Asymmetric Cationic Porphyrin as a New G-Quadruplex Probe with

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Biological and Medical Applications of Materials and Interfaces

An asymmetric cationic porphyrin as a new G-quadruplex probe with wash-free cancer-targeted imaging ability under acidic microenvironments Ran Zhang, Meng Cheng, Li-Ming Zhang, Li-Na Zhu, and Deming Kong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01901 • Publication Date (Web): 05 Apr 2018 Downloaded from http://pubs.acs.org on April 5, 2018

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ACS Applied Materials & Interfaces

An asymmetric cationic porphyrin as a new G-quadruplex probe with wash-free cancer-targeted imaging ability under acidic microenvironments

Ran Zhang,a,b Meng Cheng,a,b Li-Ming Zhang,a,b Li-Na Zhu*a,b and De-Ming Kongb,c a

State Key Laboratory of Medicinal Chemical Biology, Department of Chemisitry,

School of Science, Tianjin University, Tianjin, 30072, P R China b

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin),

Tianjin, 30072, P R China c

Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of

Chemistry, Nankai University, Tianjin, 300071, P R China

ACS Paragon Plus Environment

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ABSTRACT Porphyrins are promising candidates for nucleic acid G-quadruplex-specific optical recognition. We previously demonstrated that G-quadruplex recognition specificity of porphyrins could be improved by introducing bulky side arm substituents, but enhanced protonation tendency limits their applications in some cases, such as under acidic conditions. Here, we demonstrated that the protonation tendency of porphyrin derivatives could be efficiently overcome by increasing molecular asymmetry. To validate this, an asymmetric, water-soluble, cationic porphyrin

FA-TMPipEOPP

(5-{4-[2-[[(2E)-3-[3-methoxy-4-[2-(1-methyl-1-

piperidinyl)ethoxy]phenyl]-1-oxo-2-propenyl]oxy]ethoxy]phenyl},10,15,20-Tri{4[2-(1-methyl-1-piperidinyl)ethoxy]-

phenyl}porphyrin)

was

synthesized

by

introducing a ferulic acid (FA) unit at one side arm, and its structure was well characterized. Unlike its symmetric counterpart TMPipEOPP that has a tendency to protonation under acidic conditions, FA-TMPipEOPP remained in unprotonated monomeric form under the pH range of 2.0–8.0. Correspondingly, FA-TMPipEOPP showed better G-quadruplex recognition specificity than TMPipEOPP showed, and thus might be used as a specific optical probe for colorimetric and fluorescent recognition of G-quadruplexes under acidic conditions. The feasibility was demonstrated by two proof-of-concept studies: probing structural competition between G-quadruplex and duplex, and label-free and wash-free cancer cell-targeted bioimaging under an acidic tumor microenvironment. As G-quadruplex optical probes, FA-TMPipEOPP works well under acidic conditions, while TMPipEOPP works well


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under neutral conditions. This finding provides useful information for G-quadruplex probe research. That is, porphyrin-based G-quadruplex probes suitable for different pH conditions might be obtained by adjusting the molecular symmetry. KEYWORDS: asymmetric porphyrin, G-quadruplex, acidic microenvironment, optical probe, cancer cell-targeted imaging

INTRODUCTION G-quadruplex is a non-conventional DNA secondary structure that can be formed in many important regions of the human genome (e.g. telomere and promoters of many oncogenes). It has attracted attention in many fields including anticancer drug research, biosensor design and targeted delivery of drugs.1–4 Over the last 20 years, hundreds of small molecule ligands, such as telomestatin,5 triphenylmethane,6,7 tetraphenylethylene,8

porphyrin,9–11

funtumineguanyl

hydrazone,12

peimine,13

squaraine14 and metal complex,15,16 have been reported as candidates of G-quadruplex-targeted anticancer drugs or optical G-quadruplex probes. To achieve the best performance, a basic requirement for these ligands is the high G-quadruplex recognition specificity over other DNA structures.17,18 Although some of them are reported to show excellent G-quadruplex recognition specificity under neutral and near neutral pH conditions, highly specific G-quadruplex recognition ligands that can work well under acidic pH conditions are rarely reported.19,20 pH is an important biological parameter, and pH fluctuation is related to initiation and development of some diseases. Well-known examples are that tumor usually has an acidic



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microenvironment in the pH range from 5.5 to 7.0,21,22 and intracellular acidic organelles, such as lysosomes (pH 4.5–5.5) and endosomes (pH 5.4–6.2) play critical roles in endocytic and digestive processes.23,24 Therefore, it is conceivable that specific G-quadruplex recognition under acidic conditions can be used to guide the development of G-quadruplex-targeted drugs, and might be beneficial for understanding the biological roles of G-quadruplex formation in some diseases. Pronounced similarity to G-quartet planar structure, combined with excellent optical and photovoltaic properties,25–29 makes porphyrins excellent candidates for G-quadruplex ligands. However, most of the reported porphyrin-based G-quadruplex ligands (e.g. TMPyP4, Scheme 1) have the drawback of poor G-quadruplex recognition specificity against double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA). Our group demonstrated that the G-quadruplex recognition specificity of porphyrins could be improved by introducing bulky side arm substituents,9–11,30,31 and

the

prepared

cationic

porphyrin

derivatives

(e.g.

