Coumarin-based fluorescent 'AND' logic gate probes for the detection of homocysteine and a chosen biological analyte

Article abstract of DOI:10.1039/c9ra04908h

With this research we set out to develop a number of coumarin-based 'AND' logic fluorescence probes that were capable of detecting a chosen analyte in the presence of HCys. Probe JEG-CAB was constructed by attaching the ONOO^- reactive unit, benzyl boronate ester, to a HCys/Cys reactive fluorescent probe, CAH. Similarly, the core unit CAH was functionalised with the nitroreductase (NTR) reactive p-nitrobenzyl unit to produce probe JEG-CAN. Both, JEG-CAB and JEG-CAN exhibited a significant fluorescence increase when exposed to either HCys and ONOO^- (JEG-CAB) or HCys and NTR (JEG-CAN) thus demonstrating their effectiveness to function as AND logic gates for HCys and a chosen analyte.

Full text of DOI:10.1039/c9ra04908h

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Coumarin-based fluorescent ‘AND’ logic gate
probes for the detection of homocysteine and
Cite this: RSC Adv., 2019, 9, 26425
a chosen biological analyte†
a
a
d
Luling Wu,
Jordan E. Gardiner,
Lokesh K. Kumawat,c Hai-Hao Han,
Ruiying Guo,e Xin Li,
Adam C. Sedgwick,
Xiao-Peng He,
Steven D. Bull
Robert B. P. Elmes,
e
d
cf
*
*
*
a
b
a
*
*
*
and Tony D. James
With this research we set out to develop a number of coumarin-based ‘AND’ logic fluorescence probes that
were capable of detecting a chosen analyte in the presence of HCys. Probe JEG-CAB was constructed by
attaching the ONOOꢀ reactive unit, benzyl boronate ester, to a HCys/Cys reactive fluorescent probe, CAH.
Similarly, the core unit CAH was functionalised with the nitroreductase (NTR) reactive p-nitrobenzyl unit to
produce probe JEG-CAN. Both, JEG-CAB and JEG-CAN exhibited a significant fluorescence increase when
exposed to either HCys and ONOOꢀ (JEG-CAB) or HCys and NTR (JEG-CAN) thus demonstrating their
effectiveness to function as AND logic gates for HCys and a chosen analyte.
Received 28th June 2019
Accepted 31st July 2019
DOI: 10.1039/c9ra04908h
rsc.li/rsc-advances
Homocysteine (HCys) is a non-proteinogenic amino acid,
A number of uorescent sensors have been developed for the
formed from the de-methylation of methionine,1 which is then detection of HCys to help improve our understanding of its role in
converted into cysteine (Cys) via a vitamin B6 cofactor. Typical biological systems.8–11 However, these uorescent probes have
physiological concentrations of HCys range between 5–15 mmol focused on the detection of a single biomarker (HCys), however,
Lꢀ1.2 However, elevated levels of HCys (>15 mmol Lꢀ1), which is processes associated with HCys induced toxicity oen involve more
known as hyperhomocysteinemia (hHCys),3 have been associ- than one biochemical species. For example, it has been reported
ated with pregnancy disorders, Alzheimer's disease, cardiovas- that peroxynitrite (ONOOꢀ) and nitric oxide (NOc) play a signicant
cular disease and neurodegenerative diseases (NDs).4–6 It is role in HCys-mediated apoptosis in trigeminal sensory neurons12
believed that the main cause of HCys induced toxicity is through and HCys has been reported to induce cardiomyocytes cell death
the non-enzymatic modication of proteins. This is achieved through the generation of ONOOꢀ.13 The production of ONOOꢀ is
through irreversible covalent attachment of the predominant believed to be the result of an increased production of superoxide
metabolite of HCys, homocysteine thiolactone (HTL), to lysine (O2cꢀ) by HCys activating the enzyme NADPH oxidase.14–16 This
residues;
a
phenomenon known as ‘protein N-homo- increased production of O2cꢀ leads to a reduction in the bioavail-
cysteinylation’ that results in the loss of a proteins structural ability of NOc by increasing the formation of ONOOꢀ (NOc + O2cꢀ
integrity leading to loss of enzymatic function and aggregation.7 / ONOOꢀ).17 The reported ONOOꢀ concentrations in vivo are
believed to be approximately 50 mM but, higher concentrations of
500 mM have been found in macrophages.18,19 Furthermore, hypoxia
aDepartment of Chemistry, University of Bath, Bath, BA2 7AY, UK. E-mail: t.d.james@
bath.ac.uk; chssdb@bath.ac.uk
has been reported to facilitate HCys production in vitamin-decient
diets20 where hypoxia leads to an upregulation of nitroreductase
(NTR) – a reductive enzyme upregulated in cells under hypoxic
stress.21,22 Therefore, the development of tools that enable an
understanding of the relationship of HCys with these biologically
important species would be highly desirable.
