Synthesis of reference standards to enable single cell metabolomic studies of tetramethylrhodamine-labeled ganglioside GM1

Article abstract of DOI:10.1016/j.carres.2006.10.002

Ganglioside GM1 and its seven potential catabolic products: asialo-GM1, GM2, asialo-GM2, GM3, Lac-Cer, Glc-Cer and Cer, were labeled with tetramethylrhodamine (TMR) to permit ultra-sensitive analysis using laser-induced fluorescence (LIF) detection. The p

Full text of DOI:10.1016/j.carres.2006.10.002

Carbohydrate Research 342 (2007) 482–489
Synthesis of reference standards to enable single cell metabolomic
studies of tetramethylrhodamine-labeled ganglioside GM1
a
a
b
a
c
E. Andreas Larsson, Ulf Olsson, Colin D. Whitmore, Rita Martins, Guido Tettamanti,
d
b
a
a,
*
Ronald L. Schnaar, Norman J. Dovichi, Monica M. Palcic and Ole Hindsgaul
a
Carlsberg Laboratory, Valby-Copenhagen DK-2500, Denmark
Department of Chemistry, University of Washington, Seattle, WA 98195-1700, USA
b
c
Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, 20090 Segrate (MI), Italy
d
The Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
Received 15 September 2006; accepted 2 October 2006
Available online 7 October 2006
Dedicated to the memory of Professor Nikolay K. Kochetkov
Abstract—Ganglioside GM1 and its seven potential catabolic products: asialo-GM1, GM2, asialo-GM2, GM3, Lac-Cer, Glc-Cer
and Cer, were labeled with tetramethylrhodamine (TMR) to permit ultra-sensitive analysis using laser-induced fluorescence (LIF)
detection. The preparation involved acylation of the homogenous C18 lyso-forms of GM1, Lac-Cer, Glc-Cer and Cer with the N-
hydroxysuccinimide ester of a b-alanine-tethered 6-TMR derivative, followed by conversion of these labeled products using galac-
tosidase, sialidase, and sialyltransferase enzymes. The TMR–glycolipid analogs produced are detectable on TLC down to the 1 ng
level by the naked eye. All eight compounds could be separated within 4 min in capillary electrophoresis where they could be
detected at the zeptomole (ca. 1000 molecule) level using LIF.
ꢀ
2006 Elsevier Ltd. All rights reserved.
Keywords: Glycolipid metabolism; Ganglioside; GM1; Metabolomics; Fluorescence labeling; Membrane recycling
1
. Introduction
sphingolipids are often referred to as gangliosides, gan-
glioside GM1 (Chart 1) being a prominent member of
this family.
The surface of animal cells is covered with glycoproteins
and glycolipids where the oligosaccharide chains face
the outside of the cells and often act as recognition mol-
ecules. They are known to serve as receptors for the
binding of other cells, proteins, bacteria, toxins and
viruses and are postulated to have numerous other func-
The brain is unique among the wide array of
vertebrate cells and tissues in that >80% of the conju-
gated oligosaccharides are in the form of glycolipids.
Changes in cell-surface glycolipid structures accom-
pany embryonic development and tumor progres-
6
–9
sion, and many inborn errors of metabolism involve
enzymes in the metabolic pathways of glycolipid meta-
1
0,11
bolism, including lysosomal storage diseases.
There
1
–4
tions.
has therefore been a long-standing interest in understand-
ing the biosynthetic pathways for both glycolipid biosyn-
5
12
thesis and catabolism. Many studies have confirmed
Among the glycolipids, glycosphingolipids are the most
abundant and intriguing molecules. Their sugar chains
range from 1 to over 20 residues and are attached to
ceramide, a fatty-acyl sphingosine. Sialylated glyco-
that the glycolipids on the cell surface are not static, but
are continuously being internalized and recycled back
to the cell surface, often on a time scale of hours.
