Accurate Quantitative Isotopic 13C NMR Spectroscopy for the

Sep 30, 2009 - Alexis Gilbert, Virginie Silvestre, Richard J. Robins, and Ge´ rald S. Remaud*. Elucidation of Biosynthesis by Isotopic Spectrometry G...
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Anal. Chem. 2009, 81, 8978–8985

Accurate Quantitative Isotopic 13C NMR Spectroscopy for the Determination of the Intramolecular Distribution of 13C in Glucose at Natural Abundance Alexis Gilbert, Virginie Silvestre, Richard J. Robins, and Ge´rald S. Remaud* Elucidation of Biosynthesis by Isotopic Spectrometry Group, CNRS-University of Nantes Unit for Interdisciplinary Chemistry: Synthesis, Analysis, Modelling (CEISAM), UMR CNRS6230, 2 Rue de la Houssinie`re, BP 92208, Nantes 44322 Cedex 3, France In order to understand 13C isotope distributions in glucose and its metabolites, it is necessary to measure the internal 13C distribution at natural abundance. These data, however, are not directly accessible, even by quantitative isotopic 13C NMR spectrometry, due to anomerization at the C-1 position. A strategy has been developed that overcomes this difficulty by converting glucose via a three-step synthesis into 3,5,6-triacetyl1,2-O-isopropylidene-r-D-glucofuranose (TAMAGF). This compound provides a satisfactory molecular probe to measure the site-specific 13C/12C ratios in glucose by 13C NMR. It is shown that the isotopic 13C NMR signal gives sufficient precision (repeatability standard deviation e0.8‰) for routine use for the determination of the 13C abundance of each carbon atom position in glucose. Thus, it can be seen that the internal 13C distribution of glucose biosynthesized by the C3 and C4 metabolic pathways differs markedly. Furthermore, the method is suitable for determining the isotope ratio in the glucose moiety of sucrose and, possibly, in free fructose and the fructose moiety of sucrose. Glucose is the most important monosaccharide providing a primary carbon reserve in the form of starch and/or sucrose in many plants and is usually considered to be the product of photosynthesis. Three main metabolic pathways are recognized in plants for the assimilation of CO2: the Calvin cycle (C3), the Hatch-Slack pathway (C4), and crassulacean acid metabolism (CAM). No matter which pathway is used, glucose molecules produced by photosynthesis are 13C-depleted compared with atmospheric CO2. Typically, the molecular average (whole molecule) of the carbon isotope composition of glucose (denoted as δg13C) is about -12 to -28‰ (V-PDB scale) in C4 and C3 plants, respectively, as measured by isotope ratio mass spectrometry (IRMS).1,2 However, IRMS can only provide a δg13C value, whereas, in order to interpret fully the complete carbon * To whom correspondence should be addressed. E-mail: gerald.remaud@ univ-nantes.fr. Phone: 33 2 51 12 57 19. Fax: 33 2 51 12 57 12. (1) O’Leary, M. H.; Madhavan, S.; Paneth, P. Plant, Cell Environ. 1992, 15, 1099–1104.

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isotope distribution pattern in glucose-derived products, the determination of the 13C/12C ratio for each carbon position in glucose and other monosaccharides that are primary reserves is required. An important step toward this objective was made using IRMS on fragments of glucose obtained by partial or total degradation (chemical and/or biochemical).3 Critically, it was shown that the 13 C/12C ratio distribution in natural glucose is nonstatistical. However, this approach is both extremely tedious and incomplete, as a number of positions have to be inferred by difference. Although this work proved unequivocally the nonstatistical isotope distribution in natural glucose, it only partially solved the discrepancies in the internal 13C/12C distribution of glucose from C3 versus C4 plants. Recent indirect evidence that the internal 13C/12C profiles in glucose may differ in relation to the photosynthetic pathway (C3 and C4 plants) has been obtained from examination of ethanol produced through fermentation of C3- or C4-derived glucose.4 It has been shown that the 13C/ 12 C ratios of the CH2 and CH3 groups in ethanol is the converse in C4 plants to that in C3 plants, in which the methylene site is 13C-enriched compared with the methyl. Moreover, site-specific isotope effects in fundamental reactions involving fructose, glucose, and sucrose (e.g., the glucose/fructose conversion by isomerase, and the transformation of glucose and fructose to sucrose by invertase) have not yet been determined, although they clearly would be of great interest. Recently, a small apparent 13C kinetic isotope effect (KIE) for invertase has been measured in vitro using IRMS.5 However, such studies are at the limit of the technique, since an effect on one carbon atom is diluted by a factor of 6 in the case of glucose, and they are incapable of indicating whether more than a single carbon position expresses a KIE related to the reaction mechanism. (2) Brugnoli, E.; Farquhar, G. D. In Photosynthesis: Physiology and Metabolism; Kluwer Academic Publishers: Alphen aan den Rijn, The Netherlands, 2000, pp 399-434. (3) Rossmann, A.; Butzenlechner, M.; Schmidt, H. L. Plant Physiol. 1991, 96, 609–614. (4) Caytan, E.; Botosoa, E. P.; Silvestre, V.; Robins, R. J.; Akoka, S.; Remaud, G. S. Anal. Chem. 2007, 79, 8266–8269. (5) Mauve, C.; Bleton, J.; Bathellier, C.; Lelarge-Trouverie, C.; Gue´rard, F.; Ghashghaie, J.; Tchapla, A.; Tcherkez, G. Rapid Commun. Mass Spectrom. 2009, 23, 2499–2506. 10.1021/ac901441g CCC: $40.75  2009 American Chemical Society Published on Web 09/30/2009

