Two-Dimensional NMR Spectroscopy:

Background and Overview of the Experiments New Analytical

Methods (36)
By Horst Kessler,” Matthias Gehrke, and Christian Griesinger

An exact knowledge of the structure, dynamics, and reactions of molecules provides the key
to understand their functions and properties. NMR spectroscopy has developed, through
the introduction of two-dimensional methods, into the most important method for the in-
vestigation of these questions in solution. A great variety of different techniques is availa-
ble. However, for their successful application not only the appropriate equipment is re-
quired, but also the right choice of experiments and optimum measurement parameters, as
well as a careful evaluation of the spectra. This contribution describes the necessary back-
ground for modern NMR spectroscopy. With the aid of the so-called product operator for-
malism it is possible to understand pulsed Fourier transform N M R spectroscopy both qual-
itatively and quantitatively. Very few, readily understandable assumptions are sufficient for
confident application of these methods. This article attempts to introduce in a simple man-
ner this formalism as well as phase cycles necessary for the understanding of pulse se-
quences, and to train the reader through the discussion of several 2D NMR techniques. An
overview of the most important techniques is given in the second part of this article.

1. Introduction used, yet quite cumbersome and insufficient, vector de-

scription.
NMR spectroscopy has undergone a revolution with the
Another reason for writing this article is the experience
introduction of 2D Although several re-
which has accumulated in applications of the various ex-
views on this subject have been p ~ b l i s h e d , [ ’ , ~we
- ~ find
~ ~ it
periments and which has led to a realization of the advan-
useful to provide a readily understandable introduction to
tages and disadvantages of each technique. Thus, for in-
and application of the product operator formalism (with
stance, J - r e ~ o l v e d [ * . ~ ~and
- ~ ’spin
) echo correlated spectros-
Cartesian basis operator^).[^^-*^] In this way the experi-
copy,136-381 which once played a major role in 2D NMR
ments can be understood both qualitatively and quantita-
spectroscopy, are now employed only in a few special
tively, without recourse to the somewhat more cumber-
cases. We can of course only describe a selection of the
some quantum mechanical treatment (keyword: spin den-
different 2D NMR techniques. Some 500 pulse sequences
sity matrix). The product operator formalism makes it pos-
for high-resolution N M R spectroscopy have been pub-
sible to describe the behavior of weakly coupled spin sys-
lished. Only a small fraction of those found their way to
tems (i.e., systems in which the difference in chemical
general recognition and application, and the entire field is
shifts of two nuclei is large relative to the J coupling be-
still in flux. Thus a whole new series of better experiments
tween them) under the influence of pulse sequences by the
is now available, which quantitatively measure such param-
application of a few simple rules. Thus even nonexperts
eters as NOE values and vicinal coupling constants, neces-
may follow and apply the experiments.
sary for the determination of structure and dynamics. In
Along with the description of product operators, some
addition, the NMR spectroscopy of insensitive nuclei has
understanding of phase cycles in 2D NMR experiments is
become more and more accessible, even in large mole-
also necessary. Phase cycles suppress undesired signals in
cules, through the application of inverse technique^.[^^-^^^
a multipulse experiment. This concept, as well as the asso-
We have tried to write in such a way that the required
ciated “coherence order”128-321 will be introduced and dis-
prior knowledge of the reader be minimal. Therefore the
cussed in Section 2.2.3. Suffice it to mention, at this point,
text has been divided into two levels: for a first reading it
that by following the “coherence-transfer pathways” a
should be sufficient to cover the normal print. For further
simple and powerful tool is provided for the development
discussion and insight the small-print sections should be
of phase cycles. The reader will soon recognize that with
followed.
the product operator formalism and coherence-transfer
pathways a theory is now available which in terms of clar-
ity, simplicity, and accuracy by far exceeds the widely
2. Background

Nuclear magnetic resonance deals with the interaction

[*] Prof. Dr. H. Kessler, DipLChem. M. Gehrke
Institut fur Organische Chemie der Universitat
Niederurseler Hang, D-6000 Frankfurt SO (FRG)
Dr. C . Griesinger
Laboratorium fur Physikalische Chemie der
Eidgenossischen Technischen Hochschule, ETH-Zentrum
Universitatstrasse 22, CH-8092 Zurich (Switzerland)
between the magnetic moments of atomic nuclei and mag-
netic fields. The magnetic moment of the nucleus is asso-
ciated with the nuclear spin S. Only those nuclei for which
SfO have a magnetic moment. The high-resolution NMR
spectroscopy described in this review is used predomi-

490 0 VCH Verlagsgesellschaft mbH. 0-6940 Weinheim, 1988 0570-0833/88/0404-0490 $ 02.50/0 Angew. Chem. Inf. Ed. Engl. 27 (1988) 490-536
nantly to investigate nuclei with spin 1 / 2 , which have no
quadrupole moment and hence give rise to sharp spectral
lines. The nuclei of greatest interest for organic chemistry
and biochemistry, H, C, N, and P, all have isotopes with
spin 1/2, though for C and N the natural abundance of
these isotopes is very small. The proton, the most abun-
dant nucleus in organic compounds, has (with the excep-
tion of tritium) the largest nuclear magnetic moment and
hence is the simplest nucleus for investigation. However,
the N M R spectroscopy of the more “exotic” nuclei, which
are of greater significance in organometallic and inorganic
chemistry, has also become increasingly important.
2.1. One-Dimensional NMR Spectroscopy
The method now generally used for recording 1D N M R
spectra is the Fourier technique introduced by Ernst and
A n d e r s ~ n . ‘ ~ ’ -Since
~ ’ ~ the principles of this technique also
underlie 2D NMR spectroscopy, we shall begin with its
description.
Nuclei with spin 1 / 2 are aligned either in a parallel o r
an antiparallel orientation with respect to the applied Bo
field. Only these two states exist, which are marked a and
p, respectively. The parallel a state[*]is lower in energy
than the antiparallel p state. Therefore, the population of
the a state is greater than that of the state (Boltzmann
distribution), and a macroscopic magnetization along the
Bo field (“longitudinal magnetization”) exists due to this
population difference. The macroscopic magnetization can
be expressed in terms of the Bloch equations.[581According
to usual convention, the so-called rotating-frame coordi-
nate system is used. The coordinate system rotates about
the z axis, which is aligned with the Bo field, at the “carrier
frequency” wo of the radio frequency pulse.
Pulses are short-term high-frequency oscillating magnetic fields
of the form B, coswor. Because the magnetization precesses about
Bo during the pulse, a very complicated motion results. A simpli-
fied description is achieved in a coordinate system rotating at
wo=yB,, such that the magnetization appears to rotate about B ,
alone.
The linearly polarized B , field (e.g., only the x component) may
be divided into two circularly polarized fields [Eq. (I)]. The two
vectors rotate at equal frequencies but in opposite directions. In
the rotating frame (e:,e;) one obtains Equation (2).
The component in (2) rotating at twice the Larmor frequency
does not meet the resonance condition and can therefore be ne-
glected. Only the time-independent (!) component % B y e : is con-
sidered. Thus, the Bloch equation contains only time-independent
fields and is readily solved. The static fields (Bo and B ; ) cause
rotations of the magnetization about the corresponding axes (x or
z’, respectively). The magnetization rotates by a certain angle, the
[*] The assignment a and B for parallel and antiparallel, respectively, follows
the notation in Ref. [3], and will be used here throughout. Sometimes the
opposite assignment can be found in the literature.
Angew. Chem in!. Ed. Engl. 27 (19881 490-536
so-called flip angle, depending on the duration of the B , field (the
pulse length). Therefore pulses are often characterized by their flip
angle, rather than by their field strength and duration.
Pulses may be described as rotations by a certain angle
(flip angle) about a certain axis in the x,y plane (phase).
For example, application of a short radio frequency pulse
generally labeled /?, will rotate the magnetization about the
y axis by a n angle p from the z axis [Eq. ( 3 ) and Fig. l].[*]
Equation ( 3 ) means that the M , magnetization[*’] trans-
forms under the influence of a & pulse in part (sinp) into
M , magnetization, the other part (cosp) remaining along
the z axis.
2 z
I
I /
/
Y ’I I
M,sin p
Y
Fig. 1. The effect of an on-resonance radio frequency pulse in the y direction
in the rotating frame and a flip angle B on longitudinal magnetization. We
have rotated the coordinate system by 90” from the usual representation (x in
front) for easier understanding.
After application of the pulse, the transverse magnetiza-
tion M , begins to precess about the z axis at a precession
frequency w = y B e f f .The chemical shifts of the different
nuclei are represented by Beff.Nuclei of the same isotope
(e.g., protons) have equal y and nearly the same B e f f ,so
that their precession frequencies fall within a narrow
range. For protons this frequency varies, depending o n in-
dividual chemical shifts, within ca. lo-’ ma, o r 10 ppm. In
the rotating-frame the observer (i.e., detector) also rotates
about the z axis at the carrier frequency wo. The detector
“sees” now only difference frequencies w - wo. Henceforth
we shall term these difference frequencies precession fre-
quencies o r chemicalshijk, SZ (Fig. 2). In the rotating frame
we thus obtain Equation (4).
M, -
01
M, C O f~i t + M y sin flt (4)
The precession frequency C2 is the deviation of the Lar-
mor frequency from the carrier frequency wo, which is reg-
[*] The direction of rotation of the magnetization under the influence of
pulses follows Ref. 131, and is in accordance with the mathematical sense
of rotation. Frequently, the opposite definition is found in the literature
(in our example this would correspond to a rotation to - x).
[**I M,,My,and M, mean unit magnetizations given by the initial polariza-
tion along the corresponding axes.
49 I
 a I I i
Y

I I j positive
: single-quantum coherence (SQC)
i
.’\ : negative
b oz -olio1 8
-o* i single-quantum coherence ( - SQC)

Fig. 2. Evolution of the chemical shift during time 1. A view onto the x.y
plane in the rotating frame is shown.

istered as a signal by the detector aligned, for example, 00

with the x axis in the rotating frame. Thus a signal S ( t )
oscillating at frequency C2 is obtained, the so-calledfree in-
duction decay (FID). A Fourier transformation will convert
this signal into a spectrum with a single signal (“peak”) at
frequency C2 (see also Fig. 4).
Fourier transformation is a mathematical procedure by
which frequencies, which are detected in a n oscillating
function of time, are transformed into signals at the corre-
sponding positions o n a frequency scale.
i
d : I I ;
Result after
real Fourier transformation
Fig. 3. Principle of the Redfield technique. A single-channel detection spec-
trum, obtained after complex Fourier transformation (a) contains signals at
both positive and negative frequencies, which are shown separately in (b).
This corresponds to positive and negative single-quantum coherences. The
two spectral parts n o longer overlap (c) when the receiver rotates at fre-
quency SW/2 in the rotating frame. After folding about o,=O, , i.e., real
Fourier transformation, a spectrum is obtained (d) which contains only the
positive frequencies.

The function cosQr in Equation (4) contains the frequency

w = + Q as well as to o = -R, and hence a distinction between
these frequencies is impossible. It is however desirable to know the
sign of a given frequency, for only then can the carrier frequency
be placed in the center of the spectral range. Quadrature detec-
ti~n,[’~,~’’
by which both x and y magnetizations in the rotating
frame are detected, allows the sign discrimination of the detected
frequency. A signal cosnr is constructed in the x channel (K,)
from the x magnetization M. in (4), and a signal sinRt in the y
channel (K,) from the y magnetization M y . When the two signals
are combined, K,+i K,, the complex expression elR‘ is obtained,
which after complex Fourier transformation produces a single line
at frequency + Q (and indeed not also at -a). The disadvantage
of this procedure is that it requires simultaneous collection of two
signals.
A trick introduced by Redfield et a1.‘61,621is utilized in some in-
struments, whereby, starting with the ( + x) direction, signal detec-
tion is alternated among the (+ x), (+ y), ( - x), and ( - y) direc-
tions (Fig. 3). Hence the receiver rotates in the rotating frame with
an additional frequency QR. The recorded signal is then
cos(R+R,)f. Q, is set to one-half the spectral width (SW/2) (Fig.
3b). After a complex Fourier transformation, a spectrum is ob-
tained in which signals with positive and negative frequencies no
longer overlap (Fig. 3c). Finally, the spectrum may be “folded”
about R = 0 (Fig. 3d), or, equally well, a real Fourier transforma-
tion may be performed.
We now wish to describe the same experiment in terms
of the product operator formalism.[241In contrast to the
vector formalism discussed above, in which the macro-
scopic magnetization rotates under the influence of pulses
and/or precesses under the influence of chemical shift, the
entire state of the spins or spin system (only the observable
magnetization) is described by means of the density ma-
trix. The observable quantities must then be “read out” of
the spin state. The following short discussion of the quan-
tum mechanical formalism should be fully sufficient for
the understanding of 2D NMR experiments (for detailed
discussions of the physical background see Refs. [63-671).
The ground state of the system in thermal equilibrium is
described by a polarization along the z axis, t,. This state is
associated with a magnetization M,. Under the influence
of pulses from the y direction (henceforth termed in short
“y pulses”) I,, as well as t, and I,, transform according to
(5)-(7) (see also Fig. 1).
I, 4 I,
The Cartesian operators“’ (Ix, ty,t,)transform like mag-
netizations when a rotation is performed. The properties of
a state I,, ty,or t, can be read off directly in such simple
product operators which consist of only one operator. I, is
associated with an x magnetization, t, with a y magnetiza-
tion, and I, with a z magnetization.
We have yet to explain the notations used in Equa-
tions‘”] ( 5 ) to (7), as well as in other expressions through-
out the paper. The starting state of the spin system is on
the left-hand side of the equation. On the right is the end
[‘I We use throughout this article the symbols I (or S ) for the operator as
well as for the expectation value, and not the symbol i (or $) for the
operator.
[**I Strictly speaking one should use the term “transformations.” However,
in this article we use the term “equation” not in its strict mathematical
definition.

492 Angew. Chem. Int. Ed. Engl. 27 (1988) 490-536

state after the “propagation,” i.e., the evolution under the
influence of a particular operator responsible for a specific
interaction (e.g., for pulses, chemical shift, coupling). The
terms above the arrows are the parts of the Hamiltonian
operator (which describes the interactions of the spins with
external fields and with each other) under which the sys-
tem evolves. Thus,

(a) /?I,,yor in general DIP represents a pulse with a flip an-

Figure 4 summarizes again the principles of I D NMR
gle /? about an axis in the x,y plane, which forms a n
spectroscopy for a one-spin system. Application of a high-
angle rp with the x axis,
frequency pulse to the z magnetization (spin state Iz), ini-
(b) QI,t represents the evolution of chemical shift L2 of
tially at its Boltzmann equilibrium, produces transverse
nucleus I during time t , and
magnetization (state I J . This state, the so-called coherence
(c) 2nJI,,12,t stands for the evolution of coupling J be-
evolves, i.e., develops during the detection period t. This
tween nuclei I ’ and 1’ during time t.
variation of the intensity with time can be observed by the
detection of an induced signal in the receiver coil ( I x , I, are
Equations (8)-( 10) apply to the evolution of chemical
associated with transverse magnetization). Thus, coher-
shifts (see also Fig. 2). The operators behave here again
ences produced by product operators are displayed in the
like the magnetizations. For a single particle of spin 112
spectrum at their characteristic frequencies.
the description is equivalent to that of the vector formal-
The following discussion should serve to clarify the con-
ism.
cept of coherence. In contrast to populations (occupation
of states in the energy level scheme) the coherences de-
I, - nt i ,
I, cos R t + I, sin R t (8)
scribe transitions among different energy levels. These
transitions are characterized by a change Am, in the quan-
tum number m,. This change, Amz, is the coherence order

Iy - ntr,
Iycos R t - I, sin R t
The detection of x magnetization means that the signal
is built u p (in Eq. (9), for instance) by the coefficient
-sin Q t of I,. Fourier transformation yields a single line at
f Q .Quadrature detection, which is now available on all
spectrometers, allows the selection of only one frequency,
+ Q o r -Q.
Quadrature detection means, as we have seen earlier, the addi-
tion of the measured x components of the magnetization to i times
the corresponding y components (K,+iK,). This yields the com-
plex signal eio‘. This corresponds to the detection of I - according
to the conventions and transformations (1 1)-( 13).
(9)
p, which is essential for describing phase cycles (see Sec-
tion 2.2.3). Although in general many of the possible co-
herences occur under the influence of pulses in a pulse se-
quence, only a few are associated with observable magneti-
zations, namely those for which Am,= k 1 (the selection
rule for N M R spectroscopy). Of these so-called single-
quantum coherences only a few are detectable under weak
coupling conditions, those in which the state of exactly
one spin is changed, i.e., product operators with only one
transverse operator.
Finally, a technical remark: The induced signal in 1D
N M R experiments is not registered in a continuous man-
ner. The signal picked up by the receiver coil is sampled in
discrete time intervals At (“horizontal” time digitization)
and then digitized “vertically” by an analog-to-digital con-
verter. An F I D is obtained consisting of N points, where
N . At yields the acquisition time. The horizontal digitiza-
tion is possible according to the sampling theorem [68-733

Preparation
90;
Detection
tz
~

-- Fourier
transformation 0 Spectrum

Vector representation for the

+
y +y +yX u . . magnetization of a nucleus without coupling
m2

Fig. 4. Principle of ID N M R spectro-

90; pulse Evolution scopy. Evolution of magnetization is
I, I. during + I, wsm2 Corresponding product operators for shown in the vector description and
detection + Iysinnt, one kind of nucleus by product operators.

Angew. Chem. Int. Ed. Engl. 27 (1988) 490-536 493

 Preparation : Evolution :Magnetization I
I transfer
“c I I
---I-
r: t-.; LIZ--A
4 f-
since the spectral width, i.e., the range of possible frequen-
cies, is limited due to the nature of the sample and to ana-
log filtering. Discrete sampling is essential for 2 D N M R
spectroscopy, since it enables the definition of a second
time scale in digital form (see Section 2.2).
2.2. Two-Dimensional NMR Spectroscopy
The pulse sequence of a 2 D NMR experiment may be
divided in general into the following blocks:‘21
preparation-evolution(f,)-rnixingdetection(f2).
The detection period corresponds exactly to the one for 1 D
NMR spectroscopy (Section 2.1). The time t2 provides,
after Fourier transformation, the w2 frequency axis of a 2 D
NMR spectrum. The trick of 2 D NMR spectroscopy is that
a second time variable, the evolution time tl, is introduced.
The evolution time t l is made stepwise longer, i.e., incre-
mented, in analogy to the detection time tl. For each t l
increment a separate F I D is detected in t2. Thus a signal
is obtained which is a function of two time variables,
t l and t 2 : S ( t l , t 2 ) .A series of w2 spectra is then obtained
upon Fourier transformation of each of the FIDs. They
differ from one another in the intensities and/or phases of
the individual signals, according to the different t i incre-
ments. A second Fourier transformation over t , , “perpen-
dicular” to the w2 dimension, results in a spectrum as a
function of two frequencies (w2 horizontal, w i vertical).
The principles of 2 D N M R spectroscopy are demon-
strated in Figure 5 with a n example of proton-carbon
chemical shift correlation (H,C-COSU). First, a transverse
proton magnetization (coherence) is generated, which is
subsequently transferred to carbon during the mixing pe-
riod by means of ‘ H and I3C90” pulses. The exact mecha-
nism of this transfer will be discussed in Section 2.2.1. Fi-
nally, the carbon magnetization is detected.
Let us consider this experiment again in greater detail
using the pulse sequence of Figure 5 with product opera-
tors.“’ After the preparation, which in this case consists of
[*] Originally I and S were identified with “insensitive” and “sensitive,” re-
spectively. Nevertheless we use here I for the proton, although it is mag-
netically the more sensitive nucleus, and S for the heterospin, to corre-
spond to conventional use.
494
Detection :
;
-
4 h
Fig. 5. Principle of 2D N M R spectros-
m p ~ ,exemplified by H,C-COSY.
Note the intensity variation of the car-
bon signals after the first Fourier
transformation (lower center), which
reflects the evolution of Droton man-
1
3c netization during f,.
a 90; pulse, transverse magnetization I, (single-quantum
coherence) is generated from the equilibrium magnetiza-
tion. During the evolution time t l , I , evolves with its char-
acteristic frequency [Eq. (14)]. Through the mixing se-
quence consisting of 90” pulses in ‘H and I3C, the proton
coherence is transferred to I3C coherence (i.e., I, trans-
forms into &). The I , term is not considered further, since,
as we shall see in Section 2.2.1, it is not relevant for this
experiment. The mechanism of the coherence transfer is li-
kewise not discussed further at this point. S, evolves dur-
ing t2 with its characteristic frequency [Eq. (15)]. The state
of the spin system after a time t2 is given by Equation (16).
cos n,t, (S, cos n$, + S, sin Rst,) (16)
Thus the coefficients of the S operator describing the time
evolution in t , and t 2 contain not only its own characteris-
tic frequencies Q,, but also the I-spin frequency Q,. This is
sometimes referred to as “labeling.” S,, is labeled during t l
with the I frequency. Quadrature detection converts the
operator combination of Equation (16) into a complex sig-
nal [Eq. (17)]. Fourier transformation along tI and t2 results
in the frequencies, namely, +Q, in o1and Q, in w2. A
spectrum like the one shown schematically in Figure 6 is
obtained. A line in position (Q,, Q,) in the 2 D N M R spec-
trum will appear when the signal is modulated in t , by the
frequency Q, and in t2 by the frequency as.
The 2 D N M R spectrum is thus a representation of the
transfer processes of coherences, whereby peaks arise at
the characteristic frequencies of the interconnected coher-
ences. The intensity of the signals is determined by the ef-
Angew. Chem. l n t . Ed. Engl. 27 (1988) 490-536
 s p e c t r o s ~ o p y [ ~I ~‘7*3711
~ brought advantages not only for
the measurement of rate constants of reactions at equili-
brium (such as in vivo reactions), but also for the determi-
nation of rates and mechanisms of site exchanges in flux-
ional molecules.

