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Journal of the Chilean Chemical Society

On-line version ISSN 0717-9707

J. Chil. Chem. Soc. vol.49 no.2 Concepción June 2004

http://dx.doi.org/10.4067/S0717-97072004000200016 

 

J. Chil. Chem. Soc., 49, N 2 (2004), pags.:189-195

PULSED FIELD GRADIENTS IN NMR HIGH RESOLUTION EXPERIMENTS

Luis Espinoza*, Juan A. Garbarino, M. Cristina Chamy and Marisa Piovano

 

*Laboratorio de RMN V Región, Laboratorio de Productos Naturales, Departamento de Química, Universidad Técnica Federico Santa Maria, Casilla 110V, Valparaíso, Chile.

Dirección para correspondencia


ABSTRACT

High-resolution NMR spectra of selective 1D, 2D homonuclear and 2D heteronuclear experiments were registered of the mixtures of the natural products 3-4 and the methyl esters (5-6) and acetyl derivates (7-8) of the natural product 1-2, using Pulsed Field Gradients (PFGs) technique. Thus, it was possible to obtain high-resolution spectra, smaller time of acquisition and larger structural information.


INTRODUCTION

Since approximately 1991 actively shielded probe heads have been commercially available and consequently, many gradient-based experiments have recently been proposed and are useful for resolving many chemical questions1-3.

The advantages offered by the incorporation of PFGs into high-resolution NMR pulse sequences combined with the advanced software tools available at the present time to acquire and process multidimensional NMR experiments have changed dramatically the concept of what is routine NMR for chemists.

In this work, we report some examples of PFGs applications in RMN: 1D selective, 2D homonuclear and 2D heteronuclear experiments4,5,6 of the mixtures of the natural products 3-4 (geometrical isomers) and the methyl ester and the acetyl derivatives of 1-27 (geometrical isomers)


 
Fig. 1: Compounds used in this study.

EXPERIMENTAL

Apparatus

All spectra were recorded on a AVANCE 400 Digital NMR Bruker Spectrometer, equipped with a 5.00mm Inverse Multinuclear Detection Pulsed Field Gradients probe heat (1H-BBI, PFG-ZGRD, Z8202/0253), operating at 400.132MHz for 1H and 100.623MHz for 13C. Located at the Universidad Técnica Federico Santa María.

Organic compounds studied

Mixtures: 2b-hydroxy-9-epi-ent-labda-8(17)-13E-dien-15-al (3) with 2b-hydroxy-9-epi-ent-labda-8(17)-13Z-dien-15-al (4); methyl 2b-hydroxy-9-epi-ent-labda-8(17)-13E-dien-15-oate (5) with methyl 2b-hydroxy-9-epi-ent-labda-8(17)-13Z-dien-15-oate (6) and methyl 2b-acetoxy-9-epi-ent-labda-8(17)-13E-dien-15-oate (7) with methyl 2b-acetoxy-9-epi-ent-labda-8(17)-13Z-dien-15-oate (8)

Solvents

CDCl3, 99.5% atom of D, contains 0.1% v/v TMS (Merck)

Pulses program

The pulses program, acquisition parameters and processing of all experiments were obtained and optimized from Bruker standard parameters library and are given for each experiment6.

1D-Selectives experiments

Two types of 1D-experiments were registered with PFGs technique: selective-PFGs-1D-NOESY (sel-pfg-1D-NOESY) and selective-PFGs-1D-TOCSY (sel-pfg-1D-TOCSY).

2D Homonuclear Experiments

The 2D experiments that were recorded with PFGs technique are the following: pfg-2D-1H-1H-COSY-DQF magnitude-mode and pfg-2D-1H-1H-NOESY phase-sensitive.

2D Heteronuclear experiments

The 2D experiments that were recorded with PFGs technique are the following: pfg-2D-1H-13C-HSQC multi-edit-phase-sensitive and pfg-2D-1H-13C-HMBC magnitude-mode.

RESULTS AND DISCUSSION

As part of the structure elucidation of the mixture of geometric isomers 1-2 and 3-4 obtained from Calceolaria inamoena7, where the relative stereochemistry of H-9 could not be determined by rutinary NMR, we made an experimental sequences of high resolution spectra of selective 1D, 2D homonuclear and 2D heteronuclear experiments.

Compounds 1-2 where methylated with ethereal diazomethane affording 5-6 and this mixture was acetylated with Ac2O in pyridine, obtaining compounds 7-8.

