Highly efficient chemoenzymatic synthesis and facile purification of α-Gal pentasaccharyl ceramide Galα3nLc4βCer

Article abstract of DOI:10.1039/c7cc04090c

A highly efficient chemoenzymatic method for synthesizing glycosphingolipids using α-Gal pentasaccharyl ceramide as an example is reported here. Enzymatic extension of the chemically synthesized lactosyl sphingosine using efficient sequential one-pot mult

Full text of DOI:10.1039/c7cc04090c

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Highly efficient chemoenzymatic synthesis
and facile purification of a-Gal pentasaccharyl
Cite this:DOI: 10.1039/c7cc04090c
ceramide Gala3nLc4bCer†
Received 27th May 2017,
Accepted 30th June 2017
a
Abhishek Santra,
Yanhong Li,a Hai Yu,a Teri J. Slack,a Peng George Wangb and
a
Xi Chen
*
DOI: 10.1039/c7cc04090c
rsc.li/chemcomm
A highly efficient chemoenzymatic method for synthesizing glyco-
GSLs used in functional studies and clinical applications
sphingolipids using a-Gal pentasaccharyl ceramide as an example is have been commonly purified from mammalian cells, blood,
reported here. Enzymatic extension of the chemically synthesized and/or tissues.11–13 The inherited heterogeneity, the presence
lactosyl sphingosine using efficient sequential one-pot multienzyme of other compounds with similar properties, and potential
(OPME) reactions allowed glycosylation to be carried out in aqueous contamination by infectious agents11 make large-scale purifica-
solutions. Facile C18 cartridge-based quick (o30 minutes) purifica- tion of desired glycosphingolipids challenging, especially for
tion protocols were established using minimal amounts of green low abundant compounds.
solvents (CH3CN and H2O). Simple acylation in the last step led to the
Complex GSLs are also challenging synthetic targets despite
formation of the target glycosyl ceramide in 4 steps with an overall advances in the development of modern chemical, enzymatic,
yield of 57%.
and chemoenzymatic methods. A general strategy for synthe-
sizing GSLs has been using multistep chemical synthesis14–17 of
Glycosphingolipids (GSLs) are glycoconjugates consisting of an a trichloroacetimidate glycosyl donor16,18–21 for coupling with
oligosaccharide linked to a ceramide, a lipid consisting of a an azido derivative of the protected glycosphingosine followed
sphingoid base (sphingosine in mammalian glycosphingolipids). by reduction of the azido group, coupling with an acyl chain,
In ceramide, the amino group of the sphingoid base was coupled and deprotection. Unavoidably, the chemical synthetic approaches
to a fatty acid via an amide bond.1 GSLs are ubiquitous compo- involve multiple tedious protection and deprotection processes
nents of mammalian cell membranes and are well known for which lead to extended preparation time and low overall yields.
their important roles in human health and diseases.2–6 De novo Alternatively, glycans synthesized enzymatically and chemo-
synthesis of glycosphingolipids in nature involves the formation enzymatically have been protected and activated to generate
of ceramide7 at the cytoplasmic face of the endoplasmic glycosyl donors, such as trichloroacetimidate21 and more
reticulum (ER), transfer of the ceramide to the cytoplasmic face recently perbenzoylated glycosyl N-phenyltrifluoroacetimidate,
of the Golgi, formation of glucosylceramide and translocation for chemical glycosylation with a selectively benzoyl protected
to the luminal face of the Golgi, and subsequent extension azido-sphingosine as the glycosylation acceptor. Another chemo-
of the oligosaccharide chain by glycosyltransferases for the enzymatic strategy is an endoglycoceramidase glycosynthase strategy
formation of more complex glycosphingolipids in the Golgi, using an oligosaccharyl fluoride as the donor substrate and a
followed by delivery to the cell surface.2 All complex glyco- sphingoid base or its derivative as the acceptor substrate.11,22 Both
sphingolipids share a common lactosyl ceramide (LacbCer) chemoenzymatic strategies require pre-assembly of non-protected
core.8–10 LacbCer has a low solubility in water and is a poor oligosaccharides or oligosaccharyl fluorides in an aqueous solution
acceptor for in vitro enzymatic reactions using glycosyltrans- which involve time-consuming non-trivial purification of non-
ferases in aqueous solutions.9,10
protected glycans after each glycosylation step. The products
that can be obtained via the glycosynthase-catalyzed direct
glycosylation strategy are also limited by the substrate specifi-
cities of the enzyme mutants used towards both the glycan and
the lipid components.
