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From the Elucidation of Biosynthesis by Isotopic Spectrometry Group, Interdisciplinary Chemistry: Synthesis, Analysis, Modeling, CNRS–University of Nantes UMR6230, F-44322 Nantes, France andthe Institute of Applied Radiation Chemistry, Faculty of Chemistry, Łodź University of Technology, ul. Stefana Żeromskiego 116, 90-924 Łódź, Poland
From the Elucidation of Biosynthesis by Isotopic Spectrometry Group, Interdisciplinary Chemistry: Synthesis, Analysis, Modeling, CNRS–University of Nantes UMR6230, F-44322 Nantes, France and
From the Elucidation of Biosynthesis by Isotopic Spectrometry Group, Interdisciplinary Chemistry: Synthesis, Analysis, Modeling, CNRS–University of Nantes UMR6230, F-44322 Nantes, France and
To whom correspondence should be addressed: Elucidation of Biosynthesis by Isotopic Spectrometry Group, CEISAM, UMR6230, CNRS–University of Nantes, 2 rue de la Houssinière, BP92208, Nantes 44322, France. Tel.: 332-5112-5701; Fax: 332-5112-5712
From the Elucidation of Biosynthesis by Isotopic Spectrometry Group, Interdisciplinary Chemistry: Synthesis, Analysis, Modeling, CNRS–University of Nantes UMR6230, F-44322 Nantes, France and
* This work was supported by grants from the French Research Ministry (to K. M. R.) and the CNRS (to R. J. R.). The authors declare that they have no conflicts of interest within the contents of this article. 2 Isotopic fractionation is the selection of one isotopomer versus another during a physical or (bio)chemical process that leads to a non-statistical distribution of isotopes in the population of isotopomers within the final product and the residual substrate. 4 The notations CXA, CXASP, CXCIT, CXO, CXOX, CXT, and CXN indicate the relevant carbon atom in acetate (A), aspartate (Asp), citrate (CIT), l-ornithine (O), oxaloacetate (OX), tropine (T), or nicotine (N), respectively. 5 Δ is the difference in δ13Ci (‰) values between two carbon positions. Thus, for example, ΔC4X-C2X means the difference between the δ13Ci of the C4 position of compound X and the δ13Ci of the C2 position of compound X. For convenience, Δ is always attributed a positive sign and the difference described as “relatively depleted” or “relatively enriched.”
During the biosynthesis of natural products, isotopic fractionation occurs due to the selectivity of enzymes for the heavier or lighter isotopomers. As only some of the positions in the molecule are implicated in a given reaction mechanism, position-specific fractionation occurs, leading to a non-statistical distribution of isotopes. This can be accessed by isotope ratio monitoring 13C NMR spectrometry. The solanaceous alkaloids S-(−)-nicotine and hyoscyamine (atropine) are related in having a common intermediate, but downstream enzymatic steps diverge, providing a relevant test case to: (a) elucidate the isotopic affiliation between carbon atoms in the alkaloids and those in the precursors; (b) obtain information about the kinetic isotope effects of as yet undescribed enzymes, thus to make predictions as to their possible mechanism(s). We show that the position-specific 13C/12C ratios in the different moieties of these compounds can satisfactorily be related to their known precursors and to the known kinetic isotope effects of enzymes involved in their biosynthesis, or to similar reaction mechanisms. Thus, the pathway to the common intermediate, N-methyl-Δ1-pyrrolinium, is seen to introduce similar isotope distribution patterns in the two alkaloids independent of plant species, whereas the remaining atoms of each target compound, which are of different origins, reflect their specific metabolic ancestry. We further demonstrate that the measured 13C distribution pattern can be used to deduce aspects of the reaction mechanism of enzymes still to be identified.
Members of the plant family Solanaceae produce a range of alkaloids derived from l-ornithine or l-lysine, several of which are exploited for their recreational and/or pharmaceutical properties, despite their toxicity. S-(−)-Nicotine, the principle alkaloid of Nicotiana tabacum L. and related species has a long history of use for recreational and ethnopharmaceutical applications. Considered a component of the defensive chemical array of the genus Nicotiana (
), nicotine was widely exploited as an insecticide, eventually being phased out as less toxic neonicotinoids became available. Within a closely related group of plants occurs the tropane alkaloids, of which atropine, hyoscyamine, and scopolamine (hyoscine) have a history of use in mystical ceremonies (
). These compounds are now extensively used in modern medicine, atropine and hyoscyamine as mydriatic agents, scopolamine as an anti-emetic for controlling travel sickness.
