Phytochemistry Reviews 2: 31–43, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.
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One-dimensional 13C NMR and HPLC-1H NMR techniques for observing carbon-13 and deuterium labelling in biosynthetic studies Bernd Schneider∗ , Jonathan Gershenzon, Gerson Graser, Dirk Hölscher & Bettina Schmitt Max-Planck-Institute for Chemical Ecology, Beutenberg Campus, Hans-Knöll-Str. 8, 07745 Jena, Germany; ∗ Author for correspondence (Tel: +49-3641-571600; Fax: +49-3641-571601; E-mail:
[email protected])
Key words: biosynthesis, carbon-13, deuterium, glucosinolates, HPLC-NMR, isotopomer analysis, labelling, NMR, phenylphenalenones, phenylpropanoids
Abstract For several decades isotope labelling techniques have been the indispensable tools used to unravel pathways of secondary product biosynthesis. NMR spectroscopy, together with mass spectrometry, is the most effective measuring technique used in the analysis of metabolites enriched with stable isotopes. 2 H and 13 C are the NMR-detectable nuclides which have been most frequently employed in plant secondary metabolite synthesis. Examples from the biosynthesis of phenylpropanoids, phenylphenalenones, and glucosinolates are used when discussing some aspects of one-dimensional NMR analysis of metabolites selectively labelled with 2 H and 13 C. Besides direct NMR detection of 13 C-enriched metabolites, special emphasis is placed on indirect detection of 13 C and 2 H, especially by HPLC-1 H NMR coupling, to analyse the isotopomer pattern of compounds in low concentration. The examples discussed in this paper were obtained from studies with Anigozanthos preissii (root cultures) (Haemodoraceae) and Eruca sativa (Brassicaceae). Abbreviations: ds-4MTB – desulphated 4-methylthiobutylglucosinolate; NMR – nuclear magnetic resonance; HPLC – high performance liquid chromatography; S:N – signal-to-noise ratio.
NMR in biosynthetic labelling experiments Labelled precursors, enriched in one or more positions with stable isotopes, have been extensively used in biosynthetic studies to detect incorporation, to examine the origin, or to follow the fate of specific parts of a molecule or atoms in a pathway (Vederas, 1985; Simpson, 1998). Among the elements making up the major constituents of living organisms, carbon, hydrogen, oxygen, nitrogen, and sulphur are the most abundant. Mass spectrometric and NMR spectroscopic techniques have been developed to analyse enrichment of heavy atoms in target molecules. Mass spectrometry detects mass differences between natural abundance isotopic distribution and isotopically enriched molecules and is therefore able to detect any heavy isotope. In contrast, direct NMR analysis is restricted to nuclei which possess a nuclear magnetic moment (nuclear spin I = 0). Among the elements in
living organisms these are 1 H, 2 H, 3 H, 13 C, 14 N, 15 N, 17 O, 31 P, and 33 S. 1 H (natural abundance 99.98%), 14 N (99.63%), and 31 P (100%) are the normal isotopes (‘normatopes’) of the corresponding elements and, therefore, not feasible for labelling. However, as discussed below for 1 H, this does not mean that these nuclei are useless in the detection of labels by means of NMR. The use of 3 H (I = 12 ), although of the highest sensitivity in NMR and zero natural abundance, is limited due its radioactivity (radiolabelling is not the subject of the present paper). The very low natural abundance of 2 H (0.16%) and 17 O (0.37%) makes these nuclei suitable for labelling studies (Staunton and Hailes, 1996; Pearson and Oldfield, 1996), although sensitivity is low and, due to the nuclear quadrupole (2 H: I = 1; 17 O: I = 52 ), signals are broad. 2 H NMR in particular is widely used to investigate the fate of deuterated precursors and to study
32 mechanistic aspects of biosynthesis by means of stereo specific labelling. So far 33 S has not been used as a label in biosynthetic studies. Besides 2 H, 13 C (I = 12 ) and 15 N (I = 12 ) are the stable isotopes most frequently used to unravel precursor-product relationships. In this paper, some recent examples are given of the NMR analysis of 13 C labelling (direct detection), the 1 H NMR analysis of 13 C-labelled carbon atoms with attached protons (indirect detection), and double and multiple 2 H/13 C labelling including online detection by HPLC-NMR. The discussion is restricted to one-dimensional techniques. Direct detection of 13 C in labelled metabolites The most significant information gained from the application of selectively 13 C-labelled precursors (or multiple labelled precursors in isolated positions) in feeding experiments is the location of the label within the target molecule formed during the biosynthetic