molecules Article
A Near-Infrared Fluorescent Probe Based on a FRET Rhodamine Donor Linked to a Cyanine Acceptor for Sensitive Detection of Intracellular pH Alternations Yibin Zhang 1,2 , Jianheng Bi 1 , Shuai Xia 1 , Wafa Mazi 1 , Shulin Wan 1 , Logan Mikesell 1 , Rudy L. Luck 1, * and Haiying Liu 1, * 1
2
*
Department of Chemistry, Michigan Technological University, 1400 Townsend Drive, Houghton, MI 49931, USA;
[email protected] (Y.Z.);
[email protected] (J.B.);
[email protected] (S.X.);
[email protected] (W.M.);
[email protected] (S.W.);
[email protected] (L.M.) School of Chemistry and Chemical Engineering, Yangtze Normal University, Chongqing 408100, China Correspondence:
[email protected] (R.L.L.);
[email protected] (H.L.); Tel.: +1-906-487-3451 (H.L.)
Received: 18 September 2018; Accepted: 15 October 2018; Published: 18 October 2018
Abstract: A fluorescence resonance energy transfer (FRET)-based near-infrared fluorescent probe (B+ ) for double-checked sensitive detection of intracellular pH changes has been synthesized by binding a near-infrared rhodamine donor to a near-infrared cyanine acceptor through robust C-N bonds via a nucleophilic substitution reaction. To demonstrate the double-checked advantages of probe B+ , a near-infrared probe (A) was also prepared by modification of a near-infrared rhodamine dye with ethylenediamine to produce a closed spirolactam residue. Under basic conditions, probe B+ shows only weak fluorescence from the cyanine acceptor while probe A displays nonfluorescence due to retention of the closed spirolactam form of the rhodamine moiety. Upon decrease in solution pH level, probe B+ exhibits a gradual fluorescence increase from rhodamine and cyanine constituents at 623 nm and 743 nm respectively, whereas probe A displays fluorescence increase at 623 nm on the rhodamine moiety as acidic conditions leads to the rupture of the probe spirolactam rings. Probes A and B+ have successfully been used to monitor intracellular pH alternations and possess pKa values of 5.15 and 7.80, respectively. Keywords: near-infrared imaging; fluorescent probes; FRET; rhodamine; cyanine dye
1. Introduction Different cellular compartments regulate intracellular pH as precise control is essential for various cell functions such as vesicle trafficking, cellular metabolism, cellular signaling, cell membrane polarity, cell activation, proliferation growth, and apoptosis [1–4]. Intracellular pH values are quite different in different organelles [1–4]. Lysosomes function best under acidic pH conditions between 4.5 to 5.5 to break down a variety of biomolecules while mitochondria operate under slightly alkali pH conditions around 8.0 [4–6]. Various diseases such as neurodegenerative disease, cancer, and Alzheimer’s disease, are associated with significant deviations from normal functional intracellular pH values [1–4]. Therefore, monitoring intracellular pH levels is important in order to understand cellular functions. Fluorescence imaging is frequently used for real-time pH monitoring in biological systems due to its rapid response time, high sensitivity, non-destructive nature, operational simplicity, and high-speed spatial capabilities [4]. Recently, near-infrared pH fluorescent probes were developed to take advantage of near-infrared imaging unique features such as minimum photobleaching, deep tissue penetration, suppressed photodamage to cells and tissues, and low biological luminescence background [5–33]. Most of these near-infrared fluorescent probes that measure pH levels are based
Molecules 2018, 23, 2679; doi:10.3390/molecules23102679
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Molecules 2018, 23, 2679
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on fluorescence changes in a single near-infrared wavelength [5–28,30–33]. We have developed a near-infrared fluorescent (B+ ), Scheme 1, with unique double-checked capability to accurately on fluorescence changes probe in a single near-infrared wavelength [5–28,30–33]. We have developed a + detect intracellular pH alternations byScheme monitoring deep-red near-infrared fluorescence changes at near-infrared fluorescent probe (B ), 1, with unique and double-checked capability to accurately detect by monitoringfeature deep-red and near-infrared fluorescence changes 623 nm intracellular and 780 nm.pH Thealternations probe’s double-checked was achieved by connecting a near-infrared at 623 nm dye and as 780 nm. Theresonance probe’s double-checked achieved by connecting a near-dye rhodamine a Forster energy transferfeature (FRET)was donor to a near-infrared cyanine dye as avia Forster resonance energy linkage transfer with (FRET) donor to bonds. a near-infrared asinfrared a FRETrhodamine acceptor tethered an ethylene-diamino robust C-N Probe B+ cyanine dye tethered via an ethylene-diamino linkage robust C-Nrhodamine bonds. possesses twoas pKaaFRET valuesacceptor of 4.0 and 7.4 corresponding to spirolactam ring with opening of the + possesses two pKa values of 4.0 and 7.4 corresponding to spirolactam ring opening of the Probe B donor, and protonation of the central nitrogen atom of the cyanine acceptor respectively under rhodamine donor donor, excitation and protonation the central nitrogen atomcharacteristic of the cyaninewith acceptor rhodamine at 450ofnm. This advantageous two respectively different pKa under rhodamine donor excitation at 450 nm. This advantageous characteristic with twofluorescence different values in one system enables us to determine pH changes in a broad range with both pKa values in one system enables to cyanine determine pH changes in a broad range with both fluorescence increases of the rhodamine donorus and acceptor. We also prepared a near-infrared fluorescent increases of the rhodamine donor and cyanine acceptor. We also prepared a near-infrared fluorescent probe (A), Scheme 1, through modification of a near-infrared rhodamine dye with ethylenediamine. probe (A), Scheme 1, through modification of a near-infrared rhodamine dye with ethylenediamine. We demonstrate that there is slight overlap between the rhodamine donor emission and cyanine We demonstrate that there is slight overlap between the rhodamine donor emission and cyanine acceptor absorption for energy transfer from the rhodamine donor to the cyanine acceptor presumably acceptor absorption for energy transfer from the rhodamine donor to the cyanine acceptor through the short ethylenediamino linkage [34,35]. Probe A shows the expected fluorescence increase presumably through the short ethylenediamino linkage [34,35]. Probe A shows the expected responses to pH variance under pH stimulus from 7.4 to 3.0. Probe B+ exhibits weak fluorescence fluorescence increase responses to pH variance under pH stimulus from 7.4 to 3.0. Probe B+ exhibits from the cyanine acceptor under basic pH conditions with retention of the closed spirolactam weak fluorescence from the cyanine acceptor under basic pH conditions with retention of the closed configuration. Gradual increase in acidity from pH 9.0 to 3.0 results in fluorescence increases with spirolactam configuration. Gradual increase in acidity from pH 9.0 to 3.0 results in fluorescence the rhodamine cyanine moieties under rhodamine donorrhodamine excitation. donor This allows for accurate increases withand the the rhodamine and the cyanine moieties under excitation. This + shows excellent monitor of intracellular pH levels through two near-infrared channels. Probe B allows for accurate monitor of intracellular pH levels through two near-infrared channels. Probe B+ photostability, low cytotoxicity, low good selectivity,good and high sensitivity to pH near-infrared shows excellent photostability, cytotoxicity, selectivity, and high sensitivity to pHimaging nearfeature. These conclusions are also confirmed by theoretical studies. infrared imaging feature. These conclusions are also confirmed by theoretical studies. N O NH2 N
N
O NH2
Probe A
+ OH
O
H N
pka = 5.15 + H+
NH2
O
+ OH
H 2N
O
-
O NH
N
N
O H N
N
-
H 2N
N
pka = 7.40 and 4.00 + H+
O NH
H 2N
N
N
N
Probe B
+
Scheme 1. 1. Drawings Scheme Drawings of ofthe theprobes probesand andtheir theirprotonated protonatedversions. versions.