For both these alkaloid types, the biosynthetic pathways have been extensively investigated at the metabolic, enzymatic, and genetic levels (
). Despite superficial structural dissimilarities, the pyrrolidine moiety of nicotine and tropine (of which atropine or hyoscyamine is the tropoyl ester) have a common precursor in l-ornithine, and share four enzymatic steps to the intermediate N-methyl-Δ1-pyrrolinium salt (Fig. 1). Thereafter, a series of diverse enzymes intervene to condense this intermediate with different groups to render more elaborated structures. A number of the enzymes involved in this pathway are well described: others remain poorly understood.
FIGURE 1.Schematic representation of the biosynthetic pathway to nicotine and tropine indicating the common and unique steps. Carbon atoms on the final compounds are numbered according to the IUPAC nomenclature and this numbering is used to indicate the carbon positions from which they are derived in the precursor molecules. Numbers in normal bold font refer to tropine; numbers in italic bold font refer to nicotine. The enzymes indicated are: a, ornithine decarboxylase (EC 4.1.1.17); b, putrescine N-methyltransferase (EC 2.1.1.53); c, N-methylputrescine oxidase (EC 1.4.3.6); d, undescribed activity, probably spontaneous (
Nicotine synthase: an enzyme from Nicotiana species which catalyzes the formation of (S)-nicotine from nicotinic acid and 1-methyl-Δ′-pyrrolinium chloride.
In both these alkaloids, the reactions by which N-methyl-Δ1-pyrrolinium salt is further elaborated has resisted a number of attempts at characterization, despite its central role in the pathway. In the case of S-(−)-nicotine, a stereospecific decarboxylative condensation with a derivative of nicotinate leads to S-(−)-nicotine. An extremely weak “nicotine synthase” has been reported (
Nicotine synthase: an enzyme from Nicotiana species which catalyzes the formation of (S)-nicotine from nicotinic acid and 1-methyl-Δ′-pyrrolinium chloride.
), no details of the nature of this key enzyme nor of its mechanism are available.
In a parallel fashion, condensation with the C2 position of acetoacetate (probably activated as the CoA thioester) followed by decarboxylation (probably spontaneous) leads to tropinone (
). Similarly, despite many efforts, the enzymology of the conversion of N-methyl-Δ1-pyrrolinium to tropinone remains elusive. Tropine is produced by a stereospecific oxidoreductase (
In such cases, where enzymological approaches have proved inadequate, alternative means to probe the biochemistry are required. The development of molecular biological approaches has been fruitful (
) but is challenging where the enzymology is completely unknown. An alternative “retro-biosynthetic” approach involves the measurement and interpretation of the natural fractionation in 2H or 13C isotopes during the course of biosynthetic reactions. During the metabolic processes by which natural products are biosynthesized, isotopic fractionation
Isotopic fractionation is the selection of one isotopomer versus another during a physical or (bio)chemical process that leads to a non-statistical distribution of isotopes in the population of isotopomers within the final product and the residual substrate.
occurs, due to the sensitivity/insensitivity of the enzymes involved to the presence/absence of one or more heavy atoms in the reaction center. This is the kinetic isotope effect (KIE).
As a result, the position-specific isotope ratio in natural products is non-statistically distributed among the different positions. Of especial interest in the context of biosynthesis are the position-specific 13C/12C ratios, as these reflect the carbon skeleton(s) already present in the precursor molecule(s) and the biochemical processing each position has undergone at different steps in the pathway. Hence, a study of the position-specific 13C/12C ratios should enable the interpretation of the values observed in terms of the known biochemistry (
for an expanded discussion of these), when suitable conditions for isotope ratio monitoring by 13C NMR (irm-13C NMR) spectrometry applied to medium-sized molecules have been established.
) applying this tactic to a newly discovered natural product, we were able to propose a credible biosynthetic route based on the observed pattern of the site-specific 13C/12C ratios. However, in that study, no direct underlying biological chemistry is yet available to substantiate the proposed reaction scheme. We have now turned our attention to the natural solanaceous alkaloids, S-(−)-nicotine and tropine, to establish the degree to which observed position-specific 13C/12C ratios can be related to known biochemistry to test three principal hypotheses.
1)
Can we predict that similar reaction schemes should lead to similar isotope patterns? Isotopic fractionation should be associated with the KIEs intrinsic to the reaction mechanisms of the implied enzymes, rather than to the plant species involved. This can be tested by comparing the five carbon atoms derived from the common intermediate, N-methyl-Δ1-pyrrolinium salt, that these alkaloids have in common in their biosynthetic schemes (Fig. 1).
2)
Can we show that the differing origins of the other carbon atoms present that are derived from dissimilar origins, three in tropine and five in S-(−)-nicotine, do not display any particular common features?
3)
Can the characteristics of the position-specific 13C/12C ratios help indicate mechanistic features of the enzymes that are as yet undescribed for these pathways?
Results
Suitable conditions were established (see “Experimental Procedures”) for the acquisition of 13C NMR spectra amenable to the calculation of position-specific 13C/12C ratios. Representative spectra are illustrated in Fig. 2.
