Threonine resolution

Crystallization Method. Such methods as mechanical separation, preferential crystallisation, and substitution crystallisation procedures are included in this category. The preferential crystallisation method is the most popular. The general procedure is to inoculate a saturated solution of the racemic mixture with a seed of the desired enantiomer. Resolutions by this method have been reported for histidine (43), glutamic acid (44), DOPA (45), threonine (46), A/-acetyl phenylalanine (47), and others. In the case of glutamic acid, the method had been used for industrial manufacture (48). 

The L-threonine (EC 4.1.2.5), D-threonine (EC 4.1.2.-) or L-allothreonine aldolases (EC 4.1.2.6 synonymous to S1IMT) can be used for resolution of racemic (allo)threonine mixtures by highly selective cleavage of the unwanted isomers42, but can also efficiently direct the anabolic pathways. The substrate spectrum includes propanal, butanal and dodecanal43. 

Preferential crystallisation is one option for optical resolution on a manufacmr-ing scale. Online polarimetry and refractometry have been used to d3mamically optimise the process for resolution of DL-threonine in aqueous solution by variation of process parameters such as degree of supersaturation, seed quantity, initial enantiomeric excess and scale [148]. The method is claimed to be suitable for control of quasi-continuous processes. 

Purity Decrease of L-Threonine Crystals in Optical Resolution by Batch Preferential Ciystalli tion... 

In the optical resolution of DL-threonine mixtures by batch preferential crystallization, changes of solution concentration and crystal purity were measured. The mechanism of nucleation of the un-seeded enantiomer was discussed to explain the purity decrease of the resolved crystals. From the observation of crystallization behavior of the seed crystals of L-threonine, it was concluded that the existence of the D-enantiomer on the surface of the seed caused the sudden nucleation when they grew to attain sufficient amounts. 

This observation is in accordance with the phenomena of the crystallization in the resolution operation mentioned above in the following points. There are no clear, definite critical supersaturations above which nucleation of D-threonine occurs. Ohtsuki (2), however, reported supersolubility curve for this system, who gave the value of the supersaturation width At=7 C at 50 C. Their definition of the metastablllty was that no nucleation of the enantiomer other than seeded one was observed for two hours of resolution experiments. According to this definition, the supersolubility can be determined to lie somewhere between At=8 and 5 C from the present experimental data, this being in agreement with his result. If the crystallization proceeds further, however, D-threonine crystals may start to crystallize from the solution even if the initial supersaturation is 5 C. In this sense it is no longer the metastablllty limit. 

From the consecutive measurements of solution concentrations and crystal purities during the optical resolution by preferential crystallization, the crystallization of D-threonlne other than the seeded component (L-threonine) was observed In the later stage of the resolution. Washing of the seed crystals was found to be effective to delay the purity decrease. D-threonine was believed to be Introduced... 

Batch crystallizers, applications, 102 Batch preferential crystallization, purity decrease of L-threonine crystals in optical resolution, 251-259 Bayer proce description, 329 Benzene, hi -pressure crystallization from benzene-cydohexane mixture, 281-289... 

Sulfosalicylic acid has most commonly been used to precipitate proteins prior to ion-exchange amino acid analysis (11). In this mode, SSA allows for a very simple sample preparation that requires only centrifugation of the precipitated sample and then direct injection of the resulting supernatant solution. The supernatant solution is already at an appropriate pH for direct injection. Also, the SSA does not interfere chromatographically since it elutes essentially in the void volume of the column. It has been noted that, if an excessive amount of SSA is employed, resolution of the serine/threonine critical pair can suffer (12). The use of SSA prior to reversed-phase HPLC can be more problematic, since its presence can interfere with precolumn deriva-tization. For example, Cohen and Strydom (13) recommend the separation of the amino acids from the SSA solution on a cation-exchange resin prior to derivatization with phenylisothiocya-nate (PITC). 

Figure 33 Electropherogram of threonine and glutamine enantiomers using an amino acid derivative CSP immobilized onto silica particles. Conditions fused silica capillary, 30 cm x 75 mm i.d., packed segment is 15 cm in length, mobile phase is 30/70 5 mM phosphate (pH 2.5)/acetonitrile, field strength is 0.83 kV/cm. For resulting resolution, see Table 2. (Reprinted from Ref. 142, with permission.)...

Scheme 5.49. Resolution of fi-hydroxy-a-amino acids, using threonine aldolase from Strepto-myces amakusaensis.

Hydrogenation of certain racemic 2-substituted 3-oxocarboxylates occurs with high diastereo- and enantioselectivity via dynamic kinetic resolution involving in situ racemization of the substrates. The (R)-BINAP-Ru-catalyzed reaction of 2-acylamino-3-oxocarboxylates in dichloromethane allows preparation of threonine and DOPS (anti-Parkinsonian agent)... 

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