Structure elucidation of amide bonds with dipolar chemical shift NMR spectroscopy

Article abstract of DOI:10.1021/jp971904i

The structure of the amide bonds of gluconamide has been elucidated and compared to acetanilide by the combined application of ¹³C and ¹⁵N double- and triple-resonance solid-state NMR spectroscopy. The length of the amide bond has be

Full text of DOI:10.1021/jp971904i

J. Phys. Chem. B 1997, 101, 11265-11272
11265
Structure Elucidation of Amide Bonds with Dipolar Chemical Shift NMR Spectroscopy
G. Buntkowsky,* I. Sack, H. H. Limbach, B. Kling, and J. Fuhrhop
Institut fu¨r Organische Chemie der Freien UniVersita¨t Berlin, Takustrasse 3, D-14195 Berlin, Germany
ReceiVed: June 10, 1997; In Final Form: October 16, 1997X
The structure of the amide bonds of gluconamide has been elucidated and compared to acetanilide by the
combined application of 13C and 15N double- and triple-resonance solid-state NMR spectroscopy. The length
of the amide bond has been determined from the dipolar spectrum using a SEDOR type experiment, and the
orientation of the principal axis systems of both the 13C and 15N chemical shift tensors have been determined
by employing dipolar chemical shift NMR spectroscopy in conjunction with CSA spectroscopy. The groups
exhibit for amide bonds typical approximately 120° bond angles between -CO, -CN, -CR, and -NC,
-NH, -NR. Comparing the structure of the gluconamide with the corresponding structure of the acetanilide,
two major differences are visible: the orientations of the CSA tensors in the amide plane with respect to the
CN-bond direction are different (12° for the 13C tensor and 10° for the 15N tensor), and the directions of least
shielding and intermediate shielding are interchanged in the gluconamide as compared to the acetanilide.
Since the chemical shielding tensors of the 15N are strongly influenced by hydrogen bonding, these different
orientations are an indication of the different hydrogen bond structure of the gluconamide as compared to the
acetanilide.
1. Introduction
amides, namely, acetanilide (which has been recently studied1a)
as a reference substance for the NMR structure of amide
In recent years 13C and 15N solid-state NMR spectroscopies
have become important tools for the structure elucidation of
small peptides and proteins.1 On one hand, the chemical shift
interaction is a sensitive probe of structural features such as
the primary and secondary structure of the molecule; however,
for the interpretation of the corresponding CSA tensors one
usually has to employ ab initio calculations of the molecular
groups under investigation. Due to the importance of the amide
bond as the structure-determining part of the peptide and thus
the proteins, these tensors have been investigated in numerous
studies of amide and peptide bonds. Magnetic dipolar interac-
tions give direct geometrical information about intra- and
intermolecular distances; therefore, this type of interaction has
been used in various studies of bond length or intramolecular
distances. Due to the axial symmetry of the interaction most
information about the relative orientation of the coupled nuclei
is not accessible. It is advantageous to apply a combined
analysis of chemical shielding anisotropy and dipolar interaction.
This approach allows for an orientation of the dipolar vector in
the frame of the chemical shielding interaction. Recent studies
of 13C-CSA and 15N-CSA tensors and 13C-15N dipolar interac-
tion using various solid-state NMR techniques1 demonstrated
the usefulness of this approach. The prime result of all these
studies showed the principal components of the CSA tensors
of the amide carbon and nitrogen in the various polypeptides
investigated to be very similar.2 The chemical shielding
interaction is a local interaction that depends mostly on the
molecular and electronic structure close to the nuclei of interest.
It was found that for the 13C-CSA tensor the most shielded
direction σ33 is perpendicular to the NCO plane and the
intermediate component σ22 is approximately along the -CO
bond. For the 15N-CSA the most shielded component points
approximately into the direction of the 13C-15N bond and the
intermediate component is perpendicular to the amide plane.
In the present study 13C and 15N solid state NMR spec-
troscopies are used for comparing the structure of two different
bonds and N-1-octyl-D-gluconamide. The latter compound
assembles in bulk water to form micellar fibers in the form of
extended quadruple helixes. Such H-bonded fibers have been
isolated in the solid state. The molecular conformation of the
gluconamide headgroup region has been elucidated by solid-
state 13C CP-MAS - NMR spectroscopy3 and comparisons
made with crystal structures of the second, crystalline modifica-
tion of the N-1-octyl-D-gluconamide. It has been found that
the linear arrangement of the N-1-octyl-D-gluconamide in the
crystal (all-trans) is disturbed at C-2. The amide bond
conformation was assumed to be trans in all cases, and chemical
shift differences of the carboxamide carbon were related to
different hydrogen bond lengths. Supporting evidence came
from crystal structure and 13C NMR solid-state spectra of the
crystals.
