Long-wavelength absorbing fluorescent polymethine dyes derived from the 6-(N,N-diethylamino)-1,2,3,4-tetrahydroxanthylium system

Article abstract of DOI:10.1016/j.jphotochem.2017.07.045

Several new dyes derivatives of the “rhodamine-like” 6-(N,N-diethylamino)-1,2,3,4-tetrahydroxanthylium heterocyclic system displaying absorption within the near-infrared were synthesized. The photochemical behaviour of these molecules was evaluated in dic

Full text of DOI:10.1016/j.jphotochem.2017.07.045

Accepted Manuscript
Title: Long-wavelength absorbing fluorescent polymethine
dyes derived from the
6
-(N,N-diethylamino)-1,2,3,4-tetrahydroxanthylium system
Authors: D.S. Concei c¸ a˜ o, D.P. Ferreira, Y. Prostota, O.D.
Kachkovsky, L.F.Vieira Ferreira, P.F. Santos
PII:
S1010-6030(17)30499-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jphotochem.2017.07.045
JPC 10771
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
12-4-2017
28-7-2017
31-7-2017
Please cite this article as: D.S.Concei c¸ a˜ o, D.P.Ferreira, Y.Prostota,
O.D.Kachkovsky, L.F.Vieira Ferreira, P.F.Santos, Long-wavelength absorbing
fluorescent polymethine dyes derived from the 6-(N,N-diethylamino)-1,2,3,4-
tetrahydroxanthylium system, Journal of Photochemistry and Photobiology A:
Chemistryhttp://dx.doi.org/10.1016/j.jphotochem.2017.07.045
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Long-wavelength absorbing fluorescent polymethine dyes derived from the 6-
N,N-diethylamino)-1,2,3,4-tetrahydroxanthylium system
(
a
a
b
c
a
D.S. Conceição , D.P. Ferreira , Y. Prostota , O. D. Kachkovsky , L.F.Vieira Ferreira ,
P.F. Santosb,*
aCentro de Química-Física Molecular and IN-Institute of Nanoscience and Nanotechnology,
Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa
bCentro de Química - Vila Real, Universidade de Trás-os-Montes e Alto Douro, 5001-801 Vila
Real, Portugal
cInstitute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine, 1
Murmanska str, 02660, Kyiv, Ukraine
*
To whom correspondence should be addressed.
Email: psantos@utad.pt
Keywords: Rhodamines; 1,2,3,4-tetrahydroxanthylium; NIR-dyes; fluorescence quantum yields;
fluorescence lifetimes; singlet oxygen.
1

1
,2
1
0
0
0
0
,8
,6
,4
,2
0
6
00
700
800
900
1000
1100
1200
Wavelength (nm)

Abstract
Several new dyes derivatives of the “rhodamine-like” 6-(N,N-diethylamino)-1,2,3,4-
tetrahydroxanthylium heterocyclic system displaying absorption within the near-infrared were
synthesized. The photochemical behaviour of these molecules was evaluated in dichloromethane,
regarding their ground state absorption properties, fluorescence emission quantum yields and
fluorescence lifetimes determinations. The singlet oxygen quantum yields of the new dyes were also
obtained. The absorption and luminescence spectral data showed that the behaviour of these dyes is
strongly dependant on the nature of the (hetero)aromatic substituent introduced in the 1,2,3,4-
tetrahydroxanthylium scaffold. The performed quantum-chemical calculations indicated that those
(hetero)aromatic terminal substituents are practically planar and hence are conjugated with the
chromophoric system of the 1,2,3,4-tetrahydroxanthylium framework. Such elongation of total -
system is accompanied by regular delocalization of the frontier MOs and decreasing of the first
electron transition energy manifested itself by bathochromic effect in dyes absorption.
2

1
. Introduction
In the last few years, fluorescence-based imaging techniques emerged as one of the most
widely tools used for biomolecules monitoring in living systems. Fluorescent probes, more
specifically organic dye molecules, possess several properties which make them suitable for single-
molecule imaging [1- 3]. One of the biggest issues regarding the use of organic molecules in
biological imaging is the strong absorption in the visible range where light penetration into the
tissues is reduced. In this way, Near InfraRed (NIR) dyes have been the subject of numerous studies
concerning their application as fluorescent markers. The advantages of using NIR dyes include
minimal interfering absorption and fluorescence from biological samples, minimum photo-
damaging of the sample under study and enhanced tissue penetration depth [4,5] due to a high
molar absorption coefficient (ε) in the long wavelength region (600 – 900 nm) where tissue light
scattering is lower [6, 7].
Among the variety of organic fluorophores, polymethine dyes and related molecular systems
are one of the most commonly used class of molecules for NIR-fluorescent sensor design [6].
NIR-fluorescent dyes based on the rhodamine core (or rhodamines) have been used as laser
dyes and fluorescence standards for a long time [8] and also as fluorescence markers in many
applications [7, 8], due to their high fluorescence quantum yields, high extinction coefficients, good
photostability and usually low quantum yield of triplet state formation. It is also well known that
rhodamine dyes can present a fluorescence on-off switching mechanism (when the o-carboxylic
acid group of rhodamine dyes undergo intramolecular cyclization turning the open form into a
spirocyclic one [8], which is non-fluorescent due to the interruption of the -electron system in the
dye’s chromophoric structure).
Regarding the mentioned above, we decided to explore the synthesis of new compounds
absorbing in the NIR region, composed by an heterocyclic system which bears structural fragments
of both rhodamines and benzo[b]pyrilium salts in one single molecular skeleton, the 6-(N,N-
dialkylamino)-1,2,3,4-tetrahydroxanthylium system.
3

