Conformationally locked chromophores of CFP and Sirius protein

Accepted Manuscript Conformationally Locked Chromophores of CFP and Sirius protein Nadezhda S. Baleeva, Aleksandra S. Tsarkova, Mikhail S. Baranov PII: DOI: Reference:

S0040-4039(16)30666-9 http://dx.doi.org/10.1016/j.tetlet.2016.06.006 TETL 47733

To appear in:

Tetrahedron Letters

Received Date: Revised Date: Accepted Date:

1 April 2016 15 May 2016 1 June 2016

Please cite this article as: Baleeva, N.S., Tsarkova, A.S., Baranov, M.S., Conformationally Locked Chromophores of CFP and Sirius protein, Tetrahedron Letters (2016), doi: http://dx.doi.org/10.1016/j.tetlet.2016.06.006

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract

Conformationally Locked Chromophores of CFP and Sirius protein

Leave this area blank for abstract info.

Nadezhda S. Baleeva, Aleksandra S. Tsarkova, and Mikhail S. Baranov,

1

Tetrahedron Letters

Conformationally Locked Chromophores of CFP and Sirius protein Nadezhda S. Baleevaa,b, Aleksandra S. Tsarkovaa,b and Mikhail S. Baranova,b a b

Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow 117997, Russia Pirogov Russian National Research Medical University, Ostrovitianov 1, Moscow, 117997, Russia

A RT I C L E I N F O

A BS T RA C T

Article history: Received Received in revised form Accepted Available online

Recent works have demonstrated the potential of fluorescent protein chromophores as useful dyes with unique photophysical properties. Herein, we report the synthesis of two novel highly fluorescent dyes based on conformationally locked chromophores of the fluorescent proteins CFP and Sirius. These novel synthetic dyes demonstrate high fluorescence quantum yields and pH-independence in the physiological pH range, making them promising molecular tools for a broad range of fluorescent labeling applications.

Keywords: Fluorophore; Borylation; Imidazolone; Chromophore; Fluorescent protein

Introduction Since the early 90’s fluorescent proteins (FP) have become the object of extensive research as a useful tool in fluorescent microscopy.1 Over the years many FP were obtained from Nature, yet their true diversity was achieved artificially.1 The FP chromophores, responsible for their colour and fluorescent properties, are formed from three amino acids by intramolecular condensation and oxidation reactions, which produce the 4benzylidene-1H-imidazol-5(4H)-one core. The benzylidene fragment of the chromophore is derived from the second of the imidazolone-forming amino acids present in the corresponding protein. In natural proteins, only tyrosine plays this role. This amino acid may be artificially substituted with other aromatic residues (His, Trp, Phe) thus generating a multicoloured palette of proteins.2 The FP chromophores and its analogs have attracted special attention from various research teams as model compounds in protein studies.3 Many chromophores and their synthetic analogs possess low fluorescence quantum yield (QY) in solution due to a non-radiative relaxation process based on conformational lability.3c However, under different conditions, upon structural locking, they show an increase of fluorescence emission and were recently used as aptamer-based fluorescent labels for RNA,4 as well as polymeric5 and solid-state emitters.6 The wide range of colors, small size and high polarity as well as simple syntheses7 continue to make these molecules particularly interesting subjects of scientific research.

2009 Elsevier Ltd. All rights reserved .

Recently, we reported a new strategy for the conformational fixation of these molecules based on the introduction of a difluoroboryl bridge. This strategy was applied to the green fluorescent protein (GFP) chromophore,8 Kaede chromophore,9 and several GFP chromophore analogs,10 leading to a great improvement of quantum yields. The consequently obtained highly fluorescent derivatives were successfully used for cell membrane staining10 as well as covalent labeling of human cathepsins.11 Similar modification was carried out on hispidincontaining protein chromophore analogs;12 however, phenylalanine- and tryptophan-based chromophores of fluorescent proteins never underwent a conformation-locking functionalization. Herein, we present the synthesis of two novel highly fluorescent dyes based on the conformationally locked chromophores of cyan fluorescent protein (CFP) and Sirius protein. Spectral properties of new compounds were examined and compared with those of the original proteins. In addition, their potential as pH non-sensitive fluorescent labels was estimated. Results and Discussion Conformationally locked chromophore analogs (2a and 2b) were synthesized from non-locked derivatives 1 via a recently reported borylation reaction.13 Boron tribromide and molecular sieves were used as previously reported,8-10 but a lower excess of tribromide was used.14

———  Corresponding author. Tel.: +7 499 724 81 22; fax: +7 499 724 81 22; e-mail: [email protected]

2

Tetrahedron b

Non fluorescent, QY<0.1%.