bDepartment of Chemistry, University of Texas at Austin, 105 E 24th Street A5300,
Austin, TX 78712-1224, USA. E-mail: a.c.sedgwick@utexas.edu
cDepartment of Chemistry, Maynooth University Human Health Institute, Maynooth
University, National University of Ireland, Maynooth, County Kildare, Ireland.
E-mail: Robert.Elmes@mu.ie
dKey Laboratory for Advanced Materials, Joint International Research Laboratory of
To achieve this, a number of uorescent probes have been
Precision Chemistry and Molecular Engineering, Feringa Nobel Prize Scientist Joint developed that are capable of detecting two or more analytes.23
Research Center, School of Chemistry and Molecular Engineering, East China
These include AND logic gate based-uorescence probes, which
require both analytes to work in tandem to produce a measur-
able optical output.24–28 In our group, we have developed several
University of Science and Technology, 130 Meilong Rd, Shanghai 200237, China.
E-mail: xphe@ecust.edu.cn
eCollege of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
‘AND’ reaction-based probes for the detection of ROS/RNS and
E-mail: lixin81@zju.edu.cn
a second analyte.29–32 These ‘AND’ logic scaffolds have been used
fSynthesis and Solid State Pharmaceutical Centre, Maynooth University, Ireland
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra04908h
to detect two analytes within the same biological system.24,33
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 26425–26428 | 26425

View Article Online
RSC Advances
Paper
Owing to the pathological role of HCys, we set out to develop
the rst example of a uorescent probe for the detection of
HCys and biological related analyte. Aiming towards that target,
we became interested in a previously reported coumarin-based
uorescent probe developed by Hong et al. CAH, with a salicy-
laldehyde (Fig. 1).34 Salicylaldehyde is a known reactive unit
towards HCys/Cys, therefore we believed CAH could be used as
a scaffold for the development of ‘AND’-based systems for the
detection of HCys/Cys and a second analyte.34 In the presence of
HCys, CAH exhibited a ‘turn-on’ uorescence response which is
attributed to the nucleophilic nature of the nitrogen and sulfur
atoms resulting in thiazine ring formation (Scheme S1, Fig. S1
and S2†).34–36
We believed that CAH was a useful core unit that can be used
to introduce the chosen reactive chemical trigger on the phenol
for the detection of the corresponding analyte with HCys/Cys.
Owing to the relationship between HCys and ONOOꢀ/NTR, we
set out on the development of a HCys AND ONOOꢀ probe and
a HCys AND NTR probe.
Therefore, we set out to prepare JEG-CAB and JEG-CAN,
which are able to detect HCys/Cys and peroxynitrite (ONOOꢀ) or
nitroreductase (NTR), respectively (Scheme 1). For JEG-CAB,
a benzyl boronate ester was introduced as a ONOOꢀ reactive
unit.37 For JEG-CAN, a p-nitrobenzyl group was installed as it is
known to be an effective substrate for NTR.38–40
To afford CAH, compound 2 was synthesized by reuxing
umbelliferone and acetic anhydride at 140 ꢁC. Compound 2 was
then dissolved in triuoroacetic acid at 0 ꢁC followed by the
addition of hexamethylenetetramine (HMTA). The mixture was
heated to reux overnight and the solvent was then removed.
