Proline N-oxides: Modulators of the 3D conformation of linear peptides through nO-turns

Article abstract of DOI:10.1039/c4ob00433g

Small peptides are essential mediators of numerous physiological processes. Consequently, there is huge interest in the de novo design of peptides with a predictable folding and related biological activity. In this study, we investigate the possibility of modulating the secondary structure of tetrapeptides through proline N-oxide moieties and N-methylation of the peptide backbone. A series of tetrapeptides were synthesised to investigate the combined effect of Pro N-oxide and N-methylation of the amide bond on the (n + 1) residue in terms of cis- and trans-isomerization, as well as how these modifications direct potential intramolecular hydrogen bonding interactions. The right combination of both these parameters led to a trans to cis-conformational interconversion and a change in the nature of the hydrogen bonding interactions, as demonstrated by NMR spectroscopic, molecular modeling analysis and thermal coefficient studies. Proline N-oxide residues were proposed to induce turns we named as NO-γ-turns and NO-β-turns based on their similarity to traditional γ- and β-turns. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00433g

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PAPER
Proline N-oxides: modulators of the 3D
conformation of linear peptides through
“NO-turns”†‡
Cite this: Org. Biomol. Chem., 2014,
12, 4479
Majid D. Farahani,a Bahareh Honarparvar,a Fernando Albericio,a,b,c
Glenn E. M. Maguire,a,b Thavendran Govender,a Per I. Arvidsson*a,d and
Hendrik G. Kruger*a
Small peptides are essential mediators of numerous physiological processes. Consequently, there is huge
interest in the de novo design of peptides with a predictable folding and related biological activity. In this
study, we investigate the possibility of modulating the secondary structure of tetrapeptides through
proline N-oxide moieties and N-methylation of the peptide backbone. A series of tetrapeptides were syn-
thesised to investigate the combined effect of Pro N-oxide and N-methylation of the amide bond on the
(n + 1) residue in terms of cis- and trans-isomerization, as well as how these modifications direct potential
intramolecular hydrogen bonding interactions. The right combination of both these parameters led to a
trans to cis-conformational interconversion and a change in the nature of the hydrogen bonding inter-
actions, as demonstrated by NMR spectroscopic, molecular modeling analysis and thermal coefficient
studies. Proline N-oxide residues were proposed to induce turns we named as NO-γ-turns and
NO-β-turns based on their similarity to traditional γ- and β-turns.
Received 25th February 2014,
Accepted 9th April 2014
DOI: 10.1039/c4ob00433g
www.rsc.org/obc
Selective N-methylation of amide nitrogen atoms8–10
increases the proteolytic stability/bioavailability.11 It also dra-
Introduction
During the last few years, the number of peptide-based matically increases the aqueous solubility of the peptide,11
pharmaceutical drugs reaching the market has notably whilst simultaneously increasing the lipophilicity10 and the
increased.1 Peptide-based drugs have many advantages, such conformational rigidity of such peptides.8–10
as high potency, and they often display a more limited off-
Proline residues are important inducers of peptide folding,
target side-effect profile than traditional small molecule yielding secondary structures with β-turn or alpha helix
drugs.2 However, the oral bioavailability of peptides is a major character.12–18 The proline amide bond displays a relatively
obstacle that hinders the development of more therapeutic high cis–trans isomerization ratio (3–5% cis-isomers).19–25 The
formulations.3–5 Pharmacokinetic properties such as short rotational energy barrier for proline cis/trans-amide isomeriza-
plasma half-life and sensitivity to enzyme degradation, as well tion is normally in the range of 16–20 kcal mol−1 (ref. 26) which
as the tendency to undergo aggregation are some of the main is on the border of being achievable at room temperature;
reasons for their low bioavailability.3,6,7
however, values as low as 8 kcal mol−1 have been recorded.27,28
A change in cis/trans-isomerization ratio can be of fundamental
importance for controlling peptide folding.29,30
The synthesis of structurally constrained peptides can be
achieved with several strategies, the most popular being cycli-
sation of the peptide.31,32 Synthesis of self-assembled peptides
utilizing van der Waals forces, hydrogen bonds, hydrophobic
and aromatic π–π stacking is another way to form secondary
structures, such as turns, helices, and sheets.33–40 A γ-turn41
and a β-turn41–50 are presented in Fig. 1.
aCatalysis and Peptide Research Unit, School of Health Sciences, University of
Kwazulu-Natal, Durban 4001, South Africa
bSchool of Chemistry and Physics, University of Kwazulu-Natal, Durban 4001,
South Africa
cDepartment of Organic Chemistry, University of Barcelona, 08028 Barcelona, Spain
dScience for Life Laboratory, Drug Discovery & Development Platform & Division of
Translational Medicine and Chemical Biology, Department of Medical Biochemistry
and Biophysics, Karolinska Institutet, Stockholm, Sweden.
E-mail: kruger@ukzn.ac.za, per.arvidsson@scilifelab.se
†In memory of Professor Per Ahlberg, leading physical organic chemist and
mentor.
