Synthesis, protonation constants and biological activity determination of amino acid–salicylaldehyde-derived Schiff bases

Article abstract of DOI:10.1007/s00726-019-02816-0

Schiff bases represent a class of molecules widely studied for their importance in organic and coordination chemistry. Despite the large amount of studies on the chemical and biological properties of the Schiff bases, the different experimental conditions

Full text of DOI:10.1007/s00726-019-02816-0

Amino Acids
https://doi.org/10.1007/s00726-019-02816-0
ORIGINAL ARTICLE
Synthesis, protonation constants and biological activity determination
of amino acid–salicylaldehyde‑derived Schif bases
Claudia Fattuoni1 · Sarah Vascellari2 · Tiziana Pivetta1
Received: 24 September 2019 / Accepted: 27 December 2019
© Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract
Schif bases represent a class of molecules widely studied for their importance in organic and coordination chemistry. Despite
the large amount of studies on the chemical and biological properties of the Schif bases, the diferent experimental conditions
prevent a useful comparison to search for a correlation structure–activity. Moreover, literature is lacking in comprehensive
data on the spectroscopic characterization of these compounds. For this reason, six Schif bases, derived from salicylalde-
hyde and natural amino acids were fully characterized by nuclear magnetic resonance and infrared spectroscopy, and their
aqueous solution equilibria, antiproliferative activity and DNA-binding activity were examined. All experimental conditions
were kept constants to achieve comparable information and useful insights about their structure–activity correlation. The
synthesized compounds showed DNA binding constants in the 101–102 M−1 range, depending on the substituent present in
the amino acid side-chain, and resulted devoid of signifcant cytotoxic activity against the diferent human tumor cell lines
showing IC50 values higher than 100 µM.
Keywords Schif bases · l-Amino acids · DNA binding · Cytotoxicity · Aqueous solution equilibria
Introduction
and Aljahdali 2013; Rajesh and Ray 2014). In organic syn-
thesis, Schif bases are useful building blocks in addition,
hetero Diels–Alder (Dossetter et al. 1999) and Staudinger
(Palomo et al. 1999) reactions. The last is widely used for
the preparation of β-lactams, a widely used class of antibi-
otics, by reaction of a Schif base with ketenes (Turan et al.
2016). Other Schif bases show biological activity against
fungi and bacteria (Malik et al. 2018; Antony et al. 2019).
In addition, some derivatives have been recently proposed
for the treatment of Alzheimer’s disease, as metal binding
agents able to promote the disaggregation of amyloid-β
plaques (Gomes et al. 2014). Thanks to their soft–hard
donor character, Schiff bases are commonly used as
ligands for several metal ions. They are used to recognize
anions and cations and their application in biochemical
and environmental felds has been proposed (Dalapati et al.
2014). Moreover, complexes formed by Schif bases with
metals such as Cu (II) (Qiao et al. 2011), Cd (II) (Zhang
et al. 2012), Ru (II) (Chow et al. 2014), Zn (II) (Zhong
et al. 2006) and lanthanides (Yang et al. 2000) show anti-
tumor activity. Recently the applications of Schif bases
and their metal complexes in the feld of chiral catalysis,
functional materials and antitumor bioactive substances
Schif bases, also known as imines or azomethines and
named after their discoverer Hugo Schif, are condensa-
tion products of primary amine and aldehydes or ketones
(Qin et al. 2013). Schif bases are important intermediates
in bio-processes such as the transamination reaction or
the protein glycation and are involved in racemization and
decarboxylation reactions (Vilanova et al. 2012; El-Sherif
Handling Editor: E. R. Perez Gonzalez.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00726-019-02816-0) contains
supplementary material, which is available to authorized users.
* Claudia Fattuoni
cfattuon@unica.it
* Tiziana Pivetta
tpivetta@unica.it
1
Department of Chemical and Geological Sciences,
University of Cagliari, Cittadella Universitaria, Monserrato,
09042 Cagliari, Italy
2
Department of Biomedical Sciences, University of Cagliari,
Cittadella Universitaria, Monserrato, 09042 Cagliari, Italy
Vol.:(0123456789)
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C. Fattuoni et al.
have been reviewed (Pessoa and Mannar 2017; Liu et al.
2018; Banti et al. 2019). The potential development of
pharmaceutical applications of these compounds requires
a proper knowledge of their chemical and biological reac-
tions and also of their toxicological profle. Despite the
high level of interest in the scientifc community for these
molecules, a literature survey highlighted a general lack
of consistency. In fact, although Schif bases have been
widely studied under diferent points of view, a direct
comparison of the reported data is not possible because
of the diferent experimental conditions. This problem is
relevant for the aqueous solution equilibria study, since
protonation constants of Schif bases belonging to the
same family are often determined in diferent solvents,
temperatures, and ionic bufers (el Sherif and Aljahdali
2013; Galić et al. 1997; Metzler et al. 1980; Turkoglu et al.
2011). Due to the fundamental importance of the proto-
nation constants in the study of the complex formation,
it appears necessary that all experiments are carried out
in the same conditions at least for homologous series of
compounds. Another discrepancy in the literature data is
the lack of nuclear magnetic resonance (NMR) charac-
terization of several amino acid derived Schif bases: in
some case only the melting point was reported, or only
Scheme 1 Schif bases synthesized from salicylaldehyde and amino
acids l-alanine (a), l-valine (b), l-cysteine (c), l-serine (d), l-arginine
(e), l-histidine (f)
of human tumor cell lines (SK-MES-1, DU-145, Hep-G2,
CCRF-CEM, CCRF-SB) were also examined.
