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Glycobiology vol. 10 no. 11 pp. 1157–1169, 2000
MINI REVIEW
Complex carbohydrate synthesis tools for glycobiologists: enzyme-based approach and
programmable one-pot strategies
Kathryn M.Koeller and Chi-Huey Wong1
Department of Chemistry, The Scripps Research Institute and Skaggs Institute
for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037,
USA
Accepted on August 4, 2000
The ultimate goal in complex carbohydrate synthesis is to
develop synthetic tools which are simple and easily accessible
to glycobiologists. This review will describe methods which
have the potential to reach this goal, with particular focus
on enzymatic and computer-based one-pot approaches for the
preparation of complex carbohydrates and glycoconjugates.
Key words: oligosaccharide/glycoconjugate/enzymatic/one-pot
synthesis
Introduction
Carbohydrates play important structural and functional roles in
numerous physiological processes, including various disease
states (Varki, 1993; Dwek, 1996; Sears and Wong, 1998). The
relatively recent recognition of carbohydrates as a medicinally
relevant class of biomolecules has led to the investigation of
therapeutic agents based on either glycan structure or mimics
thereof (Figure 1; Sears and Wong, 1999). For example, cancer
cell metastasis (Hakomori and Zhang, 1997) and cell–cell
adhesion in the inflammatory response (Kansas, 1996) are
dependent on cell surface presentation of specific glycans.
Synthetic carbohydrate-based cancer vaccines (Danishefsky
and Allen, 2000) and small molecule selectin inhibitors
(Simanek et al., 1998) are therefore being pursued as potential
medicinal agents, respectively. Likewise, the initial stages of
bacterial or viral infection often rely on the recognition of host
cell glycoconjugates by the invading organism (Karlsson,
1995). Consequently, naturally occurring and designed synthetic
antibiotics frequently contain carbohydrate structures for the
disruption of these deleterious interactions (Williams and Bardsley, 1999). Additionally, xenotransplant rejection is due to an
undesirable anti-α-Gal antibody induced immune response.
Various treatment strategies based on synthetic α-Gal derivatives are under study, with the future goal that foreign tissue be
an acceptable organ source for human transplants (Chen et al.,
1999). These examples illustrate only a few of the biologically
relevant processes in which glycoconjugates are involved. As
1To
whom correspondence should be addressed
© 2000 Oxford University Press
knowledge of their diverse physiological roles increases, research
into carbohydrate-based therapeutics will continue to accelerate.
The development of carbohydrate-based therapies depends on
the synthetic accessibility of novel glycoconjugates for study.
Unfortunately, obtaining glycoconjugates has been much more
difficult than procuring other biomolecules, such as proteins
and nucleic acids. Automated solid-phase synthetic technologies
have revolutionized protein and nucleic acid science, but
decades of synthetic research have yet to provide analogous
methods for automated oligosaccharide synthesis. This impedance
is the result of intrinsic carbohydrate structure, and is mainly
due to the fact that monosaccharides contain several hydroxyl
groups of similar chemical reactivity. When attempting to
construct even a simple oligosaccharide, each hydroxyl group
must be distinguished from the others in order to obtain the
desired product, with the correct regio- and stereochemistry.
The hydroxyl differentiation process has generally been
tedious, making arduous shuffling of protecting groups and
lengthy synthetic routes the hallmarks of synthetic carbohydrate
chemistry. This is in marked contrast to the synthesis of oligopeptides, which involves a single protecting group manipulation,
followed by peptide bond formation between one activated
carboxylic acid and a sole free amine, iteratively. This synthetic
simplicity made automation of solid-phase peptide synthesis, as
well as that of nucleic acids, readily achievable.
Adaptation of oligosaccharide synthesis to the solid phase is
currently being pursued by several research groups (Ito and
Manabe, 1998), although solid-phase methods to date have
generally been more practical for the synthesis of glycopeptides
(St. Hilaire and Meldal, 2000). At the present time, solution-phase
synthesis is the standard for complex glycoconjugate
assembly, and recent advances have made this strategy a
highly efficient one.
