Hydrogen bonding and protonation effects in amino acids' anthraquinone derivatives - Spectroscopic and electrochemical studies

Article abstract of DOI:10.1016/j.saa.2019.117226

Six novel amino acid chromophores were synthesized and their spectroscopic, acid-base, and electrochemical properties are discussed in this work. In studied compounds, selected amino acid residues (L-Aspartic acid, L-Glutamic acid, L-Glutamine, L-Histidine, L-Lysine, L-Arginine) are attached to the 1-(piperazine) 9,10-anthraquinone skeleton via the amide bond between the carboxyl group of amino acid and nitrogen atom of the piperazine ring. All derivatives have been characterized using a variety of spectroscopic techniques (mass spectrometry, 1HNMR, UV–Vis, IR spectroscopy), acid-base (electrochemical and UV–Vis) titrations, and cyclic voltammetry methods. Basing on observed experimental effects, supported by quantum chemical simulations, the structure-properties links were established. They are indicative of the specific interactions within and/or in-between amino acid side groups, which are prone to form both, intra- and intermolecular hydrogen bonds as well as electrostatic interactions with the anthraquinone system.

Full text of DOI:10.1016/j.saa.2019.117226

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117226
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Hydrogen bonding and protonation effects in amino acids'
anthraquinone derivatives - Spectroscopic and electrochemical studies
a,
b,
b
b
b
Sandra Ramotowska ⁎, Dorota Zarzeczańska ⁎, Iwona Dąbkowska , Anna Wcisło , Paweł Niedziałkowski ,
b
b
b
Elżbieta Czaczyk , Beata Grobelna , Tadeusz Ossowski
Department of Bioinorganic Chemistry, Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308 Gdańsk, Poland
Department of Analytical Chemistry, Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308 Gdańsk, Poland
a
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Six novel amino acid chromophores were synthesized and their spectroscopic, acid-base, and electrochemical
properties are discussed in this work. In studied compounds, selected amino acid residues (L-Aspartic acid, L-
Glutamic acid, L-Glutamine, L-Histidine, L-Lysine, L-Arginine) are attached to the 1-(piperazine) 9,10-anthraqui-
none skeleton via the amide bond between the carboxyl group of amino acid and nitrogen atom of the piperazine
ring. All derivatives have been characterized using a variety of spectroscopic techniques (mass spectrometry,
Received 21 March 2019
Received in revised form 30 May 2019
Accepted 31 May 2019
Available online 02 June 2019
1HNMR, UV–Vis, IR spectroscopy), acid-base (electrochemical and UV–Vis) titrations, and cyclic voltammetry
Keywords:
methods. Basing on observed experimental effects, supported by quantum chemical simulations, the structure-
properties links were established. They are indicative of the specific interactions within and/or in-between
amino acid side groups, which are prone to form both, intra- and intermolecular hydrogen bonds as well as elec-
trostatic interactions with the anthraquinone system.
Anthraquinone
Amino acids
Pyroglutamic acid
Hydrogen bonding
Protonation
©
2019 Elsevier B.V. All rights reserved.
Electrochemistry
1
. Introduction
2 2
electron-donor substituents (e.g. -OH, -NH , -NHR, -NR ) evoke new ab-
sorption bands in the visible region. If the single amino group is bound
at the position 1 of a 9,10-anthraquinone ring, there appears an absorp-
tion maximum at around 475 nm, associated with the red color of such
derivatives. Additionally, the wavelength of the absorption maximum
has been for a long time postulated [20] and recently proved [21–24]
to be related to the capability of the substituents to form a hydrogen
bond with an oxygen of the anthraquinone ring. Moreover, the position
and the intensity of this peak is extremely sensitive, signaling even the
subtle changes in a distant regions of the molecules, hence may be used
as a diagnostic tool [25]. The presence of different amino acids in the
9,10-anthraquinone system differentiate the properties of these com-
pounds due to the capabilities of amino acids to form hydrogen bonds
and to electrostatic interactions.
The quinone/hydroquinone pair is also a well-known example of the
organic redox system. The electrochemical studies of quinone deriva-
tives have been widely performed for many years [26–29]. Electro-
chemical characteristics of quinoid compounds related to the
equilibrium and kinetics of electron and proton transfer provide valu-
able information about the structure of the molecules, their interactions
and the electrochemical processes' environment [30].
Aminoanthraquinone derivatives are commonly used as popular
dyes [1–3]. Furthermore, these molecules play an important role as sig-
naling devices in supramolecular structures sensitive to pH and redox
potential changes or can be used as a sensor for ions and organic mole-
cules [4–7]. The 9,10-anthraquinone moiety, due to its unique spectro-
scopic and electrochemical properties, can act as a chromophore, a
fluorophore and an electrophore and thus be used in a variety of sensor
devices [8–13].
The presence of an amino acid residue attached to the quinone sig-
naling system makes it possible to use anthraquinone derivatives in
the studies of interactions occurring in biological systems. These types
of quinone derivatives can be incorporated into the structure of poly-
peptides or proteins, giving the opportunity to track physiological pro-
cesses. In addition, due to their antineoplastic properties and an ability
to intercalate into DNA, they may act as chemotherapeutic agents
[
14–19].
The spectroscopic properties of the 9,10-anthraquinone can be eas-
ily modulated by attaching different moieties to the anthraquinone
skeleton. Electron-acceptor substituents (e.g. -NO , -CN,\\Cl) do not
change the absorption spectrum in a significant way, whereas
2
The aim of this work was to study the specific interactions, both
intra- and intermolecular, which caused the spectral differences ob-
served in different (acidic and alkaline) solutions, of six new chromo-
⁎
Corresponding authors.
E-mail addresses: sandra.ramotowska@ug.edu.pl (S. Ramotowska),
phores, that could be used as
a molecular recognition tool.
dorota.zarzeczanska@ug.edu.pl (D. Zarzeczańska).
Spectrophotometric, acid-base and voltammetric properties of a group
https://doi.org/10.1016/j.saa.2019.117226
1386-1425/© 2019 Elsevier B.V. All rights reserved.

2
S. Ramotowska et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117226
of new anthraquinone derivatives containing piperazine ring
substituted by selected amino acid were examined.
chromatography with elution: dichloromethane to dichloromethane/
methanol 5:1, v/v) to affort 1.318 g as a red residue (yield = 73%).
TLC (SiO
2
): DCM:MeOH (5:1); R
H NMR (CDCl ) (400 MHz): δ (ppm): 1.57 (s, H, -N-CH
-CH -); 3.19 (s, 8H, -N-CH -CH -NH-CH -CH -); 7.39–7.41 (dd,
2H, H-2 Ar, H-3 Ar, J = 0,8 Hz, J = 8,4 Hz); 7.62–7.66 (t, 1H, H-3 Ar,
= 8.0 Hz, J = 7.6 Hz, J = 7.8 Hz); 7.69–7.73 (t, 1H, H-7 Ar, J
8.0 Hz, J = 7.6 Hz, J = 7.8 Hz); 7.75–7.79 (t, 1H, H-6 Ar, J = 8.0 Hz,
= 7.6 Hz, J = 7.8 Hz); 7.94–7.96 (d, 1H, H-4 Ar, J = 7.2 Hz);
1
= 6.7 Hz); 8,27-8,29- (d, 1H, H-8 Ar, J =
f
= 0.22.
1
2
. Experimental
3
2 2
-CH -NH-
CH
2
2
2
2
2
2
2
.1. Materials and methods
1
2
J
1
1
2
1
=
Fmoc-protected amino acids were purchased from Sigma-Aldrich.
