Stereoisomers of aspartame

Chiral chromatography can also be used in order to obtain resolution of stereoisomers from aspartame, its precursors, and its degradation products. Lin et al. (84), using a Chiracel OD column and a mobile phase of 2-propanol -hexane (1 1, v/v), achieved complete separation of aspartame precursors, dd-, dl-, LL-, and LD-[(Z)-AspOS-Bzl)-Phe-OCH3], Motellier and Wainer (85) separated four stereoisomers of aspartame, two of diketopiperazine, and three of aspartyl-phenylalanine using a stationary phase composed of a chiral crown either coated on a polymeric support—CrownPack CR( + )—a mobile phase of aqueous perchloric acid, pH 2.8, and modified... 

Figure 12.6 Chromatogram of the four stereoisomers of aspartame (APM). Peak 1 L,L-APM. Peak 2 L,D-APM. Peak 3 D.D-APM. Peak 4 D.L-APM. The early eluting peaks are degradation compounds. Using a Crownpak CR( + ) column and propan-2-ol aqueous perchloric acid mobile phase, a temperature gradient was required to elute peaks 1 and 2 (Motellier and Wainer, 1990).

Finally, as a reminder that stabiUty studies are not restricted to pharmaceutical products, a chromatogram taken from the study of a stability study of LL-aspartame (NutrasweeO in diet cola is shown in Figure 12.14. The same conditions were used for the resolution of the stereoisomers of aspartame (Figure 12.6), and the study of the stability of LL-aspartame in distilled water and coffee sweetened with Nutrasweet. ... 

FIGURE 1-23 Stereoisomers distinguishable by smell and taste in humans, (a) Two stereoisomers of carvone R) carvone (isolated from spearmint oil) has the characteristic fragrance of spearmint (S)-carvone (from caraway seed oil) smells like caraway, (b) Aspartame, the artificial sweetener sold under the trade name NutraSweet, is easily distinguishable by taste receptors from its bitter-tasting stereoisomer, although the two differ only in the configuration at one of the two chiral carbon atoms. 

The most commonly used stationary phase for the separation of aspartame from synthesis intermediates, stereoisomers, and degradation products is the reverse-phase Cl8 column. As can be seen on Table 2, the main type of the mobile phase used is a phosphate buffer at pH ranging from 2.5 to 5.0 associated with acetonitrile (14,55,80,83). Reverse-phase HPLC with gradient elution of acetonitrile in phosphate buffer has also been used (16,78). 

Table 2 HPLC Methods for the Simultaneous Determination of Aspartame and Its Synthesis Intermediates, Stereoisomers, and Degradation Products... 

S Motellier, IW Wainer. Direct stereochemical resolution of aspartame stereoisomers and their degradation products by high-performance liquid chromatography on a chiral crown ether based stationary phase. J Chromatogr 516 365-373, 1990. 

At the present time, the reported applications of the CR CSP have been limited to the separation of amino acids and some dipeptides as bulk substances. One example of the use of the CR CSP in a complex matrix was the direct stereochemical resolution of aspartame stereoisomers and their degradation products in coffee and diet soft drinks (76). Aspartame (N-DL-a-aspartyl-DL-phenylalanine methyl ester) is a dipeptide whose L,L-isomer is a low-calorie sweetener sold under the name NutraSweet. The structure of aspartame and its major degradation products are presented in Fig. 9 aiwl the stereochemical separation of these compounds on the CR CSP in Fig. lOA. The resolutions were accomplished using a mobile phase... 

Aspartame is made by a relatively simple procedure in which two amino acids, aspartic acid and phenylalanine, are reacted with each other to form a two-amino-acid product, called a dipeptide. The carboxylic acid group in the dipeptide is then reacted with methanol (methyl alcohol CH3OH) to obtain the methyl ester of the compound. One problem that makes the preparation somewhat more difficult is that hoth aspartic acid and phenylalanine have stereoisomers. The term stereoisomer refers to two forms of a compound that contain the same kind and number of atoms, hut differ in the orientation in space ( stereo ) of some of the atoms. Because of these stereoisomers, four different kinds of aspartame are formed during the preparation described above — D D L L D L L D (the latter two are different from each other). Only one is the desired product, the one that contains only L stereoisomers. 

Organoleptic Properties. Stereochemical differences of enantiomeric excipients may influence perception by sensory organs. Kutti [17] reported as early as 1886 that the interaction of stereoisomer with chiral receptors led to chiral discrimination as a consequence of the formation of diastereomers. He observed that the dextrorotatory asparagine has a sweet taste whereas the levorotatory form is tasteless. Greenstein and Winitz [18] and Solms et al. [19] reported such differences for many amino acids. Shallenberger et al. [20] reported that for some monosaccharides, both isomers have similar sweetness. In contrast, aspartame (A-aspartylalanine methyl ester) is marketed as the l,l isomer because it is more than 100 times as sweet as sucrose. However, the l,d diastereomer of aspartame is bitter [11], It should be noted that the individual differences of perception of these properties could vary. 

FIGURE 3.6 Separation of aspartame and its precursor stereoisomers on Chiralplate. D,D-, D,L-aspartame, R[ = 0.62 L,L-, L,D-aspartame, Rf = 0.50. (Reprinted from the Macherey-Nagel Application Database. With permission.)... 

The example of an LC chiral separation shown in Figure 12.6 serves to emphasise (a) that the demand for effective chiral selectors is such that even complex synthetic chiral selectors have been commercialised, and (b) the interest in chirality extends beyond pharmaceutical applications, being widespread and in this instance being found in food analysis. Aspartame (N-DL-cx-aspartyl-DL-phenylalanine methyl ester (Figure 12.7)) can exist as four stereoisomers, DD-, LL-, DL- and LD-. On an achiral column DD- and LL- would appear as a single peak which would be separable from another single peak arising from DL- and LD-. A chiral column is needed to separate the enantiomeric pairs (i.e. DD- from LL- and DL-from LD-). The LL-isomer is used as artificial sweetener (under the brand... 

A new chiral reagent, NSP-Cl was synthesized and used to derivatize amino acids and the resulting diastereomers were resolved by TLC (166), Chiralplate with MeCN-MeOH-HjO (4 1 1) as mobile phase was used to evaluate reaction products in the synthesis of modified phe and tyr derivatives (167), and also to separate aspartame and its precursor stereoisomers (168). Tryptophans and substituted tryptophans were separated on cellulose layers developed with copper sulphate solutions (169) when excess of Cu " ions decreased the chiral discrimination of the system, and with aq a-cyclodextrin (1 to 10%) plus NaCl solutions (O.IM, 0.5M, l.OM) when the best results were... 

This is demonstrated by the case of the artificial sweetener aspartame. Only the stereoisomer (9) is sweet and the other three (1(1-12) are all slightly bitter and must be avoided in the manufacturing process. 

---