11 NMR Spectros

job:OC094361 12-10-1998 page:1 colour:0
11 NMR Spectroscopy
By B. A. SALVATORE
Department of Chemistry and Biochemistry, University of South Carolina, Columbia,
SC 29208, USA
1 Introduction
It appears that most recent breakthroughs in NMR are driven by research involving
biological macromolecules. The fact that many of these developments are occurring in
neighboring elds does not preclude their application within the realm of organic
chemistry. Asurvey of the recent literature reveals that many newNMRtechniques are
indeed important to chemists who study relatively small organic molecules. Thus, it is
instructive to distill the recent literature from a cross-section of disciplines and
pinpoint those NMR techniques which are of interest to the organic community. It is
in that spirit that this review focuses on recent advances in NMR from a variety of
areas. The material pertains to both isotropic and oriented NMR sample systems, and
the author has adopted a selective (rather than comprehensive) approach in presenting
some of the most signicant developments.
2 Monitoring chemical reactions by NMR
The utility of NMR in monitoring the progress of chemical reactions is continually
being enhanced, with recent advances demonstrating its analytical power in both
solution and solid-phase chemistry. This includes the development and renement of
new techniques and of existing hardware.
Solution-phase chemistry
NMR is particularly well-suited for detecting and identifying intermediates in chemi-
cal reactions. Organocuprate chemistry is one area in which mechanistic details
remain obscure. A recent study of several cuprates helped clarify the dichotomous
reactivity of these agents, which often participate in both electron-transfer and conju-
gate addition processes. In the reaction between Me
`
CuLi
`
and trimethyl ethylene-
tricarboxylate, the cuprate was found to be a particularly strong reducing agent, with
the reduction product predominating over the conjugate addition adduct by more
than 4: 1. Standard H-decoupled `C spectra of Me
`
CuLi
`
and trimethyl ethylene-
361
tricarboxylate [`C-enriched at C(2)], in ether at 0 C, revealed a single carbon
resonance, representing the intermediate which follows single electron-transfer from
the cuprate. However, no resonances from an intermediate along the conjugate addi-
tion pathway were detectable by NMR, for reasons that remain unclear. This is
puzzling, in light of the fact that the conjugate addition adduct still accounts for nearly
20% of the total product yield, and thus further investigations are warranted.
Chemically Induced Dynamic Nuclear Polarization (CIDNP) is a powerful NMR-
based method for probing the molecular structure of species in which a photochemi-
cally generated free-radical electron spin polarizes a nuclear spin on the same atom.
Using this technique, Giese et al. provided the rst spectroscopic proof for the
existence of a radical cation intermediate in a chemical reaction that models the
C,O-bond scission process of 4-DNAradicals.` Such radical species are believed to be
important intermediates in the cleavage of DNA by bleomycin and enediyne-based
natural products.
NMR is also a powerful tool in assessing the formation of organometallic com-
plexes. Particularly important, are new metallo-NMR methods for characterizing the
structure of ionic species in solution. Koga et al. described the 'Li and `Nanalysis of a
labeled chiral bidentate lithium amide. 'Li`N couplings established the basis for
studying these complexes in solution.` Note that 'Li is quadrupolar (nuclear spin1).
Such experiments should help in explaining the dependence of enantioselectivity on
solvent composition in proton transfer of asymmetric carbon. These investigators
concluded that an observed drop in enantioselectivity in certain solvents was caused
by formation of a dimer of the chiral lithium amide species.
Other atom-pairs have also recently been investigated by NMR. Gunther and
Bohler reported the rst 2D heteronuclear shift correlation experiment for the spin
pair, 'Li`"Si," which they used to study the structure of a silyl-substituted or-
ganolithium anion. The experiments were performed unlocked, on a doubly-tuned
probe, with proton decoupling. The retuned deuterium lock coil served as the 'Li
channel and the X-coil was used to pulse `"Si nuclei (natural abundance :5%). One-
and three-bond correlations were visible in the 2D HSQC spectrum, which displayed
two sets of 'Li`"Si scalar couplings. Such experiments are very powerful, because they
can establish the site of chelation within an ionic complex.
New hardware developments have also broadened the applications of NMR in
monitoring chemical reactions. Baumann et al. described a simple apparatus which is
particularly applicable (but not limited) to kinetic and mechanistic studies of reactions
between dissolved gases and organometallic complexes.` Such studies make it possible
to monitor the appearance and disappearance of intermediates that ordinarily cannot
be isolated. Several reactions have been studied with this apparatus, including an
unusual one, in which two molecules of ethylene add to a zirconocenealkyne complex,
displacing the alkyne.'
Other researchers have sought to ally NMR with liquid chromatographic instru-
mentation.` LC-NMR consists of linking an HPLC in series with a specially-develop-
ed NMR probe, capable of detecting ow-through samples. A temporary pause in the
ow, as the compound moves through the probe, allows the sample to remain within
the NMR coil long enough to obtain adequate signal averaging. Other implementa-
tions of this technique have also been introduced. For example, elimination of the
HPLC column and introduction of an autoinjector establishes the basis for another
362 B. A. Salvatore
new analytical system known as Versatile Automated Sample Transport (VAST)
NMR, which promises to be a very powerful tool in the analysis of combinatorial
libraries.`
Applications in solid-phase synthesis
One long-standing problem in solid-phase synthesis (SPS) involves monitoring the
progress of reactions during synthesis. FT-IRis often employed to accomplish this, but
it lacks the analytical power of H and `C NMR. The primary obstacle to NMR
analysis of SPS is local discontinuities in magnetic susceptibility that result from the
heterogeneous nature of resin-bound samples. This heterogeneity causes variations in
the eective magnetic eld around the attached molecules, resulting in chemical shift
dispersions (i.e. broad lines!). As a result, the solid-phase synthetic chemist often must
cleave some product from the resin after each step, which is used for solution NMR or
mass spectrometric analyses. This is time-consuming and wastes synthetic intermedi-
ates.
