Study on the separation mechanism of 1-phenyl-3-methyl-5-pyrazolone derivatives of aldoses in acid buffer by capillary zone electrophoresis

Abstract

Baseline separation was achieved for 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of six usual aldoses by capillary zone electrophoresis (CZE) using acid buffer (pH 2.5). The result showed that the migration times of mannose and rhamnose were much longer than those of the other four aldoses, though the molecular weights of the two monosaccharides were parallel to the others. This phenomenon is due to the intramolecular ring formation in PMP-aldoses, which impairs the conjugate in the pyrazolone ring, and further increases the alkalinity and positive charge of PMP-aldoses in acidic ambience. The aldose having 2,3-trans disposition will be favorable for ring formation and has more positive charge than that of the aldose having 2,3-cis disposition.

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Introduction

The active polysaccharides and oligosaccharides are getting more and more attention in immunotherapy for their natural and secure potency.^1–4 For example, mannan-oligosaccharides have played an important role in the research of the human immunodeficiency virus vaccine,⁵ and human immunity can be enhanced by lentinan.⁶

Now, capillary electrophoresis (CE) has been recognized as an effective technique for the analysis of carbohydrates.^7–9 Establishing a relationship between electrophoretic mobility and molecular weight by CE will be helpful to the identification, preparation and activity screening of oligosaccharide.^10,11 Recently, we have been undertaking a project to research the active fragments of traditional Chinese medicine polysaccharides by capillary zone electrophoresis (CZE). However, most carbohydrates lack chromophores and need pre-capillary derivatization to improve the detection sensitivity. The 1-phenyl-3-methyl-5-pyrazolone (PMP) derivative method has many advantages over hitherto reported methods based on reductive amination and hydrazone formation, such as the very mild condition of the derivative reaction and the strong absorbance in the ultraviolet of the derivatives.^12,13 So, in this project, PMP was used for pre-capillary derivatization of the oligosaccharide fragments and monosaccharides of traditional Chinese medicine polysaccharides by controlled degradation, and attempts were made to separate them according to their molecular weights. The result showed that PMP derivatives of maltooligosaccharides could be separated from low to high molecular weights by CZE at pH 2.5, but PMP derivatives of six usual aldoses showed unexpected separation behaviors.¹⁴ Their separation didn't follow the molecular weight discipline. The migration times of mannose and rhamnose were much longer than those of the other four aldoses, even longer than that of DP 7 maltooligosaccharide.

In this paper, the separation mechanism of PMP-aldoses by CZE at pH 2.5 was studied, and the proposed separation mechanism was also verified.

Materials and methods

Reagents

PMP was purchased from Rizhao Lideshi Chemical Reagent Company (Beijing, China). Ethyl-p-aminobenzoate (ABEE) was obtained from Beijing West Chemical Plant (Beijing, China). D-Mannose was purchased from Solarbio Technology Company (Beijing, China). D-Glucose was purchased from Tianjin Fuchen Chemical Reagent Company (Tianjin, China). High purity grade D-galactose and D-arabinose were obtained from Sigma Company (St. Louis, MO, USA). D-Xylose was purchased from Baoding Second Chemical Plant (Baoding, China). L-Rhamnose was purchased from Hefei Hiromi Biotechnology Company (Hefei, China). 2,3,4,6-tetramethyl-D-glucose, 2,3,4,6-tetramethyl-D-galactose and 2,3,4,6-tetramethyl-D-mannose were prepared^15,16 in our laboratory.

Derivatization

PMP derivatization ¹⁷. A sample of 100 μL aldoses mixture solution (200 nmol aldoses) or tetramethyl aldohexoses mixture solution (approximately 500 nmol tetramethyl aldohexoses) was added to 100 μL 0.3 mol L⁻¹sodium hydroxide solution, and then 100 μL 0.5 mol L⁻¹ methanolic solution of PMP was added, oscillated sufficiently. The mixture was left to react at 70 °C for 30 min, then neutralized with 100 μL 0.3 mol L⁻¹hydrochloric acid solution and 1 mL chloroform was added, oscillated sufficiently (1 min) and centrifugated. The supernatant was filtered through a 0.45 μm nylon membrane filter and degassed by sonication.

ABEE derivatization . A sample of 1 mg aldose was added to 40 μL methanol-acetic solution of ABEE (13.4 mg ABEE, 36.5 μL anhydrous methanol, 3.5 μL glacial acetic acid). The mixture was left to react at 80 °C for 60 min. Then, 5 mg potassium borohydride was added to the solution, and the resultant solution was left to react at 80 °C for 30 min, 0.5 mL chloroform and 0.5 mL water were added, oscillated sufficiently (1 min) and centrifugated. The supernatant was filtered through a 0.45 μm nylon membrane filter and degassed by sonication.

