Partial synthesis of ganglioside and lysoganglioside lipoforms as internal standards for MS quantification

Article abstract of DOI:10.1194/jlr.D054734

Within recent years, ganglioside patterns have been increasingly analyzed by MS. However, internal standards for calibration are only available for gangliosides GM1, GM2, and GM3. For this reason, we prepared homologous internal standards bearing nonnatural fatty acids of the major mammalian brain gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, and of the tumor-associated gangliosides GM2 and GD2. The fatty acid moieties were incorporated after selective chemical or enzymatic deacylation of bovine brain gangliosides. For modifi cation of the sphingoid bases, we developed a new synthetic method based on olefi n cross metathesis. This method was used for the preparation of a lyso-GM1 and a lyso-GM2 standard. The total yield of this method was 8.7% for the synthesis of d17:1-lyso-GM1 from d20:1/18:0-GM1 in four steps. The title compounds are currently used as calibration substances for MS quantifi cation and are also suitable for functional studies.

Full text of DOI:10.1194/jlr.D054734

Partial synthesis of ganglioside and lysoganglioside
lipoforms as internal standards for MS quantification
Martin Gantner1, Günter Schwarzmann1, Konrad Sandhoff1, and Thomas Kolter1*
1LIMES, Membrane Biology & Lipid Biochemistry Unit, c/o Kekulé-Institut für Organische Chemie und
Biochemie, Universität Bonn, Gerhardt-Domagk-Str. 1, D-53115 Bonn, Germany
Correspondence to Thomas Kolter*, Ph.D. at Life & Medical Sciences, University of Bonn, Germany. Tel.:
+49 228 735798; Fax: +49 228 737778; E-mail: tkolter@uni-bonn.de.
Running title: Non-natural ganglioside and lysoganglioside lipoforms1
1 Abbreviations: CHN analysis, carbon hydrogen nitrogen analysis; DCC, N,N‘-dicyclohexylcarbodiimide; DMAP, 4-
dimethylaminopyridine; DEAE-, diethylaminoethyl-; GC-FID, gas chromatography with flame ionization detector;
GSL, glycosphingolipid; HPTLC, high performance thin-layer chromatography; MAG, myelin-associated
glycoprotein; Neu5Ac, N-acetylneuraminic acid; NP, normal phase, RP, reversed phase; RT, room temperature;
SCDase, sphingolipid ceramide N-deacylase; VEGF, vascular endothelial growth factor.
1

Abstract
Within the recent years, ganglioside patterns have been increasingly analyzed by mass spectrometry.
However, internals standards for calibration are only available for gangliosides GM1, GM2, and GM3. For
this reason, we prepared homologous internal standards bearing non-natural fatty acids of the major
mammalian brain gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, and of the tumor associated
gangliosides GM2 and GD2. The fatty acid moieties were incorporated after selective chemical or
enzymatic deacylation of bovine brain gangliosides. For modification of the sphingoid bases, we developed
a new synthetic method based on olefin cross metathesis. This method was used for the preparation of a
lyso-GM1- and a lyso-GM2 standard. The total yield of this method was 8.7 % for the synthesis of d17:1-
lyso-GM1 from d20:1/18:0-GM11 in four steps.
The title compounds are currently used as calibration substances for MS quantification and are also suitable
for functional studies.
Supplementary key words: brain lipids · chemical synthesis · glycolipids · lipidomics · mass spectrometry
· sphingolipids · olefin metathesis
1
2

Introduction
Gangliosides are sialic acid containing glycosphingolipids (GSLs). They can be found in vertebrates and,
with a few exceptions, not in invertebrates (1). Gangliosides are especially abundant in neuronal tissues,
where their content is one to two orders of magnitude higher than in extraneural tissues (2). In the brain,
gangliosides together with other glycosphingolipids are the main glycan carriers (80 % of the total glycan
mass in adult rat brain) (3). The main gangliosides in adult mammalian brain are GM1 (Fig. 1), GD1a,
GD1b, GT1b, 9-O-Ac-GT1b, and GQ1b (4). They contain mostly C18- and C20-sphingosine acylated with
stearic acid, which constitutes more than 80 % of the total ganglioside fatty acid content in the nervous
system (4). In mammals, C20-sphingsosine-containing gangliosides can only be found in significant
amounts in the nervous system (5). In contrast to adult brain, different glycosphingolipid- and ganglioside-
series are expressed in the developing nervous system (6).
Subcellularly, the majority of gangliosides is localized in the outer leaflet of the plasma membrane. They
are anchored to the membrane by their hydrophobic ceramide part while their hydrophilic carbohydrate part
extrudes into the extracellular space. They are part of the cellular glycocalyx and interact with molecules
outside the cell (“trans” interaction) and within the same membrane (“cis” interaction) (1). Examples are
the “trans”-interaction of ganglioside GM1 with cholera toxin (7), of NeuAcα2-3Galß1-3GalNAc-termini
on axonal gangliosides such as GD1a, GT1b, and GM1b with the myelin-associated glycoprotein MAG
(8), and the “cis”-interaction of GM3 with the receptors for insulin (9), epidermal growth factor (10), or
VEGF (11).
In the past, ganglioside patterns have been largely determined by TLC followed by densitometric
quantification (12, 13). Within recent years, ganglioside determinations by mass spectrometric methods
have been increasingly applied (14). In addition to ganglioside classes, which are defined by the
carbohydrate head group, MS allows also profiling of ganglioside lipoforms (15) with different structure of
acyl chain and sphingoid base. Ganglioside quantification is applied to ganglioside pattern investigation
(16), in food analyses (17, 18), but also in the analysis of lysosomal storage diseases (19) and in quantitative
imaging mass spectrometry (20). Lysogangliosides play a major role in the pathogenesis of gangliosidoses.
3

Elevated levels of lyso-GM2 and lyso-GA2 are present in the brains of patients with GM2 gangliosidoses
(21), and elevated levels of lyso-GM1 and lyso-GA1 are present in patients with GM1 gangliosidosis (22).
Both substances are potential biomarkers for these diseases (23) and the lysoganglioside standards prepared
in this work can be used for their quantitative analysis.
Quantification of gangliosides by mass spectrometry including quantitative imaging mass spectrometry
requires calibration substances. Hereby, calibration is defined as the establishment of a correlation between
analyte concentration and mass spectrometer response. Three principle methods can be distinguished:
external calibration, standard addition calibration, and internal standard calibration. The internal standard
calibration shows the highest accuracy and precision since standard and analyte are measured in the same
sample at the same time, and the internal standard acts as a self-correcting system for analyte losses during
purification steps and for fluctuations in mass spectrometer response (24). Internal standards are chemically
and physically similar, but non-isobaric derivatives of the analyte. Suitable are stable isotope labeled
derivatives (type 2 internal standards) or homologous derivatives (type 3) of the analyte. Type 1 internal
standards, which are any non-isobaric compounds with similar physical and chemical properties, are less
accurate. Usually, the standard is added in a defined amount to the sample at an early stage of sample
handling. After purification of the sample, the ratio of the MS response of analyte and standard is measured.
The concentration of the analyte can be calculated by the following equation (24):
ꢃꢁꢂ
ꢀꢁꢂ
=
∙ ꢀꢄꢅ = ꢆꢇ ∙ ꢃꢁꢂ
ꢄꢅ
ꢃꢄꢅ
(I: intensity, an: analyte, IS: internal standard, c: concentration, CFIS: calibration factor)
We report on the preparation of a set of new ganglioside internal standards for the major mammalian brain
gangliosides and the tumor-associated gangliosides GM2 and GD2. The title substances were prepared from
a mixture of gangliosides from bovine brain, which was separated first into ganglioside classes and then
into pure lipoforms. Non-natural chain length in the fatty acid part were introduced by selective chemical
4

