DANPY (dimethylaminonaphthylpyridinium): an economical and biocompatible fluorophore

Organic &

Biomolecular Chemistry

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DANPY (dimethylaminonaphthylpyridinium):

an economical and biocompatible ﬂuorophore†

Cite this: DOI: 10.1039/c8ob02536c

a

b

a

Lewis E. Johnson, * Jason S. Kingsbury,*‡ Delwin L. Elder,

‡

c

a

c

d

Rose Ann Cattolico,* Luke N. Latimer, William Hardin, Evelien De Meulenaere,§

c

d

c

Chloe Deodato,

Griet Depotter, Sowmya Madabushi,

a

b

Nicholas W. Bigelow,

Werner Kaminsky,

Brittany A. Smolarski,

Trevor K. Hougen,

a

d

a

Koen Clays and Bruce H. Robinson

Dyes with nonlinear optical (NLO) properties enable new imaging techniques and photonic systems. We

have developed a dye (DANPY-1) for photonics applications in biological substrates such as nucleic acids;

however, the design speciﬁcation also enables it to be used for visualizing biomolecules. It is a prototype

dye demonstrating a water-soluble, NLO-active ﬂuorophore with high photostability, a large Stokes shift,

and a favorable toxicity proﬁle. A practical and scalable synthetic route to DANPY salts has been optimized

featuring: (1) convergent Pd-catalyzed Suzuki coupling with pyridine 4-boronic acid, (2) site-selective

pyridyl N-methylation, and (3) direct recovery of crystalline intermediates without chromatography. We

characterize the optical properties, biocompatibility, and biological staining behavior of DANPY-1. In

addition to stability and solubility across a range of polar media, the DANPY-1 chromophore shows a ﬁrst

hyperpolarizability similar to common NLO dyes such as Disperse Red 1 and DAST, a large two-photon

absorption cross section for its size, substantial aﬃnity to nucleic acids in vitro, an ability to stain a variety

of cellular components, and strong sensitivity of its ﬂuorescence properties to its dielectric environment.

Received 13th October 2018,

Accepted 19th February 2019

DOI: 10.1039/c8ob02536c

rsc.li/obc

fluorescent dyes can be enhanced by methods such as encap-

Introduction

1

sulation or photoswitching, chemical transformation to an

2

Fluorescent dyes are one of the principal tools for detection active form upon interaction with a substrate, conjugation

3

,4

and quantification of molecules of interest in molecular with nanoparticles, interactions with nano-engineered sur-

5

biology, clinical diagnostics, and analytical chemistry. A multi- faces, or nonlinear optical (NLO) techniques. Commonly used

6

–8

tude of such dyes have been developed to facilitate the study of NLO applications include second-harmonic generation

particular biological targets, including membranes, proteins, (SHG, often referred to as SHIM in the context of imaging/

and nucleic acids. The sensitivity and spatial resolution for microscopy), which can be performed using dyes that possess

a large first hyperpolarizability (β), and two-photon excited

9

–11

fluorescence

(TPEF), which relies on a large second hyper-

a

Department of Chemistry, University of Washington, Box 351700, Seattle,

polarizability (γ). While the structure of dye molecules can be

optimized to maximize first or second hyperpolarizability, all

molecules exhibit nonzero γ, whereas non-centrosymmetric

structures are required for nonzero β.

Development of dyes with large first hyperpolarizability has

been heavily driven by photonics due to the capability of these

WA 98195, USA. E-mail: lewisj@uw.edu

b

Department of Chemistry, Ahmanson Science Center, California Lutheran

University, 60 West Olsen Rd., Thousand Oaks, CA 91360, USA.

E-mail: jkingsbu@callutheran.edu

1

2

c

Department of Biology, University of Washington, Box 351800, Seattle, WA 98195,

USA. E-mail: racat@uw.edu

d

Department of Chemistry, Katholieke Universiteit Leuven, Leuven, Belgium B-3001

dyes to achieve large electro-optic (EO) constants (r33) and ultra-

†

Electronic supplementary information (ESI) available: Detailed synthetic and

13,14

fast response to radio-frequency signals.

While current-

1

13

experimental protocols, H and C NMR spectra, potential energy surface ana-

lysis, detailed crystallographic information, detailed methodology for DNA

generation organic electro-optic (OEO) materials are typically

binding analysis, third-party toxicology data (Ames test, MEM elution cyto- monolithic organic glasses composed of chromophores functio-

toxicity, rabbit irritation test, and mouse systemic toxicity test), nonlinear fitting nalized with pendant groups to control processability and

code, and coordinates for computed structures. CCDC 1838228. For ESI and crys-

tallographic data in CIF or other electronic format see DOI: 10.1039/c8ob02536c

13,15

thermal stability,

OEO materials based on chromophores

blended with deoxyribonucleic acids (DNA) have also been

examined due to favorable near-infrared optical properties of

DNA such as low loss and a tunable refractive index.

‡

These authors contributed equally to this work.

§

Present address: Marine Biology Research Division, Scripps Institution of

9

,16

Oceanography, La Jolla, CA 92093. USA.

The

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Fig. 1 Disperse red 1 (DR1, top left) is an older generation dye used in early organic EO devices. Ethidium bromide (bottom left) is a common

ﬂuorescent stain for nucleic acids. DAST (top center) is a cationic dye used for organic EO applications that has a large β and forms non-

centrosymmetric crystals. DANPY-1 (bottom center) combines features from ethidium bromide and DAST to retain a large β as well as compatibility

with biological environments. Isoc (top right) is a neutral EO dye that has also been explored for nucleic acid-related applications. Prodan (lower

right) is a neutral membrane dye sharing the same dimethylaminonaphthalene donor as DANPY.

DNA used in these films is often complexed with surfactants design to reduce this gap, producing a dye (DANPY-1) that

such as cetyltrimethylammonium bromide (CTAB) to form a combines a wide variety of favorable properties into a single

9

processable, alcohol-soluble, and homogeneous material. OEO molecule accessible through a short and scalable synthesis.

waveguide modulators have been demonstrated, as have other

There are two principal strategies that can be followed to

electronic devices fabricated from DNA-containing materials, accelerate the design process for a biologically compatible,

such as light-emitting diodes (LEDs) and field-eﬀect transistors NLO-active chromophore. The first approach is to start with

1

7

(

FETs). These initial electro-optic studies were usually per- existing NLO-active dyes, screen for water solubility and aﬃnity

formed with older-generation, alcohol-soluble dyes such as for targets of interest, and then modify the core structures

Disperse Red 1 (DR1, Fig. 1); substantially higher performance with pendant groups as needed. This approach has been suc-

would be needed for state-of-the-art device applications.

cessful for nucleic acid staining with isophorone derivatives

1

9

Both SHG activity and EO activity of a molecule are pro- such as Isoc (Fig. 1). These dyes are related to a class of ring-

portional to β, which requires a molecular structure that lacks locked, aminophenyltetraene-based compounds used for high-

12

inversion symmetry. The asymmetric structure creates an performance EO devices, such as the CLD-1 chromophore

2

0

anharmonic potential when the electrons in the material are developed by Zhang, Dalton, and co-workers, but have rela-

perturbed by an applied electric field. Symmetry is typically tively low aﬃnities for their intended targets. A similar

broken via a push–pull structure (Fig. 1), in which an electron- attempt at complexation with nucleic acids using other

21

donating moiety (e.g. dialkylamine or diarylamine) is connected uncharged OEO dyes met with limited success. Greater

to an electron-accepting moiety (e.g. pyridinium, nitro, or cyano) success was achieved for NLO-based imaging of membranes

2

by a conjugated bridge. Such an arrangement allows electron using glyconjugates of isophorone-based dyes. The second

density to more easily polarize in the donor → acceptor direction approach is to start with proven biological stains and screen

than in the acceptor → donor direction. Furthermore, the bulk for those that have NLO activity or could easily have their sym-

material itself must be non-centrosymmetric, and this typically metry broken to produce a NLO response. This approach has

2

3

requires aligning the dye molecules with electric field poling been applied to candidates such as ethidium bromide and

2

4

(

rotating dipoles by applying a voltage while the material is Hoechst 33342. We decided upon a hybrid approach, mixing

heated above its glass transition temperature), except in rare components from NLO-active dyes such as DAST (Fig. 1) and

cases such as DAST (4-N,N-dimethylamino-4′-N′-methyl-stilbazo- nucleic acid stains such as ethidium bromide and applying

lium tosylate; Fig. 1) where individual dye molecules crystallize design rules developed by Del Castillo et al., which require a

1

8

25

acentrically to form a ferroelectric material.

single positive charge and at least three aromatic rings.

