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All 38 posts Subject: Ephedra Species List Please login to post Down

Aurelius
(Hive Addict)
03-29-03 02:49
No 422144
      Ephedra Species List
(Rated as: excellent)

This is List of the Ephedra Species, their synonyms and Varities. It is probably (almost surely) not complete. If you find additions, errors or helpful changes, please suggest them.

E. Alata
E. Altissima (Synonymous: E. Andina, E. Fragilis, E. Americana)
E. Americana (Syn: E. Americana, E. Altissima, E. Fragilis, E. Humilis)
Var. Rupestris
E. Andina (Syn: E. Americana, E. Altissima, E. Fragilis)
E. Antisyphilitica (Syn: E. Nevadensis)
E. Aphylla
E. Arenarius
E. Arenicola
E. Aspera
http://botany.cs.tamu.edu/FLORA/BigBend/BB0116.jpg
http://botany.cs.tamu.edu/FLORA/BigBend/BB0117.jpg

E. Breana
E. Distachya (Syn: E. Gerardiana, E. Vulgaris, E. Saxatilis, E. Likiangensis)
Var. Helvetica (Synonymous: E. Helvetica)
http://www.pharmakobotanik.de/schfld/Ephedra.jpg

E. Californica
Var. Funerea
E. Campylopoda
http://131.152.161.2/FMPro?-db=b.fp5&-lay=L&-format=b/bgoogle.htm&file=3905&-find

E. Ciliata
E. Coryii
E. Cutleri
E. Equisitena (Syn: E. Shennungiana)
http://www.ibiblio.org/herbmed/pictures/p05/pages/ephedra-equisetina.htm
http://www.ibiblio.org/herbmed/pictures/p05/pages/ephedra-equisetina-1.htm

E. Fasciculata
E. Fedtschenkoi (Syn: E. Fedtschenkoae)
E. Ferganensis (Syn: E. Intermedia, E. Glauca, E. Microsperma, E. Tesquorum, E. Tibetica, E. Persica, E. Valida)

E. Flava (Syn: E. Sinica, E. Sinensis, E. Ma-Huang)
E. Fragilis (Syn: E. Altissima, E. Andina, E. Americana)
http://www.pharmakobotanik.de/systematik/7_bilder/c920/C92-1689.jpg

E. Foliata
E. Frustillata
E. Funerea (SEE: E. Californica)
E. Geradiana (Syn: E. Distachya, E. Vulgaris, E. Saxatilis, E. Likiangensis)
Var. Congesta

E. Glauca (Syn: E. Microsperma, E. Tesquorum, E. Tibetica, E. Persica, E. Valida, E. Ferganensis, E. Intermedia)

E. Gracilis
E. Humilis (Syn: E. Americana, E. Rupestris, E. Altissima, E. Fragilis)
E. Intermedia (Syn: E. Glauca, E. Microsperma, E. Tesquorum, E. Tibetica, E. Persica, E. Valida, E. Ferganensis)
Var. Glauca, Persica, Schrenkii, Tibetica
http://131.152.161.2/FMPro?-db=b.fp5&-lay=L&-format=b/bgoogle.htm&file=2767&-find

E. Kaschgarica (Syn: E. Przewalskii)
E. Lepidosperma
E. Likiangensis (Syn: E. Gerardiana, E. Distachya, E. Vulgaris, E. Saxatilis)
Var. Mairei
E. Lomatolepis
E. Macedonica
E. Major (Syn: Nebrodensis, E. Scoparia)
SubSpecies (ssp) Procera (Syn: E. Procera)
E. Ma-Huang (Syn: E. Sinica, E. Sinensis, E. Flava)
E. Microsperma (Syn: E. Tesquorum, E. Tibetica, E. Persica, E. Valida, E. Ferganensis, E. Intermedia, E. Glauca)

E. Minima (Syn: E. Monosperma, E. Regeliana)
E. Minuta
Var. Dioeca
E. Monosperma (Syn: E. Minima, E. Regeliana)
E. Nebrodensis (Syn: E. Major, E. Scorparia)
E. Nevadensis (Syn: E. Antisyphilitica)
SubVariety- Paucibracteata
E. Occidentalis
E. Pachyclada
E. Pedunculata
E. Persica (Syn: E. Valida, E. Ferganensis, E. Intermedia, E. Glauca, E. Tesquorum, E. Microsperma, E. Tibetica)
E. Procera (SEE: E. Major)
http://www.pharmakobotanik.de/systematik/7_bilder/pis/S-000087.jpg
http://www.pharmakobotanik.de/systematik/7_bilder/c920/C92-0710.jpg
http://www.pharmakobotanik.de/systematik/7_bilder/c920/C92-0711.jpg

E. Przewalskii (Syn. E. Kaschgarica)
Var. Kaschgarica
E. Regeliana (E. Monosperma, E. Minima)
Var. Disperma
E. Rupestris (Syn: E. Humilis, E. Americana, E. Fragilis, E. Altissima)
E. Rhytidosperma
E. Saxatilis (Syn: E. Gerardiana, E. Distachya, E. Vulgaris, E. Likiangensis)
E. Scoparia (Syn: E. Major, E. Nebrodensis)
E. Shennungiana (Syn: E. Equisetina)
E. Sinensis (E. Sinica, E. Flava, E. Ma-Huang)
E. Sinica (Syn: E. Sinensis, E. Flava, E. Ma-Huang)
http://www.pharmakobotanik.de/systematik/7_bilder/yamasaki/Ephedra.jpg
http://www.pharmakobotanik.de/systematik/7_bilder/yamasaki/EphedraD.jpg

E. Texana
E. Tesquorum (Syn: E. Tibetica, E. Persica, E. Valida, E. Ferganensis, E. Intermedia, E. Glauca, E. Microsperma)

E. Tibetica (Syn: E. Persica, E. Valida, E. Ferganensis, E. Intermedia, E. Glauca, E. Tesquorum, E. Microsperma)

E. Torreyana
Var. Torreyana, Powelliorum
E. Trianda
E. Trifurca
http://www.fs.fed.us/database/feis/plants/shrub/ephvir/references.html
http://ag.arizona.edu/classes/rnr202/pix/202_3.gif
http://ag.arizona.edu/classes/rnr202/pix/202_1.gif
http://botany.cs.tamu.edu/FLORA/Fern&Gym/F&G034.jpg

Male
http://ag.arizona.edu/classes/rnr202/pix/202_2.gif
http://ag.arizona.edu/classes/rnr202/pix/202_104.jpg
http://botany.cs.tamu.edu/FLORA/Fern&Gym/F&G035.jpg

Female
http://botany.cs.tamu.edu/FLORA/Fern&Gym/F&G036.jpg
http://botany.cs.tamu.edu/FLORA/Fern&Gym/F&G037.jpg

E. Tweediana
E. Valida (Syn: E. Ferganensis, E. Intermedia, E. Glauca, E. Tesquorum, E. Microsperma, E. Tibetica, E. Persica)

E. Viridis
Var. Viridis
E. Vulgaris (Syn: E. Gerardiana, E. Distachya, E. Saxatilis, E. Likiangensis)
Var. Helvetica

http://wpsm.net/Ephedra.pdf
http://bodd.cf.ac.uk/BotDermFolder/BotDermE/EPHE.html
http://hua.huh.harvard.edu/china/mss/volume04/EPHEDRACEAE.published.pdf

Aurelius
(Hive Addict)
03-29-03 04:55
No 422168
      US Patent 4277420 (Ephedrine and the like)
(Rated as: excellent)

US Patent 4227420

Ephedrine and Pseudoephedrine Precursors

Referenced Cited:

US Patents:
2458420
2458422
4061767

Abstract:

Preparation of novel prochiral olefinic compounds which can be asymmetrically hydrogenated to enantiomers, which are converted to ephedrine and pseudoephedrine by described procedures

Example 1:

1-phenyl-2-oxime-1,2-propanedione

The following procedure is essentially as reported in the literature; Org. Synth., II, 363.

Propiophenone (14.6g, 0.1mol) was diluted with 100ml anhydrous diethyl ether in a N₂ atmosphere purged 200ml, 3-necked round bottom flask which was equipped with a magnetic stirring bar, addition funnel and syringe septum. While HCl gas was bubbled through the solution, butyl nitrite (10.3g, 0.1mol) in 10ml of anhydrous diethyl ether was added dropwise at such a rate as to create a gentle reflux. Additino time was approximately forty minutes. After ten additional minutes of HCl addition, the gas was turned off and the solution was stirred at room temperature for forty-eight hours. The solvent was removed under reduced pressure and the residue drying the resulting crystals at 80*C for 1.5 hours, 9.02g (52% yield) of 1-phenyl-2-oxime-1,2-propanedione was obtained that had; MP: 113-114*C

Example 2:

N-(1-Benzoylvinyl)acetamide

In a glass 90ml hydrogenation bottle equipped with a magnetic stirring bar was placed 1.0008g (6.15mmols) of 1-phenyl-2-oxime-1,2-propanedione, 0.12g of 5% Rhuthenium on Carbon and 26ml of acetic anhydride. The solution was purged with N₂ and pressurized to 50 psi with hydrogen. After heating to 50*C, the solution was stirred at temperature for forty-eight hours. The pressure was released and the solvent removed under reduced pressure (1mm/65*C). The resulting oil was purified on a dry silica gel column (0.5”x12”) using CH₂Cl₂ as the eluant. The column was followed by thin layer chromatography and the fastest moving band collected 0.63g (63% yield) of N-(1-Benzoylvinyl)acetamide that had; MP: 73-79*C.

Example 3:

N-(alpha-methylphenacy)acetamide

In a 50ml glass hydrogenation bottle equipped with a magnetic stir bar was placed 0.7496g (3.97mmol) of the Product from Example 2 in 20ml of 100% ethanol. N₂ gas was bubbled through for ten minutes. Cyclooctadiene-1,5-[(R,R)-1,2-ethanediylbis-(o-methoxyphenyl)-phenylphosphine] Rhodium tetrafluoroborate (7.4g, 0.01mmol) was added and the bottle was evacuated and filled with 30 psi of N₂ five times. The solution was heated to 50*C and 50 psi of hydrogen was introduced. After one hour, hydrogen uptake ceased. The pressure was released and the solution was diluted to 50ml with 100% ethanol at 20*C. The solvent was removed under reduced pressure (1mm/150*C) and a chiral shift study with tris [3-heptafluoropropylhydroxymethylene)d-camphorate] Europium III was conducted. The study indicated an 85% enantiomeric excess of (S)-N-(alpha-methylphenacyl)-acetamide.

Example 4:

N-(1-benzoylvinyl)acetamide

In a flame dried, N₂ purged 100ml round bottom flask equipped with and magnetic stirrer and a reflux condensor were placed 0.5g (3.38mmol) alpha-ketopropiophenone, 0.2g (3.39mmol) acetamide, 30mg paratoluenesulfonic acid, 10mg hydroquinone and 30ml toluene. The mixture was refluxed under N₂ for twenty hours. The solution was then cooled to room temperature and the solvent was removed under reduced pressure giving a mixture of aryl containing products which were 51% alpha-ketopropiophenone, 47% N-(1-benzoylvinyl)-acetamide.

Example 5:

N-(alpha-methylphenacyl)-benzamide

In a flask as in Example 4, 12ml (152mmol) pyridine, 34g (150mmol) benzoic anhydride and 3g (34mmol) racemic alanine were placed. The solution was heated at 120-125*C for two hours then cooled to room temperature. The solution was carefully diluted with saturated sodium bicarbonate and the product taken up in ethyl acetate. The organic layer was separated and washed 10x100ml of water. The solvent was removed under reduced pressure. This residue was steam distilled for 1.5 hours and the pot residue taken up in ethyl acetate and carefully washed with sodium bicarbonate (3x100ml). The solution was filtered through a 1”x3” plug of dry silica gel and the solvent removed under reduced pressure. The resulting oil was crystallized from diethyl ether by slow evaporation to pure d,l-N-(alpha-methylphenacyl)-benzamide that had; MP: 87-89*C.

Example 6:

N-(1-benzoylvinyl)benzamide

In a flame dried nitrogen purged 50 ml round bottom flask equipped with a magnetic bar, nitrogen inlet and septum were placed 0.83 g (3.28 mmol) N-(.alpha.-methylphenacyl) benzamide (prepared as in Example 5) and 10 ml of methanol. Sodium tetraborate (0.14 g, 0.37 mmol) was added. After five minutes the solution was homogeneous and 0.56 ml (4.98) of freshly prepared t-butylhypochlorite was added. After approximately one hour the solvent was removed under reduced pressure to give a light oil which was placed on a 1/2".times.10" dry silica gel column using methylene chloride as the eluant giving 0.63 g (R.sub.f =0.53 with CHCl.sub.3) of N-(.alpha.-methylphenacyl)-N-chlorobenzamide. This material was taken up in carbon tetrachloride and 0.4 g of DABCO amine was added in one portion at room temperature. After ten minutes the solution was filtered giving virtually pure N-(1-benzoylvinyl) benzamide (R.sub.f =0.47 in CHCl.sub.3) in quantitative yield (light yellow oil).

DABCO amine is 1,4-diazabicyclo (2.2.2.) octane, also called triethylenediamine. Amines in general or other bases can also be employed to effect the dehydrohalogenation. The combination of sodium tetraborate and t-butylhypochlorite is convenient for laboratory use, but other forms of sodium hypochlorite, or hypochlorite and base, can be used.

Example 7:

ethyl N-(1-benzoylvinyl)carbamate

In a flame dried nitrogen purged 100 ml round bottom flask equipped with a magnetic stirring bar, Dean-Stark trap and a reflux condenser were added 3.0 g (20.3 mmol) .alpha.-keto-propiophenone, 3.6 g (40.5 mmol) urethane (ethyl carbamate),10 mg (0.1 mmol) hydroquinone, 60 mg (0.3 mmol) paratoluenesulfonic acid and 20 ml of toluene. The mixture was refluxed under nitrogen with vigorous stirring for 31/2 hours. The solvent was removed under reduced pressure and the residue distilled through a short path distillation column giving approximately 1 g of ethyl N-(1-benzoylvinyl)carbamate bp.perspectiveto.130.degree.-135.degree. C. (1 mm).

The hydroquinone in the above Example was employed as a polymerization inhibitor, but its use is not necessary

Example 8:

ethyl-N-(.alpha.-methylphenacyl)carbamate

In a 60 ml glass pressure bottle equipped with a magnetic stirrer were placed 25 ml of 100% ethanol, 0.3511 g of a mixture which was composed of 80%-N-(1-benzoylvinyl)urethane, 10% urethane and 9% .alpha.-keto-propiophenone, and 9.times.10.sup.-3 g of cyclooctadiene-1,5 [(R,R)-1,2-ethanediylbis(o-methoxyphenyl)phenyl phosphine] rhodium tetrafluoroborate. The solution was heated to 50.degree. C.; purged by filling the reactor with nitrogen followed by evacuation. This sequence was conducted five times. The pressure bottle was pressurized to 50 psi with hydrogen and the reaction monitored until hydrogen uptake ceased (approximately two hours). The pressure was released and the solent was removed under reduced pressure at 50.degree. C. The residue was purified on dry silica gel column using chloroform as the eluant. The column was followed by thin layer chromatography and the band with R.sub.f =0.24 collected giving over 200 mg of N-(.alpha.-methylphenacyl)urethane, [.alpha.].sub.D.sup.20 =20.3 (C,1,4 ethanol).

This, in the context of other results, indicates a large excess of the S form. At times it may be convenient to employ a mixture as described above containing the N(1-benzoylvinyl)urethane reactant, obtainable in procedures like that of Example 7, rather than the pure compound.

Example 9:

Ethyl N-(2-hydroxyl-1-methyl-2-phenylethyl)carbamate

In a flame dried, nitrogen purged 10 ml round bottom flask equipped with a nitrogen inlet and a magnetic stirring bar were placed 60.3 mg (0.273 mmol) of optically active ethyl N-(.alpha.-methylphenacyl)carbamate obtainable in procedures like Example 8, and 3 ml of 100% ethanol. Sodiumborohydride (24 mg, 1.6 mmol) was added at room temperature in one portion. The mixture was stirred at room temperature for one hour. The solvent was then removed under reduced pressure (1 mm, 40.degree. C.) yielding a residue which was taken up in deuteriochloroform and filtered. By nmr the starting material had been fully reduced to give approximately a 70/30; erythro/threo mixture of ethyl-N-(2-hydroxyl-1-methyl-2-phenylethyl) carbamate, with the S form (at the 2-carbon) being in large excess. The S form could be obtained in approximately 100% excess if the starting -methylphenacyl carbamate were converted to about 100% S form by crystallization. Alternatively, the product of the present example can be purified by crystallization to obtain the desired excess of S form. A small amount of ethanol was still present which was removed as an azeotrope with nitromethane.

