A Proline Mimetic for the Design of New Stable Secondary Structures: Solvent-Dependent Amide Bond Isomerization of (S)-Indoline-2-carboxylic Acid Derivatives

Article abstract of DOI:10.1021/acs.joc.1c00184

A thorough experimental and computational study on the conformational properties of (S)-indoline-2-carboxylic acid derivatives has been conducted. Methyl (S)-1-acetylindoline-2-carboxylate, both a mimetic of proline and phenylalanine, shows a remarkable tendency toward the cis amide isomer when dissolved in polar solvents. This behavior is opposite to the general preference of proline for the trans isomer, making indoline-2-carboxylic acid a good candidate for the design of different secondary structures and new materials.

Full text of DOI:10.1021/acs.joc.1c00184

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Article
A Proline Mimetic for the Design of New Stable Secondary
Structures: Solvent-Dependent Amide Bond Isomerization of
(S)‑Indoline-2-carboxylic Acid Derivatives
Matteo Pollastrini, Filippo Lipparini, Luca Pasquinelli, Federica Balzano, Gloria Uccello Barretta,
Gennaro Pescitelli, and Gaetano Angelici*
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ABSTRACT: A thorough experimental and computational study on the conformational properties of (S)-indoline-2-carboxylic acid
derivatives has been conducted. Methyl (S)-1-acetylindoline-2-carboxylate, both a mimetic of proline and phenylalanine, shows a
remarkable tendency toward the cis amide isomer when dissolved in polar solvents. This behavior is opposite to the general
preference of proline for the trans isomer, making indoline-2-carboxylic acid a good candidate for the design of different secondary
structures and new materials.
many peptide analogues. For example, Tomasini’s group
developed oxazolidinone-containing peptides,32,33 showing
that the Phe-D-Oxd moiety is a privileged scaffold for controlling
the formation of supramolecular materials,34,35 thanks to the
preferential trans conformation of generic Xaa-D-Oxd bonds and
the presence of phenylalanine. Moreover, they recently
INTRODUCTION
■
Conformational diversity is Nature’s way of disclosing important
properties; therefore, a deeper understanding of the intrinsic
conformational order of natural and unnatural amino acids is
essential for the rational design of biomimetic compounds.
Proline and phenylalanine are two important amino acids often
involved, respectively, in protein conformational switching and
aggregation phenomena. For proline and its analogues, the
pioneering work of Wennemers’ group showed that a deep
knowledge of the conformational properties of model peptides
in different environments,1 especially for the cis/trans isomer-
ization equilibrium, can lead to a number of applications in
catalysis2 or in the synthesis of collagene-like materials,3−5 as a
tool for polyproline II crystal structure resolution6 or in the use
of oligoprolines in supramolecular assembled materials.7,8
Regarding phenylalanine, since the groundbreaking studies by
Gazit about the aggregation properties of amyloid peptides,9,10
so many developments have taken place in nanobiotechnol-
ogy,11,12 and biomaterials13 that it seems appropriate to refer to
more extensive reviews.14−16 The deeper comprehension and
combination of the many factors that control the cis/trans
isomerization and aggregation, like n→π* interactions,17−24
π−π stacking,25,26 or steric factors,27−31 have led to the study of
developed new soft materials based on L-DOPA,36,37
a
psychoactive analogue of phenylalanine, and in collaboration
with them we could spot through chiroptical techniques the
elusive π-helix motif attained by oligomers containing
pyroglutamic acid,38 a proline mimetic. (S)-Indoline-2-carbox-
ylic acid ((2S)-Ind) is an interesting case, as it is both a mimetic
of L-proline and L-phenylalanine. (2S)-Ind can be considered as
the result of the fusion of an aromatic ring to the pyrrolidine
bond between C4 and C5 of proline. At the same time, (2S)-Ind
can also be seen as a phenylalanine with the side chain
Received: January 23, 2021
Published: June 3, 2021
© 2021 The Authors. Published by
American Chemical Society
https://doi.org/10.1021/acs.joc.1c00184
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conformationally locked in a fixed orientation. Both these
features make (2S)-Ind a good candidate for the investigation of
its conformational properties. Contrary to the many studied
proline analogues with substituents at position C4 and C5,39−43
pseudoprolines,44 or other bicyclic proline derivatives,45,46 (2S)-
Ind cannot undergo stabilization or destabilization by ring
puckering, as the 5-membered ring is intrinsically quasiplanar.
