A.J. Dirks

Bioconjugation with Strained Alkenes and Alkynes

The structural complexity of molecules isolated from biological sources has always served as an inspiration for organic chemists. Since the first synthesis of a natural product, urea, chemists have been challenged to prepare exact copies of natural structures in the laboratory. As a result, a broad repertoire of synthetic transformations has been developed over the years. It is now feasible to synthesize organic molecules of enormous complexity, and also molecules with less structural complexity but prodigious societal impact, such as nylon, TNT, polystyrene, statins, estradiol, XTC, and many more. Unfortunately, only a few chemical transformations are so mild and precise that they can be used to selectively modify biochemical structures, such as proteins or nucleic acids; these are the so-called bioconjugation strategies. Even more challenging is to apply a chemical reaction on or in living cells or whole organisms; these are the so-called bioorthogonal reactions. These fields of research are of particular importance because they not only pose a worthy challenge for chemists but also offer unprecedented possibilities for studying biological systems, especially in areas in which traditional biochemistry and molecular biology tools fall short. Recent years have seen tremendous growth in the chemical biology toolbox. In particular, a rapidly increasing number of bioorthogonal reactions has been developed based on chemistry involving strained alkenes or strained alkynes. Such strained unsaturated systems have the unique ability to undergo (3 + 2) and (4 + 2) cycloadditions with a diverse set of complementary reaction partners. Accordingly, chemistry centered around strain-promoted cycloadditions has been exploited to precisely modify biopolymers, ranging from nucleic acids to proteins to glycans. In this Account, we describe progress in bioconjugation centered around cycloadditions of these strained unsaturated systems. Being among the first to recognize the utility of strain-promoted cycloadditions between alkenes and dipoles, we highlight our report in 2007 of the reaction of oxanobornadienes with azides, which occurs through a sequential cycloaddition and retro Diels-Alder reaction. We further consider the subsequent refinement of this reaction as a valuable tool in chemical biology. We also examine the development of the reaction of cyclooctyne, the smallest isolable cyclic alkyne, with a range of substrates. Owing to severe deformation of the triple bond from ideal linear geometry, the cyclooctynes show high reactivity toward dienes, 1,3-dipoles, and other molecular systems. In the search for bioorthogonal reactions, cycloadditions of cyclic alkenes and alkynes have now established themselves as powerful tools in reagent-free bioconjugations.

Metal-Free Triazole Formation as a Tool for Bioconjugation

The development of selective and site-specific bio-orthogonal conjugation methods is an important topic in chemical biology. A wide range of methods, such as the Staudinger ligation, native chemical ligation, genetic incorporation, expressed-protein ligation, Huisgen azide–alkyne cycloaddition, and the Diels–Alder ligation are currently employed in the selective modification of proteins and other biomolecules. In recent years, the Cu-catalyzed variant of the Huisgen 1,3-dipolar cycloaddition, also referred to as “click reaction”, has been increasingly applied in various fields of chemistry as a versatile and mild ligation method. This method allows for the synthesis of complex materials, which include bioconjugates, glycopeptides, functionalized polymers, virus particles, and therapeutics. However, due to the toxicity of the copper catalyst to both bacterial and mammalian cells applications that involve in vivo ligation are limited. In order to circumvent the use of copper ions, Bertozzi and co-workers have devised a strain-promoted [3+2] cycloaddition reaction that involves azides and a strained cyclooctyne derivative. Recent reports by Ju et al. have also shown successful applications of copper-free 1,3-dipolar cycloaddition by using either elevated temperatures or electron-deficient alkynes. We envisioned that the combination of ring strain and electron deficiency, as occurs in oxa-bridged bicyclic systems 2a and 2b, could also lead to an increased reactivity toward [3+2] cycloaddition reactions. Here, we report a spontaneous tandem [3+2] cycloaddition–retro-Diels–Alder ligation method that results in a stable 1,2,3-triazole linkage. This methodology can be applied to biomacromolecules that contain various functional groups under physiological conditions. The oxabridged bicyclic systems 2a and 2b were prepared by a Diels– Alder reaction of substituted propiolates with furan (Scheme 1). Subsequent hydrolysis provided the desired carboxylic acid derivatives 3a and 3b, in excellent yield. To compare the reactivity of Diels–Alder products 2a and b with the corresponding alkynes, [3+2] cycloaddition reactions were performed under ambient conditions by using benzyl azide, and monitored over time with H NMR spectroscopy (Figure 1). The oxanorbornadienes 2a and 2b and their respective alkynes provided identical 1,4,5-substituted triazoles to the products.