2

1

However, one of the challenges in incorporating dyes with Computational screening of over 600 dyes chosen via chemi-

2

6

high nonlinear optical activity into biological systems such as cal intuition and combinatorics identified a dye now known

nucleic acids derives from a choice between divergent design as DANPY-1 (Fig. 1) that was predicted to have substantial

criteria: (1) dyes are typically optimized for high nonlinear hyperpolarizability, reasonable polarity for aqueous environ-

optical performance and stability if intended for use in photo- ments, and that was easily disconnected by retrosynthetic ana-

nics applications, or (2) dyes can be modified for aﬃnity to lysis. The dye is similar to the previously reported membrane

2

7

biological substrates, safety, and fluorescence performance if dye BNBP, but is cationic instead of zwitterionic and has

intended for biological staining. We have used theory-aided fewer long, hydrophobic alkyl chains. DANPY-1 also shares

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substantial structural commonality with the membrane stain the boronic acid in two portions improved the yield and

2

8

Prodan (Fig. 1) but with a charged methylpyridinium accep- lowered reaction time, yet heterogeneity remained a concern

tor instead of an uncharged propanone acceptor. Preliminary (entry 3). A MIDA (N-methyliminodiacetic acid) boronate was

2

6,29

characterization of DANPY-1 included aﬃnity to DNA,

tested under conditions optimal for less stable 2-heterocyclic

3

5

capability to form thin films in polymer hosts, sum-frequency nucleophiles, but conversion was incomplete in spite of the

generation (SFG) response on surfaces, including how the dye yield improving to 71% (entry 4). This could be the result

aﬀects the SFG response of DNA, genotoxicity tests in yeast,

3

0

31

of slower transmetallation for the pyridine 4-boronic ester

and exploratory characterization of potential biological appli- formed by alcoholysis in situ. In any case, Burke and co-

3

2,33

35

cations.

In the present work, we disclose a convergent, workers’ preference for DMF–alcohol solvent media proved

three-step synthesis of DANPY-1 from commercially available enabling, and a control experiment to probe the singular influ-

materials, along with extensive characterization of structural, ence of 4 : 1 DMF–EtOH as solvent diminished reaction time

photophysical, and biocompatibility-related properties in further and allowed direct crystallization of product in 50%

order to demonstrate that the DANPY fluorophore meets the yield after workup (entry 5). Final refinements in reaction

design expectations for both nonlinearity and biocompatibi- eﬃciency were achieved by catalyst/ligand screening. As shown

lity. We expect these findings to enable development of an in entry 6, a combination of 3 mol% Pd

entire family of DANPY-derived dyes for biological imaging equiv. of SPhos

2

(dba)

gave >98% conversion in 4 h and 95% of 3

by altering substituents as a way to tune absorbance/fluo- after a filtration through silica gel. Loading was further

3

and 4 catalyst

3

2

36,37

rescence properties, hyperpolarizability, and hydrophobicity.

2 3

reduced to 1 mol% Pd (dba) /4 mol% SPhos on a gram scale

with regard to 1; 64% of product 3 was isolated in a single

crop as thin needles by crystallization from CHCl (entry 7).

3

Completing

the

synthesis

required

site-selective

Synthesis

N-alkylation. We find that gentle warming of a dilute solution

Our preparative studies began with the Suzuki–Miyaura cross of 3 and 1.3 equiv. of MeI in dry acetonitrile aﬀords DANPY-1

coupling of pyridyl boronic acid 2 with 6-bromo-N,N-dimethyl- as a bright red, microcrystalline solid in high yield following

naphthalen-2-amine (1), itself accessible by dimethylation silica gel chromatography and recrystallization (Scheme 1).

of commercially available 6-bromo-naphthalen-2-amine (see The full synthesis was scaled up, producing 2.6 g of DANPY-1

Table 1); synthesis of 1 is detailed in the ESI.† An alternative to support characterization, photophysical studies, biological

synthesis in which the Suzuki coupling was performed directly staining studies, and toxicity testing. The salt is hygroscopic

on 6-bromo-2-naphthylamine followed by exhaustive methyl- and best stored under nitrogen or in a desiccator to prevent

2

6

ation proved substantially less eﬃcient due to solubility of

the starting material and/or background deactivation of the

catalyst. Two challenges to the success of the Suzuki coupling

3

4

included the electron-rich nature of electrophile 1 and the

low organic solubility of 2. Since commercial samples of 2 can

be decolorized by recrystallization from MeCN–H O, an

2

opening trial utilized 1 : 1 MeCN–EtOH as solvent, aﬀording

2

8% of 3 (Table 1, entry 1). A base screen revealed that K

2 3

CO

was of similar eﬃcacy to Na CO (Table 1, entry 2). Addition of Scheme 1 Pyridinium installation and anion exchange.

2

3

a

Table 1 Suzuki optimization

Entry

Catalyst/ligand

Base

Na CO

Solvent

t (h)

Isolated yield

1

2

3 mol% Pd(PPh

3

)

4

; none

; XPhos

; none

; SPhos

2

3

MeCN/EtOH

4 : 1 DMF : EtOH

24

12

8

4

28%

30%

39%

71% (6% rsm)

50% (crystals)

95%

3

K

Na

2

CO

3

b

3

2

CO

c

4

3 mol% Pd

2

(dba)

3

K

2

CO

3

5

6

7

3 mol% Pd(PPh

)

3 4

Na

2

CO

3

3 mol% Pd

1 mol% Pd

2

(dba)

3

CO

d

2

3

64% (crystals)

a

b

c

Reactions performed on 0.2 mmol of 1, 0.05 M, in a sealed vial under N

2

atm. 2 added in two batches. With 4-pyridinylboronic acid MIDA

d

ester. 1 g scale.

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absorption of water. Experiments suggest that the study of

other ion pairs is likely to be straightforward, either using

alternative electrophiles such as methyl tosylate or through

metathesis with silver(I) salts. For example, the iodide anion

can be converted to nitrate in quantitative yield by treatment

of DANPY-1 with 1.05 equivalents of silver nitrate (Scheme 1).

Tuning of dye properties (e.g. hydrophobicity or strength of

electron-donating or electron-accepting groups) can be readily

achieved by altering the N-substitution of the naphthylamine Fig. 2 ORTEP structure of DANPY-1 with triiodide (I ⁻) counterion,

3

2,38

1

with thermal ellipsoids at 50% probability. The space group is P2 /n

prior to the Suzuki coupling

or during quaternization of

3

2

and the cell parameters are a = 28.113(3) Å, b = 13.9070(15) Å, and

c = 20.473(16) Å.

the pyridine in the final step of the synthesis. Other DANPY

derivatives will be discussed in upcoming work; the remainder

of this article will focus on rigorous characterization and appli-

cations of DANPY-1.

torsional barrier of 25 kJ mol⁻from a relaxed potential energy

surface scan of the aryl–aryl dihedral angle (Fig. S2†). The

rotamer in which the pyridinium and naphthalene are co-

planar is about 5 kJ mol⁻higher in energy than the equili-

brium geometry and, therefore, thermally accessible at 300 K.

