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Gas-liquid chromatography of N- and O-acylated neuraminic acids
ANALYTICAL
BIOCHEMISTRY
Gas-Liquid
65,
507-514
(1975)
Chromatography
of N-
Neuraminic JORGE
and
0-Acylated
Acids
CASALS-STENZEL, HANS-PETER AND ROLAND SCHAUER
BUSCHER.
Received July 19. 1974: accepted October 30. 1974 This paper is dedicated to Prof. Dr. G. Weitzel to mark the occasion of his 60th birthday. The qualitative and quantitative analysis by gas-liquid chromatography of natural and synthetic sialic acids with special reference to O-acylated sialic acids is described. Sialic acids are derivatized by N-trimethylsilylimidazol. All hydroxyl groups including the carboxyl group are t~methylsiiylated as was shown by mass spectrometry. No elimination of the O-acyl groups occurs during the silylation reaction. The TMS-derivatives of the sialic acids are stable in the refrigerator. The TMS-sialic acids including the isomeric ~-acyl-~-acylneu~minic acids can be separated from each other on OV- 17 or OV-22. Single peaks having individual retention indices from the following sialic acids were obtained: N-acetylneuraminic acid. N-glycolylneuraminic acid, N-Auoroacetylneuraminic acid and N-chloroacetylneuraminic acid; neuraminic acid+methylglycoside or its methylester and N-bromoacetylneuraminic acid-fi-methylglycoside: N-acetyl4-O-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid and N-acetyl9-O-acetylneuraminic acid: N-acetyl-7.9-di-O-acetylneuraminic acid: N-acetyl4-~-glycolylneu~~minic acid, ~~-acetyl-9(?~-U-~~yco~ylneuraminic acid and .~-acet}~l-7(?~-~-glycolylneuraminic acid; ~-~lycolyi-4-~-acetylneuraminic acid, ~~-glycolyl-9-~-acetylneuraminic acid and ~~-glycolyl-7(?)-~-acetylneuraminic acid. On OV-1 N-acetylneuraminic acid and N-glycolylneuraminic acid can be separated from each other and from N-acyl-0-acylneuraminic acids: however. the Oacylated sialic acids elute essentially together from this material. The relative detector responses for the different sialic acids vary, as was shown for N-acetylneuraminic acid, N-glycolylneuraminic acid and N-acetyl-9-O-acetylneuraminic acid. 35% of the radioactivity from radioactive N-acetylneuraminic acid and h’-glycolylneuraminic acid eluted correspondingly with the sialic acid peaks detected by the FID. Gas-liquid chromatography enables a sensitive qualitative and quantitative analysis of different sialic acids occurring in biological materials, produced during chemical synthesis or in enzymic assays.
The increasing interest in the structure and biological functions of sialic acids as components of glycoproteins and glycolipids requires methods for a more accurate identification of the different sialic acids. In 507 Copyright @I 1975 by Academic Press, Inc. Ail rights of reproduction in any form reserved.
508
CASALS-STENZEL,
BUSCHER
AND
SCHAUER
mammals, at least twelve different sialic acids have been found which are acetylated or glycolylated at the amino group only or at both N- and O-positions (1,2). Not all of these N-acetyl-U-acetylneuraminic acids, N-acetyl-0-glycolylneuraminic acids and N-glycolyl-0-acetylneuraminic acids can be distinguished by the usual analytical methods, e.g., by thinlayer chromatography or by calorimetric methods (1). Gas-liquid chromatography is assumed to be a more sensitive tool to distinguish sialic acids which are different with regard to the nature and positions of 0-acyl groups. The qualitative and quantitative determination of N-acetylneuraminic acid and N-glycolylneuraminic acid by glc has already been carried out with success, either by trimethylsilylation of the native N-acylneuraminic acids (3) or of the corresponding p-methylketosides (4-6). Conversion of 0-acylated sialic acids to their p-methylketosides by methanolysis before trimethylsilylation, however, causes an appreciable loss of the 0-acyl groups (6); therefore, the analysis of N-acyl-Oacylneuraminic acids by glc has not yet been achieved satisfactorily. This paper describes the analysis of different sialic acids including the 0-acylated sialic acids by glc. The direct trimethylsilylation method of different natural or synthetic sialic acids avoiding preceding conversion to the corresponding p-methylketosides leads to derivatives which are distinguishable by glc. This enables the qualitative and in part also the quantitative analysis of sialic acids, either individually or in mixtures. MATERIALS
AND
METHODS
Reagents The silylation reagents trimethylchlorsilane (TMCS), hexamethyldisilazane (HMDS), N,O-bis-trimethylsilylacetamide (BSA), bis-trimethylsilyl-trifluoroacetamide (BSTFA) and N-trimethylsilyl-diethylamine (TMSDEA) were purchased from Applied Science Laboratories Inc., USA. N-trimethylsilylimidazole (TSIM) was obtained from EGAChemie KG, GFR. Sucrose and trehalose obtained from Merck AG, GFR, were used as internal standards. Insta-Gel emulsifier from Packard was used for scintillation counting. All compounds and solvents used were of analytical grade. glc Materials and Apparutus The stationary phases OV-1, OV-17, OV-22, GE-SE-54 and Apiezon L and the supports Gas Chrom Q, 60-80, 80-l 00 and 100-l 20 mesh were obtained from Applied Science Laboratories Inc. Anachrom SD, 80-100 mesh was purchased from Analabs Inc., USA. Pretested 3% OV-17 on Gas Chrom Q, 100-l 20 mesh, was obtained from Applied Science Laboratories Inc. The other column packing materials were prepared using the technique reported by Williams (7), i.e., 1% OV-1 on
GAS-LIQUID
CHROMATOGRAPHY
OF
SIALIC
ACIDS
509
Anachrom SD, 3% OV-17 on Gas Chrom Q, 3% OV-22 on Anachrom SD, and 3% GE-SE-54 on Chromosorb W. Hewlett-Packard glc apparatus Models 402 and 5750 were equipped with flame ionisation detectors (FID) and combined with a Kent integrator Model Chromalog 2. In the Model 402 U-shaped glass columns of 0.8-1.6 m length and 2 or 4 mm internal diameters (i.d.) were used. In the Model 5750 metal columns of max length 2.5 m and 2 mm i.d. were adapted. Operuting
Conditions
Routine analyses were run isothermally. (The results were not fundamentally influenced by application of temperature programs.) The oven temperatures ranged between 170” and 220°C depending on the length of the column and the nature of the stationary phase. The injection port was heated to 40°C and the detector to 50-60°C over the column temperature. The flow rates were 50 ml/min for N, (carrier gas): 30 ml/min for H, . and 300 ml/min for air. For routine analyses (including those of all figures and tables) of sialic acids glass columns were used containing 3% OV-17 on Gas Chrom Q, loo-120 mesh, 1.6 m X 2 mm i.d. and heated to 196°C. Preparation
and IdentiJcution
of Sialic Acids
Sialic acids were released by neuraminidase or by mild acid hydrolysis from glycoproteins of bovine, equine or porcine submandibular glands or from equine erythrocyte membranes, purified by ion-exchange chromatography, and fractionated by passage through a cellulose column using n-butanolln-propanol/water (1 : 2 : 1; by volume) as solvent (1,2,8). They were determined quantitatively by the periodic acid/thiobarbituric acid assay (9) and by the orcinol/Fe3+/hydrochloric acid reagent (10). The purity of the sialic acids eluting from cellulose was checked by tic in different systems (8). The sialic acids were analysed routinely on cellulose thin layers using n-butanolln-propanol/O. 1 N HCl (1: 2: 1; by volume) as solvent (1 1). Their structures were established by analysis of the nature of the N-acyl groups and of the amount, nature and position of the 0-acyl groups as described in detail in Refs. 2 and 8. In addition, mass spectrometry was used for structural analyses of the sialic acids (12). Some of the mass spectral analyses were in combination with glc (13). The following sialic acids were obtained in more than 95% purity (2): N-acetylneuraminic acid, N-glycolylneuraminic acid, N-acetyl-4-O-acetylneuraminic acid, N-acetyl-9-0-acetylneuraminic acid, N-acetyl-7,9di-0-acetylneuraminic acid, N-glycolyl-4-0-acetylneuraminic acid and
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AND
SCHAUER
N-glycolyl-9-U-acetylneuraminic acid. N-acetyl-7-U-acetylneuraminic acid could not be obtained as a pure substance as it eluted together with N-acetyl-9-0-acetylneuraminic acid from cellulose. In addition, Nacetyl-9(?)-0-glycolylneuraminic acid, N-acetyl-7(?)-O-glycolylneuraminic acid and N-glycolyl-7(?)-O-acetylneuraminic acid could not be separated from N-glycolyl-9-O-acetylneuraminic acid by cellulose chromatography. N-(:‘H)acetylneuraminic acid ( 11 mCi/mmole) and N- [ 1JdC] glycolylneuraminic acid (0.355 mCi/mmole) were synthesized according to Ref. 14. Neuraminic acid-p-methylglycoside and its methylester, N-chloroacetylneuraminic acid, N-fluoroacetylneuraminic acid and N-bromoacetylneuraminic acid-p-methylglycoside were prepared as described previously ( 15; and unpublished work).
Aqueous solutions containing 1O-l 00 Fg of sialic acids were lyophilized in tapered test tubes ( 10 X 50 mm) and 25 ~1 of TSIM added under nitrogen. The stoppered tubes were whirled for either 15 min at room temperature or 5 min at 60°C in a heating block to complete the silylation reaction which is visible by total solubilization of the substances. With the reagent mixture HMDS-TMCS-pyridine (2: 1: 10: Ref. 4) complete silylation of sialic acids was achieved within 30 min at room temperature. Silylation with BSA, BSTFA. TMSDEA or a mixture of TSIM: BSA: TMCS (3 : 3 : 2) was carried out in the same way. For comparison with the method of direct derivatization of sialic acids described above, P-methylketoside methyl-esters of various sialic acids were prepared by heating for 1 hr at 80°C with 0.02 N (6) or with 0.5 N methanolic HCl followed by trimethylsilylation with the HMDSTMCS-pyridine reagent (4) or TSIM. Qrralitrrti\v
Analysis of Sicrlic Acids
For determination of the retention indices (16) of individual TMSsialic acids, 1 ~1 of the silylation mixture described above was injected, and the times of elution were related to those of C,, and C,, normal alkanes. The preservation of the natural 0-acyl groups of sialic acids in the silylation reaction was established by conversion of these 0-acyl groups before or after derivatization of the sialic acids with TSIM to their acylhydroxamates which were determined quantitatively and qualitatively by calorimetric methods and by tic, respectively, according to Ref. 2 and 8. The nature of the de-0-acylated N-acylneuraminic acid residues was
GAS-LIQUID
CHROMATOGRAPHY
OF
SIALIC
ACIDS
511
identified by glc in the following way: Lyophilized samples of Oacylated sialic acids were exposed for 3 hr to an atmosphere of ammonia over a solution of 5 N ammonium hydroxide at room temperature to hydrolyze the 0-acyl groups. The resulting N-acylneuraminic acid samples were dried again under vacua before trimethylsilylation by TSIM. The retention indices of the sialic acids silylated before or after release of the 0-acyl groups were compared.
