2-Pyridylfuran: A New Fluorescent Tag for the Analysis of

Apr 27, 2014 - Glycomics and Glycan Bioengineering Research Center, College of Food ... Manchester Institute of Biotechnology, University of Mancheste...
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2‑Pyridylfuran: A New Fluorescent Tag for the Analysis of Carbohydrates Zhi Peng Cai,†,‡ Andrew Kevin Hagan,†,‡ Mao Mao Wang,† Sabine Lahja Flitsch,§ Li Liu,*,† and Josef Voglmeir*,† †

Glycomics and Glycan Bioengineering Research Center, College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China § Manchester Institute of Biotechnology, University of Manchester, Manchester, M13 9PL, United Kingdom S Supporting Information *

ABSTRACT: We herein report the use of 1,3-di(2-pyridyl)-1,3-propanedione (DPPD) as a fluorogenic labeling reagent for sugars. Reaction of DPPD with the anomeric carbon affords a fluorescent 2-pyridylfuran (2-PF) moiety that permits the sensitive HPLC-based detection of monosaccharides. 2-PF-labeled monosaccharides can be easily separated and analyzed from mixtures thereof, and the reported protocol compares favorably with established labeling reagents such as 2-aminobenzoic acid (2-AA) and 1-phenyl-3-methyl5-pyrazolone (PMP), ultimately allowing subfemtomole detection of the galactosederived product. Furthermore, we demonstrate the application of DPPD in the labeling of monosaccharides in complex biological matrices such as blood and milk samples. We envisage that DPPD will prove to be an excellent choice of labeling reagent in monosaccharide and carbohydrate analysis.

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n the structure elucidation of complex carbohydrates, the analysis of the monosaccharide composition is usually the first step. Since monosaccharides and carbohydrates are highly hydrophilic molecules with multiple hydroxy groups and generally lack any intrinsic fluorescent or chromophoric moieties, their detection can be challenging. Therefore, most oligosaccharide analytical techniques rely on labeling with an external fluorophore followed by HPLC(-MS) detection. The labeling step serves the dual purpose of installing a hydrophobic group on the polar sugar to improve chromatographic properties and enabling sensitive detection due to the fluorophore. The typical such method is reductive amination with an aromatic amine such as 2-aminobenzoic acid (2-AA; anthranilic acid), 2-aminobenzamide (2-AB), or 2-aminopyridine (2-AP), which react with the oligosaccharide reducing end under mild conditions with in situ reduction of the intermediate Schiff base with sodium cyanoborohydride.1−4 A second common technique is derivatization with 1-phenyl-3methyl-5-pyrazolone (PMP) to afford products containing two chromophores per product molecule and deliver a detection limit in the high femtomole range with UV detection.5,6 Many of these fluorescent/UV labeling methods result in intense HPLC peaks due to the excess reagent required, which can complicate the analysis by obscuring peaks of interest or necessitate sample cleanup steps that extend the analysis time and result in sample loss.2,3 Several fluorogenic reagents for reducing sugar analysis have also been reported in the literature dating as far back as the 1960s, including 1,2-diarylethylenediamines,7 aromatic amidines,8 o-phenylenediamine,9 2-cyanoacetamide, 10 resorcinol,11 and ethylenediamine sulfate, 12 © 2014 American Chemical Society

although the majority of such methods relied only on total fluorescence measurements in a spectrometer rather than separation of various sugars, and these protocols do not appear to have become part of the standard carbohydrate analysis toolkit. Fluorogenic reagents specific for sialic acids are also known, such as thiobarbituric acid13 and acetoacetanilide/ ammonia.14 A recently discovered method for carbohydrate labeling relies on the reaction with simple β-diketones such as pentane-2,4dione to afford C-glycosidic ketones.15,16 This has the advantages of preserving the native ring structure at the reducing end and installing a reactive ketone moiety for subsequent reaction with various reagents (e.g., fluorophores, biotin).17 During our recent investigations to develop new carbohydrate-labeling reagents, we discovered that pyridyl diketone 2c reacts with monosaccharides such as D-Gal 1 to afford fluorescent 2-pyridylfuran (2-PF) 4c. As both the monosaccharide/carbohydrate and unreacted 2c are nonfluorescent, this reagent provides a fluorescent signal only upon reaction of the carbohydrate with the diketone, thus allowing sensitive detection of the fluorescent product. In this report, we describe the reaction optimization, structural characterization, photochemical properties, and applications of 1,3-di(2-pyridyl)-1,3-propanedione (DPPD) labeling in monosaccharide analysis. This new labeling strategy has great Received: April 16, 2014 Accepted: April 27, 2014 Published: April 27, 2014 5179

