Novel bioluminescent coelenterazine derivatives with imidazopyrazinone C-6 extended substitution for: Renilla luciferase

Article abstract of DOI:10.1039/c6ob00659k

Two series of novel coelenterazine analogues (alkynes and triazoles) with imidazopyrazinone C-6 extended substitution have been designed and synthesized successfully for the extension of bioluminescent substrates. After extensive evaluation, some compounds display excellent bioluminescence properties compared with DeepBlueC in cellulo, thus becoming potential molecules for bioluminescence techniques.

Full text of DOI:10.1039/c6ob00659k

View Article Online
View Journal
Organic &
Biomol ec ular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: T. Jiang, X. Yang,
X. Yang, M. Yuan, T. Zhang, H. Zhang and M. Li, Org. Biomol. Chem., 2016, DOI: 10.1039/C6OB00659K.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/obc

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 1 of 10
View Article Online
DOI: 10.1039/C6OB00659K
Journal Name
FULL ARTICLE
Novel bioluminescent coelenterazine derivatives with
imidazopyrazinone C-6 extended substitution for
Renilla luciferase
Received 00th January 20xx,
Accepted 00th January 20xx
a
a,b
a
a
a
a
DOI: 10.1039/x0xx00000x
Tianyu Jiang , Xiaofeng Yang , Xingye Yang , Mingliang Yuan , Tianchao Zhang , Huateng Zhang
a†
and Minyong Li
www.rsc.org/
Two series (alkynes and triazoles) of novel coelenterazine analogs bioluminescent intensity, peak wavelength, half decay, etc.
with imidazopyrazinone C-6 extended substitutions have been Among them, coelenterazine 400a (DeepBlueC) is a case in
well designed and synthesized successfully for extension of point that was reported by Milton J. Cormier et al in the period
1
0, 16, 17
bioluminescence substrates. After extensive evaluation, some of 1973-1979,
and therefore, it has been a common
compounds display excellent bioluminescence properties coelenterazine analogue that widely be used in the
compared with DeepBlueC in cellulo, thus becoming potential bioluminescent assay. Compared with coelenterazine,
molecules involved in bioluminescence techniques.
DeepBlueC does not have hydroxyl groups at the C-2 and C-6
position, which leads to easier synthesis and blue-shifted
bioluminescence.
Introduction
Bioluminescence is a luminescent phenomenon that results
from an enzyme-catalyzed reaction within a living organism,
which widely exists in lots of organisms, such as bacteria,
As mentioned above, alternative substituents at the C-6
position of the imidazopyrazinone core effects on
bioluminescence properties. Most of the them are aromatic
1
-3
insects, and marine organisms . In a general bioluminescence
system, molecule named luciferin is oxidized by the
1
5
a
groups according to previously reported . To further
investigate the influence of substituents at the C-6 position of
coelenterazine, we designed and synthesized two series (A and
corresponding luciferase to produce the bioluminescence
signal with co-factors or not. For example, coelenterazine (CTZ)
is a universal substrate that can be recognized and catalyzed
by various luciferases, such as Renilla, Gaussia, and
T,
short for alkynes and triazoles) of substrates with
imidazopyrazinone C-6 extended substitution based on
DeepBlueC and investigated their bioluminescent properties
with Renilla and Gaussia luciferase in vitro herein. As for
2, 4
Oplophorus, to yield bioluminescence . Coelenterazine –
luciferase system is the simplest bioluminescence system, and
the mechanism is biocatalysis by luciferase to yield strong peak
alkynes series (
introduction of alkyne substituents at the C-6 position leads to
more rigid molecular structure. Besides, triazole series (
A) of coelenterazine derivatives (Scheme 1), the
2
,
5
emission around 480 nm without any cofactor
.
Coelenterazine was discovered and defined by the Cormier's
-12
T
)
6
and Johnson's groups . So far, a mass of coelenterazine
analogues have been prepared ever since it was chemically
compounds (Scheme 2) have the triazole substituents by
taking the place of the benzene group of DeepBlueC. The
synthesis was based on the classic synthesis of
1
3, 14
synthesized in 1975
, and the bioluminescent and
1
8-20
chemiluminescent properties of these analogues have been
well investigated as well. The modification at C-2, C-6 and C-8
positions of the imidazopyrazinone core is the most important
to influence the bioluminescence intensity and maximum
coelenterazine
. The key intermediates 1 and 3 were
1
5
emission wavelength . Some of CTZ analogues have been
reported remarkably with their individual characteristics on
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 2 of 10
View Article Online
DOI: 10.1039/C6OB00659K
COMMUNICATION
Journal Name
2
0
obtained according to the previous report . As for alkynes Zn dust (235 mg, 3.6 mmol) and I
series (A), key intermediates
via Sonogashira coupling reaction . When it comes to triazole stirred at room temperature until the brown color of I
2
(12 mg) were suspended in fresh
2
were synthesized in good yield anhydrous THF under argon atmosphere and the mixture was
2
1
2
disappeared.
series (
T
), the key intermediates
7
were produced by the click Then the anhydrous benzyl bromide was added by using a syringe
o
2
reaction .
2
and the reaction mixture was refluxed at 80 C for 3 h. After
insertion of Zn, the reaction mixture was cooled to room
temperature. Then the suspension of 2-amino-3, 5-
Methods and Materials
2 3 2
dibromopyrazine (506 mg, 2 mmol) and PdCl (PPh ) (70 mg, 0.1
Materials and instruments
mmol) in 7 mL of DMF was added. The reaction mixture was
continuously stirred overnight. Then the mixture was filtered by
celatom and the filtrate was collected and evaporated under
vacuum. The collection was dissolved in and extracted by ethyl
acetate and washed with saturated sodium chloride solution. After
dried over anhydrous sodium sulfate and concentrated under
reduced pressure, the crude product was further purified by
All reagents and solvents available were used as received unless
otherwise noted. All reactions were monitored by TLC with 0.25
mm silica gel plates (60GF-254). UV light, iodine stain, and ninhydrin
were used to visualize the spots. Silica gel was utilized for column
1
13
chromatography purification. H NMR and C-NMR were recorded
on a Bruker DRX spectrometer at 300 or 400 MHz, δ in parts per
million and J in hertz, using TMS as an internal standard. Mass
spectra were performed by the analytical and the mass
spectrometry facilities at Shandong University. HPLC tests were
measured with Agilent Technologies 1260 liquid chromatography
chromatography on silica gel (PE/EtOAc 20:3) to give a viscous
1
yellow solid (313 mg). Yield: 59%. HNMR (400 MHz, DMSO-d
6
): δ
7
.65 (s, 1 H), 7.30~7.25 (m, 5 H), 6.56 (s, 1 H), 3.98(s, 2H). ESI-MS:
m/z [M+H] + calcd for 264.01, 266. 01, found 264.2, 266.3。
(
Singapore City, Singapore). Water used for the fluorescence and
The preparation of 2-amino-3-benzyl-5- phenylethynyl -pyrazine (2-
21
bioluminescence studies was doubly distilled and further purified
with a Mill-Q filtration system (Millipore, Watertown, MA, USA).
