生物核磁共振波谱学
NMR in Biological Science
5,1D NMR Spectra
THNMR
YAN
5.1 1D 1H NMR Spectra
THNMR
YAN
? chemical shift
? spin-spin coupling
? integral area化学位移 高 —————————— >低频率 高 —————————— >低
磁场 弱 —————————— >强
Dictionary:
1H
Spin = 1/2
Natural abundance = 99.985%
Magnetogyric ratio = 26.7522
Receptivity vs,1H = 1.00
Receptivity vs.13C = 5.67x103
Shift range = -25 to +12 ppm
Shift reference,(CH3)4Si [TMS]
THNMR
YAN
Chemical-Shift Reference
Dictionary:
A chemical-shift reference is used to define the
positions of the resonances in the spectrum in
terms of parts-per-million,or ppm,of frequency,It
is a material which is often directly added to the
sample,For relatively commonly observed
nuclides,chemical-shift reference standards have
been agreed upon as indicated,In all cases,the
shift of this material is defined as d=0,For some
more obscure nuclides,no commonly accepted
standard exists,Most modern spectrometers can
perform frequency referencing operations
automatically,but if highly precise chemical shifts
are needed,it is still advisable to include the
appropriate standard and perform the frequency
referencing manually,
非极性溶剂：
TMS - 四甲基硅烷
(CH3)4Si
极性溶剂：
DSS
(CH3)4Si-CH2-CH2-CH2-SO3-Na
屏蔽常数 s和所处的化学环境有关
s = sd + sp + sa + ss
sd, 抗磁屏蔽，与核外 s电子有关
sp, 顺磁屏蔽，与核外 p,d电子有关
sa, 相邻基团各向异性的影响
ss, 溶剂、介质的影响
THNMR
YAN
THNMR
YAN
A,取代基电负性
取代基电负性越强，与取代基连接于同一碳原子上的 H的
共振频率移向低场
有机官能团的电负性均大于氢原子的电负性，故化学位移
CH > CH2 >CH3
In some cases,such as the benzene
molecule,the circulation of the
electrons in the aromatic orbitals
creates a magnetic field at the
hydrogen nuclei which enhances
the Bo field,This phenomenon is
called deshielding,In this
example,the Bo field is applied
perpendicular to the plane of the
molecule,The ring current is
traveling clockwise if you look
down at the plane,
the benzene molecule
THNMR
YAN
THNMR
YAN
B,环状共轭体系的环电流效应
C6H12
C6H6
THNMR
YAN
B,环状共轭体系的环电流效应
C6H5CH3
C6H5CH2CH3
THNMR
YAN
B,环状共轭体系的环电流效应
THNMR
YAN
C,相连键的磁各向异性
D,相连碳原子的 s-p杂化
E,溶剂、氢键 ……
F,活泼氢
THNMR
YAN
Integration
Assumptions:
? T1 and T2 values of all the
spins are equal,
? No spin decoupling being
performed,
? The S/N ratio is good,
? No sloping baseline in the
spectrum,
THNMR
YAN
Spin Systems Dictionary:
A Spin System is a group of spins that are
connected by scalar (through-bond) spin-spin
coupling J,For practical purposes,in biological
macromolecules such J couplings can usually
be observed between hydrogen atoms that are
separated by three or less covalent bonds,For
each amino acid residue the nonlabile protons
constitute one or several spin systems.
AB
AB2
ABC
AMX
Note:
磁等价 = 化学等价
+对任一核 J相等
THNMR
YAN
Spin Systems of amino acids
AX AMX
AMX AMX
THNMR
YAN
Applications ----PHB
CH CH2
CH3
THNMR
YAN
Applications ----P(HB-co-HV)
CDCL3
2 7 3+8 6
1
5
THNMR
YAN
Applications
6% 18% 24% 30%
THNMR
YAN
Applications ----P(HB-HHX)
CDCL3 TMS
—— > 13C-NMR Spectrum
THNMR
YAN
Applications ----P(HB-MC-LC)
OVERLAP for complex compounds!!!