Application of Metal‐Free Triazole Formation in the Synthesis of Cyclic RGD–DTPA Conjugates

The tandem 1,3‐dipolar cycloaddition‐retro‐Diels–Alder (tandem crDA) reaction is presented as a versatile method for metal‐free chemoselective conjugation of a DTPA radiolabel to N‐δ‐azido‐cyclo(‐Arg‐Gly‐Asp‐d‐Phe‐Orn‐) via oxanorbornadiene derivatives. To this end, the behavior of several trifluoromethyl‐substituted oxanorbornadiene derivatives in the 1,3‐dipolar cycloaddition was studied and optimized to give a clean and efficient method for bio‐orthogonal ligation in an aqueous environment. After radioisotope treatment, the resulting 111In‐labeled c(RGD)‐CF3‐triazole‐DTPA conjugate was subjected to preliminary biological evaluation and showed high affinity for αvβ3 (IC50=192 nM) and favorable pharmacokinetics.

Controlled integration of polymers into viral capsids

In this paper, we describe the controlled incorporation of two synthetic polymers with different structures in the cowpea chlorotic mottle virus (CCMV) capsid. Poly(ethylene glycol) (PEG) chains have been attached to the amine groups of lysine residues on the outer surface of the viral capsid. The functionalization of CCMV with PEG chains provoked a slow but irreversible dissociation of the virus into PEG-coat protein (CP) subunits, likely due to steric interference between the protein-protein subunits as a result of the presence of the PEG chains. This thermodynamic instability, however, can be overcome if a second polymer, such as polystyrene sulfonate (PSS), is present within the capsid. After complete disassembly of the PEG-CCMV conjugates and removal of the viral RNA, incubation of the PEG-functionalized coat proteins with PSS resulted in the formation of much more robust PSS-CCMV-PEG capsids with a diameter of 18 nm (T = 1 capsids). These are the first virus-like particles bearing synthetic organic polymers both inside and outside the viral capsid, opening a new route to the synthesis of biohybrid nanostructured materials based on viruses.

Monitoring Protein−Polymer Conjugation by a Fluorogenic Cu(I)-Catalyzed Azide−Alkyne 1,3-Dipolar Cycloaddition

The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) has recently proven to be a powerful synthetic tool in various fields of chemistry, including protein-polymer conjugation. In this article, we describe a fluorogenic CuAAC, which allows for efficient monitoring of protein-polymer conjugation. We show that profluorescent 3-azido coumarin-terminated polymers can be reacted with an alkyne-functionalized protein to produce a strongly fluorescent triazole-linked conjugate. Upon formation of the product, the evolution of fluorescence can accurately be determined, providing information about the course of the CuAAC. As a proof of concept, we synthesized several 3-azido coumarin terminated poly(ethylene glycol) (PEG) chains and investigated their conjugation with alkyne-functionalized bovine serum albumin (BSA) as a model protein. CuAAC conjugation was shown to be very efficient and proceeded rapidly. Conversion plots were constructed from measuring the fluorescence as function of reaction time. An additional benefit of the fluorogenic CuAAC is the in situ labeling of bioconjugates. We envision that the fluorogenic protein-polymer conjugation is not restricted to the reaction system reported in this work, but may also be ideal to screen for optimal reaction conditions of various other systems.

Synthesis and aggregation behavior of biohybrid amphiphiles composed of a tripeptidic head group and a polystyrene tail

The modular synthesis and self-assembly behavior of a set of peptide-polymer hybrid amphiphiles is described. Gly-Gly-Arg derivatives were conjugated to one of the ends of hetero-telechelic polystyrene (PS) via either the Cu-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reaction or conventional peptide coupling chemistry. Both conjugation strategies proved to be efficient and they were also applied sequentially to create a biohybrid ABA-type triblock copolymer, which can be considered as a macromolecular bolaamphiphile. In the synthesis of the regularly-shaped peptide-PS amphiphiles (i.e. AB-type) two polymer lengths (n = 21 and n = 49) were employed having additional variations in their end group composition. As expected, the structural variations were demonstrated not to influence the self-assembly behavior of the biohybrids in water, and comparable vesicles were formed in all cases. At the air/water interface, the structural variations had a greater impact on the self-assembly of the biohybrids, especially for the phase transition from gas to liquid because it is dominated by steric interactions between the polymer chains. In a condensed phase (which closely resembles the situation in a bilayer vesicle), the packing of various biohybrids was found to be comparable. The interfacial self-assembly behavior of the bolaamphiphile (n = 21) was also studied and this compound probably formed multi-layered structures on the water surface. In aqueous solution the bolaamphiphile formed spherical aggregates similar to the regular peptide-PS amphiphiles. Although these assemblies appeared to be vesicular, their exact nature remains unclear.