Diels–Alder Cycloaddition-Cycloreversion: A Powerful Combo in Materials Design

Authors


  • Dedicated to the warm memory of our beloved student Ali Hikmet Karayel

Abstract

Reactive polymers are increasingly becoming materials of wide interest because they offer solutions to challenges in biomolecular immobilization, drug-delivery and enzyme modifications. The Diels–Alder (DA) cycloaddition and the retro Diels–Alder (rDA) cycloreversion reaction in combination enables synthesis of maleimide based reactive polymeric materials. Also, the DA cycloaddition offer a reagent-free ‘click’ reaction to construct various macromolecular architectures. Furthermore, the thermally reversible nature of the DA adducts makes them attractive building blocks for the design of thermoreversible materials. A discussion of some of the author's own research work in this area is presented in this article within the context of recent reports in polymer chemistry employing this reaction combo.

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Introduction

Taking a step back to go two steps forward is quite natural to a synthetic organic chemist. Protection–deprotection strategies are oftentimes a routine exercise in target oriented multi-step synthesis. In recent years, the growing impact of synthetic organic chemistry methodologies in polymer chemistry is noteworthy.1 Synthetic polymer chemistry is increasingly incorporating the ‘protection–deprotection’ approach to effectively obtain well-defined materials. The adoption of this strategy allows polymerization of reactive monomers that are not feasible otherwise. The growing interest in polymers that contain reactive functional groups as side chains or chain termini has necessitated the development of methodologies that enable the polymerization of reactive monomers. Such reactive polymers can be efficiently modified to obtain a wide variety of functional materials. In recent years, reactive polymers have emerged as attractive candidates for obtaining polymer-biomolecule conjugates, soft materials for biomolecular immobilization, and soluble supports for polymer therapeutics. Among the many reactive functional groups that can be utilized for these applications, maleimide is among the best candidates for conjugation chemistry since it undergoes two types of ‘click’ reaction: a Michael-type conjugate addition with thiol containing molecules, and a [4 + 2] cycloaddition reaction with electron rich dienes (Figure 1). While the conjugation with thiol containing molecules is irreversible, the cycloadduct obtained via cycloaddition reaction is thermally reversible. Oftentimes, both the conjugation reactions proceed with high efficiency, under mild and reagent free conditions. The ability to undergo efficient reactions under reagent free conditions offers an immense practical advantage. The facile bioconjugation chemistry attracts chemists interested in conjugation of drug molecules or biomolecules to polymeric materials for applications such as drug delivery and biomolecular immobilization. On the other hand, the thermoreversible attribute of the cycloadducts make them desirable candidates for material scientists interested in ‘self-healing’ materials and thermally responsive materials.

Figure 1.

Maleimide functional group: An ideal ‘click’ substrate.

Otto Diels and Kirk Alder, in 1928, disclosed the [4 + 2] cycloaddition reaction between an electron rich diene and an electron deficient dienophile.2 This century old reaction that bears their names and what appears to be a simple and straightforward transformation has shaped the art and science of organic synthesis of complex natural products.3 In recent years, this powerful organic transformation has become a widely utilized reaction in polymer and material science. The cycloaddition reaction, apart from being a high yielding, regio- and stereo-controlled transformation under reagent free conditions, provides ‘thermoreversible’ adducts. This ‘thermoreversibility’ enables fabrication of materials incorporated with ‘self-healing’ pathways. This attribute of the cycloadducts has long been recognized by polymer scientists and a wide variety of mendable materials have been fabricated using the diene-dienophile based crosslinking of polymers.4 Recent years have witnessed utilization of the Diels–Alder cycloaddition reaction to design and obtain thermoreversible discrete macromolecular constructs like dendrimers and dendronized polymers. The realization that the ‘cycloaddition’ step provides masking of the dienophile, while the ‘cycloreversion’ step affords the unmasking has resulted in development of a methodology for taming the reactive maleimide group during polymerization and other transformations hampered by its presence (Figure 2). This combination of cycloaddition/cycloreversion reactions has opened doors for design and fabrication of polymeric materials that enable biomolecular immobilization utilizing conjugation via the thiol reactive maleimide moiety. The following sections will build upon the prelude set forth and discuss recent progress made using this chemistry by various research groups while highlighting the work done in our group.

