Assessment of the organocatalytic activity of chiral L-Proline-based Deep Eutectic Solvents based on their structural features

Article abstract of DOI:10.1016/j.molliq.2020.113573

The impact on the environment of polluting and volatile organic solvents represents nowadays a severe problem. In order to reduce the incidence on the environment of chemical applications, Deep Eutectic Solvents (DESs) represent a step ahead in this field thanks to their green properties. These novel liquids are formed by inter- and intra-molecular weak interactions between two species and they are synthesized by simply heating and mixing them without any solvent. These solvents also show interesting catalytic capabilities, since their properties are truly tuneable. In this paper, L-Proline-based Chiral Deep Eutectic Solvents (CDESs) were prepared and used as green and organocatalytic reaction media in a probe asymmetric Michael addition; in this reaction the L-Proline acts as solvent component as well as chiral organocatalyst. The results were analysed with NMR studies taking in account the availability of the L-Proline, considering the strength of the association of it with the counterpart of the liquids. With DFT studies, the geometry and energy of the adducts are shown and a qualitative rationale to the reaction stereoisomers distribution is given.

Full text of DOI:10.1016/j.molliq.2020.113573

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Assessment of the organocatalytic activity of chiral l-Proline-
based Deep Eutectic Solvents based on their structural features
Matteo Tiecco, Diego A. Alonso, Diego Ros Ñíguez, Gianluca
Ciancaleoni, Gabriela Guillena, Diego J. Ramón, Alberto Apio
Bonillo, Raimondo Germani
PII:
S0167-7322(20)30248-8
DOI:
https://doi.org/10.1016/j.molliq.2020.113573
MOLLIQ 113573
Reference:
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
13 January 2020
7 May 2020
9 June 2020
Please cite this article as: M. Tiecco, D.A. Alonso, D.R. Ñíguez, et al., Assessment of
the organocatalytic activity of chiral l-Proline-based Deep Eutectic Solvents based on
their structural features, Journal of Molecular Liquids (2020), https://doi.org/10.1016/
j.molliq.2020.113573
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ASSESSMENT OF THE ORGANOCATALYTIC ACTIVITY OF CHIRAL
L-PROLINE-BASED DEEP EUTECTIC SOLVENTS BASED ON THEIR
STRUCTURAL FEATURES
Matteo Tiecco* a, Diego A. Alonso* b, Diego Ros Ñíguez b, Gianluca Ciancaleoni* c, Gabriela Guillena
b, Diego J. Ramón b, Alberto Apio Bonillo b, Raimondo Germani a.
a Department of Chemistry, Biology and Biotechnology, Università degli Studi di Perugia, via Elce di
Sotto 8, 06124, Perugia, ITALY.
b Department of Organic Chemistry and Institute of Organic Synthesis (ISO), University of Alicante,
P.O. Box 99, 03080 Alicante, SPAIN.
c
Department of Chemistry and Industrial Chemistry, University of Pisa, via Giuseppe Moruzzi 13,
56124 Pisa, ITALY.
Email: matteotiecco@gmail.com; diego.alonso@ua.es; gianluca.ciancaleoni@unipi.it.

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ABSTRACT
The impact on the environment of polluting and volatile organic solvents represents nowadays a
severe problem. In order to reduce the incidence on the environment of chemical applications,
Deep Eutectic Solvents (DESs) represent a step ahead in this field thanks to their green properties.
These novel liquids are formed by inter- and intra-molecular weak interactions between two
species and they are synthesised by simply heating and mixing them without any solvent. These
solvents also show interesting catalytic capabilities, since their properties are truly tuneable.
In this paper L-Proline-based Chiral Deep Eutectic Solvents (CDESs) were prepared and used as
green and organocatalytic reaction media in a probe asymmetric Michael addition; in this reaction
the L-Proline acts as solvent component as well as chiral organocatalyst. The results were analysed
with NMR studies taking in account the availability of the L-Proline considering the strength of the
association of it with the counterpart of the liquids. With DFT studies, the geometry and energy of
the adducts are showed and a qualitative rationale to the reaction stereoisomers distribution is
given.
KEYWORDS
Deep Eutectic Solvents; Enantioselectivity; Green Chemistry; Asymmetric Organocatalysis; Active
Solvents.

