Differences in heterocycle basicity distinguish homocysteine from cysteine using aldehyde-bearing fluorophores

Article abstract of DOI:10.1039/c4cc03527e

We report the detection of homocysteine over cysteine based upon characteristic differences between 5- and 6-membered heterocyclic amines formed upon reaction with aldehyde-bearing compounds. Homocysteine-derived thiazinane-4-carboxylic acids are more basic than cysteine-derived thiazolidines-4-carboxylic acids. Fluorescence enhancement in response to homocysteine is achieved by tuning pH and excitation wavelength. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc03527e

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Differences in heterocycle basicity distinguish
homocysteine from cysteine using aldehyde-
bearing fluorophores†
Cite this: Chem. Commun., 2014,
50, 8219
Received 10th May 2014,
Accepted 10th June 2014
Aabha Barve, Mark Lowry, Jorge O. Escobedo, Katherine T. Huynh,
Lovemore Hakuna and Robert M. Strongin*
DOI: 10.1039/c4cc03527e
www.rsc.org/chemcomm
We report the detection of homocysteine over cysteine based upon viologens.23,24 Yoon et al. recently reported pyrene aldehyde fluor-
characteristic differences between 5- and 6-membered heterocyclic ophores to detect Hcy.25 However, the vast majority of reported
amines formed upon reaction with aldehyde-bearing compounds. optical thiol probes respond to classes of analytes rather than
Homocysteine-derived thiazinane-4-carboxylic acids are more basic individual species.15–17 Thus, the development of new and improved
than cysteine-derived thiazolidines-4-carboxylic acids. Fluorescence probes is an active area of research.
enhancement in response to homocysteine is achieved by tuning
pH and excitation wavelength.
Aldehyde-functionalized probes12–14,22,25–31 have received a great
deal of attention for the optical detection of amino thiols and
amino acids. Interestingly, this class of probes has been reported to
Changes in the abundance of glutathione (GSH), cysteine (Cys), respond to their respective analytes through either fluorescence
and homocysteine (Hcy) in human plasma are correlated with quenching,12,13,27 or fluorescence enhancement14,22,25,26,28 on a
several diseases.1–7 Variations in GSH levels are associated with case by case basis. In an early example, Glass et al. reported detection
chronic diseases such as cancer, neurodegenerative diseases, cystic of amino acids using aldehyde-bearing coumarin probes, wherein
fibrosis (CF), HIV, and aging.1,2 Elevated Cys levels have been reaction of the aldehyde with amino acids resulted in enhancement
linked to neurotoxicity.3 The improper metabolism of Hcy results of the fluorescence.26 Our group popularized the visible detection of
in elevated plasma levels,4 that have been associated with Alzhei- amino thiols with aldehyde-bearing fluorophores12,13 based upon
mer’s,5 cancer,6 and cardiovascular diseases.7 Thus, detecting the well known reaction of amino thiols to form heterocyclic
these amino thiols in human plasma is of great interest. Several thiazolidines32 and thiazinanes. Fluorescence quenching of fluor-
techniques such as high-performance liquid chromatography escein aldehyde probes following the formation of thiazolidine/
(HPLC),8,9 liquid chromatography/mass spectrometry (LC-MS),10 thiazinane heterocycles occurred upon reaction of Cys and Hcy
electrochemical,11 and optical methods12–17 have been developed under the conditions investigated.12,13 Others have subsequently
for the detection of amino thiols. However, many of these methods used this concept for the detection of Cys and Hcy. For example,
involve labour intensive steps or complex instrumentation.
Kim et al., observed fluorescence quenching of an aldehyde-
Optical methods have received a great deal of attention due to bearing amino coumarin in response to Cys/Hcy.27 Hong et al.,
their sensitivity, relative ease, and low cost; however, to date there reported an aldehyde-bearing hydroxy coumarin28 in which
have been relatively few optical methods reported which can reliably addition of Cys/Hcy resulted in fluorescence enhancement. It
distinguish GSH,18,19 Cys,20–22 and Hcy23–25 over related compounds. is therefore of utility to develop a general understanding of the
For example, we recently developed a resorufin acrylate-based probe mechanisms behind differing modes of signal transduction in
with selectivity for GSH over related analytes.18 A monochlorinated the related aldehyde-bearing probes. Such knowledge will lead to
BODIPY-based GSH sensor has also been reported.19 Probes improved probes with increased sensitivity for selected amino
reported to show preferential responses toward Cys include a styryl thiols of interests, specifically for Hcy over Cys.
