* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.
Reference：
[1] Patent: WO2007/131286, 2007, A1, . Location in patent: Page/Page column 70
[2] Chemical Communications, 2017, vol. 53, # 36, p. 5020 - 5023
[3] Chemistry - A European Journal, 2017, vol. 23, # 45, p. 10906 - 10914
2
[ 6066-82-6 ]
[ 58-85-5 ]
[ 35013-72-0 ]
Yield
Reaction Conditions
Operation in experiment
91%
at 20℃;
To a solution of (+)-biotin (0.82 mmol, 200 mg) and N-hydroxysuccinimide (0.89 mmol, 102 mg) was added 3-(ethyliminomethylidenamino)-N,N-dimethylpropan-1-aminehydrochloride (0.96 mmol, 184 mg). The reaction mixture was stirred overnight at room temperature and concentrated to give a white solid. The crude solid was mixed with isopropanol, heated up to 70 °C and cooled down. The pure product was filtered off as white powder in 91percent yield (0.75 mmol, 255 mg).
90%
With dicyclohexyl-carbodiimide In N,N-dimethyl-formamide at 20℃; for 12 h; Heating
D-Biotin (5.00 g, 2.05 mmol) and N-hydroxysuccinimide (2.36 g, 2.05 mmol) were dissolved in hot DMF (150 mL). N,N′-Dicyclohexylcarbodiimide (5.50 g, 2.67 mmol) was added to the above reaction mixture, and was stirred overnight at room temperature. The dicyclohexyl urea was filtered, and the filtrate was evaporated and triturated with ether. The white precipitate was obtained and washed with ether to give a white powder in 90percent. (6.29 g) yield (1H NMR(400 MHz, CDCl3): 1.5–1.6 (m, 2H)), 1.6–1.9 (m, 4H) 2.58–2.70 (m, 2H),2.75 (d, 1H,) 2.86 (s, 4H), 2.87–2.97 (m, 1H), 3.16 (m, 1H), 4.33 (m, 1H), 4.52 (m, 1H), 4.96 (s, 1H), 5.23 (s, 1H). m/z (ESI) found 342 (M + H)+.C14H19N3O5S calculated 341.
79%
With dicyclohexyl-carbodiimide In N,N-dimethyl-formamide at 20 - 70℃;
D-Biotin (0.819 mmol) and N-hydroxysuccinimide (0.819 mmol) were dissolved into anhydrous DMF (6 ml) at 70 00 while stirring. DCC (1.064 mmol) was added and the solution was stirred overnight at room temperature. The formed DCU was filtered off and the solvent was removed under reduced pressure. The residue was taken up into boiling isopropanol and the solution was allowed to cooldown to RT. The target compound precipitated and the product was filtered off as a white solid.Yield: 79percent, MS (ESI) m/z 364.1 [M ÷ Na]NMR (DMSO-d6, 400 MHz): 6 1.42-1.67 (m, 6H), 2.57-2.60 (d, J=12.4 Hz, 1H), 2.68 (t, J=7.3 Hz, 2H), 2.81-2.90(m, 5H), 3.11 (m, 1H), 4.14-4.17(m, 1H), 4.29-4.35 (m, 1H), 6.37(s, 1H), 6.42 (s, 1H)
70%
With dicyclohexyl-carbodiimide In N,N-dimethyl-formamide at 20 - 50℃;
1. Synthesis of Biotinyl-N-hydroxysuccinimide (Scheme I) . D-Biotin (0.49 g, 2.0 mmol, Sigma B4501) was added to N-hydroxysuccinimide (0.3 g, 2.7 mmol, Pierce HC102040) into 20 mL DMF (Sigma 227056), heated to 50°C until most of the material dissolved. A solution of Ν,Ν'- Dicyclohexylcarbodiimide (0.45g, 2.1 mmol, Sigma D80002) in 5 mL DMF was added dropwise to the aforementioned solution. The resulting solution was stirred overnight at room temperature during which time a white precipitate was formed. The reaction mixture was filtered through celite, and the filtrate was triturated with diethyl ether (Sigma 309966) . The white precipitate was vacuum filtered and then washed with diethyl ether to give a white powder. The yield was 0.52 g (~ 70 percent final efficiency) .
68%
Stage #1: With 1,1'-carbonyldiimidazole In N,N-dimethyl-formamide at 78℃; Inert atmosphere Stage #2: at 20℃;
Example 2. Biotin N-Hydroxysuccinimide Ester (5); [0157] To a solution containing 101 mg (0.41 mmol) of biotin in 2 rnL of DMF at 78 0C was added 67 mg (0.41 mmol) of l,l '-carbonyldiimidazole with continued heating until CO2 evolution ceased. The solution was equilibrated to room temperature and stirred for an additional 3 h. To the reaction mixture was added a solution of 47 mg (0.41 mmol) of N- hydroxysuccinimide in 2 mL of DMF. The solution was stirred overnight at room temperature. The solvent was removed under diminished pressure and the product was crystallized from 2-propanol and then DMF-ether to afford 5 as a fine white powder: yield 95 mg (68percent); 1H NMR (CDCl3) δ 1.43-1.66 (m, 7H), 2.58 (m, IH), 2.66 (t, 2H), 2.75 (s, 4H), 3.05 (m, IH), 4.11 (m, IH), 4.27 (m, IH), and 6.36 (d, 2H, J = 18.4 Hz); 13C NMR (CDCl3) δ 55.19, 60.68, 63.46, 65.43, 69.11, 70.08, 73.25, 75.85, 79.05, 101.35, 101.83, 126.32, 127.20, 127.81, 128.11, 128.16, 128.48, 128.72, 129.18, 137.81, and 138.24; mass spectrum (MALDI-TOF), m/z 342.1 (M + H)+.
65%
With diisopropyl-carbodiimide In N,N-dimethyl-formamide at 45℃;
[0074] To 2 grams of biotin was added 2 eq of N,N’-diisopropylcarbodiimide and 3 eq of Nhydroxysuccinimide in 0.2 M DMF at 45 °C to yield compound 6. One eq of t-Boc protected PEG amine (3) was then added in methylene chloride at room temperature to give compound 7 and deprotected using TFA to afford compound 8, followed be activation using 2 eq of N,N’-diisopropylcarbodiimide and 3 eq of N-hydroxysuccinimide to form its activated ester, compound 9. G3 dendrimer DNT-296 was added to compound 9 in methanol at room temperature to yield compound 10.
61%
With CDI In N,N-dimethyl-formamide; isopropyl alcohol
1. Preparation of Biotin-N-hydroxysuccinimide Biotin (1.0 g, 4.1 mmole) was dissolved in 10 ml DMF (dry) with heating at 80° C. in oil bath. CDI (665 mg, 4.1 mmoles) was added and the mixture was heated at 80° C. The reaction mixture was stirred at 80° C. for 30 minutes, then at room temperature for 2 hours; a white precipitate formed. N-hydroxysuccinimide (475 mg, 4.1 mmoles) was added and the reaction mixture was stirred at room temperature overnight. DMF was removed under vacuum on rotary evaporator. The solid residue was dissolved in 250 ml of refluxing isopropanol, filtered, and stored in the cold room overnight. The precipitate was filtered, washed one time with cold isopropanol and dried in vacuo at 45° C. overnight to give 870 mg (61percent yield) of the desired product.
57%
With dicyclohexyl-carbodiimide In N,N-dimethyl-formamide at 20℃; Inert atmosphere
Biotin (498mg, 2mmol) was dissolved in anhydrous DMF (10mL) by heating to approximately 70°C for 10min or until fully dissolved. The reaction mixture was allowed to cool to room temperature before adding N-hydroxysuccinimide (240mg, 2.1mmol) with stirring at room temperature. A solution of N,N′-dicyclohexylcarbodiimide (438mg, 2.13mmol) in anhydrous DMF (2mL) was added dropwise to the stirred solution. The reaction was then stirred overnight at room temperature over which time a white precipitate formed (N,N′-dicyclohexylurea). The precipitate was removed by filtration and washed with DMF. The filtrate was diluted with EtO2 until a white precipitate formed. The precipitate was collected by filtration and rinsed with EtO2 then dried to give the crude product 26 as a white solid (395mg, 57percent): m.p. (decomp.) 178–190°C; IR (νmax, ATR): 3227, 2941, 2876, 1818, 1788, 1729, 1698, 1465, 1369, 1210, 1071, 861, 739, 656cm−1; 1H NMR (600MHz, DMSO-d6): δ=6.40 (1H, s, NH), 6.35 (1H, s, NH), 4.30 (1H, m, HNCHCHNH), 4.14 (1H, m, HNCHCHNH), 3.10 (1H, m, SCH), 2.84–2.78 (5H, contains NCOCH2CH2 and SCHAHB), 2.67 (2H, t, J=7.7Hz, CH2CH2CO2N), 2.57 (1H, d, J=12.4Hz, SCHAHB), 1.64 (3H, contains CHAHBCH2CH2CH2CO2N), 1.52–1.36 (3H, contains CHAHBCH2CH2CH2COON); 13C NMR (150MHz, DMSO-d6): δ=170.3, 169.0, 162.7, 61.0, 59.2, 55.3, 40.1 (overlaps with NMR solvent peak), 30.0, 27.9, 27.6, 25.5, 24.3; HRMS (ESI): M+H+, found 342.1128. C14H20N3O5S+ requires 342.1118.
30%
Stage #1: With pyridine; dicyclohexyl-carbodiimide In N,N-dimethyl-formamide for 0.5 h; Stage #2: for 24 h;
Biotin (24.4mg, 0.1mmol) was dissolved to N, N- dimethylformamide (DMF) (10mL),Pyridine (Py) (2mL) and N, N'- dicyclohexylcarbodiimide imide (DCC) (41.2mg, 0.2mmol), after half an hour the reaction was added N- hydroxysuccinimide (N-Hydroxysuccinimide ) (13.8mg, 0.12mmol), the reaction is stopped after 24h the reaction, after removal of DMF by filtration and the filtrate evaporated under reduced pressure, then dissolved in hot isopropanol solvent and then filtered and the filtrate precipitated 2 days in a refrigerator at 4 finally filtered to give a white solid precipitated 10.2mg, yield 30percent.