Beyond small molecule construction, techniques for homogeneous glycoprotein synthesis are direly needed. Expression
systems in various cellular hosts have proven utility in protein
production. However, obtaining homogeneous glycoproteins is
not a trivial matter. Because glycosylation is a posttranslational
modification, rather than falling under direct transcriptional
control, glycan structure is subject to various environmental
considerations. Factors such as enzyme competition for the
same substrate and altered glycosylation patterns with cellular
host play a role in the structure of the resultant glycan (Jenkins et
al., 1996). Glycoproteins obtained from prokaryotic or eukaryotic
expression systems therefore exhibit glycoform microheterogeneity (Schachter, 1986). This makes the contribution of
specific glycan structure to underlying protein structure and
function nearly impossible to assess.
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Fig. 1. Examples of biologically important complex carbohydrates.
At the present time, complex carbohydrate and glycoconjugate synthesis remains much more complicated than that of
other biomolecules, and to advance glycoconjugate synthetic
technology to the automated level of proteins and nucleic acids
remains a daunting challenge. However, enzyme-based strategies
toward complex glycoconjugates and one-pot methods for the
synthesis of oligosaccharides represent emerging technologies
that have the potential to greatly simplify glycan assembly. As
described below, these approaches also obviate the need for
laborious protecting group manipulations, which greatly
decreases the amount of time spent obtaining molecules
through synthesis.
Enzymes in glycosidic bond formation
Glycosyltransferases and sugar nucleotide recycling systems
The application of enzymes to organic synthesis is a particularly
powerful approach, and in some cases a single enzymatic
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transformation can be substituted in place of numerous sequential chemical reactions. In the case of complex oligosaccharide
synthesis, the enzymatic approach is especially noteworthy for
glycosidic bond formation. Several efficient chemical glycosylation methods have been developed over the past few
decades (Barresi and Hindsgaul, 1995; Schmidt, 1986), yet
none rival the regio- and stereospecificity that results when the
catalyst is a glycosyltransferase.
Glycosyltransferases have been applied as catalysts in the
construction of numerous complex glycoconjugates (Wong et al.,
1995b). On a preparative scale, it is advantageous to employ
protocols for the regeneration of sugar nucleotides in conjunction
with glycosyltransferase catalysis (Figure 2A,B). In this manner,
the expense of sugar nucleotides, and product inhibition of the
glycosyltransferase by the resulting nucleoside di- or monophosphates (NDPs or NMPs) are overcome. Recycling systems
for UDP-Gal incorporating UDP-Gal 4-epimerase (UDPGE;
Wong et al., 1982) Gal-1-phosphate uridyltransferase (Gal-1-P
UT; Wong et al., 1992), or sucrose synthetase (Elling et al.,
1993) for use with β1,4-GalT have been reported for the largescale synthesis of N-acetyllactosamine (LacNAc). In conjunction
with α2,3-SiaT or α2,6-SiaT, CMP-NeuAc recycling systems
(Ichikawa et al., 1991a) have been utilized for the sialylation of
LacNAc-based glycoconjugates. For example, solution- and
solid-phase syntheses of complex structures such as 3′-sialylLacNAc (3′-SLN) and sialyl Lewis x (sLex; Halcomb et al.,
1994; Wong et al., 1995b) have been accomplished by these
methods. Other sugar nucleotide recycling systems have also
been explored, including GDP-Fuc (Ichikawa et al., 1992),
GDP-Man (Wang et al., 1993; Herrmann et al., 1994), UDPGlcNAc (Look et al., 1993), and UDP-GlcUA (Gygax et al.,
1991). However, these systems are not generally utilized as extensively, as some of the enzymes required for the regeneration
schemes are difficult to obtain.
