NMR Strategies for labeled Nucleic Acids
Sugar
3D HCCH-COSY3D HCCH-TOCSY
3D H(C)CH-E.COSY3D TROSY-(H)CCH-COSY3D HCC-TOCSY-CCH-COSY3D HCC-TOCSY-CCH-E.COSY
Sequential 31P-based
2D HP HETCOR2D HP HSQC2D HP HeteroTOCSY
2D HP HSQC-NOESY2D & 3D HPHeteroTOCSY-NOESY
2D P(C)H2D P(CC)H
3D P(H)CH
3D HCP 3D HCP-CCH-TOCSY
2D for Base
2D H(NC)C2D H(NCC)H2D H(CCN)H
2D H(CN)C
3D for Base
3D HbC
bN
b
3D (H)N(C)CH
3D HC(CN)H3D H6(CCC)NH3D H(CC)NH-COSY3D H(CC)NH-TOCSY3D(H)C(C)NH-TOCSY
2D Sugar-to-base
2D Hs(C
sN
bC
b)H
b
2D (Hs)C
s(N
bC
b)H
b
2D (Hb)C
b(N
bC
s)H
s
2D Hs(C
sN
b)C
b
2D Hs(C
sN
b)H
b
2D Hs(C
s)N
b
3D Sugar-to-base
3D HsC
sN
b
3D HsC
s(N
bC
b)H
b
3D Hs(C
sN
b)C
bH
b
3D Hs(C
s)N
b(C
b)H
b
3D (Hb)C
bN
b(C
s)H
s
3D (Hs)C
sN
b(C
b)H
b
3D (HsC
s)N
b(C
b)C
bH
b
Through-hydrogenBond
2D H(N)N-COSY2D H(NN)H
NOE Experiments
3D NOESY-C-HSQC
3D NOESY-N-HSQC
NMRGuide 4.3 - TOPSPIN 3.0Written by Teodor ParellaCopyright © 1998-2009 BRUKER Biospin. All rights reserved.
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NMR experiments for labeled nucleic acids http://localhost:6600/guide/eNMR/3Dtrip1.html
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H1' 5-6H2' 2.3-2.9(A,G) 1.7-2.3(T,C)H2'' 2.4-3.1(A,G) 2.1-2.7(T,C)H3' 4.4-5.2H4' 3.8-4.3H5' 3.8-4.3H5'' 3.8-4.3
H1' 5-6H2' 4.4-5.0H3' 4.4-5.2H4' 3.8-4.3H5' 3.8-4.3H5'' 3.8-4.3
C1' 83-89C2' 35-38C3' 70-78C4' 82-86C5' 63-68
C1' 87-94C2' 70-78C3' 70-78C4' 82-86C5' 63-68
RNADNA
Adenine GuanineH2 7.5-8 C2 152-156 - - C2 156H8 7.7-8.5 C8 137-142 H8 7.5-8.3 C8 131-138N6H 5-6/7-8 N6 82-84 N1H 12-13.6 N1 146-149- - - N2H 5-6/8-9 N2 72-76
C4 149-151 C4 152-154C5 119-121 C5 117-119C6 157-158 C6 161N1 220-226 N1 146-149N3 214-216 N3 167
N7 224-232 N7 228-238N9 166-172 N9 166-172
Thymidine Uridine CytidineH6 6.9-7.9 C6 137-142 H6 6.9-7.9 C6 137-142 H6 6.9-7.9 C6 136-144Me5 1.0-1.9 Me5 15-20 H5 5.0-6.0 C5 102-107 H5 5.0-6.0 C5 94-99N3H 13-14 N3 156 N3H 13-14 N3 156-162 - - N3 210- - - - - - - N4H 6.7-7.0
/8.1-8.8N4 94-98
C2 154 C2 154 C2 159C4 169 C4 169 C4 166-168C5 95-112 C5 102-107 C5 94-99N1 144 N1 142-146 N1 150-156
C
N
N
C
O
O
H
H
R
H
Adenosine Guanosine
Cytidine
aHH
C
C
C
N
N
N
N
N
H
H
R H
H
C
C
C
N
N
CN
N
N
O
H
R
H
HH
N
C
C
N
N
O
H
H
R
8888
21
75
~0.7 65203
0.2
0.3
11
20
216
10.8
907.5
15
2391
91
88
637.6
8.5
216
20
11
11
91 10.7 65179
67185
13
1219
19176
68184
13
12
~12
~8
6.5
20
86 86
55
Uridine
C
N
N
C
O
O
H
H
R
H
Adenosine Guanosine
UridineCytidine
HH
C
C
C
N
N
N
N
N
H
H
R H
H
C
C
C
N
N
CN
N
N
O
H
R
H
HH
N
C
C
N
N
O
H
H
R
15
5.2
11
7.8
8.98.5
6.0 3.7
11
7.9
~9.5 ~8~8
5.7 4.64.4
~6
15
C
C
C
N
N
N
N
N
H
H
R
H H
C
C
C
N
N
CN
N
N
O
H
R
H
H
H
N
C
C
N
N
O
H
H
R
HH
C
N
N
C
O
O
H
H
R
H
11
~8.511
125.2
11
5.1
~9
11
10.7
8.36.2
8.9
Adenosine Guanosine