The intermediate was then hydrolyzed in H2O for 30 min at 60
ꢁC. Upon isolating CAH, it was then alkylated using 4-bromo-
methylphenylboronic acid pinacol ester and K2CO3 in DMF at
r.t. to afford JEG-CAB in 51% yield. JEG-CAN was produced by
alkylating CAH using 4-nitrobenzyl bromide and K2CO3 in DMF
at r.t. to give 49% yield (Scheme 1).
Scheme 1 Synthesis of target probe JEG-CAB and JEG-CAN.
JEG-CAB was initially non-uorescent, but the addition of HCys
(1 mM) led to a small increase in uorescence intensity, the
subsequent additions of ONOOꢀ (0–24 mM) led to a signicant
increase in uorescence intensity (>17-fold, see Fig. S5†). These
results demonstrated the requirement for both ONOOꢀ ‘AND’
HCys to obtain a signicant turn ‘‘on’’ uorescence response.
The addition of HCys and ONOOꢀ were then performed in
reverse where JEG-CAB exhibited a negligible increase in uo-
rescence intensity upon addition of ONOOꢀ (16 mM). However,
in a similar manner to that shown in Fig. 2, a large increase in
uorescence intensity was produced aer the subsequent
addition of HCys (0–5.5 mM) (Fig. 3 and S6†). LC-MS experi-
ments were carried out to ascertain the reaction mechanism
and the results conrmed the sequential formation of the
thiazine ring in the presence of HCys followed by boronate ester
cleavage in the presence of ONOOꢀ or vice versa (Scheme S2 and
Fig. S19–S21†).
As expected, probe JEG-CAB was shown to have excellent
selectivity with ONOOꢀ against other ROS in the presence of
HCys (1 mM) (Fig. S9 and S10†). Furthermore, JEG-CAB
We then evaluated the ability of JEG-CAB to detect ONOOꢀ
‘AND’ HCys in PBS buffer solution (10 mM, pH 7.40). The
maximum absorption of JEG-CAB at 336 nm shied to 323 nm
with the addition of HCys and then slightly shied to 328 nm
following the addition of ONOOꢀ (Fig. S3†). As shown in Fig. 2,
Fig. 2 Fluorescence spectra of JEG-CAB (15 mM) with addition of
Fig. 1 (a) CAH – a core fluorescent unit that enables the synthesis of HCys (1 mM) and incubated for 40 min then measured. Followed by
‘AND’ based fluorescent probe for the detection of HCys/Cys and incremental additions of ONOOꢀ (0–24 mM). The data was obtained in
a second analyte. (b) JEG-CAB enables the detection of HCys/Cys and PBS buffer solution (pH 7.40, 10 mM) at 25 ꢁC. lex ¼ 371 (bandwidth
(ROS/RNS) while (c) JEG-CAN enables the detection of HCys/Cys and 20) nm. Dashed line represents JEG-CAB and Hcys addition only. Blue
NTR.
line represents highest intensity after addition of ONOOꢀ.
26426 | RSC Adv., 2019, 9, 26425–26428
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
RSC Advances
Fig. 3 Fluorescence spectra of JEG-CAB (15 mM) with addition of
ONOOꢀ (16 mM) and followed by incremental additions of HCys (0–5.5
mM) measurements were taken after 40 min of both additions. The
data was obtained in PBS buffer solution (pH 7.40, 10 mM) at 25 ꢁC. lex
¼ 371 (bandwidth 20) nm. Dashed line represents JEG-CAB and
ONOOꢀ addition only. Blue line represents highest intensity after
addition of HCys.
