* 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.
Stage #1: With sodium hydroxide In water at 0 - 5℃; for 2 h; Stage #2: With hydrogenchloride In water at 0℃; for 12 h;
L-Valine (200 g, 1.7 mol eq) is dissolved in water (500 mL) followed by the addition of NaOH (30percent, 170 mL). The mixture is cooled to 0°-5°C followed by the addition of acetic anhydride (32 ml, 1.4 eq. ). Sodium hydroxide (30percent, 34 mL) is added while keeping the temperature at 0°-5°C. Acetic anhydride and 30percent NaOH alternate additions are repeated six time while keeping the temperature (acetic anhydride, 6 x 32 mL; 30percent NaOH 6 x 34 mL). After all the additions are completed, the mixture is stirred for an additional two hours at 0°C. Hydrochloric acid (32percent, 380 mL) is added to lower the pH below 3 while keeping the temperature at 0°C. The resulting slurry is granulated for 12 hours, filter and the cake washed with HCI (0.1 N, 100 mL). The wet N-acetyl-L-valine was dried to produce 233 g (86percent yield).
Reference：
[1] New Journal of Chemistry, 2002, vol. 26, # 7, p. 834 - 843
[2] Synthetic Communications, 1992, vol. 22, # 2, p. 257 - 264
[3] Patent: WO2005/40097, 2005, A1, . Location in patent: Page/Page column 9
[4] Journal of the American Chemical Society, 2011, vol. 133, # 43, p. 17176 - 17179
[5] Journal of the American Chemical Society, 1956, vol. 78, p. 4636,4642
[6] Tetrahedron, 1991, vol. 47, # 12-13, p. 2169 - 2180
[7] Chemistry - A European Journal, 2003, vol. 9, # 17, p. 4031 - 4045
[8] Patent: WO2015/95227, 2015, A2, . Location in patent: Page/Page column 138
[9] Angewandte Chemie - International Edition, 2017, vol. 56, # 14, p. 3847 - 3851[10] Angew. Chem., 2017, vol. 129, # 14, p. 3905 - 3909,5
[11] RSC Advances, 2018, vol. 8, # 34, p. 19144 - 19151
8
[ 72-18-4 ]
[ 96-81-1 ]
Reference：
[1] Biochemical Journal, 1945, vol. 39, p. 363,366
9
[ 72-18-4 ]
[ 1415322-55-2 ]
[ 616-91-1 ]
[ 1415322-26-7 ]
Reference：
[1] Chemical Research in Toxicology, 2012, vol. 25, # 12, p. 2704 - 2714
10
[ 2185-00-4 ]
[ 72-18-4 ]
[ 1963-21-9 ]
Reference：
[1] Journal of Organic Chemistry, 1972, vol. 37, p. 327 - 329
11
[ 72-18-4 ]
[ 3190-70-3 ]
[ 13588-95-9 ]
Reference：
[1] Journal of Organic Chemistry, 1967, vol. 32, p. 3415 - 3425
12
[ 72-18-4 ]
[ 35150-08-4 ]
Reference：
[1] Tetrahedron Letters, 2007, vol. 48, # 8, p. 1465 - 1468
[2] Journal of Organic Chemistry, 1996, vol. 61, # 23, p. 8207 - 8215
Reference：
[1] Journal of the American Chemical Society, 2011, vol. 133, # 11, p. 3832 - 3835
[2] Medicinal Chemistry Research, 2016, vol. 25, # 6, p. 1148 - 1162
[3] Indian Journal of Chemistry - Section B Organic and Medicinal Chemistry, 2016, vol. 55B, # 10, p. 1239 - 1242
16
[ 72-18-4 ]
[ 77877-19-1 ]
Reference：
[1] Chemische Berichte, 1996, vol. 129, # 11, p. 1361 - 1368
[2] Journal of Organic Chemistry, 1991, vol. 56, # 7, p. 2489 - 2498
[3] Organic Syntheses, 2008, vol. 85, p. 158 - 171
17
[ 72-18-4 ]
[ 3339-44-4 ]
Reference：
[1] Synthesis, 1998, # 1, p. 67 - 70
18
[ 72-18-4 ]
[ 76186-04-4 ]
Reference：
[1] Organic Letters, 2016, vol. 18, # 11, p. 2560 - 2563
[2] European Journal of Organic Chemistry, 2016, vol. 2016, # 4, p. 688 - 692
[3] European Journal of Organic Chemistry, 2017, vol. 2017, # 1, p. 29 - 33
[4] Tetrahedron Letters, 2018, p. 624 - 627
19
[ 72-18-4 ]
[ 5041-09-8 ]
Yield
Reaction Conditions
Operation in experiment
79%
Stage #1: With (R)-Carvone In propan-1-ol at 20 - 190℃; for 0.0833333 h; Sealed tube; Microwave irradiation Stage #2: With hydrogenchloride In propan-1-ol; water at 190℃; for 0.0833333 h; Microwave irradiation
General procedure: General “One-Pot” Procedure for the Decarboxylation of Amino Acids [0045] A magnetic stir bar, 3 mL of n-PrOH, 10 mmol of R-Carvone, and 5 mmol of amino acid were charged to a pressure vessel. The vessel was heated from room temperature to 190° C. over 5 min with stirring. If necessary the reaction vessel was maintained at 190° C. for additional time until the slurry became clear. The vessel was allowed to cool to below the solvent boiling point, carefully vented to release evolved CO2, and 10 mL of 2M HCl was added. The vessel was heated to 190° C. over 5 min with stirring and allowed to cool. The aqueous reaction mixture was washed three times with 25 mL of ether and water solvent distilled off from the hydrochloride salt. The hydrochloride salt was transferred to a vacuum oven and dried overnight at 150° C. and 10 Torr. The hydrochloride salt was then weighed and analyzed via IR and NMR.; 2-methylpropan-1-amine hydrochloride, δH 0.83 6H d J=4, 1.79 1H m J=8, 2.69 2H d J=8; δC 18.8, 26.2, 46.3
General procedure: The esterifications were carried out on L amino acids withthe exception of phenylglycine, the D enantiomer of whichwas used. A mixture of amino acid (0.05 mol), p-toluenesulfonicacid (0.06 mol), benzyl alcohol (0.25 mol) andcyclohexane (30 mL) was refluxed for 4 h using a Dean-Stark apparatus to separate water that was azeotroped outas it formed. The reaction mixture was cooled to roomtemperature and ethyl acetate (80 mL) was added. Afterstirring for 1 h, the precipitate was collected by filtrationand dried to give the corresponding benzyl ester p-toluenesulfonateas a white solid. According to this procedure,the amino acids 1–6 were converted into the correspondingbenzyl ester p-toluenesulfonates 1a–6a. The benzylationof 7 was accomplished in the same manner but in thepresence of more p-toluenesulfonic acid (0.11 mol) to givethe di-p-toluenesulfonate 7a as a white solid. The p-toluenesulfonate8a separated at the end of the reaction as anoil; instead of adding ethyl acetate, the supernatant wasremoved, the oily phase was washed with cyclohexane andthen poured into dichloromethane/aqueous Na2CO3. Afterremoving the water layer and evaporating dichloromethane,the residue was treated with hydrochloric methanol to give the corresponding hydrochloride as a white solid. Thebenzylation of 9 was prolonged over night and, at the endof the reaction, 9a separated as an oil, which was pouredinto dichloromethane/water. After removing the organiclayer, the water phase was made alkaline with NaHCO3 andextracted with ethyl acetate. The organic extract was concentratedto a small volume and a slight excess of p-toluenesulfonicacid was added to precipitate 9a as a white crystallinesolid.