and enzymatic deacylation and reacylation. Furthermore, we developed a new method for the modification
of the sphingosine chain length by olefin cross metathesis (Fig. 1). The method was applied for the synthesis
of a lyso-GM1- and a lyso-GM2 standard.
5

Materials and Methods
Materials
All chemicals were of analytical grade or the highest purity available. Water used for buffers and solutions
was purified by an ultrapure water system (EASYpure UV/UF D8612, Werner Reinstwassersysteme,
Leverkusen, Ger). Solvents used in reactions of oxygen- and moisture sensitive compounds were in
anhydrous form. 1-Propanol, methanol, pyridine, and acetic anhydride were degassed before use. The native
mixture of bovine brain gangliosides Cronassial®, which consists of 21 % of GM1, 40 % of GD1a, 16 % of
GD1b, 19 % of GT1b, and 4 % of other gangliosides (25), was available in our lab. Sphingolipid ceramide
N-deacylase (SCDase) and beta-galactosidase from bovine testes were from Sigma Aldrich (Schnelldorf,
Ger). Triton™ X-100, sodium taurodeoxycholate, Grubbs catalyst 2nd gen., Hoveyda-Grubbs cat. 2nd gen.,
and Stewart-Grubbs catalyst were also from Sigma Aldrich. For NP column chromatography, silica gel 60
(0.040-0.063 mm or 0.015-0.040 mm) from Merck (Darmstadt, Ger) was used. For desalting and RP column
chromatography, LiChroprep® RP-18 (0.040-0.063 mm) from Merck was used. For TLC analysis, HPTLC
silica gel 60 F254- and HPTLC silica gel 60 RP-18 plates from Merck were used. For anion-exchange
chromatography, DEAE Sephadex™ A-25 from Amersham Pharmacia Biotech AB (Uppsala, Swe) was
used.
Purification
Desalting was performed by the method of Wiliams and McCluer (26). The usage of polar eluents for
column chromatography causes significant contamination of column material in the products. We reduced
this contamination to a minimum by the following method. NP silica gel columns of a very small column
volume (approx. 1 mL column volume per 5 mg of product) were prepared in filtration columns from
Supelco (Bellefonte, USA) using a mobile phase in which the substance is retained on the column. The
substance was applied to the column and washed with three column volumes of the same solvent. Then, the
substance was eluted by an appropriate solvent of higher polarity.
6

Analytical procedures
1
13
For H- and C-NMR spectroscopy, Bruker Avance 300, 400, or 500 instruments (Billerica, USA) were
used. For exchange of exchangeable protons by deuterium, gangliosides were dissolved in CD3OD and the
solvent was removed in a stream of nitrogen. This process was repeated three times before measurement.
For EI-MS, a MAT 95 XL sector field instrument (Thermo Finnigan MAT GmbH, Bremen, Ger) or a MAT
90 sector field instrument (Thermo Finnigan MAT GmbH) was used. For ESI-MS and ESI-MS/MS, a nano-
ESI-QTOF mass spectrometer (Micromass, Manchester, UK) was used. In general, measurements were
conducted in positive ion mode. For interpretation of the ESI-MS/MS spectra, the nomenclature for
carbohydrate fragments by Domon and Costello was applied (27). For GC-MS, a GCMS-QP2010 Ultra
(Shimadzu, Kyoto, Jpn) equipped with a ZB-5MSi column (l = 30 m, Ø = 0.25 mm, df (film thickness) =
0.25 µm) containing a 5 % phenyl and 95 % dimethylpolysiloxane phase was used. For CHN-microanalysis,
a vario EL cube (Elementar Analysensysteme GmbH, Hanau, Ger) was used.
In general, ganglioside yields were determined by dissolving the ganglioside in a defined volume of
methanol or water and subsequent sialic acid determination. Ganglioside lipoforms which were quantified
by CHN analysis before were used as external standards for the quantification of the synthesized
ganglioside derivatives. Photometric sialic acid determination was performed by the resorcinol method of
Svennerholm (28) modified by Mietinnen and Takkiluukkainen (29) using an Ultrospec III (Pharmacia
Biotech, Uppsala, Swe). Fluorimetric sialic acid determination was performed using the sialic acid assay
kit EnzyChrom™ ESLA-100 of BioAssay Systems (Hayward, USA) and a Fluoroscan II (Labsystems Oy,
Helsinki, Fin).
Purification of bovine brain gangliosides
To obtain pure natural ganglioside lipoforms as starting material, the ganglioside mixture Cronassial® was
purified by anion-exchange chromatography, followed by desalting, NP column chromatography, and RP
column chromatography. In the first step, a slightly modified method of Momoi et al. was used (30). Briefly,
5.03 g of ganglioside mixture were applied to a DEAE Sephadex™ A-25 column (2.2 × 22 cm) and
7