- “2

Fig. 6. Schematic cross signals of I and S nuclei in a hypothetical 2D NMR

2.2. I . Scalar Coupling

spectrum. The spectra on the margins represent projections along the axes. Scalar coupling underlies most of the 2 D N M R experi-
I n o2the Redfield technique (see Fig. 3) was used.
ments. It also leads to the failure of the vector description
of N M R experiments. Because of the fundamental role of
ficiency of the mixing process. The transferability of co- scalar coupling in 2 D N M R spectroscopy we shall discuss
herences depends, o n the one hand, on the properties of it first.
the spin system: on structural, dynamic, and chemical mo-
lecular properties which reflect the behavior of the magne- Scalar Coupling in the Product Operator Formalism
tically active nuclei. O n the other hand, it is determined by Scalar coupling is the interaction between two spins,
the mixing sequence of the particular 2 D N M R experi- which occurs predominantly through bonds (indirect cou-
ment. The multitude of 2 D N M R experiments relates di- pling) and only occasionally “through space.”[“81 It leads
rectly to these various transfer processes. to splitting of the resonances in the N M R spectrum (multi-
In high-resolution N M R spectroscopy there are essen- plet structure). In terms of the product operator formalism,
tially two spin-spin interactions which can be used to ef- spin-spin coupling between two nuclei 1 and 2 may be de-
fect coherence transfer: one is the through-bond scalar scribed according to (1 8)-(21). The resulting expressions
spin-spin coupling ( J coupling), well known in 1 D N M R
spectroscopy since it gives rise to the observed multiplet
splitting. It is effective only between spins which are sepa- IIXI
rated by few bonds in the molecule. Therefore it is particu-
larly useful for the elucidation of connectivities, i.e.,
bonds, between atoms, and serves for the investigation of
the constitution of organic compounds, as well as for spec-
(201
tral assignments. The magnitude of the scalar coupling is a
sensitive measure of the torsion angle about single and
double bonds, and hence provides important information (21)
about spatial structure ( c o n f ~ r m a i i o n ) . [ ~ ~ - ~ ~ ]
The other interaction is the dipolar coupling of two are quite similar to those obtained for the evolution of
spins, which operates through space. It is responsible for chemical shifts. However, here for the first time products
the dominating mechanism of relaxation in s o l ~ t i o n , [ ~ ~ - ~of~ two ~ operators are obtained, to which this formalism
but is only indirectly apparent in the 1D N M R spectrum as owes its name. We turn back for a moment to the vector
line broadening. Dipolar coupling generates also the mu- description in order to clarify the above equations. Cou-
tual relaxation between spatially neighboring nuclei, the pling between spin 1 and spin 2 is shown in the vector pic-
so-called cross relaxation. The intramolecular cross relaxa- ture as two vectors for spin 1, precessing at different angu-
tion gives rise to the nuclear Overhauser enhancement lar velocities corresponding to either the a state o r the
(NOE) effect, i.e., to a variation in the intensity of a signal state of spin 2 (Fig. 7). The x magnetization of spin 1 is
as a result of a disturbance in the populations of another
n~cleus.[~’-”~ The NOE effect depends on the distance be-
tween the cross-relaxing nuclei, and hence allows the de-
termination of intramolecular interatomic distance^.'^^-^^^
NOE values therefore provide the most important parame-
ter for the determination of three-dimensional structures. Y
The quantitative measurement of distances for larger mole-
cules became only p r a ~ t i c a b l e [ ~ ~through
- ’ ~ ’ ~ 2 D NMR
s p e c t r o s c ~ p y NOE
. ~ ~ ~effects
~~~~ and J couplings are com-
plementary to each other in their structural information
content.
A third mechanism for coherence transfer is provided by Fig. I . Evolution of scalar coupling during time 1. starting from x magnetiza-
exchange processes, such as chemical reactions.[10z~’031 In tion. The resulting anti-phase magnetization is shown in red, the remaining
in-phase magnetization in green.
contrast to the other processes mentioned above, this co-
herence transfer does not involve multispin systems. The
fact that a nucleus may have different chemical shifts when apparently modulated by c o s z J t , as is also shown in the
it is in molecule A o r B is sufficient to observe so-called first part of Equation (18). In addition there is a magneti-
exchange spectra. This was applied very early in kinetic zation along the y axis (red), which is described by
studies.[“’2-’’S1The introduction of 2 D exchange N M R Zl,,(ZT - I $ ) (the y magnetization of spin 1 points to + y for

Angew. Chem. Int. Ed. Engl. 27 (1988) 490-536 495

spin 2 in the a state, and to - y for spin 2 in the 0 state).
This y magnetization is not observable; thus, in the ma-
croscopic sense, it is not really a magnetization, since the
positive and negative components cancel exactly. The dif-
ference in populations of the CL and B levels (I”-I”) is
given by 21,. Hence, I,,(I:-IQ) is identical with 21,,IZZ,
which is obviously modulated by s i n n J t [cf. Eq. (18)].
In contrast to the evolution under chemical shift and/or
pulses, the evolution of coupling “creates” or “annihi-
lates” operators: I,x+2 I,, &,and 211, 12z+11y. Annihila-
tion of operators occurs since a squared operator always
equals the unit operator (in this case, I, I,= %). Only cou-
pling and not chemical shift can “bring other nuclei into
the scene.” This is the salient point for coherence trans-
fer,“] since polarization can only be transferred when a
second spin is involved in the product operator.
Before we can proceed to use the product operator for-
malism, we need a number of rules which regulate opera-
tor transformations:
(a) A product operator evolves under the effect of chem-
ical shift or pulses such that each operator in the product
is transformed separately [Eq. (22)-(24)]. Izdoes not evolve
under chemical shift [see Eq. (lo)].
(23)
One must be careful when dealing with scalar coupling,
since in this case also pairs of product operators of the mu-
tually coupled nuclei must be treated together. For exam-
ple, 21,,I,, evolves under a 1,3 coupling according to (25)
1251
(i.e., only the transverse operator 11,evolves [cf. Eq. (IS)]).
Under 1,2 coupling, however, 2 11, IZZevolves according to
Equation (26).
(261
llx
It is also important to note that product operators of two
transverse operators [Eq. (27) and (28)], as well as product
operators of two longitudinal operators, d o not develop
mutual coupling [Eq. (29)l.
(”1 Most often the expression “polarization transfer‘’ is used instead of “co-
herence transfer.” However, we think the latter is more precise.
496
2II?JZx 211J2z + U,J2,, (two transverse operators) (27)
U,X, dl’ U1J2z 211J2y (two transverse operators) (28)
Z,J2,, d12f?-IlJ2z ,U1J2, (two longitudinal operators) (29)
However, 21ix12ydevelops coupling to a third spin (I3=)
[Eq. (30)]. In this equation we have already made use of
rule (b).
(b) Couplings and chemical shifts operate simultaneous-
ly; in the absence of strong coupling, however, the evolu-
tion under these interactions may formally be considered
sequential. This is due to the commutation of the operators
of chemical shift and weak coupling. This does not apply
to pulses. Therefore pulses limit the periods of free preces-
sion.
The product operators only “come to life” when one re-
alizes to which signals in the spectrum they give rise. The
following is valid for weak coupling:
(a) Only product operators with one transverse operator
(I, or I,) are observable. All other products cannot be ob-
served.
(b) Product operators with one transverse operator ( I x ,
I,) and any number of longitudinal operators (Iz) evolve
into “observable” operators as long as the “transverse”
spin is scalar-coupled to all the ‘‘longitudinal’’ ones.
(c) All other product operators are not observable, and
d o not evolve into observable operators.
We turn now to discuss product operators in a three-
spin system, in which nucleus 1 is coupled to two other
nuclei, 2 and 3 (Fig. 8). I,, generates the well-known dou-
11, -
Fig. 8 Schematic in-phase double doublet (line spectrum)
ble doublet at chemical shift Q,. This results from Equa-
tion (31). We make use of the fact that only 11,and I,, are
Ilxcos n1tcos nJ,,t cos nJ13t + Ilysin R,t cos nJlzt cos nJ,,t + ... (31I
observable, and neglect all other 6 unobservable terms. Be-
fore we can show how a spectrum is constructed from this
time function we need to add a few comments on phases
and Fourier transforms: The absolute phase of a signal
cannot be defined in a significant manner. While the phase
can change owing to the electronics, it may be corrected by
Angew. Chem. Int. Ed. Engl. 27 (1988) 490-536
the experimenter as desired through zero-order phase cor-
rection. However, the definition of relative phases, which
remain invariant with respect to phase correction, is signif-
icant. The following rule may be formulated: the relative
phase equals 90” multiplied by the number of sine factors
counted in the coefficients of the x magnetizations. We
can, for example, adjust the absolute phase such that an I,
term with only cosine factors as coefficients will be in ab-
sorption. The following will then hold for the phases of the Time domain
other terms (see Fig. 9):
0, 4, . . . sine factors: a b s o r p t i o n
1, 5, . .. sine factor: dispersion
2, 6, . .. sine factors: negative a b s o r p t i o n
3, 7, . . . sine factors: negative dispersion
b Fig. 1 I . Construction of an in-phase multiplet from the two in-phase doub
C
Fig. Y. Line shapes in N M R spectra: a) absorption signal, h) dispersion sig-
nal, c) negative absorption signal (emission), d) negative dispersion signal.
The modulation of the time signal by coupling occurs
exclusively through amplitude factors of the form cosnJt
and s i n n J t . Its Fourier transform is shown in Figure 10.
Time domain
cos nJl$
Fig. 10~Signal shapes for a doublet with in-phase structure (a and b), and
antiphase structure (c and d). The spectra on the left (a, c) are in absorption,
those on the right (h, d) in dispersion.
A starting point for the construction of a signal from a
product of functions [see, for instance, Eq. (30)] is the fold-
ing principle: The Fourier transform of a product of func-
Angew. Chem. Int. Ed. Engl. 27 (1988) 490-536
tions can be constructed from the Fourier transforms of
the individual functions by convolution. In our case, the
product consists of the trigonometric functions, the Fou-
rier transforms of which are well known. The preceding
discussion, applied to the terms in Equation (31), leads to
the signals shown in Figure 1 1 .
Spectrum Product operator
at the beginning of f2
cos XIl$ I +Jl24
cos xil$ I -Jl3-l
cosnJi$.cosxilI,f u- + J l p *--JI3-
I,,
lets corresponding to J12and J , , through convolution.
Antiphase operators behave as follows: 2I,,12zis not di-
rectly detectable [rule (a)]; however, it develops observable
magnetization according to Equation (32). If the J12cou-
pling equals 0, no observable magnetization evolves [rule
(b)], as can also be seen directly in (32).
We apply again the convolution principle and obtain the
result shown in Figure 12. An iniphase doublet is obtained
Spectrum Product operator
a t the beginning of fl
I C-J13- I
Fig. 12. Construction of a double doublet with antiphase structure with re-
spect to JI2and in-phase structure with respect to J,,.
for JI3(cosine) and a n antiphase doublet for J12(sine). The
x component contains two sine factors, so that finally a
negative absorption signal is obtained, in-phase with re-
spect to JI3and antiphase with respect to J I 2 . The spectra
for the remaining product operators are likewise obtained
using the same “recipe.”
At this point we need no longer denote the time domain
signal. According to the recipe stating that euery z operator
generates an antiphase doublet and the others generate in-
497
 phase doublets, we can construct the missing spectra, which terms, but shall instead ignore the irrelevant ones, which
are shown in Figure 13. d o not lead to signals in the spectrum.
Only terms I , , , I z x , or 13xare detected at the end of the
detection period t2. Let us focus on the I,, term, since the
T i m e domain Spectrum Product operator transfer I l x + I z x gives rise to a cross signal at SZ, in w , (ver-
a t t h e beginning of fL
tical axis) and SZ, in w2 (horizontal axis). We noted earlier

cos dl$.sin 7tJ13f - 1,A

that only coupling can introduce an additional spin into
the product operator. The evolution (33) must hence take

sin (33)

place during t , . Only this product operator is converted by

Fig. 13. Double doublets with different antiphase structures: upper part, an-
tiphase with respect t o J13.Lower part, with respect t o both J , 2 and J,,. a 90; pulse completely into - 2 I , , I Z x . The transfer dia-
gram for the 1,2 cross signal in the COSY experiment is
thus given by Equation 34. There is no other way to trans-
There are rules also for the phases of signals (absorp-
tion, dispersion): All the terms with an I , operator (e.g.,
I , , , 21i,12,, 4 1 1 ,12,13z, etc.) are in absorption (an even (34)

number of sine factors). It is readily seen that product op-

erators with an I , operator are in dispersion. Absorption
fer magnetization from spin 1 to spin 2 in this pulse se-
and dispersion can be exchanged by means of a phase cor-
quence. Exactly one z operator, which will subsequently
rection. In a 2 D N M R signal there is an independent
transform into a transverse operator during t,, is required
phase for each of the frequency dimensions w,, oz.The
before the mixing. Spin 1 must be present during t , with an
phases can hence be either equal or different in both di-
x operator I I x . More than one z operator in the product
mensions.
operator would transform into several transverse opera-
The following is a discussion of the H,H-COSY experi-
tors, which are undetectable. The coefficients of these op-
ment[','' (pulse sequence P1) in greater detail. It has only
erators are obtained as follows. During t , , I , , evolves un-
two pulses and hence is one of the simplest 2 D N M R pulse
der chemical shift and coupling as in (35). All the cou-
sequences."]

90" 90" t,

(PI)
plings to nuclei that are not responsible for the transfer
2
enter as cosine factors. Here only one such coupling, to
-!. nucleus 3 , was considered. The polarization transfer takes
place through the 90; pulse, resulting in (36). This evolves
into observable magnetization according to Equations (1 8)
and ( 2 0 ) . The overall amplitude of the signal, the so-called
Historically 2 D COSY was the first 2 D N M R experi-
transfer function, is given by (37).
ment,"] and it remains the most important one. It relates to
the earlier mentioned coherence transfer. We have seen
that coupling is associated with antiphase terms such as
2ZlJk sin n1flsin nJI2tl cos nJ13fl (36)
2 I , , Izz, which can lead to observable magnetization. When
a 90; pulse is applied to an operator 21,,12, the latter
transforms into -2Z1,12, [cf. also Eq. (22)]. That means sin nltl sin nJ12t,cos nJ& sin n2tzsin nJ12t2cos nJz,tz (37)
that spin 1, which was transverse before the pulse, be-
comes longitudinal, and the longitudinal spin 2 becomes
Construction of 2 0 Multiplets
transverse. Thus the coherence of spin 1 ( 2 1 , x I , z evolves
We have discussed the construction scheme for 1D
with the chemical shift of nucleus 1) has been transferred
N M R spectra from their time-domain signals, and now ap-
into the coherence of spin 2 . The COSY experiment is
ply it to the construction of a 2D NMR spectrum. From
based on this coherence transfer.
the multiplets shown in Figure 14 in w i and w 2 the 2 D
Let us examine again a three-spin system with spins 1 , 2 ,
NMR spectrum is constructed by placing peaks at the in-
and 3. The first 90; pulse generates from the equilibrium
tersection points of the lines. The intensity is obtained
magnetization a transverse magnetization I , , 12x &. In + + from the product of the intensities of the crossing lines.
the following discussions we shall not evaluate all the
The phases in w , and w , are identical with those of the
corresponding "1 D N M R spectra" discussed above.
[*] The "staffs" given under the actual pulse sequences show the corre-
sponding coherence-transfer pathway. They will be explained separately
So far we have only dealt with a single cross peak, that
later in the text. for spins 1 and 2. The others are obtained similarly, e.g.,

498 Angew. Chem. Int. Ed. Engl. 27 (1988) 490-536


R, J2n ..........
00 00
0 0 00
+-+-
that between 2 and 3 by starting with ZZx after the 90" pulse
and detecting 13x.
We now summarize again the formation of cross signals.
During t , , coherences evolve under the chemical shift of
spin 1 ; during t 2 , under the chemical shift of spin 2. For a
coherence transfer with a 90; pulse a term 2Zl,Z2z is re-
quired, which is converted into -2ZlzZ2x; i.e., antiphase
magnetization exists before and after the coherence trans-
fer, caused by the J , 2 coupling. The J , 2 coupling is referred
to as active coupling. Couplings to other nuclei such as spin
3 appear in-phase and are termed passive. They d o not par-
ticipate in the transfer.
Diagonal signals (e.g., w , = w 2 =a,) are also formed in a
COSY spectrum. For a 1,l diagonal signal the product op-
erator must have one transverse operator for spin 1 before
the mixing, o r else no chemical shift for that spin would
evolve during tl. Likewise the operator for spin 1 must re-
main transverse after the mixing, in order to appear as a
signal at SZ, in w2. No terms with more than one I, opera-
tor can exist before the mixing pulse, for they would trans-
form into transverse operators by the 90" pulse, and thus
generate unobservable terms with more than one trans-
verse operator at the beginning of t 2 . The transfer diagram
for diagonal signals is given by (38) and the corresponding
transfer amplitude by (39).
The analysis of the multiplet structure reveals dispersive
(a single sine factor for both time dimensions) in-phase
signals (cosine factors for the couplings) in both frequency
dimensions. The diagonal signals in the COSY spectrum
are dispersive in both dimensions (double dispersive),
while the cross signals are absorptive in both dimensions
(double absorptive). Absorption and dispersion can, of
course, be exchanged also in 2D N M R spectra by a sepa-
rate phase correction in each dimension. The diagonal sig-
nal is in-phase with respect to all couplings, since they ap-
pear as cosines.
So far we have analyzed only the useful transfers (cross
and diagonal signals). The undesired signals were simply
Angew. Chem. Int. Ed. Engi. 27 (1988) 490-536
00 0 0
Fig. 14. Construction of a 2 D multiplet
from the corresponding 1D NMR spectra.
The signals in the 2 0 NMR spectrum
(right) arise at the crossing points of the
ID NMR spectra (left). Their intensities
result from the product of the intensities of
R2J237 the I D NMR spectra.
ignored. In the COSY experiment, so-called axial peaks
occur as artifacts. They arise when an I , term at the end of
t l is converted by the 90" pulse into a transverse operator
I , observable in t2. Since it is not modulated in 1 , it gives
rise to a signal at SZ, = O (axial peak). The spectroscopist
has, nevertheless, a powerful tool to suppress artifacts:
phase cycling. Before we elaborate further on this, we wish
to describe the second important process for magnetization
transfer between two spins, the dipolar coupling.
2.2.2. Dipolar Coupling
The dipolar coupling plays an entirely different role in
high-resolution N M R spectroscopy compared to the scalar
coupling. In 1 D N M R spectroscopy it operates only as a
relaxation phenomenon and gives rise to line broadening
(T2),and of course T I processes are responsible for the es-
tablishment of the Boltzmann state after a certain time.
Scalar coupling, on the other hand, leads to oscillations of
the F I D and splitting of lines in the spectrum. As ex-
plained above, J coupling generates during evolution peri-
ods operators which cause coherence transfer under the in-
fluence of pulses. Thus the scalar coupling generates a
state which can then (even without the effect of coupling
during the mixing) be converted by pulses into the desired
final state [Eq. (40)].
The situation is different for mixing through dipolar
coupling. There the dipolar coupling must be effective dur-
ing the mixing period, while it need not be effective in the
evolution o r detection times [Eq. (41)].
I,
t
I, -
NOE
s, f 2 s r (41)
Dipolar Relaxation
Dipolar coupling is the interaction of the magnetic
moments of two spins through space.165~77~78.1191The mag-
netic field BD, which is generated by spin 2 at the site of
spin 1, is orientation dependent [Eq. (42), where r I 2 is the
499
internuclear distance and OI2the angle between the inter- To get a feeling for these transition probabilities, we
atomic vector and the Bn field]. now examine the relaxation generated by polarization of
spin I (Zz). Only deviations from the Boltzmann equili-

-1, -% (I, + S,) -% (I, ~ S,)

The rapid reorientation of molecules in solution pro-

duces alternating magnetic fields at the sites of the nuclei
(Fig. 15). These lead to’ exchange of spin polarization (z
magnetization) and cause relaxation of a spin system,
which deviates from the Boltzmann distribution, back to
its equilibrium state.

Fig. 17. Population changes due lo dipolar relaxation. Note that only devia-
tions from the Boltzmann distribution are shown schematically. a) Initial
state I,, b) after relaxation under the influence of W, alone, c) after relaxa-
tion under W, alone.

brium are shown in Figure 17. Hence, the state shown in

Figure 17a corresponds to the Boltzmann equilibrium of
0 = 90” 0 = arccos (1/3)’ 0 = 0”
ED. -1 ED = 0 BD=2
spin S and saturation of spin I, resulting in a difference
magnetization - I z . We recognize that W s does not affect
Fig. 15. Dipole-dipole interaction between two spins in the field B,, and its and W’,equalizes the population difference for the I tran-
dependence on the orientation angle 0. The field reaches a maximum sition, and thus leads to a decay of -1, (relaxation). Since
at O=O, a minimum at 0 = 9 0 ° , and vanishes at the magic angle 0 =
arccos( 1 / 3 ) ” * . this applies equally to both I transitions, there is no change
