* 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] Chemische Berichte, 1907, vol. 40, p. 491
[2] Journal of Molecular Structure, 2011, vol. 994, # 1-3, p. 269 - 275
2
[ 338-69-2 ]
[ 95-92-1 ]
[ 7126-50-3 ]
Yield
Reaction Conditions
Operation in experiment
86%
With potassium <i>tert</i>-butylate In ethanol at 0 - 20℃; for 1.7 h;
Diethyl oxalate and D-alanine were weighed according to a molar ratio of 1:1.Mixing and stirring to obtain a mixture; by mass ratio1:17, potassium tert-butoxide was dissolved in absolute ethanol, mixed and cooled to 0 ° C, and the mixture was slowly added to a solution of potassium t-butoxide in ethanol.The amount of the mixture added is 9percent of the mass of the potassium t-butoxide solution.After the completion of the addition, the temperature was raised to room temperature, and the reaction was stirred for 1.7 h; after the reaction, the mixture was discharged, and the reaction product was poured into ice water. After stirring for 17 min, the pH was adjusted to 5.7 with hydrochloric acid, and a solid was precipitated, suction filtered, and the residue was collected and recrystallized. After drying, ethyl 5-formyl-1H-pyrrole-2-carboxylate was obtained, the melting point was 72.8 ° C, the purity was 99.8percent, and the yield was 86percent.
Reference：
[1] Patent: CN108314640, 2018, A, . Location in patent: Paragraph 0009; 0022-0027
3
[ 338-69-2 ]
[ 35320-23-1 ]
Yield
Reaction Conditions
Operation in experiment
82.5%
With lithium aluminium tetrahydride In tetrahydrofuran at 0℃; for 18.5 h; Reflux
The present embodiment relates to a class of long-chain ethyl piperazine sulfonamide derivative intermediate () in the general synthetic methods.Particularly to the synthesis of L- propylamine alcohol.The 150mL dry THF and 1.1gLiAlH4(29mmol) views into a three-necked flask, cooled to 0 deg.] C, the 4.9gL- alanine (55.0 mmol) portionwise into the reactor, at 30min the addition was completed, stirring was continued for 2h, gradually warming to reflux and heating continued for 16h , the reaction was stopped, the ice bath was cooled, diluted with 100mL of diethyl ether, 4.5mL water successively, 4.5mL15percent NaOH and 12mL of water into the reactor, the reaction solution was filtered through celite, the filtrate was collected, the solvent was evaporated under reduced pressure to obtain a crude product, purified by column chromatography on silica gel, eluting with methylene chloride: ethanol = 10 to yield of 3.41 g of a colorless oil, yield 82.5percent.
73%
Stage #1: With lithium aluminium tetrahydride In tetrahydrofuran at 0℃; for 6 h; Inert atmosphere; Reflux Stage #2: With sodium hydroxide In tetrahydrofuran; water for 0.25 h; Inert atmosphere
(R)-Alanine (10.0 g, 112 mmol) was added solwly to a mixture of lithium aluminium hydride (8.50 g, 224 mmol) in dry THF (300ml) at 0. The mixture was refluxed for 6h, water (8.50 ml) was added slowly, stirred for 15min. Then 15percent NaOH aqueous solution(8.5 ml) was added to the mixture. The crude product was purified by vacuum distillation (Bp: 54 (700Pa)) to give (R)-Alaniol(6.15 g) as a colorless liquid in yield of 73percent.
Reference：
[1] Patent: CN105566220, 2016, A, . Location in patent: Paragraph 0016
[2] Chemistry Letters, 2015, vol. 44, # 12, p. 1777 - 1779
[3] Tetrahedron Letters, 1987, vol. 28, # 19, p. 2083 - 2086
[4] Journal of Organic Chemistry, 1995, vol. 60, # 21, p. 6987 - 6997
[5] Helvetica Chimica Acta, 2000, vol. 83, # 9, p. 2594 - 2606
[6] Patent: US2009/170874, 2009, A1, . Location in patent: Page/Page column 58
[7] Journal of the American Chemical Society, 2010, vol. 132, # 3, p. 1151 - 1158
[8] Bioorganic and Medicinal Chemistry Letters, 2012, vol. 22, # 6, p. 2216 - 2220
[9] Bioorganic and Medicinal Chemistry, 2012, vol. 20, # 21, p. 6305 - 6312,8
[10] Journal of Agricultural and Food Chemistry, 2016, vol. 64, # 46, p. 8927 - 8934
[11] Patent: WO2005/42464, 2005, A1, . Location in patent: Page/Page column 56
4
[ 338-69-2 ]
[ 1821-02-9 ]
[ 2013-12-9 ]
[ 127-17-3 ]
Reference：
[1] Journal of Molecular Catalysis B: Enzymatic, 2013, vol. 94, p. 15 - 22
Reference：
[1] Journal of Natural Products, 2017, vol. 80, # 10, p. 2677 - 2683
13
[ 338-69-2 ]
[ 100-51-6 ]
[ 100836-85-9 ]
[ 33106-32-0 ]
Reference：
[1] Journal of Organic Chemistry, 1988, vol. 53, # 15, p. 3457 - 3465
14
[ 67-56-1 ]
[ 338-69-2 ]
[ 14316-06-4 ]
Yield
Reaction Conditions
Operation in experiment
99%
at 20℃; for 2 h;
General procedure: SOCl2 (21.8mL, 0.3mol) and (S)-alanine ((S)-19, 8.91g, 0.1mol) were added to CH3OH (100mL) and the mixture was stirred at RT for 2h. The solvent was removed in vacuo, the residue was dissolved in methanol (30mL) and the organic solvent was removed in vacuo again. This procedure was repeated twice. Colorless amorphous solid, mp 103°C (Ref. 39 mp 98–99°C), yield 14.3g (>99percent).
90%
at 0 - 20℃; for 24 h;
In a separate reaction, the intermediate reagent V(E) was prepared by suspending V(D) (100 g, 1.12 mol) in 1 L of MeOH. The mixture was cooled to 0-5 0C. 50 mL of SOCI2 was added dropwise at 0-5 0C. The reaction mixture was then stirred at rt for 24 hours. The mixture was evaporated in vacuum to give compound V(E) (141 g, 90percent) as a white solid.
3.46 g
at 20℃; for 6 h;
D-alanine (2.5 g, 27.8 mmol) was added to a dry flask.Add anhydrous anaerobic methanol (14 mL), stir at 20 ° C for 10 min,Thionyl chloride (5 mL) was slowly added thereto, and the reaction was stirred for 6 hours.After the reaction was completed, the solvent was evaporated, diluted with diethyl ether and filtered.D-Alanine methyl ester 9 was obtained as a white solid, 3.46 g.
Reference：
[1] Bioorganic and Medicinal Chemistry, 2017, vol. 25, # 17, p. 4778 - 4799
[2] Organic Preparations and Procedures International, 2002, vol. 34, # 1, p. 87 - 94
[3] Tetrahedron Letters, 1989, vol. 30, # 3, p. 313 - 316
[4] Journal of Medicinal Chemistry, 2015, vol. 58, # 15, p. 6293 - 6305
[5] European Journal of Organic Chemistry, 2010, # 22, p. 4276 - 4287
[6] Patent: WO2008/125945, 2008, A2, . Location in patent: Page/Page column 20-21
[7] Journal of Organic Chemistry, 1986, vol. 51, # 14, p. 2728 - 2735
[8] Agricultural and Biological Chemistry, 1987, vol. 51, # 2, p. 537 - 548
[9] Journal of Physical Chemistry, 1994, vol. 98, # 27, p. 6862 - 6864
[10] Heterocycles, 1988, vol. 27, # 9, p. 2077 - 2080
[11] Journal of Organic Chemistry, 1998, vol. 63, # 1, p. 92 - 98
[12] Journal of the Chemical Society - Perkin Transactions 1, 1999, # 4, p. 387 - 398
[13] Tetrahedron, 2009, vol. 65, # 45, p. 9116 - 9124
[14] Chemistry--A European Journal, 2014, vol. 20, # 36, p. 11428 - 11438,11
[15] Journal of Polymer Science, Part A: Polymer Chemistry, 2016, vol. 54, # 8, p. 1065 - 1077
[16] Journal of Medicinal Chemistry, 2017, vol. 60, # 13, p. 5889 - 5908
[17] Russian Chemical Bulletin, 2017, vol. 66, # 1, p. 136 - 142[18] Izv. Akad. Nauk, Ser. Khim., 2017, # 1, p. 136 - 142,6
[19] Patent: CN108610269, 2018, A, . Location in patent: Paragraph 0016; 0017
[20] Patent: CN108690005, 2018, A, . Location in patent: Paragraph 0024; 0025
With pyridoxal 5'-phosphate In aq. phosphate buffer; dimethyl sulfoxide at 30℃; for 24 h; Resolution of racemate; Enzymatic reaction
General procedure: All experiments were carried out keeping the protein content constant (8 mg mL−1) if not otherwise stated. One of the substrates rac-1a–d (50 mM), sodium pyruvate (50 mM) and pyridoxal-5′-phosphate monohydrate (0.2 mg mL−1) in phosphate buffer (1 mL, 0.1 M, pH 7.5) containing IPA or DMSO as a possible cosolvent (10, v/v-percent) was added to a 2 mL Eppendorf tube, containing the ω-transaminase sol–gel catalyst (25 or 50 mg). The reaction was shaken (170 rpm) at 30 °C. After 24 h the reaction was stopped by centrifuging the mixture and removing the solution by pipette. The solid catalyst was reused while conversion was monitored by taking a sample (5 μL) from the solution and diluting it with the HPLC eluent (500 μL). The sample was filtered and analyzed for conversion by HPLC. A sample (400 μL) for ee(S)−1 analysis was taken, and aqueous NaOH (2 M, 50 μL) was added followed by the extraction of the amine into ethyl acetate (400 μL). The organic phase (300 μL) was dried with Na2SO4, and after filtration the amine in the sample (200 μL) was derivatized with acetic anhydride (10 μL) to determine the enantiomeric excess of (S)-1 by GC.#10;
Reference：
[1] Process Biochemistry, 2013, vol. 48, # 10, p. 1488 - 1494
Characterization of synthesized compound(R)-4-methyloxazolidine-2,5-dione (1)
This compound was obtained by reaction of D-alanine with phosgene. It was obtained as white solid; Yield: 83 %; m.p.89-90 °C 1H NMR (DMSO-d6, 400.13 MHz): d = 9.01 (s,1H, NH), 4.47 (q, 1H, 3J = 7.2 Hz, CH), 1.33 (d, 3H,3J = 7.2 Hz, CH3); 13C NMR (DMSO-d6, 100.62 MHz)d = 172.5 (COO), 151.8 (CONH), 52.9 (CH), 16.8 (CH3).
83%
In tetrahydrofuran at 40 - 45℃; for 2h; Inert atmosphere;
(R)-4-methyloxazolidine-2,5-dione (2)
This compound was obtained by the reaction of D-alanine 1with phosgene. The mixture of 150 mL dry tetrahydrofuranand 100 mmol finely milled D-alanine was placed undernitrogen into 250 mL three-neck flask. Phosgene (250mmol) then was bubbled into rapidly stirring reactionmixture. The reaction mixture was stirred at 40-45 °C for 2h to afford homogeneous solution. The solution was cooleddown to 20 °C and purged of excess phosgene by bubblingN2 through the reaction mixture, and passing the exhaustgases through aqueous sodium hydroxide solution (15%).The solvent was removed in vacuum to afford a crude solid,which was recrystallised from hexan to afford 2 as a whitecrystalline solid; yield: 83%; m.p. 89-90 °C 1H NMR(DMSO-d6, 400.13 MHz,): δH 9.01 (s, 1H, NH), 4.47 (q,1H, 3JH-H 7.2 Hz, CH), 1.33 (d, 3H, 3JH-H 7,2 Hz, CH3);13C NMR (DMSO-d6, 100.62 MHz,) δC 172.5 (COO),151.8 (CONH), 52.9 (CH), 16.8 (CH3).
1.1 (1) Synthesis of compound 2-1 ((R)-2-(1,3-dioxoisoindolin-2-yl)propionic acid):
1 mol of D-alanine, 1 mol of compound 1-1 and 500mL of toluene were added into a 1L single-necked bottle, the condensation reaction was carried out under the condition of reflux and water separation until no more water was produced, 80% toluene was recovered by concentrating under reduced pressure, a white solid was precipitated by cooling, and the obtained filter cake was filtered at 510 . , washed with 50 mL of toluene, and dried under vacuum to constant weight to obtain compound 2-1 (white crystal, 199.91 g, yield 91.2%).
90%
With triethylamine In toluene Heating;
88%
With pyridine for 12h; Heating;
72%
Stage #1: D-Alanine With Merrifield resin bound NH2CH2COOEt
Stage #2: phthalic anhydride With mesoporous silica; tantalum pentachloride for 0.0833333h; microwave irradiation;
Stage #3: With trifluoroacetic acid In dichloromethane
64%
With glacial acetic acid for 3h; Reflux;
7.1 Step 1
To a 1 -necked round-bottom flask (200 mL) equipped with a condenser was charged with D-alanine (5.0 g, 56.2 mmol), phthalic anhydride (18.2 g, 56.2 mmol), and glacial acetic acid (100 mL) in that order. The resulting mixture was refluxed for 3 hrs. The solvent was completely removed in vacuo, and the residue was precipitated from a mixture of ethyl acetate and hexanes to give (R)-2-(l,3-dioxoisoindolin-2-yl)propanoic acid (8.0 g, 64%) as a white solid.
at 120 - 125℃; for 7h;
With glacial acetic acid at 120℃; for 12h; Reflux;
With glacial acetic acid for 4h; Reflux;
1.1.1 General procedure for preparation of compounds Q1-Q18
General procedure: Phenylalanine or alanine (10 mmol) and anhydride (10 mmol) were heatedand refluxed in 20 mL of acetic acid for 4 h and monitored by TLC. Aftercompletion of the reaction, the solution was stirred and poured into 500 mL ofice water. When the precipitate was appeared, filtering it and then dry it in vacuumdrying oven. Next, the carboxylic acid (5 mmol) which was obtained in theprevious step and podophyllotoxin (6 mmol) were dissolved in dichloromethane(25mL) and stirred for 12 h with DCC (10 mmol) as dehydrator and DMAP (1 mmol) ascatalyst at room temperature. Adding proper amount of silica gel and condensingsolvent by vacuum concentration. Then, collecting target compounds by columnchromatography (V (dichloromethane):V (acetone) =1:50).
With triethylamine In toluene Reflux;
1-2 Example 2
Add 635g of toluene, 61.5g of D-alanine, 102g of phthalic anhydride and 1g of triethylamine to the reaction flask, heat under reflux to separate water, react for 2.5-3.5h, add 82g of thionyl chloride dropwise, and control the rate of addition In order to keep the temperature in the reaction flask at 60-65°C, after dripping, the temperature was raised to 72°C. After 5 hours of reaction, the reaction was confirmed by thin-layer chromatography monitoring, and then concentrated at -0.09MPa and 60°C for 1 hour to obtain the formula (III). 164 g of the shown acid chloride compound.