1
2
1
All other chemicals and solvents were of analytical grades and used as
received (from Sigma-Aldrich or POCH S.A. Poland) without further pu-
J
1
2
1
8.22–8.24 (d, 1H, H-5 Ar, J
1
1
rification. H NMR spectra were recorded on Varian, Mercury-400BB
7.6 Hz).
spectrometer (400 MHz) and Bruker Avance III 500 MHz (500 MHz)
MALDI TOF MS: m/z 293.4 [M H], (MW = 292.33).
IR (KBr) (cm− ): 3433, 3257, 2931, 2814, 1667, 1579, 1426, 1314,
1
spectrometers at ambient temperature in CDCl
3
with tetramethylsilane
(
TMS) as internal reference. Infrared (IR) spectra were recorded using
1267, 1238, 710.
KBr pellet technique on the Bruker IFS 66 spectrometer. MALDI-TOF
mass spectra analysis were performed on a Bruker BIFLEX III TOF mass
spectrometer using 2, 5-dihydroxybenzoic acid (2, 5-DHB) as matrix
and internal standard. Flash column chromatography was carried out
using silica gel 60 (Merck 0.040–0.063 mm, 230–400 mesh). Thin
layer chromatography (TLC)was performed on precoated silica gel 60
t
RP-HPLC: R = 11.54 min.
2.2.2. A general procedure for the synthesis of amino acids and 1-
(piperazin-1-yl)-9,10-anthraquinone (AQ-ppz) derivatives
2.2.2.1. 1-(4-L-aspartic acid)piperazin-1-yl)-9,10-anthraquinone (AQ-
ppz)-Asp. 0.7 g of 1-(piperazin-1-yl)-9,10-anthraquinone (AQ-ppz)
(2.394 mmol) dissolved in 50 mL of dichloromethane was placed in a
round-bottom flask equipped with a stirring bar and the resulting solu-
tion was cooled to 0 °C. In another flask, to the solution of 1.585 g Fmoc-
Asp(OtBu)-OH (3.591 mmol) dissolved in 50 mL of N,N-
dimethylformamide (DMF), 0.453 g of diisopropylcarbodiimide (DIC)
(3.591 mmol) was added, and the resulting solution was cooled to -
10 °C for 20 min. This suspension was added dropwise to the flask of
(AQ-ppz) and the reaction mixture was stirred for 24 h. The progress
of the coupling reaction was monitored by TLC (eluent: dichlorometh-
ane: methanol 5:1, v/v). The reaction mixture was filtered to remove
the precipitate and the solvents were evaporated under reduced pres-
sure. The resulting residue was treated without purification by a mix-
ture of 10 mL of dichloromethane and 10 mL of TFA. The solution was
stirred for (from 1 h to 24 h for Fmoc-(AQ-ppz)-Arg(Pbf)-OH) and the
solvent was removed under vacuum. The product was precipitated in
(
Merck) aluminium plates with fluorescent indicator F254. Analytical
reversed-phase high-performance liquid chromatography (RP-HPLC)
analyses were performed on a Shimadzu HPLC system with a UV–Vis
detector using a reverse-phase column Phenomenex Luna C8, (100–3,
1
50 mm × 4.60 mm) at 25 °C. Eluents A: 0.1% (v/v) trifluoroacetic acid
in water. B: 0.1% (v/v) trifluoroacetic acid in acetonitrile. A linear gradi-
ent was applied from 0% to 100% solvent B at a flow rate of 1.0 mL/min
was used with UV detection at 254 and 210 nm. The time of analysis was
1
5 min, then mobile phase returned to 100% solvent A during 5 min and
reequilibrated for next 5 min. The preparative RP-HPLC analyses were
performed on a Knauer HPLC system with a UV–Vis detector using a
reverse-phase column Macherey Nagel, Nucleosil C18, (100–7,
2
1
50 mm × 16 mm) with a different linear gradient at flow rate
0 mL/min was used with UV detection at 254 or 210 nm.
2
.2. Synthesis method
2
Et O and treated by mixture of 5 mL of DCM and 5 mL of piperidine.
All compounds have been synthesized using as a substrates 1-
piperazin-1-yl)- 9,10- anthraquinone (AQ-ppz) and the N-protected
After 1 h stirring, the deprotection step was complete. The solvent
was removed and the crude product in the case of (AQ-ppz-Lys), was
purified by silica gel column chromatography with dichloromethane/
methanol (5:1, v/v). In the case of other compounds the preparative re-
versed phase chromatography RP- HPLC was used to purify the desired
compounds.
(
α-amino acids by the 9- fluorenylmethoxycarbonyl (Fmoc) additionally
containing protective groups in the side chain commonly used in the
Fmoc/tBu strategy of peptide synthesis [31]. The activation of a carbox-
α
ylic acid of N -Fmoc amino acid derivatives was performed in the pres-
ence of N,N′-Diisopropylcarbodiimide (DIC, Sigma-Aldrich). The
reactions between (AQ-ppz) and all amino acid derivatives involved
amide bond formation have been performed in dichloromethane
Yield: m = 0.556 g; 57%.
TLC (SiO
2
): DCM; R
H NMR (CDCl ) (500 MHz): δ (ppm): 2.50–2.52 (t, 2H, C((=O)
OH)-CH -CH(NH )-C(=O)- N-, J = 5 Hz, J = 5 Hz, J = 5 Hz);
3.34–3.36 (d, 4H, Ar-N-CH -CH -N(-C=O)-CH -CH -, J
3.39–3.40 (d, 4H, Ar-N-CH -CH -N(-C=O)-CH -CH -, J
3.62–3.66 (q, 1H, C((=O)OH)-CH -CH(NH )-C(=O)-N-, J
= 5 Hz, J = 10 Hz, J = 7.5 Hz, J = 5 Hz, J = 6,25 Hz); 5.52–5.53
(d, 2H, C((=O)OH)-CH -CH(NH )-C(=O)-N-, J = 10 Hz); 7.56–7.57
(d, 1H, H-2 Ar, J = 5 Hz); 7.60–7.62 (d, 1H, H-4 Ar, J = 10 Hz);
7.80–7.83 (t, 1H, H-3 Ar, J
f
= 0,1.
1
3
(
DCM, POCH S.A. Poland). Then the side chain protecting groups were
2
2
1
1
2
removed in the reaction with trifluoroacetic acid (TFA, Sigma-Aldrich).
In the next stage of synthesis the deprotection of Fmoc group was car-
ried out in the reaction with piperidine.
The crude products were purified by flash column chromatography
and then using preparative RP-HPLC or using only preparative RP-
HPLC (Scheme 1). The identification and purity of obtained fractions
were investigated using analytical RP-HPLC and MALDI-TOF analysis.
2
2
2
2
1
= 10 Hz);
1
= 5 Hz);
= 5 Hz, J
1 1
2
2
2
2
2
2
1
2
2
3
2
2
1
1
1
1
1
= 10 Hz, J
= 10 Hz, J
1
1
= 5 Hz, J
= 5 Hz, J
2
2
= 7.5 Hz);
= 7.5 Hz);
7
.87–7.90 (t, 1H, H-6 Ar, J
2
.2.1. Synthesis of 1-(piperazin-1-yl)-9,10-anthraquinone (AQ-ppz)
.500 g of 1-chloro-9,10-anthraquinone (6.18 mmol) and 798 mg of
7.91–7.93 (t, 1H, H-7 Ar, J
1
= 5 Hz, J
1
= 5 Hz, J
2
= 5 Hz); 8.14–8.15
= 5,0 Hz); 9.02
1
(d, 1H, H-8 Ar, J
(s, 1H, C((=O)OH)-CH
1
= 5 Hz); 8.18–8.19 (d, 1H, H-5 Ar, J
-CH(NH )-C(=O)-N-).
1
piperazine (9.27 mmol) dissolved in 150 mL of toluene was placed in a
three-necked round-bottom flask equipped with a stirring bar and a re-
flux condenser. The reaction mixture was stirred at 80 °C in oil bath
monitoring by TLC. The reaction was completed after 24 h, then the sol-
vent was removed under reduced pressure and the residue was dis-
solved in 250 mL of dichloromethane. The organic phase was washed
2
2
MALDI TOF MS: m/z 408.1 [M H], (MW = 407.1).
