Synthesis and characterization of bio-based quaternary ammonium salts with gibberellate or l-tryptophanate anion

Article abstract of DOI:10.1007/s00706-020-02672-9

Numerous biologically active acids can be transformed into an ionic form in a facile way and combined with appropriate quaternary ammonium cation to improve their application properties or biological activity. This study describes the synthesis of new quaternary ammonium salts with anions of gibberellic acid, a common plant growth regulator from the gibberellin group, or l-tryptophan, an important precursor of auxin biosynthesis. The surface-active tetrapentylammonium ion and natural substances such as acetylcholine, choline, and quinine were the sources of cations. Novel salts of gibberellic acid and l-tryptophan were obtained with high yields exceeding 97% as a result of the metathesis reaction or the neutralization of quaternary ammonium hydroxides. Phase transition temperatures, thermal and chemical stability, and solubility in solvents with different polarities were determined for all obtained salts. On the basis of studies regarding the influence of synthesized salts on the post-harvest longevity and quality of leaves of Convallaria majalis, it was established that the biological activity of the natural plant regulators in most cases was maintained. Therefore, it can be concluded that the conversion of the active substance into the form of a quaternary ammonium salt results in obtaining novel forms of plant growth regulators with favourable physicochemical properties while maintaining the efficacy of the biological active ingredients. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-020-02672-9

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-020-02672-9
ORIGINAL PAPER
Synthesis and characterization of bio‑based quaternary ammonium
salts with gibberellate or l‑tryptophanate anion
Daria Szymaniak1 · Juliusz Pernak1 · Tomasz Rzemieniecki · Damian Krystian Kaczmarek1
1
·
Roman Andrzejak2 · Tomasz Kosiada · Beata Janowska
2
2
Received: 30 May 2020 / Accepted: 10 August 2020
©
The Author(s) 2020
Abstract
Numerous biologically active acids can be transformed into an ionic form in a facile way and combined with appropriate
quaternary ammonium cation to improve their application properties or biological activity. This study describes the synthesis
of new quaternary ammonium salts with anions of gibberellic acid, a common plant growth regulator from the gibberellin
group, or l-tryptophan, an important precursor of auxin biosynthesis. The surface-active tetrapentylammonium ion and
natural substances such as acetylcholine, choline, and quinine were the sources of cations. Novel salts of gibberellic acid
and l-tryptophan were obtained with high yields exceeding 97% as a result of the metathesis reaction or the neutralization of
quaternary ammonium hydroxides. Phase transition temperatures, thermal and chemical stability, and solubility in solvents
with diꢀerent polarities were determined for all obtained salts. On the basis of studies regarding the inꢁuence of synthesized
salts on the post-harvest longevity and quality of leaves of Convallaria majalis, it was established that the biological activity
of the natural plant regulators in most cases was maintained. Therefore, it can be concluded that the conversion of the active
substance into the form of a quaternary ammonium salt results in obtaining novel forms of plant growth regulators with
favourable physicochemical properties while maintaining the eꢂcacy of the biological active ingredients.
Graphic abstract
Keywords Bioorganic chemistry · Ionic liquids · Natural compounds · Phytochemistry · Plant growth regulators
Introduction
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00706-020-02672-9) contains
The ageing process of plant organs is the ꢃnal phase of
supplementary material, which is available to authorized users.
ontogenesis, during which irreversible changes that result
in cell degradation and plant death are initiated [1]. Post-
harvest longevity is a species-speciꢃc trait, even at a varietal
level; hence, the ageing of cut ꢁowers occurs at diꢀerent
rates and is the main limitation for the marketing of many
species [2]. The situation is similar for species grown as ꢁo-
rist’s greens. The longevity of the leaves of many species is
unsatisfactory; hence, it is necessary to carry out additional
*
Juliusz Pernak
juliusz.pernak@put.poznan.pl
1
2
Faculty of Chemical Technology, Poznan University
of Technology, ul. Berdychowo 4, 60-965 Poznan, Poland
Faculty of Horticulture and Landscape Architecture,
Poznan University of Life Sciences, ul. Dąbrowskiego 159,
6
0-594 Poznan, Poland
Vol.:(0
1
123456789)
3

D. Szymaniak et al.
post-harvest treatments, which are designed to extend their
life after cutting. Ageing is closely associated with the action
of growth regulators that accelerate (ethylene, abscisic acid)
or delay (cytokinins, gibberellins) this process [3–8]. In the
case of leaves, compounds from the cytokinin and gibberel-
lin groups play a special role as they inhibit the degradation
of proteins and chlorophyll, which directly contributes to
delaying the ageing processes [9, 10].
