Stephen Miller - Cochemist

Rapid Access to a Broad Range of 6′-Substituted Firefly Luciferin Analogues Reveals Surprising Emitters and Inhibitors

Co-reporter:Deepak K. Sharma, Spencer T. Adams Jr., Kate L. Liebmann, and Stephen C. Miller

Organic Letters November 3, 2017 Volume 19(Issue 21) pp:5836-5836

Publication Date(Web):October 17, 2017

DOI:10.1021/acs.orglett.7b02806

Light-emitting firefly luciferin analogues contain electron-donating groups in the 6′-position, but the scope of known 6′-substitution remains narrow. A two-step route to a broad range of 6′-substituted luciferin analogues was developed to fill this void and enable more extensive study of the 6′-functionality. This chemistry allowed direct access to “caged” amide and bright azetidine analogues, but also revealed thioether inhibitors and unexpectedly luminogenic aryl amine derivatives.

Luciferase Activity of Insect Fatty Acyl-CoA Synthetases with Synthetic Luciferins

Co-reporter:David M. Mofford, Kate L. Liebmann, Ganapathy Subramanian Sankaran, G. S. Kiran Kumar Reddy, G. Randheer Reddy, and Stephen C. Miller

ACS Chemical Biology December 15, 2017 Volume 12(Issue 12) pp:2946-2946

Publication Date(Web):October 26, 2017

DOI:10.1021/acschembio.7b00813

Long-chain fatty acyl-CoA synthetases (ACSLs) are homologues of firefly luciferase but are incapable of emitting light with firefly luciferin. Recently, we found that an ACSL from the fruit fly Drosophila melanogaster is a latent luciferase that will emit light with the synthetic luciferin CycLuc2. Here, we have profiled a panel of three insect ACSLs with a palette of >20 luciferin analogues. An ACSL from the nonluminescent beetle Agrypnus binodulus (AbLL) was found to be a second latent luciferase with distinct substrate specificity. Several rigid luciferins emit light with both ACSLs, but styryl luciferin analogues are light-emitting substrates only for AbLL. On the other hand, an ACSL from the luminescent beetle Pyrophorus angustus lacks luciferase activity with all tested analogues, despite its higher homology to beetle luciferases. Further study of ACSLs is expected to shed light on the features necessary for bioluminescence and substrate selectivity.

Reductively-labile sulfonate ester protecting groups that are rapidly cleaved by physiological glutathione

Co-reporter:Adam Choi;Stephen C. Miller

Organic & Biomolecular Chemistry 2017 vol. 15(Issue 6) pp:1346-1349

Publication Date(Web):2017/02/07

DOI:10.1039/C7OB00063D

Sulfonates are frequently used to endow water solubility on hydrophobic molecules, but the repertoire of sulfonate protecting groups remains limited. Here we describe the first sulfonate esters that can be unmasked by the mild reducing conditions found in live mammalian cells. Self-immolative cleavage releases the sulfonate and the two-electron reduction product of a thioquinone methide.

Firefly Luciferase Mutants Allow Substrate-Selective Bioluminescence Imaging in the Mouse Brain

Co-reporter:Spencer T. Adams Jr.;Dr. David M. Mofford;Dr. G. S. Kiran Kumar Reddy ; Stephen C. Miller

Angewandte Chemie International Edition 2016 Volume 55( Issue 16) pp:4943-4946

Publication Date(Web):

DOI:10.1002/anie.201511350

Abstract

Bioluminescence imaging is a powerful approach for visualizing specific events occurring inside live mice. Animals can be made to glow in response to the expression of a gene, the activity of an enzyme, or the growth of a tumor. But bioluminescence requires the interaction of a luciferase enzyme with a small-molecule luciferin, and its scope has been limited by the mere handful of natural combinations. Herein, we show that mutants of firefly luciferase can discriminate between natural and synthetic substrates in the brains of live mice. When using adeno-associated viral (AAV) vectors to express luciferases in the brain, we found that mutant luciferases that are inactive or weakly active with d-luciferin can light up brightly when treated with the aminoluciferins CycLuc1 and CycLuc2 or their respective FAAH-sensitive luciferin amides. Further development of selective luciferases promises to expand the power of bioluminescence and allow multiple events to be imaged in the same live animal.

Firefly Luciferase Mutants Allow Substrate-Selective Bioluminescence Imaging in the Mouse Brain

Co-reporter:Spencer T. Adams Jr.;Dr. David M. Mofford;Dr. G. S. Kiran Kumar Reddy ; Stephen C. Miller

Angewandte Chemie 2016 Volume 128( Issue 16) pp:5027-5030

Publication Date(Web):

DOI:10.1002/ange.201511350

Abstract

Bioluminescence imaging is a powerful approach for visualizing specific events occurring inside live mice. Animals can be made to glow in response to the expression of a gene, the activity of an enzyme, or the growth of a tumor. But bioluminescence requires the interaction of a luciferase enzyme with a small-molecule luciferin, and its scope has been limited by the mere handful of natural combinations. Herein, we show that mutants of firefly luciferase can discriminate between natural and synthetic substrates in the brains of live mice. When using adeno-associated viral (AAV) vectors to express luciferases in the brain, we found that mutant luciferases that are inactive or weakly active with d-luciferin can light up brightly when treated with the aminoluciferins CycLuc1 and CycLuc2 or their respective FAAH-sensitive luciferin amides. Further development of selective luciferases promises to expand the power of bioluminescence and allow multiple events to be imaged in the same live animal.

Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity

Co-reporter:David M. Mofford; Spencer T. AdamsJr.; G. S. Kiran Kumar Reddy; Gadarla Randheer Reddy;Stephen C. Miller

Journal of the American Chemical Society 2015 Volume 137(Issue 27) pp:8684-8687

Publication Date(Web):June 29, 2015

DOI:10.1021/jacs.5b04357

Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain.

Luciferins Behave Like Drugs

Co-reporter:David M. Mofford and Stephen C. Miller

ACS Chemical Neuroscience 2015 Volume 6(Issue 8) pp:1273

Publication Date(Web):July 30, 2015

DOI:10.1021/acschemneuro.5b00195

The light emission chemistry of firefly luciferase can be harnessed to reveal otherwise invisible biological processes occurring in the brains of live animals. Though powerful, the need for the luciferase substrate D-luciferin to traverse the blood-brain barrier poses limitations on the sensitivity and interpretation of these experiments. In this Viewpoint, we discuss bioluminescent imaging probes for the enzyme fatty acid amide hydrolase (FAAH) and the broader implications for optical imaging and drug delivery in the brain.Keywords: bioluminescence imaging; blood-brain barrier; drug delivery; FAAH; luciferase; luciferin

Aminoluciferins Extend Firefly Luciferase Bioluminescence into the Near-Infrared and Can Be Preferred Substrates over d-Luciferin

Co-reporter:David M. Mofford ; Gadarla Randheer Reddy ;Stephen C. Miller

Journal of the American Chemical Society 2014 Volume 136(Issue 38) pp:13277-13282

Publication Date(Web):August 28, 2014

DOI:10.1021/ja505795s

Firefly luciferase adenylates and oxidizes d-luciferin to chemically generate visible light and is widely used for biological assays and imaging. Here we show that both luciferase and luciferin can be reengineered to extend the scope of this light-emitting reaction. d-Luciferin can be replaced by synthetic luciferin analogues that increase near-infrared photon flux >10-fold over that of d-luciferin in live luciferase-expressing cells. Firefly luciferase can be mutated to accept and utilize rigid aminoluciferins with high activity in both live and lysed cells yet exhibit 10 000-fold selectivity over the natural luciferase substrate. These new luciferin analogues thus pave the way to an extended family of bioluminescent reporters.

Latent luciferase activity in the fruit fly revealed by a synthetic luciferin

Co-reporter:David M. Mofford;Gadarla Randheer Reddy;Stephen C. Miller

PNAS 2014 111 (12 ) pp:4443-4448

Publication Date(Web):2014-03-25

DOI:10.1073/pnas.1319300111

Beetle luciferases are thought to have evolved from fatty acyl-CoA synthetases present in all insects. Both classes of enzymes activate fatty acids with ATP to form acyl-adenylate intermediates, but only luciferases can activate and oxidize d-luciferin to emit light. Here we show that the Drosophila fatty acyl-CoA synthetase CG6178, which cannot use d-luciferin as a substrate, is able to catalyze light emission from the synthetic luciferin analog CycLuc2. Bioluminescence can be detected from the purified protein, live Drosophila Schneider 2 cells, and from mammalian cells transfected with CG6178. Thus, the nonluminescent fruit fly possesses an inherent capacity for bioluminescence that is only revealed upon treatment with a xenobiotic molecule. This result expands the scope of bioluminescence and demonstrates that the introduction of a new substrate can unmask latent enzymatic activity that differs significantly from an enzyme’s normal function without requiring mutation.

Latent luciferase activity in the fruit fly revealed by a synthetic luciferin

Co-reporter:David M. Mofford;Gadarla Randheer Reddy;Stephen C. Miller

PNAS 2014 111 (12 ) pp:4443-4448

Publication Date(Web):2014-03-25

DOI:10.1073/pnas.1319300111

Beetle luciferases are thought to have evolved from fatty acyl-CoA synthetases present in all insects. Both classes of enzymes activate fatty acids with ATP to form acyl-adenylate intermediates, but only luciferases can activate and oxidize d-luciferin to emit light. Here we show that the Drosophila fatty acyl-CoA synthetase CG6178, which cannot use d-luciferin as a substrate, is able to catalyze light emission from the synthetic luciferin analog CycLuc2. Bioluminescence can be detected from the purified protein, live Drosophila Schneider 2 cells, and from mammalian cells transfected with CG6178. Thus, the nonluminescent fruit fly possesses an inherent capacity for bioluminescence that is only revealed upon treatment with a xenobiotic molecule. This result expands the scope of bioluminescence and demonstrates that the introduction of a new substrate can unmask latent enzymatic activity that differs significantly from an enzyme’s normal function without requiring mutation.

A Trifluoroacetic Acid-labile Sulfonate Protecting Group and Its Use in the Synthesis of a Near-IR Fluorophore

Co-reporter:Steven M. Pauff and Stephen C. Miller

The Journal of Organic Chemistry 2013 Volume 78(Issue 2) pp:711-716

Publication Date(Web):November 20, 2012

DOI:10.1021/jo302065u

Sulfonated molecules exhibit high water solubility, a property that is valuable for many biological applications but often complicates their synthesis and purification. Here we report a sulfonate protecting group that is resistant to nucleophilic attack but readily removed with trifluoroacetic acid (TFA). The use of this protecting group improved the synthesis of a sulfonated near-IR fluorophore and the mild deprotection conditions allowed isolation of the product without requiring chromatography.

Design and application of esterase-labile sulfonate protecting groups

Co-reporter:Laert Rusha and Stephen C. Miller

Chemical Communications 2011 vol. 47(Issue 7) pp:2038-2040

Publication Date(Web):05 Jan 2011

DOI:10.1039/C0CC04796A

Three esterase-labile, but chemically-stable sulfonate protecting groups have been designed and synthesized. One of these sulfonate esters allowed the cytoplasmic delivery and unmasking of a sulfonated dye in live cells.

Synthesis of Near-IR Fluorescent Oxazine Dyes with Esterase-Labile Sulfonate Esters

Co-reporter:Steven M. Pauff and Stephen C. Miller

Organic Letters 2011 Volume 13(Issue 23) pp:6196-6199

Publication Date(Web):November 2, 2011

DOI:10.1021/ol202619f

Near-IR oxazine dyes are reported that contain sulfonate esters which are rapidly cleaved by esterase activity to unmask highly polar anionic sulfonates. Strategies for the synthesis of these dyes included the development of milder dye condensation conditions with improved functional compatibility and the use of an alkyl halide that allows for the introduction of esterase-labile sulfonates without the need for sulfonation of the target molecule.

Identification of Mutant Firefly Luciferases that Efficiently Utilize Aminoluciferins

Co-reporter:Katryn R. Harwood, David M. Mofford, Gadarla R. Reddy, Stephen C. Miller

Chemistry & Biology 2011 Volume 18(Issue 12) pp:1649-1657

Publication Date(Web):23 December 2011

DOI:10.1016/j.chembiol.2011.09.019

Firefly luciferase-catalyzed light emission from D-luciferin is widely used as a reporter of gene expression and enzymatic activity both in vitro and in vivo. Despite the power of bioluminescence for imaging and drug discovery, light emission from firefly luciferase is fundamentally limited by the physical properties of the D-luciferin substrate. We and others have synthesized aminoluciferin analogs that exhibit light emission at longer wavelengths than D-luciferin and have increased affinity for luciferase. However, although these substrates can emit an intense initial burst of light that approaches that of D-luciferin, this is followed by much lower levels of sustained light output. Here we describe the creation of mutant luciferases that yield improved sustained light emission with aminoluciferins in both lysed and live mammalian cells, allowing the use of aminoluciferins for cell-based bioluminescence experiments.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (185 K)Download as PowerPoint slideHighlights► Mutation of luciferase can improve light emission from all aminoluciferins ► F247L luciferase improves light emission from 6′-aminoluciferin ► CycLuc1 is a better substrate for S347A luciferase than D-luciferin ► Live cell light emission is highly dependent on cell-permeability and Km