The biological importance of kinetic and thermodynamic
quantities of prolinamide conformational interconversions is
well demonstrated, for example, by the extensive work of
Fischer’s group.47−51 Cis/trans proline isomerization has indeed
often been found as the rate-limiting step in the protein-folding
processes. Regarding specifically (2S)-Ind, an earlier explorative
computational study of Torras et al.52 on the N-acetyl-N′-
methylamide derivative (Ac-(2S)-Ind-NHMe) investigated the
structural role of the aromatic ring, highlighting that its presence
further reduces the intrinsically low conformational flexibility of
proline, giving higher preference for the cis state of the peptide
bond involving the pyrrolidine nitrogen. However, the presence
of a terminal secondary amide might have a strong influence on
the conformation of the molecule, given the possibility of
hydrogen bond formation. Consequently, in the conformational
analysis of proline and its analogues, acetylated methyl esters or
N-acetyl-N′,N′-dimethylamides are often preferred.53 For this
reason we decided to investigate Ac-(2S)-Ind-OMe (1) and the
dimer Ac-(2S)-Ind-(2S)-Ind-OMe (2) (Scheme 1) to explore
the cis and trans isomers; the procedure is detailed in the
Experimental Section. The capability to distinguish each signal is
useful to study the physicochemical parameters of the
equilibrium in solution. It was possible to evaluate the
populations of cis and trans isomers, and consequently, the
value of the cis-to-trans equilibrium constant Ktrans/cis at 25 °C,
through the ratio between the integrated areas of the two peaks
1
corresponding to the Hα of the two isomers in the H NMR
spectrum, found between 4.1 and 5.2 ppm (Figure 1 and Table
Figure 1. 1H NMR in the Hα region of a 0.1 M solution of 1 in different
solvents at 25 °C.
Scheme 1. (Top) Cis−Trans Isomerization Equilibrium of the
Amidic Bond in Ac-(2S)-Ind-OMe (1) and Major Torsional
angles. (Bottom) Structure of Ac-(2S)-Ind-(2S)-Ind-OMe
(2) Shown as the Major Cis−Cis Conformer, Relative to the
Two ω Angles
Table 1. Ktrans/cis of Ac-(2S)-Ind-OMe (1) Obtained from 1H
NMR Spectroscopy of 0.1M Solutions in Different
Deuterated Solvents at 298 K
a
a
b
solvent
C6D6
CDCl3
CD2Cl2
CD3OD
CD3CN
DMSO-d6
Ktrans/cis
δ (ppm) trans
δ (ppm) cis
εr
0.72
0.87
0.63
0.47
5.06
5.17
5.10
5.15
5.04
5.02
4.14
4.91
4.93
5.22
5.04
5.30
2.3
4.8
8.9
32.7
37.5
46.7
c
0.43
0.31
a
Chemical shift values of the Hα of the trans and cis conformer.
Solvent dielectric constant. For CD3CN, Ktrans/cis was calculated
b
c
through the ratio between the integrated areas of the CH3 acetamide
signals of the two isomers.
1). The preferred species in a 0.1 M CDCl3 solution of 1 is the cis
conformer, with a corresponding Ktrans/cis = 0.87. The conforma-
tional preference seems to be independent of the concentration,
as the trans/cis ratio did not change analyzing 0.04 and 0.8 M
solutions of 1 in CDCl3. More interestingly, we observed a
marked influence of the solvent polarity on the equilibrium
(Figure 1 and Table 1). More polar solvents favor the prevalence
of the cis conformer. Another interesting phenomenon, also
evident in Figure 1, is that the signal of the Hα in the cis
conformer moves significantly to lower frequencies depending
on the polarity of the solvent, while the Hα signal corresponding
to the trans conformer is less affected.
both the methyl ester derivative and the amidic bond in the
dipeptide. Referring to Scheme 1, it was anticipated that in Ac-
(2S)-Ind-OMe angle ϕ is fixed by the pyrrolidine ring; thus, the
main degrees of conformational freedom of 1 are represented by
the torsional angles ω and ψ. The amide angle ω is expected to
assume values around 0° and 180° leading to cis and trans
isomers, respectively, distinguished by NMR. Conversely, the
rotational barrier for angle ψ is too low to be recognized by
NMR, but it is still important for the overall conformational
equilibrium.
As it can be seen in Table 1, by testing several solvents, the
trans/cis ratio spans from 0.72 in benzene-d6 to 0.31 in DMSO-
d6, depending on the dielectric constant (εr) of the solvent. This
observation can be rationalized by assuming a larger dipole
moment for the cis isomer than for the trans one, which results in
RESULTS AND DISCUSSION
■
We were pleased to see that the 1H NMR in CDCl3 of 1 showed
well-separated signals, and through a complete bidimensional
analysis it was possible to unambiguously assign each signal of
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a stabilization of the cis conformer by more polar solvents.54 The
experimental Ktrans/cis values were fitted against an empirical
correlation of the type defined by eq SI4 (see the Supporting
Information, SI), which describes a solvent-dependent con-
formational equilibrium where the solvent is seen as a uniform
dielectric with relative permittivity εr.55 A very good fit was
observed for all solvents, except benzene, demonstrating that the
equilibrium is indeed controlled by the solvent reaction field,
while benzene is possibly capable of specific interactions with
the solute aromatic ring.
the proximate acetamide group when the amidic bond is in the
cis conformation, remains anchored around 8.2 ppm, allowing
the determination of Keq at different temperatures. By plotting ln
Keq vs 1/T, ΔH° and ΔS° can be obtained (see the SI). The
estimated values are ΔH° = +3.10 kcal/mol and ΔS° = +8.2 cal/
molK for the cis-to-trans equilibrium (in DMSO). Thus, an
enthalpy/entropy compensation effect seems to be at play for
the considered conformational equilibrium.