1
) :
Bioluminescence measurements were determined with an IVIS The suspension of PdCl
Kinetic (Caliper Life Sciences, USA) equipped with a cooled charge- 0.151 mmol) and PPh (20 mg, 0.076 mmol) in 2 mL toluene was
coupled device (CCD) camera or Omega microplate reader added to the solution of 3-benzyl-5-bromo-2-amino-pyrazine (200
POLARstar Omega, Germany). Bioluminescence spectra were mg, 0.76 mmol) in 3 mL toluene under argon atmosphere. Then
2 3 2
(PPh ) (27 mg, 0.038 mmol), CuI (29 mg,
3
(
measured with F-2500 FL Spectrophotometer (HITACHI High phenylacetylene (232 mg, 2.27 mmol) and triethylamine (0.7 mL)
o
Technologies Corporation, Tokyo, Japan). Fluorescence spectra were sequentially added. The mixture was stirred at 40 C for 6 h.
were obtained with a Varioskan microplate spectrophotometer Then the mixture was filtered and the filtrate was collected and
(
Thermo Electron Corporation, Waltham, MA, USA). Recombinant evaporated under vacuum. The collection was extracted by ethyl
Renilla reniformis luciferase was purchased from RayBiotech acetate and washed with saturated sodium chloride solution. After
Norcross, GA, USA). Recombinant Gaussia luciferase was dried over anhydrous sodium sulfate and concentrated under
(
purchased from NanoLight (Pinetop, AZ, USA). ES-2 cells (human reduced pressure, the crude product was further purified by
ovarian cancers cell line) expressing Reilla luciferase (Rluc) were chromatography on silica gel (PE/EtOAc 10:1) to give a yellow solid
1
purchased from Shanghai BioDiagnosis Co., Ltd. Coelenterazine was (165 mg). Yield: 77%. HNMR (400 MHz, DMSO-d
6
): δ8.09(s, 1H),
purchased from Chemedir Biopharm-tech.Co., Ltd.
7.43~7.41(m, 6H), 7.29~7.28(m, 4H), 6.81(s, 2H), 4.04(s, 2H).
1
3
CNMR (100 MHz, DMSO-d
6
): δ 153.21, 144.57, 141.87, 138.19,
Organic synthesis
1
29.29, 129.22, 129.13, 128.78, 126.75, 122.69, 88.96, 88.32, 38.83.
+
ESI-HRMS: m/z [M+H] calcd for 286.1344, found 286.1341.
2
0
The preparation of 3-benzyl-5-bromo-2-amino-pyrazine (1) :
2
-Amino-3-benzyl-5-(4-fluorophenylethynyl)-pyrazine (2-2): yellow
Scheme 1: the synthesis route of alkynes series (A) coelenterazine analogs.
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 10
Journal Name
solid; yield 50%; HNMR (400 MHz, DMSO-d
Orga Pn l iec a s&e Bd oi o nmo to al ed cj uu sl ta mr Ca rhg ei nms istry
View Article Online
DOI: 10.1039/C6OB00659K
COMMUNICATION
1
6
): δ8.09(s, 1H), The
preparation
of
2-benzyl-8-benzyl-6-
1
8, 20
7
.61~7.58(m, 2H), 7.48~7.43(m, 2H), 7.30~7.21(m, 5H). 6.82(s, 2H), (phenylethynyl)imidazo[1,2-a]pyrazin-3(7H)-one (A1)
:
1
3
4
1
1
6
.03(s, 2H). CNMR (100 MHz, DMSO-d ): δ 163.67, 161.21, 153.23,
The mixture of f 2-amino-3-benzyl-5- phenylethynyl -pyrazine (4-1,
0 mg, 0.28 mmol) and 3-phenyl - 1,1- diethoxyacetone (3-1, 124
44.59, 138.97, 134.85, 133.97, 129.29, 128.78, 126.75, 125.15,
+
8
19.11, 116.58, 116.36, 88.03, 87.90, 38.82. ESI-HRMS: m/z [M+H]
mg, 0.5 6mmol) was dissolved in ethanol (3 mL) under argon
atmosphere and allowed to stir at room temperature for 10 min.
calcd for 304.1250, found 304.1244.
2
-Amino-3-benzyl-5-(3-fluorophenylethynyl)-pyrazine (2-3): yellow The con.HCl (0.2 mL) in ethanol (1 mL) was then added to the
1
6
solid; yield 45%; HNMR (400 MHz, DMSO-d ): δ8.11(s, 1H), mixture via syringe over 10 min. The reaction was heated to
o
7
.49~7.38(m, 3H), 7.32~7.19(m, 6H), 6.86(s, 2H), 4.04(s, 2H). refluxed at 80 C for 8 h and then allowed to cool to room
1
3
CNMR (100 MHz, DMSO-d
6
): δ 163.57, 161.14, 153.36, 144.91, temperature. The crude was concentrated under vacuum and
Cl /MeOH 50:1)
1
1
8
41.96, 138.14, 131.37, 131.28, 129.29, 128.79, 128.06, 128.04, further purified by chromatography on silica gel (CH
2
2
26.77, 124.75, 124.73, 118.30, 118.08, 116.51, 116.30, 89.32, to give a light brown solid (30 mg). Yield: 26%. Analytical RP HPLC
+
7.74, 87.71, 38.80.ESI-HRMS: m/z [M+H] calcd for 304.1250, (Phenomenex, C8, 250 x 4.6 mm column): 60% acetonitrile with 0.1%
found 304.1253.
trifluoroacetic acid, 1.0 mL/min at 380 nm, Rt: 7.448 min, 97%.
1
3
HNMR (400 MHz, CD OD): δ8.47(s, 1H), 7.58~7.24(m, 15H), 4.59(s,
1
3
2-Amino-3-benzyl-5-(2-fluorophenylethynyl)-pyrazine (2-4): yellow
2
1
1
3
H), 4.34(s, 2H). CNMR (100 MHz, CD OD): δ147.24, 137.12,
1
solid; yield 44%; HNMR (400 MHz, DMSO-d
6
): δ8.11(s, 1H),
34.94, 131.58, 129.75, 128.94, 128.60, 128.45, 128.42, 127.28,
7
7
1
1
.64~7.60(td, J=7.53Hz, 1.6Hz, 1H), 7.48~7.46(m, 1H), 7.36~7.20(m,
26.60, 120.68, 117.41, 94.22, 80.17, 35.89, 31.07. ESI-HRMS: m/z
1
3
+
6
H), 6.88(s, 2H), 4.04(s, 2H). CNMR (100 MHz, DMSO-d ): δ 163.44,
[
M+H] calcd for 416.1763, found 416.1757.
60.96, 153.39, 144.80, 138.11, 133.79, 129.28, 128.79, 126.77,
+
25.32, 116.31, 116.10, 93.23, 82.25, 38.81. ESI-HRMS: m/z [M+H]
2-Benzyl-8-benzyl-6- (4-fluorophenylethynyl)imidazo[1,2-a]pyrazin-
calcd for 304.1250, found 304.1242.
