Racemization tests

First, they compared CSPs 1 and 3 prepared by the two-step solid-phase methodology with their commercially available counterparts (CSPs 2 and 4) obtained by direct reaction of the preformed selector with a silica support. Although no exact data characterizing the surface coverage density for these phases were reported, all of the CSPs separated all four racemates tested equally. These results shown in Table 3-3 subsequently led to the preparation of a series of dipeptide and tripeptide CSPs 5-10 using a similar synthetic approach. Although the majority of these phases exhibited selectivities lower or similar to those of selectors built around a single amino acid (Table 3-3), this study demonstrated that the solid-phase synthesis was a... 

FIGURE 4.8 Racemization tests employed for acquiring information on stereomutation. Couplings are carried out, and the isomeric content of the products is determined by a variety of techniques. AAA = amino acid analyzer GLC = gas-liquid chromatography. 

N Izumiya, M Muraoka. Racemization test in peptide synthesis. J Am Chem Soc 91, 2391, 1969. 

It has been known for years that the activated residues of acyl- and peptidylamino acids enantiomerize during coupling (1.9). However, the racemization tests available (see section 4.9) did not allow for a valid comparison of the tendency of residues to isomerize because they incorporated a variety of aminolyzing residues and N-substituents. Valid demonstration of the different sensitivities of residues was provided by classical work on the synthesis of insulin. It was found that a 16-residue segment with O-tert-butyltyrosine at the carboxy terminus produced 25% of epimer in HOBt-assisted DCC-mediated coupling in dimethylformamide, and the same segment with leucine at the carboxy terminus produced no epimer. Only when series such as Z-Gly-Xaa-OH coupled with valine benzyl ester became available was it possible to compare many residues with confidence. Unfortunately, it transpires that the issue is extremely complex. 

Only few studies addressed the effect of the silica surface concentration of glycopeptides antibiotics on the chiral performances. The first was performed by Armstrong on a TE CSP toward five racemic test solutes [30]. The study evidenced a modest increase of a and Rs values with increasing the initial selector concentration in the grafting reaction mixture, but no data were reported regarding the effective... 

Similarly, reversed-phase HPLC can be used as an Eilternative to the racemization test for amino acids as developed by Manning and Moore (115). Rivier and Burgus (109) have suggested the use of L-phenylalanine, coupled via the N-carboxyanhydride method to a hydrolysate, to monitor racemization during synthesis, although other hydrophobic L-amino acids should also prove equally effective. The use of /eri-butyloxycarbonyl-L-amino acid-Af-hydroxysuccinimide esters in the separation of enantiomeric amino acids and diastereoisomeric peptides has been described (110). Ultimately, these methods may not prove as versatile as the use of chiral stationary phases made by stereoselective control of the bonding process or, alternatively, with surface-active reagents similar to the D-... 

From the observation that isobutyl chloroformate can lead to higher yields as compared to ethyl chloroformate in the MCA coupling procedure, 2-iso-butoxy-l-isobutoxycarbonyl-l,2-dihydroquinoline (BBDQ) was prepared and found a useful alternative to the etho.xy derivative of the EEDQ procedure. In the Bodanszky racemization test (quantitative amino acid analysis of D-Ile from Ac-Ile plus Gly-OMe). The BBDQ procedure gave 4% racemate while DCCI led to 27% racemate. 

In substance, ionically bonded plates had less background color than covalently bonded stationary phases and gave better resolution of racemic test material. In addition, preparation of impregnated plates was very simple and fast plates were cut into 5 X 10 cm sizes and placed in a large crystallizing dish, submerged with 30 ml tetrahydrofuran (THF) containing 1 g CS1. After 2 min, the plates were removed and washed with pure THF. 

With the dicyclohexylcarbodiimide (DCQ reagent racemization is more pronounced in polar solvents such as DMF than in CHjCl2, for example. An efficient method for reduction of racemization in coupling with DCC is to use additives such as N-hydroxysuccinimide or l-hydroxybenzotriazole. A possible explanation for this effect of nucleophilic additives is that they compete with the amino component for the acyl group to form active esters, which in turn reaa without racemization. There are some other condensation agents (e.g. 2-ethyl-7-hydroxybenz[d]isoxazolium and l-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline) that have been found not to lead to significant racemization. They have, however, not been widely tested in peptide synthesis. 

The synthesis of dextromethorphan is an outgrowth of early efforts to synthesize the morphine skeleton. /V-Methy1morphinan(40) was synthesized in 1946 (58,59). The 3-hydroxyl and the 3-methoxy analogues were prepared by the same method. Whereas the natural alkaloids of opium are optically active, ie, only one optical isomer can be isolated, synthetic routes to the morphine skeleton provide racemic mixtures, ie, both optical isomers, which can be separated, tested, and compared pharmacologically. In the case of 3-methoxy-/V-methylmorphinan, the levorotatory isomer levorphanol [77-07-6] (levorphan) was found to possess both analgesic and antitussive activity whereas the dextrorotatory isomer, dextromethorphan (39), possessed only antitussive activity. Dextromethorphan, unlike most narcotics, does not depress ciUary activity, secretion of respiratory tract fluid, or respiration. 

However, the use of a HPLC separation step enabled a remarkable acceleration of the deconvolution process. Instead of preparing all of the sublibraries, the c(Arg-Lys-O-Pro-O-P-Ala) library was fractionated on a semipreparative HPLC column and three fractions as shown in Fig. 3-2 were collected and subjected to amino acid analysis. According to the analysis, the least hydrophobic fraction, which eluted first, did not contain peptides that included valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine residues and also did not exhibit any separation ability for the tested racemic amino acid derivatives (Table 3-1). 

Fig. 3-7. Evaluation of a focused library of 71 DNB-dipeptide CSPs for enantioseparation of the test racemate 8. (Reprinted with permission from ref. [86], Copyright 1999, American Chemical Society.)...

Fig. 3-9. Preparative HPLC of 100 mg of the test racemate 8 in a single 2 mL injection using a 250 x 4.6 mm i.d. column containing (5)-Glu-(5)-Leu-DNB CSP. Conditions mobile phase ethyl acetate, flowrate 2.0 mL min , UV detection at 380 nm. Injection 2 mL of 50 mg mL racemate solution. Fractions collected before and after the indicated cut point were 98.4 % ee and 97 % ee pure, respectively. (Reprinted with permission from ref. [86]. Copyright 1999, American Chemical Society.)...

Our strategy consisted of the following steps A mixture of potential chiral selectors is immobilized on a solid support and packed to afford a complete-library column , which is tested in the resolution of targeted racemic compounds. If some separation is achieved, the column should be deconvoluted to identify the selector possessing the highest selectivity. The deconvolution consisted in the stepwise preparation of a series of sublibrary columns of lower diversity, each of which constitute a CSP with a reduced number of library members. 

The guideline on chiral active substances states that particular attention should be paid to identity and stereochemical purity. It states that specifications for a racemate should include a test to show that the substance is indeed a racemate and this is a position supported by the requirements of the European Pharmacopoeia for drug substance monographs [16]. 

For drug substances and drug products, applications for enantiomers and racemates should include a stereochemically specific identity test and/or a stereochemically selective assay. The choice of control tests should be based on the method of manufacture and stability characteristics and, in the case of the finished product, its composition. 

Methods of assessing the stereochemical integrity of enantiomeric drug substances and drug products should be included in the stability protocols for both, but stereoselective tests may not be required once it has been shown that racemization does not occur. 

---