Fortunately, this is changing with the development of fast, non-destructive NMR
methods for characterization of the products of SPS. `C NMR of solvent-swelled
resin samples (gel-phase NMR) is suitable in certain situations.` Solvating the resin
sample as much as possible before acquiring spectra increases motional freedom of the
resin-boundmolecules, thereby reducing linewidths. This is a convenient adaptation of
conventional solution NMR methods, requiring no specialized equipment. Yet, even
though this method often provides carbon NMR spectra suitable for analysis of the
products of chemical syntheses, the problem of broad lines remains and renders the
method unsuitable for high-resolution H NMR studies.
Magic-angle-spinning (MAS) of solvent-swelled resin beads drastically reduces line
widths by averaging out magnetic susceptibility dierences within the sample, thus
improving resolution and making it suitable for high-resolution H NMR applica-
tions.` This technique is useful in monitoring the progress of SPS, and eorts are being
made to optimize its utility. It was found that poly(ethylene glycol) (PEG)-tethered
resins, commonly called TentaGel, give the narrowest NMR line-widths." Spin echo
experiments and spin-locking are commonly employed to attenuate unwanted poly-
mer peaks and enhance resolution obtained with other resins. However, one disadvan-
tage of spin echoes is that J-couplings are lost as the echo refocuses. If it is important to
retain the J-coupling information, it can be recovered through 2DJ-resolved spectros-
copy. Shapiro et al. reported a useful experiment, in which an untilted 2D J-resolved
spectrumis projected along a single axis." This technique capitalizes on the benets of
the spin echo experiment by attenuating unwanted polymer resin peaks, while retain-
ing proton scalar coupling information along the chemical shift axis of a standard
one-dimensional H-spectrum.
MAS NMRis an excellent tool for analyzing the products from peptide SPS. Opella
et al. have shown that it is now possible to determine the three-dimensional structural
characteristics of resin-bound molecules. They acquired 2D NOESY spectra and
established the conformation of a resin-bound hexapeptide. Such information is
relevant to drug design, because binding assays are sometimes performed on resin-
bound libraries of ligands.
Lippens et al. employed similar techniques to investigate the structural basis for the
diculties encountered during the SPS of certain peptide sequences.` Specically,
363 NMR Spectroscopy
Fig. 1 A schematic representation of the H nano-probe. The plug for the nanoprobe
cell and the RF leads have been omitted for clarity.
(Reproduced with permission from J. Magn. Reson., Series A, 1996, 119, 65
they used NOE and chemical shift data to establish a correlation between coupling
diculties and the degree of interchain aggregation, as the synthesis progressed.
Advances have been made in hardware development, as well. Keifer et al. inves-
tigated magic-angle-spinning with new, high-resolution probes (Fig. 1) that were
optimized for very small sample volumes (ca. 40 l).` The excellent resolution ob-
tained with these nano-NMR probes demonstrates the important benets of mini-
mizing magnetic susceptibility discontinuities in probe design, as well as in the sample.
Shapiro et al. have adapted MAS NMR methods to analyze products on multipin
crowns, thus extending its utility in parallel combinatorial synthesis." Application of
MAS NMR in the solid-phase synthesis of oligosaccharides` and other non-peptide-
based combinatorial libraries" has also been documented.
Supramolecular chemistry
NMR is a powerful tool for probing non-covalent molecular assemblies, along with
the dynamic exchange processes that occur in those assemblies. Lehn et al. have used
""Ag-NMR spectroscopy to monitor the formation of a rectangular supramolecular
grid, assembled from the combination of tritopic ligand 1, ditopic ligand 2, and silver
triate in a 2: 3: 6 stoichiometric ratio.' The ""Ag NMR spectrum displayed two
signals in a 2: 1 ratio, corresponding to four peripheral and two central silver ions,
respectively. This provides evidence for formation of the complex 3 in which the signal
from the peripheral silver ions is shifted downeld by 59 ppm from the resonance
generated by the central silver ions. Thus, it was consistent with the formation of a
2 ;3 rectangular grid, via mixed-ligand recognition.
364 B. A. Salvatore
N
N
N
N
N
N
Me
Me
N
N
N
N
Me
Me
1 2
N N N N
Me
Me
N
N
N
N
N
N
Me
Me
N
N
N
N
N
N
Me
Me
N N N N
Me Me
N N N N
Me Me
3
+6
Another elegant example of NMRs utility in supramolecular chemistry was re-
ported in an investigation of the reversible dimerization and guest exchange in
C
`
-symmetric calixarenes.` O-resonance rotating frame NMR experiments provide
a useful way of discriminating between exchange and the direct cross-relaxation
transfers that are commonly witnessed in standard NOE experiments. O-resonance
spectroscopy also eliminates complications from TOCSY transfers that sometimes
arise during on-resonance rotating frame experiments. Spin locking was performed in
these experiments, such that the angle between the eective eld and the external
magnetic eld (35.6) was in between those used in standard NOE experiments
(0) and on-resonance rotating frame experiments (90). That value was
chosen because it resulted in a cancellation of the cross-relaxation contributions from
the laboratory frame and the on-resonance rotating frame for the observed protons.