obtain environmentally friendly salts. Whereas, l-tryptophan
was combined with a synthetic tetrapentylammonium cation,
since the salt thus obtained was characterized by the great-
est chemical stability under aerobic conditions. Salts with
l-tryptophanate anion and cations of natural origin degraded
faster, which was considered highly undesirable in biologi-
cal applications. The chemical structures of both gibberel-
late and l-tryptophanate anions are prone to decomposition,
thus it was important to determine the thermal and chemical
stability of the obtained salts. Since the new forms of both
active ingredients were expected to possess more favour-
able physicochemical properties compared to acidic forms
Gibberellic acid (gibberellin A , GA ) is the most com-
3
3
monly used regulator from the gibberellin group. This chem-
ical aꢀects shoot elongation [11], stimulates germination
[
12, 13] and extends the post-harvest longevity of ꢁowers
and ꢁorist’s greens [10, 14]. This compound, along with
other gibberellins, is actively synthesized in plant tissues as
well as in bacterial and fungal cells (e.g., in the case of the
of GA an l-tryptophan (e.g., ready solubility in water and
3
better thermal stability), we additionally evaluated the prod-
ucts’ eꢀect on the prolongation of post-harvest longevity of
lily of the valley leaves (C. majalis) to conꢃrm that that the
introduction of organic cations does not hinder the biological
properties of the active ingredients.
genus Fusarium) [15]. GA is a tetracyclic diterpenoid that
3
contains eight chiral carbon atoms in its structure (Fig. 1).
Due to the low eꢂciency of long-term fermentation pro-
cesses, the market price of GA with technical purity can
3
even reach $1000/kg. As indicated in literature, gibberel-
lic acid should be used at concentrations up to 100 ppm to
be eꢀective [14, 16]. Additional problems are associated
Results and discussion
−
3
with the low solubility of GA in water, which is 5 g dm ,
Synthesis
3
and the instability of its aqueous solutions, which hinders
the preparation of stable solutions used for agrotechnical
operations [15, 17]. Similar problems apply to the use of for-
mulations containing auxin growth regulators, e.g., indole-
Quaternary ammonium salts (Table 1) with selected organic
cations and anions from the group of plant growth regulators
(Fig. 1) were obtained using two methods. Salts with choline
(1) and acetylcholine (2) cations were obtained as a result of
a metathesis reaction using a potassium salt of gibberellic
acid in methanol (Scheme 1, method I). Salts with 1-eth-
ylquinine (3), 1-dodecylquinine (4), and tetrapentylammo-
nium (5) cations were obtained in a two-step reaction. In the
ꢃrst stage, the bromide anion was exchanged with a hydroxyl
ion using an ion exchange resin, followed by a neutralization
reaction with gibberellic acid or l-tryptophan in methanol
(Scheme 1, method II). The methodology of synthesis and
puriꢃcation allowed us to obtain products with high yields,
exceeding 90%. The obtained products were stored in the
presence of a drying agent (P O ). The results of elemental
3
-acetic acid (IAA) or indole-3-butyric acid (IBA), and
other auxin-stimulating compounds, e.g., l-tryptophan (Trp)
17–19]. It is, therefore, necessary to search for new ways of
[
applying plant growth regulators in new, persistent chemi-
cal forms that will allow overcoming these disadvantages.
Several recent studies have focused on the introduction of
active substances from the group of plant growth regulators
in the form of potassium salts to chemically stabilize them.
In this study, we focused on the synthesis of new plant
growth regulators in the form of quaternary ammonium
salts. Natural cations such as choline and acetylcholine (both
present in the human body) as well as cations derived from
quinine (a plant alkaloid used to treat malaria) have been
combined with gibberellic acid to increase its biological
activity. The selection of these counter ions allowed us to
4
10
analysis conꢃrmed the high purity the obtained compound
(Table S1 in Electronic Supplementary Material).
The obtained salts were solids with melting points rang-
ing from 96 to 136 °C. The exception was the salt with the
l-tryptophanate anion (5), which was a high-viscosity liquid
at 25 °C. This observation allowed us to classify 1, 4, and
5
3
as ionic liquids (ILs). The melting points for salts 2 and
were above the threshold of 100 °C, which did not allow
them to be classiꢃed as ILs.
The phase transitions and thermal stability of the obtained
salts were determined by diꢀerential scanning calorimetry
(
DSC) or thermal gravimetric analysis (TGA). The results
are presented in Table 1. In the studied temperature range,
Fig. 1 Structures of gibberellic acid (GA ) and l-tryptophan (Trp)
only the glass transition temperature (T ) was observed,
3
g
1
3

Synthesis and characterization of bio-based quaternary ammonium salts with gibberellate…
Table 1 Synthesized quaternary
Salt
1
Cation
Anion
GA3−
Yield/%
98
Tm/°C
Tg/°C
Tonset5%/°C
Tonset50%/°C
ammonium salts
100–102
78
170
236
2
3
GA3−
GA3−
97
99
133–136
109–112
110
–
180
175
252
285
4
5
GA3−
Trp−
99
98
101–106
–
–
219
190
315
211
10
GA3
Trp
–
–
–
–
–
–
229
286
–
–
243
277
298
305
T
T
melting point, T glass transition temperature, T
decomposition temperature of 5% sample,
m
g
onset5%
decomposition temperature of 50% sample
onset50%
which depended on the chemical structure. Salts character-
salts with a more complex cation (3 and 4) did not exhibit
ized by structurally “simple” cations exhibited T . Moreo-
this phase transition. Furthermore, the conversion of GA
g
3
ver, the glass transition temperature is associated with the
strong cation–anion interaction and the formation of addi-
tional hydrogen bonds [20]. This hypothesis was also con-
ꢃrmed in our studies, as ILs 1, 2, and 5 were characterized
by the occurrence of a glass transition temperature, while
into an organic salt caused a decrease in thermal stability
(T
and T
). The presence of an ionic bond in the
onset50%
onset5%
obtained salts 1–3 resulted in a decrease in the T
in
onset50%
the range of 13–62 °C relative to GA (T
=298 °C).
onset50%
3
It can, therefore, be assumed that the presence of an ionic
1
3

D. Szymaniak et al.
bond increases the resistance to thermal decomposition [21].
