RAFT Polymers: Novel Precursors for PolymerâProtein Conjugates

Chapter 41

RAFT Polymers: Novel Precursors for PolymerProtein Conjugates 1,

1

Downloaded by UNIV OF GUELPH LIBRARY on August 8, 2012 | http://pubs.acs.org Publication Date: June 26, 2003 | doi: 10.1021/bk-2003-0854.ch041

2

Christine M. Schilli , Axel H. E. Müllier *, Ezio Rizzardo , San H. Thang , and (Bill) Y. K. Chong 2

2

1Makromolekulare Chemie II, Universität Bayreuth, D-95440 Bayreuth, Germany C S I R O Molecular Science, Bag 10, Clayton South, Victoria 3169, Australia 2

Temperature- and pH-sensitive polymers and block copolymers have been synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Thiocarbonylthio compounds used as chain transfer agents in the polymerization lead to end-functionalized polymers that give the corresponding thiols upon hydrolysis. Both block copolymers with short blocks of oligo(active ester)s and thiol-terminated polymers can be used for polymer-protein conjugation. 2-Vinyl-4,4dimethyl-5-oxazolone (VO), N-hydroxysuccinimide methacrylate (NHSM), diacetone acrylamide (DAA), N-isopropylacrylamide (NIPAAm), and acrylic acid (AA) have been polymerized to homo- and block copolymers with narrow MWD in most cases, and their conjugation to model peptides has been investigated. Block copolymers of NIPAAm and AA were investigated for their response to combined external stimuli. Thus, thermo- and pH-responsive systems can be created for control of enzyme activity or molecular recognition processes.

© 2003 American Chemical Society

In Advances in Controlled/Living Radical Polymerization; Matyjaszewski, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

603


604 Recently, the synthesis of polymers via reversible addition-fragmentation chain transfer (RAFT) polymerization has gained importance due to its great versatility, its compatibility with a wide range of monomers, the control of the molecular weights, and the low polydispersities of the resultant polymers (1,2). Besides, it offers all advantages of conventional free radical polymerization. RAFT polymerizations are carried out in the presence of thiocarbonylthio com pounds of general structure Z-C(=S)S-R and result in the formation of end-functionalized polymers (3). Poly(iV-isopropylacrylarnide) (polyNIPAAm) exhibits a lower critical solu tion temperature (LCST) of 32 °C in aqueous solutions. With the conjugation of the LCST polymer to a peptide or protein, thermoresponsive systems are created. Poly(acryiic acid) can be used to enable response to pH and ionic strength. Proteins conjugated to block copolymers of NIPAAm and acrylic acid are espe cially interesting due to their response to combined external stimuli. Potential controlled release of enzymes/drugs to bioseparations and sensor or actuator systems. Two different routes for polymer-protein conjugation have been envisaged: (a) RAFT polymerization in order to obtain dithiocarbonyl endgroups which are hydrolyzed to the corresponding thiols and can be conjugated to thiol functions of proteins; (b) synthesis of block copolymers with short blocks of oligo(active ester)s for conjugation to the amino functions of proteins. Poly(2-vinyl-4,4dimethyl-5-oxazolone) (polyVO) and poiy(iV-hydroxysuccinimide methacrylate) (polyNHSM) consist of active ester units that are frequently used for conjugation to amino moieties of proteins (4,5).

Experimental Section Materials A/-Isopropylacrylamide (Aldrich, 97 %) was recrystallized twice from benzene/hexane 3:2 (v:v) and dried under vacuum prior to use. Acrylic acid was purified according to the standard procedure. The monomers 2-vinyl-4,4dimethyl-5-oxazolone (TCI Tokyo), diacetone acrylamide (Lubrizol Co.) and Nhydroxysuccinimide methacrylate (donation from Dr. Steve Brocchini, School of Pharmacy at the University of London) were used as received. Azobisisobutyronitrile (AIBN, Fluka, purum) was recrystallized from methanol. Ι,Γ-Αζοbis(cyciohexanecarbonitrile) (VAZ088, DuPont) was recrystallized from ethanoi and 4,4'-azobis(4-cyanopentanoic acid) (ACP, Fluka, £ 98 %) was recrystallized from methanol prior to use. The initiator V-70 (2,2'-azobis(4-methoxy-2,4dimethyl valeronitrile), donation from Wako Chemicals GmbH Germany) was used as received. The syntheses of the chain transfer agents are described else where (6-11).