BODIPY-based20 and a seminaphthofluorescein acrylate-based Cys
Herein we further investigate the mechanism of signal
probe,21 and other selective two-photon Cys probes.22 We have also transduction in fluorescein aldehyde probes upon reaction with
developed colorimetric assays for the selective detection of Hcy using amino thiols. Unprecedented control over signal transduction
is attained such that the probes can be tuned to respond to
Cys/Hcy through either quenching or enhancement. More
importantly, the response can be controlled in such a manner
Department of Chemistry, Portland State University, Portland, Oregon 97207, USA.
E-mail: strongin@pdx.edu; Fax: +1 503-725-9525; Tel: +1 503-725-9724
that Hcy is detected over Cys. There are several phenomena that
impact the fluorescence response of aldehyde-bearing probes.
† Electronic supplementary information (ESI) available: Detailed experimental
procedures and additional spectral data. See DOI: 10.1039/c4cc03527e
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Fig. 1 Mechanism of signal transduction in generic aldehyde-bearing amino
thiol probes. (a) PET-based fluorescence quenching following reaction of
Cys/Hcy with the aldehyde group. (b) PET inhibition through hydrogen
bonding between the amine-containing heterocycle and adjacent groups.
(c) PET inhibition through protonation of the amine-containing heterocycle.
It is important to note the differences in how aldehyde-bearing
probes react with amino thiols compared to amino acids. Amino
acids react to form Schiff bases26 whereas amino thiols such as
Cys and Hcy react to form heterocyclic thiazolidines and thiazi-
nanes respectively.12 These differences result in unique optical
responses towards amino thiols. Fig. 1 depicts the reaction of
Cys/Hcy with the aldehyde group of a generic aldehyde-bearing
probe. It is well established that the free electrons of the amino
group in the heterocycle can quench fluorescence of the probe
(Fig. 1a) through photo induced electron transfer (PET).27 PET-
induced responses have been modulated through tuning the
functionality of the fluorescent probes.25,28 For example, hydro-
gen bonding in aqueous media between the amine-containing
heterocycles and adjacent groups (Fig. 1b) has been reported to
inhibit PET leading to fluorescence enhancement upon reaction
with both Cys and Hcy with a coumarin aldehyde.28 Conversely,
in this work we demonstrate PET inhibition leading to selective
fluorescence enhancement for Hcy via a mechanism involving
protonation of the amino group of the heterocycle (Fig. 1c).
Previously we have investigated the reaction of Cys/Hcy with
fluorescein mono aldehyde 1 and fluorescein dialdehyde 2 at
pH 9.5, sodium bicarbonate buffer (Scheme 1). Reaction with
Fig. 2 Modulating the signal transduction through changing the pH and
excitation wavelength. (a) Fluorescence emission upon excitation at 480 nm
for 1 and 1 + Cys or Hcy at various pH values. (b) Fluorescence emission upon
excitation at 495 nm for 1 and 1 + Cys or Hcy at various pH values.
(c) Comparison of 1 and 2 response upon excitation at 495 nm at pH 6.0.
All solutions are composed of 4 mM of 1 or 2 with 1 mM of analyte in 100 mM
phosphate or carbonate buffer : DMSO 99 : 1 at 20 1C after 2 h, lem = 515 nm.
Cys/Hcy resulted in a yellow-to-orange colour change with a
shift in absorbance maxima from near 480 nm to ca. 500 nm. In
addition to the colour change, a quenching-based response
toward both analytes was reported.12,13 At high pH, excitation
near either peak yields a quenching-based response (Fig. 2a and
b, pH 9.5). Under basic conditions, the lone pair electrons on
the nitrogen of the heterocyclic moiety are free to quench
fluorescence through PET (Fig. 1a).