Furthermore, the rigidity of DANPY-1 is enhanced compared

to DAST due to the fused naphthalene ring. The larger fused

ring system improves compatibility with a planar, aromatic

environment such as a nucleic acid helix, increasing the poten-

tial for intercalation. Further structural details are provided in

the ESI.†

1

Structural characterization

Structural characterization of DANPY-1 was performed by high

1

13

field H and C NMR spectroscopy. NMR spectral data are

available in the ESI,† along with both thin-film and DFT-

computed infrared and Raman spectra (Fig. S1†). Furthermore,

since DANPY-1 is a salt, elemental analysis was used to identify

the counterion. CHNOX analysis performed by Galbraith labora-

tories on samples from a multi-gram batch of DANPY-1 identi-

fied the composition of the material from duplicate tests as

5.19 ± 0.16% C, 4.94 ± 0.04% H, 6.99 ± 0.05% N, with oxygen

near the detection limit of 0.5%. The large non-CHON fraction

33%) is consistent with an iodide counterion (expected: 55.4%

5

(

C, 4.91% H, 7.18% N, 35.2% I). Corroboration of iodide as the

counterion was performed by elemental analysis on a separate

Optical properties in solution

batch of DANPY-1 for which the anion had been previously We evaluated linear optical properties of DANPY-1 in water,

2

9,30

reported

as acetate due to presence of a stoichiometric methanol, dimethylsulfoxide (DMSO), chloroform, and pH 8

0.5× TAE (20 mM Tris, 10 mM acetic acid, 0.5 mM EDTA)

quantity of co-crystallized acetic acid (see ESI†).

DANPY-1 proved diﬃcult to grow X-ray diﬀraction (XRD)- buﬀer. We determined the extinction coeﬃcient of free

quality crystals of suﬃcient size with an iodide counterion. DANPY-1 in pH 8 TAE buﬀer at λmax (414 ± 2 nm) to be

Nonetheless, satisfactory crystals were eventually obtained from 16 900 ± 20 M⁻cm based on four measurements of a 60 µM

a slightly less polar chromatography fraction at the tailing edge solution of DANPY-1 prepared from 4.94 mg of dye which

of the DANPY-1 peak. This fraction was found to have a triio- had been vacuum-dried and weighed under dry nitrogen.

dide (I₃⁻) counterion. These findings suggest some oxidation of UV/Visible absorbance was measured in various solvents with

the iodomethane starting material to form molecular iodine, 15 µM concentrations; spectra are shown in Fig. 3A. The dye

although partial oxidation of the counterion on the silica gel was readily soluble at this concentration in all solvents tested.

may represent another potential source of this impurity. The Fluorescence behavior of DANPY-1 was also characterized

crystal structure (CCDC: 1838228†) is shown in Fig. 2; details of using the same set of 15 µM solutions used for the absorbance

1

−1

crystallographic characterization are reported as ESI.†

measurements; spectra are shown in Fig. 3B.

The crystallographic data confirms that unlike DAST,

The dominant absorbance peak was determined to be an

DANPY-1 with a triiodide counterion crystallizes centrosym- intramolecular charge transfer (ICT) excitation based on DFT

metrically; i.e. would not be expected to have a bulk SHG calculations; details are discussed as ESI.† The strength of the

response. It is not yet known whether it can be acentrically principal charge transfer absorbance band in DANPY-1

crystallized with a larger counterion such as tosylate. The increases substantially as solvent polarity is decreased;

crystal structure also indicates that the molecule is slightly increases in fluorescence intensity were even more dramatic.

distorted from planar, with a naphthyl–pyridyl dihedral Furthermore, we observed a large hypsochromic shift in absor-

angle of 25.9°. The small dihedral angle is consistent with bance, with the absorbance maximum moving 61 nm to the

density functional theory (DFT) calculations at the B3LYP/ blue on going from chloroform (least polar) to water (most

cc-pVTZ level of theory in an implicit methanol solvent polar). By contrast, the fluorescence maximum was observed

(

polarizable continuum model, PCM) environment. These to undergo a large bathochromic shift, moving 44 nm to the

calculations yielded a dihedral angle of 20.17° at the equili- red between chloroform and water. Absorbance and fluo-

brium geometry, consistent with the crystal structure, and a rescence data are detailed in Table 2. The nearly symmetric

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Fig. 3 Absorbance (A) and ﬂuorescence at 450 nm excitation (B) of DANPY-1 as a function of solvent. The ﬂuorescence intensity in water or pure

TAE buﬀer is very low and is shown on a logarithmic scale in Fig. S3.†

Table 2 Absorbance and ﬂuorescence properties of DANPY-1

Abs. max

(nm)

Rel. abs vs.

ε

max

Fluor. max

(nm)

Stokes shift

Rel. fluor vs.

DI H O

2

(M cm⁻¹

−

1

)

(cm

−1

)

Solvent

DI H

2

DI H O

2

O

412

414

465

442

448

473

1

18 600

16 900

18 200

30 500

35 500

621

625

609

627

618

577

8170

8160

5090

6680

6140

3810

1

0.5× TAE (pH 8)

0.91

0.98

1.64

1.91

1.2

190

23

7.3

53

TAE/DNA

DMSO

Methanol

Chloroform

solvatochromic shift is consistent with observations collected upon binding, we also calculated the eﬀective fluorescence

3

9

42

on other pyridinium-based chromophores, including BNBP,

eﬃciency,

and is indicative of reversal of the sign of the molecular dipole

upon excitation, which was confirmed by DFT calculations

Imax;solvþDNA

I_m_a_x_;_s_o_l_v

εsolvðλabsÞ

QðλabsÞ ¼

^ꢀε

ð1Þ

(

Table S3†). Coupled with the large Stokes shift (209 nm, or

ðλ_a_b_sÞ

solvþDNA

1

8

170 cm⁻, in H

2

O), the strong sensitivity of DANPY-1’s absor-

bance and fluorescence maxima to diﬀerent dielectric environ- at an excitation wavelength of 414 nm, obtaining Q = 106. The

ments shows promise for wavelength-dependent sensing of fluorescence enhancement compares favorably with the Q

4

2

substrates with diﬀerent polarities. While the magnitude and of 17.5 ± 0.3 observed by Garbett for ethidium bromide.

dielectric sensitivity of DANPY’s Stokes shift are similar to that A larger Q-factor indicates greater fluorescence enhancement

of Prodan, instead of undergoing a small bathochromic shift on binding, while a Q-factor below unity indicates quenching

in absorbance and a large bathochromic shift in fluo- of fluorescence on binding.

4

0

rescence, the signs of the solvatochromic shifts are opposed

To further explore the large diﬀerence in fluorescence of

due to intramolecular charge transfer reducing the dipole DANPY-1 in diﬀerent environments, we measured absolute

moment of the first excited state of DANPY-1. In contrast, fluorescence quantum yields (ϕ) via the integrating sphere

4

3

charge transfer in Prodan involves a transition from a neutral method and fluorescence lifetimes (τ ) via time-correlated

f

4

but polar ground state to an excited state with even greater single-photon counting. Fluorescence lifetimes were fit to a

4

1

45

charge separation.

double-exponential model using DecayFit; a₁is the weight

In addition to characterizing solvatochromic response, on the first of the two exponentials. Experimental details are

we also examined the eﬀect of adding an excess of DNA in further discussed as ESI.† To evaluate suitability for TPEF-

0

3

.5× pH 8 TAE buﬀer, such that the final solution was based techniques, we measured two-photon absorption (TPA)

6

,46

00 µM in DNA base pairs (salmon sperm DNA). The fluo- cross-sections (δ_T_P_A) by the TPEF method,

using a Ti:sap-

rescence intensity increased dramatically when DNA was phire laser with a fundamental wavelength of 800 nm and

added, with Imax,TAE+DNA/Imax,TAE = 164 (190× vs. H O). In 129 µM aqueous fluorescein at pH 11 as a reference, for which

order to correct for changes in the extinction coeﬃcient δ_T_P_Ahas been previously measured to be 36 GM. TPA values

