Conjugates of Polymers and Sequence-Defined Polypeptides via

Chapter 15

Conjugates of Polymers and Sequence-Defined Polypeptides via Controlled Radical Polymerization

Downloaded by PENNSYLVANIA STATE UNIV on August 8, 2012 | http://pubs.acs.org Publication Date: September 7, 2006 | doi: 10.1021/bk-2006-0944.ch015

Mattijs G. J. Ten Cate and Hans G. Börner

*

Max Planck Institute of Colloids and Interfaces, Colloid Department, MPI KGF Golm, 14424 Potsdam, Germany Corresponding author: [email protected] *

The assessment of macromolecular conjugates comprising polymers and sequence-defined oligopeptides via either ATRP or RAFT polymerization is described. Therefore, well-defined oligopeptide macroinitiators for the ATRP process, as well as macro chain-transfer agents for the RAFT polymerization were synthesized. These could be obtained by solid-phase supported synthesis approaches, allowing the preparation of ATRP macroinitiators and RAFT agents without the need for a chromatographic purification step. Kinetic investigations were presented, revealing an efficient control of the polymerization processes. However, inherent difficulties were also encountered. Moreover, it has been shown that both the structure and chirality of the peptide segment are not affected by the radical polymerization processes.

198

© 2006 American Chemical Society

In Controlled/Living Radical Polymerization; Matyjaszewski, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

199


Introduction The combination of both bioorganic and synthetic polymer segments in well-defined architectures has received considerable attention (1-7). Particularly, the tailored incorporation of sequence-defined oligo- or polypeptides into synthetic polymers is interesting for the design of bioactive polymeric materials having the potential to actively interact with biological systems (8-12). Furthermore, defined structure formation of the peptide segment e.g. the adoption of secondary, tertiary or quaternary structures can be used to induce and guide structure formation processes in synthetic polymers (75). Therefore, specific interactions including molecular recognition of peptide-based receptorsligand pairs could lead to self-assembled, materials such as nanowires or fibers, useful for biomedical applications. These structures might response to external stimuli and exhibit potentially auto-correction or self-healing properties due to relative soft but often cooperative interactions in between the building blocks (14). Besides the potential properties of the peptide segment, the inexpensive polymeric block contributes to the solubility and mechanical properties of the resulting materials. This is advantageous compared to exclusively peptidic or protein-based materials (15). In this chapter, the synthetic approaches to obtain conjugates between well-defined oligopeptides and polymers are described. Conjugates synthesized utilizing grafting techniques via the polymerization of oligopeptide functionalized macromonomers will be excluded (16,17). oligopeptide

synthetic polymer chain

J coupling

I

polymerization n — — * polymer-peptide conjugate

Figure 1 .Approaches to integrate sequence controlled polypeptides into synthetic polymer (e.g. X = NH , Y = COOH). 2

Two synthetic approaches to integrate sequence controlled polypeptides into synthetic polymers are described. These comprise the direct coupling (18-20) of peptides to synthetic polymers and the polymerization from a peptidic macroinitiator (Figure 1). The direct coupling approach becomes progressively more difficult (21) when the molecular weight of the polymer or peptide increases (due to an decrease in chain end-group reactivity) and when complex peptides are coupled (since the establishment of regioselectivity becomes progressively more difficult). By application of the polymerization approach,



200 these problems can be avoided. Therefore, existing polymerization techniques were adapted to establish generally applicable routes. Controlled radical polymerization (CRP)(22) techniques seem to be most suited for the synthesis of the macromolecular conjugates. Due to the control over molecular weight and molecular weight distribution as well as the high tolerance for diverse functional groups, CRP methods are promising for the production of a variety of welldefined conjugates with broad spectra in composition, topologies and architectures (22). Currently, the application of sequence-defined polypeptides as macroinitiators for various CRP methods have been described (2,23-25), including nitroxide-mediated radical polymerization (NMP (26)), atom transfer radical polymerization (ATRP (27,28)) and reversible addition-fragmentation transfer radical polymerization (RAFT (22,29,30)). The following sections describe recent developments in the use of ATRP and RAFT techniques to obtain well-defined macromolecular conjugates comprising synthetic polymers and sequence-defined oligopeptides.