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1. INTRODUCTION
Deep eutectic solvents (DESs) are a novel class of organic liquids that have increased their
relevance in the green chemistry framework due to their unique properties[1–3]. DESs can be
divided into different sub-classes depending on the molecules they are made of[4], however they
can be simply interpreted as mixtures of a hydrogen bond donor (HBD) molecule and a hydrogen
bond acceptor (HBA) molecule. These weak interactions between the two species determine a
difficult lattice organization of the structures, therefore a liquid formation[5]. Recently, Ribeiro-
Claro and co-workers, via inelastic neutron scattering measures, showed that the DESs can be
interpreted as mixtures of intra- and inter-molecular weak interactions between the two species,
leading to a better comprehension of the structures of these novel green liquids; being this of
prominent importance for novel DESs preparation[6].
Born as a “side-class” of ionic liquids, DESs share with them many properties such as: low or
absent vapour pressure (that can lead to “out of the hood” procedures, pointing out to economic
and environmental advantages); low flammability; high recycle and re-use capabilities, and so
on[7–10]. However, DESs show lower (and in some cases absent) toxicity compared to ionic liquids
with lower their environmental, just thanks to their higher biodegradability and to the
bioavailability of their components[11–14]. Moreover, their preparation is simple: generally, they
are made only by mixing and heating the two components (often solids), leading to a liquid with a
100% of yield; no solvent or other reactant are needed for their preparation, increasing their
availability compared also to other environmental friendly solvents[15].
The use of DESs presents many advantages in several research area thanks to the range of
structural differences that can be obtained by just changing their components[16–18]. If the HBD
and the HBA molecules have natural source, the liquids are named NADESs: Natural Deep Eutectic
Solvents; these liquids have an increased green impact for their bioavailability and
biocompatibility[3,19]. Some relevant examples of their uses are: the possibility of working in non-
anhydrous conditions in procedures that usually need the absence of water (thanks to the
structural role of water in those mixtures)[20,21]; the catalytic properties of those liquids (such as
Brønsted or Lewis acids)[22–24]; their reducing properties (when formed by reducing
molecules)[25]; and the excellent extraction/preconcentration capabilities[26,27]. These
properties promote their use as substituents of volatile organic compounds (VOCs) in many
applications[28]. In these years, when environmental and economic issues are at the forefront of
human and planetary health, these liquids represent a convincing alternative to VOCs[29].

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In organic synthesis, DESs are finding fruitful applications for the cleanliness, the purity of the
obtained products, and the quickness of the developed synthetic procedures. In this field, the role
of DESs can be defined as “active” or “innocent” if they participate as reactant or catalyst in the
chemical transformation or they just act as green and environmental friendly liquids[30–33].
However, so far, the potential catalytic activity of DESs is unpredictable and only can be assessed
after trial and error procedures. Therefore, it would be very desirable to find some physical-
chemical measurements that could somehow anticipate the possible activity of a given mixture.
Recently, we have reported the first enantioselective transformations using chiral Deep Eutectic
Solvents (CDESs) as both green medium and organocatalyst[34]. The two enantiomers of
camphorsulfonic acid were used as HBD in the mixtures with different HBAs, leading to room-
temperature liquids. The yields and the enantiomeric excesses observed in the conjugate addition
of indole to chalcone correlated with the properties of the liquids; in particular it was observed
that the catalytic properties of camphorsulfonic acid relied on its “freedom” or availability, which
was related to the strength of the interaction with the HBA counterpart.
In this work, the preparation of novel chiral Deep Eutectic Solvents (CDESs) based on L-Proline as
HBA and their use as green media and chiral organocatalyst in the asymmetric Michael addition is
shown. Starting from the knowledge acquired on the structure/activity experience in this
topic[5,34–36], a correlation between the observed results with the strength of the binding of the
L-proline in the DESs with the HBD counterpart and with the structural features of the liquids
obtained was performed using NMR and DFT studies. Glycerol/L-Proline and p-toluenesulfonic acid
· H2O/L-Proline mixtures were used and analysed to compare the results obtained with the novel
CDESs with the already reported liquids with L-proline as HBA[37–39].
2. EXPERIMENTAL
2.1 GENERAL
Unless otherwise noted, all commercial reagents and solvents were used without further
purification. Reactions under argon atmosphere were carried out in oven-dried glassware sealed
1
with a rubber septum using anhydrous solvents. H NMR (300 MHz) spectra were obtained on a
Bruker AC-300, using CDCl3 as solvent and TMS (0.003%) as reference, unless otherwise stated.
Chemical shifts () are reported in ppm values relative to TMS and coupling constants (J) in Hz.
Analytical TLC was performed on Merck aluminium sheets with silica gel 60 F254. Analytical TLC

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was visualized with UV light at 254 nm Silica gel 60 (0.04-0.06 mm) was employed for flash column
chromatography whereas P/UV254 silica gel with CaSO4 (28-32%) supported on glass plates was
employed for preparative TLC. Chiral HPLC analyses were performed on an Agilent 1100 Series
(Quat Pump G1311A, DAD G1315B detector and automatic injector) equipped with chiral columns
using mixtures of hexane/isopropanol as mobile phase, at 25 °C. The deep eutectic solvents were
sonicated in an ultrasounds apparatus P-Selecta (360W). The well-known conjugate addition
1
compounds were just characterized by H NMR and chiral HPLC analyses. All referenced
synthesized compounds are in accordance with the data reported in the literature.
2.2 DESs PREPARATION
Urea, choline chloride, acetamide, (1S)-(+)-10-Camphorsulfonic acid, tetramethylammonium
chloride, D-(+)-glucose, ZnCl2, 1,6-hexanediol, S-(+)-mandelic acid, picolinic acid, L-(+)-tartaric acid,
citric acid, phenol, glycolic acid, diethylene glycol, 1,4-butanediol, glycerol and L-proline were
purchased from Sigma-Aldrich, Merck or Alfa Aesar (purities >98%); the solid samples were used
after drying under vacuum over silica gel and P2O5, the liquid reactants were used without further
purifications.
In a 10 mL round-bottomed flask L-Proline was weighed, then specific molar ratios (from 0.5 to 1
to 8:1) of the counterpart were weighed in the same flask. After each addition, the mixture was
mixed with a vortex and then put at 80°C under magnetic stirring until homogeneous and stable
fluids are formed at the proper molar ratio at room temperature.
The freezing points were determined with a thermometer using an acetone/liquid nitrogen
mixture for cooling the samples in a Dewar; the measures were repeated in triplicate and the
values have errors from ±1 to ±3°C.
Water contents were measured with a Karl-Fischer titration with Metrohm 684 KF Coulometer:
(Thymol/L-Proline = 1.4% w/w; Glycolic acid/L-Proline = 2.4% w/w; Diethylene glycol/L-Proline =
5.0% w/w; 1,4-butanediol/L-Proline = 3.6% w/w; Glycerol/L-Proline = 8.6% w/w, pTSA/L-Proline
9.0% w/w); all the measures were replicated in triplicate and have errors ranging from 0.1% to
0.2%.
Thermogravimetric analyses were carried out on a METTLER TOLEDO TGA/DSC2 apparatus under
nitrogen atmosphere at 25 °C and using a heating rate of 10 °C min-1 up to 400 °C.
2.3 CONJUGATE ADDITIONS. GENERAL PROCEDURE