Although the first examples of these molecules were reported in 1979 (as side products) [9]
the dyes derivatives of this heterocyclic framework were synthesized only in 2012 [10]. These
compounds (namely unsymmetrical trimethinecyanines from 1,3,3-trimethyl-3H-indolenine and
1
,3,3-trimethyl-3H-benzo[e]indolenine) showed excellent photophysical properties, including
absorption in the NIR region (688 – 728 nm), large absorption extinction coefficients (130 –
3
-1
-1
1
40x10 M cm ), high fluorescence quantum yields (0.29 – 0.56), good photostability and
sufficient chemical stability. It was also reported that these dyes are superior to traditional
rhodamine dyes with both absorption and emission in the NIR region, while retaining a very
important structural feature such as the rhodamine-like fluorescence ON-OFF switching mechanism
[
10].
One of the most remarkable features affecting both chemical and photostability of
polymethine dyes is their conjugated chain length and type of structure (opened, bridged, etc.). The
stability of cyanines increases with the decrease of the number of “opened” methine units
(unsaturated carbons) in the conjugated chain. In this way, we decided to focus our attention in the
dyes derivatives of the 6-(N,N-diethylamino)-1,2,3,4-tetrahydroxanthylium system, bearing only
one “opened” conjugated methine atom.
Herein, we describe the synthetic route to eight different dyes and the evaluation of their
photochemical parameters namely the absorption and emission wavelength, the fluorescence
quantum yields and lifetimes and also the singlet oxygen formation quantum yields. The nature of
the electronic transitions was studied by analysing the molecular geometry of the synthesized
compounds.
2
. Materials and methods
2
.1. General
Reagents and solvents used in the synthesis of the dyes were of commercial origin and, unless
otherwise stated, used as received. Solvents used in the measurement of spectral-luminescent
properties were of spectroscopic grade. 9-(2-Carboxyphenyl)-6-(N,N-diethylamino)-1,2,3,4-
4

tetrahydroxanthylium perchlorate (1) [11], 7-(N,N-diethylamino)coumarine-3-carbaldehyde (2h)
12], 9-(2-carboxyphenyl)-6-(N,N-diethylamino)-4-[(3-methylimidazo[1,5-a]pyridin-1-
[
yl)methylidenyl]-1,2,3,4-tetrahydroxanthylium perchlorate (3e) [13] and 3-phenylimidazo[1,5-
a]pyridine (4) [14] were prepared as previously reported. All reactions were monitored by thin-layer
1
chromatography on aluminium plates pre-coated with Merck silica gel 60 F254 (0.25 mm). H and
13C NMR spectra were record on a Bruker ARX400 spectrometer at room temperature, unless
otherwise stated;  in ppm relative to SiMe or to residual solvent signals, J in Hz. IR spectra were
4
-1
obtained on a Unicam Research Series FT-IR spectrophotometer; max in cm . High and low
resolution Electrospray Ionization Time-of-Flight (ESI-TOF) mass spectra were measured with a
VG AutoSpec M spectrometer. Melting points were determined in open capillary tubes in a Buchi
5
35 melting point apparatus and are uncorrected.
2
2
.2. Synthesis of compounds
.2.1. 3-Phenylimidazo[1,5-a]pyridine-1-carbaldehyde (2f)
To a solution of 3-phenylimidazo[1,5-a]pyridine (4) (1.50 g, 7.73 mmol) in anhydrous N,N-
dimethylformamide (10 mL), under vigorous stirring at 0-5ºC, was added dropwise phosphorus
oxychloride (0.72 mL, 7.73 mmol) during 20 min. Once the addition was complete, the reaction
mixture was kept at room temperature for 3 h and then poured into 20% aqueous sodium hydroxide
(50 mL). The resulting solid was filtered off under reduced pressure, washed with water (40 mL)
and recrystallized from propan-2-ol to afford 2f as greenish-yellow flakes. Yield 82%. Mp 229-231
ºC. IR (KBr) max: 700, 768, 968, 1011, 1149, 1286, 1330, 1418, 1500, 1536, 1654, 1879, 2846,
1
3
7
071. H NMR (400.13 MHz, DMSO-d
6
): 6.80 (1H, t, J = 6.8, ArH), 7.15 (1H, t, J = 7.8, ArH),
.39-7.47 (3H, m, ArH), 7.68 (2H, d, J = 7.2, ArH), 8.20 (1H, d, J = 9.0, ArH), 8.27 (1H, d, J = 7.0,
1
3
ArH), 10.06 (1H, s, CHO). C NMR (100.62 MHz, DMSO-d
6
): 115.5, 119.5, 122.5, 126.3, 128.2,
1
28.5, 129.0, 129.6, 130.3, 134.3, 139.8, 185.6. MS (ESI-TOF): 223 [M+H]+.
5

