Bioinspired Molecular Lantern: Tuning the Firefly Oxyluciferin

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Bioinspired Molecular Lantern: Tuning the Firefly Oxyluciferin Emission with Host-Guest Chemistry Na'il Saleh, Abdul Rahman Ba Suwaid, Ahmad Alhalabi, Ahmed Z. A. Abuibaid, Oleg V. Maltsev, Lukas Hintermann, and Pance Naumov J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b06557 • Publication Date (Web): 21 Jul 2016 Downloaded from http://pubs.acs.org on July 24, 2016

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The Journal of Physical Chemistry

Bioinspired Molecular Lantern: Tuning the Firefly Oxyluciferin Emission with Host-Guest Chemistry Na’il Saleh,†* Abdul Rahman Ba Suwaid,†,# Ahmad Alhalabi,†,# Ahmed Z. A. Abuibaid,†,# Oleg V. Maltsev,‡ Lukas Hintermann,‡ Panče Naumov§* †

Department of Chemistry, College of Science, United Arab Emirates University, P.O. Box

15551, Al Ain, United Arab Emirates ‡

Department Chemie, Technische Universität München, Lichtenbergstrasse 4, 85748 Garching

bei München, Germany §

New York University Abu Dhabi, P.O. Box 129188, Abu Dhabi, United Arab Emirates

Corresponding

authors:

[email protected]

(N.S.).

Tel.:

+971-(0)3-713-6138;

[email protected] (P.N.) Tel.: +971-(0)2-628-4572

1

ACS Paragon Plus Environment


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ABSTRACT. Fireflies generate flashes of visible light via luciferase-catalyzed chemiexcitation of the substrate (luciferin) to the first excited state of the emitter (oxyluciferin). Microenvironment effects are often invoked to explain the effects of the luciferase active pocket on the emission, however the exceedingly complex spectrochemistry and synthetic burdens have precluded elucidation of the nature of these interactions. To decipher the effects of microenvironment on the light emission, here the hydrophobic interior of cucurbit[7]uril (CB7) is used to mimic the non-polar active pocket of luciferase. The hydrophobic interior of CB7 induces shift of the ground-state pKas by 1.9‒2.5 units to higher values. Upon sequestration, the emission maxima of neutral firefly oxyluciferin and its conjugate monodeprotonated base are blueshifted by 40 nm and 39 nm, respectively, resulting in visual color changes of the emitted light.

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INTRODUCTION Fireflies communicate with each other by generating flashes of light in a very efficient two-stage, fourstep reaction catalyzed by an enzyme, firefly luciferase (Luc), whereby the benzothiazolyldihydrothiazole carboxylic acid luciferin, LH2 (‘H2’ here stands for the two ionizable protons) and oxygen react to give the first excited state of the oxidation product, oxyluciferin (OxyLH2; Scheme 1).1 The chemiexcited oxyluciferin deexcites on a nanosecond time scale with emission of a photon of greenyellow light (λ ≈ 560 nm). The high efficiency of this process of generation of cold light 2 has spurred ample experimental3–12 and computational research efforts13‒24 into the photophysics and photochemistry of the emitter. The ongoing research revolves around the yet unresolved chemical form from which oxyluciferin emits light and the unknown mechanism by which some natural and genetically engineered luciferases can generate light of varying colors.9 The multiple chemical equilibria (dissociation of the two hydroxyl groups and keto-enol tautomerism of the hydroxythiazole, Chart 1) and the chemical instability of OxyLH2 in basic solutions25 have precluded direct studies into the chemistry of the firefly emitter in the enzyme. The electronic spectra, distribution with pH,11 and ground- and excited-statedissociation constants of the chemical forms of OxyLH2 in buffered model solutions have only recently been unveiled.12 Notably, the effects of polarity of the Luc active pocket on OxyLH2 spectrochemistry are central to one of the theories that is commonly invoked to explain the color variations in the emitted light.4 Early studies have concluded that the active pocket is of low polarity.1 Recent results, however, indicate that a single water molecule can affect the wavelength of emitted light 26 and thus the water dynamics could play a critical role in the OxyLH2 photophysics in vivo.17,23 Even that the collective interactions with the active pocket of Luc, often referred to as microenvironment effects, are undoubtedly relevant to the firefly photochemistry, the true nature of these interactions remains elusive.