IR (KBr) (cm− ): 3430, 3341, 2968, 2926, 2852, 1668, 1618, 1580,
1
1323, 1272, 1251, 1202, 1130, 736, 720.
t
RP-HPLC: R = 8.61.
with water (3 × 250 mL), dried over anhydrous MgSO
4
and the solvent
2.2.2.2. 1-(4-L-Glutamic acid)piperazin-1-yl)-9,10-anthraquinone (AQ-
was removed under reduced pressure. The resulting product was dis-
ppz)-Glu. Yield: 72%.
solved in dichloromethane and purified by silica gel column
2 f
TLC (SiO ): DCM; R = 0.24.

S. Ramotowska et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117226
3
Scheme 1. Synthesis of selected amino acids derivatives incorporated into 1-(piperazin-1-yl)-9,10-anthraquinone (AQ-ppz).
1H NMR (CDCl
OH)-CH -CH -CH(NH
4 Hz, J = 4 Hz, J = 4 Hz); 2.41–2.45 (t, 2H, C((=O)OH)-CH
CH(NH )-C(=O)-N-, J = 8 Hz, J = 8 Hz, J = 8 Hz); 3.00–3.04 (p,
H, C((=O)OH)-CH -CH -CH(NH )-C(=O)-N-, J = 4 Hz, J = 4 Hz,
= 4 Hz, J = 4 Hz, J = 4 Hz, J = 4 Hz, J = 4 Hz, J
Hz, J = 4 Hz); 3.62 (s, 4H, Ar-N-CH -N(-C=O)-CH -CH
.86 (s, 4H, Ar-N-CH -CH -N(-C=O)-CH
-CH -CH(NH )-C(=O)-N-); 7.58–7.59 (d, 1H, H-2 Ar, J
Hz); 7.78–7.81 (t, 1H, H-3 Ar, J = 4 Hz, J = 8 Hz, J = 6 Hz);
.84–7.86 (d, 1H, H-4 Ar, J = 8 Hz); 7.86–7.89 (t, 1H, H-6 Ar, J
Hz, J = 8 Hz, J = 6 Hz); 7.91–7.94 (t, 1H, H-7 Ar, J = 8 Hz,J
Hz, J = 6 Hz); 8.14–8.16 (d, 2H, H-8 Ar, H-5 Ar J
H, C((=O)OH)-CH -CH -CH(NH )-C(=O)-N-).
) (400 MHz): δ (ppm): 2.00–2.03 (q, 2H, C((=O)
)-C(=O)-N-, J = 4 Hz, J = 4 Hz, J = 4 Hz, J
-CH
3
t
RP-HPLC: R = 8.77.
2
2
2
1
1
1
2
=
2
3
2
2
-
2.2.2.3. 1-(4-Pyroglutamic acid)piperazin-1-yl)-9,10-anthraquinone (AQ-
ppz)-pGlu. Yield: 77%.
2
1
1
2
1
2
2
2
1
1
TLC (SiO
2
): DCM; R
H NMR (CDCl ) (500 MHz): δ (ppm): 2.13–2.15 (q, 2H, (-HN-C(=
O)-CH2-CH2-CH-)-C(=O)-N, J = 3 Hz, J = 4,5 Hz, J = 5 Hz, J
3,75 Hz, J = 4.75 Hz, J = 4.25 Hz); 2.16–2.17 (d, 1H, (-HN-C(=O)-
CH2-CH2-CH-)-C(=O)-N, J = 5 Hz); 2.34–2.39 (t, 2H, (-HN-C(=O)-
CH2-CH2-CH-)-C(=O)-N, J = 10 Hz, J = 15 Hz, J = 12,5 Hz);
f
= 0.34.
1
J
1
1
2
2
= 4 Hz, J
2
3
3
=
3
4
3
4
2
-CH
2
2
2
-);
1
1
1
2
=
2
2
2 2
-CH -); 4.50 (s, 2H, C((=O)
2
3
OH)-CH
2
2
2
1
=
1
4
7
4
4
1
1
1
2
1
1
2
1
1
1
=
=
3.75–3.76 (d, 4H, Ar-N-CH
3.83–3.84 (d, 4H, Ar-N-CH
2
2
-CH
-CH
2
-N(-C=O)-CH
-N(-C=O)-CH
2
-CH
-CH
2
-, J
-, J
1
= 5 Hz);
= 5 Hz);
1
= 3,5 Hz,
1
2
1
2
2
2
1
2
1
= 8 Hz); 9.33 (s,
4,61–4,64 (q, 1H, (-HN-C(=O)-CH2-CH2-CH-)-C(=O)-N, J
= 5 Hz, J = 5 Hz, J = 5 Hz, J = 5 Hz, J
1H, H-2 Ar, J = 5,0 Hz); 7.71–7.72 (d, 1H, H-4 Ar, J
1
2
2
2
J
1
1
2
2
3
= 5 Hz); 7.51–7.52 (d,
= 5 Hz);
MALDI TOF MS: m/z 423.1 [M 2H] , (MW = 421.45).
IR (KBr) (cm− ): 3418, 2967, 1667, 1580, 1319, 1269, 1201, 1133,
34, 798, 720, 709.
1
1
7.74–7.77 (t, 1H, H-3 Ar, J
7.80–7.83 (t, 1H, H-6 Ar, J
1
= 10 Hz, J
= 5 Hz, J
1
= 5 Hz, J
1
= 10 Hz, J
2
= 7,5 Hz);
= 7,5 Hz);
8
1
2

4
S. Ramotowska et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117226
7
.85–7.89 (t, 1H, H-7 Ar, J
1
= 10 Hz, J
1
= 10 Hz, J
2
= 10 Hz); 8.07–8.09
= 10 Hz).
Ar, J
4 Hz); 7.78–7.82 (t, 1H, H-6 Ar, J
Hz);7.86–7.87 (d, 1H, H-4 Ar, J = 4 Hz); 7.91–7.94 (t, 1H, H-7 Ar, J
= 4 Hz, J = 8 Hz, J = 6 Hz); 8.14–8.16 (d, 1H, H-8 Ar, J = 8 Hz);
8.19–8.17 (d, 1H, H-5 Ar, J = 8 Hz).
1
= 8 Hz); 7.59–7.61 (t, 1H, H-3 Ar, J
1
= 4 Hz, J
1
= 4 Hz, J
2
=
(
d, 1H, H-8 Ar, J
1
= 10 Hz); 8.12–8.14 (d, 1H, H-5 Ar, J
1
1
= 8 Hz, J
1
= 8 Hz, J
2
= 8
MALDI - TOF MS: m/z 404.2 [M H] , (MW = 403.44).
IR (KBr) (cm− ): 3417, 2923, 2853, 1701, 1650, 1579, 1468, 1443,
427, 1380, 1315, 1266, 1230, 1200, 1151, 1079, 1029, 1001, 902, 833,
01, 758, 736, 710.
1
1
1
1
2
1
1
8
1
MALDI TOF MS: m/z 449.1 [M H], (MW = 448.53).
IR (KBr) (cm− ): 3423, 2925, 1670, 1581, 1430, 1270, 1202, 1131,
1
RP-HPLC: R
t
= 9.58.
835, 800, 721, 709.
2
.2.2.4. 1-(4-L-Histidine)piperazin-1-yl)-9,10-anthraquinone (AQ-ppz)-
t
RP-HPLC: R = 8.11.
His. Yield: 45%.
