biofisica.rmn11 12.ingles

7/31/2019 Biofisica.rmn11 12.Ingles

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Biophysics

Nuclear Magnetic Resonance 1) Introduction to NMR (20, 27 of March)

2) Applications in macromolecules (27 of March)

3) Laboratory practice (17 of April)

Antxon Martinez de Ilarduya

[email protected] (UPC)

Departament dEnginyeria Qumica

Master en Ingeniera Biotecnolgica 2010-2011

2

i) Introduction

- Technique for characterization of:

- Organic compounds

- Macromolecules: - Tacticity (vinyl polymers)

- Composition and sequence distribution(copolymers)

- Conformation (biopolymers, proteins)

- Medicine: - MRI

- Evolution: - 1946 Block y Purcel : 1H NMR

- Currently : FT-RMN

- 1H, 13C, 31P, 15N

- 2D and 3D Spectra

- Solid Samples (CP-MAS)

NUCLEAR MAGNETIC RESONANCE (NMR)

NMR


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ii) Theory

Nuclei of atoms small magnets

E=0

H0

Radiacin

h = E

Observables nucleus :

a) Nuclei with rotation

- They have magnetic moment

- Can be observed by NMR

a1) Spherical charge distribution

spin I= 1/2 1H, 13C, 15N, 19F, 31P,...

a2) Non spherical charge distribution

spin I > 1/2 2H, 14N, 33S, 35Cl, ...

(They have an electric quadrupole moment)

NMR

4

b) Nucleus without rotation

- They dont have magnetic moment

- They cant be observed by NMR

Spin I = 0 12C, 16O, ...

Nucleus with spin I=1/2 (1H, 13C,...)

- Antiparallel

- In a magnetic field Ho: 2 directions

- Parallel

- Small excess in parallel direction to the magnetic field

Population m= +1/2

----------------------- = 1.0000066

Population m= +1/2

NMR


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- Condition for resonance:

Ho = Intensity of Magnetic Field

= Radiation frequency

= Magnetogyric ratio (Depends of the type of nucleus)

2 = H0

H0 (T) (1H) (MHz) (13C) (MHz) (19F) (MHz)

4.7 200 50.29 188.15

7.05 300 75.44 282.23

11.75 500 125.73 470.38

NMR

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Nuclei of interest in NMRNMR


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1H NMR

The chemical shift ( ):

- The hydrogen nucleus (proton)

0

- The hydrogen atom (proton + electron)

- The shielding effect of the electron

simplified scheme of the shielding effect in the hydrogen atom

NMR

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Effect of the substituent (between the effect in the proton and the hydrogen)

Electronegativity of the substituent Unshielding >

Paramagnetic effect

NMR


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Effect of the electronegativity of the substituent

el

ectronegati

vity

NMR

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Chemical shifts of a variety of protons referred to TMS NMR


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Calibration of NMR spectra

- We introduce a reference (TMS)

Si

CH3

H3C CH3

CH3

=e eTMS

0

ppm 12 11 10 9 8 7 6 5 4 3 2 1 0

TMS

partes por milln

NMR

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Spin-spin Coupling

Chemical shift () influenced by the neighboring nuclei

A

Interest: 1) Easier assignation of signals

2) J depends on conformation (Conformational studies)

NMR


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H A H x

H A H X

H A H X

-1/2

+1/2

H A

A

J HxHy

(p p m )

TM S

0

A C O P L A M I E N T O E S P I N - E S P I N

A(p p m )

TM S

0

J J

X

H AH X

H X

25 %

25 %

25 %

25 %

(p p m )

TM S

0

J J

X A

J

50 %

25 % 25 %

S i s te m a A X

S i s te m a A X 2

Spin-spin coupling

HxHa

Hx

HxHa

NMR

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Number of signals in coupled protons

Example: diethyl ether

NMR


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Quantitative analysis

- Integral of signals Number of protons

3

2

3

NMR

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Problem 1: Establish the chemical structure and coupling constants ofdifferent protons of this compound with the help of the molecular formulaand the1H NMR spectrum.

2.

0000

2.

9860

3.

0410

(ppm)

0.00.51.01.52.02.53.03.54.04.5

2.

0000

1247.

69

1240.

63

1233.

57

1226.

25

(ppm)

4.104.20

3.

0410

385.

63

378.

32

371.

26

(ppm)

1.20

C4H8O2

NMR


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Problem 2:Establish the chemical structure of this organic compound withthe help of FTIR and1H NMR spectra.

2 2 2 2 31

C10H12O3

NMR

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13C NMR

Disadvantages:

- Not abundant nucleus (1%)

- Low sensibility (accumulate 200- 10000 spectra)

- It is not quantitative

Advantages:

- sharp peaks (not couplings)

- Wide spectral width (250 ppm). Better resolution.