We hypothesize that at sufficiently low pH, the heterocycles
formed upon reaction with the probe must be protonated; thus
inhibiting PET and leading to fluorescence enhancement. PET
inhibition and a wavelength-dependent fluorescence response are
observed upon lowering solution pH (Fig. 2a and b). In acidic
media (pH 5.5) longer wavelength excitations near the absorption
maxima of the heterocyclic reaction products (495 nm) result in
fluorescence enhancement (Fig. 2b), unlike the quenching
observed under basic conditions. (Fig. 2b, pH 9.5)
We further hypothesize that under certain conditions, the
response can be tuned to detect Hcy over Cys due to pKa
differences caused by the differing C–N–C bond angles in 5- vs.
6-membered ring heterocycles. In the case of a 5-memebered
Scheme 1 Reaction of 1 and 2 with Cys and Hcy.
8220 | Chem. Commun., 2014, 50, 8219--8222
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Cys-derived thiazolidine ring the C–N–C bond angle is smaller
than in a 6-membered Hcy-derived thiazinane ring. This results
in greater s-character of the N–H bond in the thiazolidines,
rendering them less basic than the thiazinanes.33–35
We observe that on adjusting the pH from 5.5 to 6.0, the
signal is modulated such that Hcy enhances the fluorescence of
1 while there is minimal change upon reaction with Cys (Fig. 2b).
Dialdehyde 2 was more sensitive than monoaldehyde 1 at high
pH.12,13 This trend in sensitivity is also observed under these
new conditions with a 45 fold fluorescence enhancement of 2
observed in response to Hcy (Fig. 2c). The response of both 1 and
2 towards Hcy is easily observed in as little as 15 min and
plateaus near 45 min (Fig. S12 and S13, ESI†). For the purpose of
investigating the mechanism responsible for this unique selec-
tivity, we focused first on the simpler monoaldehyde 1.
Scheme 2 Different ionization states of the amine moieties in Cys- and
Hcy-derived heterocycles at pH 6.0.
To demonstrate the mechanism behind this pH dependent
response and Hcy-derived enhancement, a series of 5- and
6-membered 2-substituted thiazolidine/thiazinanes-4-carboxylic
acids were synthesized (see ESI†) as model compounds of 1a–b.
Cys- and Hcy-derived heterocycles of fluorescein aldehydes are
stable under the conditions previously investigated12 as well as
under the present system (Fig. S14, ESI†). However, they are
extremely polar and we were unable to isolate 1a–b.
To confirm the hypothesis that the pH dependent Hcy
enhancement was related to the differing basicity of 5- and
6-membered heterocyclic amines, we measured the pKa of the
Cys- and Hcy-derived analogues (Table 1) (Fig. S16, ESI†). Cys-
derived 3a–5a are known and their pKa values previously
published36 are in general agreement with those in Table 1.
We report and measure the pKa of Hcy-derived 3b–5b for the
first time. As seen in Table 1, the amine pKa of 6-membered
thiazinanes is higher than that of 5-membered thiazolidines by
at least one unit. Therefore based on the pKa of these analo-
gues, we expect the amine pKa of Hcy-derived 1b to be at least
1 unit greater (B6.7) than Cys-derived 1a (B5.7).
This difference in basicity results in the selectivity for Hcy
over Cys (Fig. 2c). We investigated the response of 1 towards
Hcy and Cys over a range of pH (Fig. S17, ESI†) values. At the
appropriate pH the ratio of ionization states of the amine
moiety in thiazolidine 1a would be such that fluorescence
quenching by the lone pair electrons is cancelled by PET
inhibition from the ammonium species 1a0 (Scheme 2). At this
pH the amine group in thiazinane 1b exists predominately as
the more fluorescent ammonium 1b0 (Scheme 2) because the
pKa of 1b0 is greater than that of 1a0. At pH = 6 the reaction
between 1 and Cys does not result in a change in fluorescence
while reaction with Hcy results in fluorescence enhancement.
Thus pH 6.0 is optimal for the detection of Hcy. Raising the
pH increases the ratio of amine to ammonium which leads to
quenching, while decreasing the pH leads to enhancement
(Fig. S17, ESI†). We also investigated the response of 1 towards
GSH and various amino acids at pH 6.0 (Fig. S18, ESI†). Only
Hcy promotes fluorescence enhancement.
