Polymer Biocatalysis and Biomaterials - American Chemical Society

Chapter 25

Enzyme-Catalyzed Synthesis of Hyperbranched Aliphatic Polyesters 1

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1,

Downloaded by NANYANG TECHNOLOGICAL UNIV on October 13, 2017 | http://pubs.acs.org Publication Date: February 15, 2005 | doi: 10.1021/bk-2005-0900.ch025

Ingo T. Neuner , Mihaela Ursu , and Holger Frey * 1

Institute of Organic Chemistry, Johannes-Gutenburg-University, D-55099 Mainz, Germany Department of Macromolecular Chemistry, Technical University "Gh. Asachi", Bulevardul Mangeron 71A, 6600 Iasi, Romania

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Immobilized CALB (Novozym 435) is an appropriate catalyst to synthesize polyesters. In this paper, we present the general concept of concurrent ring-opening polymerization and polycondensation to form hyperbranched aliphatic polyesters. The concept is exemplified for the synthesis of hb-poly(εcaprolactone) copolyesters. The route permits variation of the degree of branching by the ratio of ε-CL and the AB comonomer. Specific issues of hyperbranched materials such as degree of branching DB, intrinsic viscosity and molecular weight determination are addressed. 2

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© 2005 American Chemical Society Cheng and Gross; Polymer Biocatalysis and Biomaterials ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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Although the structure of polysaccharides such as dextrane and glycogen was identified to be hyperbranched already in the 1930's and Flory introduced his theoretical work on random A B polycondensates in the early 1950's , only since the beginning of the 1990's a rapidly increasing interest in dendritic and hyperbranched polymers can be noticed. The perfectly branched dendrimers have to be prepared by tedious stepwise organic synthesis that involves protecting group chemistry and purification procedures in each step. Therefore, perfectly branched structures of higher, monodisperse molecular weight involve demanding, time consuming and costintensive procedures. In contrast to dendrimers, hyperbranched polymers are not perfectly, but randomly branched, which usually reduces the synthesis protocol to a one-pot set-up . As a result, highly branched materials can be produced at low cost. In general, dendrimers and hyperbranched polymers have some properties in common that renders them different in comparison to their linear analogues, such as a rather globular or bulky shape, resulting in the absence of entanglements . These characteristics lead to low viscosity in bulk and solution, strongly hindered crystallization, high functionality, eventually at the outer sphere or throughout the whole molecule. Hyperbranched structures are usually obtained by polycondensation of either A B monomers or by polymerization of inimer-type structures, i.e., monomers bearing an initiating site. Since biocompatibility is a precondition for medical and pharmaceutical application and moreover controlled degradability, by UV radiation or in vivo, it is highly desirable for use of polymers as drug carrier in subdermal implants or film former in ointment formulations the choice of suitable co-monomers is limited. Another prerequisite for this segment of application is the complete absence of heavy-metal catalysts and other, potentially toxic, organic residues such as solvents or residual monomer in the final product that has to be addressed by the synthetic procedure. In 1999 Trollsas as well as Fréchet reported the synthesis of hyperbranched copolyesters using hydroxy-functional ε-CL as cyclic inimer ' . In 2002, our group introduced a concurrent Ring-Opening Polymerization / Polycondensation concept where an A B type co-monomer, BHB, and an AB type monomer, in this case ε-caprolactone serving as latent AB monomer are polymerized . By the use of Novozym 435 they were able to synthesize hyperbranched copolyesters of higher molecular weights (more than 5xl0 gmol* ) and avoided heavy-metal catalysts, e.g. Sn(Oct) . Post reaction enzyme removal was facilitated by its adsorption on a macroporous resin that allows easy filtration and regeneration of the enzyme coated beads for repeated use. 1

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m

3

3,4

m

5 6

2

7

3

1

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Cheng and Gross; Polymer Biocatalysis and Biomaterials ACS Symposium Series; American Chemical Society: Washington, DC, 2005.