Proline is known to be an active β-turn inducer when placed
in the (i + 1) position in peptide backbones. In an effort to
find alternative sources of β-turn inducers, our group has pre-
viously reported various cage peptides.50–52 Herein, we present
‡Electronic supplementary information (ESI) available: NMR assignments for
each compound, NMR-based thermal coefficient graphs, and NMR and HRMS
spectra. See DOI: 10.1039/c4ob00433g
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Fig. 3 N-Oxide-proline tetrapeptides. [Bz(NO)PGNF], [Bz(NO)PMeGNF],
[Bz(NO)PIVQ] and [Bz(NO)PMeIVQ].
Fig. 1 Criteria used in the identification of γ- and β-turn
characteristics.41,46,50
Fig. 2 Proposed N-oxide hydrogen bonding arrangement by O’Neil
and co-workers.54
a new tool to form β-turns by taking advantage of the capa-
bility of the N-oxide moiety to form strong hydrogen bonds
when introduced into proline.
O’Neil and co-workers studied the conformational effect of
proline N-oxides [P(NO)] using either N-alkyl-P(NO)-amide or
N-alkyl-P(NO) containing dipeptides and showed that hydro-
gen bonding takes place mainly through a six-membered ring
(Fig. 2). In addition it was reported that the formation
of hydrogen bonds between amide protons further away from
(i + 2) and the N-oxide is unlikely and, if formed, bonds would
be quite weak.53,54 The stereochemistry of the oxygen atom is
well established.53,54 Attack of the oxygen on this system
occurs exclusively from the same side as the amide group due
to the hydrogen bonding interaction between the amide and
the chloroperbenzoic acid (m-CPBA).
The observed “NO-γ-turn” consists of 6 atoms and cannot
be classified as a classic γ-turn with 7 atoms (see Fig. 1).
We therefore decided to develop a new concept of peptide
“NO-turns” based on the presence of tertiary N-oxide moieties,
with or without selective N-methylation. The scope and limit-
ations of this concept as modulators of tetrapeptide confor-
mations are reported.
Fig. 4 Peptides
characteristics.
theoretically
designed
for
optimal
β-turn
selected [Bz(NO)PIVQ]. Both isoleucine (I) and valine (V) are
known to prevent β-turn formation of short peptides.55–58
Glutamine was chosen as the final residue since it is bulkier
than asparagine. Control peptides without the N-oxide were
also synthesised. In the next level of this study, the first resi-
dues were replaced with N-methylated amino acids to investi-
gate the possibility of obtaining
a wider turn. The
conformational preferences of the designed N-oxide peptides
were studied by NMR spectroscopy, including NOE exper-
iments59,60 as well as molecular modeling.
In the case of the N-oxide and N-methylated peptides,
potential hydrogen bonding interactions between the (i + 2)
amide proton and the N-oxide group is a possibility. Such a
“NO-β-turn” has 9 atoms (Fig. 4) and cannot be classified as a
classic β-turn with 10 atoms (Fig. 1).
Peptides were synthesized via a solid-phase approach on
2-chlorotrityl-chloride (CTC)-resin with HBTU/DIEA as a coup-
ling cocktail using Fmoc protection of the amino groups. For
better control of the synthetic process, previously prepared
N-benzylproline was incorporated as the last building block.61
At the end of the synthesis the peptides were cleaved from the
resin with 40% TFA in DCM and the tertiary amine of the
proline was stereoselectively oxidized62,63 using m-CPBA and
K2CO3 in DCM at −72 °C (Scheme 1). Peptides were purified
via semi-preparative HPLC with a C8 column and the struc-
Results and discussion
Two different families of peptides were earmarked. The
sequence of the first was chosen based on a high probability
for β-turn formation and the second with amino acids known
not to induce turns. Several tetrapeptides, containing N-oxide-
N-benzylproline as the N-terminal residue (Fig. 3), were pre-
pared. For the first peptide family we chose [Bz(NO)PGNF].
Asparagine (N) and phenylalanine (F) are also known to assist
with turn formation.55,56 For the second peptide family we
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complex peak splitting should have been observed for 2b.
Therefore, these conformations are ascribed to cis/trans
isomers of the N-methylated peptide bonds, as shown in
Fig. 4. Interestingly, individual cases have been reported where
certain proline containing peptides gave up to 50% and also
complete cis-conformations.60,64–78 However, there is no defi-
nite rule with respect to the equilibrium of cis- and trans-
isomers of peptides, but solvent polarity plays a prominent
role. A polar solvent, such as DMSO with a large dipolar
moment (3.96 D), favours cis-isomers, while less polar sol-
vents, such as water (1.85 D), induce more trans-character.79,80
The cis/trans ratio around the amide bond increases
through N-methylation.81–84 There appears to be no particular
rule that governs the ratio of cis- and trans-conformations of
N-methylated peptides. The population of cis-isomers is gener-
ally higher at lower temperatures, but decreases at room temp-
erature,85,86 this equilibrium being highly dependent on
the local environment, such as peptide backbone and
solvent.80,85,87 Energy barriers between 15 and 20 kcal mol−1
can be overcome at room temperature88,89 and the energy
barrier of amide rotation for N-methylated peptides is slightly
lower than that,28,90 confirming our conclusion about cis–trans
conformations of the N-methylated peptides.