Different methods have been proposed to measure the rate
constants and the activation energy for the cis/trans isomer-
ization.58,59 However, considering the unequal population
between the two exchanging species, as well as similar spin−
lattice relaxation time T1 of the two species (T1cis = 2.39 s; T1trans
= 2.46 s) we found the approach outlined by Perrin and Dwyer
to be convenient.60 2D exchange spectroscopy (EXSY), which
provides a map of the exchanging species, was used. This
approach allows for the calculation of the rate constants for a
chemical exchange process by measuring the cross-peak to
diagonal peak intensity ratio. The ratio of the rate constants,
obtained with the optimal mixing time found (τm = 0.03 s), was
Comparing the measured Ktrans/cis with those reported for Ac-
Pro-OMe, we can see that the nature of the solvent and the
presence of the aromatic ring drastically affects the conforma-
tional equilibrium around the amide bond (Table 2). More
Table 2. Comparison of the Ktrans/cis of Ac-(2S)-Ind-OMe (1)
and Ac-Pro-OMe in Different Solvents at 298 K
Xaa
CDCl3
DMSO-d6
C6D6
Ac-Pro-OMe
3.653
3.853
∼4.956
Ac-(2S)-Ind-OMe
0.87
0.31
0.72
K
trans/cis = kc→t/kt→c = 0.31. This value is fully consistent with that
obtained from the Hα peak integral ratio at 25 °C (see above).
From the rate constants, we can also calculate the free energy of
activation for the cis to trans and the trans to cis isomerization
according to the absolute rate theory. The estimated values were
ΔG⧧c→t = 16.4 kcal/mol and ΔG⧧t→c = 15.7 kcal/mol, which lie
in the lower range of observed cis/trans barriers for N,N-
disubstituted acetamides.61 NMR spectra of 1 acquired in
CD2Cl2 to the lower temperature of −60 °C did not influence at
all the conformational equilibrium.
A well-recognized factor, which favors the trans conformer
over the cis conformer in proline analogues, is the n→π*
interaction between adjacent carbonyl groups.20,23 The nature
of the terminal group can affect the equilibrium, as esters are
more electrophilic than amides and act as better acceptors for
the n→π* interaction.53 In our case, given the cis preference of 1,
we speculated that other factors may overcome a possible n→π*
interaction. Therefore, we expected that the presence of a
tertiary amide as the acceptor group would not dramatically
change the conformational preference. For that reason, we
decided to synthesize compound 2 as model to confirm the
preference of the cis conformer in an (S)-indoline-2-carboxylic
acid derivative involved in a peptide bond (Scheme 1).
Compound 2 is short enough to be a convenient model to
investigate the impact of a tertiary amide in the backbone,
avoiding the occurrence of cooperative effects which would
show up in larger oligomers.
importantly, contrary to most proline-mimetic compounds
reported in the literature, 1 shows in solution an excess of the cis-
amide isomer which increases with the solvent dielectric
constant.
To confirm the importance of solvent polarity in the
stabilization of the cis conformer of 1, we performed a series
of titrations. We observed that adding sequential amounts of
D2O to a DMSO-d6 solution of 1 did not affect at all the
preference for the cis isomer, while adding portions of DMSO-d6
to a solution of 1 dissolved in benzene-d6 proportionally
increased the population of the cis isomer. These experiments
show that the conformational preference is dominated by the
solvent polarity, as expected, rather than by solute−solvent
hydrogen bonding.
With the aim of evaluating the thermodynamic parameters
associated with the isomerization equilibrium in DMSO-d6, we
performed variable-temperature NMR experiments to be
analyzed by the Van’t Hoff equation.57 As can be seen in Figure
2, the increase of temperature led to a coalescence of the cis/
trans Hα signals around 5.2 ppm already at 60 °C, indicating a
fast exchange between the two populations. However, the
characteristic signal of the aromatic proton HAr4cis, deshielded by
The NMR analysis in DMSO-d6 of the dimer Ac-(2S)-Ind-
(2S)-Ind-OMe (2) demonstrated for both amidic bonds a
strong preference for the cis conformation, confirming this
tendency also when a tertiary amide moiety is present as
terminal substituent. In Figure 3, it is possible to appreciate the
influence of solvent polarity in CDCl3 and DMSO-d6, while a
complete attribution of NMR signals of the predominant cis−cis
conformer of 2 in DMSO-d6 is reported in the Experimental
Section. Although compound 2 is too short to fold into a stable
secondary structure, we expect the same preference for the cis
geometry of the amide junction to be retained in longer
oligomers of indoline-2-carboxylic acid, which will be the subject
of future studies.
To investigate the cis/trans isomerization from a different
perspective, a computational study was performed to determine
the relative stability of the two isomers of 1 in various solvents.