in the population difference for the S spin. If Wo alone is
effective, however, the populations of the two middle lev-
Because of the dipolar coupling, the two spins d o not
els equalize, and the population distribution shown sche-
relax independently, but influence each other; i.e., each
matically in Figure 17b is obtained, corresponding to a
deviation of a spin state from equilibrium is transferred to
change in population of the S spin at the same time. Thus
the other spin state, which therefore also deviates from
Wo relaxation converts an I , operator in part into S,, and
equilibrium. This effect is termed nuclear Overhauser en-
hence enables magnetization transfer through dipolar cou-
hancement (NOE). Figure 16 depicts the situation for two
pling. The overall polarization remains unchanged in this
dipolar coupled spins, I and S:
process. If W, alone operates, the upper and lower levels
equalize, resulting in the situation shown in Figure 17c.
PP Magnetization is transferred again from spin I to spin S,
however this time with a change in sign, since now the
lower levels of S become more densely populated than the
higher ones. Evidently the overall polarization is not pre-
served by this process.
The effectiveness of the relaxation processes Wn and W,
is determined by the rate of the molecular reorientation.
When the molecular rotation in the external field is slow
(corresponding to a long “correlation time,” z, >a, ’),
cross-relaxation occurs mainly through the low-frequency
transitions Wo( p wl -as)[see Eq. (44)]. Conversely, the re-
am
laxation through W, (paIfa,) is only possible when the
Fig. 16. Relaxation i n a two-spin system. Zero-, single-, and double-quantum molecular reorientation is fast [Eq. (45)], corresponding to
transitions are labeled W,, W,, and W,, respectively.
a short correlation time z., Depending on whether W, or
W, dominates, the cross-relaxation rate W2- W,, may be
Equations (43)-(45)[”’ express the transition probabili- either negative or positive and hence also the NOE effect.
ties W (“rates”) under isotropic reorientation of the inter- For W2- W,, the cross relaxation rate vanishes. Conven-
atomic vector. tionally, the positive cross-relaxation rate and the en-
hanced intensity of the S-spin signal correspond to a posi-
(43) tive NOE; conversely, a negative rate and a decreased S-
signal intensity correspond to a negative NOE.[781
Just as COSY is for the coherence transfer, so is the
NOESY experiment the fundamental and most important
(44)
pulse sequence for the incoherent magnetization transfer.
We have seen that the transfer of magnetization via NOE
is achieved by creating a nonequilibrium situation and
(45)
then waiting (pulse sequence P2).[891

500 Angew. Chem. Int. Ed. Engl. 27 (1988) 490-536

 900
.. 90” 90” I,
I 1
‘ 7m
2
-1
The relevant product operators result from the following
ideas: at the beginning and the end of ,z, z magnetization
is required. It is converted into transverse magnetiza-
tion (detectable coherence), and is also generated from
transverse magnetization. Hence the transformation (46)
results. The transfer amplitude is given by (47), where
I ] c o s n J , , t , is the product‘’] of all c o s n J I , t l factors and
J , , are the scalar couplings between spin I and other spins
n
i ; cos nJs, t2 is the equivalent expression for spin S.
90”
I, -4 -Iy -!L -Iy-
90:
-I, -
NOE
-S,
90”
S,
cos R,f, 7 cos n.T,,fl sin Rsf, 7 cos nJ,,t,
As far as the relative signs of the diagonal and cross sig-
nals in the NOESY spectrum are concerned, when the
NOE is positive ( W , - Wo> 0), Z, evolves into -Sz; i.e., the
signs of the diagonal and cross signals are opposite. If the
NOE is negative ( W, - Wo< 0), however, I , evolves into S,,
and the signs of the diagonal and cross signals are the
same.
The NOESY signal is in-phase with respect to all the
couplings (only cosine factors), and can be corrected to
pure absorption. We shall later discuss in greater detail the
analysis of NOESY spectra and a few of its variants.
We now come to discuss phase cycles, which are an im-
portant means for the elimination of artifact (i.e., un-
wanted) signals from spectra. For example, it is evident
from the NOESY sequence that also the transfer (48) be-
tween two coupled spins is possible. It leads to an unde-
sired signal due to scalar coupling, which overlaps with the
NOE signal. Phase cycling allows the suppression of such
undesired signals.
2.2.3. Coherence Orders and Phase Cycles
In a phase cycle a pulse sequence is repeated, whereby
the phases of the single pulses (transmitter) and the re-
ceiver phase are varied systematically (“cycled”), while all
other parameters (delays, flip angles, etc.) remain constant.
All the FIDs obtained in this way for one t l increment are
[“I Here and later in the text the index i (or j) in the product
spins in the system which d o not appear in the equation.
Angew. Chem. Inr. Ed. Engl. 2711988) 490-536
added up. Phase cycles serve to select a desired transfer
and simultaneously to suppress undesired magnetizations.
They are only useful when the phase dependencies of the
desired and undesired transfer processes are different.
A simple example follows. Cross signals arise in the
COSY experiment due to the transfer (49). Nothing in the
(49)
product operator changes upon rotation of the phase of
the 90” mixing pulse by 180” [see (SO)].
Common artifacts in almost all 2D N M R experiments
are the so-called axial peaks, which arise from transfer of
2
t,
-S, (46)
longitudinal magnetization by the mixing sequence into
detectable transverse magnetization. This magnetization is
not modulated during t , , and hence resonates at w 1= O (the
term axial results from ‘&onthe w , = O axis”). Under 90;
(47) and 90?, pulses the axial signals transform according to
(51) and (52). Thus, in contrast to the desired cross signals,
I,z 2-LI,* (51)
9Q
I,= 4 -4* (52)
the axial signals are dependent on the phase of the second
pulse in the COSY experiment.
Therefore, the axial signals can be suppressed and the
desired signals preserved by adding u p the two experi-
ments: 90,%-90~-t, and 90,”-tI-90 ?,-iz. The axial sig-
nals are canceled, while the desired signals add up. We
turn now to describe a systematic way which will lead to a
recipe for the construction of phase cycles.
Coherence Order[29.301
As we have seen, pulse phases are related to rotation
about the z axis. Hence, let us consider now the behavior
of the various operators under such rotation [Eq. (5 3 )-(SS),
note the analogy with Eq. (8)-(10) with v=Qt]. It is appar-
I. a I, cos cp + I, sin rp (53)
I, 3 I~cos rp - I, sin rp 154)
I, 2% I, (55)
ent that the operators Z, and I , d o not transform indepen-
dently under rotation about the z axis, but rather as a mix-
ture; i.e., a pure I , operator transforms into a mixture of I ,
and I,,. The Cartesian basis is, for reasons beyond the
scope of this article, rather unsuitable for describing phase
cycles. However, there exists another basis in which opera-
denotes the tors d o transform independently. It is the basis of the one-
element operator^^'^^-'^^^ [Eq. (56) and (57)].
501
 sums of positive and negative single-quantum coherences
[Eq. (60) and (61), cf. the definitions for I + and I - in Eq.
(56) and (57)].2 IlxZ2z contains the same coherence orders
IEq. (6211.

According to the rules for product operators (successive

application of the transformations to each operator), we Ilx= % ( I ; + I ; ) (60)
obtain, for instance, Equation (58) for I : I ; . Thus the op-
erator remains invariant under rotation about the z axis. Ily= -vZi(I; - I ; ) (61)
For I : I:, on the other hand, we obtain Equation (59).

Finally we arrive at the following result: the number of

transverse operators determines the highest (p = n) and +
lowest (p = - n) coherence order present in a product op-
erator. All the values from p = - n to p = + n in increments
We always find phase factors e p i P p with the rotation of 2 are valid coherence orders [see (63)-(65)].
about the z axis. p is the so-called coherence order. It is the
number of I + operators minus the number of I - operators
in a product operator. Thus, for I : I ; , it follows that
p = 0 and this coherence is called zero-quantum coherence,
while 1: I: is a double-quantum coherence ( p = +2).
41JJ3x : +3, +1, -1, - 3 (64)
I ; I ; is likewise a double-quantum coherence (p = - 2).
Populations as well as operators that have only longitudi-
nal operators behave like zero-quantum coherences. The
significance of coherence order for the classification of co-
herences results from the fact that it is preserved during We can now see which coherence orders are present at
periods of free precession. any moment by following the product operators during a
2 D NMR experiment. This is commonly represented in a
I + is the “raising” operator and I - the “lowering” operator.
pictorial form (Fig. 19), where lines are drawn (similar to
“Raising” and ‘‘lowering’’ refer to changes in the quantum number
those in musical notation) to indicate the coherence orders
m, and not the energy. They describe processes in which a single
quantum is either emitted (D+a through I + ) or absorbed (a+D during given periods.f301The straight horizontal lines repre-
through I - ) , respectively. The number of absorbed quanta equals sent the conservation of coherence orders during periods
the number of I - minus the number of I + in the product operator of free evolution. Figure 19 represents, for example, a po-
(Fig. 18). sitive single-quantum coherence during t l and a negative
single-quantum coherence during t,.
BB L7B

Fig. 19. Representation of various coherence orders i n d ”staff.” In contrast

to free evolution, pulses do not conserve the coherence order.

Fig. 18. A graphic representation of zero-quantum coherence (ZQC, left) and

double-quantum coherence (DQC, right) in a two-spin energy level scheme. Selection of Coherence Order Jumps
Only one of the two possible transitions is shown in each case, i.e., only ab- Phase cycles select jumps in the coherence orders. These
sorption in the case of DQC. For further explanation see text.
jumps are generated by pulses. A D, pulse transforms, for
instance, z magnetization into a mixture of x and z magne-
The symmetry of the spin Hamiltonian for rotations about the z tizations (Fig. 20). The transfer amplitudes for the various
axis (axial symmetry), which is valid during free evolution, forms coherence-order jumps are not represented in this dia-
the basis for classification of coherences by coherence orders. A gram.
symmetry corresponds always to a conservation in this
case the conservation of coherence order during pulse-free peri-
ods. The symmetry breaks down under the influence of pulses; i.e.,
pulses induce coherence order jumps.

The Cartesian product operators are always associated Fig. 20. Coherence orders resulting from longitudinal magnetization after a @
with a set of coherence orders. Thus 11, and I , , represent pulse.

502 Angew. Chem In! Ed. Engl. 27 (1988) 490-536

 In general, pulses excite all the coherence orders be-
tween p = - n and p = + n, where n is the number of terms
in the product operator [Eq. (66)]. This multitude of coher-
ence-transfer processes (Fig. 2 la) can be disentangled with
phase cycling. Phase cycles act like masks, in that they
only let certain coherence order jumps happen at a certain
position. Such a mask may, for instance, act in the form
shown schematically in Figure 21b. The form of the mask
is determined by two parameters: the selectivity N of the
phase cycle and the coherence order jump Ap. There is,
however, not just one coherence order jump for a given
phase cycle, but a n infinite number incremented by N. The
selectivity is the interval between successive "holes" in the
mask (in the present example N = 3). A phase cycle allows
all the coherence order jumps A p f k N (where
k=O, 1,2, ...).
-
-Ap=5
Only the phase increments are of importance. The refer-
ence phase #('), to which the phase increments are added,
is not determined. The reference phase does not play a role
in the selection of coherence-transfer pathways; it is,
nevertheless, often significant for the effectiveness by
which the selected coherence transfers take place (e.g.,
even/odd selection of multiple-quantum coherence
through 90"-z-1 80"-t-90").
Before we go into the detailed arrangement of phase cy-
cles, we wish to point out the following two essential
facts :
(1) The detector conventionally selects negative single-
quantum coherences [cf. the detection of Z-, Eq. (11)-
(13~.
(2) The first pulse can only create a single-quantum co-
herence (positive and negative) from the thermal equili-
brium state (p =0).
Let us proceed one step further and consider now sev-
eral three-pulse experiments (Table I) with the pulse se-
quence (P3). We shall elaborate later on the individual

1
3
2
1
-1
-Ap=2

1
0
-1
-2
4- Ap = -1 -1

-3
A p = -4
I
a b C
Table 1. Various 2D NMR experiments with three 90" pulses [a].
Fig. 21. a) Coherence orders of a three-spin system generated by a pulse from
a single-quantum coherence (fSQC). Schematic representation of the phase Experiment A, A2 Comment Section
cycle mask (b), by which certain coherence order jumps (c) can be selected.
The mask selects all of the jumps of Ap fk N (k = 0, 1,2, ...). where the selec- COSY with MQF [b] I, A A is very small 3. I
tivity N in this example is 3 and Ap is - 1. Thus, from +SQC all of the Relayed COSY f, A Refocusing by a 180" pulse 3.1.6
generated coherence orders can pass the mask which correspond to a jump during the mixing time is
Ap of -4, - 1 , 2, or 5 (c). advantageous
NOESY I, r Transfer of dipolar coupling 2.2.2
If we now place this mask over the excitation scheme of during r
MQ spectroscopy A f, Refocusing in the middle of A 3.1.8
Figure 2 la, the number of possible coherence-transfer by a 180" pulse is advantageous
pathways is reduced dramatically (Fig. 21c). Only 5 of the
14 possible ways were selected. [a] Because there is no generally accepted preference for the meaning of de-
lays A and r, we use in this article mainly A if the delay evolves coupling, and
r if other processes such as relaxation or exchange are of importance. [b]
Setting Up Phase Cycles Multiple-quantum filter.
The selectivity N of a phase cycle for a pulse
(qVk)= k Aq5 +#(') with k = 0, 1 ...) is related to the phase in- techniques. They differ essentially in the function of the
crement A q : A q = 2 x / N . It follows that for the mask in second 90" pulse: In the Relayed COSY experiment it
Figure 21 with N = 3 the phase increment is 12Oo=2n/3. creates coherence transfer via single-quantum antiphase
Ap determines the receiver phase qdet: if the pulse phase is magnetization (2IkxZ1,+2Ik~I&~); in the NOESY experi-
increased by Aq, the receiver phase must also be increased ment it generates longitudinal z magnetization (Zx+Zz);
by - A p . A q , hereby - ( - 1).120"= 120". It relates to the while in DQF-COSY as well as in MQ experiments it pro-
periodicity of the trigonometric functions that any selected duces multiple-quantum coherence (21kx IfZ+2I k x Z,J. The
Ap can be chosen. In this way the desired coherence path- desired effect is determined by the direction of the second
way is followed exactly by the receiver. Other unwanted 90" pulse relative to the first one in the selection of anti-
pathways are turned off. Finally, the general formula (67) phase magnetization [see (68)].
is obtained for the calculation of phase cycles. Api is the
coherence-order jump which should be selected by the i-th
pulse.

The desired coherence order (in the example given

Thus the mask shown in Figure 21 would be realized above, zero-, single-, o r multiple-quantum coherence), and
through the following phase cycle: hence the determination of the correct phases, can be
achieved by considering the coherence-transfer pathway.
k 0 1 2 0 1 2 The derivation of the phase cycle for an H,H-COSY ex-
Pulse 0 120 240 or 3 123 243 periment with double-quantum filtering (DQF-COSY) is
Receiver 0 120 240 238 358 118 shown below.

Angew. Chem. Int. Ed. Engl. 27 (1988) 490-536 503

 Double-quantum coherence should be selected by the
second 90" pulse. The desired changes in coherence orders
are presented in the coherence-transfer pathway in Figure
22. left.
DQF-H,H-COSY NOESY
ZQF-H,H-COSY
90" 90" 90"
I, 7 I, * 90" tl 90"
+L
+1
-1
-2
Fig. 22. Coherence-transfer pathways for a phase-sensitive DQF-COSY (left)
and a NOESY experiment (right).
On the right-hand side of Figure 22 is shown, for com-
parison, the here unwanted pathway which would lead to a
NOESY experiment (selection of longitudinal magnetiza-
tion, which in the phase cycles behaves like a zero-quan-
tum coherence). This pathway is successfully suppressed
by phase cycling. Sequences with the same coherence-
transfer pathway as in NOESY, but with mixing pulses
smaller than 90°, are known as z-COSY (Section 3.1.4) and
zz spectroscopy (Section 3.4.2).[1z7-1301
In the DQF-COSY example discussed above the Ap for
the first pulse is + 1 o r -1, for the second pulse either
+ I / - 3 or + 3 / - 1, and in the third pulse + 1 or -3. The
complete phase cycle for an experiment with L pulses con-
sists of N, x Nz x . . . x N L variations, where each pulse i is
varied independently of other pulses by 2n/N, steps. We
have seen that in addition to the desired pathways with Ap,
+
also pathways having Ap, k N, are selected. The greater
N,, the more selective is the phase cycle. However, with the
increase of N, also the number of necessary accumulations
for each t , increment increases. Thus N, is kept as small as
possible; i.e., one must decide whether the undesired
transfer pathways which pass the phase cycle really matter.
Often this decision is made unnecessary by the availability
of only a 90"-phase shifter; hence only N,=2,4 are availa-
ble options. If we take N , = 4 in our example, we obtain
43=64 variations.
When phase-sensitive spectra are recorded (see Section
2.2.4) it must be noted that during t l both the positive and
the negative coherence orders (in this case, p = 1 and +
p = - 1) are necessary. Only in this way can a Fourier
transformation over t , lead to signals of pure phase (i.e.,
either absorption or dispersion). It follows that the relative
phase shifts of both pulses that limit the t , period (in this
case, with phases cp, and cpz) can only be 0 or n. Thus the
number of variations in a phase-sensitive spectrum is re-
duced by one-half ( = 32 for our case).
At first the phase of the first pulse (q,)is shifted in steps
of 90". The phase variation (qZ)of the second pulse fol-
lows from the rules for phase-sensitive detection. The
phase cp3 for the third pulse is kept constant at first. The
504
receiver phase is calculated in accord with Equation (67).
Thus the phases for the first four scans are obtained. The
phase variation for the second pulse follows under consid-
eration of the rule for phase-sensitive detection, so that all
four directions are included. After these eight scans the
phase of the third pulse can be varied. These variations are
shown explicitly, whereby the shorthand notation is used
(O=Oo, 1=90", 2=180", and 3=270"). The entire phase
7 90" f2
cycle for the above-mentioned spectrum is thus obtained
as follows:
The determination of the receiver phase qdeiis demon-
strated for the fourteenth scan, which is framed in the
above scheme [Eq. (69)].
9,3cf=-{(+1)*1+(+1)*3+(-3)*1)= -1 (69)
This result corresponds to a receiver phase shift of 3,
due to the fourfold periodicity of phase shifting
( - 1 P -90" = + 270" 3). The alternative way through
p = - 1 during f , can similarly be formulated.
Thus a phase-sensitive DQF-COSY experiment requires
a 32-scan phase cycle. This could lead to excessively long
measuring times in cases where the desired resolution calls
for many t , increments, or where sample concentrations
are high enough to permit fewer scans. A reduction of the
phase cycle can be obtained as follows:
(a) Out of the 32 phase constellations introduced above,
24 can be constructed from 8 basic phases, when at the
same time all phases are rotated by 90". This rotation of
the phases by 90" serves to compensate for imperfect re-
ceiver phase shifts. The eight basic phases are obtained by
leaving out the variation of the third pulse. This yields the
first eight experiments shown in the scheme above.
(b) Additional reduction is obtained when one realizes
that axial peaks, which are formed from z magnetization at
the end o f t , , cannot be transformed by the second pulse
into double quanta. The double-quantum filter thus sup-
presses also axial peaks. Therefore the explicit suppression
of axial peaks with the second pulse may be left out. The
remaining four phases are:
(4,: 0 1 2 3
9,: 0 1 2 3
qs: 0 0 0 0
qde,: 0 2 0 2
Experience shows, however, that pulse and phase imper-
fections are best averaged out when large phase cycles are
used. In order to cancel out differences in receiver chan-
nels it is useful to apply the CYCLOPS ~ e q u e n c e " ~in
"
both 1D and 2 D NMR spectroscopy; i.e. all the phases are
shifted by 90" increments.
2.2.4. Phase-Sensitive 2 0 NMR Spectra
The expression "phase-sensitive'' reception of 2 D N M R
spectra is somewhat unfortunate, since, in fact, every ac-
Angew. Chem. Ins. Ed. Engl. 27 (1988) 490-536
quisition is phase sensitive. It is the different method of
processing of the data which leads to either phase-sensitive
spectra or spectra which are displayed in absolute value
mode. Phase-sensitive processing is done in those cases
where some o r all of the signals in a 2 D N M R spectrum
can be corrected to pure phases. The sign information thus
obtained and the smaller line width relative to the absolute
value mode are the main reasons for doing so.
We d o not wish to elaborate extensively on the back-
ground for phase-sensitive 2 D N M R spectroscopy. For a
detailed discussion we recommend the article by J. Keeler
and D. N e ~ h a u s . [ ~Suffice
~*’ it to say that phase-sensitive
spectra require an amplitude modulation of the signal in t l .
Practically all of the most often used experiments, like
COSY, NOESY, Relayed, TOCSY, ROESY, and hetero-
nuclear shift correlation, generate amplitude-modulated
signals.
There are two methods for obtaining phase-sensitive spectra.
The method of Stores, Huberkorn, and R u b e r ~ “ ~ generates
~ . ‘ ~ ~ ] two
data sets for each f l value. In one set the signal in t , is cosine-
modulated, in the other sine-modulated (in analogy with the
synchronous two-channel detection in lz). If each data set is sub-
