Synthesis of energy transfer cassettes via click and Suzuki–Miyaura cross coupling reactions

Abstract

Novel cassettes capable of energy transfer involving simple synthetic methods viz., copper catalyzed azide–alkyne cycloaddition (click reaction) at the C-8 position and palladium catalyzed Suzuki–Miyaura cross coupling at the C-6 position have been represented. The resulting imidazo[1,2-a]pyrazine-triazole bridged coumarin cassettes are capable of energy transfer from a donor core to an acceptor moiety.

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Energy transfer (ET) cassettes involve the absorption of a photon of light by one of the chromophores, resulting in energy transfer. The donor molecule returned to the ground state along with raising the acceptor to the excited state.¹ The energy transfer process can take place in two different ways; (i) through bond energy transfer (TBET),² and (ii) through space energy transfer (fluorescence resonance energy transfer, FRET).³ Energy transfer through both pathways involved the use of a donor and an acceptor linked through a conjugated spacer in the case of TBET and a non-conjugated spacer in the case of FRET. The energy transfer through TBET does not require the criterion of spectral overlap between the absorption spectrum of the acceptor and the emission spectrum of the donor. On the other hand, the FRET phenomenon requires the criterion of spectral overlap between the absorption spectrum of the acceptor and the emission spectrum of the donor. It can also afford simultaneous recording ratio signals of two emission intensities at different wavelengths, which could provide built-in correction for the environmental effects and supply a facile method for visualizing complex biological processes at the molecular level, such as nucleic acid regulation, DNA detection, protein labelling, and other biomarkers. Consequently, studying the construction of energy donor–acceptor architectures, or “energy transfer cassettes” is very interesting and significant to many chemists, biologists, environmentalists, and medical scientists.

The major challenge currently encountered for efficient energy transfer cassettes either through space or through bonds is their syntheses, which are not straightforward^4,5 and which require harsh conditions and tedious purification to isolate the product with low to moderate yields.⁶

Click reactions⁷ and Suzuki–Miyaura cross coupling⁸ have been proven to be powerful tools to construct heterocyclic molecular frameworks. Herein, we have reported the first sequential functionalization at the C-8 and C-6 positions of imidazo[1,2-a]pyrazine by implementing “click” and “Suzuki–Miyaura cross coupling” reactions, respectively. These have been leading to energy transfer cassettes along with the determination of photophysical characterization of the synthesized compounds by depicting the quantum yields. We have introduced various electron donating substituents viz., methyl, ethyl, hydroxy, and methoxy as well as electron withdrawing substituents viz., formyl, and acetyl to the donor part of the cassette, thereby determining the overall quantum yield and energy transfer efficiency (ETE). The effect of the spacer length between the donor and the acceptor has also been studied to predict the energy transfer efficiency. To the best of our knowledge, this is the first report for the coupling of imidazo[1,2-a]pyrazine based donors with coumarin acceptors in a way that facilitate energy transfer through space.

To synthesize these energy transfer cassettes, our study was initiated with the easily available starting material 2-bromoethylamine hydrobromide 1 which was refluxed with sodium azide in water for 12 h followed by stirring with sodium hydroxide at room temperature to give 2-azidoethanamine 2.⁹ The reported tedious work up has been avoided in this case. The azide has limited thermal stability but it could be stored in ether or hexane for a longer period of time at a low temperature in a pure state. To obtain the first precursor, we have treated 6,8-dibromoimidazo[1,2-a]pyrazine 3 (obtained from bromination of 2-aminopyrazine followed by cyclization with 50% aqueous chloroacetaldehyde)¹⁰ with 2-azidoethanamine 2 in the presence of diisopropylethylamine (DIPEA) in acetonitrile at the reflux temperature for 24 h to obtain N-(2-azidoethyl)-6-bromoimidazo[1,2-a]pyrazin-8-amine 4 in 72% yield (Scheme 1).

Scheme 1 Synthesis of N-(2-azidoethyl)-6-bromoimidazo[1,2-a]pyrazin-8-amine (4).

The second precursor 4-methyl-7-(prop-2-ynyloxy)-2H-chromen-2-one 6 was obtained in 81% yield via the reaction of 7-hydroxy-4-methyl-2H-chromen-2-one 5 with 2 equivalents of a 80% solution of propargyl bromide in the presence of potassium carbonate and acetone at room temperature for 12 h (Scheme 2).

Scheme 2 Synthesis of 4-methyl-7-(prop-2-ynyloxy)-2H-chromen-2-one (6).