3(7H)-one (A2): yellow solid; yield 35%. Analytical RP HPLC
(
Phenomenex, C8, 250 x 4.6 mm column): 60% acetonitrile with 0.1%
2
-Amino-3-benzyl-5-(3-methylphenylethynyl)-pyrazine (2-5): yellow
trifluoroacetic acid, 1.0 mL/min at 380 nm, Rt: 7.842 min, 96%.
1
1
6
solid; yield 62%; HNMR (400 MHz, DMSO-d ): δ8.07(s, 1H),
3
HNMR (400 MHz, CD OD): δ8.07(s, 1H), 7.64~7.60(m, 2H),
1
3
7.36~7.19(m, 9H), 6.77(s, 2H), 4.03(s, 2H), 2.31(s, 3H). CNMR (100
7
4
1
1
8
.38~7.36(m, 2H), 7.29~7.21(m, 7H), 7.19~7.15(m, 3H), 4.42(s, 2H),
13
6
MHz, DMSO-d ): δ 153.17, 144.48, 141.87, 138.54, 138.21, 132.10,
3
.22(s, 2H). CNMR (100 MHz, CD OD): δ164.74, 162.25, 147.35,
1
8
3
29.90, 129.29, 129.10, 128.78, 128.74, 126.75, 125.38, 125.54,
40.52, 137.03, 134.98, 134.07, 134.03, 133.98, 128.85, 128.38,
28.34, 127.23, 127.10, 126.57, 117.39, 115.84, 115.61, 92.98,
+
9.11, 88.04, 38.85, 21.18. ESI-HRMS: m/z [M+h] calcd for
00.1501, found 300.1500.
+
0.05, 35.85, 30.94. ESI-HRMS: m/z [M+H] calcd for 434.1669,
found 434.1663.
2
-Amino-3-benzyl-5-(3-hydroxyphenylethynyl)-pyrazine
(2-6):
1
yellow solid; yield 62%; HNMR (400 MHz, DMSO-d
6
): δ9.69(s, 1H),
2
3
-Benzyl-8-benzyl-6- (3-fluorophenylethynyl)imidazo[1,2-a]pyrazin-
8.07(s, 1H), 7.29~7.26(m, 3H), 7.22~7.18(t, J=8Hz, 2H), 6.95(d, J=8Hz,
(7H)-one (A3): yellow solid; yield 30%. Analytical RP HPLC
13
1
H), 6.87(s, 1H), 6.82~6.79(m, 1H), 6.77(s, 2H), 4.03(s, 2H). CNMR
): δ 157.83, 153.11, 144.56, 141.78, 138.20,
(
Phenomenex, C8, 250 x 4.6 mm column): 60% acetonitrile with 0.1%
(100 MHz, DMSO-d
6
trifluoroacetic acid, 1.0 mL/min at 370 nm, Rt: 6.173 min, 97%.
1
130.34, 129.28, 128.78, 126.75, 125.31, 123.53, 122.54, 118.03, 116.
3
HNMR (400 MHz, CD OD): δ8.58(s, 1H), 7.46~7.20(m, 14H), 4.59(s,
+
1
3
68, 89.09, 87.71, 38.83. ESI-HRMS: m/z [M+H] calcd for 302.1293,
2
1
1
9
4
3
H), 4.34(s, 2H). CNMR (100 MHz, CD OD): δ163.64, 161.19,
found 302.1293.
47.39, 137.00, 134.98, 130.58, 130.49, 128.56, 128.40, 128.34,
27.75, 127.22, 126.60, 118.19, 117.95, 117.72, 117.04, 116.82,
2
0, 23
The preparation of 3-phenyl - 1,1- diethoxyacetone (3)
:
+
2.29, 81.25, 35.98, 30.83. ESI-HRMS: m/z [M+H] calcd for
34.1669, found 434.1664.
Ethyl diethoxyacetate (500 mg, 2.84 mmol) was dissolved in fresh
o
anhydrous THF, and the solution was cooled to -78 C under argon
2
3
-Benzyl-8-benzyl-6- (2-fluorophenylethynyl)imidazo[1,2-a]pyrazin-
atmosphere. Then benzylmagnesium chloride (642 mg, 4.26 mmol)
solution was added via syringe over 15 min, and the reaction was
allowed to stir for 3 h. The reaction was quenched by addition of
ammonium chloride aqueous solution and then allowed to warm to
room temperature. The reagent was evaporated under vacuum and
redissolved in ethyl acetate. After extraction by ethyl acetate, the
organic layer was washed with saturated sodium chloride aqueous
solution. The mixture was concentrated under reduced pressure
(7H)-one (A4): yellow solid; yield 14%. Analytical RP HPLC
(
Phenomenex, C8, 250 x 4.6 mm column): 65% acetonitrile with 0.1%
trifluoroacetic acid, 1.0 mL/min at 380 nm, Rt: 5.788 min, 97%.
1
HNMR (400 MHz, CD
3
OD): δ8.57(s, 1H), 7.69~7.65(td, J=7.36Hz,
1
2
1
1
3
4
.6Hz, 1H), 7.52~7.43(m, 3H), 7.34~7.22(m, 10H), 4.59(s, 2H), 4.34(s,
13
3
H). CNMR (100 MHz, CD OD): δ164.06, 161.55, 147.41, 140.59,
36.99, 134.98, 133.59, 128.80, 128.40, 128.31, 127.21, 126.60,
24.42, 124.39, 117.71, 115.55, 15.34, 109.38, 109.22, 87.15, 85.15,
and then was subjected to chromatography on silica gel (PE/EtOAc
+
5.96, 30.82. ESI-HRMS: m/z [M+H] calcd for 434.1669, found
1
20:1) to give a colorless oil (380mg). Yield: 60%. HNMR (400 MHz,
34.1662.
DMSO-d
6
): δ7.31~7.16 (m, 6H), 4.80(s, 1H) 3.87 (s, 2 H), 3.66~3.53
+
(m, 4H). ESI-MS: m/z [M+NH ] calcd for 240.16, found 240.5.
4
2
3
-Benzyl-8-benzyl-6-(3-methylphenylethynyl)imidazo[1,2-a]pyrazin-
(7H)-one (A5): yellow solid; yield 40%. Analytical RP HPLC
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 4 of 10
View Article Online
DOI: 10.1039/C6OB00659K
COMMUNICATION
Journal Name
(Phenomenex, C8, 250 x 4.6 mm column): 65% acetonitrile with 0.1% The preparation of 2-amino-3-benzyl- 5-ethynyl-pyrazine (5):
trifluoroacetic acid, 1.0 mL/min at 380 nm, Rt: 6.910 min, 95%.