To provide direct and reliable measurements of the 13C/ C ratios of individual carbon atom positions of glucose and related monosaccharides, a new analytical method is required. We have therefore developed isotopic quantitative 13 C NMR for glucose, as this technique has proved successful for determining the site-specific ratios (denoted as δi13C) in a number of compounds.6-8 NMR spectrometry has a number of properties that facilitate the measurement of positional 13Csignals: notably, separation of the signal of each site of the molecule and quantification of the amount of resonating nuclei under the peak (peak area). After overcoming initial difficulties in isotopic 13C NMR, we have been able to propose methodologies to reach appropriate accuracy in the measurement of absolute site-specific 13C/12C ratios with good long-term repeatability.4 Key features include (i) homogeneity and robustness of the 1H decoupling by using appropriate adiabatic decoupling sequences8 and (ii) the reduction of the experimental time by using relaxation reagents.9 While these aspects provide a general framework, the NMR parameters nevertheless have to be fine-tuned and the specific parameters (relaxation, line width, satellites, etc.) established for each new molecular probe in order to exploit effectively the isotopic 13C NMR tool. The present work describes a full analytical protocol that makes it possible to determine the site-specific δi13C values in glucose at natural abundance by 13C NMR. The strategy employed is based on that developed for the determination of the 2H/1H ratios as measured by 2H NMR,10,11 i.e., derivatization of glucose by acetonation. However, the rigor required for quantitative isotopic 13C NMR, typically a precision better than 1‰, necessitated the introduction of a number of modifications in order to attain the appropriate precision and trueness. 12

MATERIALS AND METHODS Chemicals. Silica (63-200 µm mesh), thin layer chromatography (TLC) plates (aluminum sheets, silica gel 60 F254), sulfuric acid (98%), acetone (99.9%), acetic acid (99%), acetic anhydride (99%), pyridine (99%) ethanol (99%), sodium chloride, magnesium sulfate, sodium thiosulfate, and sodium carbonate and solvents (>99% purity) dichloromethane (DCM), petroleum ether, diethylether, cyclohexane, ethylacetate, and ethanol were purchased from VWR (fr.vwr.com). Solvents used for the purification steps (recrystallization and column chromatography) were redistilled before use. Molecular iodine was from Sigma Aldrich (www.sigmaaldrich.com). Sugars used for the chemical synthesis were from various commercial sources. Hexadeuterated acetone (acetone-d6) was purchased from Eurisotop (www.eurisotop.fr). (6) Caytan, E.; Cherghaoui, Y.; Barril, C.; Jouitteau, C.; Rabiller, C.; Remaud, G. Tetrahedron: Asymmetry 2006, 17, 1622–1624. (7) Tenailleau, E.; Akoka, S. J. Magn. Reson. 2007, 185, 50–58. (8) Tenailleau, E.; Remaud, G.; Akoka, S. Instrum. Sci. Technol. 2005, 33, 391– 399. (9) Caytan, E.; Remaud, G. S.; Tenailleau, E.; Akoka, S. Talanta 2007, 71, 1016–1021. (10) Schleucher, J.; Vanderveer, P.; Markley, J. L.; Sharkey, T. D. Plant, Cell Environ. 1999, 22, 525–533. (11) Zhang, B. L.; Billault, I.; Lo, X. B.; Mabon, F.; Remaud, G.; Martin, M. L. J. Agric. Food Chem. 2002, 50, 1574–1580.

Figure 1. Protocol used for the derivatization of glucose, fructose, and sucrose in order to obtain δi13C on each site of the glucose molecule using TAMAGF as the molecular probe.

