Energetic contribution to both acidity and conformational stability in peptide models

Article abstract of DOI:10.1039/c5nj03611a

The acidity of N-acyl amino acids is dependent upon the rotameric state of the amide bond. In this work we systematically investigated the acidity difference of the rotamers (ΔpK_a) in the frames of various acetylated amino acids. Our results indicated a mutual interaction of two carbonyl groups of an attractive type. We observed conservative ΔpK_as for acyclic amino acids (2.2-3.0 kJ mol^-1), whereas in the case of alicyclic amino acids, the experimental values revealed a strong dependency on the structural context (1.5-4.4 kJ mol^-1). In homologous amino acids (α-, β-, γ-, etc.), the strength of the attraction decays in an exponential fashion. Furthermore, the interaction can accumulate through a chain of amide bonds in a cascade fashion, as demonstrated by an Ac-Pro-Pro dipeptide. As a result, we demonstrate that ΔpK_a is an experimental parameter to estimate increments in the carbonyl-carbonyl alignment, as determined by the amino acid or peptidyl context. This parameter is also important in understanding the roles of amino acids in both protein folding and translation in biological systems as well as their evolutionary appearance in the genetic code.

Full text of DOI:10.1039/c5nj03611a

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Energetic contribution to both acidity and conformational
stability in the peptide models
Vladimir Kubyshkin,* Patrick Durkin and Nediljko Budisa*
Biocatalysis group, Institute of Chemistry, Technical University of Berlin, Müller-
Breslau-Str., 10, 10623, Berlin, Germany
E-mails: kubyshkin@win.tu-berlin.de; nediljko.budisa@tu-berlin.de
TOC entry: The acidity difference of the amide rotamers has been revised for a large
set of N-acetyl amino acids.
Abstract: Acidity of N-acyl amino acids is dependent upon the rotameric state of the
amide bond. In this work we systematically investigated the acidity difference of the
rotamers (ΔpKa) in the frames of various acetylated amino acids. Our results indicated a
mutual interaction of two carbonyl groups of an attractive type. We observed
conservative ΔpKas for acyclic amino acids (2.2-3.0 kJ/mol), whereas in case of alicyclic
amino acids, the experimental values revealed strong dependency on the structural
1

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context (1.5-4.4 kJ/mol). In homologous amino acids (α-, β-, γ-, etc.), the strength of the
attraction decays in an exponential fashion. Furthermore, the interaction can
accumulate through a chain of amide bonds in a cascade fashion, as demonstrated by
Ac-Pro-Pro dipeptide. As a result, we demonstrate that ΔpKa is an experimental
parameter to estimate increments in the carbonyl-carbonyl alignment, as determined by
the amino acid or peptidyl context. This parameter is also important in understanding
the roles of amino acids in both protein folding and translation in biological systems as
well as their evolutionary appearance in the genetic code.
2

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1. Introduction
There are a number of non-covalent interactions that govern the folding of
polypeptides such as hydrogen bonds, electrostatic attraction (salt bridges) and
repulsion, various aromatic stacking interactions (π-π, cation-π, aliphatic-π, aromatic-
Pro or aromatic-Met), dipolar interactions and the n→π* donation. Some of these, like
hydrogen bonding, are very well described in chemistry and biology as their distances
and impact are easy to observe by conventional methods such as X-ray crystallography,
NMR, CD, IR etc. On the other hand, there are structural motifs, such as the polyproline
II helix, where the driving forces behind this organization are more difficult to understand,
rationalize and follow experimentally. The left-handed polyproline II (PPII) helix was
initially described in 19561 and later found to be a ubiquitous structure, not only in
fibrillar structures like collagen, but also in many folded and unfolded proteins.2,3,4
Conversely, the right-handed polyproline I helix (PPI) only has limited biological
relevance. 5 Although proline has high PPII propensity, this topology can also be
generated in the absence of proline residues, such as in antifreeze glycoproteins.6
Clearly, PPII is an essential secondary structure of proteins that participates in
major vital biological functions (transcription, signal transduction, ligand-receptor
interplay, protein-protein interactions etc.).7 Thus, the fundamental goal is to understand
and (if possible) to quantify the nature and extent of the forces behind its stability. This
is particularly relevant for the canonical secondary amino acid proline which has a
comparable energy of the s-trans and s-cis peptide bonds, therefore exhibiting an
equilibrium of both peptide bond isomers in solution (Scheme 1).8 Equally important is
3

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the amount of energy required for the stabilization of this structure, since entropy (at
least to a certain extent) should oppose the formation of an ordered structure.
One possible rationalization for this stabilization is the n→π* donation, a donative
effect established between the oxygen lone pair of an upstream amide bond to the
carbon atom of the downstream carbonyl. Since it represents a form of a nucleophilic
attack, it should therefore be enhanced by certain dihedral angles that optimize the
angle of donation (Βürgi-Dunitz trajectory). An essential prerequisite for n→π* donation
is the s-trans conformation (ω = 180 °) of the donating amide (Fig. 1).
