Synthesis of Thioester End-Functionalized Poly(-caprolactone) and Its

Chapter 6

Synthesis of Thioester End-Functionalized Poly(ε -caprolactone) and Its Application in Chemoselective Ligation

Tailored Polymeric Materials for Controlled Delivery Systems Downloaded from pubs.acs.org by UNIV LAVAL on 12/16/15. For personal use only.

Qiang Ni and Luping Yu

1

Department of Chemistry and The James Franck Institute, The University of Chicago, 5735 South Ellis Avenue, Chicago, IL 60637

In order to synthesize novel poly(ε-caprolactone) (PCL) conjugates, thioester functionalized PCL has been synthesized by using dimethylaluminum benzylthiolate as an initiator. The living character and quantitative introduction of thioester end in this ring opening polymerization (ROP) process have been confirmed by GPC and H NMR characterization. Furthermore, the applicability of chemoselective ligation to the thioester end has been demonstrated with compounds containing a cysteine terminal. 1

Increasing attention is being directed to the design and synthesis of polymeric drug delivery systems since they show a great potential to achieve controlled release as well as site-specific targeting (1-3). In this regard, synthetic polymers have been studied extensively along with their natural counterparts (e.g., sugars, proteins). Aliphatic polyesters, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(e-caprolactone) (PCL), and their copolymers, rank among the most promising candidates in the synthetic polymer category due to their unique property of combining biocompatibility, biodegradability, and permeability (in the case of PCL and its copolymers) (3, 4). Their incorporation into other biocompatible polymeric materials or bioactive species (e.g., peptides, enzymes) may lead to novel polymer conjugates. In fact, novel drug delivery systems (5) and potential prodrugs (6) have been developed based on these biodegradable polyesters. To this end, their heterobifunctional analogs are truly valuable intermediates, as elucidated in the model for pharmacologically active polymers by Ringsdorf (7). Ring opening polymerization (ROP) of lactones and related compounds has been the major polymerization approach to the sythesis of aliphatic polyesters (8). Numerous initiator systems based on organo aluminum, tin, and rare earth metal compounds have been developed (8-10). It is well recognized that the mechanism of the ROP using these initiators is a so called coordination-insertion mechanism (Scheme 1) and the polymer chain thus formed should carry the alkoxide (OR') group from the initiator (dialkylaluminum alkoxide, 1) while the other chain end is a hydroxyl group after hydrolysis. Accordingly, a functionalized initiator approach (e.g., using initiators like 1, where R = - C H C H ; R' = -(CH ) Br; (CH ) CH=CH ; -(CH ) NEt ) has been developed to achieve the synthesis of 2

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2

2

3

3

2

2

2

©1998 American Chemical Society

93 heterobifunctional aliphatic polyesters (11). However, this approach is limited by the available functionalities which can survive the reaction conditions. For example, attempts to synthesize primary amine-terminated P C L using an initiator like 1, where R' = -(CH ) NH , failed to yield the expected polyester due to amide linkage formation. Our interest in introducing thioester functionality into the P C L chain ends is motivated by the following considerations. First, known to be a kind of moderately activated carboxy derivative, thioester has many synthetic applications (12-16), especially in peptide synthesis. In particalar, chemoselective ligation (CSL) between two peptide segments, which possess a thioester end and N-Cys terminal, respectively, has provided direct synthetic access to polypeptide chains with the size of typical protein domains (16-18). The desirable features of this C S L lead us to reason that this approach should make a complement to the coupling methods currently employed for the synthesis of polymer conjugates (2). Secondly, living polymerization of lactones using dialkylalurninum aJkanethiolate is likely since its analog, dialkylalurninum alkoxide (1), is an excellent initiator in such cases (8). Therefore, well-defined polyesters and their copolymers with thioester end functionality can be synthesized. Furthermore, chemical modifications of these heterobifunctional polyesters (i.e., one thioester and one hydroxyl end) may provide useful materials for a variety of applications. In this paper we report a facile method for the introduction of thioester end group to P C L , and results on the reactivity of this functional group in C S L processes.


