Journal of the American Chemical Society
Communication
Analogues 2−4, like D-luciferin, contain a unique 2−2′
linkage of heteroaromatic rings. This connectivity is scarcely
observed in known natural products, and facile methods to
prepare such linkages are rare.19 In fact, the first synthesis of D-
luciferin, reported by White in 1963, is basically the same route
used to produce nearly all luciferins today.20 This synthesis
proceeds through a cyanobenzothiazole intermediate (5),
which, upon protecting group removal, can be condensed
with D-cysteine to provide the native luciferin. This
condensation is both mild and high-yielding, making it an
appealing method for late-stage introduction of the luciferin
stereocenter. Unfortunately, the White synthesis of 5 requires
seven steps and is not amenable to heteroatom substitutions.
Recognizing the utility of cyano heterocycles for luciferin
production, we aimed to identify a more expedient route to 5,
along with the analogous cyanobenzimidazole 6. Condensation
of these scaffolds with either D-cysteine or diaminopropionic
acid could provide the entire set of luciferin analogues (2−4).
To access 5 and 6 in tandem, we were drawn to the
dithiazolium chloride 9 (Scheme 1). This reagent, also known
Scheme 2. Synthesis of Luciferin Scaffolds: (a) D-Luciferin
(1) and the Imidazoline Analogue 3; (b) Benzimidazole
Analogues 2 and 4
Scheme 1. Retrosynthetic Analysis of Luciferin Analogues
Cyanobenzimidazole 6 was ultimately demethylated and
condensed with D-cysteine as above to isolate luciferin 2.
Multigram quantities of both luciferins 1 and 2 have been
produced using the routes outlined in Scheme 2, highlighting
the scalability of the approach.
The cyano heterocycles produced with Appel’s salt can be
condensed with a variety of other 1,2-disubstituted nucleophiles
in addition to D-cysteine. We exploited this mode of reactivity
to generate the imidazoline rings present in luciferins 3 and 4.
First, intermediates 12 and 14 were converted into the
corresponding imidates using standard conditions. The
imidates were not isolated but treated directly with
diaminopropionic acid to afford the desired luciferins in
reasonable yield.
as Appel’s salt, has previously been used to synthesize both
benzothiazole and benzimidazole scaffolds from anilines.21
Appel’s salt condenses readily with arylamines, and the resulting
iminodithiazoles can be easily opened with a variety of
nucleophiles.22 If the nucleophile is present on the aniline
itself (as in the case of o-aminoanilines), cyanobenzimidazole
structures can be isolated directly. In the absence of
intramolecular nucleophiles, the dithiazole adduct can be
fragmented with a variety of exogenously supplied reagents.
When amidine bases are used, dithiazole cleavage provides
thioformamides; molecules of this sort can be readily cyclized
to cyanobenzothiazoles.22
To investigate the utility of Appel’s salt for luciferin synthesis,
we first used the reagent to prepare the cyanobenzothiazole 5
(en route to D-luciferin, Scheme 2a). p-Anisidine was treated
with 9 to provide the expected dithiazole adduct 10. This
intermediate was isolable using standard chromatographic
techniques and found to be remarkably shelf stable. Treatment
of 10 with excess DBU produced cyanothioformamide 11 in
excellent yield. Palladium- and copper-mediated cyclization of
this compound generated the key cyanobenzothiazole 5.
Notably, this route to 5 is four steps shorter than the sequence
employed by White and provides the compound in markedly
better yield (84% versus 10% overall). The desired luciferin 1
was eventually isolated by removal of the methyl protecting
group from 5, followed by condensation with D-cysteine under
mild conditions. The functional activity of the isolated luciferin
was also confirmed in light emission assays with Fluc (Figure
S1).
Our initial efforts to characterize luciferins 2 and 4 were
complicated by tautomerism. Benzimidazole scaffolds are
known to undergo rapid N−H isomerization in solution
1
(Figure S2), resulting in significantly broadened H and 13C
NMR signals. We were also concerned that such rapid
tautomerization would suppress bioluminescent light emission
from the analogues. Such quenching behavior has been
observed with other electronically excited benzimidazoles.23,24
To mitigate against potential quenching effects and aid our
structural characterization efforts, we prepared a methylated
version of 2 (Scheme S1). Interestingly, only one N-methyl
regioisomer (15, Scheme S1) was formed in reasonable yield
from intermediate 6.
With the nitrogenous analogues in hand, we assayed the
compounds for light emission with Fluc. Luciferins 2−4 and 15
were incubated with the enzyme, ATP, and coenzyme A (to
reduce product inhibition) at pH 7.4.25 Light emission was
measured using a cooled CCD camera, and representative
images are shown in Figure 2. No photons were detected for
analogue 3, and only minimal light emission was observed with
the related imidazoline 4 at low substrate concentrations. These
reduced intensities may be attributed to poor binding to Fluc,
Encouraged by these results, we next investigated whether
Appel’s salt could provide access to the benzimidazole
intermediate 6 (Scheme 2b). Gratifyingly, this molecule was
isolated in a single step upon incubation of bis-aniline 8 with 9.
In this reaction, the initial dithiazole adduct is likely trapped by
the o-amino substituent of 8, providing the cyclized product.
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dx.doi.org/10.1021/ja301493d | J. Am. Chem. Soc. 2012, 134, 7604−7607