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trans-β-Nitrostyrene (0.10 mmol) was added to the corresponding CDES at 30°C and the obtained
mixture was stirred for 10 minutes at the same temperature. Then, the nucleophilic ketone (0.20
mmol) was added to the reaction mixture, which was vigorously stirred for the corresponding time
(3 or 5 days). Then, EtOAc (1 mL) was added to the reaction and the resulting mixture was
sonicated for 1 minute. The organic phase was collected, and the mixture was extracted again two
times with 1 mL of EtOAc under ultrasound irradiation. The combined organic solvents were
1
evaporated under reduced pressure to give the crude reaction product which was analysed by H
NMR and chiral HPLC.
(3S,4R)-3-methyl-5-nitro-4-phenylpentan-2-one[40].
Colourless oil; H: 0.99 (d, J = 7.4 Hz, 3H), 2.23 (s, 3H), 2.92-3.00 (m, 1H), 3.67 (td, J = 9.2, 5.4 Hz,
1H), 4.70-4.61 (m, 2H), 7.12-7.15 (m, 2H), 7.24-7.32 (m, 2H); chiral HPLC analysis: Chiracel AS
column, Hexane/iPrOH: 90/10, flow rate = 1.0 mL/min, λ = 210 nm, retention times: syn 9.7 (minor
enantiomer), 11.0 (major enantiomer) min, anti 12.2 (major enantiomer), 14.4 (minor enantiomer)
min.
(S)-2-[(R)-2-Nitro-1-phenylethyl)]cyclohexanone[40].
Yellow solid; mp 122-130 °C, H: 1.23-1.31 (m, 2H), 1.60-1.78 (m, 3H), 2.07-2.12 (m, 1H), 2.40-2.48
(m, 2H), 2.70-2.75 (m, 1H), 3.74-3.82 (td, J = 9.9, 4.6 Hz, 1H), 4.62-4.69 (dd, J = 12.5, 9.9 Hz, 1H),
4.89-4.99 (dd, J = 12.5, 4.5 Hz, 1H), 7.16-7.20 (m, 2H), 7,28-87,37 (m, 3H); chiral HPLC analysis:
Chiralcel AS-H column, Hexane/iPrOH: 95/05, flow rate = 1.0 mL/min, λ = 210 nm, retention times:
syn 13.2, 17.6 min, anti 16.1, 19.1 min.
2.4 NMR STUDIES OF HBD/L-PROLINE INTERACTIONS
1H NMR spectra were measured at 298 K on a Bruker Avance III HD spectrometer. All the spectra
were calibrated on CDCl3 solvent signal and were registered with a single scan.
2.4.1 NMR Titrations
1
The titrations were performed recording the H NMR spectra adding subsequent amounts of L-
Proline to weighed solutions of HBDs with concentrations ranging from 0.039 M to 0.045 M in the
different experiments. No internal standards were used as the integrations were calibrated on the
weighed HBD. The binding constants were determined fitting the data using

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http://supramolecular.org (Prof. Pall Thordarson and Sir Fraser Stoddart)[41,42] using 1:2 fitter, L-
BFGS-B method with non-cooperative flavour, no dilution correction and with subtraction of initial
values. Except for GA/L-Pro sample, none of thevs. [L-Pro] curves reached plateau, but the
fittings were excellent with low errors (2.4% for GA/L-Pro; 3.0% for BD/L-Pro; 5.2% for GLY/L-Pro;
1.7% for DEG/L-Pro respectively).
2.4.2 1H NMR Analysis
Stock solutions of L-Proline were prepared dissolving it in CDCl3/MeOD 75/25 v/v. The HBD
molecules were weighed in NMR tubes and the proper amount of the stock solution of L-Proline
were added with syringe in order to have the same concentration of HBD and L-Proline in each
tube. The same L-Proline stock solutions were added in another tube and analysed for the spectra
of the sample without HBD. All the samples were shaken vigorously for some minutes to prevent
bad shimmed spectra. The concentrations of the samples were ranging from 0.039 M to 0.045 M
in the different HBD experiments.
2.5 THEORETICAL CALCULATIONS
All the geometries have been optimized with ORCA 4.1.0[43,44] using the B3LYP functional in
conjunction with the def2-TZVP basis set and the def2/J auxiliary basis set. The RIJCOSX
approximation has been used. The contribution of dispersion has been taken into account by using
the D3 correction[45] with Becke-Johnson energy damping. The polar environment has been
considered using the conductor-like polarizable continuum model (C-PCM) and using the
parameters of acetone (εr = 20.7). All the thermodynamic properties have been computed at the
same level of theory. All the geometries showed no imaginary frequencies or, in some cases, vary
small and unavoidable imaginary frequencies (<15 cm-1) related to relative translational motions
between the noncovalently bound components of the adducts. The list of coordinates of the
optimized geometries of the adducts are available in the Supplementary Material section.
3. RESULTS AND DISCUSSION
3.1 PREPARATION OF NOVEL L-PROLINE-BASED DEEP EUTECTIC SOLVENTS
A large set of molecules was tested as counterpart of L-Proline (L-Pro) to prepare DESs liquids at
room temperature. In this screening, molecules that formed DESs with other counterparts