Chemical Synthesis of the Glucose Derivative (TAMAGF). The typical procedure for the three-step synthesis of 3,5,6-triacetyl1,2-O-isopropylidene-R-D-glucofuranose (TAMAGF) is depicted in Figure 1. Step 1: Synthesis of 1,2,5,6-Di-O-isopropylidene-R-D-glucofuranose (DAGF). This step is common to the protocols used for glucose, fructose, and sucrose; only the latter is presented here. A total of 2.5 g of sucrose was introduced into a 250 mL round-bottomed flask containing 125 mL of acetone. Molecular iodine (93 mg, 0.05 equiv) was added at 50 °C and the reaction mixture was refluxed at 80 °C for 6-8 h. (For the test of H2SO4 as a catalyst, 100 µL was added per 1 g of glucose.) After completion of the reaction monitored by TLC (petroleum ether/diethylether 4:1), the reaction mixture was allowed to cool to room temperature and aqueous Na2S2O3 (0.1 M) was added until complete discoloration of the solution. After evaporation, a white solid was Analytical Chemistry, Vol. 81, No. 21, November 1, 2009

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obtained to which was added aqueous Na2S2O3 (0.1 M, 75 mL) and DCM (25 mL). After separation of the organic layer, the aqueous phase was extracted twice with DCM (35 mL). The organic layers were pooled, washed with 100 mL of aqueous NaCl (10% w/v), dried over MgSO4, filtered, and evaporated to give a mixture of 1,2,5,6-di-O-isopropylidene-R-D-glucofuranose (DAGF) and 2,3,4,5-di-O-isopropylidene-β-D-fructopyranose (DAFP) as a yellowish solid. This was used in step 2 without further purification. Step 2: Synthesis of 1,2-O-Isopropylidene-R-D-glucofuranose (MAGF). The solid obtained in step 1 was treated with 80 mL of aqueous acetic acid (80% v/v). The reaction mixture was stirred for 15-18 h, and the reaction was monitored by TLC (petroleum ether/ethyl ether 4:1). After completion, ethanol (50 mL) was added to the reaction mixture, which was then evaporated to give a white solid. To this was added H2O (100 mL), and the aqueous layer was extracted twice with DCM (50 mL). The remaining aqueous layer containing MAGF was evaporated with ethanol in order to obtain a dry white solid. The organic layers containing DAFP were pooled, washed twice with 100 mL of aqueous Na2CO3 (15% w/v), dried over MgSO4, filtered, and evaporated to give a yellowish oil which crystallized on standing. Step 3: Synthesis of 3,5,6-Triacetyl-1,2-O-isopropylidene-R-Dglucofuranose (TAMAGF). MAGF from step 2 was treated with Ac2O (1.5 equiv/OH) in pyridine (10 mL/g of product). The reaction mixture was stirred overnight, and MAGF disappearance was monitored by TLC (cyclohexane/ethylacetate 2:1). After completion (15-17 h), the reaction mixture was evaporated and then coevaporated with toluene and the viscous yellow liquid obtained was dissolved in DCM. The organic layer was washed with aqueous NaHCO3 (15% w/v) and distilled water. The organic layers were pooled and dried over MgSO4, filtered, and evaporated under vacuum. The resulting crude product (yellow solid) was purified on a silica chromatography column (63-200 µm) with a mixture of cyclohexane/ethylacetate (4:1) to separate the TAMAGF from R- and β-pentaacetate glucopyranose, formed from residual glucose coming from MAGF hydrolysis. The purity of the compounds was checked by gas chromatography (GC). Products of sufficient purity were directly analyzed by 13C NMR; insufficiently pure compounds were further recrystallized from hot light petroleum ether (bp 40-65 °C). It should be noted that during the sucrose (or mixture of glucose + fructose) derivatization process, a byproduct coming from fructose is obtained which is an isomer of DAFP: 1,2,4,5-diO-isopropylidene-β-D-fructopyranose. This compound will be hydrolyzed into 1,2-O-isopropylidene-β-D-fructopyranose during step 2 and then acetylated during step 3. It can be eliminated by the recrystallization of TAMAGF in hot light petroleum ether (bp 40-65 °C). Hydrolysis of MAGF. MAGF (50 mg) was added dropwise into a 50 mL round-bottomed flask containing 20 mL of water. A total of 200 mg of Amberlyst cation exchange resin (H+ form), preconditioned with HCl (1 M), was then added, and the reaction mixture was refluxed for 36 h. The completion of the reaction was checked by TLC on silica developed with ethanol. At the end of the reaction, the resin was filtered and rinsed 8980