Fig. 5 Fluorescence spectra of JEG-CAN (15 mM) with initial addition
of nitroreductase (4 mg mLꢀ1) and NADPH (400 mM) and incubated for
60 min. Followed by addition HCys (2 mM) and measured over 90 min
in PBS buffer solution (pH ¼ 7.40, 10 mM, containing 1% DMSO). lex
¼
363 nm. Ex slit: 5 nm and em slit: 5 nm. Dashed line represents JEG-
CAN and NTR addition only. Blue line represents highest intensity after
addition of HCys.
exhibited a high degree of selectivity towards a series of amino
acids where only HCys and Cys led to a uorescence response in
the presence of ONOOꢀ. This is due to the formation of stable
ve or six-membered thiazine rings (Fig. S7 and S8†).34
We then evaluated the changes in the uorescence of JEG-
CAN with both HCys and NTR in PBS buffer solution (10 mM,
pH 7.40, containing 1% DMSO). As shown in Fig. 4, addition of
HCys led to a small increase in uorescence intensity. However,
subsequent addition of NTR (4 mg mLꢀ1) led to a large time
dependant increase in uorescence intensity. To ensure both
analytes were required, NTR and NADPH was kept constant (4
mg mLꢀ1 and 400 mM respectively) resulting in a 3.4 fold uo-
rescence increase (Fig. 5). We attribute the large initial increase
to background uorescence of NADPH.41 NTR then facilitates
reduction of the nitro group of JEG-CAN releasing the core
probe CAH via a fragmentation cascade (Scheme S3†).38,42
Subsequent addition of HCys (2.0 mM) led to a 2 fold increase in
uorescence intensity. Again, LC-MS experiments conrmed
the proposed reaction mechanism (Fig. S22†).
Kinetic studies for JEG-CAN with both NTR and HCys were
carried out (Fig. S11–S18†) where it is clear that JEG-CAN
exhibits a dose dependant uorescence increase in response of
both HCys and NTR.
Unfortunately, the probes failed to give good data in cells,
which could be due to their short excitation wavelengths or the
extremely low intracellular HCys concentrations (5–15 mM). We
are now pursuing the development ‘AND’ logic uorescence
probes with longer excitation and emission wavelengths suit-
able for in vitro and in vivo applications.
In summary, we have developed two coumarin-based ‘AND’
logic uorescence probes (JEG-CAB and JEG-CAN) for the
detection of HCys/Cys and ONOOꢀ or NTR, respectively. CAH is
a useful platform that enables easy modication for the devel-
opment of ‘AND’-based uorescent probes for the detection of
HCys/Cys and a second analyte. Both JEG-CAB and JEG-CAN
were shown to be ‘AND’-based uorescent probes.
Conflicts of interest
Fig. 4 Fluorescence spectra of JEG-CAN (15 mM) with initial addition
of HCys (2 mM) and incubated for 60 min. Followed by addition of
nitroreductase (4 mg mLꢀ1) and NADPH (400 mM) and measured over
90 min in PBS buffer solution (pH ¼ 7.40, 10 mM, containing 1%
DMSO). lex ¼ 363 nm. Ex slit: 5 nm and em slit: 5 nm. Dashed line
represents JEG-CAN and HCys addition only. Blue line represents
highest intensity after addition of NTR.
No conicts of interest.
Acknowledgements
LW wishes to thank China Scholarship Council and the
University of Bath for supporting his PhD work in the UK. JEG
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 26425–26428 | 26427

View Article Online
RSC Advances
Paper
and ACS wish to thank the EPSRC for funding their PhD 18 M. J. Mihm and J. A. Bauer, Biochimie, 2002, 84, 1013.
studentships. TDJ wishes to thank the Royal Society for a Wolf- 19 O. Stachowiak, M. Dolder, T. Wallimann and C. Richter, J.
son Research Merit Award. X.-P. He thanks the National Natural
Biol. Chem., 1998, 273, 16694.
