Sander S. van Berkel

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.

Staudinger Ligation as a Method for Bioconjugation

In 1919 the German chemist Hermann Staudinger was the first to describe the reaction between an azide and a phosphine. It was not until recently, however, that Bertozzi and co-workers recognized the potential of this reaction as a method for bioconjugation and transformed it into the so-called Staudinger ligation. The bio-orthogonal character of both the azide and the phosphine functions has resulted in the Staudinger ligation finding numerous applications in various complex biological systems. For example, the Staudinger ligation has been utilized to label glycans, lipids, DNA, and proteins. Moreover, the Staudinger ligation has been used as a synthetic method to construct glycopeptides, microarrays, and functional biopolymers. In the emerging field of bio-orthogonal ligation strategies, the Staudinger ligation has set a high standard to which most of the new techniques are often compared. This Review summarizes recent developments and new applications of the Staudinger ligation.

Single-Step Azide Introduction in Proteins via an Aqueous Diazo Transfer

The controlled introduction of azides in proteins provides targetable handles for selective protein manipulation. We present here an efficient diazo transfer protocol that can be applied in an aqueous solution, leading to the facile introduction of azides in the side chains of lysine residues and at the N-terminus of enzymes, e.g. horseradish peroxidase (HRP) and the red fluorescent protein DsRed. The effective introduction of azides was verified by mass spectrometry, after which the azido-proteins were used in Cu(I)-catalyzed [3 + 2] cycloaddition reactions. Azido-HRP retained its catalytic activity after conjugation of a small molecule. This modified protein could also be successfully immobilized on the surface of an acetylene-covered polymersome. Azido-DsRed was coupled to an acetylene-bearing protein allowing it to act as a fluorescent label, demonstrating the wide applicability of the diazo transfer procedure.

Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody–Drug Conjugates

A robust, generally applicable, nongenetic technology is presented to convert monoclonal antibodies into stable and homogeneous ADCs. Starting from a native (nonengineered) mAb, a chemoenzymatic protocol allows for the highly controlled attachment of any given payload to the N-glycan residing at asparagine-297, based on a two-stage process: first, enzymatic remodeling (trimming and tagging with azide), followed by ligation of the payload based on copper-free click chemistry. The technology, termed GlycoConnect, is applicable to any IgG isotype irrespective of glycosylation profile. Application to trastuzumab and maytansine, both components of the marketed ADC Kadcyla, demonstrate a favorable in vitro and in vivo efficacy for GlycoConnect ADC. Moreover, the superiority of the native glycan as attachment site was demonstrated by in vivo comparison to a range of trastuzumab-based glycosylation mutants. A side-by-side comparison of the copper-free click probes bicyclononyne (BCN) and a dibenzoannulated cyclooctyne (DBCO) showed a surprising difference in conjugation efficiency in favor of BCN, which could be even further enhanced by introduction of electron-withdrawing fluoride substitutions onto the azide. The resulting mAb-conjugates were in all cases found to be highly stable, which in combination with the demonstrated efficacy warrants ADCs with a superior therapeutic index.

“Clickable” elastins: elastin-like polypeptides functionalized with azide or alkyne groups

Elastin-like polypeptides (ELPs) functionalized with azide or alkyne groups were produced biosynthetically and coupled via the Cu-catalyzed azide-alkyne cycloaddition to a variety of (bio)molecules.

Diacetin, a reliable cue and private communication channel in a specialized pollination system

The interaction between floral oil secreting plants and oil-collecting bees is one of the most specialized of all pollination mutualisms. Yet, the specific stimuli used by the bees to locate their host flowers have remained elusive. This study identifies diacetin, a volatile acetylated glycerol, as a floral signal compound shared by unrelated oil plants from around the globe. Electrophysiological measurements of antennae and behavioural assays identified diacetin as the key volatile used by oil-collecting bees to locate their host flowers. Furthermore, electrophysiological measurements indicate that only oil-collecting bees are capable of detecting diacetin. The structural and obvious biosynthetic similarity between diacetin and associated floral oils make it a reliable cue for oil-collecting bees. It is easily perceived by oil bees, but can’t be detected by other potential pollinators. Therefore, diacetin represents the first demonstrated private communication channel in a pollination system.

Fluorogenic peptide-based substrates for monitoring thrombin activity

The synthesis of a series of peptides containing C‐terminal 7‐amino‐4‐methylcoumarin (AMC) for use in the thrombin generation test (TGT) is described. The lead structure in this project was H‐Gly‐Gly‐Arg‐AMC, of which the water solubility and kinetic parameters (KM and kcat) are greatly improved over those of the substrate in current use in the TGT: Cbz‐Gly‐Gly‐Arg‐AMC. A series of N‐terminally substituted Gly‐Gly‐Arg‐AMC derivatives were synthesized, as well as implementation of structural changes at either the P2 or P3 position of the peptide backbone. Furthermore, two substrates were synthesized that have structural similarities to the chromogenic thrombin substrate SQ68 or that contain a 1,2,3‐triazole moiety in the peptide chain, mimicking an amide bond. To determine the applicability of newly synthesized fluorogenic substrates for monitoring continuous thrombin generation, the KM and kcat values of the conversion of these fluorogenic substrates by thrombin (FIIa) and factor Xa (FXa) were quantified. An initial selection was made on basis of these data, and suitable substrates were further evaluated as substrates in the thrombin generation assay. Assessment of the acquired data showed that several substrates, including the SQ68 derivative Et‐malonate‐Gly‐Arg‐AMC and N‐functionalized Gly‐Gly‐Arg‐AMC derivatives, are suitable candidates for replacement of the substrate currently in use.

In-depth evaluation of the cycloaddition–retro-Diels–Alder reaction for in vivo targeting with [111In]-DTPA-RGD conjugates

INTRODUCTION The spontaneous copper-free tandem 1,3-dipolar cycloaddition-retro-Diels-Alder (tandem crDA) reaction between cyclic Arg-Gly-Asp-d-Phe-Orn(N(3)) [c(RGDfX)] and oxanorbornadiene-DTPA (o-DTPA) or methyloxanorbornadiene-DTPA (mo-DTPA) into two DTPA-c(RGDfX) regioisomers is characterized. Since there is no information on the stability and reaction rate of the tandem crDA reaction in biological media, we set out to characterize these reaction parameters. METHODS The effects of concentration of the reactants, temperature, pH and reaction environment (serum, blood) on the kinetics of the reaction were determined using (111)In-labeled oxanorbornadiene-DTPA analogs. The affinity of the radiolabeled conjugate was determined in a solid-phase alpha(v)beta(3) integrin binding assay. Furthermore, the octanol-water partition coefficient was determined and, finally, the biodistribution of the labeled compounds in mice with subcutaneous alpha(v)beta(3)-expressing tumors was determined. RESULTS Fifty percent conversion was reached after 26 h. Kinetic experiments furthermore established that the reaction rate of the tandem crDA reaction follows temperature- and concentration-dependent second-order kinetics, but is independent of the pH of the medium. Affinity of the two [(111)In]DTPA-cRGDfX conjugates for alpha(v)beta(3) integrin is 191 nM. Biodistribution studies showed specific (alpha(v)beta(3)-mediated) uptake of [(111)In]DTPA-c(RGDfX) in the tumor and in alpha(v)beta(3)-expressing tissues. CONCLUSION The tandem crDA reaction using methyl-substituted oxanorbornadiene is a versatile method for a single-step ligation that proceeds independently of pH and also proceeds in serum and blood. Currently, we are further looking into enhancement of reaction kinetics and exploitation of tandem crDA in vivo.

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.