jected separately to a complex Fourier transformation, the two
spectra shown schematically on the left-hand side of Figure 23 are
obtained. The addition of the two spectra results in the desired
spectrum (Fig. 23, right).
02
Fig. 23. The result of quadrature detection in I , according to Smtes. Huber-
korn. and Ruben. Signals modulated by C O S ~ I ,are accumulated separately
from those modulated by sinQr, for each I, value, and then, again separately,
complex Fourier transformed and added up.
The other method uses a real Fourier transformation and ap-
plies in t , the same Redfield method already described for quadra-
ture detection in t,. Detection is done at twice the rate (the t , incre-
ment is divided by two) and with a 90” phase shift after each 1 ,
increment. This phase shift in 1, is a pulse phase, which leads to
the name of the method: lime Proportional Phase Incrementation
(~pp1),1I35.~361
The two methods are equivalent. They both require twice as
many data points as needed for a non-phase-sensitive spectrum of
the same resolution (and equal measuring time).
3. Survey of 2D NMR Techniques
The most important 2 D N M R techniques in current use
are listed in Tables 2-5. Groups are arranged according to
the type of correlated nuclei (homo- o r heteronuclear) and
according to the type of correlation.“]
In previous chapters we have explained the underlying
principles required in order to understand the currently
[*] The explanations of the acronyms used in the text for pulse sequences are
given in the Appendix.
Angew. Chem. Inl. Ed. EngL 27 (1988) 490-536
used techniques. In the following discussion of pulse se-
quences we shall encounter further methods which, like
phase cycles, are designed to discriminate between coher-
ences on the basis of their different properties, in order to
either select or suppress them: multiple-quantum filter, z-
filter, editing techniques, low-pass J-filter, X-filter, and
BIRD pulses.
The time t l , which in contrast to t2 is not a “real” time,
can also be manipulated. Thus particular interactions can
be influenced o r scaled during t l . These include, among
others, w I decoupling, BIRD pulses during t , , and selective
decoupling.
Manipulation of the t Z time is relatively difficult. The
most common one is heteronuclear decoupling, which is
applied in practically all heteronuclear experiments. The
associated pulse sequences (see Section 3.2.10) are highly
developed and have reached astounding efficiency.
We shall explain these methods in the discussion of
pulse sequences and try to show their common features.
First we shall describe the most important experiments as-
sociated with homonuclear coherence transfer.
3.1. Homonuclear Experiments
3.1.1. H, H-COSY and Variants
The cross signals in a COSY experiment are caused, as
we have seen in Section 2.2.1, by two-operator terms like
211,Z2, (with a y-mixing pulse). The cross signals are anti-
phase with respect to the active J I zcoupling and in-phase
with respect to all other couplings. The diagonal signals
are formed from a transfer of the type Ily+Ily. They are
in-phase dispersion signals in both dimensions. Cross sig-
nals near the diagonal are often severely distorted and dif-
ficult to observe due to the dispersive extensions from the
diagonal signals.
This problem can be solved with the application of mul-
tiple-quantum filters (pulse sequence (P3), delays as shown
in Table 1, coherence-transfer pathways in Fig. 22):[1’8-1411
the product operator responsible for the diagonal signals
before the mixing pulse contains a single operator (I,,). In
contrast, the one responsible for the cross signals contains
two operators (2 11,Z2J. Double-quantum coherence can-
not be generated from a single operator by the mixing
pulse; it can, however, be generated from a two-operator
term. Thus a 90: pulse is applied to cause a double-quan-
tum excitation of the two-spin operator term mentioned
above, according to Equation (70) [cf. Eq. (60) and (61)].
The first term on the right-hand side represents positive
and negative double-quantum coherence and the second
term zero-quantum coherence. The double-quantum co-
herence is selected by phase cycling as described in Sec-
tion 2.3.3 and finally converted back, by a 90; pulse, into
single-quantum magnetization. This leads to filtration of
505
Table 2. Homonuclear 2D NMR techniques through mixing by means of coherence transfers.
Symbol Name Application
H-(-),H H,H-COSY Homonuclear shift correlation
a) normal Identification of spin systems,
signal assignment
b) with delay Detection of small couplings
c) DQF-COSY Identification of spin systems,
determination of J values
d) "Constant time" Decoupling in I,
COSY
e) fl-COSY Relative sign of J
f ) E.COSY Accurate measurement of J
g) P.E.COSY Accurate measurement of J
h) z-COSY E.COSY-like spectra
i) zz-COSY Solvent-signal suppression,
Determination of J
j) JR-COSY Solvent-signal suppression
k) X-Filter COSY Simplification of proton spectra,
spectroscopy of very large
molecules
I) X-Half-filter COSY Same applications as k)
MQ spectroscopy a) With emphasis on direct cou-
pling
- different representation (in
case of overlapping signals
in the COSY spectrum)
- For measurements in water
b) With emphasis on remote
coupling
TOCSY (HOHAHA) For macromolecules since cross
peaks appear also in case of
broad lines (in-phase multiplets)
x-x INADEQUATE C connectivity
INEFTINADEQUATE C connectivity
Inverse variants
the single-spin operator terms (dispersive diagonal signals)
introduced above [Eq. (71)l.
We thus obtain, through double-quantum coherences,
among others the desired transfer from spin 1 to spin 2
IEq. (7211.
The transfer 2 1 , ~ 1 ~ ~ ~ 1 / 2 ( 2which
1 i ~ 1is~ ~effected
), at
the same time, leads to the diagonal peaks in the DQF-
506
Comment Reference Pulse
(A: advantage; D: disadvantage) sequence
Most important technique
Fast routine technique
D: No pure phases [I371
Best standard technique [ 138- 1411
A: Low-intensity diagonal
signals
D: Artifacts in case of strong 1137, 1421
coupling, no pure phases,
strong dispersive contribu-
tions on the diagonal
Alternatives: f.. e. h
-I
12, 137, 1431
A: High resolution, good [ 144- 1461
suppression of dispersion
signals on the diagonal
A: Simpler method than 11471
E.COSY
D: Not as accurate as E.COSY
A: Absorptive diagonal [130, 148, 1491
D: Simultaneous formation of
NOE cross signals especially
for macromolecules
A: Absorptive diagonal [ 127- 1291
D: N o quadrature detection in Il501
wt
Only protons bound to a hetero- [151, 1521
atom give rise to cross signals
A: More flexible than k) since 11531
the filter operates only in
one frequency domain
D: J dependence of excitation 1154- 1581
efficiency, no pure phases
A: Cross peaks to resonances 13741
under the water signal
Information similar to relayed I I591
spectrum
Transfer across the total spin [ 160- 1631
system possible. This may cause
assignment problems
D: Very insensitive, requires 1.50, 164-1671
" C - Y pairs
Slightly better sensitivity, alter- 11681
native in some cases: C-relayed
H,C-COSY (see Table 5 )
COSY experiment. They are now antiphase and absorptive
as desired (like the cross peaks). We realize also that,
through the factor 0.5, the transfer is now only half as effi-
cient as in a conventional COSY.
The concept of multiple-quantum filtration can also be formu-
lated in a general form: a coherence of the type I l , f 2 , . . .fnz can
give rise maximally to an n-quantum coherence. An m-quantum
filter with m > n would hence suppress such a product operator.
Certain spin topologies may then be selected: A spin I ] , which is
coupled to n - 1 other nuclei (Jli# 0 for i = 2 , 3 , ..., n), evolves, e.g.,
after 90,"-t1, product operators with a maximum of n operators.
This means that all the peaks that originate from I ' are suppressed
in a spectrum with an m-quantum filter (m>n)."381
The cross signals in an n-quantum-filtered (nQF)-COSY are ob-
tained through the transfer (73). For the generation of the relevant
coherence before mixing, I ' must be coupled to n - 1 spins; for the
Angew Chem Int. Ed. Engl. 27 (1988) 490-536
Table 3. Heteronuclear 2D NMR techniques through mixing by means of coherence transfers

Symbol Name Application Comment Reference Pulse

(A: advantage; D: disadvantage) sequence

H-X H,X-COSY (HETCOR) Heteronuclear shift correlation

a) normal Routine method at present
b) DEPT-COSY Permits editing A: Phase-sensitive representa-
tion
c) H.X-COSY with o,decoupling
d) Modified DEPT-COSY . No advantage over b)
e) COLOC for 'JCH Short measuring time
f) Modified COLOC for 'JCH Short measuring time A : Not as strongly J depend-
ent as e)
X,H-COSY (inverse hetero- For small substance quantities, D: Requires special spectrom-
nuclear correlation) most sensitive method eter hardware
H -( H),>-X Hetero-TOCSY Transfer from proton magneti- D: Intensity loss through dis-
zation via TOCSY followed by tribution of polarization
INEPT transfer over many protons
H-(-hX H,X-COSY for long- COLOC should be preferred [ 187, 1881
range coupling
COLOC Assignment of quaternary C, in- "89-1931
direct detection of C connectiv-
ity, recognition of proton spin
systems, qualitative evaluation
of "JcH
modified COLOC For discrimination of 2JcHand
(XCORFE) 'JCH couplings to proton-bear-
ing carbons
"Freeman experiment'' Determination of "JCH D : Lack of resolution and in- [I951
tens it y
X,H correlation A: Very sensitive [50, 54, 1961
through long-range coupling
Hetero E.COSY, Exact determination of "JcH D: Relatively insensitive [143, 1971
pcosu

Table 4. 2D NMR techniques with mixing through NOE and/or exchange.

Symbol Name Application Comment Reference Pulse

(A: advantage; D: disadvantage) sequence

H.--H Homonuclear nuclear Overhausei and exchange spectroscopy

NOESY Distance information, three- Besides COSY most important [17, 89) (P2)
dimensional structure of mole- technique
cules, exchange spectroscopy
zz-NOESY Solvent-peak suppression, sepa- [127, 1281 (P36)
ration of chemical exchange and
NOE.
ROESY Separation of chemical Suppression of J cross peaks [ 198-2021 (P39)
(CAMELSPIN) exchange and NOE in medium- necessary
sized molecules without NOE
effects
H . . .X Heteronuclear nuclear Overhauser and exchange spectroscopy
H,X-NOESY, HOESY D: Low sensitivity 1203-207, 3681 (P20)

information only on some parts of the structure. Triple-quantum-

conversion of the resulting coherence into observable magnetiza-
tion, I 2 must be coupled to the same spins (J,,#O for
i = 1, 3, 4,. . ., n)."381
Cross signals are generated between two coupled spins in an n-
quantum-filtered COSY spectrum when they have in common ad-
ditional n -2 coupled nuclei. The multiplet structure of this cross
signal shows in both dimensions n - 1 couplings in antiphase.
Multiple-quantum filters with n > 2 are not very common. This is
due to the reduction of intensity by the factor 2"-'. Another rea-
son is the fact that the higher the order of the multiple-quantum
filter the fewer moieties in a molecule "pass it," so that one gets
filtered COSY spectra are used in the analysis of proteins when,
for instance, the identification of glycine units is sought; these
units are the only amino acids which give rise to cross signals in
the TQF-COSY spectrum between the NH protons and both of the
a protons.[259'
The net intensity obtained upon integration of the diag-
onal peak in a multiple-quantum-filtered COSY spectrum
( f l x + l l xZ,,,+Zly)
r is zero. Thus the problem of the intense
diagonal peaks is solved through double-quantum filtra-
tion; no dispersion signals are obtained. This is, however,
achieved at the cost of a cross-peak intensity reduced by a
factor of one-half. The advantage of the strongly reduced
diagonal peaks has nevertheless made the DQF-COSY ex-
periment the most widely used technique. An example is
shown in Figure 24.'2601

Angew. Chem. Inr. Ed. Engl. 27(1988) 490-536 507

Table 5 . Additional 2D NMR methods.

Symbol Name Application Comment Reference Pulse

(A: advantage; D: disadvantage) sequence

Methods with more than one mixing step

a) Two consecutive J mixings
H-H-H H-Relayed H,H-COSY Assignment in crowded spectral A: Stepwise elucidation of [208, 2091 (PSI
(RCT spectroscopy) or regions connectivities
H-Relayed H,H- D : Less sensitive than TOCSY I211.2121
DQF-COSY For special problems I D var-
iants I2101 are applicable
H-H-X H-Relayed H,X-COSY In overcrowded 'H-NMR sig- Mostly well replaceable by [213-2181
nals COLOC
Inverse variants 153, 2191
H-X-H X-Relayed H,H-COSY 1220, 2211
H-X-X X-Relayed H,X-COSY C connectivity in H-C-C frag- Also used in a I D variant [222-2251
ments A: Relatively insensitive, but
more sensitive than
INADEQUATE
X-H-H H-Relayed X,H-COSY D: Useful only for heteronuclei [226, 2271
with large y (e.g. "P, ''F)

b) Mixing through scalar and dipolar coupling

H-H. . . H Relayed NOESY In crowded regions, D: Useful only when large
H . . .H-H peptide sequential assignment NOE effects are observed
a) normal
b) with DQF For spectral assignment
near the diagonal
c) with antisymmetrization
H-(H),. . . H TOR0 Peptide sequencing A: No need for a-proton reso-
TOCSY-NOESY lution
H...H-(H), ROT0
H . . .H-X Hetero-relayed NOESY D: Low sensitivity

Separation of chemical shft and coupling

J H , ~ H Homonuclear Determination of J and 6 Replaced by DISCO and [5, 235-2401 (P4)
2 D J spectroscopy E.COSY
JHCJC Heteronuclear Determination of multiplicity, Generally replaceable by DEFT [24 1-2461
2D J spectroscopy determination of J
Determination of "JCH [247, 2481
Heteronuclear 2D J spectro- Determination of "JCH D: separated H signal re- I2491
scopy with selective H pulse quired, for coupling to each
H one 2D NMR experi-
ment necessary

One-dimensional equivalents of 2 0 N M R cxperiments

Excitation with Gaussian pulses
Traces from 2D NMR spectra (210, 2501
obtained
A: Time and disc-space saving
in case of few well-aimed
questions

2D N M R spectroscopy with selective excitation

Possible with all pulse se- [251, 3731
quences
A: Sometimes sensitivity
gained vs. 2D NMR
methods with nonselective
pulses

3 0 N M R experiments
3 D J.6 12521
cosY-cosY [253-2551
NOESY-COSY, and other combinations

Various
CIDNP-COSY Connection of photochemical Part of the molecule must be 12561
CIDNP-NOESY excitation with 2D NMR photochemically active (e. g. W I
spectroscopy arenes)
COCONOSY Simultaneous COSY and D: Low resolution in the 12581
NOESY experiment COSY spectrum

508 Angew. Chem. Int. Ed. Engl. 27 (1988) 490-536

 F5 F' F3,F2 F4

1
t
i
8.0 7.0 6.0 - 8 50 4.0 3.0
NH ~

Fig. 24. DQF-H.H-COSY NMR spectrum of cycle(-D-Phe-Phe,.) in

[D6]DMS0, 500 MHz. The connectivities within the phenylalanines (F' to
F') are indicated with lines. The signals in the boxes are expanded in Figure
25. The assignment of the amino acids to the peptide sequence does not fol-
low from such a spectrum.

IbS
8.6

The multiplet structure in COSY spectra contains valua-

ble information about coupling constants. The fine struc-
ture of the cross signals in Figure 24 is readily apparent 1

i " " l " " " " ' " ' " i " ' ' l ' ' ' 8
from the enlarged section of the spectrum shown in Figure 4.5 4.4 4.3 4.2 41 4.0 3.9 [ppml
25. -6
The coupling constants over three bonds depend on di-
Fig. 25. Expansions from the spectrum in Figure 24: the NH-H, region (bot-
hedral angles according to the Karplus r e l a t i o n ~ h i p . [ ~ ~ , ' ~ I tom) and Ha-H, region (top). The signals labeled 1, 2 , and 3 were used for
The magnitude of the coupling constants is thus related to the DISCO procedure (see Fig. 28).
the spatial structure of the corresponding molecule.
In 1 D N M R spectra of complex systems the coupling
constants can no longer be read out directly. Therefore 2D The Effect of 180" Pulses
N M R methods suitable for the analysis of not-too-strongly 180" pulses are often used to improve pulse sequences.
coupled systems (see Section 3.1.2) were developed. They have different effects on chemical shift and coupling,
and can therefore be utilized to manipulate interactions.
3. I . 2. 2 D J Spectroscopy We show now that the evolution of coupling is not affected
by a 180" pulse [Eq. (74) and (75)l. The relative signs of the
The first 2D N M R experiment suggested for the analysis
of spin systems was the 2 D J-resolved The
pulse sequence (P4)for this experiment is the only one, in
addition to that in the COSY experiment, with only two
pulses and is hence likewise easy to analyze.

two operators remain unchanged; Le., the evolution of

coupling, apart from a possible overall exchange of sign,
-2 remains unaffected by the 180" pulse. The chemical shift,

Angew. Chem. Int. Ed. Engl. 27 (1988) 490-536 509

 in contrast, reacts differently [Eq. (76)]. The right-hand
term in Equation (76) is identical with (77). After the 180"
pulse the system is, with respect to chemical shift, in a state
180;
I, cos nt + Iysin nt f (I, cos nt - Iysin nt)
f (I, cos n(-t) + Iysin n(-t))
as if it had evolved in the first period t backward (t has to
be replaced by -t , time reversal). In other words, further
evolution after the 180" pulse will lead after a second
period t to the starting situation, Le., to refocusing of
the chemical shift [Eq. (78)].
I, -t
I. cos nt +
180"
sin nt ---A I, cos nt - I? sin nt -I, I,
Thus chemical shift evolves in the 2 D J sequence only
during t2, while coupling evolves during both tl and t2. The
resulting signal amplitude for spin 1 is given by (79), where
~ c o s d l is
, the product over all couplings of spin 1.
Complex Fourier transformation of this expression and
absolute value representation yield the multiplets shown
schematically in Figure 26a. Projection of such 2 D multi-
a b
o1j2n
Fig. 26. a) Schematic peak i n a homonuclear2DJ-resolved NMRspectrumofa
three-spin system ;b) separationofchemical shifts(projectionon thew,axis)and
coupling constants (projection on the w I axis) after tilting.
plets on the w 1 or w2 axis affords directly the coupling
constants. After tilting according to w i = w l , ~ ; = w ~ - w ~ , [ ~ ~ ]
the spectrum shown in Figure 26b is obtained, in which
now w2 represents only chemical shifts, and w I only cou-
pling constants. 2 D J-resolved NMR spectra are phase-
modulated in t l ( Z J , is~ a~ phase)
~ and hence cannot be
corrected to pure phase. The mixed phases lead to false
line positions and make the determination of coupling
constants unreliable. Furthermore, the resolution achieved
through the reduction of multiplets to singlets along w2 in
tilted 2 D J NMR spectra is often insufficient in complex
spin systems,[26'.2621 and artifacts may appear in the pres-
ence of strong coupling.
510
3.1.3. Determination of Coupling Constantsfrom COSY
Spectra
Direct Extraction of Coupling Constants from COSY
For very simple multiplets, coupling constants can be
(76) measured directly from 2 D NMR spectra.[1361However,
this is not possible for complex spin systems.
DISCO[263-2651
( 77)
The disadvantages of 2 D J-resolved spectroscopy men-
tioned in Section 3.1.2 (impure phases, insufficient resolu-
tion) are not found in DQF-COSY. The cross signals in
DQF-COSY show a rather well resolved multiplet struc-
ture, with the active couplings antiphase and all the others
in-phase. Consider a three-spin system, in which JI3=O.
We focus on the two cross signals, the 1,2 and the 2,3 sig-
nals. The multiplet structure for the 1,2 cross signal in w2 is
(78)
described by (80) and for the 2,3 cross signal by (81). Sum-
mation of the two signals yields a signal in the time do-
main described by (82); i.e., the active coupling in this "hy-
sin nJlzt2cos nJ& 7 cos nJzit2 (80)
(79)
sin n(J12+ JB)f2 ? cos nJ,,t, (82)
brid trace" is J l 2 + J Z 3 . The number of lines in this signal
has been reduced by one-half. Through the reduction in
the number of lines, multiplet structures become easier to
analyze. The active coupling appears in DISCO spectra as
the sum of two couplings and is hence effectively in-
creased, so that cancellation of lines from antiphase sig-
nals is less likely (Fig. 27).
LIZ 2.3 CP
2-DIA
B U
*,72n t, 1
1.2 CP
2 3
DISCO decoupling DISCO of cross signals
Fig. 27. DISCO with the cross signals 01' spin 2 of a three-spin system. Note
that the diagonal signal is 90" phase-shifted (dispersive). In order to use this
signal (2-DIA) for "DISCO-decoupling," the COSY spectrum for the diag-
onal signals must be calculated with an additional 90" phase correction in
both dimensions. Upper left: schematic COSY spectrum. Upper right: cross-
sections of 1,2 and 2,) cross signals (1,2-CP, 2.3-CP) and the diagonai signal
(2-DIA) for spin 2 in 02. Lower left: DISCO decoupling through addition
and subtraction of cross signal and diagonal signal. Lower right: DISCO
through addition and subtraction of the cross signals.
Angew. Chem. lnr. Ed. Engl. 27 (1988) 490-536
 If the diagonal signals in a COSY spectrum are free, the
DISCO procedure can be applied to these diagonal signals
in combination with the corresponding cross signals with
equal chemical shifts in w2. Since the cross signals are in-
phase, Equation (83) is obtained, i.e., a multiplet without
To improve the signal-to-noise ratio, coaddition"' is per-
formed over the entire cross signal, and the resulting
pseudo 1D NMR spectra are summed together.[26s1The di-
gital resolution can be improved by a reverse Fourier
transformation, zero filling (to 64 K, for example) followed
by retransformation. The true scaling factor for the combi-
nation of two spectra is unknown and poses some diffi-
culty in the DISCO evaluation. It must be determined
from the result of the combination. This makes the appli-
cation of DISCO in complex or strongly coupled spin sys-
tems difficult.

3.1.4. E. COSY
the active coupling (decoupled multiplet), in a position
shifted by either +nJ12or -nJl,. Comparison of the two A second procedure (E. COSY['44-'461)simplifies the
spectra obtained by addition o r subtraction yields the ac- cross-signal pattern, as shown schematically in Figure 29.
tive coupling constant.
A practical example is shown in Figure 28, with a DOF-COSY E. COSY
DISCO evaluation of the cross signals due to the a protons
of the amino acid Phe' in the spectrum of the cyclic penta-
peptide from Figure 25.

1 2 3
Fig. 29. Multiplet fine structure of a cross signal in a DQF-COSY spectrum
(center) and in an E. COSY spectrum (right).

In order to understand this technique we turn back to the

multiple-quantum-filtered COSY spectra. We saw that in
the DQF-COSY spectrum only two-operator terms con-
tributed. This determines the multiplet structure in COSY