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reported in literature were tested, as well as other hydrogen-bond capable molecules; L-Pro was
tested as HBA as well as HBD since it is known its dual role[46,47]. Urea, choline chloride,
acetamide, (1S)-(+)-10-camphorsulfonic acid, tetramethylammonium chloride, D-(+)-glucose, ZnCl2,
1,6-hexanediol, S-(+)-mandelic acid, picolinic acid, L-(+)-tartaric acid, citric acid and phenol were
tested in the molar ratio range from 0.5:1 to 8:1 with L-Pro. However, none of these mixtures gave
stable liquids at room temperature, showing higher melting points, unstable mixtures during time,
non-formation of liquid phases even adding molar equivalents of water. On the contrary, 1,4-
butanediol (BD), diethylene glycol (DEG) and thymol (THY) gave stable liquids at room
temperature. Furthermore, three other L-Proline-based DESs were used and analysed in this
work[39,46,48]: Glycolic Acid and L-Proline (GA/L-Pro), Glycerol and L-Proline (GLY/L-Pro) and p-
toluenesulfonic acid · H2O and L-Proline (pTSA/L-Pro). The acronym of the mixtures, their molar
ratio and the freezing points are reported in Table 1. In the Supplementary Material section the
molar ranges for all the tested molecules are reported in Table S1, the eutectic profiles of the
novel mixtures are reported in Figure S1, and the thermogravimetric analysis of the liquids are
reported in Figure S2.
Table 1: Composition, acronyms, freezing points, molar ratios of L-Proline-based Deep Eutectic Solvents used in this
work.
HBD
glycolic acid
HBA
DES acronym
GA/L-Pro
freezing point (°C)
molar ratio (HBD/L-Pro)
L-Proline
L-Proline
L-Proline
L-Proline
L-Proline
L-Proline
-9
3/1
3/1
3/1
7/1
3/1
2/1
1,4-butanediol
diethylene glycol
thymol
BD/L-Pro
11
DEG/L-Pro
THY/L-Pro
GLY/L-Pro
pTSA/L-Pro
-49
-12
-31
12
glycerol
p-toluenesulfonic acid · H2O
All these mixtures showed freezing points lower than room temperature, with DEG/L-Pro mixture
showing the lowest freezing point in the set (-49°C). Due to the natural presence of glycolic acid
(that can be obtained and it is present in the sugar beet) and thymol (that is a molecule present in
the oil of thyme, GA/L-Pro and THY/L-Pro mixtures can be considered NADESs. Regarding the role
of the counterparts, in all of these mixtures L-Pro can be considered as an HBA[5,49–52].

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The prepared liquids were then used as active green reaction media/organocatalysts in the
Michael addition of butanone to trans-β-nitrostyrene, taking advantage of the enamine activation
capability of L-Proline.
3.2 APPLICATION OF L-PROLINE DESs AS GREEN REACTION MEDIA/ORGANOCATALYSTS
The prepared CDESs were tested as catalysts in the Michael addition of butanone to trans-β-
nitrostyrene (Scheme 1); this probe reaction is reported in literature in different media with
reaction times from 1 to 8 days[53–57], so it was performed in 5 days. The results are reported in
Table 2. As shown, when using pTSA/L-Pro as CDES (entry 1), no reaction was observed at 30°C,
after 5 days. This result is probably a consequence of the highly acidic character of the CDES that
can cause a detrimental effect in the conjugate addition of ketones to nitro-olefins maybe because
of a catalyst protonation[40]. With THY/L-Pro liquid (entry 2), no reaction conversion was
observed after five days. This could be due to the hydrophobicity of the liquid caused by the HBD
(thymol) that is present in the hydrophobic phase as demonstrated in a previous work[50]. As
depicted in Table 2, the rest of the CDES afforded the Michael addition product with low (15% for
GA/L-Pro: 3/1, entry 3) to very high conversions (almost quantitative for all the other mixtures)
and moderate syn/anti diastereoselectivities (from 60/40 to 85/15). With respect to the
enantioselectivity, only the chiral eutectic solvent GA/L-Pro showed good asymmetric induction
affording both diastereomers with high enantioselectivities (syn: 86% ee; anti: 95% ee, Table 2,
entry 3). The other three mixtures (BD/L-Pro, DEG/L-Pro and GLY/L-Pro, entries 4-6 respectively)
showed only racemates in the anti diastereomers, while low enantiomeric excesses were observed
for the syn (13% with BD/L-Pro entry 4, 26% with DEG/L-Pro entry 5). Both racemates were
observed with GLY/L-Pro mixture (entry 6).
Scheme 1: CDES-catalysed conjugate addition of butanone to trans-β-nitrostyrene.