Scheme 1 Synthetic strategy used to prepare the proposed peptides.
tures were characterized by NMR and HRMS. The HPLC
chromatogram presents a single peak for each compound,
despite the different conformations observed by NMR. No sign
of epimerization of the peptides was observed.
Comparative structural study on the peptides designed for
optimal β-turn characteristics
The four structures in the first family of peptides are presented
Comparison between compounds 1a and 1b. Peptide 1a dis-
in Fig. 4. NMR techniques were employed to identify the played only the trans-isomer. After assigning each peak in
proline N-oxide peptides and to determine the conformational 1H NMR and 2D NMR, it seemed that this peptide assumed an
preferences and stabilities of these series of molecules. Com- extended or linear conformation; none of the NMR data sup-
plete NMR assignments of all peptides were performed ported a turn structure.
through 2D NMR techniques. These assignments are pre-
The N-oxide peptide 1b appeared as two conformers (major
sented in the ESI.‡ Three aspects were monitored: (a) the ratio and minor). Since introduction of the N-oxide was proven to be
of cis/trans isomerization, (b) deshielding of the amide protons stereo-selective,53,54 we ruled out the possibility of diastereo-
(NH) due to through space deshielding by the NO oxygen and mers. ROESY NMR data indicated that both conformers are
(c) potential NOE interactions characteristic of β-turns.
The cis/trans ratio was calculated from the integration of
trans with respect to the amide bonds (available in the ESI‡).
It should be noted that the percentage of minor confor-
unique proton signals (such as the N-methyl protons). A mations in 1b was quite small (15%), hence only a weak NOE
summary of these results is presented in Table 1. interaction was observed for this conformer. An analysis of
From the NMR data, no cis conformation was observed for these two conformations was performed from the NMR data
peptides 1a or 1b, while it was clear that at least two dominant and is presented in Fig. 5.
conformations were present for peptides 2a (47 : 53) and 2b
The 1H NMR data suggest that the first amide proton (i + 1)
(29 : 71). Introduction of the N-oxide does not lead to epimeri- of the major isomer of 1b prefers to engage with the N-oxide
zation as is demonstrated from the NMR spectra for com- moiety, thus forming the NO-γ-turn. This is evident from the
pounds 1b and 3b. Also, if epimerization of the same proton downfield shift of the (i + 1) amide proton with respect to 1a
had occurred, then the ratio should have moved closer to in the proton NMR spectra. This shift is most likely the result
50 : 50 and not further away as is the case for 2b (29 : 71). If of an intramolecular hydrogen bonding interaction between
epimerization of other α-protons had taken place, then more the amide proton and the N-oxide oxygen atom, causing
through space deshielding and suggests the presence of the
projected NO-γ-turn. The minor conformation exhibited a
Table 1 cis–trans Isomerizations of proline amide bonds at the
more extended structure. The variable temperature NMR data
C-termini of peptides 1 and 2 at room temperature. Data obtained from
indicated that the major isomer (NO-γ-turn) converts to a more
proton NMR integrations in DMSO-d6
extended conformation at higher temperatures (Fig. 6).
Peptide
Sequence
cis %
trans %
The fact that the major product of 1b converts to the minor
product upon heating from 293 K to 303 K confirms our con-
clusion that the splitting of these proton signals is due to a
conformational issue (cis–trans-conformation of N-methylated
peptides) and as a result of epimerization or diastereomeric
effects from non-stereospecific N-oxygen addition.
1a
1b
2a
2b
BzPGNF
0
0
47
29
100
100a
53
Bz(NO)PGNF
BzPMeGNF
Bz(NO)PMeGNF
71
a This sequence shows two conformations (see Fig. 5).
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Fig. 5 Downfield region of 1H NMR of designed peptides for optimal β-turn characteristics. The spectra were obtained at room temperature and
DMSO-d6 was used as the solvent. (Blue = trans-isomer, red = cis-isomer, M = major and m = minor). *Two conformations were observed. The
major conformation appeared to exhibit NO-γ-turn character (prominent (i + 1) NH and NO interaction). The minor conformation is possibly a more
extended peptide conformation (lack of NH and NO (i + 1) interaction).
Table 2 NOE correlations for trans-2b at room temperature in DMSO-d6,
[Bz(NO)PMeGNF]
Atom number
Correlated hydrogen atoms
18
21
22
26
15
11, 14, 15, 26
17
17,20
Fig. 6 Thermodynamic study of peptide 1b, [Bz(NO)PGNF]. Chemical
shifts are reported in ppm. The major (M) product converts to the minor
(m) product with increasing temperature (solvent: DMSO-d6).
de-shielded (about 9 ppm) due to the proposed interaction
with the N-oxide oxygen (Fig. 5) which causes a moderate
hydrogen bonding interaction. This result suggests that the
N-oxide moiety can be used as a kind of β-turn inducer leading
Comparison between compounds 2a and 2b. Peptide 2a to formation of the new NO-β-turn, as confirmed by corre-
includes N-methylated glycine as the first amino acid following lations in the ROESY NMR spectra (Table 2).