With hydrogenchloride; sodium nitrate In water at -5 - 25℃; for 0.111167h;
18 Example 18
Hydrochloric acid (25 mL, 7.5 mol/L) was added to a beaker and cooled to -5 to 0°C.Alanine (3.3409 g, 0.0375 mol) was weighed and dissolved in the hydrochloric acid to obtain a homogeneous mixed solution and transferred to a syringe matched with the injection pump A at a flow rate of 1 mL/min.An aqueous solution (40%) of sodium nitrite (5.1735 g, 0.075 mol) was transferred to a syringe fitted with a syringe pump B at a flow rate of 0.5 mL/min.The two were mixed with a Y-type mixing valve and pumped together into a microreactor. The volume of the microreactor was 10 mL, the reaction temperature was 25° C., and the reaction time was 6.67 min. The reaction solution was α-chlorocarboxylic acid product.The resulting product was examined by liquid chromatography-mass spectrometry and had a HRMS (TOF) m/z [M-H]- of 120.9933 and a yield of 93.4%.
65%
With hydrogenchloride; sodium nitrite
With hydrogenchloride; water; sodium nitrite
With hydrogenchloride; sodium nitrite at 0 - 20℃;
With hydrogenchloride; sodium nitrite In water
21.4 g
With hydrogenchloride; sodium nitrite In water at 0℃; for 6.5h;
Preparation of (2R)-2-chloropropanoic acid
To a solution of D-alanine (35.6 g, 400 mtnol) in aqueous HC1 (6 N, 500 mL) was added NaNO2 (44 g, 640 mtnol) in small portions at 0°C in 2.5 hours with vigorous stirring. After addition, the reaction was stirred at 0°C for additional 4 hours. The solution was then extracted with ether (200 mL) three times, dried over anhydrous Na2SO4 and then concentrated in vacuo. The slightly yellow residue was fractionally distilled to afford (2R)-2-chloropropanoic acid (compound 21a) 21.4 g as a yellow oil. Compound 21a: 1H NMR (400 MHz, DMSO-d6) δ ppm: 13.08 - 13.39 (m, 1 H), 4.58 (d, J = 6.90 Hz, 1 H), 1.57 (d, J = 6.78 Hz, 3 H). MS obsd. (ESO[(M-H)-]: 107.
With hydrogen bromide; potassium bromide; sodium nitrite at 0 - 5℃; for 1h;
82%
With hydrogen bromide; potassium bromide; sodium nitrite In water at -15 - 20℃; Inert atmosphere;
Synthesis of Chiral Bromoacid
D-alanine (8.9 g, 0.1 mol) and KBr (11.9 g, 0.1 mol) was dissolved in 100 ml, of a 30% HBr water solution and kept in -15°C dry ice/ethylene glycol bath. NaNO2 (10.35 g, 0.15 mmol) were dissolved in 15 ml water and slowly dripped in the above solution under argon atmosphere. The reaction was allowed to proceed for 3 hr and to warm from -15°C to room temperature. It was then put under vacuum for 30 min. Product was extracted by diethyl ether (25 ml × 3). Organic phases were combined and dried over Na2SO4. Then the solvent was evaporated under vacuum, and the crude product was further purified by distillation at 115°C, high vacuum. The pure product was obtained as a colorless oil in 82% yield.
41%
With sulfuric acid; potassium bromide; sodium nitrite at 0 - 20℃; for 2h;
41%
With sulfuric acid; potassium bromide; sodium nitrite In water
6.A Preparation of (2R)-2-Bromopropanoic Acid
6A. Formula 9 Where R1 Is Methyl and X Is Bromo To a stirred solution of 5.00 g of D-Ala, 23.4 g of KBr in 37 mL of water and 11 mL of conc. sulfuric acid at -8° C. was added a solution of 5.14 g of NaNO2 in 2 mL of water over a period of 1 h. Stirring was continued for 1 h at the same temperature, the reaction mixture was then extracted with CH2 Cl2. The organic layer was washed with water, dried over Na2 SO4 and evaporated under vacuum to give 3.16 g of (2R)-2-bromopropanoic acid as an oil at 41% yield. Characteristic analytical data are as follows: 1 H NMR (300 MHz, CDCl3) δ4.41 (q, J=7 Hz, 1H), 1.86 (d, J=7 Hz, 3H); [α]D25 +32.6° (c=2.10, MeOH).
With water; hydrogen bromide; sodium nitrite
With sulfuric acid; potassium bromide; sodium nitrite at 0℃; for 1h;
With sulfuric acid; potassium bromide; sodium nitrite for 3h; Ambient temperature;
51.3 g
With hydrogen bromide; sodium sulfate; sodium nitrite In water at 15℃;
With hydrogen bromide; sodium nitrite
With potassium bromide; sodium nitrite In sulfuric acid at 0℃; for 1h;
With hydrogen bromide; potassium bromide; sodium nitrite
17.a a
a (R)-2-Bromopropanoic acid Following the procedure of Example 14(a) but employing D-alanine in place of (R)-aminobutanoic acid, (R)-2-bromopropanoic acid was obtained as a light yellow oil.
With sulfuric acid; potassium bromide; sodium nitrite In water
Stage #1: formaldehyd; D-Alanine With sodium dihydrogenphosphate dihydrate In water at 20℃; for 1h;
Stage #2: With zinc In water at 20℃; for 1h;
1 Example 1: Forming N,N-Dimethyl-D-Alanine
In this example, N,N-dimethyl-D-alanine was formed utilizing a process similar to step 102 of exemplary method 100 as presented in FIG. 1. At first, a solution of D-alanine had a concentration of 0.33 M with a pH level of 5.25 and was formed in a round bottom flask by dissolving 6 mmoles of D-alanine in 18 ml of deionized water. Then, formaldehyde 37% with an amount of about 24.3 mmoles was added to the solution of D-alanine to form a mixture. The mixture was stirred at room temperature for two (2) minutes. After that, sodium dihydrogen phosphate.2H2O with an amount of about 36.9 mmoles was added to the mixture and stirred at room temperature for 1 hour. Then, the round bottom flask was put in water bath with a temperature of about 20° C. and zinc dust with an amount of about 45 mmoles was added and stirred at room temperature for 1 hour. (0082) Again, formaldehyde 37% with an amount of about 24.3 mmoles was added, followed by adding 18 ml deionized water, and then stirred at room temperature 2 minutes. Then, sodium dihydrogen phosphate.2H2O with an amount of about 36.9 mmoles was added and stirred at room temperature for 1 hour. After this time, zinc dust with an amount of about 45 mmoles was added and stirred at room temperature for 1 hour. This process was repeated twice to complete the reaction of forming N,N-dimethyl-D-alanine. The reaction progress was checked by thin-layer chromatography (TLC) using n-butanol, acetic acid, and water with a ratio of 2:1:1, respectively. The TLC was visualized using ninhydrin. The reaction was completed after 24 hours and the reaction mixture was filtered and the filtrate was adjusted to a pH level of about 7 by adding an ammonia solution while stirring at this pH level for a time period of about 45 minutes. Then, the filtrate was concentrated under reduced pressure to form a residue. (0083) After that, methanol with an amount of about 100 ml was added to the residue and remained for fifteen (15) minutes. Then, the reaction mixture was mechanically stirred for one (1) hour and filtered. The filtrate was concentrated utilizing a rotary evaporator. Again, about 30 ml methanol was added to the residue and stirred for twenty (20) minutes and filtered using a filter paper. Then, the filtrate was concentrated by evaporating the solvent utilizing a rotary evaporator, leading to a crude product that recrystallized from hot ethanol/acetone. In the end, N,N-dimethyl-D-alanine with an amount of about 0.6466 gram and with a yield of about 92% was obtained. (0084) The optical purity of N,N-dimethyl-D-alanine was confirmed by its optical rotation. FIG. 3 shows a proton nuclear magnetic resonance (1H NMR) spectrum of N,N-dimethyl-D-alanine, consistent with one or more exemplary embodiments of the present disclosure. Referring to FIG. 3, the 1H NMR spectrum was consistent with the proposed structure of N,N-dimethyl-D-alanine. [α]25D=-10° (c=5, H2O), Literature Data: [α]25D=-10.5° (c=5.12, H2O), m.p=188° C., Literature Data: m.p=184° C., 1HNMR (500 MHZ, DMSO)=δ 1.24 (d, J=7.1 Hz, 3H), 2.58 (s, 6H), 3.31-3.34 (m, 1H), 4.48 (brs, 1H) ppm. Characteristic chemical shifts (δ) are given in parts-per-million using conventional abbreviations for designation of major peaks, such as s for singlet; d for doublet; t for triplet; q for a quartet; m for a multipet, and brs for broad singlet. (0085) FIG. 4 shows a Fourier-transform infrared (FTIR) spectrum of N,N-dimethyl-D-alanine, consistent with one or more exemplary embodiments of the present disclosure. Referring to FIG. 4, spectrum of N,N-dimethyl-D-alanine exhibited peaks at wavelengths 3441 cm-1, 3038 cm-1, 1625 cm-1, 1466 cm-1, 1402 cm-1, 1365 cm-1, 1094 cm-1, 1023 cm′ which may be attributed to the structure of N,N-dimethyl-D-alanine.
85%
With palladium 10% on activated carbon; hydrogen In water at 50℃; for 21h; Reflux;
1.B Example 1. B. Ν,Ν-dimethyl D-alanine (B).
General procedure: A larger scale dimethylation was carried out with 100 g D-Ala in 500 mL water with 250 mL formaldehyde, 30 g Pd/C, and 40 psi H2 pressure, and worked up in accordance with Example 1 A. Product was recrystallized from 100mL/500mL (EtOH:Acetone) to yield 110.5 g Ν,Ν-dimethyl D-alanine (B) (85% yield). Product was characterized by 1H- MR as in example 1A. To a solution of D-alanine (A) (50. Og, 561 mmol) in water (800mL) was added formaldehyde (37%, 125mL, 1,680 mmol) and Pd/C (10% Pd, 20g). After the flask was purged with Ar for 10 min and with H2 three times, the reaction mixture was stirred at 50°C under H2 (35 psi) for 20 hrs. The reaction mixture was then heated to reflux for lh and filtered while hot through a short pad of celite. The filtrate was concentrated under reduced pressure. More water (200mL each time, 3 times) was added and the reaction concentrated in order to remove the unreacted formaldehyde. Residual water was removed azeotropically with toluene (3 x lOOmL). 1H-NMR analysis revealed complete consumption of the starting material and clean and essentially quantitative production of the Ν,Ν-dimethylated product. The crude product was recrystallized with hot EtOH/acetone (50ml/250mL) at 55-60°C to give 41.36g (63.0%)) product as a white solid; another crop (11.38g, 17.3%) was obtained after the mother liquor was concentrated and the residual recrystallized from the same solvent system; further purification of the remaining product in the mother liquor was not pursued and the material was saved for future use. 1H NMR (300MHz, MeOH-13C NMR (75MHz, MeOH-i) δ 172.1, 66.0, 40.1, 11.6 ppm as shown in FIG. 8
With hydrogen
In methanol at 20℃;
16.12a
Example 16.12a. Synthesis of (/?)-2-(dimethylamino)propanoic acid A solution of (R)-2-aminopropanoic acid (5 g, 56.2 mmol, 1 equiv), excessformaldehyde solution and a catalytic amount of Pd/C in methanol (5.0 mL) was stirred at room temperature overnight. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give the desired product, which was directly used for next step without further purification. MS (ESI): 118 (MH+).
With palladium 10% on activated carbon; hydrogen In water for 16h;
4.4.3. Preparing L- and D-N,N-dimethylalanine (Choi et al., 2012)
General procedure: The L and D-alanine (8 mg) standards in H2O were individually treated with HCHO (27 μL) and 10% Pd/C (10.4 mg). The mixtures were subjected to H2 for 16 h and each reaction mixture was boiled and then dried under reduced pressure.
With triethylamine In ethanol at 50℃; for 8h; Yield given;
Stage #1: D-Alanine With triethylamine In ethanol; water at 20℃; for 0.5h;
Stage #2: Decyl-oxiran In ethanol; water at 50℃; for 9h;
Synthesis of N-(2-Hydroxydodecyl)-L-alanine (L-AlaC12OH)
General procedure: To a mixed solution of water and ethanol (40:60 by vol/vol, 20 mL in total) was added L-Alanine (502 mg, 5.63 mmol) and triethylamine (0.57 g, 5.63 mmol) and stirred for 30 min at ambient temperature. 1,2-epoxydodecane (1.05 g, 5.70 mmol) was added and stirred for nine hours at 50 °C. The solvent was evaporated and the resultant solid was recrystallized with ethanol, and colorless solid was obtained. Yield: 441 mg, (28.7 %).
General procedure: SOCl2 (21.8mL, 0.3mol) and (S)-alanine ((S)-19, 8.91g, 0.1mol) were added to CH3OH (100mL) and the mixture was stirred at RT for 2h. The solvent was removed in vacuo, the residue was dissolved in methanol (30mL) and the organic solvent was removed in vacuo again. This procedure was repeated twice. Colorless amorphous solid, mp 103C (Ref. 39 mp 98-99C), yield 14.3g (>99%).
90%
With thionyl chloride; at 0 - 20℃; for 24h;
In a separate reaction, the intermediate reagent V(E) was prepared by suspending V(D) (100 g, 1.12 mol) in 1 L of MeOH. The mixture was cooled to 0-5 0C. 50 mL of SOCI2 was added dropwise at 0-5 0C. The reaction mixture was then stirred at rt for 24 hours. The mixture was evaporated in vacuum to give compound V(E) (141 g, 90%) as a white solid.
Add 50g of methanol to the three-necked bottle, cool down to 0 C, start adding 23.8g of thionyl chloride, control the temperature -5-5 C, add the time of 25 minutes, after the completion of the addition, the heat preservation reaction for 1 hour, after the end of the heat preservation 15g of alanine was added, the temperature was controlled at 30-40 C for 3 hours, and then the temperature was raised to 40-45 C for 5 hours. After the reaction was completed, the temperature was lowered to 35 C, and the mixture was distilled under reduced pressure until the pressure was reduced to no fraction. Adding 15 g of methanol, stirring, and distilling under reduced pressure until no fraction is distilled off to obtain alanine methyl ester hydrochloride;
3.46 g
With thionyl chloride; at 20℃; for 6h;
D-alanine (2.5 g, 27.8 mmol) was added to a dry flask.Add anhydrous anaerobic methanol (14 mL), stir at 20 C for 10 min,Thionyl chloride (5 mL) was slowly added thereto, and the reaction was stirred for 6 hours.After the reaction was completed, the solvent was evaporated, diluted with diethyl ether and filtered.D-Alanine methyl ester 9 was obtained as a white solid, 3.46 g.