ganglioside classes were eluted by a step gradient of different ammonium acetate concentrations in
methanol. After desalting, the ganglioside classes were obtained as colorless solids. GQ1b was already
obtained in pure form after these steps. From 5.03 g of starting material 1.28 g of monosialogangliosides,
2.80 g of disialogangliosides, 0.674 g of trisialogangliosides, and 41.5 mg of GQ1b were obtained. Then,
proportions of these ganglioside classes were separated by NP column chromatography. The
monosialogangliosides were separated isocratically using the solvent system CHCl3/MeOH/0.2 % CaCl2
60:35:8 v/v/v to obtain GM1. The disialogangliosides were separated by a step gradient of
CHCl3/MeOH/2.5 M NH3 60:35:6 v/v/v and CHCl3/MeOH/2.5 M NH3 60:35:8 v/v/v to obtain GD1a and
GD1b, respectively. Trisialogangliosides were separated isocratically using the solvent system
CHCl3/MeOH/H2O 50:42:11 v/v/v to obtain GT1b. Proportions of these five gangliosides were separated
into their d18:1/18:0- and d20:1/18:0-lipoforms by RP column chromatography using the solvent system
MeOH/H2O in appropriate compositions (90:25 v/v for GM1, 90:49 v/v for GD1a, 90:40 v/v for GD1b,
90:40 v/v for GT1b, and 90:50 v/v for GQ1b). The obtained ganglioside lipoforms were analyzed by ESI-
MS, ESI-MS/MS, 1H-NMR, and CHN analysis (Table 1).
Chemical preparation of lysogangliosides
d18:1-Lyso-GM1·NH3 (7) was prepared from d18:1/18:0-GM1·NH3 (1) by the method of Sonnino et
al. (31). All steps were performed under an argon atmosphere. 20 mg (12.8 µmol) of 1 were dissolved in
9.2 mL of dry Propanol. Then, 2.3 mL of a 1 M potassium hydroxide solution in the same solvent were
added and the reaction mixture was heated to 90 °C for 20 h. After cooling to RT, the reaction mixture was
brought to pH 9 by adding an aq. ammonium chloride buffer. The solvent was removed in a stream of
nitrogen, the crude product was dried in vacuum and subsequently purified by RP column chromatography
(1.5 × 3.5 cm, MeOH/H2O 90:40 v/v). A second purification step was performed by NP column
chromatography (2.0 × 22 cm, CHCl3/MeOH/2.5 M NH3 60:40:9 v/v/v). After lyophilization, 7 was
obtained as a colorless powder (8.69 mg, 52 %), ESI-MS: 1280.62 [M + H]+, 651.80 [M + Na + H]2+, 640.81
[M + 2H]2+, Rf = 0.12 (CHCl3/MeOH/2.5 M NH3 60:40:10 v/v/v). The method was also applied for the
8

preparation of d18:1-lyso-GD1a·2NH3 (8) (1.23 mg, 4.8 %) from d18:1/18:0-GD1a·2NH3 (3), ESI-MS:
1571.75 [M + H]+, 797.36 [M + Na + H]2+, 794.88 [M + NH4 + H]2+, 786.37 [M + 2H]2+, Rf = 0.24
(CHCl3/MeOH/2.5 M NH3 60:44:12 v/v/v).
Enzymatic preparation of lysogangliosides
The enzymatic preparation of lysogangliosides was performed by reported methods (32, 33). Triton™ X-
100 was used instead of sodium cholate because we observed better conversions. 0.919 mg (491 nmol) of
d18:1/18:0-GD1a·2NH3 (3) were dissolved in 500 µL of 50 mM sodium acetate buffer (pH 6.0), which
contained 0.8 % of Triton™ X-100. The mixture was exposed to an ultrasonic bath for 15 s. Then, 3.6 µL
(18 mU) of a SCDase solution (in 50 mM sodium acetate buffer, pH 6.0) were added, the aqueous phase
was covered with 5 mL of n-decane to remove the fatty acid from the equilibrium, and the mixture was
incubated at 37 °C. Every 2 h the n-decane phase was replaced. To obtain a complete conversion of the
educt further 36 mU of enzyme were added over a period of 2 d. The reaction was stopped by the addition
of a few drops of 2.5 M ammonia. The solvent was removed in a stream of nitrogen, the crude product was
dried in vacuum and subsequently purified by NP column chromatography (2.0 × 23 cm, CHCl3/MeOH/2.5
M NH3 60:40:10 v/v/v). After lyophilization, d18:1-lyso-GD1a·2NH3 (8), was obtained as a colorless
powder (0.74 mg, 47 % from two preparations), ESI-MS: m/z 1571.67 [M + H]+, 915.46 [Y2α + 2H]+,
816.30 (1.6) [M + K + Na]2+, 797.32 [M + Na + H]2+, 794.84 [M + NH4 + H]2+, 786.33 [M + 2H]2+, Rf =
0.24 (CHCl3/MeOH/2.5 M NH3 60:44:12 v/v/v). The method was also applied for the preparation of d20:1-
lyso-GD1b·2NH3 (9) (1.72 mg, 53 %), from d20:1/18:0-GD1b·2NH3 (4), ESI-MS: m/z 1599.67 [M + H]+,
811.32 [M + Na + H]2+, 808.85 [M + NH4 + H]2+, 800.33 [M + 2H]2+, Rf = 0.18 (CHCl3/MeOH/2.5 M NH3
60:44:12 v/v/v), d20:1-lyso-GT1b·3NH3 (10) (1.16 mg, 61 %) from d20:1/18:0-GT1b·3NH3 (5), ESI-MS:
m/z 1890.74 [M + H]+, 956.89 [M + Na + H]2+, 945.90 [M + 2H]2+, Rf = 0.26 (CHCl3/MeOH/2.5 M NH3
50:50:16 v/v/v), and d20:1-lyso-GQ1b·4NH3 (11) (0.97 mg, 44 %) from d20:1/18:0-GQ1b·4NH3 (6), ESI-
MS: m/z 1113.92 [M + 2Na]2+, 1102.93 [M + Na + H]2+, 1099.95 [M + NH4 + H]2+, 1091.93 [M + 2H]2+,
Rf = 0.25 (CHCl3/MeOH/2.5 M NH3 50:50:16 v/v/v).
9