Recent advances in sugar nucleotide recycling systems
include efforts in engineering and chemical synthesis. Genetic
manipulation of sugar nucleotide biosynthetic pathways in
microorganisms has yielded coupled systems for the preparative
scale synthesis of UDP-Gal (Koizumi et al., 1998) and CMPNeuAc (Endo et al., 2000). Notably, this strategy allowed the
synthesis of the globotriose trisaccharide without side-products
from simple and inexpensive starting materials (Figure 3). In
addition, chemical synthesis has provided new coupling
methods (Wittmann and Wong, 1997) for high yielding
production of GDP-Fuc, GDP-Man, and UDP-Gal. Generation
of fusion enzymes such as CMP-NeuAc synthetase/α2,3-SiaT
(Gilbert et al., 1998), UDPGE/α1,3-GalT (Wang et al., 1999),
and UDPGE/β1,4-GalT (Paulson et al., unpublished observations)
are a result of progress in enzyme engineering. Furthermore,
new inexpensive kinase catalyst systems such as polyphosphate kinase/polyphosphate serve as an alternative to the
pyruvate kinase/PEP system (Noguchi and Shiba, 1998).
The second basic strategy for relief of glycosyltransferase
product inhibition is to utilize a phosphatase to break down the
inhibitory product NDP or NMP (Figure 2C; Unverzagt et al.,
1990). While sugar nucleotide regeneration schemes are by far
the most efficient method on a preparative scale, the simple
addition of a phosphatase is often more convenient on a small
scale. Representative examples in which this method has been
employed include the synthesis of complex oligosaccharides
from the glycodelins (Depre et al., 1999) as well as the
Complex carbohydrate synthesis tools for glycobiologists
Fig. 3. Large-scale production of UDP-Gal and globotriose utilizing
metabolically engineered bacterial cells.
tertiary alcohols of synthetically prepared acceptor substrates
(Qian et al., 1999).
Glycosidases
Fig. 2. Strategies to relieve product inhibition in glycosyltransferase-catalyzed
glycosylation reactions. (A) Recycling of nucleoside diphosphate sugars: E1,
glycosyltransferase; E2, pyruvate kinase; E3, sugar nucleotide
pyrophosphorylase; E4, pyrophosphatase. (B) Recycling of nucleoside
monophosphate sugars: E1, glycosyltransferase; E2, myokinase; E3, pyruvate
kinase; E4, sugar nucleotide synthetase; E5, pyrophosphatase. (C) Addition of
phosphatase: E1, glycosyltransferase; E2, alkaline phosphatase.
decasaccharide sialyl trimeric Lewis x (Koeller and Wong,
2000). In studies of multivalency, sialyl Lewis x dimers were
constructed chemoenzymatically, based on a series of glycosyltransferase-catalyzed reactions (DeFrees et al., 1995; Lin et
al., 1995b; Wittmann et al., 1998). Fluorescent sLex derivatives
were also synthesized analogously for use in cell staining
experiments (Figure 4; Wittmann et al., 2000). The finding that
mercury-labeled CMP-NeuAc served as a substrate for α2,3SiaT allowed the construction of heavy atom labeled sLex
glycans (Martin et al., 1998). Other studies with unnatural
substrates have focused on the ability of retaining glycosyltransferases to transfer carbohydrates to sterically hindered
Glycosidases can also be induced to serve as glycosidic-bond
forming catalysts, albeit their physiological function is the
cleavage of glycosidic linkages (Crout and Vic, 1998).
Glycosidases are often much less expensive, more widely
available, and more stable than glycosyltransferases, although
synthetic drawbacks include weak regiospecificity and
competing product hydrolysis. Recently, synthetic difficulties
associated with glycosidases have been overcome with newly
developed synthetic strategies.
