DOI: 10.1002/cbic.201300744

Self-Immolative Bioluminogenic Quinone Luciferins forNAD(P)H Assays and Reducing Capacity-Based CellViability AssaysWenhui Zhou,*[a] Donna Leippe,[b] Sarah Duellman,[b] Mary Sobol,[b] Jolanta Vidugiriene,[b]

Martha O’Brien,[b] John W. Shultz,[b] Joshua J. Kimball,[a] C�line DiBernardo,[a]

Leonard Moothart,[a] Laurent Bernad,[a] James Cali,[b] Dieter H. Klaubert,[a] andPoncho Meisenheimer[a]

Highly sensitive self-cleavable trimethyl lock quinone-luciferinsubstrates for diaphorase were designed and synthesized tomeasure NAD(P)H in biological samples and monitor viablecells via NAD(P)H-dependent cellular oxidoreductase enzymesand their NAD(P)H cofactors.

The pyridine dinucleotide NAD/NADH and its phosphorylatedform (NADP/NADPH) are important electron carriers. Theirfunctions as cofactors are widely recognized in multiple redoxreactions, such as NAD(P)/NAD(P)H-dependent energy meta-bolic pathways[1] or maintenance of intracellular redox status.[2]

Recent research indicates that NAD(P) is also linked to majorsignaling events,[3] DNA repair, chromatin structure/function,cell cycle progression, and transcription. Therefore, the abilityto sense perturbations in NAD(P)/NAD(P)H levels provides aconvenient tool for monitoring oxidative stress response andmetabolic homeostasis and also facilitates the elucidation ofcellular reprogramming mechanisms that include changes inchromatin structure and transcription function.

A number of methods have been utilized for quantificationof NAD(P)/NAD(P)H in various biological samples by absorp-tion,[4] fluorescence,[5] electrochemistry,[6] and HPLC.[7] The mostprevalent methods include the reduction of tetrazolium deriva-tives (MTT, MTS, MTX, etc.) or resazurin (with phenazine salt asan electron mediator or in a diaphorase-coupled enzymatic re-action). However, complicated sample preparation is often re-quired for these spectrometric methods due to three factors:1) interference from light scattering, 2) fluorescence interfer-ence from other compounds in the sample, and 3) co-absorb-ance of other compounds in crude biological samples. We rea-soned that a luciferase-coupled bioluminescent assay[8] would

eliminate steps and offer an opportunity to measure cellular ortissue NAD(P)H levels in a sensitive and simplified manner. Thisled us to target bioluminogenic diaphorase substrates condu-cive to high-throughput screening (HTS).

Quinones,[9] nitro groups,[10] and N-oxides[11] are typical sub-strates for oxidoreductases and might be reduced by accept-ing two electrons from NAD(P)H in a reaction catalyzed by dia-phorase. “Trimethyl lock quinone” substrates particularly ap-pealed to us, considering the facile intramolecular lactonizationthat could lead to the rapid release of a reporter moiety uponreduction (Scheme 1),[12] and we focused on trimethyl lock qui-

none-luciferin molecules to develop a bioluminescent NAD(P)Hdetection method.

Our first attempt was to attach aminoluciferin directly to thetrimethyl lock quinone (1). The synthesis was accomplished asshown in Scheme 2 A. The quinone propionic acid (1 a) wasprepared by lactonization of the commercially available 2,3,5-trimethylhydroquinone and methyl 3,3-dimethyl acrylate inmethanesulfonic acid, followed by oxidation with NBS.[12] Com-pound 1 a was then activated with isobutyl chloroformate and

Scheme 1. Release of luciferin from reduction of trimethyl lock quinonecompound 1.