1
Potassium carbonate (40 mg, 0.284 mmol) was added to the
solution of 2-amino-3-benzyl- 5-((trimethylsilyl)ethynyl)-pyrazine
HNMR (400 MHz, CD
3
OD): δ7.70(s, 1H), 7.40~7.18(m, 14H), 4.34(s,
13
2
1
1
1
3
4
H), 4.18(s, 2H), 2.36(s, 3H). CNMR (100 MHz, CD OD): δ147.24,
3
(
100 mg, 0.356 mmol) in methanol and the mixture was stirred at
40.63, 138.54, 138.47, 137.10, 134.99, 134.95, 134.02, 131.94,
30.95, 130.58, 128.86, 128.79, 128.72, 128.56, 128.37, 128.22,
28.12, 127.24, 126.56, 120.95, 120.57, 117.28, 94.50, 79.72, 35.73,
room temperature for 2h. Then the reagent was evaporated under
vacuum, and the crude was redissolved in ethyl acetate. The
organic layer was washed with saturated sodium chloride solution
after extraction with ethyl acetate. After dried over anhydrous
sodium sulfate and concentrated under reduced pressure, the
crude product was further purified by chromatography on silica gel
+
1.06, 20.1. ESI-HRMS: m/z [M+H] calcd for 430.1919, found
30.1914.
2
-Benzyl-8-benzyl-6-(3-hydroxyphenylethynyl)imidazo[1,2-
a]pyrazin-3(7H)-one (A6): yellow solid; yield 15%. Analytical RP (PE/EtOAc 20:3) to give a light yellow solid (50 mg). Yield: 67%.
1
HPLC (Phenomenex, C8, 250 x 4.6 mm column): 60% acetonitrile
6
HNMR (400 MHz, DMSO-d ): δ 7.98 (s, 1 H), 7.30~7.20 (m, 5H),
1
3
with 0.1% trifluoroacetic acid, 1.0 mL/min at 370 nm, Rt: 4.946 min, 6.76 (s, 2 H), 4.15 (s, 2H), 3.99(s, 2H). CNMR (100 MHz, DMSO-d
6
):
OD): δ7.69(s, 1H), 7.41~7.18(m, 14H), δ 153.38, 144.56, 141.72, 138.13, 129.30, 128.75, 126.73, 124.75,
.33(s, 2H), 4.18(s, 2H). CNMR (100 MHz, CD OD): δ157.37, 147.15, 82.36, 80.32, 38.77. ESI-HRMS: m/z [M+H +] calcd for 210.1031,
1
9
9%. HNMR (400 MHz, CD
3
1
3
4
3
137.09, 134.96, 129.59, 128.80, 128.57, 128.36, 127.25, 126.56, found 210.1019.
1
22.87, 121.52, 117.94, 117.28, 94.44, 79.38, 35.66, 31.11. ESI-
2
6
+
The preparation of 2-azidoethanol (6-1) :
HRMS: m/z [M+H] calcd for 432.1712, found 432.1701.
2
-Bromoethanol (1.76 g, 14.08 mmol) was dissolved in water (3 mL)
The preparation of 2-amino-3-benzyl- 5-((trimethylsilyl)ethynyl)-
2
4, 25
and then sodium azide (1.83 g, 28.17 mmol) was added to this
mixture with stirring. The mixture was stirred at room temperature
pyrazine (4)
:
The suspension of PdCl
2
(PPh
3
)
2
(27 mg, 0.038 mmol), CuI (29 mg, for 20 h. The mixture was extracted with diethyl ether, and the
0
.151 mmol) and PPh (20 mg, 0.076 mmol) in 2 mL toluene was organic layer was washed with saturated sodium chloride solution.
3
added to the solution of 3-benzyl-5-bromo-2-amino-pyrazine (200 After dried over anhydrous sodium sulfate and concentrated under
mg, 0.76 mmol) in 3 mL toluene under argon atmosphere. Then reduced pressure, the crude product was applied to the next
trimethylsilylacetylene (223 mg, 0.27 mmol) and triethylamine reaction without further purification.
o
(
0.8 mL) were sequentially added. The mixture was stirred at 40 C
3
-Azido-1-propanol (6-2) and 1-azido-2-fluoroethane (6-3) were
for 9 h. Then the mixture was filtered, and the filtrate was collected
and evaporated under vacuum. The collection was extracted by
ethyl acetate and washed with saturated sodium chloride solution.
After dried over anhydrous sodium sulfate and concentrated under
reduced pressure, the crude product was further purified by
prepared with similar method.
The preparation of 2-amino-3-benzyl- 5- [(1-hydroxyethyl)-1, 2, 3-
2
2
triazol-4]-pyrazine (7-1) :
chromatography on silica gel (PE/EtOAc 10:1) to give a light yellow
1
Copper sulfate pentahydrate (2 mg, 7.15 μmol) and sodium
ascorbate (4 mg, 1.45 μmol) in water (1.5 mL) were sequentially
added to the mixture of 2-amino-3-benzyl-5-ethynyl-pyrazine (30
mg, 0.143 mmol) and 2-azidoethanol (16 mg, 0.186 mmol) in THF
solid (129 mg). Yield: 60%. HNMR (400 MHz, DMSO-d
6
): δ 7.98 (s, 1
13
H), 7.29~7.20 (m, 5H), 6.79 (s, 2 H), 3.99 (s, 2H). CNMR (100 MHz,
DMSO-d ): δ 152.42, 143.94, 140.85, 137.30, 128.36, 127.92, 125.88,
6
+
124.25, 103.09, 92.93, 37.91, 0.50. ESI-HRMS: m/z [M+H] calcd for
(
1.5 mL) under argon. Then triethylamine (2 μL) was added and the
282.1426, found 282.1418.
mixture was stirred at room temperature for 8 h. Then, the mixture
was extracted with ethyl acetate and the organic layer was washed
Scheme 2: the synthesis route of triazole series (T) coelenterazine analogs.
4
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 10
Journal Name
Orga Pn l iec a s&e Bd oi o nmo to al ed cj uu sl ta mr Ca rhg ei nms istry
View Article Online
DOI: 10.1039/C6OB00659K
COMMUNICATION
with saturated sodium chloride solution. After dried over anhydrous 2-Benzyl-8-benzyl-6- [(1-fluoroethyl)-1, 2, 3- triazol-4]imidazo[1,2-
sodium sulfate and concentrated under reduced pressure, the a]pyrazin-3(7H)-one (T3): yellow solid; yield 25%. Analytical RP HPLC
crude product was further purified by chromatography on silica gel (Phenomenex, C8, 250 x 4.6 mm column): 60% acetonitrile with 0.1%
(
PE/EtOAc 20:3) to give a light yellow solid (40 mg). Yield: 95%. trifluoroacetic acid, 1.0 mL/min at 370 nm, Rt: 3.035 min, 95%.