process and, in a ‘multistep version’, being able to follow the flux of the label through biosynthetic sequences. In a 13 C NMR spectrum, the signal of the labelled carbon atom appears to be enhanced compared to that of natural abundance carbon (∼1.1% 13 C in each position). An example demonstrating incorporation of 13 C into two isolated positions of one molecule is shown in Figure 1. Administration of p-coumaric acid resulted in hydroxyanigorufone, a phenylphenalenone typical of an Anigozanthos species (Cooke and Edwards, 1981). The enhancement of carbon signals at δ 127.7 and δ 132.5 indicated selective labelling of C-5 and C-8 of hydroxyanigorufone (Hölscher and Schneider, 1995). The signals appear as singlets because (a) most of the 13 C atoms are separated in singly labelled [5-13C]- and [8-13C]isotopomers and (b) spin-spin coupling in the [5,813 C ]-isotopomer was not detectable through four 2 carbon-carbon bonds under the conditions used. A multitude of techniques rely on double or multiple labelling. Labelling in magnetically equivalent positions, such as C-2/6 of a phenyl ring, results in enhancement of the corresponding signal and hence reduction of the detection limit. This is important for samples of low concentration and, in particular, for in vivo NMR studies. However, in most cases the labels are located in magnetically non-equivalent positions. Typical examples are metabolites derived from [1,2-13C2 ]acetate, which represents the most frequently used doubly labelled precursor, especially
in polyketide biosynthesis. 13 C NMR spectra obtained from such ‘carbon-carbon bond-labelling’ experiments exhibit characteristic satellite signals due to spin-spin coupling between adjacent 13 C atoms, indicating incorporation of the intact C-2 unit. This requires, however, sufficient dilution of the bondlabelled precursor by endogenous or exogenous natural abundance acetate to prevent inter-unit coupling. An example in which [U-13C]methionine was used to study glucosinolate biosynthesis (Graser et al., 2000) is shown in Figure 2. The resonances of C3 and C-5 of an aliphatic glucosinolate, the desulphated form of 4-methylthiobutylglucosinolate (ds4MTB), appear as doublets (1 JC-3–C-4 = 34.7 Hz and 1 JC-5–C-4 = 34.7 Hz) and C-4 as a doublet of doublets with coupling constants of equal size (1 JC-4–C-3 =1 J C-4–C-5 = 34.7 Hz), demonstrating high levels of incorporation of an intact methionine carbon chain unit. The natural abundance-sized central signals of C-3 and C-5 exclude enhanced occurrence of singly labelled [3-13C]- and [5-13C]-isotopomers arguing strongly against methionine chain degradation and scrambling of the label prior to incorporation in ds-4MTB. The additional resonances of low intensity in between the lines of the doublet of doublets of C-4 (Figure 2) can be interpreted as spin-spin couplings (1 JC-4–C-3 =1 JC-4–C-5 = 34.7 Hz) in small amounts of [3,4-13C]- and [4,5-13C]-isotopomers originating as impurities during the synthesis of the [U13 C]methionine precursor used (there is still ∼1% 12 C in each position of the 99% 13 C uniformly enriched precursor). Rearrangement or disintegration of 13 C-13 C bonds of doubly labelled precursors gives rise to singlet signals without flanking satellites. Vice versa, intra- or intermolecular bond formation between two 13 C enriched positions may result in a new 13 C-13 C bond, which in the 13 C NMR spectrum is detectable from its spin-spin coupling, i.e. satellites with structuredependent coupling constants JC-C (Horak et al., 1985). 13 C-13 C bond formation may occur in an intramolecular manner (Robins et al., 1994) or through an intermolecular reaction between different precursors, but probably is more frequent between molecules formed sequentially from the same precursor in a multiple-step reaction, e.g. chain growth from monomeric units. Figure 3 demonstrates an example of the latter, which again is derived from the biosynthesis of aliphatic glucosinolates. Administration of [2-13C]acetate to Eruca sativa (Graser et al., 2000) not only gave rise to enhancement of the signals of C-1
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Figure 1. 13 C NMR partial spectra (125 MHz) of hydroxyanigorufone. A: Enhanced signals indicate selective 13 C enrichment of C-5 (marked by ) and C-8 (marked by ). This spectrum demonstrates the biosynthetic formation of [5-13 C]- and [8-13 C]-isotopomers of hydroxyanigorufone from [2-13 C]p-coumaric acid in Anigozanthos root cultures (Hölscher and Schneider, 1995). Owing to the high degree of labelling, the occurrence of the [5,8-13 C2 ]-isotopomer is likely but was not directly detectable in the spectrum. B: Spectrum of natural abundance hydroxyanigorufone.
