absorption and emission spectroscopic characterization of lumichrome in aqueous solutions

Absorption and Emission Spectroscopic Characterization of Lumichromein Aqueous Solutions†

Amit Tyagi and Alfons Penzkofer*

Fakultat fur Physik, Universitat Regensburg, Regensburg, Germany

Received 20 August 2010, accepted 6 October 2010, DOI: 10.1111/j.1751-1097.2010.00836.x

ABSTRACT

The spectroscopic behavior of lumichrome (7,8-dimethyl-allox-

azine, LC) in aqueous solutions in a pH range from )1.08 to 14.6is studied. Absorption spectra, fluorescence quantum distribu-

tions, quantum yields, and lifetimes are determined. The

ionization stage of ground-state LC changes with rising pH

from the cationic form (LCH2+) to the neutral form (LCH) with

a mid-point pH of pKc � )0.53, and to the anionic form (LC))

with a mid-point pH of pKa � 12.5. Above pH 7 a partial

ground-state tautomerization of LCH to 7,8-dimethyl-isoallox-

azine (IAH) occurs by N1–N10 intra-molecular proton transfer.

For pH > pKa � 12.5 LCH and IAH change to the anionic

forms LC) and IA), and above pH 14 LC) tautomerizes

completely to IA). In the excited state some neutral lumichrome

(LCH*) converts to cationic lumichrome (LCH2+) at low pH by

proton transfer from H3O+ to LCH*. No photoinduced excited-

state tautomerization of lumichrome was observed. LCH for

pH > 3 and IAH are reasonably fluorescent. The fluorescence

efficiencies of LC) and IA) are lower than those of LCH and

IAH. The fluorescence of LCH2+ is strongly quenched likely by

intra-molecular diabatic charge transfer and excited-state relax-

ation by potential surface touching with the ground state.

INTRODUCTION

The alloxazine dye lumichrome (7,8-dimethyl-alloxazine, 7,8-dimethyl-1H-benzo[g]pteridine-2,4-dione, general abbreviated

by LC, structural formula is shown in Fig. 1) is involved invarious biological actions like metabolism processes (1), theinhibition of flavin reductase in Escherichia coli (2), and theliver take-up of riboflavin by human-derived Hep G2 cells (3).

It is the main photodegradation product of flavins (4–11).Even one way of synthesis of lumichrome is the photodegra-dation of riboflavin in acidic aqueous solution (12–14).

In the case of long-time short-wavelength exposure of flavinbased blue-light photoreceptors (15,16) lumichrome turned outto be a dominant photodegradation product. Lumichrome was

found in photodegradation studies of the BLUFdomainmutantH44R of AppA from Rhodobacter sphaeroides (17)(BLUF = sensors of blue light using FAD, FAD = flavin

adenine dinucleotide), the blue-light-regulated phosphodiester-aseBlrP1 protein fromKlebsiella pneumonia (18), and theE149A

mutant of cryptochrome 3 from Arabidopsis thaliana (19).Recent studies on 8-amino-riboflavin indicated 8-amino-

lumichrome as a photodegradation product (20). The photode-gradation of the BLUF protein Slr1694 from Synechocystis sp.

PCC6803 with roseoflavin cofactor (roseoflavin = 8-dimethyl-amino-riboflavin) resulted in 8-dimethylamino-lumichrome and8-methylamino-lumichrome formation (21).

Photophysical properties of lumichrome and lumichromederivatives in organic solvents were investigated in (12,22–27).The molecule has several active sites for hydrogen bonding

(N1, O2, N3, O4, N5, N10) with appropriate hydrogen bondforming solvents like water (12,25,27–29), pyridine(12,23,25,27), carboxylic acids (12,25,27), phosphoric acids

(30), aliphatic and aromatic amines (22).Photoexcitation of hydrogen bonded lumichromes caused

changes of the hydrogen bond structure and proton transfer(tautomerization) from N1 to N10 forming 7,8-dimethyl-

isoalloxazine (abbreviated by IAH, structural formula shownin Fig. 1) thereby changing the lumichrome-like emission to anisoalloxazine-like emission (7,12,23,25–35). The phototauto-

merization was observed for lumichrome in pyridine (27),glacial acetic acid (27), methanol-acetic acid (34), ethanol-acetic acid (28,29,31,32), dioxane-pyridine (23,26,31,32),

water-pyridine (27), acetone-water (27), 1,2-dichloromethane-hexafluoropropanol (25), trifluoroacetic acid (25), acetic acid(25), methanol-aliphatic amine, and methanol-aromatic aminemixtures (22). It was found for N(3)-undecyllumichrome in

dodecylammonium propionate reversed micelles (33). Nophototautomerization was found for lumichrome in neutralwater (7).

Semi-empirical quantum chemical calculations couldexplain the phototautomerization by considerable electrondensity redistribution in the excited-state leading to an acidity

enhancement of N1H hydrogen and a basicity rise at the N10position (32).