Scheme 1. trans/cis equilibrium in Ac-Pro-NHMe. The s-trans rotamer is preferred over the s-cis by
approx. - 3 kJ/mol.
Figure 1. The n→π* interaction in a polypeptide chain.
Once established, the n→π* donation results in 1) an energetic stabilization of
particular secondary structural motifs,9 2) stabilization of the trans-amide bond over the
cis-isomer,10 and 3) partial pyramidalization of the carbonyl carbon atom.11 It has also
been argued, however, that a classical dipole-dipole interaction might be mistaken as
being an n→π* interaction.12 Recently, a crystal structure of the PPII helix demonstrated,
4

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on the contrary, the existence of a certain degree of pyramidalization of proline
carbonyls, which could originate from a specific donation.13 For a proline residue,
indeed, the n→π* interaction can play a significant role, since its  angle (-65±10) is
naturally fixed around a value optimal for this donation (-63±6).9 In proline-rich
structures, such as polyproline or collagen, which lack other types of non-covalent
interactions, the accumulation of the n→π* donation through a peptide chain may give
rise to a significant stabilizing effect due to entropic pre-organization.14
Figure 2. Both n→π* donation and dipole-dipole interaction will result in relative stabilization of the s-
trans amide (left). Arbitrary orientation of the C-terminal dipole is given for the plane projection here (see
also Fig. 14).
A critical question is whether the energetic contribution of this interaction can be
of any significance in terms of a single residue. From a chemical standpoint, it appears
to be doubtful, that the amide oxygen can donate enough electron density to enable a
significant structural stabilization. An alternative explanation might be the dipole-dipole
interaction established between the two carbonyl groups. Both effects, n→π* and the
dipole-dipole interactions, may potentially result in the relative stabilization of similar
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dihedral angles in a proline residue (in particular, the trans-amide bond), and from this
standpoint they cannot be distinguished (Fig. 2).
Further, by using proline analogues containing polar substituents (such as
fluorine or hydroxyl-group), additional interacting dipoles will be present which are
capable of participating in the overall interaction. Alternatively, in this case the
conformational preferences can be explained by the geometric pre-organization of the
trajectory between the interacting peptide bonds due to the changes in the pyrrolidine
conformational states (exo/endo pucker equilibrium). As a result, the trans/cis
equilibrium is known to experience dramatic variations.15
Figure 3. Peptidyl transfer reaction with peptidyl-Pro on the P-site of the ribosome.
The final outcome of this mutual interaction of the polar groups must result in a
change of the electronic state of the interacting groups, and hence their chemical
reactivities. The latter conclusion is particularly interesting, since the ribosomal
translation of proline is also known to be slow and may be as a result of the above
mentioned interactions, seen as a donative effect. Furthermore, oligoproline segments
in polypeptide sequences slow down the whole translation process, and can induce
ribosome stalling,16 in particular when accommodated on the P-site.17 Since the C-
terminal group in peptidyl-tRNA on the P-site should be attacked by an amino group in
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the course of a peptidyl transfer reaction on the ribosome (Fig. 3), the interactions that
will reduce the electrophilicity of the carbonyl group may also slow down the rate of the
reaction. Supporting this suggestion, we previously reported dramatic differences in the
translation efficiency in living cells for (4R)- and (4S)-fluoro- and hydroxyprolines.18
To determine a possible logical link between the chemical reactivity and the
folding energy, we turned our attention to the acidity of the terminal carboxyl in N-acetyl
proline and other amino acid residues. Indeed, it has been discovered in the mid-1970s
that N-acylated proline and sarcosine (Sar) exhibit different acidities of their amide
rotameric forms, where the s-trans rotamer is less acidic than s-cis (Fig. 4). 19 , 20
N-acylated Pro and Sar were convenient subjects for the studies, since their rotameric
populations are of comparable intensity in the NMR spectra, where sensitivity is a
general problem. In the times when robust water suppression techniques were still
15
absent,21 the pKa for the rotameric states were determined by means of 13C, N and
17O NMR, delivering results with a certain degree of discrepancy.19,22,23
Figure 4. Acidity difference characterized for N-acetyl proline (Ac-Pro).
These initial studies revealed, among others, that the difference of the acidity of
the two rotamers (ΔpKa) is defined by the mutual interaction of the amide and carboxyl-
groups. The origin of ΔpKa therefore can be seen in the following way: if the n→π*
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donation takes place in Ac-Pro, the electrophilicity of the terminal carbonyl group is
reduced, but only in the case when the peptide bond is in the s-trans conformation.10b,24
From that, it follows that the resulting acidity of the s-trans rotamer should be then lower
than in the s-cis (ΔpKa > 0). Consequently, if the dipole-dipole interaction contributes to
stabilization of the s-trans rotameric form, the effect should be stronger when the
terminal group is a carboxylic acid (large dipole) than salt (small dipole). Eventually the
effect should result in a reduced acidity of the trans-rotamer (ΔpKa > 0). In a similar
fashion to the conformational considerations, both interactions predict the same
outcome for the acidity as well.