Atom transfer radical polymerization ATRP is recognized as a robust polymerization technique, allowing access to a broad range of different polymers (27,31) and polymer architectures (32), while showing tolerance towards diverse functional groups (33). This section describes a two step route involving solid-phase supported synthesis of an oligopeptide macroinitiator and solution-phase controlled radical polymerization. An elegant way towards block copolymers, avoiding purification problems of the product from residual monomer, solvent and catalyst is the controlled solid-phase supported radical polymerization. This is because with this technique impurities can simply be removed by washing. However, compared to solution-phase polymerizations, diffusion limitation of the deactivation step in the ATRP is difficult to overcome (34-36). A sequence-defined macroinitiator was synthesized by coupling of an ATRP initiator to the amine terminus of a supported oligopeptide (Scheme 1). Since alkyl 2-bromopropionates are standard ATRP initiators for the acrylate polymerization, 2-bromopropionic acid was used for the amidation of the Nterminal amine group of the supported oligopeptide. An acid labile 2-chlorotrityl resin was used for the synthesis of the peptide, allowing the liberation of fully side chain protected oligopeptides from the support under gentle conditions. Therefore, die relatively sensitive ATRP initiator moiety could be introduced before liberation from the resin and critical functionalities (e.g. carboxylates or amines that interfere with the ATRP catalyst) remain reversibly protected. As primary structure of the oligopeptide, Gly-Asp-Gly-Phe-Asp (GDGFD, 1 in Scheme 1) was selected to demonstrate the process of obtaining peptide-polymer



2

/,

u

Scheme 1. Solid-phase supported synthesis of the oligopeptide macroinitiator for ATRP (Conditions: i. 2-aminoethanol-2-chlorotrityl resin, Fmoc-Aa-OH, HBTU, DIPEA, NMP; ii. 20% piperidine in NMP; iil DCQ NMP, 12 h; iv. 2% trifluoroacetic acid in DCM).

NH

SPPS Fmoc-strategy


202 conjugates via ATRP. The Asp residues exhibit bulky tert-butyl ester protecting groups improving solubility and preventing oligopeptide aggregation, while the phenyl ring from the Phe residue can act as a label for quantitative analysis. As a result of the fundamental orientation of this study we renounce the use of a biological active sequence. The macroinitiator 3 was subsequently used homogeneously in solution for the CRP of Λ-butyl acrylate. As the ATRP catalyst system pentamethyldiethylenetriamine (PMDETA) complexes of CuBr and CuBr were used. The PMDETA ligand forms a relatively stable and active catalyst, allowing moderate reaction conditions and short reaction times. The solution-ATRP reaction leading to poly(w-butyl acrylate)-Woc#-polypeptide revealed a process with high level of control, providing molecular weights that increased linearly with reaction time and relatively low polydispersities (MJM « 1.20; Figure 2). However, even though the ATRP process involving the oligopeptide 3 resulted in well-defined products, interactions between the copper catalyst and the peptide took place (2). This was indicated by the relatively slow overall polymerization rate compared to the ATRP with non-peptidic initiators and the observed curvature in the first kinetic plot (Figure 2b). This was, indeed, expected because oligopeptides have the inherent property to act as multi-dentate ligands for metal ions due to their polyamide backbone (57). These interactions were confirmed by additional experiments using various amounts of an oligopeptide with a similar amino acid sequence, but without the ATRP initiator moiety (2). Instead of the peptide macroinitiator, methyl-2bromopropionate was used to start the ATRP process, while the other reaction conditions were kept constant. A significant decrease of overall polymerization rate was observed with increasing oligopeptide concentration, leading to an inversely proportional relation between overall polymerization rate and peptide concentration. This indicates the occurrence of defined interactions between the catalyst and the peptide, which is most likely due to a ligand exchange reaction with the ATRP metal complex, resulting in partial inhibition of the catalyst. The polyamide backbone and, when relevant, the side chain functionalities of the peptide causes the molecule to function as a multidentate ligand for metal ions (57). A comparable behavior has been reported in the ATRP processes of (meth)acrylamides (38). There,frequentlythe increase of the amount of catalyst could overcome this problem (38). Initial attempts to characterize the poly(«-butyl acrylate)-6/ocA-DFGDG conjugate by MALDI-TOF mass spectroscopy failed due to difficult desorption and/or ionization properties of the peptide-polymer conjugate. However, it was possible to analyze the conjugate comprising a GGFGG peptide by MALDITOF mass spectrometry (13), indicating the influence of the peptide segment on the polymer properties (Figure 3). The mass spectrum clearly shows the poly(wbutyl acrylate)-Woc£-polypeptide conjugates. Two homologue series were observed assignable to the same molecular species with either sodium or potassium ion adducts, as indicated by the mass difference of 16 m/z.


2

R


203

(a)

ο

14000 12000

1.5

Polydispersity M .

