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Anlise de Carboidratos
Polissacardeos Oligossacardeos
Composio de monossacardeos
Metilao Posio das ligaes glicosdicas Hidrlise enzimtica com glicosidases especficas Sequncia de monossacardeos Posio das unidades monossacardicas RMN e espectrometria de massa
Sequncia de monossacardeos
Posio das unidades monossacardicas
4. Determinar a configurao absoluta (i.e. D or L) de cada resduo glicosdico (rotao especfica; C.D. absoro; susceptibilidade enzimtica; derivatizao e M.S.; NMR).
uma forma de espectroscopia que faz uso da absoro diferenciada da luz polarizada no sentido horrio ou no sentido anti-horrio. Este fenmeno rotineiramente utilizado no estudo da estrutura secundria de protenas.
6. Estabelecer a forma do anel (i.e. furanose ou piranose) de cada resduo (analise metilao etilao).
5.
glcosidica(s)
(metilao;
As hidroxilas livres das oses e demais glicdios podem ser metilados com sulfato de dimetila. A ligao ter entre a metila e os carbonos da ose no rompida pela adio de cidos, com exceo se essa ligao for com o carbono anmero.
Metilao Posio das ligaes glicosdicas Metilao Posio das ligaes glicosdicas
Hidrlise s do C anmrico
8. Determinar a configurao anomerica (i.e. ou ) de cada resduo glicosdico (NMR; susceptibilidade enzimatica). 9. Determinar os pontos anexos dos substituintes que no so carboidratos.
Diversidade Estrutural
A interpretao de espectro de RMN no muito
RMN (ressonncia nuclear magntica) uma tcnica poderosa para estudar a estrutura dos carboidratos. Todas as informaes estruturais (exceto configurao L vs D) podem ser obtidos por RMN.
RMN particularmente til para determinar uma ligao glicosdica ( vs ) e para a determinao da estrutura secundria
Configurao D ou L
Embora a maioria dos acares so D algumas excees existem e s podem ser distinguidas usando-se solventes quirais.
Tautomerismo
Monossacardeos so estabilizados por ciclizao
para formar um hemiacetal ou hemicetal.
Glicose piranoae
Deteco em 13C a 150 MHz
H OH
37,64 %
H O
H OH
61,96 %
H O
HO HO H H OH
d 92.9 1
H
HO HO H H OH H
OH
d 96.7
HO
Glicose furanose
0,28 %
H HO HO HO H H OH
0,11 %
H O H HO HO
d 103.2 OH
O HO H H OH H
d 97.6
OH
Glicose
0,004 %
0,0059 %
CH2OH
d 90.3
OH H OH OH CH2OH
D-ribose, D-altrose, D-idose e D-talose predomina o tautomeros furanose a concentrao varia entre 21 e 32 %.
H OH O HO H HO H H OH H OH H O
gua deuterada,
Solventes
CDCl3
Piridina-d6
DMSO4-d6
Referncia interna
Para solues aquosas o sinal 1H e 13C de referncia
interna pode ser:
HO H O
d 7,5 Hz
HO HO O H H OH H O P O
HO
A
H O
J1-2 = 2-4 Hz
Mudanas de sinal
E
H
no carbono anomrico
so indicativos carboidrato para est
HO HO H HO H
OH OH
axial-equatorial
ligado a quem.
A H
J1-2 = 7-9 Hz
OH
HO HO H H
OH
A H
diaxial
HO HO
2
J1-2 = 1,6 Hz
E
HO HO H HO HO HO HO H H
E
diequatorial
H
E
H OH
J1-2 = 0,8 Hz
OH
axial-equatorial
Fuc (D),
5.58 (A)
The 1H NMR spectrum (500 MHz) (Fig. 1) of this polysaccharide at 27 oC showed five anomeric proton signals at d 5.12, 5.09, 5.07, 4.94, and 4.43 ppm in a molar ratio of 1:1:1:1:1. The methoxyl proton was observed at d 3.78 ppm.
OCH3
Figure 1. 1H NMR spectrum (500 MHz, D2O, 27 C) of the polysaccharide isolated from Moringa oleifera
Figure 1. NMR spectra of the fraction BIWP2: (a) 13C NMR spectrum (150 MHz); (b) 1H NMR spectrum (600 MHz). Solvent: D2O, temperature: 20 C.
A C-1 signal for typical b-glycosidic configuration H-1 signals for typical b-glycosidic configuration
104.66 ppm in the 13C NMR spectrum 4.42 ppm in the 1H NMR spectrum.
