Article pubs.acs.org/Biomac
Short One-Pot Chemo-Enzymatic Synthesis of L‑Lysine and L‑Alanine Diblock Co-Oligopeptides Jenny Fagerland,†,‡ Anna Finne-Wistrand,*,† and Keiji Numata*,‡ †
Department of Fibre and Polymer Technology, School of Chemical Science and Engineering, KTH Royal Institute of Technology, SE-100 44, Stockholm, Sweden ‡ Enzyme Research Team, RIKEN Biomass Engineering Program, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan S Supporting Information *
ABSTRACT: Amphiphilic diblock co-oligopeptides are interesting and functional macromolecular materials for biomedical applications because of their self-assembling properties. Here, we developed a synthesis method for diblock co-oligopeptides by using chemo-enzymatic polymerization, which was a relatively short (30 min) and efficient reaction (over 40% yield). Block and random oligo(L-lysine-co-L-alanine) [oligo(Lys-co-Ala)] were synthesized using activated papain as enzymatic catalyst. The reaction time was optimized according to kinetic studies of oligo(L-alanine) and oligo(L-lysine). Using 1H NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we confirmed that diblock and random co-oligopeptides were synthesized. Optical microscopy further revealed differences in the crystalline morphology between random and block co-oligopeptides. Plate-like, hexagonal, and hollow crystals were formed due to the strong impact of the monomer distribution and pH of the solution. The different crystalline structures open up interesting possibilities to form materials for both tissue engineering and controlled drug/gene delivery systems.
■
applications.15 In addition, long hydrophobic or cationic sequences are synthesized much less efficiently by this method. Over the last few decades, protease-catalyzed chemoenzymatic synthesis of oligopeptides has been shown to be a promising option in order to obtain cost-effective, environment-friendly (without toxic chemicals), and high-purity peptides with a relatively high productivity. The reaction is either a thermodynamically controlled process, where the enzyme accelerates the attainment of equilibrium, or a kinetically controlled process. The latter reaction is more selective; the enzyme needs to react with an ester compound and form an acyl-enzyme intermediate. Then, in competition with water, it will react with an amino acid-derived nucleophile and form a new peptide bond.16 This synthesis method has been used by several researchers to obtain homooligopeptides14,17,18 and co-oligopeptides.19 Unfortunately, the method gives low yields and low-molecular-weight products. In our previous work, we improved the molecular weight and yield of homooligopeptides of L-alanine by using papain as enzymatic catalyst and found that self-assembled fibrils were formed when the pH was increased.20 The natural behavior of oligo(Lalanine) (OligoAla) to self-assemble into fibrils is an interesting feature that gives a strong natural material similar to spider dragline silk. Furthermore, we performed a chemo-enzymatic synthesis by using L-phenylalanine and tris(2-aminoethyl)amine as a monomer and terminator, respectively, and successfully obtained branched peptides.21 Combined with other amino
INTRODUCTION Synthesis and design of 3D scaffolds for tissue regeneration have been in focus ever since Langer and Vacanti first used the concept of “tissue engineering”.1 Functionalized aliphatic copolyesters2−4 and injectable thermosensitive hydrogels5 are examples of such materials, where the design and properties have been tailored to obtain better tissue regeneration. Selfassembling polypeptides are another example of a biomaterial that has been studied intensively for tissue engineering using hydrogel and scaffolds,6,7 as well as drug/gene delivery systems using micelle.8−11 Compared to synthetic polymers, peptidebased materials are naturally bioactive, resulting in better cell attachment and controlled cell biodegradability where the degradation products are metabolized. 12 However, the preparation of these materials has been challenging over the years because the synthetic reactions are limited in terms of amino acid sequence, yield, and purity of the products. Solid phase polypeptide synthesis (SPPS), ring-opening polymerization of α-amino acid N-carboxyanhydrides, and recombinant DNA methods are three major synthesis approaches that have been developed to overcome these problems. They give precise control of the monomer sequence and chain length as well as high purity. However, these methods also have some drawbacks. SPPS shows relatively low yield, is time-inefficient, and is an environmentally unfriendly method involving toxic solvents and complicated protection and deprotection chemistry.13 For N-carboxyanhydride ring-opening polymerization, high-purity monomers, and strict removal of water are required. The synthesis also requires toxic phosgene equivalents.14 Recombinant DNA methods can be laborious and give peptides that are difficult to purify to a degree suitable for in vivo © 2014 American Chemical Society
Received: October 14, 2013 Revised: January 22, 2014 Published: February 2, 2014 735
dx.doi.org/10.1021/bm4015254 | Biomacromolecules 2014, 15, 735−743
Biomacromolecules
Article