Thus, only exchange cross-peaks were detected.
Determination of acid dissociation constants by NMR
It is often a challenge to accurately measure dissociation constants of organic acids in
non-aqueous media. Pehk et al. have devised a general approach for measuring
relative acid dissociation constraints by `C NMR.` This technique is based on the
measurement of frequency dierences at varying degrees of protonation between a
known reference compound and a compound whose dissociation constant is un-
known. The degree of protonation of the reference compound (e.g. acetic acid) is
known exactly at each stage of the process. The ratio of dissociation constants for the
acid under study and the reference compound can be determined from the following
relationship in eqn. (1),
K/K (
)(
)/(
)(
) (1)
where represents the chemical shift for the partly protonated reference acid, while
and
represent the chemical shifts of its fully protonated and fully deprotonated
species, respectively. The corresponding chemical shift values for the acid under
investigation are represented by ,
, and
, accordingly. In the most convenient

implementation of this technique, one measures the chemical shift for the reference
acid and for the compound under investigation and then plots the dierence
against the degree of protonation (n), according to eqn. (2),

n(
) nK/K(
)/[1 n[(K/K) 1]] (2)

where K/K represents the ratio of dissociation constants between the reference acid
and the acid under study. The derivation of this equation (based on the law of mass-
action and two expressions relating NMR chemical shift to the distribution of molecu-
lar populations during the exchange process) was presented by the authors. Plotting
the experimental data for vs. n, one obtains bell-shaped plots which are t to
obtain the sought quantity K/K, according to eqn. (2). In principle, the dissociation
constant of a particular acid can be determined by this technique when it diers from
that of the reference acid by as little as 4 J mol. This technique has minimal
requirements for sample purity, and it can be carried out without any determination of
pH. Additional methods are available for treating the experimental data which sim-
plify the data interpretation when the chemical shift dierences between the fully
protonated and deprotonated forms of the compounds are not equal.` The authors
illustrate several practical examples, involving branched carboxylic acids.
3 Dening structure and conformation through NMR
Correlation spectroscopy
Rychnovsky et al. reported a strategy for assigning the conguration of 1,3-skipped
polyols." This method was demonstrated by analysis of a pair of polyacetonide
acetate derivatives of the natural product dermostatinA, which are frame-shifted with
respect to each other (4 and 5, Fig. 2). Thus, this strategy is particularly well-suited for
366 B. A. Salvatore
O
O O
O
O O O O O O AcO
O
O OAc
O
O O O O O O O
4
5
15
15
16
16
17
17
19
19
21
21
23
23
25
25
27
27
29
29
31
31
Fig. 2 Two isomeric (frame shifted) polyacetonide acetate derivatives (4 and 5) of
dermostatin A
polyols which contain an odd number of hydroxy groups. The technique relies on a
combination of HH COSY, H`C DQF-HSQC, and HH ROESY experi-
ments. After assigning all the protons fromCOSYspectra, HSQCand ROESYspectra
are acquired to determine which of the acetonides in 4 are syn and which are anti (Fig.
3). The relative stereochemistry between each pair of acetonides in 4 is then established
by analyzing the HSQC and ROESY spectra from an isomeric frame-shifted aceton-
ide (5, Fig. 2). A small level of `C-enrichment within the acetonides (ca. 10%) is
optimal. This is a very powerful NMR-based method for assigning the relative
stereochemistry within polyol chains. Mosher ester analysis can then be used to
subsequently establish the absolute stereochemistry.
Gervay et al. have explored long-range `CH NMR connectivity in carbohy-
drates.`" They applied a 1DInverse Detected Single QuantumLong Range (INSQLR)
experiment to establish the existence of a highly-labile sialic acid lactone moiety.
Selective excitation at the `C-labeled carbonyl in one of the possible lactones resulted
in magnetization transfer through the lactone oxygen, which was detected at a proton,
three bonds away. This signied the presence of the lactone. Had that lactone been
absent, this correlation would not have been observed, since it would have required
`CH magnetization transfer through seven -bonds.
One of the major problems in three-dimensional structure determination of
oligosaccharides by solution NMR results from the limited number of distance and
angular restraints, which are generally dened from HH NOEs, and three-bond
H`C coupling constant measurements. To alleviate this problem, Homans et al.
have enhanced existing NMR methods for deriving information from exchangeable
protons (i.e. -OH, -NH protons).` This technique was demonstrated on N-acetyllac-
tose with three experiments (TOCSY-HSQC, ROESY-HSQC, and NOESY-HSQC).
Fig. 3 ROESY (left) and HMQC (right) of the acetonide methyl group region for
compound 4. There are three syn- and two anti-acetonides [two of the axial methyl
groups in distinct syn acetonides coincidentally have the same HSQC chemical shifts
(H: 1.44 ppm and `C: 20 ppm)]. No ROE correlations appear with the equatorial
methyl groups in the syn acetonides.