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Scheme 1. Structural formulas of cucurbit[7]uril (CB7), firefly D-luciferin (LH2), oxyluciferin (OxyLH2) and derivatives used as models to assign the associated protonation and tautomerization equilibria. To investigate the effect of a non-polar environment within a binding pocket on the emission from firefly oxyluciferin, we resorted to host-guest chemistry as straightforward and inexpensive approach that bypasses the synthetic burdens associated with purposefully engineered mutant luciferases. Sequestration of fluorophores into macromolecules are known to have multiple benefits, notably prevention of the fluorescence quenching observed with solid OxyLH29 by suppression of aggregation.27 This supramolecular “isolation” by complexation could enhance the emission from OxyLH2, alleviate its instability at high pH by preventing dimerization,25 and protect its anionic forms from oxidation.27–29 Macromolecular hosts such as cucurbiturils (CBs)30 and cyclodextrins (CDs)31 appear ideally suited to encage OxyLH2 and could be considered a mimic of the hydrophobic microenvironment of Luc since they have cavities of variable size and well-defined interactions. 4


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Chart 1. Acid-base equilibria between the chemical forms of firefly oxyluciferin in solution and definition of the pKa values. The keto-enol equilibria in each protonation state are not specified for clarity. The notation of the pKa values refers to all molecules studied in this work.

The modulation of the emission properties of the guest molecules utilizing host-guest chemistry is well known in literature.28,32‒35 In this work we report, for the first time, complex formation of natural bioluminescence molecule, the firefly emitter oxyluciferin and its precursor with cucurbit[7]uril (CB7; Scheme 1), and we investigate the associated photochemistry. It is concluded that the low-polarity environment in the CB7 interior affects the multiple chemical equilibrium of OxyLH2, increases the emission energy, blueshifts the maximum of the emitted light, and extends the fluorescence lifetimes of its 5



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chemical forms. Besides providing insight into the effect of low polarity of the Luc active pocket, these results also set the path to a general concept of controlling light emission from natural and artificial bio/chemiluminescent reactions by host-guest chemistry.

MATERIALS AND METHODS The spectra were acquired with a Cray 300 spectrometer for the absorption and Cary Eclipse for steadystate data. The slit width was optimized depending on the pH, and the concentration of the analyte was kept low to attain absorbance of 0.1 in the emission spectra, and 0.3 in the absorption spectra. The samples were excited at the isobestic point obtained from the UV titrations. The evolution of the structures and dynamics of the free and CB7-bound firefly compounds was studied at different pH to clarify the associated protonation equilibria. The absorption, steady-state and timeresolved fluorescence spectra of several solutions of each chromophore were recorded at different pH (0.5–12). The absorption, emission maxima and the lifetimes were extracted for each chemical form in water and inside the cavity of CB7 (the procedure used to calculate the binding constants is described in the Supporting Information). Stock solutions of CB7 and a given analyte were prepared by dissolving 50–100 μg of each guest either in 10 mL pure water and/or in CB7-containing (25 mg) acidic aqueous solution (pH ≈ 0.5) and mixing until complete dissolution. The procedure for the pH titration by UVVisible or fluorescence spectroscopy was as follows: after measuring the pH, 3 mL solution was placed in a rectangular cuvette with 1 cm optical path length and the absorption spectra were recorded. Then, µL-volumes from 0.01 and 0.1 M NaOH solution were pipetted consecutively to achieve the target pH value. The pKa value was determined from the sigmoidal fitting of the titration data at a selected wavelength. In addition to LH2 and OxyLH2, three model compounds where some of the equilibria are selectively blocked were prepared and studied: 3'-de-aza-OxyLH2 and 3-de-aza-OxyLH2 (Scheme 1) where the 6


