Unprecedented Enzymatic Synthesis of Perfectly Structured

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Unprecedented Enzymatic Synthesis of Perfectly Structured Alternating Copolymers via “Green” Reaction Cocatalyzed by Laccase and Lipase Compartmentalized Within Supramolecular Complexes Dieter Michael Scheibel, and Ivan Gitsov Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01567 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on December 31, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Biomacromolecules

Unprecedented Enzymatic Synthesis of Perfectly Structured Alternating Copolymers via “Green” Reaction Cocatalyzed by Laccase and Lipase Compartmentalized Within Supramolecular Complexes Dieter M. Scheibel,1 Ivan Gitsov*1,2 1Department

of Chemistry, State University of New York – ESF and 2The Michael M.

Szwarc Polymer Research Institute, Syracuse, NY 13210 *Corresponding author. E-mail: [email protected]; Phone: 1-315-470-6851/Fax: 1-315-4706856 ABSTRACT: This study describes the first use of laccase-lipase enzymatic reaction for the synthesis of novel perfectly structured alternating copolymers. Initially six types of complexing agents: linear(A)linear(B),

linear(A)-linear(B)-linear(A),

linear-dendritic,

dendritic-linear-dendritic,

linear-

hyperbranched, and hyperbranched-linear-hyperbranched amphiphilic block copolymers are proven to significantly enhance enzyme activity of three different types of lipases - Penicillium camemberti, Candida rugosa, and Burkholderia cepacia (up to 1400%, 1700%, and 870% increase with respect to the native enzymes). The copolymerization is performed in several consecutive steps: a) lipase and laccase are dissolved in aqueous medium at neutral pH; b) a complexing agent is added leading to co-compartmentalization of the two enzymes within a micelle or physical network; c) the two comonomers are introduced simultaneously to the tandem enzyme complex. The reaction proceeds in the following pathway: laccase catalyzes the oxidation of catechol to oquinone followed by lipase co-mediated Michael addition of a diamine. While laccase could catalyze the entire process, addition of lipase is able to increase copolymer yield up to 30.7%. Addition of a complexing agent improves the yield further up to 67.9% (23.2% yield obtained for native laccase). Complexing agents significantly increase polymer molecular mass (Mw = 130,900 Da vs 35,500 Da for the native enzymes reaction system). The resulting copolymers are highly

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fluorescent (quantum yield up to 0.733) and demonstrate pH sensitive behavior, properties that hint toward their potential as imaging agents.

1. INTRODUCTION The development and implementation of multi-catalyst reactions is highly sought-after in all fields of chemistry due to the significant advantages such reactions afford.1 Primary benefits of these reaction systems include increased atom economy, decreased waste generation, and minimal reaction setup and workup.2 These benefits significantly enhance the financial efficiency of chemical synthesis by decreasing resource expenditure with the avoidance of multiple purification processes additionally increasing labor efficiency and leading to increased yields. As catalysts have the potential to be recycled and very low quantities are required for efficient amplification, their use in organic synthesis also increases atom economy, further decreasing waste generation. Recent advances in the production of low cost enzymes have markedly increased the use of enzymes as catalysts in organic synthesis. Low cost, minimal byproducts, and high stereo- and regioselectivity have made enzymes highly desirable as “green chemistry” alternatives to traditional transition metal catalysts.3,4 Of significant interest is the development of multi-enzyme reactions which serve to combine the advantageous traits of enzyme catalysis and multi-stage reactions affording a facile and environmentally benign means of developing complex synthetic schemes.5,6 Although numerous examples have been reported, a rather common strategy of employing multi-enzymatic reactions is using oxidoreductases whereby a second enzyme can be utilized in the regeneration of a reduced cofactor.7,8 Laccase, a benzenediol:oxygen oxidoreductase, is of specific interest due to its ability to oxidize a wide variety of substrates under very mild reaction conditions.9-15 Although laccase primarily serves to oxidize phenolic compounds in nature, the breadth of laccase catalyzed 2 ACS Paragon Plus Environment

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Biomacromolecules

oxidations is greatly increased by the addition of catalytic amounts of radical mediators such as 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic

acid)

(ABTS),

2,2,6,6-tetramethyl-1-

piperidinyloxy (TEMPO), 1-hydroxybenzotriazole (HOBT), or other nitroxide-based mediators.716

This is significant as the role of oxidant has traditionally been reserved for hazardous chemicals

which often contain heavy metals known to have widespread adverse environmental impacts and deleterious effects on human health.17 These hazardous compounds necessitate significant resource expenditure for their safe use and disposal; a trait circumvented by the use of laccase catalyzed oxidation processes. Lipase, a hydrolase/esterase, has also garnered significant interest as a biocatalyst for organic synthesis.18 Sharing similar “green chemistry” characteristics as laccase, lipase is most well-known for its ability to catalyze the esterification of alcohols with high stereospecificity allowing for facile chiral resolution.19 Lipase has also been employed to catalyze the hydrolysis and transesterification of fatty acids for the production of biofuels and in ring-opening polymerizations for the production of polyesters.20-25 However, recent work demonstrated that lipase has the ability to catalyze a variety of unnatural reactions including epoxide formation,26 aldol reactions,27 Michael additions,28,29 and thiol addition to imines.30 These newfound catalytic roles of lipases towards unnatural substrates increase their versatility and usefulness in organic synthesis. In a recent paper we have shown that previously developed hydrogel and micellar hyperbranched-linear-hyperbranched, linear(A)-linear(B)-linear(A), and dendritic-linear-dendritic (Figure S1A, S1B, S1C) block copolymers can notably improve the activity and stability of laccase.31 Linear-dendritic complexing agents were also shown to significantly increase the molecular mass of unnatural poly(tyrosine) polymerized by laccase as catalyst demonstrating their