Figure 2.

Diels–Alder/retro Diels–Alder reaction sequence.

Maleimide Containing Polymers and Dendrimers

Thermoreversible Crosslinked Materials

To date, most of the approaches towards fabrication of self-healing materials have relied on cross-linking of furan side chain containing polymers with bis-maleimide containing small molecules as cross-linkers (Figure 3).5–8 Alternatively, polymers appended with maleimides could be mixed with furan containing polymers or crosslinker molecules containing multiple furan units to obtain crosslinked materials. Molecules with multiple furan units have been mixed with multimeric maleimide containing molecules to obtain crosslinked polymeric materials that have excellent mechanical properties and are thermally remendable.9 Interestingly, there are only a few examples in the literature that involve polymers appended with maleimides towards fabrication of thermoreversible materials.10 In these studies, maleimides were appended onto the polymers as a post polymerization step. The post functionalization approach suffers from poor efficiency and reliable quantification of the extent of functionalization. Hence, development of a methodology that will allow direct access to polymers containing maleimide units as side chains will be indispensable towards the development of tailored polymers for fabrication of self-healing materials. Our work towards the synthesis of maleimide containing discrete macromolecular constructs, as described ahead expands the toolbox for self-repairing materials design.

Figure 3.

Synthetic approaches to self healing crosslinked materials.

Thermoreversible Discrete Macromolecular Constructs

Thermally responsive symmetrical dendrimers have been reported a few years ago by McElhannon, McGrath and coworkers.11, 12 The dendrimers were synthesized either by condensation of dendrons with a furan-maleimide based focal point around a core molecule or by the combination of two dendrons containing furan moiety at their focal point using a bis maleimide linker via the Diels–Alder cycloaddition reaction (Figure 4). The thermally stimulated disassembly of these dendrimers was demonstrated by heating them to 110 °C. Slow recombination of these disassembled dendrons was accomplished upon thermal treatment. More recently, the same group has extended the methodology to obtain thermoreversible dendronized step polymers.13

Figure 4.

Synthesis of thermoreversible symmetrical dendrimers via Diels–Alder cycloaddition reaction.

Observing from a different point of view, among the many attractive tenets of the ‘Click’ reactions is that they offer an excellent unification of complementary reactive groups. Noteworthy demonstration of this function was via the assembly of segment block dendrimers as shown by Hawker and coworkers.14 We asked ourselves, could the Diels–Alder cycloaddition offer a reagent free alternative approach towards the synthesis of segment block dendrimers? Furthermore, the thermally activated disassembly of such systems would provide an interesting class of thermally responsive segment block macromolecules.

To obtain segment block dendrimers, we utilized Fréchet type dendrons appended with an electron rich diene, furan, at the focal point of one of the building blocks (Figure 5).15 A complementary set of polyester dendrons containing the electron deficient maleimide group at the focal point were synthesized. Combination of these two complementary dendrons upon heating in benzene afforded segment-block dendrimers in good yields. As one would expect, we found that the yields of dendrimers were highly sensitive to the temperature. The stereochemistry of the bridging adduct (endo-exo) was also dependent on the temperature. While a high temperature was required to obtain good yields and good exo-endo selectivity, too high of a temperature lead to lower yields due to the competing retro Diels–Alder reaction. The obtained dendrimers fell apart upon heating these macromolecules to 110 °C. Recently, layered block dendrimers that are thermoreversible were reported by Kakkar and coworkers.16 They utilized the Huisgen type copper catalyzed azide-alkyne ‘click’ cycloaddition in combination with furan-maleimide Diels–Alder ‘click’ reaction to divergently construct thermoresponsive dendrimers that disassemble upon heating.

Figure 5.

Synthesis of thermoresponsive segment-block dendrimers via Diels–Alder cycloaddition reaction.