A

605 General Polymerization Procedure The reagents were mixed in a vial and aliquots were transferred to ampoules, which were degassed by threefreeze-thaw-evacuatecycles and then flame sealed under vacuum. The ampoules were immersed completely in an oil bath at the specified temperature. For GPC measurements in THF, poly(acryiic acid) was methylated using methyl iodide and 20 wt.-% Me NOH solution in methanol for 1 h at 80 °C.


4

Instrumentation Gel permeation chromatography (GPC) of the THF-soluble polymers was performed on PSS SDVgel columns (30 χ 8 mm, 5 μπι particle size) with 10 , 10 , 10 , and 10 À pore sizes using RI and U V detection (A=254 nm). THF was used as an eluent (flow rate 0.5 mL/min) at a temperature of 25 °C. For the polyNIPAAm samples, GPC was performed using THF + 0.25 wt.-% of tetrabutylammonium bromide as an eluent (flow rate 0.5 mL/min). The injection volume was 100 μL· As an internal standard, 0-dichlorobenzene was used. Poly styrene standards were used for calibration. GPC on the DMF-soluble polymers poly(NHSM) and PNIPAAm-6-PAA was performed using a series of four Styragel columns HT2, HT3, HT4, and HT5 and an oven temperature of 80 °C. The solvent was DMF + 0.05 M LiBr at a flow rate of 1.0 mL/min. A Dawn EOS light scattering detector with Optilab DSP interferometer (bothSci.at 690 nm) was used. GPC using water + 0.05 M sodium azide was conducted on PSS Suprema columns (300 χ 8 mm, 10 μπι particle size) with ΙΟ , 10 , and 10 À pore sizes. Poly(methacrylic acid) standards were used for calibration. The measurements were carried out at a flow rate of 1 mL/min at 25 °C or 60 °C, respectively, using RI and U V detection (Λ=254 nm). Cloud-point measurements were performed in 0.2 wt.-% (buffered) aqueous polymer solutions on a Hewlett Packard HP 8453 UV-visible chem station at a wavelength λ = 500 nm using a thermostatted cell with a heating rate of 0.5 K/min. Spectra were recorded in transmission and cloud points were determined as the inflection points of the transmission versus temperature plots. MALDI-TOF mass spectrometry of the poly(NIPAAm) samples was per formed on a Bruker Reflex III spectrometer equipped with a 337 nm N laser in the reflector mode and 20 kV acceleration voltage. Dithranol (Aldrich, 97 %) was used as the matrix material. Sodium or potassium trifluoroacetate was added for ion formation. Samples were prepared from THF solution by mixing matrix (20 mg/mL), sample (10 mg/mL) and salt (10 mg/mL) in a ratio of 10:1:1. The 2

3

4

5

2

3

4

2


606 number-average molecular weights, M , of the polymer samples were determined in the linear mode. H - N M R spectra were recorded on a Bruker 250 M H z instrument using TMS as internal standard. U V measurements were performed on a LambdalS UV-vis spectrophotometer (Perkin-Elmer) in the wavelength range from 190 to 550 nm. n

!


Results and Discussion Figures 1 and 2 give an overview of the chain transfer agents and monomers, respectively, used in the different RAFT polymerizations reported herein.

S

Jl S-R

1

a

Ζ

N

R

"C · *

•O-

2

e Z =

Ν

3

2

d Z = PhCH , R = CH(CH )Ph

c Z = Ph, R = C ( C H ) C N 3

R = C(CH ) Ph

b Z=

CH Ph

2

2

3

, R = CH(CH )CN 3

Figure 1. Chain transfer agents used in the RAFT polymerizations.