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Table 2: Conversion, diastereomeric and enantiomeric excesses in CDES-catalysed conjugate addition of butanone to
trans-β-nitrostyrene.
Entry
CDES
Conversion (%)a
dr (syn/anti)a
nd c
ee (syn/anti)b
nd c
1
2
3
4
5
6
pTSA/L-Pro: 2/1
THY/L-Pro: 7/1
GA/L-Pro: 3/1
BD/L-Pro: 3/1
DEG/L-Pro: 3/1
GLY/L-Pro: 3/1
< 5
< 5
15
> 95
92
nd c
nd c
60/40
75/25
85/15
80/20
86/95
13/rac
26/rac
> 95
rac/rac
aDetermined by H NMR analysis in the crude reaction mixture. Determined by chiral HPLC analysis. No product
1
b
c
observed.
In literature the L-Pro catalyzed conjugate addition of linear ketones, such as butanone and 2-
pentanone, to trans--nitrostyrene has shown from very low to moderate enantioselectivities
along with a strong solvent effect. Usually, only dipolar (protic and aprotic) solvents such as
DMSO[58] (85%, 50% de, 10% ee), MeOH[59] (70%, 97% de, 76% ee), EtOH[59] (91%, 64% de, 12%
ee), and H2O[60] (no reaction) have been used in this particular transformation. Also, L-proline has
failed to provide high enantioselectivities when using ionic liquids, such as [bmim][PF6][61] (68%,
80% de, 45% ee) as reaction medium.
3.3 NMR STUDIES OF HBD/L-PROLINE INTERACTIONS IN THE NOVEL DESs
The results observed in the addition of butanone to trans-β-nitrostyrene in the different CDESs
were analysed with structural NMR studies on the CDESs considering the strength of the
interactions between L-Proline and the HBD counterparts[34]. The amino acid is present, in fact, in
all the mixtures but the observed conversions and the enantiomeric excesses are largely different
among the novel liquids probably due to the different interactions between L-Proline and the
corresponding counterpart HBD. Therefore, the balance between the two roles of L-Pro was
analyzed in a semi-quantitative approach.
The first study performed was the determination of the association constants (K) of the HBD with
L-Pro via NMR titration[41,42,62]. THY/L-Pro and pTSA/L-Pro mixtures were not analyzed in this
study because they did not show conversion in the reaction. The analyzed CDESs have different
solubility properties, therefore, and in order to obtain a homogeneous set of data, all the
experiments were conducted in the solvent mixture CDCl3/MeOD: 75/25 (v/v). Chloroform is a
suitable solvent for determining the H-bond interactions because it is aprotic, while methanol is a

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disaggregating solvent and it can interfere with the hydrogen bonds between the species; the 25%
v/v amount of MeOD is a compromise between solubility and preservation of the CDES
interactions. However, these data presented valuable indications on the structures of the different
CDESs. In the Supplementary Material section, the graphs of  on [L-Pro] are reported.
Under the studied conditions, DEG/L-Pro, BD/L-Pro and GLY/L-Pro mixtures showed very low
association constants (lower than 10-8, 10-7 and 10-10 M-1 respectively), therefore they cannot be
considered for a quantitative and comparative analysis. However, the shift of the signals of the
HBD when adding L-Pro is an evidence of weak interactions occurring between the species.
Interestingly, GA/L-Pro CDES showed an association constant of 0.73 M-1, a value that is low but it
is far higher than the others, indicating the presence of an interaction between glycolic acid and L-
Pro which is stronger than the observed with the other HBDs. In the Michael addition of butanone
to trans-β-nitrostyrene, GA/L-Pro mixture showed the lowest conversion (15%) but the highest
enantiomeric excess for both diastereomers (86/95% ee). These data suggest that the interaction
between L-Pro and GA interferes with the reaction (i.e. an interaction or steric hindrance on the
nitrogen of L-Pro), but once the chiral enamine is formed, this “more structured” system leads to
higher enantiomeric excesses. The possibility of protonation of L-Pro by GA can be excluded due to
1
the similarities shown by the H NMR spectra of the CDES and L-Pro. These data are supported by
the pTSA/L-Pro liquid results that showed no conversion of the reactants even after five days. The
spectrum of L-Pro, in fact, is largely different when pTSA is added (see Supplementary Material) so
suggesting a stronger interaction that can inhibit the catalytic activity of the organocatalysts, even
if this liquid is reported in literature as based on weak interactions.
1
The shifts of the H NMR signals of L-Pro in the presence of the HBD molecule were then used to
determine the strength of the HBA-HBD interactions. The more the HBD interacts with L-Pro, the
higher shifts could be observed. In these experiments, the sum of the absolute values of the shifts
for each signal ( were evaluated to compare the different effects observed in the L-Pro-based
CDESs. In Table 3, the values of  and the association constants (K) are reported as well as the
conversions, the diastereomeric excesses and the enantiomeric excesses obtained for the model
conjugate addition reaction.

