. Photophysical characterization
To study this derivatization reaction in detail from a
photophysical point of view, cyclopropylamine (Cyp-NH ) was
2
selected again, this time for the electron-releasing character of its
saturated three-membered ring. Indeed, this inductive +I effect
should enhance the donor strength in the push–pull relationship
operating in the excited state with the nitro acceptor.
Fig. 1. Representative chromatograms of the HPLC purification process
recorded by the PDA detector set at A) 537 nm and B) 280 nm.
The chromatograms depicted in Fig. 1 demonstrate that a
baseline separation was obtained (Fig. 1B). The derivatized
product is the only one that can be detected in the yellow range
(
Fig. 1A) due to its extended π-conjugation induced by the
formation of an additional ring.

7
spectra displayed a certain solvatochromism (about 35 nm) since
the maxima vary from 510 nm in toluene to 544 nm for DMSO
Table 1 & Fig. 5). This moderate dependency on solvent polarity
(
revealed that charge transfer between the donor and acceptor
groups is already operating in the ground state. This results in a
color change from crimson red to purplish blue, respectively for
apolar and strongly polar media. In protic EtOH, it is noteworthy
that H-bonding with the NO
2
acceptor group induced a
hypsochromic shift of the absorption band.
Fig. 4. Investigation of the colorimetric sensing: spectrophotometric titration
of BrXa with Cyp-NH leading to CypPyXa. Inserts represent the initial (left)
2
and ending (right) solutions.
To check the chromogenic detection, the derivatization reagent
was titrated with this challenging primary amine, and the
formation of the resulting CypPyXa was monitored by UV–Vis
spectroscopy (Fig. 4). For this purpose, BrXa was dissolved in
DMF in the mM range, and changes in absorption were recorded
2
upon the gradual addition of Cyp-NH . Since the xanthone core of
BrXa absorbs at approx. 340 nm, and thus outside the visible
range, the starting solution is completely colorless (insert in Fig.
Fig. 5. Absorption observables of the derivatized adduct CypPyXa, illustrating
solvatochromic characteristics.
4
). As soon as the primary amine is added, an absorption band
emerges in the yellow range (ca. 535 nm), transducing the
appearance of the fuchsia color observed for the reaction mixture.
Once an equivalent is reached, the yellow absorbance of CypPyXa
no longer seems to change, and the solution turns into a persistent
dark magenta (insert in Fig. 4). A similar observation could be
made during the fluorescence titration where the emission signal
does not fluctuate after addition of one equivalent of 1° amine
Molar absorptivity of CypPyXa was determined to be approx.
−1
−1
13,000 M ·cm in DMF, almost twice as high as that of BrXa (
−1
−1
= 7,000 M ·cm ). That observation is consistent with the
extension of the π-system occurring during derivatization due to
the pyrrole ring formation [43]. This augurs well for the
fluorogenic sensing, given that brightness is the product of the
molar extinction coefficient and the quantum yield.
(
Figure S1). Therefore, these results highlight a 1: 1 stoichiometry
for the derivatization reaction, as reflected by the mechanism
hypothesized herein in Scheme 6.
Table 1. Photophysical properties of CypPyXa in various
solvents, representative of a wide range of polarity.
H
[
a]
Br
N
O
O
O
O
R
NO2 SN2
HBr
NO2

max (MeOH) = 13 [c]
[e]