Figure 2. 13 C NMR partial spectra (125 MHz) of desulphated 4-methylthiobutylglucosinolate (ds-4MTB). A: Enhanced signals indicate selective 13 C enrichment of C-3, C-4, and C-5. The doublets for C-3 (1 JC-3–C-4 = 34.7 Hz) and C-5 (1 JC-5–C-4 = 34.7 Hz) and the doublet of doublets for C-4 (1 JC-4–C-3 =1 J C-4–C-5 = 34.7 Hz) proved incorporation of an intact methionine carbon chain unit into aliphatic glucosinolates in Eruca sativa (Graser et al., 2000). The signal of the S-methyl group (Figure 7) was also enriched. Except for signals of low intensity from minor isotopomers originating from natural abundance distribution of 13 C and chemical synthesis of the labelled precursor (for details, see text), only signals of the [3,4,5,S-methyl-13 C4 ]-isotopomer, marked by , were found. B: Spectrum of natural abundance ds-4MTB.
34 (δ 160.7, central signal, [1-13C]isotopomer) and adjacent C-2 (δ 35.7, central signal, [2-13C]isotopomer) atoms of ds-4MTB, but, in addition, a high level of the [1,2-13C2 ]isotopomer was detected from the intense satellites (1 JC-1–C-2 = 49.0 Hz) of both signals. The conclusions drawn from this experiment were that 1) a chain elongation process is operating in the biosynthesis of aliphatic glucosinolates (due to incorporation of C-2 of acetate in two adjacent positions, C-1 and C-2 of ds-4MTB), and 2), the acetyl-CoA pool in this plant is low (due to the high proportion of the [1,2-13C2 ]isotopomer) (Graser et al., 2000). The value of 13 C-13 C coupling constants 1 JC-C is indicative of the chemical environment of the carbon atoms involved. A survey of one-bond and some longrange carbon-carbon coupling constants derived from biosynthetic studies has been published by Horak et al. (1985). In biosynthetic studies, carbon-carbon couplings between 13 C atoms in geminal and vicinal positions as well as those between carbon atoms directly attached to each other also provide useful information. Condensation of two phenylpropanoic acid units with one acetate or malonate molecule results in diarylheptanoids, which after cyclization form anigorufone or related phenylphenalenones (Hölscher and Schneider, 1995). As shown in Figure 4, spinspin coupling between enriched geminal carbon atoms (2 JC-6–C-7 = 2.7 Hz) indicates the occurrence of an [6,7-13C2 ]isotopomer of anigorufone biosynthesised from [1-13C]phenylalanine (Schmitt et al., 2000). A more complex isotopomeric mixture of phenylphenalenones originated from the administration of two different precursors, [1-13C]cinnamic acid and [2-13C]dihydrocinnamic acid, to Anigozanthos root cultures. As anticipated from former feeding experiments, [1-13C]cinnamic acid should be incorporated twice into phenylphenalenones with labels ending up in positions C-6 (δ 130.2) and C-7 (δ 136.2). Rather surprisingly, [2-13C]dihydrocinnamic acid was also incorporated symmetrically, as shown by enhanced signals of C-5 (δ 127.7) and C-8 (δ 132.5) (Figure 5). Closer consideration of the coupling patterns of the four enriched carbon signals revealed the occurrence of a variety of isotopomers of hydroxyanigorufone. The pseudo triplet signals of C-5 and C-8 are each made up of a central singlet (representing the [513 C]-, [8-13 C]- and [5,8-13 C ]-isotopomers, respect2 ively) and doublets of [5,7-13C2 ]- and [8,6-13C2 ]species. The vicinal inter-unit couplings 3 JC-5–C-7 = 3J C-6–C-8 = 5.3 Hz were shown to be larger than the geminal couplings 2 JC-6–C-7 = 2.7 Hz observed for
[6,7-13C2 ]anigorufone (Figure 4). The small coupling constant of inter-unit coupling through two bonds of the [6,7-13C2 ]-isotopomer was detected in the pseudo quintet signals of C-6 and C-7 which were further composed of singlets of singly labelled [6-13 C]and [7-13C]-isotopomers, and doublets of [5,7-13C2 ]and [8,6-13C2 ]species with vicinal coupling constants 3J 3 C-6–C-8 = JC-5–C-7 = 5.3 Hz. Differences in the size of coupling constants of one- and multiple-bond 13 C-13 C spin-spin couplings have been employed in retro biosynthetic studies (Bacher et al., 1998). This technique makes use of uniformly labelled early precursors, resulting in isotopomeric mixtures with complex coupling patterns in the 13 C NMR spectrum. Another extension of the bond-labelling concept is its use in the combination of 13 C with other nuclei, e.g. 2 H. Indirect detection of 13 C in labelled metabolites A fundamental problem in NMR spectroscopy is its inherent low sensitivity, and even modern high field spectrometers often operate at their detection limits. These limitations are especially true for biosynthetic studies. However, there are several strategies for enhancing sensitivity, that include increasing the magnetic field strength, improving probe head design, miniaturization, and cryotechnology. Inversedetection 2D NMR techniques, most efficiently used with pulsed field gradients, are now routinely used in NMR