Effect of solvent on the cis-trans conformational equilibrium of a proline imide bond of short model peptides in solution

Article abstract of DOI:10.1016/S1386-1425(00)00493-5

Proton NMR spectra of proline-containing short peptides with N-terminal sequences of N-acetyl-prolyl- (Ac-Pro-) N-tert-butoxycarbonyl-phenylalanyl-prolyl- (Boc-Phe-Pro-) and N-tert-butoxycarbonyl-leucyl-prolyl- (Boc-Leu-Pro-) were measured in mixed solvents of hexadeuterodimethylsulfoxide and deuterochloroform (CDCl₃). Population ratios of cis and trans conformers with respect to the proline imide bond and chemical shifts of NH protons were obtained as a function of a CDCl₃ fraction of solvent. With increasing fraction of CDCl₃, the trans percentages of the Ac-Pro- imide bonds increased. On the other hand, those of Boc-Phe-Pro- decreased, and those of Boc-Leu-Pro- exhibited middle tendency. From the solvent-dependent variation of the chemical shifts of the NH protons, intramolecular hydrogen bonds that stabilize the trans form of Ac-Pro- and the cis form of Boc-Phe-Pro- were discussed. For the Ac-Pro- peptides, only the trans forms are found to the compatible with 7-, 10-, and 13-membered hydrogen-bonded rings that would be similar to the ordinary secondary structures, γ- and β-turns and α-helix, respectively. For the cis form of Boc-Phe-Pro-R (R = O-methyl or glycyl-O-ethyl), the hydrogen-bonded structure is found to be similar to the type-VIa β-turn. On the other hand, for Boc-Phe-Pro-Pro-Leu-Gly-NH₂, it has been suggested that two different hydrogen bonds, which are different from that of the type-VIa β-turn, support each other and cooperatively stabilize the cis form.

Full text of DOI:10.1016/S1386-1425(00)00493-5

Spectrochimica Acta Part A 57 (2001) 1305–1316
www.elsevier.nl/locate/saa
Effect of solvent on the cis–trans conformational
equilibrium of a proline imide bond of short model peptides
in solution
Masae Sugawara, Kenji Tonan, Shun-ichi Ikawa *
Di6ision of Chemistry, Graduate School of Science, Hokkaido Uni6ersity, Sapporo 060-0810, Japan
Received 1 November 2000; accepted 10 December 2000
Abstract
Proton NMR spectra of proline-containing short peptides with N-terminal sequences of N-acetyl-prolyl- (Ac-Pro-)
N-tert-butoxycarbonyl-phenylalanyl-prolyl- (Boc-Phe-Pro-) and N-tert-butoxycarbonyl-leucyl-prolyl- (Boc-Leu-Pro-)
were measured in mixed solvents of hexadeuterodimethylsulfoxide and deuterochloroform (CDCl3). Population ratios
of cis and trans conformers with respect to the proline imide bond and chemical shifts of NH protons were obtained
as a function of a CDCl3 fraction of solvent. With increasing fraction of CDCl3, the trans percentages of the Ac-Pro-
imide bonds increased. On the other hand, those of Boc-Phe-Pro- decreased, and those of Boc-Leu-Pro- exhibited
middle tendency. From the solvent-dependent variation of the chemical shifts of the NH protons, intramolecular
hydrogen bonds that stabilize the trans form of Ac-Pro- and the cis form of Boc-Phe-Pro- were discussed. For the
Ac-Pro- peptides, only the trans forms are found to the compatible with 7-, 10-, and 13-membered hydrogen-bonded
rings that would be similar to the ordinary secondary structures, g- and b-turns and a-helix, respectively. For the cis
form of Boc-Phe-Pro-R (R=O-methyl or glycyl-O-ethyl), the hydrogen-bonded structure is found to be similar to the
type-VIa b-turn. On the other hand, for Boc-Phe-Pro-Pro-Leu-Gly-NH2, it has been suggested that two different
hydrogen bonds, which are different from that of the type-VIa b-turn, support each other and cooperatively stabilize
the cis form. © 2001 Elsevier Science B.V. All rights reserved.
Keywords: Peptides; Proline; Cis–trans; Intramolecular hydrogen bond; NMR
1. Introduction
The peptide bond preceding a prolyl residue,
* Corresponding author. Present address: Biomolecular,
NMR Laboratory, Biotechnology Research Institute, National
Research Council Canada, 6100 Royalmount Avenue, Mon-
treal, Quebec, Canada, H4P 2R2. Tel.: +81-11-7062707; fax:
+81-11-7064924.
which is called a proline imide bond, is unique
among other peptide bonds in proteins. The pep-
tide bonds that do not involve Pro residues take
predominantly the trans form and scarcely occur
in the cis form in the folded structure of proteins,
E-mail address: sikawa@sci.hokudai.ac.jp (S.-i. Ikawa).
1386-1425/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S1386-1425(00)00493-5