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nitrogen atom on the benzothiazole or thiazole is substituted by a CH group and cannot be protonated, and the molecule 1 in Chart S1, Supporting Information, which is a model compound for the bioluminescence intermediate. The procedures for preparation and purification of these compounds will be a subject of a subsequent publication. The interaction of LH2, OxyLH2 and the two model compounds in Scheme 1 with CB7 were studied at pH ≈ 5.5 where the CB7-bound and free neutral species exist. Stock solutions of CB7 and a given analyte were prepared by dissolving few tens of micrograms (50–100 μg) of each guest in 10 mL pure water and/or CB7-containing (25 mg) aqueous solution at pH ≈ 5.5. Under excitation, the polytetrafluoroethylene (PTFE)-filtered solutions emitted light with various colors (Table 3). For measurement of the lifetime a spectrometer LifeSpec II was used which utilizes the TCSPC method with time resolution of 30 ps and excitation at 375 nm. The time-resolved emission (intensity of ~1000–3000 counts s‒1) was collected up to 10,000 counts s‒1 with a red-sensitive high-speed PMT detector (Hamamatsu, H5773-04). The data were analyzed by the iterative reconvolution method using commercial software that utilizes the Marquardt-Levenberg algorithm to minimize χ2. In order to correct for the contribution of each lifetime, τi with an amplitude αi in the multiexponential model, to the steady-state intensity, the formula 𝑓𝑖 =

𝛼𝑖 𝜏𝑖 ∑𝑗 𝛼𝑗 𝜏𝑗

was used, where the sum in the denominator is over all the decay times and amplitudes. The mean decay time (average lifetime) is then calculated by 𝜏̅ = ∑ 𝑓𝑖 𝜏𝑖 𝑖

The pH was adjusted by addition of aliquots of each acid and base, and measured using WTW pH meter with SenTixMic probe (pH range 0‒14). 7



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RESULTS AND DISCUSSION Host-Guest Complex Formation and Binding Constants. The binding affinity of LH2, OxyLH2 and two model compounds that each lack one of the nitrogen atoms (3'- and 3-de-aza-OxyLH2; Scheme 1) with four cucurbiturils (CB5, CB6, CB7, and CB8) and three cyclodextrins (α-, β- and γ-CD), whose structures are illustrated in Chart S1 (Supporting Information), was assessed by UV-Visible absorption and fluorescence spectroscopies (for experimental details, complete spectra and titration curves, see the Experimental section and Figures S1–S9, Supporting Information). The assignment of the pKa of the carboxyl group of LH2 was additionally confirmed with another model compound (see Chart S1 and Figures S5, Supporting Information). As it is inferred from the binding constants in Table 1, from the hosts used in this study, OxyLH2 forms a stable 1:1 complex only with CB7; the interaction with other hosts is weak or complexes do not form. While formation of complex with α-CD was detected at low pH, the binding is very weak and was not investigated further (Figures S1‒S3, Supporting Information). Figure 1 shows the interactions between the guests and CB7 at pH ≈ 5.5 (all spectra are deposited as Figures S2 and S3, Supporting Information). Binding of LH2 (as the conjugate base LH‒, see below), 3-de-aza-OxyLH2 and 3'-de-azaOxyLH2 with CB7 is weak (Figures S2 and S3, Supporting Information). The lack of encapsulation by other cages, including β-CD which has a similar size to CB7, is due to size mismatch, more flexible cages, weaker non-bonded interactions, and higher polarity of their cavities.30 It is noteworthy that the complex of CB7 with the ion of LH2, LH‒/CB7 (K = 541 ± 125 M‒1) is less stable than that of OxyLH2, OxyLH2/CB7 (K = 1,400 ± 300 M‒1), presumably due to the steric bulk of the carboxylate group.36 The strong binding of OxyLH2 to CB7 provides supramolecular analogy to the propensity of firefly Luc for autoinhibition, where the product remains strongly bound to the enzyme after deexcitation and effectively inhibits its activity.37 8


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Figure 1. Complexation of firefly oxyluciferin and its analogues with CB7 (the structures are given in Scheme 1) determined by titration based on steady-state fluorescence (a‒c) and absorption spectra (d). The related spectra are deposited as Figures S2 and S3, Supporting Information.

