CHEMBIOCHEM REVIEWS

DOI: 10.1002/cbic.201300079 Fluorescent Amino Acids: Modular Building Blocks for the Assembly of New Tools for Andrew T. Krueger[a] and Barbara Imperiali*[b]

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Fluorescence spectroscopy is a powerful tool for probing com- building blocks into a or of interest provides plex biological processes. The ubiquity of peptide–protein and the advantage of closely retaining native function and appear- protein–protein interactions in these processes has made them ance. The development of an array of fluorescent amino acids important targets for fluorescence labeling, and to allow sensi- with a variety of properties, such as environment sensitivity, tive readout of information concerning location, interactions chelation-enhanced fluorescence, and profluorescence, has al- with other biomolecules, and macromolecular dynamics. This lowed researchers to gain insights into biological processes, review describes recent advances in design, properties and including protein conformational changes, binding events, applications in the area of fluorescent amino acids (FlAAs). The enzyme activities, and protein trafficking and localization. ability to site-selectively incorporate fluorescent amino acid

1. Introduction their widespread adoption throughout the research communi- ty as a standard tool in protein imaging. The use of fluorescence as an analytical and diagnostic tool An alternative approach for covalently labeling target pro- has become increasingly important in research ranging from teins with fluorophores is to exploit the expression of fusion fundamental studies in molecular and cellular biology to bio- that, while not intrinsically fluorescent, can undergo logical chemistry, biophysics, and the applied fields of biotech- an enzymatic reaction to covalently self-label the encoded nology and medicine. Advances is the utility of fluorescence- fusion tag with a fluorophore, thereby imparting fluorescence based approaches have been accelerated by the development to the protein target. For example, Johnsson and co-workers of advanced techniques and instruments for monitoring and have pioneered methods that exploit fusing a protein of inter- imaging fluorescence responses in diverse and complex sys- est with DNA O6-alkylguanine alkyltransferase. This enzyme ef- tems. Fluorescence is highly sensitive, and, most importantly, ficiently reacts with benzylguanine derivatives, thereby allow- there are tremendous opportunities for expanding the struc- ing covalent attachment of synthesized fluorophore–benzyl- tural variety and for fine-tuning the photophysical properties guanine analogues to the protein of interest.[10–12] Such tags of molecular frameworks that exhibit fluorescence. Additional- have been commercialized (SNAP, CLIP tags) and are now used ly, methods for the integration of fluorophores into macromo- extensively in live-cell protein imaging. In addition, Cornish lecular targets are rapidly evolving. et al. have exploited and commercialized the interaction be- In the context of peptide and protein architecture, fluores- tween Escherichia coli dihydrofolate reductase (eDHFR) and tri- cence has proven useful in probing protein structure and dy- methoprim conjugates to tag proteins in live cells (Ligand- namics, including ligand-induced conformational change, pro- Link).[13] Another tag, based on a similar strategy, is the halo- tein–protein and protein–nucleic acid interaction, protein traf- alkane dehalogenase tag (HALO-Tag), which reacts covalently ficking, and enzyme activity.[1–3] The quest for insight into these with halogenated alkanes.[14] In principle, by using this ap- ubiquitous biological processes, coupled with the limited proach any synthesized fluorophore-modified halogenated range of naturally occurring fluorescent amino acids, has led to alkane can be ligated to the fused enzyme, thereby labeling the development of versatile approaches for integrating fluo- the target protein. rescent (or profluorescent) motifs into and pro- Despite their widespread use, a drawback of fused FPs and teins.[4,5] self-modifying enzymes is their size (often greater than Currently, one of the most broadly applied methods for co- 20 kDa), which can interfere with the function, localization, and valently labeling a protein with a fluorophore is to express the native interactions of the target protein. Smaller tags have target with a naturally occurring fluorescent protein. Indeed, been developed to address this limitation, for example, amino the discovery of green fluorescent protein (GFP)[6] and elucida- acid sequences that bind small molecules or metal ions and tion of the chemistry of the GFP chromophore by Tsien and become fluorescent (FlAsH, ReASH, RhoBo, etc.),[15–17] or are co-workers has led to substantial improvements, thereby pro- recognized by enzymes that can transfer a separate substrate viding an array of multicolored and analyte-responsive fluores- moiety that is fluorescent or can be orthogonally modified cent proteins (FPs) with increased photostability.[7–9] The suc- (e.g., biotin ligase, sortase, lipoic acid ligase, formylglycine-gen- cess and applicability of fluorescent proteins is underscored by erating enzyme).[18–21] Complementary to these approaches is the application of fluorescent a-amino acid analogues. These have the advantage of being relatively nonperturbing replacements for the native [a] Dr. A. T. Krueger Department of Biology, Department of Chemistry residues, thereby maintaining the overall native structure of Massachusetts Institute of Technology a target peptide or protein. In addition, the modularity of a- 77 Massachusetts Ave. 68-395 Cambridge, MA 02139 (USA) amino acid building blocks makes them versatile components [b] Prof. Dr. B. Imperiali in the protein assembly toolbox; thus, once syntheses and Department of Biology, Department of Chemistry methods for peptide and protein incorporation are developed, Massachusetts Institute of Technology 77 Massachusetts Ave. 68-380 Cambridge, MA 02139 (USA) new applications can be readily adopted. Finally, the increasing E-mail: [email protected] versatility of methods for selective incorporation of synthetic

