Design, synthesis and evaluation of novel P450 fluorescent probes bearing α-cyanoether

Article abstract of DOI:10.1016/S0040-4039(03)00953-5

Four α-cyano-containing ethers based on 2-alkoxy-2-naphthylacetonitriles have been designed as a novel structural class of cytochrome P450 fluorescent probes. Their syntheses, fluorescence properties and evaluation in the fluorogenic assay of cytochrome P

Full text of DOI:10.1016/S0040-4039(03)00953-5

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 4331–4334
Design, synthesis and evaluation of novel P450 fluorescent
probes bearing a-cyanoether
Rong Zhang,a Kyung-Don Kang,a,b Guomin Shana,† and Bruce D. Hammocka,b,
*
aDepartment of Entomology and University of California Cancer Research Center, Davis, CA 95616, USA
bMicrobiology Program, University of California, Davis, CA 95616, USA
Received 10 January 2003; revised 20 February 2003; accepted 15 April 2003
Abstract—Four a-cyano-containing ethers based on 2-alkoxy-2-naphthylacetonitriles have been designed as a novel structural
class of cytochrome P450 fluorescent probes. Their syntheses, fluorescence properties and evaluation in the fluorogenic assay of
cytochrome P450 monooxygenase are reported. After P450 enzymatic O-dealkylation, the cyanohydrin metabolite of the four new
substrates rearranges to a fluorescent aromatic aldehyde with a larger Stokes shift, and the new substrates exhibit higher specific
activities than that of the commercial substrate 7-ethoxyresorufin (ER). © 2003 Elsevier Science Ltd. All rights reserved.
The cytochromes P450 are involved in regulating hor-
mones, steroids, and fatty acids, and in metabolism of
xenobiotics such as drugs, carcinogens, pesticides, and
diverse pollutants.1 Many laboratories in both
academia and industry routinely screen novel com-
pounds for inhibition of cytochromes P450 to guard
against drug–drug interactions.2 Numerous types of
fluorescent substrates have been designed and synthe-
sized as diagnostic probes of cytochromes P450, includ-
a-cyano-containing ester substrates had been developed
and demonstrated as novel fluorogenic esterase
reporters.7,8 The concept is that an esterase substrate
containing an a-cyano group could be designed with
little or no fluorescence background yet be transformed
to a strongly fluorescing aldehyde upon ester hydroly-
sis. Herein, a similar strategy was utilized to design
a-cyano-containing ether derivatives based on 2-alkoxy-
2-naphthylacetonitriles as a novel structural class of
fluorescent substrates for cytochrome P450 monooxyge-
nase (Scheme 1). The reported probes possess one
a-cyano group and have very low fluorescence back-
ground, undergo O-dealkylation by P450 monooxyge-
nase to form cyanohydrins, which spontaneously
convert to 2-naphthaldehyde generating larger red shift
and obvious higher fluorescence emission intensity for
detection. Four new fluorescent probes with methyl,
ethyl, pentyl and benzyl substitutents have been synthe-
sized. These are a-cyanodialkyl rather than the com-
monly used aryl-alkyl ethers and thus provide
structural diversity among spectraphotometric sub-
ing
alkoxyresorufins,
alkoxyquinolines,
alkoxy-
coumarins and their modified analogues.3–5 The com-
mon chemical structural model is that they are hetero-
cyclic
aryl-ether
derivatives.
After
enzymatic
O-dealkylation, they yield the corresponding phenolic
metabolites, which have fluorescence properties for
detection.
Over the last few years, we have been interested in
developing novel fluorescent enzyme substrates in an
effort to expand our knowledge of these enzymes and
provide sensitive and easily used assays.6 A series of
Scheme 1. Metabolism of a-cyanoether substrate to its fluorescent metabolite by P450s.
Keywords: cytochrome P450; fluorescent probes; a-cyanoether; 2-alkoxy-2-naphthylacetonitriles.
* Corresponding author. Tel.: +1-530-752-7519; fax: +1-530-752-1537; e-mail: bdhammock@ucdavis.edu
† Present address: Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)00953-5