Synthesis of 2,5-dioxopyrrolidin-1-yl tetradecanoate (12)
3.00 g (13.1 mmol) of tetradecanoic acid and 3.24 g of DCC (15.7 mmol) were dissolved in 26.2 mL of dry
tetrahydrofuran and stirred at RT for 10 min. Then, 1.81 (15.7 mmol) of N-hydroxysuccinimide in 14.1 mL
of the same solvent were added and the reaction mixture was stirred at RT for 22.5 h. TLC analysis
(CHCl3/MeOH 50:1 v/v) revealed a complete conversion of the educt. After this, the reaction mixture was
filtrated by suction. The solvent was removed under reduced pressure and the residue was recrystallized
from ethanol containing a trace of water (34). The product was obtained as a colorless solid (3.08 g, 72 %),
1H-NMR (300 MHz, CDCl3): δ [ppm] = 2.83 (s, 4H, H-3, 4), 2.60 (t, 2H, 3JH2'-H3' = 7.5 Hz, H-2'), 1.74 (tt,
2H, 3JH3'-H4' = 7.5 Hz, 3JH3'-H2' = 7.5 Hz, H-3'), 1.46 – 1.18 (m, 20H), 0.88 (t, 3H, 3JH14'-H13' = 6.7 Hz, H-14'),
13C-NMR (75 MHz, CDCl3): δ [ppm] = 169.15 (RCON, C-2, 5), 168.68 (RCO2R', C-1'), 31.90 (CH2, C-2'),
30.93 (CH2, C-12'), 29.63-28.77 (8 CH2), 25.57 (CH2, C-3, 4), 24.56 (CH2, C-3'), 22.67 (CH2, C-13'), 14.09
·
(CH3, C-14'), EI-MS: m/z 211.3 [M+· - C4H4NO3 ], 129.1 [C8H17O+], 98.2 [C7H14+·], 84.1 [C6H12+·], CHN
analysis: calc.: C, 66.43; H, 9.60; N, 4.30, found: C, 66.03; H, 9.63; N, 4.29. The method was also applied
1
for the synthesis of 2,5-dioxopyrrolidin-1-yl heptadecanoate (13) (0.897 g, 66 %), H NMR (400 MHz,
CDCl3): δ [ppm] = 2.83 (s, 4H, H-3, 4), 2.59 (t, 1H, 3JH2'-H3' = 7.5 Hz, H-2'), 1.74 (tt, 2H, 3JH3'-H4' = 7.5 Hz,
3JH3'-H2' = 7,5 Hz, H-3'), 1.45 – 1.19 (m, 26H), 0.88 (t, 3H, 3JH17'-H16' = 6.9 Hz, H-17'), 13C-NMR (75 MHz,
CDCl3): δ [ppm] = 169.15 (RCON, C-2, 5), 168.68 (RCO2R', C-1'), 31.90 (CH2, C-2'), 30.93 (CH2, C-15'),
29.66-28.78 (11 CH2), 25.57 (CH2, C-3,4), 24.56 (C-3'), 22.67 (CH2, C-16'), 14.10 (CH3, C-17'), EI-MS:
·
m/z 367.3 [M+·], 253.3 [M+·– C4H4NO3 ], 98.1 [C7H14+·], 84.1 [C6H12+·], CHN analysis: calc.: C, 68.63; H,
10.15; N, 3.81, found: C, 68.63; H, 10.14; N, 3.75.
Reacylation of lysogangliosides
All steps were performed under an argon atmosphere. 0.98 mg (0.610 µmol) of d18:1-lyso-GD1a·2NH3 (8)
and 7.6 µL (44.7 µmol) of Hünig’s base were dissolved in 62 µL of dry dimethylformamide. Then, 38 µL
of the same solvent containing 1.43 mg (3.89 µmol) of 2,5-dioxopyrrolidin-1-yl heptadecanoate (13) were
added. The reaction mixture was stirred at 30 °C for 28 h. Then, a few drops of 2.5 M ammonia were added,
10

the solvent was removed in a stream of nitrogen, the crude product was dried in vacuum and subsequently
purified by NP column chromatography (2.0 × 20 cm, CHCl3/MeOH/2.5 M NH3 60:40:10 v/v/v). After
lyophilization, d18:1/17:0-GD1a·2NH3 (16) was obtained as a colorless powder (0.98 mg, 86 %), ESI-MS:
m/z 929.51 [M + 2NH4]2+, 921.00 [M + NH4 + H]2+, 912.48 [M + 2H]2+, Rf = 0.18 (CHCl3/MeOH/2.5 M
NH3 60:40:10 v/v/v). The method was also applied for the synthesis of d18:1/14:0-GM1·NH3 (14) (6.32
mg, 28 %) from 7, ESI-MS: m/z 1490.72 [M + H]+, 764.83 [M + K + H]2+, 754.37 [M + NH4 + H]2+, 745.86
[M + 2H]2+, Rf = 0.22 (CHCl3/MeOH/2.5 M NH3 60:40:9 v/v/v), d18:1/14:0-GD1a·2NH3 (15) (0.88 mg,
42 %) from 8, ESI-MS: m/z 918.86 [M + K + NH4]2+, 910.86 [M + Na + NH4]2+, 908.39 [M + 2NH4]2+,
899.88 [M + NH4 + H]2+, 891.37 [M + 2H]2+, Rf = 0.25 (CHCl3/MeOH/2.5 M NH3 60:44:12 v/v/v),
d20:1/14:0-GD1b·2NH3 (17) (1.28 mg, 70 %) from 9, ESI-MS: m/z 1809.92 [M + H]+, 916.44 [M + Na +
H]2+, 905.45 [M + 2H]2+, Rf = 0.21 (CHCl3/MeOH/2.5 M NH3 60:44:12 v/v/v), d20:1/14:0-GT1b·3NH3
(18) (0.40 mg, 34 %) from 10, ESI-MS: m/z 1070.48 [M + K + H]2+, 1070.99 [M + Na + NH4]2+, 1062.49
[M + Na + H]2+, 1051.50 [M + 2H]2+, Rf = 0.29 (CHCl3/MeOH/2.5 M NH3 60:50:15 v/v/v), and d20:1/14:0-
GQ1b·4NH3 (19) (0.19 mg, 19 %) from 11, ESI-MS: m/z 1227.01 [M + K + Na]2+, 1216.02 [M + K + H]2+,
1208.03 [M + Na + H]2+, 1205.55 [M + NH4 + H]2+, 1197.04 [M + 2H]2+, Rf = 0.27 (CHCl3/MeOH/2.5 M
NH3 50:50:16 v/v/v).
Enzymatic degalactosylation of ganglioside standards
1.34 mg (0.889 µmol) of d18:1/14:0-GM1·NH3 (14) were dissolved in 410 µL of 100 mM McIlvaine buffer
(pH 4.3), which contained 3.9 mM sodium taurodeoxycholate. Then, 200 µL of buffer containing 3.2 mM
Triton™ X-100 and 41 µL of pure buffer were added. The mixture was exposed to an ultrasonic bath for
15 s. Then, 150 µL (134 mU) of beta-galactosidase solution were added. After 1d, TLC analysis
(CHCl3/MeOH/2.5 M NH3 50:40:9 v/v/v) revealed a conversion of approx. 50 %. Additional 150 µL of
enzyme solution were added. After a further day, the conversion was complete and the reaction was stopped
by addition of a few drops of 2.5 M ammonia. The solvent was removed in a stream of nitrogen and the
residue was redissolved in 1 mL of methanol. The enzyme was separated by centrifugation (12100 × g),
11