Diꢀerent behaviour was observed for compounds with the
l-tryptophanate anion. Salt 5 exhibited lower thermal sta-
hydrophilic structure of acetylcholine limits the solubility of
salt 2 in less polar solvents than DMSO, while salt 1 with the
choline cation does not dissolve in isopropanol and less polar
solvents. Cations with less hydrophilic properties allow salts
3–5 to dissolve in acetone and isopropanol. Therefore, com-
paring the solubility of the obtained salts with gibberellic
acid and l-tryptophan, it can be indicated that the conversion
of these compounds into ionic form has a positive eꢀect on
solubility in polar solvents.
bility than pure l-tryptophan (T
=305 °C) by 94 °C,
onset50%
which may indicate a diꢀerent mechanism of decomposition
of salts with the amino acid anion [22].
The structures of the newly obtained salts were conꢃrmed
by nuclear magnetic resonance spectroscopy and FT-IR
spectroscopy. NMR and FT-IR spectra for salts 1–5 with
corresponding descriptions are provided in the ESM (Figs.
Additionally, salt 4 exhibited good solubility in chloro-
form while maintaining low aꢂnity for other weakly polar
solvents: ethyl acetate, toluene and hexane. This fact con-
ꢃrms the hypothesis that quaternary ammonium salts dis-
S1–S15). Characteristic signals derived from GA (doublet
3
of doublets at 5.70–5.90 ppm and doublet at 6.20–6.40 ppm)
1
and Trp (signals at 2.95 and 3.26 ppm) observed in the H
NMR spectra conꢃrm the presence of the appropriate ani-
ons in the obtained salts. Signiꢃcant diꢀerences in chemical
shifts derived from the carboxyl group were observed in
solve better in solvents with higher dielectric constants (ε
r
of chloroform=4.8) than in solvents with a lower value of
this parameter (ε of toluene=2.4, ε of hexane=1.9) [23].
r
r
1
3
the C NMR spectrum. Compared to that chemical shift
The synthesized bio-based IL 5 also exhibited a high aꢂnity
for GA , the diꢀerences were 1.19 ppm for 1, 0.87 ppm for
for highly polar systems.
3
2
, 1.31 ppm for 3, and 1.22 ppm for 4. The conversion of
l-tryptophan into an IL resulted in a change in the chemical
shift of the signal associated with the carbon atom in the
carboxyl group from 172.61 to 184.26 ppm.
Chemical stability
The chemical stability of aqueous solutions of gibberellic
acid, l-tryptophan and the obtained salts 1, 2 and 5 was
1
13
Solubility
tested at 80 °C. H and C NMR spectra of the salts were
obtained before and after heating. Comparison of the NMR
spectra allowed observation and identiꢃcation of the degra-
The synthesized ILs were tested in terms of their solubility
in ten solvents, which were ordered from the highest (9.0)
to the lowest (0.0) value of the polar coeꢂcient according
to Snyder. The test results summarized in Table 2 indicate
that salts 1–3 and 5 are characterized by good solubility in
the most polar solvents (water, methanol, DMSO) and that
salt 4 is characterized by a lack of solubility in water. Gib-
berellic acid is poorly soluble in water [17]; therefore, the
obtained results indicate that its transformation into an ionic
form can signiꢃcantly facilitate the preparation of dosage
forms in the form of aqueous solutions. Depending on the
structure of the cation in the quaternary ammonium salt, the
solubility of the compounds in organic solvents varies. The
dation products, as described in Figs. S16–S21 in the ESM.
1
Analysis of the H NMR spectra of GA after heating
3
indicated the disappearance of the signal from hydro-
gen atoms in the cyclohexene ring (6.38–6.35 ppm,
5.89–5.85 ppm, and 4.00–3.99 ppm as well as the signal
at 3.23–3.20 ppm, which is associated with the hydrogen
atom in the cyclopentane ring). Furthermore, the charac-
teristic signals from the decomposition of GA have been
3
described in the literature [24, 25]: 5.08 ppm (epiallogib-
beric acid), 4.71–4.69 and 3.95–3.89 ppm (allogibberic
acid), 4.20–4.19 ppm (gibberic acid), 2.46–2.20 ppm
and 7.25–6.96 ppm (gibberic acid, epigibberic acid,
Table 2 Solubility of prepared salts at 25 °C
Salt
Water
Methanol
6.6
DMSO
6.5
Acetonitrile
6.2
Acetone
5.1
Isopropanol
4.3
Ethyl acetate
4.3
Chloroform
4.1
Toluene
2.3
Hexane
0.0
a
9
.0
1
+
+
+
–
+
–
+
+
+
+
+
+
–
+
+
+
+
+
±
–
±
–
–
–
±
–
–
+
–
+
+
+
+
–
–
–
+
+
±
–
–
–
–
–
–
–
–
–
–
–
+
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
2
3
4
5
GA3
Trp
–
–
+
Soluble,± limited solubility,−not soluble
a
Snyder polarity index
1
3

Synthesis and characterization of bio-based quaternary ammonium salts with gibberellate…
allogibberic acid, epiallogibberic acid). In contrast,
the disappearance of signals from carbon atoms in the
cyclohexene ring was visible (92.48 and 70.53 ppm) in
1
3
the C NMR spectra. Moreover, after decomposition,
additional signals from carboxyl groups in the structure
of GA decomposition products appeared in the carbon
3
spectra (179.64, 178.04 and 175.68 ppm).