5,10,15,20-tetra-{4-[2-(1-methyl-1-piperidinyl)ethoxy]phenyl}porphyrin, TMPipEOPP, Scheme 1) can optically recognize G-quadruplexes with excellent specificity under physiological pH conditions.10,30 However, such a derivative cannot work well under acidic conditions due to its tendency for protonation.20,31 Herein，to address this issue, we designed a structurally asymmetrical porphyrin derivative (FA-TMPipEOPP, Scheme 1) by inserting a ferulic acid (FA) unit in one of the four side arms of TMPipEOPP. FA is a copious and almost ubiquitous phytochemical phenolic derivative of cinnamic acid, and has attracted wide research


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interest due to its anticancer, antioxidant and UV-protective properties.32–34 The carboxylic and phenolic hydroxyl groups of FA provide a good anchor for the coupling

reactions,

allowing

the

linking

of

the

porphine

ring

and

1-(2-chloroethyl)-piperidine hydrochloride as a bridge. TMPipEOPP tends to be protonated

under

acidic

conditions

and

can

only

give

satisfactory

G-quadruplex-specific recognition with the neutral pH range. In contrast, protonation is rarely observed for FA-TMPipEOPP, even when the pH is decreased to 2.0. More interestingly, even better optical recognition specificity towards G-quadruplex was observed with the pH decrease. As a supplement of TMPipEOPP, FA-TMPipEOPP might be a useful tool for G-quadruplex-related studies under acidic conditions. As proof-of-concept, FA-TMPipEOPP was successfully used in G-quadruplex/duplex competition-probing under different pH conditions. As a label-free fluorescent probe, FA-TMPipEOPP/G-quadruplex complex was demonstrated to work well in wash-free bioimaging of living cells under an acidic tumor microenvironment. N+ O

N+

NH N

NH N +N

N+ N HN

N+

O O

O N HN

O

O

N+

OCH3

N+ O +N

TMPyP4

FA-TMPipEOPP

Scheme 1. Chemical structures of TMPyP4, TMPipEOPP and FA-TMPipEOPP



EXPERIMENTAL SECTION Chemicals and materials. The oligonucleotides (Table S1) were purchased from Sangon Biotech. Co. Ltd. (Shanghai, China). The oligonucleotide concentrations were represented as single-stranded concentrations which were determined by measuring the absorbance at 260 nm. The molar extinction coefficient was calculated using a nearest

neighbor

approximation

(http://www.idtdna.com/analyzer/Applications

/OligoAnalyzer), and the determined molar extinction coefficients of these oligonucleotides were listed in Table S1. N,N-dimethylformamide (DMF), K2CO3, CH3OH, CH2COOCH2CH3, CH2Cl2 and (CH3CH2)3N were obtained from Jiangtian Co. Ltd. (Tianjin, China). Na2EDTA (Disodium ethylenediaminetetraacetic acid), Tris (tris(hydroxymethyl)aminomethane), HCl, KCl and NaCl were obtained from Sigma. All aqueous solutions were prepared using deionized and sterilized water (resistance > 18 MΩ·cm-1). All chemical reagents were of analytical grade and used without further purification. The details of synthesis and characterization of FA-TMPipEOPP are available in Supporting Information (Figure S1-Figure S7). UV-vis absorption spectroscopy. UV-vis absorption spectra were measured on a Cary 60 UV–vis spectrophotometer (Agilent Technologies) with 1cm-path-length microquartz cell (40 µL, Starna Brand, England). Solutions containing 10 µM individual oligonucleotides, 10 mM Tris-HCl buffer, 50 mM KCl, 100 mM NaCl and 1 mM Na2EDTA were prepared. Each solution was heated at 95 °C for 5 min to remove any aggregates, then cooled rapidly to 25 °C and was allowed to incubate at this temperature for 30 min. After overnight incubation at 4 °C, 5 µM


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FA-TMPipEOPP or TMPipEOPP was added, and the absorption spectra in the range of 350–800 nm were recorded. Absorption titration experiments were carried out by varying the DNA concentration and but maintaining the FA-TMPipEOPP concentration at 5 µM. The sample solutions were prepared as aforementioned, and the absorption spectra in the range of 350–800 nm were recorded. By utilizing the absorption signal changes at 696 nm, the binding stoichiometries and affinities between FA-TMPipEOPP and G-quadruplexes under different pH conditions were calculated by the following equation.14,35 −1 = 1+ ( + 1 + − ( + 1 + − 4 2 Where A and A0 are the absorption signal intensities at 696 nm in the presence and absence of G-quadruplex, respectively. P=Amax/A0 (Amax is maximum absorption intensity in the presence of maturated G-quadruplex). M=1/(Ka·CFA-TMPipEOPP). Here, Ka is the apparent binding constant between FA-TMPipEOPP and G-quadruplex, and CFA-TMPipEOPP is the FA-TMPipEOPP concentration. x=nCquadruplex/CFA-TMPipEOPP (Cquadruplex

is

the

concentration

of

G-quadruplex,

n

is

the

putative

FA-TMPipEOPP-binding site number on the tested G-quadruplex). By fitting the A/A0~Cquadruplex plot using above equation, Ka and n can be obtained. Job plot analysis was carried out by systematic variation of the molar fraction of FA-TMPipEOPP and DNA while keeping a constant total concentration of 5 µM. The sample was prepared as above, and the absorbance signals at 696 nm were recorded.