TLC (SiO ): DCM:MeOH (5:1); R
H NMR (CDCl ) (400 MHz): δ (ppm): 3.25 (s, 2H, (-N-CH-NH-CH-C-
-CH -CH(NH )-C(=O)-N); 3.35 (s, 4H, Ar-N-CH -CH -N(-C=O)-CH
CH -); 3.39–3.41 (t, 4H, Ar-N-CH -CH -N(-C=O)-CH -CH -, J = 4 Hz,
= 4 Hz, J = 4 Hz); 3.63–3.66 (t, 1H, -N-CH-NH-CH-C-)-CH -CH
NH )-C(=O)-N-, J = 8 Hz, J = 64 Hz, J = 6, Hz); 4.88–4.90 (t, 2H,
N-CH-NH-CH-C-)-CH -CH(NH )-C(=O)-N-, J = 4 Hz, J = 4 Hz, J
4 Hz); 7.48 (s, 1H, H-2 Ar);7.58–7.59 (d, 1H, H-4 Ar, J = 4 Hz);
= 8 Hz, J = 4 Hz, J = 6 Hz); 7.71–7.74
= 8 Hz, J =,4 Hz, J = 6 Hz); 7.77–7.79 (d,
= 8 Hz); 7.87–7.88 (d, 1H, H-5 Ar, J = 4 Hz);
.96–7.97 (d, 1H, -N-CH-NH-CH-C-)-CH -CH(NH )-C(=O)-N-, J
Hz); 8.73 (s, 1H, -N-CH-NH-CH-C-)-CH -CH(NH )-C(=O)-N-).
2
f
= 0,60.
2.3. Spectroscopic measurements
1
3
)
2
2
2
2
2
-
Spectrophotometric measurements were performed in an acetoni-
trile:methanol (9:1, v/v) mixture and in methanol. All spectra were reg-
istered on Perkin Elmer Lambda 650 spectrophotometer, at 298 K
temperature. Quartz spectrophotometric cuvettes of 1 cm thickness
were used. Concentrations of analyzed compounds used for all spectro-
2
2
2
2
2
1
J
1
2
2
(
-
=
7
2
1
1
2
2
2
1
1
2
photometric measurements were between 1 × 10− M and 5 × 10 M.
4
−4
1
.66–7.69 (t, 1H, H-3 Ar, J
t, 2H, H-6 Ar, H-7 Ar, J
H, H-8 Ar, J
1
1
2
All the compounds were stable at normal temperature and pressure.
(
1
1
2
1
7
4
1
1
2.4. Acid-base titrations
2
2
1
=
2
2
Amphiprotic solvents, such as water (but also alcohols or acetoni-
trile), undergo autoprotolysis reaction. If we denote the solvent as SH,
then it is possible to write the general reaction of autoprotolysis as:
MALDI TOF MS: m/z 430.1 [M H], (MW = 429.48).
IR (KBr) (cm− ): 3414, 2968, 1669, 1582, 1433, 1384, 1202, 1134,
1
8
34, 797, 721, 708.
þ
−
RP-HPLC: R
t
= 8.09.
2SH⇄SH2 þ S
ð1Þ
2
.2.2.5. 1-(4-L-Lysine)piperazin-1-yl)-9,10-anthraquinone (AQ-ppz)-Lys.
and hence, the degree of autoprotolysis can be expressed by constant
K :
SH
Yield: 48%.
TLC (SiO
2
): DCM:MeOH (5:0.2); R
) (400 MHz): δ (ppm): 1.45–1.50 (q, 2H, NH
-CH(NH )-C(=O)-N-, J = 8 Hz, J = 8 Hz, J = 4 Hz, J
= 6 Hz, J = 7 Hz); 1.67–1.73 (p, 2H, NH -CH -CH -CH
-CH(NH )-C(=O)-N-, J = 4 Hz, J = 8 Hz, J = 8 Hz, J
6 Hz, J = 6 Hz, J = 6 Hz, J = 6 Hz, J
-CH -CH -CH -CH -CH(NH
= 4 Hz, J = 6 Hz, J = 4 Hz, J = 5 Hz); 2.97–3.00
-CH -CH -CH -CH(NH )-C(=O)-N-, J
= 6 Hz); 3,27 (s, 8H, Ar-N-CH -CH -N(-C=O)-CH
-CH(NH
-CH -CH
= 4 Hz); 7.73–7.76 (t, 1H, H-3
f
= 0.1.
1
ꢀ
þꢁ
H NMR (CDCl
-CH
8 Hz, J
3
2
-CH
2
-
−
KSH ¼ SH2 ½S ꢀ
ð2Þ
CH
=
CH
=
1
8
2
-CH
2
2
2
1
1
1
2
2
3
2
2
2
2
-
Therefore, pH measurement is also possible for non-aqueous solu-
tions [32,33].
2
2
1
1
1
= 4 Hz, J
1
2
2
2
3
3
= 6 Hz, J
4
= 6 Hz);
)-C(=O)-N-, J =
2 1
Acid dissociation constants values were determinated by pH-
spectrophotometric titrations using Cerko-Lab microtitration automatic
system with a Hamilton 1 mL syringe. After addition of each portion of
titrant, the UV–Vis spectrum was recorded in the wavelength range
from 275 to 650 nm. The pH values during measurement were obtained
by simultaneous potentiometric titration performed also using
microtitration automatic system Cerko-Lab. The Schott BlueLine N16
pH glass-electrode was calibrated on the acetonitrile:methanol (9:1 v/
v) mixture using 2,6-dinitrophenol and its tetrabutylammonium salt
buffer solution method. Electrode parameters were calculated using a
STOICHIO version CVEQUID software based on the non–linear least-
squares Gauss-Newton-Marquardt algorithm [34]. The resolution of
the voltage measurement was b0.1 mV. All measurements were per-
formed at 298 K temperature.
.90–1.95 (q, 2H, NH
Hz, J = 4 Hz, J
-CH
2
2
2
2
2
1
1
2
2
3
(
t, 2H, NH
2
2
2
2
2
2
1
= 4 Hz, J
-CH
2
)-C(=O)-N-, J
1
1
=
-);
=
8
3
4
Hz, J
.39–3.40 (d, 3H, NH
Hz); 4.57–4.58 (d, 2H, NH
2
2
2
2
2
-CH
2
-CH
2
-CH
2
-CH
2
2
2
-CH
2
-CH
2
2
2 2
-CH(NH )-C(=O)-N-,
J
1
= 4 Hz); 7.56–7.57 (d, 1H, H-2 Ar, J
Ar, J = 8 Hz, J = 4 Hz, J = 6 Hz); 7.770–7.80 (t, 1H, H-6 Ar, J
Hz, J = 4 Hz, J = 6 Hz); 7.81–7.84 (t, 1H, H-7 Ar, J = 4 Hz, J
Hz, J = 6 Hz); 7.88–7.90 (d, 1H, H-4 Ar, J
H, H-8 Ar, H-5 Ar, J = 4 Hz, J = 8 Hz, J = 6 Hz).
1
1
1
2
1
=
=
8
8
2
1
2
1
1
2
1
= 8 Hz); 8.07–8.10 (t,
1
1
2
MALDI TOF MS: m/z 421.2 [M H], (MW = 420.51).
IR (KBr) (cm−1): 3358, 2927, 1666, 1642, 1580, 1468, 1443, 1316,
1
267, 1236, 1154, 1031, 1003, 753, 710.
t
RP-HPLC: R = 8.16.
a
2.5. Calculation of the pK values
2
.2.2.6. 1-(4-L-Arginine)piperazin-1-yl)-9,10-anthraquinone (AQ-ppz)-
Acid dissociation constant values were calculated with the
Arg. Yield: 55%.
