Intramolecular hydrogen bonding and conformation of small peptides: Variable-temperature FTIR study on N-acetyl-L-Pro-L-Leu-Gly-NH2 and related compounds

Article abstract of DOI:10.1021/ja953380a

Intramolecular hydrogen bonding and conformation of tripeptide amides in CDCl₃ solutions have been examined by variable-temperature FTIR spectroscopy. Absorption in the NH stretching region of N-acetyl-L-Pro-L-Leu-Gly-NH₂ was decomposed into some component bands by least-squares fitting. Two broad bands at around 3350 and 3290 cm^-1 were assigned to intramolecularly hydrogen-bonded NH groups in different conformers. The other relatively sharp bands were assigned to hydrogen-bond-free NHs of the three different amide groups by referring to spectra of analogous amide compounds. From temperature dependence of the band intensities, it is concluded that the C-terminal amide group takes part in the hydrogen bonding in both hydrogen-bonded conformers. From comparison with spectra of N-trifluoroacetyl-L-Pro-L-Leu-Gly-NH₂ and L-Pro-L-Leu-Gly-NH₂, the 3290-cm^-1 band has been assigned to a 13-membered ring with the hydrogen bond between NH of the C-terminal amide group and CO of the N-terminal acetyl group. The 3350-cm^-1 band, on the other hand, has been assigned to a 10-membered hydrogen-bonded ring between the C-terminal amide NH and a prolyl carbonyl group. A van't Hoff analysis shows that the 13-membered hydrogen-bonded ring is lly more favorable but entropically less favorable than the 10-membered hydrogen-bonded ring.

Full text of DOI:10.1021/ja953380a

6960
J. Am. Chem. Soc. 1996, 118, 6960-6965
Intramolecular Hydrogen Bonding and Conformation of Small
Peptides: Variable-Temperature FTIR Study on
N-Acetyl-L-Pro-L-Leu-Gly-NH2 and Related Compounds
Kenji Tonan* and Shun-ichi Ikawa*
Contribution from the Department of Chemistry, Faculty of Science, Hokkaido UniVersity,
Sapporo 060, Japan
ReceiVed October 4, 1995X
Abstract: Intramolecular hydrogen bonding and conformation of tripeptide amides in CDCl3 solutions have been
examined by variable-temperature FTIR spectroscopy. Absorption in the NH stretching region of N-acetyl-L-Pro-
L-Leu-Gly-NH2 was decomposed into some component bands by least-squares fitting. Two broad bands at around
3350 and 3290 cm-1 were assigned to intramolecularly hydrogen-bonded NH groups in different conformers. The
other relatively sharp bands were assigned to hydrogen-bond-free NHs of the three different amide groups by referring
to spectra of analogous amide compounds. From temperature dependence of the band intensities, it is concluded
that the C-terminal amide group takes part in the hydrogen bonding in both hydrogen-bonded conformers. From
comparison with spectra of N-trifluoroacetyl-L-Pro-L-Leu-Gly-NH2 and L-Pro-L-Leu-Gly-NH2, the 3290-cm-1 band
has been assigned to a 13-membered ring with the hydrogen bond between NH of the C-terminal amide group and
CO of the N-terminal acetyl group. The 3350-cm-1 band, on the other hand, has been assigned to a 10-membered
hydrogen-bonded ring between the C-terminal amide NH and a prolyl carbonyl group. A van’t Hoff analysis shows
that the 13-membered hydrogen-bonded ring is enthalpically more favorable but entropically less favorable than the
10-membered hydrogen-bonded ring.
Introduction
in detail by spectroscopic methods.9,10 Infrared spectra are
particularly useful for detecting and characterizing the hydrogen
bonds11,12 which are related to the R-helix, â-turn, and so on.
Recently Gellman and co-workers13-16 have reported IR and
NMR studies on intramolecular hydrogen bonding for series of
diamides and depsipeptides in dichloromethane solutions. They
have examined various types of hydrogen-bonded rings and
discussed their thermodynamic stabilities by use of synthetic
compounds with the constitutionally restricted possibility of
hydrogen bonding. Winningham and Sogah17 have measured
IR spectra of phenoxathiin derivatives as model compounds of
â-turn nucleators and specified the hydrogen-bonding sites.
These results provide a useful guide to a study of secondary
structure formation of polypeptides. However, measurements
of real peptides also will be indispensable for further looking
into the secondary structures.
The tertiary structure of a protein molecule is formed along
the folding pathway, which seems to be determined by its amino
acid sequence and its physical and chemical environment.