Stage #1: D-Alanine With sodium hydroxide In water at 0℃; for 0.166667h;
Stage #2: di-<i>tert</i>-butyl dicarbonate In tetrahydrofuran; water at 20℃;
15.1 Step 1: (2R)-2-(tert-butoxycarbonylamino)propanoic acid (17B)
[0228] No.28 D-alanine (17A) (10 g, 112.24 mmol) and No.5 water (56 mL) were added in a 250 mL reaction flask, and the reaction solution was cooled to 0°C and then No.5 sodium hydroxide (6.73 g, 168.36 mmol) was added. After the addition, the reaction was held at 0°C for 10 minutes, and then a solution of No.2 di-tert-butyl dicarbonate (31.85 g, 145.91 mmol) in No.2 tetrahydrofuran (50 mL) was added dropwise. After the addition, the temperature was raised to room temperature and stirred overnight. The reaction solution was extracted with petroleum ether (100 mL 2) and the organic phase was discarded. The aqueous phase was acidified with 4 M hydrochloric acid solution to a pH of about 1, and then extracted with ethyl acetate (100 mL 4). The organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain No.29 (2R)-2-(tert-butoxycarbonylamino)propanoic acid (17B) as colorless oily substance (21.2 g, yield: 100%). MS m/z =212.1 [M+Na]+; 1HNMR (400 MHz, DMSO-d6) δ 12.34 (br, 1H), 7.05 (d, 1H), 3.98-3.87 (m, 1H), 1.38 (s, 9H), 1.22 (d, 3H).
98%
With sodium hydroxide In tetrahydrofuran; water at 0 - 30℃; for 4.5h;
5.1 Step 1 (2R)-2-(tert-butoxycarbonylamino)propionicacid
(2R) -2-aminopropionic acid (10 g, 0.11 mol) was dissolved in water (100 mL) at room temperature. Tetrahydrofuran (50 mL) and sodium hydroxide solid (4.9 g, 0.12 mol) were added, then cooled to 0 . Boc anhydride (27 mL, 0.12 mol) was added. The mixture was heated to room temperature and stirred for 4.5 h. The organic solvent was removed by concentration, and the aqueous phase was adjusted pH to 3 with 1 M hydrochloric acid. The mixture was extracted with ethyl acetate (150 mL× 2) . The organic phase was washed with saturated salt water (100 mL) , dried over anhydrous sodium sulfate, filtered and concentrated to give the title compound as colorless oil (21 g, 98%) .
92%
With triethylamine; sodium hydroxide In tetrahydrofuran; water at 20℃; for 10.5h;
1.2 (2)N-tert-butoxycarbonyl-D-alanine5Preparation
Add 50 mmol of D-alanine and 100 mmol of sodium hydroxide to the mixed solution (10 ml THF/25 ml H 2 O).Triethylamine 60mmol,Take 50 mmol of di-tert-butyl dicarbonate and dissolve it in 15 ml of THF under ice bath.Slowly adding the above mixed solution with a constant pressure dropping funnel,After reacting for 30 minutes, the reaction was carried out to room temperature for about 10 hours.It was cooled to 0 ° C with an ice bath, and the pH was adjusted to 2 with 2 mol/L hydrochloric acid, and the mixture was extracted three times with ethyl acetate, 20 ml each time.The organic phases were combined and washed three times with saturated brine.20 ml each time, dry with an appropriate amount of anhydrous Na2SO4, and filter.Evaporating the organic solvent to give an oil.Add a small amount of petroleum ether and stir quickly until a large amount of solids appears.Filtered to a white solid. Yield 92%,
90%
With sodium hydroxide In 1,4-dioxane; water for 20h; Ambient temperature;
90.9%
With triethylamine In tetrahydrofuran; water at 0 - 20℃;
3.1.2. N-tert. Butoxycarbonyl-D-alanine (6a)
To a solution of compound D-alanine (6.23 g, 70 mmol) and triethylamine (11.76 ml, 84mmol) in 40 ml H2O and 20 ml tetrahydrofuran, was added dropwise a solution of 15.28 g(70 mmol) di-tert-butyl dicarbonate in 20 ml tetrahydrofuran at 0 °C. The reaction mixturewas stirred for 0.5 h at 0 °C, then was reacted overnight at room temperature keeping pH 8-9.After cooling to 0 °C the mixture was acidified to pH 2-3 by the dropwise addition of 2 mol/l HCl solution and extracted with ethyl acetate. The organic phase was washedwith saturated brine and dried over anhydrous MgSO4 and concentrated with a rotaryevaporator to get the colorless oil which was stirred in petroleum ether. The white solidwas filtered as the title compound. Yield: 90.9 %; mp: 80.5-81.3 °C.
87%
With sodium hydroxide In 1,4-dioxane; water at 5 - 20℃; for 72h;
82%
75%
With triethylamine In 1,4-dioxane; water at 20℃;
74%
With sodium carbonate In tetrahydrofuran; water at 0 - 20℃; for 48h;
73%
With sodium hydroxide In tetrahydrofuran; water at 20℃; for 3h;
1.1 Step 1 (2R)-2-[(tert-butoxy)carbonyl]amino}propanoic acid (1b)
D-alanine 1a (9.0 g, 0.10 mol) was dissolved in tetrahydrofuran (100 mL)a mixed solution with 10% aqueous sodium hydroxide solution (100 mL),Di-tert-butyl dicarbonate (25 mL, 0.11 mol) was added dropwise.The reaction was carried out for 3 hours at room temperature.Extracted with ethyl acetate (100 mL×2), the aqueous phase was adjusted to pH 2 with 4mol/L hydrochloric acid, and then extracted with dichloromethane/methanol (v/v=10/1, 100mL×2).The combined organic phases were dried over anhydrous sodiumThe title compound 1b was obtained as a white solid (14 g, yield 73%).Can be used directly in the next reaction.
With sodium hydroxide In <i>tert</i>-butyl alcohol for 12h; Ambient temperature;
With sodium hydroxide In tetrahydrofuran at 20℃; for 18h;
With sodium hydroxide In 1,4-dioxane at 0℃;
With sodium hydroxide In water; <i>tert</i>-butyl alcohol at 20℃;
Stage #1: D-Alanine; di-<i>tert</i>-butyl dicarbonate With potassium hydroxide In 1,4-dioxane; water at 20℃; for 12h;
Stage #2: With sodium hydrogen sulfate In water; ethyl acetate
Stage #1: D-Alanine With sodium hydrogencarbonate In tetrahydrofuran; water for 0.25h;
Stage #2: di-<i>tert</i>-butyl dicarbonate In tetrahydrofuran; water at 20℃; for 4h;
Intermediate 30To a solution of D-alanine (4.45 g, 50 mmol) in THF (100 mL) and water (50 mL) was added a solution of NaHC03 (4.2 g, 50 mmol) in water (30 mL). After stirring for 15 minutes, a solution ofBoc-anhydride (16.35 g, 75 mmol) in THF (20 mL) was added and the mixture was stirred at room temperature for 4 hours. The solvent was evaporated and 2N HCI was used to adjust the pH=3-4.The mixture was extracted with ethyl actetate (3 times 200 mL) and the combined ethyl acetate layers were washed with brine (50 mL), dried and concentrated. The residue was recrystallized with ethyl acetate/hexane to afford the title compound as a white solid (5 g).HNMR (DMSO-d6): δ ppm 12.38 (1 H, s), 7.1 1 - 7.09 (1 H, d), 3.94 - 3.88 (1 H, m), 1.38 (9H, s), 1.22- 1.21 (3H, d).
With potassium hydroxide In 1,4-dioxane; water at 25℃; for 12h;
1.78 g
Stage #1: D-Alanine; di-<i>tert</i>-butyl dicarbonate With potassium carbonate In methanol; water at 0 - 20℃; for 23.0833h;
Stage #2: With citric acid In methanol; water
81a Intermediate 81 a: (R)-2-tert-butoxycarbonylamino-propionic acid
To a suspension of D-alanine (1g, 1 1.22 mmol) in water (5.6 mL)/MeOH (2.80 mL) (2:1 ) K2C03 (1.55 g, 1 1.22 mmol) was added at 0-5°C, followed by a solution of Boc20 (2.61 mL, 1 1.22 mmol) in MeOH (2.80 mL) over 5 min. The resulting suspension was stirred for 23 h at rt. The reaction mixture was poured into water and acidified to pH= 3 with aqueous citric acid solution. The white cloudy solution was extracted with DCM (3x). Organic layers were combined, washed with brine, dried over MgS04, filtered and the solvent removed to yield 1.78 g, 9.43 mmol (R)-2- tert-butoxycarbonylamino-propionic acid.1H NMR (400 MHz, DMSO-c 6) δ ppm: 12.14 - 12.63 (m, 1 H), 6.97 - 7.19 (m, 1 H), 3.76 - 4.00 (m, 1 H), 1.38 (s, 9 H), 1.22 (d, J=7.09 Hz, 3 H).
With sodium hydroxide In tetrahydrofuran; water at 20℃; for 0.5h;
184 (R)-2-(tert-butoxycarbonylamino)propanoic acid (2):
To a stirring solution of (R)-2-aminopropanoic acid SM1 (0.2 g, 2.25 mmol) in THF (10 mL) was added (Boc)20 (0.58 g, 2.7 mmol) and 10% NaOH aq (10 mL), the mixture was stirred at room temperature for 30 mins. The solvent was removed out in vacuo, the residue was diluted with brine and extracted with EtOAc. Combined organic extracts were dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain cmde product as an off-white solid, which was useddirectly for subsequent step. ‘HNMR (300 MHz, DMSO-d6): 12.44 (s, ‘H), 7.11 (s, 1H), 3.91 (m, 1H), 1.47 (d, J = 3.0 Hz, 3H), 1.37 (s, 9H).
With sodium hydroxide In water at 0 - 20℃;
1 Example 1 Preparation of compound (VIII)
50 g of D-alanine, 52 g of sodium hydroxide and about 40 mL of water were added to a 1000 mL single-necked flask, and about 150 g of di-tert-butyl dicarbonate was added thereto at 0 ± 10 ° C. After completion of the dropwise addition, the mixture was stirred at room temperature overnight, Methyl-N-methoxyhydrochloride, 130 g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) After the addition, the mixture was stirred overnight at room temperature, and the reaction mixture was extracted with methylene chloride. The dichloromethane was washed once more with water, and the solvent was evaporated to give compound (VIII).
In chloroform
9.6 g
With potassium hydroxide In tetrahydrofuran; water
2.1 Ν- (tert-butoxycarbonyl) -D-alanine (8a)
(50 mmol) of D-alanine, 3.8 g (55 mmol) of potassium hydroxide and 13.0 g (55 mmol) of di-tert-butyl carbonate were suspended in a mixed solvent of water (200 mL) and THF (20 mL) Followed by stirring to give 9.6 g of N-Boc-D-alanine as a white solid which was used directly in the next esterification reaction without purification.
5.07 g
Stage #1: D-Alanine With sodium hydroxide In water at 26℃; for 0.166667h;
Stage #2: di-<i>tert</i>-butyl dicarbonate In water at 26℃; for 12h;
12 Synthesis of N-Boc-D-alanine compound 8, the reaction formula is:
D-alanine (2.5 g, 27.8 mmol) was added to a dry flask.Distilled water (14 mL) and 1 M aqueous sodium hydroxide (139 mL) were added.After stirring at 26 ° C for 10 min, (Boc) 2 O (12.12 g, 55.6 mmol) was added thereto.The reaction was carried out at 26 ° C for 12 hours, and the reaction compound was lowered to 0 ° C.After adding 3M hydrochloric acid to adjust pH 2, the reaction mixture was diluted with 50 mL of ethyl acetate, and then the mixture was extracted with ethyl acetate three times, 70 mL each time, and the organic phase was combined.Dry over anhydrous sodium sulfate. The solvent is distilled off to obtain N-Boc-D-alanine 8,White solid 5.07 g.
Stage #1: D-Alanine With sodium carbonate In 1,4-dioxane; water
Stage #2: di-<i>tert</i>-butyl dicarbonate In 1,4-dioxane; water for 18h;
General procedure for the preparation of amino acidderivatives
General procedure: The amino acid derivatives were prepared employinga methodology developed by our research group.19Initially, the coupling between the tert-butyloxycarbonyl(Boc) protecting group and amino acids (L-alanine,D,L-alanine, D-alanine, L-valine, L-leucine, L-isoleucineor L-phenylalanine, 1.0 mmol) was done by adding toa solution of 1,4-dioxane/water (40:60 v/v) the aminoacid and 50 mg of Na2CO3. The mixture was stirred untilcomplete dissolution and then Boc2O (1.5 mmol) wasadded. The reaction was kept at magnetic stirring for 18 hand then purified employing literature protocols. For the amide bond formation, the previously preparedBoc amino acids (1.0 mmol) were transferred to a famedriedglassware vial with dichloromethane (5.0 mL) andthen EDC (3-dimethylaminopropyl)-N-ethylcarbodiimide)hydrochloride (1.1 mmol) was added. The reaction stirredat 0 °C for 30 min, and then washed twice with distilledwater. The organic layer was immediately transferred toa vial with the amine nucleophile (2.0 mmol) and the(±)-camphorsulfonic acid organocatalyst (0.1 mmol,10 mol%). After 24 h the dichloromethane was removedand the desired compounds purified by recrystallization,liquid-liquid extraction or column chromatography.Boc deprotection of two of the previously preparedproducts was carried out following literature protocols,21in which the derivatives (1 mmol) and trifluoroacetic acid(22 mmol) were added in dichloromethane (5 mL) and keptunder magnetic stirring for 3 h, affording the salts 33 and34 after solvent removal.
With trifluoroacetic acid; sodium nitrite In water at 20℃; for 6h;
With sulfuric acid; sodium nitrite In water at 0℃; for 2h;
With acetic acid; sodium nitrite
With hydrogenchloride; sodium nitrite at 4℃;
With sulfuric acid; sodium nitrite In water at 20℃; for 27h; Cooling with ice;
1. 4.1 D-α-hydroxy acid 10(A-C, G)
General procedure: A 60 ml of aqueous solution of sodium nitrite (20.7 g, 0.3 mol) was added into a stirred and ice-cooled solution of D-amino acids (9A-C, G, 50 mmol) in 1 M H2SO4 (100 ml, 0.1 mol) over 3 h, and the mixture was stirred for 24 h at room temperature until the completion of the reaction (monitored by ninhydrin). The mixture was adjusted to pH 6 with solid NaHCO3 and then to pH 3 with concentrated HCl followed by freeze-drying. The resulting residue was extracted with hot acetone (4×100 ml), and the extracts were concentrated and dried to offer colorless oil, to which ether (200 ml) was added and filtrated to remove insoluble solids, the filtrate was concentrated and re-crystallized in ether/hexanes mixture to afford 10(A-C, G) as white crystalline, yield 82%-92%.
With water; acetic acid; sodium nitrite at 0 - 20℃; for 24h; Inert atmosphere;
With lithium aluminium tetrahydride In tetrahydrofuran at 0℃; for 18.5h; Reflux;
1 L- propylamine alcohol (): Preparation Example 1
The present embodiment relates to a class of long-chain ethyl piperazine sulfonamide derivative intermediate () in the general synthetic methods.Particularly to the synthesis of L- propylamine alcohol.The 150mL dry THF and 1.1gLiAlH4(29mmol) views into a three-necked flask, cooled to 0 deg.] C, the 4.9gL- alanine (55.0 mmol) portionwise into the reactor, at 30min the addition was completed, stirring was continued for 2h, gradually warming to reflux and heating continued for 16h , the reaction was stopped, the ice bath was cooled, diluted with 100mL of diethyl ether, 4.5mL water successively, 4.5mL15% NaOH and 12mL of water into the reactor, the reaction solution was filtered through celite, the filtrate was collected, the solvent was evaporated under reduced pressure to obtain a crude product, purified by column chromatography on silica gel, eluting with methylene chloride: ethanol = 10 to yield of 3.41 g of a colorless oil, yield 82.5%.