FIGURE 2.13C NMR spectra acquired under quantitative conditions of (A) tropine and (B) S-(−)-nicotine. Carbon atom displacements are indicated. See ”Experimental Procedures“ for the acquisition conditions.
Subjecting these spectra to total line shape curve fitting provided the area under the peak for each carbon atom i. From this, the molar fraction was calculated, which gives the extent to which the 13C/12C ratios diverge from a statistical distribution. By combining this with the global value for the whole molecule, δ13Cg (‰), i.e. the deviation of the carbon isotopic ratio of the whole molecule relative to that of the international standard Vienna Pee Dee Belemnite, (V-PDB), values for the positional isotopic distribution, δ13Ci (‰), can be calculated (Table 1). The advantage of expressing isotopic deviation as δ13Ci (‰) is that all molecular targets are related back to the same defined international reference.
TABLE 1Data obtained by irm-13C NMR for the 13C distribution in natural tropine or natural S-(−)- nicotine
TABLE 1Data obtained by irm-13C NMR for the 13C distribution in natural tropine or natural S-(−)- nicotine
aMean of 10 spectral acquisitions obtained as two separate preparations from the same sample (5 spectra per preparation).
bAs these are independent samples, it is not appropriate to use a mean value. Rather, so as to give an indicative value for the text discussion, an average of the two samples each of nicotine and of tropine is given.
cfi/Fi is the positional isotopic distribution, i.e. the variation of the δi from the statistical distribution (see experimental Procedures for details).
dMean of five spectral acquisitions. A field-homogenizing correction factor is applied for the data for S-(−)-nicotine. (42).
For S-(−)-nicotine, the range of values obtained for the two samples is −17.3 to −52.3‰ and for tropine the range is −9.1 to −52.3‰, with standard deviations acceptable for irm-13C NMR spectrometry (∼1‰; range 0.7 to 2.7‰). These δ13Ci (‰) values are within the typical range for natural products from plants using a C3 metabolism (
). Their relative position-specific distributions are illustrated in Fig. 4, which shows the extent to which each δ13Ci (‰) value differs from the δ13Cg (‰) value.
Discussion
Following the successful development of the required protocols, it proved feasible to obtain position-specific 13C/12C ratios for all carbon atoms of the two target alkaloids. Thus, it is possible to exploit these to probe a number of aspects of the biosynthesis of these natural products.
To What Extent Are Isotope Ratios Determined by the Primary Precursor Molecules Giving Rise to the N-Methyl-Δ1-pyrrolinium Salt?
In S-(−)-nicotine the carbon atoms that compose the pyrrolidine ring, C2′N,
). Looking further back into the metabolic precursors, these carbons can be traced to citric acid, a component of the Krebs cycle, then to oxaloacetate and acetyl-CoA. Therefore, it can be deduced that the C2OX and C3OX of oxaloacetate become the C3′N and C2′N, respectively, in nicotine. Similarly, the C1A and C2A of acetate are incorporated via citrate into the C5′N and C4′N, respectively.
FIGURE 3.Schematic representation of the biosynthetic pathway to N-methyl-Δ1-pyrrolinium salt from primary precursors. Carbon atoms on the final compounds are numbered according to the IUPAC nomenclature and this numbering is used to indicate the carbon positions from which they are derived in the precursor molecules. Numbers in normal bold font refer to tropine; numbers in italic bold font refer to nicotine. Values for δ13Ci (‰) of citrate acting as a metabolic intermediate taken from Ref.
For tropine, by a similar argument, it can be deduced that the C2OX and C3OX of oxaloacetate become the C6T and C5T, respectively, whereas the C1A and C2A of acetate are incorporated into the C1T and C7T, respectively (Fig. 3). This ancestry will play a significant role in determining the position-specific 13C/12C ratios observed, as they will either essentially be retained, or will be modified due to KIEs in the enzymes involved in the pathway.
Because tropine is spectrally the simpler of the two target molecules, its isotope pattern will be analyzed first. As can be seen from Fig. 2A, the pairs of carbons showing the same degree of deshielding, C2T + C4T, C1T + C5T, and C6T + C7T result in coincident peaks in the 13C NMR spectrum. The impact of this on interpreting the 13C/12C ratios measured is, however, minimal, as the C1T + C5T and C6T + C7T pairs of positions are biosynthetically equivalent. Not only is N-methyl-Δ1-pyrrolinium a symmetrical molecule, but it is derived from the symmetrical molecule, putrescine. As there is no evidence of metabolic tunneling between l-ornithine and N-methylputrescine (
Ellis B.E. Kuroki G.W. Stafford H.A. Progress in the genetic engineering of pyridine and tropine alkaloid biosynthetic pathways of Solanaceous plants. Plenum Press,
New York1994: 1-33
), it can be presumed that methylation of putrescine can occur on either the 1- or 4-amino group with equal probability. Hence, the (C2O;C5O) and (C3O;C4O) positions in l-ornithine become indistinguishable in putrescine, hence in N-methylputrescine. Furthermore, tautomerism occurs around the quaternary nitrogen in N-methyl-Δ1-pyrrolinium (
), so no distinction is made as to at which carbon the substitution that leads to the elaborated molecules takes place.