Through the folding, some residues which are far apart in the
sequence are put spatially close to one another and provide a
specific site for interaction with ligands or other biomolecules.
Thus the protein molecules acquire their unique biological
functions. For a long polypeptide chain to fold into a compact
and globular structure, formation of secondary structures such
as R-helix and â-turn is an essential process. The R-helix
shortens the overall length of a polypeptide backbone, while
the â-turn reverses its direction. Both the R-helix and â-turn
are formed by hydrogen bonding of carbonyl groups to amide
NH groups located upward by 3 or 4 residues.1-4 Through
further folding, these secondary structural segments associate
with or separate from each other by various interactions, such
as hydrogen bonding, hydrophobic and hydrophilic interactions,
and solvation effects, and finally form the tertiary structure of
proteins.
In this study, we have measured the infrared spectra of three
kinds of tripeptide amides, N-acetyl-L-Pro-L-Leu-Gly-NH2(Ac-
PLG-NH2), N-trifluoroacetyl-L-Pro-L-Leu-Gly-NH2(Tfa-PLG-
NH2), and L-Pro-L-Leu-Gly-NH2(H-PLG-NH2) (Chart 1), and
(7) Goodman, E. M.; Kim, P. S. Biochemistry 1989, 28, 4343-4347.
(8) Tonan, K.; Kawata, Y.; Hamaguchi, K. Biochemistry 1990, 29, 4424-
4429.
The folding pathway has been one of the most important
subjects of the study of proteins and a wide variety of studies
have been performed.5 Nevertheless, it still remains largely
mysterious. In order to reveal a scheme of the folding, it is
useful to examine the mechanism of the secondary structure
formation for relatively small peptides,6-8 which can be studied
(9) Higashijima, T.; Tasumi, M.; Miyazawa, T.; Miyoshi, M. Eur. J.
Biochem. 1978, 89, 543-556.
(10) Wang, Y. S,.; Kempe, T. G.; Raina, A. K.; Mazzocchi, P. H. Int. J.
Peptide Protein Res. 1994, 43, 277-283.
(11) Pimentel, G. C.; McClellan, A. L. The Hydrogen Bond; Freeman:
San Francisco, 1960.
X Abstract published in AdVance ACS Abstracts, July 1, 1996.
(1) Pauling, L.; Coley, R. B.; Branson, H. R. Proc. Natl. Acad. Sci. U.S.A.
1951, 37, 205-211.
(2) Kendrew, J. C.; Dickerson, R. E.; Strandberg, B. E.; Hart, R. G.;
Davies, D. R.; Phillips, D. C.; Shore, V. C. Nature 1960, 185, 422-427.
(3) Leung, Y. C.; Marsh, R. E. Acta Crystallogr. 1958, 11, 17-31.
(4) Marsh, R. E.; Donohue, J. AdV. Protein Chem. 1967, 22, 235-256.
(5) Creighton, T. E. Proteins: structures and molecular properties, 2nd
ed.; Freeman and Company: New York, 1993; pp 309-328.
(6) Shoemaker, K. R.; Kim, P. S.; York, E. J.; Stewart, J. M.; Baldwin,
R. L. Nature 1987, 326, 563-567.
(12) Aaron, H. S. Top. Stereochem. 1980, 11, 1-52.
(13) Gellman, S. H.; Dado, G. P.; Liang, G. B.; Adams, B. R. J. Am.
Chem. Soc. 1991, 113, 1164-1173.
(14) Liang, G. B.; Rito, C. J.; Gellman, S. H. J. Am. Chem. Soc. 1992,
114, 4440-4442.
(15) Gallo, E. A.; Gellman, S. H. J. Am. Chem. Soc. 1993, 115, 9774-
9788.
(16) Dado, G. P.; Gellman, S. H. J. Am. Chem. Soc. 1994, 116, 1054-
1062.
(17) Winningham, M. J.; Sogah, D. Y. J. Am. Chem. Soc. 1994, 116,
1173-1174.
S0002-7863(95)03380-4 CCC: $12.00 © 1996 American Chemical Society

Intramolecular H Bonding and Conformation of Small Peptides
J. Am. Chem. Soc., Vol. 118, No. 29, 1996 6961
Chart 1
Figure 1. Infrared spectrum of 1.25 mM Ac-PLG-NH2 in CDCl3 at
25 °C obtained by subtracting the pure CDCl3 spectrum at the same
temperature: observed intensity (open circle), calculated total intensity
(solid line), and calculated component intensities (dotted line).
