[2] Synthesis of firefly luciferin and structural analogs

[2]

SYNTHESIS

OF FIREFLY

LUCIFER1N

AND

STRUCTURAL

ANALOGS

15

these can be detected for appropriate corrections by using internal controis. The pH optimum is such that one should run the reaction around pH 7.5. H o w e v e r , even p H levels as high as 8 can be used. At pH levels below 8, a red light occurs instead of the usual yellow-green, thus giving rise to an apparent inhibition. The activity of firefly luciferase is very sensitive to specific anion inhibition. ~6The order of effectiveness of inhibition by the anions, S C N - > I- -- NO:~- > Br- > CI, followed their position in the Hofmeister series. Extracts containing such anions must be diluted appropriately. ~"J. L. Denburg and W. D. McElroy, Arch. Biochem. Biophys. 141,668 (1970).

[2] S y n t h e s i s o f F i r e f l y L u c i f e r i n Structural Analogs

and

B y LEMUEL J. BOWIE

Luciferin Synthesis Firefly luciferin, o-(--)-2-(6'-hydroxy-2'-benzothiazolyl)-A2-thiazoline4-carboxylic acid, was first isolated in pure form from the American firefly P h o t i n u s p y r a l i s in 1957 by Bitler and McElroy. 1 Approximately 9 mg of crystalline luciferin from approximately 15,000 fireflies was used to perform the initial characterization of this molecule. Luciferin was subsequently chemically synthesized by White et al. 2 and further characterized.:~ The structure of luciferin (LH2) and two useful structural analogs, dehydroluciferin (L) and dehydroluciferol (LOH) are shown in Fig. 1. 4 Owing to the lability of the thiazoline portion of the luciferin molecule, synthetic approaches have generally involved the synthesis of a suitable benzothiazolyl derivative followed by condensation with cysteine to form the thiazoline ring of the luciferin molecule or derivative directly. In contrast, dehydroluciferin and dehydroluciferol have a very stable thiazolyl ring, which has made them very useful as close structural analogs of luciferin in mechanistic studies of firefly luciferase. '~,~ B. Bitler and W. D. McElroy, Arch. Biochem. Biophys. 72, 358 (1957). 2 E. H. White. F. McCapra, G. F. Field, and W. D. McEIroy, J. Am. Chem. Soc. 83, 2402 (1961). :~E. H. White, F. McCapra, and G. F. Field, J. Am. Chem. Soc. 85, 337 (1%3). 4 G. E. Blank, J. Pletcher, and M. Sax, Biochem. Biophys. Res. Commun. 42, 583 (1971). J. L. Denburg, R. T. Lee, and W. D. McElroy, Arch. Biochem, Biophys. 134, 381 (1969). " L. J. Bowie, V. Horak, and M. DeLuca, Biochemisto' 12, 1845 (1973). M E T H O D S IN E N Z Y M O L O G Y , VOL. LVll

Copyright '~ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181957-4

16

FIREFLY LUCIFERASE 4'

3'

1

7'

1'

3

[2]

LH2

L

LOH

FIG. I. Structures ofluciferin (LH2), dehydroluciferin (L), and dehydroluciferol (LOH). Rings are oriented with the N = C -- C = N system transplanar as has been demonstrated for luciferin by X-ray analysis. From G. E. Blank, J. Pletcher, and M. Sax, Biochem. Biophys. Res, Commun. 42, 583 (1971). Figure 2 summarizes the three major synthetic paths that have been reported for luciferin. The key intermediate in all these schemes is 2-cyano-6-methoxybenzothiazole. Upon removal of the methyl group at position 6, the molecule is condensed with D-cysteine to give luciferin. Method 1 was used for the first chemical synthesis by White et al. 2 but was soon replaced by the much shorter approach (method 2) 7 utilizing a commercially available benzothiazole derivative. F o r large-scale preparations of luciferin, however, method 3 by Seto et al.8 proved to be a very convenient and useful route. Therefore, in the following detailed discussion of the preparation of luciferin, this approach with minor modifications will be described. Procedure