UridineCytidine
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3D HCCH-COSY
DESCRIPTION
The 3D HCCH-COSY experiment is specifically designed to correlate side-chain aliphatic proton resonances with their attached13C resonances via 1J(CH) and 1J(CC) coupling constants. The experiment provides nearly complete assignments of all aliphatic1H and 13C resonances, with the exception of some resonances of the long aliphatic side chains (as Lys or Arg) for whichsubstantial overlap remains.
REQUIREMENTS
Implementation on AVANCE spectrometers is feasible. Improved versions using pulsed field gradients (PFGs) are also available
and, therefore, in such cases gradient technology is required. The experiment is applied on 13C-labeled proteins. Since NHprotons are not involved, this experiment is performed in D2O.
VERSIONS
The original HCCH-COSY pulse sequence ( 90JACS888 ) consisted of the following steps:
After the initial 90º 1H pulse, 1H chemical shift evolution during the variable evolution t1 period takes place.1.
Fixed evolution delay to achieve antiphase 1H magnetization with respect to 13C via 1J(CH).2.
Magnetization transfer to 13C by applying simultaneous 90º 1H and 13C pulses.3.13C chemical shift evolution during the variable evolution t2 period. followed by a fixed period to achieve antiphase 13C
magnetization with respect to its 13C neighbors via 1J(CC).
4.
A 90º 13C pulse transfers magnetization to its coupling partner.5.13C magnetization is transferred back to the protons by reversing the transfer steps described in points 4 and 2, respectively.6.
Proton acquisition under 13C decoupling.7.
Several improved versions have been proposed incorporating the following modifications:
An improved version reduces indesirable artifacts and noise-like features by using selective 180 pulses and a modifiedphase-cycle ( 90JMR620-87 ).
Incorporation of a constant-time 13C evolution period ( 91JB299 ).HCCH-E.COSY experiments to measure J(HH) ( 93JMRA321-105 and 98JMR216-135 ) and J(CC) ( 93JACS7878 ) couplingconstants .
Use of pulsed-field gradients and PEP methodology ( 94JACS2203 and 98JMR185-135 ).
3D HCCH-COSY Experiment file:///D:/home/fiala/Teaching/C7995/Guide/hcchco3d.html
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A related 2D experiment has been proposed to trace out heteronuclear connectivites at natural abundance (see 2DADEQUATE experiment)Specific 2D and 3D HCCH-COSY experiments have been reported to assign deoxyribose spin systems in doubly labeledDNA ( 98JB25 ).A 3D H(C)CH-COSY pulse sequence with two carbon-dimensions has been recently reported ( 98JMR185-135 ).A modified version has been used to measure J(CP) coupling constants in RNA ( 98JMR236-133 ).