TLC (SiO ): DCM:MeOH (5:1); R
H NMR (CDCl ) (400 MHz): δ (ppm): 1.53–1.58 (p, 2H, NH
NH)-NH-CH -CH -CH -CH(NH )-C(=O)-N-, J = 8 Hz, J = 4 Hz, J
4 Hz, J = 4 Hz, J = 6 Hz, J = 4 Hz, J = 4 Hz, J = 5 Hz, J
Hz, J = 4,5 Hz); 1.71–1.75 (q, 2H, NH -C(=NH)-NH-CH -CH
CH(NH )-C(=O)-N-, J = 8 Hz, J = 4 Hz, J = 4 Hz, J = 6 Hz, J
Hz, J = 5 Hz); 2.71 (s, 1H, NH -C(=NH)-NH-CH -CH -CH -CH
NH )-C(=O)-N) 3.13–3.15 (t 2H, NH -C(=NH)-NH-CH -CH -CH -CH
NH )-C(=O)-N-, J = 4 Hz, J = 4 Hz, J = 4 Hz); 3.17 (s, 8H, Ar-N-
-N(-C=O)-CH -CH -); 3.20 (s, 1H, NH -C(=NH)-NH-CH
-CH(NH )-C(=O)-N-); 4.47–4.48 (d, 2H, NH -C(=NH)-NH-
-CH -CH(NH )-C(=O)-N-, J = 4 Hz); 7.56–7.58 (d, 1H, H-2
Henderson–Hasselbach equation implemented into Origin Lab soft-
ware. These measurements are based on the change in absorptions in-
tensity as a function of pH of the solution [35,36]. The acid – base
2
f
= 0.11.
1
3
2
-C(=
2
2
2
2
1
1
1
a
equilibrium constant K can be described by following equations:
=
4
1
2
2
2
3
3
=
4
2
2
2 2
-CH -
2
=
BH22 ⇆BH þ Hþ
þ
þ
ð3Þ
ð4Þ
2
1
1
1
2
4
(
(
CH
CH
CH
3
2
2
2
2
ꢀ
ꢁꢀ
ꢁ
þ
þ
BH
H
2þ
2
2
2
2
2
Ka2 ¼
h
i
2
1
1
2
BH2
2
2
2
-CH
-CH
-CH
2
2
2
2
2
-
2
2
2
BH ⇆B þ Hþ
þ
ð5Þ
2
2
2
1

S. Ramotowska et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117226
5
Scheme 2. The mechanism of glutamine (Gln) cyclization to pyroglutamic acid (pGlu) in the 1-(piperazin-1-yl)-9,10-anthraquinone (AQ-ppz).
working electrode was glassy carbon electrode of 3 mm diameter, and
a platinum wire served as an auxiliary electrode. All potentials were
measured versus a double junction silver/silver chloride (Ag/AgCl) ref-
erence electrode with the aqueous sodium chloride (1 M NaCl) solution
in 0.1 M TBAP in methanol. All cyclic voltammetry measurements were
performed at 298 K temperature and the solutions were degassed by
passing argon.
ꢀ
ꢁ
þ
½
Bꢀ H
Ka1 ¼ ꢀ þꢁ
ð6Þ
BH
where B stands for compound (9,10-anthraquinone derivative) in non-
ionized form and BH , BH2
- protonated forms.
2
2
.6. Quantum-chemical simulations
3. Results and discussion
In order to understand why, despite their close resemblance of
chemical formulas, the spectral characteristics of (AQ-ppz)-Glu and
AQ-ppz)-Asp differ so distinctively (Scheme 1), we preformed quan-
The aim of this work was the synthesis and full characteristic of
(
novel chromophores, in which amino acids moieties are linked with
9,10-anthraquinone via piperazine ring, as well as the determination
of the structure-properties relationships. While characterizing these de-
rivatives, the following factors should be considered:
tum chemical simulations. We have implemented DFT (Density Func-
tional Theory) method with one of the Minnesota Functionals,
developed by the group of prof. Truhlar – MP06-2X [37] as it performs
strikingly well, consistently giving the smallest mean errors, in applica-
tions involving noncovalent interactions of organic molecules [38]. The
basis set of our choice was 6–31 G(d′,p′) – designed by Petersson and
co-workers as a part of Complete Basis Set approach [39] with an addi-
tional set of diffuse functions. Monomers and dimers of each compound
were fully optimized separately in their canonical, zwitterionic and pro-
tonated forms in vacuum and in solvent. Solvent effects have been taken
into account by means of a Polarizable Continuum Model (PCM) using
the integral equation formalism with the acetonitrile defined by the di-
electric constant ε = 35.688, as this solvent had been used by us in the
experiments. Each geometry optimization was followed by frequency
calculations in order to include zero-point vibrations and entropic fac-
tors into the interaction energies as well as to confirm that given struc-
ture is in fact a minimum on PES (potential energy surface).
1
) UV–Vis absorption bands – their number, position and intensity,
which reflects the efficiency of electron transfer within the
aminoanthraquinone skeleton (intramolecular charge transfer ICT),
All quantum chemical calculations were performed using the Gauss-
ian 16 RevB.01 software suite [40] available at Wrocław Centre for Net-
working and Supercomputing.
2
.7. Voltammetric measurements
The electrochemical measurements were carried out in a single-
compartment, three-electrode cell with an Autolab potentiostat/
galvanostat PGSTAT30 (Eco Chemie B.V., The Netherlands) controlled
by the Nova software. The analyzed compounds used for all
−3
voltammetric measurements were ca. 1 × 10 M solutions dissolved
in 0.1 M tetrabutylammonium perchlorate (TBAP) in DMSO. The
Fig. 1. UV–Vis spectra of all derivatives measured in acetonitrile: methanol (9:1) mixture.

6
S. Ramotowska et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117226
Table 1
Absorption maxima (λ
absorption bands intensities (ε
in acetonitrile-methanol (9:1) mixture and in acidic and alkaline environment. The sub-
scripts “1” or “max” refers to the maximum around 380 nm and the long-wavelength band
respectively.
ppz)-Asp: 77%, 77% and 72%, respectively. The yield for compounds
AQ-ppz)-Glu, (AQ-ppz)-Lys, (AQ-ppz)-His was 57%, 48%, 45%,
respectively.
As a result of reaction of 1-(piperazin-1-yl)-9,10-anthraquinone
(AQ-ppz) with Fmoc-Gln(Trt)-OH the Fmoc-(AQ-ppz)-Gln(Trt) was
obtained (Scheme 1). During the deprotection steps, both under influ-
ence of TFA and piperidine, performed at room temperature, the cycliza-
tion reaction of Gln took place, which led to creation of new compound:
1
and λmax), values of molar absorption coefficients (ε) and ratio of
/εmax) for AQ-ppz and its amino acid derivatives measured
(
1
a
b
a
b
a
a
Compound
λ
1
ε
1
λ
max
ε
max
ε
1
/εmax
λ
max
λ
max
(acidic) (alkaline)
(
(
(
(
(
(
(
AQ-ppz)c
AQ-ppz)-Asp
AQ-ppz)-Glu
381 1880
378 1600
381 1420
508
471
484
488
484
490
485
3200
1800
1980
2580
1400
2150
1310
0.6
0.9
0.7
0.7
0.7
0.7
0.8
468
469
482
~486
484
485
481
508
504
492
488
496
492
492
1-(4-Pyroglutamic acid)piperazin-1-yl)-9,10-anthraquinone (AQ-ppz)-
pGlu, with a yield of 77%. The reactions of cyclization of Gln to pGlu is
known in amino acid chemistry and pGlu is formed by enzymatic
AQ-ppz)-pGlu 381 1770
AQ-ppz)-His
AQ-ppz)-Lys
AQ-ppz)-Arg
381 1010
381 1500
380 1050
(
glutaminyl cyclase) or nonenzymatic reaction, and under acidic or al-
kaline, and/or high temperature conditions [41,42]. pGlu can be formed
from Gln as well as from Glu. The mechanism of pGlu formation from
Gln is described on Scheme 2 [43].
a
b
c
[
[
nm].
dm mol−1 cm−1].