2

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2

Experimental Section Materials. ε-Caprolactone (Aldrich) was dried over C a H and distilled under reduced pressure. L-Lactide (Aldrich) was recrystallized twice from ethyl acetate. Benzyl mercaptan (Aldrich) was purified by distillation. Trimethylaluminum (2.0 M) in methylene chloride (Aldrich) was used as received without further purification. Poly(ethylene glycol) methyl ether (mPEG, M n ca. 550) was purchased from Aldrich. Toluene and methylene chloride (Fisher) were dried by refluxing over C a H and distilled just before use. T H F (Fisher) was purified by distillation over sodium chips and benzophenol. A l l other chemicals were purchased from Aldrich and used as received. 2

2

1 3

Characterization. The *H and C N M R spectra were collected on a Bruker 400 MHz and a QE-300 75 M H z N M R spectrometer, respectively. The G P C measurements were performed on a Waters 410 RI system equipped with a differential refractometer detector using THF (HPLC grade) as an eluent. Molecular weight and molecular weight distributions were calculated according to a modified universal calibration curve derived from polystyrene standards and the MarkHouwink equations for both polystyrene and PCL (79). Initiator preparation (Scheme 2). Dimethylalurninum benzylthiolate (2) was prepared by reaction of benzyl mercaptan with trimethylaluminum as described by Corey et al (12). To a previously flamed and argon purged 25 mL round-bottomed flask , CH C1 (4.0 mL) and benzyl mercaptan (0.14 mL, 1.2 mmol) were introduced through a rubber septum. To this solution was added slowly a 2.0 M solution of A1(CH ) in toluene (0.60 mL, 1.2 mmol) with stirring at -5 °C. This solution was then warmed up to room temperature and kept for 2 to 4 hrs before use. 2

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Scheme 1. Coordination-Insertion Mechanism of ROP of Lactones Using Initiator R AIOR'(l) Tailored Polymeric Materials for Controlled Delivery Systems Downloaded from pubs.acs.org by UNIV LAVAL on 12/16/15. For personal use only.

2

O

initiation

6

C—OR*

propagation

OR'

ΑΙ--0 Jn+1

Scheme 2. Tailored Synthesis of Thioester End-Funtionalized P C L (CH3)3AI+ HSCH C H 2

e

-A;

(CH )2AISCH C H5 + C H 3

5

Tol/CH CI 2

2

e

4

•(CH ) AIC^ ^ j ^YSCH CeH 3

2

(1 )

2

v

0

N

2

5

Toi ε-caprolactone

(2) SCh^CeHs

acetic acid

95


Synthesis of thioester end-functionalized PCLs (Scheme 2). Atypical plymerization was exemplified by the synthesis of PCL-40 (the number 40 indicates the designed monomer to initiator ratio). To a previouslyflamedand argon purged 25 mL Schlenk tube, toluene (8.0 mL) and ε-CL (0.93 mL, 8.4 mmol) monomer were introduced through a rubber septum. To this solution was added quickly the initiator (1) solution (0.8 mL, 0.21 mmol) with vigorous stirring. The Schlenk tube was then sealed with valve and kept at room temperature for 16 hrs. An increase in viscosity was noted. After quenching the reaction by adding excess acetic acid, the polymer was precipitated into η-heptane, dissolved in THF, reprecipitated into η-heptane and then dried under vacuum for 2 days. There was no ε-CL monomer detectable by *H NMR in the residue collected from the nheptanepart The yield was quantitative. Synthesis of diblock copolymer PCL-b-PLA (Scheme 3). The living prepolymer PCL-120 was synthesized by a similar approach as described above. After 24 hrs, certain portion of the living PCL solution was transfered to another Schlenk tube containing L-lactide in toluene at 80 °C. After another 24 hrs, the reaction was quenched by adding excess acetic acid. The polymer was precipitated into η-heptane, dissolved in THF, then precipitated into methanol. The conversions of ε-CL and L-lactide monomer were 100% and 80% respectively. Synthesis of N-Cys-terminated mPEG (3d). approach is outlined in Scheme 4.