Experimental
Calf thymus DNA sodium salt, hydrochloric acid standard
solutions, diethyl ether, dimethylsulfoxide-d6 (DMSO-d6),
ethanol, l-alanine, l-arginine, l-cysteine, l-histidine, l-ser-
ine, l-valine, methanol, 1,4-piperazinediethanesulfonic acid
(PIPES), potassium bromide, salicylaldehyde, sodium bro-
mide, sodium chloride, sodium hydroxide pellets, sodium
hydroxide standard solution and sodium sulfate were pur-
chased from Sigma-Aldrich and used without any further
purifcation. Infrared spectra of the studied compounds were
recorded using a Bruker Equinox 55 spectrophotometer,
1
the H-NMR chemical shift data. Some molecules have
never been characterized, such as the salicylaldehyde/
serine or histidine derivatives. Moreover, some reported
data are incomplete or inconsistent. For the above reported
considerations, we decided to synthesize six Schif bases
from salicylaldehyde and proteinogenic l-amino acids, and
to study their properties in the same experimental con-
ditions to have comparable information on their chemi-
cal and biological behavior. In particular, we studied the
aqueous solution equilibria, the cytotoxic activity and
the DNA binding ability of l-salicylidenealanine, N-[(2-
hydroxyphenyl)methylene]-l-alanine (1a), l-salicylide-
nevaline N-[(2-hydroxyphenyl)methylene]-l-valine (1b),
l-salicylidenecysteine N-[(2-hydroxyphenyl)methylene]-
l-cysteine (1c), l-salicylideneserine N-[(2-hydroxyphe-
nyl)methylene]-l-Serine (1d), l-salicylidenearginine
N-[(2-hydroxyphenyl)methylene]-l-arginine (1e), and
l-salicylidenehistidine N-[(2-hydroxyphenyl)methylene]-
l-histidine (1f) (Scheme 1). The chosen compounds were
prepared modifying literature methods, to improve the
product yield. All compounds were fully characterized by
elemental analysis, NMR (1H- and 13C-NMR) and Infrared
(IR) spectroscopy techniques. The protonation constants
of the compounds were determined by potentiometric
and spectrophotometric titrations, carried out simultane-
ously in aqueous solution at 25 °C in 0.1 M ionic strength
(NaCl). The interaction of 1a–f bases with calf thymus
DNA (ct-DNA) and the cytotoxic activity against a panel
1
in KBr medium (pellets). H and 13C NMR spectra were
recorded on Varian 400 and 500 spectrometers. Proton and
carbon chemical shifts in DMSO-d6 were referenced to the
residual solvent signals (1H NMR, δ = 2.60 ppm; 13C NMR,
δ = 39.6 ppm). Melting points (mp) were measured using a
Kofer hot stage microscope and are uncorrected.
Synthesis of l‑salicylideneaniline (1a)
Compound 1a was prepared according to a literature method
(Hsieh et al. 2007). A mixture of l-alanine (0.8 g, 9 mmol),
salicylaldehyde (1 mL, 9 mmol) and Na2SO4 (4 g) in metha-
nol (150 mL) was stirred under refux for 12 h. The solid
was fltered, and the solvent was removed under reduced
pressure to give 1.6 g (90% yield) of light brown solid, mp
138–142 °C (dec). 1H NMR (400 MHz, DMSO-d6): δ 8.59
(s, 1H, CH=N), 7.46 (m, 1H, Ar–H), 7.35 (m, 1H, Ar–H),
6.90 (m, 2H, Ar–H), 4.21 (q, 1H, CH–CO2H, J = 6.7), 1.44
(d, 3H, CH3, J = 6.7). 13C NMR (125 MHz, DMSO-d6):
δ 19.48, 64.79, 116.60, 117.36, 118.69, 131.93, 132.69,
160.62, 166.61, 173.29. IR (KBr): ν/cm−1 3418, 3090,
2604, 1630, 1595, 1452, 1409, 1356, 1309, 1117, 853, 542.
1 3

Synthesis, protonation constants and biological activity determination of amino acid–…
Elemental analysis found (calc. for C10H11NO3): C% 61.80
yield), mp 220 °C (dec). 1H NMR (500 MHz, DMSO-d6): δ
(62.17), H% 5.64 (5.74), N% 7.21 (7.25).
3.69 (dd, 1H, CH2, J = 7.5 and 11 Hz), 3.86 (dd, 1H, CH2, J
= 4 and 11 Hz), 4.09 (dd, 1H, CH–CO2H, J = 4 and 7.5 Hz),
6.90 (m, 2H, Ar–H), 7.35 (t, 1H, Ar–H, J = 7.5 Hz), 7.46
(d, 1H, Ar–H, J = 7.5 Hz), 8.53 (s, 1H, CH=N). 13C NMR
(125 MHz, DMSO-d6): δ 62.7, 72.5, 116.7, 118.5, 118.7,
132.0, 132.7, 160.9, 167.6, 171.4. IR (KBr): ν/cm−1 3444,
3054, 2947, 1635, 1618, 1603, 1507, 1472, 1416, 1348,
1313, 1125, 1015, 615, 527. Elemental analysis found (calc.
for C10H11NO4): C% 57.46 (57.41), H% 5.25 (5.30), N%
6.31 (6.70).
Synthesis of l‑salicylidenevaline (1b)
Compound 1b was prepared as reported for 1a to give 1.22 g
(62% yield) of light yellow solid, mp 162–164 °C. 1H NMR
(500 MHz, DMSO-d6): δ 0.89 (d, 3H, CH3, J = 4 Hz), 0.91
(d, 3H, CH3, J = 4.5 Hz), 2.26 (m, 1H, CH–CH3), 3.82 (m,
1H, CH–CO2H), 6.89 (m, 2H, Ar–H), 7.35 (t, 1H, Ar–H,
J = 7.6 Hz), 7.45 (d, 1H, Ar–H, J = 7.6 Hz), 8.53 (s, 1H,
CH=N). 13C NMR (100 MHz, DMSO-d6): δ 17.8, 19.3,
31.1, 75.9, 116.6, 118.6, 118.7, 132.0, 132.7, 160.8, 167.4,
172.4. IR (KBr): ν/cm−1 3430, 2961, 2626, 1648, 1589,
1511, 1394, 1330, 757, 541. Elemental analysis found (calc.
for C12H15NO3): C% 64.98 (65.14), H% 6.84 (6.83), N%
6.21 (6.33).
Synthesis of l‑salicylidenearginine (1e)
Compound 1e was prepared as reported for 1c to give a
bright yellow solid (2.45 g, 98% yield), mp 200–202 °C. 1H
NMR (500 MHz, DMSO-d6): δ 1.58 (m, 2H, CH2CH2CH2),
1.83 (m, 1H, CHCH2), 2.01 (m, 1H, CH–CH2), 3.18 (m,
2H, CH2–NH), 3.88 (m, 1H, CH–COOH), 6.86 (m, 2H,
Ar–H), 7.36 (t, 1H, Ar–H, J = 8 Hz), 7.45 (d, 1H, Ar–H,
J = 8 Hz), 8.52 (s, 1H, CH=N), 9.60 (s, broad, 1H, NH).
13C NMR (100 MHz, DMSO-d6): δ 26.0, 31.6, 41.4, 71.3,
117.4, 118.2, 119.4, 133.2, 134.5, 157.5 (NH2-C = NH),
165.3, 166.8, 174.8. IR (KBr): ν/cm−1 3407, 3315, 3078,
2965, 2865, 1662, 1650, 1634, 1591, 1474, 1377, 1343,
1192, 1149, 777, 536. Elemental analysis found (calc. for
C13H18N4O3): C% 56.16 (56.10), H% 6.59 (6.52), N% 20.08
(20.13).