R A F T Polymerization of NIPAAm NIPAAm was polymerized using two different chain transfer agents, i.e. benzyl 1-pyrrolecarbodithioate l a and cumyl 1-pyrrolecarbodithioate l b . In both cases, low polydispersities were obtained with good agreement between calculated and experimental molecular weights (7). The results are shown in Table I. The GPC characterization of poly(NIPAAm) in THF involves various problems (12,13) due to irreversible chain aggregation after complete drying of the polymer samples (74). Nevertheless, we have obtained good results by the addition of 0.25 wt-% tetrabutylammonium bromide (TBAB) to the THF solu-


607 ΛΓ-isopropylacrylamide (NIPAAm)

CH =CH I

CH =CH 2

N-hydroxysuccinimide methacrylate (NHSM)

acrylic acid (AA)

CH

2

ι

c=o

c=o

2

!

I

c=o

OH

NH

I

I


H3C-CH I CH

3

CH =C

ο I 3

diacetone acrylamide (DAA)

2-vinyi-4,4-dimethyi5-oxazolone (VO)

CH =CH 2

I c=o I NH CH C H

2

3

- ^

Figure 2. Monomers used in the RAFT polymerizations.

tion and using PSS SDVgel columns, whereas with pure THF no Anal.ysable results could be obtained (7). The GPC traces of poly(NIPAAm) obtained with chain transfer agent l b show a high-molecular weight shoulder only at conversions higher than 90 %. This is usually seen for RAFT polymers at high monomer conversions (14), which is most likely due to combination of the growing macroradicals. GPC evaluation of the molecular weights using polystyrene standards for calibration gives significantly higher molecular weights than those obtained from M A L D I TOF Anal.ysis. Therefore, M values obtained by MALDI-TOF were used for evaluation of the effectiveness of the RAFT process. In this case, good to excel lent agreement between calculated and experimental molecular weights was found (entries 1+2 in Table I) (7). Considering the two chain transfer agents used, l a seems slightly more effective in the RAFT polymerization of NIPAAm with respect to polydispersities and control of molecular weight. This can be explained in terms of the lower stability of the benzyl radical as compared to the cumyl one, making the benzyl radical more active towards monomer addition. In N


Table I. Experimental Data for Synthesized Homopolymers PDI initiator time conv CTA (min) (%) (mM) (mM) 0 c 3900 1.13 2900 73 415 la 5300 1.28 3900 1 NIPAAm (39.2) AIBN 99 2000 9000 1.19 (1.742) (6.9) 5600 87 315 la 10300 1.37 6400 (19.6) 2000 99 1.21 7300 10100 71 462 AIBN 2 NIPAAm* lb 15200 10200 1.07 (1.742) (19.6) (6.9) 99 1085 3100 2.11 43500 89 600 AIBN 3 NHSM la 2600 2.34 41300 (10.0) (78.0) 74 (1.373) 960 3100 1.71 24100 83 4 600 NHSM AIBN lb 2300 1.78 22400 (10.0) 60 (1.376) (74.8) 960 1.52 3100 24500 81 600 5 NHSM AIBN lc 2700 1.47 24200 (5.08) (1.360) 70 (70.3) 960 2.06 2300 36200 63 600 6 NHSM V-70 Id 2200 1.95 31300 (12.16) (1.367) (77.0) 58 960 1.48 2000 1800 18 60 AA VAZ088 7 le (5.84) (0.66) (41.41) 1.19 8100 7900 80 180 8 AA VAZ088 le (5.84) (42.64) (0.66) 1.09 1300 1200 35 360 9 VO AIBN le 2200 2200 1.09 64 (3.52) (159.0) (14.63) 960 1.46 2600 2300 99 4140 AIBN 10 VO le (13.84) (3.52) (200.0) 1.29 4800 4000 49 120 AIBN DAA 11 le 6900 1.23 5900 (21.27) (3.57) (1.18) 71 240 1.23 6800 5600 70 120 12 DAA ACP le 8500 1.21 6600 89 (1.20) (21.69) 240 (4.15) Experimental conditions: entry 1+2, dioxane at 60 °C; entry 3-5, DMF at 60 °C; entry 6, DMF at 30 °C; entry 7+8, MeOH/H 0 (4:1) at 90 °C; entry 9+10, benzene at 65 °C; entry 11+12, MeOH at 65 °C. For initiator abbreviations, see experimental part. entry

monomer (M)