laboratories. However these methods still need considerable spectrometer time, especially when employed with samples of very low concentration. Indirect detection of 13 C by 1 H NMR in its 1D version was used in biosynthetic studies even before 13 C NMR became routinely available (Tanabe, 1973). The advantage of this approach, like inversedetection 2D techniques, is that it makes use of the much greater sensitivity of 1 H NMR spectroscopy as compared with 13 C NMR. However, in order to detect 13 C-labelling by means of protons attached to the 13 C atom, the second dimension is necessary only when signals remain unresolved in 1D spectra. In many cases, information about 13 C enrichment of protonated carbons can be gained directly from a 1 H NMR spectrum by evaluation of the coupling patterns. This is possible because standard 1 H spectra are recorded without decoupling. Edited 1 H NMR spectra that contain only the signals of 1 H coupled to 13 C have been
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Figure 3. 13 C NMR partial spectra (125 MHz) of desulphated 4-methylthiobutylglucosinolate (ds-4MTB). A: Enhanced signals indicate selective 13 C enrichment of C-1 and C-2. The central singlets marked by and , respectively, prove the biosynthetic formation of [1-13 C]- and [2-13 C]-isotopomers of ds-4MTB from [2-13 C]acetate acid in Eruca sativa (Graser et al., 2000). The occurrence of the [1,2-13 C2 ]-isotopomer was shown by the intense doublets for the C-1 and C-2 signals (1 JC-1–C-2 = 49.0 Hz, marked by ) indicating spin-spin coupling between 13 C atoms in both positions. B: Spectrum of natural abundance ds-4MTB.
used, for example, to study aspects of the biosynthesis of the antibiotic vancomycin (Doddrell et al., 1984). Estimation of the integral ratios of signals of protons attached to enriched carbon sites compared to those of natural abundance signals provides information about isotopomer ratios. Indirect detection of 13 C is useful even for the analysis of much diluted samples and enables detection of low or even negligible microgram amounts of a sample. The sensitivity is not only dependent on the gyro magnetic ratio and natural abundance of the respective nuclei but also on the design of the probe head used for measurement. The signal-to-noise ratio (S:N) of signals obtained from a sample containing 99% 13 C enriched carbon with an inverse detection probe head is dramatically higher for indirect than for direct 13 C. Figure 6A/B depicts 1 H NMR (indirect detection mode) and direct 13 C NMR spectra of [S-methyl-13C]methionine measured with a TXI CryoProbeTM (500 MHz). The signal of the S-13CH3 group shows signal-to-noise ratios of 693 in the 1 H NMR spectrum and only 31 in the 13 C NMR spectrum. Even in a broad-band probe head with the inner coil tuned to 13 C, a higher sensitivity (signal-to-noise ratio 79) was observed in the 1 H spectrum (400 MHz) in comparison to the 13 C spec-
trum (S:N 21) for the same sample (Figure 6C/D). Since the 1 H NMR spectra were measured without decoupling, 1 H-13 C spin-spin coupling was observed. The 1 H signal of the S-13CH3 group appears as a doublet (1 JC-H = 139 Hz), indicating attachment to 13 C. Hence, 1 H-13 C spin-spin coupling can be used as a tool to assign 13 C resonances of enriched carbon sites. From this example another essential advantage of 13 C detection through 1 H NMR becomes clear, i.e. assignment of 13 C resonances is not required prior to labelling experiments. In the 1 H NMR spectrum of ds-4MTB, biosynthesised from [U-13 C]methionine (Figure 7), 13 C enrichment of the S-CH3 group was demonstrated by indirect detection using 1 H NMR (1 JC-H = 139 Hz). An additional long-range spinspin coupling (3 JC-H = 4.1 Hz), which was visible only in the coupling satellites but not in the central signal, also indicated 13 C enrichment of C-5 in the same isotopomer. Together with the results described above (Figure 2), it can be concluded that this must be the [3,4,5,S-methyl-13C4 ]-isotopomer of ds-4MTB. Online LC coupling to NMR has become a routine method in natural product chemistry during the last decade (Schneider, 2000). However, the use of HPLCNMR coupling in biosynthetic labelling experiments
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Figure 4. 13 C NMR partial spectra (125 MHz) of anigorufone. A: Enhanced signals indicate selective 13 C enrichment of C-6 and C-7. The extended signals reveal pseudo triplet fine structures with central singlets for the [6-13 C]-isotopomer (marked by ) and the [7-13 C]-isotopomer (marked by ), and doublets for the [6,7-13 C2 ]-isotopomer (2 JC-6–C-7 = 2.7 Hz, marked by ). This labelling pattern demonstrates high levels of biosynthetic incorporation of two molecules of [1-13 C]phenylalanine into anigorufone in Anigozanthos root cultures (Schmitt et al., 2000). B: Spectrum of natural abundance anigorufone.