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versatility towards water soluble and water insoluble substrates.32 This observed increase in activity is attributed to two factors: a) regioselective adsorption of the hydrophobic blocks of the copolymers to the carbohydrate regions of the glycoprotein33 and b) the copolymer ability to selectively and efficiently sequester hydrophobic substrates facilitating a localized increase in substrate concentration around the enzyme active site (in a previous study16 we have confirmed the pathway for binding of substrates into the hydrophobic nano-pockets of the amphiphilic complexing agents and their subsequent migration to the active site of the enzyme). These immobilizing agents will therefore be utilized in this study to increase enzyme activity and robustness. O

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Figure 1. Schematic representation of laccase (yellow) and lipase (grey) randomly immobilized in a hydrogel (A) and micelles (B), formed by the self-assembly of linear-dendritic complexing agents. The present work exploits the broad substrate specificity of both laccase and lipase to synthesize a novel alternating copolymer using for the first time a laccase-lipase cocatalytic cascade reaction. Both enzymes are co-compartmentalized within supramolecular assemblies of amphiphilic block copolymers (an example is shown in Figure 1). The influence of the


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Biomacromolecules

immobilizing agents on conversion and polymer molecular mass is also investigated. The developed laccase-lipase cocatalytic system employs “green chemistry” principles, with the reaction occurring in a buffered aqueous solution (pH 7) at 45°C. The environmental friendliness of the reaction is further promoted by utilizing a one-pot reaction setup, thus requiring minimal workup procedures. The synthesized copolymer is composed of an o-quinone-m-xylylenediamine or o-quinone-p-xylylenediamine repeating units via lipase catalyzed Michael addition of m- or pxylylenediamine to o-quinone (the inherent instability of o-quinone necessitates its synthesis insitu, accomplished by laccase catalyzed oxidation of catechol). The resulting polymers show strong pH dependent fluorescence and therefore could be utilized in biomedical applications as an imaging agents with sufficient modification. It is envisioned that water soluble derivatives of the synthesized polymer may be acquired by utilizing more hydrophilic monomers, increasing the polymer’s versatility in biomedical applications. 2. EXPERIMENTAL 2.1.

Materials Catechol (99%), m-xylylenediamine (99%), and p-xylylenediamine (99%) (all from

Sigma-Aldrich, St. Louis MO) were used as received. 2,5-Dihydroxybenzoic acid (DHB, 99%) (from Sigma-Aldrich, St. Louis MO) was further purified by recrystallization with methanol and deionized water (18.1 MΩ) using standard techniques. Laccase from Trametes versicolor (≥0.5 U/mg), Amano lipase G from Penicillium camemberti (≥5,000 U/mg), lipase from Candida rugosa (≥700 U/mg), Amano lipase PS from Burkholderia cepacia (≥3,000 U/mg), and p-nitrophenyl palmitate (≥99%) (all purchased from Sigma Aldrich, St. Louis MO) were used without further purification. Linear(A)-linear(B) and linear-hyperbranched complexing agents were synthesized by atom transfer radical polymerization of either styrene or p-chloromethyl styrene initiated by αchlorophenylacetyl capped poly(ethylene glycol).31 Linear-dendritic and dendritic-linear-dendritic 5 ACS Paragon Plus Environment


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complexing agents were synthesized by coupling of preformed reactive fragments34 or via living ring-opening anionic polymerization of ethylene oxide initiated by third generation benzyl ether dendrons.35 General chemical structures, molecular mass characteristics, representative chromatograms and NMR spectra of all three groups of complexing agents are included in ESI (Figures S1-S3 and Table S1). 2.2.

Methods

2.2.1. Chromatography Size exclusion chromatography (SEC) was conducted on system consisting of a Waters 1515 isocratic pump, a Waters 1500 series manual injector, two PolyPore 5 μm 300 mm x 7.5 mm mixed bed columns, and a Waters 2414 refractive index detector. All analyses were conducted at 60°C with dimethyl sulfoxide (DMSO) as the eluent containing 0.1% (1 mg/mL) lithium bromide (LiBr) at a flow rate of 0.8 mL/min. Sample solutions were filtered using a 0.45 µm cellulose acetate filter prior to injection. Calibration was performed using 15 monodisperse poly(ethylene glycol) and poly(ethylene oxide) standards (1.98 −452 kDa) and Waters Breeze software. 2.2.2.Dynamic Light Scattering All DLS analyses were performed on a Malvern Zetasizer ZS instrument using a 633 nm laser source with a fixed backscattering detector at 173°. Size calculations were completed by a CONTIN procedure. 2.2.3 NMR Spectroscopy 1H

NMR and

13C

NMR spectra were recorded using DMSO-d6 as solvent at 22°C on a

Bruker AVANCE 600 MHz instrument with the solvent signal as the internal standard.