Experimental method

Separations were performed on a Beijing Cailu CL1020 apparatus equipped with a continuous-wavelength UV monitor. An uncoated fused-silica capillary (I.D. 50 μm, 50.0 cm, effective length 40.0 cm) was obtained from Hebei Yongnian Ruifeng Chromatography Devices Company (Hebei, China). The capillary was sequentially rinsed with 0.1 mol L⁻¹sodium hydroxide (20 min) and distilled water (10 min), and equilibrated with the running buffer before use. Gravity injection (10 cm height) was at the anodic end of the capillary. All solutions were passed through a 0.45 μm cellulose membrane filter before using. The detection was carried out at 245 nm (PMP) or 214 nm (ABEE). The experiment was operated at 28 ± 2 °C.

Results and discussion

PMP derivatives of aldoses and maltooligosaccharides were separated by CZE using 50 mM phosphate at pH 2.5, the electropherogram was shown in Fig. 1. Although the molecular weights of all PMP-aldoses were parallel, comparing with the other four aldoses (arabinose, galactose, xylose, glucose), the migration times of mannose and rhamnose were very long, even longer than that of DP 7 maltooligosaccharide.

Fig. 1 Electropherogram of PMP derivatives of aldoses and maltooligosaccharides by CZE. Running buffer, 50 mM phosphate buffer, pH 2.5; voltage, 15 kV; hydrodynamic pressure injection, 10 cm × 6 s. Peaks: Ara = arabinose, Gal = galactose, Xyl = xylose, Glc = glucose, Rha = rhamnose, Man = mannose, 2–7 = the degree of polymerization (DP) of the oligosaccharides are from 2 to 7, R = PMP.

Honda et al. had reported a similar migration behavior of PMP-aldoses in neutral buffer.¹⁷ They proposed a ring formation hypothesis (Fig. 2A) to explain the mechanism for the separation of PMP-aldoses in neutral buffer. The rings formed by hydrogen bonding between the keto group in the pyrazolone ring and the hydroxyl groups at C-2 and C-3 in the carbohydrate moiety made ionization of keto group (via the enol group) disadvantageous and reduced the negative charge of PMP-aldose. The 2,3-trans disposition (Fig. 2B) would be favorable for ring formation, so aldoses of 2,3-trans disposition would have less negative charge and migrate faster than aldoses of 2,3-cis disposition. However, this mechanism can't explain the phenomenon for the separation of PMP-aldoses in acid buffer perfectly. PMP carried a positive charge at pH 2.5 in view of the peak of PMP being in front of the peak of neutral marker. So, the ionization of the keto group had hardly occurred.

Fig. 2 Intramolecular rings and fischer structures of the six monosaccharides.

Study on the influencing factors of separation at pH 2.5

The kinds of running buffer and derivative reagent might influence the separation of aldoses at pH 2.5. Therefore, two experiments were designed to confirm the above conjecture.

First, the phosphate buffer was replaced by acetate buffer, and the other experimental parameters such as pH value and buffer concentration were the same.

Fig. 3A and 3B showed the separation of PMP-aldoses using the two different running buffers. Obviously, the two electropherograms were similar. In Fig. 3B, the migration times of mannose and rhamnose were still much longer than those of the other four aldoses. So buffer kind wasn't the reason that influenced the separation of PMP-aldoses.

Fig. 3 Electropherogram of PMP-aldoses and ABEE-aldoses by CZE. A: PMP-aldoses were separated in phosphate buffer; B: PMP-aldoses were separated in acetate buffer; C: ABEE-aldoses separated in phosphate buffer. Other conditions were the same as Fig. 1. Peaks: Ara = arabinose, Gal = galactose, Xyl = xylose, Glc = glucose, Rha = rhamnose, Man = mannose, R = PMP, R′ = ABEE.

Second, the derivative reagent PMP was replaced by ABEE, and other experimental parameters were the same as Fig. 1. Fig. 3A and 3C showed the separation of aldoses using the two different derivative reagents. Obviously, the long migration times of mannose and rhamnose didn't appear in Fig. 3C. So the PMP derivative reagent must be the key factor that led to the unusual separation behavior of aldoses at pH 2.5.