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were analyzed by TLC (1:9, MeOH/CHCl3) at 254 nm. The fluorescent products (RF = 0.2) were pooled together and lyophilized. The dry samples were dissolved in 4 mL of MeOH and mixed with 1 g of silica gel, and the sample was dried again. The sample was loaded onto the silica column, which was then eluted with MeOH/CHCl3 (1:9). Fractions were analyzed by TLC, and fractions containing the fluorescent product (RF = 0.2) were pooled together and lyophilized (76.4 mg, 19%). Photophysical Properties of 4c. Compound 4c was dissolved in 50% aqueous MeOH to different concentrations (1 pM to 10 nM). Excitation and emission spectra of the dilutions were recorded on a FluoroMax-4 fluorescence photometer (HORIBA, France). For the emission spectrum, the excitation wavelength was maintained at 330 nm and the emission wavelength was scanned from 350 to 550 nm (Figure 4). For the excitation spectrum, the emission wavelength was kept at 380 nm and the excitation wavelength was scanned from 200 to 349 nm. The molar extinction coefficient at 335 nm of compound 4c in 50% aqueous methanol was determined on a cuvette-free microphotometer (One Drop OD-2000+, Shanghai) by applying the Beer−Lambert law. NMR Analysis of 4. NMR spectroscopy was performed on a 400 MHz Bruker BioSpin NMR spectrometer fitted with a 5 mm PABBO BB-1H/D Z-GRD probe at 296 K. The data set used for the analysis consisted of one-dimensional 1H and 13C spectra, a gradient-selected 1H−1H COSY, and a 1H−13C HMBC (Figures S2−S5 in the Supporting Information). The purified 2-PF-Gal 4c (11.1 mg) was dissolved in CD3OD (500 μL). Data were processed in MestReNova version 8.1 and referenced to the residual solvent peak18 for 1D spectra and the prominent and easily distinguished furanyl H−H or H−C crosspeaks for 2D spectra. 1 H NMR (CD3OD, 400 MHz, δ): 3.68 (d, J = 6.4 Hz, 2H, H13), 3.96 (dd, J = 8.7, 1.7 Hz, 1H, H11), 4.03 (dt, J = 6.4, 1.7 Hz, 1H, H12), 4.84 (d, J = 8.7 Hz, 1H, H10), 6.67 (d, J = 3.6 Hz, 1H, H8), 7.43 (d, J = 3.6 Hz, 1H, H7), 7.62 (ddd, J = 7.6, 5.6, 1.1 Hz, 1H, H2), 8.14 (ddd, J = 8.3, 1.1, 0.8 Hz, 1H, H4), 8.29 (ddd, J = 8.3, 7.6, 1.6 Hz, 1H, H3), 8.60 (ddd, J = 5.6, 1.6, 0.8 Hz, 1H, H1). 13C NMR (CD3OD, 100.6 MHz, δ): 64.56 (C13), 68.62 (C10), 71.51 (C12), 73.25 (C11), 112.05 (C8), 115.65 (C7), 122.09 (C4), 124.44 (C2), 144.06 (C3), 145.19 (C1), 146.24 (C5), 148.34 (C6), 162.27 (C9). Comparison of 2-PF with 2-AA and PMP. For 2-PF labeling, different amounts of D-galactose in aqueous solution (1, 10, and 40 nmol) were vacuum-dried and subsequently derivatized as described above. 2-AA and PMP derivatization of 1, 10, and 50 nmol of D-galactose were carried out according to the methods described by Stepan and Staudacher (additional details can be found in the Supporting Information).19 Liberation of Monosaccharides from HRP. Samples of HRP (100 μg) were dissolved in 4 M trifluoroacetic acid (TFA, 300 μL) in 2 mL prescored glass ampules and hydrolyzed for 2 h at 115 °C after sealing. After cooling down, the ampules were opened and the reaction mixture was vacuum-dried. The dried samples were dissolved in 300 μL of methanol, dried again to remove residual TFA, and 2-PF derivatized as described above. Analysis of Monosaccharides from Milk Samples. Aliquots of defatted human and bovine milk (10 μL) were dried by vacuum centrifugation. Monosaccharide hydrolysis and 2-PF derivatization was carried out identically to the procedure described for the monosaccharide analysis of HRP.

