Excitation-dependent visible fluorescence in decameric nanoparticles with monoacylglycerol cluster chromophores

Abstract

Organic fluorescent nanoparticles, excitation-dependent photoluminescence, hydrogen-bonded clusters and lysobisphosphatidic acid are four interesting individual topics in materials and biological sciences. They have attracted much attention not only because of their unique properties and important applications, but also because the nature of their intriguing phenomena remained unclear. Here we report a new type of organic fluorescent nanoparticles with intense blue and excitation-dependent visible fluorescence in the range of 410-620 nm. The nanoparticles are composed of ten bis(monoacylglycerol)bisphenol-A molecules and the self-assembly occurs only in elevated concentrations of 2-monoacylglycerol via radical-catalysed 3,2-acyl migration from 3-monoacylglycerol in neat conditions. The excitation-dependent fluorescence behaviour is caused by chromophores composed of hydrogen-bonded monoacylglycerol clusters, which are linked by an extensive hydrogen-bonding network between the ester carbonyl groups and the protons of the alcohols with collective proton motion and HO···C=O (n->π*) interactions.

ARTICLE

Received 31 Aug 2012 | Accepted 30 Jan 2013 | Published 26 Feb 2013

Kwang-Ming Lee1, Wan-Yin Cheng1, Cheng-Yu Chen1, Jing-Jong Shyue2, Chih-Chun Nieh1, Chen-Fu Chou1, Jia-Rong Lee1, Ya-Yun Lee1, Chih-Yang Cheng1, Sarah Y. Chang1, Thomas C. Yang1, Mei-Ching Cheng1& Bi-Yun Lin3

Organic uorescent nanoparticles, excitation-dependent photoluminescence, hydrogen-bonded clusters and lysobisphosphatidic acid are four interesting individual topics in materials and biological sciences. They have attracted much attention not only because of their unique properties and important applications, but also because the nature of their intriguing phenomena remained unclear. Here we report a new type of organic uorescent nanoparticles with intense blue and excitation-dependent visible uorescence in the range of 410620 nm. The nanoparticles are composed of ten bis(monoacylglycerol)bisphenol-A molecules and the self-assembly occurs only in elevated concentrations of 2-monoacylglycerol via radical-catalysed 3,2-acyl migration from 3-monoacylglycerol in neat conditions. The excitation-dependent uorescence behaviour is caused by chromophores composed of hydrogen-bonded monoacylglycerol clusters, which are linked by an extensive hydrogen-bonding network between the ester carbonyl groups and the protons of the alcohols with collective proton motion and HO C O (n-p*) interactions.

1 Department of Chemistry, National Kaohsiung Normal University, No. 62, Shenjhong Road, Yanchao, Kaohsiung 82444, Taiwan. 2 Research Center for Applied Sciences, Academia Sinica, Taipei 11529, Taiwan. 3 Instrument Center of National Cheng Kung University, Tainan 70101, Taiwan. Correspondence and requests for materials should be addressed to K.-M.L. (email: mailto:[email protected]

Web End [email protected] ).

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DOI: 10.1038/ncomms2563 OPEN

Excitation-dependent visible uorescence in decameric nanoparticles with monoacylglycerol cluster chromophores

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2563

Over the past few years, metal-free or organic uorescent nanoparticles1 have received considerable attention because of their potential applications in biology

and the material sciences due to some of their advantages, including a low biological toxicity and being environmentally friendly. In addition, excitation-dependent photoluminescence (excitation-dependent uorescent, EDF)2, which is an intriguing photoluminescence phenomenon exhibited by carbon-based nanoparticles, has received considerable interest. Several pioneering examples, including graphene24, graphite5,6, diamonds7,8, carbon nanotubes9, combustion candle soot10 and pyromellitic diimide nanowire11, have recently been reported in the literature. Although a number of mechanisms have been suggested, the origin of the excitation-dependent photoluminescence remains unclear. The mechanisms for the formation and stabilization of spherical organelles via the self-assembly of phospholipids is one of the most important subjects in molecular cell biology. Lysobisphosphatidic acid (LBPA, Fig. 1a), which is also called bis(monoacylglycerol)phosphate, is an unconventional phospholipid that possesses two monoacylglycerol moieties, is found in elevated concentrations in the late endosomes and lysosomes and is believed to have a signicant role in the formation of the intraendosomal vesicular bodies12,13. It is not clear how the active form of LBPA adopts the less thermodynamically stable 2,20-positional rather than the 3,30-positional isomer14, or what the mechanisms are for the formation and transformation of the 2,20-LBPA.