FAB low-resolution mass spectrometric result, m/e ) 381, agreed with
an expected value for C15H23N4O4F3.
N-Isopropylacetamide. Seventeen milliliters (200 mmol) of iso-
propylamine and 28 mL (200 mmol) of triethylamine were diluted with
200 mL of dichloromethane. To this solution was added a total of
14.2 mL (200 mmol) of acetyl chloride in 70 portions at 0 °C with
stirring. After 30 min, dichloromethane was removed by evaporation
at room temperature and the resulting slurry was suspended in 100
mL of ethyl acetate. The filtrate was left standing overnight. The
supernatant was collected and concentrated by evaporation at 70 °C so
that ethyl acetate was completely removed. The residual liquid was
finally obtained (14 mL). 1H NMR (90 MHz, in CDCl3): δ 1.14
(doublet, J ) 6.6 Hz, 6H), 1.94 (singlet, 3H), 4.05 (multiplet, J ) 6.6
Hz, 1H), and 5.66 (broad, 1H). Any impurity signals were negligibly
small.
IR Measurements. Infrared spectra were measured with a BOMEM
DA3 Fourier-transform spectrometer at 2-cm-1 resolution. A variable-
temperature cell used was described previously.18 Windows used were
optically polished CaF2 discs and sample path lengths were fixed at 8
mm. Absorbance of samples was obtained by subtracting the pure
solvent spectrum measured at the same temperature.
related compounds in chloroform solutions at various temper-
atures. Two types of intramolecularly hydrogen-bonded con-
formers with 10- and 13-membered rings, respectively, have
been found. In addition, ultraviolet CD spectra were measured
and found to be correlated with the infrared bands assigned to
the 13-membered ring.
Experimental Section
Reagents. L-Pro-L-Leu-Gly-NH2(H-PLG-NH2) was purchased from
Sigma Chemical Co. Deuteriochloroform from CEA (France) was used
as solvent for IR measurements after being dried over molecular sieves
4A for more than 48 h. All other reagents except those mentioned
below were of the highest grade commercially available and were used
without further purification.
CD Measurements. CD measurements were carried out with a
JASCO J-500A spectropolarimeter. A quarts cell with a 1-mm sample
path length was used and its temperature was controlled at 20 °C.
Solvents used were 20 mM 2-(N-morpholino)propanesulfonate (MOPS)
buffer at pH 7.0, trifluoroethanol (TFE), and CDCl3. The spectral
results were given as mean residue ellipticity (deg cm2/dmol) after
subtracting a pure solvent spectrum.
N-Acetyl-L-Pro-L-Leu-Gly-NH2 (Ac-PLG-NH2). Three hundred
and fifty milligrams (1.25 mmol) of H-PLG-NH2 was dissolved in a
mixed solvent of 40 mL of N,N-dimethylformamide, 80 mL of
dichloromethane, and 4 mL of triethylamine. To this solution was
added a total of 12 mL (125 mmol) of acetic anhydride in 24 portions
at room temperature with stirring, which was continued for 1 h.
Volatiles were removed with a rotary evaporator. The resulting residue
was washed with a total of 40 mL of diethyl ether and the final product
obtained was 282 mg (862 µmol, 69% yield). The melting point was
measured to be 164-165 °C and purity was confirmed by a single
peak of reverse-phase HPLC. Amino acid composition: Pro (1), Leu
(1.03), and Gly (1.02). Elemental Anal. Found: C (54.8%), H (7.9%),
N (16.9%). Calcd: C (55.2%), H (8.0%), N (17.2%). The FAB low-
resolution mass spectrometric result, m/e 327, agreed with an expected
value for C15H26N4O4.