potential for the detection of natural and unnatural monosaccharides and oligosaccharides in glycomics analysis.

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EXPERIMENTAL SECTION Materials. D-Galactose, D-mannose, and L-fucose were obtained from Aladdin (Shanghai, China); D-gulose, D-allose, D-ribose, and D-glucuronic acid were obtained from J&K (Beijing, China). L-Rhamnose and D-xylose were obtained from Kayon (Shanghai, China); D-arabinose was obtained from BioDuly (Nanjing, China). L-Lyxose and D-altrose were from Alligator Reagent (Nanjing, China); D-talose, L-gulose, Dgalacturonic acid, D-apiose, and D-glucose were obtained from Alfa Aesar (Tianjin, China), TCI (Shanghai, China), Solarbio (Beijing, China), Carbosynth (Suzhou, China), and Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), respectively. 1,3-Di(2-pyridyl)-1,3-propanedione was obtained from TCI (Shanghai, China). Horseradish peroxidase (HRP) was obtained from BioDuly (Nanjing, China). 1-Phenyl-3-methyl5-pyrazolone (PMP) was obtained from J&K (Beijing, China); 2-aminobenzoic acid (2-AA) was obtained from Aladdin (Shanghai, China). All other chemicals were of the highest grade commercially available. Chromatography. Chromatographic analyses were performed on a Shimadzu LCMS 2020 system (Shimadzu Corporation, Kyoto, Japan) consisting of an LC-30AD pump equipped with a low-pressure gradient mixing unit, a SIL-30AC autosampler, an RF-20Axs fluorescence detector, and an ESI mass spectrometer. The analytes were separated on a reversed phase HPLC column (Phenomenex Hyperclone 5 μm ODS 120 Å, 250 × 4.60 mm) at a constant flow rate of 1.5 mL/min with fluorometric detection (excitation 330 nm, emission 390 nm). Solvent A was 50 mM NH4COOH (pH 4.5) in water, and solvent B was acetonitrile. After injection of 5 μL of sample, a linear gradient of 12−20% B was applied from 0 to 3 min; then, B was increased to 95% over 1 min and held at 95% for 2 min. B was then decreased to 12% in 1 min, and the column was equilibrated with the initial conditions for 3 min. Silica gel TLC plates were obtained from Merck KG (Type 60 F254, Darmstadt, Germany), developed using MeOH/CHCl3 (1:9 v/v), and visualized by UV at 254 nm and p-anisaldehyde staining. Derivatization. In the standard procedure, monosaccharides (100 nmol) were dissolved in 8 μL of water, and 2 μL of 0.4 mol/L NaHCO3 and 30 μL of 0.1 M DPPD 2c in methanol were added. The mixture was then incubated overnight at 110 °C. Samples were diluted to 0.2−1.0 mL prior to HPLC analysis. These reactions were typically performed in a batchwise fashion, using the apparatus shown in Figure S1 in the Supporting Information. Preparative-Scale Synthesis and Isolation of 4c. DGalactose (300 mg, 1.67 mmol), DPPD (340 mg, 1.5 mmol), sodium bicarbonate (33.5 mg, 0.4 mmol), and 4 mL of MeOH/ H2O (3:1) were introduced and mixed in a 50 mL sealable glass tube. The reaction was carried out at 110 °C overnight. The reaction was allowed to cool to room temperature and dried under reduced pressure. The sample was redissolved in H2O and purified on reversed-phase C18 SPE cartridges (Supelco) followed by flash chromatography on silica gel. The C18 reversed-phase cartridges were preconditioned with MeCN and equilibrated with water. The samples were applied to the cartridges followed by washing with water, and the cartridges were successively eluted with MeCN/water (10:90), MeCN/water (50:50), and MeCN/water (90:10). All fractions 5180