Novel donor substrates that favor trans-glycosylation rather
than hydrolysis have been identified in certain cases. For
example, when galactose oxidase was utilized in tandem with
β-galactosidase from Bacillus circulans, a 6-oxo-galactosyl
substrate was generated as an intermediate (Figure 5A). In this
system, the LacNAc end product was obtained in nearly double
the yield normally observed, due to lack of competing hydrolysis of the 6-oxo-substrate (Kimura et al., 1996). With the
polymerizing enzyme chitinase, a transition state analog of the
enzymatic hydrolysis reaction was employed as a polymerization
monomer (Figure 5B). In this case, polymerization was
observed at specified pH values, in the absence of hydrolysis
(Kobayashi et al., 1996). Mutant glycosidases devoid of hydrolyzing activity have also recently been reported (Mackenzie et
al., 1998; Malet and Planas, 1998). These enzymes lack a catalytic nucleophile in the active site, and were utilized in
conjunction with activated glycosyl donors of the opposite
configuration as the desired product. Furthermore, to circumvent issues of regioselectivity, catalyst libraries can now be
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K.M.Koeller and C.-H.Wong
Fig. 4. The multienzyme synthesis of a bivalent fluorescently labeled sLex
conjugate.
screened for the ability to form a desired specific linkage. This
was exemplified by using a thermophilic glycosidase library to
discover a Fuc(β1,2)Xyl specific enzyme (Li et al., 1998).
One-pot multi-enzyme synthesis
As a large majority of enzymes function optimally near neutral
pH, conditions for multi-enzyme one-pot oligosaccharide
synthesis can often be identified. Glycosyltransferase based
one-pot systems with the regeneration of sugar nucleotides
have been reported for the synthesis of 6′-SLN (Ichikawa et al.,
1991b), the α-Gal epitope (Figure 6A; Hokke et al., 1996; Fang
et al., 1998), and a hyaluronic acid polymer (Figure 6B; De
Luca et al., 1995). A one-pot synthesis of Lex using β1,4-GalT
and α1,3-FucT has also been achieved (Arlt and Hindsgaul,
1995). Furthermore, glycosidases and glycosyltransferases
have been applied together in one-pot syntheses. In these
cases, the glycosyltransferase removes the product from the
glycosidase-catalyzed reaction, which drives the glycosidase
equilibrium in the synthetic direction. This strategy has been
employed in the synthesis of a core 2 trisaccharide (Dudziak et
al., 1998), the sialyl-TF antigen (Kren and Thiem, 1995), and
6′-SLN (Herrmann et al., 1993).
Enzymes in glycopeptide, glycolipid, and glycoprotein
synthesis
In addition to glycosidic bond–forming catalysts, other enzymatic transformations are valuable for the facile construction
of complex glycoconjugates. Relevant posttranslational
modifications of oligosaccharides and glycopeptides often
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Fig. 5. New strategies in glycosidase-based synthesis. (A) Tandem use of
galactose oxidase and β-galactosidase for the synthesis of LacNAc.
(B) Utilization of an oxazoline transition state analog as the monomer for
chitinase-catalyzed polymerization.
provide required biological recognition elements. Enzymes
have also had great utility in protecting group removal or in the
synthesis of unnatural sugar derivatives. The combination of
glycosyltransferases with other biological catalysts or
synthetic strategies often provides an efficient route toward
complex glycoconjugates other than oligosaccharides.
Taking advantage of the wealth of knowledge and techniques provided by the exquisitely developed methods for
solid-phase peptide synthesis, solid-phase glycopeptide
synthesis has been very successful. However, one of the main
hurdles in establishing this procedure has been cleavage of the
glycopeptide product from the resin without destruction of
acid- or base-sensitive glycan functionality. When performing
enzymatic reactions, the solid phase selected also must have
specific properties, such as water compatibility and appreciable
swelling that will allow the macromolecular enzyme access to
the tethered substrate. Monitoring solid-phase glycosyltransferase reactions can also be difficult, often requiring that
cleavage from the support be achieved before reaction progress
can be assessed.
Complex carbohydrate synthesis tools for glycobiologists
Fig. 7. (A) Solid-phase chemoenzymatic synthesis of glycopeptides from
MAdCAM-1. (B) Solution-phase portion of the chemoenzymatic synthesis of
sulfated glycopeptides from PSGL-1.