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three times with distilled water. The filtrate was evaporated and then coevaporated with distilled water to remove traces of acetone formed during the hydrolysis of MAGF. The glucose syrup obtained was then concentrated under a flow of nitrogen gas. GC Experiments. The analysis of TAMAGF was performed on a base-deactivated poly(5% diphenyl/95% dimethylsiloxane) PTA-5 fused-silica capillary column (Supelco; length 30 m, inner diameter 0.32 mm, film thickness 0.5 µm). An Agilent Instruments gas chromatograph HP 6890 series equipped with a flame ionization detector (FID) was used. The carrier gas was helium with a constant flow rate of 1.2 mL/min and a split ratio of 1:50. The chromatogram was developed with the following temperature program: 100 °C for 5 min, 10 °C/min to 300 °C, isothermal at 300 °C for 5 min. NMR Spectrometry Experiments. Quantitative 13C NMR spectra were recorded using a Bruker DRX 500 spectrometer fitted with a 5 mm i.d. dual probe 13C/1H carefully tuned at the recording frequency of 125.76 MHz. Spectral Acquisition Conditions. The temperature of the probe was set at 303 K. The offsets for both 13C and 1H were set at the middle of the frequency range for each molecule. Inverse-gated decoupling was applied in order to avoid NOE. The repetition delay between each 90 °C pulse was set at 10xT1max of the molecule under investigation. The longest T1 (T1max) value measured using the sample preparation described below was 1.2 s. The decoupling sequence employed a cosine adiabatic pulse with appropriate phase cycles, as described in ref 7. For samples without relaxation agent, acquisition conditions were acquisition time 0.8 s, 480 scans with a repetition delay of 15 s, leading to a signal-to-noise ratio (SNR) ≈ 650. For samples prepared with relaxation agent, conditions were acquisition time 0.7 s, 1104 scans with a repetition delay of 10 s (T1max ) 1 s), leading to an SNR ≈ 900. Each measurement consisted of the average of three to five independently recorded NMR spectra. Sample Preparation. TAMAGF (280 mg) was dissolved in acetone-d6 (500 µL) and then submitted to ultrasonication for 15 min (to eliminate any oxygen trapped during the dissolving of the compound), before being filtered into a 5 mm o.d. tube. For measurements with relaxation agent, 50 µL of a 0.1 M chromium(III) acetylacetonate (Cr(Acac)3) solution in acetone was added to the mixture of TAMAGF and acetone before ultrasonication. NMR Data Processing. Free induction decay was submitted to an exponential multiplication inducing a line broadening of 1.6 Hz. The curve fitting was carried out in accordance with a Lorentzian mathematical model using Perch Software (Perch NMR Software, University of Kuopio, Finland). Isotopic Data. The isotopic distribution in a molecule is characterized by the actual 13C molar fractions f of a specific site i: fi ) Si/St, where Si is the area of the 13C NMR signal of i and St is the sum of the areas of all the signals for the molecule. Each Si is corrected according to the number of carbons directly connected in order to compensate for intensity losses due to satellite lines caused by the bilabeled isotopomers (see the Results and Discussion). In accordance with the 13C natural mean abundance of 1.1%, areas Si were multiplied by

(1 + n × 0.011), where n was the number of carbons directly connected.9,12 The shift from the random distribution of 13C may be expressed as the ratio fi/Fi named the reduced molar fraction, where Fi is the statistical molar fraction, i.e., Fi ) 1/6 for TAMAGF. The 13C abundance on each site Ai was obtained by multiplying the reduced molar fraction fi/Fi by the global abundance Ag given by IRMS; hence, the isotopic ratio at each carbon atom position Ri was readily determined.12,13 The δi13C values were calculated against Vienna-Pee Dee Belemnite (VPDB), the international standard for13C measurements, using the equation δ(‰) )

(

)