MALDI-TOF-MS. A MALDI-TOF-MS system (Autoflex Speed; Bruker, Bremen, Germany) was used to analyze the molecular weight and degree of oligomerization of the samples. A scoutMTP ion source was used for the analysis in reflector mode using a nitrogen laser and data were acquired with the Flex control software. α-Cyano-4-hydroxycinnamic acid was used as matrix, and the analysis was run in positiveion mode. Before analysis, a matrix solution (10 mg/250 μL) was prepared in a 1:1 mixture of acetonitrile and water with 0.1% trifluoroacetic acid. The solution was then ultrasonicated for 20 min and centrifuged for 10 min to obtain a clear, saturated solution. The samples were dissolved in a 1:1 mixture of acetonitrile and water (1 mg/mL); a ground steel plate (MTG 384 TF) was then used as target plate. Matrix and sample solutions were spotted onto the target plate by using the layer-by-layer technique. Spectra were obtained from 500 accumulations taken by a sample carrier (50 spots over a limited area [diameter 2000 μm]). The range of the mass-to-charge ratio was 0− 2000 m/z with matrix suppression (deflection 450 Da). The maximum molecular weight, which was the highest molecular weight obtained in the spectra of the samples, was determined. NMR. 1H NMR spectra were obtained with a Varian system 500 NMR spectrometer (500 MHz) with the VnmrJ software (Agilent Technologies, Santa Clara, CA). Deuterium oxide (D2O) or dimethyl sulfoxide-d6 (DMSO-d6) was used to prepare the samples, 5 mg/mL, in NMR glass tubes (5 mm). The samples were analyzed at 25 °C. Water was used as internal standard (ppm: 4.75) for the D2O samples and tetramethylsilane (ppm: 0) was used as the internal standard for the DMSO-d6 samples. The conversion was determined by 1H NMR spectroscopy. It was calculated as peak area of the oligopeptide (Aoligopeptide) divided by the peak area of the oligopeptides and monomer (Aoligopeptide + Amonomer) as follows:
acids and the right configuration, it can give promising materials for biomedical applications. Here, we present the synthesis of amphiphilic diblock cooligopeptides of L-alanine and L-lysine, oligo(Lys-b-Ala), by using activated papain. Random oligo(Lys-co-Ala) [oligo(Lys-rAla)] was also synthesized and used as negative reference. The oligomerization kinetics of monomers and the chemical structure and crystalline morphology of the synthesized cooligopeptides were analyzed by proton nuclear magnetic resonance spectroscopy (1H NMR), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and optical microscopy. We hypothesized that the properties of the amino acids together with the blockness and randomness will result in a self-assembling structure that can be used in biomedical applications.
■
EXPERIMENTAL SECTION
Materials. L-Lysine ethyl ester complexed with HCl (Lys-Et·2HCl) and L-alanine ethyl ester complexed with (Ala-Et·HCl) were purchased from Sigma Aldrich (St. Louis, MO) and used as received. Papain was purchased from Wako Pure Chemicals (Osaka, Japan). Before being used, papain was activated and purified as follows: the enzyme was suspended in Milli-Q water, followed by centrifugation at 12000 × g for 30 min. The precipitate was then separated from the enzyme, frozen, and lyophilized for 24 h. Solid-phase-synthesized diblock and alternating peptides of L-lysine and L-alanine were synthesized using the standard 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis. The peptides were purified using high-performance liquid chromatography, and the molecular weights were confirmed by MALDI-TOF-MS. Kinetic Study of Oligo(L-alanine) and Oligo(L-lysine) Syntheses. Chemo-enzymatic synthesis of OligoAla and oligo(L-lysine) (OligoLys) were performed to monitor monomer conversion, pH, and maximum and peak molecular weights for 24 h. Activated papain was used as catalyst. The synthesis of OligoAla was carried out according to a previously described method.20 Briefly, a 0.7 M solution of Ala-Et in 1.0 M phosphate buffer solution (PBS) was prepared in an EYELA ChemiStation (Tokyo Rikakikai Co., Tokyo, Japan) at 40 °C. The activated papain (7.0 mg/mL) was dissolved in 1 mL of PBS and then added to the reaction. OligoLys was prepared similar to OligoAla, except that the concentration of Lys-Et was set to 0.5 M, according to a previous study,14 to enhance the conversion. The pH of the reactions was adjusted to 7.6. To determine an optimal reaction time for cooligomer synthesis, molecular weights of the products and conversion of the monomers were monitored. A sample (20 μL) was taken from the solutions after 5, 10, 15, 20, 30, 40, 50, 60, 90, and 120 min, every hour until 8, 18, and 24 h. Synthesis of Oligo(Ala-b-Lys) and Oligo(Ala-r-Lys). Synthesis of diblock and random oligo(Ala-co-Lys) were prepared by the same synthesis method as described earlier in the kinetic study. The diblock co-oligopeptide was prepared in two steps with a monomer molar ratio of 1.4:1 (Ala/Lys). The ratio was set according to previous studies to obtain the highest monomer conversion.14,20 In the first step, 865 mg of Lys-Et was dissolved in 7 mL of 1 M PBS (pH 7.6) in a 15 mL glass tube. Activated papain (7.0 mg/mL) was then added. The reaction was allowed to proceed for 15 min; in the second step, 753 mg of Ala-Et was added to the reaction. After another 15 min, the reaction was stopped by removing the enzyme from the solution using centrifugal filters (Amicon Ultra-4, MWCO: 10000 g/mol, Merck Millipore, Germany). The product was then purified by dialysis (MWCO of 500−100 g/mol) and water was removed by lyophilization. For the preparation of the random co-oligopeptide, equal molar amounts of Ala-Et and Lys-Et were dissolved in 7 mL of PBS (pH 7.6) in a 15 mL glass tube. The reaction time was set to 30 min and the product was purified in the same way as the block co-oligopeptide. Both reaction mixtures were kept under magnetic stirring at 40 °C in an EYELA ChemiStation. The products were analyzed with MALDI-TOF-MS, 1 H NMR, and optical microscopy.