1.4

10000-1

'2


8000 H

I

6000

Ι

1.3 δο

4000 2000-1

1.2

ο α.

oJ

10

20

30

40

50

60

70

Monomer conversion [%]

(b) 1.0 0.8

I

0Λ-

0.2-I 0.0 25

50

75

100

125

Time [min] v

Figure 2. ATRP of nBA initiated by 3 at 60 °C: App. M QPC $ conversion (a) andln([M]o/[MJ) vs reaction time (b) (cond: [nBA]o/fïn]o/[CuBr]o/[CuBr ]

2



204

2000

4000 m/z

6000

Figure 3. MALDI-TOF mass spectrum showing the sodium and potassium adducts ofpoly(n-butyl acrylate)-block-GGFGG.

Furthermore, in both series, a characteristic mass difference of 128 m/z was found that is consistent with the mass of the nBA repeat unit. As end groups, bromine and the GGFGG peptide segment was found with a reminder mass of 0.5 m/z, verifying the chemical structure of the pwBA-peptide conjugate. Neither major side products nor the presence of polymers, lacking the peptide segment could be observed within the error of the MALDI-TOF-MS method. This confirms a clean incorporation of the peptide segment by the ARTP macroinitiator approach. In conclusion, the two-step synthetic route for the introduction of sequencedefined oligopeptides into synthetic polymers can be utilized for the preparation of oligopeptide-Woc£-poly(/?BA). Well-defined block copolymers with low My,/M„ and controllable M were obtained. Solution-phase ATRP was initiated by an oligopeptide macroinitiator accessible via solid-phase peptide synthesis. Interactions between the catalyst and the oligopeptide, however, were evident and could not be suppressed. Nevertheless, these interactions were not critical in terms of synthesis control of the desired copolymer. n


205


Reversible addition-fragmentation transfer radical polymerization An inherent difficulty of ATRP involving oligopeptide structures is the interaction between the copper catalyst and the peptide. These interactions cannot be suppressed (2) and likely depend strongly on the length as well as on the amino acid sequence of the oligopeptide. Therefore, the polymerization conditions have to be optimized for each applied oligopeptide, complicating the use of ATRP as a general approach for the synthesis of peptide-polymer conjugates. These ligating properties do not occur within RAFT radical polymerization since this technique does not require a metal catalyst. RAFT radical polymerization has been proven to be a valuable tool to access welldefined polymer-peptide conjugates (25). The RAFT process allows for the controlled polymerization of a large diversity of monomers and is a facile method since the involved components are not air or moisture sensitive. The components can be simply dissolved and deoxygenated prior to the polymerization. Moreover, racemization and thermal degradation is expected to be limited because the polymerizations are performed at moderate reaction temperatures and basic conditions, that might cause racemization, are strictly avoided. This section describes the straightforward solid-phase supported synthesis (SPPS) approaches to oligopeptide based RAFT agents and their utilization within the solution polymerization of «-butyl acrylate. For the SPPS of oligopeptide transfer agents, two different synthetic strategies were evaluated: i. the coupling of a preformed RAFT agent to the TV-terminus of a peptide and; ii. The transformation of an ATRP macroinitiator into a RAFT functionality (Scheme 2). The GDGFD peptide sequence, utilized in the ATRP section, was used to demonstrate the process, making the results directly comparable to the previously described ATRP study. Synthesis of the peptide macrotransfer agents A carboxylic acid functionalized RAFT agent (4 in Scheme 2) was coupled to the Af-terminus of a resin bound oligopeptide, similar to the established synthesis of oligopeptide ATRP macroinitiators. This approach would allow the regio-selective introduction of the transfer group either at the JV-terminus or at a specific sequence-position by the modification of an ε-amine group of a lysine residue (25). However, besides the oligopeptide chain transfer agent (CTA) 5 the formation of a byproduct was observed. This resulted from the nucleophilic attack of the peptide amine terminus on the dithioester, leading to a thioamide structure. The thioamide was present in significant amounts (-24%), but was not


In Controlled/Living Radical Polymerization; Matyjaszewski, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006. Ο

(6)

Scheme 2. Solid-phase supported synthesis of the oligopeptide transfer agents 5 and 6. (Conditions: i. DC NMP, room temperature; il 2% trifluoroacetic acid in DCM; Hi. dithiobenzoic acid, pyridine, THF, 60 °C).

*BuO'

r

^OiBu



207 found to interfere with the CRP process (data not shown) (25). Nevertheless, the method could be applied to the coupling of RAFT agents via hydroxyl moieties, e.g. to side chain functionalities of serine or threonine residues, where the particular side reaction is not expected. This would lead to a hydrolytically labile ester-linkage between the peptide and the polymer that might be interesting for the predefined degradation of peptide-polymer conjugates or for the liberation of peptide segments as bio-functional units in e.g. medical applications. The second method comprises the functionality switch of an ATRP macroinitiator (2) into an oligopeptide transfer agent (6). With this approach, the formation of a thioamide side product is avoided due to the absence of nucleophilic amines during the introduction of the RAFT chain transfer moiety. The oligopeptide transfer agent 6 was obtained by reaction of the resin bound ATRP initiator 2 with the pyridinium salt of the dithiobenzoic acid (25). The substitution of the bromine of 3 is quantitative and besides the formation of 6 no side reactions occurred. The solid-phase supported synthesis of 6 has shown to be convenient for the synthesis of (oligopeptide RAFT agents. Moreover, the products are readily purified, while chromatographic purifications that are usually necessary can be avoided.