Synthesis of l‑salicylidenecysteine (1c)
A mixture of l-cysteine (1.09 g, 9 mmol) and salicylalde-
hyde (1 mL, 9 mmol) in ethanol (50 mL) was stirred under
refux for 12 h (Pillai et al. 1999). The solid product was
fltered, washed with ethanol and dried. The white solid
obtained (1.75 g, 86% yield) showed in the NMR analysis
to be formed by two isomers (E and Z) of compound 1c, mp
1
166–167 °C. H NMR (500 MHz, DMSO-d6): δ (E) 2.97
(dd unresolved, 1H, CH2, J = 9.5 Hz), 3.34 (dd, 1H, CH2,
J = 7 and 9.5 Hz), 4.20 (m, 1H, CH–CO2H), 5.65 (s, 1H,
CH=N), 6.81 (t, 2H, Ar–H, J = 7 Hz), 7.13 (t, 1H, Ar–H,
J = 7.5 Hz), 7.34 (d, 1H, Ar–H, J = 7 Hz); δ (Z) 3.03 (dd,
1H, CH2, J = 5 and 10 Hz), 3.20 (dd, 1H, CH2, J = 7 and
10 Hz), 3.82 (m, 1H, CH–CO2H), 5.85 (s, 1H, CH=N), 6.76
(m, 2H, Ar–H), 7.06 (t, 1H, Ar–H, J = 7.5 Hz), 7.30 (d,
1H, Ar–H, J = 7.5 Hz). 13C NMR (100 MHz, DMSO-d6): δ
(E) 37.11 (CH2), 64.85 (CH–C=O), 65.69 (CH=N), 115.15
(CAr–H), 118.81 (CAr–H), 127.67 (CAr–C), 126.16 (CAr–H),
128.16 (CAr–H), 155.23 (CAr–OH), 172.98 (C=O); δ (Z) 38.21
(CH2), 65.26 (CH–C=O), 67.73 (CH=N), 115.74 (CAr–H),
119.10 (CAr–H), 124.30 (CAr–C), 127.94 (CAr–H), 129.09
(CAr–H),154.65 (CAr–OH),172.51 (C=O). IR (KBr): ν/cm−1
3438, 3100, 2701, 2578, 1622, 1598, 1577, 1457, 1384,
1334, 1283, 760, 679. Elemental analysis found (calc. for
C10H11NO3S): C% 53.64 (53.32), H% 5.06 (4.92), N% 6.31
(6.22), S% 13.87 (14.23).
Synthesis of l‑salicylidenehistidine (1f)
A mixture of l-histidine (0.7 g, 4.5 mmol) and salicylalde-
hyde (1 mL, 9 mmol) in ethanol (50 mL) was stirred under
refux for 24 h. The solid product was fltered, washed with
ethanol and dried. Product 1f was obtained as a bright yel-
1
low solid (0.9 g, 77% yield), mp 174–180 °C. H NMR
(400 MHz, DMSO-d6): δ 3.00 (dd, 1H, CH2, J = 8.4 and
14.6 Hz), 3.18 (dd, 1H, CH2, J = 4.8 and 14.6 Hz), 4.32 (dd,
1H, CH-CO2H, J = 4.8 and 8.4 Hz), 6.77 (s, 1H, C=CH–N),
6.85 (m, 2H, Ar–H), 7.34 (m, 2H, Ar–H), 7.59 (s, 1H,
NH–CH=N), 8.32 (s, 1H, CH=N). 13C NMR (125 MHz,
DMSO-d6): δ 31.22, 70.19, 116.57, 118.59, 119.49, 129.28,
131.86, 132.63, 134.84, 136.46, 160.62, 166.93, 172.38. IR
(KBr): ν/cm−1 3422, 3122, 3018, 2879, 1638, 1531, 1470,
1416, 1341, 1267, 1149, 838, 624, 535. Elemental analysis
found (calc. for C13H13N3O3): C% 60.19 (60.22), H% 4.97
(5.05), N% 16.24 (16.21).
Synthesis of l‑salicylideneserine (1d)
A mixture of l-serine (0.95 g, 9 mmol) and salicylaldehyde
(1 mL, 9 mmol) in ethanol (50 mL) was stirred under refux
for 2.5 h, then kept in the refrigerator overnight. The solid
product was fltered, washed with ethanol, and dried. Prod-
uct 1d was obtained as a light brown solid (1.00 g, 53%
Potentiometric and spectrophotometric titrations
Potentiometric titrations were carried out in a thermostated
vessel with a Mettler-Toledo Seven Compact pH/Ion-meter,
equipped with a Mettler-Toledo InLab Micro Pro combined
1 3

C. Fattuoni et al.
glass electrode with an integrated temperature probe. Poten-
tiometric titrations were performed at 25 °C in 0.1 M ionic
strength (NaCl) under N2 atmosphere. The glass electrode
was calibrated daily by titration of a known amount of HCl
with carbonate-free NaOH standard solution. Electrode
standard potential (E0), water ionic product (pKw), elec-
trode response and carbonate content of the titrant solution
were checked with Gran’s procedure (Gran 1952) using the
Glee software (Gans and O’Sullivan 2000). The UV–Vis-
ible (UV–Vis) measurements were carried out with a Var-
ian Cary 60 spectrometer equipped with an optical fber dip
probe with a 1 cm optical path length.
temperature. The DNA binding constants were obtained by
using the Hyperquad 2003 software (Gans et al. 1996).
Cell lines
Cell lines were purchased from the American Type Culture
Collection (ATCC) and were derived from: lung squamous
carcinoma (SK-MES-1, ATCC number HTB-SB); prostate
carcinoma (DU-145, ATCC number HTB-B1); hepatocel-
lular carcinoma (Hep-G2, ATCC number HB-8065); acute
T-lymphoblastic leukemia (CCRF-CEM, CRL-8436); acute
B-lymphoblastic leukemia (CCRF-SB, ATCC number CCL-
120). All cell lines were grown at 37 °C in a 5% CO2 atmos-
phere, in their specifc media according to ATCC instruc-
tions, in the presence of 5–10% fetal bovine serum (FBS),
antibiotic and, unless otherwise indicated, sodium pyruvate.
All cell cultures were maintained in exponential growth by
periodically splitting high density suspension cultures (i.e.
106 cell/mL) or when cell monolayers reached sub-confu-
ence (70–90% confuence). The absence of mycoplasma con-
tamination was checked periodically by the Hoechst staining
method (Latt et al. 1975).
Protonation constants
Protonation constants of compounds 1a–f were determined
by spectrophotometric and potentiometric titrations, at 25 °C
in 0.1 M ionic strength (NaCl), following the spectral vari-
ations due to the additions of the titrant. Solutions of ligand
were prepared daily by dissolving the proper amount of
the compound in freshly distilled water. Concentrations of
compounds 1a–f ranged from 2.8 × 10–4 to 1.0 × 10–3 M,
according to their absorptivity and solubility. Solutions con-
taining a known amount of compound and HCl (necessary
to fully protonate the molecule) were titrated with NaOH
standard solution. The reversibility of the involved equilibria
was checked by back-titration with standard HCl. The over-
all stability constants were determined with the Hyperquad
2003 software (Gans et al. 1996).