Considering the history of the four carbon atoms derived from N-methyl-Δ1-pyrrolinium, the C6T and C7T have undergone no bond-forming reaction since the formation of citrate (Fig. 3). As isotopic fractionation is associated with bond forming and breaking, so the opposite is true: carbon atoms not undergoing reactions are likely not to be significantly fractionated. Hence, at these positions the chances for isotopic fractionation are minimal, and this is reflected in these positions having a relatively high δ13Ci (‰) value: −11.9‰ (Fig. 4A, Table 1). The C(1 + 5)T is, however, relatively depleted, having a Δδ13Ci (‰)
Δ is the difference in δ13Ci (‰) values between two carbon positions. Thus, for example, ΔC4X-C2X means the difference between the δ13Ci of the C4 position of compound X and the δ13Ci of the C2 position of compound X. For convenience, Δ is always attributed a positive sign and the difference described as “relatively depleted” or “relatively enriched.”
value 10.4‰ lower than the C(6 + 7)T. This can be explained by two contributing factors. The C(1 + 5)T is derived from the carboxyl group (C1A) of acetyl-CoA, which in eukaryotes is enriched relative to the methyl group in conditions in which commitment of acetyl-CoA to synthesis is significant (
Biosynthetic control of the natural abundance of carbon 13 at specific positions within fatty acids in Saccharomyces cerevisiae: isotopic fractionation in lipid synthesis as evidence for peroxisomal regulation.
). The degree of this varies. In lipids of eukaryotic origin (Saccharomyces cerevisiae) carbon positions derived from the C1A are enriched relative to those from the C2A by ∼5‰ (
Biosynthetic control of the natural abundance of carbon 13 at specific positions within fatty acids in Saccharomyces cerevisiae: isotopic fractionation in lipid synthesis as evidence for peroxisomal regulation.
The intramolecular 13C-distribution in ethanol reveals the influence of the CO2-fixation pathway and environmental conditions on the site-specific 13C variation in glucose.
). In tropine, the impact of this is, however, compensated by the relative depletion at the C2 of citrate (C2C), and, based on estimated values for citrate where it is acting as a metabolic intermediate (
), a Δδ13Ci = [C(1 + 5)T–C(6 + 7)T] value of ∼9‰ can be deduced. This is close to the observed differences in tropine.
FIGURE 4.Position-specific 13C/12C ratios expressed as Δgδ13Ci (‰) for two samples each of (A) tropine and (B) S-(−)-nicotine. See ”Experimental Procedures“ for the acquisition conditions and Table 1 for absolute values. In these graphs the notation Δgδ13Ci (‰) indicates the difference at site i of δ13Ci from the mean overall value of δ13Cg (‰): i.e. Δgδ13Ci (‰) = (δ13Ci-δ13Cg).
An addition potential source of fractionation at the C(1 + 5)T value (but not the C(6 + 7)T value) is the two reactions involved in the final steps of the common pathway forming N-methyl-Δ1-pyrrolinium, in both of which bond breaking/creation occurs. The ring closure reaction (Fig. 3, step d), in which a C-N bond is formed, is considered spontaneous (
), the two compounds being in equilibrium. This step is unlikely to contribute a significant isotope fractionation as, even if there is an equilibrium isotope effect, the compound in either open or ring-closed structure is fully committed to the pathway. The previous step, in which a C-N bond is broken and a C=O bond is formed is catalyzed by the enzyme N-methylputrescine oxidase (EC 1.4.3.6) (Fig. 3, step c). An intramolecular normal KIE associated with amine oxidase have been reported (
), so a fractionation during the deamination of N-methylputrescine would select in favor of isotopomers containing 12C at the C1T position, because it involves an sp3 → sp2 conversion (
). This would lead to a 13C depletion of the C-atom. However, as the commitment of N-methylputrescine to tropine biosynthesis is very high, this intermediate being a dedicated component of the pathway, isotope fractionation due to this enzyme is unlikely to be significant (see Ref.