The general synthetic

Synthesis of nitrophenyl-terminated mPEG (3a). To a solution of mPEG (6.18 g, 11.2 mmol), Ph P (3.07 g, 11.7 mmol) and 4-nitrophenol (1.63 g, 11.7 mmol) in THF (25 mL) was added slowly diethyl azodicarboxylate (DEAD) (1.9 mL, 12.1 mmol) at 0 °C. The reaction mixture was stirred overnight at room temperature. After removal of the solvent, the residue was dissolved in distilled water and thenfiltered.Thefiltratewas extracted with CH C1 several times. The combined organic layer was washed with distilled water, dried over Na S0 , concentrated by rotary evaporation and dried under vacuum to yield a light orange oil (5.93 g, 79%). This crude product was purified by gel chromatography using hexane:ethyl acetate:methanol (5:5:1) as an eluent. 3

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4

Synthesis of aminophenyl-terminated mPEG (3b). A Parr hydrogénation bottle was charged with 3a (2.8 g, 4.2 mmol), 95% ethanol (30 mL) and 10% Pd/C (0.45 g). The bottle was pressurized with hydrogen (50 psi) and allowed to shake overnight. The filtrate was concentrated by rotary evaporation and dried under vacuum to yield a pale yellow oil (2.6 g, 97%). Synthesis of 3c. To a solution of 3b (2.6 g, 4.0 mmol), N,N'-bis(t-Boc)-Lcystine (1.9 g, 4.3 mmol) and DMAP (0.08 g, 0.6 mmol) in CH C1 (40 mL) was added dropwise 1.0 M DCC in CH C1 (4.5 mL, 4.5 mmol) at 0 °C with stirring. The reaction mixture turned to be cloudy after several minutes and it was stirred overnight at room temperature. The precipitate formed wasfilteredoff and the solution was washed with dilute hydrochloric acid and then distilled water. After the organic layer was dried over Na S0 , the CH C1 was evaporated under vacuum to yield a viscous oil (3.5 g, 80%). The crude product was purified by gel chromatography using hexane:ethyl acetate:acetone (1:3:2) and then acetone as eluents. 2

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96 Synthesis of 3d. Compound 3c (1.9 g, 1.7 mmol) was dissolved in CH C1 (15 mL) and treated with trifluoroacetic acid (4 mL) under N . After 1 hr stirring at room temperature, the solvent was removed. The residue was taken up in CH C1 (15 mL), concentrated by rotary evaporation (this procedure was repeated four times), and finally dried under vacuum for 2 hr. The resulting deprotection product was dissolved in distilled water (10 mL) under N , and pH of the solution was adjusted to be ca. 8 by adding 1 M NaOH. The solution was then treated with dithiothreitol (DTT) (0.95 g, 6.2 mmol) and stirred overnight at room temperature. The resulting reaction mixture was acidified by adding 2 M hydrochloric acid and extracted with CH C1 several times. The combined organic layer was washed with saturated aqueous NaCl, dried over N a ^ C ^ and then under vacuum to yield a viscous oil (1.4 g, quantitative). This crude product was dissolved in THF for chemoselective ligation without further purification. 2

2

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2

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Chemoselective ligation. Under various reaction conditions, chemoselective ligation using the thioester end functionalized PCLs with L-cysteine ethyl ester hydrochloride and 3d has been investigated. Two representative examples are given below. Chemoselective ligation using PCL-15 with L-cysteine ethyl ester hydrochloride. To a solution of PCL-15 (50 mg, 0.03 mmol) and 18-C-6 (4 mg) in T H F (5 mL) was added another solution of N-cysteine ethyl ester hydrochloride (8 mg, 0.05 mmol) and Κ ^ 0 (20 mg) in distilled water (1 mL) with stirring under N at room temperature. The reaction mixture was acidified and precipitated into η-heptane after 12 hr. The precipitate formed was washed with dilute hydrochloric acid then distilled water, and finally dried under vacuum to give the ligation product. 3