R - 1 × 1000 Rstd

where R is the isotope ratio of the sample and Rstd is the isotope ratio of V-PDB (Rstd ) 0.0112372). IRMS Measurements. The global isotope deviation, δg13C (‰), was determined by encapsulation and analysis using a Delta-VAdvantageisotoperatiomassspectrometer(www.thermo. com) coupled to an NA2100 elemental analyzer (www.thermo. com). Compound (0.8-1.0 mg) was sealed in a tin capsule, and the δ13C was determined by reference to a working standard of glutamic acid standardized against calibrated international reference material (IAEA-N1 or IAEA-N2, International Atomic Energy Agency, Austria). RESULTS AND DISCUSSION Selection of the Glucose Derivative (TAMAGF). Glucose, as with many reducing sugars, can adopt different forms in solution: in this case R-D-glucopyranose is in mutarotational equilibrium with β-D-glucopyranose.14,15 This rules out the possibility of performing isotopic 13C NMR directly on the sugar, due to overlapping signals in the 13C NMR spectrum. Furthermore, in matrixes of interest, such as fruit juice, honey, and maple syrup, glucose is often present with other carbohydrates, which all have very close δg13C values. Therefore, in order to determine the internal distribution of 13C in glucose, a separation-purification methodology needed to be developed that addressed these issues but ensured that the isotopic integrity of the starting glucose was maintained. The adopted choice was to block the anomeric center (C-1) by the isopropylidene group, a strategy successfully proposed for the study of 2H distribution in glucose by 2H NMR.10,11 The three-step protocol developed (Figure 1) was also designed to be applicable in the presence of fructose, frequently found with glucose and, critically, the other monomeric sugar in sucrose. The methodology enables both the derivatization of the glucose moiety of sucrose and its separation from the fructose moiety. Thus, in the following discussion, a mixture of glucose and fructose is always considered in order to justify the strategy. Although the final strategy is based on that previously described for the analysis of 2H distribution in glucose by 2H NMR,10,11 a number of key adaptations were made (for details (12) Silvestre, V.; Mboula, V. M.; Jouitteau, C.; Akoka, S.; Robins, R. J.; Remaud, G. S. J. Chromatogr., A 2009, 1216, 7043–7048. (13) Tenailleau, E.; Lancelin, P.; Robins, R. J.; Akoka, S. Anal. Chem. 2004, 76, 3818–3825. (14) Hyvo ¨nen, L.; Varo, P.; Koivistoinen, P. J. Food Sci. 1977, 42, 657–659. (15) Yamabe, S.; Ishikawa, T. J. Org. Chem. 1999, 64, 4519–4524.

see the Materials and Methods). At the end of step 1 of the protocol (Figure 1), a mixture of DAGF (glucose derivative) and DAFP (fructose derivative) is obtained with high yield (>92%). Separation of these two products by, for example, flash chromatography, could be tedious and could lead to alterations in the site-specific isotope ratios.16 Therefore, a reaction step was introduced to remove one isopropylidene group selectively from DAGF, forming MAGF, while DAFP was unaffected. Thus, MAGF could be separated from DAFP on the basis of the higher affinity for water of the former and the very good solubility in DCM of the latter. At this stage, the two molecules showed very acceptable 13 C NMR spectral properties: high solubility in NMR solvents and no overlapping peaks. However, at a concentration above ∼1.5 M, a type of gel formed that was stabilized by hydrogen bonds between the hydroxyl groups.17-19 A slow exchange may occur in the presence of mobile hydrogen atoms. When mobile deuterium atoms are available, as in the NMR deuterated solvents, 1 H and 2H are slowly exchanged at the hydrogen sites, leading to the well-known effect of 2H on the 13C chemical shift.20,21 Consequently, several aggregate structures appear in the NMR spectrum generating additional NMR peaks very close to the main signal, thereby compromising the 13C quantification for the δi13C determination (data not shown). Moreover, this affects all carbons, since β and even γ chemical shift effects are possible.20,21 Hence, the third step was introduced (Figure 1) to remove the free hydrogen atoms by esterification. In appropriate acetylation conditions, TAMAGF was obtained with high yield (>95%). TAMAGF also has a number of advantages over MAGF: it is easier to purify and to handle, it does not form a gel-aggregate, it gives less viscous NMR solutions, and it is easy to recover. Thus, the protocol produces a molecular probe well adapted to the analysis of glucose. Another pertinent feature of the protocol is that it enables the separation of glucose from fructose, whether it is present in the starting mixture or in sucrose. At step 2, it is also possible to hydrolyze MAGF further (removing the second isopropylidene group) to a glucose syrup, which can be subsequently analyzed by IRMS to obtain the global 13C content used for the calculation of each δi13C (see the Materials and Methods). In those cases where the matrix is constituted of glucose, fructose, and sucrose, as in fruit juices, sucrose can first be separated from the monomers using either preparative HPLC chromatography22,23 or a charcoal column.24,25 Constraints in Compound Purity Imposed by Quantitative 13 C NMR. As the calculation of the site-specific isotope deviation (16) Botosoa, E. P.; Silvestre, V.; Robins, R. J.; Rojas, J. M. M.; Guillou, C.; Remaud, G. S. J. Chromatogr., A, in press. DOI: 10.1016/j.chroma.2009.08.066. (17) Luboradzki, R.; Pakulski, Z.; Sartowska, B. Tetrahedron 2005, 61, 10122– 10128. (18) Gronwald, O.; Sakurai, K.; Luboradzki, R.; Kimura, T.; Shinkai, S. Carbohydr. Res. 2001, 331, 307–318. (19) Tritt-Goc, J.; Bielejewski, M.; Luboradzki, R.; Lapinski, A. Langmuir 2008, 24, 534–540. (20) Reuben, J. J. Am. Chem. Soc. 1984, 106, 6180–6186. (21) Reuben, J. J. Am. Chem. Soc. 1985, 107, 1747–1755. (22) Doner, L. W.; Ajie, H. O.; Sternberg, L. d. S. L.; Milburn, J. M.; DeNiro, M. J.; Hicks, K. B. J. Agric. Food Chem. 1987, 35, 610–612. (23) Gonzalez, J.; Remaud, G.; Jamin, E.; Naulet, N.; Martin, G. G. J. Agric. Food Chem. 1999, 47, 2316–2321. (24) Hayashi, F. J. Biochem. 1932, 16, 1–16. (25) Whistler, R. L.; Durso, D. F. J. Am. Chem. Soc. 1950, 72, 677–679.