These arguments apply equally to the equivalent four carbon atoms of the pyrrolidine ring in S-(−)-nicotine biosynthesis (C2′N, C3′N, C4′N, and C5′N). In this analyte, however, these four carbons are resolved in the 13C NMR spectrum (Fig. 2B), which enables a more detailed interpretation (see below). Once again, the C3′N and C4′N, derived from citrate essentially without undergoing further reaction, are enriched relative to the C5′N and C2′N, which have undergone deaminative oxidation and C-N bond formation, respectively (Fig. 3). Furthermore, the relative depletion at the C(2′+5′)N relative to the C(3′ + 4′)N of ∼7‰ is in the same sense and similar size to this comparison in tropine (∼10‰). Hence, it can be concluded, (i) that the values obtained in the common compound N-methyl-Δ1-pyrrolinium can be explained on the basis of the values in their distal precursors, and (ii) that this relationship is dictated primarily by the metabolic pathway, and is relatively independent of the plant species involved.
To What Extent Are Isotope Ratios Determined by the Primary Precursor Molecules Giving Rise to the Nicotinic Acid-derived Moiety of S-(−)-Nicotine?
Nicotinic acid in plants is considered to be synthesized primarily from aspartate plus dihydroxyacetone phosphate/glyceraldehyde-3-phosphate (
). Considering that nicotinate is a predominant source of precursor for S-(−)-nicotine, which is a major sink for carbon, then some characteristics can potentially be traced back to the precursor pools. C2N and C3N are derived from the C1ASP and C2ASP, respectively, whereas the C4N, C5N, and C6N are from dihydroxyacetone phosphate/glyceraldehyde-3-phosphate (Fig. 5). (Note that the interconversion of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate in primary metabolism means that the C4N and C6N are of equivalent origin.) During the synthesis of nicotinic acid, all these positions are involved in reactions, although only the C3N, C4N, and C6N positions are involved in bond formation. Again, therefore, it is these three positions that might be expected to show isotopic fractionation.
FIGURE 5.Schematic representation of the biosynthetic pathway to S-(−)-nicotine and tropine indicating the origins of the carbon atoms. Those carbon and nitrogen atoms involved in the formation of bonds and therefore the more likely to undergo isotope fractionation are indicated in italics. Carbon atoms on the final compounds are numbered according to the IUPAC nomenclature. For all other compounds, the numbering indicates the position in the analyte in which the given carbon will be found. Numbers in normal bold font refer to tropine; numbers in italic bold font refer to nicotine.
First, let us consider the three carbon positions showing the least relative depletion in the pyridine ring, C2N, C4N, and C5N. The C5N (δ13Ci = −26.7‰) is from the keto group of dihydroxyacetone phosphate and undergoes an sp2 → π transition in the formation of quinolinic acid. The C4N is the most enriched position (δ13Ci = −18.9‰) and, although this carbon is involved in the formation of a C–C bond with the C3N in the formation of quinolinic acid and an sp3 → π transition, its bonding thereafter is unchanged. In both of these positions, it is apparent that negligible fractionation has occurred, at least relative to the other three positions in the pyridine ring. The C2N is involved in the decarboxylation of quinolinic acid and has a δ13Ci ∼15‰ and ∼7‰ depleted relative to the C4N and C5N, respectively. Although decarboxylation can take place with negligible 13C KIE on the α-carbon (
), it would appear that fractionation is occurring here and that decarboxylation is probably a kinetically regulating step.
The C3N at δ13Ci ≈ −42.8‰ is ∼8‰ depleted relative to the mean for the pyridine ring. This may be due to two additive effects. The first is the origin of this carbon: the C3OX of oxaloacetate. This carbon is estimated to be markedly depleted (
) by ∼11‰ relative to the other carbons of oxaloacetate (the immediate precursor of l-aspartate). Furthermore, C3N is involved in two bond-forming and one bond-breaking reaction, each of which may lead to selection against 13C in this position (see below), hence to relative depletion.
However, the most depleted position is the C6N, which at δ13Ci = −52‰ is ∼17‰ depleted relative to the mean for the pyridine ring. This position is involved in the formation of the C=N bond created when it is joined to the imino group of iminoaspartate by quinolinate synthase (EC 2.5.1.72) (Fig. 5, boxed positions in the first row). Two mechanisms have been proposed for this enzyme, which differ essentially in the order in which the two bonds are formed: there is no experimental evidence to favor one over the other (
). However, in both cases the formation of the new bond involves the nucleophilic attack of either -NH2 or =NH2+ on a >C=O group. The degree of electrophilicity of the >C=O is considered likely to have a major influence on the 13C/12C ratio at this atom (
). Based on our data, a strong normal KIE is indicated.
To What Extent Do the Reactions Involving the Conversion of N-Methyl-Δ1-pyrrolinium to Tropinone Influence the Observed Isotope Ratios in Tropine?