During this glycolipid recycling, the molecules can
circulate through intracellular organelles and become
distributed through the lysosomes, endoplasmic reticu-
lum, Golgi apparatus and transport vesicles. This
1
3–15
*
0
Corresponding author. E-mail: hindsgaul@crc.dk
008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.10.002

E. A. Larsson et al. / Carbohydrate Research 342 (2007) 482–489
483
Chart 1. Structure and component-nomenclature of ganglioside GM1.
metabolism in healthy cells is thought to be tightly
regulated, with lysosomal degradation closely matching
the biosynthesis to produce a net stable cell-surface
glycosylation pattern. This pattern is ultimately decided
upon by the regulated distribution of glycolipids among
the organelles and the relative expression, localization
and activities of metabolic glycosidases and glycosyl-
bolic products from single cells were, however, not
analyzed.
Encouraged by observations that fluorescently tagged
lactosylceramide and GM1 analogs were indeed inter-
nalized and metabolized in a variety of cells, and that re-
cently developed technology has permitted the assay of
glycosidase activity in single cells, we decided to prepare
GM1 and all of its possible catabolites (Chart 2) tagged
with a fluorescent dye that would afford sufficient sensi-
1
2,16–19
transferases.
The glycolipid profile of a cell will
therefore report on the relative abundance of these
enzymes as well as on the integrity of the intracellular
2
9,30
tivity to analyze the metabolites in single cells.
The
2
0
organelle system and filament network.
dye chosen was the brilliant red tetramethylrhodamine,
previously shown to offer a sensitivity of detection in
The earliest studies on glycolipid recycling used radio-
actively labeled natural glycolipids structures to deduce
the rate and metabolic pathways accessed during the
ꢀ21
the zeptomol (10
mol) range when assayed in capil-
lary electrophoresis (CE) using laser-induced fluores-
cence (LIF) detection in an instrument equipped with
2
1–23
recycling (recently reviewed
). Superb data were ob-
3
1
tained, but these studies necessarily required the extrac-
tion of glycolipids from large populations of cells thus
masking any potential differences between individual
cells. Later, fluorescently tagged glycolipids (e.g., syn-
thetic BODIPY-labeled lactosylceramide analogs or
GM1 analogs) were used and the rate of internalization
and intracellular localization could be followed in single
a sheath-flow cuvette. The ultimate objective of the
present work is to see if there are significant differences
in glycolipid recycling pathways between individual
cells, such as tumor cells or stem cells, in culture. Such
a large-scale metabolomic study will ultimately require
the development of high-throughput automated cell
picking coupled to rapid high-sensitivity analysis. These
are the long-term goals of the present research program.
2
4–28
cells using confocal fluorescence microscopy.
Meta-
Chart 2. Potential catabolic and biosynthetic pathways for GM1.

484
E. A. Larsson et al. / Carbohydrate Research 342 (2007) 482–489
2
. Results and discussion
dye derivative. The starting material used for the prepa-
ration of GM1-TMR, asialo-GM1-TMR, GM2-TMR
and asialo-GM2-TMR was GM1 isolated from bovine
2
.1. Synthetic strategy
3
5
brain (Scheme 1). We used the commercially available
sphingolipid ceramide N-deacylase (Takara) to cleave
the fatty acid chain to give the lyso-GM1 structure(s)
that contained a free amino group to permit installation
of the TMR label. Mass-spectrometry confirmed that
the lyso-GM1 produced was clearly a ca. 1:1 mixture
of compounds differing in the chain length of the
sphingosine residue, one with C - and the other with
The potential flux through the catabolic and biosynthetic
pathways for GM1 is shown in Chart 2. The ‘natural’
pathway in humans is the vertical one, with sequential
glycosidase-mediated removal of single sugar residues
in the sequence GM1!GM2!GM3!Lac!Glc!Cer.
The alternate pathways going from GM1!asialo-GM1
and/or GM2!asialo-GM2 are not active in human gan-
glioside catabolism, although they have been reported in
1
8
C -sphingosine. Since single pure compounds would
2
0
3
2,33
the mouse.
Since all eight compounds may in princi-
be required as reference standards, these had to be
separated. Separation of GM1 chain-length isomers by
reverse-phase chromatography has previously been
ple be formed, the decision was made to prepare all of
them as reference standards. The biosynthesis of GM1
proceeds in the reverse direction beginning with ceramide
and using glycosyltransferases to add monosaccharides
one at a time.