The method of Seto et al.S involves the reaction of carbamoylthiocarbonylthioacetic acid (I), prepared fresh and without isolation, with p-anisidine (II) to form 4-methoxythiooxanilamide (III). This product is then cyclized to form 2-carbamoyl-6-methoxybenzothiazole (IV), followed by conversion to the corresponding nitrile (V) as seen in Fig. 3. The rest of the synthesis proceeds as outlined in Fig. 2. P r e p a r a t i o n o f 4 - M e t h o x y t h i o o x a n i l a m i d e (III)

Since carbamoylthiocarbonylthioacetic acid is fairly unstable, it is prepared and used in alkaline solution without isolation by first dissolving 7 E. H. White, H. WSrther, G. F. Field, and W. D. McElroy, J. Org. Chem. 30, 2344(1965). 8 S. Seto, K. Ogura, and Y, Nishiyama, Bull. Chem. Soc. Jpn. 36, 332 (1%3).

[2]

SYNTHESIS OF FIREFLY LUCIFER1N AND STRUCTURAL ANALOGS

~

jNH2

CH30~,'t'-,~

~N~__NH2 CH3O/~'~/~'S--

0

"t" C2Hso/C'--.C

LL

CH30- v

~ N

/0... C2 H 5

41 3STEPS[~I/NH2

2 sTEPS ~ N x x ~ •

17

"S

CH30 I V

-{SII O~ /CH2 /C.~ /NH 2 ~C "S C~ HO 0

~--C= N

HO

SH \CH2

NHz COOH

N~

S

FIG. 2. Synthetic approaches to the synthesis of luciferin: (1) E. H. White, F. McCapra, G. F. Field, and W. D. McElroy,J. Am. Chem. Soc. 85, 337 ( 1963): (2) E. H. White, H. W6rther, G. F. Field, and W. D. McElroy,J. Org. Chem. 30, 2244 ( 1965): (3) S. Seto, K. Ogura, and Y. Nishiyama, Bull. Chem. Soc. Jpn. 36, 332 (1963).

potassium hydroxide (300 g) in methanol (2000 ml). The solution is then divided into two halves: one half to be saturated with hydrogen sulfide and the other to be saturated with nitrogen. The half to be saturated with hydrogen sulfide is rapidly bubbled for approximately 3 hr with H.,S that has been dried by passage through calcium chloride. Owing to its odoriferous and toxic nature, this gas should be connected to a suitable trap. Two liters of 10% (w/v) lead acetate is very useful. As H2S is initially bubbled through the alcoholic potassium hydroxide, the solution becomes light green and bubbles slowly escape from the trap without any decoloration of the lead acetate solution. Concomitantly, a slow stream of nitrogen is passed through the other flask. After approximately 1.5 hr, a black precipitate begins to form in the trap and the methanol-potassium hydroxide solution gradually turns yellow-green. A trichloroacetamide solution is prepared at this time by adding 185 g to 1000 ml of methanol and set aside.

18

FIREFLY LUCIFERASE

KzS + C13CCONHz

(I )

S 0 II II HS--C--C~NH2 t f S 0 II II HOOC-CHE-S-C-C-NH 2 •

l~-

(]I)

(In)

[2]

C[CH2 COOH

CH30~O / NH2

HI 0I! f~I/N'c"C"NH2

0

II

CH3

I K3Fe(CN)s 0 CH30,"-~.I-,S-~EPOC[3

I Pyr. HCl (=)

/ ~ N~---C---N H0.---~.f-.S

(SZ]r)

NH2/C~COOH #"~YN~,..__.Z',S" HO~"L,%JLS,7~NLCOOH

FIG. 3. Reaction scheme for the synthesis ofluciferin according to the method of S. Seto, K. Ogura, and Y. Nishiyama, Bull. Chem. Soc. Jpn. 36, 332 (1963).

