A new synthesis of 2-cyano-6-hydroxybenzothiazole, the key intermediate of d-luciferin, starting from 1,4-benzoquinone

Article abstract of DOI:10.1055/s-0029-1217971

2-Cyano-6-hydroxybenzothiazole is the key intermediate for the synthesis of d-luciferin, the natural substrate of firefly luciferases. A new synthesis of 2-cyano-6-hydroxybenzothiazole has been realized starting from the reaction of 1,4-benzoquinone with l-cysteine ethyl ester, followed by oxidation-cyclization of the intermediate ethyl (R)-2-amino-3-(2,5-dihydroxyphenylsulfanyl)propan-oate hydrochloride to 2-carbethoxy-6-hydroxybenzothiazole. A suitable protection of this intermediate and a conversion to the corresponding nitrile gave, after deprotection, 2-cyano-6-hydroxybenzothiazole (32% yield from 1,4-benzoquinone). This nitrile reacts with d-cysteine to afford d-luciferin at room temperature in nearly quantitative yield (90-95%).

Full text of DOI:10.1055/s-0029-1217971

2682
LETTER
A New Synthesis of 2-Cyano-6-hydroxybenzothiazole, the Key Intermediate of
D-Luciferin, Starting from 1,4-Benzoquinone
S
ynthesis of 2-Cya
i
no-6-h
u
ydroxybenzo
s
thiazole eppe Meroni,a Paolo Ciana,b Adriana Maggi,b Enzo Santaniello*a
a
Department of Medicine, Surgery and Dentistry, University of Milan, Polo Universitario S. Paolo, Via A. di Rudinì 8, 20142 Milan, Italy
Fax +39(02)50323275; E-mail: enzo.santaniello@unimi.it
b
Center of Excellence on Neurodegenerative Diseases, Department of Pharmacological Sciences, University of Milan,
Via Balzaretti 9, 20133 Milan, Italy
Received 26 June 2009
COOH
H
Abstract: 2-Cyano-6-hydroxybenzothiazole is the key intermedi-
ate for the synthesis of D-luciferin, the natural substrate of firefly lu-
ciferases. A new synthesis of 2-cyano-6-hydroxybenzothiazole has
been realized starting from the reaction of 1,4-benzoquinone with L-
cysteine ethyl ester, followed by oxidation–cyclization of the inter-
mediate ethyl (R)-2-amino-3-(2,5-dihydroxyphenylsulfanyl)propan-
oate hydrochloride to 2-carbethoxy-6-hydroxybenzothiazole. A
suitable protection of this intermediate and a conversion to the cor-
responding nitrile gave, after deprotection, 2-cyano-6-hydroxyben-
zothiazole (32% yield from 1,4-benzoquinone). This nitrile reacts
with D-cysteine to afford D-luciferin at room temperature in nearly
quantitative yield (90–95%).
H2N
COOH
N
S
N
S
N
S
SH
CN
D-cysteine
HO
HO
2
1
Scheme 2 Synthesis of D-luciferin (1) from 2-cyano-6-hydroxy
benzothiazole (2)
Most of the synthetic procedures currently available for
the preparation of the key intermediate 2-cyano-6-
Key words: D-luciferin, 1,4-benzoquinone, firefly luciferase, 2-cy- hydroxybenzothiazole (2) start from p-anisidine (3)4–10
(Scheme 3).11 All these routes12 allow the preparation of
ano-6-hydroxybenzothiazole
the intermediate 2-cyano-6-methoxybenzothiazole (7),
that can be demethylated with pyridinium hydrochloride
(200 °C, 62%) to the nitrile 2.4
The luciferase from the North American firefly Photinus
pyralis (PpyLuc) catalyzes the conversion of D-luciferin
We describe here a new synthesis of 2-cyano-6-hydroxy-
[(S)-2-(6¢-hydroxy-2¢-benzothiazolyl)-D2-thiazoline-4-car-
benzothiazole (2), the immediate precursor for the synthe-
boxylic acid (1)] to oxyluciferin in the presence of ATP,
sis of D-luciferin (1), starting from 1,4-benzoquinone
Mg2+, and oxygen with production of a yellow-green light
(8).13 Our synthetic approach was planned to be more ver-
satile than the previously existing ones. In fact, starting
from 1,4-benzoquinones containing different groups, the
characterized by a broad emission spectrum and a peak at
560 nm (Scheme 1).1
preparation of variously substituted analogues of the ni-
trile 2 would become feasible. By this approach, luciferin-
like compounds, potentially endowed with new biolumi-
nescent properties in the luciferase-catalyzed light emis-
sion,14 would be available.