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.OH

CH

3

ο-α α BHP

ο

ε-CL

BHB

TMC

δ-VL

Figure 1: General Scheme of Combined Ring-opening Polymerization/Polycondensation and Possible Monomers Representing Branched and linear units. It should be pointed out that this principle is general, opening access to a large variety of hyperbranched analogues of aliphatic polyesters, as is obvious from screening experiments and subsequent studies with other lactones and trimethylene carbonate . Furthermore, enzymatic copolymerization avoids complex multistep monomer synthesis required for cyclic inimers used in previously reported self-condensing ring-opening-type polymerizations . Another concept that leads to dendritic structures containing a defined number of linear units was introduced in 1998 . It was right now investigated with respect to the influence of the length of the linear backbone segments . 8

5,6

9

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/tfr-Caprolactone Copolyesters The results presented in this paper deal with hyperbranched poly (ε-caprolactone) copolyesters (hb-PCL), based on the combination of ring-



357 opening polymerization of ε-caprolactone (ε-CL) as AB monomer and polycondensation of 2,2'-bis(hydroxymethyl) butyric acid (BHB) as branching A B 2 comonomer unit. By systematic variation of the fraction of the A B 2 comonomer BHB, series of hyperbranched copolyesters with different degree of branching have been prepared. Immobilized Lipase Β from Candida Antarctica (commercialized as Novozym 435) is known to catalyze both ring-opening polymerization of ε-CL and the concurrent polycondensation of BHB, leading to the desired hyperbranched structure (Figure 1). The copolyester synthesis was performed either in toluene or in bulk. The choice of toluene as solvent and the reaction temperature of 90 °C is based on the results published by Gross and Kumar". However, a mixture of toluene-dioxane had to be used, when the fraction of the polar A B comonomer, BHB, exceeded 20 mol% in order to solubilize BHB. As the activity of the enzyme is considerably lower in dioxane than in toluene , the amount of dioxane used was kept to a minimum. In the case of bulk polymerization it should be mentioned that BHB and 6CL form a homogenous solution above 76 °C. Both techniques, solution and bulk polymerization lead to similar results with respect to polymer properties. In terms of sustainable chemistry bulk techniques are more ecologically efficient and therefore favorizable. 2

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Table 1: Experimental data, a) determined from 'H-NMR; b) in C H C I 3 ; c) C H C 1 ; T=20 °C; d) commercial sample of Solvay Caprolactones. 3

Sample

BHB (feed)

BHB Yield (polym)

GPC

a

[mol%] [mol%]

b

VPO

[%] [g/mol] [g/mol]

d

L-PCL A&-PCL99 hb-PCL 98 hb-PCL 94 hb-PCL 90 hb-PCL 85 hb-PCL 75

1 2 6 10 15 25

1.0 1.7 4.5 8.4 14 25

c

90 95 97 85 80 73

62000 9000 7200 3500 2500 2500 1100

100300 20200 14500 6300 4100 4300 1200

[g/mol] 1.6 2.1 2.0 1.8 1.6 1.7 1.1


2000 1750 1700 1400 1350 1200

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The data compiled in Table 1 illustrate that the yield of the hyperbranched copolyesters decreases as the BHB fraction in the feed increases. This issue was already observed by Skaria et al. and ascribed to the antagonistic solubility properties of BHB and CALB. This is best described by the solubility of BHB being best in polar solvents, the difficulties in BHB homopolymerization and the decrease in C A L B s activity the more polar its close surroundings . Calculations of DB were made based on expressions described previously for copolymerization of A B - and AB monomers while values of characteristic structural information were extracted from H-NMR analysis (Figure 2a-c). In the equations below, D represents the dendritic units and L the total number of linear units in the A B / A B copolymers: 7

7

1

11


13

2

!

c o

2

11 n 111 n 11 • i n I » • 111 • ι • 1111111111111111111111111111111111 • 1111 ι ι μ ι ι 11111111111111111111111111111111 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

!

Figure 2: H-NMR of (a) Linear PCL, (b) hb-FCL, DB = 10%, (c) to-PCL, DB = 30%. Molecular weights characterization of hyperbranched polymers which usually possess a large number of hydroxyl or other end groups is problematic and can lead to erroneous results, if common linear standards are employed . 14



