fireflies are beetles 
 that have evolved the remarkable ability 
 to emit visible light based on a chemical reaction . instead of a photon 
 of light , firefly
luciferase uses adenosine triphosphate and the chemical 
 energy of oxygen to convert its substrate d - luciferin into 
 an excited - state molecule of oxyluciferin ( figure 1a ) .
this bioluminescent reaction 
 has been widely used as a biological reporter both in vitro and in vivo .
although 
 bioluminescence has much lower background than fluorescence and is 
 more sensitive for in vivo imaging , it has been limited by the relative 
 lack of luciferases and luciferins compared to the broad palette of 
 fluorescent probes . in nature ,
d - luciferin is the only 
 luminogenic substrate 
 for beetle luciferases . over the past few years , many new luciferin 
 analogues have been described , including several that yield peak light 
 emission well into the red .
synthetic luciferins thus have the potential to extend bioluminescence 
 imaging to wavelengths where tissue is more transparent to light . 

however , there is already a significant red and near - infrared component 
 to luciferase emission with d - luciferin , and shifting the 
 peak wavelength does not necessarily mean that the overall level of 
 red light has actually increased .
the synthetic luciferin 
 cycluc1 ( figure 1 ) performs better than d - luciferin for bioluminescence imaging in live mice , primarily 
 due to its improved ability to access the luciferase rather than a 
 red - shift in light emission .
therefore , 
 substrates that combine this ready access to intracellular luciferase 
 in live cells with a red - shift in total emission are expected to be 
 candidates to further improve in vivo performance .
structural differences between luciferins could also 
 potentially 
 allow the creation of orthogonal luciferases .
although mutant firefly 
 luciferases that exhibit luciferin selectivity have been reported , d - luciferin remains a light - emitting substrate .
surprisingly , recent work has identified the drosophila fatty acyl - coa synthetase cg6178 as a latent luciferase that emits 
 light with cycluc2 but not d - luciferin . while this demonstrates that it is possible to retain luciferase 
 activity in beetle luciferase homologues that are unresponsive to d - luciferin , higher rates of photon emission are desirable for 
 use as reporters . with all of these considerations in mind , 
 we synthesized compact , 
 rigid aminoluciferins modeled after cycluc1 and cycluc2 ( figure 1 ) .
we found that these new substrates 
 could greatly increase the total photon flux of near - ir light from 
 live luciferase - expressing cells over d - luciferin .
moreover , 
 high photon flux was observed from a newly identified mutant luciferase 
 that gave virtually no light emission with the natural substrate . 

chemical modification of the luciferin substrate can thus extend the 
 scope of bioluminescence beyond what is possible with d - luciferin .
( a ) firefly luciferase catalyzes the adenylation and oxidation 
 of its native substrate d - luciferin to emit a photon of light . 

the synthetic 
 aminoluciferins cycluc1 and cycluc2 have 5,6-fused 
 five - membered indoline rings ( figure 1b ) . to 
 evaluate the effect of this ring fusion on bioluminescence , we synthesized 
 new luciferin analogues with fused six - membered rings of varying composition . 

luciferin analogues cycluc3 and cycluc4 , containing a six - membered 
 oxazine ring , were readily accessed following a slight modification 
 of the cycluc1 synthesis paradigm ( scheme 1 ) .
cycluc5 and cycluc6 were designed 
 to emit light at longer wavelengths 
 and to test the scope of substrates that could be accommodated by 
 the luciferase ( scheme 2 ) .
these bulky , lipophilic 
 analogues incorporate 2,2,4-trimethyl - dihydroquinoline , a scaffold 
 that has been widely used to red - shift the emission of many classes 
 of fluorophores , including coumarins , rhodamines , and oxazines .
attempted synthesis of intermediate 16 by 
 trifluoroacetylation of the corresponding nitroarene as in scheme 1 failed , presumably due to a combination of steric 
 hindrance and electronic deactivation .
we therefore synthesized the boc - protected compound 14 , which suffers the same steric hindrance 
 but was anticipated to be less electronically deactivated .
gratifyingly , 
 trifluoroacetylation of this intermediate was successful , and subsequent tfa deprotection readily afforded 16 .
the corresponding saturated tetrahydroquinoline 
 aminoluciferin 
 analogues cycluc7 and cycluc8 were synthesized using the same general 
 synthetic route as cycluc1cycluc4 ( figure 1c and scheme 3 ) .
surprisingly , attempts to access 
 the 5,6-fused 
 ring  differing from cycluc1 by only a single methylene  primarily 
 yielded the 6,7-fused product in a > 8:1 ratio . 
 nonetheless , 
 elaboration of 32b to the 5,6-fused cyclic 
 alkylaminoluciferins cycluc7 and cycluc8 proceeded uneventfully . 