To obtain calibration curves, crystalline NeuNAc and NeuNGl were dissolved in water to a final concentration of I .O mglml. N-acetyl-O-acetylneuraminic acids, which appeared pure on tic after passage through cellulose powder, could not be obtained in crystalline form without partial decomposition. They were, therefore, determined quantitatively with the orcinollF&“/HCl reagent ( 10) using the same molar extinction coefficient as for N-acetylneuraminic acid (2). For calculation of the quantity of N-acetyl-0-acetylneuraminic acid, the content of one 0-acetyl was taken into consideration. A solution of NeuNAc-9-OAc (1.25 mglml) was used for calibration. Aliquots ranging between 10 and 100 ~1 were taken from these solutions and mixed with 10 ~1 of a solution containing 50 pg of trehalose or sucrose as internal standards. The lyophilized sialic acid and internal standard mixtures were silylated with 50 ~1 of TSIM, and 1 ~1 (containing 1 pg of the internal standard) was injected. The internal standards sucrose (RI 2732) or trehalose (RI 2827) were used for the sialic acid identification on OV-1 , and trehalose (RI 27 19) on OV-17. Trehalose appears behind NeuNGl on both liquid phases and does not interfere with the different sialic acid peaks. Sucrose (RI 2622) is not suitable for OV-17, as its peak interferes with that of NeuNGl. In some experiments, NeuNAc and NeuNGl also served as internal standards. Calibration curves have not yet been obtained from the other naturally occurring 0-acylated sialic acids as they could not be isolated in sufficient amounts as pure substances. Therefore, the analyses of these sialic acids were semiquantitative in relation of their peak areas to those sialic acids used for calibration. The areas of the glc peaks were measured by automated integration or by the triangulation method.
“{H-labeled NeuNAc and Y-labeled NeuNGl were derivatized and injected as described above. A 1: 10 split was fitted between a column of 0.8 m X 4 mm i.d. packed with 1% OV-1 or 3% OV-23 on Anachrom
512
CASALS-STENZEL,
BUSCHER
AND
SCHAUER
SD, 80-100 mesh, and the detector. Teflon tubes (1.5 mm X 150 mm) were connected to the main exit of the split. The eluted substances were completely condensed in the teflon tubes due to the temperature gradient. The tubes were changed after each peak detected by the FID. They were rinsed with 5-ml Instagel. and the radioactive substances were measured in a Nuclear Chicago, Mark I, liquid scintillation counter. RESULTS AND Derivatization
DISCUSSION
of Sialic Acids
From all silylation reagents tested, TSIM and the mixture of HMDSTMCS-pyridine (4) were found to be the best reagents for silylation of acylneuraminic acids. This was tested in detail with NeuNAc and NeuNGl which result in single and symmetrical peaks in glc. NeuNAc and NeuNGl are trimethyl-silylated at all hydroxyl groups including the carboxyl group, as could be established by mass spectrometry. (A fragment at m/e 726 was obtained from TMS-NeuNAc which corresponds to the M-15 ion fragment and confirms the expected molecular weight of 741 of the sixfold trimethyl-silylated NeuNAc. TMS-NeuNGl resulted in a fragment at m/e 814 which corresponds to the M-l 5 ion and indicates the molecular weight of 829 of the sevenfold trimethylsilylated NeuNGl.) NeuNAc or NeuNGl derivatized by BSA, BSTFA, or a mixture of TSIM-BSA-TMCS (3 : 3 : 2) result in double peaks, as was also found with BSA by Craven and Gehrke (3). One of them is probably due to an additional silylation at the hydrogen position of the amino group of NeuNAc or NeuNGl. In contrast, silylation of these sialic acids by TMCS, HMDS or TMSDEA lead to none or only to low peaks; long reaction times and higher temperatures (60°C) do not improve the silylation effectivity. The 0-acylated sialic acids can also be silylated with TSIM and the HMDS-TMCS-pyridine reagent (4). They do not lose their 0-acyl groups during this procedure. This could be established in the following way: 1. The TMS-derivatives of the different N-acyl-0-acylneuraminic acids result in single and distinct peaks in glc which differ from those of NeuNAc and NeuNGI; 2. NeuNAc- or NeuNGl-peaks as decomposition products of Oacylated sialic acids do not appear on the gas-chromatogram after injection of TMS-derivatives of N-acyl-0-acylneuraminic acids. Samples containing N-acetyl-7,9-di-0-acetylneuraminic acid result in two glc peaks (see below); however, the possible de-0-acetylated products NeuNAc, NeuNAc-7-OAc and NeuNAc-9-OAc could not be detected.
GAS-LIQUID
CHROMATOGRAPHY
OF
SIALIC
513
ACIDS
3. The quantitative and qualitative hydroxamate assay after silylation of 0-acylated sialic acids gives evidence for the preservation of the 0-acetyl- or 0-glycolyl groups during the silylation reaction; 1 mole of 0-acyl per mole of trimethylsilylated N-acyl-0-acylneuraminic acids was found. 4. It was demonstrated by mass spectrometry of the TMS-derivatives of NeuNAc-4-OAc, NeuNAc-7-OAc and NeuNAc-9-OAc that no elimination of the 0-acetyl residues had occurred. 5. NH,-treatment of the 0-acylated sialic acids before silylation gave additional evidence that the individual glc peaks are significant for individual 0-acylated sialic acids. By this method all 0-acylated sialic acids are converted to NeuNAc and NeuNGl respectively leading to the corresponding glc peaks, as will be discussed later. Derivatization of sialic acids by preparing the p-methylketoside methylesters before trimethylsilylation did not prove to be successful in the case of the 0-acylated sialic acids. Most of the 0-acyl residues were released even by using 0.02 N methanolic HCl (Table I), although Yu and Ledeen (6) have reported that de-0-acylation is largely prevented under these mild conditions. The extent of de-0-acylation was established by the hydroxamate test and by glc-analysis of the reaction products shown in Table 1. From this table it can also be seen that NeuNAc or NeuNGl can be better separated from their methylated products and from the methylester of neuraminic acid-fi-methylglycoside on OV- 17 than on OV- 1. The RI-values of the unmethylated and the methylated NeuNAc or NeuNGl are different on OV- 17, however, they are similar on OV- 1. TABLE PRODUCTSFROMMETHANOLYSISOF NeuNAc, DIFFERENTCONCENTRATIONSOF
1 NeuNG1. AND NeuNAc-POAc HCl INMETHANOL~
0.5 N methanolic HCI. I hr. 80°C Methanolysis products NeuNAc
NeuNAc
10.0
Me-NeuNAc-?-OMe NeuNGl Me-NeuNGl-?-OMe
53.0 -
NeuNAc-9-OAc Me-NeuN-2.OMe
37.0
NeuNGl 8.0 56.0
36.0
0.02 N methanohc HCI. 2.5 hr. 80°C
NeuNAc9.OAc 22.7 65 5 -
01 Il.7
NeuNAc 49.0 410 -
NeuNGl 51.0 38.0
10.0
11.0
NeuNAc9.OAc 86.8 II.5 1.7 0
WITH
RI on ov-17
RI on ov-I
2488 2557 2653 2742 2598 2319
2483 2498 2674 2703 2523 2284
L1The %-relationship of the peak areas of the TMS-derivatives on 3% OV-I7 (column 1.6 m x 2 mm i.d.: 196°C) and the RI-values on OV-17 and 1% OV-I (column 1.6 m x 2 mm i.d.: 206°C) are shown. Only the main peaks of the gaschromatograms were taken to calculate the percentage of the compounds. These were identified by reference substances (14) or according to Yu and Ledeen (6). Me-NeuN-2.OMe corresponds to the methylester of neuraminic acid-@methylglycoside. (The peak may also contain the free aad which cannot be distinguished from its methylester under the glcconditions chosen (see Table 3). Me-NeuNAc-2.OMe and Me-NeuNGI-2.OMe are taken to represent the methylesters of the /3-methylglycosides of NeuNAc or NeuNGI.
514
CASALS-STENZEL,
BUSCHER
AND
SCHAUER
The silylated sialic acids can be stored under dry nitrogen without noticeable decomposition.
at +4”C
The material of the column whether glass or metal, has no influence on the quality of the separation of sialic acids. The length of the column is of great influence on the separation, especially of the 0-acylated sialic acid isomers, as their retention indices do not differ greatly from one another. Although NeuNAc and NeuNGl can be separated satisfactorily on a column of 0.4 m length, a 2.5 m column is needed for the complete separation of, e.g., NeuNAc-7-OAc and NeuNAc-9-OAc on OV-17. However, the better resolution of sialic acids on long columns is accompanied by a higher loss of the substances resulting in an appreciable decrease of the peak areas as will be discussed below. Therefore, in the routine analyses a compromise length of 1.6 m and 2 mm i.d. was used. While 0-acylated sialic acids cannot be separated from N-acylneuraminic acids on a 60-80 mesh support covered by 3% OV-17 and under the conditions described above, the use of 100-I 20 mesh support gives sharp and symmetrical peaks of the different sialic acids. The middle polar phases OV-I 7 and OV-22 have been proven best for the resolution of the different derivatives of neuraminic acid. However, the peaks appeared more symmetrical and sharper on OV-17 than on OV-22. The nonpolar phase OV-1 (or Apiezon L or GE-SE-54) is useful only for special analyses, e.g., for mixtures of NeuNAc and NeuNGl, or for estimation of the total amount of N-acyl-O-acylneuraminic acids. 0-Acylated sialic acids cannot be separated suitably on this material. The retention indices of the naturally occurring sialic acids on OV-17 and OV-I are compared in Table 2 (see also Fig. 4). The TMS-sialic acids do not elute from polar phases. For analysis, particularly of sialic acid mixtures, we recommend the use of two differently loaded columns (e.g., OV-1 and OV-17) to avoid errors in the identification of the peaks in agreement with other authors (4,6).