1
13
Analogous observations were noted in the H and
C
NMR spectra of salts 1 and 2; the disappearance of signals
from the gibberellin anion and the appearance of signals
from its degradation products were visible. In the spec-
trum of salt 2, signals originating from the degradation
products of the acetylcholine cation to choline and acetic
Chemical shift /ppm
1
13
acid ( H NMR, 3.47 and 3.39 ppm; C NMR, 180.00,
1
Fig. 3 Comparison of the H NMR spectra of salt 5 and its degrada-
6
9.03, 57.09 and 21.96 ppm) were also visible after heat-
tion products (5
)
deg
ing in addition to signals from the GA anion degradation
3
products, as shown in Fig. 2. The conversion of gibberellic
acid to ions did not increase its chemical stability.
In the case of l-tryptophan, no shift of resonance sig-
Biological activity
1
13
nals was observed in the H and C NMR spectra, which
conꢃrms the stability of l-tryptophan under the given
conditions (Figs. S22, S23 in ESM). In the case of salt
C. majalis was the test plant in the post-harvest analysis of
leaf longevity. The obtained results are presented in Fig. 4.
The post-harvest longevity of C. majalis leaves
depended both on the type of growth regulator used and
its concentration (Fig. 4). Analysis of the obtained results
indicated that leaves in the control sample were the least
5
, the chemical shift changed in the NMR spectra (Figs.
S24, S25 in ESM), indicating the instability of this com-
1
pound. In the H NMR spectrum of salt 5 after heating, a
decrease in signal integration and intensity by more than
durable comparing with salts 1–4 and GA . The most
3
1
2
0% as well as shifts in resonance signals in the H and
C NMR spectra by ~ 0.1 ppm and 1 ppm, respectively.
durable leaves were obtained after using GA at the tested
3
1
3
concentrations and salt 1 at 100 ppm. Comparing the eꢂ-
ciency of salts with gibberellin anions (1–4), it was found
that their eꢀect was signiꢃcantly more potent when used
at a concentration of 100 ppm. The exception was salt 3,
which showed higher activity when used at a concentra-
tion of 50 ppm. This phenomenon may be associated with
the unique interactions of the quinine cation, which is not
blocked by the presence of a long alkyl chain. In addition,
it was observed that the introduction of the acetyl group
into the choline cation limited the preserving eꢀect of the
compound at both tested concentrations (Fig. 4a).
The observed diꢀerences clearly indicate a change in the
chemical structure of salt 5 after heating. The conversion
of l-tryptophan to ion reduced its chemical stability. A
1
comparison of the H NMR spectra of salt 5 and its deg-
radation products is shown in Fig. 3.
The available literature reports do not provide an answer
to the question regarding whether tryptophan can have a
positive eꢀect on the post-harvest longevity of ꢁorist’s
greens. After using l-tryptophan and salt 5 in an analogous
manner to that of the gibberellic acid derivatives, it was
found that the most durable leaves were obtained in the
control combination, regardless of the concentration used.
Compared to that of control leaves, the post-harvest leaf
longevity after using l-tryptophan and salt 5 at 0.1 and
1
.0 ppm was 8–12% lower, which indicates that salt 5 acts
analogously to l-tryptophan. The use of higher concentra-
Chemical shift /ppm
tions of both compounds decreased the leaf longevity by
2
0–33%, indicating their phytotoxicity (Fig. 4b).
1
The lowest chlorophyll a + b content in the ageing
Fig. 2 Comparison of the H NMR spectra of salt 2 and its degrada-
tion products (2
)
leaves of C. majalis was found in the control leaves.
deg
1
3

D. Szymaniak et al.
Conclusion
The use of two highly effective methods of synthesis
allowed the transformation of gibberellic acid and l-tryp-
tophan into quaternary ammonium salts. Three out of
ꢃve obtained compounds can be classiꢃed as ionic liq-
uids. Based on the spectral analysis, it was conꢃrmed that
during synthesis, neither gibberellate anions nor l-tryp-
tophanate anions are degraded. After conversion to the
ionic form, the resulting products exhibited aꢂnity for
protic (methanol, isopropanol) and highly polar (DMSO)
solvents.
Based on the conducted chemical stability tests, it can
be concluded that the transformation of gibberellic acid to
the ionic form does not increase its stability. In the case
of l-tryptophan, it has been observed that converting it
into an anion reduces its stability. The degradation of the
obtained compounds is consistent with the natural meta-
bolic pathways for plant regulators.