Effect of Encapsulation on the Chemical Equilibria. Sequestration of the guest molecules in CB7 occurs by joint action of hydrophobic and ion-dipole interactions.31 The glycoluril core of CB7 provides hydrophobic environment that fosters binding of the neutral forms of the guests or of their less polar parts. The protonated nitrogen atoms of the cationic oxyluciferins, on the other hand, are expected to interact mainly with the oxygen atoms at the two portals of CB7 by ion-dipole interactions, based on analogy with similar derivatives.38 The non-polar cavity of CB7 affects the physical and chemical properties of the encapsulated guest molecules.29 Since CBs bind preferentially protonated species, the pKa values are shifted toward higher values.38 9



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To identify the chemical forms of OxyLH2 in solution and in the interior of CB7, the protonation equilibria in the ground state were studied from the pH-dependent absorption spectra (Figures S5 and S6, Supporting Information). The pKa values were extracted from the titration plots in Figures S9, Supporting Information. Tables 2 and 3 list the absorption/emission maxima and the color of emitted light for each species. The emission maxima are attributed to the dominant emitting species in light of the chemical equilibria in the ground state, as detailed in Chart 1. It should be noted, however, that the emission is not restricted to a single species and it is very likely that more than one form contribute to each spectrum.

Table 1. Binding Constants of Firefly Luciferin, Oxyluciferin and Model Compounds with Different Macrocyclic Hosts at pH 3 and 5.5 as Determined by UV-Visible Absorption Titration (UV) and Fluorescence Titration (FL) Species CB5 CB6 CB7 CB8 α-CD βγCD CD OxyLH2

NFa

NFa

LH2b

NFa

NFa

3'-de-aza-OxyLH2

NDc

NDc

3-de-aza-OxyLH2

NDc

NDc

(1.4 ± 0.3) × 103 M‒1 (FL; pH 5.5) (541 ± 125) M‒1 (FL; pH 5.5) (1.1 ± 0.9) × 104 M‒1 (UV; pH 5.5) (7.3 ± 0.9) × 104 M‒1 (FL; pH 5.5) VWd

NFa

NDc

(42 ± 65) M‒1 NFa (FL; pH 3) NFa NFa

NDc

NDc

NDc

NDc

NDc

NDc

NDc

NDc

NFa

NFa

Complex does not form. bExists as ion, LH‒. cNot determined. dThe interaction is very weak at pH 5.5, however, the complex forms at lower pH and pKa,4 could be determined (see text). a

The first two dissociation constants of OxyLH2 correspond to single (pKa,2) and double (pKa,1) deprotonation of its neutral form (Chart 1). These two constants of OxyLH2, LH2, 3'-de-aza-OxyLH2 and 3de-aza-OxyLH2, shift to higher values upon inclusion in CB7 (Figures S5‒S9, Supporting Information). Decrease in acidity of OxyLH2 in the first dissociation step was observed in the ground state, from pKa,2 = 6.19 ± 0.06 to 7.2 ± 0.1 (Figures S5 and S6, Supporting Information). In the second dissociation step, the dissociation constant of OxyLH2 decreased slightly, from pKa,1 = 9.32 ± 0.04 to 8.98 ± 0.04 (Figures 10


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S5 and S6, Supporting Information). The difference in binding affinities of CB7 to each species, as reflected in the increase in pKa, is attributed to the preference for binding of CB7 to neutral species over anions.36 The third acid-base equilibrium (pKa,3) is assigned to protonation of the thiazole nitrogen by comparison with the model compound 3-de-aza-OxyLH2 which lacks the thiazole nitrogen as protonation site (Scheme 1). In the ground state, the first protonation of OxyLH2 in solution occurs with pKa,3 = 0.85 ± 0.05 (Figure S5, Supporting Information). In CB7, the acidity of OxyLH3+ decreases to 4.1 ± 0.1 (Figure S6, Supporting Information). By analogy with other host-guest systems,38 the pronounced increase in pKa,3 of OxyLH2 relative to 3'-de-aza-OxyLH2 upon inclusion in CB7 (ΔpKa,3 = 3.3 vs. 1.3) (see Figures S5 and S6, Supporting Information) is attributed to the more pronounced difference in binding affinity to CB7 between the monoprotonated and neutral species in the former compound. The smaller difference in the latter compound can be attributed to the substitution of the nitrogen atom with more hydrophobic group (CH). This turns its binding affinity comparable to that of the protonated form and results in smaller pKa shifts.38

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Figure 2. pH-dependent emission spectra of firefly luciferin (LH2) and oxyluciferin (OxyLH2) in solution without (a,c) and in presence (b,d) of CB7.