Figure 2. 1H NMR of a 0.1 M solution of 1 in DMSO-d6 at different
temperatures
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Table 3. Computed Relative Free Energies of the Four
Conformers of Ac-(2S)-Ind-OMe, in kcal/mol
solvent
C1
C2
T1
T2
Ktrans/cis
benzene
CHCl3
CH2Cl2
CH3OH
CH3CN
DMSO
0.0
0.0
0.1
0.3
0.3
0.3
0.1
0.0
0.0
0.0
0.0
0.0
0.4
0.4
0.5
0.6
0.6
0.6
0.4
0.3
0.4
0.5
0.5
0.5
0.55
0.52
0.53
0.48
0.48
0.48
underestimated by calculations. From the computed dipole
moments in various solvents of the cis and trans isomers,
calculated as the Boltzmann average of the dipoles of the two
conformers for each isomer, it appears that there is no definite
prevalence of either isomer as the more polar one. This is due to
the combination of the local dipoles allied with the amide and
ester moieties, which in the four isomers arrange in different
ways as depicted in Figure 4, yielding an overall dipole with
variable direction and intensity. It is clear that, being the
populations of the four conformers all very similar, and the
respective dipole moments very variable, subtle changes in the
populations may affect the average dipole moment substantially.
This may be the reason why polarizable continuum solvent
models seem not fully adequate to quantitatively describe the
solvent-dependent conformational equilibrium of Ac-(2S)-Ind-
OMe (1).
Figure 3. 1H NMR of 2 in the Hα region and in the aromatic region in
CDCl3 and DMSO-d6.
Four different conformations (called C1, C2 and T1, T2,
respectively) were considered, characterized by values of the ω
dihedral of approximately 0 and 180 deg (C and T isomers), and
of the ψ dihedral around +160 and −20 degrees (1 and 2
conformers), respectively. The optimized geometries of the C1,
C2 and T1, T2 conformers in DMSO are reported in Figure 4.
For the sake of completeness, we carried out geometry
optimizations in various solvents also on compound 2 (see the
SI). In the dimer, angles ω1, ω2, ψ1, and ψ2 (Scheme 1) are
allowed to vary, yielding 16 potential conformers of which 15
corresponded to stable minima. In this case, too, we found the
cis−cis conformers (i.e., with ω1 and ω2 ≈ 0°) to be the most
stable ones, in good agreement with the experimental data.
In the attempt to rationalize the observed behavior of (S)-
indoline-2-carboxylic acid derivatives, we estimated all of the
possible interactions that could influence their conformational
preferences by using natural bond orbital (NBO) analysis. We
first verified the extent of the n→π* interaction for compound 1,
in comparison with Ac-Pro-OMe which prefers the amide trans
conformation.17 All the typical geometrical indicators of the n→
π* interaction (CO··COO distance, CO−C Bu
̈
rgi−Dunitz
trajectory, pyramidalization of ester C),20 as well as the overlap
between the nCO and π*COO orbitals, were similar for the two
compounds in their trans conformations (see the SI). This
finding suggested that the different conformational behavior is
not related to a different extent of the n→π* interaction. In fact,
the main geometrical difference between Ac-(2S)-Ind-OMe (1)
and Ac-Pro-OMe is not in the reciprocal arrangement between
the amide and ester groups but in the fact that in the former
compound the N-acetyl moiety lies in the same plane as the
phenyl ring, as shown in Figure 5. A possible reason for the
higher stability of the cis isomer of Ac-(2S)-Ind-OMe (1) is a
subtle combination between steric and electrostatic effects
involving the N-acetyl moiety. In the trans isomer of Ac-(2S)-
Ind-OMe, a steric repulsion between CH3 and HAr4 is detectable,
which is relieved both in the cis isomer (between CH3 and CHα)
and in Ac-Pro-OMe (between CH3 and CH2δ). Conversely, the
cis isomer allows for a stabilizing attraction between CO and
Figure 4. Optimized geometries of the four conformers of 1 in DMSO
obtained using the B3LYP-D3BJ functional and the 6-311+G(d,p) basis
set and the IEFPCM continuum solvation model. Arrows represent
dipole moments: magenta, overall molecular dipole moment (the size is
proportional to the magnitude); blue and red, local dipole moments
associated to the amide and ester moiety, respectively, estimated at the
same level of calculation on N,N-dimethylacetamide and methyl
acetate.
For each starting geometry, a full optimization was performed,
followed by a frequency calculation to characterize the
optimized geometries as minima and compute, for each
conformer, its Gibbs free energy. The computed Gibbs free
energy differences between the various conformers in different
solvents are reported in Table 3, while all of the optimized
geometries and absolute Gibbs free energies are reported in the
SI.
We note that, qualitatively, DFT calculations nicely reflect the
experimental finding that the cis isomer is favored in all solvents,
and there is a tendency toward the stabilization of the cis isomer
in more polar solvents. Quantitatively, however, the impact on
the conformational equilibrium of the different solvents is
H
Ar4, although this latter effect is less important than anticipated.
