Dr Tim Claridge - Modern NMR Techniques for Chemical Structure Elucidation

Embed Size (px)

DESCRIPTION

NMR Techniques

Citation preview

  • Modern NMR techniques for chemical structure

    elucidation

    -ethanol and beyond...

    Dr. Tim Claridge

    [email protected]

  • Introduction

    Historical overview

    NMR Instrumentation

    Introducing NMR spectroscopy

    NMR techniques for structure elucidation

    Undergraduate NMR course content

  • What is NMR spectroscopy?

    Nuclear- dealing with the property of nuclear spin Magnetic- interaction of nuclear spins with applied magnetic fields

    Resonance Spectroscopy- excitation of these nuclear spins

    Why do we use it?

    Molecular structure

    conformation

    dynamics

    interactions

  • Nuclear Spin

    +

    spin

    magnetic moment

    precession B0

    External magnetic

    field

    N

    S

  • and Resonance

    DE

    a

    b S

    N External magnetic

    field

    Electromagnetic pulse of energy DE DE = hn

  • History: the beginning

    1946: Nuclear Magnetic Resonance

    (Nuclear induction)

    Purcell, Torrey and Pound- 1 kg Paraffin wax

    Bloch, Hansen and Packard- 850 ml water

  • Organic NMR spectroscopy

    1951: First published high-resolution NMR spectrum:

    Neat ethanol @ 30 MHz

    HO-CH2-CH3

  • And now

    10 mg incubation product from antibiotic biosynthesis

    pathway (700 MHz, cryogenic probe)

    NH

    HO2C

    CO2H

    Me

  • NMR instrumentation

    Magnet 400 MHz

    robot

    console

    anti-vibration platform

    preamplifier

  • 0

    200

    400

    600

    800

    1000

    1950 1960 1970 1980 1990 2000 2010

    1H

    Fre

    qu

    en

    cy (

    MH

    z)

    Year

    20 MHz/year

    Magnet development

    11.7

    14.1

    16.4

    18.8

    21.1

    8.5

    7.0

    B0 /Tesla

    23.5

  • Superconducting magnet

  • NMR probeheads

    Radiofrequency transmit & receive

    coils

    Tuning circuitry

  • Cryogenic probeheads

    Probe detection coils @ ~25K

    NMR signal preamplifier @ ~ 70K

    Sample @ ~ 300K

    Thermal noise reduced significantly

    Cold head

  • Introducing NMR Spectroscopy

    The electromagnetic spectrum

  • 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

    Hydrogen NMR spectrum (1H)

    H2C

    CH3

    1) Chemical shift

    2) Spin-coupling fine structure

    3) Absorption intensities: Peak integration

  • Introducing NMR Spectroscopy

    Features of the NMR Spectrum

    1. Chemical shifts

    Tell us about the local chemical environments of each nucleus

    2. Spin-spin couplings

    Tell us about the interactions of each nucleus with its neighbouring nuclei

    3. Peak intensities (integrals)

    Tell us the relative number of nuclei in each chemical environment

  • Chemical shifts reflect chemical environments: nuclei act as

    undercover spies reporting on their surroundings

    Feature I: Chemical Shifts (d)

    9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

    1H Spectrum

    frequency

  • The origin of chemical shifts

    +

    N External magnetic

    field

    Electron spins surround nucleus their own magnetic fields shield nucleus from external field ..differences in electronic shielding produce differences in chemical shifts

  • Feature II: Spin Spin couplings (J)

    A nucleus can sense the presence of its neighbours across bonds: nuclear spies again

    Strength of interaction reflected in the spin-spin coupling constant, J (Hz)

    1.21.31.41.51.61.71.81.92.02.12.22.32.42.52.62.72.82.9 ppm

    H2C

    CH3

    Bond to HQ. Come in HQ Im next to three hydrogensout!

    and Im next to two hydrogensout!