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R. Zhang et al. / Tetrahedron Letters 44 (2003) 4331–4334
Scheme 2. Synthetic routes to 2-alkoxy-2-naphthylacetonitriles.
strates. The fluorescence properties of these substrates
and their fluorescent product 2-naphthaldehyde were
compared with the commonly used P450 substrate 7-
ethoxyresorufin (ER) and its product resorufin, and the
parent compounds were compared as substrates in rat
microsomes fortified with NADPH. The significant
advantages found indicate that this and related series of
a-cyano-containing ethers will complement existing
spectral substrates used for the measurement of
cytochrome P450 activities.
Scheme 2 shows the synthetic routes to the target
compounds. The first synthesis was carried out using
modified literature procedures.9,10 Syntheses of dimethyl
acetal and diethyl acetal were achieved from readily
available starting materials trimethyl orthoformate and
triethyl orthoformate, respectively. Then one of the
alkoxyl groups of acetals was converted to cyano group
using cyanotrimethylsilane under the catalytic action of
BF3·OEt2 with high yield. For pentyl and benzyl ethers,
the cyanonaphthyl toluenesulfonate alcoholysis reaction
was employed for synthesis.11 Either benzenesulfonyl
chloride or p-toluenesulfonyl chloride can be used in
the reaction with a same yield. The pentyl and benzyl
ethers were also prepared according to the same proce-
dure as that of 1 and 2 by refluxing 2-naphthaldehyde
with tripentyl orthoformate or benzyl alcohol respec-
tively to give the corresponding diacetal, followed by
changing one of the alkoxy group to cyano group with
cyanotrimethylsilane and a catalytic amount of SnCl2.
All new compounds were characterized via H and 13C
1
Figure 1. Comparison of new substrates 1–4 and 2-naphth-
aldehyde fluorescent emission. 10 mM solutions in sodium
phosphate buffer (pH 7.6, 0.1 M).
NMR, MS and microanalyses.12
Fluorescent spectra of compounds 1–4 (Fig. 1) were
scanned as 10 mM solutions in sodium phosphate buffer
(pH 7.6, 0.1 M) at excitation 345 nm. All substrates
(quantum yield below 0.03) have a low intensity fluores-
cence emission peak at 370 nm, while a stronger
fluorescence emission is measured at 445 nm with the
metabolite 2-naphthaldehyde (quantum yield 0.19).
This property helps to minimize possible interference of
the assay between aldehyde and substrates. The extinc-
tion coefficient of aldehyde in Tris–malate buffer (0.1
M, pH 7.0) at 292 nm was calculated to be 12,700 M−1
cm−1, those of substrates were 2,400–10,160 M−1 cm−1
at their maximum absorbance. The Stokes shift of
2-naphthaldehyde (uex=345 nm, uem=445 nm) is also
larger than that of resorufin (uex=570 nm, uem=585
nm). When the substrates were dissolved by using
DMSO as a vehicle, the solubility of the substrates is
better than that of commercial substrate ER. These
advantages in fluorescent and solubility properties
provide a sound background for developing a-cyano-
ether compounds as novel surrogate substrates for
cytochrome P450 O-dealkylation reactions.
Enzymatic O-dealkylation of the P450 substrates were
performed by incubating rat liver microsomes (150 mg)
at 37°C in a final volume of 2 mL of 40 mM Tris–HCl
buffer (pH 7.8). The substrate was added in 10 uL of
DMSO solution to give a final substrate concentration
of 50 mM. Reactions were initiated by the addition of
10 mL of 50 mM NADPH in distilled water. After the
incubation for 30 min, the reactions were added to
tubes containing DEAE Sepharose fast flow (Pharma-
cia, Uppsala, Sweden) and mixed well to bind the
NADPH. Before use, the DEAE ion exchanger was
washed five times to equilibrate with the reaction