2.2.4. Fluorescence Spectroscopy 6 ACS Paragon Plus Environment

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Biomacromolecules

All

fluorescence

measurements

were

performed

on

a

Horiba

Fluorolog-3

spectrophotometer with a SpectrAcq system controller. The instrument used a xenon arc lamp source. All measurements were conducted at 90° using an excitation wavelength of 365 nm in dimethyl sulfoxide. Quantum yields of polymer samples were calculated using fluorescein and 7hydroxycoumarin (umbelliferone) as standards. 2.2.5. UV-Vis Spectroscopy All UV-Vis spectroscopic measurements were conducted on an Agilent 8453 UV-Vis spectrophotometer at room temperature in deionized water unless otherwise specified. 2.2.6. FT-IR Spectroscopy Fourier transform infrared spectroscopy (FT-IR) spectra were obtained on a Bruker Tensor 27 spectrophotometer with a MIR source and a DLaTGS detector. Spectra were recorded under ambient conditions at a resolution of 4 cm-1. A total of 64 scans were recorded for each spectrum in addition to the background. 2.2.7. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry MALDI-TOF MS spectra were acquired on a Bruker Autoflex III equipped with Smartbeam II laser source (Nd-YAG laser, 266 or 355 nm). Spectra were collected in linear positive mode with the attenuation set to the lowest value capable of obtaining high resolution spectra. Matrix solution was formed by dissolving DHB in deionized water (18.1 MΩ) at a concentration of 80 mg/mL. Sample solutions were prepared in deionized water (18.1 MΩ) at a concentration of 1 mg/mL. Samples were spotted by mixing matrix solution and sample solution in a 1:1 ratio and spotting 5 µL of the resulting solution on a MTP 384 target plate (polished steel, Bruker Daltronics). 2.2.8. Enzyme Activity Assay



Page 8 of 33

Activity assays of native lipase and complexed lipase solutions were conducted at 45°C at pH 5.5 using p-nitrophenyl palmitate as substrate.36 Lipase solutions of concentration 2.5 mg/mL were prepared in a 0.1 M 5.5 pH sodium acetate buffer. Buffer solution of pH 5.5 was used to limit autohydrolysis of the p-nitrophenyl palmitate substrate. 10 mL of lipase solution was then transferred to a scintillation vial. Under vigorous stirring, 10 mg of complexing agent dissolved in 100 µL THF was added dropwise. The solution was incubated at 45°C with stirring for 4 h. To 2 mL of this solution was added 50 µL of a 5 mM p-nitrophenyl palmitate solution in acetonitrile. Activity of the solution was determined by monitoring the increase in absorption at the isosbestic point of p-nitrophenol (348 nm) for 60 seconds. All activity tests were conducted in triplicates. 2.2.9. Copolymerization of Catechol and m-Xylylenediamine A typical copolymerization was conducted by first preparing a 0.1 M pH 7 sodium phosphate buffer. To 20 mL of buffer solution 20 mg of laccase and 20 mg of lipase were added. Once homogenized, 20 mg of complexing agent, dissolved in 200 μL distilled tetrahydrofuran, was added dropwise under vigorous stirring causing the mixture to become opaque or cloudy white in appearance manifesting the formation of micelles or a physical network (Figure S4). The solution was then allowed to acclimate for 4 h at 45°C. To the stirring solution, 60 mg of catechol (0.55 mmol) and 74 mg of m-xylylenediamine (0.55 mmol) were added simultaneously. The reaction was allowed to proceed for 72 h at 45°C. Polymer which had precipitated during the reaction was collected via centrifugation and washed three times with deionized water to remove the enzymes, unreacted comonomers, and any potential catechol homopolymer. The sediment was then washed with tetrahydrofuran to remove residual complexing agents. Copolymer yield was calculated from the ratio of isolated copolymer weight versus initial comonomer amounts. Copolymers of catechol and p-xylylenediamine were formed using a similar protocol.


Page 9 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

3. RESULTS AND DISCUSSION 3.1. Activity of Lipase Complexes Experiments in this study reveal that the previously developed complexing agents have a strong catalysis-enhancing effect on lipases from Burkholderia cepacia (Amano lipase PS), Penicillium camemberti (Amano lipase G), and Candida rugosa, significantly increasing their activity towards the substrate p-nitrophenyl palmitate (Figures 2 and S5-S12). It is found that G3PEO13kDa was the most universal complexing agent in significantly enhancing enzyme activity in all three enzymes. Linear-dendritic copolymers have the greatest activity effect on lipase from Candida rugosa, while both linear(A)-linear(B) and linear-hyperbranched copolymers have the greatest activity effect on lipase from Burkholderia cepacia. This may be a result of differences in micellar size among the complexing agents better accommodating enzymes of different molecular mass37-39 in addition to variations in enzyme structure changing the distance between the enzyme active site and anchoring place of the complexing agent. Micellar complexing agents were superior to hydrogels owing to the more efficient diffusion of substrate to the complexed enzyme active site and increased surface contact (Figure 1). Although there is limited literature reporting on lipase immobilized in PEG-based hydrogels, there are numerous studies which have investigated the dependence of lipase activity on PEG-based detergents. Triton X-100, Triton X-114, Tween 20, Tween 40, and Tween 80, have all found use in lipase activity protocols.40,41 Both Triton X-100 and Triton X-114 afford modest improvements in enzymatic activity, with relative activities increasing to 108.7-200% of the native enzyme,40-42 congruent with Triton X-100 results in our experiments. Even though Tween 20 did significantly enhance enzymatic activity of several prokaryotic lipases, up to 400% relative activity, the use of Tween 20, Tween 40, or amphiphilic polymers with the majority of lipases,