In a first step the 15N and 13C dipolar interaction was
determined by a variant of the SEDOR4 experiment. Then the
static powder spectra of the 15N and 13C nuclei were measured,
and their CSA tensors and the orientation of the dipolar tensors
in these CSA frames were determined. The 15N and 13C CSA
tensors of the two amides were correlated by dipolar-chemical
shift NMR spectroscopy,1a,h,i allowing one to determine the
relative orientation of the two CSA tensors. Due to the axial
symmetry of the dipolar interaction, the two tensors can be
rotated along the dipolar axis and there exists a cylindrical
ambiguity for the relative orientation.1a The usual approach to
resolve this ambiguity is the application of symmetry arguments,
comparing the system to related systems or ab initio calculations.
However one has to be careful in the application of these second-
hand arguments.
The rest of the article is organized as follows. After a brief
introduction into the theoretical background necessary for
understanding how the CSA tensors are correlated with respect
to each other, a short description of our experimental setup,
sample synthesis, and preparation is given. Then the experi-
mental results are presented and discussed and finally sum-
marized.
* Corresponding author.
X Abstract published in AdVance ACS Abstracts, December 1, 1997.
S1089-5647(97)01904-4 CCC: $14.00 © 1997 American Chemical Society

11266 J. Phys. Chem. B, Vol. 101, No. 51, 1997
Buntkowsky et al.
2. Theory
5σ can then be combined to effective shielding tensors 6ΣPAS( in
the PAS frame of 5σ, which in general are no longer diagonal:
As has been well-known for a long time, the anisotropic
interactions in solid-state NMR can be described by second rank
6ΣPAS( ) 5σPAS - D6CSA ) 5σPAS - RCDD6PASRCD
(8)
-1
tensors, i.e., real symmetric 3*3 matrixes.5 For I ) /2 nuclei
1
and nonconducting organic solids, there are the chemical
shielding tensor 5σ, the dipolar interaction tensor D6, and the
J-coupling (which, however, can be neglected in our systems),
but no quadrupolar tensor interaction. Thus for a heteronuclear
two spin system I, S the Hamiltonian Hˆ can be written as:
Transforming these effective interaction tensors into the lab
frame, the equations for the transition frequencies can be
rewritten as
υS(12,34) ) γSB0(1 - 6ΣS(
υI(13,24) ) γIB0(1 - 6ΣI(
)
(9)
zz
Hˆ ) γIBB(1 - 5σI)ˆI + γsBB(1 - 5σs)Sˆ + ˆID6Sˆ
(1)
)
zz
The matrix elements of the dipolar interaction tensor are given
as5d
While in single crystals only two lines for each spin are
observable, in a powder the average over all possible orientations
has to be taken into account. Due to the axial symmetry of the
magnetic field, it is sufficient to integrate over two angles (ϑ,φ)
only. Thus, assuming that T2 is the transversal relaxation time,
which for simplicity is assumed to be orientational independent,
the spectra can be calculated as
µ
3(bem‚br/r)(ben‚br/r) - bem‚ben
(r)3
D6mn ) - 0 pγIγS
4π
m, n ) x, y, z (2)
In high magnetic field only the secular part of the interaction is
effective. If B0 is pointing in the z-direction one obtains
T2S
S(ν) ) πdϑ sin(ϑ) 2πdæ
+
Hˆ ) γIB0(1 - σIzz)ˆIz + γSB0(1 - σSzz)Sˆz + ˆIzDzzSˆz (3)
∫
∫
2
S
2
0
0
(
1 + 4π (T2(ν - ν12(ϑæ)))
T2S
All terms in this Hamiltonian commute, and it is diagonal with
the following eigenvalues
(10)
2
S
2
)
1 + 4π (T2(ν - ν34(ϑæ)))
1
2
1
2
1
4
E1 ) + γIB0(1 - σIzz) + γSB0(1 - σSzz) + Dzz (4)
T2I
I(ν) ) πdϑ sin(ϑ) 2πdæ
+
∫
∫
2
I
2
0
0
(
1
2
1
2
1
4
1 + 4π (T2(ν - ν13(ϑæ)))
E2 ) + γIB0(1 - σIzz) - γSB0(1 - σSzz) - Dzz
T2I
2
I
2
1
2
1
2
1
4
)
1 + 4π (T2(ν - ν24(ϑæ)))
E3 ) - γIB0(1 - σIzz) + γSB0(1 - σSzz) - Dzz
The result is for each spin a superposition of two powder
patterns, where the singular values of the two patterns are
determined by the principal values of the tensors 6ΣS( or 6ΣI(.