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M. Sugawara et al. / Spectrochimica Acta Part A 57 (2001) 1305–1316
where the probability of occurrence of the cis
form is less than 0.05% [1,2]. On the contrary,
about 5% of the proline imide bond have been
found to occur in the cis form in the folded
proteins. For unstructured peptides, about 30% of
the imide bonds adopt the cis form [1,3] and, for
short peptides in solution, the amount of the cis
form can be even comparable to that of the trans
form, depending on amino acid sequences and
solvent conditions [4–6]. This unusual distribu-
tion of the proline imide bonds can be explained
by the unique structure of proline. The pyrro-
lidine ring, which covalently connects the side
chain to the backbone nitrogen atom of proline,
yields steric hindrance which is unfavorable to the
trans form and, consequently, reduces signifi-
cantly the energy difference between the trans and
cis forms [1,2]. As a result, the cis-trans equi-
librium of the proline imide bond becomes sensi-
tive to environmental conditions, being different
form that of other peptide bonds.
The above-mentioned difference in the cis/trans
ratio between the folded and unfolded structure of
proteins indicates that some change in environ-
mental conditions, which occurs on the folding
pathway, must shift the conformational equi-
librium to the trans form. Solvent condition
should be one of the important factors that influ-
ence conformation of local segments of he peptide
chains. In order to study mechanism of the sol-
vent effect, it is useful to examine short peptides
because they are free from various long-range
interactions, which would give rise to complicated
effect on the conformation of polypeptide chains.
It is known that the fraction of the trans form
of short model peptides is higher in less polar or
non-polar solvents than in polar solvents [4,5]. In
the recent IR and NMR study making use of
mixed solvents of hexadeuterodimethylsulfoxide
(DMSO-d6) [6], we have found that both in-
tramolecular hydrogen bonding and cis-to-trans
isomerization are promoted by increased in frac-
tion of CDCl3 in the mixed solvents. It was con-
cluded that the cis-to-trans isomerization is
facilitated by formation of 10 membered or 13-
membered intramolecular hydrogen-bonded rings
that include the carbonyl group precedent to the
prolyl residue.
It should be noted, however, that the above-
mentioned results were only for peptides with an
N terminus of an N-acetyl-Pro- (Ac-Pro-) or N-
tert-butoxycarbonyl-Pro- (Boc-Pro-) type. If an-
other residue exists before the prolyl residue,
situation could be changed. For imide bonds in a
sequence of -Xaa-Pro-, it has been found that the
occurrence of the cis form depends on the type of
the residue Xaa [1,7]. In addition, it has been
suggested that the cis form could be stabilized by
interaction between the pyrrolidine ring and the
aromatic side chain of the preceding residue or
both the preceding and following residues [8,9].
However, effect of solvent on the cis–trans
equilibrium of -Xaa-Pro- has not been reported so
far to our knowledge. In the present study, we
have performed NMR measurements on two se-
ries of peptides; one includes peptides with an
N-terminus of Ac-Pro- and the other includes
Boc-Xaa-Pro- where Xaa is Phe or Leu residue.
We have used mixtures of DMSO-d6 and CDCl3
at various mixing ratios as solvents for the NMR
measurements. These mixed solvents are particu-
larly useful for characterization of the cis–trans
equilibrium and the intramolecular hydrogen
bonding [6].
2. Experiments
The model peptides used for measurements are
shown in Fig. 1, where amino acid residues are
indicated by the usual one-letter abbreviations.
These are classified into two types. The peptides
in one group have a proline as the first residue
with an acetyl group at the N-terminus. In the
other group, peptides have a proline as the second
residue and a Boc group as an N-terminal sub-
stituent. These compounds were synthesized by
the ordinary liquid-phase method [10] and impuri-
ties were found to be negligible by infrared and
NMR spectra. Starting compounds for the syn-
theses were commercially available Boc-Xaa-OH
(Xaa=Pro, Phe, Leu, Gly), H-Pro-OMe (Me=
methyl), H-Leu-OMe, H-Gly-OEt (Et=ethyl),
and H-Pro-Leu-Gly-NH2. Condensation reagent
1- ethyl - 3 - (3 - dimethylaminopropyl)carbodiimide
and anti-racemization reagent 1-hydeoxybenzotri-

M. Sugawara et al. / Spectrochimica Acta Part A 57 (2001) 1305–1316
1307
tion of sample solutions.
azole from Watanabe Chemical Industries, Ltd.
were used. Deprotection of Boc-peptides was per-
formed with 4 M HCl in 1,4-dioxane. Solvents
used for spectroscopic measurements were CDCl3
and DMSO-d6 from CEA (France) and dried over
molecular sieves 4A for 48 h before the prepara-
Proton NMR spectra of 1.25 mM peptide solu-
tions in DMSO-d6/CDCl3 mixed solvents were
measured with a JEOL EX400 Fourier-transform
NMR spectrometer at 298 K. Chemical shifts
were standardized by a residual proton of DMSO
Fig. 1. Peptides used in the present study. Ac: acetyl, Boc: tert-butoxycarbonyl, P: Pro (prolyl), G: Gly (glycyl), L: Leu (leucyl), F:
Phe (phenylalanyl), Me: methyl, Et: ethyl.