Biomacromolecules

specifically those from eukaryotes, resulted in moderate to severe enzyme inhibition with some lipases experiencing a complete loss of enzymatic activity.35-38 This is in contrast to this work where all synthesized complexing agents promoted enzyme activity in both eukaryotic and prokaryotic lipases - Candida rugosa (eukaryotic, 137-1727% relative activity, Figures 2, S11, and S12), Penicillium camemberti (eukaryotic, 152-1087% relative activity, Figures S5, S6, and S7), and Burkholderia cepacia (prokaryotic, 160-1407% relative activity, Figures S8, S9, and S10). These results suggest that the synthesized complexing agents are a notable improvement upon the currently used classes of detergents in both enzyme activity enhancement and lipase versatility and would therefore be well suited either for further lipase activity testing and/or lipase catalyzed reactions.

Linear-Dendritic Complexed Candida

k 13

3

G3 -P EO

G2

-P EO 13

kG

kG

4

3 -P EO 13

-P EO

13

kG G3

3 kG

-P EO

13

5k G1

-P EG G3

G3

0 X10 on

Tr it

Na

G3

2000 1800 1600 1400 1200 1000 800 600 400 200 0

tiv e

Relative Activity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 33

Figure 2. Activity of linear-dendritic Candida rugosa lipase micellar (blue hashed) or hydrogel (red checkered) complexes. G1, G2, G3: first-, second-, third generation poly(benzyl ether) dendron; PEG: poly(ethylene glycol); PEO: poly(ethylene oxide). The greater catalytic activity of the copolymer enzyme complexes relative to the Triton X100 mixtures is most likely the result of the larger hydrophobic pockets of the copolymer complexing agents having higher binding capacity compared to those produced by the shorter alkyl chains of traditional detergents. The beneficial effects, experienced by all three enzymes, 10 ACS Paragon Plus Environment

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Biomacromolecules

demonstrate that the complexing agents do not deleteriously influence or change the enzyme function. It should be emphasized that the lipase activity data in this study and the previous report of laccase complexation31 demonstrate the high efficiency of the developed complexing strategy with different enzymes of broad molecular mass range and different mechanisms of action. This further validates the wide applicability of the synthesized complexing agents. The promising results yielded in this study have supplied the basis to extend the use of the described complexing agents to additional enzymes with potential applications including tandem reactions. 3.2. Laccase-Lipase Cocatalytic Tandem Reaction Witayakran et al. previously developed an interesting environmentally benign two-enzyme cocatalytic system for the convenient synthesis of dyes.28 In the reaction system reported, laccase is initially used for the oxidation of catechol to o-quinone followed by the lipase catalyzed Michael addition of an aniline derivative to the previously produced quinone. Additional literature has also reported lipase to be an effective catalyst for the Michael addition of benzylamines to enones.29 In this study these concepts are extrapolated to the facile synthesis of a novel alternating copolymer of catechol and m-xylylenediamine or p-xylylenediamine using both laccase and lipase as catalysts (Figure 1 and Scheme 1). To our knowledge this is the first approach to use a two-enzyme reaction for the synthesis of perfectly structured alternating copolymers. Furthermore, the complexing agents which were previously shown to significantly enhance the activity and durability of laccase and currently demonstrate an even stronger effect on lipase are investigated for their influence on reaction yield and molecular mass of the produced copolymer. Scheme 1. Laccase-Lipase cascade copolymerization of catechol and m-xylylenediamine. Reaction conditions are described in section 2.2.8.



O OH

HO

O

Page 12 of 33

OH

HO

OH

HN

NH

O

Laccase

Lipase

Lipase H2N

NH2

H2N

NH2

NH

NH2

Lipase/Laccase

HO

OH

HO

OH

HN

NH

Laccase H2N

N

N

H2N

NH2

NH2

n

n

Lipases from Burkholderia cepacia, Candida rugosa, and Penicillium camemberti are employed to investigate the catalytic effect these enzymes have on the described reaction system. The yield is found to be highest when Burkholderia cepacia is used, followed by Penicillium camemberti, and Candida rugosa, while being lowest when no lipase is added (Table 1). As Burkholderia cepacia provides the greatest yield increase, it is solely selected for further investigation. Table 1. Effect of different lipases on copolymerization yield. Entrya

Lipase

Yield (%)

Relative Yield Increase (%)

1

None

23.2 ± 2.13

-

2

Candida rugosa

24.8 ± 1.20

+6.9

3

Penicillium camemberti

25.6 ± 2.06

+10.3

4

Burkholderia cepacia

30.7 ± 3.57

+32.3

aIn

all entries the reaction is performed in triplicates without a complexing agent for 72 h. Reaction conditions are described in the Experimental Section. Relative yield calculated using the following formula (yieldLac/Lip yieldLac)/yieldLac x 100.