the 6,7-fused product 32a was similarly 
 converted into 6,7-fused cyclic alkylaminoluciferins 
 cycluc9 and cycluc10 .
finally , 5,6-fused cyclic aminoluciferins 
 cycluc11 and cycluc12 were synthesized as bulkier and more lipophilic 
 analogues of cycluc7 and cycluc8 , where the gem - dimethyl 
 substituents also direct exclusive formation of the 5,6-fused 
 isomers ( figure 1c and scheme 4 ) . at 
 the outset of this 
 work , it was anticipated that some subset of the rigid luciferin analogues 
 would not be well accommodated by the luciferase .
however , all of 
 the new aminoluciferin analogues are substrates , further underscoring 
 the generality of the light emission chemistry and the tolerance of 
 luciferase for modifications . when purified firefly luciferase 
 is treated with d - luciferin or an aminoluciferin , a 
 high initial rate of light emission ( burst ) is observed in the first 
 few seconds , which is then followed by a substantial decrease in the 
 rate of sustained light output .
this effect is more pronounced for 
 high - affinity aminoluciferins than for d - luciferin 
 itself , and all of the new substrates exhibited this same general 
 behavior under saturating substrate conditions ( figure 2 and supplementary figure 3 ) .
relative 
 to d - luciferin with the wild - type ( wt ) luciferase , the initial 
 rate of photon flux ranged from a high of 43% for cycluc4 to a low 
 of 2% for cycluc11 .
this initial rate is high compared to luciferin 
 analogues in which the benzothiazole has been replaced .
however , aminoluciferins 
 have reduced emission 1 min after substrate addition , consistent with 
 product inhibition as the primary factor limiting the light emission 
 in vitro ( figure 2 and supplementary figure 3 ) .
initial and sustained 
 bioluminescence from luciferin analogues 
 ( 250 m ) with purified wt firefly luciferase ( 0.2 nm ) : ( a ) initial 
 rates of emission relative to d - luciferin and ( b ) relative 
 emission 1 min after substrate addition .
peak bioluminescence 
 emission for the aminoluciferins ranged from 594 to 642 nm ( figure 3 , supplementary table 1 , and 
 supplementary figure 1 ) .
as expected , cycluc6 yielded strongly 
 red - shifted bioluminescence ( 636 nm ) , exceeding that of red - emitting 
 mutant firefly luciferases , railroad 
 worm ( beetle ) luciferase , and the red - shifted 
 emission from 6-me2nlh2 .
however , we were surprised to find that cycluc10 yielded 
 an even more red - shifted peak ( 642 nm ) . to determine whether these 
 differences are inherent to each luciferin , we measured the fluorescence 
 emission wavelengths of the substrates .
the fluorescence and bioluminescence 
 emission wavelengths of aminoluciferins were found to be strongly 
 correlated , suggesting that the bioluminescence wavelength is primarily 
 dictated by the photophysical properties of the luciferin ( supplementary figure 2 ) .
bioluminescence is red - shifted 
 by 5075 nm from the substrate fluorescence in phosphate - buffered 
 saline , which is expected since the oxyluciferin emitter produced 
 in the luciferase has increased conjugation with respect to the substrate .
the fluorescence emission of acyclic monoalkylated aminoluciferins 
 generally ranges from 520 to 540 nm , more 
 red - shifted than that of 6-aminoluciferin ( 517 nm ) but 
 less so than for dialkylated substrates such as 6-me2nlh2 ( 554 nm ) .
cycluc6 fluoresces at still longer wavelength 
 ( 567 nm ) , while cycluc10 is the most red - shifted of all the luciferin 
 analogues ( 576 nm ) . while this was initially surprising ,
we have previously 
 found that the luciferase active - site mutant r218k increases the rate 
 of light emission from cycluc1 , cycluc2 , 6-menhlh2 , and 6-me2nlh2 .
this effect is not particularly selective , as it is observed 
 with all of the aminoluciferins ( figures 4 and 5 , supplementary 
 figure 3 ) .
for instance , cycluc7 exhibits higher initial and 
 sustained emission rates with r218k compared to wt ( figure 4 ) .
the r218k mutant also resulted in a slight red - shift 
 in bioluminescence for most substrates , pushing the maximal emission 
 wavelength for cycluc10 to 648 nm ( supplementary 
 table 1 and supplementary figure 1 ) .
light emission from d - luciferin and cycluc7 ( 250 m ) 
 treated with equal amounts of purified wt and mutant luciferases ( 0.2 
 nm ) .
the assay was performed in triplicate and is represented as the 
 mean  sem .
initial and sustained 
 bioluminescence from each luciferin ( 250 
 m ) with equal amounts of three different purified luciferases 
 ( 0.2 nm , see text ) : ( a ) peak intensity within the first 2 s of substrate 
 addition and ( b ) sustained light emission 1 min post - injection .
the 
 assay was performed in triplicate and is represented as the mean  
 sem . in principle , chemical 
 and structural differences in luciferin substrates could be exploited 
 to create new orthogonal luciferases . however , active - site mutations 
 such as r218k and l286 m raise the km of d - luciferin but do not prevent its utilization as a substrate .
a more selective point mutant , s347a , raises 
 the km and lowers the rate of emission 
 from d - luciferin , possibly because it removes a hydrogen - bonding 
 interaction with the benzothiazole nitrogen that may be more 
 important for d - luciferin binding and orientation than for 
 aminoluciferins . yet
thus , previous work 
 has not established whether high luciferase activity can be retained 
 in luciferase mutants that do not yield light emission from d - luciferin . here
we find that the combination of r218k , l286 m , 
 and s347a renders d - luciferin essentially inactive .
the purified 
 triple - mutant luciferase dramatically reduced both the initial and 
 sustained rates of photon emission from d - luciferin by > 10  000-fold 
 ( figure 4 and supplementary 
 figure 3 ) .
gratifyingly , this is not due to a lack of luciferase 
 activity , as the photon flux from cycluc2 , cycluc7 , and cycluc11 actually 
 increased compared to that of the wt enzyme ( figure 5 and supplementary figure 3 ) .
cycluc7 
 is the optimal substrate for the triple - mutant luciferase in vitro , 
 achieving 46% of the initial rate of d - luciferin with wt 
 luciferase .
this result demonstrates that high luciferase activity 
 can be maintained in a luciferase mutant that is essentially unresponsive 
 to the native substrate d - luciferin .
furthermore , product 
 inhibition has largely been eliminated , as there is little diminution 
 in flux after the initial burst ( figures 4 and 5 and supplementary figure 3 ) .
we next compared the aminoluciferins to d - luciferin 
 in live wt luciferase - expressing chinese hamster ovary ( cho ) cells . 
 under these conditions
in marked contrast to what is observed 
 in vitro , almost all of the alkylated aminoluciferins yield 
 higher flux than d - luciferin when assayed at a concentration 
 < 30 m ( figure 6 and supplementary figure 4 ) . at high substrate concentration ( 250 
 m ) ,
the relative emission from d - luciferin is still 
 equaled or exceeded by those of cycluc1 , cycluc10 , and cycluc12 ( figure 6 ) .
if the cell membrane is removed by cell 
 lysis , d - luciferin is the superior substrate at both high 
 and low doses , suggesting that substrate access is the primary factor 
 limiting luciferins in live cells ( supplementary 
 figure 5 ) . photon flux from luciferase - expressing cho cells .
( a ) 
 live cells 
 expressing the indicated luciferase and treated with high or low doses 
 of luciferin .
( b ) comparison of total and near - ir photon flux from 
 wt luciferase with the indicated luciferins .
( c , d ) comparison of photon 
 flux from wt and triple - mutant luciferase with d - luciferin 
 or cycluc7 in live cells ( c ) or lysed cells ( d ) .
all assays were performed 
 in triplicate and are represented as the mean  sem .
to 
 assess the near - ir emission from each luciferin in live cells , we 
 measured the relative photon flux passing through a cy5.5 filter ( 695770 
 nm ) .
all of the aminoluciferins exhibited greater relative photon 
 flux in the near - ir than d - luciferin ( figure 6b and supplementary figure 6 ) . 