N-benzylproline. As expected, both cis- and trans-isomers exist
ROESY data for trans-2b are presented in Table 2. The corre-
and the isomerization ratio is 47 : 53 at room temperature (Fig. 5). lations of H-21 with H-11, H-14 and H-15 as well as the corre-
The isomers were verified from the ROESY data (available in lations of H-26 with H-20 and H-17 provide convincing proof
the ESI‡). It is clear that the trans-conformation should experience of the presence of a NO-β-turn in 2b. These correlations
a through space NOE interaction between the proline Hα and the suggest that the intramolecular hydrogen bonding interaction
methyl protons (N-Me). This was absent for the cis-isomer due to between NO and the backbone asparagine NH is able to
the methyl protons (N-Me) pointing in the opposite direction.
The complete NMR elucidation of the cis-isomer could not
induce folding of the peptide (Table 2).
Comparison between compounds 1b and 2b. When the first
be achieved due to a low signal to noise ratio and overlapping hydrogen bond donor in the position (i + 1) is blocked with a
of signals with the trans-isomer. Still, it is clear that the methyl group as in the case of 2b, the possibility of hydrogen
cis-isomer (29%) was not involved in any hydrogen bonding bonding between the amide group (i + 1) and the N-oxide
interaction (Fig. 5).
moiety is eliminated. Thus, the inherently strong NO-γ-turn
Peptide 2b is mainly present as a trans-isomer (71% popu- preference can be overcome through the introduction of an
lation); the backbone amide proton of asparagine (i + 2) was N-Me amino acid that forces the peptide to adopt the novel
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NO-β-turn “by design”. The shielding effect of the NO on the
corresponding amide NH protons (i + 2) can be seen in Fig. 5.
For 1b, the hydrogen bonding interaction between the amide
proton of glycine (i + 1) and the negative NO oxygen at the
N-terminal of proline provides a stronger deshielding inter-
action, compared to the amide proton (i + 2) of the more
distant asparagine and NO.
Table 3 cis/trans Isomers of the proline amide bonds at the C-termini
at room temperature. Data obtained from proton NMR integrations are
in DMSO-d6
Peptide
Sequence
cis %
trans %
3a
3b
4a
4b
BzPIVQ
0
0
40
77
100
100
60
Bz(NO)PIVQ
BzPMeIVQ
Bz(NO)PMeIVQ
23
Comparative structural study on peptides designed for
minimal β-turn characteristics
peptide bonds, as illustrated in Fig. 7. A summary of these
results is presented in Table 3.
The four structures in the second family of peptides are pre-
sented in Fig. 7. Complete NMR assignments for all peptides
were performed utilising 2D NMR techniques. These assign-
ments are presented in the ESI.‡ As expected, only trans-amide
conformations were observed for the non-methylated peptides
3a and 3b. The NMR spectra of 3b clearly demonstrate that epi-
merization does not occur upon introduction of the N-oxide.
At least two conformations were presented for peptides 4a
and 4b. As argued for 2a and 2b, these split NMR signals are
being ascribed to cis–trans-isomers of the N-methylated
Comparison between 3a and 3b. Comparisons of the NMR
chemical shifts for NH in the (i + 1) positions were made.
A 4 ppm difference is found between the isoleucine amide
proton signals (Fig. 8; in peptide 3a this NH appears at
7.85 ppm and in 3b at 12.05 ppm). This strong through-space
deshielding effect was a clear indication of a strong hydrogen
bonding interaction between the NH (i + 1) proton and the
negatively charged NO oxygen atom. These results are in
agreement with the report by O’Neil et al.,53,54 and mark the
presence of a NO-γ-turn.
In order to analyse the versatile nature of this NH to NO
hydrogen bonding interaction, N-methylation of the isoleucine
amide nitrogen (i + 1) was carried out. When peptide 1b is
compared with peptides 3a and 3b, it appears that the flexible
nature of glycine in the peptide backbone and the absence of a
chiral centre cause the hydrogen bonding interaction between
the N-oxide oxygen and the amide proton to be less prominent
in 1b despite the known turn-inducing element Pro-Gly.
Comparison between 4a and 4b. From Table 4, it is pro-
posed that N-methylation induces cis-isomers for both pep-
tides. The presence of the cis-isomer for 4a in a 40% ratio is
favoured by the polar NMR solvent (DMSO-d6). The trans-
isomer was recognized as the major conformer in the proton
spectrum (60%) at ambient temperature (Fig. 8). Both isomers
were observed in a ROESY NMR experiment (available in the
ESI‡).
Further investigations were conducted to observe if any
folding occurred in peptide 4a. No extraordinary ROESY
Fig. 7 Peptides designed for minimal formation of β-turn
characteristics.
Fig. 8 Downfield region of 1H NMR of designed peptides for minimal β-turn characteristics. The spectra were obtained at room temperature and
DMSO-d6 was used as the solvent.