´ ´
´
Science Foundation of China (21722801 and 21572058), the 20 S. Blaise, J.-M. Alberto, E. Nedelec, A. Ayav, G. Pourie,
´
Programme of Introducing Talents of Discipline to Universities
(B16017) and the Shanghai Rising-Star Program (16QA1401400).
J.-P. Bronowicki, J.-L. Gueant and J.-L. Daval, Pediatr. Res.,
2005, 57, 777.
LKK wishes to acknowledge the Irish Research Council for 21 K. Xu, F. Wang, X. Pan, R. Liu, J. Ma, F. Kong and B. Tang,
a Government of Ireland Postdoctoral Research Fellowship Chem. Commun., 2013, 49, 2554.
(GOIPD/2017/1091) and Science Foundation Ireland are 22 R. B. P. Elmes, Chem. Commun., 2016, 52, 8935.
acknowledged for the funding of Advion Mass Spec facilities 23 F. Yu, P. Li, B. Wang and K. Han, J. Am. Chem. Soc., 2013, 135,
through the Opportunistic Infrastructure Fund (16/RI/3399).
RBPE would like to thank Science Foundation Ireland (SFI) 24 J. L. Kolanowski, F. Liu and E. J. New, Chem. Soc. Rev., 2018,
and the European Regional Development Fund (12/RC/ 47, 195.
7674.
2275_P2). NMR characterisation facilities were provided 25 A. Romieu, Org. Biomol. Chem., 2015, 13, 1294.
through the Material and Chemical Characterisation Facility 26 C. Yan, Z. Guo, Y. Liu, P. Shi, H. Tian and W.-H. Zhu, Chem.
(MC2) at the University of Bath (http://go.bath.ac.uk/mc2). The
Sci., 2018, 9, 6176.
EPSRC UK National Mass Spectrometry Facility at Swansea 27 I. Takashima, R. Kawagoe, I. Hamachi and A. Ojida, Chem.–
University is thanked for analyses.
Eur. J., 2015, 21, 2038.
28 Y. Yue, F. Huo, F. Cheng, X. Zhu, T. Mareyi, R. M. Strongin
and C. Yin, Chem. Soc. Rev., 2019, 48, 4155.
29 A. C. Sedgwick, H.-H. Han, J. E. Gardiner, S. D. Bull, X.-P. He
and T. D. James, Chem. Sci., 2018, 9, 3672.
30 M. L. Odyniec, A. C. Sedgwick, A. H. Swan, M. Weber,
T. M. S. Tang, J. E. Gardiner, M. Zhang, Y.-B. Jiang,
G. Kociok-Kohn, R. B. P. Elmes, S. D. Bull, X.-P. He and
T. D. James, Chem. Commun., 2018, 54, 8466.
31 L. Wu, H.-H. Han, L. Liu, J. E. Gardiner, A. C. Sedgwick,
C. Huang, S. D. Bull, X.-P. He and T. D. James, Chem.
Commun., 2018, 54, 11336.
32 M. L. Odyniec, J. E. Gardiner, A. C. Sedgwick, X.-P. He,
S. D. Bull and T. D. James, Front. Chem. Sci. Eng., 2019,
DOI: 10.1007/s11705-018-1785-9.
33 S. Erbas-Cakmak, S. Kolemen, A. C. Sedgwick,
T. Gunnlaugsson, T. D. James, J. Yoon and E. U. Akkaya,
Chem. Soc. Rev., 2018, 47, 2228.
34 K.-S. Lee, T.-K. Kim, J. H. Lee, H.-J. Kim and J.-I. Hong, Chem.
Commun., 2008, 6173.