2
.2.2. 9-(2-Carboxyphenyl)-6-(N,N-diethylamino)-4-[4-(N,N-dimethylamino)benzylidenyl]-1,2,3,4-
tetrahydroxanthylium perchlorate (3a). Typical procedure.
A solution of 9-(2-carboxyphenyl)-6-(N,N-diethylamino)-1,2,3,4-tetrahydroxanthylium
perchlorate (1) (0.20 g, 0.42 mmol) and 4-(N,N-dimethylamino)benzaldehyde (2a) (0.63 g, 0.42
mmol) in freshly distilled acetic anhydride (5 mL) was heated under reflux for 5 min. After cooling
to room temperature, the precipitated solid was filtered off under reduced pressure, washed with
ethyl acetate and diethyl ether and recrystallized from glacial acetic acid/acetonitrile (5/1, v/v), to
afford chromatographically pure shinning green crystals. Yield 66%. Mp 267-269 ºC (dec.). IR
1
(
(
(
KBr) max: 619, 710, 823, 924, 1141, 1263, 1315, 1354, 1432, 1519, 1568, 1628, 1720. H NMR
400.13 MHz, DMSO-d
6
) : 1.21 (6H, t, J = 6.9, NCH
2
CH
3
), 1.65-1.89 (2H, m, CH
2
), 2.19-2.29
CH ), 6.86-
2H, m, CH ), 2.92-2.94 (2H, m, CH
2
2
), 3.09 (6H, s, NCH
3
), 3.63 (4H, q, J = 6.9, NCH
2
3
6
7
=
.91 (2H, m, ArH), 7.18 (1H, d, J = 9.0, ArH), 7.29 (1H, br s, ArH), 7.38 (1H, d, J = 7.6, ArH),
.67 (d, J = 9.0, 2H), 7.75 (1H, t, J = 7.5, ArH), 7.86 (1H, t, J = 8.0, ArH), 8.19-8.21 (2H, m, ArH +
1
3
CH), 13.18 (1H, br s, COOH). C NMR (100.62 MHz, DMSO-d ) : 12.4, 20.8, 25.4, 26.9, 45.0,
6
9
1
5.5, 111.9, 115.5, 116.5, 121.3, 122.1, 123.0, 128.9, 129.7, 130.0, 130.8, 132.9, 134.2, 134.4,
39.8, 151.7, 154.1, 157.3, 160.1, 163.1, 166.4. HRMS (ESI-TOF) m/z: 507.26409 (M+;
C
33
H
35
N
2
3
O requires 507.26422).
Dyes 3b-d and 3f-h were prepared using a similar procedure. Precipitation of compounds 3f and 3h
from the reaction mixture required cooling to 0-5 ºC overnight. Compounds 3f and 3g did not
required any additional purification.
2
.2.3.
9-(2-Carboxyphenyl)-6-(N,N-diethylamino)-4-[5-(N,N-dimethylamino)tiophene-2-yl)-
methylidenyl]-1,2,3,4-tetrahydroxanthylium perchlorate (3b)
Shining green crystals. Yield 57%. Mp 294-296 ºC (dec.). IR (KBr) max: 688, 818, 910, 1070,
1
1
133, 1171, 1251, 1371, 1400, 1446, 1623, 1724. H NMR (400.13 MHz, DMSO-d
6
) : 1.17 (6H,
6

d, J = 6.9, NCH
2
CH
), 3.50 (4H, q, J = 6.9, NCH
=CHtiophene), 6.92 (1H, d, J = 2.0, ArH), 7.30 (1H, d, J = 7.5, ArH), 7.68 (1H, t, J = 7.5, ArH),
3
), 1.65-1.81 (2H, m, CH
2
), 2.11-2.35 (2H, m, CH
2
), 2.72 (2H, br s, CH
2
), 3.35
(
6H, s, NCH
3
2
CH
3
), 6.61 (1H, d, J = 9.2, ArH), 6.78-6.81 (2H, m, ArH
+
7
.77 (1H, t, J = 7.5, ArH), 7.95 (1H, d, J = 5.0, =CHtiophene), 8.13 (1H, d, J = 7.6, ArH), 8.46 (1H, s,
1
3
=
CH), 12.82 (1H, br s, COOH). C NMR (100.62 MHz, DMSO-d ) : 12.4, 20.0, 21.0, 25.7, 25.8,
6
4
1
2.8, 44.3, 95.8, 111.0, 112.2, 112.5, 112.7, 120.1, 125.9, 127.6, 129.3, 130.2, 130.6, 132.7, 135.0,
36.0, 147.0, 151.0, 151.5, 155.3, 162.1, 166.6, 171.9, 173.5. HRMS (ESI-TOF) m/z: 513.22057
+
(
M ; C31
H
33
N
2
O
3
S requires 513.22064).
2
.2.4.
9-(2-Carboxyphenyl)-6-(N,N-diethylamino)-4-[(5-(N-pyrrolidinyl)tiophene-2-yl)-
methylidenyl]-1,2,3,4-tetrahydroxanthylium perchlorate (3c)
Shining green crystals. Yield 52%. Mp 294-296 ºC (dec.). IR (KBr) max: 710, 771, 845, 907, 1063,
1
1
111, 1154, 1220, 1254, 1310, 1372, 1515, 1546, 1624, 1724. H NMR (400.13 MHz, DMSO-d
6
) :
), 2.73 (2H, br s,
), 6.59 (1H, d, J = 9.2, ArH), 6.72 (1H,
d, J = 4.9, =CHtiophene), 6.77 (1H, dd, J = 2.2, 9.2, ArH), 6.90 (1H, d, J = 2.2, ArH), 7.31 (1H, d, J =
1
.17 (6H, t, J = 6.8, NCH
2
CH
3
), 1.72-1.80 (2H, m, CH
2
), 2.07-2.28 (6H, m, CH
2
CH
2
), 3.49 (4H, q, J = 6.8, NCH
2
CH ), 3.62 (4H, br s, CH
3
2
7
8
.6, ArH), 7.68 (1H, t, J = 7.6, ArH), 7.79 (1H, t, J = 7.5, ArH), 7.97 (1H, d, J = 4.9, =CHtiophene),
1
3
.12 (1H, d, J = 7.8, ArH), 8.48 (1H, s, =CH), 12.96 (1H, br s, COOH). C NMR (100.62 MHz,
) : 12.4, 20.3, 21.0, 25.2, 25.7, 25.8, 44.3, 52.0, 95.8, 111.8, 112.1, 112.6, 120.1, 126.0,
27.5, 129.3, 129.4, 130.3, 130.6, 132.7, 135.1, 136.1, 147.1, 150.3, 151.3, 155.1, 161.8, 166.6,
DMSO-d
6
1
1
+
69.7, 171.9. HRMS (ESI-TOF) m/z: 539.23623 (M ; C33
H
35
N
2
3
O S requires 539.23629).
2
1
.2.5. 9-(2-Carboxyphenyl)-6-(N,N-diethylamino)-4-[3-(4-N,N-dimethylaminophenyl)allylidenyl]-
,2,3,4-tetrahydroxanthylium perchlorate (3d)
Shining green crystals. Yield 79%. Mp 263-265 ºC (dec.). IR (KBr) max: 704, 1110, 1283, 1364,
1
1
435, 1532, 1628, 1725. H NMR (400.13 MHz, DMSO-d
6
) : 1.23 (t, J = 6.8, NCH
2
CH ), 1.72-
3
7

