StrategiesofNMRResonanceAssignment

forBiofluidSamples

生物體液的核磁共振譜峰歸屬900MHz1H1DNMRSpectrumofHumanUrine1HNMRspectrumofTaxolC47H51NO14PublicNMRDatabaseofBiologicalsamples-1HMDB(HumanMetabolomeDatabase)www.hmdb.ca

Version3.5Chemical,Clinical,Molecularbiology/biochemistrydata

40,444metabolites1D(1Hand13C)&2D(COSY,TOCSYandHSQC)NMRdata

MMCD

(MadisonMetabolomicsConsortiumDatabase)Chemical,physical,clinical,molecularbiology/biochemistrydata

20,306metabolites1D(1Hand13C)&2D(COSY,TOCSY,HMBCandHSQC)NMRdataPublicNMRDatabaseofBiologicalsamples-2

BMRB

(BiologicalMagneticResonanceBank)Chemicalandphysicaldata,NMRpulsesequencesPeptides,proteinandnucleicacidChemicalshift,J-couplings,relaxationrates,hydrogenexchangeratesandpKavaluesMetabolomicsdatabasePublicNMRDatabaseofBiologicalsamples-3PersonalNMRDatabaseofBiologicalsamplesApproximateprotonchemicalshifts3.7-5.0ppmAlphaHtoether3.1-4.4ppmAlphaHtohalogen2.1-2.6ppmAlphaHtoCarbonyl0.7-1.7ppmAlkaneCH2.1-3.0ppmAlphaHtoaminonitrogen2.3-2.7ppmAlphaHtoBenzylic6.5-8.0ppmAromaticH9.0-10.0ppmAldehydeH5.0-9.0ppmAmideH3.55-4.0ppmAlphaHtoaminoacid3.3-4.0ppmAlphaHtooxygenFromsampletostructure1D1H

spectrum2D1H-1HJRES2D

1H-1HCOSY2D

1H-1HTOCSY2D

1H-13CHSQC(HMQC)2D

1H-13CHMBC2D

1H-1HNOESY??Noesypr1DCPMGDiffusionWeight1D1HNMRmethodSeparatethesignalswithoutseparatingthesamples!△PFGttn1H-1HJ-resolvedspectroscopySplitpattern:n+1SpinsystemAX2

(CH-CH2)JAXd(F2)J(F1)0Hz1:2:11:1TiltAX2DJ-RevolvedSpectroscopyF1(Hz)F2(ppm)01H-1Hcouplingconstants1Henvironment

JHHrange[Hz]–CH2-CH3

3J=7~8R2–CH2

2J=-10~-18-N=CH22J=8~16

3Jtrans=12～24，3Jcis=3-18，2JG=-3～7

3JO=5～9，4Jm=2～3，5Jp=0～1JcouplingconstantsarehelpfultoobtainthestructureinformationH1H2H3H4H1H2H3H4H4H3H1H2

Correlatescalarcoupledprotons

nJHH(n=2,3)SubsequentrelationshipbetweenprotonsH1R2OR1OH2H3H4H1H2H3H4

2DCOSY:COrrelation

SpectroscopYCOSYCCCCHHHHabcdabcdabcdF1(ppm)F2(ppm)H1R2OR1OH2H3H4H1H2H3H4H1H2H3H4H4H3H1H2H1H2H3H4H4H3H1H2

Identifyprotonsinthesamescalarcouplingnetwork！4JHH

5JHH2DTOCSY(TOtal

CorrelationSpectroscopY)TOCSYCCCCHHHHabcdabcdabcdF1(ppm)F2(ppm)2D1H-13CHMQC/HSQCHeteronuclear

MultipleQuantumCoherence

Heteronuclear

SingleQuantumCoherenceCorrelate1H-13Cchemicalshiftsviaone-bond1JC-H

couplings2DHMQC/HSQCH1H3H4H2aC4

C1C2C313C(ppm)H2b1H(ppm)4CH3CCHHHCH312b32aCRRRRCORR?HMQCorHSQC?HMQC:+

Lesspulses

tolerancetoexperimentalimperfections-

Indirect1H-1HcouplingsinF1

lowerresolutionHSQC:

+

No1H-1Hcouplings

inF1

betterresolution-

Morepulses

sensitivetopulseerrorsCH3CCHHHCH31235C4OC5C3C4C1H5H4H2bH1H2aC2Maximumlongrange:3JC-H2DHMBC(Heteronuclear

MultipleBondCoherence)2DHeteronuclearCorrelatedNMRSpectroscopyF1(ppm)F2(ppm)F1(ppm)F2(ppm)HSQCHMBC2DNOESYNuclearOverhauser

EnhancedSpectroscopYHMBCTOCSYHSQCNOESYCOSYExp

F2 F1 mechanismCOSY

dH

/JHH

dH

/JHH

nJHH(n=2,3)TOCSY

dH

/JHH

dH

/JHH

nJHH(n>2)HSQC

dH

/JHH dC

1JCHHMBC

dH

/JHH dC

nJCH

(n>1)JRES

dH

nJHH(n>2)NOESY

dH

/JHH

dH

/JHH

NOE(~rHH-6)2DNMRStrategiesCCCHOCHCHHCHCH3?1D1HNMRspectroscopyJ-Resolveddd0.99ppm1.04ppm2DTOCSYJ-Resolveddd0.99ppm1.04ppm2.27ppm3.61ppmCH3CH3?“n+1”CHTOCSYCOSYCH3CH3CH2.27ppm3.61ppm0.99ppm1.04ppmCH3CH3CH?HHMBCHSQC0.99ppm1.04ppm19.5ppm20.8ppm31.9ppm63.3ppmCH3CH3CHCH??2.27ppm3.62ppm177ppm177ppmRCH3：0-40ppmRCH2：10-50ppmRCH：15-50ppmCOO160-185ppmCOONH3+Valine

TOCSYCOSY7.55ppm7.63ppm7.83ppmRR1R2R3R1R2?J-Resolved7.55ppm7.63ppm7.83ppmmmm?7.63ppm7.83ppm7.55ppm173.1ppm130.1ppm135.3ppm131.5ppm3.98ppm46.3ppm173.1