8

2

M values determined by MALDI-TOF mass spectrometry. n

contrast to that, fragmentation of the intermediate radical bearing the cumyl moiety is expected to be faster than the one bearing the benzyl moiety. Nevertheless, the radical stability factor seems to govern the process. Poly(NIPAAm) is a thermoresponsive polymer, i.e. it shows LCST behaviour in aqueous solutions (15). It was of interest to find out to what extent the


609 polymer molecular weight influences the exact temperature of this transition from hydrophilic to hydrophobic state. As can be seen from Figure 3, the cloud point, F , decreases virtually linearly with increasing reciprocal molecular weight and approaches Τ ~ 32 °C for M > 20,000 g/mol. This result seems reasonable as the hydrophobic endgroups should lower the cloud point due to an increase of overall hydrophobicity and a collapse of the intramolecular hydrogen bonds at lower temperature. This effect becomes less and less important the larger the molecular weight of the polymer, i.e. the smaller the fraction of the hydrophobic endgroups in the polymer. Nakahama et al. reported a dependence of LCST on endgroup structure for poly(N,N-diethylacrylarnide) (16). c


n

34

32

£

3

°i

28 H

26

0.00

0.05

— j — 0.10

—ι— 0.15

j 0.25

— , —

0.20

0.30

J 0.35

3

1/M [10*]

Figure 3. Cloud points ofpoly(NIPAAm) versus reciprocal molecular weight.

R A F T Polymerization of Acrylic Acid RAFT is the only known process for the controlled polymerization of acrylic acid (AA) without the need for a protecting group (2). As A A is a quite active monomer in terms of RAFT polymerization, chain transfer agents with moderate activities can be used. Rizzardo et al. used 1c as chain transfer agent and methanol as solvent (17). Here, the polymerization was conducted in a mixture of methanol/water (4:1) in order to increase solvent polarity and thereby accelerate the polymerization. 1-Cyanoethyl 2-pyrrolidone-l-carbodithioate le was used as chain transfer agent. The pyrrolidone moiety shows a moderate reactivity as for activating the C=S double bond towards radical addition. Under these conditions, best results are obtained at medium to high conversions (cf. entry 8 in Table I). Samples taken at low conversions show high polydispersities, indicating a rather slow equilibration between dormant and active polymer chains.


610 Synthesis of Block Copolymers of NIPAAm and Acrylic Acid


For the synthesis of the block copolymers, poly(acrylic acid) (PAA) was used as aMacromolecularchain transfer agent (see Table II). P A A was purified by reprecipitating its methanol solution into ethyl acetate in order to remove residual monomer and avoid formation of gradient copolymers in the subsequent block copolymerization with NIPAAm. The polydispersities obtained were quite low.

Table II. Experimental Data for Synthesized Block Copolymers monomer initiator time conv M„ />c CTA Mn.theo PDI (M) (mM) (mM) (min) (%) 1.11 2.37· 10* 15100 64 600 NIPAAm/AA AIBN PAA-le 2.75· 10 17200 1.09 82 960 (1.49) (14.87) (7.0) 1.22 10400 5800 960 59 AIBN NIPAAm/VO PVO-lc 6200 13200 1.14 1320 79 (2.24) (18.20) (1.94) Polymerization in MeOH at 60 °C; polymerization in benzene at 65 °C; Macromole cular chain transfer agents, M (PAA) = 7900 and M„(PVO) = 2200; cf. Table I fG

a)

c)

b)

c)