which was repeated five times. The supernatant was separated and evaporated in a stream of nitrogen. The
crude product was purified by preparative HPTLC (20 × 20 cm glass plate, CHCl3/MeOH/2.5 M NH3
60:40:9 v/v/v). Silica gel was removed by the method described under “Purification”. After lyophilization,
d18:1/14:0-GM2·NH3 (20) was obtained as a colorless powder (1.84 mg, 77 % from two preparations),
ESI-MS: m/z 1345.81 [M + NH4]+, 1328.78 [M + H]+, 681.49 [M + 2NH4]2+, 664.89 [M + 2H]2+, Rf = 0.27
(CHCl3/MeOH/2.5 M NH3 60:40:9 v/v/v). The method was also applied for the preparation of d20:1/14:0-
GD2·2NH3 (21) from d20:1/14:0-GD1b·2NH3 (17). For this conversion, the concentrations of detergents
and enzyme were doubled. 21 was obtained as a colorless powder (0.25 mg, 61 %), ESI-MS: m/z 846.43
[M + 2Na]2+, 835.44 [M + Na + H]2+, 841.47 [M + 2NH4]2+, 832.96 [M + NH4 + H]2+, 824.45 [M + 2H]2+,
Rf = 0.30 (CHCl3/MeOH/2.5 M aq. NH3 60:44:12 v/v/v).
Synthesis of lysoganglioside standards
(E)-Hexacos-13-ene (22)
All steps were performed under an argon atmosphere. 200 mg (1.02 mmol) of 1-tetradecene and 36.7 mg
(0.204 mmol, 20 mol%) of tetrafluoro-1,4-benzoquinone were dissolved in 2.8 mL of dry toluene. 6.33 mg
(10.1 µmol, 0.99 mol%) of Hoveyda-Grubbs catalyst 2nd gen. [1,3-bis-(2,4,6-trimethylphenyl)-2-
imidazolidnylidene]dichloro(o-isopropoxyphenylmethylene)ruthenium(II) were added and the reaction
mixture was stirred at RT for 3 h. Ethylene was removed by a stream of argon during the reaction. The
solvent was removed in a stream of nitrogen and the catalyst was removed by NP column chromatography.
The crude product was applied to the column (1.0 × 4.0 cm) in hexane/EtOAc 80:1 v/v. The column was
eluted by four column volumes of the same solvent. Product containing fractions were pooled, the solvent
was removed in a stream of nitrogen and subsequently dried in vacuum. After recrystallization from 4 °C
cold ethanol, 22 was obtained as a colorless solid (58.8 mg, 32 %), 1H NMR (400 MHz, CDCl3): δ [ppm]
= 5.38 (tt, 2H, 3JH13-H12 = 3.6 Hz, 4JH13-H11 = 1.4 Hz, H-13, 14), 1.97 (m, 4H, H12, 15), 1.26 (m, 40H), 0.88
+
+
+
(t, 6H, 3JH1-H2 = 6.8 Hz, H-1, 26), EI-MS: m/z 364.45 [M+·], 111.15 [C8H15 ], 97.10 [C7H13 ], 83.10 [C6H11 ],
12

+
+
+
69.00 [C5H9 ], 57.10 [C4H9 ], 55.05 [C4H7 ], GC-MS: tR 14.14 min, 100 %, M+· = 364.45, CHN analysis:
calc.: C, 85.63; H, 14.37, found: C, 85.21; H, 14.11.
Synthesis of pentadeca-O-acetyl-GM1 (d20:1/18:0) sialoyl-II2-lactone (23)
All steps were performed under an argon atmosphere. 30.9 mg (19.4 µmol) of d20:1/18:0-GM1·NH3 (2)
and 0.5 mg (4.09 µmol) of DMAP were suspended in 832 µL of dry pyridine. 413 µL of acetic anhydride
were added and the reaction mixture was stirred at RT for 15 h. It took a few hours for the reactants to
dissolve completely. The solvent was removed in a stream of nitrogen and the crude product was purified
by NP column chromatography (2.0 × 21 cm, toluene/acetone 5:4 v/v). After drying in vacuum, 23 was
1
obtained as a colorless solid (26.3 mg, 62 %), H NMR (400 MHz, CDCl3): see table 2, ESI-MS: m/z
2220.10 [M + MeOH + H]+, 2188.07 [M + H]+, 1127.56 [M + MeOH + 2NH4]2+, 1119.05 [M + MeOH +
NH4 + H]2+, 1110.54 [M + MeOH + 2H]2+, 1111.55 [M + 2NH4]2+, 1103.04 [M + NH4 + H]2+, 1094.53 [M
+ 2H]2+, Rf = 0.23 (toluene/acetone 5:4 v/v).
Synthesis
of
O-(tetradeca-O-acetylmonosialogangliotetraosyl)-(1"1)-(2R,3S,4E)-3-O-acetyl-2-
octadecanoylamino-5-phenylpent-4-en-1-ol sialoyl-II2-lactone (24)
All steps were performed under an argon atmosphere. 39.7 mg (18.3 µmol) of a 1:1 mixture of pentadeca-
O-acetyl-GM1 (d20:1/18:0) sialoyl-II2-lactone (23) and pentadeca-O-acetyl-GM1 (d18:1/18:0) sialoyl-II2-
lactone (M = 2173.41 gmol-1) were dissolved in 477 µL of dry dichloromethane in a 1 mL glass vial.
33.0 mg (183 µmol) of (E)-stilbene and 23.9 mg (28.2 µmol) of Grubbs catalyst 2nd gen. [1,3-
bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(phenylmethylene)(tricyclohexylphosphine)-
ruthenium(II) were added and the reaction mixture was stirred at 43 °C for 46 h. TLC monitoring
(toluene/actone 1:1 v/v) revealed a conversion of more than 90 %. The solvent was removed in a stream of
nitrogen, the crude product was dried in vacuum and subsequently purified by NP column chromatography
(2.0 × 22 cm, toluene/acetone 10:9 v/v). Traces of catalyst were removed by the method described under
“Purification”. After drying in vacuum, 24 was obtained as a colorless solid (25.7 mg, 68 %), 1H NMR (400
13

MHz, CDCl3): see table 2, ESI-MS: m/z 2085.94 [M + MeOH + H]+, 2053.90 [M + H]+, 1060.46 [M +
MeOH + 2NH4]2+, 1051.95 [M + MeOH + NH4 + H]2+, 1043.44 [M + MeOH + 2H]2+, 1044.45 [M +
2NH4]2+, 1035.94 [M + NH4 + H]2+, 1027.43 [M + 2H]2+, Rf = 0.15 (toluene/acetone 5:4 v/v).
Synthesis of pentadeca-O-acetyl-GM1 (d17:1/18:0) sialoyl-II2-lactone (25)
All steps were performed under an argon atmosphere. 15.5 mg (7.55 µmol) of 24, 55.0 mg (151 µmol) of
(E)-hexacos-13-ene (22), and 3.43 mg (31.7 µmol, 20 mol%) of p-benzoquinone were dissolved in 271 µL
of dry dichlormethane in a 1 mL glass vial. Then, 19.8 mg (31.6 µmol, 20 mol%) of Hoveyda-Grubbs
catalyst 2nd gen. were added and the reaction mixture was stirred at 40 °C. After 1 d, TLC monitoring
(toluene/actone 1:1 v/v) revealed a conversion of approx. 70 %. Further 19.8 mg of catalyst and 3.43 mg of
p-benzoquinone were added and the reaction was stirred at 40 °C for another day. After a total of 41 h, the
solvent was removed in a stream of nitrogen and the crude product was dried in vacuum. Subsequently, it
was purified by NP column chromatography (2.0 × 23 cm, toluene/acetone 10:9 v/v). Traces of catalyst
were removed by the method described under “Purification”. After drying in vacuum, 25 was obtained as
1
a colorless solid (9.91 mg, 61 %), H NMR (400 MHz, CDCl3): see table 2, ESI-MS: m/z 2178.10 [M +
MeOH + H]+, 2146.05 [M + H]+, 1106.56 [M + MeOH + 2NH4]2+, 1098.04 [M + MeOH + NH4 + H]2+,
1089.53 [M + MeOH + 2H]2+, 1090.54 [M + 2NH4]2+, 1082.02 [M + NH4 + H]2+, 1073.52 [M + 2H]2+, Rf
= 0.20 (toluene/acetone 5:4 v/v).
Preparation of d17:1-lyso-GM1·NH3 (26)
In the next step, pentadeca-O-acetyl-GM1 (d17:1/18:0) sialoyl-II2-lactone (25) was deprotected and
regioselectively deacylated by the method described under “Chemical preparation of lysogangliosides”.
Briefly, 9.40 mg (4.38 µmol) of 25 were applied. The reaction time was 19 h. The crude product was
purified by RP column chromatography (0.9 × 2.8 cm, MeOH/H2O 90:40 v/v). A second purification step
was performed by NP column chromatography (2.0 × 19 cm, CHCl3/MeOH/2.5 M NH3 60:40:10 v/v/v).
MS analysis revealed that the product contained 4.8 % of the d16:1-lyso-GM1 isomer (see results section),
14

