Chemical reporters for biological discovery

Abstract

Functional tools are needed to understand complex biological systems. Here we review how chemical reporters in conjunction with bioorthogonal labeling methods can be used to image and retrieve nucleic acids, proteins, glycans, lipids and other metabolites in vitro, in cells as well as in whole organisms. By tagging these biomolecules, researchers can now monitor their dynamics in living systems and discover specific substrates of cellular pathways. These advances in chemical biology are thus providing important tools to characterize biological pathways and are poised to facilitate our understanding of human diseases.

Figure 1

Bioorthogonal labeling of biomolecules. a) Bioorthogonal chemical reporters and chemoselective reactions allow covalent labeling of biomolecules with detection or affinity tags for imaging or proteomics applications, respectively. b) Staudinger ligation allows labeling of alkyl-azides with triarylphoshine reagents. c) Cu(I)-catalyzed cycloaddition between alkyl-azides and terminal alkynes affords triazole adducts, which can operate in both orientations. d) Activated cyclooctynes can react with alkyl-azides for Cu-free labeling. e) Tetrazine reagents can undergo selective and rapid Diels-Alder reactions with activated alkenes such as trans-cyclooctenes.

Figure 2

Applications of bioorthogonal chemical reporters. a) Pulse-labeling of isolated cells or organisms with a chemical reporter leads to the metabolic incorporation of the reporter into targeted biomolecules. Subsequent labeling of cells with the native metabolites or differentially labeled reporter enables the monitoring of biomolecule turnover rates. b) Pulse-labeling of two distinct cellular populations or organisms allows the comparative analysis of their metabolically labeled biomolecule pools. c) The application of chemical reporters in cell lysates, with purified substrates, or in combination with substrate arrays allows for enzyme-specific substrate detection.

Figure 3

Chemical reporters for nucleic acid synthesis and modifications. a) 5-ethynyl-2’-deoxyuridine (EdU) is incorporated into DNA. b) 5-ethynyluridine (EU) is incorporated into RNA. c) 5-ethynyl-2’-deoxyfluorouridine (F-ara-EdU) is incorporated in to DNA. d) 5-ethynyl-2’-deoxycytosine (EdC) in incorporated into DNA. e) N-6-propargyladenosine (N6pA) is incorporated into RNA and into mRNA polyadenylation tails. f) The DNA modification 5-hydroxymethylcytosine (5-hmc) can be enzymatically labeled with 6-azido-glucose for subsequent bioorthogonal detection.

Figure 4

Amino acid reporters for site- and residue-selective labeling of proteins. a) Analogs of the amino acid pyrrolysine (Pyl) with trans-cyclooctene (trans-ø) or strained-cyclooctyne (bicyclo[6.1.0]non-4-yn-9-ylmethanol, BCN) groups can be site-selectively incorporated into individual proteins through the expression of orthogonal aminoacyl-tRNA-synthetase and tRNA. b) Analogs of methionine (Met) can be selectively incorporated throughout the bacterial or mammalian proteome to install an alkyne (homopropargylglycine, HPG) or azide (azidohomoalanine, AHA) group. c) O-propargyl-puromycin (OP-puro) covalently labels the C-terminus of nascent peptides.

Figure 5

Glycan chemical reporters. a) Peracetylated N-azidoacetyl-D-mannosamine (Ac₄ManNAz) is metabolically incorporated into sialic acid glycans. b) Peracetylated N-azidoacetyl-D-galactosamine (Ac₄GalNAz) is metabolically incorporated into mucin type O-linked glycans. c) Peracetylated 6-ethynyl-L-fucose (Ac₄6-ethynyl-L-fucose) is metabolically incorporated into fucosylated proteins. d) Peracetylated N-alkynylacetyl-D-glucosamine (Ac₄GlcNAlk) is metabolically incorporated into O-GlcNAc modified proteins. e) O-GlcNAc modified proteins can be chemoenzymatically labeled using a β-1,4-galactosyltransferase that transfers GalNAz onto O-GlcNAc modified proteins. OGT, O-GlcNAc transferase.

Figure 6

Protein lipidation chemical reporters. a,b) Alkynyl-fatty acid reporters of different chain length enable selective metabolic labeling of fatty-acylated proteins. c) Alk-FOH allows metabolic labeling of S-farnesylated and S-geranylgeranylated proteins in mammalian cells. d) An azide analog of cholesterol can be metabolically incorporated onto the C-terminus of sonic hedgehog. NMT, N-myristoyltransferase. FT, farnesyltransferase.

Figure 7

Chemical reporters for other posttranslational modifications. a) Sodium-4-pentynoate allows metabolic labeling of acetylated proteins, while 4-pentynoyl-CoA can be used for in vitro analysis of acetyltransferase substrates. b) AdoEnYn and other SAM-based reporters have been developed to profile methyltransferase substrates. c) 6-alkynyl-NAD is a chemical reporter for mono- and poly-ADP-ribosylation. d) N-6-propargyl-ATP serves as a chemical reporter for protein AMPylation. e) N-6-benzyl-ATPγS serves as a substrate for engineered kinases and allows selective modification with a nitrobenzyl-hapten for antibody detection as well as enrichment for proteomic studies. f) The oxidative modification of cysteines to sulfenic acid can be monitored by reaction with an alkynyl-dimedone reagent for subsequent bioorthogonal detection of protein sulfenation.