6

a)

b)

c)

n

In order to investigate the influence of the P A A block on the LCST behav iour of poly(NIPAAm), cloud-point measurements were conducted at different pH values in buffered polymer solutions. Figure 4 shows that transmission decreases only slightly at pH > 5 when the temperature is increased above the LCST of poly(NIPAAm). This may suggest the presence of micelles with poly(NIPAAm) forming the micellar core at Τ > T and P A A forming the corona. Micelle formation has been confirmed by preliminary dynamic light scattering experiments. Transmission decreases substantially at pH4.5, indicating the formation of a gel due to increasing insolubility of the protonated P A A corona. Thus, the formation of this type of micelles is dependent on both pH and temperature. A doubly-responsive behaviour in aqueous media has been described by Armes et al. (18) and by Laschewsky et al. (19). Both DMF and aqueous GPC were used to determine the molecular weights and polydispersities of the polymer samples. D M F GPC gave M values that were about two orders of magnitude higher than expected (see Table II). There fore, it was assumed that some sort of micelle formation takes place. This is not quite expected as DMF should be a good solvent for both blocks. In order to clarify this assumption, aqueous GPC of the polymer samples was performed at 25 °C and at 60 °C. At 25 °C, only one peak was observed. At 60 °C, two peaks were observed, one in the high-molecular weight region and the second one at the same elution volume as the peak observed at 25 °C (Figure 5). c

n



611

Temperature [°C] Figure 4. Turbidimetry of buffered aqueous solutions ofPNIPAAm-b-PAA; (,) pH4.5,0.)pH5-7.

—Γ ΙΟ

~i

15

"»

r-

20

T"

25

30

V [mL] Figure 5. Aqueous GPC of PNIPAAm-b-PAA at 25 °C (top) and 60 °C (bottom).


612


298 Κ

318Κ

328 Κ

368 Κ

398 Κ (ppm) l

Figure 6. Temperature-sweep H~NMR spectrum of PNIPAAm-b-PAA in DMF/LiBr.


613 !

Temperature-sweep H - N M R spectroscopy in c? -DMF + 0.05 M LiBr shows an interesting behaviour of these block copolymers (see Figure 6): The methine proton ascribed to acrylic acid disappears at temperatures around 328 Κ and then reappears above 358 K. This phenomenon has not been explained yet. 7


R A F T Polymerization of Active Esters To the best of our knowledge, the active ester monomers 2-vinyi-4,4dimethyl-5-oxazolone (VO) and JV-hydroxysuecinimide methacrylate (NHSM) have been polymerized by RAFT for the first time. Figure 7 shows the plots of M (PS calibration) and PDI versus conversion for the RAFT polymerization of V O using le as a chain transfer agent. The agreement between experimental and calculated molecular weights (shown as solid line in the plot with MQTA as intercept) are excellent as well as the obtained polydispersities which are equal or smaller than 1.10. With chain transfer agent le, a broad molecular weight distribution is obtained. This result might be attributed to the pyrrolidone moiety in le which slightly deactivates the monomer in the RAFT process as compared to the phenyl substituent in le (2,6). N

1.5 2800 - J

1.0

0 0

10

20

30

40

50

80

Conversion [%] Figure 7. Plot ofM (\) and PDI (B) vs. conversion for RAFT polymerization of vinyl oxazolone using CTA lc (entry 9 in Table I); the straight line represents the calculated molecular weights. N



614 The RAFT polymerization of NHSM is much more difficult and so far, no satisfactory results have been obtained regarding polydispersities and control of molecular weight. NHSM is a rather active monomer in controlled radical polymerization and reactive radicals are produced upon initiation. Therefore, a chain transfer agent containing an equally active leaving group has to be used to ensure effective polymerization. For this purpose, RAFT agent lc seemed to be a good choice as it consists of a reactive cyanopropyl leaving group. Nevertheless, the obtained polymers showed polydispersities around 1.5 and their molecular weights were much higher than predicted (see entry 5 in Table I). The mother liquors of the precipitated polymers showed residual unreacted chain transfer agent in the *H-NMR spectra. The same findings were made for the polymerizations using RAFT agents la, lb, and Id. Chain transfer agent Id decomposes at ambient temperature (#), and the RAFT polymerization was conducted at 30 °C using the low-temperature initiator V-70. It was assumed that a lower polymerization temperature might slow down the polymerization process and a controlled polymerization would take place. This was not the case, though.