Use of an isotropic 13C mixing period instead of the 90º 13C pulse (see 3D HCCH-TOCSY experiment).
Use of the TROSY approach ( 98JACS6394 , 99JMR447-139 ) to be applied on aromatic 1H-13C spin systems. Editing andSuppression of diagonal peaks in such experiments has been also reported ( 00JMR171-144 and 01JB69-19 ).TROSY-relayed HCCH-COSYfor correlation H2/H8 resonances in 13C-labeled RNA ( 01JB173-20 )A forward-directed quantitative HCCH experiment has been proposed for the measurement of the sugar conformation inRNA oligonucleotides from CH-CH dipole-dipole cross-correlated correlation ( 99JACS1956 ).A HCC-TOCSY-CCH-E.COSY experiment to measure J(HH) for ribose protons in uniformly 13C-labeled RNA (95JACS7251 ).
EXPERIMENTAL DETAILS
The 3D HCCH-COSY experiment can be recorded in automation mode. More details on practical implementation of the 3DHCCH-COSY experiment on AVANCE spectrometers can be found in the corresponding Tutorial 3D HCCH-COSYexperiment
SPECTRA
The HCCH-COSY experiment affords a 3D spectrum in which 1H, 13C and 1H chemical shifts are displayed in three independent
dimensions. Cross-peaks are due to 1H-13C-(13C)-1H spins systems.
RELATED TOPICS
Analogs 2D HCCH-COSY experiments are also possible, yielding a COSY-type spectra. However, for larger proteins such 2Dspectra show very severe overlap.
NMRGuide 4.0 - TOPSPIN 1.3Written by Teodor ParellaCopyright © 1998-2004 BRUKER Biospin. All rights reserved.
NMRGuide 4.0 - TOPSPIN 1.3Written by Teodor ParellaCopyright © 1998-2004 BRUKER Biospin. All rights reserved.
3D HCCH-COSY Experiment file:///D:/home/fiala/Teaching/C7995/Guide/hcchco3d.html
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3D HCCH-TOCSY
DESCRIPTION
The 3D HCCH-TOCSY experiment is specifically designed to correlate side-chain aliphatic proton and 13C resonances via1J(CH) and 1J(CC) coupling constants. The experiment provides nearly complete assignments of all aliphatic 1H and 13Cresonances, with the exception of some resonances of the long aliphatic side chains (as Lys or Arg) for which substantial overlapremains.
REQUIREMENTS
Implementation on AVANCE spectrometers is feasible. Improved versions using pulsed field gradients (PFGs) are also available
and, therefore, in such cases gradient technology is required. The experiment is applied on 13C-labeled proteins. Since NHprotons are not involved, this experiment is usually recorded in D2O.
VERSIONS
The original 3D HCCH-TOCSY pulse sequence ( 90JMR425-88 ) consisted of the following steps:
After the initial 90º 1H pulse, 1H chemical shift evolution during the variable t1 period takes place.1.
Fixed evolution delay to achieve antiphase 1H magnetization with respect to 13C via 1J(CH).2.
Magnetization transfer to 13C by applying simultaneous 90º 1H and 13C pulses.3.13C chemical shift evolution during the variable t2 period.4.
An isotropic mixing 13C period transfers magnetization along the 13C side chain via 1J(CC).5.13C magnetization is transferred back to the protons by reversing the transfer steps described above.6.
Proton acquisition under 13C decoupling.7.
Several improved versions have been proposed incorporating the following modifications:
Use of pulsed-field gradients and PEP methodology ( 93JMRB333-101 and 94JACS2203 ).
3D HCCH-TOCSY Experiment file:///D:/home/fiala/Teaching/C7995/Guide/hcchto3d.html
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Use of an 90º 13C pulse instead of the isotropic 13C mixing period (see 3D HCCH-COSY experiment).Use of heteronuclear cross-polarization ( 93JMRB333-101 and 94JACS2203 ).
Use of homonuclear 13CB decoupling during the 13C evolution t2 period ( 96JMRB190-113 ).