conversion = Aoligopeptide /(Aoligopeptide + A monomer ) For the calculations of the conversion of Ala-Et, the α-proton in Ala-Et showed different shift compared to the α-proton of free alanine because of their different end groups. In the case of lysine and Lys-Et, the chemical shifts of the α-proton slightly differed. Based on the assignment shown in the results, free alanine and lysine were not included into our calculations. The number-average molecular weight (Mn) was calculated based on the peak areas of methyl protons of alanine and ε-methylene of lysine as well as the methin proton of lysine and alanine at the chain end. The equation to obtain Mn is as follows: M n = 71 g/mol × A(methyl protons of alanine) /3A(methin proton of lysine or alanine at the chain end) + 128 g/mol × A(e − methylene of lysine)/2A(methin proton of lysine or alanine at the chain end) where A indicates each peak area, 71 and 128 g/mol denote each molecular weight of alanine and lysine unit, respectively. Optical Microscopy. Optical microscopy images of the crystal morphology of the random and diblock co-oligopeptides were obtained with an Olympus BX 51 optical microscope (Olympus, Tokyo, Japan). Sample solutions (60 wt %) were analyzed at pH 3 and 7.4. Solutions of pH 7.4 were prepared by dissolution in PBS (pH: 7.4, 0.1 M) and Milli-Q water; those of pH 3 were obtained by addition of 1 mL of 5 M HCl. The pH was determined with pH indicator paper. The samples were placed directly on the glass slide and fixed with a coverslip. Images were taken with an Olympus DP 21 camera directly after preparation and after 12 h. Cell Viability. To evaluate the viability of cells cultured on diblock and random co-oligopeptides, human mesenchymal stem cells (Lonza Walkersville Inc., Walkerville, MD) were cultured in 96-well plates (ca. 8000 cells/well) coated with films and crystals of oligo(Lys-b-Ala) and oligo(Lys-r-Ala). Crystals were prepared at pH 3 and 7.4. Films of the co-oligopeptides were prepared at pH 10. The concentrations of the co-oligopeptides were 0.6, 3.0, and 6.0 mg/well, that is, 3, 15, and 30 g/L. The crystals and films of the co-oligopeptides were washed with Milli-Q twice before the cell viability assay was carried out. The cells 736
dx.doi.org/10.1021/bm4015254 | Biomacromolecules 2014, 15, 735−743
Biomacromolecules
Article
Figure 1. Reaction schemes of OligoAla (A), OligoLys (B), Oligo(Lys-b-Ala) (C), and Oligo(Lys-r-Ala) (D), (E) Picture of OligoLys (to the left) and OligoAla (to the right) in phosphate buffer solution after 24 h of synthesis. were cultured for 48 h in 100 μL of Dulbecco’s modified Eagle’s medium on co-oligopeptide crystals or films, according to previous studies.22 The film was dissolved into the media after several minutes and became a water-soluble peptides. Cell viability was measured using a standard 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2H-tetrazolium (MTS) assay (Promega, Madison, WI) according to the manufacturer’s instructions (n = 4). Cell viability was calculated according to the following equation: (Cell viability, %) = (absorbance at 490 nm of the cell culture incubated on oligo[Lys-b-Ala] or oligo[Lys-r-Ala])/(absorbance at 490 nm of the cell culture incubated on a uncoated 96-well cell culture plate [positive control]) × 100 Statistical differences in the cell viability were determined using an unpaired t-test with a two-tailed distribution. Differences were considered statistically significant at p < 0.05. Data from the cell viability assay are expressed as means ± standard deviation.