Table 2. Effect of different complexing agents on laccase-lipase cocatalytic copolymerization system of catechol and m-xylylenediamine. Entry

Catalysta

Complexing Agentb c

Yield (%)

Mn

Mw

Mpd

Mw/Mn


Page 13 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

None

None

0

--

--

--

--

2

Lac

None

23

7500

17500

9000

2.3

3

Lac-Lip

None

31

12200

30500

6700

2.5

4

Lac

G3-PEO13kDa (M)

36

9600

16700

13600

1.7

5

Lac-Lip

G3-PEO13kDa (M)

43

14500

29500

16500

2.0

6

Lac

G3-PEO13kDa-G4 (H)

31

20400

49000

19000

2.4

7

Lac-Lip

G3-PEO13kDa-G4 (H)

39

21800

55500

13200

2.5

8

Lac

PS750-PEG13k-PS750

45

15300

29500

15800

1.9

50

14500

35400

15000

2.4

39

10000

23900

9800

2.4

50

15000

32200

16000

2.1

25

15200

106800

9900

7.0

40

19400

119300

11700

6.1

37

20100

144700

8600

7.2

43

23700

130900

13400

5.5

(M)

9

Lac-Lip

PS750-PEG13k-PS750 (M)

10

Lac

PS10k-PEG13k-PS10k (H)

11

Lac-Lip

PS10k-PEG13k-PS10k (H)

12

Lac

PPCMS2.3k-PEG13kPPCMS2.3k (M)

13

Lac-Lip

PPCMS2.3k-PEG13kPPCMS2.3k (M)

14

Lac

PPCMS9k-PEG13kPPCMS9k (H)

15

Lac-Lip

PPCMS9k-PEG13kPPCMS9k (H)

aAll

Lac-Lip entries are produced using lipase from Burkholderia cepacia. Copolymerization time 72 h. bM - micelle. cH - hydrogel. dMp – molecular mass at the SEC peak apex. Additional data provided in Supporting Information.



Page 14 of 33

A

B

C

D

Figure 3. Hydrodynamic diameters, Dh, of native laccase (black, A), G3-PEO13k/laccase/lipase mixed micellar complex (green, B), G3-PEO13k micelles (blue, C) and native lipase (red, D). Peak at 200 nm is lipase aggregate. Overall a near universal increase in copolymer yield is observed upon the addition of lipase to the polymerization system (Table 1). The deviations in this trend are probably due to the effect of the macromolecular architecture of the complexing agents (see ESI for examples). Without the 14 ACS Paragon Plus Environment

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Biomacromolecules

addition of an enzyme complexing agent the highest relative yield increase of 32 % is detected when Burkholderia cepacia lipase was used as cocatalyst (Table 1, entry 3) while a relative yield increase of 74 % (based on the total yield of 40.3 % with the complex vs 23.2 %, Table 1, entry 1) is achieved when the same enzyme pair is compartmentalized within PPCMS1.5k-PEG3kPPCMS1.5k micelles (Table S2, entry 39). The observed increase achieved with lipase as cocatalyst is congruent with previously reported studies where the enzyme was found to catalyze the Michael addition of benzyl and aryl amines to enones.28,29 Addition of enzyme complexing agent is also found to increase overall yield of poly(catechol-alt-m-xylylenediamine) up to 68 % (relative yield ~193%) for PS750-PEG3k-PS750 micelles (Table S2, entry 25) and up to 65 % (relative yield ~180%) for PPCMS13k-PEG13k-PPCMS13k hydrogels (Table S2, entry 47) resulting from their ability to more efficiently sequester the substrates. Additionally, as the complexing agents are able to co-compartmentalize both laccase and lipase in a single hydrophobic pocket (Figure 3B, see also Figures S14b,c in ESI), there is a significantly shorter travel distance between the catalytic active sites of the two enzymes, allowing catechol to be rapidly converted to o-quinone and reacted with sequestered m-xylylenediamine. The increased yield afforded by the enzyme complexing agents is similarly observed for the polymerization of the p-xylylenediamine derivative (Table S3). Due to increased diffusion efficiency, micellar enzyme complexes afforded noticeably higher yields than their hydrogel counterparts. Although molecular mass of the synthesized polymer does increase when lipase is used in the native reaction system, addition of a complexing agent is found to have a much more profound effect on this copolymer characteristic (Table 2, entries 3, 7, 9, 11, 13, and 15, and Table S2). Polymer molecular masses (Mw) up to 55,500 Da (Table 2, entry 7), 51,500 Da (Table S2, entry 37) and 163,300 Da (Table S2, entry 45) are obtained for dendritic-linear-dendritic, linear(B)-


Biomacromolecules

linear(A)-linear(B) and hyperbranched-linear-hyperbranched complexing agents, respectively. The increased molecular mass afforded by the complexing agents is attributed to their ability to form hydrophobic nano-pockets at the carbohydrate domains of the glycoproteins,16,31,32 which promote a sustained supply of comonomers to the growing copolymer chain and may assist in solubilizing larger molecular mass polymers allowing for continued chain propagation. SEC Elution of Poly(catechol-alt-m-xylylenediamine) 110

Normalized Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 33

5

2

90

1

70

4

3

50 30 10 -10 8

13

18

23

28

Retention Time (min)

Figure 4. SEC traces of poly(catechol-alt-m-xylylenediamine) polymerized using laccase (1), micellar G3-PEO13kDa/laccase (2), and hydrogel G3-PEO13kDa-G4/laccase (3). Catechol comonomer (4), and m-xylylenediamine comonomer (5). Flow rate 0.8 mL/min with 0.1% LiBr in DMSO as eluent.