The RAFT polymerization The poly(«-butyl acrylate)-è/ocA;-polypeptide (7) was synthesized to examine the RAFT radical polymerization of wBA (25). The macrotransfer agent 6 was used homogeneously in solution for CRP of Η-butyl acrylate with AIBN (20 mol%) as radical source. The polymerization proceeds in a controlled manner, providing well-defined peptide-polymer conjugates with molecular weights that increase linearly with monomer consumption and low polydispersities (MJM ~ 1.1) (Figure 4a). Moreover, the semi-logarithmic plot shows a first order kinetics after a retardation period of about 8 hours (Figure 4b). Though retardation periods are frequently observed within RAFT processes, the causes are still controversially since the mechanism in the early stage of the polymerization remains difficult to access. It has been suggested that retardation within the RAFT process occurs either due to an intermediate radical termination (39-43), or due to a slow fragmentation/reinitiation (39,44). Retardation times should be taken into consideration for controlled polymerizations, especially when low molecular weight compounds are targeted. A comparable retardation period was observed when only 5 mol% of A I B N was used, excluding the possibility that potential impurities retard the polymerization of HBA with 6. The slope of the first order kinetics plot was, expectedly, 4 times smaller (dashed line in Figure 4b); i.e. 0.014 for 5% A I B N and 0.056 for 20% A I B N , indicating that the rate of polymerization directly correlates to the amount of formed radicals. n


208 To confirm the incorporation of the oligopeptide segment into the polymer a low molecular weight conjugate of 7 (A/^NMR = 4.6 kDa; DP^NMR = 29) was synthesized and precipitated multiple times in MeOH/H 0. Since this is a good solvent mixture for the oligopeptide segment, the absence of peptides that are not bound to a polymer can be ensured. The formation of the poly(w-butyl acrylate)6/ocA-polypeptide conjugate was conclusively demonstrated by H N M R spectroscopy, showing the characteristic resonances for the protons of the oligopeptide, the poly(wBA) segment, as well as the RAFT moiety (Figure 5a). Since both the RAFT and peptide end-group functionalities were quantified in a ratio 1:1, formation of the dimerization product, resulting from termination via radical coupling can be excluded within the experimental error of the analytical method. These results are supported by size exclusion chromatography (SEC) showing that 6 has a mono-modal and narrow molecular weight distribution with M / M = 1.18. In addition, the number average molecular weight was determined with M = 4.1 kDa, resembling the value calculated based on *H N M R end group analysis. Since the RAFT moiety remains quantitatively at the end of the polymer chain of the isolated conjugates, the polymer chain-end can be modified by further block extension or fiinctionality transformation. This may allow access of polymers with advanced architectures. It is essential to prove that the chirality of the oligopeptide segment is unaffected during the polymerization process, since the structural conformation and, thus, the biological function of peptides, is strongly influenced by the chirality. Circular dichroism (CD) spectroscopy showed conclusively that the chirality of the oligopeptide segment is unaffected during the polymerization process. The similar CD spectra of 6 and 7 (Figure 5b) support the effective incorporation of the peptide segment into the polymer and verify the absence of racemization. These results demonstrate a novel and straightforward synthesis route toward multifunctional RAFT agents that can be utilized within CRP to obtain well-defined peptide-polymer conjugates. Moreover, the method exhibits high potential to be extended to diverse conjugated polymer systems including polypeptides, PNA's and polysaccharides. 2


!

w

n

n > a p p

Conclusion It was shown that well-defined conjugates comprising sequence-defined oligopeptides and synthetic polymers can be obtained utilizing ATRP and RAFT radical polymerization. Solution-phase ATRP was initiated by an oligopeptide macroinitiator accessible via solid-phase peptide synthesis. Although interactions between the catalyst and the oligopeptide were evident, they were not critical in terms of synthesis control of the desired copolymer. The use of ATRP introduces the possibility to integrate oligopeptides into a variety of different polymers or



209

Monomer conversion [%]

(b)

Time [h]

Figure 4. RAFT polymerization of nBA controlled by 6 at 60 °C: vs conversion (a) andfirst-orderkinetic plot ln([M]

Conjugates of Polymers and Sequence-Defined Polypeptides via

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