2

4

7

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2

7

Table 3 Additional photophysical properties of DANPY-1

small variation with solvent, unlike BNBP, suggesting that

the interaction of the side chains with the solvent environment

has a large eﬀect on the fluorescence lifetime. While over 99%

of the fluorescence intensity is associated with this rapid relax-

ation, fitting of the fluorescence decay curves (Fig. S4†) is sub-

stantially improved by including a small fraction of a second

exponential with a longer (3–5 ns) lifetime. More substantial

double-exponential behavior has been observed for Prodan in

Quantum

yield (ϕ)

δ

TPA

(GM)

Solvent

τ

1

(ns)

a

1

τ

2

(ns)

Chloroform

DMSO

Methanol

pH 8 TAE

TAE w/DNA

0.142

0.222

0.057

0.004

2.72

0.994

0.997

0.999

0.509

3.34

3.31

4.57

4.45

0.709

0.068

0.045

0.010

0.005

0.174

71

61

a

—

b

c

—

30

5

a

polar solvents. DANPY-1 exhibited substantially longer fluo-

TPEF signal was substantially red-shifted from one-photon fluo-

b

rescence peak; see ESI. Smaller than instrument resolution; not sig- rescence lifetimes when bound to DNA, and substantial

c

nificant and included only for completeness of fit. No TPEF signal

double-exponential behavior (a ∼0.5), which may suggest that

the dye binds within multiple environments in the DNA. The

1

was detected.

increase in fluorescence lifetime is consistent with that

5

6

observed for ethidium bromide.

DANPY-1 has a substantial TPA cross section (Table 3) for its

are typically reported in Goppert–Mayer units; 1 GM = 10⁻cm s

50

4

per photon. Results are shown in Table 3, and methodology is size, approximately twice that of aqueous fluorescein when

discussed as ESI;† uncertainty in δ_T_P_Ais estimated at 25% measured in chloroform or DMSO, or comparable to aqueous

based on measurements at three laser power levels.

fluorescein when it is bound to DNA. The TPA cross sections of

DANPY-1 has a minuscule (≤0.01) fluorescence quantum DANPY-1 in each environment studied are substantially larger

57

yield in polar, protic solvents such as methanol or water. than the 19–20 GM observed for similarly-sized Prodan.

However, its quantum yield when bound to DNA is comparable Larger organic molecules can have much higher TPA cross sec-

to that of ethidium bromide (0.15) and propidium iodide tions, including several carbazole derivatives engineered for

4

8,49

6

(

0.16).

While substantially lower than Hoechst 33258 combined SHG/TPEF imaging, as can organometallic com-

4

9

49

0.42) and SYBR variants SYBR Gold (0.6) and SYBR Green plexes that incorporate large, conjugated ligands with values on

5

8

0.8), which are often used for nucleic acid visualization, the the order of 1000 GM. In sum, the small size and simplicity,

large Q and extinction coeﬃcient (3× that of ethidium synthetic approachability, and high photostability of

5

0

−1

bromide, where ε = 5450 M cm at λmax = 480 nm) suggest DANPY-1 makes it a compelling candidate for TPEF.

that DANPY-1 could be competitive for the gel electrophoresis

The photochemical stability of DANPY-1 was assessed by two

of nucleic acids or other cellular components with less polar experiments: (1) a 91-day photostability study in which the

media than water, particularly due to its relative synthetic ease maximum absorbance of DANPY-1 solutions (ambient light at

and low toxicity.

room temperature, dark at room temperature, dark at +5 °C, and

Quantum yields in polar aprotic solvents are low but are dark at −20 °C (one solution per condition) was measured by UV/

larger than in water or methanol, suggesting that hydrogen Visible spectroscopy; and (2) chemical stability toward several

bonding may have a substantial role in quenching fluo- common laboratory reagents was monitored over a period of two

rescence from DANPY-1. A major factor behind the low weeks. Photostability was excellent, with samples stored in the

quantum yield of DANPY-1 is likely non-radiative relaxation dark (ambient or +5 °C) experiencing a ∼3% decrease in the

due to rotation of the aryl–aryl bond between the donor and absorbance intensity of λ_m_a_xafter 91 days and the benchtop

5

1,52

acceptor regions of the dye.

The eﬀects of torsional flexi- (ambient light) sample experiencing a 7.3% decrease in absor-

bility were examined via a viscosity-dependent fluorescence bance intensity. Additional photostability and chemical stability

study using methanol : glycerol mixtures; integrated fluo- results are compiled and included as ESI (Fig. S6†).

rescence intensity at constant concentration increased by over

The hyperpolarizability of DANPY-1 was determined in both

a factor of 3 from methanol (low viscosity) to 1 : 2 methanol : dichloromethane (DCM) and methanol by Hyper-Rayleigh scat-

5

9

glycerol (high viscosity), strongly supporting the role of aryl– tering (HRS). HRS experiments were performed using a Ti:sap-

5

3

aryl rotation in non-radiative relaxation. Similar eﬀects have phire laser with a fundamental wavelength of 800 nm and refer-

5

4

60

been observed in BODIPY dyes with flexible aryl–aryl bonds.

enced against Crystal Violet. Signal contamination from two-

The quantum yield of DANPY-1 in chloroform is also an order photon fluorescence was suppressed using high-frequency

2

7

61

of magnitude lower than that observed from BNBP, which is demodulation. The hyperpolarizability at 800 nm was extrapo-

62–64

substituted with butyl chains at each nitrogen; the side chains lated to the static limit using the damped two-level model,

may produce drag in a dense solvent environment that inhibits

aryl–aryl rotation. However, DANPY-1 exhibits a similar depen-

dence on solvent dielectric constant to BNBP, in which the

Stokes shift increases with dielectric constant and the

quantum yield decreases with dielectric constant.

DANPY-1 in solution exhibits short (∼100 ps) fluorescence

lifetimes, consistent with other dyes with flexible aryl–aryl

bonds. The fluorescence lifetime of DANPY-1 exhibits only a

βðꢁ2ω; ω; ωÞ

βð0Þ ¼

Fðωlaser; ωmax; γÞ

ꢀ

1

þ

ðωmax þ 2ωlaser þ iγÞðωmax þ ωlaser þ iγÞ

2

ωmax

1

Fðωlaser; ωmax; γÞ ¼

ðωmax þ ωlaser þ iγÞðωmax ꢁ ωlaser þ iγÞ

3

1

ðωmax ꢁ ωlaser þ iγÞðωmax ꢁ 2ωlaser þ iγÞ

5

2

ð2Þ

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where ωmax is the wavenumber corresponding to the wavelength

of maximum absorbance for the light (λ_m_a_x), ω_l_a_s_e_ris the wave-

Nucleic acid binding

number corresponding to the wavelength of the laser used for To characterize the in vitro interactions between DANPY-1 and

the experiment (λlaser), γ is a homogeneous damping parameter DNA in solution, we performed binding titrations by absorption

of 0.1 eV (806 cm⁻), i is the imaginary unit, and ω = 2πc/λ. The spectroscopy and electronic circular dichroism (CD) spec-

hyperpolarizability component along the chromophore dipole troscopy (Jasco J-720), using salmon sperm DNA as a substrate.

1

6

5

(βzzz) assumed to be the dominant tensor element

for As these experiments used isolated materials, they only provide

DANPY-1 given its linear structure; thus β_z_z_z≈ 0.414β_H_R_S

.

evidence of the magnitude of binding aﬃnity towards the sub-

Hyperpolarizabilities are compiled in Table 4, along with strate tested and do not confer any information about possible

DFT computed values determined using analytic diﬀeren- interference from other substrates in a living cell. Spectroscopy

6

tiation at the M062X/6-31+G(d) level of theory with a PCM data were analyzed using a standard macromolecular identical

7

6

solvent; this model has proven highly applicable in comparing and independent site binding model, in which the binding is

hyperpolarizabilities among high-performance chromo- represented as a reversible reaction between a ligand (L, the

6

7

68

phores. Calculations were run using Gaussian 09. Unlike dye) and binding sites on a macromolecule (N, the DNA) to

HRS, DFT calculations are capable of determining the sign of form a complex (LN) with equilibrium constant

hyperpolarizability for a chromophore. The experimental HRS

6

9

½LNꢂ

values for DANPY-1 were previously reported

based on

K ¼

ð3Þ

½

Lꢂ½Nꢂ

acetate as a counterion but have been corrected for the now

definitive iodide anion and for the purity of the solution.