Reference：
[1] Tetrahedron Letters, 2008, vol. 49, # 49, p. 6962 - 6964
[2] Journal of the Indian Chemical Society, 2001, vol. 78, # 3, p. 137 - 141
[3] Amino Acids, 2017, vol. 49, # 5, p. 965 - 974
[4] Chirality, 2012, vol. 24, # 2, p. 188 - 192
[5] European Journal of Medicinal Chemistry, 2018, vol. 157, p. 962 - 977
21
[ 72-18-4 ]
[ 78342-42-4 ]
Reference：
[1] Tetrahedron Asymmetry, 1998, vol. 9, # 2, p. 321 - 327
[2] Angewandte Chemie, 1981, vol. 93, # 9, p. 793 - 795
[3] Tetrahedron, 1983, vol. 39, # 12, p. 2085 - 2092
[4] Organic Process Research and Development, 2005, vol. 9, # 2, p. 185 - 187
Reference：
[1] Indian Journal of Chemistry - Section B Organic and Medicinal Chemistry, 2011, vol. 50, # 9, p. 1157 - 1164
25
[ 64-17-5 ]
[ 72-18-4 ]
[ 17609-47-1 ]
Yield
Reaction Conditions
Operation in experiment
6.1 kg
at 0℃; for 18 h; Reflux; Large scale
5 kg (ie 42.7 mol) of L-valine and4kg anhydrous ethanol added to50L glass reactor, stirring to dissolve, cooling to 0 ~ 10 ,Slowly added dropwise 8.8L of thionyl chloride, dropping was completed, the reaction was warmed to reflux 18h,Concentrated to dry, at 15 ~ 20 ° C was added 15L isopropyl acetate beating 1h,Filtering with suction gave 6.1 kg of an off-white solid, intermediate 1, where R1 is ethyl.
Reference：
[1] Tetrahedron, 2005, vol. 61, # 35, p. 8423 - 8442
[2] Journal of the Indian Chemical Society, 2001, vol. 78, # 3, p. 137 - 141
[3] Journal of the American Chemical Society, 2018, vol. 140, # 22, p. 6818 - 6822
[4] Chemistry of Natural Compounds, 1994, vol. 30, # 2, p. 238 - 244[5] Khimiya Prirodnykh Soedinenii, 1994, # 2, p. 261 - 268
[6] Tetrahedron, 1990, vol. 46, # 15, p. 5325 - 5332
[7] Helvetica Chimica Acta, 1995, vol. 78, p. 109 - 121
[8] Tetrahedron, 2007, vol. 63, # 14, p. 3031 - 3041
[9] European Journal of Medicinal Chemistry, 2011, vol. 46, # 1, p. 11 - 20
[10] Indian Journal of Chemistry - Section B Organic and Medicinal Chemistry, 2011, vol. 50, # 9, p. 1157 - 1164
[11] Green Chemistry, 2012, vol. 14, # 5, p. 1350 - 1356
[12] Tetrahedron, 2012, vol. 68, # 49, p. 10218 - 10229,12
[13] Tetrahedron, 2012, vol. 68, # 49, p. 10218 - 10229
[14] Nucleosides, Nucleotides and Nucleic Acids, 2013, vol. 32, # 4, p. 161 - 173
[15] Journal of Heterocyclic Chemistry, 2013, vol. 50, # 5, p. 1067 - 1070
[16] Patent: CN107056720, 2017, A, . Location in patent: Paragraph 0058; 0059; 0060; 0061
[17] Journal of Medicinal Chemistry, 2018, vol. 61, # 7, p. 2707 - 2724
[18] European Journal of Organic Chemistry, 2018, vol. 2018, # 44, p. 6088 - 6091
26
[ 72-18-4 ]
[ 17609-47-1 ]
Reference：
[1] Liebigs Annalen der Chemie, 1985, # 9, p. 1917 - 1921
27
[ 72-18-4 ]
[ 24347-63-5 ]
Reference：
[1] Angewandte Chemie - International Edition, 2005, vol. 44, # 19, p. 2903 - 2907
[2] Journal of Chemical Research, Miniprint, 1998, # 1, p. 126 - 142
[3] Journal of the Chemical Society - Perkin Transactions 1, 1996, # 12, p. 1427 - 1433
[4] Bulletin of the Chemical Society of Japan, 1987, vol. 60, # 3, p. 1027 - 1036
[5] Journal of Organic Chemistry, 1993, vol. 58, # 7, p. 1646 - 1648
[6] Journal of Organic Chemistry, 1987, vol. 52, # 22, p. 4978 - 4984
[7] Bulletin de la Societe Chimique de France, 1983, vol. 2, # 7/8, p. 185 - 194
[8] Tetrahedron Letters, 1989, vol. 30, # 29, p. 3757 - 3760
[9] Tetrahedron Letters, 1987, vol. 28, # 10, p. 1019 - 1022
[10] Journal of the American Chemical Society, 1990, vol. 112, # 21, p. 7659 - 7672
[11] Tetrahedron Asymmetry, 2011, vol. 22, # 3, p. 294 - 299
[12] Tetrahedron Asymmetry, 2011, vol. 22, # 13, p. 1448 - 1454
[13] Tetrahedron Letters, 2017, vol. 58, # 3, p. 185 - 189
Reference：
[1] Chemistry Letters, 1986, p. 1169 - 1172
31
[ 219916-61-7 ]
[ 72-18-4 ]
[ 57224-50-7 ]
Reference：
[1] Journal of the Chemical Society - Perkin Transactions 1, 1998, # 22, p. 3657 - 3658
32
[ 72-18-4 ]
[ 7732-18-5 ]
[ 75-24-1 ]
[ 3014-80-0 ]
Yield
Reaction Conditions
Operation in experiment
49%
With hydrogenchloride; sodium hydroxide; ammonia In hexane; dichloromethane
EXAMPLE 9 Preparation of (S)-2-amino-3-methylbutanamide hydrochloride Anhydrous ammonia was bubbled through 150 mL of methylene chloride cooled to 0° C. (ice-bath) until the solution was saturated. To this mixture cooled to 5° C. and under N2 was added dropwise trimethylaluminum (136.2 mL of a 2M solution in hexane, 272.4 mmol) available from Aldrich Chemical Co., (Milwaukee, Wis.). The resultant cloudly solution was allowed to warm to room temperature and stirred for 22 h. L-Valine (10.6 g, 90.79 mmol) was added portionwise and stirred for 18 h at room temperature. To this mixture, cooled to 0° C. (ice-water bath), was then added dropwise 190 mL of 6 N HCl until the pH was 2. The resultant mixture allowed to warm and stirred for 2 hours and then made basic (pH=11-12) with 50percent aqueous NaOH. To the basic solution was added 100 mL of methylene chloride and 100 mL of H2 O. The organic layer was separated, dried over magnesium sulfate and evaporated under reduced pressure to dryness. The resultant residue was dissolved in 100 mL of methylene chloride and acidified with HCl gas. The solid that formed was filtered and dried under reduced pressure to give 6.8 g (49percent) of the title compound, mp 258°-260° C. IR (Nujol, cm-1), C=O (1686), N--H (3387, 3241). 1 H NMR and 13 C NMR (CDCl3) consistent with title product. Analysis calculated for C5 H13 ClN2 O: C, 39.35; H, 8.59; N, 18.35; Cl, 23.23; Found: C, 39.82; H, 8.52; N, 18.40; Cl, 23.13. MS: m/e 117 (M+ -Cl).
Reference：
[1] Patent: US5643855, 1997, A,
33
[ 72-18-4 ]
[ 100-51-6 ]
[ 2462-34-2 ]
Yield
Reaction Conditions
Operation in experiment
98%
Stage #1: With hydrogenchloride; iron(III) chloride In 1,2-dichloro-ethane at 20℃; for 0.5 h; Stage #2: for 3 h; Reflux
Will 11 · 5g (0 · lmol) valine,30ml of dichloroethane and 16.2g (0.1mol) of FeCl3 were put into a 250ml three-necked flask,Hydrogen chloride gas is fed at a rate of 1.25 ml/s (hydrogen chloride gas is always fed at this rate for a reaction time of 0.5 h).Reaction at room temperature for 0.5 h. Pass into HC1 total 2250ml, molar amount 0. lmolThen, 11.66 g (0.108 mol) of benzyl alcohol (proline: benzyl alcohol = 1:1.08) was put into a three-necked flask, and hydrogen chloride was introduced at 0.05 ml/s (hydrogen chloride gas was always fed at this rate for a reaction time of 3 h). , Under reflux conditions, distill off the aqueous dichloroethane (ie, the mixture distilled out during azeotropic distillation), and add anhydrous dichloroethane to the reaction system at the same rate to maintain The amount of dichloroethane in the reaction was essentially constant, and the reaction was completed in 3 hours. A total of 120 mL of azeotrope (ie, mixed liquor, ie, aqueous dichloroethane) was distilled out, and HC1 540 mL (0.024 mol) was introduced in total.The reaction solution was filtered while hot to obtain a filter residue containing a catalyst (Note: The "heat filtration reaction liquid" is for the purpose of removing the metal chloride as a catalyst, which can be used in the subsequent step 2).0032] After the filtered reaction solution was cooled to room temperature, the remaining dichloroethane solvent was removed by vacuum distillation (lOmmHg pressure, 40°C temperature), recrystallized at -10°C, and the precipitated solid was cooled with 20 ml of The solution was washed with iced dichloroethane at 0° C., filtered (retain filtrate), and the filter cake was dried at 40° C. for 5 h to obtain 18.20 g of proline benzyl ester hydrochloride. The yield was 75.3percent.[0033] 120ml of the mixed liquor having been distilled out during the water-retention process is obtained, and after dehydration treatment (water is removed with anhydrous sodium sulfate and 5g of anhydrous sodium sulfate is added per 100ml of dichloromethane), anhydrous dichloroethane (118mL) is obtained. Recycle in step 2.The retained filtrate was rotary evaporated (pressure of 10 mmHg, temperature of 40° C.) to 20 ml as a mother liquor for the next cycle (BP, as a raw material, fed into Step 2)..3percent.See also3/4
Reference：
[1] Patent: CN105061283, 2017, B, . Location in patent: Paragraph 0047-0049
[2] Journal of the Chinese Chemical Society, 2009, vol. 56, # 5, p. 1010 - 1017
[3] Bioorganic and Medicinal Chemistry, 2010, vol. 18, # 17, p. 6220 - 6229
[4] Indian Journal of Chemistry - Section B Organic and Medicinal Chemistry, 2011, vol. 50, # 9, p. 1157 - 1164
34
[ 72-18-4 ]
[ 100-44-7 ]
[ 2462-34-2 ]
Reference：
[1] Letters in Organic Chemistry, 2010, vol. 7, # 1, p. 39 - 44
General procedure: The esterifications were carried out on L amino acids withthe exception of phenylglycine, the D enantiomer of whichwas used. A mixture of amino acid (0.05 mol), p-toluenesulfonicacid (0.06 mol), benzyl alcohol (0.25 mol) andcyclohexane (30 mL) was refluxed for 4 h using a Dean-Stark apparatus to separate water that was azeotroped outas it formed. The reaction mixture was cooled to roomtemperature and ethyl acetate (80 mL) was added. Afterstirring for 1 h, the precipitate was collected by filtrationand dried to give the corresponding benzyl ester p-toluenesulfonateas a white solid. According to this procedure,the amino acids 1-6 were converted into the correspondingbenzyl ester p-toluenesulfonates 1a-6a. The benzylationof 7 was accomplished in the same manner but in thepresence of more p-toluenesulfonic acid (0.11 mol) to givethe di-p-toluenesulfonate 7a as a white solid. The p-toluenesulfonate8a separated at the end of the reaction as anoil; instead of adding ethyl acetate, the supernatant wasremoved, the oily phase was washed with cyclohexane andthen poured into dichloromethane/aqueous Na2CO3. Afterremoving the water layer and evaporating dichloromethane,the residue was treated with hydrochloric methanol to give the corresponding hydrochloride as a white solid. Thebenzylation of 9 was prolonged over night and, at the endof the reaction, 9a separated as an oil, which was pouredinto dichloromethane/water. After removing the organiclayer, the water phase was made alkaline with NaHCO3 andextracted with ethyl acetate. The organic extract was concentratedto a small volume and a slight excess of p-toluenesulfonicacid was added to precipitate 9a as a white crystallinesolid.
In water; toluene; for 24h;Reflux; Dean-Stark;
General procedure: A mixture of L-valine (1.00 g, 8.53 mmol), cyclohexylmethanol (3.67 mL, 29.87 mmol) and p-TsOH.H2O (1.948 g, 10.24 mmol) in 15.0 mL of toluene were heated to reflux using a Dean-Stark apparatus for 24 hours. Next, the reaction mixture was allowed to cool to room temperature and the solvent was evaporated. The viscous oil thus obtained was placed under high vacuum pump for several hours and recrystalized using 5% hexane in EtOAc to afford 2.985 g (7.74 mmol) of creamy white amorphous solid as a product. The crude product thus obtained was used directly for next step without further purification or characterization.
2-[10-(4-amino-butyl)-19-[2-(2-amino-propionylamino)-propionylamino]-16-benzyl-7-(4-hydroxy-benzyl)-13-(1<i>H</i>-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentaaza-cycloeicosane-4-carbonyl]-amino}-3-methyl-butyric acid[ No CAS ]
2-[10-(4-amino-butyl)-19-[2-(2-amino-propionylamino)-3-carboxy-propionylamino]-7-(4-hydroxy-benzyl)-13-(1<i>H</i>-indol-3-ylmethyl)-16-methyl-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentaaza-cycloeicosane-4-carbonyl]-amino}-3-methyl-butyric acid[ No CAS ]
2-[10-(4-amino-butyl)-19-[2-(2-amino-propionylamino)-3-carboxy-propionylamino]-16-benzyl-7-(4-hydroxy-benzyl)-13-methyl-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentaaza-cycloeicosane-4-carbonyl]-amino}-3-methyl-butyric acid[ No CAS ]
2-[10-(4-amino-butyl)-19-[2-(2-amino-propionylamino)-3-carboxy-propionylamino]-16-benzyl-13-(1<i>H</i>-indol-3-ylmethyl)-7-methyl-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentaaza-cycloeicosane-4-carbonyl]-amino}-3-methyl-butyric acid[ No CAS ]
2-[19-[2-(2-amino-propionylamino)-3-carboxy-propionylamino]-16-benzyl-7-(4-hydroxy-benzyl)-13-(1<i>H</i>-indol-3-ylmethyl)-10-methyl-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentaaza-cycloeicosane-4-carbonyl]-amino}-3-methyl-butyric acid[ No CAS ]
2-[10-(4-amino-butyl)-19-[2-(2-amino-propionylamino)-3-carboxy-propionylamino]-16-benzyl-7-(4-hydroxy-benzyl)-13-(1<i>H</i>-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentaaza-cycloeicosane-4-carbonyl]-amino}-3-methyl-butyric acid[ No CAS ]
With hydrogenchloride; sodium hydroxide; ammonia; In hexane; dichloromethane;
EXAMPLE 9 Preparation of (S)-2-amino-3-methylbutanamide hydrochloride Anhydrous ammonia was bubbled through 150 mL of methylene chloride cooled to 0° C. (ice-bath) until the solution was saturated. To this mixture cooled to 5° C. and under N2 was added dropwise trimethylaluminum (136.2 mL of a 2M solution in hexane, 272.4 mmol) available from Aldrich Chemical Co., (Milwaukee, Wis.). The resultant cloudly solution was allowed to warm to room temperature and stirred for 22 h. L-Valine (10.6 g, 90.79 mmol) was added portionwise and stirred for 18 h at room temperature. To this mixture, cooled to 0° C. (ice-water bath), was then added dropwise 190 mL of 6 N HCl until the pH was 2. The resultant mixture allowed to warm and stirred for 2 hours and then made basic (pH=11-12) with 50percent aqueous NaOH. To the basic solution was added 100 mL of methylene chloride and 100 mL of H2 O. The organic layer was separated, dried over magnesium sulfate and evaporated under reduced pressure to dryness. The resultant residue was dissolved in 100 mL of methylene chloride and acidified with HCl gas. The solid that formed was filtered and dried under reduced pressure to give 6.8 g (49percent) of the title compound, mp 258°-260° C. IR (Nujol, cm-1), C=O (1686), N--H (3387, 3241). 1 H NMR and 13 C NMR (CDCl3) consistent with title product. Analysis calculated for C5 H13 ClN2 O: C, 39.35; H, 8.59; N, 18.35; Cl, 23.23; Found: C, 39.82; H, 8.52; N, 18.40; Cl, 23.13. MS: m/e 117 (M+ -Cl).
With sodium chloride; acetic acid; sodium nitrite; In water; ethyl acetate;
a. L-2-Hydroxyisovaleric acid To a liquid mixture of 170 ml of water, 170 ml of acetic acid and 40 ml of 1,4-dioxane, were added 23.4 g (0.2 mol) of L-valine, followed by heating to 40 C. to dissolve the valine in, said liquid. To the resulting solution, a solution of 41.4 g of sodium nitrite in 50 ml of water was added dropwise. The resultant mixture was stirred and reacted at room temperature for 3 hours. Under ice cooling, the reaction mixture obtained was added to a liquid mixture of 300 ml of a saturated aqueous solution of sodium chloride and 750 ml of ethyl acetate. The resulting mixture was allowed to separate into two layers. The water layer so obtained was extracted four times with 100 ml-portions of ethyl acetate. The combined organic layers were dried over anhydrous magnesium sulfate. The magnesium sulfate was filtered off and the filtrate was concentrated under reduced pressure, whereby the title compound was obtained.
With caesium carbonate; In dimethyl sulfoxide; at 90℃;
Reference Example 114 N-(3-chloro-4-cyanophenyl)valine To a solution of <strong>[60702-69-4]2-chloro-4-fluorobenzonitrile</strong> (3.50 g) in dimethyl sulfoxide (60 mL) were added L-valine (3.16 g) and cesium carbonate (9.53 g), and the mixture was stirred at 90 C. overnight. After allowing to room temperature, ethyl acetate was added, and the mixture was extracted twice with saturated aqueous sodium hydrogen carbonate solution. The aqueous layers were combined, and acidified with citric acid, and the mixture was extracted twice with ethyl acetate. The organic layers were combined, washed with saturated brine, and dried over magnesium sulfate. The solvent was evaporated under reduced pressure to give the title compound as a brown oil (yield: 5.69 g, 100%). 1H-NMR(CDCl3)delta:1.07(6H,t,J=6.7 Hz), 2.16-2.31(1H,m), 3.91-3.99(1H,m), 4.71(1H,d,J=8.9 Hz), 6.51(1H,dd,J=8.7,2.3 Hz), 6.67(1H,d,J=2.5 Hz), 7.42(1H,d,J=8.7 Hz).
Peptide Synthesis. All the peptides were synthesised by solid phase synthesis on a MultiSynTech Syro (Witten, Germany), using Fmoc/tBu chemistry. Coupling activation was carried out by HOBt/DIPEA/HBTU (1/2/0.9) in DMF and the Fmoc-protection on amine was removed employing 40% piperidine in NMP. Side chain protecting groups were: tert-butyl ester for Glu and Asp; trityl for His, Gln and Asn; 2,2,4,6,7-pentamethyldihydro-benzofuran-5-sulfonyl (Pbf) for Arg; tent-butyl ether for Ser, Thr and Tyr; tert-butyloxycarbonyl (Boc) for Lys, Pro and Trp. Fmoc-Lys(Fmoc)-OH was the amino acid used to synthesize dimeric and tetrameric peptides. Fmoc-5-amino valeric acid (5-Ava) and Fmoc-8-amino octanoic acid (8-Aoa) were used to carry out lipidation at N- and/or C-terminus both on linear and dimeric peptides. Tetrameric, dimeric and linear peptides were prepared on Rink amide 4-benzhydrylamine resin (MBNA), evaluating by spectrophotometric measurements the final loading in free amino groups, while acid peptides were prepared on a 2-chlorotrityl chloride resin. All peptides were cleaved from the resins and deprotected by treatment with trifluoroacetic acid, water and triisopropylsilane (TIPS, 95:2.5:2.5). The crude peptides, obtained by precipitation in diethyl ether, were purified by Waters HPLC-UV (Milford, Mass.) on a C12 Phenomenex column and characterized by Bruker MALDI-TOF spectrometry (Billerica, Mass.). The peptide synthesis was performed as described above. Six linear acid peptides, having the general sequence H-Q-X1-KIRVRLSA-OH with X1F, I, L, V, W, Y, were synthesized and combined in equimolar amounts. The crude peptide mixture was purified as reported in the Example I. MS (LC-MS ESI/Ion Trap): (ID8) calcd for C55H96N18O13(X1F) is (M) 1216, found is 1217 (M+H); (ID9) calcd for C53H101N18O13(X1I) is (M) 1197, found is 1198 (M+H); (ID10) calcd for C52H96N18O13 (X1=L) is (M) 1182, found is 1183 (M+H); (ID11) calcd for C51H96N18O13 (X1V) is (M) 1168, found is 1169 (M+H); (ID12) calcd for C57H97N19O13 (X1W) is (M) 1255, found is 1256 (M+H); (ID13) calcd for C55H96N18O14(X1Y) is (M) 1232, found is 1233 (M+H).
The peptide synthesis was performed on a 2-chlorotrityl chloride resin, as described above. Six linear acid peptides, having the general sequence H-X1-KKIRVRLSA-OH with X1F, I, L, V, W, Y, were synthesized and combined in equimolar amounts. The resulting crude peptide mixture was purified on C12 HPLC column, in order to remove salts and scavengers coming from cleavage, and analysed by LC-MS (ESI/Ion Trap) coupled with a HPLC system (Agilent Technologies, Santa Clara, Calif.), eluting components with a gradient mode (B 5 - 95% in 20 min; A 0.1% TFA in water, B 0.1% TFA in acetonitrile) at a flow rate of 1.0 ml/min. MS (LC-MS ESI/Ion Trap): (ID2) calcd for C56H100N18O12 (X1F) is (M) 1216, found is 1217 (M+H); (ID3) calcd for C53H102N18O12 (X1I) is (M) 1182, found is 1183 (M+H); (ID4) calcd for C53H102N18O12 (X1=L) is (M) 1182, found is 1183 (M+H); (ID5) calcd for C52H100N18O12 (X1V) is (M) 1168, found is 1169 (M+H); (ID6) calcd for C58H101N19O12 (X1W) is (M) 1255, found is 1256 (M+H); (ID7) calcd for C56H100N18O13 (X1Y) is (M) 1232, found is 1233 (M+H). Peptide Synthesis. All the peptides were synthesised by solid phase synthesis on a MultiSynTech Syro (Witten, Germany), using Fmoc/tBu chemistry. Coupling activation was carried out by HOBt/DIPEA/HBTU (1/2/0.9) in DMF and the Fmoc-protection on amine was removed employing 40% piperidine in NMP. Side chain protecting groups were: tert-butyl ester for Glu and Asp; trityl for His, Gln and Asn; 2,2,4,6,7-pentamethyldihydro-benzofuran-5-sulfonyl (Pbf) for Arg; tent-butyl ether for Ser, Thr and Tyr; tert-butyloxycarbonyl (Boc) for Lys, Pro and Trp. Fmoc-Lys(Fmoc)-OH was the amino acid used to synthesize dimeric and tetrameric peptides. Fmoc-5-amino valeric acid (5-Ava) and Fmoc-8-amino octanoic acid (8-Aoa) were used to carry out lipidation at N- and/or C-terminus both on linear and dimeric peptides. Tetrameric, dimeric and linear peptides were prepared on Rink amide 4-benzhydrylamine resin (MBNA), evaluating by spectrophotometric measurements the final loading in free amino groups, while acid peptides were prepared on a 2-chlorotrityl chloride resin. All peptides were cleaved from the resins and deprotected by treatment with trifluoroacetic acid, water and triisopropylsilane (TIPS, 95:2.5:2.5). The crude peptides, obtained by precipitation in diethyl ether, were purified by Waters HPLC-UV (Milford, Mass.) on a C12 Phenomenex column and characterized by Bruker MALDI-TOF spectrometry (Billerica, Mass.).
(2S)-2-amino-3-(1-tert-butoxycarbonylindol-3-yl)propanoic acid[ No CAS ]
[ 126631-93-4 ]
C66H117N21O12[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
Peptide Synthesis. All the peptides were synthesised by solid phase synthesis on a MultiSynTech Syro (Witten, Germany), using Fmoc/tBu chemistry. Coupling activation was carried out by HOBt/DIPEA/HBTU (1/2/0.9) in DMF and the Fmoc-protection on amine was removed employing 40% piperidine in NMP. Side chain protecting groups were: tert-butyl ester for Glu and Asp; trityl for His, Gln and Asn; 2,2,4,6,7-pentamethyldihydro-benzofuran-5-sulfonyl (Pbf) for Arg; tent-butyl ether for Ser, Thr and Tyr; tert-butyloxycarbonyl (Boc) for Lys, Pro and Trp. Fmoc-Lys(Fmoc)-OH was the amino acid used to synthesize dimeric and tetrameric peptides. Fmoc-5-amino valeric acid (5-Ava) and Fmoc-8-amino octanoic acid (8-Aoa) were used to carry out lipidation at N- and/or C-terminus both on linear and dimeric peptides. Tetrameric, dimeric and linear peptides were prepared on Rink amide 4-benzhydrylamine resin (MBNA), evaluating by spectrophotometric measurements the final loading in free amino groups, while acid peptides were prepared on a 2-chlorotrityl chloride resin. All peptides were cleaved from the resins and deprotected by treatment with trifluoroacetic acid, water and triisopropylsilane (TIPS, 95:2.5:2.5). The crude peptides, obtained by precipitation in diethyl ether, were purified by Waters HPLC-UV (Milford, Mass.) on a C12 Phenomenex column and characterized by Bruker MALDI-TOF spectrometry (Billerica, Mass.). The amido peptide synthesis was performed on a Rink amide resin, as described above in Peptide Synthesis paragraph. After synthesis, the crude peptides were purified by HPLC-UV and characterized by MALDI-TOF, as reported in the Example I. The mass values (M+H) were: (ID15) calcd is (M) 1395, found is 1397 (M+H); (ID16) calcd is (M) 1395, found is 1397 (M+H); (ID17) calcd is (M) 1337), found is 1339 (M+H).