Constant-time 3D (H)CCH-TOCSY and H(C)CH-TOCSY experiments ( 98JB89 ). Analogs 3D (H)CCH3-TOCSY and
H(C)CH3-TOCSY experiments have been specifically designed for side-chain resonance assignment of methyl-containing
residues ( 00JMR288-142 )Editing and Suppression of diagonal peaks in such experiments has been also reported ( 01JB69-19 ).Analogs 2D HCCH-TOCSY ( 90JACS886 , 92JACS9202 and 94JACS2205 ) and 4D HCCH-TOCSY experiment ( 92JB655 ) havebeen also proposed.HCC-TOCSY-CCH E.COSY experiments (or HCCH-TOCSY-E.COSY) experiments to measure J(HH) coupling constants( 95JACS7251 , 96JACS4388 , 96JMRB160-112 )
3D HCCH-TOCSY Experiment file:///D:/home/fiala/Teaching/C7995/Guide/hcchto3d.html
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3D alpha/beta selective HC(C)H-TOCSY scheme to measure nJCHCH coupling constants in RNAs ( 01JB117-21 )
A forward-directed quantitative HCCH-TOCSY experiment has been proposed for the measurement of the sugarconformation in RNA oligonucleotides from CH-CH dipole-dipole cross-correlated correlation ( 99JB241 and 99JACS1956 ).
Z-filtered HCCH-TOCSY using adiabatic TOCSY ( 00JB199-18 ).
Carbon-detected HCCH-TOCSY pulse sequence verified in a cryoprobe ( 01CBC247 )
EXPERIMENTAL DETAILS
The 3D HCCH-TOCSY experiment can be recorded in automation mode. More details on practical implementation of the 3DHCCH-TOCSY experiment on AVANCE spectrometers can be found in the corresponding Tutorial 3D HCCH-TOCSYexperiment
3D HCCH-TOCSY Experiment file:///D:/home/fiala/Teaching/C7995/Guide/hcchto3d.html
3 of 4 22.10.2013 15:41
SPECTRA
The HCCH-TOCSY experiment affords a 3D spectrum in which 1H, 13C and 1H chemical shifts are displayed in three
independent dimensions. Cross-peaks are due to 1H-13C-(13C)n-1H spins systems.
RELATED TOPICS
NMRGuide 4.0 - TOPSPIN 1.3Written by Teodor ParellaCopyright © 1998-2004 BRUKER Biospin. All rights reserved.
NMRGuide 4.0 - TOPSPIN 1.3Written by Teodor ParellaCopyright © 1998-2004 BRUKER Biospin. All rights reserved.
3D HCCH-TOCSY Experiment file:///D:/home/fiala/Teaching/C7995/Guide/hcchto3d.html
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Go to Tutorial
3D HCN
DESCRIPTION
The 3D HCN experiment allows to obtain sugar-to-base correlations from the H1'(sugar) toN1/N9 (base) via C1'(sugar) (HsCsNb experiment)
or non-exchangeable base proton assignments from the H6/H8 (base) to N1/N9 (base) via C6/C8
(base) (HbCbNb experiment) in 13C,15N-labeled nucleic acids.
REQUIREMENTS
Implementation on AVANCE spectrometers equipped with a third channel and gradient technology.
VERSIONS
The original experiment was described in 93JB721 and 94JB129 .
3D HCN Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hcn.html
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Sensitivity-improved version using shaped pulses:
Improved sensitivity is achieved in MQC-based HCN experiments ( 97JACS7361 , 98JB373 ,98JMR119-130 , and 02JB9-22 )
and TROSY ( 00JB291 , 01JACS658 and 01JB367-21 ) experiments:
3D HCN Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hcn.html
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Modifications of the basic pulse scheme have been used to measure residual dipolar couplings inlabeled nucleic acids ( 02JB9-22 , 05JB231-31).
Other approaches:
Several 2D H(C)N schemes have been compared ( 98JB373 ).
TROSY-based 2D H(C)N have been also proposed ( 01JACS658 and 01JB367-21 ).