R. Zhang et al. / Tetrahedron Letters 44 (2003) 4331–4334
4333
buffer. The mixed tubes were centrifuged at 3000 rpm
for 5 min. The fluorescence intensities were measured in
a Fluoromax II spectrofluorometer with excitation at
345 nm and emission at 445 nm. The commonly used
commercial substrate ER was also used for a compari-
son. The results are shown in Figure 2, in which each
entry represents an average of three independent tests.
Sciences (NIEHS) Superfund Basic Research Program,
5 P42 ES04699-08, NIEHS Grant R01 ES02710, the
NIEHS Center for Environmental Health Sciences,
USDA/CRESS, 2001-35302-09919, and UC-System-
wide Mosquito Research Program c01-017-2-1.
References
As shown in Figure 2, the substrates were rapidly
dealkylated in microsomes in the presence of NADPH
with the relative rates of O-dealkylation in rat liver
1. Stegeman, J. J.; Livingstone, D. R. Comp. Biochem.
Physiol. 1998, 212C, 1–3.
2. Ortiz de Montellano, P. R. Cytochrome P450: Structure,
Mechanism, and Biochemistry; Plenum Press: New York,
1995.
3. Mayer, R. T.; Netter, K. J.; Heubel, F.; Hahnmann, B.;
Buchheister, A.; Mayer, G. K.; Burker, M. D. Biochem.
Pharmacol. 1990, 40, 1645–1655.
4. Nakamura, K.; Hanna, I. H.; Cai, H.; Nishimura, Y.;
Williams, K. M.; Guengerich, F. P. Anal. Biochem. 2001,
292, 280–286.
microsomes
of
methyl-ether>benzyl-ether>pentyl-
ether>ethyl-ether. All the new a-cyanoether substrates
exhibited higher specific activities than that of ER in
end-point assays. The rapid generation of the fluores-
cent metabolite from these new substrates supported
the general idea of designing additional a-cyanoethers
as P450s probes.
5. Onderwater, R. C. A.; Venhotst, J.; Commandeur, J. N.
M.; Vermeulen, N. P. E. Chem. Res. Toxicol. 1999, 12,
555–559.
6. Hammock, B. D.; Shan, G.; Zhang, R. Cyanohydrin
Ethers and Esters as High-Sensitivity Enzyme Substrates;
US Patent US 6,528,675 B1 (March 4, 2003).
7. Shan, G.; Hammock, B. D. Anal. Biochem. 2001, 299,
54–62.
8. Wheelock, C. E.; Karlsson, O. M.; Zhang, R.; Stok, J. E.;
Morisseau, C.; Valley, S. E. L.; Green, C.; Buckpitt, A.
R.; Hammock, B. D. Anal. Biochem. 2003, 315, 208–222.
9. Bailey, D. M.; DeGrazia, C. G.; Wood, D.; Siggins, J. J.
Med. Chem. 1974, 17, 702–708.
10. Utimoto, K.; Wakabayashi, Y.; Shishiyama, Y.; Inoue,
M.; Nozaki, H. Tetrahedron Lett. 1981, 22, 4279–4280.
11. Makosza, M.; Goetzen, T. Rocz. Chem. 1972, 46, 1059–
1067.
Figure 2. Comparison of specific activities for new substrates
1–4 and common P450 substrate ER.
12. Preparation of 2-methoxy-2-(2-naphthyl)ethanenitrile 1: A
mixture of 1.56 g (10 mmol) 2-naphthaldehyde, 1.74 g
(16.4 mmol) freshly distilled trimethyl orthoformate, 4.2
mL of absolute methanol and 10 mg of zinc chloride were
refluxed for 6 h under dry conditions. Part of solvent was
removed and an additional 0.5 mL of trimethyl orthofor-
mate was added. The reaction mixture continued to reflux
overnight and was monitored by TLC. Excess trimethyl
orthoformate and solvent was removed under reduced
pressure, the residue was treated with aqueous 5% (w/v)
NaHCO3 and extracted with diethyl ether. The ether
extract was washed with water, dried over MgSO4 and
concentrated. The crude dimethyl acetal was used for the
next step without further purification. Under nitrogen,
0.1 mL (0.7 mmol) of BF3·OEt2 was added gradually to a
mixture of the above-prepared dimethyl acetal and 1.33
mL (0.99 g, 10 mmol) of cyanotrimethylsilane at 0°C.
After stirring at room temperature for 2 h, the clear
solution turned into a cream colored slurry. TLC indi-
cated that the reaction was complete. The mixture was
worked up with dilute aqueous sodium bicarbonate solu-
tion, filtered, washed with water and dried in vacuo to
give 1.62 g (82.4% yield) of crude product, which was
recrystallized from diethyl ether to give a cream colored
solid, mp 47–48°C. TLC Rf 0.60 (hexane:EtOAc=3.5:1,
v/v). 1H NMR (CDCl3): l 3.56 (s, 3H), 5.39 (s, 1H),
In summary, we have designed, synthesized and evalu-
ated a-cyano-containing ethers based on 2-alkoxy-2-
naphthylacetonitriles as novel P450 fluorescent probes.
Because of their unique molecular structures represent-
ing a new family of fluorescent probes for P450s, their
facile synthesis high-turnover and attractive optical
properties, they may prove to be useful and attractive
tools in pharmacological and biochemical studies. Fur-
ther studies on their metabolism by recombinant
isozymes, synthesis of alternate more red shifted a-
cyano-containing ether reporters are currently under
investigation.
Acknowledgements
We thank Dr. Craig E. Wheelock for kindly providing
the microsomes, Dr. Watanabe Takaho for valuable
discussions, Dr. Jozsef Lango, Department of Chem-
istry and Superfund Analytical Laboratory for running
ESI-MS. The authors gratefully acknowledge funding
from the National Institute of Environmental Health

