Single-conformation ultraviolet and infrared spectroscopy of model synthetic foldamers: β-peptides Ac-β3-hPhe-NHMe and Ac-β3-hTyr-NHMe

Article abstract of DOI:10.1021/ja078271y

The conformational preferences and infrared and ultraviolet spectral signatures of two model β-peptides, Ac-β³-hPhe-NHMe (1) and Ac-β³-hTyr-NHMe (2), have been explored under jet-cooled, isolated molecule conditions. The mass-resolved, resonant two-photon ionization spectra of the two molecules were recorded in the region of the S ₀-S₁ origin of the phenyl or phenol ring substituents, respectively. UV-UV hole-burning spectroscopy was used to determine that two conformations of 1 are present, with the transitions due to conformer A, with S₀-S₁ origin at 34431 cm^-1, being almost 20 times larger than those due to conformer B, with S₀-S₁ origin at 34404 cm^-1. Only one conformation of 2 was observed. Resonant ion-dip infrared spectroscopy provided single-conformation infrared spectra in the 3300-3700 cm^-1 region. The spectra of conformer A of both molecules have H-bonded and free amide NH stretch infrared transitions at 3400 and 3488 cm^-1, respectively, while conformer B of 1 possesses bands at 3417 and 3454 cm^-1. For comparison with experiment, full optimizations of all low-lying minima of 1 were carried out at the DFT B3LYP/6-31+G* and RIMP2/aug-cc-pVDZ levels of theory, and single point MP2/6-31+G* calculations at the DFT geometries. On the basis of the comparison with previous studies in solution and the calculated results, conformer A of 1 and 2 were assigned to a C6 conformer, while conformer B of 1 was assigned to a unique C8 structure with a weak intramolecular H-bond. The reasons for the preference for C6 over C8 structures and the presence of only two conformations in the jet-cooled spectrum are discussed in light of the predictions from calculations.

Full text of DOI:10.1021/ja078271y

Published on Web 03/18/2008
Single-Conformation Ultraviolet and Infrared Spectroscopy of
Model Synthetic Foldamers: â-Peptides Ac-â3-hPhe-NHMe
and Ac-â3-hTyr-NHMe
Esteban E. Baquero,† William H. James, III,† Soo Hyuk Choi,‡
Samuel H. Gellman,‡ and Timothy S. Zwier*,†
Department of Chemistry, Purdue UniVersity, West Lafayette, Indiana 49707-2084, and
Department of Chemistry, UniVersity of Wisconsin, Madison, Wisconsin 53706
Received October 29, 2007; E-mail: zwier@purdue.edu
Abstract: The conformational preferences and infrared and ultraviolet spectral signatures of two model
â-peptides, Ac-â3-hPhe-NHMe (1) and Ac-â3-hTyr-NHMe (2), have been explored under jet-cooled, isolated
molecule conditions. The mass-resolved, resonant two-photon ionization spectra of the two molecules were
recorded in the region of the S0-S1 origin of the phenyl or phenol ring substituents, respectively. UV-UV
hole-burning spectroscopy was used to determine that two conformations of 1 are present, with the transitions
due to conformer A, with S0-S1 origin at 34431 cm-1, being almost 20 times larger than those due to
conformer B, with S0-S1 origin at 34404 cm-1. Only one conformation of 2 was observed. Resonant ion-
dip infrared spectroscopy provided single-conformation infrared spectra in the 3300-3700 cm-1 region.
The spectra of conformer A of both molecules have H-bonded and free amide NH stretch infrared transitions
at 3400 and 3488 cm-1, respectively, while conformer B of 1 possesses bands at 3417 and 3454 cm-1
.
For comparison with experiment, full optimizations of all low-lying minima of 1 were carried out at the DFT
B3LYP/6-31+G* and RIMP2/aug-cc-pVDZ levels of theory, and single point MP2/6-31+G* calculations at
the DFT geometries. On the basis of the comparison with previous studies in solution and the calculated
results, conformer A of 1 and 2 were assigned to a C6 conformer, while conformer B of 1 was assigned to
a unique C8 structure with a weak intramolecular H-bond. The reasons for the preference for C6 over C8
structures and the presence of only two conformations in the jet-cooled spectrum are discussed in light of
the predictions from calculations.
1. Introduction
R-peptides in having two carbon atoms rather than one linking
adjoining amide groups. This additional carbon adds flexibility
to the polypeptide backbone and changes the relative spacing
between amide groups. This backbone variation changes the
nature of the amide-amide H-bonds that dictate the propensity
toward helical or other secondary structures, relative to proteins.
These preferences can be fine-tuned by changing substituents
at the â2 and/or â3 position.10,11
In recent years, there has been a surge of interest in exploring
the properties of synthetic foldamers, that is, oligomers made
up of building blocks that differ in well-defined ways from the
R-amino acid units that comprise natural proteins.1-5 The use
of unnatural subunits in place of R-amino acid residues gives
foldamer backbones unique secondary and tertiary structural
possibilities. Foldamers are of possible practical interest because
they can display useful chemical6 and biological7 activities.
One class of synthetic foldamers that has generated particular
interest and attention are â-peptides,8-11 which differ from
† Purdue University.
‡ University of Wisconsin.
(1) Gellman, S. H. Acc. Chem. Res. 1998, 31, 173-180.
(2) Kirshenbaum, K.; Zuckermann, R. N.; Dill, K. A. Curr. Opin. Struct. Biol.
1999, 9, 530-535.
(3) Hill, D. J.; Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S. Chem.
ReV. 2001, 101, 3893-4011.
The fundamental base of knowledge regarding the inherent
conformational preferences and potential energy landscapes of
â-peptides is much less developed than that of R-peptides. One
useful strategy to address this deficiency is to study the
conformational preferences through conformation-specific spec-
troscopy of isolated â-peptides free from solvent. In this way,
(4) Huc, I. Eur. J. Org. Chem. 2004, 17-29.
(5) Hecht, S.; Huc, I. Foldamers: Structure, Properties, and Applications;
Wiley-VCH: Weinheim, 2007.
(6) Pomerantz, W. C.; Abbott, N. L.; Gellman, S. H. J. Am. Chem. Soc. 2006,
128, 8730-8731.
(7) Goodman, C. M.; Choi, S.; Shandler, S.; DeGrado, W. F. Nat. Chem. Biol.
2007, 3, 252.
(8) Dado, G. P.; Gellman, S. H. J. Am. Chem. Soc. 1994, 116, 1054-1062.
(9) Seebach, D.; Matthews, J. L. Chem. Commun. 1997, 2015-2022.
(10) Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem. ReV. 2001, 101,
3219-3232.
(11) Seebach, D.; Beck, A. K.; Bierbaum, D. J. Chem. BiodiVers. 2004, 1, 1111-
1239.
9
4784
J. AM. CHEM. SOC. 2008, 130, 4784-4794
10.1021/ja078271y CCC: $40.75 © 2008 American Chemical Society

â
-Peptides Ac-
â
3-hPhe-NHMe and Ac-
â
3-hTyr-NHMe
A R T I C L E S
the N-terminal amide (NH(1)) acts as donor in a H-bond with
the oxygen of the C-terminal carbonyl group (CO(2)). This
leaves the C-terminal NH(2) group free. Alternatively, an eight-
membered cycle (C8) can form when the N-H of the C-terminal
amide (NH(2)) hydrogen bonds to the CdO of the N-terminal
amide (CO(1)), leaving NH(1) free. As we shall see, these model
â-peptides show evidence for both H-bonded motifs, with the
C6 ring being dominant.