Fluorescence spectroscopy. Fluorescence spectra were measured on a SHIMADZU RF-5301PC spectrofluorimeter with 1 cm-path-length micro quartz cell (40 µL, Starna Brand, England). The sample solutions were prepared as aforementioned. Fixing the excitation wavelength at 470 nm, the fluorescence spectra in the range of 600-850 nm were recorded (excitation slit = emission slit = 5 nm), or fixing the excitation wavelength at 696 nm, the emission spectra in the range of 700– 900 nm were recorded (excitation slit = emission slit = 3 nm). Fluorescence titration experiments were carried out by varying the DNA concentration but maintaining the FA-TMPipEOPP concentration at 5 µM. The sample solutions were prepared as above. Fixing the excitation wavelength at 470 or 696 nm, corresponding emission spectra were collected at room temperature. Resonance light scattering (RLS) spectroscopy. RLS experiments were performed on a SHIMADZU RF-5301PC spectrofluorimeter with 1 cm-path-length micro quartz cell (40 µL, Starna Brand, England). The sample solutions were prepared as aforementioned. The excitation and emission monochromator wavelengths were coupled and adjusted to scan simultaneously in the range of 350–900 nm (excitation slit = emission slit = 3 nm). RLS titration experiments were carried out by varying the DNA concentration but maintaining the FA-TMPipEOPP concentration at 5 µM. The sample solutions were prepared as above. The excitation and emission monochromator wavelengths were coupled and adjusted to scan simultaneously in range of 250–900 nm (excitation slit = emission slit = 1.5 nm).


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Studies on competition between G-quadruplex and duplex structures. 10 µM G-rich oligonucleotides (C-MYC or KRAS) were mixed with different concentrations of their individual complementary C-rich oligonucleotides in the presence of 10 mM Tris-HCl buffer, 50 mM KCl, 100 mM NaCl and 1 mM Na2EDTA. Each mixture was heated at 95 °C for 5 min to remove any aggregates, then cooled rapidly to 25 °C and was allowed to incubate at this temperature for 30 min. After overnight incubation at 4 °C, 5 µM FA-TMPipEOPP or TMPipEOPP was added, and the absorption spectra in the range of 350-800 nm were recorded. The absorption signal intensities at 696 nm were extracted and normalized. According to the normalized A696~C-rich oligonucleotide concentration plots, the percentage of G-quadruplex structure in the mixture could be obtained at the site of G-rich/C-rich ratio of 1:1. Cell culture. CCRF-CEM (Human T cell lymphoblast-like cell line) and Ramos (Human Burkitt lymphoma) cells were cultured in RPMI 1640 (gibco) supplemented with 10% fetal bovine serum medium (gibco) and 0.5 mg/mL penicillin-streptomycin (life technologies) at 37 oC under 5 % CO2 atmosphere. Cells were washed before incubation with DPBS (Dulbecco's Phosphate Buffered Saline). Confocal laser scanning microscopy assay. Cells (5×105) were incubate with sgc8c-KRAS (5 µM) in buffer (DPBS with 50 mM KCl) for 30 min, and washed by 0.2 mL DPBS twice. After that, the cells were incubated with FA-TMPipEOPP in buffer (DPBS with 50 mM KCl) for 10 min. The steps prior to confocal imaging were performed at 4 oC to prevent receptor internalizations.36 Then, imaging was performed on confocal fluorescence microscope (Olympus FV1000, Japan). Cells bound with



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sgc8c-KRAS and FA-TMPipEOPP conjugates were excited at 458 nm and fluorescence emission from 650 to 750 nm was collected.

RESULTS AND DISCUSSION Synthesis of asymmetric cationic porphyrin.

Asymmetric porphyrin

FA-TMPipEOPP was synthesized by four steps starting from THPP (Scheme 2). The synthetic detail is given in Supporting Information. A FA group needs to be introduced on one side arm of porphyrin to form an asymmetric structure. To achieve this goal, we first synthesized an asymmetricalkyl brominated hydroxyphenyl porphyrin Br-THPP, since the halogenoalkanes can easily undergo nucleophilic substitution reactions and Br is a good leaving group. In the presence of FA, its carboxyl group will initiate a nucleophilic substitution reaction, resulting in replacement of the Br group by FA. It is worth noting that FA has both carboxylic and phenolic hydroxyl groups. All of them can perform a substitution reaction with Br-THPP. The proton-nuclear magnetic resonance (1H-NMR) spectrum of FA-THPP gave a peak (δH 9.67(s)), which corresponded to the hydroxylcinnamoyl unit of phenolic hydroxyl, thus confirming the structure shown in Scheme 2. In our previous studies,9–11,30,31 we demonstrated that porphyrin containing four bulky side arms shows increased G-quadruplex recognition specificity. To obtain an asymmetric porphyrin with four bulky

side

arms,

the

foregoing-prepared

FA-THPP

was

treated

with

1-(2-chloroethyl)-piperidine hydrochloride to give FA-TPipEOPP. This was a difficult step, and an optimal yield was obtained after reaction at room temperature for 72 h under a N2 atmosphere. Shorter reaction time or higher reaction temperature resulted


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in lower yields of FA-TPipEOPP. Finally, FA-TPipEOPP was treated with an excess concentration of methyl iodide to obtain the water-soluble, methylated product FA-TMPipEOPP. The final product and all reaction intermediates were well characterized with nuclear magnetic resonance (NMR) and mass spectrometry (MS) (Figures S1–S7).

Scheme 2. The synthesis route of FA-TMPipEOPP.