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Table 3: Association constants (K), sum of absolute values of  of the CDESs, conversion, diastereomeric ratio
and enantiomeric excesses, in the addition of butanone to trans-β-nitrostyrene catalysed by CDESs.
Entry
K
Conversion
dr
(syn/anti)a
60/40
ee

(M-1) (ppm)
CDES
(%)a
(syn/anti)b
1
2
3
4
GA/L-Pro: 3/1
DEG/L-Pro: 3/1
BD/L-Pro: 3/1
0.74
n.a.c
n.a.c
n.a.c
0.043
0.011
n.a.d
15
86/95
92
85/15
26/rac
> 95
75/25
13/rac
GLY/L-Pro: 3/1
n.a.d
> 95
80/20
rac/rac
a
1
b
c
Determined by HNMR analysis in the crude reaction mixture. Determined by chiral HPLC analysis.
Values lower than 10-8 M-1. d Values lower than 0.01 ppm.
The CDES GA/L-Pro showed the highest value of in the set (0.043 ppm), DEG/L-Pro mixture
showed a lower value (0.011 ppm), while BD/L-Pro and GLY/L-Pro mixtures showed values lower
than 0.01 ppm and therefore they were not considered. These values are really low, but still the
de-aggregating properties of MeOD must be considered. However, these experiments gave
important indications about the structural features of the liquids. As observed, the interaction
between L-Pro and the glycolic acid in the GA/L-Pro liquid was stronger than the detected between
L-Pro and the other HBDs (Table 3, entry 1). In particular, the hydrogen in position 2 (H2) of the
chiral organocatalyst showed the highest shift (downfield) of all the studied signals and
experiments, as demonstrated in Figure 1, where the superimposed 1H NMR spectra of L-Pro in the
presence and in the absence of the correspondent HBD for H2 of L-Pro are shown. All the spectra
are reported in Supplementary Material section.
L-Pro
L-Pro
GA/L-Pro
DEG/L-Pro
L-Pro
L-Pro
BD/L-Pro
GLY/L-Pro
Figure 1: 1H NMR (400 MHz, CDCl3/MeOD: 75/25 v/v) spectra of H2 of L-Proline (black) the corresponding CDES (blue).

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These NMR experiments showed results similar to those obtained from the NMR titration studies:
GA/L-Pro showed the strongest interaction between the HBD and the L-Pro. Furthermore,
analysing the data from the model asymmetric organocatalyzed reaction, it can be observed that
the stronger interaction between L-Pro and the HBD is, the lower the conversion of the reaction
(Table 3, entry 1). On the contrary, the enantiomeric excess is favoured by these structured
systems, as corroborated with the DEG/L-Pro mixture, which showed a slight lower interaction,
thus increasing the conversion (from 15 to 92%) but decreasing the ee (from 86 to 26% ee for the
major syn diastereomer) (Table 3, entries 1 and 2). Finally, for those mixtures where the HBA/HBD
interactions are weak (GLY/L-Pro and BD/L-Pro), the reaction conversions are quantitative but only
racemic diastereomers were observed.
3.4 DFT STUDIES
The results obtained from the NMR experiments suggested to investigate the interactions
occurring between L-Pro and the HBD counterpart with computational calculations. Thus, DFT
calculations were performed [B3LYP-D3/def2-TZVP/c-PCM (εr = 20.7) level of theory] on the
different adducts involved in the organocatalytic conjugate addition reaction. Considering the
nature of the interactions, a polar environment was necessary to correctly assess the energy of
the adducts. Unfortunately, the experimental determination of the polarity of a DES is not
straightforward[62], so the polarity of acetone was used (polarity index: 5.1; dielectric constant:
20.7)[63] as an educated guess and compare all the adducts under the same computational
conditions.
Figure 2 shows the optimized structure of the 1:1 adducts between L-Pro and GLY, GA, DEG and
BD. As seen in the geometry of the 1:1 GLY/L-Pro adduct, the secondary –OH groups of glycerol
points directly toward the lone pair of the nitrogen of L-Pro, whereas the carboxylic moiety of the
latter interacts with one of the primary –OH groups of the HBD. A similar HBD/HBA adduct
geometry is obtained using GA and BD, whereas in the case of DEG, the amino proton (and not the
nitrogen lone pair) is involved in the inter-molecular interaction.

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Figure 2: DFT optimized geometries of adducts formed between L-Pro and GLY, GA, DEG and BD.
The stability of the CDESs adducts, with respect to the isolated components, follows the trend
(ΔE): GA/L-Pro (-3.8 kcal/mol) < BD/L-Pro (-3.0 kcal/mol) < GLY/L-Pro (-2.7 kcal/mol) < DEG/L-Pro
(+2.2 kcal/mol). The Gibbs free energy is positive in all the cases (9.0 kcal/mol for GA/L-Pro), but it
should be remembered that in the CDES many other interactions are present, contributing to
lower ΔG. Notably, the most stable adduct is GA/L-Pro, coherently with its lowest reaction
conversion value (Table 3, entry 1).
In order to give a rationale to the different enantioselectivities obtained in the Michael addition of
butanone to -nitrostyrene with CDESs, firstly the two most stable enamines E1 (s-cis or syn
enamine) and E2 (s-trans or anti enamine) formed by reaction between L-Pro and butanone have
been optimized (Figure 3). The former resulted to be slightly less stable than the latter (0.4
kcal/mol). Interestingly, in the isolated enamine the carboxylic acid establishes a hydrogen bond
with the lone pair of the nitrogen atom (N…H distance 1.916 Å), which is not necessarily true in the
DES.