3
6,37
reported,
and it proved especially convenient to do
so here at the lyso-stage, as the enzyme reaction mixture
contained the C18 lyso-form, the C20 lyso-form along
with some unreacted GM1. The C -form cleanly eluted
3
4
We chose a well-precedented approach for installing
the required fluorescent tag on the glycolipid structures.
The fatty-acid moiety of glycosphingolipids can be
cleaved either chemically or enzymatically, producing
the so-called ‘lyso’ derivatives (Scheme 1) with a free
amino group that can be readily acylated by a reactive
1
8
first from a reverse-phase (C ) reverse-phase column,
1
8
using a stepwise methanol/water gradient, and could
easily be isolated in pure form. Later fractions contained
the C20 form followed by the much more hydrophobic
GM1. The conversion of GM1 to the lyso-GM1 having
Scheme 1. Labeling and further enzymatic conversions of GM1.

E. A. Larsson et al. / Carbohydrate Research 342 (2007) 482–489
485
a homogenous C18 sphingosine was routinely carried out
on a 20 mg scale of GM1, yielding >5 mg of homo-
genous C18 product each time (Scheme 1).
compound 2 from which the Boc-group was removed un-
der standard conditions to give 3 which was then con-
verted to the active-ester 4.
2.2. Preparation of tetramethylrhodamine-acylating
agent 4
2.3. Preparation of GM1-TMR, Lac-TMR, Glc-TMR
and Cer-TMR
Since the structures of both the dyes, and any linkers to
the dyes, are expected to importantly influence the recy-
cling properties of fluorescently tagged GM1 and its
Acylation of lyso-GM1 with 4 proved highly effective
and produced the target GM1-TMR which was isolated
in 87% yield on product scales of over 5 mg. The lyso-
forms of Lac-Cer, Glc-Cer and Cer are fortunately com-
mercially available with homogenous C -sphingosine
chains (Avanti Polar Lipids). These could be readily
functionalized, as described for the preparation of
GM1-TMR, to produce Lac-TMR (90%), Glc-TMR
3
8
metabolites, we elected a flexible procedure for label-
ing where the dye is first coupled to a spacer whose
length and structure may be varied. Attachment of the
dye to the very valuable lyso-GM1 would then occur
in a single last step. Scheme 2 shows the preparation
of the TMR-labeling reagent 4, starting with isomerically
pure 6-tetramethylrhodamine N-hydroxysuccinimide
1
8
(95%) and Cer-TMR (94%) (Scheme 3).
39
ester (1) which was coupled to a short spacer, a
Boc-protected b-alanine in the present instance, yielding
2.4. Enzymatic preparation of asialo-GM1-TMR,
GM2-TMR, asialo-GM2-TMR and GM3-TMR
The glycosidases acting in the natural biosynthetic path-
way for GM1 catabolism are complex, requiring chaper-
one proteins and operating in a defined order. For
example, most sialidases do not act on GM1 or GM2,
4
0
but only on GM3. The preparation of the missing met-
abolic standards therefore used commercial glycosidase
preparations with sufficient cross-reactivity to produce
generally small but isolable quantities of the desired
products.
Conversion of GM1-TMR to GM2-TMR was carried
out on milligram scale using b-galactosidase from bo-
vine testes (Scheme 1). This, and the other, enzyme reac-
tions were very easy to monitor by silica gel TLC where
monosaccharide removal resulted in faster moving spots
(or bands) that could be seen by the naked eye at high
sensitivity. Younger researchers (ca. 30 years old) can
readily detect about 1 ng by naked eye, while more expe-
rienced researchers (in their 50’s) commonly require
about 10 ng. GM2-TMR was thus readily separated
Scheme 2. Synthesis of TMR-labeling agent 4.
Scheme 3. Synthesis of Lac-TMR, Glc-TMR, Cer-TMR and GM3-TMR.