THNMR
YAN
Applications
VAL in D2O
A3B3MX
THNMR
YAN
Applications
LYS in D2O
THNMR
YAN
Applications
HIS in D2O AMX+AX
THNMR
YAN
Applications
THR-VAL-LEU in D2O
THNMR
YAN
Applications
OVERLAP and CROWDED for proteins!!!
5.2 1D 13C NMR Spectra
? Introduction
? Decoupling
? NOE
? 1-D Spectra
THNMR
YAN
5.2.1 Introduction to 1D 13C NMR
? Many of the molecules studied by
NMR contain carbon,
? Wide chemical shift range
? C without H bonded
? 12C does not have a nuclear spin,while
13C make up approximately one percent
of the carbon nuclei on earth,Less
sensitive than 1H-NMR
? appropriate concentration,field
strength,and pulse sequences
? the presence of 2J between a 13C and
the 1H bonded to 13C,splits the 13C
peaks and causes an even poorer S/N.
THNMR
YAN
Dictionary:
13C
Spin = 1/2
Natural abundance = 1.11%
Magnetogyric ratio = 6.7283
Receptivity vs,1H = 1.76x10-4
Receptivity vs.13C = 1.00
Shift range = -25 to +250 ppm
Shift reference,(CH3)4Si [TMS]
THNMR
YAN
5.2.2 Decoupling Dictionary:
Couplings,or interactions,
between nuclei can be either
homonuclear (such as 1H-1H) or
heteronuclear (such as 13C-1H),
In either case,they cause
splitting or broadening of the
resonances in the spectrum,
complicating interpretation,
Irradiation,at an appropriate
power level,at the frequency of
a particular nuclide or
resonance,is called decoupling,
The interaction is thus removed,
leading to a simpler spectrum.
HC-(CH2CH3)3
If the effect of the 6 -CH2- hydrogens could be removed,we would
lose the 1:6:15:20:15:6:1 splitting for the HC hydrogen and get
one peak,
CH3CH
2CH
1J ~= 100Hz
THNMR
YAN
5.2.2 Decoupling
?The process of removing the spin-
spin splitting between spins is called
decoupling,
?Decoupling is achieved with the aid
of a saturation pulse,
?A saturation pulse is a relatively low
power B1 field left on long enough
for all magnetization to disappear,
1:3:3:1 1+3+3+1=8?
In reality,we see a single line with a
relative intensity of 24,Where did the extra
factor of three come from?
THNMR
YAN
5.2.3 NOE
Dictionary
The nuclear Overhauser effect (NOE)
causes changes in peak areas,as a secondary effect of
decoupling,In most cases,it leads to an increase in
areas,the maximum value of which is governed by
the relative magnitudes of the magnetogyric ratios for
the decoupled and observed nuclides:
NOE (max) = 1- gdec/gobs
For example,the g ratio for the 1H-13C pair is about 4,
so the maximum NOE that could be encountered is 3,
Note that,for some nuclides,g < 0,leading to
negative NOE's,which actually reduce signal
intensity,The exact value of the NOE experienced by
a particular nucleus in the sample depends on the
number of directly attached decoupled nuclei,and on
its spin-lattice relaxation characteristics,For 13C,
methyl groups typically exhibit the largest NOE's,
followed by methylenes,then methines,Quaternary
carbons feel little effect,Because the enhancement is
uneven,the NOE should be suppressed when
quantitative results are desired,This can be
accomplished by gating the decoupler,i.e.,by turning
it on only during the data acquisition time,This
relatively brief period is sufficient to achieve
decoupling,but does not provide enough time for the
NOE to develop to any appreciable extent.
化学键相连 —— >
spin-spin coupling
空间相近 —— >
dipole couping (NOE effect)
THNMR
YAN
5.2.3 NOE
NOE (max) = gS/(2*gI)
同核 NOE,gS = gI,最大增强 = 50%
异核 NOE,gS = gH,gS = gC,最大增强 = 200%
Normally for 1D C-13 NMR,NOE CH3 > CH2 > CH > C
Reference:
? Noggle JH and Schirmer RE,The Nuclear Overhauser effect,New York,1971,
Acadmic Press
? Neuhaus and Williamson.The Nuclear Overhauser Effect in Structural and
Conformational Analysis,VCH (1989).