3D HCN Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hcn.html
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Simultaneous acquisition of HCN and HCP experiments ( 96JB251 ).2D H(CN)C for sugar-to-base (2D Hs(CsNb)Cb) and base (2D Hb(CbNb)Cb) resonance
assignments ( 04JB69-28 )
Related 2D and 3D sugar-to-base pulse sequences are:
2D Hs(CsNbCb)Hb
2D (Hs)Cs(NbCb)Hb
2D (Hb)Cb(NbCs)Hs
2D Hs(CsNb)Hb
3D HsCs(NbCb)Hb
3D Hs(CsNb)CbHb
3D (Hb)CbNb(Cs)Hs
3D (Hs)CsNb(Cb)Hb
EXPERIMENTAL DETAILS
SPECTRA
In the HCN spectra, all H/C pairs are sorted in a 2D spectrum as a function of 15N chemical shift inthe third dimension.
3D HCN Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hcn.html
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RELATED TOPICS
NMRGuide 4.3 - TOPSPIN 3.0Written by Teodor ParellaCopyright © 1998-2009 BRUKER Biospin. All rights reserved.
3D HCN Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hcn.html
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2D Hs(CsNbCb)Hb
DESCRIPTION
The 2D Hs(CsNbCb)HbH experiment has been proposed to achieve through-bond
intranucleotide sugar-to-base correlations in 13C,15N-labeled nucleic acids.
REQUIREMENTS
Implementation on AVANCE spectrometers equipped with a third channel and gradienttechnology.
VERSIONS
The original experiment was described in ( 93JACS11040 ).
See a list of related 2D and 3D sugar-to-base NMR experiments
2D H(CNC)H Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hcnch.html
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EXPERIMENTAL DETAILS
SPECTRA
In the H(CNC)H spectra, a conventional 2D 1H-1H map correlation is obtained only displayingsugar-to-base correlations starting from the anomeric sugar proton.
RELATED TOPICS
NMRGuide 4.3 - TOPSPIN 3.0Written by Teodor ParellaCopyright © 1998-2009 BRUKER Biospin. All rights reserved.
2D H(CNC)H Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hcnch.html
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3D (H)N(C)CH
DESCRIPTION
REQUIREMENTS
Implementation on AVANCE spectrometers equipped with a third channel and gradient technology.
VERSIONS
See a list of related 2D and 3D Nucleic-Acid NMR experiments2D H(NCCC)H or H(NC)-TOCSY-(C)-H experiment for guanosine.
3D (H)N(C)CH Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hncch.html
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Specific 2D H(NCCC)H versions for uridine and cytidine
3D H(NCCC)CH versions for cytidine
EXPERIMENTAL DETAILS
SPECTRA
3D (H)N(C)CH Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hncch.html
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RELATED TOPICS
NMRGuide 4.3 - TOPSPIN 3.0Written by Teodor ParellaCopyright © 1998-2009 BRUKER Biospin. All rights reserved.
3D (H)N(C)CH Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hncch.html
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2D HNN-COSY
DESCRIPTION
The ge-2D HNN-COSY experiment has been developed for direct observation of hydrogenbonds in 15N-labeled nucleic acid base pairs by internucleotide 2J(N,N) couplings
REQUIREMENTS
Implementation on AVANCE spectrometers equipped with gradient technology.
VERSIONS
This experiment was originally described in 98JACS8293 for Watson-Crick base pairs in RNA
2D HNN_COSY Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hnn.html
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Cross-hydrogen bond 2hJN,N coupling constants, ranging from +6 to +10 Hz, has also been
measured in:
Hoogsteen base pairs in RNA ( 98JACS8293 and 98PNAS14147)DNA ( 99JB67, 99JACS6019 and 00JB279)DNA-protein complex ( 00JB39).Between NH.....N sidechain of histidines in a protein. ( 99JACS5123)A related sequence has been proposed for the measurement of H....H/H-N hydrogen bond
length in double stranded DNA from 1J(NH) dipolar couplings ( 01JB361-19).