Qlralituti,v Identification of Siulic Acids TMS-NeuNAc and TMS-NeuNGl result in single symmetric peaks eluting from the nonpolar or middle polar stationary phases chosen. It has already been explained that the eluted substances detected by the FID represent the per-trimethylsilylated NeuNAc and NeuNGl, respectively, according to mass spectral analysis. The possibility of appreciable decomposition of these TMS-sialic acids during passage through the glc-columns which would lead to a con-
GAS-LIQUID
CHROMATOGRAPHY
OF
TABLE RErENrroN
INDICES
OF TMS-DERIVATIVES
MATERIALSON
SIALIC
515
ACIDS
2
OF SIALIC
3% OV-17 (196°C)
Sialic acids NetrNAc NeuNAc-COAc NeuNAc-7-OAc NeuNAc-Y-OAc NeuNAc-7.9-di-OAc NeuNAc-4-OGl NeuNAc-7(?)-OGl NeuNAc-9(?)-OGl NeuNGl NeuNG I-4-OAc NeuNG I-7(?)-OAc NeuNG I-9-OAc
ACIDS
AND
1%
FROM
BOVINE
AND
EQUINE
OV-1 (206°C)”
ov-17
OV-I
2488 2637 2622 2599 2702 2560 2599 2560 2653 2758 2775 2758
2483 2561 2522 2522 2648 2535 3540 2535 2674 2720 2700 2720
If Glass columns. 1.6 m x 3 mm i.d. The data are average values from two to three different preparations and from three injections each.
fusing peak pattern was further excluded by radio-glc of TMSNeuN [3H] AC and TMS-NeuN [““Cl Gl. As can be seen from Fig. 1 the bulk of the “H- or ‘“C-radioactivities eluted from the columns in accordance with the peaks recorded by the FID, and only small amounts of the radioactivity were found to appear before or after the peaks. On average, 60% of the injected radioactivity did not elute from a 1.6-m column while 30-35% of the eluted radioactivity corresponded to the
,I
I
I 10
I
m 20
[min]
FIG. I. Radio-glc of a mixture of NeuN(3H)Ac and NeuN(l-“C)Gl. 3% OV-22 on 80-100 mesh Anachrom SD, 0.8 m X 4 mm i.d.: split 1: 10; program: Initiai temperature 188°C. after 5 min delay increase of 2”C/min. The eiuting radioactivity (vertical lines) measured by the **C- or JH-channels is collected as indicated by the dotted lines.
516
CASALS-STENZEL,
BUSCHER
BEFORENHI 12%s6710
AND
SCHAUER
AFTERNng 1’
2’3’
10
20
FIG. 2. Comparison of tic on cellulose and glc on 3% OV-I 7 of sialic acids from bovine submandibular gland glycoproteins. Glass column: I .6 m x 2 mm i.d.; isothermally 196°C. The tfc-sialic acid spots (left side) are marked a-e. The gas-liquid chromatograms of the siahc acids from the tic-spots are shown in the middle part of the figure (before NH,). On the right side the same sialic acids de-f)-acylated by NH,-treatment are shown (after NH,). Peak ~denti~cation: 1, NeuNAc; 2, NeuNAc-9(?)-OGI; 3, NeuNAc-7(?)-OGI and NeuNAc-9-OAc: 4. NeuNAc-7-OAc; 5, NeuNG1: 6, unknown sialic acid: 7, NeuNAc7,9-di-OAc: 8, NeuNGl-9-OAc; 9, NeuNGl-7(?)-OAc: 1’. NeuNAc: 2’, NeuNGI: 3’. alkaline degradation product of sialic acids of unknown structure.
GAS-LIQUID
CHROMATOGRAPHY
OF
SIALIC
517
ACIDS
:d
30 [nnin)
10
20
30
[min]
FIG. 3. comparison of tic on cetlulose and glc on 3% OV-17 of sialic acids from equine subm~dibular gland glycoproteins. The method of analysis and the glc-conditions correspond to those of Fig. 2. Peak identification: 1, NeuNAc: 2. NeuNAc-4-OGI: 3. unknown sialic acid (NeuNAc-9-OAc ?); 4, NeuNAc-4-OAc; 5, NeuNG1: h, NeuNGl-4-OAc; 1’. NeuNAc: 2’. NeuNGI: 3’. unknown alkaline degradation product of sialic acids.
NeuNAc or NeuNGl peaks, a value which agrees favourably with the results obtained with neutral mono- and disaccharides by Jansen and Baglan ( 17). The individual 0-acylated siahc acids also result in single peaks. Their retention indices are different from NeuNAc and NeuNGl and are distinguishable from one another on middle polar phases as OV-17 and OV-22 (Table 2, Figs. 2-4). The identification of the glc-peaks of the Oacylated sialic acids was carried out by correlation of their relative retention times with those of the TMS-derivatives of sialic acids which had been characterized before (2). For further and direct proof of the nature of the O-acylated sialic acids their ~-acy~neuraminic acid residues
518
CASALS-STENZEL,
lrnirll
BUSCHER
AND
SCHAUER
I rn,l,
4. Gas-liquid chromatograms of sialic acids from bovine submandibular gland glycoproteins on 1% OV-1 and on 3% OV-17, 1.6 m X 2 mm i.d.: run isothermally at 206°C and 196”C, respectively. Peaks: I, NeuNAc; 2, NeuNAc-9(?)-OGl: 3, NeuNAc7(?)-OG1 and NeuNAc-9-OAc together: 4, NeuNAc-7-OAc: S. NeuNGl; 6, unknown sialic acid: 7, NeuNAc-7,9-di-OAc: 8. NeuNGL9-OAc also containing NeuNGl-7(?)-OAc. FIG.
(NeuNAc or NeuNGl) de-0-acylated with the aid of ammonia vapour were identified by glc. In Figs. 2 and 3 the analysis of sialic acids from bovine and equine submandibular gland glycoproteins by tic on cellulose is compared with the analysis by glc on OV-17. (The sialic acids elute from cellulose columns in the same sequence as they migrate on cellulose thin-layer.) While NeuNGl (“a”) or NeuNAc (“b”) spots from bovine and equine sialic acids appeared homogeneous in glc and were not influenced by ammonia vapour, spot “c” of Fig. 2 (bovine sialic acids) resulted in 4 peaks which could be de-0-acylated to NeuNAc and NeuNGl, respectively. The higher peaks of the two peak groups correspond to N-acetyl9(?)-0-glycolylneuraminic acid (RI 2560) and N-glycolyl-9-O-acetylneuraminic acid (RI 2758), respectively. The two smaller peaks with somewhat higher retention times are assumed to be the isomeric Nacetyl-7(?)-0-glycolylneuraminic acid (RI 2599) and N-glycolyl-7(?)-0acetylneuraminic acid (RI 2775), respectively. The position of the 0-acyl groups at C, is assumed in analogy to the structure of N-acetyl-70-acetylneuraminic acid isolated earlier from bovine submandibular gland glycoproteins (8); however, direct experimental proof by mass spectrometry of the assumed structures of these NeuNAc-7(?)-OGl and NeuNGl-7(?)-OAc is not yet available. The ratios of these four sialic acids were different in individual preparations. The corresponding tic spot (“c”) from equine sialic acids (Fig. 3) containing only minute amounts of sialic acids resulted in three peaks which could be converted
GAS-LIQUID
CHROMATOGRAPHY
OF
SIALIC
ACIDS
519
to NeuNAc and NeuNGl by ammonia treatment. Peak No. 2 is derived from N-acetyl-4-0-glycolylneuraminic acid (RI 2560), the identification of which was possible as it was found in individual sialic acid fractions from equine submandibular glands with 70% purity (2). Peak No. 4 contains N-acetyl-4-0-acetylneuraminic acid (RI 2637). Peak No. 6 consists of N-glycolyl-4-0-acetylneuraminic acid (RI 2758) which was obtained in nearly pure form from equine erythrocyte membrane. The tic spot “d” from the bovine sialic acids (Fig. 2) consists of the 70-acetyl- and 9-0-acetyl isomers of N-acetyl-0-acetylneuraminic acid, yielding NeuNAc after ammonia treatment. The NeuNAc-9-OAc peak (main peak; RI 2599) was established from one of the preparations, in which a few of the fractions from cellulose chromatography contained pure NeuNAc-9-OAc. The shoulder of the NeuNAc-9-OAc peak could be attributed to NeuNAc-7-OAc (RI 2622) by comparison of the RIvalue with that resulting from NeuNAc-7-OAc isolated earlier (8). The structure of the TMS-derivative of NeuNAc-7-OAc was further established by glc-mass spectrometry. The corresponding equine sialic acid spot (“d”) of Fig. 3 also contains two isomeric N-acetyl-O-acetylneuraminic acids. Among these NeuNAc-4-OAc prevails in all preparations from equine origin. However, the second N-acetyl-O-acetylneuraminic acid present in this preparation (peak No. 3) elutes with a retention index of 2606 which is similar to that of NeuNAc-9-OAc found in bovine tissues. More analytical data are required to establish the structure of this new equine N-acetyl-0-acetylneuraminic acid which was detected in several preparations. The uppermost tic spot (“e”) of Fig. 2 leads to two peaks in glc which can be degraded to NeuNAc under alkaline conditions. The main peak (No. 7) with the higher retention time (RI 2702) is due to N-acetyl-7,9di-0-acetylneuraminic acid found in some preparations as a pure substance (2). The structure of the second sialic acid present in this preparation which is doubly 0-acylated, is unknown. An unknown compound is produced in variable amounts by NH,treatment of the different sialic acids which appears after NeuNGl (peak No. 3’; RI 2690) and is believed to be a degradation product, probably a pyrrol derivative of sialic acids. Figure 4 shows the glc analysis on OV-17 or OV-1 of a mixture of sialic acids isolated from bovine submandibular gland glycoproteins. Appreciable differences in the resolution of the various sialic acids can be seen between OV-1 and OV-17. From OV-17, the TMS-sialic acids elute as distinct peaks in the following sequence: NeuNAc is eluted initially followed by its 0-acyl derivatives; NeuNAc-9(?)-OGl, then NeuNAc-7(?)-OGl and NeuNAc-9-OAc eluting together and then NeuNAc-7-OAc. (NeuNAc-4-OAc from equine submandibular gland glycoproteins elutes after NeuNAc-7-OAc.) NeuNGl is then eluted
520
CASALS-STENZEL,
BUSCHER
AND
SCHAUER
followed by NeuNAc-7,9-di-OAc and finally the O-acylated derivatives of NeuNGl: NeuNGl-9-OAc and NeuNGl-7(?)-OAc. The elution sequence of TMS sialic acids on OV-17 may be related to the polarity and the molecular size of the silylated sialic acids. In the Nacetyl-0-acetylneuraminic acid series it is remarkable that the retention indices (see Table 2) become higher if the 0-acetyl groups are closer to or even in the pyranoside ring of neuraminic acid. The nature of peak No. 6 (RI 2676) from OV-17 is unknown, as are the other small peaks which can be seen in the regions between and after NeuNAc-7,9-di-OAc and NeuNGl-9-OAc. It may be speculated that these peaks are due to hypothetical neuraminic acid derivatives with other combinations of glycolyl and acetyl groups such as, e.g., Nglycolyl-0-glycolylneuraminic acid, N-glycolyl-di-0-acetylneuraminic acid or N-acetyl-0-acetyl-0-glycolylneuraminic acid. We were not able to separate NeuNAc-9-OAc and NeuNAc-7(?)OGl which have identical retention indices on OV-17, even on columns longer than 1.6 m. The apolar liquid phase OV-1 is not qualified for the separation of most of the individual 0-acylated sialic acids as the N-acetyl-o-acetylneuraminic acids appear together, shortly after NeuNAc as a broad unsymmetrical peak, and the N-glycolyl-O-acetylneuraminic acids elute near NeuNGl (Fig. 4). OV-1 is better suited than OV-17 only for the analysis of the sialic acids from equine submandibular gland glycoproteins, as NeuNAc-4-OAc, the prevailing 0-acylated sialic acid from this material, appears on OV-1 between NeuNAc and NeuNGl as a well separated peak. On OV-17 NeuNAc4-OAc partially overlaps with NeuNGl. IdentiJication of Synthetic. Neumminic Acid Derituti\?es Three percent OV- 17 or 1% OV- 1 columns are also suitable for identification of synthetic neuraminic acid derivatives such as neuraminic acid-p-methylglycoside or its methyl ester, and some sialic acids containing N-halogenacyl groups (Table 3). These sialic acids which were TABLE RETENTION
INDICES DERIVATIVES
Sialic
3
OF TMS-DERIVATIVES ON 1% OV-1 (206°C)
OF SYNTHETIC NEURAMINIC AND 3% OV-17 (196°C)”
acids
ov-17
Neuraminic acid-/3-methylglycoside and its methylester N-fluoroacetylneuraminic acid N-chloroacetylneuraminic acid N-bromoacetylneuraminic acid-fi-methylglycoside n Glass
columns,
1.6 x 2 mm
i.d. The
values
are the mean
ov-I
2319 2442 2594 2774 of three
ACID
2284 246 1 2579
analyses.
