Amide rotation trajectories probed by symmetry

Article abstract of DOI:10.1039/c7ob01421j

Amide rotation of peptidyl-prolyl fragments is an important factor in backbone structure organization of proteins. Computational studies have indicated that this rotation preferentially proceeds through a defined transition-state structure (syn/exo). Here, we complement the computational findings by determining the amide bond rotation barriers for derivatives of the two symmetric proline analogues, meso and racemic pyrrolidine-2,5-dicarboxylic acids. The rotations around these residues represent syn/exo-syn/exo and anti/endo-syn/exo hybrid transition states for the meso and racemic diastereomer, respectively. The rotation barriers are lower for the former rotation by about 9 kJ mol^-1 (aqueous medium), suggesting a strong preference for the syn/exo (clockwise) rotation over the anti/endo (anticlockwise) rotation. The results show that both hybrid rotation processes are enthalpically driven but respond differently to solvent polarity changes due to the different transition state dipole-dipole interactions.

Full text of DOI:10.1039/c7ob01421j

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Amide rotation trajectories probed by symmetry
Vladimir Kubyshkina* and Nediljko Budisaa*
Received 00th January 20xx,
Accepted 00th January 20xx
The amide rotation of peptidyl-prolyl fragments is an important factor in backbone structure organization of proteins.
Computational studies have indicated that this rotation preferentially proceeds through a defined transition-state
structure (syn/exo). Here, we complement the computational findings by determining the amide bond rotation barriers for
the two symmetric proline analogues, meso and racemic pyrrolidine-2,5-dicarboxylic acid. The rotations around these
residues represent syn/exo – syn/exo and anti/endo – syn/exo hybrid transition states for the meso and racemic
diastereomer, respectively. The rotation barriers are lower for the former rotation by about 9 kJ mol−1 (aqueous medium),
suggesting a strong preference for the syn/exo (clockwise) rotation over the anti/endo (anticlockwise). The results show
that both hybrid rotation processes are enthalpically driven but respond differently to solvent polarity changes due to the
different transition state dipole-dipole interactions.
DOI: 10.1039/x0xx00000x
www.rsc.org/
non-conjugated state trapped by a rigid molecular scaffold (so-
called twisted amides).20
Introduction
A
B
Proline (1, Fig. 1A) plays an important role in various areas
O
O
of organic and biological chemistry. These include cellular
uptake,1 metabolism,2 translation,3 protein folding and
engineering,4 organocatalysis,5 pre-biotic chemistry,6 foldamer
research,7 drug design,8 and more. A particularly interesting
focus is the tertiary amide bond formed by peptidyl-prolyl
fragments in peptide and protein structures.9,10 The
isomerization of this backbone structure can often become the
rate-limiting step in protein folding.11 This process is frequently
facilitated by peptidyl-prolyl cis-trans isomerases in natural
systems12 alternatively by substitution of proline with its
analogues in laboratory protein expression experiments.13
Notably, peptidyl-prolyl isomerization is not necessarily slow
compared to other amide bond rotations; rather, it may
become rate limiting due to the relative thermodynamic
stability of the non-native versus the native state.14
N
N
CO2
N
X
X
H2
R
O
O
R
1
s-trans
s-cis
C
unfavored:
R
O
O
O
X
X
N
N
O
N
O
rigid scaffold
H
H
R
twisted amides
anti/exo
syn/endo
(structural model)
favored:
O
O
O
X
X
N
R
N
model?
R
H
H
O
The two ground states of the N-acyl prolyl amide bond are
s-trans and s-cis (Fig. 1B), with s-trans conformation forming
preferentially. The preference is about 3:1 in Ac-Pro-NHMe,15
syn/exo
(dominant)
anti/endo
4:1 in Ac-Pro-OH,16 5:1 in Ac-Pro-OMe,17 and 7:1 in GlyProGly-
Fig. 1 A. The structure of proline (1), B. two N-acyl-prolyl ground states, s-trans
NH2 and AcGlyGlyProGlyGlyNH2 peptides (water, 298 K),19 and s-cis, C. four possible transition states for the amide rotation, the syn/exo
18
transition state is most favored. The nomenclature is from Ref. 21.
which translates to an approximately 4-5 kJ mol−1 in energetic
preference. At the same time, the amide bond rotation should
However, these elegant structures do not definitively
proceed through one of the four theoretically possible
model the amide transition state due to the following reason.
transition states, in which the amide conjugation should be
Already in mid 1990s results from a theoretical study showed
lost (Fig. 1C). Several structures successfully have depicted the
that the amide rotation preferably proceeds via the syn/exo
transition states with antiparallel orientation of the N→lone
pair and carbonyl C→O dipoles,21 whereas in the covalent
twisted amides this orientation rather resembling parallel.
