The Basics of 2D NMRt 1part 1

heteronuclear correlations

CHM 5235, October 2009

Chemical shifts depend on the solvent, temperature, concentration.

In CDCl33

In DMSO-d6

Assignment of the structure on basis of the chemical shifts is impreciseAssignment of the structure on basis of the chemical shifts is imprecise

One can remove the overlap of the signals by changing the solvent or temperature.

2 D t 3D bj t2 D spectra are 3D objects

Cross-peaks reveal a correlation

between the frequencies on the

two frequency axes.

Correlations:

- Through bonds - based on scalar coupling.

- Through space - based on dipolar relaxation

(nOes).

The reaction of the AF4 aryne with 1,4-dimethylnaphthalene y , y p

can yield 4 isomers

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FF FF126.56.54

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141.6

43.9131.3145.4

128.6

127.5

120.4 120.1

127.4

137.6145.7

44.2137.5

130.1

143.6

18.1

6.79

5.92

6.51

7.07

2.23

7.065.87

6.55

7.13

FF FF

141.4

18.1

127.4

119.1

128.6

127.9 129.4

129.0

134.4 134.2

119.0

2.49

6.86

5.65 6.91

Major, 1p minor, 0.37p

The residual one bond H1-C13 correlations in the ghmbc spectrum show that the bridgehead carbons are protonated, therefore the regioselectivity was 5,8 and not 1,4

FF FF126.56.54

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141.6

43.9131.3145.4

128.6

127.5

120.4 120.1

127.4

137.6145.7

44.2137.5

130.1

143.6

18.1

6.79

5.92

6.51

7.07

2.23

7.065.87

6.55

7.13

FF FF

141.4

18.1

127.4

119.1

128.6

127.9 129.4

129.0

134.4 134.2

119.0

2.49

6.86

5.65 6.91

Major, 1p minor, 0.37p

The through-space correlation in the noesy spectrum between 2 49 d 5 65 l th t2.49 and 5.65 reveals that the major product is endo.

Standard Approach to Structure ElucidationStandard Approach to Structure Elucidation

H1-H1 correlation experiment @ the fragments with contiguous H1-H1 coupling.

H1-C13 one bond correlation experiment @ the CH pairs.

H1 C13 two or three bonds correlation experiment @ the carbon skeleton H1-C13 two or three bonds correlation experiment @ the carbon skeleton.

H1-H1 nOe experiment @ the stereochemistry.

One acquires an array of some sort of 1D spectra in f2One acquires an array of some sort of 1D spectra in f2

In the hmqc experiment, the 1D spectra are proton spectra of the C13 satellites.

This spectrum (top) can be obtained by suppressing the central signal (200x more

intense) from the proton spectrum (bottom).

The suppression can

be done with PFGbe done with PFG

(pulsed field

gradients) or with

h li ( tphase cycling (nt

must be a multiple of

the steps in the

phase cycle).

In hmqc, the spectrum with the C13 satellites inverted (top)In hmqc, the spectrum with the C13 satellites inverted (top) is subtracted from the normal spectrum (bottom).

A pulsed field gradient (PFG)A pulsed field gradient (PFG)

produces a linear variation of the field

along the z-axis, for a time of

miliseconds The magnetization ismiliseconds. The magnetization is

dephased in the xy plane, but it can be

refocused by a second gradient pulse of

the same duration and opposite sign.the same duration and opposite sign.

One can use PFGs in a pulse

sequence to defocus all signals and

selectively refocus only those that one

desires.

Cancellation efficiency in hmqc (top) vs. ghmqc (bottom).

The Pulse Sequence

is a sequence of pulses and delays, ending with acquisition.

it encodes the frequencies from f1 into the intensity of the signals observed in f2.

the spectra in f2 differ in an incremented time, d2.p

This pulse sequence uses two

rf channels and gradientsrf channels and gradients.

It contains a time, jtau, which

is a function of 1JCH. It is

averaged for 1JCH = 140 Hz.

Adjust jtau for 10 Hz and one

bt i k fobtains cross-peaks for

correlations over 2 or 3 bonds.

Interferogramg

After the FT in f2, one obtains an interferogram.

The spectra in f2 differ in an incremented time,

d2. The intensity of the C13 satellites as a function

of d2 oscillates at the frequency of the C13of d2 oscillates at the frequency of the C13.

One can make an FID by taking the intensity in

f2 vs. time in f1 (t1, or d2). At the frequency of the

satellite, the FT produces the frequency of C13.

Fourier Transform in 2DFourier Transform in 2D

2D FID (intensity vs. time in f1, time in f2)

1st FT (along time in f2, t2, at)

Interferogram (intensity vs. time in f1, frequency in f2)

2nd FT (along time in f1 t1 d2)2nd FT (along time in f1, t1, d2)

2D spectrum (intensity vs. frequency in f1, frequency in f2)

Digital Resolution in 2D Spectra

Calculate the digital resolution in both dimensions, and the total experiment time, for the parameter set aboveparameter set above.

Doubing the dres in f2 does not increase the total time, because the increase in at can be substracted from d1.

Doubing the dres in f1 is more than doubling the total time, because of the increase in d2.

