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

Article abstract of 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.

Full text of DOI:10.1021/acs.orglett.7b02806

Letter
Cite This: Org. Lett. XXXX, XXX, XXX-XXX
pubs.acs.org/OrgLett
Rapid Access to a Broad Range of 6′-Substituted Firefly Luciferin
Analogues Reveals Surprising Emitters and Inhibitors
Deepak K. Sharma, Spencer T. Adams, Jr., Kate L. Liebmann, and Stephen C. Miller*
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street,
Worcester, Massachusetts 01605, United States
*
S Supporting Information
ABSTRACT: 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.
1
1
he natural substrate for firefly luciferase, D-luciferin,
or palladium catalysis. One concern was that the activated
nitrile is prone to react with nucleophiles. Indeed, fear of this
possibility initially dissuaded us from exploring this route, in
T
contains an electron-donating hydroxyl group in the 6′-
1
position that is crucial for bioluminescence (Figure 1). Synthetic
5
,12
favor of reductive alkylation and other synthetic strategies.
Nonetheless, this chemistry could offer access to a wide variety of
analogues not readily accessible by other approaches.
We first synthesized 6-fluoro-2-cyanobenzothiazole 3
(
Scheme S1) and performed S Ar reactions with a variety of
N
cyclic secondary amines that could be challenging or tedious to
synthesize by the conventional N-alkylation approach (Scheme
1
). Of particular interest, azetidine-substituted fluorophores have
been reported to have higher quantum yields than those of
Scheme 1. Nucleophilic Aromatic Substitution of 6-Fluoro-2-
cyanobenzothiazole with Secondary Amines and Thiols
Figure 1. Firefly luciferase substrates all have hydroxy, amino, or
alkylamino electron donors. Examples include D-luciferin, 6′-amino-
luciferin, CycLuc1, and AkaLumine.
analogues containing amine or alkylamine donors are also
2
,3
luminogenic and can offer advantages for in vivo imaging.
However, to date most firefly luciferin substrates and “caged”
sensors have been synthesized from 6-amino or 6-hydroxy
benzothiazoles, placing inherent restrictions on the nature of the
1
,4−10
electron-donating 6′-functionality.
As such, only a relatively
narrow range of 6′-substituted luciferin analogues are known,
limiting our understanding of the chemistry of bioluminescence
and ability to take full advantage of this imaging modality. In
order to further investigate the role of this vital component, we
explored the derivatization of 6-fluoro and 6-bromo-2-cyano-
benzothiazoles via nucleophilic aromatic substitution and
Buchwald−Hartwig amination, respectively. We found that this
chemistry allowed access to new classes of luciferase substrates,
inhibitors, and their precursors, with immediate applications for
biocompatible chemistry and bioluminescence imaging.
We envisioned that 6-halo-2-cyanobenzothiazoles could be
modified at the 6-position by nucleophilic aromatic substitution
Received: September 7, 2017
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b02806
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
1
3
analogous dyes. Although the desired 6-substituted-2-cyano-
Buoyed by the success of this approach, we then sought access
to entire classes of 6′-modifications heretofore unknown in
luciferin analogues, in order to more broadly explore the range of
electron-donating groups (EDGs) that could be accommodated
in luciferin substrates or caged sensors. For example, no 6′-
arylamino luciferin analogues have been reported. Excitingly,
Buchwald−Hartwig amination with xantphos allowed ready
access to a wide variety of 6-arylamino derivatives (Scheme 2,
benzothiazoles 4−9 could be isolated in most cases, the yields
from the S Ar route were low (<20%; Scheme 1). The reaction
N
with azetidine was particularly problematic (2% yield). Primary
amines and weakly nucleophilic amines such as thiomorpholine
dioxide failed to give any desired product. However, displace-
ment with methanethiol was successful (48% yield), giving access
to a new class of analogue that was expected to be
1
7−30). Furthermore, secondary and tertiary “caged” 6-amide
1
4,15
fluorescent.
be prepared by oxidation with Oxone and mCPBA, respectively
Scheme 1). The thiophenol analogue could also be prepared,
The corresponding sulfoxide and sulfone could
analogs could also be synthesized directly in good yields under
these conditions (31−35). Although simple 6′-amidoluciferins
are all potential sensors for amidases, only the 6′-acetamide has
18
(
19
albeit in low yield (13%).
been previously described. We extended this chemistry to
carbamates and ureas such as 2-oxazolidone 39, benzyl
carbamate 36, dimethylurea 37, and trimethylurea 38. The
thiophenol derivative 13 could be prepared in improved yield.
Primary amines could also be coupled (40−42), enabling the
direct synthesis of derivatives that previously required functional
group protection (42).
We next turned to Buchwald−Hartwig amination of 14,
synthesized from 6-bromo-2-chlorobenzothiazole by heating
with KCN in DMSO, or alternatively in higher yield at room
1
6
temperature using DABCO as a catalyst (Scheme S2).
17,18
Palladium-catalyzed amination using xantphos as a ligand
allowed synthesis of the morpholine analogue in 71% yield vs
2% for the S Ar reaction (Scheme 2). Additionally, the
The new 6-substituted nitriles were all readily converted into
their respective luciferin analogues 3a−41a by reaction with D-
cysteine (Schemes S1−S2). However, it should be noted that
these nitriles are also of direct interest for their mild
biocompatible condensation with N-terminal cysteines and
1
N
thiomorpholine dioxide analogue was obtained in 74% yield, and
the azetidine, in 33% yield (low, but vastly improved over 2%).