[2]

S Y N T H E S I S OF F I R E F L Y L U C I F E R 1 N A N D S T R U C T U R A L A N A L O G S

19

The first two flasks are mixed in a 6-liter Erlenmeyer flask and equilibrated with nitrogen for a short time (5 rain). The trichloroacetamide solution is then added slowly with stirring while continuing to pass nitrogen through the mixture. As the solution gradually turns red, the temperature rises, and an ice bath should be used to maintain the temperature at ambient or below. Trichloroacetamide is again added slowly with cooling. This process is repeated until all the trichloroacetamide is added. The solution is then deep red but progressively turns reddish brown over a period of 30 rain. A solution of monochloroacetic acid (160 g) in water (1000 ml) is neutralized with solid potassium carbonate and added to the above mixture. Upon vigorous shaking a deep red color forms. A layer of inorganic salt is allowed to form for approximately 5 rain and removed by filtration. The filtrate is immediately added top-anisidine (100 g) in 50% (v/v) aqueous methanol (1000 ml). (Note: Reagent grade p-anisidine must be used: it should be tan to near white, not brown.) As nitrogen bubbling is continued, yellow crystals begin to form. After approximately 30 rain the flask is stoppered, and crystallization is allowed to continue overnight at 4 ° . The yellow crystals are removed by filtration. The yield of 4-methoxythiooxanilamide (IlI) is approximately (95-125 g) with a melting point of 183°- 185°. Recrystallization of this product is not necessary for subsequent cyclization.

Preparation of 2-Carbamoyl-6-methoxybenzothiazole (IV) 4-Methoxythiooxanilamide (50 g) is dissolved in sodium hydroxide ( 100 g in 2000 ml) and added dropwise, using a separatory funnel, to a stirred solution of potassium ferricyanide ( 1 lb, or approximately 450 g) in 1 liter of water at room temperature. Fumes that are possibly toxic are generated during this reaction, requiring the use of a fume hood. A fine precipitate forms immediately, but the reaction is allowed to proceed with stirring for I hr. The precipitate (approximately 30 g) is then removed by filtration. Repeating the above procedure with an additional 40 g of product (III) yields an additional (20-25 g) of 2-carbamoyl-6-methoxybenzothiazole (IV). The precipitates are combined and recrystallized from methanol, yielding (50-55 g) of product (IV), with a melting point of 254°-256 ° (decomp.).

Preparation of 2-Cyano-6-methoxybenzothiazole (V) The above product (50 g) is refluxed as a suspension in phosphorus oxychloride (500 ml) for 90 rain. At the end of this time the excess phosphorus oxychloride is distilled off under reduced pressure. Care should be

20

FIREFLY LUCIFERASE

[2]

taken to remove all excess POCI3 since it can interact with water with the evolution of heat. The product is cooled, and small aliquots are poured onto crushed ice (no excess liquid present) and mixed to ensure good yields. The resultant precipitate is filtered and extracted with benzene. This extract is filtered, and petroleum ether is added slowly to give a colorless precipitate. This yields approximately 30 g of 2-cyano-6-methoxybenzothiazole (V). Sublimation of the precipitate gives colorless needles (m.p. 128°-130°).

Preparation of 2-Cyano-6-hydroxybenzothiazole (VI) Removal of the methyl group at position 6 is accomplished in a pyridine hydrochloride melt at 200°-210 °. Fresh pyridine hydrochloride is most easily prepared by passing HCI gas over the surface of pyridine in a flask equipped with a reflux condenser. A controlled flow of hydrogen chloride is conveniently generated by allowing concentrated sulfuric acid to drip into a flask of ammonium chloride. The entire apparatus is shown in Fig. 4. The hydrogen chloride is first dried by passage through concentrated H2SO4. The safety bottle trap is included as a precaution against "sucking back" of contents of the reaction vessel (l-liter three-necked flask). Hydrogen chloride generated in this manner is passed over the surface drying tube

nc. H2SO 4

~ - ' - / ' ~ " NH 4 Cl

~\conc.