1 g
With dicyclohexyl-carbodiimide In N,N-dimethyl-formamide at 50℃; for 24 h; Inert atmosphere
Into a 50 mL reaction flask, 1.0 g of biotin [manufactured by Wako Pure Chemical Industries, Ltd.], 0.59 g of NHS, and 19 g of DMF were charged and the mixture was dissolved with stirring at 50° C. To this solution, 1.0 g of DCC was added and the resultant reaction liquid was stirred at 50° C. for 24 hours in a nitrogen atmosphere. Subsequently, insoluble substances were removed from the reaction liquid by filtration and then DMF was removed by distillation. The obtained residue was washed with 3 g of diethyl ether and then recrystallized with 15 g of IPA. The obtained white crystal was filtered under reduced pressure and dried under vacuum to obtain 1.0 g of biotin N-hydroxysuccinimide ester.
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3
[ 105832-38-0 ]
[ 58-85-5 ]
[ 35013-72-0 ]
Reference：
[1] European Journal of Organic Chemistry, 2015, vol. 2015, # 28, p. 6153 - 6159
[2] Patent: US2015/376246, 2015, A1, . Location in patent: Paragraph 0147
[3] Patent: WO2016/164504, 2016, A1, . Location in patent: Page/Page column 40; 41
4
[ 6066-82-6 ]
[ 35013-72-0 ]
Yield
Reaction Conditions
Operation in experiment
2.7 g
With dicyclohexyl-carbodiimide In N,N-dimethyl-formamide at 20℃; Heating
Step 1: To a round bottom flask with a stirring bar, biotin (2.0 g, 8.2 mmol) and DMF (60 mL) were added. After the solid was dissolved with heat, N-hydroxysuccinimide (0.944 g, 8.2 mmol) and DCC (2.2 g, 10.7 mmol) were added. The reaction mixture was stirred at room temperature overnight. The white solid was filtered, and the DMF was evaporated under reduced pressure. The resulting residue was further purified by re-crystallization from isopropanol to give the desired product 2 (2.7 g, M+H+ = 342.5) as white crystals.
Reference：
[1] Journal of Medicinal Chemistry, 1984, vol. 27, # 11, p. 1406 - 1410
[2] Journal of the American Chemical Society, 2009, vol. 131, # 7, p. 2438 - 2439
Reference：
[1] Organic and Biomolecular Chemistry, 2012, vol. 10, # 43, p. 8570 - 8574
10
[ 35013-72-0 ]
[ 60-32-2 ]
[ 72040-64-3 ]
Yield
Reaction Conditions
Operation in experiment
1.455 g
With sodium hydrogencarbonate In water; N,N-dimethyl-formamide at 20℃; for 16 h;
ε-Aminocaproic acid (966mg, 8.4mmol) was dissolved in 30mL 1M aqueous NaHCO3 solution. Then N-hydroxysuccinimidobiotin (3.09g) in 35mL DMF was added dropwise to the solution and the mixture was stirred at room temperature for 16h. The solution was concentrated under reduced pressure to remove partial solvent and 150mL aqueous citric acid (100g/L) was then added and stirred at 60°C for 30min. The precipitate was collected by filtration and washed with distilled water. The precipitate was dissolved in a mixture of isopropanol and water (8:2, v/v) and kept at 4°C to give pure 3 (1.455g, 50percent) as a yellowish crystal; IR cm−1 (KBr): 3200–3500, 3070, 2932, 2859, 1696, 1644, 1543, 1475, 1391, 1324, 1265, 1118; 1H NMR (600MHz, DMSO-d6): δ 7.75 (1H, t, J=5.4Hz, C6–NH), 6.45 (1H, s, C13–NH), 6.38 (1H, s, C14–NH), 4.35–4.27 (1H, m, H-14), 4.12 (1H, m, H-13), 3.09 (1H, m, H-12), 3.00 (1H, m, H-6) 2.82 (1H, dd, J=12.4, 5.1Hz, H-15a), 2.57 (1H, d, J=12.4Hz, H-15b), 2.19 (2H, t, J=7.4Hz, H-2), 2.04 (2H, t, J=7.3Hz, H-8), 1.20–1.67 (14H, m). 13C NMR (150MHz, DMSO-d6): δ 171.84 (C-1, C-7), 162.74 (C-16), 61.06 (C-13), 59.20 (C-14), 55.44 (C-12), 39.68 (C-15), 38.25 (C-6), 35.21 (C-8), 29.05 (C-2), 28.92, 28.21, 28.04, 26.04, 25.73, 25.36.
Reference：
[1] Chemical Communications, 2009, # 33, p. 5030 - 5032
[2] European Journal of Medicinal Chemistry, 2014, vol. 71, p. 219 - 228
[3] RSC Advances, 2018, vol. 8, # 57, p. 32775 - 32793
11
[ 105047-45-8 ]
[ 35013-72-0 ]
[ 146987-10-2 ]
Reference：
[1] Chemical Communications, 2006, # 7, p. 717 - 719
[2] Chemical Communications, 2017, vol. 53, # 36, p. 5020 - 5023
[3] Chemistry - A European Journal, 2017, vol. 23, # 45, p. 10906 - 10914
With dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; at 20℃; for 24h;
Disperse D-<strong>[58-85-5]biotin</strong> (0.3 g, 1.2 mmol, 1.0 equiv.) In N, N-dimethylformamide (10 mL),Heat until all solids are dissolved.Add N-hydroxysuccinimide (0.17 g, 1.4 mmol, 1.2 equiv.) In that order, andN, N'-dicyclohexylcarbodiimide (0.28 g, 1.4 mmol, 1.2 equiv.).The reaction was stirred at room temperature for 24 h. The solid precipitate was removed by filtration, and then concentrated by distillation under reduced pressure. Diethyl ether (10 mL) was added to the residue and sonicated to give a white precipitate. The pellet was settled using centrifugation and the ether was removed. The pellet was re-dispersed in ethanol, and the pellet was washed repeatedly by repeated dispersion and centrifugation 3 times. After vacuum drying, a white solid (0.40 g) was obtained in a yield of 94%.
91%
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; at 20℃;
To a solution of (+)-<strong>[58-85-5]biotin</strong> (0.82 mmol, 200 mg) and N-hydroxysuccinimide (0.89 mmol, 102 mg) was added 3-(ethyliminomethylidenamino)-N,N-dimethylpropan-1-aminehydrochloride (0.96 mmol, 184 mg). The reaction mixture was stirred overnight at room temperature and concentrated to give a white solid. The crude solid was mixed with isopropanol, heated up to 70 C and cooled down. The pure product was filtered off as white powder in 91% yield (0.75 mmol, 255 mg).
90%
With dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; at 20℃; for 12h;Heating;
D-<strong>[58-85-5]Biotin</strong> (5.00 g, 2.05 mmol) and N-hydroxysuccinimide (2.36 g, 2.05 mmol) were dissolved in hot DMF (150 mL). N,N?-Dicyclohexylcarbodiimide (5.50 g, 2.67 mmol) was added to the above reaction mixture, and was stirred overnight at room temperature. The dicyclohexyl urea was filtered, and the filtrate was evaporated and triturated with ether. The white precipitate was obtained and washed with ether to give a white powder in 90%. (6.29 g) yield (1H NMR(400 MHz, CDCl3): 1.5-1.6 (m, 2H)), 1.6-1.9 (m, 4H) 2.58-2.70 (m, 2H),2.75 (d, 1H,) 2.86 (s, 4H), 2.87-2.97 (m, 1H), 3.16 (m, 1H), 4.33 (m, 1H), 4.52 (m, 1H), 4.96 (s, 1H), 5.23 (s, 1H). m/z (ESI) found 342 (M + H)+.C14H19N3O5S calculated 341.
89%
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 20℃; for 24h;
<strong>[58-85-5]Biotin</strong> (1 mmol) and HOSu (1.07 mmol) were dissolved in 12 mL DMF.EDC.HCl (1.17 mmol) was added with stirring at room temperature, and the reaction was stirred at room temperature.After 24 hours, DMF was distilled off under reduced pressure.The resulting residue was washed 3 times with MeOH.Finally, <strong>[58-85-5]biotin</strong> activated ester,Yield: 89%.
79%
With dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; at 20 - 70℃;
D-<strong>[58-85-5]Biotin</strong> (0.819 mmol) and N-hydroxysuccinimide (0.819 mmol) were dissolved into anhydrous DMF (6 ml) at 70 00 while stirring. DCC (1.064 mmol) was added and the solution was stirred overnight at room temperature. The formed DCU was filtered off and the solvent was removed under reduced pressure. The residue was taken up into boiling isopropanol and the solution was allowed to cooldown to RT. The target compound precipitated and the product was filtered off as a white solid.Yield: 79%, MS (ESI) m/z 364.1 [M ÷ Na]NMR (DMSO-d6, 400 MHz): 6 1.42-1.67 (m, 6H), 2.57-2.60 (d, J=12.4 Hz, 1H), 2.68 (t, J=7.3 Hz, 2H), 2.81-2.90(m, 5H), 3.11 (m, 1H), 4.14-4.17(m, 1H), 4.29-4.35 (m, 1H), 6.37(s, 1H), 6.42 (s, 1H)
Ca. 70%
With dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; at 20 - 50℃;
1. Synthesis of <strong>[58-85-5]Biotin</strong>yl-N-hydroxysuccinimide (Scheme I) . D-<strong>[58-85-5]Biotin</strong> (0.49 g, 2.0 mmol, Sigma B4501) was added to N-hydroxysuccinimide (0.3 g, 2.7 mmol, Pierce HC102040) into 20 mL DMF (Sigma 227056), heated to 50C until most of the material dissolved. A solution of Nu,Nu'- Dicyclohexylcarbodiimide (0.45g, 2.1 mmol, Sigma D80002) in 5 mL DMF was added dropwise to the aforementioned solution. The resulting solution was stirred overnight at room temperature during which time a white precipitate was formed. The reaction mixture was filtered through celite, and the filtrate was triturated with diethyl ether (Sigma 309966) . The white precipitate was vacuum filtered and then washed with diethyl ether to give a white powder. The yield was 0.52 g (~ 70 % final efficiency) .