[a] Dr. W. Zhou, J. J. Kimball, C. DiBernardo, L. Moothart, Dr. L. Bernad,Dr. D. H. Klaubert, Dr. P. MeisenheimerResearch and Development, Promega Biosciences, Inc.277 Granada Drive, San Luis Obispo, CA 93401 (USA)E-mail : wenhui.zhou@promega.com

[b] Dr. D. Leippe, Dr. S. Duellman, M. Sobol, Dr. J. Vidugiriene, Dr. M. O’Brien,Dr. J. W. Shultz, Dr. J. CaliResearch and Development, Promega Corporation2800 Woods Hollow Road, Madison, WI 53711-5399 (USA)

Supporting information for this article is available on the WWW underhttp ://dx.doi.org/10.1002/cbic.201300744.

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coupled to 2-cyano-6-aminobenzothiazole to give 1 b in rela-tively low yields, which varied with the bases used. Yields of1 b improved with the use of N-methyl morpholine (~10 %)over TEA (~3 %). Cyclization of 1 b with d-cysteine under slight-ly basic conditions yielded the desired final compound 1.

Although the tetra-substituted quinone moiety survived theexcess d-cysteine without forming d-cys/quinone Michael addi-tion products, isolated compound 1 unexpectedly decom-posed in aqueous solution. The stability of 1 in acetonitrile andphosphate buffer (pH 6, 7, and 8), monitored by HPLC, indicat-ed that 1 rapidly converted to two unknown compounds witha rate of conversion and ratio that were highly pH dependent.The ratio of the two products varied from about 7:3 (pH 6), to2:8 (pH 7), to almost exclusivly one product at pH 8 after1 hour incubation (see the Supporting Information). Both com-pounds showed the same mass as compound 1. The productin pH 8 buffer was isolated and confirmed to be an amide-cyc-lization product from an intramolecular Michael addition (1 d) ;thus, the first product was likely the corresponding enol inter-mediate (1 c ; Scheme 2 B). We reasoned that, due to the elec-tron deficiency of the luciferin ring, the acidic N�H proton ofthe amide was easily deprotonated under basic conditions tofacilitate the intramolecular Michael addition to form 1 d viathe 1 c enol intermediate.

We modified target 1 to diminish the intramolecular cycliza-tion problem by reducing the acidity of the problematicamide. Compounds 2 and 3 include a p-aminobenzyl ethertraceless linker[13, 14a] between the quinone acid 1 a and the luci-ferin. (Scheme 3 A). The syntheses of quinone-luciferins 2 and 3are described in Scheme 3 B. The quinone propionic acid 1 awas coupled with p-aminobenzylalcohol to give compound 2 aand 3 a in much better yields than for 1 a under the same con-ditions. Although the standard Mitsunobu reaction of alcohol2 a and 6-hydroxyl-2-cyanobenzothiazole failed to give 2 c withPh3P and DEAD, 2 a and 3 a were converted into bromides 2 band 3 b with CBr4/Ph3P in methylene chloride. The subsequentalkylation of 6-hydroxyl-2-cyanobenzothiazole with 2 b and 3 bunder basic Ag2CO3/1,3,5-collidine conditions, followed by cyc-lization with d-cysteine, yielded final compounds 2 and 3. Sta-

bility, assessed by HPLC, showed that compound 2 had no for-mation of the Michael addition product(s) at pH 7 or belowbut about 40 % conversion at pH 8 after 1 hour incubation, in-dicating that 2 is more stable than 1 in aqueous buffer solu-tion. As anticipated, compound 3, with the methyl group in-stead of the acidic proton was stable at various pH buffer con-ditions, confirming that ring cyclization by amide Michael addi-tion was eliminated (Supporting Information).

The reactivity of compound 3 as a diaphorase substrate wasexamined in a two-step assay format (Figure 1). In the diaphor-ase reaction (step 1), classic Clostridium diaphorase (Sigma)was incubated with 3 for 30 min at room temperature in 50 mLof buffer containing 0.5 mm NADH, 10 mm HEPES (pH 7),10 mm MgCl2, and 100 mm ZnCl2. For luciferin detection

Scheme 2. A) Synthesis of trimethyl lock quinone compound 1; B) Amide cyclization of 1 through an intramolecular Michael addition under basic conditionsto form 1 d via intermediate 1 c. a) isobutyl chloroformate/N-methyl morpholine; b) d-Cys/TEA.