2

Chemoselective ligation using PCL-15 with 3d (Scheme 5). To a solution of PCL-15 (54 mg, 0.03 mmol) in T H F (8 mL), acetonitrile (6 mL), Κ £ 0 (0.6 g), K H C 0 (0.4 g) and 18-C-6 (6 mg) were introduced under N in that order. After stirring for about 10 miniutes, to this inhomogeneous solution was added a solution of 3d (60 mg, 0.08 mmol) in THF (1 mL). The reaction mixture was acidified and the solvent was removed under vacuum after stirring at room temperature for 18 hr. The residue was dissolved in THF and precipitated into η-heptane. The precipitate was washed with dilute hydrochloric acid then distilled water, and finally dried under vacuum to give the ligation product. 3

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2

Results and Discussion For the synthesis of thioester end-functionalized P C L , dialkylalurninum benzylthiolate (2) was chosen as the initiator because it is easily prepared and very effective in the synthesis of thioesters from lactones and esters. The general synthetic approach is outlined in Scheme 2. Polymerizations under different reaction conditions have been systematically conducted. Selected data on the molecular weight and molecular weight distributions (MWDs) of PCLs synthesized are collected in Table 1. While the quite narrow molecular weight distributions (1.09 - 1.34) and the linear relationship between the number-average molecular weight (Mn) at total monomer conversion and the monomer to initiator ratio (M/I) (Figure 1) imply the living character of the polymerization process, more conclusive evidence was from the polymerization resumption experiment. In this experiment, more ε-CL monomer was added after the polymerization of the first addition of εC L had gone to completion. Figure 2 shows that the molecular weight increased

97

Scheme 3. Synthesis of Diblock Copolymer PCL-b-PLA

,SCH CeH5

(H C) AIO

neCL

3

2

2

Tol.,r.t.

n-1


Ο

Ο 80 °C

,SCH CeH5 2

n-1 ii) acetic acid PCL-b-PLA Scheme 4. Synthesis of N-Cys-Terminated mPEG(3d)

P P h , DEAD 3

η

THF

3a N,N'-bis(t-Boc)-Cystine

H,

DCC/CH CI

Pd/C (10%)

2

3b t-BocNH I

J

w

H

NHt-Boc

Ο

3c i) TFA/CH CI 2

2

ii) DTT/NaOH/H 0 2

HS

3d

2


98

Scheme 5. Chemoselective Ligation between Thioester EndFunctionalized P C L and N-Cys-Terminated mPEG(3d)

Chemoselective ligation

Entropy-activated rearrangement

SH

Η

Ο


99

Ο

100

200

300

400

M/I Figure 1. Plot of number-average molecular weight (Mn) versus monomer to initiator ratio(M/I).

τ

1

j 20.00

1

1

1

1

j 30.00

1

r

Minutes Figure 2. GPC profiles of polymerization resumption experiment: peak χ PCL after addition of ε-CL, M/I = 60; peak b: PCL after second addition of ε-CL, M/I = 150.

100 for the final polymer (peak b, Mn = 20400, MWD = 1.21), relative to the first peak (peak a, Mn = 7810, MWD = 1.13), and there was no detectable residual peak. This result indicates that the polymerization proceeds in the absence of chain termination and intramolecular chain transfer since either mechanism will produce inactive polymers or cyclic structures. Since we are more concerned with the polymer chain ends, *H NMR study on this system was carried out in detail. As a typical example, shown in Figure 3, the H NMR spectrum of PCL-20 gave an intensity ratio (1.00/1.02) between He (2.58ppm, methylene groupfromε-CL at


!