Quaternary ammonium salts with gibberellate anions
extended the post-harvest longevity of C. majalis leaves. A
more than twofold increase in leaf longevity was achieved
after using choline gibberellate at a concentration of
1
00 ppm and gibberellic acid at the tested concentrations.
The biological activity of the remaining salts was lower;
however, after their application, the post-harvest longev-
ity of the leaves was higher than that of the control leaves.
However, the use of both l-tryptophan and tetrapentylam-
monium l-tryptophanate did not increase the post-harvest
longevity, which was particularly apparent when these
compounds were used at higher concentrations.
Fig. 4 Post-harvest longevity of C. majalis leaves conditioned in
solutions of GA and salts 1–4 with the gibberellate anion (b) and salt
3
5
with the l-tryptophanate anion (b); the means followed by the same
letter do not diꢀer signiꢃcantly at α=0.05
Experimental
Acetylcholine chloride (purity 99%), bromoethane (purity
99%), 1-bromododecane (purity 97%), choline chloride
(purity 99%), Dowex-Monosphere 550A anion exchange
resin, gibberellic acid (purity 90%), l-tryptophan (purity
98%), tetrapentylammonium bromide (purity 98%), qui-
nine (purity 99%) were purchased in Sigma-Aldrich
(Poznań, Poland). Potassium hydroxide (purity 99%) and
all solvents (methanol, acetonitrile, acetone, hexane, tolu-
ene, chloroform, isopropanol, DMSO and ethyl acetate)
were purchased from Avantor (Gliwice, Poland). Deion-
Solutions of GA and salts 1–4 signiꢃcantly inhibited the
3
degradation of chlorophyll in the leaves at both concentra-
tions, with no signiꢃcant diꢀerences between the concen-
trations used and no diꢀerences in the eꢀects of GA and
3
salts 1–4 (Fig. S26A in ESM). Moreover, both gibberellic
acid and salts 1–4 used in the studies inhibited protein
degradation in the leaves at both tested concentrations
(
Fig. S26B in ESM). A similar eꢀect in terms of delaying
protein ageing after application of GA was observed in
3
−
1
the leaves of Zantedeschia aethiopica and Zantedeschia
elliottiana [26]. Despite a signiꢃcant modiꢃcation of the
plant growth regulators, their metabolic activity remained
unchanged, and the majority of the obtained compounds
had a positive eꢀect on the post-harvest leaf longevity.
ized water with a conductivity < 0.1 μS cm , from dem-
ineralizer HLP Smart 1000 (Hydrolab, Poland) was used
for solubility tests. All reagents and solvents were used
without further puriꢃcation.
1
The H NMR spectra were recorded on Mercury
3
00 (Varian Inc.) and VNMR-S 400 MHz (Varian Inc.)
1
3

Synthesis and characterization of bio-based quaternary ammonium salts with gibberellate…
spectrometers operating at 300 MHz and 400 MHz,
General synthetic method of 1‑ethylquinine
1
3
respectively, using TMS as the internal standard. The
C
gibberallate, 1‑dodecylquinine gibberallate,
NMR spectra were obtained using the same instruments
operating at 75 and 100 MHz, respectively. The elemental
analyses (CHN) were performed using an Elementar Ana-
lyser Vario EL III at Adam Mickiewicz University, Poznan
tetrapentylammonium l‑tryptophanate
The synthesis of salts 3–5 was carried out in two stages.
To conduct the synthesis, 1-alkylquinine bromides obtained
analogously to the method described in the literature [27]
and puriꢃed and commercial tetrapentylammonium bro-
mide were used. In the ꢃrst stage, 1-alkylquinine bromide
or tetrapentylammonium bromide (0.02 M) was dissolved in
(
Poland). Melting points of the obtained products were
determined using MP90 Melting Point System (Mettler
Toledo). The FT-IR spectra of the samples as KBr pellets
were recorded on a spectrophotometer (IFS 66v/S, Bruker)
−
1
3
from 4000 to 400 cm .
30 cm anhydrous methanol and exchanged to a hydroxide
salt using an anion exchange resin. The reaction was carried
out for 30 min at 25 °C. The resin was then ꢃltered under
reduced pressure. A stoichiometric amount of gibberellic
acid or l-tryptophan was added to the obtained hydroxide.
The reaction was carried out for 1 h at 25 °C. Then, the sol-
vent was removed using a rotary evaporator. The obtained
salts were dried using a vacuum dryer at 40 °C for 48 h.
Synthetic method of choline gibberallate
and acetylcholine gibberellate
Salts with choline and acetylcholine cations (1, 2) were
obtained using the metathesis reaction. To conduct the
reaction, choline chloride or acetylcholine chloride
3
1
(
0.05 M) was dissolved in 30 cm anhydrous methanol.
1‑Ethylquinine gibberallate (3, C H N O ) Yield: 99%; H
4
1 50 2 8
A stoichiometric amount of potassium gibberellate was
added to the solution. The reaction was carried out for
NMR (400 MHz, DMSO-d ): δ=8.75 (d, J=4.5 Hz, 1H),
6
7.99 (d, J=9.2 Hz, 1H), 7.73 (d, J=4.6 Hz, 1H), 7.46 (dd,
3
0 min at 25 °C. The mixture was then cooled to 5 °C, the
J
= 2.6 Hz, J = 9.2 Hz, 1H), 7.30 (d, J = 2.6 Hz, 1H),
1,2 1,3
inorganic salt was separated by ꢃltration, and the solvent
was evaporated using a vacuum evaporator.