1
1
HNMR (400 MHz, CDCl
3
): δ 8.70 (s, 1H), 8.10(s, 1H), 7.34~7.28 (m,
3
HNMR (400 MHz, CD OD): δ 8.47 (s, 1H), 8.26(s, 1H), 7.41~7.20 (m,
1
3
2
1
H), 7.26~7.22(m, 3H), 4.55(t, J=4Hz, 2H), 4.49(s, 2H), 4.13(m, 4H). 10H), 4.92(m, 1H), 4.85(m, 3H), 4.46(s, 2H), 4.21(s, 2H). CNMR
3
CNMR (100 MHz, DMSO-d
6 3
): δ 153.52, 146.18, 140.71, 138.47, (100 MHz, CD OD): δ148.43, 143.66, 138.03, 136.52, 135.46, 134.00,
1
37.01, 133.86, 129.19, 128.73, 126.68, 122.12, 60.34, 52.74, 38.93. 128.86, 128.55, 128.38, 128.17, 126.88, 126.81, 124.36, 109.68,
+
+
ESI-HRMS: m/z [M+ H] calcd for 297.1464, found 297.1456.
82.24, 80.54, 37.85, 29.03. ESI-HRMS: m/z [M+H] calcd for
29.1839, found 429.1833.
4
2
-Amino-3-benzyl- 5- [(1-hydroxypropyl)-1, 2, 3- triazol-4]-pyrazine
1
(7-2): light yellow solid; yield 83%; HNMR (400 MHz, CDCl
3
): δ 8.76 Luminescence assay
(s, 1H), 8.04(s, 1H), 7.33~7.28 (m, 2H), 7.26~7.22(m, 3H), 4.59(t,
Millipore water was used to prepare all aqueous solutions.
Measurements for Renilla luciferase bioluminescent assays were
determined in 50mM Tris-HCl buffer, pH 7.42. Measurements for
Gaussia luciferase bioluminescent assays were determined in
J=6Hz, 2H), 4.48(s, 2H), 4.14(s, 2H), 3.70(m, J=6Hz, 2H), 2.19(m, 2H).
1
3
6
CNMR (100 MHz, DMSO-d ): δ 153.54, 146.29, 140.68, 138.47,
1
37.08, 133.81, 129.17, 128.73, 126.68, 121.80, 57.96, 47.21, 38.92,
+
33.37. ESI-HRMS: m/z [M+H] calcd for 311.1620, found 311.1603.
2
5mM Tris-HCl buffer, pH 7.8, with 600 mM NaCl, 1 mM EDTA, 0.05%
2
-Amino-3-benzyl- 5- [(1-fluoroethyl)-1, 2, 3- triazol-4]-pyrazine (7- BSA. The bioluminescence images were captured by using an IVIS
1
6
3): light yellow solid; yield 79%; HNMR (400 MHz, DMSO-d ): δ 8.45 Kinetic (Caliper Life Sciences, USA) equipped with a cooled charge-
(
s, 1H), 8.39(s, 1H), 7.33~7.19 (m, 5H), 6.46(s, 2H), 4.93(t, J=4Hz, 1H), coupled device (CCD) camera. The bioluminescence spectra were
1
3
4
6
.80(m, 2H), 4.73(m, 2H), 4.07(s, 2H). CNMR (100 MHz, DMSO-d ): performed with F-1200 fluorescence spectrophotometer in
δ 153.62, 146.61, 140.77, 138.44, 137.10, 133.56, 129.16, 128.64, luminescence mode. Luciferase was purchased from RayBiotech.
+
126.69, 122.08, 66.21, 50.65, 50.45, 38.92. ESI-HRMS: m/z [M+H]
m m
The bioluminescence kinetic parameters K , V and half decay life
calcd for 299.1420, found 299.1406.
were calculated using GraphPad Prism software.
The preparation of 2-benzyl-8-benzyl-6- [(1-hydroxyethyl)-1, 2, 3- Bioluminescence properties measurement (Renilla luciferase)
1
8, 20, 23
triazol-4]imidazo[1,2-a]pyrazin-3(7H)-one (T1)
:
To determine the bioluminescence properties of the novel
The mixture of 2-amino-3-benzyl- 5- [(1-hydroxyethyl)-1, 2, 3- derivatives, all compounds were freshly dissolved in 95% EtOH as
triazol-4]-pyrazine (7-1, 60 mg, 0.202 mmol) and 3-phenyl-1,1- stock and diluted to appropriate concentrations in Tris-HCl (50 mM,
diethoxyacetone (3-1, 90 mg, 0.404 mmol) was dissolved in ethanol pH 7.42) for each measurement. In all the measurements, the final
(3 mL) under argon atmosphere and allowed to stir at room ethanol concentration in the sample solution was kept constant at
temperature for 10 min. The con.HCl (0.2 mL) in ethanol (1 mL) was 0.5% (v/v) to avoid the effect of ethanol on the BL reaction. The
then added to the mixture via syringe over 10 min. The reaction Renilla luciferase was dissolved in and diluted to 1 μg/mL with Tris-
o
was heated to refluxed at 80 C for 8 h and then allowed to cool to HCl (50 mM, pH 7.42). To measure bioluminescence intensity, the
room temperature. The crude was concentrated under vacuum and solution of compound (50 µL) was added to a 96-well black flat
further purified by chromatography on silica gel (CH
to give yellow solid (60 mg). Yield: 69%. Analytical RP HPLC mixed quickly. The final concentration of luciferase was 0.5 μg/mL.
Phenomenex, C8, 250 x 4.6 mm column): 45% acetonitrile with 0.1% The final compound concentrations were 0.25, 0.5, 1, 2, 5, 10 and
trifluoroacetic acid, 1.0 mL/min at 370 nm, Rt: 3.923 min, 98%. 25 µM. Bioluminescence intensities of DeepBlueC and the
2 2
Cl /MeOH 50:1) bottom microscale plate, and luciferase (50 µL) was added and
(
1
3
HNMR (400 MHz, CD OD): δ 8.35 (s, 1H), 8.02(s, 1H), 7.24~7.05 (m, derivatives were immediately measured with an IVIS Kinetic (Caliper
1
0H), 4.44(t, J=6Hz, 2H), 4.32(s, 2H), 4.04(s, 2H), 3.86(t, J=6Hz, 2H). Life Sciences, USA) equipped with a cooled charge-coupled device
1
3
CNMR (100 MHz, CD
35.47, 133.85, 128.86, 128.54, 128.37, 128.17, 126.88, 126.80, were reported as total photon flux within an ROI in photons per
24.56, 109.73, 60.10, 52.93, 37.81, 29.08. ESI-HRMS: m/z [M+ +] second. The Michaelis constant K and maximum rate Vmax were
3
OD): δ148.40, 143.06, 138.07, 136.52, (CCD) camera. The assays were measured in triplicate. The results
1
1
m
calcd for 427.1882, found 427.1876.
estimated with the Michaelis–Menten kinetics equation using
GraphPad Prism software.
2-Benzyl-8-benzyl-6-
[(1-hydroxypropyl)-1,
2,
3-
triazol-
4]imidazo[1,2-a]pyrazin-3(7H)-one (T2): yellow solid; yield 23%.