Only very recently metal coordination based twisted amides
were reported, and these can model the antiparallel twist.22
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Since the seminal theoretical reports, numerous
Journal Name
Therefore we designed an experimental model based on
computational studies have demonstrated that the amide derivatives of pyrrolidine-2,5-dicarboxylic acid (Pdc). Pdc has
rotation around proline has a preference for the syn/exo two diastereomeric forms, meso (mPdc, with a symmetry
rotation scenario.23 These findings are further supported by plane, σ) and racemic (rPdc, with a symmetry axis, C2). We
recent theoretical24 and experimental25 studies of the catalytic assumed that the amide ground states are equivalent in the N-
mechanism of Pin1 peptidyl-prolyl cis-trans isomerase, and acyl Pdc derivatives when the acyl-moiety is not chiral. At the
experimental demonstration of the self-catalytic mechanism of same time, we expected the amide rotation to proceed via the
the amide rotation in unpolar media.26 In the syn/exo syn/exo – syn/exo hybrid state for mPdc and the anti/endo –
transition state, the nitrogen lone pair is oriented on the same syn/exo hybrid state for rPdc derivatives (Fig. 2) (using the
side and the carbonyl oxygen rotates to the opposite side with proline transition state nomenclature, Fig 1C). We therefore
respect to the proline carbonyl group, whereas the upstream synthesized a series of amides with non-chiral N-terminal acyl
alkyl moiety is oriented outward. Curiously, despite the groups (Fig. 3). Here, we report the experimental amide bond
existing consensus among theoretical studies, an proper rotation barriers for these two model situations. The results
experimental model that addresses the energetic differences obtained for proline and pyrrolidine derivatives will also be
in the relevant rotation modes is lacking. While there little shown for reference but will not be discussed in the context of
doubt that the syn/exo transition state is the most relevant, it the paper.
is not clear whether the anti/endo transition state can
contribute to the rotation or whether the properties of the
Results and Discussion
syn/exo conformation can be extrapolated to other transition
scenarios.
The ground state
We designed our study with the assumption that the amide
CO2
CO2
O2C
O2C
N
N
ground state formed by the Pdc residue will experience similar
chemical environments in both diastereomeric situations. The
similarity in the through-bond electronic environment of the
N-terminal fragment can be assumed from the acidity of the
amino acid ammonium salts, which was nearly identical for
both Pdc derivatives, as shown in Fig. 4.
H2
H2
meso-Pdc
rac-Pdc
(mPdc)
(rPdc)
COX
H
COX
H
COX
H
versus
N
O
N
O
H
R
R
COX
syn/exo- syn/exo
anti/endo - syn/exo
Fig. 2 Design of the study. Two diastereomeric forms of pyrrolidine-2,5-
dicarboxylic acid yield different rotation modes around in the N-acyl derivatives.
rPdc:
mPdc:
MeO2C
CO2Me
MeO2C
CO2Me
N
N
O
O
R
R
2a R = CH3
3a R = CH3
2b R = C(CH3)3
3b R = C(CH3)3
2c R = CH2NHAc
3c R = CH2NHAc
Fig. 4 The pKa of the ammonium group in the pyrrolidine-2,5-dicarboxylic acid
derivatives. The data on pyrrolidine and proline methyl ester are included for
comparison. Standard error: ±0.10.
Pro:
pyrrolidine:
Next, the X-ray crystal structures for both N-acetyl
derivatives 2a and 3a demonstrate fully planar amide bonds
(Fig. 5A).27 In the mPdc derivative 2a, the amide conformation
breaks the symmetry of the structure and creates two
enantiomers, which were present at a ratio of 4:2 in the cell
unit. In the rPdc derivative 3a, the amide does not break the
existent C2 symmetry of the structure, and the cell unit
contains a racemate with a 2:2 ratio of the (R,R) and (S,S)
enantiomers. The envelope conformation of pyrrolidine ring in
CO2Me
N
N
O
O
R
R
4a R = CH3
5a R = CH3
4b R = C(CH3)3
4c R = CH2NHAc
5b R = C(CH3)3
5c R = CH2NHAc
Fig. 3 Examined compounds.
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proline is usually described in terms of the exo-/endo-pucker differences are expected to become influential in unpolar
nomenclature (alternatively called up-/down-) depending on media.30
the direction of the C4-atom displacement from the plane of
Hereafter, we will not correct for these energies and will
the ring relative to the carboxyl group orientation. Following consider both ground states equivalent for simplicity of
this nomenclature, the mPdc derivative 2a adopts a hybrid of presentation.
exo- and endo-pucker, whereas the rPdc derivative 3a is a
hybrid of two endo-puckers. The crystal structure is consistent Energetic terms of the amide rotation
with the solution 1H NMR spectra, in which the two
We then analyzed the amide rotation barriers in the N-
acetyl derivatives 2a,
3a and 4a in aqueous medium by 1H EXSY
CH(CO2Me) resonances appear as a triplet and doublet of
doublets in 2a and as two doublets in 3a (Fig. 5B).28
Importantly, these NMR spectral signatures persisted in all
examined Pdc derivatives and in all employed NMR solvents
(see ‘Polar effects’).
NMR. The results are shown on Fig. 6, where the activation
energy values E≠ are presented as a function of temperature
(also see Tab. S1). As expected, the rotation barrier for the
mPdc derivative 2a was remarkably lower compared to the
rPdc derivative 3a (by 9.3-9.5 kJ mol−1; equivalent to a factor of
43-46 at 298 K).
A Crystal structures:
C4-
C4-
exo
endo
C4-
endo
For all three examined structures, the amide rotation is an
enthalpically defined process with a small negative slope −TΔS≠
(ΔS≠ > 0). The smallest entropic contribution was found for
pyrrolidine derivative 4a (ΔS≠ = +3.2±1.8 J mol−1 K−1), whereas
for the mPdc derivative 2a, this value was somehow larger (ΔS≠
= +11.9±0.9 J mol−1 K−1). We were not able to determine the
entropic value for the rPdc derivative 3a with the same level of
accuracy (ΔS≠ = +7.6±9.0 J mol−1 K−1) due to the high relative
experimental error for the very slow rates at the lower
temperature conditions. Nonetheless, this lack of accuracy
does not affect the overall conclusions, as same tendencies
Pna21
P21/c
C4-
endo
2a
B 1H NMR multiplicities of the CH-fragments (in D2O):
3a
dd, = 7.0 and 4.0 Hz
J
d, = 9.0 Hz
J
d, = 8.8 Hz
t, = 7.5 Hz
J
J
were found for the N-acetylglycyl derivatives 2c-4c. In this
case, the activation enthalpy was reduced by 5-7 kJ mol−1 and
the entropy was reduced by 6-9 J mol−1 K−1 relative to the N-
acetyl derivatives (See Tab. S2-S3).