3
In acetonitrile [20].
3.2. Spectroscopic properties
2
) the basicity of functional groups in the anthraquinone skeleton,
which can be expressed by the UV–Vis characteristics of a
chromophore,
The spectroscopic properties of the aminoanthraquinone determine
that the most diagnostic range for studying its derivatives is from 400
and 500 nm, with the most characteristic band appearing at 475 nm
[20]. This broad band is assigned to the π → π* transition, resulting
from the intramolecular charge transfer between the amino-
substituent and the 9,10-anthraquinone skeleton. The replacement of
3
) the specific interactions, both intra- and intermolecular, which cause
the spectral differences observed in acidic and alkaline solutions,
) influence of above-mentioned interactions on the redox properties
of 9,10-anthraquinone.
4
2
-NH group with a piperazine ring results in the shift of 475 nm maxi-
mum band towards red light (λmax = 508 nm in acetonitrile) [22].
This shift is also characteristic for 9,10-aminoanthraquinone derivatives
substituted with simple alkyl groups (e.g. 1-(dimethylamino)-9,10-an-
thraquinone, 1-(diethylamino)-9,10 anthraquinone) [21].
3
.1. Synthesis
All compounds have been synthesized using 1-(piperazin-1-yl)-
9
,10-anthraquinone (AQ-ppz) and Fmoc-Asp(OtBu)-OH, Fmoc-Glu
In case of anthraquinones with tertiary-amino substituents, there is
a second characteristic feature of UV–Vis spectra - an absorption peak
about 400 nm. We identified this band as the consequence of electron
transition involving the carbonyl group of 9,10-anthraquinone [5,44].
The lone pair of nitrogen atom prolongs conjugated system and there-
fore promotes this excitation. On the other hand, protonation of the ni-
trogen atom present in the aminoanthraquinone leads to a hydrogen
bond involving electron density from the oxygen, which prohibits
these excitations. Consequently, in order to observe ~400 nm band, nei-
ther lone pair from oxygen, nor lone pair from nitrogen cannot be in-
volved in any kind of bonding.
(
OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Fmoc)-
OH, Fmoc-Arg(Pbf)-OH as substrates (Scheme 1). The DIC was used as
a reagent in an amide bond formation reaction between (AQ-ppz) and
all amino acid derivatives. After completion of the coupling reaction in
all obtained products, except compound Fmoc-(AQ-ppz)-Lys(Fmoc)-
OH, the side chain protecting groups were removed in the reaction
with TFA. Then Fmoc group was removed using piperidine in high con-
centration. The crude products were purified initially by column chro-
matography in the case of (AQ-ppz)-Lys and then using preparative
RP-HPLC or using only preparative RP-HPLC in the case of rest com-
pounds (Scheme 1). The identification and purity of obtained com-
pounds were performed using analytical RP-HPLC and mass analysis.
The purity of each compound was higher than 99.8%. The maximum
yield of reaction was observed for: (AQ-ppz), (AQ-ppz)-pGlu, (AQ-
For all derivatives investigated by us, both above-described bands
are observed (Fig. 1). However, comparing the molar absorption coeffi-
cients of the studied derivatives with other tertiary amino-
anthraquinones, one can notice a significant reduction of their values
Fig. 2. Proposed chemical structure of a) (AQ-ppz)-Asp with intramolecular hydrogen bond and b) (AQ-ppz)-Glu dimer.

S. Ramotowska et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117226
7
from 4360 dm ·mol ·cm−1 for 1-(diethylamino)-9,10-anthraquinone
3
−1
(
[
3
−1
−1
19] to 2580–1310 dm ·mol ·cm for our derivatives – see Table 1).
100
The reason for the observed aberration in the intensity of long-
wavelength bands is probably related to the presence of side chains
rich in groups capable of electrostatic interactions with the anthraqui-
none system.
80
Furthermore, several meaningful differences in the UV–Vis spectra
of studied derivatives are observed. Considering the high similarity in
molecular structure of derivatives with a carboxyl group in a side
chain (AQ-ppz)-Asp, (AQ-ppz)-Glu, the differences in their spectral
characteristics are striking - we noticed unexpectedly large dissimilar-
ities in both: the position and intensities of absorption maxima. The
long-wavelength band of (AQ-ppz)-Asp is located at much shorter
wavelengths (λmax = 471 nm) than in (AQ-ppz)-Glu derivative (λmax
6
4
2
0
0
0
(
(
(
AQ-ppz)-Asp
AQ-ppz)-Glu
AQ-ppz)-pGlu
=
484 nm). At the same time, it distinguishes (AQ-ppz)-Asp from all
other compounds whose λmax values are in the 484–490 nm range
(
Table 1).
To eliminate the possible influence of residual trifluoroacetic acid
from the synthesis procedure) we additionally record all the spectra
4000
3500
3000
2500
2000
1500
1000
500
(
-1
Wavenumber cm
in an alkaline environment, where only the deprotonated forms may
be present. In this case, the long-wavelength (AQ-ppz)-Asp band occurs
at 504 nm, and again, the value of the red-shift (35 nm) is the highest.
This is another property that significantly differentiates an asparagine
derivative from other compounds for which these shifts vary between
Fig. 3. IR spectra of (AQ-ppz)-Asp, (AQ-ppz)-Glu and (AQ-ppz)-pGlu.
each of the compared compounds, although these peaks differ from
each other. In the spectra of the (AQ-ppz)-Asp two well-separated
0
and 12 nm. On the other hand, from Table 1 we see, that the addition
bands (at 1670 cm− and 1618 cm ) coming from vibrations in the
C_O groups of the quinone system and C_O amino acid residue are ob-
served. For (AQ-ppz)-pGlu derivative, which side group is in the form of
lactam ring, we also observe these two bands, however, shifted towards
1
−1
of the acid hardly affects the positions of the bands (0–5 nm). However,
its molar absorption coefficient decreases significantly (almost to zero),
for most compounds. This is accompanied by almost complete discolor-
ation of the studied derivatives' solutions.
Other characteristic signals at UV–Vis are also noteworthy. For all
1
compounds, the value (λ ) of the absorption maximum fluctuates
higher frequencies (1702 cm− and 1650 cm ), which is characteristic
for vibrations of C_O groups of intramolecular amides with five-
membered rings. While in the case of the (AQ-ppz)-Glu derivative, the
shape and intensity of the band at approx. 1670 cm− gives the opportu-
nity to observe only one wide peak. The main difference in spectra is
manifested in the 2500–3400 cm− range of frequencies, which is the
most diagnostic for assessing the presence of hydrogen bonds. Only
for the (AQ-ppz)-Glu, a broad band is present in this range, confirming
the formation of intermolecular hydrogen bonds by this derivative.
The presence of such bonds is also confirmed by quantum-chemical
simulations of the ground state PES of monomers and dimers of these
molecules. They indicate that in case of (AQ-ppz)-Glu the formation of
two intermolecular hydrogen bonds (Fig. 2b) leads to the thermody-
namically stable dimer. This results in an energy gain of 11.2 kcal/mol
with respect to the sum of the electron energies of the two monomers,
in the ΔG scale (Gibbs free energy). These bonds are equivalent, with a
length characteristic of the hydrogen bonding system between the car-
boxylic groups, which is 1.67 Å. On the contrary, in case of (AQ-ppz)-
Asp, such dimers, even though the strength of hydrogen bonds are basi-
cally the same (11.0 kcal/mol in respect to the monomers in conforma-
tion from dimers) are less stable than the sum of the most stable
monomers. This clearly demonstrates, that the intramolecular H-bond
prevails here. This is also confirmed by the analysis of entropy contribu-
tions. Creation of the (AQ-ppz)-Glu intramolecular H-bond requires
much more of a torsion tension than in case of (AQ-ppz)-Asp.