Atom charges calculated with natural population analysis reveal
a slight increase of HAr4 charge (by 10% in CHCl3) passing from
the trans to the cis isomer of Ac-(2S)-Ind-OMe, which might be
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solvents and liquid reagents with hypodermic syringes. The glassware
has been dried with a heating gun under vacuum and allowed to cool
under argon. Anhydrous solvents and liquid reagents were obtained
using standard drying techniques. Solid reagents were of commercially
available grade, used without further purification and, when necessary,
stored in a controlled atmosphere and/or at −20 °C. (S)-Indoline-2-
carboxylic acid was purchased from abcr GmbH. Reactions were
monitored by thin-layer chromatography using Merck silica gel 60 F254
plates. Visualization of the developed chromatogram was performed by
UV absorbance, aqueous potassium permanganate, or iodine. Flash
chromatography was performed using Sigma-Aldrich silica gel 60,
particle size 40−63 μm, with the indicated solvent system. NMR
spectra, unless otherwise specified, were recorded on Bruker Avance
DRx 400, 401.36 MHz for 1H and 100.92 MHz for 13C. Chemical shifts
are reported in ppm with the deuterated solvent signal as the internal
standard. Data are reported as follows: chemical shift, integration,
multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, qn =
quintet, m = multiplet and br = broad), and coupling constant in hertz.
All 13C spectra were obtained with complete proton decoupling.
Structural assignments were made with additional information from
gCOSY, gHSQC, gHMBC, and NOESY experiments. 2D-EXSY
(exchange spectroscopy) experiments, based on 2D-ROESY (rotat-
ing-frame Overhauser enhancement spectroscopy) sequence, and
NOESY experiments were recorded on a VARIAN INOVA 600 MHz
instrument. The optimized 2D map was recorded by using a relaxation
time of 15 s, a mixing time of 0.03 s; 128 increments of 4 transients of
2K points each were collected. HPLC-ESI-Q/ToF flow injection
analyses (FIA) were carried out with a 1200 Infinity HPLC (Agilent
Technologies, USA), coupled with a quadrupole−time-of-flight
tandem mass spectrometer (6530 Infinity Q-TOF; Agilent Tech-
nologies) through a Jet Stream ESI interface (Agilent). Mass Hunter
Workstation Software (B.04.00) was used to control the HPLC and the
mass spectrometer, for data acquisition, and for data analysis.
Methyl (S)-1-Acetylindoline-2-carboxylate Ac-(2S)-Ind-OMe
(1). (S)-Indoline-2-carboxylic acid (H-Ind-OH) (2.8 g, 17.2 mmol)
was dissolved in 280 mL of methanol and cooled to 0 °C with an ice
bath. To this suspension was added dropwise thionyl chloride (1.87
mL, 25.7 mmol, 1.5 equiv). The reaction mixture was stirred for 1 h and
allowed to reach room temperature. Afterward, the resulting solution
was heated at 70 °C and allowed to stir at reflux for 16 h. After being
cooled at room temperature, the reaction mixture was concentrated
under reduced pressure. The residue was dissolved in ethyl acetate
(EtOAc) and washed with an aqueous saturated solution of sodium
bicarbonate (NaHCO3) three times. The combined organic layers were
washed with brine, dried over Na2SO4, and concentrated under reduced
pressure. The resulting crude material was purified by flash
chromatography on silica gel using 10 to 50% EtOAc in hexane to
give the desired product (H-(2S)-Ind-OMe) as a white solid in an 82%
yield (2.5 g, 14.1 mmol). 1H NMR (400 MHz, CDCl3) δ = 7.12−6.99
(m, 2H), 6.79−6.68 (m, 2H), 4.39 (dd, J = 10.2, 5.5 Hz, 2H), 3.76 (s,
3H), 3.36 (m, 2H).
Figure 5. Analysis of selected van der Waals contacts (in Å) for
structures optimized at B3LYP-D3BJ/6-311+G(d)/IEFPCM
(DMSO) level of calculation, seen from two viewpoints. Conformers
C2 and T2 of Ac-(2S)-Ind-OMe (1) were considered for better
comparison with the lowest-energy conformer of Ac-Pro-OMe.
indicative of a C−HAr4··OC interaction in the cis isomer. At
the same time, however, the atom charge on CO oxygen is the
same (within 0.5% in CHCl3) for the two isomers. Moreover,
atom charges are only slightly affected when switching from
CHCl3 to DMSO; for example, the CO oxygen negative
charge increases by ∼2% (absolute value) in the more polar
solvent, while the HAr4 charge increases by ∼1%. So, although
the calculations hint at a possible C−H··OC interaction, we
must conclude that they do not highlight a clear solvent
dependence for this interaction.
CONCLUSIONS
■
The collected experimental and computational evidence
unambiguously demonstrated that (S)-indoline-2-carboxylic
acid derivatives, when involved in amide bonds, have a strong
preference for the cis conformation, especially in polar solvents.
It is interesting to note how these results confirm the findings of
Siebler et al.53 on the importance of dipole moment for proline
derivatives isomerization, although in our case the effect of the
polarity of the solvent is much more pronounced. Besides the
influence of the dipole moment on the conformational
equilibrium, a combination of steric and electrostatic
interactions may contribute to stabilizing the amide cis
geometry. Considering that small differences at a molecular
level transpose to big changes of macroscopic properties, these
findings could lead to the synthesis of longer oligomers forming
stable polyproline I like structures, or the use in peptide
sequences of (S)-indoline-2-carboxylic acid as a preferential
scaffold to form β-hairpins, or as a conformational constrain for
the design of new organo-catalysts.