The elaboration of N-methyl-Δ1-pyrrolinium to tropinone involves the introduction of the three carbons C2T, C3T, and C4T and the creation of two new C–C bonds at the C1T and C5T positions (Fig. 5). These three carbons are derived from acetate, via acetoacetyl-CoA and 4-(1-methyl-2-pyrrolidinyl)-3-oxobutyrate (probably as the CoA thioester) (
). On the basis of the above discussion of the relative enrichments in acetyl-CoA, the C3T, derived from the carboxyl in acetoacetyl-CoA, might be expected to be slightly enriched relative to the C(2 + 4)T if no other factors come into play. In fact, the Δδ13Ci = [(C3T)–(C(2 + 4)T)] of ∼13‰ implies a significant KIE associated with the formation of this moiety. This greater relative enrichment is, however, consistent with the characteristic strong 13C KIE associated with aldol reactions of acetyl-CoA in the biosynthesis of natural products when there is a high commitment of acetyl-CoA (
). In these cases, a large depletion in the carbons derived from the methylene group is observed. Similarly, in citrate extracted from fruit juice, in which there is again a high commitment of acetyl-CoA to this end product, the C2CIT is relatively depleted, by ∼11‰ (
). Hence, it is probable that these positions in acetoacetyl-CoA are more depleted relative to the carboxyl positions than in acetyl-CoA.
In addition, the condensation of N-methyl-Δ1-pyrrolinium with acetoacetyl-CoA is likely to have normal 13C KIEs for both the formation of the C1T–C2T bond in the synthesis of 4-(1-methyl-2-pyrrolidinyl)-3-oxobutyrate, and for the formation of the C4T–C5T bond created during the synthesis of the 8-azabicyclo[3.2.1]octan-3-ol ring. This should lead to a further relative depletion in the C(2 + 4)T position, compatible with the observed values. Unfortunately, due to the coincidence of the C2T and C4T in the 13C NMR spectrum (Fig. 2A), it is not possible to assess whether the first or second bond formation has a major influence on the 13C/12C ratios observed.
To What Extent Do the Reactions Involving the Conversion of N-Methyl-Δ1-pyrrolinium to S-(−)-Nicotine Influence the Observed Isotope Ratios?
The elaboration of N-methyl-Δ1-pyrrolinium to S-(−)-nicotine involves the creation of a new C–C bond linking the C3N and C2′N positions, thus joining the nicotinate-derived moiety to the N-methyl-Δ1-pyrrolinium-derived moiety (Fig. 5). Once again, the symmetry of the N-methyl-Δ1-pyrrolinium salt means that in this precursor the C2′N = C5′N and C3′N = C4′N. However, the values observed at the C2′N and C5′N are not equivalent (Fig. 4B, Table 1): the C2′N, the carbon at which the C–C bond with the nicotinate moiety is made, is found to be ∼4‰ enriched relative to the C5′N. The observed difference needs therefore to be accounted for by another cause. It is proposed that this is due to an inverse 13C KIE associated with the mechanism of the nicotine synthase reaction condensing the N-methyl-Δ1-pyrrolinium- and nicotinate-derived moieties (Fig. 6). Although the mechanism of this reaction is not known, some suggestions can be made on the basis of the isotope fractionation, hence the intrinsic KIE of the reaction. The C2′N undergoes a change of state from a semiaromatic nature with delocalized electrons to a tertiary aliphatic carbon (Fig. 6). Our data indicate that this reaction favors the 13C=N+ over the 12C=N+, as known for sp2 → sp3 conversions (
). Such a KIE will lead to relative enrichment in the position becoming the C2′N and an equivalent relative depletion in the C5′N must occur. This is clearly seen in Fig. 4B. Furthermore, the C3N position is strongly depleted, which would indicate a selection against 13C at this position, compatible with a mechanism in which the C3N undergoes an sp3 → sp2 conversion with kinetic limitation (Fig. 6). Further support for this proposal needs to be sought using specifically labeled substrates.
FIGURE 6.Schematic representation of the proposed mechanism for nicotine synthase, the last step on the biosynthetic pathway to S-(−)-nicotine, based on the observed site-specific 13C/12C isotopic fractionation. Partial enzymatic steps are given as: e1, the formation of the bond between C2′N and C3N in which the C2′N undergoes an sp2 → sp3 with kinetic limitation leading to selection for 13C at the C3N position; e2, the decarboxylation at the C3N position, in which the C3N undergoes an sp3 → sp2 with selection for 12C at the C3N position. Carbon atoms on the final compounds are numbered according to the IUPAC nomenclature. Numbers in normal bold font refer to tropine; numbers in italic bold font refer to nicotine.
What Can Be Deduced About the Introduction of the N-Methyl Group Based on the Observed Isotope Ratios?