1
−1
around 380 nm. However, there are perceivable differences in the
shape of this bands and the relation of their intensity to the long-
1
wavelength maxima. In the case of (AQ-ppz)-Asp the ratio ε
1
/εmax is
1
0
.9, which is the highest among the studied derivatives. This difference
is noticeable even when we compare this ratio for deprotonated forms,
except that its value is then the lowest (0.6), very similar to simple (AQ-
ppz) compound.
The effect of absorption decrease upon protonation also applies to
the band around 380 nm. The only exception is the (AQ-ppz)-Asp deriv-
ative, which does not show significant spectral changes neither in the
position nor in the intensity of maxima under the influence of acid.
These distinctive features of (AQ-ppz)-Asp spectral characteristics
suggest, that there are some interactions governing specifically the be-
havior of this derivative. In studied compounds, the amino acids are in-
corporated into the 1-(piperazin-1-yl)-9,10-anthraquinone (AQ-ppz)
by the amide bond between the amino acid's carboxyl group and the ni-
trogen atom of piperazine. In the case of acidic amino acids, there is one
additional carboxyl group of the side chain. Its presence opens the pos-
sibility to form hydrogen bonds with α-amino groups. However, these
interactions can be formed both, within one molecule or
intermolecularly.
Considering the length of the side chain in (AQ-ppz)-Asp, it is possi-
ble to form an intramolecular hydrogen bond creating a six-membered
ring (Fig. 2a). In (AQ-ppz)-Glu, containing one more methylene group,
it is less likely to form such hydrogen bond, due to the lower stability
of the resulting ring. For this compound, intermolecular hydrogen
bonds may be an alternative (Fig. 2b).
Table 2
a
pK values (with uncertainties) determined by the pH-spectrophotometric method in ace-
tonitrile:methanol (9:1) mixture.
One of the techniques to assess the presence of hydrogen bonds is
infrared spectroscopy. Comparing IR spectra (Fig. 3) of three deriva-
tives: (AQ-ppz)-Asp, (AQ-ppz)-Glu, (AQ-ppz)-pGlu leads to interesting
conclusions. The spectrum for asparagine derivative strikingly resem-
bles the one for the pyroglutamic one, even though the chemical struc-
ture of (AQ-ppz)-Asp is much closer to (AQ-ppz)-Glu than to (AQ-ppz)-
pGlu (containing the lactam ring). The bands assigned to vibrations of
Compound
pKa1
pKa2
(
(
AQ-ppz)-Asp
AQ-ppz)-Glu
(AQ-ppz)-pGlu
(AQ-ppz)-His
AQ-ppz)-Lys
(AQ-ppz)-Arg
8.20 ± 0.01
7.99 ± 0.01
8.84 ± 0.01
8.03 ± 0.02
7.93 ± 0.01
8.22 ± 0.02
15.38 ± 0.01
–
–
–
(
15.21 ± 0.41
−
1
–
carbonyl groups, in the 1600–1700 cm range, are clearly visible for

8
S. Ramotowska et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117226
Fig. 4. (AQ-ppz)-His: a) UV–Vis spectrum of acid titration in acetonitrile:methanol (9:1) mixture. The arrows indicate the direction of changes. b) Spectrophotometric titration curve A as a
function of pH at λ = 485 nm.
3
.3. Acid-base properties
of the basic center from the 9,10-anthraquinone ring and the specific
structures formed by molecules. All pK values obtained by us applying
a
It is well known that the pH of the solution can strongly influence
pH-spectrophotometric titrations are collected in Table 2.
the spectroscopic properties of chromophores [45]. In order to under-
stand the behavior of derivatives in acidic and alkaline solutions and
to obtain information about the protolytic forms of molecules at differ-
ent pH values, the acid dissociation constants (pK ) of studied com-
a
pounds were determined.
The basicity of presented 9,10-anthraquinone derivatives is dictated
by nitrogen atoms of the piperazine ring and amino acids' moieties. The
diversity of amino acid side chains is the main source of differences in
acid-base properties. Due to the presence of many groups capable of
accepting a proton, one can expect the occurrence of several acid-base
equilibria. However, considering studied pH range and the effectiveness
of the chromophore, we managed to determine one or two acid dissoci-
ation constant values for each derivative. The ability of a chromophore
to signal the successive stages of protonation depends on the distance
For all derivatives, pKa1 values oscillating around 8 (from 7.93 to
8.22) were determined. They are assigned to the protonation of pipera-
zine nitrogen atom directly attached to the anthraquinone system. This
is justified by the value of dissociation constant, which is clearly close to
the pKa1 of (AQ-ppz) - 7.80 in a acetonitrile:methanol mixture. It is also
confirmed by significant changes in the UV–Vis spectra during the titra-
tion. The maximum absorption associated with the ICT transition
(~480 nm) disappears almost completely, without any shifts. Similar ef-
fect is also observed for absorption band at 380 nm. See representative
spectrum in Fig. 4.
For the (AQ-ppz)-Lys and (AQ-ppz)-Asp derivatives, two values of
the dissociation constant were determined. Only in the case of the
(AQ-ppz)-Lys derivative, the occurrence of these two equilibria is visible
during the acid titration. There are two types of changes on the
Fig. 5. (AQ-ppz)-Lys: a) UV–Vis spectrum of acid titration in acetonitrile:methanol (9:1) mixture. The arrows indicate the direction of changes. b) Spectrophotometric titration curve A as a
function of pH at λ = 490 nm.

S. Ramotowska et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117226
9
Fig. 6. (AQ-ppz)-Asp: a) UV–Vis spectrum of base titration in acetonitrile:methanol (9:1) mixture. The arrows indicate the direction of changes. b) Spectrophotometric titration curve A as
a function of pH at λ = 503 nm.
spectrum (Fig. 5a). In the first stage, a slight hypo- and hypsochromic ef-
fects (5 nm) are observed, followed by almost complete disappearance
of the long-wavelength band. Presence of two equilibria is also con-
firmed by an isosbestic points observed only for a certain concentration
range and two visible equivalence points on curves A = f(pH) (Fig. 5b).
The pKa2 value for (AQ-ppz)-Lys (15.21) is indicative of the proton-
ation of a more basic nitrogen atom than the one directly linked to the
anthraquinone moiety. This is also confirmed by the slight change in
the position of long-wavelength band in this pH range. The observed
hypsochromic effect is associated with a small change in the electron
density of the chromophore nitrogen atom during the protonation of
the peripheral group. However, the ε-amino group should be excluded,
due to the distance from anthraquinone unit, that makes it difficult to
signal the attachment of a proton. Also piperazine nitrogen atom unre-
lated with chromophore, may be eliminated, due to its involvement in
an amide bond, which reduces its basicity in comparison to (AQ-ppz).
Thus, the value of the pKa2 constant corresponds to the protonation of
the α-amino nitrogen atom.
quasi-reversible process. This is assigned to the first cathode peak ob-
served at the potential value of about −1 V (Ec1). At lower potential
values (Ec2), the second step of reduction, corresponding to the forma-
tion of a quinone dianion Q , takes place, and this process is irrevers-
ible. The values of reduction and oxidation potentials for all measured
derivatives and the peak potential separation (ΔE) are shown in Table 3.