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Since the hydrodynamic volume of hyperbranched polymers is a specific property influenced by structural features as the degree of branching, the shape or the rigidity of the backbone and by chemical characteristics such as polarity and type of functionalization, appropriate GPC-calibration for molecular weight measurement is difficult. Although the hydrodynamic volume of compact hyperbranched macromolecules can be smaller than for linear chain analogues, interaction of the highly polar end groups with solvent and/or column often leads to strong overestimation of molecular weights. Number-average molecular weights (M ) determined by means of GPC (polystyrene standards) are in the range of 11Ï)0 to 9000 g/mol, weight-average molecular weights ( M ) in the range of 1200 to 20200 g/mol. All the samples showed monomodal molecular weight distributions with apparent polydispersities in the range of 1.6 to 2.1. Due to the relatively low polydispersities as well as the monomodal molecular weight distribution, formation of aggregates does not seem very likely. As determined with respect to yield, molecular weight of the synthesized A&-PCLs also exhibits a decreasing tendency the higher the fraction BHB in feed (table 1). But we are skeptic that this reflects reality but a limitation of GPC as analytical method for molecular weight determination of to-polymers. As vapor pressure osmometry (VPO) is independent of the structure of the samples, it is more appropriate for molecular weight determination of hbpolymers but limited by a maximum number average molecular weight of approximately 25-100 kDa depending on the solvent. Number average molecular weights (M ) of hb-PCL are in the range of 1200 to 2000 g/mol, depending of the amount of BHB incorporated. As observed in GPC molecular weights decrease the higher the amount of BHB although this effect is considerably less pronounced. Since all samples show excellent linear correlation in VPO measurements (Figure 3), aggregation due to polar hydroxyl groups can be excluded. Concluding, absolute molecular weights determined by VPO are more reliable compared to GPC analysis but limited by an upper maximum molecular weight. As important as elucidation of structure and actual molecular weights is determination of the materials' properties in solution by intrinsic viscosity. On the one hand information on the shape of the polymer chains in the respective solvent is obtained, but on the other hand knowledge for processing the materials is achieved as well. The dependence of the intrinsic viscosity [η] on molecular weights as well as the DB has been investigated for all copolymers. Figure 4 depicts the results of viscosity measurements in chloroform at 20 °C.



.

400A

,-Δ

hb-PCL 75

, ftb-PCL85 χ

M>-PCL90

^ /7&-PCL94 fob-PCL98 A''

ftb-PCL99

/Δ

10

15

-τ— 20

25

30

~~35

40

c[g/kg]

Figure 3. VPO measurements for HB-PCLs



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Figure 4. Reduced viscosity (Tj /c) of hyperbranched copolyesters as a function of concentration in chloroform at 20°C s

The linear relationship between r| /c and concentration demonstrates that no aggregation occurs in the investigated range which corresponds to observations discussed in the GPC section. The slope of the lines flattens out the higher the DB of the sample which means that higher branched samples have a very compact structure in solution. sp

Table 2. Intrinsic viscosities for hyperbranched polymers of different branching degrees in CHCI3 and THF solutions, at 20 and 50 °C resp. Sample

hb-PCL 99 hb-PCL 98 hb-PCL 94 hb-PCL 90 hb-PCL 85 hb-PCL 75

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Intrinsic viscosity, [η] [cm /g] Tetrahydrofuran Chloroform T=20°C T=20°C T=50°C T=50°C 15.8 40.1 35.5 17.5 10.4 24.8 15.8 15.3 7.8 16.7 10.2 11.3 7.2 6.6 8.0 9.5 4.5 6.3 7.0 8.1 3 4.0 3.3 3.8


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The interaction that exists in the hyperbranched PCL - solvent system is due to the physical interactions between the carbonyl group of hyperbranched PCL and the characteristic functional groups of the solvents. As illustrated in table 2, the intrinsic viscosity of hyperbranched PCL in CHC1 is larger than the intrinsic viscosity of hyperbranched PCL in THF at 20 and 50°C respectively which leads to the conclusion that CHC1 is a better solvent for the hyperbranched PCL than THF. Polymer-solvent interactions in CHC1 are larger than in THF, hence the coil swells in solution and the hydrodynamic volume is increased, which is translates into higher intrinsic viscosity. Raising the temperature from 20°C to 50°C enlarges the intensity of polymer-solvent interactions, which leads to a decrease of the intrinsic viscosity.. Figure 5 shows a plot of intrinsic viscosities versus degree of branching. It is obvious that the intrinsic viscosity of the hyperbranched samples decreases the higher the degree of branching, in analogy to the plot of M versus DB. 3

3

n

45n 40- • TCM 20 °C 35- ά

- O - T C M 5 0 °C —A—

30- i\

i

25-

.0

20-

Intrinsi


3

THF 20 °C

- δ - T H F 50 °C

1510-

— ~LL - .