For the recording of bioluminescence spectra, an aliquot of
Analytical RP HPLC (Phenomenex, C8, 250 x 4.6 mm column): 50% Renilla luciferase solution (0.5 mL, 1 μg/mL) was mixed with
acetonitrile with 0.1% trifluoroacetic acid, 1.0 mL/min at 370 nm, Rt: derivative solution (0.5 mL, 25 μM) in a quartz cell and the mixture
1
3
1
3
1
1
.888 min, 100%. HNMR (400 MHz, CD
3
OD): δ 8.44 (s, 1H), 8.15(s, was immediately measured with
a
F-1200 fluorescence
H), 7.35~7.12 (m, 10H), 4.57(t, J=8Hz, 2H), 4.44(s, 2H), 4.14(s, 2H), spectrophotometer in luminescence wavelength mode with the
13
.58(t, J=8Hz, 2H), 2.13(m, 2H). CNMR (100 MHz, CD
43.09, 136.51, 135.48, 133.72, 128.84, 128.55, 128.39, 128.16, The wavelengths of maximal bioluminescence intensities (λmax
26.88, 126.80, 124.19, 109.90, 65.52, 57.92, 37.84, 32.49, 29.07. were corrected by the use of coelenterazine and reported data (485
3
OD): δ148.42, lamp off at a scan rate of 3000 nm/min with response time of 2 s.
)
+
ESI-HRMS: m/z [M+H] calcd for 441.2039, found 441.2034.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 6 of 10
View Article Online
DOI: 10.1039/C6OB00659K
COMMUNICATION
Journal Name
1
5
nm) . Moreover, the quantitative bioluminescence spectra were ES-2 cells (human ovarian cancers cell line) expressing Reilla
determined using the instrument software (FL Solutions ver. 2.1).
luciferase (Rluc) were purchased from Shanghai BioDiagnosis Co.,
Ltd. The ES-2-Rluc cells were cultured in DMEM high glucose
o
For the recording of half decay life, an aliquot of Renilla
luciferase solution (0.5 mL, 1μg/mL) was mixed with derivative
solution (0.5 mL, 25 μM) in a quartz cell and the mixture was
supplemented with 10% fetal bovine serum (FBS) at 37 C in a
humidified atmosphere in a 5% CO2 incubator.
immediately
measured
with
a
F-1200
fluorescence Cell bioluminescence imaging
spectrophotometer in luminescence time scan mode with the lamp
off at a scan rate of 3000 nm/min with response time of 2s. The
measurement was last 300 s. The half decay life was calculated by
using GraphPad Prism software.
5
Cells were grown in black 96-well plates (4×10 cells per well). After
a 24-h incubation period, the medium was removed, and cells were
treated with 100 μL of various concentrations of compounds (range
0
.25 μM to 25 μM). Bioluminescence intensity was measured
Bioluminescence properties measurement (Gaussia luciferase)
immediately using an IVIS Kinetic imaging system. Luminescent
signal (photons per second) for each well was measured and
plotted as average values (experiments conducted in triplicate).
To determine the bioluminescence properties of the novel
derivatives, all compounds were freshly dissolved in 95% EtOH as
stock and diluted to appropriate concentrations in Tris-HCl (25 mM, Cell concentration dependence assay
pH 7.8, containing 600 mM NaCl, 1 mM EDTA, 0.05% BSA) for each
5
Cell were grown in black 96-well plates (0.125, 0.25, 0.5, 1, 2, 4×10
measurement. In all the measurements, the final ethanol
concentration in the sample solution was kept constant at 0.5% (v/v)
to avoid the effect of ethanol on the BL reaction. The Gaussia
luciferase was dissolved in and diluted to 0.5 μg/mL with Tris-HCl
cells per well, respectively). After a 24-h incubation period, the
medium was removed, and cells were treated with 100 μL of
compounds (20 μM). Bioluminescence intensity was measured
immediately using an IVIS Kinetic imaging system. Luminescent
signal (photons per second) for each well was measured and
plotted as average values (experiments conducted in triplicate).
(
25 mM, pH 7.8, containing 600 mM NaCl, 1 mM EDTA, 0.05% BSA).
To measure bioluminescence intensity, the solution of compound
50 µL) was added to a 96-well black flat bottom microscale plate,
(
and luciferase (50 µL) was added and mixed quickly. The final
concentration of luciferase was 0.25 μg/mL. The final compound
concentrations were 0.25, 0.5, 1, 2, 5, 10 and 25 µM.
Bioluminescence intensities of DeepBlueC and the derivatives were
immediately measured with POLABstar Omega microplate reader or
an IVIS Kinetic (Caliper Life Sciences, USA) equipped with a cooled
charge-coupled device (CCD) camera. The assays were measured in
Chemiluminescent assay
For the recording of chemiluminescence spectra, 2 mL of DMSO
with 0.5% (v/v) of NaOH (1 M) was mixed with 200 μL of derivative
solution (50 μM) in a quartz cell and the mixture was immediately
measured with an F-1200 fluorescence spectrophotometer in
luminescence wavelength mode with the lamp off at a scan rate of
m
triplicate. The Michaelis constant K and maximum rate Vmax were
3
000 nm/min with response time of 2 s. The wavelengths of
estimated with the Michaelis–Menten kinetics equation using
GraphPad Prism software.
maximal bioluminescence intensities (λmax) and the quantitative
chemiluminescence spectra were determined using the instrument
software (FL Solutions ver. 2.1).
For the recording of bioluminescence spectra, an aliquot of
Gaussia luciferase solution (0.5 mL, 0.5 μg/mL) was mixed with
derivative solution (0.5 mL, 25 μM) in a quartz cell and the mixture
Fluorescent assay
was immediately measured with
a
F-1200 fluorescence
To measure fluorescent spectra, the solution of compound (200 µL,
spectrophotometer in luminescence wavelength mode with the
lamp off at a scan rate of 3000 nm/min with response time of 2 s.
1
00 µM) was added to a 96-well black flat bottom microscale plate
and was immediately measured with Varioskan microplate
spectrophotometer (Thermo Electron Corporation, Waltham, MA,
USA).
The wavelengths of maximal bioluminescence intensities (λmax
)
were corrected by the use of coelenterazine and reported data (488
1
5
nm) . Moreover, the quantitative bioluminescence spectra were
determined using the instrument software (FL Solutions ver. 2.1).
Solubility assay
For the measurement of half decay life, the solution of
compound (50 µL) was added to a 96-well black flat bottom
microscale plate, and luciferase (50 µL) was added and mixed
quickly and was immediately measured with a POLABstar Omega
1
mg of compound A4, A6 or DeepBlueC was dissolved in deionized
water until the solution was clear. The assays were measured in
triplicate.
microplate reader with a measurement time of 300 s. The half Reasults and discussion
decay life was calculated by using GraphPad Prism software.