Cytotoxic assays
The cytotoxic efect of the compounds 1a–f was evaluated
in cell lines during the exponential growth stage. Stock solu-
tions of 1a–f were stored at 4 °C in the dark. For the evalu-
ation of cytotoxicity, solutions of compounds 1a–f were
serially diluted in growth medium specifc for the difer-
ent cell lines. Suspension cell lines were seeded in 96-well
plates at an initial density of 1 × 105 cells/mL in specifc
growth medium, with or without serial dilutions of each
compound. Adherent cell lines were seeded at an initial
density of 1 × 104 cells per well and incubated overnight
before adding serial dilutions of the test compound. Cell
viability was determined after 96 h at 37 °C, 5% CO2, by
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) method as previously described (Pauwels
et al. 1988). Cell growth at each drug concentration was
expressed as the percentage of untreated controls. The
results were expressed as IC50, i.e. the concentration of
compound required to reduce the viability of the tested cells
by 50%. The antitumor agent vincristine was used as a refer-
ence compound.
DNA binding
The binding constants (Kb) between ct-DNA and com-
pounds 1a–f were determined at 25 °C by spectrophotomet-
ric titrations in PIPES bufer 0.01 M at pH 7.0. A stock
solution of ct-DNA in 0.01 M PIPES bufer at pH 7.0 was
prepared and stored at 4 °C. This solution was used within
4 days. The concentration of DNA per nucleotide was
determined by UV absorption at 260 nm using its molar
absorption coefcient (6600 M−1 cm−1) (Reichmann et al.
1954). The purity of the DNA was checked by monitoring
the ratio of the absorbance at 260 nm to that at 280 nm. A
ratio higher than 1.8 indicates a DNA sufciently protein-
free (Murmur 1961). Twenty solutions containing a fxed
amount of the compounds (ranging from ≈ 2.03 × 10–4 to ≈
6.07 × 10–4 mmol, according to the absorptivity) and vari-
able amounts of DNA (ranging from 6.09 × 10–4 to 1.82 ×
10–3 mmol) were prepared. The stock solution of 1c was
prepared at least 39 h before the experiment due to its slow
dissolution in PIPES. Since the interactions between DNA
and compounds 1a–f are slow processes, spectra in the
200–500 nm range were recorded when equilibration was
reached (15 h). All solutions were stored in the dark at room
Results and discussion
Literature reports many diferent synthetic methods to pre-
pare amino acid-derived Schif bases (Smith et al. 1977; Rao
and Philipp 1991; Saleem et al. 2012; Ershov et al. 2013).
The classic method to synthesize Schif bases consists of
1 3

Synthesis, protonation constants and biological activity determination of amino acid–…
simply mixing equimolar amounts of amine and aldehyde in
ethanol and refuxing the mixture for some hours: the desired
product readily formed can be fltered and isolated. When
the amine role is played by an α-amino acid, the synthetic
procedure may not be so straightforward, due to the amino
acid zwitterionic character resulting in a weakly nucleophilic
amino group. Several modifcations of the classic method
have been reported. For example, potassium (Liu et al. 2012)
or sodium hydroxide (Hiskey and Jung 1963) was added
to an aqueous or methanolic solution of the amino acid to
enhance its nucleophilic character. An analogous efect is
obtained with a bicarbonate bufer (Rao and Philipp 1991).
On the other hand, the added base has the efect of promot-
ing the aldehyde self-reaction, leading to a lower yield of
the desired Schif base. We found that the simple mixing-
refux method can be applied only to cysteine and arginine.
To obtain a satisfactory yield from the other four amino
acids, the classic method must be modifed as follows: (i)
for the histidine derivative, a double amount of aldehyde
was used; (ii) for alanine and valine derivatives, anhydrous
sodium sulfate was added to the reaction mixture as reported
by Hsieh (Hsieh et al. 2007); (iii) for serine derivative, an
equimolar mixture of amino acid and aldehyde was refuxed
in ethanol for 2 h and the resulting solution was kept in
the refrigerator overnight. The high reactivity of cysteine
in the reported condition is noteworthy: in fact, after only
1 h at the refux temperature a thick white precipitate was
obtained. The NMR analysis revealed that it constituted by
two geometric isomers E and Z. The E isomer was the more
abundant, due to the lower steric hindrance: the correspond-
ence of the imino CH=N proton chemical shift value with
those reported (Maurya et al. 2008) allows its identifca-
tion. All attempts to prepare only one isomer, lowering the
reaction temperature or increasing the solvent amount, were
unsuccessful.
The protonation constants were determined by a basic
titration, measuring simultaneously the electrochemical
potential and the UV–Vis absorption. The absorbance val-
ues measured during the potentiometric titrations of 1a and
1b are reported vs wavelength and pH in Fig. 1 as an exam-
ple. Spectra recorded during the titrations of the other com-
pounds are reported in the Supporting material. The func-
tional groups involved in the protonation and the protonation
sequence were determined by comparing the pure UV–Vis
spectra of the formed species. No reliable NMR spectra in
water solution were obtained due to the low water solubility
of the compounds (< 1 mM).
Spectral evidences
By titrating the synthesized compounds, several equilibria
were spectrally evidenced in the 2–11.4 pH range: three for
Fig. 1 Absorbance as a function of wavelength and pH measured
during the titrations of 0.0240 mmol of 1a (a) and 0.0178 mmol
of 1b (b); 25 °C, 0.1 M NaCl ionic bufer, 1 cm optic path length.
Compound 1a: by titrating compound 1a with NaOH, three equi-
libria are spectrally evidenced in the pH range 2–11.4 (Fig. S1).
At pH 2, two peaks and a shoulder are present at 325, 257 and ≈
280 nm, respectively. Increasing the pH, the absorbance of the peaks
slightly decreases, not for the dilution as can be seen in the inset of
the Fig. S1B where the absorbance at 325 nm is reported corrected
for the dilution. The observed spectral variation is due to the depro-
tonation of the H3L+ species to form the H2L one. These two spe-
cies appear spectrally not well distinguishable. Increasing the pH till
8.8, the intensity of the peaks at 325 and 257 nm decreases while
two new peaks appear at 378 nm and 266 nm due to the formation
of the HL− species. Four isosbestic points are present at 340, 300,
265 and 247 nm. At pH higher than 8.8, the intensity of the peak at
378 raises while that of the peak at 265 diminishes and a shoulder if
formed at ≈ 300 nm, for the formation of the L2− species. Two isos-
bestic point are present at 283 and 259 nm. Selected spectra at spe-
cifc pH values are reported in Fig. S1; Compound 1b: the analysis
of the spectra recorded during the titration of 1b, gives evidence of
three equilibria (Fig. S2). At acidic pH two peaks are present at 252
and 325 nm. Increasing the pH till 8, the intensity of these two peaks
decreases, while two new peaks appear at 264 and 378 nm. Four isos-
bestic points are present at 342, 292, 265 and 245 nm. After pH 8,
the maximum of intensity shifts at 380 nm and three isosbestic points
are present at 342, 292 and 265 nm. After pH 9, the peaks shift at
374 nm with larger width, a poor resolved isosbestic point is present
at 332 nm. Selected spectra at specifc pH values are reported in Fig.
S2
1 3

C. Fattuoni et al.
Table 1 Protonation constants of compounds 1a–f at 25 °C and 0.1 M
1a, 1b, 1d, 1f, and four for 1c and 1e. Some spectra recorded
during the potentiometric titrations at diferent pH ranges
are reported in Figs. S1–S6. A detailed description of the
spectral variations for 1a and 1b is reported in Fig. 1 caption
as an example, while and for 1c–1f reported in the Support-
ing Fig. S7.
NaCl ionic strength
Compound
Species
Log β
pK
Overall stability constants
1a
1b
1c
HL−
10.2 (1)
10.2
8.1
< 2
9.4
8.8
H2L
18.30 (5)
19.8 (2)
9.40 (2)
18.2 (1)
21.5 (1)
10.14 (1)
18.72 (1)
26.72 (1)
29.80 (4)
11.0 (5)
20.3 (1)
27.8 (1)
28.5 (5)
9.81 (3)
18.52 (3)
26.09 (4)
28.2 (4)
10.7 (2)
20.15 (2)
27.33 (2)
29.3 (3)
H3L+
HL−
Calculation of the protonation constants
H2L
H3L+
HL2−
H2L−
H3L
3.3
From the eigenvalue analysis of the spectrophotometric data,
signifcant eigenvalues, interpreted as the number of linearly
independent absorbing species were found (Malinowski
2002). The concentration profle as a function of pH and
the pure UV–Vis spectra of all the absorbing species were
then calculated without model assumptions. The results are
shown in Fig. 2 for 1c and in Figs. S1–6 E and S1–6 F for
all the compounds.
10.14
8.58
8.00
3.08
11.0
9.3
H4L+
HL2−
H2L−
H3L
1d
1e
1f
7.5
< 2
H4L+
HL−
Three signifcant eigenvalues were found for 1a, four for
1b, 1d and 1f, and fve for 1c and 1e. Four eigenvalues were
expected for 1a and 1b and fve for the other compounds.
Any attempt to ft the experimental data considering four
eigenvalues for 1a or fve for 1d and 1f led to negative cal-
culated pure spectra. These results showed that H3L+ and
H2L species for 1a and H4L+ and H3L species for 1d and 1f
were not spectrally distinguishable. The potentiometric and
spectrophotometric data were ftted simultaneously suppos-
ing a model considering three protonation equilibria (from
L2− to H2L) for 1a and 1b, and four protonation equilibria
(from L3− to H4L+) for 1c, 1d, 1e, and 1f. The protonation
constants and the refned pure spectra were calculated. The
protonation constants are reported in Table 1. The pK values
related to the formation of the H3L+ and H4L2+ species for
1a and 1f, respectively, were outside the experimental pH
range.
9.81
8.71
7.57
≈ 2
10.7
9.45
7.18
< 2
H2L
H3L+
H4L2+
HL−
H2L
H3L+
H4L2+
The assignment of the protonation sequence was straight-
forward for compounds 1a and 1b. In fact, the frst pK was
related to the protonation of the phenolic oxygen atom, the
second one to the iminic nitrogen, and the third one to the
carboxylate group. To defne the order in which the func-
tional groups of 1c were protonated, the absorptivity spectra
Fig. 2 Model free concentration profle (a) and pure spectra (b), calculated with eigenvalues analysis of the spectrophotometric data collected
during the potentiometric titration of compound 1c (7.07 × 10–4 M, 25 °C, 0.1 M NaCl, 1 cm optical path length, 25 °C)
1 3

Synthesis, protonation constants and biological activity determination of amino acid–…
of the diferent species of 1c were compared with those of
1a. The spectra of the HL species (charges are omitted for
simplicity) of 1a and 1c were similar for the position and
absorptivity ratio of the bands. This suggests that the same
group was protonated in both species, i.e. the phenolic oxy-
gen. The spectrum of the H2L species of 1c was diferent
from that of the H2L species of 1a, where the iminic group
was protonated, and was similar to that of the HL species
of 1a, where the iminic group is still not protonated. Then
the second protonation in 1c involves the sulfur atom. The
spectra of the H3L species of 1c and that of the H2L spe-
cies of 1a being similar, it can be deduced that the third
protonation in 1c involves the nitrogen atom. Finally, the
fourth protonation occurs on the carboxylate group of 1c
(see Supporting, Scheme S1). For 1d, by comparing the pure
Fig. 3 Selected spectra recorded during the titration of 2 × 10–4 mmol
spectra of all the species formed by the studied compounds,
the protonation involves in the order: the oxygen of the side
chain, the phenolic oxygen, the imino group and lastly the
carboxylate group (Scheme S2). For compound 1e, consider-
ing the absorptivity spectra of the formed species and the pK
12.48 of the –NH2 group in the arginine side chain (Yoo and
Cui 2008), the frst protonation appears to involve the –NH2
group. The second protonation involves the phenolic oxygen,
the third one the C=N group and the last one the carboxylate
group. As regards compound 1f, comparing the spectra of
the absorbing species of all the studied compounds, and con-
sidering the protonation constants of histidine (Canel et al.
2006), the protonation involves in the order: the phenolic
oxygen, the histidine nitrogen atom, the imino group and
fnally the carboxylate group (Scheme S3).
of 1e with 0.002 M ct-DNA; 0.01 M PIPES bufer, 25 °C, 1 cm opti-
cal path length. The arrows show the absorbance changes occurred
during the titration
Table 2 DNA-binding constants
of 1a–f
Compound
Kb (M−1
)
1a
1b
1c
1d
1e
1f
48 (7)
69 (8)
229 (22)
110 (12)
148 (15)
447 (39)
DNA binding study
generic molecule interacting with DNA, D is the DNA
and LD is the formed adduct. The DNA binding constants,
defned as Kb = [LD]/[L][D], have been calculated by ft-
ting the experimental absorbance data supposing the for-
mation of the 1:1 compound-DNA adduct, according to
Pivetta (Pivetta et al. 2014, 2017). The Kb values range
from 48 to 447 (Table 2) and vary along the series 1f > 1c
> 1e > 1d > 1b > 1a.
The ability of compounds 1a–f to bind DNA has been stud-
ied by UV–Vis titration with ct-DNA. The interaction of a
molecule with DNA is known to result in a modifcation
of its UV–Vis spectrum. The type of changes (red or blue
shift, and hyper- or hypochromism) gives information on the
binding mode (Babu et al. 2007). All the studied compounds
showed, in the chosen experimental conditions, a peak at ≈
325 nm. After each addition of DNA, the band decreased in
intensity for compounds 1b–f, whereas for the compound 1a
only slight variations of the absorbance were observed. No
signifcant shift of the maximum was observed. The forma-
tion of another small band around 400 nm was also observed
for all the compounds. Selected spectra collected during the
titrations with ct-DNA are reported in Fig. 3 for 1e, and in
Fig. S8 for the other compounds. The spectral evidences
suggest that compounds 1b–f slightly interact with DNA
thorough intercalation between base pairs.
The afnity with the DNA appears related to the sub-
stituents present in the side chain. In fact, the lowest values
(48 and 69 M−1) were observed for 1a and 1b, bearing a
methyl and an iso-propyl as substituents. The presence in
the side-chain of the imidazole or the thiol group increased
the Kb by ≈ 10 and ≈ 5 times, respectively, with respect
to the compound 1a. The measured Kb values were lower
than those reported in literature for similar Schif bases
(Ali et al. 2014; Gowri and Jayabalakrishnan 2012; Arshad
et al. 2014). The diference was probably due to the pres-
ence of other substituents participating in the DNA inter-
action. The experimental evidences showed that the amino
acid moiety present in our compounds contributes, but not
heavily, to the DNA binding.
The equilibrium reaction involved in DNA binding is
described by the equation (L + D ↔ LD), where L is a
1 3

C. Fattuoni et al.
Table 4 Antiproliferative activity in vitro of Schif bases compounds
Cytotoxicity
against solid tumour-derived (SK-ME 1, DU 145, HEP-G2) cell lines
An evaluation of the cytotoxic activity of the synthetized
compounds wasn carried out to study the infuence of the
introduction of the phenolic ring on the cytotoxicity of the
amino acids. In fact, phenolic compounds are known to be
toxic due to reactive oxygen species (ROS) formation (Mich-
alowicz and Duda 2007). The antiproliferative activities of
the six studied compounds were tested against three human
solid tumor cell lines (lung squamous carcinoma (SK-
MES-1), prostate carcinoma (DU-145), and hepatocellular
carcinoma (Hep-G2)), and two human haematological tumor
cell lines (acute T-lymphoblastic leukemia (CCRF-CEM),
and acute B-lymphoblastic leukemia (CCRF-SB)). All the
studied compounds showed IC50 values higher than 100 µM
against all the tested tumor cell lines (Tables 3, 4) and were
then devoid of a signifcant cytotoxic activity (Fig. 4a–e).
Compounds
aIC50 (μM)
bSK-MES 1
cDU 145
dHEP-G2
1a
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
1b
1c
1d
1e
1f
Ref. compd
VINCRISTINE
0.17
1.18
0.005
aCompound concentration (µM) required to reduce cell proliferation
by 50%, as determined by the MTT method, under conditions allow-
ing untreated controls to undergo at least three consecutive rounds of
multiplication
bHuman lung squamous carcinoma
cHuman prostate carcinoma
Conclusions
dHuman hepatocellular carcinoma; vincristine was used as a reference
antitumor compound
The Schif bases 1a–f were synthesized and characterized by
means of elemental analysis, NMR and IR spectroscopy. The
protonation and DNA-binding constants, and the cytotoxic
activity were determined. With respect to the literature meth-
ods, reduced reaction time (for 1c and 1d), double amount of
salicylaldehyde (for 1f) and sodium sulfate addition (for 1a
and 1b) were needed for a better yield. All compounds were
obtained as a mixture of E and Z geometric isomers, the E
isomer always being more abundant. The protonation of the
carboxylate group happens at around pH 3 for 1b and 1c,
whereas for all the other compounds around 2 or lower. The
protonation of the imino group occurs between pH 7.18 and
8.8, while that of the phenolic oxygen between pH 8.71 and
10.7. The protonation of the amino acid side chain occurs
before the protonation of the phenolic oxygen only for 1d
and 1e. In all compounds the imino group is protonated after
the carboxylic group. The examined compounds do not show
cytotoxic activity, despite the presence of the phenolic ring
derived from salicylaldehyde. Nevertheless, 1a–f show DNA
binding constant in the 48–447 M−1 range. The absence of
cytotoxicity and the poor reactivity with DNA, confrm the
feasibility of these compounds for pharmaceutical applica-
tions, also in metal complexes, to exploit their unique prop-
erties. In fact, although the ligands are devoid of signifcant
activity, an antitumor activity of their metal complexes is
not totally unexpected. As an additional interesting applica-
tion, the studied compounds may be also promising green
catalysts in chiral synthesis, due to their intrinsic chirality
derived from the l-amino acid moiety.
Table 3 Antiproliferative activity in vitro of the synthesised com-
pounds against human leukaemia-/lymphoma-derived cell lines
Compounds
IC50 (μM)a
CCRF-CEMb
CCRF-SBc
1a
1b
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
1c
1d
1e
1f
Ref. compd
VINCRISTINE
0.0007
0.0009
aCompound concentration required to reduce cell proliferation by
50%, as determined by the MTT method, under conditions allowing
untreated controls to undergo at least three consecutive rounds of
multiplication
bHuman acute T-lymphoblastic leukaemia
cHuman acute B-lymphoblastic leukaemia; vincristine was used as a
reference antitumor compound
1 3

Synthesis, protonation constants and biological activity determination of amino acid–…
Fig. 4 Dose response of
antiproliferative activity of the
studied compounds (1a–1f)
against human leukemia- cell
lines CCRF-CEM (a), CCRF-
SB (b) and solid tumor cell
lines DU-145 (c), Hep-G2 (d)
and SK-MES-1 (e). The cell
lines were incubated with difer-
ent concentrations (0.045 μM,
0.13 μM, 0.41 μM, 1.23 μM,
3.7 μM, 11 μM, 33 μM,
100 μM, 1000 μM) of each
tested compound for 72 h
activities. Spectrochim Acta A Mol Biomol Spectrosc 125:440–
448. https://doi.org/10.1016/j.saa.2014.01.086
Antony R, Arun T, Manickam STD (2019) A review on applications
of chitosan-based Schif bases. Int J Biol Macromol 129:615–
633. https://doi.org/10.1016/j.ijbiomac.2019.02.047
Compliance with ethical standards
Conflict of interest The authors declare no conficts of interest.
