4. Materials and methods
4.1. General
4.1.2. General experimental details
All temperatures quoted are uncorrected. All reagents are commercially available and used without further purification. All solvents are dried over molecular sieves and used directly without further purification. All reactions were conducted under exclusion of air and moisture. Purification of monophosphates was performed on a BioLogic DuoFlow System (Bio-Rad Laboratories) with DEAE Sephadex™ A-25 (GEHealthcare Bio-SciencesAB) column using a linear gradient of triethylammonium bicarbonate buffer (TEAB, pH 7.5) (0.1 - 1.0 M, flow 2 mL/min, pH = 7.5). For medium pressure liquid chromatography (MPLC), a Büchi unit with a Büchi controller C-620, two pumps C-605, a UV monitor C-630 (λ = 254 nm) and fraction collector C-660 was used.
NMR spectra: Bruker Avance III 400 MHz spectrometer. 1H chemical shifts are reported relative to the residual solvent peak and are given in ppm (δ). Flash chromatography: Merck silica gel G60. TLC: Merck precoated plates (silica gel 60 F254). The reported yield refers to the analytically pure substance and is not optimized.
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4.2. Synthesis of NAD
+analogues
2′-O-(propagyl)-adenosine (3, 4)
Adenosine (2 g, 7.48 mmol, 1 eq) were dissolved in 40 mL of hot abs. DMF and cooled to 5 °C, before NaH (0.23 g, 9.73 mmol, 1.3 eq) and propargyl bromide (0.87 mL, 9.73 mmol, 1.3 eq) were added then the reaction mixture was reheated to 55
°C and stirred for 48 h. The solvent was removed under reduced pressure and residue was purified by column – chromatography (3 % methanol in dichloromethane), Rf h. Reaction was quenched with NaHCO3, extracted with ethyl acetate and washed with a brine, before the organic layer
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2′-O-(propagyl)adenosine (3)
To a stirred solution of 5 (0.941 g, 1.7 mmol, 1 eq) in 23 mL THF was added tetra-n-butylammonium fluoride (TBAF) (3.4 mL, 3.4 mmol, 2 eq) and stirred at room temperature for 40 min. After evaporation the residue was purified by MPLC (in 3 % methanol in dichloromethane), Rf value: 0.325 obtaining 0.43 g of 3 in 83 % yield. MS-ESI calculated for [M+H]+ 506.1, found 506.5.
1H NMR (400 MHz, Methanol-d4) δ 8.33 (s, 1H, H-2), 8.21 (s, 1H, H-8), 6.10 (d, J = 6.5 Hz, 1H, H-1′), 4.87 (dd, J = 6,4, 5,0 Hz, 1H, H-2′), 4.53 (dd, J = 4.9, 2.5 Hz, 1H, H-3′), 4.30 (dd, J = 16.1, 2.4 Hz, 1H, CH2), 4.25 (dd, J = 16.1, 2.4 Hz, 1H, CH2), 4.21 (q, J = 2.5 Hz, 1H, H-4′), 3,91 (dd, J = 12,6, 2,4 Hz, 1H, H-5′a), 3,77 (dd, J = 12,6, 2,6 Hz, 1H, H-5′b), 2.64 (t, J = 2.4 Hz, 1H, CH).
13C NMR (101 MHz, MeOD) δ 157.59 (C-6), 153.54 (C-4), 150.12 (C-2), 142.05 (C-8), 120.99 (C-5), 89.33 (C-1′), 81.71 (C-4′), 79.82 (C-2′), 76.54 (propagyl C), 71.16 (propagyl =CH), 63.44 (C-3′), 59.58 (C-5′), 58.78 (propagyl OCH2).