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Table 1. Mean Isotope Deviation (δi13C) for Each Carbon of TAMAGF (see Figure 1 for Carbon Numbering) over 10 Measurements from the Same Batch, Measured by 13C NMR Spectrometrya carbon position TAMAGF mean (‰) SD (‰)

C-1 -20.5 0.7

C-2 -23.7 0.6

C-3 -23.4 0.8

C-4 -25.8 0.6

C-5 -29.3 0.8

Table 2. Mean Isotope Deviation (δi13C) for Each Carbon of TAMAGF (see Figure 1 for Carbon Numbering) over Eight Syntheses from the Same Glucose and over Four Syntheses from the Same Sucrose, Measured by 13C NMR Spectrometrya carbon position

C-6 -26.8 0.6

TAMAGF from glucose

a The standard deviation (SD) from the 10 determinations is expressed on the δ-scale (‰).

sucrose a

δi13C is based on the reduced molar fractions fi/Fi (see the Materials and Methods), the purity of the molecular probe used for isotopic 13C NMR measurement is crucial. On the one hand, the purity should be as high as possible for the determination of δg13C by IRMS; on the other hand, its precise value does not need to be known since it is not taken into account during the δi13C calculation. In fact, it is the impurity profile that matters: an impurity as low as 0.2% but which has a signal under peak i of interest will lead to an error of 2‰ in δi13C. Furthermore, this potentially leads to incorrect values at the other carbon atom positions because of the use of molar fractions (fi/Fi) for the calculation of δi13C. Therefore, particular attention was paid to the byproducts and their chemical shifts in 13C NMR. As an illustration, the presence of R- and β-pentaacetate-glucopyranose formed (at no more than a few percent) during the third step of the derivatization (see the Materials and Methods) has to be avoided, since one of its peaks will lie under one peak of TAMAGF. Although unwanted, the fructose derivative 3,4,5-tri-O-acetyl-1,2-O-isopropylidene-β-Dfructopyranose will not lead to erroneous measurements, as none of its peaks are under the peaks of TAMAGF. Nevertheless, even byproducts that do not generate false δi13C values can decrease the quality of the measurement by being the source of an alteration of the SNR, leading to a decrease in precision. A systematic control of the profile of impurities has to be performed by gas chromatography and 1H/13C NMR. The proposed analytical protocol generates no byproducts that affect the quantitative measurements. Precision of the δi13C in Glucose via TAMAGF. The precision (see ref 26 for a standard definition) of an analytical method is usually assessed by a repeatability study. The longterm repeatability of the isotopic 13C NMR protocol has already been established for ethanol and vanillin samples at natural abundance.4 Once the configuration and the stability of the spectrometer are achieved, then the standard deviation (SD) of replicates depends solely on the SNR,4 each replicate being constituted of three to five independent NMR spectra. Thus, it can be anticipated that for an SNR ≈ 600-700, the SD should be below 1‰. This requirement has been tested on a large quantity of TAMAGF samples: 10 replicates were carried out over 3 months using the NMR conditions described in the Materials and Methods. Table 1 shows the mean value and the associated SD for each carbon of the glucose skeleton (see Figure 1 for carbon numbering). The standard deviation for the instrumental repeatability is no more than 0.8‰. (26) Menditto, A.; Patriarca, M.; Magnusson, B. Accredit. Qual. Assur. 2007, 12, 45–47.