As the effective interaction tensors 6ΣS( and 6ΣI( depend, via
1
2
1
2
1
4
E4 ) - γIB0(1 - σIzz) - γSB0(1 - σSzz) + Dzz
The allowed transitions are (1-2, 3-4) for spin S and (1-3,
2-4) for spin I with the corresponding transition frequencies
the rotations RS and RICD, on the mutual orientations of the
CD
chemical shift and dipolar interaction tensor, simulating the I
and S spin spectra (using eq 10), it is possible to orient the
dipolar vector in the coordinate frame of each shielding
interaction. Thus one obtains information about the mutual
orientation of the two shielding tensors. By successive pairwise
labeling of molecules (or more generally aggregates, since the
dipolar interaction does not depend on the existence of a direct
or indirect chemical bond between the interacting nuclei), it is
possible to map the complete geometrical structure of the
aggregate.
1
2
υS(12,34) ) γSB0(1 - σSzz) ( Dzz
(5)
1
υI(13,24) ) γIB0(1 - σIzz) ( Dzz
2
The tensors D6 and 5σ can be expressed from their diagonal
representation in their principal axis system via a rotation
R(Râγ), which can be parametrized using the Euler angles
(Râγ). In general the CSA and the dipolar interaction tensor
will have different principal axis systems; thus, for each tensor
a separate set of Euler angles is necessary for the transfor-
mation.
If second-order quadrupolar interaction is neglected, the
1
extension to nuclei with I > /2 is straightforward. Instead of
two effective tensors 6ΣS( there are now (2I + 1) tensors 6ΣSm
,
where m varies from -I to I. However, there exists a particular
problem for nuclei with integer spin I (for example 2H), as the
transitions with m ) 0 occur at the same resonance frequency
as the transitions of S nuclei bound to a proton. Thus in these
cases, to correct for imperfect isotope labeling, it is useful to
introduce a labeling factor to the m ) 0 transition. In the case
of isotopes with half-integer spin I, the spectra of the unlabeled
system will be different from all the subspectra and thus no
such problems exist.
-1
D6 ) R(RDâDγD)D6PASR(RDâDγD)-1 ) RDD6PASRD
(6)
-1
5σ ) R(RCâCγC)5σPASR(RCâCγC)-1 ) RC5σPASRC
However, it is always possible to first transform the dipolar
interaction tensor from its principal frame into the frame of the
CSA tensor (DCSA) by a rotation RCD and then do a common
transformation into the lab frame:
3. Materials and Methods
-1
D6 ) RCRCDD6PASRCD-1RC-1 ) RCD6CSARC
(7)
3.1. The Spectrometer. A scheme of our experimental
The dipolar tensor in the CSA frame D6CSA and the CSA tensor
setup is shown in Figure 1. All experiments were performed

Structure Elucidation of Amide Bonds
J. Phys. Chem. B, Vol. 101, No. 51, 1997 11267
Figure 2. SEDOR type pulse sequence for measuring heteronuclear
dipolar couplings. The simultaneous irradiation of the π-pulses at t1/2
in the 13C and 15N channels refocuses the evolution under the chemical
shift Hamiltonian at time t1. The partial echo, which is modulated by
the dipolar interaction, is converted into z-magnetization by a (π/2)y-
pulse. After dephasing of remaining transverse magnetization the
z-magnetization is read out with a (π/2)/yj-pulse. Fourier transformation
with respect to t1 gives the dipolar spectrum.
Figure 1. Scheme of experimental setup.
at a field of 6.98 T, corresponding to a proton resonance
frequency of 297.8 MHz on a standard Oxford wide bore magnet
(89 mm) equipped with a room temperature shim unit. We used
a home-built three channel NMR spectrometer, with all three
channels are controlled by a pulse programmer with 100 ns
resolution, 32-bit binary output, and 32k maximum pulse
program length. Each channel consists of a digital-controlled
(DDS) synthesizer for generating the NMR frequencies. The
necessary phases are generated by directly switching the phase
of the digitally controlled synthesizers.6 After the synthesizer,
the actual pulses were generated with fast RF-switches (10 ns
rise time) and fed into the power amplifiers. For the proton
channel a Creative Electronics 1 kW class C amplifier was used.
For the 13C-channel a 1 kW class AB and for the 15N-channel
a 2 kW class AB amplifier, both from AMT, were employed.
All amplifiers are equipped with an RF-blanking for suppressing
the noise during data aquisition. All experiments were per-
formed using a commercial Doty triple-resonance NMR probe
operating at room temperature. To improve the mutual RF-
isolation of the three channels commercial band-pass filters
(Texscan) in conjunction with home-built notch filters were
employed. The RF of the observed channel was fed through a
crossed diode duplexer, connected to the detection preamplifier
and through the filters into the probe. The other two channels
were fed directly through the filters into the probe. Typical
90° pulse width was 6.5 µs for all three channels, corresponding
to a B1-field in frequency units of ν1 ) 38 kHz. This was
sufficient to remove 1H-X dipolar line broadening. Repetition
time of the experiments was 10 s for the gluconamide and 30
s for the acetanilide. To avoid dead time problems caused by
the rather high Q of the probe, we measured all spectra using
a fully phase cycled spin-echo technique. The spectra were
measured by first cross-polarizing the observed nucleus from
the protons and then recording the spin-echo signal under
proton decoupling. Typical echo delay was 300 µs. For the
15N-13C samples the labeling was better then 99%; therefore,
no labeling factor had to be included. However for the
simulation of the 15N-2H spectra, we had to include a labeling
factor to account for imperfect deuteration of the -NH group.