Example 10:

2-(methylamino)-1-phenyl propanol

In a flame dried nitrogen purged 5 ml round bottom flask equipped with a magnetic stirring bar and a nitrogen inlet were placed 50 mg (0.22 mmol) of ethyl-N-(2-hydroxy-1-methyl-2-phenylethyl) carbamate (as prepared in Example 9) and 3 ml of anhydrous diethyl ether. Lithium aluminum hydride (LAH) was added (10 mg. 2.6 mmol) in one portion. The suspension was stirred for 21/4 hours at room temperature. The reaction was quenched with water (carefully) and the products extracted with diethylether. The reaction had gone 9% to completion. The mixture was taken up in 5 ml tetrahydrofuran (THF). LAH (30 mg, 7.8 mmol) was added and the mixture refluxed for fifteen minutes. The reaction was carefully quenched with water and extracted with diethylether. The rection had gone 40% to completion by nmr. The material was then refluxed in 5 ml of THF with 30 mg LAH for an additional 11/4 hours. The mixture was cooled, quenched with water and extracted with diethylether giving cleanly 80% of 2-(methylamino)-phenylpropanol. The material was purified by acid extraction with HCL/water, neutralization and back extraction with diethylether. With the starting carbamate being largely in the S form (at the .alpha.-carbon), the product is mainly a mixture of ephedrine and pseudoephedrine.

badbody
(Hive Bee)
03-29-03 15:30
No 422275
      Pseudo/Ephedrine Derivatives
(Rated as: excellent)

Ephedrine (2-methylamino-1phenylpropanol-1, alpha-hydroxy-beta-methylaminopropylbenzene)
C6H5-CH(OH)-CH(NH-CH3)-CH3
C10H15ON
MW 165

(-).
Present in various species of Ephedra. Hydrated cryst. From H2O m.p. 40°. b.p. 225°. Sol H2O, EtOH, Et2O, CHCl3. n_D²⁰ –6.3° in EtOH.
B, HCl: m.p. 218°. n_D²⁰ -36.6° (-34.9°) in H2O.
B, HBr: m.p. 205°.
B, H2PtCl6: needles. m.p. 186°.
B, HAuCl4: yellow needles. m.p. 128-31°.
Oxalate: m.p. 239-240°.
N-Ac: m.p. 85.5-86.5° corr. n_D²⁰ +8.1° in EtOH, -63.2° in CCl3. Hydochloride: m.p. 106-7°. n_D²⁰ +5.6° in 50% EtOH.
N-p-Nitrobenzoyl: pale yellow prisms. m.p. 187-8°. n_D²⁰ –51.77° in CHCl3.

(+).
Plates from H2O. m.p. 40-40.5°.
B,HCl: plates from EtOH. M.p. 217-18°. n_D²⁰ +34.42° in H2O. More easily sol. than (-) form.
N-p-NitroBenzoyl: Yellowish leaflets from EtOH. m.p. 187-8°. n_D²⁰+51.12° in CHCl3.

(+/-).
Needles from EtOh or pet. Ether. m.p. 76. Sol H2O, EtOH, Et2O, CHCl3, C6H6.
B, HCl: Plates from EtOH. m.p. 188-9.5°.
B,HAuCl4: yellow cryst. m.p. 115°.
B2,H2PtCl6: reddish-yellow needles or leaflets. m.p. 199° (183°) decomp.
N-Ac: m.p. 77-8.5°.   hydrochloride: m.p. 180°.
O-Ac hydrochloride: m.p. 201-201.5°.
N-p-Nitrobenzoyl: pale yellow plates from EtOH. m.p. 162°.
Methiodide: needles. m.p. 228-9°.

Nagai, Kanao, Ann., 1929, 470, 157.
Emde, Helv. Chim. Acta, 1929, 12, 365, 405.
Spath, Gohring, Monatsh., 1920, 41, 319.
Freudenburg, Braun, Schoeffel, J. Am. Chem. Soc., 1932, 54, 234.
Hoffman-La Roche A.G., D.R.P. 554,553 (Chem. Zentr., 1932, II, 1693).
Welsh, J. Am. Chem. Soc., 1947, 69, 128.

psi-Ephedrine (Isoephedrine)
C6H5-CH(OH)-CH(NH-CH3)-CH3
C10H15ON
MW 165

(-).
Prisms from Et2O. m.p. 118-118.5°. n_D²⁰ –51.93° in EtOH.
B, HCL: needles from EtOH. m.p. 182-182.5°. n_D²⁰ –61.88°.

(+).
Occurs in leaves of Ephedra vulgaris, prisms from Et2O. m.p. 117-18° n_D²⁰ +51.24° in EtOH.
Sol EtOH, Et2O. Spar. sol. cold H2O.
B,HCl: prisms from EtOH. m.p. 182-182.5°. n_D²⁰ +61.6° in H2O.
Oxalate: Needles from EtOH. m.p. 219°.
N-Ac: m.p. 103.5-104°. n_D²⁰ 110.4° in 50% EtOH, 113.8° in EtOH, 121.8° in CHCl3.
O-Ac Hydrochloride: m.p. 179.5-181°. n_D²⁰ 98.6° in H2O.
N-p-Nitrobenzoyl: yellowish cryst. from EtOH. m.p. 177°. n_D²⁰ +140.8° in CHCl3.
B,HAuCl4: m.p. 126-126.5°.

(+/-).
Needles m.p. 118°. b.p. 130°/16mm. Sol EtOH, C6H6. Spar sol. H2O, Et2O.
B, HCl: needles from EtOH.   m.p. 164°.
Oxalate: prisms from EtOH. m.p. 218° decomp.
B, HAuCl4: yellow prisms. m.p. 117°.
(B, HCl)2,AuCl3: yellow needles. m.p. 194°.
N-p-Nitrobenzoyl: prisms from EtOH. m.p. 165-6°.
Methiodide: cryst. from H2O.   m.p. 183°.
Picrate: m.p. 192°.

Spath, Koller, Ber., 1925, 58 1268
Nagai, Kanao, Ann., 1929, 470, 157
Emde, Helv. Chim. Acta., 1929, 12, 365
Bossert, Brode, J. Am. Chem. Soc.,1934, 56, 165
Stevens, J. Am. Chem Soc., 1938, 60, 3089

I photocopied this years ago. I don’t know the name of the book, but it’s on pages 1344 and 1355.

Aurelius
(Hive Addict)
03-30-03 04:04
No 422421
      Finally!

Finally! Somebody who comes through! thanks for the material, it will be added soon.

pHarmacist
(Hive Addict)
03-30-03 04:18
No 422430
User Picture       I especially like this:

I don’t know the name of the book, but it’s on pages 1344 and 1355.

Charming, needless to say wink

Accept No Imitations, There Can Only Bee One; www.the-hive.ws

Aurelius
(Hive Addict)
03-30-03 21:13
No 422579
      ????

What are you talking about (pHarmacist)? as in, who's work are you refering?

Rhodium
(Chief Bee)
03-30-03 22:13
No 422590
User Picture       BadBody's post

He is just commenting on the last line of BadBody's post, referencing the data to "Unknown book, page 1344-5". I think that's cute too wink

Aurelius
(Hive Addict)
03-31-03 20:48
No 422837
      US Patent 1799110 (Ephedrine Analogues)
(Rated as: excellent)

I wouldn't mind somebody going through and checking the nomenclature of the derivatives for this patent as it isn't my strongest point in chemistry. Much appreciated.

US Patent 1799110

Process of Producing Ephedrine and Structurally-Similar Compounds and Products of Such Process

Abstract:

The sythesis of various ephedrine derivatives by way of reductive amination with different amines and the respective ketones. The first step comprising of condensation of a dione with a primary amine. The imido-carbonyl compound from the first step is then hydrogenated in the presence of a platinum catalyst to give the ephedrine derivative.

Preparation of Phenylpropanedione:

A few drops of an alcoholic solution of HCl are added to 67 parts by weight of ethyl phenyl mono-ketone, desireably contained in a flask fitted with an inlet tube leading to the bottom. A dehydrating agent is also added as 10 parts or more of anhydrous calcium chloride, to remove any water that may be contained in the alcohol and that is formed in the reaction. The mixture is heated to about 60*C and 35 parts of dry NO₂ from a weighed tube is passed in sufficiently slowly so that any escape of the gas from the liquid is substantially prevented. The temperature should be maintained between 60-75*C and may conveniently be prevented from rising above 75*C by retarding the rate of inflow of NO₂ or by cooling the gas or the reaction mixture.

The reaction is allowed to go on until the temperature of the mixture begins to drop spontaneously, or until NO₂ begins to escape from the exit in case the amount thereof supplied was not weighed. Any excess gas is removed by drawing air through the apparatus. The reaction mix is washed with water; and then with a solution of sodium carbonate until no more CO₂ is evolved. Then the oily layer is separated from the aqueous solution and dried. The liquid is now distilled under reduced pressure, and the fraction at 20mmHg/130*C is kept. (Has some monoketone impurities)

60 parts of sodium acid sulphite is now added to the product and shaken until most of the yellow color disappears. Water is added, if necessary, to give good suspension. The sodium acid sulphite readily forms an ether-insoluble salt with the dione. This reacts only slightly with the mono-ketone. The mass is now well cooled; and is then filtered. Some ether may be run through to wash out any mono-ketone that was adsorbed on the sulphite salt. This ether rests as a separate layer on the water filtrate. This water and the ether filtrate may be saved to recover the mono-ketone, and also any dione which may be in the water layer.

The Bisulphite salt is treated with 200 parts of hot water, and about 30 parts sodium carbonate.   The material is now steam distilled until no more dione is separated from the water, then dried. The product may be redistilled under reduced pressure to obtain a purer product although this is not necessary for futher steps in this patent.

Example 1:

Ephedrine Hydrochloride

7.4 parts of the product above is treated with 50 parts of ethanol and an alcoholic solution of methylamine (3 parts). A slight temperature rise is noted, due to the formation of the imido-carbonyl compound. A small amount (0.2 parts) of a platinum catalyst is added and the flask is filled with hydrogen and the reaction takes place as is given in the literature. Upon addition of air to the flask after the hydrogenation, the catalyst readily coagulates and may then be filtered. The solvent is removed under pressure to a small volume and the product is made slightly acidic with a small volume of HCl. The solvent is then completely removed whereby ephedrine hydrochloride separates out as white crystals. Racemic ephedrine with a smaller portion of racemic pseudoephedrine is formed. The isomers may be separated by methods in the literature (fractional crystallization from chloroform is mentioned). A substancially pure racemic ephedrine hydrochloride may thus be obtained, whose melting point is about 186*C. [red]

Example 2:

1-phenyl-1-hydroxy-2-ethylaminopropane hydrochloride

[red]This process utilized ethyl amine as the amine for formation of the imido-carbonyl compound, by which upon reduction yielded the named analogue hydrochloride. The product had; MP: 198*C

Example 3:

1-phenyl-1-hydroxy-2-ethylaminobutane hydrochloride

This process utilized ethyl amine as the amine and 1-phenyl-1,2-butanedione as the dione for the formation of the imido-carbonyl compound by which upon reduction yielded the named analogue hydrochoride. The white crystalline product had; MP: 226-227*C.

Example 4:

1-(p-methylphenyl)-1-hydroxy-2-ethylaminoethane
Hydrochloride: white crystals; MP: 209-211*C.
Picrate: MP: 158*C

Example 5:

1-(p-ethylphenyl)-1-hydroxy-2-ethylaminopropane hydrochloride that had; MP: 208*C

Example 6:

1-phenyl-1-hydroxy-2-benzylaminopropane hydrochloride
Hydrochloride: white solid; sp. sol cold water, alcohol; insol. acetone; MP: 184-185*C

Example 7:

1-phenyl-1-hydroxy-2-ethanolaminopropane
Hydrochloride: white solid; readily sol. water; sol. alcohol; sp. sol. acetone; MP: 166*C

Example 8:

1-phenyl-1-hydroxy-2-phenylaminopropane
Hydrochloride: MP: 177*C

Example 9:

1-phenyl-1-hydroxy-2-(2-phenethyl)aminopropane (?) (used beta-phenethylamine for amine)
Hydrochloride: white solid; MP: 207-208*C

Example 10:

1-(2,4-dimethylphenyl)-1-hydroxy-2-ethylaminopropane
Hydrochloride: white solid; MP: 221*C

Aurelius
(Hive Addict)
03-31-03 22:16
No 422855
      US Patent 1956950 (l-Ephedrine from L-PAC)
(Rated as: excellent)

US Patent 1956950

Manufacture L-1-Phenyl-2-Methylaminopropanol-1 (l-Ephedrine)

Abstract: Manfacture of l-Ephedrine by the Reductive Amination of L-PAC

Example 1:

l-Ephedrine Hydrochloride

120 g of L-PAC are added over 2 hours into a solution of 10 g of methylamine in 500 ml of ether in the presence of 20g of activated aluminum (as in Brit. Pat. 336,412) whilst stirring. Simultaneously, 20-30g of water are added dropwise. The vigorous reaction, which at once sets in, is moderated by periodical cooling. Activated aluminum is an aluminum/mercury amalgam. When it contacts water, it liberates hydrogen and insoluble aluminum hydroxide is formed. Activated aluminum thus serves as the source for hydrogen in the reductive amination.   When the reaction is completed, the ethereal solution is filtered and the optically active base which has been formed is extracted from the filtrate by means of dilute acid. The product is worked up in the usual manner. Ephedrine Hydrochloride is obtained in a yield ranging from 25-45g depending on the nature (probably purity) of the parent material. It has; MP: 214*C. Stated to have the optical rotation as listed in literature.

Example 2:

300g of of L-PAC is reduced in the same manner as in the previous example, but with 70cc of a 1% colloidal Pt solution and 85g of 33% methylamine solution.   (It is advantageous to add ether to the mix.) After the reaction, the mix is shaken with HCl solution and the Ephedrine Hydrochloride is isolated as is normal. The yield amounts to 110g of the hydrochloride that had; MP: 214*C.

Example 3:

100g of L-PAC is dissolved into 200cc of ether, 75g of 33% methylamine solution is added and the whole is shaken for about 0.5 hours. Condensation occurs with evolution of heat. The reaction mixture is then treated with hydrogen in the presence of 70cc of a colloidal Pt of 1% strength. The product is then worked up as before. Hydrochloride; MP: 214-216*C; Freebase;MP: 40*C.

Aurelius
(Hive Addict)
04-02-03 00:41
No 423174
      US Patent 5834261 (Vicinal aminoalcohols)

US Patent 5834261

Method for the Production of Chiral Vicinal Aminoalcohols

Abstract:

The disclosure describes a method for the preparation of chiral vicinal aminoalcohols in high optical purity. The method combines the stereoselective reduction of the keto group of a beta-ketoacid, beta-ketoester, or derivative with the stereospecific rearrangement of the corresponding amide, hydroxyamic acid, or hydrazide to produce chiral vicinal aminoalcohols with control of stereochemistry at both chiral centers.

Example 1:

Production of ethyl (2R,3S)-2-ethyl-3-hydroxybutrate

Twenty grams of baker’s yeast (Sigma Chemical Company, Saccharomyces Cerevisiae, type II) was suspended in a solution of 30g of sucrose in water in a conical flask, and the mixture was placed in an orbital shaker chamber maintained at 220rpm and 30*C for 30 minutes to initiate fermentation. Two grams of ethyl –2-ethylacetoacetate was dissolved in 2 ml of 95% ethanol, the resulting solution was added to the fermenting yeast, and shaking was resumed. The reaction was followed by TLC (staining with phosphomolybdic acid in ethanolic sulfuric acid) to monitor the consumption of starting material and the production of product alcohol. After approximately 48 hours the reaction was judged complete, and the reaction was terminated by removing the flask from the shaker and adding 20-30grams of Celite to the reaction mixture. The resulting suspension was suction filtered through a pad of Celite, and the clear yellow filtrate was extracted with ethyl acetate (4x200ml). The extracts were combined, dried over MgSO₄, filtered, and rotary evaporated to leave 1.6g of a yellowish oil containing ethyl (2R, 3S)-2-methyl-3-hydroxybutyrate as the major product (80%) and ethyl (2S, 3S)-2-methyl-3-hydroxybutyrate (20%) as the minor product as judged by chiral chromatography.

Example 2:

Production of Octyl (2R, 3S)-2-ethyl-3-hydroxybutyrate

20g of baker’s yeast (Sigma Chemical Company, type II) was suspended in an aqueous solution containing 30g of sucrose in a conical flask, and the mix was placed on an orbital chaker (220rpm) at 30*C for 30 minutes to initiate fermentation. 2g of octyl-2-ethyl acetoacetate was dissolved in 2 ml of 95% ethanol, the resulting solution was added to the fermenting yeast, and shaking was resumed. The reaction was followed by TLC (staining with anisaldehyde) to monitor the consumption of starting material and the production of product alcohol. After approximately 48 hours the reaction was judged complete, and the reaction was terminated by removing from the shaker and adding 20-30g of Celite. The resulting suspension was suction filtered through a pad of Celite and the clear yellow filtrate was extracted with ethyl acetate (4x200ml). The extracts were combined, dried over MgSO₄, filtered, and rotary evaporated to leave 1.8g of octyl (2R, 3S)-2-ethyl-3-hydroxybutyrate as a yellowish oil (>96% enantiomeric excess as judged by chiral chromatography).