In a round-bottom flask, 510 mg (2.9 mmol, 1 equiv) of H-(2S)-Ind-
OMe was dissolved in 25 mL of dry dichloromethane and the resulting
solution stirred at 0 °C. A catalytic amount (0.5%) of 4-
dimethylaminopyridine (DMAP) and 1.60 mL (11.5 mmol, 4 equiv)
of triethylamine (TEA) were added to the solution. Acetic anhydride
(2.2 mL, 23 mmol, 8 equiv) was then added dropwise, and after 15 min,
the reaction mixture was warmed at room temperature and let stirring
for 16 h. The reaction mixture was then concentrated under reduced
pressure, and the residue was dissolved in dichloromethane, acidified
with HCl 1 M, and washed three times with dichloromethane. The
combined organic layer layers were dried over Na2SO4, filtered, and
concentrated under reduced pressure. The resulting crude product was
purified by flash chromatography on silica gel using 10 to 50% EtOAc in
hexane to give the desired product as a white solid in an 85% yield (538
mg, 2.45 mmol). TLC Rf: 0.6 (Hex/EtOAc = 7:3), Mp: 65 °C. 1H NMR
(400 MHz, CDCl3) δ = 8.21 (d, J = 8.1 Hz, 1Hcis), 7.28−7.12 (m, 2Hcis
+ 3Htrans), 7.03 (t, J = 7.7 Hz, 1Hcis + 1Htrans), 5.17 (d, J = 10.7 Hz,
1Htrans), 4.91 (d, J = 10.7 Hz, 1Hcis), 3.77 (s, 3Hcis), 3.73 (s, 3Htrans),
3.62 (dd, J = 16.6, J = 10.7 Hz, 1Hcis), 3.47 (dd, J = 16.6 Hz, J = 10.7 Hz,
EXPERIMENTAL SECTION
■
General Information and Materials. All nonaqueous reactions
were run in oven-dried glassware under a positive pressure of argon,
with exclusion of moisture from reagents and glassware, transferring
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1Htrans), 3.27 (d, J = 16.6 Hz, 1Hcis), 3.10 (d, J = 16.6 Hz, 1Htrans), 2.48
(s, 3Htrans), 2.17 (s, 3Hcis). 13C{1H} NMR (100 MHz, CDCl3) δ: 171.8,
168.9, 142.6, 130.9, 128.4, 128.0, 127.9, 125.7, 124.2, 124.0, 123.5,
117.4, 113.8, 61.4, 60.6, 53.0, 52.5, 33.6, 31.5, 29.7, 24.5, 23.7. HRMS
(TOF MS ES+): m/z [M + Na]+ calcd for C12H13NO3 242.0788; found
242.0784. Analytical HPLC purity 97%.
2.48 ppm, which is its acetyl group. On the contrary, Hα at 4.91 ppm
gave NOEs at both frequencies of acetyl groups. Therefore, Hα
centered at 5.17 and 4.91 ppm must be attributed to trans and cis
isomer, respectively. Starting from the frequency of Hα and based on
the analysis of scalar correlations and integration, the complete
assignment of cis and trans isomer was obtained (see the SI).
Optimization of the Mixing Time for EXSY Experiments. The
rate constants and the activation energy for the cis/trans isomerization
were determined in DMSO-d6 from 2D exchange spectroscopy (EXSY)
which provides a map of the exchanging species. Considering the
greatly unequal populations of the two exchanging species, as well as
similar spin−lattice relaxation time T1 of the two species (T1cis = 2.39 s;
Methyl (S)-1-((S)-1-Acetylindoline-2-carbonyl)indoline-2-
carboxylate, Ac-((2S)-Ind)2-OMe (2). In a round-bottom flask,
(S)-indoline-2-carboxylic acid (H-(2S)-Ind-OH) (0.6 g, 3.6 mmol, 1
equiv) was dissolved in 25 mL of dry dichloromethane and allowed to
stir and cool in an ice bath. Triethylamine (TEA) (2.0 mL, 14.4 mmol, 4
equiv) and a catalytic amount (0.5%) of DMAP were added. To this
solution, 2.7 mL of acetic anhydride (28.8 mmol, 8 equiv) was then
added dropwise. The reaction mixture was warmed at room
temperature and stirred for 16 h. The resulting mixture was then
concentrated under reduced pressure, and the residue was dissolved in
dichloromethane, acidified with HCl 1 M, and washed three times with
dichloromethane. The combined organic layer layers were dried over
Na2SO4, filtered, and concentrated under reduced pressure. The
product Ac-(2S)-Ind-OH was obtained as a white solid with a yield of
T
1trans = 2.46 s) we found the approach outlined by Perrin and Dwyer
convenient. This approach allows for calculation of rate constant for
chemical exchange by knowing the diagonal peak to cross-peak
intensity ratio. For a simple two-site exchange, the total exchange rate k
(k = kt→c + kc→t) is given by the equation
1
τm
r + 1
r − 1
k =
ln
1
85% (0.63g) and used without further purification. H NMR (400
The term r accounts for unequal populations and is defined as
MHz, DMSO-d6) δ = 8.20 (d, J = 8.0 Hz, 0.75H), 7.30−6.95 (m,
3.25H), 5.21 (d, J = 9.0 Hz, 0.75 H) and 4.87 (d, J = 9.0 Hz, 0.25H),
3.67−3.20 (m, 2H), 2.52 (s, 2.25H) and 2.19 (s, 0.75H).