In this context, we set out to study the acidity differences in various N-acetylated
amino acids in order to provide an experimental set of parameters for understanding the
carbonyl-group alignment in terms of the energy of the interaction. Additionally, we also
aimed to provide a solid experimental parameter that would make a logical link between
the conformational energy and the chemical reactivity. However, the reader should be
fully aware to draw their own conclusion from the present study concerning the
molecular origins of the discussed energetic term.
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2. Experimental Section
2.1. N-acylation of the amino acids
Typical procedure for the synthesis of N-acetyl proline:
L-proline (2.26 g; 19.7 mmol, 1 equiv.) was stirred with anhydrous dichloromethane (40
ml) and acetic anhydride (1.85 ml; 19.6 mmol, 1 equiv.) until a clear solution was
obtained (30 min). Dichloromethane was removed under reduced pressure and the
residue was dissolved in water (20 ml) and freeze-dried. Resulting solid contained
approx. 1/10 of unreacted proline, therefore it was dissolved in water (25 ml) and filtered
through a short ion-exchange column (Dowex® 50WX8, 50-100 mesh). Acidic fractions
were collected and freeze-dried to give 2.79 g of N-acetyl proline as white solid (91 %
yield).
For analytical characterization of the substances 11, 12, 15, 16, 17, 18, 19 and 20 see
ESI. For the already known compounds, 1, 2, 3, 7, and 22 spectral data are also given
in the ESI.
2.2. Determination of the carboxyl pKa in Ac-Xaa
Typical procedure was the following:
Ac-Xaa (5 mg) and potassium dihydrogenphosphate (5-6 mg) were dissolved in 10 ml of
water and titrated by potassium hydroxide and hydrochloric acid solutions to different
pH values ranging from 7 to 1. 12-15 aliquots with 500 μl volume were taken to NMR
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tubes, followed by addition of 50 μl TPS (sodium 3-(trimethylsilyl)propane-1-sulfonate)
solution in deuterium oxide (0.1 mg/ml) to each tube for lock and referencing. Final
concentration in the samples was: Ac-Xaa 2-4 mM, phosphate 7-8 mM, TPS 0.1 mM. 1H
NMR spectra were measured at either 500 or 700 MHz frequency in Watergate W5
pulse sequence with the calibrated pulses. For pH < 2 pulses were re-calibrated for
each sample due to the increased ionic strength of the solutions. TPS most upfield
resonance was set to zero, except of the spectra for the most acidic samples (pH≤1.5),
for which TPS resonances deviated due to partial protonation of the sulfonic group. In
that case we used any visible additive in the spectrum, for instance, methanol
resonance at 3.349 ppm for the referencing. Chemical shifts were read out and plotted
against pH. In particular, acetyl CH3-group resonance was taken with three digits after
the decimal in the chemical shift value. In this case s-trans-chemical shift was higher
than s-cis, with the only exception of Ac-MeAla. Chemical shift vs pH curves were fitted
according to either Boltzman or Logistic fits (OriginPro 9.1), the fits were derivatized and
the global maximum of the derivative curves was read as the pKa value. The derivative
curves were checked to exhibit a symmetric shape.
In particular, by this procedure we determined pKa values for Ac-Sar, Ac-Aze, Ac-Pip.
For fluorine containing Ac-Xaa also 19F{1H} NMR spectra were recorded in inverse-
gated decoupling experiments (no NOE enhancement).
In addition, for Ac-Pro we determined pKa values under the following conditions:
Ac-Pro ≤ 0.5 mM / citric buffer 100 mM
pKa s-cis 2.79; s-trans 3.54
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Ac-Pro 25 mM / no buffer
pKa s-cis 2.79; s-trans 3.56
Where it was seen, that the pKa values (in particular, for the s-trans form, which is more
reliable in detection) are invariant to the offered conditions of the analyte or buffer
concentrations.
For Ac-Xaa with the secondary amide (Ac-Gly, Ac-Val, Ac-Ile) we used higher
concentrations of the analytes (25, 40 or 100 mM) in order to detect the minor rotameric
form. Examples also see in ESI, section 4.
2.3. Equilibrium populations in acids and salts
We found that 140-170 mM solution of potassium hydrogen sulphate solution delivers
pH values of 1.4-1.3, also, when some amount of Ac-Pro is present in solution.
Therefore the acidic samples were prepared as following. Ac-Xaa (5 mg) and potassium
hydrogen sulphate (10-12 mg) were freeze-dried from deuterium oxide (500 μl) and then
dissolved in deuterium oxide (525 μl), and this sample was then used for the
measurements. Final concentration of Ac-Xaa was therefore 40-70 mM.