N-protonation of cationic porphyrins under acidic conditions. To investigate whether increasing structural asymmetry can overcome the protonation tendency of cationic porphyrins

or

not,

the

absorption

spectra of TMPipEOPP and



FA-TMPipEOPP were recorded and compared. As shown in Figure 1, changing pH from 8.0 to 5.0 resulted in remarkable hypochromicity of the TMPipEOPP Soret band at 419 nm, accompanied by the emergence and continuous intensity increase of the two bands at 445 and 676 nm, respectively. Such absorption spectral changes might be attributed to the protonation of the two imino nitrogens in the porphyrin core,37 and an apparent pKa≈6.1 was determined. This pKa value is much higher than that reported for TMPyP4 (around 1.8).38 The reason is that the positive charge delocalization of peripheral pyridinium moieties on the porphyrin macrocycle increases the difficulty of TMPyP4 protonation.39 In FA-TMPipEOPP, however, the positive charges are far from the porphyrin macrocycle, and they tend to localize at the peripheries, thus giving increased pKa value. Therefore, although TMPipEOPP shows much better G-quadruplex recognition specificity than TMPyP4, the increased pKa value makes it cannot work well under acidic conditions. Different form TMPipEOPP, obvious spectral changes were observed for FA-TMPipEOPP only when the pH value was decreased to 1.5, and an apparent pKa≈1.6 was determined. The greatly reduced protonation tendency might be attributed to the flexible arm on FA-TMPipEOPP. That is, the mobile piperidinium group might be positioned above the centre of the porphyrin macrocycle, thus decreasing the concentration of H+ in this microenvironment.


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TMPipEOPP

1.0

pH=5.0 pH=5.5 pH=6.0 pH=6.5 pH=7.0 pH=7.5 pH=8.0

0.8

Absorbance

(b)

0.6

0.4

FA-TMPipEOPP pH=1.0 pH=1.5 pH=2.0 pH=2.5 pH=3.0 pH=3.5 pH=4.0 pH=4.5

1.8 1.5

Absorbance

(a)

1.2 0.9 0.6

0.2

pH=5.0 pH=5.5 pH=6.0 pH=6.5 pH=7.0 pH=7.5 pH=8.0

0.3 0.0 300

400

500

600

700

800

0.0 300

400

TMPipEOPP

1000

(d)

pH=5.0 pH=5.5 pH=6.0 pH=6.5 pH=7.0 pH=7.5 pH=8.0

800 600 400

600

FA-TMPipEOPP pH=1.0 pH=1.5 pH=2.0 pH=2.5 pH=3.0 pH=3.5 pH=4.0 pH=4.5

1000

800

RLS intensity (au)

(c)

500

700

800

Wavelength (nm)

Wavelength (nm)

intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


200

600

400

pH=5.0 pH=5.5 pH=6.0 pH=6.5 pH=7.0 pH=7.5 pH=8.0

200

0 0

400

500

600

700

800

900

400

Wavelength (nm)

500

600

700

800

900

Wavelength (nm)

Figure 1. pH-dependent UV-vis absorption (a,b) and RLS (c,d) spectral changes of TMPipEOPP and FA-TMPipEOPP. [porphyrin] = 5 µM. Excitation slit = emission slit = 3 nm.

It is reported that N-protonation might induce a light distortion of the porphyrin macrocycle from planarity,37 but the peripheral aryl moieties can be converted from a twisted configuration relative to the macrocycle to a nearly coplanar one,40 thus promoting the molecular aggregation. To investigate the aggregation behaviors of the two cationic porphyrins, their resonance light scattering (RLS) spectra were recorded under different pH conditions (Figure 1). Interestingly, obviously increased RLS signals at 690–700 nm were given by the protonation of TMPipEOPP and FA-TMPipEOPP and the RLS signal continuously decreased with temperature (Figure



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S8), thus suggesting the aggregation of protonated porphyrins. The formation of porphyrin dimer is more possible. Colorimetric recognition of G-quadruplexes. Having demonstrated that FA-TMPipEOPP remained in unprotonated monomeric form as long as the solution pH is higher than 2.0, it was reasonable to predict that this porphyrin might be used for optical recognition of G-quadruplexes under acidic conditions. As expected, under acidic conditions (pH 5.0–6.5), addition of G-quadruplexes led to dramatic changes in UV-vis

absorption

spectrum

of

FA-TMPipEOPP

(Figure

2):

significant

hypochromicity of the Soret band at 422 nm accompanied by the emergence of two new bands centered at 454 and 696 nm. On the contrary, ssDNA and dsDNA showed little effect on the absorption spectrum of FA-TMPipEOPP, and the absorption signal at 696 nm (A696) was hardly affected. Therefore, by comparing the absorption signal change at 696 nm, G-quadruplexes could be easily discriminated from ssDNA and dsDNA under acidic conditions. Unlike FA-TMPipEOPP, structurally symmetrical porphyrin TMPipEOPP gave satisfactory G-quadruplex-specific recognition in the pH range of 7.0–7.5. Further acidification resulted in poorer G-quadruplex recognition specificity due to TMPipEOPP protonation under acidic conditions. Collectively, TMPipEOPP and FA-TMPipEOPP are suitable for optical G-quadruplex recognition under neutral and acidic conditions, respectively. By combining these two probes, specific G-quadruplex recognition with nearly negligible background can be achieved in the pH range of 5.0–7.5, which almost covers the whole physiological pH range.