Figure 1. The activities of diaphorase with compounds 3–5. Diaphorase(0.5 U mL�1) and 0.5 mm NADH in a volume of 50 mL were incubated with20 mm compounds 3–5 in 10 mm HEPES, pH 7. After 20 min of incubation,luciferin detection reagent (LDR, 50 mL) was added, and the luminescentsignal was measured at various time periods. Luminescent signals for com-pounds 3 (&), 4 (~), and 5 (*) in the presence of diaphorase and NADH;background controls in the absence of NADH for 3 (&), 4 (~), and 5 (*),respectively.

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(step 2), 50 mL of a luciferin detection reagent (Promega) wasadded to the diaphorase reaction, and the luminescent signalwas measured on a Veritas 96 microplate luminometer. The re-sulting luminescence in the presence of diaphorase and NADHwas significantly above the background control signal andreached a plateau within 20 min, suggesting that compound 3was a substrate for diaphorase and produced luciferin by spon-taneous lactonization and subsequent 1,6-elimination uponquinone reduction. Although compound 3 exhibited excellentreactivity toward diaphorase, the high background biolumines-cence, equivalent to 0.3 % luciferin, was possibly due to hydro-lysis of the aromatic amide of the traceless linker, leading torelease of a trace amount of luciferin. The high background of3 prompted exploration of alternative traceless linkers. Wefurther modified the quinone-luciferin target with an N,N’-di-methylethylenediamine spacer[14, 15] (Scheme 4 A).

The syntheses of compounds 4–5 are shown in Scheme 4 B.The quinone propionic acid 1 a was coupled with mono-Boc-protected diamine to give compound 4 a and 5 a under theprevious conditions. Boc was removed with TFA to yield 4 band 5 b. When 6-hydroxyl-2-cyanobenzothiazole was first acti-vated with p-nitrobenzylchloroformate or triphosgene, fol-lowed by treatment with 4 b, no desired product (4 c) was ob-tained. It was noticed that 4 b and 5 b were stable under acidicconditions but unstable under basic conditions, most likely be-cause an intramolecular Michael addition occurred under basicconditions. Solubility problems prevented the use of electro-philicly activated forms of 6-hydroxyl-2-cyanobenzo-thiazole,and an umpolung reaction was attempted. The quinone-aminewas activated with large excess of phosgene to generate com-

pounds 4 c and 5 c with excellent yields (>80 %). Compounds4 c and 5 c reacted with 6-hydroxyl-2-cyanobenzothiazole togive compounds 4 d and 5 d in acceptable yields. Finally, cycli-zation of 4 d and 5 d with d-cysteine yielded compounds 4 and5.

The reactivity of compounds 4–5 toward Clostridium dia-phorase was measured with the previously described two-stepassay. Although the luminescent signals were slightly slower toreach a plateau compared to 3, the stabilized signals after30 min were at the same levels as 3, suggesting that 4 and 5are good substrates for diaphorase, and the release of luciferinupon quinone reduction could be driven to completion after30 min. In addition, as we expected, compounds 4 and 5showed 20-fold lower bioluminescent backgrounds than 3(Figure 1), indicating that both compounds are very stable inreaction buffer.