2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 788 – 799 789 CHEMBIOCHEM REVIEWS www.chembiochem.org amino acids into peptides through solid phase peptide synthe- Also, the synthesis of the described FlAAs will not be discussed sis (SPPS) or into proteins by expressed protein ligation (EPL) in detail, but it is worth mentioning the wealth of advance- or unnatural amino acid mutagenesis (by using modified trans- ments in the synthesis of a-amino acids, including approaches lation systems) enables the tailored assembly of innumerable which exploit asymmetric reaction methodologies (e.g., alkyla- targets with precisely positioned fluorophores (Figure 1). Im- tion and hydrogenation), as well as methods that use starting portantly, the relatively small molecular framework of typical materials from the chiral pool and enzyme-catalyzed resolu- a-amino acids makes them readily amenable to modification tion.[22] through organic synthesis, thus providing a range of fluoro- phore options with a variety of properties. 3. Summary of Approaches Advances in solid-phase peptide synthesis (SPPS) have enabled 2. Scope of this Review incorporation of a wide variety of FlAAs into native peptide en- vironments. In particular, the relatively recent and more wide- This review will present an overview and analysis of the cur- spread use of Fmoc-based SPPS, together with an ever- rent literature on fluorescent a-amino acids (FlAAs) that have expanding range of commercially available and orthogonally been incorporated directly into peptides and proteins through protected amino acids, linker strategies, and coupling reagents chemical synthesis, protein semisynthesis, and protein biosyn- (which enable employment of milder reaction conditions) has thesis (by using protein machinery). In particular, further contributed to the robustness and versatility of SPPS we focus on studies in which the integrated FlAAs enabled with unnatural amino acids in general, and FlAAs in particular. new applications in the study of peptide and protein interac- The motivation for many efforts in this area has been the tions and activities. Although we will not discuss the extensive desire to develop residues with fluorescent side-chain func- applications of fluorescent labels that are conjugated to reac- tionalities that show more advantageous photophysical prop- tive naturally occurring amino acids (in particular cysteine), we erties and a broader range of applications than the naturally will additionally present new opportunities for the selective encoded fluorescent amino acid (tryptophan).[23] With the incorporation of fluorophores by using unnatural amino acids search for more diverse and powerful reporter properties, re- that can be labeled by bioorthogonal reaction chemistries. search groups took the approach of integrating known fluoro- phores into the side-chains of a-amino acids and investigating the utility of these new FlAAs in peptide and protein struc- Andrew T. Krueger received his B.S. in tures. As substrates for larger proteins or as components of chemistry from the University of Wis- binding domains, modified peptide sequences can now be consin-Madision. He obtained his Ph.D. readily synthesized for interrogating the known processes in under the guidance of Prof. Eric T. Kool which they participate, by fluorescence-based readout. FlAAs at Stanford University while exploring have also become invaluable in probing protein structure, the properties of an alternative genetic function, and interaction. For these studies, site-specific incor- system with size-expanded geometry. poration of fluorescent building blocks is crucial: one may Currently he is conducting postdoctor- want to examine the local dynamics of a particular region of al research with Prof. Barbara Imperiali a protein while avoiding perturbation of the protein’s native at MIT with an interest in chemical function (see Figure 1 for a summary of approaches for the probes for monitoring and biasing incorporation of FlAAs into proteins). protein–protein interactions in extra- With the merging of chemical synthesis and biology, the cellular signaling. ability to exploit nature’s machinery to modify the protein bio- polymer with non-natural amino acid surrogates has become Barbara Imperiali received her Ph.D. in viable. Several groups have incorporated FlAAs into full-length synthetic organic chemistry from MIT proteins to enable their detection and to interrogate biological in 1983 under the supervision of activities and interactions. In this respect, incorporation of the Satoru Masamune. She then carried FlAAs into a protein (for which synthesis is beyond the practi- out postdoctoral studies in the group cality of SPPS) is generally achieved one of two methods: of Robert H. Abeles at Brandeis Univer- 1) protein semisynthesis involving expressed protein ligation sity, and in 1986 started her independ- (EPL), in which a peptide fragment (synthesized by SPPS and ent career at Carnegie Mellon Universi- containing the unnatural amino acid) is ligated to a recombi- ty. In 1989 she became an assistant nantly expressed region of the desired protein (commonly by professor at Caltech, then returned in reaction of a C-terminal thioester with and N-terminal cys- 1999 to MIT, where she is now the teine), or 2) ribosomal machinery; here chemical acylation of Class of 1922 Professor of Biology and suppressor tRNAs is used or orthogonal tRNA/aminoacyl-tRNA Chemistry. Imperiali’s research uses chemical, physical, and bio- synthetase (AARS) systems are exploited. chemical approaches to the study of complex biological systems, Techniques based on exploiting an organism’s ribosomal including protein and cellular signaling networks. machinery can be applied in theory to any protein, and these