There is one previous report on the gas-phase rotational
spectroscopy of â-alanine,23 which finds evidence for a C6
conformer analogous to that just described. There are several
studies of small model â-peptides in dilute, nonpolar solution,
where many of the same issues regarding the inherent confor-
mational preferences of the â-peptides were explored.8,24,25 The
results from Marraud and co-workers25 and Dado and Gellman8
are of most direct relevance, since both groups used amide NH
stretch infrared spectra as a tool in assigning the observed
conformations. In particular, Marraud et al.25 compared the IR
spectra of N-acetyl-3-aminobutanoic acid-N′-methylamide (Ac-
â3-hAla-NHMe, 3), which contains a branched-chain â3 carbon,
with Ac-â-hGly-NHMe (4), in which â3 carbon is unbranched.
Figure 1. Structures for (a) 3-acetoamido-N-methyl-4-phenylbutamide (Ac-
â3-hPhe-NHMe, 1) and (b) 3-acetamido-4-(4-hydroxyphenyl)-N-methylb-
utamide (Ac-â3-hTyr-NHMe, 2). (a) The numbers in parentheses indicate
the labeling scheme used for the amide groups along the peptide chain. (b)
The indicated curves connect NH and CdO groups that can be involved in
intramolecular H-bonds. The indicated angles are the relevant Ramachandran
angles used to conduct conformational searches in â-peptides.
the inherent conformational preferences can be determined and
their spectral signatures explored. This strategy has been used
to good advantage in previous work on R-peptides, probing in
some detail their single-conformation spectroscopy up through
tetra-, pentapeptides, and beyond.12-22 Many of these studies
incorporate an aromatic chromophore that enables double
resonance spectroscopy to determine the number of conforma-
tions present and obtain their ultraviolet and infrared spectral
signatures. The amide NH stretch region of the infrared was
used as a powerful diagnostic of the H-bonded architectures
present in these molecules. The number and strength of the
H-bonds are imprinted on the NH stretch fundamentals, produc-
ing shifts in vibrational frequency and enhancements in infrared
intensity that are diagnostic of the structure. Assignments have
been made to various peptide backbone conformations, including
structures that incorporate 5-membered (C5), 7-membered (C7),
and 10-membered (C10) H-bonded rings.
In this paper, we extend such studies to two model â-peptides
containing either a phenylalanine or tyrosine side chain.
3-Acetoamido-N-methyl-4-phenylbutamide (Ac-â3-hPhe-NHMe,
1) and 3-acetamido-4-(4-hydroxyphenyl)-N-methylbutamide
(Ac-â3-hTyr-NHMe, 2) contain a single â-amino acid residue
(Figure 1), which has the flexibility to accommodate two types
of intramolecular hydrogen-bonded structures, shown in Figure
1b. A six-membered cycle (C6) can form when the N-H of
These authors assigned bands in the amide NH stretch region
to both C6 and C8 conformers. A key aspect of their assignment
is their recognition that the free amide NH stretch bands can
be used as a diagnostic of which group is not involved in the
intramolecular H-bond. In 4, both amide NH groups are attached
to unbranched carbons, producing a single peak in the free amide
NH stretch region. However, in 3, two bands appear, with the
free amide NH stretch of the branched chain lower in frequency
than that of the NHMe group by ∼30-40 cm-1. Since 1 differs
from 3 only by addition of the phenyl group to the â3 methyl
substituent, we anticipate being able to use the same diagnostic
in the present study. Indeed, we shall see that the positional
dependence of the free amide NH stretch does carry over to
isolated molecule conditions and can be used as an unequivocal
signature distinguishing C6 from C8 cycles.
In support of our experimental work, we also carry out an
extensive set of density functional theory and ab initio calcula-
tions of the structures, relative energies, vibrational frequencies,
and infrared intensities of the various conformational minima
of Ac-â3-hPhe-NHMe. These calculations build closely on the
results of previous calculations of Hofmann and co-workers26,27
on 3, which revealed several unique C6 and C8 conformers.
These authors explored the conformational surface by systemati-
cally changing dihedral angles along the peptide backbone (φ,
θ, ψ defined in Figure 1b) and optimizing the resultant structures
(12) Abo-Riziq, A. G.; Bushnell, J. E.; Crews, B.; Callahan, M. P.; Grace, L.;
De Vries, M. S. Int. J. Quantum Chem. 2005, 105, 437-445.
(13) Abo-Riziq, A.; Bushnell, J. E.; Crews, B.; Callahan, M.; Grace, L.; De
Vries, M. S. Chem. Phys. Lett. 2006, 431, 227-230.
(14) Abo-Riziq, A.; Crews, B. O.; Callahan, M. P.; Grace, L.; de Vries, M. S.
Angew. Chem., Int. Ed. 2006, 45, 5166-5169.
(15) Chin, W.; Compagnon, I.; Dognon, J. P.; Canuel, C.; Piuzzi, F.; Dimicoli,
I.; von Helden, G.; Meijer, G.; Mons, M. J. Am. Chem. Soc. 2005, 127,
1388-1389.
(16) Chin, W.; Dognon, J. P.; Canuel, C.; Piuzzi, F.; Dimicoli, I.; Mons, M.;
Compagnon, I.; von Helden, G.; Meijer, G. J. Chem. Phys. 2005, 122.
(17) Chin, W.; Dognon, J. P.; Piuzzi, F.; Dimicoli, I.; Mons, M. Mol. Phys.
2005, 103, 1579-1587.
(18) Chin, W.; Mons, M.; Dognon, J. P.; Mirasol, R.; Chass, G.; Dimicoli, I.;
Piuzzi, F.; Butz, P.; Tardivel, B.; Compagnon, I.; von Helden, G.; Meijer,
G. J. Phys. Chem. A 2005, 109, 5281-5288.
(23) McGlone, S. J.; Godfrey, P. D. J. Am. Chem. Soc. 1995, 117, 1043-
1048.
(19) Chin, W.; Piuzzi, F.; Dimicoli, I.; Mons, M. Phys. Chem. Chem. Phys.
2006, 8, 1033-1048.
(24) Gellman, S. H.; Dado, G. P.; Liang, G. B.; Adams, B. R. J. Am. Chem.
Soc. 1991, 113, 1164-1173.
(20) Compagnon, I.; Oomens, J.; Meijer, G.; von Helden, G. J. Am. Chem. Soc.
2006, 128, 3592-3597.
(25) Marraud, M.; Neel, J. J. Polym. Sci. Part C: Polym. Symp. 1975, 52, 271-
(21) Reha, D.; Valdes, H.; Vondrasek, J.; Hobza, P.; Abu-Riziq, A.; Crews, B.;
de Vries, M. S. Chem.-Eur. J. 2005, 11, 6803-6817.
282.
(26) Mohle, K.; Gunther, R.; Thormann, M.; Sewald, N.; Hofmann, H. J.
Biopolymers 1999, 50, 167-184.
(22) Dian, B. C.; Longarte, A.; Mercier, S.; Evans, D.; Wales, D. J.; Zwier, T.
S. J. Chem. Phys. 2002, 117, 10688-10702.
(27) Gunther, R.; Hofmann, H. J. HelV. Chim. Acta 2002, 85, 2149-2168.
9
J. AM. CHEM. SOC. VOL. 130, NO. 14, 2008 4785

A R T I C L E S
Baquero et al.
J ) 6.9 Hz, 1H), 2.78-2.62 (m, 3H), 2.33 (ABX, JAB ) 14.3 Hz,
JAX ) 6.0 Hz, JBX ) 7.5 Hz, ∆υ ) 0.06 ppm, 2H), 1.85 (s, 1H);
ESI-TOF MS 250.9 [M + H]+, 272.8 [M + Na]+, 522.7 [2M +
Na]+.
at the Hartree-Fock (HF) and density functional (B3LYP) levels
of theory with two different basis sets (3-21G and 6-31G*).