the homooligopeptides are presented in Figures 2 and 3. From the 1H NMR spectra, it can be concluded that OligoAla and OligoLys were successfully obtained. This was also shown in Figure 1E, which illustrates the solubility of the homooligopeptides in the buffer. OligoLys was soluble in the buffer, whereas OligoAla was insoluble. The results of the monomer conversion of Lys-Et to OligoLys and the hydrolysis of OligoAla to alanine are presented in Figures 4 and 5. The conversion of Ala-Et is not presented because complete conversion of the monomer was achieved within 15 min. The rapid conversion of Ala-Et compared to the slower conversion of Lys-Et can be explained by the properties of the enzyme catalyst, papain, used in the reaction. Papain has broad substrate specificity for peptide bonds but prefers amino acids with hydrophobic side groups (e.g., alanine, valine, and leucine). This also resulted in faster hydrolysis of the formed OligoAla. After 5 h, 50% of the oligopeptide was hydrolyzed to alanine (Figure 5). The conversion of Lys-Et to OligoLys was slow; a conversion of 40% was noted in the first 30 min and the conversion rate decreased at later time points. After 8 h, 60% conversion was achieved, and after 24 h, 80% conversion was achieved. The pH of the solutions was affected by conversion rate of the monomers. For the synthesis of OligoLys (where the
■
RESULTS Kinetic Study. Kinetic studies of OligoAla and OligoLys were performed to determine the optimal reaction parameters for the synthesis of oligo(Lys-b-Ala). The reaction scheme of the synthesis and OligoAla and OligoLys as reaction products are shown in Figure 1. Monomer conversion, pH, peak molecular weight, and maximum molecular weight were monitored over a period of 24 h in PBS at 40 °C. The conversion was determined by 1H NMR. 1H NMR spectra of 737
dx.doi.org/10.1021/bm4015254 | Biomacromolecules 2014, 15, 735−743
Biomacromolecules
Article
Figure 2. 1H NMR of OligoLys in D2O.
lysine and L-alanine. The reaction time of Ala-Et should be relatively short because complete conversion was achieved within 15 min and hydrolysis of the formed product should be avoided (Figure S2). The reaction time of Lys-Et could be longer; however, because the highest conversion rate was achieved during the first 30 min, and only small differences were noted in the molecular weight as the reaction time increased. Synthesis of Oligo(Lys-b-Ala) and Oligo(Lys-r-Ala). Oligo(Lys-b-Ala) was prepared by chemo-enzymatic synthesis using activated papain as catalyst. The reaction time and reaction temperature were 30 min and 40 °C, respectively. To obtain a block co-oligopeptide, Lys-Et was first added to the reaction and allowed to react for 15 min. Then, Ala-Et was added and the reaction was continued for another 15 min. Oligo(Lys-r-Ala) was prepared as negative reference to oligo(Lys-b-Ala). It was synthesized by adding the monomers at the same time and allowing them to react for 30 min. The reaction schemes of the co-oligopeptides can be seen in Figure 1. The products were analyzed by 1H NMR and MALDI-TOFMS (Figures 7 and 8). The pH of the reaction mixture decreased from 7.6 to 7.0 after both the reactions, which were not remarkable pH decreases to affect molecular weight and yield of the products. To confirm the successful synthesis of both random and diblock co-oligopeptides, references diblock and alternating co-oligopeptides of L-lysine and L-alanine were synthesized by SPPS and compared with the results obtained for oligo(Lys-b-Ala) and oligo(Lys-r-Ala).
conversion was slow), the pH was constant up to 50% conversion and then decreased slowly while for the synthesis of OligoAla it started to decrease as soon as all Ala-Et was converted to OligoAla (after 15 min). This is because of the HCl (complexed to the monomers), which was released to the solution as soon as the monomers reacted with the enzyme. The different conversion behaviors between OligoAla and OligoLys can be explained by the substrate specificity of papain rather than the pH effect, which will be discussed in the following Discussion section. The maximum molecular weights were determined by MALDI-TOF-MS and 1H NMR. The results are shown in Figures 6 and S1 and are consistent with the results from Figures 4 and 5. The maximum molecular weight of OligoLys was constant over 24 h, which can be explained by the slow conversion of Lys-Et to OligoLys. In contrast, OligoAla had the highest molecular weight during the first hour, but it decreased thereafter. The peak molecular weight was constant up to 8 h. Based on the conversion result, the substrate specificity was one of the important factors for the rate of conversion. According to the substrate specificity, papain prefers to hydrolyze OligoAla rather than OligoLys. Similar to the hydrolysis, papain preferred the polymerization of Ala-Et in compared with Lys-Et. Moreover, the solubility of OligoAla is lower than OligoLys in the buffer, resulting in the lower digestion rate and higher conversion rate of OligoAla. These results suggest that a short reaction time should be chosen for the synthesis of a diblock co-oligopeptides of L738
dx.doi.org/10.1021/bm4015254 | Biomacromolecules 2014, 15, 735−743
Biomacromolecules
Article
Figure 3. 1H NMR of OligoAla in DMSO-d6.