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Figure 1. Approaches for incorporating unnatural amino acids into proteins and peptides. can be particularly appropriate when the target is a large pro- a modest increase in quantum yield together with a significant tein that is not amenable to semisynthesis or difficult to site- blue shift of lem(max) (up to ~130 nm) when changing from selectively label with an exogenous reagent. Three main trans- aqueous protic to organic aprotic solvent environment.[35] An lation-based methods have become widespread: 1) exploiting a-amino acid building block containing this fluorophore, 6-(2- the promiscuity of wild-type synthetases and mutants,[24] 2) ex- dimethylaminonaphthoyl) alanine (DANA) was synthesized in pression with a chemically acylated tRNA that is then intro- 2002,[36] and has found use in peptides for monitoring pep- duced into the expression system containing mRNA that en- tide–protein and protein–protein interactions, where binding codes a nonsense 3-base or 4-base codon,[25–27] or 3) nonsense events often bring substrates into contact with more hydro- expression with an evolved orthogonal AARS pair that recog- phobic surfaces. An early study with DANA illustrated its po- nizes a nonsense codon (commonly UAG, or “amber”).[28,29] The tential by reporting the binding of a phosphoserine-containing final method has resulted in the generation of several evolved 14-3-3-binding peptide to the target 14-3-3z protein.[37] By tRNA/AARS pairs, and the incorporation of a variety of unnatu- basing the peptide design on the available structural informa- ral amino acids into proteins in E. coli, yeast, and mammalian tion, DANA was incorporated into the peptide in place of the cells.[30–34] Tyr residue that was localized at (À)2 relative to the phospho- Ser. The residue at the (À)2 position was shown by X-ray crys- tallography to be buried inside a hydrophobic pocket upon 4. Implementation binding the target protein.[38] An approximately fourfold fluo- rescence increase accompanied by a blue shift of the emission 4.1 Probing interactions with solvatochromic FlAAs maximum (from 522 to 501 nm) was observed upon binding One of the earliest extrinsic fluorophores that was applied for phosphopeptides to the 14-3-3z, relative to the unphosphory- monitoring biological processes was the highly environment- lated peptide. sensitive (solvatochromic) 6-propionyl-2-(dimethylamino)naph- Contemporarily, Cohen et al. employed the amber suppres- thalene (PRODAN, Scheme 1).[35] This fluorophore shows sion technique with a chemically charged tRNA bearing the

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FlAA in this family was based on 4-(N,N-dimethylamino)-phthali- mide (4-DMAP), a fluorophore which was known to exhibit robust changes in quantum yield

(~70-fold) and lem(max) shift (~ 100 nm) on changes in the sur- rounding media.[42] Furthermore, the corresponding FlAA, 4-(N,N- dimethylamino)-phthalimide pro- pionic acid (4-DAPA), had the ad- vantage of being a closer size- mimic of tyrosine, thus making it less likely to disrupt native pep- tide structure/interactions. In comparison with DANA, 4-DAPA gave an approximately sixfold enhancement in fluorescence emission upon binding the 14-3- 3z protein when placed at the phosphoSer(À2) Tyr position in the 14-3-3-binding peptide, with a blue shift in the emission maxi- mum from 570 to 531 nm.[43] In an elegant study comparing the 4-dimethylaminonaphthalimide FlAAs, 4-DAPA and the synthe- sized 6-N,N-dimethylamino-2,3- naphthalimide amino acid (6- DMNA) were made by incorpo- Scheme 1. Comparison of some environment-sensitive fluorophores and their solvachromatic properties. Reported fluorescence data are in the context of a Lys/Arg-rich peptide.[49] [a] Data reported for PRODAN-labeled cysteine. rating each of these FlAAs, and DnsA: Dansylalanine; NbdA: Nitrobenzooxadiazole alanine. used to track regulated cell-sur- face peptide-binding activity in primary human monocyte-de- same amino acid fluorophore (“Aladan”), to probe protein elec- rived dendritic cells. These compounds are antigenic peptides trostatics.[39] By first incorporating Aladan into selected sites of capable of binding to the major histocompatibility complex Kir2.1 and Shaker potassium channels, it was concluded that (MHC); upon binding class II MHCs, peptides containing these Aladan was not only compatible with the cellular biomachi- FlAAs showed substantial changes in fluorescence emission (> nery, but that substitutions in buried, aqueous, or lipid envi- 1000-fold for 6-DMNA, >35-fold for 4-DAPA) and fluorescence ronments would still allow proper folding and functioning of lifetime (11.8 vs. 4.8 ns; 7.3 vs. 2.2 ns (complex vs. free peptide) the protein. As a proof of utility of this FlAA to probe protein for 6-DMNA and 4-DAPA, respectively). In addition, in silico electrostatics, these researchers incorporated an Fmoc variant modeling of DANA, 6-DMNA, and 4-DAPA within crystal struc- of Aladan into the thermally stable IgG binding domain GB1 (~ tures of HLA-DR (a class II MHC) protein and the influenza he- 6 kDa) by SPPS. By observing steady-state and time-resolved magglutinin (HA) peptide showed the importance of shape fluorescence data of GB1 mutants with exposed or buried and size for hydrophobic binding of the amino acid, and po- Aladan, it was concluded that the interior of GB1 is polar and tentially of linker length (Figure 2). A crystal structure of the 4- electrostatically heterogeneous. The ability to selectively posi- DAPA-HA peptide-bound HLA-DR protein was also determined, tion Aladan—an amino acid with proportions similar to those thus confirming occupation by the 4-DAPA side chain of the of tryptophan—within specified regions of a protein makes it expected hydrophobic pocket.[44] These phthalimide- and a valuable amino acid for gaining insight into protein electro- naphthalimide-based FlAAs have also been incorporated into statics and how the variation in this property might affect pro- peptides recognizing PDZ and SH2 domains, thus illustrating tein function, interaction, and stability. their versatility in diverse applications.[45–48] To improve on the concept of environment-sensitive FlAA The success of the dimethylaminonaphthalimide family led building blocks, residues based on the dimethylaminophthali- to further exploration of their application as environment-sen- mide and dimethylaminonaphthalimide systems were later de- sitive FlAAs. For example, 4-(N,N-dimethylamino)naphthalimide veloped, with the goal of increasing the fluorescence changes amino acid (4-DMNA) was developed to complement 4-DAPA upon binding in target systems (Scheme 1).[40,41] The initial and 6-DMNA; this also yielded excellent improvements in sta-