? Http://glove.2y.net
屏蔽常数 s和所处的化学环境有关
s = sd + sp + sa + ss
sd, 抗磁屏蔽，与核外 s电子有关
sp, 顺磁屏蔽，与核外 p,d电子有关
sa, 相邻基团各向异性的影响
ss, 溶剂、介质的影响
THNMR
YAN
Note,取代基团对 C-13化学位移影响的机理与 H-1中不一样
THNMR
YAN
C-13 NMR
cyclohexane
benzene
Toluene
C6H5CH3
ethyl benzene
C6H5CH2CH3
THNMR
YAN
C-13 NMR
MEK
CH3(C=O)CH2CH3
2-butanol
CH3CH2CH(OH)CH3
Question,
How to characterize
CH3,CH2,CH,and C?
THNMR
YAN
碳原子级数的确定
? J调制法或 APT法
? INEPT法
? DEPT法
DEPT法：
450,CH3,CH2,CH为正，无 C
900,CH为正，其它无
1350,CH3,CH为正,CH2为负
450
1350
MEK
CH3(C=O)CH2CH3
THNMR
YAN
Applications
P(HB-co-HV)
CDCL3
9 4
13
2
8 567
THNMR
YAN
Applications
CDCL3
10
4
132
8 5679
—— > 1H-NMR Spectrum
5,3 1D 31P NMR
? Nucleic acids,in vivo,
? Wide chemical shift range
? 100% natural abundance
? good sensitivity
Though less sensitive than 1H-NMR
THNMR
YAN
Dictionary
31P
Spin = 1/2
Natural abundance = 100%
Magnetogyric ratio = 10.8394
Receptivity vs,1H =0.0663
Receptivity vs.13C = 3.77x102
Shift range = -500 to +600ppm
Shift reference,H3PO4 (85%)
THNMR
YAN
THANK YOU
THNMR
YAN
1H vs,13C NMR
NMR in Biological Science
5,1D NMR Spectra
THNMR
YAN
5.1 1D 1H NMR Spectra
THNMR
YAN
? chemical shift
? spin-spin coupling
? integral area化学位移 高 —————————— >低频率 高 —————————— >低
磁场 弱 —————————— >强
Dictionary:
1H
Spin = 1/2
Natural abundance = 99.985%
Magnetogyric ratio = 26.7522
Receptivity vs,1H = 1.00
Receptivity vs.13C = 5.67x103
Shift range = -25 to +12 ppm
Shift reference,(CH3)4Si [TMS]
THNMR
YAN
Chemical-Shift Reference
Dictionary:
A chemical-shift reference is used to define the
positions of the resonances in the spectrum in
terms of parts-per-million,or ppm,of frequency,It
is a material which is often directly added to the
sample,For relatively commonly observed
nuclides,chemical-shift reference standards have
been agreed upon as indicated,In all cases,the
shift of this material is defined as d=0,For some
more obscure nuclides,no commonly accepted
standard exists,Most modern spectrometers can
perform frequency referencing operations
automatically,but if highly precise chemical shifts
are needed,it is still advisable to include the
appropriate standard and perform the frequency
referencing manually,
非极性溶剂：
TMS - 四甲基硅烷
(CH3)4Si
极性溶剂：
DSS
(CH3)4Si-CH2-CH2-CH2-SO3-Na
屏蔽常数 s和所处的化学环境有关
s = sd + sp + sa + ss
sd, 抗磁屏蔽，与核外 s电子有关
sp, 顺磁屏蔽，与核外 p,d电子有关
sa, 相邻基团各向异性的影响
ss, 溶剂、介质的影响
THNMR
YAN
THNMR
YAN
A,取代基电负性
取代基电负性越强，与取代基连接于同一碳原子上的 H的
共振频率移向低场
有机官能团的电负性均大于氢原子的电负性，故化学位移
CH > CH2 >CH3
In some cases,such as the benzene
molecule,the circulation of the
electrons in the aromatic orbitals
creates a magnetic field at the
hydrogen nuclei which enhances
the Bo field,This phenomenon is
called deshielding,In this
example,the Bo field is applied
perpendicular to the plane of the
molecule,The ring current is
traveling clockwise if you look
down at the plane,
the benzene molecule
THNMR
YAN
THNMR
YAN
B,环状共轭体系的环电流效应
C6H12
C6H6
THNMR
YAN
B,环状共轭体系的环电流效应
C6H5CH3
C6H5CH2CH3
THNMR