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R. Zhang et al. / Tetrahedron Letters 44 (2003) 4331–4334
7.50–7.57 (m, 3H), 7.82–8.00 (m, 4H); 13C NMR
(CDCl3): l 57.4 (CH3), 72.7 (CCN), 117.2 (CN), 124.3,
127.1, 127.2, 127.4, 128.0, 128.5, 129.4, 130.6, 133.1,
133.9; MS (EI, 70 eV): m/z % 197 [M+, 51], 166 [(M−
OCH3)+, 100], 139 [(M−OCH3−HCN)+, 21]. ESI-MS
calcd for C13H12NO [(M+H)+]: 198.0919. Found:
198.0929.
naphthyl toluenesulfonate as a cream colored solid. The
6
above sulfonate was immediately mixed with 3.5 mL of
benzyl alcohol and heated to 80°C for 2 h, then filtered
and the solution was flash chromatographed on silica gel
using (hexane:EtOAc=3.5:1, v/v) as the eluent to give
2.07 g (75.8% yield) of the target compound as a cream
colored solid, mp 36–37°C. TLC Rf 0.68 (hex-
ane:EtOAc=5:1, v/v). 1H NMR (CDCl3): l 4.71–4.87 (m,
2H), 5.44 (s, 1H), 7.36–7.44 (m, 5H), 7.54–7.59 (m, 3H),
Preparation of 2-ethoxy-2-(2-naphthyl)ethanenitrile 2: By
a similar procedure, the target compound was prepared
in 70.6% yield, which crystallized as white plates from
diethyl ether, mp 53–54°C. TLC Rf 0.52 (hex-
7.84–8.00 (m, 4H); 13C NMR (CDCl3): l 69.9 (C
6 CN),
71.9 (CH2), 117.4 (CN), 124.5, 127.0, 127.3, 127.4, 128.0,
128.5, 128.6 (2C, phenyl), 128.7, 129.0 (2C, phenyl),
129.4, 130.8, 133.1, 133.9, 135.9; MS (EI, 70 eV): m/z %
273 [M+, 19], 166 [(M−OCH2Ph)+, 100], 139 [(M−
OCH2Ph−HCN)+, 31]. Anal. calcd for C19H15NO: C,
83.49; H, 5.53; N, 5.12. Found: C, 83.20; H, 5.74; N,
5.09.
1
ane:EtOAc=5:1, v/v). H NMR (CDCl3): l 1.32 (t, J=
6.6 Hz, 3H), 3.65–3.90 (m, 2H), 5.43 (s, 1H), 7.50–7.58
(m, 3H), 7.83–7.98 (m, 4H); 13C NMR (CDCl3): l 15.2
(CH3), 66.0 (CH2), 71.1 (C6 CN), 117.6 (CN), 124.4, 127.0,
127.1, 127.3, 127.9, 128.5, 129.3, 131.1, 133.1, 133.9; MS
(EI, 70 eV): m/z % 211 [M+, 35], 166 [(M−OCH2CH3)+,
100], 139 [(M−OCH2CH3−HCN)+, 21]. Anal. calcd for
C14H13NO: C, 79.59; H, 6.20; N, 6.63. Found: C, 79.66;
H, 6.32; N, 6.54.
Preparation of 2-(2-naphthyl)-2-pentyloxyethanenitrile 4:
The target compound was prepared as described above
with a yield of 69.3% as a golden yellow oil. TLC Rf 0.76
(hexane:EtOAc=3.5:1, v/v). 1H NMR (CDCl3): l 0.80–
0.96 (m, 3H), 1.16–1.42 (m, 4H), 1.55–1.74 (m, 2H),
3.40–3.80 (m, 2H), 5.42 (s, 1H), 7.42–7.62 (m, 3H),
7.74–8.00 (m, 4H); 13C NMR (CDCl3): l 14.4 (CH3),
Preparation
of
2-(2-naphthyl)-2-(phenylmethoxy)-
ethanenitrile 3: A 1.56 g (10 mmol) sample of 2-naphth-
aldehyde was dissolved in 2.3 mL of tetrahydrofuran, and
1.9 g (10 mmol) of p-toluenesulfonyl chloride was added.
Then 0.66 g (10 mmol) of potassium cyanide in 2 mL of
water was added dropwise with stirring at a rate sufficient
to maintain the temperature below 5°C in an ice bath.
After the mixture was stirred for 1 h at 0–5°C, the solvent
was stripped under reduced pressure washed with ice-cold
diethyl ether, dried in vacuo to give 2.52 g of a-cyano-
22.8, 28.5, 29.3, 70.5, 71.3 (C6 CN), 117.7 (CN), 124.4,
126.9, 127.0, 127.5, 128.0, 128.1, 128.5, 129.3, 130.0,
133.2; MS (EI, 70 eV): m/z % 253 [M+, 28], 166 [(M−
OC5H11)+, 100], 139 [(M−OC5H11−HCN)+, 100]. Anal.
calcd for C17H19NO: C, 80.60; H, 7.56; N, 5.53. Found:
C, 80.21; H, 7.43; N, 5.58.