Tautomerization in the ground-state was achieved for

lumichrome dissolved in acetonitrile by fluoride and acetateanions which replaced the hydrogen atoms at N3 and N1position and transferred one hydrogen atom to the N10position (36). Thereby the lumichrome absorption and emis-

sion behavior changed to an isoalloxazine like behavior (36).Tautomerization of lumichrome in the ground-state in aque-ous solution was found by addition of cucurbit[7]uril, which

has a pumpkin-shaped macrocyclic structure and binds

†This paper is part of the Symposium-in-Print on ‘‘Blue Light Effects.’’*Corresponding author email: [email protected](Alfons Penzkofer)

� 2010 The AuthorsPhotochemistry and Photobiology� 2010 The American Society of Photobiology 0031-8655/11

Photochemistry and Photobiology, 2011, 87: 524–533

524

lumichrome to its cavity (37). No ground-state tautomeriza-

tion was found for lumichrome in pure water (7).In aqueous solution the ionic stage of lumichrome depends

on the pH (27,35,38,39). Reported pK constants of the

lumichromes are pKc = )0.38 (35) for the equilibriumbetween cationic form (N10 protonated) and neutral form,pKa = 8.28 (27) for the equilibrium between neutral form andanionic form (either N3 or N1 deprotonated with similar

probability [35,39]), and pKb = 12.9 (27) for the equilibriumbetween single anionic form and bi-anionic form (N1 and N3deprotonated). The absorption and emission behavior of

lumichrome was found to be different for the different ionicforms (27,35,38,39). The absorption spectrum of anioniclumichrome was observed to be similar to those of anionic

isoalloxazines (27,35,38–40), and the absorption spectrum ofcationic lumichrome (in 8.1 N HClO4) resembled the absorp-tion spectra of cationic isoalloxazines (35).

Fluorescence lifetime studies on lumichromes in differentsolvents were carried out in (7,23,25,26,31,33,35,37,41). Lumi-chrome-characteristic and isoalloxazine-characteristic timeconstants were obtained in situations of phototautomerization.

The triplet state formation and relaxation in lumichrome wasstudied by laser flash photolysis experiments (7,24,42,43).

In this paper we study the absorption and fluorescence

behavior of lumichrome in aqueous solutions at different pHvalues in the range from )1.08 to 14.6. Absorption cross-sectionspectra, fluorescence quantum distributions, fluorescence quan-

tum yields, and fluorescence lifetimes are determined overthis pH range. For pH < pKc � )0.53 LC is found to bedominantly present in the cationic form (LCH2

+). In the range)0.53 < pH < 8 the neutral form of LC (LCH) is found to be

dominant. Between pH 7 and 12.5 the presence of LCH and thetautomer 7,8-dimethyl-isoalloxazine (IAH) is observed.For pH > pKa � 12.5 anionic lumichrome (LC)) and

7,8-dimethyl-isolloxazine (IA)) are dominantly present. AbovepH 14 only IA) was observed (tautomerisation of LC) to IA)).

MATERIALS AND METHODS

Lumichrome (LC) was purchased from Sigma-Aldrich and was usedas delivered. The dye was dissolved in aqueous solutions of differentpH. In the range from pH )1.08 to 3 differently concentratedaqueous HCl solutions (37 wt% HCl to 10)3 M HCl) and in therange of pH 11–14.6 differently concentrated NaOH solutions(10)3 M NaOH to 4 M NaOH) were used. Commercial buffersolutions from Aldrich were used (Fixanal) for pH 4 (citric

acid ⁄NaOH ⁄NaCl), pH 5 (citric acid ⁄NaOH), pH 7 (KH2PO4 ⁄Na2HPO4) and pH 9 (Na2B4O7 ⁄HCl). For pH 6, 8 and 10 self-prepared 10 mM sodium phosphate buffers with 10 mM NaCl(abbreviated by NaP) were used. The measurements were carriedout at room temperature. Transmission spectra of 37 wt% HCl, 4 MNaOH and the used buffer solutions are shown in Fig. 2. The samplelength was 1 cm. The transmissions were measured relative to doublesteam-distilled water. In the relevant visible and near ultraviolet thebuffer solutions are transparent.

The absorption spectra were measured with a commercial spectro-photometer (Cary 50 from Varian). The fluorescence emission spectrawere recorded with a commercial fluorimeter (Cary Eclipse fromVarian). The spectra were corrected for the spectral sensitivities. Forabsolute intrinsic fluorescence quantum distribution and quantumyield measurement the dyes POPOP (1,4-di-(5-phenyloxazolyl)benzene) in ethanol (fluorescence quantum yield /F = 0.85 [44]) andriboflavin in water (/F = 0.26 [45]) were used as reference standards.