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C-1

C-2

C-3

C-4

C-5

C-6

δ13C mean (‰) -6.6 -8.7 -8.0 -10.3 -12.4 -11.9 SD (‰) 0.8 0.8 0.6 0.5 0.5 0.7 δ13C mean (‰) -22.8 -26.0 -22.2 -17.8 -27.8 -31.1 SD (‰) 0.5 0.6 0.8 0.7 0.6 1.1

The standard deviation (SD) is expressed on the δ-scale (‰).

We have also tested the repeatability of sample preparation by studying the variability of δi13C carried out on replicates from eight independent syntheses of TAMAGF from the same glucose sample and from four repetitions from the same sucrose sample. As can be seen in Table 2, the SD is of the same order as for the NMR repeatability. No variability is introduced by the preparation step. The standard deviation could be further improved by increasing the SNR but to the detriment of the analysis time. On the basis of the determined instrumental repeatability (e0.8‰) and the reliability of the derivatization method, it is possible to define acceptable parameters for comparing δi13C values between sites of TAMAGF and between batches of glucose: values for δi13C that differ by 1.6 but 92%, n ) 8). The error bars are the standard deviations, obtained from the synthesis repeatability (i.e., 0.8‰). Legend: yield ) 55%, mean of 2 reactions of 50% and 60% yield (stopped at 1 h); yield > 92%, mean of 8 reactions all of >92% yield (stopped at 6 h).

Table 3. Isotope Deviation (δi13C) for Each Carbon of TAMAGF (See Figure 1 for Carbon Numbering) from Glucose Used to Test the Repeatability for the Synthesis (Table 2), under Normal Yield (>95%) and under Low Yield (20%) for Step 2 carbon position yield

TAMAGF

normal (>95%) low (20%)

δ13C(‰) δ13C(‰)

C-1

C-2

C-3

C-4

C-5

C-6

-6.6 -8.7 -8.0 -10.3 -12.4 -11.9 -5.3 -8.2 -6.3 -10.8 -14.5 -12.8

Table 4. Isotope Deviation (δi13C) for Each Carbon of TAMAGF (see Figure 1 for Carbon Numbering) from Glucose Used to Test the Instrumental Repeatability (Table 1), under Normal Yield (>95%) and under Low Yield (60%) for Step 3 carbon position yield

TAMAGF

normal (>95%) low (60%)

δ13C(‰) δ13C(‰)

C-1

C-2

C-3

C-4

C-5

C-6

-20.5 -23.7 -23.4 -25.8 -29.3 -26.8 -21.7 -22.9 -23.6 -25.7 -29.3 -26.3

The presence of water in the medium was also checked, since glucose from naturally occurring matrixes (honey, syrup, etc.) is not fully dried, which could reduce the yield in the first step. The isotopic pattern of glucose is not affected when the reaction is carried out in the presence of water, up to 30% (v/m), even at yields below 20% (data not shown). The influence of the catalyst at the first step was also investigated, as H2SO4 can be used as a potential alternative catalyst in order to obtain high yields even in the presence of water. This shows no difference with iodine as catalyst (data not shown). Yields between 20% and 60% during steps 2 or 3, although much lower than the routine conversion yield (>95%), do not affect δi13C at a 95% confidence level (see Table 3 for step 2 and Table 4 for step 3). Since no significant isotope fractionation was observed during each step of the derivatization under conditions of yield much lower than in the “normal” protocol, and since we worked at a conversion yield higher than 92%, we can conclude that the internal 13C Analytical Chemistry, Vol. 81, No. 21, November 1, 2009

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Figure 3. Isotopic 13C pattern of TAMAGF prepared from glucose of different origins. Values are described as isotope shift ∆δ (‰) (∆δ ) δi13C - δg13C). The error bars are the standard deviations from the mean value observed for the set of samples (n ) 4 for each origin).

