Simultaneous Dual Protein Labeling Using a Triorthogonal Reagent

Overview

Journal J Am Chem Soc

Publisher American Chemical Society

Specialty Chemistry

Date 2013 Oct 19

PMID 24134212

Citations 27

Authors

Mohammad Rashidian

Sidath C Kumarapperuma

Kari Gabrielse

Adrian Fegan

Carston R Wagner

Mark D Distefano

Affiliations

Soon will be listed here.

Abstract

Construction of heterofunctional proteins is a rapidly emerging area of biotherapeutics. Combining a protein with other moieties, such as a targeting element, a toxic protein or small molecule, and a fluorophore or polyethylene glycol (PEG) group, can improve the specificity, functionality, potency, and pharmacokinetic profile of a protein. Protein farnesyl transferase (PFTase) is able to site-specifically and quantitatively prenylate proteins containing a C-terminal CaaX-box amino acid sequence with various modified isoprenoids. Here, we describe the design, synthesis, and application of a triorthogonal reagent, 1, that can be used to site-specifically incorporate an alkyne and aldehyde group simultaneously into a protein. To illustrate the capabilities of this approach, a protein was enzymatically modified with compound 1 followed by oxime ligation and click reaction to simultaneously incorporate an azido-tetramethylrhodamine (TAMRA) fluorophore and an aminooxy-PEG moiety. This was performed with both a model protein [green fluorescent protein (GFP)] as well as a therapeutically useful protein [ciliary neurotrophic factor (CNTF)]. Next, a protein was enzymatically modified with compound 1 followed by coupling to an azido-bis-methotrexate dimerizer and aminooxy-TAMRA. Incubation of that construct with a dihydrofolate reductase (DHFR)-DHFR-anti-CD3 fusion protein resulted in the self-assembly of nanoring structures that were endocytosed into T-leukemia cells and visualized therein. These results highlight how complex multifunctional protein assemblies can be prepared using this facile triorthogonal approach.

Citing Articles

Targeted Drug Delivery by MMAE Farnesyl-Bioconjugated Multivalent Chemically Self-Assembled Nanorings Induces Potent Receptor-Dependent Immunogenic Cell Death.

Wang Y, Kilic O, Rozumalski L, Distefano M, Wagner C Bioconjug Chem. 2024; 35(5):582-592.

PMID: 38701361 PMC: 11633779. DOI: 10.1021/acs.bioconjchem.4c00004.

Bioorthogonal labeling and profiling of N6-isopentenyladenosine (i6A) modified RNA.

Li Y, Zhou H, Chen S, Li Y, Guo Y, Chen X Nucleic Acids Res. 2024; 52(6):2808-2820.

PMID: 38426933 PMC: 11014277. DOI: 10.1093/nar/gkae150.

Promiscuous Enzymes for Residue-Specific Peptide and Protein Late-Stage Functionalization.

Alexander A, Elshahawi S Chembiochem. 2023; 24(17):e202300372.

PMID: 37338668 PMC: 10496146. DOI: 10.1002/cbic.202300372.

Enzymatic Bioconjugation: A Perspective from the Pharmaceutical Industry.

Debon A, Siirola E, Snajdrova R JACS Au. 2023; 3(5):1267-1283.

PMID: 37234110 PMC: 10207132. DOI: 10.1021/jacsau.2c00617.

Site-specific dual encoding and labeling of proteins via genetic code expansion.

Bednar R, Karplus P, Mehl R Cell Chem Biol. 2023; 30(4):343-361.

PMID: 36977415 PMC: 10764108. DOI: 10.1016/j.chembiol.2023.03.004.

References

Guo P . The emerging field of RNA nanotechnology. Nat Nanotechnol. 2010; 5(12):833-42. PMC: 3149862. DOI: 10.1038/nnano.2010.231. View

Salgado E, Radford R, Tezcan F . Metal-directed protein self-assembly. Acc Chem Res. 2010; 43(5):661-72. PMC: 2873059. DOI: 10.1021/ar900273t. View

Chen I, Ting A . Site-specific labeling of proteins with small molecules in live cells. Curr Opin Biotechnol. 2005; 16(1):35-40. DOI: 10.1016/j.copbio.2004.12.003. View

Kim M, Kleckley T, Wiemer A, Holstein S, Hohl R, Wiemer D . Synthesis and activity of fluorescent isoprenoid pyrophosphate analogues. J Org Chem. 2004; 69(24):8186-93. DOI: 10.1021/jo049101w. View