and DQF-COSY spectra. Higher-quantum filters (e.g., a
triple-quantum filter, TQF) can be used instead of the dou-
ble-quantum filter. The pulse sequence remains the same
(P3, COSY with MQF); only the phase cycle changes. In
( 7- 1 I " I ' I i 7 - ' I "
'7'' the TQF-COSY experiment the phases and q52 go
455 4.50 4.55 4.50 455 4.50 through the values O", 60", 120°, 180°, 240", 300", and
-6
during t , alternating signals are added and subtracted. The
multiplet structure is shown schematically in Figure 30.
The cross signak are generated only by the transfer
ZlxZ2,Z3z-+ZlzZ2x13z. A factor 4 in intensity is lost in such a
spectrum, relative to the unfiltered COSY spectrum (a fac-
tor 2 in the DQF-COSY spectrum). The multiplet structure
is antiphase in both dimensions: with respect to Ji2and J I 3
in w , and with respect to JI2and J23 in w2.

DQF-COSY TQF-COSY E. COSY

L I I 1 1 1

Fig. 30. Fine structure of an E. COSY cross signal from a DQF-COSY and d
v-7' I i 1 ' ' I " -v TQF-COSY spectrum. The E. COSY spectrum is obtained, theoretically,
from weighted addition of these two spectra. When additional passive cou-
4.55 4.50 455 4.50 4.55 4.50 4.55 4.50
-6 plings render the spectrum more complex, correspondingly more multiple-
quantum spectra must be added.
Fig. 28. DISCO spectra from the spectrum of Figure 25. The signals labeled
there with I , 2, and 3 were used. Pseudo 1D spectra (top), obtained by coad-
dition of the traces, were added up or subtracted (bottom). The simplification [*I Coaddition is a special way of addition where each trace is multiplied
of multiplets is clearly evident, particularly after addition, in that the signals with the sign of a reference point in that trace in order to avoid cancella-
are well resolved. tion when summing up over antiphase multiplets.

Angew. Chem. Inr. Ed. Engl. 27 (1988) 490-536 511

 Examination of the multiplet structure in DQF- and
TQF-COSY spectra reveals that summation of the two (see
Fig. 30) greatly simplifies the multiplet pattern.
A general treatment for more than three spins can be
found in the original l i t e r a t ~ r e . [ ' ~ ~An
- ' " ~example
~ of the
extraction of coupling constants from an E. COSY spec-
trum is shown in Figure 31.
3,
sdb
~, , . , .", ~ ,.,,,. , ,,,,...,,,,,,.
46 45 44 4jabL2 41 LO 39
Fig 31 E COSY spectrum of cycle(-o-Phe-Phe,-) i n [D,]DMSO, the same
region as in Figure 25 The expansion of signal 2 is shown in the upper right
along with the associated co-added traces The passive coupling constant
3J,,32 can be measured very accurately from the distance between two
peaks
The appearance of a cross signal in an E. COSY spec-
trum resembles that in a COSY spectrum, in which a mix-
ing pulse with a small flip angle is used (p-COSY)."371
However, there are large dispersive parts in the diagonal
peaks, which make the interpretation of cross signals near
the diagonal very difficult. This disadvantage is overcome
by a technique introduced in Ref. [45] and called later P. E.
COSY."41 '41 1471 Another possibility is the introduction
of a z-filter[2661("Z-COSY").['~~] However, both methods
fail to quantitatively suppress nonconnected transfers. For
the last method cross relaxation during the z-filter delay
may occur in large molecules.
z-Filter
The z-filter is an additional method to select certain co-
herences (or in this case populations) which cannot be sep-
arated by phase cycles. We saw the multiple-quantum filter
as a "high-pass filter" for the number of operators in a
product operator, in so far as all the products with fewer
operators than the multiple-quantum order were sup-
pressed. In contrast, the z-filter (P5) selects populations
and suppresses coherences.
B
I b f (PSI
With the phase cycle of the B pulses zero-quantum co-
herences and populations are selected. The zero-quantum
512
coherences are then suppressed by variation of ,z, which
is based on the fact that they are modulated with the dif-
ference of the chemical shifts of the two involved spins.
Populations, on the other hand, are invariant under varia-
tion of z, (disregarding relaxation). Therefore, if zm is var-
ied and the resulting spectra added, the zero-quantum co-
herences average out, and ideally only the populations are
left.
The z-filter can be used, for instance, to select pure in-
1- 2.6 phase magnetization just before the detection (P6). Of all
99 90; AQ
Single quantum T,,
2 lP6)
the detectable single-quantum coherences only I, is trans-
formed by the first 90," pulse into a population and then
regenerated by the second pulse. All the antiphase terms
are suppressed, in the ideal case.
This principle is used in the 1D z-filtered COSY tech-
nique, where the antiphase terms are refocused after the
COSY transfer and undesired contributions with other
phases are suppressed by the z-filter. A pure in-phase mul-
-3 1 tiplet for spin 2, which is coupled to spin l, is obtained
through excitation of the latter. This multiplet can then be
further simplified through the DISCO
,.,. , # 32 The z-filter function is more complicated in the z-COSY
38 experiment, for here the z-filter is flanked by pulses with
small flip angles. Essentially, transformation (84) should
be selected. This then provides substantially simplified
cross signals of the E. COSY type.
8, 8,
I&JwJnz --* f i J J 3 z - J m --* I 1 J 2 J d n z (84)
3.1.5. Additional COSY Variants
Two older COSY variants are described in this section,
which were introduced only for signal assignment, i.e., for
qualitative NMR spectroscopy. They have lost some of
their significance, but are discussed here since they clarify
some important principles.
Delay COSY
The signals in a COSY spectrum are antiphase with re-
spect to the active coupling in both frequency domains
(see Section 3.1.1). Taking into account the line width, this
multiplet structure is not optimal because broad lines in
antiphase are likely to cancel partially. Another point is
that the resolution determined by t y X is often not suffi-
cient to resolve small couplings. However, if a delay is in-
troduced before and after the 90" (if necessary,
with 180" pulses in the middle of the delays to refocus
chemical shifts and compensate for field inhomogeneities:
SUPERCOSY[267-2691), in-phase portions are also obtained
[Eq. (SS)], which d o not cancel, irrespective of the ratio of
sin n%+Ai) sin W z + A z ) = (sin nlt, cos 7 ~ +3cos~d i~, sin XJAJ
(sin nlf, cos d A 2 + cos allz sin d A 2 )
(85)
= sin aJA, ...
sin XJA, cos rlr, cos dfZ+
Angew. Chem. I n t . Ed. Engl. 27 (1988) 490-536
line width to coupling constant. This type of spectrum'2701
therefore produces cross signals with mixed phases, which
is the reason why it is now seldom used. In contrast, refo-
cusing and defocusing are widely applied in heteronuclear
NMR spectroscopy.
w,-Decoupled COSY
w ,-Decoupling can be achieved almost universally
through "constant time" variants of pulse sequences.
wl-Decoupling causes complete disappearance of the
multiplet structure in w l r which means a gain in
r e s o l u t i ~ n . "The
~ ~ ~simplest
~ ~ ~ ~ sequence of this type is
the w,-decoupled COSY''371(P7), in which chemical shift
evolution is generated by a "wandering" 180" pulse.
90" 180" 90" fl
I 1,/2 I A-r,/2 [ ond mixing pulse ( -2IZzI3J, and, after refocusing of the
2 detected. Since the coherence-transfer pathways in Re-
In the 2D J N M R experiment (Section 3.1.2) 180" pulses
d o not affect the evolution of coupling, but they reverse
the evolution of chemical shifts. As a result the homonu-
clear coupling in the pulse sequence (P7) evolves during
the entire delay A between the 90" pulses, whereas chemi-
cal shift evolves during - t l / 2 + ( A - t l / 2 ) = A - t l . The
transfer amplitude for a 1,2 cross signal between two nu-
clei in constant time COSY is given by (86). The multiplet
sin 7rJ12A 7 cos n&A cos Ql(A - r l ) sin xJl2f27 cos nG1t2cos Q2t2
structure in w , is lost completely, since the signal is modu-
lated in t I only by the chemical shift (cosQ(A--t,)). How-
ever, the size of the coupling constant enters through the
intensity of the cross signal (transfer amplitude). w,-De-
coupled spectra can, with some difficulty, be corrected to
pure phase.
3.1.6. Relayed Spectroscopy
There is only one coherence transfer step in the COSY
experiment. Hence magnetization is transferred from one
nucleus to another one which is coupled to it:
2 I,, IZ7+2IlzIZx.Relayed spectroscopy introduces one o r
more additional steps of this type. The Relayed COSY
pulse sequence is (P8).~20s~2091
We consider again only those terms which are relevant
for a relayed signal. Coherence transfer is accomplished by
Angew. Chem. Inr. Ed. Engl. 27(1988) 490-536
90" pulses; therefore only two-operator terms are of inter-
est [Eq. (87)]. After preparation (90° pulse) and labeling
(evolution of chemical shifts and coupling during t l )
(2 IIyIZz)the first mixing pulse follows. Antiphase magneti-
zation of spin 2 is obtained (-21,z12y), labeled with the
chemical shift of spin 1. In the following period A l 2 -
180°-A/2 only coupling evolves, since chemical shifts are
refocused. The .Ilr coupling refocuses (- 2 11,12y-+12x)and
antiphase magnetization with respect to spin 3 evolves
(12,+2 12yZ3z).Spin 3 becomes transverse through the sec-
coupling to spin 2 during t 2 , magnetization of spin 3 can be
layed COSY correspond to those in COSY, relayed as weN
as direct COSY signals can always be found in the spec-
trum. Phase-sensitive processing is possible like in COSY.
However, only the phases of the relayed peaks are pure,
whereas those of the COSY-type peaks are mixed and de-
pend o n A. The relayed peaks have no simple multiplet
structure when more than one intermediate spin contri-
butes to a cross peak. The transfer amplitude is given by
(88).
(86)
The delay A must be optimized with respect to the mag-
nitude of the coupling constants. It must be short enough
with respect to the largest coupling, such that cosnJA $0,
for otherwise this passive coupling leads to disappearance
of the relayed signals. Long delays should also be avoided
in order to keep intensity losses due to relaxation at a min-
imum.
Relayed spectroscopy enables transfer of magnetization
from spin 1 through a relay spin to a third spin not directly
coupled with spin 1. This procedure is very useful for spec-
tral assignments. If, for example, in an AMX and a BNY
spin system the spins M and N overlap, but spins A and B
as well as X and Y d o not, assignment of A to X and B to
Y can be made with the aid of this technique. An example
is shown in Figure 32.
Experiments with several relayed-transfer steps were
successfully applied in the spectral assignment of pep-
and in the analysis of When the cou-
pling constants are known the transfer amplitudes can be
optimized by the right choice of the delays.[2091Relayed
cross signals are often formed through more than one
transfer. If there is more than one possible relay spin, the
relayed signals consist of the sum of different multiplet
structures. This problem of overlapping multiplet patterns
in relayed spectra can be solved with the 1D-relayed se-
quence through selective transfer of magnetization.["']
Such a I D relayed spectrum is shown in Figure 33.
513
 - H"

L 5 0
r -
4 5 4 0
" ' I
3 5
" " I
3 0
1 ' "I "
2 5
" I
2 0
1 ' ' I
1 5
" ' I

-6

Fig. 32. H-Relayed H,H-COSY of cycio(-Pro'-AIa2-Ala3-~-Pro'-Phes-Phe6-) in CDCI, (270 MHz). The N H region in w , is shown and the
corresponding cross signals with a and 0 protons ( 0 2 Note
). that two conformations exist in slow exchange in this solvent (two signal sets
labeled with upper- and lower-case letters). The alanine (A and a) and phenylalanine (F, 9 spin systems are readily identified through the
relayed peaks to the B protons in the right part of the spectrum. This type of spectrum does not lead to assignment of the signals to the
peptide sequence. Signals o n the counterdiagonal are due to instrument-specific artifacts.

1D Relayed COSY at t h e N H s f Phe'

I I I I

- 40
6 IHzl
0

/$

I , I , , ~ " ~ ' ~ ~ ~ " ~ ~ ~ ~ ' I ~ ~ ~ ~ I ~ ~

8.0 7.0 6.0 5.0 L O 30
-6
Fig. 33. I D Relayed spectrum of cyc/o(-o-Phe-Phe,-). The N H signal of PheS was selectively irradiated. With the right choice of the
first delay the corresponding a signal appears dispersive in-phase, while the relayed signals show the multiplet of both 0 protons in
absorptive antiphase. As the expanded region in the upper right corner shows, coupling constants can easily be determined from
this multiplet.

514 Angew. Chem. l n t . Ed. Engl. 2 7 ( 1 9 8 8 ) 490-536

3.1.7. TOCSY
A fundamentally different method for the transfer of
coupling information is utilized in the TOCSY experi-
ment."""' In contrast to the COSY experiment, in-phase
signals are obtained, i.e., the following transfer takes
place: ll,-+12x(similar to the NOESY experiment). Mixing
is achieved while a spin-lock field is applied, and ideally
the effect of chemical shift is completely removed. All the
spins are temporarily equivalent (AMX becomes AA'A").
The spin-lock sequences are termed according to Chandra-
kumar et al. as isotropic mixing (no effective field) or as
Hartmann-Hahn mixing (with effective field: HOHAHA
experiment).""" Theoretical investigations are described in
several publicati~ns.~'"'~'~~-~~"~
spectrum is ( W ) , where ~ = ( n m o d 2 ) 9 0 °and &=k(rr/n)
with k=O, 1, ..., 2 n - 1.
We first treat multiple-quantum spectra with two-spin
systems. Indeed, double-quantum spectroscopy is used
mainly for the correlation of "C nuclei (INADE-
QUATE"641). Due to the small natural abundance of this
nucleus (ca. 1%) only signals from two-spin systems are de-
tected.
Double-quantum coherence evolves under chemical
shift according to (89) and (90), i.e., the sums of the fre-
quencies evolve. In contrast, J12coupling between the
spins participating in the double-quantum coherence does
not evolve.

The isotropic Hamiltonian includes only the scalar coupling,

which is insensitive towards rotation (isotropic). If, however, a re-
sidual field remains which is equal for all the chemical shifts, the
isotropy of the Hamiltonian is lost and only axial symmetry re-
mains. This condition is termed in analogy to solid-state N M R ex-
periments "Hartmann-Hahn Match." The spins remain strongly
coupled, which is essential for the transfer.

Isotropic mixing leads to spectra with mixed phases

The INADEQUATE sequence is (PIO), whereby the
(phase modulation), whereas in the Hartmann-Hahn mix-
phase q changes through O", 90°, 180", and 270". The first
ing pure phases are obtained.L160.zx11 The multiplets, even
"sandwich" (90;-A- 18O0-A-9O;) generates double-quan-
when generated over different mixing times, give rise to
tum coherences. For q=90°, for example, (91) is ob-
combinations of different mirror-symmetric mukiplet pat-
tained :
t e r n ~ . [ ' " ~Ideally cross signals are formed between all the
nuclei in a spin system.
Several pulse sequences have been proposed for the 90" AQ,
ti
mixing in the TOCSY experiment. The most successful is
MLEV- 17,"8'-zx'1 introduced by Bax et al.,[2sz1which is also
used with heteronuclear decoupling (see Section 3.2.10). A
further technical realization is found in Ref. [365], theory =- i
-1
-2
in Refs. [366, 3671.
TOCSY is particularly useful in the analysis of peptides
in which the line widths are of the order of couplings, in
which case COSY becomes insensitive due to the anti-
phase structure of the cross signals. The number of nuclei
to which magnetization is distributed is controlled through
the duration of the spin-lock periods. Short mixing times Zero- and single-quantum coherences are suppressed by
produce cross signals practically only between directly the appropriate phase cycles. With the correctly tuned de-
coupled nuclei, whereas longer mixing times give rise also lays only molecules with two adjacent I3C nuclei give rise
to relayed and multiple-relayed peaks. to signals in INADEQUATE spectra.
Chemical shift of the double-quantum coherence,
which, as shown in Equation (89), corresponds to the sum
3.1.8. Multiple-Quantum Spectroscopy of the frequences of the single-quantum coherences of the
two spins, evolves during tI. The 90" pulse that follows
We saw in the discussion of the multiple-quantum-fil-
converts the double-quantum coherence into antiphase
tered COSY technique (Section 3.1.1) that single-quantum
single-quantum coherence, which is evenly distributed be-
coherences were mutually connected through intermediate
tween both spins [Eq. (92)J. The resulting spectrum is
multiple-quantum coherences. In multiple-quantum spec-
tra,[157.158.?86-291] h owever, multiple-quantum coherences shown schematically in Figure 34.
are correlated with single-quantum coherences.
Despite the fact that multiple-quantum coherences are
not detectable, they can be observed with the aid of 2 D (92)
NMR spectroscopy. The pulse sequence for an n-quantum

There is no mukiplet structure in wl. Each pair of cross

signals at equal w Ifrequency represents a C-C connectivi-
ty. There is hence a direct correlation between constitution

Angew. C'hem. Inr. Ed. Engl. 2711988) 190-536 515


Fig. 34. A schematic INADEQUATE spectrum for an isolated two-spin sys-
tem, as it appears for “dilute” nuclei. Pairs of antiphase doublets are ob-
tained, symmetrical with respect to the skew diagonal at w , = 2 w z .
and spectrum. For a detailed discussion see J . Buddrus and
H . B a ~ e r . [The
~ ~ INADEQUATE
~] technique offers maxi-
mum sensitivity for the correlation of nuclei of low natural
abundance. However, indirect excitation and detection of
the heteronuclei by protons as discussed later as “inverse
spectroscopy” (Section 3.2.1 1) leads to an additional in-
crease of intensity.
INADEQUATE spectra with pure phases, as well as
quadrature detection in wl, are achieved by 45” phase in-
crementation. Other variants achieve quadrature detection
in w , without such phase incrementation, using pulse an-
gles other than 90”. Details of the method can be found in
the above-mentioned o r in the original litera-
ture.
Double-quantum spectra of larger spin systems (e.g., protons)
are substantially more c ~ m p l i c a t e d . ” ~First
’ ~ we note the coupling
to other nuclei. We find (93) and (94).Thus the sum of the cou-
pling constants of the active spins with the passive spins evolves.
Direct connectivities are found in the double-quantum spectrum
according to (95), which leads to the cross-signal pair in wI at the
sum of the chemical shifts (R, +R,,R,) and (a,+R2,R2),symme-
trical about the diagonal wI = 2 0 2 .
In linear spin systems of the type 1’-12-13 additional “remote”
peaks are found according to Equation (96). They appear in w 2 at
the frequency of spin 2, but in wl at the sum of the frequencies for
spin 1 and 3 (0,+R,,R,). In contrast to the direct signals, they
have no symmetry reflection about the diagonal; they contain,
however, the relayed information.
5 16
For three spins which are all mutually coupled, both types of
signals, “direct” as well as “remote” peaks, are found in the spec-
trum. These signals have mixed phases. For example, the cross
peak with the coordinates (a, +Q2,Q1)arises from (97). This di-
rect signal is in absorption in both frequency dimensions and in-
phase with respect to the J13 and JZ3coupling in 0,. Another path-
way is (98). The resulting cross peak is dispersive in o,,
absorptive
in w,,and antiphase with respect to both couplings in w,.Both
transfers give rise to a cross peak at (Q, +R,,R,).
Similarly, for the “remote” peak at coordinates (R2+R3,Rl)one
obtains (99). The resulting signal is in absorption in both dimen-
sions and in-phase with respect to both couplings in wl. Still an-
other transfer route is conceivable [Eq. (loo)]. Now dispersion is
(99)
found in wl, absorption in w2,and antiphase with respect to both
couplings. Again, the cross peak is a mixture of multiplets with
different phases and fine structure.
Because of the different mapping of connectivities in the
spectrum, double-quantum spectroscopy is sometimes
used to simplify assignments in complex spectra. The use
of multiple-quantum spectroscopy is however not wide-
spread, mainly because of the complex multiplet structures
of the cross peaks, the dependence of the cross signal in-
tensities upon the length of the delays in the preparation
sandwich, the need for phase increments smaller than 90°,
and the inconveniency in connection with the evaluation
of the o1chemical shift. The use of a z-filter offers some
improvement with respect to the phases of the cross
peaks.‘2931
3.2. Heteronuclear Experiments
Almost all the heteronuclear I D and 2 D NMR tech-
niques are also associated with coherence transfer. Opti-
mal sensitivity of the measurement of heteroatoms is of
particular importance, for they practically all (except tri-
tium) have smaller gyromagnetic ratios y than the proton,
and their natural abundance is often very small.
We first comment on the sensitivity of heteronuclear
N M R experiments in general. The “global” sensitivity fac-
tor of any NMR experiment is y5’2B2’2. It contains a fac-
tor y, which stems from the polarization at thermal equili-
brium. For experiments which start with heteronuclear ex-
citation, the polarization at thermal equilibrium is given by
yxS,, where S stands for the heteronuclear magnetization.
Angew. Chern. Int. Ed. Engl. 27 (1988) 490-536
If, however, the experiment starts with proton magnetiza-
tion, then the corresponding polarization is yHI , ( I for pro-
ton magnetization). Thus the gain in sensitivity in experi-
ments that start with proton rather than heteronuclear
magnetization is y H / y x . The remaining factor y3" in the
global sensitivity is due to detection; when multiplied by
the gyromagnetic ratio of the excited nucleus it is propor-
tional to the signal-to-noise ratio (SIN). In conventional
heteronuclear experiments the heteronucleus is detected
and the factor is yg'. In "inverse" spectroscopy protons
are detected, and the factor is y g 2 . Thus an additional
gain in sensitivity of y3H/'/y;'' is obtained in indirectly de-
tected versus conventionally detected spectra146' (see Sec-
tion 3.2.1).
Practically all the homonuclear experiments discussed
so far have heteronuclear analogues. A proton pulse of an-
gle /3 in the homonuclear experiment is analogous to simul-
taneous pulses of angle D on both protons and heteronuclei
in the heteronuclear experiment. It follows that every ho-
monuclear 2D N M R experiment can readily be transferred
to a heteronuclear system. However, often these direct