6.51 (s, 1H), 6.27 (d, J=9.3 Hz, 1H), 5.82–5.70 (m, 2H),
5.13 (d, J=17.3 Hz, 1H), 5.04–4.98 (m, 2H), 4.60 (s, 1H),
4
.25–4.16 (m, 1H), 3.97 (s, 3H), 3.95–3.87 (m, 2H), 3.83–
1
Choline gibberallate (1, C H NO ) Yield: 98%; H NMR
3.81 (d, J=3.3 Hz, 1H), 3.74–3.62 (m, 2H), 3.43–3.31 (m,
2H), 3.06 (d, J=10.3 Hz, 1H), 2.81–2.72 (m, 1H), 2.25–2.31
(m, 2H), 2.23–2.11 (m, 2H), 2.02–1.98 (m, 1H), 1.91–1.89
(m, 2H), 1.87–1.83 (m, 1H), 1.74–1.51 (m, 6H), 1.46–1.31
2
4
35
7
(
300 MHz, CD OD): δ=6.37 (d, J=9.3 Hz, 1H), 5.85 (dd,
3
J
=3.6 Hz, J =9.4 Hz, 1H), 5.15 (s, 1H), 3.98–4.03 (m,
1,3
1
,2
2
H), 3.96 (d, J=3.5 Hz, 1H), 3.48–3.52 (m, 2H), 3.22 (s,
13
9
H), 2.54 (d, J=10.4 Hz, 1H), 2.43–2.51 (m, 1H), 2.20–2.31
(m, 4H), 1.03 (s, 3H) ppm; C NMR (100 MHz, DMSO-d ):
6
1
3
(
(
m,1H), 1.65–2.02 (m, 9H), 1.25 (s, 3H) ppm; C NMR
δ=179.71, 174.73, 158.91, 157.15, 147.46, 145.41, 143.68,
138.13, 133.26, 131.77, 131.45, 125.59, 121.25, 120.56,
116.48, 104.79, 101.92, 101.81, 91.07, 76.76, 68.59, 65.70,
62.66, 58.91, 55.54, 55.03, 53.87, 53.17, 52.96, 51.98,
75 MHz, CD OD): δ = 182.21, 179.66, 159.48, 134.20,
3
1
5
33.74, 106.67, 93.31, 78.99, 71.03, 69.19, 55.23, 54.88,
4.43, 52.15, 51.29, 46.44, 44.95, 39.96, 18.22, 15.17 ppm;
−
FT-IR:=ꢀ3346, 3033, 2967, 2933, 2875, 1759, 1476, 1452,
50.40, 49.30, 45.08, 43.35, 38.99, 37.51, 26.18, 24.70,
−
1
1
398, 1381, 1351, 1329, 1298, 1226, 1162, 1130, 1102,
20.16, 16.71, 14.50, 8.20 ppm; FT-IR:= ꢀ3370, 3278, 3038,
−
1
050, 1018, 952, 890, 864, 746, 599, 571 cm .
2968, 2937, 2878, 1758, 1659, 1622, 1576, 1510, 1454,
1
431, 1397, 1379, 1329, 1240, 1227, 1161, 1128, 1102,
1
Acetylcholine gibberallate (2, C H NO ) Yield: 97%; H
1046, 1022, 996, 970, 944, 921, 889, 862, 828, 778, 745,
2
6
37
8
−
1
NMR (300 MHz, CD OD): δ=6.37 (d, J=9.3 Hz, 1H), 5.85
721, 700, 663, 634, 599, 570, 543, 463, 430, 411 cm .
