SciFinder Page R:NaOH, S:H 2 O, rt, ph C:p-MeC 6 H 4 SO 3 H, S:PhMe, S:MeOH, 15 h, rt Reactants: 1, Catalysts: 1,

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1 SciFinder Page 1

2 SciFinder Page 2 Reaction Information R:NaOH, S:H 2 O, rt, ph R:HCl, S:H 2 O, rt, ph R: S:H 2 O, rt; rt; 4 h, rt biotransformation, enzymic, S243n/A182S mutant BMDATexpressing E. coli was used, stereoselective, Reactants: 2, Reagents: 6, Solvents: 3, Steps: 1, Stages: 4 3% R:Zn(OAc) 2, 1.4 R:D-Alanine, R:pyridoxalphosphate, rt; 24 h, 33 C Process for producing an optically active indole compound using asymmetric addition in the presence of α- amino acids and a metal ion By Kawahara, Shigeru et al From Eur. Pat. Appl., , 13 Aug 2008 Experimental Procedure General/Typical Procedure: [0105] 5.0 g of indole-3-pyruvic acid was suspended in 100 ml of water, which was dissolved by addition of 2N sodium hydroxide aqueous solution and adjusted to ph 12. Then, the solution was adjusted to ph 9 by addition of 1N hydrochloric acid, and then sodium pyruvate (8.12 g) and the solution of trans-l-hydroxyproline/zinc (II) complex (22 ml) were added. The mixture was stirred at room temperature for 4 hours, and the cross-aldol reaction was performed. To 25 ml of the obtained reaction mixture, D-Ala (1.8 g) and pyridoxal-5'-phosphate (1 mg) were added, and then 0.5 g of BMDATexpressing E. coli or S 243 N/A 182 S mutant BMDAT-expressing E. coli was added in suspension to carry out amination reaction at 33 C for 24 hours. By HPLC analysis of the reaction mixture with appropriate dilution, it was confirmed to genarate (2R,4R)-monatin or (2R,4S)-monatin as shown in Table 4. Incidentally, the BMDAT-expressing E. coli and S 243 N/A 182 S mutant BMDAT-expressing E. coli used for the amination reaction were prepared according to the method described in the following Reference Example. S 243 N/A 182 S C:p-MeC 6 H 4 SO 3 H, S:PhMe, S:MeOH, 15 h, rt Reactants: 1, Catalysts: 1, Solvents: 2, Steps: 1, Stages: 1 By Buddoo, Subash et al From U.S. Pat. Appl. Publ., , 02 Apr 2009 Experimental Procedure 3b. The process of 3a is typically carried out in 1:1 methanohtoluene at a substrate concentration of about 25% (m/v), with 10% TsOH added. This process may also be carried out in toluene at a range of substrate concentrations of about 10-20% (m/v) and adding 1% TsOH, which tends to result in less decomposition. Using this process variant, the reaction is run at room temperature for 15 hours, and the product precipitates from toluene, eliminating the need for column chromatography. This process variant typically results in a yield of 70% (from monatin), and the product is 98% pure by HPLC yield 70% 70%

3 3 1.1 S:MeCN, 6.5 h, rt, ph 7.5 biotransformation, buffered solution (phosphate), enzymic, ChiroCLEC-BL used, stereoselective, Reactants: 1, Solvents: 1, Steps: 1, Stages: 1 By Buddoo, Subash et al From U.S. Pat. Appl. Publ., , 02 Apr 2009 Experimental Procedure Enzymatic Hydrolysis 4a. The lactone from Example 3a (IV, R 1 is Me, -N(H)Prot is -NHCbz) was dissolved in 640 mlacn and 2.04 L of phosphate buffer, 0.3 M, ph ml CLEC- BL (lot: B103010A) was added. The reaction was stirred at room temperature for 6.5 hours. HPLC indicated that the hydrolysis was close to 50%. The solution was acidified to ph 2.0, and then extracted with methylene chloride (4x0.7 L). The combined methylene chloride layer was dried over Na 2 SO 4. The solvent was evaporated. The residue was dissolved in 1.2 L methylene chloride and 1.2 L saturated NaHCO 3.The phase separation was not clear because of aggregation of Vb. The organic phase was separated and the solid-aqueous phase was washed with methylene chloride (2x200 ml). HPLC indicated that the solid still contained over 5% IVa. The aqueous-solid phase was adjusted to ph 2.0, and 500 ml of methylene chloride was added. A milky suspension was formed, and the solid could not be re-dissolved. The ph ofthis milky suspension was adjusted back to 10. The organic phase was separated. The combined organic phase was washed with 200 ml saturated NaHCO 3 and evaporated to give 42 g of IVa (R,R).The solid-aqueous phase was filtered. The solid was dried to give 40.0 g of Vb (S,S) sodium salt. IVa (R,R), yield 42 g R:KOH, S:H 2 O, S:CH 2 Cl 2, 30 min, 25 C 1.2 R:AcOH, S:H 2 O, rt, ph By Buddoo, Subash et al From U.S. Pat. Appl. Publ., , 02 Apr 2009 Reactants: 1, Reagents: 2, Solvents: 3, Steps: 1, Stages: R:Et 3 N, R:H 2, C:Pd, S:MeOH, 3 h, rt Reactants: 1, Reagents: 2, Catalysts: 1, Solvents: 1, Steps: 1, Stages: 1 By Buddoo, Subash et al From U.S. Pat. Appl. Publ., , 02 Apr 2009 SciFinder Page C:Carbonicesterase, S:H 2 O, 37 C, ph 7; 20 h, rt, ph R:KOH buffered solution, Triton X-100 was used, Sodium phosphate buffer was used, Unspecified conditions were used for hydrogenation, sealed tube used, Reactants: 1, Reagents: 1, Catalysts: 1, Solvents: 1, Steps: 1, Stages: 2