с

QY of one of the brightest CFP variant - mseCFP2b

Scheme 1. Synthesis of conformationally locked analogs of CFP and Sirius chromophores 2.

As anticipated, borylated derivatives 2 possessed bright blue and green emissions and demonstrated more than a hundredfold increase of QY relative to the non-locked analogs 1 (Fig 1, Table 1).

Figure 1. The absorbance (solid) and emission (dashed) spectra of compounds 2a (black) and 2b (gray) in acetonitrile.

Previously, we observed bathochromic shifts of emission and absorbance spectra for other borylated compounds in contrast to their non-borylated derivatives.8-10 In the present work only compound 2b possessed a strong red-shifted emission and absorbance spectra compared to initial chromophore 1b and the corresponding protein CFP (Table 1).15 The spectral maxima of 2a were close to those of the Sirius protein2b and its chromophore 1a. As this effect can be explained by coordinate bond formation between boron and nitrogen atoms, the spectral properties of the borylated analog were closer to the N-protonated or N-alkylated imidazolones. We investigated the properties of 1 and found that in the case of 1а, protonation of the nitrogen atom does not produce a substantial red shift in the spectral maxima, but merely alters the peak shape, whereas in the case of 1b N-protonation leads to a 40 nm spectral shift (Table 1). Table 1. Optical properties of chromophores 2a,b in various solvents and a comparison with the related proteins. 2a

2b

Solvent

λabs (nm)

λem (nm)

QY (%)

Water

368

430

λabs (nm)

λem (nm)

QY (%)

~1-10a

476

520

59

a

482

516

66

EtOH

365

426

~5-20

CH3CN

360

420

55

461

509

68

EtOAc

362

421

63

468

504

64

Dioxane

365

422

78

472

502

65

Water

1b14

1a

pH=7

350

-b

-b

408

-b

-b

pH=2.5

352

-b

-b

446

-b

-b

Sirius2b

a

424

The QY of 2b did not depend on the solvent, while 2a showed a dramatic decrease of emission intensity in water and EtOH (Table 1). In these solvents the QY as well as spectral shape were dependent on the concentration of 2a, and therefore the formation of non-fluorescent associates can be assumed. These properties preclude the use of compound 2a as a covalently binding fluorescent label, but enable its use as an environment-sensitive dye, for instance, in cellular membrane labeling. No perceptible changes were detected in the spectra of aqueous solutions of 2 (in contrast with 1) upon acidification, as coordinate bond formation prevents protonation of the nitrogen atom. Even for chromophore 2b, comprising of an indole moiety which is potentially capable of protonation, the spectral data remained unaltered under acidic conditions. This phenomenon could be attributed to a noticeable decrease in the indole pKa value due to conjugation. However, under basic conditions chromophores 2 displayed more complex behavior (Fig. 2).

Figure 2. Absorbance spectra of aqueous solutions of 2a (right) and 2b (left) obtained at varying pH values.

With increasingly high pH two independent processes were observed in the case of compound 2b. The first process was reversible and characterized by the moderate hypsochromic shift of the absorption and fluorescent emission maxima (both 15 nm), a pKa value of 9.8-9.9 and was not accompanied by the loss of fluorescence. It can be assumed that in this case a deprotonation of the NH-group takes place, which was consistent with that previously observed for tryptophan-containing proteins.15 Further basification above pH 12 resulted in the virtually permanent loss of fluorescence and a blue shift of absorbance maxima (300 nm and 430 nm for 2a and 2b respectively) for both chromophores 2. This process can be explained by addition of the hydroxide ion to the imidazolone ring, which was previously observed in photoswitchable GFP-like proteins16 (Scheme 2).

