Varian NMR Instructions - 1D - Chemistry NMR Lab,University of
NMR Course, Lab.5 May 12,1999 Maltseva T. V.boc2.boc.uu.se/boc14www/Compendium/lab5.pdf · NMR...
Transcript of NMR Course, Lab.5 May 12,1999 Maltseva T. V.boc2.boc.uu.se/boc14www/Compendium/lab5.pdf · NMR...
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May 12,1999NMR Course, Lab.5
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Lab. Sa. Assignment of Chemical.Shifts and Conpling Constants
Aim of the session:
-Assignment of the lH and 13C chemical shifts and coupling constants of adenosine (sample 1 in D2O
solution) and a uniformly carbon-13 labelled sample ( except the methyl groups are not labelled)(sample 2)
Samples:NH2
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3jlN"""
Hs' HS-1 7../ 8C 9';<" '7;(, -
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CH3
Sample 2Sarnple 1
A) Task:
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-as signment of alllH and 13C chemical shifts of sample I and 2.
-extract the lJC-C, the 2JC-C and the lJC-H coupling constants of sample 2.
-extract the 3 JH-H coupling constants of sample I and where possible of sample 2.
-explain the multiplicity of Hl' and H2' in sample 2.
B) A vailable material:
Sample l: A: 1 H spectrum
B: lH spectrum (with different expanded regions)
C: 1 H spectrum after heating of the sample at 75°C, proton -deuterium exchange has happened at
position 8.
D 13C spectrum (IH decoupled)
E: HMQC spectrum (will be explained during the lab)
F: HMBC spectrum (will be explained during the lab)
Sample 2: A: 1 H spectrum
B: lH spectrum (with different expanded regions)
C 13C spectrum ( 1 H coupled)
D: 13C spectrum (IH coupled and with different expanded regions)
E 13C spectrum (IH decoupled)
F: 13C spectrum (IH decoupled and with different expanded regions)
G: HMQC spectrum
H: DQF-COSY spectrum
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May 12,1999NMR Courset Lab.5
Maltseva T. v.
Lab.5b
Goal of the sessionAnalysis of the ID and 2D NMR spectra, DQF COSY,TOCSY and NOESY to obtain the as signment of the sugarand nucleobase protons of each nucleotide of the compound:
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Material available
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The f1Ist step in the structure detennination of nucleic acids is the assignment of the lH-NMR spectrum
using two-dimensional J-correlated and noe experiments which show through-bonds and through-space
connectivities respectively.
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A T wo types of J-correlated experiments can be distinguished
Experiments which provide information about directly coupled spins
Experiments which connect spins which are pan of a J-coupled network in which
individual spins are not necessarily coupled to all other spins in this network.
The COSY (COrrelation SpectroscopY) experiment [A. Bax and R. Freeman, J. Magn. Reson.,
44, p 542, 1981; W. P. Aue, E. Batholdi and R. R. Ernst, J. Chem. Phys., 64, p 2229, 1976] belong to the
first, the TOCSY experiment [M. W. Edwards and A. Bax, J. Am. Chem. Soc., 108, p 918, 1986] to the
second category.
The DQF-COSY [O. w. Piantini, 0. w. Sorensen and R. R. Ernst, J. Am. Chem. Soc., 104, p
6800, 1982] is now the standard COSY experiment for correlating J-coupled spins and for distinguishing
networks of coupled spins for as signment purposes. The fine-structure of the cross peaks in phase-
sensitive COSY spectra is characterised by a multiplet pattem, from which J-couplings can be obtained.
In principle, J-coupling can be extracted directly from these multiplets, but in practice one is confronted
with t wo problems:
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The finite line width
The limited digital resolution
The line width has a profound effect on the peak separation in a multiplet. To obtain reliable coupling
constants, the line width should be a factor of t wo smaller than the coupling constants involved [K.
Wuthrich, NMR of proteins and nucleic acids, Wiley, New York, 1986].For N-type sugars, all antiphase
peaks in a HI'-H2' multiplet tend to cancel due to the small ]1'2' (2Hz), while for HI'-H2" multiplet and
for S-type sugars, only panial cancellation may occur.
The TOCSY spectrum, which shows both the direct and the relayed through-bond connectivities
along the Hl' to H5'/H5" pathway in a sugar unit, provide a means of recognising spins belonging to the
same spin system."The appearance of a 2D NOESY [J. Jeener, B. H. Meier, P. Bachffiann and R. R. Ernst, J. Chem. Phys.,
71, p 4546, 1979; S. Macura and R. R. Ernst, Mol. Phys., 41, p 95, 1980; S. Macura, Y. Huang, D. Suter
and R. R. Ernst, J. Magn. Reson., 43, p 259, 1981] spectruffi is very dependent on the 3D structure of the
nucleic acid fragment. Pair of protons having a short interproton distance will produce strong cross peaks.
The intensity of the--cross peaks depends on parameters such as the mixing time tm, the correlation time
tc, and can be very roughly described as being inversely proportional to the sixth power of the
interproton distance. Thus, noe experiments will provide infonnation on the spatial proximity's between
p. --protons and there~y-Qn the confonnation of a moleculer Noe is also used to morYtor the orientation of the
base (syn or anti) relative to the sugar ring and to monitor the confonnation of the sugar (North or South).
When a nucleotide has a sugar in the North conformation and a base in the anti confonnation, a strong
noe will be observed between the H8 (purine) I H6 (pyrimidine) of the base and the H3' of the sugar.
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