The optimization yielded a six-membered cycle (C6) as the
lowest energy structure but also found several different types
of C6 and C8 structures within a 20 kJ/mol energy window
from the global minimum. These findings were later cor-
roborated by Beke et al.28,29 on the analogous â3-monosubsti-
tuted peptide backbone without C-terminal methyl caps (HCO-
â3-hAla-NH2). This study included optimizations at a higher
level of theory (B3LYP/6-311++G(d,p)), which found the same
C6 structure to be the global minimum. The higher level
optimization also energetically rearranged structures above the
global minimum and removed some higher energy C8 and
extended structures that no longer formed stable minima.
In the present work, we extend these previous studies to
completely explore the low-lying minima of 1. It is important
to note that the addition of the phenyl ring in 1 adds an extra
degree of freedom which results in a larger number of
conformers than in 3 or 4.
Results from this study provide a foundation for experimental
and theoretical exploration of the structures formed by larger
â-peptides. This extension is taken up in the paper immediately
following this one, where two larger phenyl-containing â-pep-
tides are studied: Ac-â3-hPhe-â3-hAla-NHMe and Ac-â3-hAla-
â3-hPhe-NHMe.30 In these molecules, a rich variety of H-bonded
structures are found, including single-ring (C10) and a variety
of double-ring structures (C6/C6, C8/C8, and either C6/C8 or
C8/C12).
The experimental apparatus used in these studies has been described
in detail elsewhere.32 As a result, only a brief description will be given
here. Ac-â3-hPhe-NHMe (1) and Ac-â3-hTyr-NHMe (2) were intro-
duced into the gas phase by heating the sample to approximately 180-
190 °C in a sample holder located directly behind a pulsed valve. A
glass insert was placed inside a sample holder to minimize decomposi-
tion due to direct contact of the sample with the metal sample holder.
The samples were expanded into vacuum via a pulsed valve (Parker
General Valve, Series 9, 400 µm orifice, 20 Hz) using a 70/30 neon/
helium gas mixture as a carrier gas with a backing pressure of 1.7 bar.
The collisionally cooled molecules were interrogated in the ionization
region of a time-of flight-mass-spectrometer (TOFMS) approximately
10 cm from the nozzle. The ionization and source regions are separated
by a conical skimmer (Beam Dynamics, Inc., 55° conical angle, 2 mm
diam). Flow rates of 0.15-0.30 bar·cm3/s were maintained in order to
avoid skimmer interference.
Electronic spectra were recorded by employing one-color-resonant
two-photon ionization (R2PI), exciting the molecules with the frequency-
doubled output of an Nd:YAG (Continuum) pumped tunable dye laser
(Lambda-Physik Scanmate). The resultant ultraviolet radiation (0.1-
0.5 mJ/pulse) traverses the ion source region of the TOFMS in a
collimated beam of ∼1 mm diameter. The ions were detected by a
microchannel plate detector (RM Jordan, 2.5 cm) mounted on top of a
1 m flight tube.
Conformation specific electronic spectra were obtained using UV-
UV hole-burning (UVHB) spectroscopy. A higher power hole-burning
laser (10 Hz) was fixed on a transition observed in the R2PI spectrum,
and the probe laser (20 Hz) was scanned over the wavelength region
of interest. The two laser beams were counter-propagated, spatially
overlapped, and separated temporally so the hole-burning laser preceded
the probe by 200 ns. The hole-burning spectra were recorded by
monitoring, via active baseline subtraction, the difference between the
ion signal due to the probe laser with the hole-burning laser “on” or
“off”. All bands that originate from the same ground state level as the
transition on which the hole-burn laser is fixed appear as depletions in
the ion signal.
2. Methods
2.1. Experiment. The synthesis and characterization of the â-peptide
compounds studied followed well-established methods. The N-Boc
protected â-amino acid methyl amides were prepared from the
corresponding N-Boc-R-amino acids by a previously demonstrated, two-
step procedure.31 The R-amino acids were purchased from Novabio-
chem.
For the synthesis of Ac-â3-hPhe-NHMe (1), Boc-â3-hPhe-NHMe
was treated with 4.0 M HCl in dioxane solution for 1 h. After
evaporation of solvent, the hydrochloride salt was dissolved in aqueous
NaHCO3, and excess acetyl chloride was added with stirring. The
reaction mixture was made basic to pH 8 with NaHCO3, and the
insoluble product was collected by suction filtration. The product was
further purified by recrystallization from a methanol/ether/n-heptane
mixture: 1H NMR (300 MHz, CD3OD) δ 7.33-7.11 (m, 5H), 4.41
(quintet, J ) 6.5 Hz, 1H), 2.81 (ABX, JAB ) 13.4 Hz, JAX ) 6.1 Hz,
Conformation-specific IR spectra were obtained using resonant ion
dip infrared (RIDIR) spectroscopy.33,34 Tunable infrared radiation from
3300 to 3700 cm-1 was produced with a seeded Nd:YAG pumped
parametric converter (LaserVision, KTA based, 10 Hz). Typical infrared
laser powers were 1-5 mJ/pulse. The IR and UV probe lasers were
counter-propagated, spatially overlapped, and temporally separated so
the IR laser preceded the probe by 200 ns. The probe laser wavelength
was fixed on a transition in the electronic spectrum and the ion signal
monitored while the IR was tuned. IR transitions arising from the same
ground state level as the probe laser resonance appear as depletions in
the ion signal. As in UV-UV hole-burning, active baseline subtraction
was employed to compare the difference between IR laser “on” or “off”.
2.2. Calculations. In order to ensure a complete search of the
conformational space of the molecule, two complementary approaches
were taken to identify starting structures for structure optimizations at
higher levels of theory. In one approach, a systematic grid of starting
structures was generated using the dihedral angles (æ, θ, ψ) along the
â-peptide backbone (Figure 1b), using the same procedure as a previous
computational study of N-acetyl-3-aminobutanoic-acid-N′-methylamide
(Ac-â3-hAla-NHMe) from Hofmann et al.26 Alternatively, a random
search was also performed using the Amber force field in MACRO-
JBX ) 7.8 Hz, ∆υ ) 0.07 ppm, 2H), 2.69 (s, 3H), 2.36 (ABX, JAB
)
13.8 Hz, JAX ) 5.7 Hz, JBX ) 6.9 Hz, ∆υ ) 0.05 ppm, 2H), 1.84 (s,
3H); ESI-TOF MS 235.4 [M + H]+, 257.4 [M + Na]+, 491.7 [2M +
Na]+.
The synthesis of Ac-â3-hTyr-NHMe (2) involved an extra step
relative to the synthesis of 1. Ac-â3-hTyr(OBn)-NHMe was synthe-
sized from Boc-Tyr(OBn)-OH by a procedure analogous to that
described above. The O-benzyl group was removed via hydrogeno-
lysis with 10% palladium on activated carbon in methanol to yield the
crude product, which was further purified by recrystallization from
a methanol/ether/n-heptane mixture: 1H NMR (300 MHz, CD3OD) δ
7.02 (d, J ) 7.5 Hz, 2H), 6.69 (, J ) 7.5 Hz, 2H), 4.34 (quintet,
(28) Beke, T.; Csizmadia, I. G.; Perczel, A. J. Comput. Chem. 2004, 25, 285-
307.
(29) Beke, T.; Somlai, C.; Perczel, A. J. Comput. Chem. 2006, 27, 20-38.
(30) Baquero, E. E.; James, W. H.; Choi, S. H.; Gellman, S. H.; Zwier, T. S. J.
Am. Chem. Soc. 2008, 130, 4795-4807.