Figure 4. Conversion of Lys-Et to OligoLys (■) and pH (×) over a period of 24 h determined by 1H NMR.
Figure 5. Hydrolysis of OligoAla to alanine (○) and pH (×) over a period of 24 h determined by 1H NMR.
By referring to the 1H NMR and MALDI-TOF-MS results of the SPPS co-oligopeptide references (Figures S3 and S4), the chemical structures of oligo(Lys-b-Ala) and oligo(Lys-r-Ala) could be assigned. The monomer conversion of lysine and alanine were 47% and 100% for oligo(Lys-b-Ala) and 68 and 100% for oligo(Lys-r-Ala), respectively. The maximum molecular weight and Mn, determined by MALDI-TOF-MS and 1H NMR were 922 and 623 g/mol for oligo(Lys-b-Ala) and 851 and 624 g/mol for oligo(Lys-r-Ala), respectively. The results suggest that the diblock and random co-oligopeptides consisted of approximately two or three repeat units of lysine and three repeat units of alanine.
Figure 7 shows the 1H NMR spectra of oligo(Lys-b-Ala), oligo(Lys-r-Ala), OligoLys, and OligoAla. By comparison with the homo-oligopeptide spectra, it can be seen that the peaks of both alanine and lysine were present in the co-oligopeptides. In addition, the triplet peak and quartet peak in the area between 3.95−4.15 ppm shifted differently depending on the molecular structure of the co-oligopeptides. The distance between the triplet and quartet peaks in oligo(Lys-b-Ala) was 0.1 ppm, whereas that for oligo(Lys-r-Ala) was much smaller. This behavior was also observed in the reference 1H NMR spectra of the diblock and alternating co-oligopeptides synthesized by 739
dx.doi.org/10.1021/bm4015254 | Biomacromolecules 2014, 15, 735−743
Biomacromolecules
Article
the propagation from the amine in the R-group of lysine. The pKa value of the amine group is 10.54, which makes it fully protonated and a less effective nucleophile. The released HCl was expected to be buffered with 1 M buffers and did not yield significant pH change. If there was free alanine, free alanine would be protonated. However, almost all the alanine monomer must be Ala-Et rather than free alanine. Therefore, OligoAla was synthesized in the present study. From the MALDI-TOF-MS results of oligo(Lys-b-Ala) and oligo(Lys-r-Ala) (Figure 8), no clear differences were detected in the molecular weight between the spectra. This is also consistent with the MALDI-TOF-MS results of the SPPS reference co-oligopeptides (Figure S4). In both spectra, two strong mass spectrum patterns appear with repeat units of alanine (A). The two patterns have a mass difference that is consistent with two repeat units of lysine (K). This finding suggests that one of the patterns has one lysine and up to nine repeat units of alanine. The other pattern has three repeat units of lysine and four repeat units of alanine. These results also prove that the monomers oligomerized successfully. Generally, 1 H NMR spectra provide the average molecular weight of overall sample, while MALDI-TOF-MS shows the molecular weight at a certain part of the sample, that is, the result from MALDI-TOF-MS is not quantitative and does not represent
Figure 6. Maximum molecular weight (Mmax) of OligoLys (■) and OligoAla (○) determined by MALDI-TOF-MS.
SPPS (Figure S3). Diblock co-oligopeptides starting with lysine and ending with alanine showed the same characteristic distance between these peaks, whereas the peaks of the alternating co-oligopeptides almost overlapped in this region. The peaks of the random co-oligopeptides did not overlap but were not separated as much as the block co-oligopeptides (Figure 7A), suggesting that the structure was random (i.e., between alternating and diblock structures). The pH during propagation was maintained around neutral, which restricted
Figure 7. 1H NMR spectra of oligo(Lys-r-Ala) in D2O (A), oligo(Lys-b-Ala) in D2O (B), OligoLys in D2O (C), and OligoAla in dimethyl sulfoxided6 (D). 740
dx.doi.org/10.1021/bm4015254 | Biomacromolecules 2014, 15, 735−743
Biomacromolecules
Article
Figure 8. MALDI-TOF-MS spectra of oligo(Lys-b-Ala) (A) and oligo(Lys-r-Ala) (B). The distance labeled “A” represents the repeat units of alanine and the distance labeled “K” represents the repeat units of lysine.