transfers d o not represent the optimum in terms of sensi-
tivity. A nonselective pulse in homonuclear experiments
operates equally (neglecting off-resonance effects) on all
the nuclei. In contrast, in heteronuclear experiments the
two different nuclei can be operated on separately. This
leads in certain cases to an enormous gain in sensitivity.
The heteronuclear analogue of the homonuclear COSY
experiment (90"( 'H)-11-90"(1H,'3C)-t2) would only yield,
for an HC fragment, one-fourth of the intensity obtained
by the conventional H,C shift correlation technique.
A second property of heteronuclear systems, which is
significant for the arrangement of pulse sequences, is the
large difference in magnitude between direct couplings
over one bond, I J H X ,and what is termed in heterosystems
"long-range coupling" over n bonds (n 2 2), "JHX. There-
fore, to a first approximation, H,X units can be treated as
isolated systems. In addition, the variation in l J H Xis lim-
ited, in favorable cases, within a narrow range (the lJHp
coupling is an exception).
3.2.1. The Role of 180" Pulses
Let us first treat the role of 180" pulses in heteronuclear
pulse sequences. The effect of a 180; pulse on the evolu-
- s: 0
tion of chemical shift of spin I after time t (the chemical
shift of the heterospin S is obviously unaffected) is shown
in (101). Thus a time reversal of period t before the 180"
pulse takes place [cf. Eq. (76) and (77)].
The homonuclear I coupling remains, as we saw earlier,
unaffected. The same is true also for homonuclear S
coupling. The heteronuclear coupling, however, behaves
differently [Eq. (102)l. The corresponding expression
for the 180" (S) pulse is (103). Thus, in contrast to homo-
nuclear coupling, after time t and a 180" pulse on one
of the heteronuclear species the heteronuclear cou-
Angew. Chern. Int. Ed. En& 27 (1988) 490-536
pling has evolved backwards, just like chemical shift. I t
can hence be refocused by a sequence A-180°(I)-A or
A- 180°(S)-A.
S,cos nJt t 2SJ,sin nft
18Oq;,, ( S ) .
I1031
t (S,cos ~ J +I 2SJLsin n J f ) = t (S,cos aJ( - 1 ) + X&in nJ( - 1 ) )
Two simultaneous 180" pulses on both spins have the
same effect as one 180" pulse in a homonuclear system:
refocusing of chemical shift, no effect on homo- or hetero-
nuclear coupling.
A final comment: The rules for phase cycling can readily
be transferred to heterosystems. It is recommended to use
two "staffs" in heteronuclear systems, since the different
nuclei are separately excited by pulses.
There are essentially two 1D NMR techniques which
perform heteronuclear coherence transfer: INEPT and
DEPT.
3.2.2. INEPT
Coherence Transfer in Heteronuclear Spin Systems through
'Jcrl Couplings
The pulse sequence for is (PII). It corre-
sponds to COSY: a 90" excitation pulse and a 90" pulse on
each of the two nuclei for the coherence transfer. The 180"
pulses serve merely for the refocusing of the chemical shift
evolution.
IPI I I
I: ir! v f
-1
-2
-1
-2
We now analyze the INEPT experiment in terms of the
product operator formalism. The starting magnetization at
thermal equilibrium is y, I, + y&. The 90; 1 pulse gener-
ates transverse I-spin magnetization: yII,. The S-spin mag-
netization remains longitudinal and can be suppressed by
phase cycling (x, - x for the first pulse). During the follow-
ing period Al/2-l 80°(I,S)-Al/2, heteronuclear coupling
lJls as well as homonuclear coupling " J , , evolve, while
chemical shifts for both nuclei are refocused. If the direct
heteronuclear coupling ' J l s is large relative to the hetero-
nuclear long-range coupling "Jls and the homonuclear
coupling J,,, one obtains Equation (104). As usual only the
antiphase term is effective in the polarization transfer [Eq.
517
(IOS)]. The phase of the second 90" pulse must be shifted
by 90" relative to the first one.
The sequence that follows A2/2-180"(I,S)-A2/2 results
in refocusing of the heteronuclear coupling into detectable
in-phase magnetization of spin S [Eq. (106)].
-usy - -Upzcos nJtSA2+ S, sin nJ,,A? (106)
The broadband decoupling at the beginning of the ac-
quisition time destroys all the I-spin magnetization. This
means that all the product operators that contain I-spin
operators are destroyed, and hence can be left out.
The delays Al and A, can be set to A l = A 2 = ( 2 J ) - ' so
that all the transfer factors equal 1 (sinnJA= I,
cosnJA=O); the full factor y I / y s is gained in sensitivity
relative to a broadband decoupled spectrum (without
NOE), because of the initial excitation of the I spin. A fac-
tor 4 is obtained for 'H/I3C, and a factor 10 for 'H/"N.
When several I nuclei are bound to one S nucleus, care
must be taken that the passive I spins contribute a cosnJA2
term during the delay A2. Hence we find for an SI, group
(only relevant operators are observed) Equation (107).
This transfer function is the key to editing methods,
which enable sorting of signals according to the number of
I nuclei connected to S. We note the analogy to multiple-
quantum filters. Here, however, we are no longer dealing
with a high-pass filter, but with a band-pass filter, which
selects product operators with exactly n operators. With
n'JlsA2= 0 and neglecting the factor y,sin7c1JISA1,which
is anyhow always the same, we obtain the factor
nsinOcos"-'O for the signal intensity.
The cosine factor is negative for 0 > 90". It follows that
c o s " - ' 0 responds to the multiplicity of the S nucleus, i.e.,
to the number of I nuclei connected to S. Negative signals
are obtained for even n, and positive ones for odd n. A
selection according to multiplicity is achieved through the
combination of spectra with different 0 values. This is
done by adding three successive experiments with different
0 values (for the sake of simplicity we use here degrees
rather than radians):
@ , = +@; 0 2 = 9 0 " ; O1= 180"-@
If 0 = 345 is chosen, the coefficients shown in Table 6 are
obtained. From the Table we see that the spectrum for S13
groups is obtained according to (108),while for SI groups
518
Table 6. Relative signal intensities for the INEPT experiment as a function of
0 = nJHcA2.
n 0 0 0
45 90 0 135"
it is spectrum(90"). The spectrum for S12 groups is always
obtained by subtraction of the spectrum for 0, from the
one for O1.
It should further be noted that the quality of the editing
by INEPT depends on the assumption of equal coupling
constants. A 10%variation in coupling constants changes
the angles 0 = 4 5 " , 90", and 135" to ca. 0=40", 80",and
120°, respectively. The corresponding signal intensities are
shown in Table 7. Clearly, the signal suppression under
these conditions is no longer complete.
Table 7. Relative signal intensities with editing using the INEPT sequence
assuming a 10% deviation of the coupling from the ideal value.
Intensity of
CH CHZ CH,
C H subspectrum 0.99 0.3 0.07
C H I subspectrum - 0.2 1.88 0.43
C H 3 subspectrum 0. I -0.34 1.72
Transfer through Long-Range Couplings
Magnetization can obviously also be transferred in the
INEPT experiment from the I spin to the S spin through
long-range couplings "Jls.1i881The delays must be changed
for this purpose. In contrast with transfer through direct
couplings, homonuclear I,I couplings must also be taken
into account, for they are in general of the same order of
magnitude as the heteronuclear long-range couplings.
The amplitude for a transfer from Ik to S, in the INEPT
experiment is given by (109). The optimal setting of the de-
lays depends on the actual coupling constants. When these
are known approximately, the delays can be optimized,
whereby due to relaxation short delays should be pre-
ferred.
The excitation pathway is not evident from the INEPT
experiment; i.e., it is not known from which I spin the
measured magnetization was transferred to the S spin. If,
however, clarification of this pathway is desired for the
elucidation of connectivities, either a selective INEPT or a
2D NMR experiment must be performed.
Selective INEPT
In this method all the I pulses are replaced by selective
pulses on Ib.[295.2961 In this way all the passive I spins relate
to the S spin as well as to the Ik spin as heterospins. The
transfer amplitude is thus simplified to ( I 10); i.e., the in-
Angew. Chem. In,. Ed Enyl 27 (1988) 490-536
tensity in this spectrum is substantially higher than in
INEPT with nonselective, "hard" pulses. In addition, due
to the choice of a particular proton signal for the selective
excitation, the transfer pathway is unequivocal. A prere-
quisite is, of course, a sufficiently resolved proton spec-
trum.
3.2.3. 2 D Variants of INEPT: COLOC
The second possibility to separate the transfer pathways
is the constant time 2 D variant of INEPT, the COLOC ex-
periment.[lXY1 In the constant time 2D N M R experiment
the total evolution time is constant. Evolution of chemical
shift is achieved by a 180" pulse, which is shifted within
the delay (see Section 3.1.5). In the heteronuclear experi-
ment, after the initial INEPT excitation, two 180" pulses
are simultaneously shifted, one for protons and the other
for the heteronucleus. Hence the COLOC pulse sequence
(P12) is obtained.
(PI21
.;- -
-7-
-.a
There is a direct analogy with the o,-decoupled homo-
nuclear COSY experiment (Section 3.1.5): here, too, the
cross signals in oIare decoupled (no homonuclear I cou-
pling). The transfer amplitude is, except for the term for
chemical shift evolution, the same as for the INEPT ex-
periment. All the signals appear in one spectrum, which
makes up for the lower intensity relative to the selective
INEPT experiment. However, the selective INEPT experi-
ment is more sensitive if one is only interested in a few
correlations, ignoring the greater effort in setting u p the
experiment.
3.2.4. DEPT
We saw in Section 3.2.2 that the quality of spectral edit-
ing in INEPT depends strongly on variations in the cou-
pling constants. The DEPT (P13) does not
suffer from this disadvantage.
Let us analyze the sequence for an SI, group. Since the
two 180" pulses serve only for the refocusing of chemical
I:
s:
I: -;
-1
-2
- 6
s:
Angew. Chem. In1 Ed. Engl 27 (1988) 490-536
shift, we will ignore them in the following discussion, as
well as the evolution of chemical shift. The delay should
be A=(2'JIS)-'. Equation (111) is obtained. The cosine
term results from the coupling of protons that d o not par-
ticipate in the generation of heteronuclear antiphase terms
21ySz, which is effective in the second half of the pulse
sequence in transverse Sx,ycoherence. The transfer func-
tion is, like in the INEPT experiment, sinOcos"-'O ; how-
ever, here 0 is a tuneable pulse angle, independent of J .
So far we have not discussed the J dependence in the transfer
amplitude. J was set equal to (2A)-'. We repeat the analysis and
focus now on terms which originate from SI, groups, but appear in
the edited spectrum as an SI, group. Such a "cross talk" is possi-
ble in an n-spin system when a product operator Syly(lJm-' ap-
pears before the 0 pulse. It follows immediately that n 2 m . This
operator behaves in the editing process like an SI, group. I t is
built up as shown in (112). When we again assume the values 45",
90", and 180°[3001for 0 and a 10% deviation of the coupling con-
stants from the ideal 'Jls=(2A)-' is considered, we obtain the
editing results summarized in Table 8. This result is significantly
better in comparison with the INEPT experiment (see Table 7).
Further improvement is achieved through DEPT-GL.I3"']
Table 8. Relative signal intensities with editing using the DEPT sequence
assuming a 10%deviation of the coupling from the ideal value.
~ ~
Intensity of
CH CH2 CHI
C H subspectrum 10 0 02 0 002
C H 2 subspectrum 00 19 0 14
CH, subspectrum 00 00 2 89
3.2.5. Heteronsclear ShiB Correlation
Both INEPT and DEPT serve as the one-dimensional
precursor of heteronuclear shift correlation via 'Jls cou-
pling. A 2 D N M R experiment is formed by the introduc-
tion of a variable evolution period (t,) after the 90" prepa-
ration pulse. Decoupling of the heteronuclear coupling in
w I is achieved by a 180" pulse on the heteronuclei in the
middle o f t , . The spectra that are obtained show no hetero-
nuclear coupling in oI.Starting from the INEPT se-
quence we obtain the pulse sequence for the H,X-COSY
experiment[16z. 1 7 2 . 1 7 ~ 1(P14), and starting from the DEPT
["I We use here the term COSY (Correlated Spectroscopy) also for heteronu-
clear experiments. Often this term is used only for homonuclear experi-
ments. For H,X-COSY one often finds other expressions in use. such as
HETCOR or simply shift correlation.
5 I9
 3.2.6. Heteronuclear Relayed Experiments

H-Relayed H,X-COS Y
12
S: Other heteronuclear experiments can be designed
through combinations of pulse sequences. Thus by insert-
I
ing an additional H,H polarization transfer in the H,X-
I. 0 ! \
1 L
COSY sequence we arrive at the H-Relayed H,X-COSY
-2 sequence[213.2 141 (P17). After evolution with heterodecou-

s: 6 Labeling Homonudear INEPT transfer

i \ transfer

sequence that for the DEPT-COSY experiment is ob-

tained,[ 174- ,771 (P15) and (P16). The same spectrum is ob-
tained from pulse sequences (P15) and (P16), although
s: *r
(P16) has one pulse less. The evolution of proton chemical
shifts takes place in (P15) before the generation of trans- pling, polarization is transferred from spin I ' to other I
verse heteronuclear magnetization; in (P16) the two blocks
spins coupled to I I . The evolution of I-spin chemical shift
are exchanged.
is refocused in this relay transfer by the first 180" (I) pulse.
It is advantageous to refocus the I-spin chemical shift also
I in the INEPT transfer which f o l I o w ~ . [ ~ The " ~ ~ 180"
' ~ ~ two
I-refocusing pulses can be replaced by a single 180" pulse
in the middle of the delays (2A, and 2A2), without chang-
S:
ing the net effect. Since any addition of pulses to a se-
quence is a further possible source of artifacts, sequences
1
I: o! \ with fewer pulses are generally preferred. Thus the se-
-1
-2 quence (P18) is obtained for the heteronuclear H-Relayed
+
H,X-COSY experiment, with A = A, A2.1215.2'61 An exam-
ple of an H-Relayed H,X-COSY is shown in Figure 35b.
s: A
-1
-2

I:

S:
; I: -1A (PIS)
-2
I:

s: 0

s: 0 f \
-1
-2
The DEPT-COSY sequence is fully refocused; i.e., for
f l = f 2 = 0 no chemical shift evolves. This is an advantage
for the phase correction, since only small frequency-de-
pendent phase correction values are obtained. Spectrum
editing is equally possible as in the 1D variant.[3021In con-
trast, the INEPT sequence is less suitable for pure phase
2 D spectra, since for f , =t2=0 proton chemical shift
evolves in A, as well as heteronuclear chemical shift in A2.
This introduces a large first-order phase correction in w ,
and m2.[171. ,721
The COLOC sequence and its variant with a BIRD pulse (see
Section 3.2.8) can also be used for heteronuclear shift correlation
via one bond,""] if the requirement for resolution in I , (protons) is
sufficiently small. We only see an advantage in the constant time
variants of I N E P T and DEPT transfers if the 'JIsdependence of
the transfer amplitude is eliminated through the application of a
pair of time-shifted "wandering" 180" pulses."*"
We find that in comparison with the H,C-COSY spec-
trum (Fig. 35a) additional signals, so-called relayed peaks,
appear, which are marked by arrows. They are formed by
H'-H2-CZ fragments and hence provide indirect informa-
tion on carbon connectivity.[2151For example, all proton
signals and carbon signals of the tetrabenzylglucose ring
were assigned as shown in Figure 35. However, the signals
due to direct HZ,C2coupling are also seen in the spectrum.
This is due to the fact that relayed transfer uses single-
quantum coherence, like the magnetization remaining on
HI, and thus selection through phase cycling is impossible
(see homonuclear relayed experiment). If necessary, the
H,X-COSY parts can be filtered with a low-pass J-filter,
taking advantage of the different magnitudes of the cou-
pling constants (e.g., lJHCand "JHC)(see Section 3.2.8).
X-Relayed H,X-COS Y
A further variant of the heteronuclear relayed spectros-
copy is the X-Relayed H,X-COSY e ~ p e r i m e n t . [ ~The
'~.~~~~
pulse sequence is (P19). This experiment, like the INADE-

520 Angew Chern Int Ed Engl. 27 (1988) 490-536

 4

5
01
6 /OR 6('H)

RO*H
RO I
6
Ro OH
H
HO -
7

90
- 80
6 ('3C)
70 90
- 80
6 (13C)
70

Fig. 35. a) H.C-COSY and b) H-Relayed H,C-COSY of tetrabenzylglucose in [D,]DMSO. The CH cross-signal assignment of the sugar is given in (a). It follows
directly from the relayed spectrum (b) without additional assumptions, when the proton signal intensities are consulted (signal 6 is due to two protons attached to
one carbon and can hence serve as a starting point for the sequential assignment).

QUATE experiment, serves to resolve connections in A H' H3 B H' H3

I I I 1
which quaternary carbons participate. It also enables ex- '( Ac 12
C-
traction of 'Jcc coupling constants. I I
H2 H2
on- ono Fig. 36. Different isotopomers of a CH2CH fragmenL. The different signals in
I. 1,/2 i,/2 A, [ Az IBB A3 BB
I a "C-NMR spectrum arise from different isotopomers of the compound in-
vestigated.

BIRD
Let us consider the "pulse (P21) with
3.2.7. Heteronuclear NOESY A=(2'JIS)-'. This BIRD, pulse is essentially a 360" pulse

The heteronuclear NOESY experiment (P20)[203-207.3681

is almost a direct conversion from the homonuclear experi- lP2Ii

ment (P2). Heteronuclear NOES are often too small to pro-

s:
duce useful signals in spectra of larger molecules.
for I spins which are not directly connected to an S spin
3.2.8. Filters and meir Applications (due to the short delays, long-range and homonuclear cou-
plings can be neglected to a good approximation). For an I
The low-pass 5-filter'3031and BIRD pulses[3041serve to spin which is connected to an S spin through IJlS,however,
modify the heteronuclear pulse sequences discussed so far. we obtain (for A = (25) - I ) Equations (1 13) and (1 14). Thus
In cases of large differences between ' J l s and "JlSand ex- the BIRD, pulse acts as a 180," pulse for I spins which are
coupled with S, while for I spins which are not coupled
I: r,/2 1J2 r, BB
with S it acts as a 0" pulse.

180" 90" r,
S: I
Iy - -
99
I,
A
I,
18o;(I,s)
-1,
A
------* -I,
90"
a I, (114)

If the phase of the 180" pulse is changed to y (BIRD,,

P22), the effect of the BIRD sequence is reversed. Now I

treme dilution of the S spin, it is possible to selectively ma- lP22i

nipulate those I spins which have a 'JlScoupling with a n S

spin. In the isotopomer ( A ) in Figure 36 it is possible to
discriminate H ' and H2 from H3, since the first two have a spins with 'JlSremain unaffected, and those without 'Jls
'JcH coupling, whereas H 3 does not. The opposite is true undergo inversion. (P23) is yet another representation of
for the isotopomer B. BIRD, and (P24) of BIRD,. Applications of BIRD pulses

Anyew. Chem In1 Ed Engl 27 (1988) 490-536 52 1

are presented in the following sections, dealing with pro-
ton-carbon correlations.
(P23)
180"
s: I troscopy, to suppress direct 1,s correlations, for they gen-
(P24l
w,-Decoupled Sh$t Correlation
In H,C shift correlation the spectra of different isoto-
pomers overlap to yield the final spectrum. A line in the
I3C-NMR spectrum means that only that carbon in the
molecule is a I3C isotope. Only those protons which are
directly bound to the I3C atom can be called "carbon bear-
ing," in our way of speech; all the others are not.
w,-Decoupling in shift correlation['781(P25) is achieved
by a selective inversion of the passive protons after ? , / 2 ,
while the active protons are left ~ n a f f e c t e d . [ " ~ - 'This
* ~ ~ is
done with the BIRD, sandwich. The necessary 180°(S)
pulse gives with the 18O0(S)decoupling pulse a 360" pulse,
and hence can be left out. Homonuclear coupling between
geminal protons is not refocused, since geminal protons
are always both carbon-bearing. The same concept can be
applied in the DEPT-COSY experiment.[3051
,/2 i A3 A2 BB
COLOC with Protonated Carbons
During the refocusing period A, in the COLOC experi-
ment also the rapidly oscillating lJCHcouplings evolve
with the amplitude cosn'JCHA2. Even a small scatter in
lJCH will therefore have a strong effect on the transfer am-
plitude. The problem can be solved with a BIRD,
which inverts protons bound to 13C. In this way
the 'JcH couplings are selectively refocused in
~,,1194.195,306-3081 while the long-range couplings remain
unaffected.
The BIRD,, pulse in t l of the COLOC experiment serves
also to discriminate between 'JCHand 3JCH couplings in
long-range correlations to proton-bearing I3C nuclei
(XCORFE['94]).The pair of wandering 180" pulses in the
COLOC sequence is replaced by a BIRD, pulse. The latter
has no effect on protons directly bound to I3C (H' in the
fragment '3CH'-'2CHZ-'2CH3), as opposed to the other
protons. Now protons H2, which are coupled to H ' and
inverted by the BIRD, pulse, evolve coupling. If
3JHlH~
this coupling is large enough, then splitting of the C H 2
cross signal is observed (doublet for 13CH-"CH2, double
doublet for 13CHZ-'2CH2, etc.). However, protons that are
coupled to that carbon through 3JcH (e.g., in the fragment
H1zC-"C-'3CHA) transmit to the corresponding proton H i
only a negligibly small 45HH coupling, which does not lead
to measurable splitting."941 In this way the coupling via
522
two bonds can be discriminated from that via three
bonds.
Low-Pass J-Filter
It is sometimes desirable, in correlation experiments in-
volving long-range couplings as well as in relayed spec-
erally lead to strong cross signals and thus may interfere
with weaker signals. 'Jiscross signals can be suppressed
with the low-pass J-filterr3O3I(P26). For A=(2 ' J & ' , pro-
90"
I: I
lP26)
S: A '7.'
tons directly bound to I3C appear after the 90" S pulse as
l x S , o r as -ZxS,, depending on the pulse phase. Thus the
magnetization of the I spin directly bound to the S spin is
totally destroyed through addition. However, the magneti-
zation of I spins that are coupled to S spins with long-
range couplings is only slightly attenuated, by the factor
cos *TIJlsA.
Low-pass J-filters are easy to install in 'H,I3C correla-
tion experiments, since the ' J C H and "JCH coupling con-
stants lie within narrow ranges. Details about the optimiza-
tion of these filters can be found in Ref. [303]. They are
used in heteronuclear relayed[3031and in COLOC spectros-
copy."97]
3.2.9. Composite Pulses
So far we have worked only with ideal pulses; for exam-
ple, a 90" pulse should be exactly 90" for all of the nuclei
of one type, should have no phase distortions, and should