1
.80 (2H, m, CH
br q, J = 6.8, NCH
ArH), 7.29-7.33 (2H, m, ArH), 7.37 (1H, d, J = 7.4, ArH), 7.63 (2H, d, J = 8.8, ArH), 7.75 (1H, t, J
7.5, ArH), 7.85 (1H, t, J = 7.4, ArH), 8.03 (1H, d, J = 10.4, ArH), 8.20 (1H, d, J = 7.6, ArH),
2
), 2.22-2.31 (2H, m, CH
2
), 2.67-2.87 (2H, m, CH
2
), 3.05 (6H, s, NCH
3
), 3.63 (4H,
2
CH ), 6.77 (2H, d, J = 8.8, ArH), 6.88 (1H, d, J = 9.4, ArH), 7.16-7.20 (2H, m,
3
=
1
3
1
9
1
5
3.18 (br s, 1H, COOH). C NMR (100.62 MHz, DMSO-d ) : 12.4, 20.4, 21.0, 24.5, 25.8, 45.1,
6
5.4, 112.1, 115.7, 116.5, 120.0, 121.6, 123.6, 123.9, 129.0, 129.7, 130.0, 130.5, 130.8, 133.0,
34.4, 140.8, 146.6, 151.8, 154.1, 157.2, 160.0, 162.0, 166.4, 171.9. HRMS (ESI-TOF) m/z:
+
33.27953 (M ; C35
H
37
N
2
O requires 533.27987).
3
2
.2.6.
9-(2-Carboxyphenyl)-6-(N,N-diethylamino)-4-[3-phenylimidazo[1,5-a]pyridin-1-yl)
methylidenyl]-1,2,3,4-tetrahydroxanthylium perchlorate (3f)
Shining green crystals. Yield 47%. Mp 226-228 ºC (dec.). IR (KBr) max: 697, 824, 997,
1
1
120, 1181, 1259, 1320, 1437, 1533, 1581, 1624, 1811. H NMR (400.13 MHz, DMSO-d
6
) : 1.28
), 3.38-3.50 (2H, m,
), 6.88 (1H, d, J = 9.4, ArH), 7.14-7.17 (2H, m, ArH), 7.38-
(6H, t, J = 6.9, NCH
2
CH
3
), 1.75-1.84 (2H, m, CH
2
), 2.24-2.34 (2H, m, CH
2
CH
2
), 3.66 (4H, br q, J = 6.9, NCH
2
CH
3
4
7
8
.40 (2H, m, ArH), 7.52 (1H, t, J = 7.8, ArH), 7.57-7.66 (3H, m, ArH), 7.75 (1H, t, J = 7.6, ArH),
.83 (1H, t, J = 7.5, ArH), 7.94 (2H, d, J = 7.4, ArH), 8.20 (1H, d, J = 7.8, ArH), 8.49 (1H, s, =CH),
1
3
.54 (1H, d, J = 9.0, ArH), 8.70 (1H, d, J = 6.9, ArH), 12.96 (1H, br s, COOH). C NMR (100.62
) : 12.5, 20.6, 21.0, 25.9, 26.6, 44.9, 95.7, 115.4, 116.1, 116.5, 118.3, 121.5,
21.8, 124.6, 126.3, 128.3, 128.4, 128.8, 129.0, 129.1, 129.7, 129.8, 129.9, 130.8, 132.9, 137.9,
MHz, DMSO-d
6
1
1
+
41.7, 153.9, 157.1, 163.2, 166.4, 171.9. HRMS (ESI-TOF) m/z: 580.25865 (M ; C38
H
34
N
3
O
3
requires 580.25947).
2
.2.7. 9-(2-Carboxyphenyl)-6-(N,N-diethylamino)-4-[(N-ethylcarbazol-3-yl)methylidenyl]-1,2,3,4-
tetrahydroxanthylium perchlorate (3g)
8