The results obtained are:

sel-pfg-1D-NOESY

Figure 2 shows some sel-pfg-1D-NOESY spectra on mixture 5-6. In this case, the ultra-clean spectra allow the identification of very small NOEs effects and to eliminate undesired electronic artifact.


 
Fig. 2: 1HNMR spectrum and sel-pfg-1D-NOESY spectrum of natural product mixture 5-6 derivates of 1-2 in CDCl3, with selnogp.3 pulses sequence and mixing time of 400ms.

In fig. 2 a) when the signal of H-2,2' was selectively excited, we observed the NOEs effects between H-2,2' with CH3-19, CH3-20 and with H-1a and H-3a; we can also see the vicinal coupling between these two proton (H-1a and H-3a.)

In fig b) we can see that when the CH3-20 signal is selectively excited, the NOEs effects between CH3-20 with CH3-19, H-6a, H-11, H-1a and H-2 were observed. These experiments allowed the assignment of the stereochemical position of the OH in C-2 as b.

Some parameters used for record this experiments are summarized in table 1. In the figure 3 are illustrated the pulses sequence used in this experiments.


Table 1: Acquisition basic parameters of sel-pfg-1D-NOESY experiments. The most important variables were: d8 (mixing time) and p12 (selective 1800 pulse duration)
 


 
Fig. 3: selnogp.3 pulses sequences for sel-pfg-1D-NOESY experiment with two selective 1800 pulses, a refocus G3 gradient and four gradients with 11:17:40:-40 ratio.

sel-pfg-1D-TOCSY

This experiment gives information about scalar coupling between spin sub system concerning to determined hydro carbonated fragment chain or pertaining to a determined ring. In figure 4 a general model of this experiment is illustrate.


 
Fig. 4: General illustration of TOCSY experiment.

Sel-pfg-1D-TOCSY is a very important information of spin sub system that can be easily recognized and that are coupled by 3J.

Some sel-pfg-1D-TOCSY experiment of 3-4 and 5-6 mixtures are shown in the figures 5 and 6 respectively. We can observe scalar coupling of spin sub systems of the A rings.


 
Fig. 5: 1HNMR spectrum and sel-pfg-1D-TOCSY spectra of the mixture 3-4 in CDCl3, with a mixing time MLEV17 of 60ms.


 
Fig. 6: 1HNMR spectrum and sel-pfg-1D-TOCSY spectra of mixture 5-6 in CDCl3, with a mixing time MLEV17 of 60ms.

In this case the spin sub systems at A ring in 3, 4, 5 and 6 are almost magnetically equivalents. This experiment, together with sel-pfg-1D-NOESY, correlations 2D homo and 2D heteronuclear experiments, permitted unequivocally the assignments of the stereochemistry at the positions 2, 5, 9 and 10 in both mixtures, confirming unequivocally that compounds 1 and 2 are 9-epi-labdanes, that is a characteristic of all the diterpenes present in Calceolaria species7.Some parameters used to record these experiments are summarized in table 2. In the figure 7, the applied pulses sequences is shown.


Table 2: Acquisition basic parameters of sel-pfg-1D-TOCSY experiment. The most important variables are: d9 (mixing time) and p12 (selective 1800 pulse duration)
 


 
Fig. 7: selmlgp pulses sequence for sel-pfg-1D-TOCSY experiment with a selective 1800 pulse, refocus G2 gradient, a train of pulses MLEV17 and two gradients with 15:15 ratio.

It is very important to indicate that d9 parameter governs duration time of MLEV17 pulses train, therefore is definitive a observation scope of spin sub systems under study.

pfg-2D-1H-1H-COSY-DQF magnitude-mode

This correlation experiment, gives information about the vicinal coupling constant (3J) and the geminal coupling (J2) when these exists. In particular this version has the advantage of filtering the intense signals corresponding to the methyl groups, achieving a better observation of the CH2 and CH signals, therefore, spectra are recorded with high resolution.

In figure 8 is shown pfg-2D-1H-1H-COSY-DQF spectra of the mixture 7-8. The prosecution parameters are summarized in table 3 and the pulses sequences applied in this experiment is illustrated in figure 9.


 
Fig. 8: pfg-2D-1H-1H-COSY-DQF magnitude-mode spectrum of the mixture 7-8 in CDCl3.