Bioluminescence properties with Renilla luciferase
Cell culture
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 7 of 10
Journal Name
Orga Pn l ieca s&e Bd oi o nmo to al ed cj uu sl ta mr Ca rhg ei nms istry
View Article Online
DOI: 10.1039/C6OB00659K
COMMUNICATION
All novel compounds were initially evaluated with recombinant flexible of molecular structure may result in humble
Renilla reniformis luciferase (Rluc). As shown in Fig. 2 and
Table 1, Renilla luciferase with them displayed increasing
bioluminescence when the rise of concentrations. Moreover,
A4 is the best one of series alkynes, and T3 is the best one of
series triazoles. They emit moderate bioluminescence with
interaction with active sites of luciferase. However, the results
of luminescence spectra disclosed that the maxima of Rluc
light emission with analogs except T1 are significantly red-
shifted by 20-60 nm as compared to that with the DeepBlueC
(Fig. 2 and Table 1). The maximum emission peak of compound
Renilla luciferase, which is much lower than that of DeepBlueC. A6 with Rluc is remarkably red-shifted up to 60 nm. Moreover,
1
7, 27
However, relative bioluminescence quantum yields (RQY)
of all novel compounds seem lower than that of DeepBlueC
Table 1). With increasing of molecular rigidity resulting from
some compounds such as A4, A6, T1 and T3 with Rluc display
the slow kinetic decay of the light signal. Bioluminescence
K
m
(
values of novel analogs are in the range of 2-4 μM except T2
and T3 (12.97 and 18.43, respectively), which are slightly
higher than that of the DeepBlueC. Though the
the introduction of alkynyl group, series alkynes may have
poor activities to interact with luciferase. The 2-
fluorophenylethynyl group of compound A4 may interact with
the key active sites of luciferase firmly in contrast with other
bioluminescence
Vmax values of them are lower than that of
DeepBlueC, some of them such as the
V
max of T3 approaches
compounds containing fluoro-group. As for T series, their more that of DeepBlueC.
Table 1. Bioluminescence properties of all compounds with Renilla luciferase.
b
m
b
Compounds
Corrected
Bioluminescence Half
Bioluminescence K
Bioluminescence Vmax
Relative Bioluminescence
a
c
Bioluminescence Emission
Life (s)
(μM)
(s/p)
Quantum Yield (RQY)
(
nm)
7
DeepBlueC
388
416
416
448
42.23
8.60
0.61 ± 0.06
2.24 ± 0.15
2.08 ± 0.44
3.06 ± 0.41
(3.89 ± 0.12) × 10
1
6
A1
A2
A6
(2.98 ± 0.09) × 10
0.046
0.026
0.064
6
8.40
(1.69 ± 0.21) × 10
6
29.68
(2.69 ± 0.11) × 10
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 8 of 10
View Article Online
DOI: 10.1039/C6OB00659K
COMMUNICATION
Journal Name
6
6
A3
A4
A5
T1
T2
412
397
421
388
427
417
13.11
141.00
20.01
80.64
16.41
232.50
3.56 ± 0.28
2.68 ± 0.20
2.10 ± 0.28
4.50 ± 0.62
12.97 ± 1.02
18.43 ± 0.86
(3.53 ± 0.09) × 10
(8.14 ± 0.23) × 10
(5.25 ± 0.32) × 10
(1.82 ± 0.09) × 10
(2.44 ± 0.07) × 10
(2.08 ± 0.04) × 10
0.040
0.109
0.090
0.016
0.009
0.059
6
6
6
7
T3
a
BL half life was calculated using GraphPad Prism software and taken advantage of the values when compounds were 25 μM.
b
m
Michaelis constant K and maximum rate Vmax were estimated with the Michaelis–Menten kinetics equation using GraphPad Prism software. The values are shown by
means±SD of three independent assays performed in triplicate.
c
Relative quantum yields of bioluminescence were obtained with an IVIS Kinetic (Caliper Life Sciences, USA) equipped with a cooled charge-coupled device (CCD)
camera. The quantum yield (QY) was calculated by dividing the measured total number of photons by the number of substrate molecules in the solution.
compound A4 emits 88% bioluminescence with Gluc as
Bioluminescence properties with Gaussia luciferase
compared to that with DeepBlueC, which is the best
performance. All T series compounds have insignificant
In addition, all novel compounds were evaluated with Gaussia
luciferase (Table 2). However, all of them including DeepBlueC
had poor performance with Gluc. The bioluminescence spectra
of T series are similar with of DeepBlueC. All A series
compounds with Gluc demonstrate blue-shifted emission by
bioluminescence. Though they have lower Vmax values, their K
m
values are also worse. In a word, our compounds may not be
suitable to interact with Gaussia luciferase, which leads to
underprivileged bioluminescence. The chemiluminescence
spectra and fluorescence spectra of all novel compounds were
also measured as displayed in Table S1.
30-40 nm as compared to that of DeepBlueC. Among them,
Table 2: Bioluminescence properties of novel compounds with Gaussia luciferase.
Corrected
Bioluminescence
Bioluminescence Half
Bioluminescence K
m
Bioluminescence Vmax
Relative
Bioluminescence
a
b
b
Compounds
decay life (s)
(μM)
(s/p)
c
Emission (nm)
493
Quantum Yield (RQY)
4
DeepBlueC
27.07
--
22.97 ± 2.93
--
(1.24±0.09) ×10
1
T2
T1
T3
A1
A2
A5
A3
A4
A6
490
--
0.078
0.028
0.059
0.487
0.225
0.576
0.548
0.885
0.386
497
--
--
--
493
--
--
--
3
456
--
1.35±0.12
3.24±0.35
1.26±0.093
1.70±0.081
0.85±0.064
0.54±0.049
(1.25±0.03) ×10
2
451
--
(7.96±0.28)×10
(1.50±0.03) ×10
(1.42±0.02) ×10
(2.16±0.04) ×10
(8.93±0.20) ×10
3
3
3
2
452
--
455
--
456
--
458
--
a
BL half decay life was calculated using GraphPad Prism software and taken advantage of the values when compounds were 25 μM.
b
m
Michaelis constant K and maximum rate Vmax were estimated with the Michaelis–Menten kinetics equation using GraphPad Prism software. The values are shown by
means±SD of three independent assays performed in triplicate.
c
Relative quantum yields of bioluminescence were obtained with an IVIS Kinetic (Caliper Life Sciences, USA) equipped with a cooled charge-coupled device (CCD)
camera. The quantum yield (QY) was calculated by dividing the measured total number of photons by the number of substrate molecules in the solution.
and A6 with superior water solubility are easier to diffuse into the
cells so as to appear improved bioluminescence. Furthermore, the
Bioluminescence in cellulo
introduction of the electron-rich group, such as a hydroxyl group of
compound A6, can result in an appropriate lipid-water partition
coefficient, which promotes the bioluminescence induced by A6 in
cellular level. Hence, compounds A4 and A6 are more potential CTZ-
type bioluminescence substrates to be applied at the cellular level
compared with DeepBlueC.
Subsequently, all compounds were investigated with ES-2 cells
expressing Renilla luciferase as shown in Fig. 3. It is exciting that our
novel compounds behave improved properties in cellular level.
Light emission of compounds A4 and A6 displays 4- and 6-fold
higher emission as compared to that of DeepBlueC, respectively.
The performance of compound A2 is similar to that of DeepBlueC.