-

7~

eter

safety

H2SO4

battle trap

heating mantle

FIG. 4. Apparatus for the in situ production of pyridine hydrochloride and demethylation of 2-cyano-6-methoxybenzothiazole.

[2]

SYNTHESIS OF FIREFLY LUCIFERIN AND STRUCTURAL ANALOGS

21

of pyridine (250 ml). The pyridine is heated under reflux until the reaction temperature reaches 180°. The solution at this time becomes somewhat yellow and pasty. Product (V) (20 g) is then added, and HCI is again passed over the surface. The temperature is increased to 200°-210 ° and held there for 3 hr. Hydrogen chloride flow is then stopped, the reaction mixture is allowed to cool, and finally the flask is placed on an ice bath. Small aliquots of the product, a red paste, are removed and mixed with crushed ice (2000 ml) and sodium carbonate (10 g). The residual material in the flask is washed out with small amounts of cold water into the sodium carbonateice mixture. The pH of the final mixture should be in the range of 6.5-7.0. The product is removed by filtration and dried in air. Traces of starting material (V) are extracted by boiling for 1 hr in benzene (1000 ml) and filtering while hot. It is important to remove all traces of 2-cyano-6methoxybenzothiazole (V) at this stage since the methoxyluciferin formed by its reaction with cysteine is somewhat difficult to separate from luciferin. The residue after extraction is recrystallized from methanol to yield a fine, light brown solid with a melting point of 209°-211 °. The yield of product (VI) may vary at this stage but should be in the range of 6-10 g. Owing to the importance of eliminating small traces of any starting material from this final product, it is necessary to confirm purity by ultraviolet absorption spectroscopy or thin-layer chromatography. The absorption spectrum of compound (V) in dilute alkali has a maximum in the region around 320 nm while compound (VI) has an absorption maximum at approximately 390 nm. Therefore, the absorption spectrum of the product (VI) in dilute base should show no shoulder in the region between 320 and 360 nm. Alternatively, the product can be chromatographed on silica gel plates (Baker Chemical Co.) using chloroform/ethyl acetate (5:1) as the solvent system. The relative migration can be monitored by visualizing with a tong wavelength ultraviolet lamp. Under these conditions product (VI) has a relative mobility of 0.51. Any traces of material which chromatograph with an R s of 0.75 should be removed by further extraction with hot benzene and recrystallization of the residue from methanol.

Preparation of D-( --)-2-(6 '-Hydroxyl2 '-benzothiazolyl)- Azthiazolinecarboxylic Acid--' 'Firefly Luc(ferin" o-Cysteine hydrochloride (5 g) and potassium carbonate (4.0 g) are dissolved in 125 ml of water. The solution is adjusted to pH 7.5 if necessary and is thoroughly equilibrated with nitrogen (approximately 1 hr). Concomitantly, 2-cyano-6-hydroxybenzothiazole (5.0 g) is dissolved in methanol (150 ml) in a low-actinic glass flask (or a flask that has been masked to

22

FIREFLY LUCIFERASE

[2]

