Gas chromatographic separation of stereoisomers of non-protein amino acids on modified γ-cyclodextrin stationary phase

Journal of Chromatography A, 1411 (2015) 101–109

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Gas chromatographic separation of stereoisomers of non-protein amino acids on modified ␥-cyclodextrin stationary phase Stefan Fox a , Henry Strasdeit a , Stephan Haasmann b , Hans Brückner b,∗ a

Institute of Chemistry, Department of Bioinorganic Chemistry, University of Hohenheim, D-70599 Stuttgart, Germany Interdisciplinary Research Centre for BioSystems, Land Use and Nutrition (IFZ), Department of Food Sciences, Institute of Nutritional Science, University of Giessen, Heinrich-Buff-Ring 26-32, D-65392 Giessen, Germany b

a r t i c l e

i n f o

Article history: Received 2 March 2015 Received in revised form 20 July 2015 Accepted 22 July 2015 Available online 26 July 2015 Keywords: Amino acid enantiomers Chirality ␣,␣-Dialkyl amino acids Meteoritic amino acids Octakis(3-O-butyryl-2,6-di-O-pentyl)-␥cyclodextrin

a b s t r a c t Stereoisomers (enantiomers and diastereoisomers) of synthetic, non-protein amino acids comprising ␣-, ␤-, and ␥-amino acids, including ␣,␣-dialkyl amino acids, were converted into the respective Ntrifluoroacetyl-O-methyl esters and analyzed and resolved by gas chromatography (GC) on a commercial fused silica capillary column coated with the chiral stationary phase octakis(3-O-butyryl-2,6-di-Opentyl)-␥-cyclodextrin. This column is marketed under the trade name Lipodex® E. Chromatograms, retention times, and a chart displaying the retention times of approximately 40 stereoisomers of amino acids are presented. With few exceptions, baseline or almost baseline resolution was achieved for enantiomers and diastereoisomers. The chromatographic method presented is considered to be highly suitable for the elucidation of the stereochemistry of non-protein amino acids, for example in natural products, and for evaluating the enantiopurity of genetically non-coded amino acids used for the synthesis and design of conformationally tailored peptides. The method is applicable to extraterrestrial materials or can be used in experimental work related to abiotic syntheses or enantioselective destruction and amplification of amino acids. © 2015 Elsevier B.V. All rights reserved.

1. Introduction If used without further definitions, the term “amino acid” is often understood as synonymous with alkanoic acid having in the ␣- or 2-position an amino group, a hydrogen atom, and an alkyl or alkaryl group (that might be further substituted). The 20 common amino acids occurring in ribosomally synthesized proteins are encoded by triplets of nucleic acid bases (the “code of life”) and are, therefore, named coded or canonical amino acids or, more commonly, proteinogenic or protein amino acids. As a result of the amino group being able to occur in the ␣-position as well as the ␤- or ␥-position in carboxylic acids, a large number of natural ␤and ␥-amino acids are known. Higher homologs are denoted ␦-, ␧-, ␻-amino acids, etc. Use of such unusual or non-protein amino acids for peptide drug design, peptide based materials, and protein engineering are established research topics [1,2]. An estimated number of approximately 500 natural amino acids occurring in the free or conjugated form in microorganisms, animals, and plants were already known in 1983 [3], and about 750 non-protein amino

∗ Corresponding author. Tel.: +49 711 349919; fax: +49 641 9939149. E-mail address: [email protected] (H. Brückner). http://dx.doi.org/10.1016/j.chroma.2015.07.082 0021-9673/© 2015 Elsevier B.V. All rights reserved.