The temporal fluorescence behavior was studied by using a mode-locked titanium-sapphire laser oscillator amplifier system (Hurricanefrom Spectra Physics) and an ultrafast streak-camera (type C1587temporaldisperserwithM1952high speed streakunit fromHamamatsu)(46). Picosecond second harmonic pulses at 400 nm and Raman-shiftedpulses at 456 nm (Ramanmediumethanol) (47)were used for excitation.

N

N

N

N

H3C

H3C

O

O

H

H

12

34567

8 9 10

N

N

N

N

H3C

H3C

O

O

H

12

34567

8 9 10

H

Lumichrome (LCH, 7,8-dimethyl-alloxazine) Lumichrome tautomer (7,8-dimethyl-isoalloxazine, IAH)

Figure 1. Structural formulae of lumichrome in neutral form (LCH, 7,8-dimethyl-alloxazine) and lumichrome-tautomer in neutral form (7,8-dimethyl-isoalloxazine, IAH).

0

0.2

0.4

0.6

0.8

1

pH -1.08 (37 wt-% HCl)

pH 4 (Fixanal)

pH 5 (Fixanal)

pH 7 (Fixanal)

pH 9 (Fixanal)

Wavelength (nm)

200 250 300 350 400

Tra

nsm

issi

onT

0

0.2

0.4

0.6

0.8

1

pH 6 (NaP)

pH 8 (NaP)

pH 10 (NaP)

pH 14.6 (4 M NaOH)

Figure 2. Transmission spectra of applied buffer solutions, 37 wt%HCl, and 4 M NaOH measured in fused silica cells of 1 cm path lengthrelative to double steam distilled water.

Photochemistry and Photobiology, 2011, 87 525

RESULTS

Absorption cross-section spectra, r(k), of the studied lumi-

chrome samples in aqueous solution at different pH are shownin Fig. 3a (pH )1.08 to 6) and Fig. 3b (pH 7–14.6). They werecalculated from measured transmission spectra (r = )ln[T] ⁄ [‘N0], T: transmission, ‘: sample length, N0 molecule

number density). Molar decadic absorption coefficient spectra,e(k), are related to the absorption cross-section spectra byeðkÞ ¼ rðkÞNA=½1000 lnð10Þ�, where NA is the Avogadro

constant. At k = 354 nm the absorption cross-section oflumichrome in water at pH 6 was determined to be

r(354 nm) = 3.93 · 10)17 cm2 (e[354 nm] =1.028 · 104 cm2

mol)1) (7,8).The absorption cross-section spectra are unchanged in the

range from pH )0.3 to 7 where the neutral form of lumichrome

(LCH) is dominant. The main absorption band consists of twosub-bands centered at around 400 nm (S0–S1 transition) and355 nm (S0–S2 transition). The absorption cross-section spec-

trum at pH )1.08 is dominated by the cationic form oflumichrome (LCH2

+). The main absorption band peaks at395 nm. It has a weak shoulder at 480 nm (likely due to the

presence of a small fraction of bi-cationic lumichrome,LCH3

2+ [38]). A weak absorption extends out to 700 nm(likely due to the presence of small fractions of constitutional

isomers of cationic lumichrome and aggregates). The absorp-tion spectrum belonging to pH 8 shows the build-up of anabsorption shoulder at 445 nm. This absorption shoulder isattributed to the formation of tautomeric lumichrome (7,8-

dimethyl-isoalloxazine, IAH). The absorption spectra in therange from pH 10 to 12 are unchanged. Their IAH content isestimated to be about 40% by comparison with the absorption

spectrum at pH 14 where 7,8-dimethyl-isoalloxazine is presentin its anionic form abbreviated by IA) (assumption of similarabsorption of IAH and IA) [45]). The absorption cross-section

spectra at pH 14 and 14.6 are thought to belong to IA). Theabsorption cross-section spectrum of lumiflavin at pH 6 isincluded in Fig. 3b. It has the same spectral shape aslumichrome at pH 14, but it is about 17 nm red-shifted. This

shift is attributed to the effect of the 10-methyl group oflumiflavin (lumiflavin is 7,8,10-trimethyl-isoalloxazine).

In Fig. 4a the absorption cross-sections of lumichrome at

335, 350, 395, and 450 nm versus pH are plotted. The mid-point changes of r at pH )0.53 gives the minus decadiclogarithm of the equilibrium constant Kc between cationic and

Wavelength (nm)

300 400 500 600 700

Abs

orpt

ion

Cro

ss-S

ectio

n(c

m2 )

10-20

10-19

10-18

10-17

10-16

pH -1.08

pH -0.6

pH -0.3

pH 0.3

pH 6

(a)

(b)

Wavelength (nm)

250 300 350 400 450 500

Abs

orpt

ion

Cro

ss-s

ectio

n(c

m2 )

10-17

10-16

pH 7

pH 8

pH 10

pH 12

pH 13

pH 14

pH 14.6

Lumiflavin pH 6

Figure 3. (a and b) Absorption cross-section spectra, r(k), of lumi-chrome in aqueous solution at different pH values. For comparison theabsorption cross-section spectrum of lumiflavin aqueous solution atpH 6 is included in (b) (from [48]).