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Analysis of Human Blood. One milliliter of blood was donated by J.V. under sterile conditions, and the plasma fraction was used without further processing. Ten microliter aliquots were then vacuum-dried and derivatized with 2-PF as described above.

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RESULTS AND DISCUSSION 2-PF Labeling of Monosaccharides. The reaction of simple diketones such as 2a with reducing sugars to afford the

Figure 2. TLC analysis (254 nm) of the reaction between D-galactose and (a) DPPD 2c or (b) its diphenyl analogue 2b at different time points. The topmost (blue fluorescent) spot in (a) corresponds to the 2-PF-labeled D-galactose 4c, with the rightmost lane (P) being an isolated sample of the purified compound. (c) ESI mass spectrum of 4c.

Figure 1. Reaction of D-Gal 1 (or any arbitrary monosaccharide) with β-diketones 2 to afford C-glycosides 3 and subsequent dehydration to generate furan derivatives 4.15,16 The fluorescent 2-pyridylfuran tag of 4c is indicated, with atom numbering corresponding to that used for the NMR assignment.

conditions to afford furan and pyrrole derivatives from sugars and amino sugars, respectively, although the main target of these studies was the corresponding ketone.15,16 Subsequent results showed that water-tolerant Lewis acids dramatically increased the formation of these cyclized byproducts under mild conditions.15,21 We also explored the potential of such Lewis acids (FeCl3, CeCl3, Sc(OTf)3, Yb(OTf)3; 20 mol %) in MeOH/H2O (3:1) for promoting the formation of 4; however, in our hands, these were all substantially inferior in terms of product yield compared to our standard conditions using only NaHCO3 (Figure S6 in the Supporting Information), leading to a complex mixture of products including the intermediate noncyclized compounds 3. Optimization of Reaction Conditions. We investigated the effect of temperature, organic solvent, and NaHCO3 concentration on the reaction of D-galactose 1 with DPPD 2c by monitoring the HPLC peak area of the fluorescent 2-PF-Gal 4c (Figure 3). D-Galactose (100 nmol) was reacted with DPPD (3 μmol) in the presence of NaHCO3 (800 nmol) in acetonitrile/water (3:1) in a sealed tube at 65−125 °C overnight (Figure 3a). Yields of 4c steadily increased with temperature up to approximately 110 °C, beyond which they remained stable; we selected 110 °C as a suitable temperature for subsequent experiments. We next tested the effect of varying the organic cosolvent (Figure 3b). The limited solubility of DPPD is presumably a key factor here, although most of the solvents tested, including fully aqueous reactions, afforded significant yields of 4c. Methanol gave the best results

corresponding C-glycoside ketones 3 has been studied by Lubineau15 and Fessner16 and subsequently developed by Price et al.17 These reactions are typically performed using a slight molar excess of the diketone in aqueous sodium bicarbonate and generate the C-glycoside ketone in high yields (Figure 1, upper). During the course of our research directed at discovering new derivatization reagents for carbohydrate analysis, we discovered that reaction of simple hexoses such as D-galactose 1 with 1,3-di(pyridin-2-yl)propane-1,3-dione (DPPD) 2c led to a strongly fluorescent product during TLC analysis (Figure 2a). This appears to be a unique feature of the diketone 2c; neither the earlier reported diketone 2a15,16,20 nor the analogous diphenyl compound 2b (Figure 2b) gave rise to fluorescent products. LC-MS analysis of this compound revealed a singly charged [M + H]+ peak at 266.1 Da for simple hexoses such as D-galactose (Figure 2c), 18 Da lower than that expected for the C-glycoside ketone 3c (284.1 Da). We hypothesized that this was the conjugated 2-pyridylfuran (2-PF) derivative of D-galactose 4c (Figure 1, lower). After formation of the C-glycoside 3c by a Knoevenagel condensation followed by intramolecular oxa-Michael cyclization,16 intramolecular attack of the C2 hydroxyl group on the newly installed ketone and subsequent dehydration results in the formation of 4c. Previous investigations have also noted that intramolecular cyclization can occur under more forcing 5181