Here we report a series of bis(monoacylglycerol) decameric nanoparticles with intense blue and excitation-dependent visible uorescence. The self-assembly of nanoparticles only occurs when the concentration of 2-monoacylglycerol is increased from B17% to B61%, which is analogous to the formation of intraendosomal vesicles in the late endosomes. Therefore, the bis(monoacylglycerol) bisphenol-A molecules resemble LBPA not only because both are wedge-shaped and possess two monoacylglycerol moieties, but most important, because both are functional active when the amount of 2-monoacylglycerol increases from functional inactive 3-monoacylglycerol form in the spherical aggregation through 3,2-acyl migration. The results from the experiments and theoretical calculations indicate that the radical-induced 3,2-acyl migration reactions may occur more readily for monoacylglycerol groups. These results also highlight important roles of 2-monoacylglycerol, hydrogen-bonded monoacylglycerol clusters, collective proton motion and HO C O

(n-p*) interactions in the stabilization of nanoparticles and in the mechanism of EDF phenomenon. The concept of monoacylglycerol cluster-induced excitation-dependent visible uorescence can be generalized to polymer systems. We expect that the results we report here can provide a new direction for designing and fabrication of low-toxicity visible uorescent nanomaterials without an extended conjugated system via green synthesis. We also hope that these results may facilitate our understanding of the role of monoacylglycerol-containing lipids in biology, such as the formation and mechanism of 2,20-LBPA in late endosomes.

ResultsCharacterization and formation mechanism. The initial products, which are abbreviated as n-No-EDF (Fig. 1c) because no EDF behaviour is observed, are obtained via a ring-opening reaction by treating the diglycidyl ether of bisphenol-A epoxy resin with the 3,4,5-tris(n-alkan-1-yloxy)benzoic acids (Fig. 1b). Both the 2,20-n-BTBA and the 3,30-n-BTBA are identied through 1H, 13C, heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) NMR experiments (Supplementary Figs S1S3). The ratio of 17

and 83% is typical for kinetically controlled reactions15 because the 3-position contains less steric hindrance than the 2-position. A subsequent radical-catalysed 3,2-acyl migration reaction is conducted by treating the initial products, n-No-EDFs, with 0.13 equiv. ammonium persulfate ((NH4)2S2O8) as a catalyst under neat conditions at 150 C for 38 h. The products resulting from the migration of the 3,2-acyl, which are abbreviated as n-EDFONP (Fig. 1d), are also identied through 1H, 13C, HSQC and HMBC NMR spectra, but their NMR spectra are signicantly different from that of the n-No-EDFs. The 1H signals of the 12-EDFONP in CDCl3 at 10 mM are composed of B80% broad and 20% sharp signals (Supplementary Figs S4S11 ). The dominant broad NMR signals are assigned to 2,20-12-BTBA (40%) and 2,3-12-BTBA (40%), and they have signicant upeld or downeld shifts compared with that of the initial 12-No-EDF. The remaining 20% sharp signals correspond to free 2,20-12-

BTBA (1%), 3,30-12-BTBA (15%) and 2,3-12-diacylglycerolbisphenol-glycerol isomer (4%, a phosphatidylglycerol-like compound). These broad signals are attributed to the extremely dense aggregated structures, in which the internal conformational mobility is reduced16. In contrast, all of the 1H NMR signals corresponding to 2,20-12-BTBA and 3,30-12-BTBA in the 12-No-

EDF in CDCl3 at 10 mM are normally sharp, which indicates no dense aggregated structure being formed in the low concentration of 2,20-12-BTBA. In other words, the increased concentration of the 2-monoacylglycerol moieties in the 12-EDFONP from B17% to B61% after the 3,2-acyl migration causes the formation of dense aggregated structures with a yield of 80%. To determine whether the acyl migration is caused by the sulphate radical or the basicity of the sulphate, one reaction was performed by replacing (NH4)2S2O8 with potassium carbonate (K2CO3) under the same reaction conditions. The results indicated that only a small amount of 2,3-diacylglycerol-bisphenol-glycerol was obtained; however, no 2,20-isomer was observed (Supplementary Fig. S12).

Therefore, the radical-catalysed 3,2-acyl migration was established. The results from theoretical calculations at the M06-2x (ref. 17)/cc-pVDZ level indicate that the activation energy of the alkoxy radical-induced acyl migration mechanism is 1.8 kcal/mol less than the migration by the hydroxyl form (Supplementary Fig. S13). Therefore, the 3,2-acyl migration is readily able to occur via the alkoxy radical pathway.