(31) Seebach, D.; Abele, S.; Gademann, K.; Guichard, G.; Hintermann, T.; Jaun,
B.; Matthews, J. L.; Schreiber, J. V. HelV. Chim. Acta 1998, 81, 932-982.
(32) Shubert, V. A.; Baquero, E. E.; Clarkson, J. R.; James, W. H., III; Turk, J.
A.; Hare, A. A.; Worrel, K.; Lipton, M. A.; Schofield, D. P.; Jordan, K.
D.; Zwier, T. S. J. Chem. Phys. 2007, 127, 234315.
(33) Zwier, T. S. Annu. ReV. Phys. Chem. 1996, 47, 205.
(34) Page, R. H.; Shen, Y. R.; Lee, Y. T. J. Chem. Phys. 1988, 88, 4621.
9
4786 J. AM. CHEM. SOC. VOL. 130, NO. 14, 2008

â
-Peptides Ac-
â
3-hPhe-NHMe and Ac-
â
3-hTyr-NHMe
A R T I C L E S
MODEL.35 Lists of starting structures from the two methods were
compared and used as the basis for structural optimizations using DFT
B3LYP/6-31+G* which employed the GAUSSIAN 03 suite of
programs.36 Since B3LYP calculations do not properly account for
dispersion forces, single point MP2/6-31+G* calculations were per-
formed on the optimized DFT structures.37-40 Also, in an effort to
improve our structural assignments, selected structures were reoptimized
and the harmonic frequencies recalculated at the RIMP2/aug-cc-pVDZ
level of theory.41-50
3. Results
The R2PI spectrum of Ac-â3-hPhe-NHMe in its S0-S1 origin
region (37380-37590 cm-1) is shown in Figure 2a. This
spectrum is dominated by a strong transition at 37431 cm-1
which is 100 cm-1 lower in frequency than the lowest-frequency
conformer origin observed in phenylalanine.51,52 A UVHB
spectrum taken with the hole-burning laser fixed on transition
A is shown in Figure 2b. Several of the weak intensity transitions
appearing in the UVHB spectrum can be ascribed to vibronic
bands of conformer A. Bands labeled B, D, and W do not hole-
burn with transition A and hence are due to other species. R2PI
spectra taken while monitoring other mass channels indicate
that bands at 37465 cm-1 (labeled D) and 34576 cm-1 (labeled
W) are due to the Ac-â3-hPhe-NHMe dimer and Ac-â3-hPhe-
NHMe‚H2O complex, respectively. These assignments were
confirmed by R2PI scans monitoring the relevant cluster mass
channels (not shown).
The peak marked B at 37404 cm-1 is not due to a cluster
and did not vary in intensity with backing pressure relative
to A, indicating that it is not a hot band. We shall see shortly
that this band has a unique infrared spectrum and is
thus ascribable to a second, minor conformer of Ac-â3-hPhe-
NHMe.
The R2PI spectrum of the tyrosine-substituted analogue (Ac-
â3-hTyr-NHMe) is shown over the 35200-35700 cm-1 region
in Figure 2c). This close analogue of Ac-â3-hPhe-NHMe was
studied in order to test whether the conformational preferences
observed in 1 carried over to other close analogues sharing the
same â-peptide backbone. The most intense band of Ac-â3-
hTyr-NHMe occurs at 35318 cm-1, 168 cm-1 red of the S0-S1
origin band of tyrosine.52-54 Several intense transitions appear
Figure 2. (a) R2PI and (b) UV-UV hole-burning spectrum for Ac-â3-
hPhe-NHMe (1). (c, d) Corresponding spectra for Ac-â3-hTyr-NHMe (2).
The asterisks denote the transitions used as the hole-burn transition in each
case.
just to the high-frequency side of this band, leading to a much
more highly congested spectrum than in Ac-â3-hPhe-NHMe.
This increase in the congestion of the spectrum is consistent
with the Franck-Condon activity involving several low-
frequency vibrations, as occurs in the spectra of tyrosine and
tyrosine-substituted polypeptides.52-54 Figure 2d shows the
UV-UV hole-burning of Ac-â3-hTyr-NHMe taken over the
same wavelength region. All transitions to the blue of transition
A burn with A, indicating that the entire spectrum is ascribable
to a single conformation of Ac-â3-hTyr-NHMe. Small transitions
to the red of the origin of A may be ascribable to a second
conformer (analogous to B in Ac-â3-hPhe-NHMe); however,
they were so weak in intensity that they were not pursued
further.
RIDIR spectra of Ac-â3-hPhe-NHMe conformers A and B
in the N-H stretch region (3330-3513 cm-1) are shown in
Figure 3a,b respectively. As expected, both spectra show two
N-H stretch fundamentals associated with the two amide NH
groups. Conformer A possesses a sharp NH stretch fundamental
at 3488 cm-1 and a broadened, more intense transition shifted
(35) Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton, M.;
Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem.
1990, 11, 440-467.
(36) Frisch, M. J.; et al. Revision C.02 ed.; Gaussian, Inc.: Wallingford, CT,
2004.
(37) Zhao, Y.; Truhlar, D. G. J. Chem. Theory Comput. 2007, 3, 289-300.
(38) van Mourik, T. Chem. Phys. 2004, 304, 317.
(39) Wodrich, M. D.; Corminboeuf, C.; Schleyer, P. V. Org. Lett. 2006, 8, 3631-
3634.
(40) Perezjorda, J. M.; Becke, A. D. Chem. Phys. Lett. 1995, 233, 134-
137.
(41) RIMP2 Calculations with the Dunning basis set were performed by Dr.
Daniel P. Schofield from Prof. Kenneth Jordan’s research group, Department
of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260.
(42) Bernholdt, D. E.; Harrison, R. J. Chem. Phys. Lett. 1996, 250, 477.
(43) Dunning, T. H. J. Chem. Phys. 1989, 90, 1007.
(44) Feyerisen, M.; Fitzgerald, G.; Komornicki, A. Chem. Phys. Lett. 1993, 208,
359.
(45) Kendall, R. A.; Dunning, T. H.; Harrison, R. J. J. Chem. Phys. 1992, 96,
6796.
(46) Petersson, K. A.; Dunning, T. H. J. Chem. Phys. 2002, 117, 10548.
(47) Woon, D. E.; Dunning, T. H. J. Chem. Phys. 1993, 98, 1358.
(48) Woon, D. E.; Dunning, T. H. J. Chem. Phys. 1994, 100, 2975.
(49) Woon, D. E.; Dunning, T. H. J. Chem. Phys. 1995, 103, 4572.
(50) Vahtras, O.; Almlof, J.; Feyerisen, M. W. Chem. Phys. Lett. 1993, 213,
514.
(51) Snoek, L. C.; Robertson, E. G.; Kroemer, R. T.; Simons, J. P. Chem. Phys.
Lett. 2000, 321, 49-56.
(52) Martinez, S. J.; Alfano, J. C.; Levy, D. H. J. Mol. Spectrosc. 1992, 156,
421-430.
(53) Cohen, R.; Brauer, B.; Nir, E.; Grace, L.; Vries, M. S. d. J. Phys. Chem.
A 2000, 104, 6351-6355.
(54) Grace, L. I.; Cohen, R.; Dunn, T. M.; Lubman, D. M.; Vries, M. S. d. J.
Mol. Spectrosc. 2002, 215, 204-219.
9
J. AM. CHEM. SOC. VOL. 130, NO. 14, 2008 4787

A R T I C L E S
Baquero et al.