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Figure 3: DFT optimized geometries of E1 and E2. The relative Gibbs energy is given in parenthesis (kcal/mol).
In fact, it is important to underline that the amino group of L-Pro is heavily involved in the
interaction with the second component of the CDES. Then, the system was modelled as follows:
the enamine interacts with both the HBD and trans-β-nitrostyrene (NS) at the same time, giving a
ternary adduct. For this, one of the enantiofaces of the enamine will be occupied by the HBD,
forcing NS to interact with the other enantioface and, therefore, producing a specific enantiomer.
In conventional solvents, a high selectivity in this test reaction is explained by a HB interaction
between the carboxylic group of L-Pro and the nitro group of NS[59,64,65], but in this case also the
solvent is able to establish HBs with L-Pro, making the situation more complex. Under the
hypothesis that the activation barrier of the addition will likely be similar for all of the studied
adducts, a high ee is expected in those cases where one adduct is much more stable than the
others. Considering also the possibility to have the two different enamines (anti and syn), it means
that 8 ternary adducts have been optimized for each HBD. To differentiate them, the following
nomenclature was adopted: the suffix –nA indicates that the electrophile does not interact with
the carboxylic group of the enamine (-nB, if it does). Further, the suffix 1 indicates that the NS
interacts with the other components through its si face, 2 if through the re face. In Figure 4 are
reported the optimized geometries of GA/L-Pro adducts with the relative Gibbs energies, all the
adducts of the other mixtures are reported in Supplementary Material section. Table 4
summarizes the results, indicating the relative energy for each group of ternary adducts, setting
the most stable as the reference, and the isomer coming from that geometry.

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Figure 4: DFT optimized geometries of Glycolic Acid/L-Proline. Only selected hydrogen atoms are explicitly shown.
Relative Gibbs energies, in kcal/mol, are given in parenthesis.
The relative energies of the ternary allow to semi-quantitatively rationalize the observed
experimental results for the model Michael addition reaction. In fact, among the possible GA/E1
and GA/E2 adducts, GA/E2-nB1 and GA/E2-nB2 are quite close in energy (0.3 kcal/mol, Table 4),
but the former leads to the formation of the anti isomer, whereas the latter to the formation of
the syn one. This allows to justify the low diastereomeric excess experimentally found for this
system (60/40). Generally speaking, the parameter Δd can be defined as the energy difference
between the most stable configuration leading to the anti isomer and that leading to the syn one.
As seen for GA/E, Δd directly relates with the de of the reaction.
The same strategy can be used to give a rationale for the ee. In fact, Δanti can be defined as the
energy difference between the most stable adducts leading to the isomers anti(S,S) and anti(R,R).
In the case of GA/E, Δanti is 0.9 kcal/mol, which reflects in a 1:0.22 ratio in concentration between
the two enantiomers, qualitatively coherent with a quite high ee (experimental = 95).
Analogously, Δsyn can be defined as the energy difference between the most stable adducts
leading to the isomers syn(S,R) and syn(R,S) and it is related to the ee of the syn isomer. For GA/E,
it is 0.6 kcal/mol, leading to a lower ee (experimental = 86).

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It is useful to compare the values of Δd, Δanti and Δsyn for all the systems. By using DEG, many
adducts are close each other in energy. In this case Δd, Δanti and Δsyn are 0.5, 0.4 and 0.3
kcal/mol, respectively, allowing to predict a moderate de (experimental = 85/15) and low ee
values (experimental = rac for anti, 26 for syn).
In the case of BD, Δd, Δanti and Δsyn are 0.5, 0.3 and 0.7 kcal/mol, respectively, allowing to
predict a moderate de (experimental = 75/25), a low ee value for the anti isomer (experimental =
rac) and a moderate ee for the syn isomer (experimental = 13). Finally, for GLY, Δd, Δanti and Δsyn
are 1.3, 0.1 and 0.4 kcal/mol, respectively, allowing to predict a high de (experimental = 80/20)
and low ee values (experimental = rac for both the isomers).
This simple framework allows giving a qualitative rationale to the isomers distribution.
Table 4: Energies and energies differences (Δd, Δanti and Δsyn) between the most stable configurations of the ternary
adducts (kcal/mol). For each isomer, the most stable adduct is underlined.
Adduct
Relative
energy
1.7
isomer
produced
anti(S,S)
syn(R,S)
syn(S,R)
anti(R,R)
syn(R,S)
anti(S,S)
anti(R,R)
syn(S,R)
Adduct
Relative
energy
1.6
Enantiomer
produced
anti(S,S)
syn(R,S)
GA/E1-nA1
GA/E1-nB1
GA/E1-nA2
GA/E1-nB2
GA/E2-nA1
GA/E2-nB1
GA/E2-nA2
GA/E2-nB2
Δd
BD/E1-nA1
BD/E1-nB1
BD/E1-nA2
BD/E1-nB2
BD/E2-nA1
BD/E2-nB1
BD/E2-nA2
BD/E2-nB2
Δd
0.9
0.7
7.2
3.3
syn(S,R)
0.9
0.8
anti(R,R)
syn(R,S)
1.6
3.7
0.0
0.5
anti(S,S)
anti(R,R)
syn(S,R)
1.2
2.3
0.3
0.0
0.3
0.9
0.6
0.5
0.3
0.7
Δanti
Δanti
Δsyn
Δsyn
DEG/E1-nA1
DEG/E1-nB1
DEG/E1-nA2
DEG/E1-nB2
DEG/E2-nA1
DEG/E2-nB1
3.5
0.3
2.0
0.5
3.7
0.9
anti(S,S)
syn(R,S)
syn(S,R)
anti(R,R)
syn(R,S)
anti(S,S)
GLY/E1-nA1
GLY/E1-nB1
GLY/E1-nA2
GLY/E1-nB2
GLY/E2-nA1
GLY/E2-nB1
4.0
0.4
2.1
1.3
1.6
1.4
anti(S,S)
syn(R,S)
syn(S,R)
anti(R,R)
syn(R,S)
anti(S,S)