Salt samples were prepared as following. Ac-Xaa (10 mg) and potassium
dihydrogenphosphate (10-12 mg) were dissolved in water (1 ml) and titrated with
potassium hydroxide solution to pH 6.9-7.1. An aliquot of this solution (525 μl) was
freeze-dried, freeze-dried from deuterium oxide (2 x 500 μl) and taken into deuterium
oxide (525 μl) for the NMR measurements. Final phosphate concentration was therefore
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70-80 mM, Ac-Xaa concentration 40-70 mM. Due to the slow amide rotation is salts, it
turned out to be essential that some samples should be pre-equilibrated for at least one
day standing at the room temperature prior the measurements.
1H NMR intensities were measured at 700 MHz frequency in 90-pulse experiments in
one scan in order to ensure complete pre-relaxation of the nuclei. In particular, the most
characteristic α-CH proton resonances characterized with the longest T1 relaxation
times, and s-trans resonance relaxed slower than s-cis.
2.4. Thermodynamic analysis
Thermodynamic analysis was performed for the salt sample of Ac-Pro and Ac-β-homo-
Pro, prepared as described above, and for N-acetyl-2-methylpyrrolidine sample (see
1
ESI). Equilibrium populations were measured by H NMR at different temperatures.
Temperature heating and cooling series were performed in the range 295-355 K.
Variable temperature unit was calibrated by standard methanol and glycerol sample
measurements.25
For the final analysis we collected data points from several series and plotted them in
coordinates ΔGtrans/cis versus T, rather than Van ‘t Hoff plot coordinates. Linear fit
delivered the values of ΔH and ΔS.
2.5. Rotational barriers in different solvents
Ac-Pro-OK-18-crown-6 complex was prepared as following: Ac-Pro was titrated by
potassium hydroxide solution in the presence of
a
small potassium
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dihydrogenphosphate additive to pH 7. Followed by addition of equimolar amount of 18-
crown-6 in acetonitrile-water the mixture was freeze-dried. Obtained solid contained Ac-
Pro-OK / 18-crown-6 / H2O in 1:1:2 ratio according to the 1H NMR spectra.
Equilibrium constants for Ac-Pro-OMe and Ac-Pro-OK·18-crown-6·2H2O complex in
1
perdeuterated solvents were measured by H NMR at 298 K at 700 MHz resonance
frequency.
1
Rotational rates were measured in H 2D cross relaxation experiments with gradients
(NOESY-EXSY) with typical mixing time of 1-2 s for exchange detection and 5 ms for
referencing. Recycling delay was chosen to be 3-5·T1 of the nucleus with slowest
relaxation (usually, α-CH of the s-trans rotamer). The latter was estimated in 1D
inversion recovery experiments for each compound/solvent combination. Since relevant
spectral windows varied with the solvents, time domain was inset to give 2-4 Hz
resolution in the direct and 10-30 Hz resolution in the indirect dimensions, respectively.
On the processing step the indirect dimension was zero filled to reach the initial direct
dimension resolution. 2D frequency domain spectra were phased, baseline corrected
and integrated. Exchange rate matrices were calculated by EXSYCalc freeware
(Mestrec). Activation energies were calculated using Eyring equation. In addition to the
exchange quantification, the EXSY spectra were interpreted as NOESY for assignment
of the rotameric forms.
2.6. Equilibrium populations and pKa determination in Ac-Pro-Pro
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Ac-Pro-Pro dipeptide was synthesized both on solid support and in solution synthesis as
described in ESI. For the pKa measurements we followed pH dependences of the most
characteristic α-CH resonances. Since the spectra contained eight such resonances (2
proline residues x 4 rotameric forms) in very different intensity close to the water signal,
and some of them overlapped, the measurements of all four pKa values were conducted
in a series of 25 1H{13C} HSQC spectra (cnst 150 Hz, recycling delay 2 s) assisted by 1H
13
Watergate W5 and C{1H} dept45 experiments on samples with pH between 1 and 7.
The spectra windows were zoomed to the α-CH region in both dimensions. The sample
concentrations were: 100 mM Ac-Pro-Pro and 14 mM phosphate. Partial overlap of the
1
signals was observed in H and 13C spectra, but simultaneous overlap in both
dimensions was seldom. The titration curves and the pKa values read out were
performed as described above for Ac-Xaa (also, see ESI).