(Reproduced with permission from J. Org. Chem., 1997, 62, 2925)
The samples were prepared in an H
`
O[`H
'
]acetone mixture, which was cooled
(17C) to minimize proton exchange. The investigators employed 3D `C-editing
techniques to overcome chemical shift overlap of non-exchangeable protons, a com-
mon problem in most proton-detected 2D NMR experiments involving carbohy-
drates. Water suppression in these experiments was performed without pulsed-eld
gradients (vide infra). These experiments produced a vast number of additional dis-
tance restraints which were useful in conformational analysis of the disaccharide.
Symmetry often poses an obstacle in conformational studies with NMR, since
NOEs and ROEs normally cannot be detected between chemically-equivalent proto-
ns. Thus, fewer distance constraints are available for analysis. Wagner and Berger
reported an eective solution to this problem,`` building upon prior work, in which
HMQC-ROEtransfers were performed between two chemically equivalent protons on
a `C/`C atom-pair. Unwanted signals resulting from ROEs between protons on
`C-atoms were successfully suppressed with pulsed eld gradients. These authors also
reported an improved 1D version of this experiment, based on the selective excitation
of a single `C resonance, bearing one member of a chemically-equivalent pair of
protons (the other proton being on a `C-atom within the same molecule). Enclosing
the selective 180 pulses within a gradient sandwich facilitates calibration of the pulses.
A negatively-phased signal appears in the center of each CH doublet (representing
two equivalent protons), where an NOE exists (Fig. 4). Jimenez-Barbero et al. have
reported a similar 1D experiment for extracting NOE (ROE) information from chemi-
cally equivalent anomeric protons in the C
`
symmetric disaccharide, trehalose.`` Their
technique is based on the selective inversion of one anomeric `C resonance with a
DANTE-Z pulse train, during which time the proton magnetization is spin-locked.
368 B. A. Salvatore
Fig. 4 1D HSQC-NOESY spectrum of phenanthrene, obtained through a selective
pulse on C(4). Anegative peak in the centre of the H`Csplitting for H(4) is apparent
above. This represents an NOE between the two chemically-equivalent H(4) protons.
(Spectrum reproduced with permission from Magn. Reson. Chem., 1997, 35, 199)
Deriving information from scalar coupling constants
It is possible to garner a tremendous amount of structural and conformational
information through the accurate measurement of scalar (J) coupling constants. In a
recent review, Thomas reminds the chemical community not to lose sight of the
importance of coupling constants in conformational analysis.`" He attributes recent
neglect, in part, to the explosive development of new multidimensional NMR experi-
ments, which have relegated J-couplings to an uninteresting role. Investigators who
simply view J-couplings as the basis for multidimensional correlations, rather than a
direct source of information, as well, may be ignoring potentially valuable data.
Conformational analysis, based on scalar couplings, has, in fact, been undergoing a
renaissance recently, with many investigators gathering information about H`C
and `C`C J-values. Serianni et al. have developed synthetic and spectroscopic
methods for measuring the complete set of one-, two- and three-bond H`C and
`C`C scalar couplings in --ribofuranose and 2-deoxy--ribofuranose rings.```'
Since all the furanosyl rings within DNA (or RNA) have the same chemical structure,
comparisons between related J-couplings fromthe sugars within discrete segments can
yield important information about the topological structure of nucleic acids.
This same group has devised an empirical method for predicting the magnitude and
sign of two-bond `C`C scalar coupling constants (`J
''
) in aldopyranosyl rings.``
Fig. 5 Seriannis projection rule for determining `J
''
. To determine J
'
'`
for this
carbohydrate, one must rst inspect the angles made by each oxygen substituent on C1
and C2 with the projection anti to the C(2)C(3) bond [viewed along C(1)C(2)] and
then inspect the angles made by each oxygen substituent on C2 and C3 with the
projection anti to the C(1)C(2) bond [viewed along C(2)C(3)]. Summing the cosines
of all of these angles provides a resultant (0.5), which predicts a small negative
coupling constant (ca. 2 Hz). The measured value is 2.4 Hz
The values of such couplings have been shown to depend on the orientation of
electronegative substituents relative to the CC bond. Seriannis projection rule for
estimating `J
''
is based on an inspection of the angle that each electronegative
substituent on each of the two CC bonds makes with a projection anti to the other
CC bond (Fig. 5). Then, the cosines of all these angles are simply added together. A
small positive sum (:1.0) or a negative sum is indicative of a negative `J
''
, while a
larger positive value (91.0) predicts a positive `J
''
. This report includes data for
several dierent sugars and demonstrates that ab initio calculations of `J
''
in model
compounds agreed with the authors predictions. This empirical rule also applies to
`J
''
couplings through oxygen (i.e. COC), and this should prove particularly useful
in the conformational analysis of O-glycosidic linkages in oligosaccharides.
In structural studies of complex natural products, it is often desirable to detect
long-range H`Cscalar couplings. The standard 2DHMBCexperiment is one of the
most powerful methods for accomplishing this. Yet, it is often hampered by low
sensitivity for some long-range correlations, due to the diculty in setting a univer-
sally-optimal delay time after the rst 90 `C-pulse. As a compromise, a 5060 ms
delay is generally prescribed, but this is often not optimal, particularly for couplings
involving fast-decaying signals (e.g. methylene signals). Seto et al. have proposed a way
to overcome this commonly encountered problem, which entails displaying a 2D
projection from a three-dimensional HMBC experiment.`` The delay time following
the rst carbon pulse in the conventional 2D experiment becomes variable and is
increased uniformly in 4 ms increments from 20 ms up to 80 ms, thus establishing the
third dimension. The results of this 3D experiment are viewed as a projection of the
370 B. A. Salvatore
sum of sixteen separate 2D spectra onto the f2,f3 plane. By employing gradient-based
coherence selection, requiring only 4 scans per t
`
point, this experiment takes no
longer than a standard 2D HMBC experiment, performed with 64 scans per t
point.