exclude light) and equilibrated with nitrogen for the same period of time. The solution of D-cysteine is then added to the flask containing the benzothiazole derivative and allowed to react at room temperature with stirring and under nitrogen stream for an additional 1.5 hr. The solution is adjusted to pH 6-7 with dilute hydrochloric acid, and the precipitate is collected by filtration. After redissoiving in warm methanol, the solution is concentrated under vacuum until precipitation commences. The solution is then removed, reequilibrated with nitrogen for a short time, and allowed to precipitate overnight at 4° in a stoppered flask protected from light. The product is isolated as fine, pale yellow needles (6-7 g) with a melting point of 2000-204 ° (decomp.). Owing to the extreme lability of the product, it should be stored dry, under nitrogen atmosphere, in sealed or tightly stoppered tubes and protected from light by the use of aluminum foil, tape, etc. Storage and Handling of Luciferin Luciferin is a pale-yellow solid that recrystallizes with difficulty and sublimes with decarboxylation and decomposition. In aqueous solutions, it is sensitive to extremes in pH, estsecially in the presence of oxygen and light. Racemization occurs rapidly in certain solvents (e.g., 7% per hour at 4° in aqueous pyridine). Alkaline solutions, in the presence of oxygen, can give rise to dehydroluciferin. For these reasons, if crystalline luciferin is to be stored for extended periods of time, it should be well desiccated in light-tight containers and under nitrogen atmosphere. Under these conditions luciferin can be stored indefinitely without oxidation, racemization, or photodecomposition. Aqueous solutions at near neutral pH values can be stored at 4° safely for periods of i-2 months if protected from light, and preferably under nitrogen atmosphere. Spectral Properties of Luciferin Luciferin is a highly fluorescent molecule, exhibiting a quantum yield of 0.62 in aqueous solutions at a pH 11.9 Owing to the presence of the 6'hydroxyl group, luciferin can undergo both ground-state and excited-state ionization to form the phenolate ion. The lowest energy absorption for the ground-state phenol form occurs at 327 nm, and the corresponding absorption for the ground-state phenolate ion occurs at 385 nm, as shown in Fig. 5A. Irrespective of the ground-state species that is excited (in aqueous solutions), fluorescence emission occurs primarily from a single species, 9 R. A. Morton,T. A. Hopkins, and H. H. Seliger,Biochemistry 8, 1598(1969).

[2]

SYNTHESIS OF FIREFLY LUCIFERIN A N D STRUCTURAL ANALOGS

23

0.8327

385

0.7-

/

0.6

0.5z

",

/

"\ \

\

,

0.4o

<

/

//

\

\

268

03

,,

285

/

\ ,,

0.203250

300 350 WAVELENGTH(nm)

A

400

450

tO0 90 80 70 z LIJ

60 50 4 0 ~

LU Q:::

30 201O0

400

B

450

5()0 550 6()0 WAVELENGTH(nm)

6;0

~

700

FIG. 5. (A) Normalized absorption spectra of luciferin at p H 4 ( ) and at pH I1 ( - - - ) . (B) Fluorescence emission s p e c t r u m (uncorrected) for luciferin at pH 4. The normalized emission s p e c t r u m for luciferin at pH 9 (not shown) is superimposable.

24

FIREFLY LUCIFERASE

[2]

the excited-state phenolate (emission maximum approximately 537 nm), as seen in Fig. 5B. This results from ionization of the phenol in the excited state to form excited-state phenolate prior to fluorescence emission. In nonpolar solvents, where excited-state ionization is inhibited, emission occurs predominantly from the excited-state phenol (emission maximum at approximately 420 nm9). These properties of luciferin and some of its analogs have been very useful in studying the luciferase active site microenvironment. 6,9,10 Criteria for Purity The single most definitive test for structure and purity is examination of in vitro bioluminescence quantum yields and peak shapes for the yellow

green (565 nm) emission. If the quantum yield is less than 0.7, the luciferin should be purified by recrystallization from methanol. This approach although described in some detail 11 is not simple to perform, however, and requires the use of purified luciferase. As an alternative, a combination of thin-layer chromatography and absorption and fluorescence spectroscopy can be used to assess the general purity of the product. Luciferin migrates as a single spot with an R r of 0.31 on silica gel plates using a solvent system of ethyl acetate and methanol (10: 1). Paper chromatography using Whatman No. 3 and development with a solvent system of 95% ethanol and I M ammonium acetate, pH 7.5 (7: 3) gives rise to a single spot at an Rrof 0.55. The relative migrations can easily be visualized by observation of the developed chromatograms with a long-wavelength (365 nm) ultraviolet lamp, since luciferin and many of its derivatives are highly fluorescent. Chromatography on silica gel plates which incorporate a fluorescent indicator is even more useful, since this allows visualization, by noting areas of fluorescent quenching, of contaminants that are not fluorescent but have ultraviolet absorption. Another convenient technique for estimating purity is to examine absorption spectra of aqueous solutions of luciferin. At neutral pH, the ratio of the absorbance at 327 nm to that at 268 nm should be 2.3 or greater; and at alkaline pH, the ratio of absorbance at 385 nm and 285 nm should be 2.5. Ifa small amount of pure luciferin is available, fluorescence excitation and emission spectra of pure versus synthetic luciferin can be compared and provides another convenient means of estimating purity. If pure luciferin is not available, fluorescence excitation and emission spectra can be corrected for lamp output and phototube sensitivity and compared to pub10M. DeLuca, L. Brand, T. A. Cebula,H. H. Seliger,and A. F. Makula,J. Biol. Chem. 246, 6702 (1971). 11H. H. Seligerand W. D. McElroy,Arch. Biochem. Biophys. 88, 136 (1960).