70%
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 20 - 70℃; for 12h;
In a 100-mL flask, 2.5 g of D-<strong>[58-85-5]biotin</strong> (10.23 mmol; 1 eq) and 1.18 g of N-hydroxysuccinimide (10.23 mmol; 1 eq) are dissolved in 40 mL of anhydrous dimethylformamide. The reaction is stirred magnetically at 70 C. until the reagents have dissolved completely. 2.55 g of EDC (13.30 mmol; 1.3 eq) is then added, and the reaction mixture is stirred at room temperature for 12 h.The mixture is evaporated to dryness and then taken up in30 mL of methanol. The precipitate obtained is then filtered on a frit, and then washed with MeOH (3x30 mL) and diethyl ether (3x90 mL).The product is obtained in the form of white powder at a yield of 70% (2.46 g; 7.21 mmol).m.p.: 210 C.IR (K13r): v 3234; 1820; 1789; 1747 (CO); 1730 (CO); 1705 (CO); 1216; 1072 cm?.?H NMR (400 MHz, DMSO-d5): oe 1.39-1.67 (m, 6H);2.57 (m, 1H); 2.66 (t, 2H, 3J=7.3 Hz); 2.68 (s, 4H); 2.84 (m,1H); 3.09 (m, 1H); 4.14 (m, 1H); 4.29 (m, 1H); 6.37 (s, 1H);6.43 (s, 1H).?3C NMR (100 MHz, DMSO-d5): oe 24.3; 25.5 (2C); 27.6;27.9; 30.0; 39.9; 55.3; 59.1; 61.0; 162.8; 169.0; 174.4 (2C).
68%
Example 2. <strong>[58-85-5]Biotin</strong> N-Hydroxysuccinimide Ester (5); [0157] To a solution containing 101 mg (0.41 mmol) of <strong>[58-85-5]biotin</strong> in 2 rnL of DMF at 78 0C was added 67 mg (0.41 mmol) of l,l '-carbonyldiimidazole with continued heating until CO2 evolution ceased. The solution was equilibrated to room temperature and stirred for an additional 3 h. To the reaction mixture was added a solution of 47 mg (0.41 mmol) of N- hydroxysuccinimide in 2 mL of DMF. The solution was stirred overnight at room temperature. The solvent was removed under diminished pressure and the product was crystallized from 2-propanol and then DMF-ether to afford 5 as a fine white powder: yield 95 mg (68%); 1H NMR (CDCl3) delta 1.43-1.66 (m, 7H), 2.58 (m, IH), 2.66 (t, 2H), 2.75 (s, 4H), 3.05 (m, IH), 4.11 (m, IH), 4.27 (m, IH), and 6.36 (d, 2H, J = 18.4 Hz); 13C NMR (CDCl3) delta 55.19, 60.68, 63.46, 65.43, 69.11, 70.08, 73.25, 75.85, 79.05, 101.35, 101.83, 126.32, 127.20, 127.81, 128.11, 128.16, 128.48, 128.72, 129.18, 137.81, and 138.24; mass spectrum (MALDI-TOF), m/z 342.1 (M + H)+.
65%
With diisopropyl-carbodiimide; In N,N-dimethyl-formamide; at 45℃;
[0074] To 2 grams of <strong>[58-85-5]biotin</strong> was added 2 eq of N,N?-diisopropylcarbodiimide and 3 eq of Nhydroxysuccinimide in 0.2 M DMF at 45 C to yield compound 6. One eq of t-Boc protected PEG amine (3) was then added in methylene chloride at room temperature to give compound 7 and deprotected using TFA to afford compound 8, followed be activation using 2 eq of N,N?-diisopropylcarbodiimide and 3 eq of N-hydroxysuccinimide to form its activated ester, compound 9. G3 dendrimer DNT-296 was added to compound 9 in methanol at room temperature to yield compound 10.
62%
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 20℃;
9. Weigh 2 g of <strong>[58-85-5]biotin</strong> (2.046 mol),Dissolved in an appropriate amount of DMF solvent, and added 1.1 g (2.25 mol) of N-hydroxysuccinimide and 2.4 g (3.05 mol) of carbodiimide hydrochloride, respectively.Stir well overnight at room temperature. After the reaction is completed, the reaction mixture is slowly dropped into ice water.There is a large amount of white solid precipitated, suction filtration,Molten twice with methanol and dried in vacuum.A <strong>[58-85-5]biotin</strong> succinimide ester of 3.53 g was obtained.Yield: 62%.
61%
With CDI; In N,N-dimethyl-formamide; isopropyl alcohol;
1. Preparation of <strong>[58-85-5]Biotin</strong>-N-hydroxysuccinimide <strong>[58-85-5]Biotin</strong> (1.0 g, 4.1 mmole) was dissolved in 10 ml DMF (dry) with heating at 80 C. in oil bath. CDI (665 mg, 4.1 mmoles) was added and the mixture was heated at 80 C. The reaction mixture was stirred at 80 C. for 30 minutes, then at room temperature for 2 hours; a white precipitate formed. N-hydroxysuccinimide (475 mg, 4.1 mmoles) was added and the reaction mixture was stirred at room temperature overnight. DMF was removed under vacuum on rotary evaporator. The solid residue was dissolved in 250 ml of refluxing isopropanol, filtered, and stored in the cold room overnight. The precipitate was filtered, washed one time with cold isopropanol and dried in vacuo at 45 C. overnight to give 870 mg (61% yield) of the desired product.
57%
With dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; at 20℃;Inert atmosphere;
<strong>[58-85-5]Biotin</strong> (498mg, 2mmol) was dissolved in anhydrous DMF (10mL) by heating to approximately 70C for 10min or until fully dissolved. The reaction mixture was allowed to cool to room temperature before adding N-hydroxysuccinimide (240mg, 2.1mmol) with stirring at room temperature. A solution of N,N?-dicyclohexylcarbodiimide (438mg, 2.13mmol) in anhydrous DMF (2mL) was added dropwise to the stirred solution. The reaction was then stirred overnight at room temperature over which time a white precipitate formed (N,N?-dicyclohexylurea). The precipitate was removed by filtration and washed with DMF. The filtrate was diluted with EtO2 until a white precipitate formed. The precipitate was collected by filtration and rinsed with EtO2 then dried to give the crude product 26 as a white solid (395mg, 57%): m.p. (decomp.) 178-190C; IR (numax, ATR): 3227, 2941, 2876, 1818, 1788, 1729, 1698, 1465, 1369, 1210, 1071, 861, 739, 656cm-1; 1H NMR (600MHz, DMSO-d6): delta=6.40 (1H, s, NH), 6.35 (1H, s, NH), 4.30 (1H, m, HNCHCHNH), 4.14 (1H, m, HNCHCHNH), 3.10 (1H, m, SCH), 2.84-2.78 (5H, contains NCOCH2CH2 and SCHAHB), 2.67 (2H, t, J=7.7Hz, CH2CH2CO2N), 2.57 (1H, d, J=12.4Hz, SCHAHB), 1.64 (3H, contains CHAHBCH2CH2CH2CO2N), 1.52-1.36 (3H, contains CHAHBCH2CH2CH2COON); 13C NMR (150MHz, DMSO-d6): delta=170.3, 169.0, 162.7, 61.0, 59.2, 55.3, 40.1 (overlaps with NMR solvent peak), 30.0, 27.9, 27.6, 25.5, 24.3; HRMS (ESI): M+H+, found 342.1128. C14H20N3O5S+ requires 342.1118.
30%
<strong>[58-85-5]Biotin</strong> (24.4mg, 0.1mmol) was dissolved to N, N- dimethylformamide (DMF) (10mL),Pyridine (Py) (2mL) and N, N'- dicyclohexylcarbodiimide imide (DCC) (41.2mg, 0.2mmol), after half an hour the reaction was added N- hydroxysuccinimide (N-Hydroxysuccinimide ) (13.8mg, 0.12mmol), the reaction is stopped after 24h the reaction, after removal of DMF by filtration and the filtrate evaporated under reduced pressure, then dissolved in hot isopropanol solvent and then filtered and the filtrate precipitated 2 days in a refrigerator at 4 finally filtered to give a white solid precipitated 10.2mg, yield 30%.
With dicyclohexyl-carbodiimide; In DMF (N,N-dimethyl-formamide);
For the amination of <strong>[58-85-5]biotin</strong> (FIG. 5A), <strong>[58-85-5]biotin</strong> (1 mmol) was activated with DCC (1.25 mmol) and NHS (1.5 mmol) in DMF. <strong>[58-85-5]Biotin</strong>-NHS was collected after recrystallization (DMF/diethyl ether).
With dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; at 20℃;Heating;
D-<strong>[58-85-5]Biotin</strong> (0.200 g, 0.82 mmol, 1.0 equiv) was dissolved in anhydrous DMF (6 mL) and gently heated. N-hydroxysuccinimide (0.104 g, 0.90 mmol, 1.1 equiv) and DCC (0.203 g, 0.98 mmol, 1.2 equiv) were added. The reaction mixture was stirred overnight at room temperature. The mixture was filtered. After addition of Et2O to the filtrate, the precipitate formed was collected by filtration, and washed with cooled Et2O and iPrOH to give 8 as a colorless solid. It was directly used in the next step without further purification.
With dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; at 20℃; for 16h;
<strong>[58-85-5]Biotin</strong> (2g, 8.2mmol) and NHS (924mg, 8mmol) were dissolved in 60mL DMF. Then DCC (1.85g, 9mmol) was added and the mixture was stirred at room temperature for 16h (Scheme 1). The resulting precipitate (N,N-dicyclohexylurea, DCU) was removed by filtration and the filtrate (75mL) was concentrated to small volume (10mL) under reduced pressure. Diethyl ether (200mL) was then added and the resulting precipitate was collected by filtration and dried in the presence of CaH2 under vacuum to provide N-hydroxysuccinimido<strong>[58-85-5]biotin</strong> (3.09g) which was used in the next step.
With triethylamine; dicyclohexyl-carbodiimide; at 20℃;
<strong>[58-85-5]Biotin</strong> (5g) was dissolved in 100 ml dry DMSO, plus 2.5 ml triethylamine. N-hydroxysuccinimide (2.6 gm) was added as a powder to the <strong>[58-85-5]biotin</strong> and reacted overnight with 4.7 gm dicyclohexylcarbodiimide at room temperature. The dicyclohexylurea was removed by filtration. The DMSO was concentrated under reduced pressure and heating, and NHS-<strong>[58-85-5]biotin</strong> precipitated by adding multiple volumes of diethylether. The product, was washed several times with anhydrous ether, dried under vacuum and stored as a white powder.
With dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; at 20℃; for 24h;Inert atmosphere;
<strong>[58-85-5]Biotin</strong> was treated with NHS and DCC in a molar ratioof 1:1:1.2 in dry N,N-dimethylformamide under nitrogenatmosphere at room temperature for 24 h to give a whiteprecipitate. The reaction mixture was filtered, and the filtratewas evaporated and triturated with ether. The whiteprecipitate was filtered and washed with diethyl ether toafford a white powder. The NMR data of the <strong>[58-85-5]Biotin</strong>-NHSis shown below: 1H-NMR in d6-DMSO, (ppm): 6.42and 6.35 [ds, 2 H, (CHNH)2CO], 4.30 and 4.13 [m, 2 H,(CHNH)2CO], 3.09 (m, 1 H, SCH), 2.84-2.80 (dd, 4 H,SCH2 and CH2CON), 2.20 (t, 2 H, CH2COO), 1.66-1.26(m, 6 H, CHCH2CH2CH2).
1 g
With dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; at 50℃; for 24h;Inert atmosphere;
Into a 50 mL reaction flask, 1.0 g of <strong>[58-85-5]biotin</strong> [manufactured by Wako Pure Chemical Industries, Ltd.], 0.59 g of NHS, and 19 g of DMF were charged and the mixture was dissolved with stirring at 50 C. To this solution, 1.0 g of DCC was added and the resultant reaction liquid was stirred at 50 C. for 24 hours in a nitrogen atmosphere. Subsequently, insoluble substances were removed from the reaction liquid by filtration and then DMF was removed by distillation. The obtained residue was washed with 3 g of diethyl ether and then recrystallized with 15 g of IPA. The obtained white crystal was filtered under reduced pressure and dried under vacuum to obtain 1.0 g of <strong>[58-85-5]biotin</strong> N-hydroxysuccinimide ester.
With dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; at 25℃; for 48h;
To a solution of d-<strong>[58-85-5]biotin</strong> (14.5 mg, 60 mol in dimethylformamide (0.6 mL), 61 mol of bothDCHC and N-hydroxysuccinimide (each in ethyl acetate 1.0 mL) were added. After 2 days at 25 C,the solution was centrifuged (13,000 g at room temperature for 30 s) and this solution of <strong>[58-85-5]biotin</strong>hydroxysuccinimide ester was stored at 4 C. The amino-alkyl cytokinins 5a and 6a were reacted witha 50% excess of the ethyl acetate solution of this ester, as for the preparation of the dexamethasoneconjugates, yielding the <strong>[58-85-5]biotin</strong> conjugates 5c and 6c purified by TLC (solvent D).
With dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; at 20℃; for 16h;
The reaction mixture for 12 synthesis was prepared by dissolving 7 (0.098 g, 0.58 mmol), Et3N (82 muL, 0.58 mmol) and (+)-<strong>[58-85-5]biotin</strong> N-hydroxysuccinimide ester [11; 0.19 g, 0.55 mmol; synthesized from (+)-<strong>[58-85-5]biotin</strong> (8)] in 5 mL of DMF while stirring at rt for 5 h. The final reaction mixture was concentrated under reduced pressure, recrystallized in isopropyl alcohol (IPA), and washed with ethyl acetate (EA) to obtain the white-colored solid 12 (0.371 g, 80%). Compound 12: 1H NMR (400 MHz) (CD3OD) delta: 4.49 (1H, dd), 4.31 (1H, dd), 2.93 (1H, dd), 2.70 (1H, d), 1.60-1.31 (4H, m), 1.25 (2H, q). 13C NMR (100.67 MHz) (CD3OD) delta: 176.4, 176.1, 63.4, 61.6, 57.0, 52.1, 47.9, 41.0, 40.1, 36.8, 36.5, 29.8, 29.5, 29.4, 26.8, 24.1. HRMS (ESI) calculated for C17H29N7O3, [M+Na]+ 434.1950 (calculated), 434.1947 (found).
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 20℃; for 4h;
(3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) 3 · 83g (20 mmol)N-hydroxysuccinimide (NHS) 2.30 g (20 mmol)And D-<strong>[58-85-5]biotin</strong> (purchased from Aladdin Reagent Co., Ltd.)4.87 g (20 mmol) were mixed in 40 mL of dimethylformamide (DMF).After the reaction was stirred at room temperature for 4 hours, 200 mL of ice water was added,To obtain a <strong>[58-85-5]biotin</strong>-N-succinimidyl ester.13.9 g (120 mmol) of hexamethylenediamine was dissolved in 40 mL of DMF,6.82 g (20 mmol) of the <strong>[58-85-5]biotin</strong>-N-succinimidyl ester was slowly added dropwise,After 24 hours at room temperature, poured into 200 mL of ice water and filtered to give a pale yellow solid,And washed twice with 100 mL of water to give product 4,Yield: 77.1%.
66 mg
With dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; at 20℃; for 18h;
To 0.72 gr of <strong>[58-85-5]Biotin</strong> (2.94 mmol) 40 ml of dimethylformamide were added followed by addition of 1.69 gr NHS (14.68 mmol). To reaction solution 2.00 gr of DCC (14.80 mmol) were added and reaction was stirred for 18 hours at ambienttemperature before completion. Reaction was tested by TLC (TLC mobile phase:80% Ethyl Acetate: 20% Methanol; Staining PMA). Reaction solution was filtered and then diluted with 100 ml of solution (30% Ethyl Acetate: 70% hexane). The product was filtered to get 600 mg of product contain some traces of reagents. Then additional 50 ml of solution was added to get more precipitation. The precipitate was removed by filtration to get 66mg of pure product (tested by TLC and NMR). Then to reaction solution additional 100 ml same solution was added to get more precipitate. After isolation 600 mg of product in mixture with some urea byproduct were isolated. Pure product (66mg) was used for reactions for biological applications.
With dmap; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 12h;
To <strong>[58-85-5]biotin</strong> in CH2CI2 was added N-Hydroxysuccinimide, l-(3-Dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine. The reacting mixture was stirred at room temperature for 12 h. After filtration, the resulting solution of intermediate 7 was collected and used for the next step.
1) Preparation of <strong>[58-85-5]biotin</strong> active ester (B-NHS): <strong>[58-85-5]Biotin</strong> (0.62 g, 2.5 mmol) was dissolved in 40 ml of anhydrous DMF and added to 0.5 ml.TEA triethylamine is mixed and stirred at room temperature in the absence of light in an anhydrous environment;Then 0.51 g (2.5 mmol) of DCC and 0.28 g (2.5 mmol) of NHS were mixed.The mixture was stirred for 24 hours in the dark, and the by-product dicyclohexylurea was removed by filtration, and DMF and TEA were removed by vacuum drying at a low temperature, and the product was precipitated with diethyl ether to obtain <strong>[58-85-5]biotin</strong> active ester B-NHS.
With dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; at 20℃; for 18h;
D-<strong>[58-85-5]Biotin</strong> 58 (1.00 g, 4.09 mmol, 1.0 eq.), 1 ,3- dicyclohexylcarbodiimide (1.10 g, 5.32 mmol, 1.3 eq.) and N- hydroxysuccinimide 59 (518 mg, 4.50 mmol, 1.1 eq.) were suspended in dry dimethylformamide (30 ml_) and stirred at room temperature for 18h. Reaction mixture was filtered through cotton pad in order to remove urea byproduct derived from DCC. Solvents were evaporated under high vacuum. The residue was triturated in diethylether (100 ml_) and collected by filtration through fritte affording compound 60 (1.65g, yield >100%) as a white powder. The compound was used in the next step without any further purification.