4 Production of monatin enantiomers By Brady, Dean et al From PCT Int. Appl., , 09 Apr 2009 Experimental Procedure SciFinder Page 4 General/Typical Procedure: Example 1. Screening of Hydrolytic Enzymes for Isoxazoline Diester [0045] The screening is done in 0.1 M sodium phosphate buffer ph 7. 1% Triton X- 100 is added to the buffer and then isoxazoline diester is added slowly at 37 C. until saturation is reached. [0046] Aliquots of 1.5 ml of the above solution are placed in Eppendorff tubes and different hydrolytic enzymes are added to the tubes. A microspatula tip of the powdered enzymes is used or 100 µl of the liquid enzymes. The reactions are incubated for 20 hours on a vibrating shaker at an appropriate temperature, generally, room temperature. Example 2.Evaluation of Carboxylesterase NP (DSM) Activity for Isoxazoline Diester [0048] Only Carboxylesterase NP, was tested for activity according to the protocol in Example 1. The HPLC yielded a single product peak, and corresponding to enantiopure R- monatin according to chiral-hplc and LC-MS following hydrogenation and workup. CASREACT :. All Rights Reserved. CASREACT contains reactions from CAS and from: ZIC/VINITI database ( ) provided by InfoChem; INPI data prior to 1986; Biotransformations database compiled under the direction of Professor Dr. Klaus Kieslich; organic reactions, portions copyright John Wiley & Sons, Ltd., John Wiley and Sons, Inc., Organic Inc., and Organic Synthesis Inc. Reproduced under license. All Rights Reserved.

5 SciFinder Page 5 Substance Information

6 SciFinder Page C 3 H 4 O 3. Na Propanoic acid, 2-oxo-, sodium salt (1:1) ~ C 23 H 22 N 2 O 6 threo-pentaric acid, 2,3-dideoxy-4-C- (1H-indol-3-ylmethyl)-2- [[(phenylmethoxy)carbonyl]amino]-, 1,4-lactone, 5-methyl ester ~ C 23 H 22 N 2 O 6 L-threo-Pentaric acid, 2,3-dideoxy-4- C-(1H-indol-3-ylmethyl)-2- [[(phenylmethoxy)carbonyl]amino]-, 1,4-lactone, 5-methyl ester ~ C 22 H 22 N 2 O 7 D-Glutamic acid, 4-hydroxy-4-(1H- indol-3-ylmethyl)-n- [(phenylmethoxy)carbonyl]-, (4R)- ~3 C 24 H 26 N 2 O 7 D-Glutamic acid, 4-(1H-indol-3- ylmethyl)-4-methoxy-n- [(phenylmethoxy)carbonyl]-, 5-methyl ester, (4R)-rel- ~2 C 11 H 9 N O 3 1H-Indole-3-propanoic acid, α-oxo- ~571

7 SciFinder Page C 14 H 16 N 2 O 5 L-Glutamic acid, 4-hydroxy-4-(1Hindol-3-ylmethyl)-, (4R)- ~ C 14 H 16 N 2 O 5 D-Glutamic acid, 4-hydroxy-4-(1Hindol-3-ylmethyl)-, (4R)- ~48 C 22 H 20 N 2 O 6 D-threo-Pentaric acid, 2,3-dideoxy-4- C-(1H-indol-3-ylmethyl)-2- [[(phenylmethoxy)carbonyl]amino]-, 1,4-lactone (9CI) ~4 C 18 H 20 N 2 O 5 3,5-Isoxazoledicarboxylic acid, 4,5- dihydro-5-(1h-indol-3-ylmethyl)-, 3,5- diethyl ester ~7

8 SciFinder Page 8 Process for producing an optically active indole compound using asymmetric addition in the presence of α- amino acids and a metal ion By Kawahara, Shigeru; Amino, Yusuke; Sugiyama, Masakazu From Eur. Pat. Appl. (2008), EP A , Language: English, Database: CAPLUS The invention provides a process for producing an optically active compd. of the following formula I (including a salt form) which comprises the step of reacting a pyruvic acid with indole-3-pyruvic acid in the coexistence with an optically active α-amino acid contg. a secondary amine and a metal ion. Production of monatin enantiomers By Brady, Dean; Steenkamp, Lucia H.; Rousseau, Amanda L.; Buddoo, Subash; Steenkamp, Paul A. From PCT Int. Appl. (2009), WO A , Language: English, Database: CAPLUS Methods were disclosed for the prepn. of the high intensity sweetener monatin (2S,4S)-I, a.k.a. (2S,4S)-2-amino-4- carboxy-4-hydroxy-5-(3-indolyl)pentanoic acid, and its enantiomers via enzymic hydrolysis of an isoxazoline diester II (R1, R2 = alkyl). Methods for preferentially hydrolyzing one stereoisomer of an isoxazoline diester over another, as well as an enzyme for facilitating the preferential hydrolysis were provided. Also provided were methods for providing mixts. of (2R,4R)- and (2S,4R)-monatin as well as (2S,4S)- and (2R,4S)-monatin, which methods can include the step of stereoselectively hydrolyzing an isoxazoline diester. By Buddoo, Subash; Rousseau, Amanda Louise; Brady, Dean; Lalonde, James; Yao, Yiming; Wang, Yifong From U.S. Pat. Appl. Publ. (2009), US A , Language: English, Database: CAPLUS Methods and materials for the prodn. of the high intensity sweetener, monatin, in stereoisomerically-pure or stereoisomerically-enriched form are disclosed. For example, methods using stereoselective hydrolysis and sepn. of a monatin-derived lactone ester are disclosed.