Shining blue crystals. Yield 67%. Mp 228-230 ºC. IR (KBr) max: 624, 758, 937, 985, 1102,
1
1
163, 1233, 1259, 1342, 1377, 1441, 1529, 1572, 1629, 1725, 1812. H NMR (400.13 MHz,
DMSO-d
2H, m), 2.35-2.39 (2H, m, CH
q, J = 6.8, NCH CH ), 7.01 (1H, d, J = 9.5, ArH), 7.25-7.31 (2H, m, ArH), 7.36-7.41 (2H, m, ArH),
.53 (1H, t, J = 7.7, ArH), 7.67 (1H, d, J = 8.1, ArH), 7.74-7.80 (2H, m, ArH), 7.86-7.90 (52H, m,
6
, 50 ºC) : 1.28 (6H, t, J = 6.8, NCH
2
CH
3
), 1.38 (3H, t, J = 6.8, NCH
2
CH
3
), 1.79-1.86
(
2
), 3.10 (2H, br s, ), 3.70 (4H, br q, J = 6.8, NCH
2
CH
3
), 4.51 (2H, br
2
3
7
1
3
ArH), 8.22-8,28 (2H, m, ArH), 8.46 (1H, s, =CH), 8.56 (1H, s, ArH). C NMR (DMSO-d
6
) : 12.5,
1
1
1
3.7, 21.0, 25.4, 26.7, 37.2, 45.3, 95.5, 109.6, 109.7, 116.5, 117.7, 119.7, 120.7, 121.8, 122.2,
22.7, 124.1, 125.3, 126.3, 126.5, 128.9, 129.3, 129.4, 129.6, 130.1, 130.9, 133.0, 134.2, 138.8,
40.1, 140.3, 154.9, 157.8, 161.9, 166.4, 171.9. HRMS (ESI-TOF) m/z: 581.27858 (M+;
C
39
H
37
N
2
3
O requires 581.27987).
2
.2.8. 9-(2-Carboxyphenyl)-6-(N,N-diethylamino)-4-[7-(N,N-diethylamino)benzo[e]pyran-2-on-3-
yl)methylidenyl]-1,2,3,4-tetrahydroxanthylium perchlorate (3h)
Shining green crystals. Yield 70%. Mp 262-264 ºC (dec.). IR (KBr) max: 624, 706, 750,
1
8
11, 959, 1007, 1115, 1254, 1332, 1385, 1451, 1506, 1564, 1616, 1707. H NMR (400.13 MHz,
DMSO-d
m, CH ), 2.29-2.32 (2H, m, CH
4H, br q, J = 6.5, NCH CH ), 6.61 (1H, s, ArH), 6.79 (1H, d, J = 8.9, ArH), 6.96 (1H, d, J = 9.3,
ArH), 7.25-7.28 (2H, m, ArH), 7.40 (1H, d, J = 7.5, ArH), 7.59 (1H, d, J = 8.9, ArH), 7.77 (1H, t, J
6
) : 1.15 (6H, t, J = 6.8, NCH
2
CH
3
), 1.23 (6H, br t, J = 6.5, NCH
2
CH
3
), 1.74-1.79 (2H,
2
2
), 2.91 (2H, br s, CH
2
), 3.49 (4H, br q, J = 6.8, NCH
2
CH ), 3.68
3
(
2
3
1
3
=
7.6, ArH), 7.88 (1H, t, J = 7.5, ArH), 8.01 (s, 1H, ArH), 8.19-8.23 (2H, m, ArH + =CH). C
NMR (100.62 MHz, DMSO-d ) : 12.3, 20.8, 21.0, 25.4, 26.9, 44.3, 45.4, 95.4, 96.4, 108.4, 109.2,
14.2, 116.7, 118.0, 121.9, 128.1, 128.8, 129.5, 130.2, 130.8, 131.1, 133.1, 144.1, 151.8, 155.1,
6
1
1
+
56.2, 157.7, 160.2, 160.9, 166.4, 171.9. HRMS (ESI-TOF) m/z: 603.28346 (M ; C38
H
39
N
2
O
5
requires 603.28535).
9

2
.3. UV-Visible absorption spectroscopy
Steady-state absorption spectra were recorded with a Camspec M501 double beam scanning
UV/Visible spectrophotometer, at room temperature, in the spectral range from 190 to 1100 nm.
The optical densities (O.D.) were measured using a UV quartz cuvette (1 cm path length). All the
solutions were used at O.D. = 0.6 in dichloromethane.
2
.4. Laser-induced luminescence (LIL): fluorescence emission quantum yields determination
Schematic diagrams of the LIL system are presented in reference [15]. A N laser (PTI
2
model 2000, ca. 600 ps FWHM, ~1.0 mJ per pulse, 5Hz), was used in the laser-induced
luminescence experiments, the excitation wavelength being 337 nm. With this set-up, luminescence
spectra were easily acquired by the use of an InGaAs photodiode array detection system (PDA)
from Andor, model i-Dus, working at -60 °C. This detector allows the acquisition of signals that
arise in the NIR range (700 nm to 1700 nm), and that are not possible to acquire using a normal
UV-visible detector. Corrected spectra were obtained by applying a calibration curve to the desired
range. The optical density at the excitation wavelength for both the unknown and standard samples
was 0.6 in dichloromethane.
The fluorescence quantum yields of the dyes were calculated relative to the standard from
the respective average fluorescence peak areas, and the published quantum yield of the standard,
which in this case was HITC (1,1',3,3,3',3'-hexamethylindotricarbocyanine) iodide (Φ = 0.28 in
F
ethanol).
2
.5. Fluorescence lifetimes determination
Fluorescence lifetimes were determined using Easylife VTM equipment from OBB (Lifetime
range from 90 ps to 3 µs). This technique uses pulsed light sources from different LEDs (630 nm in
this case) and measures fluorescence intensity at different time delays after the excitation pulse. In
this case a 695 nm cut-off filter was used. The instrument response function was measured using a
1
0