2.2.Results Results 2.1. 2.1.Synthesis SynthesisofofFluorescent Fluorescent Probes Probes InInorder rhodaminedonor donortotoaacyanine cyanineacceptor acceptor which contains a reactive orderto tobind bind aa near-infrared near-infrared rhodamine which contains a reactive chloro site for chemical substitution, we prepared rhodamine dye (3) by a condensation reaction chloro site for chemical substitution, we prepared rhodamine dye (3) by a condensation reaction of of 2-(4-(diethylamino)-2-hydroxybenzoyl) acid (1) and 6-amino-3,4-dihydro-1(2H)-naphthalenone 2-(4-(diethylamino)-2-hydroxybenzoyl)benzoic benzoic acid (1) and 6-amino-3,4-dihydro-1(2H)(2) in concentrated acid under reflux acid conditions. The rhodamine dye bearing a closed naphthalenone (2)sulfuric in concentrated sulfuric under reflux conditions. The rhodamine dyespirolactam bearing a closed ring(probe with amine residue (probe was prepared by reacting dyeexcess (3) ring with spirolactam amine residue A) was prepared byA)reacting rhodamine dye (3)rhodamine with a large with aof large excess amount 1,2-diaminoethane (4) in the presence of benzotriazol-1amount 1,2-diaminoethane (4) in of the presence of benzotriazol-1-yloxytris(dimethylamino)phosphonium yloxytris(dimethylamino)phosphonium (BOP). Probe B+ was synthesized hexafluorophosphate (BOP). Probe B+ was hexafluorophosphate synthesized through a nucleophilic substitution reaction of the through a nucleophilic reaction of thering central chloro group in a rigid chlorocyclohexenyl central chloro group in asubstitution rigid chlorocyclohexenyl of cyanine dye (IR-780) with the dangling -NH2 ring of (IR-780) with the dangling -NH 2 group on probe A probes under were basic characterized conditions group on cyanine probe A dye under basic conditions (Scheme 2). All intermediates and 1H and 13C NMR and mass 1 13 (Scheme 2). All intermediates and probes were characterized by by H and C NMR and mass spectrometer as detailed in Supplementary Materials. spectrometer as detailed in Supplementary Materials.
Molecules Molecules2018, 2018,23, 23,2679 x FOR PEER REVIEW Molecules 2018, 23, x FOR PEER REVIEW
33 of of1515 3 of 15 O O
COOH H2N 2 COOH H2N O 2 H2SO4 O OH H2SO4 OH
N N
N N
BOP reagent
O O
3 3
NH2 NH2
N N
Cl Cl
NH2 NH2
O O
Probe A Probe A
N N
N N
+
O O N N
NH2 NH2
4 4 BOP reagent
HOOC HOOC
1 1
N + N
H 2N H 2N
NH2 NH2
O O H 2N H 2N
IR-780 IR-780
N N O NH O NH
+
N N
N + N
DIPEA DIPEA
+
Probe B+ Probe B
+. Scheme 2. Synthetic route to probes A and B ++. Scheme 2. Synthetic route to probes A and B Scheme 2. Synthetic route to probes A and B .
Probe A Probe A
Absorbance Absorbance
0.15 0.15
0.10 0.10
0.05 0.05
0.00 0.00
300 300
400 500 600 700 400Wavelength 500 600 (nm) 700
Wavelength (nm) (a) (a)
pH 2.4 pH2.4 2.8 pH pH2.8 3.2 pH pH3.2 3.6 pH pH3.6 4.0 pH pH4.0 4.4 pH pH4.4 4.8 pH pH4.8 5.2 pH pH5.2 5.6 pH pH5.6 6.0 pH pH6.0 6.4 pH pH6.4 7.0 pH pH 7.0 800 800
Fluorescence Intensity Fluorescence Intensity
2.2.Optical OpticalResponeses ResponesesofofFluorescent FluorescentProbes ProbestotopH pHChanges Changes 2.2. 2.2. Optical Responeses of Fluorescent Probes to pH Changes wereinvestigated investigatedinindifferent differentpH pH Theoptical opticalpH-responsive pH-responsiveproperties propertiesofofprobes probesAAand andBB + +were The in different ring pH Thecontaining optical pH-responsive properties ofintensity-based probes A and rhodamine B+ were investigated buffers 1% DMSO. Probe A is an dye with a spirolactam buffers containing 1% DMSO. Probe A is an intensity-based rhodamine dye with a spirolactam ring buffers containing 1%can DMSO. Probe A opening/closing is an intensity-based rhodamine dye with a spirolactam ring on/off switch, processes under pH stimulus. ProbeProbe A has an on/off switch, which which canundergo undergoring ring opening/closing processes under pH stimulus. A on/off switch, which can undergo ring opening/closing processes under pH stimulus. Probe A has an absorption at 300 nm but no emission with a closed spirolactam ring configuration under basic has an absorption at 300 nm but no emission with a closed spirolactam ring configuration under absorption at 300 nm no gradual emissiondecrease with a closed spirolactam ring configuration under conditions (Figure 1). but Upon in in pH, both 591 nm nmbasic and basic conditions (Figure 1). Upon gradual decrease pH, bothabsorbances absorbancesatat415 415 and and 591 and conditions (Figure 1). Upon gradual decrease in pH, both absorbances at 415 and 591 nm and fluorescence intensity at 623 nm increase due to acid-activated opening of the spirolactam structure, fluorescence intensity at 623 nm increase due to acid-activated opening of the spirolactam structure, fluorescence intensityprobe at 623 nm increase due to acid-activatedfluorescence opening of the spirolactam structure, Figure1.1.Therefore, Therefore, displays typical intensity-based responses topH pH changes, Figure probe AAdisplays typical intensity-based fluorescence responses to changes, Figure 1. Therefore, probe A displays typical intensity-based fluorescence responses to pH changes, andpossesses possessesaapK pKavalue valueofof5.15 5.15related relatedtotothe thespirolactam spirolactamring ringopening. opening.Probe ProbeAAshows showsaahigh high and a and possessesquantum a pKa value ofof5.15 related toacidic the spirolactam ring4.0). opening. Probeabsorbtivity A shows aishigh fluorescence yield 31.1% under conditions (pH The molar fluorescence quantum yield of 31.1% under acidic conditions (pH 4.0). The molar absorbtivity2.88 is fluorescence quantum yield of under acidic conditions (pH 4.0). The molar absorbtivity is 2.88 4 L·mol × 10× ·cm−−11at 591 at31.1% pH 4.0. 4 L−1 − 1 nm 2.88 10 · mol · cm at 591 nm at pH 4.0. × 104 L·mol−1·cm−1 at 591 nm at pH 4.0.
8.0x106 8.0x106
Probe A Probe A
6.0x106 6.0x106 4.0x106 4.0x106
2.0x106 2.0x106 0.0 0.0 550
550
600 650 700 600 650 700 Wavelength (nm)
pH 2.4 pH2.4 2.8 pH pH2.8 3.2 pH pH3.2 3.6 pH pH3.6 4.0 pH pH4.0 4.4 pH pH4.4 4.8 pH pH4.8 5.2 pH pH5.2 5.6 pH pH5.6 6.0 pH pH6.0 6.4 pH pH6.4 7.0 pH pH 7.0
750 750
Wavelength (nm) (b) (b)
Figure1.1.Absorption Absorption fluorescence spectra (b) of Aprobe A in different buffers under Figure (a) (a) andand fluorescence spectra (b) of probe in different pH bufferspH under excitation Figure 1. Absorption (a) and fluorescence spectra (b) of probe A in different pH buffers under excitation at 550 nm. at 550 nm. excitation at 550 nm.