A suite of 2HJNN schemes have been proposed to correlate proton-proton systems across
NH........N hydrogen bonds in nucleic acids ( 01JB289-21). These sequences affordsH(NN)H correlations in several types of RNA, DNA and peptide-RNA complexes.A modified TROSY experiment has also been proposed ( 02JB31-24)
EXPERIMENTAL DETAILS
SPECTRA
A 2D 1H-15N spectrum is obtained
2D HNN_COSY Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hnn.html
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RELATED TOPICS
NMRGuide 4.3 - TOPSPIN 3.0Written by Teodor ParellaCopyright © 1998-2009 BRUKER Biospin. All rights reserved.
2D HNN_COSY Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hnn.html
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Go to Tutorial
2D 1H-31P HETCOR
DESCRIPTION
REQUIREMENTS
Implementation on AVANCE spectrometers equipped with gradient technology.
VERSIONS
Basic sequence using gradient selection:
Constant-time version with gradient selection:
2D 1H-31P HETCOR Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hphetcor.html
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Constant-time version without gradient selection:
Constant-time version without gradient selection and jump-and-return solvent suppression:
See a list of related 2D and 3D Nucleic-Acid NMR experiments
EXPERIMENTAL DETAILS
SPECTRA
2D 1H-31P HETCOR Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hphetcor.html
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RELATED TOPICS
NMRGuide 4.3 - TOPSPIN 3.0Written by Teodor ParellaCopyright © 1998-2009 BRUKER Biospin. All rights reserved.
2D 1H-31P HETCOR Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hphetcor.html
3 of 3 22.10.2013 15:11
Go to Tutorial
3D HCP
DESCRIPTION
The 3D HCP experiment is a 1H-13C-31P triple-resonance experiment specifically designedto assign the ribose H3'/C3', H4'/C4' on the 5' side and, H4'/C4' and H5',H5''/C5' resonances
on the 3' side of the intervening phosphorus in 13C-labeled nucleic acids.
REQUIREMENTS
Implementation on AVANCE spectrometers equipped with a third channel and gradienttechnology.
VERSIONS
The 3D HCP pulse sequence ( 94JACS4983 and 94JACS6472 ) consists of the following basicssteps:
Initial transfer from 1H to 13C via 1J(CH) using an INEPT pulse sequence.1.
Transfer from 13C to 31P via nJ(CP) using another INEPT pulse sequence.2.31P chemical shift evolution during the variable evolution t1 period.3.
Transfer back to 13C from 31P using a retro-INEPT pulse sequence.4.13C chemical shift evolution during the variable evolution t2 period5.
Magnetization is finally transferred back to the 1H protons using a CH-optimized PEPmethodology.
6.
Proton acquisition under 13C decoupling.7.
3D HCP Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hcpch.html
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Other approaches:
Simultaneous acquisition of HCN and HCP experiments ( 96JB251 ).An analog 2D H(C)P experiment has been proposed ( 95JMRB89-106 ).A similar approach called HCPCH experiment has been proposed using naturalabundance samples ( 95JMRB117-109 ).Enhanced resolution in 3D HCP to measure 3J(C2'P) and 3J(C4'P) coupling constants (04JB61-30 )
Quantitative 2D and 3D HCP to measure 3J(C4'(i)-P(i)) and 3J(C4'(i)-P(i+1)) couplingconstants in oligonucleotides ( 98JB223 ).
EXPERIMENTAL DETAILS
2JC3'(i)-P(i) and 2JC5'(i+1)-P(i) coupling constants are about 3-5 Hz, and 3JC4'(i)-P(i) and
3JC4'(i+1)-P(i) coupling constants are about 8-10 Hz
SPECTRA
In the HCPCH spectra, the H-4'/C-4', H-3'/C-3', H-5',H-5''/C-5' pairs are sorted in a 2D
spectrum as a function of 31P chemical shift in the third dimension.
3D HCP Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hcpch.html
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RELATED TOPICS
3D HCP Experiment http://localhost:6600/guide/eNMR/eNMR3Dadn/hcpch.html
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