so the product was purified by isocratic RP column chromatography (1.0 × 22 cm, MeOH/H2O 90:31 v/v),
again. After lyophilization, 26 was obtained as a colorless powder (1.93 mg, 34 %), ESI-MS: m/z 1266.60
[M + H]+, 1104.55 [Y3α + 2H]+, 901.47 [Y2α + 2H]+, 652.78 [M + K + H]2+, 644.79 [M + Na + H]2+, 642.31
[M + NH4 + H]2+, 633.88 [M + 2H]2+, Rf = 0.14 (CHCl3/MeOH/2.5 M NH3 60:40:10 v/v/v).
Preparation of d17:1-lyso-GM2·NH3 (27)
d17:1-Lyso-GM2·NH3 (27) was prepared by the method described under “Enzymatic degalactosylation of
ganglioside standards”. Briefly, 1.09 mg (0.849 µmol) of d17:1-lyso-GM1·NH3 (26) and 314 mU of beta-
galactosidase were applied. The reaction time was 15.5 h. The crude product was purified by NP column
chromatography (2.0 × 19 cm, CHCl3/MeOH/2.5 M NH3 60:40:10 v/v/v). After lyophilization, 27 was
obtained as a colorless powder (0.66 mg, 69 %). ESI-MS: m/z 1126.53 [M + Na]+, 1104.54 [M + H]+,
563.76 [M + Na + H]2+, 561.28 [M + NH4 + H]2+, 552.77 [M + 2H]2+, Rf = 0.16 (CHCl3/MeOH/2.5 M NH3
60:40:9 v/v/v).
Validation of the lysoganglioside lipoforms as calibrators
Six different concentrations of pure d18:1-lyso-GM1·NH3 and of pure d18:1-lyso-GM2·NH3 (0 µM, 5 µM,
10 µM, 15 µM, 20 µM, 25 µM, and 35 µM) in methanol were prepared. The d18:1-lyso-GM1·NH3 solutions
were spiked each by 26 to a final concentration of 13.4 µM of the internal standard and the d18:1-lyso-
GM2·NH3 solutions were spiked by 27 to a concentration of 14.0 µM of the internal standard. The solutions
were measured in the full scan mode and the ratio of the peak height of the most intensive peak
([M + K + H]2+) was plotted against the concentration of the analyte (see Fig. 8 in the results section).
15

Results
Purification of bovine brain gangliosides to obtain starting materials
5.03 g of bovine brain gangliosides (Cronassial®) were separated into different ganglioside classes by anion-
exchange chromatography. Monosialogangliosides were eluted by an ammonium acetate concentration of
0.05 mol/L. Disialogangliosides and trisialogangliosides were successively eluted by an ammonium acetate
concentration of 0.15 mol/L and were separated without overlap. GQ1b was eluted by an ammonium acetate
concentration of 0.45 mol/L and was already obtained in pure form after desalting. The recovery of the
anion-exchange chromatography was 95 %. In the next step the ganglioside classes were purified by NP
column chromatography using different techniques and different solvent systems. The disialogangliosides
were separated into GD1a and GD1b. The purified gangliosides were analyzed by HPTLC, ESI-MS and
ESI-MS/MS. The NP HPTLC showed only one band for each ganglioside (Fig. 2A). The RP-TLCs showed
two distinct bands for each ganglioside (Fig. 2B, given for GQ1b), whereby the upper band corresponds to
the d18:1/18:0-lipoform while the lower band corresponds to the d20:1/18:0-lipoform. In the next step the
gangliosides were separated into their lipoforms by RP column chromatography using MeOH/H2O in
different compositions as eluent. The purity of the ganglioside lipoforms was demonstrated by RP-TLC and
ESI-MS. Their structure was analyzed by ESI-MS/MS and 1H-NMR. CHN analysis demonstrated a purity
of 86-95 % compared to the theoretical value, which is due to residual water content (Table 1). The residual
water content was 10-15 % when the gangliosides were dried by lyophilization and around 5 % when the
gangliosides were dried in vacuum. An overview about the preparations which were performed in this work
is given in figure 3.
Preparation of lysogangliosides
In this work one step preparations of lysogangliosides were applied. d18:1-Lyso-GM1·NH3 was prepared
by alkaline treatment of d18:1/18:0-GM1·NH3 in a yield of 52 %, which is comparable to the yield of 54 %
reported by Sonnino et al. (31). This method turned out to be not suitable for the regioselective deacylation
of oligosialogangliosides: d18:1-lyso-GD1a·2NH3 was obtained in a yield of only 4.8 % due to concomitant
16

