Biomacromolecules 2002, 3, 889-893
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Monitoring the Enzymatic Polymerization of 4-Phenylphenol by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry: A Novel Approach Peng Xu,† Jayant Kumar,† Lynne Samuelson,‡ and Ashok L. Cholli*,† Center for Advanced Materials, Departments of Chemistry and Physics, University of Massachusetts Lowell, Lowell, Massachusetts 01854, and Natick Soldier Center, U.S. Army SBCCOM, Natick, Massachusetts 01760 Received April 30, 2002; Revised Manuscript Received June 4, 2002
Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is a powerful tool for polymer characterization. It has been used to understand the enzymatic polymerization of 4-phenylphenol and to monitor number average molecular weight and weight average molecular weight of the polymer as a function of systematic addition of hydrogen peroxide (H2O2) in the reaction. A novel method, an introduction of internal standard for quantification of data, has been developed for MALDITOF MS to investigate the fate of each mers during the reaction. The preliminary data suggest that this approach provides new insight on the enzymatic synthesis, which is not available by other techniques. For the first time, we are able to understand the fate of several mers as a function of reaction conditions. The relative content of each mer increases with the addition of H2O2, except for dimer and trimer. For example, the concentration of dimer species decreases as a function of H2O2. On the other hand, the concentration of trimer species increases first and then decreases in the course of the reaction. Introduction Phenolic polymers are commercially important materials. A series of phenolic polymers and phenol formaldehyde resins are widely used in a number of electronic and industrial applications. Compared to traditional methods, a promising alternative approach to the synthesis of these polymers is the use of peroxide as an oxidant in the presence of enzymes as catalysts. Enzymatic polymerization reactions are advantageous in that they can offer simple and environmentally friendly reaction conditions. They are normally carried out at room temperature and atmospheric pressure, and pH ranges from 5 to 7.5.1-3 Enzymatic polymerization of 4-phenylphenol proceeds primarily through ortho couplings, along with some coupling through the hydroxyl group to form C-O-C coupling (Scheme 1). It is important to understand the molecular mechanism of enzymatically synthesized polymers so that their desired properties can be improved. Many efforts have been made to measure the molecular weight of polyphenols by various techniques.4 The most commonly used method is gel permeation chromatography (GPC). Due to the rigid nature of their backbones, polyphenols adopt a more rodlike conformation in solution.5 GPC, which is size dependent, will overestimate the molecular weight. It also suffers from additional problems related to the difficulties of obtaining standards that have characteristics similar to those being investigated. Furthermore, conditions must be such that the * To whom correspondence should be sent. † University of Massachusetts. ‡ Natick Soldier Center.
Scheme 1
stationary phase of the GPC column does not interact with the species being analyzed.4 Since its introduction, matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry has became a popular and powerful tool in determining the molecular weight of polymers and biomolecules.6,7 For polymers, it also provides polydispersity. MALDI provides a soft ionization method with very little fragmentation for nonvolatile analyte. It utilizes a pulsed laser beam to ionize the sample. The matrix separates and isolates the sample. It absorbs energy and desorbs to the analyte.8 The laser beam is focused on the surface of the sample and ionizes the molecules. The ionized molecules can be directed to the detector. With the introduction of MALDI-TOF mass spectrometry, it is possible to desorb large, low-volatile polymers up to 106 Da. Unlike other characterization methods such as GPC, high-performance liquid chromatography, gas chromatography, UV, etc., that are well developed for quantitative analysis, the quantitative aspects of MALDI-TOF mass analysis are yet to be developed fully.9 Some efforts have been made to quantitatively analyze biological samples and natural products.10-13
10.1021/bm0255600 CCC: $22.00 © 2002 American Chemical Society Published on Web 07/20/2002
