Chemistry Is Dead. Long Live Chemistry! - Biochemistry (ACS

Subscriber access provided by Kent State University Libraries

Perspective

Chemistry is dead. Long live chemistry! Luke D. Lavis Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00529 • Publication Date (Web): 13 Jul 2017 Downloaded from http://pubs.acs.org on July 14, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Chemistry is dead. Long live chemistry! Luke D. Lavis

Janelia Research Campus, Howard Hughes Medical Institute 19700 Helix Drive, Ashburn, Virginia 20147 USA email: [email protected]

Invited Perspective for the “Seeing into Cells” issue in Biochemistry

ABSTRACT: Chemistry, once king of fluorescence microscopy, was usurped by the field of fluorescent proteins. The increased demands of modern microscopy techniques on the “photon budget” requires better and brighter fluorophores. Here, we review the recent advances in biochemistry, protein engineering, and organic synthesis that have allowed a triumphant return of chemical dyes to modern biological imaging.

1

ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The ability to visualize the structure and dynamics of molecules inside cells is an essential part of unraveling fundamental biological processes. Fluorescence microscopy is uniquely suited for such efforts, given the high sensitivity of fluorescence measurements and the compatibility with live cells, tissues, and animals. The applications of fluorescence microscopy are broad, ranging from tracking of individual proteins molecules in single cells to measuring neuronal activity over vast swaths of tissue in vivo (1, 2). All of these experiments rely on fluorescent dyes and a wide variety of fluorophores have emerged as useful labels for microscopy: organic fluorophores, fluorescent proteins, lanthanide chelates, and quantum dots. Here, we review the relatively old field of small molecule fluorescent dyes, which is undergoing a renaissance to meet the needs of 21st century biochemistry and biology (3, 4). The story of fluorescence and fluorescence microscopy is one of chemistry. Fluorescence was first observed and elucidated using the fluorescent natural product quinine (1, Figure 1A). Quinine was also the synthetic target of William Perkin, who instead accidentally made the first synthetic dye, mauvine (2), in 1856. This discovery of synthetic colored compounds set off a flurry of activity and the majority of the classic fluorophores were first synthesized in the subsequent decades, including coumarins (e.g., umbelliferone, 3, ca. 1884), fluorescein (4, ca. 1871), rhodamines (e.g., tetramethylrhodamine, TMR, 5, ca. 1887), phenoxazines (e.g., resorufin, 6, ca. 1903), and cyanines (e.g., hexamethylindocyanine, 7, ca. 1924; Figure 1B) (4). These classic dyes played an important role in the development of fluorescence-based technologies such as fluorescence microscopy. For example, fluorescein (4), sparked the field of immunofluorescence, where the amine-reactive fluorescein isothiocyanate (FITC) derivative was developed specifically for preparing fluorescent antibody bioconjugates (5).

2


Page 2 of 21

Page 3 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

The growth of fluorescence microscopy and the development of other labeling strategies (e.g., fluorescence in situ hybridization, FISH) demanded more sophisticated biomolecule labels. Classic dyes such as 3–7 have several problems: poor photostability, low brightness, and relatively high hydrophobicity. Attachment of these dyes to biomolecules often had detrimental effects on the binding of an antibody or oligonucleotide in cells. To improve the performance of small molecule labels, chemists introduced different functionality, such as sulfonates to increase solubility and rigidification motifs to improve brightness and photostability. This resulted in a panel of excellent and still-widely used dyes, such as the ‘Alexa Fluor’ dyes (8–11) developed by Molecular Probes (6) and ‘CyDyes’ (e.g., Cy5, 12) developed by Alan Waggoner (7) (Figure 1C). In addition to these improved biomolecule labels, other synthetic fluorophore reagents were being developed for live cell microscopy including stains for specific organelles, fluorescent ion indicators, and photoactivatable dyes for advanced imaging experiments (3, 4). Chemistry was king… …and then everything changed. The discovery of green fluorescent protein (GFP) sparked a revolution in biological imaging (8). Cell biologists were no longer beholden to chemists and their relatively expensive synthetic fluorophores. Easily replicated DNA plasmids replaced vials of exhaustible fluorophores and cells proved perfectly capable of synthesizing fluorophore fusions on their own. The general utility of fluorescent protein fusions superseded many specific organelle markers and these labels could be multiplexed with established and emerging small molecule lipid and nucleic acid stains. Subsequent advances in fluorescent proteins have replicated many of the properties once exclusive to small-molecules, such as red-shifted spectra, ion sensitivity, and photoactivation (2, 8, 9). These important advances lead to an obvious question: In this age of GFP and its ilk, is the field of fluorophore chemistry dead?

3


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Chemical structures of early and optimized fluorophores. (A) The first small molecule fluorophore, quinine (1) and the first synthetic dye, mauvine (2). (B) Classic fluorophores: umbelliferone (3), fluorescein (4), tetramethylrhodamine (5), resorufin (6), and hexamethylindocyanine (7). (C) Examples of optimized Alexa Fluor (8– 11) and CyDye (12) fluorophores.

4


Page 4 of 21

Page 5 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Although fluorescent proteins will remain an essential part of the fluorescence microscopy toolkit, small molecule dyes cannot be ignored. There has been a resurgence in chemical fluorophores for imaging in the last decade, driven largely by advances in fluorescence microscopy techniques, which place increased demands on the “photon budget”. The photon budget equals the number of fluorophores in a given sample multiplied by the number of photons emitted by each fluorophore before photobleaching. The amount of information one can extract from a biological sample is wholly dependent on the photon budget; pushing the frontiers of fluorescence microscopy often requires more photons. For example, super-resolution localization microscopy places a large burden on the photon budget, as the number of photons emitted by the fluorophore determines how precisely individual molecules can be localized (10). Likewise, moving from transient overexpression of protein fusions to gene-edited cells with endogenous expression levels typically decreases the number of fluorophores, also compromising the photon budget. The importance of this parameter in imaging has led to the development of new microscope geometries expressly designed to capture more photons (3). In addition to optical physics methods, chemistry can also be used to improve the photon budget. Chemical fluorophores can be substantially brighter and more photostable than fluorescent proteins, providing a straightforward way to increase the number of photons emitted by a sample. Of course, ‘regressing’ to small molecule dyes seems unpleasant at best; classic methods to get otherwise cell-impermanent fluorescent conjugates in live cells involve tedious microinjection, electroporation, or bead loading. Fortunately, over the last 20 years, clever chemists and biochemists have developed techniques to make the labeling chemistry easier and more functional in complex biological environments such as live cells and tissues (Figure 2).

5


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

These flexible strategies allow the excellent photophysics of chemical dyes to be combined with the genetic specificity of fluorescent proteins. The majority of in-cell labeling strategies have two parts: (i) a genetically encoded ‘tag’ expressed as a fusion with your favorite protein and (ii) a synthetic fluorophore-containing ‘ligand’ that binds to the tag (11, 12). Instead of introducing an entire fluorescent bioconjugate into cells, one is only faced with getting a small molecule across the lipid bilayer. As with most useful ideas in biological imaging, the initial breakthrough for in-cell labeling techniques was provided by Roger Tsien, who showed that the selective interaction between a bisarsenical dye ligand (e.g., FlAsH, 13) and a short genetically encoded tetracysteine (Cys4) peptide tag could be used to label proteins in cells (Figure 2A) (13). This idea sparked other strategies to label biomolecules in cells, including the self-labeling tags such as the SNAP-tag (14) and HaloTag (15) proteins. These widely used systems consist of a genetically encoded enzyme variant tag that reacts specifically and irreversibly with a small substrate ligand motif attached to a fluorophore such as the TMR-HaloTag ligand 14 (Figure 2B). Another approach involves the use of engineered ligases, such as lipoic acid ligase, that catalyze the covalent attachment of a fluorophore ligand such as resorufin derivative 15 to a small peptide tag (Figure 2C) (16). Nonnatural amino acids such as coumarin 16 can be incorporated into a protein structure using exogenous tRNA and tRNA synthetases to place fluorophores at specific sites (Figure 1D) (11). Engineered binding motifs such as the antibody-based fluorogen activating proteins (FAPs) bind and enhance small molecule fluorogens such as the Malachite Green derivative 17 (Figure 2E) (17). Finally, new cellular stains can be created by attachment of a dye to a molecular species with high affinity for an endogenous molecular target such as paclitaxel–fluorescein conjugate 18 (Figure 2F) (18, 19).

6


Page 6 of 21

Page 7 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 2. Cellular labeling strategies using small-molecule fluorophores. (A) Labeling of tetracysteinecontaining peptides with bisarsenical 13. (B) Attachment of HaloTag ligand 14 to self-labeling HaloTag protein fusion. (C) Amidation of lipoic acid ligase tag with ligand 15 catalyzed by engineered lipoic acid ligase variant. (D) Use of non-natural amino acid technology to incorporate coumarin amino acid 16 into proteins. (E) Antibody-based fluorogen activating proteins (FAPs) bind Malachite Green derivatives such as 17. (F). Cellluar stains such as paclitaxel 18 bind endogenous proteins in a ‘GMO-free’ strategy. Light grey: protein of interest; Dark grey: exogenous tag.

7


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

All of these labeling strategies have trade-offs between the size of the genetically encoded tag, the speed and selectivity of the fluorophore attachment, the brightness of the resulting conjugate, and the complexity of the system. The bisarsenical system utilizes a small tag but the ligands such as FlAsH (13) label slowly and exhibit relatively high background staining in cells, presumably due to reaction with other endogenous cysteine residues. Self-labeling tags exhibit rapid and selective labeling with cell-permeable ligands such as 14, but are typically as large or larger than GFP, which can alter the biological activity of the fusion protein. Ligases combine a small tag with enzyme-mediated kinetics, but the system requires separate expression of an exogenous ligase and the scope of compatible fluorophores is limited to relatively small dyes such as resorufin 15. Nonnatural amino acids are perhaps the smallest possible perturbation, but require exogenous expression of two biochemical machines and the delivery of the nonnatural amino acid. In addition, only relatively small fluorophores such as coumarin 16 are compatible with the ribosome, which further limits the utility of this approach. One strategy to circumvent this problem is to incorporate a small “click chemistry” handle into the protein for subsequent biorthogonal labeling by a larger fluorophore moiety (11, 12). In the FAP system, the binding of the ligands is fluorogenic and reversible; this can improve bleaching through fluorophore exchange but also decreases overall brightness (17). Finally, development of stains is attractive as this approach dispenses with the requirement of a genetically encoded tag and is able to label endogenous proteins. Nevertheless, this system is not as generalizable as other methods, requiring a unique small molecule binding element for each target protein (19, 20). Currently, self-labeling tag systems are perhaps the best method for live cell labeling and the easiest switch from fluorescent proteins given the relative simplicity of the system, the generality of the labeling reaction with diverse chemical functionality, and the availability of fluorescent

8


Page 8 of 21

Page 9 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

and fluorogenic ligands. Of these, the HaloTag system reigns supreme, based on its rapid labeling kinetics (k2 ≈ 107 M–1s–1) and high cell-permeability of the ligand moiety, which allows labeling of live cells with low concentrations of fluorophore (~100 nM) and facilitates use in tissue and in vivo REF. Nevertheless, the larger size of the HaloTag (~33 kD) can be problematic with some protein fusions. The SNAP-tag is smaller (~20 kD) but labels slower (k2 ≈ 104 M–1s–1) and the benzyl guanine ligand motif is less cell permeable than the chloroalkane HaloTag ligand. Thus, the SNAP-tag system requires higher concentrations of ligand (~1 µM), which necessitates more rigorous washing protocols. Continued optimization and benchmarking of these systems and exploration of other protein-based self-labeling tags (e.g., dihydrofolate reductase, βlactamase, photoactive yellow protein) is needed to enable multicolor imaging in live cells using synthetic fluorophores. Given the advances in both dye and protein engineering, it would seem obvious to combine the commercial panels of advanced fluorophores (Figure 1C) with these improved live-cell labeling technologies (Figure 2). However, the majority of optimized commercial fluorophores were designed for antibody and oligonucleotide labels for fixed-cell imaging, not for live-cell applications. The polar functionality and relatively high molecular weight of these dyes (Figure 1C) complicates their use with these new labeling strategies due to poor cell membrane permeability and/or incompatibility with enzyme-mediated labeling. For this reason, many of the existing small-molecule labeling techniques have focused on classic, small, and net-neutral dyes that date back a century, as demonstrated in example labels 13–18 (Figure 2). The development of these new labeling strategies and their use in advanced microcopy experiments have necessitated revisiting and reinventing fluorophore chemistry (4). Although the focus on synthetic dyes in the 19th century yielded useful and general synthetic methods, these

9


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 21

methods utilized reagents and techniques from the earliest era of chemistry, predating canonical chemistry concepts such as organometallic reagents and transition metal catalysis. To expand the scope of dye synthesis chemists have been applying modern methods for synthesizing dyes. For example, instead of the century-old acid-catalyzed chemistry to synthesize rhodamines, the addition of aryl-Grignard or aryl-lithium species to xanthone-type derivatives allows an alternative method for preparing rhodamines and red-shifted analogs where the xanthene oxygen is replaced with a gem-dimethyl carbon (21, 22), gem-dimethyl silicon (23, 24), phosphinate (25), or sulfone (26) groups (Figure 3A). In particular, the far-red Si-substituted rhodamine scaffold has emerged as a bright and photostable framework that can substantially increase the photon budget in imaging experiments. The synthesis of these analogs would be difficult or impossible using the conventional xanthene dye syntheses that rely on acid-catalyzed condensation.

10


Page 11 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 3. Modern chemistry yields new dye derivatives. (A) Addition of metalated aryl species to xanthone analogs yields a variety of rhodamine derivatives. (B) The ‘open–closed’ equilibrium of carborhodamines and Sirhodamines is shifted towards the closed lactone form. (C) Reagents based on carborhodamines and Si-Rhodamines: SiR700 SNAP-tag ligand (21); spontaneous blinking HM-SiR (22); fluorogenic carborhodamine HaloTag ligand 23. (D) Use of Pd-catalyzed cross-coupling to synthesize known (TMR, 5) and novel (Janelia Fluor 549, 25) rhodamines from a simple fluorescein derivative (24). (E) Replacing N,N-dialkyl substituents in classic dyes yields a panel of bright, photostable ‘Janelia Fluor’ dyes (25–31) and allows fine tuning of spectral and chemical properties.

In addition to eliciting a bathochromic shift, these substitutions also affect chemical properties such as the equilibrium between the ‘open’, fluorescent, zwitterionic form and the ‘closed’, nonfluorescent, lactone form. Both the carbon-containing analogs (e.g., ‘carboTMR’, 19) and the silicon congeners (e.g., SiTMR or ‘SiR’, 20) are substantially shifted toward the closed form (Figure 3B). For SiTMR (20), the altered open–closed equilibrium ensures the free dye primarily adopts the colorless lactone form in solution, but the equilibrium can shift to the open form upon an environmental change such as binding to a polar protein surface. Discovered by Johnsson and coworkers, this property of SiTMR has allowed the synthesis of fluorogenic ligands for live cell labeling, including self-labeling tags (27), stains for endogenous cytoskeletal

11


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

proteins (19), and a fluorogenic DNA binder (20). This property appears general to Si-rhodamines and can be exploited to make fluorogenic reagents with farther red-shifted spectra (e.g., SiR700-SNAP-tag ligand 21; λmax ≈ 700 nm; Figure 3C) (28) or ‘spontaneous blinking’ dyes such as hydroxymethyl-Si-rhodamine (HM-SiR; 22) that allow super-resolution localization microscopy without photoactivation (29); these are particularly useful for imaging intracellular membranes (30). Finally, carborhodamines can also be made into fluorogenic ligands by shifting the open–closed equilibrium through subtle structural modifications. A HaloTag ligand based on 2ʹ,7ʹ-difluoro-carboTMR (23) shows a nine-fold increase in fluorescence upon binding to the HaloTag protein (31). Another modern chemical reaction that can be applied to fluorophores is Pd-catalyzed crosscoupling chemistry. Instead of synthesizing TMR (5) using the traditional acid-catalyzed condensation chemistry, the dye can be prepared by Buchwald–Hartwig cross-coupling of dimethylamine with fluorescein bistriflate (24) (Figure 3D). This application of contemporary chemistry not only allows easier access to known structures with established biological utility, such as 5, but also enables exploration of new molecules with improved properties. The use of Pd-catalyzed cross-coupling chemistry allowed our laboratory to introduce of a new auxochrome group, the four-membered azetidine ring, which would not survive the classic acid-catalyzed condensation conditions (Figure 3D). This simple modification elicited substantial improvements in the brightness and photostability of TMR (5) with the azetidinyl analog (25, ‘Janelia Fluor 549’) emitting four-fold the number of photons under the same imaging conditions in live cells, thereby substantially increasing the photon budget of experiments (24). This strategy is general, allowing the design and synthesis of Janelia Fluor (JF) dyes that span the visible spectrum (25–31; Figure 3E) and exhibit superior properties compared to the

12


Page 12 of 21

Page 13 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

N,N-dimethylamino-substituted parent dyes (24, 32). The excellent brightness of these fluorophores makes them useful scaffolds for self-labeling tags or stains for cellular imaging and can be extended to more complex biological environments such as intact animals (32, 33). Conclusion. Biologists now have a wealth of options to visualize and track molecules inside cells. The ease of use and continuing optimization of fluorescent proteins make these labels the go-to choice for the majority of fluorescence microscopy experiments. Nevertheless, the quest for higher spatial and temporal resolution in fluorescence microcopy demands increasingly large photon budgets. Innovative labeling strategies and improved fluorophores are making chemical dyes increasingly attractive and accessible to cell biologists, particularly for imaging in the far-red or for super-resolution microscopy. The continued development of fluorescent and fluorogenic stains (Figure 2F) circumvents the need for transfection, allowing ‘GMO-free’ imaging in live cells. This triumphant return of chemical dyes is driven by chemistry; the application of modern synthetic techniques will allow further structural refinements to yield dyes with better brightness, higher fluorogenicity, improved photoactivation, and enhanced bioavailability. We look forward to the next era of imaging where the frontiers of microscopy expand under the reign of both fluorescent proteins and synthetic dyes.

Acknowledgments. An earlier version of this piece was published as a blog post on addgene.org. I thank Tyler Ford (addgene), Brett Mensh (Janelia), and Jonathan Grimm (Janelia) for helpful comments. Related work in our laboratory is supported by the Howard Hughes Medical Institute.

Competing Interest Statement. L.D.L. has filed patent applications on the Janelia Fluor dyes, whose value might be affected by this publication.

13


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 21

REFERENCES 1. 2. 3. 4. 5.

6.

7.

8.

9. 10. 11. 12. 13. 14.

15.

16.

Liu, Z., Lavis, L. D., and Betzig, E. (2015) Imaging live-cell dynamics and structure at the single-molecule level, Mol. Cell 58, 644-659. Lin, M. Z., and Schnitzer, M. J. (2016) Genetically encoded indicators of neuronal activity, Nat Neurosci 19, 1142-1153. Lavis, L. D., and Raines, R. T. (2008) Bright ideas for chemical biology, ACS Chem. Biol. 3, 142–155. Lavis, L. D., and Raines, R. T. (2014) Bright building blocks for chemical biology, ACS Chem. Biol. 9, 855–866. Coons, A. H., and Kaplan, M. H. (1950) Localization of antigen in tissue cells. II. Improvements in a method for the detection of antigen by means of fluorescent antibody, J. Exp. Med. 91, 1-13. Panchuk-Voloshina, N., Haugland, R. P., Bishop-Stewart, J., Bhalgat, M. K., Millard, P. J., Mao, F., Leung, W.-Y., and Haugland, R. P. (1999) Alexa Dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates, J. Histochem. Cytochem. 47, 1179-1188. Mujumdar, R. B., Ernst, L. A., Mujumdar, S. R., Lewis, C. J., and Waggoner, A. S. (1993) Cyanine dye labeling reagents: Sulfoindocyanine succinimidyl esters, Bioconjugate Chem. 4, 105-111. Rodriguez, E. A., Campbell, R. E., Lin, J. Y., Lin, M. Z., Miyawaki, A., Palmer, A. E., Shu, X., Zhang, J., and Tsien, R. Y. (2017) The growing and glowing toolbox of fluorescent and photoactive proteins, Trends Biochem. Sci. 42, 111-129. Zhou, X. X., and Lin, M. Z. (2013) Photoswitchable fluorescent proteins: ten years of colorful chemistry and exciting applications, Curr. Opin. Chem. Biol. 17, 682-690. van de Linde, S., Heilemann, M., and Sauer, M. (2012) Live-cell super-resolution imaging with synthetic fluorophores, Annu. Rev. Phys. Chem. 63, 519-540. Lang, K., and Chin, J. W. (2014) Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins, Chem. Rev. 114, 4764-4806. Xue, L., Karpenko, I. A., Hiblot, J., and Johnsson, K. (2015) Imaging and manipulating proteins in live cells through covalent labeling, Nat. Chem. Biol. 11, 917-923. Griffin, B. A., Adams, S. R., and Tsien, R. Y. (1998) Specific covalent labeling of recombinant protein molecules inside live cells, Science 281, 269-272. Keppler, A., Gendreizig, S., Gronemeyer, T., Pick, H., Vogel, H., and Johnsson, K. (2002) A general method for the covalent labeling of fusion proteins with small molecules in vivo, Nat. Biotechnol. 21, 86-89. Los, G. V., Encell, L. P., McDougall, M. G., Hartzell, D. D., Karassina, N., Zimprich, C., Wood, M. G., Learish, R., Ohana, R. F., and Urh, M. (2008) HaloTag: A novel protein labeling technology for cell imaging and protein analysis, ACS Chem. Biol. 3, 373-382. Liu, D. S., Nivon, L. G., Richter, F., Goldman, P. J., Deerinck, T. J., Yao, J. Z., Richardson, D., Phipps, W. S., Ye, A. Z., Ellisman, M. H., Drennan, C. L., Baker, D., and Ting, A. Y. (2014) Computational design of a red fluorophore ligase for site-specific protein labeling in living cells, Proc. Natl. Acad. Sci. U. S. A. 111, E4551-4559.

14


Page 15 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

17.

18.

19.

20.

21. 22.

23.

24.

25.

26.

27.

28.

29.

30.

Szent-Gyorgyi, C., Schmidt, B. F., Creeger, Y., Fisher, G. W., Zakel, K. L., Adler, S., Fitzpatrick, J. A., Woolford, C. A., Yan, Q., Vasilev, K. V., Berget, P. B., Bruchez, M. P., Jarvik, J. W., and Waggoner, A. (2008) Fluorogen-activating single-chain antibodies for imaging cell surface proteins, Nat. Biotechnol. 26, 235-240. Evangelio, J. A., Abal, M., Barasoain, I., Souto, A. A., Lillo, M. P., Acuna, A. U., AmatGuerri, F., and Andreu, J. M. (1998) Fluorescent taxoids as probes of the microtubule cytoskeleton, Cell Motil. Cytoskeleton 39, 73-90. Lukinavičius, G., Reymond, L., D'Este, E., Masharina, A., Gottfert, F., Ta, H., Guther, A., Fournier, M., Rizzo, S., Waldmann, H., Blaukopf, C., Sommer, C., Gerlich, D. W., Arndt, H.-D., Hell, S. W., and Johnsson, K. (2014) Fluorogenic probes for live-cell imaging of the cytoskeleton, Nat. Methods 11, 731-733. Lukinavičius, G., Blaukopf, C., Pershagen, E., Schena, A., Reymond, L., Derivery, E., Gonzalez-Gaitan, M., D’Este, E., Hell, S. W., and Gerlich, D. W. (2015) SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy, Nat. Comm. 6, 8497 doi:8410.1038/ncomms9497. Arden-Jacob, J., Frantzeskos, J., Kemnitzer, N. U., Zilles, A., and Drexhage, K. H. (2001) New fluorescent markers for the red region, Spectrochim. Acta, Part A 57, 2271-2283. Grimm, J. B., Sung, A. J., Legant, W. R., Hulamm, P., Matlosz, S. M., Betzig, E., and Lavis, L. D. (2013) Carbofluoresceins and carborhodamines as scaffolds for high-contrast fluorogenic probes, ACS Chem. Biol. 8, 1303-1310. Koide, Y., Urano, Y., Hanaoka, K., Terai, T., and Nagano, T. (2011) Evolution of Group 14 rhodamines as platforms for near-infrared fluorescence probes utilizing photoinduced electron transfer, ACS Chem. Biol. 6, 600–608. Grimm, J. B., English, B. P., Chen, J., Slaughter, J. P., Zhang, Z., Revyakin, A., Patel, R., Macklin, J. J., Normanno, D., Singer, R. H., Lionnet, T., and Lavis, L. D. (2015) A general method to improve fluorophores for live-cell and single-molecule microscopy, Nat. Methods 12, 244–250. Zhou, X., Lai, R., Beck, J. R., Li, H., and Stains, C. I. (2016) Nebraska Red: A phosphinate-based near-infrared fluorophore scaffold for chemical biology applications, Chem. Commun. 52, 12290-12293. Liu, J., Sun, Y. Q., Zhang, H., Shi, H., Shi, Y., and Guo, W. (2016) Sulfone-Rhodamines: A new class of near-infrared fluorescent dyes for bioimaging, ACS Appl. Mater. Interfaces 8, 22953-22962. Lukinavičius, G., Umezawa, K., Olivier, N., Honigmann, A., Yang, G., Plass, T., Mueller, V., Reymond, L., Corrêa Jr, I. R., Luo, Z. G., Schultz, C., Lemke, E. A., Heppenstall, P., Eggeling, C., Manley, S., and Johnsson, K. (2013) A near-infrared fluorophore for livecell super-resolution microscopy of cellular proteins, Nature Chem. 5, 132–139. Lukinavicius, G., Reymond, L., Umezawa, K., Sallin, O., D'Este, E., Gottfert, F., Ta, H., Hell, S. W., Urano, Y., and Johnsson, K. (2016) Fluorogenic probes for multicolor imaging in living cells, J. Am. Chem. Soc. 138, 9365-9368. Uno, S. N., Kamiya, M., Yoshihara, T., Sugawara, K., Okabe, K., Tarhan, M. C., Fujita, H., Funatsu, T., Okada, Y., Tobita, S., and Urano, Y. (2014) A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging, Nat. Chem. 6, 681-689. Erdmann, R. S., Takakura, H., Thompson, A. D., Rivera-Molina, F., Allgeyer, E. S., Bewersdorf, J., Toomre, D., and Schepartz, A. (2014) Super-resolution imaging of the

15


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

31.

32.

33.

Golgi in live cells with a bioorthogonal ceramide probe, Angew. Chem. Int. Ed. 53, 10242-10246. Butkevich, A. N., Mitronova, G. Y., Sidenstein, S. C., Klocke, J. L., Kamin, D., Meineke, D. N., D'Este, E., Kraemer, P. T., Danzl, J. G., Belov, V. N., and Hell, S. W. (2016) Fluorescent rhodamines and fluorogenic carbopyronines for super‐resolution STED microscopy in living cells, Angew. Chem. Int. Ed. 55, 3290-3294. Grimm, J. B., Muthusamy, A. K., Liang, Y., Brown, T. A., Lemon, W. C., Patel, R., Lu, R., Macklin, J. J., Keller, P. J., Ji, N., and Lavis, L. D. (2017) A general method to finetune fluorophores for live-cell and in vivo imaging, bioRxiv, doi: 10.1101/127613. Legant, W. R., Shao, L., Grimm, J. B., Brown, T. A., Milkie, D. E., Avants, B. B., Lavis, L. D., and Betzig, E. (2016) High-density three-dimensional localization microscopy across large volumes, Nat. Methods 13, 359-365.

16


Page 16 of 21

Page 17 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

For Table of Contents Use Only

17


1 2 3 4 B5 6 HO O O 7 8 9 3 10 C11 12 H2N O O 13 CO2H –14 O3S 15 16 17 18 8 (Alexa Fluor 350)

Biochemistry

A

Page 18 of 21

N

N

OH

H2N

O

N+

NH

N 1 HO

O

O

2

N

CO2H

H 2N

O

O

HO

5 SO3–

CO2–

HO2C 9 (Alexa Fluor 488)

+

NH2

H N

SO3–

O

N

6 SO3–

CO2–

Cl

O

N

CO2–

4 SO3–

N+

O

7

10 (Alexa Fluor 546)

SO3–

–O S 3

H+ N

N

N+

O

CO2–

–O S 3

ACS Plus Environment HO2Paragon C S Cl HO C Cl

N+

N+

N –

SO3

2

11 (Alexa Fluor 594)

CO2H 12 (Cy5)

Page 19 of 21

A.

Bisarsenical Dyes

Biochemistry

B.

Self-Labeling Tags

C.

1 Cys4 motif OH 2 3 SH SH SH SH HaloTag 4 + 5 + N+ N O 6 S S S S 7 As As 8 CO2– O HO O 9 O 14 10 HN CO2H O 11 O Cl 12 13 13 14 15 N+ N O 16 17 CO2– S S S S 18 As As O O HO O 19 HN 20 O O 21 CO2H O 22 23 24 D.25 Nonnatural Amino Acids E. Fluorogen Activating Proteins F. 26 fluorogen-activating 27 protein D.28 R 29 H N CO2H 30 2 31 + 32 + HO O O 33 N N+ 34 16 35 36 H2N CO2H 17 37 38 O mutant mutant tRNA CO2H 39 tRNA synthetase 40 41 OH 42 43 44 N N+ O 45 O 46 47 ACS Paragon Plus Environment 48 O CO2H 49

Ligases ligase substrate NH2

+ HO

O

O

N

H N

CO2H

O 15

lipoic acid ligase

HN

HO

O

O

O NH

N

O

Stains

HO

O

O

CO2H

O

N H

O

O

O

O

O

O

O OBz

O

OH

O

O OH

HN O 18

small-molecule binding motif

Biochemistry

A

N

X

B

N

C

N+

X

N

Si

N

M

1 + O 2 3 M = MgBr, Li 4 5 6 X N N+ 7 8 9 10 X = O, C(CH3)2, Si(CH3)2, PO2–, SO2 11 12 OTf O TfO D13 O 14 O 15 24 16 17 + 18 R 2N NR2 O 19 20 CO2– 21 22 23 5: NR2 = N 25: NR2 = N 24

Page 20 of 21 N+

CO2–

CO2–

N

N+

Si

O HN

O

N O

O

E

F N

O

O

F N

O

CO2H 26 (JF424)

F

F

F N

HN

N+

F

O

O O

O

Cl

22

F

23

F

F N

N+

O

CO2–

CO2–

27 (JF503)

28 (JF525)

F

N

N+

O

CO2– 25 (JF549)

F

F N

F

N

N+

Si

ACS Paragon CO2– Plus Environment 29 (JF585)

CO2–

HN

CO2H

N

21

19: X = C(CH3)2 20: X = Si(CH3)2

F

OH NH2

N

X

N

N+

N

N

N+

Si

CO2–

CO2–

30 (JF635)

31 (JF646)

Page 21 of 21

1 2 3 4

Biochemistry

Y Environment X ACS X Paragon Plus Z

Chemistry Is Dead. Long Live Chemistry! - Biochemistry (ACS

Recommend Documents