Solvent
E
T
N(30) [b]
R
NH2
[d]
Abs
[f]
[g]
λ
λ
Em
–
(to derivatize)
EtOH
0.65
0.46
0.44
0.39
0.33
0.23
0.21
0.10
528
531
544
535
533
514
514
510
656
654
666
635
631
611
607
584
128
123
122
100
98
1
nucleophilic
addition
CH
3
CN
4
H+
DMSO
DMF
4
±
Br
12
15
22
65
91
H
R
N
R
H
DCE
N
O
HO
EtOAc
THF
97
NO2
NO2
Dehydration
93
H O Br-
+
–
Toluene
74
O
3
a
Scheme 6. Hypothetical mechanism of the derivatization reaction using one
equivalent of primary amine. The push–pull relationship, involving the donor
Reported values are the average of two or more independent and reproducible
measurements, ±1 nm for wavelengths. Excitation wavelength was at the
corresponding absorption maximum. Normalized Reichardt's solvent polarity
index [52]. Molar extinction coefficient in 10 M ·cm was determined in
methanol; relative standard deviations are lower or equal to 5 %. Position of
b
(blue) and acceptor (red) groups, is depicted in green.
c
3
−1
−1
d
To provide further insights into the spectroscopic features of
CypPyXa, absorption measurements were conducted in a set of
e
the absorption maximum in nm. Wavelength of the emission maximum in nm.
f
For convenience, Stokes shifts are expressed in nm for Δλ = λEm − λAbs
solvents of increasing polarity, as transcribed by the Dimroth-
−
1
g
N
(difference in cm ). Fluorescence quantum yields Φ were determined using
an excitation at the corresponding absorption maximum of the reference
standard in the considered solvent: Nile blue perchlorate in EtOH (λEx
321 nm, Φ = 27 %)[53], ±10 % mean standard deviation.
Reichardt E
T
(30) parameter [52]. This empirical scale takes into
account the dielectric constant and the H-bond donating ability of
the solvent. It is normalized from TMS (0) to water (1). Absorption
=

8
Dyes and Pigments
initial BrXa solution proved to be completely “dark” (flat curve
and insert, Fig. 7). This is an essential prerequisite for the
development of a fluorogenic derivatization method. The
2
increasing addition of Cyp-NH made emanate a bright red
emission (insert in Fig. 7) resulting from the formation of
CypPyXa. Plotting the fluorescence intensity as a function of the
incremental concentration allowed to estimate that the sensitivity
limit was located at the nanomolar scale (insert in Fig. 7, Fig. S3).
Such a low level of detection combined with the colorimetric and
fluorogenic character of this simple procedure, constitute as many
assets to promote this derivatization method of primary amines.
To prove the potential applicability of BrXa to the study of
biological samples, additional reactions were conducted with
various thiols. Indeed, this class of compounds is known to include
the strongest nucleophiles in biological media. Control
experiments were performed with 2-mercaptoethanol and a L-
cysteine derivative (Fig. S4–5). As expected, the substitution
product was mainly formed but no color and fluorescence
appearance was detected (Fig. S6–9). This confirms that the
chromogenic and fluorogenic character of this derivatization is
directly related to the sensing of 1° amines. Competition reactions
between thiol vs. amine were also carried out to demonstrate that
only a small excess of BrXa allows the detection of a primary
amine even in the presence of a reactive thiol. Since only the 1°
amine derivatization products generate colored and fluorescent
products, the slight excess of this “completely transparent” reagent
should not interfere with spectrophotometric detection.
Fig. 6. Emission spectra of the derivatized adduct CypPyXa, demonstrating
fluorescence quenching over the increasing polarity.
Fluorescence measurements were performed in the same set of
solvents (Table 1 & Fig. 6). After selective excitation, CypPyXa
exhibited significant positive solvatofluorochromism (~ 70 nm)
with maxima oscillating from 584 to 656 nm and an emission color
varying over yelloworangered (Fig. S2). Mega-Stokes shifts
(>100 nm) were even observed in polar solvents, attesting to the
strong dependance of the excited-state dipole along the increasing
polarity; typical of push–pull fluorophores. In parallel, quantum
yields fluctuate over the entire range, making the dye extremely
bright in toluene (91 %) and strongly quenched in protic medium,
like EtOH (1 %). Such a fluorescence quenching – resulting from
the steady increase of polarity – is characteristic of a substantial
push–pull relationship being established between the pyrrolo
donor (D) and the nitro acceptor (A) groups. In terms of solubility,
reactivity and brightness, DMF appeared to be the best
compromise for this derivatization reaction.
3
.5. Quantum calculations
Gaussian 09 was employed for calculating the theoretical
optical and quantum chemical properties of BrXa and the
derivatized amino product as well as for determining their
geometry in both ground and excited states. To simplify the
calculations, methylamine was chosen as the reacting species
(MePyXa). DFT and TD-DFT were used at the B3LYP/ 6-
3
11+G(d) level of theory for the geometry optimization of both
molecules in their ground and excited states, respectively, verified
by Hessian analysis. Calculations of the electronic absorption
spectra were completed by selecting singlet vertical excitation
energies and their oscillator strengths were determined by TD-
DFT. It was found that the first excited state would fluoresce,
therefore the IROOT=1 keyword was utilized. Fluorescence
spectra were calculated at the single point level of the optimized
geometry in the excited state.
Figure 8 shows the differentiation of the bond lengths upon
excitation. For the reagent, the differentiation is associated with
the benzene ring 3, with the largest elongation and de-conjugation
of the aromatic ring being apparent for the two nitro group oxygens
(
Table S1). Similarly, but to a far lesser extent, the carbonyl
oxygen partially lost its double bond character. MePyXa exhibits
a similar trend, but the main geometric changes are expanded to
rings 2 and 4, in addition to ring 3. As the structure of this
derivative presents more extended π-delocalization and
consequently increased rigidity, it should induce larger Stokes
shifts and enhanced fluorescence quantum efficiency regarding its
emission properties. The nitro group does show an important
geometrical alteration in both cases, but to a lesser extent for BrXa
(Fig. 8). All the dihedral angles of BrXa and MePyXa are >177°,
pointing out high aromaticity, both in the ground and excited
states.
Fig. 7. Fluorogenic sensing of Cyp-NH
of CypPyXa by fluorescence titration of BrXa. Inserts represent the starting
left) and final (right) solutions, respectively non-emissive and strongly red
fluorescent.
2
: determination of the sensitivity limit
(
Lastly, the sensitivity limit was determined in order to define
the scope of applicability of this derivatization process. By
exciting at the absorption maximum of CypPyXa (535 nm), the