The referencing of the tensor values was accomplished by
determining the isotropic chemical shift value from the 13C and
15N CP-MAS spectra, measured at 7 kHz spinning speed, and
adjusting the trace of the fitted tensors to this value.
Figure 3. Formula of (A) acetanilide and (B) gluconamide.
experiment,4,5a which is a variation of the 2D-experiment
described for measuring homonuclear dipolar couplings.7 The
basic sequence is shown in Figure 2. After cross-polarization
and delay t1, a 180° pulse is applied simultaneously to the 15
N
and 13C channel. As a result of these pulses after the time 2t1
a partial echo is formed, where the chemical shielding interaction
is selectively refocused, and the spins evolve only under the
13C-15N dipolar interaction. By applying a 90° pulse to the
13C, this echo is stored as z-magnetization, and after a dephasing
delay without proton decoupling of 10 ms, all remaining
transversal coherences are destroyed. The stored 13C echo signal
is then read by either a 90° pulse for recording a 2D spectra or
by a CPMG sequence for recording the dipolar FID directly.
3.2. Samples and Preparation. Acetanilide (Figure 3a) was
used as a standard reference for the experiments with the various
isotopic configurations. Acetanilide has several advantageous
features: it is rather easy to perform the selective isotope
labeling, and its crystal structure and NMR parameters are
known. The synthesis of isotopically labeled acetanilide was
performed in the standard way8a by adding a solution of acetyl
chloride (either unlabeled or 99% 13C(CO) labeled from
Chemotrade, Leipzig, Germany) in chloroform to aniline (either
unlabeled or 99% 15N labeled, also from Chemotrade, Leipzig,
Germany) solved in chloroform and stirred in an ice bath. The
addition was completed at a higher temperature of about 50
°C. The solution was stirred for 1 h, and the excess solvent
removed to yield crystals of acetanilide that were recrystallized
in water.
The synthesis of the selectively isotope labeled gluconamide
(Figure 3b) samples is described in detail elsewhere8b and will
only be briefly summarized here. For the synthesis of the 15
N
single labeled N-1-octyl-D-gluconamide 99% [15N]ammonia
produced from ammonium chloride was employed. In a first
step [15N]ammonia was condensed into a solution of octanoyl
chloride in diethyl ether at -40 °C, which lead to the
spontaneous formation of [15N]octylamide, which precipitated
from the solution. After filtering and recrystallization from
water, pure [15N]octylamide was obtained. In a second step,
by reduction with LiAlH4, [15N]octylamin was obtained. In the
last step by aminolysis with D-gluconic acid-δ-lacton the N-1-
octyl-D-gluconamide was obtained. The 15N-13C double la-
beled compound was synthesized by employing 13C-labeled
The 15N-13C dipolar coupling constant of acetanilide was
measured independently by employing a variant of the SEDOR

11268 J. Phys. Chem. B, Vol. 101, No. 51, 1997
Buntkowsky et al.
Figure 6. 13C dipolar CSA NMR spectra of 13C-15N doubly labeled
gluconamide. Experimental (lower curves) and simulated spectra (upper
curves). In the range between 100 and 0 ppm there is additional spectral
intensity from natural abundance bulk 13C.
Figure 4. Dipolar 13C NMR spectra of (A) 13C-15N doubly labeled
acetanilide and (B) 13C-15N doubly labeled gluconamide after the
SEDOR sequence (Figure 2). Experimental (lower curves) and simu-
lated (upper curves) spectrum. The intensity in the center of the
experimental spectra stems from bulk 13C nuclei, which have no 15N
partner.
Figure 7. 15N dipolar CSA NMR spectra of 13C-15N doubly labeled
acetanilide. Experimental (lower curves) and simulated spectra (upper
curves). (Left panel) The full dipolar CSA spectrum (i.e., without 13C
decoupling). (Right panel) Only the CSA is visible, i.e., the dipolar
interaction is suppressed by 13C decoupling.
Figure 5. 13C NMR spectrum of 13C-15N doubly labeled acetanilide.
Experimental (lower curves) and simulated spectra (upper curves). (Left
panel) The full dipolar CSA spectrum (i.e., without 15N decoupling).
(Right panel) Only the CSA is visible; i.e., the dipolar interaction is
suppressed by 15N decoupling.
D-gluconic acid-δ-lacton, synthesized from 98.5% 13C-labeled
D-glucose (Sigma). The deuterated samples were produced by
boiling 150 mg 15N-labeled N-1-octyl-D-gluconamide in 3 mL
of 99% D2O. The material was placed into a standard 5 mm
high-speed Doty rotor with KELF caps, which sealed the sample
sufficiently tight to prevent structural modifications or rehy-
drogenation of the deuterated sample by moist air.