1
For the H NMR measurements of the equilibrium populations the acidic and salt
samples in deuterium oxide were prepared as described for Ac-Xaa. The samples
contained 75 mM Ac-Pro-Pro and 140 mM potassium hydrogen sulphate or 140 mM
1
potassium phosphate buffer for acid and salt respectively. H NMR spectra were
measured at 700 MHz resonance frequency as described for Ac-Xaa. Quantified
13C{1H} spectra were acquired in samples containing 250 and 350 mM Ac-Pro-Pro for
1
the acid and salt respectively. The spectra were recorded in 90-pulse inverse gated H
decoupled pulse sequence at 126 MHz resonance frequency. Recycling delay was 60-
80 s to ensure complete relaxation. 512-768 transients were enough to collect spectra
with all four rotamers visible. It is important to note, that the α-CH intensity ratios
obtained in quantified 13C{1H} NMR spectra were in a good agreement (within ±1 %
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13
accuracy) with those obtained in C{1H} dept45 spectra with routine setup (recycling
delay 2 s, cnst 150 Hz). This observation justifies our approach of the pKa
measurements conducted by HSQC, where the averaged populations of salt/acid upon
the pKa transitions should deliver correct average of the chemical shifts according to
their molar ratios.
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3. Results and Discussion
3.1. N-terminal amide cis/trans rotameric ratio as a function of the C-
terminal group
It has been previously demonstrated that trans-content in N-acyl proline
derivatives is dependent from the nature of the C-terminal group. Generally, Ktrans/cis is
higher in the case of the terminal ester compared to the amide due to the differences in
the enthalpic term.26 Further evaluation (Table 1) demonstrates that the preferences
towards the s-trans conformation gradually decreases in the row X = OMe > OH >
NHMe > O-, i.e. with the decrease in the carbonyl electrophilicity (decrease in the C=O
dipole). The same occurs in the case when two C-terminal groups are integrated in the
frame of one molecule.
Table 1. Energetic terms of the amide equilibrium for Ac-Pro-X.
a)
Ac-Pro-X
X = OMe27
X = OH
X = NHMe8
X = O-
Ktrans/cis
ΔG298, kJ/mol
- 3.9±0.1
- 3.3±0.1
- 2.9±0.1
+ 0.5±0.2
b)
ΔH,
ΔS,
k,b) s-1
0.84
2.4c)
0.81
0.075
c)
kJ/mol
J/mol·K
4.8
- 5.04
±0.05
- 3.82
±0.16
3.8
-
-
3.2
- 3.97
±0.04
- 3.64
±0.08
0.81
- 1.17
±0.10
- 5.24
±0.30
a)
in aqueous medium at 298 K; cis-to-trans rotation rate at 340 K; the value is the largest because acidic medium catalyzes rotation at high
temperature due to the partial amide protonation.
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In particular, for racemic N-acetyl-pyrrolidine-2,5-dicarboxylic acid mono methyl ester
(Ac-(MeO)Pdc) the Ktrans/cis values were predicted from the proline data as Ktrans/cis(Ac-
Pro-OH/O-)/Ktrans/cis(Ac-Pro-OMe) are in good agreement with the experimental values
(Fig. 5; see also Fig. S61 for comparison in the entire pH range).
Figure 5. In N-acetyl-rac-(trans-)pyrrolidine-2,5-carboxylic acid mono methyl ester, Ac-(MeO)Pdc, two
carboxylates compete for the donation from the amide carbonyl. Resulting trans/cis equilibrium constant
can be predicted from the data on proline.
Figure 6. Effective charge on the C-terminal carbon atom as dependant from the nature of the C-terminal
group.a)
a) as simulated in MarvinSketch 15.4.27 (ChemAxon Ltd.)
In the terminal case when the C-terminal group is a carboxylate, the effective
charge on the carbonyl carbon is close to zero (Fig. 6). Therefore it can be expected
that in the salts both n→π* and the dipole-dipole interactions are substantially
attenuated, and the trans/cis equilibrium is defined by the steric effects. Subtraction of
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the ΔGtrans/cis values yields the energetic term ΔΔG which accounts for the effect of the
peptide bond alignment due to the n→π* and dipole-dipole interactions (eq. 1).
Instrumentally this value was experimentally determined by NMR in two different ways
as described in the next section.
ΔΔG = ΔGtrans/cis(acid) – ΔGtrans/cis(salt)
(eq. 1)
3.2. pKa and rotameric populations in Ac-Xaa
The experimental approach is illustrated in Fig. 7. Because the acid-salt
transition is fast on the NMR time scale, the mixture of the two ionization states provides
an averaged chemical shift value, whereas the slow amide rotation enables one to
observe the two rotamers separately (Fig. 7A). While going from basic to acidic pH
values, the chemical shifts exhibit characteristic sigmoidal transitions (Fig. 7B), which is
also accompanied by the change in the rotameric populations (Fig. 7C). The difference
in the pKa exhibited by the two rotamers (ΔpKa) corresponds to the free energy
difference ΔΔG = ΔG°(A) - ΔG°(B) - ΔG°(C) + ΔG°(D), which can also be calculated
from the equilibrium populations according to eq. 2.
Ktrans/ cis (acid)
Ktrans/ cis (salt)
(eq. 2)
G  2.30 RTpKa  RT ln
Since ΔΔG and ΔpKa values are equivalent, we can also convert equilibrium
constants to the ΔpKa* values expressed according to eq. 3.