With dmap; dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; at 20℃;
General procedure: A solution of <strong>[58-85-5]Biotin</strong> (488 mg, 2.0 mmol, 1.0 equiv) in N, N-dimethylformamide(DMF, 20 ml) was added N-hydroxysuccinimide (NHS, 276 mg, 2.4 mmol, 1.2 equiv) and dicyclohexylcarbodiimide (DCC, 824 mg, 4.0 mmol, 2.0 equiv) and 4-dimethylaminopyridine(DMAP, 610 mg, 5.0 mmol, 2.5 equiv) at room temperature. The solution was stirred overnight, and then the solvent was removed under reduced pressure. The residue was dissolved in isopropanol (40 ml) with ultrasonic. The solution was cool to 4 C and stood for 1 h. The resulted mixture was filtered to afford intermediate a. Intermediate a (341 mg, 1.0 mmol, 1.0 equiv) and 6-aminocaproic acid(157 mg, 1.2 mmol, 1.2 equiv) were dissolved in 20 ml DMF. Triethylamine (0.3 ml,2.16 mmol, 2.16 equiv) was added. The solution was stirred overnight, and then the solvent was removed under reduced pressure. The residue was dissolved in water(25 ml) and formic acid (2 ml) with ultrasonic. The solution was cooled to 4 C. The resulted mixture was filtered to afford compound 1. Intermediate b was obtained according to the similar procedure of preparing intermediate a staring from 1. Compound 2 was obtained according to the similar procedure of preparing compound 1 staring from intermediate b. To a solution of <strong>[58-85-5]biotin</strong> (400 mg, 2.0 mmol, 1.0equiv) and tributylamine (0.64 ml, 2.7 mmol, 1.3 equiv) in DMF (40 ml) was added isobutyl chloroformate (0.32 ml, 2.5 mmol, 1.2 equiv) at room temperature. The reaction mixture was stirred 10 min, and then 3-aminobenzoic acid (548 mg, 4.0 mmol,2.0 equiv) in 40 ml DMF was added slowly to the mixture at 0 C and left to stir for 2 h. The solvent was removed under reduced pressure. The residue was dissolved inwarm ethanol solution (50%, 36 ml). The pH of the mixture was adjusted to 2.0 by hydrochloric acid. The mixture was cooled to 0 C and stood for 12 h. The resulted mixture was filtered to give compound 3. Compounds 4a, 4 b and 4c were obtained according to the similar procedure of preparing 3 starting from 4-aminobenzoic acid, 4-amino-3-methylbenzoic acid and 4-amino-3-fluorobenzoic acid. The <strong>[58-85-5]biotin</strong> derivatives were all white solids. Compound 1, 270 mg, yield 75.6%. Compound 2, 340 mg,yield 72.2%. Compound 3, 465 mg, yield 62.4%. Compound 4a, 483 mg, yield 64.9%. Compound 4b, 600 mg, yield 77.6%. Compound 4c, 465 mg, yield 62.4%.
Biotin derivative 3 (0.29 mmol, 100 mg), Boc-1-amino-3,6-dioxa-8-octanamine (0.44 mmol, 109 mg) and triethylamine (0.59 mmol, 81.0 muL) were dissolved in dry DMF (5 mL). The reaction mixture was stirred overnight at room temperature. DMF was removed, the residue taken up with CH2Cl2 and washed with water. The organic layer was dried with MgSO4 and concentrated to yield the crude product. Column chromatography (CH2Cl2 + MeOH (slowly increasing from 0% to 7.5%)) yielded the desired product 4 in 86% (0.25 mmol, 118 mg) as a white sticky solid.
With dmap; triethylamine; In N,N-dimethyl-formamide;
Preparation of compound 58: To a solution of compound 57 (316 mg, 1.30 mmol) in DMF (1.3 mL) was added biotinDNHS (435 mg, 1.30 mmol), DMAP (16 mg, 0.13 mmol) and Et3N (0.178 mL, 1.30 mmol). The resulting mixture was stirred overnight. When TLC indicated completion of reaction, DMF was removed. The mixture was redissolved in CH2CI2 (30 mL) and was washed with 0.5 M HC1 (2 5 mL), 1 M K2C03 (2 5 mL) and brine (1 x 5 mL). The organic layer was dried over Na2SC>4, filtered and concentrated. The resulting product had enough purity for next step (521 mg, 84%). The crude product was a light yellow solid. Analytical TLC (silica gel 60), 15% MeOH in CH2C12, R/= 0.83; NMR (300 MHz, CDCI3) delta 6.89 (br s, 1H), 6.84 (br s, 1H), 6.16 (br s, 1H), 5.24 (br s, 1H), 4.52- 4.48 (m, 1H), 4.32-4.28 (m, 1H), 3.61 (s, 4H), 3.59-3.53 (m, 4H), 3.43 (q, / = 4.9 Hz, 2H), 3.35-3.28 (m, 2H), 3.14 (q, / = 6.1 Hz, 1H), 2.89 (dd, / = 12.8, 4.7 Hz, 1H), 2.73 (d, / = 12.8 Hz, 1H), 2.23 (t, / = 7.4 Hz, 2H), 1.78-1.67 (m, 4H), 1.50-1.40 (m, 11H); 13C NMR (75 MHz, CDCI3) delta 173.3, 164.2, 155.9, 79.1, 69.9, 61.7, 60.1, 55.6, 40.3, 40.2, 39.0, 35.8, 28.3, 28.2, 28.0, 25.5. IR (CH2C12) 3035, 2927, 2855, 1799, 1499, 1447 cm"1; LRMS (ESI) 497.0 (M++Na), 475.1 (M++H); HRMS (EI) calcd for C21H38O6N4S (M+) 474.2507, found 474.2493.
In N,N-dimethyl-formamide for 1h; Inert atmosphere;
21.2 Step 2:
To a solution of 4,7,10-trioxododecanel,13-diamine (6.7 g, 30.4 mmol) in anhydrous DMF(100 mL) was drop-wise added a solution of 2 (2.0 g, 5.86 mmol) in dry DMF (50 mL) over a period of 30 min under N2. The resulting thick white suspension was stirred for 30 min. The precipitate was filtered and washed with DMF. The combined filtrate was concentrated and diethyl ester was added. The precipitate (sticky solid) was collected and purified by flash chromatography (DCM/ MeOH = 5/1) to give desired compound 3 (2.44 g, yield 93%, M+H+ =448.5)
67%
With triethylamine In N,N-dimethyl-formamide at 20℃; for 72h;
61.5%
In N,N-dimethyl-formamide at 0℃; for 2h;
Synthesis of 3
Biotin succinimidylester (500 mg, 1.5 mmol) in 5 mL DMF was added dropwise by syringe to a solution of Diethylene Glycol Bis(3-aminopropyl) Ether (0.98 mL, 4.5 mmol) in 5 mL DMF at 0. After addition, the reaction mixture was stirred at 0 for 2h. The mixture was concentrated and the crude product was purified by silica gel column chromatography (CH2Cl2: MeOH = 50:1) to get the product 3 as colorless oil 412 mg, 61.5%. 1H NMR (400 MHz, CDCl3) δ 4.55-4.48 (m, 1H), 4.34-4.31 (m, 1H), 3.64-3.55 (m, 25H), 3.33-3.32 (m, 2H), 3.15-3.14 (m, 1H), 2.89-2.72 (m, 6H), 2.23-2.20 (m, 2H), 1.79-1.72 (m, 10H), 1.46-1.42 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 173.32, 163.98, 70.55, 70.49, 70.35, 70.13, 69.99, 69.83, 69.60, 69.46, 61.93, 60.11, 55.82, 40.62, 39.54, 37.26, 35.78, 32.75, 28.87, 28.04, 25.65. HRMS(ESI): m/z [M+H+] calculated for C20H35N4O5S: 447.2641, found: 447.2640.
15 mg
In dimethyl sulfoxide at 20℃; for 0.25h;
1 Synthesis of Biotin-PEG3-NH2
Synthesis of Biotin-PEG3-NH2 (2) Biotin (1, 13.5 mg, 55.3 pmol, 1 eq) was dissolved in 100 pL of dimethylsulfoxide (DMSO) and diisopropylethylamine (DIEA) was added (19.3 jtL, 2 eq). O-(N-succinimidyl)-N,N,N’,N’-tetramethyl-uronium (TSTU, 15.0 mg, 0.9 eq) was added and the clear solution was stirred for 10 minutes at room temperature to form the biotin-NHS estet 4,7,10-Trioxa-1,13-tride- canediamine (18 mg, 1.5 eq) was dissolved in 200 pL of dry DMSO and the biotin-NHS was added drop wise under vigorous stirring over 5 minutes. The mixture was stirred for a thrther 10 minutes at room temperature. 1.5 mL of diethyl ether was added to the clear solution and the resulting suspension was centrifuged. The supernatant ether phase was discarded and the remaining oil was purified by preparative RP-HPLC (5 mL/min, 10-100% acetonitrile in 0.1% TFA in H20). The fractions containing the product were lyophilized to afford 2 as a yellowish liquid (15mg, 67%). [HRMS (ESI):447.42 mlz (447.7 mlz expected). ‘H NMR (400 MHz, DMSO) ö 7.78 (t, 1H, J=5.6 Hz), 7.70 (m, 2H), 6.42 (d, 1H, J=0.2 Hz), 6.37 (m, 1H), 4.31 (m, 1H), 4.13 (dd, 1H, J=7.6, 4.5 Hz), 3.50 (m, 1 1H), 3.39 (t, 2H, J=6.3 Hz), 3.08 (m, 3H),2.85 (m, 3H), 2.05 (t, 2H, J=7.4 Hz), 1.78 (m, 2H), 1.61 (m, 4H), 1.49 (m, 3H), 1.30 (m, 2H). ‘3C NMR (101 MHz, DMSO) ö 172.4, 163.2, 70.2, 70.1, 70.0, 70.0, 68.6, 67.8, 61.5, 59.7, 55.9, 40.3,37.3,36.2,35.7,29.9,28.7,28.5,27.7, 25.8.]
With triethylamine; In tetrahydrofuran; at 20℃; for 18h;
Compound 60 (850 mg, 2.49 mmol, 1.0 eq.) was solubilized in dry dimethylformamide (20 ml_). After triethylamine (756 mg, 7.47 mmol, 1.0 nriL, 3.0 eq.) and propargylamine 61 (274 mg, 4.98 mmol, 0.3 ml_, 2.0 eq.) addition, reaction mixture was stirred at room temperature for 18h. Solvents were evaporated under vacuum and the residue was purified by flash chromatography [Biotage ; column AIT 40g; eluant: Cyclohexane / EtOAc; gradient: 100/0 · 100/0 (3 CV), 100/0 · 0/100 (12CV) and then 0/100 -> 0/100 (3CV)]. A second elution was done [CH2CI2 / MeOH; gradient: (3CV)], affording compound 62 (690 mg, yield 98%) as a ligh brown solid.
