Christian P. R. Hackenberger

Chemoselective Ligation and Modification Strategies for Peptides and Proteins

The investigation of biological processes by chemical methods, commonly referred to as chemical biology, often requires chemical access to biologically relevant macromolecules such as peptides and proteins. Building upon solid-phase peptide synthesis, investigations have focused on the development of chemoselective ligation and modification strategies to link synthetic peptides or other functional units to larger synthetic and biologically relevant macromolecules. This Review summarizes recent developments in the field of chemoselective ligation and modification strategies and illustrates their application, with examples ranging from the total synthesis of proteins to the semisynthesis of naturally modified proteins.

An alkaloid-mediated desymmetrization of meso-anhydrides via a nucleophilic ring opening with benzyl alcohol and its application in the synthesis of highly enantiomerically enriched β-amino acids

The cinchona alkaloid-mediated opening of prochiral cyclic anhydrides in the presence of benzyl alcohol leading to optically active hemiesters is described. Structurally diverse anhydrides are converted into their corresponding benzyl monoesters with either enantiomer being obtained with up to 99% e.e. by using quinine or quinidine as the directing additive. A simple aqueous work-up protocol permits the isolation of the products in analytically pure form and the recovery of the alkaloids almost quantitatively. These hemiesters can be converted to N-protected β-amino esters by means of Curtius degradation of the corresponding acyl azides. Subsequent cleavage of both protecting groups by a single reaction step leads to the free β-amino acids in excellent yields. The efficiency of this procedure is demonstrated by the short asymmetric synthesis of the fungicide cis-pentacin delivering the amino acid with >99.7% enantiomeric excess.

Covalent Attachment of Cyclic TAT Peptides to GFP Results in Protein Delivery into Live Cells with Immediate Bioavailability.

The delivery of free molecules into the cytoplasm and nucleus by using arginine-rich cell-penetrating peptides (CPPs) has been limited to small cargoes, while large cargoes such as proteins are taken up and trapped in endocytic vesicles. Based on recent work, in which we showed that the transduction efficiency of arginine-rich CPPs can be greatly enhanced by cyclization, the aim was to use cyclic CPPs to transport full-length proteins, in this study green fluorescent protein (GFP), into the cytosol of living cells. Cyclic and linear CPP-GFP conjugates were obtained by using azido-functionalized CPPs and an alkyne-functionalized GFP. Our findings reveal that the cyclic-CPP-GFP conjugates are internalized into live cells with immediate bioavailability in the cytosol and the nucleus, whereas linear CPP analogues do not confer GFP transduction. This technology expands the application of cyclic CPPs to the efficient transport of functional full-length proteins into live cells.

Site-specific PEGylation of proteins by a Staudinger-phosphite reaction

Current protocols in protein bioengineering allow the site-specific incorporation of chemical reporter moieties. Subsequently, these functional groups can be chemoselectively transformed to decorate proteins with charged and oversized functional units. Based on our recent report on the chemoselective reaction of azides with phosphites, we now apply the Staudinger-phosphite reaction to an efficient and metal-free PEGylation of an azide-containing protein with symmetrical phosphites. Thereby, two types of branched oligoethylene glycol scaffolds are generated, which deliver either a stable or light-cleavable protein-PEG conjugate. Furthermore, we demonstrate that the Staudinger-phosphite reaction is an efficient transformation in both aqueous media as well as in a highly crowded bacterial cell lysate.

Perturbing the folding energy landscape of the bacterial immunity protein Im7 by site-specific N-linked glycosylation

N-linked glycosylation modulates protein folding and stability through a variety of mechanisms. As such there is considerable interest in the development of general rules to predict the structural consequences of site-specific glycosylation and to understand how these effects can be exploited in the design and development of modified proteins with advantageous properties. In this study, expressed protein ligation is used to create site-specifically glycosylated variants of the bacterial immunity protein Im7 modified with the chitobiose disaccharide (GlcNAc-GlcNAc). Glycans were introduced at seven solvent exposed sites within the Im7 sequence and the kinetic and thermodynamic consequences of N-linked glycosylation analyzed. The values for glycan incorporation were found to range from +5.2 to -3.8 kJ·mol-1. In several cases, glycosylation influences folding by modulating the local conformational preferences of the glycosylated sequence. These locally mediated effects are most prominent in the center of α-helices where glycosylation negatively effects folding and in compact turn motifs between segments of ordered secondary structure where glycosylation promotes folding and enhances the overall stability of the native protein. The studies also provide insight into why glycosylation is commonly identified at the transition between different types of secondary structure and when glycosylation may be used to elaborate protein structure to protect disordered sequences from proteolysis or immune system recognition.