DANPY-1 exhibits comparable hyperpolarizability to DR1,

DAST, and isophorone derivative Isoc, indicating that sub-

stantial NLO activity is retained despite replacing the conju-

gated bridge with an aryl–aryl linkage. Its hyperpolarizability is

also competitive with carbazole dyes, and given its small π

electron count (16), is near the ‘apparent limit’ of hyperpolar-

At a wavelength where the bound and unbound dye are the

only significant absorbing species, the equilibrium constant

can be determined by titrating the macromolecule into a solu-

1

9

50,77

tion of the dye,

with the fraction of dye bound ( f

b

) related

to the absorbance as

A=½L ꢂ ꢁ ε

0

f

7

3,74

fb ¼

ð4Þ

izability in organic molecules,

indicative of very eﬃcient

nonlinear optical activity. A potential explanation for the high

ε ꢁ ε

b

f

6

2

where ε

f

is the extinction coeﬃcient of the free dye, ε

extinction coeﬃcient of bound dye, and [L

centration of dye (bound or unbound). The equilibrium con-

stant can be written in terms of the fraction of dye bound,

is the

b

] is the total con-

eﬃciency based on the two-state model is the large dipole

change between the ground and first excited states (Table S3†).

The experimentally measured hyperpolarizability of DANPY-1

is more sensitive to the solvent environment than that pre-

dicted by DFT calculations. Some discrepancies between the

experimental and DFT results can be attributed to the DFT

0

fb½L0ꢂ

K ¼

ð5Þ

ð½L0ꢂ ꢁ fb½L0ꢂÞð½N0ꢂ ꢁ nfb½L ꢂÞ

6

7

0

functional itself, application of the two-level model for fre-

quency extrapolation, or from limitations of the PCM model where [N ] is the total concentration of DNA base pairs (bound

0

7

5

versus an actual solvent. Interestingly, the trend between or unbound) and n is the number of base pairs per dye

DCM and methanol indicates that hyperpolarizability is sub- binding site (e.g. an intercalator, which sits between base

stantially suppressed by more polar environments. The pairs, has n ∼2). Eqn (4) and (5) directly relate the equilibrium

decrease in hyperpolarizability with increasing solvent polarity constant to absorbance, allowing determination of K and n via

contrasts with the behavior exhibited by neutral chromo- either a double-reciprocal linear relation (Scatchard plot) or

7

5

phores and is consistent with the hypsochromic shift in directly via nonlinear regression. Details of the binding model

absorbance. Collectively, these results imply that DANPY-1 will derivation and fitting are provided as ESI.†

exhibit a stronger SFG, SHG, or EO response if ordered in a

less polar environment.

Binding titrations were performed using fresh solutions of

DANPY-1 and salmon sperm DNA in either pH 7.2 1× TE

Table 4 Computed and experimental hyperpolarizability of DANPY-1 and comparable dyes. Hyperpolarizabilities are reported in 10⁻³⁰esu

Dye

Solvent

λlaser (nm)

λmax (nm)

|βzzz(λ)| (exp)

|βzzz(0)| (TLM)

βzzz(0) (DFT)

DANPY-1

DCM

Methanol

MeCN

CHCl

800 (HRS)

484

448

470

478

496

461 ± 4

358 ± 5

440 ± 10

138 ± 1

66 ± 1

112 ± 3

150 ± 10

166

−155

−151

−255

153

−

70

a

DAST:PF

DR1

6

800 (HRS)₇

1

b

3

780 (HRS)

468 ± 30

7

2

c

Isoc

3

1907 (EFISH)

242

160

a

b

of 0.15 × 10⁻³⁰esu. Electric field-

c

Identical cation to DAST; diﬀerent counterion. HRS referenced to chloroform solvent assuming βHRS CHCl

3

induced second harmonic generation (EFISH) measurement, uncertainty not reported by authors.

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Fig. 4 (A) Photometric titrations of DANPY-1 with salmon sperm DNA. Blue circles – titration of 2.5 mL of 49 μM DANPY-1 in pH 7.2 TE buﬀer with

0 μL aliquots of 1.52 mM DNA. Red squares – titration of 2.5 mL of 40 μM DANPY-1 in pH 7.2 TE buﬀer with 30 μL aliquots of 1.52 mM DNA. Black

3

diamonds – titration of 2.5 mL of 51 μM DANPY-1 in 0.5× pH 8.0 TE buﬀer with 30 μL aliquots of 1.71 mM DNA. DNA solutions were in the same

buﬀer and at the same pH as the corresponding dye solutions. (B) UV/Visible spectra from pH 8 titration after correcting for dilution, showing red-

shift of DANPY-1 absorbance maximum upon interaction with DNA.

buﬀer (10 mM Tris, 1 mM EDTA) or pH 8 0.5× TAE buﬀer. groove binder Hoechst 33342 has an aﬃnity to poly(dAT) of

7

−1

These conditions were chosen to represent cytoplasm (pH 7.2) 3.23 × 10 ± 1.25 × 10 M in 10 mM cacodylate buﬀer at pH

7

9

and typical gel electrophoresis buﬀers (pH 8). Solutions were 6.8, much higher than either ethidium bromide or DANPY-1

mixed on a roller to ensure homogeneity and allowed to stand at pH 7.2. The binding aﬃnity of DANPY-1 decreased to about

overnight before use. Titration curves are shown in Fig. 4, half that of ethidium bromide at pH 8 and n increased, indi-

along with the UV/Visible spectra from the pH 8 titration; cating a potential transition to a diﬀerent binding mode in

experimental details are included as ESI.†

which each binding site involves more base pairs. On the

Binding titrations were also run with ethidium bromide as other hand, n decreased with increasing pH for ethidium

a positive control; results are given in Table 5 with ethidium bromide, suggesting a greater contribution from simple

7

bromide titration curves included as ESI (Fig. S7†). RNA electrostatic attraction to the exterior of the double helix. The

binding was also observed in preliminary experiments (ESI, DANPY-1 pH 8 binding curve also shows some sigmoidal char-

Fig. S8†), but insuﬃcient data was obtained to calculate a acter, potentially indicative of multiple binding modes

reliable binding constant.

between the dye and DNA under increasingly basic conditions.

Structural information concerning the binding mode of

At pH 7.2, DANPY-1 has a DNA aﬃnity comparable to ethi-

dium bromide, and both dyes exhibit a binding stoichiometry DANPY-1 was further explored by CD spectroscopy in pH 7.2

with n ∼2, suggesting that DANPY-1 may bind by intercalation TE buﬀer, comparing against ethidium bromide and focusing

as is known for ethidium bromide at this pH. The aﬃnity of on the ultraviolet region in which DNA exhibits substantial

7

8

DANPY-1 to DNA was also similar to that observed between CD. A higher concentration of ethidium bromide was used

the iodide salt of DAST and salmon DNA in water (22 000 such that the absorbance of the two samples was similar.

−

1

M

). However, the fluorescence amplification of bound versus Results are shown in Fig. 5.

unbound dye in water was over an order of magnitude larger DANPY-1 causes a decrease in the magnitude of the

for DANPY-1 than DAST (190× vs. 9.5×), which may implicate observed CD of the DNA at 245 nm, similar to that previously

diﬀerent binding modes. The equilibrium constant for observed for isophorone derivatives Isoa and Isoc and poten-

1

9

DANPY-1 is also substantially larger than those observed for tially indicative of a partial B-DNA to A-DNA transition.

uncharged, isophorone-based dyes explored for DNA-based Combined with the strong fluorescence enhancement and

biophotonics, for which the best compound (Isoc) has an binding stoichiometry, this shift could suggest an intercalation

aﬃnity of 6890 M⁻

1 19

.