Characterization and properties of EDF organic nanoparticles. Investigations based on the combination of NMR, high-resolution transmission electron microscopy (HRTEM), dynamic light scattering (DLS) and photoluminescence spectra reveal that the densely aggregated structures of the acyl-migrated product are discrete self-assembled EDF organic nanoparticles (n-EDFONPs). The HRTEM image of 18-EDFONP with a diameter of B7.6 nm is shown in Fig. 2a. The mean diameters of 12-EDFONP to 18-EDFONP in CH2Cl2 measured using DLS are 4.8, 6.0, 8.6 and9.5 nm (Supplementary Fig. S14). All of the n-EDFONPs have a similar photoluminescence excitation and emission behaviour in CH2Cl2 and CHCl3 solvents. In the case of 12-EDFONP in CHCl3 at a concentration of 10 mM, the excitation spectrum exhibits a broad peak at 330 nm with a shoulder that extends to B650 nm (Fig. 2b). The excitation of 12-EDFONP from 360 to 600 nm results in an EDF from 415 to 660 nm, where the intensity remarkably decreases (Fig. 2b). The quantum yield is determined to be 8.3% with coumarin 1 as a reference and the uorescence lifetime measurement of the excitations of 12-No-EDF at 329 nm and 12-EDFONP at 360 nm are determined to be 0.98 and1.54 ns, respectively (Supplementary Fig. S15). Figure 1e is a uorescent photograph of 12-EDFONP in a CH2Cl2 solution, which was simultaneously excited at 405 and 532 nm and exhibits

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2563 ARTICLE

n-No-EDF

O

Ring-opening

R

O

O O

O

P

2,2-LBPA

R

R

R

H H O O

O O O O

DGEBA epoxy resin

+ 2

HO

R

O

O O

O

O

R O

R

O

O

O

O

3,4,5-tris(n-alkan-1-yloxy) benzoic acid

H

O

3

3'

2

OH

O

HO

O

O

3 3' 1'

2'

1'

1

OCnH2n+1

OCnH2n+1

1

O

O

O O

R=

OCnH2n+1

n = 12,14,16 and 18

2 2'

2,2-n-BTBA 17%

3,3-n-BTBA 83%

R

O O

II

(NH4)2S2O8 Radical-catalysed

3,2-acyl migration

n-EDFONP

X 5

X 5

I

R

Fold

O

H O

R R

OO OH

O

H

O

O

O O

O

HO

III

Great Stellated Dodecahedron

Self-assembly of decamer

2,2-n-BTBA 40%

2,3-n-BTBA 40%

O

Excitation-dependent fluorescent organic NP n-EDFONP

II

I

III

Twenty monoacylglycerol groups locate at twenty triangles of lcosahedral net

+

II II

OR

RO

OR

OR

OR'

RO

RO

RO

OR

O O

O

OR

OR

O

O

O O

O

H

O

H

H

O

O

O

O

OR'

O

O

O

H

H

O

H

O

O

O

OR'

Collective proton motion

Hydrogen-bonded monoacylglycerol clusters

Figure 1 | Self-assembly of excitation-dependent visible uorescent bis(monoacylglycerol)bisphenol-A decameric nanoparticles with monoacylglycerol cluster chromophores. (a) Structure of 2,20-lysobisphosphatidic acid (2,20-LBPA). (b) Reactants. (c) Initial products of n-No-EDF (due to no EDF behaviour): two kinetically controlled products, 2,20-n-BTBA and 3,30-n-BTBA in a ratio of 17:83, are obtained via a ring-opening reaction in the rst stage. (d) n-EDFONP: the concentration of 2-monoacylglycerol moiety of 2,20-n-BTBA and 2,3-n-BTBA is increased from 17 to 60% after the radical-catalysed 3,2-acyl migration reaction in the second stage. (e) Intense blue (up) and yellow (down) uorescences excited by a 405 nm violet and a 532 nm green light laser diodes, respectively. (f) Formation of Great Stellated Dodecahedral decamers by the self-assembly of ve 2,20-n-BTBA and ve 2,3-n-BTBA. (g) Formation of the cyclic hydrogen-bonded monoacylglycerol clusters with collective proton motion (green line and dash) within three adjacent monoacylglycerol groups (I, II and III).

intense blue and yellow uorescences in the same quartz cuvette. In contrast, the excitation spectrum of 12-No-EDF at a concentration of 10 mM displays only one excitation peak at 329 nm and one corresponding relatively shaper ultraviolet uorescence peak at 353 nm (Fig. 2c). However, when the concentrations of 12-EDFONP and 12-No-EDF are subsequently decreased from 10 to 0.01 mM by dilution, both exhibit similar excitation and emission spectra (Fig. 2c,d). Two excitation peaks at 235 and 274 nm and one emission peak at 355 mM (excited at 274 nm)

were observed, which indicated that both 12-EDFONP and 12-No-EDF dissociated into individual 3,30-12-BTBA and 2,20-12-BTBA molecules without dense aggregation when diluted to a concentration of 0.01 mM. The emission behaviour of 12-No-EDF in high and low concentrations is typical of J-aggregate emission18. However, when 12-EDFONP is reconcentrated from0.01 to 10 mM, the broad characteristic excitation and EDF reappears (spectrum in green in Fig. 2d). This result clearly indicates that the EDF behaviour is induced by the dense

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2563

Ex

Em with Ex at

274 nm

329 nm

355 nm

0.01 mM excitation0.01 mM emission ex at 274 nm

10 mM excitation

ex at 329 nm

10 mM emission

Intensity (a.u.)