9
fluorophores into D-π-D, known to exhibit a strongly quenched
and blue-shifted emission.
A)
B)
Fig. 8. Bond elongation features upon excitation for BrXa (top) and MePyXa,
the methylamine-derivatized product (bottom). Only bond length differences
larger than 0.005 Å were reported.
The HOMO and LUMO orbitals of MePyXa are delocalized,
highlighting the aromatic character of the substance. Apparently,
the LUMO orbital is more localized on certain atoms compared to
the more widespread HOMO. This observation is also reflected in
the distribution of the Mulliken charges (Table S2). Since the
calculated HOMO–LUMO energy difference turns out to be small
Fig. 9. HOMO (A) and LUMO (B) of the derivatization product of
methylamine.
(
2.76 eV), this indicates that the derivative is highly conjugated.
Excitation occurs from HOMO (Fig. 9A) towards LUMO (Fig.
B), which corresponds to λ = 490 nm affording an oscillator
9
strength of f = 0.31. A second, less intense excitation (f = 0.13) is
predicted from HOMO to the second, lower lying LUMO with an
energy gap of 3.46 eV, which corresponds to λ = 358 nm. Finally,
the dipole moment lies on the long axis of the compound showing
a total magnitude of 9.6 D. The emission from the geometry-
optimized excitation level between HOMO and LUMO occurs
with an orbital state difference of 2.46 eV corresponding to an
emission λ = 565 nm with an oscillator strength of f = 0.21 (Fig.
1
0). Smaller oscillator strength transitions were observed at 359
nm (f = 0.146), 328 nm (f = 0.167) and 316 nm (f = 0.04). The
dipole moment was calculated mainly along the x axis, affording
a value of 8.8 D, attesting to a significant charge separation in the
excited state. Thus, the derivatization product becomes noticeably
polarized upon photon absorption, which is characteristic of
molecules interacting efficiently with light.
On the other hand, the direction and magnitude of the dipole
moment vector reveal that the nitro group is essential for the
fluorescence emission by establishing a push–pull relationship
with the electron-donating N-methylpyrrole. This was verified
experimentally by reduction of the nitro group to an amine,
yielding derivatives practically devoid of fluorescence.
Specifically, this chemical reduction converts initially D-π-A
Fig. 10. Theoretical excitation (top) and emission (bottom) spectra of the
derivatized amine.
4
. Concluding remarks
Since amines are present in many naturally occurring molecules
–
including proteins, peptides, nucleic acids, or alkaloids – fishing
for this functional group in a complex mixture at low
concentrations, represents a major challenge. In this context, a