97%
In N,N-dimethyl-formamide; at 20℃; for 12h;
Dissolve N-hydroxysuccinimide biotin (100mg, 0.293mmol) in 4mL DMF,Additional propargylamine (21 mg, 0.381 mmol) was added and stirred at room temperature for 12 hours.The reaction was then stopped and dried.The residue was separated and purified by 200-300 mesh silica gel column chromatography [eluent is V (ethyl acetate): V (methanol) = 10: 0 to V (ethyl acetate): V (methanol) = 10: 1], 80 mg of pure white solid powder was obtained with a yield of 97%.
With triethylamine; In N,N-dimethyl-formamide; for 14h;
General procedure: To a solution of 5 [ 37 ]. (100 mg, 320 μmol. 1.0 equiv.) in DMF (8 mL), amino alkyne 6b [ 49 ]. (53.2 mg, 640 μmol, 2.0 equiv.) and NEt3 (160 mg, 219 μL, 1.6 mmol, 5.0 equiv.) was added. After stirring for 14 h the reaction mixture was concentrated in vacuo. Purification by column chromatography (CH2Cl2/MeOH 8:1) gave the biotin alkyne 7b (82.5 mg, 270 μmol, 84%) as a colorless solid
D-Biotin (1 gm) was dissolved at 48.8 mg/ml in DW. NHS (0.5 gm) was dissolved at 92 mg/ml in DMF and added to the biotin. Solid EDAC at ~ 4-fold excess (3.1 gm) was added as powder to the NHS/Biotin mix, while stirring rapidly. The reaction was allowed to proceed for 20 mins. At this stage complexes of Lysyl-Copper-Lysine at 100 mg/ml in 1% NaHCCbeta (5.8 gm in 58 ml) was added to Biotin-NHS and reacted overnight. The reaction mix was precipitated with acetone to 70% and then the pellet was dissolved in 100 ml 50 mM EDTA. The soluble material was loaded onto Dowex 1 X 2 resin. All of the Lys-C-Lys stuck to the column. The biocytin in the flow through was loaded onto XAD- 16 resin and washed with DW. Residual Lys-Cu-Lys washed through. The biocytin was eluted with 4 x 100 ml 75% MeOH. After which the product was rotary evaporated, resuspended in distilled water and lyophilized. Mass spectral analysis of biocytin showed a Molecular ion of 373.
With N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 20℃; Inert atmosphere;
92%
In N,N-dimethyl-formamide at 20℃; for 2h;
6.2.2
Step 2. Dissolve the product from the previous step (0.12g, 0.36mmol, 1.0equiv.) In N, N-dimethylformamide (3mL), Heat until all solids are dissolved.Add ethylenediamine (0.5mL, 3.6mmol, 20.0equiv.),The reaction was stirred at room temperature for 2h. Ether (50 mL) was added, the precipitate was settled using centrifugation, and the ether was removed. The precipitate was re-dispersed in ether, and the dispersion was repeated and centrifuged 3 times to wash the precipitate. After vacuum drying, a white solid (94.7 mg) was obtained in a yield of 92%.
92%
In N,N-dimethyl-formamide at 20℃; for 2h;
89.11%
With triethylamine In N,N-dimethyl-formamide at 20℃; for 12h;
In DMF (N,N-dimethyl-formamide) at 20℃;
1
Biotin-NHS (1 mmol) was dissolved in DMF and then mixed with ethylene diamine (20 mmol) plus 500 μg pyridine and allowed to react at room temperature overnight. The reaction was confirmed by thin layer chromatograph (TLC) analysis (silica gel plate, 2-propanol/chloroform=70/30 vol %). The product was dissolved in 1 N NaOH and then biotin-amine was precipitated and collected by filtration. The product was also dissolved in 2 N HCl and then filtered to remove insoluble materials. The solution was freeze-dried for 3 days.
With triethylamine In N,N-dimethyl-formamide at 50℃; for 8h; Inert atmosphere;
XV.B Synthesis of DOTA-Biotin conjugates
This NHS ester was reacted in situ with DOTA-aniline to give the desired product which was purified by preparative HPLC. More specifically, a mixture of D-alanine (78 mg, 0.88 mmol, 1.2 equivalents), biotin-NHS ester (250 mg, 0.73 mmol, 1.0 equivalent), triethylamine (0.30 ml, 2.19 mmol, 3.0 equivalents) in DMF (4 ml) was heated at 110° C. for 30 minutes. The solution was cooled to 23° C. and evaporated. The product solid was acidified with glacial acetic acid and evaporated again. The product biotinyl-D-alanine, a white solid, was suspended in 40 ml of water to remove excess unreacted D-alanine, and collected by filtration. Biotinyl-D-alanine was obtained as a white solid (130 mg, 0.41 mmol) in 47% yield.
With triethylamine In N,N-dimethyl-formamide
XV.B Synthesis of DOTA-Biotin Conjugates
This NHS ester was reacted in situ with DOTA-aniline to give the desired product which was purified by preparative HPLC. More specifically, a mixture of D-alanine (78 mg, 0.88 mmol, 1.2 equivalents), biotin-NHS ester (250 mg, 0.73 mmol, 1.0 equivalent), triethylamine (0.30 ml, 2.19 mmol, 3.0 equivalents) in DMF (4 ml) was heated at 110° C. for 30 minutes. The solution was cooled to 23° C. and evaporated. The product solid was acidified with glacial acetic acid and evaporated again. The product biotinyl-D-alanine, a white solid, was suspended in 40 ml of water to remove excess unreacted D-alanine, and collected by filtration. Biotinyl-D-alanine was obtained as a white solid (130 mg, 0.41 mmol) in 47% yield.
With triethylamine In N,N-dimethyl-formamide
XI.B A.
This NHS ester was reacted in situ with DOTA-aniline to give the desired product which was purified by preparative HPLC. More specifically, a mixture of D-alanine (78 mg, 0.88 mmol, 1.2 equivalents), biotin-NHS ester (250 mg, 0.73 mmol, 1.0 equivalent), triethylamine (0.30 ml, 2.19 mmol, 3.0 equivalents) in DMF (4 ml) was heated at 110° C. for 30 minutes. The solution was cooled to 23° C. and evaporated. The product solid was acidified with glacial acetic acid and evaporated again. The product biotinyl-D-alanine, a white solid, was suspended in 40 ml of water to remove excess unreacted D-alanine, and collected by filtration. Biotinyl-D-alanine was obtained as a white solid (130 mg, 0.41 mmol) in 47% yield.
With triethylamine In N,N-dimethyl-formamide
XXI.B Synthesis of DOTA-Biotin Conjugates
This NHS ester was reacted in situ with DOTA-aniline to give the desired product which was purified by preparative HPLC. More specifically, a mixture of D-alanine (78 mg, 0.88 mmol, 1.2 equivalents), biotin-NHS ester (250 mg, 0.73 mmol, 1.0 equivalent), triethylamine (0.30 ml, 2.19 mmol, 3.0 equivalents) in DMF (4 ml) was heated at 110° C. for 30 minutes. The solution was cooled to 23° C. and evaporated. The product solid was acidified with glacial acetic acid and evaporated again. The product biotinyl-D-alanine, a white solid, was suspended in 40 ml of water to remove excess unreacted D-alanine, and collected by filtration. Biotinyl-D-alanine was obtained as a white solid (130 mg, 0.41 mmol) in 47% yield.
With triethylamine In N,N-dimethyl-formamide
XX.B Synthesis of DOTA-Biotin Conjugates
This NHS ester was reacted in situ with DOTA-aniline to give the desired product which was purified by preparative HPLC. More specifically, a mixture of D-alanine (78 mg, 0.88 mmol, 1.2 equivalents), biotin-NHS ester (250 mg, 0.73 mmol, 1.0 equivalent), triethylamine (0.30 ml, 2.19 mmol, 3.0 equivalents) in DMF (4 ml) was heated at 110° C. for 30 minutes. The solution was cooled to 23° C. and evaporated. The product solid was acidified with glacial acetic acid and evaporated again. The product biotinyl-D-alanine, a white solid, was suspended in 40 ml of water to remove excess unreacted D-alanine, and collected by filtration. Biotinyl-D-alanine was obtained as a white solid (130 mg, 0.41 mmol) in 47% yield.
With triethylamine In N,N-dimethyl-formamide
XI.B Synthesis of DOTA-Biotin Conjugates
This NHS ester was reacted in situ with DOTA-aniline to give the desired product which was purified by preparative HPLC. More specifically, a mixture of D-alanine (78 mg, 0.88 mmol, 1.2 equivalents), biotin-NHS ester (250 mg, 0.73 mmol, 1.0 equivalent), triethylamine (0.30 ml, 2.19 mmol, 3.0 equivalents) in DMF (4 ml) was heated at 110° C. for 30 minutes. The solution was cooled to 23° C. and evaporated. The product solid was acidified with glacial acetic acid and evaporated again. The product biotinyl-D-alanine, a white solid, was suspended in 40 ml of water to remove excess unreacted D-alanine, and collected by filtration. Biotinyl-D-alanine was obtained as a white solid (130 mg, 0.41 mmol) in 47% yield.
1
Synthesis of adamantane-desferrioxamine B (Desferadax)NaOH (0.036 g, 0.90 mmol) and Af-[(l-adamantylcarbonyl)oxy]-succinimide (NHS-adamantane, 0.25 g, 0.90 mmol) were dissolved in MeOH (14 mL, 40 0C). DFOB-mesylate (0.29 g, 0.447 mmol) was added with stirring and the solution left at 40°C for 4 h. Alternatively, NaOH was added to DFOB mesylate prior to the addition of iV-[(l-adamantylcarbonyl)oxy]-succmimide (NHS-adamantane) as described for related NHS-esters, below. The volume of the solution was reduced in vacuo until the solution was slightly cloudy at which point, an aliquot (5 mL) of water was added to the solution which resulted in the formation of a precipitate. The suspension was kept in a refrigerator (4 0C) overnight. The white precipitate was filtered and washed with small amounts of water and air dried. Yield: 0.298 g, 92 %.