Table 1. Experimentally determined S1 r S0 Origins and IR
Bands of the Two Conformers of Ac-â3-hPhe-NHMe (1) and the
Single Conformer of Ac-â3-hTyr-NHMe (2)
(S1 r S0)
NH(1)
NH(2)
OH
ring
origins
stretch
stretch
stretch
(cm-
∆
va
1
1
1
1
1
molecule
type
(cm-
)
(cm-
)
(cm-
)
)
(cm-
)
Ac-â3-hPhe-NHMe (A) C6
Ac-â3-hPhe-NHMe (B) C8
37431
37404
35318
3400 3488
3454 3416
3400 3488 3650 -54
-54
-72
Ac-â3-hTyr-NHMe
C6
a ∆ν column represents the frequency shift with respect to the free NH
position that occurs upon hydrogen bonding. The free NH frequency position
for the C6 and C8 conformers is taken to be 3488 and 3454 respectively.
by 34 cm-1, a larger difference than the H-bonded NH stretches.
The shift to 3454 cm-1 in Figure 3b could indicate formation
of a weak H-bond, such as would occur between an amide NH
and the phenyl ring. However, the other possibility is one noted
in the introduction, namely, that the frequencies of the free
amide NH stretch of the two amide NH groups differ from one
another by virtue of their chemically distinct positions in the
â-peptide backbone. The structure of Ac-â3-hPhe-NHMe shows
that the N-terminal amide NH(1) is attached to a branched-
chain carbon atom, while the C-terminal NH(2) is bonded to a
methyl group. According to the previous studies in solu-
tion,25,57,58 NH(1) is thereby predicted to be lower in frequency
than NH(2) by about 30-40 cm-1, as observed.
On this basis, we tentatively assign the band at 3454 cm-1
to the interior amide group to which the â3 benzyl side-chain is
attached, and the 3488 cm-1 transition to the methyl-terminated
amide group. H-bonds would then necessarily involve the other
amide NH, forming a C6 H-bonded ring in conformer A, and
a C8 H-bonded ring in conformer B. Similarly, the only
observed conformer of the Tyr analogue would also be assigned
to a C6 structure. A summary of these results is shown in
Table 1.
Figure 3. RIDIR spectra in the amide NH stretch region for (a, b)
conformers A and B of Ac-â3-hPhe-NHMe, 1, respectively, and (c) the
sole conformer of Ac-â3-hTyr-NHMe, 2.
4. Calculations
down in frequency to 3400 cm-1. Both of these characteristics
of the 3400 cm-1 fundamental indicate that the N-H group
responsible for this absorption is involved in a hydrogen bond.
Similarly, conformer B shows a free N-H stretch fundamental
at 3454 cm-1 and a broadened, more intense transition ascribable
4.1. Conformational Minima. In order to learn more about
the structural possibilities available to Ac-â3-hPhe-NHMe and
to refine our assignments, we have used the search and
optimization procedure described in Section 2.B to obtain
optimized structures, harmonic vibrational frequencies, and
infrared intensities for all low-lying minima within 30 kJ/mol
of the global minimum on the potential energy surface for Ac-
â3-hPhe-NHMe.
to a H-bond at 3416 cm-1
.
Figure 3c shows the RIDIR spectrum of the single conformer
present in Ac-â3-hTyr-NHMe, taken over the 3330-3680 cm-1
region, which includes both amide NH stretch and OH stretch
fundamentals. The two fundamentals in the NH stretch region
are within 1 cm-1 of their values in conformer A in Ac-â3-
hPhe-NHMe, indicating that the most stable backbone confor-
mational preference is maintained upon substitution of Tyr for
Phe. This spectrum also shows a band at 3650 cm-1 assigned
to the O-H stretch of Tyr, appearing within a few cm-1 of its
value in phenol monomer (3657 cm-1).55,56
A preliminary assignment for the H-bonded structures
responsible for the IR spectra can be obtained immediately from
the spectra. In each case, the spectra contain a ‘free’ and a
H-bonded NH stretch fundamental. Interestingly, the ‘free’ NH
stretch fundamentals of Figure 3a,b are shifted from one another
As anticipated, all such conformations of Ac-â3-hPhe-NHMe
contain an NH‚‚‚OdC H-bond that links an NH of one amide
with the carbonyl group of the other. As previously described,
the six-membered cycle (C6) is formed when the N-H of
the N-terminal amide (NH(1)) is hydrogen bonded to the CdO
of the C-terminal amide (CO(1)). Alternatively, the eight-
membered cycle (C8) forms when the N-H of the C-ter-
minal amide (NH(2)) hydrogen bonds to the CdO of the
N-terminal amide (CO(2)). Different arrangements of the
dihedral angles along the â-peptide backbone give rise to distinct
C6 and C8 arrangements. Figure 4 shows representative
examples of the different types of C6 and C8 structures which
can be formed. These structures differ from one another
(55) Ebata, T.; Watanabe, T.; Mikami, N. J. Phys. Chem. 1995, 99, 5761-
5764.
(57) Fillaux, F.; Loze, C. Biopolymers 1972, 11, 2063-2077.
(58) Aubry, A.; Cung, M. T.; Marraud, M. J. Am. Chem. Soc. 1985, 107, 7640-
7647.
(56) Tanabe, S.; Ebata, T.; Fujii, M.; Mikami, N. Chem. Phys. Lett. 1993, 215,
347-352.
9
4788 J. AM. CHEM. SOC. VOL. 130, NO. 14, 2008

â
-Peptides Ac-
â
3-hPhe-NHMe and Ac-
â
3-hTyr-NHMe
A R T I C L E S
Figure 4. Representative structures (DFT B3LYP/6-31+G*). (Left/right) Three lowest energy C6/C8 chain arrangements. The phenyl ring has been removed
for clarity. (Center) Three possible chromophore positions for the phenylalanine or tyrosine side chain on the â3 carbon.
Table 2. Key Spectroscopic and Structural Data Calculated for the Low-Lying Minima of Ac-â3-hPhe-NHMe, 1, at the Indicated Level of
Theory
O
‚‚‚
H
calculated NH(1)b
calculated NH(2)b
∆
E
0 DFTc
∆
E
0 MP2d
∆
E
0 RIMP2e
fractionalf population
behind nozzle
1
1
conformer
æa
θa
ψa
distance (Å)
stretch (cm-
)
stretch (cm-
)
(OPT) (kJ/mol)
(SP) (kJ/mol)
(OPT) (kJ/mol)
extended(2) -118.3
17.3
-60.8 -134.9
53.0
132.6
-63.8 -148.0
67.7
143.0
-
3455.55
3411.18
3397.93
3462.11
3443.6 9
3463.88
3480.10
3453.79
3448.24
3452.87
3401.95
3379.55
3462.15
3481.48
3489.06
3490.11
3488.33
3373.86
3488.06
3348.17
3437.03
3361.00
3434.51
3390.44
3491.62
3487.29
3343.05
3373.48
36.87
0.00
5.63
7.16
7.34
8.66
9.70
9.77
10.65
11.12
12.13
14.18
16.11
20.52
45.58
0.00
8.32
7.29
9.85
7.57
4.31
8.55
5.76
8.70
11.96
4.74
18.10
17.28
-
-
C6a(1)
C6b(1)
C8a(3)
C6c(3)
C8b(1)
C8c(3)
C8b(2)
C8d(3)
C8a(2)
C6b(2)
C6a(2)
C8a(1)
C8e(3)
-147.7
-161.4
-70.0
-112.3
-109.7
50.1
-109.5
-54.4
-71.3
-161.7
-148.2
-78.2
2.08
2.13
2.05
2.18
1.96
2.34
1.99
2.31
2.22
2.12
1.98
1.97
1.94
0.00
11.55
11.34
-
0.445
0.032
0.033
0.014
0.035
0.176
0.060
0.064
0.027
0.007
0.101
0.002
0.003
108.8
-73.9
11.3
13.60
-
53.0 -108.4
68.4
-49.6
138.7
52.6
10.5
105.5
-74.1
110.5
7.49
-
7.41g
-
-48.8 -132.6
132.7
-
-49.5
82.4
-
58.0 -120.2
-
a Dihedral angles along â-peptide chain (for definition, see text and Figure 1b). Different dihedral arrangements are included in the conformer labeling
by the letters a-c for C6 conformers and a-e for C8 conformers. C6 (a, b, c) correspond to structures labeled B1, B5, B4 by Hofman.26 C8 (a,b,c,d,e)
correspond to structures labeled B2, B6, B3′, B3, B2′ by Hofman.26 b Calculated NH stretches for NH(1) and NH(2) along the â-peptide chain (free NH
stretches are italic). c Zero point corrected energies of optimized structures at DFT/6-31+G* level of theory. d Single point, zero point corrected energies at
MP2/6-31+G* level of theory of DFT/6-31+G* structures (zero point correction done with DFT harmonic frequencies). e Zero point corrected energies of
selected structures optimized at RIMP2 level. f Fractional population of conformers behind the nozzle calculated by adding the free energy available at
the stagnation temperature (453 K) to the zero point corrected MP2 single point energies. g C8a(2) Experience a major structural change upon RIMP2
optimization.