Arshad N, Ahmad M, Ashraf MZ, Nadeem H (2014) Spectroscopic,
electrochemical DNA binding and in vivo anti-infammatory
studies on newly synthesized Schif bases of 4-aminophena-
zone. J Photochem Photobiol B Biol 138:331–346. https://doi.
org/10.1016/j.jphotobiol.2014.06.014
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Babu MSS, Reddy KH, Krishna PG (2007) Synthesis, characteri-
zation, DNA interaction and cleavage activity of new mixed
ligand copper(II) complexes with heterocyclic bases. Polyhe-
dron 26:572–580. https://doi.org/10.1016/j.poly.2006.08.026
Banti CN, Hadjikakou SK, Sismanoglu T, Hadjiliadis N (2019)
Anti-proliferative and antitumor activity of organotin(IV) com-
pounds. An overview of the last decade and future perspectives.
J Inorg Biochem 194:114–152
References
Ali AQ, Teoh SG, Salhin A, Eltayeb NE, Khadeer Ahamed MB,
Abdul Majid AM (2014) Synthesis of isatin thiosemicarbazones
derivatives: in vitro anti-cancer, DNA binding and cleavage
1 3

C. Fattuoni et al.
Liu S, Peng J, Yang H, Bai Y, Li J, Lai G (2012) Highly efcient and
convenient asymmetric hydrosilylation of ketones catalyzed with
zinc Schif base complexes. Tetrahedron 68:1371–1375. https://
doi.org/10.1016/j.tet.2011.12.054
Canel E, Gültepe A, Doğan A, Kilıç E (2006) The determination of
protonation constants of some amino acids and their esters by
potentiometry in diferent media. J Solut Chem 35:5–19. https://
doi.org/10.1007/s10953-006-8934-3
Malik MA, Dar OA, Gull P, Wani MY, Hashmi AA (2018) Heterocy-
clic Schif base transition metal complexes in antimicrobial and
anticancer chemotherapy. Med Chem Commun 9:409–436. https
://doi.org/10.1039/c7md00526a
Chow MJ, Licona C, Wong DYQ, Pastorin G, Gaiddon C, Ang WH
(2014) Discovery and investigation of anticancer ruthenium–arene
Schif-base complexes via water-promoted combinatorial three-
component assembly. J Med Chem 57:6043–6059. https://doi.
org/10.1021/jm500455p
Malinowski ER (2002) Factor analysis in chemistry, 3rd edn. Wiley,
New York
Dalapati S, Jana S, Guchhait N (2014) Anion recognition by sim-
ple chromogenic and chromo-fluorogenic salicylidene Schiff
base or reduced-Schif base receptors. Spectrochim Acta Part A
Mol Biomol Spectrosc 129:499–508. https://doi.org/10.1016/j.
saa.2014.03.090
Maurya MR, Kumar U, Correia I, Adão P, Costa Pessoa J (2008) A
polymer-bound oxidovanadium(IV) complex prepared from an
l-cysteine-derived ligand for the oxidative amination of styrene.
Eur J Inorg Chem. https://doi.org/10.1002/ejic.200700662
Metzler CM, Cahill A, Metzler DE (1980) Equilibria and absorption
spectra of Schif bases. J Am Chem Soc 102:6075–6082. https://
doi.org/10.1021/ja00539a017
Dossetter AG, Jamison TF, Jacobsen EN (1999) Highly enantio- and
diastereoselective hetero-Diels-Alder reactions catalyzed by
new chiral tridentate chromium (III) catalysts. Angew Chem
Int Ed Engl 38:2398–2400. https://doi.org/10.1002/(sici)1521-
3773(19990816)38:16%3c2398:aid-anie2398%3e3.0.co;2-e
El-Sherif AA, Aljahdali MS (2013) Review: protonation, complex-
formation equilibria, and metal–ligand interaction of salicylal-
dehyde Schif bases. J Coord Chem 66:3423–3468. https://doi.
org/10.1080/00958972.2013.839027
Michałowicz J, Duda W (2007) Phenols-sources and toxicity. Polish J
Environ Stud 16:347–362
Murmur J (1961) A procedure for the isolation of deoxyribonucleic
acid from micro-organisms. J Mol Biol 3:208–218. https://doi.
org/10.1016/S0022-2836(61)80047-8
Palomo C, Aizpurua JM, Ganboa I, Oiarbide M (1999) Asymmetric
synthesis of β-lactams by Staudinger Ketene-imine cycloaddition
reaction. Eur J Org Chem. https://doi.org/10.1002/(SICI)1099-
0690(199912)1999:12%3c3223:AID-EJOC3223%3e3.0.CO;2-1
Pauwels R, Balzarini J, Baba M, Snoeck R, Schols D, Herdewijn P
et al (1988) Rapid and automated tetrazolium-based colorimetric
assay for the detection of anti-HIV compounds. J Virol Methods
20:309–321. https://doi.org/10.1016/0166-0934(88)90134-6
Pessoa JC, Mannar MR (2017) Vanadium complexes supported on
organic polymers as sustainable systems for catalytic oxidations.
Inorg Chim Acta 455:415–428
Ershov AY, Nasledov DG, Nasonova KV, Sezyavina KV, Susarova
TV, Lagoda IV, Shamanin VV (2013) Ring-chain tautomerism of
2-aryl-6-oxohexahydropyrimidine-4-carboxylic acid sodium salts.
Chem Heterocycl Compd 49:640–645. https://doi.org/10.1007/
s10593-013-1287-0
Galić N, Cimerman Z, Tomišić V (1997) Tautomeric and protonation
equilibria of Schif bases of salicylaldehyde with aminopyridines.
Anal Chim Acta 343:135–143. https://doi.org/10.1016/S0003
-2670(96)00586-7
Gans P, O’Sullivan B (2000) GLEE, a new computer program for glass
electrode calibration. Talanta 51:33–37. https://doi.org/10.1016/
s0039-9140(99)00245-3
Pillai MRA, Kothari K, Banerjee S, Samuel G, Suresh M, Sarma HD,
Jurisson S (1999) Radiochemical studies of 99mTc complexes of
modifed cysteine ligands and bifunctional chelating agents. Nucl
Med Biol 26:555–561
Gans P, Sabatini A, Vacca A (1996) Investigation of equilibria in
solution. Determination of equilibrium constants with the hyper-
quad suite of programs. Talanta 43:1739–1753. https://doi.
org/10.1016/0039-9140(96)01958-3
Pivetta T, Trudu F, Valletta E, Isaia F, Castellano C, Demartin F et al
(2014) Novel copper(II) complexes as new promising antitu-
mour agents. A crystal structure of [Cu(1,10-phenanthroline-
Gomes LMF, Vieira RP, Jones MR, Wang MCP, Dyrager C, Souza-
Fagundes EM et al (2014) 8-Hydroxyquinoline Schif-base com-
pounds as antioxidants and modulators of copper-mediated Aβ
peptide aggregation. J Inorg Biochem 139:106–116. https://doi.