the thioester chain end) and Hh (3.64ppm, methylene group from ε-CL at the hydroxy end) very close to unity, which was in agreement with our expectation that the polymer chain should be capped with one thioester end and one hydroxy end. Moreover, further study on the initiator in CDC1 (data not shown here) has shown that during the initiation step, all the initiators were reactive, and only the acyl-oxygen cleavage was involved. All these results allow us to conclude that this initiator is effective not only for the ROP of ε-CL but also for the quantitative introduction of the thioester end group as well. On the other hand, these results also suggest that this initiator might be suitable for the synthesis of pure block copolymers such as PCL-b-PLA. Consequently,the synthesis of PCL-b-PLA with 2 was carried out (Scheme 3). Both resultsfromGPC (Figure 4) (peak a, Mw = 17600, MWD = 1.21; peak b, Mw = 29100, MWD = 1.20) and *C NMR (Figure 5) characterization are consistent with our prediction (20). 3

l

Table 1. Polymerization of ε-CL Initiated with 2 in Toluene at Room Temperature.

a

Entry (PCL-n) PCL-15 PCL-20 PCL-40 PCL-80 PCL-120 PCL-150 PCL-400

tCL]

t(h)

b

conv.(%) M

1.0 1.0 1.1 1.0 1.8 1.2 1.8

12 12 16 18 18 20 18

>99 >99 >99 >99 >99 >99 90

M

w

n

2190 3090 5890 11000 15600 20500 49600

1.11 1.10 1.09 1.21 1.20 1.21 1.34

a. The number η indicates the designed degree of polymerization. b. GPC results: THF as an eluent and calculated by a modified universal calibration curve. Application of the thioester end-functionalized P C L in C S L . Since the goal of our efforts toward the introduction of thioester group into PCL is to further utilize this functionality to achieve the synthesis of novel PCL conjugates, it is vital to understand the reactivity of the thioester end. As mentioned above, the CSL using thioester functionality has been very successful in peptide synthesis. However, application of such a method to polymer synthesis is unprecedented. To test the applicability of the CSL approach, we first chose L-cysteine ethyl ester hydrochloride as a small model compound to react with the thioester endfunctionalized PCL. Under appropriate conditions (see experimental section), the reaction went smoothly. The Ή NMR spectrum of the resulting product shows a complete disappearance of the 2.58 ppm signal (methylene groupfromε-CL at the

TMS


CDCI3

1

»

7

1 1 5

I t 3

1 1

ppm

Figure 3. *H NMR spectrum of PCL-20 in CDCI3.

1 t 10

Min. Figure 4. GPC profiles of PCL-b-PLA (b) and its PCL prepolymer (

180

120 13

60

Figure 5. C NMR spectrum of PCL-b-PLA.

0 ppm


102 PCL thioester end), instead, a new peak characteristic of the formation of an amide bond appeared at 6.47 ppm. To exclude the interference caused by hydrolysis of the thioester end or the vulnerable ester backbone of PCL, the thioester endfunctionalized PCL was also treated under the same conditions without adding Lcysteine ethyl ester hydrochloride as a control. According to the *H NMR and GPC results, there were no detectable changes of Mn and MWDs from the PCL samples before and after the treatment. Therefore, the thioester at PCL chain end is indeed a reactive functional group. Furthermore, N-Cys-terminated mPEG (3d) has been synthesized (Scheme 4) to reacact with thioester end-functionalized PCL. Not only because its coupling product may be interesting for drug delivery systems, but more importantly, the resultsfromthese reactions can be readily generalized since PEG is soluble in both aqueous medium and common organic solvents. Detailed study on the CSL between thioester end-functionalized PCL and N-Cys mPEG (3d) has been carried out by systematically changing solvents, reagents and temperature as well. The results from our experiments are very encouraging. High coupling efficiencies (>80%) with regard to the benzyl thioester were observed under several different reaction conditions. For example, under room temperature, using CH CN/THF as mixing solvent and KHC0 /K2C0 /18-C-6 as reagents, the coupling reaction went to completion within 18 hr. The H NMR spectrum and GPC profile of the coupling product are shown in Figure 6 and 7. The doublet at 6.63 ppm unambiguously indicates the formation of a new amide linkage. Although the complete disappearance of the 2.58 ppm triplet, which is due to the thioester end, does not ensure a quantitative coupling, the results from its GPC profile (i.e., no residual peak and no MWD broadening) excluded the presence of any detectable side reactions under these conditions. Combined with the observation that there was virtually no reaction using ethanolamine instead of N-Cys-mPEG under the same reaction conditions, the coupling reaction is believed to proceed in a similar fashion as that in peptide synthesis (Scheme 5). 3