2′-O-(propagyl)adenosine monophosphate (6)
The 2′-O-(propargyl)adenosine 3 (100 mg, 0.327 mmol, 1eq) was dried over night with proton sponge (105 mg, 0.49 mmol, 1.5 eq) under reduced pressure. The dried starting material was dissolved in 3 mL of P(OMe)3, cooled to -20 °C and added 80 µL distilled POCl3. Reaction control through TLC (isopropanol/water/NH2OH = 6/1/1) after stirring for 4 h showed still starting material, so 80 µL of POCl3 were added
again. TLC after further 30 min showed arising side products. The reaction was stopped through addition of the 0.1 M TEAB buffer solution and purified with FPLC obtaining 2′-O-(propargyl)adenosine monophosphate (6) (92 mg, 0.241 mmol) in 73 % yield. MS-ESI calculated for [M+H]+ 385.0, found 385.9.
1H NMR (400 MHz, Deuterium Oxide) δ 8.45 (s, 1H, H-2), 8.17 (s, 1H, H-8), 6.14 (d, J
= 6.4 Hz, 1H, H-1′), 4.71 (dd, J = 6.4, 5.2 Hz, 1H, H-2′), 4.59 (dd, J = 5.2, 3.0 Hz, 1H, H-3′), 4.36 – 4.32 (m, 1H, H-4′), 4.28 (dd, J = 16.2, 2.4 Hz, 1H, CH2), 4.23 (dd, J = 16.2,
91 mmol, 0.01g) was dissolved in a mixture of water (0.25 mL) t-butanol (0.25 mL) and morpholine (10.4 µL), the reaction mixture was refluxed and DCC (0.026 g) was added slowly in t-butanol (0.37mL). Reaction was refluxed for 2 h and then
cooled to room temperature. Dicyclohexyl urea was filtered. Reaction was purified with MPLC 50 mM TEAB buffer with acetonitril giving compound in 27 % yield (0.006 mmol, 3 mg). MS-ESI calculated for [M+H]+ 455.4, found 455.9.
1H NMR (400 MHz, Deuterium Oxide) δ 8.45 (s, 1H, H-2), 8.21 (s, 1H, H-8), 6.17 (d, J mmol)in glove box. To dried overnight mixture of adenosine monophosphate imidazolidate (3 mg, 6.62 *10-3 mmol),
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μL) in formamide. The resulting suspension was stirred at room temperature for 48 h.
Reaction mixture was purified on HPLC giving dinucleotide as a triethylammonium salt in 20 % yield.
2′′mNAD+ method B
To a solution of β-NMN (12 mg, 0.0359 mmol) in dry DMF (300 µL) was added carbonyldiimidazole (22mg, 0.1356 mmol) and triethylamine (5.4 µL). The mixture was stirred at room temperature for 3 h, after which a small amount of MeOH was added to quench the excess CDI. The solvents were removed under vacume, and the residue was coevaporated three times with DMF. 2′-O-(propagyl)adenosine monophosphate (10 mg, 0.0026 mmol) was added with DMF (300 µL), and the mixture was stirred at room temperature for 4 days. The solvent was removed under reduced pressure, and the residue was purieffied with FPLC. The final product was coevaporate with MeOH and lyophilized to affored dinucleotide as a triethylammonium salt (1) (6 mg, 0.0359 mmol), in 24 % yield. MS-ESI calculated for [M+H]+ 701.1, found 701.9.
1H NMR (400 MHz, Deuterium Oxide) δ 9.28 (s, 1H, H2), 9.10 (d, J = 6.3 Hz, 1H, H6),
Under light exclusion trityl chloride (6.26 g, 22.44 mmol, 3 eq) was added to a stirred solution of adenosine (2 g, 7.48 mmol, 1 eq) in 36 mL pyridine at room temperature. The reaction mixture was stirred for two days. After evaporation the residue was purified through MPLC (in 5 % ethyl acetate in dichloromethane), Rf value 0.09. Giving protected adenosine (7)
in 78 % yield (4.39 g, 5.84 mmol). MS-ESI calculated for [M+H]+ 767.9, found 768.3.