In order to monitor pH changes and the assumed double-checked feature, probe B+ was In order to monitor apH changes donor and the feature, B+ was prepared by introducing rhodamine to assumed a cyaninedouble-checked acceptor through a very probe short ethylene prepared introducing a rhodamine donor to a cyanine a veryProbe shortBethylene + shows a spacer to by achieve high efficiency of energy transfer from theacceptor donor tothrough the acceptor. + spacer to achieve high efficiency of energy transfer from the donor to the acceptor. Probe B shows a
Absorbance
pH 2.4 pH 3.2 pH 4.0 pH 4.8 pH 5.6 pH 6.0 pH 6.8 pH 7.6 pH 8.2 pH 8.8 pH 9.5 pH 10.3 pH 10.8
Probe B+
0.08
0.06
0.04
0.02
0.00 300
400
500
600
700
Wavelength (nm)
(a)
800
Fluorescence Intensity
absorption of the cyanine acceptor (probe B+) (Figure 3, right), excitation at 450 nm allows for an effective FRET process from the rhodamine donor to the cyanine acceptor, resulting in both fluorescence increases of the rhodamine donor and the cyanine acceptor with decreases in pH (Figure 2). It is also noteworthy that the intensity of the fluorescence from the cyanine acceptor has a much more significant Molecules 2018, 23, 2679 increase than the intensity of the fluorescence from the rhodamine donor 4 of 15 presumably due to the FRET process for probe B+, which in the case of the rhodamine moiety in isolated probe A (free of the cyanine moiety) was quite substantive. Probe B+ shows a quantum yield In order monitor and theusing assumed double-checked feature, probe B+absorptivity was prepared of 12.4% (pHto4.0) underpH 450changes nm excitation human dye as standard. The molar is by introducing a rhodamine donor to a cyanine acceptor through a very short ethylene spacer to 4 −1 −1 + 1.50 × 10 L·mol ·cm at 663 nm at pH 4.0. The rhodamine donor of probe B has a pKa value of 4.0 achieve high efficiency energy transfer from the donor the acceptor. B+ shows a weak due to spirolactam ringofopening of the rhodamine donorto while the probeProbe cyanine acceptor has a absorption peak at 664 nm and an extremely weak fluorescence peak at 743 nm, Figure 2. However, higher pKa value of 7.4 arising from protonation of the central nitrogen atom of the cyanine acceptor gradual pH decreases from 10.8 to at 2.4450 causes increases the absorption peak, under rhodamine donor excitation nm. corresponding This nice feature with twoindifferent pKa values inand one results a new to absorption 413 nm, gradual increases of fluorescence at 616 systemin enables determinepeak pH at changes inand a broad range with both fluorescencepeaks increases of nm the and 743 nm under excitation at 450 nm, Figure 2. Additionally, under cyanine acceptor excitation at rhodamine donor and cyanine acceptor. The efficiency of FRET from the rhodamine donor to the 645 nm, the fluorescence intensitytoofbe the23.5% cyanine moiety also increases pH decrease,depends indicating cyanine acceptor was calculated in pH 3.2 buffer since theupon FRET efficiency on that acidic pH results in protonation of the central amine atom of cyanine acceptor and increases in not only the distance, but also the overlap between donor’s emission and acceptor’s absorption + through the spirolactam ring opening. fluorescence of probe B spectra and orientation.
3x10
5
Probe B
pH 2.4 pH 3.2 pH 4.0 pH 4.8 pH 5.6 pH 6.0 pH 6.8 pH 7.6 pH 8.2 pH 8.8 pH 9.5 pH 10.3 pH 10.8
+
2x105
1x105
0 600
700
800
Wavelength (nm)
(b)
Figure 2.2. Absorption Absorption (a) and fluorescence fluorescence spectra spectra (b) (b) of of probe probe BB++ in in different different pH pH buffers buffers under under Figure (a) and excitation at at 450 450 nm. nm. excitation
The introduction of a robust C-N bond to the cyanine moiety results in a blue shift of absorption of the cyanine acceptor with absorption around 650 nm which allows for efficient FRET processes as the absorption of the cyanine acceptor overlaps with the emission from the rhodamine donor [34,35]. Since there is significant overlap between the fluorescence of the rhodamine donor (probe A) and absorption of the cyanine acceptor (probe B+ ) (Figure 3, right), excitation at 450 nm allows for an effective FRET process from the rhodamine donor to the cyanine acceptor, resulting in both fluorescence increases of the rhodamine donor and the cyanine acceptor with decreases in pH (Figure 2). It is also noteworthy that the intensity of the fluorescence from the cyanine acceptor has a much more significant increase than the intensity of the fluorescence from the rhodamine donor presumably due to the FRET process for probe B+ , which in the case of the rhodamine moiety in isolated probe A (free of the cyanine moiety) was quite substantive. Probe B+ shows a quantum yield of 12.4% (pH 4.0) under 450 nm excitation using human dye as standard. The molar absorptivity is 1.50 × 104 L·mol−1 ·cm−1 at 663 nm at pH 4.0. The rhodamine donor of probe B+ has a pKa value of 4.0 due to spirolactam ring opening of the rhodamine donor while the probe cyanine acceptor has a higher pKa value of 7.4 arising from protonation of the central nitrogen atom of the cyanine acceptor under rhodamine donor excitation at 450 nm. This nice feature with two different pKa values in one system enables to determine pH changes in a broad range with both fluorescence increases of the rhodamine donor and cyanine acceptor. The efficiency of FRET from the rhodamine donor to the cyanine acceptor was calculated to be 23.5% in pH 3.2 buffer since the FRET efficiency depends on not only the distance, but also the overlap between donor’s emission and acceptor’s absorption spectra and orientation.
6.0x105 6.0x105 6.0x105 4.0x105 4.0x105 4.0x105
2.0x105 2.0x105 2.0x105 0.0 0.0 0.0 650 650 650
700 700
750 750
800 800
1.0 1.0 1.0 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0.0 0.0 500 550 600 650 700 750 800 0.0 500 550 600 650 700 750 800 500 550 Wavelength 600 650 700 (nm)750 800
850 850 850
Wavelength (nm) 700 750 (nm) 800 Wavelength Wavelength (nm) (a)(a) (a)
Fluor escence spectr um of pr obe A Fluor escence spectr um of pr obe A+ Absor ption spectr um ofof prpr obe B Fluor escence spectr um obe Absor ption spectr um of pr obe B+ A Absor ption spectr um of pr obe B +
Absor bance Absor bance Absor bance
8.0x10 8.0x105 8.0x105
pH 2.4 pH 2.4 pH2.4 3.2 pH pH 3.2 pH3.2 4.0 pH pH 4.0 pH4.0 4.8 pH pH 4.8 pH 5.6 pH5.6 4.8 pH pH5.6 6.0 pH pH 6.0 pH6.0 6.8 pH pH 6.8 pH 7.6 pH7.6 6.8 pH pH7.6 8.2 pH pH 8.2 pH8.2 8.8 pH pH 8.8 pH 9.5 pH9.5 8.8 pH pH9.5 10.3 pH pH 10.3 pH10.3 10.8 pH pH 10.8 pH 10.8
Excitation Excitation 645 nm Excitation 645 nm 645 nm
+ + ProbeBB Probe + Probe B
5
5ofof1515 55 of 15 5 of 15
Fluor escence intensity Fluor escence intensity Fluor escence intensity
Fluorescence intensity Fluorescence intensity Fluorescence intensity
Molecules 2018, x FOR PEER REVIEW Molecules 2018, 23,23, x FOR PEER REVIEW Molecules 2018, 23, 2679 Molecules 2018, 23, x FOR PEER REVIEW
Probe A Probe A Probe A
Fluorescence Intensity Fluorescence Intensity Fluorescence Intensity
8.0x106 8.0x106 8.0x106
pHpH 4.04.0 pH 4.0
6.0x106 6.0x106 6.0x106
4.0x106 4.0x106 4.0x106
2.0x106 2.0x106 2.0x106 0.0 0.0 0.00 0
0
2
2
4
4
6