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In this work, effort has been made to understand the mechanism of enzymatically polymerized 4-phenylphenol. The goal was to monitor the changes in molecular weight as a function of the addition of the oxidant (H2O2) and the influence of the concentration of organic solvent in the reaction mixture on the polymerization and to follow the trends for each mers growth. A novel approach has been developed to quantify the MS data by introducing an internal standard. Experimental Section Materials. Horseradish peroxidase (HRP) (EC 1.11.1.7) (200 units/mg) was purchased from Sigma with RZ > 2.2. A stock solution of 10 mg/mL in distilled water was prepared. Sodium phosphate was purchased from Sigma. A stock solution of 0.1 M at pH 6.0 was prepared. The monomer 4-phenylphenol was purchased from Aldrich and used as received. The oxidant, H2O2 (50 wt % solution in water), was purchased from Aldrich, and a diluted solution of 0.2 M was prepared. The matrix dithranol and the internal standard pentadecanoic acid were purchased from Aldrich and used as received. All other chemicals and solvents used were commercially available, of analytical grade or better, and used as received. MALDI-TOF Mass Spectrometry. All data and spectra presented in this work were obtained on a Bruker Reflex mass spectrometer. This spectrometer was equipped with a nitrogen laser of 337 nm, and 5 ns pulse width. The working vacuum was approximately 6.5 × 10-6 Torr. The detector worked in the linear mode with negative potential. An acceleration voltage of 20 kV was applied. The obtained mass spectra were generated by the accumulation of 150 shots. Calibration of the spectrometer was performed using a polystyrene standard sample with molecular weight of 2200 Da. HRP-Catalyzed Polymerization. Enzymatic polymerization of 4-phenylphenol was carried out at room temperature in a 10 mL, 50% acetone and 50% 0.01 M sodium phosphate buffer mixture, which contained 170 mg of 4-phenylphenol. To this solution, 100 µL of HRP stock solution was added. The reaction was initiated by the addition of H2O2. A stoichiometric amount of H2O2 dilute solution (0.2 M) was added incrementally under vigorous stirring over 3 h. After the addition of hydrogen peroxide, the reaction was left stirring for an additional hour. The grayish green precipitates formed during the reaction were then collected after centrifugal sedimentation, washed thoroughly with the mixed solvent of 25% acetone and 75% water (v/v) to remove any residual enzyme, sodium salt, and unreacted monomer, and then dried. MALDI-TOF Measurement. Dithranol was used as the matrix, which was dissolved in THF at a concentration of 10 mg/mL. The sample solutions were prepared at 10 mg/ mL in THF. An internal standard solution was prepared by dissolving pentadecanoic acid in THF at the concentration of 1 mg/mL. The solution for MALDI-TOF measurement was prepared by mixing solutions of matrix, internal standard, and sample at a volumetric ratio of 1:1:1. To avoid
Figure 1. MALDI-TOF mass spectrum of poly(p-phenylphenol) with internal standard of pentadecanoic acid.
fragmentation in MALDI-TOF MS measurements of poly(4-phenylphenol), the laser power required for the desorption/ ionization process was carefully adjusted slightly above the threshold. Results and Discussions Horseradish peroxidase catalyzed polymerization of phenols has been extensively studied in recent years.1,3 These studies also include understanding the kinetics aspect of the reactions. The reaction mechanism is known to involve free radical processes.3 It appears that this is a radical polymerization starting from the formation of dimer, trimer, and oligomers, etc. It is not clearly understood the distribution of these components and the influence of experimental conditions on them. The main focus of the present work is to investigate the distribution of mers and the effect of experimental conditions on the distribution. The MALDI-TOF mass spectrometer was used to monitor the reaction process. A method was designed for successive sampling by MALDI-TOF MS during the reaction period. Ten individual samples were prepared under identical experimental conditions with the addition of H2O2 from 10% to 100% of the total stoichiometric amount of H2O2. These 10 samples were designed to create a reaction system as if the continuous reaction was monitored at every incremental addition of H2O2. At the present time, it is not possible to use MALDI-TOF MS for real time in situ experiments, as development of sample handling techniques is required to perform in situ measurements. Grayish green powder samples were obtained after removing the solvent and then redissolved in THF for the MALDI-TOF measurement. Figure 1 is a typical MS spectrum showing the constituents of the sample. This spectrum gives the whole distribution of poly(4-phenylphenol) from dimer to the highest molecular weight of up to 4500 Da. The m/z value of each adjacent peak is about 168 and exactly matches the molar mass of the repeat units of the poly(4-phenylphenol). The number average and weight average molecular weight and the polydispersity can be determined by MALDI-TOF measurements. The estimated polydispersity is about 1.3. The
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Figure 2. Molecular weight and polydispersity changes during the course of enzymatic polymerization of 4-phenylphenol as a function of the amount of H2O2 added.