Page 17 of 33

SEC Elution of Poly(catechol-alt-m-xylylenediamine) Normalized Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

110

2

90

1

5 4

70

3

50 30 10 -10 8

13

18

23

28

Retention Time (min)

Figure 5. SEC traces of poly(catechol-alt-m-xylylenediamine) polymerized using laccase/lipase (1), micellar G3-PEO13kDa/laccase/lipase (2), and hydrogel G3-PEO13kDa-G4/laccase-lipase (3). Catechol comonomer (4), and m-xylylenediamine comonomer (5). Flow rate 0.8 mL/min with 0.1% LiBr in DMSO as eluent. Polydispersities (PDI) values between 2-3 are observed for polymers produced by the native enzyme reaction system and enzyme complexes using linear(A)-linear(B) and linear-dendritic type copolymers (Figures 4 and 5), typical of chain addition polymerizations. Noticeably higher PDIs are obtained for polymers produced by linear-hyperbranched enzyme complexes (Tables 2 and S2). This discrepancy is most likely due to structural irregularities encountered with hyperbranched molecules as opposed to their more ordered linear and dendritic counterparts. These irregularities produce micelles of broader size-distribution as observed by DLS analysis (Figure S14a) causing differences in monomer feed to the enzymes. A similar effect may be extrapolated to hydrogel networks whereby linear-hyperbranched hydrogels will be composed of less uniform hydrophobic pockets. Overall, hydrogel enzyme complexing agents are more prone to produce increased polymer molecular mass when compared with micellar complexes, a result of the higher hydrophobic domain contents per unit volume facilitating more efficient substrate supply (Figures 4 and 5). The same factors regarding the ability of complexing agents to co-compartmentalize both 17 ACS Paragon Plus Environment


Page 18 of 33

laccase and lipase appear to play an important role for the observed increase in the molecular mass of the synthesized copolymers (Table 2, entry 3 compared to entries 5, 7, 9, 11, 13, and 15). Similar results are achieved with the p-xylylenediamine isomer, where polymer molecular masses (Mw) as high as 60,600 Da are observed (Table S3, entry 7). The presence of multiple fractions in the copolymerization mixture from the laccase/lipase system without complexing agents (Figure 5, 1solid blue trace) is most probably caused by two factors: interrupted supply of reaction partners from the two enzymes free-floating in the solution and different reaction rates of the Michael addition proceeding as lipase-catalyzed and not lipase-catalyzed. In contrast the nearly monomodal molecular mass distribution observed with both enzymes co-immobilized in polymer complexes (Figure 1; Figure 5, 2-orange and 3-grey dotted traces) indicates a single dominant mechanism of copolymerization. 3.3. Characterization of Poly(catechol-alt-m-xylylenediamine) Characterization of poly(catechol-alt-m-xylylenediamine) synthesized using the described laccase-lipase co-catalytic system is conducted using NMR and IR spectroscopy. 1H NMR spectra of poly(catechol-alt-m-xylylenediamine) (Figure 6) display the characteristic broad peaks of relatively rigid polymers. Signals from 6.0-10 ppm are assigned to the aromatic protons Hb, Hb’, Hc, Hc’, Hd, and He and signals from 2.8-6.0 ppm are assigned to the benzyl protons Ha and Ha’ (structures shown in Figure 6). Since the copolymers are purified by at least 3 comonomerselective precipitations, (see also the SEC traces in Figures 4 and 5), the sharp peaks present in the 6.0-8.0 ppm region of 1H NMR spectra could not represent unreacted monomers occluded in the precipitated copolymer. It could be assumed that they are caused by the copolymer chain ends, which should have a higher degree of mobility.


Page 19 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

FT-IR spectra of the copolymer samples also display very broad peaks (Figure 7B). Of most significance are the signals ranging from 3600-2000 cm-1 and at 1600 cm-1 corresponding to O-H and C=N stretching bands respectively. The strong presence of both signals indicate near complete oxidation of the initial mixed dimer (quinone-diamine Michael addition product) to the final repeating unit as shown in Scheme 1. These findings are consistent with similar reports in the literature.28,44

NH2j'

Hf

Hi, Hi’

Hf, Hg, Hg’, Hh

NH2 j

Hj, Hj’

Hi

Hi' Hg

Hg' Hh

. OH

HO

Hd

N

N

×

Aromatic protons Hb, Hb’, Hc, Hc’, Hd, He

Hb'

Hb

Ha' Ha

×

Benzylic protons Ha and Ha’

n Hc'

Hc He

Hk, Hk’ Hk'O

OHk

Hl, Hl’, Hm, Hm’ Hl

Hl' Hm'

Hm

×

×

Figure 6. 600 MHz 1H NMR spectrum of m-xylylenediamine (top - green), poly(catechol-alt-mxylylenediamine) (Table 2, entry 2) (middle - red), and catechol (bottom - blue) recorded at 22°C in DMSO-d6. Solvent impurity peaks are marked with (×). For polymer peaks integration see Figure S13.