Stage #1: deferoxamine mesylate With sodium hydroxide In methanol
Stage #2: biotin N-Hydroxysuccinimide ester In methanol at 55℃; for 4h;
1
Synthesis ofhiotin-DFOB, benzo- 15-crown-5-4-carboxylate-DFOB and 4- methylphenoxyacetate-desferrioxamine BThis method was modified from a published procedure [8]. Desferoxamine B mesylate (0.293 mg, 0.447 mmol) and NaOH (18 mg, 0.447 mmol) were dissolved in MeOH (20 mL). An approximate 2-fold molar excess of the solid NHS-activated ligands (NHS-biotin, NHS-benzo-15-crown-5-4-carboxylate or NHS-4-methylphenoxyacetate) were added to the DFOB/NaOH solution and the mixtures were heated in a water bath at 55 0C for 4 h. The volumes of the solution were reduced via rotary evaporation (45 0C, 30 min) until a precipitate formed. The products were filtered, washed with water (~2 mL) and dried overnight. Yields: biotin-DFOB (68%); benzo- 15-crown-5 -4-carboxylate- DFOB (74%), 4-methylphenoxyacetate-DFOB (71%).
55%
With sodium hydroxide In methanol at 60℃; Inert atmosphere;
biotin-NHS (1 eq., 388 mg, 1.14 mmol) was dissolved in the anhydrous DMF (8.54 mL). To the resulting solution was added 6-aminocaproic acid (1 eq., 149 mg, 1.14 mmol) andthe mixture was stirred at r.t. overnight and then concentrated to yield 1:1 mixture of the product and NHS. To the product was then added 95:1 :4 mixture of EtOH-AcOH-H20. The resulting suspension was heated to boiling, filtered, and left to crystallize to yield compound 9 (305 mg, 0.854 mmol, 75 percent) as a white fluffy solid. The structure of 9 was confirmed by ESI-MS analysis (Method 1).ESI-MS m/z: 358.3 [M+H]
1.455 g
With sodium hydrogencarbonate; In water; N,N-dimethyl-formamide; at 20℃; for 16h;
epsilon-Aminocaproic acid (966mg, 8.4mmol) was dissolved in 30mL 1M aqueous NaHCO3 solution. Then N-hydroxysuccinimidobiotin (3.09g) in 35mL DMF was added dropwise to the solution and the mixture was stirred at room temperature for 16h. The solution was concentrated under reduced pressure to remove partial solvent and 150mL aqueous citric acid (100g/L) was then added and stirred at 60°C for 30min. The precipitate was collected by filtration and washed with distilled water. The precipitate was dissolved in a mixture of isopropanol and water (8:2, v/v) and kept at 4°C to give pure 3 (1.455g, 50percent) as a yellowish crystal; IR cm?1 (KBr): 3200?3500, 3070, 2932, 2859, 1696, 1644, 1543, 1475, 1391, 1324, 1265, 1118; 1H NMR (600MHz, DMSO-d6): delta 7.75 (1H, t, J=5.4Hz, C6?NH), 6.45 (1H, s, C13?NH), 6.38 (1H, s, C14?NH), 4.35?4.27 (1H, m, H-14), 4.12 (1H, m, H-13), 3.09 (1H, m, H-12), 3.00 (1H, m, H-6) 2.82 (1H, dd, J=12.4, 5.1Hz, H-15a), 2.57 (1H, d, J=12.4Hz, H-15b), 2.19 (2H, t, J=7.4Hz, H-2), 2.04 (2H, t, J=7.3Hz, H-8), 1.20?1.67 (14H, m). 13C NMR (150MHz, DMSO-d6): delta 171.84 (C-1, C-7), 162.74 (C-16), 61.06 (C-13), 59.20 (C-14), 55.44 (C-12), 39.68 (C-15), 38.25 (C-6), 35.21 (C-8), 29.05 (C-2), 28.92, 28.21, 28.04, 26.04, 25.73, 25.36.
368.3 mg
With sodium hydrogencarbonate; In water; acetone; at 20℃; for 12h;
General procedure: Biotin-NHS was ammonolyzed withlinkers to afford biotin-linker products [Biotin-AAn-COOH (n = 1, 3, 5, and 7) orBiotin-PEG(n+1)-COOH (n = 1, 2 and 3)]. The linker [AAn (n = 1, 3, 5, and 7) orNH2-PEG(n+1)-COOH (n = 1, 2 and 3), 1 eq.] was dissolved in 4 mL water solutionrespectively, and 240.0 mg NaHCO3 was added to the solution. The reaction mixturewas stirred and the Biotin-NHS (1 eq.) and acetone (500 ul) were added subsequently.After stirring for 12h at room temperature, the solvent was removed by vacuum, andthe crude was dispersed in water, adjusting the pH by 2M HCl to pH=3. The mixturesolution was filtered, washed by water and concentrated under reduced pressure toachieved Biotin-linker products [Biotin-AAn-COOH (n = 1, 3, 5, and 7) orBiotin-PEG(n+1)-COOH (n = 1, 2 and 3)]2, 3.
With triethylamine; In water; N,N-dimethyl-formamide; at 20℃;
In accordance to a described procedure ( Wang et al., 2012 ), Biotin-OSuc (14) (300 mg, 0.87 mmol) was completely dissolved in DMF (3 mL) with gentle warming (40 C). After slowly cooling the solution to room temperature without recurring precipitation, it was mixed with an aqueous solution (1 mL) containing cystamine dihydrochloride (107 mg, 0.47 mmol) and Et3N (309 muL, 2.10 mmol). The reaction proceeded at room temperature overnight with stirring. The white solid was filtered and washed with water and EtOH to obtain N,N'-(disulfanediylbis(ethane-2,1-diyl))bis(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanamide) (7) as a white solid (250 mg; 87%); 1H NMR (400 MHz, DMSO-d6): delta 8.08-8.04 (br m, 2H), 6.48 (s, 2H), 6.43 (s, 2H), 4.39-4.34 (m, 2H), 4.22-4.17 (m, 2H), 3.42-3.35 (m, 2H), 3.19-3.14 (m, 2H), 2.88-2.81 (m, 6H), 2.64 (d, J = 12.9 Hz, 2H), 2.13 (t, J = 7.3 Hz, 4H), 1.72-1.53 (m, 8H), 1.40-1.34 (m, 4H); 13C NMR (100 MHz, DMSO-d6): delta 25.7, 28.5, 28.7, 35.6, 37.8, 38.3, 40.3, 55.9, 59.7, 61.5, 163.2, 172.7.
With triethylamine; In N,N-dimethyl-formamide; at 20℃; for 24h;
Biotin succinimidyl ester (7.2 mg, 21.6 mmol) and 6-amino-6-deoxy-beta-CD (20 mg, 17.6 mmol) were dissolved in dry DMF (4 ml), TEA (3 ml, d 0.72) was added, and the reaction mixture was stirred at room temperature for 24 h. The solvent was evaporated to dryness in vacuo. The product was freeze-dried to afford pure product as a white powder. Yield: 72percent.
[0074] To 2 grams of biotin was added 2 eq of N,N?-diisopropylcarbodiimide and 3 eq of Nhydroxysuccinimide in 0.2 M DMF at 45 C to yield compound 6. One eq of t-Boc protected PEG amine (3) was then added in methylene chloride at room temperature to give compound 7 and deprotected using TFA to afford compound 8, followed be activation using 2 eq of N,N?-diisopropylcarbodiimide and 3 eq of N-hydroxysuccinimide to form its activated ester, compound 9. G3 dendrimer DNT-296 was added to compound 9 in methanol at room temperature to yield compound 10.
With triethylamine In N,N-dimethyl-formamide at 20℃; for 1h; Inert atmosphere;
Synthesis of 2
D-Biotin (500 mg, 2.1 mmol) and TSTU (742.5 mg, 2.5 mmol) were dissolved in 5 mL dry DMF, followed by addition of dry TEA (0.55 mL, 4.1 mmol) by syringe. The reaction was stirred under argon at room temperature for 1h. The mixture was concentrated in vacuo and extracted with 50 mL CH2Cl2, washed with 10 mL 1 M HCl and dried over Na2SO4. The mixture was concentrated in vacuo and the crude product was purified by column chromatography (CH2Cl2: MeOH=50:1) to afford the product 2 as white solid 650 mg, 90.7%. 1H NMR (400 MHz, DMSO) δ 6.44 (d, J = 28.4 Hz, 1H), 4.31 (s, 1H), 4.16 (s, 1H), 3.11 (d, J = 3.2 Hz, 1H), 2.83 (bs, 5H), 2.68 (t, J = 6.8 Hz, 1H), 2.60-2.50 (m, 1H), 1.63 (d, J = 15.6, 6.4 Hz, 3H), 1.51-1.42 (m, 2H). 13C NMR (100 MHz, DMSO) δ 170.76, 169.43, 163.20, 61.46, 59.63, 55.71, 40.36, 30.45, 28.29, 28.05, 25.91, 24.77. HRMS(ESI): m/z [M+H+] calculated for C14H20N3O5S: 342.1124, found: 342.1120.