360 nm 380 nm 400 nm 420 nm 440 nm 460 nm 480 nm 500 nm 520 nm 540 nm 560 nm 580 nm 600 nm

340 nm 360 nm 380 nm 400 nm 420 nm 440 nm 460 nm 480 nm 500 nm

560 nm

520 nm 540 nm

Intensity (a.u.) Intensity (a.u.)

300 650

550

350

400

450

500

250 300 350 400

Wavelength (nm) Wavelength (nm)

274 nm 330 nm

Em with Ex at

Dilution

Reconcentration

O

C16H33O

C16H33O

OC16H33

O O

O

Em with Ex at

OH HO OOH

O

O

10 mM

345 nm 365 nm 385 nm 405 nm 425 nm 445 nm 465 nm

O

O

Intensity (a.u.)

Intensity (a.u.)

10 mM

0.01 mM

250 300 350 400 450 500 550 600 600

500

350 650

550

400

450

350 550

400

450

500

600

Wavelength (nm) Wavelength (nm)

600

Wavelength (nm)

Figure 2 | Image and uorescence spectra of EDFONPs. Spectroscopy performed in CHCl3. (a) HRTEM image of 18-EDFONP with a scale bar of10 nm. (b) Excitation and emission spectra of 12-EDFONP at a concentration of 10 mM with a excitation-dependent visible uorescence from 410 to 620 nm. (c) Excitation and emission spectra of 12-No-EDF at concentrations of 0.01 and 10 mM. (d) Excitation spectra of 12-EDFONP at an initial concentration of 10 mM (red line), then diluted to 0.01 mM (blue line) and reconcentrated back to 10 mM (green line). (e) Emission spectrum of 17-EDFONP based on stearic acid at a concentration of 10 mM with a visible EDF phenomena. (f) Emission spectrum of 16-EDFONP-BGE at a concentration of 10 mM with a visible EDF phenomena.

aggregation in the self-assembled organic nanoparticles but not by the individual n-BTBA molecule itself. To determine the functional groupsincluding bisphenol, benzoate and monoacylglycerolthat are responsible for the EDF behaviour, two compounds were prepared by changing the benzoate and bisphenol-A groups to the heptadecyl carboxylate and n-butyl glycidyl ether groups, respectively (Supplementary Figs S16 and S17). Both of the modied compounds also exhibited the EDF behaviour (Fig. 2e,f), which clearly indicates that the 2-monoacylglycerol group is responsible for the EDF behaviour. In addition, this result also indicates that the EDF behaviour is a common phenomenon for the 2-monoacylglycerol-dominated compounds. Note that the bis(monoacylglycerol) clusters serve as chromophores here without any extended conjugated system.

Decamer and hydrogen-bonded monoacylglycerol cluster. Further structural characterization using matrix-assisted laser desorption ionization time-of-ight mass spectrometry (MALDITOF-MS), NMR, Fourier transform infrared (FT-IR) experiments and theoretical calculations indicate the formation of decamers that contain a hydrogen-bonded monoacylglycerol clustered structure with collective proton motion. The MALDI-TOF-MS spectra of the n-EDFONPs reveal a group of signals that exhibit the pattern of an arithmetic series corresponding from a monomer to a decamer. For example, Fig. 3a shows the MALDI-TOFMS spectrum of the 18-EDFONP, which exhibits an ion peak at m/z 21,856 ([M10-6H2O Na] ) that corresponds to the

decamer losing six water molecules. It is well known that dehydration is typical for hydroxyl-containing compounds in MALDITOF-MS experiments. In addition, a group of signals with an

arithmetic series pattern corresponding to the decamer to tetramer losing water molecules. There are six ion peaks (AF) to be observed between n-mers, which attributed to several fragment eliminations after acyl migration during the ionization process. The diffusion coefcient is measured using the NMR DOSY technique for solutions of 18-EDFONP. The spectrum shows only one diffusion species that is characterized by the diffusion coefcient, D, of 0.95 10 10 m2 s 1 (Supplementary Fig. S18) and

the hydrodynamic radius of 3.9 nm is calculated from the Stokes Einstein equation: rH kBT/6pZD, where k is the Boltzmann

constant, T is the temperature, Z is the viscosity of solution. The molecular weight of 18-EDFONP is estimated to be B24,000 from the MarkHouwinkSakaruda equation of D 2.45 (

0.04) 10 4Mw (0.550.05), which is determined by the phenolphthalein poly(ether sulphone) in chloroform solution.19 By combining this result with the MALDI-TOF-MS spectrum, it was suggested that the formation of a decamer. Because the ratio of 2,20-n-BTBA and 2,3-n-BTBA determined using 1H NMR is B1:1, the decamer should be composed of ve 2,20-n-BTBA and ve 2,3-n-BTBA, fteen 2-monoacylglycerol and ve 3-monoacylglycerol moieties, specically (Fig. 1d,f). These 20 monoacylglycerol moieties could construct a Great Stellated Dodecahedral (Fig. 1f), in which every three neighbouring monoacylglycerol groups form a triangular unit (Fig. 1f,g, 2-monoacylglycerol I, II and III). The