By contrast, the high-performing minor binding mode and a similar mechanism for fluorescence

enhancement to ethidium bromide. However, unlike DANPY-1,

ethidium bromide causes large-scale disruption of the DNA

helix, with shifts in CD observed across the entire 225 nm to

Table 5 Binding of DANPY-1 and ethidium bromide to DNA

3

75 nm wavelength range in a manner consistent with prior lit-

5

0

80

K (M⁻

1

,

n

K (M⁻¹

pH 8 TAE)

,

n

erature, including the formation of dye-associated peaks.

Dye

pH 7.2 TE)

(pH 7.2)

(pH 8 TAE) The smaller disruption to the structure of the DNA helix upon

complexation with DANPY-1 suggests that DANPY-1 is less

21 900 ± 3600 1.79 ± 0.03 10 900 ± 300 2.34 ± 0.11

DANPY-1

Ethidium bromide 19 600 ± 900 1.74 ± 0.05 22 500 ± 1300 1.49 ± 0.04

likely to exhibit genotoxic eﬀects than ethidium bromide, a

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Fig. 6 Visualization of oligomeric DNA using DANPY-1 dye.

Commercially acquired DNA ladders having sequences of diﬀerent size

distributions were loaded onto 0.8% agarose gels. When DNA was elec-

trophoretically separated, then post-stained and illuminated with blue

light, excellent visualization of: (A) 10 kb, (B) 12 kb and (C) 1.5 kb com-

ponents was observed.

Fig. 5 Electronic circular dichroism spectra for 198 μM DNA in pH 7.2

TE buﬀer (black line), 274 μM DNA + 44.4 μM DANPY-1in pH 7.2 TE

buﬀer (red line), and 292 μM DNA + 103 μM ethidium bromide in pH 7.2

TE buﬀer. DANPY-1 induces a change in DNA that decreases the magni-

tude of circular dichroism at 245 nm with no other observed eﬀects,

unlike ethidium bromide, which also alters the 275 nm DNA peak and

forms two new peaks (295 nm and 310 nm).

(

Auxvasse, MO). Complete toxicity results and detailed meth-

odologies used in their genesis are available in the “Final

conclusion further supported by results of an Ames Test (see reports” provided by these laboratories (see ESI†). Four total

Toxicity section).

tests were run – two evaluating eﬀects on mammals and two

on cultured cells.

The ISO/USP Intracutaneous Reactivity Irritation Test (Test 1)

measured the impact of DANPY-1 when applied to the skin

of Albino New Zealand White rabbits. The ISO/USP Medical

Gel electrophoresis

In addition to solution-phase measurements of binding Device Systemic Toxicity Test (Test 2) monitored the eﬀects of

aﬃnity and fluorescence, which were conducted at high DNA DANPY-1 on Albino CD-1 mouse health before, immediately

concentrations (e.g., 30 to 50 μM), gel electrophoresis was used after, and for three days following intravenous dye injection

to determine the sensitivity of DANPY-1 to micro-quantities of into the mice (e.g., clinical observations were made and body

small sized DNA sequences. Commercially available DNA refer- weight was determined). Both the ISO/USP Intracutaneous

ence ladders containing sequences of known size (from 100 to Reactivity Irritation Test and the ISO/USP Medical Device

1

5 000 bp) were electrophoretically separated on 0.8% agarose Systemic Toxicity Test animals showed no diﬀerences in

gels. DNA-dye complexes were formed using three complemen- measured parameters after DANPY-1 dye administration rela-

tary techniques: (1) adding DANPY-1 to the agarose gel while tive to those observed for the negative controls.

the agarose was in molten pre-pour phase (0.96 μg mL⁻);

1

DANPY-1 toxicity data generated from unicellular assays

(

2) adding DANPY-1 to the agarose gel running buﬀer was more ambiguous. The Ames assay (Test 3) screens for

−

1

0.32 μg mL ) or (3) post-staining gels with DANPY-1 solution potential mutagenic eﬀects on bacteria. In this test, five histi-

1.92 μg mL⁻). All methods of dye addition gave an identical dine-dependent strains of Salmonella typhimurium (TA97a,

1

response (see ESI†). The resulting DANPY-1 stained DNA frag- TA98, TA100, TA1535, and TA102) were exposed to various con-

ments were visualized using blue light (400–500 nm range). As centrations of DANPY-1 dye. The treated bacterial cultures

shown in Fig. 6, DANPY-1 complexed with DNA sequences were then monitored for successful mutagenic reversion (e.g.,

within each ladder array, regardless of nucleic acid fragment the loss of autotrophic dependence on exogenous histidine

size. Total DNA load per lane in Fig. 6 was 2.0 μg.

addition to the growth medium). No significant diﬀerences

from the negative control were observed at the concentrations

of DANPY-1 tested. However, the Minimal Essential Media

Elution test (Test 4) that monitors cytotoxicity by examining

mouse fibroblast L929 cellular morphology (e.g., the rounding

Toxicity

Third party testing of DANPY-1 genotoxicity was accomplished of the cells to their complete lysis) gave indication that

by Nelson Laboratories (Salt Lake City, UT), with some assays DANPY-1 could potentially have a negative impact on cell func-

performed in partnership with Sinclair Research Center tion. The buﬀer control for this assay showed a mild impact,

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Fig. 7 Impact of DANPY-1 on the survival of Chrysochromulina tobinii (Haptophyta). Growth curves for entire population when exposed to

diﬀerent concentrations of DANPY-1 (fraction of cells stained at 0 time is shown in the inset) (A); increase in cells within the culture populations that

were treated with either (B) 5.0 μM or (C) 18 μM DANPY-1; stained (red) and unstained (blue) cells within the culture. Note cell concentration diﬀer-

ence between B and C.

and the highest concentration of dye tested (212 μM) exhibited

moderate morphological cytotoxicity.

Biological staining

Observations concerning the impact of DANPY-1 on cul- While in vitro binding studies demonstrated aﬃnity of

tured eukaryotic cells were extended by monitoring the DANPY-1 to nucleic acids (e.g., Fig. 4), these studies did not

growth of the unicellular alga Chrysochromulina tobinii provide insight into the interaction of the dye with additional

(

Haptophyta) in the presence of the dye. Cells were exposed components of living cells. To generate an overview of in vivo

to diﬀerent concentrations of DANPY-1 (0 to 18 μM). Flow DANPY-1 staining potential, a selection of eukaryotic organ-

cytometric assessment showed that initially (time 0), the per- isms were exposed to the dye, and cellular responses were

centage of stained cells observed in a culture directly corre- examined using confocal microscopy. Organisms were chosen

lated to the amount of dye added (e.g., at 7.0 μM concen- for their broad taxonomic representation and their unique

tration, almost all cells were labeled; Fig. 7A insert). cellular structures. Microscopy experiments included both con-

Subsequent total cell counts suggests that robust cell division focal fluorescence microscopy and nonlinear (TPEF and SHG)

continued to occur in the presence of the dye for almost all confocal microscopy.

cultures, except the culture having the highest DANPY-1

addition (Fig. 7A). A closer analysis of stained versus

Confocal linear fluorescence microscopy

unstained cells revealed that at lower dye concentrations (e.g., Giardia lamblia (Sarcomastigothphora) is a eukaryote that

5

.0 μM), unstained cells dominated as the culture density parasitizes the small intestine of vertebrates. This protist cell

increased over time (Fig. 7B). In contrast, at more elevated is “reduced” – lacking typical intracellular components such as

8

3

dye treatments (e.g., 18 μM), although nearly 100% of the mitochondria and peroxisomes. DANPY-1 appeared to loca-

cells initially stain, cells apparently failed to divide and lize perinuclear and also to network throughout the cytoplasm

8

4–86

culture density decreased (Chrysochromulina cells are limited of this organism (Fig. 8A).

solely by plasma membrane and most likely lyse). are unique protists in that their chromosomes, which lack

Interestingly, after approximately one week of culture, the typical histones, are permanently condensed. Both dinoflagel-

8 μM DANPY-1 treated culture finally increased in cell late representatives chosen for this study have a complex cell

Dinoflagellates (Dinoflagellata)

a

1

number. Few cells in this newly generated population were covering (the amphiesma) that is sandwiched between an

stained (Fig. 7C). Whether the DANPY-1 dye was: (1) degraded exterior outermost membrane and the plasma membrane of

8

7

by or interacts with a component of the growth medium (the the cell. This structure is comprised of a series of sacks that

alga extrudes a broad range of organic molecules into the encase an undefined array of organic materials that are

8

1

88,89

minimal fresh water medium in which it is grown), (2) was taxonomically determined.