6
8
8
Experiment Cycle 2 4 6Cycle8 Experiment Experiment Cycle
pHpH 7.67.6 7.6 10 10pH
10
Fluorescence Intensity Fluorescence Intensity Fluorescence Intensity
Wavelength (nm) Wavelength (b) (nm) (b) (b) + Figure Fluorescence spectra cyanine acceptor probe different pH buffers , fluorescence Figure 3.3. Fluorescence spectra ofof cyanine acceptor ofof probe B+Binin different pH buffers (a)(a) , fluorescence + in different pH buffers (a), fluorescence + at + Figure 3. Fluorescence spectra of cyanine acceptor of probe B pH 4.8 (b) . spectrum of probe A, and absorption spectrum of probe B Figure 3. Fluorescence spectra of cyanine acceptor of probe B in different pH buffers (a), fluorescence + spectrum of probe A, and absorption spectrum of probe B at pH 4.8 (b). + at pH 4.8 (b). spectrumofofprobe probe and absorption spectrum probe spectrum A,A, and absorption spectrum ofof probe B+Bat pH 4.8 (b). + under 450 ProbeAAreversibly reversiblyresponds respondstotopH pHchanges changesbetween between4.0 4.0and and7.6, 7.6,Figure Figure4.4.Probe ProbeB+Bunder Probe 450 + under + Probe A reversibly responds to pH changes between 4.0 and 7.6, Figure 4. Probe B 450 nm Probe A reversibly responds to pH changes between 4.0 and 7.6, Figure 4. Probe B under 450 nm excitation also shows excellent reversible fluorescence responses at 743 nm to pH changes nm excitation also shows excellent reversible fluorescence responses at 743 nm to pH changes + are excitation also shows excellent reversible fluorescence responses atand 743 +B nm to pH changes between 4.0 nm excitation also shows excellent reversible fluorescence responses at 743 nm to pH changes between 4.0 and 10.8. The results indicate that probes A stable and demonstrate between 4.0 and 10.8. The results indicate that probes A and B are stable and demonstrate + are stable and demonstrate and 10.8. The results indicate that probes Aas and Bthat reversibility to changes between 4.0 and 10.8. The results indicate AIR-780 anddye, B+ Figure are stable reversibility to changes in pH, Figure 4, compared theIR-780 dye, Figure reversibility to changes in pH, Figure 4, as comparedprobes totothe 5.5. and demonstrate inreversibility pH, Figure 4, compared to the IR-780 Figure 5. toas changes in pH, Figure 4, asdye, compared to the IR-780 dye, Figure 5. 3.0x105 3.0x105 5 3.0x10 2.5x105 2.5x105 5 2.5x10 2.0x105 2.0x105 5 2.0x10 1.5x105 1.5x105 5 1.5x10 1.0x105 1.0x105 5 1.0x10 5.0x104 5.0x104 4 5.0x10 0.0 0.0 0 0.00
0
(a)(a) (a)
Probe B+ Probe B+ Probe B+
2 4 6 8 4 6 8 Experiment Cycle 2 4 6 8 Experiment Cycle
2
pH 4.0 pH 4.0 pH 4.0
pH 11.3 pH 11.3 pH 11.3 10
10 10
Experiment (b) Cycle
(b) (b)
1.00 1.00 1.00
Normalized Fluorescence Intensity Normalized Fluorescence Intensity Normalized Fluorescence Intensity
Normalized Fluorescence Intensity Normalized Fluorescence Intensity Normalized Fluorescence Intensity
+ Figure 4. Fluorescence intensities probes A and different pH conditions under 450 nm Figure Fluorescence intensities ofof probes (a)(a) and (b)(b) inin different pH conditions under 450 nm Figure 4.4.Fluorescence intensities of probes AA(a) and BB++B(b) in different pH conditions under 450 nm + + Figure 4.for Fluorescence intensities of probes Afor (a) and BA. excitation for probe , and 550 nm excitation for probe A. in different pH conditions under 450 nm excitation for probe and 550 nm excitation for probe A.(b) excitation probe BB++B ,, and 550 nm excitation probe excitation for probe B+, and 550 nm excitation for probe A.
1.00 1.00 1.00
0.75 0.75 0.75
IR-780 at 804 nm IR-780 at 804 nm Probe A at 623 nm Probe A at at 804 623 nm nm IR-780 Probe A at 623 nm
0.50 0.50 0.50
0.75 0.75 0.75
IR-780 at 804 nm IR-780 at 804 nm Probe+ B+ at 743 nm 743nm nm Probe B atat804 IR-780 + Probe B at 743 nm
0.50 0.50 0.50 0.25 0.25 0.25
0.25 0.25 0.25 0.00 0.00 0 0.00 0
0
30 60 90 120 150 180 30 60 90 120 150 180 Time (min) 30 60 90 120 150 180 Time (min)
Time (a)(min)
(a) (a)
0.00 0.00 0 0.00 0
0
30 60 90 120 150 180 30 60 90 120 150 180 Time (min) 30 60 90 Time (min)120 150 180
Time (min)
(b) (b) (b)
+ Figure 5.Fluorescence Fluorescence intensity probes A(a)(a) and under continual excitation 450 nm Figure 5.5.Fluorescence intensity of probes AA(a) and BB++B(b) under continual excitation of 450 nm for Figure intensity ofof probes and (b)(b) under continual excitation ofof 450 nm forfor + Fluorescence + (b) under continual excitation of 450 nm for + probe B and 550 nm for probe A. Figure 5. intensity of probes A (a) and B + probe probeBB and and 550 550 nm nm for for probe probe A. A. probe B+ and 550 nm for probe A.
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2.3. Selectivity the Probes to pH pH over Cations, Anions, and Amino Acids 2.3.2.3. Selectivity the the Probes to pH overover Cations, Anions, andand Amino Acids Selectivity Probes to Cations, Anions, Amino Acids to pH pH to metal metal ions. Essential metal ions We studied the responsiveness of probes probes A and and B++ pH WeWe studied thethe responsiveness of probes A and B+ to to metal ions. Essential metal ions to to ions. Essential metal ions studied responsiveness of A B + 2+ 2+ 2+ 2+ + 3+ 2+ 3+, and Co2+ + 2+ 2+ 2+ 2+ + 3+ 2+ 3+ 2+ ions +,, Cu including KCu Cu, 2+Mg Mg,2+Mn Mn2+ Ni2+, ,,Ag Ag+, ,,Fe Fe3+,,, Fe Fe2+,,, Al Al3+ were added to solutions solutions of of including K ,K , Ni and Co Co2+ ions were were added added to including ,, Mg ,, Mn ,, Ni Ag Fe Fe Al ,, and ions to solutions ++ at pH 4.0, 7.6, or 11.3, and, only insignificant changes of fluorescence intensity were + probe A or B of probe or B B at atpH pH4.0, 4.0,7.6, 7.6,oror11.3, 11.3,and, and,only only insignificant changes fluorescence intensity probe A A or insignificant changes of of fluorescence intensity were 2−, NO 3−, SO34− 2−, SO32− 2−, HCO − ,−−,Br − , 2− 2−3− 2− 2− −2−and 2− detected, Figure 6. Further, Further, common anions such as as Br NO were detected, Figure 6. Further, common anions such NO , ,NO ,, SO , detected, Figure 6. common anions such as II−−,, IBr , NO , NO SO42− SO43 2− ,, ,SSSO , HCO 3 , S33−,, and 2− ions also display + under pH 4.0,+ 7.6 − 2− ions also 2− + CO 3 little influence on the fluorescence intensity of probe A or B HCO , and CO display little influence on the fluorescence intensity of probe A or B CO3 3 ions also3display little influence on the fluorescence intensity of probe A or B under pH 4.0, 7.6 or 11.3, 11.3, Figure 7.orFinally, Finally, amino7.acids acids andamino nucleophilic biothiols such as as leucine, leucine, methionine, alanine, under pH Figure 4.0, 7.67. 11.3, Figure Finally, acids and nucleophilic biothiolsmethionine, such as leucine, or amino and nucleophilic biothiols such alanine, proline, arginine, arginine, threonine, glycine, cysteine,glycine, and glutathione glutathione have small effects effects on fluorescence methionine, alanine, proline, arginine, threonine, cysteine, and glutathione haveon small effects proline, threonine, glycine, cysteine, and have small fluorescence responses at pH levels of 4.0, 7.6 or 11.3, Figure 8. on responses fluorescence responses at pH levels of 4.0, 7.6 or 11.3, Figure 8. at pH levels of 4.0, 7.6 or 11.3, Figure 8.