3.3. Data Evaluation. The echo spectra after phase cor-
rection were simulated using eq 10. Instead of numerically
performing the powder integration of eq 10, the analytical
expression of the powder pattern in terms of elliptic integrals5c
was used to calculate the line shape for zero width from the
eigenvalues of the effective interaction tensors 6ΣS(. This line
was then numerically convoluted with a Lorentzian by Fourier
transforming into the time domain, multiplying with a decaying
exponential function, and transforming back into the frequency
domain to give the spectra of eq 10.
Figure 8. 15N dipolar CSA NMR spectrum of 13C-15N doubly labeled
gluconamide. Experimental (lower curves) and simulated spectra (upper
curves).
summarized in Tables 1-3 will be presented. Figure 4a shows
the experimental dipolar 13C NMR spectrum of the 13C-15N
doubly labeled acetanilide after the SEDOR sequence described
above.
4. Results and Discussion
4.1. Experimental Results. In the following the experi-
mental results obtained from the 13C and 15N spectra, that are

Structure Elucidation of Amide Bonds
J. Phys. Chem. B, Vol. 101, No. 51, 1997 11269
Figure 9. 15N dipolar CSA NMR spectra of 2H-15N doubly labeled acetanilide (A, left panel) and of 2H-15N doubly labeled gluconamide (B, right
panel). Experimental (lower curves) and simulated spectra (upper curves).
The central part of the experimental spectrum has some
additional Lorentzian contribution, which can be attributed
mainly to bulk 13C atoms. The outer parts of the spectrum
exhibit the typical Pake pattern of a heteronuclear spin pair.
Therefore, for the simulation of the experiment, the central part
of the spectrum was ignored and the theoretical dipolar powder
pattern was adjusted to the outer wings of the spectrum. This
gave a coupling constant of 1220 ( 30 Hz, which corresponds
to a CN distance of 1.35 ( 0.01 Å, which is in very good
agreement with the value of 1230 ( 40 Hz, given by ref 1a as
well as with the value of 1220 Hz calculated from the distance
obtained by crystal structure (1.354 Å).9 Figure 4B shows the
experimental dipolar 13C NMR spectrum of the 13C-15N doubly
labeled gluconamide after the same SEDOR sequence as above,
which gave the same dipolar coupling of 1220 ( 30 Hz (1.35
( 0.01 Å).
Figure 5 compares the 13C NMR spectra of the 13C-15N
doubly labeled acetanilide. By 15N decoupling, the dipolar
Figure 10. Definitions of the polar angles R and â that characterize
interaction to the 15N nucleus is suppressed (right side of the
the direction of the dipolar interaction in the coordinate frame of the
figure), and thus the pure 13C-CSA spectrum is measured. The
CSA interaction.
left figure, which is recorded without 15N decoupling, depicts
the full dipolar CSA spectrum. From the pure CSA spectrum
the principal values of the CSA tensor were obtained. These
were used in conjunction with the dipolar coupling constant
obtained from the SEDOR experiment (Figure 4A) for simulat-
ing the dipolar CSA spectra by fitting the orientation of the
dipolar vector in the frame of the CSA tensor.
termed δ11 (least shielded), δ22 (intermediate), and δ33 (most
shielded). The parameters of the various CSA NMR tensors
are summarized in Tables 1-3. To avoid asymmetry parameters
of η > 1, we defined η ) (δ22 - δ33)/(δ11 - δiso) instead of
the usual η ) (δ22 - δ11)/(δ33 - δiso) for the 15N tensors. In
the notation of bond vectors the first nuclei always refers to
the observed nuclei (i.e. -CN vector, 13C observed; -NC vector,
15N observed).
Figure 6 shows the corresponding 13C NMR spectrum of the
gel modification of the 13C-15N doubly labeled gluconamide.
The CSA parameter obtained from the spectrum with 15N
decoupling (not shown) was used in conjunction with the dipolar
coupling from the SEDOR experiment (Figure 4B) to perform
the simulation of the full dipolar CSA spectrum from which
the orientation of the dipolar vector in the CSA frame was
obtained. In the same way, the 15N spectra of the 15N-13C-
labeled acetanilide and gluconamide samples were recorded and
evaluated. The resulting spectra and simulations are shown in
Figure 7 (acetanilide) and Figure 8 (gluconamide). Figure 9
displays the 15N spectra of the 15N-2H-labeled acetanilide and
gluconamide samples. While the deuteration of the acetanilide
was better than 99% for the gluconamide, we had to adapt an
isotope labeling factor of 80 ( 5% in the simulation to account
for the partial deuteration of the sample. The spectra were
simulated using the CSA parameter from the 15N-13C-labeled
samples by adjusting the strength and orientation of the dipolar
coupling vector in the CSA frame.