A powerful attribute of the Diels–Alder reaction is the easy availability of a wide variety of dienes and dienophiles provides a handle for tuning the thermoreversibility. For example, the use of anthracene as a diene results in adducts that undergo cycloreversion over 200 °C. Hence, high yielding stable conjugation can be achieved using the combination of anthracene and maleimide. Click reaction based on this combination of diene and dienophile has led to efficient synthesis of diblock copolymers,17 and graft copolymers.18 We recently reported the synthesis of dendronized polymers using the Diels–Alder reaction (Figure 6).19 Anthracene functionalized polymer was reacted with dendrons containing furan protected maleimide groups at their focal points by refluxing in toluene. At such elevated temperature, unmasking of the reactive maleimide functional group at the focal point of the dendrons took place via the retro-DA reaction. Thus generated dienophile appended dendrons generated in situ reacted with anthracene containing copolymer via [4 + 2] cycloaddition reaction resulting in dendron grafted polymer.

Figure 6.

Synthesis of dendronized polymers via the rDA/DA sequence.

Recently, the unparalleled efficiency offered by the Diels–Alder ‘click’ reaction was demonstrated by Kowollik and coworkers. The choice of cyclopentadiene as the diene component and dithioester unit as a dienophile can result in ‘ultra-fast’ reactions that can provide diblock copolymers in a matter of few minutes via the hetero Diels–Alder cycloaddition reaction.20 This example demonstrates that the wide plethora of dienes and dienophiles available in the Diels–Alder click toolbox can provide the synthetic polymer chemist with many choices.

Reactive Maleimide Containing Macromolecules

Polymers Bearing Reactive Maleimide Groups at Chain Termini

Growing interest in polymer–protein conjugates due to their beneficial impact in health sciences and biotechnology has necessitated the development of efficient synthetic methodologies to obtain such biomolecule–macromolecule hybrids.21 To achieve site specificity during the bioconjugation process, thiol groups of cysteine residues that are either naturally present or introduced via site-directed mutagenesis are often targeted. Thus, various approaches to obtain polymers with thiol reactive end groups have been developed. Until recently, most of these methods relied on post-polymerization modification of end groups of polymers to introduce thiol reactive groups such as vinyl sulfones, disulfides and maleimide groups.22–25 An alternative approach that provides a higher level of confidence regarding the presence of end group in each individual chain is to incorporate the desired functional group into the initiator molecule. The challenge then becomes to overcome the high reactivity of these groups to participate in polymerization reactions (Figure 7). For example, the maleimide group contains a conjugated double bond that is active towards polymerization. Indeed, maleimide-based monomers have been extensively utilized to obtain polymers containing maleimide groups incorporated along the back bone. An initiator or an acrylic or vinylic monomer containing a maleimide group will result in gelation due to crosslinking during the polymerization. Hence, one needs to use a ‘protecting’ group strategy to mask the reactive double bond of the maleimide unit during the polymerization event (Figure 7).

Figure 7.

Synthetic routes to maleimide terminated polymers.

During the progress of our work, synthesis of semi-telechelic polymers with maleimide group at the chain end was reported by Haddleton and coworkers.26 They appended an initiator group onto the furan-protected maleimide moiety and utilized the initiator to synthesize water soluble polyethylene glycol methacrylate based polymers using atom transfer radical polymerization (ATRP). Recently, Maynard and coworkers adopted the furan protected maleimide group to synthesize a chain transfer agent to obtain a maleimide end functionalized poly(ethylene glycol) methyl ether acrylate based polymer.27 Telechelic polymers containing maleimide groups at both termini have been developed by the same group, via coupling of maleimide terminated polymers under atom transfer terminal coupling reaction (ATRC) conditions.28 More recently, Maynard and coworkers synthesized tetra-arm polymers containing maleimide group at the chain ends, by heating star polymers obtained by reversible addition-fragmentation chain transfer (RAFT) polymerization in the presence of a furan protected maleimide containing azo initiator.29