ICC + ITT
ICT + ITC
r = 4XCXT
− (XC − XT)2
In a two-neck round-bottom flask, 210 mg (1.02 mmol, 1 equiv) of
Ac-(2S)-Ind-OH was dissolved in dry dichloromethane under argon.
The coupling reagent 2-chloro-1-methylpyridinium iodide (Mukaiya-
ma’s reagent) (365 mg, 1.43 mmol, 1.4 equiv) and freshly distilled TEA
(0.4 mL, 2.86 mmol, 2.8 equiv) were added into the solution. The
amine H-(2S)-Ind-OMe (181 mg, 1.02 mmol, 1 equiv) was added, and
the reaction mixture was heated at reflux for 16 h before being allowed
to cool to room temperature. The reaction mixture was then diluted
with DCM and washed with HCl 1M, aqueous saturated sodium
bicarbonate, and brine. The organic layer was then dried over Na2SO4
and evaporated under reduced pressure. The resulting solid was
purified by flash chromatography on silica gel using 10−50% EtOAc in
hexane. The desired product was obtained as white solid in an isolated
yield of 46% for solubility issues (168 mg, 0.46 mmol). TLC Rf = 0.6
(DCM:MeOH = 9:1), M.P. = 175 °C (dec)1H NMR (400 MHz,
CDCl3) δ = 8.35−8.14 (m, 1H), 7.36−7.11 (m, 5H), 7.11−6.95 (m,
2H), 5.87−5.75, 5.51−5.45, 5.34−5.21 and 5.12−4.92 (four m, 2H),
3.95−3.00 (m, 7H), 2.47 and 2.18 (couple of d, 3H). 1H NMR
characterization of prevailing cis−cis species (>70%) in the stereo-
isomers mixture in DMSO-d6 on a 600 MHz is reported in the SI.
13C{1H} NMR (150 MHz, DMSO-d6) δ = 171.7, 169.7, 169.2, 143.5,
where ICC and ITT are the diagonal peak intensities of two exchangeable
resonances in the EXSY, ITC and ICT are the intensities of the exchange
cross peaks, and τm is the mixing time. XC and XT are the mole fractions
of the cis and trans forms. The choice of the mixing time τm is critical.
Kinetic effects on the cross-peak intensities will be too small to measure
accurately, if it is too short. However, the effects will be so large as to be
insensitive to the kinetic parameters, if it is too long. The optimum
mixing time should be chosen to minimize the error in the rate constant
and an approximate expression was shown to be
1
τm,opt
∼
T1−1 + kt + kc
c
t
We found that τm = 0.03 s is the closest to the optimal value.
1H NMR Characterization of Prevailing Cis−Cis Species
(>70%) of 2 in DMSO-d6 on a 600 MHz Instrument. The main
species of Ac-Ind-Ind-OMe (2) in DMSO-d6 was fully characterized by
compared analysis of homonuclear and heteronuclear scalar correla-
tions in COSY and HSQC maps and homonuclear dipolar correlations
in ROESY map (see the SI). The prevailing conformer was identified as
the cis-cis one on the basis of the significant ROE detected between the
more intense singlets at 2.05 and 3.73 ppm due to the methyl protons of
the acetyl and the ester functions, respectively. Therefore, among NMR
signals of Hα of the conformer mixture (5.00−6.00 ppm), the two more
intense ones (doublet of doublets) at 5.24 and 5.27 ppm were assigned
to the two units of the cis−cis species. In particular, the high-frequency
signal (5.27 ppm) was attributed to the Hα of the residue with the N-
acetyl terminal substituent, which is in spatial proximity of the methyl
group of acetyl moiety (ROE constraint). Accordingly, to the above
said stereochemical assignment, no ROEs between aromatic protons
and Hα of methyl ester terminal residue were detected. Starting from
each Hα, the two diastereotopic protons of their adjacent methylenes
were assigned (3.29/3.78 ppm for the N-acetyl residue and 3.34/3.76
ppm for the methyl ester residue) by means of their scalar correlations.
Har1 of each residue was identified on the basis of the ROE effects
produced by the methylene protons. Resonances of aromatic protons of
each residue were assigned on the basis of their scalar correlations,
starting from Har1. NMR characterization data for the cis−cis species
are collected in the SI.
142.2, 129.5, 128.9, 127.4, 127.2, 124.7, 124.3, 123.1, 116.0, 115.9,
115.8, 113.8, 61.2, 61.0, 60.0, 59.7, 59.6, 53.1, 52.1, 33.4, 33.0, 32.9,
30.5, 23.6, 23.5, 20.7, 14.1. HRMS (TOF MS ES+): m/z [M + Na]+
calcd for C12H20N2O4 387.1315; found 387.1317. Analytical HPLC
purity 98%.