Synthesis of Block Copolymers of VO and NIPAAm For the synthesis of the block copolymers, poly(VO) was used as a macrochain transfer agent for the polymerization of NIPAAm. The observed polydispersities were 1.22 and 1.14, respectively, the molecular weights are somewhat lower than expected (see Table II), which might be ascribed to GPC calibration with polystyrene standards. Containing a thermoresponsive NIPAAm block on one side and an active ester block on the other side, these block copolymers are of interest for the synthesis of polymer-protein conjugates. These conjugates also show thermoresponsive behaviour due to the poly(NIPAAm) block. Cloud-point measurements on the block copolymers were carried out in order to determine the LCST of this system. It is expected that the active ester block influences the LCST behaviour of NIPAAm through its polar nature. Generally speaking, hydrophobic substituents lower the LCST of poly(NIPAAm) whereas hydrophilic substituents raise it (20,21). Turbidity measurements show that the LCST is shifted to a higher temperature (37 °C) relative to the LCST reported for the homopolymer poly(NIPAAm) (32 °C)(/5), accounting for a more hydrophilic nature of the poly(VO) block.

RAFT Polymerization of Diacetone Acrylamide Diacetone acrylamide was polymerized by RAFT for the first time using l e as chain transfer agent. Two different initiators, namely A I B N and 4,4'-azobis(4cyanopentanoic acid) ACP, were employed but no significant influence was found on molecular weights and polydispersities even though ACP gives rise to faster polymerizations (entry 11 and 12 in Table I, 70% conversion after 2 h compared to same conversion after 4 h with AIBN). The monomer is especially


615 interesting for polymer-analogous reactions as it possesses the reactivity of an activated double bond, an N-substituted amide and a methyl ketone (22).


Synthesis of Polymer-Protein Conjugates Using R A F T Polymers RAFT polymers can be utilized for protein conjugation in two ways: (i) hydrolysis of the dithiocarbonyl-terminated polymers to the corresponding thiolterminated polymers to provide functional groups for disulfide formation or for bismaleimide crosslinkers between polymer and protein (Figure 8), (ii) short active ester blocks synthesized via RAFT containing functional groups that allow for amide formation between polymer and protein amino groups (Figure 9)(4,5).

proteinS

Figure 8. Bismaleimide erosslinker for protein conjugation.

The hydrolytic cleavage of the dithiocarbonyl endgroup with aqueous NaOH was confirmed by U V spectroscopy: The dithiocarbamate moiety absorbs in the UV-vis region with λ = 296 nm. After hydrolysis, only the thiol moiety is left, which does not absorb in the range measured. Additional proof of the thiol endgroup was provided by MALDI-TOF mass spectrometry and by the formation of a thioester with 2-naphthoyl chloride giving rise to an absorption shift in the U V spectrum. ΠΜΧ

peptidc-NH2 >>wv>CH2—CH^vvv

*~

vAwCH2—CH^vw

NH peptide Figure 9. Conjugation ofprimary amino groups to poly(VO).


616

The active ester block poly(VO) undergoes ring opening on addition of compounds containing active hydrogen atoms. Azlactone ring opening is especially facile with primary amines. The conjugation of a peptide with its primary amino group to poly(VO) is illustrated in Figure 9. As a model peptide, glycineleucine (Gly-Leu) was used, and the reaction was performed in dry DMF under reflux. Figure 10 shows the *H-NMR spectra of poly(VO) and its conjugate with Gly-Leu. Conjugation to the active ester N H S M has been described elsewhere


(S).

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

ô(ppm] Figure 10. *H-NMR spectra ofpoly(VO) (top) and poly(VO) conjugate to GlyLeu (bottom) in Q D 6 and CD OD, respectively. 3

Conclusions The synthesized RAFT homopolymers and block copolymers are promising candidates for protein or drug conjugation as they show narrow molecular weight distributions. This is especially important in the field of polymer therapeutics where narrow MWDs are required for defined retention times in the body.


617 Active esters copolymerized with PNIPAAm represent thermoresponsive systems that can also be used for protein conjugation. The system PNIPAAm-oP A A is both pH- and thermoresponsive and forms micelles in aqueous solutions. These micelles can be used for drug encapsulation and controlled release thereof by utilizing both pH and temperature stimuli.