Star Polymers Bearing Reactive Maleimide Groups

Polymer architecture has a profound effect on the properties of materials and interaction of polymers with other materials. It has been recently shown that multiarm macromolecular scaffolds are attractive candidates for polymer conjugated drug delivery.30 Attachment of water soluble polymeric chains to drug molecules increases bioavailability and reduces clearance rate.31 Interestingly, it was observed that the polymer drug conjugates with multiarm structures possessed longer circulation time in the body compared to the linear polymers of the same molecular weight. We recently utilized the Diels–Alder/retro Diels–Alder (rDA) reaction based strategy for the synthesis of multiarm polymers containing a maleimide functional group at the focal point.32 Dendritic initiators containing a furan protected maleimide unit at their focal point were synthesized using a divergent strategy. Atom transfer radical polymerization at 70 °C was utilized for the synthesis of poly(ethylene glycol) methacrylate based water soluble multiarm polymers. As expected, the star polymers thus obtained contained a masked maleimide core. Afterwards, retro Diels–Alder reaction was utilized to deprotect the maleimide unit to obtain the multiarm polymers with a reactive core. A thiol containing tripeptide glutathione was conjugated to the core of these multiarm polymers via the Michael type thiol-ene ‘click’ addition reaction under reagent free conditions (Figure 8).

Figure 8.

Synthetic route to multiarm polymers with maleimide based core.

Polymers Bearing Reactive Maleimide Groups as Side Chains

Few years ago, we disclosed synthesis of polymers containing thiol-reactive maleimide groups on their side chains, by utilization of a novel methacrylate monomer containing a masked maleimide (Figure 9).33 Diels–Alder reaction between furan and maleimide was adapted for the protection of the reactive maleimide double bond prior to polymerization. A methacrylate based latent-reactive monomer containing a masked maleimide group was readily obtained by treatment of a furan protected maleimide based alcohol with methacryloyl chloride in the presence of triethyl amine.

Figure 9.

Synthesis of masked maleimide monomer.

AIBN initiated free radical polymerization was utilized for the synthesis of copolymers containing the masked maleimide groups (Figure 10). A variety of polymers (P1-P4) with different feed ratio of the maleimide monomer were prepared successfully. No unmasking of the maleimide group was evident under the polymerization conditions. The maleimide groups on the side chain of the polymers were unmasked into their reactive form by utilization of retro Diels–Alder reaction. This cycloreversion was monitored by thermo gravimetric analysis (TGA), differential scanning calorimetry (DSC) and 1H and 13C NMR spectroscopy.

Figure 10.

Synthesis of polymers with maleimide side chains. Feed ratio of monomers: (MMA: latent-reactive monomer) P1 (1:1), P2 (2:1), P3 (4:1), P4 (8:1).

In the 1H NMR spectrum of the polymer, the presence of the intact masked maleimide groups as side chains were evident from proton resonances at 2.80, 5.25 and 6.50 ppm belonging to the bicyclic unit (Figure 11). Appearance of a new peak at 6.71 ppm corresponding to the vinyl protons of the maleimide group and complete disappearance of proton resonances corresponding to the bicyclic unit demonstrated quantitative and efficient activation of the maleimide groups in the polymer.

Figure 11.

1H NMR spectra of depicting quantitative activation of maleimide groups via rDA cycloreversion.

Alternatively, TGA was used to determine the thermal stability of the copolymer and monitor the activation of the maleimide groups via loss of furan during rDA reaction. TGA of the polymers (P1–P4) containing varying amounts of the masked monomers showed a weight loss starting at 120 °C (Figure 12). As expected, a consistent increase in weight loss of the polymers was observed upon increasing the amount of furan based monomer in the copolymer. As expected, no weight loss in the region of 120 °C was observed upon subjecting the polymer P5 obtained after the cycloreversion step, due to lack of any furan adducts in the side chains. Thermal cycloreversion was also probed using DSC. A broad endotherm during the first heating was observed between 115 and 165 °C, which was ascribed to the cycloreversion reaction. The cycloaddition/cycloreversion based methodology could be extended to obtain thiol reactive, water soluble biocompatible polymeric supports based upon monomers such as polyethylene glycol methacrylate. Such polymers would be instrumental in design and development of novel platforms for drug delivery.

Figure 12.

Activation of maleimide groups probed via thermal analysis.