Comment over the Stability of Indoline-2-carboxylic Acid
Derivatives. The starting material (S)-indoline-2-carboxylic acid (H-
(2S)-Ind-OH) has been purchased by abcr GmBh with a declared
purity up to 95%. In our opinion, the risk of degradation of the
carboxylic moiety is low, but on the other hand, the risk of oxidation to
form 1H-indole-2-carboxylic acid could have been higher, and we
checked regularly, over a long period of time (more than one year), the
1
purity of the SM and of the synthesized derivatives by H NMR. We
could observe that if the SM and H-Ind-OMe were not correctly stored
under argon at 0 °C they could change color to a reddish powder and
1
show the formation of a small signal on the H NMR typical of the
indole. However, we were pleased to notice then whenever the
secondary amine was protected as an acetamide or involved in a peptide
bond, compounds remain stable and pure even after 2 years.
Computational Details. Conformational analysis was run by the
systematic conformational search algorithm implemented in Spartan’18
(Wavefunction, Inc., Irvine, CA, 2018) using the Merck Molecular
Force Field (MMFF). Geometry optimizations were performed using
density functional theory with the B3LYP exchange-correlation
functional,62 augmented with Grimme’s GD3 empirical dispersion
correction dispersion,63 in conjunction with the 6-311+G(d) basis set.
Assignment of 1H NMR Signals of 1. Cis and trans stereoisomers
of 1 were attributed on the basis of NOESY analysis (see the SI). Even
though the majority of dipolar interactions suffered from exchange
processes occurring between the two species, one effect was selective for
the trans one. In particular, in CDCl3 Hα at 5.17 ppm produced NOE at
2.17 ppm (acetyl group), which is due to exchange processes, but not at
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J. Org. Chem. 2021, 86, 7946−7954

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pubs.acs.org/joc
Article
Solvation effects have been accounted for using the integral equation
formalism64,65 formulation of the polarizable continuum model (IEF-
PCM).66 Natural bond orbital (NBO) analysis was run with NBO
version 3.67 All of the DFT calculations have been performed using the
Gaussian 16 suite of programs.68
Funding
Financial support from the University of Pisa (PRA 2018_23
and PRA 2020_77) is gratefully acknowledged.
Notes
The authors declare no competing financial interest.
HPLC Analysis of 1 and 2. A 1000 ppm solution was prepared in
DMSO and then further diluted in MeOH to ca. 50 ppm and injected in
the chromatographic system. The separation was performed on an
Agilent Zorbax Extend C18 Rapid resolution HT column (50 × 2.1
mm, 1.8 μm particle size). The injection volume was 1 μL, and the
column temperature was 30 °C. Separation was obtained by using a
gradient of 0.1% formic acid in water (eluent A) and 0.1% formic acid in
acetonitrile (eluent B) programmed as follows: 90% A for 2 min,
followed by a linear gradient to 50% B in 9 min, then to 70% B in 3.3
min, finally to 90% B in 3.7 min held for 12 min at 90% B. Re-
equilibration time for each analysis was 13 min. The chromatographic
runs were performed at a flow rate of 0.2 mL/min. The eluents were all
HPLC-MS grade, Sigma-Aldrich. The MS acquisition was performed in
full scan, and the Jet Stream ESI operating conditions were: drying gas
(N2, purity >98%): 350 °C at 10 L/min; capillary voltage 4.5 kV;
nebulizer gas 35 psig; sheath gas (N2, purity >98%): 375 °C at 11 L/
min. High-resolution mass spectra were acquired in the range 100−
3200 m/z in high-resolution positive mode. The fragmentor was kept at
175 V, nozzle voltage 1000 V, skimmer 65 V, octapole RF 750 V. The
mass axis was calibrated prior analyses using the Agilent tuning mix
HP0321 (Agilent Technologies) prepared in acetonitrile and water.
ACKNOWLEDGMENTS
■
G.A. acknowledges the contribution of the COST Action
CA17120. G.A. is grateful to Prof. Ilaria Degano for HPLC-
HRMS analysis. G.P. acknowledges the CINECA award under
the ISCRA initiative for the availability of high-performance
computing resources and support.
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ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.1c00184.
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Corresponding Author
Gaetano Angelici − Dipartimento di Chimica e Chimica
̀
Industriale, Universita di Pisa, 56124 Pisa, Italy;
orcid.org/0000-0002-4397-3449;
Email: gaetano.angelici@unipi.it
Authors
Matteo Pollastrini − Dipartimento di Chimica e Chimica
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Industriale, Universita di Pisa, 56124 Pisa, Italy;
orcid.org/0000-0001-9334-2613
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Luca Pasquinelli − Dipartimento di Chimica e Chimica
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Industriale, Universita di Pisa, 56124 Pisa, Italy
Federica Balzano − Dipartimento di Chimica e Chimica
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Industriale, Universita di Pisa, 56124 Pisa, Italy;
orcid.org/0000-0001-6916-321X
Gloria Uccello Barretta − Dipartimento di Chimica e Chimica
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Industriale, Universita di Pisa, 56124 Pisa, Italy
Gennaro Pescitelli − Dipartimento di Chimica e Chimica
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.1c00184
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J. Org. Chem. 2021, 86, 7946−7954

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