Rapid and scalable assembly of firefly luciferase substrates

Article abstract of DOI:10.1039/c4ob02529f

Bioluminescence imaging with luciferase-luciferin pairs is a popular method for visualizing biological processes in vivo. Unfortunately, most luciferins are difficult to access and remain prohibitively expensive for some imaging applications. Here we report cost-effective and efficient syntheses of d-luciferin and 6′-aminoluciferin, two widely used bioluminescent substrates. Our approach employs inexpensive anilines and Appel's salt to generate the luciferin cores in a single pot. Additionally, the syntheses are scalable and can provide multi-gram quantities of both substrates. The streamlined production and improved accessibility of luciferin reagents will bolster in vivo imaging efforts. This journal is

Full text of DOI:10.1039/c4ob02529f

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Biomolecular Chemistry
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Rapid and scalable assembly of firefly luciferase
substrates†
Cite this: DOI: 10.1039/c4ob02529f
a
a
a,b,c
David C. McCutcheon, William B. Porterfield and Jennifer A. Prescher*
Bioluminescence imaging with luciferase–luciferin pairs is a popular method for visualizing biological
processes in vivo. Unfortunately, most luciferins are difficult to access and remain prohibitively expensive
for some imaging applications. Here we report cost-effective and efficient syntheses of D-luciferin and
6’-aminoluciferin, two widely used bioluminescent substrates. Our approach employs inexpensive anilines
and Appel’s salt to generate the luciferin cores in a single pot. Additionally, the syntheses are scalable and
can provide multi-gram quantities of both substrates. The streamlined production and improved accessi-
bility of luciferin reagents will bolster in vivo imaging efforts.
Received 3rd December 2014,
Accepted 9th December 2014
DOI: 10.1039/c4ob02529f
www.rsc.org/obc
4
Bioluminescence imaging is among the most powerful tech- light emission with a variety of D-luciferin analogs, including
1
,2
5–7
8,9
niques for visualizing cells and other biological features. At the 6′-amino variant (2) and related cyclic amino analogs,
4
,10–12
the heart of this technology are enzymes (luciferases) that cata- heterocyclic derivatives,
lyze the oxidation of small molecule substrates (luciferins), systems. Some of these molecules emit different colours of
releasing visible light in the process (Fig. 1). Bioluminescent light and are thus useful for multi-spectral imaging. Other
photons can penetrate tissues—even in intact rodents— analogs are more cell permeant than D-luciferin, making
making this technique well suited for imaging in vivo and them attractive for sensitive imaging applications. “Caged”
other complex environments. Indeed, bioluminescence has forms of luciferin can also be used in conjunction with Fluc to
been widely used to monitor cell proliferation and migration, measure enzyme activities and monitor cell–cell interactions
gene expression patterns, and enzyme activities in a variety of in vivo.
and luciferins with extended pi
1
3
2
14
1
5
1
6
1
,2
preclinical models.
Given their broad utility and critical roles in biological
The most prevalent luciferase–luciferin pair in biological imaging, it is surprising that luciferins remain difficult to
1
7,18
imaging originates from the firefly. Firefly luciferase (Fluc) access in an expedient and cost-effective manner.
The first
catalyzes the oxidation of D-luciferin (1), producing yellow- synthesis of D-luciferin reported by White in 1961 was seven
3
green light at room temperature (Fig. 1). Fluc can be steps and provided the compound in only 6% overall
1
9–21
expressed in many cell and tissue types, and when D-luciferin yield.
Iterative improvements to this route have been
is administered, photons are produced. Fluc can also catalyze reported over the past several decades, although the basic
strategy remains the same: generate a 2-carboxy-substituted
benzothiazole, replace the carboxylate with a cyano group, and
condense
D-cysteine.
the
resulting
cyanobenzothiazole
with
1
7,22–24
While reliable, these routes remain low yield-
ing and unnecessarily long. Functionalized cyanobenzothia-
zoles can be purchased directly from commercial suppliers
and condensed with cysteine to prepare luciferins in a single
step. However, these reagents are expensive and not practical
for studies requiring multi-gram quantities of a light-emitting
substrate.