MeO2C
N
CO2Me
MeO2C
CO2Me
N
O
O
Me
Me
2a
3a
Fig. 5 The X-ray crystal structures (A) and solution 1H NMR (in D2O) data (B) for
the N-acetyl compounds 2a and 3a are compatible. The ellipsoids represent 30 %
probability displacement, carbon – black, oxygen – red, and nitrogen – blue.
Based on this spatial organization argument we expect the
steric requirements to be identical around the methyl group of
the acetyl moiety, whereas the environment of the carbonyl
group is different. These differences are very well described in
the proline literature in terms of the pre-organization of the
n→π* interaction between the carbonyl groups.29 As the
result, some additional stability of about 1-3 kJ mol−1 can be
expected for the mPdc derivatives relative to the rPdc-derived
compounds.17
Fig. 6 Amide rotation barriers for the N-acetyl derivatives 2a, 3a and 4a as a
function of temperature, measured by 1H EXSY NMR in deuterium oxide. For
details, see Tab. S1, S3.
Another aspect, is the ψ-torsion due to the rotation of the
carboxyl-group. For the proline derivative this rotation yields
two states ψ1 ~ 170 and ψ2 ~ −20° with different polarity. In 5a
the first state is preferred by about 1.3-2.5 kJ mol−1.30 We
A small positive activation entropy is also known for the
amide rotation in the proline derivative 5a, where ΔS≠ has
been reported +9.0 for cis→trans and +12.8 J mol−1 K−1 for
trans→cis rotations, respectively.31,32 The positive entropy of
the amide rotation process may originate from the higher
molecular rigidity of the ground state (featured by the n→π*
interaction, for instance),15,24 as well as the stronger polar
interactions of the ground state with water (organization of
the solvent). For example, negative activation entropy for the
amide rotation in Ac-AA-OMe has been reported for some
modelled the carboxyl-group rotation in 2a-3a, and found that
this preference remains nearly identical for the rPdc derivative
3a (ΔΔGψ1/ψ2 ~ 1.6-2.7 kJ mol−1), whereas in 2a the two
carboxymethyl groups are more proximal, and the rotation
state difference becomes more prominent (ΔΔGψ1/ψ2 ~ 3.7-4.7
kJ mol−1). This however, produces only little energy difference
between the two ground states, and resulting polarity
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proline analogues with additional polar groups,33 nonetheless,
for some other polar proline analogues this value was also
positive.34
Fig. 7 Amide rotation barriers of the N-acyl derivatives. The values were determined in deuterium oxide solution (for AcGly-drrivatives, buffered at pH 7 with
potassium phosphate buffer) at 298 or 310 K. See ESI Tab. S4 for details.
Steric bulk of the N-acyl substituent
amide fragment moves towards the pyrrolidine ring and may
sterically interfere with carboxymethyl group in rPdc
a
We then measured the amide rotation barriers for the full
set of the prepared compounds in deuterium oxide medium
derivatives and create polar interactions with the
carboxymethyl groups in both rPdc and mPdc derivatives.
(Fig. 7, Tab. S4). The N-acetyl (2a-5a) and N-pivaloyl derivatives
(
2b-5b) feature the same high symmetry of the acyl moieties,
Polar effects
while the steric bulk of the alkyl groups is significantly
different. Due to the much higher steric size, the ground state
The polar interactions between the amide and
is largely destabilized in the N-pivaloyl derivatives, and the carboxymethyl groups were then studied by comparison of the
overall difference from the N-acetyl compounds is about 15-21 rotation barriers of the N-acetyl compounds (2a 3a 4a) in a
,
,
kJ mol−1 (equivalent to a factor of 500-5000 at 298 K). At the set of nine NMR solvents (see Tab. S5). It is well known that
same time, the bulky pivaloyl group excludes the possibility for the conjugation in the amide ground state is highly dependent
the anti/exo or syn/endo transition state situations (proline on the solvent polarity;9,35 mechanistically, this is illustrated by
-
nomenclature, see Fig. 1C), in which the tert-butyl group the change in the amide resonance contributions [O=C-N ↔
should move under the pyrrolidine ring. According to our semi- O-C=N+]. The
Reichardt-Dimroth parameter (ET) is
empirical modelling, these will be disfavored by about 11-12 solvatochromic value that quantifies the ability of a solvent to
kJ mol−1 in both N-pivaloyl derivatives 2b
3b, whereas in the N- separate intramolecular charges. Therefore, plotting rotation
acetyl compounds 2a
3a the disfavor is only by about 4-5 barriers in the E≠ – ET coordinates produces positive slopes due
kJ mol−1. Finally, the N-acetylglycyl derivatives 2c 5c represent to the differences in the ground state stabilities.16
a
-
-
-
a symmetric N-peptidyl-substitution, and the rotation barriers
here were reduced by about 3 kJ mol−1 (a factor of 3-4 at 298
K) compared to N-acetyl compounds 2a-5a. Notably, the amide
rotation barriers found for the C-terminal methyl ester
derivative of proline 5c are identical to the found for the C-
terminal peptidyl derivative AcGlyGlyProGlyGlyNH2,19 and from
this we may conclude that the C-terminal ester adequately
models the amide rotation in the polar medium such as water.