1H and 13C NMR and liquid FT-IR spectra reveal the formation of dynamic hydrogen-bonded alcohol clusters with collective proton motion within each triangular unit (Fig. 1g). Remarkable differences in both the 13C and 1H chemical shifts are observed in the comparison of the 12-EDFONP and 12-No-EDF spectra. In the spectrum for the 12-EDFONP, the 13C signals of the hydroxyl-bearing carbon of 2,20-12-BTBA and 2,3-12-BTBA undergo a

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2563 ARTICLE

downeld shift of approximately 7.0 and 7.5 p.p.m., respectively, relative to the signals of both BTBAs without dense aggregation in 12-No-EDF (Fig. 3b and Supplementary Fig. S7). The Dd 7.0 and7.5 p.p.m. downeld shifts are typical for the hydroxyl-bearing carbons of polyvinyl alcohol and are characteristic of the formation of oligomeric alcohol clusters through continuous alcohol O-H O-H hydrogen bonding20. The

1H signals of the hydroxyl protons are no longer present in the 12-EDFONP, and these signals are broadened and shifted downeld to 6.26.6 p.p.m. for the other three n-EDFONP (n 14, 16 and 18; Supplementary

Fig. S5) relative to sharp signals at 2.75 p.p.m. for the 12-No-EDF (Supplementary Fig. S1). Figure 3c shows the liquid FT-IR spectra of 12-EDPONP and 12-No-EDF in CH2Cl2. The primary difference between these two spectra is the O-H band region at 3,300 3,650 cm 1. In the 12-No-EDF spectrum, one terminal O-H band at 3,596 cm 1 and one hydroxyl dimer broadband at 3,487 cm 1 were observed21. However, in the 12-EDPONP spectrum, only three very weak bands, including one terminal O-H band at 3,593 cm 1, one hydroxyl dimer at 3,512 cm 1 and one cyclic hydroxyl trimer band at 3,414 cm 1, were observed. The dramatic decrease in the intensity of the former two bands accounts for the

formation of cooperative alcohol O-H OH hydrogen-bonding

networks with collective proton motions, which result in very weak OH stretching bands22. The results from theoretical calculations at the M06-2X/cc-pVDZ level for three acetyl 2-monoacylglycerolbenzene molecules as a static model reveal that the hydrogen-bonded cyclic alcohol trimer is 33.3 kcal mol 1 more stable than that of the three discrete acetyl 2-monoacylglycerolbenzene molecules, which is indicative of stabilization by cooperative alcohol hydrogen bonding (Fig. 4a and Supplementary Fig. S19). Therefore, the cyclic 2-monoacylglycerol trimer can serve as a triangular-shaped building block with a large curvature leading to an n-EDPONP via spherical aggregation. In contrast, the result of theoretical calculations for the 3-monoacylglycerol trimer cluster using the same UM06-2x/cc-pVDZ method used for the 2-monoacylglycerol trimer cluster (Supplementary Fig. S19d) reveals that the three 3-monoacylglycerol molecules are nearly parallel and could not contribute to the curvature leading to the formation of the spherical aggregate. Because the primary difference between 2-monoacylglycerol and 3-monoacylglycerol is the hydroxyl group, it is either a primary R2CHCH2OH or secondary R2CHOH group.

Considering the length between O and tertiary C atoms and

R R R

R

O O

O

O O O

O

2,2-18 -BTBA

O

H

O H

O

O

HO

Eliminated fragments afteracyl migration

M10 =

OO OH

O

2,3-18 -BTBA

6H2O + Na+

5

Mn = M(n-4)

+

Acyl migration m/z

M4

5

f

+

+

M5 M8

A

= M(n-4)

= M(n-4)

= M(n-4)

= M(n-4)

= M(n-4)

= M(n-4)

=

Mn - 376

Mn - 752

Mn - 911

Mn - 1287

Mn - 1663

Mn - 1822

R

OC18H37

OC18H37

OC18H37

c

f

B

d

C

+

Intensity (a.u.)

e

f

c

e

d

b

b

D

+

c

M9

a

+

a

E

e

M6

f

d

b

e

f

d

F

+

c

a

e

c

d

M7

b

b

a

H

a

f

e

M10

d

c a

b

OH OHO

O O

H O

= OC18H37O

OC18H37

OC18H37

16,000 18,000 20,000 22,000

,

10,000

C

O

62 R R R

R R

O O O

O

2,3-12 -BTBA

O

12-No-EDF 12-EDFONP

O

[afii9829](2 -gly)= 7.0 p.p.m.

12,000 14,000m/z

Free 3,3-12 -BTBA Free 2,2-12 -BTBA

3,487

64

O O O O

OH

O O

B C

O

O

HO

OHHO

R

O

O

66

3,596

3,414 3,512 3,593

3,500 3,000 2,500Wave number (cm1)

2,000 1,500 1,000

B

68

C

70

Intensity (a. u.)