N-Trifluoroacetyl-L-Pro-L-Leu-Gly-NH2 (Tfa-PLG-NH2). One
hundred milligrams (352 µmol) of H-PLG-NH2 was dissolved in 21
mL of the mixed solvent mentioned above. A total of 0.1 mL (800
µmol) of S-ethyl trifluorothioacetate was added in 10 portions to the
solution at room temperature with stirring. After the solution was stirred
for 30 min, volatiles were removed with an evaporator and the residue
was washed with a total of 50 mL of diethyl ether to afford 52 mg
(137 µmol, 39%) of the desired compound. Melting point: 174-179
°C. Elemental Anal. Found: C (46.2%), H (6.3%), N (12.8%), F
(16.3%). Calcd: C (47.4%), H (6.1%), N (14.7%), F (15.0%). The
Results
Figure 1 shows an IR spectrum of 1.25 mM Ac-PLG-NH2 in
CDCl3 at 25 °C. Four distinct peaks at frequencies higher than
3400 cm-1 are assigned to hydrogen-bond-free (HB-free) NH
groups, and a broad band at around 3350 cm-1 and its low-
frequency shoulder are assigned to intramolecularly hydrogen-
bonded (HBd) NH groups. These overlapping bands have been
resolved by least-squares fitting with a computer. It should be
noted here that curve fitting, in general involves some arbitrari-
ness. To manage the problems of how many components should
be taken into account and how to get reliable band parameters,
we have performed curve fitting in several steps. At first, the
calculation was carried out on the limited wavenumber range
from 3550 to 3485 cm-1 and parameters of three Lorentzian
functions were obtained. Next, the calculation range was
extended to the lower wavenumbers in several steps, and
parameters of newly added components were adjusted at each
step. Finally, taking all the parameter values thus obtained as
initial values, a least-squares calculation was performed on the
whole wavenumber region 3550-3250 cm-1 to refine the
parameter values. The baseline was taken to be a flat line
estimated from a higher-frequency region which is free from
the solute absorption. A Lorentzian function is used for
(18) Hashimoto, S.; Ohba, T.; Ikawa, S. Chem. Phys. 1989, 138, 63-
69.
(19) Landolt-Bo¨rnstein, Zahlenwerte und Funktionen, 6 Aufl. II/1;
Springer: Berlin, 1971.
(20) Polo, S. R.; Wilson, M. K. J. Chem. Phys. 1955, 23, 2376-2377.

6962 J. Am. Chem. Soc., Vol. 118, No. 29, 1996
Tonan and Ikawa
will be nearly independent of the temperature-dependent shift
in equilibrium. Then, the temperature dependence of the band
intensity is given only by the thermal expansion of the solution.
The temperature coefficient of band intensities due to this effect
is estimated to be -1.3 × 10-3 K-1, which is given by the
thermal expansion coefficient19 of the solvent, CDCl3. Observed
temperature coefficients of symmetric and asymmetric NH
stretching bands of acetamide, and the NH stretching bands of
N-methylacetamide, N-ethylacetamide, and N-isopropylaceta-
mide in CDCl3 solutions, which were dilute enough to neglect
intermolecular hydrogen bonding, are -3.7 × 10-3, -3.2 ×
10-3, -2.9 × 10-3, -4.2 × 10-3, and -2.9 × 10-3 K-1
,
Figure 2. Infrared spectra of 1.25 mM Ac-PLG-NH2 in CDCl3 at (a)
- 20, (b) 25, and (c) 70 °C.
respectively. Differences between these values and the above-
mentioned value, -1.3 × 10-3 K-1, are attributed to change in
the Polo-Wilson internal-field effect20 and in the solvent effect
on the transition moments, both of which will decrease with
decreasing solvent density. Then, the value of -1.3 × 10-3
K-1 can be taken as the upper limit of the temperature coefficient
for HB-free bands that are irrespective of the HBd-HB-free
conformational equilibrium. A band with a temperature coef-
ficient larger than -1.3 × 10-3 K-1 is, therefore, assigned to a
HB-free band of the NH group that is concerned with the
hydrogen bonding, because the population of the HB-free
conformer will increase with increasing temperature. Con-
versely, the temperature coefficient of a HBd band should be
representing each of the HB-free bands, in view of the fact that
single HB-free bands of acetamide and its N-alkyl derivatives
were well reproduced by a Lorentzian function. Each of the
HBd bands, on the other hand, is represented by a Gaussian
function which is adequate to reproduce an inhomogeneously
broadened band, such as the HBd bands. In addition to four
Lorentzian functions for the four distinct bands, two more
Lorentzian functions were needed to reproduce the observed
spectra. The resulting fit is shown in Figure 1.
The four distinct HB-free NH bands have been assigned by
referring to spectra of simple amide compounds in CDCl3
solutions. Acetamide is a structural analogue of the C-terminal
part of Ac-PLG-NH2. The asymmetric and symmetric NH
stretching bands of this compound are observed at 3534 and
3416 cm-1, respectively. These frequencies are in good
agreement with those of the 3524- and 3412-cm-1 bands of Ac-
PLG-NH2, which are then assigned to the asymmetric and
symmetric NH stretchings of the C-terminal amide group. Peak
frequencies of NH stretching bands of N-methylacetamide,
N-ethylacetamide, and N-isopropylacetamide are measured at
3469, 3454, and 3442 cm-1, respectively. This indicates that
the frequencies of the NH stretching become lower with an
increase in the number of substituent methyl groups on the
carbon atoms adjacent to the NH groups. From this fact, it can
be inferred that the more carbon atoms attached to the R-carbon,
the lower the frequency of the adjacent NH stretching in
peptides. Therefore, it is reasonable to assign the lower 3443-
cm-1 band to HB-free stretching of Leu-NH and the higher
3484-cm-1 band to that of Gly-NH. Assignments of 3496- and
3432-cm-1 bands will be given later.
definitely smaller than -1.3 × 10-3 K-1
.