Importantly, for all three types of tested compounds, the
rotation barrier difference between the Pdc derivatives was
about 8-10 kJ mol−1 (a factor of 25-55 at 298 K), with the
barriers always lower for the mPdc derivative. This result
confirms that the rotation barrier difference in this case
originates from the transition state structures. In both the
syn/exo and anti/endo transition states shown in Fig 1C, the
alkyl group of the acetyl moiety moves outwards and cannot
sterically interfere with the substituents in the pyrrolidine ring
(see also Fig. 2). On the other hand, the carbonyl group of the
Fig.
8 Amide rotation barriers for the N-acetyl derivatives 2a, 3a and 4a
measured in different solvents by 1H EXSY NMR (at 298 or 310 K). For details see
ESI Tab. S4.
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While the experimental slopes (Fig. 8) for the rPdc and
pyrrolidine derivatives (3a and 4a) were nearly identical
(0.076±0.006 and 0.075±0.010, respectively), the slope for the
mPdc derivative (2a) was notably larger (0.088±0.008). This
result illustrates differences in the polar effects of the
transition states. We performed semi-empirical modelling of
the amide rotation, and found that the overall molecular
dipole follows the orientation of the amide carbonyl group
C=O in the pyrrolidine derivative 4a, as well as in the rPdc
derivative 3a. In the latter compound the dipolar contribution
of both carboxymethyl-moieties is mutually compensated by
the C2-symmetry of the residue (Fig. 9). As the result, the
overall molecular dipole is defined by the orientation of the
amide carbonyl group, and effectively, both 3a and 4a yield
the same slope in the E≠ – ET coordinates.
Conclusions
In summary, we described the amide rotation properties in
N-acyl pyrrolidine-2,5-dicarboxylic acid derivatives, capped at
the C-terminal sides as methyl esters. The meso diastereomer
of this compound exhibited an amide rotation barrier of
approximately 9 kJ mol−1 lower than that of the racemic
diastereomer. This outcome was reproducible in three N-acyl
derivatives with different degrees of steric bulk of the acyl-
moieties to represent varying ground state energies.
This model system allowed us to demonstrate
experimental features of the amide rotation relevant for
peptidyl-prolyl groups: 1) the transitions are enthalpically
driven and 2) differ in dipole-dipole compensation levels in the
transition states and 3) syn/exo transition scheme prevails in
general over the anti/endo. These conclusions are consistent
with the results of computational studies, which suggested
dominance of the syn/exo over the anti/endo transition state.
At the same time, the other theoretically possible transition
states, anti/exo and especially syn/endo, are expected to be
disfavoured in peptide structures, as these would require
movement of a bulky upstream peptidyl-fragment under the
pyrrolidine ring of proline.
The orientation of the molecular dipole in 2a is more
complex. The configuration of the two carboxymethyl
substituents renders the compound more polar than 3a. For
example, the Rf of mPdc derivatives are consistently lower
compared with the equivalent rPdc derivatives (see
Experimental section). Higher polarity of the meso-
diastereomer is also inferred from the octan-1-ol/water
partitioning values as shown on Fig. 9. The amide rotation via
the putative syn/exo – syn/exo hybrid transition state partially
compensates for the intramolecular dipole-dipole in the
transition state, causing the transition state to be relatively
favored in the nonpolar solvents. As the result, the slope in the
E≠ – ET coordinates increases. Similar dipolar compensation can
be expected for the amide rotation via the syn/exo transition
state in peptidyl-prolyl fragments, and this effect might be
enhanced due to a higher polarity of the amide fragment
versus the esters reported here. As a result, the syn/exo
transition state dominance should increase in less polar
environments.
Experimental section
Synthesis
The N-benzyl derivatives of Pdc were prepared starting
from adipic acid as a mixture of two diastereomers.36 The
resulting Bn-(MeO)Pdc-OMe compounds were separated on a
silica gel column using hexane – ethyl acetate (4:1) as an
eluent (Rf = 0.48 for rPdc and 0.27 for mPdc derivative). The 1H
NMR spectra exhibit a characteristic AB system for the
diastereotopic benzyl CH2 group in the rPdc derivative,
whereas this signal is a singlet in the mPdc derivative, where
the CH2 moiety is not diasteretopic. 1H NMR (500 MHz, CDCl3),
δ: Bn-(MeO)mPdc-OMe, 7.44-7.32 (m, 5H), 4.05 (s, 2H), 3.68 (s,
6H), 3.59 (br, 2H), 2.18 (m, 4H); Bn-(MeO)rPdc-OMe, 7.44-7.32
(m, 5H), 4.11 (d, J = 13 Hz, 1H), 3.92 (br d, J = 7 Hz, 2H), 3.91 (d,
J = 13 Hz, 1H), 3.75 (s, 6H), 2.42 (m, 2H), 2.04 (m, 2H). The
removal of the N-benzyl group was performed by stirring of
the substance with palladium on charcoal (10%) in methanol
acidified with an equivalent amount of hydrochloric acid,
under hydrogen pressure (40 atm), at 35 °C. The reaction was
monitored by NMR. After the reaction was complete, the
mixture was filtered, the solvent was removed under reduced
pressure, and the residue was dissolved in water, filtered with
activated charcoal and freeze-dried. The amino acid
hydrochlorides were obtained as pinkish powders. 1H NMR
(700 MHz, CD3OD), δ: HCl∙H-(MeO)mPdc-OMe, 4.33 (t, J = 6 Hz,
2H), 3.85 (s, 6H), 2.40 (m, 2H), 2.15 (m, 2H); HCl∙H-(MeO)rPdc-
OMe, 4.22 (t, J = 6 Hz, 2H), 3.80 (s, 6H), 2.32 (m, 2H), 2.06 (m,
2H).