C

[afii9829](3 -gly) =7.5p.p.m.

4.4 4.2 4.0 p.p.m.

O O O

R

O

B O

H

72 OH OH

O

O

O

H O

C

74

O C

R

B

76 2,2-12-BTBA

78

With collective proton motion in 12-EDFONPs

Figure 3 | Characterization of decamer and hydrogen-bonded monoacylglycerol cluster. (a) MALDI-TOF-MS of 18-EDFONP shows an arithmetic series ion peaks corresponding to the decamer to tetramer with dehydration. Inset spectrum is amplication of the higher mass region. There are six ion peaks (AF) to be observed between n-mers that attributed to several fragment eliminations after acyl migration during the ionization process. (b) Part of the 1H13C HSQC spectrum of 12-EDFONP; the 13C signals of the hydroxyl-bearing carbon atoms (H-C-OH OH) of the 3- and 2-monoacylglycerol

groups in the hydrogen-bonded monoacylglycerol cluster are signicantly shifted downeld by approximately 7.5 and 7.0 p.p.m., respectively, compared with both in solution. (c) Liquid FT-IR spectra of 12-No-EDF (blue line) and 12-EDFONP (red line) at a concentration of 10 mM in CH2Cl2 at room temperature. The dramatic decrease in the intensity of the two bands at 3,596 and 3,487 cm 1 indicates the formation of cooperative hydrogen-bonded alcohol clustering with collective proton motion.

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2.038

2.038

1.969 1.969

2.170

2.170

Emission intensity (a.u.)

200 300 400 500 600

Hydrogen-bonded cyclic alcohol trimer

n* interaction

Wavelength (nm)

Pyramidalized C=O group

Figure 4 | TD-DFT calculation of trimeric monoacylglycerol cluster.(a) Optimized ground-state geometries of cyclic hydrogen-bonded acetyl 2-monoacylglycerolbenzene trimer at M06-2X/cc-pVDZ level viewed from side (left) and top (right) directions. The hydrogen atoms except that of hydroxyl groups are omitted for clarity. (b) Calculated emission spectrum. (c) Optimized excited state geometry at UM06-2x/6-31g(d) with TD-DFT method based on the optimized ground-state structureof the second excited state at UM06-2X/6-31g(d) level. One carbonyl group was pyramidalized due to two n-p* interactions with two adjacent alcohol O atoms (blue dash lines). (d) LUMO orbital and (e) HOMO orbital were dominated by carbonyl p* and non-bonding orbitals, respectively, as well as the adjacent OH non-bonding orbitals. The unit of bond length is angstrom.

exibility, the former is not only longer (2.477 in Supplementary Fig. S19c) than the latter (1.406 in Supplementary Fig. S19d), but also more rotationally exible. In addition, apparently the angles between the hydroxyl O atom, tertiary C atom and methyl C atom in the 2-monoacylglycerol and 3-monoacylglycerol trimers are 104 and 73 (Supplementary Fig. S19c,d), which clearly indicate that the 2-monoacylglycerol group is more suitable for the formation of a spherical aggregate than the 3-monoacylglycerol group.

TD-DFT calculation. We suggest that the EDF phenomenon is caused by the combination of dynamically hydrogen-bonded alcohol clusters with collective proton motion and excited-state HO C O (n-p*) interactions, which can be regarded as a

BrgiDunitz trajectory23 interaction (Nu C O interac

tions). The existence of O C O interactions have been

conrmed by protein structures2426 and small molecules27, and they have a signicant role in the stabilization of collagen28 and the a-helix29. The uorescence spectrum was calculated using the

TD-DFT/UM06-2X/6-31g(d) method based on the optimized structure of the second excited state of the cyclic acetyl-2-monoacylglycerolmethane trimer at the UM06-2X/6-31g(d) level, and it is shown in Fig. 4b. The result from the calculation predicts two emission peaks at 263 and 418 nm. The latter is consistent with the experimental value. Note that the LUMO (S1) is

dominated by the p* orbital of the C O group with a signicant

pyramidalization (Fig. 4c,d). In addition, two adjacent alcohol O atoms bond to the p* orbital of the C O group using the non-

bonding electron pair (n) to form two H-O C O

interactions, which are n(OH)-p*(CO) interactions (Fig. 4d). The HOMO (S0) primarily consists of the non-bonding orbital of the C O group (n(CO), Fig. 4e); therefore, the emission peaks

located at 418 nm should be described as a n(OH)-p*(CO)

stabilized S1-S0 relaxation. The results from the calculations indicate that the H-O C O interaction has a signicant role

in stabilizing the excited state via an n(OH)-p*(CO) interaction,

which results in a low-lying LUMO state and a red-shifted uorescence. The results are consistent with those reported by Raines and co-workers30, in which theoretical calculations and high-resolution structures of green uorescent proteins revealed the existence of a n-p* interaction between the chromophore of uorescent proteins and a main-chain oxygen, and this interaction could contribute to the photophysical properties of the chromophore. Based on the above-mentioned results, the EDF behaviour can be explained by the electronically excited state manifold of the carbonyl groups due to the oscillating HO C O interactions caused by the collective proton

motion system.