In addition to having the Δ1-pyrrolinium moiety in common, the N-methyl group in both tropine and S-(−)-nicotine is derived from the common source, S-adenosylmethionine (AdoMet) and is introduced by the enzyme putrescine N-methyltransferase EC 2.1.1.53) (
). In both compounds, this carbon is found to be depleted relative to the other putrescine-derived positions, with which it should directly be compared (rather than with the whole molecule). In tropine, the extent of depletion is the major difference between the two samples: relative to the putrescine-derive part: ∼38 and ∼22‰ in TRI-1 and TRI-3, respectively. In nicotine, similarly, when the comparison is made only with the putrescine-derived atoms, then the relative depletion is ∼12‰, rather less than in tropine but still well below the average of the other carbon atoms in this moiety.
This relative depletion in the 13C/12C isotope ratio of the N-methyl group appears to be characteristic for AdoMet-derived methylation in natural products, previously recognized for O-methyl groups (
Quantitative isotopic 13C nuclear magnetic resonance at natural abundance to probe enzyme reaction mechanisms via site-specific isotope fractionation: the case of the chain-shortening reaction for the bioconversion of ferulic acid to vanillin.
). The position-specific analysis of 13C/12C isotope ratios of N-methyl group has only recently been investigated by using irm-13C NMR and so far only for a very few alkaloids. Nonetheless, comparison can be made with the purine alkaloids, caffeine and theobromine (
Position-specific isotope analysis of xanthines: a 13C nuclear magnetic resonance method to determine the 13C intramolecular composition at natural abundance.
). In caffeine extracted from coffee the mean of the three N-methyl groups is on average ∼36‰ depleted relative to the other carbon atoms, whereas in theobromine extracted from cocoa, the mean of the two N-methyl groups is on average ∼23‰ below that of the other carbon atoms (
Position-specific isotope analysis of xanthines: a 13C nuclear magnetic resonance method to determine the 13C intramolecular composition at natural abundance.
). These values suggest that relative depletion in the N-methyl group is a general phenomenon reflecting the biochemistry of methyl group insertion. The degree of depletion varies, but in all four alkaloids so far analyzed, including tropine and S-(−)-nicotine (Fig. 4), it is marked. This also parallels the relative depletion in a number of O-methyl groups, for example, ferulic acid (
Quantitative isotopic 13C nuclear magnetic resonance at natural abundance to probe enzyme reaction mechanisms via site-specific isotope fractionation: the case of the chain-shortening reaction for the bioconversion of ferulic acid to vanillin.
), which encouraged us to consider AdoMet as its probable origin. The O-methyl group of tramadol was also relatively depleted, although only by ∼7‰. Overall, then, the depletion found in tropine and S-(−)-nicotine compares favorably with that in other N- and O-methyl groups, supporting the suggestion that this is a general phenomenon related to AdoMet metabolism (
The application of irm-13C NMR to the study of compounds of known biosynthetic pathways has made possible the interpretation of the origin of the δ13Ci (‰) values obtained for two alkaloids, S-(−)-nicotine and tropine. Generally, we can conclude that the majority of the carbon positions show an isotope fractionation that is dominated by the position-specific 13C/12C isotope ratios in the primary precursors that are exploited by the pathway. Thus, where moieties are incorporated that have other functions in the cell, for example, acetate via acetyl-CoA or acetoacetyl-CoA, then isotopic fractionation against the cellular pool can occur, as these precursors fulfill many other metabolic roles. The strength of this link will reflect the extent to which the primary precursors are committed to the synthesis of the specialized compounds, offering access to improved understanding of the primary/specialized metabolite interface in plant metabolism.
Once a major commitment of the substrate/product cascade to the final product is made, overall fractionation must be expected to have a more limited impact, as there is no branching taking place (
). Nevertheless, some intra-molecular fractionation can be observed when C–C bond formation/breaking takes place. In contrast, isotopic fractionation is minimal for those carbon atoms that undergo no bond-forming reactions. These position-specific observations make possible deductions as to the putative reaction mechanism involved, as in the present case of the nicotine synthase condensation.
Experimental Procedures
Materials
Tropine ((3-endo)-8-methyl-8-azabicyclo[3.2.1]octan-3-ol) (TRI-1, hydrate 98+%, batch number S21312-474) and S-(−)-nicotine ((S)-3-[1-methylpyrrolidin-2-yl]pyridine) (NIC-1, free base >99%, batch number 1449164V) were obtained from Sigma; tropine-free base (TRI-3, >97%, batch number 1212025) was obtained from Fluka; S-(−)-nicotine (NIC-3, free base >99.9%) was obtained from CHEMNOVATIC Ławecki Gęca Sp.j., Lublin, Poland. All these samples are of authenticated natural origin as indicated by their Certificate of Origin (note that the plant species used is not indicated) except for TRI-1, for which no certificate was available. Tris(2,4-pentadionato)chromium-(III) (Cr(Acac)3) was from Merck. Benzene-d6 and acetonitrile-d3 were obtained from Euriso-top.