Example 3:

Production of (2R,3S)-ethyl-2-allyl-3-hydroxybutyrate

20g of baker’s yeast (Sigma Chemical Company, type II) was suspended in an aqueous solution containing 30g of sucrose in a conical flask, and the mix was placed on an orbital chaker (220rpm) at 30*C for 30 minutes to initiate fermentation. 2g of octyl-2-ethyl acetoacetate was dissolved in 2 ml of 95% ethanol, the resulting solution was added to the fermenting yeast, and shaking was resumed. The reaction was followed by TLC (staining with anisaldehyde) to monitor the consumption of starting material and the production of product alcohol. After approximately 48 hours the reaction was judged complete, and the reaction was terminated by removing from the shaker and adding 20-30g of Celite. The resulting suspension was suction filtered through a pad of Celite and the clear yellow filtrate was extracted with ethyl acetate (4x200ml). The extracts were combined, dried over MgSO₄, filtered, and rotary evaporated to leave 1.6g of a yellow oil containing (2R,3S)-2-ethyl-3-hydroxybutyrate as the major product (75%) and (2S,3S)-2-ethyl-3-hydroxybutyrate (25%) as the minor product as judged by chiral chromatography.

Example 4:

Production of ethyl (2S,3S)-2-ethyl-3-hydroxybutyrate

Colletotrichum gloeosporioides (MMP 3233) was cultured according to the method of Buisson and Azerad (Tet. Lett. 27, 2631-2634), herein incorporated by reference) in one liter of a medium of glucose (30g), KH₂PO₄ (2g), corn steep liquor (10g) MgSO₄-7H₂O (0.5g), NaNO₃ (2g), FeSO₄-7H₂, and KCl (0.5g) with rotary shaking at 25*C. 2g of ethyl-2-ethyl acetoacetate was dissolved in 2 ml of 95% ethanol, the resulting solution was added to the fermenting yeast, and shaking was resumed. The reaction was followed by TLC (staining with anisaldehyde) to monitor the consumption of starting material and the production of product alcohol. After approximately 48 hours the reaction was judged complete, and the reaction was terminated by removing from the shaker and adding 20-30g of Celite. The resulting suspension was suction filtered through a pad of Celite and the clear yellow filtrate was extracted with ethyl acetate (4x200ml). The extracts were combined , dried over MgSO₄, filtered, and rotary evaporated to leave 1.7g (2S,3S)-2-ethyl-3-hydroxybutyrate as a yellow oil. The chiral purity of the product was greater than 98% as judged by chiral chromatography.

Example 5:

Production of ethyl-(2S,3S)-2-ethyl-3-hydroxybutyrate

Rhizopus Arrhizus (ATCC 11145) was cultured according to the method of Buisson and Azerad (Tet. Lett. 27, 2631-2634 (1986), herein incorporate by reference) in one liter of a medium of glucose (30g), KH₂PO₄ (1g), K₂HPO₄ (2g), corn steep liquor (10g), MgSO₄ (0.5g), NaNO₃ (2g), FeSO₄-7H₂ (0.02g), and KCl (0.5g) with rotary shaking at 25*C. 2g of ethyl-2-ethyl acetoacetate was dissolved in 2ml of 95% ethanol, the resulting solution was added to the fermenting yeast, and shaking was resumed. The reaction was followed by TLC (staining with anisaldehyde) to monitor the consumption of starting material and the production of product alcohol. After approximately 48 hours the reaction was judged complete, and the reaction was terminated by removing from the shaker and adding 20-30g of Celite. The resulting suspension was suction filtered through a pad of Celite and the clear yellow filtrate was extracted with ethyl acetate (4x200ml). The extracts were combined , dried over MgSO₄, filtered, and rotary evaporated to leave 1.6g of (2S,3S)-2-ethyl-3-hydroxybutyrate as a yellow oil. The chiral purity of the product was shown to be greater than 98% as judged by chiral chromatography.

Example 6:

Alternative Production of ethyl (2S,3S)-2-ethyl-3-hydroxybutyrate

2g of ethyl-2-ethyl acetoacetate was dissolved in 2ml of 95% ethanol, and the resulting solution was added to a solution of alcohol dehydrogenase (500 units from Rhizopus arrhizus –ATCC 11145) containing Potassium Phosphate buffer, 100mM, pH 7.0. NAD+ (100mg) was added to the solution along with 1g of sodium formate and 100 units of formate dehydrogenase (Boehringer Mannhelm). for recycling of the NAD+ cofactor. The reaction was followed by TLC (staining with anisaldehyde) to monitor the consumption of starting material and the production of product alcohol. After approximately 48 hours the reaction was judged complete, and the reaction was terminated by removing from the shaker. The resulting solution was extracted with ethyl acetate (4x200ml). The extracts were combined, dried over MgSO₄, filtered, and rotary evaporated to leave 1.8g of (2S,3S)-2-ethyl-3-hydroxybutyrate as a light yellow oil. The chiral purity of the product was greater than 99% as judged by chiral chromatography.

Example 7:

Production of (1S,2R)-ethyl-2-hydroxycyclopentanecarboxylate

25g of baker’s yeast (Saccharomyces Cerevisiae, Sigma Chemical Company, type II) was suspended in 100ml of sterilized tap water in a conical flask, and the mixture was placed on an orbital shaker (220rpm) at 30*C for 1 hour to activate the yeast. 1g of ethyl-2-oxocyclopentanecarboxylate was added, shaking was resumed, and progress of the reaction was monitored by TLC (staining with anisaldehyde). After approximately 100 hours the reaction was judged complete, and the reaction was terminated by removing from the shaker and adding 20-30g of Celite. The resulting suspension was suction filtered through a pad of Celite and the clear yellow filtrate was extracted with diethyl ether (4x100ml). The extracts were combined , dried over MgSO₄, filtered, and rotary evaporated to leave 0.7g of octyl-(1R,2S)-ethyl-2-hydroxycyclopentanecarboxylate as a yellowish oil (70% yield).

Aurelius
(Hive Addict)
04-02-03 00:44
No 423175
      more

Example 8:

Production of (1R,2S)-ethyl-2-hydroxycyclohexanecarboxlyate

25g of baker’s yeast (Saccharomyces Cerevisiae, Sigma Chemical Company, type II) was suspended in 100ml of sterilized tap water in a conical flask, and the mixture was placed on an orbital shaker (220rpm) at 30*C for 1 hour to activate the yeast. 1g of ethyl-2-oxocyclohexanecarboxylate was added, shaking was resumed, and progress of the reaction was monitored by TLC (staining with anisaldehyde). After approximately 100 hours the reaction was judged complete, and the reaction was terminated by removing from the shaker and adding 20-30g of Celite. The resulting suspension was suction filtered through a pad of Celite and the clear yellow filtrate was extracted with diethyl ether (4x100ml). The extracts were combined , dried over MgSO₄, filtered, and rotary evaporated to leave 0.6g of octyl-(1R,2S)-ethyl-2-hydroxycyclohexanecarboxylate as a yellowish oil (60% yield).

Example 9:

Production of (1S,2S)-ethyl-2-hydroxcyclopentanecarboxylate

Geotrichum Candidum (ATCC 34614) was cultured according to the method of Buisson and Azerad (Tet. Lett. 27, 2631-2634 (1986), herein incorporate by reference) in one liter of a medium of glucose (30g), KH₂PO₄ (1g), K₂HPO₄ (2g), corn steep liquor (10g), MgSO₄ (0.5g), NaNO₃ (2g), FeSO₄-7H₂ (0.02g), and KCl (0.5g) with rotary shaking at 25*C. 2g of ethyl-2-oxocyclopentanecarboxylate was dissolved in 2ml of 95% ethanol, the resulting solution was added to the fermenting yeast, and shaking was resumed. The reaction was followed by TLC (staining with anisaldehyde) to monitor the consumption of starting material and the production of product alcohol. After approximately 48 hours the reaction was judged complete, and the reaction was terminated by removing from the shaker and adding 20-30g of Celite. The resulting suspension was suction filtered through a pad of Celite and the clear yellow filtrate was extracted with ethyl acetate (4x200ml). The extracts were combined , dried over MgSO₄, filtered, and rotary evaporated to leave 1.5g of (1S,2S)-2-hydroxycyclopentanecarboxylate as a yellow oil. The chiral purity of the product was greater than 99% as judged by chiral chromatography.

Example 10:

Production of (1S,2S)-ethyl-2-hydroxycyclohexanecarboxylate

Geotrichum Candidum (ATCC 34614) was cultured according to the method of Buisson and Azerad (Tet. Lett. 27, 2631-2634 (1986), herein incorporate by reference) in one liter of a medium of glucose (30g), KH₂PO₄ (1g), K₂HPO₄ (2g), corn steep liquor (10g), MgSO₄ (0.5g), NaNO₃ (2g), FeSO₄-7H₂ (0.02g), and KCl (0.5g) with rotary shaking at 25*C. 2g of ethyl-2-oxocyclohexanecarboxylate was dissolved in 2ml of 95% ethanol, the resulting solution was added to the fermenting yeast, and shaking was resumed. The reaction was followed by TLC (staining with anisaldehyde) to monitor the consumption of starting material and the production of product alcohol. After approximately 48 hours the reaction was judged complete, and the reaction was terminated by removing from the shaker and adding 20-30g of Celite. The resulting suspension was suction filtered through a pad of Celite and the clear yellow filtrate was extracted with ethyl acetate (4x200ml). The extracts were combined , dried over MgSO₄, filtered, and rotary evaporated to leave 1.4g of (1S,2S)-ethyl-2-hydroxycyclohexanecarboxylate as a yellow oil . The chiral purity of the product was greater than 99% as judged by chiral chromatography.

Example 11:

Production of ethyl-3(S)-hydroxybutyrate

20g of baker’s yeast (Sigma Chemical Company, type II) was suspended in an aqueous solution containing 30g of sucrose in a conical flask, and the mix was placed on an orbital chaker (220rpm) at 30*C for 30 minutes to initiate fermentation. 2g of ethyl acetoacetate was dissolved in 2 ml of 95% ethanol, the resulting solution was added to the fermenting yeast, and shaking was resumed. The reaction was followed by TLC (staining with anisaldehyde) to monitor the consumption of starting material and the production of product alcohol. After approximately 48 hours the reaction was judged complete, and the reaction was terminated by removing from the shaker and adding 20-30g of Celite. The resulting suspension was suction filtered through a pad of Celite and the clear yellow filtrate was extracted with ethyl acetate (4x200ml). The extracts were combined, dried over MgSO₄, filtered, and rotary evaporated to leave 1.5g of a light yellow oil containing ethyl-3(S)-hydroxybutyrate as the major product as judged by chiral chromatography.

Example 12:

Production of (R)-ethyl-3-hydroxybutyrate

Geotrichum Candidum (ATCC 34614) was cultured according to the method of Buisson and Azerad (Tet. Lett. 27, 2631-2634 (1986), herein incorporate by reference) in one liter of a medium of glucose (30g), KH₂PO₄ (1g), K₂HPO₄ (2g), corn steep liquor (10g), MgSO₄ (0.5g), NaNO₃ (2g), FeSO₄-7H₂ (0.02g), and KCl (0.5g) with rotary shaking at 25*C. 2g of ethyl-acetoacetate was dissolved in 2ml of 95% ethanol, the resulting solution was added to the fermenting yeast, and shaking was resumed. The reaction was followed by TLC (staining with anisaldehyde) to monitor the consumption of starting material and the production of product alcohol. After approximately 48 hours the reaction was judged complete, and the reaction was terminated by removing from the shaker and adding 20-30g of Celite. The resulting suspension was suction filtered through a pad of Celite and the clear yellow filtrate was extracted with ethyl acetate (4x200ml). The extracts were combined , dried over MgSO₄, filtered, and rotary evaporated to leave 1.4g of (R)-ethyl-3-hydroxybutyrate as a yellow oil.

Example 13:

Production of (2S,3S)-2-ethyl-3-hydroxybutyramide by microbial reduction of the corresponding 2-ethylacetoacetamide

Geotrichum Candidum (ATCC 34614) was cultured according to the method of Buisson and Azerad (Tet. Lett. 27, 2631-2634 (1986), herein incorporate by reference) in one liter of a medium of glucose (30g), KH₂PO₄ (1g), K₂HPO₄ (2g), corn steep liquor (10g), MgSO₄ (0.5g), NaNO₃ (2g), FeSO₄-7H₂ (0.02g), and KCl (0.5g) with rotary shaking at 25*C. 2g of 2-ethyl-3-ketobutyramide was dissolved in 2ml of 95% ethanol, the resulting solution was added to the fermenting yeast, and shaking was resumed. The reaction was followed by TLC (staining with anisaldehyde) to monitor the consumption of starting material and the production of product alcohol. After approximately 48-72 hours the reaction was judged complete, and the reaction was terminated by removing from the shaker and adding 20-30g of Celite. The resulting suspension was suction filtered through a pad of Celite and the clear yellow filtrate was extracted with ethyl acetate (4x200ml). The extracts were combined , dried over MgSO₄, filtered, and rotary evaporated to leave 1.0g of (2S,3S)-2-ethyl-3-hydroxybutyramide as a yellowish solid.

Example 14:

Production of S-3-hydroxybutyramide

20g of baker’s yeast (Sigma Chemical Company, type II) was suspended in an aqueous solution containing 30g of sucrose in a conical flask, and the mix was placed on an orbital chaker (220rpm) at 30*C for 30 minutes to initiate fermentation. 2g of acetoacetamide was dissolved in 2 ml of 95% ethanol, the resulting solution was added to the fermenting yeast, and shaking was resumed. The reaction was followed by TLC (staining with anisaldehyde) to monitor the consumption of starting material and the production of product alcohol. After approximately 48 hours the reaction was judged complete, and the reaction was terminated by removing from the shaker and adding 20-30g of Celite. The resulting suspension was suction filtered through a pad of Celite and the clear yellow filtrate was extracted with ethyl acetate (4x200ml). The extracts were combined, dried over MgSO₄, filtered, and rotary evaporated to leave 1.4g of (S)-3-hydroxybutyramide as a light yellow solid.

Aurelius
(Hive Addict)
04-02-03 00:48
No 423176
      more

Example 15:

Production of the Hydroxamic acid of (2S,3S)-2-ethyl-3-hydroxybutyrate

(2S,3S)-ethyl-2-ethyl-3-hydroxybutyrate (1.0g) was dissolved in 5ml of absolute ethanol, followed by the addition of 0.5g of hydroxylamine. The solution was heated to reflux, and the progress of the reaction was followed by TLC. After the reaction was judged complete, the ethanol was evaporated and the resulting residue was redissolved in ethyl acetate. Hydroxylamine was removed by extraction with 1% HCl and the ethyl acetate solution was dried over MgSO₄, filtered, and rotary evaporated to leave 0.8g of the hydroxamic acid of (2S,3S)-ethyl-3-hydroxybutyrate.

Example 16:

Enzymatic production of the hydroxamic acid of (2S,3S)-2-ethyl-3-hydroxybutyrate

(2S,3S)-2-ethyl-2-ethylhydroxybutyrate (1.0g) was dissolved in 5ml of t-butyl methyl ether, followed by the addition of 0.5g of hydroxylamine. Lipase from Candida Rugosa (0.5g, Sigma L1754) was added, and the progress of the reaction was followed by TLC. After the reaction was judged complete, the ethanol was evaporated and the resulting residue was redissolved in ethyl acetate. Hydroxylamine was removed by extraction with 1% HCl and the ethyl acetate solution was dried over MgSO₄, filtered, and rotary evaporated to leave 0.8g of the hydroxamic acid of (2S,3S)-2-ethyl-3-hydroxybutyrate.

Example 17:

Alternate Production of the hydroxamic acid of (2S,3S)-2-ethyl-3-hydroxybutyrate by microbial reduction of the corresponding hydroxamic acid of 2-ethylacetoacetate.

Geotrichum Candidum (ATCC 34614) was cultured according to the method of Buisson and Azerad (Tet. Lett. 27, 2631-2634 (1986), herein incorporate by reference) in one liter of a medium of glucose (30g), KH₂PO₄ (1g), K₂HPO₄ (2g), corn steep liquor (10g), MgSO₄ (0.5g), NaNO₃ (2g), FeSO₄-7H₂ (0.02g), and KCl (0.5g) with rotary shaking at 25*C. 2g of 2-ethyl-acetoacetate hydroxamic acid was dissolved in 2ml of 95% ethanol, the resulting solution was added to the fermenting yeast, and shaking was resumed. The reaction was followed by TLC (staining with anisaldehyde) to monitor the consumption of starting material and the production of product alcohol. After approximately 48-72 hours the reaction was judged complete, and the reaction was terminated by removing from the shaker and adding 20-30g of Celite. The resulting suspension was suction filtered through a pad of Celite and the clear yellow filtrate was extracted with ethyl acetate (4x200ml). The extracts were combined , dried over MgSO₄, filtered, and rotary evaporated to leave 1.0g of the hydroxamic acid of (2S,3S)-2-ethyl-3-hydroxybutyrate as a yellowish solid.

Example 18:

Conversion of (2S,3S)-ethyl-2-ethyl-3-hydroxybutyrate to the hydrazide derivative

(2S,3S)-ethyl-2-ethyl-3-hydroxybutyrate (1.0g) was dissolved in 5ml of absolute ethanol followed by 0.5g of hydrazine. The solution was heated to reflux, and the progress of the reaction was followed by TLC. After the reaction was judged complete, the ethanol was evaporated and the resulting residue was redissolved in ethyl acetate. Hydrazine was removed by extraction with 1% HCl and the ethyl acetate solution was dried over MgSO₄, filtered, and rotary evaporated to leave 0.9g of the hydrazide of (2S,3S)-ethyl-2-ethyl-3-hydroxybutyrate.