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FA-TMPipEOPP (pH=5.5)

(a')

0.25

1.0

Absorbance

0.8 0.6

0.30 0.25

1.0

0.15

Absorbance

A696

0.20

TMPipEOPP (pH=5.5)

1.2

0.10 0.05 0.00

0.4

1 2 3 4 5 6 7 8 9 10

A696

(a)

0.8

0.15 0.10 0.00

1 2 3 4 5 6 7 8 9 10

0.4 0.2

0.0

0.0

400

500

600

700

800

400

500

Wavelength (nm)

1.4

A696

0.05 0.00

0.4

1 2 3 4 5 6 7 8 9 10

0.15

1.0

Absorbance

0.10

0.6

800

0.20

1.2

0.15

0.8

700

TMPipEOPP (pH=7.0)

(b')

0.20

A696

1.0

600

Wavelength (nm)


(b) Absorbance

0.20

0.05

0.6

0.2

0.10

0.8

0.05

0.6

0.00

1 2 3 4 5 6 7 8 9 10

0.4 0.2

0.2 0.0

0.0 400

500

600

700

800

400

(c)

FA-TMPipEOPP 0.3

pH=5.0

(c')

700

800

pH=5.0

pH=5.5

pH=6.0

pH=6.5

pH=7.0

pH=7.5

0.2

0.2

0.1

pH=6.5

pH=7.5

pH=7.0

A696

0.1 0.0 0.2

600

TMPipEOPP

0.3

pH=6.0

pH=5.5

500

Wavelength (nm)

Wavelength (nm)

A696

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.0 0.2

0.1 0.1

0.0

0.0

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 101 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 101 2 3 4 5 6 7 8 9 101 2 3 4 5 6 7 8 9 10

DNAs

DNAs

Figure 2. Colorimetric recognition of G-quadruplexes by FA-TMPipEOPP and TMPipEOPP.

(a,a′,b,b′)

representative

absorption

spectral

changes

of

FA-TMPipEOPP and TMPipEOPP in the absence (black line) or presence of dsDNAs (green lines), ssDNAs (blue lines) or G-quadruplex DNAs (red lines) at pH 5.5 or 7.0. The inserts show the absorption signal changes at 696 nm. (c,c′) pH-dependent A696 changes of FA-TMPipEOPP and TMPipEOPP in the absence or presence of different



structural DNAs. The DNAs are: Blank (1), AT (2), LD (3), GC (4), ssDNA1 (5), ssDNA2 (6), Oxy28 (7), C-MYC (8), KRAS (9) and Hum48 (10). [porphyrin] = 5 µM; [quadruplex] = [dsDNA] = [ssDNA] = 10 µM. The detail results in the pH range of 5.0-8.0 can be found in Figure S9. The oligonucleotide sequence was listed in Table S1.

Fluorescent recognition of G-quadruplexes. Fluorescent probes usually have wider applications than colorimetric ones, for example, imaging studies. We investigated the feasibility of FA-TMPipEOPP for fluorescent recognition of G-quadruplexes. Considering that G-quadruplexes can induce the emergence of two new absorption bands centered at 454 and 696 nm under acidic conditions, the fluorescence excited within these two wavelength ranges might be used for specific recognition of G-quadruplexes. To avoid the effect of marginal absorption of Soret band, 470 nm was selected instead of 454 nm as the excitation wavelength. When excited at 470 nm, free FA-TMPipEOPP emitted weak fluorescence with a maximum at 658 nm (Figure 3). ssDNA and dsDNA showed weak or negligible effects on FA-TMPipEOPP fluorescence. Addition of G-quadruplexes, however, resulted in decreased fluorescence at 658 nm, accompanied by a significant increase at 719 nm. By comparing the F719/F658 ratio, G-quadruplexes could be easily discriminated from ssDNA and dsDNA in the pH range of 5.0–6.5. In the same way, TMPipEOPP could achieve satisfactory fluorescent probing of G-quadruplexes with almost no background fluorescence interference in the pH range of 6.5–7.5. Combination of FA-TMPipEOPP and TMPipEOPP can fulfill the requirement of the whole


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physiological pH range. When 696 nm was used as the excitation wavelength, similar pH-dependent G-quadruplex recognition specificity was observed for these two porphyrin derivatives (Figure S11). FA-TMPipEOPP (pH=5.5) 60 40 20 0

1 2 3 4 5 6 7 8 9 10

100

50

0 600

F719/F658

150

100

150

Fluorescence (au)

200

TMPipEOPP (pH=5.5)

(a')

80

F719/F658

Fluorescence (au)

(a)

100

800

600

700

F719 /F658

100

6 4 2 1 2 3 4 5 6 7 8 9 10

100

50

80

30 20 10 0

1 2 3 4 5 6 7 8 9 10

60 40 20 0

700

800

900

600

pH=5.5

700

800

900

Wavelength (nm)

FA-TMPipEOPP pH=5.0

900

40

8

0

800

120

Fluorescence (au)

Fluorescence (au)

150

80

1 2 3 4 5 6 7 8 9 10

TMPipEOPP (pH=7.0)

(b')

Wavelength (nm)

(c)

20

Wavelength (nm)

10

0 600

40

50

900


200

60

0 700

F719/F658

(b)

80

0

Wavelength (nm)

pH=6.0

(c')

TMPipEOPP

100 80

pH=5.0

pH=5.5

pH=6.0

pH=6.5

pH=7.0

pH=7.5

60

60 40

40

20 0 40

pH=6.5

pH=7.0

pH=7.5

30

F719/F658

F719/F658

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20 0 60 40

20

20 10

0

0

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 101 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 101 2 3 4 5 6 7 8 9 10

DNAs

DNAs

Figure 3. Fluorescent recognition of G-quadruplexes by FA-TMPipEOPP and TMPipEOPP (λex = 470 nm). (a,a′,b,b′) representative fluorescent spectral changes of FA-TMPipEOPP and TMPipEOPP in the absence (black line) or presence of dsDNAs



Page 18 of 37

(green lines), ssDNAs (blue lines) or G-quadruplex DNAs (red lines) at pH 5.5 or 7.0. The inserts show the changes in the fluorescence intensity ratio of F719/F658. (c,c′) pH-dependent F719/F658 changes of FA-TMPipEOPP and TMPipEOPP in the absence or presence of different structural DNAs. DNAs are same to Figure 2. The detail results in pH range of 5.0-8.0 can be found in Figure S10.