Prost L, Grim J, Tonelli M, Kiessling L . Noncarbohydrate glycomimetics and glycoprotein surrogates as DC-SIGN antagonists and agonists. ACS Chem Biol. 2012; 7(9):1603-8. PMC: 3509189. DOI: 10.1021/cb300260p. View

Li Q, Hapka D, Chen H, Vallera D, Wagner C . Self-assembly of antibodies by chemical induction. Angew Chem Int Ed Engl. 2008; 47(52):10179-82. PMC: 3573144. DOI: 10.1002/anie.200803507. View

Matsuura K, Watanabe K, Matsuzaki T, Sakurai K, Kimizuka N . Self-assembled synthetic viral capsids from a 24-mer viral peptide fragment. Angew Chem Int Ed Engl. 2010; 49(50):9662-5. DOI: 10.1002/anie.201004606. View

Wang T, Kartika R, Spiegel D . Exploring post-translational arginine modification using chemically synthesized methylglyoxal hydroimidazolones. J Am Chem Soc. 2012; 134(21):8958-67. PMC: 3397831. DOI: 10.1021/ja301994d. View

Yin J, Liu F, Li X, Walsh C . Labeling proteins with small molecules by site-specific posttranslational modification. J Am Chem Soc. 2004; 126(25):7754-5. DOI: 10.1021/ja047749k. View

10.

Wen R, Tao W, Li Y, Sieving P . CNTF and retina. Prog Retin Eye Res. 2011; 31(2):136-51. PMC: 3288688. DOI: 10.1016/j.preteyeres.2011.11.005. View

11.

Hu M, Qian L, Brinas R, Lymar E, Hainfeld J . Assembly of nanoparticle-protein binding complexes: from monomers to ordered arrays. Angew Chem Int Ed Engl. 2007; 46(27):5111-4. DOI: 10.1002/anie.200701180. View

12.

Chen G, Jiang M . Cyclodextrin-based inclusion complexation bridging supramolecular chemistry and macromolecular self-assembly. Chem Soc Rev. 2011; 40(5):2254-66. DOI: 10.1039/c0cs00153h. View

13.

Kim J, Seo M, Lee S, Cho K, Yang A, Woo K . Simple and efficient strategy for site-specific dual labeling of proteins for single-molecule fluorescence resonance energy transfer analysis. Anal Chem. 2013; 85(3):1468-74. DOI: 10.1021/ac303089v. View

14.

Lai J, Fischbach M, Liu D, Walsh C . Localized protein interaction surfaces on the EntB carrier protein revealed by combinatorial mutagenesis and selection. J Am Chem Soc. 2006; 128(34):11002-3. DOI: 10.1021/ja063238h. View

15.

Ip N, Yancopoulos G . The neurotrophins and CNTF: two families of collaborative neurotrophic factors. Annu Rev Neurosci. 1996; 19:491-515. DOI: 10.1146/annurev.ne.19.030196.002423. View

16.

Hudak J, Barfield R, de Hart G, Grob P, Nogales E, Bertozzi C . Synthesis of heterobifunctional protein fusions using copper-free click chemistry and the aldehyde tag. Angew Chem Int Ed Engl. 2012; 51(17):4161-5. PMC: 3379715. DOI: 10.1002/anie.201108130. View

17.

Labadie G, Viswanathan R, Poulter C . Farnesyl diphosphate analogues with omega-bioorthogonal azide and alkyne functional groups for protein farnesyl transferase-catalyzed ligation reactions. J Org Chem. 2007; 72(24):9291-7. PMC: 2516946. DOI: 10.1021/jo7017747. View

18.

Placzek A, Gibbs R . New synthetic methodology for the construction of 7-substituted farnesyl diphosphate analogs. Org Lett. 2011; 13(14):3576-9. PMC: 3401630. DOI: 10.1021/ol201069x. View

19.

Fegan A, White B, Carlson J, Wagner C . Chemically controlled protein assembly: techniques and applications. Chem Rev. 2010; 110(6):3315-36. DOI: 10.1021/cr8002888. View

20.

Weinrich D, Lin P, Jonkheijm P, Nguyen U, Schroder H, Niemeyer C . Oriented immobilization of farnesylated proteins by the thiol-ene reaction. Angew Chem Int Ed Engl. 2010; 49(7):1252-7. DOI: 10.1002/anie.200906190. View