A standard NADH curve was measured with compound 4coupled to the classic Clostridium diaphorase in a one-stepassay format. The luminescent signal reached the maximalsignal after 40 min incubation. The signal-to-background (S/B)was linearly dependent on the concentration of NADH over

a range greater than 2 logs, indicating that generation of luci-ferin in the presence of excess substrate 4 occurs at a rate suf-ficient to maintain a linear correlation between NADH concen-tration and RLUs. (Figure 2) However, the S/B ratio of the lumi-nescent method with 4 coupled to Clostridium diaphorase wasonly enhanced sevenfold in comparison with the resazurin flu-orescent method. This motivated us to explore the possibilityof improving the sensitivity of substrate 4 by using a differentdiaphorase. To our delight, substrate 4 exhibited much greater

Scheme 3. A) Release of luciferin from reduction of modified trimethyl lock quinone compounds 2 and 3 ; B) Syntheses of modified quinone-luciferins 2 and 3.a) Isobutyl chloroformate/TEA/p-aminobenzyl alcohol/THF; b) Ph3P/CBr4/CH2Cl2; c) Ag2CO3/THF; d) d-cysteine/TEA.

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reactivity toward rat diaphorase (Promega), with a 12-fold im-provement in S/B ratio compared to Clostridium diaphorase,while maintaining the same linear dynamic range for NADHtitration. In contrast, resazurin showed slightly enhanced reac-tivity toward rat diaphorase. The limit of detection with com-pound 4 coupled to rat diaphorase was <0.02 mm, greaterthan for the corresponding Clostridium diaphorase coupledbioluminescent method (<0.07 mm), and significantly greaterthan for the resazurin method (0.15 mm). Relying on the highreactivity of compound 4 toward rat diaphorase and combinedwith NAD- or NADP-specific cycling enzymes, such as lactatedehydrogenase or glucose-6-phosphate dehydrogenase, wefurther developed a homogeneous and one-step assay for spe-cific quantification of NAD/NADH, NADP/NADPH, or totalNADH/NADPH in biological samples. Assay formulation, optimi-zation, validation, and integration of the coupled diaphoraseand cycling enzymes into a simple add-and-read format formonitoring NAD(P)H/NAD(P) levels in cells and other biologicalsamples will be published separately.

Cellular reducing capacities based on NAD(P)H-dependentcellular oxidoreductase enzymes and their NAD(P)H cofactorsprovide great potential to correlate the reduction of diaphor-ase substrates with the number of viable cells.[16] Therefore,the feasibility of compound 4 to measure cell viability was ex-amined in a two-step assay format. Unfortunately, incubation

Scheme 4. A) The release of luciferin from a N,N’-dimethyldiamine linker-modified quinone luciferins. B) Syntheses of trimethyl lock quinone compounds 4and 5. a) Isobutyl chloroformate/N-methylmorpholine/THF, b) TFA/CH2Cl2, c) COCl2/TEA, d) TEA/THF, e) d-Cys/TEA.

Figure 2. NADH titration curve with compound 4 coupled to different dia-phorase isozymes in comparison to the resazurin-diaphorase fluorescencemethod. For the bioluminescent assay, 50 mL of 40 mm compound 4 in LDRwith 2 m mL�1 Clostridium diaphorase or 30 mg mL�1 rat diaphorase wasadded to 50 mL of each NADH sample, in triplicate. The luminescent signalwas measured after 40 min. For the fluorescence assay, 50 mL of 40 mm resa-zurin in 50 mm Tris pH 7.5 buffer with 2 m mL�1 Clostridium diaphorase or30 mg mL�1 rat diaphorase was added to 50 mL of each NADH sample, in trip-licate. Fluorescence was recorded at 590 nm with excitation at 560 nm after10 min, at which time the signal had reached maximum intensity.