GAS-LIQUID
CHROMATOGRAPHY
OF
SIALIC
521
ACIDS
silylated in the same way as the naturally occurring sialic acids, resulted in single and symmetrical peaks most of them having distinct retention indices. Only neuraminic acid-p-methylglycoside and its methylester eluted together from OV-17. Glc may therefore be useful for following the synthesis of these or other sialic acids, especially for the assay of their yields and purity.
The relative detector responses (RDR)’ of NeuNAc, NeuNGl and NeuNAc-9-OAc on OV-1 and OV-17 using two column lengths are summarized in Table 4. The low RDR values of NeuNAc-9-OAc on both liquid phases when compared with those of NeuNAc or NeuNGl is striking. The RDR values decrease appreciably below those given in Table 4 at NeuNAc or NeuNGl concentrations below 0.5 pg/pl TSIM. The latter phenomenon has also been reported for NeuNAc by Yu and Ledeen (6). The calibration curves for NeuNAc. NeuNGl and NeuNAc-9-OAc on OV-1 and OV-17 are shown in Fig. 5. The calibration curves do not originate from zero when the peak areas of the TMS-sialic acids are related to TMS-sucrose or TMS-trehalose, but they are linear in the concentration ranges shown. The calibration curves intersect at zero, if NeuNAc serves as standard for the determination of NeuNGl and vice versa. However, the standard curve for NeuNAc-9-OAc does not originate from zero, even when NeuNAc is used as internal standard. While the slopes of the curves for NeuNAc and NeuNGl are identical on OV-17 and do not differ greatly for OV-1, the slope of the curve for NeuNAc-9-OAc was found to be about three times lower on both liquid phases when compared with NeuNAc. These differences may be due to adsorption phenomena on the
INFLUENCE
OF COLUMN RESPONSES
LENGTH
OF NeuNAc.
Column length (ml
NeuNAc
NeuNGl
1.6 0.8
0.402 i 0.022 0.531 t 0.030
0.294 t 0.021 0.510 f 0.035
TABLE 4 (2 mm i.d.) ON THE RELATIVE NeuNGl AND NeuNAc-9-OAc”
DETECTOR
ov-17
ov- I NruNAc-9.OAc
0.258 k 0.022 0.440 k 0.020
NeuNAc
NeuNGl
0.458 2 0.022 0.567 i 0.030
0.451 t 0.026 0.548 i 0.038
NeuNAc-Y-OAc
0.212 + 0.024 0.366 c 0.025
‘I Glc on lq, OV-I (temperatures: 1.6 m column. 206°C; and 0.8 m, 196°C) and 3% OV-17 (temperatures: 1.6 m column, 196°C: and 0.X m. 186°C) on X0-100 mesh Gas Chrom Q. The values are average* from IO independent ramples each Injected three times and ranging between 1.0 and 3.0 wg sialic aad per ~1 TSIM injected. As internal amdards trehalose on OV-17 and sucrose on OV-I were used: each I pg/pl TSIM.
’ RDR = [area (sialic acid)].
(sialic
acid)]/[area
(int.
standard)]
X
[weight
(int.
standard)]/[weight
522
CASALS-STENZEL,
BUSCHER
AND
SCHAUER
FIG. 5. Calibration curves obtained with NeuNAc, NeuNGl and NeuNAc-9-OAc on 3% OV-17 (196°C) and 1% OV-1 (206°C) columns of 1.6 m length and 2 mm i.d. See text for further details.
packing materials of the sialic acids tested. It has already been outlined above that only about 35% of the radioactivity from NeuNAc or NeuNGl is eluted from 1.6 m columns. ~o~esponding data from NeuNAc-9-OAc are not yet available. The appreciable Iower RDR values of NeuNAc-POAc may be explained by a stronger adsorption of this TMS-sialic acid to the column materials. Errors in the quantitative estimation of this sialic acid were excluded by calorimetric control of the sialic acid amounts injected. The great RDR differences between NeuNAc and NeuNAc-9-OAc cannot be explained by an altered sensitivity of the FID due to the presence of only 5 TMS groups in TMSNeuNAc-9-OAc (18). When using a 0.8 m instead of a 1.6 m cohrmn the RDR values for NeuNAc and NeuNGl relative to sucrose or trehalose increase appreciably (Table 4), probably due to reduced adsorption and decomposition. For sensitive quantitative sialic acid analyses therefore, the columns should be as short as possible. However, short columns can be used only if a small number of sialic acids with significantly different retention times are present, e.g., mixtures of NeuNAc and NeuNGl. The ratio of
GAS-LIQUID
CHROMATOGRAPHY
OF
SIALIC
ACtDS
523
NeuNAc to NeuNGl from porcine submandibular gland glycoproteins established already as 1: 10 ( 19) was confirmed by glc on OV- 17. The accuracy of this method for the quantitative analysis of acylneuraminic acids was determined by repeated injection of five equally prepared samples; the limit of error was within 7% with the 1.6 m and 5% with the 0.8 m column. The smallest amount of NeuNAc and NeuNGl which can accurately be determined under the conditions described is about 0.5 pg and that of NeuNAc-9-OAc between l.S and 2.0 pg (Fig. 5). The minimally detected amount was about 10 ng for both TMS-NeuNAc (in agreement with Ref. 3) or TMS-NeuNGl and about 20 ng for TMS-NeuNAc-9-OAc. The appreciable difference of the sensitivity in glc-analysis observed between NeuNAc and NeuNGl on the one hand and NeuNAc-9-OAc on the other indicates that calibration curves are required for the exact quantitative analysis of the various U-acylated sialic acids occurring in mixtures from biological origin. The relation of the peak areas from the various 0-acylated sialic acids to NeuNAc, NeuNGl or NeuNAc-9OAc leads to only approximate quantitative values. However, the relative amounts of the many different sialic acids occurring in biological sources, e.g., in bovine submandibular gland glycoproteins (Fig. 4) can quickly be appraised without using an internal standard. The isolation of sufficient amounts of the individual sialic acids in pure form for their quantitative analysis (determination of the RDR values, calibration of columns) is under investigation. These sialic acids are also required as reference substances for the qualitative analysis of sialic acid mixtures of unknown composition.
The experiments demonstrate the comprehensive and reproducible applicability of glc for the analyses of different types of sialic acids. This method is better than tic not only on account of the requirement of smaller amounts of substances but also with regard to the possibility to analyse the different 0-acyl isomers of sialic acids, as they lead to distinct glc-peaks. It is shown in Figs. 2 and 3 that sialic acid spots appearing homogeneous in tic in part result in different glc peaks, each peak representing an individual isomer of an 0-acylated sialic acid. Therefore, tic can be used only for an approximate estimation of the composition of a sialic acid mixture. Thus glc is shown to be a valuable method to check the purity and occurrence of the various types of sialic acids derived from biological materials, chemical syntheses or enzymatic assays. The method is, however, nearly as time consuming as tic as the sialic acids must be carefully purified by ion-exchange chromatography to obtain good resolution on
524
CASALS-STENZEL,
BUSCHER
AND
SCHAUER
glc. The advantages of glc over the usual calorimetric methods for sialic acid analysis have been discussed for NeuNAc by Craven and Gehrke (3), as other carbohydrates and fatty acids do not interfere with sialic acids in glc. The direct trimethylsilylation of sialic acids without previous conversion to the P-methyl-ketosides first used for NeuNAc by Craven and Gehrke (3) has the advantage of speed and as our experiments clearly have shown of full preservation of the 0-acyl groups. It may be expected that in the field of sialic acid chemistry more analytical problems can now be solved by application of glc, e.g., the question of migration of 0-acyl groups, which still is open (2). The sensitivity of the glc method may lead to the discovery of still more neuraminic acid derivatives occurring in nature. Combination of glc and mass spectrometry will enable the elucidation of the structures, especially concerning the position of the 0-acyl groups, of those sialic acids which have been detected only in minute amounts in sialic acid mixtures. ACKNOWLEDGMENTS We are indebted with many thanks to Mrs. G. Malske. Mrs. M. Wember and Mrs. B. Wrage for the excellent technical assistance. The financial support by the Deutsche Forschungsgemeinschaft (Grant Scha 202/l and 202/3) and by the Fonds der Chemischen lndustrie is gratefully acknowledged. We thank the Erwin Riesch Stiftung for the provision of a grant for instruments.
REFERENCES I. 2. 3. 4.
Schauer, Buscher, Craven, Sweeley, SW.
R. ( 1973) Anger. Chem. Intern. Edit. 12, 127. H.-P., Casals-Stenzel, J., and Schauer, R. (1974) Eur. J. B&hem. D. A., and Gehrke, G. W. ( 1968) J. Chromutogr. 37, 414. C. C.. Bentley. R.. Makita, M.. and Wells, W. W. (1963) J. Amer.
50,
71.
Chem.
85, 2497.
5. 6. 7. 8. 9. 10. 11. 12.
Clamp, J. R.. Dawson. G., and Hough, L. (1967) Biochim. Biophys. Ac.trt 148, 342. Yu. R. K., and Leeden, R. W. (1970) J. Lipid. Res. 11, 506. Williams, I. H. (1961) .I. Chrotnufogr. 5, 457. Schauer. R., and Faillard, H. (1968) Hoppe-Seyler’s Z. Physiol. Chem. 349, 961. Warren. L. (1959) J. Biol. Chem. 234, 197 1. Bohm, P., Dauber. St., and Baumeister. L. (1954) K/in. Wochensc~hr. 32, 289. Svennerholm, E., and Svennerholm, L. (1958) Natrtre (London) 181, 1154. Kamerling, J. P., Vliegenthart, J. F. G., Versluis. C., and Schauer, R., Carbohydrate Research, in press. 13. Wirtz-Peitz, F., unpublished results. 14. Schauer, R., and Buscher. H.-P. ( 1974) Biochim. Biophys. AM 338, 369. 15. Schauer. R., Wirtz-Peitz, F., and Faillard, H. (1970) Hoppe-Seyler’s Z. Physiol. Chetn.