Peptide Synthesis. All the peptides were synthesised by solid phase synthesis on a MultiSynTech Syro (Witten, Germany), using Fmoc/tBu chemistry. Coupling activation was carried out by HOBt/DIPEA/HBTU (1/2/0.9) in DMF and the Fmoc-protection on amine was removed employing 40% piperidine in NMP. Side chain protecting groups were: tert-butyl ester for Glu and Asp; trityl for His, Gln and Asn; 2,2,4,6,7-pentamethyldihydro-benzofuran-5-sulfonyl (Pbf) for Arg; tent-butyl ether for Ser, Thr and Tyr; tert-butyloxycarbonyl (Boc) for Lys, Pro and Trp. Fmoc-Lys(Fmoc)-OH was the amino acid used to synthesize dimeric and tetrameric peptides. Fmoc-5-amino valeric acid (5-Ava) and Fmoc-8-amino octanoic acid (8-Aoa) were used to carry out lipidation at N- and/or C-terminus both on linear and dimeric peptides. Tetrameric, dimeric and linear peptides were prepared on Rink amide 4-benzhydrylamine resin (MBNA), evaluating by spectrophotometric measurements the final loading in free amino groups, while acid peptides were prepared on a 2-chlorotrityl chloride resin. All peptides were cleaved from the resins and deprotected by treatment with trifluoroacetic acid, water and triisopropylsilane (TIPS, 95:2.5:2.5). The crude peptides, obtained by precipitation in diethyl ether, were purified by Waters HPLC-UV (Milford, Mass.) on a C12 Phenomenex column and characterized by Bruker MALDI-TOF spectrometry (Billerica, Mass.). The acid peptide synthesis was performed as described above. After synthesis, the crude peptides were purified by HPLC-UV by a gradient B 5 - 95% in 20 min and characterized by MALDI-TOF. The mass values (M+H) were: (ID1) calcd is (M) 1197, found is 1198 (M+H); (ID6) calcd is (M) 1255, found is 1256 (M+H); (ID14) calcd is (M) 1383, found is 1384 (M+H).
General Procedure: A sample of compounds 3-6 (0.1 mg each) was hydrolyzed with 6 N HCl (0.5 mL) at 110 °C for 24 h. The hydrolysate was concentrated to dryness, resuspended in H2O (100 muL), and then analyzed by chiral HPLC. Amino acid units were analyzed by HPLC/MS chiral analysis [column: Chirobiotic TAG (250 x 4.6 mm), Supelco;solvent: MeOH/10 mM NH4OAc (40:60, pH 5.6); flow rate 0.5 mL/min; detection by ESIMS in positive ion mode (MRM scan)]. The retention times (tR min; MRM ion pair, parent -> product) of the authentic amino acids were as follows: L-Pro (12.3; 116 -> 70), D-Pro (32.0), L-Val (7.7; 118 -> 72), D-Val (17.0), L-Ile (8.2;132 -> 86.1), D-Ile (20.5), L-Phe (15.1; 166.2 -> 120.2), D-Phe(22.5), N-Me-L-Phe (21.2; 180 -> 134.2), N-Me-D-Phe (37.0). The hydrolysates showed peaks corresponding to L-amino acids at tR12.3, 7.7, 8.2, 15.1 and 21.2 min. The MS parameters used were as follows: DP 32, EP 4, CE 21.8, CXP 2.8, CUR 30, CAD medium, IS 4500, TEM 700, GS1 65, and GS2 65. The absolute configurations of the alpha-hydroxy acid units were analyzed by chiral HPLC analysis under different conditions; [column: CHIRALPAK MA (+) (50 x 4.6 mm); solvent: CH3CN/2 mM CuSO4 (10:90); flow rate1 mL/min; detection by UV (254 nm)]. The retention times (tRmin) of the authentic standards were as follows: (2S,3S)-Hmpa (30.5), (2R,3R)-Hmpa (17.0), (2S,3R)-Hmpa (24.1), (2R,3S)-Hmpa (14.0), (2S)-Hiva (8.9), (2R)-Hiva (5.0). The retention times of the samples corresponded to (2S,3S)-Hmpa (30.5) in compounds 3, 4,and 6, and (2S)-Hiva (8.9) in 5.
General Procedure: A sample of compounds 3-6 (0.1 mg each) was hydrolyzed with 6 N HCl (0.5 mL) at 110 °C for 24 h. The hydrolysate was concentrated to dryness, resuspended in H2O (100 muL), and then analyzed by chiral HPLC. Amino acid units were analyzed by HPLC/MS chiral analysis [column: Chirobiotic TAG (250 x 4.6 mm), Supelco;solvent: MeOH/10 mM NH4OAc (40:60, pH 5.6); flow rate 0.5 mL/min; detection by ESIMS in positive ion mode (MRM scan)]. The retention times (tR min; MRM ion pair, parent -> product) of the authentic amino acids were as follows: L-Pro (12.3; 116 -> 70), D-Pro (32.0), L-Val (7.7; 118 -> 72), D-Val (17.0), L-Ile (8.2;132 -> 86.1), D-Ile (20.5), L-Phe (15.1; 166.2 -> 120.2), D-Phe(22.5), N-Me-L-Phe (21.2; 180 -> 134.2), N-Me-D-Phe (37.0). The hydrolysates showed peaks corresponding to L-amino acids at tR12.3, 7.7, 8.2, 15.1 and 21.2 min. The MS parameters used were as follows: DP 32, EP 4, CE 21.8, CXP 2.8, CUR 30, CAD medium, IS 4500, TEM 700, GS1 65, and GS2 65. The absolute configurations of the alpha-hydroxy acid units were analyzed by chiral HPLC analysis under different conditions; [column: CHIRALPAK MA (+) (50 x 4.6 mm); solvent: CH3CN/2 mM CuSO4 (10:90); flow rate1 mL/min; detection by UV (254 nm)]. The retention times (tRmin) of the authentic standards were as follows: (2S,3S)-Hmpa (30.5), (2R,3R)-Hmpa (17.0), (2S,3R)-Hmpa (24.1), (2R,3S)-Hmpa (14.0), (2S)-Hiva (8.9), (2R)-Hiva (5.0). The retention times of the samples corresponded to (2S,3S)-Hmpa (30.5) in compounds 3, 4,and 6, and (2S)-Hiva (8.9) in 5.
General Procedure: A sample of compounds 3-6 (0.1 mg each) was hydrolyzed with 6 N HCl (0.5 mL) at 110 °C for 24 h. The hydrolysate was concentrated to dryness, resuspended in H2O (100 muL), and then analyzed by chiral HPLC. Amino acid units were analyzed by HPLC/MS chiral analysis [column: Chirobiotic TAG (250 x 4.6 mm), Supelco;solvent: MeOH/10 mM NH4OAc (40:60, pH 5.6); flow rate 0.5 mL/min; detection by ESIMS in positive ion mode (MRM scan)]. The retention times (tR min; MRM ion pair, parent -> product) of the authentic amino acids were as follows: L-Pro (12.3; 116 -> 70), D-Pro (32.0), L-Val (7.7; 118 -> 72), D-Val (17.0), L-Ile (8.2;132 -> 86.1), D-Ile (20.5), L-Phe (15.1; 166.2 -> 120.2), D-Phe(22.5), N-Me-L-Phe (21.2; 180 -> 134.2), N-Me-D-Phe (37.0). The hydrolysates showed peaks corresponding to L-amino acids at tR12.3, 7.7, 8.2, 15.1 and 21.2 min. The MS parameters used were as follows: DP 32, EP 4, CE 21.8, CXP 2.8, CUR 30, CAD medium, IS 4500, TEM 700, GS1 65, and GS2 65. The absolute configurations of the alpha-hydroxy acid units were analyzed by chiral HPLC analysis under different conditions; [column: CHIRALPAK MA (+) (50 x 4.6 mm); solvent: CH3CN/2 mM CuSO4 (10:90); flow rate1 mL/min; detection by UV (254 nm)]. The retention times (tRmin) of the authentic standards were as follows: (2S,3S)-Hmpa (30.5), (2R,3R)-Hmpa (17.0), (2S,3R)-Hmpa (24.1), (2R,3S)-Hmpa (14.0), (2S)-Hiva (8.9), (2R)-Hiva (5.0). The retention times of the samples corresponded to (2S,3S)-Hmpa (30.5) in compounds 3, 4,and 6, and (2S)-Hiva (8.9) in 5.
180 mg, 0.27 mmol) was added AcOEt (3.6 ml_). The resulting suspension was stirred at 80 C (bath temperature) for 3 hours. The solid was filtered with a sintered funnel (porosity 4), washed with hot AcOEt (1 ml_) and dried under vacuum at room temperature. L-valine was obtained as a white powder (31 mg, 97% yield). The filtrate was washed with water (1 .5 mL), dried (MgSO4) and distilled under vacuum to yield (S)-2-hydroxy-3-methoxy-3,3- diphenylpropanoic acid as a white solid (quantitative yield, 97% ee)
(S)-1-((S)-2-((2S,3S)-2-((R)-3-(1H-imidazol-4-yl)-2-(3-methylbutanamido)-propanamido)-3-methylpentanamido)-3-methylbutanoyl)pyrrolidine-2-carboxamide formate salt[ No CAS ]
General procedure: Approximately 0.2 mg of 1-3 and 0.1 mg of 4-5 were hydrolyzed with 6 N HCl (100 muL) at 110 C for 30 min with stirring. The hydrolysates were evaporated to dryness and then the dried hydrolysates were resuspended in H2O (100 muL). The solutions were concd under reduced pressure.
General procedure: Approximately 0.2 mg of 1-3 and 0.1 mg of 4-5 were hydrolyzed with 6 N HCl (100 muL) at 110 C for 30 min with stirring. The hydrolysates were evaporated to dryness and then the dried hydrolysates were resuspended in H2O (100 muL). The solutions were concd under reduced pressure.
General procedure: Under argon protection, BSA (2.2equiv.) was added to a solution of amino acid (1.1equiv.) in anhydrous dichloromethane. After the mixture was stirred for 1-8h at 23C, a solution of N-Boc protected NHS ester (1equiv.) in dichloromethane was added. The reaction mixture was stirred at 23C under argon until all active ester was consumed as judged by TLC analysis. The reaction mixture was washed with brine, dried over Na2SO4 and concentrated in vacuo to provide a white solid. The isolated product was recrystallized from diethyl ether/n-hexane to yield the targeted dipeptide as a white solid.
2-chlorotrityl chloride polystyrene resin[ No CAS ]
[ 72-18-4 ]
[ 18822-58-7 ]
(S)-6-[(Diphenyl-p-tolyl-methyl)-amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoic acid[ No CAS ]
(S)-2-amino-5-(3-(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-ylsulfonyl)guanidino)pentanoic acid[ No CAS ]
[ 60-32-2 ]
C153H214ClN20O23PolS3[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
The peptide sequence was synthesized by Fmoc based solid phase peptide synthesis (SPPS) on a 2-Chlorotrityl Chloride. The coupling reaction of two amino acids lasted 2-4 hours at room temperature in the presence of HBTU as a coupling reagent and N, N-Diisopropylethylamine (DIPEA) as a basic catalyst, respectively. The reaction was allowed to last for 4 hours for coupling with arginine and 2 hours for other amino acids. The deprotection of Fmoc group was performed within 20% peperidine in Dimethyformamide (DMF) for 20 min. The dodecanoic acid was coupled to the N-terminus of the peptide using the same SPPS protocol.
(S)-1-(cyclohexylmethoxy)-3-methyl-1-oxobutan-2-aminium 4-methylbenzenesulfonate[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
90%
In toluene; for 24h;Reflux; Dean-Stark;
General procedure: A mixture of L-valine (1.00 g, 8.53 mmol), cyclohexylmethanol (3.67 mL, 29.87 mmol) and p-TsOH.H2O (1.948 g, 10.24 mmol) in 15.0 mL of toluene were heated to reflux using a Dean-Stark apparatus for 24 hours. Next, the reaction mixture was allowed to cool to room temperature and the solvent was evaporated. The viscous oil thus obtained was placed under high vacuum pump for several hours and recrystalized using 5% hexane in EtOAc to afford 2.985 g (7.74 mmol) of creamy white amorphous solid as a product. The crude product thus obtained was used directly for next step without further purification or characterization.