limitation of MALDI TOF is that it may not provide accurate results for polymer samples having a wide polydispersity. If the polydispersity is very wide, the high molecular weight part or the low molecular weight part of the sample may be omitted due to its sensitivity and issues associated with the dynamic range. The changes in the molecular weight and the polydispersity during the course of the reaction are plotted in Figure 2 as a function of percentage amount of H2O2 added to the stoichiometric amount. Figure 2 suggests the number average and weight average molecular weight increase quickly during the initial stage and continue up to 60% of the total amount of H2O2 until a plateau is reached. When the reaction appears to be complete, the polymers are dominant and the concentration of the monomer is very low. It is difficult for a macromolecular radical to react with a monomer in the concentrated solution. The reaction media was 50% acetone and 50% water (v/v) mixture. The molecular weight cannot grow indefinitely. When the molecular weight is high enough, polymer will precipitate from the reaction solution. In the free radical chain reaction, prolonging the reaction time will increase the yield and not the molecular weight. Therefore during the final stage of the reaction, addition of H2O2 will not increase the molecular weight. Quantitative analysis is another important aspect of the MALDI-TOF characterization and is in the early stages of development. Some efforts have been made in the past few years toward this on biomolecules or natural product analysis and some concerned polymers.15,16 To know quantitatively the presence of dimer, trimer, tetramer, etc., in an individual sample and how the quantities of each mers change with the addition of H2O2, development of a quantitative approach is very essential. The concentration of each component is reflected by the MALDI signal intensity. It is not possible to use the absolute value of the signal intensity for quantitation in the present work. Since it depends on the amount of the sample used for the measurement and the number of shots accumulated in the MALDI data collection, an internal standard is necessary. By introduction of a known amount of internal standard, the signal intensity of the analyte will be normalized to that of the internal standard and make the
Figure 3. Internal standard normalized distributions of each repeat unit for poly(4-phenylphenol) as a function of increasing addition of H2O2.
relative content of each component comparable between different samples. We have screened several potential internal standard compounds for this study. Experience gathered during this screening process of internal standards suggests that the following guidelines should be applied in selecting a suitable internal standard: (i) the MS peaks arising from the internal standard should not interfere with the spectral profile of the sample under study; (ii) the sample should not disintegrate due to laser ionization, thus avoiding interference with the spectral data of the sample; (iii) the internal standard sample should dissolve in the solvent selected for analyte and the matrix; (iv) the molecular weight of the internal standard should be between that of matrix and the dimeric species of the analyte; and (v) the molar ratio of internal standard: analyte:matrix should be 1:10:10 for optimal results. Pentadecanoic acid (with a molecular weight of 242.40) was selected as the internal standard for quantitative MALDI measurement in the present study. In Figure 1, the internal standard peak appears at 242.11. Although the desorption behavior of pentadecanoic acid differs from that of the analyte, its influence on all samples is the same. It not only is stable in MALDI measurement but also can help with charging of the analyte. Ten samples that were enzymatically synthesized with different amounts of H2O2 were measured by a MALDITOF mass spectrometer. Each spectrum was normalized for signal intensity of pentadecanoic acid. Repeat units from 2 to 15 were observed. The normalized signal intensity of each mer is plotted as a function of increasing addition of H2O2 and is presented in Figure 3. The normalization makes these spectra suitable for quantitative analysis. For a given H2O2 concentration, a distribution of each mers as shown is given in Figure 1. The concentration of each mers as a function of H2O2 increases except in the case of dimers and trimers. The relative content of dimers decreases with the addition of H2O2. The profile for trimers is an exceptional one. Its concentration quickly increases with the addition of H2O2
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Figure 4. Molecular weight and polydispersity changes in the HRPcatalyzed polymerization of 4-phenylphenol as a function of the acetone volume percentage content in the reaction media.
during the initial stage, then reaches a maximum at 0.3 (H2O2 to monomer ratio), and then decreases very quickly. This suggests that both dimers and trimers are actively consumed along with monomers in the reaction. It appears that the trimers are coupled to form hexamers or higher n-mers only after addition of 50% of the stoichiometric ratio of H2O2. On the other hand, all the n-mers (n > 4) tend to increase in concentration as a function of added H2O2. In the HRP-catalyzed polymerization of 4-phenylphenol, mixed solvent plays an important role because the HRP is water soluble and the monomer only organic solvent soluble. Many research studies indicate the influence of the solvent on the reaction. A major limitation of the enzymatic polymerization in pure aqueous solutions is that only low molecular weight oligomers are formed.3 Additional studies suggest that the solubility of monomers and the molecular weight of polymers increase significantly with introduction of organic solvent that is miscible in water, despite the decrease in the activity of HRP. In the present work, the HRP-catalyzed polymerization of 4-phenylphenol is carried out in the acetone-water media. Ten samples were prepared with the acetone content varying from 10% to 100% (volume percentage). The concentration of the monomer was 0.1 mM/mL. Saturated solution was prepared when the solubility of 4-phenylphenol is less than 0.1 mM/mL. The molecular weights of final products were determined by MALDI-TOF. Pentadecanoic acid was used as an internal standard during the measurement. Figure 4 shows the changes in the molecular weight and polydispersity that are plotted as a function of acetone concentration in the reaction media. The plots in Figure 4 suggest that the molecular weight reachs the maximum when the acetone content is 50% in the acetone-water mixture. The weight average molecular weight is over 2400 Da with 50% acetone, which coincides with the weight average molecular weight at the point when 100% of the H2O2 is added, as shown in Figure 2. Acetone is a good solvent for both the monomer and the polymer. When the acetone content was lower than 40%, 4-phenylphenol could not reach the concentration of 0.1 mM/mL. Even though 4-phenylphenol is nearly saturated in the initial stage of the reaction, its concentration is quite low due to
Figure 5. Internal standard normalized distributions of each repeat unit for poly(4-phenylphenol) as a function of the increment of acetone content in reaction media.