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Ktrans/ cis (acid)
Ktrans/ cis (salt)
(eq. 3)
pKa *  log10
Following these considerations, we determined the experimental pKa and ΔpKa*
parameters for 19 different N-acetyl amino acids with different structures (Fig. 8). In
Table 2 we summarize the obtained values. The ΔpKa and ΔpKa* values found were in
close agreement, providing a good mutual control. Doubtless, an important observation
is that ΔpKa exhibited positive values (s-trans rotamer is less acidic) in all studied cases.
In addition, the ΔpKa and ΔpKa* values for the Pdc derivative were in full agreement with
the proline data.
Figure 7. A. Equilibrium forms of Ac-Pro in aqueous medium and their interconversions; B. pH
dependence of the 1H NMR chemical shifts (CH3-groups) of the two amide rotamers of Ac-Pro. This plot is
equivalent to pH titration curves; C. pH dependence of the trans/cis equilibrium in Ac-Pro as determined
by 1H NMR spectra integration (δ-CH2 fragment). Observations are made at 298 K.
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Further careful inspection of our experimental data reveals several important
observations. Firstly, comparison of the acidity values of the s-cis forms and the
textbook acidities of the corresponding free amino acids (Pro, Gly, Ala, Phe, Val, Ile)
reveals a conservative acidity reduction of 0.8-0.9 units. Considered mechanistically,
this is the result of the absence of the compensating ammonium charge in the
N-acetylated amino acids. At the same time, ΔpKa appears to be a function of the amino
acid context.
Figure 8. The series of examined N-acetyl amino acids with the designated experimentally determined
ΔpKa = pKa (s-trans) – pKa (s-cis) values.
Secondly, the value of ΔpKa increases when going from unsubstituted residues
(Gly and Sar, ΔpKa 0.4) to β-branched amino acids (Val and Ile, ΔpKa 0.5), where the
bulky sidechain substituent may reinforce a more stable alignment of the carbonyl-
groups. The value was significantly higher for proline (ΔpKa 0.7), whereas its homologs
azetidine-2-carboxylic acid (Aze 0.5) and pipecolic acid (Pip 0.25) exhibit lower ΔpKa
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values.28 Next, incorporation of substituents at position 4 of the proline ring afforded
significant changes of ΔpKa in the range of about 0.4 units.
Table 2. pKa and amide equilibrium constants determined for the examined N-acetylated amino acids at
298 K.
a)
a)
b)
Ac-Xaa
pKa
ΔpKa
Ktrans/cis
ΔΔG298
,
ΔpKa*
kJ/mol
s-cis
s-trans
salt
acid
methyl ester
4.826
n.a.
Ac-Pro, 1
Ac-Aze, 2
Ac-Pip, 3
Ac-Gly, 4
Ac-Sar, 5
Ac-Ala, 6
2.85
3.55
0.70
0.81±0.02
0.57±0.01
1.42±0.01
56±1
3.77±0.05
1.67±0.02
2.55±0.02
141±2
-3.81±0.09
-2.66±0.08
-1.45±0.04
-2.29±0.08
-2.39±0.03
-2.50±0.37
-2.34±0.09
-2.21±0.10
-3.01±0.50
-3.06±0.32
-4.37±0.14
-2.99±0.17
-4.21±0.10
-2.21±0.15
-4.46±0.07
-3.81±0.09
-3.16±0.06
-3.43±0.11
-3.74±0.17
0.67
2.73
3.37
3.19
3.11
3.11
3.23
2.98
3.10
3.12
2.37
2.87
2.39
3.19
3.54
3.48
2.34
2.57
2.68
3.24
3.63
3.57
3.54
3.56
3.64
3.42
3.55
3.63
3.19
3.39
3.15
3.62
2.79
2.84
2.93
3.21
3.36
0.51
0.26
0.38
0.43
0.45
0.41
0.44
0.45
0.51
0.82
0.52
0.76
0.43
0.75
0.64
0.59
0.64
0.68
0.47
0.25
0.40
0.42
0.44
0.41
0.39
0.53
0.54
0.77
0.52
0.74
0.39
0.78
0.67
0.55
0.60
0.66
n.a.
n.a.
1.00±0.01
62±5
2.62±0.01
170±13
2.8
n.a.
Ac-MeAla, 7
1.37±0.03
68±1
3.53±0.05
166±4
n.a.
Ac-Phe, 8
n.a.
Ac-Val, 9
71±2
239±45
n.a.
Ac-Ile, 10
71±2
244±26
n.a.