Example 19:

Microbial Production of the Hydrazide of (2S,3S)-2-ethyl-3-hydroxybutyrate by stereospecific reduction of 2-ethyl acetoacetate hydrazide

Geotrichum Candidum (ATCC 34614) was cultured according to the method of Buisson and Azerad (Tet. Lett. 27, 2631-2634 (1986), herein incorporate by reference) in one liter of a medium of glucose (30g), KH₂PO₄ (1g), K₂HPO₄ (2g), corn steep liquor (10g), MgSO₄ (0.5g), NaNO₃ (2g), FeSO₄-7H₂ (0.02g), and KCl (0.5g) with rotary shaking at 25*C. 2g of 2-ethyl-acetoacetate hydrazide (produced by the reaction of ethyl-2-ethyl-acetoacetate with hydrazine) was dissolved in 2ml of 95% ethanol, the resulting solution was added to the fermenting yeast, and shaking was resumed. The reaction was followed by TLC (staining with anisaldehyde) to monitor the consumption of starting material and the production of product alcohol. After approximately 48-72 hours the reaction was judged complete, and the reaction was terminated by removing from the shaker and adding 20-30g of Celite. The resulting suspension was suction filtered through a pad of Celite and the clear yellow filtrate was extracted with ethyl acetate (4x200ml). The extracts were combined , dried over MgSO₄, filtered, and rotary evaporated to leave 1.0g of (2S,3S)-2-ethyl-3-hydroxybutyrate hydrazide as a yellowish solid.

Example 20:

Conversion of (2S,3S)-ethyl-2-ethyl-3-hydroxybutyrate to the amide derivative

(2S,3S)-ethyl-2-ethyl-3-hydroxybutyrate (1.0g) was dissolved in 5ml of absolute ethanol followed by the addition of 0.5g of gaseous ammonia. The solution was kept in a stoppered flask, and the progress of the reaction was monitored by TLC. After the reaction was judged complete, the ethanol was evaporated and the resulting residue was redissolved in ethyl acetate. Ammonia was removed by extraction with 1% HCl and the ethyl acetate solution was dried over MgSO₄, filtered, and rotary evaporated to leave 0.7g of (2S,3S)-ethyl-2-ethyl-3-hydroxybutyramide.

Example 21:

Production of (2S,3S)-2-amino-3-hydroxybutane by Hofmann Reaction

10g of (2R,3S)-2-methyl-3-hydroxybutyramide was dissolved in 250ml of a 0.03M NaOH and added slowly to a solution of 25g of Bromine in 300ml of 0.03M NaOH. The mixture was warmed with stirring until the reddish brown color disappeared. The solution was then cooled, extracted with methyl-t-butyl ethyl (4x250ml) and the extracts dried over MgSO₄, filtered, and rotary evaporated. The product (2R,3S)-2-amino-3-hydroxybutane is isolated as a light yellow oil.

Example 22:

Production of (2R,3S)-2-amino-3-hydroxybutane by Lossen Rearrangement

10g of (2R,3S)-2-methyl-3-hydroxybutyrohydroamic acid is reacted with benzoyl chloride under Schotten-Bauman conditions, followed by warming to reflux. Reaction process is monitored by TLC. The solution is then cooled to RT. Then extracted with methyl-t-butyl ether x250ml, and the extracts dried over MgSO₄, filtered, and the solvent removed by rotary evaporation. The product (2R,3S)-2-amino-3-hydroxybutane is isolated as a light yellow oil.

Example 23:

Production of (3S,4S)-3-amino-4-hydroxypentane by Lossen Rearrangement

10g of (2S,2S)-2-methyl-3-hydroxypentanohydroxamic acid is reacted with benzoyl chloride under Schotten-Bauman conditions, followed by warming to reflux. Reaction progress is monitored by TLC. The solution is then cooled to RT, extracted with methyl-t-butyl ether x250ml, and the extracts dried over MgSO₄, filtered, and the solvent removed by rotary evaporation. The product (3S,4S)-3-amino-4-hydroxypentane is isolated as a light yellow oil.

Example 24:

Production of (3S,4S)-3-amino-4-hydroxypentane by a modified Lossen Rearrangement

10g of (2S,3S)-2-methyl-3-hydroxypentahydroxamic acid is reacted with equimolar amounts of diethyl azodicarboxylate and triphenylphosphine in THF at RT using the procedure of Bittner, Grinberg and Kartoon (Tet. Lett. 23, 1965-8(1974)). Reaction takes place rapidly to produce the product (3S,4S)-3-amino-4-hydroxypentane. The product is isolated by acidification and extraction of the reaction mixture with ethyl acetate, followed by basification of the resulting aqueous solution with NaOH, extraction with methyl-t-butyl ether, drying of the extracts over MgSO₄, filtration, and the removal of solvent by rotary evaporation. The product (3S,4S)-3-amino-4-hydroxypentane as a light yellow oil.

Example 25:

Production of (2S,3S)-2-amino-3hydroxybutane

5g of (2S,3S)-2-methyl-3-hydroxybutyrate hydrazide is reacted with a solution of 5g of sodium nitrite in 100ml of 5% H₂SO₄. Reaction takes place rapidly to produce the product. The product is isolated by acidification and extraction of the reaction mixture with ethyl acetate, followed by basification of the resulting aqueous solution with NaOH. Then the mixture is extracted with methyl-t-butyl ether, drying of the extracts over MgSO₄, filtration, and the removal of solvent by rotary evaporation. The product (2S,3S)-2-amino-3-hydroxybutane is isolated as a light yellow oil.

Aurelius
(Hive Addict)
04-02-03 01:38
No 423189
      CA : 41, 3774g (Akabori abstract)

Abstract:

The Akabori Reaction (I) (CA: 41, 3774g) on BzH and dl-MeCH(NHMe)CO₂H (II) with and w/o pyridine and removal of the unreacted BzH by steam distillation gave dl-ephedrine and dl-y-ephedrine. Similarly, direct heating of piperonal and II gave 2 dl-1-(3,4-methylenedioxyphenyl)-2-methylamino-1-propanols. A new reaction (III), differing from I, takes place on heating BzH and dl-alanine directly; PhCH₂NH₂, PhCH(OH)CHPhNH₂ (2 dl-compounds), AcH, CO₂ are formed. It is considered that the I-type reaction occurs when the N of the amino acid is secondary and the III-type reaction when it is primary.

Aurelius
(Hive Addict)
04-02-03 01:57
No 423196
      References dealing with Akabori

References dealing with Akabori

Akabori, J Chem Soc Japan 64, 608 (1943)

Reaction between aromatic aldehydes and a-aminoacids. I New facts on the Akabori reaction. Takagi, Eiichi. J. Pharm. Soc. Japan (1951), 71, 648-651.

Takagi, et al., J. Pharm. Soc. Japan, 72, 812 (1952)

A. Lawson, H.V. Motley, J. Chem. Soc., (1955) 1695

A. Lawson, J. Chem. Soc. (1956) 307

Dose K., Ber., 1957, 90, 1251.

Belikov V. M., Izv. Akad. Nauk SSSR, Ser. Khim. (1969), 2536

J. Am. Chem. Soc. 76, 1322 (1954) same as

https://www.rhodium.ws/pdf/akabori.phcho.glycine.pdf

J. Chem. Soc. Japan, 52, 606 (1931)

Liebig's Annalen der Chemie, Vol. 284, 36 (1895)

Dehmlow, Enc. of Chem. Tech., Vol.5, pp. 62-69, 3rd ed. (1980)

Jones, Aldrich Chimica Acta, 9, 35 (1976)

http://www.pmf.ukim.edu.mk/PMF/Chemistry/reactions/akabori2.htm

Reactant BRN 471223 benzaldehyde
1720250 DL-alanine
Product BRN 3196917 (1RS,2RS)-2-amino-1-phenyl-propan-1-ol
-------------------------
Reaction Details
Reaction Classification Preparation
Temperature 140 øC
Other conditions Erwaermen des Reaktionsprodukts mit wss.-aethanol. HCl
Ref. 1 2262852; Journal; Takagi et al.; YKKZAJ; Yakugaku Zasshi; 73; 1953; 1086; Chem.Abstr.; 1954; 12021;

As promised, here are some more refs on the interesting condensation reaction between aromatic aldehydes and glycine/alanine:
BER 25: 3445 (1892) + 52 :1734 ('19)
ANN. 284: 36 + 307: 84
JCS 1943 ('26) + 2600 ('22)
JACS 76: 1322 ('54)
J.PHARM.SOC.JAP. 67: 218 ('47)
Ber.66, 143, 151 (1933)

Most of the articles are pretty old to say the least but they contain some interesting stuff on the reaction we're interested in here. I'm especially interested in the J.Pharm.Soc.Jap article, which describes the preparation of a methylenedioxy-substituted phenylserine. But the practical way to go is definitely as mentioned in a certain patent, that is using a two-phase solvent system. This prevents the benzylidene phenylserine from crystallising and makes sure that the reaction mixture can be stirred at all times.
After decarboxylation, these phenylserine derivates turn into amino alcohols, the perfect substrates for aminoxazolines. By substituting the benzaldehyde, a lot of phenylserine and amino alcohol derivates can be made and thus a lotta aminoxazolines!

Aurelius
(Hive Addict)
04-02-03 02:08
No 423201
      Experiences with the Reaction (Akabori)

Experiences with Akabori, thus far:

“direct heating of 20g N-methyl-alanine
with 50g Bnz at 150deg untill fizzing stops, produces
12g of a mixture 3g ephedrine and 9g pseudoephedrine isomers…..

….some 10x experiments with 20g alanine + 50g BnZ
a massive 15% return sux, any suggestions besides learn how to make nitroethane”

--IOC

“crap, at best 15% using 100 g BnZ, 40g l-alanine.
Heat to 140 seems best, extracted into H2o and evaperate, then clean up.
would decarboxylation of a methylamino over the amino maybe improve results?
How about a base and high bp solvent with C5H5N??
as suggested in tfse.
could some one please explain the reaction mechanics for the reaction:
,on heating BzH and DL-alanine directly; PhCH2NH2, PhCH(OH)CHPhNH2 (2 dl-compds.), AcH, and CO2 are formed.
Is this a condensation (no H2o) or a decarboxylation?
what conditions would be pref?
any ed imput would be great, cheers”

--IOC

“In the paper SWIM read, they used 50g benaldehyde and 20g N-methyl alanine. There is an abundance of benzaldehyde used, like a 2:1 ratio. The final yield was 12g of ephedrine isomers. Thats less than a 50% yield.

The procedure, as written, is to heat the benzh and alanine to 150 until CO2 bubbles cease”

--ChemicalSolution

Aurelius
(Hive Addict)
04-02-03 03:19
No 423224
      US Patent 4501919 (Akabori)

US Patent 4501919

Process for the Production of Serine Derivatives

Abstract:

The invention concerns a new process for the production of serine derivatives. More particularly the present invention relates to a process for the production of a serine derivative of the formula ##STR1## wherein R stands for phenyl, substituted phenyl such as methylphenyl, methoxyphenyl, ethoxyphenyl, halogenophenyl, hydroxyphenyl, acetylphenyl, nitrophenyl, cyanophenyl, biphenylyl, sulfamoylphenyl, and methylsulfonylphenyl, tert-alkyl such as tert-butyl or a heteroaromatic mono- or bi-cyclic radical such as pyrrolyl, thienyl, furyl, pyridinyl, pyrazinyl, quinolinyl, phthalazinyl and the like. R' represents hydrogen or (C.sub.1 -C.sub.4) alkyl and R" is hydrogen, (C.sub.1 -C.sub.4)alkyl or phenyl.

The process described in the present invention can suitably be employed for the preparation of threo-phenylserine, threo-p-nitrophenylserine, threo-p-acetylphenylserine and threo-p-methylsulphonylphenylserine which are used, for instance, as starting materials for the production of known antibiotic substances such as chloramphenicol, thiamphenicol and cetophenicol.

It is known since 1895 that phenyl-serine and homologues of this compound can be prepared by condensing glycine with an aromatic aldehyde in alkaline solution (Liebig's Annalen der Chemie, Vol. 284 (1895), pages 36 et seq.)

Notes:

Thus for example benzaldehyde can be condensed with glycine in an aqueous sodium hydroxide solution yielding first a benzylidene phenylserine and, after acid hydrolysis, the desired phenyl serine. Several other processes are known in the literature for the preparation of phenyl- and p-nitrophenyl-serine, such as for instance those described in German Pat. Nos. 839,500 and 1,086,242 wherein the condensation reaction is carried out in aqueous alcohol in the presence of alkaline earth hydroxides, such as calcium hydroxide, as condensing agents. Other methods are known which generally involve as the first reaction step, the formation of an intermediate Schiff's base of glycine with an aldehyde, which is subsequently condensed in aqueous alcohol with the suitably selected aromatic aldehyde. Treatment of the condensation product with mineral acids then affords the desired phenylserine derivative (see for instance German Pat. Nos. 960,722 and 1,140,198 and French Pat. No. 1,017,396).

It has now been found, according to the present invention, that serines may be prepared in an improved manner by condensing an alkaline salt of glycine dissolved in an aqueous solution of an alkali metal hydroxide with a suitably selected carbonyl compound located in an organic phase in the presence of a suitable catalyst according to the "phase-transfer catalysis" techniques. Phase-transfer catalysis is a new method in preparative organic chemistry in which substances located partly in an aqueous and partly in an organic medium are made to react in the presence of suitable catalysts. This new method has been developing very fast in the last few years but up to now no example of an aldol-type condensation, as described in the present invention, has been reported.

The process described in the present invention has considerable advantages over the conventional methods known in the prior-art and namely:

increased yields, which on the average range from 80 to 90, and even more, percent while in the prior-art processes they are generally lower than 80%. As an example in German Pat. Nos. 839,500 and 960,722, the yields amount to about 50%, in German Pat. No. 1,086,242 to about 75% and in French Pat. No. 1,017,396 to about 78%,

handling of less dangerous materials, since for instance the use of alcoholic solvents generally employed in the prior-art reactions, can be avoided, and

simpler workup which does not involve time-consuming operations.

More particularly the process described in the present invention runs through the following scheme: ##STR2## wherein

R, R' and R" are as defined before;

M stands for an alkali metal such as sodium, lithium or potassium;

each of the four radicals R'", which may be equal or different, represents an alkyl residue containing from 1 to 20 carbon atoms and the anion X.sup.- represents a normal anion deriving from inorganic or organic acids such as chloride, iodide, bromide, hydrogen sulfate, perchlorate, nitrate, acetate, benzoate, p-toluensulfonate, naphthalenesulfonate and the like.

Suitable quaternary ammonium salts which may be employed in the process of the invention are for instance methyltributylammoniumchloride, methyltributylammonium iodide, tetrabutylammonium hydrogen sulfate, methyltrioctylammonium chloride (tradename of technical-grade, not entirely uniform product Andogen 464.RTM.), decyltriethylammonium bromide, hexyltriethylammonium bromide and the like. H.sup.+ Y.sup.- stands for mineral acid.

The molar ratio of the reaction partners i.e., the carbonyl compound of formula II and the glycine salt of formula III is 2 to 1 respectively, as the first step of the reaction is the formation of a Schiff's base; however, the excess of carbonyl compound which, as described in the above scheme, is regenerated by acid hydrolysis, can be easily recovered at the end of the reaction and recycled.

The amount of phase-transfer catalyst employed may range between wide limits, but balancing the facts that the reaction rate is proportional to the amount of catalyst and that large amounts of catalyst may provoke undesired side reactions which lower the yields, it is preferably employed between about 5 and 10 mol % catalyst.

The concentrated alkali metal hydroxide is generally employed at least in molar ratio to the catalyst. However, it may be convenient to prepare the glycine alkaline salt directly in situ from glycine and the selected alkali metal hydroxide; in these cases the alkali metal hydroxide is employed at least in molar ratio to the glycine substrate plus the catalyst.

According to the phase-transfer catalysis technique one of the reactants, in this case the glycine salt, is dissolved in water and preferably in the minimal amount of water as it is usually optimum for PCT that working concentrations should be as high as possible, and its reaction partner, in this case the carbonyl compound, is dissolved in the organic phase. The organic solvent, named co-solvent, which is employed in this reaction must be selected with particular care taking into consideration the following facts:

according to the liquid-liquid phase-transfer catalysis conditions the co-solvent must substantially be immiscible with the aqueous phase; however in the present case it has to be considered that the aqueous phase is a very salted solution containing the glycine salt and the alkali metal hydroxide and that therefore also solvents partially miscible with water, such as for instance tetrahydrofuran or dioxane, can suitably be employed;

it should be unreactive in the presence of concentrated aqueous alkalis; for instance chloroform which is considered the solvent of choice in several phase-transfer reactions, should not be used in this case because it reacts with the alkali metal hydroxide in the presence of the phase-transfer catalyst yielding the very reactive and undesired dichlorocarbene; and finally

it should not inhibit the reaction; it has been found in fact that some solvents such as for instance nitrobenzene and some alcohols surprisingly inhibit phase-transfer reactions (see J. Dockx. Quaternary ammonium compounds in organic synthesis--Synthesis 1973, 441-456).

Solvents which may suitably be employed are therefore methylene chloride, dioxane, tetrahydrofuran, toluene, benzene, chlorobenzene, carbon tetrachloride, and other organic solvents that comply with the above requirements.