Carbono 13C
Na ausncia de modificao estrutural, um resduo de acar de um oligossacardeo tem sinal de 13C usualmente +/- 0.3 ppm do correspondente a ressonncia de monossacardeos livres.
Glicosilao leva ao aumento a frequncia de sinal entre 410 ppm para carbonos anomricos e ligao nesta posio.
O sinal qumico do carbono anmerico em ligao a ( ~ d 97- d 101) em geral menor que da ligao b (~ d 103 d 105).
Carboidratos
Gal C-1 b Gal C-1 a GLC C-1 b GLC C-1 a
150.9-MHz 2H O. 2
13C
Absoro ppm
103.18 103.15 96.01 92.07
13C
d 92.9
HO HO H
d 96.7
OH
OH H
H HO HO HO H H O
d 97.6
d 103.2
O
OH
HO HO HO H H
OH OH
H OH
6
C=O 5 4 3
2 1
2
3
Figure 3. 13C NMR spectrum (100.63 MHz) in D2O of digeneaside. The inset shows the carboxyl signal (C-1).
In the 13C NMR spectrum (125 MHz) (Fig. 2) at 27 C five anomeric signals.
OCH3
C anmerico
C=O CH3
Figure 2. 13C NMR spectrum (125 MHz, D2O, 27 C) of the polysaccharide isolated from Moringa oleifera
The 13C NMR spectrum of PSI demonstrated a regular structure. It contained signals for six anomeric carbons at d 96.4105.1, five HOCH2C groups (C-6 of Man, Glc and Gal) at d 62.262.7, one carboxyl group (C-6 of GlcA) at d 174.6 and sugar ring carbons in the region d 66.280.6.
Espectros 2-D
Como os espectros de de 1H so muito limitados em relao aos sinais os espectros de 2-D ajudam na determinao dos carboidratos.
Figure 2. The HMBC spectrum for the fraction BIWP2. Relevant crosspeaks are labeled.
Figure 4 Gradient-selected, sensitivity-enhanced HSQC spectrum (1/21JC,H, 3.45 ms; J 145 Hz) at 600.1 MHz of a 120-mM solution of lactose in 2H2O.
Infravermelho
lcoois ( carboidratos) A banda de estiramento OH, entre 3300 e 3500 cm-1, a absoro mais caracterstica de compostos desta natureza. Outras bandas presentes so de estiramento C O (entre 1250 e 1400cm-1) e de deformao OH (entre 1000 e 1200 cm-1), sua freqncia variando segundo o no de substituintes: lcoois primrios e secundrios absorvem em menores comprimentos de onda que lcoois tercirios.
estiramento OH
estiramento C = O
estiramento C O
deformao OH
glycopeptides at the low (25) picomole level during chromatography of glycoprotein digests and for differentiation of O-linked from N-linked oligosaccharides. The technique involves observation of diagnostic sugar oxoniumion fragments, particularly the HexNAc+ fragment at m/z 204, from collisionally excited glycopeptides. Collision-induced fragmentation can be accomplished in either of two regions of a triple quadrupole mass spectrometer equipped with an atmospheric pressure, electrospray (ES) ionization source. If collisions before the first quadrupole are chosen, it is possible to enhance formation of carbohydraterelated fragment ions without distorting the distribution of peptide and glycopeptide signals by increasing the collisional excitation potential only during that portion of each scan in which the low mass carbohydrate-related ions are being detected. This procedure, requiring only a single quadrupole instrument, identifies putative glycopeptide-containing fractions in the chromatogram but suffers from a lack of specificity in the case of co-eluting peptides. Increased specificity is obtained by selectively detecting only those parent ions that fragment in Q2, the second collision region of the triple quadrupole, to produce an ion at m/z 204 (HexNAc+). Only (M + H)+ ions of glycopeptides are observed in these liquid chromatography-electrospray tandem mass spectrometry (LC-ESMS/MS) parent-scan spectra. N-linked carbohydrates are differentiated from O-linked by LC-ESMS/MS analysis of the digested glycoprotein prior to and after selective removal of N-linked carbohydrates by peptide N:glycosidase F. These methods, which constitute the first liquid chromatography-mass spectrometry (LC-MS)-based strategies for selective identification of glycopeptides in complex mixtures, facilitate location and preparative fractionation of glycopeptides for further structural characterization. In addition, these techniques may be used to assess the compositional heterogeneity