73%
Stage #1: D-Alanine With lithium aluminium tetrahydride In tetrahydrofuran at 0℃; for 6h; Inert atmosphere; Reflux;
Stage #2: With sodium hydroxide In tetrahydrofuran; water for 0.25h; Inert atmosphere;
4.3 Synthesis of 1-(octyloxy)propyl-2-amine (IIc) (Scheme 4).
(R)-Alanine (10.0 g, 112 mmol) was added solwly to a mixture of lithium aluminium hydride (8.50 g, 224 mmol) in dry THF (300ml) at 0. The mixture was refluxed for 6h, water (8.50 ml) was added slowly, stirred for 15min. Then 15% NaOH aqueous solution(8.5 ml) was added to the mixture. The crude product was purified by vacuum distillation (Bp: 54 (700Pa)) to give (R)-Alaniol(6.15 g) as a colorless liquid in yield of 73%.
With lithium aluminium tetrahydride In tetrahydrofuran for 10h; Heating; Yield given;
With lithium aluminium tetrahydride In tetrahydrofuran 1.) r.t., 12 h, 2.) reflux, 18 h;
With lithium borohydride; chloro-trimethyl-silane In tetrahydrofuran at 0℃;
Stage #1: D-Alanine With lithium aluminium tetrahydride In tetrahydrofuran for 12h; Heating / reflux;
Stage #2: With sodium hydroxide; water In tetrahydrofuran at 0 - 20℃; for 0.166667h;
1.1
Step 1 (S)-Boc-2-amino-propanol D-Alanine (3.5 g, 39.3 mmol) was added in small portions to a suspension of LiAlH4 (2.89 g, 76.26 mmol) in refluxing THF. Refluxing continued for 12 hours, then the reaction mixture was cooled to 0° C., and excess reagent was quenched by careful addition of an aqueous 15% NaOH solution (3 ml) and water (9 ml). After stirring at room temperature for 10 minutes, a solution of (Boc)2O (8.31 g, 38.13 mmol) in CH2Cl2 (40 ml) was added. The reaction mixture was stirred at 60° C. for 6 hours, cooled to room temperature, filtered through a pad of anhydrous Na2SO4, and the filtrate concentrated under vacuum. Purification of the residue by silica-gel column chromatography afforded (S)-Boc-2-amino-propanol as a white solid, yield: 63%. MS (M+H)=176.
With lithium aluminium tetrahydride Inert atmosphere;
With sodium tetrahydroborate; sulfuric acid; iodine In tetrahydrofuran Reflux;
With lithium aluminium tetrahydride In tetrahydrofuran
With sodium tetrahydroborate In tetrahydrofuran at 0 - 80℃; for 12h; Inert atmosphere; Schlenk technique;
Stage #1: D-Alanine With boron trifluoride diethyl etherate In tetrahydrofuran at 70℃; for 1.5h;
Stage #2: With dimethylsulfide borane complex In tetrahydrofuran at 70℃; for 17h;
90
Example-90 4-((R)-2-Hydroxy-1-methyl-ethylamino)-2-trifluoromethyl-benzonitrile D-Alanine (36 mg, 0.40 mmol) was dissolved in THF (dry, 1 ml) and the vials were purged with N2 for 5 min. BF3-Et20 (0.050 ml 0.40 mmol) was added with syringe and the mixture was heated at 70°C for 1. 5 h. BH3-SMe2 (0.22 ml, 0.44 mmol, 2M solution) was added carefully during vigorous stirring (an exoterm was formed approx half way) (a evolution of gas was noticed). The reactions was purged with N2 and then heated at 70°C over night (17h). The reaction was allowed to cool to room temp. The excess borane was quenched by addition of 1 ml of a 1: 1 mixture of THF: H20, followed by 1 ml of NaOH (5M). The two phase system was heated at 70°C in 4h. The flask was purged with N2 to blow off the THF. CH2Cl2 (2 ml) was added and the two phase system was transformed to a Phase separator. Additional CH2Cl2 (2 ml) was added and the combined organic phases were evaporated. The crude (21 mg) was then dissolved in DMSO and the reaction was continued as in example 1. 4-Fluoro-2-trifluoromethyl- benzonitrile (19 mg, 0.1 mmol) was coupled with the formed (R)-2-amino-propan-1-ol. DIPEA (0.021 ml, 0.12 mmol), in 1 mL DMSO using the same procedure as described in Example-1. Purification on preperative HPLC gave 4 mg (16 %) of 4-((R)-2- Hydroxy-l-methyl-ethylamino)-2-trifluoromethyl-benzonitrile as a white solid.
With sodium carbonate In 1,4-dioxane; water at 20℃; for 20h;
92.7%
With sodium hydroxide In water at 0 - 20℃;
89%
Stage #1: D-Alanine; benzyl chloroformate With sodium hydroxide In water at 0 - 20℃;
Stage #2: With hydrogenchloride In water
II.29.1
II. Indole Preparations; Intermediate 29: Synthesis of tert-Butyl (2R)-2-Methyl-4-(1H-pyrrolo[3,2-b]pyridin-3-yl)piperazine-1-carboxylate; 1. Synthesis of (2R)-2-[(benzyloxy)carbonyl]amino}propanoic acid; Benzyl chloroformate (168 mmol) is added dropwise to a solution of (2R)-2-(amino)propanoic acid (169 mmol) in 2 M sodium hydroxide (96 mL) at 0° C. and the reaction mixture is maintained for 3 h at rt. The resulting solution is extracted with diethyl ether (50 mL) and the pH of the aqueous layer is adjusted to 4-5 by the addition of 2 N hydrochloric acid. The aqueous layer is extracted two times with ethyl acetate (2×100 mL) and the combined organic layers are dried (sodium sulfate) and concentrated to provide (2R)-2-[(benzyloxy)carbonyl]amino}propanoic acid in 89% yield as a white solid.
With sodium hydroxide for 0.333333h; Ambient temperature; Yield given;
With sodium carbonate In tetrahydrofuran at 20℃; for 4h;
With sodium hydroxide In water at 10 - 30℃; for 5h;
3 Example 3 General formula 6 synthesis
Add 35g (0.393mol) D-alanine (compound 4), 33g (0.825mol) sodium hydroxide, 150g water into the flask, and add 1.05 equivalent 70.4g (0.41mol) CbzCl (benzyl chloroformate) at 10-15. After the addition, the temperature was raised to 20-30°C and reacted for 5 hours. After the reaction, the pH was adjusted to 2 with 3M hydrochloric acid, and extracted with 100g of toluene for three times. The solvent was removed under reduced pressure to obtain general formula 5, which was used directly in the next step Reaction; then add 180g of toluene, 1.1 equivalent of 74.4g (0.432mol) p-toluenesulfonic acid and 11.8g (0.393mol) formaldehyde in formula 5, turn on the stirring, and react at 20-25 for 12h. After the reaction, the toluene phase is washed with water Two times, the toluene phase was collected, and the solvent was removed under reduced pressure to obtain 86.9 g of the compound of general formula 6, Y=94%, and HPLC purity 97%.
With sodium hydroxide In water at 60 - 70℃; for 0.5h;
33%
With sodium hydroxide In water at 60℃; for 6h;
Synthesis of tosyl alanine (Fig. 1) (3):
To the suspension of D-alanine (1 g, 11.2 mmol) in 25 mL water at room temperature, NaOH (1.34 g, 33.6 mmol) and p-toluene sulfonylchloride (2.56 g, 13.4 mmol) were added. The mixture was stirred at 60 °C for 6 h. The combined basic layers were cooled to 0 °C and acidified (pH = 1) by the addition of conc. HCl.The precipitates were collected by filtration, washed with cold water and air dried to give tosyl alanine as a white solid (0.90 g,33 %).
With sodium hydroxide In diethyl ether; water at 23℃; for 6h;
With sodium hydroxide at 65 - 70℃;
With N-ethyl-N,N-diisopropylamine; sodium hydroxide In water; acetone at 20℃; for 16h; Inert atmosphere;
With isopropylamine In aq. buffer at 37℃; for 24h; Enzymatic reaction;
2.7 Preparative-Scale Deracemization
General procedure: Deracemization of dl-1a in a preparative-scale was carried out in a 25 mL reaction mixture (50 mM Tris buffer, pH 7.0)charged with 0.22 g dl-1a (2.5 mmol), 0.15 g 3 (2.5 mmol),17 mg NAD+,1750 U AlaDH, 40 U ARTAmut and 250 U NOX. Reaction was carried under magnetic stirring at 37 °C.Aliquots (typically 10 μL) of the reaction mixture were takenat predetermined reaction time and mixed with 40 μL of 5 NHCl after 20-fold dilution with water. Quantitative chiral analysis of dl-1a was performed by HPLC for determination of eeD. When eeD of d-1a exceeded 99%, reaction was stopped and then product isolation was performed. The pH of the reaction mixture was adjusted to 1.0 byadding 5 N HCl and then filtered using a glass-fritted filter funnel to remove protein precipitates. The filtrate was loaded on a glass column packed with Dowex 50WX8 cation exchange resin (40 g), followed by washing with 0.1 N HCl(100 mL) and then water (50 mL). Elution was performed by 10% (v/v) aqueous ammonia solution (150 mL) and then the eluate was evaporated at 70 °C and 0.25 bar. The resulting white solid was washed with ethanol (20 mL) and dried overnight at 37 °C.
49%
With D-glucose; Bacillus subtilis glucose dehydrogenase; Symbiobacterium thermophilum meso-Diaminopimelate dehydrogenase W121L/H227I mutant; NADP; ammonium chloride In aq. buffer at 37℃; for 24h; Enzymatic reaction;
With ammonium hydroxide; D-amino acid oxidase In phosphate buffer for 10h; Electrochemical reaction;
> 99.9 % ee
With (R)-selective ω-transaminase from arthrobacter sp.; isopropylamine In aq. phosphate buffer at 37℃; for 0.2h; Enzymatic reaction;
With (R)-1-phenyl-ethyl-amine; R-selective ω-transaminase from Arthrobacter sp
Stage #1: D-Alanine; ethyl trifluoroacetate, With N,N,N',N'-tetramethylguanidine In methanol at 20℃; for 5h;
Stage #2: With hydrogenchloride In methanol; water
15.1
1) Preparation of (R)-N-trifluoroacetyl-2-amino propionic acid(R)-2-amino propionic acid (8.9 g, 0.1 mol) was dissolved in 100 ml of methanol, and 1,1,3,3-tetramethyl guanidine (15.6 g, 0.136 mol) was added. At room temperature, trifluoroethyl acetate (18.5 g, 0.13 mol) was added dropwise. After the completion of the addition, stirring is conducted to allow a reaction to proceed for 5 hours. Most solvent was removed by evaporation, and the residue was poured into 100 ml of water, acidified with 12 ml of concentrated hydrochloric acid, and extracted with ethyl acetate (3×60 ml). The organic phases were combined, washed with saturated brine, dried with anhydrous sodium sulfate, concentrated and crystallized, washed with 160 ml of hexane, filtered, and dried, obtaining 18.0 g of product. The yield was 97.3%. MS: m/z 185.1
95%
With triethylamine In methanol at 20℃; for 24h;
General procedure for the preparation of TFA-α-amino acid.
General procedure: The TFA-α-amino acid was prepared with reported procedure [1,2] with slightly modification.Triethylamine (33 mmol, 1.5 equiv.) was added to a solution of α-amino acid (22 mmol) inMeOH (22 mL). After 5 min, ethyl trifluoroacetate (29 mmol, 1.3 equiv.) was added and thereaction was allowed to stir for 24 h. The solvent was removed by rotary evaporation and theresidue that remained was dissolved in H2O (35 mL) and acidified with concentrated HCl (4 mL).After stirring for 15 min, the mixture was extracted with ethyl acetate and the organic layerswere combined and washed with brine, dried by MgSO4, filtered, and concentrated by rotaryevaporation. Further subjection into high vacuum for overnight, if needed to solidify the product(L-/D-1a-L-/D-2a, 3a, L-/D-4a-L-/D-8a).
86%
With triethylamine In methanol for 24h;
86%
With triethylamine In methanol at 20℃; for 8h;
2.1
Alternatively, N-trifluoroacetyl alanine can be prepared by suspending (2.0 g, 22 mmol) L-alanine in methanol (11 mL) and adding triethylamine (3.1 mL, 22 mmol) to the suspension with stirring. After stirring for about 5 min, ethyl trifluoroacetate (3.3 mL, 28 mmol) was added and the resulting mixture stirred for about 8 h at room temperature. The solvent was removed under reduced pressure and the resulting residue dissolved in water (50 mL), acidified with concentrated aqueous hydrochloric acid (4 mL), and stirred for about 15 min. The mixture was then extracted with ethyl acetate (2×30 mL) and the organic layers combined. The combined organic layers were then washed with brine (50 mL), dried (Na2SO4), and the ethyl acetate removed under reduced pressure to provide a solid which washed with n-hexane and dried to provide N-trifluoroacetyl alanine. (2.7 g, 86%, mp 68° C.; 1HNMR (CDCl3) δ 1.58 (d, 3H, J=7.5 Hz), 4.68 (p, 1H, J=7.5 Hz), 6.87 (bs, 1H, NH)).
80.8%
Stage #1: D-Alanine; ethyl trifluoroacetate, With triethylamine In methanol at 20℃; for 24h;
Stage #2: for 0.166667h; Acidic conditions;
1
To the mixture of D-alanine (20.00 g; 0.224 mol) and triethylamine (31.3 ml ; 0.224 mol) in absolute methanol (100 ml), ethyl trifluoroacetate (33.4 rnl ; 0.280 mol) is added and stirred at room temperature to homogenise the mixture (approx. 1 day). The solution is concentrated on a rotavapor (35 °C ; 16 mmHg) and then dissolved in the mixture THF/water (1: 1; 140 ml). Acidic ion exchanger Dowex 50W-X8 (100 g) is added, stirred for 10 minutes, filtered and concentrated again on a rotavapor (35 °C ; 16 mmHg). The residue is sublimed (80 °C ; 0.05 mmHg). The pure product in the form of colourless crystals is obtained (33.50 g; 80. 8 %).
With sodium hydrogencarbonate In water at 20℃; for 24h;
With sodium hydrogencarbonate
Stage #1: D-Alanine; chloroformic acid ethyl ester With sodium hydroxide at 15℃;
Stage #2: With phosphoric acid In water at 0 - 5℃;
2
Reference Example 2 Preparation of R-(N-Ethoxy carbonyl) alanine (III) R-Alanine (4.45 g, 0.05 moles) was dissolved in 50 ml 1 N sodium hydroxide solution and cooled to 15 °C. Ethyl chloro formate (5 ml, 0.05 mole) was added while maintaining the pH of reaction mixture at 9-9.5 by using IN sodium hydroxide solution. Reaction mixture was cooled to 0-5 °C and extracted with diethyl ether. Orthophosphoric acid was added to aqueous layer and pH was adjusted below 1.0. Product was extracted in dichloromethane. Organic layer was dried over sodium sulphate. The solvent was evaporated under reduced to give 6.5 g of R-(N-Ethoxy carbonyl) alanine as oil.