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as well as mammalian cells. Bioorthogonal labeling is discussed further in Section 4.6. Another class of environment-sensitive amino acid building blocks based on the dansyl fluorophore has been exploited for probing protein interactions and protein unfolding. Two amino acids, dansylalanine and dansylysine, show a twofold increase Figure 2. DANA, 4-DAPA, and 6-DMNA modeled in silico into the hydropho- of quantum yield and ~50 nm hypsochromic shift upon chang- bic P1 pocket, in place of tyrosine in the crystal structure of the DR1–HA ing from polar to nonpolar environments.[54] The sensitivity of peptide complex. Reprinted with permission from ref. [44]. Copyright 2007 these fluorophores makes them ideal tools for studying struc- Nature Publishing Group. tural dynamics in proteins: they can be site-specifically posi- tioned in the protein without incurring significant structural perturbation. The Schultz lab has shown, by incorporation into bility towards hydrolysis, as well as ease of synthesis.[49] When the human superoxide dismutase (hSOD) protein in yeast by integrated at position 8 in the M13 peptide (which binds using an evolved tRNA/leucyl-tRNA-synthetase pair, dansylala- avidly to calcium-activated calmodulin), 4-DMNA exhibited a re- nine fluorescence intensity can be monitored in the presence markable fluorescence enhancement. Upon binding, a signifi- of guanidinium hydrochloride to monitor protein unfolding.[55] cant enhancement (~106-fold) in emission intensity over back- More recently, a tRNA/tyrosyl-tRNA synthetase pair was used ground was observed, as well as a blue shift from 550 nm to to incorporate a PRODAN amino acid into the same protein to 505 nm. This change compares favorably with other fluoro- study conformational changes.[56] phores in the dimethylaminophthalimide class: ~26-fold (4- In a recent study involving mammalian cells, dansylalanine DAPA) and ~19-fold (6-DMNA) in the same context. Additional- has also been incorporated into neural stem cells by using an ly, relative to the established solvatochromic FlAA derivatives orthogonal tyrosyl tRNA/TyrRS-synthetase pair from E. coli.By (PRODAN (fourfold), dansyl (tenfold) and NBD (threefold)), the using a lentiviral vector delivery system (thus allowing efficient dimethylaminonaphthalimide family exhibited superior signal- expression of the orthogonal biomachinery throughout cell dif- to-background ratios in these studies (Scheme 1). Further syn- ferentiation), Wang and co-workers site-selectively incorporat- thetic efforts have yielded several thiol-reactive 4-DMN biocon- ed dansylalanine into the voltage sensitive domain (VSD) of jugation reagents with varying linker lengths, thus allowing the Ciona intestinalis voltage-sensitive phosphatase in HCN- further analysis and development of solvatochromic peptides A94 cells. Dansylalanine was able to report, through environ- in general protein-labeling experiments.[50] ment-sensitive fluorescence, the conformational change of this The solvatochromatic 4-DMNA has recently been incorporat- domain in response to membrane polarization.[57] Dansyl ed into a sensor domain for the Rho GTPase Cdc42 by protein amino acids have also been used in a scaffold for detecting semisynthesis.[51] This domain, based on a Cdc42/Rac interac- concentrations of Hg2+ that approach toxic levels, as well as tive binding (CRIB) domain of the Wiskott–Aldrich syndrome Zn2+ .[58–60] protein (WASP) was assembled by native chemical ligation be- Additionally, coumarins (known to exhibit sensitivity to sol- tween the C-terminal peptide fragment and the recombinant vent polarity and pH) have seen use in studying protein dy- N-terminal WASP domain (residues 230–268) expressed as namics. One example of direct coumarin incorporation into a GB1 fusion. Experiments with the sensor, Cdc42, and GTP-gS proteins by the Schultz lab involved the fluorescent amino (a nonhydrolyzable analogue of GTP to activate the sensor) acid l-(7-hydroxycoumarin-4-yl)ethylglycine (7HC). For incorpo- showed a greater than 17-fold increase in fluorescence emis- ration of 7HC, a mutant Methanococcus jannaschii tyrosyl sion, as well as a hypsochromic shift from 534 nm to 514 nm amber suppressor tRNA/tyrosyl-tRNA-synthetase pair was (relative to GDP-bound inactive Cdc42). In addition to semisyn- evolved, thereby incorporating the coumarin amino acid upon thesis, native Cdc42 was mutated at residue 271 to a cysteine recognition of a TAG stop codon. After incorporation into the (F271C) based on a previous sensor design,[52] and the mutant protein holomyoglobin, 7HC fluorescence was monitored and was labeled with various 4-DMN-based thiol-reactive agents correlated to helix unfolding in various concentrations of urea, with different linker lengths. These sensors exhibited fluores- thereby suggesting the utility of coumarin as a probe for local cence changes from 11- to 32-fold, thus underscoring the im- protein conformational changes.[61] portance in systematic screening, to guide the placement of Overall, solvatochromic fluorophores have become invalua- environment-sensitive fluorophores and their utility for report- ble for the interrogation of protein–protein interactions, and, ing on protein interactions. The 4-DMN fluorophore has also although notable examples are described here, there exist seen success as an alkyne analogue. In an elegant sensor ex- several environment-sensitive fluorophore scaffolds yet to be ample that utilized a pyrrolysyl-tRNA-synthetase (PylRS)/tRNA adapted to FlAA design.[62,63] A more recent example reported pair, Chen and co-workers incorporated an azidocyclopentyl by Klymchenko et al. involved the synthesis of an FlAA trypto- lysine at various sites of the pH-responsive HdeA protein.[53] phan mimic based on the two-color fluorescent moiety 2-(2- The researchers were able to demonstrate not only that HdeA furyl)-3-hydroxychromone (FHC). Not only did they find it to be mutants could be labeled in vivo with an alkyne variant of 4- a relatively conservative substitute for a tryptophan residue, DMN by CuACC labeling, but also that substantial fluorescence but also, after incorporation into the nucleocapsid (NC) protein increases could be visually observed upon pH change, in E. coli of HIV-1, they observed the FHC FlAA to be highly sensitive to

2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 788 – 799 793 CHEMBIOCHEM REVIEWS www.chembiochem.org hydration in polar environments, thus allowing investigation of the assay, a quencher molecule forms noncovalent interactions peptide–oligonucleotide complexes.[64] with the peptide substrate and the incorporated pyrene moiety. On addition of ATP to PKA and the phosphoserine- binding domain 14-3-3t, pyrene fluorescence is restored be- 4.2 Investigating by disrupting quenching cause of displacement of the quencher by the phosphoserine- of a pyrene FlAA binding domain (Scheme 2). After screening an array of An application of FlAAs from the Lawrence laboratory focused quencher molecules against peptides with the pyrene FlAA at on exploiting fluorescence enhancements of a pyrene-based various positions, a very effective probe was found when using amino acid (enhancement occurs after tyrosine phosphoryla- Rose Bengal or Aniline Blue as the quencher. With this design tion), to report on protein tyrosine kinase activity (Scheme 2). (“Deep Quench”), fluorescence increases up to 64-fold were observed.[66] Wang and co-workers have also exploited the difference in environment between phosphorylated and nonphosphorylated states by incorporating 7HC into the STAT3 (signal transducer and activator of transcription 3) protein, which is phosphorylat- ed at Tyr705. In the context of stimulated cell lysate, up to 15- fold fluorescence enhancement was observed relative to the (nonphosphorylated) Y705F mutant.[67]

4.3 Interaction of FlAAs with metal ions: lanthanide-binding tags Peptides incorporating FlAAs that are capable of interacting with metal ions have found use with lanthanide-binding pep- tides. In this context, a specific peptide scaffold together with lanthanide metal-binding properties of encoded (as well as synthetic) amino acids can be exploited to great advantage for long-wavelength protein imaging. Recently, these lanthanide-binding tags (LBTs) have been in- troduced as genetically encoded peptide tags that bind and sensitize lanthanide cations, thus imparting luminescence to a target protein upon lanthanide binding. As previously men- tioned, the size of encoded fluorescent protein tags can be Scheme 2. Pyrene FlAAs (left) and the kinase sensor design. Reprinted with problematic, as the added bulk can disrupt the native protein permission from ref. [66]. Copyright 2006 American Chemical Society. structure or function. LBTs are relatively small (17–20 amino acids), and bind the lanthanide series trivalent cations with

In this study, pyrene fluorescence (lem = 375 nm) was high affinity (nanomolar Kd) and selectivity, relative to all phys- quenched when the pyrene was involved in p–p stacking with iologically relevant divalent cations. The version of this short a proximal tyrosine residue; when this stacking was disrupted tag that comprises only naturally encoded amino acids in- due to tyrosine phosphorylation, quenching was abrogated. cludes a strategically positioned tryptophan residue, which Amino acid derivatives of pyrene with different side-chain sensitizes bound Tb3+, thus leading to luminescence at linker lengths (l-2,3-diaminopropionic acid (Dap), and l-2,4-di- 544 nm (Figure 3).[68,69] As lanthanide luminescence emission is aminobutanoic acid (Dab)) were prepared and incorporated in the visible region and the luminescence lifetimes are long into tyrosine-containing peptide sequences on either side of (milliseconds), these tags are very well suited to application in the phosphorylated tyrosine. The selected peptides were de- in vitro assays and in diagnostics. Additionally, LBTs have been signed to be substrates for the Src tyrosine kinase family, genetically encoded into, or conjugated to, a variety of pro- which phosphorylate tyrosines in this context. Incubation of teins for use in monitoring protein–peptide interactions by lu- the pyrene-FlAA-containing peptides with Src yielded increases minescence resonance energy transfer (LRET).[69] in fluorescence intensity over time (1.8- to 5-fold).[65] The in- Although the luminescence and low background of Tb3+ crease in fluorescence was attributed to disruption of the p–p -bound LBTs is very interesting, excitation of the tryptophan interaction between tyrosine and pyrene, and was corroborat- sensitizer requires UV light, which is not ideal for cellular stud- ed by 2D NOE NMR data showing marked differences in the ies, and the emission wavelengths of the indole are only com- observed NOEs between the pyrene and tyrosine protons in patible with terbium sensitization (for which the emission nonphosphorylated and phosphorylated states.[65] wavelength may not suitable for all potential applications). To The pyrene amino acid approach was further applied in address this shortcoming, two unnatural FlAAs were targeted a protein kinase detection assay that used a pyrene-containing for incorporation into LBTs, based on the fluorophores carbos- peptide substrate for protein kinase A (PKA, a serine kinase). In tyril 124 (cs124) and acridone (Acd), which exhibit emission