The HB-free bands of C-terminal NH2 of Ac-PLG-NH2 at
3524 and 3412 cm-1 exhibit positive temperature coefficients,
1.6 × 10-2 and 6.6 × 10-3 K-1, respectively. Therefore, the
proportion of the HB-free state of C-terminal NH2 increases
with increasing temperature. This fact clearly indicates that the
C-terminal amide group takes part in the intramolecular
hydrogen bonds.
The temperature coefficient of the Gly-NH band at 3484 cm-1
was -2.7 × 10-3 K-1
. This value is close to that of
N-methylacetamide mentioned above and indicates that the Gly-
NH is not involved in the hydrogen bonding. The temperature
coefficients of the Leu-NH 3443-cm-1 band and its low-
frequency neighbor the 3432-cm-1 band, being -0.61 × 10-3
and -8.9 × 10-3 K-1, respectively, are distinctly larger and
smaller than those of the Gly-NH and N-ethyl- and N-
isopropylacetamide NH bands. The former value is even larger
than -1.3 × 10-3 K-1. This fact suggests that the Leu-NH
group is in equilibrium between two different HB-free states.
The frequency of the Leu-NH group, even if in the HB-free
state, will be more or less dependent on whether the neighboring
prolyl carbonyl group acts as a hydrogen bond acceptor or not.
Therefore, the 3443- and 3432-cm-1 bands may be assigned to
HB-free Leu-NH groups of conformers without and with,
respectively, the hydrogen bond that involves the prolyl carbonyl
group. It will be shown later that the prolyl carbonyl group
takes part in a 10-membered hydrogen-bonded ring. A band
at 3496 cm-1 is probably assigned to a HB-free NH of the
C-terminal NH2 group, the other NH of which is hydrogen
bonded. This assignment is supported by the considerably large
Figure 2 shows spectra of 1.25 mM Ac-PLG-NH2 at -20,
25, and 70 °C. With increasing temperature, the HB-free bands
at 3524 and 3412 cm-1 that are assigned to the C-terminal amide
group increase in intensity, while all the other band intensities
apparently decrease. This fact indicates that Ac-PLG-NH2 in
the CDCl3 solution exists in equilibrium among a few conform-
ers and that the C-terminal amide group takes part in the
intramolecular hydrogen bonds. This is confirmed by estimating
temperature coefficients of the component band intensities,
(1/I)(dI/dT). Examination of the temperature coefficients is
useful for further looking into the HB-free bands of different
NH groups.
negative temperature coefficient of this band, -6.9 × 10-3 K-1
,
which indicates this band is given by a HB-free NH of the HBd
conformers.
The temperature coefficients of the band intensities are
thought to be affected by two factors. One is the temperature-
dependent shift in the conformational equilibrium and the other
is the thermal expansion of the solution. For a NH group which
does not take part in the hydrogen bonds, the bands of both the
HB-free and HBd conformers will almost completely overlap
and provide a single band. Therefore, its apparent band intensity
As shown in Figure 3, the spectra of Ac-PLG-NH2 exhibit
concentration dependence. Spectra of 1.25 and 10 mM solutions
are considerably different in shape at the low-frequency region
at -20 °C, though they agree fairly well with each other at 70
°C. The spectral shapes of 2.5 and 5 mM solutions were found
to be almost the same as that of the 1.25 mM solution at both
temperatures. Therefore, intermolecular hydrogen bonding

Intramolecular H Bonding and Conformation of Small Peptides
J. Am. Chem. Soc., Vol. 118, No. 29, 1996 6963
Figure 5. Infrared spectrum of 1.25 mM Ac-G-NH2 in CDCl3 at 25
°C.