(10-1

7cm

2 )

0

1

2

3

4

5

335 NM

350 NM

395 NM

450 NM

(a)

pH0 2 4 6 8 10 12 14

x

0

0.2

0.4

0.6

0.8

1

xc

xn xn

xa

(b)

Figure 4. (a) Absorption cross-sections of lumichrome in aqueoussolution versus pH for four selected wavelengths: 335, 350, 395 and450 nm. (b) Left side: mole-fractions of cationic form LCH2

+, xc, andof neutral form LCH, xn, at low pH. Right side: mole-fractions ofneutral forms LCH and IAH, xn, and of anionic forms LC) and IA), xa.

526 Amit Tyagi and Alfons Penzkofer

neutral form of LC, i.e. pKc � )0.53. The mid-point change ofr(335 nm), r(350 nm) and r(395 nm) of lumichrome at highpH is difficult to locate. The equilibrium constants Ka and thecorresponding pKa values between the neutral and anionic

form of ground-state lumichrome LC and lumichrome tauto-mer IA are assumed to be the same as the mid-point pH valuesof fluorescence emission, pKFa*, (48) which is found below to

be pKFa* � 12.5 for both LC and IA, i.e. pKa � pKFa* � 12.5for the equilibrium between LCH and LC), and between IAHand IA) (48). The mole-fractions xc of LCH2

+ and xn of LCH

at low pH are given by (45,48)

xc ¼1

1þ Kc=½Hþ�¼ 1

1þ 10�pKc=10�pH; ð1aÞ

xn ¼ 1� xc: ð1bÞ

The mole-fractions xn of LCH and IAH, and xa of LC) andIA) at high pH are given by (45,48)

xa ¼1

1þ ½Hþ�=Ka¼ 1

1þ 10�pH=10�pKa; ð1cÞ

xn ¼ 1� xa: ð1dÞ

The determined mole-fractions are displayed in Fig. 4b.Fluorescence quantum distributions, EF(k), of lumichrome

in aqueous solutions at several pH values in the range from pH)1.08 to 14.6 are shown in Fig. 5a–c. The correspondingfluorescence quantum yields, /F ¼

REFðkÞdk, versus pH are

depicted in Fig. 6.For the curves in Fig. 5a and the line-connected circles in

Fig. 6 the fluorescence excitation wavelength was

kexc = 350 nm. Coming from low pH, the fluorescence effi-ciency increased with pH up to pH 3 (LCH2

+ at low pH isonly weakly fluorescent, /F(LCH2

+) = /F,c £ 4.5 · 10)5, seebelow), and then it remained approximately constant up to pH

7 (/F[LCH] = /F,n � 0.05). At pH 4 the upper data pointbelongs to LC in 10)4 M HCl and the lower data point to LCin citric acid ⁄NaOH ⁄NaCl (Fixanal) buffer. The difference

indicates some influence of the specific buffer on the fluores-cence efficiency. For LC in Millipore water (no buffer added) afluorescence quantum yield of /F = 0.051 ± 0.002 was mea-

sured in good agreement with the fluorescence quantum yieldof LC in the buffered solutions in the range from pH 5 to 7. Ata mid-point pH of pKFc* = 1.65 the fluorescence quantum

yield is /F(pKFc*) = (/F,a + /F,n) ⁄ 2 (see [48]). It ispKFc* > pKc indicating excited-state conversion of LCH* toLCH2

+ (see below). The fluorescence spectra are rather broad(full spectral half-width D~mF � 4600 cm)1) and unstructured

indicating inhomogeneous broadening (locally different sur-roundings and hydrogen bonding interactions). In the rangefrom pH 8 to 13 the shape of the fluorescence quantum

distribution changed with some steepening of the short-wavelength emission (LCH emission peaking at �450 nm)and the formation of a new band around 530 nm (lumichrome

tautomer IAH emission). At pH 13 the fluorescence efficiencyis reduced. Around pH 12.5 (pKa � 12.5) the lumichrome partis present in neutral form (LCH) and in anionic form (LC))(see line-connected dots in Fig. 6 with /F(LC, pH

12.5) = /F(LC, pKFa*) = (/F,n[LCH] + /F,a[LC)]) ⁄ 2).

The strong reduction of the fluorescence quantum distributionaround 450 nm indicates that LC) is much less fluorescentthan LCH (/F[LC

)] = /F,a � 0.001). At pH 14 and 14.6 thereis no fluorescence emission seen around 450 nm (no LC)

contribution), the fluorescence spectra are determined by IA)

emission. The fluorescence spectra at pH 14 and 14.6 as well asthe absorption spectra in Fig. 2b show the presence of IA) and

the absence of LC). This reveals that full tautomerization(intra-molecular proton transfer) from N10 to N1 (alloxazineto isoalloxazine transfer LC) fi IA)) occurred for pH ‡ 14.