Fig. 9 Schematic representation of the dipolar effects during the amide rotation
in N-acetylated compounds. LogP values represent partitioning of the
compounds between octan-1-ol and water at 298 K.
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N-acetyl compounds were prepared as follows. A salt
Journal Name
N-pivaloyl compounds were prepared according to the
HCl∙H-(MeO)Pdc-OMe (Pdc = mPdc or rPdc, 101 mg, 0.45 procedure for 5b synthesis.37 A salt HCl∙H-(MeO)Pdc-OMe (Pdc
mmol) was stirred with acetic anhydride (0.4 ml, 4.2 mmol, 9 = mPdc or rPdc, 101 mg, 0.45 mmol) was mixed with pivaloyl
equiv.), triethylamine (0.2 ml, 1.4 mmol, 3 equiv.) and chloride (70 μl, 0.57 mmol, 1.3 equiv.) in dichloromethane (1.5
dimethylaminopyridine (5 mg, 0.04 mmol, 9 mol %) in a ml), followed by addition of triethylamine (150 μl, 1.08 mmol,
dichloromethane – tetrahydrofuran mixture (1.5 ml, 2:1) for 48 2.4 equiv.) and dimethylaminopyridine (5 mg, 0.04 mmol, 9
hours at room temperature. The solvent was gently removed mol %). The mixture was stirred at room temperature for 4
under nitrogen gas flow, and the residue was dissolved in hours. Then, it was washed with water (1x0.3 ml), 1 M
water (0.7 ml) and freeze-dried. The crude material was aqueous hydrochloric acid (1x0.3 ml) and 1 M sodium
purified on a short silica gel column (about 10 g) using an ethyl hydrogencarbonate solution (1x0.3 ml), and subsequently
acetate – methanol mixture (19:1) as an eluent (Rf = 0.49 for dried over sodium sulphate, filtered and evaporated. Crude
2a and 0.63 for 3a).
material was purified on a silica gel column (about 5 g), using a
Dimethyl (r,s)-1-acetylpyrrolidine-2,5-dicarboxylate
(
2a). hexane – ethyl acetate mixture (1:1) as an eluent (Rf= 0.41 for
1
Yellowish solid, yield 84 mg (0.37 mmol, 81 %). H NMR (700 2b and 0.51 for 3b).
MHz, D2O), δ: 4.74 (dd, J = 7.0, 4.0 Hz, 1H), 4.44 (t, J = 7.5 Hz,
Dimethyl (r,s)-1-pivaloylpyrrolidine-2,5-dicarboxylate (2b).
1
1H), 3.75 (s, 3H), 3.69 (s, 3H), 2.33-2.26 (m, 3H), 2.03 (s, 3H), Clear oil, yield 80 mg (0.29 mmol, 66 %). H NMR (700 MHz,
1.92 (m, 1H). 13C{1H} NMR (176 MHz, D2O), δ (all singlets): D2O), δ: 5.03 (br d, J = 7 Hz, 1H), 4.43 (br t, J = 8 Hz, 1H), 3.72
173.9 (C=O), 173.8 (C=O), 173.3 (C=O), 61.2 (CH), 60.1 (CH), (br s, 3H), 3.69 (br s, 3H), 2.26 (br m, 3H), 1.77 (br m, 1H), 1.13
53.1 (CH3O), 52.8 (CH3O), 29.4 (s, CH2), 27.7 (CH2), 21.2 (CH3). (s, 9H). 13C{1H} NMR (176 MHz, D2O), δ (all singlets): 180.7
HMRS (ESI-orbitrap): calcd. for [M+H]+ C10H16NO5 230.1023 Da, (C=O), 174.3 (C=O), 174.0 (C=O), 62.8 (CH), 60.8 (CH), 53.0
found 230.1028 Th. M.p. 60-65 °C.
(CH3O), 52.6 (CH3O), 39.2 (C), 31.1 (CH2), 26.7 (CH3), 25.4 (CH2).
3a). HMRS (ESI-orbitrap): calcd. for [M+1]+ C13H22NO5 272.1492 Da,
Dimethyl (r,r)-1-acetylpyrrolidine-2,5-dicarboxylate
White solid, yield 85 mg (0.37 mmol, 82 %). 1H NMR (700 found 272.1492 Th.
(
MHz, D2O), δ: 4.82 (d, J = 9.0 Hz, 1H), 4.54 (t, J = 8.8 Hz, 1H),
Dimethyl (r,r)-1-pivaloylpyrrolidine-2,5-dicarboxylate (3b).