4.2.1. Orientation of the Acetanilide 13C CSA Tensors in the
Molecular Frame. For the orientation of the acetanilide 13C
CSA tensors (Table 1) in the molecular frame, we used the
constraint that, because of the planar symmetry of the amide
group, one of the principal axis of the tensor is perpendicular
to the amide plane and that the -CN bond direction, which is
also the direction of the dipolar vector, lies in the amide plane.
The δ33-axis is the only axis perpendicular to the 13C-15N
dipolar vector. It follows that the δ33-axis is the axis perpen-
dicular to the amide plane and the -CN and -CO bonds are in
the (δ11,δ22) plane.
Since the sign of the angle RΝ is not known, there are two
possible orientations of the -CN bond in the (δ11,δ22) plane.
These are shown as vectors CNA and CNB in Figure 11a, which
shows a sketch of the 13C geometry in the (δ11,δ22) frame (the
carbonyl carbon is in the center of the coordinate system). In
configuration A the -CN vector CNA is rotated with an angle
RAN ≈ +33° with respect to the δ11 axis, and in configuration
B the -CN vector CNB is rotated with an angle RBN ≈ -33°
4.2. Discussion. In the following the notation of Lumsden1a
is used; i.e., the principal values of the CSA interaction are

11270 J. Phys. Chem. B, Vol. 101, No. 51, 1997
Buntkowsky et al.
Figure 11. Orientation of the bond directions in the (a) 13C and (b) 15N CSA frame of the acetanilide and in the (c) 13C and (d) 15N CSA frame
of the gluconamide. From these orientations the molecular geometry of the group can be constructed.
with respect to the δ11 axis. Because of the sp2-hybridization
of the carbonyl C, the bond angle between the -CN and the
-CO bond is approximately 120°, and there are two possible
orientations for the -CO bond. For configuration A the COA
vector could point either at an angle of ≈153° or at an angle of
≈273° with respect to the δ11-axis, while for configuration B
the COB vector points either at an angle of ≈87° or at an angle
of ≈207° with respect to the δ11-axis. By NMR alone it is not
possible to decide between these two configurations. It has been
shown in ref 1a that this problem can be solved by employing
additional data. Molecular orbital calculations10 have shown
that the most shielded component is perpendicular to the
carbonyl plane and the least shielded component is perpendicular
to the carbonyl bond and in the carbonyl plane. It follows that
the intermediate component δ22 is close to the carbonyl bond.
This is only fulfilled for the bond vectors COB and COA,
belonging to the configurations A or B respectively, which also
determines the direction of the bond to the -CH3 group, shown
as CH3A and CH3B respectively. The resulting two configura-
tions A and B are symmetry related via a 180° rotation around
the δ11-axis.
CSA tensor. Therefore, we used the constraint that, because
of the planar symmetry of the amide group, one of the principal
axis of the tensor is perpendicular to the amide plane and that
the -NC bond direction, which is also the direction of the 15N-
13C dipolar vector, lies in the amide plane. Using the angles
from Table 2 and Table 3 it follows that the (δ11,δ33) plane is
the amide plane and that the δ22 axis is the axis perpendicular
to that plane and that the -NC vector as well as the -ND vector
are both in the (δ11,δ33) plane. The signs of the angles âD and
âC are not known, and there are two possible orientations for
the -ND and the -NC vector in the (δ11,δ33) plane. However,
it is well-known that the 15N atom in an amide group is in a
nearly sp2-configuration with an angle of approximately 120°
between -NC and -ND vector. There are two possible
combinations of the -NC and -ND vectors to fulfill this
constraint, shown as configurations A and B in Figure 11b. In
the first combination A, the vector NCA is rotated âCA ) -22°
with respect to the δ33-axis and has to be combined with the
vector NDA stemming from the (180°-83°) rotated -ND vector.
In the second configuration B, the vector NCB is rotated âCB
)
22° with respect to the δ33-axis and has to be combined with
the vector NDB, stemming from the -(180°-83°) rotation with
respect to the δ33-axis. The resulting two configurations A and
B are symmetry related via a 180° rotation around the δ33-axis.
4.2.3. Orientation of the Gluconamide 13C CSA Tensors in
the Molecular Frame. The isotropic chemical shielding of the
gluconamide 13C CSA tensor gives the typical value of a
carbonyl carbon and the principal values of the gluconamide
13C CSA tensor as well as the 13C-15N dipolar interaction
4.2.2. Orientation of the Acetanilide 15N CSA Tensors in the
Molecular Frame. For the orientation of the 15N CSA tensors
in the molecular frame two orientation constraints are avail-
able: The direction of the -NC bond in the 15N CSA frame
from the 15N spectrum of the 15N-13C-labeled compound and
the direction of the -ND bond in the 15N CSA frame from the
15N spectrum of the deuterated compound. However, this
advantage is reduced by the nearly axial symmetry of the 15N

Structure Elucidation of Amide Bonds
J. Phys. Chem. B, Vol. 101, No. 51, 1997 11271
Figure 12. Geometrical structure of the amide group in acetanilide.