and crystals of oligo(Lys-b-Ala) and oligo(Lys-r-Ala) at pH 7.4 was evaluated using an MTS assay (Figure 10A−C). We prepared four types of crystals at pH 3.0 and 7.4, as shown in Figure 9, and films of both co-oligopeptides at pH 10. The samples were washed with Milli-Q before the cell viability assay was carried out at pH 7.4. The films were dissolved into the media within several minutes and became a soluble peptides. The cell viability calculated from a positive control, that is, cells that were seeded on an uncoated cell culture plate and incubated for 48 h, was considered 100%. The results demonstrate that both random and diblock co-oligopeptides exhibited low cell cytotoxicity at concentrations below 15 g/L. Culturing on diblock co-oligopeptides resulted in slightly lower cell viability at all concentrations compared to random cooligopeptides.
the overall sample. In the present study, therefore, the polydispersity of co-oligopeptides yielded some differences in terms of molecular weight and amino acid composition between the results by 1H NMR and MALDI-TOF-MS. Crystalline Morphology of the Co-Oligopeptides. To further define the properties of oligo(Lys-b-Ala) and oligo(Lysr-Ala), the crystal morphology was analyzed using optical microscopy. The co-oligopeptides were studied in two different environments, that is, at pH 3 and 7.4. Figure 9A,B show oligo(Lys-b-Ala) at pH 3 and 7.4, and Figure 9C,D show oligo(Lys-r-Ala) at pH 3 and 7.4. The crystal morphologies differed because of the pH and the charged R-group on the lysine repeat units. In Figure 9A, a small branch originated from the inner surface of the crystal, which strongly suggests that cubic, hollow crystals were formed. These branches cannot be seen in irregularly shaped crystals at pH 7.4 (Figure 9B). On the other hand, oligo(Lys-r-Ala) did not form any cubic or hexagonal crystals but formed plate-like crystals at pH 3 and 7.4 (Figure 9C,D). Thus, obvious differences between oligo(Lys-bAla) and oligo(Lys-r-Ala) in terms of crystal morphology and assembly behavior were observed, supporting that oligo(Lys-bAla) and oligo(Lys-r-Ala) were successfully synthesized. Cytotoxicity of the Co-Oligopeptide Crystals. The viability of human mesenchymal stem cells cultured on films
■
DISCUSSION The results from the kinetic studies of OligoLys and OligoAla demonstrate that the conversion of Lys-Et and Ala-Et were strongly dependent on the substrate specificity of the enzymatic catalyst, activated papain, used in the reaction. The strong preference of papain for hydrophobic amino acids resulted in complete conversion of Ala-Et after only 15 min, whereas only 80% conversion was achieved after 24 h in case of Lys-Et. 741
dx.doi.org/10.1021/bm4015254 | Biomacromolecules 2014, 15, 735−743
Biomacromolecules
Article
hydrolysis. Lys-Et and Ala-Et, which were complexed with HCl, released HCl to the buffer solution after an acyl-enzyme intermediate was formed. As the HCl concentration increases, the pH of the buffer decreases, which results in reduced activity of papain.14,23 In the polymerization of the co-oligopeptides, the pH of the reaction buffer decreased from 7.6 to 7.0 and did not show remarkable change. Compared to the molecular weight and the conversion (Figures 4−6), the decrease in pH did not significantly affect molecular weight and of the cooligopeptides and the conversion of the monomers. To improve the molecular weight, the product needs to be separated from the enzyme to avoid hydrolysis of the peptide bonds. This might be possible to achieve by changing the reaction solvent to a two-phase system where the product dissolves in one phase and the enzyme and monomers remains in the other phase. Clear differences between oligo(Lys-b-Ala) and oligo(Lys-rAla) in terms of crystal morphology were observed by optical microscopy (Figure 9). Oligo(Lys-b-Ala) formed independent crystals that were of cubic or hexagonal shape at pH 3.0 and of irregular shape at pH 7.4. In contrast, oligo(Lys-r-Ala) formed only plate-like crystals at both pH 3.0 and 7.4. At pH 3.0, oligo(Lys-b-Ala) appeared to be hollow. In the hollow structure, a branch was observed that originated from the surface of the crystal and advanced to the inner center. These differences in the crystal morphology were caused by differences in the monomer sequence within the cooligopeptides and the pH of the solution. The block structure together with the charged side chain in lysine forms the regular crystal. At a low pH, the charged side group will be fully protonated (pKa: 10.54) and screened by protons in the solution. Hence, the co-oligopeptides will interact each other via more hydrophobic interactions rather than ionic interactions. On the other hand, at neutral pH, there are less protons in the solution but the charged side group of lysine was still protonated, resulting in more ionic interactions between peptide molecules in compared to acidic pH. Those balance between ionic and hydrophobic interactions would affect crystallization behaviors of the oligopeptides. The cell viability was also affected by differences in the monomer sequence and self-assembly of the co-oligopeptides (Figure 10). The results revealed that cells cultured on block co-oligopeptides exhibited slightly lower viability compared to random co-oligopeptides and that cells cultured on cooligopeptides that were prepared at pH 10 exhibited the highest viability. However, after incubation for 48 h at
Figure 9. Crystalline morphology of oligo(Lys-b-Ala) at pH 3 (A), oligo(Lys-b-Ala) at pH 7 (B), oligo(Lys-r-Ala) at pH 3 (C), and oligo(Lys-r-Ala) at pH 7 (D).