3
(
dd, J =3.6 Hz, J =9.4 Hz, 1H), 5.16 (s, 1H), 4.49–4.57
1,2 1,3
(
m, 1H), 3.98–3.97 (d, J=3.6 Hz, 1H), 3.76–3.72 (m, 2H),
1‑Dodecylquinine gibberallate (4, C H N O ) Yield: 99%;
5
1 70 2 8
1
3
1
1
1
6
4
3
1
8
.27–3.12 (m,9H), 2.55 (d, J=10.5 Hz, 1H), 2.47–2.38 (m,
H NMR (400 MHz, DMSO-d ): δ = 8.75 (d, J = 4.5 Hz,
6
H), 2.18–2.28 (m, 1H), 2.11 (s, 3H), 1.98–1.72 (m, 9H),
1H), 7.99 (d, J=9.2 Hz, 1H), 7.71 (d, J=4.5 Hz, 1H), 7.46
(dd, J =2.5 Hz, J =9.2 Hz, 1H), 7.30 (d, J=2.4 Hz, 1H),
1
3
.23 (s, 3H) ppm; C NMR (75 MHz, CD OD): δ=181.89,
3
1,2
1,3
78.70, 159.09, 134.05, 133.55, 106.87, 93.06, 78.79, 70.68,
6.49 (s, 1H), 6.25 (d, J=9.3 Hz, 1H), 5.82–5.70 (m, 2H),
5.12 (d, J=17.3 Hz, 1H), 5.04–4.96 (m, 2H), 4.58 (s, 1H),
4.33–4.22 (m, 1H), 3.97 (s, 3H), 3.95–3.89 (m, 1H), 3.83 (d,
J=4.5 Hz, 1H), 3.74–3.66 (m, 3H), 3.45–3.34 (m, 2H), 3.06
(d, J=10.2 Hz, 1H), 2.80–2.72 (m, 1H), 2.31 (d, J=16.2 Hz,
1H), 2.25 (d, J=10.2 Hz, 1H), 2.23–2.12 (m, 2H), 1.97 (s,
5.94, 58.98, 55.45, 54.78, 54.56, 51.99, 51.10, 46.04,
−
4.64, 39.79, 20.84, 18.06, 15.11 ppm; FT-IR: =ꢀ3416,
030, 2964, 2929, 2871, 1748, 1651, 1568, 1476, 1449,
394, 1383, 1326, 1245, 1159, 1107, 1100, 1046, 1019, 945,
−
1
91, 861, 812, 743, 596, 568, 407 cm .
1
H), 1.93 (s, 1H), 1.89–1.77 (m, 4H), 1.73–1.51 (m, 6H),
1
3

D. Szymaniak et al.
1
3
1
1
1
5
4
2
1
1
1
9
5
.43–1.19 (m, 19H), 1.03 (s, 3H), 0.88–0.84 (t, J=6.9 Hz,
into a speciꢃc volume of solvent. The analyses were per-
formed at 25 °C. Depending on the volume of solvent used,
three outcomes have been recorded: “ready solubility”
1
3
H) ppm; C NMR (100 MHz, DMSO-d ): δ = 179.80,
6
74.94, 158.99, 157.18, 147.52, 145.66, 143.70, 138.13,
33.36, 131.70, 131.43, 125.61, 121.05, 120.56, 116.49,
04.63, 101.99, 76.77, 68.58, 66.64, 62.57,59.79, 59.47,
5.33, 54.18, 53.16, 53.13, 51.95, 50.38, 49.35, 48.60,
5.27, 43.45, 37.63, 31.36, 29.14, 29.08, 28.79, 28.69,
3
applies to salts that dissolved in 1 cm of the solvent, “lim-
3
ited solubility” applies to compounds that dissolved in 3 cm
of the solvent, and “low solubility” applies to ILs that did
3
not dissolve in 3 cm of the solvent.
6.35, 26.12, 24.69, 22.15, 22.06, 20.15, 16.80, 14.54,
−
3.98 ppm; FT-IR:=ꢀ3370, 3270, 3079, 3037, 2926, 2854,
Chemical stability
759, 1659, 1622, 1579, 1510, 1455, 1431, 1394, 1378,
328, 1298, 1240, 1227, 1160, 1128,1101, 1046, 996, 970,
43, 920, 888, 862, 827, 779, 743, 722, 662, 635, 599, 570,
To evaluate the chemical stability, gibberellic acid, l-trypto-
3
phan, and selected salts (0.1 g) were dissolved in 50 cm of
−
1
47, 500, 462, 412 cm .
water and heated at 80 °C for 2 days. The solvent was then
evaporated using a rotary evaporator. The obtained com-
pounds were dried using a vacuum dryer at 60 °C for 24 h.
Changes in the structure of the compounds were determined
on the basis of NMR spectra analysis.
Te t r a p e n t y l a m m o n i u m l ‑ t r y p t o p h a n a t e ( 5 ,
1
C H N O ) Yield: 98%; H NMR (400 MHz, D O):
3
1
55
3
2
2
δ=7.73 (d, J=7.9 Hz, 1H), 7.49 (d, J=8.3 Hz, 1H), 7.26–
7
.20 (m, 2H), 7.18–7.11 (m, 1H), 3.64–3.59 (m, 1H), 3.24
(
dd, J = 4.6 Hz, J = 14.6 Hz, 1H), 3.08–2.92 (m, 9H),
Vase life of the leaves of C. majalis
1
,2
1,3
1
1
1
6
.56–1.45 (m, 8H), 1.39–1.19 (m, 17H), 0.90 (t, J=7.1 Hz,
1
3
2H) ppm; C NMR (100 MHz, D O): δ=184.26, 138.95,
Healthy leaves of C. majalis L. without mechanical damage
were collected early in the morning from plants growing
on a bed at the University of Life Sciences in Poznan. The
leaves were conditioned in aqueous solutions of gibberellic
2
30.05, 127.15, 124.51, 121.90, 121.56, 114.56, 113.00,
0.76, 59.14, 32.78, 30.48, 24.33, 23.44, 15.91 ppm;
−
FT-IR:=ꢀ3416, 3228, 2955, 2922, 2872, 1575, 1463, 1385,
1
7
351, 1230, 1147, 1097, 1064, 1043, 1011, 915, 835, 739,
acid (GA ) and quaternary ammonium salts with the gib-
3
−
1
33, 609, 467, 431 cm .
berellate anion at concentrations of 50 and 100 ppm and
in aqueous solutions of l-tryptophan (Trp) and tetrapenty-
lammonium l-tryptophanate at concentrations of 0.5, 1.0,
Thermal analysis
5
0, and 100 ppm for 4 h in a room with a temperature of
Thermal analysis was performed based on the method
described in the literature [28, 29]. Thermal transition tem-
perature was determined by DSC, with a Mettler Toledo
18–20 °C. The leaves were then placed in distilled water.