Figure 13. Geometrical structure of the amide group in gluconamide.
strength and direction in the 13C CSA frame are comparable to
the values obtained for the acetanilide. Therefore, the same
reasoning can be used to orient the 13C CSA tensor with respect
to the molecular frame of the amide group, and again two
symmetry-related configurations are found, which are shown
in Figure 11c. Comparing this figure with Figure 11a, it is
evident that for the gluconamide the bond directions in the CSA
frame are rotated approximately 12° as compared to the
acetanilide. This indicates that there are differences in the
electronic structure of the amide groups, and these differences
are also reflected in the asymmetry parameter η ) 0.80 and in
the intermediate shielding value δ22.
its fibrous form, exists in the cis or trans form can be resolved
by employing additional data from 13C-CPMAS NMR and
infrared spectroscopy. Such data point to the trans diastomer
as the preferential configuration. Figure 13 depicts the resulting
structure of the amide group of the gluconamide, including the
directions of the CSA tensors.
5. Summary and Conclusion
The 13C and 15N solid-state NMR spectra of acetanilide and
gluconamide have been recorded, and the corresponding chemi-
cal shift and dipolar interaction tensors have been determined.
Our results on the principal values and orientations of the
acetanilide 13C tensors corroborates the data presented by
Lumsden et al.1a From these data the complete geometrical
structures of the amide group of the acetanilide and gluconamide
were determined. The groups exhibit amide and peptide bonds
of typical approximately 120° bond angles between -CO, -CN,
-CR, and -NC, -NH, -NR. Comparing the NMR results
from the two amide groups, several noteworthy differences are
evident. (i) The eigenvalues of the gluconamide 13C tensor
exhibit a weaker high field shift (4 ppm) compared to acet-
anilide. (ii) The orientation of the δ11-axis of the gluconamide
13C tensor is 12° farther away from the CN-bond direction, the
CN bond is intersecting the δ11,δ22 angle (45°) and the
anisotropy of the 13C tensor is stronger. (iii) The 15N CSA
tensor is shifted to high field and exhibits a stronger anisotropy.
(iv) The amide plane is spanned by the (δ11,δ22) components
of the 15N CSA tensor but not by the (δ11,δ33) components.
There are two possible explanations for these differences,
which are not necessarily mutually exclusive. They can be due
to intramolecular effects, such as different head and tail groups
and different conformations of the head and tail groups, or by
intermolecular effects such as N-H‚‚‚OdC intermolecular
hydrogen bonding. Differences in the 13C isotropic shift of
amide 13CO values have been attributed to different hydrogen-
bonding lengths;3,11 furthermore, it has been found that all three
components of a 15N-CS tensor are very sensitive to the
geometry of the hydrogen bonding.2g,1a In the gluconamide the
neighboring carbon atom of the amide group is in an sp3-
configuration, and in the acetanilide the neighboring carbon atom
is in an sp2-configuration, which could account for the differ-
ence. However, 15N-2H dipolar NMR experiments have
shown13 that for sp3-configurations of the neighboring carbon
atom the same tensor orientation as in the case of the acetanilide
can be found as well. This suggests that the intramolecular
4.2.4. Orientation of the Gluconamide 15N CSA Tensors in
the Molecular Frame. The values of the polar angles of the
dipolar interaction vectors for the gluconamide exhibit an
interesting difference as compared to the values of the acet-
anilide. For the gluconamide the angle â between the δ33 axis
and both the -NC (Table 2) and -ND (Table 3) bond direction
is 90°. Thus the least shielded component is perpendicular to
the amide plane and not the intermediate component δ22 of the
CSA tensor, and the amide plane is spanned by the (δ11,δ22)
components of the CSA tensor. The direction of the bonds in
the (δ11,δ22) plane is determined by the angle R. Following
the same arguments as above, there are two different orientations
corresponding to (R, the angle between the bond direction and
the δ11-axis. Employing the argument that the angle between
the -CN and -ND direction is approximately 120°, it is
possible to assign the corresponding bond axes to each other,
which are marked as configuration A or B in the Figure 11d.
The two configurations are symmetry related via a C2 rotation
around the δ11-axis of the CSA tensor.
4.2.5. Structure of the Amide Group of the Acetanilide. From
the previous discussion, the geometrical structure of the amide
group can be constructed. Rotational symmetry along the CN
bond means it is not possible by NMR alone to decide if the
molecule exists in the cis or trans form. This ambiguity can
be resolved by employing X-ray data,9 and these data show that
in the solid state the trans configuration is preferred. The
resulting structure of the amide group including the directions
of the CSA tensors is displayed in Figure 12.