deacetylation of sialic acid groups. Hence, we applied a biocatalyst to this reaction. The enzyme SCDase
(EC 3.5.1.69) catalyzes the hydrolysis of the amide bond within the ceramide moiety of various
glycosphingolipids, but not ceramides, in a reversible manner (35). We applied the method to d18:1/18:0-
GD1a·2NH3 (3), d20:1/18:0-GD1b·2NH3 (4), d20:1/18:0-GT1b·3NH3 (5), and d20:1/18:0-GQ1b·4NH3 (6)
(Table 3). Yields were in the range of 44 to 60 %. The ESI-MS spectrum of d18:1-lyso-GD1a showed no
impurities of other GD1a lipoforms. By comparing the [M + 2H]2+ peaks it was found that d20:1-lyso-
GD1b·2NH3 contained 3.1 % of d18:1-lyso-GD1b·2NH3 due to traces of d18:1/20:0-GD1b·2NH3 in the
precursor, which could not be separated by RP column chromatography. Also, d20:1-lyso-GT1b·3NH3
contained 4.7 % of its d18:1-lipoform. For d20:1-lyso-GQ1b·4NH3 no other GQ1b-lipoforms were
detected.
Reacylation of lysogangliosides
The lysogangliosides were reacylated by the method of Schwarzmann and Sandhoff (36) using N-
hydroxysuccinimide esters of tetradecanoic acid and heptadecanoic acid. The reaction was applied to d18:1-
lyso-GM1·NH3 (7), d18:1-lyso-GD1a·2NH3 (8), d20:1-lyso-GD1b·2NH3 (9), d20:1-lyso-GT1b·3NH3 (10),
and d20:1-lyso-GQ1b·4NH3 (11). Yields were in the range of 19-86 % (Table 4). The ESI-MS of 14, 15,
16 (Fig. 4), and 19 showed no traces of other ganglioside lipoforms than the desired products. The ESI-MS
of d20:1/14:0-GD1b·2NH3 (17) showed an impurity of 3.4 % of d18:1/14:0-GD1b·2NH3 and the ESI-MS
of d20:1/14:0-GT1b·3NH3 (18) showed an impurity of 4.8 % of d18:1/14:0-GT1b·3NH3 as it was observed
for their precursors.
Preparation of ganglioside standards by enzymatic degalactosylation
For the preparation of gangliosides that do not occur in larger amounts in bovine brain, we applied
glycosidase-treatment to ganglioside standards already modified in their lipid part. The commercially
available enzyme beta-galactosidase (EC 3.2.1.23) from bovine testes can hydrolyze terminal galactosyl
residues in β1-3, β1-4, and β1-6 linkage from saccharides, glycosaminoglycans, glycoproteins, and
17

glycolipids (37). The reaction was applied to the preparation of d18:1/14:0-GM2·NH3 (20) from
d18:1/14:0-GM1·NH3 (14) and d20:1/14:0-GD2·2NH3 (21) from d20:1/14:0-GD1b·2NH3 (16). The
preparations had yields of 77 % and 61 %, respectively (Table 5). The lower yield for the preparation of
GD2 derivatives is due to a slight desialylation of GD1b, which occurs at the applied pH of 4.5. This was
demonstrated by applying the reaction conditions described to GD1b in the absence of enzyme. The ESI-
MS of 20 showed no traces of other GM2 lipoforms than the desired product. The ESI-MS of 21 showed
an impurity of 3.2 % of d18:1/14:0-GD2·2NH3 as already observed for the precursor.
Modification of the sphingosine chain of gangliosides
The olefin cross metathesis is a suitable reaction for the modification of the sphingosine chain of
gangliosides and glycosphingolipids because it tolerates hard nucleophiles and electrophiles and it is
performed under neutral conditions. The following synthetic steps were performed for the synthesis of
d17:1-lyso-GM1·NH3 (26) from d20:1/18:0-GM1·NH3 (2) (Fig. 5). Our approach includes a stilbenolysis
followed by the introduction of a new alkyl chain. We did not choose an ethenolysis because reported yields
(tested by the model methyl oleate using standard Ru-based catalysts) are only in the range of 13 to 57 %
(38) and 13 to 24 % (39), respectively. In the recent years, more effective catalysts for ethenolysis have
been developed, which give yields up to 95 % (40) resp. 80 % (41) but only for (Z)-olefins. These catalysts
are much more reactive towards (Z)-olefins than towards (E)-olefins because the residues of the olefin are
forced into the same direction in the metallacyclobutane formation by bulky ligands (42). A direct reaction
of a peracetylated ganglioside lactone with 13-hexacosene is impracticable because of separation problems.
The procedure illustrated in Fig. 5 has three advantages: First, the phenyl group facilitates the
chromatographic purification because it is more polar than an alkyl group. Second, the phenyl group serves
as an UV-probe. Third, stilbene is not prone to isomerizations because the phenyl group stops the migration
of the double bond. In the first step 2 was peracetylated by a modified method of Schwarzmann et al. (43).
These reaction conditions led to the formation of lactone 23 in a yield of 62 %. In the ESI-MS spectrum
only peaks of the product and the methyl ester of the product, which is formed by opening of the lactone
18

by methanol, which is used as solvent in the measurement, were found. Compound 23 was converted to 24
1
by a tenfold excess of (E)-stilbene and 14 mol% of Grubbs catalyst 2nd gen. in a yield of 68 %. The H-
NMR spectrum demonstrated that selectively the E-isomer was formed due to the bulky residues. (E)-
Hexacosene (22) was synthesized by an olefin cross metathesis of 1-tetradecene because it is commercially
not available. It was observed that only a statistical mixture of constitutional isomers is obtained if no
hydride scavenger is used. Presence of 20 mol% of p-benzoquinone leads to an isomeric purity of 98 % as
analyzed by GC-MS. By using 20 mol% of tetrafluoro-1,4-benzoquinone an isomeric purity of 100 % was
obtained. In the 1H-NMR and 13C-NMR spectra, only one signal for the olefinic protons was observable so
we concluded that selectively the E-isomer was formed. Then, 24 was converted to 25 by a twentyfold
excess of 22 in a yield of 61 %. Hereby, also selectively the E-isomer was formed. To optimize the
conditions for the formation of 25 we tested the more effective hydride scavengers tetrafluoro-1,4-
benzoquinone and 2,6-dichloro-1,4-benzoquinone as well as the Stewart-Grubbs catalyst, which contains
N-tolyl groups instead of N-mesityl groups in the N-heterocyclic carbene ligand (Fig. 6). This catalyst is
reported to be more active than the Hoveyda-Grubbs catalyst 2nd gen. in cross metathesis reactions of
sterically challenging olefins (44). The results are given in table 6. Unexpectedly, the more active hydride
scavengers tetrafluoro-1,4-benzoquinone and 2,6-dichloro-1,4-benzoquinone suppressed the catalyst
activity even when they were added in low amounts. Furthermore, the Stewart-Grubbs catalyst showed no
activity towards 24 regardless of the hydride scavenger tested. So the most effective reaction conditions are
20 mol% of Hoveyda-Grubbs catalyst 2nd gen. and 20 mol% of p-benzoquinone. In the last step the
protecting groups and also the acyl chain were removed from 25 by using the chemical deacylation method
to prepare d17:1-lyso-GM1·NH3 (26). The yield was 34 %. The ESI-MS showed that the product contained
4.8 % of d16:1-lyso-GM1·NH3, which indicates that isomerizations could not be completely suppressed by
p-benzoquinone, but to an extent larger than 95 %. Other isomers were not found. The isomer was removed
by isocratic RP column chromatography. A proportion of 26 was used for the preparation of d17:1-lyso-
GM2·NH3 (27) by the described enzymatic degalactosylation method. The yield was 69 % (Fig. 7).
19

As a proof of principle, the synthesized lysoganglioside standards 26 and 27 were tested for the
quantification of pure natural lysogangliosides (Fig. 8). Good linearity was obtained for both compounds
in the applied concentration range. As expected, quantification of pure substances is possible in the full
scan mode but for biological samples MS/MS modes have to be applied.
20