Fireflies are only a part of the big family of biolumines-
cent insects that can emit light with wavelengths going
from yellow-green to red, but all luciferases use the same
substrate, namely D-luciferin (1).2 The chemical structure
of D-luciferin (1), isolated from firefly tails, was proposed
in 19613 and later confirmed by synthesis.4
The reaction of 1,4-benzoquinone (8) with L-cysteine eth-
yl ester affords different products, depending on the ex-
perimental conditions.15–17 However, ethyl (R)-2-amino-
3-(2,5-dihydroxyphenylsulfanyl)propanoate hydrochlo-
ride (9) has been isolated and characterized.18 Under con-
trolled conditions, the oxidation of the ester 9 can lead to
the benzothiazolyl ester 10.19 We have followed essential-
This synthetic approach (Scheme 2) remains yet the only
and unsurpassable method for the preparation of D-
luciferin (1), that is obtained in a nearly quantitative yield
(90–95%) by reaction at room temperature of 2-cyano-6-
hydroxybenzothiazole (2) with an aqueous solution of
D-cysteine.
ATP/Mg2+
O2
CO2
4'
3
N
3'
N
COOH
4
N
S
N
5'
2
light
2'
5
S
1' S
S1
luciferase
6'
HO
HO
OH
7'
oxyluciferin
D-luciferin (1)
Scheme 1 Luciferase-catalyzed production of bioluminescence
SYNLETT 2009, No. 16, pp 2682–2684
x
x
.x
x
.2
0
0
9
Advanced online publication: 09.09.2009
DOI: 10.1055/s-0029-1217971; Art ID: G21009ST
© Georg Thieme Verlag Stuttgart · New York

LETTER
Synthesis of 2-Cyano-6-hydroxybenzothiazole
2683
N
S
N
S
S
a
b
COOEt
COOEt
CN
HN
COOEt
c–f
HO
O
N
S
10
11
CONH2
N
S
N
S
d
MeO
c
2
CONH2
OMe
5
a,b
O
4
O
NH2
g
N
S
13
12
CN
MeO
Scheme 5 Synthesis of the nitrile 2 from the ester 10. Reagents and
conditions: (a) allyl bromide, DMF, K2CO3, reflux, 1 h, 96%; (b) con-
cd NH4OH–EtOH, reflux, 5 h, 95%; (c) POCl3, pyridine, imidazole,
CH2Cl2, from –10 °C to r.t., 12 h, 81%; (d) Pd(PPh3)4, NaBH4 in THF,
r.t., 24 h, 83%.
h
i, j
7
N
S
OMe
NH2
3
MeO
6
Scheme 3 Synthetic approaches to the preparation of 2-cyano-6-
methoxybenzothiazole (7) from p-anisidine (3). Reagents and condi-
tions: (a) ethyl oxalate, 180 °C, 5 min, 58%; (b) P2S5, xylene, reflux;
(c) NaOH, 0 °C; then HCl; (d) K3Fe(CN)6, NaOH, <10 °C, 15 min
(76% crude, no isolation in steps b–d); (e) CH2N2, 0 °C, 15 min, 40%;
(f) anhyd NH3–MeOH, heat, 30 min, 100%; (g) POCl3, reflux, 15 min,
56%; (h) KSCN, Br2, AcOH, 35 °C, 10 h, 87%; (i) CuBr2 in PEG 200,
MeCN, isoamyl nitrite, 65 °C, 3 h, 79.3%; (j) NaCN, DMSO, 110 °C,
5 h, 70.4%.