The temperature of the reaction, which generally is completed in from 4 to 8 hours, is kept between 0.degree. and 25.degree. C. and preferably between 5.degree. and 10.degree. C.

More particularly the reaction of the present invention is carried out by stirring a concentrated aqueous solution of the glycine salt of formula III and an alkali metal hydroxide, into a cooled organic solution of the carbonyl compound of formula II containing the selected phase-transfer catalyst. The addition may take from 5 minutes to 5 hours, however, preferably it is carried out in 3-4 hours. Stirring at low temperature is prolonged until the reaction is completed and then an aqueous solution of a strong mineral acid and preferably aqueous hydrochloric acid, is added to the reaction mixture which is moderately heated. Upon cooling to room temperature the two phases are separated: the aqueous one is concentrated to a small volume and cooled yielding the desired serine derivative of formula I in the form of its acid addition salt as a crystalline precipitate, while from the organic phase the excess of carbonyl compound, set free by the acid hydrolysis, is recovered.

Further details of the process are to be found in the following representative examples which are given to illustrate the best mode of the invention but are not to be intended as limitative of the scope of the same.

Example 1:

60.4 g (0.4 mole) of p-nitrobenzaldehyde and 4.8 g (0.02 mole) of methyltributylammonium chloride in 200 ml of CH.sub.2 Cl.sub.2 are cooled to 5.degree.-7.degree. C. 15 g (0.2 mole) of glycine and 8.8 g (0.22 mole) of sodium hydroxide are dissolved in 30 ml of water and the obtained solution is dropped into the stirred methylene chloride phase in 4 hours. Stirring at 5.degree.-7.degree. C. is continued for further three hours then 35 ml of concentrated hydrochloric acid and 200 ml of water are added. The mixture is heated to 35.degree. C. for 30 minutes and then cooled to 20.degree. C. The two phases are separated and the aqueous one is concentrated by distilling out a volume of 200 g of water. The residue is cooled to 5.degree. C. for 2 hours and the crystalline precipitate which forms is recovered by filtration and dried under vacuum. Yield 42.2 g of threo-(p-nitrophenyl)serine hydrochloride. Considering that the CH.sub.2 Cl.sub.2 phase contains 34 g of p-nitrobenzaldehyde (as determined by G.L.C. analysis) which is recycled, the percent yield in threo-(p-nitrophenyl)serine hydrochloride is 92% calculated on the p-nitrobenzaldehyde. Further 1.6 g of threo-(p-nitrophenyl)serine hydrochloride and 1.5 g of the erithro form may be obtained from the mother liquors deriving from the filtration.

Examples 2-4:

The reaction has been carried out several times by following the procedures of the foregoing example but using different phase-transfer catalysts and threo-(p-nitrophenyl)serine hydrochloride has been obtained in the following yields:

Ex.2:--with tetrabutylammoniunm hydrogen sulfate—Yield 87% calculated on p-nitrobenzaldehyde.
Ex.3:--With methyltributylammonium iodide—Yield 79% calculated on p-nitrobenzaldehyde.
Ex.4:--With ADOGEN 464.RTM.—Yield 73%--Calculated on p-nitrobenzaldehyde.

Aurelius
(Hive Addict)
04-02-03 18:13
No 423359
      US Patent 5102792 (Process for Serine Derivs)

US Patent 5102792

Selective production of L-serine derivative isomers

Abstract:

L-Serine derivatives produced by the enzyme catalyzed aldol condensation of glycine and an aldehyde in aqueous solution are recovered in high yield by extracting the aqueous product solution containing said serine derivatives with an organic phase comprising (i) an aldehyde, or (ii) mixtures of an aldehyde and a water immiscible organic solvent, followed by re-extracting the organic phase with an aqueous phase having a pH of less than about 7.0. The L-erythro isomers of L-serine derivatives such as L-phenylserine may be preferentially prepared by the use of this extraction/re-extraction procedure in combination with bioreactor reaction conditions which include a pH of from about 7.5 to 10, an aldehyde concentration of from about 1 to about 90 grams/liter, a glycine concentration of from about 10 to about 300 grams/liter, and a molar ratio of glycine to aldehyde of from about 4:1 to about 100:1.

Background of the Invention

The present invention relates to an improved process for the enzymatic preparation of L-serine derivatives by the aldol condensation of glycine with an aldehyde. It further relates to an improved process for the preparation of such L-serine derivatives whereby synthesis of the L-erythro isomer of such serine derivatives may be selectively obtained. While the present invention is thus concerned with the preparation of L-serine derivatives, it also may be used to advantage in the aldol condensation of glycine with formaldehyde to produce L-serine. Accordingly as used herein, the term "L-serine derivative" includes L-serine itself as well as the various derivatives defined hereinafter in formula (I).

Serine hydroxymethyltransferase (alternatively referred to for the purposes of the subject application as "SHMT") is widely distributed in both eucaryotes and procaryotes and has been isolated from the livers of a variety of mammals and from various bacteria such as Escherichia coli and Clostridium cylindrosporum. Genetically engineered microorganisms which overproduce this enzyme in large quantities and thereby facilitate the preparation of pure enzyme have also been reported in the literature. See, Plamann et al., Nucleic Acids Res., Vol. 11, pages 2065-2075 (1983), Schirch et al., J. Bacteriology, Vol. 163, No. 1, pages 1-7 (1985), and Hamilton et al., Trends in Biotechnology, Vol. 3, No. 1, pages 64-68 (1985).

SHMT from a variety of different sources has been reported to catalyze the reversible cleavable of betaphenylserines, including L-erythro-beta-phenylserine, to benzaldehyde (or substituted benzaldehyde) and glycine. See, Ulevitch et al., Biochemistry, Vol. 16, No. 24, pages 5342-5350 and 5356-5369 (1977); Schirch et al., J. Bacteriology, Vol. 163, No. 1, pages 1-7 (1985); and Ching et al., Biochemistry, Vol. 18, No. 5, pages 821-829 (1979).

In Nakazewa et al., U.S. Pat. No. 3,871,958, there is disclosed a process for the preparation of L-serine derivatives of the formula: ##STR1## wherein R is an organic residue having at least two carbon atoms by reacting an aldehyde with glycine in aqueous solution at a pH of 5 to 10 and a temperature of 5 to 60.degree. C. in the presence of an enzyme obtained from microorganisms belonging to the genera Escherichia, Citrobacter, Klebsiella, Aerobacter, Serratio, Proteus, Bacillus, Staphylococcus, Arthrobacter, Bacterium, Xanthomonas, Candida, Debaryomyces, Corynebacterium and Brevibacterium. It is suggested that the active enzyme in this reaction is threonine aldolase. In order to improve yields, it is recommended that the amount of glycine in the reaction system be equimolar with or in excess of the aldehyde, and that the amount of aldehyde in the reaction system be limited to from 0.1 to 10% by weight of the reaction mixture.

A similar description of threonine aldolase for the preparation of L-beta-phenylserine is set forth in Japanese patent document SHO 54-3952, published Feb. 28, 1979.

A number of authors have also suggested that glycine may be condensed with formaldehyde to give L-serine using SHMT. See, Hamilton et al., Trends in Biotechnology, Vol. 3, No. 1, pages 64-68 (1985); and U.K. Published Patent Application No. 2130216A, filed Nov. 18, 1983).

European Patent Application No. 0 220 923, published May 6, 1987, corresponding to commonly assigned copending U.S. Patent application Ser. No. 789,595, filed Oct. 21, 1985, describes the use of SHMT obtained from a genetically engineered Escherichia coli strain transformed with the pGS29 plasmid for condensing benzaldehyde and glycine methyl ester to produce betaphenylserine methyl ester. The reaction conditions employed during the condensation reaction include a pH of from 6.5 to 9, a temperature of from 10 to 65.degree. C., a benzaldehyde concentration of from 10 to 100 mM and a glycine ester from 10 to 150 mM. While at a betaphenylserine methyl ester yield of 1.48 g/l, this process produced a beta-phenylserine methyl ester product containing as much as 83% erythro isomer, it has been found that as the yield is increased the amount of threo isomer present in the product increases until at commercial rates of production substantial amounts of threo isomer are present.

While the prior art has thus recognized that various enzymes can be employed to catalyze the aldol condensation of glycine and aldehydes to make L-serine derivatives, the processes of the prior art have suffered from a number of disadvantages. The use of excess glycine in the prior art processes has resulted in a L-serine derivative product containing large amounts of residual glycine. The presence of this glycine in the product not only leads to high raw material costs, but in addition, requires the use of a troublesome separation procedure in order to separate the glycine from the L-serine derivative, further adversely effecting the process economics.

Moreover, with the processes of the prior art, it has been found that the L-seine derivative formed comprises a mixture of optical isomers containing predominantly L-threo isomer at equilibrium. While for some purpose such mixtures are satisfactory, other purposes, such as the preparation of aspartame from L-phenylserine, require the use of only the L-erythro isomer of such L-serine derivatives. In end-uses of this latter type, the l-threo isomer is either non-reactive, or leads to contamination of desirable stereoisomers with undesirable stereoisomers.

Summary of the Invention

In accordance with the present invention, there has been provided a process for the preparation of L-serine derivatives of the formula (I): ##STR2## wherein R is hydrogen, or an organic radial containing form about 1 to 25 carbon atoms, which in its broadest aspect comprises the steps of:

(a) reacting an aldehyde of the formula : RCHO(II), wherein R is as set forth above, with glycine in aqueous solution having a pH of from about 7.5 to 10 under result effective reaction conditions in the present of an amount of an enzyme effective to form an aqueous phase containing the L-serine derivative;

(b) extracting the aqueous phase of step (a) with an organic phase comprising an aldehyde of the formula RCHO(II) (which may be the same or different than the aldehyde employed in step (a)), or a mixture of such aldehyde and a water immiscible organic solvent; and

(c) extracting the organic phase of step (b) with an aqueous phase having a pH of less than about 7.0 to produce an aqueous L-serine derivative product phase.

The aqueous L-serine derivative product phase produced in step (c) contains very low residual glycine and low aldehyde, and can either be employed as is, or can be treated to recover pure L-serine derivative. In addition to these purity advantages, the claimed process also accrues enhanced yields of the L-serine derivatives. The extraction step of the invention shifts the equilibrium in favor of L-serine derivative production and away from L-serine derivative cleavage such that a greater amount of L-serine derivative product is obtained.

In a particularly preferred embodiment, the foregoing process is adapted to provide for the selective synthesis of the L-erythro isomer of the L-serine derivatives. In accordance with this embodiment, which requires the use of aldehydes wherein R is an organic radical, production of the L-threo isomer is suppressed through the use in step (a) of a set of critical reaction parameters comprising a pH of from about 7.5 to 10, a temperature of less than 60.degree. (and preferably less than 40.degree. C.), a glycine concentration of less than about 500 grams/liter, an aldehyde concentration of less than about 90 grams/liter, and a molar ratio of glycine to aldehyde of from about 4:1 to about 100:1. The resulting L-serine derivative-containing phase is then extracted with organic phase and acidic aqueous phase as set forth above in steps (b) and (c) to produce an aqueous product phase containing primarily L-erythro isomer. For purposes of the present application and appended claims, the phrase "containing primarily L-erythro isomer" means that greater than 50% on a molar basis of the L-serine derivative present in the aqueous product phase comprises the L-erythro isomer. In this embodiment of the invention, preferably at least 75%, and most preferably at least 90% on a molar basis of the L-serine derivative product comprises the L-erythro isomer.

In accordance with the present invention, it has been discovered that through the use of the foregoing preferred form of the invention, erythro/threo ratios of up to 16/1, corresponding to an erythro purity of 94%, can readily be obtained at commercially desirable rates of production. This result is particularly surprising since, as noted above, conventional enzyme catalyzed reactions yield mixtures of erythro and threo isomers containing predominantly threo isomer (approximately 3:1 threo:erythro ratio) at commercial rates of production. The fact that pH can be used to suppress L-threo synthesis is itself surprising since Ulevitch et al., Biochemistry, Vol. 16, No. 24, pages 5342-5350 and 5356-5369 (1977) indicate that the pH dependence of the reversible cleavage of the L-erythro and L-threo phenylserine isomers is the same for both isomers.

Other embodiments, features and advantages of the present invention will become apparent to those skilled in the art upon examination of the following detailed description of the invention and accompanying drawings.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic flow diagram of one embodiment of the invention directed to the preferential preparation of L-erythro-phenylserine using a continuous extraction/re-extraction procedure.

FIG. 2 is a schematic flow diagram of the apparatus employed in conducting Example 7 infra.

Detailed Description of the Invention

In its broadest form, the process of this invention comprises the steps of (a) reacting an aldehyde of the formula (II) above and glycine in the presence of enzyme under result effective reaction conditions to form an L-serine derivative of the formula (I); (b) extracting the resulting product mixture of step (a) with an aldehyde-containing organic phase; and (c) extracting the organic phase of step (b) with an acidic aqueous phase (also referred to herein as the re-extraction step) to produce an aqueous L-serine derivative product phase. Surprisingly, it has been discovered that this process not only enables the recovery of the L-serine derivatives of formula (I) in higher yields and with less glycine contamination than with the conventional enzyme catalyzed processes for the preparation of these compounds, but that this process may be adapted in accordance with the teachings of this invention to achieve the preferential formation of the L-erythro isomers of these compounds.

While not wishing to be bound by any particular theory or mechanism, it has been discovered by the instant inventors that synthesis of the L-threo isomer, relative to the L-erythro isomer, is suppressed at reaction conditions in the range of pH 7.5 to 10. This result is particularly surprising since, as noted above, studies of the reversible cleavage of the L-phenylserines indicated that the response of both the L-erythro and L-threo isomers to changes in pH was similar, suggesting that pH conditions that suppress L-threo isomer formation should also suppress L-erythro isomer formation.

pH, however, is not the only factor responsible for the ability of the process of this invention to selectively prepare the L-erythro isomers of the serine derivatives of ormula I. Other factors such as temperature and concentration of glycine and aldehyde contribute to this result.

Also critical is the use of the extraction/ re-extraction procedure of the invention. Not only does this procedure reduce the amount of residual glycine present in the aqueous L-serine derivative product phase, but the removal of L-serine derivative from the reaction mixture shifts the equilibrium of the reversible L-serine derivative synthesis reaction such that the reverse reaction (the cleavage reaction) is thermodynamically not favored. As a result, higher yields of L-serine derivative are obtained since loss due to cleavage is reduced. Moreover, minimization of the cleavage reaction prevents the L-erythro isomer from reaching its equilibrium with the l-threo isomer such that predominantly wit the L-threo isomer can be obtained in high yield despite an unfavorable equilibrium.

Aurelius
(Hive Addict)
04-02-03 18:16
No 423362
      more

The Aldol Condensation Reaction

Depending upon whether a mixture of L-erythro and L-threo isomers is to be made or whether the L-erythro isomer is to be preferentially synthesized, the reaction conditions can vary over a wide range. The aldol condensation reaction will typically be conducted in an aqueous solution containing enzyme, glycine and aldehyde at a pH of from about 7.5 to 10 (preferably from 8.0 to 10.0), and a temperature ranging from the freezing point of the reaction medium and/or reactants to about 60.degree. C., e.g., from 5 to 60.degree. C. In order to maintain the pH of the reaction system within the desired pH range during the reaction a buffer such as a phosphate, tris, Hepes (N-2-hydroxymethylpiperazine-N'-2-ethane sulfonic acid), Mes (2-(N-morpholino) ethane sulfonic acid),or ammonium chloride-ammonia, etc., buffer may be employed. Preferably, the reaction is conducted with stirring.

The pH of the reaction medium is critical to the successful practice of the process of this invention, whether a diastereoisomeric mixture of L-threo and erythro isomers is to be made or whether the L-erythro isomer is to be preferentially synthesized. While not wishing to be bound by any particular theory or mechanism of action, it is believed that the selective extraction of the L-serine derivative into the organic phase requires the formation of a L-serine derivative/aldehyde Schiff base. The L-serine derivatives themselves (like glycine) have only limited solubility in organic media. In accordance with this invention, however, it has been discovered that upon contact of the aldehyde-containing organic extractant phase with the aqueous reaction medium, wherein the pH is maintained in the range of 7.5 to 10, an aldehyde/L-serine derivative Schiff base is formed which is soluble in and readily extracted into the organic phase. In contrast, glycine has less tendency to form a Schiff base at this pH range, and its Schiff base has much less tendency to be extracted into the organic phase.

Adjustment of the pH may be made by the addition of suitable organic or inorganic acids and bases to the reaction medium. The only limitation on the selection of a suitable acid or base is that the same should not form an insoluble salt with the L-serine derivative Schiff base. Usually pH adjustment will require the addition of a base. For reasons of economy and convenience, the base is typically a basic salt of an alkali metal, for example, the lithium, sodium and potassium hydroxides, carbonates, bicarbonates, etc. Other bases, such as ammonium hydroxide, alkyl substituted ammonium hydroxide, etc., however, may also be conveniently employed.