However, the highest conversion rate was noted for Lys-Et during the first 30 min. From these findings together with the results of the peak and maximum molecular weights of the homo-oligopeptides, it was suggested that the optimal reaction time for the synthesis of oligo(Lys-b-Ala) should not be longer than 30 min. A longer reaction time would result in hydrolysis of the formed peptide oligomers and not in an increase in the conversion of Lys-Et. The successful preparation of diblock and random cooligopeptides of L-lysine and L-alanine was confirmed by 1H NMR and MALDI-TOF-MS and comparison to reference samples, that is, diblock and alternating co-oligopeptides of lysine and alanine synthesized by SPPS. The molecular weight of the co-oligopeptides was relatively low, which is likely due to hydrolysis by the activated papain. Because the product was hydrophilic, it remains soluble and responsive to the enzyme during the reaction. Peptide bonds earlier formed by the enzyme are therefore digested, resulting in the release of monomeric amino acids and a decrease in the molecular weight of the products. The pH of the solution also affects the activity of papain, because there is an optimal pH for papain-catalyzed
Figure 10. Cell viability of human mesenchymal stem cells (hMSC) seeded on crystals or films of the co-oligopeptides prepared at pH 3.0 (A), 7.4 (B), and 10 (C). The results were determined from the absorbance measured at 490 nm by using cell cultures after incubation for 48 h. *Significant difference between two groups at p < 0.05. 742
dx.doi.org/10.1021/bm4015254 | Biomacromolecules 2014, 15, 735−743
Biomacromolecules
■
concentrations below 15 g/L, no significant cell cytotoxicity was observed for random and diblock co-oligopeptides. The low cytotoxicity of co-oligopeptides and the correlation between the monomer sequence and cell viability have also been reported in earlier studies.24,25 Also, polycations like polylysine are famous cell membrane-destabilizing peptides (cell penetrating peptides).26 Hence, at high concentrations of polylysine, cell viability must be decreased. In the present study, the oligopeptides did not show significant cytotoxicity to the cells, indicating that the cationic sequence, namely, OligoLys, was not long enough to destabilize the cell membrane. The low cytotoxicity and hollow structure of these crystals open possibilities not only for their use as scaffold materials in tissue engineering but also as low molecular weight drug/gene carriers. Many materials used in drug delivery systems are prepared by complicated synthesis techniques involving several synthesis steps, template materials, and toxic chemicals.27−33 As shown in this study for oligo(Lys-b-Ala), co-oligopeptides can be prepared by a short one-pot chemo-enzymatic synthesis without any toxic components or template materials.
CONCLUSION Diblock and random co-oligopeptides of L-lysine and L-alanine were successfully synthesized by a short one-pot chemoenzymatic synthesis using activated papain as enzymatic catalyst. The amino acid sequences were verified by 1H NMR and MALDI-TOF-MS and compared to diblock and alternating co-oligopeptides synthesized by SPPS as references. Optical microscopy clearly revealed that the crystal morphology of the co-oligopeptides was dependent on the monomer distribution within the co-oligopeptide and the pH of the solution. At pH 3.0, diblock co-oligopeptides formed cubic or hexagonal crystals with a hollow structure. These findings together with the results from the cell viability assay suggest that co-oligopeptides, one of the promising materials for biomedical applications, can be prepared by a short one-pot chemo-enzymatic synthesis without any toxic components or template materials. ASSOCIATED CONTENT
S Supporting Information *
Figure S1: Peak molecular weight of OligoLys and OligoAla; Figure S2: MALDI-TOF-MS spectra of OligoLys and OligoAla; Figure S3: 1H NMR of SPPS synthesized block and alternating co-oligopeptides of lysine and alanine; Figure S4: MALDITOF-MS spectra of SPPS synthesized block and alternating cooligopeptides of alanine and lysine. This material is available free of charge via the Internet at http://pubs.acs.org.