Absorption and fluorescence titration spectra of FA-TMPipEOPP. To confirm

the

FA-TMPipEOPP

G-quadruplex-specific under

acidic

optical

recognition

conditions,

ability

of

G-quadruplex

concentration-dependent changes in absorption and fluorescence signals were recorded and compared with those induced by ssDNA and dsDNA (Figure 4). With the addition of increasing concentrations of G-quadruplexes, significant changes were observed for both absorption and fluorescence spectra. On the contrary, neither ssDNA nor dsDNA lead to spectral changes. According to the changes in A696, F722 or F719/F658, G-quadruplexes can be easily discriminated from ssDNA and dsDNA in the pH range of 5.0–6.5. F719/F658 is the ratio of the fluorescent signal intensities at two wavelengths. Using this ratio, ratiometric recognition of G-quadruplexes can be achieved, thus efficiently overcoming interference from many factors, including probe concentration, light-source intensity, instrument sensitivity and light scattering.


Hum48 (pH=5.5)

(a)

1.0

50 µM 40 µM 30 µM 20 µM 10 µM 8 µM 6 µM 4 µM 2 µM 0 µM

25 µM 0.8

Absorbance

0.6

0 0.4 0.2

0.6 0.4 0.2

0.0 600

700

400

(e)

500

600

Fluorescence (au)

50

650

700

750

800

150 100 50

(h) Fluorescence (au)

150

0 100

50

750

800

Wavelength (nm)

850

900

(f)

40

50

pH=6.0

80 70 60 50 40 30

AT

Oxy28

LD

20 10

700

750

800

C-MYC

GC

KRAS

ssDNA

Hum48

ssDNA-c

0

850

10

100

50

30

40

50

pH=6.5

(j)


150

20

DNA (µM)

GC (pH=6.5)

200

0 700

30

0 650

25 µM

0 700

20

Wavelength (nm)

Hum48 (pH=6.5)

200

10

DNA (µM)


Wavelength (nm)

(g)

0

800

GC (pH=6.0)

250

0 600

850

Oxy28 C-MYC KRAS Hum48

0.05

700

200

0

100

0 600

AT LD GC ssDNA ssDNA-c

0.10

Wavelength (nm)

25 µM

150

0.15

0.00

800

Hum48 (pH=6.0)

250

200

Fluorescence (au)

0.20

F719/F658

500

Wavelength (nm)

(d)

0.25

0.0 400

pH=5.5

0.30

150

F722

Absorbance

0.8

(c)

GC (pH=5.5)

(b)

1.0

Fluorescence (au)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A696

Page 19 of 37

100

ssDNA2 Oxy28 C-MYC KRAS Hum48

AT LD GC ssDNA1

50

0

750

800

850

900

Wavelength (nm)

0

10

20

30

40

50

DNA (µM)

Figure 4. DNA concentration-dependent absorption and fluorescence signal changes of FA-TMPipEOPP. (a, b) Hum48 and GC concentration-dependent absorption spectral changes. (c) DNA concentration-dependent changes of A696. (d, e) Hum48 and GC concentration-dependent fluorescence spectral changes (λex = 470 nm). (f) DNA concentration-dependent changes in fluorescence intensity ratio of F719/F658. (g, h) Hum48 and GC concentration-dependent fluorescence spectral changes (λex = 696 nm). (j) DNA concentration-dependent fluorescence changes at 722 nm (λex = 696 nm). [FA-TMPipEOPP] = 5 µM. Absorption and fluorescent titration spectra in the presence of other DNAs and under other pH conditions can be found in Figures S12-S20.

Binding interactions between G-quadruplexes and FA-TMPipEOPP. To



further elucidate the specific optical recognition of FA-TMPipEOPP towards G-quadruplexes under acidic conditions, the binding interactions between this porphyrin and differently structured DNAs were investigated. First, RLS spectra of the mixture of FA-TMPipEOPP and G-quadruplex (e.g. Hum48) were recorded under different pH conditions. The mixture gave a similar RLS peak at around 700 nm to free FA-TMPipEOPP at pH of 1.0 (Figure S21). In addition, under the pH condition of 6.0, titration of FA-TMPipEOPP with Hum48 gave significant RLS spectral changes (Figure 5). Taking the RLS signal at 462 nm as an example, it sharply increased when the [Hum48]/[FA-TMPipEOPP] concentration ratio was changed from 0 to 0.15, and then significantly decreased with the further increase of the concentration ratio. On the contrary, the maximum RLS signal at 700 nm continuously increased with [Hum48]/[FA-TMPipEOPP] concentration ratio, and the RLS signal at this wavelength was much higher than that at 462 nm. These results suggested that large J-type aggregates were formed by FA-TMPipEOPP with the assistance of Hum48 at the [Hum48]/[FA-TMPipEOPP] ratio of 0.15.41 With the further addition of Hum48, however, the aggregates was dissolved and converted to FA-TMPipEOPP/Hum48 complex. Since the FA-TMPipEOPP/Hum48 complex showed similar RLS signal to free FA-TMPipEOPP at pH of 1.0, we speculated that a dimer might be formed by FA-TMPipEOPP under strong acidic conditions.