[2]

SYNTHESIS OF FIREFLY LUCIFERIN AND STRUCTURAL ANALOGS

25

lished spectra, 9 although this procedure is somewhat more difficult and error prone. Table I summarizes both the spectral and chromatographic properties of luciferin, its analogs, and synthetic precursors. This information can be useful in the preliminary characterization of these products.

Synthesis of Related Analogs Dehydroluciferin Although dehydroluciferin can be easily prepared by the oxidation of alkaline aqueous solutions of luciferin by the use of potassium ferricyanide or molecular oxygen, 3 the subsequent isolation and purification of large quantities is somewhat difficult. Therefore, dehydroluciferin can be most readily prepared by direct thiazole synthesis2 2-Cyano-6methoxybenzothiazole (V) is converted to the corresponding thioamide, 2-thiocarboxamido-6-methoxybenzothiazole, by treatment with hydrogen sulfide, pyridine, and triethylamine. This thioamide readily condenses with methyl bromopyruvate to form the dimethyl derivative of dehydroluciferin. Treatment of this derivative with boiling, concentrated hydrobromic acid then yields dehydroluciferin. Dehydroluciferol Dehydroluciferol is prepared by reduction of the carboxylic acid group of dehydroluciferin. Owing to the limited solubility of dehydroluciferin in nonpolar solvents, reduction is conveniently accomplished after esterification of the carboxylic acid group and protection of the phenolic hydroxyl groupf Esterification also serves to facilitate the reduction and allows the use of less vigorous and more selective reducing agents. Dehydroluciferin is esterified by refluxing in acidified absolute ethanol. The dehydroluciferyl ethyl ester thus formed is allowed to react with dihydropyran (under nitrogen) to give the tetrahydropyranyl ether derivative, which is subsequently reduced with LiAIH4 or NaAIH2(OCH2CH2OCH3) 2 in benzene. Hydrolysis is then achieved rapidly under mild conditions (e.g.. 0.1 N HCI. 5 rain) to give dehydroluciferol. Luciferyl and Dehydroluciferyl Adenylate The synthesis of the adenylates of luciferin and dehydroluciferin is accomplished by condensation in aqueous pyridine in the presence of dicyclohexyl carbodiimide according to the method of Morton e t a l . :~ with

26

FIREFLY LUCIFERASE

[2] C~

~

o

I

II!11

;>

0 Z

o

Z

~e~l

Z

0

2 .a
X

X

X

X

X

X

c~. c~

p.

.<,,~

X X X X X . . . . .