Although it is less preferred, the aldol condensation reaction may be conducted at a pH below 7.5, e.g., in the range of pH 5.0 to 7.5. In such case, however, the pH of the aqueous reaction media must be adjusted to a pH in the range of 7.5 to 10 prior to organic extraction. Since in the preferred embodiment the aqueous reaction medium is continuously extracted with the organic phase, maintenance of a pH in the range of 5.0 to 7.5 in the reaction medium would require a further pH adjustment following the extraction step to return the pH of the aqueous reaction medium to the pH 5.0 to 7.5 range. Use of a pH below 7.5 would thus require the addition of two pH adjustment steps to the process, and accordingly is not preferred, but may be employed where desired.

The aldehyde and glycine concentrations employed during the reaction may range up to the saturation point for each of these compounds (i.e., up to 1000 grams/liter glycine and up to about 90 grams/liter aldehyde), with the glycine usually being in molar excess relative to the aldehyde. Typically, the concentration of aldehyde will range from about 1 to about 20 grams/liter, with the concentration of glycine ranging from about 10 to about 300 grams/liter. In the preferred embodiment, the glycine concentration comprises about 100 to 200 grams/ liter, e.g., about 150 grams/liter.

Where L-erythro isomer is preferentially desired, it is critical that the pH of the solution be maintained in the range of from about 7.5 to 10, preferably in the range of from about 8.5 to 10, and most preferably from about 9 to 9.5. The reaction temperature employed in this preferred embodiment of the invention is preferably maintained at less than 40.degree. C., e.g., from about 5 to 30.degree. C., and most preferably at from about 10 to 25.degree. C. In order to preferentially obtain the L-erythro isomer, a low concentration of aldehyde is required. In this embodiment, the aldehyde concentration will typically be maintained at less than 90 grams/liter, preferably in the range of from 1 to 10 grams/liter, and most preferably from about 2 to 5 grams/liter. Glycine is employed in a molar excess relative to aldehyde of from about 4:1 to about 100:1, preferably from about 10:1 to about 100:1 and most preferably from about 15 to about 25 moles of glycine per mole of aldehyde, with a concentration from about 10 to about 300 grams/liter. Most preferably, the reaction mixture will contain a glycine concentration of from about 100 to 200 grams/liter, e.g., about 150 grams/liter.

Useful aldehydes of the formula (II) include aldehydes wherein the R group is alkyl, alkenyl or alkynl of from 1 to 25 carbon atoms, preferably 1 to 15 carbon atoms and most preferably 1 to 10 carbon atoms; aryl of 6 to 10 carbon atoms; alkaryl of 1 to 25 carbon atoms; aryl substituted with hydroxy, nitro, amine or halide groups; heterocyclic aldehydes, and various other aldehydes such as salicylaldehyde; cinnamaldehyde; formal carboxylic acids such as formylacetic acid; ketoaldehydes such as glyoxal, methyl glyoxal, phenylglyoxal, etc.; succinaldehyde, acrylaldehyde, crotonaldehyde, propiolaldehyde, trichloroacetaldehyde; vanillin; as well as p-methylsulfonylaldehyde, etc. Specific examples of a wide variety of useful aldehydes are set forth in U.S. Pat. No. 3,871,958, the entirety of which is herein incorporated by reference and relied on in its entirety. Particularly preferred aldehydes for use in the process of this invention include benzaldehyde; hydroxysubstituted benzaldehydes, such as 3,4- and 2,4-dihydroxybenzaldehyde; and acetaldehyde.

The enzyme employed in this invention may comprise any of the enzymes known in the art to catalyze the aldol condensation of glycine and aldehydes. Such enzymes are generally referred to in the literature as serine hydroxymethyltransferase (SHMT), but in addition have also been referred to as threonine aldolase, serine hydroxymethylase, and allothreonine aldolase. While minor variations exist in these enzymes depending on their source, all of the enzymes of this class appear to possess similar reaction mechanisms and active site structures and are useful in the process of this invention and are intended to be encompassed thereby. For the sake of uniformity of nomenclature, therefore, for the purposes of this invention, all of the enzymes which are capable of catalyzing the glycine-aldehyde condensation reaction will be referred to as serine hydroxymethyltransferases (SHMT). As used herein, the terms "serine hydroxymethyltransferase" and "SHMT" are thus defined to include all of the various enzymes which catalyze the condensation of glycine and aldehyde to L-serine derivatives of formula (I).

As noted above, SHMT is readily available, and may be obtained from various mammalian liver extracts by art recognized techniques. In addition, this enzyme may be obtained from any of the various microorganisms described in U.S. Pat. No. 3,871,958, the entirety of which is incorporated by reference and relied on in its entirety.

In the preferred embodiment, genetically engineered microorganisms transformed with high-copy-number plasmids containing the E. coli glyA gene are used as the enzyme source. The glyA gene is contained in a 3.3 kilobase Sal I-EcoRI fragment. One known plasmid, designated pGS29, is formed by insertion of the glyA gene into the tetracycline resistance gene of pBR322. E. coli strains transformed with the pGS29 plasmid produce as much as 26 times the amount of SHMT, as compared with wild-type strains. Further details concerning the preparation of such E. coli strains are set forth in Schirch et al., J. Bacteriology, Vol. 163, No. 1, pages 1-7 (1985) and Plamann et al., Nucleic Acids Research, Vol. 11, No. 7, pages 2065-2075 (1983).

A genetically engineered Klebsiella aerogenes strain, stabilized by nutritional selection, is reported by Hamilton et al. in Trends in Biochemistry, Vol. 3, No. 1, pages 64-68 (1985). This strain was prepared by insertion of the E. coli glyA gene into the tetracycline resistance gene of pBR322. The resulting plasmid, designated pGX122, was subcloned into a plasmid with multiple restriction endonuclease sites (pGX 145) to create the plasmid pGX139. A trp operon stabilized glyA plasmid was next prepared by insertion of the glyA gene from pBX139 into the trp operon plasmid pGX110 to create the plasmid pGX2236, which was inserted into K. aerogenes GX1705, a strain containing a mutation in the tryptophan synthetase gene. As a result of this mutation, only cells that retained the plasmid are capable of growing in media lacking tryptophan.

Techniques for culturing SHMT containing microorganisms are well known to those skilled in the art, and are described, for example, in U.S. Pat. No. 3,871,958, the entirety of which is herein incorporated by reference and relied on in its entirety.

The enzyme source may comprise intact, whole cells, an aqueous suspension of ground cells, a filtrate of such suspensions, crude extracts of such cells, or the pure enzyme. Techniques for the recovery and purification of SHMT from liver and bacterial cells are well known to those skilled in the art, and are described for example in Ulevitch et al., Biochemistry, Vol. 16, No. 24, pages 5342-5350 (1977); and in Schirch et al., J. Bacteriology, Vol. 163, No. 1, pages 1-7 (1985)

The amount of enzyme present during the reaction can vary over a wide range. Typically, the enzyme will be used in an amount of from about 100 to 4,000,000 units/ liter, preferably from about 1000 to about 1,000,000 units/liter, and most preferably from about 5,000 to 500,000 units/liter. As used herein a unit of SHMT is equal to that amount of enzyme which catalyzes production of 1 micromole of benzaldehyde per minute from L-threophenylserine at 25.degree. C., neutral pH.

The reaction may be conducted by culturing a suitable SHMT microorganism source in a conventional nutrient medium containing glycine and aldehyde. Generally, however, the reaction is conducted by adding SHMT to an aqueous solution containing glycine and aldehyde. The reaction may be conducted on a batch basis or on a continuous basis by the intermittent or continuous addition of glycine and aldehyde. SHMT requires pyridoxal-5-phosphate (P-5-P) for activity. Where the reaction is conducted in the microorganism culture medium, addition of P-5-P is not required, since the microorganism is able to synthesize in vivo the P-5-P necessary for SHMT activity. In all other cases P-5-P must be added to the reaction mixture for SHMT activity. Typically, P-5-P will be employed in an amount equimolar to, or smaller than, the amount of enzyme present. Use of amounts of P-5-P greater than equimolar quantities are not preferred since the use of excess P-5-P leads to non-enzymatic synthesis and a racemic product mixture. Preferably the amount of P-5-P employed will be an amount sufficient to activate the SHMT enzyme not exceeding an equimolar amount. Where whole cells are used as the enzyme source, pyridoxine or pyridoxal may replace P-5-P. These compounds are converted in vivo by the microorganism to P-5-P.

When L-serine is to be produced by the process of the invention, tetrahydrofolate (THF) or similar folic acid derivatives are required to enhance activity in the bioreactor. The use of such folate compounds may also be desirable during the production of derivatives of L-serine in order to enhance total L-serine derivative synthesis.

The SHMT enzyme may be immobilized, if desired, using any of a variety of supports and immobilization techniques well known to those skilled in the art. Where non-immobilized enzyme is employed, in the preferred embodiment the SHMT enzyme is preferably separated from the reaction mixture by a suitable separation technique. such as dialysis, ultrafiltration, etc., prior to extraction of the L-serine derivative-containing reaction mixture with the organic phase. This expedient is desirable in order to prevent the L-erythro isomer from coming into equilibrium with tis L-threo isomer via the SHMT catalyzed cleavage and re-condensation of the same into the thermodynamically more favored L-threo isomer. Moreover, contact between the enzyme and organic phase has a deleterious effect on enzyme stability and on phase separation.

The Extraction/Re-extraction Procedure

The organic phase used to extract the L-serine derivative reaction mixture may comprise (i) an aldehyde of formula (II), which may be the same or different than the aldehyde employed in the condensation reaction, or alternatively may comprise mixtures of aldehyde and one or more water immisicible solvents. As used herein, the term "water-immisicible solvent" means that the solvent forms a two-phase system with water. Such solvents are well known to those skilled int he art; and include, by way of example, the alkyl ester of carboxylic acids such as ethyl acetate, isopropyl acetate, butyl acetate, and isobutyl acetate; lower alkyl halides such as chloroform or ethylene dichloride; detones such as methylisobutyl ketone; aromatic hydrocarbons such as benzene, toluene, or mixtures thereof; higher alcohols such as t-butanol, cyclohexanol and benxyl alcohol; and various other solvents such as butanediol, triethylene glycol, dioxane, isopropyl ether, trichloroethylene, tetrachloroethylene and the like.

The presence of aldehyde int he organic phase is a critical feature of the process of this invention and is essential to the successful extraction of the L-serine derivative into the organic phase. As noted above, in the absence of aldehyde the L-serine derivative has only limited solubility in the organic phase. While not wishing to be bound by any particular theory or mechanism of action, it is believed that the aldehyde reacts with the L-serine derivative to form the Schiff base of such compound. The Schiff base is preferentially soluble in organic media and accordingly can be readily extracted into the same from the aqueous reaction media.

Aurelius
(Hive Addict)
04-02-03 18:18
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For successful practice of the invention process, therefore, the aldehyde must be present in the organic phase in an amount at least sufficient to convert the L-serine derivative present in the aqueous reaction media to its Schiff base. Typically, the organic phase will contain from about 5 to 100% by volume of aldehyde, preferably from about 5 to 50% by volume of aldehyde, and most preferably from about 10 to 35% by volume of aldehyde.

Following extraction into the organic phase, the L-serine derivative is then contacted with an aqueous phase having a pH of less than 7.0, preferably less than 6.0, and most preferably less than 5.0, to form the aqueous L-serine derivative. The pH of the aqueous phase is also critical to the successful operation of the invention. At a pH of less than 7.0, the L-serine derivative/aldehyde Schiff base will be broken such that the free L-serine derivative is preferentially extracted into the aqueous phase.

The extraction/re-extraction procedure is conducted such that build-up of L-serine derivative in the reactor is prevented. While this objective may be achieved by intermittent extractions of adequate frequency, it is preferred that the L-serine derivative be removed from the reaction media continuously as it is synthesized by continuous extraction/re-extraction. For the preferential synthesis of L-erythro isomer, the rate of extraction of the L-erythro isomer must be greater than one-half the rate of total L-serine derivative production in the bioreactor, and most preferably approximately equal to or greater than the rate of L-serine derivative production in the bioreactor. Such rates of extraction can readily be achieved by those skilled in the art by appropriate balancing of flow rates, ratios of flow rates, ratios of volumes of extractants and aqueous reaction medium, contact times, equipment sizes, etc. The extraction and re-extraction steps may be conducted in concurrent fashion, but are preferably conducted countercurrently.

FIG. 1 is a schematic flow diagram of a preferred method for conducting the process of the invention which employs a continuous, countercurrent extraction/ re-extraction procedure. In the bioreactor 1, SHMT, glycine, aldehyde and P-5-P are reacted in aqueous solution under conditions described above to form the L-serine derivative. A portion of the aqueous reaction mixture is continuously removed through line 2 for countercurrent extraction with organic phase in extractor vessel 3. Where non-immobilized enzyme is employed, an ultrafilter or dialysis device is preferably interposed prior to the extractor vessel 3 for enzyme separation, such as is illustrated in FIG. 2 to be discussed hereinafter. In the extractor vessel 3, the L-serine derivative forms an aldehyde/L-serine derivative Schiff base which is extracted into the organic extractant phase. The L-serine derivative depleted aqueous reaction mixture, high in glycine content, is continuously returned to the reactor 1 through line 4.

The organic phase, in turn, is continuously recirculated via lines 5 and 7 through re-extractor vessel 6 where it is countercurrently contacted with a low pH aqueous phase, which is itself continuously recirculated from product vessel 9 via lines 8 and 10. As discussed above, in the re-extractor vessel 6 the aldehyde/L-serine derivative Schiff base is broken, resulting in the re-extraction of the L-serine derivative into aqueous solution. The L-serine derivative containing aqueous solution is collected in product vessel 9, and may be either used as is, or processed using known techniques such as, for example, precipitation, chromatography, ion exchange, etc., to recover pure L-serine derivative.

The L-serine derivatives produced by the process of this invention are valuable intermediates for the production of compounds such as epinephrine, norepinephrine, aspartame and various other dipeptides, chloramphenicol, as well as various pharmaceuticals.

The following Examples serve to give specific illustration of the practice of this invention, but they are not intended in any way to act to limit the scope of this invention.

In each of the examples set forth hereinafter, the SHMT enzyme source comprised a genetically engineered E. coli strain which was transformed by conventional procedures to contain the plasmid pGS29. This strain, identified by the assignee hereof as GR64/pGS29, will have been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, ATCC Deposit No. 67673, with no restrictions as to availability, and W. R. Grace & Co., the assignee hereof, assures permanent availability of the culture to the public through ATCC upon the grant hereof.

While as noted above, the process of this invention contemplates the use of whole cells, extracts, etc., as SHMT enzyme sources, in the Examples which follow SHMT obtained by fermentation of the aforementioned GR64/pGS29 E. coli strain and purified to homogeneity was employed. The GR64/pGS29 E. coli strain was cultured as follows: One liter of seed culture was first prepared by aseptically inoculating a sterilized (121.degree. C. for 25 minutes) two liter shake flask containing one liter of an aqueous culture medium comprising 60.0 grams of tryptocase soy broth (Difco Laboratories, Inc., Detroit, Michigan), 15.0 grams of K.sub.2 HPO.sub.4, and four drops of P-2000 silicone antifoam (Dow Chemical Company, Midlands, Michigan) with an ampoule of microorganism thawed under tap water (which was previously stored at -70.degree. C.). Prior to inoculation, the pH of the culture medium was adjusted to pH 7.2 to 7.5 by the addition of 1.3 ml of concentrated H.sub.2 SO.sub.4. This mixture was then incubated at 31.degree. C. for 8 to 10 hours at 250 rpm.

This seed culture was then added to a sterilized (121.degree. C., 30 minutes) 20 liter fermentation vessel containing 13.0 liters of an aqueous culture medium comprising 220 ml of separately sterilized, 63% glucose solution, 322 grams of casein protein digest (NZ Amine A, Schofield Products, Norwich, New York), 112 grams of Amberex 1003 yeast extract (Universal Foods, Hackensack, N.J.), 56 grams of (NH.sub.4).sub.2 SO.sub.4, 14 grams of MgSO.sub.4. 7H.sub.2 O, 70 grams of KH.sub.2 PO.sub.4, 220 ml of a trace mineral solution (containing 8.8 grams/liter of ZnSO.sub.4. 7H.sub.2 O, 10 grams/liter FeSO.sub.4. 7H.sub.2 O, 0.06 grams/liter CuSO.sub.4. 5H.sub.2 O, 0.12 grams/liter of CoCl.sub.2. 6H.sub.2 O, 0.055 grams/liter of CaCl.sub.2. 2H.sub.2 O, .088 grams/liter of Na.sub.2 B.sub.4 O.sub.7. 10H.sub.2 O, 0.053 grams/liter Na.sub.2 Mo.sub.2 O.sub.4. 2H.sub.2 O, and 7.5 grams/liter of MnSO.sub.4.H.sub.2 O in 6 N NH.sub.4 OH), and 1.4 ml of P-2000 silicone antifoam (Dow Chemical Company, Midlands, Michigan) to produce 14.0 liters of fermentation medium.

Prior to addition of the seed culture, the pH of the medium was adjusted to pH 7.0 by the addition of 17 ml of 50% NaOH. Fermentation thereafter proceeded over a period of about 18 hours at pH 7.0 (controlled via addition of 6N NH.sub.4 OH), a temperature of 30.degree. C., an airflow of 14 slpm, and 1200 rpms until the optical density at 640 nm of the medium reached 9.0. During the fermentation period, the medium was sampled every two hours for pH, optical density and glucose. The dissolved oxygen during the fermentation period was found to fall below 15%. Upon the attainment of an optical density of 9.0, the bacteria were harvested, and the SHMT enzyme purified to homogeneity according to the procedure of Schirch et al., Journal of Bacteriology, Vol. 163, No. 1, pages 1-7 (1985) reported at page 3 thereof, omitting, however, the hydroxylapatite and TSK 3000 HPLC treatments.