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(i + 2) in 4b appeared at approximately 10 ppm (Fig. 8). This
result indicates a relatively close distance between the hydro-
gen bond donor (i + 2) and the acceptor (N-oxide) in the case
of 4b. An even stronger hydrogen bond between the closest
amide and NO in peptide 3b was evident from the large
deshielding effect on the (i + 1) amide proton (Fig. 8). The
trans-conformation of 3b and the steric hindrance imposed by
isoleucine and valine appeared to prevent any interaction
between the other amide protons and the NO. In contrast, the
N-oxide moiety in 4b revealed a 77% cis-conformation which
was able to overcome this difficulty of inducing hydrogen
bonding between NH (i + 2) and NO.
Table 4 NOE correlations of cis-4b, [Bz(NO)PMeIVQ], at room temp-
erature in DMSO-d6
Atom number
Correlated hydrogen atoms
20
24
26
28
12, 16, 17, 18
Thermal shift coefficient NMR investigation: the nature of the
hydrogen bonding interaction between the proline N-oxide
and the backbone NH protons
12
15
13
As discussed before, the presence of this hydrogen bond can
be detected by observing the chemical shifts of backbone
1
correlations were observed; it was therefore concluded that neither
of the backbones are involved in any stable turn or fold. The
NMR spectra of 4b also presented multiple isomers. The ratio
could not be accurately established from protons of the methyl
group of the N-Me group due to overlap; however, the glutamic
acid side chain amide protons revealed an unexpected 77 : 23
cis/trans-ratio in favour of the cis-isomer (Fig. 8). The isomer
assignments were confirmed when it was established that the
NH proton of valine (i + 2) at about 9.9 ppm experienced
through space deshielding due to the proposed intramolecular
hydrogen bonding with the N-oxide moiety (Fig. 8). Further evi-
dence for this elucidation was obtained from the ROESY
spectra of 4b (available in the ESI‡).
We initially chose isoleucine and valine residues because
they have been reported to prevent β-turns.55–58 The turn
observed in the peptide cis-4b appears to be a new NO-β-turn,
created by the N-oxide moiety in the presence of these resi-
dues. To the best of our knowledge this is the first example of
such a secondary structure involving this sequence. Develop-
ment of a NO-β-turn secondary structure in the presence of
bulky amino acids, such as isoleucine and valine, is therefore
a novel and unique achievement, and appears to only occur as
a result of the (NO)P in the peptide backbone.
The NO-β-turn character of cis-4b was confirmed with
several long-range correlations from ROESY NMR spectra, as
demonstrated in Table 4. Convincing evidence for the presence
of a NO-β-turn in 4b is: (a) correlations of H-20 with H-12,
H-17 and H-18, (b) a correlation between H-23 and H-12 and
(c) a relation between H-24 and H-12, (d) the existence of NOE
correlations between H-26 and H-15, H-17 and H-18, and (e) a
correlation between H-28 and H-12. These correlations support
the presence of intramolecular hydrogen bonding interactions
between the backbone asparagine NH and NO, which induced
folding of the peptide though the NO-β-turn.
amide protons in the H NMR spectra. Furthermore, an ana-
lysis of the thermal behaviour of hydrogen bonds in the pep-
tides by NMR is an elegant technique to study the exact nature
of this interaction.91–96 Normally the intramolecular hydrogen
bonding interaction between a hydrogen bond donor (amide
proton) and an acceptor (carbonyl oxygen) in a peptide is dis-
turbed at higher temperatures; the amide proton signals also
experience a downfield shift (less through space deshielding)
in the 1H NMR spectra in aprotic solvent systems.91–96 In
aprotic solvents such as DMSO-d6, when −Δδ/ΔT > 51 ppb K−1
,
the typical intramolecular peptide hydrogen bonding is absent
if the amide protons are solvent-exposed. When −Δδ/ΔT < 3
ppb K−1 the amide proton is shielded from the solvent, due to
hydrogen bonding with any carbonyl oxygen atom.91–96
The plotted −Δδ/ΔT graphs (available in the ESI‡) for pep-
tides and N-methylated peptides in DMSO-d6 confirmed that
no intramolecular hydrogen bonds exist for peptides 1a–4a.
The presence of hydrogen bonds between the NO moiety and
hydrogen-bonded NH in different N-oxide peptides 1b–4b was
also investigated with this method. These molecules present a
low −Δδ/ΔT value (−2.64 to 0 ppb K−1) for amide protons
involving hydrogen bonding with the N-oxide moiety. When
these amide protons are not involved in hydrogen bonding
interactions, the values are >4 (ppb K−1). The results for pep-
tides 1b–4b are presented in Fig. 9–12. The negative value for
−Δδ/ΔT (which renders positive slopes) called for a more in
depth analysis.