for every substrate except 6-aminoluciferin and cycluc3 , 
 this translated to a higher total near - ir photon flux from live cells 
 than d - luciferin under both low - dose and high - dose conditions 
 ( supplementary figure 6 ) .
cycluc10 gave 
 the greatest fraction of near - ir light emission : 13.9% of the total 
 photon flux , > 10-fold higher than that of d - luciferin 
 ( figure 6b ) .
however , total near - ir flux of 
 cycluc10 was 
 slightly exceeded by that of cycluc12 .
thus , the substrate that yields 
 the highest cellular near - ir light emission is a function of substrate 
 access ( affinity and cell permeability ) as well as wavelength .
transfection 
 of cho cells with r218k luciferase instead of wt luciferase improved 
 relative photon flux from synthetic luciferins compared to d - luciferin ( figure 6 and supplementary figure 4 ) .
cycluc2 yielded the highest signal 
 in live cells , and most alkylated aminoluciferins were superior 
 to d - luciferin at both low and high substrate concentrations 
 ( figure 6 and supplementary 
 figure 4 ) . further highlighting the importance of factors other 
 than peak emission wavelength on the total emission of red - shifted 
 photons in live cells , cycluc2 yielded the highest cy5.5-filtered 
 signal at 250 m , while cycluc6 was best at low concentration 
 ( figure 6 and supplementary 
 figure 6 ) .
the longer emission wavelength of cycluc6 ( figure 3 ) and its higher cell permeability likely lead to 
 its superior flux at low concentrations , while the higher maximal 
 rate of photon emission for cycluc2 ultimately prevails at high substrate 
 concentration ( figure 5 ) .
no signal above background 
 could be measured from live cells expressing the triple - mutant luciferase 
 after treatment with d - luciferin ( figure 6c ) .
in contrast , cycluc2 , cycluc7 , and cycluc11 achieve high 
 photon flux ( figure 6 ) .
the triple mutant possesses 
 lower affinity for its substrates compared to wt or r218k luciferase , 
 yielding reduced photon flux at low substrate concentration ( figure 6 and supplementary figure 4 ) . however , in lysed cells , the lower affinity of the triple mutant 
 improved the signal from cycluc7 and cycluc11 due to its lessened 
 product inhibition ( figure 6d and supplementary figure 5 ) .
cycluc7 is the best 
 substrate in cell lysates , achieving 20% of the d - luciferin signal with the wt luciferase ( figure 6d and supplementary figure 5 ) . 