Site-specific PEGylation of Proteins: Recent Developments

The attachment of linear polyethylene glycol (PEG) to peptides and proteins for their stabilization for in vivo applications is a milestone in pharmaceutical research and protein-drug development. However, conventional methods often lead to heterogeneous PEGylation mixtures with reduced protein activity. Current synthetic efforts aim to provide site-specific approaches by chemoselective targeting of canonical and noncanonical amino acids and to improve the PEG architecture. This synopsis highlights recent work in this area, which also resulted in improved pharmacokinetics of peptide and protein therapeutics.

Live-cell MRI with Xenon Hyper-CEST Biosensors Targeted to Metabolically Labeled Cell-Surface Glycans†

The targeting of metabolically labeled glycans with conventional MRI contrast agents has proved elusive. In this work, which further expands the utility of xenon Hyper-CEST biosensors in cell experiments, we present the first successful molecular imaging of such glycans using MRI. Xenon Hyper-CEST biosensors are a novel class of MRI contrast agents with very high sensitivity. We designed a multimodal biosensor for both fluorescent and xenon MRI detection that is targeted to metabolically labeled sialic acid through bioorthogonal chemistry. Through the use of a state of the art live-cell bioreactor, it was demonstrated that xenon MRI biosensors can be used to image cell-surface glycans at nanomolar concentrations.

Current Status: Site-Specific Antibody Drug Conjugates

Antibody drug conjugates (ADCs), a promising class of cancer biopharmaceuticals, combine the specificity of therapeutic antibodies with the pharmacological potency of chemical, cytotoxic drugs. Ever since the first ADCs on the market, a plethora of novel ADC technologies has emerged, covering as diverse aspects as antibody engineering, chemical linker optimization and novel conjugation strategies, together aiming at constantly widening the therapeutic window for ADCs. This review primarily focuses on novel chemical and biotechnological strategies for the site-directed attachment of drugs that are currently validated for 2nd generation ADCs to promote conjugate homogeneity and overall stability.

Site-specifically phosphorylated lysine peptides.

Protein phosphorylation controls major processes in cells. Although phosphorylation of serine, threonine, and tyrosine and also recently histidine and arginine are well-established, the extent and biological significance of lysine phosphorylation has remained elusive. Research in this area has been particularly limited by the inaccessibility of peptides and proteins that are phosphorylated at specific lysine residues, which are incompatible with solid-phase peptide synthesis (SPPS) due to the intrinsic acid lability of the P(═O)-N phosphoramidate bond. To address this issue, we have developed a new synthetic route for the synthesis of site-specifically phospholysine (pLys)-containing peptides by employing the chemoselectivity of the Staudinger-phosphite reaction. Our synthetic approach relies on the SPPS of unprotected ε-azido lysine-containing peptides and their subsequent reaction to phosphoramidates with phosphite esters before they are converted into the natural modification via UV irradiation or basic deprotection. With these peptides in hand, we demonstrate that electron-transfer dissociation tandem mass spectrometry can be used for unambiguous assignment of phosphorylated-lysine residues within histone peptides and that these peptides can be detected in cell lysates using a bottom-up proteomic approach. This new tagging method is expected to be an essential tool for evaluating the biological relevance of lysine phosphorylation.

Versatile and Efficient Site-Specific Protein Functionalization by Tubulin Tyrosine Ligase.

A novel chemoenzymatic approach for simple and fast site-specific protein labeling is reported. Recombinant tubulin tyrosine ligase (TTL) was repurposed to attach various unnatural tyrosine derivatives as small bioorthogonal handles to proteins containing a short tubulin-derived recognition sequence (Tub-tag). This novel strategy enables a broad range of high-yielding and fast chemoselective C-terminal protein modifications on isolated proteins or in cell lysates for applications in biochemistry, cell biology, and beyond, as demonstrated by the site-specific labeling of nanobodies, GFP, and ubiquitin.