2.99

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N N

ON

N

N

N

N N

2.99

N

N N

ON

N N

N N

41.02.99

150.0

N

N N

ON

N N

N N

41.02.99

150.0

113.36.82

129.57.21

124.4

NN

N N

ON

N N

N N

41.02.99

150.0

113.36.82

129.57.21

124.4

130.2

N

N N

ON56.3

5.12

N N

N N

41.02.99

150 0

113.36.82

129.57.21

124 4

7.21130.2

6.8

105.1

153 7

NN

N N

ON150.0124.456.3

5.12

153.7

N N

N41.02.99

150 0

113.36.82

129.57.21

124 4

7.21130.2

6.81.97

105.1

153 7

NN

N N

ON150.0124.456.3

5.12

153.7

N N

N N

41.02.99

150.0

113.36.82

129.57.21

124.4

7.21130.2

6.81.97

105.1

153.7

NN

N N

ON56.3

5.12

167 1

93.3

N N25.62.62

167.1

N N

41.02.99

150.0

113.36.82

129.57.21

124.4

7.21130.2

6.81.97

105.1

153.7

N

N N

ON56.3

5.12

167 1

93.36.53

154.0

8.44155.8

N N25.62.62

167.1 8.44157.0

HETCOR (HETeronuclear CORrelation)

Observation on C13. poor resolution on H1. needs enough sample to see the C13 spectrum in 4-64 transients.

N

O

Br

H1-H1 decoupling in f1, except for the geminal coupling.O

O

HETCOR (HETeronuclear CORrelation)HETCOR (HETeronuclear CORrelation)

N

O

Br

One can give up the H1-H1 decoupling in f1 (hmult=y), in O

O

order to use the proton multiplicity.

The sensitivity (signal to-noise ratio S/N) of a two-dimensional experiment

involving nuclei with spin 1/2 can be expressed as:

S/N ~ N*A*T-1*ex*obs3/2*B03/2*T*2*(nt)1/2

where N is the number of molecules in the observed sample volume, A is a

term that represents the abundance of the NMR-active spins involved in the

i t T i th b l t t t d t thexperiment, T is the absolute temperature, ex and obs represent the magnetogyric ratios of the excited and the detected spins, respectively, B0 is

the static magnetic field, T*2 is the effective transverse relaxation time and ntg , 2is the total number of accumulated scans.

By direct observation of N15, for the same sample, to get the same Csignal to noise ratio as in a C13 spectrum, one has to acquire

(1/0.022)2 times longer. If the C13 spectrum took 30 minutes, the N15 spectrum will take 30*(1/0.022)2 = 61983 minutes = 43 days.

If the C13 spectrum took 30 minutes, the same S/N can be attained in the N15 indirect detected spectrum in 30/(0.022*306)2 = 1 minute.

Data in Table 2 represent the maximum increase in sensitivity attainable by p y yindirect detection. It assumes that the timing in the pulse sequence is optimized for that particular N15-H1 coupling constant. This is easy to do for one-bond coupling constants, which are in a narrow range of values, ca. 90 Hz. It is more difficult to do for long range coupling constants which are smaller and have adifficult to do for long-range coupling constants, which are smaller, and have a larger range of values. Besides, smaller coupling constants require longer delays in the pulse sequence, which imply loss of signal.

! !! !

41.02.99

113.36.82

129.57.21

7.21130.2

6.81.97

105.1C13 spectrum taken in 30 minutes.

NN

N N

ON150.0124.456.3

5.12

153.7

93.36.53

154.0

N

N

N25.62.62

167.1 8.44155.8

157.0

C13-H1 gHMBC spectrum taken in 30 minutes, with two relevant traces, at 6.82 and 7.21.

N N

41.02.99

150.0

113.36.82

129.57.21

124.4

7.21130.2

6.81.97

105.1

153.7

6 53

5.12

2.62NN

N N

ON150.0124.456.3

5.12285.7

202.1

16 1

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154.0

8 44155.8

214.7266.0

8.44

7.21

6.53

N N25.62.62

167.1

258.7

8.44157.0

228.8

N15-H1 gHMBC spectrum taken in 30 minutes, with five relevant traces.

Direct vs. Indirect Detection

HETCOR vs. HMQC (or LRHETCOR vs. HMBC)

An indirect detection probe (like the one on Inova 500) has the high-band coil as the inner one - this gives the maximum sensitivity on H1. Usually, with an indirect detection

probe, indirect detection experiments are the only choice for heteronuclear correlation.

With a conventional probe (like the one on the Mercury 300), LRHETCOR and HMBC produce comparable s/n The choice is based on what dimension one wants toHMBC produce comparable s/n. The choice is based on what dimension one wants to

resolve. If the indirect detection experiment works for nt=1, it should be preferred over

HETCOR (minimum nt=4).

Because the relaxation of protons is relevant in indirect detection experiments, HMBC should be preferred for correlations of quaternary carbons.

With a conventional probe, HETCOR should be preferred over HMQC when H1-H1 decoupling in f1 can be used to increase the s/n.

NN

NH2

7.22

116.0156.1237.2

79.8

209.8N15-H1 correlations allow for discrimination

N

NN

N NH9.60

8.03

4 284 071.80

140.3

150.3

160.342.9

152.3139.5

197.2159.8

between acylation in position 2 or 6. The amide

proton (9.60) couples with two Ns, while the

amino protons (7 22) with just oneO

O

4.284.07

1.23

3.36

72.1

58.5

29.3

26.0

1 23

28.11.23

amino protons (7.22) with just one.

0.8413.8

1.23

1.23

22.0

31.1