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1H NMR (400 MHz, Chloroform-d) δ 8.13 (s, 1H, H-2), 8.01 (s, 1H, H-8), 7.37 – 7.20 (m, 36H, NH, Tr), 5.99 (d, J = 5.4 Hz, 1H, H-1’), 4.71 (t, J = 5.3 Hz, 1H, H-2’), 4.41 (q, J
= 3.0 Hz, 1H, 4’),, 4.35 (dd, J = 5.1, 2.6 Hz, 1H, 3’), 3.52 – 3.46 (m, 2H, OH, H-5’a), 3.26 (dd, J = 10.7, 3.2 Hz, 1H, H-5’b).
13C NMR (101 MHz, CDCl3) δ 154.28 (C-6), 151,14 (C-2), 146,90 (C-4), 144.68 (C-Tr), 143.35 (C-Tr), 138.16 (C-8), 128.98 (6C), 128.55 (6C), 127.94 (6C), 127.91 (6C), 127.90 (6C), 127.23 (3C), 127.02 (3C), 121.12 (C-5), 91.00 (C-1’), 87.08 (C-trityl), 86.20 (C-2’), 76.00 (C-4’), 72.64 (C-3’), 71.69 (C-trityl), 63.62 (C-5’).
2,3′-O-propargyl-N6-5’-O-ditriphenylmethyladenosine (8)
Tritylated adenosine (3.27 g, 4.34 mmol, 1 eq) was dissolved in 30 mL abs. DMF and 60 % NaH (224 mg, 5.6 mmol, 1.3 eq) were added followed by addition of propargyl bromide (502.75 µL, 5.64 mmol, 1.3 eq). The reaction mixture was stirred at room temperature for two days. After evaporation the residue was purified through MPLC with 15 % ethyl acetate in dichloromethane obtaining compound (8) in 55 % yield (2.38 g)
as a white crystalline solid. MS-ESI calculated for [M+H]+ 843.4, found 843.9.
1H NMR (400 MHz, Chloroform-d) δ 8.26 (s, 1H, H-2), 7.85 (s, 1H, H-8), 6.21 (br.s, 2H NH2), 5.86 (d, J = 7.7 Hz, 1H, H1’), 4.97 (dd, J = 7.8, 5.0 Hz, 1H, H-2’), 4.51 (dd, J = 5.4, 1.0 Hz, 1H, H3’), 4.37 (br.s, 1H, H-4’), 4.35 (d, J = 2.4 Hz, 2H, propargyl-CH2), 4.09 (d, J = 2.4 Hz, 2H, propargyl-CH2), 3.92 (dd, J = 13.0, 1.7 Hz, 1H, H-5’a), 3.71 (dd, J = 13.0, 1.7 Hz, 1H, H-5’b), 2.45 (t, J = 2.4 Hz, 1H, propargyl-CH), 2.12 (t, J = 2.4 Hz, 1H, propargyl-CH).
13C NMR (101 MHz, CDCl3) δ 155.32 (C-6), 151.30 (C-4), 148.63 (C-2), 141.36 (C-8), 121.10 (C-5), 89.40 (C-1’), 86.35 (C-3’), 79.82 (C-2’), 79.04 (propargyl-C), 76.60 (C-4;) 75.50 (propargyl-CH), 63.15 (C-5’), 58.37 (propargyl-OCH2).
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2,3′-O-propargyladenosine (9)
Portected adenosine 9 (420 mg, 0.53 mmol) was added to 15 mL of 80 % acetic acid, the solution heated to 100 °C and stirred at this temperature for 100 minutes. The concentrated product was purified over medium pressure column chromatography (DCM : MeOH, 95 : 5) and re-crystallized from methanol to obtain compound 9 (130 mg, 0.43 mmol, 80 %) as white crystals. MS-ESI calculated for [M+H]+ 344.1, found 121.10 (C-5), 89.40 (C-1’), 86.35 (C-3’), 79.82 (C-2’), 79.04 (propargyl-C), 76.60 (C-4;) 75.50 (propargyl-CH), 63.15 (C-5’), 58.37 (propargyl-OCH2).