90%
In toluene; for 24h;Reflux; Dean-Stark;
Synthesis of compound 6b: A mixture of L-valine (1.00 g, 8.53 mmol), (0323) cyclohexylmethanol (3.67 mL, 29.87 mmol) and />-TsOH.H20 (1.948 g, 10.24 mmol) in 15.0 mL of toluene were heated to reflux using a Dean-Stark apparatus for 24 hours. Next, the reaction mixture was allowed to cool to room temperature and the solvent was evaporated. The viscous oil thus obtained was placed under high vacuum pump for several hours and recrystallized using 5% hexane in EtOAc to afford 2.985 g (7.74 mmol) of creamy white solid as a product. The crude product thus obtained was used directly for the next step without further purification or characterization.
With hydrogenchloride; In water; at 110℃; for 20h;
General procedure: Samples of 1 (0.3 mg) and 2 (0.1 mg) were heated with 6 NHCl (110 C, 20 h) and the hydrolysates subjected to chiral HPLCMS[column, Chirobiotic TAG (4.6 250 mm), Supelco; solvent,MeOH-10 mM NH4OAc (40:60, pH 5.12): flow rate, 0.5 mL/min;detection by ESIMS in positive or negative ion modes (MRM scan)]. The retention times (tR, min; MRM ion pair, parent?product) ofthe authentic amino acids and compound-dependent MS parameterswere listed in Tables 2 and 5. The source and gas-dependentMS parameters were as follows: CUR 50, CAD medium, IS 5500,TEM 750, GS1 65, GS2 65.
With hydrogenchloride; In water; at 110℃; for 20h;
General procedure: Samples of 1 (0.3 mg) and 2 (0.1 mg) were heated with 6 NHCl (110 C, 20 h) and the hydrolysates subjected to chiral HPLCMS[column, Chirobiotic TAG (4.6 250 mm), Supelco; solvent,MeOH-10 mM NH4OAc (40:60, pH 5.12): flow rate, 0.5 mL/min;detection by ESIMS in positive or negative ion modes (MRM scan)]. The retention times (tR, min; MRM ion pair, parent?product) ofthe authentic amino acids and compound-dependent MS parameterswere listed in Tables 2 and 5. The source and gas-dependentMS parameters were as follows: CUR 50, CAD medium, IS 5500,TEM 750, GS1 65, GS2 65.
With hydrogenchloride; In water; at 110℃; for 10h;Sealed tube;
To 1.0 mg of cichorinotoxin was added 1.0 mL of 6 N HCl, andthe mixture was frozen at -30 C and degassed under a highvacuum pump. The glass vessel was sealed and heated at110 C for 5 h, 10 h and 22 h to obtain the hydrolysates, each of which were concentrated to dryness. The residues were evaluatedusing an amino acid analyzer. Marfey?s method was employedto determine the stereochemistry of each of the aminoacids. The dried hydrolysates obtained by heating for 10 h weredissolved in 140 muL of H2O. To 25 muL of the solution wereadded 1% Marfey?s reagent ((1-fluoro-2,4-dinitro-5-fluorophenyl)-L-alaninamide, 1.8 muM) dissolved in acetone and 10 muLof 1 M NaHCO3 (10 muM). The solution was heated at 35 C for1 h, then 2 N aq HCl was added to quench the reaction, and thenit was concentrated to dryness. The residues were dissolved inDMSO and subjected to reversed-phase HPLC (C18) using amobile phase composed of 13% CH3CN/87% 50 mM triethylaminephosphate. Amino acid compositions of naturalcichorinotoxin (not Marfey?s derivatives) were analyzed byHitachi Keisoku Service Ltd (Tokyo)
(S)-1-carboxy-2-methypropan-1-aminium ((2R,3S,4R,5R)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl phosphate[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
100%
In water; at 10℃;pH Ca. 3.8 - 4;
A 50 mL 3 N RBF fitted with a water condenser and internal thermometer was charged with NMN (0.200 g, 0.598 mmol, 1 eq) and 5 ml of distilled deionised water and mixed to form a solution, a faint suspension is also ok. This solution was cooled using an ice/water hath to bring the internal temperature below 10 C. To this solution was then added dropwise 5.68 mi of 0.1M L-Valine solution in distilled deionised water slowly so as to prevent the temperature from increasing. After this addition the pH was ~3.8-4.0. The flask was then removed and the colourless solution frozen using liquid nitrogen. While the flask was frozen it was connected to the freeze dryer. This will slowly remove water. Once dried the product is rendered as a colourless to faint yellow solid. Yield: 312 mg (Quantitiative) Melting point: 148 C (degradation, corrected) outgassing at 150 C Analytical data. 1H-NMR (400 MHz, D20) d = 9.52 (s, 1H), 9.32 (d, 1H), 9.02 (d, 1H), 8.33 (dd, 1H), 6.25 (d, 1H), 4.67 (m, 2H), 4.61 (t, 1H), 4.48 (dd, 1H), 4.32 (dq, 1H), 4.17 (dq, 1H), 3.62 (d, H i), 2.30 (m, H i), 1.08 (d, 31 1), 1.02 (d, 1 1 ) ppm
The colorimetric detection of glucose was performed with rGO/CM (2:1) catalyst in preoptimized conditions. 50muL of GOx (2 mg/ml) was dissolved in an equal amount (350 muL) of different glucose concentrations (10-100 muM) in PBS buffer (pH 7.1) and incubated at 35C. 100 muL of TMB (1 mM) was dissolved in 1.5 ml of NaAc-HAc buffer (pH 4.0) containing 100 muL of 0.1 mg/ml rGO/CM (2:1) concentration. This solution was mixed with glucose-containing a solution and the mixture was incubated at 35C. The obtained blue color was detected with UV/Vis. spectrophotometer and maximum absorbance was recorded at 652 nm. For real sample analysis, glucose content in human serum samples was detected with the same methodology. The serum samples of healthy volunteers collected from Ashirvad Pathology Laboratory were first centrifuged at 12000 rpm to remove the possible aggregates in serum samples. The supernatants were diluted by 150 folds and performed the same detection methods as described above. In a control experiment, 350 muL of glucose (100 muM), 350 muL of sucrose (5 mM), 350 muL of fructose (5 mM), 350 muL lactose (5mM), 100muL of maltose (5mM), 350muL of L-Leucine (5mM) 350muL of L-Valine (5mM) and 350muL of L-sistine were mixed with 50 muL of GOx (2mg/ml) and incubated at 35C for 10 min. After that, 1.5 ml of NaAc-HAc buffer (pH 4.0) containing the appropriate amount of rGO/CM (2:1) and TMB was mixed with the above mixture for selectivity analysis. Limit of detection and linearity range were calculated using equation LOD= (3*Std)/(slope of calibration line).
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