OCH2CHCH2O

OH

CH, CH2

1

1

24

323

4

NMR


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Scheme of chemical shifts in 13C NMR

C=O

NMR

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Experimental Method

- Solvent without hydrogens (deuterated)

CDCl3 ; deuterated DMSO; CD3COCD3 ; D2O

- introduce TMS or derivative (one drop)

- NMR tubes of special glass

- Put the sample in the superconductive Magnet

- He y N2 (liquid)

- Field homogenization (Shims)

NMR


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Magnetic field direction

Irradiacin

Radiation emission(to detector)

Pulse irradiation

(All frequencies)

I

t

FID

I

(ppm)

Spectrum

FT

Relaxation of nuclei

(Radiation emission (FID)

(intensity vs. time)

FT NMR Spectrum(intensity vs. frequency)

NMR

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Pulse Diagram :

1) 1 H NMR

pulso

FID

P1 AT D1

P1 = Pulse duration

AT= Acquisition time

D1= Time delay between pulse and pulse

pulso

FID

P1 AT D1

13C

IRADIACION

CONTINUA1

HDESACOPLADOR

TRANSMISOR

2) 13C NMR (with nOe and1H decoupled)

NMR


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3) DEPT

Very useful multipulse sequence for the determination of the multiplicity of

different carbons (13C)

C, CH, CH2, CH3

Depending on the pulse duration it is possible to observe different spectra

NMR

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135

C not observed

CH2 down

CH, CH3 up

90

CH up

C, CH2, CH3 not observed

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CH , CH2, CH3 up

C not observed

13C

DEPT 135

DEPT 90

DEPT 45

CH2CH3

CH3CH2

DEPTNMR


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Examples: Isobutanol (CH3)2CHCH2OH

a) 13C

CH3

CH

CH2

c) DEPT 135 CH

CH3

CH2

CH

b) DEPT 90

NMR

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Example: Beta-Ionona

7 85,6

2

1

4

1 (CH3)

10 3

5 (CH3)

CH

CH3

CH2

CH

CH

CH2CH3

NMR


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4) 2D NMR

Very useful for the interpretation of complicated 1D NMR spectra

NMR signals overlapped or at very similar chemical shifts

Solution 2D NMR

Changing:

D1 P1, P2 Different 2D spectra t1 y t2 (COSY, HETCOR, NOESY...)

NMR

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4.1) COSY

1H-1H Homonuclear correlation

Utility

- Knowing which protons are coupled to each other

- Determination of J values

Hx

HxHa

spin-spin coupling

NMR


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Interpretation of a COSY

Symmetric respect to the

diagonal (take one side)

Diagonal (spectrum in one

dimension)

Off of diagonal: Signals ofcoupled protons

Coupled protons:

1.Trace a vertical and ahorizontal line to diagonal

2. When this line meet the

diagonal trace a vertical to thesignal

H3C C CH2CH3

O 1 23

Coupling signal

coupled

protons

1 23

NMR

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Example 2: Ethyl Benzene

CH2CH3

CH2

CH3Phenyl

Coupling signal

NMR


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Example 3: 1-propanol: CH3CH2CH2OHCH3

CH2OH OH

CH2CH3

NMR

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Example 4: 2-Chlorobutane: CH3CHClCH2CH32 4 3 1

a b c

a

b

c

NMR


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Problem 3:Assign the1H NMR signals ofthe following organic compound CH3

CH3

CH3 CH3O

54

3

2

1

7

810

6

NMR

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Problem 4:Assign the1H NMR signals of the following organiccompound (C6H12O). (FTIR absorptions at 3300 and 1640 cm

-1)

1H-RMN

13C-RMN

510

2,5

10

10

10

10

NMR


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FTIR absorptions at 3338 and 1642 cm-1

NMR

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COSY

NMR


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4.2. HETCOR

Heteronuclear correlation 1H-13C or 1H-X90

{1H}1H:

13C:

90 90

t1

- Utility:

- Correlate protons and carbons attached to each other.

- Easy assignement of 1H y 13C signals in complex compounds

NMR

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Methodology:

1) Trace from 1H peak an horizontal line to the coupling signal.2) Then trace a vertical line (direct couplings H-C)

Example 1: Polyaspartate of ethylene glycol methyl ether

NHCHCH2CO

COOCH2CH2OCH3n

c

a b

CH

a

b

cCH3

CH3b aCH c

Couplin signals 1H-13C

NMR


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Example 2: Isobutanol(CH3)2CHCH2OH

CH2

CH

CH3

1H

13C

NMR

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Problem 5:Asign the13C NMR signals of the following organic compoundwith the help of the HETCOR spectrum

CH3

CH3

CH3 CH3O

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3

2

1 7

810

6

NMR


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Problem 6:Asign the1H y13C signals of the following organic compound withthe help of COSY, DEPT 135, and HETCOR spectra. FTIR (band at 1640 cm-1) (C13H18O)

CDCl3

1H-RMN

13C-RMN

DEPT 135

20

48 8

88 8 8

NMR

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COSYHETCOR

NMR


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Other 2D experiments:

NOE-2D (NOESY): Allows the determination of the distances ofdifferent protons in a molecule (Conformational studies in proteins)

INADEQUATE: 13C-13C Correlacin. (Similar to COSY). Low sensibility

COLOC: Long distance correlation 1H-13C. (Assignments of C y C=O)

J-Resolved Homonuclear and Heteronuclear: Determination of J y .