be of zero length. In real pulses there are complications
due to off-resonance effects and B , inhomogeneity. Off-
resonance effects arise because the chemical shift is not
"turned o f f ' during the pulse. As a result the magnetiza-
tion rotates about a tilted field and by a greater angle than
the nominal pulse angle. Inhomogeneities in B , arise from
imperfect coils, which do not generate a constant field
within a given volume, and outside this volume generate
no field at all.
Composite pulses serve to remove these imperfections.
The literature is quite Yet, with the exception
of decoupling and TOCSY sequences, composite pulses
have rarely been used. We therefore limit the discussion to
a few examples.
180" Pulse
The most common pulse is 90,"-180,"-90,".[3093271 It
achieves an inversion of z magnetization over a large
chemical-shift range, and is moderately sensitive to Bi in-
homogeneities. It is applied, for instance, in Ti measure-
ments of I3C resonances and in shift correlations in the
middle of 1 , .
90" Pulse
The 90" pulse is substantially self-compensating against
offset effects. Similar compensation against B, inhomoge-
neities is achieved through the sequence 90,"-90,".L309,3'01
Note that this pulse is exactly one-half of the composite
inversion pulse.
Angew. Chem. Inr. Ed. Engl. 27 (1988) 490-536
 One must be careful in the construction of pulse se-
quences with composite pulses, since straightforward sub-
stitution of simple pulses by composite pulses does not
lead to the goal. The reason is that these pulses introduce
additional phases. An example of the construction of a
pulse sequence with composite pulses can be found in Ref.
13201. Composite pulses with essentially no phase distor-
tions, as well as pulses that compensate for both offset ef-
fects and B , inhomogeneities, are discussed in Refs. [312,
323, 3261.
3.2.10. Heteronuclear Decoupling
Most of the heteronuclear experiments work with
heteronuclear broadband decoupling during detection.
The removal of the heteronuclear coupling leads to an in-
crease in intensity, since the multiplet structure due to het-
eronuclear coupling collapses. The difficulty in broadband
heteronuclear decoupling arises from the sometimes huge
chemical shift range of the decoupled nucleus. Thus at 300
MHz, for instance, the proton shift range is ca. 3 kHz,
whereas for "C it is 15 kHz and for I9F approximately 60
kHz. On the other hand, the decoupler cannot operate at
excessively high power levels because of heating of the
sample. The main goal here is to find decoupling se-
quences which are, as much as possible, offset indepen-
dent.
This goal is, to state it in a simple way, to keep the de-
coupled spin rotating in such a way that its a and p states
are equally populated. The exchange between a and fi states
must be faster than the spin-spin interaction, since the
multiplet structure (e.g., of a I3C multiplet) depends o n the
fact that the a and p states of the coupled nucleus are well
defined.
The first method introduced for heteronuclear broad-
band decoupling through stochastic modulation of the de-
coupler field[32X1 yielded good results. The decoupled spins
are stochastically led around the unit sphere such that all
of the orientations of the magnetization are equally proba-
ble.
Soon better methods were developed, utilizing compos-
Ref. [333] provides a very good review of decoupling se-
quences.
3.2. I I . Inverse Spectroscopy
All of the heteronuclear spectroscopic methods dis-
cussed so far involve the excitation of proton magnetiza-
tion and the observation of heteronuclear magnetization.
When one estimates the sensitivity of a given pulse se-
quence, there is a factor yH(yx)3'2 in addition to the spe-
cific transfer function of the sequence. Inverse spectros-
copy starts likewise with proton magnetization, but also
detects proton magnetization, whereby ( Y ~ ) " is~ obtained
for the sequence-independent sensitivity factor. The factor
is thus eight times greater in proton-detected C,H correla-
tion than in conventional spectroscopy. It is, of course,
even greater for heteronuclei with smaller y, e. g., "N. This
substantial gain in sensitivity makes inverse spectroscopy
(sometimes called reverse spectroscopy) very attractive. In
samples with small natural abundance, however, the sup-
pression of proton signals from molecules without NMR-
active heteroatoms is problematic. Thus for I3C the sup-
pression must be 1 to 200, and in "N 1 to 600. This places
a severe stability requirement on the NMR spectrometer,
which, when not met, leads to strong / , noise (see Appen-
dix). We shall later show methods to suppress /, noise by
the modification of the NMR experiment. Often, however,
the problem can be solved by enrichment of the NMR-
active heteronucleus.
Table 9 compares the relative sensitivities of the various
detection methods. The first column gives the general sig-
nal-to-noise ratio (S/N), the second shows the resulting
amplification factor. The last two columns show, for "C
and I5N measurements, respectively, the theoretical gain in
intensity for inverse detection.
The most frequently used sequence (P27) is also the sim-
plest one.
Table 9. Comparison of sensitivities of the various heteronuclear N M R ex-
periments as a function of different excitations and detections.

ite-pulse decoupling sequences. The first of these se- Experiment S/N Amplification "C "N
quences, MLEV with 16 composite pulses, uses the com- factor
posite 180" pulse (90,"-180,"-90,")[309.3151to achieve an in- Yx(Yx)"2 1.0 1.0
a) Direct observation
version of the spins to be decoupled with as little as possi- b) INEPT YH(YX)3'2
ble offset dependence. A sophisticated e ~ p a n s i o n [ ~ * ~ ~ compared
~ ~ ~ ~ with a) (YH/YX) 4.0 9.9
c) Inverse INEPT YX(YH)"'
leads to compensation for imperfections of ever higher or- (YH/yX)'' *
*
der. The band width of MLEV-16 is about yB2, which is
compared with a)
compared with b)
d) Inverse shift correlation
(YH/YX)"*
7.9
2.0
31.0
3I
the strength of the decoupler field. YH(YH)"2
compared with a) ( ~ ~ / y ~ ) ~ ' ' 31.6 306.0
Another method, which is now generally used for decou- compared with b) lYri/YX)3'2 7.9 31.0
pling of I3C-NMR spectra, is WALTZ-16. Here, in contrast
to the MLEV sequences, 90" pulses are permuted. The ba-
sic inversion pulse is 90,"-180:-270,". WALTZ, like
MLEV, has an offset range of f yB2. However, the scaling
of the coupling is by far better. Precision measure-
ments showed I3C-NMR line broadening of only ca. 10
mHZ.[32y-?211 WALTZ IS . also significantly less sensitive to 2

phase errors than MLEV.[3331

The C A R P sequence[3321is particularly suitable for very -i
. i J lP27l

large spectral ranges. It decouples over a range of

2 2 . 5 y B Z ,and can hence be applied, for example, for I3C
decoupling in proton-detected C,H experiments.

Angew. Chem. Inr. Ed. Engl. 27 (1988) 490-536 523

 We examine only the relevant operators for an IkSlcross
signal: the transverse proton magnetization following the
first 90" pulse evolves homonuclear coupling during
+ +
(A t i t 2 ;chemical shift, however, is evolved only during
+
A tz. The proton network leads therefore to the transfer
amplitude ( I 15). This includes all the passive protons.
So far the heteronuclear coupling was disregarded, al-
though it is responsible for the cross peak. The active
heteronuclear I h , s,coupling (Jlh) evolves during h and con-
tributes a factor sin nJ,hA to the transfer amplitude. Dur-
ing t , the 180" (I) pulse prevents evolution of heteronu-
clear coupling. Finally, during tz the Jlh coupling is refo-
cused and contributes a factor sin nJ,,At,. Combination
with Equation (1 16) leads to the final transfer amplitude
( 1 17). The multiplet pattern is like that in a 2 D J spectrum
(phase-modulated in t,). It is therefore impossible to ob-
tain pure phases. In the ideal case, in which all of the cou-
pling constants are sufficiently different from each other
and greater than the line width, the pattern shown in Fig-
ure 37, left, is obtained for a CHI-CH2H3 spin system. Fig-
ure 37, right, shows the corresponding pattern which
would be obtained in a conventional spectrum.
0 .
Fig. 37. Schematic representation of the multiplet splitting in the H'-"C
cross signal of a CH'-CH2H3 fragment in an inverse spectrum (left) com-
pared with that in a conventional H,C-COSY spectrum (right).
A factor 4 in sensitivity is gained in the inverse C,H shift
correlation; the factor 8 is not gained because the spec-
trum splits into twice as many lines, as can be seen in Fig-
ure 37 (eight lines instead of the four obtained in the usual
H,C shift correlation). A factor 16 is obtained for the mea-
suring time, since the sensitivity corresponds to the square
root of the measuring time. Such factors in pulse se-
quences must always be taken into account when an exact
comparison of sensitivities is attempted. Thus in the above
example only the I3C multiplicity of the protons (in this
case 2) is of interest for the comparison of inverse/conven-
tional shift correlation spectroscopy.
A further gain in sensitivity can be reached through the
insertion of a refocusing delay after the second 90" S
pulse, followed by heterospin decoupling. In this way the
' J l Scoupling in w 2 is removed and, in addition, a factor of
2 in sensitivity is gained. Thus the inverse heteronuclear
shift correlation with a refocusing delay should be eight
times more sensitive than the conventional shift correla-
524
tion. This makes even proteins accessible to heteronuclear
measurement~.['~1
Presaturation of Protons Not Bound to NMR-Active
Heteronuclei
A BIRD pulse, which we described earlier in connection
with conventional heteronuclear spectroscopy, finds an
important use in inverse spectroscopy for reasons men-
tioned above. By installing a BIRD, pulse it is possible to
eliminate the protons which are not bound to an NMR-
active heteroatom. The BIRD, pulse inverts these protons,
and after a delay period of T F .In 2 the signal due to these
protons is minimal (passes through zero). If now the se-
quence for the inverse shift correlation is started, only co-
herences due to the protons bound to active heteronuclei
are generated. Undesired signals are hence suppressed
This procedure is only applicable for
more effe~tively.[~']
correlations through direct coupling.
Inverse Correlation through Long- Range Couplings
An increase of the delay A enables inverse correlations
through heteronuclear long-range couplings. Obviously the
common variants, like the low-pass J-filter, can be applied
here as Long-range couplings contain not only
valuable information about constitution, but also about the
molecular conformation.[3341Quantitative extraction of
heteronuclear long-range coupling constants is possible for
the first time with good sensitivity through the use of in-
verse s p e c t r ~ s c o p y . ~ ~ ~ ~ ~
Inverse Relayed Spectroscopy
Relayed spectroscopy has also been successfully carried
out in the inverse mode.[s31The suppression of signals due
to protons that are not carbon-bearing happens as follows:
after the excitation sequence 9 0 3I)-Al/2- 180"(I,S)-Al/2
the resulting antiphase magnetization 2ZxS, is transformed
by a 90,"(1) pulse into longitudinal two-spin order
( - 2Z7Sz). The remaining transverse proton magnetization
is then destroyed by a homospoil pulse (HS, see Appen-
dix), which results in extensive suppression of signals from
protons which are not heteroatom-bearing. The overall
pulse sequence is then (P28).
tPZ8t
-1
-2
2
1
0 / \
-1
'2
After a recovery period A2 the sequence described in
Equation (1 16) begins: the 90" pulses ,on both nuclei give
rise to heteronuclear double-quantum coherence
(-2ZjxSx), which is then labeled during t , with the chemi-
cal shift of the heteronucleus. This is transformed by the
Angew. Chem. I n l . Ed. Engl. 27 (1988) 490-536
9Oo(S) pulse into antiphase magnetization (2IlXSz),which The sequence (P31) is a generalized form. Product operators
is refocused during A1 to tly.During t , + A 3 + A l antiphase with nS, operators are influenced as shown in ( 1 18).
magnetization to another proton can evolve (211, Z2J, I
whereby the last 90e(I) pulse generates a coherence trans-
fer to the I' spin. With this pulse sequence a successful lP31l

[ T - N M R spectral assignment of a small protein was

achieved.[531This pulse sequence can be improved if the
90-,(I) pulse is not set directly after A2 but, instead, before
the begin of the A3 delay. Then the modulation of the sig-
S," - S," cos" fl + transverse terms (118)

nals by homonuclear coupling in t l disappears. The spectra can be edited to produce subspectra which result
from any desired number of S, operators. Depending on the pulse
3.3. Homonuclear Correlations through Heterospins phase one obtains for different n values the different coefficients
The NMR experiments described in the following sec- shown in Table 10, by which the corresponding transfer functions
tions d o not involve proton and heteroatom correlations, must be multiplied.
but correlations between protons which are, either singly Table 10. Coefficients for different numbers n of S, operators as a function
or together, coupled to a heterospin. The experiments that of the phase fl in the X-filter.
fall in this category are essentially the X-Relayed H,H-
n fl=O 60 I20 I80
COSY, as well as sequences like COSY, NOESY, and
TOCSY combined with X-filter or X-half-filter. 0 I -8 8 I
1 I -4 -4 I
3.3. I . X- Relayed H, H- COS Y 2 I -1 -I I
3 I -2 2 -I
The oldest of these experiments is the X-Relayed H,H-
COSY"20.22'1(P29). It correlates protons in a fragment H-
X-H. The magnetization is stepwise transferred from H to In the following section we shall examine a series of use-
X and back to H. I-spin magnetization that evolved during ful sequences, in which the X-filter has been installed.
t , is transferred to S magnetization by the two 90" pulses.
The second I spin is activated through the heteronuclear X-Filter COS Y
coupling and finally the S magnetization is transferred to In the sequence (P32) only protons that are coupled to a
the I spin. Consequently the sequence detects a linear H- common heterospin should give rise to cross signals. The
X-H coupling network. Application of this technique to
94 90" r2
"P-NMR spectra has been described in the litera- I 11
ture.1220.'2'I
lP32)
90" 90" 180" 90" I,
I: I f, I A/2 I A/2
S:

90" 180" 90" necessary coupling network is m12 . X-filter is thus

The
s: I A/2 1 A/2 I applied together with the mixing pulse. Heteronuclear an-
lP29l tiphase terms evolve and refocus, respectively, during the
I. n ! \ evolution and detection periods. The transfer is similar to
-2
that of a conventional COSY experiment, except that het-
eronuclear coupling must be taken into account [Eq. ( 1 19)].
-s 6 A 90,"-90," (S) pulse during the mixing pulse inverts all the
-1
-2
terms which contain an S, operator.

3.3.2. X- Filter and X- Half-Filter

Recently, the so-called X-filter and X-half-
I,, -90;
Ily - 21Ja - 411JJz
90"
2 2I& + 41J& ( 1 19)

filter[lS1-153.3361 (P30) have been introduced. They are build-
ing blocks which can be incorporated into any homonu-
I spin are formed (i.e., the cross signals are in-phase with
(P30l
clear pulse sequence. The purpose of the filter is to test at a
given point in a pulse sequence whether or not a hetero-
spin is involved in the product operator, and depending on
the result either to suppress o r to select cross signals. Note
the similarity with the low-pass J-filter. Coherences with
an S, operator remain unchanged under 90,"-90",, but
change sign under 90,"-90,". Coherences without a n S, op-
erator remain totally unaffected. Subtraction of two such
sequences suppresses all of the coherences which d o not
contain S,.
If the subspectra for 90,"-90," or for 90,"-90Yx are ad-
ded, cross signals resulting from operators without hetero-
respect to the heteronuclear coupling). If, however, the
subspectra are subtracted, only cross signals due to I spins
that are coupled to a common S spin are obtained, since
the terms without an S, operator are eliminated. The hetero-
nuclear coupling appears in antiphase. The spectrum is
substantially simplified compared with the conventional
spectrum, since now only a part of the total number of
cross signals appears.
The X-filter can obviously be applied not only with a
90" mixing pulse, but with more complicated mixing se-
quences (MQF-COSY, E. COSY). The heteronuclear cou-
pling is then present as antiphase splitting in the spec-
trum.

A n g e w . Chem lnl. Ed. Engl. 27 11988) 490-536 525

 The pulse sequence (P33) selects the same type of cou-
pling network. Here a fixed delay A=(2'Jls)-' serves to
build up heteronuclear antiphase These are se-
lected. such that at the beginning of t , only coherences of
(P33)
S-bound I spins are present. Due to the 180" pulse in the
middle of t , , no heteronuclear coupling evolves, so that co-
herence of the type 21,x12zSzis transformed by the 90"
proton mixing pulse into 2 IlzZ2xSz. This coherence refo-
cuses d u r H . The structural requirement is therefore
again an S- ' system, with .Ilz+O. The advantage of this
realization of the X-filter is that during t , no multiplet
structure develops due to the heteronuclear coupling,
which would lead to a decrease in the signal-to-noise ra-
tio.
X-Half-Filter COSY
If in the sequence (P33)1'53.3361 the 180" refocusing pulse
in the middle o f t , is left out, the heteronuclear antiphase
magnetization can already refocus in t , . This enables also
spin systems of type S-I'-12 to pass, and the X-half-filter is
90" f With the aid of one known distance rkl in a molecule
11
(P34)
IP3S)
obtained. The sequence for the (al)-half-filter COSY is
thus (P34). This filter can also be introduced at the begin-
ning of t , instead of at the beginning of t , , whereby the
(a,)-half-filter COSY (P35) is obtained. The simultaneous
90" (S) pulse and 90" (I) mixing pulse serve to select, at the
beginning of t,, only product operators which d o not con-
tain heterospin components.
The X-filter and X-half-filter techniques find more and
more use, mainly with very large proteins,[3371 in which the
resolution in conventional 2D NMR spectra is no longer
sufficient. The better resolved heterospin resonances are
used, or else the NMR-active isotope of the heteronucleus
is selectively enriched, so that subspectra of the conven-
tional spectrum are obtained, corresponding to certain
subunits of the protein near the isotopic labels. We may
expect further success in this area in the future, with the
development of ever simpler genetic-engineering methods
for the preparation of selectively enriched proteins.
3.4. Experiments Utilizing the Dipolar Coupling
3.4.1. NOESY
Dipolar relaxation and the NOESY experiment have al-
ready been discussed briefly. We return to them now in
greater detail.
526
Quantitative Evaluation of N O E Information
Cross signals in a NOESY spectrum rely on the cross
relaxation of longitudinal magnetization during the mixing
period. The intensity of the signals (the integral) contains
valuable information about internuclear distance^.^^^"^^^
The exact analysis shows that the cross relaxation rate o,
which determines the efficiency of the NOE transfer, is
given (for an isolated two-spin system IS) by Equation
(120). Thus the distance information ( r ) is built into the
cross relaxation rate.
In order to determine the cross relaxation rate from the
NOESY spectrum, one either studies the slope of the
buildup of the NOE rates for r, = 0, the so-called "initial
buildup rate" (by measuring NOE spectra for various mix-
ing times), or one makes use of the linear approximation. In
the second case it is assumed that during the selected mix-
ing time r, the system is still linear, i.e., the intensity of the
cross signals depends only on the cross relaxation rate. Re-
cently attempts have been made to extract NOE values
from a single spectrum without the initial rate approxima-
tion. These approaches consider multistep transfers, or
even solve the entire relaxation matrix.1345-3461
(e.g., two protons in a CH, group) and the corresponding
cross signal rate okl, an unknown distance rmncan be cal-
culated from a measured rate gmn(where lkland I,, are
the intensities of the corresponding cross signals) [Eq.
(121)], whereby it is assumed that the correlation times for
reorientation, r,, for the various distance vectors are equal.
This can be verified experimentally through I3C relaxation
times o r steady-state values of the heteronuclear NOES. A
different way is the simultaneous evaluation of NOESY
and ROESY, which permits the determination of rc and
o.13701
Artifacts
The phase cycling in a NOESY experiment is set such
that during the mixing time r, nonzero quantum coher-
ences are filtered out. Zero-quantum coherences are not
affected and thus are present in the NOESY spectrum just
like the desired NOE cross signals. Let us examine their
fine structure as represented by the product operator de-
scription [Eq. (122)j. Zero-quantum coherences precess in
z, and finally lead to dispersion signals with double anti-
phase structure in the spectrum. Although their integral is
zero, they can nevertheless interfere with the integration of
cross signals.
Suppression is most successful when a 180" pulse is applied in
the mixing period. This pulse is shifted within the constant mixing
Anqew. Chem. I n ! . Ed. Engl 27 (1988) 490-536
time. T,,, thus becomes {A/2- 18O0-(s,-A/2)}. The pulse refocuses 90: B
zero-quantum coherences, so that they evolve with I 4
cos(0, - f&)(rm -A). Random['471or also ~ystematic"~' variation of
A suppresses the zero-quantum coherences, because the argument
of the cosine assumes positive or negative values, depending on A, z-cosY
and thus the terms average out. Since in this experiment the mix- We mentioned this sequence earlier (Section 3.1.4) in
ing time remains constant, no additional t , noise is introduced, in connection with the E. COSY technique. By using @ pulses
contrast with the random variation of 5,. as flanking pulses for a z-filter one obtains cross signals
Another possibility is to shift the zero-quantum coherence or to
with E. COSY-like multiplet structure. If the z-filter (P38)
split it. I f T,,, is incremented synchronously with the evolution time
is made long enough to permit cross relaxation, it is possi-
by ~ t , then, the zero-quantum coherences give a signal which is
modulated by cos(R, - R , ) K ~ , . These signals are placed at
ble, in ideal cases of coupled spin systems, to separately