16. 17. 18. 19.
351, 359.
Kovats, E. ( 1958) Helv. Chim. Acta 41, 19 15. Jansen, E. F., and Baglan, N. C. (1968) J. Chromatogr. 38, 18. Ackman, R. G. (1964) J. Gaschromatogr. 2, 173. Martensson, E., Raal, A., and Svennerholm, L. (1957b) Acra Chem.
Sccltd.
11. 1604.
BIOCHEMISTRY
Gas-Liquid
65,
507-514
(1975)
Chromatography
of N-
Neuraminic JORGE
and
0-Acylated
Acids
CASALS-STENZEL, HANS-PETER AND ROLAND SCHAUER
BUSCHER.
Received July 19. 1974: accepted October 30. 1974 This paper is dedicated to Prof. Dr. G. Weitzel to mark the occasion of his 60th birthday. The qualitative and quantitative analysis by gas-liquid chromatography of natural and synthetic sialic acids with special reference to O-acylated sialic acids is described. Sialic acids are derivatized by N-trimethylsilylimidazol. All hydroxyl groups including the carboxyl group are t~methylsiiylated as was shown by mass spectrometry. No elimination of the O-acyl groups occurs during the silylation reaction. The TMS-derivatives of the sialic acids are stable in the refrigerator. The TMS-sialic acids including the isomeric ~-acyl-~-acylneu~minic acids can be separated from each other on OV- 17 or OV-22. Single peaks having individual retention indices from the following sialic acids were obtained: N-acetylneuraminic acid. N-glycolylneuraminic acid, N-Auoroacetylneuraminic acid and N-chloroacetylneuraminic acid; neuraminic acid+methylglycoside or its methylester and N-bromoacetylneuraminic acid-fi-methylglycoside: N-acetyl4-O-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid and N-acetyl9-O-acetylneuraminic acid: N-acetyl-7.9-di-O-acetylneuraminic acid: N-acetyl4-~-glycolylneu~~minic acid, ~~-acetyl-9(?~-U-~~yco~ylneuraminic acid and .~-acet}~l-7(?~-~-glycolylneuraminic acid; ~-~lycolyi-4-~-acetylneuraminic acid, ~~-glycolyl-9-~-acetylneuraminic acid and ~~-glycolyl-7(?)-~-acetylneuraminic acid. On OV-1 N-acetylneuraminic acid and N-glycolylneuraminic acid can be separated from each other and from N-acyl-0-acylneuraminic acids: however. the Oacylated sialic acids elute essentially together from this material. The relative detector responses for the different sialic acids vary, as was shown for N-acetylneuraminic acid, N-glycolylneuraminic acid and N-acetyl-9-O-acetylneuraminic acid. 35% of the radioactivity from radioactive N-acetylneuraminic acid and h’-glycolylneuraminic acid eluted correspondingly with the sialic acid peaks detected by the FID. Gas-liquid chromatography enables a sensitive qualitative and quantitative analysis of different sialic acids occurring in biological materials, produced during chemical synthesis or in enzymic assays.
The increasing interest in the structure and biological functions of sialic acids as components of glycoproteins and glycolipids requires methods for a more accurate identification of the different sialic acids. In 507 Copyright @I 1975 by Academic Press, Inc. Ail rights of reproduction in any form reserved.
508
CASALS-STENZEL,
BUSCHER
AND
SCHAUER
mammals, at least twelve different sialic acids have been found which are acetylated or glycolylated at the amino group only or at both N- and O-positions (1,2). Not all of these N-acetyl-U-acetylneuraminic acids, N-acetyl-0-glycolylneuraminic acids and N-glycolyl-0-acetylneuraminic acids can be distinguished by the usual analytical methods, e.g., by thinlayer chromatography or by calorimetric methods (1). Gas-liquid chromatography is assumed to be a more sensitive tool to distinguish sialic acids which are different with regard to the nature and positions of 0-acyl groups. The qualitative and quantitative determination of N-acetylneuraminic acid and N-glycolylneuraminic acid by glc has already been carried out with success, either by trimethylsilylation of the native N-acylneuraminic acids (3) or of the corresponding p-methylketosides (4-6). Conversion of 0-acylated sialic acids to their p-methylketosides by methanolysis before trimethylsilylation, however, causes an appreciable loss of the 0-acyl groups (6); therefore, the analysis of N-acyl-Oacylneuraminic acids by glc has not yet been achieved satisfactorily. This paper describes the analysis of different sialic acids including the 0-acylated sialic acids by glc. The direct trimethylsilylation method of different natural or synthetic sialic acids avoiding preceding conversion to the corresponding p-methylketosides leads to derivatives which are distinguishable by glc. This enables the qualitative and in part also the quantitative analysis of sialic acids, either individually or in mixtures. MATERIALS
AND
METHODS
Reagents The silylation reagents trimethylchlorsilane (TMCS), hexamethyldisilazane (HMDS), N,O-bis-trimethylsilylacetamide (BSA), bis-trimethylsilyl-trifluoroacetamide (BSTFA) and N-trimethylsilyl-diethylamine (TMSDEA) were purchased from Applied Science Laboratories Inc., USA. N-trimethylsilylimidazole (TSIM) was obtained from EGAChemie KG, GFR. Sucrose and trehalose obtained from Merck AG, GFR, were used as internal standards. Insta-Gel emulsifier from Packard was used for scintillation counting. All compounds and solvents used were of analytical grade. glc Materials and Apparutus The stationary phases OV-1, OV-17, OV-22, GE-SE-54 and Apiezon L and the supports Gas Chrom Q, 60-80, 80-l 00 and 100-l 20 mesh were obtained from Applied Science Laboratories Inc. Anachrom SD, 80-100 mesh was purchased from Analabs Inc., USA. Pretested 3% OV-17 on Gas Chrom Q, 100-l 20 mesh, was obtained from Applied Science Laboratories Inc. The other column packing materials were prepared using the technique reported by Williams (7), i.e., 1% OV-1 on
GAS-LIQUID
CHROMATOGRAPHY
OF
SIALIC
ACIDS
509
Anachrom SD, 3% OV-17 on Gas Chrom Q, 3% OV-22 on Anachrom SD, and 3% GE-SE-54 on Chromosorb W. Hewlett-Packard glc apparatus Models 402 and 5750 were equipped with flame ionisation detectors (FID) and combined with a Kent integrator Model Chromalog 2. In the Model 402 U-shaped glass columns of 0.8-1.6 m length and 2 or 4 mm internal diameters (i.d.) were used. In the Model 5750 metal columns of max length 2.5 m and 2 mm i.d. were adapted. Operuting
Conditions
Routine analyses were run isothermally. (The results were not fundamentally influenced by application of temperature programs.) The oven temperatures ranged between 170” and 220°C depending on the length of the column and the nature of the stationary phase. The injection port was heated to 40°C and the detector to 50-60°C over the column temperature. The flow rates were 50 ml/min for N, (carrier gas): 30 ml/min for H, . and 300 ml/min for air. For routine analyses (including those of all figures and tables) of sialic acids glass columns were used containing 3% OV-17 on Gas Chrom Q, loo-120 mesh, 1.6 m X 2 mm i.d. and heated to 196°C. Preparation
and IdentiJcution
of Sialic Acids
Sialic acids were released by neuraminidase or by mild acid hydrolysis from glycoproteins of bovine, equine or porcine submandibular glands or from equine erythrocyte membranes, purified by ion-exchange chromatography, and fractionated by passage through a cellulose column using n-butanolln-propanol/water (1 : 2 : 1; by volume) as solvent (1,2,8). They were determined quantitatively by the periodic acid/thiobarbituric acid assay (9) and by the orcinol/Fe3+/hydrochloric acid reagent (10). The purity of the sialic acids eluting from cellulose was checked by tic in different systems (8). The sialic acids were analysed routinely on cellulose thin layers using n-butanolln-propanol/O. 1 N HCl (1: 2: 1; by volume) as solvent (1 1). Their structures were established by analysis of the nature of the N-acyl groups and of the amount, nature and position of the 0-acyl groups as described in detail in Refs. 2 and 8. In addition, mass spectrometry was used for structural analyses of the sialic acids (12). Some of the mass spectral analyses were in combination with glc (13). The following sialic acids were obtained in more than 95% purity (2): N-acetylneuraminic acid, N-glycolylneuraminic acid, N-acetyl-4-O-acetylneuraminic acid, N-acetyl-9-0-acetylneuraminic acid, N-acetyl-7,9di-0-acetylneuraminic acid, N-glycolyl-4-0-acetylneuraminic acid and
510
CASALS-STENZEL,
BUSCHER
AND
SCHAUER
N-glycolyl-9-U-acetylneuraminic acid. N-acetyl-7-U-acetylneuraminic acid could not be obtained as a pure substance as it eluted together with N-acetyl-9-0-acetylneuraminic acid from cellulose. In addition, Nacetyl-9(?)-0-glycolylneuraminic acid, N-acetyl-7(?)-O-glycolylneuraminic acid and N-glycolyl-7(?)-O-acetylneuraminic acid could not be separated from N-glycolyl-9-O-acetylneuraminic acid by cellulose chromatography. N-(:‘H)acetylneuraminic acid ( 11 mCi/mmole) and N- [ 1JdC] glycolylneuraminic acid (0.355 mCi/mmole) were synthesized according to Ref. 14. Neuraminic acid-p-methylglycoside and its methylester, N-chloroacetylneuraminic acid, N-fluoroacetylneuraminic acid and N-bromoacetylneuraminic acid-p-methylglycoside were prepared as described previously ( 15; and unpublished work).