Page 20 of 33

A H 2N

NH2

HO

N

N-H

C-H

B

OH

N n

O-H, N-H, C-H C=N C

HO

OH

O-H

Figure 7. FT-IR spectra of m-xylylenediamine (A), poly(catechol-alt-m-xylylenediamine) (Table 2, entry 2) (B), and catechol (C). MALDI-TOF MS spectra of poly(catechol-alt-m-xylylenediamine) display the alternating structure of the copolymer (Figure 8). Oxidation of catechol to o-quinone results in the loss of two protons producing a fragment (A) of m/z 108 while Michael addition and subsequent oxidation of m-xylylenediamine causes a total loss of four protons, resulting in a fragment (B) of m/z 132. Thus the repeating unit of the alternating AB copolymer would have an m/z of 240, which is clearly evident in the MALDI-TOF traces. Oligomers with uneven number of A and B fragments are also present in the polymerization mixture, Figure 8. The absence of higher molecular mass peaks could be explained by the increasing difficulty to volatilize fractions with increasing molecular mass.


Page 21 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

A1B1

A1B2

A2B2 A3B3 A2B3 A2B3-N

A3B4 A4B4 A5B5

A3B2

A6B6

A4B5 A5B6

Figure 8. MALDI-TOF MS spectra of poly(catechol-alt-m-xylylenediamine) (Table 2, entry 2) with repeating units (A) catechol (m/z = 108) and (B) m-xylylenediamine (m/z = 132). A3B2 addition of catechol to A2B2. A2B3-N - addition of m-xylylenediamine to A2B2 with loss of amine. AnBn+1 - addition of m-xylylenediamine to AnBn. 3.4. Fluorescent Properties of Poly(catechol-alt-m-xylylenediamine) Formation of the catechol-diimine polymer repeat unit imparts fluorescent properties in the synthesized polymer. The presence of phenolic and imine groups in the polymer backbone, which can be easily deprotonated and protonated respectively, induces additional pH sensitivity of the fluorescent copolymer. The fluorescence of poly(catechol-alt-m-xylylenediamine) is at its maximum when an excitation wavelength of 365 nm is used (Figure S19). In neutral, basic, and acidic DMSO respectively, quantum yields of 0.073, 0.171, and 0.31 are observed for poly(catechol-alt-m-xylylenediamine) (Figure 9) while quantum yields of 0.305, 0.722, and 0.733 are observed for poly(catechol-alt-p-xylylenediamine) (Figure 10). The lower quantum yield achieved under neutral conditions is most likely due to self-quenching as a result of chain stacking. Differences in copolymer fluorescence and quantum yield in neutral, basic, and acidic pH can 21 ACS Paragon Plus Environment


Page 22 of 33

therefore be attributed to both changes in the electronic structure of the copolymers and also probably to chain extension due to charge repellence (Figure 11, see also DLS traces in Figures S15 and S16). The significant differences in quantum yield of the m-xylylenediamine and pxylylenediamine polymers can be explained by the intrinsic fluorescent differences of the xylylenediamine monomers with the para isomer displaying fluorescence approximately 15 times greater than the meta isomer (Figures S17 and S18).45 The difference in monomer fluorescent intensity is further amplified by the increased rigidity (thus increased fluorescence), of the pxylylenediamine repeating unit when incorporated into the polymer chain. The molecular mass of the copolymers has insignificant influence on the fluorescence intensity. For example, in acidic media the quantum yields are 0.31 for m-copolymer with Mw 17,500 and 0.281 for m-copolymer with Mp 49,000 (Table 2, entries 2 and 6, respectively). For the p-copolymers the values are 0.733 for copolymer with Mw 10,400 and 0.674 for copolymer with Mw 27,900 (Table S3, entries 2 and 6, respectively). In neutral media the quantum yields for the same m-copolymers are 0.103 and 0.116 (Table 2, entries 2 and 6 respectively), while the same p-copolymers have quantum yields of 0.305 and 0.319 (Table S3, entries 2 and 6 respectively).


Page 23 of 33

Poly(catechol-alt-m-xylylenediamine) Fluorescence Dependence on pH

Relative Intensity

800000 700000

2

600000 500000 400000

3

300000 200000

1

100000 0 380

400

420

440

460

480

500

520

540

560

580

600

620

640

660

680

700

Wavelength (nm)

Figure 9. Fluorescence spectra overlay of poly(catechol-alt-m-xylylenediamine) (Table 2, entry 2) in DMSO. Neutral - 1, acidic (0.23 M HCl) - 2, and basic (0.23 M NaOH) - 3. Copolymer concentration: 0.050 mg/mL; excitation wavelength: 365 nm. Poly(catechol-alt-p-xylylenediamine) Fluorescence Dependence on pH Relative Fluorescence Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

700000 600000 500000

2

3

400000 300000

1

200000 100000 0 380

430

480

530

580

630

680

Wavelength (nm)

Figure 10. Fluorescence spectra overlay of poly(catechol-alt-p-xylylenediamine) (Table S3, entry 2) in DMSO. Neutral - 1, acidic (0.23 M HCl) - 2, and basic (0.23 M NaOH) – 3. Copolymer concentration: 0.050 mg/mL; excitation wavelength: 365 nm.