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M. Sugawara et al. / Spectrochimica Acta Part A 57 (2001) 1305–1316
for solutions in DMSO-containing solvents and
by a residual CHCl3 proton for solutions in pure
CDCl3. The observed NMR signals were assigned
with COSY (two-dimensional correlated spec-
troscopy) and NOESY (nuclear Overhauser en-
hancement-exchange spectroscopy) spectra. These
two-dimensional spectra were measured with a
JEOL ALPHA600 Fourier-transform NMR spec-
trometer at 298 K. Each data matrix size collected
with 24–64 scans was 512×1024 and the spec-
trum was Fourier-transformed in both dimensions
using the squared sine-bell window function.
Infrared spectra were measured with a Perkin-
Elmer System-2000 Fourier-transform spectrome-
ter at
2 a room
cm−1 resolution and at
temperature. A cell with CaF2 windows and a
sample path-length of 6 mm was used. Sample
concentrations were 1.25 mM in CDCl3.
3. Results
Two different signals, assigned to the cis and
trans forms, were observed for each proton of the
peptides. Among these signals, clearly separated
pair for a Pro-a proton or a Lue-NH or Phe-NH
proton were integrated to obtain the population
ratios of the cis and trans forms. Fig. 2 shows the
resulting percentages of the trans form plotted
against the CDCl3 fraction of the mixed solvents.
For the Ac-Pro-Leu- compounds shown in Fig.
2a, the ratios of the trans forms increase with
increasing fraction of CDCl3, while the cis–trans
ratio of Ac-Pro-OMe is approximately indepen-
dent of the solvent mixing ratio. This is consistent
with the previous results for different Ac-Pro-Gly-
and Boc-Pro-Gly- compounds [6]. The increase of
the trans population has been found to be parallel
to increase in intramolecular hydrogen bonding of
the peptides. We have concluded in the previous
paper that formation of the 10-membered or 13-
membered intramolecular hydrogen-bonded ring
that includes the carbonyl group precedent to the
prolyl residue facilitated the cis–trans isomeriza-
tion. These hydrogen-bonded rings would corre-
spond to those of the usual secondary structures,
the b-turn and a-helix, respectively.
Fig. 2. The trans percentages of proline imide bonds plotted
against CDCl3 fractions of the DMSO-d6-CDCl3 mixed sol-
vents for (a) Ac-Pro- compounds, (b) Boc-Leu-Pro-Leu- com-
pounds, and (c) Boc-Phe-Pro- compounds.
For Boc-Phe-Pro-Leu- compounds, on the con-
trary, relative ratio of the trans forms decrease
with increasing CDCl3 fraction as shown in Fig.
2c, and those for the Boc-Leu-Pro-Leu- com-
pounds exhibit middle tendency as shown in Fig.
2b. The contrast between the Ac-Pro-Leu- and
Boc-Phe-Pro-Leu- compounds are clearly shown
in Fig. 3, where solvent dependent changes in the
trans percentages are compared among related
compounds of the three types. It is intriguing how
the cis–trans isomerization is related to the in-
tramolecular hydrogen bonding for the Boc-Phe-
Pro-Leu- compounds. A simple logical extension
of the previous results [6] might suggest that the
intramolecular hydrogen bonds that stabilize the
trans form could be disturbed as the CDCl3 frac-
tion increases. Another possibility is existence of

M. Sugawara et al. / Spectrochimica Acta Part A 57 (2001) 1305–1316
1309
some hydrogen-bonded structure that stabilizes
the cis form, a candidate of which would be the
type-VIa b-turn-like structure [7]. To elucidate
hydrogen-bonded structures, infrared spectra and
NMR chemical shifts are useful.
Figs. 4 and 5 show observed infrared NH
stretching absorption of Ac-Pro-and Boc-Phe-
Pro-compounds. The broad bands with peak
wave numbers below 3400 cm−1 are assigned to
the intramolecularly hydrogen-bonded NH
groups while bands at higher than 3400 cm−1 are
assigned to hydrogen-bond-free NH groups
[6,10,11]. The broad peaks at around 3350 cm−1
are assigned to the b-turn like structure with a
10-membered hydrogen-bonded ring, while the
peaks or shoulders at around 3300 cm−1 are
assigned to a 7-membered hydrogen-bonded ring.
For Ac-Pro-Leu-Gly-NH2 and Boc-Phe-Pro-Leu-
Gly-NH2, the bulge at around 3300 cm−1 can be
assigned to a 13-membered hydrogen-bonded
ring, which resembles one turn of the a-helix [10],
instead of the 7-memebered hydrogen-bonded
Fig. 4. Infrared absorption in the NH stretching region of 1.25
mM solutions of Ac-Pro- compounds in CDCl3.
ring. This is supported by the chemical shift data
that will be discussed later.
It should be noted that corresponding com-
pounds of the Ac-Pro- and Boc-Phe-Pro- groups
exhibit similar infrared NH stretching absorption
Fig. 3. Comparison of solvent-dependent changes of the trans
percentages among Ac-Pro-, Boc-Phe-Pro- and Boc-Leu-Pro-
compounds with (a) -Leu-Gly-OEt and (b) -Leu-Gly-NH2,
respectively, at the C-terminus.
Fig. 5. Infrared absorption in the NH streching region of 1.25
mM solutions of Boc-Phe-Pro-compounds in CDCl3.