2,3’-O-(propargyl)adenosine monophosphate
The two, three – prime modified adenosine (9) (100 mg, 0.291 mmol, 1eq) was dried over night with proton sponge (105 mg, 0.49 mmol, 1.5 eq) under reduced pressure. The dried starting material was dissolved in 3 mL of P(OMe)3, cooled to -20 °C through a acetone/nitrogen bath and added 80 µL destilled POCl3. Reaction control through TLC (isopropanol/water/NH2OH = 6/1/1) after stirring for 4 h
showed still starting material, so 80 µL of POCl3 were added again. TLC after further 30 min showed arising side products. The reaction was stopped through addition of the 0.1 M TEAB buffer solution and purified with FPLC obtaining compound 10 (90 mg, 0.213 mmol) in 71 % yield. MS-ESI calculated for [M+H]+ 424.1, found 424.9.
N
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1H NMR (400 MHz, Deuterium Oxide) δ 8.48 (s, 1H, H-2), 8.20 (s, 1H, H-8), 6.16 (d, J
= 6.5 Hz, 1H, 1’), 4.84 (dd, J = 6.5, 5.3 Hz, 1H, 2’), 4.58 (dd, J =5.4, 2.8 Hz, 1H, H-3’), 4.50 (p, J = 2.9 Hz, 1H, H-4’), 4.42 – 4.36 (m, 2H, propargyl-CH2), , 4.28 (d, J = 2.4 Hz, 2H, propargyl-CH2), 4.08 – 4.03 (m, 2H, H-5’a,b)3.14 (q, J = 7.3 Hz, 14H, NCH2), 2.91 (t, J = 2.4 Hz, 1H, propargyl-CH), 2.62 (t, J = 2.4 Hz, 1H, propargyl-CH), 1.22 (t, J
= 7.3 Hz, 21H, CH3).
13C NMR (101 MHz, D2O) δ 152.72 (C4), 85.53 (C3’), 80.35 (C-2’), 79.02 (propargyl C), 76.52 (C-4), 76.39 (propargyl – CH), 64.46 (C-5’), 58.26 (propargyl OCH2), 46.64 (CH2), 8.21 (CH3).
31P NMR (162 MHz, D2O) δ 0.27.
2,3′′mNAD+
To a solution of β-NMN (12 mg, 0.0359 mmol) in dry DMF (300 µL) was added carbonyldiimidazole (22 mg, 0.1356 mmol) and triethylamine (5.4 µL). The mixture was stirred at room temperature for 3 h, after which a small amount of MeOH was added to quench the excess CDI. The solvents were removed under vacuum and the residue was evaporated three times with DMF. 2,3′-O-(propagyl)adenosine monophosphate (10 mg, 0.0026 mmol) was added with DMF (300 µL), and the mixture was stirred at room temperature for 4 days. The solvent was removed under reduced pressure, and the residue was purieffied with FPLC. The final product
was coevaporate with MeOH and lyophilized to affored dinucleotide as a triethylammonium salt (6 mg, 0.0359 mmol), in 24 % yield. HRMS: calculated for:
C27H30N7O14P22-: 738.5028 ([M+Na]+), found: 763.1336.
1H NMR (400 MHz, Deuterium Oxide) δ 9.28 (t, J = 1.5 Hz, 1H, H2), 9.10 (dt, J = 6.4, 1.2 Hz, 1H, H6), 8.79 (dt, J = 8.0, 1.4 Hz, 1H, H4), 8.39 (s, 1H, H2), 8.15 (dd, J = 8.1, 6.3 Hz, 1H, H5), 8.09 (s, 1H, H8), 6.05 (d, J = 6.4 Hz, 1H, H1’), 6.02 (d, J = 5.4 Hz, 1H, H1”), 4.86 – 4.81 (m, 1H, H2”), 4.59 (dd, J = 5.5, 3.2 Hz, 1H, H3’), 4.48 (dd, J = 4.7, 2.5 Hz, 2H, OH), 4.44 (t, J = 5.3 Hz, 1H, H4’), 4.39 (d, J = 2.5 Hz, 3H, H5’, H4”), 4.28 (dd, J
= 2.5, 1.0 Hz, 2H, propagyl CH2), 4.26 – 4.13 (m, 2H, H5’a,b), 3.14 (q, J = 7.3 Hz, 6H,
96 (1.015 g) were dissolved in 11 mL of acetic anhydride and malonaldehyde dianil hydrochloride (0.885 g) were added and the mixture was heated to 110 °C for 30 min. After cooling the reaction to room temperature 1-(1-ε-carboxypenthynyl)-2,3,3-trimethylindoleninium-5-sulfonate was added with 11 mL pyridine and stirred overnight. The reaction was diluted with EtOAc to precipitate the product.