Page 20 of 37

(a)

400

0.75 µM

(b)

400

0.5 µM

350 200

10 µM

200

0

100 0

150

300

500

700

900

100 50 0

Intensity at 462 nm

300 250

400

0.15

350

300

RLS intensity (au)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


90

300 80

250 200

462 nm 700 nm

150

70

100 60 50 0

300

400

500

600

700

800

900

Intensity at 700 nm

Page 21 of 37

50 0.0

Wavelength (nm)

0.5

1.0

1.5

2.0

[Hum48]/[FA-TMPipEOPP]

Figure 5. (a) RLS titration spectra of FA-TMPipEOPP with Hum48 and (b) RLS signal changes at 462 nm and 700 nm with [Hum48]/[FA-TMPipEOPP] concentration ratio at pH of 6.0. [FA-TMPipEOPP] = 5 µM. Excitation slit = emission slit = 1.5 nm. Then, Job plot analysis was used to determine the binding stoichiometry of FA-TMPipEOPP to G-quadruplex by using the absorption signal at 696 nm (Figure S22). Although different binding stoichiometries were observed for G-quadruplexes with different structures (1:2 for parallel G-quadruplexes C-MYC and KRAS, 1:1 for antiparallel G-quadruplex Oxy28, and 1:1 for multimeric G-quadruplex Hum48 containing two G-quadruplex units), identical stoichiometry was maintained for the same G-quadruplex under different pH conditions (Table 1), suggesting that the G-quadruplex-binding mode of FA-TMPipEOPP was almost unchanged with pH. Table 1. pKa values of FA-TMPipEOPP/G-quadruplex complex, binding stoichiometry and affinity between FA-TMPipEOPP and G-quadruplex under different pH conditions. G-quadruplex

pKa

Oxy28 C-MYC KRAS Hum48

6.1 6.6 6.8 6.1

Ka (×106 M−1 ) pH 5.5

pH 6.0

pH 6.5

0.83 17.07 3.29 2.02

0.49 6.90 1.84 0.69

0.10 0.94 0.60 0.08


Stoichiometry 1:1 1:2 1:2 1:1


Similar G-quadruplex-binding stoichiometries were obtained by TMPipEOPP in our previous studies,9 thus, it is possible that a similar G-quadruplex-binding mode was adopted by FA-TMPipEOPP and TMPipEOPP. That is, they bind to G-quadruplex by an end-stacking mode via the synergy between π–π stacking (between the porphine core and terminal G-quartet(s) of G-quadruplex) and electrostatic interaction (between positively charged side arm substituents and the negatively charged phosphate backbones of DNA). Polyanionic nature of DNA can attract a large amount of positively-charged H+ ions to its surface. Tightly stacked structure of G-quadruplex makes it gather more H+ ions than ssDNA and dsDNA. When FA-TMPipEOPP end-stacks with G-quadruplexes, unusually high local concentration of H+ promotes the protonation of FA-TMPipEOPP, thus giving obvious absorption and fluorescence signal responses to G-quadruplexes. This can be demonstrated by obviously increased pKa values of FA-TMPipEOPP/G-quadruplex complexes (Table 1) compared to free FA-TMPipEOPP. Protonation converts bound FA-TMPipEOPP to a diacid form with 6 positive charges, thus further increasing the binding stability between FA-TMPipEOPP and G-quadruplex, which can be reflected by the increase in the binding affinity of FA-TMPipEOPP to G-quadruplex with pH decrease (Table 1 and Figure S22). Overall, high negative charge intensity of G-quadruplex (and thus high H+ concentration gathered on its surface) and end-stacking interaction between FA-TMPipEOPP and G-quadruplex confer FA-TMPipEOPP with excellent G-quadruplex recognition specificity against ssDNA and dsDNA.


Page 22 of 37

Page 23 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Applications of FA-TMPipEOPP as a G-quadruplex probe. The ability of specific G-quadruplex recognition under acidic conditions makes FA-TMPipEOPP applicable in some fields, such as G-quadruplex formation-probing in acidic conditions and G-quadruplex-based biosensing that needs to work under acidic conditions. As two proof-of-concept examples, the feasibility of FA-TMPipEOPP for probing competition between G-quadruplex and duplex structures and wash-free cancer cell-targeted bioimaging was tested. In the human genome, potential G-quadruplex-forming G-rich sequences usually coexist with their complementary C-rich sequences, and thus competition inevitably occurs between G-quadruplex and duplex structures. Probing such a competition is helpful to elucidate the physiological functions of G-rich genomic sequences. In addition, G-quadruplex/duplex competition-probing is also useful in biosensing fields since many G-quadruplex-based biosensors are designed on the basis of interconversion between G-quadruplexes and duplexes. Under acidic conditions, FA-TMPipEOPP showed strong optical responses to G-quadruplexes (e.g. C-MYC and KRAS). With the addition of increasing concentrations of individual complementary C-rich sequences (C-MYC-c and KRAS-c), more G-quadruplexes were converted to duplexes, accompanied by a decrease in optical signals (Figure 6). According to the constructed plots of signal output versus C-rich sequence concentration, the percentage of G-quadruplexes formed by G-rich sequences could be obtained at the G-rich to C-rich sequence ratio of 1:1. Both C-MYC and KRAS showed a pH-dependent decrease in G-quadruplex percentage, which might be



attributed to two reasons: decrease of duplex stability and formation of i-motif structures by C-rich sequences under acidic conditions. Under the same conditions, the G-quadruplex percentage of C-MYC was higher than that of KRAS, which is consistent with the higher stability of KRAS/KRAS-c duplex than C-MYC/C-MYC-c duplex (predicted Gibbs free energy ∆G: −42.37 vs −28.30 kcal/mol).

