PET-dependent fluorescence sensing of enzyme reactions using the large and tunable pKa shift of aliphatic amines

Article abstract of DOI:10.1039/c3cc46824k

A fluorescence sensing system exploiting the large and finely tunable pK_a shift of aliphatic amines was developed. The amine-containing fluorescent probes with distinct pK_a values in a wide pH range were successfully applied to detect a variety of enzyme reactions with a large and real-time fluorescence enhancement.

Full text of DOI:10.1039/c3cc46824k

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Cite this: DOI: 10.1039/x0xx00000x
PET-dependent Fluorescence Sensing of Enzyme
Reactions Using a Large and Tunable pKa Shift of
Aliphatic Amines
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
Yuji Oshikawa, Akio Ojida*
A fluorescence sensing system exploiting large and finely
tunable pKa shift of aliphatic amine was developed. The
amine-containing fluorescent probes with the distinct pKa
values in a wide pH range were successfully applied to
detect a variety of enzyme reactions with a large and real-
time fluorescence enhancement.
Photoꢀinduced electron transfer (PET) is a useful mechanism to
control luminescence of fluorescent probes. In typical fluorescent
sensing with PET, probe luminescence is restored by inhibiting the
electron transfer from the electron donor group upon binding and/or
reaction with a target analyte to emit a turnꢀon fluorescence signal.
The great versatility of PET as a fluorescence sensing mechanism
has provided numerous of fluorescent probes for various organic and
inorganic substances.1
Fig. 1 PETꢀdependent fluorescence sensing of enzyme reactions of aliphatic
amines. The pKa shift induced by an enzymatic transformation provides a
fluorescence sensing window (shown as yellow box) in a specific pH region.
fluorescence enhancements.
Aliphatic amines are effective electron donors in the PETꢀ
Initially, we prepared a series of anthracene derivatives that have
dependent fluorescent probes. Since the first example of a PET different aminomethyl groups at the 9 positions, and determined
sensor was reported by Wang and Moravetz in dibenzylamine, 2
their pKa values by pHꢀdependent fluorescence changes (Fig. 2a,b).
a
variety of biological PET probes containing an aliphatic amine have Probe 1 with a methyl group on its nitrogen has a high pKa value of
been developed for sensing H+ (pH), metal ions, and saccharides. 1, 3 9.20, while 2, possessing an electronꢀwithdrawing αꢀacetyl ester
In general, the basicity (pKa) of an aliphatic amine is sensitive to group, has a lower pKa of 5.66 in the weakly acidic pH region. The
substituents bonded to the amine nitrogen. For example, the pKa of pKa of the amino group was effectively shifted to the more acidic pH
methyl amine (10.62) shifts to 7.90 in methyl glycinate4 with an region by the introduction of an electronꢀwithdrawing cyanoalkyl
electronꢀwithdrawing ester group. It is also known that the pKa of group. For example, Probe 3, possessing a cyanopropyl group, has a
amineꢀcontaining Good’s buffers are finely controlled over the wide pKa value of 4.14. The introduction of a stronger electronꢀ
pH range of 6.15 (MES) to 10.40 (CAPS) by slight structural withdrawing cyanoethyl group created the extremely weak basic
modifications. We expected that such a large and finely tunable pKa amine 4 with a pKa value below 2. Probe 5 bearing a cyanomethyl
shift of the aliphatic amine would be useful to fluorescently detect group emitted strong fluorescence over a wide pH region without
various chemical reactions. In this sensing system, the pH region significant change. This implies that the aliphatic amine that
between two pKa values that is formed by structural change of an possesses a very strong electronꢀwithdrawing group no longer serves
amineꢀcontaining probe would be a suitable sensing window with a as an electron donor in PET.
large fluorescence enhancement (Fig. 1). We report here on a
The pKa values of the anthracene derivatives shifted to the higher
fluorescence sensing system exploiting large and finely tunable pKa pH region when their ester groups were hydrolyzed to the
shift of an aliphatic amine. The introduction of substituents on the corresponding carboxylates. For example, the pKa value of the ester
amine provides the fluorescent probes with distinct pKa values in a 2 shifted from 5.66 to 8.61 in carboxylate 6. Similarly, the pKa
wide pH range from 9 to below 2, which were successfully applied values of 3 and 4 shifted from 4.14 and < 2 to 7.16 and 5.71,
to detect a variety of enzyme reactions with the large and realꢀtime respectively, upon hydrolysis to the corresponding carboxylate
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Fig. 2 (a) Structures of the fluorescent probes with the aliphatic amino groups.
(b) pHꢀdependent fluorescent intensity changes of the aliphatic amines.