YAN
B,环状共轭体系的环电流效应
THNMR
YAN
C,相连键的磁各向异性
D,相连碳原子的 s-p杂化
E,溶剂、氢键 ……
F,活泼氢
THNMR
YAN
Integration
Assumptions:
? T1 and T2 values of all the
spins are equal,
? No spin decoupling being
performed,
? The S/N ratio is good,
? No sloping baseline in the
spectrum,
THNMR
YAN
Spin Systems Dictionary:
A Spin System is a group of spins that are
connected by scalar (through-bond) spin-spin
coupling J,For practical purposes,in biological
macromolecules such J couplings can usually
be observed between hydrogen atoms that are
separated by three or less covalent bonds,For
each amino acid residue the nonlabile protons
constitute one or several spin systems.
AB
AB2
ABC
AMX
Note:
磁等价 = 化学等价
+对任一核 J相等
THNMR
YAN
Spin Systems of amino acids
AX AMX
AMX AMX
THNMR
YAN
Applications ----PHB
CH CH2
CH3
THNMR
YAN
Applications ----P(HB-co-HV)
CDCL3
2 7 3+8 6
1
5
THNMR
YAN
Applications
6% 18% 24% 30%
THNMR
YAN
Applications ----P(HB-HHX)
CDCL3 TMS
—— > 13C-NMR Spectrum
THNMR
YAN
Applications ----P(HB-MC-LC)
OVERLAP for complex compounds!!!
THNMR
YAN
Applications
VAL in D2O
A3B3MX
THNMR
YAN
Applications
LYS in D2O
THNMR
YAN
Applications
HIS in D2O AMX+AX
THNMR
YAN
Applications
THR-VAL-LEU in D2O
THNMR
YAN
Applications
OVERLAP and CROWDED for proteins!!!
5.2 1D 13C NMR Spectra
? Introduction
? Decoupling
? NOE
? 1-D Spectra
THNMR
YAN
5.2.1 Introduction to 1D 13C NMR
? Many of the molecules studied by
NMR contain carbon,
? Wide chemical shift range
? C without H bonded
? 12C does not have a nuclear spin,while
13C make up approximately one percent
of the carbon nuclei on earth,Less
sensitive than 1H-NMR
? appropriate concentration,field
strength,and pulse sequences
? the presence of 2J between a 13C and
the 1H bonded to 13C,splits the 13C
peaks and causes an even poorer S/N.
THNMR
YAN
Dictionary:
13C
Spin = 1/2
Natural abundance = 1.11%
Magnetogyric ratio = 6.7283
Receptivity vs,1H = 1.76x10-4
Receptivity vs.13C = 1.00
Shift range = -25 to +250 ppm
Shift reference,(CH3)4Si [TMS]
THNMR
YAN
5.2.2 Decoupling Dictionary:
Couplings,or interactions,
between nuclei can be either
homonuclear (such as 1H-1H) or
heteronuclear (such as 13C-1H),
In either case,they cause
splitting or broadening of the
resonances in the spectrum,
complicating interpretation,
Irradiation,at an appropriate
power level,at the frequency of
a particular nuclide or
resonance,is called decoupling,
The interaction is thus removed,
leading to a simpler spectrum.
HC-(CH2CH3)3
If the effect of the 6 -CH2- hydrogens could be removed,we would
lose the 1:6:15:20:15:6:1 splitting for the HC hydrogen and get
one peak,
CH3CH
2CH
1J ~= 100Hz
THNMR
YAN
5.2.2 Decoupling
?The process of removing the spin-
spin splitting between spins is called
decoupling,
?Decoupling is achieved with the aid
of a saturation pulse,
?A saturation pulse is a relatively low
power B1 field left on long enough
for all magnetization to disappear,
1:3:3:1 1+3+3+1=8?