Table 3: Acquisition basic parameters of pfg-2D-1H-1H-COSY-DQF magnitude-mode experiment. The main variables are: d1 and d16.
 


 
Fig. 9: cosygpmfqf pulses sequences for pfg-2D-1H-1H-COSY-DQF experiment and three gradients with 16:17:40 ratio.

The spin sub systems at A ring in 7-8 mixture are almost magnetically and chemically equivalents, with this experiment it possible to identify spin-spin coupling (3J) of H-2,2' with H-1a1a', H-1b1b', H-3a3a' and H-3b3b'. We could also see the constant coupling 3J between H-5,5' with H-6a6a' and H-6b6b'.

Other 3J couplings were identified between H-11 with H-12 and H-11' with H-12'. Also it was possible to identify 4J coupling (allyl coupling) between H-14 with CH3-16 and CH3-16'.

The intense signals corresponding to the methyl groups were filtered, so we could achieved a spectral high resolution.

pfg-2D-1H-1H-NOESY phase-sensitive

This long distance correlation experiment, phase sensitive, allow us to differentiate positive and negative signals in the spectra. In the diagonal the positive signals are represented, that correspond to a 1D-1H spectrum, while outside of diagonal it is possible to detect the negative signals, that are assigned to the NOEs effects and the scalars coupling (3J). These phase sensitive experiments, are obtained with more resolution that those recorded with phases cycle.

In this work, the spectra recorded by phase sensitive experiments, the positive signal are represented in gray color, while the negatives are represented in red. Partial spectra pfg-2D-1H-1H-NOESY phase-sensitive of the mixture 7-8 is illustrated in figure 10. In table 4 are summarized the parameters used to record this experiment, the applied pulses sequences is shown in figure 11.


 
Fig, 10: pfg-2D-1H-1H-NOESY partial phase-sensitive spectrum of the mixture 7-8 in CDCl3.


Table 4: Acquisition basic parameters of pfg-2D-1H-1H-NOESY phase-sensitive experiment. The main variables are: d1 and d8.
 


 
Fig. 11: noesygpph pulses sequence for pfg-2D-1H-1H-NOESY phase-sensitive experiment and two gradients with G1:G2 : 40:-40 ratio.

In this zone of the spectrum it was possible to observe several NOEs effects, mainly those between H-2,2' with CH3-20, CH3-19, H-9' and CH3CO'. This fact allowed us to clarify that these hydrogen's are in the same side of the plane of the molecule; as we could deduced in experiment sel-pfg-1D-NOESY (figure 2) H-2,2' has an a orientation so the relative stereochemistry of CH3-19, CH3-20 and H-9 obeys to an a orientation.

The main applications of PFGs are the 2D heteronuclear inverse detection experiments, which are used to determine which 1H of a molecule is bonded to which 13C nuclei (13C, 15N or other nuclei (X)). The advantage of inverse experiments over X detection experiments is that with inverse experiments the nucleus with the highest g (usually 1H) is detected yielding the highest sensitivity. The challenge of an inverse chemical shift correlation experiment, however, is that the large signal from 1H that is not directly coupled to a 13C nucleus must be suppressed in a difference experiment, which posses a dynamic range problem. Common techniques for the suppression of 1H bound to 12C are BIRD-sequence in HMQC experiments and a trim pulse of 1-2ms during the first INEPT in HSQC experiments. However, the suppression is still imperfect and usually additional phase cycling is required. The introduction of PFGs in high-resolution NMR greatly improved the problem of suppressing signal from 1H bonded to 12C. The suppression is almost perfect without additional phase cycling.

pfg-2D-1H-13 C-HSQC multi-edit-phase-sensitive

Experiment of direct correlation (1J) correspond to a new version of the experiments HMQC and HSQC. However, this experiment allow to obtain high resolution that the previous version HMQC, because it is phase sensitive and also gives additional information related with the 13C nuclei multiplicity. It can differed between carbons of type CH3, CH (positive phase, gray color) and CH2 (negative phase, red color). In figure 12 is shown the spectra pfg-2D-1H-13C-HSQC multi-edit-phase-sensitive of compounds 7-8. The parameters used to record this experiment are summarized in table 5 and in figure 13 the applied pulses sequences is shown.


 
Fig. 12: pfg-2D-1H-13C-HSQC multi-edit-phase-sensitive spectrum of mixture 7-8 in CDCl3.


Table 5: Acquisition basic parameters of pfg-2D-1H-13C-HSQC multi-edit-phase-sensitive experiment. The main variables are: d1 and d16.
 