org/10.1016/j.jinorgbio.2014.04.011
5,6-dione)2(OH
2)(OClO3)](ClO4). J Inorg Biochem 141:103–113.
https://doi.org/10.1016/j.jinorgbio.2014.08.011
Pivetta T, Valletta E, Ferino G, Isaia F, Pani A, Vascellari S, Castellano
C, Demartin F, Cabiddu MG, Cadoni E (2017) Novel coumarins
and related copper complexes with biological activity: DNA
binding, molecular docking and in vitro antiproliferative activ-
ity. J Inorg Biochem 177:101–109. https://doi.org/10.1016/j.jinor
gbio.2017.09.013
Gowri M, Jayabalakrishnan C (2012) DNA binding and cytotoxicity of
newly synthesized Schif base (Z)-4-(((2-hydroxyphenyl)amino)
(phenyl)methylene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one
and its analogues. Int J Appl Biol Pharm Technol 3:327–337
Gran G (1952) Determination of the equivalence point in potentiomet-
ric titrations-Part II. Analyst 77:771
Qiao X, Ma ZY, Xie CZ, Xue F, Zhang YW, Xu JY et al (2011)
Study on potential antitumor mechanism of a novel Schif base
copper(II) complex: synthesis, crystal structure, DNA binding,
cytotoxicity and apoptosis induction activity. J Inorg Biochem
105:728–737. https://doi.org/10.1016/j.jinorgbio.2011.01.004
Qin W, Long S, Panunzio M, Biondi S (2013) Schif bases: a short sur-
vey on an evergreen chemistry tool. Molecules 18:12264–12289.
https://doi.org/10.3390/molecules181012264
Hiskey RG, Jung JM (1963) Azomethine chemistry. II. Formation of
peptides from oxazolidine-5-ones. J Am Chem Soc 85:578–582.
https://doi.org/10.1021/ja00888a021
Hsieh SH, Kuo YP, Gau HM (2007) Synthesis, characterization, and
structures of oxovanadium(V) complexes of Schif bases of beta-
amino alcohols as tunable catalysts for the asymmetric oxidation
of organic sulfdes and asymmetric alkynylation of aldehydes.
Dalton Trans. https://doi.org/10.1039/b613212j
Rajesh CM, Ray M (2014) Characterization of a meso-chiral isomer
of a hexanuclear Cu(II) cage from racemization of the l-ala-
nine Schiff base. Dalton Trans 43:12952–12960. https://doi.
org/10.1039/c4dt01443j
Latt SA, Stetten G, Juergens LA, Willard HF, Scher CD (1975) Recent
developments in the detection of deoxyribonucleic acid synthesis
by 33258 Hoechst fuorescence. J Histochem Cytochem 23:493–
505. https://doi.org/10.1177/23.7.1095650
Rao G, Philipp M (1991) Boronic acid catalyzed hydrolyses of
salicylaldehyde imines. J Org Chem 56:1505–1512. https://doi.
org/10.1021/jo00004a031
Liu X, Manzur C, Novoa N, Celedón S, Carrillo D, Hamon JR (2018)
Multidentate unsymmetrically-substituted Schif bases and their
metal complexes: synthesis, functional materials properties, and
applications to catalysis. Coord Chem Rev 357:144–172
Reichmann ME, Rice SA, Thomas CA, Doty P (1954) A further exami-
nation of the molecular weight and size of desoxypentose nucleic
1 3

Synthesis, protonation constants and biological activity determination of amino acid–…
acid. J Am Chem Soc 76:3047–3053. https://doi.org/10.1021/
ja01640a067
Yang ZY, Yang RD, Li FS, Yu KB (2000) Crystal structure and anti-
tumor activity of some rare earth metal complexes with Schif
base. Polyhedron 19:2599–2604. https://doi.org/10.1016/S0277
-5387(00)00562-3
Saleem H, Erdogdu Y, Subashchandrabose S, Thanikachalam V, Jayab-
harathi J, Ramesh Babu N (2012) Structural and vibrational stud-
ies on (E)-2-(2-hydroxybenzylidenamino)-3-phenylpropionic acid
using experimental and DFT methods. J Mol Struct 1030:157–
167. https://doi.org/10.1016/j.molstruc.2012.04.011
Yoo J, Cui Q (2008) Does arginine remain protonated in the lipid
membrane? Insights from microscopic pKa calculations. Bio-
phys J Biophys Lett 94:L61–63. https://doi.org/10.1529/bioph
ysj.107.122945
Smith HE, Burrows EP, Marks MJ, Lynch RD, Chen FM (1977) Opti-
cally active amines. 22. Application of the salicylidenimino chiral-
ity rule to α-amino acids. J Am Chem Soc 2:707–713. https://doi.
org/10.1021/ja00445a008
Zhang N, Fan Y, Zhang Z, Zuo J, Zhang P, Wang Q et al (2012) Synthe-
ses, crystal structures and anticancer activities of three novel tran-
sition metal complexes with Schif base derived from 2-acetylpyr-
idine and l-tryptophan. Inorg Chem Commun 22:68–72. https://
doi.org/10.1016/j.inoche.2012.05.022
Turan B, Şendil K, Şengül E, Gültekin MS, Taslimi P, Gulcin I,
Supuran CT (2016) The synthesis of some β-lactams and inves-
tigation of their metal-chelating activity, carbonic anhydrase and
acetylcholinesterase inhibition profles. J Enzyme Inhib Med
Chem 31 (sup1):79–88
Zhong X, Yi J, Sun J, Wei HL, Liu WS, Yu KB (2006) Synthesis
and crystal structure of some transition metal complexes with a
novel bis-Schif base ligand and their antitumor activities. Eur
J Med Chem 41:1090–1092. https://doi.org/10.1016/j.ejmec
h.2006.05.009
Türkoğlu G, Berber H, Dal H, Öğretir C (2011) Synthesis, characteriza-
tion, tautomerism and theoretical study of some new Schif base
derivatives. Spectrochim Acta A Mol Biomol Spectrosc 79:1573–
1583. https://doi.org/10.1016/j.saa.2011.04.089
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
Vilanova B, Gallardo J, Caldés C, Adrover M, Ortega-Castro J, Muñoz
F et al (2012) Formation of Schif bases of O-phosphorylethan-
olamine and O-phospho-d, l-serine with pyridoxal 5′-phosphate.
Experimental and theoretical studies. J Phys Chem A 116:1897–
1905. https://doi.org/10.1021/jp2116033
1 3