Fig. 6. One-pot enzymatic syntheses involving glycosyltransferases and sugar
nucleotide regeneration systems. (A) Synthesis of the α-Gal trisaccharide.
(B) Synthesis of a hyaluronic acid polymer; E1, hyaluronic acid synthase; E2,
UDP-Glc dehydrogenase; E3, UDP-Glc pyrophosphorylase; E4, UDP-GlcNAc
pyrophosphorylase; E5, pyruvate kinase; E6, lactate dehydrogenase; E7,
inorganic pyrophosphatase.
To resolve some of these issues, solid-phase linkers that can
be cleaved under essentially neutral conditions have been
developed (Gewehr and Kunz, 1997). The protease chymotrypsin has been utilized as a cleaving reagent following
glycosyltransferase-catalyzed assembly of a solid-phase bound
3′-SLN glycopeptide (Schuster et al., 1994). In the synthesis of
glycopeptides from MAdCAM-1, a Pd(0)-labile HYCRON
linker was employed for facile release following the enzymatic
construction of sLex attached to threonine (Figure 7A; Seitz
and Wong, 1997). A combination of chemical, solid-phase,
and enzymatic strategies can also be efficient, as was shown in
the synthesis of sulfated glycopeptides from PSGL-1 (Figure 7B;
Koeller et al., 2000a,b). In this case, a pre-glycosylated threonine residue was incorporated into the solid-phase synthesis.
Following peptide assembly, the construct was cleaved from
the resin, sulfated by chemical means, and enzymatically glycosylated in solution.
Another solution-phase strategy towards glycopeptides has
been to employ subtilisin as a peptide bond forming catalyst.
Several N- and O-linked glycopeptides have been synthesized
by this protocol (Wong et al., 1993). In order to maximize
synthetic efficiency, the positioning of the glycan in precise
subtilisin subsites has been investigated. In this case, the
preparation of N-protected peptide esters as substrates for
subtilisin-catalyzed glycopeptide condensation was accomplished
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K.M.Koeller and C.-H.Wong
on Rink Amide resin (Witte et al., 1998). The culmination of
these efforts has been a general strategy for the synthesis of
homogeneous glycoproteins (Figure 8), and was applied to
construction of a homogeneous RNase B glycoform. The
natural RNase B glycan was initially cleaved by Endo-H,
leaving the reducing terminal GlcNAc attached to the protein
surface. Further addition of desired sugars was achieved with
glycosyltransferases. In addition, ligation of glycopeptide
fragments prepared by solid-phase methods was accomplished
with subtilisin (Witte et al., 1997).
Other methods of constructing homogeneous glycoproteins
are also being pursued. Intein-mediated protein ligation has
provided an avenue for the modification of proteins through a
natural protein-splicing mechanism (Figure 9; Tolbert and
Wong, 2000). By this methodology, glycans or other molecular
probes can be appended to the C-terminus of a given protein. Alternatively, native peptide ligation (Shin et al., 1999) or endoglycosidase catalyzed trans-glycosylation (Figure 10; Haneda
et al., 1998; Wang et al., 1997) are other tools available for
glycoprotein construction and remodeling. Employing Endo-A
or Endo-M, it is possible to synthesize a protein with homogeneous N-linked glycans in a single enzymatic transformation.
New strategies toward glycolipids are also being developed.
Employing a water-soluble polymeric solid phase, glycosyltransfer enzymes have been utilized in solid-phase glycolipid
synthesis (Figure 11). Following glycosylation, cleavage from
the polymer support was accomplished by ceramide glycanase
catalyzed trans-glycosylation, resulting in the transfer of the
oligosaccharide directly from the polymer to ceramide
(Nishimura and Yamada, 1997). Polysialyltransferases have
Fig. 8. Combination of subtilisin and glycosyltransferases for the synthesis of homogeneous glycoproteins.
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Complex carbohydrate synthesis tools for glycobiologists
Fig. 10. Endo-glycosidase catalyzed trans-glycosylation for the synthesis of
homogeneous glycoproteins.
Fig. 9. Intein-mediated synthesis coupled with glycosyltransferase-catalyzed
glycosylations for the synthesis of homogeneous glycoproteins.
also recently been investigated for the ability to synthesize
polysialylated glycolipids (Shen et al., 1999). Furthermore,
posttranslationally modified gangliosides like 9-O-acetyl-GD3
serve as cancer antigens, and facile methods for their synthesis
are desirable. Employing subtilisin as a catalyst, site-specific
esterification of GD3 was accomplished (Takayama et al.,
1996).
Sulfation on either the hydroxyl group of an oligosaccharide
or the peptide to which it is attached is an important posttranslational modification that has not yet been studied in great
detail. However, methods for the recycling of PAPS, the
universal biological sulfating reagent, have been reported.
Originally, a six-enzyme recycling system was employed in
the sulfation of chito- and LacNAc-based oligomeric substrates
(Lin et al., 1995a). A simpler two-enzyme regeneration technique which utilizes aryl sulfotransferase for the same purpose
has also recently been described (Figure 12; Burkart et al., 1999).
Furthermore, the tyrosylprotein-sulfotransferase responsible for
the sulfation of the N-terminus of glycoprotein PSGL-1 has
been utilized to a limited extent for micro-scale synthesis
(Leppanen et al., 1999). The ability of protein-sulfotransferases to be employed with the regeneration of PAPS may
provide efficient synthetic methods for sulfated glycoproteins
in the future.
Enzymes in the synthesis of monosaccharides and their
analogs
Aldolases have immense utility in the production of monosaccharides and their derivatives, and synthetic applications
have been reviewed extensively (Wong and Whitesides, 1994;
Gijsen et al., 1996; Fessner, 1998; Machajewski and Wong,
2000). The use of pyruvate-dependent aldolases such as
NeuAc aldolase has allowed the preparation of various NeuAc
derivatives, including imino-sugars (Zhou et al., 1993; Fitz et al.,
1995). The NeuAc aldolase reaction is a substrate-control
process, i.e., using L-sugars as substrates, L-NeuAc derivatives
are obtained. However, employing the technique of directed
evolution, pyruvate-dependent D-KDPG aldolase has been altered
to a KDG aldolase which accepts both L- and D-glyceraldehyde
as substrate with the same facial selectivity (Fong et al., 2000).
Thus, the scope of enzymatic aldol addition reactions can be
extended through enzyme engineering.
The acetaldehyde-dependent aldolase DERA is unique in that it
catalyzes the condensation of two aldehydes. DERA-catalyzed
reactions have provided many unnatural sugar derivatives,
including iminocyclitols and deoxy sugars. DERA has also
been employed in tandem with either FDP aldolase or NeuAc
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K.M.Koeller and C.-H.Wong
Fig. 12. Two-enzyme PAPS regeneration system for the synthesis of sulfated
chito-oligomers.
Fig. 11. Enzymatic glycolipid synthesis on a water-soluble polymer,
culminating in ceramide glycanase catalyzed trans-glycosylation.
aldolase to afford novel deoxysugars and deoxy-NeuAc
derivatives (Gijsen and Wong, 1995).
DHAP-dependent aldolases have been utilized for the
synthesis of disaccharide mimetics (Eyrisch and Fessner,
1995), as well as carbohydrates containing 13C labels, heteroatoms, and deoxygenated sites. Furthermore, the condensation
of DHAP with pentoses or hexoses affords NeuAc and KDO
analogs, and iminocyclitols and L-sugars can also be readily
obtained (Wong et al., 1995a; Moris-Varas et al., 1996;
Takayama et al., 1997). Recently, a four-enzyme one-pot
synthesis of 5-deoxy-5-ethyl-xylulose utilizing FDP aldolase
as one of the catalysts has been reported (Schoevaart et al.,
1999). Although the enzymes employed in the scheme had a
range of pH optima, enzyme activities were controlled by
variation of pH over the course of the reaction.