1
0
Dyes and Pigments
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0. Nanda JS, Lorsch JR. Labeling of a protein with fluorophores
using maleimide derivitization. Methods Enzymol 2014;536:79–
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fluorescence biosensor for selective detection of tryptophan and
histidine. Analyst 2014;139:4794–8.
xanthene-based derivatization reagent BrXa was engineered,
which combines both colorimetric and fluorogenic sensing,
exclusively for primary amines. Its preparation relies on an
efficient and scalable synthesis. Given that the detection reaction
is based on a pyrrole formation, its selectivity towards primary at
the expense of secondary and tertiary amines revealed to be
extremely high. As the process is chromogenic (colorless 
magenta), this should interestingly make the method more
accessible and enable to reach a wider audience. Besides the
appearance of an easily detectable fluorescent color (dark  red),
the fluorogenic nature of this detection turns out to be a real asset.
As a matter of fact, it allows to benefit from the exquisite
sensitivity of the fluorescence spectroscopy, making it possible to
achieve the nanomolar range. Supported by quantum calculations
and photophysical characterization, the push–pull relationship
from which the red fluorescence originates, was clearly evidenced.
Combining all these attractive features with the development of a
robust LC analytical method, offers a unique turnkey solution for
the derivatization of primary amines.
8
1
1
https://doi.org/10.1039/C4AN00829D.
13. Fu Y, Yao J, Xu W, Fan T, He Q, Zhu D, et al. Reversible and
“fingerprint” fluorescence differentiation of organic amine
vapours using a single conjugated polymer probe. Polym Chem
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015;6:2179–82. https://doi.org/10.1039/C4PY01793E.
1
4. Mallick S, Chandra F, Koner AL. A ratiometric fluorescent probe
for detection of biogenic primary amines with nanomolar
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1
5. Cabanes J, Gandía-Herrero F, Escribano J, García-Carmona F,
Jiménez-Atiénzar M. Fluorescent bioinspired protein labeling with
betalamic acid. Derivatization and characterization of novel
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Acknowledgments
16. Saravanakumar M, Umamahesh B, Selvakumar R, Dhanapal J,
Ashok kumar SK, Sathiyanarayanan KI. A colorimetric and
ratiometric fluorescent sensor for biogenic primary amines based
on dicyanovinyl substituted phenanthridine conjugated probe.
Dyes Pigments 2020;178.
We thank for the PhD grants of S.V., H.-N.L., Y.W.M., G.B.,
the Agence Nationale de la Recherche (PFPImaging – 18-CE09-
JEDI
0
020-01, UCA
project: ANR-15-IDEX-01), the LIFE graduate
https://doi.10.1016/j.dyepig.2020.108346.
school (UCA) and the French Government, respectively. We are
grateful to the CNRS Emergence@INC2020 program for both the
financial support and the postdoctoral fellowship of S.M. This
research work was also funded by the Hellenic Foundation for
Research and Innovation (HFRI) under the HFRI PhD Fellowship
grant of A.D.K. (Fellowship Number: 1559/27.04.2018).
17. Abbas JM, Stortz M, Rodríguez HB, Levi V, Wolosiuk A,
Spagnuolo CC. The intramolecular self-healing strategy applied to
near infrared fluorescent aminotricarbocyanines. Dyes Pigments
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021;186. https://doi.10.1016/j.dyepig.2020.109040.
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2
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8. Gikas E, Parissi-Poulou M, Kazanis M, Vavagianis A.
MOZPhCSE, a new coumarin based fluorescent derivatization
reagent. J Liq Chromatogr Relat Technol 2004;27:2699–713.
https://doi.org/10.1081/JLC-200029226.
9. Mertens MD, Gütschow M. Synthesis and evaluation of two
coumarin-type derivatization reagents for fluorescence detection
of chiral amines and chiral carboxylic acids. Chirality
2013;25:957–64. https://doi.org/10.1002/chir.22240.
0. Li J, Zhang C, Wu S, Wen X, Xi Z, Yi L. Facile synthesis of
green-light and large Stokes-shift emitting coumarins for
bioconjugation. Dyes Pigments 2018;151:303–9.
Appendix A. Supplementary data (Material)
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.dyepig.2021.x.x.
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DYES
&
Pigments
HIGHLIGHTS
The identification of challenging amines in complex extracts of natural
products requires more and more innovative and sensitive spectroscopic
methods.

A xanthene-based derivatization reagent was engineered for the
exclusive detection of primary amines.

Thanks to its chromogenic nature (colorless  magenta), the
derivatization process becomes accessible to a broader audience.

The developed fluorogenic sensing (non-fluorescent  red) allows
the method to reach an exquisite level of sensitivity, approaching the
picomolar range.

Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: