10 research outputs found
Synthesis of Rhodamines from Fluoresceins Using Pd-Catalyzed CâN Cross-Coupling
A unified, convenient, and efficient strategy for the preparation of rhodamines and <i>N,N</i>â˛-diacylated rhodamines has been developed. Fluorescein ditriflates were found to undergo palladium-catalyzed CâN cross-coupling with amines, amides, carbamates, and other nitrogen nucleophiles to provide direct access to known and novel rhodamine derivatives, including fluorescent dyes, quenchers, and latent fluorophores
Synthesis of Rhodamines from Fluoresceins Using Pd-Catalyzed CâN Cross-Coupling
A unified, convenient, and efficient strategy for the preparation of rhodamines and <i>N,N</i>â˛-diacylated rhodamines has been developed. Fluorescein ditriflates were found to undergo palladium-catalyzed CâN cross-coupling with amines, amides, carbamates, and other nitrogen nucleophiles to provide direct access to known and novel rhodamine derivatives, including fluorescent dyes, quenchers, and latent fluorophores
General Synthetic Method for Si-Fluoresceins and Si-Rhodamines
The century-old fluoresceins
and rhodamines persist as flexible
scaffolds for fluorescent and fluorogenic compounds. Extensive exploration
of these xanthene dyes has yielded general structureâactivity
relationships where the development of new probes is limited only
by imagination and organic chemistry. In particular, replacement of
the xanthene oxygen with silicon has resulted in new red-shifted Si-fluoresceins
and Si-rhodamines, whose high brightness and photostability enable
advanced imaging experiments. Nevertheless, efforts to tune the chemical
and spectral properties of these dyes have been hindered by difficult
synthetic routes. Here, we report a general strategy for the efficient
preparation of Si-fluoresceins and Si-rhodamines from readily synthesized
bisÂ(2-bromophenyl)Âsilane intermediates. These dibromides undergo metal/bromide
exchange to give bis-aryllithium or bisÂ(aryl Grignard) intermediates,
which can then add to anhydride or ester electrophiles to afford a
variety of Si-xanthenes. This strategy enabled efficient (3â5
step) syntheses of known and novel Si-fluoresceins, Si-rhodamines,
and related dye structures. In particular, we discovered that previously
inaccessible tetrafluorination of the bottom aryl ring of the Si-rhodamines
resulted in dyes with improved visible absorbance in solution, and
a convenient derivatization through fluoride-thiol substitution. This
modular, divergent synthetic method will expand the palette of accessible
xanthenoid dyes across the visible spectrum, thereby pushing further
the frontiers of biological imaging
Evaluation of the Ser-His Dipeptide, a Putative Catalyst of Amide and Ester Hydrolysis
Efficient hydrolysis
of amide bonds has long been a reaction of
interest for organic chemists. The rate constants of proteases are
unmatched by those of any synthetic catalyst. It has been proposed
that a dipeptide containing serine and histidine is an effective catalyst
of amide hydrolysis, based on an apparent ability to degrade a protein.
The capacity of the Ser-His dipeptide to catalyze the hydrolysis of
several discrete ester and amide substrates is investigated using
previously described conditions. This dipeptide does not catalyze
the hydrolysis of amide or unactivated ester groups in any of the
substrates under the conditions evaluated
Rhodium(III)-Catalyzed Indazole Synthesis by CâH Bond Functionalization and Cyclative Capture
An efficient, one-step, and highly
functional group-compatible
synthesis of substituted <i>N</i>-aryl-2<i>H</i>-indazoles is reported via the rhodiumÂ(III)-catalyzed CâH
bond addition of azobenzenes to aldehydes. The regioselective coupling
of unsymmetrical azobenzenes was further demonstrated and led to the
development of a new removable aryl group that allows for the preparation
of indazoles without <i>N</i>-substitution. The 2-aryl-2<i>H</i>-indazole products also represent a new class of readily
prepared fluorophores for which initial spectroscopic characterization
has been performed
Virginia Orange: A Versatile, Red-Shifted Fluorescein Scaffold for Single- and Dual-Input Fluorogenic Probes
Fluorogenic molecules are important
tools for biological and biochemical
research. The majority of fluorogenic compounds have a simple inputâoutput
relationship, where a single chemical input yields a fluorescent output.
Development of new systems where multiple inputs converge to yield
an optical signal could refine and extend fluorogenic compounds by
allowing greater spatiotemporal control over the fluorescent signal.
Here, we introduce a new red-shifted fluorescein derivative, Virginia
Orange, as an exceptional scaffold for single- and dual-input fluorogenic
molecules. Unlike fluorescein, installation of a single masking group
on Virginia Orange is sufficient to fully suppress fluorescence, allowing
preparation of fluorogenic enzyme substrates with rapid, single-hit
kinetics. Virginia Orange can also be masked with two independent
moieties; both of these masking groups must be removed to induce fluorescence.