Ludox scattering solution. FelixGX software from OBB was used for fitting and analysis of the
decay dynamics.
2
.6. Singlet oxygen quantum yield determinations
The singlet oxygen measurement set-up was assembled in our laboratory. As an excitation
source we used the nitrogen laser described in 2.4. The detector was once again the InGaAs PDA
working at low temperature (-60ºC) coupled to a fixed spectrograph, model Shamrock 163i also
from Andor [16]. The solutions of the dyes were prepared in chloroform with O.D. = 0.6, and
phenazine was used as the standard for quantum yields determination.
3
. Results and discussion
3
.1. Synthesis
The synthesis of the target dyes 3a-h was carried out by condensation of 9-(2-
carboxyphenyl)-6-(N,N-diethylamino)-1,2,3,4-tetrahydroxanthylium perchlorate (1), prepared by an
improved method developed by one of us [11], with several aromatic and heteroaromatic aldehydes
(2a-h) in refluxing acetic anhydride (Scheme 1). The dyes were obtained in moderate to good
yields, crystallizing directly from the crude reaction mixture, in some cases without the need of
additional purification.
1
1

O
O
ArCHO
OH
OH
2
a-h
+
Ac O /
2
+
N
O
N
O
ClO4-
ClO4-
Ar
1
3a-h
a: Ar =
c: Ar =
e: Ar =
N
b: Ar =
S
N
N
N
d: Ar =
S
f: Ar =
N
N
N
N
H C
3
g: Ar =
h: Ar =
O
O
N
N
Scheme 1. Synthesis of dyes 3a-h.
3
.2. Photochemical characterization
All synthesized dyes 3a-h display strong absorption and emission in the NIR range as
depicted in Figure 1.
1
2

a)
b)
Fig. 1. a) Absorption spectra and b) fluorescence emission spectra of dyes 3a-h in dichloromethane.
Table 1 summarizes the photochemical parameters obtained for dyes 3a-h: wavelength of
maximum absorption (λabs) and emission (λem), molar absorption coefficient (εmax), fluorescence
emission quantum yield (Φ
F
), fluorescence lifetime (τ
F
) and singlet oxygen quantum yield (Φ ).
Δ
Table 1
Spectral data of dyes 3a-h.
a
a
-1
a
-1
-1
a
a
b
Dye λabs (nm) λem (nm) Stokes shift (cm ) εmax (M cm )
Φ
F
τ
F
(ns)
Φ
∆
689
733
738
757
766
770
147059
303030
312500
75321
175894
156309
0.04
0.04
0.04
0.25 ± 1.1x10-2
0.17 ± 8.8x10-2
0.16 ± 3.5x10-4
-
3
a
3
b
-
-
3
c
1
3

749
701
703
646
678
857
735
746
712
797
92592
294117
232558
151515
84034
68189
104000
90000
49000
39000
0.20
0.30
0.40
0.08
0.07
1.03 ± 3.0x10-2
2.21 ± 4.9x10-3
2.97 ± 2.2x10-2
0.79 ± 1.3x10-2
0.36 ± 7.8x10-3
-
3
d
3
e
0.02
0.03
0.03
0.02
3
f
3
g
3
h
a
In CH
2
Cl .
2
b
In CHCl
3
.
In a previous work from our group pertaining the study of two “rhodamine-like”
hemicyanines derived from the 6-(N,N-diethylamino)-1,2,3,4-tetrahydroxanthylium system [17], it
was found that the substitution with an aniline fragment in the conjugation chain leads to an
increase in the resonance of the molecule which is responsible for the large Stokes shift observed
when compared to classical rhodamines. Comparing the dyes under study herein with the
“rhodamine-like” dyes described in [17] a considerable deviation of the emission’s wavelength to
the red, in some cases more than 100 nm, was observed, which clearly evidences the strong increase
in the molecules resonance duo to the influence of the different (hetero)aryl substituents. The most
pronounced effect amongst this group of new NIR-absorbing dyes is evident in the spectrum of dye
3
d in which the absorption maximum is located at 749 nm and the emission maximum at 857 nm
(Stokes shift of 108 nm).
This type of dyes shows a strong solvatochromic behaviour [13,17]. One of the main
consequences of the solute-solvent interactions is, unquestionably, the intramolecular charge
transfer (ICT) mechanism, which, in high polarity solvents, can strongly affect the fluorescence
quantum yield of the molecule. However, as explored in the aforementioned works of Ferreira et al.
and Conceição et al., there should not be a direct correlation between the spectroscopic properties
exhibited by these rhodamine-like dyes with the common macroscopic polarity parameters, since
both the absorption and emission properties are a consequence of a complex function of polarity,
specific solute-solvent interactions, and polarizability of the solvent. Accordingly, dicloromethane,
1
4