Chart 2
Figure 3. Infrared spectra of 1.25 (solid line) and 10 mM (broken
line) Ac-PLG-NH2 in CDCl3 at (a) 70 and (b) -20 °C.
hydrogen bond related to this band is between the C-terminal
NH and the carbonyl oxygen of the N-terminal acetyl group,
forming a 13-membered ring (Chart 2). Trifluorination of the
acetyl group will largely reduce the proton-accepting ability of
its carbonyl oxygen owing to the considerable electron-
withdrawing property of the trifluoromethyl group. Therefore,
the hydrogen bond concerned is weaker in Tfa-PLG-NH2 than
in Ac-PLG-NH2. On the other hand, H-PLG-NH2 has no
carbonyl group at the N-terminus and no possibility of forming
the 13-membered hydrogen-bonded ring.
Figure 5 shows the spectrum of a 1.25 mM solution of
acetylglycinamide (Ac-G-NH2, Chart 1) in CDCl3 at room
temperature. This compound has an equivalent constitution to
the C-terminal half of Ac-PLG-NH2 and has the possibility of
forming a 7-membered hydrogen-bonded ring. Obviously bands
to be assigned to hydrogen bonds are not appreciable except
for a minor swelling, and this spectral feature was found to be
nearly independent of temperature. Therefore, the 7-membered
ring will not be appreciable for Ac-PLG-NH2 as well as for
Ac-G-NH2 at the experimental temperatures. In conclusion, the
hydrogen-bonded bands at around 3350 cm-1, which are
observed in common for Ac-PLG-NH2, Tfa-PLG-NH2, and
H-PLG-NH2, are assigned to a 10-membered ring with a
hydrogen bond between the C-terminal amide NH and the prolyl
carbonyl group (Chart 2). This is consistent with the temper-
ature behavior of the HB-free bands of the Leu-NH group
mentioned previously.
It is well-known that the hydrogen bonds in the R-helix and
in the â-turn of polypeptides form 13- and 10-membered rings,
respectively, which are similar to those of the tripeptide amides
of the present study. The ultraviolet CD spectrum is one of
the most useful tools for studying specific secondary structures
of polypeptides or proteins. But for short peptides such as Ac-
PLG-NH2 or its analogues, it is unknown whether the discrimi-
nation of secondary structures can be obtained from CD or not,
because only minimal CD data have been accumulated. In view
of this, we have measured ultraviolet CD spectra and compared
them with the IR spectra. The resulting CD spectra of Ac-
PLG-NH2, Tfa-PLG-NH2, and H-PLG-NH2 are shown in Figure
Figure 4. Infrared spectra (solid line) and the component bands (dotted
line) calculated by the least-squares method for 1.25 mM (a) Ac-PLG-
NH2, (b) Tfa-PLG-NH2, and (c) H-PLG-NH2 in CDCl3 at 70 °C.
becomes appreciable at high concentrations and low tempera-
tures, and the 1.25 mM concentration is dilute enough to neglect
the intermolecular hydrogen bonding in the experimental
temperature range.
Figure 4 shows spectra of 1.25 mM solutions of the three
peptides, Ac-PLG-NH2, Tfa-PLG-NH2, and H-PLG-NH2, at 70
°C. The component band of the lowest frequency, which is
assigned to the hydrogen-bonded C-terminal NH for Ac-PLG-
NH2, noticeably decreases in intensity for Tfa-PLG-NH2 and
disappears for H-PLG-NH2. This fact indicates that the

6964 J. Am. Chem. Soc., Vol. 118, No. 29, 1996
Tonan and Ikawa
Figure 7. Logarithmic plots of the intensity ratios If(3412)/Ib(3350) (closed
circle) and If(3412)/Ib(3290) (open circle) against inverse absolute temper-
ature for 1.25 mM Ac-PLG-NH2 in CDCl3.
Table 1. IR Spectroscopic Results of the Thermodynamic
Properties for the Intramolecular Hydrogen Bonding of Three
Tripeptides
Figure 6. CD spectra of 1.25 mM Ac-PLG-NH2 (a), Tfa-PLG-NH2
(b), and H-PLG-NH2 (c) in CDCl3 (solid line) and Ac-PLG-NH2 in
TFE (broken line) and in a MOPS (pH 7) buffer solution (dotted line).