Robust Light Emission from Cyclic Alkylaminoluciferin Substrates for Firefly Luciferase

Co-reporter:Gadarla Randheer Reddy ; Walter C. Thompson ;Stephen C. Miller

Journal of the American Chemical Society 2010 Volume 132(Issue 39) pp:13586-13587

Publication Date(Web):September 9, 2010

DOI:10.1021/ja104525m

Firefly luciferase utilizes the chemical energy of ATP and oxygen to convert its substrate, d-luciferin, into an excited-state oxyluciferin molecule. Relaxation of this molecule to the ground state is responsible for the yellow-green light emission. Synthetic cyclic alkylaminoluciferins that allow robust red-shifted light emission with the modified luciferase Ultra-Glo are described. Overall light emission is higher than that of acyclic alkylaminoluciferins, aminoluciferin, and the native substrate d-luciferin.

Profiling Sulfonate Ester Stability: Identification of Complementary Protecting Groups for Sulfonates

Co-reporter:Stephen C. Miller

The Journal of Organic Chemistry 2010 Volume 75(Issue 13) pp:4632-4635

Publication Date(Web):June 1, 2010

DOI:10.1021/jo1007338

Sulfonation is prized for its ability to impart water-solubility to hydrophobic molecules such as dyes. This modification is usually performed as a final step, since sulfonated molecules are poorly soluble in most organic solvents, which complicates their synthesis and purification. This work compares the intrinsic lability of different sulfonate esters, identifying new sulfonate protecting groups and mild, selective cleavage conditions.

Leveraging a Small-Molecule Modification to Enable the Photoactivation of Rho GTPases

Co-reporter:Katryn R. Harwood ;Stephen C. Miller

ChemBioChem 2009 Volume 10( Issue 18) pp:2855-2857

Publication Date(Web):

DOI:10.1002/cbic.200900546

Inside Cover: Leveraging a Small-Molecule Modification to Enable the Photoactivation of Rho GTPases (ChemBioChem 18/2009)

Co-reporter:Katryn R. Harwood ;Stephen C. Miller

ChemBioChem 2009 Volume 10( Issue 18) pp:

Publication Date(Web):

DOI:10.1002/cbic.200990083

Labeling Tetracysteine-Tagged Proteins with a SplAsH of Color: A Modular Approach to Bis-Arsenical Fluorophores

Co-reporter:Anjan K. Bhunia Dr.;Stephen C. Miller

ChemBioChem 2007 Volume 8(Issue 14) pp:

Publication Date(Web):10 AUG 2007

DOI:10.1002/cbic.200700192

Everything nicely tied up. We have synthesized a non-fluorescent bis-arsenical targeting moiety that can be tethered to a wide variety of different fluorescent payloads. This strategy greatly broadens the scope of dyes that can be used to label tetracysteine-tagged proteins.

Design and application of esterase-labile sulfonate protecting groups

Co-reporter:Laert Rusha and Stephen C. Miller

Chemical Communications 2011 - vol. 47(Issue 7) pp:NaN2040-2040

Publication Date(Web):2011/01/05

DOI:10.1039/C0CC04796A

Three esterase-labile, but chemically-stable sulfonate protecting groups have been designed and synthesized. One of these sulfonate esters allowed the cytoplasmic delivery and unmasking of a sulfonated dye in live cells.

Reductively-labile sulfonate ester protecting groups that are rapidly cleaved by physiological glutathione

Co-reporter:Adam Choi and Stephen C. Miller

Organic & Biomolecular Chemistry 2017 - vol. 15(Issue 6) pp:NaN1349-1349

Publication Date(Web):2017/01/19

DOI:10.1039/C7OB00063D

Sulfonates are frequently used to endow water solubility on hydrophobic molecules, but the repertoire of sulfonate protecting groups remains limited. Here we describe the first sulfonate esters that can be unmasked by the mild reducing conditions found in live mammalian cells. Self-immolative cleavage releases the sulfonate and the two-electron reduction product of a thioquinone methide.