acids are listed in an monograph chapter by Hunt [4]. However, the number of possible synthetic amino acids is almost limitless. The general structures of the synthetic ␣-, ␤-, and ␥-amino acids analyzed in this work are presented in Fig. 1. Owing to the presence of one or more stereogenic centers, these compounds occur as mirror images (enantiomers) or mixtures of enantiomers and diastereoisomers. As shown in the figure, substitution of the C␣ –H-atom in chiral ␣-aminoalkanoic acids by alkyl groups leads to the formation of ␣,␣-dialkyl amino acids, also referred to in the literature as ␣-alkyl amino acids, ␣-alkyl-␣-amino acids or C␣,␣ -dialkylglycines [5–7]. The simplest members of this group are achiral ␣-aminoisobutyric acid (2-amino-2-methylpropanoic acid, Aib) and chiral isovaline (2-amino-2-methylbutanoic acid or 2-ethylalanine, Iva). Aib and Iva, besides further non-protein and protein amino acids, are principal constituents of a fairly common group of fungal peptides produced by many genera of filamentous fungi. For these peptides the acronym peptaibiotics became established defined as peptides containing Aib and exerting (anti)biotic activities [5,8]. As of May 2015, the number of peptaibiotics compiled in the “Peptaibiotics Database” already exceeded 1300 [9], and many more are expected to be discovered in the near future [5]. If present in these peptides, Iva occurs as d- or l-enantiomer, or even as both enantiomers in the same molecule (e.g. in peptaibiotics neoefrapeptins and integramides) [10,11].

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S. Fox et al. / J. Chromatogr. A 1411 (2015) 101–109

Fig. 1. Amino acid structural types investigated in the present study: (A) ␣-Amino acids (proteinogenic; R1 = H; R2 = alkyl, carboxyalkyl), ␣,␣-dialkyl ␣-amino acids (R1 = CH3 , C2 H5 ; R2 = alkyl, carboxyalkyl, CH2 OH, (CH2 )2 SCH3 , (CH2 )3 NH2 , CH2 C6 H5 ); (B) ␤-Amino acids (R1–4 = H, CH3 , C2 H5 ); (C) ␥-Amino acids (R1–3 = H, CH3 ); for a ␦-amino acid see Table 1.

Almost 90 different abiotically synthesized amino acids have been detected in carbonaceous meteorites and are compiled, for example, in [12]. Depending on the class of carbonaceous meteorite, these “meteoritic” amino acids occur in varying number, proportions, and concentrations. But only in a minority of cases have they been characterized unambiguously with respect to stereochemistry and enantiomeric ratio. For an overview and discussion on amino acid enantiomers in meteorites, see for example [13–16]. Among the ␣,␣-dialkyl amino acids in meteorites, Aib and Iva are the most abundant. The quantities of the others decrease with increasing alkyl chain length, chain branching, and number of Catoms [17]. This indicates, together with the presence of more or less racemic amino acids, abiotic synthesis in the extraterrestrial environment [17,18]. Subsequent chiral discrimination as result of aqueous alteration [19] or physico-chemical processes [12,14,20] have been postulated. In carbonaceous meteorites, Iva shows varying enantiomeric excesses mostly of the l-form but is also present as racemate, depending on the specimen and meteorite type analyzed [15,16,18,19]. The determination of the stereochemistry of amino acids occurring in natural, extraterrestrial or synthetic materials requires effective methods for the separation and characterization of enantiomers and epimers or diastereoisomers (if several stereogenic centers are present). For the direct separation of derivatized and thus volatile amino acid enantiomers, gas chromatography (GC) employing capillary columns coated with chiral stationary phases is among the most effective methods. The most frequently used commercially available columns for the complete separation of mixtures of protein l-amino acids and their corresponding d-enantiomers by GC are wall-coated opentubular (WCOT) fused silica (FS) capillary columns marketed under the name Chirasil-l-(or d-)Val. The chiral selector is a copolymer of poly(dimethylsiloxane) together with (2-carboxypropyl)siloxane functionalized with l-(or d-)valine tert-butylamide [21]. This phase was also used for the separation of Iva enantiomers and the assignment of the configuration in polypeptide antibiotics [22]. Capillary columns coated with various modified ␤- and ␥cyclodextrins also enable the gas chromatographic enantiomer separation of many classes of compounds including amino acids [23–26]. In particular, columns coated with octakis(3-O-butyryl2,6-di-O-pentyl)-␥-cyclodextrin [27] provide excellent resolution of enantiomeric pairs of dl-amino acids and multicomponent mixtures in standards [28–31] as well as in microbial metabolites and foodstuffs [32]. Lipodex E, which belongs to this type of column, enables also the separation of stereoisomers of N-perfluoroacyl dipeptide esters [33]. The efficiency of this phase was further improved by chemical binding to poly(dimethylsiloxane) via a spacer group [29,30]. The resulting chiral phase, dubbed Chirasil␥-Dex, is currently not commercially available. Various modified ␤- and ␥-cyclodextrins together with various volatile derivatives were found to be complementary to Chirasil-lVal for the enantioseparation of dl-amino acids as briefly outlined in the context of this work in the following:

Vandenabeele-Trambouze et al. [34] analyzed standard mixtures of protein dl-amino acids to which Aib and dl-Iva had been added and compared the separation of the TFA-O-2-propyl esters on a commercial FS Lipodex E column and a Chirasil-l-Val column. dl-Iva was well resolved on the former column but not on the latter. Other non-protein amino acids were not investigated and some of the protein dl-amino acids were not completely separated. Pizzarello et al. [18,35] used 25-m Chiradex CB columns (representing heptakis(2,3,6-tri-O-methyl)-␤-cyclodextrin bonded to poly(dimethylsiloxane) and a 50-m Lipodex E column for the separation of dl-Iva and some other meteoritic non-protein amino acids which were unsatisfactorily resolved as TFA-O-2-propyl esters on a 50-m Chirasil-l-Val column. Zampolli et al. [36] analyzed standard mixtures of protein dl-amino acids as various (N,O,S)-perfluoroacyl-O-perfluoroalkyl esters comparatively on Chirasil-l-Val and Rt-G-DEXsa (representing 2,3-di-acetoxy-6-O-tert-butyl dimethylsilyl-␥-cyclodextrin doped into 14% cyanopropylphenyl/86% dimethyl polysiloxane). The separations achieved were only partially satisfactory. Furthermore, the formation of by-products and racemization phenomena were observed under the derivatization conditions used. Non-protein amino acids were not analyzed. Freissinet et al. [37] derivatized a standard mixture of protein dl-amino acid using N,N-dimethylformamide dimethyl acetale and separated the volatile enantiomers on a CP-Chirasil-DEX CB capillary column (representing heptakis(2,3,6-tri-O-methyl)␤-cylclodextrin as chiral selector). Ten out of 19 enantiomeric pairs of amino acids could be resolved, but 5 amino acids were partly racemized under the derivatization conditions used. No nonprotein amino acids were analyzed, and the allo-forms of Ile and Thr were also not considered. Meinert and Meierhenrich [6] resolved N-ethoxycarbonyl-Oheptafluorobutyryl esters of dl-Iva and some other ␣-methylated and non-protein amino acids by 2D-GC on a 50-m Chirasil-l-Val column coupled to a short DB Wax (polyethylenglycol) secondary column. FS capillary columns coated with Chirasil-l-Val, Chirasil-Dex-CB and Cyclodextrin G-TA (representing octakis(2,6-di-O-pentyl-3O-trifluoroacetyl)-␥-cyclodextrin), together with five non-chiral capillary columns, were installed in the Philae Lander of the Rosetta Mission [14,38]. The various derivatization approaches and capillary columns installed on space probes for in situ chiral discrimination of organic molecules are compiled and critically discussed by Pietrogrande and Basaglia [39], and Poinot and Geffroy-Rodier [40]. König and co-workers used their own, self-made, deactivated Pyrex glass capillary columns of 50 m length, coated with the undiluted chiral stationary phase for the separation of various kinds of amino acids, including ␣-dialkylated amino acids, and showed that this phase is complementary to or surpasses in terms of versatility the performance of Chirasil-Val columns [27,28]. Nowadays, fragile glass capillary columns are rarely used and are replaced by flexible FS capillary columns surface-coated with a polyimide.

S. Fox et al. / J. Chromatogr. A 1411 (2015) 101–109

To summarize, none of the various derivatization procedures or modified cyclodextrin columns used for GC provided overall resolution of common protein dl-amino acids as well as non-protein amino acids as reported for TFA-O-alkyl esters on the modified ␥cyclodextrin selector Lipodex E, employed either in undiluted [27], silicone oil blended (e.g. OV-1701) [29,30], or chemically bonded form [30]. Since limited information is available on the separation of amino acids using FS capillary columns marketed under the trade name Lipodex® E, here we report on the separation of more than 40 non-protein amino acids as TFA-O-methyl esters using this column.

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2. Experimental 2.1. Gas chromatography GC was carried out using a Model HRGC 5160 chromatograph with flame-ionization (FID) detector (Carlo Erba, Rodano, Italy) equipped with a C-R3A integrator (Shimadzu, Kyoto, Japan). The column used was a 25 m × 0.25 mm i.d. column coated with a solution of octakis(3-O-butyryl-2,6-di-O-pentyl)-␥-cyclodextrin in modified polysiloxane similar to OV-1701 with a film thickness of 0.25 ␮m (commercially available as Lipodex® E from Macherey-Nagel, Düren, Germany). Hydrogen (99.999% purity,

Table 1 GC analysis of N-trifluoroacetyl-O-methyl esters of stereoisomers of non-protein ␣-, ␤-, ␥-, and ␦-amino acids and ␣,␣-dialkyl ␣-amino acids (A) on Lipodex® E. See Section 2 for the temperature program used. No.

Class

Amino acid

1a

A

2a 3

␤ ␤

4 5a 6

␤ ␤ ␣

2-Amino-2-methylpropanoic acid (␣-Aminoisobutyric acid, Aib) 3-Aminopropanoic acid 3-Amino-2-methylpropanoic acid (␤-Aminoisobutyric acid, ␤-Aib) 3-Amino-2-ethylpropanoic acid 3-Amino-2,2-dimethylpropanoic acid 2-Aminobutanoic acid (␣-Aminobutyric acid, Abu or Aba) 2-Amino-2-methylbutanoic acid (Iva)

Net retention time of derivatives (min) t1

7 (1b ) b

A

8 (8 )

A

9a 10

A A

11

␣

12 13 14a 15a

␤ ␤ ␤ ␥

16 17 18 19 (2b )

␥ ␥ ␣ A

20 (9b )

A

21 (7b )

A

22

␣

23 24 25 26 27 28 29 30a 31 32

A ␣ ␣ ␣ ␣ ␤ ␥ ␦ ␣ ␣

33 (3b ) 34 35 36 37

A ␣ ␣ ␣ ␣

2-Amino-2-methylbutanedioic acid (␣-Me-Asp) 2-Amino-2-ethylbutanoic acid (diethylglycine) 2-Amino-2,3-dimethylbutanoic acid (␣-Me-Val) 2-Amino-3-methylbutanedioic acid (␤-Me-Asp) 3-Aminobutanoic acid 3-Amino-2-methylbutanoic acid 3-Amino-3-methylbutanoic acid 4-Aminobutanoic acid (␥-Aminobutyric acid, GABA) 4-Amino-2-methylbutanoic acid 4-Amino-3-methylbutanoic acid 2-Aminopentanoic acid (Nva) 2-Amino-2-methylpentanoic acid (␣-Me-Nva) 2-Amino-2-methylpentanedioic acid (␣-Me-Glu) 2-Amino-2,4-dimethylpentanoic acid (␣-Me-Leu) 2-Amino-3-methylpentanoic acid (dl-Ile + allo-dl-Ile) 2-Amino-2-ethylpentanoic acid 2-Amino-3-ethylpentanoic acid 2-Amino-3,3-dimethylpentanoic acid 2-Amino-3,4-dimethylpentanoic acid 2-Amino-4,4-dimethylpentanoic acid 3-Aminopentanoic acid 4-Aminopentanoic acid 5-Aminopentanoic acid 2-Aminohexanoic acid (Nle) 2-Aminohexanedioic acid (2-Aminoadipic acid, Ada) 2-Amino-2-methylhexanoic acid 2-Amino-3-methylhexanoic acid 2-Amino-5-methylhexanoic acid 2-Aminoheptanoic acid 2-Aminoheptanedioic acid (2-Aminopimelic acid)

t2

t3

t4

13.17 23.38 21.50 29.64 21.13 17.18 (R) 11.72 (S) 22.41

21.83 29.92 18.92 (S) 15.81 (R) 22.85

9.67 17.51

19.20

24.52

25.00

25.55

25.56

22.79 21.55 21.40 27.03

23.46 21.77 ns

22.56

22.96

26.41 26.10 18.88 14.59

26.52 ns 20.84 15.38

25.60

ns

12.62

13.20

21.39 (2R,3S) 11.71 19.31 18.19 19.20 21.05 23.13 25.71 28.81 20.45 28.86

21.60 (2R,3R) ns 20.88 19.80 20.01 21.60 23.41 26.00

21.91 (2S,3R)

21.99 (2S,3S)

20.32

20.60

17.17 19.79 21.18 22.56 30.60

17.32 20.43 21.54 22.89 ns

21.26 28.96

21.33

Numbers (No.) correspond to selected chromatograms shown in Fig. 3. Superscript ‘a’ on No. refers to achiral compounds (not shown in Fig. 3). Superscript ‘b’: No. in Table 2 and Fig. 3. A = ␣,␣-dialkyl ␣-amino acid, ␣ = ␣-amino acid, ␤ = ␤-amino acid, ␥ = ␥-amino acid, ␦ = ␦-amino acid. Some common or frequently used names and abbreviations are given in parenthesis. Included in the table are net retention times of achiral amino acids and stereoisomers that are not separated (ns). Assignment of the elution order of No. 6, 7, and 22 according to own measurements. For a chromatogram of no. 7 see no. 1 in Fig. 4. For a chart of amino acid stereoisomers and corresponding retention times see Fig. 5. d-Iva = (R)-Iva, l-Iva = (S)-Iva; d-Ile = (2R,3R), l-Ile = (2S,3S), d-allo-Ile = (2R,3S)-Ile, l-allo-Ile = (2S,3R)-Ile.

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Table 2 Separation of racemic ␣-methyl ␣-amino acids (as TFA-O-methyl esters) on Lipodex® E using isothermal conditions. No.

dl-Amino acid

t1

t2

1 2 3 4 5 6 7 8 9 10 11 12 13

2-Amino-2-methylbutanoic acid (Iva) 2-Amino-2-methylpentanoic acid 2-Amino-2-methylhexanoic acid 2-Amino-2-methylheptanoic acid 2-Amino-2-methyloctanoic acid 2-Amino-2,3-dimethylbutanoic acid (␣-Me-Val) 2-Amino-2,4-dimethylpentanoic acid (␣-Me-Leu) 2-Amino-2-methylbutane-2,4-dioic acid (␣-Me-Asp) 2-Amino-2-methylpentane-2,5-dioic acid (␣-Me-Glu) 2-Amino-2-methyl-3-hydroxypropanoic acid (␣-Me-Ser) 2-Amino-2-methyl-4-methylmercaptobutanoic acid (␣-Me-Met) 2,5-Diamino-2-methylpentanoic acid (␣-Me-Orn) 2-Amino-2-methyl-3-phenyl-propanoic acid (␣-Me-Phe)

6.08 (S) 7.93 11.35 10.58 11.65 4.14 7.37 5.40 10.01 5.40 (S) 15.78 12.40 9.05

7.64 (R) 8.50 11.66 ns ns 4.93 7.59 5.84 ns 5.84 (R) 16.15 12.68 9.17

T (◦ C) 90 90 90 100 110 105 90 120 125 130 125 165 95

˛ 1.26 1.07 1.03 1.00 1.00 1.19 1.03 1.08 1.00 1.08 1.02 1.02 1.01

t1,2 = net retention time (min) of first (t1 ) and second (t2 ) eluted enantiomer; separation factor ˛ = t2 /t1 , determined at isothermal temperature T; ns = not separated; numbers (No.) correspond to chromatograms in Fig. 2; assignment of the elution order of no. 1 and no. 10 according to [27,28], no. 1 confirmed by own measurements.

Messerschmidt-Griesheim, Germany) was used as carrier gas at a pressure of 0.6 bar (60 MPa). The temperature program applied for the amino acids listed in Table 1 was 5 min at 70 ◦ C, ramped at 2.5 ◦ C min−1 to 100 ◦ C, then 3.5 ◦ C min−1 to 190 ◦ C, then 5 min at 190 ◦ C, followed by a cooling phase down to 70 ◦ C. This temperature program was selected in order to be applicable to the separation of amino acid stereoisomers expected or known to occur in meteorites. The amino acids compiled in Table 2 were analyzed using isothermal conditions as indicated in order to achieve optimal resolution and to calculate the separation factors.

amino acid was added to 200 ␮l of HCl in MeOH (prepared by mixing MeOH and AcCl 8:2, v/v) [41]. The reaction mixture was subjected to 110 ◦ C for 1 h. The derivatization chemistry is presented in Fig. 2. The mixture was then evaporated to dryness under a stream of nitrogen. Subsequently, 200 ␮l of DCM and 50 ␮l of TFAA were added, and the mixture was heated for 20 min at 100 ◦ C. The solution was evaporated to dryness under a stream of nitrogen at room temperature. The residue was dissolved in 200 ␮l of DCM, and 2-␮l aliquots were injected manually in the port of the GC in the split mode (split ratio ∼1:20). The experiments were performed over a prolonged period of time.

2.2. Sources and abbreviations of amino acids and reagents The amino acids analyzed represent diastereomeric and/or racemic mixtures. Common protein amino acids and the corresponding C␣ -alkylated derivatives are abbreviated according to the three-letter nomenclature; rational names and special abbreviations are displayed in the tables. Donation of a series of racemic aminoalkanoic acids to H.B. by J.R. Cronin (Arizona State University, USA) is acknowledged. Origin and characterization of these amino acids have been reported in detail [17]. The late E. Gil-Av (Rehovot, Israel) provided racemic ␣-Me-Val, ␣-Me-Nva, and ␣-Me-Nle, and P.M. Hardy (Exceter, UK) diethylglycine. C␣ -methylated protein amino acids and dl-Ile + allo-dl-Ile were purchased from Sigma–Aldrich (Deisenhofen, Germany) [7]. The homologous series of racemic 2-amino-2-methylbutanoic acid to 2-amino-2-methyloctanoic acid was synthesized in our laboratory according to the Strecker procedure; l-Iva was purchased from Acros Chemicals (Geel, Belgium). Methanol (MeOH), dichloromethane (DCM), and the derivatizing reagents acetyl chloride (AcCl) and trifluoroacetic acid anhydride (TFAA) were of analytical grade and were purchased from Merck (Darmstadt, Germany). 2.3. Derivatization procedures Amino acids were converted into the corresponding Ntrifluoroacetyl-O-methyl esters as follows. Approximately 1 mg of

3. Results and discussion The rational names of the amino acids, together with the amino acid types (␣-, ␤-, ␥-, and ␣,␣-dialkyl amino acids) and the elution times of their derivatives from the Lipodex E column under the conditions described in Section 2, are compiled in Tables 1 and 2. Focus was put on the resolution power of Lipodex E for a broad range of non-protein amino acids. Owing to the lack of homochiral standards in most cases, assignment of the elution order of stereoisomers is confined to a rather limited number of amino acids. The amino acids in Table 1 are listed according to the principle alkanoic acid, ranging from propanoic to heptanoic acid, in which the positions of the amino group and alkyl groups are indicated. This allows the rational and unambiguous assignment of the compounds analyzed and correlation with the structures and chromatograms displayed in Fig. 3. However, owing to the frequent use of trivial names (for a selection see [12]), a few common names such as Aib, Iva, Nva, and Nle for ␣-aminoisobutyric acid, isovaline, norvaline, and norleucine, respectively, are included in the tables. Because of the importance of synthetic C␣ -methylated protein amino acids for peptide drug design, the respective acronyms are added in parenthesis as ␣-Me-Aaa (Aaa = protein amino acid). The chromatograms obtained from GC-FID measurements of the TFA-amino acid-Omethyl esters are displayed in Fig. 3 together with the chemical structures of the amino acids. Racemic amino acids that have one chiral center show two peaks for the corresponding enantiomers.

Fig. 2. Reaction steps of the derivatization procedure exemplified by dl-isovaline: (A) Esterification; the MeOH/HCl mixture was produced in situ from acetyl chloride and an excess of methanol (see Section 2); (B) N-trifluoroacetylation. TFAA = trifluoroacetic anhydride, TFA = trifluoroacetic acid.

S. Fox et al. / J. Chromatogr. A 1411 (2015) 101–109

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Fig. 3. Chromatograms (GC-FID) and underlying chemical structures of amino acids analyzed on Lipodex E as N-TFA-O-methyl esters using increasing temperature gradient conditions. Numbers correspond to numbers in Table 1.

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Fig. 3. (Continued ).

Amino acids possessing two chiral centers yield 4 peaks resulting from two pairs of enantiomers, provided that all stereoisomers are resolved. Chromatograms of achiral amino acids, which elute as single peaks from the column, are not shown, but their retention times are included in Table 1. The series of 2-amino-2-methylalkanoic acids (formerly also named 2-ethylalanine to 2-octylalanine) [7] and some other selected alkylated ␣-amino acids derived from protein amino acids were analyzed under isothermal conditions at relatively low temperatures, enabling higher separation factors (˛) [30]. Data are compiled in Table 2, and chromatograms of the derivatives are presented in Fig. 4. With increasing chain length in the series of 2amino-2-methylalkanoic acids, a decrease of resolution is observed. Whereas the enantiomers of 2-amino-2-methylbutanoic acid (Iva) and 2-amino-2-methylpentanoic acid are baseline resolved and 2-amino-2-methylhexanoic acid is satisfactorily resolved, the enantioseparation of 2-amino-2-methylheptanoic acid and 2-amino-2-methyloctanoic could not be achieved under these chromatographic conditions. Among the C␣ -methylated protein amino acids, ␣-Me-Ser and ␣-Me-Val are baseline resolved, while ␣-Me-Orn and ␣-Me-Leu show acceptable resolution. Remarkably, enantiomers of ␣-Me-Asp are baseline resolved, but those of ␣-MeGlu are not. The enantiomers of ␣-Me-Met and ␣-Me-Phe display partial separation. The broad eluting peaks are the result of the low temperature employed to achieve separation. Owing to the importance of non-protein and ␣,␣-dialkyl amino acids in meteorites, a chart displaying retention times of the amino acids analyzed is presented in Fig. 5. It allows identifying possible interferences at a glance under the temperature program applied. From Lipodex E, the d(R)-enantiomers of TFA-O-methyl esters of common protein amino acids are eluted prior to the corresponding l-enantiomers (imino acids show the reversed elution order

[27]). However, in the case of the C␣ -methylated amino acids ␣-MeSer and 2-amino-2-methylbutanoic acid (Iva), the l(S)-enantiomers elute prior to the d(R)-enantiomers (see Tables 1 and 2). Thus, the significance of the structural aspects of the analytes with respect to the mechanisms and forces involved in their separation is difficult to assess. However, evaluation of the resolution of homologous series or pairs of amino acids provides some insight into the mechanisms of separation involved. In the series of 2-amino-2-methylbutanoic acid to 2-amino2-methylhexanoic acid, a decrease of the resolution and a lack of resolution of 2-amino-2-methylheptanoic acid and 2-amino-2methyloctanoic acid are observed (see Table 2 and Fig. 3). This indicates that the increasing size and flexibility of the C␣ -alkyl side chain together with increasing hydrophobic interactions surpass the influence of the C␣ -methyl group. This is also observed by comparison of ␣-Me-Glu (not resolved) and ␣-Me-Asp (baseline resolve), distinguished only by an additional methylene group of the former. Analogously, ␣-Me-Val shows better resolution in comparison to the homologous ␣-Me-Leu. We have also studied the series of chiral aminopentanoic acids. In this series, the shift of the amino group from the C-2 atom to C3, and concomitant shift of the stereogenic center, results in lower resolution of the 3-aminopentanoic acid in comparison to the 2aminopentanoic acid. Further shift of the amino group from the 3to the 4-position, however, does not impede the baseline resolution of enantiomers. This indicates that the stereogenic effects of the Catom and the conformational effects of the methyl side chains of the ␤- and ␥-amino acid are counterbalanced. As a general rule with regard to Lipodex E and for TFA-O-methyl esters, it can be said that substitution of the C␣ -hydrogen in ␣amino acids by a homologous series of alkyl groups results in decreasing resolution. In contrast, the shift of the methyl group

S. Fox et al. / J. Chromatogr. A 1411 (2015) 101–109

Fig. 4. Chromatograms (GC-FID) of ␣-methyl ␣-amino acids analyzed on Lipodex E as N-TFA-O-methyl esters using isothermal conditions. Numbers correspond to numbers in Table 2.

107

Fig. 5. Chart of the elution times of stereoisomers of amino acids compiled in Table 1 using the temperature program described in Section 2.

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from the ␣-position to the ␤- or ␥-position provides better resolution. Further, the shift of the amino group from the 2- to the 3or 4-position has little effect on the enantioseparation. Mixtures of enantiomers and diastereoisomers are mostly well resolved; only 2-amino-3-methylhexanoic acid shows co-elution of 2 of its 4 isomers. Taking all structural variabilities into account, it is difficult to evaluate a general resolution mechanism and to predict the elution order of enantiomers, even for homologous series. The definitive assignment of the individual peaks depends on the availability of standards of defined stereochemistry. The separation mechanism for enantiomers of amino acids on chiral stationary phases that are based on functionalized amino acids, such as Chirasil-Val, is mainly based on hydrogen-bridged intermediate diastereomeric associates. In modified cyclodextrin stationary phases, inclusion of the analytes in the chiral cavity, formation of diastereomeric complexes, hydrophobic and dipole–dipole interactions, and van der Waals forces, varying in their relevance depending on the chosen derivatization of the amino acid, play a role. Furthermore, techniques of coating the capillary column and dilution of the chiral stationary phase with modified achiral polysiloxanes, in combination with temperature and carrier gas, influence the resolution of derivatives and might lead to a reversal of the elution order of enantiomers even within a homologous series [42]. 4. Conclusions and perspectives From our study on the gas-chromatographic separation of nonprotein amino acid stereoisomers, the following conclusions have been drawn: a) The commercial Lipodex E column enables the enantiomeric and diastereomeric separation of approximately 40 non-protein amino acids, resulting in baseline separation of the derivatives in the majority of cases. b) In particular, the direct enantioresolution of derivatized ␣,␣dialkyl amino acids, which is difficult to achieve by GC on standard FS Chirasil-Val columns, is possible with baseline separation for most enantiomers. c) A graph displaying retention times of stereoisomers of nonprotein amino acids, with emphasis on “meteoritic” amino acids, immediately indicates possible interferences in the chromatogram (Fig. 5). d) The generally excellent separation of stereoisomers of amino acids makes Lipodex E well suited for the precise determination of the enantioratio of these compounds in natural or synthetic peptides as well as meteorites or other extraterrestrial materials. Lipodex E is also considered to be suitable for the analysis of amino acids resulting from, for example, simulated prebiotic [43–45] and astrochemical/astrophysical experiments related to enantioselective discrimination or amplification [12,14,46,47]. Columns containing this stationary phase are also recommended to be installed in advanced GC–MS payloads of future space missions with the objective of searching for extraterrestrial chiral amino acids [40] or even small peptides [33,48]. References [1] P. Balaram, Non-standard amino acids in peptide design and protein engineering, Curr. Opin. Struct. Biol. 2 (1992) 845–851. [2] D. Seebach, A.K. Beck, D.J. Bierbaum, The world of ␤- and ␥-peptides comprised of homologated proteinogenic amino acids and other components, Chem. Biodivers. 1 (2004) 1111–1239. [3] Wagner, H. Musso, New naturally occurring amino acids, Angew. Chem. Int. Ed. 22 (1983) 816–828. [4] S. Hunt, The non-protein amino acids, in: G.C. Barret (Ed.), Chemistry and Biochemistry of the Amino Acids, Chapman and Hall, London, 1985, pp. 55–138.

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