486
E. A. Larsson et al. / Carbohydrate Research 342 (2007) 482–489
Figure 2. Separation of standards by CE: (1) asialo-GM1-TMR; (2)
GM1-TMR; (3) asialo-GM2-TMR; (4) GM2-TMR; (5) GM3-TMR; (6)
Lac-TMR; (7) Glc-TMR; (8) Cer-TMR.
Figure 1. Electrospray mass spectra for GM1-TMR and its expected
natural catabolites. The mass spectra were acquired in the positive-ion
mode except for GM1-TMR and GM2-TMR. GM3-TMR exhibits a
peak at 710.7 corresponding to the doubly charged ion.
35 mM sodium deoxycholate and 5 mM methyl-b-cyclo-
dextrin in 20 lM i.d. capillaries. Details of the custom-
designed and built instrumentation will be reported else-
where. The limit of detection was found to be in the
range 2–5 zeptomoles.
from unreacted GM1-TMR using preparative TLC with
simple visual detection of the bands.
Incubation of GM1-TMR with commercial sialidase
A (Arthrobacter ureafaciens) for 6 days yielded asialo-
GM1-TMR which could be isolated in 65% yield. Fur-
ther incubation of asialo-GM1-TMR with b-galactosi-
dase yielded asialo-GM2-TMR in 58% yield on a small
4
. Conclusion
In summary, we have prepared highly fluorescent TMR-
derivatives of GM1 and seven of its potential catabolites
using a combined chemical-enzymatic strategy. This
approach was greatly simplified by performing the
enzymatic reactions on compounds that were already
labeled, permitting the monitoring of the progress of
the reactions by TLC using the naked eye and, similarly,
aiding in the isolation and quantitation of the products.
The synthetic approach chosen will easily permit the
installation of different spacer molecules and different
fluorescent dyes, which may have important conse-
quences on the uptake and recycling of these artificial
glycosphingolipid analogs. Finally, a rapid separation
of all eight compounds was achieved within 4 min by
CE, with detection sensitivities in the fmol range. Preli-
minary results indicate that GM1-TMR is indeed taken
up by PC12-cells, yielding several metabolites co-eluting
with the reference standards prepared in this work,
suggesting the utility of these compounds in single-cell
metabolic studies. These results will be reported in detail
elsewhere.
(
0.1 mg) scale (Scheme 1). The missing GM3-TMR
was readily produced on a >5 mg scale from Lac-
TMR in 96% yield using CMP–sialic acid and a recom-
4
1
binant a2,3-sialyltransferase.
Representative mass-spectra for GM1-TMR and its
expected natural catabolitic products are shown in Fig-
ure 1, confirming their expected structures. The level of
purity of each of the compounds was assessed by routine
CE analysis, where their retention times spanned from
2
0 to 40 min. In each case, the major peak integrated
1
for over 95% of the fluorescence signal. The H NMR
spectra displayed very broad lines in a variety of sol-
vents and were thus not useful for product
characterization.
3
. Separation of the eight standards by capillary
electrophoresis
As stated in the introduction, a major challenge of the
present project is to achieve rapid separation of the eight
metabolites synthesized in the present work, in combina-
tion with the efficient selection of single cells for analy-
sis. Preliminary conditions were found that achieved
the required separation within 4 min (Fig. 2) using a
running buffer consisting of 10 mM sodium tetraborate,
5. Experimental
5.1. General experimental
Methyl-b-cyclodextrin was product 66292 from Fluka,
with a methylation extent of 1.7–1.9 per glucose residue.

E. A. Larsson et al. / Carbohydrate Research 342 (2007) 482–489
487
ESIMS was recorded on a Bruker Esquire 3000-Plus Ion
Trap instrument with samples injected as solutions in
either MeOH or MeOH–CH CN–H O (1/1) mixtures.