With triethylamine In dimethyl sulfoxide at 20℃; for 0.5h;
With N-ethyl-N,N-diisopropylamine In dimethyl sulfoxide at 20℃; for 0.166667h;
1
Synthesis of Biotin-PEG3-NH2 (2) Biotin (1, 13.5 mg, 55.3 pmol, 1 eq) was dissolved in 100 pL of dimethylsulfoxide (DMSO) and diisopropylethylamine (DIEA) was added (19.3 jtL, 2 eq). O-(N-succinimidyl)-N,N,N’,N’-tetramethyl-uronium (TSTU, 15.0 mg, 0.9 eq) was added and the clear solution was stirred for 10 minutes at room temperature to form the biotin-NHS estet 4,7,10-Trioxa-1,13-tride- canediamine (18 mg, 1.5 eq) was dissolved in 200 pL of dry DMSO and the biotin-NHS was added drop wise under vigorous stirring over 5 minutes. The mixture was stirred for a thrther 10 minutes at room temperature. 1.5 mL of diethyl ether was added to the clear solution and the resulting suspension was centrifuged. The supernatant ether phase was discarded and the remaining oil was purified by preparative RP-HPLC (5 mL/min, 10-100% acetonitrile in 0.1% TFA in H20). The fractions containing the product were lyophilized to afford 2 as a yellowish liquid (15mg, 67%). [HRMS (ESI):447.42 mlz (447.7 mlz expected). ‘H NMR (400 MHz, DMSO) ö 7.78 (t, 1H, J=5.6 Hz), 7.70 (m, 2H), 6.42 (d, 1H, J=0.2 Hz), 6.37 (m, 1H), 4.31 (m, 1H), 4.13 (dd, 1H, J=7.6, 4.5 Hz), 3.50 (m, 1 1H), 3.39 (t, 2H, J=6.3 Hz), 3.08 (m, 3H),2.85 (m, 3H), 2.05 (t, 2H, J=7.4 Hz), 1.78 (m, 2H), 1.61 (m, 4H), 1.49 (m, 3H), 1.30 (m, 2H). ‘3C NMR (101 MHz, DMSO) ö 172.4, 163.2, 70.2, 70.1, 70.0, 70.0, 68.6, 67.8, 61.5, 59.7, 55.9, 40.3,37.3,36.2,35.7,29.9,28.7,28.5,27.7, 25.8.]
With triethylamine In N,N-dimethyl-formamide at 30℃; for 3h; Inert atmosphere;
(2S,7R,12R,17S)-2-amino-17-carboxy-7,12-bis((carboxymethyl)carbamoyl)-5,14,19-trioxo-23-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-9,10-dithia-6,13,18-triazatricosanoic acid[ No CAS ]
5-(((R)-1-((carboxymethyl)amino)-3-(nitrosothio)-1-oxopropan-2-yl)amino)-5-oxo-2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)pentanoic acid[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
43%
With triethylamine In tetrahydrofuran; water at 0℃; for 2h; Darkness;
43%
With triethylamine In tetrahydrofuran; water at 0℃; for 2h; Darkness;
II Biotin-GSNO. 5-(((R)-1-((carboxymethyl)amino)-3-(nitrosothio)-1-oxopropan-2-yl)amino)-5-oxo-2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)pentanoic Acid (16)
A 25 mL scintillation vial was charged with 11 mg of Biotin-NHS ester (30 μmol, 1.1 eq), 10 mg S-nitroso-glutathione (Cayman, 30 μmol, 1 eq.), and 17 μL triethylamine (119 μmol, 4 eq) in water/THF (2 mL) and stirred in the dark for 2 hours. The mixture was then purified by HPLC in the dark. The purified product (retention time 13 minutes) was lyophilized in the dark to obtain compound 16 in 43% yield. HRMS (ESI positive): Calculated m/z: [M+Na]+=585.1408, Found m/z [M+Na]+=585.1407. FIG. shows HPLC trace of reaction mixture of biotin-GSNO, compound 16; Peak at minute 13.4 represents product as confirmed by MS analysis.
2. Synthesis of Biotin-Jeffamine intermediate (Scheme II) . 2, 2 '- (Ethylenedioxy) bis (ethylamine) (Sigma 385506, henceforth referred to as jeffamine) (0.9 mL, 5.8 mmol) was dissolved in 500 mL acetonitrile (MeCN, Sigma 34888) . Biotinyl-N-hydroxysuccinimide (B-NHS, 0.3 g, 0.88 mmol) was dissolved in MeCN (100 mL) , this solution was added into a dropping funnel. The B-NHS solution was then added dropwise to the jeffamine solution and stirred overnight, at 0-5 C under N2. The reaction was filtered, the solid washed with MeCN (2x 10 mL) and then with diethyl ether (2 x 10 mL) . Finally pulled dry under N2 to yield a white hygroscopic solid. TLC (10% MeOH/DCM + 5 dps NH4OH ) does not show apparent starting material after reaction. The solid was dissolved in MeOH (30 mL) at 40 C, dry, loaded on silica and eluted with 10 % MeOH/DCM + 1 % Et3N. Fractions 25-45 were collected and evaporated to a yellow solid/gel. Final yield: 0.257 g (~ 80% final efficiency) . This product is henceforth referred to as BJl .
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 12h;
Compound 7. N,N-dimethylformamide (400 muL), Biotin NHS (72 mg, 0.211 mmol), and N,N-diisopropylethylamine (33 mg, 0.25 mmol) were added to 15 (42 mg, 0.19 mmol) for 12 h at RT. The reaction was concentrated under reduced pressure and purified on silica gel using flash chromatography (2-5% methanol in CH2Cl2) to afford the product as an oil (84 mg, 0.189 mmol, 98% yield).
With triethylamine In N,N-dimethyl-formamide at 20℃;
With N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide
11
Probe substrate BP was prepared as shown above, starting by making the NHS ester of biotin carboxylic acid (i.e., compound 21 from Example 1). BP is formed by preparing the amide of the NHS ester (compound 21) by contacting the NHS ester with an amine (i.e., tyramine) in the presence of a non-nucleophilic based (DIPEA).
Stage #1: N-BOC-1,2-diaminoethane; biotin N-Hydroxysuccinimide ester In N,N-dimethyl-formamide at 20℃;
Stage #2: With hydrogenchloride In 1,4-dioxane; methanol
1 iV-(2-aminoethyl)-5-(2-oxo-l,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl)pentanamide (22)
Neat tert-butyl Ar-(2-aminoethyl)carbamate was added to a suspension of NHS-ester 21 in anhydrous DMF (8.0 mL) and the reaction stirred at rt overnight. Upon consumption of starting material EtzO was flowed in the mixture chilled to -20 °C overnight. The resultant precipitate was collected via vacuum filtration and dried under suction to yield Boc-protected amine (1.18 g, 78%) as a white powder. The intervening carbamate was stirred in MeOH containing 4M HC1 to provide the amine after evaporation of the solvent, which was taken on without further purification. lH NMR (500 MHz, DMSO-d6) d 6.45 (s, 1H), 6.39 (s, 1H), 4.30 (dd, J= 7.7, 5.1 Hz, 1H), 4.13 (ddd,J= 7.8, 4.4, 1.9 Hz, 1H), 3.28 (q, J= 6.2 Hz, 2H), 3.10 (ddd,J= 8.6, 6.1, 4.4 Hz, 1H), 2.86 - 2.78 (0269) (m, 3H), 2.57 (d, J= 12.4 Hz, 1H), 2.10 (t, J= 7.5 Hz, 2H), 1.65 - 1.41 (m, 4H), 1.39 - 1.22 (m, 2H). 13C NMR (125 MHz, DMSO-d6) d 172.78, 162.75, 61.05, 59.22, 55.42, 38.59, 36.39, 35.16, 28.26, 28.08, 25.03.
Stage #1: 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22-heptaoxa-4-azapentacosan-25-oic acid; C33H44N5O10Pol With O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 20℃;
Stage #2: With piperidine In N,N-dimethyl-formamide at 20℃;
Stage #3: biotin N-Hydroxysuccinimide ester Further stages;
A portion of the resin-bound intermediate 7 (1.0 g, 0.70 mmol, 1.00equiv.) was swollen in 20 mL DMF for 20 min. The solvent was removedby filtration. A solution of Fmoc-PEG6-COOH (1.62 g, 2.80 mmol, 4.00equiv.), HBTU (1.06 g, 2.80 mmol, 4.00 equiv.) and DIEA (0.49 mL,2.80 mmol, 4.00 equiv.) in DMF (20 mL) was added to the resin. Theresulting mixture was agitated at room temperature overnight.Completion of reaction was confirmed with ninhydrin test. The supernatantwas removed by filtration. The resin was then washed with DMF(15 mL × 3), followed by agitation with 20% piperidine in DMF (15 mL,10 min × 2). The supernatant was removed by filtration, and the resinwas washed with DMF (15 mL × 3). The obtained resin-bound MDPPEG6intermediate 8 was divided into portions for further modifications.4.1c. Synthesis of MDP-PEG6-biotin (11).A solution of biotin-NHS ester (0.48 g, 1.40 mmol, 4.00 equiv.) andDIEA (0.24 mL, 1.40 mmol, 4.00 equiv.) in DMF (15 mL) was added toresin-bound MDP-PEG6 intermediate 8 (0.5 g, 0.35 mmol, 1.00 equiv.).The resulting mixture was agitated at room temperature for 2 h.Ninhydrin tested indicated completion reaction. The supernatant wasremoved by filtration. The resin was washed with DMF (15 mL × 3) and DCM (15 mL × 3).A portion of the obtained resin-bound intermediate 9 (50 mg) wasagitated with 95% TFA in DCM (1 mL) at room temperature for 1 h. Thesupernatant was collected by filtration, and the resin was washed withDCM (0.5 mL × 2). The combined filtrate was concentrated underreduced pressure to dryness. The residue was taken up with 4 mL 50%methanol/water, and Pd/C (10%, 40 mg) was added. The resultingmixture was agitated under a hydrogen atmosphere overnight, followedby filtration. The filtrate was concentrated on a rotary evaporator todryness. The residue was purified by RP-HPLC to afford MDP-PEG6-Biotin (11) as a white solid (14 mg, yield 32%).
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