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M. Sugawara et al. / Spectrochimica Acta Part A 57 (2001) 1305–1316
the cis form [designated by Leu-NH(c)] of Ac-
Pro-Leu-OMe because its chemical shift shows a
very similar variation. On the contrary, Leu-
NH(t) shows distinctly smaller chemical shift vari-
ation. This proton is able to form an
intramolecular hydrogen bond with the acetyl car-
bonyl group in the trans configuration. The Leu-
NH(c) proton, on the other hand, can not from
sterically any intramolecular hydrogen bond.
Then the infrared hydrogen-bonded band of Ac-
Pro-Leu-OMe seen in Fig. 4 is assigned to the
trans form, and moreover, the small chemical shift
variation of Leu-NH(t) can be assumed to be
caused by formation of the intramolecular hydro-
gen bond in the CDCl3-rich solvent. That is to
say, the decay of the hydrogen bonds between
DMSO and Leu-NH(t) may be compensated by
the intramolecular hydrogen bonds between
DMSO and Leu-NH(t) may be compensated by
the intramolecular hydrogen bonding, and the
variation of the chemical shift is significantly re-
duced. Thus, the chemical shift variations in the
DMSO–CDCl3 mixed solvents are useful to char-
acterize the NH protons by their ability to form
intramolecular hydrogen bonds.
Fig. 6. Chemical shifts plotted against CDCl3 fractions in the
DMSO-CDCl3 mixed solvents for NH-protons of Ac-Pro-Leu-
OMe and Ac-Gly-OEt.
to each other except for Ac-Pro-OMe and Boc-
Phe-Pro-OMe. Although Boc-Phe-Pro-OMe has a
Phe-NH proton, it proves to be free from in-
tramolecular hydrogen bonds like Ac-Pro-OMe
that has no NH proton. Then the degrees of the
intramolecular hydrogen bonding of the Boc-Phe-
Pro- compounds are similar to the corresponding
Ac-Pro- compounds. Infrared spectra, however,
can not distinguish the cis and trans forms, and
therefore, can not provide information about cor-
relation between the hydrogen-bonded structures
and the cis–trans ratios. To obtain further insight,
we need to know to what degree each of the NH
protons of the peptides takes part in the in-
tramolecular hydrogen bonding. For this purpose,
the NMR chemical shifts are useful [6].
Fig. 7 shows chemical shifts of four different
types of NH protons of Boc-Phe-Leu-OMe plot-
ted against the CDCl3 percentage of the DMSO-
CDCl3 mixed solvents. It is found that both
Phe-NH(t) and Phe-NH(c) show large variation of
about the same magnitude. This is reasonable
because the Phe-NH proton has no chance to
Fig. 6 shows an example of how the chemical
shifts of NH protons vary with the CDCl3 frac-
tion in the DMSO–CDCl3 mixed solvents. The
large chemical shift variation for the Gly-NH
proton of Ac-Gly-OEt is interpreted as follows.
Since DMSO is a strong hydrogen-bond acceptor,
the Gly-NH proton is considered to be hydrogen-
bonded to DMSO in the DMSO-rich solvents.
These hydrogen bonds will decrease as the CDCl3
fraction increases and completely disappear in
100% CDCl3 solvent, which does not act as a
hydrogen-bond acceptor but a very weak hydro-
gen-bond donor. As a result, signals of the Gly-
NH proton show a large high-field shift as the
CDCl3 fraction increases from 0 to 100%. This
will be also the case for a Leucyl amide proton of
Fig. 7. Chemical shifts plotted against CDCl3 fractions in the
DMSO-CDCl3 mixed solvents for NH-Protons of Boc-Phe-
Pro-Leu-OMe.