Stage #1: D-Alanine With sodium hydroxide In water at 15℃;
Stage #2: chloroformic acid ethyl ester In water
Stage #3: With phosphoric acid In water
2
R-Alanine (4.45 g, 0.05 moles) was dissolved in 50 ml 1 N sodium hydroxide solution and cooled to 15° C. Ethyl chloroformate (5 ml, 0.05 mole) was added while maintaining the pH of reaction mixture at 9-9.5 by using 1N sodium hydroxide solution. Reaction mixture was cooled to 0-5° C. and extracted with diethyl ether. Orthophosphoric acid was added to aqueous layer and pH was adjusted below 1.0. Product was extracted in dichloromethane. Organic layer was dried over sodium sulphate. The solvent was evaporated under reduced to give 6.5 g of R-(N-Ethoxy carbonyl)alanine as oil.
N-(2,4-dinitro-5-fluorophenyl)-L-alaninamide[ No CAS ]
D-Ala-FDAA[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
With sodium hydrogencarbonate In water; acetone at 40℃; for 1h;
With sodium hydrogencarbonate In acetone at 45℃; for 1.5h;
With sodium hydrogencarbonate In water; acetone at 40℃; for 1h;
With sodium hydrogencarbonate In acetone at 40℃; for 1h;
With sodium hydrogencarbonate In acetone at 45℃; for 1h;
With sodium hydrogencarbonate In water; acetone at 40℃; for 1h;
With sodium hydrogencarbonate In acetone at 40℃; for 1h;
With sodium hydrogencarbonate In water; acetone at 40℃; for 1h;
With sodium hydrogencarbonate In water at 40℃; for 1.33333h;
With sodium hydrogencarbonate In acetone at 37℃; for 1h;
Stereochemistry of amino acids constituting verlamelin A and B
General procedure: One milligram of aliquots of 1 or 2 were hydrolyzed by heating at 115 1C for8 h in 10 ml of 6M HCl. After cooling to room temperature, theywere completely dried in vacuo and dissolved in 150 ml of water. Marfey’sreagent (300 ml of 10mgml1 solution in acetone) (Na-(2,4-dinitro-5-fluorophenyl)-L-alaninamide) (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan)was added, followed by the addition of 70 ml of 1M NaHCO3. The reactionsproceeded at 37 1C for 1h and were quenched by addition of 70ml of 1M HCl.The resulting mixture was dried in vacuo and then dissolved in 1ml of DMSOto be analyzed by HPLC. Marfey’s derivatives of amino acids as a standard wereprepared by reacting 50mM of amino acids in the same manner as describedabove. HPLC analysis was carried out on a Shiseido Capcell-Pak C18 column(4.6250mm) with a linear gradient of CH3CN from 10% to 50% in 0.05%aqueous TFA solution (60min from 10% to 50%, 5 min at 50%, 3 min from50% to 10%), at a flow rate of 1.0mlmin1, with detection at 340 nm. Theglutamine (Gln) residues of 1 and 2 should be converted by acid hydrolysis toglutamic acid (Glu). Retention times (min) of Marfey’s derivatives used asstandards were as follows: L-Ser (25.6), D-Ser (26.0), L-Thr (27.0), D-Thr(31.8), L-alloThr (27.5), D-alloThr (29.3), L-Glu (30.1), D-Glu (31.9), L-Pro(34.3), D-Pro (36.0), L-Ala (33.2), D-Ala (37.2), L-Val (42.1), D-Val (48.1),L-Tyr (57.9) and D-Tyr (40.4, 62.2).
With sodium hydrogencarbonate In water; acetone at 43℃; for 1h;
With sodium hydrogencarbonate In water; acetone at 80℃; for 0.166667h;
With sodium hydrogencarbonate In water; acetone at 40℃; for 1h;
With sodium hydrogencarbonate In water at 40℃; for 1.5h;
3.4. Hydrolysis of Compounds 1-2 and HPLC Analysis by Marfey’s Method
Compounds 1 (1.0 mg) and 2 (1.4 mg) were dissolved in 6 N HCl (1 mL), and heated at 110 °C for18 h. After cooling to room temperature, the hydrolysates were dried under reduced pressure and resuspended into 100 μL of H2O.Then they were treated with 1 M NaHCO3 (25 μL), and reacted with100 μL of 1% (w/v) FDAA in acetone at 40 °C for 1.5 h. After cooling to room temperature, the mixture was added with 1 M HCl (25 μL) to neutralize and terminate the reaction. MeOH was then added to the quenched reaction to afford a total volume of 500 μL; 10 μL of each hydrolysate derivatization reaction was used for HPLC analysis using an Agilent C18 column (150 × 4.6 mm,5 μM) with a solvent gradient from 15% to 45% solvent B (solvent A: CH3COOH/H2O, 0.05/99.95,solvent B: CH3CN) over the course of 30 min and UV detection at 340 nm at a flow rate of 1 mL/min.Similarly, 10 μL of the standard amino acids in H2O (4 μM) were added to 1 M NaHCO3 (20 μL) and each mixture was treated with 1% (w/v) FDAA (50 μL) for 1.5 h at 40 °C. Derivatization reactions were terminated with 1 M HCl (20 μL) and diluted to a total volume of 500 μL with MeOH. Of these standard amino acid derivatization reactions, 10 μL was subjected to HPLC analysis and used as structural standards in the elucidation of structures 1 and 2.
With sodium hydrogencarbonate In water; acetone at 40℃; for 1h;
With sodium hydrogencarbonate In acetone at 40℃; for 90h;
General procedure: Each amino acid was added with 0.1% solution of Nα-(5-fluoro-2,4-dinitrophenyl)-l-alaninamide (l-FDAA, Marfey's reagent, 200 μL) in acetone and 0.5M NaHCO3 (100 μL) followed by heating at 40 °C for 90 min. After cooling to room temperature, the reaction mixture was neutralized with 2N HCl (25 μL) and diluted with MeOH (300 μL). The solution was subjected to reversed-phase HPLC [Cosmosil 5C18-AR-II (4.6×250 mm), MeOH/20 mM AcONa=55:45 (solvent A) or 45:55 (solvent B) at 1.0 mL/min, UV detection at 340 nm].
With sodium hydrogencarbonate In water; acetone at 40℃; for 1.5h;
With sodium hydrogencarbonate In water; acetone at 45℃; for 1h;
Stage #1: D-Alanine With sodium hydrogencarbonate In water
Stage #2: N-(2,4-dinitro-5-fluorophenyl)-L-alaninamide In water; acetone at 60℃; for 1h;
Step 1 D-alanine amide D-alanine (10 g, 71.6 mmol) was dissolved in EtOH in a pressure vessel. The solution was cooled to -78. NH3 (l) (90 mL) was condensed into the reaction mixture. The pressure vessel was sealed and stirred at room temperature for 2 days. The excess ammonia was released from the vessel. The resulting suspension was concentrated to yield the title compound as a white solid which was dried under hi-vacuum for 16 hours overnight. 1 H NMR (400 MHz, CDCl3) delta (12.52 (bs, 1H); 7.37 (d, 1H); 7.14 (d, 1H); 2.49 (s, 3H)
Stage #1: D-Alanine; 2-Chloroethyl chloroformate With sodium hydrogencarbonate In 1,4-dioxane; water
Stage #2: With hydrogenchloride In water
1
To a solution Of NaHCO3 (9.43 g, 112 mmol) and H-D-AIa-OH (2 0 g, 22.5 mmol) m water (110 mL) was slowly added a solution of 2-chloroethyl chloroformate (2.55 mL, 24.7 mmol) in dioxane (100 mL). The reaction mixture was stirred overnight. Dioxane was removed by evaporation and the resulting aqueous solution was acidified with 1 N HCl to pH = 3. The solution was extracted three times with ethyl acetate (3 x 175 mL) The combined organic layers were dried over Na2SO4, filtered, and concentrated to dryness, which gave 2 (4.29 g, 21. 9 mmol, 97%) as a viscous oil. - 1H NMR (300 MHz, CDCl3) δ: 1.49 ppm (d, 3H, J = 7.2 Hz, CH3-AIa), 3.68 (t, 2H, J = 5.7 Hz, CH2Cl), 4.32-4.42 (m, 3H, CH2OR + α-H)
r Methods of Preparing Compounds of the Invention
D-alanine (1) was reacted with 2-chloroethyl chloroformate to provide N-(2-chloroethoxycarbonyl)-D-Ala-OH (2), which was transformed into AEC-D-Ala-OH (3) through a substitution reaction with sodium azide in DMF and then activated to AEC-D-Ala-OSu (4) with DCC and N-hydroxysuccinimide.
With sodium hydrogencarbonate In 1,4-dioxane; water
1 Example 1
Example 1 N-(2-Chloroethyloxycarbonyl)-D-Ala-OH (2): To a solution of NaHCO3 (9.43 g, 112 mmol) and H-D-Ala-OH (2.0 g, 22.5 mmol) in water (110 mL) was slowly added a solution of 2-chloroethyl chloroformate (2.55 mL, 24.7 mmol) in dioxane (100 mL). The reaction mixture was stirred overnight. Dioxane was removed by evaporation and the resulting aqueous solution was acidified with 1 N HCl to pH=3. The solution was extracted three times with ethyl acetate (3*175 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated to dryness, which gave 2 (4.29 g, 21.9 mmol, 97%) as a viscous oil. 1H NMR (300 MHz, CDCl3) δ: 1.49 ppm (d, 3H, J=7.2 Hz, CH3-Ala), 3.68 (t, 2H, J=5.7 Hz, CH2Cl), 4.32-4.42 (m, 3H, CH2OR+α-H).
(R)-2-tertbutoxycarbonylamino-1-propanol[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
63%
Stage #1: D-Alanine With lithium aluminium tetrahydride In tetrahydrofuran for 12h; Heating / reflux;
Stage #2: With sodium hydroxide; water In tetrahydrofuran at 0 - 20℃; for 0.166667h;
Stage #3: di-<i>tert</i>-butyl dicarbonate In tetrahydrofuran; dichloromethane; water at 60℃; for 6h;
4.1
Preparation 4: (5)-2-Methoxy-l-methyl-ethylamine; The synthetic procedure used in this preparation is outlined below in Scheme F. Step 1 (5)-Boc-2-amino-propanol; D-Alanine (3.5g, 39.3 mmol) was added in small portions to a suspension Of LiAlH4 (2.89g, 76.26 mmol) in refluxing THF. Refluxing continued for 12 h, then the reaction mixture was cooled to 00C, and excess reagent was quenched by careful addition of an aqueous 15% NaOH solution (3 ml) and water (9 ml). After stirring at RT for 10 min, a solution of (Boc)2O (8.3 Ig, 38.13 mmol) in CH2Cl2 (40 ml) was added. The reaction mixture was stirred at 600C for 6 h, cooled to RT, filtered through a pad of anhydrous Na2SO4, and the filtrate concentrated under vacuum. Purification of the residue by silica- gel column chromatography afforded (S)-Boc-2-amino-propanol as a white solid, yield: 63%. MS (M+H) = 176.
63%
Stage #1: D-Alanine With lithium aluminium tetrahydride In tetrahydrofuran for 12h; Heating / reflux;
Stage #2: With sodium hydroxide; water In tetrahydrofuran at 0 - 20℃; for 0.166667h;
Stage #3: di-<i>tert</i>-butyl dicarbonate In tetrahydrofuran; dichloromethane; water at 60℃; for 6h;
5.1
Step 1 (S)-Boc-2-amino-propanol; D-Alanine (3.5 g, 39.3 mmol) was added in small portions to a suspension of LiAlH4 (2.89 g, 76.26 mmol) in refluxing THF. Refluxing continued for 12 hours, then the reaction mixture was cooled to 0° C., and excess reagent was quenched by careful addition of an aqueous 15% NaOH solution (3 ml) and water (9 ml). After stirring at room temperature for 10 minutes, a solution of (Boc)2O (8.31 g, 38.13 mmol) in CH2Cl2 (40 ml) was added. The reaction mixture was stirred at 60° C. for 6 hours, cooled to room temperature, filtered through a pad of anhydrous Na2SO4, and the filtrate concentrated under vacuum. Purification of the residue by silica-gel column chromatography afforded (S)-Boc-2-amino-propanol as a white solid, yield: 63%. MS (M+H)=176.
63%
Stage #1: D-Alanine With lithium aluminium tetrahydride In tetrahydrofuran for 12h; Heating / reflux;
Stage #2: With sodium hydroxide; water In tetrahydrofuran at 0 - 20℃; for 0.166667h;
Stage #3: di-<i>tert</i>-butyl dicarbonate In dichloromethane; water at 60℃; for 6h;
1.1
Step 1 (S)-Boc-2-amino-propanol; D-Alanine (3.5 g, 39.3 mmol) was added in small portions to a suspension of LiAlH4 (2.89 g, 76.26 mmol) in refluxing THF. Refluxing continued for 12 hours, then the reaction mixture was cooled to 0° C., and excess reagent was quenched by careful addition of an aqueous 15% NaOH solution (3 ml) and water (9 ml). After stirring at room temperature for 10 minutes, a solution of (Boc)2O (8.31 g, 38.13 mmol) in CH2Cl2 (40 ml) was added. The reaction mixture was stirred at 60° C. for 6 hours, cooled to room temperature, filtered through a pad of anhydrous Na2SO4, and the filtrate concentrated under vacuum. Purification of the residue by silica-gel column chromatography afforded (S)-Boc-2-amino-propanol as a white solid, yield: 63%. MS (M+H)=176.
63%
Stage #1: D-Alanine With lithium aluminium tetrahydride In tetrahydrofuran for 12h; Heating / reflux;
Stage #2: di-<i>tert</i>-butyl dicarbonate With sodium hydroxide; water In tetrahydrofuran; dichloromethane at 60℃; for 6h;
1.1
Step 1 (S)-Boc-2-amino-propanol D-Alanine (3.5 g, 39.3 mmol) was added in small portions to a suspension of LiAlH4 (2.89 g, 76.26 mmol) in refluxing THF. Refluxing continued for 12 hours, then the reaction mixture was cooled to 0° C., and excess reagent was quenched by careful addition of an aqueous 15% NaOH solution (3 ml) and water (9 ml). After stirring at room temperature for 10 minutes, a solution of (Boc)2O (8.31 g, 38.13 mmol) in CH2Cl2 (40 ml) was added. The reaction mixture was stirred at 60° C. for 6 hours, cooled to room temperature, filtered through a pad of anhydrous Na2SO4, and the filtrate concentrated under vacuum. Purification of the residue by silica-gel column chromatography afforded (S)-Boc-2-amino-propanol as a white solid, yield: 63%. MS (M+H)=176.
63%
Stage #1: D-Alanine With lithium aluminium tetrahydride In tetrahydrofuran for 12h; Reflux;
Stage #2: With water; sodium hydroxide In tetrahydrofuran at 0 - 20℃; for 0.166667h;
Stage #3: di-<i>tert</i>-butyl dicarbonate In tetrahydrofuran; dichloromethane; water at 60℃; for 6h;
1.1
Step 1 (S)-Boc-2-amino-propanolD-Alanine (3.5 g, 39.3 mmol) was added in small portions to a suspension of LiAlH4 (2.89 g, 76.26 mmol) in refluxing THF. Refluxing continued for 12 hours, then the reaction mixture was cooled to 0° C., and excess reagent was quenched by careful addition of an aqueous 15% NaOH solution (3 ml) and water (9 ml). After stirring at room temperature for 10 minutes, a solution of (Boc)2O (8.31 g, 38.13 mmol) in CH2Cl2 (40 ml) was added. The reaction mixture was stirred at 60° C. for 6 hours, cooled to room temperature, filtered through a pad of anhydrous Na2SO4, and the filtrate concentrated under vacuum. Purification of the residue by silica-gel column chromatography afforded (S)-Boc-2-amino-propanol as a white solid, yield: 63%. MS (M+H)=176.
63%
Stage #1: D-Alanine With lithium aluminium tetrahydride In tetrahydrofuran for 12h; Reflux;
Stage #2: With water; sodium hydroxide In tetrahydrofuran at 0 - 20℃; for 0.166667h;
Stage #3: di-<i>tert</i>-butyl dicarbonate In tetrahydrofuran; dichloromethane; water at 60℃; for 6h;
1.1
Step 1 (S)-Boc-2-amino-propanolD-Alanine (3.5 g, 39.3 mmol) was added in small portions to a suspension of LiAlH4 (2.89 g, 76.26 mmol) in refluxing THF. Refluxing continued for 12 hours, then the reaction mixture was cooled to 0° C., and excess reagent was quenched by careful addition of an aqueous 15% NaOH solution (3 ml) and water (9 ml). After stirring at room temperature for 10 minutes, a solution of (Boc)2O (8.31 g, 38.13 mmol) in CH2Cl2 (40 ml) was added. The reaction mixture was stirred at 60° C. for 6 hours, cooled to room temperature, filtered through a pad of anhydrous Na2SO4, and the filtrate concentrated under vacuum. Purification of the residue by silica-gel column chromatography afforded (S)-Boc-2-amino-propanol as a white solid, yield: 63%. MS (M+H)=176.
63%
Stage #1: D-Alanine With lithium aluminium tetrahydride In tetrahydrofuran for 12h; Reflux;
Stage #2: With water; sodium hydroxide In tetrahydrofuran at 0 - 20℃; for 0.166667h;
Stage #3: di-<i>tert</i>-butyl dicarbonate In tetrahydrofuran; dichloromethane at 60℃; for 6h;
1.1 (S)-2-Methoxy-1-methyl-ethylamine
D-Alanine (3.5 g, 39.3 mmol) was added in small portions to a suspension of LiAlH4 (2.89 g, 76.26 mmol) in refluxing THF. Refluxing continued for 12 hours, then the reaction mixture was cooled to 0° C., and excess reagent was quenched by careful addition of an aqueous 15% NaOH solution (3 ml) and water (9 ml). After stirring at room temperature for 10 minutes, a solution of (Boc)2O (8.31 g, 38.13 mmol) in CH2Cl2 (40 ml) was added. The reaction mixture was stirred at 60° C. for 6 hours, cooled to room temperature, filtered through a pad of anhydrous Na2SO4, and the filtrate concentrated under vacuum. Purification of the residue by silica-gel column chromatography afforded (S)-Boc-2-amino-propanol as a white solid, yield: 63%. MS (M+H)=176.
63%
Stage #1: D-Alanine With lithium aluminium tetrahydride In tetrahydrofuran for 12h; Reflux;
Stage #2: di-<i>tert</i>-butyl dicarbonate With sodium hydroxide In tetrahydrofuran; dichloromethane; water at 0 - 60℃; for 6.17h;
4.1
Step 1 (S)-Boc-2-amino-propanol D-Alanine (3.5 g, 39.3 mmol) was added in small portions to a suspension of LiAlH4 (2.89 g, 76.26 mmol) in refluxing THF. Refluxing continued for 12 hours, then the reaction mixture was cooled to 0° C., and excess reagent was quenched by careful addition of an aqueous 15% NaOH solution (3 ml) and water (9 ml). After stirring at room temperature for 10 minutes, a solution of (Boc)2O (8.31 g, 38.13 mmol) in CH2Cl2 (40 ml) was added. The reaction mixture was stirred at 60° C. for 6 hours, cooled to room temperature, filtered through a pad of anhydrous Na2SO4, and the filtrate concentrated under vacuum. Purification of the residue by silica-gel column chromatography afforded (S)-Boc-2-amino-propanol as a white solid, yield: 63%. MS (M+H)=176.
Stage #1: D-Alanine; N-ethyl-benzene-1,2-diamine With hydrogenchloride In water for 144h; Heating / reflux;
Stage #2: With sodium hydroxide; water at 0℃;
5-([l-(l-ethyl-l£T-imidazo[4,5-^]pyridin-2-yl)-l-methylethyl]amino}sulfonyl)pyridine-2- carboxamide Intermediate 1 [(12?)-l-(l-ethyl-l£T-benzimidazol-2-yl)ethyl]amineiV-Ethyl-l,2-phenylenediamine (Starting Material (SM) Ih, 14.7 mmol) and D-Alanine (2.2 g , 22.0 mmol)were taken into 6N HCl (15.0 mL) and the mixture was refluxed for 6 d. The reaction mixture was cooled in ice-bath, basified using 2N NaOH and extracted with EtOAc (3 x 50 mL). The organic layer was washed with brine (10 mL), dried and concentrated in EPO vacuo to give a dark brown viscous glue, which was purified by flash column chromatography using silica gel and CHCl3/MeOH (95:5) as eluent to give Intermediate 1 as a brown oil in 81% yield. 1H NMR (300 MHz, CDCl3) δ 7.78-7.72 (m, IH), 7.37-7.21 (m, 3H), 4.36-4.17 (m, 3H), 1.81 (br s, 2H), 1.61 (d, J = 6.9 Hz, 3H), 1.45 (t, J= 7.1 Hz, 3H). M/Z =189Starting
Stage #1: D-Alanine; isopropyl alcohol With thionyl chloride at 85℃; for 4h; Inert atmosphere;
Stage #2: With hydrogenchloride
1 Compound A7:
To a solution of D-Alanine (56.12 mmol) in isopropanol (2.3 mL/mmol) was added dropwise at room temperature under nitrogen thionyl chloride (280.61 mmol). The reaction mixture was stirred 4 hours at 85° C. The reaction was monitored by TLC (eluent: DCM/MeOH: 10%, developing bath: ninhydrine). The reaction mixture was concentrated under reduced pressure and the crude compound was dried under vacuo overnight. The compound was triturated in diisopropylether, filtered, washed with pentane and diisopropylether, filtered and dried under vacuo at 40° C. for 4 hours to afford the pure compound as HCl salt in 98% yield. 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 1.22 (m, 6H), 1.40 (d, J=7.19 Hz, 3H), 3.95-4.01 (m, 1H), 4.98 (heptuplet, J=6.21 Hz, 1H), 8.53-8.58 (m, 3H).
98%
With thionyl chloride at 85℃; for 4h; Inert atmosphere;
1.A2 Compound A2
To a solution of D-Alanine (56.12 mmol) in isopropanol (2.3 mlV mmol) was added at room temperature, under nitrogen, thionyl chloride (280.61 mmol) dropwise. The reaction mixture was stirred at 85 °C for 4 hours. The reaction was monitored by TLC (eluent: DCM/MeOH: 10%, developing bath: ninhydrine). The reaction mixture was concentrated under reduced pressure and the crude compound was dried under vacuo overnight. The compound was triturated in diisopropylether, filtered, washed with pentane and diisopropylether, filtered and dried under vacuo at 40°C for 4 hours to afford the pure compound as HC1 salt in 98% yield. 1H NMR (DMSO-^, 400MHz) δ (ppm) 1.22 (m, 6H), 1.40 (d, J= 7.19 Hz, 3H), 3.95-4.01 (m, 1H), 4.98 (heptuplet, J = 6.21 Hz, 1H), 8.53-8.58 (m, 3H).
98%
With thionyl chloride at 20 - 85℃; for 4h; Inert atmosphere;
1c Compound 17
To a solution of D-Alanine (56.12 mmol) in isopropanol (2.3 mL/mmol) was added at room temperature under nitrogen thionyl chloride (280.61 mmol) dropwise. The reaction mixture was stirred at 85°C for 4 hours. The reaction was monitored by TLC (eluent:DCM/MeOH: 10%, developing bath: ninhydrine). The reaction mixture was concentrated under reduced pressure and the crude compound was dried under vacuo overnight. The compound was triturated in diisopropylether, filtered, washed with pentane and diisopropylether, filtered and dried under vacuo at 40°C for 4 hours to afford the pure compound as HC1 salt in 98% yield. ‘H NMR (DMSO-d6, 400MHz) ö (ppm) 1.22 (m, 6H), 1.40 (d, J= 7.19 Hz, 3H), 3.95-4.01 (m, 1H), 4.98 (heptuplet, J= 6.21 Hz, 1H), 8.53-8.58 (m, 3H).
92%
With thionyl chloride at 0℃; Reflux;
With hydrogenchloride
Stage #1: isopropyl alcohol With thionyl chloride In neat (no solvent) at 25℃; for 0.0333333h;
Stage #2: D-Alanine In neat (no solvent) at 85℃; for 16h;
(R)-isopronyl 2aminopropanoate hydrochloride:
A neat solution of isopropanol (51.6 ml, 673 rnmol) was treated slowly with thionyl chloride (901 ml, 123 mrnoi) at room temperature over a 2-mm period. The mixture was treated with D-alanine (10 g, 112 mmoi) and fitted with a reflux condenser. This suspension was heated to 85 °C (reflux) and stirred overnight. The solution was concentrated to dryness, Oily solids were suspended in hexanes and concentrated. The resultingoil crystallizes upon standing and drying under high vacuum to provide the title compound:LC/MS: [(M+i)] = 132.0.
With chloro-trimethyl-silane at 70℃; for 39h;
18 Alternate Preparation of Compound 11
A 1 L flask was charged with D-Alanine (10, 50 g, 561 mmol) and iPrOH (432 mE). To the resulting solution was added TMS-Cl (108 mE, 842 mmol, 1.5 eq) and the reaction was allowed to stir at 70° C. for 19 hours. The mixture was then concentrated in vacuo at 35° C. to 189 g total weight, then diluted with iPrOH (350 mE). The mixture was concentrated in vacuo to 118 g weight, then diluted with iPrOH (390 mE). TMS-Cl (36 mE, 281 mmol, 0.50 equiv) was added and the resulting reaction was allowed to stir at 70° C. for 20 hours. The reaction mixture was then concentrated in vacuo to 140 g weight, then concentrated in vacuo with IPAC (four portions of 250 mE) to 124 g final weight. The resulting residue was diluted with 250 mE IPAC to provide a mixture containing compound 2. A portion of the mixture, compound 2, was used without thrther purification.
(S)-2-(3,4-dichloro-phenylamino)-propionic acid[ No CAS ]
[ 1206599-39-4 ]
Yield
Reaction Conditions
Operation in experiment
With potassium phosphate In N,N-dimethyl-formamide at 80℃; for 40h;
11
Alternative Preparation of 2-(3,4-dichloro-phenylamino)-propionic Acid; A mixture of 3,4-dichloroiodobenzene (1.50 g, 5.50 mmol), L-alanine (734 mg, 8.25 mmol), copper(I) iodide, 2-hydroxybenzaldehyde phenylhydrazone (233 mg, 1.10 mmol), tri-potassium phosphate (3.50 g, 16.5 mmol), and N,N-dimethylformamide (8 mL) was stirred at 80° C. for 40 h, then after cooling diluted with water and acidified to pH 3 by addition of 37% aq. HCl solution. The mixture was extracted with EtOAc, the organic phase was washed with brine, dried (MgSO4), filtered, and evaporated. Chromatography (SiO2; EtOAc) afforded the title compound (1.08 g, 67%) as a 71:29 mixture of the (S) and (R) stereoisomers (accordingly, when D-alanine was used instead of L-alanine as starting material, the title compound was obtained as a 29:71 mixture of the (S) and (R) stereoisomers). Brown solid, MS: 232.1 (M-H)-.
With potassium phosphate; copper(l) iodide; 2-hydroxybenzaldehyde phenylhydrazone In N,N-dimethyl-formamide at 80℃; for 16h; Inert atmosphere;
(R)-2-(3,4-Dichloro-phenylamino)-propionic acid (62% ee) A mixture of 6.00 g (22.00 mmol) 3,4-dichloroiodobenzene, 2.94 g (33.00 mmol) D-alanine, 0.42 g (2.20 mmol) copper(I) iodide, 0.96 g (4.40 mmol) 2-hydroxybenzaldehyde phenylhydrazone, 14.00 g (66.00 mmol) tri-potassium phosphate in 32 ml of N,N- dimethylformamide was stirred at 80°C for 16 h under argon, then after cooling diluted with water and acidified to pH 3 by addition of 25% aq. HC1 solution. The mixture was extracted with EtOAc (3x), the organic phases were washed with brine, dried (Na2S04), filtered and evaporated. Flash chromatography (Si02; dichloromethane/MeOH 95:5 to 4:1) afforded 3.11 g (60%)) of the title compound (as 81 :19 mixture of the (R) and (S) stereoisomers, BGB- 175*0.25 column) as light brown solid. MS: 231.9 (M-FT, 2C1).
62 % ee
With potassium phosphate; 2-hydroxybenzaldehyde phenylhydrazone In N,N-dimethyl-formamide at 80℃; for 16h; Inert atmosphere;
31 (R)-2-(3,4-Dichloro-phenylamino)-propionic acid (62% ee)
(R)-2-(3,4-Dichloro-phenylamino)-propionic acid (62% ee) A mixture of 6.00 g (22.00 mmol) 3,4-dichloroiodobenzene, 2.94 g (33.00 mmol) D-alanine, 0.42 g (2.20 mmol) copper(I) iodide, 0.96 g (4.40 mmol) 2-hydroxybenzaldehyde phenylhydrazone, 14.00 g (66.00 mmol) tri-potassium phosphate in 32 ml of N,N-dimethylformamide was stirred at 80° C. for 16 h under argon, then after cooling diluted with water and acidified to pH 3 by addition of 25% aq. HCl solution. The mixture was extracted with EtOAc (3*), the organic phases were washed with brine, dried (Na2SO4), filtered and evaporated. Flash chromatography (SiO2; dichloromethane/MeOH 95:5 to 4:1) afforded 3.11 g (60%) of the title compound (as 81:19 mixture of the (R) and (S) stereoisomers, BGB-175*0.25 column) as light brown solid. MS: 231.9 (M-H-, 2Cl).