Having identified the optimal conditions for the discrimina-
tion of Hcy over Cys, we investigated the response of the more
sensitive dialdehyde 2 towards normal healthy concentrations of
amino thiols in pH 6.0 buffer. The fluorescence response of
2 towards Hcy increased linearly with increasing Hcy concen-
tration and the limit of detection was found to be as low as 2.5 mM
(Fig. S19, ESI†). No response was observed toward 250 mM Cys,
Table 1 pKa values of the amine moiety of 2-substituted thiazolidine/
thiazinane-4-carboxylic acids
Compound
pKa exp, (pKa lit
)
Compound
pKa exp
NA,a (5.6736
)
6.67b
5.50,a (5.3136
)
6.85b
7.76
6.44, (6.1936
)
a
Fig. 3 Optical sensing behavior of 2 towards Cys and Hcy in deprotei-
nized bovine plasma at pH 6.0. Solutions are composed of 25 mM of 2 with
250 mM of Cys and 100 mM of Hcy in phosphate buffered plasma (100 mM,
pH 6.0) : DMSO 99 : 1 at 20 1C after 2 h, lex = 495 nm.
3a and 4a were found to be relatively unstable showing signs of
decomposition in aqueous solution during the course of the titration
(Fig. S15 and S16, ESI). 3b and 4b showed no indication of decom-
position (Fig. S15 and S16, ESI).
b
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14 S. Lim, J. O. Escobedo, M. Lowry, X. Xu and R. Strongin, Chem.
Commun., 2010, 46, 5707–5709.
15 J. O. Escobedo, O. Rusin, W. Wang, O. Alpturk, K. K. Kim, X. Xu and
R. M. Strongin, in Reviews in Fluorescence 2006, ed. C. D. Geddes and
J. R. Lakowicz, Springer, US, 2006, pp. 139–162.
16 H. Peng, W. Chen, Y. Cheng, L. Hakuna, R. Strongin and B. Wang,
Sensors, 2012, 12, 15907–15946.
while 15 mM Hcy resulted in a B10% increase in fluorescence
(Fig. S20, ESI†). Importantly, 2 was also shown to function
similarly in deproteinized bovine plasma detecting an elevated
level of Hcy (Fig. 3 and Fig. S21, ESI†).
In conclusion, we report a new approach for the selective
detection of Hcy using fluorescein aldehyde-based probes. This
method is based on intrinsic differences between the pKa of
5- and 6-membered thiazolidines and thiazinanes formed upon
reaction with the aldehyde-bearing probes. Selective Hcy probes
based on the mechanism described herein with improved
sensitivity are currently being developed in our laboratory.
This work was supported by the National Institutes of Health
(R15EB016870).
17 H. S. Jung, X. Chen, J. S. Kim and J. Yoon, Chem. Soc. Rev., 2013, 42,
6019–6031.
18 Y. Guo, X. Yang, L. Hakuna, A. Barve, J. O. Escobedo, M. Lowry and
R. M. Strongin, Sensors, 2012, 12, 5940–5950.
19 L. Y. Niu, Y. S. Guan, Y. Z. Chen, L. Z. Wu, C. H. Tung and
Q. Z. Yang, J. Am. Chem. Soc., 2012, 134, 18928–18931.
20 J. Shao, H. Guo, S. Ji and J. Zhao, Biosens. Bioelectron., 2011, 26,
3012–3017.
21 X. Yang, Y. Guo and R. M. Strongin, Org. Biomol. Chem., 2012, 10,
2739–2741.
22 Z. Yang, N. Zhao, Y. Sun, F. Miao, Y. Liu, X. Liu, Y. Zhang, W. Ai,
G. Song, X. Shen, X. Yu, J. Sun and W.-Y. Wong, Chem. Commun.,
2012, 48, 3442–3444.
References
23 J. O. Escobedo, W. Wang and R. M. Strongin, Nat. Protoc., 2006, 1,
2759–2762.
1 D. M. Townsend, K. D. Tew and H. Tapiero, Biomed. Pharmacother.,
2003, 57, 145–155.