∆Sb
∆Gc
∆Ha (kJ mol-1
)
(J mol-1 K-1
[10 f 13]
)
(kJ mol-1
)
6. Ellipticity could not be measured at wavelengths shorter than
230 nm in CDCl3 solutions due to strong absorption of CDCl3
in this region. Therefore, we did not observe the double
minimum of mean residue ellipticity at 222 and 208 nm,
characteristic of the R-helix. However, the CD spectrum of
Ac-PLG-NH2 in CDCl3 shows a rapid increase in negative
ellipticity at wavelengths shorter than 250 nm. This spectral
feature diminished for Tfa-PLG-NH2 and disappeared for
H-PLG-NH2. Interestingly enough, the difference in the CD
spectrum among the three peptides is consistent with that in
the IR intensity of the 3290-cm-1 band that is assigned to the
13-membered hydrogen-bonded ring. In spite of its ability to
form the 10-membered ring, H-PLG-NH2 exhibits almost a flat
CD spectrum in the same wavelength region. These findings
suggest that the increase in the negative ellipticities at wave-
lengths shorter than 250 nm in CDCl3 solutions of Ac-PLG-
NH2 and Tfa-PLG-NH2 becomes a useful sign of a specific
conformation of the 13-membered ring. As seen in Figure 6,
this spectral feature of Ac-PLG-NH2 disappears in the aqueous
MOPS solution, while the TFE solution exhibits intermediate
ellipticity between those in the CDCl3 and aqueous solution. In
the aqueous environment, Ac-PLG-NH2 may adopt a random
structure. Of course, the CD spectra alone indicates nothing
about the R-helical nature of the 13-membered rings. However,
the hydrogen-bonded sites determined from the IR spectra for
the present 13-membered rings are exactly the same as those
of the R-helix. It is, therefore, reasonable to suggest that the
13-membered ring may have a structure similar to that of a one-
loop segment of the R-helix.
compds
[free f 10] [free f 13]
[10 f 13]
Ac-PLG-NH2 -8.1 ( 0.8 -12 ( 3
Tfa-PLG-NH2 -4.4 ( 0.6 -4.7 ( 2.9
H-PLG-NH2 -8.1 ( 1.3
a Mean values of ∆Hs obtained by using the upper limit and the
lower limit of cb. b Errors are three times the standard deviations.
c Values at 295 K.
-22 ( 2
-34 ( 5
2.5 ( 2.9
9.7 ( 3.5
However, we have to take account of the fact that the molar
integrated intensities depend on temperature in somewhat
different ways particularly between HB-free and HBd NH bands.
In this case, the logarithm of the intensity ratio can be written
as21
ln(If/Ib) ) ∆H/RT + (cf - cb)T + constant
(1)
where cf and cb are the temperature coefficients of the molar
integrated intensities of HB-free and HBd bands, respectively.
Since the concentration of the conformers changes with tem-
perature, the values of cf and cb cannot be obtained from the
observed band intensities of the molecule concerned. We have
assumed, therefore, that the intrinsic cf value of the 3412-cm-1
band is given by that of the symmetric NH stretching of
acetamide, -3.7 × 10-3 K-1. For estimation of the cb value,
it is difficult to find a model compound with an analogous
intramolecular hydrogen bond but without the HBd-HB-free
equilibrium. However, we can take the observed cb value of
the band concerned as the lower limit of the intrinsic cb value,
while the upper limit is given by the previously mentioned value,
-1.3 × 10-3 K-1. With this restriction, the ∆H value has been
obtained by least-squares fitting of eq 1 to the observed plots.
The same analysis has been carried out for Tfa-PLG-NH2 and
H-PLG-NH2. The resulting mean values are listed in Table 1
together with the errors estimated from the both limits of cb.
Entropy difference between two conformers is given by12
Discussion
Thermodynamic properties of the HBd conformers are
obtained from analysis of the band intensities as a function of
temperature.12,21 Figure 7 shows van’t Hoff plots for Ac-PLG-
NH2 in CDCl3. Intensity ratios of the 3412-cm-1 band, which
is assigned to the HB-free NH of the C-terminal amide, to each
of the two hydrogen-bonded bands, If/Ib, were plotted against
inverse absolute temperature. Here, two-state conformational
equilibrium has been assumed between the HB-free conformer
and each of the HBd conformers with the 10- and 13-membered
rings. Usually, the slopes of the logarithmic plots provide the
enthalpy changes, ∆H, for formation of the 10- and 13-
membered hydrogen-bonded rings from the HB-free conformer.
∆S ) ∆H/T - R{ln(I1/I2) - ln(R1/R2)}
(2)
if the ratio of the molar integrated intensities, R1/R2, is given.
This ratio could be given by a slope of a I1 vs I2 plot, but
unfortunately it is usually difficult to estimate owing to
temperature dependence of the molar integrated intensities.21 It
is well-known that the molar integrated intensities of the N-H
or O-H stretching bands increase remarkably when the NH or
OH groups take part in hydrogen bonds.11 In addition, the local
environment of the NH or OH bonds changes with the hydrogen
(21) Yasuda, T.; Okuyama, M.; Tanimoto, N.; Ikawa, S. J. Chem. Soc.,
Faraday Trans. 1995, 91, 3379-3383.