In reality,we see a single line with a
relative intensity of 24,Where did the extra
factor of three come from?
THNMR
YAN
5.2.3 NOE
Dictionary
The nuclear Overhauser effect (NOE)
causes changes in peak areas,as a secondary effect of
decoupling,In most cases,it leads to an increase in
areas,the maximum value of which is governed by
the relative magnitudes of the magnetogyric ratios for
the decoupled and observed nuclides:
NOE (max) = 1- gdec/gobs
For example,the g ratio for the 1H-13C pair is about 4,
so the maximum NOE that could be encountered is 3,
Note that,for some nuclides,g < 0,leading to
negative NOE's,which actually reduce signal
intensity,The exact value of the NOE experienced by
a particular nucleus in the sample depends on the
number of directly attached decoupled nuclei,and on
its spin-lattice relaxation characteristics,For 13C,
methyl groups typically exhibit the largest NOE's,
followed by methylenes,then methines,Quaternary
carbons feel little effect,Because the enhancement is
uneven,the NOE should be suppressed when
quantitative results are desired,This can be
accomplished by gating the decoupler,i.e.,by turning
it on only during the data acquisition time,This
relatively brief period is sufficient to achieve
decoupling,but does not provide enough time for the
NOE to develop to any appreciable extent.
化学键相连 —— >
spin-spin coupling
空间相近 —— >
dipole couping (NOE effect)
THNMR
YAN
5.2.3 NOE
NOE (max) = gS/(2*gI)
同核 NOE,gS = gI,最大增强 = 50%
异核 NOE,gS = gH,gS = gC,最大增强 = 200%
Normally for 1D C-13 NMR,NOE CH3 > CH2 > CH > C
Reference:
? Noggle JH and Schirmer RE,The Nuclear Overhauser effect,New York,1971,
Acadmic Press
? Neuhaus and Williamson.The Nuclear Overhauser Effect in Structural and
Conformational Analysis,VCH (1989).
? Http://glove.2y.net
屏蔽常数 s和所处的化学环境有关
s = sd + sp + sa + ss
sd, 抗磁屏蔽，与核外 s电子有关
sp, 顺磁屏蔽，与核外 p,d电子有关
sa, 相邻基团各向异性的影响
ss, 溶剂、介质的影响
THNMR
YAN
Note,取代基团对 C-13化学位移影响的机理与 H-1中不一样
THNMR
YAN
C-13 NMR
cyclohexane
benzene
Toluene
C6H5CH3
ethyl benzene
C6H5CH2CH3
THNMR
YAN
C-13 NMR
MEK
CH3(C=O)CH2CH3
2-butanol
CH3CH2CH(OH)CH3
Question,
How to characterize
CH3,CH2,CH,and C?
THNMR
YAN
碳原子级数的确定
? J调制法或 APT法
? INEPT法
? DEPT法
DEPT法：
450,CH3,CH2,CH为正，无 C
900,CH为正，其它无
1350,CH3,CH为正,CH2为负
450
1350
MEK
CH3(C=O)CH2CH3
THNMR
YAN
Applications
P(HB-co-HV)
CDCL3
9 4
13
2
8 567
THNMR
YAN
Applications
CDCL3
10
4
132
8 5679
—— > 1H-NMR Spectrum
5,3 1D 31P NMR
? Nucleic acids,in vivo,
? Wide chemical shift range
? 100% natural abundance
? good sensitivity
Though less sensitive than 1H-NMR
THNMR
YAN
Dictionary
31P
Spin = 1/2
Natural abundance = 100%
Magnetogyric ratio = 10.8394
Receptivity vs,1H =0.0663
Receptivity vs.13C = 3.77x102
Shift range = -500 to +600ppm
Shift reference,H3PO4 (85%)
THNMR
YAN
THANK YOU
THNMR
YAN
1H vs,13C NMR