Fluorescence Fluorescence Intensity Intensity
3.0x1055 3.0x10 2.5x1055 2.5x10
550ex -- pH4.0 pH4.0 550ex 550ex -- pH7.6 pH7.6 550ex
Probe A A Probe Fluorescence Fluorescence Intensity Intensity
450ex-pH4.0 450ex-pH4.0 450ex-pH11.3 450ex-pH11.3
Probe B B++ Probe
6.0x1066 6.0x10
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5.0x1044 5.0x10 0.0 0.0
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l3+ k g22+ a22+ o22+ u2+ n22+ Ni2i + Znn2+ Agg++ K K+ Fee2+ Fee3+ A ank Mg Ca Co Cu Mn N F F Al Z A Bllan M C C C M B
2 2 2 3 2 + l33+ k g22+ a22+ o22+ u2+ n22+ Ni2i + Znn2+ Agg++ K K+ Fee2+ Fee3+ A ank Mg Ca Co Cu Mn N F F Al Z A Bllan M C C C M B
(a) (a)
(b) (b)
Probe B B++ Probe
450ex-pH4.0 450ex-pH4.0 450ex-pH10.8 450ex-pH10.8
3.0x1055 3.0x10
550ex -- pH4.0 pH4.0 550ex 550ex -- pH7.6 pH7.6 550ex
Probe A A Probe Fluorescence Fluorescence Intensity Intensity
Fluorescence Fluorescence Intensity Intensity
Figure 6. Fluorescence Fluorescence intensity of probes probes A and and B++ the in the the absence and presence of µM 50 µM µM different Figure 6. intensity of A B in absence presence of 50 different Figure 6. Fluorescence intensity of probes A and B+ in absence andand presence of 50 different + (a) and under 550 nm excitation + + metal ions at pH 4.0 and 11.3 under 450 nm excitation for probe B metal at 4.0 pHand 4.0 and under 450excitation nm excitation for probe (a)under and under nm excitation metal ionsions at pH 11.3 11.3 under 450 nm for probe B (a)Band 550 nm550 excitation for forA probe A (b). (b). for probe probe (b). A
6.0x1066 6.0x10
2.5x1055 2.5x10 2.0x1055 2.0x10
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22- 22--22-22O22 O44 S O33- SO O33 SO O33 CO O33 NO NO CO S S N HC S N C H
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0.0 0.0 k ank Bllan B
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Brr B
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O22 O33NO NO N N
22- 22-22-22O44 S O33 SO O33 CO O33 SO CO S S HC S C H
(b) (b)
Figure 7. Fluorescence intensity of probes A and B+ in absence andand presence of 50 different Figure 7. Fluorescence Fluorescence intensity of probes probes A and and B++ the in the the absence and presence of µM 50 µM µM different Figure 7. intensity of A B in absence presence of 50 different anions at pH 4.0 4.0 and 11.311.3 under excitation of 450 nm nm for probe B+ (a) andand under 550550 nmnm excitation for for anions at pH pH 4.0 and and 11.3 under excitation of 450 450 nm for probe probe B++ (a) (a) and under 550 nm excitation for anions at under excitation of for B under excitation probe A (b). probe A (b). probe A (b).
pH4.0 - 450ex pH10.8 -450ex
Pr obe B +
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s et s k u H ro la yr rg lan L-Le5 L-Cy L-M Gly L-P L-A L-T L-A L-Cy GS B2.0x10 D D D D D D D
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nk eu ys et ly Pro Ala Tyr Arg ys SH Bla DL-L DL-C DL-M G DL- DL- DL- DL- L-C G
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Figure 8. Fluorescence intensity of probes A and absence presence of µM 50 µM different Figure 8. Fluorescence intensity of probes A and B+ in thethe absence andand presence of 50 different 5.0x10 ++ (a) amino acid and biothiols at pH 4.0 and 11.3 under excitation of 450 nm for probe B and under 550 0.0 amino acid and biothiols at pH 4.0 and 11.3 under excitation of 450 nm for probe B (a) and under 0.0 nk eu ys et ly Pro Ala Tyr Arg ys SH s et s k u H ro la yr rg Bla DL-L DL-C DL-M G DL- DL- DL- DL- L-C G lan L-Le L-Cy L-M Gly L-P L-A L-T L-A L-Cy GS B D D DA D D probe 550nm nmexcitation excitationDfor for probe A(b). (b). D 4
2.4.2.4. Theoretical Results Theoretical Results
(a)
(b)
Figure 8. Fluorescence intensity of probes A and B in the absence and presence of 50 µM different +
TheThe drawings andand data in Figure 9 summarize pertinent results calculations based amino acid biothiols atin pHFigure 4.0 and 11.3 under excitation of 450 nm forfrom probe theoretical B+ (a) and under 550 drawings and data 9 summarize pertinent results from theoretical calculations nm excitation for probe A (b). on based density functional theory DFT-APFD [36], at the 6-311+G(2d, p) [37–39] level implemented using on density functional theory DFT-APFD [36], at the 6-311+G(2d, p) [37–39] level implemented Gaussian 16 [40]. We16find reasonable with the experimental calculated transitions for using Gaussian [40]. We findagreement reasonable agreement with theand experimental and calculated 2.4. Theoretical Results thetransitions probes as listed in Figure 9. The results suggest that the transition for probe A consists of electron for drawings the probes as listed in Figure 9. The results suggest that the transition A The and data in Figure 9 summarize pertinent results from theoretical calculations for probe movement from the diethylamino moiety onto the[36], spirolactam sectionp) of the molecule. With probe AH+ consistsbased of electron from the diethylamino moiety onto the spirolactam on densitymovement functional theory DFT-APFD at the 6-311+G(2d, [37–39] level implementedsection of the which does not contain spirolactam ring, eventhe π-delocalization pertains, by the lack + which using Gaussian 16AH [40]. We find reasonable agreement with the experimental and judging calculated molecule. With probethe does notmore contain spirolactam ring, more even π-delocalization transitions for the probesalso as listed in Figure 9. The results suggest thatmoiety, the transition for probe Aand occurs of intense blue. The transition emanates from the diethylamino namely ES 1, pertains, judging by the lack of intense blue. The transition also emanates from the diethylamino of electron movement fromspirolactam the diethylamino ontoathe section thewith 99.5% at 513 nm.consists Probe B+ES which contains the ring+moiety revealed ESspirolactam transition at 580 ofnm moiety, namely and occurs atdoes 513not nm. Probe which contains ring revealed a + which molecule. With 1, probe AH contain theB spirolactam ring, morethe evenspirolactam π-delocalization + 2+ results in percent localization on the cyanine moiety. Protonation of probe B to produce probe BH ES transition 580 nm 99.5% percent cyanine moiety. Protonation of probe pertains,atjudging by with the lack of intense blue.localization The transitionon alsothe emanates from the diethylamino + which three ESproduce transitions of suitable oscillator strength toBbe considered, see Table S8.strength Therevealed transition ES 2 at moiety, namely ES occursinatthree 513 nm. contains the spirolactam ring a considered, B+ to probe BH1,2+and results ESProbe transitions of suitable oscillator to be ESatransition at 580 on nm π with 99.5% localizationon on the moiety. Protonation of probe 585see nmTable has 94.5% toES π*2orbitals localized thecyanine cyanine moiety the rest (i.e., 4.8%) S8. Thebasis transition atpercent 585 nm has a 94.5% basis on π to π*with orbitals localized on the + to produce probe BH2+ results in three ES transitions of suitable oscillator strength to be considered, B from π to π* orbitals localized on the rhodamine moiety. This situation is reversed with the ES 3 cyaninesee moiety with rest (i.e., π ato94.5% π* orbitals on the rhodamine Table S8. Thethe transition ES 24.8%) at 585from nm has basis onlocalized π to π* orbitals localized on the moiety. This transition with a 92.5% composition from the rhodamine sections and 4.7% from the cyanine. This is situation is reversed with transition with a 92.5% composition from the rhodamine sections cyanine moiety with thethe rest ES (i.e.,34.8%) from π to π* orbitals localized on the rhodamine moiety. This 2+ that are colored light blue in Figure 9. Clearly in an clearly observable in the sections of probe BH 2+ situation is reversed with the ES 3 transition with a 92.5% composition from the rhodamine sections and 4.7% from the cyanine. This is clearly observable in the sections of probe BH that are colored and 4.7% from theabsorption cyanine. Thisspectrum, clearly observable in the sectionswould of probeoverlap BH2+ thatand are colored experimentally obtained these contributions resultcontributions in a broad light blue in Figure 9. Clearly in anisexperimentally obtained absorption spectrum, these light bluenm in Figure 9.664 Clearly in an experimentally obtained absorption spectrum, these consisting contributionsof a lower transition at 550 (expt nm), see Figure S28. A higher energy transition would would overlap andand result ininaabroad transition at 550 nm 664 (expt 664 overlap result broad transition at 550 nm (expt nm), see nm), Figuresee S28.Figure A higherS28. A higher energy π-delocalize (i.e., HOMO-3) orbital to the LUMO localized on the rhodamine section energy energy transition consisting energy π-delocalize (i.e., HOMO-3) orbital tooccurring the LUMO transition consistingofof aa lower lower energy π-delocalize (i.e., HOMO-3) orbital to the LUMO at 398 nm (expt 413 nm) is also calculated. The results of this calculation do not reveal any localized onrhodamine the rhodaminesection section occurring at 398 nm (expt 413 nm) is also The resultstransitions localized on the occurring at 398 nm (expt 413 nm)calculated. is also calculated. The results 2+ . Thistoadds of this calculation do not reveal any transitions from theBH rhodamine the cyanine moieties probe from the rhodamine to the cyanine moieties in probe credence to in the likelihood of of this calculation do not reveal any transitions from the rhodamine to the cyanine moieties in probe BH2+. as This adds credence to of thethe likelihood of FRET transfer as an explanation of the aforementioned FRET transfer an explanation aforementioned absorption and fluorescence data. 2+ BH . This adds credence to the likelihood of FRET transfer as an explanation of the aforementioned absorption and fluorescence data.