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of compound 4 with HepG2 cells did not result in the increaseof luminescent signal above the background signal. (Figure 3)We reasoned that the negatively charged carboxylic anion onthe luciferin scaffold might prevent compound 4 from diffus-ing into cells. The simple way to mask the negative charge ofcompound 4 was to convert its carboxylic acid into methylester 6 or directly utilize its cyanobenzothiazole precursor (4 d).The bioluminescent signals of 6 and 4 d in HepG2 cells weremeasured with the two-step assay. To remove the methyl esterof 6, a sufficient amount of porcine liver esterase was includedin the luciferin detection reagent to completely convert theester into acid during the detection step. For compound 4 d,excess d-cysteine was added to the luciferin detection reagentto drive the completion of 6-hydroxyl cyanobenzothiazole toluciferin during the detection step. As anticipated, incubationof compounds 6 and 4 d with HepG2 cells generated signifi-cant bioluminescent signals, suggesting that compounds 6and 4 d can be effectively reduced in viable cells. (Figure 3)However, compound 6 showed very poor linear correlation ofluminescent signals with cell numbers. It was suspected thatthis poor correlation might be attributed to the partial hydroly-sis of ester 6 to form acid 4 by cellular esterase and that 4might have lower reactivity toward cellular oxidoreductase en-

zymes than ester 6. Clearly, the bioluminescent signals of com-pound 4 d increased with increasing cell numbers at low celldensity (<10 000 cells), suggesting that compound 4 d wouldbe the preferable substrate for measuring viable cells.

Human A549 lung carcinoma cells were treated with the an-ticancer drugs doxorubicin[17a] or etoposide,[17b] and viable cellswere measured with compound 4 d after treatment. The IC50

values obtained for doxorubicin and etoposide were 1.1 mm

and 175 mm, similar to the values obtained by the MTS method(0.8 mm and 108 mm, respectively). Compound 4 d showeda faster and greater response in viable cells than MTS, with S/Bratios for 4 d after 30 min incubation that were tenfold greaterthan S/B ratios for MTS after 2 h incubation. (Figures S4 and S5in the Supporting Information). The IC50 value obtained fromA549 cells treated with digitonin[17c] was 18 mm, comparable tothe value of 11 mm obtained by the resazurin fluorescencemethod. The S/B ratios after 1 h incubation were more than 50times greater than the resazurin method after the same incu-bation time. (Figure S6) Based on these promising viable cellmeasurements with compound 4 d, we developed a new setof quinone profurimazine substrates for a NanoLuc luciferaseassay system, which enables measurement of viable cells inreal-time mode (detailed report is in preparation).

Figure 3. Luminescent signals generated with compounds 4, 6, and 4 d in various quantities of HepG2 cells. HepG2 cells were added to the wells of a 96-wellplate in 25 mL EMEM medium, plus a medium-only control, in triplicate. The cells were allowed to adhere for 2.5 h, and 25 mL of 100 mm 4, 6, and 4 d in mediawas added to the wells. After 30 min at 37 8C in a humidified, 5 % CO2 atmosphere and 30 min at room temperature, 50 mL LDR was added. Porcine esterasewas included in the LDR for compound 6, and 10 mm d-cysteine was included in the LDR for compound 4 d. Luminescent signals were read after 20 min incu-bation at room temperature. Compound 4 appeared to be cell-impermeable.

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In summary, a bioluminescent assay provides advantages formeasuring NAD(P)H/NAD(P) in various biological samples andfurther provides an opportunity for measuring viable cells bycellular reducing capacity. A series of quinone-luciferins weredemonstrated as bioluminogenic diaphorase substrates formeasuring NAD(P)H. The quinone-luciferins were modulatedwith different self-cleavable linkers to enhance the stabilityand signal needed for a sensitive assay. In particular, N,N’-dime-thylethylenediamine-linked quinone-luciferin 4 exhibited excel-lent reactivity toward rat diaphorase and had a low back-ground signal. The S/B ratio was 50 times greater than for theresazurin fluorescence method, and the detection limit was10 times more sensitive. In addition, compound 4 d, a precursorof compound 4, can be effectively reduced in viable cells withluminescent signals proportional to cell numbers, indicating itssuitability for cell viability assays. Compound 4 d exhibiteda faster and greater response in viable cells than when usingtraditional methods and motivated us to develop a real-timecell viability assay.

Keywords: diaphorase · NAD(P)H · quinones · reducingcapacity · self-immolative linkers

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Received: November 26, 2013Published online on March 3, 2014

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