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Figure 3. Exploiting acridonyl (Acd) and carbostyril 124 (cs124) FlAAs in lanthanide sensitization for imparting luminescence to proteins with lanthanide bind- ing tags (LBTs).

properties compatible with the sensitization of europium ions fonamido-oxime (Sox; previously applied to sense divalent (Figure 3).[70] The lower energy excitation properties of these zinc),[71] was synthesized initially as an alanine variant (Sox), FlAAs (337 and 370 nm for cs124 and Acd, respectively), as and then as a cysteine variant (C-Sox, Scheme 3) to improve well as known sensitization of europium (longer emission recognition and specificity. These FlAAs can be incorporated wavelength) make these amino acids desirable. In one study, into kinase-recognition peptide motifs and can report on cs124 and Acd were incorporated into LBTs by SPPS, and were serine/threonine or tyrosine phosphorylation events through shown not only to sensitize at longer wavelengths but also to enhanced fluorescence emission in the presence of Mg2+.[72] sensitize Eu3+, which luminesces at a longer wavelength than The emission signal (~485 nm) arises from the formation of Tb3+ (620 vs. 550 nm). After observing that LBTs with synthetic a chelate between the 8-hydroxyquinoline and the installed FlAAs also showed nanomolar binding with lanthanides, a syn- phosphoryl group, which shows an affinity for Mg2+ 15 to 20 thesized tag was conjugated to the Crk SH2 domain by native times greater than that of the nonphosphorylated hydroxyl chemical ligation, thus potentially making it an effective tool group. Peptide probes containing Sox/C-Sox amino acids for for monitoring protein interactions and trafficking in vivo.[70] a variety of kinases have been synthesized and implemented for kinase measurements with recombinant enzymes and in unfractionated cell lysates, and have yielded fluorescence en- 4.4 Sulfonamido-oxine (Sox) amino acids, and chelation-en- hancements of approximately two- to 12-fold.[72–74] It was also hanced fluorescence found that replacement of the sulfonamide moiety on C-Sox Developing probes for kinase activity has attracted much interest because of the crucial roles played by kinases in cellular signaling. The sensitivity of fluores- cence-based output has afforded FlAAs an important role in the development of kinase probes and activity assays, as illustrated by the application of pyrene- based FlAAs (Section 4.2). Although several FlAAs rely on changes in the environment to elicit a response to kinase activity, the Imperiali group has developed a fluorescent amino acid for which the emission is derived from metal chelation (“chelation-enhanced fluorescence”, CHEF; Figure 4). The chromophore, sul- Scheme 3. Sulfonamido oxine and 8-hydroxyquinoline (Oxn) FlAAs.

Figure 4. Kinase activity sensing mechanism of FlAAs by chelation-enhanced fluorescence (CHEF; bottom).

2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 788 – 799 795 CHEMBIOCHEM REVIEWS www.chembiochem.org with various substituted triazole groups (installed by click By using an a-naphthyl group as a fluorescence donor and chemistry) gave rise to a multitude of novel Mg2+ -chelating a dansyl fluorophore as the acceptor, energy transfer as a func- hydroxyquinoline FlAAs with fluorescence emission maxima tion of distance was observed, thereby demonstrating the po- that are shifted by up to 40 nm.[75] These new click derivatives tential of monitoring local and specific protein conformational were incorporated into peptide substrates for the study of changes by fluorescence. MAPK-activated protein kinase 2 (MK2), and were shown to be Since 1967, methods to incorporate unnatural amino acids as efficiently turned over as C-Sox-containing peptides.[75] have advanced considerably: various unnatural entities have The ability to selectively target members of the MAK kinase been used within peptides and proteins. For example, acri- family, including ERK1/2 and p38a, that are known to be dis- done-based FlAAs employing acrydonylalanine (which was regulated in disease (and thus important therapeutic targets) used as an LBT Eu3+ sensitizer) and benzoacridone (“badAla” relies on recognition elements that involve extensive protein– FlAA) have been incorporated into peptides for use as a FRET protein interactions—more complicated than those for the pair, to report on protease activity of caspase-3.[80] In an im- short peptide sequences on which many kinase probes have pressive recent example by Hohsaka and co-workers, amino been based. In this context, a semisynthetic approach involv- acid derivatives of BODIPY fluorophores (BODIPYFL) were in- ing conjugation (by native chemical ligation) of a Sox-contain- corporated into calmodulin by the tRNA chemical-acylation ing peptide containing an ERK1/2 consensus recognition se- approach pioneered by Sisido, with a four-base anticodon quence to a recombinant N-terminal pointed (PNT) domain of tRNA.[25,27,81] After a screen of several BODIPYFL amino acid-acy- transcription factor Ets-1. The added domain (~11 kDa) allows lated tRNAs in a cell-free translation system with streptavidin specific docking of ERK1/2 to the C-Sox-based sensing module, mRNA containing a four-base codon insert, it was determined with enhanced affinity over that of the shorter Thr-Pro consen- that aminophenylalanine (AF) derivatives were most efficiently sus peptide motif. This Sox-PNT sensor was capable of specifi- incorporated. When applying this method to the calmodulin cally detecting ERK1/2 activity in cell lysate, with a four- to ten- protein, two different BODIPYFL-AF amino acids (BODIPYFL-AF, fold increase in fluorescence readout relative to the more pro- BODIPY558-AF, Scheme 4) were simultaneously incorporated miscuous C-Sox-peptide.[76] Based on this scaffold approach (at the N and C termini), by using two different four-base and the benefits of the C-Sox amino acid, further sensors for codons. Fluorescence spectra of the double-labeled mutant specific kinases (p38, JNK, and other members of the MAPK with excitation at 490 nm showed a decrease in donor (BODI- family) have been developed, thereby providing insight into PYFL-AF) fluorescence at 515 nm and an increase in acceptor the modulation of kinase activities in diseased tissue.[77] The fluorescence at 575 nm, thus indicating FRET. Further calmodu- Sox-derived kinase sensor has been commercialized under the lin mutants with varied donor positions indicated that FRET tradename “Omnia”. could be used to monitor conformational changes of calmodu- Chelating FlAAs have found use in other applications as lin upon Ca2+-dependent peptide binding.[81] well. For example, Wang and co-workers have reported the synthesis and incorporation of the metal-chelating 2-amino-3- (8-hydroxyquinoline-3-yl)propanoic acid (HQ-Ala) into proteins, not only as a CHEF fluorescent reporter, but also as a handle for the introduction of a heavy metal ion for X-ray crystallogra- phy phase determination.[78] By using an orthogonal tyrosyl amber suppressor tRNA/tRNA-synthetase (tRNA/RS) pair, incor- poration of HQ-Ala into the Z-domain of Staphylococcal pro- tein A (SpA) resulted in a protein that showed a significant increase in fluorescence (at ~540 nm) upon excitation at 400 nm, when titrated with as little as 10 mm Zn2+. HQ-Ala was also introduced into the protein O-acetylserine sulfhydrylase, incubated with Zn2+, and crystallized. Solving of the crystal structure showed no differences between the wild-type struc- ture and the Zn-coordinated HQ-Ala mutant, thus suggesting this as potential alternative to other techniques, such as sele- nomethionine incorporation, for providing phase information.