A

HO

OH

HN

NH

D

Page 24 of 33

HO

NH

OH

HN

n

B

HO

N

OH

n

E

N

HO

N

OH

N

n

C

O

O

N

N

n

F

O

O

N

N

n

n

Figure 11. Protonated (A), neutral (B), and deprotonated (C) poly(catechol-alt-mxylylenediamine) and protonated (D), neutral (E), and deprotonated (F) poly(catechol-alt-pxylylenediamine). 4. CONCLUSIONS AND OUTLOOK Previously synthesized amphiphilic linear(A)-linear(B), linear(B)-linear(A)-linear(B), linear hyperbanched, hyperbranched-linear-hyperbranched, linear-dendritic, and dendritic-lineardendritic block copolymers composed of PEG and either PS, PPCMS, or benzyl ether dendrons were further investigated as complexing agents for a different family of enzymes. In this study the aforementioned block copolymers were used to immobilize lipases from Burkholderia cepacia, Candida rugosa, and Penicillium camemberti in both micelles and hydrogel networks. Complexation of the three lipase enzymes was found to significantly increase their activity (up to 1727% with G3-PEO13kDa) when compared to both native enzymes and their Triton X-100 mixtures. The substantial increase in the observed enzymatic activity might be attributed to the 24 ACS Paragon Plus Environment

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Biomacromolecules

ability of the complexing agents to selectively bind and accumulate the substrate near the active site of the enzymes. The developed enzyme complexing strategies for both lipase and laccase were successfully combined to form a one-pot cocatalytic reaction system capable of polymerizing catechol and either m- or p-xylylenediamine to produce perfectly structured alternating fluorescent copolymers. In this new reaction system laccase initially oxidized catechol as a prerequisite for copolymerization to occur via Michael addition of xylylenediamines to the in-situ produced oquinone. Addition of lipase was found to catalyze the aforementioned Michael addition leading to an increase in conversion (polymer yield). The use of complexing agents was found to further induce a slight increase in polymer yields. However, these complexing agents most notably facilitated the formation of copolymers of significantly greater molecular masses. The achieved enhancement could be attributed not only to their capacity to selectively bind substrates, but also to their ability to co-compartmentalize laccase and lipase in a close proximity to each other leading to decreased substrate(s) travel distance between the active sites of the two enzymes. Analysis of the synthesized poly(catechol-alt-m(p)-xylylenediamine) by fluorescence spectroscopy revealed the copolymers fluorescence to be heavily dependent on pH. Quantum yield of the copolymers was found to be greatest in acidic and basic environments with quantum yields of respectively 0.31 and 0.171 for the meta isomer while the para isomer yielded quantum yields up to 0.733. The significant increase in quantum yield of the ionized copolymers can be attributed to both changes in the electronic copolymer structure and increased chain rigidity and extension due to charge repellence.

ASSOCIATED CONTENT Supporting Information 25 ACS Paragon Plus Environment


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The Supporting Information is available free of charge on the ACS Publications website at DOI: Materials and methods; complexed lipase activity; copolymerization data; copolymer fluorescence spectra; monomer fluorescence spectra; enzyme MALDI-TOF MS spectra, dynamic light scattering (DLS) of copolymers.

AUTHOR INFORMATION Corresponding Author *(I.G.) E-mail: [email protected] ORCHID Ivan Gitsov:

0000-0001-7433-8571

Notes The authors declare no competing financial interest. 5. ACKNOWLEDGEMENTS The authors gratefully acknowledge Dr. Arthur Stipanovic (State University of New York – ESF) for access to SEC instrumentation. Partial funding for this study was provided by State University of New York Network of Excellence on Materials and Advanced Manufacturing (Biomaterials focus area).

REFERENCES


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Table of Contents Graphic

O

O OO

O O O O O O OO O O O O O O O O O O O O O O O O O

O O O O O O OO

O O O OO O O O O O O O O O O O O O OH

O O O HO O O O

OO O O O O O O O

O

O

O O

O

O

O

O

O

OO

O

HO

OH

O O

O

O O O

O

O

O O O O

OO

O O

O O

O

O

O O

O

O

O

O

O O

O

O

O O

O

O

O

O O

O

O

NH2

H2N

O O O O O O O O O

O

O

O O O O O O O O O O OO O O O O O O O O O O O OO O O O O O O O O O O O O O O OO O O O O O O O O O OH O O O O

Catechol

O OO

O

O O

O O

O

OO

O O O

O O O O O O O O O O O O O O O O O O O O O O O O OO O O O O O O O O O O O O O O O O O O O O O O O O O O O HO O O O

O O O

p-Xylylenediamine

O O O O O O O O O O O O O O O O

O

O

O

OOO O

O

O

O O

O

O

O

O

O

O

O O

OO

O

O O O O

O

OO O

O

O

O O

O O

O

HO O O

O O O O O O O O O O O

O O O

O O

O

O

O

O

O

OO

O O

O O

OO

O

O

O O O O O O OO

O

HO

OH

O O O

OO O OO

O OO O O O O O O O O OO O O O O O O OO O HO O O O O O O O O O O O O O O O O O O O O O O O O

Laccase-Lipase Polymer Complex

H2N

N

NH2

N

n

Poly(catechol-alt-p-xylylenediamine)

For Table of Contents use only


Unprecedented Enzymatic Synthesis of Perfectly Structured

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