3.75 (s, 3H), 3.69 (s, 3H), 2.36 (m, 1H), 2.22 (m, 2H), 2.05 (m, White solid, yield 86 mg (0.32 mmol, 70 %). 1H NMR (700 MHz,
1H), 2.00 (s, 3H). 13C{1H} NMR (176 MHz, D2O), δ (all singlets): D2O), δ: 5.14 (d, J = 8.5 Hz, 1H), 4.52 (d, J = 9.8 Hz, 1H), 3.74 (s,
174.2 (C=O), 174.0 (C=O), 173.7 (C=O), 61.1 (CH), 59.5 (CH), 3H), 3.67 (s, 3H), 2.38 (m, 1H), 2.19 (dd, J = 13.2, 6.6 Hz, 1H),
53.4 (CH3O), 53.0 (CH3O), 29.2 (s, CH2), 27.0 (CH2), 21.1 (CH3). 2.09 (m, 1H), 1.92 (dd, J = 13.5, 7.2 Hz, 1H), 1.12 (s, 9H). 13C{1H}
HMRS (ESI-orbitrap): calcd. for [M+H]+ C10H16NO5 230.1023 Da, NMR (176 MHz, D2O), δ (all singlets): 180.4 (C=O), 175.1 (C=O),
found 230.1030 Th. M.p. 95-100 °C.
174.8 (C=O), 61.9 (CH), 60.9 (CH), 53.4 (CH3O), 52.8 (CH3O),
The proline derivative Ac-Pro-OMe (5a) was prepared by 39.1 (C), 30.3 (CH2), 26.5 (CH3), 24.8 (CH2). HMRS (ESI-
mixing commercial racemic HCl∙Pro-OMe with an equivalent orbitrap): calcd. for [M+1]+ C13H22NO5 272.1492 Da, found
amount of acetic anhydride in dichloromethane solution. 272.1494 Th. M.p. 80-85 °C.
Solvent was removed under reduced pressure, and the crude
The synthesis of 5b was fully reproduced37 starting from
material was purified using a hexane – ethyl acetate gradient 0.5 g of the racemic HCl∙H-Pro-OMe (3.0 mmol, 1/10 scale of
elution (from 1:1 to 0:1). The substance was obtained as a the reported synthesis). The product was a colorless oil. Yield
1
clear oil, which crystallized upon long time storage at 4 °C. 0.60 g (2.8 mmol, 92 %). H NMR (700 MHz, D2O), δ (s-trans
White solid, m.p. 35-40 °C. 1H NMR (700 MHz, D2O), δ (two rotamer only, Ktrans/cis = 46±2 at 298 K): 4.34 (dd, J = 8.5, 6.0 Hz,
rotamers, Ktrans/cis 4.95±0.05 at 298 K): 4.62 (s-cis, dd, J = 8.8, 1H, α-CH), 3.79 (dt, J = 10.6, 6.5 Hz, 1H, δ-CH), 3.74 (dt, J =
2.6 Hz) and 4.36 (s-trans, dd, J = 8.8, 4.6 Hz, 1H, α-CH), 3.72 (s- 10.6, 6.7 Hz, 1H, δ-CH), 3.67 (s, 3H, CH3O), 2.14 (m, 1H, β-CH),
cis, s) and 3.67 (s, s-trans, 3H, CH3O), 3.59 and 3.55 (s-trans, 1.98 (m, 1H, γ-CH), 1.92 (m, 1H, γ-CH), 1.79 (m, 1H, β-CH), 1.17
two m) and 3.46 and 3.39 (s-cis, two m, 2H, δ-CH2), 2.28 and (s, 9H, (CH3)3C).
2.15 (s-cis, two m) and 2.23 and 1.94 (s-trans, two m, 2H, β-
CH2), 2.05 (s-trans, s) and 1.93 (s-cis, s, 3H, CH3C=O), 1.95 (s- 12.2 mmol) was mixed with triethylamine (3.4 ml, 24.4 mmol,
trans, m) and 1.90 and 1.80 (s-cis, two m, 2H, γ-CH2). 2 equiv.) in dichloromethane (20 ml). Pivaloyl chloride (1.6 ml,
Compound 4b was prepared as follows. Pyrrolidine (1 ml,
N-acetyl pyrrolidine 4a was prepared as follows. Pyrrolidine 13.0 mmol, 1.07 equiv.) was added dropwise at the room
(0.25 ml, 3 mmol) was mixed with acetic anhydride (0.29 ml, 3 temperature (CAUTION: vigorous reaction!), and the mixture
mmol, 1 equiv.) in dichloromethane (5 ml) (CAUTION: vigorous was stirred for an additional 30 min. The solution was washed
reaction!). The mixture was stirred at room temperature for 30 with water (1x5 ml), 1:2 aqueous hydrochloric acid (1x5 ml)
min. Sodium carbonate (0.3 g) was added, and this mixture and 1 M sodium hydrogen carbonate (1x5 ml); subsequently, it
was shaken for about 15 hours at room temperature. The was dried over sodium sulphate, filtered and evaporated. Pure
mixture was filtered, and the solvent was removed under 4b was obtained as a colorless solid after crystallization from
reduced pressure to give 4a as a clear liquid. Yield 0.26 g (2.3 dichloromethane. Yield 1.80 g (11.6 mmol, 95 %). 1H NMR (700
1
mmol, 75 %). H NMR (500 MHz, D2O), δ: 3.41 (t, J = 7.0 Hz, MHz, D2O), δ: 3.62 (t, J = 6.1 Hz, 2H), 3.34 (t, J = 6.7 Hz, 2H),
2H), 3.28 (t, J = 7.0 Hz, 2H), 1.97 (s, 3H), 1.86 (m, 2H), 1.79 (m, 1.86 (m, 2H), 1.73 (m, 2H), 1.16 (s, 9H). M. p. 57-63 °C.