Aqueous solutions containing 1O-l 00 Fg of sialic acids were lyophilized in tapered test tubes ( 10 X 50 mm) and 25 ~1 of TSIM added under nitrogen. The stoppered tubes were whirled for either 15 min at room temperature or 5 min at 60°C in a heating block to complete the silylation reaction which is visible by total solubilization of the substances. With the reagent mixture HMDS-TMCS-pyridine (2: 1: 10: Ref. 4) complete silylation of sialic acids was achieved within 30 min at room temperature. Silylation with BSA, BSTFA. TMSDEA or a mixture of TSIM: BSA: TMCS (3 : 3 : 2) was carried out in the same way. For comparison with the method of direct derivatization of sialic acids described above, P-methylketoside methyl-esters of various sialic acids were prepared by heating for 1 hr at 80°C with 0.02 N (6) or with 0.5 N methanolic HCl followed by trimethylsilylation with the HMDSTMCS-pyridine reagent (4) or TSIM. Qrralitrrti\v
Analysis of Sicrlic Acids
For determination of the retention indices (16) of individual TMSsialic acids, 1 ~1 of the silylation mixture described above was injected, and the times of elution were related to those of C,, and C,, normal alkanes. The preservation of the natural 0-acyl groups of sialic acids in the silylation reaction was established by conversion of these 0-acyl groups before or after derivatization of the sialic acids with TSIM to their acylhydroxamates which were determined quantitatively and qualitatively by calorimetric methods and by tic, respectively, according to Ref. 2 and 8. The nature of the de-0-acylated N-acylneuraminic acid residues was
GAS-LIQUID
CHROMATOGRAPHY
OF
SIALIC
ACIDS
511
identified by glc in the following way: Lyophilized samples of Oacylated sialic acids were exposed for 3 hr to an atmosphere of ammonia over a solution of 5 N ammonium hydroxide at room temperature to hydrolyze the 0-acyl groups. The resulting N-acylneuraminic acid samples were dried again under vacua before trimethylsilylation by TSIM. The retention indices of the sialic acids silylated before or after release of the 0-acyl groups were compared.
To obtain calibration curves, crystalline NeuNAc and NeuNGl were dissolved in water to a final concentration of I .O mglml. N-acetyl-O-acetylneuraminic acids, which appeared pure on tic after passage through cellulose powder, could not be obtained in crystalline form without partial decomposition. They were, therefore, determined quantitatively with the orcinollF&“/HCl reagent ( 10) using the same molar extinction coefficient as for N-acetylneuraminic acid (2). For calculation of the quantity of N-acetyl-0-acetylneuraminic acid, the content of one 0-acetyl was taken into consideration. A solution of NeuNAc-9-OAc (1.25 mglml) was used for calibration. Aliquots ranging between 10 and 100 ~1 were taken from these solutions and mixed with 10 ~1 of a solution containing 50 pg of trehalose or sucrose as internal standards. The lyophilized sialic acid and internal standard mixtures were silylated with 50 ~1 of TSIM, and 1 ~1 (containing 1 pg of the internal standard) was injected. The internal standards sucrose (RI 2732) or trehalose (RI 2827) were used for the sialic acid identification on OV-1 , and trehalose (RI 27 19) on OV-17. Trehalose appears behind NeuNGl on both liquid phases and does not interfere with the different sialic acid peaks. Sucrose (RI 2622) is not suitable for OV-17, as its peak interferes with that of NeuNGl. In some experiments, NeuNAc and NeuNGl also served as internal standards. Calibration curves have not yet been obtained from the other naturally occurring 0-acylated sialic acids as they could not be isolated in sufficient amounts as pure substances. Therefore, the analyses of these sialic acids were semiquantitative in relation of their peak areas to those sialic acids used for calibration. The areas of the glc peaks were measured by automated integration or by the triangulation method.
“{H-labeled NeuNAc and Y-labeled NeuNGl were derivatized and injected as described above. A 1: 10 split was fitted between a column of 0.8 m X 4 mm i.d. packed with 1% OV-1 or 3% OV-23 on Anachrom
512
CASALS-STENZEL,
BUSCHER
AND
SCHAUER
SD, 80-100 mesh, and the detector. Teflon tubes (1.5 mm X 150 mm) were connected to the main exit of the split. The eluted substances were completely condensed in the teflon tubes due to the temperature gradient. The tubes were changed after each peak detected by the FID. They were rinsed with 5-ml Instagel. and the radioactive substances were measured in a Nuclear Chicago, Mark I, liquid scintillation counter. RESULTS AND Derivatization
DISCUSSION
of Sialic Acids
From all silylation reagents tested, TSIM and the mixture of HMDSTMCS-pyridine (4) were found to be the best reagents for silylation of acylneuraminic acids. This was tested in detail with NeuNAc and NeuNGl which result in single and symmetrical peaks in glc. NeuNAc and NeuNGl are trimethyl-silylated at all hydroxyl groups including the carboxyl group, as could be established by mass spectrometry. (A fragment at m/e 726 was obtained from TMS-NeuNAc which corresponds to the M-15 ion fragment and confirms the expected molecular weight of 741 of the sixfold trimethyl-silylated NeuNAc. TMS-NeuNGl resulted in a fragment at m/e 814 which corresponds to the M-l 5 ion and indicates the molecular weight of 829 of the sevenfold trimethylsilylated NeuNGl.) NeuNAc or NeuNGl derivatized by BSA, BSTFA, or a mixture of TSIM-BSA-TMCS (3 : 3 : 2) result in double peaks, as was also found with BSA by Craven and Gehrke (3). One of them is probably due to an additional silylation at the hydrogen position of the amino group of NeuNAc or NeuNGl. In contrast, silylation of these sialic acids by TMCS, HMDS or TMSDEA lead to none or only to low peaks; long reaction times and higher temperatures (60°C) do not improve the silylation effectivity. The 0-acylated sialic acids can also be silylated with TSIM and the HMDS-TMCS-pyridine reagent (4). They do not lose their 0-acyl groups during this procedure. This could be established in the following way: 1. The TMS-derivatives of the different N-acyl-0-acylneuraminic acids result in single and distinct peaks in glc which differ from those of NeuNAc and NeuNGI; 2. NeuNAc- or NeuNGl-peaks as decomposition products of Oacylated sialic acids do not appear on the gas-chromatogram after injection of TMS-derivatives of N-acyl-0-acylneuraminic acids. Samples containing N-acetyl-7,9-di-0-acetylneuraminic acid result in two glc peaks (see below); however, the possible de-0-acetylated products NeuNAc, NeuNAc-7-OAc and NeuNAc-9-OAc could not be detected.
GAS-LIQUID
CHROMATOGRAPHY
OF
SIALIC
513
ACIDS
3. The quantitative and qualitative hydroxamate assay after silylation of 0-acylated sialic acids gives evidence for the preservation of the 0-acetyl- or 0-glycolyl groups during the silylation reaction; 1 mole of 0-acyl per mole of trimethylsilylated N-acyl-0-acylneuraminic acids was found. 4. It was demonstrated by mass spectrometry of the TMS-derivatives of NeuNAc-4-OAc, NeuNAc-7-OAc and NeuNAc-9-OAc that no elimination of the 0-acetyl residues had occurred. 5. NH,-treatment of the 0-acylated sialic acids before silylation gave additional evidence that the individual glc peaks are significant for individual 0-acylated sialic acids. By this method all 0-acylated sialic acids are converted to NeuNAc and NeuNGl respectively leading to the corresponding glc peaks, as will be discussed later. Derivatization of sialic acids by preparing the p-methylketoside methylesters before trimethylsilylation did not prove to be successful in the case of the 0-acylated sialic acids. Most of the 0-acyl residues were released even by using 0.02 N methanolic HCl (Table I), although Yu and Ledeen (6) have reported that de-0-acylation is largely prevented under these mild conditions. The extent of de-0-acylation was established by the hydroxamate test and by glc-analysis of the reaction products shown in Table 1. From this table it can also be seen that NeuNAc or NeuNGl can be better separated from their methylated products and from the methylester of neuraminic acid-fi-methylglycoside on OV- 17 than on OV- 1. The RI-values of the unmethylated and the methylated NeuNAc or NeuNGl are different on OV- 17, however, they are similar on OV- 1. TABLE PRODUCTSFROMMETHANOLYSISOF NeuNAc, DIFFERENTCONCENTRATIONSOF
1 NeuNG1. AND NeuNAc-POAc HCl INMETHANOL~
0.5 N methanolic HCI. I hr. 80°C Methanolysis products NeuNAc
NeuNAc
10.0
Me-NeuNAc-?-OMe NeuNGl Me-NeuNGl-?-OMe
53.0 -
NeuNAc-9-OAc Me-NeuN-2.OMe
37.0
NeuNGl 8.0 56.0
36.0
0.02 N methanohc HCI. 2.5 hr. 80°C
NeuNAc9.OAc 22.7 65 5 -
01 Il.7
NeuNAc 49.0 410 -
NeuNGl 51.0 38.0
10.0
11.0
NeuNAc9.OAc 86.8 II.5 1.7 0
WITH
RI on ov-17
RI on ov-I
2488 2557 2653 2742 2598 2319
2483 2498 2674 2703 2523 2284
L1The %-relationship of the peak areas of the TMS-derivatives on 3% OV-I7 (column 1.6 m x 2 mm i.d.: 196°C) and the RI-values on OV-17 and 1% OV-I (column 1.6 m x 2 mm i.d.: 206°C) are shown. Only the main peaks of the gaschromatograms were taken to calculate the percentage of the compounds. These were identified by reference substances (14) or according to Yu and Ledeen (6). Me-NeuN-2.OMe corresponds to the methylester of neuraminic acid-@methylglycoside. (The peak may also contain the free aad which cannot be distinguished from its methylester under the glcconditions chosen (see Table 3). Me-NeuNAc-2.OMe and Me-NeuNGI-2.OMe are taken to represent the methylesters of the /3-methylglycosides of NeuNAc or NeuNGI.
514
CASALS-STENZEL,
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AND
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The silylated sialic acids can be stored under dry nitrogen without noticeable decomposition.
at +4”C
The material of the column whether glass or metal, has no influence on the quality of the separation of sialic acids. The length of the column is of great influence on the separation, especially of the 0-acylated sialic acid isomers, as their retention indices do not differ greatly from one another. Although NeuNAc and NeuNGl can be separated satisfactorily on a column of 0.4 m length, a 2.5 m column is needed for the complete separation of, e.g., NeuNAc-7-OAc and NeuNAc-9-OAc on OV-17. However, the better resolution of sialic acids on long columns is accompanied by a higher loss of the substances resulting in an appreciable decrease of the peak areas as will be discussed below. Therefore, in the routine analyses a compromise length of 1.6 m and 2 mm i.d. was used. While 0-acylated sialic acids cannot be separated from N-acylneuraminic acids on a 60-80 mesh support covered by 3% OV-17 and under the conditions described above, the use of 100-I 20 mesh support gives sharp and symmetrical peaks of the different sialic acids. The middle polar phases OV-I 7 and OV-22 have been proven best for the resolution of the different derivatives of neuraminic acid. However, the peaks appeared more symmetrical and sharper on OV-17 than on OV-22. The nonpolar phase OV-1 (or Apiezon L or GE-SE-54) is useful only for special analyses, e.g., for mixtures of NeuNAc and NeuNGl, or for estimation of the total amount of N-acyl-O-acylneuraminic acids. 0-Acylated sialic acids cannot be separated suitably on this material. The retention indices of the naturally occurring sialic acids on OV-17 and OV-I are compared in Table 2 (see also Fig. 4). The TMS-sialic acids do not elute from polar phases. For analysis, particularly of sialic acid mixtures, we recommend the use of two differently loaded columns (e.g., OV-1 and OV-17) to avoid errors in the identification of the peaks in agreement with other authors (4,6).
