Elsevier

Tetrahedron

Volume 67, Issue 6, 11 February 2011, Pages 1150-1157
Tetrahedron

Suzuki–Miyaura coupling for general synthesis of dehydrocoelenterazine applicable for 6-position analogs directing toward bioluminescence studies

https://doi.org/10.1016/j.tet.2010.12.001Get rights and content

Abstract

Synthesis of coelenterazine analogs is in recent demand to supply more luminescent compounds with reasonable stability as substrate for the photoprotein manipulated in a living cells or particular organelle. There are limited methods for the synthesis of 6-substituted coelenterazine due to the route and instability of the compounds under the existing conditions. This paper describes six examples including Suzuki–Miyaura cross coupling reaction with reactive triflate (unstable) and stable tosylate intermediates of the imidazo[1,2-a]pyrazin-3-one. Five examples of 2-amino-3-benzyl-5-O-Tf-pyrazine are also discussed. The product coelenterazine analogs are obtained in the form of dehydrocoelenterazine, which is the substrate of a squid photoprotein, symplectin.

Introduction

Bioluminescence of marine organisms has widely been recognized among those people, who visit the seashore to observe strong emission of blue or green light. Coelenterazine 1 has been one of the most popular substrates for the marine bioluminescent systems,1 which was found in organisms such as jellyfish Aequoria victoria,2 sea cactus Cavernularia obesa,3 sea pansy Renilla reniformis4, 5, 6, deep sea shrimp Oplophorus gracilirostris,7, 8, 9 obelin, Obelia longissima,10 and oceanic squids Symplectoteuthis oualaniensis.11, 12 Some examples as C. obesa or the tiny squid Watasenia scintillans uses the corresponding sulfate of 1.3, 11, 12, 13, 14, 15 Dehydrocoelenterazine (DCL) 2, however, is an oxidized chromophore of coelenterazine 1. But 2 is not luminescent by itself under chemiluminescence condition (strong alkaline in dipolar aprotic solvent) due to the oxidation stage. We have reported that DCL 2 shows strong bioluminescence as a quite unique chromophore in the photoprotein, symplectin of the squid, S. oualaniensis (Tobi-Ika, Japanese name meaning flying squid).16, 17, 18, 19 We have elucidated the chromophore structure as 2 but not 1 due to the fact that only 2 gives the bioluminescence with its apo-symplectin. The molecular mechanism of the bioluminescent system in symplectin has been solved in such a way that DCL or its 13C analog binds with an HS-residue of cysteine to form a conjugate adduct 3 as reported by Isobe’s group (Scheme 1).20, 21, 22 In 2008, the active center cysteine was determined to be 390-Cys in the 501 amino acids of symplectin.22 Recently, Kuse independently reported that a commercially available photoprotein pholasin, from Pholas dactylus, showed an increase in the bioluminescence by addition of DCL.23

Synthesis of coelenterazine or dehydrocoelenterazine has been well established since 1990 until recent 2009.18, 20, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 This is largely due to the fact that increase of sample-request has become much more in order to supply as the substrates for the photoproteins, which has been expressed on living cells or organelle through gene transcription for chemical biology studies.34 A more stable coelenterazine, a fluorinated-analog, was selected to exhibit stronger light amount at plant organelle, on which aequorin is often expressed to monitor increasing amount of Ca2+ ions in the living cells.35 Besides these applications, fluorinated-analog has been playing important roles to elucidate the molecular mechanism of the bioluminescence of symplectin photoprotein.22

Many important syntheses of coelenterazine 1 and its analogs have been reported by several research groups including ours. However, most of the synthetic routes are focused on either modifying the aryl group on the 2-position (R1) or introducing only limited diversified groups at the 6-position (R2) and the 8-position (R3) starting from 2-aminopyrazine analogs as shown in Scheme 2. This fact largely depends on the synthetic route, which allows finalizing the synthesis by condensing aminopyrazine (e.g., G) with ketoaldehyde equivalent as shown in Scheme 2. There still exists limitation for synthesizing diversified R2 analogs due to most of the routes, which starts from introduction of 4-methoxyphenyl group at the R2 from the beginning. Nakamura et al.36 in 2001 and Adamczyk et al.37, 38 in 2003 reported the synthesis of analogs of 1 from 3,5-dibromo-2-aminopyrazine on the basis of Suzuki–Miyaura and Negishi coupling. In 2009, Knochel reported an elegant synthesis of 1 on the basis of the Pd-mediated multiple cross coupling reactions starting from 2,5-dichloropyrazine in eight steps.32 These new synthetic routes donated advantages for synthesizing analogs of R2 and R3 groups, but we still have limitation for diversified synthesis. Herein we want to report a new route, which enables the easy synthesis of unstable coelenterazine analogs.

In 2004, we reported a new route, which may allow the diversified synthesis of R2 at the 6-position of coelenterazines A. This had been left unexplored. Instead of the fact that R2 had to be selectively introduced at the early stage of the synthesis, the attempted R2 introduction at a relatively later stage was achieved to provide two kinds of coelenterazine analogs.39 The coupling of 5-O-triflyl-3-benzyl-2-aminopyrazine E (X=OTf) from 4 as a precursor for cross coupling (G from E) at the corresponding position.39 In this paper, we report more examples of the cross coupling reaction with 2-amino-3-benzyl-5-O-triflate to form five aromatic compounds as summarized in Table 1.

For the purpose of the long time awaited synthesis route for A via the direct introduction of the R2 group into imidazopyrazinone 6 (X=OTf) at the last step of the synthesis for CL, amino-O-triflylpyrazine E was condensed with the ketoaldehyde to afford 6. The attempted R2 introduction was implemented with six kinds of aromatic system, and the results are summarized in Table 2. In addition, the product was not A, but B from C.

These successful couplings were eventually found to be very inconvenient for extremely low stability of 6, so that one have to finish the cross coupling reaction of the triflate 6 (X=O-Tf) within 24 h to have good yields even if 6 was stored in a freezer at −30 °C. To solve such a problem, we prepared the corresponding tosylate 6 (X=O-Ts) with better stability. So we went back to explore the coupling of 5-imino-O-tosy-3-benzyl-2-aminopyrazine E (X=O-Ts) for the Suzuki–Miyaura reaction for variety of the R2 with organoboron reagents. This iminotosylate showed good results having electron donating and electron withdrawing group attached to the aromatic ring of the boronates. In 2009, Makarasen and Isobe reported the palladium-mediated cross coupling reaction with 2-amino-3-benzyl-5-O-tosyl-pyrazine E (X=OTs, R3=Bn).31 This product aminopyrazine G has to be further derivatized via the acid-mediated condensation with aryl-α-ketoaldehyde in acetal form F to obtain the DCL analogs. We now envision that the Suzuki–Miyaura reaction could be achieved even in much later stage; thus, Suzuki–Miyaura coupling (S–M-coupling) of imidazopyrazinone heterocyclic systems (C or D) at the last step of the synthesis to A or B. We describe the details of the results as follows first with 6 (X=O-triflyl) and then with 6 (X=O-tosyl).

Section snippets

Preparation of imidazopyrazinone 6-O-triflate 11

As summarized in the retrosynthesis routes in Scheme 2, the challenging issue is the synthesis of A from C for providing diversified analog syntheses. The necessary triflate 9 was prepared in accordance to our previous paper; thus, 5-pyrazinone 7 was converted to 8 and N-tosyl group was hydrolyzed to amino-benzylpyrazin-O-triflate 9.39 To this aminopyrazine, a ketoaldehyde equivalent 10 was subjected for condensation to obtain imidazopyrazinone 6-O-triflate 11 (Scheme 3).

Pd-mediated cross coupling reactions with benzyl-aminopyrazine 5-O-triflate 9

The 2-amino-3-benzyl-5-O

Conclusion

We have established a successful route to synthesize dehydrocoelenterazine analogs at the 6-position at the last step of the synthesis through the Suzuki–Miyaura reaction with the 6-O-triflate and 6-O-tosylate with the arylborone compounds. Due to the unstable nature of the final coelenterazine-equivalent compounds, the cross coupling with the tosylate was better implemented with the oxidized heteroaromatic system through the most stable synthetic intermediates. Further synthetic studies are in

General procedures

UV–vis spectra were obtained on a JASCO V-570 spectrometer. Fluorescence spectra and chemiluminescence spectra were measured with a JASCO FP-777 spectrometer. IR spectra were recorded on a JASCO FT/IR 6100 spectrometer. Proton NMR spectra were recorded on a JEOL GSX 270 for 270 MHz and a JEOL A 400 for 400 MHz. Chemical shift (δ) are given in parts per million relative to DMSO­d6 (δ 2.49) or CD3OD (δ 3.30) as internal standard and coupling constants (J) in hertz. Carbon NMR spectra were recorded

Acknowledgements

Authors are indebted to National Science Foundation and National Tsing Hua University for the generous financial supports to the current studies. Part of the works was contributed for the earlier stage by the ex-Isobe’s Laboratory in Nagoya University, to whom thanks are due.

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