M. Sugawara et al. / Spectrochimica Acta Part A 57 (2001) 1305–1316
1311
make any intramolecular hydrogen bond both in
the trans and cis configurations. On the other
hand, variations for Leu-NH(t) and Leu-NH(c)
are significantly smaller, and moreover, Leu-
NH(c) shows distinctly smaller variation than
Leu-NH(t). These facts indicate that the Leu-NH
proton takes part in some intramolecular hydro-
gen bonds both in the trans and cis forms, and
intriguingly the hydrogen-bond of the cis form
could be more stable than that of the trans form.
The hydrogen-bonded structure of the trans con-
former is thought to be the 7-membered hydro-
gen-bonded ring formed by the Leu-NH(t) and
the Phe-carbonyl group. This type of hydrogen-
bonded structure, is, however, sterically incom-
patible with the cis configuration. Then the
hydrogen bond partner of the Leu-NH(c) is re-
stricted to the Boc-carbonyl group, and they form
10-membered hydrogen-bonded ring. This struc-
ture, being compatible with the cis configuration,
has the same hydrogen-bonded pattern as that of
the type-VIa b-turn.
It proved that the values of the NH protons for
the cis forms of Ac-Pro-Leu-compounds are
closed to 1. This is consistent with the steric
incompatibility of intramolecular hydrogen bond-
ing with the cis configuration for these com-
pounds, as mentioned before for an example of
Ac-Pro-Leu-OMe. For Ac-Pro-Leu-Gly-NH2, Dl
values for the cis form were not obtained because
the cis form completely disappears in the pure
CDCl3 solvent as shown in Fig. 2a. On the other
hand, the Dl/DlRef, values of the NH protons for
the trans form of Ac-Pro-Leu-compounds are dis-
tinctly smaller than unity. This fact indicates that
these protons are involved in some intramolecular
hydrogen bonds. The degrees of the involvement
in hydrogen bonding for different NH protons
could be compared by using magnitudes of the
Dl/DlRef values; smaller the values the larger the
involvement.
For Ac-Pro-Leu-Gly-OEt, both Leu-NH(t) and
Gly-NH(t) are involved in the intramolecular hy-
drogen bonding. The former is thought to make
the same type of hydrogen-bonded structure, the
7-membered ring, as that of Ac-Pro-Leu-OMe.
On the other hand, the hydrogen-bonded struc-
ture for the Gly-NH(t) is considered to be a
10-membered ring with the acetyl carbonyl group.
Another possibility of Gly-NH(t) for forming 7-
membered ring with prolyl carbonyl group can be
neglected, because the 7-membered ring that does
not cross a prolyl residue is not stable enough [6].
The fact that the Dl/DlRef values of Gly-NH(t) is
smaller than that of Leu-NH(t) suggests that the
7-membered ring might be more stable than the
10-membered ring. However, the difference in the
Dl/DlRef values seems not large enough to discuss
conclusively.
4. Discussion
The observed chemical shifts of all the com-
pounds studied are listed in Table 1. The first five
compounds have no or little possibility of in-
tramolecular hydrogen bonding in the whole
range of the mixed solvents. Therefore, their
chemical shift variations, Dl=l(DMSO)−
l(CDCl3), are considered to be solely due to
change in the intermolecular DMSO-peptide hy-
drogen bonds. Consequently, these values can be
adopted as standards for estimating the degree to
which each of the amide protons of peptides is
involved in the intramolecular hydrogen bonding
in pure CDCl3 solvent. Since the chemical shift
variations may more or less depend on structural
configuration close to the NH group concerned,
in addition to the intramolecular hydrogen bond-
ing, we have adopted several standards for differ-
ent NH protons. The ratio of the chemical shift
variation of each amide proton of the peptides to
that of a suitable standard, Dl/DlRef, is indicated
in the fifth column of Table 1.
Three amide protons of Ac-Pro-Leu-Gly-NH2,
Leu-NH(t), Gly-NH(t) and anti-NH(t), show con-
siderably smaller Dl/DlRef values than unity, and
are thought to significantly take part in the in-
tramolecular hydrogen bonding. Among others
the anti-NH(t) is most heavily involved in the
hydrogen bonding. According to the previous
study [10], it forms mainly a 13-membered hydro-
gen-bonded ring with the acetyl-carbonyl group,
and to a less extent a 10-membered ring with the
prolyl carbonyl group. The Leu-NH(t) and Gly-

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M. Sugawara et al. / Spectrochimica Acta Part A 57 (2001) 1305–1316
Table 1
Chemical shifts of the amide protons of peptidesa
Compound proton
l (DMSO)
l (CDCl3)
Dl
Dl/Dl (Ref.)
Ac-Leu-OEt
Leu-NH
8.17
8.25
7.92
8.06
5.78
6.08
6.02
6.22
2.39
2.17
1.90
1.84
(1)
(2)
(3)
(4)
Ac-Gly-OEt
Gly-NH
Ac-Leu-NHMe
Leu-NH
Aac-Gly-NHMe
Gly-NH
Ac-NH2
Anti-NH
Syn-NH
7.25
6.97
5.38
5.24
1.87
1.73
(5)
(6)
Ac-Pro-Leu-OMe
Leu-NH (t)
Leu-HN (c)
8.15
8.43
7.33
6.17
0.82
2.26
0.34 (1)
0.95 (1)
Ac-Pro-Leu-Gly-OEt
Leu-NH (t)
Leu-NH (c)
Gly-NH (t)
Gly-NH (c)
7.89
8.21
8.13
8.37
7.11
6.30
6.82
6.35
0.78
1.91
1.31
2.02
0.41 (3)
1.01 (3)
0.60 (2)
0.93 (2)
Ac-Pro-Leu-Gly-NH2
Leu-NH (t)
Leu-NH (c)
Gly NH (t)
8.02
8.28
7.92
8.09
7.08
7.19
7.08
7.06
7.03
–
7.07
–
6.63
–
5.25
–
0.99
–
0.85
–
0.45
–
1.83
–
0.52 (3)
0.46 (4)
0.24 (5)
1.06 (6)
Gly-NH (c)
Anti-NH (t)
Anti-NH (c)
Syn-NH (t)
Syn-NH (c)
Boc-Phe-Pro-Leu-OMe
Phe-NH (t)
Phe-NH (c)
Leu-NH (t)
Leu-NH (c)
7.02
6.86
8.18
8.44
5.202
4.97
7.07
7.96
1.82
1.89
1.11
0.48
0.96 (3)
0.99 (3)
0.46 (1)
0.20 (1)
Boc-Phe-Pro-Leu-Gly-Oet
Phe-NH (t)
Phe-NH (c)
Leu-NH (t)
Leu-NH (c)
7.05
6.82
7.90
8.17
8.17
8.30
5.17
5.11
6.54
7.58
6.87
7.05
1.88
1.71
1.36
0.59
1.30
1.25
0.99 (3)
0.90 (3)
0.72 (3)
0.31 (3)
0.60 (2)
0.58 (2)
Gly-NH (t)
Gly-NH (c)
Boc-Phe-Pro-Leu-Gly-NH2
Phe-NH (t)
Phe-NH (c)
Leu-NH (t)
Leu-NH (c)
7.14
6.94
8.13
8.44
8.06
8.01
5.00
5.23
6.63
6.89
7.29
7.51
2.14
1.71
1.50
1.55
0.77
0.50
1.13 (3)
0.90 (3)
0.79 (3)
0.82 (3)
0.42 (4)
0.27 (4)
Gly-NH (t)
Gly-NH (c)