Unless noted otherwise, in the Examples which follow, L-erythro and L-threo phenylserine were monitored by reverse phase high performance liquid chromatography using a Supercosil C-18 column (Supelco, Inc., Bellefonte, Pennsylvania), Shimadzu LC-4A HPLC, SPD 2AS U.V. detector, Sil-2AS autosampler and CR3A integrator, and as elutants aqueous phosphate buffer (prepared from HPLC grade ultra-pure water) and HPLC grade acetonitrile. Wavelength detection was at 220 nm. 2.0 grams/liter of L-erythro and L-threo phenylserine in HPLC grade methanol, and 3:4, 1:2, 1:4 and 1:10 dilutions thereof were used as standards. Samples were typically diluted 1:15 in HPLC grade methanol to adjust the concentrations within the range of the standards.

Example 1:

This example demonstrates the effect of benzaldehyde concentration on the relative rates of L-erythro and L-threo-phenylserine synthesis. 50.times.10.sup.-6 M pyridoxal-5-phosphate, 4,000 units/liter of purified SHMT, 88 grams/ liter glycine, and an amount of benzaldehyde as indicated in Table I below in 0.05 M HEPES (N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid) buffer at pH 7.0 were reacted at 25.degree. C. in a 50-ml. glass round bottom flask equipped with an agitator and heated with a temperature controlled bath. Samples of the reaction mixture were taken and analyzed hourly for L-erythro and L-threophenylserine concentration. The results of this experiment are set forth below.
TABLE I
______________________________________
Benzaldehyde (grams/liter)
   0.106 0.21 0.33
______________________________________
Rate of L-erythro phenylserine
   0.405 0.78 1.04
synthesis (grams/liter/hour)
Rate of L-threo phenylserine
   0.013 0.054   0.10
synthesis (grams/liter/hour)
______________________________________

As can be seen from Table I, the rate of L-erythrophenylserine synthesis is proportional to benzaldehyde concentration while the rate of L-threo-phenylserine synthesis appears proportional to the square of benzaldehyde concentration. This result indicates that the preferential formation of the L-erythro-isomer of phenylserine is enhanced by the use of low concentrations of aldehyde.

Example 3:

This example demonstrates the effect of pH on L-erythro-/L-threo-phenylserine synthesis. A saturating concentration of .sup.13 C labelled benzaldehyde (5.6 grams/ liter), and 1.8 M (158 grams/liter) glycine were reacted at 25.degree. C. for 12 hours at pH 7.6 and pH 9.6 in 0.7 ml NMR tubes. The reaction mixture at pH 7.6 contained 0.05 M HEPES buffer. The pH 9.6 reaction was self-buffered by the 1.8 M (158 grams/liter) glycine. Both reactions were started by adding a concentrated solution of purified SHMT (70,000 units/liter of SHMT in pH 8 Tris buffer) until the final concentration of enzyme in the reaction mixture was 1,000 units/liter SHMT. During this period, each solution was analyzed every 0.5 hours for L-erythro and L-threo-phenylserine production by .sup.13 C-NMR. The results are reported in Table II.
TABLE II
______________________________________
pH 7.6   9.6
______________________________________
Rate of L-erythro-phenylserine
0.6   0.3
synthesis (grams/liter/hour)
Rate of L-threo-phenylserine
0.1   0.01
synthesis (grams/liter/hour)
______________________________________

As can be seen from the above data, at pH 9.6, the rate of L-erythro isomer synthesis is about 1/2 that at pH 7.6. In contrast, the rate of L-threo isomer synthesis at pH 9.6 is only 1/10 that at pH 7.6, and approximately 1/30 the rate of L-erythro isomer production at this pH.

Example 3:

This example demonstrates the criticality of the presence of aldehyde in the organic extractant phase. In this experiment, 1 ml of an aqueous solution containing 1.5 M glycine (132 grams/liter), pH 9.1 (adjusted with KOH), and 0.05 M D,L-threo-phenylserine (purchased commercially from Sigma Chemical, St. Louis, Missouri), corresponding to 9 grams/liter, was extracted with 5 ml of the organic solvents indicated in Table III below. The amino acids in the organic phase (after extraction) were determined by HPLC. The percentage of amino acid extracted is presented in Table III.
TABLE III
______________________________________
Solvent Composition
% Glycine % phenylserine
______________________________________
Ethyl Acetate/butanol (4:1)
<1%    1.5%
Ethyl acetate/butanol/
<1%    15%
benzaldehyde (3:1:1)
Ethyl Acetate/butanol/
<1%    25%
benzaldehyde (2:1:2)
Ethyl Acetate/benzyl alcohol
<1%    <1%
(4:1)
Ethyl Acetate/benzyl alcohol/
<1%    15%
benzaldehyde (3:1:1)
______________________________________

As can be seen from Table III, solvent media lacking aldehyde were unable to effectively extract phenylserine from the aqueous reaction media.

Aurelius
(Hive Addict)
04-02-03 18:19
No 423364
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Example 4:

This example demonstrates the effect of pH on the extraction of phenylserine from aqueous media. In this example, 1 ml samples of a series of aqueous solutions containing 1.5M (132 grams/liter) glycine and 0.05M phenylserine (the commercially purchased D,L-threo phenylserine of Example 3), corresponding to 9 grams/ liter of phenylserine, with the pH adjusted with KOH as indicated in Table IV below, were extracted with an ethyl acetate/benzyl alcohol/benzaldehyde (3:1:1) organic solvent mixture. Following extraction, the organic phase was analyzed for % glycine and % phenylserine extracted by HPLC. The percentage of amino acid extracted at each pH used is set forth in Table IV.
TABLE IV
______________________________________
pH    % Glycine % Phenylserine
______________________________________
7.6 <1% 4%
8.6 <1% 6%
9.2 <1%    15%
9.6 <1%    18%
______________________________________

Taken together, the data in Table III and IV indicate that with the organic extractants used in the process of this invention, it is possible to preferentially extract phenylserine from glycine under the same conditions of pH which suppress production of L-threo-phenylserine and favor production of L-erythrophenylserine by SHMT.

Example 5:

This example demonstrates the effect of temperature on the relative rates of L-erythro- and L-threophenylserine synthesis. To a one-liter glass round bottom flask equipped with an agitator and heated with a temperature controlled bath, one liter of reaction solution containing 175 grams/liter glycine at pH 8 and 4000 units/liter of SHMT were added and brought to the reaction temperatures indicated in Table V. Benzaldehyde was then added to the agitated solution at a rate of 16 grams/liter/hour. Samples were taken periodically to monitor the ratio of the isomers and their concentrations. Table V below presents the results of these experiments and shows that lower temperatures favor the production of L-erythro-phenylserine, and that at such lower temperatures a higher ratio of L-erythro- to L-threo-phenylserine can be achieved at high L-erythrophenylserine concentrations.
TABLE V
______________________________________
Effect of Temperature on the Ratio of Erythro to Threo-
Phenylserine
Temperature
.degree.C.
3 g/L 5 g/L   7 g/L 10 g/L
15 g/L
______________________________________
5    15    9    7 6    5
10 10    7    5 4    3
35 7 5    3 2    --
45 4 2    1 0.8   --
______________________________________

Example 6:

Following the procedure of Example 5, batch reactions (no solvent extraction) were run at a temperature of 10.degree. C., an SHMT concentration of 10,000 units/liter, 175 grams/liter of glycine and a benzaldehyde feed rate of 15 grams/liter/hour until the concentration of L-erythro-phenylserine in the reaction mixture reached 16 grams/liter. As indicated in Table VI below, the pH in each of these reactions was varied from 7 to 9.5 in order to determine the effect of pH on the L-erythro/L-threo ratio at high rates of production (i.e., at a rate of L-erythro-phenylserine production of 16 grams/liter/hour).
TABLE VI
______________________________________
pH
7    8    8.7    9.1 9.5
______________________________________
Ratio of L-erythro-
0.5    2.3   5.6   8.9 10.1
to L-threo-phenylserine
______________________________________

The data in Table VI indicate that at commercial rates of production, pH strongly influences the ratio of L-erythro to L-threo isomer, and that for the preferential formation of L-erythro-phenylserine, the pH of the reaction should be within the range of from about 7.5 to 10, preferably from about 8.5 to 10, and most preferably from about 9 to 9.5.

Example 7:

This example illustrates a process for the preferential preparation of L-erythro-phenylserine using a continuous, countercurrent extraction procedure in accordance with this invention. The apparatus used in conducting this example is illustrated in FIG. 2. This apparatus comprises a one liter, glass round-bottom flask 20, equipped with a temperature control jacket 21, agitator 22, pH control 27 (Chem Cadet Model R-5984); a hollow fiber dialysis module 30 (Enka C-10 1.1 m.sup.2 hollow fiber dialysis unit); two 11/2 inch inner diameter, three foot long glass columns 35 and 36 equipped with Teflon stoppers on each end; Masterflex pumps 26, 29, 33, 39 and 41; a 100 ml glass mixing vessel 42; a benzaldehyde source 24; an acid source 46; and pH control 45.

In operation, glycine and benzaldehyde, continuously fed via line 25 and pump 26 from benzaldehyde source 24, were condensed in the reactor 20 in the presence of SHMT enzyme. The pH of the reaction mixture was continuously monitored by pH control 27 and electrode 23. A portion of the reaction mixture was continuously removed via line 28 and pump 29 to the dialysis unit 30 wherein enzyme was separated from the reaction mixture and returned to the reactor 20 via line 34. The phenylserine containing ultrafiltrate was circulated through the extraction column 35 by the pump 33 via lines 31 and 32.

In the extraction column 35, the phenylserine containing ultrafiltrate was contacted with an aldehydecontaining organic phase using a countercurrent flow to extract the phenylserine product into the organic phase. The organic extractant phase used in this example comprised a 2:2:1 by volume mixture of 1-butanol, propylacetate and benzaldehyde. The organic phase formed an upper layer identified as 54 in FIG. 2, with the aqueous ultrafiltrate phase forming lower layer 55. Countercurrent extraction was achieved by feeding the organic phase into the lower aqueous phase 55 through line 37, and withdrawing it through the upper organic phase 54 via line 38 and pump 39 so that the organic phase continuously migrated through extraction column 35 from bottom to top.

Organic phase removed from the extraction column 35 via line 38 and pump 39 was then introduced on a continuous basis into the bottom of extraction column 36 where it contacted acidic aqueous phase 56 which extracted the phenylserine from the organic phase. In this example, the acidic aqueous phase comprised an aqueous sulfuric acid solution having a pH of 4. The organic phase formed an upper organic layer 57 in the extraction column 36, and was recirculated via line 37 to extraction column 35, thereby providing a flow of organic phase which was countercurrent to the aqueous phase 56.

For pH control in the extraction column 36, a portion of the aqueous phase 56 was continuously circulated to the mixing vessel 42 via lines 40 and 43 and pump 41. As required, acidic solution (aqueous sulfuric acid, one molar) was fed from supply 46 and pH control 45 through line 44 to the mixing vessel 42.

The specific reaction conditions employed in this example were as follows:

(a) bioreactor: pH 9.4 (adjusted with NaOH), temperature 10.degree. C., a SHMT concentration of 10,000 units/liter, a glycine concentration of 144 grams/liter, a benzaldehyde concentration of 3.4 grams/liter, and a pyridoxal-5-phosphate concentration of 1.times.10.sup.-5 M.

(b) organic phase: 2:2:1 1-butanol/propyl acetate/ benzaldehyde mixture.

(c) acidic aqueous phase: aqueous sulfuric acid, pH 4.

Upon start-up, the concentration of total phenylserine in the reactor increased to about 6.7 grams/liter in the first 6 hours, corresponding to a rate of about 1 gram/ liter/hour, and then operated at near steady state for close to 30 hours. Under steady state conditions, the ultrafiltrate present as layer 55 in extraction column 35 contained about 144 grams/liter glycine and a saturating amount of benzaldehyde.

In the aqueous product phase 56 the phenylserine concentration increased at a a constant rate of about 1.9 grams/liter/hour, indicating that the extraction procedure was able to match the rate of product synthesis. A final concentration of L-erythrophenylserine of over 21 grams/liter, with an erythro to threo ratio of 16/1, was obtained after 36 hours of operation in the aqueous product phase 56. In contrast, the final concentration of L-erythro-phenylserine in the reactor 20 was approximately 12 grams/liter. The higher concentration of L-erythro-phenylserine in the aqueous product phase 56 as compared with that in the reactor 20 demonstrates the salutory effect of the extraction procedure of this invention on product yield. The results of this example should also be compared with the results obtained without the invention extraction procedure. Absent the use of the invention extraction procedure, even when the SHMT reaction is optimized in accordance with the instant teachings for L-erythrophenylserine production, the best erythro/threo ratio that can be obtained at 20 grams/liter L-erythrophenylserine production is 3/1.

Further details concerning the results of this experiment are set forth in Table VII. As can be seen from this data, the process of this invention shows several advantages over conventional processes. Since the reactants are continuously removed from solution, both the ratio of the erythro to threo isomer in the product and the obtainable phenylserine concentration can be improved over batch reactions. Erythro/threo ratios of up to 16/1 (94% erythro) were obtained with this process at erythro concentrations as high as 21 g/L in the aqueous product phase 56. Furthermore, the aqueous product phase contains very low glycine and is also lower in benzaldehyde than expected for an aqueous solution saturated with neat benzaldehyde.
TABLE VII
__________________________________________________________________________
REACTOR g/L    PRODUCT g/L
Time (hr)
Glycine
   Erythro
Threo
Benz(a)
   Ratio(b)
Glycine
   Erythro
Threo
Benz(a)
   Ratio(b)
__________________________________________________________________________
1    144 2.6 0.2 3 13   0 ND 0 0   4
3    132 4.0 0.3 4 13   0 ND 0.5 0.1 4 5
6    127 6.1 0.5 5 12   0 ND 3.3 0.3 4 11
9    124 8.6 0.9 4 10   0 ND 5.2 0.3 --   17
12 118 7.3 0.8 5 9 0 ND 8.3 0.6 --   14
18 106 8.2 0.6 4 14   0 ND 10.4 1.0 --   10
27 83   9.6 0.7 3 14   0 ND 16.3 1.3 4 13
36 79   11.6 1.2 4 10   0 ND 21.2 1.3 3 16
__________________________________________________________________________
   Footnote
   (a)Benz = Benzaldehyde
   (b)Ratio of Lerthro-phenylserine/L-threo-phenylserine
   ND -- not detectable, <5 g/L

Aurelius
(Hive Addict)
04-02-03 19:12
No 423372
      US Patent 3871958 (Bio. prod. Serine and serinol )

US Patent 3871958

Biological Method of Producing Serine and Serinol Derivatives

Abstract:

Serine-derivatives and serinol-derivatives are obtained by the action of an enzyme produced by certain microorganisms, on an aldehyde and glycine or ethanolamine.

Example 1:

An aqueous culture medium was prepared to contain 2.0g/dl L-threonine, 0.2g/dl KH₂PO₄, 0.1g/dl MgSO₄ and 0.4g/dl (NH₄)₂SO₄, and was adjusted to pH5.0. 50ml batches of said medium were placed in 500ml shaking flasks and inoculated with the microorganisms shown on Table 2 which were previously cultured on bouillon agar slants at 31*C for 20 hours, and cultivation was carried out for 24 hours with shaking. The microbial cells in 100ml of each cultured broth were harvested by centrifuging, washed and suspended in 50ml of reaction solutions (A),(B),(C), and (D) whose compositions are shown in Table 1 at pH 8.5 and at 31*C for 20hours.

Table 1:
Composition of the reaction solution A B C D
p-nitrobenzaldehyde g/dl 2.0 2.0 - -
isobutyraldehyde g/dl - - 2.0 2.0
glycine g/dl 2.0 - 2.0 -
ethanolamine g/dl - 2.0 - 2.0
SORPOL W-200 g/dl 0.2 0.2 0.2 0.2

SORPOL W-200: Brand name of surface active agent (polyoxyethylene alkyl phenol ether)

B-p-Nitrophenyl-L-serine, B-p-nitrophenyl-L-serinol, B-isopropyl-L-serine, and B-isopropyl-L-serinol were produced in solutions (A),(B),(C) and (D) respectively in the yields shown in Table 2.

Table 2:
Microorganism Used Yields for each solution (in g/dl)

A B C D
E. Coli 0.2 0.1 0.2 0.2
ATCC 15289

Citrobacter Fueundii 0.8 0.7 0.5 0.6
ATCC 6750

Klebsiella Pneumoniae 0.5 0.4 0.3 0.3
ATCC 10031

Candida Rugosa 0.6 0.5 0.5 0.4
ATCC 10571

Candida Utilis 0.7 0.6 0.5 0.4
ATCC 9950

There were other microorganisms listed- I have only included the first couple and the ones easiest to obtain

Example 4:

The microbial cells in 1L of the broth obtained by cultivation of C. Humicola ATCC 14438 as in Example 1 were harvested by centrifuging and suspended in 1L of a reaction solution containing 30g glycine, 30g benzaldehyde, 2g SORPOL W-200 and 0.1g pyridoxal-4-phosphate.