Analyzing the presented values, one can notice, that the potential of
the AQ-ppz-Glu anodic signal (−0.735 V) and consequently, the value of
2−
1
ΔE (282 mV) stands out from the others. However, when registering
only the first reduction process, the potential of the anodic response
stays at a typical value for other derivatives (−0.936 V). This means
that the dianion (Q2 ), formed in the second reduction process, un-
dergoes a chemical transformation into a compound which oxidases at
much higher potential. For quinone derivatives an anode peak at
−
−
about −0.735 V is assigned to the oxidation of the protonated HQ
−
−
2−
form. The HQ is a product of a radical (Q• ) or dianion (Q ) reaction
with traces of water in the solvent [48]. The presence of this peak only
when the dianion is formed, indicates that it is the Q2 form that reacts
with water molecules according to the equation:
−
In view of the strong bathochromic shift observed in the alkaline en-
vironment in the case of the (AQ-ppz)-Asp derivative, the titration was
also carried out with the tetrabutylammonium hydroxide solution
Q2 þ H2O⇄HQ þ OH
−
−
−
ð7Þ
(
(
Fig. 6). The calculated pKa2 constant (15.38) is very similar to the
AQ-ppz)-Lys derivative. This confirms the assignment of this acid-
Voltammograms registered in DMSO solutions containing different
amounts of strong acid (methanesulfonic) or base
base equilibrium to the α-amino nitrogen atom. The specific intense
spectral changes accompanying proton dissociation in the alkaline envi-
ronment, once again distinguishes the (AQ-ppz)-Asp derivative from all
other studied compounds and confirms the dissimilarity between the
aspartic and glutamine derivative structures.
(tetrabutylammonium hydroxide in methanol), show a series of con-
secutive changes in the electrochemical behavior of the studied deriva-
tives. For all compounds, the increasing concentration of hydrogen ions
in the solution causes shifting of the cathodic and anodic peaks towards
the positive values of the potential. However, the origin and intensity of
these shifts are different. We can recognize three types of them.
3
.4. Cyclic voltammetry
In a non-aqueous medium, in aprotic solvents like acetonitrile or di-
Table 3
methyl sulfoxide, the reduction of quinones, including 9,10-anthraqui-
none, takes place in two stages. The mechanism of the electrochemical
process consisting of two consecutive electron transfer steps is called
the EE mechanism [27,46]. However, the literature reports that this
mechanism is not always operative for quinones [47].
The values of the reduction (cathodic) potential peaks (E
c
), oxidation (anodic) potential
peaks (E ), and the peak potential separations (ΔE) for studied derivatives.
a
Compound
E
c1 [V]
Ea1 [V]
ΔE
1
[mV]
Ec2 [V]
(
(
AQ-ppz)-Asp
AQ-ppz)-Glu
−1.047
−1.017
−1.060
−1.017
−1.027
−0.997
−0.946
−0.735
−0.969
−0.926
−0.937
−0.916
101
282
91
91
90
−1.510
−1.510
−1.583
−1.510
−1. 561
−1.500
For all studied derivatives two separate cathodic peaks were ob-
served in alkaline environment, resulting from two successive one-
electron reduction steps, characteristic for anthraquinones. The first
(AQ-ppz)-pGlu
(AQ-ppz)-His
(AQ-ppz)-Lys
−
(AQ-ppz)-Arg
81
step, corresponding to the formation of a semiquinone radical Q• , is a

10
S. Ramotowska et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 222 (2019) 117226
which, when protonated, affects the acid-base equilibrium of the qui-
none system.
Another set of molecules with distinctive electrochemical features
-
5
-
1,0x10
consists of (AQ-ppz)-pGlu, (AQ-ppz)-Lys and (AQ-ppz)-Arg (Fig. 8).
For these compounds, the increase of hydrogen ions' concentration re-
sults in the appearance and gradual intensification of only one reduction
signal. The direction of the shift is the same in each case - towards pos-
itive values of the potential, but they appear at significantly different po-
tentials: −0.366 V, −0.896 V and −0.806 V for the (AQ-ppz)–pGlu, (AQ-
ppz)-Lys and (AQ-ppz)-Arg derivatives respectively.
Different values of the ΔE (between the original Ec1 and the prior sig-
nals recorded in excess of acid) indicate that the nature of the prior
peaks is completely different. The low value of ΔE for (AQ-ppz)-Lys
and (AQ-ppz)-Arg indicates the formation of hydrogen bonded forms
only. Whereas, in the case of the (AQ-ppz)-pGlu derivative, this differ-
ence is much greater and amounts to 0.694 V. This suggests the reduc-
tion of the protonated form of the molecule. The absence of additional
signals indicates the lack of hydrogen bonded structures, which may
be due to the rigidness of the lactam ring present in this compound.
Within this group, different effects are also observed for anodic re-
sponses. In the case of the (AQ-ppz)-pGlu derivative, the first anode
peak shifts towards the higher potential values and occurs in the tested
range of H ion concentrations at three or four positions.
-
6
-
5,0x10
0
,0x10
,0x10
,0
alcaline pH
-6
5
1
-5
acidic pH
-1,5
0,0
-0,5
-1,0
E vs. Ag/AgCl [V]
Fig. 7. Cyclic voltammograms of (AQ-ppz)-Asp registered in different pH.
On voltammograms registered at various pHs for three derivatives
(AQ-ppz)-Asp, (AQ-ppz)-Glu and (AQ-ppz)-His), we observed a simi-
(
lar type of changes. The deprotonated form of (AQ-ppz)-Asp is charac-
terized by two reduction peaks, at −1.047 V and − 1.510 V. The
addition of an acid in the first stage results in the appearance of the an-
odic response at the potential of –0.745 V. In the next step we observe
the disappearance of the second reduction peak and moreover - the ap-
pearance of a prior peak at the more positive values of the potential
4. Conclusions
Six newly designed and synthesized amino acids' derivatives of
piperazine-anthraquinone show unprecedented differences in spectro-
scopic and electrochemical characteristics, which originate from intra-
molecular and intermolecular hydrogen bonds. Particularly
noteworthy are the derivatives of dicarboxylic amino acids: (AQ-ppz)-
Asp and (AQ-ppz)-Glu. Considering their similarity in molecular struc-
ture, we noticed unexpectedly large dissimilarities in their spectral fea-
tures. The most stable-form of the (AQ-ppz)-Asp derivative is stabilized
by strong intramolecular hydrogen bonds. Whereas, in case of the (AQ-
ppz)-Glu - the dimeric form is favorable, which is confirmed by the re-
sults of both: quantum-chemical simulations and IR spectra.
Another important finding is relates to the signaling potential of
studied compounds. Namely, the specific architecture of the asparagine
derivative positively affects the sensitivity of the chromophore, en-
abling spectroscopic observation of the deprotonation of peripheral
amino acid groups.
The voltammetric measurements, in turn, show the wide variety of
effects. They are associated with both – the protonation and
hydrogen-bonding. Despite the fact, that only the anthraquinone moi-
ety undergoes the redox reactions, our studies clearly demonstrate
that the presence of (even distant) amino acids' substituents modulates
the electrochemical signals. This precise reflection of structure-
properties linkage makes these derivatives even more promising agents
for application in sensors.
(
−0.815 V). In the excess of methanesulfonic acid, that signal also van-
ishes and a reduction peak at the potential of –0.704 V occurs (Fig. 7).
Intermolecular and intramolecular hydrogen bonds are two differ-
ent types of interactions which have been described for quinone deriv-
atives [28,49,50]. The possibility of hydrogen bonding stabilization of
electrochemically generated quinone radical anions provokes a shift of
the reduction potentials towards more positive potentials [28,51]. Anal-
ysis of the interaction of quinone derivatives with strong acids, carried
out by Gupta [28], showed that the first prior peak of the original reduc-
tion is related to a hydrogen bonding interactions. In turn, the appear-
ance of a second prior peak is associated with the reduction of
hydroquinone in the excess of acid. The difference in its position, in
the reference to the original reduction peak, amounts to about 0.66 V.
In our studies, for (AQ-ppz)-Asp derivative we observed the similar
changes, but the value of potential shift is only 0.353 V. This may be
due to the presence of peripheral acid-base groups in the molecule,
-5
-
1,0x10
5,0x10
-6
-
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0
,0x10
,0x10
,0
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