possessing a Dimroth's ET parameter of 41.1 and a Kosower's Z-value of 64.2, was selected as
solvent of choice to minimize these processes due to its relatively low polarity. The fluorescence
quantum yields of all compounds under study were calculated relative to HITC iodide as standard.
The highest values of Φ
F
were obtained for dyes 3d-f, ranging from 0.20 to 0.40. All dyes exhibited
monoexponential fluorescence lifetime decays in dicloromethane in the order of nanoseconds. Dye
3
f presented the highest fluorescence lifetime (~ 3 ns).
The determination of the singlet oxygen formation quantum yield was performed for all dyes
in chloroform by comparison with the singlet oxygen phosphorescence emission of phenazine (Φ
Δ
=
0
.84) [18] at 1270 nm. The values obtained are invariably very low and, in some cases, there is no
evidence at all of singlet oxygen generation. As reported in literature, rhodamine dyes in general are
poor candidates for the formation of excited triplet state because their intersystem crossing ability is
very limited [19]. The functionalization of these polymethine dyes seems to have no effect in singlet
oxygen production.
The strong absorption and emission in the NIR region and the relatively good fluorescence
quantum yields and lifetimes, however, turn dyes 3d-f good candidates to be used as fluorescence
markers in molecular bioimaging techniques.
3
.3. Spectral properties and the nature of the electronic transitions
In parallel with the spectral study, quantum-chemical calculations for dyes 3a-h were also
carried out. The optimized molecular geometry of the dye molecules in the ground state was
obtained by the DFT/6-31G(d,p)/B3LYP method, while the electron-transition characteristics were
calculated by the semi-empirical ZINDO/S method using a package Gaussian 03 [20].
The optimized structures of dyes 3a-h are shown in Figure 2.
1
5

3
a
3b
3
c
3d
3
e
3f
1
6

3
g
3h
Fig. 2 Molecular geometry of dyes 3a-h.
In all cases the benzoic acid moiety is highly twisted and essentially positioned vertically in
relation to the 1,2,3,4-tetrahydroxanthylium core, with a dihedral angle of nearly 90°; both -
electron fragments are disposed orthogonally and hence no electronic conjugation exists between
them. Contrastingly, following from calculations the aryl substituents and the 1,2,3,4-
tetrahydroxanthylium fragment are closely co-planar, excepting the trimethylene bridge. Also, the
o
aryl residues in 3g and 3h are somewhat twisted (18 ). The phenyl ring in the imidazo[1,5-
o
a]pyridine residue of 3f is twisted 32 . The replacement of the N,N-dimethylamino group in the
thiophene substituent of 3b by the pyrrolidinyl group in 3c causes only a slight decrease of the
o
o
valence angle:  (CH
3
-N-CH
3
) = 120 (3b) and  (-CH
2
-N-CH -) = 112 (3c).
2
In spite of these slight distortions of the planar conformation, the strong delocalization of -
electrons is accomplished along the whole quasi-planar conjugated systems, which can be treated as
polymethine chromophores, similarly to the well-known cyanine dyes with their equalized carbon-
carbon bond lengths. According to the colour theory of polymethine/cyanine dyes, going from dye
1
7

3
a to its vinylog 3d should cause a bathochromic shift of the maximum absorption wavelength of
approximately 100 nm (the so-called “vinylene shift”) [21-23]. In this case an experimental
bathochromic shift of 60 nm was observed. An alternative way of shifting dyes absorption towards
the NIR, avoiding the loss of stability in long open polymethine chains because of twisting around
C-C bonds, is the variation of the “effective length” of the terminal groups, for example, replacing
the 4-(N,N-dimethylamino)phenyl group of dye 3a by 5-(N,N-dimethylamino)thiophenyl in 3b. The
same is observed with the introduction of the 3-methylimidazo[1,5-a]pyridine residue (in dye 3e) or
its phenyl-substituted analogue (in dye 3f). The phenyl group in the aryl substituent of 3f does not
provide experimentally an appreciable spectral effect, although calculations predict a bathochromic
shift of 21 nm (Table 2). The carbazole and the coumarine moieties (in dyes 3g and 3h,
respectively) are not capable of causing a deviation of the dyes absorption towards the NIR, despite
both aryl substituents consist of bulky conjugated systems. Inversely, hypsochromic shifts were
obtained, reaching -43 nm in the case of dye 3b; calculations predict a somewhat lower value in this
case (-38 nm).
Such modifications of the constitution of the polymethine chromophore result in shifting of
electron levels, first of all, the frontier levels involved in the lowest electron transition. The
positions of some levels, as well as the shape of the corresponding MOs, are illustrated for dyes
3
a,b and 3d in Figure 3.
1
8

E, eV
-
3
LUMO+1
-
5
LUMO
-
7
HOMO
HOMO-1
HOMO-2
-
9
3
a
3b
3d
Fig. 3 Positions of electron levels and shape of corresponding MOs for dyes 3a, 3b and 3d.
It is apparent that the lowest vacant orbital and the three highest occupied MOs are
uniformly delocalized along the whole chromophore, whereas the next vacant MO (LUMO+1) is
1
9