In a comparison, run with the natural product, monazomycin, the results of a 3D
HMBC were far better than those derived from a standard 2D HMBC. This experi-
ment is particularly useful for detecting long-range J-couplings, involving protons that
are broadened in complex spin systems.
Fructose exhibits a complex mutarotational equilibrium between ve isomeric
forms, including the pyranose, furanose, and open (straight chain) forms. In an eort to
develop receptors that will selectively bind to one isomeric form in solution, Eggert
and Norrild characterized the various boronic acid complexes of fructose on the basis
of one-bond `C`C coupling constants (J
''
).`" Their work is based on the import-
ant observation that exceptionally low J
''
(3540Hz) result when the OCCO
fragment in a vicinal diol is incorporated into a ve-membered ring (as in vicinal cyclic
boronic esters). It is believed that this eect results from a change in the orientation of
the oxygen lone pairs with respect to the CC bond. Since this species is selectively
bound (albeit, in DMSO), as its 2,3: 4,5-bis(p-tosylboronate) ester, the authors believe
that they may be able to design a bis-boronic acid-based receptor that selectively binds
to the --fructopyranose anomer in water.
Structureactivity relationships by NMR (SAR by NMR)
Individuals engaged in drug discovery today have the choice of pursuing rational
design methods or concentrating on the many recently-developed combinatorial
approaches. The latter techniques have rapidly gained popularity, because the rational
approach to drug design continues to be hampered with diculties, such as those
involved in predicting the enthalpic and entropic eects of ligand binding to drug
targets. These factors are key in determining the stability of most drug-protein com-
plexes. For example, water molecules are often released upon ligand binding or,
alternatively, they move to ll gaps at the binding site in unpredictable ways. Problems
of this sort make life dicult for those pursuing a strictly rational approach to drug
discovery. However, combinatorial chemists also encounter diculties, due to the
limited sensitivity of most biological assays, which generally facilitate the identication
of only the most active compounds in a given library. Weaker binding is often
obscured by backgroundsignals that result fromhigh ligand concentrations. Thus, it is
likely that many key lead compounds are missed in high throughput combinatorial
assays. While the debate continues between those espousing combinatorial methods
for drug discovery and those who remain rmly entrenched with rational drug design,
Fesik et al. devised a strategy which blends these two strategies into a very powerful
new approach. This is the so-called, StructureActivity Relationship by NMR (SAR
by NMR) approach to drug discovery. This technique, which has been previously
reviewed,`" is very powerful, yet elegant in its simplicity. It blends the advantages of
rational drug design and combinatorial chemistry with NMR studies of `N-enriched
proteins.
The H`N pairs in folded proteins generally yield well-resolved HSQC spectra.
Addition of a substrate with a moderate anity for the protein results in a shift of the
HSQC NMR signals for all the H`N atom pairs within the binding site. The chief
advantage of SARby NMR is that it allows one to obtain reliable SARfor compounds
N
O
O
OCH
3
OCH
3
H
3
CO
OCH
3
O
N
O
H
OH
HO
N
O
O
OCH
3
OCH
3
H
3
CO
O
O
N
O
H
OH
O
K
d
= 2 M K
d
= 100 M
K
d
= 19 nM
Fig. 6 Two fairly weak-binding FKBP ligands discovered through SAR by NMR
screening (top). These two ligands bind to two distinct sites on FKBP. Linkage of the
two ligands produced a much stronger-binding ligand (in box)
which bind to the target with low anity (millimolar range). In their seminal paper on
this technique, Fesik et al. designed a ligand which binds to FKBP in the nanomolar
range by rst identifying two ligands that bind to distinct sites on FKBP with
moderately weak binding constants (Fig. 6).` A key feature of this technique is the use
of H`N HSQC spectra to detect the binding of small ligands, and to dierentiate
between multiple binding sites on the protein surface. Due to `N-spectral editing, no
signal from the ligands is observed in the spectra, just changes in the proton and
nitrogen resonances for protein H`N atom pairs within the binding site(s). NOEs
between the ligands and specic H`Npairs on the protein can also be used to assess
the manner in which the ligands bind to the protein. Fesik et al. acquired HSQC data
with help from an automated sample-changer. Selection of the strongest binding
ligands was then accomplished by considering a weighted average of the chemical shift
changes for H and `N, upon addition of each ligand. The overall strategy involves
372 B. A. Salvatore
ve steps: (1) screening of binding of rst ligand by NMR, (2) optimization of binding
of the rst ligand, (3) screening of binding of second ligand by NMR, (4) optimization
of binding of the second ligand, (5) linking the rst and second ligands. When two or
more ligands which bind to a protein at distinct sites are linked together, one obtains a
total free energy of binding based on the sum of the two individual binding energies,
plus an additional negative free-energy term associated with the entropy decrease that
results from linking the two ligands together [eqn. (3)].
GG
G
`
G
"'''
(3)
A similar approach was adopted to identify powerful inhibitors of stromelysin, a
zinc-dependent matrix metalloproteinase.`` Fesik et al. also applied SAR by NMR to
nd initial leads for inhibitors of the E2 protein from the human papilloma virus,
which binds DNA at a single site.`` In this case, rather than physically linking the two
independently-binding ligands together, SAR from two distinct series of weakly-
binding lead compounds were gathered and merged in the design of a single lead that
displayed an IC
`"
in the micromolar range.