5.1.3. 6-Tetramethylrhodamine-(N-hydroxysuccinimidyl-
b-alanine)-amide 4. Compound 3 (7.34 mg, 14.6 lmol),
1.6 equiv, N,N -disuccinimidyl carbonate (DSC, 5.90 mg,
0
3
2
Routine CE was performed using an automated PrinCE
-lift, model 560 CE system (Prince Technologies, The
Netherlands). Separations were carried out in uncoated
23.0 lmol), and 1.7 equiv 4-dimethylaminopyridine
(DMAP, 3.01 mg, 24.6 lmol) were weighed into three
separate Eppendorf tubes. Dry DMF (200 lL) was
added to each tube and they were vortexed vigorously
to assist dissolution. The DSC and DMAP solutions
were added to the solution of 3 in succession with rapid
vortexing between each addition. The reaction tube was
sealed with Parafilm, covered with aluminum-foil and
placed on a shaker. The reaction was monitored by
2
fused-silica capillary of 75 lm i.d. with a length between
5
0 and 80 cm using 50 mM borate, pH 9.3 and 25 mM
sodium dodecyl sulfate (SDS) as running buffer. TMR-
labeled compounds were detected and quantitated using
an Argos 250B fluorescence detector (Flux Instruments,
Switzerland) equipped with an excitation filter of
5
46.1/10 nm and an emission filter of 570 nm. All exper-
TLC (2:1 CHCl –DMF) and ESIMS showed that ester
3
iments were carried out at a normal polarity, that is,
inlet anodic. Preparative TLC was performed on silica
plates (E. Merck Prod. No.1.05745, 20 · 20 cm, 2 mm
thickness). lyso-Lac-Cer, lyso-Glc-Cer and C -sphingo-
4 had formed after 2.5 h. This DMF solution was then
used directly for labeling of the lyso-glycosphingolipids.
ESIMS: m/z 599.21 [M+H] .
+
1
8
sine were from Avanti Polar Lipids (AL, USA).
5.1.4. C -lyso-GM1. GM1 (20 mg, 12.8 lmol, an
1
8
b-Galactosidase and Sialidase A were from Prozyme
approximately equimolar mixture of C18 and C20 sphin-
gosine isomers) was dissolved in 2 mL of 100 mM so-
dium acetate buffer (pH 5.8) followed by the addition
of 5.15 mL water and 0.8 mL taurodeoxycholate
(TDC, 8 mg/mL, Sigma). Sphingolipid ceramide N-
deacylase (SCDase), 50 lL of a 5 mU/lL solution
(Pseudomonas sp. Takara) was added, the reaction was
covered with 40 mL of decane and incubated at 37 ꢁC
for 2 weeks. Aliquots were periodically analyzed by
(
WA, USA).
5
.1.1. 6-Tetramethylrhodamine-(tert-butyl b-alanine)-
amide 2. 6-Tetramethylrhodamine N-hydroxy-
succinimide ester (1, 28.5 mg, 53.8 lmol) and b-alanine
t-butyl ester hydrochloride (16 mg, 88.1 lmol) were dis-
solved in 500 lL dry DMF in a 1.5 mL Eppendorf tube.
The sample was vortexed for 1 min, 15 lL diisopropyl-
ethylamine (DIPEA) was added and the sample was
covered with aluminum foil and placed on a shaker.
TLC (1:1 CH Cl –MeOH) and ESIMS indicated almost
TLC (60:39:10 CHCl –MeOH–0.25% KCl), visualized
3
by spraying with orcinol and heating. When the reaction
was nearly complete, the mixture was frozen at ꢀ20 ꢁC
and the decane was removed. The thawed mixture was
applied to a column (1.2 · 12 cm) packed with Bond-
2
2
complete conversion to the product after 10 min. After
h, the reaction mixture was transferred to a round bot-
tom flask and concentrated to near dryness. Water
25 mL) was added and the solution was applied to
1
˚
apak C18 resin (125 A 37–55 lm, Waters). The column
(
eluent was monitored at 206 nm using a UV-detector
(LKB). The column was washed with water until absor-
bance reached baseline levels, followed by development
with 60% aqueous methanol to remove the TDC and
75% aqueous methanol to elute the lyso-GM1. The
lyso-GM1 came out as two peaks (C -lyso-GM1 fol-
two tandem reverse-phase C18 Sep-Pak cartridges. The
cartridges were washed with water (30 mL), followed
by elution of product with 18 mL fractions of 10% aq
MeOH, 20% MeOH, 3 · 18 mL 30% MeOH, 50%
MeOH and finally 50 mL of 100% MeOH. TLC and
ESIMS analysis showed that the 50% and 100% frac-
tions contained pure product and these fractions were
pooled and concentrated to give the 6-TMR-derivative
1
8
lowed by C -lyso-GM1). Methanol (100%) was used
2
0
to elute any unreacted GM1 from the column. The
fractions containing the separated lyso-GM1 isomers
were pooled, concentrated to dryness and the products
were analyzed by ESIMS. C -lyso-GM1 (5.45 mg,
2
(28.95 mg, 51.9 lmol, 96%). ESIMS: m/z 558.25
+
[
M+H] .