5-

n 0.00

1

0.05

0.10 Degree of

-A.-.. - Δ

1

0.15

•

1

0.20

•

1

0.25

Branching

Figure 5. Plots of intrinsic viscosities vs. degree of branching of hyperbranched polycaprolactones in CHCl and THF at 20 and 50 °C resp. 3

As before the values of intrinsic viscosity of the Aè-PCLs with different DB are lower than those of linear PCL. This is due to the higher molecular weight of linear PCL and also related to the different structure and architecture of the linear and hyperbranched samples. Intrinsic viscosity measures the ratio of



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hydrodynamic volume to molecular weight. The structures of hyperbranched polymers are more densely packed, resulting in smaller hydrodynamic volumes compared to those of linear polymers with comparable molecular weights, leading to smaller viscosities. Thermal properties of the hyperbranched copolyesters have some importance for their applications and have been investigated using differential scanning calorimetry (DSC). A typical DSC diagram obtained after repeated heating/cooling cycles is shown in Figure 6. All the DSC curves showed a characteristic double peak up to DB = 0.35, from which the more intensive upper peak was confirmed to be the melting peak by microscopy. The shape of the double peak results from an annealing or reorganization process in the DSC, presumably through a melting - recrystallization - final melting mechanism that is due to a recrystallization point at 30 °C . 15

Figure 6: DSC curves for hb-PCl of different DB Further more the dependence of the melting points on the branching degree is easily observed. As expected, raising the incorporation of the A B monomer from 1 to 33 % mol causes a decrease of the melting point from 61 to 27 °C and a decrease of the melting enthalpy from 83 to 60 J/g. We attribute these trends to the influence of hydroxyl end groups on the crystallinity of copolyesters. Also 2


364 the values of T showed slightly increasing tendency the higher the degree of branching. g

Conclusions The universal concept of concurrent ring-opening polymerization / polycondensation was introduced for branched polymers and two routes to green hyperbranched poly(E-caprolactone) as ecologically friendly and potential medical polymer were discussed. The sustainable approach of this chemistry lies in the use of immobilized CALB (Novozym 435) and the eventual sacrifice of solvents during synthesis and work-up. In the discussion of characterization techniques that were used to describe ftè-poly(e-caprolactone) samples, systematic phenomena such as yield and molecular weight dependence on the branching co-monomer's feed (BHB) as molecular weight determination in general were addressed.


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Acknowledgements We are grateful to the SFB 428 of the DFG for financial support. Immobilized CALB was kindly provided by Novozymes A/S (Novozym 435™).

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Geddes, R. In The Polysaccharides; Aspinall, G. O., Ed.; Academic Press: London, New York, 1985; Vol. 3, p 209. Flory, P. J. J. Am. Chem. Soc. 1952, 74, 2718-2723. Newkome, G. R. Advances in Dendritic Molecules; JAI Press; Greenwich, CT, 1994; Vol. 1. Wooley, K. L.; Frechet, J. M . J.; Hawker, C. J. Polymer 1994, 35, 44894495. Liu, M . ; Vladimirov, N . ; Frechet, J. M . J. Macromolecules 1999, 32, 68816884. Trollsas, M . ; Loewenhielm, P.; Lee, V. Y.; Moeller, M . ; Miller, R. D.; Hedrick, J. L. Macromolecules 1999, 32, 9062-9066. Skaria, S.; Smet, M . ; Frey, H. Macromol. Rapid Commun. 2002, 23, 292296. Neuner, I.; Ursu, M . ; Frey, H. Polym. Mat. Sci. Eng. 2003, 88, 342. Trollsas, M . ; Hedrick, J. L. Macromolecules 1998, 31, 4390 - 4395. Choi, J.; Kwak, S.-Y. Macromolecules, submitted 2003.


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11. 12. 13. 14.

Gross, R. Α.; Kumar, Α.; Kalra, Β. Chem. Rev. 2001, 101, 2097 - 2124. Kumar, A. G., R.A. Biomacromolecules 2000, 1, 133 - 138. Frey, H.; Holter, D. Acta Polym 1999, 50, 67-76. Burgath, Α.; Hanselmann, R.; Holter, D.; Frey, H. Abstr Pap Am Chem S 1997, 214, 150-Pmse. 15. Runt, J.; Harrison, I. R. Methods Exp. Phys. 1980, 16B. 16. Note: Combined ring-opening and polycondensation has been previously employed for the synthesis of linear polymers: Namekawa, S.; Uyama, H.; Kobayashi, S. Biomacromolecules 2000, 1, 335.


Polymer Biocatalysis and Biomaterials - American Chemical Society

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