Fig. 1 Firefly luciferase (Fluc) catalyzes the oxidation of D-luciferin (1),
its native substrate, and various analogs, including the 6’-amino com-
pound (2). Light is produced during the enzymatic reactions.
We recently reported an alternative method to synthesize
1
0
D-luciferin. This approach relies on benzothiazole formation
from aniline starting materials and the dithiazolium chloride
salt 3 (also known as Appel’s salt, Scheme 1). Appel’s salt con-
denses readily with arylamines, and the resulting iminodithia-
zoles can be opened with a variety of nucleophiles, generating
cyanothioformamides; these moieties can then be cyclized to
a
Departments of Chemistry, University of California, Irvine, CA, USA.
E-mail: jpresche@uci.edu
b
Molecular Biology & Biochemistry, University of California, Irvine, CA, USA
c
Pharmaceutical Sciences, University of California, Irvine, CA, USA
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4ob02529f
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also applicable to synthesizing D-luciferin analogs, including
aminoluciferin (2).
In early attempts to optimize our published synthesis, we
focused on the dithiazole fragmentation procedure (step 2,
Scheme 2). This reaction employed DBU, a reagent that is
notoriously difficult to remove from reaction mixtures via
extraction or flash column chromatography. Indeed, in our
hands, multiple chromatographic separations were required to
isolate 5 from residual DBU upon scale up; this purification
scheme quickly became impractical. Since DBU functions to
break apart the dithiazole, we reasoned that other, more tract-
able nucleophiles could be used in its place. Initial attempts
with triphenyl phosphine and sodium sulphide, though,
proved unsuccessful. Triphenyl phosphine (and the corres-
ponding oxide) was similarly difficult to remove from the reac-
tion mixture. Sodium sulphide, by contrast, was easy to
Appel’s salt can remove, but converted 5 to an N-aryl dithiooxamide (S1, ESI
Scheme 1 Retrosynthetic analysis of D-luciferin (1) and its 6’ amino
analogue (2). Both molecules can be accessed from aniline starting
materials and Appel’s salt (3).
2
5–27
afford benzothiazole products (Scheme 1).
also be prepared in bulk quantities (>500 g from simple Scheme S1†) instead of the desired formamide 6. We attribu-
addition of sulphur monochloride to chloroacetonitrile) and ted this result to sulphide being a strong, yet sterically unen-
stored indefinitely, making it attractive for large-scale work. To cumbered nucleophile, capable of additional reactivity with
prepare D-luciferin via this approach, we treated p-anisidine (4) cyanothioformamides. Fortunately, a less nucleophilic anion—
2
−
with Appel’s salt and fragmented the resulting dithiazole with thisosulfate (S O )—provided 6 in excellent yield (Scheme 3)
2
3
DBU. The product, thioformamide 6, was then cyclized using with no dithiooxamide observed. Compound 6 also precipi-
modern C–H activation chemistry to afford cyanobenzothiazole tated from the reaction mixture and could be collected by fil-
7
. Subsequent protecting group removal and cysteine conden- tration, eliminating the need for column chromatography. We
sation ultimately provided the desired luciferin 1. Altogether, further discovered that both the formation and fragmentation
this route provided D-luciferin in just 5 steps and 42% overall of 5 (with thiosulfate) could be performed in a single pot,
yield, marked improvements over previous syntheses. We were eliminating another onerous purification step (Scheme 3).
also able to use this procedure to access unique luciferin ana- With 6 in hand, we completed the synthesis of D-luciferin (1)
1
0
logues. For example, when ortho-substituted anilines (e.g., using our previously published method. Collectively, this
o-NH and o-OH) were used in place of 4, benzimidazole and route improved the overall yield of D-luciferin (60%) and shor-
benzoxazole luciferins were isolated.
Despite the initial successes and notable advances of our entirely, along with two chromatographic separations.
published route, limitations remained. The synthesis
In addition to the fragmentation step, we recognized that
2
1
0,12
tened the time involved by eliminating one synthetic step
employed expensive metal reagents and large volumes of formation of cyanobenzothiazole 7 in the original synthesis
solvent. Several time-intensive purification steps were also (step 3, Scheme 2) was not optimal. This step required expen-
required. These features precluded the easy preparation of sive metal reagents, non-ideal solvents, excess TBAB, and
2
8
29–31
large quantities (>10 g) of 1. Here we report an improved, dilute conditions.