Ac-(4R)-Flp, 11
Ac-(4S)-Flp, 12
Ac-(4R)-Hyp, 13
Ac-(4S)-Hyp, 14
Ac-(4S)-Mep, 15
Ac-(4R)-Mep, 16
Ac-F2Pro, 17
0.91±0.02
0.68±0.04
0.95±0.02
0.82±0.03
1.03±0.01
0.85±0.01
0.79±0.01
0.90±0.02
0.18±0.01
5.30±0.20
2.27±0.02
5.20±0.09
2.00±0.05
6.24±0.11
3.95±0.10
2.83±0.03
3.59±0.09
0.81±0.02
6.826
2.526
6.115
2.415
7.415
3.715
3.326
4.029
= 1
Ac-(4S)-TfmPro, 18
Ac-(MeO)Pdc, 19
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a) the error of pKa and ΔpKa determination is ≤0.05; b) calculated from the equilibrium constants according to eq. 1.
Scheme 2. Hydrogen bond that stabilizes the protonated carboxylate in the s-trans conformation in non-
polar media.
In the two cases of (4R)-Flp and (4R)-Hyp, higher ΔpKa values are accompanied
with the higher absolute acidity of both rotameric forms. In contrast, (4S)-TfmPro and
F2Pro higher acidity values do not correlate with the increments in ΔpKa. This is indeed
an important observation which contradicts the presence of the hydrogen bond between
the carboxyl-group and amide carbonyl that might potentially stabilize the s-trans
conformation (Scheme 2). Previously in the 1980s, it was reported that “a hydrogen
bond between the terminal carboxyl group and the preceding amide carbonyl could be
ruled out in polar media”19b based on considerations of heteronuclear NMR. The fact,
that ΔpKa does not correlate with the absolute acidity further strengthens such
conclusions (see also the Pdc example). It further indicates that the effect of donation is
not driven by the carboxyl-group electrophilicity alone, but it also clearly accounts for
conformational preferences of the proline ring.
Noteworthy, (4S)-Hyp, which exhibited low ΔpKa (as well as Ktrans/cis) turned out to
also be the least acidic. It could be that the hydrogen bond between the carboxyl-group
and 4-OH group shifts the absolute acidity without affecting ΔpKa. Another explanation
would be the presence of the n→π* donation between these two functionalities. The
latter has been reported in simulations in the gas phase.30 The first explanation seems
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to be more plausible, since the absolute acidity shift was not observed in (4S)-Flp.
Indeed, the hydroxyl-group may engage in specific hydrogen bonding with the carboxyl,
since a hydroxyl-group is more donative than water, which is not the case for an
aliphatic fluorine substituent.31
3.3. ΔpKa and the rotameric populations in esters
Figure 9. Dominance of the s-trans rotamer varies in 4-substituted prolines.15,27
Susbequently, we compared the ΔpKa values found with the already known
trans/cis preferences in the methyl esters of the N-acetylated prolines bearing
substitutions in position 4 of the pyrrolidine ring (Fig. 9). We found that the ΔpKa aligns
with their ester population preferences with a decent agreement (Fig. 10). According to
the linear free energy correlation this would indicate, that the same phenomenon is
underlying the changes in both values. This correlation therefore provides a clear link
between ΔpKa and the electronic properties of the esters.
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Figure 10. Linear free energy correlation between the Ac-Xaa rotameric acidity differences and Ac-Xaa-
OMe equilibrium rotameric populations in 4-substituted proline analogues.
In addition, highly similar ΔpKa values found for N-acetylated canonical amino
acids (Gly and Ala) and their N-methylated analogues (Sar and MeAla) support the
conclusion that ΔΔG originates from geometric alignment of the carbonyl-groups. Thus
the ΔΔG values can equally well characterize the peptide bond alignment energy as the
values of ΔGtrans/cis for the structurally similar proline analogues. However, the
subtraction of the steric effects in ΔΔG values allows comparison of a large variety of
the amino acids, and is not restricted to structurally similar systems.
3.4. Effects of the charged carboxylate
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Scheme 3. Alternative explanations for the dominance of s-cis-Ac-Pro-O- rotamer in the salt form: A.
destabilization of s-trans-Ac-Pro-O- due to the repulsion of the polar groups; B. stabilization of s-cis-Ac-
Pro-O- due to the backward n→π* donation.
It has also been previously reported that the Ac-Pro-O- salt exhibits dominance of
the s-cis rotameric form, that was interpreted as the repulsion between the two polar
groups, when the terminal carboxyl is charged (Scheme 3A).32,33 Another hypothetical
explanation for the s-cis dominance in the salt would be an establishment of a backward
n→π* donation from the nucleophilic carboxylate to the carbonyl-group of the amide
(Scheme 3B).34 In the case, when such interactions are present, they will also contribute
to the ΔΔG values.