in the relative orientation of the peptide groups involved in the
H-bond created by different dihedral angles in the â-peptide
backbone. The different ring types are labeled a-c for C6
conformers and a-e for C8 conformers in Table 2. The torsional
dihedral angles associated with the structures listed in Table 2
have been previously observed in computational work by
Hofman and others.26-29 In addition, for each â-peptide
backbone structure, there are three distinct positions, numbered
1 through 3, for the phenyl ring. In some cases, not all three
phenyl ring positions give stable minima, producing five C6
structures and eight C8 structures with energies within 20 kJ/
mol of the global minimum. The key dihedral angles and relative
energies of the lowest energy (<20 kJ/mol) optimized structures
are listed in Table 2.
4.2. Vibrational Frequencies. In order to assess whether the
calculations predict a difference in frequency for the two amide
NH stretch modes, a completely extended structure for the
â-peptide backbone was optimized and the free amide NH
stretch fundamentals calculated for branched-chain and methyl-
terminated amide NH groups. As can be seen from Table 2,
the methyl-terminated free NH(2) stretch has a calculated
frequency of 3490 cm-1 (after scaling by 0.96 to match the
9
J. AM. CHEM. SOC. VOL. 130, NO. 14, 2008 4789

A R T I C L E S
Baquero et al.
Figure 5. (a, b) Top traces: RIDIR spectra of conformer A and B of Ac-â3-hPhe-NHMe, 1 in the NH stretch region. The stick diagrams below the
experimental spectra are calculated, scaled harmonic vibrational frequencies, and infrared intensities (DFT B3LYP/6-31+G*) of representative C6 (top) and
C8 (bottom) â-peptide backbone arrangements. Structures on both sides are connected to their calculated IR spectrum. The C8d(3) and C8a(2) are structures
with unusually weak H-bonds due to an acute angle or large distance of the C8 H-bond.
calculated highest-frequency free NH to the one observed
experimentally), while the branched-chain free NH(1) stretch
is 30 cm-1 lower in frequency. Furthermore, this difference in
frequency is seen to be a general feature of all conformational
minima. Whenever the methyl terminated chain NH(2) is free,
its frequency is in a range from 3487 to 3490 cm-1, while the
branched-chain free NH(1) frequency is predicted to fall
somewhere in the range from 3448-3481 cm-1. The stick
spectra shown in Figure 5a illustrate this for a representative
set of C6 and C8 structures. Note that the two regions are
nonoverlapping, and hence diagnostic of which NH group is
free. Therefore, the calculations support the assignment of con-
former A to a C6 structure and conformer B to a C8 structure.
The calculations also predict a close match between experi-
ment and calculation in the frequencies and relative infrared
intensities of the H-bonded NH stretch modes of the C6
conformers. The full set of calculated frequencies are given in
Table 2, while Figure 5a shows the calculated spectra of the
lowest two C6 conformers, which differ in the type of C6 ring
(a vs b, Figure 2) but share the same phenyl ring position
(position 1, Figure 2). We cannot distinguish between these two
C6 conformations based on the infrared spectra, but the fact
that C6a(1) is calculated to be lowest in energy at all levels of
theory supports this assignment.
conformer. A survey of Table 2 indicates that the great majority
of C8 conformers have calculated, scaled frequencies for the
H-bonded NH stretch that are lower in frequency than the
H-bonded NH stretch of the C6 conformers, at odds with
experiment. The stick spectrum of the calculated, lowest-energy
C8 conformer is included in Figure 5b, illustrating this fact.
This led us to search carefully for C8 conformers with a weaker
H-bond, leading to a smaller H-bonded NH stretch frequency
shift. The structures and calculated stick spectra for two such
C8 conformers are shown in Figure 5b. The C8d(3) conformer
has a highly unusual C8 ring (labeled ‘d’) that forms an NH‚
‚‚OdC H-bond in which the two amide planes are at an acute
angle that leads to a strongly bent H-bond, which is thereby
weakened. The C8a(2) conformer has an unsually large H-bond
distance caused by the presence of the phenyl ring in the 2
position, where both branches of the â-peptide backbone are in
gauche positions relative to the phenyl ring. The NH(1) group
is positioned so that it can interact with the phenyl ring to form
a stabilizing NH‚‚‚π interaction, thereby opening up the C8 ring.
Because DFT is known37-40 not to account for dispersive
interactions properly, the C8a(2) structure and several others
were optimized at the RIMP2/aug-cc-pVDZ level of theory.
While most structures showed only modest shifts in relative
energies or vibrational frequencies, the C8a(2) structure opened
up the C8 ring in order to accommodate a stronger dispersive
interaction between the NH(1) group and the π cloud, leading
The comparison between experiment and theory is much less
satisfying for the H-bonded NH stretch of conformer B, the C8
9
4790 J. AM. CHEM. SOC. VOL. 130, NO. 14, 2008

â
-Peptides Ac-
â
3-hPhe-NHMe and Ac-
â
3-hTyr-NHMe
A R T I C L E S
Figure 6. Relative energies for Ac-â3-hPhe-NHMe (1) conformers at different levels of theory. The structural details and quantitative energies for each
structure are given in Table 2. DFT(OPT): Fully optimized structures computed using DFT B3LYP/6-31+G*. MP2(SP): Single point MP2/6-31+G*
calculations at the optimized DFT geometries. ∆G corrected energies: Relative free energies at the preexpansion temperature (453 K) based on the MP2(SP)
relative energies. All calculations are zero point corrected utilizing DFT calculated harmonic frequencies. RIMP2(OPT): Fully optimized relative energies
for a select set of structures at the RIMP2/aug-cc-pVDZ level of theory.
to a significant shift of the H-bonded NH stretch toward higher
frequency. In fact, the RIMP2 calculations still do not match
up with experiment quantitatively, in this case predicting that
the C8 H-bond is essentially broken. This result highlights the
sensitivity of this unique C8 H-bond to the proper description
of dispersive interactions.
Condon activity in low-frequency vibrations, we do not
anticipate large differences in the Franck-Condon factors or
oscillator strengths associated with the origin transitions. If these
differences are small, the observed intensity ratio (22:1) indicates
that about 95% of the population of Ac-â3-hPhe-NHMe is in
the C6 conformer and the remaining 5% in the C8 conformer.