Conditions: [probe] = 1 ꢀM, 5 mM MES ꢀ 5 mM HEPES ꢀ 5 mM CHES buffer, λex
= 367 nm, 25 °C. (c) Realꢀtime fluorescence monitoring of the PLEꢀcatalyzed
hydrolysis of 2 to 6. Conditions: [2] = 5 ꢀM, [PLE] = 0.025(●), 0.0125(■),
0.0063(▲), and 0 (□) units/mL, 50 mM HEPES buffer, pH 7.4, λex = 366 nm,
37 °C.
Fig.
3
(a) Realꢀtime fluorescent monitoring of nitrile hydratase activity.
Conditions: [8] = 1 ꢀM, [nitrile hydratase] = 20.6 (●), 10.3 (■), 5.16 (▼), 2.58 (▲),
and 0 (□) units/mL, 20 mM Phosphate buffer, pH 7.0, λex = 367 nm, 40 °C.; (b)
Realꢀtime fluorescent monitoring of the carbonyl reductase activity. Conditions:
[10] = 1 ꢀM, [carbonyl reductase] = 0.066 (●), 0.033 (■), 0.016 (▲), and 0 (□)
mg/mL, 50 mM HEPES buffer, pH 7.4, λex = 367 nm, 25 °C. To avoid the effect
of NAD(P)H as a fluorescent enzyme coꢀfactor, the fluorescence change was
monitored at 398 nm.
derivatives (Fig. S1, ESI†). Such large pKa shifts would provide
fluorescenceꢀsensing windows for detection of enzymeꢀcatalyzed
hydrolysis of carboxyl ester. In a proofꢀofꢀprinciple experiment, we
performed the realꢀtime fluorescence monitoring of the esteraseꢀ
dcatalyzed hydrolysis reaction of 2. As shown in Fig. 2c, the
fluorescence intensity increased up to 40ꢀfold in a timeꢀdependent
manner upon treatment with porcine liver esterase (PLE) at pH 7.4.
The initial rate of the fluorescence increase depended on the amount
of PLE (Fig. S2, ESI†), and the production of carboxylate 6 was
confirmed by electrospray ionization mass spectroscopy (Fig. S3,
ESI†). The Km and Vmax value was evaluated to be 3.22 ꢁM and
0.0931 ꢁM/min, respectively, by MichaelisꢀMenten kinetic analysis
(Fig. S4, ESI†). In a similar manner, the hydrolysis of the
alkylammonium ester 7 (pKa=5.13) to 6, catalyzed by acetylcholine
esterase, was detected with a 40ꢀfold increase in fluorescence
intensity (Fig. S5, ESI†).
The large and tunable pKa shift of aliphatic amines was further
applicable to the fluorescence monitoring of other enzymatic
reactions. Nitrile hydratase is an important class of enzyme that
catalytically hydrolyzes a nitrile group to carboxyamide,5 and is
indispensable for the industrial production of acrylamide from
acrylonitrile.6 In pH titration, the pKa value of the amino group of 8
that bears a propionitrile group was 5.52, and shifted to 7.43 for the
corresponding carboxyamide derivative 9 (Fig. S6, ESI†). This large
pKa shift provides a sufficiently wide pH window for fluorescence
sensing of the nitrile hydrolysis of 8. Fig. 3a plots realꢀtime
monitoring of the hydrolysis reaction with nitrile hydratase derived
from Rhodocuccus rhodochrus J1 at pH 7.0. The anthracene
fluorescence increased 25ꢀfold during the enzymatic reaction, the
rate of which depends on the amount of the enzyme (Fig. S7, ESI†).
The Km and Vmax value was evaluated to be 308 ꢁM and 2.88
ꢁM/min, respectively (Fig. S8, ESI†). Another detectable activity
was the reduction of carbonyl groups by carbonyl reductase, which
catalyzes the reduction of a ketone or aldehyde into the
corresponding alcohol using nicotinamide adeninedinucleotide
phosphate (NAD(P)H) as a coꢀfactor.7 This class of enzymes plays
crucial roles in drug metabolism.8 Fig. 3b plots fluorescence during
the reduction of ketone 10 into the corresponding alcohol 11, with
pKa values of 6.68 and 8.06, respectively (Fig. S9, ESI†). In the
enzymatic reaction at pH 7.4, the fluorescence intensity increased
over 7ꢀfold, and the initial reaction rate clearly depended on the
amount of carbonyl reductase (Fig. S10, ESI†). The Km and Vmax
value was evaluated to be 7.14 ꢁM and 8.02 nM/min, respectively
(Fig. S11, ESI†).