Langner and Zundel97 reported that in acid–base equilibria
(AH⋯B− ⇔ A−⋯HB), where ΔpKa is defined as [pKa B −
pKa AH],98,99 in systems with an increasing ΔpKa, the proton from
the hydrogen bond donor moves closer to the acceptor, which
causes deshielding of the mentioned proton. These results are
also consistent with an entropy (S) argument and shifting of
the proton in this system is due to increase in the amount of
the negative entropy.97
Comparison between 3b and 4b. The potential of the
N-oxide group to induce hydrogen bonding can be clearly seen
in 3b and 4b (Fig. 7). The amide proton of isoleucine (i + 1)
in 3b appeared at about 12 ppm, whereas the amide of valine
The hydrogen bond donor (AH) in this study was hydrogen-
bonded amides and the acceptor’s (B) role was played by the
N-oxide moiety. The proton chemical shift for several of these
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Fig. 11 The hydrogen bond investigation with thermal coefficient plots
for each amide of trans-3b, [Bz(NO)PIVQ]. (Solvent: DMSO-d6), (temp-
erature: 293–333 K): (1) HN-Ile (−Δδ/ΔT = −0.66 ppb K−1)4, (2) HN-Val
Fig. 9 The hydrogen bond investigation with thermal coefficient plots
for each amide protons of trans-1b, [BzP(NO)GNF]. (Solvent: DMSO-d6),
= −2.64 ppb K
−1),
(−Δδ/ΔT
= = )
5.98 ppb K−1), (3) HN-Gln (−Δδ/ΔT 5.04 ppb K−1
(temperature: 293–333 K): (1) HN-Gly (−Δδ/ΔT
(2) HN-Asn (−Δδ/ΔT = 4.56 ppb K−1), (3) HN-Phe (−Δδ/ΔT = 4.50 3 ppb K−1
)
(R2 > 0.959 for all graphs).
(R2 > 0.997 for all graphs).
Fig. 10 The hydrogen bond investigation with thermal coefficient plots
for each amide protons of trans-2b, [Bz(NO)PMeGNF]. (Solvent: DMSO-
d6), (temperature: 293–333 K): (1) HN-Asn (−Δδ/ΔT = −1.49 ppb K−1),
(2) HN-Phe (−Δδ/ΔT = 7.29 ppb K−1) (R2 > 0.951 for all graphs) (graphs
for cis-2b are available with the ESI‡).
Fig. 12 The hydrogen bond investigation with thermal coefficient plots
for each amide of cis-4b, [Bz(NO)PMeIVQ]. (Solvent: DMSO-d6), (temp-
erature: 293–333 K): (1) HN-Val (−Δδ/ΔT = 0 ppb K−1), (2) HN-Gln (−Δδ/
ΔT = 6.11 ppb K−1) (R2 > 0.999 for all graphs) (graphs for trans-4b are
available in the ESI‡).
amides appeared in a region that is known for acid functional
The slopes of these graphs for normal peptides are usually
groups and this gives strong evidence for the acidic character negative.91–96 The positive slope of this gradient (i + 1)
of such amides. The ΔpKa value for this system (amide and suggests that the proton transfer in this system was tempera-
N-oxide) is expected to be low. It is important to note that ΔpKa ture sensitive and the down field proton shift (≈10 ppm)
is directly related to temperature according to the Van’t Hoff suggests that the amide proton has probably started to transfer
and Helmholtz equations.
to the acceptor atom (N-oxide oxygen). There are two opposing
With this background in mind, the amide proton NMR factors playing a role in this case. First, the ΔpKa value is
shifts were measured at different temperatures in order to directly proportional to the temperature and this causes the
determine the hydrogen bond character between the amide amide proton (i + 1) to be transferred to the N-oxide oxygen
proton and the N-oxide oxygen atom. For trans-1b, [Bz(NO)- atom. Second, the glycine residue causes less steric hindrance
PGNF], the −Δδ/ΔT graphs are presented in Fig. 9. The hydro- and provides greater flexibility to the peptide at higher temp-
gen bonding interaction between the amide proton (i + 1) and eratures. This flexible nature makes transfer of the amide
the N-oxide oxygen renders a positive slope (gradient = 2.64). proton more difficult/less possible.
This result is typical of normal acid–base equilibrium
The non-hydrogen-bonded amides (i + 2 and i + 3) seem to
be slightly more shielded at higher temperatures with the
behaviour.97
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expected negative slopes (4.56 and 4.50). These values are
In summary, the results from the NMR spectroscopic
typical of peptide amide protons in the absence of hydrogen studies show that the NO modification leads to a strong hydro-
bonding interactions.91–96
gen bond with the closest backbone NH. Peptides 1b and 3b
The −Δδ/ΔT plots for both of the backbone amides of trans-2b, presented NO-γ-turns as a trans-isomer. N-methylation of the
[Bz(NO)PMeGNF], are presented in Fig. 10. The hydrogen first amide, as in peptides 2b and 4b, leads to novel
bonding interaction between the amide proton (i + 2) and the NO-β-turns. Utilization of N-methylated amino acids in the
N-oxide oxygen gave a positive slope (gradient = 1.49). This designed backbones leads to cis- and trans-isomerization.
value verifies that the hydrogen bond was not as strong as for Peptide 2b, with less sterically hindered residues, tends to
1b (i + 1). The observed gradient for the (i + 1) system in 1b is form a NO-β-turn through the trans-isomer (cis/trans ratio
higher (gradient = 2.64) than for the (i + 2) system in 2b (gradi- 29/71). In contrast, peptide 4b creates the NO-β-turn via the
ent = 1.49); the latter exhibits less temperature dependence, cis-isomer (cis/trans ratio 77/23), because the cis-isomer is
most possibly due to the more flexible nature of the larger less sterically hindered than the trans-isomer.