What’s more, bioluminescence emitted by A4 and A6 can last Conclusions
longer in contrast with that of DeepBlueC (Fig. 3C). Moreover,
We have successfully designed and synthesized two series of CTZ-
bioluminescence emitted by compounds A4 and A6 could increase
with the rise in cell concentration (Fig. 3D). However, all T series
compounds are inferior to DeepBlueC (Fig. S2). Moreover, the mass
type derivatives based on the structure of DeepBlueC and then
investigated their bioluminescence properties with Renilla luciferase
and Gaussia luciferase in vitro. Most of the compounds display
moderate bioluminescence in the presence of Renilla luciferase and
Gaussia luciferase. However, they are inferior to DeepBlueC. All
compounds of series A behave better in combination with ES-2 cells
expressing Rluc. It is of great significance that the compounds A4
solubility of A4, A6 and DeepBlueC were determined, and the
results were display in Table S2. It is evident that both A4 and A6
were easier to dissolve in water at the room temperature as
compared to DeepBlueC. The results indicated that compounds A4
8
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 9 of 10
Orga Pn l ieca s&e Bd oi o nmo to al ed cj uu sl ta mr Ca rhg ei nms istry
View Article Online
DOI: 10.1039/C6OB00659K
Journal Name
COMMUNICATION
and A6 that have brighter and longer emission with Rluc in the
This work was supported by grants from the National Program on
cellular level are superior to DeepBlueC. The results indicate that Key Basic Research Project (no. 2013CB734000), the National
introduction of the alkynyl bond could lead to the enhancement of Natural Science Foundation of China (no. 81370085), the Program
molecular rigidity, which could affect the entrance into the pocket for Changjiang Scholars and Innovative Research Team in University
and interaction with active sites of luciferase. The 2- (No. IRT13028), the Major Project of Science and Technology of
fluorophenylethynyl moiety of compound A4 may interact with Shandong Province (No. 2015ZDJS04001), the Shandong Key
some of the key active sites of luciferase firmly in contrast with Research & Development Project (No. 2015GSF118166), and the
other compounds containing fluoro group, which makes it better Independent Innovation Foundation of Shandong University, IIFSDU
than others. Moreover, appropriate lipid-water partition coefficient (no. 2014JC008).
plays an important role in bioluminescence in cellulo. The better
mass solubility of our novel compounds promotes them to diffuse
Notes and references
into cells in order to induce bioluminescence with Rluc. The
introduction of electron-rich groups such as a hydroxyl group of
1
.
J. Li, L. Chen, L. Du and M. Li, Chemical Society reviews,
2013, 42, 662-676.
compound A6 can make it have an appropriate lipid-water partition
coefficient, which promotes the bioluminescence induced by A6 in 2.
O. Shimomura, Bioluminescence: Chemical Principles and
Methods, World Scientific Publishing Co. Pte. Ltd, 2006.
S. H. Haddock, M. A. Moline and J. F. Case, Annual review
of marine science, 2010, 2, 443-493.
K. Hori, H. Charbonneau, R. C. Hart and M. J. Cormier,
cellulo. Both of A4 and A6 are suitable and potential bioluminescent
3
4
.
.
substrates that can be applied in cellulo with the ability to probe
some biological activities. Moreover, these compounds can be
further modified to become probes with the use of “caged” strategy
1
,
15
Proceedings of the National Academy of Sciences, 1977,
,
thus becoming favorable molecules involved in
7
4
, 4285-4287.
O. Shimomura and K. Teranishi, Luminescence, 2000, 15
1-58.
bioluminescence technique. We consider that our compounds rich
the diversity of CTZ-type substrates and the knowledge gained from
this work is helpful for the development of novel CTZ-type analogs
with superior bioluminescent properties.
5
6
.
.
,
5
M. J. Cormier, The Journal of biological chemistry, 1962,
237, 2032-2037.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 10 of 10
View Article Online
DOI: 10.1039/C6OB00659K
COMMUNICATION
Journal Name
7
8
9
1
1
1
1
1
1
.
M. J. Cormier, Light and life, Johns Hopkins Press,
Baltimore, 1961.
O. Shimomura, F. H. Johnson and Y. Saiga, Journal of
Cellular and Comparative Physiology, 1962, 59, 223-239.
O. Shimomura and F. H. Johnson, Biochemistry, 1972, 11
.
.
,
1602-1608.
0.
1.
2.
3.
4.
5.
K. Hori, J. E. Wampler, J. C. Matthews and M. J. Cormier,
Biochemistry, 1973, 12, 4463-4468.
O. Shimomura and F. H. Johnson, Tetrahedron Letters,
1
973, 14, 2963-2966.
O. Shimomura, F. H. Johnson and H. Morise, Biochemistry,
974, 13, 3278-3286.
S. Inoue, S. Sugiura, H. Kakoi, K. Hasizume, T. Goto and H.
Iio, Chemistry Letters, 1975, , 141-144.
1
4
S. Inoue, H. Kakoi, M. Murata, T. Goto and O. Shimomura,
Tetrahedron Letters, 1977, 18, 2685-2688.
T. Jiang, L. Du and M. Li, Photochemical & photobiological
sciences : Official journal of the European Photochemistry
Association and the European Society for Photobiology,
2016, 15, 466-480.
1
6.
7.
K. Hori, J. M. Anderson, W. W. Ward and M. J. Cormier,
Biochemistry, 1975, 14, 2371-2376.
K. Niwa, Y. Ichino, S. Kumata, Y. Nakajima, Y. Hiraishi, D.-i.
Kato, V. R. Viviani and Y. Ohmiya, Photochemistry and
photobiology, 2010, 86, 1046-1049.
1
1
8.
9.
M. Adamczyk, D. D. Johnson, P. G. Mattingly, Y. Pan and R.
E. Reddy, Organic Preparations and Procedures
International, 2001, 33, 477-485.
1
M. Adamczyk, S. R. Akireddy, D. D. Johnson, P. G.
Mattingly, Y. Pan and R. E. Reddy, Tetrahedron, 2003, 59
129-8142.
,
8
2
2
0.
1.
US Pat., WO2012040105A2, 2012.
Y. Liang, Y.-X. Xie and J.-H. Li, The Journal of Organic
Chemistry, 2006, 71, 379-381.
22.
23.
24.
25.
26.
L. Du Ý, N. Ni Ý, M. Li and B. Wang, Tetrahedron Letters,
2
010, 51, 1152-1154.
E. Lindberg, S. Mizukami, K. Ibata, T. Fukano, A. Miyawaki
and K. Kikuchi, Chemical Science, 2013, , 4395-4400.
N. Schultheiss, C. L. Barnes and E. Bosch, Crystal Growth &
Design, 2003, , 573-580.
Y. P. Xu, R. H. Hu and M. Z. Cai, Chinese Chemical Letters,
008, 19, 783-787.
4
3
2
M. Swetha, P. V. Ramana and S. Shirodkar, Organic
Preparations and Procedures International, 2011, 43, 348-
353.
27.
Y. Ando, K. Niwa, N. Yamada, T. Irie, T. Enomoto, H.
Kubota, Y. Ohmiya and H. Akiyama, Photochemistry and
photobiology, 2007, 83, 1205-1210.
1
0 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins