Covalent Enantiomeric Derivatization of Some Important Functional Groups Liquid Chromatography

Covalent Enantiomeric Derivatization of Some Important Functional Groups

Tue, 27 Sep 2011 00:50:41 | Liquid Chromatography

Derivatization of amines The reagents suitable for the chiral derivatization of amines can be categorized as follows:

Activated carboxylic acids These are usually car-boxylic chlorides and the reaction with primary and secondary amines leads to diastereomeric carbox-amides. The classical reagent R( + )-a-methoxy-a(tri-fluoromethyl)phenylacetyl chloride is still in use for the preparation of gas chromatographically separable diastereomers. Some others ('dual-purpose' reagents with strong UV absorption or fluorescence) include 1-(4-nitrophenylsulfonyl)-l-prolyl chloride, dansyl-l-proline activated by triethylamine/diethyl phos-

10 20 30 40 50 60 70

Time (min)

: Time (min)

10 20 30 40 50 60 70

Time (min)

Figure 2 High-performance liquid chromatographic (HPLC) elution profile of the mixture of L and D-amino acids (L: D = 2: 1), glycine and L-homo-arginine (internal standard) derivatized as the isoindoles with (A) o-phthalaldehyde-A/-isobutyryl-D-cysteine and (B) o-phthalaldehyde-W-isobutyryl-L-cysteine; (C) amino acids from an ethanolic extract of Lactobacillus acidophilus, derivatization with o-phthalaldehyde-W-isobutyryl-L-cysteine. Column, Hypersil ODS (250 x4 mm, 5 ^m); mobile phase, gradient elution; A = 23 mM sodium acetate (pH 5.95); B = methanol-acetonitrile (600:50v/v), linear gradient from 0% B to 53.5% B in 75 min; flow rate 1 mL min-1; fluorescence detection, 230 nm excitation, 445 nm emission. Reproduced with permission from Bruckner H, Haasmann S, Langer M, Westhauser T and Godel H (1994) Journal ofChromatographyA 666: 259, copyright Elsevier.

10 20 30 40 50 60 70

Time (min)

phorocyanidate, N-[4-(6-methoxy-2-benzoxazolyl)] benzoyl-l-phenylalanine or -proline, activated by 2,2-dipyridyl disulflde/triethylphosphine, N-ben-zyloxycarbonyl-l-phenylalanine, activated by acetic anhydride( + )-2-methyl-2fi-naphthyl-1,3-benzo-dioxole-4-carboxylic acid chloride, (S)-( + )-naproxen chloride, (S)-( + )-flunoxaprofen chloride, (S)-( + )-be-noxaprofen chloride, etc. Their strong fluorescence enables the enantiomers of chiral drugs such as tranylcypromine, tocainide, carvedilol, baclofen and propranolol to be determined. (The isocyanate, isothiocyanate and chloroformate derivative of these compounds have also been introduced as chiral de-rivatizing agents as shown in the subsequent sections.)

An on-line solid-phase derivatization reagent is fluorenylmethyloxycarbonyl-l-proline (FMOC-l-proline), bonded to beads of a styrene-divinylbenzene copolymer as the active ester of a 4-hydroxy-3-nitrobenzophenone moiety. By positioning the HPLC column after the reaction column, the transformation of chiral amines (e.g. amphetamine) to their highly fluorescent diastereomeric FMOC-l-prolyl derivatives and their separation was achieved.

formate is that it is suitable for the derivatization of the tertiary amine promethazine via demethylation of the dimethylamino moiety.

Isocyanates These reagents form diastereomeric urea derivatives with chiral primary and secondary amines. (R)-(-)- and (S)-( #)-1-(1-naphthyl)ethyl isocyanate and (R)-a-methylbenzyl isocyanate are among the classical chiral derivatizing reagents. Isocyanate derivatives of the drugs mentioned in one of the previous sections as the carboxylic chlorides and (R)-N-3,5-dinitrobenzoyl)phenyl glycine have also been used for the derivatization of ^-blockers and other amines. Eqn [1] shows the reaction between nadolol and (R)-(-)-1-(1-naphthyl)ethyl isocyanate, while the separation of the four dias-tereomers of the two racemates is depicted in Figure 1. It is remarkable that by selecting a suitable reagent and proper chromatographic conditions not only can the four diastereomers be separated but the interference from dog plasma can also be eliminated.

Chloroformâtes The above-mentioned FMOC group has been incorporated into another types of chiral reagents: ( + )-1-(9-fluorenyl)ethyl chlorofor-mate is one of the most widely used derivatization reagents for the chiral HPLC of amino acids, fi-blockers, to form the corresponding carbamate derivatives.

An interesting feature of another widely used reagent of the chloroformate type, ( — )-menthyl chloro-

Isothiocyanates Of the isothiocyanates forming thiourea derivatives with primary and secondary amines, GITC is most widely used. If the four acetyl groups are replaced by benzoyl groups the sensitivity of the detection is greatly improved. Other reagents of this type leading to highly fluorescent derivatives include (R)-( — )- and (S)-( + )-4-(3-isothiocyanatopyr-rolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole and its 7-(N,N-dimethylaminosulfonyl) analogue as well as DDITC ((1R,2R)- and (1S,2S)-N-[(2-isothio-

cyanato)cyclohexyl]-3,5-dinitrobenzoylamide, which excels with the high chemical stability, high UV activity and the excellent separability of the dia-stereomeric derivatives. The equation of the reaction of GITC and DDITC with the ^-blocker metoprolol and the separation of the derivatives are depicted in eqn [2] and Figure 3, respectively.

valinamide analogue of the Marfey reagent gives even better resolution.

o-Phthalaldehyde +chiral thiols This dual derivatization reaction leading to fluorimetrically highly active isoindole derivatives is another generally used method in the chiral analysis of amino acids (see eqn

N-Haloarylamino acid derivatives Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-l-alaninamide) is one of the most generally used reagents in the analytical control of racemization during peptide synthesis. The peptides are split either by hydrochloric acid or en-zymatically. The nucleophyllic attack of the a-amino group of the amino acids on the C-F bond activated by the two nitro groups on the aromatic ring results in a smooth reaction to form diastereomeric aniline derivatives with goodUVdetectability (see eqn [4]). The

[3]). As the chiral thiol N-acetyl-l-cysteine is most widely used but the use of 2,3,4,6-tetra-0-acetyl-1-thio-^-d-glucopyranoside and N-isobutyryl-l-cys-teine (and d-cysteine) have been found to be advantageous in the resolution of the diastereomers. The reaction of amino acids with o-phthalaldehyde and the latter reagent is shown in eqn [3], while in Figure 1 the separation of as many as 36 enantiomers of 18 amino acids and amino acid analogues as well as glycine is depicted together with a practical applicaion.

Enzymatic deamination Chiral HPLC of a mixture of amino acid enantiomers with and without selective oxidative deamination of d-amino acids with the aid of the enzymes d-amino acid oxidase and catalase enables d-amino acids to be identified in the mixture.

Derivatization of the carboxyl group

Esteriftcation with chiral alcohols (+)- or (— )-2-octanol, ( + )-1-phenylethanol, ( —)-menthol, ( + )- or (— )-2-butanol are the classical reagents for the chiral analysis of carboxylic acids. The reactions usually require harsh conditions and for this reason the danger of racemization should be taken into consideration.

Amidation with chiral amines Prior to their reactions with chiral amines the carboxyl group should

UV or fluorometric properties are also in use, e.g. (R)-a-methyl-4-nitrobenzylamine, (R)-( + )-1-(1-naphthyl)ethylamine, (— )-1-(1-anthryl)ethylamine, R-( —)- and (s)-( + )-amphetamine, (1R,2R)-( —)- or (1S ,2S)-( + )-2-amino-(4-nitrophenyl)-1,3-pro-panediol, l-leucinamide, l-alanine-^-naphthylamide, l- or d-0-(4-nitrobenzyl)tyrosine methyl ester, ( —)-2-[4-(1-aminoethyl)-phenyl]-6-methoxybenzoxazole and other related derivatives where the 1-aminoethyl group is replaced by l-leucyl or d-phenylglycyl groups, drug-related amines (flunoxaprofen amine, benoxaprofen amine and naproxen amine), (R)- and (S)-1-(4-dansylaminophenyl)ethylamine, etc. The reaction of ibuprofen and pranoprofen with the last reagent and the separation of the diastereomeric car-boxamide derivatives are shown in eqn [4] and Figure 4, respectively.

be activated. Possibilities for this are, e.g. reaction with thionyl chloride to form carboxylic chlorides, with chloroformates to form mixed anhydrides, with 1,1-carbonyldiimidazole to form reactive N-acylimidazoles and with the classical coupling agent dicyclohexylcarbodiimide to form the reactive N-acylurea derivatives. The classical but still widely used amine reagent is (S)-( — )-a-methylbenzylamine, but several others with excellent separation power,

Derivatization of the alcoholic and phenolic hydroxyl groups The most frequently used general method for the derivatization of the hydroxyl group of chiral alcohols and phenols is esterification. A great variety of chiral carboxylic acids have been used for this purpose such as R( +)- and S( — )-a-methoxy-a(tri-fluoromethyl) phenylacetic acid (Mosher's acid), R( + )-trans-chrysanthemic acid, (— )-menthenylo-xyacetic acid for the gas chromatographic or HPLC

Figure 3 Resolution of (RS)-metoprolol derivatized as (R,R)-DDITC- and GITC-thioureas. Column, Hypersil ODS (125 x 4 mm, 5 |im); mobile phase, acetonitrile-20 mM ammonium acetate 55:45, v/v; flow rate 1 mL min-1 detection at 254 nm. Reproduced with permission from Kleidernigg OP, Posch K and Lindner W (1996) Journal of Chromatography A 729: 33, copyright Elsevier.

separation with UV detection and (— )-(1S,2R,4R)-endo-1,4, 6,7,7-hexachlorobicyclo[2.2.1]-hept-5-ene-2-carboxylic acid and (S)-( + )-2-tert-butyl-2-methyl-1,3-benzodioxolo-4-carboxylic acid for HPLC separation with fluorimetric detection, etc. Dicyclohexylcarbodiimide can be used as the coupling agent, but the use of in situ transformation of the acids to their chlorides by the addition of thionyl chloride is more widespread. The direct enzymatic d-( + )-glucuronidation reaction of phenols has also been described.

Acyl chlorides, anhydrides and acyl cyanides can be used directly. For example (— )-camphanic acid, R( + )-a-methoxy-a(trifluoromethyl)phenylacetyl chloride, ^-naphthylsulfonyl-l-prolyl chloride, flunoxaprofen chloride, (R,R)-0,0-diacetyl- (or di-p-toluoyl-) tartaric anhydride and ( —)-2-methyl (or methoxy)-1,1 '-binaphthalene-2'-carbonyl cyanide lead to well-separable, UV or fluorimetrically active diastereomeric ester derivatives with chiral alcohols.

As an illustration, the reaction of delmopinol and its analogue used as the internal standard in the determination of the drug in plasma with the reagent (R,R)-0,0-di-p-toluoyl-tartaric anhydride is shown in eqn [5]. The separation is depicted in Figure 5.

Time (min)

Figure 4 Resolution of (A) (RS)-ibuprofen and (B) (RS)-pranoprofen as the carboxamides formed with (S)-1-(4-dan-sylaminophenyl)ethylamine. Column ODS-80tm (150 x4.6 mm, 5 |im); mobile phase: 50 mM sodium acetate (pH 6.5)-acetonitrile (A) 30: 70, v/v and (B) 45:55, v/v, and flow rate 1 mL min"1; fluorescence detection, 338 nm excitation, 535 nm emission. Reproduced with permission from Iwaki K, Bunrin T, Kameda Y and Yamazaki M (1994) JournalofChromatographyA 662: 87, copyright Elsevier.

Time (min)

(ff,fl)-di-p-toluoyl tartaric acid anhydride n = 2(±)-Delmopinol 3(±)-lnternal standard

Further derivatization reactions include the use of (R)-1-(1-naphthyl)ethyl isocyanate for the derivatization of, e.g. diacylglycerol derivatives to form the corresponding carbamates. The lower reactivity of the hydroxyl group compared with the amino group can be overcome by using a suitable catalyst (4-pyrrolidinopyridine). (— )-Menthyl chloro-formate, which has already been mentioned, has also found application here as a reagent for amines; the reaction products with chiral alcohols are carbonates.

Figure 5 Resolution of ( — )- and ( # )-delmopinol (peaks 1 and 2) and ( — )- and ( # )-internal standard (peaks 3 and 4) and their determination in human plasma extracts. Derivatization with (R,R)-O,O'-di-p-toluoyl tartaric acid anhydride to form the esters. (A) Blank plasma; (B) plasma spiked with 6 ng of each enantiomer; (C) authentic plasma sample. Column, Hypersil ODS (125 x 4 mm, 5 ^m); mobile phase, 100 mM ammonium acetate-acetonitrile 35:65, v/v, pH 5.7; flow rate, 0.8mLmin~1; electrochemical detection. Reproduced with permission from Egginger G, Blaschke E, Lindner Wand Olsson A-M (1994) Journal of Chromatography A 666: 275, copyright Elsevier.

Derivatization of the aldehyde group The importance of this kind of chiral derivatization reactions is much smaller than those described for amines, carboxylic acids and alcohols. For example, aldoses can be transformed with l-cysteine methyl ester to diastereomeric thiazolidine derivatives which can be separated by GC after trimethyl-silylation. The two aldehyde groups of gossypol were transformed with (R)-( — )-2-amino-1-propanol to the diastereomeric Schiff's bases separable by HPLC.

Derivatization of epoxides The oxirane ring of chiral epoxides is opened by sodium sulfide to form a vicinal hydroxythiol derivative. In the second step of the derivatization reaction the thiol group reacts with o-phthalaldehyde and a chiral amino acid to form the diastereomeric isoindole derivative mentioned in the section dealing with the derivatization of the amino group. Another ring-opening reagent is 2-propylamine. The secondary amino group of the 1,2-amino alcohol is then reacted with 2,3,4,6-tetra-O-acetyl (or benzoyl)-ft-d-glucopyranosyl iso-thiocyanate to form the diastereomeric thiourea derivatives.

Derivatization of the thiol group The formation of diastereomeric isoindole derivatives from chiral amino acids, chiral thiols and o-phthalaldehyde (already mentioned in the section dealing with the de-rivatization of the amino group; see eqn [4]) can also be used for the enantioseparation of thiols. Reagents of other types are (R,R)-dinitrobenzoyldiamino-cyclohexyl isothiocyanate and N-[(2-isothiocyanato)-cyclohexyl]-pivalinoyl amide which transform the thiol compound to their diastereomeric dithiocarba-mate derivatives.

Derivatization of Enantiomers With Achiral Reagents to Improve Their Chromatographic Properties and Their Separation on Chiral Columns

Engineering Schemes

» Liquid Chromatography