Qlralituti,v Identification of Siulic Acids TMS-NeuNAc and TMS-NeuNGl result in single symmetric peaks eluting from the nonpolar or middle polar stationary phases chosen. It has already been explained that the eluted substances detected by the FID represent the per-trimethylsilylated NeuNAc and NeuNGl, respectively, according to mass spectral analysis. The possibility of appreciable decomposition of these TMS-sialic acids during passage through the glc-columns which would lead to a con-
GAS-LIQUID
CHROMATOGRAPHY
OF
TABLE RErENrroN
INDICES
OF TMS-DERIVATIVES
MATERIALSON
SIALIC
515
ACIDS
2
OF SIALIC
3% OV-17 (196°C)
Sialic acids NetrNAc NeuNAc-COAc NeuNAc-7-OAc NeuNAc-Y-OAc NeuNAc-7.9-di-OAc NeuNAc-4-OGl NeuNAc-7(?)-OGl NeuNAc-9(?)-OGl NeuNGl NeuNG I-4-OAc NeuNG I-7(?)-OAc NeuNG I-9-OAc
ACIDS
AND
1%
FROM
BOVINE
AND
EQUINE
OV-1 (206°C)”
ov-17
OV-I
2488 2637 2622 2599 2702 2560 2599 2560 2653 2758 2775 2758
2483 2561 2522 2522 2648 2535 3540 2535 2674 2720 2700 2720
If Glass columns. 1.6 m x 3 mm i.d. The data are average values from two to three different preparations and from three injections each.
fusing peak pattern was further excluded by radio-glc of TMSNeuN [3H] AC and TMS-NeuN [““Cl Gl. As can be seen from Fig. 1 the bulk of the “H- or ‘“C-radioactivities eluted from the columns in accordance with the peaks recorded by the FID, and only small amounts of the radioactivity were found to appear before or after the peaks. On average, 60% of the injected radioactivity did not elute from a 1.6-m column while 30-35% of the eluted radioactivity corresponded to the
,I
I
I 10
I
m 20
[min]
FIG. I. Radio-glc of a mixture of NeuN(3H)Ac and NeuN(l-“C)Gl. 3% OV-22 on 80-100 mesh Anachrom SD, 0.8 m X 4 mm i.d.: split 1: 10; program: Initiai temperature 188°C. after 5 min delay increase of 2”C/min. The eiuting radioactivity (vertical lines) measured by the **C- or JH-channels is collected as indicated by the dotted lines.
516
CASALS-STENZEL,
BUSCHER
BEFORENHI 12%s6710
AND
SCHAUER
AFTERNng 1’
2’3’
10
20
FIG. 2. Comparison of tic on cellulose and glc on 3% OV-I 7 of sialic acids from bovine submandibular gland glycoproteins. Glass column: I .6 m x 2 mm i.d.; isothermally 196°C. The tfc-sialic acid spots (left side) are marked a-e. The gas-liquid chromatograms of the siahc acids from the tic-spots are shown in the middle part of the figure (before NH,). On the right side the same sialic acids de-f)-acylated by NH,-treatment are shown (after NH,). Peak ~denti~cation: 1, NeuNAc; 2, NeuNAc-9(?)-OGI; 3, NeuNAc-7(?)-OGI and NeuNAc-9-OAc: 4. NeuNAc-7-OAc; 5, NeuNG1: 6, unknown sialic acid: 7, NeuNAc7,9-di-OAc: 8, NeuNGl-9-OAc; 9, NeuNGl-7(?)-OAc: 1’. NeuNAc: 2’, NeuNGI: 3’. alkaline degradation product of sialic acids of unknown structure.
GAS-LIQUID
CHROMATOGRAPHY
OF
SIALIC
517
ACIDS
:d
30 [nnin)
10
20
30
[min]
FIG. 3. comparison of tic on cetlulose and glc on 3% OV-17 of sialic acids from equine subm~dibular gland glycoproteins. The method of analysis and the glc-conditions correspond to those of Fig. 2. Peak identification: 1, NeuNAc: 2. NeuNAc-4-OGI: 3. unknown sialic acid (NeuNAc-9-OAc ?); 4, NeuNAc-4-OAc; 5, NeuNG1: h, NeuNGl-4-OAc; 1’. NeuNAc: 2’. NeuNGI: 3’. unknown alkaline degradation product of sialic acids.
NeuNAc or NeuNGl peaks, a value which agrees favourably with the results obtained with neutral mono- and disaccharides by Jansen and Baglan ( 17). The individual 0-acylated siahc acids also result in single peaks. Their retention indices are different from NeuNAc and NeuNGl and are distinguishable from one another on middle polar phases as OV-17 and OV-22 (Table 2, Figs. 2-4). The identification of the glc-peaks of the Oacylated sialic acids was carried out by correlation of their relative retention times with those of the TMS-derivatives of sialic acids which had been characterized before (2). For further and direct proof of the nature of the O-acylated sialic acids their ~-acy~neuraminic acid residues
518
CASALS-STENZEL,
lrnirll
BUSCHER
AND
SCHAUER
I rn,l,
4. Gas-liquid chromatograms of sialic acids from bovine submandibular gland glycoproteins on 1% OV-1 and on 3% OV-17, 1.6 m X 2 mm i.d.: run isothermally at 206°C and 196”C, respectively. Peaks: I, NeuNAc; 2, NeuNAc-9(?)-OGl: 3, NeuNAc7(?)-OG1 and NeuNAc-9-OAc together: 4, NeuNAc-7-OAc: S. NeuNGl; 6, unknown sialic acid: 7, NeuNAc-7,9-di-OAc: 8. NeuNGL9-OAc also containing NeuNGl-7(?)-OAc. FIG.
(NeuNAc or NeuNGl) de-0-acylated with the aid of ammonia vapour were identified by glc. In Figs. 2 and 3 the analysis of sialic acids from bovine and equine submandibular gland glycoproteins by tic on cellulose is compared with the analysis by glc on OV-17. (The sialic acids elute from cellulose columns in the same sequence as they migrate on cellulose thin-layer.) While NeuNGl (“a”) or NeuNAc (“b”) spots from bovine and equine sialic acids appeared homogeneous in glc and were not influenced by ammonia vapour, spot “c” of Fig. 2 (bovine sialic acids) resulted in 4 peaks which could be de-0-acylated to NeuNAc and NeuNGl, respectively. The higher peaks of the two peak groups correspond to N-acetyl9(?)-0-glycolylneuraminic acid (RI 2560) and N-glycolyl-9-O-acetylneuraminic acid (RI 2758), respectively. The two smaller peaks with somewhat higher retention times are assumed to be the isomeric Nacetyl-7(?)-0-glycolylneuraminic acid (RI 2599) and N-glycolyl-7(?)-0acetylneuraminic acid (RI 2775), respectively. The position of the 0-acyl groups at C, is assumed in analogy to the structure of N-acetyl-70-acetylneuraminic acid isolated earlier from bovine submandibular gland glycoproteins (8); however, direct experimental proof by mass spectrometry of the assumed structures of these NeuNAc-7(?)-OGl and NeuNGl-7(?)-OAc is not yet available. The ratios of these four sialic acids were different in individual preparations. The corresponding tic spot (“c”) from equine sialic acids (Fig. 3) containing only minute amounts of sialic acids resulted in three peaks which could be converted
GAS-LIQUID
CHROMATOGRAPHY
OF
SIALIC
ACIDS
519
to NeuNAc and NeuNGl by ammonia treatment. Peak No. 2 is derived from N-acetyl-4-0-glycolylneuraminic acid (RI 2560), the identification of which was possible as it was found in individual sialic acid fractions from equine submandibular glands with 70% purity (2). Peak No. 4 contains N-acetyl-4-0-acetylneuraminic acid (RI 2637). Peak No. 6 consists of N-glycolyl-4-0-acetylneuraminic acid (RI 2758) which was obtained in nearly pure form from equine erythrocyte membrane. The tic spot “d” from the bovine sialic acids (Fig. 2) consists of the 70-acetyl- and 9-0-acetyl isomers of N-acetyl-0-acetylneuraminic acid, yielding NeuNAc after ammonia treatment. The NeuNAc-9-OAc peak (main peak; RI 2599) was established from one of the preparations, in which a few of the fractions from cellulose chromatography contained pure NeuNAc-9-OAc. The shoulder of the NeuNAc-9-OAc peak could be attributed to NeuNAc-7-OAc (RI 2622) by comparison of the RIvalue with that resulting from NeuNAc-7-OAc isolated earlier (8). The structure of the TMS-derivative of NeuNAc-7-OAc was further established by glc-mass spectrometry. The corresponding equine sialic acid spot (“d”) of Fig. 3 also contains two isomeric N-acetyl-O-acetylneuraminic acids. Among these NeuNAc-4-OAc prevails in all preparations from equine origin. However, the second N-acetyl-O-acetylneuraminic acid present in this preparation (peak No. 3) elutes with a retention index of 2606 which is similar to that of NeuNAc-9-OAc found in bovine tissues. More analytical data are required to establish the structure of this new equine N-acetyl-0-acetylneuraminic acid which was detected in several preparations. The uppermost tic spot (“e”) of Fig. 2 leads to two peaks in glc which can be degraded to NeuNAc under alkaline conditions. The main peak (No. 7) with the higher retention time (RI 2702) is due to N-acetyl-7,9di-0-acetylneuraminic acid found in some preparations as a pure substance (2). The structure of the second sialic acid present in this preparation which is doubly 0-acylated, is unknown. An unknown compound is produced in variable amounts by NH,treatment of the different sialic acids which appears after NeuNGl (peak No. 3’; RI 2690) and is believed to be a degradation product, probably a pyrrol derivative of sialic acids. Figure 4 shows the glc analysis on OV-17 or OV-1 of a mixture of sialic acids isolated from bovine submandibular gland glycoproteins. Appreciable differences in the resolution of the various sialic acids can be seen between OV-1 and OV-17. From OV-17, the TMS-sialic acids elute as distinct peaks in the following sequence: NeuNAc is eluted initially followed by its 0-acyl derivatives; NeuNAc-9(?)-OGl, then NeuNAc-7(?)-OGl and NeuNAc-9-OAc eluting together and then NeuNAc-7-OAc. (NeuNAc-4-OAc from equine submandibular gland glycoproteins elutes after NeuNAc-7-OAc.) NeuNGl is then eluted
520
CASALS-STENZEL,
BUSCHER
AND
SCHAUER
followed by NeuNAc-7,9-di-OAc and finally the O-acylated derivatives of NeuNGl: NeuNGl-9-OAc and NeuNGl-7(?)-OAc. The elution sequence of TMS sialic acids on OV-17 may be related to the polarity and the molecular size of the silylated sialic acids. In the Nacetyl-0-acetylneuraminic acid series it is remarkable that the retention indices (see Table 2) become higher if the 0-acetyl groups are closer to or even in the pyranoside ring of neuraminic acid. The nature of peak No. 6 (RI 2676) from OV-17 is unknown, as are the other small peaks which can be seen in the regions between and after NeuNAc-7,9-di-OAc and NeuNGl-9-OAc. It may be speculated that these peaks are due to hypothetical neuraminic acid derivatives with other combinations of glycolyl and acetyl groups such as, e.g., Nglycolyl-0-glycolylneuraminic acid, N-glycolyl-di-0-acetylneuraminic acid or N-acetyl-0-acetyl-0-glycolylneuraminic acid. We were not able to separate NeuNAc-9-OAc and NeuNAc-7(?)OGl which have identical retention indices on OV-17, even on columns longer than 1.6 m. The apolar liquid phase OV-1 is not qualified for the separation of most of the individual 0-acylated sialic acids as the N-acetyl-o-acetylneuraminic acids appear together, shortly after NeuNAc as a broad unsymmetrical peak, and the N-glycolyl-O-acetylneuraminic acids elute near NeuNGl (Fig. 4). OV-1 is better suited than OV-17 only for the analysis of the sialic acids from equine submandibular gland glycoproteins, as NeuNAc-4-OAc, the prevailing 0-acylated sialic acid from this material, appears on OV-1 between NeuNAc and NeuNGl as a well separated peak. On OV-17 NeuNAc4-OAc partially overlaps with NeuNGl. IdentiJication of Synthetic. Neumminic Acid Derituti\?es Three percent OV- 17 or 1% OV- 1 columns are also suitable for identification of synthetic neuraminic acid derivatives such as neuraminic acid-p-methylglycoside or its methyl ester, and some sialic acids containing N-halogenacyl groups (Table 3). These sialic acids which were TABLE RETENTION
INDICES DERIVATIVES
Sialic
3
OF TMS-DERIVATIVES ON 1% OV-1 (206°C)
OF SYNTHETIC NEURAMINIC AND 3% OV-17 (196°C)”
acids
ov-17
Neuraminic acid-/3-methylglycoside and its methylester N-fluoroacetylneuraminic acid N-chloroacetylneuraminic acid N-bromoacetylneuraminic acid-fi-methylglycoside n Glass
columns,
1.6 x 2 mm
i.d. The
values
are the mean
ov-I
2319 2442 2594 2774 of three
ACID
2284 246 1 2579
analyses.