4.2.6. Structure of the Amide Group of the Gluconamide.
Once more, due to the rotational symmetry with respect to the
CN bond, it is not possible by NMR alone to decide whether
the amide group occurs in the cis or trans form. X-ray data of
the molecule are only available for the crystalline modification
of the gluconamide, where the trans form is preferred, but not
for the fibrous form. The question of whether the molecule, in
TABLE 1: Parameters from the 13C-15N-Labeled 13C Spectra
13C-15N
δ
11 (ppm)
δ22 (ppm)
δ33 (ppm)
δiso (ppm)
η
R
â
D (Hz)
AA
AAb
GAc
248(2)
247(1)
243(1)
175(1)
173(1)
183(1)
90(2)
94(1)
100(1)
171
171
175
0.90
0.95
0.80
30(5)
33
43
90(3)
90
90
1230(40)
1220
1220
a Acetanilide data from ref 1a. b Acetanilide, own results. c Gluconamide.

11272 J. Phys. Chem. B, Vol. 101, No. 51, 1997
Buntkowsky et al.
TABLE 2: Parameters from the 15N-13C Labeled 15N Spectra
15N-13C
δ
11 (ppm)
δ22 (ppm)
δ33 (ppm)
δiso (ppm)
η
R
â
D (Hz)
AAa
AAb
GAc
247(1)
245(1)
239(1)
90(1)
89(1)
76(1)
77(1)
77(1)
57(1)
138
137
124
0.12
0.11
0.17
0(10)
0
100
20(3)
22.1
89
1230(40)
1220
1220
a Acetanilide data from ref 1a. b Acetanilide, own results. c Gluconamide.
TABLE 3: Parameters from the 15N-2H-Labeled 15N Spectra
15N-2H
δ
11 (ppm)
δ22 (ppm)
δ33 (ppm)
δiso (ppm)
ηd
R
â
D (Hz)
AAa
AAb
GAc
247(1)
245(1)
239(1)
90(1)
89(1)
76(1)
77(1)
77(1)
57(1)
138
137
124
0.12
0.11
0.17
0(3)
0
15
83(3)
81
90
1690(40)
1640
1600
a Acetanilide data from ref 1a. b Acetanilide, own results. c Gluconamide. d To remain consistent with the notation of the principal values used
by Lumsden,1a but avoid values of η > 1, we defined η ) (δ22 - δ33)/(δ11 - δiso) instead of the usual η ) (δ22 - δ11)/(δ33 - δiso).
effects are not responsible for the differences in the local
electronic structure, which is reflected in the differences in the
shielding parameters. The N-H‚‚‚OdC intermolecular hydro-
gen bond is the main structural element responsible for the
formation of the extended quadruple helixes in the fibrous
modification of the gluconamide molecules. Therefore, we
conclude that differences in the intermolecular hydrogen bonding
are the principal reason for the differences in the tensor
orientations. This suggests that to obtain reasonable results from
ab initio calculations of amide and peptide bonds, it is important
not only to include the local molecular configuration but also
to include information about the neighboring molecules.
It has been demonstrated that dipolar chemical shift NMR
spectroscopy can give valuable information about the structure
of small molecular groups. The possible information gain from
this method can be greatly enhanced by combining this form
of NMR spectroscopy with additional NMR methods, for
example, SEDOR experiments for directly measuring the
strength of dipolar couplings or selectively switching on and
off heteronuclear dipolar couplings by decoupling of the second
X-nuclei and CP-MAS techniques for determining the isotropic
shielding values. These additional methods are demanding
concerning the necessary experimental setup, because at least
a three-channel NMR spectrometer is needed. It has been shown
that acetanilide, because of its favorable NMR parameters and
simple labeled synthesis, is a convenient standard for setting
up these experiments. As an application to a larger molecule,
the structure of the amide group of the N-1-octyl-D-gluconamide
in its fibrous modification has been determined.
Finally the question arises whether these methods permit the
study of larger structures, for example, small peptides or
oligonucleotides, where several bonds have to be correlated to
each other to give the complete geometrical structure of the
molecule. For these cases, the absence of the isotropic chemical
shift resolution (which would be given, for example, by MAS
type experiments) is a strong restriction for the applicability of
the method, because it demands pairwise selective isotopic
labeling, for example, of the sequence of amino acids, which is
very demanding concerning the synthetic expense. Therefore,
for these types of systems, it is advantageous to apply MAS
techniques combined with dipolar recoupling sequences.12
However, these experiments provide only very indirect access
to the orientational correlation of dipolar interactions, which
represent bond directions and chemical shift tensors that
represent the local electronic structure. If this information is
desired, it is therefore necessary to perform the labeled synthesis
and apply the techniques described in this paper.
Acknowledgment. This work was supported by the German
Israel Foundation under Grant G.I.F. Research Grant No.
I-297.092.05/93.
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