M. Sugawara et al. / Spectrochimica Acta Part A 57 (2001) 1305–1316
1313
Table 1 (Continued)
Compound proton
l (DMSO)
l (CDCl3)
Dl
Dl/Dl (Ref.)
Anti-NH (t)
Anti-NH (c)
Syn-NH (t)
Syn-NH (c)
7.21
7.20
7.18
7.16
6.59
6.55
5.21
5.21
0.62
0.65
1.97
1.95
0.33 (5)
0.35 (5)
1.14 (6)
1.13 (6)
Boc-Leu1-Pro-Leu2-Ome
Leu1-NH (t)
6.88
6.73
8.13
8.40
5.12
5.12
7.21
8.14
1.76
1.61
0.92
0.26
0.93 (3)
0.85 (3)
0.38 (1)
0.11 (1)
Leu1-NH (c)
Leu2-NH (t)
Leu2-NH (c)
Boc-Leu1-Pro-Leu2-Gly-Oet
Leu1-NH (t)
6.92
6.50
7.84
–
8.16
–
5.03
4.99
7.01
7.45
6.67
–
1.89
1.51
0.83
–
1.49
–
0.99 (3)
0.79 (3)
0.44 (3)
Leu1-NH (c)
Leu2-NH (t)
Leu2-NH (c)
Gly-NH (t)
Gly-NH (c)
0.69 (2)
Boc-Leu1-Pro-Leu2-Gly-NH2
Leu1-NH (t)
Leu1-NH (c)
Leu2-NH (t)
Leu2-NH (c)
Gly-NH (t)
Gly-NH (c)
Anti-NH (c)
Anti-NH (c)
Syn-NH (t)
6.95
–
8.11
-
7.97
–
–
4.98
5.10
7.04
6.84
7.24
7.75
6.61
6.63
5.23
5.19
1.97
1.04 (3)
–
0.56 (3)
1.07
–
0.73
0.40 (4)
–
–
–
–
Syn-NH (t)
a Chemical shifts were standardized by a residual proton of DMSO or a residual CHCl3 proton. Alphabets in parentheses mean
trans (t) and cis (c).
NH(t) are thought to form a 7-membered ring
and a 10-membered ring, respectively, with the
acetyl carbonyl group. These hydrogen-bonded
structures are considered to be similar to the
g-turn and the b-turn, respectively.
For Boc-Phe-Pro-Leu- compounds, in contrast
to Ac-Pro-Leu- compounds, the NH protons of
both trans and cis conformers take part in some
intramolecular hydrogen bonds. The Leu-NH(c)
proton as well as the Leu-NH(t) proton of Boc-
Phe-Pro-Leu-OMe exhibits a significantly small
Dl/DlRef value, which indicates existence of a
stable intramolecular hydrogen bonds in the cis
configuration. This is also the case for Boc-Leu1-
Pro-Leu2-OMe in which the Leu2-NH(c) proton is
involved in the intramolecular hydrogen bonds.
As mentioned previously, a hydrogen bond part-
ner of Leu-NH(c) is restricted to the Boc carbonyl
group and a likely candidate for the hydrogen-
bonded structure is the type-VI b turn. A ball-
and-stick
model
of
this
structure
for
Boc-Phe-Pro-Leu-OMe is examined on the basis
of molecular mechanics calculation with a MM+
force field [12], and the optimized structure is
shown in Fig. 8. Comparison of the Dl/DlRef
values suggests that the hydrogen bond in the cis
configuration is more stable than that in the trans
configuration. However, it does not mean that the
cis form is more stable than the trans form. In
fact, the cis/trans ratios of Boc-Phe-Pro-Leu-
OME and Boc-Leu1-Pro-Leu2-OMe are 32/68 and
15/85, respectively, in CDCl3. These facts indicate
that non-hydrogen-bonded structures are less sta-
ble in the cis configuration than in the trans