Scheme 4. Rotational barrier increases in more polar solvents with higher ability to separate charges due
to a shift of the contributions of their mesomeric structures in the ground states. 35
In order to test the presence of the carboxylate effects we first studied the
dependence of the amide rotational barriers on the charge separation strength of the
solvent (ET values).36 For the ester form, both activation barriers exhibited the same
slope (Fig. 11A) due to the solvent effect on the amide ground state (Scheme 4),
irrespective of the s-trans and s-cis rotameric form.37
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Figure 11. Correlations between the amide rotational barriers and Reichardt-Dimroth solvent parameter
(defined as the ability of the solvent to separate charges): A. For 60 mM Ac-Pro-OMe at 298 K; B. For 20
mM Ac-Pro-OK-18-crown-6 complex at 310 K (in dichloromethane at 298 K, in water at 340 K). Straight
line, solid circles – activation barriers for the trans-to-cis isomerization; dashed line, opened circles – for
the cis-to-trans isomerization. C. Correlation of the equilibrium free energy differences in Ac-Pro-O- and
the solvent acceptor number.
In the case of the salt Ac-Pro-O- the interaction between the charged carboxylate
and the solvent can attenuate the carboxylate interactions shown in Scheme 3. When
comparing the salt to the ester, the slopes in the ET-Ea coordinates will change: 1) for
trans-cis towards an increase in the case of the carboxylate-carbonyl repulsion
(Scheme 3A) which is a destabilizing effect, and 2) for cis-trans towards a decrease in
the case of the backward n→π* donation (Scheme 3B) which is a stabilizing effect. Our
experimental observations presented in Fig. 11B revealed that the first scenario
appeared to be valid, whereas there was no support for the hypothesis that the
backward n→π* donation was occurring. As one possible interpretation, the equilibrium
was strongly shifted towards the s-cis rotamer in the solvents with a low acceptor
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number (Fig. 11C). 38 Thus, highly polar solvents reduce the carboxylate-carbonyl
repulsion. In the terminal case, water seems to quench the repulsion, leading to nearly
equilibrated s-trans and s-cis populations. This is supported by the observation that the
salt trans/cis ratios found in water (0.81) and water-acetonitrile mixture (0.79) are
indistinguishable from the value in trifluoroethanol (0.79) (for comparison of the full
solvent set see Fig. S67).
Figure 12. Enthalpic terms of the trans/cis equilibrium determined for the salt of N-acetyl proline (1), N-
acetyl-β-homoproline (20) and in N-acetyl-2-methylpyrrolidine (21) reveals their close similarity.
The absence of the carboxylate effects is further supported by the finding that
ΔHtrans/cis values are nearly identical in substrates similar to Ac-Pro-O- (Fig. 12).
Therefore, it seems to be evident that the ΔΔG values allow quantification of the
carbonyl alignment in the esters and acids, whereas the carboxylate effects are
opposed by strong water solvation.
3.5. ΔpKa in non-α-amino acids
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Figure 13. Energy difference between s-trans and s-cis rotational states in unsubstituted N-acetyl amino
acids. The attractive effect exponentially decays with increasing chain length.
While in the frame of N-acetyl α-amino acids, the ΔpKa values were found to vary
in the range of 0.4-0.8 units, an increase of the chain length between the functionalities
should lead to an attenuation of the interaction between the carbonyl groups. Due to the
specific nature of the n→π* interaction, we would not expect it to occur with increasing
chain length, with this effect already being drastically diminished in the frame of an N-
acetyl β-amino acid. The dipole-dipole interaction should also vary dramatically in the
homologous N-acetyl amino acids, decreasing a stabilizing interaction with increasing
chain length. The question, why this should be the case was the addressed through
experiment.
Experimentally we found in the case of Ac-βAla both ΔpKa and ΔpKa* to be
approximately 0.2, which is about a half of the values of Ac-Gly. Next, for Ac-GABA the
ΔpKa* was about 0.1 (Table 3). We observed an exponential decrease of the effect by a
factor of 2 for each subsequent CH2 unit. It is further seen from the Fig. 13, that the
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effect originates in the uncharged compounds: acids and esters. Only in these species
did we expect a significant C=O dipole (Fig. 6). Then the alignment of this dipole with
that of the amide is stronger in the case of the s-trans conformation, arising from their
closer proximity, as illustrated in Fig. 14.
Table 3. Rotameric acidity differences in the amino acids with increasing chain length.
Xaa
Ac-Xaa structure
pKa of Xaa
pKa of Ac-Xaa
ΔpKa
ΔpKa*
s-cis
s-trans
Gly
2.34
3.63
4.08
4.37
2.0
3.19
3.57
4.33
4.65
4.69
3.55
0.38
0.18
-
0.40±0.02
0.22±0.08
0.10±0.04
0.08±0.05
0.67±0.02
22
(α)
βAla
(β)
4.15
23
GABA
-
-
24
(γ)
ACA
-
25
(ε)
Pro
2..85
0.70
(α)
1
βHPro
(β)
3.58
3.93
4.36
0.43
0.40±0.02
20
Figure 14. The distance between two interacting dipoles should be closer in the s-trans conformation of
an N-acetyl amino acid.
29