Stage #1: D-Alanine With lithium aluminium tetrahydride In tetrahydrofuran for 12h; Reflux;
Stage #2: di-<i>tert</i>-butyl dicarbonate In tetrahydrofuran; dichloromethane; water at 60℃; for 6h;
5.1 Step 1 (S)-Boc-2-amino-propanol
Preparation 5 (S)-2-Methoxy-1-methyl-ethylamine The synthetic procedure used in this preparation is outlined below in Scheme G. D-Alanine (3.5 g, 39.3 mmol) was added in small portions to a suspension of LiAlH4 (2.89 g, 76.26 mmol) in refluxing THF. Refluxing continued for 12 hours, then the reaction mixture was cooled to 0° C., and excess reagent was quenched by careful addition of an aqueous 15% NaOH solution (3 ml) and water (9 ml). After stirring at room temperature for 10 minutes, a solution of (Boc)2O (8.31 g, 38.13 mmol) in CH2Cl2 (40 ml) was added. The reaction mixture was stirred at 60° C. for 6 hours, cooled to room temperature, filtered through a pad of anhydrous Na2SO4, and the filtrate concentrated under vacuum. Purification of the residue by silica-gel column chromatography afforded (S)-Boc-2-amino-propanol as a white solid, yield: 63%. MS (M+H)=176.
Stage #1: D-Alanine With (R)-N-(2-benzoylphenyl)-1-(3-fluorobenzyl)pyrrolidine-2-carboxamide; sodium methylate; nickel(II) nitrate In methanol at 50℃;
Stage #2: benzyl bromide With sodium hydroxide In N,N-dimethyl-formamide at 45 - 50℃; for 0.166667h; Inert atmosphere; optical yield given as %ee; enantioselective reaction; Further stages;
With pyridoxal 5'-phosphate In aq. phosphate buffer; dimethyl sulfoxide at 30℃; for 24h; Resolution of racemate; Enzymatic reaction; enantioselective reaction;
2.4 Kinetic resolution of rac-1a-d
General procedure: All experiments were carried out keeping the protein content constant (8 mg mL-1) if not otherwise stated. One of the substrates rac-1a-d (50 mM), sodium pyruvate (50 mM) and pyridoxal-5′-phosphate monohydrate (0.2 mg mL-1) in phosphate buffer (1 mL, 0.1 M, pH 7.5) containing IPA or DMSO as a possible cosolvent (10, v/v-%) was added to a 2 mL Eppendorf tube, containing the ω-transaminase sol-gel catalyst (25 or 50 mg). The reaction was shaken (170 rpm) at 30 °C. After 24 h the reaction was stopped by centrifuging the mixture and removing the solution by pipette. The solid catalyst was reused while conversion was monitored by taking a sample (5 μL) from the solution and diluting it with the HPLC eluent (500 μL). The sample was filtered and analyzed for conversion by HPLC. A sample (400 μL) for ee(S)-1 analysis was taken, and aqueous NaOH (2 M, 50 μL) was added followed by the extraction of the amine into ethyl acetate (400 μL). The organic phase (300 μL) was dried with Na2SO4, and after filtration the amine in the sample (200 μL) was derivatized with acetic anhydride (10 μL) to determine the enantiomeric excess of (S)-1 by GC.
With pyridoxal 5'-phosphate; recombinant Lactobacillus salivarius UCC118 D-amino acid aminotransferase; In aq. phosphate buffer; at 30℃; for 0.0166667h;pH 7.5;Enzymatic reaction;
General procedure: Amino acceptor specificity was determined using a modification of the salicylaldehyde method by measuring the rate of pyruvate formation from D-alanine in the presence of an alpha-keto acid. The standard reaction mixture contained 100 mM potassium phosphate buffer (pH 7.5), 50 mM D-alanine, 20 mM alpha-keto acid, 0.05 mM pyridoxal-5?-phosphate and purified D-AAT. The reaction was run at 30 C for 1 min in a total volume of 1 ml, and the reaction was stopped by adding 1 ml of 60% potassium hydroxide, after which 0.5 ml of 2% salicylaldehyde dissolved in 99% ethanol was added, and the mixture was incubated for 30 min at 30 C. Thereafter, 1.5 ml of cold distilled water was added to the orange colored reaction mixture and absorbance at 480 nm was measured. The reaction mixture was also incubated for 1 min without the purified enzyme. The enzyme concentration was determined using the Bradford method with Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, CA, USA). All enzyme assays were performed more than 3 times.
With pyridoxal 5'-phosphate; recombinant Lactobacillus salivarius UCC118 D-amino acid aminotransferase; In aq. phosphate buffer; at 30℃; for 0.0166667h;pH 7.5;Enzymatic reaction;
General procedure: Amino acceptor specificity was determined using a modification of the salicylaldehyde method by measuring the rate of pyruvate formation from D-alanine in the presence of an alpha-keto acid. The standard reaction mixture contained 100 mM potassium phosphate buffer (pH 7.5), 50 mM D-alanine, 20 mM alpha-keto acid, 0.05 mM pyridoxal-5?-phosphate and purified D-AAT. The reaction was run at 30 C for 1 min in a total volume of 1 ml, and the reaction was stopped by adding 1 ml of 60% potassium hydroxide, after which 0.5 ml of 2% salicylaldehyde dissolved in 99% ethanol was added, and the mixture was incubated for 30 min at 30 C. Thereafter, 1.5 ml of cold distilled water was added to the orange colored reaction mixture and absorbance at 480 nm was measured. The reaction mixture was also incubated for 1 min without the purified enzyme. The enzyme concentration was determined using the Bradford method with Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, CA, USA). All enzyme assays were performed more than 3 times.
With pyridoxal 5'-phosphate; recombinant Lactobacillus salivarius UCC118 D-amino acid aminotransferase; In aq. phosphate buffer; at 30℃; for 0.0166667h;pH 7.5;Enzymatic reaction;
General procedure: Amino acceptor specificity was determined using a modification of the salicylaldehyde method by measuring the rate of pyruvate formation from D-alanine in the presence of an alpha-keto acid. The standard reaction mixture contained 100 mM potassium phosphate buffer (pH 7.5), 50 mM D-alanine, 20 mM alpha-keto acid, 0.05 mM pyridoxal-5?-phosphate and purified D-AAT. The reaction was run at 30 C for 1 min in a total volume of 1 ml, and the reaction was stopped by adding 1 ml of 60% potassium hydroxide, after which 0.5 ml of 2% salicylaldehyde dissolved in 99% ethanol was added, and the mixture was incubated for 30 min at 30 C. Thereafter, 1.5 ml of cold distilled water was added to the orange colored reaction mixture and absorbance at 480 nm was measured. The reaction mixture was also incubated for 1 min without the purified enzyme. The enzyme concentration was determined using the Bradford method with Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, CA, USA). All enzyme assays were performed more than 3 times.
With pyridoxal 5'-phosphate; recombinant Lactobacillus salivarius UCC118 D-amino acid aminotransferase; In aq. phosphate buffer; at 30℃; for 0.0166667h;pH 7.5;Enzymatic reaction;
General procedure: Amino acceptor specificity was determined using a modification of the salicylaldehyde method by measuring the rate of pyruvate formation from D-alanine in the presence of an alpha-keto acid. The standard reaction mixture contained 100 mM potassium phosphate buffer (pH 7.5), 50 mM D-alanine, 20 mM alpha-keto acid, 0.05 mM pyridoxal-5?-phosphate and purified D-AAT. The reaction was run at 30 C for 1 min in a total volume of 1 ml, and the reaction was stopped by adding 1 ml of 60% potassium hydroxide, after which 0.5 ml of 2% salicylaldehyde dissolved in 99% ethanol was added, and the mixture was incubated for 30 min at 30 C. Thereafter, 1.5 ml of cold distilled water was added to the orange colored reaction mixture and absorbance at 480 nm was measured. The reaction mixture was also incubated for 1 min without the purified enzyme. The enzyme concentration was determined using the Bradford method with Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, CA, USA). All enzyme assays were performed more than 3 times.
With pyridoxal 5'-phosphate; recombinant Lactobacillus salivarius UCC118 D-amino acid aminotransferase In aq. phosphate buffer at 30℃; for 0.0166667h; Enzymatic reaction;
General procedure: Amino acceptor specificity was determined using a modification of the salicylaldehyde method by measuring the rate of pyruvate formation from D-alanine in the presence of an α-keto acid. The standard reaction mixture contained 100 mM potassium phosphate buffer (pH 7.5), 50 mM D-alanine, 20 mM α-keto acid, 0.05 mM pyridoxal-5′-phosphate and purified D-AAT. The reaction was run at 30 °C for 1 min in a total volume of 1 ml, and the reaction was stopped by adding 1 ml of 60% potassium hydroxide, after which 0.5 ml of 2% salicylaldehyde dissolved in 99% ethanol was added, and the mixture was incubated for 30 min at 30 °C. Thereafter, 1.5 ml of cold distilled water was added to the orange colored reaction mixture and absorbance at 480 nm was measured. The reaction mixture was also incubated for 1 min without the purified enzyme. The enzyme concentration was determined using the Bradford method with Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, CA, USA). All enzyme assays were performed more than 3 times.
With pyridoxal 5'-phosphate; recombinant Lactobacillus salivarius UCC118 D-amino acid aminotransferase; In aq. phosphate buffer; at 30℃; for 0.0166667h;pH 7.5;Enzymatic reaction;
General procedure: Amino acceptor specificity was determined using a modification of the salicylaldehyde method by measuring the rate of pyruvate formation from D-alanine in the presence of an alpha-keto acid. The standard reaction mixture contained 100 mM potassium phosphate buffer (pH 7.5), 50 mM D-alanine, 20 mM alpha-keto acid, 0.05 mM pyridoxal-5?-phosphate and purified D-AAT. The reaction was run at 30 C for 1 min in a total volume of 1 ml, and the reaction was stopped by adding 1 ml of 60% potassium hydroxide, after which 0.5 ml of 2% salicylaldehyde dissolved in 99% ethanol was added, and the mixture was incubated for 30 min at 30 C. Thereafter, 1.5 ml of cold distilled water was added to the orange colored reaction mixture and absorbance at 480 nm was measured. The reaction mixture was also incubated for 1 min without the purified enzyme. The enzyme concentration was determined using the Bradford method with Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, CA, USA). All enzyme assays were performed more than 3 times.
With pyridoxal 5'-phosphate; recombinant Lactobacillus salivarius UCC118 D-amino acid aminotransferase; In aq. phosphate buffer; at 30.0℃; for 0.0166667h;pH 7.5;Enzymatic reaction;
General procedure: Amino acceptor specificity was determined using a modification of the salicylaldehyde method by measuring the rate of pyruvate formation from D-alanine in the presence of an alpha-keto acid. The standard reaction mixture contained 100 mM potassium phosphate buffer (pH 7.5), 50 mM D-alanine, 20 mM alpha-keto acid, 0.05 mM pyridoxal-5?-phosphate and purified D-AAT. The reaction was run at 30 C for 1 min in a total volume of 1 ml, and the reaction was stopped by adding 1 ml of 60% potassium hydroxide, after which 0.5 ml of 2% salicylaldehyde dissolved in 99% ethanol was added, and the mixture was incubated for 30 min at 30 C. Thereafter, 1.5 ml of cold distilled water was added to the orange colored reaction mixture and absorbance at 480 nm was measured. The reaction mixture was also incubated for 1 min without the purified enzyme. The enzyme concentration was determined using the Bradford method with Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, CA, USA). All enzyme assays were performed more than 3 times.
With sulfuric acid; sodium nitrite; In water; at 0 - 20℃;
A solution of NaNO2 (23.35 g, 338 mmol) in H2O (200 mL) was added to a stirred solution of (R)-alanine 1 (20.0 g, 225 mmol) in 0.5 M H2SO4 (344 mL, 172 mmol) over a 2 h period, with the temperaturekept at 0-5 C. The reaction mixture was then stirred and warmed to r.t. overnight. Solid NaHCO3 was then added to the reaction mixture to achieve pH 6, then conc. HCl was added until pH 3 was reached. The solution was then concentrated to roughly a fifth of its originalvolume (120 mL) under reduced pressure and then extracted withEtOAc (9 × 100 mL). The combined organic extracts were dried with MgSO4 and the solvent was removed under reduced pressure to give (R)-lactic acid.
With hydrogenchloride; In water; at 110℃; for 24h;Sealed tube;
The stereochemistry of the amino acids was determined by analysis of the acidic hydrolysate from compound 2. Compound 2 (5.0 mg) was dissolved in 6 N HCl (5 mL) and heated at 110 C, in a furnace, for 24 h in a sealed glass tube. After cooling to room temperature, the solution was dried under N2 for 24 h, reconstituted in methanol for HPLC-MS (200 muL), filtered through a 4 mm PTFE Syringe Filter F2504-4 of 0.2 mum pore size (Thermo Scientific, Mumbai, India), and then analyzed by HPLC equipped with a chiral column.
With sodium hydrogencarbonate In water; N,N-dimethyl-formamide at 20℃; for 2h;
6 Step 6. Synthesis of Compound 100
To a solution of D-alanine (1.09 g, 0.012 mmol, 1.00 equiv) and NaHCO3 (3.09 g, 0.04 mmol, 3.00 equiv) in water (50.00 mL) was added a solution of 2,5-dioxopyrrolidin-1-yl (2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]propanoate (Compound 99, 5.00 g, 12.24 mmol, 1.00 equiv) in DMF (50.00 mL,). The resulting mixture was stirred at room temperature for 2 h. LCMS indicated the reaction was completed. The reaction was adjusted to pH value to 2-3 with 2 N HC1. The resulting mixture was extracted with EtOAc (100.00 mLx3), and the combined organic layer was washed with brine (100.00 mLx3), dried over anhydrous Na2SO4 and concentrated to dryness under vacuum to give (2R)-2-[(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]-amino]propanamido]propanoic acid (Compound 100, 4.00 g, 71%) as a white solid. LCMS (ES, m/z): 383 [M+H]+
1.3 g
With sodium hydrogencarbonate In 1,2-dimethoxyethane; water for 2h;
With potassium carbonate In ethanol; water at 100℃; for 10h;
4.1.1. General procedure for the preparation of 2a-f
General procedure: To a solution of 4-bromo-2-fluoro-1-nitrobenzene (1g, 4.55mmol), K2CO3 (0.78g, 5.68mmol) and different amino acid (5.45mmol) in ethanol (5ml) and water (4ml) were refluxed at 100°C for 10h. After cooling to the room temperature, pH value was adjusted to 2 with 1M HCl solution. The mixture was filtered and the precipitate was collected to afford 2a-g as golden yellow solid. 4.1.1.1 (5-Bromo-2-nitrophenyl)-d-alanine (2a) (0013) Golden yellow solid, yield 98.2%; ESI-MS m/z 287.9 [M+H]+; 1H NMR (300MHz, DMSO-d6) δ: 8.39 (d, J=6.8Hz, 1H), 8.02 (d, J=9.1Hz, 1H), 7.23 (s, 1H), 6.90 (dd, J=9.1, 1.7Hz, 1H), 4.62-4.46 (m, 1H), 1.46 (d, J=6.9Hz, 3H).
91%
With potassium carbonate In ethanol; water for 10h; Reflux;
4.2. (5-bromo-2-nitrophenyl)-D-alanine (2)
To a solution of 4-Bromo-2-Fluoronitrobenzene (2.0 g, 9 mmol) and Potassium carbonate (1.38 g, 10 mmol) in Ethanol (15 mL) and water (6 mL) was added D-alanine (1.0 g,11 mmol), the mixture was heated to reflux for 10 h. after the reaction was completed, the solution was cooled to room temperature and pH value was adjusted to 2 with 1 M HCl solution. The precipitate was collected by filtration to afford (5-bromo-2-nitrophenyl)-D-alanine as golden yellow solid (2.37 g, 91 %) without further purification. ESI-MS m/z 289.2 [M + H]+; 1H NMR (300 MHz, DMSO) 8.39 (d, J = 6.8Hz, 1H), 8.02 (d, J = 9.1 Hz, 1H), 7.23 (s, 1H), 6.90 (dd, J =9.1, 1.7 Hz, 1H), 4.62-4.46 (m, 1H), 1.46 (d, J = 6.9 Hz,3H).