The pKa values of aliphatic amines can also be modulated by the
attached fluorophore. As shown in Fig. 4, the pKa of coumarin ester
12 and boronꢀdipyrromethene (BODIPY) ester 13 were 4.14 and
3.24, respectively, which are significantly lower than that of the
anthracene probe 2 (pKa=5.66). The low pKa values might be
attributed to the stronger electronꢀwithdrawing properties of hetero
atomꢀcontaining coumarin and BODIPY fluorophore compared to
anthracene. Similar to the case of 2, the pKa of 13 changed from 3.24
to 6.41 when hydrolyzed to the corresponding carboxylate 14.
Taking advantage of this large pKa shift in the weak acidic region
and the long emission wavelength (> 500 nm) preferable for inꢀcell
bioimaging, 13 was used in the detection of esteraseꢀactivity in
acidic cellular lysosomes. Prior to bioimaging, the sensing ability of
13 was examined in vitro using PLE as a hydrolysis enzyme (Fig.
S12, ESI†). The 10ꢀfold fluorescence enhancement was observed in
a timeꢀdependent manner when the reaction was conducted under
slightly acidic conditions (pH 5.0), whereas a negligible fluorescence
change was observed under neutral pH conditions, even though 13
was efficiently hydrolyzed to 14 by PLE. This suggests that 13 was
Fig. 4 pHꢀdependent fluorescence intensity changes of the aliphatic amines
tethered to different fluorophores; 2 (▲, ex. 366 nm), 12 (●, ex. 359 nm), 13 (■,
ex. 519 nm), 14 (□, ex. 517 nm). Conditions: [probe] = 1 ꢀM, 5 mM MES ꢀ 5 mM
HEPES ꢀ 5 mM CHES buffer, 25 °C.
2 | J. Name., 2012, 00, 1-3
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Graduate School of Pharmaceutical Sciences, Kyushu University,
3ꢀ1ꢀ1, Maidashi, Higashiꢀku, Fukuoka, 812ꢀ8582, Japan.
Fax: (+81) 92ꢀ642ꢀ6601
Eꢀmail: ojida@phar.kyushuꢀu.ac.jp
We thank Dr. K. Murao, DiaꢀNitrix Co., Ltd. for kindly providing nitrile
hydratase extracted from Rhodocuccus rhodochrus J1. Y. O.
acknowledges the JSPS Research Fellowships for Young Scientists.
† Electronic Supplementary Information (ESI) available: [synthesis of the
probes, analysis of fluorescence enzyme reactions, and detailed
experimental procedure of the enzyme assays and imaging studies]. See
DOI: 10.1039/c000000x/
‡The cells treated with PMSF for 13 h (79% of viability determined by
trypane blue assay) showed the normal staining pattern of lysosomes with
Lysotracker Red (Fig. S15, ESI†), suggesting that the PMSFꢀtreated cells
maintain the membrane permeability and the acidic lysosomal
environment.
1
(a) K. Kubo in Topics in Fluorescence Spectroscopy, Vol. 9, (Eds: C.
D. Geddes, J. R. Lakowicz, Springer, USA, 2005, pp. 219ꢀ247.); (b)
A. P. de Silva, T. S. Moody, G. D. Wright, Analyst, 2009, 134, 2385;
Fig. 5 Detection of lysosomal esterase activity in living HeLa cells that were
incubated with 13 (3 ꢀM) in Dulbecco's modified Eagle's medium for 30 min (a,
b), 4 h (c, d), and 13 h (e, f). (g) Lysosome detection by LysoTracker Red. (h)
Merged image of (f) and (g). (i, j) HeLa cells were incubated for 13 h with 13 (3
ꢀM) in the presence of PMSF (0.5 mM). Scale bars: 50 ꢀm.
(c) T. Nagano, J. Clin. Biochem. Nutr.
,
2009, 45, 111; (d) D. W.
, 168; (e)
Domaille, E. J. Que, C. J. Chang, Nat. Chem. Biol., 2008,
4
Y. Urano, M. Kamiya, K. Kanda, T. Ueno, K. Hirose, T. Nagano, J.
Am. Chem. Soc., 2005, 127, 4888; (f) J. F. Callan, A. P. de Silva, D.