The two main disadvantages of SAR by NMR are that it requires a minimum of
200 mg of `N-labeled protein, overall, and the size of the protein should not exceed
the 30 kDa limit imposed by solution NMR. However, those problems are largely
overcome with relaxation- and diusion-edited NMR screening techniques.`" These
new methods are complementary to the HSQC-based technique, since spectra of the
ligands (instead of the protein) are observed. The spectra acquired by these methods do
not allow characterization of the protein binding site. However, they minimize the
amount of protein required, eliminate the need for isotopically labelled protein and
they are amenable to binding studies with very large proteins.
Advances in water suppression
For NMR structural studies involving water-soluble compounds (e.g. carbohydrates,
peptides) from which information about exchangeable protons is gathered, H
`
O is
often a necessary component of the sample, as well as a signicant source of problems
during spectral acquisition. For studies in which the sample concentration is in the m
range, the water proton concentration may be up to ve orders of magnitude higher.
This large dierence in concentrations complicates the acquisition process. Most
problematic is the fact that the huge water resonance makes it impossible to set the
receiver gain at a level suitable for analyzing the sample. Secondly, the water resonance
often overlaps with some of the sample resonances. For these reasons, the development
of ecient water suppression methods continues to be an active research area.
Today, the most popular solvent suppression techniques involve selective excitation
of the water resonance. This is particularly useful in (but not limited to) protein NMR
applications, since protein resonances have shorter relaxation times than water and
thus return to equilibrium much faster. Radiation damping can be a serious problem.
This phenomenon results when transverse water magnetization induces a current in
the receiver coil. The induced current results in a magnetic eld that causes the water
proton magnetization to return to equilibrium at a rate much faster than that pre-
scribed by its true T
, thus ruining the solvent suppression. For this reason, a lot of

research has been done to develop gradient-enhanced, frequency-selective water sup-
pression techniques. The general utility of pulsed-eld gradients in NMR has been
recently reviewed by Canet.`` Gradient-based solvent suppression methods are gen-
erally classied into two categories: (1) frequency-selective excitation followed by
dephasing (i.e. spoiling) with gradient pulses, and (2) frequency-selective refocusing
anked by gradient pulses which dephase unwanted transverse magnetization.
A recent straightforward application of the rst category is the WANTED (water-
selective DANTE using gradients) sequence.`' Here, radiation damping is suppressed
during a water-selective pulse train by keeping the transverse water magnetization
defocused during the period in between each DANTE pulse. In homonuclear 2D
NMR, however, frequency selective refocusing of the solvent resonance is more
common. This is usually achieved by inserting a WATERGATEsequence near the end
of a pulse sequence, prior to acquisition. However, splicing this segment within the
middle of a pulse sequence often complicates the phase cycling and timing within the
entire sequence. Thus, each experiment must be developed and optimized indepen-
dently. For example, Ni et al. recently reported a new gradient-enhanced WATER-
GATE-TOCSY experiment in which pulsed-eld gradients were used to maintain
precise control of the water magnetization vector.`` This experiment demonstrated
marked improvements over a standard z-ltered TOCSY, which used water presatura-
tion, but one should not underestimate the eort needed to develop and optimize
similar experiments of this nature.
Several more sophisticated methods for solvent suppression have been developed
which, like WATERGATE, are based on the frequency-selective refocusing of the
water resonance, where the refocusing RFpulses are anked by gradients that dephase
unwanted transverse magnetization. These are the so-called, excitation sculpting and
MEGA techniques`` and they are less sensitive to ip-angle errors than WATER-
GATE. This alleviates the phase problems commonly encountered when water mag-
netization is spin-locked. In these experiments, the water magnetization is fully re-
turned to equilibrium prior to each acquisition. This improves water suppression,
alleviates attenuation of the sample signal from saturation, and eliminates radiation
damping.
As an aside, it is instructive to point out that excitation sculpting has other useful
applications besides solvent suppression. Frenkiel et al. have extended it to selective
excitation of other (i.e. non-solvent) resonances, and it is particularly applicable in
identifying correlations between specic protons in small molecules.`" Such tech-
niques often allow one to get the same amount of information from a 1D experiment
that would otherwise require 2D NMR.
Depending on the sample, however, simpler water-suppressiontechniques are some-
times adequate or even more desirable. As previously mentioned, Homans et al.`
opted not to use a gradient-based water suppression technique. This was due to the
small dierence in chemical shift between water and the anomeric protons in the
selected carbohydrates. This made it virtually impossible to selectively suppress the
water resonance without also suppressing the anomeric protons in the disaccharide.
Thus, these investigators opted for a technique which used long water-selective purge
pulses, after all the disaccharide proton magnetization had been temporarily transfer-
red to `C.
Other solvent suppression techniques sometimes have distinct advantages over
pulsed-eld gradient-based methods. The water-PRESS sequence is deployed just
before the main part of a pulse sequence."" In this technique, a -RFpulse inverts all of
374 B. A. Salvatore
the magnetization (i.e. sample and water resonances), and then any transverse magnet-
ization is removed with a homospoil. During a delay, both the water and protein
resonances relax along the z-axis. However, since the T
for water is inherently much

longer than the T
for the sample, the delay is chosen such that the water magnetiz-
ation is approximately zero when the protein resonances are almost all fully relaxed.