With both precursors in hand, the copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction of imidazo[1,2-a]pyrazine 4 and coumarin 6 was performed. To explore the feasibility of the [2 + 3] cycloaddition of the azide–alkyne reaction, various catalysts and solvents were screened (Table S1, ESI†). Among the various catalysts (CuSO₄·5H₂O, CuBr, CuI and CuCl₂), CuSO₄·5H₂O showed the highest efficiency in the presence of sodium ascorbate at room temperature (Table S1,† entry 8). Several solvents were also tested; the ethanol and water mixture was proven to be the most effective solvent (Table S1,† entries 4, 8, 12). Increasing the temperature of the reaction did not lead to any further improvement in the yield (Table S1,† entries 13, 14). A slight decrease in the yield of the desired product was observed upon increasing the loading of CuSO₄ from 5 to 15 mol% (Table S1,† entries 15, 16).

With the optimized reaction conditions, compound 4 was treated with 4-methyl-7-(prop-2-ynyloxy)-2H-chromen-2-one 6 in the presence of 5 mol% CuSO₄·5H₂O and 10 mol% sodium ascorbate in ethanol : water (8 : 2) at room temperature for 2 h to give 1,4-disubstituted triazole 7 in 91% yield. However, when the reaction was typically carried out with heating without using any catalyst, mixtures of 1,4-(7) and 1,5-(8) disubstituted triazole derivatives in a ratio of 2 : 1 were obtained (Scheme 3). It has been observed that thermal [2 + 3] dipolar cycloaddition of alkyne to azide is not a regiospecific reaction.¹¹

Scheme 3 Thermal [2 + 3] cycloaddition reaction of 4 and 6.

Strong Nuclear Overhauser Effect (NOE) was observed between the H-5′ triazole proton and the protons of the substituted alkyl groups present at the N-1′ and C-4′ positions in compound 7, suggesting that the triazole proton and the alkyl groups are in close proximity, and confirmed the formation of the 1,4-disubstituted triazole. The NOE spectrum of 1,5-disubstituted triazole 8 showed no correlation between the singlet of the triazole at the C-4′ position and the triplet of the substituted alkyl group present at the N-1′ position. The expected NOE correlation of the triazole protons at the C-4′ and C-5′ alkyl groups was observed (Scheme 3).

1,4-Disubstituted triazole 7 was further used for the synthesis of a library of compounds with Suzuki–Miyaura cross coupling reactions at the C-6 position of imidazo[1,2-a]pyrazine. To identify the optimal reaction conditions, various reported Pd-catalysts, along with different combinations of bases and organic solvents were examined in the reaction of imidazo[1,2-a]pyrazine 7 with thiophen-2-yl boronic acid. A previous study¹² showed that the base was a key reaction parameter, so, different bases (Cs₂CO₃, K₂CO₃, and DIPEA) and solvents (CH₃CN : H₂O, dioxane : H₂O and DME : H₂O) were reviewed first. Both affected the product yields as K₂CO₃ and dioxane : water gave the best results. With the identification of the base and solvent, several Pd catalysts were screened (Table S2†). Using [Pd(PPh₃)₄] as the catalyst, 9 was obtained in 73% yield (Table S2,† entry 19), while [Pd(PPh₃)₂Cl₂], [Pd(dba)₃] and [PdCl₂(dppf)]·DCM had lower yields of the desired products (Table S2,† entries 17, 18, 20). The best yield was obtained when the model reaction was catalyzed with 5 mol% [Pd(PPh₃)₄] and using 1.0 equivalent of thiophen-2-yl boronic acid, and 1.0 equivalent of K₂CO₃ in dioxane : water (9 : 1) at the reflux temperature for 6–8 h in a sealed tube (Table S2,† entry 19).

With these optimized reaction conditions, the scope of the reaction was further examined with various aryl boronic acids (Table 1). The Suzuki reaction with imidazo[1,2-a]pyrazine 7 proceeded well to give the desired products irrespective of the substitution on the phenyl ring. It has been observed that the five member rings were well compatible with the optimized conditions, affording the corresponding products in moderate yields (9–11, 58–75% yields). Both electron withdrawing substituents viz., fluoro (13), chloro (14), bromo (15), trifluoromethyl (16) and acetyl (24) as well as electron donating substituents such as methyl (17), ethyl (18) and methoxy (21 and 22) were also compatible with the protocol, providing the arylated imidazo[1,2-a]pyrazine in moderate to good yields. The change in directing groups from ortho to para or vice versa did not effect the reactivity. However, with the 2-hydroxyphenyl substrate, the reactivity decreased significantly, and the arylated products 20 was obtained in only 53% yield (Table 1, entry 12), perhaps because of the combination of both steric hindrance and strong electron deficiency.

Table 1 Suzuki–Miyaura cross coupling reaction of ethyl triazole bridged imidazo[1,2-a]pyrazine–coumarin conjugates

Entry	Time (h)	ArB(OH)2	Product	Yield (%)
1	8			9 (62)
2	8			10 (75)
3	8			11 (58)
4	6			12 (71)
5	7			13 (80)
6	7			14 (82)
7	7			15 (64)
8	6			16 (71)
9	6			17 (65)
10	6			18 (66)
11	8			19 (74)
12	8			20 (53)
13	7			21 (90)
14	6			22 (91)
15	6			23 (92)
16	8			24 (90)

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After screening the 2-azidoethanamine linker, another alkyl chain, 3-azidopropanamine, was investigated. 3-Bromopropylamine hydrobromide 25 was first reacted with sodium azide to give 26 followed by substitution at the C-8 position of 6,8-dibromoimidazo[1,2-a]pyrazine 3 to give 27 in 65% yield. Compound 27 was then coupled with coumarin 6 via a copper-catalyzed azide–alkyne cycloaddition reaction to give 28 in 82% yield (Scheme 4).

Scheme 4 Synthesis of propyl triazole bridged imidazo[1,2]-pyrazine–coumarin conjugate (28).

Compound 28 was further coupled with various aryl boronic acids via Suzuki–Miyaura cross coupling reactions using 5 mol% [Pd(PPh₃)₄], K₂CO₃ and dioxane : water at reflux temperature for 6–8 h to obtain compounds 29–38 in 56–90% yields (Table 2).

Table 2 Suzuki–Miyaura coupling reactions of propyl triazole bridged imidazo[1,2-a]pyrazine–coumarin conjugates

Entry	Time (h)	ArB(OH)2	Product	Yield (%)
1	8			29 (72)
2	8			30 (70)
3	8			31 (76)
4	6			32 (70)
5	7			33 (80)
6	7			34 (81)
7	7			35 (56)
8	6			36 (76)
9	6			37 (68)
10	6			38 (90)

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Various spectroscopic measurements (Fig. S63–S64†) were recorded for the molecules to test the cassettes, based on the quantification of their efficiencies (Tables 3 and 4). Molar absorptivities (€) and quantum yields (Φ_f) for all 28 compounds were determined in acetonitrile. All of the derivatives have similar absorption maxima (between 312 to 319 nm). However, compounds 23, 24 and 38 containing electron withdrawing groups such as formyl and acetyl attached to the phenyl ring exhibited two absorption peaks at 275 nm and 317 nm. A similar emission maximum was observed for all derivatives (between 375 to 390 nm). However, emission maxima to longer wavelengths (red shift) i.e. from 415 nm to 500 nm were observed for compounds 15, 19, 23, 24 and 38. Compounds 17, 18, 20, 21 and 22 containing the electron donating groups have lower quantum yields (Φ_f = 0.016 to 0.36) in comparison to compounds 23 and 24 (Φ_f = 0.40 to 0.48) with electron withdrawing groups 4-formyl and 4-acetyl attached to the phenyl ring at the C-6 position of imidazo[1,2-a]pyrazine. A similar pattern of quantum yield was observed in the case of the propyl chain in which the electron withdrawing group in 38 showed the highest quantum yield (Φ_f = 0.47). Overall, it was inferred that compounds with electron donating groups showed lower quantum yields in comparison to compounds with electron withdrawing groups. An increase in the linker length also resulted in similar or lower quantum yields. Energy transfer efficiencies (ETE) based on the fluorescence quantum yields have also been determined. The efficiency of the energy transfer (η_ETE) was evaluated using the equation¹³ η_ETE = 1 − Φ_{f(donor in dyad)}/Φ_{f(free donor)}. Here, Φ_{f(donor in dyad)} is the fluorescence quantum yield of the donor part in the dyad and Φ_{f(free donor)} is the fluorescence quantum yield of the donor when not connected to the acceptor.

Table 3 Photophysical characterization of the absorption and fluorescence properties of compounds 8–24 in acetonitrile^a

Compd	λ^absmax (nm)	λ^emimax (nm)	€	Φf	ETE (%)
a N.D: not determined.
7	315	380	39 600	0.12	73.9
9	315	375	49 700	0.22	52.1
10	316	375	44 600	0.24	47.8
11	316	377	25 350	0.20	56.5
12	315	377	22 400	0.15	67.3
13	312	378	19 850	0.18	60.8
14	315	385	39 050	0.30	34.7
15	317	415	49 250	0.34	17.3
16	316	385	33 400	0.32	15.2
17	314	378	26 950	0.15	67.3
18	315	378	49 800	0.13	71.7
19	312	415	26 150	0.36	N.D
20	319	378	28 000	0.016	96.5
21	314	375	20 350	0.20	56.5
22	316	375	30 900	0.16	65.2
23	275, 317	495	31 400	0.48	N.D
24	275, 317	475	40 800	0.40	N.D

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Table 4 Photophysical characterization of the absorption and fluorescence properties of compounds 28–38 in acetonitrile^a

Compd	λ^absmax (nm)	λ^emimax (nm)	€	Φf	ETE (%)
a N.D: not determined.
28	315	380	26 250	0.11	76.0
29	316	376	29 450	0.11	76.0
30	314	375	27 900	0.13	71.7
31	315	380	28 050	0.14	69.5
32	317	380	29 700	0.27	41.3
33	316	390	30 850	0.35	15.2
34	315	378	31 300	0.13	71.7
35	312	377	27 400	0.15	67.3
36	315	375	27 550	0.16	65.2
37	317	380	33 250	0.17	0.5
38	275, 317	500	31 400	0.47	N.D

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Compounds 7 and 28 with a bromo group at the C-6 position of imidazo[1,2-a]pyrazine (at the donor part) acted as a good energy transfer cassette with ETE values of 73.9% and 76%, respectively. The introduction of an electron donating groups such as methyl (17 and 34) and ethyl (18 and 35) also showed comparable ETE values (67.3% and 71.7%), while substitution with halogen to a phenyl ring on the donor part of the cassettes (13, 14, 15, 16, 31, 32 and 33) displayed low ETE values (15.2–69.5%). The presence of electron donating groups viz., methoxy in compounds 21, 22 and 36, showed 56.5%, 65.2% and 65.2% ETE, respectively. But the presence of 2-hydroxyphenyl (20) on the donor moiety showed significant energy transfer efficiency (96.5%). The presence of a naphthyl group (19) and the electron withdrawing 4-formyl (23 and 38) and 4-acetyl (24) groups on the aromatic ring did not show any energy transfer. Thus, compound 20 showed the maximum energy transfer efficiency of 96.5% which indicated that this compound behave as an energy transfer cassette to transfer energy from the imidazo[1,2-a]pyrazine donor to the coumarin acceptor. Thus, the emission spectra of imidazo[1,2-a]pyrazine (donor) and the absorption spectra of coumarin (acceptor) showed significant spectral overlap for energy transfer (Fig. 1).

Fig. 1 Spectral overlap of the emission spectrum of imidazo[1,2-a]pyrazine (donor) and the absorption spectrum of coumarin (acceptor).

The energy transfer cassettes of imidazo[1,2-a]pyrazine–coumarin conjugates were also compared with different known molecular hybrids. It has been observed that compound 20 (ETE = 96.5%) showed better or comparable results with fluorescein–coumarin cassettes for the detection of phosphodiesterase I activity (ETE = 94%),¹⁴ fluorescein–coumarin cassettes for ATP complexes (ETE = 76% and 83%),¹⁵ Bodipy with phenanthroline and terpyridine cassettes for light harvesting system (ETE = 82%),¹⁶ and Bodipy–cyanine based cassettes (ETE = 43–90%).¹⁷

Here, we have reported the first examples of a new structural strategy to improve the ETE efficiency in molecular hybrids involving imidazo[1,2-a]pyrazine as a key linking the acting chromophore. This design consists of imidazo[1,2-a]pyrazine–coumarin conjugates with a variable spacer linker (ethyl and propyl chain) via a click reaction at the C-8 position and a Suzuki–Miyaura cross coupling reaction at the C-6 position of imidazo[1,2-a]pyrazine. The goal of this simple design was to keep the conformational motion of the involved donor–acceptor chromophores restricted and tightly fixed to ensure efficient ETE via FRET. Another important goal was the straight-forward synthetic access to these conjugate cassettes from imidazo[1,2-a]pyrazine and acceptor-based coumarins, which ensures an excellent potential for developing future smarter hybrid cassettes for valuable fluorescence applications (bioimaging, chemosensing, optoelectronics etc.). Thus, further experimental and computational studies are now in progress, directed to determine the key structural factors ruling the energy transfer efficiency in these hybrid cassettes. We are convinced that we herein communicated a new structural design to boost ETE, which has great potential for inspiring the future development of molecular cassettes on the basis of molecular hybridization.

Acknowledgements

KP thanks DST, New Delhi (EMR/2014/000669) and UGC, New Delhi (41-322/2012, SR) for providing funds. RG is indebted to CSIR for SRF (09/677(0020)/2013.EMR-I). SAI labs, Thapar University is also acknowledged for recording NMR spectra.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra07861c

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