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3

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Conclusions A facile method for the introduction of thioester end group in the ROP of ε-CL using dimethylaluminum benzylthiolate (2) as an initiator has been developed. The living character shown in the polymerization process has enabled us to synthesize novel thioester end-functionalized PCL as well as diblock copolymer PCL-b-PLA in a controlled way. More importantly, the chemoselective ligation method has also been demonstrated to be applicable to the thioester end-functionalized PCL. This may provide a new approach to the design and synthesis of novel PCL conjugates such as PCL-peptide conjugate. Moreover, it is worth pointing out that recent extension work (18) on the CSL using thioester functionality will certainly make this approach more promising in polymer synthesis along with the development of novel synthetic methods that allow convenient incorporation of thioester functionality into either polymer chain ends or side chains. Acknowledgments This work was supported by the National Science Foundation. Support from the National Science Foundation Young Investigator program is gratefully acknowledged.

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Putnam, D.; Kopecek, J. Adv. in Polym. Sci. 1995,122,55. Hayashi, T. Prog. Polym. Sci. 1994,19,663. Chiellini, E.; Solaro, R. Adv. Mater. 1996, 8, 305. Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V.; Langer, R. Science, 1994, 263, 1600. Kricheldorf, H. R.; Kreiser-Saunders, I. Polymer 1994, 35, 4175. Ringsdorf, H. J. Polym. Sci. Symp. 1975, 51, 135. Lofgren, Α.; Albertsson, A. C.; Dubois, Ph.; Jerome, R. J.M.S. - Rev. Macromol. Chem. Phys. 1995, C35(3), 379. Abdel-Fattah, T. M.; Pinnavaia, T. J. J. Chem. Soc., Chem. Commun. 1996, 665. Yamashita, M.; Takemoto, Y.; Ihara, E.; Yasuda, H. Macromolecules 1996, 29, 1798. Dubois, Ph.; Jerome, R.; Teyssie, Ph. Makromol. Chem., Macromol. Symp. 1991, 42/43, 103. Corey, E. J.; Beames, D. J. J. Am. Chem. Soc. 1973, 95, 5829. Masamune, S.; Kamata, S.; Schilling, W. J. Am. Chem. Soc. 1975, 97, 3515. Mihara, H.; Maeda, S.; Kurosaki, R.; Ueno, S.; Sakamoto, S.; Niidome, T.; Hojo, S.; Aimoto, S.; Aoyagi, H. Chem. Lett. 1995, 397. Zhang, L.; Tam, J. P. J. Am. Chem. Soc. 1997,119,2363. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. Β. H. Science 1994, 266, 776. Lu, W.; Qasim, Μ. Α.; Kent, S. Β. H. J. Am. Chem. Soc. 1996, 118, 8518. Canne, L. E.; Bark, S. J.; Kent, S. Β. H. J. Am. Chem. Soc. 1996, 118, 5891. Chindler, Α.; Hibionada, Y. M.; Pitt, C. G. J. Polym. Sci., Polym. Chem. Ed. 1982, 20, 319. Jacobs, C.; Dubois, Ph.; Jerome, R.; Teyssie, Ph. Macromolecules 1991, 24, 3027.

Synthesis of Thioester End-Functionalized Poly(-caprolactone) and Its

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