absorption and fluorescence data.
A-ES3, 308 (300) nm 127 → 130 6.2% 128 → 130 88.0%
A-ES3, 308 (300) nm 127 → 130 6.2% 128 → 130 88.0%
AH+-ES1, 513 (591) nm 128 → 129 98.7%
AH -ES1, Figure 9. 513 Cont.(591) nm 128 → 129 98.7% +
B+-ES1, 580 (664) nm 264 → 265 99.5%
B+-ES1, 580 (664) nm 264 → 265 99.5%
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BH2+-ES2, 585 (664) nm 263 → 265 4.8% 2+-ES2, 264 →BH 266 94.5% 585 (664) nm 263 → 265 4.8%
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BH2+-ES3, 513 (664) nm 263 → 265 92.5% 2642+-ES3, → 266 4.7%nm BH 513 (664) 263 → 265 92.5%
BH2+-ES7, 398 (413) nm 261 → 265 93.6% BH2+-ES7, 398 (413) nm 261 → 265 93.6%
Figure 9. Current density difference illustrations as iso-surfaces of the probes as indicated for the 264 → 266 4.7% 264 → 266 94.5% excited states (ES) and the calculated and (experimental) wavelength. The composition of that specific Figure 9. Current density difference illustrations as iso-surfaces of the probes as indicated for the ES together withdensity percentage contribution is also indicated. of Drawings ofasthe numbered linear Figure 9. Current difference illustrations as iso-surfaces the probes indicated for the excited excited states (ES) and the calculated and (experimental) wavelength. The composition of that specific combination of atomic orbitals (LCAOs) are available in Supporting Information. Red areas indicate states (ES)ESand the calculated and (experimental) wavelength. The composition of that specific ES together with percentage contribution is also indicated. Drawings of the numbered linear −5 and blue are for 5.000 × 10−5, see scale on top of values for thepercentage different density of (LCAOs) −5.000 ×are10 together with contribution is also indicated. of the numbered linear combination combination of atomic orbitals available inDrawings Supporting Information. Red areas indicate values for(LCAOs) the different of −5.000 × 10−5 and blue are for 5.000Red × 10−5areas , see scale on top of illustration. of atomic orbitals aredensity available in Supporting Information. indicate values for the illustration. different density of −5.000 × 10−5 and blue are for 5.000 × 10−5 , see scale on top of illustration.
2.5. Cellular Fluorescence Imaging 2.5. Cellular Fluorescence Imaging 2.5. Cellular Fluorescence Imaging A confocal fluorescence microscope was used cellular fluorescence imaging A confocal fluorescence microscope was usedtotoconduct conduct cellular fluorescence imaging of HeLaof HeLa +. The + increases A confocal microscope was used to intensity conduct cellular imaging of HeLa incubated with fluorescence probe B+. The cellular fluorescence ofprobe probe B+ increases with probe incubated with probe B cellular fluorescence intensity of Bfluorescence with probe +1.μM + increases concentration from to 10cellular μM as deep-red fluorescence from nm to 650 nm,Bnm, and near-infrared incubated with probe Bto fluorescence intensity ofnm probe with probe concentration from 1 μM 10The μM as deep-red fluorescence from600 600 to 650 and near-infrared fluorescence nm tonm 775 nm can clearly observed from under rhodamine 440 near-infrared nm fluorescence from nm725 775 be be clearly observed under rhodamine at 440 nm concentration from725 1from µM toto10 µM as can deep-red fluorescence 600 nm toexcitation 650excitation nm,atand with 10-μM concentration level of probe+ B+, Figure 10. Strong cellular near-infrared fluorescence can with 10-μM concentration ofnm probe , Figure Strong cellular fluorescence cannm fluorescence from 725 nm level to 775 canBbe clearly10. observed under near-infrared rhodamine excitation at 440 be observed under cyanine acceptor excitation at 635 nm with 1 μM concentration of probe B+ due to + due to + , Figure be observed under cyanine acceptor excitation at 635 nm with 1 μM concentration of probe B + with 10-µM concentration level of probe B 10. Strong cellular near-infrared fluorescence can high fluorescence quantum yield under acidic pH condition. These results demonstrate that probe B + + high fluorescence quantum yield under acidic pH condition. These results demonstrate that probe B effectively stain the cells. We also conducted a colocalizaton imaging experiment of probe B with be observed under cyanine acceptor excitation at 635 nm with 1 µM concentration of probe B due to Lysosensor Green HeLa, andconducted obtained a high Pearson collocalization of 0.93 effectively stain the cells.inWe also a pH colocalizaton experiment ofbetween probethat B with high fluorescence quantum yield under acidic condition.imaging These coefficient results demonstrate probe probe B and Lysosensor Green, indicating that probe B is located in lysosomes in HeLa cells, Figure + Green in HeLa, and obtained a high Pearson collocalization coefficient of 0.93 between BLysosensor effectively stain the cells. We also conducted a colocalizaton imaging experiment of probe B with 11. probe B and Lysosensor Green, indicating that probe B is located in lysosomes in HeLa cells, Figure Lysosensor Green in HeLa, and obtained a high Pearson collocalization coefficient of 0.93 between 11. B and Lysosensor Green, indicating that probe B is located in lysosomes in HeLa cells, Figure 11. probe
Figure 10. Live-cell fluorescence images of HeLa cells incubated with probe B+ for 2 h and then with Hoechst stain for 1 h with scale bars at 20 μm. The excitation of Hoechst is 405 nm and the fluorescence collect window is from 425 to 475 nm.
+ for Figure 10. 10. Live-cell Live-cell fluorescence with probe B+ Bfor 2 h2and then with Figure fluorescenceimages imagesofofHeLa HeLacells cellsincubated incubated with probe h and then with Hoechst stain for 1 h with scale bars at 20 μm. The excitation of Hoechst is 405 nm and the fluorescence Hoechst stain for 1 h with scale bars at 20 µm. The excitation of Hoechst is 405 nm and the fluorescence collect window window is is from collect from 425 425 to to 475 475nm. nm.
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Figure B++ for Figure 11. 11. Live-cell Live-cell fluorescence fluorescence images images of of HeLa HeLa cells cells incubated incubated with with probe probe B for 22 hh and and then then with with Figure 11. Live-cell fluorescence images of HeLa cells incubated with probe B+ for 2 h and then with Lysosensor Green for 1 h with scale bars at 20 μm. The excitation of Lysosensor green is 405 Lysosensor Green for 1 h with scale bars at 20 µm. The excitation of Lysosensor green is 405 nm nm and and Lysosensor Green for 1 h with scale bars at 20 μm. The excitation of Lysosensor green is 405 nm and the to 550 nm. the fluorescence fluorescence collect collect window window is is from from 500 500 to 550 nm. the fluorescence collect window is from 500 to 550 nm.