C. Magri, Tetrahedron, 2005, 61, 8551.
useful for selectively detection of the hydrolytic activity of esterase
in the acidic cellular environment. When HeLa cells were treated
with 13 (3 ꢁM in DꢀMEM), BODIPY fluorescence was scarcely
observed in the cells. However, after 13 h at 37 °C, bright
fluorescent particles were observed at localized regions (Fig. 5). This
fluorescence overlaps that of LysoTracker Red, a selective marker of
lysosomes, with a high Pearson correlation coefficient (r = 0.793). In
contrast, the accumulated BODIPY fluorescence diminished when
the esterase activity was inhibited by phenylmethylsulfonyl fluoride
(PMSF)‡ and pH inside the lysosomes (typically pH 4.5~6) was
neutralized by chloroquine (Fig. S13, ESI†).9 These results clearly
indicate that 13 was hydrolyzed by endosomal and lysosomal
esterases to form the membraneꢀimpermeable carboxylate 14 (Fig.
S14, ESI†), which exhibited bright fluorescence in the weakly acidic
compartments.
In conclusion, we have demonstrated that the large and tunable
pKa shift of aliphatic amines is useful for the detection of various
enzymeꢀcatalyzed reactions. The important features of this
fluorescenceꢀsensing system are: (1) The probe can be rationally
designed based on the pKa of the aliphatic amine. (2) The structure
of the fluorescent probe is simple and the elaborated molecular
design is not required. (3) The sensing mechanism is compatible
with various types of fluorophores, which potentially allows
simultaneous monitoring of multiple enzymatic reactions with
multiple emission changes. (4) To our knowledge, this is the first
example of an enzyme reaction monitoring system that utilizes the
large and tunable pKa shift of aliphatic amines. These important
features represent the utility of the present sensing system
complementary to the known fluorescent probes for enzyme
reactions.10 Further applications of the present system to various
enzymatic and nonꢀenzymatic reactions are ongoing.
2
3
Y. Wang and H. Morawetz, J. Am. Chem. Soc., 1976, 98, 3611.
(a) T. Myochin, K. Kiyose, K. Hanaoka, H. Kojima, T. Terai, T.
Nagano, J. Am. Chem. Soc., 2011, 133, 3401; (b) S, Uchiyama, K.
Iwai, A. P. de Silva, Angew. Chem. Int. Ed., 2008, 47, 4667; (c) A. P.
de Silva, S. S. K. de Silva, N. C. W. Goonesekera, H. Q. N.
Gunaratne, P. L. M. Lynch, K. R. Nesbitt, S. T. Patuwathavithana, N.
L. D. S. Ramyalal, J. Am. Chem. Soc., 2007, 129, 3050; (d) W. Ni, G.
Kaur, G. Springsteen, B. Wang, S. Franzen, Bioorganic chemistry
,
2004, 32, 571; (e) T. D. James, K. R. A. S. Sandanayake, S. Shinkai,
Nature, 1995, 374, 345.
4
5
6
7
8
R. C. Knowlton, L. D. Byers, J. Org. Chem., 1988, 53, 3862.
Y. Asano, Y. Tani, H. Yamada, Agrical. Biol. Chem., 1980, 44, 2251.
T. Nagasawa, H. Yamada, Trends in Biotechnol., 1889, 7, 153.
G. L. Forrest, B. Gonzalez, Chem. Biol. Interact., 2000, 129, 21.
(a) N. Kassner, K. Huse, H. Martin, U. GödtelꢀArmbrust, A. Metzger,
I. Meineke, J. Brockmöller, K. Klein, U. M. Zanger, E. Maser, L.
Wojnowski, Drug Metab. Dispos., 2008, 36, 2113; (b) M. Kishimoto,
R. Kawamori, T. Kamada, T. Inaba, Drug Metab. Dispos., 1997, 22
,
367.
9
V. Sée, P. Free, Y. Cesbron, P. Nativo, U. Shaheen, D. J. Rigden, D.
G. Spiller, D. G. Fernig, M. R. H. White, I. A. Prior, M. Brust, B.
Lounis, R. Lévy, ACS Nano, 2009, 3, 2461.
10 Recent examples of fluorescent probes for enzyme reactions: (a) X.
Wang, L. Cui, N. Zhou, W. Zhou, R. wang, X. Qian, Y. Xu, Chem.
Sci., 2013, 4, 2936; (b) M. Sakabe, D. Asanuma, M. Kamiya, R. J.
Iwatate, K. Hanaoka, T. Terai, T. Nagano, Y. Urano, J. Am. Chem.
Soc., 2013, 135, 409; (c) M. K. Froemming, D. Sames, J. Am. Chem.
Soc., 2007, 129, 14518; (d) M. Halim, D. J. Yee, D. Sames, J. Am.
Chem. Soc., 2008, 130, 14123; (e) G. Chen, D. J. Yee, N. G.
Gubernator, D. Sames, J. Am. Chem. Soc., 2005, 127, 4544.
Notes and references
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