is still hampered by the fact that direct 13 C detection is not yet possible with commercially available LC probe heads, which have been designed, and are most efficient, for inverse detection. This drawback turns into an advantage for indirect detection through 1 H NMR enabling highly sensitive analysis of samples enriched with 13 C. Figure 8 shows, as an example from plant secondary product biosynthesis, the HPLC-1 H NMR spectra of 13 C-enriched trace phenylpropanoic acids in extracts of Anigozanthos root cultures. [213 C]Dihydrocinnamic acid was administered as a precursor and the root extract was analysed by HPLC1 H NMR coupling (Schmitt and Schneider, 1999). Dihydro-p-coumaric acid, p-coumaric acid, and ferulic acid were identified and the occurrence of [2-
13 C]-isotopomers
of each compound was demonstrated by 1 H-13 C spin-spin coupling (1 JH-2–C-2 = 129 Hz in [2-13C]dihydro-p-coumaric acid; 1 JH-2–C-2 ∼ 160 Hz in [2-13 C]p-coumaric and [2-13 C]ferulic acid) in addition to unlabelled isotopomers with 3J H-2–H-3 only. Integration of the central signal of H-2 of the unlabelled isotopomers and the 1 H -13 C coupling satellite signals of the [2-13C]-isotopomers revealed decreasing 13 C enrichment in the biosynthetic sequence dihydrocinnamic acid to dihydro-p-coumaric acid to p-coumaric acid to ferulic acid (further intermediates not shown), which is due to increasing dilution by the endogenous metabolites. In these experiments the analysis of phenylpropanoids was performed on
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Figure 5. 13 C NMR partial spectra (125 MHz) of hydroxyanigorufone. A: Enhanced signals indicate selective 13 C enrichment of C-6 and C-7 (from [1-13 C]cinnamic acid as a precursor) and C-5 and C-8 (from [2-13 C]dihydrocinnamic acid) into hydroxyanigorufone in Anigozanthos root cultures (Schmitt and Schneider, 1999). The contribution of [1-13 C]cinnamic acid gives rise to the [6-13 C]-isotopomer (central singlet in the extension of the pseudoquintet signal of C-6, marked by ), the [7-13 C]-isotopomer (central singlet in the extension of the pseudoquintet signal of C-7, marked by ), and the doublets of the [6,7-13 C2 ]-isotopomer (2 JC-6–C-7 = 2.7 Hz, marked by ). Isotopomers labelled in positions C-5 and/or C-8 are derived from [2-13 C]dihydrocinnamic acid as a precursor. The extended signals of these carbons show central singlets of the [5-13 C]-isotopomer (marked by ) and [8-13 C]-isotopomer (marked by ). The [5,8-13 C2 ]-isotopomer (marked by ) may also contribute to these singlets, but long-range coupling through four bonds is not detectable in the spectrum. Doubly labelled [5,7-13 C2 ]- and [6,8-13 C2 ]-isotopomers must be of mixed origin from [1-13 C]cinnamic acid and [2-13 C]dihydrocinnamic acid. They can be recognized in the extension of signals of each labeled carbon by the three-bond coupling (3 JC-5–C-7 = 5.3 Hz, marked by , 3 JC -6–C -8 = 5.3 Hz, marked by ). B: Spectrum of natural abundance hydroxyanigorufone.