its solubility. The polymer chain cannot grow further in such a dilute solution, even when the HRP has a relatively higher activity, because the macromolecular radical has difficulty finding the monomer radical to continue to grow. When the acetone content is above 60%, the molecular weight decreases because the activity of the HRP decreases dramatically when the organic solvent content is high in the reaction media. Due to the decreased activity of the enzyme, the reaction rate is very slow. It is more apparent when the acetone content is beyond 80% where the molecular weight decreases dramatically. The internal standard method was also used to monitor the concentration changes of each mers as a function of acetone content in the mixed solvent medium. These data are presented in Figure 5. The maximum relative content is obtained from oligomers with eight or nine units. For each mer, its relative content increased with the increment of the acetone content and reached the maximum at 50% and then decreased. Only the dimer and trimer increased again after the acetone content reached 80%. The exceptional high contents of dimer and trimer may be due to the dramatic decrease in the enzyme activity. Under these reaction conditions, many dimers and trimers are left in the solution and cannot be converted into oligomers or polymers. Conclusions MALDI-TOF mass spectrometry, a powerful tool for polymer characterization, has been applied to monitor the enzymatic polymerization of 4-phenylphenol. A novel method, introduction of internal standard, has been developed for MALDI-TOF MS to investigate the fate of each mers during the reaction. The changes of the molecular weight and polydispersity are monitored during the course of the polymerization. The preliminary results suggest that 50% of acetone in the reaction media is probably the optimized
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reaction condition for HRP-catalyzed polymerization of 4-phenylphenol. Acknowledgment. We thank the National Science Foundation (NSF) for funding this project through research grant (DMR-9986644). We sincerely acknowledge Dr. David Kaplan and Dr. Amarjit Singh (Department of Chemical & Biological Engineering, Tufts University). We express our gratitude to Professor Kaplan for the use of his MALDITOF MS research facilities. We also thank Dr. Singh for MALDI instrument training. This work could not be accomplished without their help. References and Notes (1) Bruno, F. F.; Nagarajan, R.; Sidhartha, J. S.; Yang, K.; Kumar, J.; Tripathy, S.; Samuelson, L. Proceedings of the 58th Annual Technical Conference & Exhibits 2000, 2336. (2) Akkara, J. A.; Senecal, K. J.; Kaplan, D. L. J. Polym. Sci. Polym. Chem. 1991, 29, 1561-1574. (3) Liu, W.; Bian, S.; Li, L.; Samuelson, L.; Kumar, J.; Tripathy, S. K. Chem. Mater. 2000, 12, 1577.
Biomacromolecules, Vol. 3, No. 5, 2002 893 (4) Folch, I.; Borros, S.; Amabilino, D. B.; Veciana, J. J. Mass. Spectrom. 2000, 35, 550. (5) Liu, J.; Loewe, R. S.; McCullough, R. D. Macromolecules 1999, 32, 5777. (6) Hillenkamp, F.; Karas, M. Int. J. Mass Spectrom. 2000, 200, 7177. (7) Karas, M.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1989, 92, 231. (8) Hanton, S. D. Chem. ReV. 2001, 101, 527. (9) Remmers, M.; Muller, B.; Martin, K.; Rader, H. J.; Kohler, W. Macromolecules 1999, 32, 1073. (10) Sarracino, D.; Richert, C. Bioorg. Med. Chem. Lett. 1996, 6, 2543. (11) Bartsch, H.; Koenig, W.; Strabener, M.; Hintze, U. Carbohydr. Res. 1996, 286, 41. (12) Benard, S.; Arnhold, J.; Lehnert, M.; Schiller, J.; Arnold, K. Chem. Phys. Lipids 1999, 100, 115. (13) Tang, X.; Sadeghi, M.; Olumee, Z.; Vertes, A.; Braatz, J. A.; Mcllwain, L. K.; Dreifuss, P. A. Anal. Chem. 1996, 68, 3740. (14) De Angelis, F.; Fregonese, P.; Veri, F. Rapid Commun. Mass Spectrom. 1996, 10, 1304-1308. (15) McEwen, C. N.; Jackson, C.; Larsen, B. S. Int. J. Mass Spectrom. Ion Processes 1997, 160, 387-394. (16) Zhu, H.; Yalcin, T.; Li, L. J. Am. Soc. Mass Spectrom. 1998, 9, 275-281.
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