Chemical one-pot programmable oligosaccharide synthesis
It is readily apparent that enzymes have great utility in glycoconjugate synthesis. In certain cases, complex glyco-structures
can be assembled in one pot. However, this is not invariably
the case, since enzymatic reaction conditions are not always
compatible with one another. Furthermore, enzymes that
catalyze every specific desired linkage in a carbohydrate chain are
not currently available, and enzymes may also be unpredictable
1164
with unnatural structures as substrates. As such, chemical and
chemoenzymatic carbohydrate syntheses remain valuable
pursuits. If a general reaction condition for chemical glycosylation can be identified, chemical synthesis may be more
globally amenable to one-pot strategies. Toward this end, the
recently reported one-pot programmable synthesis of oligosaccharides establishes a protocol for the accurate construction
of multiple sequential glycosidic bonds. It is hoped that eventually these chemical glycosylation methods will be developed
to mirror the specificity of enzyme-catalyzed glycosylation
reactions.
Solution-phase chemical methods for one-pot oligosaccharide
synthesis have been explored over the past decade by a number
of research groups (Raghavan and Kahne, 1993; Ley and
Priepke, 1994; Grice et al., 1997). The majority of one-pot
synthetic techniques undertaken to date have been largely
qualitative. Through various strategies, multiple glycosyl
donors have been selected to react in a specific order, thus
resulting in a single oligosaccharide product. Techniques identified for the control of glycosyl donor reactivity have included
careful selection of hydroxyl protecting groups (Fraser-Reid et
al., 1992), manipulation of thioglycoside donor steric bulk
(Geurtsen et al., 1997), use of several glycosyl donors of
sequentially increasing reactivity (Yamada et al., 1994), or
variation of the activating reagent (Chenault and Castro, 1994).
An orthogonal protection/deprotection strategy was also
reported for the synthesis of an oligosaccharide library, but the
Complex carbohydrate synthesis tools for glycobiologists
Fig. 13. Optimer-designed synthesis of a linear tetrasaccharide
extensive time commitment precluded its use as a general practical method (Wong et al., 1998).
The notion that glycosyl donor reactivity could be quantitated,
however, was the innovation that was necessary in order for
one-pot synthesis to advance to the programmable stage.
Quantitative tactics were first applied by Ley to explain the
results observed in one-pot syntheses with fully protected
mannose and rhamnose glycosyl donors. Mannosyl and rhamnosyl donors were assigned relative reactivity values (RRV)
which were determined by NMR (Douglas et al., 1998). RRVs
describe the product ratio when two glycosyl donors compete
for a single reference acceptor. These values could in turn be
utilized to predict the product outcome in a one-pot synthesis
where multiple glycosyl donors were present, and could also
aid in the selection of donor sugars.
Subsequently, the Wong group took an alternative and more
convenient route to determining glycosyl donor reactivity by
HPLC (Zhang et al., 1999). Donors and donor-acceptors
(i.e., thioglycosides with one hydroxyl exposed) evaluated
contained various protecting group patterns that allowed trends
in reactivity to be identified within a given series of carbohydrates. These trends were tabulated to create a database,
search engine, and computer program. The Optimer program
contains information for each protected monosaccharide
building block, such as the name of the residue, the position of
unprotected hydroxyl groups, and whether the 2′-substituent
directs glycosylation in an α- or β-linkage. After the user
selects an oligosaccharide structure of synthetic interest, the
program lists the 100 best combinations of reagents for its
Fig. 14. Optimer-designed synthesis of a branched tetrasaccharide.
preparation, and the predicted yield. To prove its usefulness,
Optimer was originally employed to design one-pot syntheses
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K.M.Koeller and C.-H.Wong
Fig. 15. Comparison of current solid-phase methods and one-pot programmable techniques for the synthesis of oligosaccharides.
for five different oligosaccharides, and has been successfully
applied to the preparation of linear (Figure 13) and branched
(Figure 14) oligosaccharides in a controlled fashion (Ye and
Wong, 2000).