35 Y. Kim, S. V. Mulay, M. Choi, S. B. Yu, S. Jon and
D. G. Churchill, Chem. Sci., 2015, 6, 5435.
Notes and references
1 J. Selhub, Annu. Rev. Nutr., 1999, 19, 217.
2 D. B. Dalto and J.-J. Matte, Nutrients, 2017, 9, 189.
3 Z.-Z. Yang and A.-P. Zou, Kidney Int., 2003, 63, 1012.
4 P. Ganguly and S. F. Alam, Nutr. J., 2015, 14, 6.
5 R. C. Austin, S. R. Lentz and G. H. Werstuck, Cell Death
Differ., 2004, 11, S56.
6 S. Seshadri, A. Beiser, J. Selhub, P. F. Jacques,
I. H. Rosenberg, R. B. D'agostino, P. W. Wilson and
P. A. Wolf, N. Engl. J. Med., 2002, 346, 476.
7 H. Jakubowski, Biomed. Pharmacother., 2001, 55, 443.
8 H. Y. Lee, Y. P. Choi, S. Kim, T. Yoon, Z. Guo, S. Lee,
K. M. K. Swamy, G. Kim, J. Y. Lee, I. Shin and J. Yoon,
Chem. Commun., 2014, 50, 6967.
9 J. Zhang, X.-D. Jiang, X. Shao, J. Zhao, Y. Su, D. Xi, H. Yu,
S. Yue, L.-j. Xiao and W. Zhao, RSC Adv., 2014, 4, 54080.
10 Y.-W. Wang, S.-B. Liu, W.-J. Ling and Y. Peng, Chem.
Commun., 2016, 52, 827.
11 N. Zhao, Q. Gong, R. X. Zhang, J. Yang, Z. Y. Huang, N. Li and
B. Z. Tang, J. Mater. Chem. C, 2015, 3, 8397.
12 H. M. Williams, H. Lippok and G. H. Doherty, Neurosci. Res.,
2008, 60, 380.
13 S. Levrand, P. Pacher, B. Pesse, J. Rolli, F. Feihl, B. Waeber
and L. Liaudet, Biochem. Biophys. Res. Commun., 2007, 359,
445.
36 S. V. Mulay, Y. Kim, M. Choi, D. Y. Lee, J. Choi, Y. Lee, S. Jon
and D. G. Churchill, Anal. Chem., 2018, 90, 2648.
37 A. Sikora, J. Zielonka, M. Lopez, J. Joseph and
B. Kalyanaraman, Free Radic. Biol. Med., 2009, 47, 1401.
38 L. Cui, Y. Zhong, W. Zhu, Y. Xu, Q. Du, X. Wang, X. Qian and
Y. Xiao, Org. Lett., 2011, 13, 928.
14 Z. Ungvari, A. Csiszar, J. G. Edwards, P. M. Kaminski,
M. S. Wolin, G. Kaley and A. Koller, Arterioscler., Thromb.,
Vasc. Biol., 2003, 23, 418.
15 X. Zhang, H. Li, H. Jin, Z. Ebin, S. Brodsky and
M. S. Goligorsky, Am. J. Physiol. Renal. Physiol., 2000, 279,
F671.
16 V. E. R. Edirimanne, C. W. H. Woo, Y. L. Siow, G. N. Pierce,
J. Y. Xie and K. O, Can. J. Physiol. Pharmacol., 2007, 85, 1236.
17 P. Pacher, J. S. Beckman and L. Liaudet, Physiol. Rev., 2007,
87, 315.
39 A. C. Sedgwick, A. Hayden, B. Hill, S. D. Bull, R. B. P. Elmes
and T. D. James, Front. Chem. Sci. Eng., 2018, 12, 311.
40 X. Ao, S. A. Bright, N. C. Taylor and R. B. P. Elmes, Org.
Biomol. Chem., 2017, 15, 6104.
41 G. H. Patterson, S. M. Knobel, P. Arkhammar, O. Thastrup
and D. W. Piston, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 5203.
42 Y. Shi, S. Zhang and X. Zhang, Analyst, 2013, 138, 1952.
26428 | RSC Adv., 2019, 9, 26425–26428
This journal is © The Royal Society of Chemistry 2019