After centrifugation at 4000 rpm for 10 min and washing of
the precipitate 2 times with EtOAc the crude product was dissolved in 23 mL of MQ under nitrogen at 60 °C. After 90 min the reaction was left to cool down and EtOAc was added to precipitate the product. The precipitate was separated from the solution via centrifugation at 400 rpm for 10 min. After discarding the solution the precipitate was washed with EtOAc and dried under vacuo giving 78 % yield (88
97 mixed with 1-amino-3-azido-propan (3 eq, 87 umol, 8.7 mg) and Et3N (3 eq, 12 μL) in 2.5 mL DMF.
EtOAc was added to precipitate the product that was then separated from the solution via centrifugation at 4000 rpm and 10 min. The solution was then discarded and the crude product washed with EtOAc. Afterwards the product was dried under vacuum and dissolved in 10 mL MQ for
RP-HPLC purification. The solvents from the collected fractions were then removed under reduced pressure and the product lyophilized twice, yielding Azido-Cy5 in 49 % (14.2 μmol). MS-ESI calculated for [M+H]+ 738.2, found 738.9.
1H NMR (400 MHz, DMSO) δ[ppm]: 8.36 (t, J = 13.2 Hz, 2H, β -H), 7.91 – 7.75 (m, 2H,
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13C NMR (101 MHz, DMSO) δ[ppm]: 119.36 (Ar), 110.39 (Ar), 102.69 (α-C), 39.26, 44.03 (N-CH2), 48.86 (N+-CH2), 36.36 (CH2 – N3), 45.09 (CONH-CH2), 40.23 (CH2 -CONH), 34.17 (N-CH2-CH2), 20.83 (CH3), 26.81 (CH2-CH2-CONH), 27.59 (CH2-CH2 -N3), 26.25 (N-CH2-CH2-CH2), 13.03 (N+-CH2-CH3).
Synthesis of BTTAA
To a 10 mL round-bottom flask were added N,N-bis((1-tert-butyl-1H-1, 2,3-triazol-4-yl)methyl)prop-2-yn-1-amine (0.1 g, 0.03 mmol, 1.0 eq) and 2-azidoacetic acid (0.045 g, 0.045 mmol, 1.5 eq) in 3 mL of THF. To the mixture were added N,N’-diisopropylethylamine (0.062 g, 0.048 mmol, 1.6 eq) and tris(triphenylphosphine)copper(I)
bromide (0.028 g, 0.003 mmol). The reaction mixture was stirred vigorously at 60 °C overnight under argon. To the reaction mixture were added 2 mL water and 0.2 g CupriSorbTM. The mixture was stirred for additional 30 min and then filtered. The crude product was concentrated in vacuo and was purified by RP-MPLC (in gradient acetonitrile : water) to provide 0.109 g of product (yield: 75 %) as a white solid.
1H NMR (600 MHz, D2O) δ8.00 (s, 2H, 2xCH), 7.92 (s, 1H, CH), 5.05 (s, 2H, CH2COOH), 3.92 (s, 2H, CH2), 3.90 (s, 4H, 2xCH2), 1.65 (s, 18H, CH3).
13C NMR (150 MHz, D2O) δ173.2 (COOH), 143.0 (2xC), 142.6 (C), 126.3 (2CH), 122.8 (CH), 60.3 (C(CH3)3), 53.1 (2xCH2), 48.0 (CH2), 47.9 (CH2COOH), 28.9 (CH3).