Isotope Ratio Monitoring by Mass Spectrometry
The global value for the whole molecule, δ13Cg (‰), is the deviation of the carbon isotopic ratio Rs relative to that of the international standard Vienna Pee Dee Belemnite, (V-PDB), Rv-PDB. It is determined by isotope ratio monitoring by mass spectrometry and calculated from, where the value of RV-PDB is 0.0111802 (
Isotope Ratio Monitoring by 13C NMR Acquisition Conditions
Prior to preparation of samples for irm-13C NMR, purity was confirmed to be superior to 98% by recording a 1H NMR spectrum. Tropine-free base from Fluka (TRI-3) was re-crystallized from diethyl ether before use.
For the analysis of nicotine, 250 μl of S-(−)-nicotine was homogenized in 500 μl of acetonitrile-d3. To this was added 100 μl of a solution of relaxation agent Cr(Acac)3 (0.1 m) prepared by dissolving 10.5 mg of Cr(Acac)3 in 300 μl of acetonitrile-d3. Under these conditions, T1max = 3.74 s. Spectral acquisition was with AQ = 1.0 s for 152 scans, giving a signal-to-noise ratio of ∼700.
For the analysis of tropine, 150 mg of tropine was dissolved in 600 μl of benzene-d6. As T1max = 1.53 s is relatively short in these conditions, relaxation agent was not required. Spectra acquisition was with AQ = 0.95 s for 400 scans, giving a signal-to-noise ratio ∼550.
Quantitative 13C NMR spectra were recorded at 100.6 MHz using a Bruker 400 Avance I NMR spectrometer fitted with a 5-mm 1H/13C dual+ probe. For S-(−)-nicotine, spectra were also measured on a Bruker 400 Avance III NMR spectrometer fitted with a 5-mm BBFO probe. The temperature of the probe was set at 303 K for nicotine and 313.2 K for tropine (to decrease the viscosity of the sample). The offsets for both 13C and 1H were set at the middle of the frequency range for each analyte. Inverse-gated decoupling was applied and the repetition delay between each 90° pulse was set at 10 × T1max of the analyte to avoid the nuclear Overhauser effect and achieve full relaxation of the magnetization. The decoupling sequence used adiabatic full-passage pulses with cosine square amplitude modulation (ν2max = 17.6 kHz) and offset independent adiabaticity with optimized frequency sweep (
). Each measurement consisted of the average of five independently recorded NMR spectra.
Spectral Data Processing
To obtain Si, the area under the 13C-signal in the irm-13C NMR spectrum for C-atom in position i, curve fitting based on a total line shape analyses (deconvolution) was carried out with a Lorentzian mathematical model using Perch Software (PerchTM NMR Software). In this procedure, line shape parameters are optimized in terms of intensities, frequencies, line width, and line shape (Gaussian/Lorentzian, phase, asymmetry) by iterative fitted to a minimal residue. For each target compound, spectral phasing and line fitting was performed using the same parameters. The positional isotopic distribution, fi/Fi, was then calculated from the values of Si, hence the δ13Ci (‰) value, as described previously (
). For S-(−)-nicotine, due to the large range of its spectral shifts, a correction was made to data obtained on the Avance I NMR spectrometer using the data from the Avance III NMR spectrometer (see Ref.
Conditions to obtain precise and true measurements of the intramolecular 13C distribution in organic molecules by isotopic 13C nuclear magnetic resonance spectrometry.
for a detailed explanation). It should be noted that, because the coupling constants J(13C-15N) are of the order of 10 Hz, no correction for these is required as they are included within the curve fitting applied to the peak.
Author Contributions
R. J. R. and G. S. R. designed the study. K. M. R. and V. S. conducted most of the experiments. R. J. R., G. S. R., and P. P. analyzed and interpreted the data. R. J. R. and K. M. R. wrote the paper.
Acknowledgments
We thank Anne-Marie Schiphorst and Mathilde Grand for help with irm-MS and Jacques Lebreton for discussions about reaction mechanism. We thank CHEMNOVATIC (Ławecki Gêca Sp.j., Lublin, Poland) for their generous gift of pure natural nicotine.
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The intramolecular 13C-distribution in ethanol reveals the influence of the CO2-fixation pathway and environmental conditions on the site-specific 13C variation in glucose.
Quantitative isotopic 13C nuclear magnetic resonance at natural abundance to probe enzyme reaction mechanisms via site-specific isotope fractionation: the case of the chain-shortening reaction for the bioconversion of ferulic acid to vanillin.
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Conditions to obtain precise and true measurements of the intramolecular 13C distribution in organic molecules by isotopic 13C nuclear magnetic resonance spectrometry.
where the value of RV-PDB is 0.0111802 (
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