On the basis of these calculations, we make a tentative
assignment of the C8a(2) structure as the observed C8 con-
former. If correct, the overcompensation of the RIMP2 calcula-
tions must be attributed to this level of theory overemphasizing
dispersive interactions, as has been noted previously.59,60 We
anticipate that calculations carried out at higher levels of theory
would lead to vibrational frequencies somewhere between the
DFT and RIMP2 ‘limits’, in keeping with experiment.
A first question that should be addressed, then, is why this
preference for C6 over C8 structures exists. Figure 6 presents
an energy level diagram that summarizes the relative energies
of the low-energy conformers of Ac-â3-hPhe-NHMe calculated
at the DFT B3LYP/6-31+G* level of theory. The figure also
includes the results of MP2 single point calculations at the
B3LYP-optimized geometries, and RIMP2/aug-cc-pVDZ opti-
mizations on a select subset of structures. All levels of theory
predict C6a(1) as the lowest energy structure. Above this, C6
and C8 conformers are close in energy and intermingled with
one another.
5. Discussion
5.1. Conformational Preferences and Infrared Spectral
Signatures. A primary finding of the present study is that the
spectra of the expansion-cooled, isolated â-peptides Ac-â3-hPhe-
NHMe and Ac-â3-hTyr-NHMe are dominated by transitions
assigned to a C6 structure, forming a six-membered, H-bonded
cycle between NH(1) and CO(2). A second, minor conformer
has been assigned to a C8 structure, with an intramolecular
H-bond formed between NH(2) and CO(1). In the present
circumstances, it seems likely that the relative intensities of the
S0-S1 origins are a reasonable measure of their relative
populations. Since neither conformer shows significant Franck-
Figure 6 also shows free-energy corrected relative energies
of the same conformers computed at the temperature of the
sample prior to supersonic expansion (453 K). The free energy
correction was computed using harmonic vibrational frequencies
from the B3LYP/6-31+G* calculations. C6a(1) remains the
most populated conformer and thereby a likely candidate for
the observed C6 conformer. However, C6b(1) displays a better
match between the experimental and calculated H-bonded NH
stretch. Our confidence in the accuracy of the calculated
vibrational frequencies or relative energies/fractional populations
is insufficient to make a firm assignment on the basis of either
one.
(59) Hopkins, B. W.; Gregory, S. T. Chem. Phys. Lett. 2005, 407, 362-367.
(60) Levy, J. B.; Martin, N. H.; Hargittai, I.; Hargittai, M. J. Phys. Chem. A
1998, 102, 274-279.
9
J. AM. CHEM. SOC. VOL. 130, NO. 14, 2008 4791

A R T I C L E S
Baquero et al.
these barriers are small compared to the average internal energy
available to the molecules, collisional cooling could redistribute
the population during the early portion of the expansion, thereby
funnelingthepopulationtowardthelowestenergyconformers.62,68-72
From an experimental standpoint, this calls for direct experi-
mental measures of the barriers separating the isomers, using
either SEP or IR population transfer spectroscopy.73-75 Since
calculations at both DFT and RIMP2 levels of theory consis-
tently predict the C6a(1) structure to be most stable, this
conformer would be the natural recipient of population from
other conformers during the cooling process. It is less clear why
a single C8 conformer is also observed, despite the fact that
several other structures, both C6 and C8, are calculated to be
lower in energy than the weakly H-bonded C8 conformer which
is most consistent with the experimental infrared spectrum. If
the potential energy surface divides into two subspaces involving
C6 and C8 conformers with large barriers separating them, then
collection of population from the C8 family into a single well
is plausible. Ultimately, a full exploration of the barriers
separating minima (leading to a disconnectivity diagram)76,77
and simulation of the expansion cooling will be needed before
a quantitative account of the observed populations is possible.
Another primary result of the present study is the determi-
nation of the amide NH stretch infrared spectral signatures for
the observed C6 and C8 conformers under jet-cooled, isolated
conditions. Interestingly, the key to our assignment is the free
amide NH stretch fundamental rather than the H-bonded NH
stretch. One might have thought that the size of the H-bonded
ring would present the most obvious and characteristic difference
in the NH stretch region, which is exquisitely sensitive to its
H-bonding environment. However, the H-bonded NH stretch
transitions of conformers A and B are within 20 cm-1 of one
another, making it difficult to secure an assignment as C6 or
C8 on this basis alone.
Figure 7. Calculated shifts of the scaled, H-bonded C6 or C8 NH stretch
vibrational frequencies (in cm-1
)
of Ac-â3-hPhe-NHMe (1) versus
NH‚‚‚‚OC distance. The zero of the relative frequency scale is the scaled
frequency of the free NH stretch of 1 at 3489 cm-1 for C6 and 3458 cm-1
for C8 conformers.
The dominance of the C6 conformer (both in the Phe and
Tyr analogues) occurs despite the fact that even the weak
H-bond formed in the observed C8 conformer is stronger than
that in the C6 conformer, based on the magnitude of the
frequency shift induced by the intramolecular H-bond, which
was greater for the C8 (-72 cm-1) than C6 (-54 cm-1). As
Figure 7 shows, the frequency shift is inversely correlated with
the H-bond distance, another oft-used measure of the strength
of the H-bond.
This counterintuitive result is in keeping with previous
deductions24 that observed conformational preferences represent
a delicate balance between H-bond strength, ring strain, and
other, less-specific stabilizing factors such as dispersive interac-
tions. This result is driven home by the fact that the calculations
show a much wider spread in energies among C6 or C8
conformers than separates the two families (Figure 6).
Given the results of Figure 6, it is also intriguing that only
one C6 and one C8 conformer are observed experimentally.
Table 2 lists the fractional populations in the preexpansion gas
mixture predicted by the calculations. The average internal
energy of predicted conformers is calculated at the preexpansion
sample temperatures (453 K) using unscaled B3LYP frequencies
and single point MP2 energies at the DFT-optimized geometries.
The energy distribution obtained from this calculation was used
to determine the fractional population of the predicted conform-
ers. These populations are much more broadly distributed than
experiment, indicating either that the calculated preexpansion
populations are not accurate, or that significant transfer of
population into the lowest energy C6 and C8 conformational
wells occurs during the expansion cooling. Since one of the
goals of these studies is to provide benchmark data for
calculations, it is important for calculations of these quantities
to be carried out at higher levels of theory until convergence is
reached.
5.2. Comparison with Solution. One interesting comparison
that is worth making is the one between the conformation-
specific infrared spectra obtained in the present study and the
infrared spectrum of the same molecule in room-temperature,
nonpolar solution.
Several previous studies of the conformational preferences
of closely analogous molecules have been carried out. Those
most relevant to the present work are the studies of â-peptides
by Marraud et al.25 and Dado and Gellman.8 The study of 3
and 4 by Marraud et al. was mentioned earlier as the basis of
the assignment of the NH group responsible for a given free
(64) Kaczor, A.; Reva, I. D.; Proniewicz, L. M.; Fausto, R. J. Phys. Chem. A
2006, 110, 2360-2370.
(65) Kim, D.; Baer, T. Chem. Phys. 2000, 256, 251-258.
(66) Pitts, J. D.; Knee, J. L.; Wategaonkar, S. J. Chem. Phys. 1999, 110, 3378-
3388.
(67) Reva, I. D.; Stepanian, S. G.; Adamowicz, L.; Fausto, R. Chem. Phys. Lett.
2003, 374, 631-638.
It will also be important to learn more about the barriers that
separate the conformational isomers listed in Figure 6. Several
past studies on other flexible biomolecules have concluded that
the downstream populations are representative of the preex-
pansion populations, as would occur if collisional cooling were
fast compared to isomerization.61-68 However, in cases where
(68) Ruoff, R. S.; Klots, T. D.; Emilsson, T.; Gutowsky, H. S. J. Chem. Phys.