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Figure 6. Comparison of signal-to-noise ratios (S:N) of 13 C and 1 H signals of the methyl group of [S-methyl-13 C]methionine (100 µg, 600 µl MeOH-d4 /D2 O 1:1) measured with direct and indirect acquisition techniques. The total measuring time was 7.5 min in each case. A: 1 H NMR spectrum obtained with a TXI CryoProbeTM at 500 MHz (acquisition time: 10.2 s, rel. delay: 1 s). B: 13 C NMR spectrum obtained with a CryoProbeTM at 125 MHz (acquisition time = 1.42 s, rel. delay: 2 s). C: 1 H NMR spectrum obtained with a broadband probe at 400 MHz (acquisition time: 10.2 s, rel. delay: 1 s). D: 13 C NMR spectrum obtained with a broadband probe at 100 MHz (acquisition time = 1.42 s, rel. delay: 2 s). Identical parameters were used for the processing of 1 H and 13 C NMR spectra obtained from the two spectrometers.
unpurified compounds present in quantities of a few micrograms or even less. Indirect detection of 2 H in labelled metabolites Missing 1 H signals and 1 H-2 H spin-spin coupling Direct 2 H NMR is a useful tool in biosynthetic studies and has been employed to elucidate many precursorproduct relationships (Staunton and Hailes, 1996). Due to its low natural abundance, 2 H is suitable for selective labelling and is of special importance when seeking to unravel the stereospecificity of metabolic processes. However, it suffers from low sensitivity
Figure 7. 1 H NMR partial spectrum (500 MHz) of desulphated 4-methylthiobutylglucosinolate (ds-4MTB) biosynthetically obtained from [U-13 C]methionine. The coupling satellites (1 JH-6–C-6 = 139 Hz), marked with indicate 13 C enrichment of the S-methyl group. The small coupling constants (3 JH-6–C-5 = 4.1 Hz) in the same signals are due to long-range proton-carbon coupling and indicate 13 C enrichment of C-5. Together with the 13 C NMR data (Figure 2) this proves the occurrence of a [3,4,5,6-13 C4 ]-isotopomer of ds-4MTB. The central singlet is due to [S-methyl-12 C]-isotopomers of ds-4MTB.
and therefore analysis of low-concentration samples requires extensive acquisition time. Indirect detection of 2 H by 1 H- or 13 C NMR is a suitable alternative for some applications. The position of 2 H in labelled compounds can be detected using information from missing 1 H resonances. As for other nuclei, detection of 2 H is possible indirectly via spin-spin coupling. Both of these aspects will be discussed using examples from plant secondary products. Spin-spin coupling between 1 H and 2 H follows the same mechanism as 1 H-1 H coupling and coupling between other magnetic nuclei. The coupling constants JH-D can be calculated from JH-H and the gyromagnetic ratios of 1 H and 2 H. Owing to the relation JH-D =JH-H /6.514, spin-spin coupling between hydrogen and deuterium nuclei is relatively small and, in 1 H NMR spectra these 1 H signals appear as broad singlets. The large coupling constant JH-2–H-3 =16 Hz of phenylpropanoic acids represents a suitable example to demonstrate, by means of an 1 H NMR spectrum of an [2-2 H]-isotopomer, both 1 H-2 H spin-spin coupling
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Figure 8. HPLC-1 H NMR partial spectra of isotopomeric mixtures of [2-13 C]phenylpropanoic acids biosynthetically obtained from [2-13 C]dihydrocinnamic acid. The occurrence of [2-13 C]-isotopomers is indicated by the large coupling constants JH-2–C-2 . The different ratio of [2-13 C]- and unlabelled isotopomers (central doublets) is due to increasing dilution with unlabelled metabolites during the biosynthetic process (Schmitt and Schneider, 2001). A: [2-13 C]Dihydro-p-coumaric acid. B: Mixture of [2-13 C]- and unlabelled p-coumaric acid. C: Mixture of [2-13 C]- and unlabelled ferulic acid.