4.5 FlAAs employed in FRET experiments Fluorophores and FlAAs have experienced much success as di- agnostic and basic-science tools because of their sensitivity and ease of readout. Additional information can be extracted by using more than one FlAA, for example in FRET. The use of FLAAs in FRET was first demonstrated by Stryer and Haugland Scheme 4. FlAAs with BODIPY fluorophores and unnatural AAs (orthogonally in 1967 in the context of poly-l-proline oligomeric peptides.[79] labeled) used for FRET monitoring of conformational changes in calmodulin.

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More recently, a study by Park et al. demonstrated simulta- conjugated cyclooctyne for imaging mammalian cells.[87] Addi- neous incorporation of the unnatural amino acids p-acetylphe- tional studies by the Tirrell lab have involved MetRS mutants nylalanine (AcF) and alkynyllysine (AlK) by the previously re- that accept unnatural amino acids such as azidonorleucine ported pair and the newly evolved AlKtRNA/RS pair.[82] The (NLL).[88] By using cells bearing a plasmid copy of NLL-MetRS, it strategy allowed orthogonal dual labeling of a calmodulin-AcF- was demonstrated that only proteins made in these cells could AlK mutant, with hydrazide and azide variants of Cy3 and Cy5 be tagged with affinity reagents (such as biotin-FLAG-alkyne) dyes, respectively. FRET was observed (efficiency 0.85), thereby or fluorescent dyes (dimethylaminocoumarin alkyne, TAMRA- indicating conformational changes in both ensemble and alkyne) and imaged or enriched in complex cellular mixtures single-molecule experiments upon addition of M13 peptide (Scheme 5).[88] Recently, additional MetRS mutant-screening and Ca2+ . Because of the commercial availability of both the strategies have been employed to further improve efficiency amino acids and dyes, this approach (as well as the dual-label- and selectivity for NLL.[89] Overall, the simplicity of the expres- ing approach in general) is likely to find widespread use in sion protocols for incorporating these analogues, as well as studying protein dynamics.[82–84] the useful yields of modified protein, make this a suitable method for fluorescent protein labeling, both in vitro and in vivo. 4.6 Fluorescent coumarins, alkyne analogues, and bio- Lemke et al. demonstrated that 3-azido-7-hydroxycoumarin orthogonal labeling can also be incorporated into proteins site-specifically with Tirrell and co-workers have taken advantage of the ability of metal-free click chemistry, by using an unnatural ring-strained- some wild-type aminoacyl tRNA synthetases to charge tRNAs conjugated lysine amino acid based on cyclooctyne.[90,91] To with unnatural amino acid substrates similar in shape to their accomplish this, they employed a pyrrolysine Methanosarcina natural counterparts. With this relatively simple approach, they incorporated several amino acid analogues of methionine with reasonable efficiency by using the less-discriminating methion- yl-tRNA synthetase (MetRS) from M. jannaschii in E. coli.[85] One of these methionine analogues, homopropargylglycine, has re- ceived attention because of its efficient incorporation and abili- ty to undergo click chemistry, thus essentially providing a handle for site-selective modifi- cation of a protein. In an elegant example of selective protein la- beling in bacterial cells, Beatty et al. showed that in methionine auxotrophic E. coli cells contain- ing a plasmid expressing the Barstar protein (two Met sites), click chemistry with the profluor- escent dye 3-azido-7-hydroxy- coumarin produced fluorescent enhancement only in the alkyn- yl-labeled protein-containing cells.[86] This specific, in vivo la- beling approach was also em- ployed with an alkynyl analogue of phenylalanine (ethynylpheny- lalanine), by using an auxotro- phic strain that overexpressed a phenylalanyl-tRNA synthetase mutant. A later study employed Scheme 5. Cell-selective proteome labeling with incorporated azidonorleucine (NLL) by using NLL-MetRS and bio- the same method with azidoho- orthogonal chemistry with biotin-FLAG-alkyne, profluorescent dimethylaminocoumarin, or TAMRA-alkyne. Reprint- moalanine (Aha) and a BODIPY- ed with permission from ref. [88]. Copyright 2009 Nature Publishing Group.