1314
M. Sugawara et al. / Spectrochimica Acta Part A 57 (2001) 1305–1316
bered hydrogen-bonded ring that does not cross a
prolyl residue is not stable enough and can be
neglected as mentioned before. In the trans configu-
ration, therefore, the possible hydrogen bonded
structures for Gly-NH(t) are 10- and 13-membered
rings with Phe- and Boc-carbonyl groups, respec-
tively. The anti-NH(t) proton has possibilities of
forming 10-, 13- and 16-membered rings with Pro-,
Phe-, and Boc-carbonyl groups, respectively. The
10- and 13-membered rings, which may be similar
to the b-turn and a-helix structures, are quite
common for short peptides in solution and proba-
bly coexist with each other in the present solutions.
On the other hand, it is not clear whether the
16-membered ring exists to an appreciable degree
or not.
Fig. 8. A ball-and-stick model of type-VIa b-turn structure for
Boc-Phe-Pro-Leu-OMe. The distance between the Boc-CO
The Phe-carbonyl group of Boc-Phe-Pro-Leu-
Gly-NH2 in the cis form is difficult to make any
intramolecular hydrogen bonds due to steric hin-
drance. Consequently, the hydrogen-bonding part-
ner of the Gly-NH(c) is restricted to the
Boc-carbonyl group, while the Pro-carbonyl group
is excluded by the reason mentioned above. The
C-terminal anti-NH(c) is able to make 10-mem-
bered ring with the Pro-carbonyl, while a possibility
of a 16-membered ring with the Boc-carbonyl could
not be excluded. At present we have not enough
knowledge to conclusively discuss details of the
hydrogen-bonded structure in the cis form. How-
ever, examination with a ball-and-stick model on
the basis of molecular mechanics calculations sug-
gests that a 10-membered hydrogen-bonded ring of
anti-NH(c) and a 13-membered ring of Gly-NH(c)
can be formed simultaneously. If the 10-membered
ring is like the type-I b-turn, the Gly-NH(c) group
is protruded from the ring toward sterically possi-
ble region for the Boc-carbonyl group in the cis
form. This hydrogen-bonded structure of Boc-Phe-
Pro-Leu-Gly-NH2 optimized with the MM+ force
field is shown in Fig. 9. These two intramolecular
hydrogen-bonded rings seem to support each other,
and may cooperatively contribute to stabilization
of the cis form of Boc-Phe-Pro-Leu-Gly-NH2. Each
of hydrogen bonds could be less stable than that
of the type-VIa b-turn structure. However, molec-
ular mechanics calculations indicated that the
structure with the two hydrogen-bonded rings
shown in Fig. 9 was considerably more stable than
a type-VIa b-turn structure for this peptide. Al-
,
oxygen and the Leu-NH proton is 2.15 A as shown in the
figure.
configuration. As a result, the relative stability of
the hydrogen-bonded structure is larger in the cis
configuration than in the trans configuration. Nev-
ertheless the trans form as a whole is more stable
than the cis form.
For Boc-Phe-Pro-Leu-Gly-OEt, similarly, the
hydrogen-bonded ring that involves the Leu-NH
proton is more stable in the cis form than in the
trans form in CDCl3. The hydrogen bond partner
of the Leu-NH(c) proton should be the Boc-car-
bonyl group as for Boc-Phe-Pro-Leu-OMe and a
candidate for this cis hydrogen-bonded structure is
also the type-VIa b-turn. Unfortunately, chemical
shift of the Leu2-NH(c) of Boc-Leu1-Pro-Leu2-Gly-
OEt in 100% DMSO could not be obtained by
overlap of other signals. However, this compound
may have a similar cis hydrogen-bonded structure
to that of Boc-Phe-Pro-Leu-Gly-OEt.
For Boc-Phe-Pro-Leu-Gly-NH2, on the con-
trary, the Leu-NH proton is less involved in any
intramolecular hydrogen bonding both in the trans
and cis forms as compared with those of Boc-Phe-
Pro-Leu-OMe and Boc-Phe-Pro-Leu-Gly-OEt
mentioned above. Instead, mainly the Gly-NH and
anti-NH protons take part in some hydrogen
bonding both in the trans and cis forms. There are
some possible hydrogen-bonded structures for
these amide protons. Among those, the 7-mem-