We propose an alternative chemoenzymatic strategy for
synthesizing GSLs. As shown in Scheme 1, we envision that
complex glycosphingosines can be readily obtained using
sequential glycosyltransferase-dependent one-pot multienzyme
a Department of Chemistry, University of California, One shields Avenue, Davis, CA,
95616, USA. E-mail: xiichen@ucdavis.edu; Fax: +1 530 752-8995;
Tel: +1 530 754-6037
b Department of Chemistry and Center of Diagnostics & Therapeutics,
Georgia State University, Atlanta, GA 30303, USA
† Electronic supplementary information (ESI) available: Experimental details for
chemical and enzymatic synthesis, NMR and HRMS data, and NMR spectra. See
DOI: 10.1039/c7cc04090c
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Scheme 2 Synthesis of sphingosine acceptor 12. Reagents and conditions:
(a) TfN3, CuSO4, Et3N, MeOH, CH2Cl2, H2O, r.t., 6 h, 490%; (b) TBDPSCl,
Et3N, DMAP, CH2Cl2, r.t., 8 h, 98%; (c) SO2Cl2, Et3N, CH2Cl2, 0 1C, 0.5–1 h;
(d) RuCl3ꢀ3H2O, NaIO4, CCl4 : CH3CN: H2O (1: 1 : 1), r.t., 2 h; 89% in two
steps (e) (i) Bu4NI, DBU, toluene, reflux, 4 h; (ii) H2SO4/H2O/THF, r.t., 45 min;
85% in two steps (f) BzCl, DMAP, Et3N, CH2Cl2, 0 1C to r.t., 12 h, 95%; (g) HFꢀ
pyridine, THF, 0 1C to r.t., 12 h, 97%.
Scheme 1 An efficient chemoenzymatic strategy for synthesizing complex
glycosphingolipids by enzymatic extension of lactosyl sphingosine (LacbSph, 3)
using sequential one-pot multienzyme (OPME) reactions with C18-cartridge
purification after each glycosylation reaction followed by a simple acyla-
tion process. The structures of target a-Gal pentasaccharyl sphingosine
Gala3nLc4bSph (2) and a-Gal pentasaccharyl ceramide Gala3nLc4bCer (1)
are also shown.
to using the azido protecting group, benzoyl protection of the
secondary alcohol in sphingosine was designed to improve the
regioselectivity of glycosylation. Therefore, 2-azido-3-O-benzoyl
sphingosine (12) (Scheme 2) was chosen as the glycosylation
acceptor for the formation of lactosyl sphingosine. An efficient
(OPME) systems to extend the glycan chain in lactosyl sphingosine strategy33 was chosen to synthesize 12 from inexpensive D-erythro-
(LacbSph), a glycolipid that is readily soluble in aqueous sphingosine (or phytosphingosine, 6) by converting its amino
solutions. The sphingosine (lipid) component in the glycosyl group to an azido group by treating with freshly prepared triflic-
sphingosine products can be used as a hydrophobic tag, allow- azide in the presence of catalytic CuSO4 and triethylamine to form
ing facile purification of the product using a C18 cartridge with compound 7 in a quantitative yield without chromatographic
simple green solvents such as acetonitrile and water. The purification. The primary hydroxyl of 7 was selectively protected
acylation of the glycosyl sphingosine product with a fatty acid by the tert-butyldiphenylsilyl (TBDPS) group33 to produce 8 in 98%
will form the desired glycosphingolipids. The method allows yield. The conversion of the 3,4-vicinal diol in 8 to its cyclic sulfate
the enzyme-catalyzed glycosylation reactions to occur in aqueous (9) was achieved in high yield (92%) by using thionyl chloride
solutions and facile purifications via solid-phase extraction.23 An in the presence of triethylamine followed by oxidation with
additional advantage is that the intermediates for long chain RuCl3/NaIO4. The selective opening of the cyclic sulfate by
complex glycosphingosines can be acylated to form other tetrabutylammonium iodide and 1,8-diazabicyclo[5.4.0]undec-
naturally occurring glycosphingolipids.
7-ene (DBU), dehydrohalogenation to form an alkene, followed
For a proof-of-concept experiment, a-Gal pentasaccharyl by acidic hydrolysis to remove the allylic O-sulfate group were
ceramide (Gala3Galb4GlcNAcb3Galb4GlcbCer) or a1-3-galactosyl- carried out in one pot33 to furnish compound 10 in 85% yield.
lacto-N-neotetraosyl b-ceramide Gala3nLc4bCer (1) (Scheme 1) Conventional benzoylation of compound 10 produced 11 in
was chosen as a target for synthesis. Gala3nLc4bCer (1) was 95% yield. The removal of the O-sialyl ether of 11 was carried
initially identified from rabbit red blood cells.24 This was later out using HFꢀpyridine to produce the glycosylation acceptor 12
found to be the major non-acid glycosphingolipid in the pig (2.3 grams) in 97% yield. The total yield for the chemical
kidney25 and the major a-Gal structure in the pig aorta.26 Together synthesis of compound 12 from phytosphingosine 6 was 61%
with other a-Gal epitopes, it binds to naturally existing human in six steps.
anti-Gal antibodies and is a major cause for organ rejection in pig
to human xenotransplantation.27–29
The glycosylation of 12 with per-O-benzoyl lactosyl trichloro-
acetimidate (13)34 in the presence of BF3ꢀOEt2 in CH2Cl2
For economic and effective chemoenzymatic synthesis of
glycosphingolipids using the proposed method, the first step is
to identify an efficient chemical method for large-scale synthesis
of lactosyl sphingosine (LacbSph) from commercially available
inexpensive phytosphingosine (6) and lactose.