1
8
+
4.26 lmol, 33%) expected: [M+H] 1280.62, found:
5
.1.2. 6-Tetramethylrhodamine-(b-alanine)-amide 3.
1280.6; C -lyso-GM1 (4.80 mg, 3.67 lmol, 29%) ex-
pected: [M+H] 1308.65, found: 1308.6.
2
0
+
Compound 2 (28.95 mg, 51.9 lmol) was dissolved in
CH Cl (5 mL) followed by addition of 1 mL of trifluo-
2
2
roacetic acid . After 3 h, TLC (1:1 CHCl –MeOH) and
5.1.5. GM1-TMR. C -lyso-GM1 (12.0 mg, 9.37 lmol)
3
18
ESIMS indicated that all the starting-material was con-
sumed. The reaction mixture was concentrated, dried
over high vacuum overnight, and the crude residue
was purified on a 10 · 2.5 cm Iatrobead column
was dissolved in 10:12:21 DMF–CHCl –water
3
(1.33 mL). To this solution, 1.55 equiv of NHS-ester 4
in DMF (600 lL) was added, followed by DIPEA
(30 lL). The reaction tube was sealed with Parafilm,
covered with aluminum foil and placed on a shaker.
(
(
CHCl –MeOH gradient from 10/1 to 1/3) to give 3
3
25.1 mg, 50.0 lmol, 96%). ESIMS: m/z 502.19
Analysis by TLC (60:35:8 CHCl –MeOH–0.25% KCl)
3
+
[
M+H] .
and ESIMS after 1 h showed a significant amount of

488
E. A. Larsson et al. / Carbohydrate Research 342 (2007) 482–489
product together with excess unreacted 4. The reaction
was therefore allowed to stand overnight (15 h) to go
to completion and to hydrolyze the reactive dye to facil-
itate product purification. The reaction mixture was
diluted with water (30 mL) and then applied onto two
tandem C18 Sep-Pak cartridges. The cartridges were
washed with water (30 mL), then 20 mL fractions of
each of 25% aqueous MeOH, 33% MeOH, 40% MeOH,
5.2.2. GM2-TMR. GM1-TMR (1.33 mg) was dissolved
in 40 lL of 5· galactosidase-reaction buffer (Prozyme)
and 140 lL water, then 20 lL of b-galactosidase (5 mU/
lL, bovine testes, Prozyme) was added and the sample
was mixed gently. The tube was covered with aluminum
foil and the reaction was incubated at 37 ꢁC for 4 day.
GM2-TMR (0.65 mg, 0.41 lmol, 54%) was obtained after
ꢀ
preparative TLC, ESIMS: m/z 1599.5 [MꢀH] .
5
0% MeOH, 75% MeOH and MeOH. The fractions
were analyzed by TLC (60:35:8 CHCl –MeOH–0.25%
3
5.2.3. Asialo-GM1-TMR. GM1-TMR (1.31 mg) was
dissolved in 40 lL of 5· sialidase reaction buffer (Pro-
zyme) and 140 lL water. When the GM1 was dissolved,
KCl) and ESIMS. The 25–40% fractions contained
hydrolyzed 4 and the 50–100% fractions contained
labeled GM1. The glycolipid containing fractions
were pooled and evaporated to give GM1-TMR
2
0 lL of sialidase A (5 U/mL) was added. The tube was
shaken, then covered with aluminum foil and the reac-
(
14.45 mg, 8.19 lmol, 87%). ESIMS: m/z 1763.80
tion was incubated at 37 ꢁC for 6 days. Preparative
+
[
M+H] .