CFP

Water 355

Both compounds 2a and 2b were highly soluble in a wide range of organic solvents and, contrary to the data previously presented,10 showed limited wavelength maxima diversity in the examined solvents (Table 1, ESI). The greatest bathochromic shifts were observed for polar and protic solvents concurring with our previous results.10

24

434

The QY depends on concentration (see discussion).

474

40с

Scheme 2. Possible mechanism of compound 2b hydrolysis.

3 All the pH-dependent processes observed for compounds 2 lie outside the physiological range and do not affect their possible use as fluorescent dyes in live cells.

Conclusion Thus, we have presented the synthesis of two novel conformationally locked chromophore analogs related to CFP and Sirius protein. The compounds have high QYs and are not pH-sensitive in the physiological range. These dyes can therefore be considered as good candidates for a wide spectrum of fluorescent labeling applications. In contrast with the data previously obtained8 for GFP chromophore and its borylated analog the QYs of the related proteins were lower than those of the conformationally locked analogs 2. This fact showed the potential of research on novel proteins with similar chromophores.

Acknowledgments This work was funded by RFBR, according to the research project No. 16-33-60116 mol_а_dk. This research was carried out using equipment provided by the IBCH core facility (CKP IBCH). References and notes 1. Chudakov, D. M.; Matz M. V.; Lukyanov S.; Lukyanov K. A. Physiol Rev. 2010, 90, 1103-1163. 2. (a) Heim R.; Prasher D. C.; Tsien R. Y. Proc. Natl. Acad. Sci. USA 1994, 91,12501-12504; (b) Tomosugi1 W.; Matsuda T.; Tani1 T.; Nemoto T,; KoteraI.; Saito K.; Horikawa K.; Nagai1 T. Nature Methods 2009, 6, 351353; (c) Shaner N. C.; Campbell R. E.; Steinbach P. A.; Giepmans B. N.; Palmer A. E.; Tsien R. Y. Nat. Biotechnol. 2004, 22, 1567-1572. 3. (a) Yampolsky I. V.; Balashova T. A.; Lukyanov K. A. Biochemistry 2009, 48, 8077-8082; (b) Yampolsky I. V.; Remington S. J.; Martynov V. I.; Potapov V. K.; Lukyanov S. А.; Lukyanov K. A. Biochemistry 2005, 44, 5788-5793; (c) Ivashkin P. E.; Yampolsky I. V.; Lukyanov K. A. Russ. J. Bioorg. Chem. 2009, 35, 652-669. 4. (a) Paige J. S.; Ngyuen-Duc T.; Song W.; Jaffrey S. R. Science 2012, 335, 1194; (b) Song W.; Strack R. L.; Jaffrey S. R. Nat. Methods 2013, 10, 873875; (c) Strack R. L.; Song W.; Jaffrey S. R. Nat. Protoc. 2014, 9, 146-155; (d) Song W.; Strack R. L.; Svensen N.; Jaffrey S. R. J. Am. Chem. Soc. 2014. 136, 1198-1201. 5. Deng H.; Zhu Q.; Wang D.; Tu C.; Zhu B.; Zhu X. Polym. Chem. 2012, 3, 1975-1977. 6. (a) Huang G.; Zhang G.; Wu Y.; Liao Q.; Fu H.; Zhang D. Asian J. Org. Chem. 2012, 1, 352-358; (b) Fery-Forgues S.; Veesler S.; Fellows W. B.; Tolbert L. M.; Solntsev K. M. Langmuir 2013, 29, 14718-14727. 7. Baranov M. S.; Lukyanov K. A.; Yampolsky I. V. Russ. J. Bioorg. Chem. 2013, 3, 223-244. 8. Baranov M. S.; Lukyanov K. A.; Borissova A. O.; Shamir J.; Kosenkov D.; Slipchenko L. V.; Tolbert L. M.; Yampolsky I. V.; Solntsev K. M. J. Am. Chem. Soc. 2012, 134, 6025-6032. 9. Baleeva N. S.; Myannik K. A.; Yampolsky I. V.; Baranov M. S. Eur. J. Org. Chem. 2015, 26, 5716-5721. 10. Baranov M. S.; Solntsev K. M.; Baleeva N. S.; Mishin A. S.; Lukyanov К. A.; Yampolsky I. V. Chem. Eur. J. 2014, 20, 13234-13241. 11. Frizler M.; Yampolsky I. V.; Baranov M. S.; Stirnberga M.; Gütschow M. Org. Biomol. Chem. 2013, 11, 5913-5921. 12. Wu L.; Burgess K. J. Am. Chem. Soc. 2008, 130, 4089-4096. 13. Ishida N.; Moriya T.; Goya T.; Murakami M. J. Org. Chem. 2010, 75, 8709-8712. 14. Reaction of imidazolones 1 with boron tribromide: Imidazolone 1 (6 mmol) was dissolved in dry dichloroethane (200 mL), molecular sieves 4Å (20 g) and 3Å (20 g) were added, followed by a solution of boron tribromide in CH2Cl2 (1M, 24 mL, 24 mmol), and the reaction mixture stirred for 10 days under an inert atmosphere at room temperature. The mixture was filtered and the molecular sieves washed with EtOH (2 × 50 mL) and CHCl3 (2 × 100 mL). The solution was mixed with HF (20%, 20 mL) and stirred for 30 min. The mixture was dissolved in EtOAc (200 mL), washed with K2CO3 (5%, 2 × 100 mL), water (2 × 100 mL) and brine (2 × 100 mL) and dried over Na 2SO4. The solvent was evaporated and the product purified by column

