title: Photoinducible bioorthogonal chemistry: a spatiotemporally controllable tool to visualize and perturb proteins in live cells.
abstract: Visualization in biology has been greatly facilitated by the use of fluorescent proteins as in-cell probes. The genes coding for these wavelength-tunable proteins can be readily fused with the DNA coding for a protein of interest, which enables direct monitoring of natural proteins in real time inside living cells. Despite their success, however, fluorescent proteins have limitations that have only begun to be addressed in the past decade through the development of bioorthogonal chemistry. In this approach, a very small bioorthogonal tag is embedded within the basic building blocks of the cell, and then a variety of external molecules can be selectively conjugated to these pretagged biomolecules. The result is a veritable palette of biophysical probes for the researcher to choose from. In this Account, we review our progress in developing a photoinducible, bioorthogonal tetrazole-alkene cycloaddition reaction ("photoclick chemistry") and applying it to probe protein dynamics and function in live cells. The work described here summarizes the synthesis, structure, and reactivity studies of tetrazoles, including their optimization for applications in biology. Building on key insights from earlier reports, our initial studies of the reaction have revealed full water compatibility, high photoactivation quantum yield, tunable photoactivation wavelength, and broad substrate scope; an added benefit is the formation of fluorescent cycloadducts. Subsequent studies have shown fast reaction kinetics (up to 11.0 M(-1) s(-1)), with the rate depending on the HOMO energy of the nitrile imine dipole as well as the LUMO energy of the alkene dipolarophile. Moreover, through the use of photocrystallography, we have observed that the photogenerated nitrile imine adopts a bent geometry in the solid state. This observation has led to the synthesis of reactive, macrocyclic tetrazoles that contain a short "bridge" between two flanking phenyl rings. This photoclick chemistry has been used to label proteins rapidly (within ∼1 min) both in vitro and in E. coli . To create an effective interface with biology, we have identified both a metabolically incorporable alkene amino acid, homoallylglycine, and a genetically encodable tetrazole amino acid, p-(2-tetrazole)phenylalanine. We demonstrate the utility of these two moieties, respectively, in spatiotemporally controlled imaging of newly synthesized proteins and in site-specific labeling of proteins. Additionally, we demonstrate the use of the photoclick chemistry to perturb the localization of a fluorescent protein in mammalian cells.
authors: Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14260, USA.
authors: Reyna K V
authors: RK
authors: Lim
authors: Qing
authors: Q
authors: Lin
references: Org Lett. 2008 Sep 4;10(17):3725-8
references: 18671406
references: Angew Chem Int Ed Engl. 2009;48(38):6974-98
references: 19714693
references: Science. 2005 Mar 18;307(5716):1746-52
references: 15705808
references: J Am Chem Soc. 2008 Jul 30;130(30):9654-5
references: 18593155
references: Angew Chem Int Ed Engl. 2009;48(29):5330-3
references: 19544336
references: Nat Methods. 2011 Jan;8(1):26-9
references: 21191368
references: J Neurochem. 2001 Apr;77(1):23-33
references: 11279258
references: Chem Commun (Camb). 2010 Mar 14;46(10):1589-600
references: 20177591
references: J Biol Chem. 2001 Jul 27;276(30):28242-51
references: 11350960
references: Proc Natl Acad Sci U S A. 2007 Oct 23;104(43):16793-7
references: 17942682
references: Angew Chem Int Ed Engl. 2007;46(36):6849-51
references: 17685371
references: Chem Commun (Camb). 2009 Oct 7;(37):5588-90
references: 19753366
references: Org Lett. 2009 Aug 20;11(16):3570-3
references: 19637915
references: Angew Chem Int Ed Engl. 1998 Dec 17;37(23):3281-3284
references: 29711420
references: Annu Rev Biochem. 2010;79:413-44
references: 20307192
references: J Org Chem. 2008 Aug 1;73(15):5663-74
references: 18489158
references: Angew Chem Int Ed Engl. 2010;49(5):935-7
references: 20025009
references: ACS Chem Biol. 2010 Sep 17;5(9):875-85
references: 20666508
references: Chemistry. 2010 Dec 3;16(45):13325-9
references: 21031376
references: J Am Chem Soc. 2009 Dec 23;131(50):18036-7
references: 19928921
references: Bioorg Med Chem Lett. 2011 Sep 1;21(17):5033-6
references: 21570845
references: Angew Chem Int Ed Engl. 2009;48(31):5612-26
references: 19565590
references: J Am Chem Soc. 2007 Sep 5;129(35):10646-7
references: 17685614
references: Chem Commun (Camb). 2010 Nov 14;46(42):7993-5
references: 20865197
references: Chem Biol. 2008 Nov 24;15(11):1187-97
references: 19022179
references: Org Lett. 2007 Oct 11;9(21):4155-8
references: 17867694
references: Biochemistry. 1994 Mar 15;33(10):3014-22
references: 8130214
references: Mol Biosyst. 2010 Sep;6(9):1576-8
references: 20436975
references: Proc Natl Acad Sci U S A. 2006 Jun 20;103(25):9482-7
references: 16769897
references: Nat Rev Mol Cell Biol. 2003 May;4(5):373-84
references: 12728271
references: Nat Neurosci. 2010 Jul;13(7):897-905
references: 20543841
references: Nat Methods. 2011 Jan;8(1):35-8
references: 21191370
references: J Am Chem Soc. 2008 Aug 6;130(31):10187-98
references: 18613669
references: J Am Chem Soc. 2003 Apr 2;125(13):3799-812
references: 12656613
references: Bioorg Med Chem Lett. 2011 Mar 1;21(5):1472-5
references: 21277201
references: Angew Chem Int Ed Engl. 2002 Aug 2;41(15):2840-2
references: 12203503
references: J Am Chem Soc. 2009 Jun 17;131(23):8121-33
references: 19459632
references: J Am Chem Soc. 2010 Oct 27;132(42):14812-8
references: 20919707
references: ACS Chem Biol. 2006 Nov 21;1(10):644-8
references: 17175580
references: Angew Chem Int Ed Engl. 2008;47(15):2832-5
references: 18311742