TLC gave Asialo-GM1-TMR (0.75 mg, 0.58 lmol,
+
6
5%), ESIMS: m/z 1472.2 [M+H] .
0
5
.1.6. Lac-TMR. D-Lactosyl-b-1,1 -D-erythro-sphingo-
sine (C18 isomer, 0.50 mg, 0.80 lmol) was treated as
5.2.4. Asialo-GM2-TMR. Asialo-GM1-TMR (0.1 mg)
was dissolved in 20 lL of 5· reaction buffer (Prozyme),
and 70 lL water and 10 lL of b-galactosidase (5 mU/
lL) were added and the sample was gently mixed. The
tube was covered in aluminum foil and the reaction
was incubated at 37 ꢁC for 4 days. The TLC suggested
ca. 50% conversion, and the product was isolated by
preparative TLC. CE quantitation of the purified prod-
described for lyso-GM1, giving Lac-TMR (0.80 mg,
+
0
.72 lmol, 90%). ESIMS: m/z 1107.57 [M+H] .
0
5
.1.7. Glc-TMR. D-Glucosyl-b-1,1 -D-erythro-sphingo-
sine (4.83 mg, 10.46 lmol) was treated as described for
lyso-GM1, giving Glc-TMR (9.43 mg, 9.98 lmol, 95%).
+
ESIMS: m/z 945.5 [M+H] .
+
uct indicated a 58% yield, ESIMS: m/z 1310.6 [M+H] .
5
.1.8. Cer-TMR. D-erythro-Sphingosine (3.11 mg,
5
.2.5. GM3-TMR. CMP–sialic acid (10.4 mg) was dis-
solved in 500 lL 40 mM Tris–HCl buffer (pH 7.5). This
was transferred to tube containing Lac-TMR
6.16 mg, 5.56 lmol). After dissolving the Lac-TMR,
1
0.38 lmol) was treated as described for lyso-GM1, giv-
ing Cer-TMR (7.69 mg, 9.82 lmol, 95%). ESIMS: m/z
+
a
7
83.5 [M+H] .
(
1
00 lL 100 mM MgCl , 3 lL alkaline phosphatase
2
5.2. Enzymatic conversions of TMR-labeled sphingolipid
analogs
(Roche No. 108 138, 1500 21 U/lL), 197 lL water and
2
00 mU (1 U/mL) of a2,3-sialyltransferase (MalE
4
1
fusion-protein from Campylobacter jejuni ) were added
and the mixture was mixed gently. The tube was covered
with aluminum foil and the reaction was incubated at
room temperature overnight. TLC showed complete
conversion, and the product was isolated without need
5
.2.1. General procedures. The progress of enzyme
incubations was monitored by TLC with visual detec-
tion of the red starting materials and products. After
the progress of the reaction was judged to be adequate,
the products were purified as follows. Water (typically
for preparative HPTLC. GM3-TMR (7.5 mg, 5.36 lmol,
1
0 mL) was added and the reaction mixture was applied
+
9
6%), ESIMS: m/z 1398.7 [M+H] .
to reverse-phase C18 Sep-Pak cartridges that had been
conditioned by washing with HPLC-grade MeOH
(
10 mL) followed by water (50 mL). After sample appli-
Acknowledgements
cation, the cartridges were washed with 100 mL water
then TMR-labeled products were eluted with MeOH
This work was supported by the Grant No. 1 R21
DK07317-01 from the National Institutes of Health,
USA. a2,3-Sialyltransferase was a generous gift from
Dr. Warren Wakarchuck, National Research Council,
Ottawa, Canada.
(
10–20 mL) which was evaporated. When more than
one fluorescent compound was present, they were sepa-
rated by preparative TLC using 60:39:10 CHCl3–
MeOH–0.25% KCl as eluent. The red bands were
scraped off the plates and extracted with MeOH until
the silica became colorless. After evaporation of the
MeOH, the residue was dissolved in water (5 mL) passed
through a 0.45 lm Millex-GV PVDF filter to remove
particulates and then lyophilized.
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