Discussion
Partial synthesis of non-natural ganglioside lipoforms
For the synthesis of internal ganglioside standards, we chose a partial synthesis rather than a total synthesis
because of the high effort necessary for the latter. Starting materials were obtained by purification of a
ganglioside mixture from bovine brain. It was confirmed by ESI-MS that bovine brain gangliosides mainly
contain d18:1/18:0- and d20:1/18:0-lipoforms. Usually, the purity of gangliosides is determined by
fluorimetric (45) or photometric (28, 29) sialic acid determination, sphingoid base determination by HPLC
(46), GC-FID (30) or densitometry (47). We analyzed the purified ganglioside lipoforms by elemental
analysis and used these substances as external standards for fluorimetric and photometric sialic acid
determination. The results of the CHN analysis demonstrated that there is residual water content of 10-15 %
in the gangliosides after lyophilization. By drying in vacuum, it can be reduced to 5 %. For modification of
the fatty acid acyl chain, we followed a chemical and an enzymatical deacylation approach. The results of
the chemical deacylation demonstrated that this method is appropriate for the selective deacylation of GM1,
but inappropriate for the preparation of oligosialolysogangliosides. The enzymatic deacylation turned out
to be more appropriate: yields were in the range of 44 to 60 % for the preparation of lyso-GD1a (8), lyso-
GD1b (9), lyso-GT1b (10), and lyso-GQ1b (11). This is comparable to the results of Ando et al. (48), who
reported yields of 62 % resp. 52 % for the preparation of lyso-GM1 and lyso-GM2. Due to the occurrence
of traces of d18:1/20:0-lipoforms in the starting material, which could not be separated from the d20:1/18:0-
lipoforms by RP column chromatography, the prepared d20:1-lyso-GD1b·2NH3 (9) and d20:1-lyso-
GT1b·3NH3 (10) contained 3.1 % resp. 4.7 % of their d18:1-lipoform. Since they do not disturb the mass
spectrometric application of the standards, we decided not to remove these minor byproducts. The
reacylation of the lysogangliosides was carried out in yields in the range of 19 to 86 %. In general, the
reaction works accurate for monosialolysogangliosides and disialolysogangliosides with yields up to 86 %.
For lyso-GT1b and lyso-GQ1b, the yields drops to 34 % and 19 %, respectively. An optimization of the
reaction conditions for these substrates should be performed in the future.
21

For the enzymatic degalactosylation of d18:1/14:0-GM1·NH3 (7) a yield of 77 % was obtained, which is
higher than the yield of 54 % obtained by Larsson et al. (49) for a fluorescence labeled GM1 substrate. For
the degradation of d17:1-lyso-GM1·NH3 (26) to d17:1-lyso-GM2·NH3 (27), a comparable yield of 69 %
was obtained. The method is also applicable to GD1b derivatives although we observed a slight degradation
to GM1 due to the low pH value. Hereby, a sufficient yield of 61 % was obtained for the degradation of
d20:1/14:0-GD1b·2NH3 (17) to d20:1/14:0-GD2·2NH3 (21). As far as we know, this method has not been
applied to lyso-GM1 and GD1b derivatives in the literature.
Development of a new synthetic method to modify the sphingosine chain of gangliosides
We developed a synthetic method based on the olefin cross metathesis for the preparation of gangliosides
and lysogangliosides with homogenous and, if required, artificial sphingoid bases. Advantages of this
method are a simplified chromatographic separation by introduction of a phenyl group, the opportunity to
use gangliosides heterogeneous in their sphingosine part as educts, and the applicability of the method for
the synthesis of lysoganglioside standards. A drawback of the method is that isomerizations can only be
suppressed in a range of 95 %. A direct modification of lysogangliosides by an olefin metathesis is
impracticable because the reaction does not tolerate amino groups.
We are aware of only two methods for the partial synthesis of ganglioside lipoforms as MS-standards in
the literature (Fig. 9). The method by Neuenhofer et al. (36, 50) has a total yield of approx. 29 % for the
synthesis of, for example, d18:1/17:0-GM1 from d18:1/18:0-GM1. It includes a total deacylation of the
ganglioside except the N-acetylgalactosamine residue followed by a regioselective protection of the amino
group in the sphingosine part. Subsequently, the remaining amino groups are reacetylated and the protecting
group is removed. The enzymatic deacylation (32, 51) followed by chemical reacylation has the highest
overall yield of approx. 42 %. The olefin metathesis method for the synthesis of, for example, d17:1/18:0-
GM1 from GM1 has a total yield of approx. 23 % if the yield for the removal of the protecting groups is
estimated by 90 %. But in contrast to our method the other methods require homologous pure gangliosides
as starting materials. Current experiments in our group indicate that the method is applicable to
22

peracetylated disialogangliosides, but eventually not to trisialogangliosides because they might be too
unreactive in olefin metathesis reactions. Furthermore, an application to neutral glycosphingolipids and
ceramides should be possible with even better yields because they are more reactive in olefin metathesis
reactions.
Conclusion
Analytical and functional studies of ganglioside lipoforms is an emerging area of research (1, 14, 15). We
provide a protocol for the preparation of artificial ganglioside and lysoganglioside lipoforms starting from
a ganglioside mixture. It relies on selective chemical and enzymatical deacylation steps, reacylation of the
sphingosine part and also selective degalactosylation. These methods were used for the preparation of new
internal standards for GM1, GM2, GD2, GD1a, GD1b, GT1b, and GQ1b. We also developed a new olefin
metathesis method, which was used for the synthesis of a lyso-GM1 and a lyso-GM2 standard. These
compounds are suitable as calibrator substances for the mass spectrometric determination of gangliosides
and lysogangliosides and can also be applied for functional studies.
Up to know, no method was reported for the modification of the sphingosine part of gangliosides. The
method should also be suited for isotope labeling of the sphingosine part of sphingolipids and gangliosides
because the phenyl group in intermediates of the metathesis reaction facilitates the chromatographic
separation of educts and products. Furthermore, this method may also be used to incorporate fluorescent
dyes into the sphingosine part.
23

Acknowledgments/grant support
Financial supported by the European Community (7th framework program “Lipidomic Net”, proposal No
202272) is gratefully acknowledged. We thank for the scholarship of the “NRW International Graduate
Research School LIMES Chemical Biology”. We thank graduate engineer Heike Hupfer for the MS
measurements.
24

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Footnotes to text
1This nomenclature designates the sphingosine part of gangliosides by the number of hydroxyl groups (m
= mono, d = di, t = tri) and the number of carbon:number of double bonds. The same is applied to the N-
linked fatty acid, whose description is separated by a slash. Furthermore, the ganglioside nomenclature
recommended by Svennerholm and the IUPAC-IUBMB nomenclature is used.
30