DiscussionBased on the results mentioned above, the n-EDFONPs are formed through radical-catalysed 3,2-acyl migration and stabilized by the hydrogen-bonded monoacylglycerol clusters with a collective proton motion and HO C O (n-p*) interac

tions. We suggest that the n-EDFONPs system could be considered as a model system of 2,20-LBPA-rich organelles and could provide some biological implications in their formation and stabilization mechanisms, and biofunctions in late endosomes and lysosomes. The concept of 2-monoacylglycerol-induced EDFONP can be generalized to polymer systems. Replacing the reactant of 3,4,5-tris(n-alkyloxy) benzoic acid with the poly (ethylene glycol) bis(carboxymethyl) ether (Mn 600) resulted in

an alternating copolymer, which is abbreviated as EDF-Polymer (Fig. 5a and Supplementary Fig. S20a). The molecular weight of the EDF-Polymer was estimated by gel-permeation chromatography and the number-average-molecular weight (Mn) of the

EDF-Polymer was B12,000 Da. Emission spectrum of EDF-Polymer in CHCl3 exhibits an EDF behaviour, which is similar to that of the n-EDFONPs (Fig. 5b), and the quantum yield is determined to be 6.1% with coumarin 1 as a reference. The HRTEM image reveals a aggregated nanoparticle with an approximately 58 nm diameter (Fig. 5c). We think that the nanoparticle of EDF-Polymer is constructed through a self-assembly process with bisphenol-A aggregates and poly(ethylene glycol) moieties serving as core and shell compartments, respectively (Fig. 5d). Future work will focus on the investigation of EDFONP as a biomimetic model of mono- or diacylglycerol-containing lipids and the potential applications of EDFONP and EDF-Polymer, especially for the fabrication of metal-free, low-

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2563 ARTICLE

H

O

OH

Diacylglycerol-bisphenol

O

On m

m

O

O OH O O OH OH

O O

O

O

O

HO

or

OHOH

O OH HO

O

O O

O

Excitation

Intensity (a.u.)

370 nm 390 nm 410 nm 430 nm 450 nm 470 nm 490 nm 510 nm 530 nm 550 nm 570 nm

400 500 600 700

Wavelength (nm)

Figure 5 | Excitation-dependent uorescent polymer. (a) Chemical structure of the EDF-Polymer. (b) Emission spectrum of the EDF-Polymerwith a visible EDF phenomena. (c) HRTEM image shows an aggregated nanoparticles with a scale bar of 10 nm. (d) A cartoon model of nanoparticle with bisphenol-A aggregates and poly(ethylene glycol) moieties serving as core and shell compartments, respectively.

cost organic/polymeric biosensing, bioimaging and optoelectronics materials.

Methods

Preparation of n-EDFONPs. n-EDFONPs are prepared by two sequential reactions. The rst reaction is a ring-opening reaction and the second reaction is a radical-catalysed 3,2-acyl migration reaction.

General method for the synthesis of 12-No-EDF via ring-opening reaction. The preparation of 12-No-EDF has been selected as a representative example, because the same procedure was systematically used for all the members of the series. A solution of bisphenol-A diglycidyl ether (0.68 g, 2.00 mmol), 3,4,5-tris(dodecyloxy) benzoic acid (2.80 g, 4.15 mmol) and tetra-n-butylammoium bromide (0.32 g,1.00 mmol) in mix-solvent of acetonitrile (40 ml) and xylene (20 ml) was reux at 85 C for 24 h. The solvent was removed under reduced pressure. The crude product was recrystallized twice from ethanol, and a white solid is obtained (2.30 g, 68%) which is composed of 2,20-12-BTBA and the 3,30-12-BTBA positional isomers. 1H NMR (600 MHz, CDCl3): d 7.26 (s, 4H), 7.14 (d, 4H, J 7.8 Hz), 6.83 (d,

4H, J 7.8 Hz), 5.41 (m, 0.34H, 2,20-12-BTBA), 4.51 (m, 3.32H, 3,30-12-BTBA),

4.34 (m, 1.66 H, 3,30-12-BTBA), 4.26 (m, 0.68H, 2,20-12-BTBA), 4.08 (m, 3.32H, 3,30-12-BTBA), 4.02 (m, 12.68H,), 2.75 (b, 2H), 1.78 (m, 12H), 1.63 (s, 6H), 1.47 (m, 12H), 1.34 (m, 96H), 0.88 (t, 18H, J 6.6 Hz); 13C NMR (150 MHz, CDCl3): d

166.8, 156.2, 152.9, 143.8, 142.8, 127.6, 124.1, 114.0, 108.4, 77.1, 73.5, 69.3, 68.9 (3,30-12-BTBA), 68.8(3,30-12-BTBA), 66.4 (2,20-12-BTBA), 66.1(3,30-12-BTBA),62.3 (2,20-12-BTBA), 41.8, 31.9-22.7, 14.1; analysis (calcd., found for C107H180O14): C (76.02, 76.01), H (10.73, 10.79). 14-No-EDF, analysis (calcd., found for C119H204O14): C (76.89, 76.87), H (11.06, 11.03). 16-No-EDF, analysis (calcd., found for C131H228O14): C (77.61, 77.23), H (11.34, 11.28). 18-No-EDF, analysis (calcd., found for C143H252O14): C (78.23, 77.94), H (11.57, 11.58). The DEPT-135, HSQC and HMBC spectra are shown in Supplementary Figs S1S3.

General method for the synthesis of 12-EDFONPs via radical-catalysed 3,2-acyl migration reaction. The preparation of 12-EDFONP has been selected as a representative example because the same procedure was systematically used for all the members of the series. 12-No-EDF (0.7402 g, 0.44 mmol) and ammonium persulphate (0.013 g, 0.057 mmol) were heated and stirred under N2 at 150 C for

38 h. The crude product was then disolved in CH2Cl2 and ltered. The ltrate was dried under vacuum to give a brown product with a yield of 97% (0.72 g,0.43 mmol), which is composed of 2,20-12-BTBA, 2,3-12-BTBA, 3,30-12-BTBA and 2,3-12-diacylglycerol-bisphenol-glycerol isomers, and no any non-acyl-migrated side product is found. 1H NMR (600 MHz, CDCl3): d 7.27 (b, 4H), 7.11 (b, 4H),6.88 (b, 4H), 5.73 (s, 0.04H), 5.52 (s, 1.2H), 5.42 (s, 0.02H), 4.78 (m, 0.04H), 4.66 (m, 0.04H), 4.52 (b, 1.4H), 4.36 (b, 0.3H), 4.11 (b, 18.96H), 1.76 (m, 12H), 1.60 (s, 6H), 1.46 (m, 12H), 1.27 (m, 96H), 0.87 (t, 18H, J 6.6 Hz); 13C NMR

(150 MHz, CDCl3): d 170.9, 160.1, 143.8, 143.1, 127.8, 124.2, 114.4, 108.2, 77.1,73.5, 71.8, 70.2, 69.2, 68.9, 68.7, 68.9, 67.9, 66.3, 64.4, 63.3, 62.3, 41.8, 31.9, 31.1,30.4, 29.6, 26.1, 22.7, 14.1; analysis (calcd., found for C107H180O14): C (76.02,76.74), H (10.73, 10.74). 14-EDFONP, analysis (calcd., found for C119H204O14):C (76.89, 77.34), H (11.06, 11.07). 16-EDFONP, analysis (calcd., found for C131H228O14): C (77.61, 77.91), H (11.34, 11.38). 18-EDFONP, analysis (calcd., found for C143H252O14): C (78.23, 78.62), H (11.57, 11.57). The 1H, 13C DEPT-135,

1H13C HSQC and HMBC spectra are shown in Supplementary Figs S4S12.

Synthesis and compound characterization. Synthesis and characterization of 17-EDFONP, 16- BGE-EDFONP- and EDF-Polymer are described in Supplementary Methods.

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Acknowledgements

We thank Prof H.-Y. Chen (Kaohsiung Medical University) for helpful discussions on TD-DFT calculation, Prof Y.-T. Tao, Prof S.-S. Sun and M.-M. Lee (Academia Sinica) for the measurement of uorescence lifetime and Dr Y.-T. Hsieh (Daxin Materials Corp.) for helpful discussions on characterizations of EDF-Polymer. We are grateful to the National Center for High-performance Computing for computer time and facilities. This work was supported by the National Science Council of Taiwan, Republic of China (NSC-101-2113-M-017-004-MY2) and National Kaohsiung Normal University.

Author contributions

K.-M.L. was involved in design, direction and supervision of the study, analysed data, performed calculations and wrote the manuscript. W.-Y.C., C.-Y.C., C.-C.N., C.-F.C.,J.-R.L., Y.-Y.L. and C.-Y.C. synthesized the compounds and performed NMR (400 MHz), photoluminescence and IR measurements. J.-J.S. performed HRTEM and DLS measurements. S.Y.C., T.C.Y. and M.-C.C. performed MALDI-TOF-MS measurements.B.-Y.L. performed NMR (600 MHz) measurements.

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How to cite this article:Lee, K-M. et al. Excitation-dependent visible uorescence in decameric nanoparticles with monoacylglycerol cluster chromophores. Nat. Commun. 4:1544 doi: 10.1038/ncomms2563 (2013).

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