This allows facile construction of multi-input fluorogenic probes
for sophisticated sensing regimes and genetic targeting of latent
fluorophores to specific cellular populations
Cell-Specific Chemical Delivery Using a Selective NitroreductaseâNitroaryl Pair
<p>The utility of<b> </b>small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. âMaskingâ the activity of small molecules using a general chemical modification and âunmaskingâ it only within target cells could overcome this limitation. To this end, we have developed a selective enzymeâsubstrate pair consisting of engineered variants of <i>E. coli</i> nitroreductase (NTR) and a 2ânitro-<i>N</i>-methylimidazolyl (NM) masking group. To discover and optimize this NTRâNM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.</p
Carbofluoresceins and Carborhodamines as Scaffolds for High-Contrast Fluorogenic Probes
Fluorogenic molecules are important
tools for advanced biochemical
and biological experiments. The extant collection of fluorogenic probes
is incomplete, however, leaving regions of the electromagnetic spectrum
unutilized. Here, we synthesize green-excited fluorescent and fluorogenic
analogues of the classic fluorescein and rhodamine 110 fluorophores
by replacement of the xanthene oxygen with a quaternary carbon. These
anthracenyl âcarbofluoresceinâ and âcarborhodamine
110â fluorophores exhibit excellent fluorescent properties
and can be masked with enzyme- and photolabile groups to prepare high-contrast
fluorogenic molecules useful for live cell imaging experiments and
super-resolution microscopy. Our divergent approach to these red-shifted
dye scaffolds will enable the preparation of numerous novel fluorogenic
probes with high biological utility
Distinct Substrate Selectivity of a Metabolic Hydrolase from <i>Mycobacterium tuberculosis</i>
The transition between dormant and
active <i>Mycobacterium
tuberculosis</i> infection requires reorganization of its lipid
metabolism and activation of a battery of serine hydrolase enzymes.
Among these serine hydrolases, Rv0045c is a mycobacterial-specific
serine hydrolase with limited sequence homology outside mycobacteria
but structural homology to divergent bacterial hydrolase families.
Herein, we determined the global substrate specificity of Rv0045c
against a library of fluorogenic hydrolase substrates, constructed
a combined experimental and computational model for its binding pocket,
and performed comprehensive substitutional analysis to develop a structural
map of its binding pocket. Rv0045c showed strong substrate selectivity
toward short, straight chain alkyl esters with the highest activity
toward four atom chains. This strong substrate preference was maintained
through the combined action of residues in a flexible loop connecting
the cap and ι/β hydrolase domains and in residues close
to the catalytic triad. Two residues bracketing the substrate-binding
pocket (Gly90 and His187) were essential to maintaining the narrow
substrate selectivity of Rv0045c toward various acyl ester substituents,
as independent conversion of these residues significantly increased
its catalytic activity and broadened its substrate specificity. Focused
saturation mutagenesis of position 187 implicated this residue, as
the differentiation point between the substrate specificity of Rv0045c
and the structurally homologous ybfF hydrolase family. Insertion of
the analogous tyrosine residue from ybfF hydrolases into Rv0045c increased
the catalytic activity of Rv0045 by over 20-fold toward diverse ester
substrates. The unique binding pocket structure and selectivity of
Rv0045c provide molecular indications of its biological role and evidence
for expanded substrate diversity in serine hydrolases from <i>M. tuberculosis</i>
Measuring the Global Substrate Specificity of Mycobacterial Serine Hydrolases Using a Library of Fluorogenic Ester Substrates
Among the proteins required for lipid
metabolism in <i>Mycobacterium
tuberculosis</i> are a significant number of uncharacterized
serine hydrolases, especially lipases and esterases. Using a streamlined
synthetic method, a library of immolative fluorogenic ester substrates
was expanded to better represent the natural lipidomic diversity of <i>Mycobacterium</i>. This expanded fluorogenic library was then
used to rapidly characterize the global structure activity relationship
(SAR) of mycobacterial serine hydrolases in <i>M. smegmatis</i> under different growth conditions. Confirmation of fluorogenic substrate
activation by mycobacterial serine hydrolases was performed using
nonspecific serine hydrolase inhibitors and reinforced the biological
significance of the SAR. The hydrolases responsible for the global
SAR were then assigned using gel-resolved activity measurements, and
these assignments were used to rapidly identify the relative substrate
specificity of previously uncharacterized mycobacterial hydrolases.
These measurements provide a global SAR of mycobacterial hydrolase
activity, a picture of cycling hydrolase activity, and a detailed
substrate specificity profile for previously uncharacterized hydrolases