For Fig. 5b and the dashed-line connected triangles inFig. 6 the fluorescence was excited at kexc = 450 nm. Data areshown for the range from pH 8 to 14.6. In the range from pH 8

to 12 the fluorescence originates from IAH. For pH 14 and pH14.6 the fluorescence comes from IA) emission. The fluores-cence quantum yield of IAH is /F = /F,n � 0.19. It reducesto /F = /F,a � 0.075 for IA). The pH mid-point of the

change from neutral to anionic fluorescence emission of IA isat pH 12.5 = pKFa* of IA (there /F[IA] = [/F,n(IAH) +/F,a(IA

))] ⁄ 2).The fluorescence behavior of LC at pH )1.08 is shown in

Fig. 5c where lumichrome is dominantly in its cationic form. Inthe top part fluorescence quantum distributions, EF(k), are

depicted for various fluorescence excitation wavelengths in therange from kexc = 230 nm to kexc = 670 nm. In the bottompart the solid curve shows the total fluorescence quantum yield/F of LC at pH )1.08 versus excitation wavelength, and the

dashed curve shows the absorption cross-section spectrum ofLC at pH )1.08. The fluorescence spectra peaking at around400 nm for excitation at 230, 280 and 300 nm are thought to

belong to impurities (this fluorescence contribution is also foundat other pH values as verified for pH 4). The emission from themain absorption band with absorption maximum around

395 nm is very weak (see dotted fluorescence curve forkexc = 400 nm, fluorescence quantum yield of this band isfound to be/F = 4.5 · 10)5 by integrationEF(k) over the rangefrom 440 to 660 nm). The fluorescence emission in the long-wavelength excitation part (kexc ‡ 440 nm) with bands around550, 670 and 720 nm, belongs to lumichrome species formed atthe low pH. These species may be different constitutional

isomers, aggregates, and double protonated LC. These speciesare not present at higher pH (verified for pH 4).

Normalized fluorescence signal decay curves, SF(t) ⁄SF,max,

of lumichrome in aqueous solution at several pH values aredepicted in Fig. 7a,b.

In Fig. 7a the fluorescence excitation wavelength was

kexc = 400 nm and curves are shown in the range from pH)1.08 to 4. The fluorescence detection region was restricted to450 nm < kF,det < 505 nm by optical filters. Within experi-mental accuracy the fluorescence decays single exponentially,

i.e. SF(t) ⁄SF,max = exp()t ⁄ sF). The determined fluorescencelifetime versus pH is displayed in Fig. 8. At pH 4 thefluorescence decay was measured for LC in 10)4 M HCl

(upper data point) and for LC in citric acid ⁄NaOH ⁄NaCl pH4buffer (Fixanal, lower data point). For LC in the pH 4 citricacid buffer a somewhat shorter fluorescence lifetime was

measured than for LC in 10)4 M HCl. This indicates someinfluence of the specific buffer on the fluorescence emission aswas also observed in the fluorescence quantum yield measure-

ments (Fig. 6). Fluorescence lifetime and fluorescence quan-tum yield decrease with decreasing pH for pH < 3. The pH


midpoint, pKFc*, of lifetime shortening and fluorescencequantum yield reduction occurs at pKFc* � 1.65. The pH of

ground-state equilibrium concentration of cationic and neutrallumichrome is at pKc � )0.53. The situation of pKFc* > pKc

indicates hydronium (H3O+) induced protonation of LCH* to

the low-fluorescent LCH2+ cation (see below and [48]). For

LC in 37 wt% HCl (pH )1.08) and fluorescence detectionin the range from 450 to 505 nm the true fluorescence

lifetime could not be resolved because of the limited timeresolution of the streak-camera detection system. A value ofsF,c < sF,limit � 1.5 ps was found. The fluorescence decay of

lumichrome at pH )1.08 was also measured for excitation at400 nm and fluorescence detection above 695 nm. There a

signal with decay time of sF � 1.4 ns was measured (curve notshown). This emission likely comes from structural cationiclumichrome isomers and aggregates.

In Fig. 7b fluorescence decay curves for kexc = 456 nm areshown. For pH ‡ 8 the fluorescence was detected forkF,det > 500 nm. The curves belong to lumichrome tautomer

emission since normal lumichrome is transparent at this long-wavelength excitation. The curves at pH 8 and 12 are due toneutral IAH emission. The emission at pH 13 is dominated by

10-6

10-5

10-4

pH -0.3

pH 0.3

pH 1

pH 2

pH 3

pH 7

Wavelength (nm)500 600 700

Flu

ores

cenc

eQ

uant

umD

istr

ibut

ion

EF

(nm

-1)

10-5

10-4 pH 8

pH 9

pH10

pH 11

pH 12

pH 13

pH 14

pH 14.6

exc = 350 nm

Wavelength (nm)500 550 600 650 700 750

Flu

ores

cenc

eQ

uant

umD

istr

ibut

ion

EF

(nm

-1)

10-4

10-3

pH 12

pH 13

pH 14

pH 14.6

10-4

10-3

pH 8

pH 10

pH 11

exc = 450 nm

EF

(nm

-1)

10-7

10-6

10-5

10-4

230 nm 280 nm

exc = 300 nm

400 nm

440 nm

460 nm480 nm

500 nm

520 nm

540 nm 600 nm

670 nm

Wavelength (nm)300 400 500 600 700 800

F,

(10-1

5cm

2 )

10-5

10-4

10-3

10-2

10-1

F

(a)

(c)

(b)

Figure 5. Fluorescence quantum distributions, EF(k), of lumichrome in aqueous solution at different pH values. (a) Fluorescence excitationwavelength kexc = 350 nm. (b) kexc = 450 nm, (c) Fluorescence behavior of lumichrome in 37 wt% HCl (pH )1.08). Top part: EF(k) for someexcitation wavelengths. Bottom part: fluorescence quantum yield, /F(kexc), and absorption cross-section spectrum, r(k).


IA) emission (xa � 0.76, xn � 0.24; see Fig. 4b). At pH 14 the

fluorescence results from IA) emission (xa � 0.97). For thecurve belonging to pH )1.08 the fluorescence was detected inthe range 560 nm < kF,det < 660 nm. A bi-exponential decay

is observed. The short component may result form LCH2+,

the slow component (time constant �2.6 ns) likely belongsdominantly to bi-cationic lumichrome.

The measured fluorescence lifetime versus pH forkexc = 456 nm and pH ‡ 8 is shown by the dot-connectedtriangles in Fig. 8.

DISCUSSION

Radiative lifetimes and absorption strengths

The S1–S0 radiative lifetime may be obtained from itsfluorescence lifetime, sF, and its fluorescence quantum yield,/F, by the relation

srad ¼sF/F

: ð2aÞ

The S1–S0 radiative lifetime is related to the S0–S1 absorp-tion strength, �r ¼

RS0�S1

½rðkÞ=k�dk, by the Strickler-Berg

formula (49–51)

srad ¼nA�k3F

8pc0n3F�r; ð2bÞ

where nA and nF are the average refractive indices of the solutionin the S0–S1 absorption and emission region, respectively

(nA � nF � 1.33 in aqueous solution), c0 is the speed of light invacuum, and �kF ¼

REFðkÞk3dk=

REFðkÞdk

� �1=3is the mean

fluorescence wavelength.For LCH at neutral pH we obtain srad � 45 ns (sF � 2.4 ns,

/F � 0.053) and �r � 2.3 · 10)18 cm2 (�kF = 516 nm). Thisabsorption strength gives a lower wavelength border of the S0–S1 transition of ku = 379 nm. For IA) at pH 14 we obtain

srad � 25 ns (sF � 1.8 ns, /F � 0.073) and �r � 5.3 · 10)18

cm2 (�kF = 569 nm) and ku = 375 nm. For LCH2+ at

pH )1.08 we find roughly srad � 33 ns (sF £ 1.5 ps,

pH

0 2 4 6 8 10 12 14

Flu

ores

cenc

eQ

uant

umY

ield

F

10-4

10-3

10-2

10-1

exc = 350 nm, F(LC) + F(IA)

exc = 450 nm, F(IA)

exc = 350 nm, F(LC)

F,c,limit

Figure 6. Dependence of fluorescence quantum yield, /F, of lumi-chrome on pH value of aqueous solution. Solid-line-connected circlesbelong to excitation wavelength kexc = 350 nm and give total fluores-cence emission /F,tot = /F(LC) + /F(IA) (for pH ‡ 7). The line-connected dots belong to kexc = 350 nm and show the fluorescenceemission of LC (/F[LC]). The dashed-line-connected triangles belongto kexc = 450 nm and give the fluorescence efficiency due to IAemission (/F[IA]). Filled triangle gives the upper limit of thefluorescence quantum yield of LCH2

+. Single triangle at pH )1.08belongs to kexc = 450 nm and emission from bicationic lumichrome,constitutional isomers of cationic lumichrome and aggregates.

Time t (ps)

0 200 400 600 800 1000

Nor

mal

ized

Flu

ores

cenc

eS

igna

lS

F/S

F,m

ax

0

0.2

0.4

0.6

0.8

1exc = 400 nm

pH -1.08

pH -0.3pH 0.3

pH 1

pH 1.3

pH 4

Time t (ps)

0 1000 2000 3000 4000

Nor

mal

ized

Flu

ores

cenc

eS

igna

lS

F/S

F,m

ax

0

0.2

0.4

0.6

0.8

1

pH -1.08

pH 8

pH 12

pH 13

pH 14

exc = 456 nm

pH 8

pH 12

pH 14

pH 13

pH -1.08

(a)

(b)

Figure 7. Normalized fluorescence traces, SF(t) ⁄SF,max, of lumichromein aqueous solution at different pH values. (a) Picosecond laserexcitation wavelength kexc = 400 nm, pulse duration Dtexc = 4 ps,detection wavelength range kF,det ‡ 500 nm. (b) kexc = 456 nm,Dtexc = 4 ps, kF,det > 500 nm for pH ‡ 8, and kF,det = 560–660 nmfor pH )1.08.


/F £ 4.5 · 10)5) and �r � 3.2 · 10)18 cm2 (�kF � 520 nm) andku � 402 nm. The lower S0–S1 wavelength border of

ku � 402 nm indicates that the main absorption band peakingat 395 nm comprises the S0–S1 and S0–S2 transition. For thelongest emitting component of lumichrome at pH )1.08 withabsorption maximum at 669 nm we have srad � 7.8 ns

(Eq. (2a) with sF � 1.4 ns, /F � 0.18) and �r � 3.9 ·10)17 cm2 (from Eq. (2b) with �kF � 740 nm). The apparentabsorption strength in Fig. 3a (ku � 625 nm) is �rapp �1.2 · 10)21 cm2 giving a mole-fraction of the long-wavelengthabsorbing component of x ¼ �rapp=�r � 3 · 10)5.

pH dependent composition

The pH dependent absorption and fluorescence behavior oflumichrome shows the dominant presence of LCH2

+ forpH < pKc � )0.53, the dominant presence of LCH in therange pH )0.53 to 8, the presence of LCH and IAH in the

range of pH 8–12.5 � pKa, the conversion of LCH to LC) andof IAH to IA) around pH = pKa, and the tautomerization ofLC) to IA) above pH 13 (practically complete conversion for

pH ‡ 14). At pH )1.08 lumichrome is dominantly present incationic form (xc � 0.78, xn � 0.22, see Eqs. 1a, 1b andFig. 4b). A small fraction of other species with reasonable

fluorescence efficiency was found. A small amount ofbi-cationic lumichrome (LCH3

2+) is expected to be present(38). In Scheme 1 the pH dependence of the molecular

structures of lumichrome is sketched.The ground-state tautomerization of lumichrome in aque-

ous solution (intra-molecular ground-state proton transferfrom N1 to N10) has not been reported before. In (27) the

absorption spectral change around 450 nm in the range of pH

8–9 was attributed to a changeover from neutral to single-anionic lumichrome. The absorption spectral change aroundpH 13 was attributed to a changeover from single anionic tobi-anionic lumichrome. The absorption and emission spectro-

scopic studies presented here with two absorption components(Fig. 3b) and two fluorescence components (Fig. 5a) in therange from pH 7 to 13 indicate the presence of LCH and IAH

in this pH region.

Excited-state dynamics

Information on the excited-state dynamics is obtained from

the performed fluorescence studies. For pH < pKc � )0.53the fluorescence efficiency was found to be very smallbecause of very low fluorescence efficiency of LCH2

+

(/F[LCH2+] £ 4.5 · 10)5). Below pH 3, the fluorescence

efficiency of LCH decreased with lowering pH. The mid-point pH value pKFc* where /F(pKFc*) = (/F,c + /F,n) ⁄ 2 ispKFc* = 1.65 > pKc. This behavior indicates a conversion

of LCH* to LCH2+ according to the bi-molecular excited-

state reaction

LCH� þH3Oþ ! LCH2

þ þH2O; ðR1Þwith bimolecular rate constant kn*c (intermolecular protontransfer from H3O

+ to LCH*). An illustration of the dynamics

is shown by Scheme 2. A data analysis described in (48)(Eqs. (7a,b) and (8) there) gives kn*c � 1.8 · 1010 mol)1 s)1

(sF,n = sF,0,n ⁄ 2 at pH = pKFc* = 1.65, and sF,0,n � 2.5 ns).

At high pH the change of fluorescence efficiency coincides withthe change of lumichrome from LCH to LC) and of IAH toIA) at pH = pKFa* = pKa � 12.5. This coincidence indicates

that within the excited-state lifetimes of LCH*, IAH*, LC)*,and IA)* no measurable inter-conversion of the ionic stage ofthe molecules takes place (48).

The short fluorescence lifetime and the low fluorescencequantum yield of LCH2

+ is thought to be determined bybarrier-less diabatic intra-molecular electron transfer(46,48,52) between locally excited state and charge-transfer

state and fast relaxation to the ground-state by surfacetouching of charge-transfer state and ground-state (46,48,53).

The fluorescence behavior of LCH for pH > 3 is thought to

be determined by excited-state adiabatic optical electrontransfer (46,48,52) where the S1-state potential energy surfaceis determined by vibronic relaxation and solvation dynamics

due to excited-state dipole moment changes (54–58). Thefluorescence lifetime and the fluorescence quantum yield aredetermined photophysically by internal conversion and inter-

system crossing.The fluorescence behavior of LC) and IA) is thought to be

determined by intra-molecular electron transfer (diabaticelectron transfer) (46,48,52) between locally excited state and

charge-transfer state, and moderately fast relaxation to theground-state by thermal activated sloped conical intersectionof charge-transfer state and ground-state (48,53). The faster

relaxation of LC) than of IA) indicates a higher sloped conicalintersection barrier for IA) than for LC).

Comparison with other flavins

The pH dependence of the ionization stage of flavins wasanalyzed in (27,35,38,39,48,59–61). Detailed studies on the

pH

0 5 10 15

Flu

ores

cenc

eLi

fetim

eF

(ps)

100

101

102

103

exc = 400 nm

exc = 456 nm

F,c,limit

Figure 8. Fluorescence lifetimes, sF, of lumichrome in aqueous solu-tion versus pH. Line-connected circles belong to excitation wavelengthkexc = 400 nm and detection wavelength region kF,det ‡ 500 nm forpH £ 5 and kF,det = 450–505 nm for pH ‡ 6. Dot-connected trianglesbelong to kexc = 456 nm and kF,det > 500 nm. Single triangle belongsto kexc = 456 nm and kF,det = 560–660 nm. Filled circle gives upperlimit of fluorescence lifetime of LCH2

+.


absorption and emission behavior of flavins as a function ofpH were carried for riboflavin (45) (pKc = 0.4, pKa = 9.75),

FAD (62) (pK values not determined because of hydrolysis atlow pH and high pH [61]), and lumiflavin (48) (pKc = 0.38,pKa = 10.8). The pK values of lumichrome determined hereare lower for the cationic-neutral equilibrium (pKc � )0.53)

and higher for the neutral-anionic equilibrium (pKa � 12.5).Only for ground-state lumichrome in the alkaline pH region

tautomerization occurs by intra-molecular N1–N10 protontransfer since only for lumichrome the N10 position is notoccupied by a substituent, and at the N1 position a H atom ispresent for transfer.

N

N

N

N

H3C

H3C

O

O

H

HH

H

LCH32+

H3O+

H2O

H3O+

H2O

N

N

N

N

H3C

H3C

O

O

H

HH

N

N

N

N

H3C

H3C

O

O

H

HH

N

N

N

N

H3C

H3C

O

OH

HH

LCH2+ LCH2

+ isomer LCH2+ isomer

H3O+

H2O

H3O+

H2OpKc = -0.53

N

N

N

N

H3C

H3C

O

O

H

H

pH 8 – pH 12,5

N

N

N

N

H3C

H3C

O

O

H

H

LCH IAH

OH-

H2O

OH-

H2OpKa = 12.5

OH-

H2O

OH-

H2OpKa = 12.5

N

N

N

N

H3C

H3C

O

O

H

> pH 12.5

N

N

N

N

H3C

H3C

O

O

H

LC- AI -

Scheme 1. pH dependence of molecular structure of lumichrome.


CONCLUSIONS

The absorption and emission behavior of lumichrome inaqueous solution was studied over a pH range from )1.08 to

14.6. For pH < pKc � )0.53 lumichrome is dominantlypresent in cationic form. Its fluorescence is strongly quenchedlikely by barrier-less intramolecular diabatic charge transferand excited-state relaxation likely by potential surface touch-

ing with the ground state. The neutral molecule form is presentover the range )0.53 � pKc £ pH £ pKa � 12.5. The fluores-cence behavior is determined by adiabatic optical electron

transfer (fluorescence Stokes shift determined by vibronicrelaxation and solvation dynamics, fluorescence lifetime andquantum yield determined by internal conversion and inter-

system crossing). In the range from pH 9 to 12 approximately40% of lumichrome was found to be converted to7,8-dimethyl-isoalloxazine by intra-molecular ground-stateproton transfer (tautomerization) from N1 to N10. For

pH > pKa � 12.5 the molecules were found to be dominantlyin the anionic form. Anionic lumichrome converted fullyto anionic 7,8-dimethyl-isoalloxazine for pH ‡ 14. The

fluorescence of the anionic forms of LC) and IA) was foundto be reduced compared with the neutral forms likely becauseof intra-molecular charge transfer and excited-state relaxation

via a thermally activated sloped conical intersection.

Acknowledgements—The authors thank the Deutsche Forschungs-

gemeinschaft (DFG) for support in the Graduate College GK 640

‘‘Sensory Photoreceptors in Natural and Artificial Systems’’ and in the

Research Group FOR 526 ‘‘Sensory Blue Light Receptors’’. A. P. is

grateful to Profs. F. J. Gießibl and J. Repp for their kind hospitality.

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Scheme 2. Reaction dynamics of photoexcited lumichrome at low pH(pH < 7).


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absorption and emission spectroscopic characterization of lumichrome in aqueous solutions

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