Future developments will include additional sugars in the
database, more options for branched oligosaccharide synthesis,
and examination of alternative glycosyl donors and activation
strategies. All building blocks are currently prepared separately as stable entities, and will soon become commercially
1166
available. The end goal is to generate the largest data base
possible, so that any desired oligosaccharide structure can be
formed in a single one-pot reaction designed by Optimer.
Tedious protection and deprotection manipulations necessary
for stepwise solid-phase synthesis can therefore be eliminated
(Figure 15). Furthermore, the reducing terminal acceptor in the
programmable synthesis could be attached to a solid support,
allowing the one-pot synthesis products to be adapted to the
solid-phase for facile purification or further elaboration. With
Complex carbohydrate synthesis tools for glycobiologists
continuing development, one-pot programmable synthesis will
provide a valuable tool for biologists and chemists alike in the
study of carbohydrate function.
Future directions
Innovations in enzymatic and chemical methods for the
synthesis of complex oligosaccharides and glycoconjugates
have had great value in the advancement of glycobiology.
These techniques have greatly simplified the synthesis of
carbohydrate-based structures, making automated carbohydrate synthesis a more realistic goal. Enzymatic methods
will gain increased utility as more glycosyltransferases become
available and substrate cost decreases. Moreover, as homogeneous glycoproteins are being more readily constructed, the
effects of specific glycan structure on protein function will be
possible. Posttranslational modifications are also important
additions to glycoconjugate structure, and studying the roles of
sulfation, phosphorylation, and esterification are interesting
prospects. Overall, although carbohydrates remain poorly
understood when compared to other biomolecules, general
themes in glycoconjugate function are beginning to emerge.
Further progress in glycobiology will be greatly aided by
techniques that allow facile synthetic access to specific glycoconjugates. The methods described herein represent the most
readily available synthetic tools for glycobiologists at the
present time.
Abbreviations
NDP, nucleoside diphosphate; NMP, nucleoside monophosphate;
UDP-Gal, uridine 5′-diphospho-α-galactose; UDPGE, UDP-Gal
4-epimerase; Gal-1-P UT, Galactose1-phosphate uridyltransferase; GalT, galactosyltransferase; LacNAc, N-acetyllactosamine; SiaT, sialyltransferase; CMP-NeuAc, cytidine 5′-monophospho-β-N-acetylneuraminic acid; SLN, sialyl-N-acetyllactosamine; GDP-Fuc, guanosine 5′-diphospho-β-L-fucose;
sLex, sialyl Lewis x [NeuAcα2,3Galβ1,4(Fucα1,3)GlcNAc];
FucT, fucosyltransferase; GDP-Man, guanosine 5′-diphosphoα-mannose; UDP-GlcNAc, uridine 5′-diphospho-α-Nacetylglucosamine; UDP-GlcUA, uridine 5′-diphospho-α-glucuronic acid; PEP, phospho(enol)pyruvate; Pyr, pyruvate; Xyl,
xylose; Lex, Lewis x, [Galβ1,4(Fucα1,3)GlcNAc]; MAdCAM-1,
mucosal addressin cell adhesion molecule-1; PSGL-1, P-selectin
glycoprotein ligand-1; GlcNAc, N-acetylglucosamine; RNase
B, ribonuclease B; PAPS, 3′-phosphoadenosine-5′-phosphosulfate; KDPG aldolase, 2-keto-3-deoxy-6-phosphogluconate
aldolase; KDG, 2-keto-3-deoxy-D-glucarate; FDP aldolase,
fructose 1,6-diphosphate aldolase; DHAP, dihydroxyacetone
phosphate; DERA, 2-deoxyribose 5-phosphate aldolase; KDO,
2-keto-3-deoxyoctanoate.
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