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Figure 9. 1 H NMR partial spectra (500 MHz) of cinnamic acid. A: Mixture of [2-2 H]cinnamic acid and unlabeled cinnamic acid. The broad signal of H-3 (JH-3–D-2 = 2.5 Hz not resolved) indicates the [2-2 H]-isotopomer. The diminished doublets of H-2 and H-3 are due to unlabelled cinnamic acid. B: Unlabelled cinnamic acid.
and missing 1 H signals. In this 1 H NMR spectrum (Figure 9A), the signal for H-2 represents only the minor nondeuterated isotopomer. The resonance of H3 of [2-2 H]cinnamic acid appears as a broad singlet in between the doublet of the unlabelled isotopomer. Another example from phenylpropanoid metabolism in Anigozanthos root cultures can be used to demonstrate dissection of an isotopomeric mixture derived from a precursor bearing a completely labelled 13 C2 H -methylene group with an addtitional 2 H in 2 vicinal position. Ferulic acid isotopomers, biosynthetically formed from [2,2,3-2H,2-13C]dihydrocinnamic acid, were analysed by means of HPLC-1 H NMR coupling (Schmitt and Schneider, 1999, 2001). The spectrum shown in Figure 10, in addition to endogenous ferulic acid, indicated a mixture of three 2-13 Clabelled isotopomers with one or two 2 H in the side chain. The monodeuterated isotopomers are recognized from the coupling pattern. The singlet for H-3 (δ 7.62), broadened due to a deuterium-induced shift of 3 Hz partially overlapping with the right line of the H3 doublet of the undeuterated isotopomers, is evidence of the [2-2H,2-13 C]-isotopomer. The occurrence of the [3-2 H,2-13C]-isotopomer is shown by the doublet of H-2 (JH-2–C-2 = 162 Hz), which is also subject to an isotope shift of 3 Hz to a higher field. The two lines of the doublet are broadened because of 1 H-2–2 H-3 spin-spin coupling. The [2,3-2H2 ,2-13C]-isotopomer is not detectable from the coupling pattern or isotope-
induced shifts but only indirectly by the missing contribution to the 1 H signals of the ferulic acid side chain. While the three signals (δ 6.88, d, 8.1 Hz; 7.14, d, 8.1 Hz; 7.23, s) of the aromatic ring are due to contributions of all isotopomers (100% integral area each), the integral areas of the side chain resonances are diminished. The difference represents the proportion of the doubly deuterated isotopomer. A [2-13 C]isotopomer, which has completely lost all three deuterium atoms of the precursor, was not detectable from this HPLC-1 H NMR spectrum. From this labelling experiment and the isotopomer analysis, a reversible interconversion between phenylpropanoids and dihydrophenylpropanoids in Anigozanthos was proposed, operated by a dehydrogenation/hydrogenation mechanism. Deuterium-induced shift While enhanced signals and spin-spin coupling patterns represent the most important sources of information that can be derived from the NMR spectra of multiple labelled compounds, changes of chemical shifts can be highly informative as well. The upfield shift of resonances of contiguous 13 C atoms as compared with 13 C atoms attached to 12 C is due to their altered chemical surroundings. This is caused by an isotope effect, due to the higher mass of the adjacent nucleus, which can also be observed for neighbouring isotopes other than 13 C. The detection of 18 O by
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Figure 10. HPLC-1 H NMR partial spectra of ferulic acid biosynthetically obtained from [2,2,3-2 H,2-13 C]dihydrocinnamic acid (Schmitt and Schneider, 1999). A: Mixture of two monodeuterated, a doubly deuterated and an unlabelled isotopomer. The broad singlet of H-3 and the broad doublet of H-2 are due to the monodeuterated [2-2 H,2-13 C]- and [3-2 H,2-13 C]-isotopomers, respectively. 13 C in position 2 of these isotopomers is indicated by the large coupling constant of the signal of H-2 (JH-2–C-2 = 162 Hz) and an isotope-induced shift of both H-2 and H-3. The [2,3-2 H2 ,2-13 C]-isotopomer is detectable only indirectly by the missing contribution to the 1 H signals of the ferulic acid side chain (for details see text) and the unlabelled isotopomer from normal ring and double bond signals. B: Unlabelled ferulic acid.