By this procedure, the allyl protecting group remained es-
sentially intact and the nitrile 13 was obtained in 81%
yield. The final deprotection was successfully achieved
following a described procedure30 (83% yields).31,32
In conclusion, we have prepared 2-cyano-6-hydroxyben-
zothiazole (2), the pivotal intermediate for the preparation
of D-luciferin (1), by a more versatile approach with re-
spect to the classical ones that start from p-anisidine (3).4–
10
ly both procedures18,19 with minor modifications of the
published protocols (Scheme 4). Briefly, we have pre-
pared the ester 9 in methanol–water, and the residue from
workup20 was directly oxidized with K3Fe(CN)6 in an
aqueous ethanol solution of NaOH.21 The residue ob-
tained after workup (90% yield) was dissolved in ethanol
and treated with 1 N HCl. The required ester 10 was ob-
tained in 60% yields after column chromatography.22
In our approach, starting from 1,4-benzoquinone (8),
the 6-O-allyl ester 11 has been prepared and this interme-
diate transformed into the nitrile 2 (32% overall yield).
These yields can favorably be compared with previous
procedures (6–29%), considering also that a few of these
protocols involve the use of poisonous cyanide.8–10
Acknowledgment
This work has financially been supported by the European Project
EMIL (European Molecular Imaging Laboratories, LSHC-Ct-2004-
503569) entitled ‘Molecular Imaging for Early Detection of Tumors
and Monitoring of Treatment’. We thank Dr. Giangiacomo Beretta
(Dipartimento di Scienze Farmaceutiche ‘Pietro Pratesi’, Facoltà di
Farmacia, University of Milan) for NMR spectra of compounds.
OH
O
N
S
a
b,c
COOEt
COOEt
NH2⋅HCl
S
HO
10
OH
O
8
9
Scheme 4 Synthesis of ester 10 from 1,4-benzoquinone (8).
Reagents and conditions: (a) L-cysteine ethyl ester hydrochloride,
MeOH–H2O, r.t., 1 h, 96% (crude); (b) K3Fe(CN)6, NaOH, aq EtOH,
r.t., 1 h, 90% (crude); (c) 1 N HCl, EtOH, r.t., 24 h, 60% (after column
chromatography).
References and Notes
(1) White, E. H.; Rapaport, E.; Seliger, H. H.; Hopkins, T. A.
Bioorg. Chem. 1971, 1, 92.
(2) Viviani, V. R. Cell. Mol. Life Sci. 2002, 59, 1833.
(3) White, E. H.; McCapra, F.; Field, G. F.; McElroy, W. D.
J. Am. Chem. Soc. 1961, 83, 2402.
We then studied different protections of the ester 10 bear-
ing in mind that these groups should be resistant to the
conditions required for the transformation of an amide
into a nitrile. Also the final deprotection of the intermedi-
ate protected nitrile was considered with attention, due to
sensitivity of the nitrile group to hydrolytic and reducing
conditions.23 It turned out that the 6-O-allyl ether was the
most suitable protecting group, meeting all previous re-
quirements.24 The synthetic approach is described in
Scheme 5.25 Accordingly, the 6-O-allyl ester 11 was pre-
pared from the ester 10 and converted to the amide 12
smoothly and almost quantitatively using concentrated
ammonia in ethanol.26,27 Then, the amide 12 was trans-
formed into the nitrile 13 using phosphorus oxychloride as
dehydrating agent under controlled conditions.28,29
(4) White, E. H.; McCapra, F.; Field, G. F. J. Am. Chem. Soc.
1963, 85, 337.
(5) Seto, S.; Ogura, K.; Nishiyama, Y. Bull. Chem. Soc. Jpn.
1963, 36, 332.
(6) Bowie, L. J. Methods Enzymol. 1978, 57, 15.
(7) Stuckwisch, G. C. J. Am. Chem. Soc. 1949, 71, 3417.
(8) White, E. H.; Woelther, H.; Field, G. F.; McElroy, W. D.
J. Org. Chem. 1965, 30, 2344.
(9) Suzuki, N.; Nomoto, T.; Kanamori, N.; Yoda, B.; Saeki, A.
Biosci. Biotechnol. Biochem. 1993, 57, 1561.
(10) Toya, Y.; Takagi, M.; Nakata, H.; Suzuki, N.; Isobe, M.;
Goto, T. Bull. Chem. Soc. Jpn. 1992, 65, 392; in this paper,
the nitrile 7 was prepared by a direct Sandmeyer cyanation
of compound 6 (HNO2, CuCN, KCN; 41% yield).