distribution measured in TAMAGF is the same as occurs in the parent glucose. Use of a Relaxation Agent. Ten experiments were conducted on the same TAMAGF used for NMR repeatability measurements with the addition of the relaxation agent chromium(III) acetylacetonate (Cr(Acac)3). The δi13C values were the same as those obtained without relaxation agent (data not shown). However, the SD for 10 experiments was above 0.8‰ even at an SNR ≈ 900 for each spectrum. This is probably due to a decrease in precision during the sample preparation and/or overlap between the peaks and their satellites as a result of broader peaks induced by faster transverse relaxation times T2. Moreover, the gain in experimental time was only ≈ 20%. Overall, no advantage was seen to accrue from adding relaxation agent, and therefore measurements were subsequently made without adding (Cr(Acac)3). Preliminary Applications. Although the aim of this work is not to discuss the factors that influence the isotopic 13C patterns of natural glucose, the ability of the method to distinguish between these patterns is presented to demonstrate that the method can be successfully applied to real samples. Glucose from a C3 plant (wheat) and a C4 plant (maize) and sucrose from a C3 plant (beet) were analyzed, on four different samples for each origin. The results expressed as ∆δi13C (δi13C - δg13C) are presented in Figure 3, where the error bars are the standard deviation from the mean value (n ) 4). The intramolecular isotope distribution in natural glucose appears to be reproducible for each origin (SD < 1‰ for each carbon atom, except for the C-4, 1.5‰), within the limited population studied in the present work. The relatively high SD value obtained for the carbon-4 (C-4) in starch may be due to the industrial process involved, based on a continuous batch reactor. This incomplete hydrolysis could be a source of isotope fractionation at C-4, notably since this is one of the carbons by which the glucose units are linked in starch. Moreover, the isotopic distribution in glucose obtained with our method is consistent with the results obtained by 8984

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Rossmann et al.3 who found a 13C enrichment in the C-3 and C-4 and a 13C depletion on the C-6. As already inferred from ethanol, the internal isotopic distribution in glucose differs depending on the metabolism by which it has been formed (i.e., C3 or C4 plant origin). Differences are located principally on the C-1 and C-6 of glucose, both being 13C-enriched in C4 starch compared to C3 starch. Although these results need more investigation to be understood, they are consistent with the observations made in a previous study on ethanol, that the methyl group (coming from the C-1 and C-6 through fermentation of glucose) is more 13C-enriched in C4 plants than in C3 plants compared with the methylene group (coming from the C-2 and C-5 of glucose). Glucose from sucrose, the other storage metabolite in plants, shows 13C-enrichment at the C-4 position and depletion at the C-2 position compared with starch from the same metabolism. These observations could be explained by the different fate and fluxes of glucose within the plant, which include sucrose formation/breakdown, involving the C-2 of glucose, and starch formation/breakdown, involving the C-4 of glucose. Further investigation to test these hypotheses is underway. CONCLUSIONS The method herein described for determining the distribution of 13C within glucose is shown to fulfill the necessary criteria for application in metabolic studies. It has both a repeatability below 0.8‰ for an SNR ∼700 and high robustness in relation to the three-step derivatization. Thus, the distribution of 13C within glucose can be obtained by measuring the δi13C in TAMAGF, providing a unique means by which to probe the role of each carbon position in the metabolism of this key molecule. The method described provides an effective tool for the detection of small variations in 13C/12C ratios in glucose associated with C3/C4 metabolism or with different forms of carbohydrate storage. Moreover, free glucose and fructose can be separated from a matrix and glucose and fructose obtained individually from sucrose. In addition, it will be possible to measure isotope effects at each site of the glucose molecule for fundamental enzyme reactions that take place during carbohydrate synthesis and degradation, as described recently for ferulic acid biotransformation to vanillin.31 The majority of studies of isotope fractionation in physiology use the global isotope deviation δg13C measured by IRMS. This approach is likely to underestimate, or even miss completely, isotope effects for two principal reasons. First, the information is diluted through the other carbons present that do not take part in the reaction, making it technically difficult to determine the isotope shift ∆δg13C with sufficient precision by IRMS. Second, opposed (normal and inverse) isotope effects will cancel out, masking individual isotope shifts for each isotopomer (each carbon of the molecule). That both inverse and normal isotope shifts ∆δi13C occur simultaneously was demonstrated for the biotransformation of ferulic acid.31 The analytical protocol using isotopic 13C NMR (31) Botosoa, E. P.; Blumenstein, C.; MacKenzie, D. A.; Silvestre, V.; Remaud, G. S.; Kwiecien´, R. A.; Robins, R. J. Anal. Biochem. 2009, 393, 182–188.

circumvents these problems. Further experiments are currently in progress, both to study glucose-related metabolites and to adapt the methodology to other sugars of interest.

Nantes, France) for linguistic correction. Dr. Isabelle Billault is also acknowledged for her valuable advice and for fruitful discussions.

ACKNOWLEDGMENT Alexis Gilbert thanks the Scientific Council of the Pays de la Loire Region (France) and the CNRS for a cofunded doctoral bursary. We thank Carol Wrigglesworth (Scientific English,

Received for review July 1, 2009. Accepted September 12, 2009. AC901441G

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