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DEG/E2-nA2
DEG/E2-nB2
Δd
4.4
0.0
anti(R,R)
syn(S,R)
GLY/E2-nA2
GLY/E2-nB2
Δd
2.6
0.0
anti(R,R)
syn(S,R)
0.5
0.4
0.3
1.3
0.1
0.4
Δanti
Δanti
Δsyn
Δsyn
Finally, the Michael addition of cyclohexanone to trans--nitrostyrene was also studied using the
chiral deep eutectic mixtures (Scheme 2). As depicted, GA/L-Pro afforded again the corresponding
conjugate addition product with the lowest yield (9%). However, both diastereoselectivity and
enantioselectivity were very similar for all the tested eutectic mixtures.
Surprisingly, cyclohexanone is the model nucleophile in organocatalyzed conjugated additions in
organic solvents due to the high enantioselectivity it typically shows[54,55]. The fact that this is
not the case in the studied CDESs, demonstrates again the strong substrate dependence that
asymmetric organocatalysis usually involves.
Scheme 2: CDES-catalyzed conjugate addition of cyclohexanone to -nitrostyrene.
4. CONCLUSIONS
In this paper L-Proline-based chiral Deep Eutectic Solvents (CDESs) were prepared and used as
green and organocatalytic reaction media in a model Michael addition. The results were
interpreted with NMR studies in terms of the strength of the bind of L-Proline with the

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counterpart in the different liquids. The “freedom” of the L-Proline had a key role for its reactivity
in terms of conversion of the reactants, but also conversely in the enantiomeric excesses: stronger
interactions of L-Proline to the HBD (such as the case of GA/L-Pro) led to adducts in the DESs that
lowered the conversions to 15% (because of the non-availability of the nitrogen of L-Proline) but
increased the enantiomeric excesses (up to 86%/95% for the two diastereomers) thanks to the
higher structuration of the adducts. On the other hand, weaker interactions of L-Proline with the
counterpart (such as the case of the other mixtures DEG/L-Pro, BD/L-Pro and GLY/L-Pro) led to
quantitative conversion values but showed lower enantiomeric excesses or also racemic products
(from 26% to racemates). The interactions occurring between the CDESs species and the energies
of the adducts of the Michael addition were analysed with DFT studies, that gave a rationale of the
isomers’ distribution.
The data coming from the use of the CDESs in another Michael addition (cyclohexanone to trans-
β-nitrostyrene) showed results that are promising for further studies on the use of those liquids in
asymmetric synthesis as the data are still coherent with the results of the structural features of
these liquids, but with different entities.
Thus, this work represents an approach to the activity of the DESs in terms of the strength of the
binding between the two components of the liquids. This could facilitate the interpretation of the
activity of these sustainable liquids and help in the design of novel structured liquids of this class.
DECLARATION OF INTEREST
The authors declare no conflict of interest.
AKNOWLEDGEMENTS
This work was supported by the University of Pisa through the PRA_2018_36 grant, and by the
Spanish Ministerio de Ciencia, Innovación y Universidades (MICINN, PGC2018-096616-B-I00) and
the University of Alicante (VIGROB-173).
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ASSESSMENT OF THE ORGANOCATALYTIC ACTIVITY OF CHIRAL
L-PROLINE-BASED DEEP EUTECTIC SOLVENTS BASED ON THEIR
STRUCTURAL FEATURES
AUTHORS CONTRIBUTION
Matteo Tiecco: conceptualization, methodology, investigation, resources, writing - original draft,
Project administration, visualization;
Diego Alonso: methodology, validation, resources, writing - original draft, investigation;
Diego Ros Niguez: investigation;
Gianluca Ciancaleoni: methodology, software, formal analysis, investigation, writing - original
draft, funding acquisition;
Gabriela Guillena: Writing - Review & Editing, funding acquisition;
Diego Ramon: Writing - Review & Editing, funding acquisition;
Alberto Apio Bonillo: investigation;
Raimondo Germani: supervision, Writing - Review & Editing.

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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as
potential competing interests:
NONE

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Graphical abstract

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ASSESSMENT OF THE ORGANOCATALYTIC ACTIVITY OF CHIRAL
L-PROLINE-BASED DEEP EUTECTIC SOLVENTS BASED ON THEIR
STRUCTURAL FEATURES
HIGHLIGHTS
 Novel L-Proline-based Chiral Deep Eutectic Solvents (CDESs) were prepared
 The CDESs were used as green and organocatalytic reaction media
 A semi-quantitative NMR approach based on the features of the liquids is proposed
 Organocatalytic activities (ee up to 95%) correlated with HBD-HBA association strength
 DFT studies gave a qualitative rationale of stereoisomers distribution