N

M

~E v

r~

>

0 0

G~ 0

[2]

SYNTHESIS OF FIREFLY LUCIFERIN AND STRUCTURAL ANALOGS

27

minor modifications. Luciferin (10 mg) and adenylic acid (30 mg) are dissolved in 1 ml of dry pyridine containing 0.15 ml of 0.5 N HCI. Dicyclohexylcarbodiimide (400 mg) in I ml of dry pyridine is added, and the reaction is allowed to proceed for 90 rain at 4 °. The product is then precipitated by the addition of 6 volumes of cold acetone (-20°), filtered, and washed with cold acetone until all traces of pyridine are removed. The precipitate is then dissolved in 10 mM sodium acetate containing 40 mM sodium chloride, pH 4.5, and the solution is placed on a 1 cm × 30 cm Sephadex G-25 (fine) column after removal of the undissolved residue. Elution with the same buffer gives rise to adenylic acid, followed by

TABLE II STRUCTURAL ANALOGSOF LUCIFERIN Compound

Structure

R1

R2

R4

R5

trans-5-Methylluciferin

OH OH NH 2 OCH3 OH H H OH OH

H H H H H H H H H H H H CH3 CH3 CH:~ H H CH 3

H CO-AMP" COOH COOH H COOH COOH COOH COOH

COOH H H H H H H H H

5,5-Dimethylluciferyl adenylate 5,5-Dimethyloxyluciferin

OH OH

CHz CH~ CH~ CH:~

CO-AMP" H O -(as ketone)

RI

R2

e-(+)-Luciferin Luciferyl adenylate 6'-Aminoluciferin 6'-Methoxyluciferin Decarboxyluciferin Deshydro×yluciferin 5,5-Dimethylluciferin cis-5-Methylluciferin

Oxyluciferin Oxyluciferin diacetate Dehydroluciferyl adenylate

OH H CHACO H OH H

R3

Referencesb

3 12 7 7 7 7 7 13 13 14 15

R3 OH CH3CO CO-AMP "

15 15 12

" CO-AMP, mixed anhydride formed from the carboxylic acid group of luciferin (or derivative) and the phosphoric acid group of adenosine 5'-phosphate. t, Numbers refer to text footnotes.

28

FIREFLY LUCIFERASE

[3]

luciferyl adenylate, and finally free luciferin in the eluates. Owing to the small amounts of product in the eluates, luciferyl adenylate and luciferin can be most conveniently assayed utilizing the bioluminescent reaction with luciferase or luciferase plus ATP-Mg, respectively. Because of the instability ofluciferyl adenylate (50% hydrolysis in 24 hr), it should be used as soon as possible. Dehydroluciferyl adenylate can be synthesized and purified in an analogous manner. Other Analogs Many additional analogs have been synthesized and have proved to be very useful in probing the molecular mechanism of firefly bioluminescence. A summary of the most useful of these is given in Table II 3,7,12-1~along with references that include descriptions of their synthesis. Of necessity, certain luciferyl analogs have been omitted along with a number of benzothiazole derivatives. However, descriptions of their preparation and properties can generally be found among the references listed, notably references cited in footnotes 5, 7, and 12. ,2 W. C. R h o d e s and W. D. McElroy, J. Biol. Chem. 233, 1528 (1958). '~ E. H. White, E. Rapaport, T. A. Hopkins, and H. H. Seliger, J. Am. Chem. Soc. 91, 2178 (1969). ,4 T. A. Hopkins, H. H. Seliger, E. H. White, and M. W. C a s s , J. Am. Chem. Soc. 89, 7148 (1967). ,5 N. Suzuki, M. Sato, K. Nishikawa, and T. Goto, Tetrahedron Lett. 53, 4683 (1969).

[3] P r e p a r a t i o n o f P a r t i a l l y P u r i f i e d Firefly Luciferase Suitable for Coupled Assays

By HANS N. RASMUSSEN The purification of extracts of firefly tails for use in the analysis of ATP requires methods that can be applied to rather small amounts of material. The fractionation requirements are, however, well defined: (1) removal of ATP and other substances of low molecular weight to lower the blank values, and (2) removal of interfering enzymes to decrease the systematic errors of the analysis. The substances of low molecular weight are easily removed by column gel filtration on low-porosity Sephadex gels.' Luciferin and dehydro' R. Nielsen and H. N. R a s m u s s e n , Acta Chem. Scand. 22, 1757 (1968).

METHODS IN ENZYMOLOGY, VOL. LVI1

Copyright © 1978by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181957-4