NO-β-turn structure.
An important difference between the (i + 2) data for 1b and
2b is that the sign of the slopes is indicative of substantial
Computational studies
After NMR spectroscopic characterization of these series of
molecules by NMR, we undertook a computational investi-
gation to gain additional understanding of the structure of
these novel kinds of turns. The free distance of the hydrogen
bonding interactions between the NH proton and the NO
oxygen for γ- and β-turns was calculated by density functional
theory [B3LYP/6-31+G(d)] using a DMSO solvation model.
Compounds 1b and 3b displayed NO-γ-turns (compound 1b
is shown in Fig. 13). The calculated distance between NH
(i + 1) and oxygen of NO is 1.76 Å. In the case of 3b, the corres-
ponding calculated distance was 1.71 Å.
The optimized structure for trans-2b is provided in Fig. 14,
indicating the intramolecular hydrogen bonding interactions
between NH (i + 2) and N-oxide oxygen that illustrates the
NO-β-turn.
The optimized geometry for cis-4b is presented in Fig. 15,
indicating a hydrogen bonding interaction between NH (i + 2)
and N-oxide oxygen. The relative short distance between the
hydrogen bond donor and the acceptor (2.35 Å) suggests a
tight turn in this molecule.
hydrogen bonding for 2b. Blocking of the (i + 1) amide proton
through N-methylation therefore induces stronger hydrogen
bonding interactions between the (i + 2) amide proton and the
N-oxide oxygen atom. The unusual positive slope of the
HN-Asn plot is related to an acid–base equilibrium between
the amide proton and the N-oxide moiety. For the next amide
(i + 3) of 2b, this slope was negative (−7.29 ppb K−1) suggesting
that this NH does not participate in a hydrogen bonding inter-
action with NO.
The −Δδ/ΔT plots for the backbone amides of trans-3b,
[Bz(NO)PIVQ], are presented in Fig. 11. The hydrogen bonding
interaction between the amide proton (i + 1) and the N-oxide
oxygen resulted in a positive slope (gradient = 0.66). This small
value combined with the deshielding experienced by this
proton (appeared at about 12 ppm) suggests that the proton is
being completely transferred from the donor to the acceptor.
In other words, it suggests hydrogen bonding in the absence
of acid–base equilibrium between the N-oxide moiety and the
amide proton. In comparison with 1b, the slope for the (i + 1)
system of 3b is more shallow. A potential reason for that is the
more rigid nature of peptide 3b, allowing for substantial trans-
fer of the amide proton to NO, even at higher temperatures. It
therefore becomes an entropy driven effect where the corres-
ponding system in 3b is more ordered.
The presence of sterically hindered amino acids normally
prevents the formation of hydrogen bonding interactions for
weak acceptors such as carbonyl groups. However, it can be
concluded that for a strong acceptor like N-oxide oxygen when
it is combined with N-methylation of the (i + 1) amide proton,
the existence of bulky side chains in amino acids appears to
The sign and values of the slopes of trans-3b for (i + 2) and
(i + 3) clearly show that these backbone amide protons are not
involved in hydrogen bonding interactions.
The case of 4b turned out to be unusual in terms of the be-
haviour observed so far. The proton NMR spectrum for trans-
4b exhibits no unusual deshielding of any of the amide
protons (Fig. 8). On the other hand, the cis-isomer displays an
amide proton at about 10 ppm. The −Δδ/ΔT plots for cis-4b,
[Bz(NO)PMeIVQ], illustrate a hydrogen bonding interaction
between the NH (i + 2) and the N-oxide oxygen, but it is not as
strong as that observed for the corresponding system in 3b
(Fig. 12). The slope of this system is zero, indicating a strong
hydrogen bond, but in the absence of acid–base equilibrium.
The sign and value of the slope of cis-4b for (i + 3) indicates
that the protons do not experience hydrogen bonding inter-
actions, similar to the non-hydrogen bonded amides presented
before.
Fig. 13 The NO-γ-turn and the presence of the intramolecular hydro-
gen bonding interaction in the optimized structure of 1b. (The 3D struc-
tures of 1b and 3b are available in the ESI‡.)