Boc-piperazine was similarly accessed, where the Boc group
could be retained or later removed with TFA.
20−23
related aminothiols.
A set of 36 new luciferin analogues was then evaluated in burst
bioluminescence assays with purified firefly luciferase (Figures 2;
Scheme 2. Buchwald−Hartwig Substitution of 6-Bromo-2-
cyanobenzothiazole with a Wide Variety of Partners
Figure 2. Bioluminescence emission from WT luciferase with (A)
alkylamino luciferins; (B) putative “caged” luciferins; (C) arylamino
luciferins; (D) arylamino luciferins incubated with R218K mutant
luciferase. Note differences in scale between panels.
1
2,24
S1−S2; Table S1).
This assay was used to determine whether
the new analogues have the capacity for light emission.
Unsurprisingly, monoalkyl amine 40a was the brightest, while
azetidine 4a led the new cyclic secondary amines (Figure 2a, 3).
25
Although disparate structures such as azepane 7a, thiomorpho-
line 9a, piperazine 16b, and trifluoroethylamine 41a were all
good emitters, the thiomorpholine dioxide analogue 15a was
unexpectedly only weakly luminescent (Figures 2a; S1−S2),
despite its fluorescence (Table S2). Potentially, accommodation
of the bulky sulfone group requires twisting of the amine within
the enzyme pocket, lowering the quantum yield. Such differences
could be exploited for the development of orthogonal luciferase
2
4,26−28
substrates
and for the creation and modulation of
8
selective luminogenic reporters.
B
DOI: 10.1021/acs.orglett.7b02806
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
brightest of the new substrates in this context was the allylamine
4
0a (Figure S5). None of the new dialkylamines was brighter
than CycLuc2, but the azetidine, azepane, and thiomorpholine
analogues 4a, 7a, and 9a yielded higher photon flux than D-
5
luciferin or the 6′-dimethylamino analogue. The polar morpho-
line and piperazine analogues 8a and 16b are substantially
weaker emitters in cells, perhaps reflective of their relatively poor
permeability across the cell membrane, and in the case of
piperazine, a higher K than the more lipophilic aminoluciferins
m
(
Table S3). In cells expressing the R218K luciferase, bio-
luminescence emission from D-luciferin could be exceeded by
many of the alkyl aminoluciferins. Impressively, this includes the
unconventional 2,6-dimethylaniline derivative 20a, even though
it is a relatively weak emitter.
In summary, we have developed a two-step synthesis of
luciferin analogues from a common intermediate. The essential
2
5
Figure 3. Newly synthesized luminogenic substrates.
6
′-donating group can be easily modified, enabling investigation
of its role and supporting the development of many new
substrates, inhibitors, and potential probes. Novel 6′-aniline
analogues can be inhibitors or luminescent substrates, depending
on the nature of the aryl substituent, and also hold potential as
In contrast to alkylamines, the presence of arylamine EDGs in
dyes is generally associated with negligible fluorescence.
However, fluorescence can be observed in rigid, viscous, or
11,15,29,30
15,31−33
7
,30
nonpolar environments (Table S2).
We suspected that
sensors of oxidative species.
Furthermore, numerous other
the luciferase active site might serve as a suitably rigid
environment for luminescence. Although most of the aniline
analogues were essentially nonluminescent, the subset of ortho-
substituted anilines, expected to experience hindered rotation,
was indeed emissive: 2,6-dimethylaniline 20a, indoline 29a, and
“
caged” luciferins can be directly accessed, and importantly not
all are dark. This work thus pushes the boundaries of what can be
considered a luciferin and suggests new avenues to exploit this
light-emitting chemistry for detecting and imaging chemical
reactivity and biological processes.
2-ethylaniline 18a (Figures 2c, 3). Furthermore, a substantial
increase in luminescence for 20a was observed with a mutant
luciferase that is known to better accommodate many synthetic
luciferins (Figures 2d, S2), suggesting that further enhancement
of 6′-arylamine luminescence is possible by alteration of the
substrate pocket.
ASSOCIATED CONTENT
■
*
S
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.7b02806.
Interestingly, not all “caged” analogs are completely dark
Experimental procedures, supplemental tables and figures,
and compound spectra (PDF)
(
Figure 2b; Figures S1−S2). In particular, the dimethylurea
derivative 37a was luminescent and brighter than 15a (Figures
S1−S2). Several carboxamide analogues also had weak emission
AUTHOR INFORMATION
(
>1000-fold weaker than D-luciferin, but still >10-fold over
■
*
background). In contrast, all of the new thio analogues are dark,
Corresponding Author
despite the fluorescence of the S-methyl analogue 10a (Table
E-mail: Stephen.miller@umassmed.edu.
1
4,15
S2).
Indeed, thioethers 10a and 13a are potent luciferase
ORCID
inhibitors, as are the nonemissive aryl amines (Figure S3). On the
other hand, the thiomethyl sulfoxide 11a and sulfone 12a are
only weakly inhibitory, suggesting a potential strategy for
constructing bioluminescent sensors of S-oxidation based on
relief of luciferase inhibition.
Stephen C. Miller: 0000-0001-7154-7757
Notes
The authors declare no competing financial interest.
Finally, we evaluated the new panel of luciferin analogues
Figure 3) as substrates for luciferase in live Chinese hamster
ovary (CHO) cells (Figures 4, S4). Unlike the in vitro assay, this
requires the luciferin analogue to cross the cell membrane. The
ACKNOWLEDGMENTS
(
■
This work was supported by the US National Institutes of Health
(DA039961 and EB020243). S.T.A. was supported by
F31GM016586.
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DOI: 10.1021/acs.orglett.7b02806
Org. Lett. XXXX, XXX, XXX−XXX