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Fluorescent unnatural amino acids (Uaas), when genetically incorporated into proteins, can provide unique advantages for imaging biological processes in vivo. Synthesis of optically pure L-enantiomer of fluorescent Uaas is crucial for their effective application in live cells. An efficient six-step synthesis of L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (L-Anap), a genetically encodable and polarity-sensitive fluorescent Uaa, has been developed. The synthesis takes advantage of a high-yield and enantiospecific Fukuyama-Mitsunobu reaction as the key transformation.
The past decade has witnessed the dramatic progress on site-specific incorporation of unnatural amino acids (Uaas) into proteins in live cells through the expansion of the genetic code.1–4 Using engineered tRNA-synthetase pairs orthogonal to endogenous counterparts in the host cell, more than 70 Uaas have been incorporated into proteins in Escherichia coli, yeast, or mammalian cells, which enable new opportunities to study biological problems with expanding non-proteinogenic chemistries. Fluorescent Uaas (Figure 1), including L-dansylalanine (1),5–7 L-(7-hydroxycoumrin-4-yl)ethylglycine (2)8 and L-Anap (3),9 are of particular interest for molecular and cellular biology, which may complement and enchance the widely used fluorescent proteins (FPs).10 These fluorescent Uaas can be incorporated at virtually any site of a protein, whereas the fusion of FPs is often limited to the N- or C-terminus or certain loop regions. In addition, fluorescent Uaas are much smaller than FPs, which helps to mitigate undesired perturbations to the structure and function of the target protein. Moreover, when developing fluorescent reporters, sensitivity to different environment cues such as pH and polarity can be chemically designed and synthesized11 into the fluorescent Uaas, a flexibility not readily available for FPs.
Despite the potential advantages, effective application of fluorescent Uaas in live cells faces multiple challenges. Efficient synthesis of optically pure Uaas is a foremost demand. When racemic Uaas are fed to cells, the D-enantiomer competes for cellular uptake yet cannot be incorporated into proteins by the ribosome.2 This reduces the net intracellular concentration of the bioactive L-enantiomer, leading to lower incorporation efficiency. For fluorescent Uaas in particular, the intracellular D-enantiomer further increases background fluorescence, which is a critical issue for cell imaging with Uaas. Synthetic routes to enantiomerically pure 1 and 2 have been developed,5,12 but only a racemic synthesis is available for Uaa 3.9 Anap is sensitive to polarity with changes in intensity and emission wavelength.9 Uaa 3, when selectively incorporated into proteins, has the potential to fluorescently report protein conformational changes, interactions, modifications, and activities in live cells. Herein, we report a concise and efficient synthetic approach of L-Anap, which offers significant improvements over the current synthesis.
L-Anap has been incorporated into proteins in Saccharomyces cerevisiae by Schultz and co-workers.9 They developed a synthetic approach of racemic anap, which gives 8.1% of total yield over 8 steps (Scheme 1A). The synthesis was based on the nucleophilic substitution of ethyl bromopyruvate oxime (6) developed by Gilchrist and co-workers.13,14 Presumably due to the low nucleophilicity of the Burcherer reaction product of 4 and 5, compound 7 was synthesized in only 26% yield over two steps from compound 4.15 Another drawback of this synthesis resides in the nonstereoselective reduction of 7, which leads to the final racemic product.
To synthesize sufficient amounts of L-anap, we decided to develop an efficient and enantiospecific synthetic pathway toward L-anap (Scheme 1B). Our synthetic strategy involves Fukuyama-Mitsunobu reaction16–18 as the key step, which couples compound 10 with N-trityl-L-serine methyl ester 1119 to give intermediate 12. This approach has two features: First, both the nucleophile and the electrophile are channeled to facilitate the intermolecular coupling by tuning the electronic effect of the protecting groups. o-Nitrobenzenesulfonyl group makes 10 a good nucleophile under Mitsunobu condition and can be removed easily under mild conditions. Trityl group of compound 11 can conduct the reactive intermediate through the intermolecular coupling and circumvent β-elimination as the side reaction. Second, the Mitsunobu reactions of 11 with different nucleophiles have been proved to be free of racemization,19 therefore intermediate 12 are expected to be synthesized enantiospecifically.