NMR spectroscopy is based exclusively on isotopeinduced shifts because this is not a magnetic nucleus by itself (Risley, 1996). Some aspects of the application of deuterium-induced shifts of 13 C resonances in biosynthetic studies are described below. Deuterium induces a shift to the signal of the 13 C atom to which it is attached by 0.3 to 0.6 ppm to a higher field in comparison with the non-deuterated compound. The extent of this α-shift is additive for each 2 H. In the case of 2 H being two bonds distant from 13 C, a smaller effect of up to 0.1 ppm (β-shift) can frequently be observed. Deuterium atoms alpha to a 13 C not only generate an isotope shift but, due
to spin-spin coupling, also change the multiplicity of the adjacent 13 C. The most commonly known example is CDCl3 , which is widely used as a solvent in NMR spectroscopy. It appears as a triplet in the 13 C NMR spectrum. Correspondingly, CD2 results in a quintet and CD3 in a septet. Deuterium-induced shifts have been broadly used in labelling experiments (Garson and Staunton, 1979). Recent examples of this technique have been described by Arigoni et al. (1999). Figure 11 demonstrates the suitability of 2 H-induced shifts, gaining information on the isotopomer pattern of phenylphenalenones biosynthesized from [2,2,3-2H,2-
42
Figure 11. 13 C NMR partial spectra (125 MHz) of hydroxyanigorufone. A: Enhanced signals indicate selective 13 C enrichment of C-5 (marked by ) and C-8 (marked by ). The extension of the signal of C-8 exhibited a triplet shifted for 0.3 ppm to a higher field (2 H-induced α-shift), indicating 2 H attached to 13 C-8 ([8-2 H,8-13 C]-isotopomer). The extension of the signal of C-5 revealed a small signal of the [4-2 H,5-13 C]-isotopomer which, due to the 2 H-induced β-shift, appears 0.1 ppm upfield of the large singlet of the [5-13 C]-isotopomer. The enhancement of 13 C signals of C-5 and C-8, together with additional 13 C signals owing to 2 H-induced shifts indicated partial retention of 2 H specifically in positions 4 and 8, and complete loss of two methylene group-derived 2 H atoms in position 5, during biosynthetic incorporation of [2,2,3-2 H,13 C]dihydrocinnamic acid into hydroxyanigorufone in Anigozanthos root cultures (Schmitt and Schneider, 1999). The occurrence of [5,8-13 C2 ]-, [8-2 H,5,8-13 C2 ]-, [4-2 H,5,8-13 C2 ]-, and [4,8-2 H2 ,5,8-13 C2 ]-isotopomers cannot be excluded but was not directly detectable in the spectrum. B: Spectrum of natural abundance hydroxyanigorufone.
13 C]dihydrocinnamic acid.
The 2 H-induced α-shift of the signal of C-8, which appeared as a triplet at δ 132.2, was evident from the occurrence of an [8-13C,82 H]-isotopomer, indicating (partial) retention of 2 H from [2,2,3-2H,2-13C]dihydrocinnamic acid in position 8 of hydroxyanigorufone. In the same spectrum, the signal of the 13 C-enriched C-5 appears as a large singlet at δ 127.7 arising from the [5-13C]-isotopomer
and a small singlet, β-shifted 0.1 ppm to a higher field. The missing triplet of an α-shifted C-5 indicated the lack of a [5-13C,5-2 H]-isotopomer and was interpreted as demonstrating the complete loss of 22 H of [2,2,3-2H,2-13 C]dihydrocinnamic acid en route 2 to hydroxyanigorufone.
43 Conclusions and future developments Despite the astonishingly successful use of molecular methods in recent years to elucidate metabolic processes in plants, labelling techniques are still indispensable tools for elucidating pathways of secondary product biosynthesis and will continue to be so in future. Various NMR spectroscopic techniques have been developed and employed in biosynthetic studies, and the sensitivity of NMR has been improved dramatically over recent years. Nowadays trace amounts of metabolites and amounts at even lower microgram levels are accessible to 1 H NMR analysis. In contrast, the detection limit of nuclei such as 13 C and 2 H, which are of special importance for biosynthetic studies, is much higher even if 99% enriched samples are being used. In many cases, indirect detection of these nuclei, employing a sensitive one-dimensional 1 H measurement, can serve as an alternative to heteronuclear correlation spectroscopy. As discussed for phenylpropanoic acids in this review, HPLC-NMR spectroscopy is a sensitive tool for the detection of labelled intermediates at the submicrogram scale. In addition, HPLC-NMR has been demonstrated to be feasible for 13 C/2 H isotopomer analysis and estimation of deuterium loss and/or retention in biosynthetic studies. The recent introduction of CryoProbeTM technology with its unequalled sensitivity has opened new perspectives in NMR spectroscopy and has impact also on labeling studies. The combination of HPLC with CryoProbeTM technology will probably enable direct online-detection of 13 C and further decrease the limit for indirect detection methods.
Acknowledgements The Deutsche Forschungsgemeinschaft, the MaxPlanck Society, and the Fonds der Chemischen Industrie are thanked for their financial support.
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