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underscores the power of fluo- rescent amino acids in real-time imaging and the importance of imparting fluorescence to a pro- tein without perturbing its native function. Even more re- cently, Schuman and co-workers were able to show that zebrafish larvae could metabolically incor- porate azidohomoalanine into newly synthesized protein, which then could be modified Scheme 6. Ring-strained unnatural amino acids (cyclooctyne, norbornene) for bioorthogonal metal-free click chemistry with profluorescent substrates (coumarin azide, tetrazine-TAMRA-X). with an Alexa Fluor 488-labeled alkyne by using click chemistry and then imaged.[98] As chemis- barkeri/Methanosarcina mazei tRNA/pylRS pair evolved by Chin try and biology become further intertwined, FlAAs will contin- and co-workers.[92,93] Lemke evolved the pair to accommodate ue to be at the forefront in aiding researchers to gain insights the extra bulk of the cyclooctyne amino acid, and used it to into fundamental questions concerning life’s essential biologi- incorporate the cyclooctyne moiety into the mCherry protein cal functions involving protein interactions, recognition, and (Scheme 6). Labeling with the fluorogenic coumarin azide con- synthesis. firmed incorporation of the cyclooctyne amino acid, thus sug- gesting the potential to tag any protein with a variety of Keywords: bioorthogonal conjugation · fluorescence · cargos in vivo. Most recently, Chin has evolved this tRNA/pylRS protein–protein interactions · solvatochromic fluorophores · pair to incorporate a norbornene-linked amino acid into sfGFP, synthetic amino acids myoglobin, and T7 lysozyme.[93] This study showed that nor- bornene is able to undergo a highly efficient metal-free click reaction with a variety of tetrazine–fluorophore conjugates [1] R. W. Sinkeldam, N. J. Greco, Y. Tor, Chem. Rev. 2010, 110, 2579. [2] L. D. Lavis, R. T. Raines, ACS Chem. Biol. 2008, 3, 142. (TAMRA, BODIPY-TMR, BODIPY-FL), all of which show quenched [3] A. R. Katritzky, T. Narindoshvili, Org. Biomol. Chem. 2009, 7, 627. fluorescence until clicked to norbornene (Scheme 6). By encod- [4] D. C. Dieterich, Curr. Opin. Neurobiol. 2010, 20, 623. ing the norbornene amino acid into an EGFR-GFP fusion, [5] K. M. Marks, G. P. Nolan, Nat. Methods 2006, 3, 591. metal-free labeling with a TMR-conjugated tetrazine on a mam- [6] O. Shimomura, F. H. Johnson, Y. J. Saiga, J. Cell. Comp. Physiol. 1962, 59, 223. malian cell surface could be visualized. Ongoing research in [7] R. Y. Tsien, Annu. Rev. Biochem. 1998, 67, 509. bioorthogonal protein labeling has seen further development [8] R. Heim, A. B. Cubitt, R. Y. Tsien, Nature 1995, 373, 663. of unnatural amino acids with enhanced reaction kinetics.[94–96] [9] A. B. Cubitt, L. A. Woollenweber, R. Heim, Methods Cell Biol. 1998, 58, 19. [10] A. Gautier, A. Juillerat, C. Heinis, I. R. CorrÞa, M. Kindermann, F. Beaufils, 5. Summary/Outlook K. Johnsson, Chem. Biol. 2008, 15, 128. [11] A. Keppler, H. Pick, C. Arrivoli, H. Vogel, K. Johnsson, Proc. Natl. Acad. This review of FlAAs illustrates the importance of having a vast Sci. USA 2004, 101, 9955. 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Am. proved fluorophore scaffold designs and advanced methods Chem. Soc. 2009, 131, 438. for multiple-FlAA incorporation, but also greater integration of [18] I. Chen, M. Howarth, W. Lin, A. Y. Ting, Nat. Methods 2005, 2, 99. [19] M. Fernndez-Surez, H. Baruah, L. Martnez-Hernndez, K. T. Xie, J. M. imaging techniques, to observe dynamic yet delicate cellular Baskin, C. R. Bertozzi, A. Y. Ting, Nat. Biotechnol. 2007, 25, 1483. functions in more complex systems. For example, Chapman [20] P. Wu, W. Shui, B. L. Carlson, N. Hu, D. Rabuka, J. Lee, C. R. Bertozzi, Proc. et al. have incorporated 7-hydroxycoumarin amino acid into Natl. Acad. Sci. USA 2009, 106, 3000. the bacterial tubulin homologue cytoskeleton protein FtsZ, to [21] M. W.-L. Popp, H. L. Ploegh, Angew. Chem. 2011, 123, 5128; Angew. Chem. Int. Ed. 2011, 50, 5024. [97] visualize subcellular localization. FtsZ assembles into a con- [22] Asymmetric Synthesis and Applications of a-Amino Acids (Eds.: V. A. 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