When Prorocentrum micans was

metabolized by the alga, or (3) a dye-refractile sub-population DANPY-1 stained, not only were large chromosomes readily

of Chrysochromulina tobinii cells was generated (see Lakeman visible, but additional cellular target sites were also seen,

8

2

et al. for discussion) requires further study.

including a sharp delineation of the amphiesma and apical

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In summary, it appears that DANPY-1 cellular staining is

not limited to one cellular component, but aﬃnity of this dye

may be determined by both the concentration and molecular

composition of the target site. Following the broad imaging

survey presented above, future cytological studies can now

focus on co-localization of DANPY-1 with commercially avail-

able organelle-specific markers.

Confocal nonlinear microscopy

Preliminary evaluation of DANPY-1 as a dye for nonlinear

microscopy techniques was conducted using HeLa cells in

Dulbecco’s Modified Eagle Medium (DMEM) and a Zeiss

inverted confocal microscope with 810 nm excitation and

linear polarization of the incident laser. Cells were examined

at 10° polarization increments; images are shown for both

horizontal and vertical polarization orientations. In contrast

with the linear confocal experiments, DMSO was used as a

solubility/penetration aid, consistent with prior experiments

6

on carbazole derivatives. Penetration of the dye into the cells

and emergence of the SHG signal was followed in real time.

Total experiment time was 50–60 min, with 7 μM dye added at

minute 1 of imaging.

TPEF (Fig. 9, horizontal polarization in panel A and vertical

polarization in panel B) appeared rapidly, with signal observed

from inside the cells within the first minute after addition, stabi-

lizing quickly in the endoplasmic reticulum, mitochondria, and

some signal was observed in the denser parts of the nucleus.

TPEF in these regions remained stable for the next

30–35 minutes, during which time no SHG signal was detectable.

Fig. 8 Localization of DANPY-1 in eukaryotic cells. (A) Giardia lamblia

SHG signals (Fig. 9, horizontal polarization in panel C and

stained with 0.5 μM DANPY-1; (B) Prorocentrum micans (15 μM); vertical polarization in panel D) were initially detected in the

(

C) Prorocentrum minimum (1 μM); (D) Saccharomyces cerevisiae (10 μM);

E) Phaeodactylum tricornuta (Hoechst staining; 0.5 μM DANPY-1 did not

endoplasmic reticulum after 30–35 minutes of dye application,

but this signal only persists for 5–10 minutes. After this point,

the SHG signal becomes more prominent in the plasma mem-

branes and persists until the end of the experiment

enter cell). The scale is identical in panels A–D.

spine (Fig. 8B). Prorocentrum minimum staining showed a more (50–60 min). Superpositions of the TPEF and SHG channels at

subtle intracellular DANPY-1 distribution, suggesting endo- 45 minutes are shown in panels E (horizontal) and F (vertical)

plasmic reticulum (ER) targeting and vesicular dye aﬃnities of Fig. 9 and the time evolution of the SHG signal is shown in

(

Fig. 8C). Staining of the cell covering was minimum. Fig. S9.† DANPY-1 has considerable hydrophobic character,

Chromosomes were not visible most likely since explaining aﬃnity for membranes once inside a cellular

Prorocentrum minimum has ∼7.0 pg DNA per cell while a environment. Notably, SHG signal strength can also vary with

–

9

0

93

Prorocentrum micans cell contains ∼217 pg DNA per cell.

membrane density and ordering and DMSO can alter mem-

9

4

When the yeast Saccharomyces cerevisiae (Ascomycota) was brane morphology. However, a discussion of the eﬀects of

exposed to DANPY-1, a fine labyrinth of labeling was observed solubilization/penetration enhancers on SHG is beyond the

throughout the cell, suggesting ER localization (see West scope of the present work. The appearance of SHG in the

et al. for discussion of ER distribution in this organism). The plasma membrane was not correlated with cell death. In pre-

plasma membrane also seemed to be targeted (Fig. 8D).

91

6

vious studies, SHG has sometimes been observed shortly

Notably, all of our studies were performed without the use before cell death, when compromised membrane integrity

of a dye carrier molecule (e.g., DMSO), which might be allows dye molecules to enter the membranes more easily. The

required for eﬀective staining of some cell types. For example, weaker SHG signal observed in the region that most likely con-

the

unicellular

diatom

Phaeodactylum

tricornutum tains organelles such as the endoplasmic reticulum and mito-

(

3

Heterokonta) did not allow DANPY-1 penetration after chondria (Fig. S9,† left column) is not very commonly seen,

5 minutes of treatment, even though the vital dye Hoechst but also not unprecedented. Because SHG can also be gener-

6

readily stained the nuclei of this cell (Fig. 8E). The cell wall (or ated by chiral well-ordered structures such as collagen and

theca) of this organism is comprised mainly of a sulphated myosin, even cultured cells can occasionally show “auto-SHG”

9

2

95,96

glucuronomannan.

in the absence of a dye.

The source of the signal in the

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SHG signal may be linked to the shape and conformational

dynamics of the DANPY-1 molecule.

The applicability of DANPY-1 for SHG microscopy can be

contrasted with the voltage-sensitive dye di-8-ANEPPS, which is

a member of the ANEP (aminonaphthylethyleneylpyridinium)

family of dyes. Unlike the broad range of cellular compart-

ments targeted by DANPY-1, di-8-ANEPPS targets the plasma

membrane specifically. Like DANPY-1, di-8-ANEPPS appears

rapidly in the TPEF channel, about 1 minute after adding dye.

However, unlike DANPY-1, a SHG signal appears rapidly after

addition di-8-ANEPPS, about another minute after the appear-

9

7

ance of the TPEF signal. A significant disadvantage of the

ANEP family of dyes is that they need an antioxidant (e.g. cata-

9

8

lase) in the medium to reduce cytotoxicity. In comparable

experiments to those performed with DANPY-1 cells stained

with di-8-ANEPPS without antioxidant survive for 8 minutes

or less while being imaged. In contrast, cells stained

with DANPY-1 remained intact throughout the experiment

(

∼60 minutes). These experiments suggest that DANPY-1 may

have lower phototoxicity versus the ANEP family of dyes.

Conclusion and outlook

The development of DANPY-1 as a dye represents an intriguing

outgrowth of rational molecular design. It was first developed

for DNA-based electro-optic modulation, optimized to meet

design criteria involving hyperpolarizability, compatibility with

a nucleic acid environment, and photostability, but it also

exhibits substantial one and two-photon fluorescence, making

Fig. 9 TPEF (A, B, red), SHG (B, C, green) and combined TPEF/SHG (E, F) it of value for imaging. The fluorophore’s strong solvatochrom-

micrographs of HeLa cells stained with 7 μM DANPY-1 at 45 minutes ism, large Stokes shift, and SHG activity (large β) make it a

after dye exposure, using an excitation wavelength of 810 nm. The laser

is horizontally polarized in the panels on the left and vertically on the

The combination of high photostability and low phototoxicity

right. The blue arrows indicate a cell in which a SHG signal is observed

useful core for the development of future fluorescent probes.

of DANPY-1 could prove advantageous for imaging techniques

from nuclear region.

in which higher laser intensities are required. While binding

of the first-generation dye (DANPY-1) is not specific for DNA,

nucleus of the cell highlighted with the blue arrows in Fig. 9 is its high aﬃnity, together with its hyperpolarizability and fluo-

inconclusive and could be SHG from DANPY-1 bound to DNA rescence enhancement on binding, outperform prior attempts

1

9

or auto-SHG from microtubules of the mitotic spindle.

at nonlinear nucleic acid probes. The sensitivity of DANPY-1

Since non-centrosymmetric ordering of the dye molecules is further substantiated by gel electrophoresis experiments,

is required for SHG, the presence of a SHG signal indicates and it has been demonstrated to enter and stain a diverse

that the dye molecules are aligning within the membranes. range of cell components in several species of eukaryotes.