    J

  • Spin coupling patterns and multiplicities

    doublet (d) double-

    doublet (dd)

    triplet (t)

    triple doublet (td)

    double triplet (dt)

  • 1.52.02.53.03.54.04.55.05.56.06.57.07.5 ppm

    Feature III: Resonance intensities

    Total resonance intensity reflects the number of nuclei in that environment: the number of spies present

    Calculated by integration of peak areas

    H2C

    CH3

    5 2 3

  • NMR techniques

    for chemical structure elucidation

    NMR

  • NMR in chemical research Hydrogen (1H) NMR spectra

    O

    H

    BnO

    H

    BnO

    H

    H

    OBnH

    CH2

    BnO

    O

    H

    O

    H

    BnO

    H

    H

    OBnH

    CH2

    STol

    BnO

  • Multi-pulse NMR- spin gymnastics

  • Carbon NMR and editing 12C is NMR inactive, 13C is NMR active but only 1% abundant

    Standard carbon spectra hide multiplicity information (eg CH vs CH2 vs

    CH3)- simple stick appearance

    Chemical shifts indicate chemical environments

    Carbon editing experiments indicate protonation state

  • Two-dimensional (2D) NMR Spectroscopy

    Maps or correlates nuclear interactions within molecules:

    Through-bond coupling (J)

    Indicates presence of bonding connectivity

    Through-space coupling (nOe)

    Indicates close spatial proximity -> 3-dimensional shapes of molecules & stereochemistry

    2D as two frequency axes

    8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

    1D

  • 2D contour presentation

    Top-down view: Contour plot

  • ppm

    0.51.01.52.02.53.03.54.04.5 ppm

    5

    4

    3

    2

    1

    H2N CH C

    CH

    OH

    O

    OH

    CH3

    Threonine

    coupled hydrogen

    spin system

    2D 1H-1H Correlation

    Spectroscopy (COSY)

    Maps spin-spin coupled partners

  • 2D COSY

  • 2D 1H-1H Total Correlation Spectroscopy (TOCSY)

    Maps all spin-spin interactions within molecular fragments or even complete molecules

    A B C D

    F2

    F1

    COSY

    A B C D

    F2

    F1

    TOCSY

  • 2D TOCSY

  • HH

    H

    H

    A

    B

    C

    D

    12

    3

    Heteronuclear correlation methods: 1H-13C

  • 2D Heteronuclear correlation

    13C

    1H

  • Biological chemistry: Peptides of 19 amino-acids

  • Chemical biology: Protein of 110 amino acids

  • 1H-15N correlation: 110 amino acids Isotopically 15N labelled protein

    1H

    15N

    Structure Drug binding Protein-protein interactions

  • 3D NMR methods

    for protein structures: triple-resonance NMR 1H/13C/15N

    HNCA

    1H

    15N

    1H

    15N

    13C

    N

    C

    C

    N

    H

    H

    C

    C

    O

    O

    N

    C

    C

    N

    H

    H

    C

    C

    O

    O

    N

    C

    C

    N

    H

    H

    C

    C

    O

    O

    HNCACO HNCO

    N

    C

    C

    N

    H

    H

    C

    C

    O

    O

  • 13C/15N labelled

    Protein

  • Medicine:

    Magnetic Resonance Imaging

    Functional MRI Unit, Oxford

  • Undergraduate NMR spectroscopy

    Often part of Organic Spectroscopy courses (+UV, IR, MS)

    YEAR Material

    2nd 1H & 13C chemical shifts, spin-spin couplings, resonance intensities

    Origins of chemical shifts and couplings Correlating chemical shifts with structural environments Correlating spin-spin couplings with structural features

    Influences on NMR spectra: stereochemistry, conformation, dynamics

    Properties of other NMR active nuclei: 19F, 31P, 11B Instrumentation & Fourier transform NMR

    3rd Through-space correlations: nuclear Overhauser effects

    defining stereochemistry and 3D shapes of molecules

    2D NMR methods

    1H-1H through-bond correlations 1H-13C through-bond correlations 1H-1H through-space correlations

    Physical behaviour of nuclear spins: relaxation

  • Thank you for your attention

    [email protected]