mutant and wt luciferase protein expression is equivalent in transfected 
 cells by western blot ( supplementary figure 7 ) , and the differences between luciferases in cell lysates ( supplementary figure 5 ) generally mirrors what 
 is observed with equal concentrations of purified proteins in vitro 
 ( figure 5 ) .
the triple - mutant luciferase is 
 thus a significant step toward orthogonal bioluminescent reporters 
 of gene expression in both live and lysed cells , as it yields high 
 light output with ( alkylated ) aminoluciferins but little to 
 no signal with d - luciferin .
we speculate that the triple 
 mutant primarily discriminates between substrates by lowering substrate 
 affinity and removing an interaction important for orienting the native 
 substrate ( s347 ) .
given that the mutations 
 that comprise the triple mutant were originally identified to individually 
 improve luciferase activity with cycluc1 , it is likely that further improvements in selectivity and function 
 are possible for this broadened palette of luciferins .
ironically , 
 the identification of a luciferase that emits strongly with d - luciferin but does not respond to synthetic luciferins has been 
 more elusive .
luciferase 
 reporter pairs will therefore require further work , perhaps by eschewing d - luciferin altogether in favor of two or more synthetic substrates .
there is reason to expect that substrate performance 
 in live cells , 
 rather than with purified protein or cell lysates , is more predictive 
 of in vivo behavior . in recent collaborative work
we have found that 
 cycluc1 allows dramatically improved bioluminescence imaging in live 
 mice compared to the standard imaging conditions with d - luciferin .
tumor cells can be imaged with 20200-fold 
 less substrate than d - luciferin , and luciferase expression 
 deep in the brain that can not be detected with d - luciferin 
 is detectable with cycluc1 .
many of the 
 substrates described here provide higher total and red - shifted photon 
 flux in live cells , suggesting that they may also have superior properties 
 for in vivo imaging .
differences in substrate affinity , lipophilicity , 
 and functionality are also anticipated to affect the pharmacokinetics 
 of the luciferins in vivo , perhaps allowing tuning of bioluminescent 
 half - lives and/or tissue distribution .
moreover , we have found that 
 mutation of luciferase can essentially eliminate light output from 
 the native d - luciferin substrate while retaining or improving 
 light emission from one or more aminoluciferin substrates to levels 
 comparable to or , in live cells , superior to that of d - luciferin 
 with the wt luciferase .
thus , these synthetic luciferins and mutant 
 luciferases not only expand the palette of luminogenic molecules but 
 transcend the emission properties of d - luciferin and firefly 
 luciferase .
they are therefore expected to have significant potential 
 for bioluminescence imaging applications both in vitro and in vivo .
 firefly 
 luciferase adenylates and oxidizes d - luciferin 
 to chemically generate visible light and is widely used for biological 
 assays and imaging . here 
 we show that both luciferase and luciferin 
 can be reengineered to extend the scope of this light - emitting reaction . 
 d - luciferin can be replaced by synthetic luciferin analogues 
 that increase near - infrared photon flux > 10-fold over that of d - luciferin in live luciferase - expressing cells . 
 firefly luciferase 
 can be mutated to accept and utilize rigid aminoluciferins with 
 high activity in both live and lysed cells yet exhibit 10  000-fold 
 selectivity over the natural luciferase substrate . 
 these new luciferin 
 analogues thus pave the way to an extended family of bioluminescent 
 reporters . 
Introduction
Results and Discussion
Conclusion