Study on the separation mechanism of PMP-aldoses at pH 2.5

In the acid buffer, the positive charge of PMP or PMP-aldose was provided by H⁺ in the running buffer combining with nitrogen atoms in the pyrazolone ring.¹⁸

In the structure of PMP, conjugated double bonds in the pyrazolone ring will make the enol group be the preferred conformation to the keto group.¹⁹ Moreover, the conjugated double bonds will combine with a nitrogen atom in the pyrazolone ring which has a lone pair of electrons on the 2p orbital to form a big π bond conjugate system.¹⁸ This π bond conjugate will weaken the H⁺ combining with nitrogen atoms, and further impair the positive charge at the nitrogen atoms and accordingly decrease the total positive charge of PMP-aldose. However, there are rings¹⁷ between the keto group in the pyrazolone ring and the hydroxyl groups at C-2 and C-3 in the carbohydrate moiety, and the ring will make the keto group advantageous and enol group disadvantageous. Then, the combining capacity of nitrogen atoms with H⁺ will be promoted by the ring formation, and the positive charge of PMP will be enhanced. PMP-aldose with 2,3-trans disposition will be favorable for ring formation and have more positive charge. As Fig. 2B showed, comparing with the other four aldoses, the C-2 and C-3 hydroxyl groups of mannose and rhamnose were cis-configuration, so they should have less positive charge and their migration times would be longer than that of the other four aldoses (Fig. 4).

Fig. 4 Charged mechanisms of PMP-mannose and PMP-glucose in the acidic buffer.

Comparing Fig. 3A with Fig. 5A, we could see that the reduced mobility of PMP derivatives of 2,3-cis aldoses compared to those of 2,3-trans aldoses was more apparent at low pH than at pH 7.5. At pH 7.5, the dissociation of the keto group in PMP-aldoses made them carry negative charges, and this led to their reverse migration. However, the dissociation in weaker alkaline running buffer might be very weak, and it was significantly different from their carrying more positive charges at pH 2.5. Then, even though there was a difference in intramolecular ring formation between the PMP derivatives of 2,3-cis aldoses and 2,3-trans aldoses which affected the dissociation of the keto group, the mobility would be more or less similar. So the reduced mobility of PMP derivatives of 2,3-cis aldoses compared to 2,3-trans aldoses was less apparent at pH 7.5 than at pH 2.5. In addition, the stronger EOF at pH 7.5 might be another reason that led to the phenomenon.

Fig. 5 Electropherogram of PMP-aldohexoses and PMP-tetramethyl aldohexoses by CZE. A: PMP-aldohexoses; B: PMP-tetramethyl aldohexoses. Running buffer, 50 mM phosphate buffer, pH 7.5; voltage, 15 kV; hydrodynamic pressure injection, 10 cm × 2 s. Peaks: Gal = galactose, Glc = glucose, Man = mannose, MGal = 2,3,4,6-tetramethyl-D-galactose, MGlc = 2,3,4,6-tetramethyl-D-glucose, MMan = 2,3,4,6-tetramethyl-D-mannose, R = PMP.

Verification of the intramolecular ring formation

The intramolecular ring formation was a hypothesis and needs further evidence. Honda's H¹NMR data²⁰ and Yamamoto's computer simulation analysis²¹ could verify the exist of an intramolecular ring to a certain extent. Now, we have taken another experiment to verify it further.

The electropherogram of PMP derivatives of three tetramethyl aldohexoses was shown in Fig. 5B. Comparing with the aldohexoses (Fig. 5A), the peaks of the three tetramethyl aldohexoses were overlapped completely, and the long migration time of PMP-mannose had disappeared when mannose was methylated.

Comparing with the C-2 and C-3 hydroxyl groups of aldohexose, C-2 and C-3 are methoxy groups in tetramethyl aldohexose. Without C-2 and C-3 hydroxyl groups, there will be no intramolecular rings in PMP-tetramethyl aldohexose. So the migration times of PMP-tetramethyl mannose, PMP-tetramethyl galactose and PMP-tetramethyl glucose were equivalent. So, it could be confirmed that the long migration time of PMP-mannose was due to the rings forming by hydrogen bonding between the keto group in the pyrazolone ring and the hydroxyl groups at C-2 and C-3 in the carbohydrate moiety.

Conclusions

The phenomenon that the migration times of mannose and rhamnose were even longer than that of DP 7 maltooligosaccharide was due to PMP derivatization which made monosaccharides with different C-2 and C-3 hydroxyl groups carrying different amount of positive charge. So when traditional Chinese medicine oligosaccharides are separated by CZE, we should notice that the component having a long migration time might be not the oligosaccharide of high molecular weight, it might be mannose or rhamnose. In addition, although an oligosaccharide with a mannose or rhamnose end-group has a low molecular weight, it might have a long migration time. At present, research to identify the kind of end-group monosaccharide of oligosaccharides by CE is being carried out. Combined with the molecular weights screening of oligosaccharides, it will promote the quantitative structure-active relationship (QSAR) study of active oligosaccharide fragments.

Acknowledgements

G. H. Z. is grateful for support from National Natural Science Foundation of China under grant No. 20905019 and No. 21011140338, Natural Science Foundation of Hebei Province under grant No. B2010000209 and Natural Science Foundation of Hebei University under grant No. 2007-111.

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