Jennifer A. Prescher

Copper-free click chemistry for dynamic in vivo imaging

Dynamic imaging of proteins in live cells is routinely performed by using genetically encoded reporters, an approach that cannot be extended to other classes of biomolecules such as glycans and lipids. Here, we report a Cu-free variant of click chemistry that can label these biomolecules rapidly and selectively in living systems, overcoming the intrinsic toxicity of the canonical Cu-catalyzed reaction. The critical reagent, a substituted cyclooctyne, possesses ring strain and electron-withdrawing fluorine substituents that together promote the [3 + 2] dipolar cycloaddition with azides installed metabolically into biomolecules. This Cu-free click reaction possesses comparable kinetics to the Cu-catalyzed reaction and proceeds within minutes on live cells with no apparent toxicity. With this technique, we studied the dynamics of glycan trafficking and identified a population of sialoglycoconjugates with unexpectedly rapid internalization kinetics.

Chemical remodelling of cell surfaces in living animals

Cell surfaces are endowed with biological functionality designed to mediate extracellular communication. The cell-surface repertoire can be expanded to include abiotic functionality through the biosynthetic introduction of unnatural sugars into cellular glycans, a process termed metabolic oligosaccharide engineering. This technique has been exploited in fundamental studies of glycan-dependent cell–cell and virus–cell interactions and also provides an avenue for the chemical remodelling of living cells. Unique chemical functional groups can be delivered to cell-surface glycans by metabolism of the corresponding unnatural precursor sugars. These functional groups can then undergo covalent reaction with exogenous agents bearing complementary functionality. The exquisite chemical selectivity required of this process is supplied by the Staudinger ligation of azides and phosphines, a reaction that has been performed on cultured cells without detriment to their physiology. Here we demonstrate that the Staudinger ligation can be executed in living animals, enabling the chemical modification of cells within their native environment. The ability to tag cell-surface glycans in vivo may enable therapeutic targeting and non-invasive imaging of changes in glycosylation during disease progression.

Copper-free click chemistry in living animals

Chemical reactions that enable selective biomolecule labeling in living organisms offer a means to probe biological processes in vivo. Very few reactions possess the requisite bioorthogonality, and, among these, only the Staudinger ligation between azides and triarylphosphines has been employed for direct covalent modification of biomolecules with probes in the mouse, an important model organism for studies of human disease. Here we explore an alternative bioorthogonal reaction, the 1,3-dipolar cycloaddition of azides and cyclooctynes, also known as “Cu-free click chemistry,” for labeling biomolecules in live mice. Mice were administered peracetylated N-azidoacetylmannosamine (Ac4ManNAz) to metabolically label cell-surface sialic acids with azides. After subsequent injection with cyclooctyne reagents, glycoconjugate labeling was observed on isolated splenocytes and in a variety of tissues including the intestines, heart, and liver, with no apparent toxicity. The cyclooctynes tested displayed various labeling efficiencies that likely reflect the combined influence of intrinsic reactivity and bioavailability. These studies establish Cu-free click chemistry as a bioorthogonal reaction that can be executed in the physiologically relevant context of a mouse.

Finding the Right (Bioorthogonal) Chemistry

Bioorthogonal chemistries can be used to tag diverse classes of biomolecules in cells and other complex environments. With over 20 unique transformations now available, though, selecting an appropriate reaction for a given experiment is challenging. In this article, we compare and contrast the most common classes of bioorthogonal chemistries and provide a framework for matching the reactions with downstream applications. We also discuss ongoing efforts to identify novel biocompatible reactions and methods to control their reactivity. The continued expansion of the bioorthogonal toolkit will provide new insights into biomolecule networks and functions and thus refine our understanding of living systems.

Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models

To examine the role of breast cancer stem cells (BCSCs) in metastasis, we generated human-in-mouse breast cancer orthotopic models using patient tumor specimens, labeled with optical reporter fusion genes. These models recapitulate human cancer features not captured with previous models, including spontaneous metastasis in particular, and provide a useful platform for studies of breast tumor initiation and progression. With noninvasive imaging approaches, as few as 10 cells of stably labeled BCSCs could be tracked in vivo, enabling studies of early tumor growth and spontaneous metastasis. These advances in BCSC imaging revealed that CD44+ cells from both primary tumors and lung metastases are highly enriched for tumor-initiating cells. Our metastatic cancer models, combined with noninvasive imaging techniques, constitute an integrated approach that could be applied to dissect the molecular mechanisms underlying the dissemination of metastatic CSCs (MCSCs) and to explore therapeutic strategies targeting MCSCs in general or to evaluate individual patient tumor cells and predict response to therapy.

Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids

The incorporation of noncanonical amino acids into recombinant proteins in Escherichia coli can be facilitated by the introduction of new aminoacyl-tRNA synthetase activity into the expression host. We describe here a screening procedure for the identification of new aminoacyl-tRNA synthetase activity based on the cell surface display of noncanonical amino acids. Screening of a saturation mutagenesis library of the E. coli methionyl-tRNA synthetase (MetRS) led to the discovery of three MetRS mutants capable of incorporating the long-chain amino acid azidonorleucine into recombinant proteins with modest efficiency. The Leu-13 → Gly (L13G) mutation is found in each of the three MetRS mutants, and the MetRS variant containing this single mutation is highly efficient in producing recombinant proteins that contain azidonorleucine.

Functionalized Cyclopropenes As Bioorthogonal Chemical Reporters

Chemical reporters are unique functional groups that can be used to label biomolecules in living systems. Only a handful of broadly applicable reporters have been identified to date, owing to the rigorous demands placed on these functional groups in biological settings. We describe here a new chemical reporter-cyclopropene-that can be used to target biomolecules in vitro and in live cells. A variety of substituted cyclopropene scaffolds were synthesized and found to be stable in aqueous solution and in the presence of biological nucleophiles. Furthermore, some of the cyclopropene units were metabolically introduced into cell surface glycans and subsequently detected with covalent probes. The small size and selective reactivity of cyclopropenes will facilitate efforts to tag diverse collections of biomolecules in vivo.

Chemical Technologies for Probing Glycans

Glycans are central to many biological processes, but efforts to define their functions at the molecular level have been frustrated by a lack of suitable technologies. Here we highlight chemical tools that are beginning to address this need.

Selective uptake of single-walled carbon nanotubes by circulating monocytes for enhanced tumour delivery

In cancer imaging, nanoparticle biodistribution is typically visualised in living subjects using ‘bulk’ imaging modalities such as magnetic resonance imaging, computerized tomography and whole-body fluorescence. As such the nanoparticle influx is observed only macroscopically and the mechanisms by which they target cancer remain elusive. Nanoparticles are assumed to accumulate via several targeting mechanisms, particularly extravasation ie, leakage into tumour. Here we show that, in addition to conventional nanoparticle uptake mechanisms, single-walled carbon nanotubes are almost exclusively taken up by a single immune cell subset, Ly-6Chi monocytes (almost 100% uptake in Ly-6Chi monocytes, below 3% in all other circulating cells), and delivered to the tumour in mice. Next, we demonstrate that a targeting ligand (RGD) conjugated to nanotubes significantly enhances the number of single-walled carbon nanotube-loaded monocytes reaching the tumour (p<0.001, day 7 p.i.). The remarkable selectivity of this tumour targeting mechanism demonstrates an advanced immune-based delivery strategy for enhancing specific tumour delivery with substantial penetration.

Identification of palmitoylated mitochondrial proteins using a bio-orthogonal azido-palmitate analogue

Increased levels of circulating saturated free fatty acids, such as palmitate, have been implicated in the etiology of type II diabetes and cancer. In addition to being a constituent of glycerolipids and a source of energy, palmitate also covalently attaches to numerous cellular proteins via a process named palmi‐toylation. Recognized for its roles in membrane tethering, cellular signaling, and protein trafficking, palmi‐toylation is also emerging as a potential regulator of metabolism. Indeed, we showed previously that the acylation of two mitochondrial proteins at their active site cysteine residues result in their inhibition. Herein, we sought to identify other palmitoylated proteins in mitochondria using a nonradioactive bio‐orthogonal azido‐palmitate analog that can be selectively derivat‐ized with various tagged triarylphosphines. Our results show that, like palmitate, incorporation of azido‐palmi‐tate occurred on mitochondrial proteins via thioester bonds at sites that could be competed out by palmitoyl‐CoA. Using this method, we identified 21 putative palmitoylated proteins in the rat liver mitochondrial matrix, a compartment not recognized for its content in palmitoylated proteins, and confirmed the palmitoyl‐ation of newly identified mitochondrial 3‐hydroxy‐3‐methylglutaryl‐CoA synthase. We postulate that cova‐lent modification and perhaps inhibition of various mitochondrial enzymes by palmitoyl‐CoA could lead to the metabolic impairments found in obesity‐related diseases.—Kostiuk, M. A., Corvi, M. M., Keller, B. O., Plummer, G., Prescher, J. A., Hangauer, M. J., Bertozzi, C. R., Rajaiah, G., Falck, J. R., Berthiaume, L. G. Identification of palmitoylated mitochondrial proteins using a bio‐orthogonal azido‐palmitate analogue. FASEB J. 22, 721–732 (2008)