88%
With potassium carbonate In ethanol; water at 80℃; for 8h;
1.a Example 1
(a) Compound 1A (25g, 113.64mmol), D-alanine (11.12g, 125.0mmol), potassium carbonate (17.25g, 125.0mmol) are dissolved in 500ml ethanol: water = 3:1 mixed solvent, 80 Heat to reflux at for 8 hours, monitor the reaction with a TLC plate, cool to room temperature after the reaction, evaporate the solvent, dissolve in water, adjust the pH to 1-2 with 1N HCl, a large amount of yellow solid precipitates, filter, and wash with 200ml petroleum ether The solid was dried in a vacuum drying oven to obtain 28.7 g of a yellow solid, namely compound 1B, with a yield of 88%.
87%
With potassium carbonate In ethanol; water at 80℃; for 8h;
45.56 g
With potassium carbonate In ethanol; water for 16h; Reflux;
1 N-(5-bromo-2-nitrophenyl)-D-alanine
A solution of 35.6 g of 4-bromo-2-fluoronitrobenzene (CAS No. 321-23-3), 14.4 g of D-alanine and 27.95 g of potassium carbonate in 395 ml of ethanol and 175 ml of water was heated under reflux for 6 hours. After cooling to room temperature, the reaction mixture was acidified by addition of 1 N hydrochloric acid and the product formed was filtered off as a precipitate. This gave 45.56 g of N-(5-bromo-2-nitrophenyl)-D-alanine1H NMR (400 MHz, CDCl3): δ=1.46 (d, 3H); 4.52-4.62 (m, 1H); 6.89 (dd, 1H); 7.22 (d, 1H); 8.01 (d, 1H); 8.38 (d, 1H).
17.36 g
With potassium carbonate In ethanol; water for 6h; Reflux;
Intermediate 11: N-(5-bromo-2-nitrophenyl)-D-alanine
The 13.57g 4-bromo-2-fluoro-nitrobenzene, 5.49g D-alanine and 10.66g potassium carbonate solution in 150ml of ethanol and 60ml water was heated under reflux for 6 hours. After cooling to room temperature, by addition of 1M hydrochloric acid and the reaction mixture is acidified, and the product was isolated as a precipitate generated. To give 17.36g N-(5-bromo-2-nitrophenyl)-D-alanine.
With potassium carbonate In ethanol; water at 20℃; for 12h;
With potassium carbonate In ethanol; water for 8h; Reflux;
Resin)(Loading of 2-Chlorotrityl Chloride Resin with Fmoc-fOH, Fmoc Removal and Determination of the Loading of the2-Chlorotrityl chloride resin (5.0 g, 8.01 mmol) was reacted with a solution of Fmoc-f-OH (3.10 g, 8.01 mmol) in DCM (50 mE) and DIPEA (5.59 mE, 32.0 mmol) in analogy to the general procedure described above to give Intermediate 21a (5.87 g, loading=0.897 mmol/g). (Assembly of Linear Peptide)Intermediate 21a (0.100 mmol) was subjected to solidphase peptide synthesis on the LibertyTM microwave peptide synthesizet Coupling was performed as follows: (Cleavage from the Resin with Concomitant Protecting Group Removal then Purification)A mixture of 95% aq. TFAEDT/DTT (95:2.5:2.5) (2 mE)was added to Intermediate 21b (0.1 mmol). The suspensionwas shaken at it for 2 h, then the cleavage solution was filtered off. The resin was then again treated with 95% aq. TFAEDT/ TIS (95:2.5:2.5) (2 mE), shaked at it for 1 h, and filtered. The combined cleavage and washing solutions were poured ontoa mixture of cold heptane/diethyl ether (1:1) (11 mL), giving a precipitate. The suspension was centriffiged and the supernatant poured off. Diethyl ether (10 mL) was added to the residue, the suspension was vortexed for 3 mm and centrifuged, and the supernatant was poured off. The washing pro-cess was repeated twice. The solid was dried in high vacuum The crude was purified by preparative HPLC and lyophilized from ACN/H20 to afford Intermediate 21c (55 mg, 0.030 mmol).
D-[Cu(1,10-phenanthroline)(alanine)(H2O)](NO3)*1.5H2O[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
80%
With ammonium hydroxide; sodium hydrogencarbonate In methanol; water at 50℃;
Preparation of [Cu(phen)(ala)(H2O)](NO3) and reactionwith HCHO
The D- and L-[Cu(phen)(ala)(H2O)](NO3) complexes (1a and 1b, respectively) were synthesized following the procedure for preparing the D- and L-[Cu(phen)(L-threo)(H2-O)](NO3), except that threo was replaced with ala [7]. A typical example is given for the preparation of [Cu(phen)(L-ala)(H2O)]NO3. A solution of L-ala (0.09 g,1 mmol) in 1 M NH3 solution (10 ml) was added to a water-methanol mixture (10 ml) of Cu(NO3)2*3H2O(0.25 g, 1 mmol) and 1,10-phenanthroline (0.20 g,1 mmol) to yield a dark blue solution (20 ml). The pH ofthe solution was raised to pH 6.0-7.0 with NaHCO3 solution. On heating the resulting solution at 50 C overnight, ablue crystalline product was obtained.
Determination of absolute configuration by the advanced Marfey’s method [1,2]
General procedure: 1.0 mg turnagainolide C (2) was dissolved in 6 N HCl (1 ml) and heated at 110 °C for 24 h, respectively. The hydrolyzate was evaporated to dryness, and the residue was redissolved in 100 μL of acetone. To each a half portion (50 μL) were added 20 μL NaHCO3 (1 M) and 100 μL 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide or 1-fluoro-2,4-dinitrophenyl-5-D-leucinamide (L-FDLA or D-FDLA, 1% in acetone), and the mixture was heated at 45°C for 1.5 h. Reaction was cooled to room temperature, and then acidified with 2 N HCl (10 μL), dried and dissolved in 50% aqueous CH3CN. About 5 μL of each solution of FDLA derivatives were analyzed by LC/MS.
With potassium <i>tert</i>-butylate In ethanol at 0 - 20℃; for 1.7h;
2
Diethyl oxalate and D-alanine were weighed according to a molar ratio of 1:1.Mixing and stirring to obtain a mixture; by mass ratio1:17, potassium tert-butoxide was dissolved in absolute ethanol, mixed and cooled to 0 ° C, and the mixture was slowly added to a solution of potassium t-butoxide in ethanol.The amount of the mixture added is 9% of the mass of the potassium t-butoxide solution.After the completion of the addition, the temperature was raised to room temperature, and the reaction was stirred for 1.7 h; after the reaction, the mixture was discharged, and the reaction product was poured into ice water. After stirring for 17 min, the pH was adjusted to 5.7 with hydrochloric acid, and a solid was precipitated, suction filtered, and the residue was collected and recrystallized. After drying, ethyl 5-formyl-1H-pyrrole-2-carboxylate was obtained, the melting point was 72.8 ° C, the purity was 99.8%, and the yield was 86%.
Stage #1: 1-decanoic acid With 1,1'-carbonyldiimidazole In dichloromethane at 20℃; for 0.5h;
Stage #2: D-Alanine In dichloromethane for 12h;
(21) General Procedure for Amide Coupling
General procedure: To a stirred solution of the fatty acid (1.0 mmol, 1.0 equiv.) inCH2Cl2 (5 mL) was added CDI (0.178 g, 1.1 mmol, 1.1 equiv.).After 30 min at room temperature, the amine (1.1 mmol, 1.1equiv.) was added. After 12 h, CH2Cl2 (25 mL) was added, followedby saturated aqueous NH4Cl. The mixture was acidified topH 2 by addition of HCl, the organic phase was separated, andthe aqueous layer was further extracted with CH2Cl2 (3 × 10mL). The organic phases were combined, dried over Na2SO4, filtered,and concentrated in vacuo, to give the amide.
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)
Stage #1: D-Alanine With triethylamine; isobutyl chloroformate In tetrahydrofuran at 0 - 5℃; for 2h;
Stage #2: With hydroxylamine hydrochloride In tetrahydrofuran; methanol at 0 - 5℃; for 2h;
1 Example 1:Condensation of a compound of formula I with hydroxylamine to give a compound of formula II;Wherein RCOOH is D-alanine.
147.8 g of the compound of formula I is dissolved in 450 mL of tetrahydrofuran,Add 79.5g of triethylamine,Cool down to 0-5 ° C, add 106.5 g of isobutyl chloroformate,A large amount of solid precipitated and kept for 2 hours;81.75 g of hydroxylamine hydrochloride was dissolved in 750 mL of methanol.Cool down to 0-5 ° C, add sodium hydroxide in batches, a large amount of solid precipitated,After 2 h of incubation, the filtrate of step 1 was added and stirring was continued for 2 h. Filtering,The filtrate was concentrated under reduced pressure and 1 L ethyl acetate was added.A white solid precipitated, suction filtered, and the filter cake was washed with ethyl acetateConcentrate to dryness. Beating with 750 mL of dichloromethane, suction filtration, drying at 45 ° C,Yield 137.2 g of a white solid,The yield was 86%.
N-(((4-azidobenzyl)oxy)carbonyl)-D-alanine[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
90%
With triethylamine In N,N-dimethyl-formamide at 0 - 20℃; for 72h; Inert atmosphere;
1 Synthesis of N-(((4-azidobenzyl)oxy)carbonyl)-D-alanine (Compound TS19, Acbz-D-Ala-OH)
Under a nitrogen atmosphere, DMF (1.0 mL) was added at room temperature to a mixture of D-alanine (H-D-Ala-OH) (44.5 mg, 0.50 mmol), and 4-azidobenzyl (4-nitrophenyl)carbonate (157 mg, 0.50 mmol) synthesized by the method described in the literature (Bioconjugate Chem. 2008, 19, 714). After stirring at 0° C. for five minutes, triethylamine (174 μL, 1.25 mmol) was added at 0° C. The reaction mixture was stirred at room temperature for 72 hours and then purified by reverse-phase silica gel column chromatography (0.1% aqueous formic acid solution/0.1% formic acid-acetonitrile solution) to give N-(((4-azidobenzyl)oxy)carbonyl)-D-alanine (Compound TS19, Acbz-D-Ala-OH) (119.1 mg, 90%). LCMS (ESI) m/z=263 (M-H)- Retention time: 0.61 minutes (analysis condition SQDFA05)
(4-nitrophenyl) 4-(2-(4-fluorophenyl)acetamide)benzyl carbonate[ No CAS ]
(((4-(2-(4-fluorophenyl)acetamido)benzyl)oxy)carbonyl)-D-alanine[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
87%
With triethylamine In dimethyl sulfoxide at 0 - 20℃; for 20h; Inert atmosphere;
1 Synthesis of (((4-(2-(4-fluorophenyl)acetamide)benzyl)oxy)carbonyl)-D-alanine (Compound TS22, F-Pnaz-D-Ala-OH)
Under a nitrogen atmosphere, DMSO (3.74 mL) was added at room temperature to a mixture of D-alanine (H-D-Ala-OH) (100 mg, 1.12 mmol) and (4-nitrophenyl)-4-(2-(4-fluorophenyl)acetamide)benzyl carbonate (Compound ts11) (476 mg, 1.12 mmol). After stirring at room temperature for five minutes, triethylamine (3134, 2.25 mmol) was added at 0° C. The reaction mixture was stirred at room temperature for 20 hours and then purified by reverse-phase silica gel column chromatography (0.1% aqueous formic acid solution/0.1% formic acid-acetonitrile solution) to give (((4-(2-(4-fluorophenyl)acetamide)benzyl)oxy)carbonyl)-D-alanine (Compound TS22, F-Pnaz-D-Ala-OH) (366 mg, 87%). LCMS (ESI) m/z=373 (M+H)- Retention time: 0.62 minutes (analysis condition SQDFA05)
Stage #1: o-chlorocinnamic acid With 1-hydroxy-pyrrolidine-2,5-dione; dicyclohexyl-carbodiimide In 1,4-dioxane at 20℃; for 5h;
Stage #2: D-Alanine With sodium hydrogencarbonate In 1,4-dioxane; water at 20℃; for 24h;
2.2. General Procedure for the Synthesis of Nsubstituted-D-alanine (4a-4h, 5a-5d, 6a-6i)
General procedure: To a solution of corresponding carboxylic acid (30mmol) and N-hydroxysuccinimide (32 mmol) in dioxane (46ml), N, N-dicyclohexylcarbodiimide (32 mmol) was added.The mixture was reacted for 5 h at room temperature. Afterfiltration, the filtrate was added to a solution of NaHCO3 (34mmol) and D-alanine (34 mmol) in H2O (48 mL). After reactionfor 24 h at room temperature, the solvent was distilledoff under reduced pressure. The residual was dissolved inwater and the insoluble material was filtered off. The aqueouslayer was extracted with EtOAc (3 × 30 ml) and acidifiedwith concentrated HCl (pH 2). The precipitate formedwas filtered, recrystallized from methanol to afford the targetcompounds
Stage #1: ortho-bromophenylacetic acid With 1-hydroxy-pyrrolidine-2,5-dione; dicyclohexyl-carbodiimide In 1,4-dioxane at 20℃; for 5h;
Stage #2: D-Alanine With sodium hydrogencarbonate In 1,4-dioxane; water at 20℃; for 24h;
2.2. General Procedure for the Synthesis of Nsubstituted-D-alanine (4a-4h, 5a-5d, 6a-6i)
General procedure: To a solution of corresponding carboxylic acid (30mmol) and N-hydroxysuccinimide (32 mmol) in dioxane (46ml), N, N-dicyclohexylcarbodiimide (32 mmol) was added.The mixture was reacted for 5 h at room temperature. Afterfiltration, the filtrate was added to a solution of NaHCO3 (34mmol) and D-alanine (34 mmol) in H2O (48 mL). After reactionfor 24 h at room temperature, the solvent was distilledoff under reduced pressure. The residual was dissolved inwater and the insoluble material was filtered off. The aqueouslayer was extracted with EtOAc (3 × 30 ml) and acidifiedwith concentrated HCl (pH 2). The precipitate formedwas filtered, recrystallized from methanol to afford the targetcompounds
With N-ethyl-N,N-diisopropylamine In methanol at 25℃; for 5h;
General procedure a for the synthesis of hydrazones and 1-substituted 1,2,3-triazoles
General procedure: To a dried Schlenk flask, amines (0.4 mmol, 2.0 eq), difluoroacetaldehyde N-tosylhydrazone (0.2 mmol, 49.6 mg, 1.0 eq), base (3 eq or 6.0 eq), and MeOH (4 mL) were added. Then the resultant solution was stirred at room temperature for 5 h, then diluted with 5 mL of CH2Cl2 and filtered through a plug of celite, followed by washing with 20 mL of CH2Cl2. The combined residue was concentrated under reduced pressure, and then the resulting crude product was purified by silica gel column chromatography.
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