Table 2. Dissociation Constants in the Ground State (pKa), and Absorption/Emission Maxima of Firefly Luciferin (LH2) and Oxyluciferin (OxyLH2), and Their Analogs (3-de-aza-OxyLH2 and 3'de-aza-OxyLH2) and Complexes with CB7 Dissociation constantsa Host

‒

CB7

‒

‒

CB7

CB7

‒

CB7

pKa,4

pKa,4

pKa,3

pKa,3

pKa,2

pKa,2

pKa,1

pKa,1

OxyLH2

˂0

2.4

0.9

4.1

6.2

7.2

9.3

9.0

LH2

˂0

˂0

2.8

1.8

5.6

8.0

8.5

˂0

3'-de-aza

b

b

NA

NA

0.3

1.6

7.2

7.7

9.7

9.8

3-de-aza

NDc

2.1

NAb

NAb

8.2

8.3

9.9

9.9

Maximum absorption, λabs / nm Species LH4 OxyLH2 LH2 3'-de-aza

318 ND

c

b

NA

2+

LH4 /CB 7

LH3+

LH3 /CB 7

LH2

LH2/CB7

LH‒

LH‒/CB 7

L2‒

L2‒/CB7

337

342

340

347

338

338

335

420

403

396

337

330

330

325

321

328

385

385

307

460

272

337

460

478

355

342

2+

b

NA

+

12


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3-de-aza

The Journal of Physical Chemistry NDc

NAb

395

NAb

350

352

390

381

410

407

d

Maximum emission, λem / nm Species LH4 OxyLH2 LH2 3'-de-aza 3-de-aza

2+

VWe ND

c

b

NA ND

c

LH4 /CB 7

LH3+

LH3 /CB 7

LH2

LH2/CB 7

LH‒

LH‒/CB 7

L2‒

L2‒/CB7

460

511

548

509

512

502

532

530

529

527

2+

+

560, 480

465

550

c

568, 480

560

560

b

f

ND NA

477

467

f

508

b

b

NA

NA

473 , 487

496

426

426

a

b

473, 482 511 c

g

492

482

515

527 g

503

471 503

d

Rounded to one decimal. The standard deviations are not shown. Not applicable. Not determined. λex = 330, 342, 350, 370, 375 or 382 nm (for details, see Supporting Information). eVery weak emission. fλex = 292 nm. gλex = 414 nm.

Table 3. Emission Colors of the Chemical Forms of Oxyluciferin, with and without CB7, Based on the Emission Maxima in Table 2 Compound

Chemical form LH42+ a

LH42+/CB7

LH3+

+

LH3 /CB7

LH2

LH2/CB7

LH‒

LH‒/CB7

L2‒

L2‒/CB7

VW

Blue

Yellowgreen

Blue

Yellowgreen

Pale green

Green

Pale green

Pale green

Pale green

LH2

NDb

NDb

Yellowgreen

Yellowgreen

Yellowgreen

Green

Green

Green

Green

Green

3'-de-aza

NAc

NAc

Blue

Pale green

Cyan

Cyan

Blue

Cyan

Blue

Blue

3-de-aza

b

Blue

c

Pale blue

Pale blue

Pale green

Pale green

Pale green

Pale green

OxyLH2

ND

NA

c

NA

a

Very weak emission. bNot determined. cNot applicable.

The value of pKa,4 associated with protonation of the benzothiazole nitrogen is assigned by comparison with 3-de-aza-OxyLH2, where the thiazole nitrogen is substituted for a CH group and protonation of thiazole is not possible. This constant is negative in both LH2 and OxyLH2 in the ground state. In presence of CB7, pKa,4 of OxyLH2 increases from

Bioinspired Molecular Lantern: Tuning the Firefly Oxyluciferin

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