We commenced with the synthesis of 6-acyl-2-naphthylamine (13) via two pathways (Scheme 2). In approach A, the selective Heck reaction20–22 and Cu(I)-catalyzed C-N23,24 bond formation were employed to introduce the acetyl and amino groups. 6- Bromo-2-naphthol (14) was transformed to triflate 15, which was coupled with butyl vinyl ether through Cabri’s procedure,20–22 followed by treatment with acid, to give intermediate 16. Under Buchwald’s condition,25 compound 16 underwent the C-N bond formation to give intermediate 17 in 75% yield. After deprotection, compound 13 was obtained in 54% yield over 4 steps. Although this approach provided gram-scale synthesis of 13 with satisfactory yield, approach B, which took three steps less than A, was explored in parallel. Approach B features the direct conversion of hydroxyl group of compound 4 to amino group. Although this transformation via Burcherer reaction26 has been reported by Cho and his coworkers,27 the procedure involves stirring the reaction mixture at 140 °C for 96 hours in a steel-bomb reactor, which is not viable in many biology laboratories. Inspired by recent advances in one-pot alkylation-Smiles rearrangment-hydrolysis28 sequence, we applied Mizuno and Yamano’s procedure to the transformation of 4 to 13. Monitoring the reaction with TLC showed the complete conversion of the alkylation and Smiles rearrangement steps. However, the in situ hydrolysis of 19 under basic condition was incomplete and the final product 13 was difficult to separate from intermediate 19. Therefore, the crude product of 19 was separated and hydrolyzed under acidic condition, which provided compound 13 in 73% yield.
With compound 13 in hand, we set out to explore the Fukuyama-Mitsunobu reaction (Scheme 3). Compound 13 was treated with o-nitrobenzenesulfonyl chloride and pyridine to give compound 10. In the presence of diisopropyl azodicarboxylate and triphenylphosphine, compounds 10 and 11 were coupled to give intermediate 12 in 88% yield over two steps. Sequential deprotection of trityl group and o-nitrobenzensulfonyl group furnished L-anap methyl ester (20). Deprotection of 20 under acidic condition29,30 furnished L-anap as its hydrochloride salt. The enantiomeric purity of 3 was examined with the Mosher method,31 and the dr value was above 40:1, which indicates that less than 3% racemization had occurred during deprotection (see Supporting Information).
In conclusion, we developed an efficient synthetic route to enantiomerically pure L-anap, which gives 51% of total yield. Compared to the previous method, our synthetic approach not only afforded a bioactive L-enantiomer but also enabled a 12-fold increase in yield with fewer steps. All the reactions of our approach proceeded under mild conditions as well as in gram-scale. The general approach demonstrated here can also be applied to the synthesis of other 3-N-aryl-2,3-diaminoproprionic acids, which serve as important building blocks for medicinal small molecules.32–34 The application of L-anap to image protein modifications and activities in mammalian cells is undergoing and will be reported in due course.
To a solution of 6-bromo-2-naphthol 14 (2.231 g, 10 mmol) in CH2Cl2 (50 mL) was added DIPEA (1.92 mL, 11 mmol) and PhNTf2 (3.930 g, 11 mmol) at room temperature. The reaction mixture was stirred under nitrogen for 12 hours and concentrated under vacuum. The residue was filtered through a short silica gel column and eluted with EtOAc/hexanes (1/19). The solution was concentrated under vacuum to give the crude product of 15 as yellow oil. 15 was dissolved in DMF (20 mL). Et3N (2.79 mL, 20 mmol), butyl vinyl ether (6.47 mL, 50 mmol), dppp (113.4 mg, 0.275 mmol) and Pd(OAc)2 (56.1 mg, 0.25 mmol) were added to the solution sequentially. The mixture was stirred under nitrogen at 80 °C for 2 hours and then cooled to 0 °C. 2 M HCl solution (20 mL) was added to the reaction mixture slowly and the mixture was stirred for half an hour. Water (120 mL) and EtOAc (50 mL) were added to the mixture. The two phases were separated and the aqueous phase was washed twice with EtOAc (50 mL). The organic phases were combined and washed with brine (30 mL), dried over anhydrous Na2SO4, concentrated and purified with flash chromatography (EtOAc/hexanes = 1/7) to give compound 16 (1.798 g, 72%) as white solid. Rf = 0.23 (EtOAc/hexances = 1/6). 1H NMR (500 MHz, CDCl3): δ = 8.41 (s, 1 H), 8.05 (d, J = 1.5 Hz, 1H), 8.04 (s, 1H), 7.82 (d, J = 9.0 Hz, 1H), 7.79 (d, J = 8.5 Hz, 1H), 7.62 (dd, J = 9.0, 2.0 Hz, 1H), 2.71 (s, 3H); 13C NMR (125 MHz, CDCl3): δ = 197.8, 136.6, 134.9, 131.2, 131.1, 130.4, 130.1, 127.6, 125.2, 123.0, 26.8. HRMS (EI): calcd for C12H9BrO: 247.9831, found 247.9832.
To the mixture of compound 16 (1.993 g, 8 mmol), tert-butyl carbamate (1.125 g, 9.6 mmol), CuI (76.2 mg, 0.40 mmol) and K2CO3 (2.211 g, 16 mmol) was added N,N′-dimethylethylenediamine (86.1 μL, 0.80 mmol) and toluene (16 mL). The mixture was stirred under nitrogen at 110 °C for 24 hours. After cooling to room temperature, the reaction mixture was filtered and washed with EtOAc (20 mL). The filtrate was concentrated and purified by flash chromatography (EtOAc/hexanes = 1/3) to give compound 17 (1.719 g, 75%) as white solid. Rf = 0.29 (EtOAc/hexanes = 3/7). 1H NMR (500 MHz, CDCl3): δ = 8.37 (s, 1H), 8.07 (s, 1H), 7.99 (dd, J = 9.0, 1.5 Hz, 1H), 7.86 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 8.5 Hz, 1H), 7.42 (dd, J = 8.5, 2.0 Hz, 1H), 6.85 (s, 1H), 2.70 (s, 3H), 1.56 (s, 9H); 13C NMR (125 MHz, CDCl3): δ = 198.1, 152.7, 138.5, 136.7, 133.3, 130.7, 130.0, 128.9, 127.9, 124.8, 119.9, 114.0, 81.2, 28.5, 26.7. HRMS (ESI-FT): [M+H]+ calcd for C17H20NO3, 286.1438; found 286.1441.
To a solution of compound 17 (1.427 g, 5 mmol) in CH2Cl2 (15 mL) at 0 °C was added TFA (5 mL) dropwise. The reaction mixture was stirred at room temperature for 3 hours and concentrated under vacuum. The residue was dissolved in dichloromethane (100 mL) and washed with saturated Na2CO3 aqueous solution and brine. The organic phase was dried over Na2SO4 and concentrated to give compound 13 (0.919 g, 99%) as yellow solid. 1H NMR (500 MHz, CDCl3): δ = 8.31 (d, J = 1.5 Hz, 1H), 7.92 (dd, J = 8.5, 1.5 Hz, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.58 (d, J = 8.5 Hz, 1H), 6.97 (dd, J = 8.5, 2.5 Hz, 1H), 6.95 (d, J = 2.0 Hz, 1H), 4.08 (br s, 2 H), 2.66 (s, 3 H); 13C NMR (125 MHz, CDCl3): δ = 198.0, 146.9, 137.8, 131.5, 131.3, 130.6, 126.6, 126.1, 124.8, 118.9, 108.0, 26.6.
To a solution of compound 4 (558.6 mg, 3 mmol) in DMA (6 mL) was added NaOH (360 mg, 9 mmol). The resulting mixture was stirred at room temperature for one hour. 2-Bromo-2-methylpropanamide (1.494 g, 9 mmol) was added to the mixture. After stirring at room temperature overnight, TLC showed complete conversion of compound 4. NaOH (1.080 g, 27 mmol) was added and the resulting mixture was stirred at 50 °C for 5 hours. Water (60 mL) was added to the mixture and was extracted with CH2Cl2 (25 mL) for three times. The combined organic phase was concentrated and redissolved in ethanol (15 mL) and 6 M HCl solution (15 mL). The mixture was refluxed until TLC showed complete conversion of compound 19. After the mixture was cooled to room temperature, most of EtOH was removed under reduced pressure. The residue was diluted with EtOAc and water. The aqueous solution was neutralized with 1 M NaOH solution. The mixture was extracted with EtOAc for three times. The combined organic layers were dried over anhydrous Na2SO4, concentrated and purified by flash chromatography (EtOAc/hexanes = 1/1) to give 13 (406.8 mg, 73%) as yellow solid.
To a solution of 13 (555.7 mg, 3 mmol) in CH2Cl2 (30 mL) was added pyridine (0.27 mL, 3.3 mmol) and o-NsCl (698.1 mg, 3.15 mmol) sequentially at 0 °C. The reaction was allowed to stir at room temperature overnight. The reaction mixture was washed with 1 M HCl solution (20 mL), water and brine, and dried over Na2SO4. The solution was concentrated and the resulting red solid was dissolved in toluene (30 mL) and stirred at 0 °C. To this was added 11 (2.169 g, 6 mmol) and PPh3 (1.574 g, 6 mmol). DIAD (1.18 mL, 6 mmol) was added dropwise to the solution. The reaction mixture was stirred overnight at room temperature. The solution was concentrated and purified by flash chromatography (EtOAc/hexanes = 2/3) to give compound 12 (1.892 g, 88 %) as red solid. Rf = 0.28 (EtOAc/hexanes = 1/1). 1H NMR (500 MHz, CDCl3): δ = 8.43 (s, 1H), 8.06 (dd, J = 8.5, 1.5 Hz, 1H), 7.89 (d, J = 9.0 Hz, 1H), 7.88 (s, 1H), 7.60 (dd, J = 8.0, 1.5 Hz, 1H), 7.56 (dt, J = 8.0, 1.0 Hz, 1H), 7.45 (dd, J = 8.5, 2.0 Hz, 1H), 7.42 (dd, J = 8.0, 1.0 Hz, 1H), 7.33-7.29 (m, 1H), 7.28-7.26 (m, 6H), 7.11-7.05 (m, 9H), 4.36 (dd, J = 15.0, 4.5 Hz, 1H), 4.29 (dd, J = 14.5, 7.0 Hz, 1H), 3.53 (br s, 1H), 3.16 (s, 3 H), 2.72 (s, 3H), 2.66 (br s, 1H); 13C NMR (125 MHz, CDCl3): δ = 197.8, 173.0, 145.5, 138.6, 135.7, 135.4, 133.9, 132.3, 131.8, 131.7, 131.2, 131.0, 129.7, 128.8, 128.69, 128.66, 128.0, 127.9, 127.0, 126.5, 125.0, 124., 71.4, 56.7, 56.6, 52.0, 26.9. HRMS (ESI-FT): [M+Na]+ calcd for C41H35N3O7SNa, 736.2089; found 736.2086. [α]D23 + 69.6 (c 1.00, CHCl3).
To a solution of 12 (1.428 g, 2 mmol) in CH2Cl2 (16 mL) was added TFA (2.0 mL) and water (2.0 mL) at 0 °C. The reaction mixture was stirred at room temperature for 3 hours and concentrated under vacuum. The residue was dissolved in DMF (10 mL). Thiophenol (440.4 mg, 4.0 mmol) and K2CO3 (2.76 g, 20 mmol) were added to the solution sequentially. The reaction mixture was stirred at room temperature for two hours. Water (80 mL) was added to the mixture and the solution was extracted with EtOAc (40 mL) for three times. The combined organic phase was washed with brine (40 mL), dried over Na2SO4 and filtered. The filtrate was concentrated and purified by flash chromatography (EtOAc/hexanes = 1/1, then MeOH/MeOH = 1/20) to give compound 20 (459.6 mg, 80%) as yellow solid. Rf = 0.15 (MeOH/CH2Cl2 = 1/20). 1H NMR (500 MHz, CDCl3): δ = 8.29 (s, 1 H), 7.92 (dd, J = 9.0, 2.0 Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H), 7.61 (d, J = 8.5 Hz, 1H), 6.95 (dd, J = 8.5, 2.0 Hz, 1H), 6.84 (d, J = 2.0 Hz, 1H), 4.75 (br s, 1H), 3.82 (br s, 1H), 3.78 (s, 3H), 3.65-3.63 (m, 1H), 3.35 (dd, J = 12.0, 7.5 Hz, 1H), 2.66 (s, 3H), 1.70 (br s, 2H); 13C NMR (125 MHz, CDCl3): δ = 197.9, 147.9, 138.0, 131.2, 131.0, 130.4, 126.3, 126.2, 124.9, 118.9, 104.4, 53.6, 52.5, 46.9, 26.5. HRMS (ESI-FT): [M+H]+ calcd for C16H19N2O3, 287.1390; found 287.1395. [α]D24 +74.0 (c 1.00, CHCl3).
A solution of compound 16 (286.3 mg, 1 mmol) in 2 M HCl solution (10 mL) was stirred at 60 °C for 8 hours. The reaction mixture was lyophilized to give yellow solid. After washing with diethyl ether, the solid was dried under vacuum to give L-anap (306.8 mg, 99%) as yellow solid. 1H NMR (500 MHz, D2O): δ = 7.82 (s, 1H), 7.45 (dd, J = 8.5, 2.0 Hz, 1H), 7.39 (d, J = 8.5 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 6.87 (dd, J = 8.5, 2.0 Hz, 1H), 6.71 (s, 1H), 3.53-3.51 (m, 1H), 3.47 (dd, J = 13.0, 4.5 Hz, 1H), 3.23 (dd, J = 13.0, 7.0 Hz, 1H); 13C NMR (125 MHz, DMSO-d6): δ = 197.1, 169.5, 148.0, 137.6, 130.5, 130.4, 130.2, 125.7, 125.5, 124.1, 119.0, 102.9, 51.5, 42.9, 26.4. HRMS (ESI-FT): [M+H]+ calcd for C15H17N2O3, 273.1234; found 273.1239. [α]D25 + 74.2 (c 1.07, DMSO).
We thank Dr. Michael Burkart, Mr. Christopher Vickery and Dr. Jing Xu for help with the HRMS and optical rotation. This work was supported by the March of Dimes Foundation (5-FY08-110), California Institute for Regenerative Medicine (RN1-00577-1), and National Institutes of Health (1DP2OD004744-01).