1990, 93, 3142-3150.
(69) Basu, S.; Knee, J. L. J. Phys. Chem. A 2001, 105, 5842-5848.
(70) Godfrey, P. D.; Brown, R. D.; Rodgers, F. M. J. Mol. Struct. 1996, 376,
65-81.
(71) McCoustra, M. R. S.; Hippler, M.; Pfab, J. Chem. Phys. Lett. 1992, 200,
451-458.
(72) Miller, T. F.; Clary, D. C.; Meijer, A. J. Chem. Phys. 2005, 122.
(73) Clarkson, J. R.; Baquero, E.; Shubert, V. A.; Myshakin, E. M.; Jordan, K.
D.; Zwier, T. S. Science 2005, 307, 1443-1446.
(61) Macleod, N. A.; Simons, J. P. Phys. Chem. Chem. Phys. 2004, 6, 2878-
(74) Clarkson, J. R.; Baquero, E.; Zwier, T. S. J. Chem. Phys. 2005, 122.
(75) Dian, B. C.; Clarkson, J. R.; Zwier, T. S. Science 2004, 303, 1169-1173.
(76) AdVances in Chemical Physics; Wales, D. J.; Doye, P. K.; Miller, M. A.;
Mortenson, P. N.; Walsh, T. R., Eds.; Wiley: New York, 2000; Vol. 115.
(77) Becker, O. M.; Karplus, M. J. Chem. Phys. 1997, 106, 1495.
2884.
(62) Felder, P.; Gunthard, H. H. Chem. Phys. 1982, 71, 9-25.
(63) Fraser, G. T.; Suenram, R. D.; Lugez, C. L. J. Phys. Chem. A 2001, 105,
9859-9864.
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4792 J. AM. CHEM. SOC. VOL. 130, NO. 14, 2008

â
-Peptides Ac-
â
3-hPhe-NHMe and Ac-
â
3-hTyr-NHMe
A R T I C L E S
amide NH stretch in branched-chain versus unbranched-chain
amides. In CCl4 solution, they observed a set of four bands in
the amide NH stretch region, which were attributed to a mixture
of C6 and C8 structures, with the former possessing a free NH
at 3469 cm-1 and a H-bonded NH stretch at 3393 cm-1, while
the latter had its analogous bands at 3437 and 3340 cm-1
.
Dado and Gellman carried out a complementary study of the
â-diamides 5-7, shown below.
By appropriate substitution, diamides 5 and 6 are capable of
forming only C6 or C8 H-bonded rings, respectively, while 7
can form both. In dilute CH2Cl2 solution, amide NH stretch IR
spectra of 5 in CH2Cl2 showed a single band peaked at 3440
cm-1, taken as evidence that no C6 H-bonded rings were
present, while 6 showed two bands at 3455 and 3295 cm-1
,
signaling the presence of and competition between non-H-
bonded structures and those containing C8 rings. The band at
3295 cm-1, attributed to the C8 ring, is much lower in frequency
than the one observed in the gas-phase RIDIR spectrum of
conformer B of 1 (3416 cm-1). The shift to higher frequency
in the gas-phase experiment cannot be ascribed solely to solvent
effects but points to the fact that the C8 conformer observed in
the jet has an unusually weak H-bonded arrangement. Finally,
in 7, a single peak at 3453 cm-1 was assigned to non-H-bonded
NH groups, indicating that neither C6 nor C8 rings were
substantially populated in room temperature solution.
This body of results illustrates how sensitive the conforma-
tional preferences of small â-peptides are to substitution and to
solvent. As a result, we decided to record solution-phase infrared
spectra of 1, with its phenylalanine substituent, for direct
comparison with the jet-cooled, isolated molecule results.
Figure 8 presents this comparison. The room-temperature,
solution-phase spectrum of a 1 mM solution of 1 in CH2Cl2
solvent is shown in the figure, shifted to higher frequency by
35 cm-1 in order to match the free amide NH stretch transitions
in solution with the C6 gas-phase result. The solution-phase
spectrum shows a shoulder 30 cm-1 below the main free amide
NH stretch transition which is ascribable to the free amide NH
stretch transition of NH(1), the interior branched-chain NH
group. Below this shoulder, the absorption tails off toward lower
frequency, with no further well-defined peaks.
In the isolated molecule, the H-bonded NH stretch funda-
mentals of both conformers occur within the low-frequency wing
in the solution-phase spectrum. As a result, we ascribe some or
all of the intensity of the low-frequency wing to C6 or C8 rings.
The lack of a well-defined band in this region probably arises
from broadening of the H-bonded NH stretch in room-
temperature solution, due to modulation of the strength of the
C6 H-bonded ring by the solvent and internal energy effects.
The integrated intensity of this low-frequency wing is only about
50% of the methyl-terminated and branched free NH, indi-
cating that the majority of molecules in solution are not
H-bonded.
Figure 8. Comparison of single-conformation amide NH stretch infrared
spectra obtained in the present study to solution-phase FT-IR spectra of a
1 mM solution of Ac-â3-hPhe-NHMe (1) in CH2Cl2. Bands are labeled A
C6 and B C8 for the two observed conformers of 1. The solution-phase
trace is shifted 35 cm-1 to higher frequency to match the free NH stretch
band of A in the isolated molecule spectra.
since the free NH stretch frequency depends primarily on local
chemical structure and not its chain conformation. As a result,
the intensity ratio of the two free NH peaks reflects the total
number of free NH groups of each type in solution, quantita-
tively so if the two free NH stretch modes have identical
oscillator strengths. In the limit that no intramolecular H-bonded
rings are formed, the spectrum would then consist of two free
NH stretch peaks of roughly equal intensity. We therefore
interpret the observed large intensity of the methyl-terminated
free NH in the spectrum as a reflection of the dominance of
non-H-bonded and C6 H-bonded rings in solution.
6. Conclusions
In this study, double resonance spectroscopy has been used
to record the infrared and ultraviolet spectra of single conforma-
tions of two model â-peptides, Ac-â3-hPhe-NHMe and Ac-â3-
hTyr-NHMe. The characteristic amide NH stretch frequencies
for both free amide NH and H-bonded amide NH will serve as
useful benchmarks for analogous studies of longer â-peptide
chains. In particular, the adjoining paper describes a study of
â-peptides Ac-â3-hAla-â3-hPhe-NHMe and Ac-â3-hPhe-â3-
hAla-NHMe which builds directly upon the present work.
Acknowledgment. E.E.B., W.H.J., and T.S.Z. gratefully
acknowledge support for this work from the National Science
Foundation (CHE-0551075). The authors also gratefully ac-
knowledge Daniel Schofield and Kenneth D. Jordan from the
University of Pittsburgh for optimizing the structures of a select
subset of conformational minima of Ac-â3-hPhe-NHMe at the
RIMP2/aug-cc-VDZ level of theory. They also gratefully
By contrast, the free amide NH stretch bands can have
contributions both from non-H-bonded and H-bonded structures,
9
J. AM. CHEM. SOC. VOL. 130, NO. 14, 2008 4793

A R T I C L E S
Baquero et al.
acknowledge V. Alvin Shubert for helping set up systematic
explorations of the conformational phase space of these
molecules. S.H.G. and S.H.C. acknowledge support under NSF
CHE-0551920. S.H.C. was supported in part by the Samsung
Scholarship Foundation.
Supporting Information Available: Complete ref 36. This
material is available free of charge via the Internet at
http://pubs.acs.org.
JA078271Y
9
4794 J. AM. CHEM. SOC. VOL. 130, NO. 14, 2008