According to several previous reports30–32 for the synthesis of
glucosyl, galactosyl, or lactosyl sphingosine, the azido-derivative of
sphingosine was a better acceptor than other N-protected
sphingosine derivatives for glycosylation with trichloroacetimidate
glycosylation donors. For example, our attempts for glycosylation
using a N-tetrachlorophthaloyl (N-TCP) protected acceptor30 and
lactosyl trichloroacetimidate led to very low yields. In addition
Scheme 3 Synthesis of lactosyl sphingosine (LacbSph, 5). Reagents and
conditions: (a) BF3ꢀOEt2, CH2Cl2, ꢁ18 1C, 3 h, 90%; (b) NaOMe, MeOH, r.t.,
14 h; (c) 1,3-propanedithiol, Et3N, pyridine–water (1 : 1 v/v), 50 1C, 36 h,
94% in two steps.
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at ꢁ18 1C produced per-O-benzoyl lactoside (14)35 in 90% yield Gala3nLc4bSph (2) was synthesized by enzymatic extension
(Scheme 3). After the removal of all benzoyl protecting groups from LacbSph (5) using a sequential OPME strategy involving
´
under the Zemplen conditions, several methods were tested to three OPME reactions (Scheme 4). The formation of trisaccharyl
reduce the azido group while keeping the alkene group intact. sphingosine Lc3bSph (GlcNAcb3Galb4GlcbSph, 4) from LacbSph
Methods using PPh3,36 PMe3,37 or a Lindlar catalyst38 led to (5) was achieved using a one-pot four-enzyme N-acetylglucosamine
poor yields (30–50%). The combination of 1,3-propanedithiol (GlcNAc) activation and transfer system containing Bifidobacterium
and triethylamine39 was found to be the most efficient approach longum (strain ATCC55813) N-acetylhexosamine-1-kinase
for the selective reduction of the azido group to produce LacbSph (BLNahK),46 Pasteurella multocida N-acetylglucosamine uridylyl-
(5) in an excellent yield (94%). The total yield for the glycosyla- transferase (PmGlmU),47 Pasteurella multocida inorganic pyro-
tion was 85% in three steps and the total yield for the chemical phosphatase (PmPpA),48 and Neisseria meningitidis b1-3-N-
synthesis of LacbSph (5) was 52% in nine steps.
acetylglucosaminyltransferase (NmLgtA)49 in Tris–HCl buffer
Lactosyl sphingosine (LacbSph, 5) was readily soluble in aqueous (100 mM, pH 8.0) at 37 1C for 52 hours. The BLNahK, PmGlmU,
solutions for up to 30 mM, allowing it to be used efficiently as a and PmPpA allowed in situ formation of uridine 50-diphosphate-
starting glycosyltransferase acceptor for enzymatic extension using N-acetylglucosamine (UDP-GlcNAc), the sugar nucleotide donor
one-pot multienzyme (OPME) reactions23,40–45 for the synthesis substrate, efficiently and directly from monosaccharide GlcNAc
of more complex structurally diverse glycosphingosines. The for NmLgtA-catalyze formation of Lc3bSph (4). Upon the comple-
sphingosine (lipid) component of the acceptor and the product tion of the enzymatic reaction as monitored by thin-layer
can be used as an anchor to allow facile purification of the chromatography (TLC) and high-resolution mass spectrometry
glycosphingosines by reverse phase column chromatography (HRMS), the reaction mixture was diluted with the same
such as simple C18 cartridge-based purification.
volume of ethanol. The solution was incubated at 4 1C for
For the synthesis of the target a-Gal pentasaccharyl 30 minutes and centrifuged to remove precipitates. The super-
ceramide Gala3nLc4bCer (1), a-Gal pentasaccharyl sphingosine natant was concentrated and the residue was dissolved in
2–3 mL of water at 40–45 1C. The solution was directly loaded
to a pre-conditioned C18 cartridge. The cartridge was then
washed with 0.01% TFA in water (10 mL) using a syringe. The
unreacted sugar, adenosine 50-triphosphate (ATP), uridine
50-triphosphate (UTP), adenosine 50-diphosphate (ADP), uridine
50-diphosphate (UDP), UDP-sugar, and salts were completely
removed in this step. The product Lc3bSph (4) was eluted with
37% acetonitrile in 0.01% TFA/H2O and the unreacted starting
material was eluted with 50% acetonitrile in 0.01% TFA/H2O.