■
REFERENCES
(1) Langer, R.; Vacanti, J. Science 1993, 260, 920−926. (2) Finne, A.; Albertsson, A. C. J. Polym. Sci., Part A: Polym. Chem. 2004, 42, 444−452. (3) Tyson, T.; Målberg, S.; Wåtz, V.; Finne-Wistrand, A.; Albertsson, A. C. Macromol. Biosci. 2011, 11, 1432−1442. (4) Xing, Z.; Pedersen, T. O.; Wu, X.; Xue, Y.; Sun, Y.; FinneWistrand, A.; Kloss, F. R.; Waag, T.; Krueger, A.; Steinmüller-Nethl, D.; Mustafa, K. Tissue Eng., Part A 2013, 19, 1783−1791. (5) Moon, H. J.; Ko, du Y.; Park, M. H.; Joo, M. K.; Jeong, B. Chem. Soc. Rev. 2012, 41, 4860−4883. (6) Zhang, S. Nat. Biotechnol. 2003, 21, 1171−1178. (7) Nowak, A. P.; Breedveld, V.; Pakstis, L.; Ozbas, B.; Pine, D. J.; Pochan, D.; Deming, T. J. Nature 2002, 417, 424−428. (8) Nitta, S. K.; Numata, K. Int. J. Mol. Sci. 2013, 14, 1629−1654. (9) Numata, K.; Kaplan, D. L. Adv. Drug Delivery Rev. 2010, 62, 1497−1508. (10) Numata, K.; Yamazaki, S.; Naga, N. Biomacromolecules 2012, 13, 1383−1389. (11) Numata, K.; Subramanian, B.; Currie, H. A; Kaplan, D. L. Biomaterials 2009, 30, 5775−5784. (12) Aggeli, A.; Bell, M.; Boden, N.; Keen, J. N.; Knowles, P. F.; McLeish, T. C.; Pitkeathly, M.; Radford, S. E. Nature 1997, 386, 259− 262. (13) Merrifield, R. B. In Adv. Enzymol. Relat. Areas Mol. Biol.; Rockefeller Univ.: New York, 1969; Vol. 32, pp 221−296. (14) Qin, X.; Xie, W.; Su, Q.; Du, W.; Gross, R. A. ACS Catal. 2011, 1, 1022−1034. (15) Löfblom. Biotechnol. J. 2011, 6, 1115−1129. (16) Chaiken, I. M.; Komoriya, A.; Ohno, M.; Widmer, F. Appl. Biochem. Biotechnol. 1982, 7, 385−399. (17) Viswanathan, K.; Omorebokhae, R.; Li, G.; Gross, R. A. Biomacromolecules 2010, 11, 2152−2160. (18) Fukuoka, T.; Tachibana, Y.; Tonami, H.; Uyama, H.; Kobayashi, S. Biomacromolecules 2002, 3, 768−774. (19) Li, G.; Raman, V. K.; Xie, W.; Gross, R. A. Macromolecules 2008, 41, 7003−7012. (20) Baker, P. J.; Numata, K. Biomacromolecules 2012, 13, 947−951. (21) Ageitos, J. M.; Baker, P. J.; Sugahara, M.; Numata, K. Biomacromolecules 2013, 14, 10−16. (22) Numata, K.; Katashima, T.; Sakai, T. Biomacromolecules 2011, 12, 2137−2144. (23) Li, G.; Vaidya, A.; Viswanathan, K.; Cui, J.; Xie, W.; Gao, W.; Gross, R. A. Macromolecules 2006, 7915−7921. (24) Numata, K.; Kaplan, D. L. Biochemistry 2010, 49, 3254−3260. (25) Numata, K.; Kaplan, D. L. Macromol. Biosci. 2011, 11, 60−64. (26) Wagner, E.; Ogris, M.; Zauner, W. Adv. Drug Delivery Rev. 1998, 30, 97−113. (27) Yang, J.; Lee, J.; Kang, J.; Lee, K.; Suh, J. S.; Yoon, H. G.; Huh, Y. M.; Haam, S. Langmuir 2008, 24, 3417−3421. (28) Chun Zeng, H. Curr. Nanosci. 2007, 3, 177−181. (29) Tjandra, W.; Ravi, P.; Yao, J.; Tam, K. C. Nanotechnology 2006, 17, 5988−5994. (30) Su, F.; Zhao, X. S.; Wang, Y.; Wang, L.; Lee, J. Y. J. Mater. Chem. 2006, 16, 4413−4419. (31) Lin, Z. Y.; Duan, Z. X.; Guo, X. D.; Li, J. F.; Lu, H. W.; Zheng, Q. X.; Quan, D. P.; Yang, S. H. J. Controlled Release 2010, 144, 190− 195. (32) Edlund, U.; Albertsson, A. C. J. Polym. Sci., Part A: Polym. Chem. 1999, 37, 1877−1884. (33) Guo, B.; Finne-Wistrand, A.; Albertsson, A. C. Biomacromolecules 2011, 12, 2601−2609.
■
■
Article
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (K.N.);
[email protected] (A.F.W.). Tel.: +81-48-467-9525 (K.N.); +46-8-790 8924 (A.F.-W.). Fax: +46-8-10 07 75 (A.F.-W.). Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The authors thank RIKEN Biomass Engineering Program (K.N.) and Sweden-Japan Foundation’s Gadeliusstipendium for the financial support of this work. 743
dx.doi.org/10.1021/bm4015254 | Biomacromolecules 2014, 15, 735−743