(11) The nitrile 7 has also been prepared from the corresponding
N-aryliminodithiazole under microwave irradiation; see:
Synlett 2009, No. 16, 2682–2684 © Thieme Stuttgart · New York

2684
G. Meroni et al.
LETTER
Bénéteau, V.; Besson, T.; Rees, C. W. Synth. Commun.
1997, 27, 2275.
(12) For a review on the synthesis of D-luciferin derivatives or
analogues and related in vitro/in vivo luciferase-catalyzed
bioluminescent activity, see: Meroni, G.; Rajabi, M.;
Santaniello, E. ARKIVOC 2009, (i), 265.
(13) Santaniello, E.; Meroni, G.; Maggi, A.; Ciana, P. IT
MI2009A000294, 2009.
(14) The wavelength of the emitted light in the luciferase-
catalyzed bioluminescence can be modulated by
modification of the substrate structure; for a recent review,
see: Santaniello, E.; Meroni, G. Minerva Biotechnol. 2009,
21, 77.
stirred at r.t. (30 min). After addition of allyl bromide (0.746
mL, 8.07 mmol) the reaction was refluxed for 1 h. After H2O
addition, extraction with EtOAc, washing of the organic
solution with aq NaCl, drying, and evaporation of solvent,
the product is purified by chromatography (PE–EtOAc, 9:1).
The 6-O-allyl ester 11 is obtained as a yellow solid (1.353 g,
96% yield); mp 97–99 °C. 1H NMR (500 MHz, CDCl3): d =
1.48 (t, 3 H, J = 8.7 Hz), 4.53 (q, 2 H, J = 8.7 Hz), 4.63 (d, 2
H, J = 5.2 Hz), 5.35 (dd, 1 H, J = 1.5, 10.5 Hz), 5.45 (dd, 1
H, J = 1.8, 17.5 Hz), 6.05 (m, 1 H), 7.18 (dd, 1 H, J = 2.4, 9.1
Hz), 7.36 (d, 1 H, J = 2.4 Hz), 8.10 (dd, 1 H, J = 2.4, 9.1 Hz).
13C NMR (125 MHz, CDCl3): d = 14.90, 63.00, 69.50,
104.50, 118.05, 118.50, 126.30, 132.90, 139.00, 148.00,
156.00, 158.90, 161.00.
(15) Kuhn, R.; Beinert, H. Ber. Dtsch. Chem. Ges. 1944, 77, 606.
(16) Prota, G.; Ponsiglione, E. Tetrahedron Lett. 1972, 13, 1327.
(17) Crescenzi, O.; Prota, G.; Schultz, T.; Wolfram, L. J.
Tetrahedron 1988, 44, 6447.
Amide 12
Compound 11 (1.10 g, 4.198 mmol) was added to a solution
containing concentrated aq NH3 (20 mL) in EtOH (50 mL)
and the solution refluxed (5 h). After evaporation at reduced
pressure, EtOAc was added and the solution passed through
a column of Florisil (20 g). Evaporation of the solvent left
the pure amide 12 as a yellow solid (0.929 g, 95% yield); mp
173–175 °C.
(18) Crescenzi, O.; Prota, G.; Schultz, T.; Wolfram, L. J. Gazz.
Chim. Ital. 1990, 120, 21.
(19) Löwik, D. P. W.; Tisi, L. C.; Murray, J. A. H.; Lowe, C. R.
Synthesis 2001, 1780.
(20) According to the original procedure, extraction with EtOAc
removed most of the impurities, and evaporation of the
solvents afforded the hygroscopic ester 9. In our hands,
samples of different preparations showed variable purity for
the final product that contained also variable amount of H2O.
Therefore, the recovery (96%) refers to the weight of the
final product.
(21) In the case of 4-hydroxybenzothiazole derivatives similar to
compound 9, the oxidation proceeds efficiently with
K3Fe(CN)6, in the presence of ZnSO4; see: Greco, G.;
Panzella, L.; Napolitano, A.; D’Ischia, M. Tetrahedron Lett.
2009, 50, 3095.
(28) Van der Veken, P.; Senten, K.; Kertèsz, I.; De Meester, I.;
Lambeir, A.-M.; Maes, M.-B.; Scharpé, S.; Haemers, A.;
Augustyne, K. J. Med. Chem. 2005, 48, 1768.