Based on work from Rees,
we
streamlined synthesis of D-luciferin (1) that requires only two reasoned that 7 might be attainable directly from 5 via thermo-
steps from inexpensive starting materials. This chemistry is lysis. This would eliminate the need for metal-mediated cycli-
10
Scheme 2 Previously reported synthesis of D-luciferin.
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Scheme 3 Synthesis of D-luciferin (1) using an initial one-pot condensation/fragmentation procedure.
Scheme 4 Synthesis of D-luciferin (1) via Appel’s salt condensation and thermolysis. The iminodithiazole adduct 5 can be isolated following treat-
ment of p-anisidine (4) with Appel’s salt (lower) or carried on directly into the cyclization and deprotection steps (upper).
zation entirely and shorten the overall synthesis (Scheme 4, be routinely produced in a single reaction step), and the route
lower). However, initial attempts to cyclize 5 in refluxing DMF is compatible with a variety of phenol protecting groups.
resulted in degradation or afforded thioformamide 6 as the
We next attempted to apply the improved route to the syn-
major product. The cyclization yield improved dramatically at thesis of aminoluciferin 2. This analogue and related variants
higher temperatures using sulfolane and sealed tubes. Under are attractive for bioluminescence imaging owing to their ease
these conditions, we also observed trace amounts of depro- of derivatization and, in some cases, enhanced cell per-
4
,6,14
tected phenol 8. This prompted us to explore whether 5 could meability.
Initial attempts to access 2 from p-phenylene-
be cyclized and deprotected in a single pot (Scheme 4, lower). diamine, though, were unsuccessful and afforded a complex
Indeed, upon heating 5 in sulfolane and subsequent treatment mixture of products. We therefore moved forward with nitroa-
with pyr·HCl, 8 was isolated in moderate yield (61%), along niline 9 and condensed this material with Appel’s salt 3
with the protected phenol 7 (20%). Re-subjecting 7 to pyr·HCl (Scheme 5A). Attempts to generate benzothiazole 11 via
increased the total yield of 8. Excitingly, the cyclization/depro- thermal cyclization of 10 were low yielding, likely due to the
tection sequence could also be coupled with the first synthetic electron-deficient nature of the aromatic ring. Benzothiazole
step: formation of imine 5 (as in Scheme 3). When 4 was 11 was accessible on gram-scale, though, using a route similar
treated with Appel’s salt, followed by rigorous heating and to the one shown in Scheme 3. Aniline 9 was treated with
pyr·HCl, 8 and 7 were isolated in 51 and 21 percent yields, Appel’s salt (3), followed by sodium thiosulfate (to fragment
respectively (Scheme 4, upper). Subsequent condensation of 8 the dithiazole intermediate) to provide 10. Subsequent Pd-
with D-cysteine provided luciferin 1. Thus, D-luciferin (1) can mediated cyclization generated 11 in 74% yield (Scheme 5C).
be prepared from inexpensive starting materials in only two Reduction of the nitro group followed by cysteine conden-
steps and 44% overall yield. To our knowledge, this represents sation ultimately afforded aminoluciferin 2.
the shortest synthesis of D-luciferin from simple anilines to
We were able to further improve the aminoluciferin syn-
date. Moreover, these conditions are scalable (>20 g of 8 can thesis drawing inspiration from our earlier work. We pre-
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Scheme 5 Synthesis of aminoluciferin 2. (A) Cyanobenzothiazole intermediate 11 was accessed via palladium-mediated cyclization (top) or metal-
free cyclization (bottom). (B) A reduction-condensation sequence was used to prepare 2.
viously demonstrated that ortho-nucleophiles can trap Appel’s Overman, Jarvo, Rychnovsky, and Nowick labs at UCI for
1
0
salt adducts to afford cyclized products. Thus, we reasoned reagents, along with members of the Prescher lab for helpful
that installing a thiol ortho to the amine in 9 would enable discussions.
more facile access to 6′-aminoluciferin (2, Scheme 5). Attempts
to prepare aminothiol 14 from the corresponding ortho-chloro
aniline via nucleophilic aromatic substitution were not suc-
cessful. However, 14 could be generated in situ by incubating Notes and references
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2
2
3
4
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