2H).
N-acetylglycyl (N-aceturyl) compounds were prepared by
using solution peptide coupling conditions. Aceturic acid (78
6 | Org. Biomol. Chem., 2017, 00, 1-9
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mg, 0.67 mmol, 1.3 equiv.) was mixed with benzotriazol-1-yl- Physical chemistry
oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP,
The NMR measurements were performed by 700 and 500
MHz 1H NMR detection. The variable temperature unit was
calibrated using acidified methanol sample measurements.38
The pKa measurements were performed at 298 K as
described.16 Buffer samples were taken at different pH values.
Deuterium oxide was added (1/10 to the buffer volume), and
the analyte (pyrrolidine or methyl ester hydrochlorides of the
amino acids) solution in a little amount of deuterium oxide was
added 1-2 min prior to the 1H NMR measurement. The 1H NMR
spectra were recorded using a W5 water suppression pulse
tray. The final analyte concentration was 0.5-1 mM. The
measurements were performed in 10 mM phosphate buffer
for pyrrolidine, 50 mM/60 mM phosphate/glycine buffer for
HCl∙H-Pro-OMe, and 20 mM/3 mM phosphate/citric buffer for
HCl∙H-(MeO)Pdc-OMe analytes. The standard error of the
measured pKa values is ±0.10. For pyrrolidine the obtained
value 11.41 is consistent with the previously reported 11.35.39
Partitioning was measured as follows. 5 mg of a compound
was shaken with octan-1-ol (1.00 ml) and deionized water
(1.00 ml) at 298±2 K for 24 hours. Aliquotes (250 μl) were
taken to same type NMR tubes and diluted with acetonitrile-d3
(200 μl) for locking and shimming. The concentration of the
samples were measured in single scan 90-degree pulse 1H
NMR spectra recorded at 298 K. Only the 90-degree pulses and
zero-phase values were re-adjusted between the
measurements. The method is similar to the recently reported
by Linclau et al. based on 19F NMR detection.40 Found logP
values: 2a −0.74±0.05, 3a −0.43±0.03, 4a −0.20±0.07, 5a
−0.44±0.05.
The amide rotation kinetics were measured in 1H cross-
relaxation experiments (NOESY/EXSY with z-gradients) as
described.16,19 The analyte concentration in the NMR samples
was 30-80 mM. Between 2-5 different mixing times (15 ms – 5
s) were used for the exchange detection. The time domain in
the direct dimension was 2048 points, whereas in the indirect
dimension, this was inset to yield sufficient resolution of the
analysed resonances (160-512 points, resolution 5-30 Hz).
Recycling delay was ≥2∙T1 of the analysed resonances. Note
that the use of a high recycling delay is not necessary but
improves the precision of the measurements. The exchange
rate matrices were calculated using EXSYCalc (Mestrec), and
the kinetic constants were converted to the activation energy
values using the Eyring equation. The error of the activation
energy values was calculated using the rate constant error and
does not take into account the temperature calibration error.
346 mg, 0.66 mmol, 1.3 equiv.) and diisopropylethylamine
(120 μl, 0.69 mmol, 1.35 equiv.) in dichloromethane (2 ml).
After about 10 min, the resulting solution was added to the
mixture of HCl∙H-(MeO)Pdc-OMe (Pdc = mPdc or rPdc, 113 mg,
0.51 mmol) with diisopropylethylamine (220 μl, 1.26 mmol, 2.5
equiv.) in dichloromethane (4 ml). The mixture was stirred for
about 10 hours. It was then washed with 1:2 aqueous
hydrochloric acid (1x0.5 ml) and 1 M sodium hydrogen
carbonate (1x0.5 ml); subsequently, it was dried over sodium
sulphate, filtered and evaporated. The resulting oil was
extracted with water (1.5 ml). The extract was filtered through
a short cation-exchange column (1.5 ml), and elution was
performed with water (about 5 ml). The aqueous fractions
were collected and freeze-dried. Final purification was
afforded by silica gel chromatography (about 10 g) using an
ethyl acetate – methanol gradient elution (19:1 – 4:1 – 0:1).
The resulting compounds were colorless glassy materials and
were slightly acidic. Therefore, the NMR samples in deuterium
oxide were buffered with potassium phosphate buffer (70
mM, pH 7) and contained 35 mM of the analytes.
Dimethyl (r,s)-1-(acetylglycyl)pyrrolidine-2,5-dicarboxylate
1
2c). Yield 34 mg (0.12 mmol, 24 %). H NMR (700 MHz, D2O),
(
δ: 4.78 (dd, J = 7.9, 2.0 Hz, 1H), 4.47 (t, J = 7.8 Hz, 1H), 4.08 (d, J
= 17.3 Hz, 1H), 3.93 (d, J = 17.1 Hz, 1H), 3.75 (s, 3H), 3. 68 (s,
3H), 2.34-2.24 (m, 3H), 1.98 (s, 3H), 1.89 (m, 1H). 13C{1H} NMR
(176 MHz, D2O), δ (all singlets): 174.6 (C=O), 173.8 (C=O),
172.8 (C=O), 170.5 (C=O), 60.6 (CH), 59.9 (CH), 53.2 (CH3O),
52.9 (CH3O), 41.5 (NH-CH2), 29.4 (CH2), 27.2 (CH2), 21.6 (CH3).