The control sample consisted of leaves from the beginning
of the experiment placed in distilled water. The post-harvest
stability of leaves was determined in a room with a tempera-
ture of 18–20 °C, a 12-h photoperiod and ꢁuorescent light
e
Star DSC1 (Mettler Toledo) unit, under nitrogen. Samples
between 4.5 and 7.0 mg were placed in aluminum pans and
−1
−1 −2
heated from 25 to 120 °C at a heating rate of 10 °C min and
with a quantum radiation intensity of 25 μmol s m , and
−
1
cooled with an intracooler at a cooling rate of 10 °C min
the relative humidity of the air was maintained at 70%. Dur-
ing the experiment, the water was changed twice a week.
Leaf longevity was determined in days. The loss of deco-
rative values was determined by the moment of yellowing
and/or wilting of 30% of the leaf blade area. After complet-
ing the experiment in which the post-harvest longevity of
to −100 °C and then heated again to 120 °C. Thermal gravi-
e
metric analysis was performed using a Mettler Toledo Star
TGA/DSC1 unit (Mettler Toledo) under nitrogen. Samples
between 4.0 and 5.0 mg were placed in aluminium pans and
−
1
heated from 30 to 450 °C at a heating rate of 10 °C min .
leaves was assessed after the application of GA and gib-
3
Solubility
berellates, the content of chlorophyll a +b and proteins in
leaves was determined. The content of protein in the leaves
was determined using the Bradford method [31]. During the
Water and nine popular organic solvents of varying polarity
were selected for the solubility test and arranged in order of
decreasing Snyder polarity index: water, 9.0; methanol, 6.6;
DMSO, 6.5; acetonitrile, 6.2; acetone, 5.1; isopropanol, 4.3;
ethyl acetate, 4.3; chloroform, 4.1; toluene, 2.3 and hexane,
3
assay, 2 cm of a solution of Coomassie Brilliant Blue G-250
3
(CBB) in 85% orthophosphoric acid was added to 100 mm
of a diluted extract, with the extraction in a phosphorate-
potassium buꢀer (pH 7.0). After 10 min, the absorbance was
measured at a wavelength of 595 nm. The protein content
was determined from a reference curve plotted for albumin.
The level of chlorophyll was determined following the
method described in the literature after extraction with
0
.0. The solubility of the obtained salts in organic solvents
was determined according to the methodology previously
described in Vogel’s Textbook of Practical Organic Chemis-
try [30]. The sample of salt (0.1± 0.0001 g) was introduced
1
3

Synthesis and characterization of bio-based quaternary ammonium salts with gibberellate…
DMSO without tissue maceration [32]. Weighed portions
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3
9:47
(
100 mg) were treated with 5 cm of DMSO and incubated
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in a water bath at 65 °C for 60 min. The levels of chloro-
phyll in the obtained extracts were determined spectrophoto-
metrically at a suitable wavelength. For chlorophyll a, the
absorbance of the extract was measured at a wavelength of
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9
. Janowska B, Stanecka A (2011) Acta Agrobot 64:91
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0. Janowska B, Stanecka A, Czarnecka B (2012) Acta Sci Pol Hor-
torum Cultus 11:121
6
63 nm, and for chlorophyll b, it was measured at 645 nm.
The content of the pigments was calculated by means of
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12. Chen S-Y, Kuo S-R, Chien C-T (2008) Tree Physiol 28:1431
−
1
Arnon’s formulae and given in mg g FW [33].
1
3. Urbanova T, Leubner-Metzger G (2016) Gibberellins and seed
germination. In: Hedden P, Thomas SG (eds) The gibberellins.
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The experiment in which gibberellic acid and ammonium
salts with gibberellate anion was used consisted of 11 com-
binations with 3 replicates, with 5 leaves in each, whereas
the experiment in which l-tryptophan and tetrapentylammo-
nium l-tryptophanate were used consisted of 9 combinations
and the same number of repetitions and leaves in each of
them. One combination (conditioning solution × concentra-
tion) included 15 leaves.
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The experimental results were subjected to a two-factor
analysis of variance. The means were grouped using the
Newman–Keuls test at the level of signiꢃcance α = 0.05.
The Statistica 13.1 program was used.
1
8. Nakhooda M, Watt PM, Mycock D (2011) Plant Growth Regul
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Acknowledgements This work was supported by Polish Ministry of
1. Yang R, Xiao C-F, Guo Y-F, Ye M, Lin J (2018) Mater Sci Eng C
Science and Higher Education, as a research project under the program
9
1:475
“
Diamond Grant” (Grant no. DI2016018446).
2. Patron L, Marinescu G, Culita D, Diamandescu L, Carp O (2008)
J Therm Anal Calorim 91:627
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