M. Sugawara et al. / Spectrochimica Acta Part A 57 (2001) 1305–1316
1315
enough. Interaction between the aromatic side
chain and the pyrrolidine ring has been sometimes
suggested [1,7,9]. However, conformational study
by Kang et al. [8] suggested that the interaction is
not strong enough. The present molecular mechan-
ics calculations also do not imply any specific
interaction between the Phe-aromatic ring and the
pyrrolidine ring. For Boc-Phe-Pro-Leu-OMe, an-
other conformation with a different dihedral angle
about the Ca–Cb axis of the Phe residue and a
closer distance between the two rings is slightly
higher in energy than the optimized structure
shown in Fig. 8. The same is true for Boc-Phe-Pro-
Leu-Gly-NH2. Although this could be due to an
insufficiency of the molecular mechanics force field
used in the present study, we may need to take
account of another factor such as solvent effect. In
general, an aromatic side chain is considered to be
somewhat stronger in interaction with solvent than
an aliphatic side chain, probably owing to a
smaller ionization potential of the aromatic ring
than that of the aliphatic side chain. This will cause
difference in interaction with solvent and could
yield different conformational preference in solu-
tion.
though, such cooperative actions of different hy-
drogen bonds on the structures of proteins and
polypeptides are commonly observed [13], they
have so far not been reported for such a short
peptide as composed of four residues to our knowl-
edge. It should be noted here that the above-men-
tioned cooperativity of hydrogen bonds is different
in nature from the usual hydrogen-bond coopera-
tivity that is essentially caused by electronic induc-
tion effect in a succession of hydrogen bonds [13].
The former may be called conformational cooper-
ativity of hydrogen bonds.
Finally, we briefly discuss difference in the cis/
trans ratio between imide bonds of the Phe-Pro and
Leu-Pro sequences. As seen in Fig. 2 and Fig. 3, the
cis form of Boc-Phe-Pro-Leu- compounds are
more stable than those of Boc-Leu-Pro-Leu- com-
pounds in CDCl3-rich solvent. This fact indicates
that the Phe residue stabilize more the cis imide
bond than the Leu residue. This is consistent with
a statistical preference of proline imide bonds in
proteins. Probability of occurrence of the cis imide
bond is somewhat larger in -Xaa-Pro- sequence,
where Xaa indicates a residue with an aromatic
side chain, than in other -Yaa-Pro- sequence [1,8].
The reason for this, however, seems not yet clear
5. Summary
This cis–trans conformational equilibrium of a
proline imide bond of different short peptides in
DMSO-CDCl3 mixed solvents has been studied as
a function of solvent mixing ratio by proton NMR.
It is found that, with increasing fraction of CDCl3,
the trans percentage of Ac-Pro- imide bonds in-
creases, but that of Boc-Phe-Pro- decreases and
that of Boc-Leu-Pro- exhibits middle tendency.
From the solvent-dependent change of chemical
shifts of NH protons, intramolecular hydrogen
bonds that stabilize the trans form of Ac-Pro and
the cis form of Boc-Phe-Pro- were discussed. It is
found that formation of 7-, 10-, and 13-membered
intramolecular hydrogen-bonded rings stabilizes
the trans form of Ac-Pro- imide bonds. On the
other hand, the cis of Boc-Phe-Pro-R (R=OMe or
Gly-OEt) is stabilized by formation of a 10-mem-
bered hydrogen-bonded ring similar to that of the
type-VIa b-turn. For Boc-Phe-Pro-Leu-Gly-NH2,
Fig. 9. A ball-and-stick model of hydrogen-bonded structure
for the cis form of Boc-Phe-Pro-Leu-Gly-NH2. The distance
,
between the Boc-CO oxygen and the Gly-NH proton is 2.71 A
and that between the Pro-CO oxygen and the C-terminal
,
anti-NH proton is 2.76 A as shown in the figure.

1316
M. Sugawara et al. / Spectrochimica Acta Part A 57 (2001) 1305–1316
[2] T.E. Creighton, Proteins: Structures and Molecular Prop-
erties, second edn, Freeman and Company, New York,
1993, p. 221.
[3] J.F. Brandts, H.R. Halvorson, M. Brennan, Biochemistry
14 (1975) 4953.
[4] V. Madison, J. Schellman, Biopolymers 9 (1970) 511.
[5] T. Higashijima, M. Tasumi, T. Miyazawa, Biopolymers
16 (1977) 1259.
on the other hand, it is suggested that the cis form
is stabilized by cooperative action of two different
hydrogen-bonded rings, both of which are differ-
ent from the type-VIa b-turn structure. Difference
in the cis/trans ratio between the Boc-Phe-Pro-
and Boc-Phe-Pro- sequences was briefly discussed.
[6] B. Ishimoto, K. Tonan, S. Ikawa, Spectrochim. Acta Part
A 56 (2000) 201.
[7] M.W. MacArthur, J.M. Thornton, J. Mol. Biol. 218
(1991) 397.
Acknowledgements
[8] Y.K. Kang, J.S. Jhon, S.J. Han, J. Peptide Res. 53 (1999)
30.
The NMR measurements were performed at the
High Resolution NMR Laboratory, Graduate
School of Science, Hokkaido University.
[9] J. Yao, V.A. Feher, B.F. Espejo, M.T. Reymond, P.E.
Wright, H.J. Dyson, J. Mol. Biol. 243 (1994) 736.
[10] K. Tonan, S. Ikawa, J. Am. Chem. Soc. 118 (1996) 6960.
[11] B. Ishimoto, K. Tonan, S. Ikawa, Spectrochim. Acta Part
A55 (1999) 2321.
References
[12] J.T. Sprague, J.C. Tai, Y. Yuh, N.L. Allinger, J. Compt.
Chem. 7 (1987) 581.
[1] D.E. Stewart, A. Sarkar, J.E. Wampler, J. Mol. Biol. 214
(1990) 253.
[13] G.A. Jeffrey, W. Saenger, Hydrogen Bonding in Biologi-
cal Structures, Springer, Berlin, 1991.
.