The homospoil succeeds in dephasing spurious amounts of water magnetization in the
transverse plane, thus preventing the onset of damping during the delay. A subsequent
read pulse is then applied to observe the sample magnetization, while ipping the
water magnetization back to the z-axis. Since the water-PRESS suppression module
is appended in front of the pulse sequence, and not spliced within it, the main part of
the experiment begins with the sample magnetization at thermal equilibrium. This
eliminates the phase errors often seen with gradient-based techniques, particularly
when some of the pulse-widths or gradients are not calibrated properly. This technique
is useful because it facilitates the observation of sample resonances that lie under-
neath the water resonance, something which cannot be achieved with selective-
inversion methods, like WATERGATE. In contrast to most other methods, the
Water-PRESS technique is extremely simple to implement and optimize. It does not
require accurately calibrated RF pulses, nor excellent lineshape. Moreover, there is no
loss of sample intensity from diusional eects, which are especially problematic for
small molecules. One disadvantage of the Water-PRESSmethod is the length of time it
adds to each acquisition, but this may be outweighedby some of the above advantages.
Using computers in structure determination
Traditional automated-NMR-resonance-assignment strategies are based on grouping
resonances into spin systems that represent distinct components of a molecule (e.g.
amino acid residues within a protein). This is followed by the identication of these
segments and the sequential connection of the spin systems, ultimately allowing
assignment of all the NMR spectral resonances. Advances in double isotopic labeling
(`C, `N) techniques have greatly facilitated this process for proteins. Now, the
eciency is being further increased by computers. Montelione et al. have developed a
computer program for assigning NMR resonances in proteins, called AUTOAS-
SIGN." It requires the amino acid sequence and input data generated from H`N
HSQC, as well as data from the eight most common 3D triple-resonance NMR
experiments [H(CA)(CO)NH, CA(CO)NH, CBCA(CO)NH, HNCO, H(CA)NH,
CANH, CBCANH, and HN(CA)CO]. The program employs ve sequential stages of
analysis. Depending on the stage, dierent methods and criteria are used to designate
chemical shifts and establish sequential links between individual spin systems. Each
stage uses constraint-based matching, which progressively relaxes the criteria used in
designating chemical shifts and sequential links along the protein carbon backbone.
Prestegard et al."` have pursued an alternative approach which employs a neural
network to make connections between input data and output structural assignments
from `N-edited TOCSY-HSQC spectral data. This approach is more exible, in that
probabilities are evaluated at each stage, resulting in several choices, instead of just one
denitive choice. One disadvantage of a neural network approach, however, is the
need for a large number of correctly assigned examples to train the network. This can
ultimately be oset by the small amount of data required to make the actual assign-
ments.
Emerenciano et al. have applied a similar heuristic approach to the structure
determination of natural products by `C NMR. This has resulted in a collection of
modular programs, based on the main program, called SISTEMAT. This program
operates on the premise that virtually all natural products are divisible into three
components: the skeletal atoms, the heteroatoms directly bound to these atoms, and
the carbon side-chains. These researchers recently reported a subroutine which is
suited for the identication of common side-chains (e.g. angelate, tiglate, etc.), attached
to any of the atoms in a natural product."` This module identies subspectra of the
carbon atoms representing specic substituent groups, amid the raw `C NMR
spectral data. Thus, side-chain peaks are identied and distinguished from the skeletal
carbons, whose values, in turn, can be fed to SISTEMAT to identify the carbon
skeleton. SISTEMAT, itself, was recently upgraded to identify aromatic molecules,
and it was trained with a library of over 700 avinoids, which represent 72 distinct
skeletal types."" Given a set of `C chemical shifts for an unknown avinoid, the
program was able to suggest a list of probable carbon skeletons, eliminating 70 of the
72 possible carbon skeletons in one demonstration.
Another useful application of computers in the structure identication of natural
products (albeit one that does not utilize AI methods) involves the simulation of
complex NMR spectra, based on molecular mechanics-derived structural data. Laa-
tikainen et al. have developed one such spectral-simulation program, called
PERCH."` They demonstrated this programs usefulness by performing a complete
H NMR spectral analysis of -pinene, which possesses a highly complex spin system,
containing 16 coupled protons."'
Oriented-sample NMR techniques
It is ultimately desirable to study the properties of a natural molecule in an environ-
ment that resembles its native environment. Unfortunately, this is more easily said
than done. Natural oligosaccharides, for example, are often xed on the outer phos-
pholipid bilayer of cells, and yet conformational information is still generally derived
from these molecules through solution NMR experiments, in which they tumble
isotropically. However, newNMRmethods for studying such molecules in membrane-
like environments have been developed over recent years. Isotropic micelles oer one
option for studying these molecules by NMR,"` and magic angle spinning of multi-
lamellar liposomes has facilitated the acquisition of some high-resolution NMR
spectra."` Most appealing, though, are techniques which not only allow one to study
such molecules at membrane surfaces, but to also extract information about structure
and conformation which is not available from solution or MAS NMR experiments.