The purification process took less than 30 min in contrast to
several hours using standard silica gel chromatography. A yield
of 83% was achieved after purification.
The obtained Lc3bSph (4) was used for synthesizing
nLc4bSph (3) using an improved OPME galactose (Gal) activation
and transfer system50 containing Streptococcus pneumoniae TIGR4
galactokinase (SpGalK),51 Bifidobacterium longum UDP-sugar pyro-
phosphorylase (BLUSP),50 PmPpA, and Neisseria meningitidis
b1-4-galactosyltransferase (NmLgtB)48,49 in Tris–HCl buffer
(100 mM, pH 8.0) at 37 1C for 30 hours. The SpGalK, BLUSP,
and PmPpA allowed in situ formation of uridine 50-diphosphate-
galactose (UDP-Gal), the donor substrate of NmLgtB, from mono-
saccharide galactose (Gal) for the formation of LNnTbSph (3).
A similar C18-cartridge purification procedure was carried out as
described above for Lc3bSph (4) except that 35% acetonitrile in
0.01% TFA/H2O was used as an eluent to purify nLc4bSph (3). The
acceptor was completely consumed. After purification, a yield of
92% was obtained.
The last OPME reaction was carried out to convert the
obtained nLc4bSph (3) to Gala3nLc4bSph (2) using a galactose
activation and transfer system containing SpGalK, BLUSP, PmPpA,
Scheme 4 High-yield synthesis of a-Gal pentasaccharyl ceramide
and a recombinant bovine a1-3-galactosyltransferase (Ba1-3GalT)23,52
Gala3nLc4bCer (1) by enzymatic extension of lactosyl sphingosine
(LacbSph, 5) using sequential one-pot multienzyme (OPME) reactions with
in Tris–HCl buffer (100 mM, pH 7.5) at 37 1C for 48 hours. The donor
substrate UDP-Gal was generated from galactose in situ as
described in the previous step for stereo-selective production
C18-cartridge purification for the formation of a-Gal pentasaccharyl
sphingosine Gala3nLc4bSph (2) followed by a simple acylation reaction.
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14 Y. Wang, X. Huang, L. H. Zhang and X. S. Ye, Org. Lett., 2004, 6,
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of the desired Gala3nLc4bSph (2). The enzymatic introduction of
the terminal a1-3-linked galactoside by the Ba1-3GalT was
especially advantageous as the 1,2-cis-glycosylation is more
challenging to achieve via chemical glycosylation strategies.
The product (88% yield) was purified by C18-cartridge with
elution using 32% acetonitrile in 0.1% TFA in H2O and the
unreacted acceptor was eluted with 35% or a higher concen-
tration (50%) of acetonitrile in 0.1% TFA/H2O.
It is worth mentioning that 1.5 equivalents of nucleoside
triphosphates (ATP and UTP) were used in each OPME reaction
to optimize the glycosylation yields. With decreased costs of
these compounds in situ recycling of ATP and UTP was not
necessary for preparative or gram-scale reactions.
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The target Gala3nLc4bCer (1) was readily obtained in 85%
yield via the N-acylation of Gala3nLc4bSph (2) with palmitic
acid in the presence of EDCꢀHCl, HOBt and Et3N.18 Overall, the
chemoenzymatic route provided the target Gala3nLc4bCer (1)
with an overall 30% yield in 13 steps. Although Gala3nLc4bCer
(1) had low solubility in methanol as noticed previously,15 its
solubility in CD3OD was sufficient to allow detailed nuclear
magnetic resonance characterization of the product.
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In conclusion, using a-Gal pentasaccharyl ceramide synthesis
as an example, we have demonstrated that efficient sequential
one-pot multienzyme (OPME) chemoenzymatic systems can be
combined with facile C18-purification processes for high-yield
production of glycosphingosines and glycosylceramides. The
strategy can be extended to the synthesis of other complex
glycosphingolipids. The method and the established protocols
will allow non-specialists to synthesize, purify, and study desired
glycosphingolipids of interest in their own labs with a general
research lab setting.
This work was supported by NIH grant U01GM120419 and
NSF grant CHE-1300449. Bruker Avance-800 NMR spectrometer
was funded by NSF grant DBIO-722538. Y. L., H. Y., and X. C.
are co-founders of Glycohub, Inc., a company focused on the
development of carbohydrate-based reagents, diagnostics, and
therapeutics. Glycohub, Inc. played no role in the design,
execution, interpretation, or publication of this study.
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