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Figure 5. (a) Derivatization of different amounts of D-galactose (from top to bottom are 50, 10, and 1 nmol, respectively) with equal amounts of 2-AA. (b) Derivatization of different amounts of Dgalactose (from top to bottom are 50, 10, and 1 nmol, respectively) with equal amounts of PMP. (c) Derivatization of different amounts of D-galactose (from top to bottom are 40, 10, and 1 nmol, respectively) with equal amounts of DPPD. R = unreacted reagent.

Figure 3. Effect of various (a) reaction temperatures, (b) solvents on the yield of 2-PF-Gal 4, and (c) NaHCO3/D-galactose molar ratios, as measured by HPLC fluorescence peak area. Reactions were performed with D-galactose (100 nmol), diketone 2c (3 μmol), organic solvent (30 μL), and water (10 μL). Unless otherwise stated, reactions were performed at 110 °C using acetonitrile as organic cosolvent and a NaHCO3/D-galactose molar ratio of 8:1.

DPPD (Figure 3c). The use of 8 equiv. compared to Dgalactose proved to be a suitable compromise. Structural Characterization. In order to obtain sufficient material for structural and photochemical characterization, we next turned our attention to a preparative-scale synthesis of 4c. D-Galactose was reacted with DPPD, and compound 4c was isolated in 19% yield by C18 SPE and column chromatography on silica gel. Detailed 2D-NMR analysis by 1H−1H COSY and 1 H−13C HMBC was consistent with the proposed pyridylfuran structure of 4c (Figure 1). In particular, HMBC correlations between the furan- and pyridine-ring quaternary carbons and the nearby protons supported the proposed structure. We also subjected 4c to fragmentation by in-source collision-induced dissociation; although it did not fragment particularly well, we clearly detected an ion at m/z 188.1 corresponding to cleavage of the pyridine group from the molecular ion. Photophysical Properties. The excitation and emission spectra of 4c at 1 nmol/L concentration are shown in Figure 4, revealing excitation and emission maxima at 335 and 381 nm, respectively. The extinction coefficient of 4c was measured as 2770 M−1 cm−1. The HPLC detection and quantification limits of isolated 4c under our experimental conditions were

Figure 4. Excitation and emission spectra of isolated 4c.

and was used in later reactions. Finally, the ratio of NaHCO3 to D-galactose was tested; while NaHCO3 was necessary for the highest yields, a large excess led to rapid decomposition of the 5182

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Table 1. Retention Times and m/z Values of Monosaccharides after Labeling with DPPDa

a

Notes: Products separated by double lines are chromatographically separable. Products between the same pair of double lines give the same retention times under our chromatographic conditions, either because these sugars lead to identical (e.g., D-Glc, D-Man) or enantiomeric (e.g., D-Gul, L-Gul) products upon reaction with DPPD or because they generate distinct but coeluting products (e.g., D-Gal, D-Alt). HPLC fluorescence peak areas are listed relative to D-Gal at equal concentrations (1.95 × 106 units for a 500 pmol sample under our conditions). The standard error (n = 3) for peak areas was less than ±7% for all sugars except L-rhamnose (±7.1%) and D-apiose (±8.1%). The standard error (n = 3) for the retention times was less than ±0.5% for all sugars.