HRMS (ESI-orbitrap): calcd. for [M+H]+ C12H19N2O6 287.1238
Da, found 230.1240 Th.
Dimethyl (r,r)-1-(acetylglycyl)pyrrolidine-2,5-dicarboxylate
1
3c). Yield 37 mg (0.13 mmol, 26 %). H NMR (700 MHz, D2O),
(
δ: 4.85 (d, J = 8.8 Hz, 1H), 4.56 (d, J = 9.4 Hz, 1H), 4.03 (d, J =
17.3 Hz, 1H), 3.84 (d, J = 17.3 Hz, 1H), 3.75 (s, 3H), 3.69 (s, 3H),
2.36 (m, 1H), 2.26 (dd, J = 13.6, 6.8 Hz, 1H), 2.17 (m, 1H), 2.04
(dd, J = 13.5, 7.0 Hz, 1H), 1.97 (s, 3H). 13C{1H} NMR (176 MHz,
D2O), δ (all singlets): 174.5 (C=O), 174.0 (C=O), 173.5 (C=O),
170.4 (C=O), 60.0 (CH), 59.7 (CH), 53.5 (CH3O), 53.0 (CH3O),
41.5 (NH-CH2), 29.3 (CH2), 26.4 (CH2), 21.5 (CH3). HRMS (ESI-
orbitrap): calcd. for [M+H]+ C12H19N2O6 287.1238 Da, found
230.1239 Th.
The other two peptides 4c and 5c were prepared using
analogous conditions, by employing N,N,N',N'-tetramethyl-O-
(N-succinimidyl)uronium hexafluorophosphate (HSTU) as the
1
The results are summarized in ESI in Tab. S1 (E≠ for 2a
different temperatures), Tab. S2 (E≠ for 2c
4c at different
temperatures), S3 (energetic terms for 2a 4a and 2c
4c), S4 (E≠
in all compounds, D2O), S5 (E≠ for 2a
4a in different solvents).
-4a at
coupling reagent. 4c, H NMR (700 MHz, D2O), δ: 3.93 (s, 2H),
3.40 (t, J = 6.9 Hz, 2H), 3.33 (t, J = 6.9 Hz, 2H), 1.98 (s, 3H), 1.90
(m, 2H), 1.80 (m, 2H). HRMS (ESI-orbitrap): calcd. for [M+H]+
-
-
-
1
C8H15N2O2 171.1128 Da, found 171.1129 Th. 5c, H NMR (700
-
MHz, D2O), δ (s-trans rotamer only, Ktrans/cis = 6.15±0.27 at 298
K): 4.41 (dd, J = 9.0, 4.4 Hz, 1H, α-CH), 4.03 (d, J = 17.3 Hz, 1H),
3.98 (d, J = 17.5 Hz, 1H), 3.68 (s, 3H, CH3O), 3.58 (m, 1H, δ-CH),
3.54 (m, 1H, δ-CH), 2.22 (m, 1H, β-CH), 1.98 (s, 3H), 1.97 (m,
2H, γ-CH2), 1.96 (m, 1H, β-CH). HRMS (ESI-orbitrap): calcd. for
[M+H]+ C10H17N2O4 229.1183 Da, found 229.1182 Th.
Molecular modelling was performed in Scigress Modelling
3.2 (Fujitsu, Poland). PM6-water algorithm was used for semi-
empirical calculations, and B88-PW91 GGA functional was
employed for the DFT optimizations.
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X-ray diffraction measurements were performed at 150 K.
The R-factor for the calculated structures was 0.0257 (2a) and Bansode, M. V. Sonar and K. N. Ganesh, Chem. Commun., 2016,
52, 4884-4887.
0.0346 (3a).
8 I. E. Sampaio-Dias, C. A. D. Sousa, X. Garcia-Mera, J. F. da Costa,
O. Caamaño and J. E. Rodriguez-Borges, Org. Biomol. Chem.,
2016, 14, 11065-11069.
9
Acknowledgements
cis-trans-Isomerization in Biochemistry, ed. C. Dugave, Wiley,
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VK acknowledges DFG research group 1805 for a postdoctoral
position and Synpeptide EU consortium for financial support.
The authors are also grateful to Dr Marcie Jaffee (Atlanta, USA)
for generous proof-reading of the manuscript.
10
32, 13830-13837; b) C. Cox and T. Lectka, Acc. Chem. Res., 2000,
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The article is dedicated to the memory of Prof Yuriy Kholin
(1962-2017), who nurtured a generation of young talented
Ukrainian chemists.
13
Examples of folding kinetics affected by incorporation of
fluoroprolines: in β2-microglobulin, a) V. Yu. Torbeev, D. Hilvert,
Proc. Natl. Acad. Sci. USA, 2013, 110, 20051-20056; in
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Conflict of Interest
ChemBioChem, 2015, 16, 2162-2166.
a) V. Kubyshkin and N. Budisa, submitted; b) D. Kern, M.
There is no conflict of interest to declare to this article.
14
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The early computation analysis of AcProNHMe suggested that
21
the syn/exo and anti/endo transition states are most favoured,
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8 | Org. Biomol. Chem., 2017, 00, 1-9
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27
The structures have been deposited in Cabridge
Crystallographic Data Centre under the deposition numbers: 2a
– CCDC 1554475, 3a – CCDC 1554476.
28 M. Cai, Y. Huang, J. Liu and R. Krishnamoorthi, J. Biomol. NMR,
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Somehow higher activation entropy values have been
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An experimental model for probing energetics of the amide rotation around proline is described.

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