NMR

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Main Applications of NMR in Biopolymers

1) Determination of the chemical structure (type of

comonomers) and microstructure (comonomers sequence

distribution ).

2) Study the conformation in solution

NMR


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1) Determination of the chemical structure (type of comonomers)

and microstructure (comonomers sequence distribution ).

NMR: Very useful tool

In most of the cases it is required to assign the signals by

2D NMR spectra:

(1H-1H COSY, 1H-13C HETCOR, 1H-1H NOESY y 1H-13C COLOC)

NMR

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1.

0000

0.

1822

1.

9779

0.

1955

0.

1943

2.

9787

Integral

(ppm)

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5

Example 1: PHB (Biopolymer synthesized by bacteria)

CH CH2 C

O

O

CH3

O CH2CH2CH2 C

O

x y

1

2

3 1' 2' 3'

1

3

2

1 3 2

CHCl3

TMS

Composition:

1=K1x

0.18=K2y

x+y=100

x=91.7% y=8.3%

NMR


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(ppm)

0102030405060708090100110120130140150160170

CH CH2 C

O

O

CH3

O CH2CH2CH2 C

O

x y

1

2

3 1' 2' 3'

4 4'


1

3

2

1 3 2

CDCl34

4

NMR

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(ppm)

62.563.063.564.064.565.065.566.066.567.067.568.068.569.0

Automatic Deconvolution

Fit type : lorentz

No. Position (Hz/ppm) Width (Hz/ppm) Intensity Integral(rel./abs.)

1 5100.62/ 67.5860 2.36/ 0.0312 3.161070e+008 83.66/1.074359e+009

2 5084.78/ 67.3761 2.14/ 0.0284 3.243536e+007 7.81/1.002685e+008

3 4799.54/ 63.5965 2.61/ 0.0346 2.910169e+007 8.54/1.096137e+008

CH CH2 C

O

O

CH3

O CH2CH2CH2 C

O

x y


2

CH

CH2

A B

AA

AB

BAAB

BA

Microstructure:

AA: 83.7 % AB+BA: 16.3 % BB: 0 %

Degree of randomness: 1/(((AA+0.5(AB+BA))/0.5(AB+BA)) + 1/(((BB+0.5(AB+BA))/0.5(AB+BA))

Degree of randomness = 1.09 (Random copolymer)

Microstructure:NMR


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2) Study the conformation in solution

NMR: Frequenly used for the study of the conformation of

proteins.

1H NMR:

a) : depends on the type of conformation (helix, -

sheet,)

b) 3J CH-NH: depends on the dihedral angle between

the protons.

c) Dipolar coupling (nOe effect). Used to calculate the

internuclear proton distances.

NMR

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a) : depends on the type of conformation (helix,

-sheet,)

helix-sheet

coil

NMR


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b) 3J CH-NH: depends on the diedral angle of protons:

Hlix

-Sheet

NMR

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c) Dipolar coupling (nOe effect). Used to calculate

internuclear proton distances

Conantokin-G: Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2

NOESY:

CH

NH

MOLECULAR

DINAMICS

Helical Conformation

NMR


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Poly(-peptides)

NC

CH2

C

H

OROOC H

n

R: Alkyl

-They form structures with the main chain in helix conformation

NMR

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- Helix-coil transition (solvent effect)

NMR


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- Helix-coil transition (effect of temperature)

NH

CH

H

H

C

C

This kind of polymers have the main chain in helix conformation that breaks with the

addition of acids or by thermal effects.

NMR

Bibliography"Spectrometric Identification of Organic Compounds," R.M. Silverstein, G.C. Bassler and T.C. Morrill, John Wiley &

Sons, New York, 5th Ed. 1991.

"NMR and Chemistry; An Introduction to Nuclear Magnetic Resonance Spectroscopy," J. Akitt, Chapman and Hall,

London, 1973.

"Nuclear Magnetic Resonance for Organic Chemistry," D. W. Mathieson, Academic Press, London, 1967.

"Applications of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry," L. M. Jackman and S. Sternhell,Pergamon Press, Oxford, 1969.

One-dimensional and Two-dimensional NMR Spectra by Modern Pulse Techniques, K. Nakanishi, University Science

Books, California, 1990.

"NMR of Proteins and Nueclic Acids K. Wthrich, John Wiley, 1986.

"Nuclear Magnetic Resonance Spectroscopy," F. A. Bovey, Academic Press, 2nd Ed., 1988.

"Tables of Spectral Data for Structure Determination of Organic Compounds," E. Pretsch, T. Clerc, J. Seibl, W. Simon,

2nd Ed. Springer-Verlag, 1989.

"Magnetic Resonance of Biomolecules," P. F. Knowles, D. Marsh, and H. W. E. Rattle, Wiley, London, 1976.

"13C NMR Spectroscopy, High Resolution Methods and Applications in Organic Chemistry and Biochemistry," E.

NMR

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