monosaccharide analysis, we investigated the labeling of varying amounts of D-galactose with 2-PF, 2-aminobenzoic acid (2-AA, anthranilic acid), and 1-phenyl-3-methyl-5-pyrazolone (PMP) (Figure 5).1 In terms of detection limits, we found that 2-PF

determined to be 625 amol and 3.125 fmol, respectively, as defined by United States Pharmacopeia guidelines.22 Comparison with Other Monosaccharide Labels. In order to compare our new 2-PF label with existing methods for 5183

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Figure 7. HPLC chromatograms of C2-epimeric monosaccharides, which form identical pyridylfurans upon reaction with DPPD and hence give the same retention times.

believe 2-PF to be an excellent labeling choice for monosaccharide analysis. Applications. We next investigated the scope of 2-PF labeling by reacting DPPD with a range of monosaccharides. We tested 17 monosaccharides in total, including aldohexoses (D-Gal, D-Gul, L-Gul, D-Man, D-Tal, D-All, D-Glc), aldopentoses (D-Ara, D-Xyl, D-Rib, L-Lyx), and branched chain (D-Api), deoxy (L-Fuc, L-Rha), and sugar acids (D-GalA, D-GlcA), as listed in Table 1. LC-MS analysis confirmed the presence of the corresponding fluorescent 2-PF derivative in each case. The separation of eight of the clearly resolved 2-PF-labeled monosaccharides can be seen in Figure 6a (D-Gal, D-Gul, D-Man, D-Ara, D-Api, D-Xyl, LFuc, L-Rha). It can be seen that the various sugars exhibited different labeling efficiencies and/or fluorescence responses, with the D-Xyl derivative for example giving a 3-fold higher peak area than the L-Fuc derivative at identical starting monosaccharide concentrations (Table 1). The reason for this is under further investigation, although similar effects have also been observed for other labeling reagents such as 2-AA.19 Nonetheless, for a particular monosaccharide, the peak areas were very consistent, though precise quantification will require individual standard solutions to correct for the different fluorescence responses after 2-PF labeling. Calibration curves for the DPPD labeling of different amounts of the three representative monosaccharides D-Gal, D-Glc, and D-Rib in the range from 100 pmol to 1 μmol demonstrated high linearity, clearly confirming the utility of DPPD labeling (Figure S7 in the Supporting Information). We next applied our labeling method to the detection of monosaccharides from various biological sources. The glycoprotein horseradish peroxidase (HRP) was subjected to acid hydrolysis to release the monosaccharides and then reacted with DPPD, allowing the detection of D-Gal, D-Man, D-Ara, DXyl, and L-Fuc (Figure 6b). In particular, D-Man, D-Xyl, and LFuc were detected to be the abundant monosaccharides in HRP, which is in agreement with that reported previously.23 We also detected trace amounts of D-Ara in our HRP sample, as

Figure 6. (a) HPLC chromatograms of 2-PF-labeled (top to bottom) D-galactose, D-gulose, D-mannose, D-arabinose, D-apiose, D-xylose, Lfucose, and L-rhamnose. (b) 2-PF-labeled monosaccharides of acidhydrolyzed HRP, showing D-galactose, D-mannose, D-arabinose, Dxylose, and L-fucose. (c) 2-PF-labeled human blood sample. (d) 2-PFlabeled monosaccharides from acid-hydrolyzed human and bovine milk, along with an equimolar standard of D-galactose, D-glucose, and L-fucose.

labeling of D-Gal was at least competitive with 2-AA (1 nmol) and 10-fold more sensitive than PMP (10 nmol). With an analysis time of 10 min, our method was also considerably shorter than the standard methods for either 2-AA or PMP (both 40 min run time under optimized conditions).19 The fluorogenicity of our labeling reagent means that it does not suffer from intense reagent peaks as observed for 2-AA (Figure 5a). Such peaks can interfere with the analysis of more complex monosaccharide mixtures and/or necessitate sample cleanup steps, which lead to sample loss and longer sample preparation times.2 Considering its very competitive sensitivity, short analysis times, and easy sample preparation, we therefore 5184

