Journal of the American Chemical Society
Article
restricted rotation. PhOH-Luc comprises a phenol moiety that
can engage in an intramolecular hydrogen bond that modulates
C2′-C2″ rotation.43 The introduction of the hydroxy group
increased the predicted rotational barrier (Figure S1b). The
attenuated rotational flexibility could potentially provide a
more accessible planarized π-system. Like Ph-Luc, the
phenolic scaffold was predicted to exhibit red-shifted emission
(Figure S1a). Both molecules were also structurally distinct
from most D-luc analogs synthesized to date. Such unique
architectures could potentially expedite the search for
orthogonal pairs.
We envisioned accessing Ph-Luc and PhOH-Luc from a
common brominated synthon. Late stage Suzuki-Miyaura
coupling and D-cysteine condensation (Scheme 1) would
provide both analogs. Brominated intermediate 1 was accessed
in two steps from commercial materials (Scheme S1).
Coupling this fragment with pinacol boronate 2 forged the
key biaryl C−C bond en route to Ph-Luc. Benzonitrile product
3 was ultimately condensed with D-cysteine to provide Ph-Luc
in good yield. PhOH-Luc was similarly prepared. Earlier
attempts to isolate PhOH-Luc from a more streamlined route
(via a late-stage double demethylation, Scheme S2) were
unsuccessful.
With the π-extended chromophores in hand, we evaluated
their propensity to form planar structures via fluorescence
spectroscopy. The relative emission profiles of Ph-Luc and
PhOH-Luc would inform on whether intramolecular H-
bonding was an effective rotational lock. Measurements were
performed with the luciferin scaffolds instead of the oxy-
luciferin products. These latter molecules are notoriously
difficult to isolate and analyze,44,45 and the key conformational
parameter (rotation about C2′−C2″) could be directly assayed
using the parental luciferins. Ph-Luc exhibited an emission
maximum (λem) of 442 nm (Figure S2a). This wavelength was
characteristic of an isolated benzothiazole chromophore,
suggesting a twisted excited-state geometry. By contrast,
PhOH-Luc exhibited a λem of 529 nm. This red-shifted
emission suggested that PhOH-Luc is likely more conjugated
in the excited state compared to Ph-Luc. Methylated analogs
further revealed that the pendant hydroxyl group was key to
confining the chromophore in a planar geometry (Figure S2b,
Scheme S3). Ph-Luc exhibited a second emission peak at 595
nm in water, which was not observed for PhOH-Luc (Figure
S2a). This emission is likely due to a solvent-mediated
intermolecular proton transfer at the 6’−OH, which is well
documented for D-luc and related chromophores.46 Solvent-
mediated proton transfer was less likely for PhOH-Luc due to
competing intramolecular processes (Figures S2,S3).
Analyzing Bioluminescent Light Emission with π-
Extended Luciferins. We next evaluated the bioluminescent
properties of the π-extended analogs with Fluc. Surprisingly, no
steady state light emission was observed for either luciferin
when incubated with recombinant luciferase (Figure S4). This
result was in contrast with analogs comprising intervening vinyl
units, which were previously reported as robust emitters.35 It is
possible that Fluc processed the π-extended analogs, but only
extremely low levels of light were produced. In any case, the
photon output was incompatible with screening. Since Ph-Luc
and PhOH-Luc were already “dark” with Fluc, they would be
excellent candidates for orthogonal probes. Identifying
complementary enzymes was expected to be difficult, though,
due to Fluc inactivity. Luciferases can be readily evolved to
process unnatural luciferin analogs.19,30,36 However, the
evolving of new enzymatic functions requires a starting point
(i.e., an enzyme with some basal level of activity).47 Indeed,
initial screens of small Fluc mutant libraries did not reveal any
functional hits with the π-extended analogs (Figure S5).
Identifying a Starting Point via RosettaDesign. The
lack of robust emission in our initial screens suggested that a
major redesign of the luciferase active site was needed. To
identify enzymes that could provide more photons with the π-
extended analogs, we took cues from previous efforts to
engineer lipoic acid ligase (LplA).48 LplA catalyzes the
adenylation of a dithiolane-containing lipid and its subsequent
attachment to target peptides. Initial attempts to modify this
enzyme to accept an elongated structure (resorufin) were
challenging, as LplA did not exhibit any activity toward the
molecule. Rosetta-guided enzyme design was ultimately used
to engineer the LplA active site to accept unnatural substrates.
Beyond this example, Rosetta software has enabled the de novo
design of other challenging enzyme active sites49 and protein
interfaces.50,51
RosettaDesign has also been used to craft enzymes capable
of binding distinct rotamers.52 In a key example, Schultz and
co-workers identified an engineered aminoacyl-tRNA synthe-
tase capable of “locking” a flexible, biphenyl amino acid into a
flat geometry. We aimed to use the platform to similarly
identify Fluc mutants that could maximize packing interactions
between a π-extended luciferin and surrounding amino acids.
Ideally, the mutations would provide luciferases with active
sites that not only facilitate light emission but also restrict
substrate geometry. Because the mutants would be designed to
complement the π-extended scaffolds, they would also be less
likely to accept other luciferin analogs and, thus, be orthogonal.
To identify luciferase variants that could stabilize the
extended π-systems in a planar conformation, the Rosetta-
Match53 and RosettaDesign54 algorithms were used (see
Supplementary Note for detailed protocols). The modified
luciferins were first generated in silico and docked into the
luciferase enzyme (PDB ID: 4g36) using the RosettaMatch
protocol. The RosettaDesign algorithm was then used to
optimize the residues surrounding the extended substrates.
Inspection of the calculation outputs indicated that many
mutations suggested by Rosetta were likely necessary to
accommodate the excess bulk of the new luciferin substrates.
These residues served as a guide for the development of
luciferase libraries that were generated as described below.
Mutants from the Rosetta calculations were ranked by shape
complementarity between the designed residues and luciferin
analogs. We developed a mutant library targeting the top
residues predicted by Rosetta (Figure 2a). This library was
prepared via targeted combinatorial codon mutagenesis
(Figure S6).55 On the basis of a small sample, ∼3−4 sites
were mutated in each library member. The library was
screened for light emission with Ph-Luc and PhOH-Luc via
a two-tiered approach (Figure S7).19 In brief, the library was
introduced into bacteria, and the transformants were plated on
agar plates embedded with one of the analogs. Light-emitting
colonies were identified and picked for a secondary screen.
Fluc was included in the second screen, and any mutants that
exhibited light emission on par or greater than that of Fluc
were considered “hits.”
Gratifyingly, we were able to identify a functional mutant
(G1) that exhibited improved photon outputs compared to
those of Fluc with PhOH-Luc (Figure 2b). This mutant
appeared to be selective toward the partially rigidified
C
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