0,i(Q, - 0 , ) in ~ w , . rather than at a,,and are thus shifted out of observe the W,, W , , and W2 rates between two
the region of interest through this p r 0 c e d ~ r e . I ~ ~ ' ~

3.4.2. Longitudinal Multiple-Spin Orders

Through the use of 90" mixing pulses in the NOESY ex-

periment, exclusively z magnetization is generated. Opera-
tors with more than one z operator cannot be formed from
transverse magnetization by 90" pulses. The situation is
different when pulses with small flip angles are used. In
this case also 21,,12, operators can be generated from
2 I,, 12,by a y pulse. This so-called longitudinal multiple-
spin order is the cornerstone for several pulse sequences.
zz Spectroscopy
Longitudinal two-spin order can be used to distinguish
between certain exchange and cross relaxation signals, as
well as between inter- and intramolecular NOES."~'. 12']
Through phase cycling only those cross signals are selected
3.4.3. ROESY
We mentioned earlier (Section 2.2.2) that unfavorable
correlation times may lead to W2- W ,=0, and hence to a
situation in which there is no cross relaxation. The solution
for these cases is provided by the CAMELSPIN experi-
~nent,['~*Iintroduced by Bother-By et al., which we shall
term ROESY after a suggestion by B U X . [ ' The
~ ~ ] mixing in
the ROESY experiment (P39) is done in a spin-lock
period.
cP391
I I,
Spin lock

which correspond to the conversion of the operator 2 IlzIzZ

to 213,14,. The spins I3 and I4 must likewise be coupled
with scalar coupling, like, for instance, for cross signals NOESY relates to the mutual cross relaxation of z mag-
netization. In ROESY, transverse components cross-relax,
generated by chemical exchange.
The selection of zz operators (product operators with a n which are prevented by the spin-lock field from precessing
about the z axis. For magnetization which is locked along
even number of z operators) can be achieved by the appli-
cation of a 180" pulse in every second scan. The resulting the x axis, for instance, one finds the relaxation rates
signals are added together. Operators with an odd number (123)-( 125) for a two-spin system.[77.''*.2021 I n contrast to
of z operators are inverted, whereas zz operators are not.
Another possibility for the selection of zz operators is
the use of a particular phase cycle with the pulse sequence
(P36). Here equals 45Otn90" and the receiver phase is
2icj.
90" B AQ,
I 4 ', (P36)

zz- cos Y
The pulse sequence is the same as in zz spectroscopy.
However, in zz-COSY one is only interested in the
"COSY"-type cross signals which are formed in zz spec-
troscopy; hence z, is set correspondingly short. zz Opera-
tors are selected as above. The transfer efficiency is
%sin22[j; i.e., @=45" corresponds to the maximum inten-
sity, which is, however, only one-quarter of that of a con-
ventional COSY experiment. The sequence is nevertheless
useful, for it offers an effective method for suppression of
water signals.'1291By applying a homospoil pulse the unde-
sired transverse magnetization of water is destroyed. The
remaining longitudinal magnetization is suppressed, since
it cannot evolve into longitudinal multiple-spin order. The
pulse sequence is (P37).
NOE, the ROE is always positive (negative cross signals
with respect to the diagonals). This is shown in Figure 38,
which gives a graphic representation of the maximum
NOE and ROE as a function of the correlation time z, in a
two-spin system.
In addition, the ROE build-up rates are always (except
for z,=O) greater than the NOE rates, as shown in Figure
39. However, the time for which the linear approximation
is valid is also shorter than in NOESY.

Angew. Chem In! Ed. Engl. 27 (1988) 490-536 521

 NOE
0.5
0.25 /I
I::;/&
I 00
c-0 <-
-0.25
-0.5
%Ut/%Ot
- 0.5
-10
-2 -1 0 7
lg('%)
Fig. 39. The relation of the cross relaxation rates u in a 2D NOESY and a 2D
ROESY spectrum. For r,+O the rates are equal. Otherwise uRob
greater than uNoE.
The quantitative evaluation of ROESY spectra[3491is
similar to that of NOESY spectra. When the correlation
times are near the value z,= (5/4)"2/w0, the intensities of
NOESY signals are small and can therefore be determined
only inaccurately. In addition, cross-peak intensities are
very sensitive to different individual correlation times
within the same molecule. This can easily lead to errors in
the interpretation of molecular distances. In the ROESY
experiment, in comparison, this effect is less significant,
due to the smaller z, dependence of the intensity of the
cross signals. The lock field must be weak in order to avoid
contributions to the spectrum from transfer through scalar
coupling. These contributions are stronger near the diag-
onal and near the counterdiagonal. A reasonable recom-
mended value is a lock field of the order of one-half of the
spectral width [Eq. (127)l. For a quantitative evaluation
one must take into account the ROE dependence on the
distance of the resonance from the lock frequency (offset
effect).L2021
The spin lock can equally well be generated by a C W
irradiation with low power or by a series of pulses (pz),.
should be set as small as and z is calculated
from Equation (128), where D is given in radians and z, is
the B pulse length in seconds.
In an improved version with higher sensitivity, 90"
pulses are applied before and after the spin-lock peri-
Od.12021
528
ROE
05
0.25
00 1 Fig. 38. Maximum intensity ;f of a cross signal in 2D
NOESY and 2D ROESY spectra for a rwo-spin system, nor-
0 Ig:?/:'/4 malized for It,, the intensity of a diagonal signal with a mix-
- 0 25 ing time 7,=0. I n the NOESY spectrum the intensity of the
cross signal at r,-0 is ca. 20% of that of a diagonal signal, it
Tc--
vanishes at onre= I , and reaches 50% of the diagonal signal
-0.5 intensity for 4- m . In the ROESY spectrum, in contrast,
I I I
the maximum intensity is about 20% for r'+O, and ca. 34%
-1 0
Iglwr,l -1
for L- m .
3.4.4. Relayed NOESY
The combination of NOESY and relayed-pulse se-
quences is of some significance.Lz2s1Like in the other re-
layed experiments, one often achieves through the relayed
transfer an unequivocal assignment of NOE cross signals
in overcrowded spectral regions. The introduction of a
double-quantum filter (P40) after the relayed transfer can
be useful in order to filter out the (now dispersive) diag-
onal s i g n a I ~ . ' ~ ~ ~ ]
is always
A second possibility with slightly different spectra is of-
fered by the antisymmetrized pulse ~ e q u e n c e . ~ ~The~".~~'~
pulse sequence shown above is identical with the NOESY
sequence u p to the third 90" pulse, which precedes the re-
layed transfer period. It thus generates in w I absorptive in-
phase signals, modulated by the chemical shift of the first
spin. After t , , however, the magnetization is transferred
through dipolar coupling to a second spin (NOE transfer),
and then transferred again through scalar coupling to a
third spin (relayed transfer). Antiphase magnetization of
the third spin is thus detected in t,. Therefore the signal in
w2 is antiphase. It should be further noted that the spec-
trum is also asymmetric with respect to the position of the
cross s i g n a l ~ . [ ~ ~ ' l
The combination of TOCSY and ROESY (P41) has be-
(127)
come particularly useful for medium-sized molecules. It is
easily achieved through variation of the mixing se-
I 1, MLEV ROESY (P41l
q ~ e n c e ' ~ ~(e.g.,
' ' 30 ms TOCSY with MLEV-17, followed
by 170 ms of a series of pulses with small flip angles). The
short TOCSY transfer provides COSY-type in-phase corre-
lations (the transfer goes preferentially via one coupling),
which can be used directly for the ROE transfer. The
asymmetry of the spectrum directly provides, for example
in the N H region of peptides, both the sequence and its
direction even when the a-proton region is not resolved.
For larger molecules the TOCSY-NOESY combination
can be applied.[2331
Angew. Chem. Int. Ed. Engl. 27 (1988) 490-536
3.5. Exchange Spectroscopy
A different possibility for the transfer of magnetization
from one spin to another is offered by chemical exchange.
Two isomers or topomers1102.103.350i must exchange moder-
ately rapidly in a chemical equilibrium. Depending on the
rate of exchange, either separate sets of signals (slow ex-
change) or one set of average signals (fast exchange) may
be observed in the N M R spectrum. The variation in line
shapes i n the intermediate region serves to determine the
exchange kinetic^.^^^^.^^^^''^. '13.114.3511
In the slow exchange
range polarization can be transferred from one site to the
other (e.g., saturation transfer135z1).The NOESY and
ROESY sequences[353~3541 can be used to determine such
exchange processes. The advantage of the 2 D N M R tech-
nique is straightforward: mutual assignments can be com-
pared,'3cc.27'1 exchange mechanisms are immediately visi-
ble,"5(>-3 5 8 ) and quantitative evaluations are p o ~ s i b l e . ~ ~ ~ ~netic
Cross signals due to chemical exchange, as well as those
due to negative NOE effects (large molecules, viscous sol-
vent, low temperature, high field), have the same sign as
the diagonal signals in spectra with pure phases. In
NOESY spectra of small molecules in low-viscosity sol-
vents (positive NOE effects), and in all ROESY spectra,
exchange signals1361-3621 as well as NOEs[201,3631 over sev-
eral nuclei are readily distinguished from direct NOE cross
signals due to the different signs.
An example is shown in Figure 40. This proline-contain-
ing cyclic hexapeptide exchanges between two conforma-
tions, in which the proline amide bond is either in the trans
f6F2 f5 f3 F5 F) F6
I1 I 1
Fig. 40. Section from a 300-MHr ROESY spectrum of cyclo(-o-Pro'-Phe'-
Phe'-Pro'-Phe'-Phe"-) in [D,JDMSO, 320 K, fl pulse angle 24", mixing time
200 ms. The molecule is present in two conformations. The exchange be-
tween them IS evident from the positive cross signals (black). NO€ effects
between N H signals give rise to negative (red) cross signals (F'-FZ and FT-
F"). Phenylalanine= F, f (the major isomer is labeled with capital letters).
Angew. Chem. Int. Ed. Engl. 27 (1988) 490-536
or cis configuration. The exchanging NH signals of the two
conformations are correlated by positive (black) cross sig-
nals (with respect to positive diagonals), while the negative
(red) signals represent N O E in the rotating frame (ROE).
3.6. Further 2D NMR Experiments
A number of additional 2 D N M R experiments are ob-
tained by combinations of the techniques discussed above.
Variants of these can be created by the introduction of fil-
t e r ~ pulse
, ~ ~sandwiches,
~ ~ ~ various decoupling schemes,
and similar means, and are frequently found in the litera-
ture. Conversions of homonuclear techniques into hetero-
nuclear experiments have also been realized (e.g., hetero-
TOCSY11"~'861).Furthermore, photochemical excitation
has also been successfully associated with nuclear mag-
. ~ ~ resonance
~' ( ~ ~ O ~ O - C I D N P ) . ~ ~ ~ ~ . ~ ~ ~ ~
Recently, a series of 1D variants of 2 D NMR tech-
niques have been introduced121o1 through the use of semi-
selective pulses with programmed line shapes, which, for
simple problems, open novel aspects. The introduction of
semiselective pulses into 2D[251.335.3721 and 3 D NMR
spectra~253-2551opens yet another branch for methodologi-
cal research. A more detailed description of these newest
developments would, however, range beyond the scope of
this article.
4. Conclusion
In this article we have laid the theoretical foundations
for the most important 2 D N M R experiments. Thus we
now have at our disposal the tool which should enable use-
ful application of these techniques, even though not all of
the possible variants could be fully treated. The goal of
this article has been to review today's most widely used
techniques and to provide experimentalists with the neces-
sary understanding of the theory, so that they can be in a
position to:
- select the right experiment for their problem,
- select and set the optimal delays and phase cycles which
are most suitable for their particular problem, and
- recognize artifacts in the spectrum and possibly avoid o r
minimize them.
77ianks to many discussions with various colleagues and
the co-workers of our group the manuscript has been consid-
erably improved. However. we especially acknowledge Prof.
Dr. D. Kost, Beer Sheva. Israel, for his stimulating discus-
sions and his contributions during the translation into Eng-
lish. Some of the figures were kindly provided from our co-
workers U . Anders (Fig. 32), Dr. H. Kogler (Fig. 35). K .
Wagner (Fig. 40). and M . Will (Figs. 24, 25. 31. 33). Finan-
cial support f o r the scientific results of our laboratory was
provided by the Deutsche Forschungsgemeinschaft and the
Fonds der Chemischen Industrie. C . G. thanks Prof. Dr. R .
R . Ernst. Zurich, for support.
Received: May 12, 1987;
revised: January 19, 1988 [ A 662 IE]
German version: Angew. Chern. 100 (1988) 507
529
Appendix 1

Important concepts and abbreviations used in the text:

@I The first frequency domain in a 2 D NMR spectrum, obtained by Fourier transformation in iI

w2 Second frequency domain

ID One dimensional
2D Two dimensional
Acquisition, AQ Reception of the NMR signal; in 2 D NMR experiments it takes place during the time t,
Antiphase Multiplet structure with opposite phases; the multiplet lines change signs
B,I Static magnetic field, oriented along the z axis according to convention
BI Generally an additional transverse magnetic field, used for instance for pulses
B2 Another magnetic field, used for decoupling sequences, spin lock, etc.
“Band-pass filter” Filter for the selection of a specified coherence order p
BB Broadband decoupling of a spin type. Used in heteronuclear experiments to destroy the magnetization of one
of the spin types, and generally employed during acquisition time. Nowadays commonly achieved through
highly developed composite pulse sequences
Effective magnetic field, caused by the electronic environment around the nucleus (chemical shift). Often
also: the residual field in the rotating frame, generated in cases of incomplete decoupling
Carrier frequency wo, Transmitter frequency of the high-frequency pulses
Chemical shift The difference in precession frequencies between a nucleus and the carrier frequency. It is often referred to a
standard and divided by the carrier frequency (in ppm)
Coherence Corresponds to a transition between two eigenstates and is an extension of the expression “transverse mag-
netization,” which relates only to allowed transitions. From a quantum mechanical point of view a coherence
corresponds to an off-diagonal element in the spin density matrix
Coherence order p The difference in magnetic quantum numbers Am, of two energy levels which participate in the same coher-
ence (e.g., zero-quantum coherence, single-quantum coherence)
Coherence transfer Transfer of coherence from one nucleus type to another
Coherence-transfer pathway Progress of a coherence order in a pulse sequence
Composite pulse A pulse composed of a number of pulses. I t is employed in order to compensate for phase errors, B , field
inhomogeneities or offset effects
Constant time In constant-time variants of NMR experiments, the total duration of each l1
experiment is constant, since f l is
limited by pulses within a fixed delay
Convolution A mathematical operation
+ m

F*G=
~
I F(x-y)G(y)dy

Its significance for NMR spectroscopy results from the following relation: F * G =F(f.g), whereby F ( f ) is the
Fourier transform o f f . That is, the Fourier transform of a product of two functions equals the convolution of
the Fourier transforms of the functions
Correlation time The correlation time T, is the mean time required for a rotation of a molecule. It is responsible for relaxa-
tion
Cross relaxation Describes the mutual relaxation (inter- or intramolecular) of magnetically active nuclei, for instance through
dipolar relaxation
Density matrix Describes the state of a quantum mechanical object
Dipolar coupling A through-space interaction between two spins. It is orientation dependent, and averages out in solution due
to rapid reorientation of the molecule. Responsible for relaxation and NOE

DQF Double-quantum filter

Editing By setting a few parameters in a pulse sequence (e.g., pulses, delays), a set of subspectra is obtained, which
deliver different information, for instance about multiplicities of signals (like subspectra for ”CH, I3CH,,
and ”CH3)
FID Free Induction Decay of the NMR signal
Flip angle The angle by which the magnetization vector is rotated
Folding Used in connection with Fourier transformation. Folding about o1= O means
Sr(W1,02) =S ( @ l , W d + s ( -@ l , @ 2 )

530 Angew. Chem. Inr. Ed. Engi. 2711988) 490-536

Fourier transformation A mathematical method which determines the frequencies contained in a time-dependent function. The Fou-
rier transform F(o) in a complex Fourier transformation is given by
+ar

F(o) = j e-’“‘f(r)d/
-ar

and in a real Fourier transformation by

+ar

F(o) = 1 f(r)cos(wt+rp)dt
- m

“High-pass filter“ Filter for the selection of coherence orders 2p

Homospoil pulse, HS A pulse which generates short-time field inhomogeneities. Serves to eliminate undesired coherences (trans-
verse magnetizations)
In-phase Multiplet structure with equal phase, multiplet lines d o not change signs
Inverse techniques Heteronuclear experiments which detect the heteronucleus indirectly through proton magnetization. Also
called “reversed techniques”
Longitudinal magnetization Magnetization along the Bo field, i. e., along the z axis
Long-range coupling Scalar coupling over several bonds
“Low-pass filter” Filter for the selection of coherence orders 5 p
Multiplicity The splitting of NMR signals by scalar coupling into doublets, triplets, etc.; in I3C-NMR spectra it is also
used synonymously to describe the large splitting through direct coupling in order to distinguish quaternary
“C nuclei, CH, CH2, and CH3 groups
NOE Nuclear Overhauser Enhancement. An intensity change of an NMR signal induced by another nucleus in a
nonequilibrium state. It is caused by dipolar coupling
Peak Signal in an NMR spectrum; also used for a part of a multiplet
Phase cycle Pulse sequences used for NMR spectra are mostly combinations of sequences which differ from one another
only in the phases of the individual pulses. Systematic variation of the phases constitutes the phase cycle
which suppresses unwanted coherences and artifacts
Polarization It is based on the population difference of energy levels. A polarization must not always be connected with
magnetization
Polarization transfer Transfer of polarization from one nucleus type to another: it is often used synonymously with coherence
transfer, even if coherences are transferred
Precession frequency Larmor frequency w of the nucleas precessing about the z axis. I n the rotating frame it describes only the
difference frequency a, from an individual Larmor frequency of the nuclei w,= yBCfrand the carrier fre-
quency w o = y B o
Product operator Set of orthogonal operators which can be connected to each other by a product
Product operator formalism Formalism to describe NMR experiments (see extensive explanation in the text)
Pulse Application of an additional high-frequency magnetic field with a carrier frequency wo for a short time
interval
Pulse sequence Carefully designed series of radio frequency pulses and times of free evolution (delays) with subsequent
detection of the produced transverse magnetization
Quadrature detection A procedure for the detection of N M R signals which allows discrimination of signs of frequencies (in con-
trast: one-channel detection)
ROE Rotating frame NOE. NOE caused by relaxation in the rotating frame TI,
Rotating frame Frame rotating at frequency w o ; simplifies the description of pulses and periods of free precession
Scan A single running of an N M R experiment
Scalar coupling Mutual interaction between two spins, which is transmitted through chemical bonds. Also called J coupling.
It is scalar, i.e., independent of the orientation, and yields the multiplet splittings of the NMR signals
Evolution time. Incremented time in a 2 D NMR experiment. Yields after Fourier transformation the first
frequency domain
12 Detection time, generates after Fourier transformation the second frequency domain
t , noise Noise which appears in 2 D N M R spectra parallel to the w , axis, and is particularly visible where there are
intense signals in the spectrum. It is mostly due to technical factors associated with the instrument such as
random variations of phases, pulses, temperature, Bo inhomogeneity
Thermal equilibrium A state in which the energy levels are populated according to the Boltzmann distribution:
p ( ~ ,=) -E,’h.r/ Ce- E . / h T
Tilting Transformation of a spectrum by w ; = w , o ; = w , + k - w ,
TQ F Triple-quantum filter

Anyew. Chrm. In!. Ed. Engl 27f1988) 490-536 53 I

Transverse magnetization Magnetization in the x,y plane
Transverse operator Designates I, or I y
Weak coupling J coupling constant for the coupling between two spins is small compared with the difference of their chem-
ical shifts; it causes first-order spectra
Zero-filling Addition of zeros to the FID in order to improve the digital resolution

Appendix 2
E x p l a n a t i o n of acronyms used in the text:
BIRD Bilinear Rotation Decoupling
CAMELSPIN Cross-Relaxation Appropriate for Minimolecules Emulated by Locked Spins
CIDNP Chemically Induced Dynamic Nuclear Polarization
COCONOSY Combined Correlated and Nuclear Overhauser Enhancement Spectroscopy
COLOC Correlation via Long-range Couplings
COSY Correlated Spectroscopy
DEPT Distortionless Enhancement by Polarization Transfer
DE PT-G L DEPT-Grand Luxe
DISCO Differences and Sums in COSY spectra
E. COSY Exclusive COSY
CARP Globally Optimized Alternating Phase Rectangular Pulse
HETCOR Hereronuclear Correlation
HOESY Heteronuclear 2 D NOE Spectroscopy
HOHAHA Homonuclear Hurtmann-Huhn experiment
I N A DEQU ATE Incredible Natural Abundance Double Quantum Transfer Experiment
INEPT Insensitive Nuclei Enhanced by Polarization Transfer
MLEV Malcom Leuitt
NOESY Nuclear Overhauser Enhancement and Exchange Spectroscopy
P. E. COSY Primitive E. COSY
RCT Relayed Coherence Transfer
ROESY Rotating Frame Nuclear Overhauser Effect Spectroscopy
ROT0 ROESY-TOCSY
TOCSY Total Correlation Spectroscopy
TOR0 TOCSY-ROESY
WALTZ Wideband Alternating Phase Low-Power Technique for Zero Residue Splitting
XCORFE X-Nucleus Correlation with Fixed Evolution Time

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