24 L. Hakuna, J. O. Escobedo, M. Lowry, A. Barve, N. McCallum and
R. M. Strongin, Chem. Commun., 2014, 50, 3071–3073.
25 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,
6967–6969.
26 E. K. Feuster and T. E. Glass, J. Am. Chem. Soc., 2003, 125,
16174–16175.
27 T.-K. Kim, D.-N. Lee and H.-J. Kim, Tetrahedron Lett., 2008, 49,
4879–4881.
28 K. S. Lee, T. K. Kim, J. H. Lee, H. J. Kim and J. I. Hong, Chem.
Commun., 2008, 6173–6175.
29 Y. Wang, J. Xiao, S. Wang, B. Yang and X. Ba, Supramol. Chem., 2010,
22, 380–386.
30 J. Mei, J. Tong, J. Wang, A. Qin, J. Z. Sun and B. Z. Tang, J. Mater.
Chem., 2012, 22, 17063–17070.
31 J. Mei, Y. Wang, J. Tong, J. Wang, A. Qin, J. Z. Sun and B. Z. Tang,
Chem. – Eur. J., 2013, 19, 613–620.
32 T.-R. Kim, S.-J. Yun and B.-B. Park, Bull. Korean Chem. Soc., 1986, 7,
25–29.
2 G. Y. Wu, Y. Z. Fang, S. Yang, J. R. Lupton and N. D. Turner, J. Nutr.,
2004, 134, 489–492.
3 X. F. Wang and M. S. Cynader, J. Neurosci., 2001, 21, 3322–3331.
4 Homocysteine in health and disease, ed. R. Carmel and
D. W. Jacobsen, Cambridge University Press, 2001.
5 S. Seshadri, A. Beiser, J. Selhub, P. F. Jacques, I. H. Rosenberg,
R. B. D’Agostino, P. W. F. Wilson and P. A. Wolf, N. Engl. J. Med.,
2002, 346, 476–483.
6 Y. Ozkan, S. Yardim-Akaydin, H. Firat, E. Caliskan-Can, S. Ardic and
B. Simsek, Anticancer Res., 2007, 27, 1185–1189.
7 A. G. Bostom, H. Silbershatz, I. H. Rosenberg, J. Selhub,
R. B. D’Agostino, P. A. Wolf, P. F. Jacques and P. W. Wilson, Arch.
Intern. Med., 1999, 159, 1077–1080.
8 T. D. Nolin, M. E. McMenamin and J. Himmelfarb, J. Chromatogr. B:
Anal. Technol. Biomed. Life Sci., 2007, 852, 554–561.
9 M. E. McMenamin, J. Himmelfarb and T. D. Nolin, J. Chromatogr. B:
Anal. Technol. Biomed. Life Sci., 2009, 877, 3274–3281.
10 X. M. Guan, B. Hoffman, C. Dwivedi and D. P. Matthees, J. Pharm.
Biomed. Anal., 2003, 31, 251–261.
33 K. Yoshikawa, M. Hashimoto and I. Morishima, J. Am. Chem. Soc.,
1974, 96, 288–289.
34 D. H. Aue, H. M. Webb and M. T. Bowers, J. Am. Chem. Soc., 1972, 94,
4726–4728.
11 M. Baron and J. Sochor, Int. J. Electrochem. Sci., 2013, 8, 11072–11086.
12 O. Rusin, N. N. St Luce, R. A. Agbaria, J. O. Escobedo, S. Jiang,
I. M. Warner, F. B. Dawan, K. Lian and R. M. Strongin, J. Am. Chem.
Soc., 2004, 126, 438–439.
35 T. Ohwada, H. Hirao and A. Ogawa, J. Org. Chem., 2004, 69,
7486–7494.
36 P. Butvin, J. Al-Ja’Afreh, J. Svetlik and E. Havranek, Chem. Pap.-
Chem. Zvesti, 1999, 53, 315–322.
13 W. H. Wang, O. Rusin, X. Y. Xu, K. K. Kim, J. O. Escobedo,
S. O. Fakayode, K. A. Fletcher, M. Lowry, C. M. Schowalter,
C. M. Lawrence, F. R. Fronczek, I. M. Warner and R. M. Strongin,
J. Am. Chem. Soc., 2005, 127, 15949–15958.
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