chromatography. (Z)-4-(2-(Difluoroboryl)benzylidene)-1,2-dimethyl-1Himidazol-5(4H)-one (2a) Yellow solid (0.29 g, 20%); m.p. ~250 °C with 1 decomposition; H NMR (DMSO, 700 MHz) δ 2.76 (s, 3H), 3.24 (s, 3H), 7.36 (t, 1H, J = 7.4 Hz), 7.45 (t, 1H, J = 7.2 Hz), 7.59 (d, 1H, J = 7.3 Hz), 7.61 (d, 1H, J = 7.6 Hz), 7.65 (s, 1H). 13C NMR (DMSO, 176 MHz) δ 13.1, 26.6, 126.9, 127.8, 128.2, 131.2, 131.3, 131.4, 132.9, 163.1, 167.4. HRMS (m/z) calcd. C12H11BFN2O for [M-F]+ 229.0954, found 229.0942. (Z)-4-((2(Difluoroboryl)-1H-indol-3-yl)methylene)-1,2-dimethyl-1H-imidazol-5(4H)one (2b) Red solid (0.26 g, 15%); m.p. ~275 °C with decomposition; 1 H NMR (DMSO, 700 MHz) δ 2.73 (s, 3H), 3.24 (s, 3H), 7.15 (t, 1H, J = 7.0 Hz), 7.18 (t, 1H, J = 7.0 Hz), 7.41 (d, 1H, J = 7.8 Hz), 7.92 (d, 1H, J = 7.6 Hz,), 8.09 (s, 1H), 12.29 (s, 1H). 13C NMR (DMSO, 176 MHz) δ 12.8, 26.4, 112.4, 115.2, 118.8, 120.2, 121.4, 122.9, 124.9, 125.8, 139.2, 161.4, 162.2. HRMS (m/z) calcd. C14H12BFN3O for [M-F]+ 268.1063, found 268.1032. 15. Sarkisyan K. S.; Yampolsky I. V.; Solntsev K. M.; Lukyanov S. A.; Lukyanov K. A.; Mishin A. S. Scientific Reports 2012, 2, 608. 16. Brakemann T.; Stiel A. C.; Weber G.; Andresen M.; Testa I.; Grotjohann T.; Leutenegger M.; Plessmann U.; Urlaub H.; Eggeling C.; Wahl M. C.; Hell S. W.; Jakobs S. Nature Biotechnol. 2011, 29, 942-947.

4

Tetrahedron

Two “locked” analogs of the chromophores of fluorescent proteins were synthesised. More than a hundredfold increase of fluorescence intensity was shown. The dyes demonstrate pH-independence in physiological pH range.