Probe weak base base bearing bearing an an amine amine residue, residue, should should function lysosome-targeting Probe A A as as aa weak function as as aa lysosome-targeting Probe A as a weak base bearing an amine residue, should function as a lysosome-targeting imaging agent to selectively stain lysosomes in in live live cells. cells. In order to test this hypothesis, imaging agent to selectively stain lysosomes In order to test this hypothesis, we we imaging agent to selectively stain lysosomes in live cells. In order to test this hypothesis, we conducted colocalization experiments by using commercial Lysosensor Green to determine the conducted by using using commercial commercialLysosensor LysosensorGreen Greentotodetermine determine conducted colocalization colocalization experiments experiments by thethe intracellular location of probe A. Intracelluar pH values were adjusted by incubating HeLa cells intracellular location of probe A. Intracelluar pH values were adjusted by incubating HeLa intracellular location of probe A. Intracelluar pH values were adjusted by incubating HeLa cells cells in in + +ionphore, which is employed to promote different pH buffers containing 10 µM nigericine, H indifferent different pH buffers containing 10 µM nigericine, H ionphore, which is employed to promote pH buffers containing 10 µM nigericine, H+ ionphore, which is employed to promote equilibration pH values [7,9,29,41,42]. [7,9,29,41,42].Probe Probe responds equilibration between intracellular and extracellular pH pH values values [7,9,29,41,42]. Probe A responds equilibrationbetween between intracellular intracellular and and extracellular extracellular AA responds sensitively to intracellular pH decreases from 7.01 to 3.50 with gradual fluorescence enhancement sensitively to intracellular pH decreases from 7.01 to 3.50 with gradual fluorescence enhancement sensitively to intracellular pH decreases from 7.01 to 3.50 with gradual fluorescence enhancement due with significantly enhanced π-conjugation, Figure due totoacid-activated acid-activated spirolactam ringopening openingwith withsignificantly significantlyenhanced enhanced π-conjugation, Figure 12. dueto acid-activatedspirolactam spirolactam ring ring opening π-conjugation, Figure 12.12. The respective Pearson’s colocalization coefficents between Lysosensor Green andprobe probe were The Pearson’s colocalization coefficents between Lysosensor Green andand probe A were higher Therespective respective Pearson’s colocalization coefficents between Lysosensor Green AA were higher than 0.86 under acidic 3.50 or 4.03,13, Figure 13, indicating indicating that probeAAselectively selectively stains than 0.86 under acidic pH 3.50 pH or indicating that probe A probe selectively stains lysosomes higher than 0.86 under acidic pH4.03, 3.50Figure Figure 13, that stains lysosomes in live cells with pH-sensitive responses while Lysosensor Green is insensitive to in livepH-sensitive cells with pH-sensitive responses while Lysosensor Green is insensitive to pHpH inlysosomes live cells with responses while Lysosensor Green is insensitive to pH changes, see changes, Figure The plot average fluorescence fluorescence intensities ofprobe probe Ainin HeLa cells versus changes, see Figure 12.the The plotof of the the intensities HeLa cells versus Figure 12.see The plot 12. of average fluorescence intensities of probe Aofin HeLaAcells versus pH gave a pH gavea aof pKa valueof of5.20. 5.20. pH pKa value pK value 5.20. agave
Figure 12. Confocal fluorescence images of HeLa cells incubated with probe A and Lysosensor Green Figure 12. Confocal fluorescence images of HeLa cells incubated with probe A and Lysosensor Green in Figure 12. Confocal fluorescence images of HeLa cells incubated andThe Lysosensor in different pH buffers containing 10 µM nigericine with scalewith barsprobe at 50 A μm. excitationGreen of different pH buffers containing 10 µM nigericine with scale bars at 50 µm. The excitation of Lysosensor inLysosensor different pH buffers 10 µM nigericine scale bars 500 at 50 μm.nm. The excitation of green is 405 containing nm and the fluorescence collect with window is from to 550 green is 405 nm and the fluorescence collect window is from 500 to 550 nm. Lysosensor green is 405 nm and the fluorescence collect window is from 500 to 550 nm.
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Figure 13.Enlarged Enlargedconfocal confocal fluorescence images of cells HeLaincubated cells incubated with probe A and Figure 13. fluorescence images of HeLa with probe A and Lysosensor Lysosensor Green in pHconfocal 3.50 buffer containing 10 µM nigericine Figurewith 12. probe A and Figure 13. Enlarged fluorescence images of HeLa cellsfrom incubated Green in pH 3.50 buffer containing 10 µM nigericine from Figure 12. Lysosensor Green in pH 3.50 buffer containing 10 µM nigericine from Figure 12.
We also investigated investigated whether whether probe probe B B++ could detect intracellular intracellular pH changes with with We also could be be used used to to detect pH changes + could be used to detect + in + We also investigated whether probe B intracellular pH changes with double-checked feature by incubating HeLa cells with probe B different pH buffers containing 10 double-checked feature by incubating HeLa cells with probe B in different pH buffers containing + in different pH buffers containing 10 double-checked feature by incubating HeLa cells with probe B µMµM nigericine, Figure 14.14. Gradual decreases ofofintracellular cause 10 nigericine, Figure Gradual decreases intracellularpH pHvalues valuesfrom from9.00 9.00 to to 3.01 3.01 cause µM nigericine, Figure 14. Gradualindecreases of intracellular pH values from 9.00 in to 3.01 cause increases increases of of deep-red deep-red fluorescence fluorescence in channel channel 11 and and near-infrared near-infrared fluorescence fluorescence in channel channel 22 under under increases of deep-red fluorescence in channel 1 and near-infrared fluorescence in channel 2 under rhodamine donor excitation excitationat at440 440nm. nm. In In addition, addition,intracellular intracellularnear-infrared near-infraredfluorescence fluorescenceininchannel channel3 rhodamine donor rhodamine donor excitation at 440 nm. In addition, intracellular near-infrared fluorescence in channel 3under under cyanine excitation nm also increases withgradual the same decreases, gradual pH decreases, cyanine excitation at 635at nm635 alsonm increases with thewith same demonstrating 3 under cyanine excitation at 635 also increases the same pH gradual pH decreases, + can sensitively monitor intracellular pH changes. + demonstrating that probe B thatdemonstrating probe B canthat sensitively intracellular changes.pH changes. probe B+ monitor can sensitively monitorpH intracellular
Figure 14. Confocal fluorescence images of HeLa cells incubated with probe B+ in different pH buffers
Figure 14. Confocal fluorescence images of HeLa cells incubated with probe B+ in different pH buffers containing 10 µM nigericine with scare bars at 50 μm. containing 10 µM nigericine with scare bars at 50 µm.
Figure 14. Confocal fluorescence images of HeLa cells incubated with probe B+ in different pH buffers containing 10 µM nigericine with scare bars at 50 μm.