(29) 2-Cyano-6-O-allyloxybenzothiazole (13)
A solution containing compound 12 (0.950 g, 4.077 mmol)
and imidazole (0.277 g, 4.077 mmol) in anhyd pyridine (25
mL) was cooled to –10 °C under nitrogen. Through a
syringe, a solution of phosphorous oxychloride (0.746 mL,
8.154 mmol) in CH2Cl2 (5 mL) was slowly added, and the
resulting solution was kept in ice under stirring (1 h). The
temperature was slowly raised to ambient and stirred for
additional 12 h, then CH2Cl2 and H2O were sequentially
added. The organic phase was separated, washed with H2O,
dried over Na2SO4 and evaporated. The crude was purified
by chromatography (PE–EtOAc, 9:1), obtaining compound
13 (0.713 g, 81%) as a yellow solid; mp 74–75 °C. 1H NMR
(500 MHz, CDCl3): d = 4.62 (d, 2 H, J = 5.2 Hz), 5.35 (dd,
1 H, J = 1.5, 10.5 Hz), 5.45 (dd, 1 H, J = 1.8, 17.5 Hz), 6.07
(m, 1 H), 7.21 (dd, 1 H, J = 2.4 Hz), 7.36 (d, 1 H, J = 2.4, 9.1
Hz), 8.05 (dd, 1 H, J = 2.4, 9.1 Hz). 13C NMR (125 MHz,
CDCl3): d = 69.50, 104.10, 113.70, 118.20, 118.90, 126.20,
132.80, 133.50, 137.80, 147.00, 159.70.
(22) The one-pot procedure for the preparation of the
benzothiazolyl ester 10 starting from 1,4-benzoquinone 8
proceeds less satisfactorily (35–45%).
(23) Among protecting groups, we have tested tert-butyldi-
methylsilyl, tetrahydropyranyl, and 2-methoxy ethoxy-
methyl (MEM) ethers that did not survive or were partially
hydrolyzed in the conditions required for the conversion of
the 2-carboxyamide into the 2-nitrile function. Benzyl was
an effective protecting group in the transfor-mation of amide
12 into nitrile 13, but removal of the protecting group by
hydrogenolysis of 2-cyano-6-O-benzyl-oxy benzothiazole
caused the reduction of the nitrile moiety.
(30) Beugelmans, R.; Neuville, L.; Bois-Choussy, M.; Chastanet,
(24) For an application of allyl ethers as protecting groups and to
the methods available for their removal, see: Hu, Y.-J.;
Dominique, R.; Das, S. K.; Roy, R. Can. J. Chem. 2000, 78,
838; and references cited therein.
(25) The progress of all reactions and column chromatography
were monitored by TLC (PE–EtOAc, 8:2) using silica gel 60
J.; Zhu, J. Tetrahedron Lett. 1995, 36, 3129.
(31) LiBH4 was also used as a reducing agent and reaction was
faster, as described in ref. 31. However, with this stronger
reducing reagent a competing reduction of the nitrile was
observed.
(32) 2-Cyano-6-hydroxybenzothiazole (2)
F
254 precoated plates with a fluorescent indicator (Merck).
To a solution of compound 13 (0.600 g, 2.777 mmol) in
anhyd THF under N2, Pd(PPh3)4 (0.02 mmol) and NaBH4
(0.422 g, 11.1 mmol) were added sequentially. After 24 h at
r.t., the reaction was filtered and the solvent evaporated to
leave a residue that was purified by column chromatography
(PE–EtOAc, 8:2). The nitrile 2 was obtained as a pale yellow
solid (0.406 g, 83%). Chemico-physical data were in
agreement with those reported previously.9,10
Purification of products by chromatography was performed
using silica gel 60 (230–400 mesh, Merck). All compounds
gave satisfactory elemental analysis.
(26) Liso, G.; Trapani, G.; Latrofa, A. J. Heterocycl. Chem. 1987,
24, 1683.
(27) Ester 11
To a solution of the ester 10 (1.20 g, 5.38 mmol) in DMF (15
mL), K2CO3 was added (1.11 g, 8.07 mmol) and the mixture
Synlett 2009, No. 16, 2682–2684 © Thieme Stuttgart · New York