concentrated in the perpendicular substituent. The comparison of the same orbital for the various
dyes shows that their shapes remain practically similar.
Contrastingly, the positions of the electron levels are sensitive to the lengthening of the
chain and to the structural variation of the terminal groups. Though, the change in the energy of the
lowest vacant level is negligible (for example LUMO = -5,145 eV in 3a and -5,050 eV in 3b). The
shifting of the occupied levels is shown in Figure 3 to be more appreciable. Also, the energies of the
occupied -MOs located completely within the twisted fragment are lower than the orbitals
presented in Figure 3.
To study the nature of the electron transitions in connection with the spectral bands in the
visible region, the calculations of the excited states were performed by the semi-empirical ZINDO
method (with variation of the Overlap Weight Factor - OWF). It is well known that the TD DFT
method gives a significant divergence between the calculated and the experimental wavelengths for
the first electronic transitions in polymethine dyes, especially in molecules with extensive linear -
systems [24,25]. It should also be noted that the semi-empirical ZINDO approximation gives more
realistic values of the spectral characteristics. A careful comparison of the quantum-chemical results
and the experimental data allows obtaining the necessary information for the analysis of the nature
of the electron transitions and their intensities. The calculated characteristics for dyes 3a-h are
collected in Table 2.
2
0

Table 2
Absorption maxima (max) and oscillator strengths (f) of the lowest electron transitions of dyes 3a-h
calculated by the ZINDO/S method (OWF = 0.45).
b
Dye
Transition

max (nm)
 (nm)
f
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
 S
 S
 S
 S
 S
 S
 S
 S
 S
 S
 S
 S
 S
 S
 S
 S
 S
 S
 S
 S
 S
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
678 [689]a
407
-
1.098
0.059
0.092
1.086
0.130
0.102
1.152
0.206
0.075
1.553
0.035
0.327
1.295
0.259
0.530
1.321
0.104
0.463
0.999
0.006
0.218
3
a
-
396
-
+ 54 [+ 44]a
+9
732 [733]a
416
3
b
400
+4
734 [738]a
421
+ 56 [+ 49]a
+ 14
3
c
404
+ 8
776 [749]a
444
+ 98 [+ 60]a
+37
3
d
394
-2
712 [701]a
391
+ 34 [+ 12]a
- 16
3
e
372
- 24
733 [703]a
400
+ 55 [+ 14]a
- 7
3
f
378
- 18
640 [646]a
456
- 38 [- 43]a
+ 49
3
g
400
+ 4
2
1

3
h
S
S
S
0
0
0
 S
 S
 S
1
2
3
680 [678]a
424
+ 2 [- 11]a
+ 17
1.033
0.405
0.033
390
- 6
The lowest electron transitions are described for all dyes by the following configurations: S
0
S > 
1

HOMOLUMO>; S
0
S
2
>  HOMO-1LUMO>; S
0
S
3
>  HOMO-2LUMO>.
Cl
a
Values in square brackets refer to experimental dyes absorption maxima in CH
2
2
.
b

 = max (3b-h) - max (3a).
It can be see that the first electron transition, S
0
S , is described in all dye molecules
1
practically by one single excited configuration involving HOMO and LUMO with the larger
oscillator strength. Similarly, the one-configurational nature of the first electron transition was
shown to be typical for cyanine dyes [23]. The changes in the calculated wavelength of the S
0
 S
1
transition with the variation of the chemical constitution of the main chromophore agrees (with
reasonable accuracy) with the shifts of the long-wavelength band observed in the absorption
spectra. Thus, the spectral outcome of replacing the 4-(N,N-dimethylamino)phenyl terminal group
in 3a by 5-(N,N-dimethylamino)thiophenyl in 3b is comparable to the effect of lengthening the
polymethine chain from dye 3a to 3d. Yet, the first way is preferable once the long-wavelength
absorption band becomes more sharp and intense, what can be explained by the decrease of
vibrational structures in the dyes with shorter open polymethine chains.
Table 2 also shows the spectral effects for the higher electron transitions, but their values are
lower, which is in accordance with the experimental results.
4
. Conclusions
Several new dyes, derivatives of the “rhodamine-like” 6-(N,N-diethylamino)-1,2,3,4-
tetrahydroxanthylium heterocyclic system, were prepared by condensation of 9-(2-carboxyphenyl)-
-(N,N-diethylamino)-1,2,3,4-tetrahydroxanthylium perchlorate with several aromatic and
heteroaromatic aldehydes.
6
2
2

The photochemical characterization of these dyes was obtained regarding the absorption and
fluorescence spectra, fluorescence lifetimes and quantum yields and also singlet oxygen formation
quantum yield. The new synthesized dyes display strong absorption and emission in the NIR region
(between 646 and 749 nm) and reasonably good fluorescence quantum yields and lifetimes in three
cases, 3d, 3e and 3f, the latter presenting the highest values (Ф = 0.40, τ = 3 ns). The singlet
F
F
oxygen quantum yields are very low which is in accordance with the fact that this family of dyes
presents very low values of triplet state quantum yields.
As suggested by quantum-chemical calculations, the (hetero)aromatic substituents introduced
in the 1,2,3,4-tetrahydroxanthylium framework are nearly planar and hence conjugated with its
chromophoric system. Consequently, the spectral properties of these dyes are strongly dependant on
the nature of the substituents.
All the mentioned before turn some of the synthesized dyes good candidates to be used as
fluorescence markers for bioimaging techniques.
Acknowledgments
Thanks are due to FCT (Portugal’s Foundation for Science and Technology) and FEDER for
financial support through the research unit Centro de Química - Vila Real (CQ-VR
(
UID/QUI/00616/2013). FCT is also acknowledged for the PhD fellowships for D. P. Ferreira
SFRH/BD/95359/2013) and D. S. Conceição (SFRH/BD/95358/2013).
(
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