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3. Discussion Fluorescence imaging with molecular fluorescent probes serves as an important tool in biological and medical research. Near-infrared imaging provides many advantages with prolonged fluorescence with less photobleaching problems, deep tissue penetration, and low interference from biological fluorescence backgrounds. Fluorescent probe A was measured to have a high pKa value of 5.15 corresponding to spirolactam ring opening although only 1,2-diaminoethylene was used to modify the deep-red rhodamine dye. Probe A effectively targets lysosomes in live cells and can detect pH changes in lysosomes with a very suitable pKa value since lysosomes are membrane-encased organelles with optimal pH from 4.5 to 5.0 for the enzyme activity in hydrolysis to degrade biological species, Figure 12. Compared with probe A, a commercial lysosensor is insensitive to pH changes although it can specifically target lysosomes in live cells. We further employed deep-red rhodamine as a FRET donor and near-infrared cyanine as a FRET acceptor to detect intracellular pH changes in live cells with a double-checked feature. Since the emission of the rhodamine donor has significant overlap with the absorption of the cyanine acceptor, efficient energy transfer from the rhodamine donor to the cyanine acceptor occurs through a very short ethylene spacer under acidic pH conditions. Probe B+ containing the cyanine and rhodamine moieties, shows corresponding fluorescence increases with pH decreases to achieve the double-checked capability. Finally, we have developed ratiometric near-infrared fluorescent probes by introducing a spirolactam on/off switch to a cyanine acceptor which when activated results in a deep-red rhodamine donor connected to a cyanine acceptor through an ester bond instead of a spirolactam ring with an amide bond. 4. Materials and Methods 4.1. Synthesis of Probe A After compound 3 [43] (439 mg, 1 mmol), ethylenediamine (180 mg, 3 mmol), BOP reagent (530 mg, 1.2 mmol) and trimethylamine (1 mL) were added to dry dichloromethane (10 mL), the mixture was stirred at room temperature for 16 h. The mixture was diluted with dichloromethane, washed with water and brine, dried with anhydrous Na2 SO4 , and filtered, and the filtrant concentrated under reduced pressure. The resulting residue was purified by using flash column chromatography through gradient elution with methanol ratio to dichloromethane from 5% to 10%. Probe A was obtained as blue solid. 1 HNMR (300 MHz, CDCl3 ) δ: 7.78 (d, J = 7.2 Hz, 1H), 7.59 (d, J = 8.2 Hz, 1H), 7.38 (p, J = 7.2 Hz, 2H), 7.09 (d, J = 7.1 Hz, 1H), 6.55 (d, J = 8.2 Hz, 1H), 6.45–6.28 (m, 3H), 6.23 (d, J = 8.7 Hz, 1H), 4.02 (s, 4H), 3.43–3.65 (m, 1H), 3.25–3.30 (m, 5H), 2.78–2.56 (m, 2H), 2.46–2.56 (m, 2H), 1.73–1.41 (m, 2H), 1.11 (t, J = 6.9 Hz, 6H); 13CNMR (75 MHz, CDCl3) δ: 169.61, 152.90, 151.57, 148.86, 147.50, 138.31, 132.71, 131.45, 128.55, 128.49, 123.71, 123.63, 123.03, 120.14, 114.21, 112.68, 108.95, 104.77, 100.44, 98.06, 67.20, 44.57, 42.05, 41.18, 28.52, 21.40, 12.93. LCMS (ESI): calculated for C30 H32 N4 O2 [M]+ 481.2, found 481.5. 4.2. Synthesis of Probe B+ Probe A (240 mg, 0.5 mmol), cyanine dye (IR-780) (400 mg, 0.6 mmol), N,N-diisopropylethylamine (DIPEA) (129 mg, 1 mmol) were added to acetonitrile (10 mL). The mixture was refluxed for 2 h and the reaction solution concentrated under reduced pressure. The resulting residue was purified by using flash column chromatography through gradient elution with methanol ratio to dichloromethane from 0% to 5%. Probe B+ was obtained as blue solid. 1HNMR (300 MHz, CDCl3 ) δ: 9.28 (s, 2H), 7.83 (d, J = 7.5 Hz, 2H), 7.65–7.47 (m, 5H), 7.18–7.31 (m, 5H), 7.06 (t, J = 7.5 Hz, 2H), 6.87 (d, J = 7.8 Hz, 2H), 6.60 (d, J = 8.2 Hz, 1H), 6.54 (s, 1H), 6.44–6.24 (m, 2H), 5.60 (d, J = 12.7 Hz, 2H), 3.95–4.19 (m, 4H), 3.84–3.69 (m, 4H), 3.65–3.51 (m, 2H), 3.50–3.40 (m, 2H), 3.34 (t, J = 7.4 Hz, 4H), 2.65–2.52 (m, 2H), 2.52–2.39 (m, 4H), 1.84–1.71 (m, 6H), 1.54 (d, J = 10.4 Hz, 10H), 1.14 (t, J = 7.0 Hz, 6H), 0.98 (t, J = 7.4 Hz, 6H); 13CNMR (75 MHz, CDCl3) δ: 170.89, 167.51, 153.27, 149.07, 148.07, 143.25, 140.17, 137.44, 133.54, 129.01, 128.38, 128.24, 124.11, 123.86, 123.01, 122.10, 119.26, 114.15, 112.83, 109.09, 108.97, 98.26, 94.64,
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68.13, 53.02, 47.84, 45.14, 44.71, 41.75, 28.92, 28.82, 26.46, 21.85, 21.43, 20.42, 13.00, 12.11. LCMS (ESI): calculated for C66 H75 N6 O2 [M]+ 983.5, found 983.5. 4.3. Theoretical Calculations Chemdraw structures of probes A, AH+ , B+ and BH2+ were optimized initially with the MM2 functionality in Chem3D and then further with Avogadro [44,45]. Calculations were then conducted using density functional theory (DFT) with spherical atom dispersion terms, namely APFD [36], with all electron basis sets at the 6-311+G(2d, p) [37–39] level implemented using the Gaussian16 suite of programs [40] for the full geometry optimization and frequency calculations of the probes. Imaginary frequencies were not obtained in any of the frequency calculations. The first six excited states (more if required) were assessed on the basis of TD-DFT optimizations [46] in a Polarizable Continuum Model (PCM) of water [47]. Results were interpreted using GaussView [48] for all data and figures. The diagrams and listings of atomic positions from the calculations, calculated IR and NMR spectra in some cases, listings of excited states with drawings of referenced LCAOs are supplied as supporting information. 4.4. Cell Culture and Cell Imaging Procedures HeLa cells were cultured in modified Eagle’s medium (DMEM, Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, fisher Scientific, Hampton, NH, USA) at 37 ◦ C in humidified air with 5% CO2 . HeLa cells were subcultured with 0.25% trypsin (w/v) every 2–3 day reached at 80% confluence. For confocal live cell imaging, HeLa cells were seeded into the 35 nm glass-bottom culture dishes (MatTek, Ashland, MA, USA) and allowed 1 day to reach 50% confluence. After 24 h of incubation, the cell culture medium was replaced by freshly prepared FBS-free medium with 1, 5, and 10 µM of probe B for 1 h 37 ◦ C under 5% CO2 followed by using PBS buffer to rinse three times. For the live cell fluorescence imaging at different pH, the HeLa cells were treated with 10 µM probe A or B 37 ◦ C under 5% CO2 for 1 h. The cells were rinsed with PBS buffer twice before they were treated with 10 µM nigericin in citric buffer with pH values at 3.50, 4.03, 4.54, 5.00, 5.52, 6.02, 6.51, 7.01 for probe A and 3.01, 4.54, 6.02, 7.51, 9.00 for probe B, respectively, for 30 min to equilibrate the intracellular and extracellular pH for 30 min. The cells were rinsed with PBS buffer twice again before imaging. 5. Conclusions A new FRET-based near-infrared fluorescent probe (B+ ) for pH sensing with double-checked feature was successfully prepared by conjugating a near-infrared rhodamine donor to a cyanine acceptor via a robust C-N bond connection with a short ethylene tethered spacer. Probe A was also prepared by introducing 1,2-diaminoethylene to rhodamine forming a closed spirolactam ring structure. Probe B+ responds to pH decreases with fluorescence increases of both deep-red fluorescence of rhodamine donors and near-infrared fluorescence of the cyanine acceptor under rhodamine donor excitation at 450 nm. Supplementary Materials: The following are available online. Author Contributions: Conceptualization, H.L.; Methodology, S.X. and L.M.; Computation Chemistry, R.L.L.; Validation, S.X., J.B., and W.M.; Formal Analysis, J.B.; Investigation, S.X., J.B., S.W. and L.M.; Resources, H.L.; Data Curation, S.X. and J.B.; Writing—Original Draft Preparation, W.M.; Writing—Review & Editing, H.L. and R.L.L.; Visualization, S.X.; Supervision, H.L.; Project Administration, H.L.; Funding Acquisition, H.L. Funding: This research was funded by the National Institute of General Medical Sciences of the National Institutes of Health with grant number R15GM114751. Acknowledgments: Funding from the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM114751 (to H.Y.L.) is gratefully acknowledged. The first-principles calculations were computed using RAMA and Superior, the high performance-computing cluster at Michigan Technological University.
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Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are all available from the authors. © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).