1.0

G-quadruplex percentage pH 5.5 (59%) pH 6.0 (47%) pH 6.5 (41%) pH 7.4 (26%)

0.8 0.6 0.4 0.2

(c)

1.0

G-quadruplex percentage 0.8

Normalized A696

(b) Normalized A696

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 37

pH 5.5 pH 6.0 pH 6.5 pH 7.4

0.6 0.4

(17%) (17%) (11%) (5%)

0.2 0.0

0.0 0

5

10

15

20

25

30

35

40

0

5

[C-MYC-c]/(µM)

10

15

20

25

30

[KRAS-c]/(µM)

Figure 6. G-quadruplex/duplex competition-probing by FA-TMPipEOPP. (a) Working mechanism for G-quadruplex/duplex competition-probing. (b,c) Absorption spectral titration of C-MYC/FA-TMPipEOPP complex or KRAS/FA-TMPipEOPP complex with corresponding C-rich complementary strands. The G-quadruplex percentages were labeled in the figures in red. [FA-TMPipEOPP] = 5 µM, [C-MYC] = [KRAS] = 10 µM. TMPipEOPP was used in the experiments at pH of 7.4 instead of FA-TMPipEOPP.


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Increased metabolism, together with reduced oxygen and nutrient supply make the tumor microenvironment highly acidic compared to that of normal tissues.42,43 Acidic tumor microenvironment not only promotes angiogenesis but also enhances the metastatic potential of cancer cells.44 Since FA-TMPipEOPP shows excellent G-quadruplex recognition specificity against ssDNA and dsDNA, we used FA-TMPipEOPP/G-quadruplex complexes as a signal reporter to image living cells. To achieve specific recognition of cancer cells, a G-rich sequence (KRAS) was linked at one end of sgc8c, an aptamer that targets the cell membrane protein tyrosine kinase-7 (PTK7) over-expressed in various kinds of cancers.45–48 Clear cell shapes were observed for CEM cells with over-expressed PTK7 (Figure 7), suggesting that FA-TMPipEOPP emits bright fluorescence upon binding to G-quadruplexes but does not hinder the interaction between the aptamer and its target. On the contrary, only weak fluorescence was observed by the PTK7-negative Ramos cells, thus confirming the cancer cell recognition specificity of this label-free imaging system.



Figure

7.

Wash-free

cancer

cell-targeted

imaging

Page 26 of 37

under

acidic

tumor

microenvironment using G-quadruplex/porphyrin as the fluorescent probe. (a) Working mechanism. (b,c) Fluorescence microscopic images of PTK7-positive CEM cells (b) and PTK7-negtive Romas cells (c) using G-quadruplex/FA-TMPipEOPP as the fluorescent probe.

The aforementioned cancer cell-imaging experiments were conducted in a wash-free mode. That is, excess FA-TMPipEOPP did not need to be separated before recording images, since free FA-TMPipEOPP has a different excitation wavelength from FA-TMPipEOPP/G-quadruplex complex. Under excitation at 458 nm, FA-TMPipEOPP gave no fluorescence emission and nearly negligible background will be given. Compared to persistently luminescent probes, such a wash-free probe simplifies experiments and makes in vivo applications possible.

CONCLUSION


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In summary, we demonstrated that the protonation tendency of porphyrin derivatives could be efficiently overcome by increasing molecular asymmetry. As a result, our designed asymmetrical, water-soluble, cationic porphyrin FA-TMPipEOPP had better G-quadruplex recognition specificity than its symmetrical counterpart TMPipEOPP, thus showing great potential for specific optical probing of G-quadruplexes under acidic conditions. The feasibility was demonstrated by the proof-of-concept tests of probing competition between G-quadruplex and duplex structures, as well label-free cancer cell-targeted imaging under acidic tumor microenvironments in a simple wash-free manner. In addition, the finding that asymmetrical FA-TMPipEOPP works well under acidic conditions and symmetrical TMPipEOPP works well under neutral conditions suggests that porphyrin-based G-quadruplex probes suitable for different pH conditions might be obtained by adjusting the molecular symmetry.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: Oligonucleotides used in this work; instruments for structural characterization; synthesis and characterization of FA-TMPipEOPP; Temperature-dependent RLS spectral changes of TMPipEOPP at pH of 5.0; colorimetric recognition of G-quadruplexes; fluorescent recognition of G-quadruplexes (λex = 470 nm);



Page 28 of 37

fluorescent recognition of G-quadruplexes (λex = 696 nm); absorption titration spectroscopy; fluorescent titration spectroscopy (λex = 470 nm); fluorescent titration spectroscopy (λex = 696 nm); pH-dependent RLS spectra of the mixture of FA-TMPipEOPP

and

Hum48;

Job

Plot

analysis

for

the

interaction

of

FA-TMPipEOPP with G-quadruplex; binding affinity between FA-TMPipEOPP and G-quadruplex.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] (L.-N. Zhu)； Fax: +86-22-27403475

Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 21371130), the Natural Science Foundation of Tianjin [No. 15JCYBJC48300, 16JCYBJC19900] and the Innovation Fund of Tianjin University.

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395x218mm (300 x 300 DPI)


Asymmetric Cationic Porphyrin as a New G-Quadruplex Probe with

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