 
Fig. 13: inviedgpph pulses sequence for pfg-2D-1H-13C-HSQC multi-edit-phase-sensitive experiment. In the 13C channel, at the end of the sequence, broad band decoupling is applied. Three gradients with G1:G2:G3 : 30:80:20.1 ratio.

The characteristics of this experiment (high resolution, phase sensitive and edited multiplicity) are that they allowed the assignments of the heteronuclear correlations 1H-13C observed for both isomers, and the identification signal CH3, CH (positive phase, gray color) and CH2 (negative phase, red color), which helped in great measure to the structural elucidation of geometrical isomers.

pfg-2D-1H-13C-HMBC magnitude-mode

Long range correlation experiment (2J and 3J mainly) also delivery important information of those carbons that don't possess hydrogen (quaternary). The spectra of mixture 7-8 is illustrated in the figure 14. In table 6 the parameters used to record this experiment are summarized and the applied pulses sequences are shown in figure 15.


 
Fig. 14: pfg-2D-1H-13C-HMBC magnitude-mode spectrum of mixture 7-8 in CDCl3.


Table 6: Acquisition basic parameters of pfg-2D-1H-13C-HMBC magnitude-mode experiment. The main variables are: d1 and d16.
 


 
Fig. 15: invi4gplrndqf pulses sequence for pfg-2D-1H-13C-HMBC magnitude-mode. Three gradients with G1:G2:G3 : 50:30:40.1 ratio.

The main use of this experiment was, the assignment of quaternary carbons that were not observed in the previous experiments. Through the observation of heteronuclear indirect correlations between the couplings 1H-13C at 2J and 3J we could identified the carbons C-4, C-8, C-10, C-13, C-15 and CH3CO, of both isomers.

CONCLUSSIONS

The information provided in combined form, using all the experiments described previously, allowed to identify and to establish the chemical structure and the relative configurations completely in C-2, C-5, C-9 and C-10 in unequivocal form, of the mixtures of the natural products 3-4 (geometrical isomers) and the methyl ester and the acetyl derivates of 1-2. Confirming that compound 1-2, 3-4, are 9-epi-labdanes.

In all the examples shown in this work, the application of NMR pfg-sel-1D, 2D homonuclear and heteronuclear experiments were recorded with high resolution.

In all NMR experiments were the pulses sequences with PFGs were applied we could observed a reduction of the number of phase-cycling steps for the suppression of undesired electronic artifacts of subtraction. Also, in most of the cases a good signal/noise ratio was achieved, and the acquisition time experimental was reduced significantly.

In 2D heteronuclear experiments of inverse detection (HSQC and HMBC) the incorporation of PFGs sequences, is a fundamental requirement for the acquisition and record time of spectra, due to the practical nature of these experiments, which are based on the detection of low sensibility nuclei (attributed to a low natural abundance or small gyro magnetic constant as 13C and 15N) via the detection of high sensibility nuclei, usually 1H.

 

REFERENCES

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2. A. Bax, P. G. de Jong, A. F. Mehlkopf and J. Smidt, Chem. Phys. Lett. 69, 567 (1980)         [ Links ]

3. G. Bodenhausen, H. Kogler and R.R Ernst, J. Magn. Reson. 58, 370 (1984)         [ Links ]

4. T. Parella, Magn. Reson. Chem. 36, 467 (1998)         [ Links ]5. S. Braun, H. O. Kalinowski and S. Berger, 150 and More Basic NMR Experiments, edited by Wiley-VCH Verlag Gmbh, D-69469 Weinheim (FRA) p. 465-500 (1998)         [ Links ]

6. T. Parella, NMRGuide & Encyclopedia, BRUKER Copyright © Analytik GMBH, (1998-2000)         [ Links ]

7. M.C. Chamy, M. Piovano, L. Espinoza, E. Belmonte and J. Garbarino Phytochemistry (2004) in pres.         [ Links ]

 

ACKNOWLEDGEMENTS

L E thanks support to Dirección General de Investigación y Postgrado (DGIP) of the Universidad Técnica Federico Santa María, Fundación Andes, Fondo Nacional para el Desarrollo Regional (FNDR) and the Programa de Naciones Unidas para el Desarrollo (PNUD) and Fondecyt (# 1020070).

 

Correspondencia a:e-mail: luis.espinoza@usm.cl

 

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