Such a response is further illustrated by the 90° shift in the Though further studies are warranted, preliminary third-party

SHG signal when the laser polarization was rotated from hori- toxicity tests indicate that DANPY-1 has an acceptable safety

zontal to vertical (compare Fig. 9, panels C and D). In contrast, profile for routine laboratory use – certainly an added benefit.

no significant change is observed for the orientation-indepen- This powerful combination of valuable optical properties, bio-

dent TPEF signal (compare Fig. 9, panels A and B). The delay logical utility in profiling live cells, and a readily accessible,

in the visibility of any SHG signal can potentially be due to the gram-scale synthesis present DANPY-1 as a promising imaging

time required for the dye to go from a random or centro- platform for a variety of linear and nonlinear techniques and

symmetric distribution to a non-centrosymmetric distribution across a range of appropriately conjugated targets.

within a single leaflet of the membrane. Furthermore, the

diﬀerence in timing between appearance of signals in the

TPEF and SHG imaging channels rules out that the signal

observed in the SHG channel is TPEF leaking into the SHG fil-

Conﬂicts of interest

tered channel. The slow response time for the appearance of a The authors declare no competing financial interests.

Org. Biomol. Chem.

View Article Online

Organic & Biomolecular Chemistry

Paper

resolution imaging, FEBS Lett., 2014, 588(19), 3603–

Acknowledgements

3612.

We are grateful for financial and technical support from many

parties. Work conducted in the Chemistry Department at UW

was supported by Air Force Research Laboratory (FA8659-06-

D-5401), the Air Force Oﬃce of Scientific Research (FA9550-15-

2 Z. Guo, S. Park, J. Yoon and I. Shin, Recent progress in the

development of near-infrared fluorescent probes for bio-

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Aqueous Systems by Combined and Enhanced Photonic

and Surface Eﬀects in Patterned Hollow Sphere Colloidal

Photonic Crystals, Langmuir, 2017, 33(19), 4840–4846.

6 E. De Meulenaere, W.-Q. Chen, S. Van Cleuvenbergen,

M.-L. Zheng, S. Psilodimitrakopoulos, R. Paesen,

J.-M. Taymans, M. Ameloot, J. Vanderleyden, P. Loza-Alvarez,

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1

-0319), and the National Science Foundation (DMR-1303080

and DMR-0120967); computing resources were supported by

the UW Student Technology Fund. Any opinions, findings, and

conclusions or recommendations expressed in this article

are those of the author(s) and do not necessarily reflect the

views of the National Science Foundation. Part of this work

was conducted at the Molecular Analysis Facility (MAF), a

National Nanotechnology Coordinated Infrastructure site at

the University of Washington which is supported in part by the

National Science Foundation (grant NNCI-1542101), the

University of Washington, the Molecular Engineering

&

Sciences Institute, and the Clean Energy Institute. Work con-

ducted in the Chemistry Department at California Lutheran

University was supported by startup funds awarded to JSK; LEJ

and JSK further thank the Pomona College Chemistry

Department for temporary research space and supplies for

some initial reactions performed in 2013. Research in the

University of Washington Department of Biology was sup-

ported by the UW Commercialization Gap Fund (CGF 2014-

6

596), as well the US Department of Energy under contract

DE-EE0003046 awarded to the National Alliance for Advanced

Biofuels and Bioproducts and by NOAA NA070AR4170007 to

RAC. We recognize Dr Alexander Paredez and Dr Jennifer

Nemhauser (UW Biology) who graciously provided confocal

microscope access and Sacchromyces cerviciae cells, respec-

tively. Work at KU Leuven was supported by internal funding.

Independent toxicity studies were conducted by Nelson Labs

8 J. E. Reeve, H. A. Collins, K. D. Mey, M. M. Kohl,

K. J. Thorley, O. Paulsen, K. Clays and H. L. Anderson,

Amphiphilic Porphyrins for Second Harmonic Generation

Imaging, J. Am. Chem. Soc., 2009, 131(8), 2758–2759.

9 J. G. Grote, N. Ogata, J. A. Hagen, E. Heckman, M. J. Curley,

P. P. Yaney, M. O. Stone, D. E. Diggs, R. L. Nelson,

J. S. Zetts, F. K. Hopkins and L. R. Dalton,

Deoxyribonucleic Acid (DNA) based nonlinear optics, Proc.

SPIE, 2003, 5211, 53–62.

(Salt Lake City, Utah), and funded by the UW CGF. The

authors thank Prof. Marcel Ameloot and Dr Rick Paesen

(University of Hasselt) for use of their confocal microscope 10 D. Kim, H. G. Ryu and K. H. Ahn, Recent development of

and HeLa cells, and Kristine Parra (UW WCET) and Dr Liam

Bradshaw (MAF) for experimental assistance with fluorescence

two-photon fluorescent probes for bioimaging, Org. Biomol.

Chem., 2014, 12(26), 4550–4566.

lifetime and TPA measurements. We further acknowledge 11 J. Qi, W. Qiao and Z. Y. Wang, Advances in Organic Near-

Dr Stephanie Benight, Dr Andreas Tillack, Dr Martin Sadilek,

Zachary Watanabe-Gastel, Derek Frei, Wes Winn, and Hannah

Infrared Materials and Emerging Applications, Chem. Rec.,

2016, 16(3), 1531–1548.

Zeitler (UW Chemistry); Dr Mike Casford and Prof. Paul Davies 12 L. R. Dalton, P. Gunter, M. Jazbinsek, O.-P. Kwon and

(

University of Cambridge); Prof. Malkiat Johal, Yun-Jun Kim,

and Conner Kummerlowe (Pomona College); and Aric Rininger

UW Biology) for helpful discussion and technical assistance.

P. A. Sullivan, Organic Electro-Optics and Photonics,

Cambridge University Press/Materials Research Society,

Cambridge, UK, 2015.

(

Finally, the authors wish to dedicate this work to Dr Emily 13 W. Heni, Y. Kutuvantavida, C. Haﬀner, H. Zwickel,

Heckman and Dr James Grote of Air Force Research

Laboratory, whose pioneering studies on DNA-based electronic

and optical systems enabled the search for new dyes for these

applications that led to the development of DANPY-1.

C. Kieninger, S. Wolf, M. Lauermann, Y. Fedoryshyn,

A. F. Tillack, L. E. Johnson, D. L. Elder, B. H. Robinson,

W. Freude, C. Koos, J. Leuthold and L. R. Dalton, Silicon–

Organic and Plasmonic–Organic Hybrid Photonics, ACS

Photonics, 2017, 4(7), 1576–1590.

1

4 L. R. Dalton, S. J. Benight, L. E. Johnson, D. B. Knorr,

I. Kosilkin, B. E. Eichinger, B. H. Robinson, A. K. Y. Jen

and R. M. Overney, Systematic Nanoengineering of Soft

Matter Organic Electro-optic Materials†, Chem. Mater.,

2011, 23(3), 430–445.

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Article Doi

DANPY (dimethylaminonaphthylpyridinium): an economical and biocompatible fluorophore

DOI: 10.1039/C8OB02536C

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Full text of DOI:10.1039/C8OB02536C

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