After adjustment of the pH to 8.5, the reaction was carried out at 37*C for 20 hours. 10g of crystalline L-beta-phenylserine were recovered from the solution after removal of the microbial cells.

Selections from:

Table 3:
Aldehyde Yield (g/dl)
Serine deriv. Serinol Deriv
Myristaldehyde 0.3 0.2
Benzaldehyde 0.7 0.5
p-Cl-Benzaldehyde 0.4 0.3
p-Br-Benzaldehyde 0.4 0.3
p-tolualdehyde 0.3 0.2
vanillin 0.5 0.5
p-nitrobenzaldehyde 1.0 0.8
3,4-o-methylbenzaldehyde 0.8 0.7
3,4-dihydroxybenzaldedhyde 0.8 0.7
2,4-dihydroxybenzaldehyde 0.7 0.6
3,5-dimethylbenzaldehyde 0.4 0.3
3,4-di-Br-benzaldehyde 0.8 0.7
2,4-di-Br-benzaldehyde 0.7 0.6
3,4-dinitrobenzaldehyde 0.8 0.8
3,4-di-Cl-benzaldehyde 0.5 0.4
p-Cl-benzaldehyde 0.6 0.7
beta-naphthaldehyde 0.8 0.7

Rhodium
(Chief Bee)
04-03-03 02:14
No 423469
User Picture       Information overload!

Hey Aurelius, could you please slow down somewhat?

Posting page after page of patents are not useful to us, especially not when the syntheses you post lack direct relevance to any psychoactive synthesis we are interested in here. Reposting literature at the Hive is supposed to be about cherry-picking the best parts of articles and the most useful examples from patents and then condense these excerpts into a post, glueing together the re-posted syntheses by adding explanatory notes here and there about what they are good for, and what you want to achieve by posting these excerpts.

In most of the above posts, there are no explanatory notes whatsoever, and I think that only 1% of the members here can directly understand what the purpose is of many of the synthesis examples above. For example, take the following series:

Post 423174 (Aurelius: "US Patent 5834261 (Vicinal aminoalcohols)", Stimulants)
Post 423175 (Aurelius: "more", Stimulants)
Post 423176 (Aurelius: "more", Stimulants)

This is completely ridiculous to post! Can you imagine anyone here having any use for that long (50 kb) patent being posted here? It does not contain a single substance of interest to our cause, even though the methods used may be - but then it is lightyears more reasonable to simply post "Patent US5834261 - Method for the Production of Chiral Vicinal Aminoalcohols", and at most also post the abstract! The same thing can be said about the Serine rant further down, even thought it contains at least some substance as it discusses the Akabori Reaction - but still it is too long, if you sat down for a few minutes and edited that information, you could have condensed it to 5 kilobytes and still been able to include 100% of all pertinent information.

Next, take a look at the posts below. They are next to verbatim copies of earlier posts by other bees:

Post 423189 (Aurelius: "CA : 41, 3774g (Akabori abstract)", Stimulants) has been copied from Post 278820 (ChemicalSolution: "alanine and Akabori???", Chemistry Discourse)
Post 423196 (Aurelius: "References dealing with Akabori", Stimulants) has been copied from Post 245572 (IOC: "Re: Akabori run", Novel Discourse)
Post 423201 (Aurelius: "Experiences with the Reaction (Akabori)", Stimulants) has been copied from several posts.

This breaks Hive netiquette for several reasons:

* You have not provided any links to the original source, or even mentioned that they are copies (in the first two), effectively making it plagiarism.

* It is completely unnecessary to make three posts in a row on exactly the same topic, such things should be condensed into a single post, maybe with [ hr ] markup lines inbetween. If you found new information after having submitted the first post on the topic, then please make the effort of using the Edit button instead of posting again and again. It makes it harder to read and harder to find in the search engine (as it can only search one post at a time for word occurrences).

* Finally, the posts should not have been posted by you in the first place, as they are already available at the site. Never repost a post already present in the Hive database, link to it instead! Aside from being completely unnecessary, it prevents effective use of the search engine as redundant hits containing identical information pops up when making a search on any word being present in the post - so by just making one redundant repost, you force people using the search engine to get yet another redundant hit in TFSE which they much sift through to find what they are looking for.

I do not understand why you keep doing this, as I have told you exactly this (but not as detailed) more than once before in connection to your Digest compilations. People do not have use for loads of information, they become overwhelmed, it takes them time to sift through it all to find things of value, and it becomes much harder to search for specific information, not only with TFSE, but also when reading a single post containing 15 kilobytes of compact patent text, where none of the useless parts has been edited out.

I value your work here highly, with all the comprehensive Digests you write and your tireless typing of articles, so because of that I will give you another chance and I won't downrate any of your posts in this thread, but I ask you to edit them according to what I have written above within this week, or I'll do it myself when I have the time.

Also, in the future I will rate posts of yours as "redundant", "insignificant" or whatever is suitable if they look like the ones I have discussed above, because this can't continue like this if the board database is to function effectively, I'm sorry. I know you very well can do things properly, just look at all your 37 posts with an "excellent" rating and use them as a guide for what posting style really is appreciated.

Thanks for listening.

Richy
04-21-03 07:07
      Ephedrine HCl from L-PAC
(Rated as: UTFSE!)

Chewbacca
(Newbee)
07-19-03 10:52
No 448410
User Picture       What yielding what

N-methyl-alanine yields ephedrine isomers
l-alanine yields what? PPA?
if so, then it could bee reduced to methamphetamine with a HI/P just like pseudo/ephedrine, right?

A friend with weed is a friend indeed, a friend with speed is better

Aurelius
(Active Asperger Archivist)
07-19-03 14:12
No 448430
      akabori

the ephedrine isomers give meth,
PPA reduces to amphetamine

Act quickly or not at all.

adnagi
(Stranger)
12-18-03 20:19
No 477672
      Quality and safety of products containing Ephedra

Quality and safety of products containing
Ephedra Herba on the Dutch market
Cool link Information .

http://www.chez.com/adnagi/Hive/ephedra.pdf

Good reduction.

cycosyince
(Stranger)
12-19-03 06:41
No 477791
      uh wow what a read!

The only thing left would be

Which Ephedra species are the ephedrine/psudoephedrine alkaloids (the good ones that is) found in, the percentage, the locals in which they are found, and the time of year best known for harvest (when the E and Psudo are at peak levels)

I know there are quite a great number of pheddy bush, never-the-less, relatively few tote the goods.

Imagine how bummed the crew became when in the southwest it was 2 days into November. Just as the the planned adventure was being packed and prepped the info gleaned from the PC instructed that OCTOBER not November was the harvest time. At any rate at least prior to the first frost as that signals the plant to begin its dormancy. Frosty had struck about 3-4 days prior...

04 we are ready. At least vacation slots are open at the end of Sept. and most of the harvest month!

aury, are you like "robochemist" ? My hands would be claws with that much typing, and two fingers at least would sport callouses...

uh, you know, stuff.

biotechdude
(Hive Bee)
12-19-03 09:42
No 477803
      " I have a dream..."

There are a few good species; but the most popular (used) specie is E.Sinica (aka E.Sinensis, E.Major etc - from top of thread). The wildtype variety has low alkaloid content max 1.5%, good preserved lines from china can be up round 3.5%, and the genetically engineered (just wack on a few alkaloid gene promoters!) can pump out around 8%. Growing your own from seed would cost a fortune and would take 20yrs to reach an appreciable yield-quantity. Best bet is to hunt the herb and health food stores for commercial products, or go the raw herb - which has already been harvested at the right time and treated the right way.

Alkaloid production is enhanced when a plant is stressed, however, this activates other pathways that stunt growth (win/lose situation).

Swix's dream is to remove the alkaloid production gene family from the ephedra plant and integrate it into a faster growing, more fleshy plant (like a pea vine); creating easier extraction and higher/faster alkaloid production. It would also be great to isolate only the pseudo/eph pathways so that other unwanted (for meth production) alkaloids are removed. Additionally, it would be cool to isolate alkaloid production only in seed bodies; so that extraction only need to remove some other proteins from the seed and not the whole gaaked plant.

Basically the dream is to have a nice quick growing green pea vine that you pick your pea pods, crack them open, mush the peas up, remove protein, obtain pseudo/eph. Make a billion dollars.

Even put the alkaloid production gene into a bacterial/yeast/virus vector system and ferment the large scale culture...and extract and whammo!

Curious, how do pill companies make their pseudo? And why do they want to add gaaks to stuff us up? If Swix was a pharmaco boss he'd bee making the easiest to extract pill and reap the rewards.

<<"..Mum, do i havto eat my peas??!!..">>

stratosphere
(Hive Bee)
12-19-03 18:23
No 477865
      as cycosyince elluded to, it would be great if

as cycosyince elluded to, it would be great if on that database a catergory of ephedra alkaloid percentage and growing conditions could be added.
can someone offer a online database where such info could be found?

biotechdude
(Hive Bee)
12-21-03 00:56
No 478081
      Stinky Links

https://www.rhodium.ws/pdf/chinese.ephedra.quantitative.analysis.pdf =======> pg 3

http://www.ephedra.demon.nl/stories/ephedra.htm =======> Table 2

There u go lads :)

Master_Alchemist
(Stranger)
02-06-04 09:40
No 486865
      my god

Well i'll be damned, i am so goddamn tired after reading that.
to make it worse i started reading the next page to discover in horror 10 lines later, that page 2 is CC of page 1.
Aurielis, your post has got the better of me tonight!
goodnight all i think im about to haemorage.

Rhodium
(Chief Bee)
04-08-04 06:54
No 499668
User Picture       Analysis of Ephedrine Analogues from Ephedra
(Rated as: good read)

Quantitative Analysis of Ephedrine Analogues from Ephedra Species Using ¹H-NMR
Hye Kyong Kim, Young Hae Choi, Wen-Te Chang and Robert Verpoorte
Chemical and Pharmaceutical Bulletin 51(12), 1382-1385 (2003) (http://www.jstage.jst.go.jp/article/cpb/51/12/1382/_pdf)
DOI:10.1248/cpb.51.1382

Abstract
Four ephedrine analogues such as ephedrine, pseudoephedrine, methylephedrine, and methylpseudoephedrine were determined by ¹H-NMR from Ephedra species. In the region of δ 5.0—4.0, the signals of H-1 attached to the same carbon with a hydroxyl, were well separated from each other in CDCl₃H. The amount of each alkaloid was calculated by the relative ratio of the intensity of H-1 signal to the known amount of internal standard, 200 μg of anthracene. This method allows rapid determination of the quantity of four ephedrine alkaloids from Ephedra species. The amount of these alkaloids was in the range of 1.0—2.0% of dry weight depending on the plant materials.

^{The Hive - Clandestine Chemists Without Borders}

mellow
(Hive Bee)
04-08-04 07:48
No 499680
      Don't forget E. equisetina

biotechdude

Don't forget E. equisetina - an excellent plant which grows in temperate climes, resists frost well and produces proflific amounts of ephedrine.

obelisk
(Stranger)
04-11-04 08:37
No 500173
      ephedra

I have done the work on three positively identified north american species and there is not one trace of usable alkaloid present.
These species centered around northern nevada.

Rhodium
(Chief Bee)
09-13-04 13:54
No 531144
User Picture       Ephedra Alkaloid Content in Dietary Supplements

Concentrations of Ephedra Alkaloids and Caffeine in Commercial Dietary Supplements
Haller C.A.; Duan M.; Benowitz N.L.; Jacob III P.
Journal of Analytical Toxicology 28(3), 145-151 (2004)

Abstract
Dietary supplements that contain Ma Huang (ephedra alkaloids) and guarana (caffeine) are widely marketed and used in the U.S. for weight loss and athletic performance enhancement, despite a lack of adequate research on the pharmacology of these botanical stimulants. We developed and applied a novel liquid chromatography–tandem mass spectrometry (LC–MS–MS) method to quantitate the various ephedra alkaloids found in dietary supplements that contain Ephedra species. The quantities of ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedine, methylpseudoephedrine, and caffeine were determined for 35 commercial dietary supplements and compared with the amounts listed on the product labels. The total ephedra alkaloid content ranged from 5.97 mg to 29.3 mg per serving. Two supplement brands did not list the quantity of ephedra alkaloids on the label, and four did not list the amount of caffeine per serving. Of the products tested, 31% contained > 110% of the total ephedra alkaloids listed on the label, and 6% of the supplements contained < 90% of the listed amount. For caffeine, 86% of the product lots that listed the caffeine amount contained less than 90% of the labeled quantity. No products contained > 110% of the declared caffeine content. The total ephedra alkaloid content varied significantly from lot to lot in 5 of 9 products. Three product brands contained proportions of alkaloids that exceeded amounts reported for E. sinica, including one that was 98% ephedrine, one that had 10% norpseudoephedrine, and one that contained an average of 13% methylephedrine. We conclude that product inconsistency is common among some commercially available dietary supplements that contain ephedra alkaloids and caffeine.

^{The Hive - Clandestine Chemists Without Borders}

Rhodium
(Chief Bee)
10-11-04 18:44
No 535382
User Picture       Determination of Ephedra Alkaloids
(Rated as: excellent)

Determination of Ephedra Alkaloids by Liquid Chromatography/Tandem Mass Spectrometry
Darryl Sullivan, James Wehrmann, John Schmitz, Richard Crowley, and Jeffrey Eberhard
Journal of AOAC International Vol. 86, No. 3, 471-475 (2003) (https://www.rhodium.ws/pdf/ephedra.analysis.aoac-1.pdf)

Abstract
In conjunction with an AOAC Task Group on dietary supplements, a liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was validated for measurement of 6 major alkaloids in raw ephedra sinica herb, ephedra extracts, ephedra tablets, complex dietary supplements containing ephedra, and a high-protein drink mix containing ephedra. The amount of ephedrine-type alkaloids present was determined by LC with mass selective detection. Six replicates of each matrix were analyzed on 3 separate occasions. The presence of 6 ephedrine-type alkaloids was detected at a level >0.5 µg/g based on a 0.5 g sample. The standard curve range for this assay is from 0.02 to 1.0 µg/mL. Appropriate dilutions covered a wide range of specific alkaloid concentrations. The calibration curves for all 6 analytes had correlation coefficients >0.995.
____ ___ __ _

Determination of Ephedrine Alkaloids in Dietary Supplements and Botanicals by Liquid Chromatography/Tandem Mass Spectrometry: Collaborative Study
William A. Trujillo and Wendy R. Sorenson
Journal of AOAC International Vol. 86, No. 4, 657-668 (2003) (https://www.rhodium.ws/pdf/ephedra.analysis.aoac-2.pdf)

Abstract
An interlaboratory study was conducted to evaluate the accuracy and precision of a method for ephedrine-type alkaloids [i.e., norephedrine (NE), norpseudoephedrine (NPE), ephedrine (E), pseudoephedrine (PE), methylephedrine (ME), and methylpseudoephedrine (MPE)] in dietary supplements and botanicals. The amount of ephedrine-type alkaloids present was determined using liquid chromatography with tandem mass selective detection. The samples were diluted to reflect a concentration of 0.0200 to 1.00 µg/mL for each alkaloid. An internal standard was added and the alkaloids were separated using a 5 µm phenyl LC column with an ammonium acetate, glacial acetic acid, acetonitrile, and water mobile phase. Eight blind duplicates of dietary supplements or botanicals were analyzed by 10 collaborators. Included was a negative control, ephedra nevadensis, and negative controls fortified at 2 different levels with each of the 6 ephedrine-type alkaloids. The spike levels were approximately 100 and 1000 µg/g for NE, 100 and 600 µg/g for NPE, 6500 and 65 000 µg/g for E, 1000 and 10 000 µg/g for PE, 300 and 3000 µg/g for ME, and 100 and 1000 µg/g for MPE. On the basis of the accuracy and precision results for this interlaboratory study, it is recommended that this method be adopted Official First Action for the determination of 6 different individual ephedrine-type alkaloids in dietary supplements and botanicals.

^{The Hive - Clandestine Chemists Without Borders}

Xaja
(Hive Bee)
11-03-04 01:45
No 539305
User Picture       Biotechdudes post

Nice idea. Would be interesting to know how many genes are involved in the process. Hopefully only a few, and not many genes located on different chromosomes, which would make things difficult!

But yeah, BAC inserts via Agrobacterium tumefaciens can now deliver QTLs or multigene families directly into the plant genome, so its not out of the question, providing you can get access to a decent lab that uses the technology.

Expression in bacteria would be too complicated in my view. Well, still possible but a lot of work creating the vector, then introducing the genes then selecting for transformants. Also the gene products could be toxic to the bacteria or insoluble in their cytoplasm etc etc.

Although having the eph produced in a bacterial system would be a dream come true. Imagine how fast it would be!!! cool

What? The Land of the Free? Whoever taught you that is your enemy! - Rage Against The Machine

Xaja
(Hive Bee)
11-03-04 01:49
No 539306
User Picture       Reference

Just for the sake of good science, here's a reference that shows the direct plant transformation by Agobacterium with BAC inserts:

http://www.siu.edu/~pbgc/publications/6.pdf

What? The Land of the Free? Whoever taught you that is your enemy! - Rage Against The Machine

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