One such technique is liquid crystal NMR, where molecules are studied in a eld-
orientable liquid crystalline matrix that facilitates the measurement of dipolar coup-
lings. These through-space couplings do not exist in isotropic or MAS samples,
because they average to zero as the molecules tumble randomly or spin at the magic
angle. Liquid crystal samples are often prepared by adding the compound of interest to
a concentrated aqueous lipid-micelle solution. Dimyristoyl phosphatidylcholine
(DMPC) and dihexanoyl phosphatidylcholine (DHPC), (or alternatively DMPC and
the bile salt, CHAPSO) are commonly used to form orientable micelle solutions. The
resulting bilayer micelles (i.e. bicelles) are planar and thus possess an anisotropic
magnetic susceptibility which causes them to orient in a magnetic eld, such that the
376 B. A. Salvatore
HO
O
NH
O
OH
O
OH
NH
O
HO
HO HO
O
O
HO
O
NH
OH
O
OH
NH
O
HO
HO HO
O
O
O
bilayer normal
B
o
Fig. 7 Incorporation of GM4-lactam glycolipid molecules into a DMPCCHAPSO
bicelle. Note that the bicelles bilayer normal aligns perpendicular to the static magnet-
ic eld of the spectrometer (B
"
)
normal to the bicelle surface, on average, lies perpendicular to the external magnetic
eld (Fig. 7).
In liquid crystal bicelle systems, unlike solid crystals, dipolar couplings are partially
averaged (scaled down) by the motion of the bicelles, and are hence termed, residual
dipolar couplings. The size of a particular residual dipolar coupling D
'
is dened by
eqn. (4),
D
'
'
h
2`r`
S
'''
S
`''
3 cos`
'
1
2
(4)
where
'
and
are the gyromagnetic ratios of the two nuclei, r is the distance between
the nuclei,
'
is the angle that the internuclear vector makes with the external magnetic
eld, and the two S terms are order parameters whose product is sample-dependent
and can be measured experimentally. The angular quantity (
'
) is the key term on the
right side of the equation, where the brackets denote the average value of the enclosed
quantity on the NMR timescale. The presence of this angular term indicates that a
relationship exists between D
'
and
'
. Thus one can infer that a correlation also exists
between D
'
and a molecules orientation/conformation in an oriented system. This is
the basis for liquid crystal NMR. The data are generally analyzed in terms of an order
matrix, using NMR dipolar (or quadrupolar) coupling data along with structural
models from which distance information (r) is derived.
Prestegardet al. employed oriented planar bilayer micelles (i.e. bicelles) to determine
oligosaccharide headgroup orientation at membrane surfaces. Recent studies explored
the conformations of GM4-lactam glycolipid"" (Fig. 7), a ganglioside analog with
potential applications in the development of cancer vaccines, as well as sul-
foquinovosyldiacylglycerol, a glycolipid with strong inhibitory activity against HIV-
1.`" These investigations were based on measurements of H`C, `C`C, and
H`N residual dipolar coupling measurements, as well as site-specic `C- and
`N-chemical shift anisotropy measurements. The dipolar coupling values were de-
rived from labeled samples, mainly via direct detection of `C spectra (proton-de-
coupled 1D `C`C INADEQUATE and 2D `C`C DQF-COSY), and molecular
mechanics-minimized structures were used to obtain distance (r) values.
Vold and Prosser reported a modied bicelle system for which the diamagnetic
anisotropy, and hence the sense of bicelle orientation, is ipped. Thus the bilayer
normal axis aligns parallel to the magnetic eld (S
'''
90).` This was achieved by
doping a DMPCDHPC solution with lanthanide salts (e.g. EuCl
`
), and it resulted in
a two-fold increase in the order parameter (S
'''
), as determined by quadrupolar
splitting measurements in oriented-chain perdeuterated DMPC. This modied bicelle
systemenhances the resolution of dipolar couplings in NMRspectra and makes bicelle
systems more applicable to the study of large, slow tumbling molecules, like proteins.
Bax and Tjandra found that even greater resolution spectra are attainable, even
without metals, by simply diluting the bicelle concentration (down to 3%w/v).`` This
substantial decrease in bicelle concentration does not disrupt orientation, but rather,
results in much higher resolution spectra which facilitate the measurement of H`N,
H`N, and H`C dipolar couplings. Moreover, this bicelle system is amenable to
indirect detection, which provides far greater sensitivity than the direct `C- and
`N-detection methods employed previously. In such dilute bicelle systems, it is
unlikely that the sample molecules are incorporated into the individual bicelles, but
they are still inuenced, and thus oriented, by the cooperative alignment eects within
the bicelle matrix. These results expand the power of oriented-bicelles, making them
more applicable to small molecules which are not necessarily membrane-anchored.
The same investigators also reported a newpulse sequence for determining the residual
dipolar contributions to CH splittings within methinyl groups, based on the quanti-
tative measurement of peak intensities in H`C HSQC spectra.`` They accom-
plished this with a modied constant time HSQC experiment, in which the proton
pulse is xed in at the center of the constant time evolution period, and only the carbon
pulse is stepped through this period.
4 Miscellaneous
On a nal note, one paper which did not conveniently fall into any one of the above
sections, and yet is potentially applicable to all of them, is a comprehensive list of the
H- and `C-chemical shift data of virtually all common laboratory solvents as trace in
impurities in a variety of deuterated NMR solvents.`" Such a useful compendium will
nd application in the interpretation of a myriad of NMR spectra of organic com-
pounds.
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job:OC094361 12-10-1998 page:1 colour:0

, accordingly. In the most convenient

)/[1 n[(K/K) 1]] (2)

, thus ruining the solvent suppression. For this reason, a lot of

for water is inherently much

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