Intramolecular H Bonding and Conformation of Small Peptides
J. Am. Chem. Soc., Vol. 118, No. 29, 1996 6965
bonding and so does the temperature coefficients of the
intensities.21 Therefore, it is difficult to estimate ∆S between
HB-free and HBd conformers. However, between the two HBd
conformers, it is possible to estimate ∆S. The states of the
hydrogen bonds in the 10- and 13-membered rings will not be
much different from each other, and we can assume R1 ) R2
without introducing any significant error. Then the ∆S value
can be obtained from the intercept of the van’t Hoff plots. The
values of entropy difference thus obtained and free energy
difference, ∆G, between the conformers with 10- and 13-
membered rings are listed in Table 1.
and collapse of transient local secondary structures, through the
folding pathway to the biologically active structure of proteins.
Even after being fully folded, a protein molecule may alter its
local secondary structure with an environmental change or
fluctuation. Such a local conformational change may lead to a
rather drastic change in the whole structure of the protein
molecule, and may affect its biological activity. An intriguing
example is a potential-dependent ion channel which consists
of a protein molecule embedded in a cell membrane. Its
secondary structure may vary with a change in the membrane
potential, which results in the opening and closing of the ion
channel.23 Therefore, elucidating the mechanism of the forma-
tion and stabilization of secondary structures provides a key
for understanding the biological function of the protein molecule.
At 295 K, the ∆G for the change from the 10- to the 13-
membered ring for Ac-PLG-NH2 is 2.5 ( 2.9 kJ mol-1
,
indicating that the 10-membered ring may be a little more stable
than the 13-membered ring at 295 K. The values of ∆H for
formation of the 13-membered ring from the HB-free conformer
are larger than those for the 10-membered ring. Therefore, the
13-membered ring is enthalpically more favorable than the 10-
membered ring. On the other hand, the ∆S value for change
from the 10- to the 13-membered ring is negative, and
consequently, the 13-membered ring is entropically less favor-
able than the 10-membered ring. These differences in enthalpy
and entropy between two conformers can be explained by the
difference in dipole-dipole interactions with solvent chloroform
molecules. If the 13-membered ring has a similar structure to
that of a one-loop segment of the R-helix, all the group dipole
moments of the four peptide units point parallel,22 and should
be cumulative. As a result, the dipole moment of the 13-
membered ring may be considerably larger than that of the 10-
membered ring. Therefore, the 13-membered ring exhibits
stronger dipole-dipole interaction with polar solvents and makes
its solvation structure more rigid, as compared with the 10-
membered ring. Thus the 13-membered ring is enthalpically
more favorable and entropically less favorable than the 10-
membered ring.
Conclusion
Infrared spectra of three kinds of tripeptide amides, Ac-PLG-
NH2, Tfa-PLG-NH2, and H-PLG-NH2, and related compounds
have been measured in chloroform solutions at various tem-
peratures. For Ac-PLG-NH2 and Tfa-PLG-NH2, the absorption
in the NH stretching region is decomposed into six hydrogen-
bond-free NH bands and two broad hydrogen-bonded NH bands.
The latter two bands have been assigned to hydrogen-bonded
conformers with 10- and 13-membered hydrogen-bonded rings,
respectively. Thermodynamic properties of these conformers
have been obtained from van’t Hoff analyses of the temperature
dependence of the component intensities. It is shown that the
13-membered ring is enthalpically more favorable but entropi-
cally less favorable than the 10-membered ring.
Acknowledgment. We thank Prof. Shigehiro Konaka for
valuable discussions. We are grateful to Prof. Fumi Morita and
Dr. Masumi Eto for allowing us to use the HPLC apparatus
and the JASCO J-500A spectropolarimeter. Elemental analysis,
amino acid composition analysis, and FAB low-resolution
spectrometric measurements were performed at the Center for
Instrumental Analysis, Hokkaido University.
These findings imply that the secondary structure of polypep-
tides is easily transformed by a mild change in temperature or
other surrounding conditions. We can, therefore, imagine that
the polypeptides experience a large number of steps of formation
JA953380A
(23) Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K.; Watson, J.
D. Molecular Biology of the Cell; Garland Publishing: New York, 1983;
pp 1024-1026.
(22) Wada, A. AdV. Biophys. 1976, 9, 1-63.