Acknowledgement. This work was supported by the Deutsche Forschungsgemeinschaft (Mu 896/13-1). The authors would also like to thank Dr. Michael Lanzendôrfer for the MALDI-TOF measurements.

References 1. 2.

3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Chong, Y . K.; Le, T. P. T.; Moad, G.; Rizzardo, E.; Thang, S. H . Macromolecules 1999, 32, 2071-2074. Rizzardo, E.; Chiefari, J.; Mayadunne, R. T. A. In Controlled/Living Radical Polymerization - Progress in ATRP, NMP, and RAFT, ACS Symposium Series 768; Matyjaszewski, K., Ed.; American Chemical Society: Washington, DC, 2000; Vol. 768, pp 278-295. Chiefari, J.; Chong, Y . K.; Ercole, F.; Krstina, J.; Jeffery, J.; Le, T. P. T.; Mayadunne, R. T. Α.; Meijs, G. F.; Moad, C. L.; Moad, G.; Rizzardo, E.; Thang, S. H . Macromolecules 1998, 31, 5559-5562. Zimmermann, J.; Mülhaupt, R. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 2000, 41(1), 764-765. Godwin, Α.; Hartenstein, M . ; Müller, A. H . E.; Brocchini, S. Angew. Chem. Int. Ed. 2001, 40, 594. Chiefari, J.; Mayadunne, R. T. Α.; Moad, G.; Rizzardo, E.; Thang, S. H . invs.: DuPont de Nemours Company USA, 1999, WO 99/31144. Schilli, C.; Lanzendörfer, M . G.; Müller, A. H . E. Macromolecules 2002, 35, 6819-6827. Quinn, J. F.; Rizzardo, E.; Davis, T. P. Chem. Commun. 2001, 1044-1045. Takeshima, T.; Ikeda, M . ; Yokoyama, M . ; Fukada, N . ; Muraoka, M . J. Chem.Soc.,Perkin Trans. 1 1979, 692-695. Mayadunne, R. T. Α.; Rizzardo, E.; Chiefari, J.; Chong, Y . K.; Moad, G.; Thang, S. H. Macromolecules 1999, 32, 6977-6980. Moad, G.; Chiefari, J.; Chong, Y . K.; Krstina, J.; Mayadunne, R. T. Α.; Postma, Α.; Rizzardo, E.; Thang, S. H . Polym. Int. 2000, 49, 993-1001. Cole, C.-A.; Schreiner, S. M . ; Monji, N . Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 1986, 27(1), 237-238. Yang, H. J.; Cole, C.-A.; Monji, N . ; Hoffman, A. S. J. Polym. Sci., Part A: Polym. Chem. 1990, 28, 219-226. Ganachaud, F.; Monteiro, M . J.; Gilbert, R. G.; Dourges, M.-A.; Thang, S. H.; Rizzardo, E. Macromolecules 2000, 33, 6738-6745. Schild, H . G. Prog. Polym. Sci. 1992, 17, 163-249.



618 16. Kobayashi, M.; Okuyama, S.; Ishizone, T.; Nakahama, S. Macromolecules 1999, 32, 6466-6477. 17. Le, T. P.; Moad, G.; Rizzardo, E.; Thang, S. H. invs.: Du Pont De Nemours and Co., USA; Le, Tam Phuong; Moad, Graeme; Rizzardo, Ezio; Thang, San Hoa, 1998, WO 98/01478. 18. Liu, S.; Billingham, N. C.; Armes, S. P. Angew. Chem., Int. Ed. 2001, 40, 2328-2331. 19. Arotcarena, M.; Heise, B.; Ishaya, S.; Laschewsky, A. J. Am. Chem. Soc. 2002, 124, 3787-3793. 20. Licea-Claverie, Α.; Salgado-Rodriguez, R.; Arndt, K.-F. Polym. Prepr. (Am. Chem.Soc.,Div. Polym. Chem.) 2001, 42(1), 564-565. 21. Stile, R. Α.; Healy, Κ. E. Biomacromolecules 2001, 2, 185-194. 22. Coleman, L. E.; Bork, J. F.; Wyman, D. P.; Hoke, D. I. J. Polymer Sci., Part A 1965, 3, 1601-1607.


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