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(Germany). Analytical reversed-phase HPLC was performed on
a C18 column (4.6 × 150 mm, 5 μm, YMC Triart, UK) with a
LC-MS 2020A system (Shimadzu, Kyoto, Japan). Solvents A and
B were 0.01% (v/v) formic acid in Milli-Q water and CH3CN,
respectively. Elution was achieved with linear 0–90% gradients
of solvent B to A over 15 minutes at 1 ml min−1 flow rate, with
UV detection at 200 nm. Preparative HPLC was performed with
a C8 column (150 × 21.2 mm, 10 μm, Hichrom) attached to a
Shimadzu LC-8A instrument. Solvents A and B were 0.1%
formic acid (v/v) in water and MeOH, respectively, and a linear
gradient of solvent B to A over 40 minutes at 15 mL min−1 flow
rate was applied. The UV detection wavelength was set at
200 nm. Purified fractions (>95%) by HPLC were pooled and
lyophilized on a Virtis SP scientific Sentry 2.0. Purified
peptides and conjugates were characterized by HRMS, on a
BrukermicrOTOF-Q II instrument operating at ambient temp-
erature with 1 ppm sample. DMSO-d6 was used as a solvent for
Fig. 14 The NO-β-turn and the presence of intramolecular hydrogen
bonding in the optimized structure of trans-2b. (The 3D structure of
trans-2b is available in the ESI‡.)
1
sample preparation of NMR. H NMR, 13C NMR, COSY, HSQC,
HMBC and ROESY experiments were performed at ambient
temperature on an AVANCE III 600 MHz NMR (Bruker,
Germany). The chemical shifts are expressed in ppm downfield
from TMS as the internal standard. To achieve better resolu-
tion in the NMR spectra, the water peak was suppressed in
necessary cases.
Fig. 15 The NO-β-turn intramolecular interaction presented in the
optimized structure of cis-4b. (The 3D structure of cis-4b is available in
the ESI‡.)
General procedure for the synthesis of the tetrapeptides
Peptides were manually synthesized on a 0.1 mmol scale on
CTC resin (0.6 mmol g−1). The resin was activated using 10%
thionylchloride (v/v) in dry DCM, after which incorporation of
the C-terminal amino acids was performed with a minimum
of four equivalents of Fmoc-amino acid and six equivalents of
DIEA in dry DCM for 3 hours. Elongation of the peptide chains
was achieved following standard Fmoc protocols. The coupling
conditions were 5-fold molar excess of Fmoc-amino acid and
HBTU and double molar excess of DIEA in DMF. Deprotection
was achieved with 20% piperidine in DMF (3 × 10 min). Coup-
ling of N-benzylproline was achieved employing the same
method. After chain assembly, total deprotection and cleavage
were carried out with TFA–DCM (40 : 60) for one hour and the
peptides were precipitated by adding chilled diethyl ether.
After evaporation to dryness, the solid forms of the crude pep-
tides were obtained. The crude peptides were purified via semi
preparative HPLC and characterized by NMR and HRMS.
induce a stronger hydrogen bonding interaction for the (i + 2)
system. The possibility of more prominent cis-conformations is
also possible.
Conclusion
A series of novel proline N-oxide peptides including less steri-
cally hindered residues as well as sterically hindered amino
acids were synthesised. NMR studies appear to reveal that a
proline N-oxide residue enforces hydrogen bonding inter-
actions with neighbouring backbone amide protons. Hydrogen
bonding with the (i + 1) amide bond leads to a quasi γ-turn
which we named as a NO-γ-turn. When the (i + 1) amino acid
is N-methylated, the peptide experience cis- and trans-isomeri-
zation around this bond and instead forms a novel quasi
β-turn, correspondingly named as a NO-β-turn. Computational
results support the proposed explanation of the experimental
results. Overall, N-oxide and N-methylated residue modifi-
cations allow the peptide backbone to assume predictable
folded conformations, an outcome that may play an important
role in de novo modulation of peptide secondary structures.
General procedure for the preparation of N-oxide tetrapeptides
Crude peptide (0.1 mmol) and K2CO3 (2.5 equiv.) were dis-
solved in dry DCM (10 ml). M-CPBA (1.6 equiv.) was added to a
cooled solution mixture (−72 °C). The reaction was stirred for
four hours under the same conditions and warmed up to
room temperature over two hours. Finally, the solvent was
evaporated and the product was purified by preparative HPLC
and characterized by NMR and HRMS.
Experimental section
Fmoc-protected amino acids, CTC resin and HBTU were pur-
chased from GL-Biochem (Shanghai, China). HPLC-grade
Molecular modelling method
CH3CN, MeOH, peptide synthesis-grade DMF, CH2Cl2, DIEA, The conformational models of four cases that demon-
TFA and all other reagents were purchased from Sigma-Aldrich strate NO-γ-turns and NO-β-turns were investigated using
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Gaussian09.100 These compounds were trans-1b-γ-turn, trans-
3b-γ-turn, trans-2b-β-turn and cis-4b-β-turn. The structures
were first optimized (PM6)101 with constrained bond lengths
of about 3 Å between the corresponding amide proton and the
oxygen of NO. After that, the optimized structures were again
optimized without any constraints, using B3LYP102,103 with the
6-31+G(d) basis set in DMSO (ε = 46.826) as an intrinsic
solvent using the polarizable continuum model (PCM)104 The
second-derivative analytical vibration frequency of each opti-
mized structure was calculated at the same theoretical level to
find the stationary point without imaginary frequencies.
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Acknowledgements
We thank the National Research Foundation, South Africa and
UKZN for financial support. We are also grateful for the
helpful support of CHPC (http://www.chpc.ac.za) for their com-
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