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the development of fluorescence during the reaction of monosaccharides with DPPD. We expect that DPPD will become a valuable method for the sensitive detection of monosaccharides. Further development of this work, including its application in oligosaccharide and amino sugar analysis, is under way.

was confirmed by 2-AA labeling. We then reacted 10 μL of human blood with DPPD and were able to detect peaks corresponding to the 2-PF-labeled D-glucose (Figure 6c). After compensating for the dilution and HPLC injection volume used, the final volume of blood used was only 50 nL, which still gave a strong fluorescence signal. It should be noted that sample preparation for this assay consisted solely of centrifugation of the blood, reaction with DPPD, dilution, and analysis, with no requirement for complex cleanup or separation procedures. Finally, we labeled acid-hydrolyzed human and bovine milk and observed in both cases HPLC peaks corresponding to D-Gal and D-Glc, as would be expected for hydrolyzed lactose (Figure 6d). The difference in labeling efficiency for D-Gal and D-Glc can again be observed here, with the former giving an approximately 3-fold higher peak area than the latter even for the equimolar monosaccharide standard consisting of D-Gal, D-Glc, and L-Fuc. Furthermore, a smaller fluorescence peak could be detected in the human but not in the bovine milk sample, which elutes at the same retention time as the L-Fuc standard. These data are in agreement with human and bovine milk monosaccharide compositions reported previously.24 It should be noted that C2 epimers of monosaccharides are not distinguishable by our method, as the C2 stereocenter is lost during formation of the furan ring (Figure 1). The 2-PF derivatives of D-Man and D-Glc, for example, are therefore identical, as shown by the fact that they exhibit the same retention times during HPLC analysis (Figure 7a). The same phenomenon was observed for other C2 epimers, namely, Dgalactose/D-talose (Figure 7b), arabinose/ribose, and xylose/ lyxose (Table 1). While this might be considered a limitation of DPPD labeling, it also provides further support for our proposed mechanism for the generation of fluorescence by formation of the pyridylfuran moiety. Furthermore, neither 2deoxyglucose, methyl-α-glucose, nor the ketosugars D-xylulose or D-tagatose led to fluorescent products, as would be expected due to their lack of the reactive aldehyde needed for pyridylfuran formation.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Phone: +86-150-62270463. Fax: +86-25-84399553. E-mail: [email protected]. *Phone: +86-25-84399512. Fax: +86-25-84399553. E-mail: [email protected]. Author Contributions ‡

Z.P.C. and A.K.H. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the National Natural Science Foundation of China (Project 31371739 to L.L. and J.V.), the NJAU Academian, and Departmental Startup Initiative (to both L.L. and J.V.) for funding and the Royal Society (Wolfson Award to S.L.F.).

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REFERENCES

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CONCLUSIONS We herein report a new fluorogenic labeling reagent (DPPD) for carbohydrate analysis. Reaction of this compound with reducing sugars results in the formation of a fluorescent pyridylfuran moiety (2-PF) and allows sensitive detection and separation of labeled monosaccharides. We obtained subfemtomole (625 amol) detection of the D-galactose pyridylfuran derivative 4c, which compares well with values obtained using standard monosaccharide-labeling reagents such as 2-AA (65 fmol) or PMP (3.2 pmol).19 At only 10 min, our LC-MS method is also faster than typical literature methods using 2-AA or PMP and requires little sample cleanup prior to injection, since the fluorophore is only generated after reaction with the monosaccharides. We have demonstrated the utility of our method using hydrolyzed glycoproteins, human blood, and milk, showing that the DPPD reagent works well even in the presence of complex biological matrices. Again, due to the combination of HPLC separation and sensitive and specific fluorescence detection, these species do not interfere with detection of the labeled monosaccharides. A limitation of our method is that it does not allow discrimination of C2 epimers such as D-glucose/D-mannose, since this stereocenter is lost during formation of the pyridylfuran. This observation, however, does further support our proposed mechanism for 5185

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dx.doi.org/10.1021/ac501393a | Anal. Chem. 2014, 86, 5179−5186