Maleimide Containing Biofunctionalizable Hydrogels

Biomolecular immobilization is at the heart of biosensor and microarray technologies. Moreover, it is increasingly important for tissue engineering, cell growth control and areas such as biomedical coating for stents and other implants. Covalent attachment of drugs, peptides and large biomolecules into polymeric matrices enables control over the degree of functionalization and their spatial location. The covalent approach also provides a handle to tailor stimuli responsive release profiles, an especially attractive characteristic for controlled drug delivery platforms. While there have been considerable advancements in the development of functionalizable hydrogels and polymeric surfaces in recent years, most of the research has focused upon fabrication of hydrogels containing functional groups conducive with radical polymerization. This limitation has hampered the design and development of thiol reactive bioactive materials. Recently, we discovered that the Diels–Alder/retro Diels–Alder reaction combination can be utilized to obtain hydrogels and reactive polymeric surfaces that incorporate predetermined amount of maleimide group.34 The ‘click’ nature of thiol addition to maleimide unit allows efficient functionalization of the reactive groups, resulting in a control over degree of immobilizations. Poly (ethylene glycol) methacrylate based hydrogels containing masked maleimide functional groups were synthesized using AIBN initiated thermal polymerization. The masked maleimide groups were directly introduced during the copolymerization with a furan protected maleimide containing monomer. During the polymerization, the thermal deprotection of the maleimide groups in some of the monomer results in the generation of an in situ crosslinker. After gelation, the protected maleimide groups can be activated to their reactive forms via the thermal cycloreversion step (Figure 13). The efficiency of the gel formation, maleimide incorporation and functionalization of the hydrogel was investigated. While the effectiveness of gel formation can be obtained gravimetrically, the extent of incorporation of maleimide can be quantified using thermal gravimetric analysis by monitoring the amount of weight loss corresponding to furan evolved upon cycloreversion.

Figure 13.

Fabrication of maleimide based thiol reactive hydrogel using DA/rDA strategy.

The potential of these hydrogels to act as templates for bioimmobilization of enzymes in a controlled manner was evaluated. Thiol containing biotin was covalently attached to the gels and their availability for immobilization of streptavidin was investigated. Hydrogels having different degree of maleimide groups were reacted with excess thiol containing biotin. After washing off excess biotin from the hydrogel to remove any unbound biotin, hydrogels were exposed to FITC labelled streptavidin. After washing off physiabsorbed streptavidin from the hydrogel, extent of immobilization was investigated using fluorescence microscopy. As expected, gels containing higher amounts of covalently bound biotin were able to immobilize more streptavidin. Thus hydrogels incorporated with varying density of thiol reactive maleimide groups could be fabricated using the DA/rDA reaction sequence.

Conclusion

The potential of these elegant, versatile and powerful transformations: the Diels–Alder cycloaddition and the retro Diels–Alder cycloreversion reactions, individually or as a combination in the synthesis of polymeric materials is recently realized. In a short period of time, the methodologies based on these reactions have the enabled synthesis of various macromolecular constructs difficult to realize otherwise. The richness of this reaction ‘combo’ will continue to unravel new methodologies to fabricate functional materials for the coming years.

Acknowledgements

Financial support from the Turkish Academy of Sciences under the TUBA-GEBIP program is gratefully acknowledged.

Biographical Information

Amitav Sanyal obtained his undergraduate degree from the Indian Institute of Technology at Kanpur, India in 1994. He completed his Ph.D. in 2001 from Boston University (USA) under the supervision of Prof. J. K. Snyder in the area of synthetic organic chemistry. During post-doctoral work under the direction of Prof. Vincent M. Rotello at University of Massachusetts at Amherst (USA), he worked in the area of design and fabrication of novel renewable polymer coated surfaces and polymer-nanoparticle composites materials using molecular recognition. He joined Bogazici University, Istanbul, Turkey as an assistant professor in 2004. His research focuses on the development of reactive polymers and coatings for biomolecular immobilization, thermoreversible materials, functional nanoparticles, as well as fabrication of functional materials using molecular recognition. He was the one of the recipients of the young investigator award administered by the Turkish Academy of Sciences in 2008.

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