GAS-LIQUID
CHROMATOGRAPHY
OF
SIALIC
521
ACIDS
silylated in the same way as the naturally occurring sialic acids, resulted in single and symmetrical peaks most of them having distinct retention indices. Only neuraminic acid-p-methylglycoside and its methylester eluted together from OV-17. Glc may therefore be useful for following the synthesis of these or other sialic acids, especially for the assay of their yields and purity.
The relative detector responses (RDR)’ of NeuNAc, NeuNGl and NeuNAc-9-OAc on OV-1 and OV-17 using two column lengths are summarized in Table 4. The low RDR values of NeuNAc-9-OAc on both liquid phases when compared with those of NeuNAc or NeuNGl is striking. The RDR values decrease appreciably below those given in Table 4 at NeuNAc or NeuNGl concentrations below 0.5 pg/pl TSIM. The latter phenomenon has also been reported for NeuNAc by Yu and Ledeen (6). The calibration curves for NeuNAc. NeuNGl and NeuNAc-9-OAc on OV-1 and OV-17 are shown in Fig. 5. The calibration curves do not originate from zero when the peak areas of the TMS-sialic acids are related to TMS-sucrose or TMS-trehalose, but they are linear in the concentration ranges shown. The calibration curves intersect at zero, if NeuNAc serves as standard for the determination of NeuNGl and vice versa. However, the standard curve for NeuNAc-9-OAc does not originate from zero, even when NeuNAc is used as internal standard. While the slopes of the curves for NeuNAc and NeuNGl are identical on OV-17 and do not differ greatly for OV-1, the slope of the curve for NeuNAc-9-OAc was found to be about three times lower on both liquid phases when compared with NeuNAc. These differences may be due to adsorption phenomena on the
INFLUENCE
OF COLUMN RESPONSES
LENGTH
OF NeuNAc.
Column length (ml
NeuNAc
NeuNGl
1.6 0.8
0.402 i 0.022 0.531 t 0.030
0.294 t 0.021 0.510 f 0.035
TABLE 4 (2 mm i.d.) ON THE RELATIVE NeuNGl AND NeuNAc-9-OAc”
DETECTOR
ov-17
ov- I NruNAc-9.OAc
0.258 k 0.022 0.440 k 0.020
NeuNAc
NeuNGl
0.458 2 0.022 0.567 i 0.030
0.451 t 0.026 0.548 i 0.038
NeuNAc-Y-OAc
0.212 + 0.024 0.366 c 0.025
‘I Glc on lq, OV-I (temperatures: 1.6 m column. 206°C; and 0.8 m, 196°C) and 3% OV-17 (temperatures: 1.6 m column, 196°C: and 0.X m. 186°C) on X0-100 mesh Gas Chrom Q. The values are average* from IO independent ramples each Injected three times and ranging between 1.0 and 3.0 wg sialic aad per ~1 TSIM injected. As internal amdards trehalose on OV-17 and sucrose on OV-I were used: each I pg/pl TSIM.
’ RDR = [area (sialic acid)].
(sialic
acid)]/[area
(int.
standard)]
X
[weight
(int.
standard)]/[weight
522
CASALS-STENZEL,
BUSCHER
AND
SCHAUER
FIG. 5. Calibration curves obtained with NeuNAc, NeuNGl and NeuNAc-9-OAc on 3% OV-17 (196°C) and 1% OV-1 (206°C) columns of 1.6 m length and 2 mm i.d. See text for further details.
packing materials of the sialic acids tested. It has already been outlined above that only about 35% of the radioactivity from NeuNAc or NeuNGl is eluted from 1.6 m columns. ~o~esponding data from NeuNAc-9-OAc are not yet available. The appreciable Iower RDR values of NeuNAc-POAc may be explained by a stronger adsorption of this TMS-sialic acid to the column materials. Errors in the quantitative estimation of this sialic acid were excluded by calorimetric control of the sialic acid amounts injected. The great RDR differences between NeuNAc and NeuNAc-9-OAc cannot be explained by an altered sensitivity of the FID due to the presence of only 5 TMS groups in TMSNeuNAc-9-OAc (18). When using a 0.8 m instead of a 1.6 m cohrmn the RDR values for NeuNAc and NeuNGl relative to sucrose or trehalose increase appreciably (Table 4), probably due to reduced adsorption and decomposition. For sensitive quantitative sialic acid analyses therefore, the columns should be as short as possible. However, short columns can be used only if a small number of sialic acids with significantly different retention times are present, e.g., mixtures of NeuNAc and NeuNGl. The ratio of
GAS-LIQUID
CHROMATOGRAPHY
OF
SIALIC
ACtDS
523
NeuNAc to NeuNGl from porcine submandibular gland glycoproteins established already as 1: 10 ( 19) was confirmed by glc on OV- 17. The accuracy of this method for the quantitative analysis of acylneuraminic acids was determined by repeated injection of five equally prepared samples; the limit of error was within 7% with the 1.6 m and 5% with the 0.8 m column. The smallest amount of NeuNAc and NeuNGl which can accurately be determined under the conditions described is about 0.5 pg and that of NeuNAc-9-OAc between l.S and 2.0 pg (Fig. 5). The minimally detected amount was about 10 ng for both TMS-NeuNAc (in agreement with Ref. 3) or TMS-NeuNGl and about 20 ng for TMS-NeuNAc-9-OAc. The appreciable difference of the sensitivity in glc-analysis observed between NeuNAc and NeuNGl on the one hand and NeuNAc-9-OAc on the other indicates that calibration curves are required for the exact quantitative analysis of the various U-acylated sialic acids occurring in mixtures from biological origin. The relation of the peak areas from the various 0-acylated sialic acids to NeuNAc, NeuNGl or NeuNAc-9OAc leads to only approximate quantitative values. However, the relative amounts of the many different sialic acids occurring in biological sources, e.g., in bovine submandibular gland glycoproteins (Fig. 4) can quickly be appraised without using an internal standard. The isolation of sufficient amounts of the individual sialic acids in pure form for their quantitative analysis (determination of the RDR values, calibration of columns) is under investigation. These sialic acids are also required as reference substances for the qualitative analysis of sialic acid mixtures of unknown composition.
The experiments demonstrate the comprehensive and reproducible applicability of glc for the analyses of different types of sialic acids. This method is better than tic not only on account of the requirement of smaller amounts of substances but also with regard to the possibility to analyse the different 0-acyl isomers of sialic acids, as they lead to distinct glc-peaks. It is shown in Figs. 2 and 3 that sialic acid spots appearing homogeneous in tic in part result in different glc peaks, each peak representing an individual isomer of an 0-acylated sialic acid. Therefore, tic can be used only for an approximate estimation of the composition of a sialic acid mixture. Thus glc is shown to be a valuable method to check the purity and occurrence of the various types of sialic acids derived from biological materials, chemical syntheses or enzymatic assays. The method is, however, nearly as time consuming as tic as the sialic acids must be carefully purified by ion-exchange chromatography to obtain good resolution on
524
CASALS-STENZEL,
BUSCHER
AND
SCHAUER
glc. The advantages of glc over the usual calorimetric methods for sialic acid analysis have been discussed for NeuNAc by Craven and Gehrke (3), as other carbohydrates and fatty acids do not interfere with sialic acids in glc. The direct trimethylsilylation of sialic acids without previous conversion to the P-methyl-ketosides first used for NeuNAc by Craven and Gehrke (3) has the advantage of speed and as our experiments clearly have shown of full preservation of the 0-acyl groups. It may be expected that in the field of sialic acid chemistry more analytical problems can now be solved by application of glc, e.g., the question of migration of 0-acyl groups, which still is open (2). The sensitivity of the glc method may lead to the discovery of still more neuraminic acid derivatives occurring in nature. Combination of glc and mass spectrometry will enable the elucidation of the structures, especially concerning the position of the 0-acyl groups, of those sialic acids which have been detected only in minute amounts in sialic acid mixtures. ACKNOWLEDGMENTS We are indebted with many thanks to Mrs. G. Malske. Mrs. M. Wember and Mrs. B. Wrage for the excellent technical assistance. The financial support by the Deutsche Forschungsgemeinschaft (Grant Scha 202/l and 202/3) and by the Fonds der Chemischen lndustrie is gratefully acknowledged. We thank the Erwin Riesch Stiftung for the provision of a grant for instruments.
REFERENCES I. 2. 3. 4.
Schauer, Buscher, Craven, Sweeley, SW.
R. ( 1973) Anger. Chem. Intern. Edit. 12, 127. H.-P., Casals-Stenzel, J., and Schauer, R. (1974) Eur. J. B&hem. D. A., and Gehrke, G. W. ( 1968) J. Chromutogr. 37, 414. C. C.. Bentley. R.. Makita, M.. and Wells, W. W. (1963) J. Amer.
50,
71.
Chem.
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